in 1984 , it was first reported that h. pylori , a gram negative , spiral shaped , microaerophilic bacterium that colonizes the stomach and is involved in the pathogenesis of duodenal ulceration , gastric cancer , gastric ulceration , and active chronic gastritis [ 1 , 2 ] .
dyspepsia is a common term used for abdomen pain and complimented with other gastrointestinal symptoms .
nonulcer dyspepsia is also known as functional dyspepsia which found to be associated with h. pylori infection [ 3 , 4 ] .
h. pylori infection was found to be associated with gastric cancer , peptic ulcer , duodenal ulcer , gastric carcinoma [ 58 ] , and so forth .
prolidase ( ec 3.4.13.9 ) , proline dipeptidase , degraded dipeptides with hydroxyproline or proline as c - terminal amino acid [ 9 , 10 ] .
its activity has been reported in leukocytes , erythrocytes , plasma , and the various organs such as brain , heart , kidney , uterus , thymus , and dermal fibroblasts .
prolidase activity has been reported in various disorders , like osteoarthritis , chronic liver disease , and osteoporosis [ 1215 ] . 

it has been observed that increased oxidative stress associated with gastroduodenal mucosa inflammation in h. pylori infected individuals .
it has been reported that h. pylori infection induced inflammation and can cause gastric atrophy and it is related to increased oxidative stress [ 18 , 19 ] .
serum prolidase activity and oxidative status in h. pylori infected individuals were studied by aslan et al . in 2007 , among turkey population .
the prevalence of h. pylori infection varies from country to country by geographic area , ethnicity , age , race , and socioeconomic status [ 21 , 22 ] and consists of large diversity of strains [ 2227 ] .
due to its different prevalence and diversity of strains , we aimed to observe oxidative stress and serum prolidase activity in patients with h. pylori infected nonulcer dyspepsia among indian subjects . with serum prolidase activity
, we also estimated total antioxidant capacity ( taoc ) , total oxidant status ( tos ) , and oxidative stress index ( osi ) in the above cases .
this study was done in the department of biochemistry , department of gastroenterology , and department of pathology , institute of medical sciences , banaras hindu university , varanasi , india .
a total of 238 subjects were selected after informed consent . out of 238 , 188 subjects were patients of nonulcer dyspepsia ( nud ) and 50 healthy individuals followed up examination . among nonulcer
dyspeptic patients , 106 h. pylori positive and 82 h. pylori negative patients were observed .
patients with h. pylori positive nud were considered as cases , while patients with h. pylori negative nud were considered as control-1 group .
one was fixed with 10% formalin for histopathological examination in the department of pathology , and the other was treated with 10% urea solution containing phenol red indicator ( 0.2% ) for the rapid urease test .
the serum was separated from the cells by centrifugation at 3000  rpm for 10  min .
serum samples for the measurement of tos , taoc , and prolidase activity were stored at 80c until they were used . for histopathological and gastritis scoring standard
positive rapid urease test indicated h. pylori infection , and negative result indicated negative h. pylori infection .
a positive reaction was dark pink , and a negative reaction was either a yellow or an orange color .
the media consisted of 10% urea solution1.5  ml and phenol red indicator ( 0.2%)2 drops , ph 6.46.8 . for measurement of total antioxidant capacity reagent 1 and 2
reagent 1 : 3.17  gm of orthodianisidine dihydrochloride and 0.01764  gm of fe(nh4)2(so4)26h2o were dissolved in 1000  ml of clark and lubs solution ; reagent 2 : 0.641  ml of h2o2 solution ( 35% ) was diluted to 1000  ml with clark and lubs solution .
taoc of serum was measured by the use of an automated measurement method developed by erel ( 2004 ) [ 29 , 30 ] .
reagent 1 : 114  mg of xylenol orange and 8.18  gm of nacl were dissolved in 900  ml of h2so4 solution 25  mm .
the final reagent was composed of 150  mm xylenol orange , 140  mm nacl , and 1.35  m glycerol , ph 1.75 ; reagent 2 : 1.96  gm of ferrous ammonium sulfate and 3.17  gm of o - dianisidine dihydrochloride were dissolved in 1000  ml of h2so4 solution 25  mm .
tos of serum was measured by a method developed by erel ( 2004 ) [ 29 , 30 ] .
osi values were calculated according to the following formula : 
 ( 1)osi  ( arbitrary  unit )  = tos  ( mol  h2o2  equiv./l)taoc  ( mmol  trolox  equiv./l ) .
chinard 's reagent ( 25  g of ninhydrin was dissolved in 600  ml of glacial acetic acid and 400  ml of 6  mol / l orthophosphoric acid at 70c ) , standard proline solution ( 650  mol / l solution in 0.45  mol / l trichloroacetic acid ) , and gly - l - pro / sigma chemical co. ( 94 
mmol / l in 0.05  mol / l tris hcl buffer , ph 7.8 , containing 1  mmol of mncl2 per liter ) were prepared and stored at 4c .  
