nitric oxide ( no ) is a soluble gaseous molecule with numerous biological activities that has multiple influences on the physiology and pathophysiology of the cells including cytotoxic and cytoprotective effects ( 1 ) . in human and animals bodies , it is produced by nitric oxide synthase ( nos ) enzyme isoforms and by no donors . three isoforms of nos are so far introduced ; calcium dependent and membrane associated endothelial nos , calcium dependent neuronal nos ( nnos ) , and calcium independent cytosolic inducible nos ( inos ) . these enzymes oxidize l - arginine to produce no and l - citrulline ; nadph is electron donor and acts as co - substrate and is strictly required for this enzymatic reaction ( 2 ) . pancreatic -cells constitutively express nnos , which is required for survival and physiological function ( 3 ) . inducible nos is not usually expressed in these cells , but produces constantly large amount of no in responses to inflammatory mediators ( 4 ) . no participates in regulation of - cell mass in islets of pancreas ( 5 ) . in this context , it should be mentioned that relation to its concentration and origin , no has a dual biological functions ; both proliferative ( 6 ) and apoptotic ( 7 ) . low amount of no derived from nnos has beneficial effects , but higher concentration resulting from inos activity induces inflammatory process , and is toxic both for host cells and invading biological organisms [ d1](8 , 9 ) . exposure to pathogens increases with aging ; on the other hand metabolic activities especially carbohydrate metabolism deteriorates during aging processes ( 10 ) . also , the prevalence of glucose intolerance and type ii diabetes is markedly increased with aging . in this situation , furthermore , inos activity in pancreatic islets of type ii diabetes noticeably increases ( 12 ) . according to research based theory , increase in inos activity and subsequent production of nitric oxide is associated with aging ( 13 ) . on the other hand , no can be considered a true inflammatory mediator ( 14 ) . in the course of inflammation , consequently increase in inos activity has been reported in the wide range of inflammatory condition including ; type2 diabetes , metabolic syndrome and alzheimer s disease ( 16 ) . due to important role of inos activity in above mentioned conditions and especially in type 2 diabetes , which increases during aging processes , the aim of this study was to find out the possible relation between changes in inos activity and aging and also establish / modify a rapid and sensitive hplc method of assay for nos activity in tissue homogenate . a total of twelve male wistar rats ( 15 weeks old , 200 to 250 g ) were supplied from pasture institute , tehran , iran and then were divided into two experimental groups ; adult young group : six rats of four months of age ; and six of four months age rats in old groups were kept in animal house for extra 8 months that in final became 12 months of age . the animals were housed under standard conditions ( 12 hr light12 hr dark cycle starting at 07:00 am at 24c in a controlled humidity ) with free access to food and water during one week of adaptation to laboratory situation . this study was approved by the local ethics committee of the research institute for endocrine sciences , shahid beheshti university of medical sciences , tehran , iran , and all experiments were conducted in accordance with standard ethical guidelines . animals either after two weeks of adaptation or 8 months of housing ( old group ) were anesthetized by intraperitoneal injection of sodium pentobarbital , 60 mg / kg body weight after an overnight fasting ( 17 ) . blood samples were obtained from heart for nox , insulin and glucose measurement , and then followed by median laparotomy for pancreatic islets isolation . for nos assay , groups of 200 islets were collected in ice - cold lysis buffer ( 200 l ) containing hydroxyethyl piperazine ethane sulfonic acid ( hepes ; 20.0 mm , ph 7.4 , sigma ) as a buffer , l - dithiothreitol ( l - dtt ; 1.0 mm , sigma ) and cocktail of protease inhibitor with edta supplement ( 1tablet dissolved in 10 ml buffer , cat . no.11836153001 , roche company germany ) and then aliquots were stored at -80c ( 18 ) . insulin and glucose were measured using glucose oxidase and elisa procedures , respectively . insulin and glucose kits were supplied from zistchimi ( iran ) and mercodia ( sweden ) companies , respectively . the intra - assay coefficients of variations for insulin , glucose and nox were 8.42% , 2.5% and 3.87% , respectively . for islet isolation , 10 ml ice - cold hanks balanced salt solution ( hbss ) ( ph , 7.4 ; containing nacl , 136 ; kcl , 5.36 ; cacl2 , 1.26 ; mgso4  7h2o , 0.8 ; na2hpo4  2h2o , 0.33 ; kh2po4 , 0.44 ; nahco3 , 4.16 ) all in mm concentration including 0.5 mg / ml of collagenase p ( roche , cat . number 1213 , germany ) was injected into the pancreatic duct , and then the islets were removed , and transferred to a sterile 50 ml falcon tube and then digested in a 37c water bath for 15 min ( 19 ) . reaction was terminated by adding ice - cold hbss , and then tube was shaken for 1 min . after 2 washes with cold hbss , the suspension was filtered through a 500 m plastic mesh to discard any undigested tissue particles . followed by one washing , then the islets were hand - picked under a stereomicroscope ( kyowa optical , sdz - tr - pl model , japan ) . for evaluation of insulin secretion in vitro , batches of 5 islets ( three replicas for each condition from 4 animals ) were incubated in tubes containing 1 ml of krebs  ringer solution [ ( ph , 7.4 ) ; nacl 115 ; kcl 5 ; mgcl2 6h2o 1 ; cacl2 2.5 ; nahco3 24 and hepes , 16 all in mm ] and 5 g / dl bovine serum albumin ( bsa ; fluka , usa ) ( 19 ) with different glucose concentrations ( 5.6 , 8.3 , 16.7 mm ) for 60 min in 37 c water bath , and gassed with 95% o2 /5% co2 for 5 min at the beginning , groups of five islets were incubated for 60 min with various concentration of glucose containing 5 mm of nos inhibitor l - ng - nitro arginine methyl ester ( l - name , sigma ) ( 20 ) . for indirect measurement of nos activity briefly , serum proteins were precipitated using znso4 ( merck ) , and then the reduction of nitrate to nitrite was performed by saturated solutions of vcl3 ( vanadium chloride ) ( 0.8% w / v ) in 1 m hcl ( merck ) and then the color generating solution containing n - l(naphthyl ) ethylenenndiamine dihydrochloride ( nedd ; 0.1% w / v , merck ) in h2o and sulfanilamide ( 2% w / v , merck ) in 5% hcl ( griess reagents ) were added to the reaction vessels . after 20 min of incubation at room temperature , the absorbance was read in 540 nm ( 21 ) using spectrophotometer . after centrifugation at 35,000 g for 10 min , the supernatant was kept on ice for determination of protein concentrations using bradford method for protein and enzyme assay ( 22 ) . for inos activity assay , 100 l of supernatant was added to reaction mixture containing hepes , 20.0 mmol / l ( ph 7.4 ) , 0.2 mm l - arginine ( sigma ) and 2 mm nadph ( sigma ) in a total volume of 0.5 ml and was then incubated under constant air bubbling ( 1 ml / min ) at 37c for 120 min . inducible nitric oxide synthase activity was calculated from the difference between samples and blank reading . chromatographic separation was performed using an agilent 1100 series high - performance liquid chromatography ( hplc ) system . the accompanying agilent lc chemstation software program was used for instrument control , data acquisition , and processing . an analytical nucleodur rp- column ( 100 - 5 c18 ec , 1504.6 mm machery - nagel , germany ) gave the best results . hplc analysis was performed by isocratic elution with a flow rate of 1 ml / min . the mobile phase was 10 mm kh2po4 buffer solution containing acetonitrile - methanol - tetrahydrofuran ( 76:11.5:11.5:1 all in v / v ) . the temperature was maintained at 20c and the excitation and emission wavelengths in the fluorescence detector were set to 338 and 455 nm , respectively . for determination of l - citrulline , first 100 l of the incubated pancreatic islets extract were derivatized with o - phtaldialdehyde ( opa ) . both the extracts and l - citrulline ( sigma ) standards were separately injected ( injection volume : 100 l ) into the c18-reverse phase column immediately or after 2 , 10 and 15 min of derivatization in room temperature or 37c . l - citrulline quantification was carried out using comparison of retention times of the pancreatic islets extracts with standards of citrulline and also spiking the known concentration of standards to the sample . calibration curves were constructed by plotting the measured peak in the range of 10 to 500 pmol of l - citrulline standards . linearity of the standard curves was assessed by the coefficient of determination ( r ) , equally 0.995 in this analysis . accuracy was determined using high purity standard and precision was expressed as the relative standard deviation which was calculated in percentage for each of the replicate concentrations ( n = 3 ) . 2-mercaptoethanol ( 2-me ) , opa and the other minerals were > 95% purity from merck chemicals ( darmstadt , germany ) . all the solvents ( hplc gradient grade ) were purchased from samchun chemical ( pyeongtaek , korea ) . ultrapure water ( from a direct q uv-3 millipore system ) was used in all experiments . all analysis were validated by dagostino and pearson ( omnibus k2 test performed with prism version 5 ) normality test . statistical study between groups of data was assessed by using unpaired student s t - test or , where applicable , one way analysis of variance ( anova ) followed by bonferroni post- hoc tests using graph pad prism software ( version 5 ) . a total of twelve male wistar rats ( 15 weeks old , 200 to 250 g ) were supplied from pasture institute , tehran , iran and then were divided into two experimental groups ; adult young group : six rats of four months of age ; and six of four months age rats in old groups were kept in animal house for extra 8 months that in final became 12 months of age . the animals were housed under standard conditions ( 12 hr light12 hr dark cycle starting at 07:00 am at 24c in a controlled humidity ) with free access to food and water during one week of adaptation to laboratory situation . this study was approved by the local ethics committee of the research institute for endocrine sciences , shahid beheshti university of medical sciences , tehran , iran , and all experiments were conducted in accordance with standard ethical guidelines . animals either after two weeks of adaptation or 8 months of housing ( old group ) were anesthetized by intraperitoneal injection of sodium pentobarbital , 60 mg / kg body weight after an overnight fasting ( 17 ) . blood samples were obtained from heart for nox , insulin and glucose measurement , and then followed by median laparotomy for pancreatic islets isolation . for nos assay , groups of 200 islets were collected in ice - cold lysis buffer ( 200 l ) containing hydroxyethyl piperazine ethane sulfonic acid ( hepes ; 20.0 mm , ph 7.4 , sigma ) as a buffer , l - dithiothreitol ( l - dtt ; 1.0 mm , sigma ) and cocktail of protease inhibitor with edta supplement ( 1tablet dissolved in 10 ml buffer , cat . no.11836153001 , roche company germany ) and then aliquots were stored at -80c ( 18 ) . insulin and glucose were measured using glucose oxidase and elisa procedures , respectively . insulin and glucose kits were supplied from zistchimi ( iran ) and mercodia ( sweden ) companies , respectively . the intra - assay coefficients of variations for insulin , glucose and nox were 8.42% , 2.5% and 3.87% , respectively . for islet isolation , 10 ml ice - cold hanks balanced salt solution ( hbss ) ( ph , 7.4 ; containing nacl , 136 ; kcl , 5.36 ; cacl2 , 1.26 ; mgso4  7h2o , 0.8 ; na2hpo4  2h2o , 0.33 ; kh2po4 , 0.44 ; nahco3 , 4.16 ) all in mm concentration including 0.5 mg / ml of collagenase p ( roche , cat . number 1213 , germany ) was injected into the pancreatic duct , and then the islets were removed , and transferred to a sterile 50 ml falcon tube and then digested in a 37c water bath for 15 min ( 19 ) . reaction was terminated by adding ice - cold hbss , and then tube was shaken for 1 min . after 2 washes with cold hbss , the suspension was filtered through a 500 m plastic mesh to discard any undigested tissue particles . followed by one washing , then the islets were hand - picked under a stereomicroscope ( kyowa optical , sdz - tr - pl model , japan ) . for evaluation of insulin secretion in vitro , batches of 5 islets ( three replicas for each condition from 4 animals ) were incubated in tubes containing 1 ml of krebs  ringer solution [ ( ph , 7.4 ) ; nacl 115 ; kcl 5 ; mgcl2 6h2o 1 ; cacl2 2.5 ; nahco3 24 and hepes , 16 all in mm ] and 5 g / dl bovine serum albumin ( bsa ; fluka , usa ) ( 19 ) with different glucose concentrations ( 5.6 , 8.3 , 16.7 mm ) for 60 min in 37 c water bath , and gassed with 95% o2 /5% co2 for 5 min at the beginning . , groups of five islets were incubated for 60 min with various concentration of glucose containing 5 mm of nos inhibitor l - ng - nitro arginine methyl ester ( l - name , sigma ) ( 20 ) . for indirect measurement of nos activity , nitrite concentration was measured using griess protocol . briefly , serum proteins were precipitated using znso4 ( merck ) , and then the reduction of nitrate to nitrite was performed by saturated solutions of vcl3 ( vanadium chloride ) ( 