Clinically Significant Vitamin B12 Deficiency Secondary to Malabsorption of ProteinBound Vitamin B12 CHARLES E. KING, MD, JOHN LEIBACH, MD, and PHILLIP P. TOSKES, MD
Protein- (chicken serum) bound [~rCo]cyanocobalamin absorption was evaluated in five hypochlorhydric patients who had developed B 12deficiency despite having normal absorption of unbound crystalline vitamin B 12. All five patients had decreased urinary excretion of protein-bound B 12 (0.06-0.34%) as compared to twelve normal controls (0.61-5.6%), P < .001. Improvement in protein-bound B12 absorption in four of the five patients occurred with the exogenous administration of hydrochloric acid, pepsin, gastric intrinsic factor, or a combination thereof. Vitamin B12 deficiency developing in the setting of hypOchlorhydria may result from deficiency of acid-peptic digestion of B 12bound to protein and~or a relative deficiency of intrinsic factor. This digestive defect is not detected with tests which measure the absorption of unbound crystalline B 12but is detected by a simple test which employs B12 bound to chicken serum as the form of protein-bound Bx2. The absorption of vitamin B12 is usually thought of in terms of events occuixing in the stomach, in the lumen of the small intestine, and at the ileal receptor for the vitamin B12-gastric intrinsic factor complex. As analyzed by the usual tests of absorption of unbound crystalline vitamin B12, the primary abnormalities felt to participate in the malabsorption of vitamin B12 have included deficiency of gastric intrinsic factor; pancreatic insufficiency; bacterial or parasitic infestation of the small intestine; and damage, resection, or congenital abnormalities of the specific ileal receptor for the intrinsic factor-vitamin B12 complex (1). From the Veterans Administration Hospital and Department of Medicine, University of Florida College of Medicine, Gainesville, Florida. Dr. King is the recipient of a Research Associate Award from the Veterans Administration. During the performance of these studies Dr. Toskes was a Clinical Investigator with the Veterans Administration. Supported in part by Grant RR-82 from the National Institutes of Health. Presented in part at the Annual Meeting of the American Gastroenterology Association in Toronto, Canada, May 1977. Address for reprint requests: Dr. P.P. Toskes at the Gastroenterology Section, Veterans Administration Hospital, Gainesville, Florida 32602.
The assimilation of vitamin B12 from foods requires an additional step: that of freeing vitamin B12 from the food. Despite the predominant proteinbound state of dietary vitamin B12, it has been generally accepted that vitamin Blz deficiency does not occur as a result of maldigestion of protein-bound vitamin B12 (2). Vitamin B12 deficiency following partial gastrectomy has been thought to be due primarily to intrinsic factor deficiency and secondarily to small intestinal infestation (3, 4). Although Deller's study (3) is often quoted to support the importance of intrinsic factor deficiency in patients who have had partial gastric resections, the finding that 13 of 26 patients in that study with subnormal levels of vitamin Bi2 in the serum absorbed vitamin B12 normally is not well appreciated. The fact that these 13 vitamin Ba2 -deficient subjects had normal absorption of unbound crystalline vitamin B12 suggests that they may have become deficient in the vitamin because of a failure to absorb proteinbound vitamin B12. The suspected lack of acid and pepsin secretion in these patients who had gastric surgery may have led to the malabsorption of protein-bound vitamin B12 due to an inability to cleave the vitamin B~2 from
Digestive Diseases and Sciences, Vol. 24, No. 5 (May 1979)
0163-2116/79t0500-0397503.00/19 1979DigestiveDiseaseSystems, Inc.
397
KING ET AL TABLE 1. PATIENT DATA S e r u m B 12
Stage I crystalline B 12
(normal >200 pg/ml)
absorption (normal>8%)
Patient
Age (years)
Sex
1 2
68 67
M M
75 118
25 21
3 4 5
59 66 51
M F M
107 159 177
14.4 19 20
Gastric
acid output (mEq/hr) BAO/MAO 0/1.3 0/0
0/0 2.0/14.1" 0/0
Neurological symp!oms
Dementia, ataxia
Memory loss, ataxia, dysesthesias Dysesthesias Dysesthesias
Hct (%)
30 45 35 36 43
*Alkaline, bile-stained residual gastric fluid prior to collection of basal specimen.
its protein bond. M a h m u d et al (5) d e m o n s t r a t e d malabsorption of unbound crystalline vitamin B lz in only '/3 of vitamin B l~-deficient p o s t g a s t r e c t o m y patients, with the other 2/3 (of 62 patients) having normal unbound crystalline B~2 absorption. Further support for the i m p o r t a n c e of protein-bound vitamin B12 m a l a b s o r p t i o n in the d e v e l o p m e n t of vitamin B12 deficiency was seen in the recent report of Rygvold, w h o noted low serum vitamin BI2 levels in 67 of 351 patients who had undergone a Billroth II partial g a s t r e c t o m y (6). In those patients with low s e r u m B12 levels, 31% were noted to have normal a b s o r p t i o n o f u n b o u n d c r y s t a l l i n e B~2 ( s t a g e I Schilling test) implying malabsorption of vitamin Bar from food as the etiology of the vitamin B12 deficiency. Only 33% had Schilling test results supporting intrinsic factor deficiency as the cause for the low serum vitamin Ba2 levels. The present study evaluates the absorption of protein-bound vitamin B~2 by 5 patients who were referred to us for evaluation of vitamin B,2 deficiency developing in the face of normal absorption of unbound crystalline vitamin B~2. Vitamin B,2 bound to chicken serum (7) was utilized as the test of protein-bound vitamin B12 absorption.
