Effect
of oral
D. P. Rose, and H. M.
and
M.D., Ph.D., J. E. Lek/ern. Linkswi/er, Ph.D.
ABSTRACT
Oral
The
tests
after pridoxine excretion
reduced
tolerance levels.
tolerance
of
carbohydrate consequent
were
the
the
repeated
after
loss
of
biological Clin.
controls. the
Nuir.
Informed consent was obtained from all subjects before commencing the study. The details of the experimental procedure, and the vitamin B,-deficient diet,
American
Journal
of Clinical
was
In
Nutrition
have were
oral
group
addition,
of
plasma
despite
diet.
oral
again
xanthurenic or
elevated
no change
acid in the plasma
in the
pyridoxine-responsive with
in four
and
a deterioration
normal was
insulin
pyridoxal and
B,-deficient with
Th.re
observed
and
contraceptives
by an increased
associated
pyridoxine. The
28: 872
materials
The
as judged
complexing
activity.
In two previous publications, we have descnibed the changes in urinary 4-pyridoxic acid, plasma pyridoxal phosphate and erythrocyte aminotransferases ( I ),and tryptophan and niacin metabolism (2), which occurred when a group of oral contraceptive users and female controls of similar age were fed a vitamin B6-deficient diet for 4 weeks, and were then repleted with various doses of pynidoxine. Oral contraceptives alter carbohydrate metabolism in some women, the changes being characterized by impaired glucose tolerance and abnormally elevated serum insulin levels after oral or intravenous glucose loads (3). Recently, it has been observed that the glucose tolerance of oral contraceptive users can be improved by pyridoxine administration (4, 5). In this paper, we report the results of oral glucose tolerance tests which were performed on some of the oral contraceptive-treated women and control subjects included in the previously reported studies (1, 2) when they were rendered vitamin B6 deficient.
872
by
excretion,
of a vitamin
steroid-treated
involve
acid taking
ingestion
phosphate,
reversed
B,-deficient
Am.J.
gluconeogensis.
pyridoxal
Ph.D.,
women
B6 deficiency,
Vitamin
was may
Brown,
xanthurenie
in nine 4 weeks
contraceptive
vitamin
R.
urinary’
determined
abnormality
metabolism
R.
tolerance,
were
plasma
of This
2
Ph.D.,
supplementation.
and
glucose insulin
glucose
concentrations
controls.
and
B6
on carbohydrate
phosphate
Methods
vitamin
glucose
alteration
xanthurenic
contraceptives
acid may
in with
a
enhance
878. 1975.
been described elsewhere ( I ): the part of this larger investigation.
present
subjects
In summary, the subjects received a diet containing 0.19 mg ofpyridoxine activity daily. supplemented daily with an additional 0.8 mg of py’ridoxine hydrochloride (PN ‘ HCI) for 5 days. The PN HCI supplement was then withdrawn and the vitamin B,-deficient diet fed for 28 day’s. Thereafter, the diet was continued for a further 28-day period, but supplemented with 0.8 mg. 2 mg or 20 mg of py’ridoxine hydrochloride dails. Nine women who had been taking an estrogen-containing oral contraceptive for at least 6 months, and 4 controls were included in the study of carbohydrate metabolism (Table I ). Alterations in metabolic functions arising from the reduced dietary vitamin B, intake ssere monitored by the urinary excretion of tryptophan metabolites Lifter an ral 2-g L-tryptophan load (2), urinary cystathionine excretion after a 3-g oral dose of L-methionine (Leklem, Linkswiler, Brown and Rose-unpublished observations), and assay of erythrocyte alanine and aspartate aminotransferase activities ( I ). In addition, plasma py ridoxal phosphate and urinar .4-ps ridoxic acid were determined ( I). In all instances the changes were those expected to arise from vitamin B, depletion. and they were reversed by PN ‘ HCI supplements.
‘From the Division of Clinical Oncologs. University of Wisconsin Medical School and Department of Nutritional Sciences, University of Wisconsin College of Agricultural and Life Sciences, Madison. Wisconsin 53706. 2 Supported in part by Wisconsin College of Agricultural and Life Sciences, Contract NIH. NICHD 72-2782 with the National Institute of Child Health and Human Development, and Grant no. CA-l3302 from the National Cancer Institute, Bethesda, Maryland 20014.
28:
AUGUST
1975.
pp.
872
878.
Printed
in U.S.A.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/8/872/4732955 by East Carolina University user on 12 January 2019
deficiency
contraceptives
ORAL Before ingesting
commencing
the
the
B,-deficient
vitamin
CONTRACEPTIVES
deficiency
period, diet
AND
but
plus
when
0.8
sample vored
mg
Subj n
PN
Oralcontraceptive
0.
1 2 3 4 5 6 7 8 9 10 II 12 13
Control Control Control Control Orthonovum Orthonovum Orthonovum Orthonovum Orthonovum Ovral’ Ovral Norlestrin2l Demulenh a This
is the
dose
used,
ofpyridoxine
hydrochloride
tion
Table and
acid
I shows plasma
excretions
Plasma glucose was for true glucose (6), (7). differences between or paired Student’s
PLP
IS 35 20 27 1,174 423 230 91 741 131 73 367 604
10.57 5.84 25.32 12.14 9.18 10.22 9.32 14.72 7.27 4.82 8.19 8.92 7.90
daily
the xanthurenic acid pynidoxal phosphate
Peak deficiency
XA”
given
100 g of lemon-flafurther blood sam-
XA
during
451 1,079 708 944 2,157 1,803 1,684 1,481 1,819 1,427 1,088 1,417 1,771 the
excrelevels
and
Predeficiency
0.8 2.0 2.0 2.0 0.8 2.0 2.0 2.0 20.0 0.8 2.0 0.8 20.0
I/SO”' 1/50 l/80’ 1/80 1/50
of venous blood was obtained, glucose was given orally, and
Results
xanthurenic
. HCI. a mg
873
pIes were taken I and 2 hours later. determined by an automated method and plasma insulin by radioimmunoassay The statistical significance of groups was assessed by unpaired t-tests, as appropriate.
tolerance and urinary xanthurenic acid excretion were also determined in 12 other young women who were not taking an oral contraceptive and who were consuming a self-selected diet but without vitamin supplements (Table 2). The subjects were fasted overnight for the oral glucose tolerance tests. Next morning, after resting for 30 mm, a
contraceptives concentrations
B, DEFICIENCY
28-day
After
PLP
XA
2.30 2.01 6.30 3.93 2.81 1.92 2.27 3.88 3.85 2.53 2.50 2.60 5.09
23 25 17 20 914 75 81 55 42 230 35 377 34
repletion
period.
PN
.
