Specificity of the Correction of Vitamin B12 Malabsorption by Pancreatic Extract and its Clinical Significance

GASTROENTEROLOGY 65: 199- 204, 1973 Copyright© 1973 by The Williams & Wilkins Co .

Vol. 65, No . 2

Printed in U.S.A.

SPECIFICITY OF THE CORRECTION OF VITAMIN B 12 MALABSORPTION BY PANCREATIC EXTRACT AND ITS CLINICAL SIGNIFICANCE PHILLIPP. TosKES, M.D. , Juuus J. DEREN, M.D., JAN FRUITERMAN, M.D. , AND MARCEL E. CONRAD, M.D.

Department of Hema tology, Walter Reed Army Institute of R esearch , Washington, D .C., and the Department of Medicine , University of Pennsylvania , School of Medicine, Philadelphia, Pennsylvania

Two grams of pancreatic extract increased the urinary excretion of labeled vitamin B 12 from 2.0% ± 0.5 to 15.4% ± 2.0 (mean ± SE) in 7 patients with pancreatic exocrine insufficiency, but had no effect when administered to normal volunteers or to patients with vitamin B 12 malabsorption associated with pernicious anemia, bacterial over~ growth, or drug-induced disease. The administration of pancreatic extract was useful in clarifying the cause of vitamin B 12 malabsorption unresponsive to gastric intrinsic factor in 2 patients. One patient had a history of acute pancreatitis, but no clinical or laboratory evidence of chronic pancreatic exocrine insufficiency. In a 2nd patient with pernicious anemia, vitamin B 12 absorption was restored to normal only when pancreatic extract and gastric intrinsic factor were administered concomitantly. The presence of pancreatic dysfunction was confirmed by the demonstration of impaired bicarbonate output after secretin stimulation. Thus, pancreatic extract responsive-vitamin B 12 malabsorption is specific for the vitamin B 12 malabsorption associated with pancreatic disease , can occur in the absence of overt pancreatic disease , and may be the only indication of pancreatic dysfunction. The occurrence of vitamin B 12 malabsorption in association with clinical pancreatic injury 1- 5 and after partial pancreatectomy in the rat 6 has been docuReceived January 26, 1973. Accepted March 27, 1973. Address requests for reprints to: Dr. Phillip Toskes, Department of Hematology , Walter Reed Army Institute of Research, Washington, D.C. 20012. Supported in part by the United States Army Research and Development Command ; Grant 5M01RR 40 from the Division of Research Facilities and Resources , Clinical Research Center; Training Grant (T01-AM5462) from the Nat ional In stitutes of Health; and the Viobin Company, Monticello, Illinoi s. The authors appreciate the expert technical help of Miss N ina Shopa, Miss Bonnie Mefferd , M. T. (ASCP) , and Mr. George Smith; the clinical assist199

mented. Vitamin B 12 malabsorption occurring in these settings is improved by the concomitant administration of pancreatic extract but not with gastric intrinsic factor or broad spectrum antibiotics. a-s The specificity of the response to pancreatic extract has not, however, been evaluated in a variety of diseases associated with vitamin B 1 2 malabsorption . The purpose of this study is to (a) demonstrate that pancreatic extract enhances vitamin B 12 absorption only in patients with pancreatic insufficiency, and (b) to report 2 patients with unexplained ance of Dr. Ronald Blanck; and the cooperation of Miss Cordilia Shute and the nursing staff of the Clinical Research Center, University of Pennsylvania, School of Medicine , Philadelphia , Pennsylvania.

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vitamin B, 2 malabsorption in whom the use of the response to pancreatic extract helped clarify the pathogenesis of the absorptive defect.