MATERIALS A N D M E T H O D S All studies were carried out under carefully controlled conditions in a clinical research center ward. Informed consent was granted by all subjects, and all investigations were approved by the University of Florida Health Center Committee for the Protection of Human Subjects. Serum vitamin B12 levels were determined by the Euglena gracilis assay (8). Absorption of unbound crystalline vitamin BI~ was determined by the urinary excretion test of Schilling (9). Oral administration of pTCo]vitamin B12 was followed by a flushing parenteral dose of 1000/zg of unlabeled cyanocobalamin and 24-hr collection of
398
urine. Urinary excretion of [57Co]vitamin B,2 was calculated, following determination of 57Co content of an aliquot of well-mixed urine in a standard well-type gamma counter. Absorption of unbound crystalline vitamin B12 was determined with oral administration of 1 p.g (0.6/zCi) [57Co]cyanocobalamin (Rubratope, E.R. Squibb and Sons, Inc., Princeton, New Jersey). Protein-bound vitamin B,2 doses were made by mixing 4 ml of sterilized, pathogen-free chicken serum (Grand Island Biological Co., Grand Island, New York) with approximately 0.5 ~Ci and 1 /zg of [~TCo]cyanocobalamin (Amersham, England). Dialysis for 72 hr was followed by checking for adequacy of removal of free B,2 with a modification of the charcoal assay of Gottlieb et al (10). Administration of gastric intrinsic factor with protein-bound vitamin B,2 was performed by mixing the contents of one capsule of intrinsic factor (IF) concentrate (1 NF XI unit per capsule, E.R. Squibb and Sons, Inc., Princeton, New Jersey) with the protein-B,2 solution. Acid and/or pepsin was administered via oragastric tube (100 ml of 0.1 N hydrochloric acid and/or 500 mg of 2 • crystallized hog gastric pepsin) (Sigma Chemical Co., St. Louis, Missouri), followed by extubation and immediate oral administration of the protein-vitamin B,2 solution. Gastric acid output was determined following nasogastric intubation, with fluroscopic guidance of the tube into the antrum. Following aspiration of residual contents, four 15-min basal aspirations were followed by six 15-min aspirations after the subcutaneous administration of 6/zg/ kg body weight pentagastrin (Peptavlon, Ayerst, New York, New York) Aliquots of strained gastric aspirate were titrated to pH 7 with 0.1 N sodium hydroxide. Basal acid output (BAO) was computed from the total titratable acidity for the hour basal collection. Maximal acid output (MAO) is expressed as the sum of the maximal four (of six) consecutive 15-min acid outputs in the poststimulation period. Patients. Data on the five patients who presented with vitamin B12 deficiency and normal absorption of unbound crystalline vitamin B,~ are shown in Table 1. Patients 1 and 2 were admitted with central and peripheral neuropathic symptoms, patient 1 manifesting severe dementia prompting admission to the Neurology Service. Following the discovery of severely depressed serum vitamin B,2 levels in each, parenteral vitamin B12 therapy was begun, Digestive Diseases and Sciences, Vol. 24, No. 5 (May 1979)
PROTEIN-BOUND VITAMIN Ba2 MALABSORPTION RESULTS
-
NORMAL SUBJECTS
B12
_
VITAMIN DEFICIENT SUBJECTS WITH NORMAL CRYSTALLINE VITAMIN
B12 ABSORPTION -
LU
o
|
o.
I, uJ D_
0
CONTROLS
4-
PATIENTS
Fig 1. Absorption of protein-bound vitamin B12 by twelve normal controls and five patients with vitamin B12 deficiency and normal absorption of unbound crystalline vitamin B~2.
leading to full remission of neurologic signs and symptoms. Patient 1 had, in addition, anemia of combined etiology, including vitamin B12 deficiency and chronic disease. Patient 3 had the confusing picture of vitamin B12 deficiency, Graves' disease with circulating antiintrinsic factor antibody, and achlorhydria, all felt to represent classical pernicious anemia until unbound crystalline vitamin BI~ absorption tests were repeatedly normal at > 14%. Serum folate was elevated at 22 ng/ml and decreased to 6 ng/ml during parenteral vitamin BI~ therapy, as is often noted in 9 vitamin B12 deficiency. Anemia was felt to be due to iron deficiency and possible vitamin B12 deficiency, responding to concurrent therapy with iron and vitamin B12. Patients 4 and 5 demonstrated primarily mild peripheral neuropathic symptoms, with associated anemia of chronic disease and alkaline gastric reflux in patient 4 and hypochlorhydria secondary to a Billroth II patrial gastrectomy in patient 5. None of the patients had diabetes, alcohol abuse, or folate deficiency as etiologic factors for their neuropathic symptoms and/or anemia, and all had normal dietary Ba~ intake by history. Four of the five patients demonstrated achlorhydria or severe hypochlorhydria on gastric analysis, and one Patient had alkaline, bile-strained basal gastric secretions at the time of two of three gastric intubations. Patient 5 was the only patient with previous gastric surgery. Data Evaluation. Groups were compared using Student's t test and the Wilcoxon-Mann-Whitney rank sum test (11). All data are expressed as mean --_ SEM. Digestive Diseases and Sciences, Vol. 24, No. 5 (May 1979)
Figure 1 shows the results of the urinary excretion test following the oral administration of chicken serum-[~TCo]Bi2 to twelve normal controls and the five patients. Controls excreted 2.32 _+ 0.42% (mean -+ SEM) of the dose over 24 hr, with a range of 0.61-5.6%. The five patients excreted from 0.06 to 0.34% of the dose, P < 0.01 in comparison to the controls. Figure 2 compares the range of [SrCo]B12 excretion in controls to that of the five patients when the patients had the protein-bound vitamin B12 administered concomitantly with exogenous intrinsic factor, hydrochloric acid, pepsin, or a combination thereof. One of the five patients reached the lownormal range with the administration of intrinsic factor alone, with a further remarkable increase with the exogenous administration of acid and pepsin along with the IF. In two of four patients tested, the administration of acid alone corrected the malabsorption of protein-bound B~, with a further increase in one with the additional administration of pepsin. Patient 3 had slight increases over baseline (still below the normal range) with the addition of I F alone, acid alone, and with acid plus pepsin, and had a synergistic effect into the clearly normal range when all three gastric factors were administered together. Patient 5, who had the Billroth II anastomosis, failed to normalize protein-bound Ba2 absorption with any combination tested, implying ina d e q u a t e in vivo f u n c t i o n o f the e x o g e n o u s facilitators of protein-bound B12 absorption, possibly due to rapid gastric emptying. DISCUSSION This report, along with the data of others (3, 57), demonstrates the development of vitamin BI2 deficiency due to vitamin B12 malabsorption undetected by the usual means of assessing absorption of this vitamin. All five patients had depressed serum B 1~levels, three with central and/or peripheral neuropathic symptoms responsive to parenteral B12 therapy, despite normal absorption of unbound crystalline vitamin B12 (stage I Schilling test). In addition, one patient had the clinically enigmatic pres e n t a t i o n t h a t l o o k e d like c l a s s i c a l p e r n i c i o u s anemia by several criteria e x c e p t for one thing, the lack of malabsorption of unbound crystalline vitamin B12. Low-grade anemia seen in two of the patients may have been partially related to vitamin B ~ deficiency; inadequate evaluation of anemia took
399
KING ET AL
6.0-
5.0.......
