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Intestinal absorptive and digestive function in pernicious anemia
Intestinal Absorptive and Digestive Function in Pernicious Anemia
CONSTANTINE CHARLES
ARVANITAKIS,
M.D.
L. LYFORD, M.D.‘
JOHN FOLSCROFT,
B.S.
Kansas City, Kansas
From the Departmentof Medicine, Division of Dastroenterology,Kansas University School of Medicine,
Kansas City, Kansas. This study was by funds from the Kansas University Endowment Association (Grant No. 2550). General Research Support (Grant No. 2553 and 1522-14) and, in pan, by a Grant (No. RR-828) from the GeneralClinicalResearch Centers Program of the Division of Research Resources, National Institutes of Health, Bethesda, Maryland. Requests for reprints should be addressed to Dr. Constantine Arvanitakis, Department of Medicine, Kansas University Medical Center, Rainbow Boulevard at 39th Street. Kansas Citv. Kansas 66103. Manuscript accepted March i7, 1977. Present address: 167 South Conwell, Casper, Wyoming 8260 1. supported
l
We examined the digestive and absorptive function of the small intestinal mucosa in three patients with pernicious anemia to determine the functional correlates of the morphologic changes previously described. Digestive brush border enzymes (disaccharidases and leucyl-naphthylamidase) in jejunal biopsy specimens from the patients followed a normal distribution compared with those in the control group. With the exception of one patient with mild steatorrhea, the rest of the absorption test results were within the normal range. Jejunal perfusion studies, however, with glucose, sodium and water showed intestinal secretion of sodium and water, i.e., net movement of sodium and water from plasma to lumen, in the presence of normal glucose absorption. Follow-up studies in two patients after treatment with vitamin B12 did not reveal any significant improvement in the absorption rates from the pretreatment period. This abnormality of sodium and water transport in pernicious anemia represents another intestinal defect of a systemic disease which is not corrected by vitamin B12 replacement therapy. Morphologic alterations of the epithelial cells of the gastrointestinal tract, similar to macrocytosis in the peripheral blood and megaloblastosis in the bone marrow, have been described in patients with pernicious anemia [i-3]. Jejunal mucosal abnormalities include shortening of villi, decreased number of mitoses in the crypts and increased cellular infiltrate in the lamina propria [4,.5]. Since the immature intestinal epithelium and the reduction of the absorptive surface may interfere with normal digestive and absorptive processes, it was of interest to further examine the functional significance of these morphologic abnormalities. The purpose of this study was, therefore, to examine certain indices of intestinal digestion (brush border enzymes) and absorption (d-xylose, vitamin B12). In addition, jejunal perfusion studies were performed, utilizing a test solution of glucose, sodium and water, to investigate the pattern of intestinal transport of these nutrients. PATIENTS AND METHODS The diagnosis of pernicious anemia abnormal Schilling test, i.e., urinary
was based on the following criteria: (1) excretion of less than 10 per cent of an as evidenced B 12; (2) gastric achlorhydria,
oral dose of 57Co-labeled vitamin by the absence of gastric acid secretion,
i.e., failure of pH in gastric aspirate
to fall below
(3) serum vitarnin
6 after maximal
than 200 pg/ml
(normal
stimulation;
range 200 to 1,000 pg/ml)
in the bone marrow
and macrocytosis
December
The American
1977
B12 level of less
and (4) megaloblastosis
in the peripheral
Journal of Medicine
blood.
Volume 63
959
INTESTINAL ABSORPTIVE AND DIGESTIVE FUNCTION IN PERNICIOUS ANEMIA-ARVANITAKIS
TABLE I
Results of Hematologic Studies Normal Values
Case 1
Case 2
Case 3
12-16 80-100 31-36
7.3 136 33
4.4 131 31
8.0 102 34
60-50 235-450 200-l ,000
77 234 100
30 230 50
155 195 58
6-15
19 2
4 6
9 Megaloblastic changes with giant forms
10 Marked megaloblastic erythropoiesis
6 2 18
Studies Hemoglobin (g/dl) Mean corpuscular volume (p3) Mean corpuscular hemoglobin concentration (%) Serum iron (wgldl) Total iron-binding capacity (pg/dl) Serum vitamin 8,s (pg/dl) Serum folate (ng/dl)
&hilling test (%) Schilling test with IF
10 10 Normal erythropoiesis
Bone marrow
Two male and one female patient with pernicious anemia who were not previously treated with vitamin B12 were studied. They ranged in age from 28 to 50 years. Common presenting manifestations were anemia and peripheral neuropathy. Gastrointestinal symptoms, i.e., epigastric discomfort and weight loss, occurred in two patients. Renal function, as determined by creatinine clearance, serum creatinine, blood urea nitrogen and microscopic examination of the urine, was within normal limits in all patients. Informed consent was obtained from each patient prior to the study. The following determinations were made: hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration, serum iron, total iron-binding capacity and serum folate [6]; serum vitamin B12 assay [7] and bone marrow aspiration were performed. A Schilling test was performed after the oral administration of 0.5 &i of 57Colabeled vitamin B12 [8]. The test was repeated on another day with the concomitant administration of intrinsic factor. Follow-through x-ray films of the upper gastrointestinal tract and small bowel were performed in a standard fashion. Gastric analysis was performed in all patients with the standard method. The tube was placed in the most dependent part of the stomach and collections were obtained before and after stimulation with secretagogue. The subcutaneous administration of betazole hydrochloride (Hi&log)@ (1.5 mg/kg) or histamine acid phosphate (0.04 mg/kg) was used for maximal stimulation of gastric acid secretion. Intestinal absorption tests included the determination of serum carotene [9], d-xylose [IO] in a 5 hour urine collection after a 25 g loading dose, and fecal fat [ 1 l] in a 72 hour stool collection during a daily fat intake of 100 g. Following an overnight fast, peroral jejunal biopsies were performed with a multipurpose suction biopsy tube [ 121. After
TABLE II
860
ET AL
Hypercellular with marked erythroid hyperplasia
