IMPAIRED ASSIMILATION OF EGG C057 VITAMIN B12 IN PATIENTS WITH HYPOCHLORHYDRIA AND ACHLORHYDRIA AND AFTER GASTRIC RESECTION

Transcription

1 GASTROENTEROLOGY 64: , 1973 Copyright 1973 by The Williams & Wilkins Co. Vol. 64, No.5 Printed in U.S.A. IMPAIRED ASSIMILATION OF EGG C057 VITAMIN B12 IN PATIENTS WITH HYPOCHLORHYDRIA AND ACHLORHYDRIA AND AFTER GASTRIC RESECTION ALFRED DOSCHERHOLMEN, M.D., PH.D., AND WILLIAM R. SWAIM, M.D. Departments of Medicine and Laboratory Medicine, Veterans Administration Hospital, and the Department of Medicine, University of Minnesota, Minneapolis, Minnesota Patients who have undergone gastric resection and those with simple gastric hypochlorhydria and achlorhydria may have low serum B12 concentrations with normal absorption of radiocyanocobalamin. Impaired assimilation of food vitamin B 12, but not of the crystalline C0 57 B 12, may be the cause for this phenomenon. This hypothesis was tested by comparing the assimilation of C0 57B 12 incorporated into eggs in vivo with that of crystalline C0 57B 12 As judged by the urinary excretion test, 10 patients with gastric resection having low serum B12 values and normal absorption of crystalline B 12, absorbed on the average 9% of that observed in 11 normal control subjects when the egg C0 57B 12 was administered. Similarly, 5 patients with achlorhydria or severe hypochlorhydria absorbed on the average 12% of that seen in normal subjects. There was no difference in the absorption of the crystalline C0 57B12 in these groups. An equally poor assimilation of nonlabeled food B12 may explain the low serum B12 concentrations found in postgastrectomy patients, and achlorhydric subjects with low serum B12 and normal absorption of crystalline radiocyanocobalamin. Patients with pernicious anemia and those with gastric resection showing low serum B12 concentrations and B12 absorption values had extremely poor assimilation of both food C0 57B 12 and crystalline C 0 57B 12 Decreased serum B12 concentration with normal absorption of crystalline radiocyanocobalamin is commonly observed in patients who have had gastric resection. 1 Similar findings have been noted in subjects who have simple gastric achlorhydria or hypochlorhydria. 2 The assumption has been made that these individuals must have an adequate ' intrinsic factor (IF) secretion since their absorption of crystal- Received June 30, Accepted November 24, Address requests for reprints to: Dr. A. Doscherholmen, Department of Medicine, Veterans Administration Hospital, Minneapolis, Minnesota The authors wish to acknowledge the technical assistance of Jean McMahon and Donna Ripley. 913 line radiocyanocobalamin is normal. 1, 2 Low serum vitamin B12 concentration with normal B12 absorption is typical of persons subsisting on a vitamin B 12 -deficient diet. 3-7 However, when the diet contains adequate vitamin B12 the possibility of impaired assimilation of food vitamin B12 should be considered as a cause of the low serum B 12 level. We explored this possibility by testing the absorption of radiolabeled B12 incorporated into food protein. This report compares the absorption of C0 57B 12 incorporated into eggs with that of crystalline radiocyanocobalamin in 15 patients with either gastric resection, or with hypochlorhydria and achlorhydria who had low serum B12 levels and normal B12 absorption. Eleven normal control sub-

2 914 DOSCHERHOLMEN AND SWAIM Vol. 64, No.5 jects, 5 patients with Addisonian pernicious anemia, and 5 with gastric resection and low serum B12 concentration and impaired B12 absorption were similarly studied. A preliminary abstract of this work has been published. 