0.8% w / v ) in 1 m hcl ( merck ) and then the color generating solution containing n - l(naphthyl ) ethylenenndiamine dihydrochloride ( nedd ; 0.1% w / v , merck ) in h2o and sulfanilamide ( 2% w / v , merck ) in 5% hcl ( griess reagents ) were added to the reaction vessels . after 20 min of incubation at room temperature , the absorbance was read in 540 nm ( 21 ) using spectrophotometer . after centrifugation at 35,000 g for 10 min , the supernatant was kept on ice for determination of protein concentrations using bradford method for protein and enzyme assay ( 22 ) . for inos activity assay , 100 l of supernatant was added to reaction mixture containing hepes , 20.0 mmol / l ( ph 7.4 ) , 0.2 mm l - arginine ( sigma ) and 2 mm nadph ( sigma ) in a total volume of 0.5 ml and was then incubated under constant air bubbling ( 1 ml / min ) at 37c for 120 min . the blank sample contained all reaction components except nadph . inducible nitric oxide synthase activity was calculated from the difference between samples and blank reading . chromatographic separation was performed using an agilent 1100 series high - performance liquid chromatography ( hplc ) system . the accompanying agilent lc chemstation software program was used for instrument control , data acquisition , and processing . an analytical nucleodur rp- column ( 100 - 5 c18 ec , 1504.6 mm machery - nagel , germany ) gave the best results . hplc analysis was performed by isocratic elution with a flow rate of 1 ml / min . the mobile phase was 10 mm kh2po4 buffer solution containing acetonitrile - methanol - tetrahydrofuran ( 76:11.5:11.5:1 all in v / v ) . the temperature was maintained at 20c and the excitation and emission wavelengths in the fluorescence detector were set to 338 and 455 nm , respectively . for determination of l - citrulline , first 100 l of the incubated pancreatic islets extract were derivatized with o - phtaldialdehyde ( opa ) . both the extracts and l - citrulline ( sigma ) standards were separately injected ( injection volume : 100 l ) into the c18-reverse phase column immediately or after 2 , 10 and 15 min of derivatization in room temperature or 37c . l - citrulline quantification was carried out using comparison of retention times of the pancreatic islets extracts with standards of citrulline and also spiking the known concentration of standards to the sample . calibration curves were constructed by plotting the measured peak in the range of 10 to 500 pmol of l - citrulline standards . linearity of the standard curves was assessed by the coefficient of determination ( r ) , equally 0.995 in this analysis . accuracy was determined using high purity standard and precision was expressed as the relative standard deviation which was calculated in percentage for each of the replicate concentrations ( n = 3 ) . 2-mercaptoethanol ( 2-me ) , opa and the other minerals were > 95% purity from merck chemicals ( darmstadt , germany ) . all the solvents ( hplc gradient grade ) were purchased from samchun chemical ( pyeongtaek , korea ) . ultrapure water ( from a direct q uv-3 millipore system ) was used in all experiments . all analysis were validated by dagostino and pearson ( omnibus k2 test performed with prism version 5 ) normality test . statistical study between groups of data was assessed by using unpaired student s t - test or , where applicable , one way analysis of variance ( anova ) followed by bonferroni post- hoc tests using graph pad prism software ( version 5 ) . as shown in table 1 , fasting plasma glucose and nox levels of aged rats was significantly increased compared to adult rats ( p<0.01 ) , but aging had no significant effect on plasma insulin level . plasma glucose , insulin and nox levels in different experimental groups statistical comparison between groups was made using an unpaired t test , values are mean  sem , n= 6 in adult ( 4 month old ) and old ( 12 month old ) . figure 1 shows that the basal glucose concentration ( 5.6 mm ) has no significant influence on insulin secretion in each condition ( adults and aged ) of experimental groups . stimulation of pancreatic islets with 8.3 mm glucose increased insulin secretion in both experimental groups . furthermore , l - name a nos inhibitor improved insulin secretion , but this improvement was not statistically significant . the results also showed that insulin secretion , especially in supra physiological high concentration of glucose ( 16.7mm / l ) did not increase in senescent rats , in which insulin release was