Effect of oral contraceptives on nutrients III.
Vitamins
ANANDA K.
Y.
S.
KAMRAN
S. C.
DONALD Detroit
PRASAD,
M.D.,
PH.D.,
F.A.C.P
PH.D.*
LEI,
JOANNE
B6, B12, and folic acid
MOGHISSI, STRYKER,
OBERLEAS, and .4&n
Park,
M.D. M.D. PH.D.
Michigan
The interactions of oral contraceptive agents (OCA’s) with vitamins were studied in a large population of women. In the upper socioeconomic groups, higher incidences of abnormal clinical .signs related to vitamin deficiencies were seen in OCA users than in the control subj’ects. Plasma firidoxal phosphate and red cell and serum folate were lower in subjects using OCrls in the upper socioeconomic group as compared to Levels in the control subjects. Reduction in erythrocyte glutamic oxalacetic transaminase (EGOT) activity and elevation in the EGOT-stimulation test were observed in subjects us&g OCAi in both upper and louler socioeconomic groups. These observations suggest a relatively deicient state with respect to vitamins Be and folk acid in OCA users. No signzficant effect on serum vitamin B I2 was observed as a result of OCA administration.
ALTERATIONS in the metabolism of vitamins as a result of the administration of oral contraceptive agents (OCA’s) have been reviewed by several authors. Lumeng and associates’ and Hontz and colleagues* observed a significant reduction of plasma pyridoxal phosphate (PLP) levels in OCA users. Our preliminary data3 suggested that in the high-income groups plasma PLP levels were lower in OCA users as compared to control subjects. However, Brown and co-worker? found no differences in plasma PLP levels between oral contraceptive users and nonusers receiving controlled intakes of vitamin B6.
Table I. Number of subjects in the clinical biochemical studies
No. of studies Group No OCA Pill 1 Pill 2
‘4ccepted January
Ju(v 30, 1975.
21, 1976. 22, 1976.
Reprint requests: Dr. Ananda S. Prasad, Department Medicine, Waylzp State University School of Medicine, 540 E. Canfield, Detroil, Michigan 48201.
A
B
127 37 44 97
103 112 123 105
Wertalik and associate@ and Davis and Smith6 observed a reduction in vitamin B12 levels in women using OCA’s. Reductions in serum folate levels in OCA users have been reported by Shojania and colleagues7 and Streiff.’ Streiff also found that naturally occurring folate polyglutamates were poorly absorbed in volunteers using OCA’s, although the monoglutamate was utilized normally. On the contrary, McLean and coworkers9 and Pritchard and associates” found no alterations in the serum folate levels in OCA users. Our preliminary data indicated that in the high socioeconomic groups erythrocyte folate levels were lower in OCA users as compared to control subjects.3 The objective of this paper is to document further
Supported by Contract No. NIH-NICHHD-NO1 -HD-2-2786, Center for Population Research, National Institute of Child Health and Human Development.
Revised Januaq
use
Resumption of pill (RP)
From the Departments of Medicine and Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, and the Department of Medicine, Veterans Administration Hospital, Allen Park.
Receizwd for publication
and
of
1063
1064
Prasad
et al.
Table
II. Incidence
of clinical
abnormalities Socioeconomic
Abnormality Hair Dry,
PUP
NO OCA
Pill 1
Pill 2
RP
A B A B
15.0 33.0 9.4 22.3
24.3 36.3 18.9 36.6
18.2 16.3 13.6 12.2
5.2 30.5 3.1 24.8
A B
1.6 14.5
10.8 17.9
15.9 8.9
2.1 23.8
0.02
0.001 -
A B A B
7.9 48.5 8.7 48.0
45.9 54.5 35.1 54.5
40.9 41.3 31.8 27.6
7.2 51.4 7.2 44.7
0.001
0.001 -. 0.001
A B A B
1.6 7.8 0 1.9
0.0 6.3 0 1.8
2.3 5.7 2.3 2.4
1.0 8.6 0 4.8
A L* D* B L* D* A L* D* B L* D* A L* D* B L” D*
5.5 0.0 33.0 5.8 2.4 0.0 17.8 5.8 4.7 0.0 10.7 4.9
35.1 2.7 29.5 15.2 16.2 8.1 30.4 5.4 18.9 2.7 15.2 4.5
31.8 2.3 27.6 8.1 22.7 6.8 22.0 8.1 6.8 6.8 17.1 6.5
4.1 1.0 26.7 14.3 3.1 1.0 20.0 6.7 1.0 0.0 8.6 4.8
A B
2.4 12.6
10.8 19.6
9.1 X.1
3.1 19.0
brittle
Easily Lips Angular Teeth Caries
pluckable
lesions
4+
Debris or calculus Tongue Filiform papillary, atrophy Fungiform papillary, hypertrophy Gums Marginal redness or swelling
Swollen papillae
Bleeding
red
gum
Skin Dry, scaling (xerosis) *L = Localized;
(per cent of abnormalities)
-. --
0.005
0.03
0.003 0.03 --
0.00 1
-
0.001
-
-.
