CONTRACEPTION MULTIVITAMIN SUPPLEMENTATION IN ORAL CONTlhCEPTlVE
PIM NM MOOIJ’,
CHRIS MG THOMAS’,
WIM H DOESBURG’,
’ DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY, OF MEDICAL STATISTICS, ACADEMIC HOSPITAL NIJMEGEN, THE NETHERLANDS
USERS
TOM KAB ESKES’
AND a DEPARTMENT ST RADBOUD,
ABSTRACT
The effects of oral contraceptives (OC) containing 30 ug of ethinyl oestradiol and of subsequent multivitamin and folic acid supplementation on vitamin A, total 82 [including its three individual constituents, i.e. riboflavine, RB; flavine-mono-nucleotide, FMN; and flavine-adeninedinucleotide, FAD], 812, C and folate concentration in serum and red blood cells have been studied in a group of 59 non-pregnant female volunteers. The group taking OC comprised 28 women while 31 women were included in the group of non-OC users serving as the controls. The women were studied for four cycles. Blood samples were taken on days 3 and 23 of the first cycle to obtain baseline values of each analyte. Multivitamin and folic acid supplementation started on day 1 of the second cycle and this was continued daily throughout three consecutive cycles until the end of the study. Vitamin A levels were significantly higher and vitamin 812 levels were significantly lower in the group using OC. Comparison of the baseline values of vitamin total 82, FAD, C, serum and red blood cell folate as determined on days 3 and 23 of the first cycle of the two groups compared revealed no significant differences. Multivitamin and folic acid supplementation did not affect the concentrations of vitamin A and vitamin B12 with either group, whereas all other vitamins increased significantly in both groups. The consistency of each effect of multivitamin supplementation between the two groups was also tested. The degree of these effects was not statistically different between both groups. The results suggest that the vitamin status is indeed affected by OC treatment, but the effects of multivitamin supplementation are not different in OC and non-OC users. Supplementation during OC use or just after discontinuing treatment cannot be justified for healthy young women. However, in the case of women with a critical vitamin balance or higher folate needs, multivitamin supplementation may be considered.
Reprint requests to: PIM NM Academic Hospital ST Radboud, Submitted for publication Accepted for publication
MOOIJ, Department of Obstetrics and Gynaecology, PO BOX 9101, 6500 HB Nijmegen, the Netherlands
April 8, 1991 July 15, 1991
SEPTEMBER 1991 VOL. 44 NO. 3
277
INTRODUCTION During the last decade much attention has been paid to the role of folate and vitamin levels in early pregnancy. One focus of interest is the teratogenic effect of high doses of vitamin A in man. The teratogenic potential of vitamin A in animals had been recognised more than 30 years ago (1). Deficiencies of many individual vitamins during pregnancy in animals have been shown to have devastating effects on the embryo and may result in growth retardation, intrauterine death and major congenital abnormalities (2-5). Obsenrationai studies suggested vitamin deficiencies as an aetiological factor for neural tube defects (NTDs) in man (6,7). Three studies on the possible association between the
vitamin intake of pregnant women and the presence of neural tube defects have been conducted in the USA and have given differing results (8,9,10). Although there is strong evidence for an association between maternal vitamin status and the outcome of pregnancy, this is not universally accepted, due to imperfections of study design (11). Since the introduction of oral contraceptives (OC) by Pincus in 1956, much controversy exists about the general and nutrient metabolic side effects of the contraceptive steroids. In recent years some of the metabolic side effects have been linked to vitamin nutritional status (12). A number of papers have described alterations of vitamin status in users of oestrogen-containing OC (13-15). Several authors suggest that women using combined OC tend to have raised plasma concentrations of vitamin A and reduced concentrations of vitamin 812 and foiic acid (12,15,16). A possible association between vitamin 812 deficiency and infertility has been reported recently (17). Because high levels of vitamin A can be teratogenic and may induce fetal abnormalities,, and folic acid deficiency may lead to failures in the process of neurulation, one could postulate that conception in women immediately after discontinuing OC treatment could have an adverse effect on reproduction. The aim of the present investigation was to evaluate the vitamin and folic acid status in fertile women and the effects of OC treatment on these parameters. The effects of multivitamin and folic acid supplementation on vitamin and foiate status in both groups were also studied.