8 Materials and Methods Eggs labeled with Co57B12 were obtained as follows: 12 J.LC (1 J.Lg) of C0 57B 12 were injected in divided doses over a period of 3 days into the breast muscle of an egg-laying hen. The eggs were collected and checked for radioactivity in a Tobor scintillation counter. The radioactivity began to appear in the eggs 2 to 3 days after the first injection of C0 5 7 B 12, reaching a peak activity at 6 to 10 days and then gradually tapering off. When 13 to 18 radioactive eggs had been obtained, they were homogenized for 10 min in a high speed blender, scrambled (cooked in a Teflon pan) with constant stirring until a medium dry texture was obtained, packaged in 50-g cellophane bags, and stored in a freezer at -20 C until served. Four batches of eggs were used in these studies. Since the radioactivity of individual eggs differed, two tests were made to verify the radioactive uniformity of the homogenized-scrambled mixtures. In the first test, 1 J.LC of 1311 was added to 12 nonradioactive eggs, which were homogenized and scrambled; five I-g random samples of the scrambled eggs were placed in a scintillation counter which gave the following counts per minute: 2,826, 2,812, 2,815, 2,949, and 2,882, respectively. In the second test, two I-g samples were taken from each batch of C0 57B 12 scrambled eggs and were similarly counted. The variability in the two counts was within ±1.0%. The vitamin B12 content of the scrambled eggs was determined by the Euglena gracilis assay,9 after extraction of the vitamin according to the method of Biggs et al. 10 After 1 g of scrambled eggs was diluted to 50 ml with the extraction fluid and homogenized in the highspeed blender, the B'2 was released by autoclaving and measured. The mean B12 content in the extraction fluid of the four batches of C0 57B 12 scrambled eggs was 563 pg per ml (range 500 to 670 pg per ml). Before the radiolabeled eggs were administered, a sample from each batch was cultured to ensure against the presence of pathogenic bacteria. At 8 AM on the test day, 45 to 56 g of the scrambled eggs containing 0.56 J.Lg of vitamin B12 and 0.4 to 0.5 J.LC of C0 57 were heated in a microwave oven for 30 sec and were served with a cup of black coffee and a piece of toast to each fasting subject. No other food was allowed until lunchtime. An E. gracilis assay showed that freezing, thawing, and reheating had no deleterious effect on the B12 content of the scrambled eggs. The absorption of radiocyanocobalamin was measured by three techniques: stool excretion," urinary excretion, 12 and 8-hr plasma absorption of radioactivity.13 Stools were collected quantitatively in individual daily containers for 7 days and the radioactivity was determined in a Tobor scintillation counter. Counting was carried out for 30 min, with a counting error of less than 5%. A cellophane package containing a serving of scrambled eggs was similarly counted to represent the ingested dose of radioactivity. The 7-day fecal radioactivity was expressed as a percentage of the ingested dose. Six hours after the ingestion of the scrambled eggs, the optimal time interval in the Schilling urinary excretion test, 13 1 mg of nonradioactive vitamin B'2 was given parenterally. Immediately afterwards, a 24-hr quantitative urine collection was started. Ten milliliters of urine were counted by scintillation spectrometry in a well1;ype Nuclear-Chicago model 4233 automatic scintillation counter; the total counts in the 24-hr collection were expressed as a percentage of the oral dose. The 8-hr plasma radioactivity in a 10-ml sample of plasma was similarly determined, with a O-hr plasma sample as background. The plasma absorption was expressed in picograms of C0 57B12 per ml of plasma Both plasma and urine were counted for 100 min, or 100,000 counts. The ingested radioactivity for the plasma absorption and the Schilling tests was obtained as follows: 1 g of scrambled eggs was placed in a counting vial and was completely digested at 37 C in 10 ml of a pepsin-containing buffer solution with a ph of 2.0. The radioactivity of this material was counted by the method used for counting the urine and plasma. The total ingested radioactivity was then calculated from the amount of scrambled eggs ingested. The counting error was less than 4% for urine and 7% for plasma. At 8 AM, 1 to 4 weeks after the first phase of the experiment, 0.56 J.Lg of crystalline B'2 containing 0.5 J.LC of C0 57 was dissolved in water and given to the fasting subjects. No food was allowed for at least 2 hr thereafter. The collection of stool, urine, and plasma and the parenteral injection of "cold" B12 were carried out as in phase 1. In the fecal excretion test, the total dose of

