Apolipoprotein Localization and Quantitation in the Human Intestine

GASTROENTEROLOGY 1982;83:1223-30 Apolipoprotein Localization and Quantitation in the Human Intestine PETER H. R. GREEN, JAY H. LEFKOWITCH, ROBERT M.G...
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GASTROENTEROLOGY 1982;83:1223-30

Apolipoprotein Localization and Quantitation in the Human Intestine PETER H. R. GREEN, JAY H. LEFKOWITCH, ROBERT M.GLICKMAN, JOHN W. RILEY, ELAINE QUINET, and CONRAD B. BLUM Departments of Medicine and Pathology, College of Physicians & Surgeons, Columbia University, New York, New York

Apolipoproteins B, A-I, and A-N were localized in human intestinal epithelium using immunoperoxidase techniques. Staining was most obvious in villus tip cells. Lipid absorption resulted in an increase in intraepithelial staining for each apoprotein. The pattern for apo-B in the biopsy specimens taken after lipid absorption revealed a marked redistribution of staining to the intercellular spaces and an increase in the supranuclear staining of apo-A-I and apo-A-N. After lipid absorption, staining appeared to extend further down the villus than in the fasting biopsy specimens. Quantitation of apo-A-I and apoA-N in isolated epithelial cells confirmed that the mass of these apoproteins increases in response to lipid absorption. Apolipoprotein Band apo-A-I were absent in the epithelium of 3 patients with abetalipoproteinemia while apo-A-N was present in 2 patients. These studies demonstrate differences in the localization and quantitation of apoproteins in the villus-crypt unit as well as differences in the localization pattern of the different apoproteins. The intestine has recently been recognized as an important site of lipoprotein formation. Studies in rats and humans have revealed that chylomicrons and very low density lipoproteins (VLDL) are secreted by the intestine (1-4). In addition, rat intestine secretes at least one variety of nascent high density lipoproteins (HDL) (5,6). Human and rat intestine actively synthesizes major apolipoproteins, apo-B, apo-A-I, apo-A-II, and apo-A-IV (7-12). Quantitation of the output of apo-A-I, the principal apoprotein of Received February 17, 1982. Accepted July 13, 1982. Address requests for reprints to: Peter H. R. Green, Departments of Medicine, College of Physicians & Surgeons, Columbia University, 630 West 168th Street, New York, New York 10032. This work was supported by grants AM 21367-04, HL 21006-05 Sc.F, and HL 26500-01. © 1982 by the American Gastroenterological Association 0016-5085/82/121223-08$02.50

plasma HDL, in chylous urine (13) and thoracic duct lymph (14) has revealed that the intestine is a major source of apo-A-I for plasma HDL in humans. The importance of intestinal apo-B synthesis during lipid absorption is underscored by the disease abetalipoproteinemia in which there is a genetically determined failure of apo-B synthesis (11) and resultant inability to secrete chylomicrons and VLDL (15). We have previously localized apoproteins in isolated human intestinal mucosal cells using immunofluorescence techniques (10-12). This method, however, does not provide information as to the apoprotein distribution in the intestinal epithelium (crypt vs. villus). We therefore used immunoperoxidase techniques (16) on fixed tissue sections in order to localize apoproteins in human intestinal biopsy specimens and have correlated the findings with quantitation of apoproteins in isolated intestinal epithelial cells.

Methods Intestinal biopsies were performed on 12 male volunteers after informed written consent was obtained. The studies were approved by the Institutional Review Board of Columbia University College of Physicians & Surgeons. Biopsy specimens were obtained with either a Crosby capsule or a four-port Rubin tube that was positioned radiologically at the ligament of Trietz. Biopsy specimens were obtained after a 12-h fast and again 45-60 min after 100 ml of corn oil was instilled directly into the duodenum. Biopsy specimens were either placed in 10% formalin for fixation and were used for immunoperoxidase localization studies or placed into cold phosphate-buffered saline (PBS, pH 7.2) for isolation of intestinal epithelial cells (17). Intestinal biopsy specimens were also obtained from 3 patients with abetalipoproteinemia. The diagnosis was confirmed by the immunochemical absence of apo-B in plasma. These patients were under the care of Dr. Robert

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Glickman, Dr. Claude Roy, and Dr. Fred Daum, and have not previously been reported. Ileal biopsy specimens were obtained from 3 patients with ileostomies using the Rubin tube. Two patients had colectomies in association with ulcerative colitis and 1 because of Crohn's disease. In the latter patient, there was no evidence of recurrent ileal disease. Biopsy specimens were obtained approximately 15 cm from the stoma.

Immunoperoxidase Localization of Apoproteins Apoproteins were localized in fixed tissue sections of intestinal biopsy specimens using a modification of the peroxidase-antiperoxidase (PAP) technique (16). Tissue sections were initially deparaffinized and hydrated by sequential immersion in xylenes and graded alcohols (18). The sections were incubated in a hydrogen peroxide, methanol solution (0.3% H 20 2 in 95% methanol) for 30 min to eliminate endogenous peroxidase activity (16). The tissue was first incubated with undiluted normal goat serum containing 50 JLl/ml Trasylol (FBA Pharmaceuticals, New York, N.Y.) at 37°C for 15 min. After rinsing in PBS, the primary antibody was applied, followed by sequential application of goat anti rabbit immunoglobulin G (IgG) (Cappel Lab Inc., Cochranville, Pa.) and rabbit PAP (Cappel Lab) with extensive washing in PBS between applications. The peroxidase was localized by immersing the sections for 8 min at room temperature in a saturated solution of 3-3/ -diaminobenzidine (50 mg of free base, Sigma Chemical Co., St. Louis, Mo.) in 100 ml of PBS with added hydrogen peroxide (0.003%) (18). Sections were then washed and lightly counterstained with hematoxylin. In order to localize apo-B, two antisera were used. Antisera to apo-B obtained from Behring Diagnostics, Somerville, N.J., and antisera prepared in rabbits to native low density lipoprotein (LDL) (11) were used. Each of the antisera gave a single arc of identity when reacted against purified normal LDL, and normal serum. Antiserum was absorbed with normal LDL as a control until no further reaction was seen on Ouchterlony analysis. Apolipoprotein A-I and apo-A-IV were localized using antisera prepared to purified apo-A-I and apo-A-IV (10,12). Antisera which had been absorbed with the appropriate apoprotein (purified apo-A-I, apo-A-IV), nonimmune rabbit serum, and phosphate-buffered saline were substituted for the primary antisera to serve as controls. The slides were examined by two observers. Biopsy specimens were also later coded and examined blindly.

GASTROENTEROLOGY Vol. 83, No.6

a Willem Polytron (Brinkman Instruments, Westbury, N.Y.). An aliquot of the homogenized cells was removed for protein determination using the method of Lowry et al. (19). The homogenate was then centrifuged at 100,000 g for 60 min in a Beckman L5-75 ultracentrifuge using a 50 Ti rotor. In order to quantitate apo-A-I, cells were homogenized in a 50 mM sodium decyl sulfatelPBS buffer and apo-A-I was quantitated by radioimmunoassay. Apolipoprotein A-I was purified by diethylaminoethanol (DEAE) cellulose chromatography from the apoproteins of HDL (20). The apo-A-I was radioiodinated by the chloramine T procedure as described (21). All samples were preincubated overnight in a solution of 50 mM sodium decyl sulfate before assay. The assay was performed in a final concentration of 5 mM sodium decyl sulfate, 40 mM sodium phosphate, 100 mM NaCl, 0.02% sodium azide, 0.04% nonimmune rabbit serum, 0.04% antiserum to apo-A-I, pH 7.4, and 30,000 cpm of 125I_apo_A_I. Goat antirabbit serum was added after a 48-h incubation, and the following day the assay was harvested by centrifugation. Previous deli pidation with organic solvents did not affect the immunoassayable apo-A-I in plasma or lipoprotein fractions, probably reflecting the fact that the samples were routinely incubated in sodium decyl sulfate before assay. Standard curves were prepared from a calibrated plasma pool which was stored in ampoules at -80°C. The plasma was calibrated using a primary standard of purified apo-A-I that had been quantitated by the method of Lowry et al. (20). The within assay coefficient of variation was 6% and the coefficient of variation for systemic between assay variability was 9.0%. The sensitivity of the assay was 2 ng. Cross-reactivity from apo-A-II, apo-C-III, apo-B, and apo-E were

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