Reversal of protein-bound vitamin B12 malabsorption with antibiotics in atrophic gastritis

GASTROENTEROLOGY

1991;101:1039-1045

Reversal of Protein-Bound Vitamin B,, Malabsorption With Antibiotics in Atrophic Gastritis PAOLO

M. SUTER,

FRANK

D. MORROW,

BARBARA

B. GOLNER,

and ROBERT

BARRY

R. GOLDIN,

M. RUSSELL

United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University and Tufts University School of Medicine, Boston, Massachusetts

The role of bacteria in the bioavailability of proteinbound vitamin B,, was examined in eight elderly subjects who had atrophic gastritis and in eight normal controls. On separate days and in random order, vitamin B,, absorption tests were performed using either radiolabeled crystalline or proteinbound vitamin B,,. At the same time, bacterial samples were collected from the upper gastrointestinal tract. The tests and gastrointestinal aspirates were performed before and during tetracycline therapy. Crystalline vitamin B,, was absorbed to the same extent in the two study groups. Atrophic gastritis subjects absorbed significantly less proteinbound vitamin B,, than normal controls (mean + SEM, 0.7% + 0.2% vs. 1.9% + 0.5%, respectively). However, protein-bound vitamin B,, absorption in these subjects normalized after antibiotic therapy. These results suggest that the small amounts of vitamin B,, released from the protein binders is readily absorbed (as shown in vitro) and/or metabolized by bacteria.

I

some patients, vitamin B,, (cobalamin) deficiency may be responsible for age-related neurological degenerative changes such as ataxia, sensory loss, and dementia (l-3). Despite low dietary intakes of vitamin B,, by elderly people, dietary causes of vitamin B,, deficiency are uncommon in this age group (4). It is generally believed that pernicious anemia remains the most important cause of vitamin B,, deficiency in humans in all age groups (5). In patients with hypochlorhydria/achlorhydria, absorption of crystalline vitamin B,, is normal, whereas absorption of vitamin B,, bound to food protein is abnormally low (6-15). Moreover, King et al. (15) have shown that protein-bound vitamin B,, malabsorption is often correctable in subjects with achlorhydria by the administration of either acid or pepsin, or a n

combination of them, along with the protein-bound vitamin. These investigators suggested that vitamin B,, deficiency in some individuals can result from an impairment of gastric digestion of food peptides from the vitamin B,, molecule; thus, vitamin B,, would not become available for binding to intrinsic factor. Atrophic gastritis with hypochlorhydria/achlorhydria occurs in a substantial proportion of humans > 50 years In hypochlorhydric patients in whom of age (1640). acid-pepsin digestion is not totally impaired, mechanisms such as bacterial overgrowth could also limit vitamin B,, bioavailability. We have studied vitamin B,, absorption in elderly subjects with low gastric acid output (i.e., hypochlorhydria or achlorhydria) using both crystalline and protein-bound vitamin B,, absorption tests. Further, we specifically tested the hypothesis that bacterial overgrowth is a major mechanism limiting the bioavailability of dietary vitamin B,, absorption in these patients. Materials

and Methods

Subjects Eight subjects with hypochlorhydria and eight normal subjects were studied. We selected all subjects from a pool of healthy, free-living participants in a nutrition research program who resided in the greater Boston area. Several volunteers had been identified as probably being hypochlorhydric on the basis of serum pepsinogen levels (21) obtained while subjects were participating in other research studies (221. However, the confirmed presence of hypochlorhydria was made on the basis of an abnormal

Abbreviations used in this paper: BAO, basal acid output; CFU, colony-forming units; IF, intrinsic factor; MAO, maximal acid output; PAO, peak acid output. o 1991 by tbe American Gastroenterological Association OOM-5085/91/$3.00

