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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 present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or pub l i ca t ion of th is thes is for f i n a n c i a l gain sha l l not be allowed without my writ ten pe rm i ss i on . V I V I S ' I O A N Bfpartmeint of A/uMA<V NUTR-IT/ON , SC2//OQL OF / - / O M E EdoNoM /CS The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 - 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 combi-nation-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 - i i -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—-for 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 in the control group than in the oral contraceptive group. This difference was statistically significant (p<0.05) for the serum folate values at day 5 of the menstrual cycle, but not at day 20 of the cycle. There was no significant difference in the red cell folate levels-"between these two groups of subjects although, again, the control women had higher levels than did the oral contra-ceptive users. Within each of the two groups, serum and red cell 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 cell folate levels were found to increase over the course of pregnancy. However, the rise in serum'folate was not statistically significant, and the increase in red cell folate was significant between the f i r s t and second trimesters (p<0.05) but not between the second and third trimester. It is not clear why the folate status of the pregnant subjects improved during the course of pregnancy. - i i i -There was no difference in the levels of dietary folate intake among the three groups of subjects. The degree of correlation between the serum folate levels and dietary folate intake was consistently higher in the con-t r o l women than in the women using oral contraceptives. The difference i n correlation coefficients 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 at day 5 of the cycle. In the group of pregnant women 6 the correlation between serum folate and dietary folate was higher in the f i r s t trimester than in the third trimester; but the difference in correla-tion coefficients was not s t a t i s t i c a l l y significant. There was no s i g n i f i -cant correlation between red c e l l folate levels and dietary folate intake in any of the three groups of subjects. These results indicate that oral contraceptive use reduces serum folate levels, and that this decline in serum folate i s Independent of dietary folate intake. Thus, there appears to be some direct effect of synthetic sex hormones in reducing serum folate levels. Whether this i s an effect at the level of intestinal absorption or tissue u t i l i z a t i o n remains unclear. I f women using oral contraceptives are not able to f u l l y absorb or u t i l i z e their dietary folate, i t may be that their recommended daily allowance for folate should be increased. - iv -ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Joseph Leichter, for his direc-tion 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 statis-t ical analysis of results. My appreciation is 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 v i i i 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 folic acid 6 Reduction and methylation of absorbed folates 8 Folate Deficiency and Megaloblastic Anemia 9 Definition of folate deficiency and megaloblastic anemia 9 Etiology of folate deficiency 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 folic 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 in the assay of food folates 31 Problems in estimating dietary folate intake 33 Conclusion 34 III MATERIALS AND METHODS 36 Collection of Blood 38 Treatment of blood samples 39 Collection of Dietary Data 41 Collection of food at the time of consumption for analysis of folate content 41 Analyses 42 Analysis of blood samples 42 Evaluation of diet records 43 Analysis of food samples 43 Statistical Analysis of the Data 43 IV RESULTS 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 - v i i -Dietary Folate Intake 55 Comparison of dietary folate intake among controls, oral contraceptive users, and pregnant subjects 55 Relationship between dietary folate intake and serum and red cell folate levels 57 Correlation between the calculated and assayed values of folate for food samples collected at the time of consumption 59 V DISCUSSION 61 BIBLIOGRAPHY 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 83 E. Schedule for Collecting Blood Samples from Pregnant Subjects 8k F. Mean Daily Dietary Intake of Folate Calculated from Two 3-Day Diet Records for Each Subject 85 G. Serum Folate Concentrations of Each Subject 8? H. Red Cell Folate Concentrations of Each Subject 89 I. Differences in Hematological and Dietary Parameters Measured at Two Phases of the Menstrual Cycle in Control Women and Women Using Oral Contraceptives , 91 J. Differences in Hematological and Dietary Parameters Measured During Each Trimester of Pregnancy 93 K. Calculated and Assayed Values for Folate for Duplicate Food Samples Collected at the Time of Consumption 9k - v i i i -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 for folic acid, which may result in reduced levels of serum and red cell 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 cell folate concentrations among women using oral contraceptives. The role of dietary folate intake in determining serum or red cell 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 al., 197^ ; Fleming et al., i97**a)»and low intake of dietary folate (Hibbard and Hibbard, 1972» Cole et al., 197kj Fleming et al., 1974a). Oral contraceptives are said to simulate some of the physiological changes associated with early pregnancy. Because of this, recent research - 2 -has 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 cell folate levels in oral contraceptive users (Shojania et al., 1971» Roetz and Nevinny-Stickel, 1973l Smith et al., 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 al . , 1972; Paine et al., 1975l Prasad et al., 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 ln some oral contra-ceptive 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 al . , 1972i Shojania et al., 1975), an increased urinary excretion of folates (Shojania et al., 1975)» and the presence of a folic 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 cell 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 is 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 for the body to reestablish i t s folate stores between pregnancies (Hibbard and Hibbard, 1972; Cole et al., 197k). He would not expect this to be the case in 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 folic 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 "It i s not possible to assess the clinical 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 relation-ship between serum and erythrocyte folate levels, and dietary folate intake ln women who are pregnent or using oral contraceptives, as well as in 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 deter-mined by measuring serum folate, red cell folate, and dietary folate intake at two different stages in 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 cell 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 clarify the extent to which serum and red cell 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 folic acid and its subsequent transformation to the raetabolically active forms is incomplete. A review of this area is requisite for evaluating the literature concerned with folate metabolism.during pregnancy and oral contraceptive therapy. Physiological indices of folate status. Folic acid, in its 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 folic acid in DNA synthesis and cellular growth is vital, and folates are utilized exten-sively in tissues where there is 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 will 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 cell 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 folic acid levels reflect recent nutritional - 6 -status with respect to folate intake, whereas the amount of folate in red blood cells more specifically indicates folate stores in the body (Chanarln, 1969)* Serum folate i s believed to be derived from liver and other tissue stores by a displacement mechanism (Rosenberg and Godwin, 19711 Gerson and Cohen, 1972). Absorbed folic acid enters these tissues and thus acts to displace endogenous 5-aethyltetrahydrofolate to the plasma. If dietary intake of folic acid Is reduced this phenomenon i s diminished, resulting in a rapid decrease in serum folate levels. A reduction of the folate in