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Folic acid nutritional status of British Columbia Indian populations Porritt, Barbara 1976

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FOLIC ACID NUTRITIONAL STATUS OF BRITISH COLUMBIA INDIAN POPULATIONS by BARBARA PORRITT B.Sc., U n i v e r s i t y o f B r i t i s h Columbia, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIPEMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the D i v i s i o n of HUMAN NUTRITION SCHOOL OF HOME ECONOMICS We accept t h i s t h e s i s as conforming t o the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST, 1976 (c) Barbara P o r r i t t , 1976 In p resent ing t h i s t he s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree tha t permiss ion fo r ex tens i ve copying o f t h i s t he s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r ep re sen ta t i ve s . It i s understood that copying or p u b l i c a t i o n of t h i s t he s i s f o r f i n a n c i a l ga in s h a l l not be al lowed without my w r i t t e n permis s ion. Department of The Un i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date ^ T ^ S > ^ i ABSTRACT . Recent s t u d i e s suggest t h a t f o l i c a c i d n u t r i t i o n a l s t a t u s may be poor among Canadian In d i a n s , p a r t i c u l a r l y among those l i v i n g i n i s o l a t e d areas. However the prevalence and causes of f o l i c a c i d d e f i c i e n c y have not been assessed. The present study was conducted i n order t o assess the magnitude of the problem among B r i t i s h Columbia Indians and t o examine the p o s s i b l e r e l a t i o n s h i p between low d i e t a r y i n t a k e s of f o l i c a c i d and the occurrence of low b l o o d f o l a t e v a l u e s . Using a 24-hour d i e t r e c a l l , d i e t a r y f o l a t e i n t a k e s were estimated a t f o u r r e l a t i v e l y i s o l a t e d Indian reserves (106 subjects) and a t three reserves adjacent t o urban c e n t r e s (144 s u b j e c t s ) . A more d e t a i l e d study, i n v o l v i n g e s t i m a t i o n o f d i e t a r y f o l a t e i n t a k e , measurement of serum and r e d b l o o d c e l l f o l a t e , and examination of r e l a t e d hematological parameters was undertaken a t one i s o l a t e d reserve (Fort Ware, 28 subjects) and two n o n - i s o l a t e d reserves (Necoslie and S e c h e l t , 63 subjects) as w e l l as a t a s c h o o l residence (70 c h i l d r e n , age 6 t o 16 y e a r s ) . Meal samples were c o l l e c t e d and assayed f o r f o l i c a c i d , i n order to v e r i f y the r e c a l l c a l c u l a t i o n s . R e s ults i n d i c a t e t h a t c a l c u l a t e d and assayed f o l a t e values are s i m i l a r and are s i g n i f i c a n t l y c o r r e l a t e d (r=.9694). T o t a l f o l a t e consumption i s s i g n i f i c a n t l y h i g h e r a t n o n - i s o l a t e d r e s e r v e s than a t i s o l a t e d r e s e r v e s , and males consume s i g n i f i c a n t l y more f o l i c a c i d than do females. D i e t a r y f o l i c a c i d i n t a k e i s higher a t the residence than a t the r e s e r v e s . i i Serum f o l a t e values are s i g n i f i c a n t l y c o r r e l a t e d w i t h d i e t a r y f o l a t e i n t a k e . Serum values are lower a t F o r t Ware than a t N e c o s l i e and Sech e l t . C h i l d r e n l i v i n g on reserves have lower serum f o l a t e values than do c h i l d r e n l i v i n g i n residence, and have a l a r g e r p r o p o r t i o n o f c h i l d r e n c l a s s i f i e d as "at r i s k " . On the b a s i s o f r e d c e l l f o l a t e v a l u e s , 16 t o 45% o f the subjects a t the three reserves are c l a s s i f i e d as "at r i s k " , however, no evidence o f m e g a l o b l a s t i c anemia i s i n d i c a t e d from the hematological examinations. I t i s concluded t h a t many i n d i v i d u a l s are e i t h e r b o r d e r i n g on o r are d e f i c i e n t w i t h r e s p e c t t o f o l i c a c i d . This appears t o be a more se r i o u s problem a t i s o l a t e d reserves than a t those adjacent t o urban centres and i t i s suggested t h a t t h i s i s a consequence of the a v a i l a b i l i t y , v a r i e t y and s e l e c t i o n o f foods. i i i TABLE OF CONTENTS Page ABSTRACT i ACKNOWLEDGEMENTS i i i LIST OF TABLES v i i LIST OF FIGURES i x Chapter I REVIEW OF THE LITERATURE 1 F o l i c A c i d Deficiency and Megaloblastic Anemia . . . . 1 Assessment o f F o l i c A c i d Status 2 A. I n d i r e c t measures of f o l a t e status Urinary FIGLU 3 Urinary f o l a t e 3 Plasma f o l a t e clearance 4 Leucocyte f o l a t e l e v e l s . . . . 4 B. D i r e c t measure of f o l a t e status 4 C. Iron i n d i c e s r e l a t e d t o f o l a t e status 6 D. The f o l a t e assay 7 Dietary Folates 7 A. The nature and assay o f d i e t a r y f o l a t e s . . . . 8 B. The estimation of d i e t a r y f o l a t e consumption . . 10 Studies Assessing Folate Status 11 A. Population surveys . 11 B. The N u t r i t i o n Canada Survey . . 16 C. F o l i c a c i d status of native Indians 18 II INTPODUCTION 20 iv Page Chapter -III MATERIALS AND METHODS 23 Sample Population • 23 Dietary Recalls, Food Collections and Analysis . . . 28 A. Dietary recalls and analysis 28 B. Collection of food samples and analysis . . . 28 Blood Collection, Treatment and Analysis 30 A. Collection and treatment of blood samples . . 30 B. Blood folate analysis 31 St a t i s t i c a l Analysis 31 IV RESULTS • • • 33 Dietary Folate Data • 33 A. Assayed vs. calculated values 33 B. Comparison of mean folate intakes for the seven reserves 36 C. Comparison of mean folate intakes for subjects involved i n the comprehensive part of the survey 39 Hematological Data 43 A. Control sample values '. 43 B. Comparison of means for hematological variables 44 C. Risk distributions . . . 48 Regression Analysis 56 V DISCUSSION 58 VI SUMMARY 69 v' Page BIBLIOGRAPHY 71 APPENDICES A. Consent Form 80 B. Assayed and Calculated Results For T o t a l Folate For The 24-Hour Food C o l l e c t i o n s 81 C. Canadian Recommended D a i l y Nutrient Intake 82 D. Guidelines For Interpretation Of Serum Folate, Red C e l l Folate, and Hemoglobin Levels 83 Legend to Appendices E. F, and G 84 E. Raw Data - Individual Nutrient Values Calculated From 24-Hour Dietary Recalls For Subjects From The Seven Reserves. 85 F. Raw Data - I n d i v i d u a l Nutrient Values Calculated From 24-Hour Dietary Recalls For Subjects P a r t i c i p a t i n g In The Comprehensive Part Of The Study .91 G. Raw Data - Ind i v i d u a l Hematological Values For Subjects P a r t i c i p a t i n g In The Comprehensive Part Of The Study' • 96 v i LIST OF TABLES Table • Page I C l a s s i f i c a t i o n Of Serum F o l a t e Values For The General, Indian and Eskimo P o p u l a t i o n s : Percentage High R i s k Values In D i f f e r e n t Age Categories 17 I I Indian Reserves In B r i t i s h Columbia Involved In The Assessment Of F o l a t e Status 27 I I I Person Product-Moment C o r r e l a t i o n C o e f f i c i e n t : Test o f C o r r e l a t i o n Between Assayed and C a l c u l a t e d Values For T o t a l F o l a t e From 24-Hour Food C o l l e c t i o n s 33 IV M u l t i p l e Range Test Of Mean Scores For T o t a l F o l a t e Intake For The Seven Reserves Under Study . . . . 36 V M u l t i p l e Range Test Of Mean Scores For Food F o l a t e Intake For Sex And Age Groupings For The Seven Reserves Studied 37 VI T-Test o f Mean Scores For F o l a t e Intake For The Seven Reserves With Respect To I s o l a t i o n And Sex . . . 38 V I I Mean D a i l y Intake Of F o l a t e , C a l o r i e s , And Ir o n For Subjects From The Seven Reserves 39 V I I I M u l t i p l e Range Test Of Mean Scores For Food F o l a t e Intake For Subjects Involved I n The Comprehensive F o l a t e Study For S e c h e l t , N e o o s l i e , /And F o r t Ware Reserves 40 IX T-Test Of Mean Scores For D i e t a r y F o l a t e Intake For Subjects Involved In The Comprehensive F o l a t e Study From The Three Reserves 41 X Mean D a i l y Intake Of T o t a l F o l a t e , C a l o r i e s , And Iron For L o c a t i o n And Age Groupings For Subjects Involved In The Comprehensive P a r t Of The Study 42 XI Comparison Of Serum And Red C e l l F o l a t e Values For D u p l i c a t e Samples Ran As A C o n t r o l Measure 43 X I I M u l t i p l e Range Test o f Mean Scores For Serum F o l a t e , Red C e l l F o l a t e , and Hemoglobin For The Three Reserves Involved I n The Comprehensive P a r t Of The Study . . . . 44 v i i ' Table . Page XIII Multiple Range Test of Mean Scores For Serum Folate Red C e l l Folate, and Hemoglobin For Age And Sex Groupings For The Three Reserves Involved In The Comprehensive Part Of The Study 46 XTV T-Test Of Mean Scores For Serum And Red Ce l l Folate Variables For Location And Sex Groupings For Subjects Involved In The Comprehensive Part Of The Study 47 XV SL-Test Of Comparison Of Regression Equations With Serum Folate Or Red C e l l Folate As Dependent Variables And Dietary Folate Intake The Independent Variable, With Equations In The Form Of y=A+Bx 56 XVI Regression Line Constants With Serum Folate Or Red C e l l Folate As Dependent Variables And Dietary Folate Intake As The Independent Variable, With Equations In The Form Of y=A+Bx 57 v i i i LIST OF FIGURES Figure Page 1 F o l i c A c i d And I t s Polyglutamates 8 2 B r i t i s h Columbia Indian Reserves Involved In The Study'Of F o l i c A c i d N u t r i t i o n a l Status . . . . 25 3 Scatter Diagram Showing The Degree Of C o r r e l a t i o n Between Calculated And Assayed Values Of T o t a l Folate From 24-Hour Food C o l l e c t i o n s 35 4 D i s t r i b u t i o n Of Hematological Variables Into Risk Categories For The Three Reserves 50 5 D i s t r i b u t i o n Of Hematological Variables Into Risk Categories For Residence And Reserve Subjects (0 to 17 Years Old) 53 6 C l a s s i f i c a t i o n Of Serum Folate Values For Reserve Subjects From This Study And For Two Groups From The N u t r i t i o n Canada Survey i . e . National Indian Survey and B. C. P r o v i n c i a l Survey 55 ix AOTNDWLEDGEMENTS I would l i k e to thank my research d i r e c t o r Dr. Melvin Lee, f o r his guidance and support throughout the course of t h i s study; Dr. Roy Pratt for h i s assistance and advice with the hematological measurements and thesis preparation; and Dr. Joseph Leichter for his advice i n the thesis preparation. Thanks are also expressed to the personnel of the P a c i f i c Region, Medical Services Branch, Department of National Health and Welfare, with special thanks to Dr. Pyper, Dr. Butler, and Dr. Murie, for t h e i r assistance selecting the reserves and i n execution of the study; Public Health Nurses Ruby Siemens and Maxine Ingles and personnel from Saint Paul's Hospital, with a special thanks to Luce Mauw, for t h e i r advice and technical assistance; the community Health A u x i l i a r i e s who accompanied us on the reserves and introduced us to the people, along with the Indian c h i e f s , councillors and band members from the reserves; Yolanda Stepien f o r her invaluable assistance conducting the 24-hour r e c a l l s ; and Tom Edwards for his encouragement through-out the course of t h i s project and hi s advice i n the thesis preparation. I am especially grateful to the residents of the seven reserves and Sechelt Residential School, whose p a r t i c i p a t i o n and cooperation has made t h i s study possible. 1 CHAPTER I _ REVIEW OF THE LITERATURE F o l i c A c i d D e f i c i e n c y and M e g a l o b l a s t i c Anemia F o l i c a c i d (monopteroylglutamic acid) i s a v i t a m i n which serves as a o o f a c t o r i n most one-carbon t r a n s f e r systems. F o l a t e i s the c o l l e c t i v e term t h a t comprises f o l i c a c i d and i t s d e r i v a t i v e s , i . e. coenzymes and t h e i r p r e c u r s o r s which have a ptero y l g l u t a m a t e s t r u c t u r e . F o l i c a c i d d e f i c i e n c y i s widespread throughout the w o r l d , ranging from a m i l d d e f i c i e n c y t o severe m e g a l o b l a s t i c anemia (Herbert, 1962). The m a j o r i t y o f cases o f m e g a l o b l a s t i c anemia r e s u l t from f o l i c a c i d and/or Vi t a m i n d e f i c i e n c y (Johns and B e r t i n o , 1965 and Herbert, 1968). The e a r l i e s t i n d i c a t i o n o f f o l a t e d e f i c i e n c y i s a f a l l i n serum f o l a t e a c t i v i t y , f o l l o w e d by n u c l e a r hypersegmentation o f the n e u t r o p h i l i c polymorphonuclear leu c o c y t e s . The normal nu c l e a r lobe count o f two t o f i v e i n c r e a s e s t o s i x o r more. Red blood c e l l f o l a t e l e v e l s decrease along w i t h decreases i n l i v e r f o l a t e s t o r e s . Macrocytes appear i n the p e r i p h e r a l b l o o d , as macrcovalocytosis becomes d e f i n e d and mean corpuscular volume i n c r e a s e s . The eventual development of o v e r t m e g a l o b l a s t i c anemia i s c h a r a c t e r i z e d by the presence o f megaloblasts i n the bone marrow, which r e l a t e s , d i r e c t l y t o the degree o f anemia present. A f o l a t e d e f i c i e n c y , per se, 2 may be defined by any stage in the series of changes leading to the development of megaloblastic anemia. As the changes approach the stage of anemia they indicate more severe or intense depletion of folic acid. Herbert (1962) summarized the sequence of events which occurs when the body is depleted of folate as a result of a daily intake of 5 pg. or less. IiEMATOLOGIC AND BIOCHEMICAL SEQUENCE OF CHANGES IN DIETARY FOLIC ACID DEPRIVATION IN MAN (HERBERT, 1962) Sequence of Changes Time of Change (Days) Low serum folate (3ng/ml) 22 Hypers egmentation 49 High urine FIGLU 95 Low REC folate 123 (20ng/ml) Macroovalocytosis 127 Megaloblastic marrow 134 Anemia 137 Assessment of Folic Acid Status Many biochemical measurements are available which provide information concerning the folic acid status of individuals or popula-tion groups. For adequate interpretation of results i t is important to understand that different parameters reflect different aspects of 3 folate metabolism. A. Indirect measures of folate status 1. Urinary FIGLU Several indirect tests for identifying folate deficiency are based on the measurement of urinary formiminoglutamic acid (FIGLU) and i t s precursor, urocanic acid (Herbert, 1967). Under normal conditions formiminoglutamic acid i s formed as a result of histidine degradation and i s subsequently converted to glutamic acid by a tetrahydrofolate -dependent reaction. Folate deficiency inhibits this conversion, leading to increased levels of urinary FIGLU and urocanic acid, especially after the oral adininistration of an L-histidine load (Chanarin, 1964 and Herbert, 1967). The drawback of using a urinary FIGLU test as an index of folate status i s i t s lack of specificity (Sauberlich et a l , 1974), since folate deficiency i s not distinguishable from numerous forms of metabolic blockage which also result i n high FIGLU excretions. Abnormal FIGLU excretions are seen i n subjects with l i v e r damage, protein malnutrition, and congenital formiminotransferase deficiency. 2. Urinary folate According to Ccoperman et a l (1970) urinary folate excretion averages approximately 1% of the dietary intake.• This varies from 1 to 10 ug per day and i s not a sensitive measure of dietary folate consumption. However, a comparison of urinary folate excretion after injected and oral administration of f o l i c acid i s valuable i n the diagnosis of folate malabsorption states (Johns and Bertino, 1965). 4 3. Plasma f o l a t e clearance .. The r a t e o f plasma clearance of i n j e c t e d doses of f o l i c a c i d i s abnormally r a p i d i n sub j e c t s w i t h primary f o l i c a c i d d e f i c i e n c y (Johns and B e r t i n o , 1965). The use of t h i s r a t e measure as an index o f f o l a t e d e f i c i e n c y i s i m p r a c t i c a l f o r survey a p p l i c a t i o n , s i n c e s e r i a l measurements o f plasma f o l a t e must be taken t o determine the cle a r a n c e r a t e . 4. Leucocyte f o l a t e l e v e l s F o l a t e l e v e l s i n the leucocytes c o r r e l a t e w i t h r e d c e l l f o l a t e l e v e l s and are used as an index o f f o l a t e d e f i c i e n c y (Hoffbrand and Newcombe, 1967). Leucocyte values w i l l not d i s t i n g u i s h between s u b j e c t s w i t h f o l a t e d e f i c i e n c y and those w i t h p e r n i c i o u s anemia (Hoffbrand and Newcombe, 1967), and those w i t h d i e t a r y v i t a m i n B^ 2 d e f i c i e n c y ( V i t l e r e t a l , 1963), Measurement o f leucocyte f o l a t e o f f e r s no advantage over measurement o f serum and r e d c e l l f o l a t e , and i s t e c h n i c a l l y more d i f f i c u l t t o perform (Hoffbrand and Newcombe, 1967). B. D i r e c t measure o f f o l a t e s t a t u s Measurements o f serum and r e d c e l l f o l a t e are w i d e l y used i n d i c e s o f f o l a t e s t a t u s (Johns and B e r t i n o , 1965). Serum f o l a t e l e v e l s r e f l e c t t r a n s i e n t changes i n d i e t a r y f o l a t e i n t a k e s ( J o i n t FAO/WHO Expert Group, 1970), t h e r e f o r e , d i s t i n c t l y low l e v e l s are not n e c e s s a r i l y evidence o f t i s s u e d e p l e t i o n o r p r o t r a c t e d f o l a t e d e f i c i e n c y ( J o i n t FAO/WHO Expert Group, 1970 and H a l l e t a l , 1975). The f o l a t e s t a t u s and in c i d e n c e o f d e f i c i e n c y i n a pop u l a t i o n cannot be determined from serum f o l a t e values alone. 