1040 SUTER ET AL.

GASTROENTEROLOGY Vol. 101,No.4

pentagastrin stimulation test (Peptavlon; Ayerst Laboratories, New York, NY). We judged hypochlorhydria to be present when the peak acid output (PAO) after pentagastrin stimulation was < 2.0 mmol/h and the maximal acid output (MAO) was < 1.0 mmol/h. The hypochlorhydric group was composed of two men and six women, and the control group was composed of three men and five women. All subjects were more than 60 years old; the mean ? SEM age of the subjects with hypochlorhydria was 71 2 3 years vs. 68 f 1 years for controls (P = NS). All subjects were well nourished by standard anthropometric criteria (23). The subjects were screened to have absent intrinsic factor (IF) antibodies, normal hematologic parameters (red and white blood cell counts, hematocrit, hemoglobin, mean corpuscular volume, and platelet count), normal values for serum albumin, serum folate, and liver serum tests, (i.e., alkaline phosphatase and transferases), and kidney function tests [blood urea nitrogen (BUN) and creatinine]. Subjects with a history of current medication use with a potential vitamin B,, interaction (24,25), excessive alcohol consumption, any form of intestinal surgery, pancreatic disease, or diabetes mellitus (type I or II) were excluded from the study. We obtained written informed consent from all subjects under the guidelines established by the Human Investigation Review Committee of the New England Medical Center and Tufts University, Boston, MA.

Experimental

Protocol

All subjects were admitted to the metabolic research unit of the U.S. Department of Agriculture Human Nutrition Research Center at Tufts University for a 19-day resident stay. Each subject underwent two test periods, one before and one during tetracycline treatment. Each period consisted of two 3-day vitamin B,, absorption tests (i.e., urine was collected for 3 days after each test dose) using crystalline or protein-bound vitamin B,, administered in a mixed design, random order. The two study periods were separated by a 5-day interval during which the subjects received tetracycline treatment (4 x 250 mg Sumycin; Squibb Inc., Princeton, NJ) and then continued to receive it throughout the remainder of the study. Before the first absorption test, a mercury-weighted Dobbhoff tube (Biosearch Medical Products Inc., Somerville, NJ) was passed into the stomach of each subject. After emptying the stomach of residual fluid and discarding it, a gastric sample was obtained for bacterial cultures and in vitro proteolysis studies. A pentagastrin stimulation test was then performed. Basal acid output (BAO), PAO, and MAO were calculated as described by Powell and Drossman (26). Samples of gastric juice were collected again at the end of the second test period (i.e., a morning sample 12 hours after the final tetracycline dose) for repeat bacterial counts. In five of the atrophic gastritis subjects, intestinal intubation was performed to obtain cultures from intestinal fluid for in vitro vitamin B,, binding and proteolysis studies. A mercury-weighted Entriflex tube (Biosearch Medical Products Inc., Somerville, NJ) was placed at the ligament of Treitz. Placement was confirmed by bile return, followed by fluoroscopy. A 100 g fat per day diet was maintained throughout the

study and, in all but one subject, 72-hour stools were collected twice for fecal fat measurement (27). Vitamin B,, absorption tests were performed according to the guidelines of the International Committee for Standardization in Hematology (28) before and during tetracycline treatment. At the same time, each subject received an oral dose of 1 kg of vitamin B,, with 0.57-0.60 t.Ki of “Covitamin B,, (Amersham Searle, Arlington Heights, IL). The vitamin B,, was either in the crystalline form dissolved in 5 mL of distilled water or bound to chicken serum protein (see preparation of oral doses). To avoid potential intraluminal tetracycline-vitamin B,, interactions, the antibiotic was not given for I2 hours before ingestion of the test dose of vitamin B,,. On each of the 2 days before the first oral test doses of periods 1 and 2, a loading dose of 1000 p,g vitamin B,, was given IM. In addition, flushing doses of the same magnitude were given on each day of the 3-day urinary collection periods. Absorption was expressed as the percentage of the test dose excreted over the 72 hours after administration.

