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The effect of oral contraceptives on folate metabolism
The effect of oral contraceptives on folate metabolism A.
MAJID
GLORIA
SHOJANIA, J.
PHILIP
H.
Winnipeg,
Manitoba,
M.D
HORNADY, BARNES,
R.T. M.D
Canada
We have studied the eBect of oral contraceptives on folate metabolism, in order to test the hypothesis that folate deficiency of pregnant women may be partly due to impairment of folate metabolism. Serum folate, red cell folate, and urinary excretion of formiminoglutamic acid (FIGLU) were determined in 176 women receiving combination-type oral contraceptives (the Pill) and 140 normalcontrol subjects. The group taking the Pill had significantly lower serum and red cell folate and higher urinary FIGLU excretion. There was a rise in serum and red cell folate and a fall of urinary FIGLU excretion within 3 months after stopping the Pill. The data demonstrate that women taking the Pill may have impaired folate metabolism and suggest that sex hormones may play a role in the development of folate dejiciency in pregnancy.
DEFICIENCY iscommonlyseen during pregnancy, and the topic has been recently reviewed. 1 This folate deficiency is mainly attributed to parasitization by the fetus.2l 3 However, other associated factors such as impaired folate absorption in pregnancy4 and impaired folate metabolism596 have been considered as contributory factors. If the latter two factors play a significant role in the development of folate deficiency of pregnant women, one would expect to see a similar effect in women taking oral contraceptives which are known to produce a
“pseudopregnancy state.” In a preliminary study,’ we determined the serum folate levels (SF) in a group of women receiving oral contraceptives (the Pill) and compared them to those of normal control subjectsand found that the women taking the Pill had significantly lower serum folate- values than the control group. This led us to undertake a more detailed study of the effect of oral contraceptives on folate metabolism. The present paper is the first part of the report which indicates that, by all criteria, women taking the Pill have impaired folate metabolism.
FOLATE
From the Department of Hematology, St. Boniface General Hospital, the Department of Pediatrics and the Department of Obstetrics and Gynecology, University of Manitoba.
Materials
and
methods
Subjects. The subjects studied were the women who were taking a combination-type oraI contraceptive and were attending the private clinic of an obstetrician and gynecologist (P. H. B.) . Two groups of control subjects were used. One group was from the same clinic (private clinic control), and the other group was made up of female hospital personne1 (hospital controls) . Control sub-
These studies were supported in part by grants from the National Health Research of Canada (606-7-189), Ortho Pharmaceutical (Canada),. Ltd., and the Medical Research Councal of Canada (MA-3069). Presented in part at the XIXIth International Congress of Hematology, Munich, August 2-8, 1970.
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jects had not been taking oral contraceptives for at least a year and were of reproductive age. All subjects were interviewed to obtain information about diet, intake of vitamins or other medications, and the presence of diseasesthat may affect folate metabolism. Subjects who had received folic acid therapy at any time in the past or had been delivered of infants within a year prior to this study or were on a diet to prevent weight gain were excluded. No subjects were excluded from the control group because of poor dietary folate intake. However, 4 women who had low serum folate and poor dietary folate intake, one who had low serum folate and was found to have a mild malabsorption syndrome, and one who had a high urinary formiminoglutamic acid (FIGLU) excretion but had high serum glutamic oxaloacetic acid (SGOT) and bromsulphalein (BSP) retention were excluded from the Pill group. Subjects from the private clinic were generally of good or moderate income and did not include indigent patients. Originally, blood samples were taken for serum folate only, but later on, when a more detailed study was planned, further investigations were made. These included red cell folate (RF), urinary excretion of FIGLU and folate, hemoglobin, hematocrit, blood smear morphology, reticulocyte count, liver function tests (serum bilirubin, SGOT, serum alkaline phosphatase, cephaline flocculation, thymol turbidity, and BSP retention), and serum vitamin B,,. Techniques. Serum folate was determined by the L. casei microbiological assay of Waters and Mollin* with minor modifications. All serums were kept undiluted at -20° F. and were reconstituted with L. ascorbic acid (5 mg. per milliliter) on the day of assay. Blood samples for red cell folate determination were collected in tubes containing ethylenediaminetetra-acetic acid. The blood was then diluted 1: 10 in deionized water and kept frozen at -20° F. until the day of assay when it was reconstituted with L. ascorbic acid (5 mg. per milliliter of diluted blood) and assayed as serum. In our laboratory, the normal se-
Oral
contraceptive
effect on folate
metabolism
783
rum folate range is 3 to 15 ng. per milliliter, and normal red cell folate is 100 to 500 ng. per milliliter. The reproducibility of serum folate in the same assay was determined in 9 replicates of 3 serums with mean serum folate values of 7, 8.3, and 14.1 ng. per milliter. The coefficients of variability were 2.2, 3, and 6 per cent, respectively. The variation between duplicate samples determined on two different days was determined in 45 duplicate samples.The per cent variation from the mean of the duplicates was 0 to 19 per cent (mean = 7.3 per cent). A pooled sample of serum which was stored at -20° F. and reassayed 47 times during 10 months of storage showed no detectable loss of folate activity and no more variation than that seen between duplicate samples (the mean serum folate of the pooled serum was 5 ng. per milliliter and the coefficient of variability was 12.7 per cent). Urinary FIGLU was determined in a 12 hour urine sample following histidine loading (15 Gm. of histidine monohydrochloride). FIGLU was determined by an enzymatic method with the use of a FIGLU test kit (Sigma Chemical Co., St. Louis, Missouri), which is based on the method of Tabor and Wyngarden.O The variations between the duplicate samplesassayedon two different days were determined in 9 samples. The per cent variation from the mean of the duplicates ranged between 0.2 and 20 per cent (mean 6 per cent). Results
Effect of the Pill on serum folate. Serum folate was determined in 176 women taking the Pill, 39 private clinic control subjects, and 101 hospital control subjects of similar sex and ages. Fig. 1 shows the serum folate of these groups. Only one serum folate is presented for each subject. In caseswhere more than one serum folate was available for each subject, the latest serum folate representing the longest duration of oral contraceptive therapy is shown on the figure. The mean + S.D. for private clinic control subjects is 6.1 + 2.23 ng. per milliliter, which
784
Shojania,
Hornady,
and
November 15, 1971 Am. J. Obstet. Gynecol.
Barnes
PRIVATE CLINIC ON ORAL CONTRACEPTIVES.
IL'
.
1
0
1 DURATION
.
2 3 (YEARS)
4
$5
Fig.