HCI PLP
3.16 12.00 15.50 20.80 4.00 12.50 9.74 14.80 84.20 3.70 17.40 3.50 148.0
Xanthurenic
“
acid
(XA)
excretion in zmoles/24 hr after 2-g t.-try’ptophan load. The upper limit of normal (mean ± 2 SD) obtained for the I 2 women on self-selected diets who were not taking an oral contraceptive was 52 pmoles/24 hr. Plasma pyridoxal phosphate concentration (PLP) in ng/ml. d 0.05 mg mestranol: I mg norethindrone. ‘ 0.08 mg mestranol: I mg norethindrone. I #{216}#{216}5 mg ethinyl estradiol: 0.5 mg norgestrel. 0.05 mg ethinyl estradiol: 2.5 mg norethindrone acetate. “ 0.1 mg mestranol: 0.5 mg ethy’nodiol diacetate. Comparison of predeficiencv and peak deficiency’ results using Student’s paired t-test showed a significant increase in XA excretion by controls when deficient (0.01 > P > 0.001) and significant decrease in OC users PLP levels (P < 0.001).
TABLE 2 Oral glucose self-selected experimental
tolerance and urinary xanthurenic acid excretion in women diet, and controls and oral contraceptive users when receiving diet supplemented with py’ridoxine (predeficiency’)
Normal
subjects,
self-
selected diets (12) Controls, predeficieney (4) Oral contraceptive users, predeficiency (9) Results
are
0.01: “ 0.05 lecteddiets,0.0l
Pias
Age. years
Group
mean >
±
SI).
P > 0.02; > P > 0.001.
Plasma C
ma
glucose.
fast
ingesting the
mg/I
00
a
ml
I hr
Xanthurenic
23
±
3
90
±
5
136
±
33
98
±
IS
27
22 23
±
3 4
86 82
±
7 8
86 1 15
±
16” 37
77 97
±
±
±
20” 22
24 426
glucose xanthurenic
±
significantly acid
excretion
different
±
from
significantly
women different
acid.
,moIe/24
2 hr
on self-selected from
normal
diets:
±
8
±
9 363’
±
a
subjects
hr
0.02
> on
P >
self-se-
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/8/872/4732955 by East Carolina University user on 12 January 2019
PN . HCI per day. oral glucose tolerance was assessed, and the tests of vitamin B6 nutrition carried out. These metabolic studies were repeated after 28 days on the vitamin B,-deficient diet (“peak deficiency”) and after 4 weeks supplementation with PN’HCI. Oral glucose
TABLE I Experimental subjects, oral plasma pyridoxal phosphate
VITAMIN
ROSE
874
ET AL.
oral
contraceptive-treated
TABLE 3 Effect of vitamin supplementation
B, deficiency with pyridoxine
group
(P
on oral HC1:
glucose plasma
The results show that supplementation with 0.8 mg of PNHCl a day was insufficient to restore the plasma pynidoxal phosphate to predeficiency levels, although this dose did produce reductions in xanthurenic acid excretion. Table 2 compares the plasma glucose levels and urinary xanthurenic acid excretions of the controls and oral contraceptive users before the induction of vitamin B6 deficiency with those of the I 2 young women on self-selected diets who were not taking an oral contraceptive. The 4 controls receiving the vitamin B6-deficient diet supplemented with pyridoxme had significantly lower plasma glucose levels than the 12 ingesting their regular diet at both I and 2 hours after the glucose load; the oral contraceptive users had lower fasting plasma glucose levels than these same 12 women. The xanthurenic acid values for the two groups who were not taking oral contraceptives were virtually identical (Table 2); the oral contraceptive-treated group excreted significantly higher levels of xanthurenic acid than nonusers on a self-selected diet, but the number of nonusers on the controlled diet was too small for any statistical difference to be demonstrated.
The plasma glucose tamed during the oral are shown in Tables
0.001).
Significantly
P >
0.001)
±
different and
after
88 77 75 76 74 82 86 97 80 Si) 82
±
99 69 99 76 7 86
±
58 151 164 98 81 119 160 112 88 8 115
5 6 7 8 9 10 II 12 13
Oral contraceptive users
I hr
by Student’s pridoxine
2 hr 91 53 96 69 16 77
±
92 96 99 93 84 lOS 135 114 54 37 97±
±
paired (P
±
P > 0.001). All of the oral contraceptive users excreted increased quantities of xanthurenic acid before the onset of vitamin B6 deficiency, although there was considerable variation among individual subjects. Despite the further marked increases in xanthurenic acid excretion which occurred when they were receiving the vitamin B6-deficient diet, the changes did not reach statistical significance. Treatment of the oral contraceptive users with pynidoxine reduced xanthurenic acid excretion, but the levels remained elevated in the subjects receiving 0.8 mg daily (nos. 5, 10, 12), and in two instances (nos. 6, 7) were still above the normal range after daily treatment with 2.0 mg of the vitamin. Vitamin B6 depletion was also reflected in the changes in plasma pynidoxal phosphate concentrations, although the reduced levels were statistically significant only in the larger
ORAL TABLE
Effect
4 of vitamin
supplementation
B, deficiency with
CONTRACEPTIVES
on oral
pyridoxine
HCI:
glucose plasma
AND
tolerance, insulin
VITAMIN
and
B, DEFICIENCY
its reversal
875
by
in MU/ml
Predeliciency
Peak deficiency
After
I 2 3 4 mean
Oral tive
a
Significantly
pyridoxine
(0.05
different >
I hr
25.0 25.2 21.1
43,8 151.8 39.2
2hr
fast
I hr
2hr
fast
I hr
2hr
42.5 64.3 35.3
25.0 29.5 21.5
51.4 81.3 87.7
45.2 83.7 39.8
24.3 23.9 21.7
35.2 86.7 52.6
42.3 53.3 45.3
±
SI)
23.8 ±2.3
78.3 ±63.7
47.4 ±15.1
25.3 ±4.0
73.5 ±19.4
56.2 ±23.9
23.3 ±1.4
58.2 ±26.2
47.0 ±5.7
27.0 19.5 23.0 21.2 26.8 24.1 22.7 21.5 22.2 23.1 ±2.5
74.1 134.1 42.4 52.9
69.2 141.0 81.3 44.8 34.0 52.7 94.6 76.9 54.0 72.1 ±32.2
24.1 23.4 24.0 23.8 26.0 22.0 30.7 18.6 24.0 24.1 ±3.2
104.2 108.0 62.9 69.2 31.4 53.5 136.3 64.8 45.0 75.0 ±34.0
87.9 118.8 92.6 62.2 80.6 43.4 143.7 61.3 92.8 87.1” ±30.6
25.1 26.2
101.4 103.7
80.6 113.0
±
S 6 7 8 9 10 II 12 13 SI)
22.7 27.5 23.3 29.0 19.7 27.6 25.1 ±8.0
66.3 101.7 43.2 124.2 48.7 80.1 83.6 ±28.9
54.8 59.6 49.2 92.0 53.1 38.4 67.6 ±25.2
contracepusers
mean
fast
by Student’s
47.9 134.9 74.6 72.6 79.2 ±36.3 paired
t-test
from
oral
contraceptive
users
2-hr
values
after
repletion
with
P > 0.02).
