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Folate status and dietary folate intake of women during oral contraceptive use and pregnancy Pietarinen, Gloria Jean 1975

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FOLATE STATUS AND DIETARY FOLATE INTAKE OF WOMEN DURING ORAL CONTRACEPTIVE USE AND PREGNANCY  GLORIA JEAN PISTARINEN B.A., Queens College, City University of New York, 1968  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Division of HUMAN NUTRITION SCHOOL OF HOME ECONOMICS  We accept this thesis as conforming to the required standard.  THE UNIVERSITY OF BRITISH COLUMBIA July, 1975  In p r e s e n t i n g t h i s  thesis  in p a r t i a l  fulfilment  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 the L i b r a r y s h a l l make i t I further  freely  available  that  f o r reference and study.  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s  thesis  f o r s c h o l a r l y purposes may be granted by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . of  this  It  thesis for financial  i s understood that copying o r p u b l i c a t i o n gain s h a l l not be allowed without my  w r i t ten pe rm i ss i on .  V I V I S ' I O A  Bfpartmeint of  N  A/uMA<V  NUTR-IT/ON  The U n i v e r s i t y o f B r i t i s h Columbia  2075 Wesbrook Place Vancouver, Canada V6T 1W5  ,  SC2//OQL OF  / - / O M E  EdoNoM/CS  - i -  ABSTRACT  During pregnancy, the expansion of fetal and maternal tissues increases the demand for folic acid and may result in reduced levels of serum and red cell folate in the mother. If dietary intake of folate is also poor, maternal folate stores may "be further depleted.  Recent research has investigated the  possibility of a similar alteration in folate status among women using oral contraceptives.  Although there have been several reports in the literature  demonstrating reduced serum folate concentrations in women taking the pill, other studies have contradicted these findings.  Furthermore, it has not been  established to what extent dietary folate intake determines the serum or red cell folate levels of women using oral contraceptives. A cross-sectional survey was designed to determine the relationship between dietary folate intake and serum and erythrocyte folate concentrations in women who are pregnant or using oral contraceptives, as compared with a group of control women. In addition, this study was intended to examine any differences or similarities in folate status among the three groups, A sample of 48 women of similar age, parity, and socio-economic status was selected from the Vancouver area.  These subjects were classified into  three groupst a control group of 18 women, who had neither been pregnant nor using oral contraceptives for at least six months prior to being in the study; an oral contraceptive group of 22 women, all of whom had been taking combination-type oral contraceptives for at least four months prior to being in the study; and a pregnant group of 8 women, who were in their third or fourth month of pregnancy at the time of commencing their participation in the study. All subjects were in good health and were not taking folic acid supplements. Any woman was excluded from the study if she had a condition (other than  - ii pregnancy) that might alter folate metabolism, or i f she was using a drug (other than oral contraceptives) known to interfere with folate metabolism. For the oral contraceptive users and control women, fasting blood samples were taken at two different phases of the menstrual cycle---day 5 and day 2 0 — - f o r two consecutive cycles.  In the group of pregnant women, three  fasting blood samples were taken, one during each trimester of pregnancy. Each subject also kept two three-day diet records---one for the three days preceding the f i r s t blood sample, and the other for the three days preceding the last blood sample.  The mean daily dietary folate intake was  calculated from these records using food composition tables. The accuracy of these calculated estimates for folate\;was tested by assaying food samples collected by 15 of the subjects.  Folate was measured microbiologically  with Lactobacillus casei. The results indicate that serum folate concentrations were higher i n the control group than i n the oral contraceptive group.  This difference was  statistically significant (p<0.05) f o r the serum folate values at day 5 of the menstrual cycle, but not at day 20 of the cycle.  There was no significant  difference i n the red c e l l folate levels-"between these two groups of subjects although, again, the control women had higher levels than did the oral contraceptive users.  Within each of the two groups, serum and red c e l l folate con-  centrations did not vary significantly with the time of the menstrual cycle. In the group of pregnant women studied, both serum folate and red c e l l folate levels were found to increase over the course of pregnancy.  However,  the rise i n serum'folate was not statistically significant, and the increase in red c e l l folate was significant between the f i r s t and second trimesters (p<0.05) but not between the second and third trimester. I t i s not clear why the folate status of the pregnant subjects improved during the course of pregnancy.  - iii -  There was no difference the three groups of subjects. f o l a t e l e v e l s and dietary  i n the l e v e l s o f dietary f o l a t e intake among The degree of correlation between the serum  f o l a t e intake was consistently higher i n the con-  t r o l women than i n the women using o r a l contraceptives.  The difference i n  correlation c o e f f i c i e n t s between the two groups was s t a t i s t i c a l l y s i g n i f i cant (p<0.0i) only a t day 5 of the cycle.  In the group of pregnant women  6  the correlation between serum f o l a t e and dietary f o l a t e was higher i n the f i r s t trimester than i n the t h i r d trimester; but the difference t i o n c o e f f i c i e n t s was not s t a t i s t i c a l l y s i g n i f i c a n t .  i n correla-  There was no s i g n i f i -  cant correlation between red c e l l f o l a t e l e v e l s and dietary f o l a t e intake i n any of the three groups of subjects. These r e s u l t s indicate that o r a l contraceptive use reduces serum f o l a t e l e v e l s , and that t h i s decline i n serum f o l a t e i s Independent of dietary f o l a t e intake.  Thus, there appears t o be some d i r e c t e f f e c t of synthetic  sex hormones i n reducing serum f o l a t e l e v e l s .  Whether t h i s i s an e f f e c t a t  the l e v e l of i n t e s t i n a l absorption or tissue u t i l i z a t i o n remains unclear. I f women using o r a l contraceptives are not able t o f u l l y absorb o r u t i l i z e t h e i r dietary f o l a t e , i t may be that t h e i r recommended d a i l y allowance f o r f o l a t e should be increased.  - iv -  ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Joseph Leichter, for his direction during the course of the study? Dr. Roy Pratt for his assistance and advice in the planning of the study and preparation of the thesisj and Dr. Melvin Lee for his advice during the thesis preparation. I am also grateful to Dr. R. Percival-Smith, Dr. Robert McNaughton, and Dr. Jon Schonblom for their assistance in obtaining subjects for the studyj the laboratory technicians at St. Paul's Hospital in Vancouver, Mrs. Jean Sutton, R.T. and Mr. Barrett Benny for their technical assistance; and Virginia Greene for her help with the computer programming and statist i c a l analysis of results. My appreciation i s extended to a l l the women who participated in the study as subjects for their interest and cooperation. Finally, I am thankful to my husband, Alan Sponberg, for his encouragement throughout the past year and his assistance in the typing of this manuscript. This study was supported by Grant No. A62^9 from the National Research Council of Canada,  -  V  -  TABLE OP CONTENTS  ABSTRACT  i  ACKNOWLEDGEMENTS  iv  LIST OP TABLES  viii  Chapter I  INTRODUCTION  i  II  REVIEW OF THE LITERATURE  5  Absorption and Metabolism of Folic Acid  5  Physiological indices of folate status  5  Absorption of f o l i c acid  6  Reduction and methylation of absorbed folates  8  Folate Deficiency and Megaloblastic Anemia  9  Definition of folate deficiency and megaloblastic anemia Etiology of folate deficiency  9 11  The Effect of Pregnancy on Folate Status  13  Anemias of pregnancyt iron and folate interrelationships  13  Folic acid deficiency of pregnancy  16  Assessment of folate status in pregnancy  18  Maternal and fetal complications associated with folate deficiency in pregnancy  20 9  Supplementation with f o l i c acid in pregnancy  20  The .-Effect of Oral Contraception on Folate Status  21  Megaloblastic anemia associated with oral contraceptive use  22  Oral contraceptive use and folate deficiency  23  Review of the studies finding a reduced serum folate in oral contraceptive users  23  - vi -  Review of the studies finding no difference in serum folate between oral contraceptive users and control women  25  Related factors of unknown significance  2?  Summary of the mechanisms by which oral contraceptives may act to alter folate status  30  The Assessment of Dietary Folate Status  31  Problems inherent i n the assay of food folates  31  Problems i n estimating dietary folate intake  33  Conclusion III  MATERIALS AND METHODS  36  Collection of Blood  38  Treatment of blood samples  39  Collection of Dietary Data Collection of food at the time of consumption for analysis of folate content  41  Analyses  42  41  Analysis of blood samples  42  Evaluation of diet records  43  Analysis of food samples  43  Statistical Analysis of the Data IV  34  RESULTS  43 45  Serum Folate, Red Cell Folate, and Other Hematological Parameters  45  The effect of oral contraceptives  45  The effect of type of oral contraceptive used and duration of oral contraceptive use on serum folate levels  4?  The effect of pregnancy  49  Comparison of pregnant subjects with controls and oral contraceptive users  53  - vii -  Dietary Folate Intake  V  55  Comparison of dietary folate intake among controls, oral contraceptive users, and pregnant subjects  55  Relationship between dietary folate intake and serum and red c e l l folate levels  57  Correlation between the calculated and assayed values of folate for food samples collected at the time of consumption  59  DISCUSSION  BIBLIOGRAPHY  61 71  APPENDICES A.  Consent Form  79  B. Letter to Prospective Subjects Explaining the Study  80  C. Preliminary Questionnaire  81  D.  Brand of Oral Contraceptive Used and Duration of Use Among Subjects Taking Oral Contraceptives  E.  Schedule for Collecting Blood Samples from Pregnant Subjects  F.  Mean Daily Dietary Intake of Folate Calculated from Two 3-Day Diet Records for Each Subject  83 8k  85  G.  Serum Folate Concentrations of Each Subject  8?  H.  Red Cell Folate Concentrations of Each Subject  89  I.  Differences i n Hematological and Dietary Parameters Measured at Two Phases of the Menstrual Cycle i n Control Women and Women Using Oral Contraceptives , Differences i n Hematological and Dietary Parameters Measured During Each Trimester of Pregnancy Calculated and Assayed Values for Folate for Duplicate Food Samples Collected at the Time of Consumption  J. K.  91 93 9k  - viii -  LIST OF TABLES Table 1.  Procedure for Collecting Blood Samples  2.  Serum Folate Concentrations Compared at Two Phases of the Menstrual Cycle in Control Women and Women Using Oral Contraceptives  3.  Red Cell Folate Concentrations Compared at Two Phases of the Menstrual Cycle in Control Women and Women Using Oral Contraceptives  4. Differences in Hemoglobin, MCV, Serum Iron, and Total Iron Binding Capacity Between Control Women and Women Using Oral-Contraceptives 5. SerumcFolate Concentrations at Two Phases of the Menstrual Cycle in Women Using Different Brands of Oral Contraceptive 6. Differences in Serum Folate Concentrations Measured at Each Trimester of Pregnancy ?.  Differences in Hemoglobin, MCV, Serum Iron, and Total Iron Binding Capacity Measured at Each Trimester of Pregnancy  8. Differences in Serum Folate, Red Cell Folate, Serum Iron, and Total Iron Binding Capacity Between Controls or Oral Contra ceptive users and Pregnant Women for Each Trimester of Pregnancy 9.  One-Way Analysis of Variance Comparing Dietary Folate Intake Among Control Women, Women Using Oral Contraceptives, and Pregnant Women  10.  Pearson Correlation Coefficients Comparing Serum or Red Cell Folate Levels with Dietary Folate Intake in Control Women and Women Using Oral Contraceptives  11.  Differences between First and Third Trimesters of Pregnancy in Pearson Correlation Coefficients Comparing Serum or Red Cell Folate Levels and Dietary Folate Intake  12;  Spearman's Rank Order Test of Correlation between Calculated and Assayed Values of Folate for Food Samples Collected at the Time of Consumption  - 1-  CHAPTER I INTRODUCTION It i s known that daring pregnancy there i s an increased demand f o r f o l i c acid, which may result in reduced levels of serum and red c e l l folate. Moreover, the depletion of maternal folate stores may be further aggravated i f the pregnant woman's dietary intake of folate i s low.  There i s also  evidence in the literature of a similar reduction in serum and red c e l l folate concentrations among women using oral contraceptives.  The role of  dietary folate intake in determining serum or red c e l l folate levels in these women has, however, not been established. There has been extensive research into the effect of pregnancy on folate status. Low serum and erythrocyte folate values are most frequently encountered during the last trimester of pregnancy when expansion of maternal tissues and fetal growth rate are the greatest.  Therefore this decline in  folate status has been attributed primarily to fetal demand and Increased erythropoiesls.  However, other factors more directly associated with folate  metabolism have also been implicatedi increased rate of folate clearance from the plasma (Landon and Hytten, 1972), decreased renal reabsorption of folates (Hytten and Leitch, 1971» Fleming, 1972), poor folate stores prior to pregnancy (Cole et a l . , 197^; Fleming et a l . , i97**a)»and low intake of dietary folate (Hibbard and Hibbard, 1972» Cole et a l . , 197kj Fleming  et a l . , 1974a). Oral contraceptives are said to simulate some of the physiological changes associated with early pregnancy.  Because of this, recent research  - 2has focused on investigating whether changes ln folate status and metabolism analogous to those in pregnancy occur among women taking oral contraceptives. Although several studies have revealed a tendency towards decreased serum and red c e l l folate levels i n oral contraceptive users (Shojania et a l . , 1971» Roetz and Nevinny-Stickel, 1973l Smith et a l . , 1975)» other researchers have not found these values to be significantly lower than those of control women not using the p i l l (Stephens et a l . , 1972; Paine et a l . , 1975l Prasad  et a l . , 1975). Investigation into the possible mechanisms behind these altered folate levels in the bipod has  implicated several factors.  There appears to be a  decreased absorption of polyglutamate forms of folate l n some oral contraceptive users (Streiff, 1970| Necheles and Snyder, 1970), but this does not seem to be true for a l l women (Shojania and Hornady, 1973) and may not be sufficient to account for the reduction in folate levels.  Other factors have  also emerged which could be of comparable importances an Increased rate of folate clearance from the plasma (Stephens et a l . , 1972i Shojania et a l . , 1975), an increased urinary excretion of folates (Shojania et a l . , 1975)» and the presence of a f o l i c acid binding protein in the serum and leukocytes of oral contraceptive users which could reduce folate availability (da Costa and Rothenberg, 19741 Waxman and Screiber, 1974). However, there has been a lack of research, in both pregnant women and women using oral contraceptives, concerning the extent to which dietary folate intake determines their serum and red c e l l folate values.  It may be that an  inadequate level of dietary folate i s contributing to the development of low folate concentrations in the blood of some of these women. Low dietary intake of folate has been Implicated as a factor in the development of folate /deficiency (Herbert, 1962). In economically undeveloped  - 3 areas of the world where nutrition i s below the recommended standards, the incidence of folate deficiency and megaloblastic anemia i s greatly increased. This i s especially true among women who have the additional stress of one pregnancy after another with l i t t l e chance f o r the body to reestablish i t s folate stores between pregnancies (Hibbard and Hibbard, 1972; Cole et a l . , 197k).  He would not expect this to be the case i n better-nourished societies. This i s confirmed by comparative statistics which show that the  incidence of magaloblastic anemia due to insufficient dietary folate i s rare in Industrially developed countries where nutrition i s supposedly adequate (Weir, 1973).  However, evidence presented by the recent Nutrition Canada  Survey demonstrates that there are very large segments of the Canadian population at risk with respect to f o l i c acid; 67% of adult men and women in the general population are at risk, Increasing to 97% of Eskimo men. Contrary to what one might expect, a smaller proportion of the pregnant women studied—only  to 63$—were found to be at risk.  Nutrition Canada  however, based these percentages almost solely on serum folate measurements. The authors themselves suggest1 " I t i s not possible to assess the c l i n i c a l significance and public health consequences of these findings without further hematological studies and an evaluation of dietary folate intakes."* The primary purpose of the present study was to examine the relationship between serum and erythrocyte folate levels, and dietary folate intake ln women who are pregnent or using oral contraceptives, as well as i n a group of control women. The study was also designed to investigate differences and similarities in folate status among the three groups of subjects.  ^Nutrition Canada1 National Survey. 1973, P. Ilk.  - 4 For oral contraceptive users and controls, folate status was determined by measuring serum folate, red c e l l folate, and dietary folate intake at two different stages i n the menstrual cycle.  Statistical tests were  performed to evaluate any variations in these parameters within and between the groups of women at the two phases of the cycle. group of pregnant women was also selected and their folate status was assessed; serum and red c e l l levels of folate and dietary folate intake were determined during each of the three trimesters.  These parameters of  folate status were also compared statistically with the same parameters ln controls and women using oral contraceptives. The results of this study should help to c l a r i f y the extent to which serum and red c e l l folate concentrations are a function of dietary levels of folate, and what effect oral contraceptive use or pregnancy has on this relationship.  - 5 -  CHAPTER II REVIEW OP THE LITERATURE Absorption and Metabolism of Folic Acid Current knowledge concerning the absorption of dietary f o l i c acid and i t s subsequent transformation to the raetabolically active forms i s incomplete.  A review of this area i s requisite for evaluating the literature  concerned with folate metabolism.during pregnancy and oral  contraceptive  therapy. Physiological indices of folate status.  Folic acid, in i t s reduced  forms, i s a vitamin of major importance as a cofactor in a l l one-carbon transfer systems, the most significant being the biosynthesis of purine and pyrimidine nucleotides.  Therefore, the role of f o l i c acid in DNA  synthesis and cellular growth i s v i t a l , and folates are utilized extensively in tissues where there i s a great turnover oj: multiplication of cells.  This i s evidenced by the increased requirement for the vitamin  during growth and pregnancy, as well as by the observation that a folate deficiency w i l l be most readily reflected in those tissues undergoing a continuous, rapid replication of cells.  The tissues primarily affected  are the bone marrow and blood, the intestinal mucosa and, in women, the uterine and cervical cells.  Although any of these may show morphological  changes due to a lack of folate, such changes are most pronounced in the marrow and peripheral blood (Blakley, 1969).  Alterations in c e l l mor-  phology, however, are a late sign of folate deficiency. The most common measures of folate status are levels of the vitamin in serum and whole blood.  Plasma f o l i c acid levels reflect recent nutritional  - 6status with respect to folate intake, whereas the amount of folate in red blood c e l l s more specifically indicates folate stores in the body (Chanarln, 1969)* Serum folate i s believed to be derived from l i v e r and other tissue stores by a displacement mechanism (Rosenberg and Godwin, 19711 Gerson and Cohen, 1972).  Absorbed f o l i c acid enters these tissues and thus acts to  displace endogenous 5-aethyltetrahydrofolate to the plasma.  If dietary  intake of f o l i c acid Is reduced this phenomenon i s diminished, resulting in a rapid decrease i n serum folate levels. A reduction of the folate in red blood c e l l s requires a longer period of deprivation since erythrocyte folate i s tightly bound and not released u n t i l the c e l l i t s e l f i s destroyed at the end of i t s l i f e cycle (Hoffbrand, 1971)*  Therefore measurement of red c e l l folate i s indicative  of long-term folate status. Whereas erythrocyte folate levels are used as a measure of folate stores. It i s l i v e r supplies of folate which actually form the largest reservoir of the vitamin in the body (O'Broin et a l . , 1975) • A person's folate stores are theoretically sufficient to provide folate for several months i f dietary Intake i s reduced to an inadequate level (Herbert, 1962). Absorption of f o l i c acid.  The adequacy of folate nutrition depends  not only on the availability of adequate folate-rich foods, but also on the Individual's a b i l i t y to absorb sufficient folate from these dietary sources. Some disagreement s t i l l surrounds the mechanism of folate absorption, both with regard to the mode of transport across the intestinal mucosa and the form In which dietary folates are absorbed. 1,  Site of absorption.  Absorption of folates has been shown to take place along the entire  - 7length of the small intestine although the greatest proportion i s absorbed from the proximal jejunum (Bernstein et a l . , 1970). In general, the rate of absorption i s inversely related to the length of the Y-glutamyl side chain on the folate molecule (Gerson and Cohen, 1972). Only deconjugated folates—monoglutaraates  and some diglutamates—-will eventually reach  the portal circulation.  Hydrolysis of the glutamic acid residues occurs  by the;action of intestinal folate conjugase ( -carboxypeptidase, referred Y  to as pteroylpolyglutamyl hydrolase) present in the lysosomal fraction of intestinal mucosal cells (Rosenberg and Godwin, 1971). Most evidence indicates that this i s the site of hydrolysis although i t i s not clear how the larger molecules of folate reach the inside of the mucosal cells. There i s perhaps some deconjugation in the lumen i t s e l f by the action of conjugase present i n sloughed-off mucosal cells, but this remains hypothetical (Gerson and Cohen, 1972). 2. Mode of transport across the mucosa. The mode of transport of folates across the intestinal wall (i.e.i whether active or passive) i s s t i l l unresolved.  The earliest research with  rats yielded conflicting results regarding transport and i t was not until more refined techniques for studying absorption were developed that i t became possible to examine the process in more detail. Hepner et a l . i n 1968, using an intestinal perfusion technique on humans, concluded that crystalline pterpylglutamic acid (PGA) was absorbed by a saturable transport mechanism. However, their findings have not been substantiated by others.  Gerson et a l . i n 1971» using a similar method  but employing ^HPGA, showed that glucose enhanced the absorption of f o l i c acid, probably by a solvent-drag effect secondary to sodium and water flow. This implicated a passive process and corroborated the work of Smith et a l .  - 8 (1970) using everted gut sacs from rats.  Gerson points out, however, that  although this i s strong evidence for a passive transport of f o l i c acid, i t does not exclude the possibility of a parallel system Involving either active transport or facilitated diffusion. Reduction and methylation of absorbed folates.  The majority of  folate derivatives in the body are formylated or methylated, and reduced, these changes being prerequisite for the coenzyme function. The length of the glutamyl side chain appears to also be a factor in coenzyme activity, although the effect of chain length in this regard i s not yet completely understood (Krumdieck et a l . , 1975). There has been some dispute concerning the site of conversion of absorbed folates to their metabolically active forms (primarily 5-methyltetrahydrofolate). While some researchers have suggested that reduction and methylation occur in the gut wall during absorption (Chanarin and Perry,  1969;  Perry and Chanarin, 1970; Whitehead et a l . , 1972), other work in this area contradicts these findings. Studies by Baugh and associates (1971, 1975) with intestinal loops in dogs showed no evidence of conversion of folates to reduced or methylated forms during passage through the mucosal cells. Rather, there has been some research indicating that these changes are accomplished i n the liver.  Whitehead-and Cooper (1967) demonstrated that  orally administered PGA appears unaltered in the portal circulation, and only after passage through the l i v e r does the folate emerge as 5-methyltetrahydrofolate.  In the process, hepatic folate i s evidently exchanged  for the more recently absorbed folates entering from the portal circulation (Melikian et a l . , 1971). This has been conf irmedln studies using ^CPGA (Butterworth et a l . , I969) and 3HPGA (Pratt and Cooper, 1971). Whitehead et a l . (1972) have suggested that reduced dietary folates are absorbed differently from f o l i c acid (PGA), which could account for  - 9 the discrepant results in the above studies. Whereas PGA may not he methylated until i t reaches the l i v e r , reduced folates appear to he methylated in the intestine. Thus, both the hydrolysis of the polyglutamate side chain and the methylation of dietary folates (which are primarily reduced) occur prior to the entry of folates into the hepatic portal system, and probably take place in the intestinal mucosa. A deficiency of folate may be caused by any condition which interferes with the above processes.  Inadequate intake of folate could  lead to a depletion of l i v e r supplies; a disturbance of intestinal function could impair absorption; disorders of the l i v e r could prevent formation of the functional derivatives of folate; the use of certain drugs can impair absorption of utilization of folates; and certain conditions such as hemolysis can place an inordinate demand on body stores of the vitamin. If such situations remain untreated they can result in the development of a megaloblastic anemia. Folate Deficiency and Megaloblastic Anemia Definition of folate deficiency and megaloblastic anemia.  It i s  relevant at this point to distinguish between a f o l i c acid deficiency and an outright megaloblastic anemia which i s an expression of extreme folate deficiency. Herbert's classic work (1962) has outlined the sequence of changes which take place when the body i s deprived of sufficient f o l i c acid (i.e., limited to an intake of 5.0 his findings as followsj  folate per day or less).  Herbert summarized  - 10  -  Dietary Folic Acid Deprivation In Mant Biochemical and Hematologic Sequence of Events Sequential Changes  Time of Occurrence (weeks)  Low serum folate (<3'ng*/mL«)  3  Hypersegmentation of Neutrophils  7  High urinary Figlu excretion  14  Low red blood c e l l folate (<20 ng./ml.)  17  Macroovalocytosis  18  Megaloblastic marrow  19  Anemia  20  The formation of megaloblasts in the bone marrow and macrocytes i n the peripheral blood reflects a state of lmbalanced growth due to impaired synthesis of DNA precursors. The result Is that, although cells continue to synthesize RNA and protein, there i s a delay at certain points ln mitosis which causes the formation of giant cells (Chanarin, 1969; Hoffbrand, 1971). There are also changes i n the production of white blood cells. One type of leukocyte, the neutrophil, will show an increased number of lobes in Its nucleus. to  There are normally two to five;  this w i l l increase  six or more. A megaloblastic anemia, by definition, i s characterized by the  presence of megaloblasts l n the marrow. However, a folate deficiency may be defined by the presentation of any change from a low serum folate to the appearance of macrocytes i n the peripheral blood.  Most often i t i s  the f i r s t four signs outlined by Herbert which have been used to establish the existence of a folate deficiency.  And i t i s the existence of marginal  levels of these parameters which have concerned most researchers investigating folate status.  - 11 Etiology of folate deficiency.  There are numerous factors which may  act singly, hut which more often occur together in some combination to cause a f o l i c acid deficiency that may,  i f untreated, lead to a megalo-  blastic anemia. The relative significance of these factors has not been established, so they are discussed here in an arbitrary order. 1.  Impaired absorption.  A defect in absorption of f o l i c acid i s most commonly due to some disorder of,tor damage to, the jejunal mucosa as ln sprue, steatorrhea, coeliac disease, or following a jejunal resection (Bernstein et a l . , 1970). Malabsorption has also been implicated as a mechanism by which other conditions may act to alter folate status; these include pregnancy, oral contraceptive use, anticonvulsant therapy, and 2.  alcoholism.  Alcoholism.  The occurrence of f o l i c acid deficiency i n alcotelics i s common and i s probably the result of poor dietary habits (Halsted et a l . , 196ft.; 1971 j Stebbins et a l . , 1973)*  However, absorption may also be impaired due to  damage to the intestinal mucosa, a direct effect of the alcohol i t s e l f (Stebbins et a l . , 1973J Weir, 1973). 3. Liver disease. If cirrhosis occurs in connection with alcoholism, or i f there i s some other damage to l i v e r function, this may hamper the a b i l i t y of the l i v e r to store or exchange folates (Brown et a l . , 1973)*  Such a condition  can also result in increased urinary loss of folates (Weir, 1973). k.  Increased requirement for f o l i c acid.  Any condition i n which there i s a marked increase in c e l l multiplication, as in pregnancy, lactation, growth, malignancy, or hyperthyroidism w i l l increase the need for f o l i c acid above the normal Recommended Daily  - 12 Allowance (Hoffbrand, 1971). This may also occur under conditions where the utilization of folate i s blocked as l n homocystlnuria. 5.  Increased loss of f o l i c acid.  There may be an abnormally rapid catabollsm of f o l i c acid i n situations where hemolysis or hemorrhage occur, as well as when a hemolytic anemia exists (sickle c e l l anemia, thalassemia, or congenital spherocytosis) (Weir, 1973). In addition, a person undergoing hemodialysis may lose a significant amount of f o l i c acid which, i n conjunction with the anorexia and vomiting frequently accompanying dialysis, could precipitate a folate deficiency (Hoffbrand, 1971). 6.  Inflammatory disease and Infection.  Such conditions as arthritis, tuberculosis, and psoriasis have been known to contribute to a folate deficiency.  This i s probably due to a  combination of poor appetite with an increased requirement for folate induced by increased leukocyte turnover (Hoffbrand, 1971). 7.  Drug Interferences.  There are many drugs which interfere with folate metabolism, usually by a disruption of availability or utilization of folate at the cellular level (stebbins et a l . , 1973)*  Folate antagonists, most notably methotrexate  and aminopterin, used l n treatment of cancer, have a great affinity f o r dihydrofolate reductase and act by displacing f o l i c acid from this enzyme. Pyrimethamine, an antimalarial drug, i s also, an inhibitor of dihydrofolate reductase but i s f a r less potent than methotrexate.  Anticonvulsants, the  most common being  dlphenylhydantoln, aot by some mechanism which has not  been established.  There may be a competitive Inhibition of folate enzymes,  an Impairment of polyglutamate absorption by conjugase inhibition, or a displacement of folate from i t s transport proteins.  Oral contraceptive  - 13 agents also interfere with the metabolism of f o l i c acid, although the process by which this happens remains unclear.  The effect of oral contraceptives  on folate status w i l l be discussed below in greater detail. 8. Poor dietary intake of folates. A low>level^ of dietary folate may occur in conjunction with any of the above conditions and thus contribute to the development of a folate deficiency.  The significance of inadequate dietary folate i s often over-  looked but i t may be of importance i n infancy, old age, poor socio-economic conditions, or any instance where anorexia occurs.  Diet has also been  implicated as a factor in determining the folate status of pregnant women (Hibbard and Hibbard, 1972; Cole et a l . ,  197*0,  but its: significance to the  folate status of women using oral contraceptives remains to be clarified. The present research was concerned specifically with examining pregnancy, oral contraceptive use, and dietary folate Intake as factors i n the etiology of folate deficiency. The Effect of Pregnancy on Folate Status Pregnancy induces some striking physiological alterations in the female's body i n the course of adapting to the needs of the developing fetus. Much of the nutrient intake of the woman, as well as a large portion of her body stores of nutrients, are directed toward the placenta and fetus. In addition, maternal energy and nutrient supplies must be conserved i n anticipation of the demands of labor, delivery, and subsequent nursing. Anemias of pregnancyi Iron and folate interrelationships. The requirement for both iron and f o l i c acid i s greatly increased in pregnancy and i t i s pertinent to look at these together.  They have a common role  in hematopoiesis, which i s accelerated during pregnancy, and may be interrelated i n other ways which have not been f u l l y elucidated. An iron  - Ik -  deficiency i s generally associated with a reduced serum folate level (Velez et al., I9661 Toskes et al., 1974), and Toskes et al. suggest that an iron deficiency may actually predispose a person to folic acid deficiency. It i s known that iron deficiency and folic acid deficieny do frequently occur together, and the risk of developing both of these i s especially increased during pregnancy (Pritchard, 1970). The alterations in both iron and folate metabolism that take place ln pregnancy may contribute to the development of anemia in some women. In discussing anemias of pregnancy i t Is critical to distinguish between what i s called the "physiological anemia of pregnancy" and those pathological conditions which are of more serious concern. During pregnancy there Is a remarkable Increase In plasma volume which tapers off during the last six weeks of gestation. However, the maximum increase in red blood cells does not occur until the woman Is near term. Consequently, most of pregnancy i s spent ln a state of hemodilution, where the plasma volume i s expanding without a comparable, simultaneous rise in red cell volume. And, since the size of the cells and their hemoglobin content normally show no change, the net effect is that the hemoglobin concentration and hematocrit f a l l (Hytten and Leitch, 1971). Some believe that this physiological alteration i s a true state of iron deficiency anemia and cite the evidence that iron given in therapeutic doses will prevent the f a l l ln hemoglobin concentration (Pritchard, 1970). Others reason that these changes do not represent a pathological state because the increased oxygen requirements of pregnancy are met even with the lowered hemoglobin levels. Therefore, the lowered hemoglobin does not in itself indicate impaired erythropoiesls but may simply be a consequence  - 15 of the proportionately greater increase ln plasma volume over red blood cell mass (Hytten and Leitch, 1971X Hall, 1974). In addition to the increase in plasma volume during pregnancy, there i s also an unrelenting drain on maternal iron stores by the fetus. This demand i s always met even at the expense of the mother. Thus, i f by the time the greatest increase in red cell production i s occurring in late pregnancy, iron stores have already been depleted, the desirable increase in red cell volume may be limited and a true anemia may result. However, an anemia is not ordinarily brought about simply by fetal demand. And the case has been made that a healthy, well-nourished woman does not require iron supplementation in pregnancy (Izak et a l . , 1973? Besforges, 1973)• Usually i t i s the presence of some additional stress acting to deplete maternal iron stores (such as hemorrhage, infection, or hemolytic anemia), or the existence of poor iron stores prior to conception which forces the woman into a state of negative iron balance (Desforges, 1973). This may, in turn, have a bearing on the woman's folate status. Although the association between iron balance and folate status remains obscure, two pieces of evidence have emerged which suggest that there is some relationship between them. First there i s the well-documented fact that a decrease in serum folate occurs in conditions associated with iron deficiency (Velez et al., 1966} Toskes et al., 1974). Secondly, and conversely, i t has been shown that pregnant women suffering from a folate-responsive megaloblastic anemia have a higher incidence of iron deficiency than those without such an anemia (Hoffbrand, 1971). The basis for these changes i s uncertain although the explanation may be quite simple.  