type 2 diabetes ( t2 dm ) and obesity have become epidemic in malaysia . according to the latest national health and morbidity surveys , 14.9% of malaysian adults aged 30 years and above are diabetic and they are often obese or overweight [ 1 , 2 ] . moreover , diabetic nephropathy ( dn ) is the most common cause of end - stage renal disease ( esrd ) and contributes to 57% of patients with t2 dm in this country .
although t2 dm represents a preventable and treatable cause of esrd , the number of esrd cases caused by t2 dm has increased and accounts for more than 50% of incident dialysis patients [ 35 ] .
pentraxin 3 ( ptx3 ) is an acute - phase glycoprotein and a soluble receptor acting as an opsonin .
thereby , its levels may reflect more directly the inflammatory status of the vasculature [ 6 , 7 ] .
recently , several clinical investigations have demonstrated that elevated plasma ptx3 levels are associated with cardiovascular [ 8 , 9 ] and chronic kidney diseases ( ckd ) [ 10 , 11 ] .
furthermore , plasma ptx3 levels are inversely associated with body mass index ( bmi ) suggesting that ptx3 may play a role in obesity and metabolic syndrome [ 12 , 13 ] .
interestingly , with the approach of genome - wide scan and linkage analysis , chromosome 3q is found to be linked with diabetes and dn in many ethnic groups [ 1416 ] .
the gene encoding for ptx3 protein is located in chromosome 3q25.32 and resides in the linkage region .
have shown that ptx3 is positively associated with proteinuria in turkish subjects with t2 dm hypertensive patients , while renin angiotensin system blockade lowers plasma ptx3 levels in the patients [ 17 , 18 ] . however , there are gender and racial differences of plasma ptx3 levels [ 7 , 19 ] , by which the association of ptx3 with kidney dysfunction may be influenced .   in the present study , we examined plasma ptx3 levels in a malay cohort , including ngt subjects , t2 dm patients with and without dn .
the aim of our study was to investigate the association of plasma ptx3 levels with t2 dm and dn in this malay population .
data from our study are also useful for better understanding the different effects of ptx3 in dn and ckd .
we collected the samples of subjects with ngt and patients with t2 dm from the collaborating centers all over malaysia .
the ethnic distribution of our study subjects was 67.6% malay , 15.3% indian , 14.8% chinese , and 2.3% indigenous sabahans and sarawakians . to avoid the error caused by ethnic stratification , indian , chinese , indigenous sabahans , and sarawakians were excluded in the present study .
finally , 103 ( 50 males/53 females ) malay individuals with ngt ( controls ) and 193 ( 99/94 ) malay patients with t2 dm ( cases ) were included into the analyses .
diagnoses of t2 dm were done based on the world health organization ( who ) criteria .
the diagnoses of dn were based on urine albumin - to - creatinine ratio ( acr ) suggested by ada .
the patients with t2 dm and normoalbuminuria ( acr < 3.5  mg / mmol ) were considered as controls for dn , while the patients with macroalbuminuria ( acr  35  mg / mmol ) and esrd who needed dialysis were included as the cases for dn . except for the patients with esrd ,
all other subjects were required to give urine under fasting conditions early in the morning .
clinical characteristics of all malay subjects with ngt and t2 dm with and without dn are summarized in table 1 .
informed consent was obtained from all subjects , and the study was approved by the local ethical committees .
data and materials transfer agreement from the institute for medical research , malaysia to karolinska institutet , sweden was signed prior to the study .
body weight and height were measured using a calibrated digital scale ( seca , birmingham , uk ) .
the who / international association for the study of obesity ( iaso)/international obesity task force ( iotf ) has proposed bmi cut - off values of 23.024.9  kg / m for classification of overweight and of 25.0  kg / m for obesity for adult asians . based on the malaysian clinical practice guidelines , subjects with bmi value 23.0  kg / m are considered as overweight .
systolic and diastolic blood pressures were measured using a digital sphygmomanometer ( omron healthcare , inc . ,
creatinine in serum and urine were measured using randox assayed multisera ( randox laboratories ltd . ,
urine or serum was mixed with sodium hydroxide in biochemistry analyzer ( selectra e ) .
the amount of the complex formed was measured at wavelength of 490 to 510  nm .
estimate of glomerular filtration rate ( gfr ) in the dialysis patient was calculated by the mean of renal urea and creatinine clearance from a 24-hour urine correction .  
a total of 25  ml venous blood samples were collected from each subject early in the morning after an overnight fasting and then stored at 80c .
plasma ptx3 concentrations were determined using a commercial enzyme - linked immunosorbent assay kit ( quantakine dptx 30 ; r&d systems inc . ,
briefly , 20  l of standard and plasma samples was assayed duplicate in the microtiter plate wells coated with a specific ptx3 monoclonal antibody followed by incubation at room temperature for 2 hours .
