SHORT-TERFLEFFXCT OF OVRAL AND NORGtiSTRZL ON THE VITAIQIN Be AND Bl STATUS OF WOLl3N
USHA M. JOSHI, ANURUPA LAHIHI, SHAILA KORA, SUHASINI S. DIKSHIT, and KATAYUN VIRKAR
Institute for Research in Reproduction (Indian Council of Medical Research) Jehangir Merwanji Street, Parel, Bombay-400 012. INDIA
ABSTRACT Effect of Ovral. a combination tvoe of
contracepti.ve,a.nd norgestrel, a low-doe&-progeetogen, on the Vitamin BL and B1 statue was studied in women taking these drugs for 4 cycles. Urinary excretion of xanthurenic acid increaeed in 6 out of 10 women on Ovral after a tryptophan load. No eignificant change8 in the Erythrocyte Aspartate and Alanine Aminotraneferasee(EAST and EAlT) or in their In vitro stimulations with Pyridoxal Phosphate (PALPO) were noted. In the women taking norgestrel, no effect on any of the parameter8 mentioned above, was noted. Measurement of erythrocyte transketolase, which is indicative of Vitamin Bl StatU8,did not reveal any significant change in either group of women. Accepted for
OCTOBER
1975
publication August 8, 1975
VOL. 12 NO. 4
425
CONTRACEPTION
INTRODUCTION As the modern methods of contraception like the "Pill" find wider acceptance all over the world, more and more workers are investigating the possible metabolic hazards that may arise due to the use of these steroidal compounds. Evidences are now growing that these compounds alter the requirements for several vitamins and minerals (1, 2). iiose (3), Price et al. (4), Brown et al. (51, and Aly et al. (6) h ave reported high levels of abnormal
tryptophan metabolites in the urine of subjects taking orai contraceptives, following a test dose-of L-tryptophan. Since excessive excretion of xanthurenic acid and other metabolites, following an oral load of tryptophan, is the most common indicator of Vitamin Bg deficiency, the question naturally arises as to whether the steroid contraceptives increase the requirement for Vitamin B6. Besides tryptophan metabolites, other indications of a changed Vitamin B6 status such as a change in the erythrocyte aminotransferases have been reported by various workers (7-10). Recently, Faizy et al. (11) have reported a decrease in the basal activity of the erythrocste transketolase enzyme, which requires thiamine as a co,-factor,in women taking combination type oral contraceptives. Levels of this enzyme are known to decrease in cases of Vitamin B deficiency (12, 13). Briggs and Briggs (14) observed an induction of a mild thiamine hypovitaminosis by women using combination type oral contraceptives. In a developing country like India, where the majority of the population is already undernourished, deficiency of vitamins caused by hormonal contraceptives would further worsen the nutritional status. Thus, with an intention of determining the implications of the steroidal contraceptives on the utilisation of Vitamins Bl and Bg, this study was undertaken. MATERIALS AED NETHODS Subjects were selected from the women who clinics of our Institute. attended the family planni They were of ages between ?!! 2 and 38 years. All of them
426
CONTRACEPTION
had delivered at least one year prior, and were not lactating. They were not taking any external source of vitamin, at least for four weeks before the study, and were asked not to take vitamins during the study period. They were asked to bring a 24.hour sample of urine at their first visit. At this visit, a fasting blood sample of 5 ml (heparinised)was drawn for the estimation of erythrocyte aminotransferases and traneketolaee. They were then given 2 gm of L-tryptophan in 'Limca', a popular eoft drink, and were asked to colJ,ecturine for a further period of 24 hours. Then they were started on either Ovral (500,ugm dl-norgestrelend 50~ ethinyl estradiol) or norgestrel (50,ugm or 75~gm dl-norgestrel)for a period of 3 oycles. The type of drug given was not disclosed to the laboratory workers. After 3 cycles, blood collection and tryptophan load tests were repeated. Urine collection was done with the addition of toluene and urine was analysed on the same day of completion of urine collection. Xanthurenic acid was estimated by a slight modification of the method of Waschetein and.Uudaitis (15). It was found necessary to adjust all urine samples to pH 7.8 to 8.1 and filter them before assay. Also, since the recoveries ranged between 55-85$, correction8 for recoveries were neceeeary. The response to tryptophan load was meaeured as the difference in the xanthurenic acid values before and after tryptophan load. PREPARATION OF ERYTHROCYTR HEMOLYSATES FOR ENZYMR AHAIIYSIS Erythrocytes were properly washed with saline, packed and then hemolyeed. Erythrocyte alanine aminotranaferaee(EAlT) and aapartate aminotransferase (EAaT) were measured by the method of Reitman and Frankel (16) as suggested by Aly et a . (6). A 1:5 hemolgeate was used for EAlT end +1: 0 hemolyeate for of pyridoxal phosphate waa used for EAST. Twentythe in vitro stimulation as this was found to give a maxim stinnalation. Erythrocyte transkefolase (ETK) was estimated on lrl hemolyrate by the method of Dreyfue (17). Colour development of the sedoheptuloeewas done according to the method of Dische (18). The "TPP" effect was measured by adding 75,ugm of thiamine pyrophoephate.
OCTOBER
1975 VOL. 12 NO. 4
427
CONTRACEPTION
RESULTS Data on the effect of Ovral on erythrocyte aminotransferasesand on the response to tryptophan load is presented in Table I and that of norgestrel in Table II. Table I shows that 7 out of 11 women on Ovral showed an abnormal response to tryptophan load as indicated by abnormal values of xanthurenic acid excretion (values>control + 2 S.D.; mean f S.D. for control = 9.26 f 16.49). Responses of 4 women were normal (values within control f 2 S.D.). Table II shone that responses of all 11 women on norgeetrel to tryptophan loads were normal values within control f 2 S.D.). One women (case No.5 I who showed initially abnormal valuea of xanthurenic acid after tryptophan load showed normal value8 after norgeatrel therapy. In another woman (case No.71, both the pre-therapy and post-therapy responses to tryptophan loads were above normal limits, but the difference between the two values was negligible. Analysis by Student's t test of EAlT, EAsT and percent stimulations of both Ovral and Norgestrel cases (Tables I & II) revealed that there was no significant difference between pre-therapy and posttherapy level8 in any of these parameters (P valuea)0.05). Results on erythrocyte transketolaseactivity of Ovral treated women are presented in Table III and of norgestrel treated women are presented in Table IV. The changea in basal levels in both the groups were statisticallyfound to-be not significant (P> 0.05) when the results were analysed by Wilcoxon matchedpairs signed ranks test. DISCUSSION Effect of Ovral and Nornestrel on Vitamin "6 Status Increased xanthurenic acid excretion after tryptophan load has been considered as an indication of Vitamin B6 deficiency. Thus, based on this single parameter, seven out of eleven Ovral cases showed a deterioratedVit. B6 status within 3 cycles of therapy. None of the norgestrel cases showed this abnormality.
OCTOBER
1975 VOL.
12 NO. 4
CONTRACEPTION
TABLE I EFFECT OF OVRAL ON ERYTHROCYTEALANINE AND ASPARTATE AMINOTRANSWRASES & ON XANTHURENICACID EXCRlGTIONAFTER TRYPTOPHANLOAD IN WOKEN
Case No.
EAST Sample Basala $ St&
1
Pre-T :'6': Post-T .
2
Pre-T 13.8 Post-T 27.1
z77.2
Pre-T 35.4 Poet-T 21.75
3 4
pp'o:;:T$:: /
I
Response to EAlT tryptophanload Basala' $ Stim. ;giganthrnc urn/4 Nil 23.8
11.3 166.35
:'a6
Nil 57
28.0 118.8
48.9 31.0
2.2 Nil
Nil Nil
28.4 29.3
:;
Nil Nil
Nil Nil
16.0 201.2
31 Nil
-1.0 262.5
23.5 Nil
-6.0 8
(
1 ;;:5
5
Pre-T 27.0 Post-T 29.8
19.8 Nil
8
Pre-T 25.5 Post-T 13.5
8.55 I 4.8 37.1 ~ 1.0
9
Pre-T
Conta-/ Nil mina- ’ tion of tube 28.5 1.12
15.63
Post-T 12.27
Nil
14.65
5.5
78.0 17.0 20.7
10
a =,u moles pyruvate/mlpacked cells/hr.
OCTOBER
1975
VOL. 12 NO. 4
429
CONTRACEPTION
TABLE:II EFFdCT OF NOKGASTKL ON ERYTHROCYTBALANINE AND ASPARTATE AF~NOTRANSPERASESBeON XANTIVJRENIC ACID EXCRETION AFTER TRYPTOPHANLOAD IN WONEN
EAsT Case No.
sample
Basal'
Nil. 27.8
20.5 1.5
19.4 14.0
z5
Nil. Nil.
11.5 -1.5
37.2 25.5
;z
4.6 1.62
t"8.: .
10.2 1.4
22.32 32.4
z;.; .
2.0 1 .65
Nil. 20.0
-6.1 -9.5
19.2
Post-T
42.0
Pre-T Post-T I
";z5 .
Pre-T Post-T
4
Pre-T Post-T
5
Pre-T I Post-T /
6
Pre-T 1 Post-T I
7
Pre-T / , Post-T / Pre-T 1 Post-T 1
3
Basala $ Stim.