red blood cells requires a longer period of deprivation since erythrocyte folate i s tightly bound and not released until the cell itself is destroyed at the end of its l i f e cycle (Hoffbrand, 1971)* Therefore measurement of red cell folate i s indicative of long-term folate status. Whereas erythrocyte folate levels are used as a measure of folate stores. It i s liver supplies of folate which actually form the largest reservoir of the vitamin in the body (O'Broin et al., 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 folic acid. The adequacy of folate nutrition depends not only on the availability of adequate folate-rich foods, but also on the Individual's ability 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 - 7 -length of the small intestine although the greatest proportion is absorbed from the proximal jejunum (Bernstein et al. , 1970). In general, the rate of absorption is 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 ( Y-carboxypeptidase, referred 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 is not clear how the larger molecules of folate reach the inside of the mucosal cells. There i s perhaps some deconjugation in the lumen itself by the action of conjugase present in sloughed-off mucosal cells, but this remains hypo-thetical (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) is 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 al. in 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 al. in 1971» using a similar method but employing ^ HPGA, showed that glucose enhanced the absorption of folic 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 al. - 8 -(1970) using everted gut sacs from rats. Gerson points out, however, that although this i s strong evidence for a passive transport of folic 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 is not yet completely understood (Krumdieck et al., 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 methyla-tion occur in the gut wall during absorption (Chanarin and Perry, 1969; Perry and Chanarin, 1970; Whitehead et al., 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 in the liver. Whitehead-and Cooper (1967) demonstrated that orally administered PGA appears unaltered in the portal circulation, and only after passage through the liver does the folate emerge as 5-methyl-tetrahydrofolate. In the process, hepatic folate i s evidently exchanged for the more recently absorbed folates entering from the portal circulation (Melikian et al., 1971). This has been conf irmedln studies using ^CPGA (Butterworth et al., I969) and 3HPGA (Pratt and Cooper, 1971). Whitehead et al. (1972) have suggested that reduced dietary folates are absorbed differently from folic acid (PGA), which could account for - 9 -the discrepant results in the above studies. Whereas PGA may not he methy-lated until i t reaches the liver, 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 liver supplies; a disturbance of intestinal function could impair absorption; disorders of the liver 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 is relevant at this point to distinguish between a folic acid deficiency and an outright megaloblastic anemia which is an expression of extreme folate deficiency. Herbert's classic work (1962) has outlined the sequence of changes which take place when the body is deprived of sufficient folic acid (i.e., limited to an intake of 5.0 folate per day or less). Herbert summarized his findings as followsj - 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 cell folate (<20 ng./ml.) 17 Macroovalocytosis 18 Megaloblastic marrow 19 Anemia 20 The formation of megaloblasts in the bone marrow and macrocytes in 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 is a delay at certain points ln mitosis which causes the formation of giant cells (Chanarin, 1969; Hoffbrand, 1971). There are also changes in the production of white blood cells. One type of leukocyte, the neutrophil, will show an increased number of lobes in Its nucleus. There are normally two to five; this will increase to six or more. A megaloblastic anemia, by definition, i s characterized by the presence of megaloblasts ln 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 in 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 investi-gating 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 folic 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 folic 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 al., 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 alcoholism. 2. Alcoholism. The occurrence of folic acid deficiency in alcotelics is common and is probably the result of poor dietary habits (Halsted et al . , 196ft.; 1971 j Stebbins et al., 1973)* However, absorption may also be impaired due to damage to the intestinal mucosa, a direct effect of the alcohol itself (Stebbins et al., 1973J Weir, 1973). 3. Liver disease. If cirrhosis occurs in connection with alcoholism, or i f there i s some other damage to liver function, this may hamper the ability of the liver to store or exchange folates (Brown et al . , 1973)* Such a condition can also result in increased urinary loss of folates (Weir, 1973). k. Increased requirement for folic acid. Any condition in which there i s a marked increase in cell multi-plication, as in pregnancy, lactation, growth, malignancy, or hyperthyroidism will increase the need for folic 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 ln homocystlnuria. 5. Increased loss of fo l i c acid. There may be an abnormally rapid catabollsm of folic acid in situations where hemolysis or hemorrhage occur, as well as when a hemolytic anemia exists (sickle cell anemia, thalassemia, or congenital sphero-cytosis) (Weir, 1973). In addition, a person undergoing hemodialysis may lose a significant amount of folic acid which, in 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 al . , 1973)* Folate antagonists, most notably methotrexate and aminopterin, used ln treatment of cancer, have a great affinity for dihydrofolate reductase and act by displacing folic acid from this enzyme. Pyrimethamine, an antimalarial drug, i s also, an inhibitor of dihydrof olate reductase but i s far 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 folic acid, although the process by which this happens remains unclear. The effect of oral contraceptives on folate status will 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 is often over-looked but i t may be of importance in 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 al. , 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 in the etiology of folate deficiency. The Effect of Pregnancy on Folate Status Pregnancy induces some striking physiological alterations in the female's body in 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 in anticipation of the demands of labor, delivery, and subsequent nursing. Anemias of pregnancyi Iron and folate interrelationships. The requirement for both iron and folic 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 inter-related in other ways which have not been fully elucidated. An iron - Ik -deficiency is 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 is known that iron deficiency and folic acid deficieny do frequently occur together, and the risk of developing both of these is especially increased during pregnancy (Pritchard, 1970). The alterations in both iron and folate metabolism that take place ln pregnancy may contri-bute to the development of anemia in some women. In discussing anemias of pregnancy i t Is critical to distinguish between what is 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 is spent ln a state of hemo-dilution, where the plasma volume is 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 fall (Hytten and Leitch, 1971). Some believe that this physiological alteration is a true state of iron deficiency anemia and cite the evidence that iron given in therapeutic doses will prevent the fal 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 is also an unrelenting drain on maternal iron stores by the fetus. This demand is always met even at the expense of the mother. Thus, i f by the time the greatest increase in red cell production is 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 al., 1973? Besforges, 1973)• Usually i t is the presence of some additional stress acting to deplete maternal iron stores (such as hemorrhage, infec-tion, 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 is the well-docu-mented fact that a decrease in serum folate occurs in conditions asso-ciated 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 is uncertain although the explanation may be quite simple. In iron deficiency there