proper orientation of intestinal mucosa, one piece of the tissue was submitted for light microscopy and two to three pieces were homogenized for the assay of intestinal mucosal enzymes. The following enzymes were assayed: sucrase, maltase, lactase, isomaltase, trehalase, alkaline phosphatase and leucyl-naphthylamidase. Disaccharidases were measured according to the method of Dahlqvist [ 131, leucyl-naphthylamidase, as described by Goldbarg and Rutenburg [ 141, and alkaline phosphatase using 0-nitrophenyl phosphate in a buffer solution containing cobalt, manganese and magnesium [ 151. Protein content was measured by the method of Lowry et al. [ 161. Specific enzyme activity was expressed in micromoles of substrate hydrolyzed per min per gram of protein. Twelve healthy volunteer subjects (seven men and five women), ranging in age from 21 to 50 years, without anemia, gastrointestinal, metabolic, pancreatic or hepatic disease served as a control group for comparison of the histologic jejunal biopsy findings and brush border enzyme activity. Three patients with pernicious anemia underwent jejunal perfusion studies with a triple-lumen tube according to the technic described by Cooper et al. [ 171. Following the positioning of the tube in the proximal jejunum, 40 mM of Dglucose with 140 mM sodium chloride (osmolality 290 f 10 mosm) and 0.5 per cent polyethylene glycol, as the nonabsorbable marker, were perfused at a constant rate of 14 ml/min. After a 20-minute equilibration period, three 20minute collections were obtained from the proximal and distal aspirating sites. Samples were subjected to deproteinization and the filtrate was assayed for glucose, sodium and polyethylene glycol content. Glucose was measured by the glucose oxidase methed [ 181, and polyethylene glycol by the method of Malawer and Powell [ 191. Glucose, sodium and
Results of Intestinal Absorption Tests
Case No.
Age (yr)
Serum Carotene (WJt)
1 2 3
44 28 50
58 45 74
December 1977
D-Xylose Urinary Excretion (915 hr)
72-hour Fecal Fat (g/72 hr)
Serum Calcium (rWdf)
4.8 4.0 5.5
18 45 16
9.5 9.0 9.0
The American Journal of Medicine
Volume 63
Serum Albumin Wf) 3.8 3.8 3.6
Jejunal Biopsy (light microscopy) Normal Normal Normal
INTESTINAL
TABLE
III
Group Control Pernicious P value
anemia
Speclflc Actlvlty No. 12 3
’ Values represent mean f standard N.S. = not significant. + LNA = leucyl-naphthylamldase.
ABSORPTIVE
AND DIGESTIVE
Lactase 3Sf 35 f N.S.
Sucraso 85 f 92 f N.S.
18 9
error of the mean.
13’ 16
Maltass 314 f 345 f N.S.
All values
240 46
Before treatment After treatment
ET AL.
Glucose, Sodium and Water Absorption
No. 3 2
NOTE: No statistically significant difference was detected between indicates net absorption; - indicates net secretion, i.e., movement Values represent mean f SEM.
Alkaline PhO8phst880
196 f 35 191 f 35 N.S.
67f 15 62f21 N.S.
390 f !96 293 l 110 N.S.
treatment
hydrolyzed
LNAt 37 f 39 f N.S.
5 6
per mln per g protein.
period
(Table IV).
COMMENTS
Several studies have previously shown that intestinal malabsorption may occur in patients with pernicious anemia [ 2 l-231. However, considerable disagreement exists regarding the nutrients which iare affected and the clinical significance of these abnormalities. Lindenbaum et al. [ 231, in a prospective study of 28 patients with pernicious anemia, found malabsorption of d-xylose in 29 per cent, fat in 9 per cent and vitamin B12 with intrinsic factor in 75 per cent. Malabsorption of d-xylose and vitamin B12 may be due to intestinal mucosal disease, bacterial overgrowth or both. Vitamin B12 malabsorption is a well documented intestinal defect in pernicious anemia which occurs rather frequently [23,24]. Lack of vitamin B t2 may contribute to epithelial mucosal dysfunction and impairment of intestinal absorption. Pena et al. [5] examined the morphologic and digestive enzyme abnormalities in jejunal biopsy specimens from untreated patients with pernicious anemia. They found shortening of villi and reduction of the absorptive surface, megalocytic changes of intestinal epithelial cells and a significant depression of mucosal
In Pernlclous
Glucose (mmollhrl segment) +14.5 +13.2
Trthats8s
pernicious anemia were similar to those in the control group. However, there was a marked net secretion of sodium and water into the intestinal lumen, a statistically significant difference compared with sodium and water net absorption rates in the control group (p <0.025). Perfusion studies after treatment with vitamin B12 in two patients demonstrated that in both patients glucose absorption was normal, but sodium and water absorption was inhibited, a pattern similar to that in the pre-
The results of the hematologic studies are shown in Table I. All patients presented with megaloblastic, macrocytic type of anemia of varying degree, low serum vitamin B12 levels and abnormal Schilling test results (excretion of 57Co-vitamin B12 10 per cent). It was of interest that in two patients 57Co-vitamin B12 excretion was only partially corrected with intrinsic factor, indicating secondary malabsorption of vitamin B12. Gastric achlorhydria, i.e., failure of maximal gastric pH to decrease below 6.0, was present in all patients. The results of intestinal absorption tests are shown in Table II. The only abnormality was steatorrhea’ in one patient. The rest of the absorption test results were within the normal range. Light microscopy of the jejunal mucosal biopsy specimens did not reveal any significant abnormalities. The results of the specific activity of brush-border enzymes are summarized in Table III. Since mucosal disaccharidase activity follows a log distribution, statistical analyses of logt, disaccharidase activity were performed using Student’s t test (unpaired data). There was no significant difference in any of the brush-border enzyme activity in patients with pernicious anemia as compared with those in the control group. The results of the jejunal perfusion studies are shown in Figure 1. Glucose absorption rates in patients with
Tlme of Study
ANEMIA-ARVANITAKIS
Isomaltass
are given In pmol of substrate
RESULTS
IV
IN PERNICIOUS
of Brush Border Enzymes*
water absorption rates were calculated using standard formulas [20]. In two patients with pernicious anemia, jejunal perfusion studies were repeated after treatment with monthly vitamin B12 injections (100 pg). The treatment period was approximately 18 months, and there was considerable hematologic and clinical improvement as compared to the pretreatment period.