MATERIAL
AND
METHODS
SUBJECTS. Two groups of non-pregnant, non-lactating female volunteers participated in the study after giving informed consent. Approval was given first by the ethics committee of the University Hospital. All women were between 19 and 39 years of age. The following criteria were met for inclusion: nuliigravidae under 40 years of age; no recent blood transfusion; no history of gastrointestinal or kidney disease; no recent surgery; no special diet; and in good general health. Population characteristics are given in Table I. All subjects were questioned about their general health condition, family history, drinking habits, use of drugs, social class and menstrual cycle. The subjects were studied for four consecutive cycles, a pill cycle was considered as the period from the first day of pill intake until the next first day of intake. The women used mono-, bi- or multiphasic OC containing 30 ug of estrogen. In order to obtain comparative baseline data, blood samples were taken during cycle 1 on days 3 and 23 before vitamin supplementation was started on day one of the second cycle and given daily until the end of the study. The supplement offered was Gravitamon (Chefaro BV Oss, The Netherlands) one tablet per day and folic acid 5 mg per day orally. Gravitamon contains vitamin A [retinol] 1000 IU, Bl [thiamine] 3 mg, 82 [riboflavine] 2 mg, 83 jnicotinamide] 110 mg, B6 [pyridoxine] 1 mg, 812 (cyanocobalamine] 1 ug, rutoside 10 mg, vitamin C
278
SEPTEMBER
1991 VOL. 44 NO. 3
CONTRACEPTION Table I. The general characteristics
of the groups studied
group taking oral contraceptives
No. of subjects Age [mean] range Body weight [mean] range Height [mean] range
group not taking oral contraceptives
(years)
28 29.6
(years) (kg)
19-36 62
21 - 39 61
l”s m
48 - 78 1.68
39 - 110 1.67
(m)
1.57
- 1.84
31 30.6
1.49
- 1.84
[ascorbic acid] 50 mg, vitamin D [cholecalciferol] 400 IU, vitamin E [tocoferylacetate] 1 mg, calcium carbonate 500 mg, ferrous fumurate equivalent to 16 mg Fe, copper carbonate 0.1 mg and zinc sulphate 1 mg. To study the effects of multivitamin supplementation after one cycle of treatment, blood samples were taken on days 3 and 23 of the 3rd and 4th cycle to assess vitamins A, total 62 (including its three individual constituents, i.e. riboflavin, RB; flavine-mononucleotide, FMN; and Ravine-adeninedinucleotide, FAD), B12, C, and vitamin Bl 1 as folic acid in serum and red blood cells. The blood samples were collected into a vacuum tube from an antecubital vein with the use of a tourniquet. All samples were taken between 9 AM and noon under similar conditions.
VITAMIN ASSAYS. The concentrations of the vitamins A, total 82 (i.e., its three constituents FIB, FMN and FAD), and total C (ascorbic acid and dehydro-ascorbic acid) were determined with high-performance detection after modification of procedures previously described (18). The equipment used comprised automatic sample injectors with cooling module (Waters Wisp model 71108 and 712B, Waters Assoc., Milford, MA, USA), HPLC liquid solvent pumps (models 510, 590, Waters Assoc.), spectrofluorometers (RF-530, and RF-540, Shimadzu Corp., Kyoto, Japan), and integrators CR3A (Shimadzu). The assay of vitamin A used heparinized plasma and retinol acetate as the internal standard (18). Fluorescence excitation and emission wavelengths were 348 nm and 470 nm. The characteristics of the assay including precision (19) are given in Table II. Vitamin 82 (RB, FMN, and FAD) was assayed by the procedures described previously (20-22). Heparinized whole blood samples were denatured with trichloroacetic acid (TCA) and after centrifugation, the supernatant was injected onto a polar-bonded phase column (Chrompack) which was in line with a Chromsep Hypersil APB-2 column @urn particles, 100 x 4.6 mm I.D., Chrompack). The mobile phase consisted of 0.175 M NaH,PO, in 10% methanol and had a flow rate of 0.4 mllmin. The fluorescence wavekngths of excitation and emission were 470 nm and 520 nm. The characteristics of the assay are given in Table II. The assays of total vitamin C in heparinized whole blood were performed as described by Speek et al. (23). The characteristics of the assay are given in Table II.
SEPTEMBER 1991 VOL. 44 NO. 3
279
CONTRACEPTION
Table II. Characteristics of the vitamin assays applied in the present study
Vitamin’
Sample
Dimension
Linearity
Minimum Cone
Precision’ Runs
Mean
CVw
CV.
A
plasma
umol/L
0.06-40
0.06
24
0.96
1.1%
5.3%
82, FMN
blood’
nmol/L
1o-1000
10
24
90
3.9%
8.2%
FAD
blood’
nmol/L
1o-1 000
10
24
507
4.0%
6.0%
812
plasma
pmol/L
40-l 000
40
25
293
2.3%
5.3%
24
910
1.9%
2.9%
25
5.5
2.1%
6.0%
25
35
1.3%
2.9%
25
30
5.2%
9.1%
Folic acid
plasma
nmol/L
1 .1-E
Folic acid
ery’s cells3
nmol/L
48
C
blood’
umol/L
0.3-200
1.1
0.3
‘Vitamin A as retinol; 82 as the three vitamins riboflavine, RB; flavine-monocleotide, FMN and flevine-adeninedinucleotide, FAD; 812 as cyanocobalamine; total C as ascorbic and dehydroascorbic acid. ‘Heparinized whole blood. ‘Red blood cell concentration of folic acid calculated from whole blood hemolysate and plasma. ‘Calculated for the means of duplicate determinations and given as the percent coefficient of variation (CV) of both within (CV,) and between (CV,) assay variabilities.
Vitamin 812 and folic acid were determined simultaneously with the Dualcount SPB (Solid Phase Boil) Radioassay (Diagnostic Products Corporation, Los Angeles, CA, USA) in plasma and in the case of folic acid also in red cells. The assay makes use of a dual label tracer solution (57Co-vitamin 812 and ‘*‘I-folic acid), and a suspension of the solid phase binders for vitamin 812 and folic acid. After incubation and bound-free separation by centrifugation, the radioactivity of the bound fraction is counted and the concentrations of’ the analytes are calculated from logit-log transformation of the data. The characteristics of the assay are given in Table II.
