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Pyridoxine treatment of chemical diabetes in pregnancy
Pyridoxine treatment of chemical diabetes in pregnancy M.
D. G. GILLMER
1). MAZIBUKO DwLm~,
Thirteen excessive pyridoxine restored tolerance change therapy.
South
Africa
women with chemical diabetes diagnosed in late pregnancy were found to excrete amount of urinary xanthurenic acid after a tryptophan load, indicative of a relative (vitamin 6,) deficiency. Treatment with 100 mg pyridoxine daily for 14 to 23 days the urinary xanthurenic acid excretion to normal in all patients. Improvement of glucose was observed in only two of the patients studied, deterioration in six, and no significant in the remaining five. The insulin response to glucose was unaltered during pyridoxine (AM. J. OBSTET. GYNECOL. 133:499, 1979.)
ABNORMAL TRYPTOPHAN metabolism due to pyridoxine (vitamin B,) deficiency has recently been implicated as a cause of chemical diabetes in pregnant women’. ’ and those treated with oral contraceptives.3s ’ It has been suggested that this is due to the production of excessive amounts of xanthurenic acid, an abnormal metabolite of tryptophan which forms a stable complex with insulin,” and has been demonstrated to halve the hypoglycemic action of the hormone in animals.” Pyridoxine treatment of pregnant women with chemical diabetes has been shown to produce an improvement in carbohydrate tolerance, and it has been proposed that this is due to a reduction in xanthurenic acid formation.‘. ’ The present study was performed to confirm these reports and to determine the relationship between the plasma insulin and urinary xanthurenic acid concentrations in pregnant women with chemical diabetes given pyridoxine in doses previously shown to correct glucose tolerance.
Material and methods Thirteen chemical diabetic women who had a 100 gm oral glucose tolerance test (GTT) during late pregnancy because of potential diabetic features were studied. The diagnosis of chemical diabetes was made be-
cause the venous plasma glucose at 2 hours exceeded 8 mmol/L.’ All subjects were admitted to the hospital at least 1 week before the start of the study and remained in the hospital until delivery; during this time they received a diet containing 150 gm of carbohydrate each day. The study was started between 31 and 37 weeks of pregnancy (mean ? 1 SD, 33.5 ? 1.5). After an overnight fast the 100 gm oral GTT was repeated on the first day of the study with the patient awake and semirecumbent. A blood sample was taken from an antecubital vein before drinking the glucose solution and thereafter at half-hour intervals for 3 hours. The plasma was separated immediately after the collection of each blood sample and divided into two aliquots. One was stored at 4” C and the glucose concentration was measured within 24 hours by means of a manual glucose oxidase-peroxidase method (GOD period, Boehringer). The second aliquot was stored at -20” C and used to measure the plasma insulin concentration by means of the Insulin RIA kit of The Radiochemical Centre, Amersham, England. On the second day of the study 2 gm of L-tryptophan dissolved in a glass of milk flavored with fruit juice concentrate was taken by mouth, after the patient had emptied her bladder. All urine passed during the next 8 hours was collected, the volume measured, and a 100 ml sample stored at -20” C for determination of urinary xanthurenic acid excretion.” The following day treatment with pyridoxine tablets, 25 mg four times a day, was started and continued for between 14 and 23 days (mean ? 1 SD. 17.3 + 2.8). At this time the oral GTT and L-tryptophan load were repeated during therapy. 499
500
Glllmer
and Mazibuko
12. llPLASMA
‘0
GLUCOSE
9-
1 ““t
T
T
T
T
,,w
1
PLASMA
r ’
4 F
30
60 TIME
90
120
150
180
(minutes)
Plasma glucose and insulin concentrations during 100 gm oral glucose tolerance tests performed before and during treatment with pyridoxine in 19 chemical diabetic patients in the last trimester of pregnancy. Fig.
pyridoxitle are shown in Fig. 1. The mean ;~rea unclc~ rhe (,I-al (;‘l”l‘ curve \Y;U higher after tl.eatmcnt (.‘I.8 + 10.6.~) I ban before (49.3 t 4.76). but this difle~w~c-e nas not significant. ‘I’here \\a5 ;IISO no GgIiihcant tlitYcwricc in the nwaii insulin response to glucost’ atiel. p\ ridoxinc Il’ciltlIlcIlI. ‘I’he glucow ;~iid insulin area of the oral (;Tl‘ and 111~ Ilriliarv wnthurenic ,icitl excretion in each palicnt before and dul-itig 1~1ridoxiiie therap\ ‘1l.t’ show1 iii I‘ablc I. The. III iiiarv sanrhurenic acid cs( lretion Iv35 ;ibnornial (GM) ~nio1/8 tiou1-s) before watnit’nt in AI 5tlbjecrs but W;J\ wstorccl to normal by pyridosine thctap! (t = 5.93: I, < 0.0001). ,1 significant improvement (p < O.Oli) in the glucose tolerance area \vas obscr\ed in 0111~ t&V0 !\~(JlllC~ll (CilhCS I illttl 2). Ueterioration 0~ clirred in six patient5 (cases 8 to 13) and 110 signihcan~ ctlange bxs ohscr\wI in the remaining five (cases 3 ro 7). .A
1.
Nine of the patients studied were Negresses and four were Caucasians of Indian origin. Their mean age was 31.0 2 6.4 years, their weight was 65.8 * 12.3 kg, and their height was 132.2 + 3.72 cm. All patients gained weight during the study and the mean weekly increase was 0.43 ? 0.17 kg. To assess the change in glucose tolerance before and after treatment in each individual the area under the oral GTT curve has been calculated as described previ0us1p.~ Logarithmic transformation of the plasma insulin results has been used to achieve a normal distribution. Student’s paired t test has been used to assess the significance of the differences observed after treatment with pyridoxine. Spearman’s rank correlation coefficient has been used to determine whether the change in the glucose and insulin area of the oral CT?‘ and the change in urinary xanthurenic acid excretion after pyridoxine therapy are correlated. Results The mean plasma glucose and insulin concentrations of the 13 chemical diabetic women treated with
Comment Contrary to previous reports,‘, ” correction of the pyidosine deficiency of the pregnant chemical diabetic \\omen in this study did not consistently improve glucose tolerance. The discrepancy between this finding and those of’ pre\ious workers may, however. be due to the method used for diagnosing pryidoxine depletion. Thus, although measurement of urinary xanthurenic acid excretion after a tryptophan load is a recognized technique for identifying patients with pyridoxine deficiency, I” Adams and co-workers* have recent]) questioned it5 validity. They tound excessive xanthurenic acid excretion after a tryptophan load, identical to that used in the present study. in all women taking oral contraceptives. Itrlprovement in glucose tolerance after pvridoxine treatment, however, only occurred in those kvomen with evidence of reduced tissue levels of pyridoxine such as diminished 4-pyridoxic acid excretion (the major excretion product of pyridoxine) or impaired er) throcytz: alanine and aspartate aniinotratisfera5e activities (enq,mes requiring pyritloxal phosphate as coenzyme). These observations would explain IQ-h!. onl> two of’ the patients in the present stud) shobved ;I signihcant improvement in glucose tolerance during pvridoxine treatment. It is also of interest that, although Coelingh-Bennink and Schreurs’ c.laimetl that gIucose tolerance returned to normal in
Volume Number
133 5
Pyridoxine treatment of chemical diabetes
Table I. Total glucose and insulin hours after a 2 gm oral tryptophan with pyridoxine in late pregnancy No. 1
Total glucose area Pretherapy 57.6 During therapy 36.3 Change -21.3 Total insulin area Pretherapy 1107 During therapy 393 Change -714 L‘l-inarb xanl hurenic acid escrctiou (/.Lmol/X 111.) Pretherapy 129 During therapy 18
areas under the oral GTT curve and urinary xanthurenic load in 13 chemical diabetic patients before and during
8
No. 13
No. 3
No. 4
No. 5
No. 6
No. 7
No. 8
No. Y
No. 10
No. 11
No. 12
50.8 35.9 - 14.9
47.2 46.0 -1.2
41.8 41.5 -0.3
44.2 44.6 +0.4
56.1 57.5 +1.4
45.8 48.6 +2.a
45.8 51.5 +5.7
53.0 60.7 +7.7
51.3 61.7 +10.4
50.1 62.0 +11.9
45.2 57.8 + 12.6
52.1 69.6 + 17.5
776 370 -406
480 514 +34
617 866 +249
632 609 -23
640 678 +38
1688 1438 -250
274 332 +58
1133 766 -367
486 601 +115
382 466 +84
847 1381 +534
1397 1569 +172
342 39
631 59
220 31
870 38
123 6
139 10
215 23
478 11
391 20
629 16
680 53
479 17
REFERENCES
1. Coelingh-Bennink, H. J. T., and Schreurs. W. H. P.: lmpl-ovement of oral glucose tolerance in gestational diabetes by pyridoxine. Br. Med. J. 413, 1975. 2. Spellacy, W. N., Buhi, W. C., and Birk, S. A.: Vitamin B,; treatment of gestational diabetes mellitus, AM. J. OBSTET. 127:599,
acid excretion treatment
2
No.