deficiency period one of the oral contraceptive users (no. I I ) had a plasma glucose level of 135 mg/lOO ml at 2 hours after the load (Table 3). This value was elevated compared with those of the 4 controls or the I 2 women consuming a self-selected diet, in spite of a relatively high plasma insulin (Table 4). The oral cont racept ive-t reated subjects showed a significantly higher mean 2-hour plasma glucose when they were vitamin B6 deficient compared with the corresponding value both before commencing the depletion period (0.01 > P > 0.001), and after pynidoxine supplementation (P < 0.001). This increased plasma glucose was also significantly higher than the 2-hour value for the vitamin B6-deficient control group (0.01 > P > 0.001), and for the women consuming a self-selected diet (0.01 > P > 0.001). The plasma insulin levels of the oral contraceptive users before and at the height of the deficiency were not significantly different (Table 4), but after pynidoxine administration the levels were lower than they were during the vitamin B6-deficiency period (0.05 > P > 0.02). Two oral cont racept ive-t reated women (nos. 8, 1 1) had both high I-hour and 2-hour plasma glucose levels when they were vitamin B6 deficient, compared with either of the two
groups
of
elevated
control
glucose
values concentrations
in Table were
I
.
These
present
levels of plasma insulin. In the altered glucose tolerance was reversed by treatment with 2.0 mg of pyridoxme HC1 daily for 4 weeks. Subject no. 10, who was treated with only 0.8 mg of pynidoxine HC1 a day and continued to excrete an increased quantity of xanthurenic acid, was restudied after the daily administration of 100 mg ofthe vitamin for4 days. On this occasion the xanthurenic acid excretion was 29 zmoles/ 24 hours. The results for the fourth oral glucose tolerance test were: fasting plasma glucose 97 mg/lOO ml; I hour, l28mg/lOOml;and2hours, 103 mg/lOO ml, and the corresponding plasma insulin levels: 21.6, 52.6 and 52.5 zU/ml. despite
both
increased
cases
Discussion Feeding the vitamin B6-deficient diet produced abnormalities in tryptophan and methionine metabolism, and reductions in erythrocyte aminotransferase activities, all of which were reversed by PN.HCI administration. We recognize that these indices do not provide knowledge of the pynidoxal phosphate concentrations in the various tissues, but they do indicate that insufficient coenzyme was
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/8/872/4732955 by East Carolina University user on 12 January 2019
Controls
pyridoxine
.
No.
876
ROSE
available to maintain activities at a normal least, both the controls users
became
vitamin
at least some metabolic level. In this sense at and oral contraceptive B6 deficient during the
period. the induction of vitamin B6 deficiency, both the controls and the oral contraceptive users had somewhat lower plasma glucose levels in response to the l00-g glucose load than did the normal women taking a self-selected diet. This may have resulted from the effect of their standardized, high, carbohydrate intake for 2 or 3 days before the test. The increases in blood sugar after a glucose load are known to be less in normal subjects who have received a high carbohydrate diet for several days before the assessment of glucose tolerance. Wilkerson et al. (8) have commented that the majority of patients attending for glucose tolerance tests normally ingest less than 250 g/day when they select their own diet, and that many find it difficult to achieve this level of intake. Similar differences in carbohydrate consumption may explain our findings.
The changes in plasma glucose which we observed in the oral contraceptive users after the induction of vitamin B, deficiency were not large, and were significantly different from their predeficiency and postdeficiency values only at the 2-hour time point after glucose ingestion. However, it should be stressed that the experimental subjects were all receiving exactly the same diet throughout the study, apart from the level of vitamin B6 intake, and that each was acting as her own control. Intersubject variation was thereby eliminated. In addition, however, the 2-hour plasma glucose of the vitamin B6-deficient oral contraceptive users was significantly higher than the corresponding value for the vitamin B6-deficient controls, and for the women consuming a self-selected diet. We conclude from these observations that a deterioration in glucose tolerance did take place when the influence of vitamin B6 deficiency was combined with oral contraceptive administration. The use of estrogen-containing oral contraceptives is frequently accompanied by an elevation in the xanthurenic acid excretion, at least after a tryptophan load, and because this appears to be a hepatic rather than a renal
tubular defect (9) there is most likely an increased level of this metabolite circulating in the plasma. Eighteen years ago Kotake (10) reported that feeding rats a high tryptophan-fatty acid diet resulted in an elevated xanthurenic acid excretion, hyperglycem ia, glycosuria, and histological evidence of damage to the /3-cells of the pancreatic islets. An identical picture was produced by a high tryptophan-vitamin B6-deficient diet, and also by intraperitoneal injection of xanthurenic acid into rats receiving a normal diet. In subsequent studies it was found that xanthurenic acid and insulin will form a complex in vitro, with a resulting loss of hormonal activity (I 1 On the basis of these data Kotake et al. (1 1) postulated that any condition which causes a high circulating level of xanthurenic acid may impair insulin function because of complex formation between the hormone and the ).
metabolite.
The question arises, therefore, of whether altered carbohydrate metabolism in oral contraceptive users is the result of combination between xanthurenic acid and insulin. Such an effect could conceivably produce impaired glucose tolerance in the presence of circulating insulin levels which are normal or elevated as assessed by radioimmunoassay, but of reduced biological activity. Abnormal glucose tolerance with hyperinsulinemia does occur
in
some
women
(3),
plasma
insulin
oral
and levels
in
contraceptive-treated
the observed
present were
study
the
compati-
ble with this hypothesis. An apparent objection is that there was no correlation between the urinary xanthurenic acid excretion and glucose tolerance in the oral contraceptive users, and that changes in carbohydrate metabolism were not seen in the control subjects although they also excreted very high xanthurenic acid levels during the vitamin B6-deficiency period. But, the xanthurenic acid level in urine may not correlate well with, nor necessarily reflect, high plasma and tissue concentrations of the metabolite. Elucidation of this point, and of the effect of hormones on renal handling of xanthurenic acid, must await the development of a satisfactory method for plasma assays. Two previous studies support a role for altered tryptophan metabolism or vitamin B6 function in the impaired glucose tolerance
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/8/872/4732955 by East Carolina University user on 12 January 2019
depletion Before
ET AL.