STATISTICS. Comparison of the bivariate distribution of both baseline values (days 3 and 23 of cycle 1) between the two groups was done by means of Hoteling’s T2-test. If the values on day 3 hardly differed from those measured on day 23, a comparison by means of Student’s two-sample t-test of the avaraged baseline values over these two
SEPTEMBER 1991 VOL. 44 NO. 3
CONTRACEPTION
days between the group using OC and the control group of non-OC users was made. The population means on the successive times of measurements are designated ml through m6. The effect (E) of multivitaminand folic acid supplementation was defined as E = 0.25.[(m3+m4+m5+m6) - 2(ml +m2)]. Within both groups, the statistical significance of the estimate for this effect was tested by means of Student’s one-sample test. Between both groups, this effect is compared by means of Students two-sample test. The level of significance was 0.05.
RESULTS For both groups, the mean baseline values of vitamin A, total 82, 82 components RB, FMN and FAD, 812, C, and folic acid in serum and red blood cells are presented in Figure 1 as well as the changes in vitamin levels during the study. Comparison of the baseline values of vitamin A as determined on days 3 and 23 of the first cycle of the two groups revealed significantly higher levels in the OC group, whereas vitamin 812 levels were significantly lower in the OC group. Vitamin total 82, RB, FMN, FAD, and C did not differ between the two groups. OC use did not alter the levels of folic acid in serum and red blood cells, since comparison of the baseline values revealed no significantly different levels between the two groups. The estimate of the effects of multivitamin and folic acid supplementation (E + sem) on the vitamin and folate status within each group showed unaltered levels of vitamin A and vitamin 812 in each of the two groups, whereas all other vitamins tested showed significant increases (pcO.01). All the observed effects were consistent with either group, since no significant differences could be demonstrated between the groups (p>O.lO). Supplementation also increased significantly the folate concentration in serum and red blood cells for both groups, but no significant differences could be demonstrated between the groups. The increase of the red blood cell folate concentrations continued during the whole study, while serum folate levels reached a steady-state level after one month of supplementation. The increase of the serum folate levels was higher in the non-OC group. There was no significant correlation between the mean serum folate concentrations and its increase (E) during supplementation within either group (p=O.85 for OC users and p=O.95 for non-OC users).
DISCUSSION This study was performed in two groups of healthy non-pregnant Caucasian women with similar population characteristics. The study was conducted during four months, so the possible effect of seasonal vatiations was the same for both groups, In the Netherlands, multivitamin preparations are relatively seldom used, no subject used vitamins on a regular basis previous to the study. In this report we have shown that women using OC have significantly higher vitamin A serum levels and significantly lower vitamin 812 levels. Supplementation did not affect the levels of vitamin A and vitamin 812, and this is a rather suprising observation, since both vitamins were part of the supplementation. The rise in vitamin A level in OC users is probably due to an increase in retinol binding protein, which is due to the oestrogen-containing oral contraceptive (24,25). little effect on plasma retinol levels during vitamin supplementation among generally well-nourished individuals has been reported earlier (26). In the latter study, 10,ooO IU vitamin A was
SEPTEMBER 1991 VOL. 44 NO. 3
281
CONTRACEPTION
baseline
vitamin
ntotion
upplem
1 .o 3
23
3
cycle 1
cycle 2
23
cycle 3
3
23
cycle 4
375:A--AOC O-0
$ s2
350 -
+ z E
325
+ Fz
300
non-lx
/
T t-
1,
’
,
,’ /’
Y,-’ 0 z -i
i,? 275
’
CT? .z 0
baseline
vitamin
Supplementation
256 7
F
3 cycle
35
23 1
3 cycle 2
23
cycle 3
3
23
cycle 4
i boseline
vitamin
supplementation
307 3 cycle
Fig 1.
23 1
3 cycle 2
23
cycle 3
3
23
cycle 4
Changes in serum vitamin A, total B2 and C before and during multivitamin supplementation in women with (A-A ) and without (0-e ) oral contraceptive use. (pg 1 of 3).
SEPTEMBER 1991 VOL. 44 NO. 3
CONTRACEPTION
3 cycle
300 .A-AOC O-0
23
I
1
cycle 3
i3
;
2’3 cycle 4
non-ac
vitamin
baseline
ntotion
upplem
225 3
23
cycle
1
3 cycle 2
23
3
23
cycle 4
cycle 3
60 :A---A ‘*--0
DC non-oc
55.
baseline
vitamin
9 upplementation
30, 3 cycle
Fig 1.
23 1
3 cycle 2
23
cycle 3
3
23
cycle 4
Changes in serum vitamin 62 components RB, FMN and FAD before and during multivitamin supplementation in women with ( A-A ) and without ( 0-O ) oral contraceptive use. (continued, pg 2 of 3).
SEPTEMBER 1991 VOL. 44 NO. 3
283
CONTRACEPTION
300
oc non-cc
L-A
:i B-0
2 E c
250
I *-
!/ i
200 I I
1
I I I I /
/’ /’ baseline
c ‘,
0
a-m 3
;
upplerr 3
cycle 2
cycle 1
% 0 D : m
itamin
23
23
!ntation 3
23
cycle 4
cycle 3
zooo-
1500-
P K .G 0 4
lOOObaseline
.z
9
9-m
,vitamin
upplem
500 3
3
23
cycle 1
23
cycle 3
cycle 2
iI ntation
3
23
cycle 4
I
‘-0
I
I‘\A
I
itominlsupplen ntation
3 cycle
Fig 1.
284
23 1
3 cycle 2
23
cycle 3
3
2’3
cycle 4
Changes in serum folate concentration, red blood cell folate concentration and serum vitamin 812 before and during multivitamin supplementation in women with ( A-A ) and without ( 0-O ) oral contracepYve use. (continued, pg 3 of 3).