plasma was diluted six - fold with buffer mixture containing 0.05  mmol / l tris hcl buffer ( ph 7.8 ) in 2  mmol of mncl2/l and was incubated for 2  hrs at 37c .
after incubation , prolidase reaction was initiated by adding 100  l of the preincubated mixture to 100  l of 94  mm gly - l - pro solution .
after reaction initiation , it was incubated for 30  min at 37c ; reaction was stopped by adding 1  ml of 0.45  mol / l trichloroacetic acid .
the released proline was measured by addon of 0.5  ml of supernatant to 2  ml of 1  :  1 mixture of glacial acetic acid  :  chinard 's reagent and incubated for 10  min at 90c .
a blank and standard were run under the same condition . instead of the supernatant ,
serum prolidase activity was measured by the method developed by myara et al . , 1984 .
50 healthy individuals and 188  consecutive patients with nonulcer dyspepsia who agreed to participate in the study were included in our study .
dyspepsia is characterized by upper abdominal pain or discomfort , nausea , vomiting , bloating , or any other symptom related to the upper gastrointestinal tract .
nonulcer dyspepsia was diagnosed when dyspeptic symptoms were present for at least 4 weeks unrelated to exercise for which no organic lesion , such as peptic ulcer , reflux esophagitis , and gallstones , or any systemic disease , was found to be responsible .
the following patients were excluded from our study : patients with coexistent medical illness like chronic heart failure , chronic renal failure , diabetes mellitus , and so forth , pregnant females , patients with continued use of nonsteroidal antiinflammatory drugs , patients with history of use of h2-receptor antagonist / proton pump inhibitors during the previous 3 months , patients with evidence of any organic gastrointestinal / or hepatobiliary diseases ( diagnosed by abdominal ultrasound or upper gi endoscopy ) , patients that their informed consents are not available , and patients who did not agree on followup at the regular interval . patients with history of alcohol intake and antioxidant supplement consumption were excluded .
parametric methods were used ; a p value of less than 0.05 ( p < 0.05 ) was considered significant .
most of cases in our study were of moderate inflammation 34 ( 32.1% ) on histopathological examination , while mild inflammation was present in 32 ( 30.2% ) cases .
22 cases ( 20.8% ) had severe inflammation , while 18 ( 17% ) cases had zero grade inflammation of the gastric mucosa .
grading index for control-1 and control-2 of chronic inflammation was zero ( table 1 ) .
activity is denoted by intensity of neutrophilic infiltration . in our study , most of 36 ( 34% ) h. pylori positive subjects show moderate neutrophilic infiltration . while 30 ( 28.2% ) , 20 ( 18.9% ) , and 20 ( 18.9% ) cases showed mild , severe , and no neutrophilic infiltration activity , respectively .
serum prolidase activity was found to be higher in h. pylori positive subjects ( 37.91  3.19  mmol  min 
l ) as compared to h. pylori negative ( 32.19  3.43  mmol  min  l ) and healthy subjects ( 31.92  3.10  mmol  min 
l ) . therefore , serum prolidase activity was significantly higher in cases as compared to controls ( p < 0.0001 ) .
it was observed that control-1 had increased serum prolidase activity than control-2 , but this was nonsignificant ( p = 0.6083 ) ( table 2 ) .
total antioxidant capacity ( taoc ) in h. pylori positive , h. pylori negative , and healthy subjects were 1.39  0.36 , 1.79  0.37 , and 1.88  0.21  mmol trolox eq / l , respectively .
thus , the taoc was statistically lower in h. pylori positive cases than controls ( p < 0.0001 ) .
control-1 had decreased taoc than control-2 , which was nonsignificant ( p = 0.1186 ) ( table 2 ) .
total oxidant status ( tos ) in cases , control-1 , and control-2 were 13.29  1.29 , 11.57  1.06 , and 10.65  0.21  mol h2o2 equiv./l , respectively .
thus , total oxidant status was significantly higher in cases as compared to controls ( p < 0.0001 ) .
significantly increased tos was observed more in control-1 than in control-2 ( p < 0.0001 ) ( table 2 ) .
osi was found to be significantly higher in cases ( 10.25  3.12 ) as compared to control-1 ( 6.71  1.47 ) and control-2 ( 5.72  0.56 ) ( p < 0.0001 ) .
increased osi was observed more in control-1 than in control-2 , which was significant ( p < 0.0001 ) ( table 2 ) .
weak , negative , and nonsignificant correlation was observed between serum prolidase activities and taoc in cases ( r = 0.131 , p = 0.181 ) , while weak , positive , and nonsignificant correlation was observed between serum prolidase activity and tos and osi ( r = 0.029 , p = 0.77 , and
this above type of weak , nonsignificant correlation was also observed for controls ( table 3 ) .