ORAL
CONTRACEPTIVES
AND
tration,
whereas
no
significant
change
oc-
curred in those without evidence of deficiency. In the present investigation, the oral contraceptive users, but not the controls, showed a clear-cut deterioration in oral glucose tolerance when they were vitamin B6 deficient although xanthurenic acid excretion was increased in both groups. Consideration of the findings of these two studies suggests that a combination of vitamm B6 deficiency and an independent metabolic effect of contraceptive steroids may be required to produce a pyridoxine-respon-
sive
abnormality
women The
of
using these postulated
volve
an
Estrogens
glucose
preparations. metabolic
enhanced
enzymes
concerned
the
of
effect
may
in
in-
gluconeogenesis.
activities of several in the catabolism of gluconeogenic amino acids, notably alanine aminotransferase and tyrosine aminotransferase (12). The reports of reduced plasma levels of alanine, phenylalanine and arginine (13), and tyrosine (14) are consistent with such an effect, as is the finding of enhanced blood pyruvate and plasma insulin levels after alanine loading in oral contraceptive users (Rose et al., unpublished observations). Furthermore, estrogens raise the level of circulating corticosteroids (15), and cortisol increases the activity of hepatic phosphoenolpyruvate carboxykinase (16), a key enzyme in gluconeogenesis. Apart from the possible formation of a xanthurenic acid-insulin complex, which has liver
increase
rate
tolerance
B, DEFICIENCY
877
already been discussed, it is difficult to postulate a role for vitamin B6 deficiency in the production of impaired glucose tolerance. Vitamin B6 deficiency and oral contraceptives increase the urinary excretion of quinolinic acid (17) and it has been suggested that hormonal control of this tryptophan metabolite may function in the regulation of gluconeogenesis (18). However, both in the isolated perfused rat liver (19) and in man (20), quinolinic acid and its precursors, including L-tryptophan, inhibit phosphoenolpyruvate carboxykinase and reduce glucose production. Thus, the influences of vitamin B6, tryptophan metabolism , and contraceptive steroids on carbohydrate tolerance are cornplex and their elucidation must await further investigation. We thank their skilled
Pat Stauber, Heidi technical assistance.
Kan
and
R. A. Arend
for
References I.
BRowN, R. R., D. P. ROSE, J. E. LEKt.Ei. H. LINKswiLER AND C. R. ANAND. Urinary 4-pridoxic acid, plasma pyridoxal phosphate, and erythrocyte aminotransferase levels in oral contraceptive users receiving controlled intakes of vitamin B,. Am. J. Clin. Nutr. 28: 10, 1975. 2. LEKLEM, J. E., R. R. BROWN, D. P. ROSE, H. LINKS\ILER ANt) R. A. AREND. Metabolism of tryptophan and niacin in oral contraceptive users receiving controlled intakes of vitamin B,. Am. J. Clin. Nutr. 28: 146. 1975. 3. WYNN, V., AND J. W. H. DOAR. Effects of oral contraceptives on carbohydrate metabolism. J. Clin. Pathol. 23: Suppl. 3: 9, 1970. 4. SPELLACY, W. N., W. C. BLIII AND S. A. BIRK. The effects of vitamin B, on carbohydrate metabolism in women taking steroid contraceptives: Preliminary report. Contraception 6: 265, 1972. 5.
ADAMS,
WYNN.
P. W., D. G. The
effect
on
CRAMP, carbohydrate
D. P.
ROSE
AM)
metabolism
V. of
correcting pyridoxine (B,) deficiency in women taking oral contraceptive steroids. Excerpta Mcdica, International Congress Series, 280, VIII Congress of Amsterdam, 6. SuEDLTII, Automation
the
International Diabetes 1973, p. 69. N. C., J. R. Wiuisii ANt) of glucose measurement
Federation, J.
L. MOORE. using ortho-
toluidine reagent: by automated methods. Am. 7.
8.
Comparison of values determined ferricy’anide and ortho-toluidine J. Clin. Pathol. 53: 181. 1970. HALES. C. N., AND P. J. RANDI.E. Immunoassay insulin with insulin-antibody precipitate. Biochem. 88: 137, 1963. WILKERSON,
C.
H. L. C., H. HYMAN, M. KAUFMAN, A.
AND J. 0’S. FRANCIS. Diagnostic evaluation of oral glucose tolerance tests in non-diabetic subjects after various levels of carbohydrate intake. New EngI. J. Med. 262: 1047, 1960. MCCUIST10N
of J.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/8/872/4732955 by East Carolina University user on 12 January 2019
seen during oral contraceptive administration. Spellacy, Buhi and Birk (4) have recently published a preliminary report of the effect of pyridoxine administration on 12 women whose glucose tolerance had deteriorated while taking oral contraceptives. The group as a whole showed an improvement in their glucose tolerance after treatment with 25 mg of PN ‘ HCI a day for 1 month, although in 4 subjects there was no benefit. However, no attempt was made to classify the subjects according to their vitamin B6 nutritional status in this preliminary investigation. Adams et al. (5) studied 3 1 oral contraceptive users consuming self-selected diets and divided them into those with or without biochemical evidence of vitamin B6 deficiency. Glucose tolerance of the deficient group was improved by pyridoxine adminis-
VITAMIN
ROSE
878 9.
10.
ROSE, D. HARDING.
P., R.
the effect tives on
of oestrogen-containing tryptophan metabolism
requirements. KOTAKE,
Y.
STRONG. Experimental
Clin. Sci. Xanthurenic
P. W. vitamin
42:
AD.s\is ANI) B, deficiency oral and
465, acid,
P. F.
1972. an abnormal
14. B, me-
12.
BRAIDMAN,
AND
D. P.
RoSE.
Effects
13.
ALY,
H. E., E. A.
D0NAi.o
ANI)
M. H. W.
of
sex
enzymes in rat SIMPSON.
IS.
16.
17.
ROSE, plasma
contraceptives J. Clin. Nutr.
D. P., tyrosine
24:
and vitamin 297, 1971.
AM) D. by oral
G. CRAMP. contraceptives
B,
metabolism. Reduction of and oestro-
gens: A possible consequence of tyrosine aminotransferase induction. Clin. Chim. Acta 29: 49, 1970. BURKE, C. W. The effect of oral contraceptives on cortisol metabolism. J. Clin. Pathol. 23: Suppl. 3: II, 1970. FOSTER, D. 0., P. D. RAY ANt) H. A. LARDY. Studies on the mechanisms underlying adaptive changes in rat liver phosphoenolpyruvate carboxykinase. Biochemistry 5: 555, 1966. ROSE, D. P., AND P. A. T0SEI.AND. Urinary excretion of quinolinic acid and other tryptophan metabolites after deoxypyridoxine or oral contraceptive administration. Metabolism 22: 165, 1973.
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II.
tabolite of tryptophan and the diabetic symptoms caused in albino rats by its production. J. Vitaminol., Kyoto I: 157, 1955. KOTAKE, Y., T. SOTOKA’AA. E. MURAKAMI, A. HISTATAKE, M. ABE ANI) Y. IKEDA. Studies on the xanthurenic acid-insulin complex II. Physiological activities. J. Biochem., Tokyo 63: 578, 1968.