i  In iron deficiency there i s a decrease in the  -  16  -  survival of peripheral red blood cells and a concomitant Increase In heme catabolism.  According to Toskes et a l . (1974), this in i t s e l f may  sufficient to account for the decreased serum folate levels and  be  the  increased folate requirement. Paradoxically, once an iron deficiency anemia i s established, treatment with an iron supplement may cause a further decrease in serum folate because of the additional demand on folate stores ln the I n i t i a l spurt of new erythrocyte formation (Hoffbrand, 1971)• Perhaps the major point to be aware of in c l i n i c a l practice i s that the morphological changes in the blood associated with Iron deficiency may mask evidence of a coexisting folate deficiency (Toskes et a l . , 1974). Therefore, i f a pregnant woman develops an iron deficiency, i t may be a wise precaution to also determine her serum and red c e l l folate levels. Folic acid deficiency of pregnancy.  51  Reduced serum and red c e l l  folate concentrations are a common accompaniment of pregnancy. There i s l i t t l e question that lowered maternal folate levels must be primarily attributed to fetal demand. This i s substantiated by the very high concentration of folate found in cord blood at delivery when compared to maternal folate concentration (Landon and Hey,  1974;  Baker et a l , 1975).  However, other factors relating more specifically to folate metabolism have also been implicated in the etiology of folate deficiency ln pregnancy. 1.  Decreased renal reabsorption of folates. urinary loss of many nutrients Is Increased In pregnancy (Hytten  and Leitch, 1971)»  The loss of folates by this route could cause a  depletion of the dialyzable fraction of serum folate and thus account somewhat for the lowered serum folate characteristic of pregnancy  - 17 (Fleming, 1972). However, there Is wide variability in the amount of urinary folate excreted and urinary loss i s probably not sufficient to cause a folate deficiency (Landon and Hytten, 1971)* 2. Increased rate of folate clearance from the plasma. The increased rate of plasma folate clearance i s a definite change observed during pregnancy. However, i t i s influenced by other factors which are also altered by pregnancy, (absorption, tissue uptake. and excretion of folate, as well as volume of extracellular fluid) and cannot be taken as an accurate determinant of folate status in pregnancy (Landongand Hytten, 1972). 3.  Folic acid binding protein in the blood. A f o l i c acid binding protein (FABP) has been found i n the serum  and leukocytes of women who are pregnant or using oral contraceptives (de Costa and Rothenberg, 1974). The significance of this FABP i s not known, but i t may act to bind folates so they cannot be utilized' (Waxman and Sehreiber, 1974). 4. :Poor ^6la^.e .stores prlor^to'pregi^cyi. ;  5. Poor Intake of dietary folate. With pregnancy's added strain on endogenous folate stores, exogenous folate intake assumes greater improtance.  Host studies of nutritional  folate status have made only perfunctory examination of the dietary practices of the subjects.  This variable may be of more significance i n  determining folate status than i s often supposed. Food availability, economic advantage, and ethnic custom are of particular importance l n this regard. Research among pregnant women, primarily iA Australia (Cole et a l . , 1974| Fleming et a l . , 1974a) and South Africa (Colman et a l , 1974), has  - 18 shown striking differences ln folate status among women from different ethnic and socio-economic groups. Cooking practices and traditional food preferences were implicated as factors in determining folate status. Large amounts of lightly cooked green vegetables as eaten by the Chinese in Malaysia (Hibbard and Hibbard, 1972) or the beans and lentils which form a staple in the diet of Mediterranean immigrants to Australia (Cole et a l . , I974) appear to exert a protective effect against folate deficiency. Assessment of folate status ln pregnancy. Estimates of the Incidence of folate deficiency in pregnancy vary within a wide range. This can be mainly attributed to the use of different criteria for judging folate status. The methods used to assess folate status reflect different f  aspects of folic acid metabolism. Therefore i t is not surprising that the results of different tests should lead to different conclusions. It is Important to be familiar with these criteria in order to accurately Interpret what they t e l l us about folic acid metabolism in pregnancy; Hansen (19^8) discusses the relative merits of the different methods. The Figluexcretion test measures the amount of formimlnoglutamlc acid (Figlu) excreted in the mrine after a histidine load. If there Is insufficient folate circulating to pick up the formimino group, then Figlu accumulates and i s excreted in larger amounts than normal. Whereas this test is a reliable and sensitive measure of folic acid deficiency In most instances, the results seem to be quite unreliable in pregnancy. This Is probably due to the increased amounts of histidine excreted during pregnancy, which could create a falsely high level of Figlu excretion. Plasma clearance of folates i s another commonly used test of deficiency, and i t i s also altered in pregnancy. There i s some question  - 19 however, as to whether or not an Increased rate of clearance necessarily Indicates a deficiency in pregnancy. It may simply be another of the physiological adaptations associated with normal pregnancy. The neutrophil lobe average, when i t i s increased, i s Indicative of a severe degree of folate depletion (Hibbard and Hibbard, 1971)• However i t also seems to increase in a normal pregnancy when other signs of folate deficiency are absent, so its usefulness as a measure of deficiency has been questioned. ' Serum folate reflects the balance between dietary intake and plasma turnover of folates (Gerson and Cohen, 1972). A steady decrease in serum folate i s commonly observed in the course of pregnancy. This should return to normal non-pregnant levels by six weeks postpartum (Hanson, 19^8). A low serum folate may be due to the increased plasma clearance of folates and not necessarily diagnostic of a deficiency. A decrease in red cell folate, however, i s of more value in determining the existence of a folate deficiency in pregnancy since i t i s definitely associated with a depletion of folic acid stores. However, low red cell folate i s a relatively late sign of deficiency, and i t may not be wise to wait for this change to appear before diagnosing a deficiency. There i s a very high incidence of low serum folate and accelerated plasma clearance of folate among pregnant women (Benjamin et al., 1966; Hansen, 19681 Pritehard, 1970* Rae and Robb, 1970| Roetz and Hampel, 1972; Cole et al., 1974). It i s s t i l l debatable whether these should be regarded ' as indicative of a folate deficiency or i f they are normal changes associated with pregnancy, like the decrease in hemoglobin concentration. Red cell folate levels seem to be the most reliable Indicator of a significant deficiency, and also the factor least disturbed by a coexisting iron deficiency (Roberts et al., 1971). But to delay diagnosis  -  2 0  -  of folate deficiency u n t i l a low red c e l l folate appears may present a risk to both mother and child. Maternal and fetal complications associated with folate deficiency in pregnancy.  A folate deficiency in pregnancy has been associated with  abruptio placentae, toxemia, and premature labor in the mother (Hibbard, 1964; Streiff and L i t t l e , 1967)»  a  s  well as low birth weight, fetal malformations,  and impaired contra! nervous system development "in the newborn  (Stone, 1968:  Gross et a l , 1974). The majority of these studies however, are retrospective and therefore not truly representative of the incidence with which such complications occur.  Moreover^ the presence of a folate deficiency severe  enough to cause these abnormalities may indicate the existence of some other underlying factor which could be just as strongly implicated (Hall, 1 9 7 2 ; Hall, 1972b). Although i t remains to be proven that maternal folate deficiency, per se, i s responsible for these complications, s t i l l the risk -of incurring them should be avoided i f at a l l possible. Supplementation with f o l i c acid in pregnancy.  Folate deficiency  and i t s extreme form, megaloblastic anemia, are almost always the result of more than one factor contributing to a folate imbalance.  In:pregnancy, i t  i s the woman faced with some additional complication who i s most susceptible to such a deficiency. She may be considered to be at risk in this regard i f she has a multiple pregnancy or i f her pregnancy i s accompanied by any of the following conditions» hemolytic anemia, thalassemia, epilepsy being treated with anticonvulsants, alcoholism, infection, or poor diet.  In these  situations a f o l i c acid supplement may be warranted to preclude the possibility of any maternal or fetal complications (Fleming et a l . , 1974b). This practice would not be challenged by most obstetricians. %  Rather, i t i s the question of routine f o l i c acid supplementation for a l l pregnant women which i s controversial.  The position can be argued  21 that for the majority of women who are healthy and who meet the Recommended Daily Allowance for dietary folate a f o l i c acid supplement i s unnecessary. Some clinicians, however, advocate the administration  of such supplements  to a l l pregnant women. There i s perhaps some wisdom in this attitude, which Hansen (1968) expresses, ", . . a normal serum folate level probably i s the best guarantee that the different dividing cells receive an adequate amount of f o l i c acid, which must be assumed to be of special significance for normal growth in pregnancy." The decision to prescribe a f o l i c acid supplement to a pregnant patient i s l e f t to the individual obstetrician and opinions concerning the practice of supplementation vary widely. Considering the additional burden placed on a woman's folate stores by pregnancy, i t would be prudent to examine the future mother's folate, status prior to conception.  There i s evidence that oral contraceptives  may adversely influence folate status.  The c l i n i c a l significance of low  serum folate levels and other signs of altered folate status should not be underrated.  Following the discontinuation  a woman could easily become pregnant.  of hormonal contraception  I f , at this point, there i s insuf-  ficient saturation of her folate stores,, a folate deficiency could develop in the sensitive period of pregnancy. The Effects of Oral Contraception on Folate Status Oral contraceptives have been said to induce a state i n the body analogous to pregnancy.  The fixed level of estrogen provided by the p i l l ,  along with the absence of ovulation, acts to simulate the hormonal conditions of early pregnancy.  These two situations are therefore comparable i n some  respects, and one would expect some similar alterations in physiology to exist.  - 22 There i s evidence that o r a l contraceptives have an e f f e c t on f o l a t e status which i s s i m i l a r i n many respects t o that found i n pregnancy. Whether t h i s e f f e c t i s the r e s u l t of an etiology common to both conditions i s unclear. Megaloblastic anemia associated with o r a l contraceptive use.  There  has been extensive work recently published documenting the possible adverse e f f e c t s of hormonal contraception on f o l a t e status. A survey of the l i t e r a t u r e reveals numerous incidences of megalob l a s t i c anemia occurring i n association with o r a l contraceptive use (Necheles and Snyder, 1970} S t r e i f f , 1970; Buhac and Finn, 1971; Ryser e t a l . , 1971? T o g h i l l and Smith, 1971; Flury and Angehm, 1972; S a l t e r , 1972; Johnson et a l . ,  1973; Shojania and Hornady, 1973; Lewis, 1974? Meguid and Loebl,  197k). These, however, are a l l based on i n d i v i d u a l case studies and do not represent c o n t r o l l e d experimental s i t u a t i o n s .  In reviewing these reports, i t  Is of significance that those which included a t e s t f o r malabsorption d i d , i n f a c t , f i n d evidence f o r an underlying i n t e s t i n a l disorder that could have affected f o l a t e absorption Smith,  ( S t r e i f f , 1970;  Ryser et a l . , 1971; T o g h i l l and  1971? Johnson e t a l , , 1973). Moreover, i n most of the other studies,  i t was stated that an occult malabsorption syndrome o f some kind was not, and should not, be excluded as a possible contributing f a c t o r (Necheles and Snyder, Lewis,  1970; Flury and Angehrn, 1972? Shojania and Hornady, 1973;  1974? Meguid and Loebl, 1974). In a l l cases, discontinuation of  o r a l contraceptive therapy and i n i t i a t i o n of f o l i c a c i d supplementation were s u f f i c i e n t to cure the anemia. Thus i t seems that a woman using o r a l contraceptives must also have some other condition a f f e c t i n g f o l a t e metabolism before changes w i l l develop.  megaloblastic  Conversely, the e f f e c t of such a condition might  - 23 -  not become apparent except for the added stress of treatment with synthetic hormonal contraceptives.  Therefore, i f there already exists some  unmanifested form of malabsorption such as a tendency towards coeliac disease, the further insult of oral contraceptives may be sufficient to precipitate an anemia. Oral contraceptive use and folate deficiency. Megaloblastic anemia occurring ln conjunction with oral contraceptive use i s a rare event relative to the large number of women at risk.  A more common observation  among women using oral contraceptives i s the tendency towards reduced serum folate concentrations. finding.  However, this has been a highly variable  I t appears that certain women may have an enhanced suscepti-  b i l i t y to lowered folate levels when using oral contraceptives, whereas others suffer no such adverse effects.  Even a careful perusal of the  research published i n this area makes i t d i f f i c u l t to draw conclusions applicable to a l l individuals. Review of the studies finding a reduced serum folate ln oral contraceptive users.  Of those studies which have established some positive  correlation between low serum folate levels and oral contraceptive use, the most significant are those by Shojania and associates in 1971 and t  Roetz and Nevinny-Stickel in 1973* The latter researchers showed that serum folate levels decline over time after the initiation of oral contraceptive therapy.  Looking at the converse situation, Shojania et a l .  found that the serum folate concentration improved within three months after discontinuing oral contraception.  The conclusion of these workers  was that there i s an Impairment of folate metabolism directly related to the use of hormonal contraceptives.  However, they also make the q u a l i f i -  cation that the effect on folate metabolism—-whatever the mechanism may  -  2k  -  be-—Is very mild, and that i t takes a large number of subjects with a long history of oral contraceptive use to demonstrate any effect. The major criticism of the above studies i s that they never specif i e d whether or not serum folate determinations were made on fasting blood samples.  This can make a great difference in folate levels since  eating a meal with folate-rich foods w i l l elevate the serum folate for several hours afterwards. The mechanism by which oral contraceptives might act to decrease serum folate has been a matter of some speculation.' There have been a few studies implicating a direct effect of synthetic orally-administered hormones on absorption of folates from the intestine (Snyder and Necheles, 1969J Streiff, I970).  H  Strelff's findings have formed the primary basis  for the theory that oral contraceptives act to inhibit the activity of intestinal conjugase. Streiff studied nine oral contraceptive users and nine control women? a l l had normal serum folate levels and none were anemic. He observed the changes i n serum folate levels after these women were given an oral dose of either monoglutamic folate or polyglutamic folate. The rise i n serum folate was comparable in the two groups when the monoglutamic form.was given, but after administration of polyglutandc folate the rise i n serum folate i n the oral contraceptive group was about 50% of that i n the control group.  