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
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
