Measurement of Prostaglandin E2 in Rectal Mucosa in Human ... · Radioimmunoassay. The 112511 PGE2...

Vol. 4, 239-244, April/May 1995 Cancer Epidemiology, Biomarkers & Prevention 239

3 The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug;PGE2, prostaglandmn E2; RIA, radioimmunoassay.

Measurement of Prostaglandin E2 in Rectal Mucosa in HumanSubjects: A Method Study1

;;I�i;:I-R. Finley, Cheryl 1. Bogert, David S. Alberts,2Janine Einspahr, David 1. Earnest, Gina Blackwell,and Ken Girodias

Section of Clinical Pathology, Department of Pathology (P. R. Fl. Arizona

Cancer Center IC. L. B., D. S. A., J. E., G. B., K. G.l, Section ofHematology and Oncology, Department of Medicine ID. S. Al,

and Section of Gastroenterology, Department of Medicine ED. L. E., G. B.l,

University of Arizona, College of Medicine, Tucson, Arizona 85724

Abstract

It has been demonstrated and confirmed that certainnonsteroidal anti-inflammatory drugs which inhibitcyclooxygenase and the synthesis of prostaglandins andother eicosanoids, can reduce the formation of bothcolon polyps and cancers in experimental animals givenknown carcinogens. Additionally, the results of severalepidemiologic studies have suggested that nonsteroidalanti-inflammatory drugs may reduce the risk of colonpolyp occurrence and/or colon cancer mortality. Wehave carried out a study to evaluate the methodology ofthe measurement of prostaglandin E2 (PGE2) in humancolonic mucosa because its concentration may serve as avaluable intermediate marker of the pharmacologicalactivity in Phase II studies of nonsteroidal anti-inflammatory drugs as colon cancer preventive agents.We studied all aspects of the actual measurement ofPGE2 including the extradion efficiency of the PGE2from the mucosa, the precision of the assay andcalculation of the PGE2 content in terms of milligrams ofprotein in the sample, the inhibition of PGE2 byindomethacin over time, the reproducibility of themeasurement within one homogenate, the rate of PGE2production over time, the effect of adding indomethacinversus snap freezing on PGE2 production, the stability ofPGE2 �fl tissues over time stored in liquid nitrogen, andthe variability of the measurement of PGE2 in separatebiopsies from one individual. Our studies indicated thatthe most reliable method for accurate and consistentmeasurements of PGE2 was to add the mucosal tissueinstantly after biopsy to an indomethacin buffer thateffectively inhibited the in vitro formation of PGE2.

Introduction

Cancer of the colon is the second most prevalent malig-nancy in the United States. There is strong evidence thatadenomatous colon polyps are precursor lesions of colon

cancer. Such polyps are found in approximately 10% of

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

Center, 1 501 N. Campbell Avenue, Tucson, AZ 85724.

adults with incidence increasing to as high as 50% in

persons over 70 years of age (1 ). Multiple studies haveshown that certain NSAIDs3 inhibit cyclooxygenase and thesynthesis of prostaglandmns and can reduce the formation ofboth colon polyps and cancers in experimental animalsgiven known carcinogens (2, 3). Two recent case-controldrug surveillance studies and one large cohort study found

that patients with regular aspirin use had a reduced mci-dence or decreased death rate from colorectal cancer (4-6). Most recently, the results of a randomized clinical trialshowed that the NSAID Sulindac promotes regression andinhibits recurrence of adenomatous colon polyps in patientswith familial adenomatous polyposis (7).

Further evidence that there is a relationship betweenthe concentration of PGE2 in tissue and the development ofcolonectal cancer was reported by Earnest et a!. (8). Tissueconcentration of PGE2 was significantly increased in cob-rectal adenomas compared to normal flat mucosa and waseven greater (P< 0.05) in malignant colon polyps and grosscancer. Nanisawa et a!. (9) showed a high level of PGE2 inlocal venous blood-draining colon carcinomas and in theperipheral blood in patients with liver or lung metastases.Tissue analysis showed a significantly larger amount ofPGE2 production in carcinomatous versus normal cobonicmucosal tissue. These investigators suggested that increasedlocal blood PGE2 could enhance metastasis formation, andthat increased peripheral blood PGE2 might be useful in thedetection of such metastases in colorectab cancer.