is a decrease in the i - 16 -survival of peripheral red blood cells and a concomitant Increase In heme catabolism. According to Toskes et al. (1974), this in itself may be sufficient to account for the decreased serum folate levels and the increased folate requirement. Paradoxically, once an iron deficiency anemia is established, treatment with an iron supplement may cause a further decrease in serum folate because of the additional demand on folate stores ln the Initial spurt of new erythrocyte formation (Hoffbrand, 1971)• Perhaps the major point to be aware of in clinical practice i s that the morphological changes in the blood associated with Iron defi-ciency 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 cell folate levels. Folic acid deficiency of pregnancy.51 Reduced serum and red cell 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 concen-tration 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 meta-bolism 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 folic acid binding protein (FABP) has been found in 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 prac-tices of the subjects. This variable may be of more significance in determining folate status than is often supposed. Food availability, economic advantage, and ethnic custom are of particular importance ln 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 al., I974) appear to exert a protective effect against folate deficiency. Assessment of folate status ln pregnancy. Estimates of the Inci-dence 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.f The methods used to assess folate status reflect different 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 tell 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 is 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 is another commonly used test of deficiency, and i t is also altered in pregnancy. There is 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 physi-ological adaptations associated with normal pregnancy. The neutrophil lobe average, when i t is increased, is Indicative of a severe degree of folate depletion (Hibbard and Hibbard, 1971)• How-ever 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 is 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, is of more value in deter-mining the existence of a folate deficiency in pregnancy since i t is definitely associated with a depletion of folic acid stores. However, low red cell folate is 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 is 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 is s t i l l debatable whether these should be regarded ' as indicative of a folate deficiency or i f they are normal changes asso-ciated 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 until a low red cell 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 Little, 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 retrospec-tive 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, is 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 folic acid in pregnancy. Folate deficiency and its extreme form, megaloblastic anemia, are almost always the result of more than one factor contributing to a folate imbalance. In:pregnancy, i t is the woman faced with some additional complication who is 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 is accompanied by any of the following conditions» hemolytic anemia, thalassemia, epilepsy being treated with anticonvulsants, alcoholism, infection, or poor diet. In these situations a folic acid supplement may be warranted to preclude the possibility of any maternal or fetal complications (Fleming et al., 1974b). This practice would not be challenged%by most obstetricians. Rather, i t i s the question of routine folic acid supplementation for a l l pregnant women which is 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 folic acid supplement is 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 is the best guarantee that the different dividing cells receive an adequate amount of folic acid, which must be assumed to be of special significance for normal growth in pregnancy." The decision to prescribe a folic acid supplement to a pregnant patient is left 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 clinical significance of low serum folate levels and other signs of altered folate status should not be underrated. Following the discontinuation of hormonal contraception a woman could easily become pregnant. If, at this point, there is 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 in 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 in some respects, and one would expect some similar alterations in physiology to exist. - 22 -There i s evidence that oral contraceptives have an effect on folate status which i s similar in many respects to that found in pregnancy. Whether this effect i s the result of an etiology common to both conditions i s unclear. Megaloblastic anemia associated with oral contraceptive use. There has been extensive work recently published documenting the possible adverse effects of hormonal contraception on folate status. A survey of the literature reveals numerous incidences of megalo-blastic anemia occurring i n association with oral contraceptive use (Necheles and Snyder, 1970} Str e i f f , 1970; Buhac and Finn, 1971; Ryser et a l . , 1971? Toghill and Smith, 1971; Flury and Angehm, 1972; Salter, 1972; Johnson et a l . , 1973; Shojania and Hornady, 1973; Lewis, 1974? Meguid and Loebl, 197k). These, however, are a l l based on individual case studies and do not represent controlled experimental situations. In reviewing these reports, i t Is of significance that those which included a test for malabsorption did, in fact, find evidence for an underlying intestinal disorder that could have affected folate absorption (Streiff, 1970; Ryser et a l . , 1971; Toghill and Smith, 1971? Johnson et a l , , 1973). Moreover, in most of the other studies, i t was stated that an occult malabsorption syndrome of some kind was not, and should not, be excluded as a possible contributing factor (Necheles and Snyder, 1970; Flury and Angehrn, 1972? Shojania and Hornady, 1973; Lewis, 1974? Meguid and Loebl, 1974). In a l l cases, discontinuation of oral contraceptive therapy and i n i t i a t i o n of f o l i c acid supplementation were sufficient to cure the anemia. Thus i t seems that a woman using oral contraceptives must also have some other condition affecting folate metabolism before megaloblastic changes w i l l develop. Conversely, the effect of such a condition might - 23 -not become apparent except for the added stress of treatment with syn-thetic 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 rela-tive to the large number of women at risk. A more common observation among women using oral contraceptives is the tendency towards reduced serum folate concentrations. However, this has been a highly variable finding. It appears that certain women may have an enhanced suscepti-bi 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 in this area makes i t difficult 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 1971t and Roetz and Nevinny-Stickel in 1973* The latter researchers showed that serum folate levels decline over time after the initiation of oral contra-ceptive therapy. Looking at the converse situation, Shojania et al. 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 qualifi-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 speci-fied 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 will 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 in serum folate levels after these women were given an oral dose of either monoglutamic folate or polyglutamic folate. The rise in serum folate was comparable in the two groups when the monoglu-tamic form.was given, but after administration of polyglutandc folate the rise in serum folate in the oral contraceptive group was about 50% of that in 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 hor-mones with conjugase activity. His hypothesis was also based on an in 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 is 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 ln 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 in this connection has been that of Stephens and associates in 1972 and, more recently, Paine et al, (1975)* Paine et al. surveyed a very large sample of women (N=»526) and found no significant difference in serum folate levels between oral contra-ceptive users and controls. They obtained the same result when blood samples were analyzed by either the L. easel microbiological method or the new radloassay for folate. The strength of their findings lies in 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 al. 