3 May 1973 ASSIMILATION OF EGG VITAMIN B Co57BI2 ingested was counted in a 10-ml vial placed in the Tobor counter. In the urinary excretion test and the plasma absorption of radioactivity, a 1% aliquot of the ingested Co57BI2 solution in 10 ml of water was measured in the well counter. The radioactivity of stool, urine, and plasma was expressed similarly to the radioactivity of egg. Hemograms were obtained by a Coulter Counter model S. Serum B12 was determined by E. gracilis,9 serum folate by the Lactobacillus casei assay, IS and serum iron and total iron binding capacity by the methods of Ramsay. 16, 17 Five groups of subjects were studied. The first group consisted of 10 patients, 29 to 60 years old, who had had Billroth I (3 patients) or Billroth II (7 patients) surgery for benign peptic ulcer 2 to 26 years earlier. They had low serum BI2 levels, ranging from 84 to 160 pg per ml (average 122), with normal absorption of crystalline C0 57B 12, ranging from 2.3 to 7.4 pg per ml. The second group consisted of 5 patients, 48 to 65 years old. A 1-hr collection of gastric juice obtained immediately after the injection of 100 mg of Histalog showed complete achlorhydria in 3 patients (ph above 7.0) and severe hypochlorhydria in 2 (ph of3.7 and 3.8, with 0.8 and 0.4 meq of hydrochloric acid per hr, respectively). Serum BI2 ranged from 100 to 145 pg per ml (average 121) and the 8-hr plasma absorption of crystalline C0 57B I2 was normal, ranging from 2.8 to 5.6 pg per ml before the investigation began. i The third group included 5 patients, 43 to 78 years old, who had had Billroth II surgery for benign peptic ulcer 11 to 22 years earlier. They had low serum BI2 values, ranging from 50 to 140 pg per ml (average 87), and low 8-hr plasma absorption of crystalline C0 57 B 12 (0.2 to 1.0 pg per ml), corrected by the addition of hog IF concentrate (2.2 to 5.1 pg per ml). Thus, their condition was similar to that of patients with pernicious anemia. The fourth group consisted of 5 patients, 44 to 79 years old, who had been diagnosed as having Addisonian pernicious anemia. All had megaloblastic anemia, with response to B12 therapy; histamine-fast achlorhydria; low serum vitamin BI2 values, ranging from 11 to 56 pg per ml (average 33); and low 8-hr plasma absorption of crystalline Co 57 B I2 (0.1 to 0.5 pg per ml), corrected by the addition of hog IF concentrate (1.7 to 4.8 pg per ml). The fifth group consisted of 11 healthy subjects, 23 to 55 years old, with intact stomachs and normal HCl secretion, as determined by intubation in 3 subjects and by Diagnex test in 8. Serum B12 levels were normal, ranging from 200 to 525 pg per ml (average 379). The patients receiving BI2 therapy were not studied until 4 weeks after their last BI2 injection. Hematologic data were normal except for iron deficiency in 1 and mild anemia in 3 patients in group 1. Mild folate deficiency was found in 1 subject in groups 3 and 5 each. Results Figures 1, 2, and 3 show the results of the three absorption parameters. In the post gastrectomy patients who developed low serum B12 concentration despite a normal absorption of crystalline radiocyanocobalamin, the mean fecal, urinary, and plasma absorption values after ingestion of egg-co 57 B 12 were 80.9%, 0.30%, and 0.16 pg per ml. The corresponding values after ingestion of crystalline radiocyanocobalamin were 45.4%, 17.5%, and 6.3 pg per ml. Subjects with simple gastric hypochlorhydria and achlorhydria showed mean fecal, urinary, and plasma absorption values of 79.5%, 0.42%, and 0.28 pg per ml after ingestion of egg C0 57B 12 and 33.8%, 26.7%, and 8.7 pg per ml when crystalline radiocyanocobalamin was administered. Post- N E.gg CO S7 BI2 Crystallin CoS'B,. 100.J ['j id i. rii 80, 0 u E ; 40.. " o 111[[[ 1'1, 1[[[ u c'f OL- L- -L Gastric Achlorhydria Gastric Pernicious Normals Resection RQ.sQ.ction Anemia sl AN 5L AL FIG. 1. Comparison of fecal radioactivity after ingestion of scrambled eggs containing 0.56 JJ.g of vitamin B.. (0.4 to 0.5 JJ.c of Co") and 0.56 JJ.g of crystalline radiocyanocobalamin (0.5 JJ.C of Co") in patients with abnormal B12 metabolism and in control subjects. Not all subjects were willing to collect feces. SL indicates low serum B 12, AL low absorp tion, and AN normal absorption of crystalline B...