, the inflammation induced by h. pylori infection has a chronic active gastritis which means that both lymphocytes and neutrophils infiltrate the mucosa in a characteristic manner .
apart from the inflammatory infiltrate , foci of intestinal metaplasia with atrophy , lymphatic aggregates , and lymphoid follicles were formed , and the foveolar epithelium was replaced by regenerative epithelium with correspondingly reduced mucus secretion . because it is found throughout the gastric mucosa from pylorus to cardia , h. pylori gastritis is usually pangastritis although in severity it may vary considerably between the antrum and corpus . in the present study ,
majority of the cases ( 32.0% ) had moderate inflammation , while mild and severe inflammation was seen in 30.2% and 20.8% of cases , respectively .
on analyzing activity , mild activity observed in 28.2% of cases , while 34.8% and 18.9% of cases had moderate and severe activity , respectively .
18.9% of cases had no activity ( table 1 ) , while in control-1 and control-2 , zero grading was observed for both chronic inflammation and neutrophilic infiltration .
thus , h. pylori infected nonulcer dyspeptic ( nud ) patients seem to have more chronic inflammation and neutrophilic infiltration than h. pylori negative nud and healthy subjects .
tos , taoc , and osi are used as biomarkers of oxidative stress and reflect the redox between oxidation and antioxidation .
it is well known that oxidative stress can be defined as an increase in oxidants or a decrease in antioxidant capacity , and various oxidants and antioxidants have additive effects on oxidative stress .
although the concentration of plasma level of oxidants and antioxidants can be measured individually , it may not accurately reflect the oxidative status [ 3335 ] . here , we observed oxidative stress in the study population by using tos , taoc , and osi .
tos in h. pylori infected dyspeptic subjects was found to be higher when compared to h. pylori negative subjects with functional dyspepsia and healthy subjects . in turkey population
, taoc was found to be significantly lower in h. pylori positive subjects as compared to control , while oxidative stress index was found to be higher .
among 238 eastern indian subjects , mean  sd of taoc was found to be significantly lower in h. pylori positive subjects as compared to h. pylori - negative nonulcer dyspeptic and healthy individuals ( p < 0.001 ) . oxidative stress index was found to be higher in cases as compared to controls ( p < 0.001 ) .
here we observed that in h. pylori infected nud as oxidative stress ( tos , osi ) increases , the taoc value significantly decreases as compared to h. pylori negative nud and healthy subjects .
thus , h. pylori infection associated with increased oxidative stress and decreased antioxidant level in nonulcer dyspeptic patients .
, serum prolidase activity had been showed to increase especially in early stage of fibrosis .
it has been suggested that plasma prolidase activity might be useful to evaluate the fibrotic processes in chronic liver disease in human .
a study by horoz et al . from turkey represented that serum prolidase activity is to be significantly higher in patients with nash in comparison to controls ( p = 0.016 ) .
they also found a significant correlation between serum prolidase activity and fibrosis score ( r = 0.661 , p < 0.001 ) . increased serum prolidase activity
was well documented in certain cancers like pancreatic cancer , lung carcinoma , breast cancer , stage 1 endometrial cancer , stomach cancer , ovarian cancer [ 3840 ] , and so forth . in our study population , increased serum prolidase activity was found in patients with h. pylori positive nud as compared to patients with h. pylori negative nud and healthy subjects .
( 2007 ) , who also observed that prolidase activity is to be higher in patients with h. pylori positive subjects with mean serum prolidase activity of 44.11  3.71 
this study concluded that in patients with nonulcer dyspepsia of eastern indian subjects increased oxidative stress and increased serum prolidase activity may be associated with h. pylori infection and their association may help to grant a better understanding regarding the pathogenesis of h. pylori infection .
thus , serum prolidase activity may be used as biomarker for h. pylori infection in patients with nonulcer dyspepsia .
it was also observed that chronic inflammation and neutrophilic infiltration of gastric mucosa are significantly related to h. pylori infection in patients with nonulcer dyspepsia .
 
 helicobacter pylori ( h. pylori ) infection is associated with increased oxidative stress and serum prolidase activity ( spa ) in many diseases . 
 we aimed to observe spa and oxidative stress in nonulcer dyspepsia ( nud ) infected with and without h. pylori among eastern indians . 
 106 patients with h. pylori positive nud , 82 patients with h. pylori negative nud , and 50 healthy individuals were selected . 
 spa , total antioxidant capacity ( taoc ) , and total oxidant status ( tos ) were measured with the use of spectrophotometer and an automated measurement method . 
 spa , tos , and oxidative stress index ( osi ) were significantly higher in patients with h. pylori positive than h. pylori negative nud and healthy individuals ( all p < 0.0001 ) , whereas taoc was significantly lower ( p < 0.0001 ) . 
 nonsignificant , increased spa ( p value = 0.6083 ) and decreased taoc ( p value = 0.1186 ) were observed in patients with h. pylori negative nud than healthy individuals , while increased tos and osi were significant ( p < 0.0001 ) . 
 weak , nonsignificant correlations were observed between serum prolidase activity and taoc , tos , and osi in h. pylori positive cases . 
 thus , increased spa along with increased oxidative stress was observed , which seem to be closely associated with h. pylori infection . spa and oxidative stress seem to be used as biomarkers for h. pylori infection in nud . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion