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Malabsorption of vitamin B12 in megaloblastic anaemia among Africans and Indians in Durban
445 TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICAL MEDICINE AND HYGIENE. Vol. 57. No. 6. November, 1963.
M A L A B S O R P T I O N O F V I T A M I N B12 I N M E G A L O B L A S T I C A N A E M I A A M O N G AFRICANS AND INDIANS IN DURBAN BY
W. H I F T AND E. B. ADAMS*
(From the Department of Medicine, University of Natal, and King Edward VIII Hospital, Durban) In a previous paper we attempted to classify megaloblastic anaemia as seen among Africans and Indians in D u r b a r (ADAMS and HIFT, 1961). Pregnancy and poor nutrition appeared to be the main factors in causation while only one case out of 48 was regarded as being due to intestinal malabsorption on the basis of the vitamin A test (as a screen), and subsequent fat balance studies. Since then we have seen three further patients with malabsorption among 50 cases of megaloblastic anaemia. T w e n t y patients had impaired vitamin A absorption, and in 6 of the 10 in whom the test was repeated after treatment there was apparent improvement. In the same series of 50 a radioactive vitamin B12 absorption test was done in 11 cases and found to be abnormally low in five and doubtful in three. Similar results have been reported from India and Malaya (KAuL et al., 1959 ; TASKER, 1961). In another paper one of us reported a striking increase in serum vitamin B12 levels in the majority of patients with megaloblastic anaemia treated with folic acid only, but could not decide whether this was due to temporary malabsorption of vitamin BI~, corrected by treatment with folic acid or to mobilization of vitamin B12 from the tissues (HIFT, 1963). It therefore seemed important for us to study the role of malabsorption further. In this paper we report a preliminary study of vitamin B12 absorption in the common megaloblastic anaemias seen in Durban. MATERIALS AND METHODS W e investigated 18 patients with megaloblastic anaemia, admitted consecutively, and diagnosed by examination of the bone marrow. T w o were excluded because there was achlorhydria after maximal stimulation with histamine, and although there were atypical features, pernicious anaemia could not be ruled out. T h e remaining 16, consisting of 2 Indians and 14 Africans, all with free acid in the gastric juice, form the basis of this report. The mean haemoglobin on admission was 3.6 g./100 ml. and the range 1.9 g. to 7.1 g./100 ml. Ten patients with iron-deficiency anaemia, established by serum iron estimation and examination of the bone marrow for iron stores, were used as controls. Anaemia was comparable in degree (mean haemoglobin on admission 5.2 g./100 ml., range 3.4 g. to 8.1 g./100 ml.). Serum vitamin Bt2 levels Were determined by the method of HUTNERet al. (1956), and folic acid deficiency was assessed by the formiminoglutamic acid test as described by KNOWLES et al. (1960). Radioactive vitamin B:, absorption was estimated by a modification of the method of SCHILLING (1953), using 5 ml. samples of urine in a well-crystal scintillation counter and 0.5 Vc. Co6°-labelled * This work was supported by grants from the Council for Scientific and Industrial Research. We wish to thank Dr. T. M. Adnams, Medical Superintendent of King Edward VIII Hospital, for facilities, and Miss E. M. St. George, Mrs. M. Walters and Miss D. van Heusden for technical assistance.
446
MALABSORPTION OF VITAMIN B12 IN MEGALOBLASTICANAEMIA
vitamin Ble. In each case the test was p e r f o r m e d within 3 days of admission. T h e patient was then treated with folic acid (30 mg. daily), and the test repeated at intervals of about 2 weeks until discharge, except in those in w h o m the first test was clearly normal. M o s t workers in this field w o u l d agree that a urinary excretion (without intrinsic factor) of m o r e than 10 per cent. is normal, while less than 5 per cent. is abnormal.