4 916 DOSCHERHOLMEN AND SWAIM Vol. 64, No c 30 Eggeo S7 512, Cry,tallin. C0 57 5'2.. ".. '.. lii1 1 i!! -.l!ll.,'. '. GastrIC Achlorhydria GastrIC Pornicious Normals Re.section 'Resection Anemia SL AN 5L AL FIG. 2. Comparison of urinary radioactivity after ingestion of scrambled eggs containing 0.56 /lg of vitamin B' 2 (0.4 to 0.5 /lc of Co") and 0.56 /lg of crystalline radiocyanocobalamin (0.5 /lc of Co") in patients with abnormal B' 2 metabolism and control subjects. SL indicates low serum B. 2, AL low absorption, and AN normal absorption of crystalline B. 2 1(, 14 0: 12 '5 E 10 '", ; 8.3 b '" 4 -. z. Egg C057812, Crystalline C0 57 B'2 :""I IjlJLJ.""--'---:-----'[ 1111", Gastric AchlorhydrlO Gastric Pern,cious Normal, Resection Re.se.ction Anemia SL AN SL AL FIG. 3. Comparison of the 8-hr plasma radiocyanocobalamin after ingestion of scrambled eggs containing 0.56 /lg of vitamin B' 2 (0.4 to 0.5 /lc of Co") and 0.56 /lg of crystalline radiocyanocobalamin (0.5 /lc of Co") in patients with bnormal B' 2 metabolism and control subjects. SL indicates low serum B. 2, AL low absorption, and AN normal absorption of crystalline B. 2 gastrectomy patients who developed low serum B12 concentration with low B12 absorption of crystalline radiocyanocobalamin had mean fecal, urinary, and plasma absorption values of 92.2%, 0.14%, and 0.08 pg per ml after ingestion of the C0 57 B 12 containing scrambled eggs and 84.1 %, 2.4%, and 1.4 pg per ml after administration of crystalline radiocyanocobalamin. In pernicious anemia patients, the mean fecal, urinary, and plasma absorption values after ingestion of the egg C0 57B 12 were 91.4%, 0.03%, and 0.02 pg per ml and after crystalline radio-b %, 0.8%, and 0.2 pg per ml. The normal control subjects showed mean fecal, urinary, and plasma absorption values of 72.5%, 3.37%, and 1.34 pg per ml after ingestion of egg C0 57B 12, and 38.8%, 18.1 %, and 7.3 pg per ml after crystalline radiocyanocobalamin. After ingestion of the scrambled eggs, the mean fecal excretion value in the normal subjects was not statistically different from those of the other four groups. However, under the same circumstances, the mean urinary excretion value in the normal control subjects was significantly higher than those of the other four groups (P < 0.001). Similarly, the mean plasma absorption value in the normal control subjects was statistically higher than those in the other four groups (P < 0.01). Discussion Our findings show that patients who had undergone gastric resection and subsequently developed low serum B12 concentration despite normal absorption of crystalline radiocyanocobalamin had impaired assimilation of C0 57B12 incorporated into eggs. The same was also true for subjects with simple gastric hypochlorhydria and achlorhydria. This was most evident from the studies of the urinary excretion and plasma absorption of radioactivity, in which no overlap between these two groups and the normal control subjects was found. The mean urinary excretion values in this postgastrectomy group and the hypo- and achlorhydric subjects were only 9% and 12% of that seen in normal control subjects. Similarly, the mean plasma absorption values in these same groups were 12 and 21% of that observed in the normal control subjects. Overlap did occur in the stool excretion values. The mean fecal radioactivity values in the patients with gastric resection having low serum B12 values and normal absorption of crystalline radi-

5 May 1973 ASSIMILATION OF EGG VITAMIN B ocyanocobalamin (80.9%) and the hypochlorhydric and achlorhydric patients (79.5%) were, however, higher than that in the normal control subjects (72.5%). Because of the small number of subjects in each group, these differences are not statistically significant. The overlap in the fecal excretion test is unexplained. Among the possible causes is a more incomplete stool collection by the subjects in groups 1 and 2 than by the normal control subjects although none of the patients admitted any loss of stool. In contrast to the poor assimilation of egg C0 57B 12 was the normal assimilation of crystalline B12 in patients with gastric resection in group 1 and those with hypochlorhydria and achlorhydria. In fact, both the urinary excretion and plasma absorption values indicated amazingly good absorption, especially in the hypochlorhydric and achlorhydric patients. The fecal excretion tests in these groups (1 and 2) showed stool radioactivity of less than 55%, a figure near the upper limit of the usual value in normal control subjects. 