Studies of the Small-Intestinal Bacterial Flora and of Intestinal Absorption in Pernicious Anemia WILLIAM C. SHERWOOD, M.D.,* FRANZ GOLDSTEIN, M.D., FARID I. HAURANI, M.D., and C. WILI~{ERWIRTS, M.D,

HE INTESTINAL bacterial flora in patients with pernicious anemia has long attracted the interest of investigators, but few recent studies have been reported. Tile present investigation was undertaken to reappraise the significance of the bacterial flora of the small intestine and to correlate bacterial growth with intestinal absorption in patients with pernicious anemia. T h e implication of intestinal bacteria in the pathogenesis of pernicious anemia began with the intestinal-toxin theories of William H u n t e r ill ]888.1 T h e r e followed a long series of investigations of the gastrointestinal flora in pernicious anemia with tmsuccessful attempts to demonstrate excesses of specific types of bacteria in this disease. Davidson,'-' in 1928, studied the bacterial flora of the stomach and colon and found m a r k e d quantitative increases of microorganisms in these areas in patients with pernicious anemia. Ahhough. the u p p e r gastrointestinal flora was frequently colonic in type, no particular organism was consistently present. H e r t e r a found a marked increase of B. welchii in the stool, but Nye a noted the same in patients with adllorhydria withottt anemia. Excessive numbers of Escherichia coli in colonic contents have been described by Moench. ~ Knott a found increased n u m b e r of Streptococcus" longus, and H u r s t ~ found excessive numbers of hemolytic micro,cocci in gastric and duodenal secretions. Many other investigators have noted a proliferation of fecal organisms in the u p p e r gastrointestinal tract of patients with perniciotts anemia. 2. ~. r 9 More recently, Levanto .~

T

From the Department of Medicine, 1)ivisions of Gastroenterology and Hematology, Jefferson Medical College, Philadelphia, Pa. T h e work reported in this paper was supported in part by Research Grant A-2804 (C-B) and bv Gastroenterologic Training Grant 2A-5164 (C-4) from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md. *Now Capt. ( M C ) , U . S. Armv.

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American Journal of Dicjestlve Diseases

ln÷esfinM Studies in Anemia

expressed the opinion that the findings of E. coti in gastric contents is characteristic of the disease. Until 1929, many held the view that intes. tinal organisms were liberating a toxin which passed into the bloodstream and caused anemia by hemolytic or marrow-depressive properties. With the discovery of the "liver principle" by Minot in 1926, and the subsequent description of "intrinsic factor" by Castle, the bacterial-toxin theory was abandoned and pernicious anemia was classified as a deficiency disease. T h e r e a f t e r less attention was directed to the intestinal flora, but in 1949, T e r n b e r g and Eakin 10 found that "erythein" (extrinsic factor) and "apoerythein" (intrinsic factor) combined into a nondialyzable complex, and that the combined form appeared to be unavailable to microorganisms which ordinarily utilized or absorbed u n b o u n d extrinsic factor. In 1952, Burkholder 1I found that many strains of bacteria recovered from the upper gastrointestinal tract of patients with pernicious anemia had a distinct ability to bind vitamin By,. This bacterial-binding ability could be inhibited by prior binding of the vitamin with intrinsic factor. H e suggested that intestinal bacteria were taking up the vitamin not protected by the binding properties of normal gastric juice. T h e importance of this observation was supported by the finding that orally administered broad-spectrmn antibiotics were able to produce varying degrees of hematologic remission in some patients with pernicious anemia in relapse. ~e However, in most patients the remission was not complete, and in some no response at all was observed even with vigorous antibiotic therapy, la Ungtey, la working with isolated washed segments of intestine in dogs, could not demonstrate vitamin BI,, absorption without the use of intrinsic factor, which suggests that intrinsic factor exerts its influence by properties other than mere protection from bacterial binding. M A T E R I A L S AND M E T H O D S Nine patients with pernicious anemia were studied. Of these, 8 were initially studied during a period of hematologic relapse. In 5, follow-up data were available at various intervals during hematologic remission. T h e remaining patient was studied only during complete remission. T h e diagnosis was established by measuring the urinary excretion of Co~0-1abeled vitamin B12 with and without the use of intrinsic factor of known potency. ~4 Patients fasted overnight before the test, in which 0.5 #c. Co6°-labeled Br_, were given orally and simultaneously, an intravenous dose of 1000 ug. nonradioactive vitamin B~., was administered. Urine was collected for 24 hr. T h e radioactivity of the collected urine specimen was measured, and the result was expressed in per cent of the administered dose. In a New Series, Vol. 9, No. 6, 1964