0.001
0.00 I
-
-
0.003
0.01
-
-
-
-
D = diffused.
the effects of OCA’s on vitamins B6 and BiZ and folate metabolism in a large number of subjects representing different groups which were classified based on the use of different OCA’s and the socioeconomic levels of the groups.
In this study, female subjects between the ages of 18 and 45 years were divided into eight groups in a factorial arrangement with two socioeconomic levels and four different hormonal statuses (Table I). The criteria of Myrianthopoulos and French” with an income adjustment were utilized in determining the division between the higher (A) and lower (B) socioeconomic classes. Subjects who were not using OCA’s belonged to A-none or B-none groups. A-l and B-l groups consisted of those subjects who used Norinyl* ( 1
mg. of norethindrone and 50 wg of mestranol) for 3 months or more. A-2 and B-2 groups of sub.jects received Ovral* (0.5 mg. of norgestrel and 50 kg of ethinyl estradiol) for three months or more. A-RP and B-RP groups consisted of women who resumed OCA use within five weeks after pregnancy and during lactation. Subjects in each group were divided further into “supplemented” and “nonsupplemented” subgroups according to whether or not they were using vitamin and/or mineral supplementations, according to their own admission. PLP was determined by a simpIe enzymatic assay.‘” Erythrocyte glutamic oxalacetic transaminase @GOT) was measured in a hemolysate of red blood cells. The cells were washed with 0.85 per cent sodium chloride solution and diluted with saline in a proportion of 0.8
*Syntex Laboratories, Alto, California 94304.
*Wyeth Box 8299,
Material and methods
Inc.,
Stanford
Industrial
Park,
Palo
Labs., Div. Philadelphia,
American Home Products Pennsylvania 19101.
Corp.,
P. 0.
Volume Number
125 8
ml. of saline to 1 ml. of packed cells. The test was carried out on 0.1 ml. of a 1: 20 hemolysate. For measurement of PLP stimulation, 0.1 ml. of hemolysate was incubated with 0.1 ml. of PLP (0.5 mg. per milliliter) for 20 minutes at 37” C. prior to the calorimetric determination of EGOT. Erythrocyte and plasma folic acid were determined by a microbiological assay with the use of Lactobacillus cuspi. Serum vitamin Brz was measured by a microbiological assay with the use of Lactobacillus leichmanii. Nutritional and biochemical data were analyzed statistically by analysis of variance.r3 Means were ranked by Duncan’s new multiple range testI Clinical data were statistically analyzed by the chi-square method.
Results Dry skin, easily pluckable hair, angular lesions of the mouth, caries and debris of the teeth, marginal redness, swelling, and bleeding of the gums, filiform papillary atrophy, fungiform papillary hypertrophy of the tongue and glossitis, and scaling of the skin were more frequently observed in the B groups than in the A groups. In the A groups some of the above clinical signs were most common in the nonsupplemented groups of subjects. In the A groups, higher incidences of angular lesions of the mouth, caries and debris of the teeth, and marginal redness, swelling, and bleeding of the gums were seen in the OCA users than in the control group (Table II). The incidences of easily pluckable hair, angular lesions of the mouth, and debris or calculus of the teeth were more frequently observed in the B-l group as compared to the B-none group. A higher incidence of angular lesions of the mouth was seen in the B-RP group as compared to the B-none group. In general, the intake of vitamin Bs and folic acid by OCA users did not differ from that in the control subjects. However, the A-RP group of subjects had a higher intake of vitamin Bc and folic acid than A- 1 and A-2 subjects. Although Group A-supplemented subjects had a higher intake of vitamin Bs and folic acid than the nonsupplemented group, this difference was not very clear in Group B subjects. In general, the intake of vitamin Bs and folic acid was higher in Group A subjects as compared to that in Group B. Results of PLP, EGOT. and per cent stimulation of EGOT are presented in Table III. In Group A, the plasma PLP level was decreased due to OCA administration. A supplementation effect was seen mainly in the A groups. Plasma PLP was found to be lower in the A-RP group than in the A-l and A-2 groups.
Effect
of oral contraceptives
on nutrients.
III
1065
EGOT activities were found to be lower in the RP and OCA groups. This reduction in activities due to OCA was greater in the B groups as compared to the A groups. A supplementation effect was seen in both groups. The per cent stimulation of EGOT was found to be greater in the OCA and RP groups. No socioeconomic or supplementation effect was seen so far as the EGOT stimulation test was concerned. Table IV shows the results of erythrocyte and serum folate and vitamin Br2. Both plasma and red cell folate levels were higher in Group A than in Group B. A supplementation effect was seen in both groups. Erythrocyte and serum folate in the RP groups were higher than in the OCA groups. Duncan’s new multiple range test indicated that the A-RP subjects had higher red cell and serum folate levels than A-none subjects (Table V). Erythrocyte folate in A-l and A-2 groups was decreased in comparison to that in subjects in A-none and A-RP groups. Serum folate in the A-2 group only was decreased when compared to A-none and A-RP groups. Serum vitamin Br2 levels in A-l and B-l groups were lower than those in A-2 and B-2 groups. No other significant differences were found with respect to serum vitamin Blz levels.