% Stim.
2.27 2.25
Pre-T
2
Response to tryptophan load as xenthurenic acid ud24 hr
EAlT
:;*?I .
.
.
/ ::*:.
/
::ii:
/
‘$;:;
/
2:;*;.
11.3 34.8
53.0 14.4
Nil. Nil.
Nil. Nil.
-11.6 24.0
47.5 51.25
1.05 18.0
3.75 3.75
53.3 55.1
48.8 51.1
29.05 30.0
55.0 23.0
a =p moles pyruvate/ml packed cells/hr.
430
OCTOBER
1975 VOL. 12 NO. 4
CONTRACWTION
TBBLE III SHORT-TERM EFFECT OF OVRLL ON THE ERYTHROCYTE TRANSKETOLASE (ETP) ACTIVITY & IN VITRO STIMULATION WITH TPP IN WOMEN
No.
ETK o_%g eedoheptuloee/& packed RBC/lrr) Before therapy
After therapy
Basal
I
1
1 746.1
1
0.95
I
500
2
1 429.6
1
3.5
1
402.5
1
1.2
3
1 869
1
1.44
I
629.9
I
-3.75
4
1 561.8
1
15.22
1
467.4
1
44.25
5
1800
I
0.43
j
I
-0.3
I
6
1 678.0
/
15.02
\
518.0
1
6.75
7
/ 666
/
11.4
I
628.7
I
3.39
8
1020
17.65
!
702.8
3.7
9
368
5.4
583.9
23.63
695.6
3.6
I! , i
688.4
-3.6
10
P
Stimulation
I
Basal
1006
I
/ $
-1
StimZatLon 7.16
I
431
CONTRACEPTION
TABLE IV SHORT-TERN EFFECT OF NORGESTRES ON THF: ERYTHROCYTE TRANSKETOLASE (ETR) ACTIVITY AND IN VITRO STIMUTJATION WITH TPP IN WOMEN
ETK (,u& sedoheptuloee/mlpacked RBC/hr) .-Before therapy After therapy
No. Basal 1
496
2
549.8
% StimuLation
Basal
% Stimulation
11.00
741.6
25.48
5.0
440.0
2.25
12.4
1 729.0
I
28.67
3
I
654
4
I
568
5
I
770
19.5
1 620.0
6
340
18.8
691.0
60.5
7
854
1.04
624.3
17.95
8
567.5
7.05
i 627.0
41.39
432
3.32
OCTOBER
I
-5.65
1975 VOL. 12 NO. 4
CONTRACEPTION
However, data on EAlT and EAeT showed no particular trend. Firstly, basal levels for both these enzymes varied considerably (13.8 to 35.4&m/ml/hr in the case of EAsT and 0 to ll.O@m/ml/hr in the case of EAlT). Since the levels of these enzymes and their in vitro stimulation are decided by both the apoensyme and coenzyme levels, the pattern obtained In each case will have to be considered individually. Combination pills are reported to induce the apoenzyme for EAsT (6,8,9,11). However, if coenzyme availability is reduced substantially,the baeal level may decrease and in these cases in vitro stimulation may be higher. However, if the coenzyme level6 are enough to eaturate at least Part of the induced apoenzyme, basal activity would be higher than before therapy. Percent stimulation will then depend upon availability of coen5yme. If a higher stimulation is obtained, we find higher requirements of the vitamin. Thus, the requirement of vitamin will be considered to be higher in two groups: a) Lowered basal levels with higher in vitro stimulation, and b) higher basal levels with higher in vitro etimulation. Based on this hypothesis, requirements for Vit. B6 might have changed in 5 out of 11 cases on Ovral (cases 1,2,,7,8,9). The results we obtained on EAlT enzyme with Ovral failed to confirm those of Doberenz (7) who reported decreased levels of EAlT enzyme with increased in vitro stimulation with combination pills. If we consider that a change in any two parameters, viz. higher in vitro stimulation of EAST, higher in vitro stimulation of EAlT and an abnormal response to tryptophan load, indicative of higher requirements for Vit. B 5 out of 11 oases on Ovral (case Nos. 1,2,7,9,11)&uld be considered as having changed Vitamin B6 status. Applying similar reasoning, only 1 case on norgestrel (No.1, Table II) seemed to have higher requirements for Vit. B6 after therapy. Effect of Ovral and Norgestrel on Vitamin B1 Status The basal levels for transketolase (ETK) reported in our paper (range 340-1020 units, mean 590 units) are much lower than the ones reported by Ysrkkanen and balliomaki (12) (range 860-1580 units, mean 1060 units). These workers found that the TPP
OCTOBER 1975
VOL. 12 NO. 4
433
CONTRACEF’I’ION
effect was an unreliable measure for detecting deficiencies of Vit. B but STK by itself was a useful parameter. Brin (13) on the otder hand, observed on an average, 106 stimulation of the enzyme in control subjects, which rose gradually as the enzyme level dropped in Vit. B, dificiency. Only two groups of workers (11,14) have so far reported on the effect of oral contrace tives on the ETK activity of women. Faizy et al.(llP found decreases of basal activity in mosro‘f-the women on combination type contraceptives. But the results obtained on our small series failed to confirm these findings. It was most surprising to note that whereas basal levels of ETK decreased in 7 out of 10 women on Ovral, they increased in 5 out of 8 women on norgestrel therapy. Further, in 4 of these women, a very high in vitro stimulationwith TPP was simultaneouslyobserved. Whether this indicates an induction of the apoenzyme by the norgestrel and hence higher requirements for Vitamin B, for saturation of the enzyme is not clear. This certainly warrznts more investigation. It has been suggested that the biochemical parameters for Vitamin B status should include other parameters such as urina1y thiamine, blood pyruvic acid and oC-ketoglutaricacid along with transketolaaeactivity. Future studies should involve these estimations along with the load tests with B in order to assess the Vitamin B, status under contracebtive therapy. Acknowledgements Our thanks are due to the health educators and social workers of the Institute for motivating the subjects for this study.