1. The serum folate of 176 women taking oral contraceptives, 39 control subjects from the same clinic, and 101 control subjects from hospital personnel. The solid horizontal lines represent the means of Pill and control groups. The dotted lines represent the means of subgroups (control groups according to their source and Pill groups according to the duration of oral contraceptive therapy).
is not significantly different from those of hospital control subjects (6.3 2 2.6 ng. per milliliter). Although the private clinic control subjects have slightly Iower mean serum folate than the hospital control subjects, this does not mean that they have worse folate intake or stores than the hospital control group. Not +only is the difference between the two means not statistically significant, but, actually, there are fewer subnormal serum folates in the private clinic control subjects than in the hospital control subjects (2.5 per cent of private clinic control subjects and 5.9 per cent of hospital control subjects had serum folate levels of less than 3 ng. per milliliter). The mean + S.D. for 176 women on the Pill is 4.2 t 1.77 ng. per milliliter which is significantly lower than that of either the private clinic control subjects or the hospital control subjects (P < 0.001 with the use of Student’s t test). Similarly, in the frequency distribution curve the mode for the group who were taking the Pill for more than one year is between 3 and 4 ng. per milliliter which is lower than
the mode for control subjects (5 to 6 ng. per milliliter.) The effect of duration of oral contraceptive therapy on serum foIate. In order to study the relation of the duration of oral contraceptive therapy to the serum folate, the group taking the Pill was divided into 5 groups according to the duration of oral contraceptive therapy. Fig. 1 and Table I show that the mean serum folate falls with the continuation of oral contraceptives. An analysis of variance shows that each of the 5 groups taking the Pill has a significantly lower serum folate than the control group from the same clinic (with the use of oneway analysis of variance and test of the least significant difference). Table I also shows that those who have been taking the Pill for more than two years have a significantly lower mean serum folate than those who have been taking the Pill for less than a year (with the use of the test of the least significant difference). Similarly, it can be seen that not only the mean serum folate falls but that the percentage of subjects with
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contraceptive
effect on folate metabolism
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Table I. Changes in mean serum folate and percentage of subjects with low serum folate according to the duration of oral contraceptive therapy. All subjects were from the same clinic attending one physician (P. H. B.)
I Control I %--I>
Serum folate mean + S.D. (ng./ml.)
>4
39 6.1 _+2.2
5.0 ?r 2.0
4.3 + 1.7
4.1 2 1.9
3.7 2 1.0
3.8 211.7
< 0.02
< 0.001
< 0.001
< 0.001
< 0.001
< 0.1
< 0.05
< 0.01
< 0.01
27
32
42
9 -
low serum folate (less than 3 ng. per milliliter) increases with the duration of oral contraceptive therapy. Only 2 per cent of the control group had low serum folate (less than 3 ng. per milliliter) whereas 9 per cent of the group who were taking the Pill for lessthan one year, 21 per cent of the group who were taking the Pill for 1 to 2 years, 27 per cent of the group who were taking the Pill for 2 to 3 years, 32 per cent of the group who were taking the Pill for 3 to 4 years, and 42 per cent of the group who were taking the Pill for more than 4 years had a serum folate of lessthan 3 ng. per milliliter. The rise in percentage of subnormal folate with the duration of oral contraceptive therapy is statistically significant (P < 0.01 with the useof the chi-square test). The effect of the dose of oral contraceptives on serum folate. Among the group taking the Pill, there were 39 women who were taking the Pill containing mestranol, 0.05 mg., + norethindrone, 1 mg.*t The mean serum folate (+ SD.) for this group is 4.4 t 1.87 ng. per milliliter which is significantly lower than that of control subjects (P < 0.001 with the use of the Student’s t test). There were 42 women who were taking
Laboratories,
1
6 33
2
Pharm.
1 3-4> 5 28
< 0.02
Ortho
1 2-3>
(year)
4 36
Percentage with low S.F. (less than 3)
‘Ortho-Novum I, Jersey. +Norinyl 1, Syntex fornia.
contraceptives
3 47
P vs. Group 1 P vs. Group 2
1 I-2>
of oral
2 32
I
No. of patients
Duration
Corp., Inc.,
Raritan, Palo
Alto,
21
preparations* which contain twice the amount of mestranol and norethindrone previously mentioned. The mean serum folate (+ SD.) for this group is 3.8 + 1.6. Although this mean is lower than that of the group receiving the small amounts of mestranol and norethindrone, with the number of subjects studied the difference is not statistically significant. The effect of oral contraceptives on red cell folate. Red cell folate was determined in 95 women who were taking the Pill for more than one year (private clinic) and 63 control subjects (private and hospital control subjects). The mean + S.D. for the group taking the Pill is 130 + 62 ng. per milliliter which is significantly lower than 175 + 69 ng. per milliliter for the control group (P < 0.001 with the use of Student’s t test). (The mean RF for 10 private clinic control subjects was 176 ng. per milliliter, and that of 53 control subjects was 174 ng. per milliliter.) The effect of oral contraceptives on winary FIGLU excretion. Urinary FIGLU excretion was determined in 58 women taking the Pill and 35 control subjects (private and hospital control subjects). The mean + S.D. for the Pill group is 91 + 84 PM per 12 hr. which is significantly higher than 51
New Cali-
‘Norinyl 2, Syntex Laboratories, Inc. Palo Alto, fornia. OrtheNovum II, Ortho Pharm. Corp., Raritan, Jersey.
CaliNew
786
Shojania,
Hornady,
and Barnes
tl PILL
(NO
~C~NTR~L
URINARY
FIGLU
The effect of the oral contraceptives on hemoglobin and hematocrit. Hemoglobin and hematocrit were determined in 123 women taking the Pill and 71 control subjects. The mean hemoglobin (f SD.) for the Pill group is 13.2 + 0.95 Gm. per cent which is not significantly different from that of the control subjects (13.0 + 0.83 Gm. per cent). Similarly, the mean hematocrit it S.D. for the Pill group is 39.8 + 2.94 per cent which is not significantly different from 39.8 + 2.69 per cent for the control group. Among the women receiving oral contraceptives, there was no significant difference between the hemoglobin and hematocrit of those who had subnormal serum folate (less than 3 ng. per milliliter) and those who had normal serum folate.
(No ‘35)
(/-Jmole/12hours)
Fig. 2. The frequency distribution of urinary FIGLU excretion women taking oral contraceptives and 35 control subjects. + 45 PM per 12 hr. for the control group (P < 0.02 with the use of Student’s t test). The frequency distribution of urinary FIGLU is shown in Fig. 2. It can be noted that 7 out of 58 (12 per cent) women taking the Pill had a urinary FIGLU excretion higher than the highest FIGLU seen in the control group.