Comment In the higher socioeconomic group, greater frequencies of abnormal clinical signs, indicative of malnutrition, were observed in OCA users as compared to control subjects. However, these signs, except for angular lesions of the mouth and debris or calculus of the teeth, were not observed in the lower socioeconomic group of OCA users. An increase in incidence of dental caries in OCA users of the higher socioeconomic group was also observed. In rats injected with OCA, elevated incidences of carious lesions proportional to increased doses of OCA have been reported by Liu and Lin.“’ Furthermore, these authors postulated that the increased dental caries activity of OCA-treated rats may be caused by a decrease in the plasma zinc concentration, since such a reduction has been observed in human subjects and rats receiving OCA. The reduction in the PLP level due to OCA’s was seen only in the upper socioeconomic group of subjects in this study. The subjects in the lower socioeconomic group did not show this effect. The fact that the reduction in EGOT activities due to OCA was greater in the lower than the upper socioeconomic group of subjects tended to support this finding. The activity of EGOT is dependent on the concentration of the coenzyme, PLP. Addition of PLP to the assay mixture
1066
Prasad et al.
Table
III. Levels of PLP and EGOT,
and per cent stimulation
of EGOT
(mean
2 SD.) Plasma PLP (ng. lml. )
croup A-none A-l A-2 A-RP B-none B-l B-2 B-RP P zraluesfor treatment comparisons in nnnlysis of variance P, = none vs. Norinyl, Ovral, and RP P2 = RP vs. Norinyl and Ovral Ps = Norinyl vs. Ovral Income (I) Supplement(S) Interactions I x P, I x P, s x P, IXS IXSXP, IxSxP* *Raitman-Frankel tS, Supplemented;
Table
27.3 9.5 11.7 6.2 9.3 6.1 7.6 12.3 Degrer
2 15.2 r 5.7 IT 8.6 2 5.1 It 4.3 + 3.0 t 5.0 t 10.3
12.6 10.6 7.8 6.6 11.3 11.9 10.1 8.0
k i t k iI r & i
7.3 4.6 2.8 3.0 5.7 6.3 4.0 3.7
of
flwdonl 1
0.001 0.001
-
1 1 1 10 1 1 1 1 1 1
n.05 0.00 1 0.01 0.01 0.025 0.01 0.025
units per milliliter of plasma on a basis of 45 per cent hematocrit NS, nonsupplemented; Avg., average.
IV. Levels of erythrocyte
and serum folic acid and serum BIz (mean 2 S.D.)
Group A-none A-l A-2 A-RP B-none B-l B-2 B-RP P values for treatment comparisons in analysis of variance Pi = None vs. Norinyl, Ovral, and RP P, = RP vs. Norinyl and Ovral P3 = Norinyl vs. Ovral Income (I) Supplement (S) Interactions I x Pz
s 318 204 253 457 23Ort 222 199 263 Degree of freedom 1 1 1 1 1 10 1
~fr 170 + 77 2 119 t 313 81 2 160 1?: 83 lr 214
NS 264 175 197 260 181 200 172 234
t 107 lr 56 t 107 YZ 165 r 73 -c 91 r 65 r 160
0.00 1 0.001 0.001 0.01
‘4Vg 295 135i. 208 430 192 204 176 214
+ 150 64 k 110 r 304 + 77 t 107 + 69 f 180
T&d t&jet 102 28 36 X8 63 55 106 82
t.s
comr-3
nmr-3
CQOl.mm*Tr
+I tl
owwu)cnmn
mom*-em-l*
-3 tt ti
1066
Prasad
et al.
Table
V. Duncan’s
new
Folic acid
multiple
test * for
range
erythrocyte
and
serum
folic
acid
in OCA
A-RP
A-none
B-RP
24-2
B-l
B-Ml?le
430
295
244
208
204
192
Erythrocyte (ng./ml.)
users
(P < 0.05}
.4-l
B-1
185
lib _I_-
Serum (ng./ml.)
A-RP
A-none
B-RP
A-l
A-2
B-l
B-?
7.50
6.30
4.7
4.6
4..55
4.09
3.96
B-lrrlrle 3 .i 3 --_
*Any two means not underscored not significantly different. increases lated
the
cells
and
vitamin tion
to the
so may
in both
socioeconomic
This
indicate
may
subjects.
was higher
deficiency
the the
per
OCA
in our vitamin
cent
and
those
EGOT-stimulation
red
of the stimula-
RP subjects
study, in
is re-
in the
indicator
The
in the groups
of that
activation of PLP
as an additional
of the
a relative
The
concentration
serve
Bs status of EGOT
of EGOT.
activity
inversely
by the same line are significantly
suggesting subjects. test
is a
different.
Any
two means
underscored
by the same line are
blood was sampled at the first visit after the pill \\as started. Our data indicate that the A-RP group ~;ts relatively acid,
deficient
in vitamin
suggesting
adequate
that
adequate
intake
of
in folic l&
was
in-
in this group.