I. P..
Oral Am.
and
contracepvitamin
hormones on three glucocorticoid-inducible concerned with amino acid metabolism liver. Endocrinology 89: 1250, 1971.
ET AL.
of oral
D. P. Rose, and H. M.
and
M.D., Ph.D., J. E. Lek/ern. Linkswi/er, Ph.D.
ABSTRACT
Oral
The
tests
after pridoxine excretion
reduced
tolerance levels.
tolerance
of
carbohydrate consequent
were
the
the
repeated
after
loss
of
biological Clin.
controls. the
Nuir.
Informed consent was obtained from all subjects before commencing the study. The details of the experimental procedure, and the vitamin B,-deficient diet,
American
Journal
of Clinical
was
In
Nutrition
have were
oral
group
addition,
of
plasma
despite
diet.
oral
again
xanthurenic or
elevated
no change
acid in the plasma
in the
pyridoxine-responsive with
in four
and
a deterioration
normal was
insulin
pyridoxal and
B,-deficient with
Th.re
observed
and
contraceptives
by an increased
associated
pyridoxine. The
28: 872
materials
The
as judged
complexing
activity.
In two previous publications, we have descnibed the changes in urinary 4-pyridoxic acid, plasma pyridoxal phosphate and erythrocyte aminotransferases ( I ),and tryptophan and niacin metabolism (2), which occurred when a group of oral contraceptive users and female controls of similar age were fed a vitamin B6-deficient diet for 4 weeks, and were then repleted with various doses of pynidoxine. Oral contraceptives alter carbohydrate metabolism in some women, the changes being characterized by impaired glucose tolerance and abnormally elevated serum insulin levels after oral or intravenous glucose loads (3). Recently, it has been observed that the glucose tolerance of oral contraceptive users can be improved by pyridoxine administration (4, 5). In this paper, we report the results of oral glucose tolerance tests which were performed on some of the oral contraceptive-treated women and control subjects included in the previously reported studies (1, 2) when they were rendered vitamin B6 deficient.
872
by
excretion,
of a vitamin
steroid-treated
involve
acid taking
ingestion
phosphate,
reversed
B,-deficient
Am.J.
gluconeogensis.
pyridoxal
Ph.D.,
women
B6 deficiency,
Vitamin
was may
Brown,
xanthurenie
in nine 4 weeks
contraceptive
vitamin
R.
urinary’
determined
abnormality
metabolism
R.
tolerance,
were
plasma
of This
2
Ph.D.,
supplementation.
and
glucose insulin
glucose
concentrations
controls.
and
B6
on carbohydrate
phosphate
Methods
vitamin
glucose
alteration
xanthurenic
contraceptives
acid may
in with
a
enhance
878. 1975.
been described elsewhere ( I ): the part of this larger investigation.
present
subjects
In summary, the subjects received a diet containing 0.19 mg ofpyridoxine activity daily. supplemented daily with an additional 0.8 mg of py’ridoxine hydrochloride (PN ‘ HCI) for 5 days. The PN HCI supplement was then withdrawn and the vitamin B,-deficient diet fed for 28 day’s. Thereafter, the diet was continued for a further 28-day period, but supplemented with 0.8 mg. 2 mg or 20 mg of py’ridoxine hydrochloride dails. Nine women who had been taking an estrogen-containing oral contraceptive for at least 6 months, and 4 controls were included in the study of carbohydrate metabolism (Table I ). Alterations in metabolic functions arising from the reduced dietary vitamin B, intake ssere monitored by the urinary excretion of tryptophan metabolites Lifter an ral 2-g L-tryptophan load (2), urinary cystathionine excretion after a 3-g oral dose of L-methionine (Leklem, Linkswiler, Brown and Rose-unpublished observations), and assay of erythrocyte alanine and aspartate aminotransferase activities ( I ). In addition, plasma py ridoxal phosphate and urinar .4-ps ridoxic acid were determined ( I). In all instances the changes were those expected to arise from vitamin B, depletion. and they were reversed by PN ‘ HCI supplements.
‘From the Division of Clinical Oncologs. University of Wisconsin Medical School and Department of Nutritional Sciences, University of Wisconsin College of Agricultural and Life Sciences, Madison. Wisconsin 53706. 2 Supported in part by Wisconsin College of Agricultural and Life Sciences, Contract NIH. NICHD 72-2782 with the National Institute of Child Health and Human Development, and Grant no. CA-l3302 from the National Cancer Institute, Bethesda, Maryland 20014.
28:
AUGUST
1975.
pp.
872
878.
Printed
in U.S.A.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/8/872/4732955 by East Carolina University user on 12 January 2019
deficiency
contraceptives
ORAL Before ingesting
commencing
the
the
B,-deficient
vitamin
CONTRACEPTIVES
deficiency
period, diet
AND
but
plus
when
0.8
sample vored
mg
Subj n
PN
Oralcontraceptive
0.
1 2 3 4 5 6 7 8 9 10 II 12 13
Control Control Control Control Orthonovum Orthonovum Orthonovum Orthonovum Orthonovum Ovral’ Ovral Norlestrin2l Demulenh a This
is the
dose
used,
ofpyridoxine
hydrochloride
tion
Table and
acid
I shows plasma
excretions
Plasma glucose was for true glucose (6), (7). differences between or paired Student’s
PLP
IS 35 20 27 1,174 423 230 91 741 131 73 367 604
10.57 5.84 25.32 12.14 9.18 10.22 9.32 14.72 7.27 4.82 8.19 8.92 7.90
daily
the xanthurenic acid pynidoxal phosphate
Peak deficiency
XA”
given
100 g of lemon-flafurther blood sam-
XA
during
451 1,079 708 944 2,157 1,803 1,684 1,481 1,819 1,427 1,088 1,417 1,771 the
excrelevels
and
Predeficiency
0.8 2.0 2.0 2.0 0.8 2.0 2.0 2.0 20.0 0.8 2.0 0.8 20.0
I/SO”' 1/50 l/80’ 1/80 1/50
of venous blood was obtained, glucose was given orally, and
Results
xanthurenic
. HCI. a mg
873
pIes were taken I and 2 hours later. determined by an automated method and plasma insulin by radioimmunoassay The statistical significance of groups was assessed by unpaired t-tests, as appropriate.
tolerance and urinary xanthurenic acid excretion were also determined in 12 other young women who were not taking an oral contraceptive and who were consuming a self-selected diet but without vitamin supplements (Table 2). The subjects were fasted overnight for the oral glucose tolerance tests. Next morning, after resting for 30 mm, a
contraceptives concentrations
B, DEFICIENCY
28-day
After
PLP
XA
2.30 2.01 6.30 3.93 2.81 1.92 2.27 3.88 3.85 2.53 2.50 2.60 5.09
23 25 17 20 914 75 81 55 42 230 35 377 34
repletion
period.
PN
.