significantly lower compared to adult rats ( figure 2 , p<0.05 ) . in contrast , using nos inhibitor - l - name ( 5 mm for 60 min ) concomitantly with glucose ( 16.7 mm / l ) in incubation medium of pancreatic islets , markedly increased islet insulin secretion ( figure 2 , p<0.05 ) . this enhancement was considerably prominent in aged rats compared to adults rats ; 42.39%  4.73% versus 15.27%  5.41% in adult rats ( p=003 ) . calibration curve of the opa derivative of l - citrulline lu : arbitrary luminescence unit glucose stimulated insulin secretion on isolated islets of langerhans . values are mean  sem for 8 cups ( 2 cups each containing 5 islets for each condition from each animal ; 4 animals in each group ) ; * p < 0.05 statistically significant differences between adult ( 4- month- old ) and old ( 12- month- old ) . # p<0.05 , statistically significant differences between inhibitor and glucose 16.7 mm / l the inos activity in intact pancreatic islets of aged rats compared to adult rats was significantly increased ( 1.082  0.084 and 6.277  0.475 pmol / min per mg protein in adult and aging rats respectively , p<0.001 , figure 3 ) . the specific activity of the enzyme in pancreatic islets of adult rats was too low , which in one of the samples it was not detectable with current assay . statistical comparison between groups was made using an unpaired student t - test . * * * p<0.001 , statistically significant differences between groups . n=3 in adult ( 4- month- old ) and n= 4 in old ( 12- month- old ) groups time ( immediately or after 2 , 5 , 10 and 15 min ) and temperature ( room temperature and 37c ) of incubation had no effect on the fluorescence intensity . retention time of citrulline was 8.5 to 8.9 min and samples were run 15 min . column was cleaned and regenerated by sequentially washing with water and methanol 50% ( 15 min with each ) . this procedure had the best results on column cleaning and citrulline detection compared to other solvent such as acetonitrile . concentrations of 25 , 50 , 100 , 500 pmols of l - citrulline were used to establish a calibration curve ( figure 1 ) . precision is the measure of the degree of repeatability of the assay which is performed by using enter and intra assays coefficient of variation ( cv % ) . in our study , intra and inter assay of cv for l - citrulline assay were 2.976% ( 0.654 ) and 7.645% ( 1.325 ) , respectively . as shown in table 1 , fasting plasma glucose and nox levels of aged rats was significantly increased compared to adult rats ( p<0.01 ) , but aging had no significant effect on plasma insulin level . plasma glucose , insulin and nox levels in different experimental groups statistical comparison between groups was made using an unpaired t test , values are mean  sem , n= 6 in adult ( 4 month old ) and old ( 12 month old ) . figure 1 shows that the basal glucose concentration ( 5.6 mm ) has no significant influence on insulin secretion in each condition ( adults and aged ) of experimental groups . stimulation of pancreatic islets with 8.3 mm glucose increased insulin secretion in both experimental groups . furthermore , l - name a nos inhibitor improved insulin secretion , but this improvement was not statistically significant . the results also showed that insulin secretion , especially in supra physiological high concentration of glucose ( 16.7mm / l ) did not increase in senescent rats , in which insulin release was significantly lower compared to adult rats ( figure 2 , p<0.05 ) . in contrast , using nos inhibitor - l - name ( 5 mm for 60 min ) concomitantly with glucose ( 16.7 mm / l ) in incubation medium of pancreatic islets , markedly increased islet insulin secretion ( figure 2 , p<0.05 ) . this enhancement was considerably prominent in aged rats compared to adults rats ; 42.39%  4.73% versus 15.27%  5.41% in adult rats ( p=003 ) . calibration curve of the opa derivative of l - citrulline lu : arbitrary luminescence unit glucose stimulated insulin secretion on isolated islets of langerhans . values are mean  sem for 8 cups ( 2 cups each containing 5 islets for each condition from each animal ; 4 animals in each group ) ; * p < 0.05 statistically significant differences between adult ( 4- month- old ) and old ( 12- month- old ) . the inos activity in intact pancreatic islets of aged rats compared to adult rats was significantly increased ( 1.082  0.084 and 6.277  0.475 pmol / min per mg protein in adult and aging rats respectively , p<0.001 , figure 3 ) . the specific activity of