Impairment
of
abnormality
found
related
Bs but
nutritional
to vitamin
glucose
tolerance
is an
in some OCA Bs deficiency.
important
users which
Increased
ma\ bc
resistance
to
The reduction of erythrocyte and serum folate due to OCA’s was observed mainly in the upper socioeconomic group of subjects. This was not seen in the lower
insulin by , peripheral tissues, such as pal-ametrial adipose tissues and diaphragm muscle, in vitro anti ilr vivo was found to be responsible for the irnpairmen~ of. glucose tolerance in experimental animals treated with OCA’s.‘” Murakami” suggested that xanthurenic ac id
socioeconomic
is capable
more than
sensitive plasma
index PLP
of alterations
in vitamin
Bs status
or EGOT.
group.
Our data are consistent with the hypothesis that the subjects in lower socioeconomic groups were already marginally deficient with respect to vitamin B6 and folic acid and, as such, further reduction due to OCA’s could not be detected by our assay. Although an effect of OCA’s on the levels of vitamin B6 and folate was observed in subjects of the upper socioeconomic group, the clinical significance of this observation is not clear. Further studies are needed in order to define the clinical importance of these changes. In our study, groups of subjects resuming the “pill” after pregnancy (A-RP and B-RP) demonstrated mainly the effects of pregnancy inasmuch as they resumed OCA’s within five weeks post partum and the
REFERENCES
1. Lumeng, L., Cleary, R. E., and Li, T. K.: Effect of oral contraceptives on the plasma concentration of pyridoxal phosphate, Am. J. Clin. Nutr. 27: 326, 1974. 2. Hontz, A. C., Gyorgy, P., Balin, H., Rose, C. S., and Shan, D. L., Jr.: Interaction of contraceptive steroids with metabolic functions of vitamin Bg, Am. J. Clin. Nutr. 27: 440, 1974. (Abst.) 3. Prasad, A. S., Lei, K. Y., Oberleas, D., Moghissi, K. S., and Stryker, J. C.: Effect of oral contraceptive agents on nutrients: II. Vitamins, Am. J. Clin. Nutr. 28: 385, 1975.
of attaining
a complex
with
insulin.
thereby
reducing its biological activity. Indeed, a small group of. women in whom carbohydrate tolerance had become impaired
while
using
OCA’s
improved
al’trr
sitamin
Us
administration. Several investigators have suggested that large doses of vitamin B6 should be added to OCA’s indiscriminately. However, the adlrlinistratiolI of a coenzyme may increase apoenzyme synthesis, and this may enhance the plasma amino acicl-lowering effect
of
OCA’s,
which
may
produce
an
unto\cartl
effect in communities where protein Inalnutrition exists. At present. it would appear that the idea of supplementation of B, should be approachcrl ‘autiously since the long-term effect of such therapy is uncertain.
4. Brown, R. R., Rose, D. P., Leklem, J. E.. Linkswiler-. H.. and Anand, R.: Urinary 4-pyridoxic acid. plasma pyridoxal phosphate, and erythrocytc aminotransferase levels in oral contraceptive users receiving controlled intakes of vitamin Be, Am. J. Clin. Nutr. 28: 10, 1975. 5. Wertalik, L. F., Metz, E. N., Lobuglio, A. F., and Balcerzak. S. P.: Decreased serum F&2 levels with oral contrac.eptive use. J. A. M. A. 221: 1371, 1972. 6. Davis, R. E., and Smith, B. K.: Pyridoxal, vitamin B,, and folate metabolism in women taking oral contraceptive agents, S. Afr. Med. J. 48: 1937. 1974.
Volume 125 Number8
Effect of oral contraceptives on nutrients. Ill
7. Shojania. 8. 9.
10.
11.
12.
A. M., Hornady, G., and Barnes, P. H.: Oral contraceptives and serum folate level, Lancet 1: 1376, 1968. Streiff, R. R.: Folate deficiency and oral contraceptives, J. A. M. A. 214: 105, 1970. McLean, F. W., Heine, M. W., Held, R., and Streiff, R. R.: Relationship between the oral contraceptive and folic acid metabolism, AM. J, OBSTET. GYNECOL. 104: 745, 1969. Pritchard, J. A., Scott, D. E., and Whalley, P. J.: Maternal folate deficiency and pregnancy wastage. IV. Effects of folic acid supplements, anticonvulsants, and oral contraceptives, AM. J. OBSTET. GYNECOL. 109: 341, 1971. Myrianthopoulos, N., and French, K.: An application of the U.S. bureau of the Census Socioeconomic Index to a large, diversified patient population, Sot. Sci. Med. 2: 283, 1968. Chabner. B. A., and Livingston, D. M.: A simple enzy-
13. 14.
15.
16.
1069
matic assay for pyridoxal phosphate, Anal. Biochem. 34: 413, 1970. Winer, B. J.: Statistical Principles in Experimental Design, New York, 1962, McGraw-Hill, Book Company, Inc. Liu, F. T. Y., and Lin, S.: Effect of the contraceptive steroids norethynodrel and mestranol on dental caries activity in young adult female rats, J. Dent. Res. 52: 753, 1973. Lei, K. Y., Yang, M. G., Oberleas, D., and Prasad, A. S.: Oral contraceptives: effects on plasma insulin response to glucose and on the response to insulin and P-deoxyglucose uptake by peripheral tissue, Proc. Sot. Exp. Biol. Med. 149: 417, 1975. Murakami, E.: Studies on the xanthurenic acid-insulin complex. I. Preparation and properties, J. Biochem. 63: 573. 1968.