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Materials and Methods Urinary excretion tests were performed after an overnight fast as described by Schilling 7 using a dose of 0.5 fJ.g (0.5 fJ.C of 57 CoB 12 ) followed within l!2 hr by a parenteral flushing dose of 1000 fJ.g of vitamin B 12 . Urine was collected for 24 hr and counted in a large well -y spectrometer with the normal value in our laboratory being greater than 8% of the orally administered labeled vitamin B, 2 appearing in the urine in 24 hr. Hog gastric intrinsic factor (1 National Formulary X1 U per capsule, E. R. Squibb and Sons, New York, N. Y.) was assayed for its vitamin B 12 -binding capacity• and its ability to stimulate vitamin B 12 uptake in guinea pig ileal tissue 9 before being administered to patients. Pancreatic extract in the form of Viokase (VioBin Corp., Monticello, Ill.) was dissolved in 0.9% sodium chloride (1 g per 10 ml) and administered concomitantly with labeled vitamin B 12 • Fecal fat was determined with the patient's ingesting 100 g of fat per day by the method of Van de Kamer et al. 10 Secretin stimulation (1.5 gastrointestinal hormone units per kg) was performed with 70 mM which is considered the lower limit of normal for bicarbonate concentration in the duodenal aspirate in our laboratory. 11 Augmented betazole hydrochloride (Histalog) stimulation tests were performed (1.5 mg per kg) and the results expressed as maximum acid output in milliequivalents per hour. 12 In addition, aliquots of the gastric aspirate were titrated to pH 10 for 20 min to destroy peptic activity, and retitrated to pH 7 and assayed for gastric intrinsic factor by the method of Gottlieb et a!. 8 The serum was tested for the presence of blocking antibodies to intrinsic factor by means of a charcoal assay system .• Serum vitamin B 12 was measured by the method of Lau et a!. 13 (normal 200 to 900 pg per ml), and serum folate levels with the use of Lactobacillus casei as described by Cooperman (normal > 5 ng per ml). 14

Results Figure 1 demonstrates that 2 g of pancreatic extract did not alter vitamin B, 2 absorption in control subjects or in patients with vitamin B, 2 malabsorption secondary to pernicious anemia, bacterial

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FIG. 1. The effect of 2 g of pancreatic extract on the vitamin B 12 urinary excretion test in normal volunteers and in patients with vitamin B, 2 malabsorption secondary to pernicious anemia, bacterial overgrowth, p-aminosalicylic (PAS) acid administration, and pancreatic insufficiency. e , absorption tests with labeled B 12 ; 6, absorption tests with labeled B 12 and pancreatic extract.

overgrowth, or p-aminosalicylic acid administration. However, pancreatic extract increased vitamin B, 2 absorption from 2.2 ± 0.5% to 15.4 ± 2.0% (mean ± SE) in 7 patients with well documented (abnormal secretin test and/or pancreatic calcification) pancreatic exocrine insufficiency.

Case Reports Case 1 F . J ., a 54-year-old male , was admitted to the Philadelphia Veterans Administration Hospital with the diagnosis of chronic brain syndrome . On admission he was confused and his family reported that he had been drinking heavily . The

VITAMIN B 12 AND THE PANCREAS

August 1973

patient had a past history of several episodes of acute pancreatitis with transient elevations of serum amylase and lipase. No abnormalities were found on physical examination. Pertinent laboratory findings were a hemoglobin of 9.0 g per 100 ml and a megaloblastic bone marrow . Serum folate was 2 ng per ml and serum B, 2 was 160 pg per mi. A Schilling test disclosed 3% excretion in 24 hr , without improvement when hog gastric intrinsic factor was ad ministe~ed (3.2%) (fig. 2). The patient was treated wtth vitamin B 12 and folic acid. Eight months later the patient's capacity to absorb vitamin B,2 was reevaluated. At this time the patient's hemoglobin was 12 g per 100 ml, serum B 12 800 pg per ml, serum folate 30 ng per ml, and gastric analysis showed 1.4 mEq per hr in the basal specimen, and 27.8 mEq per hr after betazole hydrochloride. Stool fat excretion was 3 g p~r day on a 100-g fat intake per day . After secretm stimulation, the bicarbonate concentration was 120 mM in the duodenal aspirate. Upper gastrointestinal series was normal and no pancreatic calcification was noted on f1at plate of the abdomen. The jejunal f1uid was sterile for aerobic and anaerobic bacteria. He excreted only 3% of the labeled B 12 in 24 hr and showed no improvement when gastric intrinsic factor was administered with the radiolabeled vitamin B, 2. When 2 g of pancreatic extract were administered with the labeled B,2, the urinary excretion rose from 3.4 to 27%. One week later the patient again excreted 3% of the labeled vitamin B, 2 in the urine. A repeat Schilling test

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Frc. 2. Seq uential vitam in B12 urinary excretion t es ts in patient F. J . I. F. , intrinsic factor.

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with pancreatic extract increased the excretion to 18%. Comment. This patient demonstrates that vitamin B, 2 malabsorption responsive to pancreatic extract may occur in the absence of overt manifestations of pancreatic exocrine insufficiency. Cas e 2 K. E. , a 23-year-o ld female, was admitted to the Walter Reed General Hospital because of increasing fatigue, weakness , and diarrhea of several weeks ' duration. The patient had been an insulin-dependent diabetic for the past 8 years with numerous hospitali zations for diabetic ketoacidosis . Approximately 2 years previously she was noted to be anemic and received blood transfusions and parenteral vitamin E,2. There was no family history of diabetes , anemia, or pancreatic disease. The patient never consumed alcohol, denied biliary tract symptoms, and had no history of repeated pulmonary infections. Physical examination was within normal limits. Pertinent laboratory findings were a hemoglobin of 8.4 g per 100 m l and a white blood cell count of 12,200 per mm •. The peripheral smear showed macrocytosis and prominent hypersegmentation of the poly~or­ phonuclear leukocytes. Bone marrow exammation revealed a megaloblastic marrow . Serum B, 2 was 160 pg per ml, serum folate 13 ng per ml , and serum lactate dehydrogenase 615 international units. Fasting blood sugar was 180 mg per 100 mi. Serum amylase was 50 ~omogyi units and quantitative immunoglobulin levels were within normal limits . Serum carotene was 80 11-g per 100 mi. Five-hour urinary o-xylose excretion after a 25-g oral dose was 4.5 g and fecal fat excretion was less than 5 g of fat per day in a 72-hr collection. An upper gastrointestinal series including examination of the small bowel demonstrated loss of the normal gastric rugae but no other abnormalities. C':llture. of secretions from the proximal small mtestme disclosed 10 6 total aerobes and 10 6 total anaerobes per mi. Histological examination of the small intestine (proximal) was normal. A gastric biopsy specimen revealed gastric atrophy and on gastric analysis a basal pH of 8.0 was obtained with no response to Histalog stimulation. There was no immunoreactive intrinsic factor present in the gastric juice, and high titers of blockin g antibodies to gastric intrins.ic factor were present in the serum. A secretm stimulation test demonstrated 53 mM of bicarbonate in the duodenal aspirate. A sweat chloride test was within normal limits .

TOSKES ET AL.

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FIG. 3. Sequential vitamin B 12 urinary excretion tests in patient K. E.

Figure 3 shows the resul ts of sequential vitamin B, 2 absorption tests. On two separate occasions the patient excreted less than 1% of the labeled B, 2 in the urine in 24 hr. There was no improvement in vitamin B 12 absorption with hog gastric intrinsic factor , or hog gastric intrinsic factor complexed to the labeled B, 2 in vitro. However, when the patient was given both hog gastric intrinsic factor and 2 g of hog pancreatic extract, her urinary excretion was 22%. Four days and 27 days later the second-stage Schilling test (labeled B 12 and gastric intrinsic factor) was repeated an d found to be abnormal (2.4 and 2.0%) . The administration of pancreatic extract a lone also did not improve vitamin B 12 absorption. After parenteral vitamin B 12 , a brisk reticulocytosis ensued, and by the time of discharge her hematocrit had increased to 38 from her admission level of 26%. Comment. This patient presented with classical features of pernicious anemia but did not res pond to gastric intrinsic factor administration. The correction of vitamin B, 2 malabsorption with the concomitant administration of gastric intrinsic factor and pancreatic extract led to the detection of subclinical pancreatic exocrine ins ufficiency. Thus this patient manifested the lack of both gastric intrinsic factor and the pancreatic factor required for optimal vitamin B, 2 absorption.

Discussion Patients with pancreatic exocrine insufficiency frequently have vitamin B 12 malabsorption that can be corrected by the administration of pancreatic extract .3 - 5

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The exact role that the pancreas plays in the absorption of this vitamin and the precise characterization of the vitamin B1zenhancing factor remain to be defined. Current evidence suggests that a . pancreatic factor with proteolytic properties may act on an intraluminal phase of vitamin B 1 2 absorption. 15 ··. ' The correction of vitamin B 12 malabsorption by the administration of pancreatic extract is specific for the vi~a,min B 12 malabsorption associated with pancreatic dysfunction. Pancreatic extract did not increase vitamin B 12 absorption · in control subjects, nor did it correct the vitamin B 12 malabsorption associated with other disease states such as pernicious anemia, the blind loop syndrome, or 'paminosalicylic acid administration. In.contrast, when pancreatic extract was given to patients with pancreatic exocrine dysfunction, there was marked improvement in vitamin B 12 absorption. The specificity of pancreatic extraCt enhancement of vitamin B 12 absorption · is a useful clinical adjunct in the evaluation of patients with vitamin B 12 malabsorption. In patients with vitamin B 12 malabsorption not improved by gastric intrinsic factor (second stage) or broad spectrum antibiotics (third stage), an attempt should be made to improve the absorptive defect with pancreatic extract administration (fourth stage). Pancreatic extract is readily available and its effect on vitamin B 12 absorption can be evaluated in 1 day. The value of determining the effect of pancreatic extract on vitamin B 12 absorption is exemplified by these 2 patients. F. J. was observed to have vitamin B 12 malabsorption that did not respond on two occasions to gastric intrinsic factor. Although he had several attacks of acute pancreatitis with elevated serum amylase and lipase levels, he had no evidence of chronic pancreatic insufficiency (i.e ., pancreatic calcification, steatorrhea, or impaired bicarbonate response to secretin stimulation). It has been claimed, however, that all patients with recurrent attacks of acute alcoholic pancreatitis have chronic pancreatic injury. 16 The currently available

August 1973

VITAMIN B , AND THE PANCREAS

pancreatic function tests , although clinically quite helpful, are not abnormal in all patients with pancreatic insufficiency . A number of patients have been described with pancreatic calcification with either normal fecal fat excretion or normal bicarbonate concentration in the duodenal aspirate after secretin stimulation. 17 - 1 9 The fact that vitamin B 1 2 absorption improved on two occasions when pancreatic extract was administered suggests that the vitamin B 12 malabsorption observed in this patient was related to a deficiency in a pancreatic factor in spite of the absence of other evidence of pancreatic dysfunction. K . E. presented with a megaloblastic anemia secondary to vitamin B 12 deficiency . She was achlorhydric after Histalog stimulation, and had gastric atrophy on biopsy and high titers of serum-blocking antibodies to gastric intrinsic factor ; hence it was felt that she had vitamin B 1 2 malabsorption secondary to pernicious anemia. However, vitamin B 12 absorption failed to improve after the administration of hog gastric intrinsic factor. Failure of patients with pernicious anemia to respond to hog gastric intrinsic factor is occasionally seen and may be related to several factors 20 including biologically inactive hog gastric intrinsic factor, bacterial overgrowth, 2 1 the presence of antibodies to intrinsic factor in the digestive secretions, 22 and transient injury of the ileal receptor consequent to vitamin B 1 2 depletion. 23 These possibilities were clearly eliminated by the correction of vitamin B 12 malabsorption when both hog gastric intrinsic factor and pancreatic extract were administered together, and the demonstration of continued vitamin B 12 malabsorption unresponsive to hog gastric intrinsic factor 4 days and 27 days later. The response to pancreatic extract prompted the performance of a secretin stimulation test. The abnormal secretin test confirmed the presence of pancreatic injury although other parameters of pancreatic dysfunction were not present. In addition, patient K. E . with combined pernicious anemia and pancreatic dysfunction helps to clarify the pathogenesis of the vitamin B 1 2 malabsorption associated with

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pancreatic insufficiency. It has been demonstrated that free gastric intrinsic factor can bind to the ileal receptor and block the subsequent attachment of the gastric intrinsic factor-vitamin B 1 2 complex. 24 Gastric intrinsic factor is secreted in 100-fold excess by the normal stomach 25 and is susceptible to enzymatic digestion in contrast to the appreciable resistance of the gastric intrinsic factor-vitamin B 12 complex. 26 This suggested that in patients with pancreatic insufficiency and vitamin B 12 malabsorption , free gastric intrinsic factor is not digested within the lumen of the upper gastrointestinal tract and reaches the ileum where it may interfere with the attachment of the gastric intrinsic factor-vitamin B 1 2 complex to the ileal receptor. The occurrence of vitamin B 12 malabsorption in a patient with pernicious anemia and pancreatic dysfunction clearly eliminates this explanation. Thus the administration of pancreatic extract-which improves vitamin B 1 2 absorption only in patients with pancreatic disease-should be included in the evalua tion of a patient with vitamin B 12 malabsorption unresponsive to gastric intrinsic factor or antibiotics. As illustrated by the case reports , vitamin B 12 malabsorption may occur in association with minimal evidence of pancreatic disease without overt manifestation of pancreatic insufficiency, and t he correction of vitamin B12 malabsorption by pancreatic extract may lead to the detection of subclinical pancreatic dysfunction. REFERENCES 1. Mcintyre PA, Sac hs MV , Krevans JR, et a l: P athogenes is a nd t reatment of macrocytic anemia: information obtained with radioactive v ita min B 12 • Arch In tern Med 98:541-549, 1956 2. Perman G, G ullberg R , Reizenstein PG , et a l: A study of absorption patterns in malabsorption syndromes. Ac ta Med Scand 168:117- 125 , 1960 3. Veeger W, Abels J, Hellemans N, et a l: Effect of sodium bicarbonate and pancreati n on the absorption of vitam in B, 2 a nd fat in pancreatic insuffi ciency . N Eng! J M ed 267: 1341- 1344, 1962 4. LeBa uer E , Smith K, Greenberger NJ: Pancreatic insufficiency and v itam in B, malabsorption. Arch In tern Med 122:423- 425, 1968

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5. Toskes PP, Hansell J, Cerda J , et al: Vitamin B 12 malabsorption in chronic pancreatic insufficiency. Studies suggesting the presence of a pancreatic "'intrinsic factor". N Eng! J Med 284:627-632, 1971 6. Toskes PP, Deren JJ: The role of the pancreas in vitamin B 12 absorption in partially pancreatectomized rats . J Clin Invest 51:216-223, 1972 7. Schilling RF: Intrins ic factor studies. II. The effect of gastric juice on the urinary excretion of radioactivity after the oral administration of radioactive vitamin B 12 • J Lab Clin Med 42:860-866, 1953 8. Gottlieb C, Lau KS , Wasserman LR, et al: Rapid charcoal assay for intrinsic factor (IF) , gastric juice unsaturated B 12 binding capacity, antibody to IF, and serum unsaturated B 12 binding capacity. Blood 25:875-884, 1965 9. Sullivan LW, Herbert V, Castle WB: In vitro assay for human intrinsic factor. J Clin Invest 42:1443- 1458, 1963 10. Van de Kamer JH , Huinink HTB, Weyers HA: Rapid method for the determination of fat in feces . J Bioi Chern 177:347-355, 1949 11. Cerda J , Brooks F: Relationship between steatorrhea and an insufficiency of pancreatic secretion in the duodenum in patients with chronic pancreatitis. Am J Med Sci 253:38-44, 1967 12. WardS , Gillespie IE, Passaro FP, et al: Comparison of histalog and histamine as stimulants for maximal gastric secretion in human subjects and in dogs. Gastroenterology 44:620-626, 1963 13. Lau KS , Gottlieb C, Wasserman LR, et al: Measurement of serum vitamin B 12 using a radioisotope dilution and coated charcoal. Blood 26:202-2 14, 1965 14. Cooperman JM: Microbiological assay of serum and whole blood folic acid activity. Am J Clin

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Nutr 20:1015-1024, 1967 15. Toskes PP, Ginsberg AL, Conrad ME, et a!: Physical-chemical properties and mode of action of pancreatic intrinsic factor. Blood 38 :809, 1971 16. Strum WB , Spiro HM: Chronic pancreatitis. Ann Intern Med 74:264-277, 1971 17. Sarles H , Sarles JC, Camatte R , et al: Observations on 205 confirmed cases of acute pancreatitis, recurring pancreatitis, and chronic pancreatitis. Gut 6:545-559, 1965 · 18. Dreiling DA, Janowitz HD : The measurement of pancreatic secretory function , The Exocrine Pancreas, Boston , Little, Brown and Co, 1961, p 225-258 19. Marks I, Banks S: Etiology , clinical features , and diagnosis of pancreatitis in the Southwestern Cape. S Afr Med J 37:1039-1053, 1963 20. Deren JJ: Pernicious anemia: A digestive disorder. Viewpoints Dig Dis 4:2, 1972 21. Badenoch H , Bedford DD, Evans JR: Massive diverticulosis of the small intestine with steatorrhea and megaloblastic anemia. Q J Med 24:321-330, 1955 22. Rose MS, Chanarin 1: Intrinsic factor antibody and the absorption of vitamin B 12 in pernicious anemia. Br Med J 1:25-26, 1971 23. Carmel R, Herbert V: Correctable intestinal defect of vitamin B 12 absorption in pernicious anemia. Ann Intern Med 67:1201-1207, 1967 24. Abels J, Vegter JJM , Woldring MG, et al: The physiological mechanism of vitamin B 12 absorption. Acta Med Scand 165:105- 113, 1959 25. Ardman S, Chanarin 1: Assay of intrinsic factor in the diagnosis of Addisonian pernicious anemia. Br J Haematol 11:305-3 14, 1965 26. Abels J , Schilling R: Protection of intrinsic factor by vitamin B 12 . J Lab Clin Med 64:375-384, 1964