An attempt has been made to separate different types of megaloblastic anaemia, but among our patients this is by no means easy, since most live on diets that would be regarded as poor by Western standards. Eight patients had recently been pregnant. The remaining eight, grouped together as "nutritional megaloblastic anaemia," included one who had cirrhosis of the liver and another who was a vegetarian and suffered from epilepsy, for which he had been taking anticonvulsants for many years. One patier]t had concomitant iron deficiency anaemia, but complicating diseases were otherwise absent in this series. RESULTS
Serum vitamin B12 levels were above 100 ~ g . / m l . in 13 out of 14 patients, but more than half had less than 200 ~z~zg./ml. The formiminoglutamic acid test was positive in all eight patients in whom it was done. Folio acid deficiency, therefore, appears to be the main cause of megaloblastic anaemia in this series, but vitamin B12 deficiency may co-exist in some patients. The radioactive vitamin Bt2 excretion test ranged from 0 to 19.0 per cent., with a mean of 6.5 per cent. It was clearly normal (above 10 per cent.) in only three patients, while in eight it was in the doubtful range (5 per cent.-10 per cent.). In the remaining five patients there was vitamin B12 malabsorption (excretion less than 5 per cent.). Although pernicious anaemia was ruled out in all patients by the presence of acid in the stomach, there remained a remote possibility of an acquired intrinsic factor deficiency as the main cause of their malabsorption. The excretion test was therefore repeated, with addition of intrinsic factor, in five patients in whom the clinical condition warranted a further delay before treatment with folic acid. In none of them did intrinsic factor produce a significant improvement in excretion. Serial tests in those excreting less than 10 per cent. Co6°-labelled vitamin B12 showed well-marked rises in two patients (4.9 per cent. to 16.4 per cent., and 6,7 per cent. to 20.0 per cent.), both with megaloblastic anaemia related to pregnancy. They appear to demonstrate temporary malabsorption of vitamin B12. Three patients with initial excretions of 0, 1.1 per cent. and 0.6 per cent. showed no significant rises after treatment with folic acid and must be regarded as cases of vitamin BI~ malabsorption ; one of these had recently been pregnant. Minor changes occurred in some of the others with poor initial levels, but these are not commented upon further, since variations in the range from 5 per cent. to 10 per cent. are difficult to interpret. In the qontrol group of 10 patients with iron-deficiency anaemia, all but one excreted more than 10 per cent. of the test dose of radioactive vitamin B~2. The range was 9.1 per cent. to 33.3 per cent. and the mean 20.3 per cent. DISCUSSION
In countries where dietary standards are poor it is difficult to classify cases of megaloblasdc anaemia because the part played by malnutrition cannot readily be assessed. Using criteria we adopted previously (ADAMS and HIFT, 1961), we observed that 11 of these 16 patients had subsisted on diets poor in sources of folio acid, or vitamin B12, or both ; while
W. HIFT AND E. B. ADAMS
447
in none could the diet be regarded as good. Megaloblastic anaemia therefore appeared to have a nutritional background in most of them, although there were other contributory factors such as recent pregnancy and cirrhosis. Serum vitamin BI~ levels above 100 ~ g . / m l . in 13 out of 14 patients and the presence of formiminoglutamic acid in all eight patients tested suggest that folic acid deficiency was mainly responsible for the anaemia. Hitherto we have observed that the incidence of malabsorption was low when judged on fat-balance studies, and we concluded that steatorrhoea was seldom a causative factor. What we have not known is whether there is malabsorption of other substances such as vitamin B12. The work of METZ et al. (1960) in Johannesburg suggested that malabsorption of vitamin BI~ does not play a significant role in the pathogenesis of megaloblastic anaemia associated with