Prostaglsndins Ixukotrienes and Essential ~Longman Group UK Ltd 19%l
Fatty Acids (NO)
41, 167-171
0952.3278/90/0041-0167/$10.00
The Effect of Essential Fatty Acid Deficiency on Eicosanoid Production in the Inflamed Rat Colonic Mucosa R. M. Short, S. W. Weidman, S. M. Sabesin and P. Sharon Department of Medicine, Section of Digestive Diseases, Rush University, 1725 W. Harrison, 256, Chicago, Illinois 60612, USA (Reprint requests to PS)
Suite
Eicosanoids are potent mediators of inflammation and are synthesized in increased quantity in active ulcerative colitis. To elucidate the role of prostaglandin ES, thromboxane A2 , prostaglandin I*, and leukotriene B2 in acute chemical colitis induced by 4% acetic acid, we utilized an animal model which has a deficiency of arachidonic acid, the precursor of eicosanoids due to an essential fatty acid deticient diet. Fortyeight hours after colitis was induced, mucosal synthesis of the cyclooxygenase products, prostaghuuiin E2, thromboxane A;?, and prostaglandin 12, was significantly decreased in essential fatty acid deficient rats compared to normal controls. However, the Wipoxygenase product, leukotriene Bq, was not different between groups. The decrease in cyclooxygenase-products did not correlate with any change in the severity of colonic inflammation as assessed by gross morphology, histology, or myleoperoxidase activity. Thus inhibition of formation of the cyclooxygenase products of arachidonate metabolism does not appear to improve the degree of inflammation under the experimental conditions employed in this study.
ABSTRACT.
INTRODUCTION
Based on these considerations, the aim of this study was to determine the effects of the EFA deficient state on the degree of acute inflammation in a rat model of chemical colitis, the acetic acidinduced colitis; and to investigate the effects of the EFA deficient state on the content and production of eicosanoids by the inflamed colon. Although the EFA deficient state decreased the cyclooxygenase products of AA prostaglandin Ez (PGE2), thromboxane TxA2 (TxAz), and prostacyclin (PGQ, no effect on the lipoxygenase product, LTB4, and on the degree of inflammation caused by acetic acidinduced colitis were observed. These findings may provide some information about the relative significance of LTB4 compared to the cyclooxygenase products in acute colitis.
Ulcerative colitis (UC) is a chronic inflammatory disease characterized by recurrent exacerbations of acute colitis. Arachidonic acid metabolites (AAM) formed through the cyclooxygenase and lipoxygenase pathways are elevated in active disease and appear to be largely responsible for the clinical and histological changes seen in the disease (l-4). Moreover, drugs that have been proven to be beneficial for UC appear to exert their therapeutic effect by blocking the synthesis of these mediators (5). Further, until the etiologic agents of UC are identified it is likely that advances in medical therapy will concentrate on the regulation of the synthesis of the soluble mediators of inflammation. Essential fatty acid (EFA) deficiency has been shown to be a useful tool to study the role of arachidonic acid (AA) and its metabolites in certain inflammatory disorders, where AAM are presumably detrimental to the inflammatory process (6). The mechanism of this effect may in part be mediated through a decrease in eicosanoid synthesis secondary to a lack of available precursors (i.e. AA).
MATERIALS AND METHODS Animal and EFA deficiency Female Sprague-Dawley rats initially weighing 150175 grams were maintained for at least 14 weeks on a diet deficient in all fatty acid (Purina Lab Chows, Indiana). Control animals received commercial rat chow (Purina).