=58)
after histidine
loading in 58
Changes in serum and red cell folate and FIGLU after discontinuation of the Pill. Serum folate was determined in 31 women before and at monthly intervals after they stopped taking the Pill. These women were advised not to become pregnant and not to change their dietary habits for at least 3 months. The serum folate was determined in each woman once after each collection and again at the end of 3 or 4 months when all the samples from one subject were assayed simultaneously. The changes in SF of this group are shown in Fig. 3. It can be seen that serum folate tends to rise within 3 months in the majority of the women who stopped taking the Pill. The rise is more marked in the group who had low serum folate (less than 3 ng. per milliliter) before stopping the Pill. In 16 women who had low serum folate, the mean SF prior to stopping the Pill was 2.1 ng. per milliliter and rose to 2.6, 3.1, and 3.3 ng. per milliliter at 1, 2, and 3 months after stopping the Pill (a rise of 55 per cent in 3 months). The rise of serum folate after stopping the Pill is statistically significant. Using factorial
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Oral
ORAL
CONTRACEPTIVE
contraceptive
CONTROL
GROUP SF
effect on folate metabolism
)3
787
GROUP
w/ml /
6
I
z
6 3
MONTHS.
Fig. 3. The changes in serum folate in 31 women who stopped taking the oral contraceptives (16 with low and 15 with normal serum folate). The control group shows the serumfolate in 6 control women at monthly intervals. All sera for each subject were assayed simultaneously. The broken lines represent the mean.
analysis of variance and 2 factors (the serum folate and the time), we found the rise of serum folate to be significant at the one per cent level. Using the least-significantdifference procedure and Duncan’s multiple range test, we found the rise of mean serum folates at 2 and 3 months to be significant at the 5 per cent level. The changes are not due to storage effect because the patterns of changes were the same in samples assayed separately, shortly after collection, and there was no significant change in the serum folate of control subjects stored for the same period. In the 15 women who had normal serum folate prior to stopping the Pill, the mean serum folate was 5.0 before and 5.0, 5.2, and 5.8 ng. per milliliter at 1, 2, and 3 months after stopping the Pill (a rise of 16 per cent in 3 months). This rise of serum folate is not statistically significant. The pattern of changes in the red cell folate of women who stopped taking the Pill is the same as that seen in the serum. As
shown in Fig. 4, the red cell folates tend to rise in the majority of the 22 women who stopped taking the Pill. The mean red cell folate rose from 115 ng. per milliliter before to 137, 146, and 143 ng. per milliliter at 1, 2, and 3 months after stopping the Pill. This rise is statistically significant at the one per cent level (with the use of factorial analysis as mentioned above). The rise in red cell folate was also more marked in the group who had lower RF prior to stopping the Pill. There were 8 women whose red cell folate was lower than 90 ng. per milliliter. The red cell folate in all of these 8 women rose after stopping the Pill. Urinary FIGLU excretion was studied in 3 women before and after stopping the Pill. There was a fall of urinary FIGLU in all 3 women after they stopped taking the Pill. In one woman who was taking norethindrone, 5 mg., plus mestranol, 0.075 mg. for 5 years, the urinary FIGLU excretions were 350 and 495 PM per 12 hr. at 5 and 3 months prior to stopping the Pill. This re-
788
Shojania,
Hornady,
and Barnes
I 0
I M’O
N
T H
S
I
1
2
3
Fig. 4. The changes in red cell folate in 24 women who stopped taking oral contraceptives. Each sample was assayed within 2 weeks after collection. The broken line represents the mean.
turned to normal at 2 months (120 PM per 12 hr.) and fell further at 4 months (59 ,uM per milliliter) after stopping the Pill. In another woman, the urinary FIGLU excretion fell from a high level of 178 PM per 12 hr. to a normal level of 67 PM per 12 hr. 4 months after stopping the Pill. In the third woman who had normal urinary FIGLU excretion (112 PM per 12 hr.) the excretion decreased to 65 PM per 12 hr. 4 months after stopping the Pill. Comment
In this report we have shown that women on combination-type oral contraceptives have significantly lower serum and red cell folate (Fig. 1 and Table I) and higher urinary FIGLU excretion (Fig. 2) than normal control subjects. The fact that serum folate progressively falls with the continuation of oral contraceptives (Fig. 1 and Table I) and parameters of folate deficiency (low SF and RF and high urinary FIGLU) improve following the discontinuation of oral contra-
ceptive therapy (Figs. 3 and 4) indicates that the impaired folate metabolism shown in oral contraceptive users is directly related to the effect of the Pill. Following our preliminary report of finding low serum folate in oral contraceptive users,’ there have been 6 reports to the contrary.*O-I5 Our criticism of these reports is that the number studied is too small, the control subjects have not been from the same source, or the duration of oral contraceptive therapy has been too short. Oral contraceptives have been in use for more than a decade, and if they could severely impair folate metabolism their effect would have been noticed much earlier. We believe that their effect on folate metabolism is very mild and that a large number of subjects and a long duration of therapy is needed to see the effect, unless there are some other associated factors such as malabsorption, dietary folate deficiency, infection, etc. Spray’O compared the serum and red cell folate of 19 women taking the Pill attending
Volume Number
111 6
a family planning association to those of 34 healthy control subjects (source not stated) and did not find any difference between the mean serum and red cell folate of the two groups. McLean and associatesll compared the serum folate of 39 women receiving two different oral contraceptives with those of 17 control subjects from a rural north central Florida clinic population and could not find any difference between serum folate levels of the two groups. However, since Streiff,16 one of the authors of this report, had previously reported the presence of folate polyglutamate malabsorption in oral contraceptive users, McLean and associates stated that women taking oral contraceptives, despite having folate malabsorption, absorb enough folate from their diets to keep the serum folate within normal limits. Castren and Ross12compared the serum folate levels of 30 women before taking the Pill (3 different types) and 3 months after stopping the Pill. The mean serum folate after 3 months was lower than that before taking the Pill. However, since the difference was not statistically significant, the authors claim that oral contraceptives do not impair folate metabolism. They state that our finding of lower serum folate in oral contraceptive users could be due to dieting by the women to prevent weight gain. We have excluded this possibility by inter&wing the women regarding their diets. Also, the fact that parameters of folate deficiency improved within 1 to 3 months after stopping the Pill without any changes in diet indicates that the impaired folate metabolism in our subjects is not due to dieting. Kahn and colleaguesI3 have compared the serum folate and average lobe score of 14 clinic patients taking oral contraceptives and 5 control subjects with those of pregnant women. Since the incidence of low serum folate and high average lobe count in the Pill group and pregnant group was not the same, the authors claim that the hormones in the Pill do not alter folate metabolism in a manner similar to pregnancy. We cannot find the basison which this conclusion is made. Considering the fact that the pregnant woman