5 Red blood c e l l f o l a t e a c t i v i t y i s a better indicator of deficiency than serum f o l a t e a c t i v i t y (Liu, 1974). In the absense of deficiency red c e l l f o l a t e a c t i v i t y i s a quantitative index of the severity of folate deficiency (Herbert, 1965 and Hoffbrand et a l , 1966) and an indicator of the fola t e content of body tissues (Omar et aL, 1970). In vitamin B^^ deficiency, serum fol a t e levels increase while red c e l l folate decreases (Herbert and Zulusky, 1962; Cooper and Lowenstein 1964? and Hoffbrand et a l , 1966). In subjects with t h i s pattern suspected of folate deficiency further evaluation should therefore be made to eliminate the p o s s i b i l i t y of pernicious anemia or dietary vitamin B^2 deficiency (Sauberlich et a l , 1974). The presence of megaloblastic anemia and low serum and red c e l l folate i s strong evidence that f o l a t e deficiency e x i s t s (Cooper and Lowenstein, 1966 and Hoffbrand e t a l , 1966) but does not ru l e out the p o s s i b i l i t y that iron deficiency could be a coexisting cause of the anemia (Hall et a l , 1975). Saraya et a l (1971) reported that i r o n deficiency resulted i n decreased serum fol a t e values although t h i s was not confirmed by others (Omer et a l , 1970 and Saraya et a l , 1973). Omer et a l (1970), Roberts et a l (1971) and Saraya et a l (1973) reported s i g n i f i c a n t increases i n red c e l l f o l a t e i n conditions of iro n deficiency while Hershko et a l , (1975) reported that red c e l l f o l a t e was unaffected by a coexistent i r o n deficiency. The e f f e c t of iro n deficiency on fo l a t e metabolism i s poorly understood (Hershko e t a l , 1975) and the data are contradi ctory. 6 The proof o f symptomatic f o l a t e d e f i c i e n c y u s u a l l y depends on a combination o f evidence r a t h e r than a s i n g l e t e s t , although the prompt response o f me g a l o b l a s t i c anemia t o p h y s i o l o g i c amounts o f f o l i c a c i d would be the most c o n c l u s i v e evidence ( H a l l e t a l , 1975). C. Iron i n d i c e s r e l a t e d t o f o l a t e s t a t u s An examination o f f o l a t e s t a t u s should i n c l u d e measurements i n d i c a t i v e o f i r o n s t a t u s , s i n c e the e x i s t e n c e o f i r o n d e f i c i e n c y c o u l d have a s i g n i f i c a n t e f f e c t on f o l a t e assessment. Hemoglobin values are used as an index o f the s e v e r i t y of i r o n d e f i c i e n c y anemia (WHO S c i e n t i f i c Group on N u t r i t i o n a l Anemias, 1968). Hemoglobin con c e n t r a t i o n s w i l l decrease when f o l a t e i s d e f i c i e n t s i n c e f o l i c a c i d i s u t i l i z e d i n hemoglobin s y n t h e s i s . The mean cor p u s c u l a r hemoglobin c o n c e n t r a t i o n (IVCHC) * r e f l e c t s b l o o d c e l l morphology and i s a st r o n g i n d i c a t o r o f i r o n d e f i c i e n c y when values are low. The mean co r p u s c u l a r volume (MCV)** increases i n c o n d i t i o n s o f f o l a t e d e f i c i e n c y f o l l o w i n g the development o f macrcovalocytosis (Herbert, 1967; Armstrong e t a l , 1974; and Colman e t a l , 1975). When comparing index values t o refe r e n c e standards i t i s important t h a t the standards being used are a p p l i c a b l e t o the p o p u l a t i o n under study. S i g n i f i c a n t r a c i a l and geographic d i f f e r e n c e s i n hemoglobin and hematocrit norms have been r e p o r t e d (Gam e t a l , 1974 and Owen e t a l , 1975). * MCHC (%) = HEMOGLOBIN (g/100 ml) HEMATOCRIT (%) ** MCV (u 3) = 10 x HEMATOCRIT (%) RED CELL COUNT (MILLIONS/MM3) 7 In summary, i r o n i n d i c e s which are important f o r assessment of f o l i c a c i d n u t r i t i o n a l s t a t u s i n c l u d e measures o f hemoglobin c o n c e n t r a t i o n , h e m a t r o c r i t , mean cor p u s c u l a r hemoglobin c o n c e n t r a t i o n (MCHC) and mean corpuscular volume (MCV). D. The f o l a t e assay Rad i o a c t i v e methods o f f o l a t e a n a l y s i s have been developed and are being used i n c r e a s i n g l y . A t present, f o l a t e l e v e l s i n serum, r e d c e l l s and ot h e r b i o l o g i c a l samples are most commonly measured by m i c r o b i o l o g i c a l assay procedures, u t i l i z i n g L a c t o b a c i l l u s c a s e i , Streptococcus f a e c a l i s , o r Pediococcus cerevesiae. L a c t o b a c i l l u s c a s e i i s the choice organism f o r e v a l u a t i n g human f o l a c i n n u t r i t i o n a l s t a t u s s i n c e i t responds t o the f o l a t e forms present i n serum and red blood c e l l s (Sauberlich e t a l , 1974). There i s good agreement w i t h i n and between l a b o r a t o r i e s f o r L. c a s e i f o l a t e assay r e s u l t s (WHO Group, N u t r i t i o n a l Anemias, 1972) , y e t due t o the assay's complexity, constant monitoring and the r e g u l a r use o f reference preparations are necessary. G u i d e l i n e s have been s e t f o r the i n t e r p r e t a t i o n of serum f o l a t e and r e d c e l l f o l a t e v a l u e s . They must be considered i n terms o f the m i c r o b i o l o g i c a l assay procedure and the t e s t organism employed (Refer t o Appendix D f o r g u i d e l i n e s ) . D i e t a r y F o l a t e s F o l i c a c i d d e f i c i e n c y commonly r e s u l t s from an inadequate 8 d i e t , u s u a l l y a s s o c i a t e d w i t h a l c o h o l i s m , i n c r e a s e d p h y s i o l o g i c a l requirements, food faddism, poverty, o r ignorance ( S u l l i v a n , 1967). I t i s important t h a t d i e t a r y f o l a t e l e v e l s are evaluated when diagnosing f o l i c a c i d d e f i c i e n c i e s . S a n t i n i and Corcino (1974) s t r e s s e d t h a t d i e t a r y s t u d i e s are e s s e n t i a l f o r determining both the e t i o l o g y and the proper treatment of n u t r i t i o n a l anemias. A. Ihe nature and assay o f d i e t a r y f o l a t e s F o l a t e s are present i n a wide v a r i e t y of foods, but f o l i c a c i d ( p t e r o y l g l u t a m i c a c i d , PGA) c o n s t i t u t e s o n ly 5% o f the t o t a l f o l a t e (Hurdle, 1973). F o l a t e s c o n s i s t mainly o f f o l i c a c i d bound t o a peptide chain o f one to s i x L-gluatamic a c i d r esidues i n v-carboxy l i n k a g e (Bernstein e t a l , 1970) 5' 6' Folic acid Polyglutamates of Folic acid n. $ 5 Figure 1: F o l i c A c i d and i t s Polyglutamates* M i c r o b i o l o g i c a l assay i s the most common method o f a n a l y s i s of * B e r n s t e i n e t a l (1970) 9 food folate activity. The different forms of folate vary with respect to their a v a i l a b i l i t y for assay organisms. According to Hoppner et a l (1972) and O'Broin et a l (1975), L. casei responds to most of the known pteroylglutamates with up to three L-glutamate residues. Food folate can be classified into "free", "conjugated" and "total" fractions (Hoppner et a l , 1972). The folate available prior to enzymatic hydrolysis with conjugase i s referred to as "^ree" folate, while that which i s active only after enzymatic hydrolysis i s referred to as "conjugated". "Total" folate consists of both the "free" and "conjugated" forms. There i s uncertainty concerning the availability of different polyglutamate forms for absorption, and once absorbed, for u t i l i z a t i o n (Tamara and Stokstad, 1973). During digestion intestinal conjugase cleaves polyglutamyl folates to monoglutamyl forms i n preparation for absorption (Butterworth et a l , 1969), while similar cleavage also occurs i n the food i t s e l f due to endogenous conjugase activity (O'Broin et a l , 1975). Baugh et a l (1971) and Baugh et a l (1975) confirmed the biological availability of monoglutamyl forms of folate in a mammalian system, and showed that polyglutamyl forms are also readily absorbed as such from the lumen in the proximal jejunum of the dog. Butterworth et a l (1969) demonstrated that ingested polyglutamates of f o l i c acid are cleaved to the monoglutamate form i n man i n the process of absorption, although the site of cleavage cannot be stated with certainty. There can be l i t t l e doubt that man i s capable of deriving his nutritional folate requirement from conjugated forms present i n the diet (Butterworth et a l , 1969). The "total" folate value of food cannot be regarded as the amount available 10 for absorption, since i n vivo and i n vitro conjugase activity are not necessarily the same. Pietarinen (1975) suggested that possibly the amount of available folate l i e s somewhere between the values representing the "free" and "total" fractions. B. The estimation of dietary folate consumption The determination of folate levels i n food may be affected by many factors other than the assay i t s e l f such as the r e l i a b i l i t y of food collections, the interest, intelligence, and education levels of subjects involved i n doing the collection, the storage f a c i l i t i e s for the food samples and the storage time (Moscovitch and Cooper, 1973). Heat lab i l e , water soluble folates are lost during cooking and processing of food, with losses increasing when large amounts of water are used (Herbert, 1962). There are few reliable food composition tables for estimating dietary folate consumption (Thenen, 1975), and values are available for a limited amount of foods. Food folate values reported prior to the use of ascorbate i n the assay procedure are exceptionally low (Chanarin et a l , 1972), since ascorbic acid prevents the oxidation of labile folates, keeping them available for the test organism. Hurdle (1973) reported folate values up to 40 times higher i n foods assayed with ascorbate as compared to without. The nutrient values for food items derived from direct analysis of the food do not necessarily agree exactly with values calculated from food tables since they provide somewhat different information (Whiting and Leverton, 1970). Chemical analysis provides a precise measure of the nutrients in the foods as eaten, whereas calculation provides an average nutritive value of a diet made up of similar foods. 11 The 24-hour r e c a l l i s considered one of the most s u i t a b l e methods f o r c o l l e c t i n g d i e t a r y data i n l a r g e surveys s i n c e i t i s r e a d i l y a p p l i e d t o wide p o p u l a t i o n groups, i r r e s p e c t i v e of age, education and i n t e l l i g e n c e (Mongeau, 1973). S i g n i f i c a n t l i m i t a t i o n s o f the 24-hour r e c a l l are i t s heavy dependence on the sharpness o f the s u b j e c t s memory and i t s questionable r e p r e s e n t a t i o n o f the h a b i t u a l d i e t . A p p l i e d t o Indian p o p u l a t i o n s , Sabry (1975) questioned the value o f 24-hour r e c a l l s , as Indians are accustomed t o famine or f e a s t e a t i n g p a t t e r n s , where the a v a i l a b i l i t y o f food i s h i g h l y v a r i a b l e . According t o Fidanza (1974) 24-hour r e c a l l s have l i m i t e d use when e v a l u a t i n g the n u t r i t i o n a l s t a t u s of i n d i v i d u a l s . I t i s more v a l i d t o estimate group i n t a k e s u s i n g mean values from 24-hour r e c a l l s . Studies A s s e s s i n g F o l a t e Status Laboratory f o l a t e analyses s u i t a b l e f o r use i n community surveys have only r e c e n t l y become p e r f e c t e d , so t h a t few surveys of f o l a t e n u t r i t i o n a l s t a t u s have been undertaken (WHO Group, N u t r i t i o n a l Anemias, 1972). There i s general agreement t h a t measurement o f serum and red c e l l f o l a t e i s the most p r a c t i c a l and meaningful procedure f o r e v a l u a t i n g f o l a t e s t a t u s i n p o p u l a t i o n groups. F o l a t e r e s u l t s should be i n t e r p r e t e d i n terms other than mean values alone, s i n c e t h i s c o u l d obscure the e x i s t e n c e of a s u b s t a n t i a l number o f low f o l a t e values i n d i c a t i v e o f widespread d e f i c i e n c y (Herbert, e t a l , 1975). F o l a t e r e s u l t s could be expressed i n terms of percent subnormal o r d e f i c i e n t v a l u e s , o r by r i s k c a t e g o r i e s w i t h percent d i s t r i b u t i o n i n each r i s k category. A. P o p u l a t i o n surveys A Barbados N u t r i t i o n Survey (1972) assessed the f o l a t e s t a t u s o f 12 415 male and female s u b j e c t s o f a l l age groups. Serum f o l a t e values O ng/ml and/or r e d c e l l f o l a t e 4.Q0 ng/ml were c l a s s e d as i n d i c a t i n g d e f i c i e n c y . T h i r t y - t h r e e percent of the preschool c h i l d r e n were anemic, due almost e q u a l l y t o i r o n and f o l a t e d e f i c i e n c y . Twenty percent o f the a d u l t women were anemic, and w h i l e most o f the anemias were due t o i r o n d e f i c i e n c y , there was a l s o a s i g n i f i c a n t i n c i d e n c e of f o l a t e d e f i c i e n c y anemia. Very few o l d e r c h i l d r e n and a d u l t men had anemia, suggesting t h a t hookworm i n f e s t a t i o n s o r other non-dietary f a c t o r s were not l i k e l y important cases of anemia, s i n c e they would have a f f e c t e d the d i f f e r e n t age and sex groups more e q u a l l y . Food consumption and socioeconomic f i n d i n g s i n d i c a t e d t h a t anemia was l a r g e l y due t o d i e t a r y i n s u f f i c i e n c y of food f o l a t e and i r o n . The v i t a m i n and f o l a t e s t a t u s of 52 a d u l t s i n a Himalayan v i l l a g e of Nepal was s t u d i e d i n e a r l y f a l l (Adams and Man S h r e s t a , 1974). Despite the r e s t r i c t e d food a v a i l a b i l i t y there was no evidence o f f o l a t e o r v i t a m i n B^ 2 d e f i c i e n c y based on serum and red c e l l measurements. I n a study conducted the previous s p r i n g i n a s i m i l a r high a l t i t u d e v i l l a g e 25% o f the 67 subjects had d e f i n i t e polymorphonuclear hypersegmentation, suggesting B^ o r f o l a t e d e f i c i e n c y . The seasonal a v a i l a b i l i t y o f f o l a t e and v i t a m i n B^ 2 i n these h i g h a l t i t u d e areas was suggested as having a s i g n i f i c a n t i n f l u e n c e on d i e t a r y i n t a k e s and deserved f u r t h e r e v a l u a t i o n . The v a r y i n g i n c i d e n c e of megaloblastosis found i n other s t u d i e s has been a t t r i b u t e d t o d i e t a r y f o l a t e d e f i c i e n c y i n off-season p e r i o d s when the supply o f f r e s h f r u i t s and vegetables i s low (Gatenby, 1956; Thompson, 1957; Coyle and Geoghegan, 1962; P e r e i r a and Baker, 1966 and Chanarin e t a l , 1968). 13 D i e t a r y and plasma f o l a t e s were evaluated i n h e a l t h y adolescents i n Birmingham, Alabama (Daniel e t a l , 1975). The data were examined i n r e l a t i o n t o age and sex m a t u r i t y r a t i n g s (SMR) according t o c l a s s i f i c a t i o n s s e t by Tanner (1966). Females had h i g h e r plasma f o l a t e than males across a l l sex m a t u r i t y r a t i n g s , y e t t h e i r d i e t a r y f o l a t e i n t a k e s were lower. In both sexes, i n c r e a s e d m a t u r i t y was a s s o c i a t e d w i t h a decrease i n plasma f o l a t e v a l u e s , y e t d i e t a r y f o l a t e i n t a k e i n c r e a s e d , p o s s i b l y i n d i c a t i n g g r e a t e r t i s s u e and c e l l u l a r f o l a t e demands a s s o c i a t e d w i t h adolescent growth. V i t a m i n B ^ arid f o l a t e s t a t u s was assessed i n a group of 562 Seventh-Day A d v e n t i s t s i n A u s t r a l i a (Armstrong e t a l , 1974). Seventy-seven percent of the s u b j e c t s were v e g e t a r i a n . Vegetarians had a s i g n i f i c a n t l y h i g h e r mean serum f o l a t e than the non-vegetarians. From a study r e p o r t i n g s i m i l a r f i n d i n g s ( E l l i s and M o n t e g r i f f o , 1970), i t was p o s t u l a t e d t h a t e l e v a t e d serum f o l a t e i n vegetarians was a consequence o f impaired f o l a t e metabolism, r e s u l t i n g from d e f i c i e n c y . I n the study by Armstrong e t a l (1974) serum v i t a m i n B^ l e v e l s were s i g n i f i c a n t l y lower i n vegetarians than s u b j e c t s who consumed meat and eggs, w h i l e those w i t h subnormal serum B^ values ( i . e . 