From this he concluded that, since the poly-  glutamic forms of folate must be deconjugated in the small intestine before they can be absorbed, there might be some direct interference of the hormones with conjugase activity. His hypothesis was also based on an i n vitro /  — *  •—™—•  demonstration of"the inhibitory effect of mestranol (a synthetic estrogen) on conjugase activity (Streiff and Greene, 1970).  - 25 Streiff was however, reluctant to Implicate oral contraceptives as the exclusive cause of this malabsorption*  He suggested that i f folate  absorption i s already hampered by some other condition, or i f dietary intake of folates i s inadequate, a further Inhibition of absorption (such as that imposed by oral contraceptives) could be enough to precipitate an overt deficiency or anemia. Review of the studies finding no difference l n serum folate between oral contraceptive users and control women. There have been numerous studies which contradict the above results but which do not, significantly, dismiss the possibility that some alteration of folate status may be incurred by oral contraceptive use.  The research of most interest i n  this connection has been that of Stephens and associates i n 1972 and, more recently, Paine et a l , (1975)* Paine et a l . surveyed a very large sample of women (N=»526) and found no significant difference i n serum folate levels between oral contraceptive users and controls.  They obtained the same result when blood  samples were analyzed by either the L. easel microbiological method or the new radloassay f o r folate.  The strength of their findings l i e s i n  the size of the sample studied.  However, they did not state whether  they used fasting blood samples and this may have skewed their results. The work of Stephens et a l .  appears to have been more carefully  controlled and logically planned than any of the other studies.  These  authors looked at both folate status and folate absorption in a series of experiments. F i r s t they examined serum folate levels i n fasting blood from a group of oral contraceptive users and a group of control women. They found no statistically significant difference l n the serum folate values between the two groups, and no significant variation in serum folate levels at different stages of the menstrual cycle.  - 26 This was followed by absorption studies on the same women to test for differences In their ability to absorb different forms of folate. Although they were able to confirm Streiff's finding of reduced polyglutamate absorption in the oral contraceptive users, further investigation showed that, after saturation of the subjects with f o l i c acid, the d i f f e r ences between the groups disappeared.  Stephens explained their results  by suggesting that oral contraceptives may cause an increase i n the rate of clearance of folates from the plasma. This accelerated clearance i s apparent when the subject's tissues are not f u l l y saturated with folates. But when tissues are pre-saturated prior to the absorption test, the rate of clearance becomes the same for both groups of women. This finding may be interpreted to mean that there i s no true malabsorption of folates in women taking, oral contraceptives. Rapid plasma clearance of folates in p i l l users may be caused by a mechanism similar to the one which results in increased clearance during pregnancy. The implication here i s that there i s some hormonal effect common to both situations. The last part of the study by Stephens et a l . involved an i n vitro examination of the effect of sex hormones on conjugase activity.  Although  previous work by Streiff and Green (1970) had indicated that me strand, a synthetic estrogen, inhibited conjugase activity in vitro, Stephens et al . 1  found no inhibition of intestinal conjugase by any of the three synthetic hormones they tested (estradiol, progesterone and estrone).  In fact,  there has been recent evidence to suggest that estrogen has an enhancing effect on conjugase activity (Krumdieck et a l . , 1975). This was, however, shown i n rat uterus and i s not necessarily applicable to human Intestinal conjugase.  A direct effect of steroid hormones on conjugase activity  remains to be proven.  -  27 -  Further research attempting to clarify these results.  In a paper  published ln 1973 Shojania and Hornady attempted to reconcile the question of malabsorption induced by oral contraceptives.  They compared folate  absorption in women on the p i l l who had a low serum folate and in women who were also using the p i l l but who had normal serum folate levels. They found no consistent malabsorption of folates in oral contraceptive users, but when they made the above distinction between two populations of women a pattern did emerge.  One population consisted of those  who had a normal fasting serum folate and showed normal polyglutamate absorption, whereas the population that had low fasting serum folate levels showed  reduced absorption of polyglutamates.  In 1975, Sho jania et a l . extended their investigation to include a study of plasma clearance and urinary excretion of folates.  In the group  of women they studied, plasma clearance of an Injected dose of pteroylglutamic acid was shown to be much faster among the oral contraceptive users than in controls. One possible explanation given for this difference was that tissues are less saturated with folates ln oral contraceptive users, thus enhancing uptake from the plasma. In this same study I t was also discovered that there was a direct correlation between urinary folate excretion and serum or red c e l l folate levels in both oral contraceptive users and control women. However, the p i l l users excreted more folate for a given level of serum folate or red c e l l folate than did their control counterparts.  The authors f e l t that  this Increased excretion of folates among women taking hormonal contraceptives may partially account for their frequently lower blood folate levels. Related factors of unknown significance. 1.  F o l i c acid binding protein (FABP).  Da Costa and Rothenberg (1974) have reported the existence of a specific f o l i c acid binding protein (FABP) in the blood of both women who  - 28 axe pregnant and women using oral contraceptives.  A factor was isolated  from the serum and leukocytes of these women which effectively bound unreduced folates and dihydrofolates. Significantly, this folate binder was absent from the blood of control women, suggesting that i t s production may be hormonally induced. It may be that this i s not a new factor in the blood but simply an already existing FABP which undergoes an increase i n concentration. Such a shift i n the relative concentration of plasma FABP's has been shown to occur inpregnancy (Karkkanen et a l . , 1973)• Whichever i s the case, the exact function of such a binding protein and i t s position i n the etiology of reduced folate status i n p i l l users have yet to be resolved. Da Costa and Rothenberg  (197*0  could not find a correlation between  the presence of the binder and the serum folate concentration.  Their  findings imply that this factor may act to sequester folates from the metabolically active pool of coenzymes and thereby contribute to the altered folate status of both pregnant women and women using the p i l l . In a related study, Waxman and Schreiber (1974) looked at the effect of FABP on  - l a b e l l e d folates.  They found that those folates  bound to FABP were less available for uptake by tissues than folates not attached to the binding factor.  Uptake was shown to be invexsely related  to the amount of FABP i n the serum. These authors suggest that this FABP may be responsible for cellular uptake and distribution, ^rather than serum transport, of folates. Shojania et al. (1975) have postulated that i t i s the change i n plasma folate binders that i s responsible for the increased urinary excretion of folates they observed among oral contraceptive users.  - 29 2.  -  Megaloblastic changes in the cervical epithelium.  There i s another observed effect of oral contraceptive use  on  folate utilization, the significance of which i s s t i l l unclear, but which warrants inclusion here.  This i s the finding by several workers  (Klaus 19711  1973*  f  Whitehead*et a l . ,  Lindenbaum et a l . , 1975)  that oral  contraceptive use i s associated with distinctly megaloblastic changes in the cervical c e l l s which can be observed from Papanicolaou smears. I t Is disturbing to note that these morphological changes i n the cervical epithelium are in no way correlated with other evidence of a systemic folate deficiency (such as reduced serum or red c e l l folate, macrocytosis, or hypersegmented neutrophils).  In fact, the serum folate  levels were found to be similar In women-.;with either normal or abnormal smears (Lindenbaum et a l . , 1975)* However, f o l i c acid therapy was shown to significantly revert the cervical morphology towards normal within three weeks, and a discontinuation of the f o l i c acid supplement resulted ln a recurrence of the abnormalities (Whitehead et a l . , 1973 ; Lindenbaum et a l . , 1975)•  This  strongly implicates some direct effect of oral contraceptives in altering folate metabolism at the level of the target organ. Sex steroids are known to stimulate DNA  synthesis and c e l l p r o l i -  feration in their target tissues (O'Malley and Means, 1974). Therefore, one would expect an Increased utilization of folate coenzymes In these tissues and a resultant depletion of folate stores in the localized target area.  The subsequent impairment of DNA synthesis^may ;be'evidenced  by the presence of megaloblastic changes.  s  The f u l l significance of these  cervical changes cannot be elaborated on without further elucidation of the dynamics behind their development.  -  30  -  Summary of the mechanisms by which oral contraceptives may act to alter folate status.  In summary, the possible mechanisms of action of  oral contraceptives in altering folate status may be enumerated as followst 1.  Decreased absorption of polyglutamate forms of folate by  direct inhibition of intestinal conjugase activity. 2.  Increased rate of plasma clearance of folates.  3.  Increased urinary excretion of folates.  4. [^Presence of a FABP which binds folates so they axe not available i n their metabolically active forms. 5.  Interference with folate utilization at the cellular level  in sex steroid target tissues. While i t i s not as yet possible to evaluate the relative importnace of the factors implicated above, i t i s nevertheless evident that they must a l l be considered significant clues to the physiological action of oral contraceptives on folate metabolism. It i s significant that i n almost a l l the studies o f i f o l i c acid status i n pregnant women and oral contraceptive users the influence of dietary intake of folates has been totally neglected.  This seems a i  serious omission since dietary folate i s known to directly influence serum levels of the vitamin and must be accounted for before conclusions can be made about the influence of other factors on serum folate levels. However, i t i s not surprising that this problem has been avoided since the d i f f i c u l t i e s involved in assessing dietary folate intake are numerous. Consideration of this problem involves introducing the additional variables of food folate analysis and dietary evaluation.  These  variables increase the complexity of any investigation into folate status and require careful examination before attempting the design of further studies in this area.  The Assessment of Dietary Folate Status Nutritional adequacy with regard to folates w i l l depend on food availability, dietary habits, and physiological absorptive capacity.  The  assessment of folate status must therefore include both a dietary e s t i mation of folate intake and an examination for factors which might interfere with i t s absorption. This discussion w i l l center on the d i f f i c u l t i e s surrounding evaluation of dietary folate intake and w i l l encompass two areas of concern! (1) Problems inherent in the assay of food folates which one should be aware of i n assessing available data on the folate content of foods.  (2) Problems in estimating dietary folate Intake.  Problems Inherent in the assay of food folates.  Derivatives of  f o l i c acid are found i n a wide variety of foods, most commonly as reduced polyglutamates.  Folic acid (pteroylmonoglutamlc acid), Itself, generally  constitutes only a small portion (about 5$-10%) of dietary folates and i s apparently present only because of the oxidation of reduced folates during processing, storage and cooking (Hurdle, 1973). I t i s the multip l i c i t y of forms of folate l n food and the differing availability of these forms to various test organisms that makes the evaluation of food folates a d i f f i c u l t task (C^Broin et a l . , 1975). Those folate derivatives which are detected by microbiological assay without prior enzymatic hydrolysis with conjugase are called "free"; whereas those which are micrpbiologically active- Only^afteir such hydrolysis are "conjugated" (i.e., the p^yglutamates).  "Total" folate  i s a measure of both the free and conjugated forms together.  Different  test microorganisms w i l l be able to u t i l i z e different forms of folate and therefore, their growth-response curves will^vary for the same  - 32 food sample.  -  Growth of Lactobacillus easel, the most commonly used  organism, i s supported by most folates with up to three glutamate r e s i dues (Hoppner et a l . , 1972). The determination of dietary f o l i c acid presents additional problems because the significance of "free" and "total" folates as determined by microbiological assay i s poorly understood i n the context of biological availability i n mammals. We cannot equate "total" folate i n food with "available" folate because the i n vivo action of conjugase i s not completely understood and cannot be assumed to be the same as that in vitro.  For this reason, some researchers have suggested that assaying  food folates without conjugase treatment (i.e., "free" folate) gives a more accurate indication of available folate i n the diet (Herbert, 1963). The measurement of truly available folate probably l i e s somewhere between the values f o r "free" and "total." In addition to the basic problem of defining available folate, there are other factors which may influence laboratory determinations of folate activity1 1.  Use of ascorbic acid as an antioxidant.  Many folate composition tables were compiled before the protective effect effect of ascorbate against oxidation of reduced folates was recognized.  Use of ascorbic acid makes more folates available to  the test organism and can give values up to forty times higher (Hurdle, 1973). 2.  Inhibitors and binders of folate present i n foods.  Variability i n folate values may also be partially accounted f o r by the presence of certain known and unknown inhibitors of conjugase (as in yeast) and binders of folate (as ln milk) and the extent of their a c t i v i t y (Taimjra and Sto^  - 33 3. The effect of cooking. Processing and cooking, especially i n large quantities of water, w i l l destroy heat-labile folates (Herbert and Bertino, 19®?), and can result i n losses of 90% or more of folate activity (Herbert, 19^3« Hurdle, 1973). 4.  Sensitivity to pH.  Folates are available for absorption l n a very narrow pH range (Butterworth, 1968). Buffering i s essential to the assay of folates as they w i l l be destroyed by extremes of pH, especially acidity (O'Broin  et a l . , 1975). 5. The effect of storage. Folates may be oxidized during storage and this can, account for a significant loss of activity (Tamura and Stokstad, 1973). Problems l n estimating dietary folate intake. Dietary assesst  n  ment i s a d i f f i c u l t task, largely because i t i s so dependent on subjective evaluations. Unless dietary habits can be observed without the knowledge of the subject, some personal bias w i l l enter ins and unless Intake of food items i s measured precisely, an estimation of dietary intake i s likely to be distorted.  Consequently, the limitations of the  available methodology for assessing nutritional status must be taken into account when evaluating dietary studies (see Fidanza, 1974). The determination of dietary folate intake i s further complicated by factors peculiar to f o l i c acid.  The accuracy of a measurement of  dietary folate intake w i l l be most influenced byi 1. The interest, Intelligence, and education of the subjects. 2. The choice of method for determining intake (e.g., 24 hour recall, diet record, or diet history). 3. The r e l i a b i l i t y of food collections.  - 3k -  k.  The choice of food composition tables.  The relative merit of these tables depends on the precautions which were taken to insure accurate measurements of food folate (as outlined i n the previous section), A good model for the application of this knowledge can be found i n the study by Moscovitch and Cooper  (1973)*  Their examination of the  folate content of the diets of pregnant women involved the use of both a dietary record and direct assay of food samples.  A similar methodology  was adapted to the design of the present research. Conclusion We have seen that reports regarding the folate status of women who are pregnant or using oral contraceptives are contradictory. I t has been d i f f i c u l t to evaluate and compare the research in the literature, mainly because the methodology has varied, often considerably, from one study to another.  Obviously, the value of any further research ln this  area w i l l depend upon choosing those parameters which may be s i g n i f i c a n t variables and insuring adequate investigation of these variables. The major factors which have emerged as important variables in the literature reviewed here are. 1. Choice of population studied. 2.,Presence of malabsorption i n subjects. 3. Choice of parameters f o r measuring folate status. 4. -, Fasting or post-absorptive blood samples. 5.  Dietary intake of folates.  Certainly the least adequately investigated factor, in studies of both pregnant women and oral contraceptive users, has been the role  - 35 that dietary Intake of folates has in determining their blood folate levels.  