417

Sherwood et ah

series of normal individuals, more than 5e' / O of the administered dose was recovered in urine. I f urinary excretion was less than 5%, the test was repeated in the same m a n n e r but with the addition of 0.5 /,g. intrinsic factor. All patients were shown to have gastric achlorhydria after histam i n e stimulation. Complete gastrointestinal X-ray examinations were p e r f o r m e d in all patients; no significant abnormalities were found. Q u a n t i t a t i v e small-intestinal cultures were performed according to the m e t h o d previously described,a~ with some modifications. A MillerA b b o t t tube soaked in 70% ethanol and rinsed with sterile saline was introduced per os into the stomach. "~Vith fluoroscopic guidance, the tip was passed through the pylorus, the balloon was then inflated, and the tube was allowed to descend the small-intestinal tract. N o attempt was made to seal the distal tip of the tube. T h e proximal opening was clamped. T h e tube was advanced to the ileum, whenever possible, over a period of 3-4 hr. An estimation of the position of the tip was made by fluoroscopy and by measuring the distance it had progressed from the lips. I n t e r m i t t e n t gentle suction to the proximal tube opening was then applied by an electric G o m c o p u m p . Usually a free flow of intest;nal iuice occurred within 5-8 min. T h e first 5 ml. were discarded. Five to 10 ml. of material were then removed, immediately placed in an ice bath, and p r o m p t l y inoculated on culture media. T h e tube was then flushed with 10 ml. of sterile saline, emptied by the injection of air, and the p r o x i m a l end reclamped. T h e tip was withdrawn under fluoroscopic control, and material was obtained in a similar m a n n e r at one or more proximal levels. Cultures from two or three levels were obtained in most patients. Bacterial counting was performed as described previously, a'~ Culture media used for this study were MacConkey's agar, blood azide, and telIurite agar for quantitative aerobic cultures. In addition, a qualitative anaerobic culture was obtained in each instan'ce in thio~lycollate broth. T h e bacterioloaic data were compared with a previously stndied control g r o u p of patients without gastrointestinal disease and with a g r o u p of patients who had postgastrectomy m a l a b s o r p t i o n secondary to afferent-loop stasis, aa T h e vitamin B ~ - b i n d i n g capacity ot the xarious strains of bacteria removed from 5 patients during relapse was determined. Each strain was incubated with increasing concentrations of inert cyanocobalamin to which a tracer dose of CoaT-labeled vitamin g,._, was added (specific activity: I /,c./0.035/,g). A duplicate system, differing only by the addition of intrinsic factor, was tested simultaneously. Incubations were carried out in vitamin B~e assay media, U.S.P. (Difco), with [ H ad.}usted to