13 Thus, an adequate gastric IF secretion must have been present. In postgastrectomy patients who developed low serum B12 concentration with decreased absorption of crystalline radiocyanocobalamin, and in patients with Addisonian pernicious anemia, the absorption of C0 57B 12 from the scrambled eggs was even more impaired than in the first two groups. The very poor absorption of crystalline radiocyanocobalamin in these same patients reflects their lack of IF secretion. Assuming that the assimilation of nonradioactive vitamin B12 from eggs and other animal food proteins is similar to that of C0 57B 12 in the scrambled eggs, our findings explain the decreased serum B12 values obtained in the postgastrectomy patients and achlorhydric subjects with low serum B12 values and normal absorption of crystalline radiocyanocobalamin. Furthermore, the more severely impaired absorption of egg C0 57B12 in pernicious anemia patients and those with gastric resection who had low serum B12 values as well as low absorption of crystalline radiocyanocobalamin explains their more severely depressed serum B12 concentration, i.e., the serum B12 values seem to correlate well with the values for the assimilation of egg C 0 57B 12. All subjects in groups 1 and 2 could not be expected to show decreased serum B12 values for two reasons: (a) the ingestion of self-administered vitamin tablets containing crystalline vitamin B 12, which these patients can absorb normally, and (b) the differences in the interval between surgery or appearance of hypochlorhydria or achlorhydria and the sampling of serum for vitamin B12 determination. The normal body store of vitamin B 12 lasts for several years, and usually after gastric resection a few years may pass before serum B12 levels fall below normal. 1, 18, 19 This probably is also true in patients who become hypochlorhydric or achlorhydric. Because patients with gastric resection having low serum B12 and normal absorption of crystalline B12 and those with simple gastric achlorhydria and hypochlorhydria behaved similarly, a common denominator for their poor assimilation of food B12 should be suspected. In these subjects the secretion of gastric HCI is decreased or absent, with concomitant loss of peptic activity. In vitro studies suggest that both gastric HCl and pepsin may have a role in releasing the food B 12,20, 21 which is the first step in the assimilation of vitamin B These findings, however, strongly suggest that the gastric HCl and/or pepsin also are important in vivo in maintaining a normal serum B12 level from food sources, although a prior study was inconclusive as to the role of pepsin in the absorption of vitamin B The importance of the gastric IF in the absorption of vitamin B12 has been long recognized. 23 Patients who have had gastric resection and those with gastric hypochlorhydria and achlorhydria have decreased secretion of IF. 24, 25 The quantity of IF needed for the absorption of physiologic doses of crystalline radiocyanocobalamin has been established,26 but not that for food B 12. The possibility

6 918 DOSCHERHOLMEN AND SWAIM Vol. 64,No. 5 should, therefore, also be considered that in these conditions the diminished secretion of IF, although sufficient for the absorption of the small amount of crystalline C0 57B12 administered, may not be adequate for the assimilation of an identical amount of food B 12. Our studies have indicated a relatively poor assimilation of egg C0 57B 12, even in normal subjects. The approximately 10% better absorption in control subjects, reported by Schade and Schilling,22 probably is due to the ingestion of homogenized eggs, a predigestive procedure which we avoided by giving the labeled B12 in cooked scrambled eggs. The relatively poor. assimilation of radiocyanocobalamin from eggs in our normal control subjects contrasts with the excellent absorption of Co6 B 12 incorporated into meat protein observed in normal volunteers by Heyssell et al. 27 This discrepancy suggests that the assimilation of B12 from varying protein sources may differ in normal persons. Impaired assimilation of radioactive iron incorporated into food despite normal absorption of iron salts has been demonstrated in patients who have had gastric resection. 