The purpose of this study was to establish strict criteria

for the measurement of PGE2 in normal appearing rectalmucosal biopsies and to control all ofthe variables that mayaffect the accuracy and precision of the assay. Such accu-racy and precision are critical to the validity of prospectivePhase II cancer prevention trials that use tissue bevels ofPGE2 as an intermediate marker of NSAID pharmacologicaleffect.

Materials and Methods

Colon Biopsy Sample Collection. Samples were collectedby one of several assisting gastroenterobogists working withus. Patients were prepped using two 1 50-mb tap waterenemas 30 min-2 h before the procedure. After the sig-moidoscope was inserted into the rectum, biopsies weretaken perpendicular to the mucosal surface from the upperhalf of the rectum (1 2-1 8 cm from the anus). Because theamount of endogenous PGE2 can be determined by thedepth of the biopsy (1 0), routine-sized forceps were used toensure a continuity of biopsy depth into the mucosa.Samples were placed in either empty cryovials or cryoviabs

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containing aqueous indomethacin and snap frozen in liquid [3H]prostaglandin tracers (1 2). Appropriate dilutions werenitrogen 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 Plusindomethacin 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 standardconcentration 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 orextraction. A single biopsy had an approximate wet weightof 1 7.9 mg (range, 1 2.0-22.0 mg). Only biopsies stored inaqueous indomethacin were weighed.

Protein Assay. Protein aliquots were analyzed using thebicinchoninic acid assay (Pierce, Rockfond, IL). When com-pared to other protein determination methods, the bicin-choninic acid assay provides improved sensitivity, easily

Tissue Homogenization. Tissue was homogenized in eitherice-cold (0-4#{176}C) extraction buffer or ice-cold indometha-

modified assay parameters, less difficulty with interferingsubstances, 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 concentratedhydrochloric acid (FisherChem, Fair Lawn, NJ). Indometha-

sides saving on reagents and providing a greater number ofprotein 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 theprocedure to be followed. Tissue was homogenized byhand in a siliconized (reagent grade Sigmacote; Sigma)Duall glass-glass tissue grinder (Kontes Glass, Vineland, CT)for approximately 30 s or until there were no visible piecesof tissue. When multiple rectal mucosal biopsies were ho-mogenized together, they immediately were diluted afterhomogenization in additional indomethacin buffer to attain

than the manufacturer’s standard test tube assay procedure(1 4). The standard protein concentration ranges used were200-1200 and 10-200 pg/mI. Appropriate dilutions weremade to the samples as needed to fit the values within anassay curve. The plates were incubated at 37#{176}Cfor 30 mmand read immediately on the Biomek 1000 (Beckman In-struments, Inc., Fullerton, CA) at an absorbance wavelengthof 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 theprocedure outlined by Powell (1 1 ). The homogenate wasplaced in a 50.0-mb polypropylene tube. Two milliliters1 00% ethanol (Quantum Chemical Corp., Cincinnati, OH)was added and the sample was vortexed and allowed tostand on ice for 5 mm. A 100-mg barge capacity reservoirC18 column (Alltech Associates, Inc., Deerfield, IL) waswashed with i00% ethanol (20 ml) followed by double

deionized water (20 ml) to remove excess ethanol. Afteradding 10.3 ml ice-cold double deionized water to thetissue homogenate to give a final concentration of 1 5%

RIA kit was diluted with extraction buffer to a final volumeof 2.0 ml. Two 0.1 -ml aliquots were placed in two test tubesand capped (total counts). Two additional 0.1-mb aliquotswere diluted with extraction buffer to a final volume of 1 .0ml and extracted. Both samples were ebuted with 10.0 mlmethyl fonmate into three separate tubes. Each tube wasevaporated to dryness under nitrogen and all tubes werecounted. The three values for the three tubes from eachsample were added. The two values from the total countswere averaged. The two values from the extracted sampleswere averaged. The extraction efficiency was calculated.