Vitamins
ANANDA K.
Y.
S.
KAMRAN
S. C.
DONALD Detroit
PRASAD,
M.D.,
PH.D.,
F.A.C.P
PH.D.*
LEI,
JOANNE
B6, B12, and folic acid
MOGHISSI, STRYKER,
OBERLEAS, and .4&n
Park,
M.D. M.D. PH.D.
Michigan
The interactions of oral contraceptive agents (OCA’s) with vitamins were studied in a large population of women. In the upper socioeconomic groups, higher incidences of abnormal clinical .signs related to vitamin deficiencies were seen in OCA users than in the control subj’ects. Plasma firidoxal phosphate and red cell and serum folate were lower in subjects using OCrls in the upper socioeconomic group as compared to Levels in the control subjects. Reduction in erythrocyte glutamic oxalacetic transaminase (EGOT) activity and elevation in the EGOT-stimulation test were observed in subjects us&g OCAi in both upper and louler socioeconomic groups. These observations suggest a relatively deicient state with respect to vitamins Be and folk acid in OCA users. No signzficant effect on serum vitamin B I2 was observed as a result of OCA administration.
ALTERATIONS in the metabolism of vitamins as a result of the administration of oral contraceptive agents (OCA’s) have been reviewed by several authors. Lumeng and associates’ and Hontz and colleagues* observed a significant reduction of plasma pyridoxal phosphate (PLP) levels in OCA users. Our preliminary data3 suggested that in the high-income groups plasma PLP levels were lower in OCA users as compared to control subjects. However, Brown and co-worker? found no differences in plasma PLP levels between oral contraceptive users and nonusers receiving controlled intakes of vitamin B6.
Table I. Number of subjects in the clinical biochemical studies
No. of studies Group No OCA Pill 1 Pill 2
‘4ccepted January
Ju(v 30, 1975.
21, 1976. 22, 1976.
Reprint requests: Dr. Ananda S. Prasad, Department Medicine, Waylzp State University School of Medicine, 540 E. Canfield, Detroil, Michigan 48201.
A
B
127 37 44 97
103 112 123 105
Wertalik and associate@ and Davis and Smith6 observed a reduction in vitamin B12 levels in women using OCA’s. Reductions in serum folate levels in OCA users have been reported by Shojania and colleagues7 and Streiff.’ Streiff also found that naturally occurring folate polyglutamates were poorly absorbed in volunteers using OCA’s, although the monoglutamate was utilized normally. On the contrary, McLean and coworkers9 and Pritchard and associates” found no alterations in the serum folate levels in OCA users. Our preliminary data indicated that in the high socioeconomic groups erythrocyte folate levels were lower in OCA users as compared to control subjects.3 The objective of this paper is to document further
Supported by Contract No. NIH-NICHHD-NO1 -HD-2-2786, Center for Population Research, National Institute of Child Health and Human Development.
Revised Januaq
use
Resumption of pill (RP)
From the Departments of Medicine and Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, and the Department of Medicine, Veterans Administration Hospital, Allen Park.
Receizwd for publication
and
of
1063
1064
Prasad
et al.
Table
II. Incidence
of clinical
abnormalities Socioeconomic
Abnormality Hair Dry,
PUP
NO OCA
Pill 1
Pill 2
RP
A B A B
15.0 33.0 9.4 22.3
24.3 36.3 18.9 36.6
18.2 16.3 13.6 12.2
5.2 30.5 3.1 24.8
A B
1.6 14.5
10.8 17.9
15.9 8.9
2.1 23.8
0.02
0.001 -
A B A B
7.9 48.5 8.7 48.0
45.9 54.5 35.1 54.5
40.9 41.3 31.8 27.6
7.2 51.4 7.2 44.7
0.001
0.001 -. 0.001
A B A B
1.6 7.8 0 1.9
0.0 6.3 0 1.8
2.3 5.7 2.3 2.4
1.0 8.6 0 4.8
A L* D* B L* D* A L* D* B L* D* A L* D* B L” D*
5.5 0.0 33.0 5.8 2.4 0.0 17.8 5.8 4.7 0.0 10.7 4.9
35.1 2.7 29.5 15.2 16.2 8.1 30.4 5.4 18.9 2.7 15.2 4.5
31.8 2.3 27.6 8.1 22.7 6.8 22.0 8.1 6.8 6.8 17.1 6.5
4.1 1.0 26.7 14.3 3.1 1.0 20.0 6.7 1.0 0.0 8.6 4.8
A B
2.4 12.6
10.8 19.6
9.1 X.1
3.1 19.0
brittle
Easily Lips Angular Teeth Caries
pluckable
lesions
4+
Debris or calculus Tongue Filiform papillary, atrophy Fungiform papillary, hypertrophy Gums Marginal redness or swelling
Swollen papillae
Bleeding
red
gum
Skin Dry, scaling (xerosis) *L = Localized;
(per cent of abnormalities)
-. --
0.005
0.03
0.003 0.03 --
0.00 1
-
0.001
-
-.
0.001
0.00 I
-
-
0.003
0.01
-
-
-
-
D = diffused.
the effects of OCA’s on vitamins B6 and BiZ and folate metabolism in a large number of subjects representing different groups which were classified based on the use of different OCA’s and the socioeconomic levels of the groups.