pregnancy, although five of their 30 patients excreted less than 5 per cent. of the test dose. The patients in our series, on the whole, absorbed radioactive vitamin B12 poorly (mean excretion 6.5 per cent.) when compared with a control group suffering from iron-deficiency anaemia (mean 20.3 per cent.), the difference between the means being statistically significant (p <0.001). Low initial excretion levels and no rise after treatment showed that three patients suffered from vitamin BI2 malabsorption. Two other patients showed temporary malabsorption of vitamin Ble, corrected by treatment. Judging from the results obtained in our control group, anaemia per se does not appear to interfere with absorption of vitamin Bxz. Despite the experimental work of Hsu et al. (1962) on rats, demonstrating that induced folic acid-deficiency does not disturb vitamin B~ absorption, the results in these two patients and the rises we have previously observed in serum vitamin Bt2 levels in some patients with megaloblastic anaemia treated only with folic acid (HIFr, 1963) suggest that folic acid-deficiency may cause malabsorption of vitamin Bx2. SUMMARY
1) The absorption of radioactive vitamin B12 was investigated in 16 African and Indian patients with megaloblastic anaemia due to folic acid deficiency. 2) The mean urinary excretion (6.5 per cent.) was poor compared with a control group of patients with iron-deficiency anaemia (mean 20.3 per cent.). 3) Serial tests showed that definite vitamin B12 malabsorption was persistent in three patients, while in two others it was temporary, corrected by treatment. REFERENCES ADAMS,E. B. & HIFT, W. (1961). Trans. R. Soc. trop. Med. Hyg., 55, 374. HIFT, W. (1963') S. Aft. ft. lab. clin. Med., 9, 24. Hsu, J. M., TANTENOCO,V. & CHOW, B. F. (1962). J. clin. Invest., 41, 532. HOTNEa, S. H., BACH,M. K. & Ross, G. I. M. (1956). J. Protozool., 3, 101. I~UL, S., DAS GUPTA,A. K., & KmSHNAN,N. R. (1959). Indian J. med. Res., 47, 139. KNOWLES,J. P., PRANKSRD,T. A. J. & WESTALL,R. G. (1960). Lancet, 2, 347. METZ., J., LEwis, S, M., KEELEY,K. J. & HART, D. (1960). J. clin. Path., 13, 394. SCHILLING, R. F. (1953). jT. Lab. clin. Med., 42, 860. TASKER, P. W. G. (1961). Trans. R. Soc. trop. Med. Hyg., 55, 272.
M A L A B S O R P T I O N O F V I T A M I N B12 I N M E G A L O B L A S T I C A N A E M I A A M O N G AFRICANS AND INDIANS IN DURBAN BY
W. H I F T AND E. B. ADAMS*
(From the Department of Medicine, University of Natal, and King Edward VIII Hospital, Durban) In a previous paper we attempted to classify megaloblastic anaemia as seen among Africans and Indians in D u r b a r (ADAMS and HIFT, 1961). Pregnancy and poor nutrition appeared to be the main factors in causation while only one case out of 48 was regarded as being due to intestinal malabsorption on the basis of the vitamin A test (as a screen), and subsequent fat balance studies. Since then we have seen three further patients with malabsorption among 50 cases of megaloblastic anaemia. T w e n t y patients had impaired vitamin A absorption, and in 6 of the 10 in whom the test was repeated after treatment there was apparent improvement. In the same series of 50 a radioactive vitamin B12 absorption test was done in 11 cases and found to be abnormally low in five and doubtful in three. Similar results have been reported from India and Malaya (KAuL et al., 1959 ; TASKER, 1961). In another paper one of us reported a striking increase in serum vitamin B12 levels in the majority of patients with megaloblastic anaemia treated with folic acid only, but could not decide whether this was due to temporary malabsorption of vitamin BI~, corrected by treatment with folic acid or to mobilization of vitamin B12 from the tissues (HIFT, 1963). It therefore seemed important for us to study the role of malabsorption further. In this paper we report a preliminary study of vitamin B12 absorption in the common megaloblastic anaemias seen in Durban. MATERIALS AND METHODS W e investigated 18 patients with megaloblastic anaemia, admitted consecutively, and diagnosed by examination of the bone marrow. T