26% o f the vegetarians) had s i g n i f -i c a n t l y h i g h e r MCV and MCHC and lcwer r e d c e l l f o l a t e than the r e s t of the group. S i m i l a r f i n d i n g s were a l s o r e p o r t e d by E l l i s and M o n t e g r i f f o (1970). Colman e t a l (1975) r e p o r t e d widespread f o l a t e d e f i c i e n c y from a study o f 469 Negro a d u l t s i n a d i s t r i c t o f Nqutu, Kwa Zulu, South A f r i c a . The s u b j e c t s chosen were h e a l t h y , e x c l u d i n g pregnant women w i t h hemoglobins l e s s than l l g / 1 0 0 ml from the study and e x c l u d i n g non-pregnant s u b j e c t s r e c e i v i n g medical a t t e n t i o n . The i n c i d e n c e o f subnormal r e d c e l l f o l a t e s 14 ( 160ng/ml) i n men, non-pregnant women, and pregnant women was 18.6%, 32.1% and 43.8% r e s p e c t i v e l y . F o l i c a c i d d e f i c i e n c y anemia was not repo r t e d , nor were any unequivocal cases o f v i t a m i n d e f i c i e n c y . From these r e s u l t s i t was recommended t h a t maize meal be f o r t i f i e d a t such a l e v e l t h a t an average o f 200 pg f o l i c a c i d would be consumed per day. This would safeguard a g a i n s t f o l a t e d e f i c i e n c y i n a l l segments of the po p u l a t i o n . A n u t r i t i o n survey conducted i n K i r y a t Shmoneh, an Upper G a l i l e e community i n I s r e a l , r e p o r t e d a h i g h i n c i d e n c e o f f o l a t e - a n d i r o n -d e f i c i e n c y anemia i n women o f c h i l d b e a r i n g age and i n c h i l d r e n (Levy e t a l 1975). From 26 f a t h e r s , 196 mothers and 160 c h i l d r e n s t u d i e d , the prevalence o f sub-normal r e d c e l l f o l a t e s was 0%, 47% and 53% r e s p e c t i v e l y . Mean d a i l y i n t a k e s f o r the same groups were 265 pg, 160 pg, and 158 pg t o t a l f o l a t e , the i n t a k e f o r a d u l t men being s i g n i f i c a n t l y g r e a t e r than f o r women or c h i l d r e n . S i m i l a r trends were ev i d e n t f o r i r o n , where low i n t a k e s i n women and c h i l d r e n c o r r e l a t e d w i t h subnormal serum i r o n values. T h i s survey s u b s t a n t i a t e d previous assumptions t h a t f o l a t e and i r o n - d e f i c i e n c y anemia was r e l a t e d , a t l e a s t i n p a r t , t o low i n t a k e s of i r o n and f o l i c a c i d . The n u t r i t i o n a l s t a t u s o f 41 f a m i l i e s was assessed i n Macon County, Alabama (Prothro e t a l , 1976). The sample o f 102 i n d i v i d u a l s was 76% b l a c k and 24% nonblack. Mean serum f o l a t e s f o r a l l age groups were low (^5ng/ml) , w i t h 20% o f the s u b j e c t s showing serum values ^3ng/ml. Based on serum v a l u e s , f o l a t e d e f i c i e n c y was the most e x t e n s i v e n u t r i e n t d e f i c i e n c y . The lengthy cooking processes i n v o l v e d i n food p r e p a r a t i o n were thought t o be r e s p o n s i b l e f o r e x t e n s i v e d e s t r u c t i o n and l e a c h i n g o f food f o l a t e s , s i g n i f i c a n t l y l owering the v i t a m i n ' s a v a i l a b i l i t y . 15 Kaufman e t a l (1975) s t u d i e d the n u t r i t i o n a l s t a t u s of F l o r i d a seasonal farmworkers. The sample p o p u l a t i o n i n c l u d e d 973 households, about o n e - t h i r d Spanish-Americans and two-thirds b l a c k s . Iron d e f i c i e n c y was the most s e r i o u s n u t r i t i o n a l problem, y e t f o l a t e d e f i c i e n c y was a l s o s i g n i f i c a n t . Approximately 40% o f the b l a c k and 35% o f the Spanish-Americans had subnormal red c e l l f o l a t e v a l u e s . F o l a t e s t a t u s was s t u d i e d i n h e a l t h y white h o s p i t a l p e r s o n n e l , b l a c k c l i e n t e l e o f an urban h e a l t h c e n t r e , and b l a c k migrant F l o r i d a farm workers ( H a l l e t a l , 1975). I n search o f a refer e n c e group i t was r e a l i z e d t h a t plasma f o l a t e l e v e l s can vary s i g n i f i c a n t l y from one he a l t h y p o p u l a t i o n t o another, and d i r e c t comparisons are not n e c e s s a r i l y meaningful. I f in a p p r o p r i a t e reference values are used, a t e s t c o u l d r e a d i l y m i s l e a d more than inform (Bech, 1974), such t h a t f o l a t e l e v e l s of a p a r t i c u l a r p o p u l a t i o n c o u l d appear hig h o r low. C e r t a i n segments o f a p o p u l a t i o n show i n d i c a t i o n s o f be i n g p a r t -i c u l a r l y prone t o f o l a t e d e f i c i e n c y : ( i . e . ) pregnant women i n both developed and developing c o u n t r i e s ( S a u b e r l i c h e t a l , 1974; Baker e t a l , 1975, and Herbert e t a l , 1975) , low b i r t h weight i n f a n t s (Mathoth e t a l , 1964 b ) , a r t i f i c i a l l y f e d , as opposed t o b r e a s t f e d , i n f a n t s (Matoth e t a l , 1964 a ) , g e r i a t r i c p a t i e n t s (Hurdle and W i l l i a m s , 1966 and Hurdle, 1968) , and v i r t u a l l y a l l a l c o h o l i c s (Herbert and Zulusky, 1961 and Baker e t a l , 1975 b ) . Reports by Shojania e t a l (1971) , P i e t a r i n e n (1975) and Prasad e t a l (1975) i n d i c a t e t h a t women t a k i n g o r a l c o n t r a c e p t i v e s are prone t o f o l a t e d e f i c i e n c y w h i l e Spray (1968), P r i t c h a r d e t a l (1971) and Paine e t a l (1975) r e p o r t c o n t r a d i c t o r y evidence i n d i c a t i n g t h a t t h i s i s not so. 16 B. The Nutrition Canada Survey The Nutrition Canada Survey (1973) evaluated serum folate levels in a wide distribution of population groups i n Canada. Serum values of ^2.5 ng/ml were classified as high risk values, 2.5 to 5.0 ng/ml were moderate risk, and >5.0 ng/ml were low risk. According to the national report serum folate values were not significantly different between male and female, and children i n general had higher values than adults. Approximately one-half of the population was cla s s i f i e d at moderate risk and 10 to 20% of teenagers and adults at high risk. The percentage of Indians cl a s s i f i e d at both moderate and high risk was greater than for the general population i n most age categories (Nutrition Canada Indian Report, 1975). Indians i n remote centres showed a significantly higher proportion of risk values than Indians closer to urban centres. A higher percentage of Eskimos were at risk with respect to f o l i c acid than either the Indian or General Population groups (Nutrition Canada Report, 1975). 17 TABLE I C l a s s i f i c a t i o n of serum f o l a t e values f o r the general, Indian and Eskimo populations: Percentage high r i s k values i n d i f f e r e n t age categories (Nut. Can. Report, 1973) AGE AND SEX CATEGORIES '2S5ATICN 0-4 5-9 10-19 20-39 40-64 a 65+ a 10-19 20-39 40-64 a 65+ a MF MF M M M M F F F F General Population 9.6 4.8 10.3 11.1 14.0 17.7 13.5 21.2 12.5 13.7 Indian Population 3.1 7.7 14.3 14.8 24.4 19.5 12.1 23.1 19.5 26.8 Eskimo Po p u l a t i o n 8 . 5 27.2 51.5 69.6 58.1 77.2 40.9 84.4 65.5 77.8 a. Indian and Eskimo population (40-54 M&F, 54+ M&F). The survey report i n d i c a t e d that the low serum f o l a t e values of the Canadian people should be viewed with concern, although low values are not n e c e s s a r i l y i n d i c a t i v e of a protracted f o l a t e d e f i c i e n c y (Nutrition Canada Report, 1973). Lew serum values were not associated with the c l i n i c a l manifestation of f o l a t e - vitamin d e f i c i e n c y anemia. According t o Sabry (1975) the high incidence of low serum values was unexpected and therefore a d d i t i o n a l measurements f o r i n t e r p r e t i n g serum f o l a t e values were not emphasized. Red c e l l f o l a t e values are important as i n d i c a t o r s of body stores and the prevalence of f o l a t e d e f i c i e n c y (Omer e t a l , 1970 and L i u , 1974), blood smears give evidence of changes i n c e l l morphology (Sauberlich e t a l , 1974), and an estimate of d i e t a r y f o l a t e intake from an accurate set of food tables i s important f o r determining the cause of low serum values. 18 (Herbert, 1962 and Hoppner e t a l , 1972). There i s general agreement t h a t the f o l a t e s t a t u s o f Canadian people i s s t i l l i n qu e s t i o n and i s an area o f concern t h a t deserves vigorous follow-up s t u d i e s (Sabry, 1975). C. F o l i c a c i d s t a t u s of n a t i v e Indians White men have had both p o s i t i v e and negative e f f e c t s on the l i v e s o f our n a t i v e people (Haworth, 1975), w h i l e many changes i n Indian l i f e s t y l e have a d i r e c t i n f l u e n c e on t h e i r n u t r i t i o n a l s t a t u s (Lee e t a l , 1971). Indian c h i l d r e n across Canada are r a i s e d i n such d i v e r s e s o c i a l s e t t i n g s t h a t i t i s d i f f i c u l t t o g e n e r a l i z e w i t h r e s p e c t t o t h e i r n u t r i t i o n a l s t a t u s and speak o f a " t y p i c a l Indian c h i l d " (Smith, 1975). The v a r i a b i l i t y i n the n u t r i t i o n a l h a b i t s depends on the r e l a t i v e importance o f c e r t a i n i n f l u e n c e s such as the b a s i c t r a d i t i o n a l economy, the settlement p a t t e r n s , the a v a i l a b i l i t y o f white man's food, and the impact o f t h e i r t e a c h i n g (Smith, 1975). The n u t r i t i o n a l s t a t u s o f d i f f e r e n t North West Coast Indian populations v a r i e s enough t h a t i t would be mi s l e a d i n g t o g e n e r a l i z e from one group t o another (Desai and Lee, 1974). Foods known t o be h i g h i n f o l i c a c i d such as f r e s h f r u i t s and vegetables are l a c k i n g i n many Ind i a n and Eskimo d i e t s . A survey o f Bush Indians i n Northern Manitoba i n 1942 found t h e i r d i e t s d e f i c i e n t i n almost a l l e s s e n t i a l n u t r i e n t s , w i t h i n t a k e s o f f r u i t s and vegetables b e i n g extremely low (Moore, 1946). Indian c h i l d r e n from A l e r t Bay, B r i t i s h Columbia consumed very few green and y e l l c w vegetables (Dong and Feeney, 1968) and d i e t s o f Indians from i s o l a t e d reserves i n B r i t i s h Columbia were p a r t i c u l a r l y low i n f r e s h vegetables (Lee e t a l , 1971). According t o 19 Smith (1975) the s e l e c t i o n o f f r e s h produce i s o f t e n r e s t r i c t e d i n i s o l a t e d p l a c e s , canned vegetables are o f t e n not a v a i l a b l e , and n a t i v e vegetables are seldom used. The recent N u t r i t i o n Canada Survey has served as an important focus on p o t e n t i a l and r e a l problems i n the n u t r i t i o n o f Indian people (Fraser, 1975). According t o the Indian Survey Report (1975) the f o l a t e s t a t u s , as i n d i c a t e d by the h i g h i n c i d e n c e o f low serum f o l a t e v a l u e s , was g e n e r a l l y poor f o r the t o t a l I n d i a n p o p u l a t i o n but tended t o be lower f o r Indians i n i s o l a t e d areas as compared t o those c l o s e r t o urban c e n t r e s . Beaton (1975) proposed t h a t extremely low serum f o l a t e values i n the Eskimo people c o u l d be a t t r i b u t e d t o the l a c k of f r e s h f r u i t s and vegetables i n t h e i r d i e t . The N u t r i t i o n Canada Survey d i d not analyze o r c a l c u l a t e d i e t a r y f o l a t e i n t a k e s ( N u t r i t i o n Canada Report, 1973) so one can o n l y speculate t h a t low serum f o l a t e values were a consequence of low d i e t a r y f o l a t e i n t a k e s . 20 CHAPTER II INTRODUCTION The native population of Br i t i s h Columbia consists of about 52,000 people, l i v i n g primarily on reserves. Their way of l i f e i s based on a culture and tradition which tends to maintain them as a separate people. On the other hand, internal and external pressures are causing radical changes in the Indian l i f e s t y l e and are creating new problems and tensions for them. Interaction between Indian and non-Indian communities is increasing with the continuous development of highways and transportation f a c i l i t i e s throughout the province. Indians are being forced to adjust from 'living o f f the land' to l i v i n g the l i f e of a wage-earner. As Bryans (1967) stated: "The native Canadian i s i n a state of turmoil, trying to grasp the future with one hand and hold on to the old values with the other, moving from an ancient culture to the 20th century without the opportunity to evolve slowly over several generations." Changes which affect the Indians 1 l i f e s t y l e w i l l also have direct influence on their nutritional status. If nutritional problems of Indians livi n g on reserves were identified and characterized, nutrition services and education programs could be more readily established to tend with the problems effectively. Although st a t i s t i c s are available concerning the morbidity and mortality of Canadian Indians i n general, less i s known about their health as related to nutritional practices. 21 The recent Nutritional Canada Survey (1973) has served to point out some major problems i n the nutrition of Canadian people. Results indicate that the nutritional status of native Indians in Canada i s poorer than i s that of the general population. F o l i c acid i n particular was suggested as being a nutrient of major concern. There was a high incidence of low serum folate values i n the general population, but an even higher incidence i n the Indian population. Folate values were lower for Indians on isolated as compared to non-isolated reserves. Since the prevalence of folate deficiency cannot be measured from serum values alone, i t was recommended that further studies be conducted to more f u l l y assess the problem. Few studies have been published which assess the nutritional status of British Columbia Indians,and none, other than the Nutrition Canada Survey, have directly studied their folate status. Therefore, a need is indicated for further research i n this area. This present study was conducted to assess the folate status of native Indians i n British Columbia. Two hundred and f i f t y subjects from seven reserves throughout the province and 70 subjects from an Indian Residential School i n Sechelt, B. C. participated. Not a l l subjects participated i n a l l parts of the study. This study primarily set out to determine i f there really was a problem with respect to folate status among these people. If poor folate status was indicated by a high prevalence of subnormal serum folate values (i.e. less than 5 ng/ml) then the data reported by Nutrition Canada would be confirmed. Additional hematological factors, including red c e l l folate determinations, thin blood smear preparations for microscopic examination, 22 hemoglobin and hematocrit determinations and white blood cell and red blood cell counts were included as essential for the adequate assessment of folate status. Twenty-four hour dietary recalls were conducted and the data were used to calculate total folate, calorie, and iron intakes from food composition tables. In addition, food collections representing a 24-hour consumption pericd were obtained and total folate values were derived by direct folate analysis and calculation from food tables. 23 CHAPTER I I I MATERIALS AND METHODS Sample Population The study of f o l a t e status i n B r i t i s h Columbia Indians was d i v i d e d i n t o two parts. In the f i r s t p a rt of the study, a 24-hour r e c a l l was used to assess d i e t a r y f o l a t e status at seven reserves throughout B r i t i s h Columbia i . e . Sechelt Indian Reserve, Sechelt; F o r t Ware Indian Reserve, Ware; Necoslie Indian Reserve, F o r t Saint James; Nanaimo Indian Reserve, Nanaimo; Nazko Indian Reserve, Quesnel; and Lake Babine and Takla Lake Reserves. (See Figure 2 f o r l o c a t i o n s ) . Reserves were c l a s s i f i e d with respect to t h e i r s tate of ' i s o l a t i o n 1 where ' i s o l a t i o n ' was assessed i n terms of the distance from urban centres. Reserves located i n o r adjacent to urban centres of le s s than 5,000 people were classed as i s o l a t e d and those located i n or adjacent to urban centres of greater than 5,000 people were classed as non-isolated. Isolated reserves had, a t most, one general s t o r e with a l i m i t e d a v a i l a b i l i t y and s e l e c t i o n of food s t u f f s . A t l e a s t two food stores or supermarkets were- located on or adjacent to the non-isolated reserves, o f f e r i n g a wider s e l e c t i o n of food s t u f f s . Permanent hea l t h personnel o r h o s p i t a l f a c i l i t i e s were a v a i l a b l e f o r the non-isolated but not the i s o l a t e d reserves. The second part o f the study was a more comprehensive assessment o f f o l a t e status, i n v o l v i n g the use o f d i e t a r y r e c a l l s , 24-hour food sample c o l l e c t i o n s , and serum and whole blood f o l a t e measurements. This was 24 Figure 2: B r i t i s h Columbia Indian Reserves Involved i n the Study o f F o l i c A c i d N u t r i t i o n a l S t a t u s . 