SEPTEMBER 1991 VOL. 44 NO. 3
CONTRACEPTION
used and these authors noted only a small but measurable increase in retinol levels. Smaller amounts of vitamin A supplementation is not likely to change the serum retinol levels, as can be concluded from our results. A daily dose of 25,000 IU vitamin A each day for the first 3 months of pregnancy resulted in urogenital abnormalities in man (27). We conclude that a daily supplementation of 1808 IU vitamin A cannot cause toxic or teratogenic levels. Low mean serum vitamin 812 levels are usually measured in OC users (15,16,28,29). In the present study, the vitamin 812 concentration in OC users was significantly lower than in the control group. It is difficult to explain the mechanism behind these observations. No sign of anaemia was seen in both groups; mean haematocrlt values did not differ. Some authors suggest that OC treatment increases the tissue avidity for vitamin 812, resulting in a redistribution of the vitamin (16). OC might inhibit the production of transcobalamin 1, which is involved in vitamin 812 transport in plasma (12). Lower vitamin 812 concentrations in OC users are seen both in populations with adequate and inadequate nutritional conditions. Absorption and excretion of vitamin 812 in OC users are not different from other women (29). Multivitamin supplementation did not change the vitamin 812 concentration both in OC users and controls, suggesting that the lower vitamin 812 concentration in OC users does not reflect a situation of higher vitamin 812 demand, which can be seen during pregnancy usually due to an underlying condition (30). In our study, sex steroids caused no differences in concentrations of vitamin total B2, 82 components FIB, FMN and FAD, and C. Also the effects of supplementation were the same in OC users and non-users. FAD comprised 80% of the concentration of total vitamin 82 and is therefore the most important vitamin 82 component. Supplementation affects both FAD and total vitamin 82 similarly. Much controversy exists about the effects of OC treatment on folate metabolism. Several authors reported significantly lower serum and red blood cell folate levels in OC users (15,31,32), others have failed to confirm these observations (3334). We could not demonstrate differences in folate levels both in plasma and red blood cells between OC users and controls. Folate levels in plasma and red blood cells increased significantly after a daily oral supplementation of 5 mg during 4 weeks, but the observed effects were consistent with either group. Folate concentrations in red blood cells increased logistically in both groups during supplementation. This confirms earlier observations (35). We conclude that there is no evidence that OC treatment per se induces folate deficiency and that no folate supplementation is required for women during OC treatment and after discontinuing OC use. Oral supplementation might be useful in women with increased folate needs caused by repeated pregnancies, iron deficiency, malnutrition, blood loss or anticonvulsant use. As we have shown, vitamin A levels are significantly increased in OC users. Caution is needed regarding daily supplementation of vitamin A especially periconceptionally to avoid toxic or teratogenic levels; supplemetation of 1000 IU of vitamin A does not approach these levels. The effects of supplementation was not altered by OC use.
REFERENCES
1.
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2.
Wilson JG, Warkany J. Cardiac and aortic arch anomalies in the offspring of vitamin Adeficient rats correlated with similar human anomalies. Pediatrics 1950;5:708-25.
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Nelson MM, Wright HV, Asling CW, Evans HM. Multiple congenital abnormalities resulting from transitory deficiency of pteroylglutamic acid during gestation in the rat. J Nutrition 195556349-70.
4.
Davis SD, Nelson T, Shepard TH. Teratogenicity of vitamin BGdeficiency: omphalocele skeletal and neural defects and splenic hypoplasia. Science 1970; 169: 1329-30.
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Laurence KM, James N, Miller MH, Tennant GB, Campbell H. Double-blind randomised controlled trial of folate treatment before conception to prevent recurrence of neural tube defects. Br Med J 1981;282:1509-11.
8.
Milunsky A, Jick H, Jick SS, et al. Multivitamin/folic acid supplementation in early pregnancy reduces the prevalence of neural tube defects. J Am Med Assoc 1989;262:2847-52.
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Mills JL, Rhoads GG, Simpson JL, et al. The absence of a relation between the periconceptional use of vitamins and neural tube defects. N Engl J Med 1989;321:430-35.
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Mulinare J, Corder0 JF, Erickson JD, Berry RJ. Periconceptional use of multivitamins and the occurrence of neural tube defects. J Am Med Assoc 1968;260:3141-45.
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Cockroft DL. Vitamin deficiencies and neural-tube studies. Hum Reprod 1991;6:148-57.
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Wynn V. Vitamins and oral contraceptive use. Lancet 1975;i:561.
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Ahmed F, Bamji MS, lyeugar L. Effects of oral contraceptive 812 metabolism. Am J Clin Nutr 1975;28:606.
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Thorp VJ. Effect of oral contraceptive agents requirements. J Am Diet Assoc 1980;76:581-84.
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Shojania AM. Oral contraceptives Can Med Assoc J 1982;126:244-47.
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Larsson-Cohn U. Oral contraceptives Gynecol 1975;121 (1):&t-9.
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SanfiliPPO JS, Liu YK. Vitamin B12 deficiency and infertility: Report of a case. lnt J Fertil 1991;36:36-38.
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Collins CA, Chow CK. Determination of vitamin A and vitamin A acetate by highPerformance liquid chromatography with florescence detection. J Chromatography 1984;317:349-54.
defects:
on
human
and animal
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vitamin
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mineral
: effect on folate and vitamin 812 metabolism. and vitamins
: A review. Am J Obstet
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CONTRACEPTION
19.
Rodbard D. Statistical quality control radioimmunoassay and immunoradiometric
20.
Speek AJ, van Schaik F, Schrijver J, Scheurs WHP. Determination of the 82 vitamer flavin-adenine dinucleotide in whole blood by high-performance liquid chromatography with fluorometric detection. J Chromatography 1982;228:31 l-1 6.
21.
Floridi A, Palerini CA, Fini C. High-performance liquid chromatography analysis of flavin-adenine dinucleotide in whole blood. Int J Vit Nutr Res 1985;55:187-91.
22.
lchinose N, Adachi K. Determination of 82 vitamers in the body fluid of plankton using high-performance liquid chromatography with fluorescence detection. Analyst 1986;i 11:391-94.
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Speek J, Schrijver J, Scheurs WHP. Fluorometric determination of total vitamin C in whole blood by HPLC with precolumn derivatization. J Chromatography 1984;305:53-60.
24.
Yeung DL. Effects of oral contraceptives and the rat. Am J Clin Nutr 1974;27:125.
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Rossner S, Larsson-Cohn U, Carlsson LA, Boberg J. Effects of an oral contraceptive agent on plasma lipids, plasma lipoproteins, the intravenous fat tolerance and the post-heparin lipoprotein lipase activity. Acta Med Stand 1971 ;l go:301 -05.
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Willet WC, Stampfer HJ, Underwood BA, et al. Vitamin A supplementation and plasma retinol levels : A randomised trial among women. J NCI 1984;73:144549.
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Bernhardt IB, Dorsey DJ. Hypervitaminosis A and congenital the human infant. Obstet Gynaecol 1974;43:750-55.
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Wertalik LF, Metz EN, Lobuglio AF, Balcerzak SP. Decreased with oral contraceptive use. J Am Med Assoc 1972221 :1371.
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Hjelt K, Brynskov J, Hippe E, Lundstrom P, Munck 0. Oral contraceptives and the cobalamin (vitamin 812) metabolism. Acta Obstet Gynecol Stand 1985$4:59X%
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Shojania AM. Folic acid and vitamin 812 deficiency neonatal period. Clin Perinat 1984;11:433-59.
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Martinez 0, Roe DA. Effect of oral contraceptives pregnancy. Am J Obstet Gynecol 1977;128:255-61.
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Grace E, Emans J, Drum DE. Hematologic abnormalities take oral contraceptive pills. J Ped 1982;101:771-74.
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Spray GH, 1972;2:167.
Burns DG. Folate deficiency
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on vitamin A metabolism
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on blood
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folate
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34.
Stephens HEM, Craft I, Peters TJ, Hoffbrand metabolism. Clin Sci 1972;42:405-14.
AV. Oral contraceptives
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Heseker H, Schmitt G. Effect of longterm supplementation of folate status in plasma and etythrocytes. J Nutr Sci Vitamin01 1987;33:163-68.
and folate
on folate
SEPTEMBER 1991 VOL. 44 NO. 3
PIM NM MOOIJ’,
CHRIS MG THOMAS’,
WIM H DOESBURG’,
’ DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY, OF MEDICAL STATISTICS, ACADEMIC HOSPITAL NIJMEGEN, THE NETHERLANDS
USERS
TOM KAB ESKES’
AND a DEPARTMENT ST RADBOUD,
ABSTRACT
The effects of oral contraceptives (OC) containing 30 ug of ethinyl oestradiol and of subsequent multivitamin and folic acid supplementation on vitamin A, total 82 [including its three individual constituents, i.e. riboflavine, RB; flavine-mono-nucleotide, FMN; and flavine-adeninedinucleotide, FAD], 812, C and folate concentration in serum and red blood cells have been studied in a group of 59 non-pregnant female volunteers. The group taking OC comprised 28 women while 31 women were included in the group of non-OC users serving as the controls. The women were studied for four cycles. Blood samples were taken on days 3 and 23 of the first cycle to obtain baseline values of each analyte. Multivitamin and folic acid supplementation started on day 1 of the second cycle and this was continued daily throughout three consecutive cycles until the end of the study. Vitamin A levels were significantly higher and vitamin 812 levels were significantly lower in the group using OC. Comparison of the baseline values of vitamin total 82, FAD, C, serum and red blood cell folate as determined on days 3 and 23 of the first cycle of the two groups compared revealed no significant differences. Multivitamin and folic acid supplementation did not affect the concentrations of vitamin A and vitamin B12 with either group, whereas all other vitamins increased significantly in both groups. The consistency of each effect of multivitamin supplementation between the two groups was also tested. The degree of these effects was not statistically different between both groups. The results suggest that the vitamin status is indeed affected by OC treatment, but the effects of multivitamin supplementation are not different in OC and non-OC users. Supplementation during OC use or just after discontinuing treatment cannot be justified for healthy young women. However, in the case of women with a critical vitamin balance or higher folate needs, multivitamin supplementation may be considered.