434
OCTOBER
1975
VOL. 12 NO. 4
CONTRACEPTION
References 1.
Theuer, R.C. Bffect of oral contraceptive agents on vitamin and mineral needs. A review. Jour. He-prod. pled. 8:l (1972)
2.
Larsson - Cohn U. Oral contraceptives and vitamins: A review. Am. J. OBst. Gynec. 121; 84.(1975)
3.
Rose, G.P. Excretion of xanthurenic acid in the urine of women taking progesto en-oestrogen preparations. Nature 210:196 'T1966)
4.
Price, J.Tfl., Thronton, h.J., and Maellor, L.X. Tryptophan metabolism in women using steroid hormones for ovulation control. Am. Jour. Clin. Nutr. 20:452 (1967)
5.
Brown, R.R., Rose, D.P., Price, J&A. and Wolf, H. Tryptophan metabolites as affected by anovulatory agents. Ann. N.Y. Acad. Sci. 166:44 (1949)
6.
Aly, H.E., Donald, E.A., Simpson, J.W. Oral contraceptives and vitamin B6 metabolism. Am. Jour. Clin. Nutr. 24: 297 (1971)
7.
Doberenz, A.R., Van, Miller, J.P., Green, J.R. and Beaton, J.R. Vitamin B depletion in women using oral contraceptives a8 8etermined by erythrocyte glutamic pyruvic trensaminase activities. Proc. Sot. Exptl. Biol. Ned. 137:llOO (1971)
8.
Rose, D.P., Strong, R., Adams, P.W., and Harding, P.E. Experimental vitamin B deficiency and the effect of estrogen containing or6: 1 contraceptives on tryptophan metabolism and vitamin B6 requirements. Clin. Sci. 42:465 (1972)
9.
Reinken, L., Dapunt, O., and Kammerlander, H. Vitamin B6-Verarmg bei einnahme oraler contraceptiva. Int. Jour. for Vit. and Nutr. Res. 43:20 (1973)
10.
Salkeld, R.K., Knorr, K., and Rorner, W.F. The effect of oral contraceptives on vitamin B6 status. Clin. Chim. Acta 49:195 (1973)
11.
Faizy, A., Iyengar, L., and Bamji, M.S. Paper presented at the Fifth Asia dc Oceania Congress of Endocrinology, Chandigarh, India. Jan. 28th Feb 1st 1974. Abstr. No. 172.
OCTOBER
1975 VOL. 12 NO. 4
435
CONTRACEPTION
12.
Markkanen, T. and Kalliomaki, J.L. Transketolase activity of blood cells in various clinical conditions. Am. J. Fled. Sci. 252:564 (1966)
13.
Brin, H. Erythrocyte tranaketolase activity in early thiamine deficiency. Ann. N.Y. Acad. Sci. 98:528 (1962)
14.
Briggs, M.H. and Briggs, M. Thiamine status and oral contraceptives. Contraception 11; 151.Cl975)
15.
Waschstein, M. and Gudaitis, A. The detection of vitamin B6 deficiency - an improved method for the determination of xanthurenic acid in urine. Am. J. Clin. Path. 22:652 (1952)
16.
Reitman, S. and Prankel, S. A calorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Path. 28:56 (1957)
17.
Dreyfus, P. Clinical application of blood transketolase. New Engl. J. Ned. 267:596 (1962)
18.