the enzyme in pancreatic islets of adult rats was too low , which in one of the samples it was not detectable with current assay . statistical comparison between groups was made using an unpaired student t - test . * * * p<0.001 , statistically significant differences between groups . n=3 in adult ( 4- month- old ) and n= 4 in old ( 12- month- old ) groups time ( immediately or after 2 , 5 , 10 and 15 min ) and temperature ( room temperature and 37c ) of incubation had no effect on the fluorescence intensity . retention time of citrulline was 8.5 to 8.9 min and samples were run 15 min . column was cleaned and regenerated by sequentially washing with water and methanol 50% ( 15 min with each ) . this procedure had the best results on column cleaning and citrulline detection compared to other solvent such as acetonitrile . concentrations of 25 , 50 , 100 , 500 pmols of l - citrulline were used to establish a calibration curve ( figure 1 ) . precision is the measure of the degree of repeatability of the assay which is performed by using enter and intra assays coefficient of variation ( cv % ) . in our study , intra and inter assay of cv for l - citrulline assay were 2.976% ( 0.654 ) and 7.645% ( 1.325 ) , respectively . in accordance to the documented studies on human ( 23 ) and animals ( 11 ) , hyperglycemia increases with aging . variation in nox level in relation with aging has shown controversial results on previous studies . some of the studies are in favors of the results obtained in this study ( 24 , 25 ) , and some other studies shows different contrast results ( 26 ) . controversy in the results might dependent on the kind of studies and or change in other parameters associated with aging such as body weight and fat . change in plasma insulin level in this study ( table 1 ) associated with decreased insulin secretion may be followed by decreased insulin clearance as have previously been reported ( 27 , 28 ) . the study also showed decrease in insulin secretion , especially in high concentration of glucose ( 16.7 mm / l ) , which was significantly lower in aged rats ( figure 2 , p<0.05 ) . pancreatic islet size and beta cell number increase with age ( 29 ) , which compensate islet insulin secretion in basal state . despite these structural changes in the endocrine pancreas , islet insulin capacity , insulin content and insulin mrna levels decrease in aging animals ( 27 , 29 ) . with regard to above explanation , in high concentration of glucose , it seems that the reduced insulin secretion of pancreatic islets in this condition is inevitable and no may be involved in this process . use of nos inhibitor increased insulin secretion in both groups , especially in aged rats . according to no as a negative modulator of islet insulin release ( 20 ) , inos activity is induced and increased in high glucose concentration  10 mm / l ( 31 ) . it is suggested that no production from inos is prominent in aged rats , and it is responsible for decrease in insulin secretion , which is compensated by nos inhibitor l - name . in respect to inos activity , the previous studies had investigated an increase in inos activity and the gene / protein expression on aorta and heart of aged 14- month- old mice , respectively ( 32 , 33 ) . inducible nos activity is involved in the antiproliferative /apoptotic effect of proinflamatory cytokines that cause islet destruction and inhibit insulin secretion from pancreatic -cells ( 6 , 34 ) . based on the oxidation - inflammation theory of aging ( 35 ) , these conditions are associated with an impairment in the physiological functions , especially in homeostatic systems including the immune system . therefore , rise in inos activity which results in no production is associated with decreased islet insulin secretion , which is implicated in impaired glucose metabolisms . this presumably increases the prevalence of type ii non - insulin dependent diabetes during aging . in this study for the first time we have established a modification in the procedure of the nos activity assay in rat islets . in our laboratory , we have employed the l - citrulline assay for measuring no synthase activity in rat islets with minimal sample manipulation ; without stop solution and using a cation exchange column . although in his procedure also l - arginine was used as the substrate , but the enzymatic reaction was stopped by the addition of acidic solution and finally aliquots of incubation medium were passed through the additional steps using an amprep