HCI PLP
3.16 12.00 15.50 20.80 4.00 12.50 9.74 14.80 84.20 3.70 17.40 3.50 148.0
Xanthurenic
“
acid
(XA)
excretion in zmoles/24 hr after 2-g t.-try’ptophan load. The upper limit of normal (mean ± 2 SD) obtained for the I 2 women on self-selected diets who were not taking an oral contraceptive was 52 pmoles/24 hr. Plasma pyridoxal phosphate concentration (PLP) in ng/ml. d 0.05 mg mestranol: I mg norethindrone. ‘ 0.08 mg mestranol: I mg norethindrone. I #{216}#{216}5 mg ethinyl estradiol: 0.5 mg norgestrel. 0.05 mg ethinyl estradiol: 2.5 mg norethindrone acetate. “ 0.1 mg mestranol: 0.5 mg ethy’nodiol diacetate. Comparison of predeficiencv and peak deficiency’ results using Student’s paired t-test showed a significant increase in XA excretion by controls when deficient (0.01 > P > 0.001) and significant decrease in OC users PLP levels (P < 0.001).
TABLE 2 Oral glucose self-selected experimental
tolerance and urinary xanthurenic acid excretion in women diet, and controls and oral contraceptive users when receiving diet supplemented with py’ridoxine (predeficiency’)
Normal
subjects,
self-
selected diets (12) Controls, predeficieney (4) Oral contraceptive users, predeficiency (9) Results
are
0.01: “ 0.05 lecteddiets,0.0l
Pias
Age. years
Group
mean >
±
SI).
P > 0.02; > P > 0.001.
Plasma C
ma
glucose.
fast
ingesting the
mg/I
00
a
ml
I hr
Xanthurenic
23
±
3
90
±
5
136
±
33
98
±
IS
27
22 23
±
3 4
86 82
±
7 8
86 1 15
±
16” 37
77 97
±
±
±
20” 22
24 426
glucose xanthurenic
±
significantly acid
excretion
different
±
from
significantly
women different
acid.
,moIe/24
2 hr
on self-selected from
normal
diets:
±
8
±
9 363’
±
a
subjects
hr
0.02
> on
P >
self-se-
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/8/872/4732955 by East Carolina University user on 12 January 2019
PN . HCI per day. oral glucose tolerance was assessed, and the tests of vitamin B6 nutrition carried out. These metabolic studies were repeated after 28 days on the vitamin B,-deficient diet (“peak deficiency”) and after 4 weeks supplementation with PN’HCI. Oral glucose
TABLE I Experimental subjects, oral plasma pyridoxal phosphate
VITAMIN
ROSE
874
ET AL.
oral
contraceptive-treated
TABLE 3 Effect of vitamin supplementation
B, deficiency with pyridoxine
group
(P
on oral HC1:
glucose plasma
The results show that supplementation with 0.8 mg of PNHCl a day was insufficient to restore the plasma pynidoxal phosphate to predeficiency levels, although this dose did produce reductions in xanthurenic acid excretion. Table 2 compares the plasma glucose levels and urinary xanthurenic acid excretions of the controls and oral contraceptive users before the induction of vitamin B6 deficiency with those of the I 2 young women on self-selected diets who were not taking an oral contraceptive. The 4 controls receiving the vitamin B6-deficient diet supplemented with pyridoxme had significantly lower plasma glucose levels than the 12 ingesting their regular diet at both I and 2 hours after the glucose load; the oral contraceptive users had lower fasting plasma glucose levels than these same 12 women. The xanthurenic acid values for the two groups who were not taking oral contraceptives were virtually identical (Table 2); the oral contraceptive-treated group excreted significantly higher levels of xanthurenic acid than nonusers on a self-selected diet, but the number of nonusers on the controlled diet was too small for any statistical difference to be demonstrated.
The plasma glucose tamed during the oral are shown in Tables
0.001).
Significantly
P >
0.001)
±
different and
after
88 77 75 76 74 82 86 97 80 Si) 82
±
99 69 99 76 7 86
±
58 151 164 98 81 119 160 112 88 8 115
5 6 7 8 9 10 II 12 13
Oral contraceptive users
I hr
by Student’s pridoxine
2 hr 91 53 96 69 16 77
±
92 96 99 93 84 lOS 135 114 54 37 97±
±
paired (P
±
P > 0.001). All of the oral contraceptive users excreted increased quantities of xanthurenic acid before the onset of vitamin B6 deficiency, although there was considerable variation among individual subjects. Despite the further marked increases in xanthurenic acid excretion which occurred when they were receiving the vitamin B6-deficient diet, the changes did not reach statistical significance. Treatment of the oral contraceptive users with pynidoxine reduced xanthurenic acid excretion, but the levels remained elevated in the subjects receiving 0.8 mg daily (nos. 5, 10, 12), and in two instances (nos. 6, 7) were still above the normal range after daily treatment with 2.0 mg of the vitamin. Vitamin B6 depletion was also reflected in the changes in plasma pynidoxal phosphate concentrations, although the reduced levels were statistically significant only in the larger
ORAL TABLE
Effect
4 of vitamin
supplementation
B, deficiency with
CONTRACEPTIVES
on oral
pyridoxine
HCI:
glucose plasma
AND
tolerance, insulin
VITAMIN
and
B, DEFICIENCY
its reversal
875
by
in MU/ml
Predeliciency
Peak deficiency
After
I 2 3 4 mean
Oral tive
a
Significantly
pyridoxine
(0.05
different >
I hr
25.0 25.2 21.1
43,8 151.8 39.2
2hr
fast
I hr
2hr
fast
I hr
2hr
42.5 64.3 35.3
25.0 29.5 21.5
51.4 81.3 87.7
45.2 83.7 39.8
24.3 23.9 21.7
35.2 86.7 52.6
42.3 53.3 45.3
±
SI)
23.8 ±2.3
78.3 ±63.7
47.4 ±15.1
25.3 ±4.0
73.5 ±19.4
56.2 ±23.9
23.3 ±1.4
58.2 ±26.2
47.0 ±5.7
27.0 19.5 23.0 21.2 26.8 24.1 22.7 21.5 22.2 23.1 ±2.5
74.1 134.1 42.4 52.9
69.2 141.0 81.3 44.8 34.0 52.7 94.6 76.9 54.0 72.1 ±32.2
24.1 23.4 24.0 23.8 26.0 22.0 30.7 18.6 24.0 24.1 ±3.2
104.2 108.0 62.9 69.2 31.4 53.5 136.3 64.8 45.0 75.0 ±34.0
87.9 118.8 92.6 62.2 80.6 43.4 143.7 61.3 92.8 87.1” ±30.6
25.1 26.2
101.4 103.7
80.6 113.0
±
S 6 7 8 9 10 II 12 13 SI)
22.7 27.5 23.3 29.0 19.7 27.6 25.1 ±8.0
66.3 101.7 43.2 124.2 48.7 80.1 83.6 ±28.9
54.8 59.6 49.2 92.0 53.1 38.4 67.6 ±25.2
contracepusers
mean
fast
by Student’s
47.9 134.9 74.6 72.6 79.2 ±36.3 paired
t-test
from
oral
contraceptive
users
2-hr
values
after
repletion
with
P > 0.02).