Dische, Z. Qualitative and Quantitative calorimetric determination of heptoses. J. Bio. Chem. 204:983 (1953)
436
OCTOBER 1975
VOL. 12 NO. 4
USHA M. JOSHI, ANURUPA LAHIHI, SHAILA KORA, SUHASINI S. DIKSHIT, and KATAYUN VIRKAR
Institute for Research in Reproduction (Indian Council of Medical Research) Jehangir Merwanji Street, Parel, Bombay-400 012. INDIA
ABSTRACT Effect of Ovral. a combination tvoe of
contracepti.ve,a.nd norgestrel, a low-doe&-progeetogen, on the Vitamin BL and B1 statue was studied in women taking these drugs for 4 cycles. Urinary excretion of xanthurenic acid increaeed in 6 out of 10 women on Ovral after a tryptophan load. No eignificant change8 in the Erythrocyte Aspartate and Alanine Aminotraneferasee(EAST and EAlT) or in their In vitro stimulations with Pyridoxal Phosphate (PALPO) were noted. In the women taking norgestrel, no effect on any of the parameter8 mentioned above, was noted. Measurement of erythrocyte transketolase, which is indicative of Vitamin Bl StatU8,did not reveal any significant change in either group of women. Accepted for
OCTOBER
1975
publication August 8, 1975
VOL. 12 NO. 4
425
CONTRACEPTION
INTRODUCTION As the modern methods of contraception like the "Pill" find wider acceptance all over the world, more and more workers are investigating the possible metabolic hazards that may arise due to the use of these steroidal compounds. Evidences are now growing that these compounds alter the requirements for several vitamins and minerals (1, 2). iiose (3), Price et al. (4), Brown et al. (51, and Aly et al. (6) h ave reported high levels of abnormal
tryptophan metabolites in the urine of subjects taking orai contraceptives, following a test dose-of L-tryptophan. Since excessive excretion of xanthurenic acid and other metabolites, following an oral load of tryptophan, is the most common indicator of Vitamin Bg deficiency, the question naturally arises as to whether the steroid contraceptives increase the requirement for Vitamin B6. Besides tryptophan metabolites, other indications of a changed Vitamin B6 status such as a change in the erythrocyte aminotransferases have been reported by various workers (7-10). Recently, Faizy et al. (11) have reported a decrease in the basal activity of the erythrocste transketolase enzyme, which requires thiamine as a co,-factor,in women taking combination type oral contraceptives. Levels of this enzyme are known to decrease in cases of Vitamin B deficiency (12, 13). Briggs and Briggs (14) observed an induction of a mild thiamine hypovitaminosis by women using combination type oral contraceptives. In a developing country like India, where the majority of the population is already undernourished, deficiency of vitamins caused by hormonal contraceptives would further worsen the nutritional status. Thus, with an intention of determining the implications of the steroidal contraceptives on the utilisation of Vitamins Bl and Bg, this study was undertaken. MATERIALS AED NETHODS Subjects were selected from the women who clinics of our Institute. attended the family planni They were of ages between ?!! 2 and 38 years. All of them
426
CONTRACEPTION
had delivered at least one year prior, and were not lactating. They were not taking any external source of vitamin, at least for four weeks before the study, and were asked not to take vitamins during the study period. They were asked to bring a 24.hour sample of urine at their first visit. At this visit, a fasting blood sample of 5 ml (heparinised)was drawn for the estimation of erythrocyte aminotransferases and traneketolaee. They were then given 2 gm of L-tryptophan in 'Limca', a popular eoft drink, and were asked to colJ,ecturine for a further period of 24 hours. Then they were started on either Ovral (500,ugm dl-norgestrelend 50~ ethinyl estradiol) or norgestrel (50,ugm or 75~gm dl-norgestrel)for a period of 3 oycles. The type of drug given was not disclosed to the laboratory workers. After 3 cycles, blood collection and tryptophan load tests were repeated. Urine collection was done with the addition of toluene and urine was analysed on the same day of completion of urine collection. Xanthurenic acid was estimated by a slight modification of the method of Waschetein and.Uudaitis (15). It was found necessary to adjust all urine samples to pH 7.8 to 8.1 and filter them before assay. Also, since the recoveries ranged between 55-85$, correction8 for recoveries were neceeeary. The response to tryptophan load was meaeured as the difference in the xanthurenic acid values before and after tryptophan load. PREPARATION OF ERYTHROCYTR HEMOLYSATES FOR ENZYMR AHAIIYSIS Erythrocytes were properly washed with saline, packed and then hemolyeed. Erythrocyte alanine aminotranaferaee(EAlT) and aapartate aminotransferase (EAaT) were measured by the method of Reitman and Frankel (16) as suggested by Aly et a . (6). A 1:5 hemolgeate was used for EAlT end +1: 0 hemolyeate for of pyridoxal phosphate waa used for EAST. Twentythe in vitro stimulation as this was found to give a maxim stinnalation. Erythrocyte transkefolase (ETK) was estimated on lrl hemolyrate by the method of Dreyfue (17). Colour development of the sedoheptuloeewas done according to the method of Dische (18). The "TPP" effect was measured by adding 75,ugm of thiamine pyrophoephate.