ethanol, the sample was vortexed vigorously and centni-fuged at 4#{176}Cat 3000 rpm for 10 mm. The supemnatant wasremoved, the pellet discarded, and the pH of the supemna-

x 100

tant adjusted to 3.0 with 0.25 M hydrochloric acid. Thesample was then applied to the prepared column and ebuted The extraction efficiency for this experiment was 92%.under gentle vacuum with a Vac-Elut (Vanian Sample Prep-aration Products, Harbor City, CA). The column was rinsedwith 1 5% ethanol (20 ml) followed by 20.0 ml reagentgrade petroleum ether (EM Science, Gibbstown, NJ). The

Extradion Efficiency [3H]. 1 5 p1 [3HIPGE2 (DuPont NewEngland Nuclear) was diluted with extraction buffer to afinal volume of 4.0 ml. One 1 .0-mI aliquot was placed in ascintillation vial and 10.0-mb scintillation cocktail was

PGE7 was gravity eluted with 1 0.0 ml reagent grade methylformate (Sigma) into a siliconized glass or polypropylenetest tube. The entire elution process occurred at a rate of

added. Two additional 1 .0-mI abiquots were extracted. Thetwo samples were eluted into two separate scintillation vialsand 1 0.0-mI scintillation cocktail were added to each. Each

approximately 0.5 mI/mm. The methyl formate was dividedinto four 2.5-mI aliquots and evaporated to dryness under agentle stream of nitrogen using a Mini-Vap Six-port con-centrator/evaporator (Chemical Research Supplies, Addi-son, IL). Samples were stored at -80#{176}Cuntil the time oftheexperiment when they were reconstituted in assay bufferfrom the RIA kit. All tubes and glassware used during the

of the three samples was counted for 1 mm on a BeckmanLS 3801 liquid scintillation counter (Beckman Instruments,

Inc.). The values for the two eluted samples were averagedand the extraction efficiency was calculated. Extractionefficiency was calculated in the same manner as the 1251

efficiency experiment. The extraction efficiency for thisexperiment was 88%.

extraction were polypropylene or siliconized glass.

Radioimmunoassay. The 112511 PGE2 RIA kit (DuPont NewEngland Nuclear, Boston, MA) was used to determine thePGE2 content in the extracted samples and was performedas per the manufacturer’s instructions. An iodinated PGE2RIA was used due to a 25-30-fold increased sensitivity over

gas liquid chromatography coupled to mass spectrometryand a 5-10-fold increase in sensitivity as compared to

PGE2 Controls. Five milliliters each of 25, 100, and 200pg/mb PGE2 standard from the RIA kit were extracted.After the elution step, the methyl formate eluent wasplaced in 0.5-mI aliquots in labeled microfuge tubesusing a positive displacement pipettor. The samples wereevaporated to dryness under nitrogen and stoned at-20#{176}C. Each time an experiment was run, one samplefrom each concentration was removed from the freezer,

240 Measurement of PGE2 in Rectal Mucosa in Human Subjects

Extraction efficiency =Total extracted counts

Total counts



3000C

.� 25000� 2000

E 1500

�1o00

�500

0 5 10 15 20 25 30 35 40 45 50 55 60

TIme (MInutes)

Fig. 1. PGE2 production rate in rectal mucosal tissue with addition of 5 or

10 pg/mI indomethacin. Five or 10 pg/mI indomethacin was added after 0,

5, 1 5, 30, or 60 mm of incubation.

Cancer Epidemiology, Biomarkers & Prevention 241

- 0- 5p�?mL Indomethacln

-O-lOpglmL Indomethecln

reconstituted in 0.25 ml RIA assay buffer, and assayedwith the extracted samples.