Date received 14 March 1990
Date accepted 24 June 1990 167
168
Prostaglandins Leukotrienes and Essential Fatty Acids
Induction of colitis
Rats were fasted for 36 h and anesthetized with ether. A midline abdominal incision was made, the colon was isolated, and the junction of the cecum and ascending colon was ligated. Two ml of 4% acetic acid was injected into the proximal lumen of the colon through a 27 gauge needle, followed immediately by 5 ml of air which cleared most of the acetic acid from the colon. The midline incision was then closed. Forty eight hours later the animals were bled; serum saved for fatty acid analysis, and the colons removed (4). Assessment
of colonic inflammation
Colonic inflammation was assessed by 3 methods. The colons were immediately examined for gross inflammation, and any visible damage was scored on a scale ranging from 0 (normal) to 5 (severe) (9). Colonic samples for histologic assessment were taken 1 cm proximal to the anus and evaluated for infiltration of inflammatory cells in all layers of the bowel wall. Colonic myleoperoxidase (MPO) activity was determined as a further index of inflammation as described by us (10). Colonic specimens (100-200 mg) were minced in 0.5% hexadecyltrimethylammonium bromide buffer in 5 mM phosphate buffer, pH 6.0 and homogenized with Polytron homogenizer. The homogenates were sonicated for lOs, freeze-thawed three times and the tissue levels of MPO activity were determined by utilization of 0.0005% hydrogen peroxide as a substrate for the MPO. A unit of MPO activity was defined as that converting 1 umol of hydrogen peroxide to water in 1 min at 25°C. Serum lipid analysis
Serum lipids were extracted by the method of Folch (7). Fatty acids were transmethylated by heating the lipid-extraction in 14% boron trifluoride in methanol (8). The methyl esters were separated and quantified using a glass column (6 ft by 4 mm) packed with 10% SP-2340 on 100/120 Chromosorb WAW Sup,elco, Inc. (Bellefonte) with a Hewlett-Packard Model 5890 gas chromatograph. The nitrogen flow rate was 40 ml/min and the injector and flame ionization detector were maintained at 280°C. The column was operated isothermally at 200°C. Fatty acid methyl esters were identified by co-migration with authentic standards. Fatty acid methyl ester retention times and quantification were computed by a Hewlett-Packard model 3392A integrator interfaced with the gas chromatograph.
described (l), to study the production of AAM by the inflamed mucosa over a period of time. Within minutes of removal 3-5 full thickness biopsies, 40 mg each, per rat were taken and incubated in organ culture. The culture medium was RPM1 1640 supplemented with penicillin (100 U/ml) and streptomycin (100 ugr/ml) (Gibco, Inc) in the presence and absence of ionophore A23187 (2 uM) (Calbiotech, Inc.). After 30 min of incubation, the medium was withdrawn, fresh medium added, and the culture was continued for 6 h. At the end of the culture period the medium was saved at -90°C for up to 4 wks before analyses for eicosanoids. Approximately 200 mg of scraped inflamed colonic mucosa was incubated in RPM1 1640 medium in the presence of ionophore A23187 (2 uM) for 5 min at 37°C in a volume of 5 ml. The incubation was ended by placing the test tube on ice. The colonic mucosa was immediately homogenized and lipids were extracted with 2.5 ml of chloroform/methanol/lO% formic acid (1.2: 1.2: :O.l) per 1 ml of buffer (3). The chloroform layer was saved at -90°C under nitrogen and later dried under nitrogen, resuspended in (pH 7.0) phospate buffer saline and analyzed for eicosanoids. Finally, colonic specimens (20-40 mg) were saved at -90°C for determination of eicosanoids content in the tissue. Before assay, biopsies were homogenized in 2.5 ml of 20 mM Tris HCl, pH 7.0, with 0.02M NazEDTA. Samples were extracted twice with ether, and allowed to sit overnight at 4°C for evaporation of the ether. Radioimmunoassays
Eicosanoids were measured without prior extraction in the organ culture medium or after extraction from the tissue as described above, by specific radioimmunoassay. LTB4 was determined with kits obtained from Amersham (Arlington Height, IL) and PGE2, TxB2 and 6-keto PG, (the last two are the stable metabolites of TxAz and PGI,) were determined with kits obtained from Du Pont (Boston, MA). The amounts of eicosanoids formed are expressed as pg per mg of wet tissue. Data analysis
The significances of the differences between EFAD and control rats parameters were determined by Student’s unpaired t-test.
RESULTS Evidence of induction of EFA-deficiency
Organ culture
Organ
culture
was
performed
as
previously
Rats fed on EFA diet over 14 wks manifested scaly dermatitis of the tails and feet characteristic of EFA
Effect of Essential Fatty Acid Deficiency on Eicosanoid Production
169
Table 1 Prostanoid production and content in inflamed colonic mucosa TxB2
PGE2
Condition
6-ketoPGFr
No.
pg/mg
No.
pg/mg
No.