2 5 26 conducted among a subsample o f the s u b j e c t s a t S e c h e l t I n d i a n Reserve, F o r t Ware Reserve, and N e c o s l i e Reserve, as w e l l as among a group o f c h i l d r e n l i v i n g a t the S e c h e l t Indian R e s i d e n t i a l School, S e c h e l t , B. C. Seventy c h i l d r e n ranging from 5 t o 16 years (34 male and 36 female), r e p r e s e n t i n g 90% o f the t o t a l e nrollment, p a r t i c i p a t e d from the R e s i d e n t i a l School. A summary o f reserves i s presented i n Table I I , i n d i c a t i n g the po p u l a t i o n s i z e o f each r e s e r v e , the sample s i z e f o r both p a r t s o f the f o l a t e study, and the s t a t e o f ' i s o l a t i o n ' , l i n g u i s t i c group, and c u l t u r a l area o f each reserve. Subjects and reserves were not s e l e c t e d randomly, and were t h e r e f o r e not considered t o be r e p r e s e n t a t i v e o f a l l B r i t i s h Columbia reserve Indians. The S e c h e l t Indian residence and the seven reserves were s e l e c t e d i n c o n s u l t a t i o n w i t h M e d i c a l Services,Department o f N a t i o n a l Health and Welfare, i n order t o assure a c c e s s a b i l i t y and l o c a l cooperation. Approval f o r t h i s research was obtained from the Health Sciences Screening Committee, U n i v e r s i t y o f B r i t i s h Columbia, from the reserve Band C o u n c i l s , and from each s u b j e c t who p a r t i c i p a t e d . Consent forms were s i g n e d by an a d u l t o r r e s p o n s i b l e f a m i l y member o f each household which p a r t i c i p a t e d i n the more comprehensive p a r t o f the study, which r e q u i r e d both a d i e t a r y assessment and blood samples from each s u b j e c t . (See Appendix A f o r a copy o f the consent form). TABLE II Indian Reserves i n British Columbia involved i n the assessment of folate status Sample Size Linguistic Cultural State of Population 1. Dietary folate 2. Comprehensive Folate Group Area Isolation Size* study: 24-hr. Study: Dietary and Recalls Hematological Measures Reserve Sechelt Salishan Pacific Coast Non-isolated 380 53 30 Fort Ware Athapaskan MacKenzie R. Isolated 165 32 28 Necoslie Athapaskan Plateau Non-isolated 534 35 33 Nanaimo Salishan Pa c i f i c Coast Non-isolated 415 57 • Nazko Athapaskan Plateau Isolated 100 23 Lake Babine Athapaskan Plateau Isolated 115 21 Takla Lake Athapaskan Plateau Isolated 265 30 Represents the number of status Indians presently l i v i n g on the reserve 28 D i e t a r y R e c a l l s , Food C o l l e c t i o n s and A n a l y s i s A. D i e t a r y r e c a l l s and a n a l y s i s Twenty-four hour d i e t a r y r e c a l l s were conducted on the seven reserves. Tne m a t r i a r c h i a l head o f the household was i n t e r v i e w e d and asked t o r e c a l l a l l food items consumed by h e r s e l f and o t h e r members i n the preceding 24 hours. Other a d u l t s and o l d e r c h i l d r e n present a t the time o f the i n t e r v i e w were a l s o i n t e r v i e w e d . * The 24-hour r e c a l l data were coded i n t o D i e t a r y A n a l y s i s Coding B o o k l e t s , together w i t h the s u b j e c t ' s sex, age and i d e n t i f i c a t i o n number. T o t a l f o l a t e , i r o n and c a l o r i c i n t a k e s were c a l c u l a t e d f o r each s u b j e c t . D i e t a r y f o l a t e was c a l c u l a t e d u s i n g values from the p u b l i s h e d t a b l e s o f B u t t e r f i e l d and Calloway (1972), Hoppner e t a l (1972) , and Hurdle e t a l (1968). Food items not l i s t e d i n the composition t a b l e s were i n d i v i d u a l l y analyzed f o r t o t a l f o l a t e as di s c u s s e d i n the f o l l o w i n g s e c t i o n . Food values f o r i r o n and c a l o r i e s were c a l c u l a t e d from U.S. Department o f A g r i c u l t u r e N u t r i e n t Composition values. The 24-hour r e c a l l d a t a were com-puted f o r the comprehensive f o l a t e s t a t u s study as w e l l , s i n c e the s u b j e c t s i n v o l v e d were a subgroup from the l a r g e r d i e t a r y study sample. B. C o l l e c t i o n o f food samples and a n a l y s i s For the more comprehensive p a r t o f the study, i n a d d i t i o n t o 24-hour d i e t a r y r e c a l l s , food samples r e p r e s e n t i n g a 24-hour consumption p e r i o d were c o l l e c t e d from S e c h e l t Residence and from S e c h e l t and N e c o s l i e reserves. *A11 24-hour d i e t a r y r e c a l l s were conducted by Yolanda St e p i e n , Masters student i n Human N u t r i t i o n , School o f Home Economics, U. B. C. 29 There was l i t t l e variety i n the foods consumed at Fort Ware and consistent eating patterns were seldom followed. Therefore, instead of collecting 24-hour food samples at Fort Ware, samples were collected of the major food items consumed i n the diet, as determined from the 24-hour recalls. Plastic buckets containing 500 ml phosphate buffer (pH6.1) and 150 mg% ascorbic acid were used to collect the 24-hour food samples. Samples from the residential school were prepared by observing the meals and snacks eaten by the children and collecting duplicate food samples representative of a child's total consumption over the 24-hour period. Records were kept of a l l food items added to the bucket. The 24-hour food samples from the two reserves were collected and recorded by women from each household. They were instructed to add to the bucket duplicate samples of a l l foods they had eaten for one 24-hour period, and to keep a written record of the amount of every item that was added. A 24-hour r e c a l l for the collection period was used to cross-check the written record. Food buckets were kept refrigerated during the sample collection. After collection the food samples were homogenized for one minute in a Waring Blender. Aliquots of homogenate were immediately refrigerated i n a i r tight containers to prevent oxidation and frozen at -20°C within 24 hours. These samples were used for food folate analysis. Individual food samples from Fort Ware were homogenized and stored i n a similar manner. Total folate values for the 24-hour food collections and individual food samples were determined by the Lactobacillus casei microbiological method of Herbert (1963) , u t i l i z i n g chicken pancreas conjugase.* Bacto-Lactobacilli Broth AOAC** was used as the maintenance * Difco Chicken pancreas, from Difco Laboratories, Detroit, Michigan ** Difco cer t i f i e d , from Difco Laboratories, Detroit, Michigan. 30 c u l t u r e and Bacto F o l i c A c i d C a s e i Medium* was the assay medium. Two groups o f pooled s e r a were run as c o n t r o l s w i t h each assay, a 'low f o l a t e 1 p o o l (1.2 t o 2.0ng/ml) and a 'high f o l a t e ' p o o l (10.5 t o 12.0ng/ml). F o l i c a c i d ( p t e r o y l - g l u t a m i c acid) "Baker Grade"** was used t o prepare standard f o l a t e s o l u t i o n s . Blood C o l l e c t i o n , Treatment, and A n a l y s i s A. C o l l e c t i o n and treatment o f b l o o d samples The comprehensive p a r t o f the f o l a t e study i n v o l v e d c o l l e c t i o n s o f blood samples from each s u b j e c t . Blood was c o l l e c t e d a f t e r a minimum f a s t o f 3 hours. Three venous blood samples were obtained from each s u b j e c t ; one i n t o a 10ml v a c u t a i n e r tube*** w i t h no a d d i t i v e , the second i n t o a 4.5ml v a c u t a i n e r c o n t a i n i n g 0.5ml o f 3.8% sodium c i t r a t e , and the t h i r d i n t o a 7ml v a c u t a i n e r c o n t a i n i n g 10.5 mg. disodium ethylenediamine t e t r a a c e t i c a c i d (EDTA). The whole blood c o l l e c t e d without a n t i c o a g u l a n t was kept a t room temperature f o r approximately 1 hour t o a l l o w f i r m c l o t formation. I t was then c e n t r i f u g e d a t 2000 rpm f o r 15 minutes and the serum was t r a n s -f e r r e d t o p l a s t i c tubes **** w i t h a pasteur p i p e t t e . The s e r a were f r o z e n a t -20°C w i t h i n 8 hours o f c o l l e c t i o n and s t o r e d f o r serum f o l a t e a n a l y s i s . A microhematocrit was performed on c i t r a t e d blood samples and the remaining whole blood was f r o z e n a t -20°C w i t h i n 8 hours o f c o l l e c t i o n and s t o r e d f o r red c e l l f o l a t e a n a l y s i s . * D i f c o c e r t i f i e d , from D i f c o L a b o r a t o r i e s , D e t r o i t , Michigan. ** J . T. Baker Chemical Co., New J e r s e y L o t No. 1-755 *** Vacutainer tubes, Becton, Dickenson and Co., Canada, L i m i t e d **** Falcon p o l y s t y r e n e tubes. 31 A t h i n blood smear was prepared from samples c o l l e c t e d i n t o EDTA - c o n t a i n i n g v a c u t a i n e r s and was used f o r microscopic examination. Hemoglobin, hematocrit and white b l o o d c e l l counts, red blood c e l l counts, and r e d c e l l i n d i c e s were determined on the EDTA-treated blood samples, w i t h a Model S C o u l t e r Counter. Blood samples were r e f r i g e r a t e d immediately a f t e r c o l l e c t i o n and were packed i n i c e d u r i n g t r a n s p o r t . B. Blood F o l a t e a n a l y s i s Serum and red c e l l f o l a t e s were assayed u s i n g L a c t o b a c i l l u s  C a s e i m i c r o b i o l o g i c a l methods. Serum f o l a t e was determined by the method o f Baker e t a l (1959) and r e d c e l l f o l a t e by the m o d i f i e d methods of Hoffbrand e t a l (1966) and Spray (1969). Serum and whole bl o o d samples were thawed f o r a n a l y s i s and immediately d i l u t e d w i t h sodium phosphate b u f f e r (pH 6.1) c o n t a i n i n g 150 mg% a s c o r b i c a c i d . The m a t e r i a l s used i n p r e p a r a t i o n o f the maintenance c u l t u r e , assay medium, and standard curve were those d e s c r i b e d f o r the assay o f food f o l a t e . The pooled c o n t r o l s e r a were run w i t h each assay. As a f u r t h e r c o n t r o l measure, s e l e c t e d serum and whole blood samples were a l s o assayed i n an independent l a b o r a t o r y . * S t a t i s t i c a l A n a l y s i s M l s t a t i s t i c a l analyses were performed w i t h an IBM 360/370 computer a t the computing centre o f the U n i v e r s i t y of B r i t i s h Columbia. A l i n e a r c o r r e l a t i o n a n a l y s i s was used t o measure the r e l a t i o n s h i p between the assayed and c a l c u l a t e d f o l a t e values from the 24-hour food c o l l e c t i o n s . The Pearson Product-Moment C o r r e l a t i o n C o e f f i c i e n t was c a l c u l a t e d . * Hematology Department, S a i n t Paul's H o s p i t a l , Vancouver, B. C. 32 The T r i p /360 program was employed t o compute the mean, standard d e v i a t i o n and c o r r e l a t i o n m a t r i x of the biochemical and d i e t a r y parameters, which were grouped according to sex, l o c a t i o n , and s p e c i f i c age c a t e g o r i e s . Regression a n a l y s i s was conducted f o r blood and food f o l a t e d a ta from three reserves i n v o l v e d i n the comprehensive p a r t of the study. Regression equations were t e s t e d f o r e q u a l i t y between sexes and the d i f f e r e n t l o c a t i o n s by means o f an S:SL Test ( E q u a l i t y of Slope T e s t ) . The TRIP r e g r e s s i o n program t e s t e d the goodness o f f i t f o r the r e g r e s s i o n l i n e s , t e s t i n g the r e l a t i o n s h i p between serum and r e d c e l l f o l a t e l e v e l s and d i e t a r y f o l a t e i n t a k e . The Student's t - t e s t w i t h p=.05 was used t o t e s t , f o r a g iven v a r i a b l e , s t a t i s t i c a l s i g n i f i c a n c e between two sample means. Comparisons were made f o r hematological and d i e t a r y v a r i a b l e s , grouping the means i n terms of sex, l o c a t i o n , or degree of i s o l a t i o n . A one-way a n a l y s i s of v a r i a n c e w i t h p-0.05 was chosen t o t e s t , f o r a given v a r i a b l e , s t a t i s t i c a l s i g n i f i c a n c e among three or more sample;:; means. Again, comparisons were made f o r hematological and d i e t a r y v a r i a b l e s , grouping the means by l o c a t i o n , sex and age c a t e g o r i e s . A Duncan's M u l t i p l e Range Test was performed, when means were s i g n i f i c a n t l y d i f f e r e n t , t o determine both homogeneous subsets w i t h i n the group of means and the means which d i f f e r e d s i g n i f i c a n t l y from the r e s t of the group. 33 CHAPTER IV RESULTS Di e t a r y F o l a t e Data A. Assayed v s . c a l c u l a t e d values R e s u l t s o f the Pearson Product - Moment Test o f C o r r e l a t i o n between assayed and c a l c u l a t e d t o t a l f o l a t e values f o r the 24-hour food c o l l e c t i o n s are presented i n Table I I I . An r-value o f .9694 suggests a h i g h l y s i g n i f i c a n t l i n e a r c o r r e l a t i o n between the assayed and c a l c u l a t e d v a l u e s , suggesting t h a t the computed f o l a t e values are a r e l i a b l e estimate o f the a c t u a l f o l a t e i n t a k e s . The s c a t t e r diagram f o r assayed and c a l c u l a t e d t o t a l f o l a t e values from the 24-hour food c o l l e c t i o n s (Figure 3) i l l u s t r a t e s the c o r r e l a t i o n between the v a r i a b l e p a i r s . The i n d i v i d u a l f o l a t e values are l i s t e d i n Appendix B. TABLE I I I Pearson Product-Moment C o r r e l a t i o n C o e f f i c i e n t : Test of c o r r e l a t i o n between assayed and c a l c u l a t e d values f o r t o t a l f o l a t e from 24-hour food c o l l e c t i o n s (21 p a i r e d v a l u e s ) . V a r i a b l e P a i r C o e f f i c i e n t s r * T o t a l F o l a t e Value Assayed vs. C a l c u l a t e d .9694 .9397 Measure o f l i n e a r C o r r e l a t i o n between the two v a r i a b l e s -100 x r % o f the v a r i a t i o n i n the values of one v a r i a b l e may be accounted f o r by the l i n e a r r e l a t i o n s h i p w i t h the other v a r i a b l e . 34 Figure 3: Scatter diagram showing the degree of correlation between calculated and assayed values of to t a l folate from 24-hour food collections. 3 5 4 0 0 H c 300 I fcxO Ok 3 o u_ I I — o 200 100 —^  • • •. 100 200 300 TOTAL FOLATE JJtig), MEASURED 36 B. Comparison o f mean f o l a t e i n t a k e s f o r the seven reserves A M u l t i p l e Range Test was performed on mean f o l a t e scores c a l c u l a t e d frcm the 24-hour d i e t a r y r e c a l l s conducted on the seven re s e r v e s . As shown i n Table IV mean i n t a k e s range from 125.4 ug t o t a l f o l a t e f o r F o r t Ware t o 236.2ug f o r Nanaimo. The h i g h e s t and lowest means are s i g n i f i c a n t l y d i f f e r e n t a t P<0.05. I n d i v i d u a l values f o r n u t r i e n t i n t a k e s are l i s t e d i n Appendix E. A M u l t i p l e Range Test was performed on mean f o l a t e scores f o r a l l seven reserves f o r d i f f e r e n t age and sex groupings (Table V ) . R e s u l t s i n d i c a t e t h a t mean f o l a t e i n t a k e s do not d i f f e r s i g n i f i c a n t l y among the three age c a t e g o r i e s i n the p o p u l a t i o n as a whole. S i g n i f i c a n t d i f f e r e n c e s are only seen f o r males, where 10 t o 19 year o l d s have a s i g n i f i c a n t l y lower mean i n t a k e than males 20 years o f age or o l d e r (P<0.05). I n g e n e r a l , these r e s u l t s i n d i c a t e t h a t f o l a t e consumption does not s i g n i f i c a n t l y i n c r e a s e w i t h age. TABLE IV M u l t i p l e Range Test o f mean scores f o r t o t a l f o l a t e i n t a k e f o r the seven reserves under study. L o c a t i o n N T o t a l F o l a t e Intake (ug/Day) F o r t Ware 26 a* 125.4 Takla Lake 30 153.5^ S e c h e l t 53 171.6^ Nazko 23 186.9^ N e c o s l i e 35 189.9^ Lake Babine 21 ah 200.9 Nanaimo 56 236.2b *Means not s h a r i n g the same s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t a t P(0.05 37 TABLE V Multiple Range Test of mean scores for food folate intake for sex and age groupings for the seven reserves studied. Age (Years) Sex N Total Folate Intake (pg/Day) 0-9 M&F 53 189.7a* 10-19 51 190.9a 20+ 146 191.6a 0-9 Male 17 248.6^ 10-19 25 210.6a 20+ 45 280.3 0-9 Female 36 161.8a 10-19 26 171.9a 20+ 101 152.l a *Means not sharing the same superscript within each group of 3 means are significantly different at P<0.05. A t-test was performed comparing mean scores for food folate intake for the seven reserves with respect to isolation and sex groupings. As Table VI shows, mean intake of total folate i s significantly greater on the non-isolated reserves as compared to the isolated reserves, both for the subjects as a whole, and for male and female subjects separately. The mean folate intake for males i s significantly greater than the mean for females (P<0.05). Generally the results indicate that diets are poorest with respect to dietary folate intakes for females on isolated reserves and are better for males, particularly on non-isolated reserves. As Table VII shows, the mean total folate intake for subjects from a l l seven reserves i s below the Recommended Daily Allowance for total 38 f o l a t e (200 ug f o r persons 1 t o 12 years o l d and 400 pg f o r those 13 years o f age and o l d e r ) . The mean c a l o r i e i n t a k e i s low as compared t o recommendations, f o r the females as a whole and f o r males from the i s o l a t e d r e s e r v e s . I r o n i n t a k e s are adequate except f o r females on n o n - i s o l a t e d r e s e r v e s , where the mean i s s l i g h t l y below recommendations. (Refer t o Appendix C f o r recommendations.) TABLE VT T-test o f mean scores f o r f o l a t e i n t a k e f o r the seven reserves w i t h r e s p e c t t o i s o l a t i o n and sex. Reserve T o t a l F o l a t e T-Test Values Grouping Sex N Mean + S. D. (pg/Day) T-Prob* F-Prob** Non-i s o l a t e d M&F 144 211.7+117.1 .000 .000 I s o l a t e d M&F 106 163.1± 76.2 Non - i s o l a t e d M 58 273.1+132.8 .046 I s o l a t e d M 29 . 216.2±106.6 Non- i s o l a t e d F 86 170.3± 83.2 .011 .000 I s o l a t e d F 77 143.1+ 48.5 A l l Reserves M 87 254.1+126.9 .000 .000 A l l Reserves F 163 157.4± 70.2 * P r o b a b i l i t y values P^0.05 i n d i c a t e s i g n i f i c a n t d i f f e r e n c e between sample means. Dashes (-) i n d i c a t e no s i g n i f i c a n t d i f f e r e n c e . ** P r o b a b i l i t y values P<0.05 i n d i c a t e s i g n i f i c a n t d i f f e r e n c e between sample v a r i a n c e s . Dashes (-) i n d i c a t e no s i g n i f i c a n t d i f f e r e n c e . 39 TABLE VTI Mean d a i l y i n t a k e o f f o l a t e , c a l o r i e s , and i r o n f o r subjects from the seven reserves. Group T o t a l F o l a t e I r o n Mean N (jag/Day) C a l o r i e s * (mg/Day* 1-12 Years 70 194.11103.2 1 1919±687 14.119.4 13+ Years 180 190.2+106.I 2 1979+788 15.018.2 Females: N o n - i s o l a t e d reserves 86 170.3+ 83.2 17271383 10.513.4 Males: No n - i s o l a t e d reserves 58 273.1+132.8 2803±848 17.716.3 Females: I s o l a t e d Reserves 77 143.1± 48.5 1673+542 15.215.3 Males: I s o l a t e d Reserves 29 216.2+106.6 22291549 19.117.7 *See Appendix C f o r Canadian Recommended Intake. WHO Recommended D a i l y Allowance = 200 ug T o t a l F o l a t e WHO Recommended D a i l y Allowance = 400 ug T o t a l F o l a t e C. Comparisons o f mean f o l a t e i n t a k e s f o r s u b j e c t s i n v o l v e d i n the comprehensive p a r t o f the survey Table V I I I shows the r e s u l t s o f a M u l t i p l e Range Test o f means f o r t o t a l f o l a t e i n t a k e f o r reserve s u b j e c t s i n v o l v e d i n the comprehensive p a r t o f the study ( i . e . ) subgroup s e l e c t e d from S e c h e l t , N e c o s l i e and F o r t Ware reserves. The mean i n t a k e f o r F o r t Ware i s s i g n i f i c a n t l y lower than f o r S e c h e l t o r N e c o s l i e reserves (P(0.05). 