The control values as well as all extracted sampleswere corrected using an average 90% extraction efficiencyas determined by the 1251 and 3H extraction efficiencyexpeni ments.

pg PGE2/mg proteinCorrected sample value =

0.9

Calculation of PGE2/mg protein

= pg PGE2/ml sample mg protein/mb sample

Results

Reproducibility, Sensitivity, and Linearity. The corrected25 pg/mI control value was 25.7 ± 2.0 (1 SD). Thecorrected 1 00 pg/mI control value was 93.7 ± 7.82 (1SD). The corrected 200 pg/mI control value was 199 ±

23.1 (1 SD). These values were obtained over a period of1 8 months with 23 separate runs. The lowest detectablelimit was demonstrated to be 2.5 pg/mI. The analyticalrange of the RIA is 2.5-250 pg/mI. Several suitable dilu-tions of the homogenate are made in each run to insurethat results fall within the measurable analytical range.Serial dilutions of homogenates from many patient sam-pIes show linearity on the assay within the confines of theanalytical range.

PGE2 Inhibition by Indomethacin over Time. Fifteen rectalmucosal biopsies from one patient were homogenized in3.5-mI ice-cold (0-4#{176}C) extraction buffer for 1 .0 mm, andthe homogenate was vortexed. A 2.0-mb homogenate ali-quot was immediately placed in 4.0 ml extraction buffer(sample 1 ). This aliquot remained on ice for the duration ofthe incubation. At 0, 5, 15, 30, and 60 mm, two 0.5-mbabiquots were removed from sample 1 . One aliquot wasplaced in 0.5 ml 10 pg/mI indomethacin buffer and theother placed in 0.5 ml 20 pg/mI indomethacin buffer. Afterthe final time point was reached, five SO-p1 abiquots wereremoved from the remaining sample 1 homogenate forprotein determination. The protein abiquots were diluted

with 50 p1 double deionized water to give the same tissueconcentrations as the time point samples. All samples wereextracted the same day.

Fig. 1 indicates the effects of different concentrationsof indomethacin on PGE2 production at a series of timepoints. There is no significant difference between 5 and

10 �jg/ml indomethacin in the ability to inhibit prostag-landin production.

Rate of PGE2 Produdion over Time. Five rectal mucosalbiopsies from one patient were homogenized in 1 .0 mlice-cold (0-4#{176}C) extraction buffer and diluted with addi-

tional extraction buffer to a final volume of 6.0 ml. At time0, 5, 15, 30, 45, 60, 90, 120, and 180 mm, 0.5 ml ofhomogenate was diluted in a microfuge tube in 0.5 mlindomethacin buffer (20 pg/mb). The homogenate remainedat room temperature during the 180-mm incubation. Eachsample was vortexed, snap frozen in liquid nitrogen, andstoned at -80#{176}C.At a later date, the samples were thawed

and two SO-p1 aliquots were removed from each for proteindetermination. The remaining 0.9 ml was transferred to a

SO-mi polypropylene tube and extracted. (The undilutedhomogenate remained at room temperature during the en-

tine procedure.) This experiment was repeated using tissuefrom the same patient. The tissue was homogenized at roomtemperature but was incubated at 37#{176}Cfor the remainder ofthe procedure.

Fig. 2 indicates the production of PGE2 over time in amucosal homogenate incubated either at room temperatureor at 37#{176}Cwith prostaglandin production inhibited by in-

domethacin added at specific time points. As shown, thereis no difference in PGE2 production between the two incu-

bation temperatures. Prostaglandmn production does not ap-pear to be stimulated by an increase of temperature aboveroom temperature. Figs. 1 and 2 both indicate an apparentplateau of approximately 30-60 mm after tissue removalfor maximum PGE2 production. Both figures show the rapidbut consistent linear rise in PGE2 content over the first20-30 mm of incubation. PGE2 content appears to remainstable for at least 2.5 h after the plateau is reached.