,.
z
4.0-
........
t.a3 .~.....
3.0-
C:::'...~y.)
/i"
o.
B
...,:.:.>.
2.0-
.i:'.'.:'.'"-:":
~i.;:?!:?)
t.o
:...~....,..
.:..::..:.;:.
1.0-
CONTROL (RANGE)
BASELINE
INTRINSIC FACTOR
ACID
ACID PLUS PEPSIN
ACID PEPSIN I,F.
Fig 2. Effect of intrinsic factor, acid, pepsin, or a combination thereof on protein-bound vitamin B12 absorption in five vitamin Bi2-deficient subjects.
place in all cases prior to commencement of parenteral vitamin B12 therapy and referral to us for evaluation of vitamin B12 malabsorption. The evaluation of protein-bound vitamin B12 absorption was made in the present study utilizing chicken serum-bound [~TCo]cyanocobalamin(7). Binding of vitamin BI~ in chicken serum probably involves at least two vitamin B12 binders (12, 13). Although this form of liquid protein-bound vitamin B12 meal is certainly not the same as a meal in which protein-bound vitamin BI~ is incorporated in solid food, the results of this study demonstrate its abilitY to detect a digestive defect in the absorption of protein-bound vitamin B12. In fact, it is probable that no one test of protein-bound vitamin B12 absorption would clearly represent the absorption of all forms of protein-bound vitamin B12 because of the variable requirement for proteolytic release of proteinbound vitamin B12 depending on the animal (beef, pig, sheep, or chicken) and/or organ (liver, muscle, kidney) that is employed (14-18) and the variability of absorption and/or postabsorptive metabolism depending on the chemical form of vitamin B12 (coenzyme B12, hydroxycobalamin, cyanocobalamin, etc) that is ingested (18-20).
400
In the present study, malabsorption of proteinbound vitamin BI~ was corrected in four of the five patients with the administration Of hydrochloric acid, pepsin, gastric intrinsic factor and/or a combination of two or three of these gastric facilitators of protein-bound B12 absorption. In vitro and in vivo data regarding release of vitamin B 12from protein in the presence of acid-pepsin is controversial, showing a requirement for acid-peptic activity in some studies (7, 17, 21), no requirement in others (16), and variable results in others (14, 22). Our data support a requirement for acid,peptic activity in the absorption of protein-bound B12. Further studies with other types of protein-bound B 12 meals employing exogenous administration of acid-peptic activity will be interesting. The normal absorption of unbound crystalline vitamin B12 in all five of our patients implies that there is no deficiency (by the commonly accepted criterion) of gastric intrinsic factor in these patients. However, the results of protein-bound vitamin B12 absorption in two of our patients (wh 9 had synergistic improvement in protein-bound B12 absorption when administered exogenous IF, acid, and pepsin) implies a possible relative deficiency of IF in these Digestive Diseases and Sciences, Vol. 24, No. 5 (May 1979)
P R O T E I N - B O U N D V I T A M I N B12 M A L A B S O R P T I O N p a t i e n t s . I t is a p p r e c i a t e d t h a t g a s t r i c i n t r i n s i c fact o r is n o r m a l l y s e c r e t e d in g r e a t e x c e s s b y h u m a n s , w i t h this e x c e s s d e c r e a s i n g f o l l o w i n g g a s t r i c surg e r y o r d e v e l o p m e n t o f g a s t r i c a t r o p h y (23, 24). I t is p o s s i b l e t h a t this e x c e s s is n o r m a l l y s e c r e t e d in o r d e r to c o u n t e r a c t n o r m a l c o m p e t e t i v e b i n d i n g o f v i t a m i n B12 b y o t h e r v i t a m i n B12 b i n d e r s (25) a n d / o r degradation or binding of intrinsic factor by constituents of the meal and/or secretions of the mouth, stomach, or small intestine. Perhaps the relative IF d e f i c i e n c y n o t e d in o u r t w o p a t i e n t s (as m a n i f e s t e d b y p r o t e i n - v i t a m i n B12 m a l a b s o r p t i o n i m p r o v e d b y e x o g e n o u s I F ) is a p h a s e in t h e d e v e l o p m e n t o f m o r e o v e r t I F d e f i c i e n c y , ie, m a l a b s o r p t i o n o f u n b o u n d c r y s t a l l i n e v i t a m i n B 12 w i t h c o r r e c t i o n b y e x o g e n o u s I F a d m i n i s t r a t i o n . I n t h e s a m e light, o u r patient with Graves' disease, anti-IF antibodies, and achlorhydria may represent an early phase of what may become more classical pernicious anemia in t h e f u t u r e . I t will b e i n t e r e s t i n g to s e e if p a t i e n t s w i t h this r e l a t i v e d e f i c i e n c y o f i n t r i n s i c f a c t o r p r o g r e s s to m o r e o v e r t d e f i c i e n c y o f I F (as m e a s u r e d b y both correctible malabsorption of unbound crystalline v i t a m i n B12 a n d m e a s u r e d o u t p u t o f g a s t r i c int r i n s i c f a c t o r ) . E v e n t h o u g h all p a t i e n t s in this s t u d y c o u l d a b s o r b u n b o u n d c r y s t a l l i n e v i t a m i n B12 n o r m a l l y , t h e r a p y w i t h o r a l c r y s t a l l i n e v i t a m i n B12 would be somewhat risky, because of the potential s u b t l e d e v e l o p m e n t o f m o r e o v e r t I F d e f i c i e n c y at a later time. The present study suggests that malabsorption of p r o t e i n - b o u n d v i t a m i n B~2 m a y o c c u r in t h e s e t t i n g o f a c i d - p e p s i n d e f i c i e n c y , b e it s p o n t a n e o u s l y d e veloping or following gastric surgery. Whether appropriate care of patients with hypochlorhydria should involve periodic determination of vitamin B~2 s t o r e s in t h e b o d y , p e r i o d i c a s s e s s m e n t o f s o m e t y p e o f p r o t e i n - b o u n d v i t a m i n B~2 a b s o r p t i o n t e s t ( s u c h as t h e o n e d e s c r i b e d in this r e p o r t ) , a n d / o r p r o p h y l a c t i c v i t a m i n B12 t h e r a p y r e m a i n s to b e d e termined.