deficiency period one of the oral contraceptive users (no. I I ) had a plasma glucose level of 135 mg/lOO ml at 2 hours after the load (Table 3). This value was elevated compared with those of the 4 controls or the I 2 women consuming a self-selected diet, in spite of a relatively high plasma insulin (Table 4). The oral cont racept ive-t reated subjects showed a significantly higher mean 2-hour plasma glucose when they were vitamin B6 deficient compared with the corresponding value both before commencing the depletion period (0.01 > P > 0.001), and after pynidoxine supplementation (P < 0.001). This increased plasma glucose was also significantly higher than the 2-hour value for the vitamin B6-deficient control group (0.01 > P > 0.001), and for the women consuming a self-selected diet (0.01 > P > 0.001). The plasma insulin levels of the oral contraceptive users before and at the height of the deficiency were not significantly different (Table 4), but after pynidoxine administration the levels were lower than they were during the vitamin B6-deficiency period (0.05 > P > 0.02). Two oral cont racept ive-t reated women (nos. 8, 1 1) had both high I-hour and 2-hour plasma glucose levels when they were vitamin B6 deficient, compared with either of the two
groups
of
elevated
control
glucose
values concentrations
in Table were
I
.
These
present
levels of plasma insulin. In the altered glucose tolerance was reversed by treatment with 2.0 mg of pyridoxme HC1 daily for 4 weeks. Subject no. 10, who was treated with only 0.8 mg of pynidoxine HC1 a day and continued to excrete an increased quantity of xanthurenic acid, was restudied after the daily administration of 100 mg ofthe vitamin for4 days. On this occasion the xanthurenic acid excretion was 29 zmoles/ 24 hours. The results for the fourth oral glucose tolerance test were: fasting plasma glucose 97 mg/lOO ml; I hour, l28mg/lOOml;and2hours, 103 mg/lOO ml, and the corresponding plasma insulin levels: 21.6, 52.6 and 52.5 zU/ml. despite
both
increased
cases
Discussion Feeding the vitamin B6-deficient diet produced abnormalities in tryptophan and methionine metabolism, and reductions in erythrocyte aminotransferase activities, all of which were reversed by PN.HCI administration. We recognize that these indices do not provide knowledge of the pynidoxal phosphate concentrations in the various tissues, but they do indicate that insufficient coenzyme was
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/8/872/4732955 by East Carolina University user on 12 January 2019
Controls
pyridoxine
.
No.
876
ROSE
available to maintain activities at a normal least, both the controls users
became
vitamin
at least some metabolic level. In this sense at and oral contraceptive B6 deficient during the
period. the induction of vitamin B6 deficiency, both the controls and the oral contraceptive users had somewhat lower plasma glucose levels in response to the l00-g glucose load than did the normal women taking a self-selected diet. This may have resulted from the effect of their standardized, high, carbohydrate intake for 2 or 3 days before the test. The increases in blood sugar after a glucose load are known to be less in normal subjects who have received a high carbohydrate diet for several days before the assessment of glucose tolerance. Wilkerson et al. (8) have commented that the majority of patients attending for glucose tolerance tests normally ingest less than 250 g/day when they select their own diet, and that many find it difficult to achieve this level of intake. Similar differences in carbohydrate consumption may explain our findings.
The changes in plasma glucose which we observed in the oral contraceptive users after the induction of vitamin B, deficiency were not large, and were significantly different from their predeficiency and postdeficiency values only at the 2-hour time point after glucose ingestion. However, it should be stressed that the experimental subjects were all receiving exactly the same diet throughout the study, apart from the level of vitamin B6 intake, and that each was acting as her own control. Intersubject variation was thereby eliminated. In addition, however, the 2-hour plasma glucose of the vitamin B6-deficient oral contraceptive users was significantly higher than the corresponding value for the vitamin B6-deficient controls, and for the women consuming a self-selected diet. We conclude from these observations that a deterioration in glucose tolerance did take place when the influence of vitamin B6 deficiency was combined with oral contraceptive administration. The use of estrogen-containing oral contraceptives is frequently accompanied by an elevation in the xanthurenic acid excretion, at least after a tryptophan load, and because this appears to be a hepatic rather than a renal
tubular defect (9) there is most likely an increased level of this metabolite circulating in the plasma. Eighteen years ago Kotake (10) reported that feeding rats a high tryptophan-fatty acid diet resulted in an elevated xanthurenic acid excretion, hyperglycem ia, glycosuria, and histological evidence of damage to the /3-cells of the pancreatic islets. An identical picture was produced by a high tryptophan-vitamin B6-deficient diet, and also by intraperitoneal injection of xanthurenic acid into rats receiving a normal diet. In subsequent studies it was found that xanthurenic acid and insulin will form a complex in vitro, with a resulting loss of hormonal activity (I 1 On the basis of these data Kotake et al. (1 1) postulated that any condition which causes a high circulating level of xanthurenic acid may impair insulin function because of complex formation between the hormone and the ).
metabolite.
The question arises, therefore, of whether altered carbohydrate metabolism in oral contraceptive users is the result of combination between xanthurenic acid and insulin. Such an effect could conceivably produce impaired glucose tolerance in the presence of circulating insulin levels which are normal or elevated as assessed by radioimmunoassay, but of reduced biological activity. Abnormal glucose tolerance with hyperinsulinemia does occur
in
some
women
(3),
plasma
insulin
oral
and levels
in
contraceptive-treated
the observed
present were
study
the
compati-
ble with this hypothesis. An apparent objection is that there was no correlation between the urinary xanthurenic acid excretion and glucose tolerance in the oral contraceptive users, and that changes in carbohydrate metabolism were not seen in the control subjects although they also excreted very high xanthurenic acid levels during the vitamin B6-deficiency period. But, the xanthurenic acid level in urine may not correlate well with, nor necessarily reflect, high plasma and tissue concentrations of the metabolite. Elucidation of this point, and of the effect of hormones on renal handling of xanthurenic acid, must await the development of a satisfactory method for plasma assays. Two previous studies support a role for altered tryptophan metabolism or vitamin B6 function in the impaired glucose tolerance
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/8/872/4732955 by East Carolina University user on 12 January 2019
depletion Before
ET AL.