Effed of Adding Indomethacin versus Snap Freezing onPGE2 Produdion. Five rectal mucosal tissue biopsies fromthe same patient were homogenized in 1 .0-mI ice-cold(0-4#{176}C) extraction buffer, and the final volume was broughtup to 5.0 ml with additional extraction buffer. The homo-

genate remained at room temperature during the incubationperiod. At time 0, 5, 60, and 180 mm, two 0.5-mI aliquotswere removed. The first was diluted in a 1 .5-mI microfugetube in 0.5 ml aqueous indomethacin and snap frozen inliquid nitrogen. The second was diluted in a 1 .5-mI mi-crofuge tube in 0.5 ml extraction buffer and snap frozen inliquid nitrogen. All samples were thawed enough to enable

dropping the frozen pellet directly into 2.0 ml of 100%ethanol. The remainder of the extraction procedure wasperformed. Of the remaining homogenate (0.2 ml) wasdiluted with 0.2 ml extraction buffer and eight SO-pb ali-quots made for protein determination. This experiment wasrepeated three times using tissue from separate patients.

There was no significant difference in the inhibition ofPGE2 production whether indomethacin was added before

snap freezing the tissue in liquid nitrogen. The values forthe three experiments showed a wide range for 2,189.4-1 3,231 .7 pg PGE2/mg protein. When PGE2 content wasconverted to percentage of control for each of the threeexperiments (control is the PGE7 content at the 1 80-mmtime point for the snap frozen sample in that experiment),the three experiments showed similar production rates. Theuse of indomethacin allows the investigator some freedomduring the manipulation of the rectal mucosal tissue. Stor-ing the tissue(s) in indomethacin is preferable for procedural



16000

.� 14000

� 12000

0. iooooE 8000

��6000

�4000

� 2000

0

0 20 40 60 80 100 120 140 160 180 A.

Control ..

- Continuing ..- -S

Inhibition

100

�10

0803.

�

� 10

#0

g

3

I�70

0503.

E4�3

�2O

� 10

B. �

� 180

�80

g�70

050

a.

�20

� 10

C. � o

0 10 20 � 40 50 60

This (minutes)

Control #{163}

- ContinuingInhibition

�- -*-

0 10 20 � 40 50 60

Time (minutes)

...........

....�-.

� _________

0 10 20 30 40 50 60

TIme (minutes)

Fig. 3. Continuous effect of indomethacin on PGE2 production during a

60-mm incubation. PGE2 production curves were produced by adding in-domethacin after 0, 1 5, 30, and 60 mm of incubation. Continuous inhibitionby indomethacin was determined from aliquots of control curve samples atvarious time points during the incubation.


. D- 3�C

-0-Room Temperature

TIme (MInutes)

Fi�,’. 2. PGE production rate in rectal mucosal tissue homogenate incu-l)ated for 3 h at room temperature or 37CC. Indomethacin 10 �ig/ml) was

added after 0, 5, 15, 30, 45, 60, 90, 120, and 180 mm of incubation.

Production plateaus between 30-60 mm.

ease and also eliminates the potential danger of an acci-dental thaw initiating prostaglandin synthesis.

Continuous Inhibitory Effect of Indomethacin on PGE2Production over Time. Four rectal mucosal biopsies fromone patient were homogenized in 1 .0 ml ice-cold (0-4#{176}C)extraction buffer for 1 minute, and the total volume wasbrought up to 4.0 ml. A control curve for prostaglandinproduction was performed (shown as dotted lines on Fig. 3)by removing aliquots from the homogenate at 0, 1 5, 30, and60 mm and placing them in equivalent volumes of 1 0 pg/mIindomethacin buffer. Additional abiquots were removedfrom the control curve samples at specific time points (0,1 5, 30, and 60 mm) during the 1 -hour incubation to deter-mine if the inhibitory effect of indomethacin was completeand continuous over time (shown as solid lines on Fig. 3).The original homogenate remained at room temperatureduring the incubation period. Two SO-pb aliquots were me-moved from each of the ten samples after the 60 mm wascompleted for protein determination and the remaining 0.9ml was extracted. This experiment was repeated three timesas designated as panels A, B, and C on Fig. 3.

Fig. 3 indicates that indomethacin does inhibit pros-taglandin production and that this inhibition appears tocontinue over time. Due to the almost 1 0-fold difference invalues among the three experiments for the same timepoints (range for 60-mm time point, 1 ,693.4-i 1 ,61 3.3 pgPGE2/mg protein), the results are shown as a percentage ofthe maximum production for each run with the maximumproduction being defined as the prostaglandin content atthe 60-mm time point.