Reprint requests to: PIM NM Academic Hospital ST Radboud, Submitted for publication Accepted for publication
MOOIJ, Department of Obstetrics and Gynaecology, PO BOX 9101, 6500 HB Nijmegen, the Netherlands
April 8, 1991 July 15, 1991
SEPTEMBER 1991 VOL. 44 NO. 3
277
INTRODUCTION During the last decade much attention has been paid to the role of folate and vitamin levels in early pregnancy. One focus of interest is the teratogenic effect of high doses of vitamin A in man. The teratogenic potential of vitamin A in animals had been recognised more than 30 years ago (1). Deficiencies of many individual vitamins during pregnancy in animals have been shown to have devastating effects on the embryo and may result in growth retardation, intrauterine death and major congenital abnormalities (2-5). Obsenrationai studies suggested vitamin deficiencies as an aetiological factor for neural tube defects (NTDs) in man (6,7). Three studies on the possible association between the
vitamin intake of pregnant women and the presence of neural tube defects have been conducted in the USA and have given differing results (8,9,10). Although there is strong evidence for an association between maternal vitamin status and the outcome of pregnancy, this is not universally accepted, due to imperfections of study design (11). Since the introduction of oral contraceptives (OC) by Pincus in 1956, much controversy exists about the general and nutrient metabolic side effects of the contraceptive steroids. In recent years some of the metabolic side effects have been linked to vitamin nutritional status (12). A number of papers have described alterations of vitamin status in users of oestrogen-containing OC (13-15). Several authors suggest that women using combined OC tend to have raised plasma concentrations of vitamin A and reduced concentrations of vitamin 812 and foiic acid (12,15,16). A possible association between vitamin 812 deficiency and infertility has been reported recently (17). Because high levels of vitamin A can be teratogenic and may induce fetal abnormalities,, and folic acid deficiency may lead to failures in the process of neurulation, one could postulate that conception in women immediately after discontinuing OC treatment could have an adverse effect on reproduction. The aim of the present investigation was to evaluate the vitamin and folic acid status in fertile women and the effects of OC treatment on these parameters. The effects of multivitamin and folic acid supplementation on vitamin and foiate status in both groups were also studied.
MATERIAL
AND
METHODS
SUBJECTS. Two groups of non-pregnant, non-lactating female volunteers participated in the study after giving informed consent. Approval was given first by the ethics committee of the University Hospital. All women were between 19 and 39 years of age. The following criteria were met for inclusion: nuliigravidae under 40 years of age; no recent blood transfusion; no history of gastrointestinal or kidney disease; no recent surgery; no special diet; and in good general health. Population characteristics are given in Table I. All subjects were questioned about their general health condition, family history, drinking habits, use of drugs, social class and menstrual cycle. The subjects were studied for four consecutive cycles, a pill cycle was considered as the period from the first day of pill intake until the next first day of intake. The women used mono-, bi- or multiphasic OC containing 30 ug of estrogen. In order to obtain comparative baseline data, blood samples were taken during cycle 1 on days 3 and 23 before vitamin supplementation was started on day one of the second cycle and given daily until the end of the study. The supplement offered was Gravitamon (Chefaro BV Oss, The Netherlands) one tablet per day and folic acid 5 mg per day orally. Gravitamon contains vitamin A [retinol] 1000 IU, Bl [thiamine] 3 mg, 82 [riboflavine] 2 mg, 83 jnicotinamide] 110 mg, B6 [pyridoxine] 1 mg, 812 (cyanocobalamine] 1 ug, rutoside 10 mg, vitamin C
278
SEPTEMBER
1991 VOL. 44 NO. 3
CONTRACEPTION Table I. The general characteristics
of the groups studied
group taking oral contraceptives
No. of subjects Age [mean] range Body weight [mean] range Height [mean] range
group not taking oral contraceptives
(years)
28 29.6
(years) (kg)
19-36 62
21 - 39 61
l”s m
48 - 78 1.68
39 - 110 1.67
(m)
1.57
- 1.84
31 30.6
1.49
- 1.84
[ascorbic acid] 50 mg, vitamin D [cholecalciferol] 400 IU, vitamin E [tocoferylacetate] 1 mg, calcium carbonate 500 mg, ferrous fumurate equivalent to 16 mg Fe, copper carbonate 0.1 mg and zinc sulphate 1 mg. To study the effects of multivitamin supplementation after one cycle of treatment, blood samples were taken on days 3 and 23 of the 3rd and 4th cycle to assess vitamins A, total 62 (including its three individual constituents, i.e. riboflavin, RB; flavine-mononucleotide, FMN; and Ravine-adeninedinucleotide, FAD), B12, C, and vitamin Bl 1 as folic acid in serum and red blood cells. The blood samples were collected into a vacuum tube from an antecubital vein with the use of a tourniquet. All samples were taken between 9 AM and noon under similar conditions.
VITAMIN ASSAYS. The concentrations of the vitamins A, total 82 (i.e., its three constituents FIB, FMN and FAD), and total C (ascorbic acid and dehydro-ascorbic acid) were determined with high-performance detection after modification of procedures previously described (18). The equipment used comprised automatic sample injectors with cooling module (Waters Wisp model 71108 and 712B, Waters Assoc., Milford, MA, USA), HPLC liquid solvent pumps (models 510, 590, Waters Assoc.), spectrofluorometers (RF-530, and RF-540, Shimadzu Corp., Kyoto, Japan), and integrators CR3A (Shimadzu). The assay of vitamin A used heparinized plasma and retinol acetate as the internal standard (18). Fluorescence excitation and emission wavelengths were 348 nm and 470 nm. The characteristics of the assay including precision (19) are given in Table II. Vitamin 82 (RB, FMN, and FAD) was assayed by the procedures described previously (20-22). Heparinized whole blood samples were denatured with trichloroacetic acid (TCA) and after centrifugation, the supernatant was injected onto a polar-bonded phase column (Chrompack) which was in line with a Chromsep Hypersil APB-2 column @urn particles, 100 x 4.6 mm I.D., Chrompack). The mobile phase consisted of 0.175 M NaH,PO, in 10% methanol and had a flow rate of 0.4 mllmin. The fluorescence wavekngths of excitation and emission were 470 nm and 520 nm. The characteristics of the assay are given in Table II. The assays of total vitamin C in heparinized whole blood were performed as described by Speek et al. (23). The characteristics of the assay are given in Table II.