While the examination of this role w i l l be one of the p r i n c i -  pal aims of the present study, a l l of the above variables are s i g n i f i cant and each was considered in the methodological design of this study.  - 36 -  CHAPTER III MATERIALS AND METHODS  For the purpose of studying the folate status of young women as affected by oral contraceptives or pregnancy a sample of 48 women was selected from the Vancouver area.  These subjects were classified into  three groupst a control group of 18 women, mean age 22.6 (rangei 18-29 years), who had neither been pregnant nor using oral contraceptives for at least six months prior to being in the study? an oral contraceptive group of 22 women, mean age 22.5 (range« 19-28 years), a l l of whom had been using combination-type oral contraceptives for at least four months prior to being in the study? and a pregnant group of 8 women, mean age 26.5 (range« 22-33 years), who were in their third or fourth month of pregnancy at the time of commencing their participation on the study.  Approval for  this research was received from the Health Sciences Screening Committee of the University of British Columbia and informed consent was obtained in writing from each participant (Appendix A). A l l subjects i n the conrol group and oral contraceptive group were women enrolled as students at the University of British Columbia in Vancouver, British Columbia.  They were informed of the study i n  one of several waysj through personal contact, by notices posted on university bulletin boards, by announcements given to several classes, or through the Birth Control Clinic at the Student Health Services on campus.  - 37 Pregnant women were contacted with the cooperation of several private obstetricians i n the City of Vancouver who had agreed to distribute the preliminary questionnaire to interested patients.' The obstetricians were restricted to choosing only women l n early pregnancy who were i n good health and who had not been prescribed a f o l i c a d d supplement. Any interested woman was given a letter explaining the purpose of the research and what would be expected of her (Appendix B ), as well as a preliminary questionnaire which was designed to gather necessary information regarding her health and medical history (Appendix C ). The volunteers were screened by means of this questionnaire and were excluded or selected as subjects on the basis of the following c r i t e r i a ! Agei The minimum acceptable age was 18, the maximum. 35* Medical historyi Evidence of any chronic inflammatory disease or infection, l i v e r disease, kidney dysfunction, epilepsy, diabetes, tuberculosis, malaria, or an anemia within the past year were grounds for elimination from the study. Drug usei I f any woman was receiving regular treatment with antibiotics, anticonvulsants, antimalarial drugs, or other drugs (besides oral contraceptives) known to interfere with f o l i c acid metabolism, she was not included ln the study.  I f a woman was undergoing temporary  treatment with anti-  biotics she was used as a subject only after antibiotic therapy had been discontinued. Blood donation! I f a woman had recently donated blood she was asked to wait at least one month before being included i n the study. Dietary information! Any severe dietary limitation, including caloric restriction to less than 1200 kilocalories a day, was cause for exclusion from the study.  -  Use of supplementsi  38 -  Subjects were carefully questioned regarding their  use of nutritional supplements.  No one taking a vitamin supplement that  included f o l i c acid was allowed to participate i n the study. tation was permitted since a very  Iron supplemen-  large proportion of the women were using  such a supplement. Use of oral contraceptivesi A detailed history of past and present oral contraceptive use was obtained from a l l subjects.  This included the  specification of types (brands) of oral contraceptives taken and the length of time in months that each one was used.  (See Appendix D for a breakdown  of the number of subjects i n the oral contraceptive group who were using each type of p i l l and the length of time each subject had been taking the p i l l . ) Obstetrical Historyi A history of past pregnancies, including those which had ended in miscarriage or therapeutic abortion, was obtained from each woman. I f a woman had been pregnant within the last six months she was not admitted as a subject to either the oral contraceptive or control group. A l l subjects in these two groups were nulliparous, except for three women who had one child each, and one woman with two children. For each woman i n the pregnant group the approximate date of conception and day of delivery were established.  Of the pregnant women, 4 had no  previous children, 3 had one child each, and one had 3 children. Collection of Blood For the oral contraceptive users and control women fasting blood samples were taken at two different phases of the menstrual cycle, for two consecutive cycles. Figuring day 1 i n control women as the f i r s t day of the menstrual flow, venous blood samples were drawn on day 5, when sex steroid levels i n the blood were low, and day 20, when hormone levels were relatively  -  high.  39 -  In women using oral contraceptives, day 5 was taken as the last day  before beginning a new cycle of p i l l s since most of the synthetic hormones from the previous cycle had been excreted by this time, and day 20 was counted from there. a l . , 1972.)  (This sampling procedure was adopted from Stephens et  Depending on the intervention of weekends and holidays, days  3 to 6 of the cycle were considered "day 5" and days 18 to 21 were considered "day 2 0 . " In the group of pregnant women, fasting venous blood samples were taken three times during the course of pregnancy.  The f i r s t sample was drawn  as soon as possible after the woman had returned her questionnaire} this was sometime during the third or fourth month of pregnancy.  The following two  samples were taken at approximately 8 to 10 week intervals.  In this way i t  was possible to have a blood sample from each trimester of pregnancy. (See Appendix E for the exact weeks of sampling for each subject.) Blood was collected in the morning following an overnight fast. The samples were drawn by venipuncture and collected into Vacutainer Tubes.* Approximately 10 to 15 mis. of blood was collected for each sample, the amount depending on which analyses were to be done on the sample. sampling  was as outlined in Table 1.  The schedule for  Whenever hemolysis occurred, a note  was made of i t and the degree of hemolysis estimated on a relative scale (severe, moderate, slight, very slight).  I f hemolysis was severe, a fresh  blood sample was drawn on the following day. Treatment of blood samples.  The blood collected into the tube  containing no additive was allowed to stand at room temperature for at least one hour i n order to form a firm clot.  This tube was then centrifuged at  2000 rpm for 15 minutes and the serum removed with a Pasteur pipette.  *Vacutainer tubes from Becton, Dickinson and Go., Canada, Ltd.  - 40 Aliquots of the serum were placed i n small plastic tubes* as followst 1.5 mis. f o r serum folate assay (for a l l samples), and an additional 1.0 ml. f o r iron assay (for a l l samples from the pregnant women, but only f o r the f i r s t and last samples from the oral contraceptive and control groups). A l l tubes of sera were kept frozen at ~20°G. until the day of assay. Table 1 Procedure f o r Collecting Blood Samples  Additive ln Vacutainer tube  No additive (for serum) Q. 5 ml. 3.:8#  Na citrate  M i l l i l i t e r s of blood collected  Cycle  Cycle  I  II  All Samples  day 5  day 20  day 5  day 20  1st sample  2nd sample  3rd sample  4th sample  8  5  5  8  8  4  4  4  4  4  4  0.07 ml. 15S EDTA  Pregnant Group  Control and Oral Contraceptive Groups  4  1  1  A microhematocrlt was performed on the blood collected into the tube containing sodium citrate as soon as possible after the blood had been drawn. The citrated whole blood was kept frozen at -20°C. u n t i l the day of assay. This blood was used only f o r the determination of red c e l l folate levels.  •Falcon polystyrene tubes.  - kl Whole "blood treated with EDTA was run through the Model S Coulter Counter and was used to make blood smears, which were subsequently stained* for microscopic examination. Collection of dietary data.  Each subject was asked to keep two  three-day diet records, one for the three days before the f i r s t blood sample was taken and the other for the three days prior to the last blood sample. This procedure was the same for a l l three groupsibf subjects. Women were Instructed i n how to record food items accurately, and were asked not to change their usual eating habits. The subjects were not informed of the specific nutrients under examinationt this was done i n order to avoid some of the bias i n their food intake during the time they were keeping the diet records. Collection of food at the time of consumption for analysis of folate content.  To evaluate the accuracy of estimations of food folate made from  the available food composition tables, the folate levels i n meal samples were determined f o r comparison with calculated values.  Fifteen women  volunteered to collect duplicate samples of a l l the food they ate for one 24 hour period. This food was then used f o r a direct assay of the free and total folate content.  The women also kept a written record of the food  they collected so that an estimation could be made of the folate values using food tables. In order to protect the labile forms of folate from oxidation the food was collected into a plastic bucket containing 500 mis. of phosphate buffer (pH 6.10) and 150 mg. of ascorbic acid per 100 mis. buffer.  During  the course of collection the food samples were kept refrigerated and on the  •Cameo Quick Stain, buffered differential Wright's Stain.  - kz day following collection they were homogenized.  The homogenate was stored  in 10 ml. aliquots at -20°C. until the day of assay.  A l l of the samples were  assayed for folate within one month of the time they were collected. Analyses Analysis of blood samples.  Hemoglobin, hematocrit, and blood c e l l  counts were determined with the Model S Coulter Counter.  Blood films were  given only cursory examination unless there was reason to suspect the existence of morphological abnormalities} in such a case the slide was more thoroughly examined for evidence of macrocytosis. Serum folate was assayed using the Lactobacillus easel microbiological method of Baker et a l . (I959),i using ascorbic acid to prevent folate oxidation.  Red cell folate was determined using the L. easel method as  modified by Hoffbrand (1966) and Spray (1969)» with the exception that the blood was not treated with ascorbic acid prior to freezing.  This was deemed  unnecessary as there was no significant loss'of: folate activity in citrated blood when the ascorbate treatment was omitted.*  Both serum and whole blood  were added to phosphate buffer containing 150 mg. of ascorbic acid per 100 mis. buffer on the day of assay, immediately after being thawed.  Folate determi-  nations were done in triplicate for each sample. For control purposes, pooled sera with serum folate values ln the low (1.5-2.0 ng/ml), mid-normal (5.0-6.0ng/ml) and high-normal (11.0-12.0 ng/ml) ranges were kept frozen in aliquots and determined each time a folate assay was performed.  Normal values for serum folate were in the range of 3.0 to  16.0 ng/ml, and for red c e l l  folate were in the range of 190-700 ng/ml.  *Dr. R.F. Pratt, Hematology Department, St. Paul's Hospital, Vancouver, British Columbia, personal communication.  -  k3 -  A standard curve was determined with each assay using F o l i c Acid "Baker Grade."*  F o l i c Acid Casei Medium** and L a c t o b a c i l l i Broth AOAC** were used  i n l i e u of preparing the assay medium and maintenance culture broth ourselves. Serum i r o n and t o t a l iron binding capacity were determined using the Hycel K i t f o r "Serum Iron and Iron Binding Capacity Tests."***  For women  from the o r a l contraceptive and control groups, the iron assays were performed on pooled serum from the f i r s t and l a s t blood samples.  For pregnant women the  iron assays were done on each of the three blood samples. Evaluation o f d i e t records.  The d a i l y intake of free and t o t a l f o l a t e  was calculated from the d i e t records using the tables published by Hoppner et a l , (1972), and Hurdle (1968),  Portion sizes and amounts were estimated  using the tables of Bowes and Church as revised by Church and Church (1970). Analysis of food samples.  The f r e e and t o t a l f o l a t e o f the c o l l e c t e d  food samples was determined using the L. casei microbiological method adapted f o r food analysis by Herbert (1963), with the exception that the concent r a t i o n of ascorbic acid used was 150 mg. per 100 mis. of buffer. f o l a t e was assayed using chicken pancreas conjugase.****  Total  For control pur-  poses, the pooled sera f o r low, mid-normal, and high-normal serum f o l a t e l e v e l s were determined with each assay.  S t a t i s t i c a l Analysis of the Data The raw data were analyzed s t a t i s t i c a l l y by computer a t the Computing Centre of the University of B r i t i s h Columbia.  The SPSS computer program  *J.T. Baker Chemical Co., New Jersey, Lot no. 1-7555. **"Difco" C e r t i f i e d , from Difco Laboratories, Detroit, Michigan. ***Hycel #HY294, from Hycel, Inc., Houston, Texas. ****DifcovChicken Pancreas, from Difco Laboratories, Detroit, Michigan.  - Mf package (Kita and Morley, 1973) was employed to draw up a program for the desired analyses. The Student's t-test with p = 0.05 was used to test for the statist i c a l significance of differences among the three groups of subjects regarding each of the hematological and dietary variables.  The statistical significance  was also determined for differences in these variables at different phases of the menstrual cycle and in the different trimesters of pregnancy. Tests of correlation among the variables within and between the groups of subjects were done using the Pearson Test for Correlation,  Those corre-  lations of most interest were the ones between serum and red c e l l folate levels and dietary folate intake.  The differences between the groups of subjects  in the correlation coefficients were tested for significance using Fisher's z-transformatipn. Comparison of the assayed values of the food samples with the calculated ("expected") values for these samples :was i n i t i a l l y done using linear regression.  The Pearson Product Moment Correlation Coefficient was calcu-  lated and i t s significance determined.  This procedure was carried out on a l l  15 food samples and again on 13 of these samples after excluding two values which were considerably higher than the others (due to inclusion of l i v e r in these two food samples).  Because these two outlying values appeared to be  skewing the results, i t was decided to also perform a test of correlation using Spearman's Rank Order Test.  This i s a non-parametric test of corre-  lation for situations where normal distribution i s not a valid assumption.  - 45 -  CHAPTER IV RESULTS Serum Folate, Red Cell Folate, and Other Hematological Parameters The effect of oral contraceptives. The results in Table 2 show that women taking oral contraceptives had significantly lower levels of serum folate than did controls at day 5 of the menstrual cycle (p<C0.05), although the difference between the two groups failed to reach a statistically s i g n i f i cant level at day 20 of the cycle.  Within each of the two groups of subjects  there was no significant difference in serum folate levels measured at the different phases of the menstrual cycle. Table 2 Serum Folate Concentrations Compared at Two Phases of the Menstrual Cycle in Control Women and Women Using Oral Contraceptives Serum Folate (ng/ml) Mean + S.D. Day 5 Controls  Day 20  P*  Overall Mean  (N - 18)  7.5^ + 3.58  8.14 + 4.97 > 0.05  7.84 + 4.04  0.C. Users (N - 22)  5. 3^ ± 2.20  5.93 ± 2.46 > 0.05  5.63 + 2.20  P*T  <0.05  >0.05  < 0.05 i  •Two-tailed, using Student's t-test. ^Separate variance estimate (F ratio significant, p<0.05).  - 46 Table 3 shows that there was no significant difference in red c e l l folate concentrations between the controls and p i l l users. any statistically significant difference  Neither was there  in red c e l l folate levels at the two  different phases of the menstrual cycle within either of the groups. Table 3 Red Cell Folate Concentrations Compared at Two Phases of the Menstrual Cycle in Control Women and Women Using Oral Contraceptives Red Cell Folate (ng/ml) Mean + S.D. Day 20  Day 5  Overall Mean  P*  Controls (N - 18)  210.1  + ?3.6  199.7  + 74.0  >  0.10  204.9 ± 71.8  O.G. Users (N 22)  184.2  + 74.1  184.0  + 72.2  >  0.10  184.1  P*t  >  0.10  >  0.10  >  + 71.7  0.10  *Two-tailed, using Student's t-test. ^Pooled variance estimate (F ratio not significant, p > 0 . 0 5 ) . In controls there was a statistically significant correlation between serum folate levels and red c e l l folate levels (p <0.05) at both day 5 (r » 0.6171) and day 20 (r » 0.5181) of the menstrual cycle.  However, in the  oral contraceptive group this correlation failed to reach levels of s t a t i s t i cal significance at either phase of the  cycle.  The comparisons for hemoglobin, M.C.V., serum iron, and total iron binding capacity between the control women and women taking oral contraceptives are found in Table 4.  No difference was found in hemoglobin concentra-  tion between the two groups, but M.C.V. was significantly higher in the oral contraceptive users at day 20 of the cycle ( p < 0 . 0 5 ) .  Therefore, although  -  4? -  the amount of hemoglobin did not vary with oral contraceptive use, the volume of the red blood cells was somewhat greater among p i l l users than in control subjects.  Although serum iron concentrations were higher among the women  using oral contraceptives than i n controls, the difference was not quite significant statistically (p » 0.057).  However, subjects taking oral contracep-  tives demonstrated significantly higher values for total iron binding capacity than did their control counterparts (p < 0 . 0 5 ) .  