12 of their 14 gestational diabetics, this only represented a major improvement in glucose tolerance in about half of the cases. Furthermore, as only eight of their patients had reduced blood pyridoxine levels before treatment, it would be interesting to know whether they were the patients who benefitted most from treatment. Spellacy, Buhi, and Birk’ have also demonstrated a significant improvement in the intravenous glucose tolerance of gestational diabetics treated with pyridoxine. The individual changes in the 13 patients studied and their pyridoxine status were not, however, reported. Perkins,” on the other hand, found no improvement in glucose tolerance in four gestational diabetic women treated with pyridoxine for 3 weeks but again no assessment of pyridoxine status was made in this study. Pyridoxine treatment during pregnancy thus appears to be associated with improvement of carbohydrate tolerance in some, but not all, patients and the reason for this is unknown. The present data do nol, however, support the concept of insulin inactivation b! xanthurenic acid,‘, “. L 6 as there was no consistent change in the plasma insulin response to glucose despite a decline in the urinary xanthurenic acid excretion from grossly pathologic levels to the normal range in all patients during pyridoxine therapy. Cornish and Tesoriero’z have also expressed doubt about this hlpothesis, as they did not find any alteration in glucose
GYNEC~L.
501
1977.
3. Spellacy, W. N.. Buhi, W. C., and Birk. S. A.: The effects
tolerance when pure xanthurenic acid was administered intraperitoneally to rats or by intravenous injection to a rabbit. The reason for the deterioration in glucose tolerance in six of the patients in the present study is difficult to explain. Considerable individual variation in the response to an oral GTT is known to occur when the test is repeated at weekly intervals during pregnancyI and some of the changes observed may be explained by this phenomenon. Furthermore, the stress imposed on maternal carbohydrate metabolism by pregnancy itself may be responsible for the progressive decline in glucose tolerance observed in some of the patients. The role of pyridoxine in the treatment of chemical diabetes arising during pregnancy therefore remains uncertain. There is definitely no place for routine therapy even of those patients with excessive urinary xanthurenic acid excretion and further studies are necessary to determine whether treatment will be beneficial when the more specific criteria of pyridoxine deficiency described by Adams and co-workers’ are fulfilled. We thank Miss Annamieke van Middelkoop and Mr. A. Bedford for their technical assistance, and Dr. P. W. Adams, Professor R. W. Beard, and Dr. N. W. Oakley for their helpful advice. We are also grateful to Miss Cynthia Knight for preparing the illustrations.
of vitamin B6 on carbohydrate metabolism in women taking steroid contraceptives: Preliminar! repel-t. Contraception, 6~265, 1972. 4. Adams, P. W., Wynn, V., Folkard, J. and others: lnfluence of oral contraceptives, py-t-idoxine (vitamin B8). and tryptophan on carbohydrate metabolism. Lancet 1:759, 1976. 5. Murakami, E.: Studies on xanthurenic acid-insulin com-
502
6.
7.
8.
9.
Gillmer
and Mazibuko
plex. I. Preparation and prcyel tit:,, J. Biochrm. 63:57:1. 1968. Kotake, XI’., Sotokawa. ‘T., Murakami. E. and uthers: Studies on the xanthurenic acid-insulin complex. II. Phyaiw logical activities, J. Biochem. 63:578, 1968. World Health Organization: Diabetes hlellitus. Report 01 a WHO Expert Committee, Technical Report Series No. 310, Geneva. 1965. W’achstein, X1., and Gudaitis. A.: Detection rot vitamin B,j deficiency: Utilization of an improved method for rapid determination of santhurenic acid in urine, .-im. J, Clin. Pathol. 22:652, 19.5?. Gillmer, M. D. G., Beard, R. It’.. Brooke, F. 51.. and others: Carbohydrate metaholism in pregnancv. 1. Di urnal plasma glucose profile in normal and diabetic IYOITIen. Br. Med. J. 3:399. 1975.