Stability of PGE2 in Tissue over Time Stored in LiquidNitrogen. Fifteen rectal mucosal tissue biopsies from onepatient were homogenized in 1 .0 ml 10 pg/mI indometha-cm buffer at 4#{176}C.The homogenate was diluted with anadditional 14.0 ml of indomethacin buffer (1 0 pg/mb). Four-teen 1 .0-mI aliquots were snap frozen simultaneously andstored in liquid nitrogen. At different intervals, one aliquotwas removed from the freezer, thawed slightly, placed in1 .0 ml indomethacin buffer, and vortexed until completelythawed and homogeneous. A 1 .0-mI aliquot was placed ina 50-mb polypropylene tube and two SO-pb aliquots wereremoved for protein determination. The remaining 0.9 ml ofhomogenate was extracted.

Table 1 indicates that PGE, is quite stable in tissuehomogenate with indomethacin stored in liquid nitrogen

over an extended period of time.

Control- Continuing

Inhibition

Table 1 Stability of PGE2 in tissue s tored in liquid nitrogen

Aliquot Date extracted pg PGE2/mg protein

1

2

3

4

5

6

7

8

9

10

12/3/91

1/7/92

2/10/92

8/5/92

12/9/92

2/3/93

12/17/93

12/28/93

1/12/94

1/14/94

2307.8

2332.2

2386.7

2265.5

2390.7

2826.5

2411.0

2864.3

2149.4

2663.3

2459.7 ± 241.6

CV” = 9.8%)

-, CV, coefficien t of variation.

Variability of Measurements in One Patient. Five rectalmucosal tissue biopsies from a single subject were placed inseparate tubes Containing aqueous indomethacin and snap



Cancer Epidemiology, Biomarkers & Prevention 243

Table 2 Variability of measurements in one patient. Five separate

biopsies immersed in indomethacin

Bio sp y

pg PGE2/ml of sample

Protein mg/

ml of samplepg PGE2/

mg of protein

1 117.84 0.360 363.7

2 54.36 0.345 175.1

3 77.94 0.350 247.4

4 67.12 0.285 261.7

5 38.06 0.325

0.333 ± 0.030

(CVa 9.0%)

130.1

235.6 ± 89

(CV = 38.1%)

-, CV, coo fficient of variation.

Table 3 Precision of PGE2 assay based on repeat analysis of same rectal

mucosal homogenate

Five rectal mucosal biopsies obtained from patient materials described in

Table 2 were combined for this assay precision analysis.

Aliquot pg PGE2/ Protein mg/ml of sample ml of sample

pg PGE2/

mg of protein

1 70.32 0.270

2 60.16 0.255

3 61.74 0.230

4 61.20 0.280

0.259 ± 0.022

(CV’ = 8.5#{176}I�)

289.3

262.1

298.2

242.9

273.1 ± 25.3

(CV = 9.3%)

-‘ CV, coefficient of variation.

frozen. Each sample was thawed and homogenized in 1 .0ml ice-cold (0-4#{176}C) extraction buffer. Two SO-pI aliquotswere removed from each sample for protein determination.The remaining 0.9 ml of each sample was extracted.

Table 2 shows that mucosal biopsies obtained from thesame patient within a small region of the rectum mayexhibit a wide range of prostaglandin contents (1 30.1-363.7 pg PGE2/mg protein) despite maintaining a relatively

consistent protein value (0.285-0.360 mg/mI of sample).Five additional rectal mucosal biopsies from the same

patient as above were placed in one tube containing aque-ous indomethacin and snap frozen. The biopsies werethawed and homogenized in 1 .0 ml indomethacin buffer.Indomethacin buffer was added to bring the volume to 5.0ml. Four aliquots of the homogenate were placed in sepa-rate tubes. Two SO-pI aliquots were removed from eachsample for protein determination. The remaining 0.9 ml ofeach sample were extracted.