40 The mean t o t a l f o l a t e consumption f o r c h i l d r e n a t the S e c h e l t R e s i d e n t i a l School i s c o n s i d e r a b l y g r e a t e r than a t any of the three reserves. ( I n d i v i d u a l values f o r nutrient, i n t a k e s are presented i n Appendix F.) TABLE V I I I M u l t i p l e Range Test o f mean scores f o r food f o l a t e i n t a k e f o r s u b j e c t s i n v o l v e d i n the comprehensive f o l a t e study from S e c h e l t , N e c o s l i e , and F o r t Ware Reserves ** L o c a t i o n N T o t a l F o l a t e Intake (ug/Day) F o r t Ware 20 130.6 a* Sech e l t 30 165.6 b N e c o s l i e 33 173.6^ Residence 70 310.1 * Means not s h a r i n g the same s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t a t P^O.05. ** Mean reserve values were c a l c u l a t e d from 24-hour r e c a l l s . The residence mean was c a l c u l a t e d from a 3-day food r e c o r d r e p r e s e n t i n g the average i n t a k e o f a l l s u b j e c t s . The r e s u l t s o f a t - t e s t o f mean scores f o r d i e t a r y f o l a t e i n t a k e f o r s ubjects i n v o l v e d i n the comprehensive p a r t o f the f o l a t e study are presented i n Table IX. The values i n d i c a t e t h a t the mean f o l a t e i n t a k e f o r F o r t Ware, an i s o l a t e d r e s e r v e , i s s i g n i f i c a n t l y l e s s than the mean i n t a k e f o r the n o n - i s o l a t e d r e s e r v e s , S e c h e l t and N e c o s l i e . The mean f o l a t e i n t a k e f o r females from the group as a whole i s s i g n i f i c a n t l y l e s s than the mean 41 f o r males (P^0.95). The r e s u l t s i n d i c a t e t h a t i n g e n e r a l , d i e t s are poorest w i t h respect t o d i e t a r y f o l a t e i n t a k e f o r females from the i s o l a t e d reserve. TABLE IX T-t e s t o f mean scores f o r d i e t a r y f o l a t e i n t a k e f o r s u b j e c t s i n v o l v e d i n the comprehensive f o l a t e study from the three reserves. Means Compared N T o t a l F o l a t e Mean + S.D. (ug/Day) T-te s t Values T-Prob* F-Prob** S e c h e l t & N e c o s l i e F o r t Ware 63 20 169.8+79.8 130.6+53.2 .041 Reserve Males Reserve Females 34 49 185.1194.4 143.2+54.4 .023 .001 * P r o b a b i l i t y value P<0.05 i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e between the sample means. Dashes (-) i n d i c a t e no s i g n i f i c a n t d i f f e r e n c e . ** P r o b a b i l i t y value P<0.05 i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e between the sample v a r i a n c e s . Dashes (-) i n d i c a t e no s i g n i f i c a n t d i f f e r e n c e . As shown i n Table X, the mean t o t a l f o l a t e i n t a k e f o r reserv e s u b j e c t s i n v o l v e d i n the comprehensive p a r t o f the study i s w e l l below the WHO Recommended D a i l y Allowance f o r t o t a l f o l a t e (200 pg f o r persons 1 t o 12 years and 400 pg f o r those 13 years of age and o l d e r ) . The mean i n t a k e f o r residence s u b j e c t s o f 310.0 pg. t o t a l f o l a t e places the c h i l d r e n from 1 t o 12 years o l d i n the recommended range f o r f o l a t e consumption. Mean c a l o r i e i n t a k e s are g e n e r a l l y low f o r reserve s u b j e c t s . I n most cases the mean i n t a k e f o r the residence i s more than 200 c a l o r i e s higher than the reserve means, and TABLE X Mean d a i l y i n t a k e o f t o t a l f o l a t e , c a l o r i e s , and i r o n f o r l o c a t i o n and age groupings f o r su b j e c t s i n v o l v e d i n the comprehensive p a r t o f the study. L o c a t i o n Age (Years) N T o t a l F o l a t e (pg/Day) C a l o r i e s * I r o n (mg)* S e c h e l t , N e c o s l i e 1-12 18 163. ,8± 91.5 1 1980+853 10.615.0 & F o r t Ware 13+ 65 159. .41 71.7 2 18931622 12.314.6 S e c h e l t & N e c o s l i e 1-12 14 165. ,5± 93.7 1 1846+888 9.715.2 13+ 49 171. ,1± 76.4 2 1917+606 12.314.3 F o r t Ware 1-12 4 158. . l i l O l . O 1 2450+905 13.916.1 13+ 16 123. ,8± 81.2^ 18211698 12.514.7 Residence 1-12 14 310. ,1 2227 13.4 13+ 56 * See Appendix C f o r Canadian Recommended Intake 1 WHO Recommended D a i l y Allowance = 200 ug T o t a l F o l a t e 2 WHO Recommended D a i l y Allowance = 400 pg T o t a l F o l a t e 43 Hematological Data . A l l hematological d a t a were obtained from the subgroup s e l e c t e d from S e c h e l t , N e c o s l i e and F o r t Ware reserves and from S e c h e l t Residence c h i l d r e n . When data are presented i n terms of s p e c i f i c r e s e r v e s u b j e c t s they represent the values f o r the subgroup s e l e c t e d from the r e s e r v e s . A. C o n t r o l sample values Table XI shows the f o l a t e concentrations of the s e l e c t e d serum and whole bl o o d samples, which were analyzed i n d u p l i c a t e ( i . e . i n t h i s l a b o r a t o r y and i n an independent l a b o r a t o r y ) . Comparison o f serum and red c e l l f o l a t e values f o r d u p l i c a t e samples run as a c o n t r o l measure. TABLE XI Sample Values Reported (ng/ml) This Laboratory Independent Laboratory* Serum F o l a t e 1 2 3 4 5 6 2.0 12.0 3.9 5.7 14.9 8.2 2.3 13.0 4.6 5.0 14.0, 8.0, 17.0 9-.0 Red C e l l F o l a t e 1 2 3 4 3 210 117 94 198 64 199 103 109 181 54 * Hematology Department, S a i n t Paul's H o s p i t a l , Vancouver, B. C. The r e s u l t s i n d i c a t e t h a t values f o r the p a i r e d samples f o r both 44 serum and red c e l l f o l a t e are i n reasonable agreement. The p a i r e d values are s i m i l a r enough t h a t one can be c o n f i d e n t t h a t very low values . ( i . e . 2.0 and 2.3 ng/ml) represent a sample w i t h very lew f o l a t e l e v e l s , and c o n v e r s e l y , high values ( i . e . 14.9, 14.0 and 17.0 ng/ml) t r u e l y represent a h i g h f o l a t e c o n t a i n i n g sample. B. Comparison of means f o r hematological v a r i a b l e s Table X I I gives the r e s u l t s o f a M u l t i p l e Range Test performed on mean scores f o r serum f o l a t e , red c e l l f o l a t e and hemoglobin measurements f o r s ubjects from the three reserves. ( I n d i v i d u a l values f o r hematological v a r i a b l e s are l i s t e d i n Appendix G.) TABLE X I I M u l t i p l e range t e s t o f mean scores f o r serum f o l a t e , red c e l l f o l a t e and hemoglobin f o r the three reserves i n v o l v e d i n the comprehensive p a r t of the study. Serum F o l a t e Red C e l l F o l a t e Hemoglobin L o c a t i o n N (ng/ml) (ng/ml) g/lOOml) Se c h e l t 30 6.d°* 251 b 13.3 a F o r t Ware 20 4.1 a 179 a 1 4 . l a N e c o s l i e 33 5."/° 189 a 13.3 a Residence 69 8.3 124 12.2 *Means not s h a r i n g the same s u p e r s c r i p t under each v a r i a b l e are s i g n i f i c a n t l y d i f f e r e n t a t P<0.05. The mean serum f o l a t e f o r F o r t Ware i s s i g n i f i c a n t l y lower than f o r S e c h e l t o r N e c o s l i e Reserves, whose means are not s i g n i f i c a n t l y d i f f e r e n t from one another (P<0.05). The mean red c e l l f o l a t e f o r S e c h e l t i s 45 s i g n i f i c a n t l y h i g h e r than f o r N e c o s l i e o r F o r t Ware, whose means are not s i g n i f i c a n t l y d i f f e r e n t . Mean hemoglobin values are not s i g n i f i c a n t l y d i f f e r e n t among the three reserves (P<0.05). The mean serum f o l a t e f o r S e c h e l t R e s i d e n t i a l School i s con s i d e r a b l y higher than the three reserve means, w h i l e the mean r e d c e l l f o l a t e i s c o n s i d e r a b l y lower. Table X I I I gives the r e s u l t s o f a M u l t i p l e Range Test o f mean scores f o r hemoglobin and f o l a t e v a r i a b l e s grouped i n t o age and sex ca t e g o r i e s f o r s u b j e c t s from the three reserves. The serum f o l a t e mean f o r 0 t o 9 year o l d s i s s i g n i f i c a n t l y g r e a t e r than f o r the o l d e r age c a t e g o r i e s (P<0.05), w i t h values f o r females c o n t r i b u t i n g most t o the v a r i a b i l i t y between the age groups. Mean r e d c e l l f o l a t e i s s i g n i f i c a n t l y h i g h e r f o r 0 t o 9 year o l d s than f o r o l d e r subjects (P(0.05) w i t h values f o r male subjects c o n t r i b u t i n g most t o the v a r i a b i l i t y . Hemoglobin means are not s i g n i f i c a n t l y d i f f e r e n t f o r the group as a whole, although a d u l t males have a s i g n i f i c a n t l y h i g h e r mean than males from 0 t o 9 years o l d and a d u l t females have a s i g n i f i c a n t l y h i g h e r mean than females from 10 t o 19 years o l d (P<0.05). The r e s u l t s o f a t - t e s t performed on serum and r e d c e l l f o l a t e means are presented i n Table XIV. Both the serum and red c e l l f o l a t e means f o r F o r t Ware are s i g n i f i c a n t l y lower than the j o i n t means f o r S e c h e l t and N e c o s l i e reserves (P<0.05), i n d i c a t i n g t h a t f o l a t e s t a t u s i s s i g n i f i c a n t l y lower on the i s o l a t e d reserve as compared t o the n o n - i s o l a t e d reserves. A comparison o f means between reserve males and females i n d i c a t e s there i s no s i g n i f i c a n t d i f f e r e n c e f o r e i t h e r serum o r r e d c e l l f o l a t e v a r i a b l e s . TABLE X I I I M u l t i p l e range t e s t o f mean scores f o r serum f o l a t e , red c e l l f o l a t e , and hemoglobin f o r age and sex groupings f o r the three reserves i n v o l v e d i n the comprehensive p a r t o f the study. Age (Years) Sex N Serum F o l a t e (ng/ml) Red C e l l F o l a t e (ng/ml) Hemoglobin (g/100 ml) 0-9 10-19 20+ Male & Female 7 26 50 5.6 a 5.4 a 243 b 212 a 203 a 12.7 a 13.3 a 13.5 a 0- 9 10-19 20+ Male 2 19 13 5.8 a 5.2 a 6.4 a 207 b 153 a 12.7 a, 14. O f 15.0 b 0- 9 10-19 20+ Female 5 7 37 8.5 b 6.5* 5.0 a 227 a 224 a 2 2 1 a 1 2 . 7 * 11.4? 13.0 b * Means not s h a r i n g the same s u p e r s c r i p t under each v a r i a b l e , i n each grouping o f three means, are s i g n i f i c a n t l y d i f f e r e n t a t P<0.05. TABLE XTV T- t e s t o f mean scores f o r serum and r e d c e l l f o l a t e v a r i a b l e s f o r l o c a t i o n and sex groupings f o r s u b j e c t s i n v o l v e d i n the comprehensive p a r t o f the survey. V a r i a b l e Means Compared N Mean + S.D. T- t e s t Values T-Prob* F-Prob** Serum F o l a t e (ng/ml) S e c h e l t & N e c o s l i e F o r t Ware 63 20 6.2±2.4 4.1+1.5 .000 .031 Reserve Males Reserve Females 34 49 5.9+2.6 5.612.3 Red C e l l F o l a t e (ng/ml) S e c h e l t & N e c o s l i e F o r t Ware 63 20 218+82 179+54 .015 .047 Reserve Males Reserve Females 34 49 190+64 222+84 * P r o b a b i l i t y v a l u e P(0.05 i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e between the sample means. Dashes (-) i n d i c a t e no s i g n i f i c a n t d i f f e r e n c e . P r o b a b i l i t y value P<0.05 i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e between the sample va r i a n c e s . Dashes (-) i n d i c a t e no s i g n i f i c a n t d i f f e r e n c e . 48 C. Risk d i s t r i b u t i o n s Serum f o l a t e , r ed c e l l f o l a t e , and hemoglobin values are d i s t r i b u t e d i n t o r i s k c a t e g o r i e s as i l l u s t r a t e d i n Fi g u r e 4. Standards used t o designate the three r i s k grouping are presented i n Appendix D. Subjects are c l a s s e d "at r i s k " w i t h r e s p e c t t o a v a r i a b l e i f the value o f the v a r i a b l e t e s t e d i s i n the range o f e i t h e r the high o r moderate r i s k c a t e g o r i e s . Results i n d i c a t e t h a t 70% o f the s u b j e c t s from F o r t Ware are "at r i s k " w i t h respect t o serum f o l a t e w i t h 10% h i g h and 60% moderate r i s k v a lues. N e c o s l i e has 45% "at r i s k " w i t h 6% high and 39% moderate r i s k values w h i l e S e c h e l t has 30% "at r i s k " , w i t h a l l values i n the moderate r i s k category. F o r t y - f i v e percent o f F o r t Ware's s u b j e c t s are "at r i s k " w i t h respect t o red c e l l f o l a t e , w i t h 25% high and 20% moderate r i s k v a l u e s . N e c o s l i e has 24% and 15% o f i t s values i n high and moderate r i s k c a t e g o r i e s r e s p e c t i v e l y , w h i l e S e c h e l t has j u s t 3% i n high r i s k and 13% i n moderate r i s k c a t e g o r i e s . Although F o r t Ware has the poorest f o l a t e s t a t u s w i t h the g r e a t e s t percentage o f s u b j e c t s "at r i s k " , i t has the l e a s t s u b j e c t s "at r i s k " w i t h r e s p e c t t o hemoglobin ( i . e . 5%). Ten percent and 18% o f the sub j e c t s from S e c h e l t and N e c o s l i e r e s p e c t i v e l y are "at r i s k " , w i t h 3% from both reserves having h i g h r i s k v a l u e s . 4 9 F i g u r e 4: D i s t x i b u t i o n o f hematological v a r i a b l e s i n t o r i s k c a t e g o r i e s f o r the three r e s e r v e s . N S e c h e l t 30 I I F o r t Ware 20 f | N e c o s l i e 33 100-8 0 -6 0 -40-20H SERUM FOLATE 50 High Moderate RISK CATEGORIES 100-8 0 -6 0 -4 0 -2 0 -RED CELL FOLATE r r r r | ' f vr I High Moderate RISK CATEGORIES IB Low 100-8 0 -6 0 -4 0 -2 0 -HEMOGLOBIN i f 1 High Moderate RISK CATEGORIES Low 51 Hematolcgical variables are distributed into risk categories for subjects 0 to 17 years old from Sechelt Residential School and from the three reserves, as i l l u s t r a t e d i n Figure 5. The percentage of reserve children with serum folate values i n high and moderate risk categories are 3% and 38% respectively. Thirteen percent of the residence children are "at risk" with values i n the moderate risk category only. Seventy-seven percent of the red c e l l folate values for residence children and 28% for reserve children are i n high and moderate risk categories. No children have high risk hemoglobin values, although 25% of the residence children and 3% from reserves have values i n the moderate risk category. The risk distribution of serum folate values for this study and for two groups from the Nutrition Canada Survey (i.e. National Indian Survey and B r i t i s h Columbia Provincial Survey) i s i l l u s t r a t e d i n Figure 6. A greater percentage of subjects 5 to 9 years old were "at risk" in the National Indian Survey and British Columbia Provincial Survey as compared to this study, i.e. 60% and 58% as compared to 17%. Approximately 70% of the subjects 10 to 19 years old were "at risk" i n the National Indian and B. C. Provincial Surveys as compared to 48% i n this study. For subjects 20 years of age and older, 50% are "at risk" i n this study as compared to 65% and 60% for the National Indian Survey and B. C. Provincial Survey respectively. Overall there was a greater incidence of high risk values for the two groups from the Nutrition Canada Survey as compared to this study, particularly for the National Indian Survey. 52 Figure 5: Distribution of hematological variables into risk categories for Residence and Reserve Subjects (0 to 17 Years old) N | | Reserve subjects (0 to 17 Years) 29 [ | Residence Subjects (0 to 17 Years) 69 % DISTRIBUTION 100-80-60-40-20-SERUM FOLATE 5 3 High 1 Moderate RISK CATEGORIES Low % DISTRIBUTION 100-80-60-40-20-RED CELL FOLATE High 1 Moderate RISK CATEGORIES Low % DISTRIBUTION 100-80-60-40-20-HEMOGLOBIN High I Moderate RISK CATEGORIES Low 54 Figure 6: C l a s s i f i c a t i o n o f serum f o l a t e values f o r reserve s u b j e c t s from t h i s study and f o r two groups from the N u t r i t i o n Canada Survey i . e . N a t i o n a l Indian survey and B. C. P r o v i n c i a l survey. R i s k Categories N High Moderate Age Categories (Years) 5-9 10-19 20+ 1. This Study | | 2. N a t i o n a l Indian Survey | [ 3. B . C . P r o v i n c i a l Survey | | • 7 217 253 26 437 548 50 815 1301 100-i 8 0 -6 0 -% DISTRIBUTION 4 0 -2 0 -AGE CATEGORIES (YEARS) 2 3 10-19 1 2 3 OVER 20 Ul 56 Regression A n a l y s i s A SL-Test o f r e g r e s s i o n equations was conducted t o compare equations f o r d i f f e r e n t groups o f s u b j e c t s i n v o l v e d i n the comprehensive p a r t o f the survey, w i t h equations i n the form of (y=A+Bx). Serum and red c e l l f o l a t e were s e t as the dependent v a r i a b l e s and d i e t a r y f o l a t e the independent v a r i a b l e . R e s u l t s from Table XV i n d i c a t e there i s not a common equation f o r the three reserves f o r both serum and r e d c e l l f o l a t e v a r i a b l e s . A common r e g r e s s i o n equation i s i n d i c a t e d between male and female s u b j e c t s , f o r serum f o l a t e o n l y (P^0.05). TABLE XV SL-Test o f comparison o f r e g r e s s i o n equations w i t h serum f o l a t e o r r e d c e l l f o l a t e as dependent v a r i a b l e s and d i e t a r y f o l a t e i n t a k e the independent v a r i a b l e , w i t h equations i n the form o f (Y=A+Bx). Dependent V a r i a b l e (Y) Group Equations Compared F-Value* Serum F o l a t e S e c h e l t v s . F o r t Ware vs. N e c o s l i e Reserve Males vs. Reserve Females 0.00 Red C e l l F o l a t e S e c h e l t v s . F o r t Ware v s . N e c o s l i e Reserve Males v s . Reserve Females ~ *F-Value ^0.05 i n d i c a t e s there i s a common p r e d i c t i n g equation f o r the groups compared. Dashes (-) i n d i c a t e a common equation does not e x i s t . A r e g r e s s i o n a n a l y s i s was performed t o t e s t the l i n e a r r e l a t i o n s h i p between blood f o l a t e values ( i . e . serum and red c e l l ) and d i e t a r y f o l a t e 57 i n t a k e f o r reserve subjects from S e c h e l t , N e c o s l i e and F o r t Ware. R e s u l t s from Table XVT i n d i c a t e there i s no s i g n i f i c a n t r e l a t i o n s h i p between red c e l l f o l a t e values and d i e t a r y f o l a t e i n t a k e f o r the three groups t e s t e d . A s i g n i f i c a n t r e l a t i o n s h i p i s i n d i c a t e d between serum f o l a t e values and d i e t a r y f o l a t e i n t a k e (P<0.05), f o r the su b j e c t s as a whole and f o r male sub j e c t s alone. Therefore, serum f o l a t e v a l u e s are s i g n i f i c a n t l y and d i r e c t l y r e l a t e d t o d i e t a r y f o l a t e i n t a k e . TABLE XVI Regression L i n e constants w i t h serum f o l a t e o r red c e l l f o l a t e as dependent v a r i a b l e s and d i e t a r y f o l a t e i n t a k e as the independent v a r i a b l e , w i t h equations i n the form o f (y=A+Bx). Dependent V a r i a b l e I n t e r c e p t A Slope B (nq/ml) Sex (ng/mlj (ng/ml)(ng/Day) F-Prob* Serum M & F 4.344 .0085 .0133 F o l a t e Male 3.898 .0110 .0172 Female 5.095 .0032 -Red C e l l M & F 202.9 .0372 F o l a t e Male 165.4 .1336 _ Female 216.9 .0349 -* P r o b a b i l i t y value P<0.05 i n d i c a t e s there i s a s i g n i f i c a n t r e l a t i o n s h i p given by the equation. Dashes (-) i n d i c a t e there i s no s i g n i f i c a n t r e l a t i o n s h i p . 