Preparation

of the Oral Doses

Crystalline vitamin B,, was given in an aqueous solution (see above). The protein-bound vitamin B,, was prepared as previously described (15). Briefly, 0.57-0.60 t.&i of “Co-vitamin B,, and 1 mL of unlabeled vitamin B,, (1 t.@ mL aqueous solution) were added to 5 mL of sterile chicken serum (Gibco Laboratories, Grand Island, NY) and mixed by vortexing at intervals for 10 minutes. The results of charcoal binding experiments (28) showed near complete binding ( > 99%) of the crystalline vitamin B,, to the chicken serum. The same procedure was performed with the crystalline preparations to exclude any unexpected binding (no binding was found). Ninety-nine percent of the radioactivity was recovered in the resuspended charcoal. To study potential interactions between tetracycline and crystalline vitamin B,, and between tetracycline and vitamin B,, bound to chicken serum, in vitro experiments were performed using the same procedure as that described in the preparation of oral doses, except that 250 mg of tetracycline was also added. Further, to determine if tetracycline frees vitamin B,, from R factor or IF (Sigma, St. Louis, MO), an aqueous solution of either R factor or IF was substituted for chicken serum as the source of protein. No interactions could be detected, i.e., no freeing of vitamin B,, from either the chicken serum protein or from the IF and/or R factor.

Microbiology The number of colony-forming units (CFU) per milliliter in the gastric aspirate was obtained by serial dilution through the 10th tube (- lOI"). A 0.1~mL aliquot of each dilution was plated on blood agar plates and incubated in an anaerobic chamber or in a CO, jar (for determination of facultative anaerobes) at 37°Cfor 48 hours.

In Vitro Binding Studies To determine the binding capacity of the bacteria for vitamin B,,, aliquots of intestinal aspirate were transferred

VITAMIN B,, ABSORPTION IN ATROPHIC GASTRITIS 1041

October 1991

to a

brain heart infusion media, pH 7 (Difco Laboratories, Detroit, MI) and cultured anaerobically for 24 hours. After several washings,

the bacterial

pellet was suspended

in 5

mL phosphate-buffered saline (PBS) (pH 71, and 1 mL of the dispersion was added to 2 mL M-9 solution (pH 7) with 0.5 mL of either PBS or sterile chicken serum (30). Five microliters of 57Co-vitamin B,, from a stock solution of IO.5 pCi/mL was added to all tubes; then after a 6-hour incubation under anaerobic conditions at 37°C the washed pellets and supernatant fractions [including washings) were counted in a gamma counter. The percentage of binding was then calculated. These binding studies were repeated at various pHs between 3.0 and 7.0.

In Vitro Proteolysis Studies To determine whether gut flora can effectively inhibit the release of labeled vitamin B,, from protein binders during intraluminal digestion, a series of in vitro incubations were performed using both small intestinal and gastric aspirates. Fresh aspirates from patients with atrophic gastritis were diluted 1:6 with sterile saline, and the pH was adjusted to 3.0 (gastric) or pH 6.0 [small intestinal). Onehalf of each preparation was then centrifuged at 5°C (20 minutes at 2500 x g) to sediment the bacteria. The supernatant fraction from the centrifuged preparations was regarded as bacteria free, whereas the uncentrifuged aspirates were regarded as bacteria laden. To each preparation, chicken serum-bound radiolabeled vitamin B,, was added to reach a final activity of 0.2 &i/mL. To allow for in vitro proteolysis by gastric and intestinal enzymes, each preparation [either with or without bacteria) was then incubated at 37°C for 3 hours. At the completion of the incubation, each aspirate was briefly centrifuged to remove particulate matter, and 0.5 mL of the supernatant fraction was applied to columns (0.2 x 10 cm) packed with BioGel P-30 (Bio-Rad Laboratories, Richmond, CA) previously equilibrated with Tris-HCl buffer (5 mmol/L, pH 7.4). Twenty fractions of 0.3 mL were collected and counted in a gamma counter. The same experiment was also performed on gastric and intestinal aspirates in which crystalline vitamin B,, label was substituted for the chicken serum-bound vitamin B,, label. Retention volumes for protein-bound and monomeric vitamin B,, label were established in separate chromatographic runs using chicken serum-bound label or crystalline vitamin B,,, respectively, in the absence of gastrointestinal aspirates.

Statistics Statistical comparisons of the data were performed using Student’s t test for paired data. Logged data were used for bacterial concentrations. A P value < 0.05 was considered significant.

Results

Table 1 shows the characteristics of the two study groups regarding the gastric secretory tests (BAO, PAO, and MAO), pepsinogen I and II levels,

Table 1. Gastric Acid Outputs (Basal and With Pentagastrin Stimulation) of Serum Pepsinogens I and II, Pepsinogen I/II Ratios, and Serum Vitamin B,, Values in Normal and Atrophic Gastritis Subjects Atrophic gastritis n=8

P

12.5 k 1.8

0.3 2 0.2

0.00003

9.2 2 1.5 0.5 2 0.1

0.03 + 0.02

0.0001

Normal n=8

PA0 (mEq HClIh) MAO (mEq HCl/h) BAO (mEq HCllh) PG I (ng/mL) PG II (ng/mL) PG I/II ratio Vitamin B,, @g/mL)

1412 22 27 It 7 7.9 rt 1.3

479 k 00

0 42 + 17 2 2.9 f 367 +

6 3 0.5 98

0.001 0.001 NS 0.003

NS

NOTE. Data are expressed as mean t SEM. PG, pepsinogen.

pepsinogen I/II ratios, and serum vitamin B,, levels. Significant differences were found for PAO, MAO, BAO, pepsinogens I and II, and the pepsinogen I/II ratio. Also the mean + SEM intragastric baseline pH was significantly higher in the atrophic gastritis group than in the normal controls (7.2 ? 0.2 vs. 2.3 + 0.7, respectively; P < 0.0001). Serum vitamin B,, levels and pepsinogen II levels were lower in the atrophic gastritis group than in the controls, but the differences were not statistically significant. Two subjects in the atrophic gastritis group had serum vitamin B,, levels below the normal range (i.e., < 150 pmol/L). The hematocrit values of these two subjects were 44% and 42% whereas their mean corpuscular volumes were 85 fL and 91 fL. None of the subjects had a low serum folate level. The mean f: SEM serum folate level was 9.7 2 0.6 ng/mL in the group with atrophic gastritis and 9.9 + 1.1 in the normal controls (P = NS). There was no statistical difference in fecal fat excretion levels (mean 2 SEM) between the two groups (atrophic gastritis group, 4.6 + 0.8 g/day; controls, 3.1 2 0.3 g/day; P = NS). Further, tetracycline did not change the fecal fat levels in either the atrophic gastritis group or in the controls. Figures 1 and 2 illustrate the results of the vitamin B,, absorption tests (crystalline and protein-bound) without and with antibiotic treatment. Results are expressed as 72-hour urinary radioactivity after each test dose, although 24- and 48-hour values showed the same result with the exception that the urinary excretion of vitamin B,, after the crystalline dose did not change significantly as a result of antibiotic treatment (see below). Comparing the atrophic gastritis group with the control group, the only significant difference among the vitamin B,, absorption tests was for protein-bound vitamin B,, before antibiotics (0.7% f 0.2% vs. 1.9% + 0.5%, P < 0.039). In all but one case, the protein-bound vitamin B,, absorption test result was < 1.0%. However, this malabsorption

1042 SUTER ET AL

GASTROENTEROLOGY

Normal

with antibiotic

without antibiotic

without antibiotic

with antiblotlc

p=NS

p
Figure 1. Vitamin B,, recovered in urine over a 7%hour period in eight normal control subjects and in eight subjects with atrophic gastritis after a crystalline vitamin B,, dose before and after tetracycline treatment. The mean is depicted by the horizontal bar.

of the protein-bound vitamin B,, was reversed in seven of the eight subjects with atrophic gastritis by the administration of tetracycline (Figure 2). That is, the absorption of the protein-bound vitamin B,, in the atrophic gastritis group increased from 0.7% + 0.2% before antibiotic treatment to 2.1% + 0.3% after the treatment (P < O.O04), no longer being significantly