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. First they examined serum folate levels in fasting blood from a group of oral contraceptive users and a group of control women. They found no statistically significant difference ln 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 polyglu-tamate absorption in the oral contraceptive users, further investigation showed that, after saturation of the subjects with folic acid, the differ-ences between the groups disappeared. Stephens explained their results by suggesting that oral contraceptives may cause an increase in the rate of clearance of folates from the plasma. This accelerated clearance i s apparent when the subject's tissues are not fully 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 al. involved an in 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 al. , 1975). This was, however, shown in 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 contra-ceptive users, but when they made the above distinction between two popu-lations 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 al. 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 pteroyl-glutamic 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 It was also discovered that there was a direct correlation between urinary folate excretion and serum or red cell 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 cell folate than did their control counterparts. The authors felt that this Increased excretion of folates among women taking hormonal contra-ceptives may partially account for their frequently lower blood folate levels. Related factors of unknown significance. 1. Folic acid binding protein (FABP). Da Costa and Rothenberg (1974) have reported the existence of a specific folic 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 in concentration. Such a shift in the relative concentration of plasma FABP's has been shown to occur inpregnancy (Karkkanen et al., 1973)• Whichever i s the case, the exact function of such a binding protein and i t s position in the etiology of reduced folate status in 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 - labelled 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 in 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 in plasma folate binders that i s responsible for the increased urinary excre-tion 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 (Klausf 19711 Whitehead*et al . , 1973* Lindenbaum et a l . , 1975) that oral contraceptive use i s associated with distinctly megaloblastic changes in the cervical cells which can be observed from Papanicolaou smears. It Is disturbing to note that these morphological changes in the cervical epithelium are in no way correlated with other evidence of a systemic folate deficiency (such as reduced serum or red cell 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 al., 1975)* However, fol i c acid therapy was shown to significantly revert the cervical morphology towards normal within three weeks, and a discon-tinuation of the folic acid supplement resulted ln a recurrence of the abnormalities (Whitehead et a l . , 1973 ; Lindenbaum et al., 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 cell proli-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^mays;be'evidenced by the presence of megaloblastic changes. 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 in 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 impor-tnace 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 in almost a l l the studies ofifolic acid status in 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 is 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 difficulties involved in assessing dietary folate intake are numerous. Consideration of this problem involves introducing the addi-tional 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 will depend on food availability, dietary habits, and physiological absorptive capacity. The assessment of folate status must therefore include both a dietary esti-mation of folate intake and an examination for factors which might interfere with i t s absorption. This discussion will center on the difficulties surrounding evaluation of dietary folate intake and will encompass two areas of concern! (1) Problems inherent in the assay of food folates which one should be aware of in 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 folic acid are found in 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 is apparently present only because of the oxidation of reduced folates during processing, storage and cooking (Hurdle, 1973). It i s the multi-plicity of forms of folate ln food and the differing availability of these forms to various test organisms that makes the evaluation of food folates a difficult task (C^Broin et al., 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 will be able to utilize 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, is supported by most folates with up to three glutamate resi-dues (Hoppner et a l . , 1972). The determination of dietary folic acid presents additional pro-blems because the significance of "free" and "total" folates as deter-mined by microbiological assay is poorly understood in the context of biological availability in mammals. We cannot equate "total" folate in food with "available" folate because the in 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 in the diet (Herbert, 1963). The measurement of truly available folate probably l i e s some-where between the values for "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 protec-tive 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 in foods. Variability in folate values may also be partially accounted for 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 activity (Taimjra and Sto^ - 33 -3. The effect of cooking. Processing and cooking, especially in large quantities of water, will destroy heat-labile folates (Herbert and Bertino, 19®?), and can result in losses of 90% or more of folate activity (Herbert, 19^ 3« Hurdle, 1973). 4. Sensitivity to pH. Folates are available for absorption ln a very narrow pH range (Butterworth, 1968). Buffering is essential to the assay of folates as they will 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 ln estimating dietary folate intake. Dietary assess-t n ment i s a difficult task, largely because i t i s so dependent on subjec-tive evaluations. Unless dietary habits can be observed without the knowledge of the subject, some personal bias will enter ins and unless Intake of food items is 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 folic acid. The accuracy of a measurement of dietary folate intake will 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 reliability 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 out-lined in the previous section), A good model for the application of this knowledge can be found in 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. It has been difficult 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 will depend upon choosing those parameters which may be significant 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 in subjects. 3. Choice of parameters for 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 will be one of the princi-pal aims of the present study, a l l of the above variables are signifi-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). All subjects in 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 in 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 in the City of Vancouver who had agreed to distribute the preliminary questionnaire to interested patients.' The obstetricians were restricted to choosing only women ln early pregnancy who were in good health and who had not been prescribed a folic add 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 criteria! Agei The minimum acceptable age was 18, the maximum. 