58 CHAPTER V DISCUSSION T o t a l f o l a t e values f o r the 24-hour d i e t a r y r e c a l l s , c a l c u l a t e d from food composition t a b l e s , are s i m i l a r and are s i g n i f i c a n t l y c o r r e l a t e d w i t h the t o t a l f o l a t e values obtained by d i r e c t assay, f o r the 24-hour food sample c o l l e c t i o n s . This o f f e r s confidence t h a t our computed f o l a t e values are r e l i a b l e f o r the assessment o f d i e t a r y f o l a t e s t a t u s on the seven r e s e r v e s , and supports the 24-hour d i e t a r y r e c a l l as bein g a r e l i a b l e method f o r e s t i m a t i n g f o l a t e consumption f o r the Indian groups i n v o l v e d i n t h i s study. A c l o s e agreement between assayed and c a l c u l a t e d f o l a t e values was a l s o obtained by Mosoovitch and Cooper (1973) and P i e t a r i n e n (1975) u s i n g s i m i l a r methods. Since most composition t a b l e s are i n a c c u r a t e f o r c a l c u l a t i n g food f o l a t e , the importance o f s e l e c t i n g the proper values and bein g c o n f i d e n t t h a t the f o l a t e values are r e l i a b l e i s fundamental t o any study o f t h i s nature (Thenen, 1975 and Hurdle, 1973). A comparison o f t o t a l f o l a t e i n t a k e s f o r the seven r e s e r v e s , as c a l c u l a t e d from 24-hour r e c a l l s , r e v e a l s t h a t the i n t a k e o f d i e t a r y f o l a t e i s s i g n i f i c a n t l y l e s s on i s o l a t e d reserves as compared t o the n o n - i s o l a t e d r e s e r v e s . This i s t r u e f o r the subgroup i n v o l v e d i n the comprehensive p a r t o f the study as w e l l , where the mean f o l a t e i n t a k e a t F o r t Ware, an i s o l a t e d r e s e r v e , i s s i g n i f i c a n t l y l e s s than mean i n t a k e s a t S e c h e l t and N e c o s l i e r e s e r v e s , which are c l a s s i f i e d as n o n - i s o l a t e d . Some authors have 59 repo r t e d t h a t foods known t o be h i g h i n f o l i c a c i d , such as f r e s h f r u i t s and vegetables, are l a c k i n g o r c o n s t i t u t e a very s m a l l p a r t o f the Canadian Indian d i e t (Moore, 1946, Dong and Feeney, 1968 and Lee e t a l , 1971). This i s e s p e c i a l l y t r u e f o r Indians l i v i n g i n more i s o l a t e d areas where the s e l e c t i o n o f f r e s h produce i s r e s t r i c t e d (Lee e t a l , 1971 and Smith, 1975) and canned f r u i t s and vegetables are o f t e n u n a v a i l a b l e (Smith, 1975). A t one time, before the Indians moved t o r e s e r v a t i o n s , they obtained a g r e a t d e a l o f food from the lan d . The Indians i n B r i t i s h Columbia consumed a v a r i e t y o f b e r r i e s such as r e d h u c k l e b e r r i e s , t h i m b l e b e r r i e s , b l a c k b e r r i e s , s a l a l b e r r i e s and salmonberries, a v a r i e t y , o f vegetables, r o o t crops, and other f r u i t s such as c l o v e r r o o t , f e r n r o o t , skunk cabbage, w i l d t u r n i p s , w i l d peas, a r t i c h o k e s , choke c h e r r i e s , crabapples and w i l d plums. P o s s i b l y many o f these foods are s t i l l consumed b u t are not reporte d by the people, s i n c e they are not regarded as important food items, o r con v e r s e l y , these foods no longer make up a s i g n i f i c a n t p a r t o f the Indian d i e t . W i l d h u c k l e b e r r i e s and soapberries are gathered and preserved by some f a m i l i e s i n t h i s study, although there was no mention o f other w i l d f r u i t s o r vegetables being consumed. D i e t a r y f o l a t e i n t a k e s do not d i f f e r s i g n i f i c a n t l y w i t h age f o r the group o f s u b j e c t s as a whole, however d i f f e r e n c e s i n f o l a t e consumption are observed between male and female s u b j e c t s . For s u b j e c t s from the t o t a l r e s e r v e p o p u l a t i o n and f o r the reserv e subgroup, the males consume s i g n i f i c a n t l y more d i e t a r y f o l a t e than the females; ( i . e . 251 ug t o t a l f o l a t e as compared t o 157.4 ug r e s p e c t i v e l y f o r the t o t a l p o p u l a t i o n ) . This i s i n agreement w i t h other s t u d i e s which r e p o r t a higher d i e t a r y f o l a t e 60 consumption i n male adolescents as compared t o females (Daniel e t a l , (1975) and a d u l t males as compared t o a d u l t females (Levy e t a l , 1975). In the study reported by Levy e t a l , ( 1 9 7 5 ) , low f o l a t e consumption i n women o f c h i l d - b e a r i n g age was r e l a t e d t o the co n s i d e r a b l y h i g h e r i n c i d e n c e o f f o l a t e d e f i c i e n c y anemia i n women as compared t o men. Lower d i e t a r y f o l a t e i n t a k e s f o r women i n t h i s study are l i k e l y p l a c i n g them a t hig h e r r i s k i n terms o f f o l a t e s t a t u s , e s p e c i a l l y f o r those who are considered t o be p a r t i c u l a r l y prone t o f o l a t e d e f i c i e n c y ; i . e . women t a k i n g o r a l c o n t r a c e p t i v e s ( P i e t a r i n e n , 1975); women who are pregnant ( S a u b e r l i c h e t a l , 1974 and Herbert e t a l , 1975) o r c h r o n i c a l c o h o l i c s (Herbert and Zulusky, 1961 and Baker e t a l , 1975). Patt e r n s o f d i e t a r y f o l a t e i n t a k e among the d i f f e r e n t groups tend t o f o l l o w p a t t e r n s o f c a l o r i e consumption, i n t h a t groups w i t h the h i g h e s t c a l o r i e i n t a k e s ( i . e . males from n o n - i s o l a t e d and i s o l a t e d reserves) a l s o have the hi g h e s t d i e t a r y f o l a t e i n t a k e s . S i m i l a r l y , the lowest groups w i t h respect t o c a l o r i e consumption ( i . e . women on i s o l a t e d and n o n - i s o l a t e d reserves) a l s o have the lowest i n t a k e s o f d i e t a r y f o l a t e . Therefore, i f the foods which c o n s t i t u t e the d i e t o f these I n d i a n people are adequate i n c a l o r i e s , they are l i k e l y t o c o n t r i b u t e c o n s i d e r a b l y more d i e t a r y f o l a t e than foods which c o n s t i t u t e a low c a l o r i e d i e t . E i t h e r l a r g e r volumes o f the same food are consumed i n h i g h c a l o r i e as compared t o low c a l o r i e d i e t s , o r a d d i t i o n a l foods are intro d u c e d which i n c r e a s e the d i e t a r y f o l a t e and c a l o r i e i n t a k e s t o more adequate l e v e l s . On the reserves where food s e l e c t i o n i s l i m i t e d , men tend t o consume l a r g e r volumes o f the food which i s a v a i l a b l e t o everyone. Women reported o m i t t i n g meals o r e a t i n g v e r y s m a l l amounts o f b r e a k f a s t and lunch, w h i l e men seldom missed a meal. 61 R e s u l t s f o r the hematological measurements from the reserves and Sec h e l t R e s i d e n t i a l School p o i n t out important d i f f e r e n c e s between the groups w i t h respect t o t h e i r f o l a t e s t a t u s . Mean serum f o l a t e i s s i g n i f i c a n t l y lower f o r F o r t Ware, the i s o l a t e d r e s e r v e , than f o r S e c h e l t o r N e c o s l i e reserves. F o r t Ware a l s o has the h i g h e s t percentage o f low serum v a l u e s , w i t h 70% o f the s u b j e c t s "at r i s k " . N u t r i t i o n Canada (1973) a l s o found t h a t the i n c i d e n c e o f low serum f o l a t e values was h i g h e r f o r Indians on i s o l a t e d as compared t o n o n - i s o l a t e d r e s e r v e s . I t i s i n t e r e s t i n g t o note t h a t a t F o r t Ware, where d i e t a r y f o l a t e i n t a k e s are the lowest, the serum f o l a t e values are a l s o the lowest. Conversely, the mean d i e t a r y f o l a t e i n t a k e f o r S e c h e l t R e s i d e n t i a l School i s c o n s i d e r a b l y higher than the mean i n t a k e s f o r the three r e s e r v e s , w h i l e the serum f o l a t e values are a l s o c o n s i d e r a b l y h i g h e r . This i s c o n s i s t e n t w i t h r e p o r t s t h a t serum f o l a t e values are r e f l e c t i o n s o f recent d i e t a r y f o l a t e i n t a k e s (Herbert, 1962, J o i n t FAO/WHO Expert Group, 1970, L i u , 1974 and H a l l e t a l , 1975). The r e s u l t s from the r e g r e s s i o n a n a l y s i s f u r t h e r support t h i s f i n d i n g , i n d i c a t i n g t h a t serum f o l a t e values are d i r e c t l y and s i g n i f i c a n t l y r e l a t e d t o d i e t a r y f o l a t e i n t a k e s . S i m i l a r r e s u l t s have been rep o r t e d by Hurdle (1968) and P i e t a r i n e n (1975). N u t r i t i o n Canada (1973) d i d not a t t r i b u t e low serum f o l a t e values i n t h e i r study t o low d i e t a r y f o l a t e i n t a k e s , s i n c e d i e t a r y f o l a t e s t a t u s was not assessed, although Beaton (1975) proposed t h a t t h i s was l i k e l y a major f a c t o r , e s p e c i a l l y i n i s o l a t e d areas. Since serum f o l a t e values i n t h i s study are d i r e c t l y r e l a t e d t o the l e v e l o f f o l a t e consumed, the h i g h i n c i d e n c e o f low serum f o l a t e values a t F o r t Ware reserve can be a t t r i b u t e d , i n p a r t , t o low d i e t a r y f o l a t e i n t a k e s . Conversely, the s i g n i f i c a n t l y 62 higher dietary folate consumption f o r subjects at Sechelt and Necoslie reserves i s a factor responsible f o r t h e i r higher serum fol a t e values. The dietary intake of t o t a l f o l a t e i s below the WHO Recommended Daily Allowance (200 pg f o r 1 to 12 year olds, and 400 ug for persons 13 years of age and older f o r almost everyone i n each group of subjects, except children from Sechelt Residential School, yet the l e v e l of f o l a t e consumed i s adequate to maintain normal serum levels i n 70%, 65% and 30% of the reserve subjects from Sechelt, Necoslie, and Fort Ware respectively. Since there i s uncertainty concerning the a v a i l a b i l i t y and u t i l i z a t i o n of d i f f e r e n t forms of dietary folates (Tamura and Stokstad, 1973), subjects consuming less t o t a l folate than recommended are not necessarily "at r i s k " . There i s further d i s p a r i t y between the f o l a t e intake required to maintain normal serum and erythrocyte levels of the vitamin, and the smaller quantity necessary to prevent c l i n i c a l i l l n e s s (Dietary Standards for Canada, 1975). This was shown by Cooper and Lowenstein (1966) i n a study where patients with megalo-b l a s t i c anemia responded c l i n i c a l l y and hematologically to 100 pg per day of f o l i c a cid, but d i d not increase t h e i r serum or erythrocyte f o l a t e l e v e l s . I t was estimated that considerably fewer than h a l f of the general population with lew serum folate w i l l have c l i n i c a l manifestations of deficiency (Dietary Standards f o r Canada, 1975), ind i c a t i n g that serum fol a t e levels alone are not a good index of fo l a t e deficiency. I f serum values of greater than 5 ng/ml are i n d i c a t i v e of adequate dietary fo l a t e intakes, t h i s study shows that 300 pg t o t a l folate d a i l y i s adequate f o r residence children, and according to dietary data from the reserves, even less i s required. Further research i s necessary, as t h i s study indicates, to investigate the current standards for t o t a l f o l a t e requirements. 63 Although there i s a lower incidence of subjects i n t h i s study as a whole "at r i s k " with respect to serum f o l a t e , as compared to the n a t i o n a l Indian population, as reported i n the N u t r i t i o n Canada Survey (1973) , the incidence of r i s k values from For t Ware alone i s comparable, i n d i c a t i n g f o l a t e inadequacy f o r t h i s group of people. According to the n a t i o n a l r e s u l t s f o r N u t r i t i o n Canada, serum values f o r males and females were s i m i l a r , and the l e v e l s f o r c h i l d r e n tended to be higher than adult l e v e l s . A s i m i l a r pattern i s apparent i n t h i s study, where mean serum f o l a t e values f o r males and females are not s i g n i f i c a n t l y d i f f e r e n t , and c h i l d r e n 0 to 9 years o l d have a s i g n i f i c a n t l y higher mean serum f o l a t e than the o l d e r age categories. Daniel e t a l (1975) found th a t increased maturity i n adolescents was associated with a decrease i n plasma f o l a t e concentrations without a concurrent decrease i n d i e t a r y f o l a t e intakes. Reduced f o l a t e l e v e l s were a t t r i b u t e d , i n p art, to greater t i s s u e and c e l l u l a r f o l a t e demands. Possibly t h i s could be r e l a t e d to t h i s study, i n that f o l a t e demands are greater during and a f t e r maturation, as WHO and Canadian recommendations i n d i c a t e , y e t d i e t a r y f o l a t e consumption does not increase to meet these demands, consequently the l e v e l o f c i r c u l a t i n g f o l a t e i n the serum decreases. The observation that males consume s i g n i f i c a n t l y more d i e t a r y f o l a t e than females y e t t h e i r serum f o l a t e l e v e l s are not s i g n i f i c a n t l y higher, might a l s o be explained by an increased f o l a t e need. The incidence of low serum f o l i c a c i d values i s not as great i n t h i s study as i n the N u t r i t i o n Canada Survey (1973) , yet there i s d e f i n i t e cause f o r concern i n c e r t a i n segments of the population, i . e . f o r i s o l a t e d reserves (such as F o r t Ware) and f o r the adult population, e s p e c i a l l y f o r women, whose die t a r y f o l a t e intakes are considerably lower than f o r men. 64 . Mean red c e l l f o l a t e l e v e l s a t For t Ware are lower than at the other reserves, although not s i g n i f i c a n t l y lower than the mean at Necoslie. Twenty-five percent and twenty-four percent of the values from F o r t Ware and Necoslie r e s p e c t i v e l y were c l a s s i f i e d as high r i s k , and 20% and 15% as moderate r i s k values f o r red c e l l f o l a t e . Instead of r e f l e c t i n g recent f o l a t e status, red c e l l f o l a t e a c t i v i t y i s a q u a n t i t a t i v e index of the s e v e r i t y of f o l a t e d e f i c i e n c y and i s considered to be i n d i c a t i v e of long-term f o l a t e status and t i s s u e stores (Herbert, 1965 and Hoffbrand e t a l , 1966). On t h i s b a s i s , the r e s u l t s i n d i c a t e that close to 50% of the subjects from F o r t Ware and 40% from Necoslie have inadequate f o l a t e stores and are at r i s k of being d e f i c i e n t . Sechelt Reserve, on the other hand, has a s i g n i f i c a n t l y higher mean red c e l l f o l a t e than Necoslie or F o r t Ware, with only 16% of the subjects c l a s s i f i e d as "at r i s k " . There i s evidence that i r o n d e f i c i e n c y may increase f o l a t e requirements (Velez e t a l , 1966 and Taskes e t a l , 1974) and consequently a l t e r serum and red c e l l f o l a t e l e v e l s (Omer et a l , 1970, Roberts e t a l , 1971, Saraya e t a l , 1971, and Saraya e t a l , 1973). Population studies have demonstrated a high prevalence of f o l a t e d e f i c i e n c y concurrent with i r o n d e f i c i e n c y anemia; (i.e.) National Food and N u t r i t i o n Survey of Barbados (1972), Kaufman e t a l , (1975), and Levy e t a l , (1975); The low incidence o f low hemoglobin and hematrocrit values f o r t h i s study i n d i c a t e s that i r o n d e f i c i e n c y anemia i s not an important problem f o r the subjects, and i s therefore not l i k e l y a complicating f a c t o r i n the et i o l o g y of the low blood f o l a t e values. Mean d a i l y intakes of i r o n a l s o support t h i s conclusion. Values f o r MCV and MCHC are w i t h i n a normal range and the majority of the t h i n blood smears examined show no s i g n i f i c a n t changes i n c e l l 65 morphology, i n d i c a t i n g t