Atrophic gastritis

Vol. 101, No. 4

different from the normal control group. In the subjects with atrophic gastritis, the amount of the radiolabe1 recovered in the urine after the crystalline vitamin B,, dose without the antibiotic treatment did not differ from that recovered after the antibiotic treatment (mean + SEM, 19.8% r 1.6% vs. 17.0% + 1.7%; P = NS; Figure 1). Mean f SEM crystalline vitamin B,, recovered in the urine of the control group without antibiotic treatment was 20.9% + 2.3% but decreased significantly to 15.8% + 1.9% after the antibiotic treatment (P < 0.04). The absorption of proteinbound vitamin B,, did not change in normal subjects before or after antibiotic treatment (mean f SEM, 1.9% + 0.5% and 1.6% + 0.3%, respectively; P = NS). In the control group, no bacterial growth from gastric aspirates could be detected. However, in the atrophic gastritis subjects there was bacterial overgrowth in the stomach; the organisms were predominantly facultative anaerobes, as evidenced by the close agreement in numbers between the facultative and the total anaerobic count. This is normally the case for gastric flora, because the predominant organisms are lactobacilli and Escherichia coli, which are both facultative anaerobes that grow either in CO, or in an anaerobic chamber. The mean + SEM count (expressed as CFU per milliliter) of the anaerobic bacteria equals 1.2 x lo7 +- 0.5 x 10’ and that of facultative anaerobic bacteria, 1.1 x lo7 + 0.6 x 107. Antibiotic treatment significantly reduced bacterial counts for facultative anaerobes. The data are summarized in Table 2. Because of lack of significant amounts of bacteria in the normal control group, no vitamin B,, binding could be shown. Although binding of crystalline vitamin B,, to bacteria could be detected over a wide range of concentrations (i’%-92%) in all of the evaluated atrophic gastritis subjects (n = 5), there was no binding to bacteria of protein-bound vitamin B,,. Changes in pH in the range 3.0-7.0 had no effect on binding.

Table 2. Bacterial Concentration (CFlJJmL) in Gastric Aspirates in Seven Subjects With Atrophic Gastritis Before antibiotic Subjects

without antiblotic

with antibiotic

p=NS

01

‘

without antibiotic

with antibiotic

p 4 0.004

Figure 2. Vitamin B,, recovered in urine over a Z&hour period in eight normal control subjects and in eight subjects with atrophic gastritis after a protein-bound vitamin B,, dose before and after tetracycline treatment. The mean is depicted by the horizontal bar.

1 2 3 4 5 6 7 Mean (2 SEM)

After antibiotic

Anaerobes

Facultative anaerobes

anaerobes

Facultative anaerobes

3.7 x 2.9 x 1.0 x 8.7 X 8.1 x 1.2 x 2.5 X 1.2 x (0.5 x

4.1 x 10’ 1.6 x 10' 8.6 x lo5 4.0 x lo5 7.4 x lo3 1.0 x 10' ND” 1.1 x lo7 (0.6 x 107)

7.0 x 1.2 x 3.1 x 0 0 0 1.0 x 5.5 x (4.4 x

5.0 x 2.4 X 2.0 x 0 0 0 2.0 x 1.0 x (0.7 x

“Not determined.

10’ 10’ lo6 lo5 lo3 lo7 10" 10' 10')

lo5 lo4 lo6

lo3 lo5 105)

10h 10' lo6

lo2 lo6 10')

October

1991

The in vitro proteolysis studies comparing bacteriafree vs. bacteria-laden gastrointestinal aspirates showed no difference between the two preparations with regard to the release of monomeric (i.e., free) vitamin B,, from the protein-bound cobalamin. Overall, < 2% of the label was recovered in the free vitamin B,, region of the chromatographic profile regardless of the bacterial content of the incubated preparation. Moreover, no differences were observed in the elution profiles when crystalline vitamin B,, was substituted for chicken serum-bound vitamin B,, label.