35* Medical historyi Evidence of any chronic inflammatory disease or infec-tion, liver disease, kidney dysfunction, epilepsy, diabetes, tuberculosis, malaria, or an anemia within the past year were grounds for elimination from the study. Drug usei If any woman was receiving regular treatment with antibiotics, anticonvulsants, antimalarial drugs, or other drugs (besides oral contra-ceptives) known to interfere with folic acid metabolism, she was not included ln the study. If a woman was undergoing temporary treatment with anti-biotics she was used as a subject only after antibiotic therapy had been discontinued. Blood donation! If a woman had recently donated blood she was asked to wait at least one month before being included in 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. - 38 -Use of supplementsi Subjects were carefully questioned regarding their use of nutritional supplements. No one taking a vitamin supplement that included folic acid was allowed to participate in the study. Iron supplemen-tation was permitted since a very 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 in 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 pill.) Obstetrical Historyi A history of past pregnancies, including those which had ended in miscarriage or therapeutic abortion, was obtained from each woman. If 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. All 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 in the pregnant group the approximate date of concep-tion 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 in 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 in the blood were low, and day 20, when hormone levels were relatively - 39 -high. 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. (This sampling procedure was adopted from Stephens et al., 1972.) 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 fi 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. The schedule for sampling was as outlined in Table 1. 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). If 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 in 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 in small plastic tubes* as followst 1.5 mis. for serum folate assay (for a l l samples), and an additional 1.0 ml. for iron assay (for a l l samples from the pregnant women, but only for 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 for Collecting Blood Samples Additive ln Vacutainer tube Milliliters of blood collected Control and Oral Contraceptive Groups Pregnant Group All Samples Cycle I Cycle II day 5 1st sample day 20 2nd sample day 5 3rd sample day 20 4th sample No additive (for serum) 8 5 5 8 8 Q. 5 ml. 3.:8# Na citrate 4 4 4 4 0.07 ml. 15S EDTA 1 1 4 4 4 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. until the day of assay. This blood was used only for the determination of red cell 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 in 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 in order to avoid some of the bias in 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 in meal samples were determined for 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 for 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. All 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 cell 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 microbio-logical method of Baker et al. (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 cell 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 Folic Acid "Baker Grade."* Folic Acid Casei Medium** and Lactobacilli Broth AOAC** were used in l i e u of preparing the assay medium and maintenance culture broth ourselves. Serum iron and total iron binding capacity were determined using the Hycel Kit for "Serum Iron and Iron Binding Capacity Tests."*** For women from the oral contraceptive and control groups, the iron assays were performed on pooled serum from the f i r s t and la s t blood samples. For pregnant women the iron assays were done on each of the three blood samples. Evaluation of diet records. The daily intake of free and total folate was calculated from the diet 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 free and total folate of the collected food samples was determined using the L. casei microbiological method adapted for food analysis by Herbert (1963), with the exception that the concen-tration of ascorbic acid used was 150 mg. per 100 mis. of buffer. Total folate was assayed using chicken pancreas conjugase.**** For control pur-poses, the pooled sera for low, mid-normal, and high-normal serum folate levels were determined with each assay. St 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 at the Computing Centre of the University of Brit i s h Columbia. The SPSS computer program *J.T. Baker Chemical Co., New Jersey, Lot no. 1-7555. **"Difco" Certified, 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 statis-tical 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 cell 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 calcu-lated ("expected") values for these samples :was in i t i a l l y done using linear regression. The Pearson Product Moment Correlation Coefficient was calcu-lated and its 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 liver 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 is a non-parametric test of corre-lation for situations where normal distribution is 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 signifi-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 Day 20 P* Overall Mean Controls (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 i < 0.05 •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 cell folate concentrations between the controls and p i l l users. Neither was there any statistically significant difference in red cell 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 5 Day 20 P* Overall Mean 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 + 71.7 P*t > 0.10 > 0.10 > 0.10 *Two-tailed, using Student's t-test. ^Pooled variance estimate (F ratio not significant, p >0.05). In controls there was a statistically significant correlation between serum folate levels and red cell 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 statisti-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 contracep-tives 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<0.05) . 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 in controls, the difference was not quite sign-ificant 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 bind-ing capacity with the time of the menstrual cycle. There was a significant difference (p<0.01) in white cell 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 in leukocyte count during the cycle was not found among control subjects. Additional data for the other hematological parameters tested (red cell count, hematocrit, M.C.H., M.C.H.C.) are found in Appendix I. Only those findings of statistical 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 in Hemoglobin, MCV, Serum Iron, and Total Iron Binding Capacity Between Control Women and Women Using Oral Contraceptives Hemoglobin (g/100 ml) Mean + S.D. MCV fyi3) Mean + S.D. Serum Iron (y^g/dl) T.I.B.C. (/*g/dl) Day 5 Day 20 Overall Mean Day 5 Day 20 1 Overall , Mean Mean ± S.D. Mean ± S.D. 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 ±65.7 * P > 0.102 >0.102 > 0.102 > 0.102 <0 . 0 5 1 >0 . 0 5 1 > 0 . 0 5 2 < 0 . 05 2 Two-tailed, using Student's t-test. 4 Separate variance estimate (P ratio significant, p<0.05) Pooled variance estimate (F ratio not significant, p>0 .05 ) - k9 -Subjects using oral contraceptives were separated into groups depen-ding 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 cell folate levels increase. However, the rise in serum folate is not statistically significant, and the increase in red cell folate is significant between trimesters 1 and 2 , and trimesters 1 and 3 » but not between trimesters 2 and 3 « Therefore the greatest increase in red cell folate in this group of women occurred before the third trimester. The correlation between serum folate and red cell 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 in Women Using Different Brands of Oral Contraceptives Oral Contraceptive Agent Subject Serum Folate (ng/ml) Day 5 Day 20 Overall Mean Ortho-Novum 1/50 (N orethinodrone, 1.0 mg) (Mestranol, 0.05 mg) A.T. J.M. J.P. N.B. CH. M.D. K.H. CM. S.M. Mean + S.;D.' 9.15 1.90 9.23 7.23 4.20 5.63 6.03 10.95 5.30 6.62 + 2.82 9.53 3.96 8.85 7.08 2.85 6.80 4.18 12.25 7.50 7.00 ± 3.00 9.34 2.93 9.04 7.15 3.53 6.21 5.11 11.60 6.40 6.81 + 2.82 Ovral (Norgestrel, 0.50mg) (Ethinyl estradiol, 0.05 L.H. J.T. CO. C.S. A.C L.P. W.B. P.M. Mean + S.D. 4.35 5.65 3.80 4.78 4.53 4.07 4.85 3.70 4.47 + 0.64 5.98 5.40 5.25 5.08 4.90 6.80 4.05 , 5.85 5.41 + 0.82 Norlestrln (Norethindrone acetate, 2.5 mg) (Ethinylestradiol, 0.05 mg) V.B. CM. 4.69 3.68 5.70 3.90 Ortho-Novum 1/80 (Norethindrone, 1.0 mg) (Mestranol, 0.08 H.R. 5.65 10.00 5.16 5.53 4.53 4.93 4.71 5.43 4.45 4.78 4.94 + 0.40 5.20 3.79 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 estradiol, 0.05 mg) B.M. 3.25 3.60 3.43 Table 6 Differences in Serum Folate and Red Cell Folate Concentrations Measured at Each Trimester of Pregnancy (K = 8) Trimester Serum Folate (ng/ml) Mean + S. D. Red Cell Folate (ng/ml) ; Mean + S.D. Pearson Correlation Coefficient between Serum and Red Cell Folate 1 5.76 + 2.66 167.6 + 44.8 0.5972* 2 5 . i i ± 1.881 268.9 + 82.4'^  0 . 