ORAL
CONTRACEPTIVES
AND
tration,
whereas
no
significant
change
oc-
curred in those without evidence of deficiency. In the present investigation, the oral contraceptive users, but not the controls, showed a clear-cut deterioration in oral glucose tolerance when they were vitamin B6 deficient although xanthurenic acid excretion was increased in both groups. Consideration of the findings of these two studies suggests that a combination of vitamm B6 deficiency and an independent metabolic effect of contraceptive steroids may be required to produce a pyridoxine-respon-
sive
abnormality
women The
of
using these postulated
volve
an
Estrogens
glucose
preparations. metabolic
enhanced
enzymes
concerned
the
of
effect
may
in
in-
gluconeogenesis.
activities of several in the catabolism of gluconeogenic amino acids, notably alanine aminotransferase and tyrosine aminotransferase (12). The reports of reduced plasma levels of alanine, phenylalanine and arginine (13), and tyrosine (14) are consistent with such an effect, as is the finding of enhanced blood pyruvate and plasma insulin levels after alanine loading in oral contraceptive users (Rose et al., unpublished observations). Furthermore, estrogens raise the level of circulating corticosteroids (15), and cortisol increases the activity of hepatic phosphoenolpyruvate carboxykinase (16), a key enzyme in gluconeogenesis. Apart from the possible formation of a xanthurenic acid-insulin complex, which has liver
increase
rate
tolerance
B, DEFICIENCY
877
already been discussed, it is difficult to postulate a role for vitamin B6 deficiency in the production of impaired glucose tolerance. Vitamin B6 deficiency and oral contraceptives increase the urinary excretion of quinolinic acid (17) and it has been suggested that hormonal control of this tryptophan metabolite may function in the regulation of gluconeogenesis (18). However, both in the isolated perfused rat liver (19) and in man (20), quinolinic acid and its precursors, including L-tryptophan, inhibit phosphoenolpyruvate carboxykinase and reduce glucose production. Thus, the influences of vitamin B6, tryptophan metabolism , and contraceptive steroids on carbohydrate tolerance are cornplex and their elucidation must await further investigation. We thank their skilled
Pat Stauber, Heidi technical assistance.
Kan
and
R. A. Arend
for
References I.
BRowN, R. R., D. P. ROSE, J. E. LEKt.Ei. H. LINKswiLER AND C. R. ANAND. Urinary 4-pridoxic acid, plasma pyridoxal phosphate, and erythrocyte aminotransferase levels in oral contraceptive users receiving controlled intakes of vitamin B,. Am. J. Clin. Nutr. 28: 10, 1975. 2. LEKLEM, J. E., R. R. BROWN, D. P. ROSE, H. LINKS\ILER ANt) R. A. AREND. Metabolism of tryptophan and niacin in oral contraceptive users receiving controlled intakes of vitamin B,. Am. J. Clin. Nutr. 28: 146. 1975. 3. WYNN, V., AND J. W. H. DOAR. Effects of oral contraceptives on carbohydrate metabolism. J. Clin. Pathol. 23: Suppl. 3: 9, 1970. 4. SPELLACY, W. N., W. C. BLIII AND S. A. BIRK. The effects of vitamin B, on carbohydrate metabolism in women taking steroid contraceptives: Preliminary report. Contraception 6: 265, 1972. 5.
ADAMS,
WYNN.
P. W., D. G. The
effect
on
CRAMP, carbohydrate
D. P.
ROSE
AM)
metabolism
V. of
correcting pyridoxine (B,) deficiency in women taking oral contraceptive steroids. Excerpta Mcdica, International Congress Series, 280, VIII Congress of Amsterdam, 6. SuEDLTII, Automation
the
International Diabetes 1973, p. 69. N. C., J. R. Wiuisii ANt) of glucose measurement
Federation, J.
L. MOORE. using ortho-
toluidine reagent: by automated methods. Am. 7.
8.
Comparison of values determined ferricy’anide and ortho-toluidine J. Clin. Pathol. 53: 181. 1970. HALES. C. N., AND P. J. RANDI.E. Immunoassay insulin with insulin-antibody precipitate. Biochem. 88: 137, 1963. WILKERSON,
C.
H. L. C., H. HYMAN, M. KAUFMAN, A.
AND J. 0’S. FRANCIS. Diagnostic evaluation of oral glucose tolerance tests in non-diabetic subjects after various levels of carbohydrate intake. New EngI. J. Med. 262: 1047, 1960. MCCUIST10N
of J.
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seen during oral contraceptive administration. Spellacy, Buhi and Birk (4) have recently published a preliminary report of the effect of pyridoxine administration on 12 women whose glucose tolerance had deteriorated while taking oral contraceptives. The group as a whole showed an improvement in their glucose tolerance after treatment with 25 mg of PN ‘ HCI a day for 1 month, although in 4 subjects there was no benefit. However, no attempt was made to classify the subjects according to their vitamin B6 nutritional status in this preliminary investigation. Adams et al. (5) studied 3 1 oral contraceptive users consuming self-selected diets and divided them into those with or without biochemical evidence of vitamin B6 deficiency. Glucose tolerance of the deficient group was improved by pyridoxine adminis-
VITAMIN
ROSE
878 9.
10.
ROSE, D. HARDING.
P., R.
the effect tives on
of oestrogen-containing tryptophan metabolism
requirements. KOTAKE,
Y.
STRONG. Experimental
Clin. Sci. Xanthurenic
P. W. vitamin
42:
AD.s\is ANI) B, deficiency oral and
465, acid,
P. F.
1972. an abnormal
14. B, me-
12.
BRAIDMAN,
AND
D. P.
RoSE.
Effects
13.
ALY,
H. E., E. A.
D0NAi.o
ANI)
M. H. W.
of
sex
enzymes in rat SIMPSON.
IS.
16.
17.
ROSE, plasma
contraceptives J. Clin. Nutr.
D. P., tyrosine
24:
and vitamin 297, 1971.
AM) D. by oral
G. CRAMP. contraceptives
B,
metabolism. Reduction of and oestro-
gens: A possible consequence of tyrosine aminotransferase induction. Clin. Chim. Acta 29: 49, 1970. BURKE, C. W. The effect of oral contraceptives on cortisol metabolism. J. Clin. Pathol. 23: Suppl. 3: II, 1970. FOSTER, D. 0., P. D. RAY ANt) H. A. LARDY. Studies on the mechanisms underlying adaptive changes in rat liver phosphoenolpyruvate carboxykinase. Biochemistry 5: 555, 1966. ROSE, D. P., AND P. A. T0SEI.AND. Urinary excretion of quinolinic acid and other tryptophan metabolites after deoxypyridoxine or oral contraceptive administration. Metabolism 22: 165, 1973.
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II.
tabolite of tryptophan and the diabetic symptoms caused in albino rats by its production. J. Vitaminol., Kyoto I: 157, 1955. KOTAKE, Y., T. SOTOKA’AA. E. MURAKAMI, A. HISTATAKE, M. ABE ANI) Y. IKEDA. Studies on the xanthurenic acid-insulin complex II. Physiological activities. J. Biochem., Tokyo 63: 578, 1968.
I. P..
Oral Am.
and
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ET AL.