IO. Spr-incr, 1-I.. I.ONV, R. S.. Folsomr.
D. G. GILLMER
1). MAZIBUKO DwLm~,
Thirteen excessive pyridoxine restored tolerance change therapy.
South
Africa
women with chemical diabetes diagnosed in late pregnancy were found to excrete amount of urinary xanthurenic acid after a tryptophan load, indicative of a relative (vitamin 6,) deficiency. Treatment with 100 mg pyridoxine daily for 14 to 23 days the urinary xanthurenic acid excretion to normal in all patients. Improvement of glucose was observed in only two of the patients studied, deterioration in six, and no significant in the remaining five. The insulin response to glucose was unaltered during pyridoxine (AM. J. OBSTET. GYNECOL. 133:499, 1979.)
ABNORMAL TRYPTOPHAN metabolism due to pyridoxine (vitamin B,) deficiency has recently been implicated as a cause of chemical diabetes in pregnant women’. ’ and those treated with oral contraceptives.3s ’ It has been suggested that this is due to the production of excessive amounts of xanthurenic acid, an abnormal metabolite of tryptophan which forms a stable complex with insulin,” and has been demonstrated to halve the hypoglycemic action of the hormone in animals.” Pyridoxine treatment of pregnant women with chemical diabetes has been shown to produce an improvement in carbohydrate tolerance, and it has been proposed that this is due to a reduction in xanthurenic acid formation.‘. ’ The present study was performed to confirm these reports and to determine the relationship between the plasma insulin and urinary xanthurenic acid concentrations in pregnant women with chemical diabetes given pyridoxine in doses previously shown to correct glucose tolerance.
Material and methods Thirteen chemical diabetic women who had a 100 gm oral glucose tolerance test (GTT) during late pregnancy because of potential diabetic features were studied. The diagnosis of chemical diabetes was made be-
cause the venous plasma glucose at 2 hours exceeded 8 mmol/L.’ All subjects were admitted to the hospital at least 1 week before the start of the study and remained in the hospital until delivery; during this time they received a diet containing 150 gm of carbohydrate each day. The study was started between 31 and 37 weeks of pregnancy (mean ? 1 SD, 33.5 ? 1.5). After an overnight fast the 100 gm oral GTT was repeated on the first day of the study with the patient awake and semirecumbent. A blood sample was taken from an antecubital vein before drinking the glucose solution and thereafter at half-hour intervals for 3 hours. The plasma was separated immediately after the collection of each blood sample and divided into two aliquots. One was stored at 4” C and the glucose concentration was measured within 24 hours by means of a manual glucose oxidase-peroxidase method (GOD period, Boehringer). The second aliquot was stored at -20” C and used to measure the plasma insulin concentration by means of the Insulin RIA kit of The Radiochemical Centre, Amersham, England. On the second day of the study 2 gm of L-tryptophan dissolved in a glass of milk flavored with fruit juice concentrate was taken by mouth, after the patient had emptied her bladder. All urine passed during the next 8 hours was collected, the volume measured, and a 100 ml sample stored at -20” C for determination of urinary xanthurenic acid excretion.” The following day treatment with pyridoxine tablets, 25 mg four times a day, was started and continued for between 14 and 23 days (mean ? 1 SD. 17.3 + 2.8). At this time the oral GTT and L-tryptophan load were repeated during therapy. 499
500
Glllmer
and Mazibuko
12. llPLASMA
‘0
GLUCOSE
9-
1 ““t
T
T
T
T
,,w
1
PLASMA
r ’
4 F
30
60 TIME
90
120
150
180
(minutes)
Plasma glucose and insulin concentrations during 100 gm oral glucose tolerance tests performed before and during treatment with pyridoxine in 19 chemical diabetic patients in the last trimester of pregnancy. Fig.