0 6 4 6 * 3 8.71 + 7 . 2 1 1 ' 2 327.9 + 168.72'3 0.75^4 Not significantly different from folate level in 1 trimester (p>0,05). 2Not significantly different from folate level in 2 n d trimester (p>0.05). Significantly different from folate level in 1 s t trimester (p<0.05). *T>0.05 Table ? Differences in Hemoglobin, MCV, Serum Iron, and Total Iron Binding Capacity-Measured at Each Trimester of Pregnancy (N «* 8) Trimester Hemoglobin (g/lOOml) Mean + S.D. MCV (yU3) Mean + S.D. Serum Iron (/*g/dl) Mean ± S.D. T.I.B.C. (yug/dl) Mean + S.D. 1 11.8 ± 1.0 87.5 + 3.3 97.6 + 29.4 335. ? ±61.9 2 11.5 + 0.81 89.0 ± 2.83 101.9 ± 24.51 ^08.7 ± 35.63 3 1 4 12.0 + Q.81'^ 88.3 ± 3.31'2 104.0 ± 48.21'2 463.4 ± 38.53'^ Not significantly different from value in 1 trimester (p>0.05), ^ o t significantly different from value in 2 n d trimester (p>0.05). -^Significantly different from value in l ? t trimester (p<0.05). Significantly different from value in 2^  trimester (p<0.05). - 53 -These findings indicate that, whereas the volume of red blood c e l l s showed the greatest increase before the third trimester, the hemoglobin concen-tration did not rise significantly u n t i l the last trimester. There was a s t a t i s t i c a l l y significant increase i n total iron binding capacity throughout the course of pregnancy; serum iron increased during pregnancy, but not to a s t a t i s t i c a l l y significant degree. The result was a net increase in the unsaturated iron binding capacity as pregnancy progressed. The raw data, t-values, and levels of significance for a l l the hemotological variables 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 significant have been included in the results section. Comparison of pregnant subjects with controls and oral contracep-tive users. Table 8 outlines the results of t-test comparisons for serum folate, red c e l l folate, serum iron, and total iron binding capacity among the three groups of subjects. In the f i r s t trimester of pregnancy there were no significant differences i n any of these parameters when compared with controls or oral contraceptive users. The pregnant women, at this point in pregnancy, appeared comparable to non-pregnant women with regard to the parameters tested. During the second trimester, pregnant subjects had serum folate concentrations which were not significantly different from those of oral contraceptive users, but which were s t a t i s t i c a l l y lower (p<0.05) than those of control women. However, by the l a s t trimester, serum folate values had increased i n the pregnant women and were no longer different from the control levels. During both the second and third trimesters pregnant women demon-strated higher red c e l l folate concentrations than either 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 Pregnant Women Mean ± S.D. Mean ± S.D. Mean ± S.D. ' st 1 v Trimester 2n d Trimester 3 r d Trimester Serum Folate (ng/ml) 7.84 ±4 .04 5.64 ±2.20 5 . 7 6 ± 2 . 6 61 * * 2 + t 5 . U ± 1 . 8 8 3 * 2 t t 8.71 + 7.211*21" Red Cell Folate (ng/ml) 204.9 + 71.8 184.1 ±71 .7 I 6 7 i 6 ± 4 4 . 8l * * 2 1 t 2 6 8 . 9 ± 8 2 . 4 1 ^ t t 327.9± 168.71*Z4't Serum Iron (>A€/dl) - 84.0 + 22.8 100.7 ±29.6 85.4 + 44.01* 2 t t 102.7 ± 22. S1**2"^ 107.9 ± 4 5 . 9 * * 2 t t T.I.B.C. (/Ag/dl) 327.6 ± 7 3 . 5 382.5 ±65.7 335.7±6l.91**2 t t 408 .9±35.6 3 * * 2 t t k6 3.4 + 3 8 . 5 3 * * W ANot significantly different from control value (p>0.05). 2Not significantly different from O.C. users value (p>0,05). 3Significantly 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. - 55 -or oral contraceptive users. These differences were only significant "between the pregnant women and p i l l users, however,; The difference "between preg-nant and control values was not statistically significant. Serum iron levels were higher among the pregnant subjects in the last two trimesters than in 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 cell 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 contracep- tive users, and pregnant subjects. As demonstrated in Table 9» no statis-tically 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 Variable Group Mean + S.D. Analysis of Variance Contrast Coefficient Matrix F-ratio * P ** t * P Free Folate Intake (yAg/day) Controls (N - 18) O.C. Users (N - 21) Pregnant (N « 8) 171.7 + 81.01 150.5 ± 60.31 165.6 + 80.92 0.436 0.639 . -,0.161 0.873 Free Folate Intake (^g/day) Controls (N = 18) O.C. Users (N - 21) Pregnant (N = 8) 300.8 + 97.1 272.1 + 79.6 315.8 + 99.8 0.870 0.429 -0.838 0.406 * Two-tailed. ** 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. It i s of interest to note that in 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 cell  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 cell folate with free and total folate intake. The slgnificaiicevof'the difference between the correlation coef-ficients of controls and oral contraceptive;users was figured using Fisher's z-transformationj the results of this test are shown in Table 10. We found a consistently higher correlation between the serum folate levels and dietary folate intake in 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 in correla-tion coefficients between these two groups was only statistically signi-ficant 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 cell folate levels and dietary folate intake was not of statistical 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 Day of Cycle Pearson Correlation Coefficient P* Controls (N - 18) O.C. Users (N - 21) Serum Folate 5 0.?208 -0.0229 .0072 and Free Folate Intake 20 o.6727 0.2155 .0872 Serum Folate 5 0.6189 -0.0832 .0198 and Total Folate Intake 20 0.5850 0.1741 .1528 Red Cell Folate 5 0.2302 - 0.1907 .2150 and Free Folate Intake 20 0.0719 -0.2157 .4010 Red Cell Folate 5 0.1122 - 0.1384 .4654 and Total Folate Intake 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 cor-relation between serum folate and dietary folate between the f irst and third trimesters of pregnancy. These differences were not statistically signi-ficant, but nevertheless there was a higher correlation in the f irst t r i -mester. Again, there was no comparable relationship found between red cel l folate levels and dietary folate intake in the pregnant women. In a l l groups of subjects there was a consistently stronger corre-lation between free folate intake and serum or red cel l folate than there was between total folate intake and serum or red cel l folate. - 59 -Table 11 Differences between First 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 P* 1 s t Trimester 3 r d Trimester 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 for free and total folate for each food sample are listed in Appen-dix K. The results of the Spearman's Rank Order Test between the calcu-lated 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 al. (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 in this study. It was found that the ratio of free folate to total folate was i t 1.77 for both the assayed and calculated folate values. Table 12 Spearman's Rank Order Test of Correlation Between Calculated and Assayed Values of Folate for Food Samples Collected at the Time of Consumption Variable Pair Spearman's Correlation Coefficients N - 15 N e* 13* Free Folate Calculated Value 0.67141 0.49452 vs. Assayed Value Total Folate 4 0.62642 Calculated Value 0.7571 vs. Assayed Value •Values for food samples containing l i v e r omitted. p< 0 . 0 1 2 P< 0 . 