Within each of the two groups  there was no variation in hemoglobin, M.C.V., serum iron, or total iron binding capacity with the time of the menstrual cycle. There was a significant difference (p<0.01)  in white c e l l count  between the two groups, the oral contraceptive users showing the higher values (Appendix I ) . In addition, leukocyte count in subjects using oral contraceptives was significantly higher on day 20 than on day 5 of the cycle (p<0.0l).  A similar variation i n leukocyte count during the cycle was not  found among control subjects. Additional data for the other hematological parameters tested (red c e l l count, hematocrit, M.C.H., M.C.H.C.) are found in Appendix I.  Only  those findings of s t a t i s t i c a l significance have been discussed here. The effect of type of oral contraceptive used and duration of oral contraceptive use on serum folate levels.  Since the results showed that  serum folate concentrations were lower in oral contraceptive users than in controls, i t was decided to examine more closely the effect of different hormonal preparations and their duration of use on the serum folate levels. The Pearson test of correlation was performed between serum folate values and the number of months of oral contraceptive use.  No significant  relationship was found between the duration of oral contraceptive therapy and serum folate concentration (r = - 0,0549, p > 0 , l 0 ) .  Table 4 Differences i n Hemoglobin, MCV, Serum Iron, and Total Iron Binding Capacity Between Control Women and Women Using Oral Contraceptives Hemoglobin (g/100 ml) Mean + S.D. Day 5  fyi )  MCV  3  Mean + S.D.  Day 20  Overall Mean  Day 5  Day 20  1  Serum Iron (y^g/dl)  T.I.B.C. (/*g/dl)  Mean ± S.D.  Mean ± S.D.  Overall , Mean  Controls (N = 18)  13.0 + 0.7  13.2 + 0.5  13.1 + 0.6  86.1 + 2.6  86.0 + 2.2  86.0 ±2.3  84.0 + 22.8  327.6 ± 73.5  0. C. Users (N - 22)  13. k + 0.8  13.3 + 0.6  13-3 + 0.6  87.5 + 3.8  88.0 + 3.8  87.8 ±3.8  100.7 ± 29.6  382.5 ± 6 5 . 7  * P  > 0.10  2  >0.10  2  > 0.10  2  > 0.10  2  <0.05  1  >0.05  1  >  0.05  2  Two-tailed, using Student's t-test. 4  Separate variance estimate (P ratio significant, p < 0 . 0 5 )  Pooled variance  estimate (F ratio not significant, p > 0 . 0 5 )  <  0.05  2  -  k9  -  Subjects using oral contraceptives were separated into groups depending on the type of hormonal preparation they were taking.  The mean serum  folate concentrations at two different phases of the menstrual cycle were calculated for each of these groups of oral contraceptive users and are shown in Table 5. The difference in serum folate levels between the women using Ortho-Novum l/jiO and those using Ovral was not statistically significant at either phase of the cycle.  There was also no significant variation  in the serum folate measured at different times in the menstural cycle among women taking Ortho-Novum 1/50. In those using Ovral, however, the mean serum folate concentration at day 20 of the cycle was statistically higher than at day 5 (p<0.05). The effect of pregnancy.  The results in Table 6 show that as  pregnancy progresses the serum folate and red c e l l folate levels increase. However, the rise in serum folate i s not statistically significant, and the increase in red c e l l folate i s significant between trimesters 1 and 2 , and trimesters 1 and 3 » but not between trimesters 2 and 3 «  Therefore  the greatest increase in red c e l l folate in this group of women occurred before the third trimester. The correlation between serum folate and red c e l l folate was positive throughout pregnancy, but became statistically significant only  in the third trimester (r » 0.?5kk, p<0.05). The variation in hemoglobin, MCV and iron values during pregnancy are shown in Table 7 .  Hemoglobin values were significantly higher in the  third trimester as compared with the second trimester.  Mean corpuscular  volume (MCV) generally increased with the duration of pregnancy, although i t was only significantly higher in trimester 2 as compared with trimester 1,  - 50 Taole 5 Serum Folate Concentrations at Two Phases of the Menstrual Cycle i n Women Using Different Brands of Oral Contraceptives  Oral Contraceptive Agent  Ortho-Novum 1/50 (N orethinodrone, 1.0 mg) (Mestranol, 0.05 mg)  Subject  A.T. J.M. J.P. N.B. CH. M.D. K.H. CM. S.M. Mean + S.;D.'  Ovral (Norgestrel, 0.50mg) (Ethinyl e s t r a d i o l , 0.05  L.H. J.T. CO. C.S. A.C L.P. W.B. P.M.  Serum Folate (ng/ml) Day 5  Day 20  9.15 1.90 9.23 7.23 4.20 5.63 6.03 10.95 5.30  9.53 3.96 8.85 7.08 2.85 6.80 4.18 12.25 7.50  9.34 2.93 9.04 7.15 3.53 6.21 5.11 11.60 6.40  6.62 + 2.82  7.00 ± 3.00  6.81 + 2.82  4.35 5.65 3.80 4.78 4.53 4.07 4.85 3.70  5.98 5.40 5.25 5.08 4.90 6.80 4.05 , 5.85  5.16 5.53 4.53 4.93 4.71 5.43 4.45 4.78  Overall Mean  4.47 + 0.64  5.41 + 0.82  4.94 + 0.40  V.B. CM.  4.69 3.68  5.70 3.90  5.20 3.79  (Norethindrone, 1.0 mg) (Mestranol, 0.08  H.R.  5.65  10.00  7.83  Ortho-Novum 2 mg. (Norethindrone, 2.0 mg) (Mestranol, 0.10 mg)  M.S.  2.79  3.14  2.96  Demulen (Ethynodiol diacetate, 1.0 mg) (Ethinyl e s t r a d i o l , 0.05 mg)  B.M.  3.25  3.60  3.43  Mean + S.D.  Norlestrln  (Norethindrone acetate, 2.5 mg) ( E t h i n y l e s t r a d i o l , 0.05 mg)  Ortho-Novum 1/80  Table 6 Differences in Serum Folate and Red Cell Folate Concentrations Measured at Each Trimester of Pregnancy (K = 8) Serum Folate (ng/ml) Trimester  Mean + S. D.  Red Cell Folate (ng/ml) ;  1  5.76 + 2.66  2  5 . i i ± 1.88  3  8.71 + 7 . 2 1 '  1  1  2  Mean + S.D. 167.6 + 44.8  0.5972*  268.9 + 82.4'^  0.0646*  327.9 + 168.7 ' 2  Not significantly different from folate level in 1 2  Not significantly different from folate level in 2  S i g n i f i c a n t l y different from folate level in 1 *T>0.05  Pearson Correlation Coefficient between Serum and Red Cell Folate  s t  3  0.75^4  trimester (p>0,05). n d  trimester (p>0.05).  trimester (p<0.05).  Table ? Differences i n Hemoglobin, MCV, Serum Iron, and Total Iron Binding CapacityMeasured at Each Trimester of Pregnancy (N «* 8) Hemoglobin (g/lOOml) Mean + S.D.  Trimester  MCV (yU3) Mean + S.D.  Serum Iron (/*g/dl) Mean ± S.D. 97.6 + 29.4  1  11.8 ± 1.0  2  11.5 + 0.8  89.0 ± 2.8  3  1 4 12.0 + Q.8 '^  88.3 ± 3.3 '  87.5 + 3.3 1  1  1  significantly different from value i n 2  -^Significantly different from value in l  ?  t  ^08.7 ± 35.6  1  2  Not significantly different from value in 1 ^ot  335. ? ±61.9  101.9 ± 24.5  3  3  104.0 ± 48.2 ' 1  2  trimester (p>0.05), n d  trimester (p>0.05).  trimester (p<0.05).  Significantly different from value i n 2^ trimester (p<0.05).  T.I.B.C. (yug/dl) Mean + S.D.  463.4 ± 38.5 '^ 3  -  53 -  These findings indicate that, whereas the volume of red blood c e l l s showed the greatest increase before the t h i r d trimester, the hemoglobin concent r a t i o n d i d not r i s e s i g n i f i c a n t l y u n t i l the l a s t trimester. There was a s t a t i s t i c a l l y s i g n i f i c a n t increase i n t o t a l i r o n binding capacity throughout the course of pregnancy; serum iron increased during pregnancy, but not t o a s t a t i s t i c a l l y s i g n i f i c a n t degree. The r e s u l t was a net increase i n the unsaturated i r o n binding capacity as pregnancy  progressed.  The raw data, t-values, and l e v e l s of s i g n i f i c a n c e f o r a l l the hemotological  v a r i a b l e s compared during pregnancy are found i n Appendix J .  Only those findings that were s t a t i s t i c a l l y s i g n i f i c a n t have been included i n the r e s u l t s section. Comparison o f pregnant subjects with controls and oral t i v e users.  contracep-  Table 8 o u t l i n e s the r e s u l t s of t - t e s t comparisons f o r serum  f o l a t e , red c e l l f o l a t e , serum i r o n , and t o t a l iron binding capacity among the three groups of subjects.  In the f i r s t trimester of pregnancy  there were no s i g n i f i c a n t differences i n any of these parameters when compared with controls o r o r a l contraceptive users.  The pregnant women,  at t h i s point i n pregnancy, appeared comparable to non-pregnant women with regard t o the parameters tested. During the second trimester, pregnant subjects had serum f o l a t e concentrations which were not s i g n i f i c a n t l y d i f f e r e n t from those of o r a l contraceptive users, but which were s t a t i s t i c a l l y lower those of control women.  (p<0.05) than  However, by the l a s t trimester, serum f o l a t e values  had increased i n the pregnant women and were no longer d i f f e r e n t from the c o n t r o l l e v e l s . During both the second and t h i r d trimesters pregnant women demonstrated higher red c e l l f o l a t e concentrations than e i t h e r the controls  Table 8 Differences in Serum Folate, Red Cell Folate, Serum Iron, and Total Iron Binding Capacity Between Controls or Oral Contraceptive Users and Pregnant Women for Each Trimester of Pregnancy  Variable  Controls  O.C. Users  Mean ± S.D.  Mean ± S.D.  Pregnant Women Mean ± S.D. ' st 1 v Trimester  2 n d Trimester  3 r d Trimester  8.71 + 7.21 1 * 21 "  Serum Folate (ng/ml)  7.84 ±4.04  5.64 ±2.20  5.76±2.661**2+t  5.U±1.883*2tt  Red Cell Folate (ng/ml)  204.9 + 71.8  184.1 ±71.7  I67i6±44.8l**21t  268.9±82.41^tt  Serum Iron  - 84.0 + 22.8  100.7 ±29.6  85.4 + 4 4 . 0 1 * 2 t t  102.7 ± 22. S ** "^  107.9 ± 4 5 . 9 * *  327.6 ±73.5  382.5 ±65.7  408.9±35.63**2tt  k6 3 .4 + 3 8 . 5 * *  (>A€/dl)  T.I.B.C. (/Ag/dl)  A  335.7±6l.9 ** 1  2tt  1  2  327.9± 168.71*Z4't  3  Not significantly different from control value (p>0.05).  2  Not significantly different from O.C. users value (p>0,05).  3  Significantly different from control value (p<0.05).  ^Significantly different from O.C. users value (p<0.05). Separate variance estimate (F ratio significant, p<0.05), compared with controls. Pooled variance estimate (F ratio not significant, pt>0.05), compared with controls, tSeparate variance estimate (F ratio significant, p<0.05)t compared with O.C. users. Pooled variance estimate (F ratio not significant, p>0.05), compared with O.C. users.  2 t t  W  - 55 or oral contraceptive users.  These differences were only significant "between  the pregnant women and p i l l users, however,; The difference "between pregnant and control values was not statistically significant. Serum iron levels were higher among the pregnant subjects in the last two trimesters than i n controls, but the difference was not statistically significant.  Total iron binding capacity in both the second and third  trimesters was significantly higher than in controls; this variable was also significantly higher in pregnant women than in oral contraceptive users, although only during the third trimester. When comparing pregnant women with controls, the only consistently significant finding was that the total iron binding capacity increased and became significantly higher than control levels as pregnancy progressed. The most impressive relationship observed when comparing pregnant women with oral contraceptive users was the continuous rise in red c e l l folate during the course of pregnancy to levels which were significantly higher than those in the p i l l users. Dietary Folate Intake Comparison of dietary folate Intake among controls„ oral contraceptive users, and pregnant subjects.  As demonstrated in Table 9» no statis-  t i c a l l y significant difference was found in dietary intake of folate among the three groups of subjects.  A one-way analysis of variance revealed that  there was no significant difference in the sample variances for free folate or total folate intake in the three groups. Two tests for homogeneity of variance were also performed (Cochran's test and the Bartlett-Box test) and i t was determined that the variances in folate intake among the groups of subjects were homogeneous.  Therefore,  a pooled variance estimate was used to perform a t-test contrasting the  Table 9 One-Way Analysis of Variance Comparing Dietary Folate Intake Among Control Women, Women Using Oral Contraceptives, and. Pregnant Women  Analysis of Variance Variable  Group  Mean + S.D. F-ratio  Free Folate Intake (yAg/day)  Free Folate Intake (^g/day)  Controls (N - 18)  171.7 + 81.0  O.C. Users (N - 21)  150.5 ± 60.3  Pregnant (N « 8)  165.6 + 80.9  Controls (N = 18)  300.8 + 97.1  O.C. Users (N - 21)  272.1 + 79.6 315.8 + 99.8  Pregnant (N = 8)  *  **  *  P  t  P  0.436  0.639  . -,0.161  0.873  0.870  0.429  -0.838  0.406  1  1  2  *  Two-tailed.  **  Contrast Coefficient Matrix  Pooled variance estimate. Canadian Recommended Daily Allowance 1 200 jj-g free folate. Canadian Recommended Daily Allowance 1 250 ^wg free folate.  -  57 -  dietary folate intake of the controls and oral contraceptive users with the intake of the pregnant group (Contrast Coefficient Matrix).  There was  no significant difference among the three groups of subjects with respect to their dietary intake of either free folate or total folate.  I t i s of  interest to note that i n a l l groups of subjects the mean daily folate intake was below the Canadian Recommended Daily Allowance of 200.~micrograms free folate (250/*g free folate for pregnant women). Relationship between dietary folate intake and serum and red c e l l folate levels.  For each group of subjects the Pearson Correlation Coef-  ficients were determined comparing serum folate with free and total folate intake and red c e l l folate with free and total folate intake. The slgnificaiicevof'the difference between the correlation coefficients of controls and oral contraceptive;users was figured using Fisher's z-transformationj the results of this test are shown i n Table 10.  We  found a consistently higher correlation between the serum folate levels and dietary folate intake i n the controls than in the oral contraceptive users.  This was the case whether serum values were correlated with free  folate intake or total folate intake.  However, the difference i n correla-  tion coefficients between these two groups was only statistically significant at day 5 of the menstrual cycle. In general, i t can be seen that serum folate concentration appears to be directly and significantly  related to dietary folate intake among  control women, whereas a comparable relationship does not exist i n women using oral contraceptives. The correlation between red c e l l folate levels and dietary folate intake was not of s t a t i s t i c a l significance in either the controls or the p i l l users,  - 58 Table 10 Pearson Correlation Coefficients Comparing Serum or Red Cell Folate Levels with Dietary Folate Intake in Control Women and Women using Oral Contraceptives  Variable Pair  Serum Folate and Free Folate Intake Serum Folate and Total Folate Intake Red Cell Folate and Free Folate Intake Red Cell Folate and Total Folate Intake  Day of Cycle  Pearson Correlation Coefficient Controls (N - 18)  P*  O.C. Users (N - 21)  5  0.?208  -0.0229  .0072  20  o.6727  0.2155  .0872  5  0.6189  -0.0832  .0198  20  0.5850  0.1741  .1528  5  0.2302  - 0.1907  .2150  20  0.0719  -0.2157  .4010  5  0.1122  - 0.1384  .4654  20  0.0667  - 0.1406  .5486  *Two-tailed test using Fisher's z-transformatlon. Table 11 shows the results of the same tests for correlation and significance applied to the pregnant women. There was a difference in correlation between serum folate and dietary folate between the f i r s t and third trimesters of pregnancy.  These differences were not statistically signi-  ficant, but nevertheless there was a higher correlation in the f i r s t t r i mester.  Again, there was no comparable relationship found between red c e l l  folate levels and dietary folate intake in the pregnant women. In a l l groups of subjects there was a consistently stronger correlation between free folate intake and serum or red c e l l folate than there was between total folate intake and serum or red c e l l folate.  - 59 Table 11 Differences between F i r s t and Third Trimesters of Pregnancy in Pearson Correlation Coefficients Comparing Serum or Red Cell Folate Levels and Dietary Folate Intake  Variable Pair  Pearson Correlation Coefficient 1  st  Trimester  3  P*  Trimester  rd  Serum Folate and Free Folate Intake  0.4563  -0.1300  .3222  Serum Folate and Total Folate Intake  0.4429  -0.0599  .3954  Red Cell Folate and Free Folate Intake  0.3024  0.2563  .9362  Red Cell Folate and Total Folate Intake  0.1769  0.2477  -.9044  *Two-tailed test using Fisher's z-transformation. Correlation between the calculated and assayed values of folate for food samples collected at the time of consumption.  The calculated and assayed  values f o r free and total folate for each food sample are l i s t e d in Appendix K. The results of the Spearman's Rank Order Test between the calculated and assayed folate values are found in Table 12.  The correlation  coefficients between the calculated and assayed values were statistically significant for both free folate and total folate. These correlations remained significant when the two outlying values were omitted.  Our  findings indicate that the values for folate calculated from the folate composition tables of Hoppner et a l . (1972) and Hurdle (1968) are comparable to the values for folate obtained by direct assay of the food. The ratio of free folate to total folate in foods has been variously reported from 1 1 2 to 1 J 4.  Therefore, this ratio was calculated for both  - 60 the assayed values and the values obtained from food tables i n t h i s study. I t was found that the r a t i o of free f o l a t e to t o t a l f o l a t e was i t 1.77 f o r both the assayed and calculated f o l a t e values. Table 12 Spearman's Rank Order Test of Correlation Between Calculated and Assayed Values of Folate f o r Food Samples Collected a t the Time of Consumption Spearman's Correlation C o e f f i c i e n t s Variable P a i r N - 15 Free Folate Calculated Value vs. Assayed Value  0.6714  Total Folate Calculated Value vs. Assayed Value  0.7571  1  4  •Values f o r food samples containing l i v e r omitted. p<0.01 2  P<0.05  N  e*  13*  0.4945  0.6264  2  2  - 61 -  CHAPTER V DISCUSSION  When comparing controls with oral contraceptive users i t was found that, although the level of dietary folate intake was similar in the two groups, the women taking oral contraceptives demonstrated a significantly (p<0.05) lower mean level of serum folate than did the controls.  