TABLE
FUNCTION
f 3.0 f 2.6
Anemia’ Sodlum (meqlhrl segment) -42.0 -31.6
f 22 f 26
Water (mllhrl segment) -426 -372
f f
126 165
the pre- and post-treatment absorption rates of sodium and water. of solute and water from plasma to lumen.
i-
l
December 1977
The American Journal of Medicine
Volume 63
661
INTESTINAL ABSORPTIVE
AND DIGESTIVE FUNCTION IN PERNICIOUS ANEMIA-ARVANITAKIS
GLUCOSE
ET AL
SODIUM 300
-I-
200
m
CONTROLS
D
PA
I
I
disaccharidases. These histologic and enzymatic defects diminished after treatment with vitamin B12. We were unable, however, to demonstrate any significant reduction in any of the brush-border enzymes in jejunal biopsy specimens. It may be that alterations in the brush-border enzymes are related to the severity and duration of pernicious anemia, the nutritional status of the patients and their dietary intake. These factors probably did not play a significant role in our patients. Since nutritional changes and morphologic abnormalities of the intestinal mucosa may affect the functional integrity of the intestinal epithelial cell, we examined glucose, sodium and water absorption utilizing the jejunal perfusion technic. We did not find any difference in jejunal glucose absorption rates between the patients with pernicious anemia and the control group. This is in contrast to the findings reported by Groen in 1938 [25], who demonstrated impaired intestinal glucose absorption in three patients with pernicious anemia. In his study, in which the Miller-Abbott technic of intestinal absorption was used, glucose absorption was restored to normal after vitamin B12 replacement therapy. There are several explanations to account for the difference in the results of our study. First, we utilized the segmental, triple-lumen intestinal perfusion technic which has been extensively evaluated in regard to its
662
December 1977
The American Journal of Medicine
‘-600
Figure 1. Jejunal glucose, sodium and water absorption rates (mean f SEM) in control subjects and patients with pernicious anemia (PA) in the pretreatment period. Glucose absorption rates in the control group were 14.5 f 3.0 versus 15.0 f 10 mmollhourf segment in pernicious anemia (N.S); sodium absorption rates in the control group were +27.7 f 2.0 versus -42.0 f 22 meqlhourlsegment in pernicious anemia (p CO.025) and water absorption in the control group was +2 12 f 26 versus - 428 f 728 mllhourlsegment in pernicious anemia (p < 0.025), indicating net secretion into the intestinal lumen.
reproducibility and accuracy. The methodologic differences in the two studies are probably too important to allow any comparison of the results. Second, it was indicated that impaired glucose absorption in pernicious anemia was a nonspecific effect, possibly related to the low caloric intake or poor nutritional status of the patients [25], a factor which was probably not significant in our patients. The most striking observation in our study, a finding not previously reported in pernicious anemia, was the demonstration of marked inhibition of sodium and water absorption in the jejunum, in the presence of a normal jejunal mucosal biopsy specimen. Not only sodium and water absorption was completely inhibited, but also a significant net secretion of sodium and water, i.e., net loss into the intestinal lumen occurred in all three patients. Luminal accumulation of sodium and water was apparent during the intestinal perfusion studies when our patients experienced abdominal pain and distress, evidently due to luminal distention. This phenomenon has been recognized in perfusion studies in which dihydroxy bile acids were used in the perfusate resulting in intestinal fluid secretion and distention of the intestinal wall [26]. Follow-up perfusion studies in two patients after a period of treatment with vitamin B12 did not show any improvement in the absorption of sodium and water, although glucose absorption was normal as in the pretreatment period.
Volume 63
INTESTINAL ABSORPTIVE AND DIGESTIVE FUNCTION IN PERNICIOUS ANEMIA-ARVANITAKIS
Sodium and water secretion into the lumen may occur in several clinical and experimental situations and in the absence of structural changes in the jejunal biopsy specimen. There are two conditions in pernicious anemia which may provide a reasonable explanation for our results. First, it is known that bacterial overgrowth in the jejunum commonly occurs in pernicious anemia [27]. Overgrowth in the lumen results in deconjugation of bile acids [28] which exert an inhibitory effect on intestinal electrolyte and water absorption [29]. Second, bacterial overgrowth may contribute to the formation of hydroxy-fatty acids [30], which are not ordinarily present in the diet, but they originate from bacterial hydroxylation of free fatty acids. Hydroxy-fatty acids exert a cathartic effect on intestinal mucosa associated with increased cellular permeability and intestinal fluid secretion [31]. It is of interest that glucose absorption was not influenced by sodium secretion, as may have been expected, because of a proposed stoichiometric relationship between glucose and sodium absorption. It is possible that the sites of glucose absorption in the absorptive epithelial cell may be different from those of sodium secretion, since glucose absorption may con-
ET AL.
tinue to occur in the presence of sodium secretion into the intestinal lumen [32]. The clinical significance of our observation is not clear, since none of the patients presented with secretory diarrhea or other electrolyte disorder. The fact, however, that this absorptive defect was not a reversible abnormality after treatment with vitamin B12 suggests that it represents a primary intestinal manifestation of pernicious anemia, not necessarily related to the anemia per se. A recent study by Cook [33], showed that the presence of hypochromic or megaloblastic anemia does not affect glucose, glycine or folic acid absorption, whereas xylose absorption is impaired. This observation lends some support ‘to the contention that luminal events (i.e., bacterial overgrowth, hydroxy-fatty acid formation or bile salt deconjugation) may account for the secretion of sodium and water in pernicious anemia. ACKNOWLEDGMENT
We wish to thank Dr. William Larson for permission to study patients under his care and Mrs. Sandra Walker for her secretarial assistance.
REFERENCES
2. 3.
4. 5.
6. 7. 8.
9. 10.
11
12.
Graham RM. Rheault MD: Characteristic cellular changes in epithelial cells in pernicious anemia. J Lab Clin Med 43: 235, 1954. Massey BW, Rubin CE: Stomach in pernicious anemia. Am J Med Sci 227: 481, 1954. Sauli S, Astaldi, G, Malossini L: Histopathology of intestinal mucosa obtained by Crosby’s capsule in pernicious anemia before and after vitamin B12 treatment. Acta Vitamin01 (Milano) 17: 143, 1963. Foroozan P. Trier JS: Mucosa of the small intestine in pernicious anemia. N Engl J Med 227: 553, 1967. Pena AS, Callender ST, Truelove SC, et al.: Small intestinal mucosal abnormalities and disaccharidase activity in pernicious anemia. Br J Haematol 23: 313, 1972. Herbert V: The assay and nature of folic acid activity in human serum. J Clin Invest 40: 8 1, 196 1. Ross GIM: Vitamin Bf2 assay in body fluids using Euglena gracilis. J Clin Pathol 5: 250, 1952. Schilling RF: Intrinsic factor studies. II. The effect of gastric juice on the urinary excretion of radioactivity after oral administration of radioactive vitamin 8,s. J Lab Clin Med 42: 860, 1953. Carr FH, Price EA: Colour reactions attributed to vitamin A. Biochem J 20: 497, 1925. Roe JH, Rice EW: Photometric method for the determination of free pentoses in animal tissues. J Biol Chem 173: 507, 1948. Van De Kamer JH, Ten Bokkel Huinink H, Weyers HA: Rapid method for the determination of fat in feces. J Biol Ghem 177: 347.1949. Brandborg LL, Rubin GE, Quinton WE: A multi-purpose instrument for suction biopsy of the esophagus, stomach, small bowel and colon, Gastroenterology 27: 1, 1959.
13. 14.
15
16.
17.
18.
19.
20.
21.
22.
23.
December 1977
Dahlqvist A: Assay of intestinal disaccharidases. Anal Biothem 22: 99, 1968. Goldbarg JA, Rutenburg AM: The colorirnetric determination of leucine amino peptidase in urine and serum of abnormal subjects and patients with cancer (and other diseases. Cancer 11: 283, 1958. Eichholz EA, Crane RK: Studies on the organization of the brush-border in intestinal epithelial cells. I. Tris disruption of isolated hamster brush borders and density gradient separation of proteins. J Cell Biol 26: 687, 1965. Lowry OH, Rosenbrough NJ, Farr AL, et al.: Protein measurement with folin phenol reagent. J iBiol Chem 193: 265, 1951. Cooper H, Levitan R, Fordtran JS, et al.: A method for studying absorption of water and solute from the human small intestine. Gastroenterology 66: 1, 1966. Huggett A St G, Nixon DA: Use of glucose oxidase peroxidase and odianisidine in determination of blood and urinary glucose. Lancet 2: 368, 1957. Malawer SJ, Powell DW: An improved turbidimetric analysis of polyethylene glycol utilizing an emulsifier. Gastroenterology 53: 250, 1967. Whalen GE, Harris JA, Geenen JE, et al.: Sodium and water absorption from the human small intestine. Gastroenterology 51: 975, 1966. Berzman A, Kinnear DG, Zamcheck N: D-xylose and potassium iodide absorption and serum carotene in pernicious anemia. J Lab Clin Med 53: 226, 1959. Brcdy EA. Estren S, Herbert V: Coexistent pernicious anemia and malabsorption in four patients: including one whose malabsorption disappeared with vitamin B12 therapy. Ann Intern Med 64: 1246, 1966. Lindenbaum J, Peuimenti JF, Shea N: Small intestinal function
The American Journal of Medlclne
Volume 63
883
INTESTINAL
24. 25. 26. 27. 28. 29.
864
ABSORPTIVE
AND DIQESTIVE
FUNCTION
IN PERNICIOUS
In vltamln B12 deflclency. Ann Intern Med 80: 326, 1974. Carmel R, Herbert V: Correctable lntestlnal defect of vltamln Bll malabsorption In pernlclous anemla. Ann Intern Med 67: 1201, 1967. Ciroen J: The absorptlon of glucose from the small lntestlne In deflclency disease. N Engl J Med 218: 247, 1938. Wlngate DL, Hyams A, Phllllps SF: Lumlnal dlstentlon as a possible consequence of experlmental Intestinal perfuslon. Gut 15: 728, 1974. Sherwood WC, Goldstein F, Hauranl FE, et al.: Studles of the small lntestlnal bacterial flora and of lntestlnal absorptlon In pernicious anemla. Am J Dig Dls 9: 416, 1984. Tabaqchall S, Hatzloannou J, Booth CC: Bile salt deconjugatlon wlth steatorrhea In patlents wlth stagnant-loop syndrome. Lancet 2: 12, 1968. Russell RI, Allan JG, Gerskowltch VP, et al.: The effect of
December 1977
The American Journal of Medicine
ANEMIA-ARVANITAKIS
30. 31.
32.
33.
Volume 63
ET AL.
conjugated and unconjugated bile acids on water and electrolyte absorptlon In the human Jejunum. Clln Scl45: 301, 1973. Thomas PJ: ldentlflcatlon of some enterlc bacteria which convert olelc acid to hydroxystearlc acid In vltro. Qastroenterology 62: 430, 1972. Ammon HV, Thomas PJ, Phllllps SF: Effects of olelc and rlclnolelc acids on net jejunal water and electrolyte movement. Perfuslon studies In man. J Clln Invest 53: 374, 1974. Wlngate DL, Phllllps SF, Hofmann AF: Effect of glyclne conjugated bile acids wlth and wlthout lecithin on water and glucose absorption In perfused human jejunum. J Clln Invest 52: 1230, 1973. Cook GG: Absorption of xylose, glucose, glyclne and follc (pteroylglutamlc) acid In Zamblan Africans wlth anemia. Gut 17: 604, 1976.
CONSTANTINE CHARLES
ARVANITAKIS,
M.D.
L. LYFORD, M.D.‘
JOHN FOLSCROFT,
B.S.
Kansas City, Kansas
From the Departmentof Medicine, Division of Dastroenterology,Kansas University School of Medicine,
Kansas City, Kansas. This study was by funds from the Kansas University Endowment Association (Grant No. 2550). General Research Support (Grant No. 2553 and 1522-14) and, in pan, by a Grant (No. RR-828) from the GeneralClinicalResearch Centers Program of the Division of Research Resources, National Institutes of Health, Bethesda, Maryland. Requests for reprints should be addressed to Dr. Constantine Arvanitakis, Department of Medicine, Kansas University Medical Center, Rainbow Boulevard at 39th Street. Kansas Citv. Kansas 66103. Manuscript accepted March i7, 1977. Present address: 167 South Conwell, Casper, Wyoming 8260 1. supported
l
We examined the digestive and absorptive function of the small intestinal mucosa in three patients with pernicious anemia to determine the functional correlates of the morphologic changes previously described. Digestive brush border enzymes (disaccharidases and leucyl-naphthylamidase) in jejunal biopsy specimens from the patients followed a normal distribution compared with those in the control group. With the exception of one patient with mild steatorrhea, the rest of the absorption test results were within the normal range. Jejunal perfusion studies, however, with glucose, sodium and water showed intestinal secretion of sodium and water, i.e., net movement of sodium and water from plasma to lumen, in the presence of normal glucose absorption. Follow-up studies in two patients after treatment with vitamin B12 did not reveal any significant improvement in the absorption rates from the pretreatment period. This abnormality of sodium and water transport in pernicious anemia represents another intestinal defect of a systemic disease which is not corrected by vitamin B12 replacement therapy. Morphologic alterations of the epithelial cells of the gastrointestinal tract, similar to macrocytosis in the peripheral blood and megaloblastosis in the bone marrow, have been described in patients with pernicious anemia [i-3]. Jejunal mucosal abnormalities include shortening of villi, decreased number of mitoses in the crypts and increased cellular infiltrate in the lamina propria [4,.5]. Since the immature intestinal epithelium and the reduction of the absorptive surface may interfere with normal digestive and absorptive processes, it was of interest to further examine the functional significance of these morphologic abnormalities. The purpose of this study was, therefore, to examine certain indices of intestinal digestion (brush border enzymes) and absorption (d-xylose, vitamin B12). In addition, jejunal perfusion studies were performed, utilizing a test solution of glucose, sodium and water, to investigate the pattern of intestinal transport of these nutrients. PATIENTS AND METHODS The diagnosis of pernicious anemia abnormal Schilling test, i.e., urinary
was based on the following criteria: (1) excretion of less than 10 per cent of an as evidenced B 12; (2) gastric achlorhydria,
oral dose of 57Co-labeled vitamin by the absence of gastric acid secretion,
i.e., failure of pH in gastric aspirate
to fall below
(3) serum vitarnin
6 after maximal
than 200 pg/ml
(normal
stimulation;
range 200 to 1,000 pg/ml)
in the bone marrow
and macrocytosis
December
The American
1977
B12 level of less
and (4) megaloblastosis
in the peripheral
Journal of Medicine
blood.