4]8

American Journal of Digestive Diseases

In÷esfinal SCudies in Anemia

7.0. After 24 hr. incubation at 37 ° C., the broth was centrifuged at 3000 rpm at 10 ° C. for 30 rain. T h e sediment, containing bacteria, was washed once with distilled water and recentrifuged. All fractions were then counted in a well-type scintillation counter. T h e percentage of the original radioactivity f o u n d in the sediment was interpreted to represent the percentage of inert vitamin BI2 b o u n d by the bacteria. Quantitative estimations of the bacterial content of the sediment and supernatant were made by colony-counting of pour-plates prepared by the serial-dilution method. T h e quantity of vitamin B12 b o u n d per million organisms was then calculated and compared with the a m o u n t b o u n d when intrinsic factor was added to the system. Studies of intestinal absorption were performed on all but one patient, in an attempt to correlate the degree of intestinal bacterial contamination with absorptive function. IW~-triolein-excretion measurements, and quantitative fecal-fat determinations of 3-day stool collections were performed by the method of van de Kamer et al. ~s Serum c'arotene concentration was measured photocolorimetrically with the use of antimony trichloride to produce the Carr-Price reaction, a7 T h e d-xylose urinaryexcretion test was carried out according to the modifications of Christiansen et aIJ s RESULTS In a control group of patients without gastrointestinal disease previously reported from this laboratory? 9 duodenal aspirates revealed bacterial counts ranging from no growth to 4.0 X 10 a colonies/ml., with a mean of 4.0 X 104 colonies/mI. T h e organisms recovered were chieflv gram-positive cocci. These values are in agreement with the results obtained by other workers, e. 0_0-22 Afferent-loop secretions of a group of patients with intestinal malabsorption following partial gastrectom,~, disclosed counts ran~in~ from 4.7 X 10a to 7.5 X I0 s colonies/inf., with a mean of 1.6 X 10s colonies/ml. T h e organisms recovered from this group were often fecal in nature, a'~ In Fig. 1, the bacterioloeic data from the d u o d e n u m and upper ]eiun u m of the group of patients with pernicious anemia are comoared with the other groups. T h e colony counts of patients with pernicious anemia were consistently elevated above the normal range. However, they tended to be lower than those of oatients with postgastrectomy steatorrhea. There was no significant difference in the bacterial counts between patients with pernicious anemia in relapse and those in remission. T h e most abundant and most frequently recovered oreanism was an ~-hemolytic Streptococcus. Gram-negative organisms such as P r o t e u s , A e r o b a c t e r , E. coli, and Pseudomo~Tas species were also present to an New Series, Vol. 9, No. 6, 1964

419

Sherwood e# a/.

excessive degree. Occasionally, Staphylococci and yeast forms were obtained in smaller numbers. T h e only anaerobic organism recovered was an anaerobic Streptococcus noted in one patient. In the 2 patients with the highest colony counts, the entire growth was coliform in type. T h e r e

I0" 0 O

iO" I0' I0"

0 O

IOs

• •

IO" i0 ~,

DOODI 0O

•

Fig. 1. Results of quantitatixe bacterial cultures of upper small-intestinal aspirates from n o r m a l controls, patients with postgastrectomy steatorrhea, and patients with pernicious anemia (P. A.) in relapse and in rcmis-

sion. •

•

I0' 10 0

.....

Normal

P 1. Relapse

P.I. Putpttrtttn He-.issiol Stoaton'ha

were no consistent differences in the quantitative or qualitative wdues obtained from various small intestinal levels in each individual patient. In T a b l e 1 are recorded the results of the intestinal absorption studies. In 2 patients during relapse, there was a mild increase in the stool-fat excretion, which returned to normal during remission. T h e I'a~-triolein and d-xylose excretion tests were normal. T h e serum carotene concentration was slightly depressed in 4 patients during relapse. T w o patients with marked coliform c o n t a m i n a t i o n (in ranges comparable to that noted in postgastrectomy steatorrhea) had normal intestinal absorption. In T a b l e 2 are recorded the largest quantities of bacterial-bound vitam i n By, obtained for the m a j o r organisms recovered. T h e gram-negative flora disclosed a greater avidity for vitamin B,e-binding than did the ,~-hemo!ytic Streptococci. T h e availability of the vitamin for bacterial binding was considerably diminished when the vitamin was previously b o u n d to intrinsic factor. However, the intrinsic factor-vitamin B12 complex could be b o u n d by bacteria, as has been previously shown by Donaldson and co-workers '-'a in studies with bacteria isolated from self-filling 420

American Journal of Digestive Diseases

Intestinal Studies in Anemia TABLE 1, INTESTINAL ABSORPTION DATA FROM P A T I E N T S 1,VITH PERNICIOUS ANEMIA IN RELAPSE AND IN REMISSION Stool-fat* excretion

