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Nutritional assessment of agricultural migrant workers in southern Brazil Swann, Marjorie Anne 1979

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NUTRITIONAL ASSESSMENT OF AGRICULTURAL MIGRANT WORKERS IN SOUTHERN BRAZIL MARJORIE ANNE SWANN B . S c , U n i v e r s i t y of B r i t i s h Columbia r 197 6 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY" OF GRADUATE STUDIES i n HUMAN NUTRITION SCHOOL OF HOME ECONOMICS We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, 197 9 © Marjorie Anne Swann , In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department nf A / r t / ^ e . E<LOr\Om<CS The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 - i -ABSTRACT With the urbanization phenomenon, a population of u n s k i l l e d migrant workers commonly known as Boia- Frias has r a p i d l y grown up i n slums on the peripheries of B r a z i l i a n c i t i e s . This study was ca r r i e d put to assess the food habits and n u t r i t i o n a l status of 100 Boia-Fria families of V i l a Recreio, a slum area on the edge of Ribeirao Preto, S.P., B r a z i l , using dietary, anthropometric, and biochemical investigations. Qual-i t a t i v e l y , the Boia-Fria diet was monotonous and simple, consisting b a s i c a l l y of polished r i c e , beans, white bread, and coffee with sugar. In general, the foods which were lacking were: milk products, meats, f i s h , eggs, poultry, non-refined grain products, and f r u i t s and vegetables r i c h i n vitamins A and C. Foods of low n u t r i t i o n a l value such as starchy gruels, sugar-water, herb tea, coffee with sugar and soft drinks were commonly used as weaning foods. Although dietary practices of pregnant and l a c t a t i n g women were poor, breastfeeding was s t i l l practiced by most mothers. According to 24-hour dietary r e c a l l data, conditions existed which were conducive to the development of n u t r i t i o n a l problems, esp e c i a l l y with respect to calcium, vitamin A, r i b o f l a v i n , n i a c i n , vitamin C, and iron , of the nutrients tested and with respect to quantitative intake of food. - 11 -Biochemical data confirmed the presence of ear ly m a l n u t r i t i o n , p r e - c l i n i c a l i n nature for about 257o of the populat ion wi th respect to v i tamin A, carotene, and i r o n . Plasma c h o l e s t e r o l , t o t a l l i p i d and v i tamin E values were found to be normal . Anthro-pometric examinations revea led c l e a r signs of c l i n i c a l u n d e r n u t r i t i o n among men and women as w e l l as some de-gree of obes i ty among women. C h i l d m o r t a l i t y data provided evidence of some advanced c l i n i c a l m a l n u t r i t i o n among c h i l d r e n . Bas ic causes of m a l n u t r i t i o n among the B o i a - F r i a s inc luded the f o l l o w i n g e c o l o g i c a l f a c t o r s : recent u r b a n i z a t i o n ; hous ing , s a n i t a t i o n , and env iron-mental c o n d i t i o n s , a s soc ia ted wi th ser ious i n f e c t i o n problems; poverty; i l l i t e r a c y ; and an ignorance of what cons t i tu tes good n u t r i t i o n . Recommendations for i n t e r -vent ion and "long-range" n u t r i t i o n programs to minimize the harsh e f fec t s of poverty and upheaval on these migrant workers of B r a z i l were suggested. - i i i -ACKNOWLEDGEMENT S To my research director, Dr. I.D.. Desai, I ex-tend my sincere thanks for his guidance and encourage-ment. Also, I would l i k e to thank the following co-workers on thi s project for without them, this study could not have been conducted: M.L. Garcia Tavares, B.S. Dutra de O l i v e i r a , F.A.M. Duarte, and J.E. Dutra de O l i v e i r a . I would also e s p e c i a l l y l i k e to thank Mr. Lewis James for his computer analysis of the data, and Dr. W. Powrie and Dr. H. Kuhnlein for t h e i r advice. Gratefully, I acknowledge the generous collabora-t i o n and help received from many i n making this research possible: Maria Luiza Mestriner of Fundacao Legiao B r a s i l e i r a de As s i s t ^ n c i a ; Elizabeth Regina Negri of Organizacao Vida Nova; Catarina Boseret of V i t a et Pax Colegio; Vera Lucia F a r i a Fernandes of Centro Educational SESI No. 346; Else Duarte and Maria Lucia Robazzi of the University of Sao Paulo Nursing School; Dr. J.E. dos Santos, Lina Azoubel, R.A.S. V e l l u t i n i and s t a f f of the C l i n i c a l Medicine Department; L.A.F. Bezerra of the University of Sao Paulo Computer Center; Dr. A.S. T r o c o l i of the University of Sao PauloHospital Laboratory; Prof. 0. Barachini of Microbiology and C l i n i c a l Laboratory; Dr. J.L. Nogueira, of the Dep. Medicina S o c i a l , Hospital das C l i n i c a s ; Pedro - i v -Veneziano of the D i e t e t i c s Department, Hospital das C l i n i c a s ; Dr. Francisco E. Martinez of the Pediatrics Department, University of Sao Paulo Medical School, Ribeirao Preto; and Paula Beatriz M. Carvalho of the C l i n i c a l Medicine Department, University of Sao Paulo, Ribeirao Preto. Also, I give my thanks to the National Research Council of Canada for providing a post-graduate fellow-ship and funds for the p a r t i a l support of this research under the supervision of Dr. I.D. Desai. F i n a l l y , I would l i k e to thank the Boia-Fria families of V i l a Recreio for t h e i r w i l l i n g p a r t i c i p a t i o n and cooperation i n this study. v -TABLE OF CONTENTS PAGE ABSTRACT i ACKNOWLEDGEMENTS . . , . i i i LIST OF FIGURES v i LIST OF TABLES v i i Chapter I INTRODUCTION 1 II REVIEW OF LITERATURE 7 A. Malnutrition Assessment Procedures ..... 8 B. Previous Relevant N u t r i t i o n a l Surveys .. 34 C. Malnutrition Ecology 59 D. National Corrective Measures .. 64 III MATERIALS AND METHODS 71 A. Sample Population 72 B. Ecological-Factors 77 C. Food Habit Information and Dietary Data . 77 D. Anthropometric Determinations .......... 90 E. Biochemical Assessment 91 F. Computer Analysis 142 IV RESULTS 144 A. Ecological Factors 145 B. Dietary Analysis 156 C. Anthropometric Assessment 170 D. Biochemical Examination 173 V DISCUSSION .... 181 A. Boia-Fria N u t r i t i o n a l Status Assessment . 182 B. Basic Causes of Malnutrition among Boia-Frias 206 C. Corrective Measures 213 VI CONCLUSIONS AND RECOMMENDATIONS 219 BIBLIOGRAPHY , 226 - v i -LIST OF FIGURES FIGURE PAGE I I - l Methods of N u t r i t i o n a l Assessment and the i r Relationship to the Natural History of Disease 9 II-2 Comparison cf Average Weight by Age of Adults of Same Height i n Northeast B r a z i l and i n North America 45 I I I - l Typical Boia-Frias at Work .............. 73 III-2 Typical Boia-Frias being Transported to Work 73 III-3 Interview i n Progress at Boia-Fria Home .. 74 III-4 Map of Ribeirao Preto Showing Location of V i l a Recreio 74 III-5 Map of South America showing B r a z i l , Sao Paulo State, Sao Paulo City and Ribeirao Preto . . 75 III-6 /5-Carotene Standard Curve . 100 III-7 Vitamin A Standard Curve .. 106 III-8 T r i f l u o r o a c e t i c Acid-Carotene Standard Curve 110 III-9 Standard Curve - Vitamin E Determination . 117 III-10 Standard Curve - Cholesterol Determination. 124 IU-11 Standard Curve - Total Lipids Determination 133 III-12 Standard Curve - Hemoglobin Determination. 143 IV- 1 Typical Infant Feeding Practices Among 'Boia F r i a ' Families 162 IV- 2 Biochemical P r o f i l e of Adult Boia-Frias: Plasma Vitamin A, Carotene, and Vitamin E , 177 IV- 3 Biochemical P r o f i l e of Adult Boia-Frias: Plasma Cholesterol, Total L i p i d s and Hemoglobin and Hematocrit 178 - v i i -LIST OF TABLES TABLE PAGE II-1 Plasma Tocopherol Levels i n Normal Human Adult Populations 36 II-2 Plasma Tocopherol Levels i n Infants of Normal Human Populations 37 II-3 B r a z i l i a n Largescale Adult Dietary Survey Results, 1961-1964 :.• 39 II-4 Adult Anthropometric Results -ICNND Northeast B r a z i l N u t r i t i o n Survey . . ... 46 II-5 Average Daily Consumption and Requirements for Various Nutrients i n South of Sao Paulo State, 1970 48 II-6 Secretaria de Planejamento da Presid^ncia da Republiea Dietary Survey - Sao Paulo Results 50 II-7 Percentage Contribution of Food Groups to Nutrient Intake for Urban Areas of the State of Sao Paulo 51 II-8 Secretaria de Planejamento da Presidencia da Republiea Anthropometric Survey -Sao Paulo Results 52 II-9 Biochemical Survey Results of Adults -Sao Paulo State 54 11-10 Mortality from S p e c i f i c Types of N u t r i t i o n a l Deficiency i n Children Under 5 Years by Age Group i n Ribeirao Preto i n 1970-1971. 55 11-11 Underlying and Associated Causes of Death i n Children Under 5 Years for Ribeirao Preto (City) and for Sherbrooke, Canada for Comparison (per 100,000 population) 56 11-12 Age D i s t r i b u t i o n of the 330 Deaths Grouped According to Their Cause i n Ribeirao Preto and Dumont 58 - v i i i -LIST OF TABLES Continued... TABLE PAGE I I I - l Quantidades de Comidas e Bebidas Consumidas no Dia Anterior (Ultimas 24 Horas) . 80 III-2 ^-Tocopherol Content of Butter 86 III - 3 Sample Calculation for the Component Method of Vitamin E Content Estimation Using Tomato, Onion, G a r l i c Sauce as the Example ........... 87 ; III - 4 Sample Coding of Food Intake Information Corresponding to 2 Tea Cups of Coffee with Milk and Sugar ......... 88 III-5 Coding of Food Nutrient Composition Table: Card Design 89 III-6 Preparation of yS-Carotene Working Standards: Procedure for D i l u t i o n with Petroleum Ether 99 III-7 ^-Carotene Standard Curve Data 99 III - 8 y9-Carotene Recovery Test 102 III - 9 ^-Carotene Recovery Test Data ........... 102 III-10 Vitamin A Working Standard Preparation: Procedure for D i l u t i o n with Chloroform 103 i n - 1 1 Vitamin A Standard Curve Data .. . . . . . 105 111-12 Vitamin A Recovery Tests: Tube Contents . 105 111-13 ^ -Carotene Chloroform Working Standards: Procedure for D i l u t i o n with Chloroform . 107 111-14 Vitamin A Recovery Tests Data 108 III-15 T r i f l u o r o a c e t i c Acid - Carotene Standard Curve Data 109 - ix. -LIST OF TABLES Continued ... TABLE PAGE 111-16 Standard Curve and Sample Preparation; Typical Absorbance Readings . . . . , . , 116 111-17 Standard Curve and Sample Preparation; Typical Absorbance Readings .......... 123 III-18 Total Lipids Working Standards; Procedure for D i l u t i o n with Chloroform , . . ,, 131 III-19 Standard Curve, Recovery Tests and Sample Preparation; Typical Absorbance Readings 132 IV-1 Typical Conditions of Boia-Fria Habitation . . 146 IV-2 Food Cost Survey . 149 IV-3 Health Personnel i n V i l a Recreio ....... 154 IV-4 Health F a c i l i t i e s and N u t r i t i o n i s t s i n Ribeirao Preto 155 IV-5 Typical Diet of Boia-Frias i n B r a z i l ..... 157 IV-6 Food Consumption of Boia-Frias by S p e c i f i c Food Group 158 IV-7 Average Daily Nutrient Intakes of Adult Male and Female Boia-Frias . . . . 164 1V-8 Numbers and Percentages with Daily Intakes Less Than 2/3 and 1/3 of the Standard , , . 165 IV-9 Percentage Contribution of Food Groups to Nutrient Intake , . 167 IV-10 Calorie Breakdown According to Source .. 169 IV-11 Fat Breakdown According to Type 169 IV-12 Anthropometric P r o f i l e of Adult Boia-Frias . 171 - x -LIST OF TABLES Continued ... TABLE PAGE IV-13 Percentage D i s t r i b u t i o n of Boia-Frias According to Standard Anthropometric C r i t e r i a . 172 IV-14 Anthropometric P r o f i l e of Adult Boia-Frias According to Age 174 IV-15 Biochemical P r o f i l e of Adult Boia-Frias .:. . . 17.5 IV-16 Biochemical P r o f i l e of Adult Boia-Frias According to Age 180 TV-1 Good Food Sources of Nutrients Most Deficient . . 189 V-2 D i s t r i b u t i o n of Plasma Vitamin E Levels of Boia-Fria.and Hyperlipemic Subjects, 199 V-3 Plasma Vitamin E D i s t r i b u t i o n i n Terms of Total L i p i d Levels for Boia-Fria and Hyperlipemic Subjects ........ , , . , 200 V-4 Biochemical L i p i d P r o f i l e of Subjects with High L i p i d Levels Compared with P r o f i l e of Boia-Frias 200 V-5 S i g n i f i c a n t Correlations between Levels of Fat-Soluble Vitamins and L i p i d Components i n Plasma 202 V-6 Different N u t r i t i o n a l Assessment Methods-S i g n i f i c a n t Correlations ......... . . . , 205 V-7 S t a t i s t i c a l l y S i g n i f i c a n t P r o b a b i l i t y Levels Relating N u t r i t i o n a l Status Data to Family Information 210 - 1 -CHAPTER 1 INTRODUCTION B r a z i l i s a vast country, the f i f t h largest i n the world and i s endowed with a wealth of yet u n u t i l i z e d natural resources. I t s population i s expected to reach over 12 0 m i l l i o n by 198 0 and has been contributed to by almost every ethnic group i n the world. About 65% of the population i s of European o r i g i n , mainly from Portugal, I t a l y and Germany. About 10% i s black, 1% Indian, less than 1% Japanese, and the remaining 24% have mixed blood (Brazil-Canada Chamber of Commerce 197 6). The national language i s Portuguese and r e l i g i o n i s predominantly Roman Catholic. B r a z i l , i n i t s development, concentrated on agriculture and remains one of the world's largest farming countries, despite evidence of growing i n d u s t r i a l i z a t i o n i n recent years. Agriculture has been the basis of the economy for centuries and even today earns foreign exchange necessary for growth i n the i n d u s t r i a l sector. In 1975, basic a g r i c u l t u r a l products and manufactured products of a g r i c u l t u r a l o r i g i n accounted for 60% of the t o t a l value of the country's export. As well, 40% of the labor force i s employed i n a g r i c u l t u r e . B r a z i l i s the world's leading producer of coffee and sugar cane and second in the production of soybeans, a crop introduced to B r a z i l as recently as a decade ago. Other main crops include r i c e , beans, corn, manioc, wheat and c i t r u s f r u i t s . B r a z i l ' s c a t t l e - 2 -population, numbering 97 m i l l i o n i n 1975, i s one of the world's largest. Also, B r a z i l i s a large scale supplier of cocoa, castor beans, natural waxes and cotton. The state of Sao Paulo i s one of the most r a p i d l y growing i n a l l respects, with an estimated population of 24 m i l l i o n by 198 0, and booming i n agriculture and industry. The climate of the c e n t r a l , west, and northeast regions of the state i s t r o p i c a l with humid summer seasons and r e l a t i v e l y dry winters. The s o i l i s very f e r t i l e i n most places, and when i t i s not, f e r t i l i z e r can make i t so. The state of Sao Paulo produces 7 0% of the country's cotton, 50% of i t s sugar, 50% of i t s f r u i t exports, l a r g e l y c i t r u s , 33% of i t s coffee, and 33% of i t s r i c e . Sao Paulo also produces potatoes, groundnuts, bananas, corn, beans, tomatoes, manioc, c a t t l e , and poultry. The B r a z i l i a n agrarian structure, as elsewhere i n Latin America, i s characterized at one end of the scale by a number of small plots that barely produce what i s needed for subsistence, and at the other end by large fazendas . In the past, most farm workers l i v e d i n the r u r a l area where they worked. However, i n B r a z i l , as elsewhere i n the world, the urbanization phenomenon has manifested i t s e l f vigorously. Now, migrant workers are transported every day by trucks from the c i t y to work i n the r u r a l areas and return to the c i t y i n the evening. These laborers are popularly referred to as Boia-Frias meaning "cold-meal eaters". The wages of these migrant workers are very low and opportunities for advancement li m i t e d . In B r a z i l , t h i s urbanization trend has resulted i n migration from North to South and from r u r a l to urban areas of u n s k i l l e d workers who end up l i v i n g i n favelas or shanty-towns on the periphery of the big c i t i e s . The urban percentage of the B r a z i l i a n population has ri s e n from 36% in 1950 to 45% i n 1960 to 53% i n 1970. Many factors are operational i n motivating t h i s trend toward urbanization including: ease of transportation; increased education and awareness of the need for increased education combined with more opportunity of receiving i t i n the c i t y ; increased awareness of middle class standards i n other countries and desire to match these same standards i n B r a z i l ; hope of r i s i n g from a lower layer of society to a higher one, which has only recently become possible; labor laws which make the conditions of the i n d u s t r i a l laborer more a t t r a c t i v e ; and f a i t h i n i n d u s t r i a l i z a t i o n as a means to grow and develop. A l l of these motivations which are common to developing countries are mixed with purely B r a z i l i a n incentives such as the expansion of p r o f i t a b l e coffee, sugar cane, and cotton production near big c i t i e s or the f l i g h t from other areas such as the Northeast which are beleaguered by droughts floods, and other calamities. The state of Sao Paulo has had considerable i n f l u x of migrants from other areas i n the l a s t 4 0 years. In 1975, i t - 4 -had a p p r o x i m a t e l y 3 m i l l i o n i n h a b i t a n t s , o n e - f i f t h o f i t s p o p u l a t i o n , born e l sewhere i n B r a z i l . R i b e i r a o P r e t o , where the s tudy p r e s e n t e d i n t h i s t h e s i s was c a r r i e d o u t , i s s i t u a t e d i n the n o r t h e a s t e r n p a r t o f the s t a t e o f Sao Pau lo , 310 km from Sao Paulo c i t y , the s t a t e c a p i t a l . Wi th an e s t i m a t e d p o p u l a t i o n o f 259,000 i n 1975 and a p o p u l a t i o n d e n s i t y o f 245 i n h a b i t a n t s per km , R i b e i r a o P r e t o i s a major c i t y o f the a r e a a c t i n g as d i s t r i b -u t i n g c e n t e r f o r the i n t e r i o r o f Sao Pau lo s t a t e and c e r t a i n d i s t r i c t s i n Minas G e r a i s , G o i a s , and Mato G r o s s o . R i b e i r a o P r e t o i s a l s o the s i t e of one o f the m e d i c a l s c h o o l s o f the U n i v e r s i t y of Sao P a u l o , w i t h i t s a t t a c h e d g e n e r a l h o s p i t a l , and has a c o n c e n t r a t i o n o f h e a l t h f a c i l i t i e s t h a t s erve the s u r r o u n d i n g r e g i o n . The r o l l i n g t e r r a i n o f the a r e a i s w e l l s u i t e d to a g r i c u l t u r a l a c t i v i t i e s , p r o d u c i n g m a i n l y c o f f e e , c o t t o n , sugar , soybean, r i c e , and c a t t l e . R i b e i r a o P r e t o i s an a t t r a c t i v e c i t y t o u n s k i l l e d l a b o r e r s from o t h e r a r e a s f o r a l l o f the f a c t o r s p r e v i o u s l y l i s t e d . V i l a R e c r e i o , a f a v e l a or shanty town, on the p e r i p h e r y of R i b e i r a o P r e t o , has become the home o f many B o i a - F r i a m i g r a n t worker f a m i l i e s . No s t u d i e s have been p u b l i s h e d which a s s e s s the n u t r i t i o n a l s t a t u s o f these f a m i l i e s , or examine t h e i r food h a b i t s . A l s o , no s t u d i e s have been p u b l i s h e d r e g a r d i n g the v i t a m i n E s t a t u s o f the B r a z i l i a n . T h e r e f o r e , a need i s i n d i c a t e d f o r r e s e a r c h i n these a r e a s . - 5 -This present study assesses the food habits and n u t r i t i o n a l status of 100 Boia-Fria migrant worker families using dietary, anthropometric, and biochemical investigations. Its purposes were: to evaluate the diet of these people using a questionnaire and the 24-hour r e c a l l procedure; to determine the n u t r i t i o n a l p r o f i l e of the matriarchal and p a t r i a r c h a l heads of these families by com-paring t h e i r anthropometric measurements with standards; to assess the n u t r i t i o n a l status of the family heads with respect to vitamin E, vitamin A, carotene, iron , cholesterol and t o t a l l i p i d s by biochemical blood analysis and comparison with standards; to correlate findings of the above and to rel a t e them to the ecolo g i c a l s i t u a t i o n of these f a m i l i e s ; and to give recommendations based on these r e s u l t s . Vitamin E status evaluation was given special attention because of the need for c l a r i f i c a t i o n of the c r i t e r i a used for this purpose. This study was part of an entire survey conducted by Dr. I.D. Desai of the University of B r i t i s h Columbia and his associates at the University of Sao Paulo Medical School i n Ribeirao Preto, B r a z i l (Desai et al. 1978a; Desai. et a l . 1978b; Desai et a l . 1980). The author of this thesis collaborated with other members of the survey team i n gathering ecological information, conducting home interviews, and c o l l e c t i n g blood samples. P a r t i c u l a r l y , the author was involved i n 24-hour r e c a l l data analysis and blood sample - 6 -treatment and analysis. Anthropometric measurements were performed by others of the survey team and used for the purpose of this thesis. - 7 -CHAPTER II REVIEW OF LITERATURE Chapter Outline Section Page A. Malnutrition Assessment Procedures 8 1. Methodology . 8 . a) Dietary 10 b) Anthropometric .-. 13 c) Biochemical ^ 2 . Standardization ^4 3. Interpretation 18 a) Dietary 18 b) Anthropometric 19 c) Biochemical 22 i ) Vitamin A and Carotene . . . . . 22 i i ) Vitamin E 24 i i i ) Plasma Lipids and Hematology ..... 33 ;B. Previous Relevant N u t r i t i o n a l Surveys 34 1. World 34 a) A g r i c u l t u r a l Workers - General Surveys 34 b) Normal Human Plasma Tocopherol Levels. 35 2 . B r a z i l 35 a) National/Largescale 35 i ) Dietary 35 i i ) Anthropometric 44 i i i ) Biochemical . . . .... 46 b) S3o Paulo State . 47 i ) Dietary 47 i i ) Anthropometric 49 i i i ) Biochemical 53 c) Ribeirao Preto 53 C. Malnutrition Ecology — . . . . . 59 D. National Corrective Measures 64 - 8 T~ According to J e l l i f f e (1966) The p r i n c i p a l aim of the n u t r i t i o n a l assessment of a community i s . . . to map out the magnitude and geographical d i s t r i b u t i o n of malnutrition as a public health problem, to discover and analyse the e c o l o g i c a l factors that are d i r e c t l y or i n d i r e c t l y responsible, and, where possible, to suggest appropriate corrective measures, preferably capable of being applied with continuing community p a r t i c i p a t i o n . With t h i s i n mind, malnutrition assessment procedures (including methodology, standardization, and interpretation of r e s u l t s ) , previous relevant n u t r i t i o n a l surveys, malnutrition ecology, and presently employed corrective measures are i n need of discussion. A. Malnutrition Assessment Procedures 1. Methodology Figure IT^-1 ,taken from Behar (1976) shows the methods of n u t r i t i o n a l assessment and t h e i r r e l a t i o n s h i p to the natural history of disease. The d i f f e r e n t methods operate at d i f f e r e n t l e v e l s of malnutrition, and complement one another. For example, food balance sheets, dietary surveys, and socioeconomic studies, define the s i t u a t i o n i n the prepathogenic stage, indicating whether conditions conducive to the development of n u t r i t i o n a l problems are present or not. Biochemical methods are e s p e c i a l l y valuable at the pathogenic but p r e - c l i n i c a l stage where they provide a basis for diagnosis of early malnutrition including exhausted reserves and physiological and Prepathogen ic P e r i o d P e r i o d o f Pathogenes i s D i m i n i s h i n g r e s e r v e s Food b a l a n c e sheets D i e t a r y surveys Socioeconomic/ s t u d i e s Reserves exhausted P h y s i o l o g i c a l and M e t a b o l i c A l t e r a t i o n s Non-s p e c i f i c s i gns and symptoms I l l n e s s Permanent damage Death G 0 Z o M o r t a l i t y d a t a C l i n i c a l 's igns and m o r b i d i t y d a t a A n t h r o p o m e t r i c s t u d i e s B i o c h e m i c a l s t u d i e s F i g u r e II-1 : Methods o f N u t r i t i o n a l Assessment and t h e i r R e l a t i o n s h i p to the N a t u r a l H i s t o r y o f D i sease (BShar 1976) - 10 -metabolic alte r a t i o n s . Anthropometric studies are useful i n assessing the problem i n i t s c l i n i c a l stages, however, they do not necessarily indicate whether the problem i s present at the moment of conducting the survey because they may merely record the permanent damage r e s u l t i n g from previous n u t r i t i o n a l d e f i c i e n c i e s . C l i n i c a l signs and morbidity and mortality data also assess the problem i n i t s advanced c l i n i c a l stages. In the present study, references w i l l be made to most of these types of methodology i n order to define the s i t u a t i o n as comprehensively as possible. a) Dietary Numerous types of dietary survey have been documented including: the 7-day record, the 24-hour r e c a l l , repeated 24-hour r e c a l l s , dietary history, the recipe method, and the food composite analysis method. Each has i t s own unique advantages and disadvantages. For this study, the 24-hour r e c a l l was chosen for i t s s i m p l i c i t y , i t s non^expected nature, i t s r e l a t i v e l y small expense, i t s easy application to populations who are i l l i t e r a t e , and i t s requirement for minimal cooperation of the subject. However, this method also has the following weaknesses: dependance on the subject's memory and honesty, and questionable representation of habitual diet, p a r t i c u l a r l y with respect to vitamins A and C which vary markedly from day to day. Gam et a l . (1975) strongly urge that analyses of one-day dietary - 11 -intakes be limited to estimating group trends. Chalmers et a l . (1952) write that a dietary record need consist of only one day when characterizing the dietary intake of a group, and that i n order to estimate the mean intake for a group with greater precision, taking more subjects rather than more days gives greater e f f i c i e n c y . They suggest that a 24-hour r e c a l l may be more representative than a one day record for then there i s no danger of the subject subconsciously eating a d i e t other than usual because of the survey. For s t a t i s t i c a l purposes, they suggest using six t y people or more for group studies to put 95% confidence l i m i t s on either side of the mean, equal to 10% of the National Research Council standard. Another factor of consideration in determining sample size i s that the more homogeneous the d i e t , the smaller the error, and the smaller the required sample s i z e . Schaeffer (1966) found that the 24-hour r e c a l l method, when compared with the recipe method wherein food items were weighed p r i o r to and a f t e r cooking and food consumption was also weighed, gave 8% less c a l o r i c intake for Ecuadorian c i v i l i a n s . Both of these survey types used food composition tables. When Schaeffer compared the 24-hour r e c a l l method with the food composite analysis method, i n which the food and waste was weighed and the food was sampled and analyzed chemically for - 12 -c a l o r i e s , the 24-hour r e c a l l method gave 15% less c a l o r i c intake. According to Whiting and Leverton (1960), i t i s important to r e a l i z e that while laboratory determinations can be expected to report within known l i m i t s the composition of the actual samples analyzed, food tables i n common use are constructed to report average food values. Also, the more common or usual the foods i n the di e t and the more simply they are prepared, then the moire representative w i l l be the values calculated for them from food tables. Oh the basis of a summary of approximately 300 cases from 20 or more l i t e r a t u r e sources, they found that comparison of re s u l t s from the two methods of processing the same data, laboratory analysis and food table c a l c u l a t i o n , showed that for protein and c a l o r i e s the calculated values for more than 50% of the t o t a l number of cases f e l l within a range of 10% of the analyzed value. However, for f a t , calculated values tended to be considerably higher than analyzed values, presumably because of losses of f a t during meat preparation. Linusson e t a l . (1974) evaluated the v a l i d i t y of the 24-hour r e c a l l method by comparing the quantity of food consumed by 86 l a c t a t i n g women as determined by weighing with the quantity as determined by the r e c a l l method. For a l l food groups, they found a tendency to overestimate actual intake when consumption was low and underestimate i t when i t was high. The v a l i d i t y - 13 -c o e f f i c i e n t , ranging from 0.28 (salad) to 0.72 (breakfast''cereal) , showed a low co r r e l a t i o n for most foods. In eight of the food groups the biases were s i g -n i f i c a n t l y large to inva l i d a t e the method. In general, underestimation was greater than overestimation. They concluded that the r e c a l l method appears f a i r l y accurate for q u a l i t a t i v e estimation of average for a population group but not highly v a l i d for ascertaining quantity of food con-sumed. In gathering trends i n dietary patterns from sizeable population groups possibly to serve a;s baseline date i n applied n u t r i t i o n and public health programs, using the 24-hour r e c a l l method would be v a l i d , and quantitative limitations might be lessened i n a developing country where less v a r i a t i o n i n the food intake exists. b) Anthropometric N u t r i t i o n a l anthropometry i s the measurement of the variations of the physical dimensions and the gross composition of the human body at d i f f e r e n t age levels and degrees of n u t r i t i o n . The apparent s i m p l i c i t y and o b j e c t i v i t y of such measurements i s deceiving. Methods and measurements employed can vary greatly i n number and complexity and must be chosen to s u i t the purpose or objectives of the study. To allow for comparison each technique must be standardized,, and employed so as to give uniform r e s u l t s . The measurements chosen for our study, weight, height, arm circumference, - 14 -and triceps s k i n - f o l d , were obtained following the standard recognized procedures of J e l l i f f e (1966). c) Biochemical For the most part, biochemical measurements rep-resent the most objective assessment of the n u t r i t i o n a l status of, an i n d i v i d u a l . However, one must remember that blood levels of nutrients may r e f l e c t recent dietary history rather than the preferred long-term true n u t r i t i o n a l status esp e c i a l l y i f the subjects have not fasted p r i o r to blood c o l l e c t i o n . In the present study, the following biochemical measurements were made; plasma vitamin A, plasma carotene, plasma vitamin E, plasma cholesterol, plasma t o t a l l i p i d s , blood hemoglobin and hematocrit. 2. Standardization A l l b i o l o g i c a l standards exhibit a range of v a r i a t i o n within zones of apparent normality. Therefore, i n defining standards, two fundamental questions must be answered. F i r s t l y , what l e v e l of health i s to be sought and secondly, what degree of coverage of individuals i s to be achieved? With regard to the f i r s t question, d i f f e r e n t levels of health, ranging from prepathogenic to pre-c l i n i c a l pathogenic to c l i n i c a l pathogenic, are associated with d i f f e r e n t levels of chronic nutrient intake, biochemical data and anthropometric r e s u l t s , as i l l u s t r a t e d by Figure I I - l . One approach.to this - 1 5 -s i t u a t i o n i s to say that the i n d i v i d u a l who i s depleted of a nutrient but does not show c l i n i c a l signs may or may not exhibit s t i l l unrecognized stigmata of malnutrition, e f f e c t s too subtle to be d i f f e r e n t i a t e d from other environmental e f f e c t s . Perhaps more important, he or she i s much more susceptible to acute malnutrition should an additional stress be imposed. V i r t u a l l y a l l bodies that put forward recommendations on suitable standards have i n mind the prevention of the depletion of body nutrients, not just the prevention of c l i n i c a l l esions. Further complicating t h i s issue however i s the body's a b i l i t y to adapt to less than optimal n u t r i t i o n . The body has a normal homeostatic mechanism involving ph y s i o l o g i c a l , enzymatic, and metabolic adjustments ' which produce changes i n the t h r i f t i n e s s i n the use of a varying nutrient supply, and/or changes i n body weight and/or composition. These adjustments, i n turn, act to impede further body change. The hereditary background of an i n d i v i d u a l may a f f e c t his a b i l i t y to e f f e c t i v e l y adapt to n u t r i t i o n a l stress. Relevant studies which indicate the occurrence of n u t r i t i o n a l adaptation include the following: 1) Hegsted et a l . (1952) from an extensive investigation of the calcium economy of men who had subsisted for years on a low-calcium d i e t , defend the proposition that a l l estimates - 16 -of calcium requirements represent primarily a study of the previous dietary calcium intake; 2) Baumann et a l . (1934), working with rats found that the number of units of vitamin A l o s t d a i l y from the l i v e r on a depletion d i e t varied d i r e c t l y with the amount of storage; i n other words, the lesser the amount of the vitamin available to the tissues, the l e s s wasteful was i t s u t i l i z a t i o n i n covering the t i s s u e requirements; 3) M i t c h e l l (1944) c i t e s instances of the type i n which dietary surveys of communities of low socio-economic status have revealed an absence of symptoms of undernutrition that might well have been expected from an assessment of the nutrient intake; 4) In growing rats, a r e s t r i c t i o n i n protein to an amount just s u f f i c i e n t to maintain a youthful body weight induced a d e f i n i t e l y more economical use of both protein and energy (Jackson 1937); 5) Human studies indicate that as the energy supply decreases,body weight, basal metabolic rate, and the energy required to move the body a l l decrease , (Mitchell 1964) . Deviations from average requirements due to adaptation to an external stress are not predictable i n any precise manner by any known method. Therefore, nutrient needs secured under non-stress conditions set a « 17 -lower l i m i t to the corresponding dietary requirement below which n u t r i t i v e disaster may r e s u l t or adaptive processes be i n i t i a t e d that, temporarily at least, may impair health and productivity (Mitchell 1964). The trend today i s to consider those adapted to a lesser nutrient intake to be in i l l health, and that the adjustments made are undesirable, though the issue i s argued i n academic c i r c l e s (Beaton and Patwardhan 1976) . Now the second question, what degree of coverage of i n d i v i d u a l s i s to be achieved? In s t a t i s t i c a l terms, i n d i v i d u a l nutrient requirements are assumed to be normally d i s t r i b u t e d about the mean because of b i o l o g i c a l v a r i a b i l i t y . Therefore, commonly, the recommended intake for the nutrient i s set at the mean plus two standard deviations except for the case of energy where the mean i s used. As a r e s u l t , most in d i v i d u a l s w i l l have actual vitamin, mineral and protein requirements below the recommended intake. The d e f i n i t i o n of recommended intake according to FAO/WHO reports i s "the amount considered s u f f i c i e n t for the maintenance of health i n nearly a l l people." The FAO/WHO recommended intakes are used i n t h i s study (Beaton and Patwardhan 1976) for a l l nutrients except for vitamin E, for which the WHO did not o f f e r an estimate of the magnitude of human need because they f e l t that too l i t t l e was known on the subject. Instead, - 18 -U.S. (National Research Council, Food and Nu t r i t i o n Board 1974) and Canadian (Committee for the Revision of the Canadian Dietary Standard 1976) standards are used for the recommended intake of vitamin E, even though they are tentative. Reference standards for the evaluation of anthropometric data are from J e l l i f f e (1966). The biochemical standards chosen for this study are given along with t h e i r source i n Table IV-15. 