Volume 63
959
INTESTINAL ABSORPTIVE AND DIGESTIVE FUNCTION IN PERNICIOUS ANEMIA-ARVANITAKIS
TABLE I
Results of Hematologic Studies Normal Values
Case 1
Case 2
Case 3
12-16 80-100 31-36
7.3 136 33
4.4 131 31
8.0 102 34
60-50 235-450 200-l ,000
77 234 100
30 230 50
155 195 58
6-15
19 2
4 6
9 Megaloblastic changes with giant forms
10 Marked megaloblastic erythropoiesis
6 2 18
Studies Hemoglobin (g/dl) Mean corpuscular volume (p3) Mean corpuscular hemoglobin concentration (%) Serum iron (wgldl) Total iron-binding capacity (pg/dl) Serum vitamin 8,s (pg/dl) Serum folate (ng/dl)
&hilling test (%) Schilling test with IF
10 10 Normal erythropoiesis
Bone marrow
Two male and one female patient with pernicious anemia who were not previously treated with vitamin B12 were studied. They ranged in age from 28 to 50 years. Common presenting manifestations were anemia and peripheral neuropathy. Gastrointestinal symptoms, i.e., epigastric discomfort and weight loss, occurred in two patients. Renal function, as determined by creatinine clearance, serum creatinine, blood urea nitrogen and microscopic examination of the urine, was within normal limits in all patients. Informed consent was obtained from each patient prior to the study. The following determinations were made: hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration, serum iron, total iron-binding capacity and serum folate [6]; serum vitamin B12 assay [7] and bone marrow aspiration were performed. A Schilling test was performed after the oral administration of 0.5 &i of 57Colabeled vitamin B12 [8]. The test was repeated on another day with the concomitant administration of intrinsic factor. Follow-through x-ray films of the upper gastrointestinal tract and small bowel were performed in a standard fashion. Gastric analysis was performed in all patients with the standard method. The tube was placed in the most dependent part of the stomach and collections were obtained before and after stimulation with secretagogue. The subcutaneous administration of betazole hydrochloride (Hi&log)@ (1.5 mg/kg) or histamine acid phosphate (0.04 mg/kg) was used for maximal stimulation of gastric acid secretion. Intestinal absorption tests included the determination of serum carotene [9], d-xylose [IO] in a 5 hour urine collection after a 25 g loading dose, and fecal fat [ 1 l] in a 72 hour stool collection during a daily fat intake of 100 g. Following an overnight fast, peroral jejunal biopsies were performed with a multipurpose suction biopsy tube [ 121. After
TABLE II
860
ET AL
Hypercellular with marked erythroid hyperplasia
proper orientation of intestinal mucosa, one piece of the tissue was submitted for light microscopy and two to three pieces were homogenized for the assay of intestinal mucosal enzymes. The following enzymes were assayed: sucrase, maltase, lactase, isomaltase, trehalase, alkaline phosphatase and leucyl-naphthylamidase. Disaccharidases were measured according to the method of Dahlqvist [ 131, leucyl-naphthylamidase, as described by Goldbarg and Rutenburg [ 141, and alkaline phosphatase using 0-nitrophenyl phosphate in a buffer solution containing cobalt, manganese and magnesium [ 151. Protein content was measured by the method of Lowry et al. [ 161. Specific enzyme activity was expressed in micromoles of substrate hydrolyzed per min per gram of protein. Twelve healthy volunteer subjects (seven men and five women), ranging in age from 21 to 50 years, without anemia, gastrointestinal, metabolic, pancreatic or hepatic disease served as a control group for comparison of the histologic jejunal biopsy findings and brush border enzyme activity. Three patients with pernicious anemia underwent jejunal perfusion studies with a triple-lumen tube according to the technic described by Cooper et al. [ 171. Following the positioning of the tube in the proximal jejunum, 40 mM of Dglucose with 140 mM sodium chloride (osmolality 290 f 10 mosm) and 0.5 per cent polyethylene glycol, as the nonabsorbable marker, were perfused at a constant rate of 14 ml/min. After a 20-minute equilibration period, three 20minute collections were obtained from the proximal and distal aspirating sites. Samples were subjected to deproteinization and the filtrate was assayed for glucose, sodium and polyethylene glycol content. Glucose was measured by the glucose oxidase methed [ 181, and polyethylene glycol by the method of Malawer and Powell [ 191. Glucose, sodium and
Results of Intestinal Absorption Tests
Case No.
Age (yr)
Serum Carotene (WJt)
1 2 3
44 28 50
58 45 74
December 1977
D-Xylose Urinary Excretion (915 hr)
72-hour Fecal Fat (g/72 hr)
Serum Calcium (rWdf)
4.8 4.0 5.5
18 45 16
9.5 9.0 9.0
The American Journal of Medicine
Volume 63
Serum Albumin Wf) 3.8 3.8 3.6
Jejunal Biopsy (light microscopy) Normal Normal Normal
INTESTINAL
TABLE
III
Group Control Pernicious P value
anemia
Speclflc Actlvlty No. 12 3
’ Values represent mean f standard N.S. = not significant. + LNA = leucyl-naphthylamldase.
ABSORPTIVE
AND DIGESTIVE
Lactase 3Sf 35 f N.S.
Sucraso 85 f 92 f N.S.
18 9
error of the mean.
13’ 16
Maltass 314 f 345 f N.S.
All values
240 46
Before treatment After treatment
ET AL.
Glucose, Sodium and Water Absorption
No. 3 2
NOTE: No statistically significant difference was detected between indicates net absorption; - indicates net secretion, i.e., movement Values represent mean f SEM.
Alkaline PhO8phst880
196 f 35 191 f 35 N.S.
67f 15 62f21 N.S.
390 f !96 293 l 110 N.S.
treatment
hydrolyzed
LNAt 37 f 39 f N.S.
5 6
per mln per g protein.
period
(Table IV).