Patients 1 (J. S.) 2 (B. M.) 3 (J. O.) 4 (M. R.) 5 6 7 8

(L. L,) ('W. F.) (H, G.) (J. M.) *Normal -~Normal +Normal §Normal

Relapse Remission Relapse Remission Relapse Remission Relapse Remission Relapse Retap~" Relapse Remission range: range: range: range:

1.6 2.3 8.4 2,7 6.7 1.2 3.8 1,3 1.3 2.3 4,0 2.8

Triolein excretion+,

1)-xylose excretion~

);erlt.~pt carotene§ concentration

6.2

21,0

0.03

2.7 5.8

6.2

0.09

3.5 3.4

12. l

0.04 0.04

2.6 6.5 32 1.8

13.l 6.4

0.05 0.03

Up to 5 gin./24 hr. Up to 5% of dose. > 6 gin. in 5-hr. urine. > 0.05 mg.%.

TABLE 2, VITAMIN Bv.,-BINDING CAPACITIES OF VARIOUS BACTERIA RECOVERED I:ROM T H E UPPER SMALL INTESTINE OF PATIENTS W I T H PERNICIOUS ANEMLk Vitamin B~a-binding capacity (amg./lO 6 bacteria Bacterial species

tVithout added intrinsic [actor

With added intrinsic [actor

a-Hemolytic Streptococcus a-Hemolytic Streptococcus a-Hemolytic Streptococcus Aerobacter aerogenes Pseudomonas aeruginosa E,scherichia coli* l{scherichia coli*

0,54 0.04 1.52 12.2 26.5 26.6 23.2

0.30 0.0003 0.84 7.8 13.9 4.1 6.8

i n t e s t i n a l d i v e r t i c u l a . N o n e of t h e s p e c i e s r e m o v e d tory requirement for the vitamin.

displayed

an obliga-

DISCUSSION Several workers have noted mild abnormalities of intestinal absorption i n s o m e p a t i e n t s w i t h p e r n i c i o u s a n e m i a d u r i n g r e l a p s e 2 4-'-'7 A r e v e r s i b l e d e f e c t o f v i t a m i n B~._, a b s o r p t i o n i n p e r n i c i o u s a n e m i a w a s p r e v i *Different strains,

New Series, Vol. 9, No. 6, 1964

421

Sherwood efa/.

ously noted by Schloesser and Schilling, 28 as well as by us (unpublished observations). Our present experience in regard to fat absorption has been similar, as evidenced by 2 of 8 patients studied who had minimal steatorrhea while in relapse, with return to normal absorption during remission following treatment with vitaInin B~2. A comparison of the bacteriologic data with the absorption data suggests that there is little, if any, direct relationship between small-intestinal sepsis and impaired intestinal absorption in this group of patients. In patients in whom intestinal sepsis was most nlarked, no absorptive defect was noted. In patients in whom mild steatorrhea was present, the bacterial flora remained unchanged during remission although the absorptive defect was corrected. Although the bacterial concentrations of 2 patients approached those t0und in patients with postgastrectomy steatorrhea, the volumes of intestinal juice were not excessive. It is likely that the total quantities of bacteria present in the small intestine of patients with pernicious anemia were far lower than the quantities of bacteria present in the afferent loops of gastrojejunostomies from which up to 200 ml. of material was recovered. Dysplasia of epithelial cells, in addition to bone-marrow changes, has been noted in vitamin B~2 deficiency. 2o-a* These changes are ordinarily reversible with replacement therapy. Conceivably, the mild intestinal malabsorption occasionally noted in pernicious anemia during relapse may be related to intestinal mucosai changes produced by deficiency of the vitamin. T h e finding of excessive bacterial growth, with similar quantitative values at u p p e r and lower small-intestinal levels, would suggest that microorganisms entering the d u o d e n u m from above are allowed to descend the small intestinal tract and are not affected to any significant extent by its secretions. Dixon ae has made similar observations in studies of bacterial survival rates in the small intestine of lower animals. T h e gastric secretions have been shown to possess no other germicidal activity than those exhibited by aqueous solutions of hydrochloric acid of the same degree of acidityY It appears that the excessive n u m b e r of smallintestinal flora in this group of individuals is the result of both the loss of the gastric-acid barrier and of intestinal stasis, although stasis was not apparent, clinically, in these patients and is difficult to prove. T h e absence of the acid barrier alone does not necessarily lead to intestinal sepsis, as shown by the absence of an abnormal small-intestinal bacterial flora in some patients with achlorhydria studied by Varro e t al. a4 and by our group. 19 Gram-negative bacilli manifested a greater avidity for binding vitamin B~2 than did the gram-positive cocci, but were able to take up amounts 422

