The long-term relationship between serum vitamin B12 and total body vitamin B12

Transcription

1 The long-term relationship between serum vitamin B12 and total body vitamin B12 Keith Boddy, B.Sc., M.Sc., Ph.D., F. Inst. P., and J. F. Adams,2 V.R.D., M.D., F.R.C.P.E., F.R.C.P.G. There is a considerable body of evidence supporting the concept that, in the majority of instances, an unequivocally low serum vitamin B12 level is a manifestation of depleted body stores of vitamin B12. Otherwise, little evidence is available concerning the relationship between serum and total body vitamin B12 in man and, in particular, the extent to which serum vitamin B12 reflects the total body status. Studies in animals have shown that the serum vitamin B12 level falls more rapidly than does the hepatic vitamin B12 concentration when the capacity to absorb vitamin B12 is impaired by total gastrectomy (1). Also the correlation between serum and hepatic vitamin B12 content in man has recently been reviewed (2). The object of the studies reported here was to examine the relationship between the serum vitamin B12 level and total body vitamin B12 in man in a situation similar to that in the animal studies. To this end, patients were studied who, for the most part, had evidence of vitamin B12 deficiency due to impairment of the capacity to absorb the vitamm. Materials and methods Fifteen patients were included in the study. Eleven were diagnosed as having pernicious anemia on the basis of a low serum vitamin B12 level and megaloblastic erythropoiesis before treatment, a histamine- or pentagastrin-fast achlorhydria, and an inability to absorb radioactive cyanocobalamin unless intrinsic factor were given. Two patients had previously undergone partial gastrectomy and had come under observation with anemia due to iron deficiency; after treatment with iron they were found to have equivocal serum vitamin B12 levels and minimal megaloblastic change. One patient had been treated for iron deficiency and another, having an irrelevant disease, was subsequently shown to be able to absorb cyanocobalamin normally but she had an equivocal serum vitamin B level. No patient showed evidence of folate deficiency, as judged by serum folate levels, before or after treatment with vitamin B12. Of the 15 patients, 1 1 were given a single intravenous dose of 57Co cyanocobalamin, hydroxocobalamin, or coenzyme vitamin B, and four were given a series of 5 or 6 daily intravenous doses of 57Co hydroxocobalamin as indicated in Table 1. The mass of the doses ranged from 1,500 to 5,000 jg and the activities from 1 to 5 jtici. Radioactive cyanocobalamin and hydroxocobalamin were obtained from the Radiochemical Centre, Amersham, and radioactive coenzyme B, prepared from radioactive hydroxocobalamin by Dr. L. Mervyn of Glaxo Limited, using the method of Johnson et al. (3). Doses for administration were prepared by mixing appropriate amounts of radioactive and stable compounds and sterilizing by Millipore filtration. Particular care was taken to ensure that coenzyme B12 solutions were not exposed to other than dull red light. Cyanocobalamin, hydroxocobalamin, and coenzyme vitamin B12 were used to compare the metabolism of the analog forms. Single large intravenous doses were chosen in preference to more physiological doses because a greater amount of vitamin (microgram) was retained, although it was only S to 10% of the administered dose. This reduced the possibility of hematological or neurological relapse in the patients during this protracted study, bearing in mind the long biological half-life of vitamin B12 in man. Whole-body radioactivity was measured in the Merlin whole-body monitors (4, 5). Prior to administration of the dose, or the first dose of a series, the counting rate due to natural body radioactivity was obtained and each subject was measured shortly after the cobalamin dose, giving a counting rate corresponding to 100% retention in those given a single dose and the appropriate incremental value in those given multiple doses. Thereafter, whole-body monitoring was repeated at intervals of 2 to 4 weeks for periods ranging from 166 to 639 days after the initial count. All counting rates 1 Senior Lecturer (Health Physics and Nuclear Medicine), Scottish Universities Research Reactor Centre, East Kilbride, Glasgow, Scotland. 2 Consultant Physician, Southern General Hospital, Glasgow, S.W.1. The American Journal of Clinical Nutrition 25: APRIL 1972, pp Printed in U.S.A. 395