COMMENTS
Several studies have previously shown that intestinal malabsorption may occur in patients with pernicious anemia [ 2 l-231. However, considerable disagreement exists regarding the nutrients which iare affected and the clinical significance of these abnormalities. Lindenbaum et al. [ 231, in a prospective study of 28 patients with pernicious anemia, found malabsorption of d-xylose in 29 per cent, fat in 9 per cent and vitamin B12 with intrinsic factor in 75 per cent. Malabsorption of d-xylose and vitamin B12 may be due to intestinal mucosal disease, bacterial overgrowth or both. Vitamin B12 malabsorption is a well documented intestinal defect in pernicious anemia which occurs rather frequently [23,24]. Lack of vitamin B t2 may contribute to epithelial mucosal dysfunction and impairment of intestinal absorption. Pena et al. [5] examined the morphologic and digestive enzyme abnormalities in jejunal biopsy specimens from untreated patients with pernicious anemia. They found shortening of villi and reduction of the absorptive surface, megalocytic changes of intestinal epithelial cells and a significant depression of mucosal
In Pernlclous
Glucose (mmollhrl segment) +14.5 +13.2
Trthats8s
pernicious anemia were similar to those in the control group. However, there was a marked net secretion of sodium and water into the intestinal lumen, a statistically significant difference compared with sodium and water net absorption rates in the control group (p <0.025). Perfusion studies after treatment with vitamin B12 in two patients demonstrated that in both patients glucose absorption was normal, but sodium and water absorption was inhibited, a pattern similar to that in the pre-
The results of the hematologic studies are shown in Table I. All patients presented with megaloblastic, macrocytic type of anemia of varying degree, low serum vitamin B12 levels and abnormal Schilling test results (excretion of 57Co-vitamin B12 10 per cent). It was of interest that in two patients 57Co-vitamin B12 excretion was only partially corrected with intrinsic factor, indicating secondary malabsorption of vitamin B12. Gastric achlorhydria, i.e., failure of maximal gastric pH to decrease below 6.0, was present in all patients. The results of intestinal absorption tests are shown in Table II. The only abnormality was steatorrhea’ in one patient. The rest of the absorption test results were within the normal range. Light microscopy of the jejunal mucosal biopsy specimens did not reveal any significant abnormalities. The results of the specific activity of brush-border enzymes are summarized in Table III. Since mucosal disaccharidase activity follows a log distribution, statistical analyses of logt, disaccharidase activity were performed using Student’s t test (unpaired data). There was no significant difference in any of the brush-border enzyme activity in patients with pernicious anemia as compared with those in the control group. The results of the jejunal perfusion studies are shown in Figure 1. Glucose absorption rates in patients with
Tlme of Study
ANEMIA-ARVANITAKIS
Isomaltass
are given In pmol of substrate
RESULTS
IV
IN PERNICIOUS
of Brush Border Enzymes*
water absorption rates were calculated using standard formulas [20]. In two patients with pernicious anemia, jejunal perfusion studies were repeated after treatment with monthly vitamin B12 injections (100 pg). The treatment period was approximately 18 months, and there was considerable hematologic and clinical improvement as compared to the pretreatment period.
TABLE
FUNCTION
f 3.0 f 2.6
Anemia’ Sodlum (meqlhrl segment) -42.0 -31.6
f 22 f 26
Water (mllhrl segment) -426 -372
f f
126 165
the pre- and post-treatment absorption rates of sodium and water. of solute and water from plasma to lumen.
i-
l
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The American Journal of Medicine
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661
INTESTINAL ABSORPTIVE
AND DIGESTIVE FUNCTION IN PERNICIOUS ANEMIA-ARVANITAKIS
GLUCOSE
ET AL
SODIUM 300
-I-
200
m
CONTROLS
D
PA
I
I
disaccharidases. These histologic and enzymatic defects diminished after treatment with vitamin B12. We were unable, however, to demonstrate any significant reduction in any of the brush-border enzymes in jejunal biopsy specimens. It may be that alterations in the brush-border enzymes are related to the severity and duration of pernicious anemia, the nutritional status of the patients and their dietary intake. These factors probably did not play a significant role in our patients. Since nutritional changes and morphologic abnormalities of the intestinal mucosa may affect the functional integrity of the intestinal epithelial cell, we examined glucose, sodium and water absorption utilizing the jejunal perfusion technic. We did not find any difference in jejunal glucose absorption rates between the patients with pernicious anemia and the control group. This is in contrast to the findings reported by Groen in 1938 [25], who demonstrated impaired intestinal glucose absorption in three patients with pernicious anemia. In his study, in which the Miller-Abbott technic of intestinal absorption was used, glucose absorption was restored to normal after vitamin B12 replacement therapy. There are several explanations to account for the difference in the results of our study. First, we utilized the segmental, triple-lumen intestinal perfusion technic which has been extensively evaluated in regard to its
662
December 1977
The American Journal of Medicine
‘-600
Figure 1. Jejunal glucose, sodium and water absorption rates (mean f SEM) in control subjects and patients with pernicious anemia (PA) in the pretreatment period. Glucose absorption rates in the control group were 14.5 f 3.0 versus 15.0 f 10 mmollhourf segment in pernicious anemia (N.S); sodium absorption rates in the control group were +27.7 f 2.0 versus -42.0 f 22 meqlhourlsegment in pernicious anemia (p CO.025) and water absorption in the control group was +2 12 f 26 versus - 428 f 728 mllhourlsegment in pernicious anemia (p < 0.025), indicating net secretion into the intestinal lumen.
reproducibility and accuracy. The methodologic differences in the two studies are probably too important to allow any comparison of the results. Second, it was indicated that impaired glucose absorption in pernicious anemia was a nonspecific effect, possibly related to the low caloric intake or poor nutritional status of the patients [25], a factor which was probably not significant in our patients. The most striking observation in our study, a finding not previously reported in pernicious anemia, was the demonstration of marked inhibition of sodium and water absorption in the jejunum, in the presence of a normal jejunal mucosal biopsy specimen. Not only sodium and water absorption was completely inhibited, but also a significant net secretion of sodium and water, i.e., net loss into the intestinal lumen occurred in all three patients. Luminal accumulation of sodium and water was apparent during the intestinal perfusion studies when our patients experienced abdominal pain and distress, evidently due to luminal distention. This phenomenon has been recognized in perfusion studies in which dihydroxy bile acids were used in the perfusate resulting in intestinal fluid secretion and distention of the intestinal wall [26]. Follow-up perfusion studies in two patients after a period of treatment with vitamin B12 did not show any improvement in the absorption of sodium and water, although glucose absorption was normal as in the pretreatment period.
