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Measurement of Prostaglandin E2 in Rectal Mucosa in Human Subjects: a Method Study1

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Measurement of Prostaglandin E2 in Rectal Mucosa in Human Subjects: a Method Study1 Vol. 4, 239-244, April/May 1995 Cancer Epidemiology, Biomarkers & Prevention 239 Measurement of Prostaglandin E2 in Rectal Mucosa in Human Subjects: A Method Study1 ;;Ii;:I-R. Finley, Cheryl 1. Bogert, David S. Alberts,2 adults with incidence increasing to as high as 50% in Janine Einspahr, David 1. Earnest, Gina Blackwell, persons over 70 years of age (1 ). Multiple studies have and Ken Girodias shown that certain NSAIDs3 inhibit cyclooxygenase and the Section of Clinical Pathology, Department of Pathology (P. R. Fl. Arizona synthesis of prostaglandmns and can reduce the formation of Cancer Center IC. L. B., D. S. A., J. E., G. B., K. G.l, Section of both colon polyps and cancers in experimental animals Hematology and Oncology, Department of Medicine ID. S. Al, given known carcinogens (2, 3). Two recent case-control and Section of Gastroenterology, Department of Medicine ED. L. E., G. B.l, drug surveillance studies and one large cohort study found University of Arizona, College of Medicine, Tucson, Arizona 85724 that patients with regular aspirin use had a reduced mci- dence or decreased death rate from colorectal cancer (4- Abstract 6). Most recently, the results of a randomized clinical trial showed that the NSAID Sulindac promotes regression and It has been demonstrated and confirmed that certain inhibits recurrence of adenomatous colon polyps in patients nonsteroidal anti-inflammatory drugs which inhibit with familial adenomatous polyposis (7). cyclooxygenase and the synthesis of prostaglandins and Further evidence that there is a relationship between other eicosanoids, can reduce the formation of both the concentration of PGE2 in tissue and the development of colon polyps and cancers in experimental animals given colonectal cancer was reported by Earnest et a!. (8). Tissue known carcinogens. Additionally, the results of several concentration of PGE2 was significantly increased in cob- epidemiologic studies have suggested that nonsteroidal rectal adenomas compared to normal flat mucosa and was anti-inflammatory drugs may reduce the risk of colon even greater (P< 0.05) in malignant colon polyps and gross polyp occurrence and/or colon cancer mortality. We cancer. Nanisawa et a!. (9) showed a high level of PGE2 in have carried out a study to evaluate the methodology of local venous blood-draining colon carcinomas and in the the measurement of prostaglandin E2 (PGE2) in human peripheral blood in patients with liver or lung metastases. colonic mucosa because its concentration may serve as a Tissue analysis showed a significantly larger amount of valuable intermediate marker of the pharmacological PGE2 production in carcinomatous versus normal cobonic activity in Phase II studies of nonsteroidal anti- mucosal tissue. These investigators suggested that increased inflammatory drugs as colon cancer preventive agents. local blood PGE2 could enhance metastasis formation, and We studied all aspects of the actual measurement of that increased peripheral blood PGE2 might be useful in the PGE2 including the extradion efficiency of the PGE2 detection of such metastases in colorectab cancer. from the mucosa, the precision of the assay and The purpose of this study was to establish strict criteria calculation of the PGE2 content in terms of milligrams of for the measurement of PGE2 in normal appearing rectal protein in the sample, the inhibition of PGE2 by mucosal biopsies and to control all ofthe variables that may indomethacin over time, the reproducibility of the affect the accuracy and precision of the assay. Such accu- measurement within one homogenate, the rate of PGE2 racy and precision are critical to the validity of prospective production over time, the effect of adding indomethacin Phase II cancer prevention trials that use tissue bevels of versus snap freezing on PGE2 production, the stability of PGE2 as an intermediate marker of NSAID pharmacological PGE2 fl tissues over time stored in liquid nitrogen, and effect. the variability of the measurement of PGE2 in separate biopsies from one individual. Our studies indicated that the most reliable method for accurate and consistent Materials and Methods measurements of PGE2 was to add the mucosal tissue Colon Biopsy Sample Collection. Samples were collected instantly after biopsy to an indomethacin buffer that by one of several assisting gastroenterobogists working with effectively inhibited the in vitro formation of PGE2. us. Patients were prepped using two 1 50-mb tap water enemas 30 min-2 h before the procedure. After the sig- moidoscope was inserted into the rectum, biopsies were Introduction taken perpendicular to the mucosal surface from the upper Cancer of the colon is the second most prevalent malig- half of the rectum (1 2-1 8 cm from the anus). Because the nancy in the United States. There is strong evidence that amount of endogenous PGE2 can be determined by the adenomatous colon polyps are precursor lesions of colon depth of the biopsy (1 0), routine-sized forceps were used to cancer. Such polyps are found in approximately 10% of ensure a continuity of biopsy depth into the mucosa. Samples were placed in either empty cryovials or cryoviabs Received 6/28/94; revised 12/21/94; accepted 12/21/94. This work was supported in part by NIH Grants CA-41 1 08 and CA-23074. 