Discussion There is no demonstrable age-related decline in free vitamin B,, absorption in normal, healthy subjects (31-36). However, the age-related decline in blood vitamin B,, levels observed in some populations may be caused by malabsorption of food-bound vitamin B,, caused by atrophic gastritis (6-8,37-39). The prevalence of atrophic gastritis increases with age, and the condition exists in up to 50% of elderly people 2 60 years of age (16-20). In our study, as in other investigations (6-9,15), crystalline vitamin B,, absorption was not affected by atrophic gastritis, although protein-bound vitamin B,, was malabsorbed. Thus, lack of IF does not play a role in causing malabsorption of food-bound vitamin B,, in most patients with atrophic gastritis, because in milder forms of atrophic gastritis IF is secreted in excess over the minimal amount needed to supply the body with the daily vitamin B,, requirement (40). In our study, antibiotics completely reversed malabsorption of protein-bound vitamin B,, in subjects with atrophic gastritis. In view of this finding and the absence of any evidence for an interaction between tetracycline and vitamin B,,, it is clear that overgrowth of bacteria in the stomach and/or the small intestine (as a result of reduced gastric acid production) is an important cause of the malabsorption of protein-bound vitamin B,, observed in patients with hypochlorhydria. Several mechanisms could explain the specific malabsorption of protein-bound vitamin B,, caused by bacterial overgrowth in atrophic gastritis. First, bacteria could specifically bind protein-bound vitamin B,,, thereby effectively making the vitamin unavailable. However, using bacteria derived from intestinal intubations in five of our atrophic gastritis subjects, we were unable to show any binding of protein-bound vitamin B,, in vitro. Conversely, free (i.e., crystalline) vitamin B,, was avidly bound by bacteria derived from the intestinal lumen of atrophic gastritis subjects. These data suggest that the defect in the absorption of food-bound vitamin B,, may occur subsequently to acid-pepsin digestion. Our in vitro proteolysis studies did not shed light on the question

VITAMIN B,, ABSORPTION

IN ATROPHIC GASTRITIS

1043

whether bacteria can inhibit proteolysis of the foodbound vitamin B,,, because very little cobalamin was released into a monomeric fraction under the conditions used in the experiment. Nevertheless, previous studies have shown impaired acid-pepsin digestion in atrophic gastritis subjects (6-15,41), suggesting that the pool of free absorbable vitamin B,, may be diminished. Thus, binding of free vitamin B,, by luminal bacteria may be of particular importance in atrophic gastritis. The oral administration of crystalline vitamin B,,, on the other hand, presumably overwhelms the total binding capacity of the bacteria, thereby allowing for essentially normal amounts of vitamin B,, uptake by the mucosal cell. Another mechanism that might explain the diminished absorption of labeled vitamin B,, from protein binders is the dilution of the administered label caused by production of vitamin B,, by intestinal microorganisms. This hypothesis is untenable in light of the chronically lower serum vitamin B,, levels observed in atrophic gastritis subjects than in normal subjects (16). Finally, it is possible that analogues of vitamin B,, (i.e., cobamides) are produced in excess in atrophic gastritis patients as a result of the large number of bacteria present in the proximal gastrointestinal tract. Attachment of the analogue to the ileal receptor could block the uptake of the vitamin B,,-IF complex, thus further impairing vitamin B,, absorption (3,41-45). In our study, analogue levels were not measured; thus, we were unable to evaluate this possible mechanism. King et al. (15) concluded that the normalization of the protein-bound vitamin B,, malabsorption in atrophic gastritis with acid was a result of hydrolysis of the protein-vitamin B,, bond caused by improved acid-pepsin digestion. Moreover, Corral and Carmel have recently reported impaired transfer of (egg yolk) protein-bound cobalamin to R binders in vitro in the absence of acid and pepsin (47). Nevertheless, our results clearly show that with a reduction in the bacterial counts of the upper gastrointestinal tract, an improvement of protein-bound vitamin B,, absorption is achieved (without affecting protein digestion). There are two possible explanations for these apparently disparate findings. First, by giving acid, the vitamin B,, binding ability of the bacteria may be altered. However, our in vitro data show that the magnitude of binding of crystalline vitamin B,, by intestinal bacteria is not variable over a wide pH range (pH 3.0-7.0). Another possibility is that by giving acid, gastric and proximal small intestinal bacteria are killed; thus, more of the freed vitamin B,, (i.e., freed from the chicken serum binders) is available for intestinal absorption. In our normal elderly subjects, crystalline vitamin B,, absorption decreased significantly before vs. after and during antibiotic treatment (P < 0.04), although