Arnerlcan Journal of DigesHve D;seases

Intestinal Studies in Anemia

which are small in terms of the quantities of the vitamin normally present in the diet or given therapeutically. Waife and associates 3a have shown that a daily dose of 300 /,g. vitamin B12 given orally is necessary to maintain hematologic remission in the absence of intrinsic factor. Of this amount, less than 3/o is absorbed. On the basis of the concentrations of bacteria present in the small-intestinal tract in these patients, and of the determined vitamin B12-binding capacity, great volumes of contaminated intestinal juice would be necessary to bind the remainder of the ingested vitamin. For the most avidly binding organism recovered, in the greatest concentrations determined, approximately 130 L. of similarly contaminated intestinal juice wouht be required. Hence, bacterial binding of vitamin BI~ does not appear to be a major factor in tile pathogenesis of the disease. SUMMARY T h e small-intestinal bacterial flora of 9 patients with pernicious anemia was studied by means of quantitative culture technics. Five of these patients were studied during both relapse and remission. Intestinal absorption of fat, Pa~-triolein and d-xylose excretion were measured in the same group of patients, and the resuhs of the absorption studies were (orrelated with the bacteriologic data. T h e vitamin B~,_,-binding capacity of the major organisms recovered was determined quantitatively, and an attempt was made to estimate the significance of intestinal-bacterial binding of vitamin B~ in the pathogenesis of pernicious anemia. T h e small-intestinal bacterial flora in patients with pernicious anemia was found to be quantitatively increased in comparison with the flora of a group of control subjects. However, the elevations in total colony counts tended to be lower than those noted in patients with postgastrectomy malabsorption secondary to afferent-loop stasis. Although the predominatin~ organism was an ~-hemolytic Streptococcus in most patients, a distinct increase in gram-negative flora was also noted. T h e r e were no consistent differences in quantitative bacterial counts or types of bacteria at different levels of the small intestinal tract in each individual patient. Intestinal absorption measurements revealed minimal steatorrhea in 2 patients in relapse; the steatorrhea subsided after vitamin B~_~ therapy. Dysplasia of intestinal-mucosal cells and functional impairment of the intestinal mucosa caused by vitamin B,_~ deficiency were considered as likely causes of the steatorrhea. T h e r e appeared to be no direct relationshil~ between the degree of intestinal contamination in the ranges found and impaired intestinal absorption. T h e isolated bacteria were able to bind vitamin B1,_,. T h e bacterial binding was partially prevented by the addition of intrinsic factor. T h e quantitative resuhs indicate that this New Series, Vo]. 9, No. 6, 1964

423

Sherwood et el. phenomenon B~2 a b s o r p t i o n

does not

contribute

in patients

with

significantly pernicious

to the

failure

of vitamin

anemia. F. G. Jefferson Medical College 1025 lValnut St. Philadelphia, Pa.