Oral
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has to supply folate to the fetus and the fact that the amount of hormones produced in pregnancy is much higher than that used for contraception, one should expect to see a lower serum folate in pregnant women than in oral contraceptive users. ManiegoBautista and BassanoI4 have claimed that there was no difference between serum folate levels of 51 women who were taking oral contraceptives for more than 6 months and those of 48 control subjects, but they have not published their data. Pritchard and coworkersI could not find any difference between the serum folate of 55 control subjects and 55 women receiving oral contraceptives. In our study we have excluded many of the factors that might have been responsible for the failure of others to see the impairment of folate metabolism in oral contraceptive users.Our subjects are not only taken from the same socioeconomicgroup and age but are attending the same physician for their care. They have been personally interviewed regarding their diets and medications, and their charts were reviewed. The number of subjects studied is large enough to make a meaningful statistical analysis, and there are enough subjects receiving long-term oral contraceptive therapy to see the accumulated effect of very mild impairment of folate metabolism. One other factor that may be responsible for the discrepancy between our findings and those of others10-15is the lower average dietary folate intake by the population that we have studied. Our subjects are living in Winnipeg where, because of the very long cold seasonand very short summer, the consumption of vegetables and fruits is generally low and the average folate intake is lower than that of a similar group of subjects in a similar socioeconomic group in other countries. Consequently, any impairment of folate metabolism will be demonstrated in our subjects sooner than in those studied by others where average dietary folate intake is higher. Streiff17 and Snyder and Nechelesl* have shown the presence of folate polyglutamate malabsorption in oral
790
Shoiania,
Hornady,
and
Barnes
and Greene’” contraceptive users. Streiff have shown that mestranol, norethindrone, and chlormadinone have an inhibitory effect on human intestinal conjugase activity in an in vitro system. We have studied the absorption of folate polyglutamate in a few control subjects and oral contraceptive users, and we have not been able to find a low folate polyglutamate absorption in all oral contraceptive users as found by Streiff”? and Snyder and Necheles.18 To date we have tested the absorption of folate polyglutamate in 7 women taking oral contraceptives and 5 control subjects. We studied the folate polyglutamate absorption after saturating the tissues with a loading dose of folic acid (pteroylglutamic acid). In 5 women taking the Pill who had normal serum folate, the polyglutamate absorption was similar to that of the control subjects. In 2 women taking the Pill who had lower serum folate prior to a folate loading dose, the absorption of folate polyglutamate was less than normal. These 2 women both had normal pteroylglutamic acid absorption and did not have any other evidence to suggest a malabsorption syndrome. However, the absorption of folate polyglutamate remained abnormal in these two women when they were tested again 2 months after discontinuation of the Pill. We are not certain whether this malabsorption of folate polyglutamate is due to the effect of the Pill or these two cases represent the mildest form of malabsorption syndrome. The pathogenesis of impaired folate metabolism in oral contraceptive users is not clear. The finding of folate polyglutamate malabsorption in oral contraceptive users17, Is may in part be responsible for this biochemical evidence of folate deficiency in the women using the Pill. However, based on our own experience, we do not think that this impaired folate absorption is the only responsible factor. We have been unable to
find any correlation between the cvidence oi impaired folatc metabolism in the Pill group and the impaired liver function tests (serun~ bilirubin, SGOT, serum alkaline phosphatase, thymol turbidity, and BSP retention) I However, this does not nega.te the role of the liver in the pathogenesis of impaired folate metabolism in oral contraceptive users. The finding of impaired folate metabolism in oral contraceptive users has practical and theoretical significance. Since the pregnant woman is predisposed to the development of folate deficiency and incidence of folate deficiency increases with increasing gestation,‘-” it would be logical to think that if a woman becomes pregnant shortly after stopping the Pill she would have a higher chance of developing folate deficiency and complications attributed to this deficiency.‘m3 Based on our findings, we recommend that serum folate be determined in any woman who stops taking oral contraceptives to become pregnant. Our findings also suggest that the fall in serum folate during pregnancy is not due to parasitization by the fetus alone but is also due to an associated impairment of folate metabolism caused by hormonal changes associated with pregnancy. We are grateful to Mr. R. Rollwagen from the Computer Department of the University of Manitoba for his help in statistical analysis of the data.
Addendum Since submission of this paper, there have been 2 published abstracts in which the authors have indicated that women on oral contraceptives have impaired folate metabolism. Luhby and associates (Fed. Proc. 30: 239, 1971. [Abst.]) have reported that 5 out of 20 subjects on oral contraceptives had biochemical evidence of folate deficiency, and Wertalik and colleagues (Am. J. Clin. Nutr. 24: 603, 1971) have reported that women on oral contraceptives have significantly lower serum folate levels than control subjects.
REFERENCES
1. Rothman, 2.
D.: Ant. J. 108: 149, 1970. Chanarin, I., MacGibbon,
W. OBSTET.
3. B. M.,
O’Sulivan,
J.,
and
Mollin,
D.
L.:
Lancet
2:
634,
D.:
Br.
1959.
GYNECOL.
Hibbard, B. M., and Hibbard, Med. Bull. 24: 10, 1968,
E.
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Chanarin, I., Anderson, B. B., and Mollin, D. L.: Br. J. Haematol. 4: 156, 1958. R. H.: Quart. J. Med. 25: 87, 5. Girdwood, 1956. 6. Witts, L. J.: J. Obstet. Gynaecol. Br. Commonw. 69: 714, 1962. A. M., Hornady, G., and Barnes, 7. Shojania, P. H.: Lancet 1: 1376. 1968. 8. Waters, A. H., and Mollin, D. L.: J. Clin. Pathol. 14: 335, 1961. 9. Tabor, H., and Wyngarden, L.: J. Clin. Invest. 37: 824, 1958. 10. Spray, G. H.: Lancet 2: 110, 1968. F. W., Heine, M. W., Held, B., 11. McLean, and St&f, R. R.: AM. J. OBSTET. GYNECOL. 104: 745, 1969. 12. CastrCn, 0. M., and Ross, R. R.: J. Obstet.
Oral
contraceptive
4.
13.
14. 15.
16. 17. 18. 19.
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metabolism
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Gynaecol. Br. Commonw. 77: 548, 1970. Kahn, S. B., Fein, S., Rigberg, S., and Brodsky, I.: AM. J. OBSTET. GYNECOL. 108: 931, 1970. Maniego-Bautista, L. P., and Bassano, G.: T. Lab. Clin. Med. 74: 988. 1969. fAbst.1 Pritchard, J. A., Scott, D. ‘E., and ‘Whalley, P. J.: AM. J. OBSTET. GYNECOL. 109: 341, 1971. Streiff, R. R.: Clin. Res. 17: 345, 1969. Abst. Streiff, R. R.: J, A. M. A. 214: 105, 1970. Snyder, L. M., and Necheles, T. F.: N. Engl. J. Med. 282: 858, 1970. Streiff, R. R., and Greene, B.: Presentation at the XIIIth International Congress of Hematology, Munich, August 2-8, 1970.
MAJID
GLORIA
SHOJANIA, J.
PHILIP
H.
Winnipeg,
Manitoba,
M.D
HORNADY, BARNES,
R.T. M.D
Canada
We have studied the eBect of oral contraceptives on folate metabolism, in order to test the hypothesis that folate deficiency of pregnant women may be partly due to impairment of folate metabolism. Serum folate, red cell folate, and urinary excretion of formiminoglutamic acid (FIGLU) were determined in 176 women receiving combination-type oral contraceptives (the Pill) and 140 normalcontrol subjects. The group taking the Pill had significantly lower serum and red cell folate and higher urinary FIGLU excretion. There was a rise in serum and red cell folate and a fall of urinary FIGLU excretion within 3 months after stopping the Pill. The data demonstrate that women taking the Pill may have impaired folate metabolism and suggest that sex hormones may play a role in the development of folate dejiciency in pregnancy.
DEFICIENCY iscommonlyseen during pregnancy, and the topic has been recently reviewed. 1 This folate deficiency is mainly attributed to parasitization by the fetus.2l 3 However, other associated factors such as impaired folate absorption in pregnancy4 and impaired folate metabolism596 have been considered as contributory factors. If the latter two factors play a significant role in the development of folate deficiency of pregnant women, one would expect to see a similar effect in women taking oral contraceptives which are known to produce a
“pseudopregnancy state.” In a preliminary study,’ we determined the serum folate levels (SF) in a group of women receiving oral contraceptives (the Pill) and compared them to those of normal control subjectsand found that the women taking the Pill had significantly lower serum folate- values than the control group. This led us to undertake a more detailed study of the effect of oral contraceptives on folate metabolism. The present paper is the first part of the report which indicates that, by all criteria, women taking the Pill have impaired folate metabolism.
FOLATE
From the Department of Hematology, St. Boniface General Hospital, the Department of Pediatrics and the Department of Obstetrics and Gynecology, University of Manitoba.
Materials
and
methods
Subjects. The subjects studied were the women who were taking a combination-type oraI contraceptive and were attending the private clinic of an obstetrician and gynecologist (P. H. B.) . Two groups of control subjects were used. One group was from the same clinic (private clinic control), and the other group was made up of female hospital personne1 (hospital controls) . Control sub-
These studies were supported in part by grants from the National Health Research of Canada (606-7-189), Ortho Pharmaceutical (Canada),. Ltd., and the Medical Research Councal of Canada (MA-3069). Presented in part at the XIXIth International Congress of Hematology, Munich, August 2-8, 1970.
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jects had not been taking oral contraceptives for at least a year and were of reproductive age. All subjects were interviewed to obtain information about diet, intake of vitamins or other medications, and the presence of diseasesthat may affect folate metabolism. Subjects who had received folic acid therapy at any time in the past or had been delivered of infants within a year prior to this study or were on a diet to prevent weight gain were excluded. No subjects were excluded from the control group because of poor dietary folate intake. However, 4 women who had low serum folate and poor dietary folate intake, one who had low serum folate and was found to have a mild malabsorption syndrome, and one who had a high urinary formiminoglutamic acid (FIGLU) excretion but had high serum glutamic oxaloacetic acid (SGOT) and bromsulphalein (BSP) retention were excluded from the Pill group. Subjects from the private clinic were generally of good or moderate income and did not include indigent patients. Originally, blood samples were taken for serum folate only, but later on, when a more detailed study was planned, further investigations were made. These included red cell folate (RF), urinary excretion of FIGLU and folate, hemoglobin, hematocrit, blood smear morphology, reticulocyte count, liver function tests (serum bilirubin, SGOT, serum alkaline phosphatase, cephaline flocculation, thymol turbidity, and BSP retention), and serum vitamin B,,. Techniques. Serum folate was determined by the L. casei microbiological assay of Waters and Mollin* with minor modifications. All serums were kept undiluted at -20° F. and were reconstituted with L. ascorbic acid (5 mg. per milliliter) on the day of assay. Blood samples for red cell folate determination were collected in tubes containing ethylenediaminetetra-acetic acid. The blood was then diluted 1: 10 in deionized water and kept frozen at -20° F. until the day of assay when it was reconstituted with L. ascorbic acid (5 mg. per milliliter of diluted blood) and assayed as serum. In our laboratory, the normal se-
Oral
contraceptive
effect on folate
metabolism
783
rum folate range is 3 to 15 ng. per milliliter, and normal red cell folate is 100 to 500 ng. per milliliter. The reproducibility of serum folate in the same assay was determined in 9 replicates of 3 serums with mean serum folate values of 7, 8.3, and 14.1 ng. per milliter. The coefficients of variability were 2.2, 3, and 6 per cent, respectively. The variation between duplicate samples determined on two different days was determined in 45 duplicate samples.The per cent variation from the mean of the duplicates was 0 to 19 per cent (mean = 7.3 per cent). A pooled sample of serum which was stored at -20° F. and reassayed 47 times during 10 months of storage showed no detectable loss of folate activity and no more variation than that seen between duplicate samples (the mean serum folate of the pooled serum was 5 ng. per milliliter and the coefficient of variability was 12.7 per cent). Urinary FIGLU was determined in a 12 hour urine sample following histidine loading (15 Gm. of histidine monohydrochloride). FIGLU was determined by an enzymatic method with the use of a FIGLU test kit (Sigma Chemical Co., St. Louis, Missouri), which is based on the method of Tabor and Wyngarden.O The variations between the duplicate samplesassayedon two different days were determined in 9 samples. The per cent variation from the mean of the duplicates ranged between 0.2 and 20 per cent (mean 6 per cent). Results
Effect of the Pill on serum folate. Serum folate was determined in 176 women taking the Pill, 39 private clinic control subjects, and 101 hospital control subjects of similar sex and ages. Fig. 1 shows the serum folate of these groups. Only one serum folate is presented for each subject. In caseswhere more than one serum folate was available for each subject, the latest serum folate representing the longest duration of oral contraceptive therapy is shown on the figure. The mean + S.D. for private clinic control subjects is 6.1 + 2.23 ng. per milliliter, which
784
Shojania,
Hornady,
and
November 15, 1971 Am. J. Obstet. Gynecol.
Barnes
PRIVATE CLINIC ON ORAL CONTRACEPTIVES.
IL'
.
1
0
1 DURATION
.
2 3 (YEARS)
4
$5
Fig.