0 5 - 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 contra-ceptive 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)» al 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 factors other than oral contraceptives which are known to influence folate status. A criticism of the study by Stephens et a l . (1972) i s that there was l i t t l e information given regarding the sample of women studied or their dietary folate intake. Paine et a l . (1975) also f a i l e d to examine the dietary practices of their subjects. Although our findings indicate that serum folate was not directly correlated with dietary folate intake in oral contraceptive users, nevertheless, dietary folate w i l l directly influence the serum folate levels of control women. For this reason folate intake must be considered an important variable. In the study by Paine et a l . (1975) a s well as that of Prasad et a l . (1975) the authors did not specify whether their determinations for serum folate were made on fasting blood samples, and this may have skewed their results, folate-rich foods are known to elevate the level of serum folate for several hours post-prandial, Prasad et a l . did, however, make a contribution towards clarifying the ambiguous findings in this area. They made-a distinction among their subjects regarding socio-economic status, and found that those women in the lower socio-economic group had significantly lower serum and red c e l l folate concentrations than those from the higher socio-economic group. This finding implicates socio-economic status as a factor which may influence folate status. It may be that what i s found to be true for one sample population cannot be extrapolated to another population of women. Factors such as the socio-economic status, dietary habits, ethnic custom, age, and parity of the subjects, as well as the seasonal a v a i l a b i l i t y of folate-rich roods, have been suggested as determinants of folate status. These should be controlled for as far 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 (all 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. This study was also some-what limited in that i t was a cross-sectional survey. It 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 is at the level of folate absorption in the intestine or uptake by the cells remains a matter of speculation. If folate absorption is 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 is the polyglutamate forms of folate 1 which are poorly absorbed, as sug-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 folic 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 respons-ible for cellular uptake rather than serum transport of folates (Waxman and Schreiber, 1974). Therefore, Shojania et al. (1975) have suggested that i t may account for the increased rate of plasma folate clearance found in oral contraceptive users. The induction of this FABP by both oral contraceptive therapy and pregnancy implies some hormonal influence common to both situa-tions. 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 fluc-tuation of circulating hormone levels in the menstrual cycle, in either the controls or oral contraceptive users. This i s in agreement with the findings of Stephens et al. (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 is most pronounced when endogenous secretion or exogenous intake of sex steroids was the lowest is difficult 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 itself exerts some as yet undefined effect on serum folate levels, although this remains speculative. - 65 -Among the oral contraceptive subjects in 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 al. (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 statistical significance. These oral contraceptive preparations differ 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 cell folate concentrations. This i s in contrast to the work of Prasad et al. (1975) and Smith et al. (1975). Prasad et al. examined the folate status of women from a high socioeconomic group and found oral contraceptive users to have red cell folate levels that were significantly lower than controls; there was no comparable difference among women from a low socio-economic group. The authors offered no explanation for these results. Smith et al. 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 cell 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 cell 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 al., 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 cell count among oral contraceptive users in this study has been previously associated with steroid therapy (Beck, 1973)• It cannot be taken as an indicator of pathological disturbance since leukocy-tosis is 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 statis-tically 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. It 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 is commonly associated with pregnancy, i t is 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 cell folate among our pregnant subjects as pregnancy progressed is more difficult to explain. The fact that the preg-nant women shovred an-increase over time in correlation between serum folate and red cell 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 likely hypothesis is 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, of-course, 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 is evidence that a defic-iency of iron may increase the folate requirement (Velez et al , , 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 cell folate in the pregnant - 68 -subjects does not substantiate this hypothesis. The relationship between iron balance and folate status should be more thoroughly examined. It 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 is 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 is assumed that a low serum folate level i s indicative of marginal folate status and a low red cell folate level is 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 folic acid may be epidemiological support for this finding. The increase, as pregnancy progressed, in hemoglobin concentration, serum iron, and MCV is 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 is 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 calcu-lated 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 in close agreement with that obtained by Moscovitch and Cooper (1973) - 69 -In a similar study. This finding lends confidence to the accuracy of our estimations of dietary folate intake calculated from food tables. I t also supports the assumption that diet records were a reliable method of measuring folate intake in the subjects who participated i n this study. Nevertheless, there i s s t i l l strength in the argument that estimation of folate intake as free and total folate i s not indicative of the actual biological a v a i l a b i l i t y of dietary folates. Among a l l three groups of subjects tested, free folate intake was consistently more highly correlated with serum and red c e l l folate levels than was total folate intake. This substantiates the hypothesis of Herbert in 1963 that free folate may be the more accurate measure of available folate. More sophisticated studies involving the assess-ment of biological a v a i l a b i l i t y of food folates appear necessary. The early work of Herbert (I962) indicated that the serum folate level i s very sensitive to changes in the f o l i c acid intake. Hansen (1968) also suggested that serum folate reflects the balance between the daily intake and plasma turnover of f o l i c acid. Although the dietary intake of folate was below the Canadian Recommended Daily Allowance in a l l our groups of subjects, this level of consumption was apparently adequate for the maintenance of normal serum folate levels among the control women and the pregnant women studied. Among the subjects using oral contraceptives, however, the same level of dietary folate appears to be inadequate to sustain their serum folate at concentrations comparable to those in controls. Although the women on oral contraceptives did not demonstrate d e f i -cient concentrations of serum folate, these findings may well imply that they are at risk with respect to folate. Should their dietary folate intake be even more restricted, or should there be some additional demand on their folate stores, their chances of developing a folate deficiency would be - 70 -further increased. Women under oral contraceptive therapy would be prudent to take in the fu l l RDA of 200 yug free folate daily. This practice may be sufficient to insure maintenance of normal serum folate levels in oral contra-ceptive users who are in good health. It should be pointed out, however, that there is no evidence that the quantity of dietary folate required to maintain a certain serum folate level in women taking oral contraceptives is greater than in women not using the: p i l l . The question of folic 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. 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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. Irish J. Med. Sci. I43t 3-20, 1974. 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. Intestinal Conversion of Folinic Acid to 5-Methyltetrahydrofolate in Man. Brit. J. Haematol. 221 63-72, 1972. 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 nutritional study of women who are pregnant, or oral contraceptive users, and agree to participate in the described study. 