pyridoxitle are shown in Fig. 1. The mean ;~rea unclc~ rhe (,I-al (;‘l”l‘ curve \Y;U higher after tl.eatmcnt (.‘I.8 + 10.6.~) I ban before (49.3 t 4.76). but this difle~w~c-e nas not significant. ‘I’here \\a5 ;IISO no GgIiihcant tlitYcwricc in the nwaii insulin response to glucost’ atiel. p\ ridoxinc Il’ciltlIlcIlI. ‘I’he glucow ;~iid insulin area of the oral (;Tl‘ and 111~ Ilriliarv wnthurenic ,icitl excretion in each palicnt before and dul-itig 1~1ridoxiiie therap\ ‘1l.t’ show1 iii I‘ablc I. The. III iiiarv sanrhurenic acid cs( lretion Iv35 ;ibnornial (GM) ~nio1/8 tiou1-s) before watnit’nt in AI 5tlbjecrs but W;J\ wstorccl to normal by pyridosine thctap! (t = 5.93: I, < 0.0001). ,1 significant improvement (p < O.Oli) in the glucose tolerance area \vas obscr\ed in 0111~ t&V0 !\~(JlllC~ll (CilhCS I illttl 2). Ueterioration 0~ clirred in six patient5 (cases 8 to 13) and 110 signihcan~ ctlange bxs ohscr\wI in the remaining five (cases 3 ro 7). .A
1.
Nine of the patients studied were Negresses and four were Caucasians of Indian origin. Their mean age was 31.0 2 6.4 years, their weight was 65.8 * 12.3 kg, and their height was 132.2 + 3.72 cm. All patients gained weight during the study and the mean weekly increase was 0.43 ? 0.17 kg. To assess the change in glucose tolerance before and after treatment in each individual the area under the oral GTT curve has been calculated as described previ0us1p.~ Logarithmic transformation of the plasma insulin results has been used to achieve a normal distribution. Student’s paired t test has been used to assess the significance of the differences observed after treatment with pyridoxine. Spearman’s rank correlation coefficient has been used to determine whether the change in the glucose and insulin area of the oral CT?‘ and the change in urinary xanthurenic acid excretion after pyridoxine therapy are correlated. Results The mean plasma glucose and insulin concentrations of the 13 chemical diabetic women treated with
Comment Contrary to previous reports,‘, ” correction of the pyidosine deficiency of the pregnant chemical diabetic \\omen in this study did not consistently improve glucose tolerance. The discrepancy between this finding and those of’ pre\ious workers may, however. be due to the method used for diagnosing pryidoxine depletion. Thus, although measurement of urinary xanthurenic acid excretion after a tryptophan load is a recognized technique for identifying patients with pyridoxine deficiency, I” Adams and co-workers* have recent]) questioned it5 validity. They tound excessive xanthurenic acid excretion after a tryptophan load, identical to that used in the present study. in all women taking oral contraceptives. Itrlprovement in glucose tolerance after pvridoxine treatment, however, only occurred in those kvomen with evidence of reduced tissue levels of pyridoxine such as diminished 4-pyridoxic acid excretion (the major excretion product of pyridoxine) or impaired er) throcytz: alanine and aspartate aniinotratisfera5e activities (enq,mes requiring pyritloxal phosphate as coenzyme). These observations would explain IQ-h!. onl> two of’ the patients in the present stud) shobved ;I signihcant improvement in glucose tolerance during pvridoxine treatment. It is also of interest that, although Coelingh-Bennink and Schreurs’ c.laimetl that gIucose tolerance returned to normal in
Volume Number
133 5
Pyridoxine treatment of chemical diabetes
Table I. Total glucose and insulin hours after a 2 gm oral tryptophan with pyridoxine in late pregnancy No. 1
Total glucose area Pretherapy 57.6 During therapy 36.3 Change -21.3 Total insulin area Pretherapy 1107 During therapy 393 Change -714 L‘l-inarb xanl hurenic acid escrctiou (/.Lmol/X 111.) Pretherapy 129 During therapy 18
areas under the oral GTT curve and urinary xanthurenic load in 13 chemical diabetic patients before and during
8
No. 13
No. 3
No. 4
No. 5
No. 6
No. 7
No. 8
No. Y
No. 10
No. 11
No. 12
50.8 35.9 - 14.9
47.2 46.0 -1.2
41.8 41.5 -0.3
44.2 44.6 +0.4
56.1 57.5 +1.4
45.8 48.6 +2.a
45.8 51.5 +5.7
53.0 60.7 +7.7
51.3 61.7 +10.4
50.1 62.0 +11.9
45.2 57.8 + 12.6
52.1 69.6 + 17.5
776 370 -406
480 514 +34
617 866 +249
632 609 -23
640 678 +38
1688 1438 -250
274 332 +58
1133 766 -367
486 601 +115
382 466 +84
847 1381 +534
1397 1569 +172
342 39
631 59
220 31
870 38
123 6
139 10
215 23
478 11
391 20
629 16
680 53
479 17
REFERENCES
1. Coelingh-Bennink, H. J. T., and Schreurs. W. H. P.: lmpl-ovement of oral glucose tolerance in gestational diabetes by pyridoxine. Br. Med. J. 413, 1975. 2. Spellacy, W. N., Buhi, W. C., and Birk, S. A.: Vitamin B,; treatment of gestational diabetes mellitus, AM. J. OBSTET. 127:599,
acid excretion treatment
2
No.