3. Interpretation a) Dietary In understanding and inte r p r e t i n g the results of the 24-hour r e c a l l t e s t s , one major possible source of inaccuracy to be remembered i s over-reporting out of vanity or shame of poverty or under-reporting to detract attention from wealth or i n the hope that r e l i e f w i l l be forthcoming (Rey 1962). Application of the recommended energy intakes to the 24-hour r e c a l l r esults for the group i s appropriate since the recommended energy intakes are based on averages and man appears toaeat food i n order to s a t i s f y his energy needs, providing there are no s o c i a l or other constraints to the d i s t r i b u t i o n and i n d i v i d u a l selection of food intake. For the other nutrients, comparing average intakes of the population with recommended intakes i s a very crude i n d i c a t i o n of the possible s i t u a t i o n since the recommended intakes are based on averages plus two standard deviations and also, man does not seem to na t u r a l l y adjust his nutrient intake to his nutrient - 1.9. -r e q u i r e m e n t a s h e d o e s w i t h r e s p e c t t o e n e r g y . T h e r e -f o r e , i n a s s e s s i n g t h e r i s k o f d e f i c i e n c y o f t h e o t h e r n u t r i e n t s , o n e m u s t c o n s i d e r t h e v a r i a t i o n o f n u t r i e n t r e -q u i r e m e n t s among i n d i v i d u a l s i n t h e g r o u p a n d a l s o t h e v a r i a -t i o n o f h a b i t u a l n u t r i e n t i n t a k e among i n d i v i d u a l s . I n t e r -p r e t a t i o n o f t h e s e r e s u l t s i s d i f f i c u l t a n d a t b e s t , n e e d s c a n o n l y b e s p e c u l a t e d ( B e a t o n a n d P a t w a r d h a n 1976). A s a c o m p a r i s o n c r i t e r i a , t w o - t h i r d s o f t h e r e c o m m e n d e d i n t a k e w a s u s e d t o c o r r e c t f o r t h e e f f e c t o f a d d i n g t w o s t a n d a r d d e v i a t i o n s . b ) A n t h r o p o m e t r i c J e l l i f f e QL966) w r i t e s t h a t c o m m u n i t y s t a n d a r d s f o r a n t h r o p o m e t r i c m e a s u r e m e n t s a r e d i f f i c u l t t o d e f i n e s i n c e t h e d e s i r e d e n d - p r o d u c t o f o p t i m a l n u t r i t i o n a t p r e s e n t l a c k s d e f i n i t i o n . T h e r e h a s b e e n a c o n s t a n t s e c u l a r t r e n d i n t h e p a s t c e n t u r y t o w a r d s h e a v i e r a n d t a l l e r p o p u l a t i o n s i n t h e w e s t e r n w o r l d t h a t h a s made p r e v i o u s " s t a n d a r d s " p r o g r e s s i v e l y o u t - o f - d a t e . T h e p a r a l l e l b e t w e e n t h i s a n d i m p r o v e m e n t i n n u t r i t i o n a n d o t h e r e n v i r o n m e n t a l i n f l u e n c e s , s u c h a s d i s e a s e c o n t r o l , seems c e r t a i n . H o w e v e r , a t t h e same t i m e t h e r e may w e l l b e d e v e l o p i n g a n u n d e s i r a b l e r e l a t i o n b e t w e e n l a r g e r , e a r l y - m a t u r i n g , p o s s i b l y o v e r f e d p o p u l a t i o n s a n d a s u b s e q u e n t d i s e a s e p a t t e r n i n a d u l t h o o d t h a t i n c l u d e s among o t h e r t h i n g s , a n i n c r e a s i n g i n c i d e n c e o f a t h e r o -s c l e r o s i s a n d o b e s i t y . O p t i m u m g r o w t h l e v e l s c a n b e e v a l u a t e d l o g i c a l l y o n l y i n r e l a t i o n t o p r e s e n t a n d - 20 -f u t u r e h e a l t h . T h i s i s the b a s i s f o r the " d e s i r a b l e " s t a n d a r d s of weight f o r a d u l t s l a i d down by the S o c i e t y o f A c t u a r i e s which are r e l a t e d to c a r d i o v a s c u l a r d i s e a s e and l o n g e v i t y . J e l l i f f e (1966) d e r i v e d h i s s t a n d a r d s f o r h e i g h t and weight from t h e s e . A c c o r d i n g t o Garn (1965) , r e f e r e n c e s t a n d a r d s f o r a g i v e n p o p u l a t i o n sh ou ld be those a p p r o p r i a t e t o t h a t p o p u l a t i o n , making use of e c o n o m i c a l l y f a v o r e d s t r a t a o r more f o r t u n a t e subgroups to p r o v i d e an i n d i c a t i o n o f optimum growth f o r c h i l d and the measures o f adequate n u t r i t i o n i n a d u l t s . D i f f e r e n c e s i n body b u i l d and f a t -f r e e mass c o m p l i c a t e the u n i v e r s a l a p p l i c a t i o n o f s imple s t a n d a r d s o f h e i g h t and weight o r v a r i o u s r a t i o s i n v o l v i n g the two. J e l l i f f e (1966) agrees t h a t the u l t i m a t e aim o f n u t r i t i o n i s t s s h o u l d be t o p r e p a r e and use l o c a l s t a n d a r d s f o r d i f f e r e n t e t h n i c groups w i t h p o t e n t i a l l y d i f f e r e n t p a t t e r n s o f growth . Growth i s i n f l u e n c e d by b i o l o g i c a l d e t e r m i n a n t s , i n c l u d i n g sex , i n t r a u t e r i n e env ironment , b i r t h o r d e r , b i r t h - w e i g h t i n s i n g l e and m u l t i p l e p r e g n a n c i e s , p a r e n t a l s i z e , and g e n e t i c c o n s t i t u t i o n , and by e n v i r o n m e n t a l i n f l u e n c e s , i n c l u d i n g c l i m a t e , season, s o c i o - e c o n o m i c l e v e l , i l l n e s s and n u t r i t i o n . The environment seems t o produce i t s e f f e c t m o s t l y by the presence (or absence) o f i n f e c t i v e , p a r a s i t i c , and p s y c h o l o g i c a l i l l n e s s e s and , above a l l , by the p lane o f n u t r i t i o n . S i n c e B r a z i l i s composed o f such a m i x t u r e - 2 1 ~ of ethnic groups ,. similar i n nature to those of North America, general non-local standards of reference compiled by J e l l i f f e (1966) were used. Selected anthropometric measurements i n adults have a useful place i n assessing past or present protein-c a l o r i e malnutrition, or overnutrition from an excessive intake of c a l o r i e s , c l i n i c a l l y presenting as obesity. Low body weights for heights may be related to a number of factors, for example, a l i g h t bone structure. However, for p r a c t i c a l purposes they are p r i n c i p a l l y a r e f l e c t i o n of body thinness due to subnormal amounts of subcutaneous f a t and muscle, the r e s u l t either of poor development, or tissue wasting, or a combination of the two. On the other hand, high body weights for heights are associated with excessive c a l o r i c intakes, together with i n s u f f i c i e n t exercise. Calorie reserves are indicated by thickness of subcutaneous f a t , usually measured, fo r convenience, at the t r i c e p s s k i n - f o l d ( J e l l i f f e 1966). The development of subcutaneous f a t i n males i s characterized by slow apposition, while i n females, i s continuous throughout childhood, adolescence and adulthood, reaching a higher l e v e l than i n males. Sexual dimorphism i n t r i c e p s s k i n - f o l d i s defined by the age of three years, and by adulthood, females exceed males by 83% (Frisancho 1974). Protein inadequacy i s best r e f l e c t e d by thin musculature, due either to poor - 22 -development or to wasting. This may be assessed by calcul a t i n g the arm-muscle circumference ( J e l l i f f e 1966). Sexual dimorphism i n upper arm muscle area i s defined by the age of 13 years, and by adulthood, males exceed females by 56% based on a sample of 12,396 Caucasians i n the U.S. (Frisancho 1974). The arm circumference, a measurement used i n the c a l c u l a t i o n of arm-muscle circumference, pro-vides a rough index of both protein and c a l o r i e reserves ( J e l l i f f e 1966). c) Biochemical In most instances, the c r i t e r i a for establishing the dietary standards have been based on biochemical a l t e r a t i o n . Therefore, i n order to provide a basis for inte r p r e t a t i o n of dietary and biochemical results with respect to vitamins A and E, detailed summaries of i n f o r -mation obtained from numerous studies of well-fed healthy people, of people obviously c l i n i c a l l y i l l with the p a r t i c u l a r form of malnutrition and of people i n between these two extremes are included. Carotene, cholesterol, and t o t a l l i p i d s are also discussed, i ) Vitamin A and Carotene Plasma levels of vitamin A and carotene are re l a t e d to the dietary intakes of these nutrients. Yet, because the body can store large reserves of vitamin A i n the l i v e r which can be drawn upon when dietary intake of the vitamin i s inadequate, plasma levels of vitamin A do not necessarily r e f l e c t recent intakes of the vitamin. Prolonged low intakes of vitamin A correlate with low - 23 -plasma vitamin A levels C< 20/ig per 100 ml) which r e f l e c t not only a long-term low intake of the nutrient but also depleted l i v e r stores. However, low serum vitamin A levels can also be caused by low protein c a r r i e r l e v e l s . Plasma vitamin A levels i n the range of 20 to 30yUg per 100 ml are less interpretable because of the uncertainty of the associated tissue reserves of vitamin A. Higher plasma vitamin A levels can generally be considered to be associated with an appreciable tissue reserve of the vitamin. Hodges and Kolder (1971) noted that i n vitamin A depleted adult men, when the intake of r e t i n o l was increased to 150 yUg per day, the serum r e t i n o l l e v e l was s t i l l less than 10yUg per 100 ml. At an intake of 300JJ. g. per day, the serum r e t i n a l l e v e l was approximately 19 jAg per 100 ml. With daily intakes of 600jug of r e t i n o l , skin lesions associated with vitamin A deficiency were corrected. Arroyave (1971) c i t e s the S h e f f i e l d study as a major sources of data to be used i n setting standards for vitamin A. This study found that a f a l l , i n the average plasma l e v e l to below 15jUg per 100 ml i n every case preceded the deterioration of dark adaptation by a few weeks, and no abnormal values for dark adaptation were noted i n any subject as long as the plasma l e v e l of vitamin A remained above 15jug per 100 ml. At an average plasma l e v e l of 12yUg per 100 ml, the deterioration of night v i s i o n was pronounced. Furthermore, none of the - 24 -deprived individuals whose dark adaption remained normal had average plasma levels below per 100 ml. From the S h e f f i e l d experiments, i t can be deduced that i n -takes of about 390jU.g per day of r e t i n o l would place the subjects i n a r i s k s i t u a t i o n where plasma levels are i n the range of 20 to 30yU-g per 100 ml and l i v e r reserves are un-certain. It i s assumed that intakes below th i s l e v e l would eventually lead to depletion of hepatic reserves. Plasma ^3-carotene measurements provide l i m i t e d information concerning vitamin A n u t r i t u r e , since, for the most part, they r e f l e c t recent dietary intakes of the nutrient. However, when low p l a s m a s-carotene levels are found i n association with low plasma vitamin A l e v e l s , the evidence for inadequate vitamin A n u t r i t u r e i s quite strong. Also, the ^ -carotene analysis procedure i s not s p e c i f i c and includes i n the measurement other non-convertible yellow pigments i n plasma, r e s u l t i n g i n f a l s e l y r a i s ed ^ -carotene values, i i ) Vitamin E Evaluation of human vitamin E status i s s t i l l inaccurate. U n t i l not very long ago vitamin E was described as a "vitamin i n search of a disease", and at present c l i n i c a l evidence of vitamin E deficiency i s almost e n t i r e l y r e s t r i c t e d to premature infants, who demonstrate edema and anemia according to some researchers (National Research Council, Food and N u t r i t i o n Board 1974). 25 -A vitamin E deficiency i n adult man i s d i f f i c u l t to produce because of the considerable tissue storage of the vitamin and the consequent extended period required for depletion ( B i e r i 1976). In the only long term study, 19 adult men receiving a d a i l y basal d i e t which contained approximately 3 mg <*-tocopherol for a period of 2.5 years showed no symptoms. Plasma tocopherol lev e l s had reached a plateau for the average subject of about 0.5 mg per 100 ml. At that time, 30 g of stripped corn o i l , which had been heated for about 2 hours at 95 C to obtain a peroxide number of 50 i n order to remove more of the tocopherol present, was substituted for equal amounts of the stripped l a r d . Nine months l a t e r when i t became obvious that adding more polyunsaturated acids would more than l i k e l y safely lower the plasma tocopherol further, the amount of stripped corn o i l added was doubled to 60 g,and a f t e r 3 years on t h i s diet,plasma tocopherol l e v e l s had dropped to less than 0.2 mg per 100 ml. The ef f e c t of low plasma tocopherol l e v e l s on the survival rate of the erythrocytes i n man was determined on those subjects who had remained on the basal d i e t for the longest period. The differences obtained i n erythrocyte l i f e span were small and probably within normal range and no anemia was manifested (Horwitt 1961). Therefore, guidelines delineated for vitamin E assessment are s t i l l somewhat tenuous and are based on the prevention ^ 26 of erythrocyte i n s t a b i l i t y and the prevention of depletion of body vitamin E i n man. According to Horwitt et aJL. (1961) , these experiments may be interpreted to show that c<-tocopherol requirements of adult man vary from a minimum of less than 5 mg per day to a maximum of about 3 0 mg per day depending on the l e v e l of unsaturated l i p i d s i n the d i e t . Since vitamin E acts as an anti-oxidant and unsaturated l i p i d s are peroxidizable, one would expect the general agreement of today that the tocopherol requirement i s related in some manner to the dietary intake of polyunsaturated f a t t y acids and the polyunsaturated f a t t y acid content of tissue l i p i d s . In an i n i t i a l e f f o r t to r e l a t e these two dietary variables, Harris and Embree (1963) suggested that a fixed r a t i o of dietary <*-tocopherol: polyunsaturated f a t t y acids might be established to define dietary vitamin adequacy. The value they proposed, 0.6, as the c r i t i c a l l e v e l below which d i e t s would be d e f i c i e n t i n vitamin E has subsequently been found inapplicable with several species i n a va r i e t y of experimental conditions because factors i n the d i e t other than the polyunsaturated f a t t y acid content also e f f e c t the vitamin E status ( B i e r i 1976). As well, t h e i r work i s c r i t i c i z e d for i t was based on foods available for consumption rather than foods consumed. The text of the 8th edi t i o n of the Recommended Dietary Allowances (National Research Council, Food and - 27 -N u t r i t i o n Board 1974) notes that s a t i s f a c t o r y d i e t s i n the United States have r a t i o s that average about 0.4. Since man i s capable of synthesizing the poly-unsaturated f a t t y acid eicosatrienoic acid from acetate, he w i l l have a f i n i t e vitamin E requirement even i n the t o t a l absence of dietary polyunsaturated f a t t y acids (Witting 1972). Data on f a t t y acid a l t e r a t i o n s i n man have been reported (Horwitt 1962) to show that whereas the f a t t y acids of plasma l i p i d s are r a p i d l y altered to give an i n d i c a t i o n of short-term dietary e f f e c t s (days), the erythrocyte f a t t y acids provide an estimation of what has been consumed during recent weeks or months. The depot f a t s change quite slowly and give an estimate of what has accumulated during past years. Horwitt (1974) reports that the l e v e l of l i n o l e i c acid obtained i n the depot f a t s , tends to approach the percentage i n the d i e t i f the d i e t i s fed for a s u f f i c i e n t period of time. He proposes that three factors be considered i n evaluating the requirement for vitamin E: f i r s t l y , the synthesis and incorporation of large amounts of polyunsaturated f a t t y acids into v i t a l compounds even when the amount of them i n the d i e t i s low; secondly, the percentage of polyunsaturated f a t t y acids i n the dietary f a t t y acids; and t h i r d l y , the amount of poly-unsaturated f a t t y acids consumed. The formula which he suggests for c a l c u l a t i n g the tocopherol equivalent - 28 -r e q u i r e d per day i s 0.2 5 (% PUFA + g PUFA) + 4 = d-°<-tocopherol e q u i v a l e n t (mg) , where "% PUFA" r e p r e s e n t s the percentage of p o l y u n s a t u r a t e s i n the t o t a l l i p i d s i n the d i e t , and "g PUFA" r e p r e s e n t s the t o t a l amount of pol y u n s a t u r a t e d f a t t y a c i d s obtained by m u l t i p l y i n g the t o t a l d i e t a r y f a t by the percentage of po l y u n s a t u r a t e d f a t t y a c i d s . Four mg of d-<x-tocopherol i s chosen as the amount t h a t should be allowed f o r t i s s u e s y n t h e s i s of p e r o x i d i z a b l e compounds i n the male a d u l t even when the d i e t i s p r a c t i c a l l y d e f i c i e n t i n po l y u n s a t u r a t e d f a t t y a c i d s . The constant, 0.25, i s the estimate o b t a i n e d when the formula i s a p p l i e d to data from experimental s t u d i e s of the v i t a m i n E requirements of a d u l t men. However, t h i s formula does not c o n s i d e r the time f a c t o r r e q u i r e d f o r depot f a t l i p i d to achieve e q u i l i b r i u m w i t h d i e t a r y f a t i n terms of p o l y u n s a t u r a t e d f a t t y a c i d percentages. Switching from a normal mixed d i e t to a d i e t high i n po l y u n s a t u r a t e d f a t t y a c i d s , an i n d i v i d u a l would be expected t o have a l e s s e r than "normal" requirement f o r s e v e r a l years i n terms of p o l y u n s a t u r a t e d f a t t y a c i d i n t a k e and v i c e v e r s a , a g r e a t e r than "normal" requirement f o r the i n d i v i d u a l who has decreased the poly u n s a t u r a t e d f a t t y a c i d content of h i s d i e t . G e n e r a l l y , i t i s to be noted t h a t as the p o l y u n s a t u r a t e d f a t t y a c i d content of d i e t s i n c r e a s e s , so does the v i t a m i n E content - 29 -( B i e r i 1976) . To g i v e g r e a t e r v a l i d i t y t o t h e s t a n d a r d s , W i t t i n g and Lee (1975b) s ugge s t t h a t a d i p o s e t i s s u e l i n o l e a t e l e v e l s i n the g e n e r a l p o p u l a t i o n be u sed as a b a s e l i n e f o r t he p e r i o d i c e v a l u a t i o n and r e v i s i o n o f the recommended d i e t a r y a l l o w a n c e f o r v i t a m i n E. They s ugge s t u s i n g 0.6 IU v i t a m i n E a c t i v i t y w h i c h i s 0.4 mg d -c< - tocophero l p e r g l i n o l e a t e i n 100 g a d i p o s e t i s s u e f a t t y a c i d s as the recommended d i e t a r y a l l o w a n c e . W i t h r e g a r d t o b i o c h e m i c a l c r i t e r i a f o r v i t a m i n E s t a t u s e v a l u a t i o n , S a u b e r l i c h e t a l . (1974) w r i t e t h a t e r y t h r o c y t e h e m o l y s i s t e s t s p r o v i d e i n d i r e c t i n f o r m a t i o n c o n c e r n i n g v i t a m i n E s t a t u s and t h a t more d i r e c t i n f o r m a t i o n can be o b t a i n e d by m e a s u r i n g t o c o p h e r o l l e v e l s i n t h e serum o r p l a s m a . However, t h e y c a u t i o n t h a t whe the r any o f t h e s e i n d i c a t e t he ( ^ - t o c o p h e r o l s t a t u s o f t he o t h e r t i s s u e s o f t he body i s u n c e r t a i n . A c c o r d i n g t o H o r w i t t e t . a l . ( 1968 ) , t h e p e r o x i d e h e m o l y s i s t e s t i n a g i v e n s u b j e c t i s n o t h i n g more t h a n an app rox ima te i n . v i t r o t i t r a t i o n o f t h e amount o f t o c o p h e r o l i n t h e e r y t h r o c y t e and any o t h e r l i p i d a n t i o x i d a n t t h a t can p e n e t r a t e t h e c e l l membrane i s e f f e c t i v e i n i n h i b i t i n g t h i s r e a c t i o n . A l s o , t hey w r i t e , hyd rogen p e r o x i d e i n s u f f i c i e n t amounts o r i n s u f f i c i e n t t ime i s c a p a b l e o f h e m o l y z i n g any h e a l t h y c e l l and i s t o o s t r o n g a r e a g e n t t o g i v e t he k i n d o f d e f i n i t i v e t e s t o f c e l l r e s i s t a n c e t o b i o l o g i c a l - 30 -oxidations that i s required. A plasma l e v e l of t o t a l tocopherols below 0.5 mg per 100 ml i s generally considered undesirable, although according to B i e r i (197 6), blood l e v e l s of ^-tocopherol may not accurately r e f l e c t either l e v e l of intake or tissue storage. The majority of plasma vitamin E i s car r i e d by the lipoproteins, which also account for most of the blood l i p i d s . Thus, in normal individuals, there i s a high c o r r e l a t i o n between plasma t o t a l l i p i d s and plasma tocopherol concentration, as well as plasma y S - l i p o p r o t e i n and plasma tocopherol concentration (Rubinstein et a l . 1969).vitamin E had no s i g n i f i c a n t e f f e c t on serum l e v e l s of cholesterol or lipoproteins when taken o r a l l y by normal individuals at the l e v e l of 300 or 600 mg per day for a period of 4 weeks, though serum low-density li p o p r o t e i n l e v e l s of vitamin E were elevated (Harman 1960) . On the other hand, physiological, pharmacological, genetic and dietary factors that change the l e v e l of the serum l i p i d s produce a concomitant change i n the l e v e l of -tocopherol i n the serum. Therefore, conclusions on the state of tocopherol n u t r i t i o n based on serum tocopherol l e v e l s alone are inadequate, and should be accompanied by values for plasma l i p i d s . Current information appears to support a r a t i o of 0.8 mg t o t a l tocopherols per gram t o t a l l i p i d s as i n d i c a t i v e of adequate n u t r i t i o n a l status (Horwitt et a l . 1972). They note that data from newborn 31 <-f u l l - t e r m infants who had low serum tocopherol l e v e l s produced a r a t i o of 0.8 or s l i g h t l y higher. Ng and Chong (197 5) found that mean serum tocopherol values of 63 healthy Malaysians were s i g n i f i c a n t l y lower than those of 63 hyperlipoproteinaemic pat ients who were on normal d i e t s or d i e t s low i n c h o l e s t e r o l - r i c h foods, but there was no d i f ference between the groups when the values were expressed as mg tocopherol per g t o t a l serum l i p i d s . For these reasons, plasma cho le s t ero l and plasma t o t a l l i p i d s were a lso determined i n t h i s study. Samples from newborn f u l l - t e r m infants and ..from -subjects, with high l i p i d l eve l s were a lso c o l l e c t e d and analyzed. The concept that changes i n serum l i p i d l eve l s can change « - t o c o p h e r o l l eve l s may also be appl i cab le to other f a t - s o l u b l e v i tamins , for example, v i tamin A. Estimates of the amount of ^-tocopherol considered to be a v a i l a b l e i n the t y p i c a l food supply inc lude: 7.4 mg (mean) and 2.6-15.4 mg (range), reported by Bunnell et a l . (1965); 9.0 mg (mean) and 4.4-12.7 mg (range), reported by B i e r i and Evarts (1972); 6.4 mg (mean) i n a composite Canadian d i e t providing 2,78 0 k c a l , reported by Thompson et aJL. (1973) ; 5 mg per 2,500 kca l i n meals ingested by B r i t i s h hosp i ta l pat ients , reported by Smith e t a l . (1971) ; and 7.5 + 3.5 mg (mean) and 2.5-15.3 mg (range) i n a 2,500 kcal d i e t containing 96 + 26 g fa t of which 19.5 + 1.8% was l i n o l e a t e , - 3 2 -reported by Witting and Lee (1975a). Witting and Lee (1975a) also reported that blood samples obtained from 26 subjects consuming t h i s d i e t contained adequate l e v e l s of plasma t o t a l vitamin E, 1.09 + 0.25 mg per 100 ml, despite the observation that 71% and 65% of the di e t s analyzed did not meet the value tabulated i n the 8th edition of the Recommended Dietary Allowances (National Research Council, Food and N u t r i t i o n Board 1974) i n terms of -tocopherol or t o t a l vitamin E a c t i v i t y , respectively. Total vitamin E a c t i v i t y , expressed as °<-tocopherol equivalent, i s calculated by multiplying values for «*-tocopherol by 1.2, to account for the les s active vitamin E compounds,^-, Y-, and 6-tocopherols and tocotrienols present i n t y p i c a l balanced U.S. d i e t s . In B r a z i l , the predominant f a t source i s soybean o i l , which contains about six times more Y - than <* -tocopherol. Even though Y-tocopherol has only about 10% of the b i o l o g i c a l a c t i v i t y of <*-tocopherol, the large amount of Y-tocopherol i n the i r d i e t could contribute substantially to the t o t a l vitamin E a c t i v i t y . Therefore, values for c* -tocopherol should be mult i p l i e d by a factor of at lea s t 1.2 to give a more accurate estimation of th e i r t o t a l vitamin E a c t i v i t y expressed as -tocopherol equivalent. In summary, the controversial and unsettled nature of the U.S. and Canadian recommended intake standards for - 33 -vitamin E i s to be recognized, and taken into consideration. Clearly, more research i s needed before vitamin E status i s f u l l y understood, and therefore this study and others l i k e i t measuring biochemical vitamin E data are of p a r t i c u l a r value. Since the tocopherols i n the blood are only a tiny part of the t o t a l tocopherols i n the body, addi-t i o n a l studies are required to r e l a t e tissue tocopherol to vitamin E n u t r i t i o n . i i i ) Plasma Lipids and Hematology Plasma cholesterol and plasma t o t a l l i p i d s are also of i n t e r e s t as r i s k factors for heart disease. Hyperlipidemia and hypercholesterolemia, cholesterol being the most ex-tensively measured l i p i d , i s strongly associated with coronary heart disease, the degree of r i s k r i s i n g i n pro-portion to the concentration of cholesterol, and usually manifested by myocardial i n f a r c t i o n rather than angina pectoris. Hypertriglyceridemia has also been associated with increased prevalence of coronary heart disease. The problems r e l a t i n g to accurate measurement of t r i g l y c e r i d e levels and the frequent association of hypertriglyceridemia with hyper-cholesterolemia have presented d i f f i c u l t i e s i n the clear i d e n t i f i c a t i o n of hypertriglyceridemia alone as an i n d i c a t i o n of s u s c e p t i b i l i t y to premature coronary heart disease (Goodhart and Shils 1973). Hematocrit, the packed c e l l volume of whole blood, i s often used as a diagnostic for n u t r i t i o n a l i r o n 34 -deficiency, which lowers the hematocrit because of i n s u f f i c i e n t hemoglobin formation r e s u l t i n g i n microcytic hypochromic red blood c e l l s . Measurement of hemoglobin i t s e l f i s a more d i r e c t means of estimating iron i n s u f f i c i e n c y because of the ultimate r o l e of the element in t h i s molecule. However, hemoglobin l e v e l s f a l l also in n u t r i t i o n a l megaloblastic anemia caused by deficiency of f o l a t e or B 1 2- The c r i t e r i a used to evaluate the hematological data obtained i n t h i s study were the ones used by the extensive n u t r i t i o n survey conducted during 1965 to 1967 on populations of Central America and Panama by the Center for Disease Control, Atlanta,Georgia (1972) i n conjunction with the Ins t i t u t e of N u t r i t i o n of Central American and Panama. (iNCAP), B. Previous Relevant N u t r i t i o n a l Surveys 1. World a) A g r i c u l t u r a l Workers - General Surveys A study by Oyemade and Olugbile (1977) c a r r i e d out i n western Nigeria revealed a high mortality, 2 5%, among children under 4 years of age of a g r i c u l t u r a l workers f a m i l i e s . Causes suggested were malnutrition, i n f e c t i o n and low standards of c h i l d care. Also noted were poor housing conditions, poor environmental sanitation, poor use of available health services, and a lack of "health" knowledge. - 35 -Wiesmann e t al_. (1975) s t u d y i n g a g r i c u l t u r a l workers i n the n o r t h o f the s t a t e o f P a r a n a , and i n the c o a s t a l and i n l a n d r e g i o n s o f the s t a t e o f Sao P a u l o , i n B r a z i l , r e p o r t e d t h a t no m a l n u t r i t i o n was f o u n d . However, men were about 10 cm s h o r t e r and 7 kg l i g h t e r than Swiss c o u n t e r p a r t s . b) Normal Human Plasma T o c o p h e r o l L e v e l s A summary o f plasma t o c o p h e r o l l e v e l s i n normal human a d u l t p o p u l a t i o n s , as de termined by numerous r e s e a r c h e r s , i s g i v e n i n T a b l e H - l . I n f a n t plasma t o c o p h e r o l l e v e l s are summarized i n T a b l e .11-2. 2. B r a z i l a) N a t i o n a l / L a r g e s c a l e i ) D i e t a r y An e x t e n s i v e but l i t t l e known work by G e r a l d o J . da Rosa e S i l v a r e g a r d i n g food and underdevelopment i n B r a z i l i s c i t e d by Shrimpton (197 5) as an e x c e l l e n t r e v i e w o f n u t r i t i o n a l surveys c a r r i e d out i n B r a z i l i n the p e r i o d 193 0 to 1960. I t r e v i e w s 5 s t u d i e s i n the n o r t h , 21 i n the n o r t h e a s t , 13 i n the s o u t h e a s t , 8 i n the sou th , and 6 i n the midwest . The s t u d i e s were l a r g e l y o f food h a b i t s , q u a l i t a t i v e i n n a t u r e and t h e r e f o r e c o n c l u s i o n s were more i m p r e s s i o n s than hard s t a t i s t i c s . The r e v i e w c o n c l u d e d however t h a t the economic and food s i t u a t i o n o f the B r a z i l i a n p o p u l a t i o n Table I I - l Plasma Tocopherol Levels l n Normal Human Adult Topulation No. of Subjects 122 14 74 20 12 188 116 35 70 17 583 10 30 21 13 30 '23 132 197 70 5* 66 • 3* 99 25 10 63 166 178 379 ? ? 290 71 46 159 195 102 26 24 29 13 Location Holland New York C i t y Nashville I t a l y New York C i t y S t. Louls Hungary Stellacoom Birmingham Rochester England Durham Philadelphia Nashville Rochester Boston New Haven Washington, D.C. Rochester, N.Y. Puerto Rico Walnwrlght (Alaska) Point Hope (Alaska) Kaslgluk (Alaska) Taiwan Baltimore, Maryland I t a l y Malaysia Japan Japan Vancouver/Chilliwack, B.C. East Pakistan Montreal Fort St. John Upper L l a r d , Yukon Ross River, Yukon Ahousat, B.C. Anaham, B.C. Pakistan Texas C a l i f o r n i a C a l i f o r n i a C a l i f o r n i a  Plasma Tocopherol (mg/lOOml3) 0.77 4 0.35 0.78 0.39 0.89 + 0.20 0.92 t 0.25 O.96 i 0.33 0.98 0.30 0.99 t 0.25 1.04 ± 0.25 1.04 t 0.30 1.05 0.27 1.05 + 0.23 1.06 i 0.06 1.08 t 0.29 1.09 + 0.17 1.20 •i 0.22 1.20 •fc 0.22 1.23 + 0.31 1.05 ± 0.26 1.05 * 0.32 0.81 4 0.30 1.23 4 0.27 1.23 4 0.27 1.27 •fc 0.33 1.05 • 0.47 0.84 0.77 4 0.14C 0.92 t 0.36 0.99 £ 0.25° 1.06 ± 0.26° 0.97 t 0.29 0.76t 0.30 0.77 0.92 0.76* 0.31 0.91* O.32 1.21 4 0.30 0. oO t 0.28 0.761 0.30° I.09 4 0.25 1.03* 0.20 1.21* 0.28 0.90 4 0.20 Reference b Engel. Hlllman and RosHer. Ferguson et a l . Rindl and P e r r l . Wechsler e_t a l . ' C n l e f f i and Kirk. Kramer. Van Bruggen and StraumfJord. Harris et a l . Scrimshaw et a l . Leitner et a l . Darby et al_. Urbach et a l , Lemley et a l . Harris and (iualfe. P o s t e l . K l a t s k i n . . B i e r l et a l . Harris et. a l . Ramirez et. a l . Wei Wo and Draper. Wei Wo and Draper. Wei Wo and Draper. • Ciien et a l . U977) Cordon et a l . (1958) S c h e t t l n i et a l . (1977) Ng and Chong (1975) S h i t a r a e t a l . (1976) Shitara'et. a l . (1976) Desai * (1968) Rahman et a l . (1964) Ooldbloom H96O) Desai and Lee Desai and lee Desai and Lee Desai and Lee Desai and Lee Ranman et. a l . Witting and Lee (1975) Lewis et a l . (1973) Lewis et a l . (1973) Lewis et a l . (1973) 1974*} 1974 c) 1974 c) (1964) a Mean * SD. b F i r s t 23 data c i t e d from Chen et al.(1977) c . Serurru l e v e l o . d Adults and children, 10 - 37 years old. Table II- 2 : Plasma Tocopherol Levels i n Infants of Normal Human Populations No. of Subjects Location Plasma Tocopherol Levels (mg/100inla) Reference 174 19 New Jersey Scotland 0.38 t 0.1 Baker et a l . (1975) McWhirter (1975) 217 Sherbrooke, Que. 0.24 b Vobeoky et a l . (1976) 217 Sherbrooke, Que. 0.37° Vobecky et. a l . (1976) 195 Ireland b 0.238 t 0.02 Leonard et a l . (I972) 118 Ireland 0.233 * o.iob Leonard et a l . (.1972) 86 Ireland 0.2521 o.09b Leonard et al,. (1972) 59 Ireland 0.215 t o.09b Leonard et a l . (1972) 82 Ireland 0.236 * 0.09b Leonard et. a l . (1972) 56 Oregon 0.34 * 0.12 b Straumf Jord and Quaife (1946) 33 P a r i s , Prance 0.394 Minkowski et a l . (1958) 9 Baltimore, Maryland b 0.45 t 0.14 Nltcwsi&y ei. al.(1962) 53 Hartford, Connecticut 0.24 1 0 . l 4 b Moyer (1950) 122 Baltimore, Maryland 0.23 Gordon et a l . (1958) 40 Rochester, N.Y. 0.38 s 0.l8 d •Wright et a l . (1951) 37 Rochester, N.I. O.37 * 0.l5 d Wright et a l . (1951) 18 Par i s , France b 0.10 Varangot (1944) ? Holland b 0.190 Engel (1949) ? Montreal b,c 0.19 Ooldbloom (i960) 57 Osaka, Japan 0.413 * 0.0296 Hlno and Nlshlno (1973) a Mean * SD. b Newborns at 1 P a r t u r i t i o n . c Infants-3 days o l d . d Infants-2 days o l d . e Serum l e v e l s . - 38 -was and continued t o be v e r y d i f f i c u l t (Shrimpton, 1975). R e s u l t s o f B r a z i l i a n l a r g e s c a l e a d u l t d i e t a r y surveys conducted d u r i n g the years 1961 to 1964 a r e summarized i n Table II-3. The numerous d e f i c i e n c i e s found support the above c o n c l u s i o n . With r e g a r d to maternal d i e t a r y b e l i e f s and h a b i t s , W i t t (1971), q u e s t i o n i n g 184 women and g i r l s s t u d y i n g domestic s u b j e c t s a t 3 c e n t e r s i n Sao Paulo i n 1968, found t h a t over 50% thought t h a t f o r the baby's sake pregnant women should eat what they wished and a few b e l i e v e d t h a t i t was necessary to eat enough f o r two. Most p u p i l s c o n s i d e r e d t h a t a s p e c i a l d i e t f o r mothers a f t e r p a r t u r i t i o n was necessary; a c c o r d i n g t o 53% of them i t should be f o l l o w e d f o r from 4 0 to 6 0 days. Although o p i n i o n s v a r i e d , m i l k , malt beer, f r u i t and a soup made from white maize were suggested most f r e q u e n t l y as means of ensuring an adequate supply of b r e a s t m i l k , and pork and f i s h were o f t e n mentioned as u n s u i t a b l e . Many p u p i l s were unable to answer the q u e s t i o n s . The Interdepartmental Committee on N u t r i t i o n f o r N a t i o n a l Development (1965) summarizing b e l i e f s c oncerning d i e t i n pregnancy and l a c t a t i o n i n n o r t h e a s t B r a z i l s t a t e d t h a t most o f the mothers questioned b e l i e v e d t h a t a l l foods were good d u r i n g pregnancy. Some women avoided cassava, beans, and c e r t a i n meats. S i m i l a r l y , n e a r l y a l l foods •were b e l i e v e d to be b e n e f i c i a l i n l a c t a t i o n . Table II-3 B r a z i l i a n Largescale Adult Dietary Survey Results, 1 9 6 1 - 1 9 6 4 Nutrient Intake Status Survey Extent Location,Target Group Reference Calo r i e s 15 towns northeast Chaves (1964) Protein. d e f i c i e n c y 15 towns northeast Chaves ( 1 9 6 4 ) Vitamin A def i c i e n c y 15 towns northeast Chaves ( 1 9 6 4 ) Thiamin def i c i e n c y 15 towns northeast Chaves ( 1 9 6 4 ) R i b o f l a v i n d e f i c i e n c y 15 towns northeast Chaves ( 1 9 6 4 ) R i b o f l a v i n low 9,125 families low Income/all B r a z i l Jansen et a l . (1977) a Calcium low 7 , 3 0 9 families low income/urban B r a z i l Jansen et al.(1977) 8 Thiamine low 7 , 3 0 9 families low income/urban B r a z i l Jansen et al.(1977) S C a l o r i e s 75% d e f i c i e n t > 1000 : families northeast USDA Economic Research Service U 9 7 0 ) a Protein 20JS d e f i c i e n t > 1 0 0 0 : families northeast USDA Economic Research Service ( 1 9 7 0 ) a C a l o r i e s d e f i c i e n t > 1000 : families east USDA Economic Research Service U 9 7 0 ) a Protein &% d e f i c i e n t > 1000 : families east USDA Economic Researcn Service U 9 7 0 ) a C a l o r i e s 29# d e f i c i e n t > 1000 ; families south USDA Economic Research Service ( I 9 7 0 ) a Protein \% d e f i c i e n t > 1000 1 families south USDA Economic Researcn Service U 9 7 0 ) a C a l o r i e s I n s u f f i c i e n t 5 5 3 8 people northeast 1CNND U 9 6 5 ) b Protein I n s u f f i c i e n t 5 5 3 8 people northeast 1CNND ( 1 9 6 5 ) b Vitamin A low 963 people . ( 0 - 5 years') . northeast iCiiwD (1965) b a S u r v e y was conducted l n the period 1 9 6 1 - 1 9 6 3 . b Survey was conducted ln the period March-Hay I 9 6 3 . - 40 -Infant feeding practices in B r a z i l have been discussed by numerous researchers. Santos (1976) reports that i n Recife, of 1029 children under 5 years of age, 64.7% belonging to the lowest socioeconomic group, only 24%: were breastfed for one month or longer. Similar weaning patterns were found by Rosenburg (1973), studying infants admitted to a welfare hospital in Sao Paulo, B r a z i l . The date, for 667 infants in 1969 and 249 i n 1972, are summarized as follows: i n the f i r s t month, breast-feeding t o t a l l e d 23.21% i n 1969 and 23.07% i n 1972; i n the second month, there was a steep f a l l , with 4.06% and 3.33% breastfeeding i n 1969 and 1972 respectively. Trigo et a l . (1978) , studying 204 mothers with 287 children under 6 years old of low socioeconomic status in Joao Pessoa, Paraiba, found that breast feeding ceased for 50% of the infants during the neonatal period, and by the end of the f i r s t month of l i f e , 62% of the infants were being given mixed or a r t i f i c i a l food. Ulloa (1973) observed that 73% of the mothers from a poor socioeconomic area of Sao Paulo stopped breastfeeding before the second month. Puffer and Serrano (1973) also reported trends of early termination of breast-feeding i n urban areas of Sao Paulo and Ribeirao Preto in the Inter-American Investigation of M o r t a l i t y i n Childhood. In a study of a poor area of Ribeirao Preto, breastfeeding of short duration was also reported - 41 -(Shrimpton, 1975) . The Interdepartmental Committee on Nu t r i t i o n for National Development (1965) also noted early weaning of infants. As c i t e d by Shrimpton (1975), the following researchers also found short durations of breast-feeding: Waterlow and Vergara noted i n 1955 that i n the urban areas of Rio de Janeiro, Belo Horizonte and Recife, the period of breastfeeding was around 2 to 5 months; i n 1966, Monteiro and Santos drew attention to the precocious breastfeeding common i n Rio de Janeiro; Coelho and Bazante, and Coelho et a l . found that most children had stopped breastfeeding i n the towns of Primavera and Belem de Maria by 3 months of age i n 1972; i n 197 5 i n Encruzilhada, one of the poorest parts of Recife, in a health center catering to the poorest 50% of the people, 8 0% of the mothers stopped breastfeeding by the end of the f i r s t month of the c h i l d ' s l i f e ; Monteiro studying 3 towns i n Pernambuco i n 1974 found that 63%, 57% and 71% stopped breastfeeding i n the f i r s t three months. On the other hand, longer durations of breastfeeding were found i n the indigenous B r a z i l i a n Indians of the Amazonas; i n the r u r a l i n t e r i o r populations l i v i n g i n the v a l l e y of the River Sao Francisco i n the l a t e s i x t i e s ; i n the Amazonas i n 1956; and i n is o l a t e d a g r i c u l t u r a l communities i n the State of Santa Catarina in 1969, as - 42 -c i t e d by Shr impton (1975). A l s o , s t u d y i n g l a c t a t i o n i n women o f the Sao Pau lo D i s t r i c t o f B r a z i l i n 1965, Yunes and Roncheze l (1975) found t h a t most women s u c k l e d t h e i r i n f a n t s , the percentage v a r y i n g from 87% f o r p a r i t y 4 to 89% f o r p a r i t y 2. The mean d u r a t i o n o f l a c t a t i o n v a r i e d from 3 0 weeks f o r p a r i t y 1 t o 35 weeks f o r p a r i t y 5, and decreased as the age o f the mother d e c r e a s e d . I g n o r i n g p a r i t y , t h e r e was a tendency f o r women w i t h a h i g h e r s tandard o f e d u c a t i o n to have s h o r t e r l a c t a t i o n s , and d u r a t i o n o f l a c t a c t i o n f e l l a l s o as income r o s e . Sousa (1975) a s s o c i a t e d s h o r t d u r a t i o n o f l a c t a t i o n w i t h s t r e s s caused by the b i r t h , o f a baby, c a u s i n g a n x i e t y and a d e c r e a s e d volume of b r e a s t m i l k . T h i s , i n t u r n , would cause the mother to b e l i e v e t h a t b r e a s t m i l k was not n u t r i t i o n a l l y adequate . Bomfim e t a l . (1974), working w i t h 220 mothers i n B r a z i l , found t h a t the most common e x p l a n a t i o n f o r not b r e a s t f e e d i n g (29.2%) was t h a t b r e a s t m i l k i s 'weak' and the next i n f r e q u e n c y (24.0%) was l a c k o f m i l k . T r i g o e t a l . (1978) r e p o r t t h a t the two most common reasons g i v e n f o r ending b r e a s t f e e d i n g were l a c k o f m i l k and r e j e c t i o n o f the b r e a s t by the i n f a n t . Other reasons f o r e a r l y weaning suggested by Sousa e t aJL. (1975a) a r e commerc ia l a d v e r t i s i n g o f powdered m i l k and i g n o r a n c e o f h e a l t h and m e d i c a l w o r k e r s . The I n t e r d e p a r t m e n t a l Committee on N u t r i t i o n f o r N a t i o n a l - 43 -Development (1965) stated that i n the northeast the most common reason for early weaning was i n s u f f i c i e n t breast milk, which they attributed to the general food d e f i c i t . F ioravanti et a_l. (1960) related the incidence of kwashiorkor i n children of the Rio de Janeiro area to some extent to i n f e c t i o u s and contagious diseases, perhaps caused by the early changeover from breast milk to gruels of maize and r i c e f l o u r with small amounts of powdered milk added. Bomfim et a_l. (1974) reported that a r t i f i c i a l feeds, as supplements to breast milk or replacing i t , erred i n excess of cereal or sugar and i n s u f f i c i e n c y of milk. Sousa et a l . (1975b) attributed a r i s e i n diarrhoeal disease and protein-energy malnutrition i n the f i r s t year of l i f e to unnecessary bottle feeding i n poorly sanitized areas of B r a z i l . Most infants suffering with diarrhoea were from very poor f a m i l i e s . Rosenburg (1973) concluded that where supplementary food i s supplied, there i s a tendency, aggravated by lack of purchasing power, for feeding to deteriorate and malnutrition to increase and to occur at e a r l i e r ages. As c i t e d by Shrimpton (197 5), i n a study i n Sao Paulo i n 1972, powdered milk was introduced into the d i e t of 33% of the children studied i n the f i r s t month. By the end of the second month about 7 0% of the children were -. 44 -receiving cow's milk, either fresh or powdered. Following cow's milk, the next most common introduction to the ch i l d ' s d i e t was f r u i t juices, i n the majority around the t h i r d month, then r i c e and the l i q u i d that kidney beans had been cooked i n , beginning i n the fourth month. The age of one year seems to be pretty universal i n B r a z i l for when a c h i l d can eat f r e e l y from the adult menu as reported i n the majority of studies reviewed by Shrimpton (1975) . i i ) Anthropometric The only B r a z i l i a n largescale adult anthropometric r e s u l t s reported i n the l i t e r a t u r e are from the Northeast B r a z i l N u t r i t i o n Survey conducted i n 1963 by the Interdepartmental Committee on Nu t r i t i o n for National Development (1965). They concluded that the population can be c l a s s i f i e d generally as a lean one as confirmed by measurements of skinfold thickness, which indicated r e l a t i v e l y low le v e l s of subcutaneous f a t . As well both men and women above age 4 5 exhibited a steady decline i n body weight, contrasted with continuing increases i n body weight i n North American females through age 6 5 and i n males through age 55. The average weight of men and women at maturation and by decade of age i n adulthood i n northeast B r a z i l are plotted i n FigureII-2. For purposes of comparison, the average weight by age of North Americans of a body height equal to the average height of B r a z i l i a n men and - 45 -7 0 , / / / / o ' I 1 1 j 1 - , 15 25 35 45 55 65 Age (Years) Figure I I - 2 - ; Comparison of Average Weight by A,ge °f Adults of Same Height i n Northeast B r a z i l and i n North America (ICNND. 1965) (North American data from Society of Actuaries build and blood pressure study) - 46 -women i n t h e s u r v e y i s a l s o p r e s e n t e d i n F i g u r e 1 1 - 2 . O t h e r d e s c r i p t i o n s o f t h e s u r v e y s a m p l e a r e i n c l u d e d i n T a b l e I I - 4 . T a b l e I I - 4 : A d u l t A n t h r o p o m e t r i c R e s u l t s - I C N N D Northeast B r a z i l Nutrition Survey Parameter Males Females N o . o f s u b j e c t s 673 1 , 5 9 3 M e a n a g e ( y e a r s ) 3 2 . 3 3 3 . 2 M e a n h e i g h t (cm) 1 6 0 . 6 1 5 0 . 4 . M e a n w e i g h t (kg) 5 5 . 0 4 9 . 2 M e a n % s t a n d a r d w e i g h t 9 1 . 5 9 7 . 6 a k C a l c u l a t e d f o r g r o u p a b o v e a g e 17 y e a r s . E s t i m a t e d b y r e f e r e n c e t o N o r t h A m e r i c a n s t a n d a r d t a b l e s , s i n c e B r a z i l i a n s t a n d a r d s w e r e n o t a v a i l a b l e . c r e f e r t o ICNND (1965) i i i ) B i o c h e m i c a l A g a i n , t h e o n l y B r a z i l i a n l a r g e s c a l e a d u l t s u r v e y w i t h b i o c h e m i c a l r e s u l t s r e p o r t e d i n t h e l i t e r a t u r e i s t h e N o r t h e a s t B r a z i l N u t r i t i o n S u r v e y c o n d u c t e d i n 1 9 6 3 b y t h e I n t e r d e p a r t m e n t a l C o m m i t t e e o n N u t r i t i o n f o r N a t i o n a l D e v e l o p m e n t ( 1965 ) . T h e i r b i o c h e m i c a l d a t a s u p p o r t t h e c o n c l u s i o n t h a t a s u b s t a n t i a l p o r t i o n o f t h e p o p u l a t i o n i s c o n s u m i n g l e s s t h a n o p t i m u m a m o u n t s o f p r o t e i n , a n d v i t a m i n A a n d i t s p r e c u r s o r s . F o r a d u l t s , a b o u t 9% o f s e r u m v i t a m i n A l e v e l s w e r e " d e f i c i e n t " a n d a n o t h e r 24% " l o w " . B i o c h e m i c a l e v i d e n c e a l s o s u g g e s t s - 47 -that thiamine and r i b o f l a v i n intakes may be marginal. Anemia prevalence and severity may be more related to protein deficiency and to p a r a s i t i c i n f e s t a t i o n than to a deficiency of dietary iron. b) Sao Paulo State i) Dietary Szarfarc (1972), studying 6 communities i n the south of the state of Sao Paulo, calculated mean iron intakes, as a percentage of requirement, as follows: 63% and 81% i n two v i l l a g e s , Pontal do Ribeira and Icapara, respectively; and 91%, 99%, 122%, and 125% i n the other communities, Iguape, Ribeira, A p i a i , and Barra do Chapeu, respectively. Surveying 154 f a m i l i e s i n 3 of the same towns, Ribeira, A p i a i , and Barra do Chapeu, i n 1970, Miguel and Bon (1974) found the r e s u l t s given i n Table II-5. Intakes of nearly a l l nutrients, according to FAO recommendations, were d e f i c i e n t , e s p e c i a l l y calcium, and vitamins A and C and r i b o f l a v i n . Despite the importance of purchasing power, i t was thought that education would play the main r o l e i n improving n u t r i t i o n . I t i s i n t e r e s t i n g that Patrick and Simoes (1971) found i d e n t i c a l r e s u l t s i n Goias, B r a z i l where diets of a l l income groups were most d e f i c i e n t i n calcium, and vitamins A and C and r i b o f l a v i n . The towns, Iguape, Icapara, and Pontal do Ribeira, previously mentioned were also studied by Roncada (1972) . Composition of the respective average Table II-5 : Average Dally Consumption and Requirements for Various Nutrients in South of SSo Paulo State, 1970b' Energjr Protein Calcium Iron Vitamin A Tniawine Riboflavin Niacin Vitamin A Town (Cals) (goto (mg> tog) (Ag) fag) (nig) (mg) (mg) Requirement 2207 62.3 1020 11.4 1270 1.1 1.6 11.1 69.4 Ribeira Consumption 1798 51.6 252 11.3 284 0.8 0.7 11.4 34.9 Requirement 2244 64.2 1013 11.7 1310 1.1 1.6 11.3 70.8 Barra do Chapeu Consumption 2087 61.5 298 14.6 405 1.2 0.8 12.5 37.6 Requirement 2209 63.5 1037 11.9 ->308 1.1 1.6 11.1 70.3 Apiai Consumption 2156 66.2 401 14.5 642 1.1 l.o 13.1 64.8 St According to F.A.O. recommended allowances, adjusted for population structure. b Refer to Miguel and Bon (1974). - 49 -d i e t s was e s t i m a t e d , per head, a s : energy 2409, 2335, ' and 2169 k c a l ; t o t a l p r o t e i n 71 .5 , 76.0 and 64.1 g , w i t h 49, 42 and 66% o f a n i m a l o r i g i n ; f a t 6 3 . 7 , 41.3 and 29.4 g; and t o t a l v i t a m i n A 497, 126, and 131 /xg . The d i e t s were c o n s i d e r e d adequate i n energy and p r o t e i n , but the i n t a k e o f v i t a m i n A averaged o n l y 38, 10 and 10% of the r e q u i r e m e n t . J u s t over 30% of v i t a m i n A a c t i v i t y was from the a c t u a l v i t a m i n and 54%, 43% and 43% from / ^ - c a r o t e n e . W i l s o n e t a_l. . (1977) r e p o r t e d t h a t n u t r i t i o n a l surveys d u r i n g 1969 to 1973 i n 11 communit ies i n the s t a t e o f Sao Pau lo r e v e a l e d t h a t more than 5 0% o f the f a m i l i e s s t u d i e d had a r i b o f l a v i n i n t a k e l e s s than 60% o f t h a t recommended. , A r e c e n t major d i e t a r y survey conducted by the S e c r e t a r i a de Planejamento da P r e s i d e n c i a da R e p u b l i e a (1977) e n t i t l e d Es tudo N a c i o n a l da Despesa F a m i l i a r -ENDEF, Dados P r e l i m i n a r e s , i s summarized f o r r u r a l , urban and m e t r o p o l i t a n a r e a s o f the s t a t e o f Sao Paulo i n T a b l e II-6. Percentage c o n t r i b u t i o n o f food groups to n u t r i e n t i n t a k e i s summarized f o r urban a r e a s o f the s t a t e o f Sao Paulo i n T a b l e I I - 7 . i i ) A n t h r o p o m e t r i c The S e c r e t a r i a de Planejamento da P r e s i d e n c i a da R e p u b l i e a (1977) a l s o c o l l e c t e d a n t h r o p o m e n t r i c d a t a which i s summarized f o r r u r a l , u r b a n , and m e t r o p o l i t a n a r e a s o f the s t a t e of Sao Pau lo i n T a b l e I I - 8 . Table II-6 : Secretaria de Planejamento da PresldSncia da Republiea Dietary Survey-SSo Paulo Results b Mean Percentage of Requirement Wutrlents Area of Sao Paulo Calories Protein Calcium Iron Vitamin A Thiamine Riboflavin Niacin Vitamin C Sao Paulo, Whole 1 0 2 . 5 5 2 0 9 . 1 0 96 .35 1 1 5 . 3 5 7 4 . 0 3 142.193 98.844 2 3 3 . 2 8 2 3 1 . 3 8 Metropolitan areas 1 0 0 . 3 ? 2 1 5 . ^ 1 1 0 3 . 3 5 118.04 84 .29 1 3 5 . 5 6 6 107.462 233.92 2 6 3 . 0 9 Urban areas, not metropolitan 1 0 2 . 9 2 2 0 3 . 2 0 90 . 9 8 1 1 1 . 0 ? 7 1 . 0 7 1 3 7 . 6 8 9 94.411 2 2 9 . 1 3 218.56 Rural areas 1 0 6 . 6 2 2 0 5 . 0 6 89.7k 1 1 6 . 9 0 5 4 . 6 1 166.657 8 6 . 1 2 0 2 3 9 . ^ 6 179.06 a PAO StandardB. Refer to Secretaria de Planejamento da--Preside"ncla da Republiea (1977). Table H-7 Percentage C o n t r i b u t i o n of Food Groups to N u t r i e n t _ a Intake f o r Urban Areas of the St a t e of Sao Paulo Food Groups N u t r i e n t Eggs and D a i r y P r o t e i n Sources Animal P r o t e i n Sources Legumes Vegetables F r u i t s G r a i n Products F a t s and O i l s C a l o r i e s 6.46 8 .74 8 .25 2.97 1.83 40 .19 15.81 P r o t e i n 13.39 30.01 18 .05 3 .06 ,0.83 32. 98 0.05 Calcium 49.32 6.99 10.00 7 .38 3.53 9.63 0.22 Iron 5.20 18 .49 30.10 9.47 3.93 26 .94 0.01 V i t a m i n A 32.23 28 .87 0.34 24 .61 3.06 1.19 9.57 Thiamine 7.91 15.33 28 .10 9.32 4 .12 32.17 0.01 R i b o f l a v i n 36.48 23.67 10.64 7.55 3.33 14.36 0.02 N i a c i n 8.10 31.69 11.11 5.50 1.13 33.09 0. 02 Vi t a m i n C 2.60 0.75 2.79 52.48 38 .94 2.35 0.00 aRefer to Secretaria de Planejamento da Presidencia da Republica (1977). » a Table II-8 : Secretaria de Planejamento da Presidencia da Republiea Anthropometric Survey - Sao Paulo Results Age (Years) Mean Anthropometric Measurements by Sex Group M F No. of obser-vations Weight (kg) Height (cm) Arm Circum-ference (cm) No. of obser-vations Weight (kg) Height (cm) Arm Circum-ference (cm) 20 - 24 1 3 3 4 6 1 . 9 1 7 0 . 0 2 7 . 2 1293 5 3 . 4 1 5 8 . 0 2 5 . 5 25 - 29 1 1 3 1 64.6 I 6 9 . 8 28 .1 1 0 5 8 5 5 . 4 1 5 7 . 5 2 6 . 3 30 - 3 9 1771 66'. 2 1 6 9 . 4 28.6 1746 ' 5 8 . 0 1 5 7 . 4 27 .4 40 - 4 9 1441 "67.0 1 6 8 .7 2 8 .6 1 5 0 6 6 0 . 2 1 5 6 .6 2 8 . 1 5 0 - 5 9 9 2 3 66.7 1 6 7 . 5 28 .4 977 6 1 . 4 1 5 5 . 8 28.6 6o - 69 6 0 8 64.4 1 6 6 . 7 2 7 .7 635 6 0 .6 1 5 4 . 8 28 .2 > 70 310 6 2 . 0 165-5 2 6 . 4 349 5 6 . 8 1 5 3 . 0 2 6 . 7 Refer to Secretaria de Planejamento da Presidencia da Republics (1977). - 53 -i i i ) B i o c h e m i c a l A d u l t b i o c h e m i c a l survey r e s u l t s from the s t a t e o f Sao Paulo are summarized i n T a b l e II-9. S z a r f a r c (1972) c o n c l u d e d t h a t i r o n - d e f i c i e n c y anemia was common i n P o n t a l do R i b e i r a and I c a p a r a , but not i n the o t h e r communi t i e s . Roncada (1972) c o n c l u d e d t h a t v i t a m i n A d e f i c i e n c y was a problem i n the a r e a s t u d i e d i f the v i t a m i n A l e v e l o f 20yUg per 100 ml plasma i s the s t a n d a r d of s u f f i c i e n c y . c) R i b e i r a o P r e t o The f i n d i n g s of P u f f e r and Serrano (1973) i n the I n t e r - A m e r i c a n I n v e s t i g a t i o n o f M o r t a l i t y i n C h i l d h o o d i m p l i c a t e s n u t r i t i o n a l d e f i c i e n c y as the most i m p o r t a n t c o n t r i b u t o r t o e x c e s s i v e m o r t a l i t y of c h i l d r e n under 5 y e a r s o f age i n L a t i n A m e r i c a . One o f t h e i r 15 p r o j e c t s was conducted i n R i b e i r a o P r e t o , and r e s u l t s from i t are summarized i n T a b l e s l i-10,and 11-11. S t a t i s t i c s from Sherbrooke , Canada are a l s o i n c l u d e d f o r c o m p a r i s o n . T e r u e l e t a l . (1973) i n a s tudy i n R i b e i r a o P r e t o o f 5,808 deaths of c h i l d r e n between 0 and 5 y e a r s o f age i n the p e r i o d 1950 to 1970, a t t r i b u t e d d i a r r h o e a as the cause o f dea th i n 27%, d i a r r h o e a and m a l n u t r i t i o n j o i n t l y i n another 5%, and m a l n u t r i t i o n the s o l e cause i n another 3%. In a s e q u e n t i a l a n a l y s i s of a l l these deaths by month of the y e a r , they found t h a t d i a r r h o e a had a peak i n the months o f June and J u l y , w h i l e m a l n u t r i t i o n peaked Table II - 9 Biochemical Survey R e s u l t s of A d u l t s -Sao Paulo S t a t e Percentage (No.) i n C a t e g o r i e s Reference Biochemical Parameter : ' . — * : ~ -: -L o c a t i o n P o n t a l do "iifcjuape- Icapara: A p i a i .Ribeira Barra R i b e i r a - do Chapeu Vitamin A ^Ag/100ml) d e f i c i e n t <10 low 10-19 Carotene (Ag/100ml) d e f i c i e n t <20 low 20-39 Hemoglobin (g/100ml) <= 10 > 10 i 12 10.9 (6) 16.4 (9) 6.4 (13) 3.9 ( 8) 7.8 .9 (45) 22.3 (46) 19.3 (11) 57.3 (118) 41 (35) 11 (30) 41 (35) 39 (99) 10.5 (9) 18.6 (16) 73.7 (;7 0) 24.2 (23) 46 (54) 43 (51) 4 (3) 9 (6) 0 (0) 4 (3) 10 (7) 0 (0) Roncada (197 2) Roncada (1972) Roneada (1972) Roncada (1972) S z a r f a r c (1972) S z a r f a r c (1972) Hematocrit (%) < 33 1 33 < 36 19 (16) 15 (38) 32 (36) 16 (11) 17(12) 10 (4) S z a r f a r c (1972) 24 (20) 9 (29) 19 (21) 10 ( 7) 29(20) 15 (6) S z a r f a r c (1972) T a b l e H - 1 0 M o r t a l i t y from S p e c i f i c Types of N u t r i t i o n a l D e f i c i e n c y i n C h i l d r e n Under 5 Years by Age Group i n R i b e i r a o P r e t o i n 1970-1971 a Age Group T o t a l V i t a m i n P r o t e i n D e f i c i e n c y M a l n u t r i t i o n N u t r i t i o n a l Marasmus Other N u t r i t i o n a l Def i c i e n c y <5 y e a r s (per 100,000 490.9 p o p u l a t i o n ) <1 year (per 100,000 1,639.3 l i v e b i r t h s ) 1 year (per 100,000 292.9 p o p u l a t i o n ) 2-4 y e a r s (per 100,000 116.7 p o p u l a t i o n ) 0.0 0.0 0.0 0.0 37.6 16.4 82.0 29.2 137.0 487.9 58.6 29.2 316.4 1,134.9 152. 3 58.3 tn uv aRefer to Puffer and Serrano (1973) T a b l e i i - 1 1 U n d e r l y i n g and A s s o c i a t e d Causes of Death i n C h i l d r e n Under 5 Years f o r R i b e i r a o Preto (City) and f o r Sherbrooke, Canada f o r Comparison (per 100,000 p o p u l a t i o n ) 3 Cause R i b e i r a o Preto (City) Underlying A s s o c i a t e d Sherbrooke^ Canada Underlying A s s o c i a t e d N u t r i t i o n a l d e f i c i e n c y V i t a m i n d e f i c i e n c y P r o t e i n m a l n u t r i t i o n N u t r i t i o n a l marasmus Other n u t r i t i o n a l d e f i c i e n c y 16.4 0.0 14.1 2.3 0.0 358.9 0.0 23.5 100.9 234.6 0.0 0.0 0.0 0.0 0.0 13.2 0.0 0.0 4.4 8.8 ^ e f e r to Puffer and Serrano (1973) - 57 -i n August. They concluded that undernourished childr e n are most susceptible to death at the beginning of the periods of occurrence of diarrhoea and that there i s a worsening of malnutrition at the end of the diarrhoea cycles. Teruel (1967), studying deaths of children,of-0 to 14 years,^in RibeirSo Preto and Dumont calculated a c o e f f i c i e n t of mortality (x 1000) of 10.97 for V i l a Recreio and a neighboring d i s t r i c t , Ipiranga, compared with a c o e f f i c i e n t of 5.79 for the two m u n i c i p a l i t i e s i n t o t a l . The age d i s t r i b u t i o n of the deaths grouped according to t h e i r cause i s given i n Table 11-12.It i s worthy of note that the peak age for deaths due to malnutrition as a basic cause was 1 year to 2 years; for deaths due to malnutrition as a contributory cause was 6 months to 1 year;and for deaths due to diarrhea or diarrhea with dietary error was 28 days to 6 months. Perhaps t h i s indicates a progression from diarrhea or diarrhea with dietary error to malnutrition as a contributory a f f l i c t i o n and f i n a l l y to malnutrition as a basic a f f l i c t i o n . As c i t e d by Shrimpton (1975), i n a cross-sectional study on breastfeeding i n a poor area of Ribeirao Preto, only 4.5% of the mothers studied were breastfeeding t h e i r children a f t e r 12 months of age. In the f i r s t month, 59% breastfed t h e i r children while 28% a r t i f i c i a l l y fed exclusively. By the t h i r d month, only 26% were exclusively breastfeeding while 50% were a r t i f i c i a l l y feeding. Of the Table 1 1 - 1 2 : Age D i s t r i b u t i o n of the 330 Deaths Grouped According co Their Cause i n Ribeirao Preto and a Dumont Cause of Death Axe Groups 0 to 28 28 days to 6 dentils to 1 year to 2 years to 5 years to Total days 6 montns 1 year 2 years 5 years 15 years Malnutrition (basic cause) 0 1 4 10 3 0 18 Malnutrition (contributory cause) 0 3 9 5 5 1 23 Diarrhea 3 2 8 • 4 2 4 0 41 Diarrhea (with dietary error) 1 20 1 0 0 0 22 Pneumonia 3 12 3 2 0 1 21 Congenital Defects 11 10 2 2 1 0 2 6 Neoplasms 0 0 0 1 4 0 11 Infectious Diseases l 4 3 ' 4 4 5 21 Other Causes 3 5 1 0 2 7 18 Undetermined 1 4 1 2 2 1 11 Sickness of Primary Childhood • 110 0 0 0 0 0 110 Accidents and Violence l 1 0 0 . 0 6 8 A l l Causes 134 8 8 28 28 25 27 330 aRefer to Teruel (1967) - 59 -reasons for discontinuing, 50% were i n s u f f i c i e n t or "weak" milk. The study concluded that breastfeeding was of short duration because of f a u l t y orientation of the mother i n 66% of the cases. One quarter of the children in the survey actually l e f t the maternity home already being fed from a bottl e . A Mother and Child Health Cente i n the same poor area were gaining encouraging r e s u l t s in prolonging breastfeeding by educational means. C. Malnutrition Ecology Non-food factors play a d e f i n i t e role i n the occurrence of malnutrition. In the tropics and subtropics, physico-chemical factors such as steady high temperatures and humidity, abundant r a i n f a l l , and/or drought tend to diminish the productivity and c r e a t i v i t y of man; to increase nutrient losses thus augmenting body demands; and to encourage the p r o l i f e r a t i o n of insects, parasites, viruses, bacteria, and other infe c t i o u s agents with pathogenic pot e n t i a l (Mata 1977). High environmental temperatures also reduce c a l o r i c requirement s l i g h t l y . Causes of malnutrition have also been traced to poverty, ignorance, t r a d i t i o n a l b e l i e f s , unhygienic and unsanitary conditions, i n s u f f i c i e n t use of l o c a l low-cost and e a s i l y available foods, waste of foods and nutrients through unsatisfactory and improper cooking methods, undesirable food habits, and fads (Devadas 1970). -r 60 -Food habits may be viewed as a part of culture. "Food" i s always defined c u l t u r a l l y , f or the same plant may be defined as edible by one society and inedible by another. These d e f i n i t i o n s however have no reference to the n u t r i t i o n a l value of the material. Mead has said: "In most s o c i e t i e s , food i s the focus of emotional associations, a channel for interpersonal r e l a t i o n s , for the communication of love or discrimination or disapproval; i t usually has a symbolic reference." Food i s associated with family sentiments, a media through which attitudes and sentiments are communicated. Culture also sets the value or p r i o r i t y of a h e a l t h f u l d i e t (Fathauer 1960). Additional factors influence the food choice of those recently urbanized (Sai 1976) . The population pressure of the c i t y discourages the c u l t i v a t i o n of vegetable gardens, a common practice in r u r a l areas, and convenience foods, a l c o h o l i c beverages, cigarettes, and other advertised "prestige" products become r e a d i l y available, and highly desirable. Confronted with a money economy and demands such as rent, d a i l y transport charges, payments for f u e l , l i g h t i n g and water, the minimal wage earner i s often compelled to choose the simplest and cheapest of staple foods, and i f he has additional money, i t may be spent on alcohol, cigarettes or a t e l e v i s i o n . Martins (1972), studying the socioeconomic s i t u a t i o n and nutrient intake of communities of the Vale do Ribeira, - 6 1 ^ Sao Paulo, B r a z i l , discovered that for v i l l a g e r s dependent on f i s h i n g and subsistence a g r i c u l t u r e , there was no c o r r e l a t i o n between energy or protein adequacy and income but for town-dwellers, casual laborers, there was a clear c o r r e l a t i o n between energy or protein adequacy and income, attributed to the need of town-dwellers to buy t h e i r food i n an urban market, i l l u s t r a t i n g the e f f e c t of urbanization. Jansen et a l . (1977) also studied the e f f e c t of urbanization on d i e t i n B r a z i l . They found that urban areas were uniformly worse off than r u r a l areas i n c a l o r i e and protein consumption. Similar r e s u l t s were founds'in the south of B r a z i l by the Getulio Vargas Foundation survey of 1961 to 1963 (USDA Economic Research Service 1970) where the r u r a l population was apparently well nourished while 40% of the urban population was energy d e f i c i e n t and 11% protein d e f i c i e n t . The urban-rural r e l a t i o n s h i p was also studied by Maldonado (1968) . In the Sao Francisco v a l l e y i n B r a z i l , he examined c l i n i c a l l y 3524 school children and concluded that even with the aid of a school lunch, those from suburban d i s t r i c t s were in a poorer n u t r i t i o n a l state than those l i v i n g i n r u r a l d i s t r i c t s f a r from urban centers without n u t r i t i o n a l aid. In both groups, the commonest sign of malnutrition was dental c a r i e s , and the next most common was enlargement of the thyroid gland. Thirty-eight signs of malnutrition were recorded i n t o t a l . Studying l a c t a t i o n i n women of the Sao Paulo D i s t r i c t , B r a z i l , Yunes and Ronchezel (1975) found that women born i n r u r a l areas have longer lact a t i o n s than women born i n the c i t y of Sao Paulo. According to Mellor (1973), the basic problem and primary cause of malnutrition i s i n s u f f i c i e n t income. Numerous B r a z i l i a n studies have shown the key e f f e c t of income on di e t . Siqueira and Lennini (1968) found that between 1963 and 1967 i n Minas Gerais, the state neighboring Sao Paulo to the north, a low-cost n u t r i t i o n a l l y adequate d i e t would cost a low paid worker with a family of 5 up to 25% more than he earned. Working with randomly selected f a m i l i e s i n C r i s t a l i n a , Goias, B r a z i l , Patrick and Simoes (1971) reported that the very poor families appeared to spend more than t h e i r income on food, the poor, about 8 0% and the well-to-do about 41%, while the diet of the average family f a i l e d to meet any of the recommended nutrient allowances. Campino e t a l . (1975) related the n u t r i t i o n a l status of pre-school childr e n i n the municipality of Sao Paulo to income l e v e l . Five hundred fa m i l i e s were divided, depending on income, into f i v e socioeconomic classes. Of the children i n those classes, i n the order lowest to highest, 46, 33, 21, 15, and 11% had some form of malnutrition. Because of the close association between income and occupation, one would expect occupation also to - 63 -a f f e c t d i e t i n B r a z i l . Lopes (1962) studying working men i n the c i t y of Maceio i n northeast B r a z i l , divided them into three groups. The f i r s t included fishermen, laborers, and factory workers; the second, state o f f i c i a l s , and businessmen; and the t h i r d , bankers and professional men. In the groups, i n order as above, d a i l y c a l o r i c intakes were 1840, 2527, and 3006 kc a l . Occupation i n turn i s influenced by education. According to Livingston (1971), the education of the wage earner i n the family i s a major factor i n determining the socioeconomic l e v e l of the family. In turn, t h i s a f f e c t s the education and n u t r i t i o n a l status of the c h i l d and, thus, a v i c i o u s cycle often i s perpetuated. This was demonstrated by the r e s u l t s of a study by T u r i n i et a l . (1978). Surveying 450 children from 7 to 8 years old attending 10 schools i n suburban areas of Londrina, B r a z i l , they discovered that of the under-nourished childr e n only 56.69% performed s a t i s f a c t o r i l y i n school compared with 73.8 8% of the normal children. More of the undernourished children than of the normal children belonged to f a m i l i e s with a low monthly income. Poor performance i n school was more c l o s e l y related to monthly expenditure on food than to gross monthly income. Ignorance of what constitutes good n u t r i t i o n , and what foods w i l l contribute to i t i s also a cause of malnutrition. Livingston (1971) c i t e s Briggs as follows: - 64 -... many people i n s i s t that a l l that i s needed to solve hunger and malnutrition i n t h i s country [U.S.3 i s a minimum guaranteed income for a l l f a m i l i e s and free or low-cost food for a l l needy persons. Such people have no f a i t h i n n u t r i t i o n education and other proven means of preventing hunger on a long-term basis . . . . Unless people know what foods provide good n u t r i t i o n and how to spend t h e i r money wisely and economically, we cannot expect malnutrition to be erased regardless of the a v a i l a b i l i t y of food. The need for n u t r i t i o n education as well as income i s demonstrated by the r e s u l t s of a study by Jansen et a l . (1977), wherein quantitative and q u a l i t a t i v e aspects of the dietary patterns i n B r a z i l were examined as influenced by income l e v e l . In order to look at quantitative and q u a l i t a t i v e aspects of the d i e t s separately, c a l o r i e and protein intakes were reported d i r e c t l y and the nutrient l e v e l s were presented per 1000 kcal and compared to the average of the INCAP dietary standards expressed s i m i l a r l y . They found that both protein and c a l o r i e intakes increased considerably as income increased, but that the n u t r i t i o n a l q u a l i t y of the food patterns i n terms of the nutrient to c a l o r i e r a t i o s was l i t t l e affected by income l e v e l . To feed themselves properly, people need not only a means to obtain food but also enough knowledge to make the ri g h t food choices. D. National Corrective Measures Any attempt to analyze a n u t r i t i o n a l s i t u a t i o n i s incomplete without some mention of the p o l i t i c a l framework 6 0 / i n which i t i s l o c a t e d and to which i t i s c l o s e l y r e l a t e d . In 1970, a c c o r d i n g to the P o p u l a t i o n Reference B u r e a u , the GNP o f B r a z i l was U . S . $250 per c a p i t a . In 1974, the GNP was p r e d i c t e d to be U . S . $748 per c a p i t a and a c c o r d i n g to the Second N a t i o n a l P l a n o f Development the t a r g e t GNP f o r 1979 i s U . S . $1000 per c a p i t a . Thus , the B r a z i l i a n model f o r development i s an e s s e n t i a l l y economic one. In o r d e r f o r the GNP to grow, the Second N a t i o n a l P l a n o f Development r e q u i r e d a y e a r l y i n c r e a s e i n e x p o r t s o f 20%, from 8 m i l l i o n U . S . d o l l a r s i n 1974 to 20 m i l l i o n U . S . d o l l a r s i n 1979. R e l i a n c e on i n d u s t r y to boos t e x p o r t s was t o be m i n i m a l because i t depends h e a v i l y on e x t e r n a l f i n a n c e , mach inery and know-how p l u s i n v o l v e s a t ime d e l a y i n r e a c h i n g p r o d u c t i o n . The 20% r i s e s per year i n e x p o r t s a r e l a r g e l y hoped to o r i g i n a t e i n the a g r i c u l t u r a l s e c t o r through the c u l t i v a t i o n o f new l a n d s i n the Amazon and M i d - w e s t , the use o f more t r a c t o r s and f e r t i l i z e r s , and the development o f r u r a l i n d u s t r y f o r p r o c e s s i n g and p r e s e r v a t i o n o f these p r o d u c t s f o r d i s t r i b u t i o n (Shrimpton 1975) . The wage d i s t r i b u t i o n o f B r a z i l i s top heavy, such t h a t a c c o r d i n g to the 1970 census the upper d e c i l e o f the p o p u l a t i o n , e c o n o m i c a l l y s t r a t i f i e d , earned a p p r o x i m a t e l y h a l f o f the t o t a l n a t i o n a l wage. A s tudy i n Sao P a u l o c l a i m e d t o show a drop i n the r e a l v a l u e of the minimum s a l a r y between 1959 and 1970 of 37% ( P i a t t 1962) . In the - 66 -decade 1960-70 only the upper decil e of the population showed any r e l a t i v e gain i n income. Rural-urban migration of un s k i l l e d workers tends to keep wages of that group from r i s i n g r a p i d l y . In contrast, the supply of s k i l l e d workers i s very i n e l a s t i c so that increases i n demand r e s u l t i n sharply higher wages (Shrimpton 1975) . In 1975, i n the guidelines for p o l i c y of the Ministry of Home A f f a i r s as c i t e d by Shrimpton (1975), the following passage regarding s o c i a l aspects of development p o l i c i e s was set down: In the s o c i a l sphere of well-being of the people i t i s important to repeat that the strategy of development adopted i n which the e s s e n t i a l p r i o r i t y i s given to growth of the GNP, i s concerned extra-o r d i n a r i l y , with the equal d i s t r i b u t i o n of benefits achieved, further to which the high increase hoped for the income per capita of the country, for t h i s decade of the order of 100%, not to be just an abstract indicator, but to convey a r e a l improvement i n the pattern of l i f e of a l l sections of the community. Nut r i t i o n programs i n B r a z i l at the national l e v e l were c l a s s i f i e d as follows by Shrimpton (1975) : 1. Governmental A. The Ministry of Education and Culture i) The B r a z i l i a n School Feeding Campaign ( CNAE) i i ) Pre-School Feeding Schemes B. The Ministry of Health i) National Ins t i t u t e of Food and Nutrit i o n (INAN) i i ) Coordination for Mother and C h i l d Protection (CPMI) C. The Ministry of Social Welfare - 67 -i) B r a z i l i a n Legion of Assistance (LBA) D. The Ministry of Agriculture i) National Commission for A g r i c u l t u r a l Research and Technical Assistance and Rural Extension (COMPATER) a) A g r i c u l t u r a l research (EMBRAPA) b) A g r i c u l t u r a l extension (EMBRATER) i i ) B r a z i l i a n Food Company (COBAL) i i i ) National Superintendency of Supply (\SUNAB) 2. Other A. National Conference of Bishops of B r a z i l , Caritas B r a s i l e i r a B. Evangelical Churches i n B r a z i l , Diaconia C. Social and Educational Service of Industry (SESI) The stated objectives of the B r a z i l i a n School Feeding Campaign are to give a food supplement and n u t r i t i o n education to the children receiving free compulsory education at government schools between 7 and 14 years of age. This supplement should supply about 15% of the d a i l y requirements for the p r i n c i p l e nutrients. Contributions come from four sources: the Federal Government, the State Government, the Municipal budget and the School Community. Despite the stated objectives of the program, the educational element i s minimal. The n u t r i t i o n education given to school children i s i n a package of health and hygiene lessons taught to teachers i n tr a i n i n g colleges and i s not within the sphere of influence of the program administration. Thus, the program has almost purely a d i s t r i b u t i v e function (Shrimpton 1975) . - 68 -In 1975, there were two pre-school feeding p i l o t projects, the CEAPE project of the School of Public Health i n Sao Paulo and the GURI project of the education o f f i c e of the Federal D i s t r i c t . The National Ins t i t u t e of Food and N u t r i t i o n was formed to a s s i s t the government i n the formulation of a national p o l i c y for food and n u t r i t i o n , including n u t r i t i o n education, and to execute, supervise, evaluate, and i f necessary, revise t h i s p o l i c y . Institute members include representatives from the M i n i s t r i e s of Health, Agriculture, Education, Labour, Social Welfare, and Planning i n addition., to the executive body which c a r r i e s out the p o l i c i e s . Broadly the objectives are: to reach the c h i l d between three and six years of age as well as the student from seven to fourteen years of age through extension of the school feeding program to pre-school feeding a c t i v i t i e s ; to reach the c h i l d between b i r t h and two years of age, the pregnant mother, and the l a c t a t i n g mother through the d i s t r i b u t i o n of supplementary food by the health systems in accordance with the program for the protection of mother and c h i l d ; to provide food supplements for the worker with low income and families earning not more than two minimum wages; to bring modern and e f f i c i e n t methods to the food production system, giving incentives to the small producer;- to stimulate food technology advancement; to combat s p e c i f i c n u t r i t i o n a l d e f i c i e n c i e s - 69 *-through food enrichment projects; to stimulate research i n the area of food and n u t r i t i o n ; to develop the adequate infra-structure for food d i s t r i b u t i o n ; and to t r a i n required personnel to carry out the above subprograms (Shrimpton 1975; Souza 1977). The Coordination for Mother and Child Protection (CPMI) aims: to combat hunger and malnutrition, as previously mentioned; to lower infant mortality rates; and to t r a i n mothers i n better n u t r i t i o n and hygiene habits for themselves and th e i r f a m i l i e s , a l l by motivating and a s s i s t i n g state health o f f i c e s to provide f a c i l i t i e s for those segments of the population not covered by existing services. The B r a z i l i a n Legion of Assistance has a strong maternal and c h i l d care commitment, with coverage over the whole of B r a z i l . There i s potential for state health services to combine with a g r i c u l t u r a l extension a c t i v i t i e s thus providing short-term food and basic preventive medicine in the r u r a l areas i n conjunction with educational inputs e s s e n t i a l l y as a community development project. The B r a z i l i a n Food Company (COBAL) i s an organ of the Ministry of Agriculture that was created i n order for the state to take a firmer control of the food d i s t r i b u t i o n system. COBAL buys i n areas of production and then d i s t r i b u t e s through i t s network of supply centers - 70 that i t has created i n urban areas. The National Superintendency of Supply (SUNAB). i s another organ of the Ministry of Agriculture. It controls and inspects market mechanisms of buying and s e l l i n g , establishes and checks r e t a i l prices for basic foods, and adopts a l l necessary measures to guarantee the supply of e s s e n t i a l commodities for consumption by the population. Although the Social and Educational Service of Industry (SESI) ex i s t s i n a l l states, the state of Sao Paulo has the most extensive programs i n n u t r i t i o n . Linked to the Ministry of Work, SESI i s funded at the state l e v e l by industries related to the State Federation of Industries, and provides s o c i a l services to a l l i t s employees. A c t i v i t i e s include the provision of lunches of about 16 00 c a l o r i e s for workers, and n u t r i t i o n education of people involved i n food preparation, as well as the worker and his wife. These services would be valuable in adapting migrant r u r a l people to a cash economy, d i f f e r e n t foods and an understanding of basic food hygiene. The impact of these corrective measures at the l o c a l V i l a Recreio l e v e l i s described and discussed i n d e t a i l l a t e r . - 71 -CHAPTER III MATERIALS AND METHODS Chapter Outline Section Page A. Sample Population 72 B. Ecolog i c a l Factors 77 C. Food Habit Information and Dietary Data ... 77 1. Food Habit Information 77 2. Dietary Data 78 a) C o l l e c t i o n 78 b) Analysis 79 D. Anthropometric Determinations 90 1. Weight 90 2. Height 90 3. Mid-upper-arm Circumference 90 4. Triceps Skin-fold , 90 5. Mid-upper-arm-muscle Circumference ... 91 E. Biochemical Assessment ... 91 1. Blood C o l l e c t i o n and Treatment 91 a) Boia-Fria Samples 91 b) High L i p i d Samples 92 c) Infant Samples 93 2. Methods of Blood Analysis 94 a) Plasma Vitamin A and Carotene ... 94 b) Plasma Vitamin E I l l c) Plasma Cholesterol 119 d) Plasma Total Lipids 126 e) Plasma Triglycerides 137 f) Hemoglobin 141 g) Hematocrit 142 F. Computer Analysis 142 - 72 -A. Sample Population The subjects of this study were B r a z i l i a n Boia-Frias or a g r i c u l t u r a l migrant workers and t h e i r f a m i l i e s , residing i n V i l a Recreio, a peripheral favela or slum or Ribeirao Preto. (Figures I I I - l , III-2, III-3, and III-4). Located i n the northeastern part of the state of Sao Paulo, Ribeirao Preto i s 310 km by road from the c i t y of Sao Paulo i n southern B r a z i l . Figures III-5 and III-4, maps of B r a z i l and the c i t y of Ribeirao Preto, are included to c l a r i f y the location of V i l a Recreio. Boia-Fria families were i d e n t i f i e d with the help of: l o c a l community workers at Plimec, a c h i l d care, educational and food supplementation center; agents taking Boia-Frias to work; and the owners of the corner-stores i n V i l a Recreio. The fami l i e s , randomly selected as described by Desai et a l . (1978a), were estimated to compose approximately 57o of the t o t a l population i n V i l a Recreio. A t o t a l of 100 families were v i s i t e d , and i n each home, the matriarchal head was interviewed using a questionnaire designed by Desai et a l . (1978a) to c o l l e c t e c o l o g i c a l and food habit information. Also, she was asked to r e c a l l a l l food items consumed by h e r s e l f and her husband i n the preceding 24 hours. Interviews were conducted i n Portuguese by a l o c a l s o c i a l worker (Figure III-3). At the end of each interview, the homemaker and her Boia-Fria husband were i n v i t e d to - 73 -F i g u r e I I I - l : T y p i c a l B o i a - F r i a s a t Work F i g u r e I I I - 2 : T y p i c a l B o i a - F r i a s b e i n g T r a n s p o r t e d t o Work - 7 4 -F i g u r e I I I - 4 : Map o f R i b e i r a o P r e t o S h o w i n g L o c a t i o n o f V i l a R e c r e i o - 75 -F i g u r e III-5:Map o f South America showing B r a z i l , S a o Paulo S t a t e , Sao Paulo C i t y and R i b e i r a o P r e t o - 76 -a t t e n d a s p e c i a l c l i n i c s e t up i n V i l a R e c r e i o f o r the purpose of blood sample c o l l e c t i o n and anthropometric examination. Of the 100 f a m i l i e s , t here were 20 s i n g l e p a r e n t s , 19 female and 1 male. A t o t a l of 124 attended the c l i n i c p r o v i d i n g b i o c h e m i c a l and anthropometric data f o r a n a l y s i s . Of these, 85 were female and 39 were male. V i l a R e c r e i o ' s p o p u l a t i o n was estimated to be 20,000 of mixed e t h n i c background. Acc o r d i n g t o Desai et a l . (1978a), the average f a m i l y u n i t of B o i a - F r i a s i n V i l a R e c r e i o c o n s i s t e d of 3 a d u l t s (range 1 to 6), an a d u l t being d e f i n e d as anyone 17 years of age or over, and 4 l i v i n g c h i l d r e n (range 0 to 9) thus making a t o t a l o f 7 f a m i l y members (range 2 to 13) i n the house. There-f o r e , of the B o i a - F r i a f a m i l i e s s t u d i e d , 57% of the members were l e s s than 17 years of age, i n d i c a t i n g a young p o p u l a t i o n . F o r the e n t i r e c i t y of R i b e i r a o Preto i n 1978, the average f a m i l y s i z e was estimated to be 4.6, and 39% of the p o p u l a t i o n was estimated to be l e s s than 17 years of age. In g e n e r a l , the people of V i l a R e c r e i o appeared to be n e i g h b o r l y and f r i e n d l y . Sounds of c h i l d r e n p l a y i n g or c r y i n g added to the clamor of the sound of r a d i o s b l a r i n g i n the background. Attempts had been made to fence i n some yards w i t h r i c k e t y wooden fen c e s , however, the c h i l d r e n and domestic animals, dogs, c a t s and c h i c k e n s , seemed f o r the most p a r t , f r e e to roam the s t r e e t s . - 77 -According to the study of Desai et a l . (1978a), on the average, each Boia-Fria family had 1 abortion (range 0 to 6) and 1 dead c h i l d (range 0 to 8). Almost a l l deaths among children were reported to have occurred before the age of f i v e and many under one year of age. Malnutrition and i n f e c t i o n were presumed to be the causes of mortality among these children. B. Ecolo g i c a l Factors A detailed questionnaire was designed, translated into Portuguese and used to c o l l e c t the following e c o l o g i c a l information from the female head of the household: family background; socio-economic data; l i v i n g conditions; educational l e v e l ; and public assistance received (Desai et a l . 1978a). The author of this thesis also conducted a b r i e f food cost survey of corner-stores and street vendors i n V i l a Recreio and the market-place i n the center of Ribeirao Preto. Additional information regarding transportation, industry and commerce, and l o c a l corrective measures was obtained by observation and v i s i t a t i o n with the aid of Paula Beatriz M. Carvalho of the C l i n i c a l Medicine Department, University of Sao Paulo,Ribeirao Preto and M.L. Garcia Tavares, of the Fundacao Legiao B r a s i l e i r a de Assistencia, Ribeirao Preto. C, Food Habit Information and.Dietary Data 1. Food Habit Information - 78 -Information regarding food habits, both.qualita-t i v e and quantitative aspects, infant feeding practices, and maternal dietary habits during pregnancy and l a c t a -t i o n , was c o l l e c t e d using the questionnaire designed by Desai-et .al. (1978a) during interviews conducted i n Portuguese by a l o c a l s o c i a l worker. 2, Dietary Data a) C o l l e c t i o n Appendix 1 of the questionnaire consisted of 2 tables e n t i t l e d , "Quantities of Foods and Beverages Consumed the Preceding Day (Previous 24 hours)" as seen i n Table I I I - l . The 24 hours were broken down into the following subdivisions: breakfast, between breakfast and lunch, lunch, between lunch and supper, supper, night. These tables were used to record food consumption as r e c a l l e d by the female head of the household f o r h e r s e l f and the p a t r i a r c h a l head of the family. Foods were recorded i n Portuguese by the interviewer, along with food quantities expressed i n terms of the following types of B r a z i l i a n measures: colher de sopa, or soup spoon; espumadeira, or skimmer; - 79 -concha, or l a d l e ; copo, or g l a s s ; x i c a r a de cafe, or c o f f e e cup; x i c a r a de cha, or tea cup; p r a t o raso, or dinner p l a t e ; g a r r a f j n h a , or s m a l l b o t t l e ; g a r r a f a , or b o t t l e . Any supplements consumed were a l s o r e c o r d e d along w i t h g e n e r a l i n f o r m a t i o n such as f a m i l y number, name, date o f i n t e r v i e w , age, and sex. S p e c i a l o b s e r v a t i o n s of i n t e r e s t were a l s o recorded. b) A n a l y s i s T r a n s l a t i o n of food i n t a k e i n f o r m a t i o n i n t o n u t r i e n t i n t a k e data i n v o l v e d the f o l l o w i n g s t e p s : 1) Breakdown of the foods l i s t e d i n t o t h e i r v a r i o u s component p a r t s ; 2) Conversion of the v a r i o u s p o r t i o n s i z e s i n t o m e t r i c u n i t s ; 3) Conversion of the v a r i o u s p o r t i o n s i z e s i n t o m e t r i c u n i t s i n terms of the v a r i o u s component p a r t s ; 4) G i v i n g each food component a p a r t i c u l a r food code and food group code; 5) Development o f a m o d i f i e d food n u t r i e n t composition t a b l e f o r B r a z i l i a n foods i n terms of the v a r i o u s food com-ponents encountered i n t h e i r d i e t ; - 80 -Table Ili-1;QUANTIDADES DE COMIDAS E BEBIDAS CONSUMIDAS NO DIA ANTERIOR (ULTIMAS 24 HORAS) Numero da Familiar Data: Nome: Idade: Sexo: HORA.RIO ALIMENTOS QUANTIDADE OBSERVACOES Cafe da Manna Entre Cafe e Aim oco » Aimoco Entre Almo9o e Jantar '. Jantar '. Noite ;Suplemento Vitaminas: Ferro: Calcio: - 81 -6) Coding of food intake information i n terms of various food components' food codes and metric portion size units; 7) Coding of food nutrient composition table i n terms of food components1 food and food group codes and nutrient composition i n standard units; and 8) Computer analysis to a r r i v e at food nutrient intake data. 1) Breakdown of the foods l i s t e d into t h e i r various component parts.  Information with regard to the various component parts of foods was gathered from many sources including: those interviewed v i a the interviewer; Pedro Veneziano, head d i e t i c i a n of the l o c a l hospital; B r a z i l i a n recipe books; B r a z i l i a n cooks i n l o c a l i n s t i t u t i o n s , and B r a z i l i a n housewives. For example, tea with milk and sugar was estimated to contain approximately 29% tea brew, 58% milk, and 13% sugar by weight. A more complicated example would be soup of corn and macaroni. This was estimated to contain approximately 3.1% soya o i l , 29% cooked corn, 43% cooked macaroni, 0.6% g a r l i c , 1.1% onion and the remaining 23% water. The foods, r i c e and beans, staples of the d i e t were estimated to contain the follow-- 82 -ing components i n determining t h e i r nutrient compositions. Beans were estimated to contain 92% cooked beans of the mature dry type and 8% soya o i l . Rice was estimated to contain 96% cooked white r i c e and 4% soya o i l . Since r i c e and beans were such common dietary items, they were l i s t e d and coded d i r e c t l y as such. However, a l l other foods were l i s t e d and coded as th e i r various components. 2) Conversion of the various portion sizes into metric units . ' ' '  B r a z i l i a n food items were prepared and/or co l l e c t e d for weighing. Food portions, expressed in terms of B r a z i l i a n measures previously described, were weighed in duplicate or t r i p l i c a t e and averaged for increased accuracy i n determining metric weights. For example, one tea cup of tea with milk and sugar weighed 114 g; one large la d l e of beans weighed 181 g; one lad l e of soup weighed 150 g; one soup spoon of ground meat and potatoes i n tomato sauce weighed 24 g; one piece of f r i e d meat, 5 x 8 cm,weighed 44 g. 3) Conversion of the various portion sizes into metric units i n terms of t h e i r various component parts.  The various portion sizes were then broken down into t h e i r component parts, and the weight of each was determined i n metric units. For example, one tea cup of tea with milk and sugar was calculated to contain approximately the follow-ing amounts of the various component parts: 33 g tea, brew, 66 g of milk, and 15 g sugar giving a t o t a l of 114 g. - 83 -Another example would be one ladle of corn and macaroni soup, which was calculated to contain approximately the following amounts of the various component parts: 5 g soya o i l , 43 g cooked corn, 64 g cooked macaroni, 1 g g a r l i c , 2 g onion, with the remainder being water. 4) Giving each food component a p a r t i c u l a r food code and food group code.  Each food component was given a unique three d i g i t food code, as well as a three d i g i t food group code, as taken from the table of food group codes, pages 5-8 in the United States Department of Agriculture Manual ARS 62-10-1 e n t i t l e d "Calculating the N u t r i t i v e Value of Diets" (Davenport 1964). 5) Development of a modified food nutrient composition table for B r a z i l i a n foods. Information for t h i s table was computed from data from food composition tables developed by: Leung and Flores (1961) ; I C N N D (1965) ; Watt and M e r r i l l (1963) ; and the Secretaria de Planejamento da Presidencia da Republica, I B G E (1977) . A l l nutrient compositions were expressed i n terms of 100 g of the food. If a food was l i s t e d i n more than one of the tables, the table with greatest regional significance was used. If the exact food item could not be found, the food item with greatest s i m i l a r i t y was chosen. For example, the B r a z i l i a n s eat a green leafy vegetable c a l l e d rucula not l i s t e d i n t h e i r food composition tables. To estimate - 84 -i t s nutrient composition, the nutrient composition of endive, a similar food item, was used as taken from the INCAP/ICNND table by Leung and Flores (1961) for use i n La t i n America. If values for a p a r t i c u l a r cooked food item were only avail a b l e for i t s raw counterpart, corrections were made for losses of vitamins and loss or gain of water during cooking. For example, the nutrient composition of cooked c o l l a r d s was based on the nutrient composition of raw co l l a r d s as taken from the INCAP/ICNND table by Leung and Flores (1961) for use i n Latin America and was modified according to the nutrient composition for c o l l a r d s cooked i n much water from the USDA Manual by Watt and M e r r i l l (1963). Modifications included reductions i n active vitamin A, thiamine, r i b o f l a v i n , n i a c i n , and vitamin C. If values for a p a r t i c u l a r f r i e d food item were only available for i t s boiled counterpart, corrections were made for less loss of water-soluble vitamins, greater loss of water during cooking, and o i l uptake during f r y i n g . For example, the nutrient composition of deep-fat f r i e d p rimitive b r i n j a l , j e l o , a vegetable similar to eggplant was based on the nutrient composition of raw eggplant as taken from the INCAP/ICNND table by Leung and Flores (1961) for use i n La t i n America and was modified according to the change i n nutrient composition i n the preparation of potato chips from raw potatoes as taken from the USDA Manual by Watt and M e r r i l l (1963). Modifications included: - 85 -a d r a s t i c reduction i n water content; increase i n c a l o r i e s due to loss of water and f a t uptake;- a three-fold increase i n protein, carbohydrate and n i a c i n content due to the concentration e f f e c t of losing water combined with some loss during cooking; and uptake of 40 g of fat per 100 g of deep-fried product; a f o u r - f o l d increase i n calcium and iron content due to the concentration e f f e c t of losing water; and increase i n vitamin A content due to the soya o i l uptake, but with a large portion destroyed due to the high temperatures; a two-fold increase .in t h i a -mine and r i b o f l a v i n due to the concentration e f f e c t of losing water combined with cooking losses; a s l i g h t decrease i n vitamin C content due to cooking losses p a l l i a t e d due to the concentration e f f e c t of losing water. The vitamin E values for foods were derived from a table compiled by Bauernfeind and refereed by Desai (1977). Instead of t o t a l tocopherol, tocopherol values of food were chosen for use i n t h i s study because the U. S. 1974 Recommended Dietary Allowances (NRC Food and N u t r i t i o n Board 1974) stated that i n c a l c u l a t i n g the vitamin E content of mixed diets, values for ^-tocopherol should be m u l t i p l i e d by a factor of 1.2 to give a more accurate estimation of t h e i r t o t a l vitamin E a c t i v i t y , expressed as PC-tocopherol equivalent. The following method was used i n deriving the o<-tocopherol values for the various food items. If many values for one - 86 -food item were l i s t e d , then an average of values was taken, weighted to correct for the number of samples analyzed i n determining the value. For example, the values l i s t e d for butter were as given i n Table III-2. Table m-2: * - Tocopherol Content of Butter Number of Samples •a ^-Tocopherol Content (mg/10 Og) <* -Tocopherol Content of A l l Samples (mg) 1 1 20 1 3.30 x 1 1.00 x 1 1.68 x 20 2.65 x 1 - 3.30 1. 00 33.60 2.65 Total 2 3 40.55 Average 40.55 . 23 ~ 1 ' 7 6 afrom Bauernfeind and Desai (1977) For those food items which could not be found i n t h i s table, the <*-tocopherol value was estimated by one or several of the following methods: i) on the basis of f a t content and the vitamin E content of the f a t or of a similar type of f a t ; i i ) on the basis of the various components of the food item; or i i i ) on the basis of a similar product. i) An example for which the f i r s t method of estimation was used was cooked beef r i b . From the USDA Manual by - 87 -Watt and M e r r i l l (1963), the f a t content was found to be 39 g per 100 g cooked beef r i b . From the t a b l e by Bauernfeind and Desai (1977), cooked ground beef c o n t a i n e d 0.37 mg oL-tocopherol per 100 g. Assuming t h a t the ground beef was approximately 2 0% beef f a t , the ot-tocopherol content of cooked beef f a t would be 1.8 2 mg per 100 g, and the t o c o p h e r o l content of cooked beef r i b would be 0.71 mg per 100 g. i i ) An example f o r which the second method of e s t i m a t i o n was used was a tomato, onion and g a r l i c sauce. T a b l e I I I - 3 shows the v a r i o u s components of t h i s sauce, t h e i r propor-t i o n a t e amounts per 100 g sauce, the estimated oc-tocopherol content of each component i n mg per 100 g, the e*-tocopherol content c o n t r i b u t e d by each component i n 100 g sauce and the t o t a l ^ - t o c o p h e r o l c o n t e n t of the sauce i n mg per 100 g. Table 111-3'Sample C a l c u l a t i o n f o r the Component Method of V i t a m i n E Content E s t i m a t i o n Using Tomato, Onion, G a r l i c Sauce as the Example Components Amount per 100 g Sauce (g) a oi-Tocopherol Content (mg/100 g of each component) oC-Tocopherol Content Con-t r i b u t e d by each Component Tomatoes Onion G a r l i c Soya O i l , cooked 83.2 8.7 1.1 7.0 0.40 0.21 0.17 9.10 0.333 mg 0.018 mg 0.002 mg 0.637 mg Tomato Sauce 0.990 mg/100 g afrom Bauernfeind and Desai (1977) - 88 -i i i ) An example for which the t h i r d method of estima-t i o n was used was cooked pumpkin. Since the oc-tocopherol value for cooked pumpkin was not l i s t e d , the value for cooked strained squash was used. 6) Coding of food intake information. The previous 24-hour food intake information co l l e c t e d for the Boxa.'-Frias was coded in terms of the various food components' food codes and the metric portion size u n i t s . For example, i f a person consumed 2 tea cups of coffee with milk and sugar for breakfast, the following information would be coded as shown on Table III-4. Table m-4: Sample Coding of Food Intake Information Corresponding to 2 Tea Cups of Coffee with Milk & Sugar Food Component Food Code Metric Portion Size (g) Coffee 848 66 Milk 700 132 Sugar 805 30 7) Coding of food nutrient composition table. The food nutrient composition information was coded i n terms of food components' food and food group codes and nutrient composition i n standard units. The information was arranged in computer card columns accord-ing to the design given i n Table III-5, - 89 -Table II1-5 :Coding of Food Nutrient Composition Table: Card Design Card Columns Description 1 - 3 Food Item Code Number 4 - 6 Food Group Code Number 7 - 1 0 Weight, g ( 1 decimal) 1 1 - 1 2 Water Content, percent (whole number) 1 3 - 1 6 Food Energy, Calories (whole number) 1 7 - 1 9 Protein, g (whole number) 2 0 - 2 2 Fat, t o t a l l i p i d , g (whole number) 2 3 - 2 5 Total Saturated Fatty Acids, g (whole number) 2 6 - 2 8 Unsaturated, Oleic Fatty Acid, g (whole number) 2 9 - 3 0 Unsaturated, L i n o l e i c Fatty Acid, g (whole number) 3 1 - 3 3 Carbohydrate, g (whole number) 3 4 - 3 7 Calcium, mg (whole number) 3 8 r 4 0 Iron, mg (one decimal) 4 1 - 4 4 Vitamin A, active,/4.g (whole number) 4 5 - 4 7 Thiamine, mg (two decimals) 4 8 - 5 0 Riboflavin, mg (two decimals) 5 1 - 5 3 Niacin, mg (one decimal) 5 4 - 5 6 Ascorbic Acid, mg (whole number) 5 7 - 6 1 <*-Tocopherol, mg (two decimals) 8 ) Computer analysis to give food nutrient intake data. Computer programs for estimating d a i l y intake of various nutrients by using the above mentioned food composition table and dietary data obtained through 2 4 - h o u r r e c a l l were developed by Lewis James of the University of B r i t i s h Columbia S t a t i s t i c a l Centre. - 90 -D. Anthropometric Determinations The anthropometric measurements included i n this survey for the assessment of body size and n u t r i t i o n a l status were obtained i n accordance with the standard re-commended procedures ( J e l l i f f e 1966). One hundred and twenty-four adults were examined for the following para-meters: weight, height, mid-upper-arm circumference, t r i -ceps s k i n - f o l d and mid-upper-arm-muscle circumference. 1. Weight Individuals were weighed to the nearest one-tenth of a kg before meals, with a minimum of indoor clothing and without footwear, 2. Height To measure height, a standardized platform-type beam balance with a telescopic v e r t i c a l measuring rod and a horizontal cross-bar was used. The height was measured to the nearest one-half cm while subjects were standing on the platform of the balance. 3. Mid-upper-arm Circumference Mid-^upper-arm circumference was measured to the nearest mm with a s p e c i a l l y made f l e x i b l e c e l l u l o i d tape which was s l i d over the hanging, relaxed l e f t arm and raised to the point midway between the t i p , o f the, acromion and the olecranon process where the measurement was made. 4. Triceps Skin-Fold The thickness of triceps s k i n - f o l d was measured to - 91 -the nearest tenth of a mm with a Lange Skin-fold Caliper, which had a uniform pressure of 10 g per mm2 of contact surface area. The measurement was taken on the back of the l e f t arm and midway between the point of the acromium and the olecranon process with the arm muscles f u l l y relaxed. 5. Mid-upper-arm-muscle Circumference The estimate of muscle size was derived from the following formula ( J e l l i f f e 1966), using actual values of arm circumference and tri c e p s s k i n - f o l d thickness. MUAMC = MUAC 7T X TSF WHERE : MUAMC = Mid-upper-arm-muscle circumference i n cm MUAC = Mid-upper-arm circumference i n cm AND TSF r = Triceps skin - f o l d i n cm Biochemical Assessment 1. Blood C o l l e c t i o n and Treatment a) Boia-Fria Samples Subjects were asked to give blood a f t e r an overnight f a s t . Approximately 20 ml of the venous blood drawn with a s t e r i l i z e d glass syringe were placed i n a c e l l u l o i d centrifuge tube containing 14 drops of 1% disodium ethylene-diamine t e t r a a c e t i c acid (EDTA), and approximately 5 ml were placed i n a glass rubber-stoppered storage v i a l , containing 3 drops of 1% disodium ethylenediamine t e t r a a c e t i c acid (EDTA). Blood samples were gently shaken and immediately - 92 -refr i g e r a t e d . The 5 ml aliquot was analyzed within 24 hours for hemoglobin and hematocrit. The blood placed i n the centrifuge tube was then centrifuged at 2500 revolutions per minute at 5°C for 10 minutes, and the plasma was transferred to glass rubber-stoppered storage v i a l s using a Pasteur pipet with a rubber bulb. The rubber stoppers were well-protected with parafilm to prevent the plasma from contacting reducing substances possibly present i n the rubber. The plasma was divided into 2 v i a l s , one for r e f r i g e r a t e d storage at 5° C and one for frozen storage at -20° C. Vitamin A, carotene, vitamin E, and cholesterol were analyzed i n the fresh r e f r i g e r a t e d plasma within one week of c o l l e c t i o n , and t o t a l l i p i d was analyzed i n the frozen plasma within 6 weeks of c o l l e c t i o n . b) High L i p i d Samples Thirty high l i p i d plasma samples, made available through the Lipids Laboratory of the University of S&o Paulo Hospital, were co l l e c t e d from hyperlipemic subjects who had been instructed to take nothing by mouth other than water for 12 to 16 hours p r i o r to sampling. These were analyzed for comparison purposes - 93 -with respect to vitamin E. Again, disodium ethy-lenediamine tet r a a c e t i c acid (EDTA) was used as an anticoagulant and the plasma was separated from the c e l l s using a Pasteur pipet with.a rubber bulb, following cooling and centrifugation. Triglycerides and cholesterol were analyzed i n fresh r e f r i g e r a t e d plasma samples and t o t a l l i p i d and vitamin E also i n the same samples after they had been held i n frozen;:storage for less than 2 months. c) Infant Samples Nine newborn infant.cord blood samples, made available by Dr. Francisco E. Martinez of the Pediatrics Department, University of S2fo Paulo Medical School, Ribeirao Preto, were s p e c i a l l y c o l l e c t e d f o r vitamin E analysis, using disodium ethylenediamine t e t r a a c e t i c acid (EDTA) as an a n t i -coagulant. They were then cooled, centrifuged, and immediately frozen p r i o r to analysis for vitamin E, arid t o t a l l i p i d s , which was ca r r i e d out within 4 weeks. - 94 -2. Methods of Blood Analysis '•:,} The author analyzed the Boia-Fria blood samples for plasma vitamin A, carotene, vitamin E, cholesterol, and t o t a l l i p i d s and the high l i p i d blood samples for plasma vitamin E and t o t a l l i p i d s . Hematological analyses were carried out by the Barachini C l i n i c a l Laboratory i n Ribeirao Preto. The high l i p i d blood samples were analyzed for cholesterol and t r i g l y c e r i d e s by the C l i n i c a l Medicine Department of the University of Sao Paulo, Ribeirao Preto. Infant blood sample analyses were performed by Dr. Francisco E. Martinez of the Pediatrics Department, University of Sao Paulo Medical School, Ribeirao Preto. a) Plasma Vitamin A and Carotene Plasma vitamin A and carotene were analyzed accord-ing to the T r i f l u o r o a c e t i c Acid Method of Neeld and Pearson (1963). Reagents - ethyl alcohol, 95%, a n a l y t i c a l grade, (Carlo Erba Do B r a s i l , ;S.A.) - petroleum ether, a n a l y t i c a l grade, b o i l i n g point 20-60°C, d i s t i l l e d at 40°C to i s o l a t e the lower b o i l i n g point f r a c t i o n , (E. Merck, Darmstadt, Germany). - chloroform, a n a l y t i c a l grade. - 95 -- t r i f l u o r o a c e t i c acid, a n a l y t i c a l grade, (Eastman Organic Chemicals). - acetic anhydride, a n a l y t i c a l grade, (E, Merck, Darmstadt, Germany). - ^ - c a r o t e n e , pure, ( B r i t i s h Drug House), stored under nitrogen i n dark i n freezer. - vitamin A acetate, pure, (Eastman Organic Chemicals and Hoffman La Roche Incorporated), ampules stored i n dark i n freezer, used when freshly opened. Reagent Solutions £>- Carotene Standard Solution (400/Mg per M ) : prepared by diss o l v i n g 20 mg^-carotene i n a few ml of chloroform and bringing t h i s solution to a f i n a l volume of 50 ml with petroleum ether. Vitamin A Acetate Standard Solution (350y&(g. per ml) : prepared by diss o l v i n g 17.5 mg vitamin A acetate i n 50 ml of chloroform. ^-Carotene Chloroform Solution (500y<><g per ml): prepared by dissolving 25.0 mg^-carotene i n 50 ml ^chloroform. Vitamin A Acetate Petroleum Ether Solution (J/u g per ml) : prepared by bringing 0.2 ml of the 350yUg per ml vitamin A acetate solution to a volume of 10 ml with petroleum ether. - 96 -A n a l y t i c a l Procedure To determine plasma vitamin A and carotene, the plasma samples were treated i n the following way: 2 ml of plasma were pipetted into a 15-ml glass-stoppered test tube; 2 ml of 95% ethanol were added and the tube was stoppered and shaken vigorously for 8 seconds. Then, 3 ml of petroleum ether were added and the tube was shaken vigorously for 2 minutes, and then centrifuged, with stopper i n place, at low speed for 3 minutes. The ethanol served to p r e c i p i t a t e the proteins of the plasma, and the caroteneand vitamin A were extracted into the petroleum ether. Leaving the tube stoppered in the centrifuge prevented evaporation of the petroleum ether. Following centrifugation, 2 ml of the upper petroleum ether layer were c a r e f u l l y pipetted o f f and placed i n a 10 x 75 mm cuvette which was promptly-stoppered to-prevent evaporation. Carotene was then read at 450 run against a petroleum ether blank i n a Coleman Junior spectro-photometer. Readings were converted i n t o - c a r o t e n e values using standard curve data. The cuvettes were then un--stoppered a na p i a c e d i n a 47 °C water-bath u n t i l the petroleum ether was evaporated to dryness, at which time the residue was immediately taken up i n 0.1 ml chloroform, followed by 0.1 ml acetic anhydride. Again, the tube was stop-pered- t° prevent evaporation. Vitamin A was then measured, following reaction of the sample with 1.0 ml - 97 -of the chromogen s o l u t i o n , c o n s i s t i n g of one p a r t of t r i f l u o r o a c e t i c a c i d and two p a r t s of chloroform, prepared j u s t p r i o r to use. To read f o r V i t a m i n A, the Coleman J u n i o r spectrophotometer was f i r s t s e t a t zero absorbance a t 62 0 nm wit h a blank tube c o n t a i n i n g 0.1 ml ch l o r o f o r m and 1.0 ml of the chromogen s o l u t i o n . The 1.0 ml of chromogen s o l u t i o n was added t o the blank and sample c u v e t t e s using a s p e c i a l home-made r a p i d d e l i v e r y 1.0 ml p i p e t c o n s i s t i n g of a 1 ml v o l u m e t r i c p i p e t a t t a c h e d t o a medical s y r i n g e . The chromogen s o l u t i o n was r a p i d l y e x p e l l e d i n t o the sample c u v e t t e and a t 30 seconds a f t e r a d d i t i o n of the chromogen the re a d i n g was made. These r e a d i n g s were converted i n t o v i t a m i n A v a l u e s u s i n g standard curve data, c o r r e c t i n g f o r ^ - c a r o t e n e , which a l s o r e a c t s w i t h the t r i f l u o r o a c e t i c a c i d reagent, and c o r r e c t i n g f o r l o s s e s of v i t a m i n A d u r i n g the a n a l y s i s procedure as determined by r e c o v e r y t e s t s . These l o s s e s were kept u n i f o r m l y t o a minimum by p r o t e c t i n g the sample from l i g h t and unnecessary heat exposure throughout. prepared a c c o r d i n g to the procedure o u t l i n e d i n Table I I I - 6 were p l a c e d i n c u v e t t e s and t h e i r absorbances read a t 4 50 nm a g a i n s t a petroleum ether blank. The r e s u l t i n g Standard Curve Two-ml a l i q u o t s of ^ - c a r o t e n e working standards. - 98 -data and standard curve are shown i n Table I I I - 7 and F i g u r e I I I -6. C a l c u l a t i o n s The corresponding y^-carotene plasma c o n c e n t r a t i o n i n ^Ug per 100 ml was c a l c u l a t e d as f o l l o w s : Working Standard ^ - C a r o t e n e C o n c e n t r a t i o n (/ug/ml pet, ether) x (3 ml pet. ether) (100 ml plasma) 2 ml plasma T h e r e f o r e , f o r working standard #1 the corres p o n d i n g plasma c o n c e n t r a t i o n would be 4.0 x 3 x 100 — 2 or 600 jmq per 100 ml plasma. The f a c t o r which when m u l t i p l i e d by absorbance w i l l g i v e yL^ g carotene per 100 ml plasma was desi g n a t e d the F f a c t o r and was c a l c u l a t e d from the standard curve data (Table IE-7) as f o l l o w s f o r working standard #1 as an example: F f a c t o r = 600 jug = 950.87 /jg (0.631) 100 ml 100 ml Recovery T e s t A r e c o v e r y t e s t was a l s o performed to ensure t h a t the /^-carotene was being n e i t h e r l o s t nor c o n c e n t r a t e d d u r i n g the a n a l y s i s procedure. Tubes c o n t a i n i n g the m a t e r i a l s l i s t e d i n Tab];e III-8 were prepared a c c o r d i n g t o the p r e v i o u s l y d e s c r i b e d procedure. R e s u l t i n g absorbance r e a d i n g s and TableIIL-6: Preparat ion of y2-Carotene Working Standards: Procedure for D i l u t i o n with Petroleum Ether Std, _ J _ 1 2 3 4 /3-Carotene Concentration (ug/ml) I n i t i a l F i n a l Volume of Solution(ml) 400 4.0 4.0 4.0 4.0 2.0 1.0 0.4 I n i t i a l 0.5 5.0 2.5 1.0 F i n a l 50 10 10 10 Table III-7: ^ - C a r o t e n e Standard Curve Data Std . # Carotene /*g/ml Cone entrat ion Corresponding / ig / lObml plasma Absorbance at 450 nm F Factor (. g/lOOml) ytg/ml-r Absorbance 1 4.0 6ob 0.631 950.87 6.339 2 2.0 300 0.321 934.58 6.231 3 1.0 150 0.168 892.86 5. 952 4 0.4 60 0.064 937.50 6. 250 Averages. 928.95 6.193 - 100 -- 101 -calculated y9-carotene values are shown i n Table III-9. Plasma ^ -carotene concentration was calculated by multiplying the absorbance by the average F factor based on the ^ -carotene standard curve data. The carotene recovery tube had an additional 3 ml of 0.4 yug per ml ^-carotene solution or 1.2 jug ^ -carotene per 2 ml plasma or 60 Jug y5-carotene per 100 ml plasma. Therefore, the percentage recovery was 1 1 6 , 1 ~ 5 2 , 5 x 100% = 106% 6 0 This recovery test procedure does not t e s t for the extraction of carotene since the added carotene was already dissolved i n ether at the s t a r t . However, the test does indicate no loss or s i g n i f i c a n t concentration of ^-carotene during the analysis. Vitamin A Standard Curve For preparation of the standard curve, 0.1 ml aliquots of the vitamin A working standards #1 to #5, prepared as described i n Table III-1Q were pipetted into cuvettes and then reacted with 1.0 ml of the t r i f l u o r o a c e t i c acid chromogen as previously described. The r e s u l t i n g data and standard curve are found i n Table 111-11 and Figure 11,1-7. Table III-8 ^-Carotene Recovery Test Tube Contents (ml) Tube Serum #1 Ethanol Petroleum Ether Carotene-Petroleum Ether Solution( 0. 4/tg/mly Serum #1 a Serum #1 b Carotene Recovery #1 2 2 2 Table III-9 : yS -Carotene Recovery Test Data Tube Serum #1 a Absorbance 0.056 Plasma y^-carotene Concentration (/*g/100ml) Serum #1 b 0.057 Carotene Recovery #1 0.125 52.0 ^1 53.0 116.1 ' average = 52.5 Table III-10 : V i t a m i n A Working Standard P r e p a r a t i o n : Procedure f o r D i l u t i o n w i t h Chloroform Working Volume of a Standard Vitamin A Acetate ^Vitamin A Standard D i l u t e d t o 10 ml. Co n c e n t r a t i o n C o n c e n t r a t i o n # (ml) (/<g/ml) (/cg/ml) 1 0.2 7.00 6.10 2 0.4 14.00 12.20 3 0.6 21.00 18.30 4 0.8 28.00 24-.40 5 1.0 35.00 30.50 ...a V i t a m i n A a c e t a t e standard ( 3 5 0 j u g / m l ) b Since the m o l e c u l a r weights of v i t a m i n A and v i t a m i n A acetate are 286 and 328 r e s p e c t i v e l y , the v i t a m i n A ac e t a t e c o n c e n t r a t i o n must be m u l t i p l i e d by 286/328 or 0.872 to o b t a i n the v i t a m i n A c o n c e n t r a t i o n . - 104 -Cal c u l a t i o n s The corresponding vitamin A plasma concentration i n JUq per 100 ml was calculated as follows: Working Standard Vitamin A Con- (3 ml centr a t i o n (0.1 ml pet- (100 ml (/ug/ml chloroform) x chloroform) ether) x plasma) (2 ml plasma) (2 ml pet. ether) Therefore, for working standard #1, the corresponding plasma concentration would be (6.10) (0.10) (3) (100) (2) (2) or 4 5.75 y^g per 100 ml plasma. The factor which when m u l t i p l i e d by absorbance w i l l giveyUg vitamin A per 100 ml plasma was designated the F f a c t o r and was calcula t e d from the standard curve data (Table ffi-ll) as follows f o r working standard #1 as an example. F f a c t o r = 45 .75 >ug- 278.96 JLtq (0.164) 100 ml 100 ml Recovery Tests For recovery t e s t s , tubes containing the materials l i s t e d i n Table 111-12 were prepared according to the previously described procedure. The o r i g i n a l 350 /jg vitamin A acetate per ml chloroform solution from which the petroleum ether vitamin A solut i o n was made was stored f o r 3 days i n the r e f r i g e r a t o r at 5 "c p r i o r to being used for t h i s purpose and had deteriorated to about 75% of i t s o r i g i n a l potency. Table I I I - 1 1 s V i t a m i n A Standard Curve Data V i t a m i n A C o n c e n t r a t i o n Absorbance F f a c t o r , y^g/ml Corresponding y£<g/100ml Plasma at 620 rim non-corrected Cag/100ml) 6.10 12.20 18. 30 24. 40 30. 50 45.75 91.50 137.25 183.00 228.75 0.164 0. 349 0.533 0.717 0.841 278.96 262.18 257.50 255!;23 272.00 Average= 2 65.17 Table i n - 1 2 :. . . V i t a m i n A Recovery T e s t s : Tube Contents -Tube (u<3 Vitamin A) Water Alcohol Petroleum Petroleum Ether (ml) (ml) Ether Vitamin A Solution (ml) (.6..10/cg/ml) 0.61 2.0 . ' 2.0 2.9 0.1 l;22 2.0 ' 2.0 2.8 0.2 1.83 2.0 ' 2.0 2.7 0.3 3.05 2.0 2.0 2.5 0.5 - 106 -- 107 -On the b a s i s of the r e s u l t s presented i n Table III-14, the 356 3 7 percentage r e c o v e r y i s about ' ^ x 100% or . 78%. T h i s was taken i n t o c o n s i d e r a t i o n and the standard curve 26 5 X 7 F f a c t o r , 265 .17 was c o r r e c t e d becoming yg— or 34 0.0. A -Carotene C o r r e c t i o n S i n c e ^ - c a r o t e n e a l s o r e a c t s w i t h the t r i f l u o r o a c e t i c a c i d chromogen t o g i v e a blue c o l o r , a t r i f l u o r o a c e t i c a c i d - carotene standard curve was a l s o made. The f o l l o w -i n g procedure o u t l i n e d i n Table III-13 was used i n p r e p a r i n g the y^-carotene c h l o r o f o r m s o l u t i o n s r e q u i r e d . Table '.^-Carotene Chloroform Working Standards: III-13 Procedure f o r D i l u t i o n w i t h Chloroform Working Standard # Volume o f a S t a n d a r d D i l u t e d t o 10 ml (ml) ' /9-Carotene C o n c e n t r a t i o n ' r b ' •• (Corresponding yUg/ml petroleum (/ug/ml) ether) 1 - 500 25. 0 2 7.0 350 17 .5 3 5.0 250 12.5 4 4.0 200 10.0 5 2.0 100 5.0 6 1.0 50 2.5 a ^ - c a r o t e n e i n c h l o r o f o r m standard (500 yug/ml) . b Note t h a t the carotene i s co n c e n t r a t e d 2 0 - f o l d d u r i n g ether e v a p o r a t i o n and subsequent uptake i n c h l o r o f o r m . For standard curve p r e p a r a t i o n , 0.1 ml a l i q u o t s of the s i x working standards were r e a c t e d w i t h the t r i f l u o r o a c e t i c Table ITI-,14: V i t a m i n A Recovery T e s t s Data Tube Corresponding V i t a m i n A j/xg V i t a m i n A) C o n c e n t r a t i o n yUg/100ml pla.sma) Absorbance at 620 nm Recovery tubes 0.61 1.22 1.83 3 .05 Standards 0.61 1.83 3 .05 30.50 61.00 91.50 152.50 45.75 137.25 228.75 F F a c t o r (^g/100ml) 0.062 0.125 0.243 0.326 0.123 0.404 0.640 491.94 488.00 376.54 467.79 average= 4 56.07 371.95 339.73 357.42 average =356.37 - 109 -a c i d c h r o m o g e n a s p r e v i o u s l y d e s c r i b e d . T h e r e s u l t i n g d a t a a n d s t a n d a r d c u r v e a r e f o u n d i n T a b l e III-15 a n d F i g u r e III-8, Table-III-15 : . T r i f l u o r o a c e t i c A c i d - C a r o t e n e S t a n d a r d C u r v e D a t a W o r k i n g A b s o r b a n c e C o r r e c t i o n S t a n d a r d T F A R e a c t i o n C o r r e s p o n d i n g F a c t o r # a t 62 0 nm a t 450 nm l a 0 . 7 2 1 4 . 0 3 7 0 . 1 7 8 6 l b 0 . 8 1 0 4 . 0 3 7 0 . 2 0 0 6 2 0 . 5 4 2 2 . 8 2 6 0 . 1 9 1 8 3 0 . 3 7 8 2 . 018 0 . 1 8 7 3 4 0 . 290 1 . 6 1 5 0 . 1 7 9 6 5 0 . 1 4 7 0 . 8 0 7 0 . 1 8 2 2 6 0 . 0 7 3 0 . 4 0 4 0 . 1 8 0 7 A v e r a g e = 0 . 1 8 5 8 To calculate the required correction factor, the t i c i -f l u o roacetic acid reaction absorbance at 620 nm i s divided by the corresponding carotene absorbance at 4 50 nm. One determines t h i s corresponding absorbance by dividing the corresponding ^ -carotene concentration i n petroleum ether (/a.g per ml) by 6.193, the factor derived from the^3-carotene standard curve data for t h i s purpose as shown i n TableIII-7. For example using working standard #2 data, correction factor = (0.542) _ (0.542) _ (17.5 >r 6.193) " (2.826) ~ u.iyxa From the foregoing ca l c u l a t i o n s and the volumes of reagents used, the amounts ofyf?-carotene and vitamin A i n - 1 1 0 -F i g u r e I I I - 8 : T r i f l u o r o a c e t i c Acid-Carotene Standard Curve jS- Carotene C o n c e n t r a t i o n (^tg/ml) - I l l -the sample are calculated as follows: (1) Carotene Absorbance 4 , - Q X 928.95 = jxq carotene/100 ml (2) Vitamin A Absorbance ~ (Absorbance ^ 0 x .186) x 340. 0 = U^g Vitamin A/100 ml b) Plasma Vitamin E Plasma vitamin E was analyzed according to a modified version of the Fabianek method (Fabianek et a l . 1968) . Reagents - ethanol, p u r i f i e d absolute, obtained by d i s t i l l a -t i o n of commercial absolute ethanol to which were added some p e l l e t s of calcium chloride (J. T. Baker Chemicals, S.P.) and c r y s t a l s of potassium permanganate (General Chemical Di v i s i o n , Baker and Adamson, N.Y.). - orthophosphoric acid, 85%, a n a l y t i c a l grade. - xylene, a n a l y t i c a l grade p u r i f i e d by d i s t i l l a t i o n , (E. Merck, A G - Darmstadt Germany) . - acetone, a n a l y t i c a l grade p u r i f i e d by d i s t i l l a t i o n . - 4, 7 - diphenyl - 1, 10 - phenanthroline or batho-phenanthroline, (BP)(Eastman Kodak Company, Rochester, N.Y.). - 112 -- f e r r i c chloride, hexahydrate, a n a l y t i c a l grade (Fisher S c i e n t i f i c Company). - d l —oc— tocopherol reference standard, 98.7% purity (Hoffmann La Roche), i n sealed ampules stored i n freezer, used when fre s h l y opened. Reagent Solutions Bathophenanthroline Solution: prepared by d i s -solving 40.0 mg bathophenanthroline i n p u r i f i e d absolute ethanol to give a f i n a l volume of 10 ml. F e r r i c Chloride Solution: prepared by d i s s o l v i n g 6.0 mg f e r r i c chloride i n p u r i f i e d absolute ethanol to give a f i n a l volume of 10 ml. This solution was prepared fresh on each day of analysis. Orthophosphoric Acid Solution: prepared by d i l u t i n g 0.25 ml orthophosphoric acid with p u r i f i e d absolute ethanol to give a f i n a l volume of 5 0 ml. Of-Tocopherol Stock Solution: prepared by diss o l v i n g 10.1 mg of dl-£X-tocopherol reference standard i n p u r i f i e d absolute ethanol to give a f i n a l volume of 10 ml. This solution was stable for at least 1 week and generally was stored in the r e f r i g e r a t o r to retard evaporation. oi -Tocopherol Working Standard: For c a l i b r a t i o n of the standard curve, 0.2 ml of the fX-tocopherol stock solu-t i o n was d i l u t e d to 10 ml with p u r i f i e d absolute ethanol - 113 -giving a f i n a l concentration of. (10 1 mg) (0. 987) (0 2 ml) (1000 = 1 9 . 9 3 .g per ml ( 10 ml ) ( 1 0 ml) ( mg ) c The p u r i f i c a t i o n of organic solvents was important to eliminate reducing compounds which i n t e r f e r e with the analysis. A l l solvents and reagents were c a r e f u l l y protected from l i g h t and stored i n amber bottles i n the r e f r i g e r a t o r . A n a l y t i c a l Procedure Stage 1: To determine plasma vitamin E, the samples were treated i n the following way. Into a 10-ml glass test-tube, was pipetted 0.6 ml of plasma (or d i s t i l l e d water for xylene blank and standards), followed by 0.6 ml of p u r i f i e d absolute ethanol (or ethanol plus standard solution for standards) for protein p r e c i p i t a t i o n . The contents were immediately mixed with a Vortex mixer for 10 seconds. Then, 0.6 ml of xylene was added and the test-tube was again mixed, t h i s time for 30 seconds, and then centrifuged at 3000 rpm for 10 minutes. Stage 2: After centrifugation, 0.4 ml of the upper xylene layer which contains the extracted tocopherol was pipetted from the surface of the l i q u i d i n the tube to another 10 ml test-tube containing 0.2 ml of bathophenan-throline solution which w i l l l a t e r become part of the color complex. Also, t h i s w i l l prevent the reduction of - 114 -f e r r i c ions by carotene extracted from the blood by xylene at the same time as tocopherol. This second t e s t -tube was then mixed for 10 seconds using a Vortex mixer. At t h i s point, a 0.2 ml pipet containing 0.2 ml of ortho-phosphoric acid solution was prepared with the help of a rubber suction bulb. Then, 0.2 ml of f e r r i c chloride solution was added to the test-tube containing the tocopherol extract. The tocopherol present reduces the f e r r i c ions to ferrous ions which i n turn form a pink complex with 4, 7-diphenyl - 1, 10-phenanthroline. The timer was immediately set at time 0, and the test-tube simultaneously began to be mixed with a Vortex. At time 9 seconds, the tube was removed from the mixer and at time 2 0 seconds, 0.2 ml of orthophosphoric acid was added from the pre-prepared pipet. The use of orthophosphoric acid as a chelating agent reduces carotene interference to a minimum, preventing i t s oxidation. As well, at the same time, the orthophosphoric acid serves to s t a b i l i z e the color by tying up excess f e r r i c ions, preventing t h e i r photochemical reduction. At time 30 seconds, the tube was again mixed with a Vortex u n t i l time 4 0 seconds, when the solution was transferred to a 1 ml capacity quartz cuvette. The absorbance was read at time 3 minutes on the timer using either a G i l f o r d Spectrophotometer 240 or a Zeiss -PMQll Spectrophotometer set at 536 nm and using a blank of water. A xylene blank, using 0.6 ml water instead of plasma - 115 -for analysis and standards were also run, and t h e i r read-ings were used to calculate the vitamin E value. Throughout the procedure, the plasma and the tocopherol extract reaction mixture were protected from contact with l i g h t , and rubber surfaces which may contain reducing sub-stances. The f e r r i c chloride solution was also c a r e f u l l y protected from l i g h t which could cause photochemical degradation. Plasma samples and vitamin E stock solutions were read within 1 week of c o l l e c t i o n or preparation to avoid autoxidation. Also, a l l glassware used was scrupu-lously cleaned using sulfoehromic acid, and rinsed thoroughly with d i s t i l l e d water, g l a s s - d i s t i l l e d water, and dried. I f the tube had to be held at some stage during the procedure f o r more than 30 minutes, i t was corked with an aluminum foil-covered cork stopper to prevent evaporation. Rubber stoppers were not used since they might contain reducing substances extractable with organic solvents, and the use of a marking p e n c i l for labeling purposes was avoided since traces of crayon could a f f e c t the reaction. Standard Curve Standard curve and sample reading tubes containing the materials l i s t e d i n Table 111-16 were prepared according to the a n a l y t i c a l procedure. Resulting t y p i c a l absorbance readings are also shown i n Table III-16 and the standard curve i s graphed i n Figure III-9. Table III—16: Standard Curve and Sample Preparation; t y p i c a l Absorbance Readings Tube Name Tube Contents (ml) Correspond Absorbance Corrected F Factor Plaai na D i s t i l l e d Water {(-Tocopherol Working Standard Absolute Etnanoi Xylene Plasma oC-Tocopherol Concentration ( mg/lOOml) at 5 3 6 nm Absorbance (mg/lOOml) Xylene Blank 0 .6 — 0.6 0 .6 0 O .O93 ~ — Standard #1 0 .6 0 .1 0 .5 ' • 0 .6 0.332 O . 2 3 1 O . 1 3 8 2.41 Standard #2 0 .6 0 .2 0.4 0 .6 0 . 6 6 5 0.375 0.282 2.35 Standard #3 0 .6 o.3 0.3 • 0 . 6 . 0.997 . 0-523 0 . 4 3 0 2 . 3 2 Standard #4 0 .6 0 . 4 0 .2 0 .6 1.33 0.649 0.556 2.39 Standard #5 0 .6 0 . 6 0 0.6 1 . 9 9 0 . 9 0 5 0.812 2.45 Average 2.38 Sample f l 0 , .6 — 0.6 0.6 1 . 0 0 0 . 5 1 5 0.422 — - 117 -F i g u r e I I I - 9 : Standard Curve-Vitamin.E Determination 0.9 1 0 0.4 0.8 1.2 1.6 2.0 Plasma V i t a m i n E c o n c e n t r a t i o n (mg/100 ml) - 118 -The xylene blank enables one to c o r r e c t each time f o r background readings caused by impure s o l v e n t s . A l s o , v i t a m i n E l o s s e s or c o n c e n t r a t i o n e f f e c t s due t o s o l v e n t e v a p o r a t i o n were c o r r e c t e d f o r s i n c e the standards under-went the same procedure as the samples. The xylene blank and a t l e a s t 2 standards were run wit h each set of samples to make p o s s i b l e t h i s c o r r e c t i o n . C a l c u l a t i o n s For each standard, the corres p o n d i n g plasma o<-tocopherol c o n c e n t r a t i o n i n mg per 100 ml was c a l c u l a t e d as f o l l o w s : Standard tube o<-tocopherol , . content - _ stage 1 -i 2 X 1 mg i or, n i n c n i •• t nnn x 100 ml plasma. 0.6 ml plasma 1000 Mg * The stage 1 standard tube rX-tocopherol content i n jag was c a l c u l a t e d by m u l t i p l y i n g the oc-tocopherol working standard c o n c e n t r a t i o n , 19.93yUg per ml, by the volume of standard i n the tube. T h e r e f o r e , f o r standard #1, the corresponding plasma (X-tocopherol c o n c e n t r a t i o n i n mg per 100 ml was /19.93 Ug\ (0.1 ml) 1 mg 100 ml _ n V miy (0.6 ml) X 1000 /jg X ~ U - J J ^ Each standard and sample absorbance rea d i n g was c o r r e c t e d f o r background by s u b t r a c t i n g the xylene blank absorbance r e a d i n g . The f a c t o r which when m u l t i p l i e d by the absorbance r e a d i n g c o r r e c t e d f o r background w i l l g i v e mg o(-tocopherol per 100 ml plasma was designated the F f a c t o r . I t was - 119. c a l c u l a t e d from the standard curve data; (Table III-1.6) as f o l l o w s f o r standard #1 .as an example. F f a c t o r = 0.332 mg _ „ 4 1 (0.138) 100 ml - 2 ' 4 i ^ fclSL The OC-tocopherol or v i t a m i n E c o n c e n t r a t i o n o f a plasma sample i n mg per 100 ml was then c a l c u l a t e d by m u l t i p l y i n g i t s absorbance c o r r e c t e d f o r background by the average F f a c t o r f o r the standards of the same run. For sample #1 f o r example, the plasma C<-tocopherol con-c e n t r a t i o n would be c) Plasma C h o l e s t e r o l Plasma C h o l e s t e r o l was analyzed a c c o r d i n g to a m o d i f i e d A b e l l - K e n d a l l method ( A b e l l e t a l . 1952). - e t h y l a l c o h o l , 95%, a n a l y t i c a l grade, C a r l o Erba Do B r a s i l , S.A.). - g l a c i a l a c e t i c a c i d , a n a l y t i c a l grade, (E. Merck, Darmstadt, Germany). - s u l f u r i c a c i d , 9 5 - 97%, a n a l y t i c a l grade, (E. Merck, Darmstadt, Germany). - a c e t i c anhydride, a n a l y t i c a l grade, (E. Merck, Darmstadt, Germany). - petroleum ether, a n a l y t i c a l grade, b o i l i n g p o i n t 40-60 °C, (E. Merck, Darmstadt, Germany). (0.422) 1.00 mg 100 ml Reagents - 120 -- potassium hydroxide, a n a l y t i c a l grade, (E. Merck, Darmstadt, Germany). - ch o l e s t e r o l , dry c e r t i f i e d , (Fisher S c i e n t i f i c Company) bott l e stored i n a dessicator. Reagent Solutions Aqueous Potassium Hydroxide Solution: prepared by d i s s o l v i n g 33.0 mg potassium hydroxide i n d i s t i l l e d water to give a f i n a l volume of 100 ml of solution. Alcoholic Potassium Hydroxide Solution: prepared by d i l u t i n g 6 ml 33% aqueous potassium hydroxide described above, to 100 ml with ethyl alcohol. This solution was prepared fresh on the day of analysis. Cholesterol Standard Solution: prepared by d i s -solving 40.0 mg dry c e r t i f i e d cholesterol i n ethyl alcohol to give a f i n a l volume of 100 ml of solution. This solution was very stable, and was stored at room temperature i n a t i g h t l y sealed bottle to avoid evaporation. Liebermann-Burchard Reagent: prepared by placing 20 volumes of acetic anhydride i n an Erlenmeyer flask and securing the flask i n an ice bath. To t h i s , 1 volume of s u l f u r i c acid was slowly added while swirling the fla s k , and the swirling i n the i c e bath was continued for 10 minutes. The f l a s k was then removed from the - 121 -ice bath and 10 volumes of acetic acid were added again c a r e f u l l y swirling the f l a s k . When the re-agent reached room temperature i t was ready to use. An a l y t i c a l Procedure To determine plasma ch o l e s t e r o l , the samples were treated in the following way. Into a 50-ml glass-stoppered centrifuge tube was pipetted 0.50 ml plasma, followed by 5 ml of a l c o h o l i c KOH. Standards, recoveries and blank tubes were set up at t h i s point by using d i s t i l l e d water instead of plasma, and by introducing cholesterol v i a the alc o h o l i c KOH. The alcoholic KOH served to p r e c i p i t a t e the proteins of the plasma. In order to hydrolyze any e s t e r i f i e d c h o l e s t e r o l , a l l the tubes were then incubated i n a water bath uniformly at 45 °C for 55 minutes. They were then re-moved and allowed to cool for 5 minutes. The next step was to extract the cholesterol into petroleum ether. Ten ml petroleum ether were added to each tube a f t e r which each was immediately capped to prevent evaporation. Five ml of water were then added to each tube; the tube was recapped and shaken vigorously for 1 minute by hand. The tubes were then allowed to stand 10 minutes to complete separation of the petroleum ether and water phases. A 4.0-ml aliquot of the upper petroleum ether layer containing the cholesterol was then transferred by pipet to a clean dry 50-ml beaker, taking care to avoid the lower phase and the sides of the tube. A l l - 122 -samples were then evaporated uniformly to dryness i n an oven, at about 40 °C. The samples were then ready for color development, reaction with the Liebermann-Burchard reagent, which was prepared as the next step, according to the procedure described e a r l i e r . The 50-ml beakers containing samples were placed i n a tray containing water at 2 5 °C. Six ml of the Liebermann-Burchard reagent were added to the blank; then the timer was started and at regular i n t e r v a l s , every minute, 6 ml of reagent were added to each beaker, swirling the beaker c a r e f u l l y to wash the inner surface. Each sample absorbance was read against the blank at 620 nm exactly 3 0 minutes a f t e r adding Liebermann-Burchard reagent, using a Coleman Junior spectrophotometer. With each set of samples, a blank and some standards were run, ensuring uniform treatment for a l l and the r e s u l t i n g readings were used to calc u l a t e the cholesterol i n mg per 100 ml plasma. Standard Curve Standard curve and sample reading tubes containing the materials l i s t e d i n Table HT-17 .were prepared according to the a n a l y t i c a l procedure. Resulting t y p i c a l absorbance readings are also shown i n Table; IliE-17. The standard curve i s graphed i n Figure 111-10. Reading each sample absorbance against the blank corrects for any background reading due to impure solvents. Also, cholesterol losses or concentra-ti o n e f f e c t s due to non-planned solvent evaporation were Table 111-17 Standard Curve and Sample Preparat ion; T y p i c a l Absorbance Readings Plasma D i s t i l l e d A l c o h o l i c Aqueous E t h y l Cho les tero l .Petroleum D i s t i l l e d Llebermann Corresponding Absorbance J? Factor Water K O H K O H Alcoho l Standard Ether Water Burchard Plasma • Reading l«g/100ml) S o l u t i o n So lu t ion S o l u t i o n Reagent Choles tro l At 620nm Concentration (OK/IOOOU.) Blank 0.50 5-0 - - - 10.0 5.0 6.0 Standard #1 - O.50 - ' 0.30 - 4.7 10.0 5.0 6.0 376.0 O.876 429.2 Standard § 2 - 0.50 - O.30 0.7 4.0 10.0 5.0 6.0 320.0 0.7^ 5 429.5 Standard #3 - 0.50 - 0.30 1.? 3.0 10.0 5.0 6.0 240.0 0.557 V30.9 Standard #4 - 0.50 - O.30 2.7 2.0 lo .o 5.0 6.0 160.0 O.378 ^23.3 Standard #5 - 0.50 - 0.30 3.7 1.0 10.0 5.0 6.0 80.0 0.186 416:1 Average = 428 .6 Sample #1 0.50 - 5.0 - - - 10.0 5.0 6.0 201.0 0 . 469 CO - 124 -- 125 -c o r r e c t e d f o r s i n c e the standards underwent the same procedure as the samples. The blank and standard were run w i t h each set of samples to make p o s s i b l e t h i s c o r r e c t i o n . C a l c u l a t i o n s . For each standard, the corresponding plasma c h o l e s t e r o l c o n c e n t r a t i o n i n mg per 100 ml was c a l c u l a t e d as f o l l o w s : standard tube c h o l e s t e r o l content _—=—^m<^ , — x 100 ml plasma 0.5 ml plasma ^ The standard tube c h o l e s t e r o l content i n mg was c a l c u l a t e d by m u l t i p l y i n g the c h o l e s t e r o l standard s o l u t i o n c o n c e n t r a -t i o n , 40 mg per 100 ml, by the number of ml i n the standard tube. For example, f o r standard #1, 4.7 ml were used g i v i n g 4.7 x 40 -r 100 or 1.88 mg c h o l e s t e r o l . T h e r e f o r e , f o r standard #1, the corresponding c h o l e s t e r o l c o n c e n t r a -t i o n was 1.88 mg 100 ml _ 376.0 mg_ 0.5 ml 100 ml The f a c t o r which when m u l t i p l i e d by the absorbance r e a d i n g w i l l g i v e mg c h o l e s t e r o l per 100 ml plasma was des i g n a t e d the F f a c t o r . I t was c a l c u l a t e d from the standard curve data '(Table .IK-1-7) as f o l l o w s f o r standard #1 as an example. F f a c t o r = 376.0 mg = 429.2 mg (0.876) 100 ml 100 ml - 126 -The cholesterol concentration of a plasma sample in mg per 10 0 ml was then calculated by multiplying i t s absorbance by the average F factor for the standards of the same run. For sample #1 for example, the plasma cholesterol concentration would be ( 0 . 4 6 9 ) Z428.6 mg_ \. = 2 0 1 . 0 mg V 100 ml/ 100 ml d) Plasma Total L i p i d s Plasma t o t a l l i p i d s were analyzed according to a modified version of the Amenta method (Amenta 1 9 7 0 ) . Reagents - chloroform, a n a l y t i c a l grade fr e s h l y d i s t i l l e d to ensure purity. - methanol, a n a l y t i c a l grade. - sodium chloride, a n a l y t i c a l grade. - calcium chloride, a n a l y t i c a l grade (J. T. Baker Chemicals, S.P.). - potassium dichromate, a n a l y t i c a l grade (Coleman and B e l l Company, Norwood, 0., U.S.A.). - s u l f u r i c acid, H 2S0 4, concentrated, (E. Merck, Darmstadt, Germany). - l e c i t h i n , L-oc-phosphatidyl choline from egg yolk, 100 mg per ml i n chloroform-methanol ( 9 : 1 ) , (Sigma Chemical Company), con-tainers stored i n dark i n freezer, used when fre s h l y opened. - 127 -- p a l m i t i c a c i d , 99% p u r i t y , a n a l y t i c a l grade, (Sigma Chemical Company), s t o r e d i n d e s s i c a t o r . Reagent S o l u t i o n s Chloroform-methanol s o l u t i o n : prepared by mixing 50 ml of d i s t i l l e d c h l o r o f o r m w i t h 60 ml of methanol. T h i s s o l u t i o n was s t o r e d i n a g l a s s - s t o p p e r e d b o t t l e t o prevent e v a p o r a t i o n . Sodium c h l o r i d e s o l u t i o n : prepared by d i s s o l v i n g 90 mg sodium c h l o r i d e i n 10 ml d i s t i l l e d water. Calcium c h l o r i d e s o l u t i o n : prepared by d i s s o l v i n g 2.5 g c a l c i u m c h l o r i d e i n 500 ml d i s t i l l e d water. Stock a c i d dichrornate reagent: prepared by d i s s o l v i n g 1.25 g potassium dichromate i n 12.5 ml d i s t i l l e d water, w i t h the a p p l i c a t i o n o f heat. T h i s s o l u t i o n was then d i l u t e d to 250 ml a t 20 °C with c o n c e n t r a t e d s u l f u r i c a c i d . L i p i d standard: prepared u s i n g l e c i t h i n and p a l m i t i c a c i d i n a 2:3 r a t i o by weight, d i s s o l v e d i n c h l o r o f o r m . T h i s r a t i o approximates the p h o s p h o l i p i d p o r t i o n normally found i n plasma and p r o v i d e s a reasonably a c c u r a t e standard f o r the t o t a l l i p i d s , both w i t h i n and s l i g h t l y above the normal range. To prepare work-ing standard #1, 24.25 mg p a l m i t i c a c i d (99% p u r i t y ) or 24.0 mg p l u s 0.16 ml l e c i t h i n (100 mg per ml) or 16 mg were d i s s o l v e d i n f r e s h l y d i s t i l l e d c h l o r o f o r m - 128 -to g i v e a f i n a l volume of 10 ml, and c o n c e n t r a t i o n o f 4.0 mg per ml. Stored i n t i g h t - f i t t i n g ground g l a s s - s t o p p e r e d c o n t a i n e r s i n the r e f r i g e r a t o r t o r e t a r d e v a p o r a t i o n , the working standards were prepared f r e s h weekly t o a v o i d c o n c e n t r a t i o n e f f e c t s due to e v a p o r a t i o n . A n a l y t i c a l Procedure To determine plasma t o t a l l i p i d s , the samples were t r e a t e d i n the f o l l o w i n g way. To a 10-ml g l a s s - s t o p p e r e d c e n t r i f u g e tube, 2.2 ml of the chloroform-methanol s o l u -t i o n were added. For the re c o v e r y t e s t s , the l i p i d was i n c o r p o r a t e d i n t o the c h l o r o f o r m f r a c t i o n here. Then, 0.3 0 ml of plasma (or sodium c h l o r i d e s o l u t i o n f o r the standards, r e c o v e r i e s , and the method blank) was. added dropwise, shaking g e n t l y t o produce a f i n e l y clumped p r e c i p i t a t e . The methanol served to p r e c i p i t a t e the p r o t e i n s of the plasma, and the l i p i d was then e x t r a c t e d from the plasma i n t o the chloroform-methanol s o l v e n t d u r i n g a f i v e minute p e r i o d of shaking the stoppered t e s t -tubes v i g o r o u s l y . A covered t e s t - t u b e rack was developed to shake numerous tubes a t one time, thus saving time and energy, and ensuring t h a t a l l of the tubes, samples, r e c o v e r i e s , standards, and blanks, were shaken u n i f o r m l y . To ensure complete e x t r a c t i o n , the tubes were l e f t t o stand f o r a t l e a s t 1 hour, mixing twice d u r i n g t h i s i n t e r v a l . 1,29 -Then, 6.0 ml of c a l c i u m c h l o r i d e s o l u t i o n were added to each tube, and the tubes were a g a i n shaken f o r 5 minutes, com-p l e t e l y mixing the two l i q u i d phases. T h i s aqueous c a l c i u m c h l o r i d e s o l u t i o n wash co n c e n t r a t e d the l i p i d s i n t o the c h l o r o f o r m phase which became d i s t i n c t from the methanol, water, c a l c i u m c h l o r i d e , n o n - l i p i d i m p u r i t y phase. The tubes without stoppers were then c e n t r i f u g e d a t about 4000 rpm f o r 15 minutes to completely separate the s o l i d and the two l i q u i d phases d e s c r i b e d above. The p r o t e i n p r e c i p i t a t e formed a t h i n l a y e r a t the i n t e r f a c e of the two l i q u i d phases. Most of the supernatant methanol, water, c a l c i u m c h l o r i d e , n o n - l i p i d i m p u r i t y phase was s u c t i o n e d o f f u s i n g a s p e c i a l l y d e v i s e d vacuum pump apparatus, l e a v i n g the lower c h l o r o f o r m phase topped w i t h a t h i n p r o t e i n p l u g . Then, 0.10 ml of the lower c h l o r o f o r m l i p i d e x t r a c t phase was t r a n s f e r r e d by p i p e t i n t o a c l e a n 15-ml t e s t tube pushing the p r o t e i n plug a s i d e t o a l l o w easy entrance of the p i p e t . The separate phases were not d i s r u p t e d by t h i s procedure. Care was taken to prevent e v a p o r a t i o n of the c h l o r o f o r m p r i o r to i t s t r a n s f e r . Standards were prepared a t t h i s step by p l a c i n g 0.1 ml of the t o t a l l i p i d standard s o l u t i o n i n a s i m i l a r t e s t tube. Then, the c h l o r o f o r m was evaporated completely under a stream of n i t r o g e n f o r two minutes. A l l the c h l o r o f o r m must be evaporated f o r any r e s i d u a l c h l o r o f o r m w i l l have a p o s i t i v e e f f e c t on the f i n a l r e s u l t . Immediately, 2.0 ml - 130 r-of stock a c i d diehrornate reagent were added and the tube mixed w i t h a Vortex mixer f o r 1 minute. A reagent blank was s t a r t e d a t t h i s p o i n t , adding the stock a c i d dichromate reagent to a c l e a n empty 15-ml tube. A l l the tubes, samples, r e c o v e r i e s , standards and blanks were then heated i n an oven a t about 110 °C, u n i f o r m l y f o r about 12 minutes, a t which time each tube was a g a i n mixed with a Vortex mixer, t h i s time f o r 3 0 seconds. T h i s h e a t i n g and mixing was then repeated, and f i n a l l y the tubes were heated s i m i l a r l y one more time. Thus, the r e a c t i o n of the l i p i d p r e s e n t w i t h the stock a c i d dichromate reagent i s enhanced, the l i p i d r e d u c i n g the dichromate, causing i t to change c o l o r from orange-red to o l i v e green. The dichromate s o l u -t i o n was then poured i n t o a 20 ml t e s t - t u b e c o n t a i n i n g 10 ml d i s t i l l e d water and the r e s u l t i n g mixture poured back and f o r t h between the tubes u n t i l i t was w e l l mixed, about 2 times. The absorbance a t 430 nm of the r e s u l t i n g s o l u t i o n was then read a g a i n s t a water blank u s i n g e i t h e r a G i l f o r d Spectrophotometer 24 0 or a Z e i s s - PMOll Spectrophotometer. With each s e t of samples, a method blank, reagent blank, and some standards were run, ensuring uniform treatment f o r a l l and the r e s u l t i n g r e a d i n g s were used to c a l c u l a t e the t o t a l l i p i d s i n mg per 100 ml plasma. A l l glassware used i n t h i s procedure was acid-washed w i t h s u l f o c h r o m i c a c i d , r i n s e d thoroughly w i t h d i s t i l l e d * 131, -water, and dried. The tubes used during the acid reaction were f i l l e d with sulfochromic acid and heated at 110 °C for at least 30 minutes as well to ensure removal of i n t e r -fering substances. Soap film, for example, reacts with the acid dichromate reagent and f a l s e l y elevated values r e s u l t . Standard Curve Total l i p i d s working standards were prepared according to the plan of Table I I I - 1 8 . Table. ' .111-18 •: Total L i p i d s for D i l u t i o n Working Standards; with Chloroform Procedure Working Standard Diluted to 10 ml Total Lipids Concentration Standard # Working Standard Used Volume Used (ml) (mg/ml) Plasma; /Corresponding] •Wg/100 ml / 1 #1 a__ 4.00 1333 2 #1 7.5 3.00 1000 3 #2 7.5 2 .25 750.0 4 #3 7.0 1.58 525.0 5 #4 6.0 0.945 315. 0 a See Reagent Solutions For preparation of the standard curve, and recovery tes t s , tubes containing the materials l i s t e d i n Table 111-19 were prepared according to the a n a l y t i c a l procedure up to the addition of acid dichromate. Typical absorbance read-ings produced at the completion of the a n a l y t i c a l procedure are also shown in 'able 111-19, and the standard curve i s graphed i n Figure I'll-11. T a b l e II1-19: S t a n d a r d C u r v e , R e c o v e r y T e s t s a n d Sample P r e p a r a t i o n ; T y p i c a l A b s o r o a n c e R e a d i n g s Tube Name Tube C o n t e n t s (ml ) C h l o r o f o r m -M e t h a n o l S o l u t i o n Method B l a n k Reagent B l a n k S t a n d a r d #1 9Z #3 #4 #5 Sample # l a Sample # l b e R e c o v e r y #1 f R e c o v e r y #2 2 . 2 2 . 2 2 . 2 W o r k i n g .. . . M e t h a n o l p l a s m a S t a n d a r d 1 . 0 1 . 0 N a C i Solution 0.30 1 .2 0 . 3 0 0 . 3 0 0 . 3 0 0.30 C a C l 2 S o l u t i o n 6.0 6.0 6 . 0 6 . 0 6 . 0 C o r r e s p o n d i n g • W o r k i n g S t a n d a r d 0 . 1 0 . 1 0 . 1 0 . 1 0 . 1 A b s o r b a n c e a t 430nm U n a d j u s t e d 6 A d j u s t e d 0 . 8 8 6 ' O . 8 9 3 0 . 3 9 7 0 . 5 1 3 0 .624 0 . 7 0 6 0 -777 0 . 6 8 3 0 . 6 ? 2 0 . 5 7 7 0 - 7 6 5 0 . 4 9 6 0 . 3 8 0 0 . 2 6 9 0 . 1 8 7 0 . 1 1 6 C a l c u l a t e d R e s u l t F f a c t o r ( m g / l O O m l ) 2 6 8 7 . 5 2 6 3 1 . 6 2 7 8 8 . 1 2 8 0 7 . 5 2715 .5 ro a v e r a g e = 2 7 2 6 . 0 P l a s m a T o t a l Lipid (mg / lOOml)  0 . 2 0 3 553. '+ 0 . 214 5 8 3 . 4 0 . 3 0 9 8 4 2 . 3 0 - J 2 1 ^29.6 a ( 0 . 9 4 5 m g / m l ) . b See T a b l e . c S t o r e d 6 weeks I n r e f r i g e r a t o r , d S t o r e d 6 weeks l n f r e e z e r , e U s i n g Sample # l a . f U s l n y mechod b l a n k . g To a d j u s t , s u b t r a c t the a b s o r b a n c e o f t h e unknown f rom t h e a b s o r b a n c e o f the method b l a n k a n d t h e a b s o r b a a c e of the s t a n d a r d f r o m t h e a b s o r b a n c e o f t h e r e a g e n t b l a n k . Corresponding Plasma Total L i p i d Concentration (mg/100ml) - 134 -The method blank should read no more than 0.03 absorbance units less than the reagent blank and thus serves as a check to ensure that there i s no contamina-t i o n of materials and that the glassware i s scrupulously clean. Calculations For each working standard, the corresponding plasma t o t a l l i p i d s concentration i n mg per 100 ml was calculated as follows: standard tube t o t a l l i p i d s chloroform content (mg) , n n , , 1.0 ml phase -x—=, =j r — 2 J — x 100 ml plasma x •_—-= , . ., ^ 0.3 ml plasma ^ 0.1 ml chloroform phase where: standard tube = working standard x 0.1 ml , t o t a l l i p i d s t o t a l l i p i d content (mg) concentration (mg/ml) and 1.0 ml chloroform phase . , . . . c . Ti-^i ; =-r= 2 ^ r - = extraction correction factor. 0.1 ml chloroform phase The extraction correction factor was necessary since of the 1.0 ml chloroform containing the extracted t o t a l l i p i d s only 0.1 ml was transferred to the next stage i n the analysis procedure. Therefore, for working standard #1, the correspond-ing plasma t o t a l l i p i d s concentration i n mg per 100 ml was /4.00 mg\ (0.1 ml) i r i „ , -.-.oo \ — T - ) / T T - T T\ x 100 ml x 10 = 1333. V ml/ (0.3 ml) Since the standards did not undergo the same procedure as the samples, recovery tests were run to ensure that t o t a l l i p i d s were not l o s t or concentrated due to solvent evapora-t i o n s i g n i f i c a n t l y during the procedure. For both recovery - 135 -t e s t s , 0.945 mg t o t a l l i p i d s were i n c o r p o r a t e d i n t o the c h l o r o f o r m f r a c t i o n of the chloroform-methanol s o l u t i o n , which i s the e q u i v a l e n t of 315.0 mg per 100 ml plasma. 0.945 mg _ 315.0 mg  0.3 ml plasma 10 0 ml plasma T h e r e f o r e , the r e c o v e r y r e s u l t s were as f o l l o w s : mg mg Recovery #1 (842.3 100 ml - 553.4 100 ml) 100% = 91.7% 315.0 ^ mg 100 ml - 0 315.0 100 ml mg 100 ml) 100% = 104.7% mg 100 ml The average r e c o v e r y was 91.7% + 104.7% = 98.2%. 2 T h i s allowed the c o n c l u s i o n t h a t t o t a l l i p i d s were not l o s t or c o n c e n t r a t e d due to s o l v e n t e v a p o r a t i o n s i g n i f i -c a n t l y d u r i n g the procedure. Since the orange-red c o l o r being measured decreases as the dichromate i s reduced by the t o t a l l i p i d p resent, i t i s the decrease i n absorbance t h a t i s d i r e c t l y p r o p o r t i o n -a l to the l i p i d p r e s e n t . T h e r e f o r e , the absorbance i s sub-t r a c t e d from the proper blank r e a d i n g , the method blank being used f o r the unknown samples and the reagent blank being used f o r the standards, s i n c e then the treatment of the blank matches the treatment of the samples. T h i s s u b t r a c t i o n r e s u l t or decrease i n absorbance i s then used i n subsequent c a l c u l a t i o n s . - 136 -The f a c t o r which when m u l t i p l i e d by the absorbance r e a d i n g s u b t r a c t e d from the proper blank r e a d i n g w i l l g i v e mg t o t a l l i p i d s per 100 ml plasma was designated the F f a c t o r . I t was c a l c u l a t e d from the standard curve data (Table iLII-19) as f o l l o w s f o r working standard #1 as an example. F f a c t o r = 1333 _ 1333 = „ f i R 7 ,-(0.893-0.397) 0.496 zov/.s The t o t a l l i p i d s c o n c e n t r a t i o n of a plasma sample i n mg per 100 ml was then c a l c u l a t e d by m u l t i p l y i n g i t s absorbance s u b t r a c t e d from the method blank by the average F f a c t o r f o r the standards of the same run. For sample #la f o r example, the plasma t o t a l l i p i d s c o n c e n t r a t i o n would be (0.886 - 0.683) (2726.0 mg ) ( 100 ml) (0.203) (2726.0 mg_ ) = 553.4 mg ( 100 ml) 100 ml The normal range f o r plasma t o t a l l i p i d s c o n c e n t r a t i o n as determined by t h i s method i s 400-800 mg per 100 ml. As expected, the range f o r t o t a l l i p i d s obtained by t h i s method i s s l i g h t l y higher than t h a t r e p o r t e d f o r some methods t h a t i n c l u d e o n l y the c h o l e s t e r o l and the t o t a l e s t e r i f i e d f a t t y a c i d s , s i n c e t h i s method measures p h o s p h o l i p i d , c h o l e s t e r o l , t r i g l y c e r i d e , and c h o l e s t e r o l e s t e r s . Since the amount of dichromate reduced by d i f f e r e n t l i p i d s v a r i e s , plasma samples from p a t i e n t s w i t h s t r i k i n g hyper-l i p i d e m i a should a l s o be analyzed f o r the v a r i o u s l i p i d - 137 -f r a c t i o n s i n case the t o t a l l i p i d standard no l o n g e r a c c u r a t e l y approximates the composition of the t o t a l l i p i d i n terms of i t s reducing a b i l i t y . e) Plasma T r i g l y c e r i d e s Plasma t r i g l y c e r i d e s were analyzed by a m o d i f i e d v e r s i o n of the C a r l s o n method (Carls o n 1963). Reagents - s u l f u r i c a c i d , a n a l y t i c a l grade, c o n c e n t r a t e d and 1 N s t r e n g t h s , (E. Merck, Darmstadt, Germany). - c h l o r o f o r m , a n a l y t i c a l grade. - s i l i c i c a c i d , a n a l y t i c a l grade, (Quimitra, S.A_.). - potassium hydroxide, a n a l y t i c a l grade, (E. Merck, Darmstadt, Germany). - e t h y l a l c o h o l , 95%, a n a l y t i c a l grade, (Carlo Erba Do B r a s i l , S. A . ) , p u r i f i e d by d i s t i l l a t i o n . - sodium meta-periodate, a n a l y t i c a l grade, (E. Merck, Darmstadt, Germany). - sodium m e t a - a r s e n i t e , a n a l y t i c a l grade, (E. Merck, Darmstadt, Germany). - 4,5 - dihydroxynapthalene - 2, 7 - d i s u l p h o n i c a c i d disodium s a l t (chromotropic a c i d disodium s a l t ) , d i h y d r a t e , a n a l y t i c a l grade, (E. Merck, Darmstadt, Germany). - g l y c e r o l s o l u t i o n , 88.25%, a n a l y t i c a l grade, (Backer). - 138 -Reagent S o l u t i o n s 0.4% E t h a n o l i c Potassium Hydroxide S o l u t i o n : prepared by mixing 1.0 ml of a 2% e t h a n o l i c potassium hydroxide s o l u t i o n w i t h 4.0 ml e t h y l a l c o h o l . 0.2 N S u l f u r i c A c i d S o l u t i o n : prepared by mixing 1.0 ml 1 N s u l f u r i c a c i d with 4.0 ml d i s t i l l e d water. 0.25 M. Sodium P e r i o d a t e S o l u t i o n : prepared by d i s -s o l v i n g 535.0 mg sodium p e r i o d a t e i n d i s t i l l e d water t o g i v e a f i n a l volume of 100 ml. 0.50 M. Sodium A r s e n i t e S o l u t i o n : prepared by d i s -s o l v i n g 2.25 g sodium hydroxide and 5.00 g of a r s e n i o u s a c i d i n d i s t i l l e d water to g i v e a f i n a l volume of 100 ml. Chromotropic a c i d S o l u t i o n : prepared by the f o l l o w i n g procedure,' 0.5 g o f chromotropic a c i d (4,5 - dihydroxy-naphthalene-2, 7 - d i s u l p h o n i c a c i d disodium s a l t ) was d i s -s o l v e d i n 50 ml of d i s t i l l e d water. To t h i s was then added a 2:1 mixture of co n c e n t r a t e d s u l f u r i c a c i d and d i s t i l l e d water t o g i v e a f i n a l volume of 250 ml of s o l u t i o n . The v o l u m e t r i c f l a s k was p l a c e d i n an i c e bath f o r t h i s a d d i t i o n . T h i s reagent was s t o r e d i n a dark b o t t l e to p r o t e c t i t from l i g h t . Stock Standard S o l u t i o n : prepared by d i s s o l v i n g 1.3056 g of g l y c e r o l s o l u t i o n (88.25%) i n d i s t i l l e d water t o g i v e a f i n a l volume of 500 ml and c o n c e n t r a t i o n of 0.025 M. S i l i c i c a c i d : p l a c e d i n a p e t r i d i s h i n an oven a t 115 °C f o r one hour, and then allowed t o c o o l i n a d e s s i c a t o r - 139 -p r i o r t o weighing and subsequent use. A n a l y t i c a l Procedure To determine plasma t r i g l y c e r i d e s , the samples were t r e a t e d i n the f o l l o w i n g way. Into a 1 5 r - m l g l a s s - s t o p p e r e d c e n t r i f u g e tube, 0 . 2 g s i l i c i c a c i d was weighed, 1 . 0 ml of c h l o r o f o r m was added, and the tube was then shaken. T h i s was f o l l o w e d by the a d d i t i o n of 0 . 1 ml plasma and 1 . 0 ml of c h l oroform, and then 2 minutes of shaking. T h i s mixture was l e f t t o r e s t f o r 1 5 minutes, and then c e n t r i f u g e d f o r 5 minutes a t 2 0 0 0 rpm. P h o s p h o l i p i d s were absorbed to the s i l i c a g e l d u r i n g t h i s p r o c e s s , and were then removed from the l i p i d e x t r a c t by p a s s i n g the mixture through f i l t e r paper. An a l i q u o t of 1 . 0 ml of the f i l t r a t e was then p l a c e d i n a 1 5 - m l g l a s s - s t o p p e r e d t e s t - t u b e , and the c h l o r o -form was evaporated i n a water bath a t 6 0 - 8 0 °C. The tubes were not allowed to remain l o n g e r than necessary i n the water bath. To the r e s i d u e , 0 . 5 ml of 0 . 4 % e t h a n o l i c potassium hydroxide s o l u t i o n was added, the tubes were stoppered, and the t r i g l y c e r i d e s were hydrolyzed f o r 2 0 minutes a t 6 0 - 6 5 C i n a water bath. The tubes were then removed from the water bath, and 0 . 5 ml of 0 . 2 N. s u l f u r i c a c i d was added. Returning the tubes to the water bath, without stoppers, they were heated to 9 7 °C f o r about 3 0 minutes or u n t i l the odor of a l c o h o l disappeared. The - 140 -r e s u l t was the p r o d u c t i o n of f r e e g l y c e r o l . At t h i s stage of the procedure, a blank was prepared by p i p e t t i n g 0.5 ml d i s t i l l e d water i n t o a s i m i l a r g l a s s - s t o p p e r e d tube, and as w e l l , the standards were prepared by p i p e t t i n g 0.5 ml of the standard s o l u t i o n s i n t o s i m i l a r tubes. The standard s o l u t i o n s commonly used were: #1) a one hundredth d i l u t i o n of the stock standard s o l u t i o n #2) a one h a l f d i l u t i o n of the standard #1 s o l u t i o n #3) a one f o u r t h d i l u t i o n of the standard #1 s o l u t i o n . These correspond t o : #1) 0.125 /unoles g l y c e r o l , #2) 0. 0625 /tmoles g l y c e r o l , and #3) 0. 03125 /jmoles g l y c e r o l per tube. To a l l of the tubes, i n c l u d i n g blanks and standards, 0.05 ml of the 0.25 M s o l u t i o n o f sodium p e r i o d a t e was added. Then, a f t e r w a i t i n g 10 minutes, 0.05 ml of the 0.50 M. s o l u t i o n of sodium a r s e n i t e was added, and the tubes were h e l d a t room temperature f o r 10 minutes. At t h i s p o i n t , 5.0 ml of the chromotropic a c i d s o l u t i o n was added and the tubes were shaken, p r e f e r a b l y i n a room without d i r e c t d a y l i g h t . The tubes were then heated i n a b o i l i n g water bath f o r 30 minutes and f i n a l l y , allowed t o c o o l to room temperature. Each absorbance was read a g a i n s t the blank a t 570 nm, u s i n g a Coleman J u n i o r Spectrophotometer. The r e s u l t i n g r e a d i n g s were used t o c a l c u l a t e the t r i g l y c e r i d e s i n mg per 100 ml plasma. - 141 -C a l c u l a t i o n s mg of t r i g l y c e r i d e s per 100 ml plasma = 0.21875 /imol x sample absorbance x 2.0 x 1. 0 (R'.S.) 1.0 0.1 x 806 yUg x 1 mg x 100 ml yUmol 1000 jmq where: - 0.21875yQmol = the sum of the q u a n t i t i e s of the 3 standards = 0.125y0;mol + 0.0625y(jmol + 0. 03125 /.(mol ; - (R.S.) = the sum of the absorbances of the 3 standards; - 2.0 1.0 = c o r r e c t i o n f a c t o r f o r e x t r a c t i o n procedure, wherein 2.0 ml c h l o r o f o r m was used to e x t r a c t the l i p i d s and o n l y 1.0 ml was c a r r i e d through d u r i n g the r e s t of the a n a l y s i s ; - 10 = f a c t o r r e q u i r e d s i n c e o n l y 0.1 ml of plasma 0.1 was analyzed; ~ 806 /i g = m o l e c u l a r weight of t r i p a l m i t i n ; yUmol ~ 1 m g = c o n v e r s i o n f a c t o r , changing jQg to mg; 1000 yu,g - 100 ml = f a c t o r r e q u i r e d i n order to express t r i g l y c e r i d e s i n terms of 100 ml plasma. f) Hemoglobin Blood hemoglobin was analyzed a c c o r d i n g to the Cyanmethemoglobin Method (ICNND 1963). A 0.02 ml a l i q u o t of well-mixed blo o d was added to 5.0 ml of potassium f e r -r i c y a n i d e s o l u t i o n u s i n g Adams d i s p o s a b l e S a h l i A/1 hemoglobin - 142 -p i p e t . The p i p e t was r i n s e d s e v e r a l times with the s o l u t i o n i n the same t e s t tube. A f t e r proper mixing, tubes were allowed to stand f o r 10 minutes and the chromophore which developed was read i n the Turner Spectrophotometer a t 54 0 nm u s i n g the mixed f e r r i -c yanide s o l u t i o n as the blank. Cyanmethemoglobin was used to prepare the standard curve, shown i n F i g u r e III-12 and the f i n a l v a l u e s were expressed as g of hemoglobin per 100 ml of blood. g) Hematocrit Hematocrit was determined by the method d e s c r i b e d i n the Manual f o r N u t r i t i o n Surveys (ICNND 1963). A small amount of f r e s h b l o o d was i n t r o d u c e d i n t o a M i c r o -Hematocrit c a p i l l a r y tube ( F i s h e r Brand), care being taken to a v o i d a i r bubbles. The tube was s e a l e d a t one end with p l a s t i c c l a y ( C r i t a s e a l ) and c e n t r i f u g e d i n a M i c r o -C a p i l l a r y C e n t r i f u g e (IEC Model MB) a t 3,000 rpm f o r f i v e minutes or u n t i l complete packing of the c e l l s was obtained. Red c e l l volume i n percent was read d i r e c t l y from the M i c r o - C a p i l l a r y Reader. F.V Computer A n a l y s i s A l l computer a n a l y s i s was c a r r i e d out by Lewis James of the U n i v e r s i t y of B r i t i s h Columbia S t a t i s t i c a l C enter. - 143 -F i g u r e M-1.2: Standard Curve - Hemoglobin Determination 5 10 15 20 25 Hemoglobin (g/100 ml) - 144 -CHAPTER IV RESULTS Chapter Outline Section Page A. Ecological Factors 145 1-. Description' of V i l a Recreio Community . . 145 a) Living Conditions 145 b) Transportation . 147 c) Industry and Commerce 147 d) Economic Status 147 e) Food Costs : 148 f) Literacy 150 g) Local Corrective Measures 150 B. Dietary Analysis 156 1. Qualitative 156 2. Quantitative 163 C. Anthropometric Assessment 170 D Biochemical Examination 173 - 145 -A. E c o l o g i c a l 1 F a c t o r s 1. D e s c r i p t i o n of V i l a R ecreio Community a) L i v i n g C o n d i t i o n s V i l a R e c r e i o may be c o n s i d e r e d a f a v e l a or shanty town l o c a t e d on the p e r i p h e r y of the c i t y of R i b e i r a o 2 P r e t o . I t i n c l u d e s a l a n d area o f about 0.75 km , g e n e r a l l y f l a t w i t h a s l i g h t s l o p e . The r e d d i s h c o l o r s o i l appeared f e r t i l e , though l i t t l e v e g e t a t i o n was grown. There were no lawns or f l o w e r s and v e r y few t r e e s to p r o v i d e shade from the hot sun. The shanty houses of the area were g e n e r a l l y s m a l l and d i r t y with a c l a y f l o o r , c o n s t r u c t e d of e i t h e r c o n c r e t e b l o c k s or o l d wooden planks, unpainted, and w i t h s l o p i n g r o o f s o f t e n made of t i l e or t i n . The houses had rooms too small and too few f o r the s i z e of t h e i r f a m i l i e s , inadequate l i g h t i n g and v e n t i l a t i o n , and no sewage system. A c c o r d i n g to Desai e t al_. (1978a) , t y p i c a l c o n d i t i o n s of Boia'-Fria h a b i t a t i o n as observed i n V i l a R e c r e i o were as presented i n Table IV-1. The houses were b u i l t c l u s t e r e d c l o s e together along unpaved roads, which were noted f o r t h e i r r u t s and r o c k s . S a n i t a t i o n was a s e r i o u s problem i n V i l a R e c r e i o . With no sewage system, open g u t t e r s a t the s i d e s of the roads c a r r i e d d i r t y wash water away from the houses. Dotted throughout the area were vacant l o t s , wide open spaces w i t h paths worn from f o o t t r a v e l and w i t h accumulations - 146 -T a b l e IV-1: T y p i c a l C o n d i t i o n s of B o i a - F r i a H a b i t a t i o n Item P o s s e s s i o n No, Housing: House: owned re n t e d No. of rooms: E l e c t r i c i t y : N a t u r a l l i g h t i n g and v e n t i l a t i o n : Running water: 77 23 69 61 61 2.5 S a n i t a t i o n : T o i l e t i n house: Out-house p i t : Sewage or g u t t e r system' 7 93 0 none Cooking F a c i l i t y : Gas: Firewood: 75 25 Domestic Equipment: Radio: T e l e v i s i o n : P r e s s u r e cooker: E l e c t r i c blender: R e f r i g e r a t o r : E l e c t r i c Fan: 64 44 76 20 18 52 a f r o m Desai et a l . (1978a) - 147 * of garbage. L i t t e r a l s o c l u t t e r e d the s t r e e t s and the e n t i r e area sraelled of r o t t i n g garbage. In a dry p e r i o d , the area was extremely dusty and i n a wet p e r i o d , extremely muddy. Accor d i n g to Desai e t al_. (1978a) , p a r a s i t i c i n f e c t i o n was v e r y common among t h i s p o p u l a t i o n . S t o o l examination of 136 c h i l d r e n i n V i l a R e c r e i o r e v e a l e d t h a t about 60% of the c h i l d r e n had p a r a s i t e s , the most predominant types being; A sc ar1s lumbr I C Qides, T r i c h o c e p h a l u s  t r i c h i u r u s , Ancylostoma, Hymenolepis nana, G i a r d i a Iambiia and S t r o n g y l o i d e s s t e r c o r a l i s . D i a r r h e a was one of the most common i n f e c t i o u s d i s e a s e s among c h i l d r e n . b) T r a n s p o r t a t i o n Means of t r a n s p o r t a t i o n i n V i l a R e c r e i o were r a t h e r l i m i t e d . Women o f t e n walked to t h e i r d e s t i n a t i o n and men were o f t e n seen on b i c y c l e s . Horse and buggy, or motor b i k e were l e s s common. Seldom was there seen a c a r parked i n f r o n t of a house. On the other hand, the c i t y bus s e r v i c e d i d p r o v i d e p u b l i c t r a n s p o r t a t i o n i n the a r e a . However, i t s c o s t may have p r o h i b i t e d i t s e x t e n s i v e use by many l o c a l r e s i d e n t s . c) I n d u s t r y and Commerce There were no major i n d u s t r i e s or i n d u s t r i a l or commercial employment o p p o r t u n i t i e s i n V i l a R e c r e i o . The c e n t e r of R i b e i r a o P r e t o , approximately 10 km away, was the heart of i n d u s t r y and commerce f o r the c i t y . d) Economic Status - 148 -The minimum monthly s a l a r y f o r a worker a t the time of t h i s study i n B r a z i l was 1560 c r u z e i r o s (about $80 U.S.). T h i s was the e q u i v a l e n t of 6.50 c r u z e i r o s per hour (about 33 c e n t s U.S.). A c c o r d i n g to Desai e t a l . (1978a), the male B o i a - F r i a s u s u a l l y worked as r u r a l farm l a b o r e r s c u t t i n g sugar cane or p i c k i n g c o f f e e and c o t t o n d u r i n g the h a r v e s t . During o f f - s e a s o n s they worked as urban l a b o r e r s doing odd jobs i n c o n s t r u c t i o n or l o a d i n g and unloading goods on t r u c k s . Female a d u l t s of the B o i a - F r i a f a m i l i e s w i t h small c h i l d r e n u s u a l l y stayed home, whereas other women worked along with t h e i r male c o u n t e r p a r t s . Often, grown-up c h i l d r e n a l s o worked as r u r a l farm l a b o r e r s to supplement the f a m i l y income. Average monthly income of the p a t e r n a l head of the B o i a -F r i a f a m i l i e s was 1469 c r u z e i r o s (about $75.33 U.S.), (range 0-4000 c r u z e i r o s ) which was l e s s than the minimum monthly s a l a r y . Average monthly income f o r the e n t i r e f a m i l y was 2051 c r u z e i r o s (about $105.18 U.S.), (range 0-6800 c r u z e i r o s ) ( p e s a i e t a l . (1978a). Seasonal unemployment d i d seem t o be a problem among the B o i a - F r i a f a m i l i e s . A l s o , the people were under-employed, working v e r y hard and long hours f o r l i t t l e e a r n i n g s . e) Food Costs The r e s u l t s o f a food c o s t survey i n R i b e i r a o Preto a t the time of our study are summarized i n Table IV-2. Table IV-2: Food Cost Survey a M o n t h l y D i e t C o s t A c c o r d i n g to L o c a t i o n (Cr) Cost per Month According to of a Worker L o c a t i o n (Cr)  C i t y Center V i l a Recreio C i t y Center V i l a R e c r e i o Meat ^sausage] (6kg) 20.0/kg 28.0/kg 120 .0 168.0 M i l k (7.51) 4.9/1 5.0/1 36 .8 37.5 Beans (4.5kg) 16.0/kg 15.0/kg 72 .0 67.5 Rice (3kg) 7.5/kg 8.5/kg 22 .5 25.5 Wheat f l o u r (1.5kg) 3.2/kg 5.5/kg 4 .7 8.2 Potatoes (6kg) 8.0/kg 9.0/kg 48 .0 54.0 Tomatoes (9kg) 5.0/kg 7.0/kg 45 .0 63 .0 Bread (6kg) 4.5/400g 4.5/4 00g 67 .5 67.5 C o f f e e (0.6kg) 60.0/kg 64.8/kg 36 .0 38 .9 Sugar (3kg) 5.7/kg 6.5/kg 17 .0 19.5 Bananas (7.5dozen) 5.3/dozen 7.0/dozen 39 .8 52.5 B u t t e r D[margarine] 2.4/100g 3.0/100g 18 .0 22.5 (0.75kg) Lard (0.7 5kg) b [soya o i l ] (8 06ml) 15.9/900ml 18.0/900ml 14 .2 16 .1 T o t a l 541.5 640.8 Defi n e d by B r a z i l i a n law i n 1948. Cost based on product i n square brackets. - 150 -Food c o s t s i n the c i t y c e n t e r and i n V i l a R e c r e i o are compared, and the c o s t f o r the monthly d i e t of a worker d e f i n e d by B r a z i l i a n law i n 1948 i s computed. Food c o s t s are 18.3% higher i n V i l a R e c r e i o than i n the c i t y c e n t e r f o r the monthly d i e t of a worker. f) L i t e r a c y The l e v e l of e d u c a t i o n among B o i a - F r i a s i s low. About 40% of the f a t h e r s and 60% of the mothers had had no education, and none of the B o i a - F r i a parents had had any more than primary education ; (-Desai e t al. 1978a). g) L o c a l C o r r e c t i v e Measures There was no p u b l i c a s s i s t a n c e i n the form of money g i v e n to needy f a m i l i e s from a w e l f a r e - t y p e government department. However, a l l f a m i l i e s r e c e i v e d a f a m i l y allowance of 78 c r u z e i r o s (about $4.00 U.S.) per month per c h i l d l e s s than 14 years o l d . A l s o , food a s s i s t a n c e programs were s e t up by the f e d e r a l , p r o v i n c i a l and c i t y governments through the p u b l i c and p r i v a t e s c h o o l s f o r the c h i l d r e n ; through Parque I n f a n t i l Bandeirantes, a day c a r e c e n t e r ; and through PLIMEC (Plan of I n t e g r a t i o n of the C h i l d i n the Community), a p r i v a t e e d u c a t i o n a l and s o c i a l s e r v i c e c e n t e r s e t up by the Organizacao V i d a Nova. . These government food a s s i s t a n c e school and day care programs were sponsored by the f o l l o w i n g agencies: B r a z i l i a n School Feeding Campaign, (CNAE),, f e d e r a l ; School H e a l t h S e r v i c e (S.S.E.) , s t a t e ; M u n i c i p a l - 151 -Sector of School Feeding, c i t y ; and the M u n i c i p a l P r e f e c t u r e , c i t y . A l s o , c o n t r i b u t i n g food were the Parent-Teachers A s s o c i a t i o n of each s c h o o l , and the S o c i a l and E d u c a t i o n a l S e r v i c e of I n d u s t r y f o r the Centro E d u c a c i o n a l SESI 34 6, a p r i v a t e s c h o o l operated by the S o c i a l S e r v i c e of I n d u s t r y . A l l c h i l d r e n t h a t attended s t a t e , m u n i c i p a l or p r i v a t e school r e c e i v e d some food t h e r e , i n the form of school lunches. Day care c h i l d r e n r e c e i v e d food 2 or 3 times per day, i n the form of b r e a k f a s t , l u n c h and perhaps a snack. PLIMEC p r o v i d e d one meal per day, two days a week f o r about 250 c h i l d r e n of ages 3 to 15 y e a r s . As w e l l , PLIMEC p r o v i d e d f o u r meals per day f o r 60 young c h i l d r e n who remained the e n t i r e day a t the c e n t e r , and two meals per day f o r a group o f 100 a d u l t s and c h i l d r e n . E l i g i b i l i t y c r i t e r i a f o r PLIMEC 1s food a s s i s t a n c e program were based on need. The f o l l o w i n g c a t e g o r i e s of c h i l d r e n are i n order of descending p r i o r i t y of acceptance; 1) p o v e r t y s t r i c k e n , 2) unemployed and not i n s c h o o l , 3) employed and not i n school , 4) employed f o r minimal wages and i n school., 5) unemployed and i n s c h o o l . P a r t i c i p a n t s i n the PLIMEC program o f t e n came from broken homes,- or l a r g e f a m i l i e s w i t h small incomes, or f a m i l i e s where the c h i l d r e n were home alone d u r i n g the day. -152-The d i r e c t o r estimated t h a t 46 to 7 5% of those r e g i s t e r e d would show up f o r t h e i r meal. However, many were not r e g i s t e r e d t h a t would q u a l i f y a c c o r d i n g to the e l i g i b i l i t y c r i t e r i a . The d i r e c t o r estimated t h a t PLIMEC served approximately 68 0 young c h i l d r e n i n t o t a l , a small p r o p o r t i o n of the t o t a l p o p u l a t i o n of c h i l d r e n i n the V i l a R e c r e i o area estimated t o be about 9000. The types of food d i s t r i b u t e d through these programs i n c l u d e d those commonly consumed by the B r a z i l i a n s ; r i c e , beans, macaroni, bread, manioc, sugar, some ve g e t a b l e s , f r u i t s , meat, and eggs and s a l t as a s p i c e . As w e l l , these programs.served powdered m i l k and e n r i c h e d f a r i n a s , o f t e n soybean-productsv . Other than these government food d i s t r i b u t i o n programs, there were two programs to help those i n need sponsored by v o l u n t e e r community s e r v i c e o r g a n i z a t i o n s . One of these c a l l e d L ar E s p i r i t a C r i s t a o gave out soup f o r more than 500 f a m i l i e s per day, each a f t e r n o o n . The soup t y p i c a l l y c o n t a i n e d legumes, v e g e t a b l e s , corn meal, macaroni, r i c e and small amounts of scrap meat and bones. T h i r t y to f o r t y v o l u n t e e r s donated time and community members donated some of the food, the r e s t being a c q u i r e d by the E s p x r i t a C r i s t S o group. To be e l i g i b l e t o r e c e i v e the soup, the f a m i l y had t o be poor, and t h i s was confirmed by home v i s i t s . S p e c i a l c l a s s e s and a food supplement of 1 kg of skim m i l k per week was - 153 -also given to 20 pregnant women. The second community service organization c a l l e d Organizacao Vida Nova had set up an experimental l i v e - i n school for problem boys less than 14 years old. The school, c a l l e d Casa das Mangueiras, House of the Mango Trees, was located i n V i l a Recreio although boys from the entire c i t y of Ribeirao Preto q u a l i f i e d to p a r t i c i p a t e . Twenty-nine boys slept i n the house, and of these, 12 worked i n the community and 17 spent th e i r entire day i n the house. Meals were provided and consisted of: bread and butter, milk and coffee for breakfast; r i c e , beans and a vegetable f o r lunch; j u i c e or cracker for mid-afternoon snack; and macaroni.soup or r i c e and beans for supper. The food was donated by the community or acquired by the Organizacao Vida Nova. Health personnel working i n V i l a Recreio are summarized i n Table IV-3. The nearest health center was located i n Ipiranga, a neighboring d i s t r i c t . State operated, i t s size depended on the number of people served. Formally, i t should have had 11 doctors, but i t had only 6, along with one nurse and one health care teacher. S t a t i s t i c s regarding health f a c i l i t i e s and n u t r i t i o n i s t s or d i e t i c i a n s i n the entire c i t y of Ribeirao Preto (> 259 ,000; population) are presented i n Table IV-4. The information regarding l o c a l corrected measures pre-sented i n this section was obtained by observation and v i s i t a -t i o n with the aid of Paula Beatriz M. Carvalho and M.L. Garcia Tavare s. Table IV-3. Health Personnel i n V i l a Recreio Location Doctors D e n t i s t s Nurses N u t r i t i o n i s t s 1 Escola Municipal de V i l a Recreio Escola Estadual de 1° Grau Francisco Bonfim Centro Educacional SESI 346 Casas de Betania (Ipiranga) Parque I n f a n t i l Bandeirantes Escola Experimental Casa das Mangueiras Casa do Vovo Lar E s p i r i t a Cristao none none none 1 volunteer 1 1 1 1 volunteer 1 when needed when needed none 1 day/week none none none none 1 none none 1 paediatrician 1 2 doctors sporadically none 1 day/week none none none none none none none none i n f o r m a t i o n collected by personal v i s i t a t i o n . - 155 -T a b l e IV-4: Heal t h F a c i l i t i e s and N u t r i t i o n i s t s i n R i b e i r a o P r e t o a H e a l t h Resource Number Comments H o s p i t a l s General: State P r i v a t e 1 6 500 beds 941 beds S p e c i a l i z e d : Mental H e a l t h : S t a t e 1 P r i v a t e 2 N o n - P r o f i t 1 C h i l d r e n ' s : N o n - P r o f i t 1 84 7 beds 120 beds 162 beds 104 beds Hea l t h Centers ( P u b l i c H e a l t h Sector) S t a t e : Emergency Centers M u n i c i p a l : General 4 C h i l d r e n ' s 1 o n l y o u t - p a t i e n t 3 0 beds P u b l i c H e a l t h Lab 1 N u t r i t i o n i s t s / D i e t i c i a n s 5 f o r r e g i o n -only a t h o s p i t a l s - p o s i t i o n s f o r 6 more a v a i l a b l e but not f i l l e d Source: P e r s o n a l communication wi t h Dr. J.L. Nogueira, Dep. Medicina S o c i a l , H o s p i t a l das C l i n i c a s , R i b e i r a o P r e t o . - 156 -D i e t a r y A n a l y s i s 1. Q u a l i t a t i v e The t y p i c a l meal p a t t e r n of B o i a - F r i a s i n southern B r a z i l i s summarized i n Tab l e IV-5 , P r i n c i p a l d i e t a r y components i n c l u d e ; c o f f e e with sugar, white bread, pinga or pure white rum, p o l i s h e d r i c e , and leguminous beans. Once a week, o f t e n on Sunday, animal p r o t e i n sources were consumed. F r u i t s and v e g e t a b l e s were sometimes eaten but not w i t h r e g u l a r i t y . Weekly, m i l k was consumed i n most homes and was r e s e r v e d mainly f o r c h i l d r e n . In g e n e r a l , B o i a - F r i a d i e t e x h i b i t e d l i t t l e v a r i e t y . Food consumption a c c o r d i n g t o food group i s o u t l i n e d i n Table IV-6. o f the many a v a i l a b l e d a i r y products l i k e m i l k powder, condensed m i l k , cheese, yogurt and b u t t e r , o n l y p a s t e u r i z e d m i l k which i s b o i l e d and consumed hot with sugar was a component of the B o i a - F r i a d i e t . M i l k i n t o l e r a n c e was not r e p o r t e d . Of animal p r o t e i n sources eggs, beef, c h i c k e n , and sausage were eaten sometimes. F i s h was too expensive and organ meats were f o r e i g n to t h e i r d i e t . Legumes, p r i n c i p a l l y the kidney bean b o i l e d served as a ve g e t a b l e p r o t e i n source. Although many other v a r i e t i e s of legume such as soybean, l e n t i l s , garbanzo bean, and d r i e d peas were grown i n B r a z i l , they were not consumed. B o i a - F r i a s d i d not u s u a l l y eat most v e g e t a b l e s even - 157 -b, Table IV-5: Typical Diet of Boia-Frias i n B r a z i l Meal Breakfast Daily rFoods and Beverages Consumed " Weekly Coffee, .-with sugar White bread Hot milk with sugar Sometimes Lunch Pinga (pure white rum) Rice and beans Beer F r i e d beef or chicken F r i e d egg Raw salad: Raw salad: lettuce tomato wild chicory onion Boiled vegetables: potato j i l o chuchu cassava Dinner Pinga (pure white rum) Rice and beans Beer Macaroni-soup White bread Fried sausage Snacks Coffee with sugar Tea with sugar Soft drinks Limeade with sugar F r u i t s : banana orange b consumed mainly by children from Desai et al. (19.78a). Table IV-6 : Food Consumption Food Group % of Families c v ^ g ^ g ^ Consuming By wnom Milk Products Eggs Meats Legumes Vegetables 84 93 92 100 N.S infants, small children aN.S. N.S . N.S . N.S. F r u i t s N.S. N.S. Cereals 100 - N.S. N.S. especially children N.S. N.S. N.S. N.S. N.S. N.S. Fats and O i l s most N 5 0 N.S. N.S. N.S. Sweets and ugar 100 N.S. b of Boia-Frias by Spe c i f i c Food Group Type pasteurized, boiled milk f r i e d preferred beef, chicken,sausage kidney bean tomatoes, onions raw lettuce raw wild chicory boiled potatoes boiled j i l o boiled r i h n c h n boiled cassava bananas oranges limeade with sugar polished r i c e non-enriched white bread macaroni maize cassava flo u r soybean o i l pork lard margarine refined sugar Frequency or Average Consumption 121± 8 6 ml/child/day i r r e g u l a r l y sometimes d a i l y weekly sometimes sometimes sometimes sometimes sometimes sometimes i r r e g u l a r l y i r r e g u l a r l y sometimes d a i l y • d a i l y weekly sometimes sometimes 38 t 23 ml/person/day 19 i - 21 ml/per son/day very l i t t l e 7 0 + 3 0 g/person/day Table IV-6 Continued Beverages 100 N.S, > 50 adults, children children adults M adults M F Nuts Condiments, Sauces and Salt none aN.S, N.S, N.S, N.S, • N,S , N.S. N.S. N.S. N.S. N.S. a N.S. = not spe c i f i e d , ^refer to Desai et al. (1978a)_ coffee with sugar carbonated soft drinks pinga (pure white rum) pinga (pure white rum) cerveja •. (beer) cerveja (beer) onion g a r l i c s a l t red pepper tomato sauce 4 5+ 4 5 ml/adult/day frequently 7 5ml/male/weekend day 5Oml/male/weekday l i t t l e 24 0 ml/male/weekend day 3 5ml/male/weekday 70 ml/female/weekend day 10 ml/female/weekday d a i l y d a i l y 9 + 6 g/person/day sometimes sometimes - 160 -though a wide v a r i e t y of them were abundantly a v a i l a b l e a l l throughout the year. O c c a s i o n a l l y , tomatoes, onions, w i l d c h i c o r y and l e t t u c e were eaten raw i n s a l a d s , and potatoes, j i l o , chuchu and cassava were eaten b o i l e d . B o i a - F r i a s n e i t h e r used canned v e g e t a b l e s nor d i d they attempt t o preserve them. F r u i t s , l i k e w i s e , were consumed i r r e g u l a r l y , l imes as limeade with sugar, and a l s o bananas and oranges. F r u i t p r e s e r v e s a l s o were not made or consumed by B o i a - F r i a s . G e n e r a l l y , c e r e a l products eaten were h i g h l y r e f i n e d i n nature i n c l u d i n g p o l i s h e d r i c e , non-enriched white bread, and macaroni. Maize and cassava f l o u r were used by some f o r making p o r r i d g e or g r u e l . Soybean o i l was the most common p l a n t o i l used f o r cooking and i n s a l a d s . A l s o used f o r cooking by about h a l f of the f a m i l i e s was pork l a r d . Margarine ("Doriana" brand), e n r i c h e d w i t h v i t a m i n A (20,000 IU/kg) and v i t a m i n D (2,000 IU/kg), was used v e r y l i t t l e . B o i a - F r i a s d i d not eat sweets as such but d i d consume r e f i n e d sugar i n beverages. Black c o f f e e w i t h sugar was the most common hot beverage f o r a l l i n the f a m i l y . Carbonated s o f t d r i n k s l i k e "Q-suco", "Coca-cola" and P e p s i - c o l a " were f r e q u e n t l y consumed by c h i l d r e n while a d u l t s , e s p e c i a l l y males, drank a l c o h o l i c beverages r e g u l a r l y . Nuts, grown commonly i n B r a z i l , i n c l u d i n g peanuts and coconuts were not consumed by B o i a - F r i a s . Onion, g a r l i c and s a l t were consumed - 161 -d a i l y as condiments, and r e d pepper and tomato sauce consumed sometimes. The B o i a - F r i a women unanimously s t a t e d t h a t they ate no s p e c i a l foods d u r i n g pregnancy and l a c t a t i o n . However, a few mentioned r i c e , beans, c o f f e e , onion, g a r l i c or red pepper as foods to be avoided d u r i n g pregnancy. Almost a l l mothers d i d not know of any foods u n d e s i r a b l e f o r l a c t a t i o n . T y p i c a l i n f a n t f e e d i n g p r a c t i c e s are shown i n F i g u r e IV - 1 . About 30% of the B o i a - F r i a mothers terminated b r e a s t f e e d i n g by 6 months, about 50% more by 12 to 18 months, and the l a s t 20% by 18 t o 36 months. F o r t u n a t e l y , the t r e n d seemed to be toward longer d u r a t i o n s of b r e a s t f e e d i n g . Weaning foods i n s u c c e s s i o n as added to the i n f a n t ' s d i e t were: s t a r c h y g r u e l s , made from corn s t a r c h , arrow r o o t f l o u r , cassava f l o u r , r i c e f l o u r or oats i n water from the day of l e a v i n g the h o s p i t a l a f t e r d e l i v e r y ; sugar-water and herb tea from the v e r y f i r s t week f o r 80% of the mothers; c o f f e e w i t h sugar and s o f t d r i n k s w i t h i n the f i r s t two weeks f o r 60% of the mothers; u n d i l u t e d cow's milk with sugar w i t h i n the f i r s t 3 months f o r 67% of the mothers; pureed f r u i t s and v e g e t a b l e s at about 3 months; cooked and mashed egg, meat, r i c e and beans a t about 4 months; bread and soup around 5 months; and the r e g u l a r a d u l t menu a t one year. F i g u r e IV-1 -.TYPICAL INFANT FEEDING PRACTICES AMONG BOIAFRIA FAMILIES CONVENTIONAL ADULT DIET RICE t BEAN soup WHITE BREAD EGG OR MEAT FRUITS VEGETABLES COWS MILK CORN STARCH CASSAVA FLOUR RICE FLOUR OAT GRUEL SUGAR WATER SOFT DRINKS COFFEE TEA BREAST MILK V//////A V//////////////7777» V///////////////777777> V////////////////////////)> V///////////////////////////A V, V 0 2 1 2 -WEEKS—• < 3 U 5 6 7-9 10-12 13-18 19-24 MONTHS : • AGE from Desai et a l . (1978a) -163 -2. Quantitative Average d a i l y nutrient intakes of adult male and. female Boia-Frias-are shown i n Table IV - 7 . The mean c a l o r i c and nutrient intakes for females were s l i g h t l y more than half of the male intakes. Standard deviations were p a r t i c u l a r l y high for calcium, vitamin A, r i b o f l a v i n , and vitamin C and therefore the mean intakes for these nutrients are subject to greater error as estimates of the true population means. These nutrients also exhibited the most extreme range values. The only nutrient which was completely absent from the d i e t of some people was vitamin C. To give a better understanding of the dietary adequacy of the adult population as a whole, numbers and percentages of Boia-Frias with d a i l y intakes less than two-thirds and one-third of the d a i l y intake standards are shown in Table IV-8. i n general from t h i s Table one can see that food was l i m i t e d i n quantity with mean c a l o r i c intakes for 81.2% of the population less than two-thirds of the standard and 17.9% of the population less than one-third of the standard. The amount of food and choice of foods normally consumed by Boia-Frias did not meet the recommended intake l e v e l s for energy and a l l the other nutrients essential for maintaining optimum health. Extreme d e f i c i e n c i e s were observed i n calcium and iron for females, and vitamin A, r i b o f l a v i n , n i a c i n and vitamin C for the o Table IV-7 - Average Dally Nutrient Intakes of Adult Male and Female Bola-Frlas Nutrient M F Both Mean. - SD Rami e Mean SD RanRe Mean. SD Range Calories (kcal) 2 0 5 0 . 9 2 3 (315*- 5 0 7 5 ) 1 0 7 1 . <+0i. (456 - 3 1 0 6 ) 1 5 0 1 . 837 ( 3 1 0 - 5075) Protein (g) 5 1 . 9 ' 31.7 ( 5 . 6 -I 6 5 .0) 2 8 . 2 14 .9 ( 7 . 7 - 84.0) 38 . 6 2 6 . 5 (5.6 - 1 6 5 . 0 ) 1 Fat (g) 82.8 42 .5 ( 1 1 . 2 - 2 7 3 - 3 ) 4 4 . 1 1 9 . i , (10.6 - 1 2 5 - 5 ) 61 .1 36.9 (10 . 6 - 2 7 3 - 3 ) I-1 Saturated Fatty Acids (g) 18.5 14 .0 (1 . 9 - 1 0 6 . 8 ) 9.9 5 . 1 I 3.0 - 2 9 . 9 ) 13.7 1 0 . 9 ( 1 . 9 - 1 0 6 . 8 ) £~ Oleic Acid (g) 24 .0 14 .9 ( 2 . 8 - I O 6 . 3 ) 1 2 . 9 6 . 2 I 3-5 - 3 9 . 8 ) 1 7 . 8 1 2 . 2 ( 2 . 8 - 1 0 6 . 3 ) 1 Llnolelc Acid (g) 33-8 14 .9 ( 5 . 6 - 7 8 . 4 ) 18.0 7.8 (3 . 0 - 46.7) 24 .9 13.9 ( 3 . 0 - 78.4) Carbohydrate (g) 2 3 2 . 9 90.7 ( 3 6 . 8 - 552.7) ' 140.4 5 6 . 5 ( 4 7 . 4 - 451.1) 181.0 8 6 . 5 (36 .8 - 5 5 2 . 7 ) -Calcium (mg) 25^.6 2 5 1 . 2 (28 . 1 - 1664.0) 1 6 8 . 2 146.8 ( 3 1 . 0 - 904 .5) 206.1 2 0 3 . 4 (28 .1 - 1 6 8 4 . 0 ) Iron (.mg) 10.64 5 . 6 1 (1 . 2 9 - 2 6 . 9 8 ) 5.59 2 . 3 5 (1.24 - 14.16) 7.80 4.81 (1.24 • - 2 6 . 9 8 ) Active Vitamin A <Jxg) 243.5 2 5 4 . 6 ( 1 1 . 4 - 1586.5) 171.8 1 7 9 . 0 ( 1 0 . 5 - 1 1 2 3 . 6 ) 2 0 3 . 2 2 1 7 . 7 I 1 0 . 5 • - 1 5 8 6 . 5 ) Thiamine (mg) 0 . 7 7 0 O . 3 8 9 (0.082 -1 . 9 1 6 ) 0.406 0 . 1 9 8 (0.080 - 1.218) 0.566 0 . 3 4 7 (0.080 - 1 . 9 1 6 ) Riboflavin (mg) 0 . 6 8 3 0 . 9 1 7 ( 0 . 0 5 4 - 7.464) 0 . 3 9 4 . 0 . 3 6 5 (0 . 0 5 2 - 2.949) 0 . 5 2 1 0 . 6 7 9 ( 0 . 0 5 2 - 7.464) Niacin (mg) > 9 . 0 3 6.04 (0 . 8 3 - 29.42) 5-29 3 - 3 6 ( 1 . 3 1 • - 18.62) 6.93 5.06 (0.83 - 29.42) Vitamin C (mg) 2 1 . 4 5 6 . 4 (0 . 0 0 - 399.5) 14.9 34.8 (0.00 - 2 3 8 . 3 ) 1 7 . 8 45.5 ( 0 . 0 0 • - 399 .5) ^-Tocopherol (mg) 7.38 ( 1 . 1 2 - 20.84) 4.04 2 . 0 6 ( 1 . 1 2 • - 1 7 . 0 7 ) 5.51 3 . 3 0 (1.12 -- 20.84) Table IV - 8 Numbers and Percentages with Dally Intakes Less Than 2^3 and V 3 of the Standard Dally Intake Standard Cases wltn Dally Intake < 2 / 3 Standnr^ Cases with Dally Intake < V 3 Standard Nutrient F Both M F Both - ., Source ., JH. F , No.. k . .Ho. S. .-.' . No. -" _ * . No. i No. No. % Calories (kcal) aFAO b 3 0 0 0 c 2 2 0 0 23 6 5 . 7 72 8 7 . 8 95 . 81 . 2 2 5 . 7 19 2 3 . 2 21 1 7 . 9 Protein (g) FAO d 5 3 e41 12 3 4 . 3 47 5 7 . 3 5 9 5 0 . 4 2 5 . 7 13 1 5 . 9 15 1 2 . 8 Calcium (mg) FAO 400 400 57 7 6 . 0 . 81 84.4 1 3 8 80.7 20 2 6 . 7 5 8 6 0 . 4 7 8 4 5 . 6 Iron (mg) FAO f 5 , 6 or 9 f14 , 1 9 or 28 8 1 0 . 7 94 9 7 . 9 .102 5 9 . 6 3 4 . 0 71 7 4 . 0 74 4 3 . » 3 Active Vitamin A J/Mg) FAO 750 750 66 8 8 . 0 9 2 9 5 . 8 1 5 8 ; 92 . 4 48 64 . 0 7 6 7 9 . 2 124 7 2 . 5 Thiamine (mg) FAO 1.2 0 . 9 . 4 4 5 8 . 7 87 9 0 . 6 . 1 3 1 7 6 . 6 10 1 3 . 3 34 3 5 . 4 4 4 2 5 . 7 R i b o f l a v i n (mg) FAO 1 . 8 1 . 3 67 8 9 . 3 89 9 2 . 7 15.6, ' 9 1 . 2 48 64 . 0 69 7 1 . 9 117 68 . 4 Niacin ^(equivalents) FAO 1 9 . 8 14 . 5 64 8 5 . 3 8 8 9 1 . 7 . 1 5 2 8 8 . 9 3 1 41 . 3 55 5 7 . 3 86 5 0 . 3 Vitamin C (mg) FAO 3 0 3 0 57 7 6 . 0 79 8 2 . 3 1 3 6 • " 7 9 . 5 47 6 2 . 7 60 6 2 . 5 107 62.6 o( -Tocopherol (mg) *\j.S. RDA 10 8 25 3 3 - 3 5 5 5 7 . 3 80 " 46 . 8 5 6 . 7 6 6 . 3 11 6 . 4 c<-Tocopherol (mg) ^Canadian RDI- J 9 or 8 6 37 4 9 . 3 8 2 8 5 . 4 • * 119 6 9 . 6 10 ' 1 3 - 3 19 1 9 . 8 29 1 7 . 0 a Food and Agriculture Organization/World Health Organization Standards, (Beaton and Patwardham 1 9 7 6 ) . b I f weight Is known, 46 per kg. c I f weight Is known, 40 per kg. d I f weight Is known, 0.81 per kg. e I f weight Is known, 0 . 7 4 per kg. f Depending on l f diet contained> 2 5 # , 10 - 2 5 # , or C10% energy from animal sources or soybean, g Niacin equivalent = 1 mg ni a c i n or 60 mg tryptophan. h U.S., Food and N u t r i t i o n Board National Academy of Sciences - National Kesearcn Council Recommended Dally Dietary Allowances, Revised 1 9 7 4 . 1 Canadain Department of National Health and Welfare Recommended Daily Nutrient Intakes, Revised 1 9 7 5 . J Depending on l f 1 9 - 3 5 years or 3 6 - 5 0 years of age. - 166 -p o p u l a t i o n i n g e n e r a l , w i t h g r e a t e r than 50% of the p o p u l a t i o n having i n t a k e l e v e l s l e s s than o n e - t h i r d of the standard. On the b a s i s of 24-hour r e c a l l of d i e t a r y i n t a k e s , females appeared to be more d e f i c i e n t i n a l l n u t r i e n t s than males, e s p e c i a l l y w i t h r e s p e c t to i r o n . A g r e a t e r percentage of people were d e f i c i e n t i n c a l o r i e s than i n p r o t e i n i n d i c a t i n g t h a t the s t a p l e d i e t of r i c e and beans, supplemented w i t h small amounts of meat, eggs, and m i l k had an adequate p r o t e i n - c a l o r i e balance. C a l o r i e and n u t r i e n t sources a c c o r d i n g to food group a r e o u t l i n e d i n T a b l e XV-9, C a l o r i e s were obtained mainly from g r a i n and legume sources, p r i n c i p a l l y r i c e and beans. The n u t r i e n t composition of these s t a p l e d i e t a r y components was c a l c u l a t e d on an as consumed b a s i s , w i t h the v e g e t a b l e o i l added d u r i n g p r e p a r a t i o n i n c l u d e d . T h e r e f o r e , the percentage of c a l o r i e s o b t a i n e d from f a t s and o i l s r e f e r s o n l y to f a t and o i l sources consumed d i r e c t l y such as margarine, or s a l a d o i l . Approximately o n e - t h i r d of the p r o t e i n was obtained from g r a i n sources, o n e - t h i r d from legume sources and o n e - t h i r d from animal and d a i r y p r o t e i n sources. For c a l c i u m , and <K-tocopherol more than h a l f of the n u t r i e n t i n t a k e o r i g i n a t e d from legume or g r a i n sources, the m a j o r i t y of the «-tocopherol being l o c a t e d i n the o i l added to r i c e and beans d u r i n g p r e p a r a t i o n . Only one-t e n t h of the c a l c i u m came from d a i r y sources. For i r o n and thiamine, about h a l f of the n u t r i e n t i n t a k e came from Table I V - 9 : Percentage Contribution of Food Groups to Nutrient Intake Nutrient Food Groups Dairy Animal Legumes Vegetables F r u i t Grain Fats and Protein Sources Protein Sources O i l s Calories M 0 .7 1 0 . 0 26.8 2 . 1 0 . 7 47 .4 2 .6 F 1.7 10 .6 23.6 3 . 1 0 . 7 48 .5 * . 5 Both 1 . 2 1 0 . 3 2 5 . 0 2.7 0 . 7 48 .0 3-6 Protein M 1 . 5 2 6 . 5 3 5 . * 2 . 0 0 . 3 3 * . 2 0 . 1 F 3 . 9 28 .0 2 9 .9 3 . 5 0 . 3 3 4 . 2 0 . 2 Both 2 .8 27.*+ 3 2 . 3 2 . 8 0 . 3 3 4 . 2 0 . 2 Calcium M 6 . 4 1 3 . 5 3 2 .9 5 . 0 1 . 1 3 6 . 1 o.l F 11.9 1 5 . * 2 5 .6 7 . 0 1 . 0 3 2 . 9 0 . 2 Both 9 . 5 14.6 2 8 . 8 6 . 1 1 . 0 3 * . 3 0 . 2 Iron H 0.7 18 .4 5 * . 7 4 . 6 1 . 0 1 7 . 5 0 . 1 F • 1.9 18.6 47.I 7 . 1 1 . 2 18.9 0 .2 Both 1 . 3 18.5 5 0 . 4 6 .0 1 . 1 18 .3 0 .1 Active Vitamin A M 1 .6 24.8 42 .8 2 0 .8 2 . 9 0 . 1 7 . 1 F 5 . 0 24 .4 3 3 . 6 2 3 . 1 3 . 0 l . l 9 . 9 Both 3 . 5 24 .5 37.6 2 2 . 1 2 . 9 0 . 7 8.6 Thiamine H 1 . 2 15 .7 5 * . 2 5 - 3 1 . 4 2 0 . 8 0 . 0 F 3 - 1 15 A 4 7 . 1 8 . 1 1 . 7 2 1 . 2 0 . 1 Both 2 . 3 1 5 - 5 5 0 . 2 6.9 1 .6 2 1 . 0 0 . 1 R i b o f l a v i n M 4 . 1 32 .4 3 * . 2 5 . 6 1 . 0 1 6 . 7 o.7 F 9-3 3 1 . 1 ;• 2 7 . 8 7 . 8 1 . 2 16.3 0 . 9 Both 7 . 0 31-7 3 0 . 6 6 . 8 • 1 . 1 I 6 . 5 0.8 Niacin M 0 . 3 21 .8 2 1 . 3 5-6 1 . 1 3 7 . 3 0 . 1 F 0 .8 2 0 . 1 1 7 . * 7:9 0 . 9 3 * . 9 0 . 2 Both 0 .6 2 0 . 8 19.1 6.9 1 . 0 36.O 0 . 1 Vitamin C M 6 .1 1 . 8 C O 76 . 6 11 .6 1 . 8 • 0 . 0 F 8.7 1 . * 0 . 0 7 6 . 0 1 0 . 3 3 . 3 0 . 0 Both 7.6 1 .6 0 . 0 7 6 . 2 1 0 . 9 2.7 0 . 0 oi -Tocopherol M 0 . 2 7.* 4 3 . 1 . 3 - 7 1 . 1 3 6 . 8 7.1 F o . 5 9 . 2 3 7 . 0 4 . 0 1 . 2 3 5 . * 1 1 . 2 Both 0 . 4 8 . 4 3 9 .7 3 - 9 1 . 2 3 6 . 0 9.* - 168 -legume sources. Major sources of v i t a m i n A were animal p r o t e i n foods, legumes and v e g e t a b l e s ; of r i b o f l a v i n were animal p r o t e i n foods and legumes; and of n i a c i n were animal p r o t e i n foods, legumes, and g r a i n products. T h r e e - q u a r t e r s of the v i t a m i n C i n t a k e was d e r i v e d from v e g e t a b l e s and o n l y one-tenth from f r u i t . There were no o u t s t a n d i n g d i f f e r e n c e s between male and female i n the percentage c o n t r i b u t i o n of the v a r i o u s food groups to t h e i r n u t r i e n t i n t a k e . C a l o r i c breakdown a c c o r d i n g t o p r o t e i n , carbohydrate or f a t source i s g i v e n i n T a b l e i v - i o . P r o t e i n s , carbohydrates and f a t s c o n t r i b u t e d about 10, 50, and 40% of the t o t a l c a l o r i e s r e s p e c t i v e l y f o r both males and females. R e f i n e d sugar s u p p l i e d 3.5% and 6.6% of the t o t a l c a l o r i e s f o r males and females r e s p e c t i v e l y ; carbonated d r i n k s s u p p l i e d 0.2% and 0.4% r e s p e c t i v e l y ; and a l c o h o l i c beverages 5.8% and 0.1% r e s p e c t i v e l y i Carbonated and a l c o h o l i c beverages added minimal c a l o r i e s t o the d i e t s of women and carbonated beverages minimal c a l o r i e s t o the d i e t s of men. A l s o , one must remember t h a t the consumption of a l c o h o l i c beverages i n c r e a s e s on weekend days and t h a t the 24-hour surveys were a l l taken d u r i n g the week. Saturated f a t t y a c i d s p r o v i d e d 8.5% of the t o t a l c a l o r i e s , o l e i c a c i d 11.2% of the t o t a l c a l o r i e s , and l i n o l e i c a c i d 16.1% of the t o t a l c a l o r i e s . F a t breakdown a c c o r d i n g to type i s shown i n T a b l e IV-11. R e s p e c t i v e l y , s a t u r a t e d f a t t y a c i d s , o l e i c a c i d and l i n o l e i c T a b l e IV-10 C a l o r i e Breakdown A c c o r d i n g to Source Source 24-Hour C a l o r i c C o n t r i b u t i o n M F Both k c a l % of t o t a l c a l o r i e s k c a l % o f t o t a l c a l o r i e s k c a l % o f t o t a l c a l o r i e s P r o t e i n - t o t a l 213 10. .13 116 10, .66 158 10, .42 C a r b o h y d r a t e - t o t a l 955 49. .10 576 53. .95 742 51. .82 s u g a r , r e f i n e d 61 3 , .46 70 6, .60 66 5. , 22 c a r b o n a t e d d r i n k s 5 0, .25 4 0, .41 4 0. .34 a l c o h o l i c beverages 164 . 5. .84 1 0. .12 73 2. . 63 F a t - t o t a l 787 38 . .34 419 39. .04 581 . 38 . .73 s a t u r a t e d f a t t y a c i d s 176 8, .32 94 8 , .71 130 8, .54 o l e i c a c i d 228 10, .96 123 11. .37 169 11. .19 l i n o l e i c a c i d 321 .16. .11 171 16, .05 237 16, . 08 T a b l e IV-11 F a t Breakdown A c c o r d i n g to Type Type S a t u r a t e d O l e i c A c i d L i n o l e i c A c i d Mean P e r c e n t a g e o f T o t a l F a t 21.94 28.75 41.86 - 170 -acid constituted 21.9, 28.8, and 41.9% of the t o t a l f a t i n the Boia-Fria diet making i t very high i n unsaturated l i p i d s . C.Anthropometric Assessment The anthropometric p r o f i l e of adult Boia-Frias i s given i n Table IV-12. Mean ages for males and females i n years were 42.5 + 9.1 and 37.6 + 8.0, with ranges 25 to 59 and 20 to 56 respectively. Mean heights i n cm were 164.4 ^6 .2 for males and 153.4 JI 5.2 for females. Obtained from J e l l i f f e (1966), the standards for comparison were based on American data. A l l anthropometric mean resu l t s were less than the standards except female weight f o r mean height, and male mid-arm muscle circumference. To characterize the population as a whole, the percentage d i s t r i b u t i o n of adult Boia-Frias according to the same standard anthropometric c r i t e r i a i s outlined i n Table IV-13. Female Boia-Frias tended toward overweight with 22.37, of them above the 1207, of standard value mark for weight for height and 2k.TL of them above the 1207o of standard value mark for triceps s k i n - f o l d . However, at the other extreme, a few, 67,, of them were below the 807, of standard value mark for weight for height and 54.27o of them below the 807. of standard value mark for triceps s k i n - f o l d . With regard to arm circumference, 88.37o of the female were within the 80 to 1207, of standard value marks, and 95.27, were within the 80 to 1207o of standard - 171 Table IV-12: Anthropometric P r o f i l e o f A d u l t B o i a - F r i a s Parameter Standards M Mean - S D B o i a - F r i a s M Age (years) Height (cm) Weight f o r mean heig h t (kg) T r i c e p s • s k i n -f o l d (mm) Arm circum-f e r e n c e (cm) Mid-arm-muscle cir c u m f e r e n c e (cm) 62.9 42.5 + 9.1 37.6 + 8.0 164.4 + 6.2 153.4 + 5.2 52.0 59.4 + 8.3 56.5 + 12.0 12.5 16.5 4.7 + 2.0 14.3 + 7.8 29.3 28.5 27.2 + 3.0 27.6 + 3.8 25.3 23.2 25.6 + 2.9 23.1 + 2.4 Standards f o r comparison were o b t a i n e d from J e l l i f f e (1966) Table IV" 13 . Percentage D i s t r i b u t i o n of Boia-Frias According to aStandard Anthropometric C r i t e r a % of "Standard Value Weight fo r Height Triceps Skin-fold Arm Circumference Mid-arm- muscle Clrcumferenc e M F M F M F H F > 175 0 0 0 5 - 9 0 0 "'0 0 150 - 1 7 * . 9 0 5 - 9 0 * . 7 0 0 0 0 140 - 149 . 9 0 * . 7 0 0 0 1 . 2 0 0 130 - 1 3 9 . 9 0 3 - 5 0 * . 7 0 0 2 . 6 0 . 0 120 - 1 2 9 . 9 5 . 1 8 . 2 0 9 . * 2 . 6 3 . 5 0 . 0 3 . 5 110 - 1 1 9 . 9 5 - 1 2 1 . 2 5 . 1 0 2 . 6 1 1 . 8 2 3 . 1 12.9 100 - 109.9 2 5 . 6 2 0 . 0 0 t . 7 2 3 . 1 2 1 . 2 3 3 . 3 3 5 - 3 90 - 9 9 . 9 3 0 . 8 1 6 . 5 0 7 . 1 3 0 . 8 2 8 . 2 3 0 . 8 32.9 8 0 - 8 9 . 9 2 3 - 1 14 . 1 0 9.4 3 3 . 3 2 7 . 1 1 0 . 3 14 . 1 70 - . 7 9 . 9 1 0 . 3 2.4 2 . 6 14 . 1 7 . 7 7 . 1 0 1.2 60 - 6 9 . 9 0 2.4 2 . 6 5 . 9 0 , 0 0 0 50 - 5 9 . 9 0 0 7 . 6 1 1 . 8 0 0 0 0 25 - * 9 . 9 0 1 . 2 6 6 . 7 2 1 . 2 0 . 0 0 0 0 - 24 . 9 0 0 1 5 . 4 1 . 2 0 ' 0 0 0 Mean % 9 5 . 2 1 0 7 . 9 3 9 . 9 8 6 . 7 92 . 7 9 6 . 9 •101.4 99 . 6 Range % 7 5 - 1 2 6 46 - 1 6 9 2 0 - 1 1 2 2 1 - 2 7 3 7 2 - 1 2 3 70-140 8 0 - 1 3 7 7 9 - 1 2 9 Standards f o r comparison were obtained from J e l l i f f e (I966). - 173 -v a l u e marks f o r mid-arm^muscle cir c u m f e r e n c e . Male B o i a - F r i a s , on the other hand, tended toward underweight w i t h 10.3% of them below the 80% of standard v a l u e mark f o r weight f o r h e i g h t and 94.9% of them below the 8 0% of standard v a l u e mark f o r t r i c e p s s k i n - f o l d . For arm c i r c u m f e r e n c e and mid-arm-muscle circ u m f e r e n c e r e s p e c t i v e l y , 89.8% and 97.5% of the males were d i s t r i b u t e d w i t h i n the 80 to 120% of standard v a l u e marks. The e f f e c t of age on anthropometric standing i s shown i n Table: IV-14. Examining the data f o r t r e n d s , one f i n d s t h a t males peaked i n weight f o r h e i g h t , t r i c e p s s k i n - f o l d , arm c i r c u m f e r e n c e and mid-arm muscle circum-f e r e n c e a t the 30 t o 39 years age group. Females, on the other hand, dropped s h a r p l y i n a l l f o u r c r i t e r i a a t the 50 to 59 years age group. D. B1oc hemic a1 Exam i n a t i o n B i o c h e m i c a l averages and i n t e r p r e t i v e standards along with t h e i r source are g i v e n i n Table 1V-L5. The o n l y standard r e q u i r i n g ; e x p l a n a t i o n i s the plasma v i t a m i n E standard expressed as mg per g l i p i d . H o r w i t t e t a l . (1972) suggested the standard of d e f i c i e n c y as being l e s s than 0.8 mg per g l i p i d . To c o n s t r u c t low and a c c e p t a b l e c a t e g o r i e s as w e l l , the form of the plasma v i t a m i n E standard l e v e l s expressed as mg per 100 ml was repeated, simply by m u l t i p l y i n g by a f a c t o r of 1.6. Table IV-14 Anthropometric P r o f i l e of Adult Bola-Frlas According to Age Mean % of a Standard Anthropometric C r i t e r i a  Age Qroup Weight for Height Triceps Skin-Fold Arm Circumference Mid-Arm Muscle Circumference M F_ Both M F Both M F Botn M F Both 20 - 29 94 .0 108.4 1 0 6 . 1 3 0 . 7 1 0 2 . 8 9 1 . 4 9 2 . 7 9 9 . 7 9 8 . 6 1 0 2 . 7 9 9 . 5 1 0 0 . 0 30 - 39 9 8 . 6 110.9 1 0 7 . 5 49-5 8 5 . 0 7 5 . 4 ' 9 7 . 7 9 6 . 0 9 6 . 5 1 0 5 - 5 9 8 . 9 • 1 0 0 . 7 40 - 49 9 3 - 5 1 0 8 . 0 103 .4 3 5 . 4 8 7 . 4 70 .9 9 0 . 7 99 .2 9 6 . 5 9 9 . 6 1 0 2 . 3 1 0 1 . 5 5 0 - 59 9 3 . 3 8 8 . 7 9 1 . 6 3 6 . 0 5 0 . 0 41 .2 8 8 .9 8 3 . 5 86.9 9 8 . 0 9 1 . 2 9 5 . 4 a Standards f o r comparison were obtained from J e l l i f f e CI966). Table IV-15 Biochemical P r o f i l e of Adult Bola-Frlas Parameter Standards Source ICNND (1963) ICNND ( 1 9 6 3 ) 0 'Neal et a l . ( 1 9 7 0 ) fauberllch et a l . 1 9 7 * ) Horwltt et al.(1972) WHO (1968) Plasma Vitamin A jXAg/lOOml) Plasma Carotene (/4g/100ml) Plasma Vitamin E (mg/lOOml) Plasma Vitamin E (mg/g l i p i d ) Hemoglobin (g/lOOml) Hemoglobin (g/lOOml) Hematocrit (%) Plasma Cholesterol (mg/lOOml) Henry et al.(1974) Plasma Total L i p i d s (mg/lOOml) Amenta ( 1 9 7 0 ) Q u a l i f i c a t i o n s Categories D e f i c i e n t Low Acceptable High — 10 10 co 19 20 — 20 20 to 39 40 -— 0.5 0.5 to 0.7 0.7 _ — 0.8 0.8 to 1.12 1 . 1 2 -M 13 F 12 — - — -18 -44 years M 1 2 . 1 12.1 to 14.0 14.1 F 10.1 10.1 Do 11.0 11.1 _ 4 5 - 6 * years 11.1 11.1 to 12.5 12.6 _ 65 years 1 0 . 9 1 0 . 9 to 12.3 12.4 -18-44 years M 35-5 35-5 to 41.0 41.1 F 29.6 29.6 to 32.2 32.3 -45 - 64 years 32.6 32.6 to 3 6 . 6 36.7 -65 years 32.0 3 2 . 6 to 3 6.I 36.2 _ Normal 20 - 2 9 years 144 144 to 275 2 7 5 . 1 3 0 - 3 9 years 165 165 to 295 2 9 5 . 1 40 - 4 9 years 170 170 to 315 315.1 5 0 years 177 177 to 340 3 4 0 . 1 Normal — — 400 400 to 800 800.1 Meant SD H Both . 2 7 . 5 . ± 1 0 . 3 26 . 0 + 7 . 7 26 . 5 ±8.6 62.3t48 . 3 6 0 . 4 * 3 1 . 1 61 . 0 ^ 3 7 . 2 1 . 1 6 * 0 . 3 5 1 . 1 4 * 0 . 3 3 1 . 1 4 * 0 . 3 3 2.40 * 0 . 6 9 2 . 2 0 * 0 . 4 3 2 . 2 7 1 0 . 5 3 *K.8 * 1 . 4 1 2 . 8 * 1 . 4 1 3 . 4 * 1 . 7 *** 45. 6 * 3.2 39.0 * 3 . 9 41.0*4. 1 5 6 * 3 0 1 6 0 * 3 3 1 5 8 1 3 2 4 9 2 * 9 8 5 1 8 * 1 2 6 510 ±118 -^ 1 . Center for Disease Control, Atlanta, Oeorgla (1972). •••Differences between male and female s t a t i s t i c a l l y s i g n i f i c a n t (P <0.001), - 176 -A l l the b i o c h e m i c a l means were w i t h i n the a c c e p t a b l e or normal ranges of the standards. There were no s i g n i f i c a n t d i f f e r e n c e s between the means f o r males and females w i t h the e x c e p t i o n s o f hemoglobin and hematocrit means which were s i g n i f i c a n t l y d i f f e r e n t a t the p r o b a b i l i t y l e v e l of 0.001 u s i n g the t w o - t a i l T - t e s t , with female means l e s s than male means. To c h a r a c t e r i z e the p o p u l a t i o n as a whole, the percentage d i s t r i b u t i o n s of a d u l t B o i a - F r i a s based on the b i o c h e m i c a l standards p r e v i o u s l y l i s t e d a re i l l u s t r a t e d i n F i g u r e s IV-2 and IV-3, Almost 25% of the p o p u l a t i o n examined had low plasma v i t a m i n A v a l u e s , while about 10% and 20% were d e f i c i e n t and low r e s p e c t i v e l y i n plasma c a r o t e n e . Male-female d i f f e r e n c e s were minor. Plasma v i t a m i n E l e v e l s , when expressed as mg per 100 ml, p l a c e d about 10% of the p o p u l a t i o n examined i n the low category, however, when expressed as mg per g t o t a l l i p i d i n d i c a t e d t h a t a l l were i n the a c c e p t a b l e range. O v e r a l l , the B o i a - F r i a p o p u l a t i o n was designated to be i n b e t t e r v i t a m i n E n u t r i t i o n a l s t a t u s when u s i n g the standards expressed i n terms of t o t a l l i p i d l e v e l s than when u s i n g the standards expressed i n terms of plasma volume, because of t h e i r g e n e r a l l y low plasma t o t a l l i p i d l e v e l s , as seen i n F i g u r e TVT-3, Again, male-female d i f f e r e n c e s were minor. With r e s p e c t t o plasma c h o l e s t e r o l and plasma t o t a l Ffercent Distribution P p * At > w R 2 P (/I 3 p <: 3 rn wzzzzzzzzzzzm j 13 3-Percent Pl'striWhon t» -n o • S" 2 S 3 P 3 5' m <r (» \ / / / / / / / / / / / / / / / / / / X J |VP X * * * * K l V « » » * "5 5 SI'S. ' * x a. Percent Pistribution e * $ i s 2 Z 2 21 > IN) "2. o J \7ZZZZZZZZZZZZA 1 « » X * X » I A. J> N D O 9 2 • 'I VP YZZX > ti cr (» ~ x 5 5 s » in X y > x v » x | 8 5 s> u 3 P s 3 5 3 Percent Pistribution 8 * 5 8 I 3? 3 P 3 3 2 31 P 3 5' I c ro H <! CD 5 n P I D 3 — • V. 3 Qu P ftreant D i ' s t r i b u + i o n * tt * 8 * $ I i 1 i 1 ' w - ~r 3. -=i S 7ZZA \ Z l IZ) I • L Percent Distribution __2 S * ? » * P 0' <•' 3 -+> I -> R P i o o -l» I 1 1 r r ] • 1 I x x x x V * * ) , > *l Percent Distribution O 8 * 8 i f— V///////////A 1 P o l 1/) • X X » * X | « « X * » 1 3 p „r ''////A n I rt. 'I 1 — \9 IA I X § Percent Distribution c » t ? 5 ^ r 7ZA 1 X) p 3 0 oi ? ! co » r\ V//////////////A 1 I -LI Q x x » V v x x 1 •6 tl-l / l v X B i o o 3" c ] 3 8 a. - 179 -l i p i d s , almost a l l of the subjects were i n the normal or low categories, which are acceptable and not at r i s k . The d i s t r i b u t i o n was si m i l a r for both males and females. F i n a l l y , hemoglobin and hematocrit values placed a small percentage of the population examined i n the de f i c i e n t and low categories at r i s k of iron deficiency anemia. According to hemoglobin standards, 6 and 15% of the subjects were found to be i n the d e f i c i e n t and low categories respectively while according to hematocrit standards only 3 and 97a were found to be i n the d e f i c i e n t and low categories. Considering that female&means were s i g n i f i c a n t l y less than male means for these parameters, and that female standards are also lower than male standards for these parameters, i t i s not surprising that male-female differences i n category d i s t r i b u t i o n were minor. The e f f e c t of age on the various blood parameters i s displayed i n Table IV-16. Plasma cholesterol was the only parameter which gave a s t a t i s t i c a l l y s i g n i f i c a n t c o r r e l a t i o n with age using the Pearson Correlation C o e f f i c i e n t . This r e s u l t supports the use of age related normality standards for cholesterol as outlined i n Table IV-15. Table IV-16 Biochemical P r o f i l e of Adult Boia-Frias According to Age Mean SD Age Groups (years) Parameter Plasma Vitamin A ^ug/100ml) 20 - 29 26.6 t 11.9 30 - 39 25.9 ± 7.8 40 - 49 27.0 ± 8.7 -50 - 59 26.6 ± 6.7 Plasma Carotene, ^ug/100ml) 59.3 t 25.9 71.4 ± 46.3 51.5 * 3 0 . 2 56.4 i 27.9 Flasma Vitamin E (mg/lOOml) 1.10 ± 0.38 1.21 ± 0.32 1.08 ± 0.33 1.18 * O.30 Plasma Vitamin E (mg/g l i p i d ) 2.28 t 0.56 2.39 i 0.49 2.14 i 0.54 2.22 i 0.56 M Hemoglobin P (g/lOOml) Both 15.2 ± 0.6 13.2 ± 1.1 13.5 * 1.3 14.6 + 1.6 12.5 * 1.2 13.1 i 1.6 14.5 * 1.6 12.8 ± 1.6 13.3 * 1.7 15.5 * 0.8 13.7 1.8 14.8 * 1.5 M Hematocrit (%) F Both 46.3 * 1.5 39-7 i 3-6 40.8 i 4.1 45.0 i 3-5 38.2 i 3-9 40.1 1 4.8 45.7 t 3 . 4 39.4 * 3.9 41.3 1 4 .7 46.0 ± 3 .2 40.2 ± 5 . 0 43.? * 4 . 8 * Plasma Cholesterol (mg/lOOml) 155 * 32 157 * 35 156 * 28 173 * 30 Plasma Total Lipids (mg/lOOml) 486 - 116 511 * 133 509 ± 107 537 * 101 00 o 'Correlation with age s t a t i s t i c a l l y significant ( P< 0 . 0 5 ) . - 181 -CHAPTER V DISCUSSION Chapter Outline Section Page A. Boia-Fria N u t r i t i o n a l Status Assessment , 182 1. Dietary Analysis 182 a) Qua l i t a t i v e , 182 b) Quantitative . . 183 2. Anthropometric Assessment 190 3. Biochemical Examination , -j.9 3 a) Plasma Vitamin A and Carotene -^ g^  b) Plasma Vitamin E 194 c) Plasma Cholesterol and Plasma Total L i p i d s 201 d) Hematology 201 4. Co r r e l a t i o n Between Assessment Methods... 204 B. Basic Causes of Mal n u t r i t i o n among Boia-Frias.. 206 C. Corrective Measures 213 - 182 -A . B o i a - F r i a N u t r i t i o n a l S t a t u s Assessment 1. D i e t a r y A n a l y s i s a) Q u a l i t a t i v e The o u t s t a n d i n g q u a l i t a t i v e c h a r a c t e r i s t i c o f the B o i a - F r i a d i e t was i t s monotony and l a c k o f v a r i e t y t y p i c a l l y c o n s i s t i n g o f r i c e , beans.-, c o f f e e w i t h sugar and white b r e a d . Food groups l a c k i n g i n p a r t i c u l a r i n c l u d e d ; d a i r y p r o d u c t s , an imal p r o t e i n sources such as meats and eggs, v e g e t a b l e s and f r u i t s . Even the c e r e a l p r o d u c t s which were r e g u l a r l y consumed, such as p o l i s h e d r i c e , u n e n r i c h e d whi te b r e a d , and m a c a r o n i , were h i g h l y r e f i n e d and t h e r e f o r e o f l e s s n u t r i t i o n a l v a l u e . The h i g h c o n -sumption o f " e m p t y - c a l o r i e " foods such as c o f f e e w i t h sugar , a l c o h o l i c beverages and s o f t d r i n k s was a cause f o r c o n c e r n c o n s i d e r i n g the f i n a n c i a l c o n s t r a i n t on food consumpt ion . The l a c k o f knowledge c o n c e r n i n g m a t e r n a l d i e t a r y needs agrees w i t h the r e s u l t s o f W i t t (1971) i n Sao Paulo where he found t h a t many p u p i l s were unab le to answer the q u e s t i o n s c o n c e r n i n g m a t e r n a l d i e t a r y b e l i e f s and h a b i t s . B r e a s t f e e d i n g p r a c t i c e s were to be commended. Most mothers c o n t i n u e d b r e a s t f e e d i n g beyond one year i n c o n t r a s t to the f i n d i n g s o f numerous o t h e r B r a z i l i a n surveys (Santos 1976; Rosenburg 1973; T r i g o e t a l 1978; U l l o a 1973; P u f f e r and^Serrano 1973; Shr impton 1975; ICNND 1965) . Shrimpton (1975) reve iwed a s tudy which - 183 -r e p o r t e d b r e a s t f e e d i n g of s h o r t d u r a t i o n i n a poor area of R i b e i r a o Preto and y e t r e p o r t e d encouraging r e s u l t s i n p r o -l o n g i n g b r e a s t f e e d i n g by e d u c a t i o n a l means. Perhaps t h i s was the reason f o r the e x c e l l e n t b r e a s t f e e d i n g p r a c t i c e s observed i n the present study. Weaning foods l e f t room f o r improvement however. The f i r s t foods i n t r o d u c e d to i n f a n t s w i t h i n 2 weeks of d e l i v e r y were of low n u t r i t i o n a l v a l u e i n c l u d i n g : s t a r c h y g r u e l s , made from corn s t a r c h , arrow r o o t f l o u r , cassava f l o u r , and r i c e f l o u r ; sugar-water; herb t e a ; c o f f e e w i t h sugar; and s o f t d r i n k s . b) Q u a n t i t a t i v e With the l i m i t a t i o n s of the 24 hour d i e t a r y r e c a l l i n mind, the q u a n t i t a t i v e r e s u l t s w i l l be d i s c u s s e d . In ; g e n e r a l , average d a i l y i n t a k e s of most n u t r i e n t s were be-low the requirement. Since the recommended energy i n t a k e s are based on mean requirements and s i n c e man appears to eat food a c c o r d -ing to h i s energy needs, p r o v i d i n g t h e r e are no c o n s t r a i n t s to the food i n t a k e , the 24-hour r e c a l l r e s u l t s should be d i r e c t l y comparable wi t h the requirements to i n d i c a t e the adequacy of the q u a n t i t y of food consumed by the i n d i v i d u a l s of the p o p u l a t i o n . However, the wide range of c a l o r i e i n t a k e s (310 - 507 5 k c a l ) suggests t h a t t h e r e maybe f a c t o r ( s ) i n f l u e n c i n g adequate food i n t a k e such as f i n a n c e s , as d i s -cussed l a t e r . - 184 -T h e r e f o r e , t o b e t t e r p o r t r a y the d i s t r i b u t i o n o f c a l o r i c i n t a k e , 77.1%, 65.7%, and 5.7% o f the males and 98.8%, 87.8%, and 23.2% o f the females had i n t a k e s l e s s than the FAO s tandard (Beaton and Patwardhan 1976), two-t h i r d s o f the FAO s t a n d a r d and o n e - t h i r d o f the FAO s t a n d a r d , r e s p e c t i v e l y . The amount o f food consumed appears to be low f o r the m a j o r i t y of B o i a - F r i a s b o t h male and female , w i t h the female d i e t a p p a r e n t l y l e s s adequate than t h a t of the m a l e . The apparent s e v e r i t y o f the d e f i c i e n c y may be reduced i f one c o n s i d e r s t h a t the 24 -hour r e c a l l method may s l i g h t l y u n d e r e s t i m a t e c a l o r i e i n t a k e ( S c h a e f f e r 1966) and t h a t those surveyed may have u n d e r - r e p o r t e d i n the hope t h a t r e l i e f may have been f o r t h c o m i n g (Rey 1962). W i t h r e g a r d to the o t h e r n u t r i e n t s , comparing average i n t a k e s o f the p o p u l a t i o n w i t h recommended i n t a k e s i s a v e r y c r u d e i n d i c a t i o n o f the p o s s i b l e s i t u a t i o n s i n c e the recommended i n t a k e s a r e based on average r e q u i r e m e n t s p l u s two s tandard d e v i a t i o n s and a l s o i n d i v i d u a l s o f t e n v a r y c o n s i d e r a b l y i n the n u t r i t i o n a l q u a l i t y o f the d i e t they consume. To c o r r e c t f o r the e f f e c t o f ad d in g two s t a n d a r d d e v i a t i o n s , t w o - t h i r d s o f the recommended i n t a k e was c o n -s i d e r e d the d e f i c i e n c y l e v e l , and f o r p r o t e i n and a l l v i t a m i n s and m i n e r a l s , g r e a t e r than h a l f o f the B o i a - F r i a p o p u l a t i o n was l e s s than t h i s . l e v e l . However, does t h i s i n d i c a t e t h a t the n u t r i e n t b a l a n c e was poor or r a t h e r t h a t j u s t the i n t a k e l e v e l was poor? F o r b o t h males and f e m al e s , the p e r c e n t a g e o f peop le d e f i c i e n t - 185 -in 5 vitamins and minerals was greater than the percentage de f i c i e n t i n calories which indicates that the qual i t y of their diet i s also poor and needs to be improved. Protein qu a l i t y appears adequate at least for adults. A greater percentage of people were de f i c i e n t i n calories than i n protein and the amino acid patterns of r i c e or white bread • and beans tend to complement one another, the f i r s t l i m i t i n g amino acid i n white bread and r i c e being lysine and i n legumes being methionine. In addition to meeting a s p e c i f i c needs for tissue replacement for\adults, dietary proteins also ultimately serve as a source of energy. When energy intake i s severely limited, dietary proteins and tissue proteins are catabolized as energy sources and the subject enters a period of net protein loss from the body. When both protein and energy intakes are low i n terms of the suggested requirements there would appear to be limi t e d benefit either i n increasing energy intake without ensuring adequate protein or i n increasing protein without ensuring adequate energy intakes (Beaton and Patwardhan 1976).. According to Shrimpton (1975), the B r a z i l i a n r i c e and bean diet would meet both protein and energy requirements i f i t were eaten i n suff-i c i e n t quantity. Of the nutrients analyzed, most severely lacking were calcium, vitamin A, r i b o f l a v i n , n i a c i n and vitamin C for the general population and iron as well for females. In comparison with the l i t e r a t u r e review results as shown i n Table I I - 3 , - 186 -s i m i l a r v i t a m i n s and m i n e r a l s were found d e f i c i e n t espec-i a l l y c a lcium, v i t a m i n A, and r i b o f l a v i n . P a t r i c k and Simoes (1971) studying the d i e t s of 23 randomly s e l e c t e d f a m i l i e s i n C r i s t a l i n a , Goias, B r a z i l , r e p o r t e d t h a t a d i e t c o n s i s t i n g b a s i c a l l y of r i c e , sugar, d r y beans and c o f f e e was most d e f i c i e n t i n c a l c i u m , r i b o f l a v i n , v i t a m i n A, and a s c o r b i c - a c i d . S i m i l a r l y , Miguel and Bon (1974), surveying towns i n the south of the s t a t e o f Sao.Paulo, found t h a t a c c o r d i n g to FAO recommendations, i n t a k e s of n e a r l y a l l n u t r i e n t s were d e f i c i e n t , e s p e c i a l l y c a l c i u m , and v i t a m i n s A and C and o r i b o f l a v i n . D e f i c i e n c i e s were not as severe as observed i n t h i s study however. Roncada (1974) a l s o r e p o r t e d v i t a m i n A d e f i c i e n c i e s i n towns of southern Sao Paulo s t a t e . He observed t h a t j u s t over 3 0% of the v i t a m i n A a c t i v i t y was from the a c t u a l v i t a m i n and almost 50% fromy^-carotene. Wilson e t a l (1977) d i s c o v e r e d t h a t i n 11 communities i n the s t a t e of Sao Paulo more than 50% of the f a m i l i e s s t u d i e d had a r i b o f l a v i n i n t a k e l e s s than 60%.of t h a t recommended. Thus, the same n u t r i e n t s were r e p e a t e d l y r e p o r t e d as the most l a c k i n g i n the d i e t which i s not s u r p r i s i n g c o n s i d e r i n g the s i m p l i c i t y and i n v a r i a b i l i t y of the d i e t . Yet, the g e n e r a l n i a c i n d e f i c i e n c y and the i r o n d e f i c i e n c y i n females were not observed by other r e s e a r c h e r s , and d e f i c i e n c i e s t h a t were observed were never as severe as found i n t h i s study. A r e c e n t nationwide food consumption survey i n B r a z i l ( S e c r e t a r i a de Planejamento da P r e s i d e n c i a - 187 -da Republiea 1977) w i t h consumption based on the d i f f e r -ence between food a c q u i r e d and food l e f t - o v e r s as determined by weighing, r e p o r t e d no severe d e f i c i e n c i e s i n any nut-r i e n t , and o n l y a m i l d d e f i c i e n c y i n v i t a m i n A i n t a k e i n the s t a t e of Sao Paulo. T h i s b r i n g s one t o q u e s t i o n the d i e t a r y inadequacies of the p o o r e s t segment ( B o i a - F r i a s ) of the p o p u l a t i o n i n the s t a t e of Sao Paulo. In s p i t e o f the l i m i t a t i o n s of the 24-hour r e c a l l method f o r the quan-. t i t a t i v e e s t i m a t i o n of food i n t a k e (L.inusson e t a l . 1974) , i t appears t h a t the d i e t a r y i n t a k e of n u t r i e n t s f o r most of the B o i a - F r i a s i n the s t a t e of Sao Paulo i s poor and r e q u i r e s s e r i o u s c o n s i d e r a t i o n s f o r improvement. The n u t r i e n t n i a c i n deserves s p e c i a l mention. The d e s c r i p t i o n of n i a c i n requirements i s s l i g h t l y c omplicated by the f a c t t h a t the e s s e n t i a l amino a c i d tryptophan i n the, i n g e s t e d . p r o t e i n can be, and i s , converted to n i a c i n w i t h -i n the body, 60 mg tryptophan g i v i n g r i s e t o 1 mg n i a c i n . The FAO standard f o r n i a c i n i n t a k e i s expressed i n terms of n i a c i n e q u i v a l e n t s , -the sum of the preformed n i a c i n and the amount of n i a c i n t h a t c o u l d be d e r i v e d from tryptophan. However, s i n c e t h e r e i s a need f o r tryptophan over and above i t s use as a p r e c u r s o r of n i a c i n , and s i n c e the B o i a - F r i a s were consuming o n l y small amounts of meats, eggs or d a i r y products, the i n c l u s i o n of p o t e n t i a l n i a c i n from tryptophan as n i a c i n e q u i v a l e n t s i n the food n u t r i e n t com-p o s i t i o n t a b l e was c o n s i d e r e d i n a p p r o p r i a t e . T h e r e f o r e , f o r those i n d i v i d u a l s with s u r p l u s tryptophan i n t a k e , the - 188 -actual n i a c i n n u t r i t i o n a l status would be better than indicated by the results of Table IV-8. Calorie and nutrient sources according to food groups shown i n Table IV-9 compare well with the results of the survey of urban areas of the state of Sao Paulo by the Secretaria de Planejamento da Presidencia da Republiea (1977) summ-arized i n Table II-7. For comparison purposes, eggs, dairy and animal protein sources must be combined i n one group and the o i l added to beans and r i c e during preparation must be considered i n the legumes and grain products groupings. On a percentage basis, Boia-Frias consumed proportionately more legumes, and less eggs, dairy, and animal protein sources than those i n urban areas of the state of Sao Paulo. Thus, almost h a l f of the calcium consumed by those i n urban areas came from dairy products while for Boia-Frias, only one-tenth came from this source. Although the f r u i t consumption of both groups was low, more than one-third of the vitamin C consumed by those i n urban areas came from f r u i t s while for Boia-Frias, only one-tenth came from f r u i t s . Another notable difference was that those i n urban areas of the state of Sao Paulo consumed about 157o of t h e i r calories as refined sugar while Boia-Frias consumed only about 570 of t h e i r calories i n this form. Good food sources of the nutrients most severely lacking that are l o c a l l y available are shown i n Table V - l . In general, the foods which are lacking are the milk products, meats, f i s h , eggs, poultry, non-refined grain products, and f r u i t s and vegetables r i c h i n vitamins A and C. P a r t i c u l a r - 189 -T a b l e V - l : Good Food Sources of N u t r i e n t s Most D e f i c i e n t N u t r i e n t Good Food Sources c a l c i u m v i t a m i n A r i b o f l a v i n n x a c i n v i t a m i n C i r o n m i l k and m i l k p r o d u c t s , s a r d i n e s , c o l l a r d s , s p i n a c h . l i v e r , c a r r o t s , pumpkin, c o l l a r d s , spinach, r u c u l a , w i l d c h i c o r y , papaya, yams. l i v e r , kidney, h e a r t , sausages, p o u l t r y , f i s h , c r i s p animal f a t , eggs, m i l k , soybean, avocado, sp i n a c h . brown r i c e , whole g r a i n s , peanuts, l i v e r , meat, p o u l t r y , f i s h , avocado, c a r r o t s , s p i n a c h , c o l l a r d s . c i t r u s f r u i t s , cashew f r u i t , tomatoes, c o l l a r d s , s p i n a c h , cabbage, p o t a t o e s , o k r a . l i v e r , kidney, heart, meat, p o u l t r y , f i s h , c r i s p animal f a t , legumes, (soybean i r o n has h i g h b i o a v a i l a b i l i t y ) , eggs, c o l l a r d s , w i l d c h i c o r y , yams. - 190 -food items, the n u t r i t i o n a l v a l u e of which may be overlooked, i n c l u d e : v a r i e t i e s o f green l e a f y v e g e t a b l e s , pumpkin, papaya, soybean, avocado, peanuts, casher f r u i t and yams. N u t r i t i o n e d u c a t i o n to promote the consumption of the foods of Table V - l would be d e s i r a b l e , keeping i n mind the f i n a n c i a l c o n s t r a i n t s of these people, and t h e i r t r a d i t i o n a l d i e t a r y b e l i e f s and taboos. 2. Anthropometric Assessment On the b a s i s o f the d i e t a r y 24-hour r e c a l l data, the women appeared to be more d e f i c i e n t i n c a l o r i c i n t a k e than the men. Acc o r d i n g to anthropometric data, the male B o i a - F r i a s tended toward underweight w h i l e some female Boia-F r i a s tended toward overweight w i t h 22.370 of them above the 1207o of standard v a l u e mark f o r weight f o r h e i g h t and 24. TU of them above the 120% of standard v a l u e mark f o r t r i c e p s s k i n - f o l d . A c c o r d i n g to J e l l i f f e (1966), high body weights f o r h e i g h t s are a s s o c i a t e d w i t h e x c e s s i v e c a l o r i c i n t a k e s , together w i t h i n s u f f i c i e n t e x e r c i s e . T r i c e p s s k i n - f o l d , measuring t h i c k n e s s o f subcutaneous f a t , a l s o i n d i c a t e s c a l o r i e r e s e r v e s . The f o l l o w i n g f a c t o r s h elp to e x p l a i n these r e s u l t s : the men were much more p h y s i c a l l y a c t i v e than the women, working long hours at p h y s i c a l l a b o r w h i l e the women remained at home, t h e i r sedentary l i f e s t y l e pro-pagated by freq u e n t pregnancies, hot t r o p i c a l weather, i s o l a t i o n and l a c k of t r a n s p o r t a t i o n (the B r a z i l i a n c u l t u r e c o n s i d e r s i t unacceptable f o r women to r i d e b i c y c l e s ) ; - 191 -and m e t a b o l i c d i f f e r e n c e s , or m a l f u n c t i o n s . At the o t h e r extreme, however, 6% of the women were below the 8 0% of standard v a l u e mark f o r weight f o r h e i g h t and 54% of them below the 80% of standard v a l u e mark f o r t r i c e p s s k i n - f o l d . Of the men, 10% were below the 8 0% of standard v a l u e mark f o r weight f o r height and 95% were below the 8 0% of standard v a l u e mark f o r t r i c e p s s k i n - f o l d . J e l l i f f e (1966) w r i t e s t h a t low body weights f o r h e i g h t s are p r i n c i p a l l y a r e f l e c t i o n of body t h i n n e s s due to subnormal amounts of subcutaneous f a t and muscle, the r e s u l t e i t h e r of poor development or t i s s u e wasting or a combination of the two. Greater than 95% of the males and females s t u d i e d were w i t h i n the 8 0 t o 1^2 0 % of standard v a l u e marks f o r mid-arm-muscle c i r c u m f e r e n c e . T h e r e f o r e , one can conclude t h a t the body t h i n n e s s r e f l e c t e d by low body weights f o r h e i g h t s was due p r i m a r i l y to subnormal amounts of subcutaneous f a t r a t h e r than muscle. Since p r o t e i n inadequacy i s best r e f l e c t e d by t h i n musculature ( J e l l i f f e 1966), these r e s u l t s are i n agreement wi t h d i e t a r y data i n d i c a t i n g low c a l o r i c i n t a k e but an adequate p r o t e i n -c a l o r i e balance. The anthropometric r e s u l t s are i n t e r n a l l y coherent as i n d i c a t e d by the s i g n i f i c a n t c o r r e l a t i o n found, between t r i c e p s s k i n - f o l d and weight f o r h e i g h t , both expressed as percentages of standard v a l u e s , a t the p r o b a b i l i t y l e v e l of 0.001 u s i n g the o n e - t a i l e d Pearson C o r r e l a t i o n C o e f f i c i e n t T e s t . - 192 -Comparing the r e s u l t s of t h i s study w i t h the ICNND (1965) survey r e s u l t s from Northeast B r a z i l , i n d i c a t e s t h a t the B o i a - F r i a p o p u l a t i o n s t u d i e d was s l i g h t l y o l d e r , t a l l e r and he a v i e r i n a b s o l u t e terms and i n terms of per-cent of standard weight than t h e i r n o r t h e a s t e r n c o u n t e r -p a r t s . The ICNND (1965) survey found t h a t both men and women above age 45 e x h i b i t e d a steady d e c l i n e i n body weight, c o n t r a s t e d w i t h c o n t i n u i n g i n c r e a s e s i n body weight i n North American females through age 6 5 and i n males through age 55. T h i s study r e v e a l e d t h a t the B o i a - F r i a males f o l l o w e d the n o r t h e a s t e r n B r a z i l p a t t e r n w i t h weight peaking a t 35 years w h i l e the B o i a - F r i a females from 2 0 t o 4 9 years of age maintained a p l a t e a u of overweight even by North American standards which dropped s h a r p l y a t the 50 t o 59 years age group t o even l e s s than the Northeast B r a z i l l e v e l . Comparison of the B o i a - F r i a r e s u l t s w i t h the r e c e n t S e c r e t a r i a de Planejamento da P r e s i d e n c i a da Republica (1977) anthropometric survey r e s u l t s from Sao Paulo s t a t e showsthat i n g e n e r a l the B o i a - F r i a p o p u l a t i o n was s l i g h t l y s h o r t e r , l i g h t e r i n weight, and males were of sm a l l e r arm cir c u m f e r e n c e than t h e i r c o u n t e r p a r t s of the g e n e r a l pop-u l a t i o n of the s t a t e of SSo Paulo. A c o n t i n u i n g i n c r e a s e i n body weight through age 45 i n males and through age 55 i n females was e x h i b i t e d by the g e n e r a l Sao Paulo s t a t e p o p u l a t i o n . - 193 -3. Biochemical Examination Biochemical studies are espe c i a l l y valuable at the pathogenic but p r e - c l i n i c a l stage where they provide a basis for diagnosis of early malnutrition including ex-hausted reserves and physiological and metabolic a l t e r a -tions. For the most part, biochemical measurements re-present the most objective assessment of the n u t r i t i o n a l status of an i n d i v i d u a l . a) Plasma Vitamin A and Carotene Almost 257. of the population examined had low plasma vitamin A values which correlates with a prolonged low intake of vitamin A and r e f l e c t s also a depleted l i v e r store. This i s expected considering the, population mean vitamin A intake of 203.2 i 217.7 g. Hodges and Kolder (1971) noted that i n vitamin A depleted adult men, when the intake of r e t i n o l was increased to 150Jj-g per day, the serum r e t i n o l l e v e l was s t i l l less than 10y6tg per 100 ml; when the intake was 300/^-g per day, the serum l e v e l was about 19jUg per 100 ml. According to the S h e f f i e l d study c i t e d by Arroyave (1971), intakes of about 390y^g per day of r e t i n o l would place normal subjects i n a r i s k s i t -uation where plasma levels are i n the range of 20 to Jj.g per 100 ml and l i v e r reserves are uncertain. It i s assumed that intakes below th i s l e v e l would eventually lead to depletion of hepatic reserves. About 107. and 207. of the population examined were def i c i e n t and low respectively i n plasma carotene, r e f l e c t -- 194 -i n g r e c e n t low d i e t a r y i n t a k e s oJr c a r o t e n e - r i c h green, orange and y e l l o w v e g e t a b l e s and f r u i t s , as p r e d i c t e d by the 24-hour r e c a l l and d i e t a r y survey r e s u l t s . When low plasma y9-carotene l e v e l s are found i n a s s o c i a t i o n w i t h low plasma v i t a m i n A l e v e l s , the evidence f o r inadequate vitamin.A n u t r i t u r e i s st r o n g . Other B r a z i l i a n surveys have a l s o found low plasma v i t a m i n A and carotene l e v e l s . The ICNND (1975) survey of n o r t h e a s t B r a z i l r e p o r t e d 9% of serum v i t a m i n A l e v e l s d e f i c i e n t and another 24% low. Roncada (1972) concluded t h a t v i t a m i n A d e f i c i e n c y was a problem i n the s t a t e of Sao Paulo, f i n d i n g ; between 6 and 11% w i t h plasma vitamin"; & d e f i c i e n t , between 4 and 19% wit h plasma v i t a m i n A l e v e l s low> between 22 and 79% wit h plasma carotene l e v e l s d e f i c i e n t , and between 19 and 57% wit h plasma carotene l e v e l s low, as he surveyed 3 towns i n the south of the s t a t e , b) Plasma V i t a m i n E Mean plasma v i t a m i n E l e v e l of the B o i a - F r i a pop-u l a t i o n was 1.14 + 0.33 mg per 100 ml. T h i s compares f a v o r a b l y w i t h the r e s u l t s of ot h e r n u t r i t i o n surveys measuring v i t a m i n E i n plasma i n numerous p a r t s of the world which are summarized i n Table II--l,of the Review of L i t e r a t u r e . The v i t a m i n E s t a t u s of v a r i o u s B r a z i l i a n p o p u l a t i o n s has y e t t o be evaluated, and r e p o r t e d i n the l i t e r a t u r e . Plasma v i t a m i n E l e v e l s , when expressed as mg per 100 ml, p l a c e d about 10% of the p o p u l a t i o n examined i n - 195 -the low category, however when expressed as mg per g t o t a l l i p i d , i n d i c a t e d t h a t a l l were i n the a c c e p t a b l e range. T h i s i s worthy of note c o n s i d e r i n g t h a t the mean o C-tocopherol i n t a k e of the p o p u l a t i o n was 5.51 + -3.30 mg and 46.8% and 69.6% of the p o p u l a t i o n consumed l e s s than t w o - t h i r d s of the U.S. and Canadian recommended i n t a k e s r e s p e c t i v e l y a c c o r d i n g to the d i e t a r y i n t a k e data by 24-hour r e c a l l , which a d m i t t e d l y may g i v e a s l i g h t l y low estimate o f a c t u a l consumption. The mean <x-tocopherol i n t a k e , however, compared f a v o r a b l y w i t h the r e s u l t s of o t h e r r e s e a r c h e r s . For example, B u n n e l l e t a l (1965) r e p o r t e d a mean of 7.4 mg; B i e r i and E v a r t s (1972), 9.0 mg; Thompson e t a l (1973), 6.4 mg; Smith e t a l (1971), 5 mg per 2,500 k c a l ; and W i t t i n g and Lee (1975a), 7.5 mg. On a 2,500 k c a l b a s i s ,the c a l c u l a t e d mean oc-tocopherol i n t a k e of the B o i a - F r i a p o p u l a t i o n would be 9.18 mg. A c c o r d i n g to Horwitt e t a l (1961), the o<-tocopherol requirements of a d u l t man may v a r y from a minimum of l e s s than 5 mg per day to a maximum of about 3 0 mg per day, i n c r e a s i n g w i t h i n c r e a s i n g amounts of u n s a t u r a t e d l i p i d s i n the d i e t . The B o i a - F r i a d i e t was v e r y h i g h i n u n s a t u r -a t e d l i p i d s , w i t h d i e t a r y f a t c o n s i s t i n g of 21.9% s a t u r -ated f a t t y a c i d s , 28.8% o l e i c a c i d , and 41.9% l i n o l e i c a c i d . A c c o r d i n g to Horwitt (1974), the d.- <* - t o c o p h e r o l e q u i v a l e n t (mg) r e q u i r e d per day i s 0.25 (% PUFA + g PUFA) + 4 - 196 -'ii I I where %PUFA r e p r e s e n t s the "percentage of p o l y u n s a t u r a t e s i n the t o t a l l i p i d s i n the d i e t , "ii 11" and g PUFA r e p r e s e n t s the t o t a l amount of p o l y u n s a t u r a t e d f a t t y a c i d s consumed per day. On the b a s i s of t h i s formula the B o i a - F r i a p o p u l a t i o n would r e q u i r e 23.86 mg d- <* - t o c o p h e r o l e q u i v a l e n t or 19.88mg c* - t o c o p h e r o l per day, because of t h e i r h i g h d i e t a r y p o l y u n s a t u r a t e d l i p i d c o n t e n t . Yet, none of the B o i a - F r i a p o p u l a t i o n was c l a s s e d as d e f i c i e n t i n terms of plasma vitamin" E l e v e l . W i t t i n g and Lee (1975a) l i k e w i s e r e p o r t e d c o n t r a d i c t o r y r e s u l t s of t h i s type i n a study of u n i v e r -s i t y students i n Texas. Using the same formula of Horwitt (1974), t h i s p o p u l a t i o n would r e q u i r e 16.78 mg d-oC-toco-p h e r o l e q u i v a l e n t or 13.98 mg o c-tocopherol per day. T h e i r a c t u a l i n t a k e was 7.5 ± 3.5 mg o<.-tocopherol per day and t h e i r plasma v i t a m i n E l e v e l s were a l l i n the a c c e p t a b l e range w i t h a mean of 1.09 * 0.25 mg per 100 ml. One p o s s i b l e e x p l a n a t i o n f o r t h i s apparent c o n t r a -d i c t i o n would be t h a t these p o p u l a t i o n s had r e c e n t l y i n -c r e a s e d the percentage of p o l y u n s a t u r a t e d f a t t y a c i d s i n t h e i r d i e t and t h a t t h e i r depot f a t l i p i d had not y e t achieved e q u i l i b r i u m w i t h d i e t a r y f a t i n terms of i t s p o l y u n s a t u r a t e d f a t t y a c i d content, r e s u l t i n g i n a l e s s e r than "normal" requirement i n terms of p o l y u n s a t u r a t e d f a t t y a c i d i n t a k e . T h i s may have been the s i t u a t i o n f o r the B o i a - F r i a p o p u l a t i o n , s w i t c h i n g from the use of l a r d to soybean o i l which has r e c e n t l y been adopted as common - 197 -d i e t a r y f a t i n B r a z i l . W i t t i n g and Lee (1975b) suggested u s i n g 0.4 mg d - oc - t o c o p h e r o l per g l i n o l e a t e i n 100 g adipose t i s s u e f a t t y a c i d s i n the g e n e r a l p o p u l a t i o n as the formula f o r s e t t i n g the recommended d i e t a r y allowance f o r v i t a m i n E. Assuming adipose t i s s u e l i n o l e a t e percentag approximating the l i n o l e a t e percentage of l a r d , and adopt-ing t h i s formula , the recommended d a i l y i n t a k e of v i t a m i n E f o r the B o i a - F r i a p o p u l a t i o n would be 4.0 mg id - <x - t o c o p h e r o l per day. Another f a c t o r which may help to e x p l a i n t h i s app-ar e n t c o n t r a d i c t i o n f o r the B o i a - F r i a p o p u l a t i o n i s t h a t a t the time of the survey, the predominant source of d i e t a r f a t f o r the B o i a - F r i a p o p u l a t i o n was soybean o i l , r i c h i n Y - t o c o p h e r o l which c o u l d c o n t r i b u t e s u b s t a n t i a l l y t o the t o t a l v i t a m i n E a c t i v i t y of t h e i r d i e t , perhaps even more than t h a t allowed f o r by the 1.2 f a c t o r f o r c o n v e r t i n g o(-tocopherol to ot- t o c o p h e r o l e q u i v a l e n t . The use of.plasma v i t a m i n E l e v e l s alone f o r n u t r i -t i o n a l s t a t u s e v a l u a t i o n i s g e n e r a l l y c o n s i d e r e d inadequate s i n c e p h y s i o l o g i c a l , pharmocological, g e n e t i c , and d i e t a r y f a c t o r s t h a t change the l e v e l of plasma l i p i d s produce a concomitant change i n the l e v e l o f ^ - t o c o p h e r o l i n the plasma. For t h i s reason, Horwitt et a l (1972) ad v i s e d to express plasma v i t a m i n E l e v e l s i n terms of mg per g t o t a l l i p i d f o r n u t r i t i o n a l s t a t u s e v a l u a t i o n . T h i s concept may a l s o be a p p l i c a b l e to other f a t - s o l u b l e v i t a m i n s , such as v i t a m i n A. \ - 198 -Plasma v i t a m i n E d i s t r i b u t i o n s i n terms of mg per 100 ml and mg per g t o t a l l i p i d a r e presented i n Ta b l e s V - 2 andV-3for B o i a - F r i a s and a l s o f o r a group of 30 B r a z i l i a n s u b j e c t s w i t h h i g h plasma l i p i d l e v e l s (hyperlipemics) f o r comparison. The b i o c h e m i c a l l i p i d p r o f i l e of these p a t i e n t s i s i n c l u d e d i n Tab l e V-4. For plasma v i t a m i n E (mg per 100 m l ) , plasma c h o l e s t e r o l , and plasma t o t a l l i p i d , the means f o r the hy p e r l i p e m i c s u b j e c t s were almost twice the means f o r the B o i a - F r i a s u b j e c t s . Yet, means of plasma v i t a m i n E, when expressed as mg per g l i p i d f o r both groups, were almost i d e n t i c a l . T h i s supports the view t h a t plasma v i t a m i n E expressed i n terms of mg per g l i p i d i s a b e t t e r i n d i c a t o r of n u t r i t i o n a l s t a t u s than when ex-pressed i n terms of mg per 100 ml of plasma or serum. Ng and Chong (1975) found s i m i l a r r e s u l t s when comparing 63 h e a l t h y Malaysians w i t h 63 h y p e r l i p o p r o t e i n a e m i c p a t i e n t s . A l s o , to help c l a r i f y the i s s u e r e g a r d i n g the method of e x p r e s s i o n of plasma v i t a m i n E, i n f a n t blood samples were c o l l e c t e d from newborns of low socioeconomic c l a s s a t the l o c a l h o s p i t a l and analyzed f o r v i t a m i n E and t o t a l l i p i d s . Based on c o r d b l o o d samples, the mean plasma v i t a m i n E l e v e l f o r 9 babies was 0.336 mg per 100 ml, comparing w e l l w i t h the l i t e r a t u r e v a l u e s of T a b l e IT-2. The 5 blood samples lowest i n v i t a m i n E were a l s o a n a l y z e d f o r t o t a l l i p i d s . For these 5 babies, the mean plasma v i t a m i n E l e v e l was 0.183 mg per 100 ml, wit h a l l 5 v a l u e s i n the d e f i c i e n t range. However, when expressed i n terms - 199 -Table V-2 : D i s t r i b u t i o n o f Plasma V i t a m i n E L e v e l s of B o i a - F r i a and Hyperlipemia S u b j e c t s Plasma V i t a m i n E B o i a - F r i a s H y p e r l i p e m ics (mg/lOOml) No. . % No. -0.50. - 0.59 3 2.4 •" 0.60 - 0.69 7 5.6 0.70 - 0.79 6 4.8 0.80 - 0.89 13 10.5 0.90 - 0.99 16 12.9 1. 00 - 1. 09 18 14 .5 1.10 - 1.19 13 10.5 1.20 - 1.29 14 11.3 1.30 - 1.39 10 8.1 1 3.3 1.40 - 1.49 4 3.2 3 10.0 1.50 - 1.59 7 5.6 1 3.3 1.60 - 1.69 4 3.2 4 13.3 1.70 - 1.79 5 4.0 3 10.0 1.80 - 1.8 9 1 0.8 1 3.3 1.90 - 1.99 1 0.8 2 6.7 2.00 - 2.09 1 0.8 3 10.0 2.10 - 2.19 0 0.0 0 0.0 2.20 - 2.2 9 1 0.8 3 10.0 2.30 - 2.39 3 10.0 2.40 - 2.49 1 3.3 2.50 - 2 .59 0 0.0 2.60 - 2.69 2 6.7 2.70 - 2.79 0 0.0 2.80 - 2.89 1 3.3 2.90 - 2.99 0 0.0 3.00 - 3.49 1 3.3 3 .50 - 3.99 0 0.0 4.00 - 4.49 1 3.3 - 200 -Table V-3 : Plasma V i t a m i n E D i s t r i b u t i o n i n Terms of T o t a l L i p i d L e v e l s f o r B o i a - F r i a and Hyperlipemic S u b j e c t s Plasma V i t a m i n E L e v e l B o i a - F r i a s H y p e r l i p e m i c s (mg/g • l i p i d ) No. % No. o. "5 1.00 - 1.24 2 1.6 0 0.0 1.25 - 1.49 4 3.2 1 3.3 1.50 - 1.74 10 8.1 3 10. 0 1.75 - 1.99 25 20.2 7 23.3 2.00 - 2.24 30 24.2 7 23.3 2.25 - 2.49 13 10.5 6 20.0 2.50 - 2.74 17 13.7 2 6.7 2.75 - 2.99 12 9.7 2 6.7 3.00 - 3.24 4 3.2 1 3.3 3.25 3.49 . 5 4.0 0 0.0 3 .50 - 3.74 1 0.8 1 3.3 3.75 - 3.99 1 0.8 0 0.0 Tabl e V-4 : B i o c h e m i c a l L i p i d P r o f i l e of Subjec t s w i t h High L i p i d L e v e l s Compared wi t h P r o f i l e of B o i a - F r i a s Parameter Mean * S 'D Hype r l i p e m i c s B o i a - F r i a s Plasma v i t a m i n E (mg/100 ml) 2.08 + 0.11 1.14 + 0.33 Plasma v i t a m i n E (mg/g l i p i d ) 2.21+0.08 2.27+0.53 Plasma c h o l e s t e r o l (mg/100 ml) 272 + 19 158 + 32 Plasma t o t a l l i p i d (mg /100 ml) 947 + 41 510 + 118 Plasma t r i g l y c e r i d e (mg/100 ml) 195 + 1 0 - 201 -of t o t a l l i p i d s , the mean plasma l e v e l was 1.025 mg per g l i p i d , w i t h no v a l u e s i n the d e f i c i e n t range, 3 i n the low range, and 2 i n the a c c e p t a b l e range. S i m i l a r r e s u l t s were r e p o r t e d by Horwitt e t a l (1972). These f i n d i n g s , t h e r e f o r e , c l e a r l y demonstrate the need f o r measuring plasma t o t a l l i p i d l e v e l s as w e l l as plasma v i t a m i n E l e v e l s f o r the assessment of v i t a m i n E s t a t u s i n humans.. V i t a m i n E i s c a r r i e d by the l i p o p r o t e i n s i n the blood and so are the blood l i p i d s . Thus, i n normal i n - • d i v i d u a l s , t h e r e i s a h i g h c o r r e l a t i o n between plasma l i p i d s and plasma t o c o p h e r o l c o n c e n t r a t i o n . Numerous c o r r e l a t i o n s of t h i s type were found i n t h i s study and are l i s t e d i n T ableV-5. Note t h a t plasma v i t a m i n A a l s o gave a h i g h l y s i g n i f i c a n t c o r r e l a t i o n w i t h plasma t o t a l l i p i d s . c) Plasma C h o l e s t e r o l and Plasma T o t a l L i p i d s Most of the B o i a - F r i a s u b j e c t s were i n the a c c e p t -a b l e range and not a t r i s k of coronary h e a r t d i s e a s e w i t h r e s p e c t to plasma c h o l e s t e r o l and t o t a l l i p i d s . The v a l i d -i t y and p r e c i s i o n of the methods was a f f i r m e d by the s i g n -i f i c a n t c o r r e l a t i o n found between plasma c h o l e s t e r o l and plasma t o t a l l i p i d s at the p r o b a b i l i t y l e v e l of 0.001 u s i n g the o n e - t a i l e d Pearson C o r r e l a t i o n C o e f f i c i e n t T e s t . d) Hematology Hemoglobin and hematocrit v a l u e s are u s e f u l i n d i c e s i n the d i a g n o s i s of i r o n d e f i c i e n c y anemia. A c c o r d i n g to standards used by an e x t e n s i v e n u t r i t i o n survey conducted Table V-5 a S i g n i f i c a n t C o r r e l a t i o n s between L e v e l s of F a t - S o l u b l e Vitamins and L i p i d Components i n Plasma Subjec t s V a r i a b l e s C o r r e l a t e d P r o b a b i l i t y L e v e l B o i a - F r i a s V i t a m i n V i t a m i n V i t a m i n V i t a m i n V i t a m i n V i t a m i n (mg/100 ml) (mg/100 ml) (mg/100 ml) (mg/g l i p i d ) (mg/g l i p i d ) (yug/100 ml) T o t a l L i p i d s (mg/lOOml) C h o l e s t e r o l (mg/100 ..ml) V i t a m i n E (mg/g l i p i d ) T o t a l L i p i d s (mg/100 ml) Vita m i n E (mg/100 ml) T o t a l L i p i d s (mg/100 ml) 0. 