pg/mg
Organ culture EFA-deficiency Control
10 II
60 + 18’ 121 + 40
7 7
114 * 37** 199 f 76
6 4
284 f 36** 326 + 25
Scraped mucosa EFA-deficiency Control
6 7
23 + 2* 42 f 6
Colonic content EFA-deficiency Control
12 11
110 z!I 41 118 + 67
-
Colonic biopsies or scraped cofonic mucosa were incubated either for 6 h or 5 min. respectively. Accumulation of cyclooxygenase metabolites in the medium was determined by radioimmunoassay, and calculated as picogram per mg of wet tissue. The content of PGE, was determined after direct extraction from the tissue. Results are expressed as mean + SD. * p
Fatty Acids (NO)
41, 167-171
0952.3278/90/0041-0167/$10.00
The Effect of Essential Fatty Acid Deficiency on Eicosanoid Production in the Inflamed Rat Colonic Mucosa R. M. Short, S. W. Weidman, S. M. Sabesin and P. Sharon Department of Medicine, Section of Digestive Diseases, Rush University, 1725 W. Harrison, 256, Chicago, Illinois 60612, USA (Reprint requests to PS)
Suite
Eicosanoids are potent mediators of inflammation and are synthesized in increased quantity in active ulcerative colitis. To elucidate the role of prostaglandin ES, thromboxane A2 , prostaglandin I*, and leukotriene B2 in acute chemical colitis induced by 4% acetic acid, we utilized an animal model which has a deficiency of arachidonic acid, the precursor of eicosanoids due to an essential fatty acid deticient diet. Fortyeight hours after colitis was induced, mucosal synthesis of the cyclooxygenase products, prostaghuuiin E2, thromboxane A;?, and prostaglandin 12, was significantly decreased in essential fatty acid deficient rats compared to normal controls. However, the Wipoxygenase product, leukotriene Bq, was not different between groups. The decrease in cyclooxygenase-products did not correlate with any change in the severity of colonic inflammation as assessed by gross morphology, histology, or myleoperoxidase activity. Thus inhibition of formation of the cyclooxygenase products of arachidonate metabolism does not appear to improve the degree of inflammation under the experimental conditions employed in this study.
ABSTRACT.
INTRODUCTION
Based on these considerations, the aim of this study was to determine the effects of the EFA deficient state on the degree of acute inflammation in a rat model of chemical colitis, the acetic acidinduced colitis; and to investigate the effects of the EFA deficient state on the content and production of eicosanoids by the inflamed colon. Although the EFA deficient state decreased the cyclooxygenase products of AA prostaglandin Ez (PGE2), thromboxane TxA2 (TxAz), and prostacyclin (PGQ, no effect on the lipoxygenase product, LTB4, and on the degree of inflammation caused by acetic acidinduced colitis were observed. These findings may provide some information about the relative significance of LTB4 compared to the cyclooxygenase products in acute colitis.
Ulcerative colitis (UC) is a chronic inflammatory disease characterized by recurrent exacerbations of acute colitis. Arachidonic acid metabolites (AAM) formed through the cyclooxygenase and lipoxygenase pathways are elevated in active disease and appear to be largely responsible for the clinical and histological changes seen in the disease (l-4). Moreover, drugs that have been proven to be beneficial for UC appear to exert their therapeutic effect by blocking the synthesis of these mediators (5). Further, until the etiologic agents of UC are identified it is likely that advances in medical therapy will concentrate on the regulation of the synthesis of the soluble mediators of inflammation. Essential fatty acid (EFA) deficiency has been shown to be a useful tool to study the role of arachidonic acid (AA) and its metabolites in certain inflammatory disorders, where AAM are presumably detrimental to the inflammatory process (6). The mechanism of this effect may in part be mediated through a decrease in eicosanoid synthesis secondary to a lack of available precursors (i.e. AA).
MATERIALS AND METHODS Animal and EFA deficiency Female Sprague-Dawley rats initially weighing 150175 grams were maintained for at least 14 weeks on a diet deficient in all fatty acid (Purina Lab Chows, Indiana). Control animals received commercial rat chow (Purina).