ACKNOWLEDGMENTS The authors gratefully acknowledge the expert technical assistance of Mrs. Cheryl Curington, Ms Debbie Duncan, Mr. George Smith, Mrs. Eugenia Ahmed, and Mr. Marshall Sunderland; the secretarial effort of Mrs. Frances T. Tucker; and the cooperation of Mrs. Shirley Bloodworth and the Nursing Staff of the Clinical Research Center, Shands Teaching Hospital, Gainesville, Florida. Digestive Diseases and Sciences, Vol. 24, No. 5 (May 1979)
REFERENCES 1. Toskes PP, Deren JJ: Vitamin B12 absorption and malabsorption. Gastroenterology 65:662-683, 1973 2. Donaldson RM, Jr: Mechanisms of malabsorption of cobalamin. Cobalamin. M Babior (ed). New York, John Wiley and Sons, 1975, p 337-368 3. Deller DJ, Richards WCD, Witts LJ: Changes in the blood after partial gastrectomy with special reference to vitamin BI~. II. The cause of the fall in serum vitamin B12. Q J Med 31:89-102, 1962 4. Hines JD, Hoffbrand AV, Mollin DL: The hematological complications following partial gastrectomy. Am J Med 43:555-569, 1967 5. Mahmud K, Ripley D, Doscherholmen A: Vitamin B12 absorption tests: Their unreliability in postgastrectomy states. J Am Med Assoc 216:1167-1171, 1971 6. Rygvold O: Hypovitaminosis B12 following partial gastrectomy by the Billroth II method. Scand J Gastroenterol 9 (Suppl 29):57-64, 1974 7. Streeter AM, Balasubramaniam D, Boyle R, O'Neill BJ, Pheils MT: Malabsorption of vitamin B12 after vagotomy. Am J Surg 128:340-343, 1974 8. Ross GSM: Vitamin B~2 assay in body fluids using Euglena gracilis. J Clin Pathol 5:250-256, 1952 9. Schilling RF: Intrinsic factor studies. II. The effect of gastric juice on the urinary excretion of radioactivity after the oral administration of radioactive vitamin B12. J Lab Clin Med 42:860-866, 1953 10. Gottlieb C, Lau K-S, Wasserman LR, Herbert V: Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated B~2 binding capacity, antibody to IF, and serum unsaturated B~2 binding capacity. Blood 25:875-884, 1965 11. Snedecor GW, Cochran WG: Statistical Methods, 6th ed. Ames, Iowa, Iowa State University Press, 1967 12. Kidroni G, Grossowicz N: Purification and properties of two vitamin BI~ binders from chicken serum. Biochim Biophys Acta 118:113-123, 1969 13. Sonneborn DW, Hansen HJ: Vitamin Bx2 binders of chicken serum and chicken proventriculus are immunologically similar. Science 168:591-592, 1970 14. Cooper BA, Castle WB: Sequential mechanisms in the enhanced absorption of vitamin B12 by intrinsic factor in the rat. J Clin Invest 30:199-214, 1960 15. Reizenstein PF: Effect of digestive enzymes on bound vitamin B12. Acta Med Scand 165:481-486, 1959 16. Heyssel RM, Bozian RC, Darby WJ, Bell MC: Vitamin B~2 turnover in man: The assimilation of vitamin BI~ from natural foodstuff by man and estimates of minimal daily dietary requirements. Am J Clin Nutr 18:176-184, 1966 17. Adams JF, Kennedy EH, Thompson J, Williamson J: The effect of acid peptic digestion on free and tissue-bound cobalamins. Br J Nutr 22:111-114, 1968 18. Doscherholmen A, McMahon J, Ripley D; Vitamin B~ assimilation from chicken meat. Am J Clin Nutr 31:825-830, 1978 19. Adams JF, Ross SK, Mervyn L, Boddy K, King P: Absorption of cyanocobalamin, coenzyme B12, methylcobalamin and hydroxocobalamin at different dose levels. Scand J Gastroenterol 6:249-252, 1971 20. Herbert V, Sullivan LW: Activity of coenzyme Blz in man. Ann NY Acad Sci 112:855-870, 1964 40 1
KING ET AL 21. Okuda K, Takara I, Fujii T: Absorption of liver-bound vitamin B 1~in relation to intrinsic factor. Blood 32:313-323, 1968 22. Schade SG, Schilling RF: Effect of pepsin on the absorption of food vitamin B12 and iron. Am J Clin Nutr 20:636-640, 1967 23. Ardeman S, Chanarin I: Assay of intrinsic factor in the diagnosis of Addisonian pernicious anaemia. Br J Haematol 11:305-314, 1965
402
24. Ardeman S, Chanarin I: Intrinsic factor secretion in gastric atrophy. Gut 7:99-101, 1966 25. Allen RH, Seetharam B, Allen NC, Podell ER, Alpers DH: Correction of cobalamin malabsorption in pancreatic insufficiency with a cobalamin analogue that binds with high affinity to R protein but not to intrinsic factor. J Clin Invest 61:1628-1634, 1978
Digestive Diseases and Sciences, Vol. 24, No. 5 (May 1979)