In this study, female subjects between the ages of 18 and 45 years were divided into eight groups in a factorial arrangement with two socioeconomic levels and four different hormonal statuses (Table I). The criteria of Myrianthopoulos and French” with an income adjustment were utilized in determining the division between the higher (A) and lower (B) socioeconomic classes. Subjects who were not using OCA’s belonged to A-none or B-none groups. A-l and B-l groups consisted of those subjects who used Norinyl* ( 1
mg. of norethindrone and 50 wg of mestranol) for 3 months or more. A-2 and B-2 groups of sub.jects received Ovral* (0.5 mg. of norgestrel and 50 kg of ethinyl estradiol) for three months or more. A-RP and B-RP groups consisted of women who resumed OCA use within five weeks after pregnancy and during lactation. Subjects in each group were divided further into “supplemented” and “nonsupplemented” subgroups according to whether or not they were using vitamin and/or mineral supplementations, according to their own admission. PLP was determined by a simpIe enzymatic assay.‘” Erythrocyte glutamic oxalacetic transaminase @GOT) was measured in a hemolysate of red blood cells. The cells were washed with 0.85 per cent sodium chloride solution and diluted with saline in a proportion of 0.8
*Syntex Laboratories, Alto, California 94304.
*Wyeth Box 8299,
Material and methods
Inc.,
Stanford
Industrial
Park,
Palo
Labs., Div. Philadelphia,
American Home Products Pennsylvania 19101.
Corp.,
P. 0.
Volume Number
125 8
ml. of saline to 1 ml. of packed cells. The test was carried out on 0.1 ml. of a 1: 20 hemolysate. For measurement of PLP stimulation, 0.1 ml. of hemolysate was incubated with 0.1 ml. of PLP (0.5 mg. per milliliter) for 20 minutes at 37” C. prior to the calorimetric determination of EGOT. Erythrocyte and plasma folic acid were determined by a microbiological assay with the use of Lactobacillus cuspi. Serum vitamin Brz was measured by a microbiological assay with the use of Lactobacillus leichmanii. Nutritional and biochemical data were analyzed statistically by analysis of variance.r3 Means were ranked by Duncan’s new multiple range testI Clinical data were statistically analyzed by the chi-square method.
Results Dry skin, easily pluckable hair, angular lesions of the mouth, caries and debris of the teeth, marginal redness, swelling, and bleeding of the gums, filiform papillary atrophy, fungiform papillary hypertrophy of the tongue and glossitis, and scaling of the skin were more frequently observed in the B groups than in the A groups. In the A groups some of the above clinical signs were most common in the nonsupplemented groups of subjects. In the A groups, higher incidences of angular lesions of the mouth, caries and debris of the teeth, and marginal redness, swelling, and bleeding of the gums were seen in the OCA users than in the control group (Table II). The incidences of easily pluckable hair, angular lesions of the mouth, and debris or calculus of the teeth were more frequently observed in the B-l group as compared to the B-none group. A higher incidence of angular lesions of the mouth was seen in the B-RP group as compared to the B-none group. In general, the intake of vitamin Bs and folic acid by OCA users did not differ from that in the control subjects. However, the A-RP group of subjects had a higher intake of vitamin Bc and folic acid than A- 1 and A-2 subjects. Although Group A-supplemented subjects had a higher intake of vitamin Bs and folic acid than the nonsupplemented group, this difference was not very clear in Group B subjects. In general, the intake of vitamin Bs and folic acid was higher in Group A subjects as compared to that in Group B. Results of PLP, EGOT. and per cent stimulation of EGOT are presented in Table III. In Group A, the plasma PLP level was decreased due to OCA administration. A supplementation effect was seen mainly in the A groups. Plasma PLP was found to be lower in the A-RP group than in the A-l and A-2 groups.
Effect
of oral contraceptives
on nutrients.
III
1065
EGOT activities were found to be lower in the RP and OCA groups. This reduction in activities due to OCA was greater in the B groups as compared to the A groups. A supplementation effect was seen in both groups. The per cent stimulation of EGOT was found to be greater in the OCA and RP groups. No socioeconomic or supplementation effect was seen so far as the EGOT stimulation test was concerned. Table IV shows the results of erythrocyte and serum folate and vitamin Br2. Both plasma and red cell folate levels were higher in Group A than in Group B. A supplementation effect was seen in both groups. Erythrocyte and serum folate in the RP groups were higher than in the OCA groups. Duncan’s new multiple range test indicated that the A-RP subjects had higher red cell and serum folate levels than A-none subjects (Table V). Erythrocyte folate in A-l and A-2 groups was decreased in comparison to that in subjects in A-none and A-RP groups. Serum folate in the A-2 group only was decreased when compared to A-none and A-RP groups. Serum vitamin Br2 levels in A-l and B-l groups were lower than those in A-2 and B-2 groups. No other significant differences were found with respect to serum vitamin Blz levels.