REFERENCES 1. HUXT~;R, "W. Pernicious Anaemia. C h a r l e s Griffin. I.ondon. 1900. 2. DAVIDSON, L. S. P. T h e gastrointestinal flola in pernicious a n a e m i a . . 1 , Path. & Bact. 31:557, 1928. 3. Cited by CORNrLL B. S, T h e etiology of pernicious anemia. Medicine 6:375, 1927. 4. MOE>;Clt, L. M., KAHN, M, C., a n d TORRFV, J. C. Analysis of the fecal flora in t h i r D - t h r e e cases of pernicious a n e m i a with particular reference to 11. welchii. J. Infect. Dis. 37:161, 1925, 5. KNOTT, F. A. A d d i s o n ' s a n a e m i a and s u b a c u t e c o m b i n e d degeneration of the cord: T h e role of a c h l o r h w t r i a in intestinal infection. Guy's Hos#. Retd. 77:t. 1927, 6. HLRST, A. F. A d d i s o n ' s (pernicious) a n a e m i a a n d s u b a c u t e c o m b i n e d degeneration of t h e spinal cord. Brit. M . . l . 1:93. 1924. 7. DICK, G. F. T h e bacteriologic e x a m i n a t i o n of the s t o m a c h contents in pernicious a n e m i a , Am..1. Digest, Dis. 8:255, 1941. 8. VFNABL~S, J. F.. a n d KNOTT, F. A. T h e investigation of the d u o d e n a l contents and bile in m a n . Guy's Hosp. Rep. 74:245. 1924. 9. LEVANTO, A. A study of gastric Escherichia strains with special reference to perniciotts a n e m i a . A n n . reed, int. Fenniae 43:1, 1954. I0. T~:RXBr:R(;, J. L., a n d EAKIN, R. E. Ervthein and apoerythein a n d their relation to t h e a n t i p e r n i c i o u s a n e m i a principle. J. A m . Chem. Sot. 71:3858. 1949. 11. BURKHOI.DER, P. R. Microbiological studies on m a t e r i a l s which p(~tentiate oral x i t a m i n B~._, t h e r a p y in A d d i s o n i a n anemia. Arch. Bioehem. 39:322, 1952. ]2. LICHI'MAN, H., GINSBERG, V,, a n d WATSOX. J. T h e r a p e u t i c effect of aureomycin in pernicious a n e m i a . Proe. Soe. Exl)er. Biol. & Med. 74:884, 1950. 13. Ux~;l.~w, C. C. A b s o r p t i o n of ~ i t a m i n B~._, in pernicious anaemia. B~it. M. J. 2:905, 1950. 14. SCHILL1NC, R, F, T h e eft'ect of gastric iuice on the u r i n a r y excretion of radioactivity after t h e oral a d m i n i s t r a t i o n of radioactive v i t a m i n B,._,. J. L(~b, & Clin. ;lied. 42:860, 1953. 15. GOI,DS'IEIN. F., 1,~rlRTS, C. ~',, and KR:k3.IER, ,~i. T h e r e l a t i o n s h i o of afferent l i m b stasis a n d bacterial flora to the p r o d u c t i o n of postgastrectoniy stcatorrhea. Gashoenterology 40:47, 1961. 16. VAN Dr: KAMER, J. H., TEN BOKKEL HUININK, H., a n d x~rEIIERS. H. A. R a p i d nletho(l for d e t e r m i n a t i o n of fat in the feces. J. Biol. Chem. 177:347, 1949. 17. K~MBLE, M. S. T h e photocolorimetric d e t e r m i n a t i o n of ~itamin A an(1 carotene in h u m a n plasma, 1. Lab. & Ctin. Med. 24:1055, 1939. 18. CHRISTIANSEN, P_ KIRSNER, J. B., a n d At.R~ZA, J. D-xvlose a n d its uses in the (liagnosis of m a l a b s o r p t i o n states. A m . . [ . Med. 27:443. 1957, 19. GOLDSTr:IX, F.. WIRTS. C. ~V., a n d toslzeHS, L. T h e bacterial flora of the smalI intestine. Gastroemerolog)' 42:755, 1962. 20. MARTINI, G. A., PHEAR, E, A., Rt:EBXFR, B., a n d S~ERIOCK. S. T h e bacterial count of the small intesthle in n o r m a l a n d cirrhotic snhjects: relation to m e t h i o n i n e toxicity. Clin. So. 16:35, 1957. 21. T n o M s o x , A. E., EINHORN..~,1., and COLICX~.*N. W. Studies on the flora of the iutestinal tract. 31..l. L* Rec. 132:417, 1930. 22. NICHOLS, A. C., a n d GLENN, P. ~[. I n t u b a t i o n studies of the h u m a n small intestine.