h a t o v e r t m e g a l o b l a s t i c anemia i s not p r e v a l e n t among the s u b j e c t s . Although s u b j e c t s are not anemic, the presence o f low b l o o d f o l a t e values r e p r e s e n t s , by d e f i n i t i o n , a f o l a t e d e f i c i e n c y (Herbert, 1965) and i s important as an index o f a degree o f m a l n u t r i t i o n (Hurdle and W i l l i a m s , 1966) which c o u l d l e a d t o megaloblastosis i f the body i s challenged by d i s e a s e s , a l c o h o l i s m , i n f e c t i o n s , o r o t h e r p a t h o l o g i c a l s t a t e s which i n c r e a s e the f o l a t e requirements (Mathoth e t a l , 1964b and Hurdle and W i l l i a m s , 1966). According t o B u t l e r (1975) the major c u r r e n t h e a l t h problems of Indians i n B r i t i s h Columbia are: a c c i d e n t s and v i o l e n c e , w i t h a l c o h o l abuse; p e r i n a t a l m o r t a l i t y , w i t h inadequate p r e n a t a l care; r e s p i r a t o r y d i s e a s e s , e s p e c i a l l y i n the young; and g a s t r o - i n t e s t i n a l diseases due, i n p a r t , t o inadequate water and s a n i t a t i o n s e r v i c e s . Simpson (1974) p o i n t e d out t h a t there i s an extremely high i n f e c t i o n r a t e i n the young which can be a t t r i b u t e d t o poor s a n i t a t i o n and h e a l t h care. According t o Simpson and Dormaar (1974) there i s no q u e s t i o n t h a t Indian h e a l t h i s much poorer than t h a t o f the m a j o r i t y o f the Canadian white popula-t i o n . The a d d i t i o n a l s t r e s s imposed upon the I n d i a n , due t o the f a c t o r s j u s t mentioned, c o u l d i n c r e a s e the f o l a t e demands of the body, consequently i n c r e a s i n g d i e t a r y f o l a t e requirements. Red c e l l f o l a t e values f o r the c h i l d r e n from S e c h e l t R e s i d e n t i a l School are extremely low. Seventy-seven percent o f these c h i l d r e n are "at r i s k " as compared t o only 28% of the c h i l d r e n l i v i n g on res e r v e s . These low values are i n c o n s i s t e n t w i t h the serum f o l a t e , d i e t a r y f o l a t e , MCV, and MCHC values which i n d i c a t e t h a t the f o l a t e s t a t u s o f these c h i l d r e n i s adequate and l i k e l y s u p e r i o r t o the s t a t u s o f reserve c h i l d r e n . The t h i n 66 blood smear preparations showed no evidence of changes i n c e l l morphology ch a r a c t e r i s t i c of megaloblastic anemia. Although serum and red c e l l values r e f l e c t d i f f e r e n t aspects of f o l a t e metabolism, t h i s cannot explain such a great discrepancy between the serum and red c e l l f o l a t e values for the children from Sechelt residence. Red c e l l f o l a t e values r e f l e c t folate status from at least four months previous to the time the blood sample i s drawn (herbert, 1962), yet there i s no reason to suspect that the residence d i e t had changed d r a s t i c a l l y i n t h i s space of time. Duplicate blood samples assayed i n t h i s laboratory and i n an independent laboratory gave comparable values, suggesting that the assay method was not at f a u l t . These residence blood samples were stored with a l l the other samples, so that i f fo l a t e a c t i v i t y was l o s t i n storage i t would have occurred f o r a l l groups, which was not the case. Either the fo l a t e values f o r the blood samples are a true indication of the childrens' red c e l l f o l a t e status, or f o l a t e a c t i v i t y was, i n some way, reduced before the residence samples were frozen. Since the red c e l l f o l a t e values for Sechelt Residence subjects are questionable, very l i t t l e weight has been put on them for interpreting the hematological r e s u l t s . As previously discussed, serum fol a t e values, t h i n blood smear examination, blood c e l l counts, and dietary f o l a t e assessment indicates that dietary f o l a t e consumption for the residence children i s adequate and there i s no evidence of fo l a t e deficiency anemia. Since i t i s assumed that values from these measurements are r e l i a b l e , the folate status of the residence children i s considered acceptable. In summary, t h i s study shows that the f o l a t e status of Indians i n B r i t i s h Columbia i s variable, with more problems indicated i n some groups than others. Dietary folate intakes are s i g n i f i c a n t l y higher i n males than 67 females and a t n o n - i s o l a t e d as compared t o i s o l a t e d r eserves. C h i l d r e n from S e c h e l t R e s i d e n t i a l School consume c o n s i d e r a b l y more d i e t a r y f o l a t e than reserve s u b j e c t s . Low c a l o r i e d i e t s are observed f o r women w i t h low d i e t a r y f o l a t e i n t a k e s , w h i l e men w i t h higher c a l o r i e d i e t s tended t o consume higher l e v e l s o f d i e t a r y f o l a t e , i n d i c a t i n g t h a t g e n e r a l l y low food consumption i s one f a c t o r c o n t r i b u t i n g t o the low d i e t a r y f o l a t e v a l u e s . I r o n i n t a k e i s g e n e r a l l y adequate f o r both the reserve and residence s u b j e c t s . Serum f o l a t e values f o r the residence c h i l d r e n are c o n s i d e r a b l y h i g h e r than values f o r the c h i l d r e n from the r e s e r v e s . Serum f o l a t e v a l u e s are s i g n i f i c a n t l y lower a t F o r t Ware reserve than a t S e c h e l t o r N e c o s l i e . Red c e l l f o l a t e values are a l s o the lowest a t F o r t Ware, s i g n i f i c a n t l y lower than values a t S e c h e l t , although not s i g n i f i c a n t l y lower than N e c o s l i e . Serum f o l a t e values are s i g n i f i c a n t l y and d i r e c t l y r e l a t e d t o d i e t a r y f o l a t e i n t a k e s f o r the s u b j e c t s as a whole. Therefore, low serum f o l a t e values are i n d i c a t o r s o f , o r can be a t t r i b u t e d i n p a r t , t o low f o l a t e - c o n t a i n i n g d i e t s . For example, the a v a i l a b i l i t y and consumption o f f o l a t e - r i c h foods a t F o r t Ware i s low and the serum values are a l s o g e n e r a l l y low. E x t e n s i v e n u t r i t i o n education programs should not be the o n l y approach t o c o r r e c t i n g n u t r i t i o n a l problems f o r Indians a t i s o l a t e d l o c a t i o n s such as F o r t Ware. There i s a l s o a need f o r more resources, w i t h a wider s e l e c t i o n and a v a i l a b i l i t y o f food s t u f f s , so t h a t people have the o p p o r t u n i t y t o e a t a balanced d i e t . Fresh f r u i t s and vegetables which are r i c h i n f o l i c a c i d would u l t i m a t e l y improve t h e i r f o l a t e s t a t u s . D i f f e r e n t problems are apparent i n n o n - i s o l a t e d areas, such as S e c h e l t and N e c o s l i e , where a wide v a r i e t y o f n u t r i t i o u s foods are r e a d i l y a v a i l a b l e , but are o f t e n not consumed. N u t r i t i o n education programs should emphasize the importance 68 of good nutrition for health and well being, and what foods can be selected and prepared at minimal costs, yielding optimal nutrition. The significance of good nutrition must be understood before people can be expected to improve nutritional practices. Nutrition counselling and services are needed to promote better nutritional practices among British Columbia Indians. The participation and enthusiasm of the Indian people themselves i s essential i f improvements i n nutritional status are to be achieved. Large differences which exist i n the levels of health of the Indian population and the general Canadian population have been documented, and, according to Butler (1975), many developments have been, and are being, introduced i n an attempt to bring the health of the native people up to national standards. These developments should include expanded nutrition education and counselling services i f this goal i s to be attained. 69 CHAPTER VI SUMMARY This study was conducted t o assess the f o l a t e s t a t u s o f n a t i v e Indian groups i n B r i t i s h Columbia. D i e t a r y f o l a t e s t a t u s was assessed, by use of a 24-hour r e c a l l , a t t h r e e n o n - i s o l a t e d and f o u r i s o l a t e d reserves throughout B r i t i s h Columbia. A subsample s e l e c t e d from the seven r e s e r v e s , comprised o f s u b j e c t s from S e c h e l t , N e c o s l i e and F o r t Ware Reserves, was i n v o l v e d i n a more comprehensive assessment o f f o l a t e s t a t u s , where d i e t a r y and hematological parameters of f o l a t e s t a t u s were measured. Subjects from S e c h e l t R e s i d e n t i a l School were a l s o i n v o l v e d i n the comprehensive p a r t of t h i s study. D i e t a r y f o l a t e consumption i s s i g n i f i c a n t l y lower on i s o l a t e d as compared t o n o n - i s o l a t e d r e s e r v e s , and i s c o n s i d e r a b l y higher f o r residence as compared t o res e r v e s u b j e c t s . Males consume s i g n i f i c a n t l y more d i e t a r y f o l a t e than do females. Serum f o l a t e values f o r the s u b j e c t s as a whole are d i r e c t l y and s i g n i f i c a n t l y r e l a t e d t o d i e t a r y f o l a t e i n t a k e s , and r e d c e l l f o l a t e values show no s i g n i f i c a n t c o r r e l a t i o n . Serum f o l a t e values are lower a t F o r t Ware than a t S e c h e l t o r N e c o s l i e r e s e r v e s , and values f o r c h i l d r e n l i v i n g on reserves are lower than f o r c h i l d r e n l i v i n g i n residence. On the b a s i s of red c e l l v a l u e s , 16 t o 45% o f the s u b j e c t s a t the three reserves are c l a s s i f i e d as "at r i s k " , however no evidence of m e g a l o b l a s t i c anemia i s i n d i c a t e d from the hematological examinations. 70 Findings from this study are comparable to those from the Nutrition Canada Survey (1973), i n that serum folate values for males and females are not significantly different, "and are significantly higher for children than for adults, with a larger proportion of the adult population classified as "at risk". Folate status appears to be lower at isolated reserves than at non-isolated reserves, yet a need for improvement i s indicated i n both groups. Many individuals are either bordering on or are deficient with respect to f o l i c acid. I t i s suggested that this appears to be a more serious problem at isolated reserves due to the poor selection and avai l a b i l i t y of foods. 71 BIBLIOGRAPHY Adams, W. 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Signature: Date: 81 APPENDIX B Assayed and calculated results for total folate for the 24-hour food collections from Sechelt and Necoslie Reserves and Sechelt Residence. Sample No. Assayed Values Values C a l c u l a t e d from Food Tables T o t a l F o l a t e T o t a l F o l a t e : (iBl . 1 360 323 2 300 265 3 258 233 4 277 246 5 79 90 6 159 137 7 134 105 8 223 190 9 299 238 10 205 159 11 84 71 12 175 139 13 198 143 14 209 168 15 199 170 16 159 121 17 201 154 18 119 132 19 239 182 20 256 232 21 230 189 82 APPENDIX C Canadian Recommended Daily Nutrient Intake* Age Energy Iron Free Folate (Years) Sex (Kcal) (mg) (ug) 1-3 Both 1400 8 100 4-6 Both 1800 9 100 7-9 M 2200 10 100 F 2000 10 100 10 - 12 M 2500 11 100 F 2300 11 100 13 - 15 M 2800 13 200 F 2200 14 200 16 - 18 M 3200 14 200 F 2100 14 200 19 - 35 M 3000 10 200 F 2100 14 200 36 - 50 M 2700 10 200 51 + F 1900 14 200 M 2300 10 200 F 1800 9 200 * Committee for Revision of the Canadian Dietary Standard. Ottawa, 1975. 83 APPENDIX D Guidelines for Interpretation of serum folate, red cell folate, and hemoglobin levels. Less than acceptable (at risk) Variable Deficient (High risk) Low (Moderate risk) Acceptable (Low risk) ng/ml ng/ml ng/ml Serum Folate 3 (All ages) 2.5 2.5-5.0 5.0 Red Cell Folate b 140 (All ages) 140-159 160 g/100 ml g/100 ml g/100 ml Hemoglobin0 Age Group (Years) Sex 0 - 1 M&F 9.0 9-10 10 2 - 5 M&F 10 10-11 11 6-12 M&F 10 10-11.5 11.5 13 - 16 M 12 12-13 13 13 - 16 F 10 10-11.5 11.5 17+ M 12 12-14 14 17+ F 10 10-12 12 a WHO Tech. Rep. Ser. No. 405 (1968) b O'Neal et al, (1970) and WHO Tech. Rep. Ser. No. 405 (1968) c ICNND Manual for Nutrition Surveys (1963) LEGEND TO APPENDICES E, F, AND G (Codes for isolation and sex) Variable Code Isolation 0 = No classification 1 = Non-Isolated Reserve 2 - Isolated Reserve Sex 6 8 = Male = Female APPENDIX E Raw Data - Individual Nutrient Values Calculated From 24-Hour Dietary Recalls For Subjects From The Seven Reserves. Total Folate (ug/Day), Calories, and Iron (mg/Day) CODE LOCATION ISO SEX AGE * * * * DIETARY* * * * MUM LAT FOLATE CALORIES IRON ION UG/DAY MG/DAY 10 01 SECHELT 1 8 41 245.7 1549 4.6 10 02 SECHELT 1 8 12 175.5 1516 8,8 1003 SECHELT 1 6 14 193.8 2115 8.7 1004 SECHELT 1 6 15 383.0 2650 11.4 1005 SECHELT 1 8 42 106,7 1205 8.3 1006 SECHELT 1 6 52 137.1 12 88 10.4 1007 SECHELT 1 6 15 125.0 1702 6.2 1008 SECHELT 1 8 66 105.2 1457 8.8 1009 SECHELT 1 6 16 204. 4 1 963 11.2 1010 SECHELT 1 6 28 255. 5 2515 17.2 1011 SECHELT 1 8 27 136.4 1624 14.0 1012 SECHELT 1 6 4 134.6 1940 26.9 1013 SECHELT 1 8 5 99.8 1303 8*6 1014 SECHELT 1 8 26 93.1 13 87 7.8 1015 SECHELT 1 3 7 63.5 1365 4.4 1016 SECHELT. 1 6 6 140.4 1671 4.9 1017 SECHELT 1 8 25 180.9 965 7.2 1018 SECHELT 1 6 29 306.6 2 546 17.5 1019 SECHELT 1 8 6 190.1 1445 8.2 1020 SECHELT 1 6 5 144.2 1026 5.7 1021 SECHELT 1 8 22 196.1 1548 7.2 10 22 SECHELT 1 6 25 314.4 2600 16.8 1023 SECHELT 1 8 55 131*5 2 019 17.0 1024 SECHELT 1 8 19 150.7 1650 16.7 1025 SEC HELT 1 8 44 88.6 1444 8.1 1026 SECHELT 1 6 48 108.0 1407 10.1 1027 SECHELT 1' 6 10 129.6 1554 9.6 1028 SECHELT 1 8 5 65.6 870 4.7 1029 SECHELT 1 6 13 15 7.0 1718 14.7 1030 SECHELT 1 6 15 140.0 1780 11 .8 1031 SECHELT 1 8 16 202.2 1813 12.2 1032 SECHELT 1 8 21 63.6 788 6.9 1033 SECHELT 1 8 16 219.2 2 533 11.9 1034 SECHELT 1 8 18 174,9 1937 12.5 1035 SECHELT 1 6 12 91. 8 770 5.5 1036 SECHELT 1 6 13 142.9 2157 8.0 1037 SECHELT 1 8 45 164.4 1429 9.3 1038 SECHELT 1 6 44 422.0 4189 25.1 1039 SECHELT 1 6 18 370.4 3702 19.5 1040 SECHELT 1 6 8 309.9 2444 12.4 1041 SECHELT 1 8 19 159.7 1476 8.6 1042 SECHELT 1 6 2 184.7 1691 11.5 10 43 SECHELT 1 8 1 108.3 850 2.1 1044 SECHELT 1 8 34 95.3 1245 11.0 1045 SECHELT 1 6 11 144.5 2630 12.6 1046 SECHELT 1 6 12 99.3 2570 13.9 1047 SECHELT 1 8 5 132-7 1671 7.9 1048 SECHELT 1 8 18 135.5 1736 15.1 1049 • SECHELT 1 6 20 278. 5 2 400 24.4 1050 SECHELT 1 8 35 85.4 1822 14.5 CODE LOCATION ISO SEX AGE NUM LAT ION 1051 SECHELT 1 8 22 1052 SECHELT 1 6 21 1053 SECHELT 1 8 1 1054 WARE 2 8 61 1055 WARE 2 6 69 1056 WARE 2 8 18 1057 WARE 2 8 33 1058 WARE 2 6 48 1059 WARE 2 8 7 1060 WARE 2 8 4 1061 WARE 2 8 86 1062 WARE 2 8 3 1063 WARE 2 8 21 1064 WARE 2 8 5 1065 WARE 2 8 32 1066 WARE 2 8 8 1067 WARE 2 8 22 1068 WARE 2 8 1 1069 WARE 2 6 2 1070 WARE 2 8 31 1071 WARE 2 8 50 1072 WARE 2 8 31 1073 WARE 2 6 8 1074 WARE 2 6 10 1075 WARE 2 8 14 1076 WARE 2 8 22 1077 WARE 2 6 25 1078 WARE 2 8 5 1079 WARE 2 8 33 1080 NECOSLIE 1 8 30 1081 NECOSL IE 1 8 7 1082 NECOSLIE 1 6 8 1083 NECOSLIE 1 8 21 1084 NECOSLIE 1 6 26 1085 NECOSLIE 1 8 25 1086 NECOSLIE 1 6 37 1087 NECOSLIE 1 8 2 1088 NECOSLIE 1 8 21 1089 NECOSLIE 1 6 36 1090 NECOSLIE 1 8 4 1091 NECOSLIE 1 8 61 1092 NECOSLIE 1 8 44 1093 NECOSLIE 1 6 44 1094 NECOSLIE 1 8 27 1095 NECOSLIE 1 6 36 1096 NECOSLIE 1 8 8 1097 NECOSLIE 1 8 51 1098 NECOSLIE 1 8 24 1099 NECOSLIE 1 8 2 1100 NECOSL IE 1 8 4 * * * * DIETARY* Of. $ i FOLATE CALORIES IRON UG/DAY MG/DA' 132.3 1454 7.7 417.5 4084 28.5 56.7 641 2.8 42. 8 870 30.1 42.8 870 30.1 90.1 1976 25.7 47.8 553 9.6 144.6 1856 20.3 61.3 892 9.2 26.0 544 1.9 80.2 988 10.8 99.8 1007 10.6 135.6 1433 17.5 136.7 1711 13.7 46.5 1800 10.0 158.3 1709 10.4 161.6 17 28 18.3 103.5 1242 15.9 169.2 2270 15.0 78.6 818 4.2 136.9 1273 8.7 187.7 2102 24.9 212.4 2110 89.1 299.0 3304 17.4 162.8 3400 26.2 156.5 1326 9.1 180.8 1929- 14.6 130.7 1228 8.3 168.4 2038 9.3 104.3 1800 8 .3 222.8 2813 13.8 436.6 4416 24.4 150.5 1823 10.3 251.3 2750 16.4 131.9 1828 14.0 274.6 3667 25.5 141.3 1687 5.9 117.2 1259 9.2 174.0 1315 15.6 148.1 1870 10.2 121.0 .1163 6.1 177.7 1887 12.7 289.2 3417 25.8 141.5 1666 7.4 311.4 3639 17.9 183.0 2130 23.7 153.7 1751 17.9 232.2 2 734 15.0 165.5 1475 8.2 187.6 1937 11.0 CODE LOCATION ISO SEX AGE * * * * DI ETARY* $ $ * NUM LAT FOLATE CALORIES IRON ION UG/DAY MG/DAY 1101 NECOSLIE 1 8 39 112.6 1316 18.2 1102 NECOSLIE 1 6 38 342.0 3 781 36.7 1103 NECOSLIE 1 6 5 250.0 2888 35.3 1104 NECOSLIE 1 8 27 175.5 1159 8.5 1105 NECOSLIE 1 6 6 184.0 1601 6.6 1106 NECOSLIE 1 8 54 141.0 1955 18.5 1107 NECOSLIE 1 8 20 63.2 1179 7.0 1108 NECOSLIE 1 8 52 125. 9 1803 9.4 1109 NECOSLIE 1 8 37 170.1 2337 14.7 1110 NECOSLIE 1 8 74 82.2 1096 17.4 1111 NECOSLIE 1 8 9 332. 8 2799 18.0 1112 NECOSLIE 1 8 26 265.4 2545 14.1 1113 NECOSLIE 1 8 42 98.8 1914 10.6 1114 NECOSLIE 1 6 46 188.0 2 867 16.9 1115 NANAIMO 1 8 28 214.9 2426 10.4 1116 NANAIMO I 6 31 280. 9 2292 11.6 1117 NANAIMO 1 6 3 215.5 2601 9.5 1118 NANAIMO 1 8 33 570. 6 1773 18.8 1119 NANAIMO 1 6 38 799.4 2498 31.4 1120 NANAIMO 1 6 10 542.6 2922 24.5 1121 NANAIMO 1 6 8 388.3 2263 20.6 1122 NANAIMO 1 8 46 154.4 .1175 7.0 1123 NANAI MO 1 8 25 177.6 2447 14.1 1124 NANAIMO 1 6 24 398. 