w o were excluded because there was achlorhydria after maximal stimulation with histamine, and although there were atypical features, pernicious anaemia could not be ruled out. T h e remaining 16, consisting of 2 Indians and 14 Africans, all with free acid in the gastric juice, form the basis of this report. The mean haemoglobin on admission was 3.6 g./100 ml. and the range 1.9 g. to 7.1 g./100 ml. Ten patients with iron-deficiency anaemia, established by serum iron estimation and examination of the bone marrow for iron stores, were used as controls. Anaemia was comparable in degree (mean haemoglobin on admission 5.2 g./100 ml., range 3.4 g. to 8.1 g./100 ml.). Serum vitamin Bt2 levels Were determined by the method of HUTNERet al. (1956), and folic acid deficiency was assessed by the formiminoglutamic acid test as described by KNOWLES et al. (1960). Radioactive vitamin B:, absorption was estimated by a modification of the method of SCHILLING (1953), using 5 ml. samples of urine in a well-crystal scintillation counter and 0.5 Vc. Co6°-labelled * This work was supported by grants from the Council for Scientific and Industrial Research. We wish to thank Dr. T. M. Adnams, Medical Superintendent of King Edward VIII Hospital, for facilities, and Miss E. M. St. George, Mrs. M. Walters and Miss D. van Heusden for technical assistance.
446
MALABSORPTION OF VITAMIN B12 IN MEGALOBLASTICANAEMIA
vitamin Ble. In each case the test was p e r f o r m e d within 3 days of admission. T h e patient was then treated with folic acid (30 mg. daily), and the test repeated at intervals of about 2 weeks until discharge, except in those in w h o m the first test was clearly normal. M o s t workers in this field w o u l d agree that a urinary excretion (without intrinsic factor) of m o r e than 10 per cent. is normal, while less than 5 per cent. is abnormal.
An attempt has been made to separate different types of megaloblastic anaemia, but among our patients this is by no means easy, since most live on diets that would be regarded as poor by Western standards. Eight patients had recently been pregnant. The remaining eight, grouped together as "nutritional megaloblastic anaemia," included one who had cirrhosis of the liver and another who was a vegetarian and suffered from epilepsy, for which he had been taking anticonvulsants for many years. One patier]t had concomitant iron deficiency anaemia, but complicating diseases were otherwise absent in this series. RESULTS
Serum vitamin B12 levels were above 100 ~ g . / m l . in 13 out of 14 patients, but more than half had less than 200 ~z~zg./ml. The formiminoglutamic acid test was positive in all eight patients in whom it was done. Folio acid deficiency, therefore, appears to be the main cause of megaloblastic anaemia in this series, but vitamin B12 deficiency may co-exist in some patients. The radioactive vitamin Bt2 excretion test ranged from 0 to 19.0 per cent., with a mean of 6.5 per cent. It was clearly normal (above 10 per cent.) in only three patients, while in eight it was in the doubtful range (5 per cent.-10 per cent.). In the remaining five patients there was vitamin B12 malabsorption (excretion less than 5 per cent.). Although pernicious anaemia was ruled out in all patients by the presence of acid in the stomach, there remained a remote possibility of an acquired intrinsic factor deficiency as the main cause of their malabsorption. The excretion test was therefore repeated, with addition of intrinsic factor, in five patients in whom the clinical condition warranted a further delay before treatment with folic acid. In none of them did intrinsic factor produce a significant improvement in excretion. Serial tests in those excreting less than 10 per cent. Co6°-labelled vitamin B12 showed well-marked rises in two patients (4.9 per cent. to 16.4 per cent., and 6,7 per cent. to 20.0 per cent.), both with megaloblastic anaemia related to pregnancy. They appear to demonstrate temporary malabsorption of vitamin B12. Three patients with initial excretions of 0, 1.1 per cent. and 0.6 per cent. showed no significant rises after treatment with folic acid and must be regarded as cases of vitamin BI~ malabsorption ; one of these had recently been pregnant. Minor changes occurred in some of the others with poor initial levels, but these are not commented upon further, since variations in the range from 5 per cent. to 10 per cent. are difficult to interpret. In the qontrol group of 10 patients with iron-deficiency anaemia, all but one excreted more than 10 per cent. of the test dose of radioactive vitamin B~2. The range was 9.1 per cent. to 33.3 per cent. and the mean 20.3 per cent. DISCUSSION