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American Journal of Digestive Diseases

Intestinal Studies in Anemia

23.

24.

25.

26. 27.

28. 29.

30. 31. 32. 33. 34.

35.

T h e bacterial flora of the ileum c o m p a r e d with t h a t of the throat a n d s t o m a c h in n o r m a l subjects. ]. Lab. U Cli~. 31ed. 25:388, 1939. I)OX.XL~SON, R. M., CORRI(;AN, H., a n d N.~TSlOS, G. M a l a b s o r p t i o n of ('.n+~+lahelled xitanain B,e in rats with intestinal diverticula. II. Studies on contents of the diverticula. Gastroenterology 43:282, 1962. SfCRM.X, M., H.XKKH.~, R.. M.XkEL~, T . E., a n d H.xrzg.tt..~,. J. Fat a l ) ~ r p t o i n in pernicious anemia. Studies with I ~a~ labelled triolein a n d pat lahelled oleic acid. An~l. reed. i~H, Fe~niae 49:225, 1960. MOLLLX, D. L., BOOXH. C. C,, a n d BAKER, S. J. T h e absorption of x i t a m i n B~e in control subjects, in A d d i s o n i a n pernicious a n a e m i a and in the m a l a b s o r p t i o n syndrome. Brit. J. Haematot. 3:412, 1957. Bat:.xLxlrm P., and HARRL J. P l a s m a v i t a m i n A level a n d the ~itamin A absorption test in achlorhydria. Acta reed. seandinav, 167:287, 1960. HLVIH, C. W., a n d FL'I.Cl~rox, H. W. T h e rate of ahsorption of iodide a n d glycine from the gastrointestinal tract of n o r m a l persons a n d in disease conditions. J. Clb~, Ira,est. 14:475, 1935. SCI"ILOESSER, L. L , a n d SCmLLL',a;, R. Y. V i t a m i n B~ absorption studies in xegetartans with megaloblastic a n e m i a . A m . J. Ctin. Nutrition t2:70, 1963. Bocx, S. T,, Mct.HcvscN, J. A., a n d SI"CCXBERt;r:X, J. N u c l e a r c h a n g e s in oral epithelial cells in s u b a c u t e c o m h i n e d degeneration of the spinal cord d u e to vitam i n B,~ deficiency. Lancet 2:294, 19:58. MAssl-;Y, B. W., a n d Rc'r~Ix, C. E. T h e s t o m a c h in pernicious a n e m i a : a cytologic study. A m . J. iU. So. 227:481, 1954. Bor~Dt.x(;rox, M. M., a n d SeRm¢;S, A. I. T h e epithelial cells in megalohlastic anemias. [. Clim Path. 12:228, 1959. DIXON, J. M. S. T h e fate of bacteria in the small intestine. ]. Path. d° Bact. 79:131, 1960. BARTI.E, H. J.. anti HARKINS. *I. J. T h e gastric secretion: its bactericidal value to m a n . A m . J. M. Se. t69:373, 1925. VARR6,V., SZARVAS.F., CSERttATY, I., a n d BAt.;,zs. V. {'her (lie B e z i e h u n g e n zwischen tier M a g e n s / i u r e a u s s c h e i d t m g u n d der Bakterienflora im oheren Tell tics Verdaumlgstraktes. Gastroen terologia 94:315, 1960. W M v L S. O., JAxsEx. C. J., JR., CRABTREE, R. E., GRINXAN, E. I,., and Fouws. P. J. Oral v i t a m i n B~, w i t h o u t intrinsic factor in t h e t r e a t m e n t of pernicious a n e m i a . A~tn. Int. Med, 5,*:810, 1963,

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425