Protein- (chicken serum) bound [~rCo]cyanocobalamin absorption was evaluated in five hypochlorhydric patients who had developed B 12deficiency despite having normal absorption of unbound crystalline vitamin B 12. All five patients had decreased urinary excretion of protein-bound B 12 (0.06-0.34%) as compared to twelve normal controls (0.61-5.6%), P < .001. Improvement in protein-bound B12 absorption in four of the five patients occurred with the exogenous administration of hydrochloric acid, pepsin, gastric intrinsic factor, or a combination thereof. Vitamin B12 deficiency developing in the setting of hypOchlorhydria may result from deficiency of acid-peptic digestion of B 12bound to protein and~or a relative deficiency of intrinsic factor. This digestive defect is not detected with tests which measure the absorption of unbound crystalline B 12but is detected by a simple test which employs B12 bound to chicken serum as the form of protein-bound Bx2. The absorption of vitamin B12 is usually thought of in terms of events occuixing in the stomach, in the lumen of the small intestine, and at the ileal receptor for the vitamin B12-gastric intrinsic factor complex. As analyzed by the usual tests of absorption of unbound crystalline vitamin B12, the primary abnormalities felt to participate in the malabsorption of vitamin B12 have included deficiency of gastric intrinsic factor; pancreatic insufficiency; bacterial or parasitic infestation of the small intestine; and damage, resection, or congenital abnormalities of the specific ileal receptor for the intrinsic factor-vitamin B12 complex (1). From the Veterans Administration Hospital and Department of Medicine, University of Florida College of Medicine, Gainesville, Florida. Dr. King is the recipient of a Research Associate Award from the Veterans Administration. During the performance of these studies Dr. Toskes was a Clinical Investigator with the Veterans Administration. Supported in part by Grant RR-82 from the National Institutes of Health. Presented in part at the Annual Meeting of the American Gastroenterology Association in Toronto, Canada, May 1977. Address for reprint requests: Dr. P.P. Toskes at the Gastroenterology Section, Veterans Administration Hospital, Gainesville, Florida 32602.
The assimilation of vitamin B12 from foods requires an additional step: that of freeing vitamin B12 from the food. Despite the predominant proteinbound state of dietary vitamin B12, it has been generally accepted that vitamin Blz deficiency does not occur as a result of maldigestion of protein-bound vitamin B12 (2). Vitamin B12 deficiency following partial gastrectomy has been thought to be due primarily to intrinsic factor deficiency and secondarily to small intestinal infestation (3, 4). Although Deller's study (3) is often quoted to support the importance of intrinsic factor deficiency in patients who have had partial gastric resections, the finding that 13 of 26 patients in that study with subnormal levels of vitamin Bi2 in the serum absorbed vitamin B12 normally is not well appreciated. The fact that these 13 vitamin Ba2 -deficient subjects had normal absorption of unbound crystalline vitamin B12 suggests that they may have become deficient in the vitamin because of a failure to absorb proteinbound vitamin B12. The suspected lack of acid and pepsin secretion in these patients who had gastric surgery may have led to the malabsorption of protein-bound vitamin B12 due to an inability to cleave the vitamin B~2 from
Digestive Diseases and Sciences, Vol. 24, No. 5 (May 1979)
0163-2116/79t0500-0397503.00/19 1979DigestiveDiseaseSystems, Inc.
397
KING ET AL TABLE 1. PATIENT DATA S e r u m B 12
Stage I crystalline B 12
(normal >200 pg/ml)
absorption (normal>8%)
Patient
Age (years)
Sex
1 2
68 67
M M
75 118
25 21
3 4 5
59 66 51
M F M
107 159 177
14.4 19 20
Gastric
acid output (mEq/hr) BAO/MAO 0/1.3 0/0
0/0 2.0/14.1" 0/0
Neurological symp!oms
Dementia, ataxia
Memory loss, ataxia, dysesthesias Dysesthesias Dysesthesias
Hct (%)
30 45 35 36 43
*Alkaline, bile-stained residual gastric fluid prior to collection of basal specimen.
its protein bond. M a h m u d et al (5) d e m o n s t r a t e d malabsorption of unbound crystalline vitamin B lz in only '/3 of vitamin B l~-deficient p o s t g a s t r e c t o m y patients, with the other 2/3 (of 62 patients) having normal unbound crystalline B~2 absorption. Further support for the i m p o r t a n c e of protein-bound vitamin B12 m a l a b s o r p t i o n in the d e v e l o p m e n t of vitamin B12 deficiency was seen in the recent report of Rygvold, w h o noted low serum vitamin BI2 levels in 67 of 351 patients who had undergone a Billroth II partial g a s t r e c t o m y (6). In those patients with low s e r u m B12 levels, 31% were noted to have normal a b s o r p t i o n o f u n b o u n d c r y s t a l l i n e B~2 ( s t a g e I Schilling test) implying malabsorption of vitamin Bar from food as the etiology of the vitamin B12 deficiency. Only 33% had Schilling test results supporting intrinsic factor deficiency as the cause for the low serum vitamin Ba2 levels. The present study evaluates the absorption of protein-bound vitamin B~2 by 5 patients who were referred to us for evaluation of vitamin B,2 deficiency developing in the face of normal absorption of unbound crystalline vitamin B~2. Vitamin B,2 bound to chicken serum (7) was utilized as the test of protein-bound vitamin B12 absorption.