cbs cation - exchange column- and then processed samples were used for hplc analysis . finally l - citrullin elated from the amprep column was determined by hplc analysis ( 18 ) . the direct measurement of no is complex and difficult because no is unstable and highly reactive molecule ( the half - life of a few tenths of a sec ) . nonetheless , several indirect methods have been used to measure no metabolites including : nox measurement by griess reagent , which can be measured by spectrophoto - metric procedure ( 21 ) , and chemiluminescence procedure in which no is assayed by measurement of the difference in the absorption spectrum for oxyhemoglobin ( 36 ) ( no oxidize oxyhemoglobin to methemoglobin ) . in addition , nos activity can be determined with great reliability , sensitivity , and specificity by conversion of l - arginine to l - citrulline . l - citrulline is a stable by - product and is equimolar of no production from l - arginine . due to its stability , the measurement of l - citrulline production provides a much more accurate assessment of nos enzymatic activity . previous studies have shown that the inos activity measurement is more sensitive and superior to protein measurement using western blotting or elisa methods ( 31 , 37 ) . for hplc analysis , incubation with opa is performed in room temperature ( temperature of incubation had no effect on the fluorescence intensity ) . as mentioned previously , opa required a few min of reaction time ( 38 ) . column conditioning before analysis is a critical step to obtain good recovery and better separation ( 39 ) of the l -- citrulline . in this study , the column was initially conditioned by methanol solution ( 50% ) for 50 min at a flow rate of 0.5 ml / min followed by 60 min the mobile phase . in chromatography , thus , in order to prevent contamination of the column and possibly memory effect , after each sample run ( 15 min ) , the columns were washed sequentially with water / methanol 50% ( 15 min with each ) . wu and meininger ( 38 ) used column washing with methanol solution for satisfactory separation of l - arginine and l - citrulline in plasma and biological fluids . also schwarz et al ( 40 ) for analysis of plasma amino acids by hplc with fluorescence detection used a mixture of 90% isopropanol and 10% methanol in order to maintain column integrity . our results showed that inos activation and increase in no production might be responsible for decreased in insulin secretion in aging . the study emphasizes the link between reduced capacity of carbohydrate metabolism and increase in no signaling pathway in senescence . inducible nitric oxide synthase activity assay using l - citrulline measurement by hplc method was achieved by minimum sample handling , short time of assay , and maximum precision and accuracy .
objective(s):some pathologic situations such as diabetes and metabolic syndrome are associated with alternation in nitric oxide level . 
 incidence of these condition increases with aging . on the other hand , 
 insulin secretion is modulated by nitric oxide , and nitric oxide synthase ( nos ) activity is also altered in diabetes . in this study , modification in the enzyme activity associated with aging and also optimized procedure for islet nos assay was investigated.materials and methods : male wistar rats were randomly divided in two experimental groups : a : adult rats ; were 4 month old and b : old rats ; were 12 month old . in all groups , plasma glucose , insulin and nox ( nitrite + nitrate = nox ) were measured , and also insulin secretion in isolated pancreatic islet with or without l - name was investigated . 
 furthermore , the inducible nos activity with l - citrulline measurement in islets was measured.results:l-citrulline was quantified using one step hplc column . 
 aging induced hyperglycemia ( p<0.05 ) and excess plasma nox ( 17.74  1.664 and 26.25 
 2.166 mol / l in a and b groups respectively , p<0.05 ) with unaltered plasma insulin . 
 islet insulin secretion was significantly reduced in aging rats . 
 l - name induced islet insulin secretion especially in aging rats ( p=0.003 ) . 
 inducible nos activity in islets of aging rats was significantly higher than adult rats ( 1.082  0.084 and 6.277  0.475 pmol / min per mg protein in adult and aging rats , respectively , p<0.001).conclusion : these findings show that decreased in islet insulin secretion may be related to increase in inos activity in islets , which follows impaired carbohydrate metabolism in aging .