2 396 BODDY AND ADAMS TABLE 1 Clinical data, treatment schedules, rates of loss of vitamin B,2 from the whole-body and serum Whele-bedy excretion, Serum vitamin 1112 Subject Clinical condition Pretreatment serum Bi,, pg/mi Administered dose, jig %/day Rate turnover, Rate %/day lf 79 PA 2F 50 PA 3F 65 PA 4F 67 PA SM 49 PA 6M 73 PA 7M 58 PA 8F 69 Normal 9F 79 Fedef. 1OM 41 PG llm 43 PG 12F 60 PA J3M 62 PA 14M 54 PA 15M 45 PA 38 < ,000 C 6 X 5,000 H 5,000 C 1,500 E 5,000 C 2,000 C 6 X 5,000 H 2,000 H 5 X 5,000 H 5 X 5,000 H 0.197(0.023) (0.016) 0.209(0.020) 0.175(0.012) 0.275(0.014) 0.129(0.015) 0.207(0.036) 0.177(0.019) 0.282(0.015) 0.082(0.005) 0.126(0.009) 0.212(0.019) 0.119(0.011) 0.218(0.019) 0.178(0.012) 0.255(0.053) 0.167(0.034) 0.210(0.070) 0.192(0.057) 0.221(0.047) 0.212(0.045) ) 0.138O.03O) 0.217(0.030) 0.094(0.018) 0.135(0.033) 0.089(0.037) 0.216(0.039) 0.050(0.059) 0.027(0.014) F = female. M = male. PA = pernicious anemia. Fe def. = iron deficiency. PG = partial gastrectomy. C = cyanocobalamin. H = hydroxocobalamin. 0 Numbers in parentheses indicate SE. were corrected for background, natural body radioactivity, and radioactive decay. Blood was obtained and serum separated for vitamin B assay before treatment and at each attendance for whole-body monitoring. Samples were stored at -20 C and at the end of the observation period all samples from each patient were assayed by the method of Hutner, Bach and Ross (6) using Euglena gracilis Z. strain as the test organism. The means of the three assays were used in the calculations. Exponential rates of loss from the body and from serum vitamin B were calculated by the method of least squares and included at least six data points for each patient from equilibration of administered and native vitamin B (at approximately 100 to iso days after treatment) up to the final measurement. Results Relevant clinical details, treatment schedules, and exponential rates of loss from the body and from serum are summarized in Table I. The rate of loss of vitamin B12 from the body was significantly greater than zero (P < 0.05) for each patient. The mean rates of loss for each of the administered analog forms of the vitamin were not significantly different from one another (P> 0.05). The rate of loss of vitamin B12 from the serum was significantly greater than zero (P < 0.05) in 13 patients but not in two of them (cases 14 and 15). Results for cases 14 and 15 were therefore excluded from further analysis. In the remainder, the mean rates of loss for each of the administered analog forms were not significantly different from one another (P > 0.05). The rate of loss from the serum in each of these cases individually was not significantly different from the rate of loss from the body with the exception of cases 12 and 13. Discussion Whether the serum vitamin B11 does or does not reflect the magnitude of body stores of vitamin B12 is a problem that is difficult to study directly in the absence of an acceptable method for measuring the mass of body stores. Even if such a method were available it seems likely that the problem would resolve into a series of problems in view of the considerable evidence, recently reviewed by Chanarin (2), that the serum vitamin B12 level can be influenced, apparently independently of stores, in a wide variety of conditions. One situation in which the relationship between serum vitamin B12 and body vitamin B12 has been studied is that following total

3 SERUM AND TOTAL BODY VITAMIN B,j 397 gastrectomy in rats. Here the absorption of vitamin B12, which may in itself be a factor in maintaining the serum vitamin B12 level, is suddenly grossly impaired and the obligatory loss of vitamin B12 continues, leading to depletion of stores. Booth and Spray (1) working with rats found that the concentration of vitamin B12 in the livers of totally gastrectomized rats fell more slowly than in serum and concluded that the serum vitamin B12 did not provide a true assessment of vitamin B12 in this instance. This conclusion has not been confirmed by evidence for a relationship between serum and hepatic vitamin B12 in vitamin B12-deficient subjects, by evidence marshalled by Chanarin (2) from work by Joske (7), Anderson (8), and Stahlberg, Radncr and Norden (9) and for a relationship between serum vitamin B12, hepatic vitamin B,2, and total body vitamin B12 reported by Adams, Tankel and MacEwan (10). The current study embodies the assumption of equilibrium between administered radioactive vitamin B12 and native vitamin B12, the justification of which has been reviewed (11, 12). The situation after equilibration of the retained fraction