This  relationship was further evidenced "by the statistically lower correlation between dietary folate intake and serum folate levels in the oral contraceptive users. From these results i t appears that oral contraceptive use reduces serum folate levels, and that this decline in serum folate is independent of dietary folate intake.  Therefore, i t cannot be concluded that low dietary  folate intake is the cause of decreased serum folate levels in women using oral contraceptives.  Rather, there appears to'4be some direct effect of the  synthetic sex hormones in reducing serum folate concentrations. The finding that serum folate concentrations were lower among the women using oral contraceptives is supported by the work of Shojania et al. (1971) and Roetz and Nevinny-Stickel (1973). "both of which were longitudinal studies, as well as Smith et al. (1975) •  It is contradicted, however, by  the research of Stephens et al. (1972), Paine et al. (1975) and Prasad et al. (1975)» a l l of whom found no statistically significant difference in serum folate levels between controls and oral contraceptive users. The reason for the discrepancy in results in this area remains obscure.  It  could be due to not only differences in methodology, but also a lack of  - 62 control f o r f a c t o r s other than o r a l contraceptives which are known t o influence f o l a t e status. A c r i t i c i s m of the study by Stephens e t a l .  (1972) i s that there  was l i t t l e information given regarding the sample of women studied o r t h e i r dietary f o l a t e intake.  Paine e t a l .  practices o f t h e i r subjects.  (1975) also f a i l e d t o examine the dietary  Although our findings i n d i c a t e that serum f o l a t e  was not d i r e c t l y correlated with dietary f o l a t e intake i n o r a l contraceptive users, nevertheless, dietary f o l a t e w i l l d i r e c t l y influence the serum f o l a t e l e v e l s of control women.  For t h i s reason f o l a t e intake must be considered  an important variable. In the study by Paine e t a l .  (1975)  a  s  well as that o f Prasad e t a l .  (1975) the authors d i d not specify whether t h e i r determinations f o r serum f o l a t e were made on f a s t i n g blood samples, and t h i s may have skewed t h e i r results,  f o l a t e - r i c h foods are known to elevate the l e v e l of serum f o l a t e  f o r several hours post-prandial, Prasad e t a l . d i d , however, make a contribution towards c l a r i f y i n g the ambiguous f i n d i n g s i n t h i s area.  They made-a d i s t i n c t i o n among t h e i r  subjects regarding socio-economic status, and found that those women i n the lower socio-economic group had s i g n i f i c a n t l y lower serum and red c e l l f o l a t e concentrations than those from the higher socio-economic group.  This f i n d i n g  implicates socio-economic status as a f a c t o r which may influence f o l a t e status. I t may be that what i s found to be true f o r one sample population cannot be extrapolated t o another population of women.  Factors such as the  socio-economic status, dietary habits, ethnic custom, age, and p a r i t y o f the subjects, as well as the seasonal a v a i l a b i l i t y o f f o l a t e - r i c h roods, have been suggested as determinants of f o l a t e status.  These should be c o n t r o l l e d  f o r as f a r as possible i n the choice of a target population.  - 63 Control and oral contraceptive subjects in the present study were drawn from a relatively homogeneous sample population (university  students)  within a narrow range of age (18 to 29-years) and parity ( a l l were n u l l i parous except for three subjects who had one child each and one subject who had two children).  While this careful control of subject selection lends  strength to our findings, conclusions drawn from these results may only be applicable to women from a similar population. what limited in that i t was a cross-sectional  This study was also somesurvey.  I t would be of value  to design a similar study on a longitudinal basis using subjects as their own controls, and following their folate status over a more extended period of time, both prior to and during oral contraceptive therapy. The mechanism by which serum folate levels are reduced among women using oral contraceptives i s unclear.  Whether the effect of these exogenous  hormones i s at the level of folate absorption i n the intestine or uptake by the cells remains a matter of speculation. If folate absorption i s disturbed in some way by oral contraceptives, one would expect a lower serum folate level among p i l l users for the same intake of dietary folate.  This was confirmed by our results.  However, i f  i t i s the polyglutamate forms of folate which are poorly absorbed, as sug1  gested by Streiff (19?0), then one might expect a greater decrease in serum folate relative to total folate intake than to free folate intake.  This was  not observed in the present study; rather, i t was the correlation between serum folate and free folate intake which was most reduced among the oral contraceptive users.  These results may be interpreted as evidence supporting  the hypothesis of an interference of oral contraceptives at the level of folate absorption, but not specifically a hormonal inhibition of conjugase activity.  Research corroborating this hypothesis has not been reported  in the literature.  - 64 If the alteration in folate metabolism i s at the level of tissue uptake, i t may be connected with the presence of the f o l i c acid binding protein (FABP) found in women who are pregnant or using oral contraceptives (da Costa and Rothenberg, 1974). This binding protein appears to be responsible for cellular uptake rather than serum transport of folates (Waxman and Schreiber, 1974). Therefore, Shojania et a l . (1975) have suggested that i t may account for the increased rate of plasma folate clearance found i n oral contraceptive users.  The induction of this FABP by both oral contraceptive  therapy and pregnancy implies some hormonal influence common to both situations.  Clarification of the nature of this factor, the mechanism of i t s  production, and i t s physiological function may  contribute to our under-  standing of the process by which sex steroids act to alter folate status. Serum folate levels were not found to vary with the normal f l u c tuation of circulating hormone levels in the menstrual cycle, i n either the controls or oral contraceptive users.  This i s in agreement with the  findings  of Stephens et a l . (1972), the only other researchers to examine serum folate changes in the cycle. However, the difference in serum folate between the controls and oral contraceptive users was statistically significant at day 5 of the cycle and failed to reach statistical significance at day 20.  The fact that this  difference i s most pronounced when endogenous secretion or exogenous intake of sex steroids was the lowest i s d i f f i c u l t to explain.  If there i s an effect  of the hormones in reducing serum folate levels, one would expect i t to be most evident when these hormones are present in the greatest relative amounts. Perhaps menstruation i t s e l f exerts some as yet undefined effect on serum folate levels, although this remains speculative.  - 65 Among the oral contraceptive subjects i n this study, the duration or oral contraceptive therapy (rangej 4 to 120 months) was found to not be significantly correlated with serum folate concentrations.  This confirms  the results of Paine et a l . (1975) who also found no relationship of this Kind in a larger group of subjects (280 oral contraceptive users). Our findings also indicate that women using different hormonal preparations did not have statistically significant differences in serum folate.  The subjects taking Ortho-Novum i/50, however, did show higher  serum folate levels than those using Ovral; the difference in serum folate between these two groups was very close to s t a t i s t i c a l significance.  These  oral contraceptive preparations d i f f e r with regard to both the type of progestogen and synthetic estrogen and the relative concentrations of these hormones.  Since the groups of women studied here were so small, i t would  be advisable to further investigate the folate status of larger groups of women using various hormonal contraceptives. Results of the present study show that there was no adverse effect of oral contraceptive use on red c e l l folate concentrations.  This i s in  contrast to the work of Prasad et a l . (1975) and Smith et a l . (1975).  Prasad  et a l . examined the folate status of women from a high socioeconomic group and found oral contraceptive users to have red c e l l folate levels that were significantly lower than controls; there was no comparable difference among women from a low socio-economic group. for these results.  The authors offered no explanation  Smith et a l . studied a homogeneous sample of oral contra-  ceptive users with controls matched for age, parity, and body weight; the p i l l users were found to have significantly lower red c e l l folate values. They also gave no reason for these findings.  - 66 Since "both of these studies involved subjects within a wider age range than in the present study, i t may be that they were looking at women who had undergone a much longer period of oral contraceptive therapy. In the present study only three subjects had been taking oral contraceptives for more .than three years (Appendix D).  One could speculate that a reduction  in red c e l l folate levels occurs only after long-term oral contraceptive use, i.e., of five or more years duration. Serum iron and total iron binding capacity were significantly higher among the women using oral contraceptives in our study than in the controls. This has been found by other researchers as well (Doar, 1973. Roetz and Nevinny-Stickel,  1973? Smith et a l . , 1975)» and may be explained by the  reduced blood loss during menstruation that i s commonly associated with oral contraceptive therapy.  It should be pointed out, however, that some of the  subjects in this study were taking iron supplements, and i t may not be valid to compare these women with others not using an iron supplement . The elevated white c e l l count among oral contraceptive users in this study has been previously associated with steroid therapy (Beck,  1973)• I t  cannot be taken as an indicator of pathological disturbance since leukocytosis i s a common result of stress or drug use, and the leukocyte counts in our oral contraceptive users were not above the normal range. Serum folate levels in the pregnant subjects studied were not s t a t i s t i c a l l y different from those of the controls. In addition, there was no change in the levels of serum folate or dietary folate intake over the course of pregnancy.  I t may be inferred that, although intake of dietary folate was  below the RDA in these women, i t was sufficient to maintain normal serum folate levels during pregnancy. While the tendency towards reduced serum  - 67 folate concentrations i s commonly associated with pregnancy, i t i s also a highly variable finding and values for serum folate in pregnant women f a l l within a wide range (Hansen, 1968). The sharp rise in red c e l l folate among our pregnant subjects as pregnancy progressed i s more d i f f i c u l t to explain.  The fact that the preg-  nant women shovred an-increase over time i n correlation between serum folate and red c e l l folate may partially account for their improved erythrocyte folate levels.  One may speculate that there i s a compensatory physiological  adaptation in pregnancy that allows for greater availability and storage of folates.  The most l i k e l y hypothesis i s that the high concentration of FABP  found in pregnant women enables them to store greater amounts of folate by increasing cellular uptake (Waxman and Schreiber, 1974), This assumes, ofcourse, that there are adequate levels of circulating folate, which in turn depends on adequate folate intake and the absence of conditions that would further increase the demand for folate.  Our pregnant subjects were in good  health, as judged by their obstetricians, and a l l were from middle-class households.  These factors may have also contributed to their superior folate  status during pregnancy. It may also be postulated that, since there i s evidence that a deficiency of iron may increase the folate requirement (Velez et a l , , I9665 Taskes et a l , , 19?k), conversely, a supply of iron adequate to meet the increased needs of pregnancy might exert a sparing effect on folate.  Perhaps the use  of an iron supplement during pregnancy prevents the ineffective erythropoiesis and excessive heme catabolism' associated with iron deficiency anemia, and would thereby relieve some of the increased demand for folate in pregnancy. However, the absence of a positive correlation between serum iron or total iron binding capacity and serum folate or red c e l l folate in the pregnant  -  68 -  subjects does not substantiate this hypothesis. The relationship between iron balance and folate status should be more thoroughly examined.  I t may be that  there i s a more complex synergy between these nutrients than has been supposed. In particular, i t would be of value to determine the consequences of iron supplementation on folate status.  If there i s a sparing effect of iron on  folate, this may mean that iron supplementation in pregnancy would preclude the use of a folate supplement in women with otherwise normal folate requirements. If i t i s assumed that a low serum folate level i s indicative of marginal folate status and a low red c e l l folate level i s the most reliable index of a true folate deficiency, then the pregnant women in this study would not be considered at risk with respect to folate.  The evidence in the  Nutrition. Canada National Survey that a smaller percentage of pregnant women were at risk with respect to f o l i c acid may be epidemiological support for this finding. The increase, as pregnancy progressed, in hemoglobin concentration, serum iron, and MCV i s common among women who are taking iron supplements in pregnancy (Pritchard, 1970). Total iron binding capacity was also found to increase, and this i s a normal adaptation to the increased need for iron in the pregnant woman (Hytten and Thomson, 1970). In addition, the rise in leukocyte count over the course of pregnancy i s a usual occurrence (Hytten and Thomson, 1970). Therefore, these were a l l changes to be expected. The values for food folate as determined from diet records and calculated from food composition tables were shown to be comparable to the folate values obtained by direct assay of the food.  The degree of correlation  between the calculated and assayed values using the Spearman's Rank Order Test was i n close agreement with that obtained by Moscovitch and Cooper (1973)  - 69 In a s i m i l a r study.  This f i n d i n g lends confidence t o the accuracy of our  estimations o f dietary f o l a t e intake calculated from food tables.  I t also  supports the assumption that d i e t records were a r e l i a b l e method o f measuring f o l a t e intake i n the  subjects who p a r t i c i p a t e d  i n t h i s study.  Nevertheless, there i s s t i l l strength i n the argument that estimation of f o l a t e intake as free and t o t a l f o l a t e i s not i n d i c a t i v e o f the b i o l o g i c a l a v a i l a b i l i t y of dietary f o l a t e s . tested, free f o l a t e intake was consistently  actual  Among a l l three groups of subjects more highly correlated with serum  and red c e l l f o l a t e l e v e l s than was t o t a l f o l a t e intake.  This substantiates  the hypothesis of Herbert i n 1963 that free f o l a t e may be the more accurate measure of available f o l a t e .  More sophisticated  studies involving the assess-  ment of b i o l o g i c a l a v a i l a b i l i t y of food f o l a t e s appear necessary. The  early work of Herbert  (I962) indicated that the serum f o l a t e l e v e l  i s very sensitive t o changes i n the f o l i c a c i d intake.  Hansen  (1968) also  suggested that serum f o l a t e r e f l e c t s the balance between the d a i l y intake and plasma turnover of f o l i c acid.  Although the dietary intake of f o l a t e was  below the Canadian Recommended Daily Allowance i n a l l our groups of subjects, t h i s l e v e l of consumption was apparently adequate f o r the maintenance of normal serum f o l a t e l e v e l s among the control women and the pregnant women studied.  Among the subjects using o r a l contraceptives, however, the same  l e v e l of dietary f o l a t e appears t o be inadequate t o sustain t h e i r serum f o l a t e at concentrations comparable t o those i n  controls.  Although the women on o r a l contraceptives did not demonstrate d e f i cient concentrations o f serum f o l a t e , these findings may well imply that they are a t r i s k with respect t o f o l a t e .  Should t h e i r dietary f o l a t e intake  be even more r e s t r i c t e d , o r should there be some additional demand on t h e i r f o l a t e stores, t h e i r chances of developing a f o l a t e deficiency would be  - 70 further increased.  Women under oral contraceptive therapy would be prudent  to take in the f u l l RDA of 200 yug free folate daily.  This practice may be  sufficient to insure maintenance of normal serum folate levels in oral contraceptive users who are in good health.  It should be pointed out, however,  that there i s no evidence that the quantity of dietary folate required to maintain a certain serum folate level in women taking oral  contraceptives  i s greater than in women not using the: p i l l . The question of f o l i c acid supplementation remains controversial, but deserves clarification since i t is-of great practical concern to both pregnant women and the large population of women taking oral contraceptives. Further research designed to investigate the current Canadian Recommended Daily Allowance for folate seems advisable.  It should be determined whether  or not this allowance i s sufficient to support normal serum folate concentrations in women during oral contraceptive therapy and pregnancy.  In addition,  i t should be specified under what conditions this allowance i s not adequate so that appropriate revisions may be recommended.  - 71 BIBLIOGRAPHY Baker, H., 0. Frank, A.D. Thomson, A. Longer,.E.D.Munves, B. De Angelis, and H.A. Kaminetzky. Vitamin P r o f i l e of 1?4 Mothers and Newborns a t P a r t u r i t i o n . Am. J. Glin. Nutr. 28t 59-65, 1975. Baker, H., V. Herbert, 0. Frank, I. Pasher, S.H. Hutner, L.R. Wasserman, and H. Sobotka. A Microbiological Method f o r Detecting F o l i c Acid Deficiency i n Man. Glin. Chem. 5t 275-280, 1959. Baugh, CM., C.L. Krumdieck, H.J. Baker, and C.E. Butterworth, Jr. Absorption of F o l i c Acid P o l y - - g l u t a m a t e s i n Dogs. J . 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Toghill, P.J. and P.G. Smith.  J. i t 608-609, 1971.  Folate Deficiency and the P i l l .  Brit. Med.  Tolomelll, B,, C. Bovina, C. Rovinetti, and M. Marchetti. Studies on the Folate Coenzyme Metabolism in the Castrated Rat and Treated with 17/3-Estradiol. Proc. Soc. Exp. Biol. Med. I4li 436-439, 1972. Toskes, P., G.W. Smith, T.A. Bensinger, R.A. Giannella, and M.E. Conrad. Folic acid Abnormalities in Iron Deficiency! the Mechanism of Decreased Serum Folate Levels in Rats. Am. J. Clin. Nutr. 271 355-361, 1974. Velez, H., A. Restrepo, J.J. Vitale, and E.E. Hellerstein. Folic Acid Deficiency Secondary to Iron Deficiency in Mani Remission with Iron Therapy and a Diet Low in Folic Acid. Am. J. Clin. Nutr. 19! 27-  36, 1966.  - 78 Waxman, S. and C. Schreiber. Role of FABP in the Cellular uptake of Folates. Proc. Soc. Expt. Biol. Med. 1471 760-764, 1974. Weir, D.G. The Pathogenesis of Folic Acid Deficiency in Man. Med. Sci. I43t 3-20, 1974.  Irish J.  Whitehead, V.M. and B.A. Cooper. Absorption of Unaltered Folic Acid from the Gastro-Intestinal Tract in Man. Brit. J. Haemat. 13i 679-686, I967. Whitehead, V.M., R. Pratt, A. Viallet, and B.A. Cooper. of Folinic Acid to 5-Methyltetrahydrofolate in Man.  221 63-72, 1972.  Intestinal Conversion Brit. J. Haematol.  Whitehead, N., F. Reyner, and J. Lindenbaum. Megaloblastic Changes in the Cervical Epithelium1 Association with Oral Contraceptive Therapy and Reversal with Folic Acid. J. Amer. Med. Assoc. 2261 1421-1424, 1973.  - 79 -  APPENDIX A.  DATE NAME.  ADDRESS 'PHONE  CONSENT FORM  I have read the accompanying statement and questionnaire concerning the n u t r i t i o n a l study of women who are pregnant, or o r a l contraceptive users, and agree to p a r t i c i p a t e  i n the described study.  I was made aware of the fact that: 1.  the study does not represent! a health r i s k  2.  a l l information w i l l be held i n confidence  3.  I have the r i g h t to withdraw from the study at any time.  Signature  1  - 80 APPENDIX B.  Division of Human Nutrition School of Home Economics University of British Columbia June, 1974  We are undertaking a project this year to study some of the effects of oral contraceptives and pregnancy on nutrition. This research w i l l help to clarify the special nutritional requirements of women who are using the p i l l or who are pregnant. We would like to know i f you are interested in participating in this study. Three groups of women are neededi (1) Oral Contraceptive Usersi If you are now taking birth control p i l l s and have been using them for at least three months you could be in this group. We would need to be able t© contact you four times within two consecutive months (these two months may be anytime between July and January). (2) Pregnant Womeni If you are not more than three months pregnant you would be able to participate as a member of this group. We would need to see you three times during your pregnancy, in the 3rd, 5th and 8th months. (3) Control Groupt This will be a group of women (age 18-35) who are neither pregnant nor currently taking birth control p i l l s . It doesn't matter i f you have taken the p i l l in the past but we would prefer that i t had been at least 6 months since you last used the p i l l . We would need to contact you four times within two consecutive months (sometime between July and January). What w i l l you need to do? If you are interested in participating f i r s t f i l l out the P r e l i minary Questionnaire attached to this letter (this i s our one and only piece of red tape). I will then contact you personally. Each woman who agrees to take part in the study w i l l then keep a dietary record for 7 days (you w i l l be instructed in how to do this) and w i l l be asked to give a series of blood samples. Women who are in the control group or the oral contraceptive group w i l l give four blood samples over a period of two months} the samples w i l l be taken at times to be determined according to each woman's individual menstrual cycle. Pregnant women w i l l be asked to give three blood samples, one each In their 3rd, 5th arid 8th months. The amount of blood taken w i l l not be sufficient to cause any significant depletion. We are unable to t e l l you specifically what we are looking for until after you have completed your participation. This i s done f o r control purposes, to avoid prejudicing your food intakei we don't want you to change your usual eating habits because that would bias the results of the study. We w i l l however l e t you know the outcome of our research when i t i s completed. Your attention and consideration i s appreciated. We can't do i t without you. If you have any questions please feel free to c a l l me. Sincerely, Jean Pietarinen  -  81 -  APPENDIX C. PRELIMINARY QUESTIONNAIRE This questionnaire i s designed to obtain information about you which we w i l l need in assessing your blood values and dietary record. We also wish to determine i f anyone should be excluded from the study for medical reasons. A l l information will be held in confidence. Please answer a l l questions as f u l l y as you can,  *********  Date Name Address  Phone at home When i s the best time to c a l l you? General Medical Age  Height  Weight  In the past five years have you had any disease (specify) Illness (specify) serious accident (specify)  ______________________  Do you now have any chronic illness or condition? _______ If so, are you being treated for i t ?  '  Specify any drugs you take in treatment!  In the last 6 months have you taken any prescription drugs (for example, antibiotics) other than birth control p i l l s ? If so, specify what When last taken  ________________________^^  Have you had any previously diagnosed anemia? If so, specify how i t was treated Have you had any recent transfusions? When was the last time you donated blood?  - 82 Dietary Information Are you now on any kind of special diet (including weight-reducing diet)? Specify Are there any foods which you must restrict or avoid for medical reasons?  Are there any foods to which you are allergic?  Do you take any nutritional supplements (for example, vitamin p i l l s or iron)? If so, specify what How much? How often? Are any of these supplements prescribed by a doctor?  -  Gynecological/Obstetrical Are your menstrual periods regular?  .  How long i s i t usually between the start of one period to the start of the next one? When was the f i r s t day of your last menstrual flow? Do you use birth control p i l l s now? If so, what kind  Since when?  Have you used birth control p i l l s in the past? If so, what kind(s)  Are you now pregnant?  When?  How many months?  Have you ever been pregnant before? Please l i s t each pregnancy by year, noting l i v e births, miscarriages and abortions * * * * * * * •* * Please return completed questionnaire to me in the enclosed envelope. Thank you.  - 83 -  APPENDIX D.  Brand of Oral Contraceptive Used and Duration of Use Among Subjects Taking Oral Contraceptives  Subject  Oral Contraceptive Agent  Duration of Use (months)  M.S.  Ortho-Novum 2 mg.  k  A.T.  Ortho-Novum 1/50  k  L.H. V.B.  Ovral Norlestrin  9 9  C.S.  Ovral  10  P.M.  Ovral  12  A.C.  Ovral  15  S.M. W.B. CM.  Ortho-Novum l/50 Ovral Norlestrin  15  16 17  K.H.  Ortho-Novum I/50  18  CH.  Ortho-Novum 1/50  18  L.P. J.P. B. M. M.D. J.T.  Ovral Ortho-Novum 1/50 Demulen Ortho-Novum 1/50 Ovral  19 22 23 36 36  J.M.  Ortho-Novum 1/50  36  N.B.  Ortho-Novum 1/50  36  C.O. H.R. C. M.  Ovral Ortho-Novum 1/80 Norlestrin  60 96  120  - 84 APPENDIX E. Schedule for Collecting Blood Samples from Pregnant Subjects  Subject  Week of Pregnancy when Blood was Drawn 1st sample  2nd sample  3rd sample  J.S.  13  23  32  F.K.  15  26  36  G.B.  16  30  36  S.M.  18  30  37  V.T.  12  24  32  L.D.  *7  28  36  J.M.  15  25  34  A.S.  20  31  40  - 85 APPENDIX F. Mean Daily Dietary Intake of Folate Calculated from Two 3-Day Diet Records for Each Subject  Group  Subject  First >Day Record* Free Folate Intake  y"g/day)  Total Folate Intakes (wg/day)  Oral Contraceptive Users  A.T.  100.13  223.78  L.H.  237.58  471.79  H.R.  (N - 21)  J.M.  148.63 95.02  267.95 228.35  J.T.  132.28  J.P.  101.61  N.B.  191.33 225.40 101.63  250.07 192.98 321.00 383.88  C.H. CO. V.B.  Second >Day Record** Free Folate Intake  (^g/day)  Total Folate Intake  66.55  252.95 104.68  C*g/day) 134.39 430.54  67.72  241.69 152.01  198.62  379.85 407.79 324.49  169.52  296.55 159.07 87.25 75.42  167.50  57.50  148.52  280.87 282.77  194.50 176.00 342.07 275.08  356.53 322.13 415.61 400.10  174.43 162.88  B.M.  70.32 161.66  M.S.  149.52  CS.  199.22 258.0?  329.34  73.72 184.33  141.73 314.47  119.95  184.70  80.37  75.67 230.45  181.83  202.39  200.13  341.98  P.M.  180.69 98.01  378.75 269.04  105.71 70.00  200.58 179.66  214.45  130.17  S.M.  96.23  232.52  147.33  274.05 312.43  A.C L.P. CM. W.B. K.H. CM.  Mean+S. D. 148.2+ 59.8  378.85  270.5± 85.2  152.7± 83.8 . 273.7±102.0 (continued)  •Three days prior to day 5 of the menstrual cyclei or 1st trimester. **Three days prior to day 20 of the menstrual cycle* or 3rd trimester.  - 86 -  F i r s t 3-Day Record* Group  Subject ,  Free Folate Intake ^g/day)  Second 3-Day Record**  Total Folate Intake ^ug/day)  Free Folate Intake ^g/day)  Total Folate Intake ^eg/day)  Controls  E.S.  430.13  490.62  147.83  264.53  (N = 18)  J.T.  219.90  339.68  267.80  450.70  B.J.  67.83  160.25  121.22  235.98  •G.B.  55.75  164.70  159.88  350.75  J.P.  235.18  426.67  288.78  406.59  K.M.  149.74  326.69  57.20  205.33  C.J.  157.96  351.85  203.18  406.24  s.v.  370.79  508. 37  250.93  381.93  E.L.  138.83  236.43  332.67  468.57  P.P.  317.90  480.57  238.65  433.98  J.H.  100.37  223.37  99.31  191.29  B.L.  8O.96  185.93  146.18  237.49  V.M.  40.22  IIO.67  53.82  113.48  L.M.  233.10  353.13  196.43  315.95  M.P.  151.35  283.87  83.69  191.35  S.R.  167.94  270.93  135.37  282.26  J.S.  92.35  198.95  128.47  281.74  S.W.  156.20  278.94  103.13  218.45  Mean+S.D. Pregnant Women (N - 8)  175.9+108.8  299.5±H9.9  167.5± 81.4  302.0+104.4  J.S.  106.55  288.83  93.12  261.03  F.K.  237.69  381.51  261.05  465.79  G.B.  398.19  529.35  175.22  378.83  S.M.  93.15  220.82  96.00  231.25  V.T.  54.12  187.85  127.60  288.34  L.D.  177.59  331.12  205.20  350.17  J.M.  284.42  439.42  156.87  332.29  A.S.  74.49  135.07  108.43  230.48  Mean+S.D.  178.3±120.6  314.3+133.2  152.9+ 5 9 . 0  317.3+ 81.1  - 87 APPENDIX G.  Group  Subject  Oral . Contraceptive • Users' (N = 22)  •  Serum Folate Concentrations of Each Subject  Serum Folate (ng/ml) , Day 5  Day 20  Overall Mean  A.T.  9.15  L.H.  4.35  9.3k 5.16  H.R.  5.65  9. 53 5.98 10.00  J. M.  1.90  3.96  J.T.  5.65  5.40  J.P.  9.23  8.85  2.93 5.53 9.04  N.B.  7.23  7.08  7.15  C.H.  2.85  CO.  4.20 3.80  V.B.  4.69  M.D. o  5.63  5.70 6.80  3.53 4.53 5.20 6.21  B.M.  3.25  3.60  3.^3  M.S.  3.14 5.08  2.96  c.s.  2.79 4.78  A.C.  4.53  L.P.  4.07  4.90 6.80  CM.  3.68  3.90  W.B.  4.85  4.05  5.^3 3.79 4.45  K.H.  6.03  4.18  5.11  CM.  10.95  12.25 5.85 7.50  11.60  P.M. S.M.  Mean+S. D.  3.70 5-30  5.3k +2.20  5.25  5.93 + 2.46  7.83  4.93 4.71  4.78 6.40 5.64 + 2.20 (continued)  - 88 -  Group  Subject  Serum Folate (ng/ml) Overall Mean  Day 5  Day 20  13.45  10.33  11.89  Controls  E.S.  , (N - 18)  • J.T.  5.28  5.03  5.15  B.J.  4.70  4.63  4.66  G.B.  5.45  7.73  6.59  J.P.  9.70  12.80  11.25  K.M.  4.55  5.15  4.85  C.J.  5.93  6.23  6.08  S.V.  14.20  11.28  12.74  E.L.  14.90  18.88  16.89  P.P.-  10.25  9.15  9.70  J.H.  6.00  5.63  5.82  B.L.  5.00  4.33  4.67  V.M.  5.55  3.90  4.73  L.M.  3.20  2.53  2.87  M.P.  6.58  7.10  6.84  S.R.  9.63  20.30  14.97  J.S.  5.68  6.75  6.22  S.W.  5.73  4.88  5.31  8.14 + 4.97  7.84 + 4.04  1st Trimester  2nd Trimester  3rd Trimester  J.S.  3.25  3.60  7.45  F.K.  11.00  6.70  5.90  G.B.  7.30  4.25  5.45  S.M.  3.75  7.70  11.70  V.T.  7.30  7.60  5.45  L.D.  4.45  3.35  3.80  J.M.  5-55  3.85  25.50  A.S.  3.50  3.80  4.40  Mean+S.D.  Pregnant Women (N = 8)  Mean+S.D.  7.54 + 3.58  5.76 + 2.66  !  5.11 ± 1 . 8 8  8.71 ± 7.21  - 89 -  APPENDIX H.  Group  Oral Contraceptive Users (N m 22)  Subject  Red Cell Folate Concentrations of Each Subject  Red C e l l Folate (ng/ml) Day 5  Day 20  A.T.  315.9  242.3  279.1  L.H.  158.2  157.4  157.8  H.R.  201.1  197.6  199.3  J.M.  86.2  87.5  86.8  J.T.  182.3  189.7  186.0  J.P.  187.6  148.4  168.0  N.B.  199.8  I65.6  182.7  C.H.  136.4  137.8  137.1  CO.  131.2  190.8  161.0  V.B.  145.7  151.8  148.8  M.D.  170.1  141.3  155.7  B.M.  131.6  144.4  138.0  M.S.  137.9  160.0  149.0  c.s.  121.1  138.8  129.9  A.G.  137.2  146.0  141.6  L.P.  254.6  268.8  261.7  CM.  222.9  234.2  228.5  W.B.  161.7  183.7  172.7  K.H.  193.0  226.1  209.6  CM.  198.0  173.**  185.7  P.M.  130.9  137.6  134.3  S.M.  429,0  445.1  437.0  Mean+S. D. 184.2 + 74.1  184.0 + 72.2  Overall Mean  184.1 + 71.7  (continued)  -  9 0  -  Red C e l l Folate (ng/ml) Group  Subject Day 5  Day 20  Overall Mean  195.2 143.1  225.7 172.4  Controls  E.S.  (N - 18)  J.T.  256.3 201.7  B.J.  141.1  119.0  130.0  G.B.  283.1  313.8  J.P.  188.3  K.M.  174.5 125.0  298.5 181.4  133.6  129.3  C.J.  174.7  140.0  157.3  s.v.  279.6  280.7  280.2  E.L.  253.7 201.3  294.8  P.F.  335.9 206.4  J.H.  172.6  171.6  172.1  B.L.  180.3  179.3  V.M.  178.3 166.0  155.4  160.7  L.M.  84.4  87.0  85.7  M.P.  296.0  S.R.  336.7 268.2  294.0  316.3 281.1  J.S.  130.3  126.3  128.3  S.W.  267.5  315.0  291.2  Mean+S.D.  Pregnant Women (N = 8)  203.8  210.1 + 73.6  199.7 ± 74.0  204.9 ± 71.8  1st Trimester 4  2nd Trimester  3rd Trimester  J• s#  116.6  96.5  73.3  F. K.  235.2  198.5  276.8  G. B.  180.4  364.1  S.M.  103.6  277.7 298.0  V.T.  310.3  323.9  L.D.  156.3 194.6  350.2  J.M.  148.7  35L7 318.2  679.0  A.S.  205.0  300.4  248.1  I67.6 +44.8  268.9 ±82.4  Mean+S.D.  307.7  327.9 ± 168.7  - 91 APPENDIX I.  Differences' in Hematological and Dietary Parameters Measured at Two Phases of the Menstrual Cycle in Control Women and Women Using Oral Contraceptives  Variable  Serum Folate (ng/ml)  Red Cell Folate (ng/ml)  Hemoglobin (g/lOOml)  Hematocrit ( % )  Control Group  Oral Contraceptive Group  Mean + S.D.  ' Mean :+,S.D.  5 20  7.54 + 3.58 8.14 + 4.97  5.3k + 2.20 5.93 ± 2.46  P  N.S.  N.S.,  5 20  210.1 + 73.6  P  N.S.  Day of Cycle  199.7 ± 74.0  184.2 + 74.1 184.0 + 72.2  1.  P  <0.05 N.S.  N.S. N.S.  N.S.  P  13.0 + 0.7 13.2 + 0.5 N.S.  13.4 + 0.8 13.3 ± 0.6 N.S.  N.S. N.S.  5 20  38.3 + 2.3 38.6 + 1.8  P  N.S.  39.2 + 2.0 38.9 ± 1.7  N.S. N.S.  5 20  4.6 + 0.8 4.9 + 1.1  6.1 + 1.4 7.1 + 1.9  P  N.S.  <0.01  5 20  4.44 + 0.23 4.48 + 0.19  4.50 + 0.28 4.43 + 0.27  P  N.S.  N.S.  5 20  .  N.S.  V  White Cell Count (x 103)  Red Cell Count (x 10 ) 6  M.C.V. (^3)  5 20  P M.C.H. (/*/*s)  5 20  P  86.1 +2.6 86.0 + 2.2  N.S. 29.3 + 0.9 29.4 + 1.0  N.S.  87.5 + 3.8 88.0 + 3.8  N.S. 29.7 + 1.4 30.0 + 1.4 N.S.  <0.01 <0.01  N.S. N.S.  N.S. <o.05  N.S. N.S.  - 92 APPENDIX I.  (continued)  Variable  M. G.H. G.  ( %  )  Day of Cycle  5 20 P  Serum Iron  Control Group  Oral Contraceptive Group  Mean + S.D.  Mean + S.D.  33.7 ± 0.7 33.5 ± 0 . 7  33.4 + 0.7  N.S.  33.6 + 0 . 6  P  N.S. N.S.  N.S.  84.0 + 22.8  100.7 ± 29.6  N.S.  Total Iron Binding Capacity (/xg/di)  327.6 + 73.5  382.5 ± 65.7  <0.05  Unsaturated Iron Binding Capacity (/*g/dl)  243.6 + 76.8  281.9 ± 78.2  N.S.  5 20  175.9 + 108.8 167.5 ± 81.4  148.2 + 59.8 152.7 ± 83.9  N.S. N.S.  P  N.S.  N.S.  5 20  299.5 + H 9 . 9  270.5 + 85.2  P  N.S.  N.S.  (/Ag/dl)  Free Folate Intake (/Ug/day)  T o t a l Folate Intake (/*g/day)  302.0 + 104.4  N.S. - Not S i g n i f i c a n t , P > 0 . 0 5  273.7 ± 102.0  N.S. N.S.  -  APPENDIX J.  93 -  Differences i n Hematological and Dietary Parameters Measured During Each Trimester of Pregnancy  Mean + S.D. Variable Serum Folate (ng/ml)  1st Trimester  2nd Trimester  3rd Trimester  5.76 + 2.66  5.11 ±1.88  3  8.71 ± 7.21 327.9 ± 168.7  3 , 4  Red C e l l Folate (ng/ml)  167.6 +44.8  268.9 ±82.4 1  Hemoglobin (g/lOOml)  11.8 + 1.0  11.5 ± 0.8  3  12.0 + 0.8  Hematocrit ( %  34.3 ± 2.9  33.9 ± 2.0  3  34.7 ± 2.6 '^  7.9 ±2.0  8.9 ± 2.0  1  3.93 ± 0.40  3.78 ±0.31  87.5 ± 3.3  89.0 + 2.8  30.1 + 1.0  3O.3 ± 0.9  a+  33.6 ± 0.6  )  White C e l l Count  (x 103)  Red C e l l .Count  (x  io6)  M.C.V. M.C.H. S)  M.C.H.C.  (%) Serum Iron (/^g/dl)  33.  0.6  ^  2  ,  3  3  9.0 + 1.9 1  1  ^  3.93 ± 0.34 88.3 ± 3.3  3 , k  3  3  30.5 + 1.5 ' 3  k  33.8 ±0.7  97.6 + 29.4  101.9 ± 24.5  3  104.0 + 48.2  Total Iron Binding Capacity (/*g/cU)  335.7 ± 6 1 . 9  408.9 ± 35.6  1  463.4 + 38.5  Unsaturated Iron Binding Capacity (/*g/dl)  225.9 ± 69.5  309.3 ± 40.8  1  362.3 + 63.5  1  1,Z  Free Folate Intake (^g/day)  178.3 ± 120.6  -  152.9 ±59.0  3  Total Folate Intake (^g/day)  314.3 ±133.2  -  317.3 + 81.1  3  ,  2  *'  S i g n i f i c a n t l y ( P < 0.05) d i f f e r e n t from value i n 1st trimester; 2nd trimester. -'Not s i g n i f i c a n t l y (P>0.05) d i f f e r e n t from 1st trimester; ^ d trimester.  - 94 APPENDIX K.  Calculated and Assayed Values f o r Folate f o r Duplicate Food Samples Collected at the Time of Consumption  Sample No.  Values Calculated from Food Tables  Assayed Values  Free Folate (/*«)  T o t a l Folate (/>• g)  Free Folate >g)  Total Folate (>g)  1  123.20  198.40  151.67  301.68  2  101.25  208.90  136.70  219.61  3  66.05  131.10  106.05  204.57  4  35.20  111.50  106. 32  193.30  5  179.70  284.70  233.26  511.60  6  60.27  150.80  220.09  418.48  7  65.35  105.45  95.02  174.43  8  155.35  305.10  137.05  216.48  9  74.50  213.95  122.94  307.36  10  142.20  208.80  142.18  275.56  11  140.80  190.14  102.96  189.66  12  144.30  254.85  122.18  290.32  13  4?.16  137.00  86.93  210.15  14  512.10  606.50  908.52  1,029.73  15  720.48  832.00  1,074.82  1,15^.58  

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