12 of their 14 gestational diabetics, this only represented a major improvement in glucose tolerance in about half of the cases. Furthermore, as only eight of their patients had reduced blood pyridoxine levels before treatment, it would be interesting to know whether they were the patients who benefitted most from treatment. Spellacy, Buhi, and Birk’ have also demonstrated a significant improvement in the intravenous glucose tolerance of gestational diabetics treated with pyridoxine. The individual changes in the 13 patients studied and their pyridoxine status were not, however, reported. Perkins,” on the other hand, found no improvement in glucose tolerance in four gestational diabetic women treated with pyridoxine for 3 weeks but again no assessment of pyridoxine status was made in this study. Pyridoxine treatment during pregnancy thus appears to be associated with improvement of carbohydrate tolerance in some, but not all, patients and the reason for this is unknown. The present data do nol, however, support the concept of insulin inactivation b! xanthurenic acid,‘, “. L 6 as there was no consistent change in the plasma insulin response to glucose despite a decline in the urinary xanthurenic acid excretion from grossly pathologic levels to the normal range in all patients during pyridoxine therapy. Cornish and Tesoriero’z have also expressed doubt about this hlpothesis, as they did not find any alteration in glucose
GYNEC~L.
501
1977.
3. Spellacy, W. N.. Buhi, W. C., and Birk. S. A.: The effects
tolerance when pure xanthurenic acid was administered intraperitoneally to rats or by intravenous injection to a rabbit. The reason for the deterioration in glucose tolerance in six of the patients in the present study is difficult to explain. Considerable individual variation in the response to an oral GTT is known to occur when the test is repeated at weekly intervals during pregnancyI and some of the changes observed may be explained by this phenomenon. Furthermore, the stress imposed on maternal carbohydrate metabolism by pregnancy itself may be responsible for the progressive decline in glucose tolerance observed in some of the patients. The role of pyridoxine in the treatment of chemical diabetes arising during pregnancy therefore remains uncertain. There is definitely no place for routine therapy even of those patients with excessive urinary xanthurenic acid excretion and further studies are necessary to determine whether treatment will be beneficial when the more specific criteria of pyridoxine deficiency described by Adams and co-workers’ are fulfilled. We thank Miss Annamieke van Middelkoop and Mr. A. Bedford for their technical assistance, and Dr. P. W. Adams, Professor R. W. Beard, and Dr. N. W. Oakley for their helpful advice. We are also grateful to Miss Cynthia Knight for preparing the illustrations.
of vitamin B6 on carbohydrate metabolism in women taking steroid contraceptives: Preliminar! repel-t. Contraception, 6~265, 1972. 4. Adams, P. W., Wynn, V., Folkard, J. and others: lnfluence of oral contraceptives, py-t-idoxine (vitamin B8). and tryptophan on carbohydrate metabolism. Lancet 1:759, 1976. 5. Murakami, E.: Studies on xanthurenic acid-insulin com-
502
6.
7.
8.
9.
Gillmer
and Mazibuko
plex. I. Preparation and prcyel tit:,, J. Biochrm. 63:57:1. 1968. Kotake, XI’., Sotokawa. ‘T., Murakami. E. and uthers: Studies on the xanthurenic acid-insulin complex. II. Phyaiw logical activities, J. Biochem. 63:578, 1968. World Health Organization: Diabetes hlellitus. Report 01 a WHO Expert Committee, Technical Report Series No. 310, Geneva. 1965. W’achstein, X1., and Gudaitis. A.: Detection rot vitamin B,j deficiency: Utilization of an improved method for rapid determination of santhurenic acid in urine, .-im. J, Clin. Pathol. 22:652, 19.5?. Gillmer, M. D. G., Beard, R. It’.. Brooke, F. 51.. and others: Carbohydrate metaholism in pregnancv. 1. Di urnal plasma glucose profile in normal and diabetic IYOITIen. Br. Med. J. 3:399. 1975.
IO. Spr-incr, 1-I.. I.ONV, R. S.. Folsomr.