OCTOBER
1975 VOL. 12 NO. 4
427
CONTRACEPTION
RESULTS Data on the effect of Ovral on erythrocyte aminotransferasesand on the response to tryptophan load is presented in Table I and that of norgestrel in Table II. Table I shows that 7 out of 11 women on Ovral showed an abnormal response to tryptophan load as indicated by abnormal values of xanthurenic acid excretion (values>control + 2 S.D.; mean f S.D. for control = 9.26 f 16.49). Responses of 4 women were normal (values within control f 2 S.D.). Table II shone that responses of all 11 women on norgeetrel to tryptophan loads were normal values within control f 2 S.D.). One women (case No.5 I who showed initially abnormal valuea of xanthurenic acid after tryptophan load showed normal value8 after norgeatrel therapy. In another woman (case No.71, both the pre-therapy and post-therapy responses to tryptophan loads were above normal limits, but the difference between the two values was negligible. Analysis by Student's t test of EAlT, EAsT and percent stimulations of both Ovral and Norgestrel cases (Tables I & II) revealed that there was no significant difference between pre-therapy and posttherapy level8 in any of these parameters (P valuea)0.05). Results on erythrocyte transketolaseactivity of Ovral treated women are presented in Table III and of norgestrel treated women are presented in Table IV. The changea in basal levels in both the groups were statisticallyfound to-be not significant (P> 0.05) when the results were analysed by Wilcoxon matchedpairs signed ranks test. DISCUSSION Effect of Ovral and Nornestrel on Vitamin "6 Status Increased xanthurenic acid excretion after tryptophan load has been considered as an indication of Vitamin B6 deficiency. Thus, based on this single parameter, seven out of eleven Ovral cases showed a deterioratedVit. B6 status within 3 cycles of therapy. None of the norgestrel cases showed this abnormality.
OCTOBER
1975 VOL.
12 NO. 4
CONTRACEPTION
TABLE I EFFECT OF OVRAL ON ERYTHROCYTEALANINE AND ASPARTATE AMINOTRANSWRASES & ON XANTHURENICACID EXCRlGTIONAFTER TRYPTOPHANLOAD IN WOKEN
Case No.
EAST Sample Basala $ St&
1
Pre-T :'6': Post-T .
2
Pre-T 13.8 Post-T 27.1
z77.2
Pre-T 35.4 Poet-T 21.75
3 4
pp'o:;:T$:: /
I
Response to EAlT tryptophanload Basala' $ Stim. ;giganthrnc urn/4 Nil 23.8
11.3 166.35
:'a6
Nil 57
28.0 118.8
48.9 31.0
2.2 Nil
Nil Nil
28.4 29.3
:;
Nil Nil
Nil Nil
16.0 201.2
31 Nil
-1.0 262.5
23.5 Nil
-6.0 8
(
1 ;;:5
5
Pre-T 27.0 Post-T 29.8
19.8 Nil
8
Pre-T 25.5 Post-T 13.5
8.55 I 4.8 37.1 ~ 1.0
9
Pre-T
Conta-/ Nil mina- ’ tion of tube 28.5 1.12
15.63
Post-T 12.27
Nil
14.65
5.5
78.0 17.0 20.7
10
a =,u moles pyruvate/mlpacked cells/hr.
OCTOBER
1975
VOL. 12 NO. 4
429
CONTRACEPTION
TABLE:II EFFdCT OF NOKGASTKL ON ERYTHROCYTBALANINE AND ASPARTATE AF~NOTRANSPERASESBeON XANTIVJRENIC ACID EXCRETION AFTER TRYPTOPHANLOAD IN WONEN
EAsT Case No.
sample
Basal'
Nil. 27.8
20.5 1.5
19.4 14.0
z5
Nil. Nil.
11.5 -1.5
37.2 25.5
;z
4.6 1.62
t"8.: .
10.2 1.4
22.32 32.4
z;.; .
2.0 1 .65
Nil. 20.0
-6.1 -9.5
19.2
Post-T
42.0
Pre-T Post-T I
";z5 .
Pre-T Post-T
4
Pre-T Post-T
5
Pre-T I Post-T /
6
Pre-T 1 Post-T I
7
Pre-T / , Post-T / Pre-T 1 Post-T 1
3
Basala $ Stim.