Table 3 demonstrates the precision of the PGE2 assay.The coefficient of variation was less than 1 0% for fourrepeat analyses of the same homogenate obtained by com-bining five separate biopsies from the same patient. Thisresult indicates that the variability seen in Table 2 appearsdue to factors other than the precision of the assay. Anadditional set of six aliquots of one homogenate from adifferent patient was run (data not shown). The range invalues was 2203-2537 pg PGE2/mg protein. The mean was2332 ± 1 20.7 (1 SD) and the coefficient of variation was5.9%.

Discussion

The literature contains a few studies ofthe effects of variousNSAIDs on the inhibition of prostaglandin synthesis in therectal mucosa and other tissues (1 5). The technical details

of the actual assay measurement are lacking, to a certainextent, in many of these papers. Our goal was to examineall of the assay factors that could affect the measurement ofPGE2 in order to maximize assay accuracy and precision.The concentration of PGE2 was standardized by relating itto the protein content of the biopsy.

The extraction efficiency was carried out using both1251 and 3H. It was found to be approximately the same forboth radioisotopes with the efficiency for 1251 being 92%and the efficiency for 3H being 88%. Three different con-centrations of PGE2 were extracted from large volumes andwere assayed in each experiment. These levels were ad-justed to 25, 1 00, and 200 pg/mI, respectively. It was found(see “Results”) that when connected for extraction efficiency,all three levels of PGE2 controls were very close to theexpected levels indicating the accuracy of the assay. Whenthe stability of PGE2 in a single homogenate of rectal mu-cosal tissue was measured over a period of 1 5 months, theassay showed a coefficient of variation of 1 0% on less.

The addition of indomethacin to the tissue homoge-nates at various time points (see Figs. 1 and 2) shows thatthe production of PGE2 from endogenous anachidonicacid is fairly linear. Also, immediate immersion of therectal mucosal specimens in 5 pg/mI aqueous indo-methacin solution as soon as the biopsy is taken preventsin vitro prostaglandin production. Because PGE2 remainsstable for at least 1 5 months when stoned in liquid nitno-gen (see Table 1) and production is effectively inhibitedby indomethacin, it is reasonable to assume that theconcentration of PGE2 measured reflects the actual con-centration in vivo. Although there is no significant dif-ference in the stability of PGE2 in rectal mucosal biopsytissue oven time when stoned in liquid nitrogen withoutindomethacin versus storage with indomethacin, wechose to use the immediate immersion of the biopsy inindomethacin as the stabilizing procedure of choice be-cause this allows more freedom in the subsequent ma-nipulation of the tissue. The various experiments indicatethat the inhibition of PGE2 production is complete andthat degradation does not appear to be a problem intissue stoned in this manner.

The variability ofthe concentration of PGE2 in multiplerectal mucosal biopsies obtained at the same level of therectum from a single individual during a single sigmoidos-copy is well illustrated in Table 2. Even though the PGE2concentrations were adjusted for the protein concentrationof the sample, the variability is substantial (coefficient ofvariation, 38.1%). This large assay variability does not ap-pear related to PGE2 assay precision. As shown in Table 3,the coefficient of variation was less than 10% for four repeatanalyses of the same homogenate obtained by combiningfive biopsies from the same patient.

The intenbiopsy variability within a single patientbrings forward some important considerations. Variabilitycould be accounted for in as simple a manner as a fewsecond difference in time between biopsy removal andplacement in indomethacin. We feel, however, that wehave developed a reliable method for determining prosta-glandin content in colonectal mucosal biopsies.

The protocol that we recommend is: each colonicmucosal biopsy, as soon as taken, is immediately placed inan individual vial containing S pg/mI aqueous indometha-cm. The biopsy-containing vials are capped and instantlysnap frozen in liquid nitrogen and maintained in liquidnitrogen. Mucosal biopsies are later thawed and homoge-




nized in 5 pg/mI indomethacin buffer at a ratio of 1-2biopsies/i ml buffer. Protein determination aliquots aretaken. Extraction and radioimmunoassay are then pen-formed.

AcknowledgmentsBecause this manuscript represents the last scientific contribution of Dr. PaulR. Finley before his death, we acknowledge him as a scientist, an inspira-

tional leader, and a gentleman of the first order. We also wish to acknowl-

edge the scientific and clinical assistance of Dr. Lee Hixson.