1. The serum folate of 176 women taking oral contraceptives, 39 control subjects from the same clinic, and 101 control subjects from hospital personnel. The solid horizontal lines represent the means of Pill and control groups. The dotted lines represent the means of subgroups (control groups according to their source and Pill groups according to the duration of oral contraceptive therapy).
is not significantly different from those of hospital control subjects (6.3 2 2.6 ng. per milliliter). Although the private clinic control subjects have slightly Iower mean serum folate than the hospital control subjects, this does not mean that they have worse folate intake or stores than the hospital control group. Not +only is the difference between the two means not statistically significant, but, actually, there are fewer subnormal serum folates in the private clinic control subjects than in the hospital control subjects (2.5 per cent of private clinic control subjects and 5.9 per cent of hospital control subjects had serum folate levels of less than 3 ng. per milliliter). The mean + S.D. for 176 women on the Pill is 4.2 t 1.77 ng. per milliliter which is significantly lower than that of either the private clinic control subjects or the hospital control subjects (P < 0.001 with the use of Student’s t test). Similarly, in the frequency distribution curve the mode for the group who were taking the Pill for more than one year is between 3 and 4 ng. per milliliter which is lower than
the mode for control subjects (5 to 6 ng. per milliliter.) The effect of duration of oral contraceptive therapy on serum foIate. In order to study the relation of the duration of oral contraceptive therapy to the serum folate, the group taking the Pill was divided into 5 groups according to the duration of oral contraceptive therapy. Fig. 1 and Table I show that the mean serum folate falls with the continuation of oral contraceptives. An analysis of variance shows that each of the 5 groups taking the Pill has a significantly lower serum folate than the control group from the same clinic (with the use of oneway analysis of variance and test of the least significant difference). Table I also shows that those who have been taking the Pill for more than two years have a significantly lower mean serum folate than those who have been taking the Pill for less than a year (with the use of the test of the least significant difference). Similarly, it can be seen that not only the mean serum folate falls but that the percentage of subjects with
Volume Number
Oral
111 6
contraceptive
effect on folate metabolism
785
Table I. Changes in mean serum folate and percentage of subjects with low serum folate according to the duration of oral contraceptive therapy. All subjects were from the same clinic attending one physician (P. H. B.)
I Control I %--I>
Serum folate mean + S.D. (ng./ml.)
>4
39 6.1 _+2.2
5.0 ?r 2.0
4.3 + 1.7
4.1 2 1.9
3.7 2 1.0
3.8 211.7
< 0.02
< 0.001
< 0.001
< 0.001
< 0.001
< 0.1
< 0.05
< 0.01
< 0.01
27
32
42
9 -
low serum folate (less than 3 ng. per milliliter) increases with the duration of oral contraceptive therapy. Only 2 per cent of the control group had low serum folate (less than 3 ng. per milliliter) whereas 9 per cent of the group who were taking the Pill for lessthan one year, 21 per cent of the group who were taking the Pill for 1 to 2 years, 27 per cent of the group who were taking the Pill for 2 to 3 years, 32 per cent of the group who were taking the Pill for 3 to 4 years, and 42 per cent of the group who were taking the Pill for more than 4 years had a serum folate of lessthan 3 ng. per milliliter. The rise in percentage of subnormal folate with the duration of oral contraceptive therapy is statistically significant (P < 0.01 with the useof the chi-square test). The effect of the dose of oral contraceptives on serum folate. Among the group taking the Pill, there were 39 women who were taking the Pill containing mestranol, 0.05 mg., + norethindrone, 1 mg.*t The mean serum folate (+ SD.) for this group is 4.4 t 1.87 ng. per milliliter which is significantly lower than that of control subjects (P < 0.001 with the use of the Student’s t test). There were 42 women who were taking
Laboratories,
1
6 33
2
Pharm.
1 3-4> 5 28
< 0.02
Ortho
1 2-3>
(year)
4 36
Percentage with low S.F. (less than 3)
‘Ortho-Novum I, Jersey. +Norinyl 1, Syntex fornia.
contraceptives
3 47
P vs. Group 1 P vs. Group 2
1 I-2>
of oral
2 32
I
No. of patients
Duration
Corp., Inc.,
Raritan, Palo
Alto,
21
preparations* which contain twice the amount of mestranol and norethindrone previously mentioned. The mean serum folate (+ SD.) for this group is 3.8 + 1.6. Although this mean is lower than that of the group receiving the small amounts of mestranol and norethindrone, with the number of subjects studied the difference is not statistically significant. The effect of oral contraceptives on red cell folate. Red cell folate was determined in 95 women who were taking the Pill for more than one year (private clinic) and 63 control subjects (private and hospital control subjects). The mean + S.D. for the group taking the Pill is 130 + 62 ng. per milliliter which is significantly lower than 175 + 69 ng. per milliliter for the control group (P < 0.001 with the use of Student’s t test). (The mean RF for 10 private clinic control subjects was 176 ng. per milliliter, and that of 53 control subjects was 174 ng. per milliliter.) The effect of oral contraceptives on winary FIGLU excretion. Urinary FIGLU excretion was determined in 58 women taking the Pill and 35 control subjects (private and hospital control subjects). The mean + S.D. for the Pill group is 91 + 84 PM per 12 hr. which is significantly higher than 51
New Cali-
‘Norinyl 2, Syntex Laboratories, Inc. Palo Alto, fornia. OrtheNovum II, Ortho Pharm. Corp., Raritan, Jersey.
CaliNew
786
Shojania,
Hornady,
and Barnes
tl PILL
(NO
~C~NTR~L
URINARY
FIGLU
The effect of the oral contraceptives on hemoglobin and hematocrit. Hemoglobin and hematocrit were determined in 123 women taking the Pill and 71 control subjects. The mean hemoglobin (f SD.) for the Pill group is 13.2 + 0.95 Gm. per cent which is not significantly different from that of the control subjects (13.0 + 0.83 Gm. per cent). Similarly, the mean hematocrit it S.D. for the Pill group is 39.8 + 2.94 per cent which is not significantly different from 39.8 + 2.69 per cent for the control group. Among the women receiving oral contraceptives, there was no significant difference between the hemoglobin and hematocrit of those who had subnormal serum folate (less than 3 ng. per milliliter) and those who had normal serum folate.
(No ‘35)
(/-Jmole/12hours)
Fig. 2. The frequency distribution of urinary FIGLU excretion women taking oral contraceptives and 35 control subjects. + 45 PM per 12 hr. for the control group (P < 0.02 with the use of Student’s t test). The frequency distribution of urinary FIGLU is shown in Fig. 2. It can be noted that 7 out of 58 (12 per cent) women taking the Pill had a urinary FIGLU excretion higher than the highest FIGLU seen in the control group.