1044

SUTER ET AL.

it remained within the normal range. There is no clear explanation for this decrease in the absorption of crystalline vitamin B,, as a result of tetracycline therapy. In vitro, we excluded any significant direct drug nutrient interaction. However, interaction in later stages of the absorptive process (e.g., by altering the pH of the terminal ileum) could not be ruled out. Other investigators have reported no significant effect of tetracycline on crystalline vitamin B,, absorption (48). Our study shows that the malabsorption of proteinbound vitamin B,, in atrophic gastritis can be reversed by antibiotic treatment. We hypothesize that the malabsorption of protein-bound vitamin B,, observed in atrophic gastritis arises from bacterial overgrowth of the stomach and small intestine secondary to hypochlorhydria and achlorhydria. In our studies, vitamin B,, bound to chicken serum was used as the model for testing bioavailability of the protein-bound vitamin. The bioavailability of vitamin B,, bound to other proteins will need to be investigated to see how generalizable our results are. Nevertheless, in view of the high.prevalence of atrophic gastritis in the elderly, the role of vitamin B,, in the etiology of neurological and cognitive deficits should be thoroughly understood.

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27. Van de Kamer JH, Huinink HTB, Weyers HA. Rapid method for the determination of fat in feces. J Biol Chem 1949;177:347352. 28. International Committee for Standardization in Hematology. Recommended methods for the measurement of vitamin B,, absorption. J Nucl Med 1981;22:1091-1093. 29. Gottlieb C, Lau K-S, Wasserman LR, Herbert V. Rapid charcoal assay for intrinsic factor (IF], gastric juice unsaturated B,, binding capacity, antibody to IF, and serum unsaturated B,, binding capacity. Blood 1965;6:875-883. 30. Adams MH. Bacteriophages. New York: Interscience, 1964: 446. 31. Swendseid ME, Gasster M, Halsted JA. Absorption of vitamin B,, in individuals with a functioning gastric mucosa (abstr). Fed Proc 1954;13:308. 32. Chow BF, Gilbert JP, Okuda K, Rosenblum C. The urinary excretion test for absorption of vitamin B,,. I. Reproducibility

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VITAMIN

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45.

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Received September 26,199O. Accepted March 4, 1991. Address requests for reprints to: Robert M. Russell, M.D., United States Department of Agriculture Human Nutrition Research Center on Aging, Tufts University School of Medicine, 711 Washington Street, Boston, Massachusetts 02111. This project has been supported with Federal funds from the U.S. Department of Agriculture, Agricultural Research Service, under contract no. 53-3K06-5-10. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, and mention of trade names, commercial products, or organizations does not imply endorsement by the US. Government. Part of this research was reported in abstract form (Gastroenterology 1987;92:1606). Dr. Suter’s present affiliation is: Institute of Physiology, University of Lausanne, 1011 Lausanne, Switzerland. The authors thank Dr. I. Michael Samloff for performing the pepsinogen assays; Dr. James Sadowski for his assistance with methods development; the recruitment department of the Human Nutrition Research Center (HNRC); the staffs of the nursing and dietary departments of the HNRC; the HNRC Nutrition Evaluation Laboratory staff for providing biochemical data on the subjects: Dr. Gerard Dallal for providing statistical support; and Pamela Tero for her expertise in the preparation of the manuscript.