Absorption of vitamin B12 in nutritional megaloblastic anaemia

272 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 55. No. 3. May, 1961.

ABSORPTION

OF V I T A M I N

B,2 IN N U T R I T I O N A L ANAEMIA*

MEGALOBLASTIC

BY The Late P. W. G. T A S K E R , M.D.t

Reports of studies of the absorption of vitamin BIa in tropical megaloblastic anaemia are few. BAKER(1958), in a series of 60 Indian patients studied in Vellore, found low serum vitamin BI2 levels and malabsorption of the vitamin which was not corrected by intrinsic factor ; these patients were nearly all men and probably suffered from tropical sprue. KAUL et al. (1959) found malabsorption in seven of 14 patients with nutritional megaloblastic anaemia. FoY et al. (1955) presented data from seven representative studies of African patients suffering from megaloblastic anaemia; the absorption studies were carried out after a course of penicillin, and indicated normal or only slightly impaired absorption. MEYER et al. (1953) found a malabsorption in two of seven patients with tropical sprue ; and MOLLIN et al. (1957) in five of seven patients, in some of whom absorption was improved by antibiotics. DHOPESHW~mKamet al. (1956) studied five Indian vegetarians, with low serum vitamin B12 levels, and found that the levels were increased by a course of aureomycin. TAsiom et al. (1958) showed that about a quarter of their Malayan patients, suffering from nutritional and pregnancy megaloblastic anaemia, had a low serum vitamin B12 level ; the proportion showing low levels was greatest in the men, less in the women, and least in the pregnant women. Subsequent assays, some of which are reported in the present paper, have confirmed these resuhs. A malabsorption of vitamin BI~ can arise from lesions of the stomach, resulting in the loss of secretion of intrinsic factor, as in pernicious anaemia and following total gastrectomy ; in these conditions absorption can usually be restored to normal with intrinsic factor (GLASS, 1956 ; MOLLIN et al., 1957). Malabsorption can also arise from disturbances at the absorbing areas of the small intestine either by removal of, or damage to, the intestinal wall ; or by changes in the environment of the lumen (COOKE et al. 1957 ; BOOTH and MOLLIN, 1959). In these conditions absorption is not improved by the administration of intrinsic factor (BADENOCH et al., 1955 ; HALSTEADet al., 1956 ; GLASS, 1956 ; GLASS and BOYD, 1957 ; MOLLIN et al., 1957 ; OXENHOaN et al., 1958), but may be improved by the use of broad spectrum antibiotics (HALSTEADet al., 1956 ; MOLLIN et al., 1957 ; OXENHORNet al., 1958). * Requests for reprints to the Institute for Medical Research, Kuala Lumpur. t I am indebted to the staff of the General Hospital, Kuala Lumpur, who allowed me to study patients under their care ; to Dr. Lester-Smith of Glaxo Laboratories who kindly supplied radioactive vitamin BI2 before it was obtainable from the Radioehemical Centre, Amersham, and to the American Cyanamid Company who supplied the intrinsic factor (blend 182 (7-6475)).

e. w. G. TASKEa

273

T h e importance of impaired absorption, as compared with the various other factors involved, in causing a vitamin B12 deficiency in nutritional megaloblastic anaemia is still not known ; the purpose of the present study was to determine whether there was any relationship between a deficiency and a malabsorption of vitamin BI~ and, where malabsorption was found, to determine if possible the cause. T h e absorption of vitamin B12, therefore, was studied in relation to such factors as race, sex, age, the serum vitamin B12 level, gastric acidity, the absorption of folic acid and of xylose, and the histological appearance of intestinal biopsies. Where absorption was impaired the effects of intrinsic factor, aureomycin, penicillin, and prednisolone were investigated in some patients. MATERIAL AND METHODS T h e patients studied were all adults admitted to the General Hospital, Kuala L u m p u r , and suffering f r o m megaloblastic anaemia as demonstrated by marrow biopsy. The absorption of vitamin BI~ was estimated in 66 patients by giving 1 ~tg of the vitamin labelled with slightly less than 1 ~C of radioactivity (usually between 2,000-3,000 c/s on the scintillation counter used) ; the recovery of the tracer in the stools was used as the index of absorption. The patients were asked to keep all their stools in waxed paper cartons. This was done for from 4 to 7 days before the tests, to check their understanding and co-operation, and then during the tests. Occasionally it was suspected that a stool specimen had been discarded after a test dose, the test was then either repeated or cancelled. The stools were macerated, weighed, and three amounts of approximately 10 g. were counted. The proportion of radioactivity recovered was presumed to represent unabsorbed vitamin Btz, although there is a tendency for the radioactive vitamin BI~ to break down during storage (LEsTERSMITH, 1959). The tracer was used within a few weeks of receipt, but the radiochemical purity was not further checked in this laboratory. The serum vitamin BI~ levels were assayed using Lactobacillus leichmanii in the microbiological method suggested by SPRAY (1953). Folic acid absorption was estimated by determining the 24 hour urinary excretion following a 5 mg. loading dose by mouth, after previous " body saturation " by treatment with folic acid ; Streptococcus faecalis R was used as the test organism (GIRDWOOD, 1956). The absorption of xylose was estimated by determining the amount excreted in the urine for 5 hours after a loading dose of 5 grammes in 500 ml. water given by mouth (RoE and RICE, 1948). The presence of achlorhydria was assessed, in the earlier stages, after giving 0.5 mg. histamine subcutaneously ; later the augmented test was used, the dose being 0.04 mg./kg, body weight, or 2 mg., whichever was the greater. In some patients intestinal biopsies were undertaken. The effect of the various drugs on some of the patients shown to have impaired absorption was assessed after using the following doses by mouth : aureomycin 250 mg., t.d.s., penicillin G. 200,000 units daily, prednisolone 15 mg. t.d.s., all given for 5 days, and intrinsic factor 100 rag. given with the test dose. RESULTS I n 30 of the 66 patients the percentage of radioactivity recovered in the stools was more than 50, and in 11 of these 30 the recovery was 75 per cent. or over ; the lowest level of recovery was 4 per cent. T h e T a b l e gives details of the serum vitamin BI~ levels, and the percentages of radioactivity recovered before and after treatment with drugs, in 24 patients selected f r o m those with an initial recovery of radioactivity of more than 40 per cent. ; no patients with a lower initial recovery were selected for drug treatment. T h e r e was no relationship between the degree of absorption and the serum vitamin Bx~ levels, nor could the absorption of the vitamin be correlated with the absorption of folic acid, or of xylose, or with the amount of gastric acidity. Fifty-six of the 66 patients

274 TABLE.

ABSORPTION OF VITAMIN B12 IN NUTRITIONAL MEGALOBLASTIC ANAEMIA

Showing results of tests of absorption on 24 patients with nutritional megaloblastic anaemia.

Race, sex age S. Indian f. 50 N. Indian f. 37 *S. Indian f. 29 *S. Indian f. 23 N. Indian f. 39 *S. Indian f. 19 S. Indian f. 32 *S. Indian f. 42 S. Indian f. 34 *S. Indian f. 24 S. Indian f. 48 S. Indian f. 44

% radioactivity Serum recovered vit. BI~ tz~tg/ml Initial After drugs

Race, sex, age

80

90

80

89

95

86

625

84

Aureomycin int. factor Aureomycin Prednisolone Aureomycin int. factor Aureomycin

90 86 80 50 37 63 30

81

Prednisolone 62

310

80

int. factor

540

80

Aureomycin 55

145

76

Prednisolone

490

75

Penicillin

175

70

Aureomycin 15

90

68

795

65

int. factor 79 Aureomycin 9 Prednisolone 63

33

9 47

S. Indian f. 20 *S. Indian f. 34 N. Indian m. 55 S. Indian m. 59 S. Indian f. 45 *S. Indian f. 28 *S. Indian f. 38 S. Indian f. 19 *S. Indian f.? S. Indian f. 47 S. Indian f. 69 S. Indian f. 19

Serum vit. B1, ~tg/ml

% radioactivity recovered ....