=58)
after histidine
loading in 58
Changes in serum and red cell folate and FIGLU after discontinuation of the Pill. Serum folate was determined in 31 women before and at monthly intervals after they stopped taking the Pill. These women were advised not to become pregnant and not to change their dietary habits for at least 3 months. The serum folate was determined in each woman once after each collection and again at the end of 3 or 4 months when all the samples from one subject were assayed simultaneously. The changes in SF of this group are shown in Fig. 3. It can be seen that serum folate tends to rise within 3 months in the majority of the women who stopped taking the Pill. The rise is more marked in the group who had low serum folate (less than 3 ng. per milliliter) before stopping the Pill. In 16 women who had low serum folate, the mean SF prior to stopping the Pill was 2.1 ng. per milliliter and rose to 2.6, 3.1, and 3.3 ng. per milliliter at 1, 2, and 3 months after stopping the Pill (a rise of 55 per cent in 3 months). The rise of serum folate after stopping the Pill is statistically significant. Using factorial
Volume Number
111 6
Oral
ORAL
CONTRACEPTIVE
contraceptive
CONTROL
GROUP SF
effect on folate metabolism
)3
787
GROUP
w/ml /
6
I
z
6 3
MONTHS.
Fig. 3. The changes in serum folate in 31 women who stopped taking the oral contraceptives (16 with low and 15 with normal serum folate). The control group shows the serumfolate in 6 control women at monthly intervals. All sera for each subject were assayed simultaneously. The broken lines represent the mean.
analysis of variance and 2 factors (the serum folate and the time), we found the rise of serum folate to be significant at the one per cent level. Using the least-significantdifference procedure and Duncan’s multiple range test, we found the rise of mean serum folates at 2 and 3 months to be significant at the 5 per cent level. The changes are not due to storage effect because the patterns of changes were the same in samples assayed separately, shortly after collection, and there was no significant change in the serum folate of control subjects stored for the same period. In the 15 women who had normal serum folate prior to stopping the Pill, the mean serum folate was 5.0 before and 5.0, 5.2, and 5.8 ng. per milliliter at 1, 2, and 3 months after stopping the Pill (a rise of 16 per cent in 3 months). This rise of serum folate is not statistically significant. The pattern of changes in the red cell folate of women who stopped taking the Pill is the same as that seen in the serum. As
shown in Fig. 4, the red cell folates tend to rise in the majority of the 22 women who stopped taking the Pill. The mean red cell folate rose from 115 ng. per milliliter before to 137, 146, and 143 ng. per milliliter at 1, 2, and 3 months after stopping the Pill. This rise is statistically significant at the one per cent level (with the use of factorial analysis as mentioned above). The rise in red cell folate was also more marked in the group who had lower RF prior to stopping the Pill. There were 8 women whose red cell folate was lower than 90 ng. per milliliter. The red cell folate in all of these 8 women rose after stopping the Pill. Urinary FIGLU excretion was studied in 3 women before and after stopping the Pill. There was a fall of urinary FIGLU in all 3 women after they stopped taking the Pill. In one woman who was taking norethindrone, 5 mg., plus mestranol, 0.075 mg. for 5 years, the urinary FIGLU excretions were 350 and 495 PM per 12 hr. at 5 and 3 months prior to stopping the Pill. This re-
788
Shojania,
Hornady,
and Barnes
I 0
I M’O
N
T H
S
I
1
2
3
Fig. 4. The changes in red cell folate in 24 women who stopped taking oral contraceptives. Each sample was assayed within 2 weeks after collection. The broken line represents the mean.
turned to normal at 2 months (120 PM per 12 hr.) and fell further at 4 months (59 ,uM per milliliter) after stopping the Pill. In another woman, the urinary FIGLU excretion fell from a high level of 178 PM per 12 hr. to a normal level of 67 PM per 12 hr. 4 months after stopping the Pill. In the third woman who had normal urinary FIGLU excretion (112 PM per 12 hr.) the excretion decreased to 65 PM per 12 hr. 4 months after stopping the Pill. Comment
In this report we have shown that women on combination-type oral contraceptives have significantly lower serum and red cell folate (Fig. 1 and Table I) and higher urinary FIGLU excretion (Fig. 2) than normal control subjects. The fact that serum folate progressively falls with the continuation of oral contraceptives (Fig. 1 and Table I) and parameters of folate deficiency (low SF and RF and high urinary FIGLU) improve following the discontinuation of oral contra-
ceptive therapy (Figs. 3 and 4) indicates that the impaired folate metabolism shown in oral contraceptive users is directly related to the effect of the Pill. Following our preliminary report of finding low serum folate in oral contraceptive users,’ there have been 6 reports to the contrary.*O-I5 Our criticism of these reports is that the number studied is too small, the control subjects have not been from the same source, or the duration of oral contraceptive therapy has been too short. Oral contraceptives have been in use for more than a decade, and if they could severely impair folate metabolism their effect would have been noticed much earlier. We believe that their effect on folate metabolism is very mild and that a large number of subjects and a long duration of therapy is needed to see the effect, unless there are some other associated factors such as malabsorption, dietary folate deficiency, infection, etc. Spray’O compared the serum and red cell folate of 19 women taking the Pill attending
Volume Number
111 6
a family planning association to those of 34 healthy control subjects (source not stated) and did not find any difference between the mean serum and red cell folate of the two groups. McLean and associatesll compared the serum folate of 39 women receiving two different oral contraceptives with those of 17 control subjects from a rural north central Florida clinic population and could not find any difference between serum folate levels of the two groups. However, since Streiff,16 one of the authors of this report, had previously reported the presence of folate polyglutamate malabsorption in oral contraceptive users, McLean and associates stated that women taking oral contraceptives, despite having folate malabsorption, absorb enough folate from their diets to keep the serum folate within normal limits. Castren and Ross12compared the serum folate levels of 30 women before taking the Pill (3 different types) and 3 months after stopping the Pill. The mean serum folate after 3 months was lower than that before taking the Pill. However, since the difference was not statistically significant, the authors claim that oral contraceptives do not impair folate metabolism. They state that our finding of lower serum folate in oral contraceptive users could be due to dieting by the women to prevent weight gain. We have excluded this possibility by inter&wing the women regarding their diets. Also, the fact that parameters of folate deficiency improved within 1 to 3 months after stopping the Pill without any changes in diet indicates that the impaired folate metabolism in our subjects is not due to dieting. Kahn and colleaguesI3 have compared the serum folate and average lobe score of 14 clinic patients taking oral contraceptives and 5 control subjects with those of pregnant women. Since the incidence of low serum folate and high average lobe count in the Pill group and pregnant group was not the same, the authors claim that the hormones in the Pill do not alter folate metabolism in a manner similar to pregnancy. We cannot find the basison which this conclusion is made. Considering the fact that the pregnant woman