28,29 The assimilation of food vitamin B12 versus crystalline B12 thus resembles what is known about the assimilation of iron in this condition, Although this study shows impaired assimilation of food B12 in patients with hypochlorhydria or achlorhydria and in patients with gastric resection and normal absorption of crystalline B 12, it does not automatically endorse wide-scale vitamin B12 therapy for these patients, It does suggest, however, that a carefully controlled study be made to learn whether subjects with decreased serum B12 levels, as observed in these patients, function below optimal capacity and therefore might benefit from therapy, In a previous study, 30 patients with gastric resection and low serum B12 levels were benefited by vitamin B12 therapy, whereas in a study of elderly subjects 31 who had low serum B12 levels but did not have pernicious anemia, showed no statistical improvement with B12 therapy. The secretion of pepsin and gastric HCl was not reported for the latter group. The reason for the difference in therapy results is not clear; however, the duration of B12 deficiency, or other factors, may have been influential in the latter study. The value of B 12 therapy, in patients with hypochlorohydria or achlorhydria and in patients who have had gastric resection and subsequently develop a low serum B12 concentration despite a normal absorption of crystalline radiocyanocobalamin, must await further investigation. REFERENCES 1. Mahmud K, Ripley D, Doscherholmen A: Vitamin B.. absorption tests. Their unreliability in postgastrectomy states. JAMA 216: , Chang S, Ripley D, Swaim W, et al: Gastric achlorhydria and hypochlorhydria with low serum B.. levels and normal B'2 absorption. Clin Res 19:655, Winawer SJ, Streiff RR, Zamcheck N: Gastric and histological abnormalities in a vegan with nutritional vitamin B.. deficiency: effect of oral vitamin B 12 Gastroenterology 53: , Hines JD: Megaloblastic anemia in an adult vegan. Am J Clin Nutr 19: , Pollycove M, Apt L, Colbert MJ: Pernicious anemia due to dietary deficiency of vitamin B 12 ' N Engl J Med 255: , Harrison RJ, Booth CC, Mollin DL: Vitamin B'2 deficiency due to defective diet. Lancet 1: , Connor PM, Pirola MC: Nutritional vitamin B'2 deficiency. Med J Aust 2: , Doscherholmen A, McMahon J, Ripley D: Impaired absorption of egg vitamin B'2 in postgastrectomy and achlorhydric patients. J Lab Clin Med 78: , Ross GIM: Vitamin B'2 assay in body fluids using Euglena gracilis. J Clin Pat hoi 5: , , Biggs JC, Mason SLA, Spray GH: Vitamin B" activity in red cells. Br J Haematol 10:36-49, Heinle RW, Scharf V, Meacham GC, et al: Studies of excretion (and absorption) of.oco labeled vitamin B" in pernicious anemia. Trans Assoc Am Physicians 65: , Schilling RF: Intrinsic factor studies. II. The effect of gastric juice on the urinary excretion of radioactivity after the oral administration of radioactive vitamin B'2' J Lab Clio Med 42: , 1953

7 May 1973 ASSIMILATION OF EGG VITAMIN B Doscherholmen A: Studies in the Metabolism of Vitamin B'2' Minneapolis, University of Minnesota Press, 1965, p 99, 89, 11, Doscherholmen A: The fundamental basis for the augmented 8-hour plasma absorption test in the diagnosis of pernicious anemia. Proceedings of the Ninth Congress of the European Society of Haematology, 1963, p Waters AH, Mollin DL: Studies on the folic acid activity of human serum. J Clin Pathol 14: , Ramsay WNM: The determination of iron in the blood plasma or serum. Clin Chim Acta 2: , Ramsay WNM: Determination of total ironbinding capacity of serum. Clin Chim Acta 2: , Deller DJ, Witts LJ: Changes in the blood after partial gastrectomy with special reference to vitamin B'2' 1. Serum vitamin B'2, haemoglobin, serum iron and bone marrow. Q J Med 31:71-88, Hines JD, Hoffbrand AV, Mollin DL: The hematologic complications following partial gastrectomy. A study of 292 patients. Am J Med 45: , Cooper BA, Castle WB: Sequential mechanisms in the enhanced absorption of vitamin B'2 by intrinsic factor in the rat. J Clin Invest 39: , Adams JF, Kennedy EA, Thompson J, et al: The effect of acid peptic digestion on free and tissuebound cobalamins. Br J Nutr 22: , Schade SG, Schilling RF: Effect of pepsin on the absorption of food vitamin B'2 and iron. Am J Clin Nutr 20: , Castle WB: Current concept of pernicious anemia. Am J Med 48: , Ardeman S, Chanarin I: Gastric intrinsic factor secretion after partial gastrectomy. Gut 7: , Ardeman S, Chanarin I: Intrinsic factor secretion in gastric atrophy. Gut 7:99-101, Ardeman S, Chanarin I: Assay of gastric intrinsic factor in the diagnosis of Addisonian pernicious anemia. Br J Haematol 11: , Heyssell RM, Bozian RC, Darby WJ, et al: Vitamin B'2 turnover in man. The assimilation of vitamin B'2 from natural foodstuff by man and estimates of minimal daily dietary requirements. Am J Clin Nutr 18: , Chodos RB, Ross JF, Apt L, et al: The absorption of radioiron labeled foods and iron salts in normal and iron-deficient subjects and in idiopathic hemochromatosis. J Clin Invest 36: , Baird 1M, Wilson GM: The pathogenesis of anemia after partial gastrectomy. II. Iron absorption after partial gastrectomy. Q J Med 28:35-41, Williams JA, Baume PE, Meynell MJ: Partial gastrectomy. The value of permanent vitamin B'2 therapy. Lancet 1: , Hughes D, Elwood PC, Shinton NK, et al: Clinical trial of the effect of vitamin B'2 in elderly subjects with low serum B'2 levels. Br Med J 2: , 1970