MATERIALS A N D M E T H O D S All studies were carried out under carefully controlled conditions in a clinical research center ward. Informed consent was granted by all subjects, and all investigations were approved by the University of Florida Health Center Committee for the Protection of Human Subjects. Serum vitamin B12 levels were determined by the Euglena gracilis assay (8). Absorption of unbound crystalline vitamin BI~ was determined by the urinary excretion test of Schilling (9). Oral administration of pTCo]vitamin B12 was followed by a flushing parenteral dose of 1000/zg of unlabeled cyanocobalamin and 24-hr collection of
398
urine. Urinary excretion of [57Co]vitamin B,2 was calculated, following determination of 57Co content of an aliquot of well-mixed urine in a standard well-type gamma counter. Absorption of unbound crystalline vitamin B12 was determined with oral administration of 1 p.g (0.6/zCi) [57Co]cyanocobalamin (Rubratope, E.R. Squibb and Sons, Inc., Princeton, New Jersey). Protein-bound vitamin B,2 doses were made by mixing 4 ml of sterilized, pathogen-free chicken serum (Grand Island Biological Co., Grand Island, New York) with approximately 0.5 ~Ci and 1 /zg of [~TCo]cyanocobalamin (Amersham, England). Dialysis for 72 hr was followed by checking for adequacy of removal of free B,2 with a modification of the charcoal assay of Gottlieb et al (10). Administration of gastric intrinsic factor with protein-bound vitamin B,2 was performed by mixing the contents of one capsule of intrinsic factor (IF) concentrate (1 NF XI unit per capsule, E.R. Squibb and Sons, Inc., Princeton, New Jersey) with the protein-B,2 solution. Acid and/or pepsin was administered via oragastric tube (100 ml of 0.1 N hydrochloric acid and/or 500 mg of 2 • crystallized hog gastric pepsin) (Sigma Chemical Co., St. Louis, Missouri), followed by extubation and immediate oral administration of the protein-vitamin B,2 solution. Gastric acid output was determined following nasogastric intubation, with fluroscopic guidance of the tube into the antrum. Following aspiration of residual contents, four 15-min basal aspirations were followed by six 15-min aspirations after the subcutaneous administration of 6/zg/ kg body weight pentagastrin (Peptavlon, Ayerst, New York, New York) Aliquots of strained gastric aspirate were titrated to pH 7 with 0.1 N sodium hydroxide. Basal acid output (BAO) was computed from the total titratable acidity for the hour basal collection. Maximal acid output (MAO) is expressed as the sum of the maximal four (of six) consecutive 15-min acid outputs in the poststimulation period. Patients. Data on the five patients who presented with vitamin B12 deficiency and normal absorption of unbound crystalline vitamin B,~ are shown in Table 1. Patients 1 and 2 were admitted with central and peripheral neuropathic symptoms, patient 1 manifesting severe dementia prompting admission to the Neurology Service. Following the discovery of severely depressed serum vitamin B,2 levels in each, parenteral vitamin B12 therapy was begun, Digestive Diseases and Sciences, Vol. 24, No. 5 (May 1979)
PROTEIN-BOUND VITAMIN Ba2 MALABSORPTION RESULTS
-
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VITAMIN DEFICIENT SUBJECTS WITH NORMAL CRYSTALLINE VITAMIN
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Fig 1. Absorption of protein-bound vitamin B12 by twelve normal controls and five patients with vitamin B12 deficiency and normal absorption of unbound crystalline vitamin B~2.
leading to full remission of neurologic signs and symptoms. Patient 1 had, in addition, anemia of combined etiology, including vitamin B12 deficiency and chronic disease. Patient 3 had the confusing picture of vitamin B12 deficiency, Graves' disease with circulating antiintrinsic factor antibody, and achlorhydria, all felt to represent classical pernicious anemia until unbound crystalline vitamin BI~ absorption tests were repeatedly normal at > 14%. Serum folate was elevated at 22 ng/ml and decreased to 6 ng/ml during parenteral vitamin BI~ therapy, as is often noted in 9 vitamin B12 deficiency. Anemia was felt to be due to iron deficiency and possible vitamin B12 deficiency, responding to concurrent therapy with iron and vitamin B12. Patients 4 and 5 demonstrated primarily mild peripheral neuropathic symptoms, with associated anemia of chronic disease and alkaline gastric reflux in patient 4 and hypochlorhydria secondary to a Billroth II patrial gastrectomy in patient 5. None of the patients had diabetes, alcohol abuse, or folate deficiency as etiologic factors for their neuropathic symptoms and/or anemia, and all had normal dietary Ba~ intake by history. Four of the five patients demonstrated achlorhydria or severe hypochlorhydria on gastric analysis, and one Patient had alkaline, bile-strained basal gastric secretions at the time of two of three gastric intubations. Patient 5 was the only patient with previous gastric surgery. Data Evaluation. Groups were compared using Student's t test and the Wilcoxon-Mann-Whitney rank sum test (11). All data are expressed as mean --_ SEM. Digestive Diseases and Sciences, Vol. 24, No. 5 (May 1979)
Figure 1 shows the results of the urinary excretion test following the oral administration of chicken serum-[~TCo]Bi2 to twelve normal controls and the five patients. Controls excreted 2.32 _+ 0.42% (mean -+ SEM) of the dose over 24 hr, with a range of 0.61-5.6%. The five patients excreted from 0.06 to 0.34% of the dose, P < 0.01 in comparison to the controls. Figure 2 compares the range of [SrCo]B12 excretion in controls to that of the five patients when the patients had the protein-bound vitamin B12 administered concomitantly with exogenous intrinsic factor, hydrochloric acid, pepsin, or a combination thereof. One of the five patients reached the lownormal range with the administration of intrinsic factor alone, with a further remarkable increase with the exogenous administration of acid and pepsin along with the IF. In two of four patients tested, the administration of acid alone corrected the malabsorption of protein-bound B~, with a further increase in one with the additional administration of pepsin. Patient 3 had slight increases over baseline (still below the normal range) with the addition of I F alone, acid alone, and with acid plus pepsin, and had a synergistic effect into the clearly normal range when all three gastric factors were administered together. Patient 5, who had the Billroth II anastomosis, failed to normalize protein-bound Ba2 absorption with