Oral
contraceptive
effect on folate metabolism
789
has to supply folate to the fetus and the fact that the amount of hormones produced in pregnancy is much higher than that used for contraception, one should expect to see a lower serum folate in pregnant women than in oral contraceptive users. ManiegoBautista and BassanoI4 have claimed that there was no difference between serum folate levels of 51 women who were taking oral contraceptives for more than 6 months and those of 48 control subjects, but they have not published their data. Pritchard and coworkersI could not find any difference between the serum folate of 55 control subjects and 55 women receiving oral contraceptives. In our study we have excluded many of the factors that might have been responsible for the failure of others to see the impairment of folate metabolism in oral contraceptive users.Our subjects are not only taken from the same socioeconomicgroup and age but are attending the same physician for their care. They have been personally interviewed regarding their diets and medications, and their charts were reviewed. The number of subjects studied is large enough to make a meaningful statistical analysis, and there are enough subjects receiving long-term oral contraceptive therapy to see the accumulated effect of very mild impairment of folate metabolism. One other factor that may be responsible for the discrepancy between our findings and those of others10-15is the lower average dietary folate intake by the population that we have studied. Our subjects are living in Winnipeg where, because of the very long cold seasonand very short summer, the consumption of vegetables and fruits is generally low and the average folate intake is lower than that of a similar group of subjects in a similar socioeconomic group in other countries. Consequently, any impairment of folate metabolism will be demonstrated in our subjects sooner than in those studied by others where average dietary folate intake is higher. Streiff17 and Snyder and Nechelesl* have shown the presence of folate polyglutamate malabsorption in oral
790
Shoiania,
Hornady,
and
Barnes
and Greene’” contraceptive users. Streiff have shown that mestranol, norethindrone, and chlormadinone have an inhibitory effect on human intestinal conjugase activity in an in vitro system. We have studied the absorption of folate polyglutamate in a few control subjects and oral contraceptive users, and we have not been able to find a low folate polyglutamate absorption in all oral contraceptive users as found by Streiff”? and Snyder and Necheles.18 To date we have tested the absorption of folate polyglutamate in 7 women taking oral contraceptives and 5 control subjects. We studied the folate polyglutamate absorption after saturating the tissues with a loading dose of folic acid (pteroylglutamic acid). In 5 women taking the Pill who had normal serum folate, the polyglutamate absorption was similar to that of the control subjects. In 2 women taking the Pill who had lower serum folate prior to a folate loading dose, the absorption of folate polyglutamate was less than normal. These 2 women both had normal pteroylglutamic acid absorption and did not have any other evidence to suggest a malabsorption syndrome. However, the absorption of folate polyglutamate remained abnormal in these two women when they were tested again 2 months after discontinuation of the Pill. We are not certain whether this malabsorption of folate polyglutamate is due to the effect of the Pill or these two cases represent the mildest form of malabsorption syndrome. The pathogenesis of impaired folate metabolism in oral contraceptive users is not clear. The finding of folate polyglutamate malabsorption in oral contraceptive users17, Is may in part be responsible for this biochemical evidence of folate deficiency in the women using the Pill. However, based on our own experience, we do not think that this impaired folate absorption is the only responsible factor. We have been unable to
find any correlation between the cvidence oi impaired folatc metabolism in the Pill group and the impaired liver function tests (serun~ bilirubin, SGOT, serum alkaline phosphatase, thymol turbidity, and BSP retention) I However, this does not nega.te the role of the liver in the pathogenesis of impaired folate metabolism in oral contraceptive users. The finding of impaired folate metabolism in oral contraceptive users has practical and theoretical significance. Since the pregnant woman is predisposed to the development of folate deficiency and incidence of folate deficiency increases with increasing gestation,‘-” it would be logical to think that if a woman becomes pregnant shortly after stopping the Pill she would have a higher chance of developing folate deficiency and complications attributed to this deficiency.‘m3 Based on our findings, we recommend that serum folate be determined in any woman who stops taking oral contraceptives to become pregnant. Our findings also suggest that the fall in serum folate during pregnancy is not due to parasitization by the fetus alone but is also due to an associated impairment of folate metabolism caused by hormonal changes associated with pregnancy. We are grateful to Mr. R. Rollwagen from the Computer Department of the University of Manitoba for his help in statistical analysis of the data.
Addendum Since submission of this paper, there have been 2 published abstracts in which the authors have indicated that women on oral contraceptives have impaired folate metabolism. Luhby and associates (Fed. Proc. 30: 239, 1971. [Abst.]) have reported that 5 out of 20 subjects on oral contraceptives had biochemical evidence of folate deficiency, and Wertalik and colleagues (Am. J. Clin. Nutr. 24: 603, 1971) have reported that women on oral contraceptives have significantly lower serum folate levels than control subjects.
REFERENCES
1. Rothman, 2.
D.: Ant. J. 108: 149, 1970. Chanarin, I., MacGibbon,
W. OBSTET.
3. B. M.,
O’Sulivan,
J.,
and
Mollin,
D.
L.:
Lancet
2:
634,
D.:
Br.
1959.
GYNECOL.
Hibbard, B. M., and Hibbard, Med. Bull. 24: 10, 1968,
E.
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Chanarin, I., Anderson, B. B., and Mollin, D. L.: Br. J. Haematol. 4: 156, 1958. R. H.: Quart. J. Med. 25: 87, 5. Girdwood, 1956. 6. Witts, L. J.: J. Obstet. Gynaecol. Br. Commonw. 69: 714, 1962. A. M., Hornady, G., and Barnes, 7. Shojania, P. H.: Lancet 1: 1376. 1968. 8. Waters, A. H., and Mollin, D. L.: J. Clin. Pathol. 14: 335, 1961. 9. Tabor, H., and Wyngarden, L.: J. Clin. Invest. 37: 824, 1958. 10. Spray, G. H.: Lancet 2: 110, 1968. F. W., Heine, M. W., Held, B., 11. McLean, and St&f, R. R.: AM. J. OBSTET. GYNECOL. 104: 745, 1969. 12. CastrCn, 0. M., and Ross, R. R.: J. Obstet.
Oral
contraceptive
4.
13.
14. 15.
16. 17. 18. 19.
effect on folate
metabolism
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Gynaecol. Br. Commonw. 77: 548, 1970. Kahn, S. B., Fein, S., Rigberg, S., and Brodsky, I.: AM. J. OBSTET. GYNECOL. 108: 931, 1970. Maniego-Bautista, L. P., and Bassano, G.: T. Lab. Clin. Med. 74: 988. 1969. fAbst.1 Pritchard, J. A., Scott, D. ‘E., and ‘Whalley, P. J.: AM. J. OBSTET. GYNECOL. 109: 341, 1971. Streiff, R. R.: Clin. Res. 17: 345, 1969. Abst. Streiff, R. R.: J, A. M. A. 214: 105, 1970. Snyder, L. M., and Necheles, T. F.: N. Engl. J. Med. 282: 858, 1970. Streiff, R. R., and Greene, B.: Presentation at the XIIIth International Congress of Hematology, Munich, August 2-8, 1970.