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279
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Table II. Characteristics of the vitamin assays applied in the present study
Vitamin’
Sample
Dimension
Linearity
Minimum Cone
Precision’ Runs
Mean
CVw
CV.
A
plasma
umol/L
0.06-40
0.06
24
0.96
1.1%
5.3%
82, FMN
blood’
nmol/L
1o-1000
10
24
90
3.9%
8.2%
FAD
blood’
nmol/L
1o-1 000
10
24
507
4.0%
6.0%
812
plasma
pmol/L
40-l 000
40
25
293
2.3%
5.3%
24
910
1.9%
2.9%
25
5.5
2.1%
6.0%
25
35
1.3%
2.9%
25
30
5.2%
9.1%
Folic acid
plasma
nmol/L
1 .1-E
Folic acid
ery’s cells3
nmol/L
48
C
blood’
umol/L
0.3-200
1.1
0.3
‘Vitamin A as retinol; 82 as the three vitamins riboflavine, RB; flavine-monocleotide, FMN and flevine-adeninedinucleotide, FAD; 812 as cyanocobalamine; total C as ascorbic and dehydroascorbic acid. ‘Heparinized whole blood. ‘Red blood cell concentration of folic acid calculated from whole blood hemolysate and plasma. ‘Calculated for the means of duplicate determinations and given as the percent coefficient of variation (CV) of both within (CV,) and between (CV,) assay variabilities.
Vitamin 812 and folic acid were determined simultaneously with the Dualcount SPB (Solid Phase Boil) Radioassay (Diagnostic Products Corporation, Los Angeles, CA, USA) in plasma and in the case of folic acid also in red cells. The assay makes use of a dual label tracer solution (57Co-vitamin 812 and ‘*‘I-folic acid), and a suspension of the solid phase binders for vitamin 812 and folic acid. After incubation and bound-free separation by centrifugation, the radioactivity of the bound fraction is counted and the concentrations of’ the analytes are calculated from logit-log transformation of the data. The characteristics of the assay are given in Table II.
STATISTICS. Comparison of the bivariate distribution of both baseline values (days 3 and 23 of cycle 1) between the two groups was done by means of Hoteling’s T2-test. If the values on day 3 hardly differed from those measured on day 23, a comparison by means of Student’s two-sample t-test of the avaraged baseline values over these two
SEPTEMBER 1991 VOL. 44 NO. 3
CONTRACEPTION
days between the group using OC and the control group of non-OC users was made. The population means on the successive times of measurements are designated ml through m6. The effect (E) of multivitaminand folic acid supplementation was defined as E = 0.25.[(m3+m4+m5+m6) - 2(ml +m2)]. Within both groups, the statistical significance of the estimate for this effect was tested by means of Student’s one-sample test. Between both groups, this effect is compared by means of Students two-sample test. The level of significance was 0.05.
RESULTS For both groups, the mean baseline values of vitamin A, total 82, 82 components RB, FMN and FAD, 812, C, and folic acid in serum and red blood cells are presented in Figure 1 as well as the changes in vitamin levels during the study. Comparison of the baseline values of vitamin A as determined on days 3 and 23 of the first cycle of the two groups revealed significantly higher levels in the OC group, whereas vitamin 812 levels were significantly lower in the OC group. Vitamin total 82, RB, FMN, FAD, and C did not differ between the two groups. OC use did not alter the levels of folic acid in serum and red blood cells, since comparison of the baseline values revealed no significantly different levels between the two groups. The estimate of the effects of multivitamin and folic acid supplementation (E + sem) on the vitamin and folate status within each group showed unaltered levels of vitamin A and vitamin 812 in each of the two groups, whereas all other vitamins tested showed significant increases (pcO.01). All the observed effects were consistent with either group, since no significant differences could be demonstrated between the groups (p>O.lO). Supplementation also increased significantly the folate concentration in serum and red blood cells for both groups, but no significant differences could be demonstrated between the groups. The increase of the red blood cell folate concentrations continued during the whole study, while serum folate levels reached a steady-state level after one month of supplementation. The increase of the serum folate levels was higher in the non-OC group. There was no significant correlation between the mean serum folate concentrations and its increase (E) during supplementation within either group (p=O.85 for OC users and p=O.95 for non-OC users).
DISCUSSION This study was performed in two groups of healthy non-pregnant Caucasian women with similar population characteristics. The study was conducted during four months, so the possible effect of seasonal vatiations was the same for both groups, In the Netherlands, multivitamin preparations are relatively seldom used, no subject used vitamins on a regular basis previous to the study. In this report we have shown that women using OC have significantly higher vitamin A serum levels and significantly lower vitamin 812 levels. Supplementation did not affect the levels of vitamin A and vitamin 812, and this is a rather suprising observation, since both vitamins were part of the supplementation. The rise in vitamin A level in OC users is probably due to an increase in retinol binding protein, which is due to the oestrogen-containing oral contraceptive (24,25). little effect on plasma retinol levels during vitamin supplementation among generally well-nourished individuals has been reported earlier (26). In the latter study, 10,ooO IU vitamin A was
SEPTEMBER 1991 VOL. 44 NO. 3
281
CONTRACEPTION
baseline
vitamin
ntotion
upplem
1 .o 3
23
3
cycle 1
cycle 2
23
cycle 3
3
23
cycle 4
375:A--AOC O-0
$ s2
350 -
+ z E
325
+ Fz
300
non-lx
/
T t-
1,
’
,
,’ /’
Y,-’ 0 z -i
i,? 275
’
CT? .z 0
baseline
vitamin
Supplementation
256 7
F
3 cycle
35
23 1
3 cycle 2
23
cycle 3
3
23
cycle 4
i boseline
vitamin
supplementation
307 3 cycle
Fig 1.
23 1
3 cycle 2
23
cycle 3
3
23
cycle 4
Changes in serum vitamin A, total B2 and C before and during multivitamin supplementation in women with (A-A ) and without (0-e ) oral contraceptive use. (pg 1 of 3).
SEPTEMBER 1991 VOL. 44 NO. 3
CONTRACEPTION
3 cycle
300 .A-AOC O-0
23
I
1
cycle 3
i3
;
2’3 cycle 4
non-ac
vitamin
baseline
ntotion
upplem
225 3
23
cycle
1
3 cycle 2
23
3
23
cycle 4
cycle 3
60 :A---A ‘*--0
DC non-oc
55.
baseline
vitamin
9 upplementation
30, 3 cycle
Fig 1.
23 1
3 cycle 2
23
cycle 3
3
23
cycle 4
Changes in serum vitamin 62 components RB, FMN and FAD before and during multivitamin supplementation in women with ( A-A ) and without ( 0-O ) oral contraceptive use. (continued, pg 2 of 3).
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283
CONTRACEPTION
300
oc non-cc
L-A
:i B-0
2 E c
250
I *-
!/ i
200 I I
1
I I I I /
/’ /’ baseline
c ‘,
0
a-m 3
;
upplerr 3
cycle 2
cycle 1
% 0 D : m
itamin
23
23
!ntation 3
23
cycle 4
cycle 3
zooo-
1500-
P K .G 0 4
lOOObaseline
.z
9
9-m
,vitamin
upplem
500 3
3
23
cycle 1
23
cycle 3
cycle 2
iI ntation
3
23
cycle 4
I
‘-0
I
I‘\A
I
itominlsupplen ntation
3 cycle
Fig 1.
284
23 1
3 cycle 2
23
cycle 3
3
2’3
cycle 4
Changes in serum folate concentration, red blood cell folate concentration and serum vitamin 812 before and during multivitamin supplementation in women with ( A-A ) and without ( 0-O ) oral contracepYve use. (continued, pg 3 of 3).
SEPTEMBER 1991 VOL. 44 NO. 3
CONTRACEPTION
used and these authors noted only a small but measurable increase in retinol levels. Smaller amounts of vitamin A supplementation is not likely to change the serum retinol levels, as can be concluded from our results. A daily dose of 25,000 IU vitamin A each day for the first 3 months of pregnancy resulted in urogenital abnormalities in man (27). We conclude that a daily supplementation of 1808 IU vitamin A cannot cause toxic or teratogenic levels. Low mean serum vitamin 812 levels are usually measured in OC users (15,16,28,29). In the present study, the vitamin 812 concentration in OC users was significantly lower than in the control group. It is difficult to explain the mechanism behind these observations. No sign of anaemia was seen in both groups; mean haematocrlt values did not differ. Some authors suggest that OC treatment increases the tissue avidity for vitamin 812, resulting in a redistribution of the vitamin (16). OC might inhibit the production of transcobalamin 1, which is involved in vitamin 812 transport in plasma (12). Lower vitamin 812 concentrations in OC users are seen both in populations with adequate and inadequate nutritional conditions. Absorption and excretion of vitamin 812 in OC users are not different from other women (29). Multivitamin supplementation did not change the vitamin 812 concentration both in OC users and controls, suggesting that the lower vitamin 812 concentration in OC users does not reflect a situation of higher vitamin 812 demand, which can be seen during pregnancy usually due to an underlying condition (30). In our study, sex steroids caused no differences in concentrations of vitamin total B2, 82 components FIB, FMN and FAD, and C. Also the effects of supplementation were the same in OC users and non-users. FAD comprised 80% of the concentration of total vitamin 82 and is therefore the most important vitamin 82 component. Supplementation affects both FAD and total vitamin 82 similarly. Much controversy exists about the effects of OC treatment on folate metabolism. Several authors reported significantly lower serum and red blood cell folate levels in OC users (15,31,32), others have failed to confirm these observations (3334). We could not demonstrate differences in folate levels both in plasma and red blood cells between OC users and controls. Folate levels in plasma and red blood cells increased significantly after a daily oral supplementation of 5 mg during 4 weeks, but the observed effects were consistent with either group. Folate concentrations in red blood cells increased logistically in both groups during supplementation. This confirms earlier observations (35). We conclude that there is no evidence that OC treatment per se induces folate deficiency and that no folate supplementation is required for women during OC treatment and after discontinuing OC use. Oral supplementation might be useful in women with increased folate needs caused by repeated pregnancies, iron deficiency, malnutrition, blood loss or anticonvulsant use. As we have shown, vitamin A levels are significantly increased in OC users. Caution is needed regarding daily supplementation of vitamin A especially periconceptionally to avoid toxic or teratogenic levels; supplemetation of 1000 IU of vitamin A does not approach these levels. The effects of supplementation was not altered by OC use.
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