of the administered radioactive vitamin B12 was very similar to that observed after total gastrectomy, and the absence of significant differences between serum vitamin B12 turnover rate and wholebody turnover rate in 11 out of 13 sets of results (in which both values were significant) provides evidence that, in these cases, the decline in stores was reflected by an insignificantly different decline in serum vitamin B12 level. There were two patients in whom both serum and whole-body turnover rates were significant, but in whom the values differed significantly. One patient had a serum turnover rate less than the whole-body turnover rate that suggests a situation in which the serum level is maintained at the expense of stores. The other had a serum turnover rate greater than the whole-body turnover rate that suggests clearance from serum to tissue. It does not necessarily follow, however, that the serum vitamin B12, measured routinely in the clinical situation as picograms per milliliter, is the same fraction of the total body stores of the vitamin from one patient to another. This point was examined further. At equilibrium, the pretreatment body stores (B1) and serum level (S1) and the posttreatment body stores (B2) and serum level (S2) in an individual patient would have the relationship: B2/B, = S2/S, It can be reasonably expected in the present patients that the pretreatment body stores are small in comparison with the amount of the administered dose retained (D). In this case, the retained dose is essentially the total posttreatment body stores so that D = B1 and the relationship can be re-written as D = B,(S,/S,) (I) The regression of D on (S2/S1), representing the correlation of total body vitamin B12 and serum vitamin B12, should be linear, passing through the origin (i.e., with an insignificant intercept). The regression coefficient, or slope of the line (B1), will be an estimate of the pretreatment body stores. Also, the serum level and body stores in the individual will be related by B1 = KS1 and D = KS2, where K is a constant representing the total body stores (micrograms vitamin B12)/ serum vitamin B12 (picograms per milliliter). The relationship (equation 1) was shown to have an insignificant intercept for each patient, except subject 1 3M, and the regression was recomputed to pass through the origin. The findings are summarized in Table 2 and show highly significant correlation between total body vitamin B12 and serum vitamin B12 in all patients (if through 1 1M) in whom the rates of loss of the vitamin from the body and from serum were not significantly different. In these patients, the correlation coefficient ranged from 0.69 to 0.96 with a mean correlation coefficient of A limitation of the accuracy of the estimated pretreatment body stores is the dependency on both pre- and posttreatment serum vitamin B12 levels because of the variability in the microbiological assay procedure. This approach was adopted, nevertheless, because radioactive assay of vitamin B12 in serum over the extended period of this study would have required administration of larger doses of radioactivity than was considered acceptable. Variations were minimized by replicate assays of all samples en bloc. There remained in some cases inevitable uncertainty in the

4 398 BODDY AND ADAMS TABLE 2 Correlation of posttreatment total body vitamin B1, (D) and posttreatment serum vitamin B,, (5,) Subject and clinical condition IF PA 2F PA 3F PA 4F PA SM PA 6M PA 7M PA 8F Normal 9F Fe def. lom PG llm PG 12F PA 13M PA Pretreatment stores, Bi ± SE, % dose 2.13(0.46) 0.2l8( (0.66) 1.06(0.28) 2.90 (0.49) 0.90 (0.20) 2.06(0.60) 2.16(0.29) 11.25(2.36) (0.71) 6.35(1.26) 1.68(0.47) 0.94 (0.21) Correlation coefficient Stores, jig B,, K jig total body pg/mi The estimates of pretreatment total body vitamin B5, (B1) and standard error (in parentheses) were derived from the relationship D = (S,/S1) B,, where S, is the pretreatment serum vitamin B,, level. The estimates of the constant K (micrograms total body/picogram/milliliter serum) were derived from the equation D = KS,. PA = pernicious anemia. Fe def. = iron deficiency. PG = partial gastrectomy. assay of the extremely low pretreatment serum vitamin B12. Despite these reservations, the results are encouraging. In the nine cases of pernicious anemia the estimated body vitamin B12 ranged from 41 to 235 g, only two being greater than 84 g and the overall mean value 83 /Lg. These values seem to be of the right magnitude as judged by the hepatic vitamin B12 concentrations in similar patients (8, 9, 13-18). The values of 210 and 318.ag in the two postgastrectomy patients who had serum vitamin B12 levels of 100 pg/mi and 157 pg/mi and megaloblastic erythropoiesis are similar to the reported values of hepatic vitamin B12 values in such patients (8, 9, 13, 17, 19). Finally, the value of 650 jg in the patients with the serum vitamin B12 level of 171 pg/mi is compatible with the pattern seen in control subjects (10). The accuracy of the estimated K values (total body vitamin B,2/serum vitamin B12 (picograms per milliter) was not influenced by the uncertainty in the pretreatment serum vitamin B12 determinations. Although K is a constant for each individual, it varies widely from one individual to another (range 0.8 to 9.4, mean 2.97 ± 0.82 (sem)). Thus a serum vitamin B12 value of 100 pg/mi could be associated with total body stores of vitamin B12 of 80 /Lg in one subject and 940.tg in another. This finding offers at least a partial cxpianation of the equivocality of serum vitamm B12 levels over a certain range with respect to normoblastic or megaloblastic erythropoiesis. In a study (10) of the total body vitamin B12, determined by isotope dilution and microbiological assay procedure on the assumption of equilibrium, a linear relationship was found between the arithmetic values for serum vitamin B12 and the total body vitamin B12, the regression equation being: Total body vitamin B,, (pg) = (pg B, 2/ml serum) The constant ( ) is of interest because this regression line should also pass through the origin or have an insignificant intercept, if the assumption of equilibrium is justified. In accordance with the present interpretation, the constant is not significant (P > 0.05). The regression coefficient, , corresponds to the constant K in the present nomenclature, namely, total body B12 per picogram vitamin B12 per milliliter serum. The equation might suggest that this constant does not vary from individual to individual but the regression coefficient has a standard error of approximately ±2.4 in accordance with the wide individual range of 2.0 to 9.7. Further, the mean value and standard error were 5.66 ± 0.59 in the earlier group that was largely comprised of patients with various diseases but excluding frank vitamin B12 deficiency, and the corresponding values were 2.97 ± 0.82 in the present patients most of whom were suffering from pernicious anemia. The difference is significant (P < 0.05), which could imply that the fraction of the total body vitamin B12 that is in serum is greater in pernicious anemia. If this were so, it would involve a disproportionate fall in body stores vis-#{224}-visserum vitamin B12. This might seem unlikely in view of the correlations found between the serum vitamin B12 and whole-body vitamin B12, but this could occur in the latent period of devel-

5 SERUM AND TOTAL BODY VITAMIN B opment of pernicious anemia and would not have been observed in this series of patients because the duration of the study was short compared with the years involved in the transition from normality to frank vitamin B12 depletion. Finally, it is of interest to note that neither the mean rate of loss from the body nor from the serum of each analog form of administered vitamin B12 was significantly different from that of the other analogs. This finding is compatible with the finding (20-23) that administered hydroxocobalamin and cyanocobalamin are converted to coenzyme form in the body and, consequently, a common form is presented to the excretory system (12). In this case, the rates of loss would be independent of the administered form of the vitamin at least when massive parenteral doses are given, as found in this and in an earlier study (24). Summary Following parenteral administration of large doses of labeled vitamin B12, the longterm rate of loss of the vitamin from the whole body and from the serum were measured, mainly in patients with pernicious anemia. In each patient, these rates of loss could be adequately described by a single exponential term and, in 11 of the 15 patients, the rate of loss from the body was not significantly different from the rate of loss from the serum. The retained dose was effectively the total body vitamin B12 and was shown to be significantly correlated with the serum vitamin B12, with the exception of two patients in whom disparities could be demonstrated. From these data, the pretreatment total body content of vitamin B12 was estimated in each patient. In the patients suffering from pernicious anemia, the total body content ranged from 41 