% Stim.
2.27 2.25
Pre-T
2
Response to tryptophan load as xenthurenic acid ud24 hr
EAlT
:;*?I .
.
.
/ ::*:.
/
::ii:
/
‘$;:;
/
2:;*;.
11.3 34.8
53.0 14.4
Nil. Nil.
Nil. Nil.
-11.6 24.0
47.5 51.25
1.05 18.0
3.75 3.75
53.3 55.1
48.8 51.1
29.05 30.0
55.0 23.0
a =p moles pyruvate/ml packed cells/hr.
430
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1975 VOL. 12 NO. 4
CONTRACWTION
TBBLE III SHORT-TERM EFFECT OF OVRLL ON THE ERYTHROCYTE TRANSKETOLASE (ETP) ACTIVITY & IN VITRO STIMULATION WITH TPP IN WOMEN
No.
ETK o_%g eedoheptuloee/& packed RBC/lrr) Before therapy
After therapy
Basal
I
1
1 746.1
1
0.95
I
500
2
1 429.6
1
3.5
1
402.5
1
1.2
3
1 869
1
1.44
I
629.9
I
-3.75
4
1 561.8
1
15.22
1
467.4
1
44.25
5
1800
I
0.43
j
I
-0.3
I
6
1 678.0
/
15.02
\
518.0
1
6.75
7
/ 666
/
11.4
I
628.7
I
3.39
8
1020
17.65
!
702.8
3.7
9
368
5.4
583.9
23.63
695.6
3.6
I! , i
688.4
-3.6
10
P
Stimulation
I
Basal
1006
I
/ $
-1
StimZatLon 7.16
I
431
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TABLE IV SHORT-TERN EFFECT OF NORGESTRES ON THF: ERYTHROCYTE TRANSKETOLASE (ETR) ACTIVITY AND IN VITRO STIMUTJATION WITH TPP IN WOMEN
ETK (,u& sedoheptuloee/mlpacked RBC/hr) .-Before therapy After therapy
No. Basal 1
496
2
549.8
% StimuLation
Basal
% Stimulation
11.00
741.6
25.48
5.0
440.0
2.25
12.4
1 729.0
I
28.67
3
I
654
4
I
568
5
I
770
19.5
1 620.0
6
340
18.8
691.0
60.5
7
854
1.04
624.3
17.95
8
567.5
7.05
i 627.0
41.39
432
3.32
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I
-5.65
1975 VOL. 12 NO. 4
CONTRACEPTION
However, data on EAlT and EAeT showed no particular trend. Firstly, basal levels for both these enzymes varied considerably (13.8 to 35.4&m/ml/hr in the case of EAsT and 0 to ll.O@m/ml/hr in the case of EAlT). Since the levels of these enzymes and their in vitro stimulation are decided by both the apoensyme and coenzyme levels, the pattern obtained In each case will have to be considered individually. Combination pills are reported to induce the apoenzyme for EAsT (6,8,9,11). However, if coenzyme availability is reduced substantially,the baeal level may decrease and in these cases in vitro stimulation may be higher. However, if the coenzyme level6 are enough to eaturate at least Part of the induced apoenzyme, basal activity would be higher than before therapy. Percent stimulation will then depend upon availability of coen5yme. If a higher stimulation is obtained, we find higher requirements of the vitamin. Thus, the requirement of vitamin will be considered to be higher in two groups: a) Lowered basal levels with higher in vitro stimulation, and b) higher basal levels with higher in vitro etimulation. Based on this hypothesis, requirements for Vit. B6 might have changed in 5 out of 11 cases on Ovral (cases 1,2,,7,8,9). The results we obtained on EAlT enzyme with Ovral failed to confirm those of Doberenz (7) who reported decreased levels of EAlT enzyme with increased in vitro stimulation with combination pills. If we consider that a change in any two parameters, viz. higher in vitro stimulation of EAST, higher in vitro stimulation of EAlT and an abnormal response to tryptophan load, indicative of higher requirements for Vit. B 5 out of 11 oases on Ovral (case Nos. 1,2,7,9,11)&uld be considered as having changed Vitamin B6 status. Applying similar reasoning, only 1 case on norgestrel (No.1, Table II) seemed to have higher requirements for Vit. B6 after therapy. Effect of Ovral and Norgestrel on Vitamin B1 Status The basal levels for transketolase (ETK) reported in our paper (range 340-1020 units, mean 590 units) are much lower than the ones reported by Ysrkkanen and balliomaki (12) (range 860-1580 units, mean 1060 units). These workers found that the TPP
OCTOBER 1975
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433
CONTRACEF’I’ION
effect was an unreliable measure for detecting deficiencies of Vit. B but STK by itself was a useful parameter. Brin (13) on the otder hand, observed on an average, 106 stimulation of the enzyme in control subjects, which rose gradually as the enzyme level dropped in Vit. B, dificiency. Only two groups of workers (11,14) have so far reported on the effect of oral contrace tives on the ETK activity of women. Faizy et al.(llP found decreases of basal activity in mosro‘f-the women on combination type contraceptives. But the results obtained on our small series failed to confirm these findings. It was most surprising to note that whereas basal levels of ETK decreased in 7 out of 10 women on Ovral, they increased in 5 out of 8 women on norgestrel therapy. Further, in 4 of these women, a very high in vitro stimulationwith TPP was simultaneouslyobserved. Whether this indicates an induction of the apoenzyme by the norgestrel and hence higher requirements for Vitamin B, for saturation of the enzyme is not clear. This certainly warrznts more investigation. It has been suggested that the biochemical parameters for Vitamin B status should include other parameters such as urina1y thiamine, blood pyruvic acid and oC-ketoglutaricacid along with transketolaaeactivity. Future studies should involve these estimations along with the load tests with B in order to assess the Vitamin B, status under contracebtive therapy. Acknowledgements Our thanks are due to the health educators and social workers of the Institute for motivating the subjects for this study.