any combination tested, implying ina d e q u a t e in vivo f u n c t i o n o f the e x o g e n o u s facilitators of protein-bound B12 absorption, possibly due to rapid gastric emptying. DISCUSSION This report, along with the data of others (3, 57), demonstrates the development of vitamin BI2 deficiency due to vitamin B12 malabsorption undetected by the usual means of assessing absorption of this vitamin. All five patients had depressed serum B 1~levels, three with central and/or peripheral neuropathic symptoms responsive to parenteral B12 therapy, despite normal absorption of unbound crystalline vitamin B12 (stage I Schilling test). In addition, one patient had the clinically enigmatic pres e n t a t i o n t h a t l o o k e d like c l a s s i c a l p e r n i c i o u s anemia by several criteria e x c e p t for one thing, the lack of malabsorption of unbound crystalline vitamin B12. Low-grade anemia seen in two of the patients may have been partially related to vitamin B ~ deficiency; inadequate evaluation of anemia took
399
KING ET AL
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place in all cases prior to commencement of parenteral vitamin B12 therapy and referral to us for evaluation of vitamin B12 malabsorption. The evaluation of protein-bound vitamin B12 absorption was made in the present study utilizing chicken serum-bound [~TCo]cyanocobalamin(7). Binding of vitamin BI~ in chicken serum probably involves at least two vitamin B12 binders (12, 13). Although this form of liquid protein-bound vitamin B12 meal is certainly not the same as a meal in which protein-bound vitamin BI~ is incorporated in solid food, the results of this study demonstrate its abilitY to detect a digestive defect in the absorption of protein-bound vitamin B12. In fact, it is probable that no one test of protein-bound vitamin B12 absorption would clearly represent the absorption of all forms of protein-bound vitamin B12 because of the variable requirement for proteolytic release of proteinbound vitamin B12 depending on the animal (beef, pig, sheep, or chicken) and/or organ (liver, muscle, kidney) that is employed (14-18) and the variability of absorption and/or postabsorptive metabolism depending on the chemical form of vitamin B12 (coenzyme B12, hydroxycobalamin, cyanocobalamin, etc) that is ingested (18-20).
400
In the present study, malabsorption of proteinbound vitamin BI~ was corrected in four of the five patients with the administration Of hydrochloric acid, pepsin, gastric intrinsic factor and/or a combination of two or three of these gastric facilitators of protein-bound B12 absorption. In vitro and in vivo data regarding release of vitamin B 12from protein in the presence of acid-pepsin is controversial, showing a requirement for acid-peptic activity in some studies (7, 17, 21), no requirement in others (16), and variable results in others (14, 22). Our data support a requirement for acid,peptic activity in the absorption of protein-bound B12. Further studies with other types of protein-bound B 12 meals employing exogenous administration of acid-peptic activity will be interesting. The normal absorption of unbound crystalline vitamin B12 in all five of our patients implies that there is no deficiency (by the commonly accepted criterion) of gastric intrinsic factor in these patients. However, the results of protein-bound vitamin B12 absorption in two of our patients (wh 9 had synergistic improvement in protein-bound B12 absorption when administered exogenous IF, acid, and pepsin) implies a possible relative deficiency of IF in these Digestive Diseases and Sciences, Vol. 24, No. 5 (May 1979)
P R O T E I N - B O U N D V I T A M I N B12 M A L A B S O R P T I O N p a t i e n t s . I t is a p p r e c i a t e d t h a t g a s t r i c i n t r i n s i c fact o r is n o r m a l l y s e c r e t e d in g r e a t e x c e s s b y h u m a n s , w i t h this e x c e s s d e c r e a s i n g f o l l o w i n g g a s t r i c surg e r y o r d e v e l o p m e n t o f g a s t r i c a t r o p h y (23, 24). I t is p o s s i b l e t h a t this e x c e s s is n o r m a l l y s e c r e t e d in o r d e r to c o u n t e r a c t n o r m a l c o m p e t e t i v e b i n d i n g o f v i t a m i n B12 b y o t h e r v i t a m i n B12 b i n d e r s (25) a n d / o r degradation or binding of intrinsic factor by constituents of the meal and/or secretions of the mouth, stomach, or small intestine. Perhaps the relative IF d e f i c i e n c y n o t e d in o u r t w o p a t i e n t s (as m a n i f e s t e d b y p r o t e i n - v i t a m i n B12 m a l a b s o r p t i o n i m p r o v e d b y e x o g e n o u s I F ) is a p h a s e in t h e d e v e l o p m e n t o f m o r e o v e r t I F d e f i c i e n c y , ie, m a l a b s o r p t i o n o f u n b o u n d c r y s t a l l i n e v i t a m i n B 12 w i t h c o r r e c t i o n b y e x o g e n o u s I F a d m i n i s t r a t i o n . I n t h e s a m e light, o u r patient with Graves' disease, anti-IF antibodies, and achlorhydria may represent an early phase of what may become more classical pernicious anemia in t h e f u t u r e . I t will b e i n t e r e s t i n g to s e e if p a t i e n t s w i t h this r e l a t i v e d e f i c i e n c y o f i n t r i n s i c f a c t o r p r o g r e s s to m o r e o v e r t d e f i c i e n c y o f I F (as m e a s u r e d b y both correctible malabsorption of unbound crystalline v i t a m i n B12 a n d m e a s u r e d o u t p u t o f g a s t r i c int r i n s i c f a c t o r ) . E v e n t h o u g h all p a t i e n t s in this s t u d y c o u l d a b s o r b u n b o u n d c r y s t a l l i n e v i t a m i n B12 n o r m a l l y , t h e r a p y w i t h o r a l c r y s t a l l i n e v i t a m i n B12 would be somewhat risky, because of the potential s u b t l e d e v e l o p m e n t o f m o r e o v e r t I F d e f i c i e n c y at a later time. The present study suggests that malabsorption of p r o t e i n - b o u n d v i t a m i n B~2 m a y o c c u r in t h e s e t t i n g o f a c i d - p e p s i n d e f i c i e n c y , b e it s p o n t a n e o u s l y d e veloping or following gastric surgery. Whether appropriate care of patients with hypochlorhydria should involve periodic determination of vitamin B~2 s t o r e s in t h e b o d y , p e r i o d i c a s s e s s m e n t o f s o m e t y p e o f p r o t e i n - b o u n d v i t a m i n B~2 a b s o r p t i o n t e s t ( s u c h as t h e o n e d e s c r i b e d in this r e p o r t ) , a n d / o r p r o p h y l a c t i c v i t a m i n B12 t h e r a p y r e m a i n s to b e d e termined.
ACKNOWLEDGMENTS The authors gratefully acknowledge the expert technical assistance of Mrs. Cheryl Curington, Ms Debbie Duncan, Mr. George Smith, Mrs. Eugenia Ahmed, and Mr. Marshall Sunderland; the secretarial effort of Mrs. Frances T. Tucker; and the cooperation of Mrs. Shirley Bloodworth and the Nursing Staff of the Clinical Research Center, Shands Teaching Hospital, Gainesville, Florida. Digestive Diseases and Sciences, Vol. 24, No. 5 (May 1979)
REFERENCES 1. Toskes PP, Deren JJ: Vitamin B12 absorption and malabsorption. Gastroenterology 65:662-683, 1973 2. Donaldson RM, Jr: Mechanisms of malabsorption of cobalamin. Cobalamin. M Babior (ed). New York, John Wiley and Sons, 1975, p 337-368 3. Deller DJ, Richards WCD, Witts LJ: Changes in the blood after partial gastrectomy with special reference to vitamin BI~. II. The cause of the fall in serum vitamin B12. Q J Med 31:89-102, 1962 4. Hines JD, Hoffbrand AV, Mollin DL: The hematological complications following partial gastrectomy. Am J Med 43:555-569, 1967 5. Mahmud K, Ripley D, Doscherholmen A: Vitamin B12 absorption tests: Their unreliability in postgastrectomy states. J Am Med Assoc 216:1167-1171, 1971 6. Rygvold O: Hypovitaminosis B12 following partial gastrectomy by the Billroth II method. Scand J Gastroenterol 9 (Suppl 29):57-64, 1974 7. Streeter AM, Balasubramaniam D, Boyle R, O'Neill BJ, Pheils MT: Malabsorption of vitamin B12 after vagotomy. Am J Surg 128:340-343, 1974 8. Ross GSM: Vitamin B~2 assay in body fluids using Euglena gracilis. J Clin Pathol 5:250-256, 1952 9. Schilling RF: Intrinsic factor studies. II. The effect of gastric juice on the urinary excretion of radioactivity after the oral administration of radioactive vitamin B12. J Lab Clin Med 42:860-866, 1953 10. Gottlieb C, Lau K-S, Wasserman LR, Herbert V: Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated B~2 binding capacity, antibody to IF, and serum unsaturated B~2 binding capacity. Blood 25:875-884, 1965 11. Snedecor GW, Cochran WG: Statistical Methods, 6th ed. Ames, Iowa, Iowa State University Press, 1967 12. Kidroni G, Grossowicz N: Purification and properties of two vitamin BI~ binders from chicken serum. Biochim Biophys Acta 118:113-123, 1969 13. Sonneborn DW, Hansen HJ: Vitamin Bx2 binders of chicken serum and chicken proventriculus are immunologically similar. Science 168:591-592, 1970 14. Cooper BA, Castle WB: Sequential mechanisms in the enhanced absorption of vitamin B12 by intrinsic factor in the rat. J Clin Invest 30:199-214, 1960 15. Reizenstein PF: Effect of digestive enzymes on bound vitamin B12. Acta Med Scand 165:481-486, 1959 16. Heyssel RM, Bozian RC, Darby WJ, Bell MC: Vitamin B~2 turnover in man: The assimilation of vitamin BI~ from natural foodstuff by man and estimates of minimal daily dietary requirements. Am J Clin Nutr 18:176-184, 1966 17. Adams JF, Kennedy EH, Thompson J, Williamson J: The effect of acid peptic digestion on free and tissue-bound cobalamins. Br J Nutr 22:111-114, 1968 18. Doscherholmen A, McMahon J, Ripley D; Vitamin B~ assimilation from chicken meat. Am J Clin Nutr 31:825-830, 1978 19. Adams JF, Ross SK, Mervyn L, Boddy K, King P: Absorption of cyanocobalamin, coenzyme B12, methylcobalamin and hydroxocobalamin at different dose levels. Scand J Gastroenterol 6:249-252, 1971 20. Herbert V, Sullivan LW: Activity of coenzyme Blz in man. Ann NY Acad Sci 112:855-870, 1964 40 1
KING ET AL 21. Okuda K, Takara I, Fujii T: Absorption of liver-bound vitamin B 1~in relation to intrinsic factor. Blood 32:313-323, 1968 22. Schade SG, Schilling RF: Effect of pepsin on the absorption of food vitamin B12 and iron. Am J Clin Nutr 20:636-640, 1967 23. Ardeman S, Chanarin I: Assay of intrinsic factor in the diagnosis of Addisonian pernicious anaemia. Br J Haematol 11:305-314, 1965
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24. Ardeman S, Chanarin I: Intrinsic factor secretion in gastric atrophy. Gut 7:99-101, 1966 25. Allen RH, Seetharam B, Allen NC, Podell ER, Alpers DH: Correction of cobalamin malabsorption in pancreatic insufficiency with a cobalamin analogue that binds with high affinity to R protein but not to intrinsic factor. J Clin Invest 61:1628-1634, 1978
Digestive Diseases and Sciences, Vol. 24, No. 5 (May 1979)