to 135 zg, with only two values being greater than 84 g. In two patients who had previously undergone partial gastrectomy, the estimated total body vitamin B12 was 210 and 318 g and in a subject with an irrelevant disease it was 650 g. Although the total body vitamin B12 (micrograms)/serum vitamin B12 (picograms per milliliter), or the K value, was constant in individual subjects, this ratio varied widely from one individual to another. The K value was significantly smaller in the present patients than in control subjects. Finally, the mean rate of loss from the body and from the serum vitamm B12 was apparently independent of the analog form in which the vitamin was administered. We wish to thank Professor H. W. Wilson for his interest and encouragement and Dr. P. C. King, Miss B. A. Holmes, and Miss M. Mahaffy for their technical assistance. The study was supported, in part, by a grant from the Scottish Hospital Endowments Research Trust which is gratefully acknowledged. References 1. Boom, M. A., AND 0. H. Siux. Vitamin B,2 activity in the serum and liver of rats after total gastrectomy. Brit. I. Haematol. 6: 288, CHANARJN, I. The Megaloblastic Anaemias. Oxford: Blackwell, 1969, p Jonrisor, A. W., L. MERVYN, N. SLIAw AND E. L. SMITh. A partial synthesis of the vitamin B,, coenzyme and some of its analogues. J. Chem. Soc. 4146, BODDY, K. The development and performance of a prototype shadow-shield whole-body monitor. Phys. Med. BioI. 12: 43, BODDY, K. A high sensitivity shadow-shield whole-body monitor with scanning-bed and tilting-chair geometries, incorporated in a mobile laboratory. Brit. J. Radio!. 40: 631, HUTNER, S. H., M. K. BACH AND 0. I. M. Ross. A sugar-containing basal medium for vitamin B2 assay with Euglena: application to body fluids.j. Prozozool. 3: 101, Josiu, R. A. The vitamin B12 content of human liver tissue obtained by aspiration biopsy. Gut 4:231, ANDERSON, B. B. Investigations into the Euglena method of assay of vitamin B,2: the results obtained in human serum and liver using an improved method of assay (Ph.D. Thesis). Univ. of London, STAHLBFRG, K. G., S. RADNER AND A. NORDEN. Liver in subjects with and without vitamin B12 deficiency. A quantitative and qualitative study. Scand. I. Haematol. 4: 312, ADAMS, J. F., J. I. TANKEL AND F. MACEWAN. Estimation of the total body vitamin B2 in the live subject. Clin. Sci. 39: 103, BODDY, K., AND J. F. ADAMS. The validity of the assumption of tracer equilibrium with respect to the excretion of vitamin B12. Phys. Med. Biol. 13: 55, 1968.

6 400 BODDY AND ADAMS 12. BODDY, K. A physicist s interpretation of some aspects of vitamin B12 metabolism. J. NucI. Mcd. 6: 275, DROUET, P.-L., R. WOLFF, R. KARLIN AND E. RAUBER. Etudes de la vitamin B12 hepatique per Ia ponction biopsie. Premiers resultats dans la maladie de Biermer. Bull. Mem. Soc. Med. Hop. (Paris) 67: 281, WOLFF, R., P.-L. DROUET AND R. KARLIN- WEISSMAN. L emploi de la ponction biopsie pour l #{233}tude de la vitamine B12 hepatique chez l homme. Compi. Rend. A cad. Sci. 232: 568, GIRDWOOD, R. H. The occurrence of growth factors for Lactobacillus, Leichmannii, Streptococcus faecalis and Leuconostoc citrovorum in the tissues of pernicious anaemia patients and and controls. Biochem. J. 52: 58, SWENDSEID, M. E., E. Hv0LB0LI, 0. Scmcx AND J. A. HALSTED. The vitamin B12 content of human liver tissue and its nutritional significance. A comparison study of various age groups. Blood 12: 24, RoSs, G. I. M., AND D. L. MOLLIN. Vitamin B12 in tissues in pernicious anaemia and other conditions. In: Vitamin B12 and Intrinsic Factor, edited by H. C. Heinrich. Stuttgart: Enke, 1957, p NELSON, R. S., AND V. M. DOCTOR. The vitamin B12 content of human liver as determined by bio-assay of biopsy material. Ann. internal Med. 49: 1361, SPRAY, 0. H. The estimated and significance of the level of vitamin B12 in serum. Postgrad. Med : 55, ROSENBLUM, C. Stability of cyanocobalamin in living systems. Scand. I. Haematol. 3: 48, Ucumio, H., Y. YAGARJ, T. YOSHIMO, M. KONDO AND 0. WAKISAKE. Conversion of cyano- and hydroxocobalamin in vivo into coenzyme form of vitamin B,, in the rat. Nature 205: 176, PAWELK!EWIEZ, J., M. FORNA, W. PENRYCH AND S. MAYAS. Conversion of cyanocobalamin in vivo and in vitro into its coenzyme form in humans and animals. Ann. N. Y. A cad. Sci. 112: 641, REL STEIN, P. Conversion of cyanocobalamin to a physiologically occurring form. Blood 29: 494, BODDY, K., AND J. F. ADAMS. Excretion of cobalamins and coenzyme B,2 following massive parenteral doses. Am. I. Clin. Nutr. 21: 657, 1968.