Date received 14 March 1990
Date accepted 24 June 1990 167
168
Prostaglandins Leukotrienes and Essential Fatty Acids
Induction of colitis
Rats were fasted for 36 h and anesthetized with ether. A midline abdominal incision was made, the colon was isolated, and the junction of the cecum and ascending colon was ligated. Two ml of 4% acetic acid was injected into the proximal lumen of the colon through a 27 gauge needle, followed immediately by 5 ml of air which cleared most of the acetic acid from the colon. The midline incision was then closed. Forty eight hours later the animals were bled; serum saved for fatty acid analysis, and the colons removed (4). Assessment
of colonic inflammation
Colonic inflammation was assessed by 3 methods. The colons were immediately examined for gross inflammation, and any visible damage was scored on a scale ranging from 0 (normal) to 5 (severe) (9). Colonic samples for histologic assessment were taken 1 cm proximal to the anus and evaluated for infiltration of inflammatory cells in all layers of the bowel wall. Colonic myleoperoxidase (MPO) activity was determined as a further index of inflammation as described by us (10). Colonic specimens (100-200 mg) were minced in 0.5% hexadecyltrimethylammonium bromide buffer in 5 mM phosphate buffer, pH 6.0 and homogenized with Polytron homogenizer. The homogenates were sonicated for lOs, freeze-thawed three times and the tissue levels of MPO activity were determined by utilization of 0.0005% hydrogen peroxide as a substrate for the MPO. A unit of MPO activity was defined as that converting 1 umol of hydrogen peroxide to water in 1 min at 25°C. Serum lipid analysis
Serum lipids were extracted by the method of Folch (7). Fatty acids were transmethylated by heating the lipid-extraction in 14% boron trifluoride in methanol (8). The methyl esters were separated and quantified using a glass column (6 ft by 4 mm) packed with 10% SP-2340 on 100/120 Chromosorb WAW Sup,elco, Inc. (Bellefonte) with a Hewlett-Packard Model 5890 gas chromatograph. The nitrogen flow rate was 40 ml/min and the injector and flame ionization detector were maintained at 280°C. The column was operated isothermally at 200°C. Fatty acid methyl esters were identified by co-migration with authentic standards. Fatty acid methyl ester retention times and quantification were computed by a Hewlett-Packard model 3392A integrator interfaced with the gas chromatograph.
described (l), to study the production of AAM by the inflamed mucosa over a period of time. Within minutes of removal 3-5 full thickness biopsies, 40 mg each, per rat were taken and incubated in organ culture. The culture medium was RPM1 1640 supplemented with penicillin (100 U/ml) and streptomycin (100 ugr/ml) (Gibco, Inc) in the presence and absence of ionophore A23187 (2 uM) (Calbiotech, Inc.). After 30 min of incubation, the medium was withdrawn, fresh medium added, and the culture was continued for 6 h. At the end of the culture period the medium was saved at -90°C for up to 4 wks before analyses for eicosanoids. Approximately 200 mg of scraped inflamed colonic mucosa was incubated in RPM1 1640 medium in the presence of ionophore A23187 (2 uM) for 5 min at 37°C in a volume of 5 ml. The incubation was ended by placing the test tube on ice. The colonic mucosa was immediately homogenized and lipids were extracted with 2.5 ml of chloroform/methanol/lO% formic acid (1.2: 1.2: :O.l) per 1 ml of buffer (3). The chloroform layer was saved at -90°C under nitrogen and later dried under nitrogen, resuspended in (pH 7.0) phospate buffer saline and analyzed for eicosanoids. Finally, colonic specimens (20-40 mg) were saved at -90°C for determination of eicosanoids content in the tissue. Before assay, biopsies were homogenized in 2.5 ml of 20 mM Tris HCl, pH 7.0, with 0.02M NazEDTA. Samples were extracted twice with ether, and allowed to sit overnight at 4°C for evaporation of the ether. Radioimmunoassays
Eicosanoids were measured without prior extraction in the organ culture medium or after extraction from the tissue as described above, by specific radioimmunoassay. LTB4 was determined with kits obtained from Amersham (Arlington Height, IL) and PGE2, TxB2 and 6-keto PG, (the last two are the stable metabolites of TxAz and PGI,) were determined with kits obtained from Du Pont (Boston, MA). The amounts of eicosanoids formed are expressed as pg per mg of wet tissue. Data analysis
The significances of the differences between EFAD and control rats parameters were determined by Student’s unpaired t-test.
RESULTS Evidence of induction of EFA-deficiency
Organ culture
Organ
culture
was
performed
as
previously
Rats fed on EFA diet over 14 wks manifested scaly dermatitis of the tails and feet characteristic of EFA
Effect of Essential Fatty Acid Deficiency on Eicosanoid Production
169
Table 1 Prostanoid production and content in inflamed colonic mucosa TxB2
PGE2
Condition
6-ketoPGFr
No.
pg/mg
No.
pg/mg
No.
pg/mg
Organ culture EFA-deficiency Control
10 II
60 + 18’ 121 + 40
7 7
114 * 37** 199 f 76
6 4
284 f 36** 326 + 25
Scraped mucosa EFA-deficiency Control
6 7
23 + 2* 42 f 6
Colonic content EFA-deficiency Control
12 11
110 z!I 41 118 + 67
-
Colonic biopsies or scraped cofonic mucosa were incubated either for 6 h or 5 min. respectively. Accumulation of cyclooxygenase metabolites in the medium was determined by radioimmunoassay, and calculated as picogram per mg of wet tissue. The content of PGE, was determined after direct extraction from the tissue. Results are expressed as mean + SD. * p