2 To whom requests for reprints should be addressed, at Arizona Cancer 3 The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; Center, 1 501 N. Campbell Avenue, Tucson, AZ 85724. PGE2, prostaglandmn E2; RIA, radioimmunoassay. Downloaded from cebp.aacrjournals.org on October 1, 2021. © 1995 American Association for Cancer Research. 240 Measurement of PGE2 in Rectal Mucosa in Human Subjects containing aqueous indomethacin and snap frozen in liquid [3H]prostaglandin tracers (1 2). Appropriate dilutions were nitrogen with approximately the same time period between made to the samples to fit the values into the curve range. biopsy removal and freezing for each biopsy. Aqueous Samples were counted on the Micromedic 1 0/6000 Plus indomethacin is Indocin IV. (Merck, Sharp, & Dohme, Apex Series gamma counter (Micromedic Systems Division, West Point, PA) dissolved in double distilled water to a final ICN Biomedicals, Inc., Costa Mesa, CA). The standard concentration of 5 pg/mI. All tissue samples remained curve was reduced using four-parameter logit curve fitting. stored in liquid nitrogen until the time ofexpenimentation or Protein Assay. Protein aliquots were analyzed using the extraction. A single biopsy had an approximate wet weight bicinchoninic acid assay (Pierce, Rockfond, IL). When com- of 1 7.9 mg (range, 1 2.0-22.0 mg). Only biopsies stored in pared to other protein determination methods, the bicin- aqueous indomethacin were weighed. choninic acid assay provides improved sensitivity, easily Tissue Homogenization. Tissue was homogenized in either modified assay parameters, less difficulty with interfering ice-cold (0-4#{176}C) extraction buffer or ice-cold indometha- substances, and increased stability in the working reagent cm buffer depending on the experiment to be performed. (1 3). Due to the small amount of sample taken for protein Extraction buffer is 0.05 M Tnizma base (Sigma Chemical determination, the microtiter plate protocol was used. Be- Co., St. Louis, MO) titrated to pH 7.4 with concentrated sides saving on reagents and providing a greater number of hydrochloric acid (FisherChem, Fair Lawn, NJ). Indometha- protein values per sample (6 wells/sample), this protocol cm buffer is Indocin IV. dissolved in extraction buffer to a allows for a sensitivity that is approximately 10-fold greaten final concentration of 5, 1 0, or 20 pg/mb depending on the than the manufacturer’s standard test tube assay procedure procedure to be followed. Tissue was homogenized by (1 4). The standard protein concentration ranges used were hand in a siliconized (reagent grade Sigmacote; Sigma) 200-1200 and 10-200 pg/mI. Appropriate dilutions were Duall glass-glass tissue grinder (Kontes Glass, Vineland, CT) made to the samples as needed to fit the values within an for approximately 30 s or until there were no visible pieces assay curve. The plates were incubated at 37#{176}Cfor 30 mm of tissue. When multiple rectal mucosal biopsies were ho- and read immediately on the Biomek 1000 (Beckman In- mogenized together, they immediately were diluted after struments, Inc., Fullerton, CA) at an absorbance wavelength homogenization in additional indomethacin buffer to attain of 540 nm. a consistent ratio of 1-2 biopsies/mI buffer. Extradion Efficiency [1251]. 125I-PGE2 (0.1 ml) from the Sample Extraction. The extraction procedure used was the RIA kit was diluted with extraction buffer to a final volume procedure outlined by Powell (1 1 ). The homogenate was of 2.0 ml. Two 0.1 -ml aliquots were placed in two test tubes placed in a 50.0-mb polypropylene tube. Two milliliters and capped (total counts). Two additional 0.1-mb aliquots 1 00% ethanol (Quantum Chemical Corp., Cincinnati, OH) were diluted with extraction buffer to a final volume of 1 .0 was added and the sample was vortexed and allowed to ml and extracted. Both samples were ebuted with 10.0 ml stand on ice for 5 mm. A 100-mg barge capacity reservoir methyl fonmate into three separate tubes. Each tube was C18 column (Alltech Associates, Inc., Deerfield, IL) was evaporated to dryness under nitrogen and all tubes were washed with i00% ethanol (20 ml) followed by double counted. The three values for the three tubes from each deionized water (20 ml) to remove excess ethanol. After sample were added. The two values from the total counts adding 1 0.3 ml ice-cold double deionized water to the were averaged. The two values from the extracted samples tissue homogenate to give a final concentration of 1 5% were averaged. The extraction efficiency was calculated. ethanol, the sample was vortexed vigorously and centni- fuged at 4#{176}Cat3000 rpm for 10 mm.
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