434
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1975
VOL. 12 NO. 4
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References 1.
Theuer, R.C. Bffect of oral contraceptive agents on vitamin and mineral needs. A review. Jour. He-prod. pled. 8:l (1972)
2.
Larsson - Cohn U. Oral contraceptives and vitamins: A review. Am. J. OBst. Gynec. 121; 84.(1975)
3.
Rose, G.P. Excretion of xanthurenic acid in the urine of women taking progesto en-oestrogen preparations. Nature 210:196 'T1966)
4.
Price, J.Tfl., Thronton, h.J., and Maellor, L.X. Tryptophan metabolism in women using steroid hormones for ovulation control. Am. Jour. Clin. Nutr. 20:452 (1967)
5.
Brown, R.R., Rose, D.P., Price, J&A. and Wolf, H. Tryptophan metabolites as affected by anovulatory agents. Ann. N.Y. Acad. Sci. 166:44 (1949)
6.
Aly, H.E., Donald, E.A., Simpson, J.W. Oral contraceptives and vitamin B6 metabolism. Am. Jour. Clin. Nutr. 24: 297 (1971)
7.
Doberenz, A.R., Van, Miller, J.P., Green, J.R. and Beaton, J.R. Vitamin B depletion in women using oral contraceptives a8 8etermined by erythrocyte glutamic pyruvic trensaminase activities. Proc. Sot. Exptl. Biol. Ned. 137:llOO (1971)
8.
Rose, D.P., Strong, R., Adams, P.W., and Harding, P.E. Experimental vitamin B deficiency and the effect of estrogen containing or6: 1 contraceptives on tryptophan metabolism and vitamin B6 requirements. Clin. Sci. 42:465 (1972)
9.
Reinken, L., Dapunt, O., and Kammerlander, H. Vitamin B6-Verarmg bei einnahme oraler contraceptiva. Int. Jour. for Vit. and Nutr. Res. 43:20 (1973)
10.
Salkeld, R.K., Knorr, K., and Rorner, W.F. The effect of oral contraceptives on vitamin B6 status. Clin. Chim. Acta 49:195 (1973)
11.
Faizy, A., Iyengar, L., and Bamji, M.S. Paper presented at the Fifth Asia dc Oceania Congress of Endocrinology, Chandigarh, India. Jan. 28th Feb 1st 1974. Abstr. No. 172.
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CONTRACEPTION
12.
Markkanen, T. and Kalliomaki, J.L. Transketolase activity of blood cells in various clinical conditions. Am. J. Fled. Sci. 252:564 (1966)
13.
Brin, H. Erythrocyte tranaketolase activity in early thiamine deficiency. Ann. N.Y. Acad. Sci. 98:528 (1962)
14.
Briggs, M.H. and Briggs, M. Thiamine status and oral contraceptives. Contraception 11; 151.Cl975)
15.
Waschstein, M. and Gudaitis, A. The detection of vitamin B6 deficiency - an improved method for the determination of xanthurenic acid in urine. Am. J. Clin. Path. 22:652 (1952)
16.
Reitman, S. and Prankel, S. A calorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Path. 28:56 (1957)
17.
Dreyfus, P. Clinical application of blood transketolase. New Engl. J. Ned. 267:596 (1962)
18.
Dische, Z. Qualitative and Quantitative calorimetric determination of heptoses. J. Bio. Chem. 204:983 (1953)
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