Comment In the higher socioeconomic group, greater frequencies of abnormal clinical signs, indicative of malnutrition, were observed in OCA users as compared to control subjects. However, these signs, except for angular lesions of the mouth and debris or calculus of the teeth, were not observed in the lower socioeconomic group of OCA users. An increase in incidence of dental caries in OCA users of the higher socioeconomic group was also observed. In rats injected with OCA, elevated incidences of carious lesions proportional to increased doses of OCA have been reported by Liu and Lin.“’ Furthermore, these authors postulated that the increased dental caries activity of OCA-treated rats may be caused by a decrease in the plasma zinc concentration, since such a reduction has been observed in human subjects and rats receiving OCA. The reduction in the PLP level due to OCA’s was seen only in the upper socioeconomic group of subjects in this study. The subjects in the lower socioeconomic group did not show this effect. The fact that the reduction in EGOT activities due to OCA was greater in the lower than the upper socioeconomic group of subjects tended to support this finding. The activity of EGOT is dependent on the concentration of the coenzyme, PLP. Addition of PLP to the assay mixture
1066
Prasad et al.
Table
III. Levels of PLP and EGOT,
and per cent stimulation
of EGOT
(mean
2 SD.) Plasma PLP (ng. lml. )
croup A-none A-l A-2 A-RP B-none B-l B-2 B-RP P zraluesfor treatment comparisons in nnnlysis of variance P, = none vs. Norinyl, Ovral, and RP P2 = RP vs. Norinyl and Ovral Ps = Norinyl vs. Ovral Income (I) Supplement(S) Interactions I x P, I x P, s x P, IXS IXSXP, IxSxP* *Raitman-Frankel tS, Supplemented;
Table
27.3 9.5 11.7 6.2 9.3 6.1 7.6 12.3 Degrer
2 15.2 r 5.7 IT 8.6 2 5.1 It 4.3 + 3.0 t 5.0 t 10.3
12.6 10.6 7.8 6.6 11.3 11.9 10.1 8.0
k i t k iI r & i
7.3 4.6 2.8 3.0 5.7 6.3 4.0 3.7
of
flwdonl 1
0.001 0.001
-
1 1 1 10 1 1 1 1 1 1
n.05 0.00 1 0.01 0.01 0.025 0.01 0.025
units per milliliter of plasma on a basis of 45 per cent hematocrit NS, nonsupplemented; Avg., average.
IV. Levels of erythrocyte
and serum folic acid and serum BIz (mean 2 S.D.)
Group A-none A-l A-2 A-RP B-none B-l B-2 B-RP P values for treatment comparisons in analysis of variance Pi = None vs. Norinyl, Ovral, and RP P, = RP vs. Norinyl and Ovral P3 = Norinyl vs. Ovral Income (I) Supplement (S) Interactions I x Pz
s 318 204 253 457 23Ort 222 199 263 Degree of freedom 1 1 1 1 1 10 1
~fr 170 + 77 2 119 t 313 81 2 160 1?: 83 lr 214
NS 264 175 197 260 181 200 172 234
t 107 lr 56 t 107 YZ 165 r 73 -c 91 r 65 r 160
0.00 1 0.001 0.001 0.01
‘4Vg 295 135i. 208 430 192 204 176 214
+ 150 64 k 110 r 304 + 77 t 107 + 69 f 180
T&d t&jet 102 28 36 X8 63 55 106 82
t.s
comr-3
nmr-3
CQOl.mm*Tr
+I tl
owwu)cnmn
mom*-em-l*
-3 tt ti
1066
Prasad
et al.
Table
V. Duncan’s
new
Folic acid
multiple
test * for
range
erythrocyte
and
serum
folic
acid
in OCA
A-RP
A-none
B-RP
24-2
B-l
B-Ml?le
430
295
244
208
204
192
Erythrocyte (ng./ml.)
users
(P < 0.05}
.4-l
B-1
185
lib _I_-
Serum (ng./ml.)
A-RP
A-none
B-RP
A-l
A-2
B-l
B-?
7.50
6.30
4.7
4.6
4..55
4.09
3.96
B-lrrlrle 3 .i 3 --_
*Any two means not underscored not significantly different. increases lated
the
cells
and
vitamin tion
to the
so may
in both
socioeconomic
This
indicate
may
subjects.
was higher
deficiency
the the
per
OCA
in our vitamin
cent
and
those
EGOT-stimulation
red
of the stimula-
RP subjects
study, in
is re-
in the
indicator
The
in the groups
of that
activation of PLP
as an additional
of the
a relative
The
concentration
serve
Bs status of EGOT
of EGOT.
activity
inversely
by the same line are significantly
suggesting subjects. test
is a
different.
Any
two means
underscored
by the same line are
blood was sampled at the first visit after the pill \\as started. Our data indicate that the A-RP group ~;ts relatively acid,
deficient
in vitamin
suggesting
adequate
that
adequate
intake
of
in folic l&
was
in-
in this group.
Impairment
of
abnormality
found
related
Bs but
nutritional
to vitamin
glucose
tolerance
is an
in some OCA Bs deficiency.
important
users which
Increased
ma\ bc
resistance
to
The reduction of erythrocyte and serum folate due to OCA’s was observed mainly in the upper socioeconomic group of subjects. This was not seen in the lower
insulin by , peripheral tissues, such as pal-ametrial adipose tissues and diaphragm muscle, in vitro anti ilr vivo was found to be responsible for the irnpairmen~ of. glucose tolerance in experimental animals treated with OCA’s.‘” Murakami” suggested that xanthurenic ac id
socioeconomic
is capable
more than
sensitive plasma
index PLP
of alterations
in vitamin
Bs status
or EGOT.
group.