5 4479 27.3 1125 NANAIMO 1 8 24 130.5 2129 12.0 1126 NANAIMO I 8 46 178.1 1956 9.2 1127 NANAIMO 1 8 25 211.2 2055 12.2 1128 NANAIMO 1 8 40 282.9 1618 4.7 1129 NANAIMO 1 6 42 280.6 1475 5.7 1130 NANAIMO 1 8 18 191.5 1918 9.3 1131 NANAIMO 1 8 13 178.3 2354 9.7 1132 NANAIMO 1 8 42 163.9 1470 9.4 1133 NANAIMO 1 6 44 46 3.1 3 941 26.5 1134 NANAIMO 1 8 3 424.3 2505 13.0 1135 NANAIMO 1 8 5 424.5 1962 10.9 1136 NANAIMO 1 8 60 149.1 1728 6.4 1137 NANAIMO 1 6 64 174.7 1975 5.6 1138 NANAIMO 1 8 17 197.2 1767 6.8 1139 NANAIMO 1 8 84 110.1 1406 5.2 1140 NANAIMO 1 6 46 335.0 4302 19.0 1141 NANAIMO 1 8 61 22 8.8 1459 9.4 1142 NANAIMO 1 8 48 159.3 1961 18.0 1143 NANAIMO 1 6 50 300.7 3861 20.6 1144 NANAIMO 1 6 24 128.1 1548 7.0 1145 NANAIMO 1 8 3 8 65.2 1845 14.4 1146 NANAIMO 1 6 40 191.5 2586 19.9 1147 NANAIMO 1 6 17 155.8 3051 17.5 1148 NANAIMO 1 8 57 124.7 1113 11.8 1149 NANAIMO 1 8 85 102.4 1044 6.2 1150 NANAIMO 1 8 29 81.3 1520 3.9 89 CODE LOCATION ISO SEX AGE NUM LAT ION 1151 NANAIMO 1 6 29 1152 NANAIMO 1 8 25 1153 NANAIMO 1 6 24 1154 NANAIMO 1 8 56 1155 NANAIMO 1 6 63 1156 NANAIMO 1 6 29 1157 NANAIMO 1 8 27 1158 NANAIMO 1 6 24 1159 NANAIMO 1 8 26 1160 NANAIMO 1 8 24 1161 NANAIMO 1 8' 4 1162 NANAIMO 1 6 2 1163 NANAIMO 1 8 17 1164 NANAIMO 1 8 41 1165 NANAIMO 1 6 41 1166 NANAIMO 1 8 28 1167 NANAIMO 1 8 27 1168 NANAIMO 1 6 6 1169 NANAIMO 1 8 23 1170 NANAIMO 1 6 23 1171 NAZKO 2 8 36 1172 NAZKO 2 6 41 1173 NAZKO 2 8 26 1174 NAZKO 2 8 4 1175 NAZKO 2 8 12 1176 NAZKO 2 8 20 1177 NAZKO 2 6 2 1178 NAZKO 2 8 29 1179 NAZKO 2 8 11 1180 NAZKO 2 8 44 1181 NAZKO 2 8 8 1182 NAZKO 2 8 51 1183 NAZKO 2 6 17 1184 NAZKO 2 6 13 1185 NAZKO 2 8 11 1186 NAZKO 2 8 14 1187 NAZKO 2 6 11 1188 NAZKO 2 6 11 1189 NAZKO 2 6 16 1190 NAZKO 2 6 14 1191 NAZKO 2 6 11 1192 NAZKO 2 8 33 1193 NAZKO 2 6 7 1194 BAB INE 2 8 32 1195 BABINE 2 6 35 1196 BABINE 2 6 10 1197 BABINE 2 8 8 1198 BABINE 2 8 59 1199 BABINE 2 8 20 1200 BABINE 2 8 10 * * * * DIETARY* * * • FOLATE CALORIES IRON UG/DAY MG/ DA 431. 5 3885 15.9 297.4 2296 17.1 373.3 2615 19.2 201.1 1438 8.0 415.3 3 490 13.0 422. 9 3047 17.6 169.9 1613 23.2 270.6 3 466 13-9 265, 7 1936 12.4 177.5 1216 8.6 232.1 1462 7.9 358.5 2493 14.0 277.5 2010 10,0 177-5 1895 12.9 323.0 4120 20.9 259. 5 1420 13.3 141.2 1653 8.5 391.7 2 713 .14.2 217.1 1105 8.0 159. 5 3269 17.7 155.3 1633 16.9 212.9 2234 21.9 247.4 2443 24.2 167.6 1346 8.2 207.2 1234 11.0 156. 1 2332 19.4 165.1 1443 10.7 148.3 1679 11.2 169.3 2359 19.3 125.4 1410 8.3 220.3 2769 12.8 173.9 1046 11.0 33 7.0 2 627 14.7 200.9 1658 10.5 122.6 1621 10.5 180.1 15 92 13.4 178.8 2483 11.8 238.9 2067 13.9 218.1 2640 17.7 81.7 1800 5.5 156.2 1850 7.3 200. 1 1945 8.8 236.6 2557 11.1 146.9 1777 15.4 187.4 2305 40.3 291.5 2412 32.8 225.8 1702 27.8 116.6 1809 14.1 117.5 1924 17.3 86.8 1641 11-2 90 CODE LOCATION ISO SEX AGE NUM LAT ION 1201 SABINE 2 8 71 12 02 BABINE 2 6 27 1203 BAB INE 2 6 20 1204 BABINE 2 6 22 1205 BABINE 2 6 . 28 1206 BABINE 2 8 42 1207 BABINE 2 8 14 1208 BABINE 2 8 17 1209 BABINE 2 8 9 1210 BABINE 2 8 14 1211 BABINE 2 8 35 1212 BABINE 2 8 40 1213 BABINE 2 8 2 1214 BABINE 2 6 9 1215 TAKLA 2 8 45 1216 TAKL A 2 8 20 1217 TAKLA 2 8 33 1218 TAKLA 8 6 1219 TAKLA 2 8 12 1220 TAKLA 2 8 56 1221 TAKLA 2 8 15 1222 TAKLA 2 8 27 1223 TAKLA 2 8 7 12 24 TAKLA 2 8 35 1225 TAKLA 2 8 11 1226 TAKLA 2 6 14 1227 TAKLA 2 8 36 1228 TAKLA 2 8 9 1229 TAKLA 2 8 46 1230 TAKLA 2 6 60 1231 TAKLA 2 8 6 1232 TAKLA 2 8 23 1233 TAKLA 2 8 3 1234 TAKLA 2 8 28 123 5 TAKLA 2 8 50 1236 TAKLA 2 6 52 1237 TAKLA 2 8 28 1238 TAKLA 2 8 35 1239 TAKLA 2 8 12 1240 TAKLA 2 8 26 1241 TAKLA 2 8 20 1242 TAKLA 2 8 13 1243 TAKLA 2 8 53 1244 TAKLA 2 6 10 7 = 0.6 7 * * * * DIETARY* FOLATE CALORIES IRON UG/DAY MG/DA 110.7 1236 12.1 92.0 1616 11.5 219. 5 2888 31.4 340.3 2911 '31.6 635.7 3097 19.7 137.3 1464 13.6 217.6 2098 15.0 174.0 2143 20.9 209.0 2406 18.3 124.8 1025 15.0 151.5 2090 17.4 149.9 1612 17.2 178.7 1647 12.6 304.7 2289 26.7 120.1 1673 13.8 124.9 1618 16.7 99.8 1234 9.9 135.3 1615 10.8 146.5 1839 14.3 145.1 1667 19.2 107.9 1215 16.0 83.7 1103 4.9 150.3 2493 11.2 112.5 1377 19.2 158.3 2027 14.1 159.8 2009 18.1 86.8 772 12.1 122.8 1321 16.3 162.4 2194 19.2 175.6 2311 27.6 176.4 1820 13.0 204.4 2627 15.6 161.0 2287 16.6 167.8 1488 19.3 133.5 1439 14.0 159.0 16 97 18.4 121.5 1092 14.5 23 7.1 1827 22.0 288.6 2136 20.3 141 .3 1983 13.3 173.8 2 358 14.6 172.8 2 5 73 20.5 152.5 1990 15.6 223.8 2457 13.6 APPENDIX F Raw Data - Individual Nutrient Values Calculated From 24-Hour Dietary Recalls For Subjects Participating In The Comprehensive Part Of The Study. Total Folate (ug/day), Calories, and Iron (mg/day) 92 CODE LOCATION I SO SEX AGE * * * * DI ETARY* # * * NUM LAT FOLATE CALORIES IRON ION UG/DAY MG/DAY 11 SECHELT 1 8 41. 14 245.7 1549 4.6 13 SECHELT 1 6 11.80 175.5 1516 8.8 14 SECHELT 1 6 13.78 193.8 2115 8.7 15 SECHELT 1 6 14. 78 383.0 2650 11.4 41 SECHELT 1 8 27.42 136.4 1624 14.0 51 SECHELT 1 8 26.48 93 . 1 1387 7.8 53 SECHELT 1 8 7.78 6 3.5 1365 4.4 54 SECHELT 1 6 6.48 140.4 1671 4.9 61 SECHELT 1 8 24. 56 180.9 965 7.2 62 SECHELT 1 6 29.62 306.6 2546 17.5 71 SECHELT 1 8 22.54 196.1 1548 7.2 91 SECHELT 1 8 44.46 88.6 1444 8.1 92 SECHELT 1 6 48.38 108.0 1407 10.1 93 SECHELT 1 8 9.62 129.6 15 54 9.6 94 SECHELT 1 8 5.08 65.6 870 4.7 95 SECHELT 1 6 12.68 157.0 1718 14.7 96 SECHELT 1 6 14. 88 140.0 1780 11.8 97 SECHELT 1 8 16.28 202.2 1813 12.2 98 SECHELT 1 8 20.82 63.6 788 6.9 103 SECHELT 1 8 16. 02 219.2 2 533 11.9 104 SECHELT 1 8 17.94 174.9 1987 12.5 105 SECHELT 1 6 11.74 91.8 7 70 5.5 106 SECHELT 1 6 13.08 142.9 2157 8.0 107 SECHELT 1 8 8.74 164.4 1936 9.1 108 SECHELT 1 8 23.92 71.0 2091 10.2 109 SECHELT 1 6 19.04 99.9 2 3 80 14.0 121 SECHELT 1 8 19.18 159.7 1476 8 .6 122 SECHELT 1 6 22.38 184.7 2197 13.3 161 SECHELT 1 8 21.60 132.3 1454 7.7 162 SECHELT 1 6 2 0. 82 417.5 4084 28.5 174 WARE 2 6 12.60 90.1 2100 13.2 185 WARE 2 6 11.28 99.9 2098 11.2 211 WARE 2 8 85. 92 80.2 988 10.8 215 WARE 2 8 20.90 135.6 1433 17.5 216 WARE 2 8 32.44 46.5 989 5.0 217 WARE 2 6 11.76 103.1 2298 13.7 231 WARE 2 8 21.68 161.6 1728 18.3 241 WARE 2 8 30.46 78.6 818 4.2 242 WARE 2 6 27.32 97.3 2615 16.1 2 51 WARE 2 8 50.00 136.9 1273 8.7 252 WARE 2 6 63. 00 131.0 1672 10.2 253 WARE 2 6 20.00 107.9 2381 13.1 254 WARE 2 6 13.00 117.1 2381 13.1 2 64 WARE 2 6 10.24 299.0 3304 17.4 265 WARE 2 8 14.00 162.8 3400 26.2 266 WARE 2 6 19.32 101.3 2103 12.9 271 WARE 2 8 22.04 156.5 1326 9.1 272 WARE 2 6 25.30 180.8 1929 - 14.6 281 WARE 2 8 33.02 168.4 2038 9.3 2 83 WARE 2 6 13.62 93.6 2065 10.9 93 CODE LOCATION ISO SEX AGE $ $ $ $ DIETARY* * * * NUM LAT FOLATE CALORIES IRON ION UG/DAY MG/DAY 2 93 NECOSLIE 1 8 7.14 222.8 2813 13.8 294 NECOSLIE ' 1 6 8.42 436. 6 4416 24.4 3 01 NECOSLIE 1 8 21.30 150.5 18 23 10.3 311 NECOSLIE 1 8 2 5.12 131.9 1828 14.0 314 NECOSLIE 1 8 24.00 151.8 18 60 13.9 3 21 NECOSLIE 1 8 20.58 117.2 1259 9.3 3 22 NECOSLIE 1 6 36. 06 174.0 1315 15.6 331 NECOSLIE I 8 61.32 121.0 1163 6.1 333 NECOSLIE 1 6 25.00 187.3 2375 15.2 341 NECOSLIE 1 8 43. 66 177. 7 1887 12,7 343 NECOSLIE 1 8 16.00 99.9 1988 9.8 344 NECOSLIE 1 6 11.66 251.3 1937 9.8 351 NECOSL IE 1 8 27.04 141.5 1666 7.4 361 NECOSLIE 1 8 51.06 153.7 1751 17.9 362 NECOSLIE 1 6 50.00 99.9 2410 16.1 3 63 NECOSLIE 1 8 25.00 171.6 1975 13.8 364 NECOSLIE 1 8 10.78 143.4 2015 10.2 371 NECOSLIE 1 8 24. 28 232.2 2 734 15.0 381 NECOSLIE 1 8 39.42 112.6 1316 18.2 3 84 NECOSLIE 1 8 45.00 149.1 1510 17.0 385 NECOSL IE 1 6 16.00 342.0 2418 16.7 402 NECOSLIE 1 6 58.36 211.0 2892 15.7 40 3 NECOSLIE 1 8 23.00 63.0 1179 7.0 404 NECOSLIE 1 8 25.00 103.3 15 81 11.0 405 NECOSLIE 1 6 10.32 143.1 1636 8,1 406 NECOSLIE 1 6 11. 86 131.8 1636 8.1 411 NECOSLIE 1 8 51.90 125.9 1803 9.4 421 NECOSLIE 1 8 37.42 170.1 2 337 14.7 423 NECOSLIE 1 8 73.50 82.2 1096 17.4 431 NECOSLIE 1 8 25.68 265.4 2545 14.1 433 NECOSL IE 1 8 24.00 281.7 2481 13.7 441 NECOSLIE 1 8 42.44 98.8 1914 10.6 442 NECOSLIE 1 6 45.44 188.0 2867 16.9 851 RESIDENCE 0 8 10,38 310.1 2227 13.4 861 RESIDENCE 0 8 12.82 310.1 2227 13.4 871 RESIDENCE 0 8 12.26 310.1 2227 13.4 881 RESIDENCE 0 8 10.66 310.1 2227 13.4 891 RESIDENCE 0 8 11.50 310.1 2227 13.4 901 RESIDENCE 0 8 13.24 310.1 2227 13.4 911 RESIDENCE 0 8 11.34 310. 1 2227 13.4 921 RESIDENCE 0 8 14.54 310. 1 2227 13 .4 931 RESIDENCE 0 8 13.24 310. 1 2227 13.4 941 RESIDENCE 0 8 14.28 310.1 2227 13.4 951 RESIDENCE 0 8 13. 12 310.1 2227 13.4 961 RES IDENCE 0 8 11.50 310.1 2227 13.4 971 RESIDENCE 0 8 13.90 310.1 2227 13.4 981 RESIDENCE 0 8 13.56 310.1 2227 13.4 991 RESIDENCE 0 8 12.50 310. 1 2227 13.4 1001 RESIDENCE 0 8 11.72 310.1 2227 13.4 10 11 RESIDENCE 0 8 12.76 310.1 2227 13.4 94 CODE LOCATION NUM 1021 RESIDENCE 1031 RESIDENCE 1041 RESIDENCE 1051 RESIDENCE 1061 RESIDENCE 1071 RESIDENCE 1081 RESIDENCE 1091 RESIDENCE 1101 RESIDENCE 1111 RESIDENCE 1121 RESIDENCE 1131 RESIDENCE 1141 RESIDENCE 1151 RESIDENCE 1161 RESIDENCE 1171 RESIDENCE 1181 RESIDENCE 1191 RESIDENCE 1201 RESIDENCE 1211 RESIDENCE 1221 RESIDENCE 1231 RESIDENCE 1241 RESIDENCE 1251 RESIDENCE 1261 RESIDENCE 1271 RESIDENCE 1281 RESIDENCE 1291 RESIDENCE 1301 RESIDENCE 1311 RESIDENCE 1321 RESIDENCE 1331 RESIDENCE 1341 RESIDENCE 1351 RESIDENCE 1361 RESIDENCE 1371 RESIDENCE 1381 RESIDENCE 1391 RESIDENCE 1401 RESIDENCE 1411 RESIDENCE 1421 RESIDENCE 1431 RESIDENCE 1441 RESIDENCE 1451 RESIDENCE 1461 RESIDENCE 1471 RESIDENCE 1481 RESIDENCE 1491 RESIDENCE 1501 RESIDENCE 1511 RESIDENCE ISO SEX AGE LAT ION 0 8 11.20 0 8 12.84 0 8 9.04 0 8 16.48 0 8 15.30 0 8 9. 75 0 8 8.32 0 8 9. 08 0 8 10.08 0 8 8.38 0 8 8. 92 0 8 6.88 0 6 14.08 0 6 11.62 0 6 12.98 0 6 15.00 0 6 15.46 0 6 13.16 0 6 10.78 0 6, 13.36 0 8 6.64 0 6 10.00 0 6 10.46 0 8 9.06 0 6 11.42 0 6 12.62 0 8 7.84 0 8 5.54 0 8 7.84 0 8 7. 94 0 8 9.48 0 6 10.48 0 6 11.34 0 6 11.00 0 6 11.76 0 6 11.42 0 6 9.00 0 6 10.14 0 6 9.48 0 6 9. 12 0 6 10.24 0 6 10.26 0 6 8.36 0 6 9.14 0 6 9.44 0 6 7.38 0 6 9.88 0 6 9.54 0 6 11.18 0 6 8.16 * * * * D I E T A R Y * * * * FOLATE CALORIES I RON UG/DAY MG/OA^  310.1 2 227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2 227 13.4 310.1 2 227 13.4 310.1 2227 13.4 310.1 2 227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310. 1 2 227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310,1 2 227 13.4 310.1 2227 13.4 310. 1 2227 13.4 310.1 2227 13.4 310.1 2 22 7 13.4 310.1 2227 13.4 310.1 2227 13 .4 310. 1 2227 13.4 310. 1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2 227 13.4 310. 1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2 227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2 227 13.4 310.1 2227 13.4 310. 1 2227 13.4 310.1 2227 13.4 310. 1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2227 13.4 310.1 2 227 13.4 95 CODE LOCATION ISO SEX AGE •M ;jc # # DIETARY* * £ * NUM LAT FOLATE CALORIES I RON ION UG/DAY MG/DAY 15 21 RESIDENCE 0 6 6.14 310. 1 2227 13.4 1531 RESIDENCE 0 6 7.98 310.1 2227 13.4 15 41 RESIDENCE 0 6 7. 94 310.1 2227 13.4 APPENDIX G Raw Data - Individual Hematological Values For Subjects Participating In The Comprehensive Part Of The Study. Serum Folate (ng/ml) Red Cell Folate (ng/ml), 3 Hemoglobin (g/100 ml), Mean Corpuscular Volume (p ), and Mean Corpuscular Hemoglobin Concentration (%) CODE LOCATION ISO SEX AGE NUM LAT ION 11 SECHELT 1 8 41.14 13 SECHELT I 6 11.80 14 SECHELT 1 6 13. 78 15 SECHELT I 6 14.78 41 SECHELT 1 8 27.42 51 SECHELT 1 8 26.48 53 SECHELT 1 8 7.78 54 SECHELT 1 6 6.48 61 SECHELT I 8 24.56 62 SECHELT 1 6 29.62 71 SECHELT 1 8 22. 54 91 SECHELT 1 8 44.46 92 SECHELT 1 6 48.38 93 SECHELT 1 8 9.62 94 SECHELT 1 8 5.08 95 SECHELT 1 6 12.68 96 SECHELT 1 6 14.88 97 SECHELT 1 8 16.28 98 SECHELT 1 8 20. 82 103 SECHELT 1 8 16.02 104 SECHELT 1 8 1 7. 94 105 SECHELT 1 6 11.74 106 SECHELT 1 6 13.08 107 SECHELT 1 8 8. 74 108 SECHELT 1 8 23.92 109 SECHELT 1 6 19-04 121 SECHELT 1 8 19. 18 122 SECHELT 1 6 22.38 161 SECHELT 1 8 21.60 162 SECHELT 1 6 20.82 174 WARE 2 6 12.60 185 WARE 2 6 11.28 211 WARE 2 8 85.92 215 WARE 2 8 20.90 216 WARE 2 8 32.44 217 WARE 2 6 11.76 231 WARE 2 8 21.68 241 WARE 2 8 30.46 242 WARE 2 6 27.32 251 WARE 2 8 50.00 2 52 WARE 2 6 63.00 253 WARE 2 . 6 20.00 254- WARE 2 6 13.00 264 WARE 2 .6 10.24 265 WARE 2 .8 14.00 266 WARE 2 6 19.32 271 WARE 2 8 22.04 272 WARE 2 6 25.30 281 WARE 2 8 33.02 2 83 WARE 2 6 13.62 97 SERUM RED HB MCV MCHC FOL- CELL ATE FOL. 5.8 519 13.4 84 34.0 6.1 152 12.6 79 34.2 7.7 330 12.8 80 35.0 4.8 171 14.8 80 34.4 4.5 187 12.7 80 34.5 4.3 294 14.2 78 34. 1 5.4 248 13.5 73 34.0 7.0 323 12.6 74 34.9 5.0 374 9.8 77 31.9 10.2 203 15.3 85 34.1 6.7 281 13.0 78 34.4 3.5 209 14.1 79 33.4 3.2 188 14.0 78 34.3 6.4 352 12.2 78 34.4 10.2 241 11.7 72 34.6 5.0 147 14.0 79 35.0 6. 1 233 13.6 81 34.6 3.9 179 12.2 76 34.0 6.7 259 14.1 86 34.2 3.9 139 14.3 83 34.2 8.5 354 11.2 71 32.2 8.5 288 14. 2 73 34. 8 6.4 274 13.1 74 34.1 10.2 221 13.2 74 34.2 6.7 306 13.6 81 33.3 6.4 259 15.6 84 34.8 9.8 245 13.0 80 34.4 9.8 148 14.5 83 34.5 8.5 147 12.2 82 34.0 12.0 249 14.9 86 34.7 3.0 125 15.6 85 34.4 4.0 177 14.1 77 34.3 6.3 265 13.7 89 33.8 3.2 185 14.0 81 34.7 3.2 183 12.8 83 33.0 2.3 179 13.9 88 33. 0 2.3 159 16.0 82 33.0 6.0 220 14.2 84 32.7 6.3 126 14.7 81 33. 1 2.6 187 12.0 78 32.3 4.0 123 14.4 82 33.6 2.8 102 15.3 86 33.8 5.3 272 99.9 80 99.9 7.3 294 13-0 76 34.3 3.2 147 13.8 82 34.0 3.7 156 16.i 83 33-8 5.6 236 13.3 81 32-5 4.2 141 14.8 83 33.0 2.8 126 12. 7 80 33.2 3.2 171 14.0 80 33.5 CODE LOCATION I SO NUM LAT ION 293 NECOSLIE 1 294 NECOSLIE i 301 NECOSLIE 1 311 NECOSLIE 1 314 NECOSLIE 1 321 NECOSLIE 1 322 NECOSLIE 1 331 NECOSLIE 1 333 NECOSLIE 1 341 NECOSLIE 1 343 NECOSLIE 1 344 NECOSLIE 1 351 NECOSLIE 1 361 NECOSLIE 1 362 NECOSLIE 1 363 NECOSLIE 1 364 NECOSLIE 1 371 NECOSLIE 1 381 NECOSLIE 1 3 84 NECOSLIE 1 385 NECOSLIE 1 402 NECOSLIE 1 403 NECOSLIE 1 404 NECOSLIE 1 405 NECOSLIE 1 406 NECOSLIE 1 411 NECOSLIE 1 421 NECOSLIE 1 423 NECOSLIE 1 431 NECOSLIE 1 433 NECOSLIE 1 441 NECOSLIE 1 442 NECOSLIE I 851 RESIDENCE 0 861 RESIDENCE 0 871 RESIDENCE 0 881 RESIDENCE 0 891 RESIDENCE 0 901 RESIDENCE 0 911 RESIDENCE 0 921 RESIDENCE 0 931 RESIDENCE U 941 RESIDENCE 0 951 RESIDENCE 0 961 RESIDENCE 0 971 RESIDENCE 0 981 RESIDENCE 0 991 RESIDENCE 0 1001 RESIDENCE 0 1011 RESIDENCE 0 SEX AGE SERUM FOL-ATE 8 7.14 8.3 6 8.42 4.6 8 21.30 5.0 8 25.12 5.2 8 24.00 6.5 8 20. 58 6.0 6 36-06 11-0 8 61.32 5.0 6 25.CO 6.9 8 43.66 6.9 8 16.00 5.2 6 11.66 3.5 8 27.04 5.7 8 51.06 7.3 6 50.00 5.7 a 25.00 9.7 8 10.78 11.5 8 24.. 28 6.3 8 39. 42 4.3 8 45.00 4.0 6 16. 00 10.3 6 58.36 3.2 8 23.00 2.0 8 25. 00 2.0 6 10.32 8.3 6 11.86 5-0 8 51.90 3.2 8 37.42 4.3 8 7.3.50 5.2 8 25.68 5.2 8 24.00 3.7 8 42.44 4.3 6 45.44 4.0 8 10.38 12.2 8 12. 82 2.7 8 12.26 5.2 8 10.66 6.5 8 11.50 8.5 8 13.24 7.1 8 11.34 8.2 8 14.54 4.1 8 13.24 6.5 .8 14.28 7.8 8 13.12 6.1 8 11. 50 9-8 8 13.90 3.0 8 13.56 4.1 8 12.50 6.5 8 11.72 5.8 8 12.76 5.8 98 RED HB MCV MCHC CELL FOL. 198 13. 1 86 32.9 226 12.6 83 34.4 259 11.1 74 32.6 168 14.0 85 34*3 191 11.8 86 34.2 321 13.7 91 33.6 97 15.8 91 34.3 216 12.9 ,81 33.3 141 15.8 87 34.2 274 11.7 83 31.9 99 13.4 85 32.8 158 13.8 76 34.2 194 13.4 83 33.6 197 13.fi 92 33.2 206 15.5 89 34*6 400 1 1 . i ,95 33.9 275 12.8 83 33.7 127 12.9 88 33.8 207 15.4 96 33-6 149 13.9 91 33.6 212 14.6 85 34.3 131 15.5 89 33.4 275 12.6 ,79 32.9 171 13.6 83 33.7 198 13.8 79 33.8 137 12. 9 81 33.8 85 12.7 93 33.9 109 14. 1 91 3.3.2 145 13.0 92 33.0 153 12.1 81 32.4 214 9.9 82 32.9 184 12.8 81 32.5 12 7 13.7 87 33.5 19 5 13.1 79 33.7 114 13.1 79 33.9 104 12.2 84 34.3 197 12.9 .80 34.8 100 12.1 83 34.0 100 11.5 75 33.5 65 12.8 81 34.6 999 12.2 80 34.3 207 11.8 74 33.6 85 12.5 81 33.9 81 12.9 82 35.0 88 12.8 84 34.7 77 11.6 83 33.9 71 12.6 83 33.7 165 11.7 82 34.3 999 12.9 81 33. 9 84 12.2 84 33.6 99 CODE LOCATION ISO SEX AGE SERUM RED HB MCV MCHC NUM LAT FOL- CELL ICN ATE FOL-1021 RESIDENCE 0 8 11.20 3.9 119 II-0 81 33. 3 1031 RESIDENCE 0 8 12.84 8.2 102 12.2 86 34..1 1041 RESIDENCE 0 8 9-04 6.9 191 12. 0 79 34.6 1051 RESIDENCE .0 8 16.48 8.2 114 12.1 82 34.3 1061 RESIDENCE 0 8 15.30 9.3 133 12.5 82 33.9 1071 RESIDENCE 0 8 9.75 7.8 170 13.0 76 34.9 1081 RESIDENCE 0 8 8.32 9.7 119 12.5 .82 34.2 1091 RESIDENCE 0 8 9.08 9.2 246 11.7 81 33.9 1101 RESIDENCE 0 8 10.08 7.6 i l l 12-4 83 34. 0 1111 RESIDENCE 0 8 8.38 8.4 177 12.0 81 33-9 1121 RESIDENCE 0 8 8.92 6.5 89 12.4 82 34.0 1131 RESIDENCE 0 8 6.88 8.0 241 12.5 83 34.1 1141 RESIDENCE 0 6 14.08 9.2 86 12.3 83 33.8 1151 RESIDENCE 0 6 11.62 8.8 132 12-2 82 34.5 1161 RESIDENCE 0 6 12. S8 6.1 129 12.9 75 34.3 1171 RESIDENCE 0 6 15.00 8.8 86 13.5 84 34. 5 1181 RESIDENCE 0 6 15.46 9.1 130 13.3 86 34.3 1191 RESIDENCE 0 6 13. 16 6.1 191 12.3 81 34.5 1201 RESIDENCE 0 6 10.78 8.8 266 12.2 78 34-8 1211 RESIDENCE 0 6 13.36 6.5 222 12.5 74 34.2 1221 RESIDENCE 0 8 6.64 11.8 152 11.6 76 33 . 9 1231 RESIDENCE 0 6 10.00 8.8 127 11.9 84 34.6 1241 RESIDENCE 0 6 10.46 8.4 110 12.6 81 34-8 1251 RESIDENCE 0 8 9.06 3.5 170 12.3 80 35.1 1261 RESIDENCE 0 6 1.1.42 8.8 112 11.4 84 34.3 1271 RESIDENCE 0 6 12.62 11.5 140 12.6 81 34.3 1281 RESIDENCE 0 8 7.84 10-0 120 11.0 74 33.9 1291 RESIDENCE 0 8 5.54 3.8 68 11.4 77 34.9 1301 RESIDENCE 0 8 7.84 10.5 84 11.3 79 35.4 1311 RESIDENCE 0 8 7.94 5.8 158 12-2 81 35.3 1321 RESIDENCE 0 8 9.48 10.0 204 11.7 79 33-7 1331 RESIDENCE 0 6 10.48 9.2 90 12. i 81 33.5 1341 RESIDENCE 0 6 11.34 5.8 95 11.2 79 34. 5 13 51 RESIDENCE 0 6 11.00 14.0 92 12-3 82 35.6 1361 RESIDENCE 0 6 11.76 15.5 71 12.4 83 34.6 1371 RESIDENCE 0 6 11.42 10.2 74 11-6 74 34.8 1381 RESIDENCE 0 6 9.00 7.8 96 11.3 78 34.0 1391 RESIDENCE 0 6 10.14 9.5 122 12.2 81 35.1 14 01 RESIDENCE 0 6 9.48 5.8 146 12.4 81 35.5 1411 RESIDENCE »o 6 9.12 12.5 123 11. 8 81 3 5.2 1421 RESIDENCE 0 6 10.24 4.7 98 12.6 7.7 35.4 1431 RESIDENCE 0 6 10.26 12.2 63 11.9 81 35.4 1441 RESIDENCE 0 6 8.36 4. 7 99 11.1 78 34.5 1451 RESIDENCE 0 6 9. 14 5.8 105 13.0 77 35.8 1461 RESIDENCE 0 6 9.44 18.5 124 12-1 79 34.7 1471 RESIDENCE 0 6 7.38 11.3 107 12.0 81 34-5 14 81 RESIDENCE 0 6 9.88 9.2 91 12.3 83 34.2 1491 RESIDENCE 0 6 9.54 9.5 64 12.1 75 34.4 1501 RESIDENCE 0 6 11.18 10-7 110 11.7 76 35.2 1511 RESIDENCE 0 6 8. 16 12-5 140 11.4 75 34.6 100 COOE LOCATION ISO SEX AGE SERUM RED H8 MCV MCHC NUM LAT FOL- CELL I ON ATE FOL. 1521 RESIDENCE 0 6 6,14 10.7 55 11.3 82 34.2 1531 RESIDENCE 0 6 7.98 9. 5 88 11.5 77 34.4 1541 RESIDENCE 0 6 7.94 10.2 116 12.3 77 33. 9 

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