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INTESTINAL ABSORPTIVE AND DIGESTIVE FUNCTION IN PERNICIOUS ANEMIA-ARVANITAKIS
Sodium and water secretion into the lumen may occur in several clinical and experimental situations and in the absence of structural changes in the jejunal biopsy specimen. There are two conditions in pernicious anemia which may provide a reasonable explanation for our results. First, it is known that bacterial overgrowth in the jejunum commonly occurs in pernicious anemia [27]. Overgrowth in the lumen results in deconjugation of bile acids [28] which exert an inhibitory effect on intestinal electrolyte and water absorption [29]. Second, bacterial overgrowth may contribute to the formation of hydroxy-fatty acids [30], which are not ordinarily present in the diet, but they originate from bacterial hydroxylation of free fatty acids. Hydroxy-fatty acids exert a cathartic effect on intestinal mucosa associated with increased cellular permeability and intestinal fluid secretion [31]. It is of interest that glucose absorption was not influenced by sodium secretion, as may have been expected, because of a proposed stoichiometric relationship between glucose and sodium absorption. It is possible that the sites of glucose absorption in the absorptive epithelial cell may be different from those of sodium secretion, since glucose absorption may con-
ET AL.
tinue to occur in the presence of sodium secretion into the intestinal lumen [32]. The clinical significance of our observation is not clear, since none of the patients presented with secretory diarrhea or other electrolyte disorder. The fact, however, that this absorptive defect was not a reversible abnormality after treatment with vitamin B12 suggests that it represents a primary intestinal manifestation of pernicious anemia, not necessarily related to the anemia per se. A recent study by Cook [33], showed that the presence of hypochromic or megaloblastic anemia does not affect glucose, glycine or folic acid absorption, whereas xylose absorption is impaired. This observation lends some support ‘to the contention that luminal events (i.e., bacterial overgrowth, hydroxy-fatty acid formation or bile salt deconjugation) may account for the secretion of sodium and water in pernicious anemia. ACKNOWLEDGMENT
We wish to thank Dr. William Larson for permission to study patients under his care and Mrs. Sandra Walker for her secretarial assistance.
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Dahlqvist A: Assay of intestinal disaccharidases. Anal Biothem 22: 99, 1968. Goldbarg JA, Rutenburg AM: The colorirnetric determination of leucine amino peptidase in urine and serum of abnormal subjects and patients with cancer (and other diseases. Cancer 11: 283, 1958. Eichholz EA, Crane RK: Studies on the organization of the brush-border in intestinal epithelial cells. I. Tris disruption of isolated hamster brush borders and density gradient separation of proteins. J Cell Biol 26: 687, 1965. Lowry OH, Rosenbrough NJ, Farr AL, et al.: Protein measurement with folin phenol reagent. J iBiol Chem 193: 265, 1951. Cooper H, Levitan R, Fordtran JS, et al.: A method for studying absorption of water and solute from the human small intestine. Gastroenterology 66: 1, 1966. Huggett A St G, Nixon DA: Use of glucose oxidase peroxidase and odianisidine in determination of blood and urinary glucose. Lancet 2: 368, 1957. Malawer SJ, Powell DW: An improved turbidimetric analysis of polyethylene glycol utilizing an emulsifier. Gastroenterology 53: 250, 1967. Whalen GE, Harris JA, Geenen JE, et al.: Sodium and water absorption from the human small intestine. Gastroenterology 51: 975, 1966. Berzman A, Kinnear DG, Zamcheck N: D-xylose and potassium iodide absorption and serum carotene in pernicious anemia. J Lab Clin Med 53: 226, 1959. Brcdy EA. Estren S, Herbert V: Coexistent pernicious anemia and malabsorption in four patients: including one whose malabsorption disappeared with vitamin B12 therapy. Ann Intern Med 64: 1246, 1966. Lindenbaum J, Peuimenti JF, Shea N: Small intestinal function
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INTESTINAL
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864
ABSORPTIVE
AND DIQESTIVE
FUNCTION
IN PERNICIOUS
In vltamln B12 deflclency. Ann Intern Med 80: 326, 1974. Carmel R, Herbert V: Correctable lntestlnal defect of vltamln Bll malabsorption In pernlclous anemla. Ann Intern Med 67: 1201, 1967. Ciroen J: The absorptlon of glucose from the small lntestlne In deflclency disease. N Engl J Med 218: 247, 1938. Wlngate DL, Hyams A, Phllllps SF: Lumlnal dlstentlon as a possible consequence of experlmental Intestinal perfuslon. Gut 15: 728, 1974. Sherwood WC, Goldstein F, Hauranl FE, et al.: Studles of the small lntestlnal bacterial flora and of lntestlnal absorptlon In pernicious anemla. Am J Dig Dls 9: 416, 1984. Tabaqchall S, Hatzloannou J, Booth CC: Bile salt deconjugatlon wlth steatorrhea In patlents wlth stagnant-loop syndrome. Lancet 2: 12, 1968. Russell RI, Allan JG, Gerskowltch VP, et al.: The effect of
December 1977
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ANEMIA-ARVANITAKIS
30. 31.
32.
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ET AL.
conjugated and unconjugated bile acids on water and electrolyte absorptlon In the human Jejunum. Clln Scl45: 301, 1973. Thomas PJ: ldentlflcatlon of some enterlc bacteria which convert olelc acid to hydroxystearlc acid In vltro. Qastroenterology 62: 430, 1972. Ammon HV, Thomas PJ, Phllllps SF: Effects of olelc and rlclnolelc acids on net jejunal water and electrolyte movement. Perfuslon studies In man. J Clln Invest 53: 374, 1974. Wlngate DL, Phllllps SF, Hofmann AF: Effect of glyclne conjugated bile acids wlth and wlthout lecithin on water and glucose absorption In perfused human jejunum. J Clln Invest 52: 1230, 1973. Cook GG: Absorption of xylose, glucose, glyclne and follc (pteroylglutamlc) acid In Zamblan Africans wlth anemia. Gut 17: 604, 1976.