Our data are consistent with the hypothesis that the subjects in lower socioeconomic groups were already marginally deficient with respect to vitamin B6 and folic acid and, as such, further reduction due to OCA’s could not be detected by our assay. Although an effect of OCA’s on the levels of vitamin B6 and folate was observed in subjects of the upper socioeconomic group, the clinical significance of this observation is not clear. Further studies are needed in order to define the clinical importance of these changes. In our study, groups of subjects resuming the “pill” after pregnancy (A-RP and B-RP) demonstrated mainly the effects of pregnancy inasmuch as they resumed OCA’s within five weeks post partum and the
REFERENCES
1. Lumeng, L., Cleary, R. E., and Li, T. K.: Effect of oral contraceptives on the plasma concentration of pyridoxal phosphate, Am. J. Clin. Nutr. 27: 326, 1974. 2. Hontz, A. C., Gyorgy, P., Balin, H., Rose, C. S., and Shan, D. L., Jr.: Interaction of contraceptive steroids with metabolic functions of vitamin Bg, Am. J. Clin. Nutr. 27: 440, 1974. (Abst.) 3. Prasad, A. S., Lei, K. Y., Oberleas, D., Moghissi, K. S., and Stryker, J. C.: Effect of oral contraceptive agents on nutrients: II. Vitamins, Am. J. Clin. Nutr. 28: 385, 1975.
of attaining
a complex
with
insulin.
thereby
reducing its biological activity. Indeed, a small group of. women in whom carbohydrate tolerance had become impaired
while
using
OCA’s
improved
al’trr
sitamin
Us
administration. Several investigators have suggested that large doses of vitamin B6 should be added to OCA’s indiscriminately. However, the adlrlinistratiolI of a coenzyme may increase apoenzyme synthesis, and this may enhance the plasma amino acicl-lowering effect
of
OCA’s,
which
may
produce
an
unto\cartl
effect in communities where protein Inalnutrition exists. At present. it would appear that the idea of supplementation of B, should be approachcrl ‘autiously since the long-term effect of such therapy is uncertain.
4. Brown, R. R., Rose, D. P., Leklem, J. E.. Linkswiler-. H.. and Anand, R.: Urinary 4-pyridoxic acid. plasma pyridoxal phosphate, and erythrocytc aminotransferase levels in oral contraceptive users receiving controlled intakes of vitamin Be, Am. J. Clin. Nutr. 28: 10, 1975. 5. Wertalik, L. F., Metz, E. N., Lobuglio, A. F., and Balcerzak. S. P.: Decreased serum F&2 levels with oral contrac.eptive use. J. A. M. A. 221: 1371, 1972. 6. Davis, R. E., and Smith, B. K.: Pyridoxal, vitamin B,, and folate metabolism in women taking oral contraceptive agents, S. Afr. Med. J. 48: 1937. 1974.
Volume 125 Number8
Effect of oral contraceptives on nutrients. Ill
7. Shojania. 8. 9.
10.
11.
12.
A. M., Hornady, G., and Barnes, P. H.: Oral contraceptives and serum folate level, Lancet 1: 1376, 1968. Streiff, R. R.: Folate deficiency and oral contraceptives, J. A. M. A. 214: 105, 1970. McLean, F. W., Heine, M. W., Held, R., and Streiff, R. R.: Relationship between the oral contraceptive and folic acid metabolism, AM. J, OBSTET. GYNECOL. 104: 745, 1969. Pritchard, J. A., Scott, D. E., and Whalley, P. J.: Maternal folate deficiency and pregnancy wastage. IV. Effects of folic acid supplements, anticonvulsants, and oral contraceptives, AM. J. OBSTET. GYNECOL. 109: 341, 1971. Myrianthopoulos, N., and French, K.: An application of the U.S. bureau of the Census Socioeconomic Index to a large, diversified patient population, Sot. Sci. Med. 2: 283, 1968. Chabner. B. A., and Livingston, D. M.: A simple enzy-
13. 14.
15.
16.
1069
matic assay for pyridoxal phosphate, Anal. Biochem. 34: 413, 1970. Winer, B. J.: Statistical Principles in Experimental Design, New York, 1962, McGraw-Hill, Book Company, Inc. Liu, F. T. Y., and Lin, S.: Effect of the contraceptive steroids norethynodrel and mestranol on dental caries activity in young adult female rats, J. Dent. Res. 52: 753, 1973. Lei, K. Y., Yang, M. G., Oberleas, D., and Prasad, A. S.: Oral contraceptives: effects on plasma insulin response to glucose and on the response to insulin and P-deoxyglucose uptake by peripheral tissue, Proc. Sot. Exp. Biol. Med. 149: 417, 1975. Murakami, E.: Studies on the xanthurenic acid-insulin complex. I. Preparation and properties, J. Biochem. 63: 573. 1968.