Initial

After drugs

63

Penicillin

76

225

63

int. factor

72

50

62

Prednisolone 73

50

59

Prednisolone 11

50

59

int. factor

20

420

59

Penicillin

73

50

59

int. factor

64

75

58

Prednisolone 29

85

57

Aureomycin 18

300

45

Penicillin

250

41

int. factor 67 Aureomycin 17 Prednisolone 65

40

38

*Pregnant.

studied were South Indians, 27 of t h e m pregnant women, so that correlation with race, sex, age, or pregnancy would have little significance. T h e gastric biopsies in the present series of patients, as in other similar patients w h o m I studied nearly always showed inflammatory changes, often to a severe degree, but rarely any atrophy. T h e effects of the various drugs u p o n absorption were as follows : Intrinsic factor : Eight patients were studied, six of w h o m had achlorhydria. I n two, absorption appeared to have been improved ; one was a young Indian pregnant w o m a n with a normal s e r u m vitamin BI~. level, the other was an elderly Indian w o m a n with a very low s e r u m vitamin BI~ level b u t with free acid secretion and no gastric atrophy. Aureomycin : Nine patients were studied, in seven the absorption was markedly improved. I n one of the remaining two patients intrinsic factor also failed to improve absorption, although there was a very low s e r u m vitamin BI~ level and achlorhydria ; in the other patient absorption was improved by prednisolone. Penicillin : F o u r patients were studied, in only one of w h o m was there m u c h improvem e n t in absorption. Prednisolone : Eight patients were studied, in three of w h o m absorption appeared to be m u c h improved.

P. W. G. TASKER

275

Although diarrhoea was present in some of these patients, clinical tropical sprue was not seen. The absence of tropical sprue, at least as a major factor, in the aetiology of these megaloblastic anaemias was further indicated by the results of the folic acid and the xylose absorption tests. Fifteen normal adult members of the laboratory staff gave folic acid recoveries of 2.7 -4- s.e. 0.17 mg. while 29 patients with megaloblastic anaemia, those in the present series and others of similar type, gave recoveries of 2.1 + 0.17 rag. In the xylose absorption test, 21 controls gave recoveries of 1.43 4- s.c. 0.12 g. while 36 patients with megaloblastic anaemia gave recoveries of 0.71 4- 0.07 g. DISCUSSION

The data presented show that a malabsorption of vitamin BI~ was rarely severe. Since patients with a deficiency of vitamin BI~ did not necessarily show an impairment of absorption this was unlikely to have been a major cause of the deficiency. The results also suggest that tropical sprue was unlikely to have been a major factor in causing either the deficiency or the malabsorption of the vitamin ; not only was there no clinical evidence of sprne, but the absorption of vitamin B12, of folic acid, and of xylose were not as impaired as might have been expected had this condition been present. However, these absorption tests gave evidence of some defect in intestinal function. As remarked by BAKER (1938), a nutritional megaloblastic anaemia associated with tropical sprue cannot always be clearly distinguished. The patients seen in this series are better defined as having nutritional megaloblastic anaemia, for intestinal malfunction was not severe. Intestinal biopsies commonly showed inflammatory changes, often severe, and the administration of aureomycin and, less certainly, of prednisolone appeared to correct the malabsorption of vitamin Bm. This suggests that inflammatory changes in the intestinal mucosa and/or alteration in the intestinal bacterial flora might have significance in causing a malabsorption of vitamin B19. However, since the malabsorption was not related to a deficiency of vitamin B12, it is possible that malabsorption was only a temporary episode in the course of the disease. Further studies of intestinal function in these anaemias are required. Since some of the patients in this series showed low serum vitamin B12 levels associated with malabsorption, the possibility of pernicious anaemia existed, particularly in the elderly. This diagnosis was not tenable in any of these patients, for they either had a secretion of free acid following histamine or, where there was achlorhydria, the absorption was not improved by giving intrinsic factor. SUMMARY

1) The vitamin B12 deficiency found in some patients with nutritional megaloblastic anaemia seen in Malaya did not appear to be directly related to a malabsorption of the vitamin. 2) This, and other evidence, suggested that tropical sprue was unlikely to have been a major cause for this deficiency. It was also noted that pernicious anaemia was not encountered. Malabsorption, when it did occur, might have been related to changes in the intestinal bacterial flora and/or inflammatory changes in the intestinal mucosa, for the malabsorption was readily corrected by aureomycin and sometimes by prednisolone.

276

ABSORPTION OF VITAMIN B12 IN NUTRITIONAL MEGALOBLASTIC ANAEMIA

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