001 0. 001 0. 001 0. 018 0.001 0. 001 Hype r l i p e m i c s V i t a m i n E (mg/100 ml) : T o t a l L i p i d s (mg/100 ml) 0. 001 V i t a m i n E (mg/100 ml) : C h o l e s t e r o l (mg/100 ml) 0. 001 V i t a m i n E (mg/100 ml) : T r i g l y c e r i d e s (mg/100 ml) 0. 040 V i t a m i n E (mg/100 ml) : V i t a m i n E (mg/g l i p i d ) 0. 001 V i t a m i n E (mg/g l i p i d ) : V i t a m i n E (mg/100 ml) 0. 001 a Using the one-^tailed Pearson C o r r e l a t i o n C o e f f i c i e n t T e s t . - 203 -d u r i n g 1965 t o 1*967 on p o p u l a t i o n s of C e n t r a l America and Panama, (Center f o r Disease C o n t r o l , A t l a n t a , G e o r g i a 1972) hemoglobin l e v e l s were found to be d e f i c i e n t and low i n 6 and 15% of the B o i a - F r i a s u b j e c t s r e s p e c t i v e l y w h i l e hematocrit v a l u e s were found t o be d e f i c i e n t and low i n 3 and 9% of the s u b j e c t s r e s p e c t i v e l y . Since i r o n was one of the most l a c k i n g n u t r i e n t s i n the d i e t , these d e f i c i e n c i e s are not s u r p r i s i n g . P a r a s i t i c i n f e s t a t i o n may a l s o be a c o n t r i b u t o r y cause of the anemia. Other r e s e a r c h e r s have a l s o noted the presence of anemia i n B r a z i l i n c l u d i n g ; ICNND (1965) i n n o r t h e a s t B r a z i l , and S z a r f a r c (1972) i n the towns of P o n t a l do R i b e i r a and Icapara i n the south of the s t a t e of Sao Paulo. The v a l i d i t y and p r e c i s i o n of the hemoglobin and hemat o c r i t methods was a f f i r m e d by the s i g n i f i c a n t c o r r e -l a t i o n found between these parameters a t the p r o b a b i l i t y l e v e l of 0.001 u s i n g the o n e - t a i l e d Pearson C o r r e l a t i o n C o e f f i c i e n t T e s t . - 204 -4. Correlation Between Assessment Methods As presented i n the Review of Literature and i n Figure II-1, the various methods of n u t r i t i o n a l assessment operate at d i f f e r e n t levels of malnutrition, and comple-ment one another. For example, dietary surveys define the s i t u a t i o n i n the prepathogenic stage, i n d i c a t i n g whether conditions conducive to the development of n u t r i t i o n a l problems are present or not. Biochemical methods provide a basis for diagnosis of early malnutrition at the path-ogenic but p r e - c l i n i c a l stage including exhausted reserves and physiological and metabolic a l t e r a t i o n s . Anthropometric studies assess c l i n i c a l malnutrition, though not i n d i c a t i n g whether the problem i s a present one or permanent damage from past d e f i c i e n c i e s . Mortality data assess the problem i n i t s advanced c l i n i c a l stages. The complementary nature of the various assessment methods was demonstrated by the numerous s i g n i f i c a n t correlations r e s u l t i n g between related data obtained by dif f e r e n t assessment methods, as shown i n Table V-6. This study c l e a r l y demonstrated that: according to dietary data, conditions existed which were conducive to the development of n u t r i t i o n a l problems, esp e c i a l l y with respect to calcium, vitamin A, r i b o f l a v i n , n i a c i n , vitamin C, and iron , of the nutrients tested and with respect to quantit-ative intake of food (kcal); according to biochemical data, the presence of early malnutrition, p r e - c l i n i c a l i n nature was confirmed for a portion of the population Table V - 6 D i f f e r e n t N u t r i t i o n a l Assessment M e t h o d s - S i g n i f i c a n t C o r r e l a t i o n s V a r i a b l e s C o r r e l a t e d P r o b a b i l i t y L e v e l D i e t a r y i r o n intake/requirement D i e t a r y i r o n intake/requirement D i e t a r y v i t a m i n A i n t a k e Cug) D i e t a r y ^ - t o c o p h e r o l i n t a k e (mg) D i e t a r y <x-tocopherol i n t a k e (mg) D i e t a r y s a t u r a t e d f a t t y a c i d i n t a k e (g) D i e t a r y o l e i c a c i d i n t a k e (g) Bioche m i c a l c h o l e s t e r o l c a t e g o r y B i o c h e m i c a l c h o l e s t e r o l c a t e g o r y B i o c h e m i c a l c h o l e s t e r o l (mg/lOOml) Bio c h e m i c a l t o t a l l i p i d (mg/lOOml) Bioche m i c a l t o t a l l i p i d (mg/lOOml) Biochemical hemoglobin Biochemical hematocrit Biochemical Biochemical Biochemical Biochemical Biochemical (g) (%) v i t a m i n A ^txg/100ml) v i t a m i n E (mg/100ml) v i t a m i n E (mg/g l i p i d ) c h o l e s t e r o l (mg/lOOml) c h o l e s t e r o l (mg/lOOml) Anthropometric weight (% Anthropometric s k i n - f o l d Anthropometric weight (% Anthropometric weight (% Anthropometric s k i n - f o l d of (% of of (% std) of s t d std) std) of std a a a a a a a b ,b a. , a 0, 0, 0. 0, 0, 0. 0, 0, 0. 0. 0 0. 001 001 001 003 021 033 050 011 048 005 001 047 a Using the o n e - t a i l e d Pearson C o r r e l a t i o n C o e f f i c i e n t T e s t b Using the o n e - t a i l e d Spearman C o r r e l a t i o n C o e f f i c i e n t Test - 206 -with respect to the nutrients vitamin A, carotene, and iron; according to anthropometric data, the presence of some degree of c l i n i c a l undernutrition or f i r s t degree malnutrition among men and women and also some degree of obesity among women was indicated; and according;, to c h i l d mortality data presented i n the Review of Lite r a t u r e for Ribeirao Preto and gathered for t h i s Boia-Fria population by Desai et a l (.1978a) , the presence of some advanced c l i n i c a l malnutrition was evidenced among children. The severe d e f i c i e n c i e s suggested by the 24-hour r e c a l l data may have been s l i g h t l y exaggerated through misreporting; may have yet to display themselves pre-c l i n i c a l l y or c l i n i c a l l y ; or may remain v e i l e d through adaptation mechanisms as described i n the Review of Lit e r a t u r e . B-. Basic Causes of Malnutrition among Boia-Frias One of the key ecological factors that appears to be i n d i r e c t l y responsible for the malnutrition observed among the Boia-Frias was the recent urbanization of these migrant workers. According to Sai (1976), accompanying c i t y l i f e i s : the population pressure, which discourages the growth of vegetable gardens; the society pressure, which encourages the buying of cer t a i n r e a d i l y a v a i l a b l e and highly "prestigeous" products, such as t e l e v i s i o n s , radios, convenience foods, a l c o h o l i c beverages and c i g -arettes; and the f i n a n c i a l pressure of a money-based economy, which compels the choice of the simplest and r- 207 T cheapest of s t a p l e foods. Among the B o i a - F r i a p o p u l a t i o n , home growing of f r u i t s and v e g e t a b l e s or domestic prod-u c t i o n of animal foods such as eggs and c h i c k e n was not common. S i x t y - f o u r percent o f the f a m i l i e s had r a d i o s and 44% t e l e v i s i o n s . Seventy percent of the men and 30% of the women smoked, wit h h a l f o f them consuming from one to two packages of c i g a r e t t e s per day. D r i n k i n g was common among B o i a - F r i a s , and a l c o h o l i s m was a s e r i o u s problem e s p e c i a l l y among men (Desai et a l , 1979). Other n u t r i t i o n a l surveys i n B r a z i l showing the d e t r i m e n t a l e f f e c t of u r b a n i z a t i o n , o n d i e t and n u t r i t i o n a l s t a t u s are presented i n the Review of L i t e r a t u r e and were performed by M a r t i n s (1972), Jansen e t a l . (1977), USDA Economic Research S e r v i c e (1970), Maldonaldo (1968) and Yunes and Ronchezel (1975) . U n s k i l l e d workers who migrate t o c i t i e s u s u a l l y end-up l i v i n g i n f a v e l a s o r slums on the p e r i p h e r y of the c i t i e s . The B o i a - F r i a s were no ex c e p t i o n . T h e i r i s o l a t i o n on the edge of the c i t y and l a c k of easy t r a n s -p o r t a t i o n , c o n t r i b u t e d to a sedentary l i f e - s t y l e among women, and a l a c k of easy access to a v a r i e t y of foods. The unhygienic and u n s a n i t a r y c o n d i t i o n s of f a v e l a l i f e , i n combination w i t h steady h i g h temperatures caused a p r o l i f e r a t i o n of i n f e c t i o u s agents w i t h pathogenic p o t -e n t i a l . D e sai et a l . (1978a) r e p o r t e d t h a t 60% of the c h i l d r e n i n the area had p a r a s i t e s , and t h a t d i a r r h o e a i s one of the most common i n f e c t i o u s d i s e a s e s among c h i l d r e n . - 208 -P u f f e r and Serrano (1973), T e r u e l (1_967) , and T e r u e l e t al... (1973) r e p o r t e d m a l n u t r i t i o n and i n f e c t i o n i n R i b e i r a o Preto as s y n e r g i s t i c . :causes of c h i l d m o r t a l i t y . High temperatures, i n f e c t i o n and m a l n u t r i t i o n tend to d r a i n the p r o d u c t i v i t y and c r e a t i v i t y of man, thus propagating more i n f e c t i o n and m a l n u t r i t i o n . T h i s b r i n g s us to another c y c l e of need c o n t r i b u t i n g to m a l n u t r i t i o n , the p overty - i l l i t e r a c y c y c l e . A c c o r d i n g to L i v i n g s t o n (19 71), e d u c a t i o n l e v e l a f f e c t s s o c i o e c o -nomic l e v e l which i n t u r n a f f e c t s n u t r i t i o n a l s t a t u s and the e d u c a t i o n l e v e l of the next g e n e r a t i o n . T u r i n i e t a l . (1978) i n a B r a z i l i a n n u t r i t i o n a l survey o f f i r s t grade c h i l d r e n found t h a t socioeconomic l e v e l a f f e c t e d n u t r i t i o n a l s t a t u s , and t h a t the n u t r i t i o n a l s t a t u s , i n t u r n , a f f e c t e d the c h i l d ' s s c h o l a s t i c achievement. The key e f f e c t o f income on d i e t and n u t r i t i o n a l s t a t u s i n B r a z i l was de-monstrated by S i q u e i r a and L e n n i n i (1968) , Patri<ck:_and Simoes (1971) and Campino e t a l . (1975), and the key e f f e c t of o c c u p a t i o n on d i e t i n B r a z i l was shown by Lopes (1962). The income l e v e l s r e p o r t e d by B o i a - F r i a s i n t e r v i e w e d i n t h i s survey were f a r below the minimum monthly s a l a r y a t the time, of 1560 c r u z e i r o s per person. The mean t o t a l income of a l l the working members i n a f a m i l y came t o 2051 c r u z e i r o s per month. On the b a s i s of 7 members per f a m i l y , the average per c a p i t a income was 293 c r u z e i r o s per month. Comparing t h i s w i t h the c o s t of a monthly d i e t of - 209 -a worker, as o u t l i n e d by the B r a z i l i a n government, which comes to 542 c r u z e i r o s i n RibeirSfo Preto c i t y c e n t e r or 641 c r u z e i r o s i n V i l a R e c r e i o , i n d i c a t e s the f i n a n c i a l p r e s s u r e experience by the B o i a - F r i a f a m i l y . Note t h a t food c o s t s were 18% higher i n V i l a R e c r e i o . With r e g a r d to l e v e l of education, 40% of the f a t h e r s and 60% of the mothers had no education, and none of the f a t h e r s or mothers had more than primary e d u c a t i o n (Desai e t a_l. 1978a). In an attempt t o r e l a t e d i e t a r y , anthropometric, and b i o c h e m i c a l n u t r i t i o n a l s t a t u s data with f a m i l y i n f o r m a t i o n i n c l u d i n g economic s t a t u s , education l e v e l and s i n g l e v e r s u s double p a r e n t i n g , o n e - t a i l e d Pearson C o r r e l a t i o n C o e f f i c i e n t T e s t , o n e - t a i l e d Spearman C o r r e l a t i o n C o e f f i c -i e n t T e s t , o n e - t a i l e d T - T e s t or C h i - Square p r o b a b i l i t i e s were performed. D i e t a r y n u t r i e n t i n t a k e s were expressed as per requirement; anthropometric data as percentages of standard v a l u e s ; and b i o c h e m i c a l data w i t h r e s p e c t to standard c a t e g o r i e s , d e f i c i e n t , low, and a c c e p t a b l e . F a m i l y i n f o r m a t i o n c r i t e r i a i n c l u d e d : s a l a r y per .number o f a d u l t s and l i v i n g c h i l d r e n , e d u c a t i o n l e v e l of mother, educ a t i o n l e v e l o f f a t h e r , and s i n g l e v e r s u s double parent f a m i l i e s . Of these s t a t i s t i c a l t e s t s , the ones t h a t were s i g n i f i c a n t w i t h a p r o b a b i l i t y l e s s than 0.05 are l i s t e d i n TableV-7 w i t h t h e i r p r o b a b i l i t y v a l u e s . From these r e s u l t s , one can see t h a t s a l a r y per i n d i v i d u a l and e d u c a t i o n of f a t h e r d i d have a high p o s i t i v e c o r r e l a t i o n w i t h nut-r i e n t i n t a k e l e v e l , as expected. On the other hand, ed u c a t i o n of mother d i d not s i g n i f i c a n t l y c o r r e l a t e * w i t h Table V-7 : S t a t i s t i c a l l y S i g n i f i c a n t P r o b a b i l i t y L e v e l s R e l a t i n g N u t r i t i o n a l S t a t u s Data to Family Information - Compiled from the data of Desai e t a l . (1978a) N u t r i t i o n a l S tatus Data D i e t a r y Expressed as per requirement: c a l o r i e p r o t e i n i r o n c a l c i u m v i t a m i n A thiamine • r i b o f l a v i n n i a c i n v i t a m i n C a v i t a m i n E hvitamin E Anthropometr i c Expressed as~% of standard: Weight f o r Height Arm Circumference Muscle Circumference B i o c h e m i c a l Expressed as c a t e g o r i e s : Plasma V i t a m i n A Hematocrit Family Information C r i t e r i a S a l a r y per I n d i v i d u a l c c c c c c 0.014 0.024 0.008 0.009 0.001 0.001 c 0.001 c 0.001 d 0.003 Education of Father d d d d d d d d d d 0.029 0.001 0.001 0.001 0.007 0.001 0.004 0.031 0.001 0.001 d0v006 d0.026 Education of Mother d0.012 d0.016 d0.015 a A c c o r d i n g to U.S. Requirement. b A c c o r d i n g to Canadian Requirement. c O n e - t a i l e d Pearson C o r r e l a t i o n C o e f f i c i e n t Test. d O n e - t a i l e d Spearman C o r r e l a t i o n C o e f f i c i e n t Test.. e O n e - t a i l e d T-Test. f Chi-Square Test. S i n g l e v s Double Parent e 0.045 e 0.000 e 0.020 e 0.005 e 0.049 e 0.017 e 0.004 0.000 - 211 -n u t r i e n t i n t a k e l e v e l . T h i s perhaps i n d i c a t e s the need f o r b e t t e r n u t r i t i o n e d u c a t i o n i n the schools even a t the primary l e v e l . A s i g n i f i c a n t d i f f e r e n c e between s i n g l e and double parents w i t h r e s p e c t t o n u t r i e n t i n t a k e l e v e l was c a l c u l a t e d f o r some n u t r i e n t s , w i t h double parents having higher i n t a k e l e v e l s . With r e s p e c t to anthropometric n u t r i t i o n a l s t a t u s data, education of mother c o r r e l a t e d p o s i t i v e l y w i t h weight, and education of f a t h e r and mother with arm c i r c u m f e r e n c e and muscle c i r c u m f e r e n c e . However, because t h e r e was seme measure of o b e s i t y i n the p o p u l a t i o n , i n c r e a s e s i n anthropometric c r i t e r i a may not n e c e s s a r i l y have been d e s i r a b l e . A s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n was a l s o o b t a i n e d between plasma v i t a m i n A l e v e l s and s a l a r y per i n d i v i d u a l , which i s worthy of note c o n s i d e r i n g the presence of some p r e - c l i n i c a l v i t a m i n A d e f i c i e n c y i n the p o p u l a t i o n as determined by plasma v i t a m i n A and carotene l e v e l s . A s i g n i f i c a n t d i f f e r e n c e i n p r o p o r t i o n of the sample p o p u l a t i o n i n the d e f i c i e n t , low, and a c c e p t a b l e hematocrit groups was c a l c u l a t e d between s i n g l e and double p a r e n t s , w i t h s i n g l e parents showing more d e f i c i e n c y . T h i s may have been simply due to g r e a t e r female i r o n d e f i c i e n c y , s i n c e most s i n g l e parents were female. A l a c k of e d u c a t i o n a l s o i n d i c a t e s an ignorance of what c o n s t i t u t e s good n u t r i t i o n . The need f o r n u t r i t i o n e d u c a t i o n as w e l l as income improvement i n B r a z i l was dem-o n s t r a t e d by r e s u l t s of a study by Jansen e t a l . (1977), i n which q u a n t i t a t i v e and q u a l i t a t i v e a s p e c t s of d i e t a r y - 212 -p a t t e r n s i n B r a z i l were examined as i n f l u e n c e d by income l e v e l . P r o t e i n and c a l o r i e i n t a k e s were found to i n c r e a s e as income i n c r e a s e d , however n u t r i t i o n a l q u a l i t y , (or n u t r i e n t s per 1000 k c a l ) , was not improved by income im-provement. S i m i l a r l y , i n t h i s study, none of the n u t r i e n t s except thiamine, when expressed per 1000 k c a l , c o r r e l a t e d s i g n i f i c a n t l y w i t h s a l a r y per i n d i v i d u a l . Thus, to feed themselves p r o p e r l y , the B o i a - F r i a p o p u l a t i o n needs not o n l y a means t o o b t a i n food but a l s o enough knowledge to make the r i g h t food c h o i c e s . N u t r i t i o n educators must c o n s i d e r the t r a d i t i o n a l d i e t a r y b e l i e f s of the p o p u l a t i o n they seek to a s s i s t . The B o i a - F r i a p o p u l a t i o n i n d i c a t e d no food r e s t r i c t i o n s being p r a c t i c e d due to r e l i g i o u s reasons. Taboos a g a i n s t mixing c e r t a i n foods, l i k e cucumber wi t h m i l k or egg or o i l or pinga or l i k e mango wit h m i l k d i d e x i s t . E a t i n g r i c e and beans or f r i e d egg a t n i g h t was c o n s i d e r e d bad. B e l i e f s i n i'hot" and " c o l d " foods were a l s o i n d i c a t e d . Pork meat, pork l a r d , s p i c e s such as r e d pepper, and c e r e a l s were c o n s i d e r e d by some as "hot" foods w h i l e f i s h , f r u i t s such as oranges, and v e g e t a b l e s such as l e t t u c e and tomato were c o n s i d e r e d " c o l d " foods (Desai e t a l . ( 1 9 7 8 a ) . None of these b e l i e f s need stand i n the way of good n u t r i t i o n , f o r t u n a t e l y . C u l t u r e i s key i n determining what foods are a c c e p t -a b l e and a l s o i n s e t t i n g the v a l u e or p r i o r i t y of a h e a l t h -f u l d i e t (Fathauer 1960). Examples of good d i e t a r y sources - 213 -of the n u t r i e n t s most d e f i c i e n t i n the B o i a - F r i a d i e t l i s t e d i n TableV-lwhich have d o u b t f u l a c c e p t a b i l i t y i n -c l u d e : organ meats, l i k e l i v e r , kidney, and heart; soybean, which i s d i f f i c u l t t o cook t o the c o n s i s t e n c y and t a s t e p r e f e r r e d by the B r a z i l i a n ; brown r i c e ; whole g r a i n bread; c o l l a r d s ; and spinach. N u t r i t i o n e d u c a t i o n e f f e c t i v e l y employed c o u l d modify the a t t i t u d e s , knowledge and p r a c t i c e s of the B r a z i l i a n B o i a - F r i a w i t h r e g a r d to d i e t and h e a l t h . C o r r e c t i v e Measures There appears to be a d i s c r e p a n c y between the p r o-j e c t e d n a t i o n a l c o r r e c t i v e measures of the B r a z i l i a n gov-ernment presented i n the L i t e r a t u r e Review and the actual, l o c a l c o r r e c t i v e measures o p e r a t i v e i n V i l a R e c r e i o a t the time of t h i s study. In 1975, the g u i d e l i n e s f o r p o l i c y of the M i n i s t r y of Home A f f a i r s gave e s s e n t i a l p r i o r i t y t o growth of the GNP w i t h concern expressed t h a t the b e n e f i t s achieved be e q u a l l y d i s t r i b u t e d to a l l t h a t a l l sections- c£ the community might experience r e a l improvement i n t h e i r p a t t e r n of l i f e . However, the r u r a l - u r b a n m i g r a t i o n of u n s k i l l e d workers tends to keep the wages of t h a t group low s i n c e l a b o r supply i s c o n t i u a l l y i n c r e a s e d . A l s o , t h e r e d i d not appear to be any program whereby the B o i a - F r i a s c o u l d r i s e out of the p o v e r t y - i l l i t e r a c y - m a l n u t r i t i o n c y c l e . ! At the l o c a l l e v e l , government a i d was p r i m a r i l y focussed i n a food d i s t r i b u t i o n program through the M i n i s t r y of E d u c a t i o n and C u l t u r e v i a the school system, of which - 214 -the e d u c a t i o n a l element i s minimal. T h i s program i n the long term c o u l d help to break the -poverty - / i l l i t e r a c y m a l n u t r i t i o n c y c l e , though i t s e f f e c t i v e n e s s would i n -c r e a s e i f i t s e d u c a t i o n a l element was e n l a r g e d . The N a t i o n a l I n s t i t u t e of Food and N u t r i t i o n of the M i n i s t r y of H e a l t h i n c l u d e d as i t s broad o b j e c t i v e s : to p r o v i d e food supplements f o r the worker wi t h low income and f a m i l i e s earning not more than two minimum wages; to combat s p e c i f i c n u t r i t i o n a l d e f i c i e n c i e s through food en-richment p r o j e c t s ; and to t r a i n r e q u i r e d personnel to c a r r y out t h e i r n u t r i t i o n programs. C l e a r l y , these o b j e c t i v e s i d e n t i f i e d needs i n B r a z i l , however, they were not c a r r i e d through to. :program implementation a t the l o c a l l e v e l . .The o n l y food a s s i s t a n c e which the B o i a - F r i a f a m i l i e s r e c e i v e d was from the Lar E s p i r i t a C r i s t a o , f o r about 20% of the f a m i l i e s , i n the form of handouts such as soup. A l s o , a t the time of the survey, t h e r e were no compulsory food enrichment p r o j e c t s except f o r the i o d i z a t i o n of s a l t . The need f o r t r a i n e d personnel was e v i d e n t i n R i b e i r a o Preto where o n l y 5 of the 11 a v a i l a b l e n u t r i t i o n i s t p o s i t i o n s were f i l l e d . Shrimpton (1975) r e p o r t e d t h a t few m e d i c a l s c h o o l s i n B r a z i l of f e r e d ,-courses i n n u t r i t i o n which were o r i e n t e d to the community and which were e d u c a t i o n a l and p r e v e n t i v e i n nature. A branch of the M i n i s t r y of S o c i a l Welfare, the B r a z i l i a n Legion of A s s i s t a n c e (LBA) was a c t i v e i n V i l a R e c r e i o , c o n t r i b u t i n g to the PLIMEC program, o f f e r i n g c l a s s e s - 215 -and food supplements to pregnant women through the Lar E s p i r i t a C r i s t a o , and a l s o h e l p i n g -to conduct home v i s i t s and i n t e r v i e w s f o r t h i s study. With r e g a r d to the a g r i c u l t u r a l r e s e a r c h of the M i n i s t r y of A g r i c u l t u r e EMBRAPA program, a c c o r d i n g to Pastore (1977), B r a z i l served as an example of a country i n which .there^was well-developed r e s e a r c h f o r key export c r o p s , c o f f e e , sugar, and c o t t o n , but not f o r home s t a p l e foods, r i c e , beans and maize. For m a l n u t r i t i o n t o ber-avoided, he suggested t h a t r e s e a r c h on export crops should be f i n a n c e d mostly p r i v a t e l y and on home food crops should be f i n a n c e d e n t i r e l y and h e a v i l y by the government. Ac c o r d i n g to Shrimpton (1975), the b a s i c consumer products, r i c e , maize, cassava and kidney beans were l a r g e l y c u l t i v a t e d i n smal l p r o d u c t i o n u n i t s t h a t used t r a d i t i o n a l and not v e r y e f f i c i e n t methods. As an o p t i o n w i t h p o s s i b l e n u t r i t i o n a l impact, he s t a t e d t h a t a program of s p e c i a l i n c e n t i v e s aimed p u r e l y a t small farmers t h a t would cause them t o produce more of the b a s i c food commodities i n a more e f f i c i e n t manner, would r e s u l t i n the combined b e n e f i t s of an i n c r e a s e d supply of these b a s i c food crops i n the f a c e of c o m p e t i t i o n from export crops, and a t the same time, a g r e a t e r income i n the s u b s i s t e n c e s e c t o r of the a g r i c u l -t u r a l community. Two exte n s i o n s of the M i n i s t r y of A g r i c u l t u r e , the B r a z i l i a n Food Company (COBAL) and the N a t i o n a l S u p e r i n -tendency of Supply (SUNAB) were r e s p o n s i b l e f o r ensu r i n g - 216 -the supply of e s s e n t i a l commodities f o r p o p u l a t i o n con-sumption, and f o r d i s t r i b u t i n g , marketing and p r i c i n g these commodities. In 1974, the c o s t of the e s s e n t i a l r a t i o n f o r a B r a z i l i a n worker to e x i s t and work f o r a month ( o u t l i n e d i n TableIV - 2 )was about t w o - t h i r d s of the minimum s a l a r y , and a t the time of t h i s study, e a r l y 1978, was about one-t h i r d of the minimum s a l a r y . These f i g u r e s i n d i c a t e t h a t COBAL and SUNAB have .improved the s i t u a t i o n since.1974, however, more improvement i s r e q u i r e d as shown by the f i n a n c i a l p r e s s u r e experienced by the B o i a - F r i a f a m i l i e s . Progress f o r these people c o u l d be prompted through: food s u b s i d i e s t o m a i n t a i n low p r i c e l e v e l s of b a s i c n u t r i t i o u s food commodities a t r e t a i l l e v e l , p o s s i b l y f i n a n c e d by t a x i n g " p r e s t i g e " commodities used mainly by the upper socioeconomic b r a c k e t s ; b e t t e r d i s t r i b u t i o n and marketing of food products a t the l o c a l l e v e l so t h a t lower income s e c t i o n s of the c i t y would not be r e q u i r e d t o pay more f o r a food product i n t h e i r area than they would f o r the same product i n the c i t y c e n t e r ; and/or g r e a t e r e a r n i n g power f o r these people. The B o i a - F r i a f a m i l i e s a l s o d i d not seem to b e n e f i t from the S o c i a l and E d u c a t i o n a l S e r v i c e of I n d u s t r y (SESI) program. I t p r o j e c t e d a c t i v i t i e s i n c l u d i n g the p r o v i s i o n of lunches of about 1600 c a l o r i e s f o r workers, and n u t r i -t i o n e d u c a t i o n o f . t h e worker and h i s wife c o u l d a i d B o i a - F r i a f a m i l i e s tremendously i n adapting t o t h e i r urban s e t t i n g , - 217 -and i n h e l p i n g them to break out of the p o v e r t y - i l l i t e r a c y -m a l n u t r i t i o n c y c l e . C l e a r l y , the p r o j e c t e d "equal b e n e f i t f o r a l l " p o l i c y of the M i n i s t r y of Home A f f a i r s i n 1975 was e a s i e r s a i d than done. A c a r e f u l examination of the problem r e -v e a l s t h a t to improve income d i s t r i b u t i o n i n harmony wi t h m a i n t a i n i n g h i g h r a t e s of economic growth, i s a p rocess which demands time and r a t i o n a l i z a t i o n . T r y i n g to de-c r e a s e i n d i v i d u a l i n e q u a l i t i e s by the l a v i s h readjustment of nominal s a l a r i e s i s d e s t i n e d to f a i l u r e by the g e n e r a t i o n of i n f l a t i o n a r y t e n s i o n s of l i m i t i n g the o p p o r t u n i t i e s f o r employment, and by the m u t i l a t i o n of the p o t e n t i a l of saving and of development. The improvement of a person's income must come, f i r s t l y from h i s betterment through edu c a t i o n and from the p o l i c y of development and c r e a t i o n of employment. Then, i n a d d i t i o n to t h i s f o u n d a t i o n , tax and customs p o l i c i e s can be improved, workers saving schemes can be c r e a t e d and s o c i a l s e c u r i t y b e n e f i t s can be implemented. B r a z i l i s going through a phenomenally r a p i d p e r i o d of t r a n s i t i o n from an underdeveloped a g r i c u l t u r a l s o c i e t y to a developed i n d u s t r i a l one. Consequently, many people have experienced c o n s i d e r a b l e upheaval w i t h d i s l o c a t i o n and a l t e r e d l i f e s t y l e . In aiming f o r the most r a p i d r a t e of t r a n s i t i o n p o s s i b l e , the government has opted f o r the m i n i m i z i n g of d u r a t i o n of t h i s t r aumatic p e r i o d , r a t h e r than the m i n i m i z i n g of p r e s e n t extent of trauma. In the - 218 -meantime, th e r e i s a need f o r p u b l i c h e a l t h , hygiene, and n u t r i t i o n e d u c a t i o n and i n t e r v e n t i o n programs i n order to minimize the harsh e f f e c t s of poverty and upheaval on c e r t a i n s e c t i o n s of the p o p u l a t i o n . - 219 -CHAPTER VI CONCLUSIONS AND RECOMMENDATIONS The conclusions and recommendations presented here are based on the ecol o g i c a l , dietary, anthropometric and biochemical re s u l t s discussed i n this study. A. Conclusions 1. The magnitude of malnutrition as a public health problem was considerable. According to dietary data, conditions existed which were conducive to the develop-ment df n u t r i t i o n a l problems, e s p e c i a l l y with respect to calcium, vitamin A, r i b o f l a v i n , n i a c i n , vitamin C, and iron of the nutrients tested, and with respect to quantitative intake of food (kcal). Biochemical data confirmed the presence of early malnutrition, p r e - c l i n i c a l i n nature for a portion of the population with respect to the nutrients vitamin A, carotene, and iron. The presence of some degree of c l i n i c a l undernutrition or f i r s t degree malnutrition among men and women was indicated by the anthropometric data as well as some degree of obesity among women. According to c h i l d mortality data for Ribeirao Preto, some advanced c l i n i c a l malnutrition was evidenced among children. 2. Q u a l i t a t i v e l y , the Boia-Fria diet was monotonous and simple, with l i t t l e v ariety, consisting b a s i c a l l y of polished r i c e , beans, white bread, and coffee with sugar. In general, the foods which were lacking are: milk products, meats, f i s h , eggs, poultry, non-refined grain products, - 220 -and f r u i t s and vegetables r i c h i n vitamins A and C. 3. There appeared to be a lack of knowledge concern-ing maternal dietary needs. Breastfeeding practices were to be. commended although there was need for improve-ment i n weaning foods, considering that the f i r s t foods introduced to infants within 2 weeks of delivery were of low n u t r i t i o n a l value, such as starchy gruels, sugar-water, herb tea, coffee with sugar and soft drinks. These additional foods could i n t e r f e r e with the mothers' continued success i n breastfeeding. 4. Good food sources of the nutrients most d e f i c i e n t i n the Boia-Fria diet which are also thought to be within their f i n a n c i a l and a c c e p t a b i l i t y constraints include: green leafy vegetables such as collards (for calcium, vitamin A, r i b o f l a v i n , n i a c i n , vitamin C and i r o n ) ; powder-ed milk (for calcium); orange or yellow f r u i t s or vegetables such as carrots, pumpkin, papaya and yams (for vitamin A);; eggs (for r i b o f l a v i n , and i r o n ) ; avocado (for r i b o f l a v i n and n i a c i n ) ; peanuts (for n i a c i n ) ; c i t r u s f r u i t s , cashew f r u i t , tomatoes, cabbage, potatoes, and okra (for vitamin C); and yams (for i r o n ) . Many of these foods, such as f r u i t s , vegetables and eggs could be home-produced. The consumption of other n u t r i t i o u s foods such as soy-bean; brown, undermilled, or parboiled r i c e ; whole grain products; and organ meats could be promoted through n u t r i t i o n education to enhance t h e i r a c c e p t a b i l i t y . - 221 -5. Basic causes of malnutrition among Boia-Frias i n -cluded the following ecological factors: recent urban-i z a t i o n ; housing, sanitation and environmental conditions, associated with serious i n f e c t i o n problems; povery; i l l i t e r a c y ; and an ignorance of what constitutes good n u t r i t i o n . 6. The Boia-Fria population indicated no food r e s t r i c -tions, taboos, or b e l i e f s which need stand in the way of good n u t r i t i o n . Therefore, n u t r i t i o n education, e f f e c t -i v e l y employed might be expected to s i g n i f i c a n t l y im-prove the n u t r i t i o n a l status of t h i s population. 7. There appears to be a discrepancy between the pro-jected national corrective measures of the B r a z i l i a n government and the actual l o c a l corrective measures operative in V i l a Recreio at the time of t h i s study. A need exists for short and long term e f f o r t s to min-imize the harsh e f f e c t s of povertyand upheaval on the migrant workers of B r a z i l during the phenomenally rapid t r a n s i t i o n of t h i s country from an underdeveloped a g r i c u l t u r a l society to a developed i n d u s t r i a l one. Recommenda t ion s The following recommendations regarding improve-ment of the food habits and n u t r i t i o n a l status of the Boia-Frias are made with the ultimate goal of helping these people help themselves. 1. Intervention Programs Intervention programs, though o f f e r i n g immediate - 222 -r e l i e f , can only p a r t i a l l y or temporarily deal with the problem. Nevertheless, the following types of programs are recommended to help minimize the harsh ef f e c t s of poverty and upheaval on the n u t r i t i o n a l status of the Boia-Fria population. a) Food d i s t r i b u t i o n programs should be set up, the target group being the bottom portion of the socio-economic st r a t a . b) Those demonstrating p r e - c l i n i c a l or c l i n i c a l vitamin and/or mineral d e f i c i e n c i e s should be treated with suitable supplements, for example, iron or vitamin; A supplements. 2. "Long-range" Programs "Long-range" programs are fundamental measures that w i l l correct with time the basic causes of malnutrition. Examples applicable to the Boia-Fria setting include the following. a) An attempt to slow down r u r a l to urban mig-ratio n i n B r a z i l would be advisable. For example, a program of special incentives aimed purely at small farmers that would cause them to produce more of the basic n u t r i t i o u s food commodities i n a more e f f i c i e n t manner, would r e s u l t i n the combined benefits of an increased food supply and at the same time, a greater income i n the subsistence sector of the a g r i c u l t u r a l community. b) The sanitary f a c i l i t i e s and l i v i n g conditions - 223 -of the B o i a - F r i a s i n V i l a R e c r e i o need s e r i o u s c o n s i d e r a t i o n s f o r improvement. B a s i c hygiene e d u c a t i o n and i n c e n t i v e s to improve t h e i r environment would be d e s i r a b l e . c) Improvement i n the income of the B o i a - F r i a s must come f i r s t l y by t h e i r betterment through education, and an i n c r e a s e d r a t e of growth of employment opport-u n i t i e s . Night school programs p r e s e n t l y o p e r a t i v e a r e to be commended. In a d d i t i o n to t h i s f o u n d a t i o n , tax and customs p o l i c i e s c o u l d be improved, workers saving schemes c r e a t e d and s o c i a l s e c u r i t y b e n e f i t s implemented. d) To i n c r e a s e the a v a i l a b i l i t y of n u t r i t i o u s foods f o r the B o i a - F r i a . and to m a i n t a i n low p r i c e l e v e l s , n u t r i t i o u s food commodities should be s u b s i d i z e d a t the r e t a i l l e v e l , p o s s i b l y f i n a n c e d by t a x i n g " p r e s t i g e " commodities used mainly by the upper socioeconomic b r a c k e t s . B e t t e r d i s t r i b u t i o n and marketing of food products a t the l o c a l l e v e l would a l s o be b e n e f i c i a l s i n c e food c o s t s were h i g h e r i n V i l a R e c r e i o than i n the ce n t e r of the c i t y . As w e l l , b a s i c food commodities of " h i g h - r i s k " f a m i l i e s such as B o i a - F r i a s should be f o r t i f i e d w i t h n u t r i e n t s l a c k i n g i n the d i e t . For example, white f l o u r used f o r making bread and macaroni c o u l d be e n r i c h e d w i t h thiamine, r i b o f l a v i n , n i a c i n , and i r o n ; r i c e , e n r i c h e d w i t h thiamine, r i b o f l a v i n and n i a c i n ; skim m i l k powder f o r t i f i e d w i t h v i t a m i n s A and D; v e g e t a b l e o i l , w i t h w i t h v i t a m i n s A a n d D; and bread, with v i t a m i n A, c a l c i u m T> 224 -and iron. Whole wheat f l o u r and bread should be made availab l e as a choice instead of white f l o u r and bread, as Well as brown, undermilled, or parboiled r i c e as opposed to polished white r i c e . e) Malnutrition r e s u l t i n g from ignorance of what constitutes good n u t r i t i o n should be combatted by nut-r i t i o n education programs of the person-to-person, group-teaching, and mass-media type, i n order to reach adults and children a l i k e . N u t r i t i o n education i n schools at the primary l e v e l i s advised because of the early drop-out tendency and because children are p a r t i c u l a r l y impressionable and can adopt changes without i n h i b i t i o n s and carry them to the i r homes. The willingness of the Boia-Fria homemakers to par t i c i p a t e in t h i s study indicates that they probably would be w i l l i n g to be involved i n group discussions and counselling sessions regarding n u t r i t i o n and good health. The high percentage of families with radios and t e l e v i s i o n s j u s t -i f i e s the use of the media for combatting popular adver-t i s i n g . Through these educational methods amd techniques, the consumption of ce r t a i n foods which are good sources of the nutrients lacking i n the d i e t should be promoted; food preparation techniques discussed; special dietary needs for physical work, gestation, l a c t a t i o n , and growth i n infants and children emphasized; the growing of home gardens and small livestock to increase con-sumption of f r u i t s , vegetables, meat, and eggs encour-- 225 -aged; and the establishing of l o c a l cooperatives or f a i r price shops for basic n u t r i t i o u s food commodities urged. Through challenge and stimulus of t h i s type, the Boia-F r i a s could be motivated to take steps to help themselves. A l l i n s t r u c t i o n must express utmost respect for t h e i r views; should highlight the best i n t h e i r c u l t u r a l back-ground; should be modified to suit t h e i r needs and l i t e r a c y l e v e l ; and should make suggestions only within t h e i r f i n a n c i a l means. To carry out these education programs, trained personnel are required, and thus there i s a need for com-munity n u t r i t i o n education courses for nurses, nurses' aids, and s o c i a l workers and a need for t r a i n i n g and em-ployment of community n u t r i t i o n i s t s . C learly, there i s no easy solution to the complex d i f f i c u l t s i t u a t i o n of the Boia-Fria population and thus an integrated approach i s a necessity for success i n im-proving t h e i r n u t r i t i o n a l status. - 226 -BIBLIOGRAPHY Ab e l l , L.L. , B.B.. Levy, B.B. Brodie' and F.E. K e n d a l l . 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