References1 . Williams, A. R., Balasooriya, B. A., and Day, D. W. Polyps and cancer of

the large bowel: a necropsy study in Liverpool. Gut, 23: 835-842, 1982.

2. Kudo, T., Narisawa, 1., and Abo, S. Antitumor activity of indomethacin onmethylazoxymethano-1-induced large boweltumors in rats. Gann, 71:260-

264, 1980.

3. Reddy, B., Maruyama, H., and Kelloff, G. Dose related inhibition of colon

carcinogenesis by dietary piroxicam, a nonsteroidal anti-inflammatory drug,

during different stages of colon tumor development. Cancer Res., 47: 5340-5346, 1987.

4. Rosenberg, L., Palmer, J., Zanber, A. G., et al. A hypothesis: nonsteroidal

anti-inflammatory drugs reduce the incidence of large-bowel cancer. I. NatI.

Cancerlnst., 83:355-358, 1991.

5. Kung, G., Kune, S., and Watson, L. F. Colorectal cancer risk, chronic

illnesses, operations, and medications: case control results from the Mel-

bourne Colorectal Cancer Study. Cancer Res., 48: 4399-4404, 1988.

6. Thun, M., Namboodiri, M., and Heath, C. W. Aspirin use and reducedrisk offatal colon cancer. N. EngI. J. Med., 325: 1593-1596, 1991.

7. Giardello, F. M., Hamilton, S. R., Krush, A. J., et a!. Treatment of colonicand rectal adenomas with sulindac in familial adenomatous polyposis. N.EngI. J. Med., 328: 1313-1316, 1993.

8. Earnest, D. L., Hixson, L. J., Finley, P. R., Blackwell, G. G., Einspahr, J.,Emerson, S. S., and Alberts, D. S. Arachidonic acid cascade inhibitors in

chemoprevention of human colonic cancer: Preliminary studies. In: L.

Wattenberg, M. Lipkin, C. W. Boone, and G. J. Kelloff, (eds.), Cancer Che-moprevention, pp. 165-180. Boca Raton, FL: CRC Press, Inc., 1992.

9. Narisawa, 1., Kusaki, H., Yamasaki, Y., et a!. Relationship between bloodplasma prostaglandin E2 and liver and lung metastases in colorectal cancer.

Dis. Colon Rectum, 33: 840-845, 1990.

10. Lee, D. Y., Lupton, J. R., and Chapkin, R. S. Prostaglandin profile and

synthetic capacity ofthe colon: Comparison oftissue sources and subcellularfractions. Prostaglandins, 43: 1 43-1 64, 1992.

1 1 . Powell, W. Rapid extraction of arachidonic metabolites from biological

samples using octadecylsilyl silica. In: W. E. Lands, W. L. Smith (eds.),Methods in Enzymology, pp. 467-477. New York: Academic Press, Inc.,1982.

12. Maclouf, J., Pradel, M., Pradelles, P., and Dray, F. 1251 derivatives ofprostaglandins: a novel approach in prostaglandin analysis by radioimmu-

noassay. Biochim. Biophys. Acta, 431: 139-146, 1976.

1 3. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner,F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., andKlenk, D. C. Measurement of protein using bicinchoninic acid. Anal. Bio-

chem., 150:76-85, 1985.

14. Redinbaugh, M. G., and Turley, R. B. Adaptation of the bicinchoninic

acid protein assay for use with microtiter plates and sucrose gradient frac-tions. Anal. Biochem., 153: 267-271 , 1986.

15. Earnest, D. L., Hixson, L. S., and Alberts, D. S. Piroxicam and other

cyclo-oxygenase inhibitors: potential for cancer chemoprevention. J. Cell.Biochem., 16 1(Suppl.): 1 56-1 66, 1992.



1995;4:239-244. Cancer Epidemiol Biomarkers Prev P R Finley, C L Bogert, D S Alberts, et al. subjects: a method study.Measurement of prostaglandin E2 in rectal mucosa in human

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