In countries where dietary standards are poor it is difficult to classify cases of megaloblasdc anaemia because the part played by malnutrition cannot readily be assessed. Using criteria we adopted previously (ADAMS and HIFT, 1961), we observed that 11 of these 16 patients had subsisted on diets poor in sources of folio acid, or vitamin B12, or both ; while
W. HIFT AND E. B. ADAMS
447
in none could the diet be regarded as good. Megaloblastic anaemia therefore appeared to have a nutritional background in most of them, although there were other contributory factors such as recent pregnancy and cirrhosis. Serum vitamin BI~ levels above 100 ~ g . / m l . in 13 out of 14 patients and the presence of formiminoglutamic acid in all eight patients tested suggest that folic acid deficiency was mainly responsible for the anaemia. Hitherto we have observed that the incidence of malabsorption was low when judged on fat-balance studies, and we concluded that steatorrhoea was seldom a causative factor. What we have not known is whether there is malabsorption of other substances such as vitamin B12. The work of METZ et al. (1960) in Johannesburg suggested that malabsorption of vitamin BI~ does not play a significant role in the pathogenesis of megaloblastic anaemia associated with pregnancy, although five of their 30 patients excreted less than 5 per cent. of the test dose. The patients in our series, on the whole, absorbed radioactive vitamin B12 poorly (mean excretion 6.5 per cent.) when compared with a control group suffering from iron-deficiency anaemia (mean 20.3 per cent.), the difference between the means being statistically significant (p <0.001). Low initial excretion levels and no rise after treatment showed that three patients suffered from vitamin BI2 malabsorption. Two other patients showed temporary malabsorption of vitamin Ble, corrected by treatment. Judging from the results obtained in our control group, anaemia per se does not appear to interfere with absorption of vitamin Bxz. Despite the experimental work of Hsu et al. (1962) on rats, demonstrating that induced folic acid-deficiency does not disturb vitamin B~ absorption, the results in these two patients and the rises we have previously observed in serum vitamin Bt2 levels in some patients with megaloblastic anaemia treated only with folic acid (HIFr, 1963) suggest that folic acid-deficiency may cause malabsorption of vitamin Bx2. SUMMARY
1) The absorption of radioactive vitamin B12 was investigated in 16 African and Indian patients with megaloblastic anaemia due to folic acid deficiency. 2) The mean urinary excretion (6.5 per cent.) was poor compared with a control group of patients with iron-deficiency anaemia (mean 20.3 per cent.). 3) Serial tests showed that definite vitamin B12 malabsorption was persistent in three patients, while in two others it was temporary, corrected by treatment. REFERENCES ADAMS,E. B. & HIFT, W. (1961). Trans. R. Soc. trop. Med. Hyg., 55, 374. HIFT, W. (1963') S. Aft. ft. lab. clin. Med., 9, 24. Hsu, J. M., TANTENOCO,V. & CHOW, B. F. (1962). J. clin. Invest., 41, 532. HOTNEa, S. H., BACH,M. K. & Ross, G. I. M. (1956). J. Protozool., 3, 101. I~UL, S., DAS GUPTA,A. K., & KmSHNAN,N. R. (1959). Indian J. med. Res., 47, 139. KNOWLES,J. P., PRANKSRD,T. A. J. & WESTALL,R. G. (1960). Lancet, 2, 347. METZ., J., LEwis, S, M., KEELEY,K. J. & HART, D. (1960). J. clin. Path., 13, 394. SCHILLING, R. F. (1953). jT. Lab. clin. Med., 42, 860. TASKER, P. W. G. (1961). Trans. R. Soc. trop. Med. Hyg., 55, 272.