1 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 in confidence 3. I have the right to withdraw from the study at any time. Signature - 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 will 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 pi 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 preg-nant 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 will you need to do? If you are interested in participating f i r s t f i l l out the Preli-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 will then keep a dietary record for 7 days (you will be instructed in how to do this) and will be asked to give a series of blood samples. Women who are in the control group or the oral contraceptive group will give four blood samples over a period of two months} the samples will be taken at times to be determined according to each woman's individual menstrual cycle. Pregnant women will be asked to give three blood samples, one each In their 3rd, 5th arid 8th months. The amount of blood taken will 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 is done for 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 will however let you know the outcome of our research when i t is completed. Your attention and consideration is appreciated. We can't do i t without you. If you have any questions please feel free to call me. Sincerely, Jean Pietarinen - 81 -APPENDIX C. PRELIMINARY QUESTIONNAIRE This questionnaire is designed to obtain information about you which we will 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. All information will be held in confidence. Please answer a l l questions as fully as you can, ********* Date Name Address Phone at home When is the best time to call 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 it? ' 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 pills? 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 is 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) When? Are you now pregnant? How many months? Have you ever been pregnant before? Please l i s t each pregnancy by year, noting live 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. Ovral 9 V.B. Norlestrin 9 C.S. Ovral 10 P.M. Ovral 12 A.C. Ovral 15 S.M. Ortho-Novum l/50 15 W.B. Ovral 16 CM. Norlestrin 17 K.H. Ortho-Novum I/50 18 CH. Ortho-Novum 1/50 18 L.P. Ovral 19 J.P. Ortho-Novum 1/50 22 B. M. Demulen 23 M.D. Ortho-Novum 1/50 36 J.T. Ovral 36 J.M. Ortho-Novum 1/50 36 N.B. Ortho-Novum 1/50 36 C.O. Ovral 60 H.R. Ortho-Novum 1/80 96 C. M. Norlestrin 120 - 84 -APPENDIX E. Schedule for Collecting Blood Samples from Pregnant Subjects Week of Pregnancy when Blood was Drawn Subject 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* Second >Day Record** Free Folate Intake y"g/day) Total Folate Intakes (wg/day) Free Folate Intake (^ g/day) Total Folate Intake C*g/day) Oral A.T. 100.13 223.78 66.55 134.39 Contra-ceptive L.H. 237.58 471.79 252.95 430.54 Users H.R. 148.63 267.95 104.68 241.69 (N - 21) J.M. 95.02 228.35 67.72 152.01 J.T. 132.28 250.07 198.62 379.85 J.P. 101.61 192.98 296.55 407.79 N.B. 191.33 321.00 159.07 324.49 C.H. 225.40 383.88 87.25 174.43 CO. 101.63 169.52 75.42 162.88 V.B. 70.32 167.50 57.50 148.52 B.M. 161.66 280.87 194.50 356.53 M.S. 149.52 282.77 176.00 322.13 CS. 199.22 329.34 342.07 415.61 A.C 258.0? 378.85 275.08 400.10 L.P. 73.72 141.73 119.95 184.70 CM. 184.33 314.47 80.37 200.58 W.B. 75.67 181.83 105.71 179.66 K.H. 230.45 378.75 70.00 202.39 CM. 180.69 269.04 200.13 341.98 P.M. 98.01 214.45 130.17 274.05 S.M. 96.23 232.52 147.33 312.43 Mean+S. D. 148.2+ 59.8 270.5± 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 -Group Subject , First 3-Day Record* Second 3-Day Record** Free Folate Intake ^g/day) 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. 175.9+108.8 299.5±H9.9 167.5± 81.4 302.0+104.4 Pregnant Women J.S. 106.55 288.83 93.12 261.03 (N - 8) 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+ 59.0 317.3+ 81.1 - 87 -APPENDIX G. Serum Folate Concentrations of Each Subject Group Subject Serum Folate (ng/ml) , Day 5 Day 20 Overall Mean Oral . A.T. 9.15 9. 53 9.3k Contraceptive • Users' L.H. 4.35 5.98 5.16 (N = 22) H.R. 5.65 10.00 7.83 J. M. 1.90 3.96 2.93 J.T. 5.65 5.40 5.53 J.P. 9.23 8.85 9.04 N.B. 7.23 7.08 7.15 C.H. 4.20 2.85 3.53 CO. 3.80 5.25 4.53 V.B. 4.69 5.70 5.20 M.oD. 5.63 6.80 6.21 B.M. 3.25 3.60 3.^ 3 M.S. 2.79 3.14 2.96 c.s. 4.78 5.08 4.93 A.C. 4.53 4.90 4.71 L.P. 4.07 6.80 5.^ 3 CM. 3.68 3.90 3.79 W.B. 4.85 4.05 4.45 K.H. 6.03 4.18 5.11 CM. 10.95 12.25 11.60 • P.M. 3.70 5.85 4.78 S.M. 5-30 7.50 6.40 Mean+S. D. 5.3k +2.20 5.93 + 2.46 5.64 + 2.20 (continued) - 88 -Group Subject Serum Folate (ng/ml) Day 5 Day 20 Overall Mean Controls E.S. 13.45 10.33 11.89 , (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 Mean+S.D. 7.54 + 3.58 ! 8.14 + 4.97 7.84 + 4.04 1st Trimester 2nd Trimester 3rd Trimester Pregnant Women J.S. F.K. 3.25 11.00 3.60 6.70 7.45 5.90 (N = 8) 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. 5.76 + 2.66 5.11 ± 1 . 8 8 8.71 ± 7.21 - 89 -APPENDIX H. Red Cell Folate Concentrations of Each Subject Group Subject Red Cell Folate (ng/ml) Day 5 Day 20 Overall Mean Oral A.T. 315.9 242.3 279.1 Contraceptive Users L.H. 158.2 157.4 157.8 (N m 22) 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 + 72.2 184.1 + 71.7 (continued) - 9 0 -Group Subject Red Cell Folate (ng/ml) Day 5 Day 20 Overall Mean Controls E.S. 256.3 195.2 225.7 (N - 18) J.T. 201.7 143.1 172.4 B.J. 141.1 119.0 130.0 G.B. 283.1 313.8 298.5 J.P. 174.5 188.3 181.4 K.M. 125.0 133.6 129.3 C.J. 174.7 140.0 157.3 s.v. 279.6 280.7 280.2 E.L. 335.9 253.7 294.8 P.F. 206.4 201.3 203.8 J.H. 172.6 171.6 172.1 B.L. 178.3 180.3 179.3 V.M. 166.0 155.4 160.7 L.M. 84.4 87.0 85.7 M.P. 336.7 296.0 316.3 S.R. 268.2 294.0 281.1 J.S. 130.3 126.3 128.3 S.W. 267.5 315.0 291.2 Mean+S.D. 210.1 + 73.6 199.7 ± 74.0 204.9 ± 71.8 1st Trimester 4 2nd Trimester 3rd Trimester Pregnant Women (N = 8) J• s# F. K. G. B. 116.6 235.2 180.4 96.5 198.5 277.7 73.3 276.8 364.1 S.M. 103.6 298.0 307.7 V.T. 156.3 310.3 323.9 L.D. 194.6 35L7 350.2 J.M. 148.7 318.2 679.0 A.S. 205.0 300.4 248.1 Mean+S.D. I67.6 +44.8 268.9 ±82.4 327.9 ± 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 Day of Cycle Control Group Oral Contraceptive Group 1. P Mean + S.D. ' Mean :+,S.D. Serum Folate (ng/ml) 5 20 7.54 + 3.58 8.14 + 4.97 5.3k + 2.20 5.93 ± 2.46 <0 .05 N.S. P N.S. N.S., Red Cell Folate (ng/ml) 5 20 210.1 + 73.6 199.7 ± 74.0 184.2 + 74.1 184.0 + 72.2 N.S. N.S. P N.S. N.S. Hemoglobin (g/lOOml) 5 20 13.0 + 0.7 13.2 + 0.5 13.4 + 0.8 13.3 ± 0.6 N.S. N.S. P N.S. N.S. Hematocrit ( % ) 5 20 38.3 + 2.3 38.6 + 1.8 39.2 + 2.0 38.9 ± 1.7 N.S. N.S. P N.S. . N.S. V White Cell Count (x 103) 5 20 4.6 + 0.8 4.9 + 1.1 6.1 + 1.4 7.1 + 1.9 <0.01 <0.01 P N.S. <0.01 Red Cell Count (x 106) 5 20 4.44 + 0.23 4.48 + 0.19 4.50 + 0.28 4.43 + 0.27 N.S. N.S. P N.S. N.S. M.C.V. (^3) 5 20 P 86.1 +2.6 86.0 + 2.2 N.S. 87.5 + 3.8 88.0 + 3.8 N.S. N.S. <o.05 M.C.H. (/*/*s) 5 20 29.3 + 0.9 29.4 + 1.0 29.7 + 1.4 30.0 + 1.4 N.S. N.S. P N.S. N.S. - 92 -APPENDIX I. (continued) Variable Day of Cycle Control Group Oral Contraceptive Group P Mean + S.D. Mean + S.D. M. G.H. G. ( % ) 5 20 33.7 ± 0.7 33.5 ± 0 . 7 33.4 + 0.7 33.6 + 0 . 6 N.S. N.S. P N.S. N.S. Serum Iron ( / A g/dl) 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 . 0 5 Unsaturated Iron Binding Capacity (/*g/dl) 243.6 + 76.8 281.9 ± 78.2 N.S. Free Folate Intake (/Ug/day) 5 20 P 175.9 + 108.8 167.5 ± 81.4 N.S. 148.2 + 59.8 152.7 ± 83.9 N.S. N.S. N.S. Total Folate Intake (/*g/day) 5 20 P 299.5 + H 9 . 9 302.0 + 104.4 N.S. 270.5 + 85.2 273.7 ± 102.0 N.S. N.S. N.S. N.S. - Not Significant, P > 0 . 0 5 - 93 -APPENDIX J. Differences i n Hematological and Dietary Parameters Measured During Each Trimester of Pregnancy Variable Mean + S.D. 1st Trimester 2nd Trimester 3rd Trimester Serum Folate (ng/ml) 5.76 + 2.66 5.11 ±1.88 3 8.71 ± 7.21 3 , 4 Red Cell Folate (ng/ml) 167.6 +44.8 268.9 ±82.4 1 327.9 ± 168.7 ^ Hemoglobin (g/lOOml) 11.8 + 1.0 11.5 ± 0.8 3 12.0 + 0.8 2 , 3 Hematocrit ( % ) 34.3 ± 2.9 33.9 ± 2.0 3 34.7 ± 2.6 3'^ White Cell Count (x 103) 7.9 ±2.0 8.9 ± 2.0 1 9.0 + 1.9 ^ Red Cell .Count (x io6) 3.93 ± 0.40 3.78 ±0.31 1 3.93 ± 0.34 M.C.V. 87.5 ± 3.3 89.0 + 2.8 1 88.3 ± 3.3 3 , k M.C.H. S) 30.1 + 1.0 3O.3 ± 0.9 3 30.5 + 1.5 3' k M.C.H.C. (%) 33. a + 0.6 33.6 ± 0.6 3 33.8 ±0.7 Serum Iron (/^g/dl) 97.6 + 29.4 101.9 ± 24.5 3 104.0 + 48.2 Total Iron Binding Capacity (/*g/cU) 335.7 ±61.9 408.9 ± 35.6 1 463.4 + 38.5 1 , 2 Unsaturated Iron Binding Capacity (/*g/dl) 225.9 ± 69.5 309.3 ± 40.8 1 362.3 + 63.5 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 Significantly (P< 0.05) different from value in 1st trimester; 2nd trimester. -'Not significantly (P>0.05) different from 1st trimester; ^ d trimester. - 94 -APPENDIX K. Calculated and Assayed Values for Folate for Duplicate Food Samples Collected at the Time of Consumption Sample No. Values Calculated from Food Tables Assayed Values Free Folate (/*«) Total 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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