anti - ptx3 polyclonal antibody conjugated to alkaline phosphatase was added to each well and incubated for two hours at room temperature . after washing step , 200  l of substrate solution was added to each well followed by incubation for 30 minutes at room temperature .
the solution of 2  n sulfuric acid was added to each well to stop the reaction .
absorbance was measured at 450  nm with corrections set at 540  nm using microplate reader .
the values of plasma ptx3 levels were extrapolated from a curve drawn using a standard ptx3 .  
all data were expressed as mean ( 95% ci ) for normally distributed variables and as geometric means ( 95% ci ) for nonnormally distributed variables .
normal probability plots were created and parameter distributions were transformed to the common logarithm for obtaining a normal distribution before performing statistical analysis .
the one - way analysis of variance ( anova ) was used for comparisons involving more than two groups or independent t - test for comparison between two groups .
pearson and spearman analyses were conducted to determine correlations with continuous and noncontinuous variables , respectively .
all analyses were performed using pasw statistic base 18 ( spssinc , chicago , usa ) .
in both males and females , there was no difference in age , waist circumference , and hba1c between t2 dm patients and ngt subjects .
although plasma ptx3 levels in males and females differed significantly , ptx3 had no relationship with age in males ( p = 0.647 ) or females ( p = 0.626 ) .
thus , all subsequent analyses of plasma ptx3 levels were done separately in males and females .
our analyses indicated that plasma ptx3 levels in t2 dm patients with and without dn were lower as compared with ngt subjects in males ( 2.62 versus 3.98  ng / ml ; p = 0.021 ) but not in females ( 3.24 versus 3.09  ng / ml ; p = 0.748 ) .  
figure 1(a ) showed that plasma ptx3 levels were consistently decreased from ngt to t2 dm without dn and to the patients with dn in males ( 3.98 , 2.78 , and 1.63  ng / ml ; p = 0.008 anova test ) . among males ,
the patients with dn had lower ptx3 levels compared to t2 dm without dn ( 1.63 versus 3.08  ng / ml ; p = 0.013 ) . in females , however , there was no statistically significant difference of the mean values of plasma ptx3 levels among ngt and t2 dm with and without dn ( 3.09 , 3.55 , and 2.11  ng / ml ; p = 0.262 , anova test ) ( figure 1(b ) ) .  
there was a negative correlation between plasma ptx3 levels and bmi in male subjects with ngt ( r = 0.390 ; p = 0.012 ) ( figure 2 ) but not in females ( p = 0.330 ) .
the correlation between ptx3 and bmi was not found in all male and female t2 dm patients with and without dn . in malaysia , the adults with bmi value  23.0  kg / m are considered to be overweight . to further understand whether the association between ptx3 and dn in t2 dm was influenced by bmi
, we performed the comparative analyses in the patients with overweight ( bmi  23  kg / m ) and lean patients ( bmi < 23  kg / m ) , respectively . in males with overweight
, we found that plasma ptx3 levels were gradually decreased from subjects with ngt to t2 dm patients without dn and to the patients with dn ( 3.68 , 2.60 and 1.42  ng / ml ; p = 0.044 , anova test ) ( figure 3 ) . in lean males and also in all females , plasma ptx3 levels of ngt and t2 dm with and without dn were varied but not with any statistical significance .
in the present study , we analyzed plasma ptx3 levels in malay subjects with ngt and t2 dm with and without dn .
lower levels of ptx3 were found to be associated with t2 dm and dn in males but not in females .
furthermore , ptx3 was found to be inversely associated with bmi in males with ngt .
the correlation was not observed both in males and females with t2 dm and dn .
males have a higher prevalence of t2 dm and dn in many populations including malaysians [ 1 , 35 ] .
epidemiologic reports have demonstrated that dn is 30% more frequent in males than in females .
genetic studies have also showed that dna polymorphisms in the genes of sex - determining region y - box 2 , angiotensin ii type 1 , and type 2 receptors are associated with dn with gender - specific effects [ 2628 ] .
previously , yamasaki et al . observed that plasma ptx3 levels between males and females in a healthy japanese population are different . in the present study
, we demonstrate that plasma ptx3 levels gradually decreased from ngt to t2 dm without dn to t2 dm with dn particularly among males but not in females .
furthermore , an inverse correlation between ptx3 and bmi was found in male subjects with ngt .
this correlation was not seen in all females and males with t2 dm and dn .
taking together , data from previous and present studies implicate that ptx3 most likely has gender - specific effects in t2 dm and dn , which should be taken into our consideration in further investigations .  
several studies have reported that increased ptx3 levels are associated with impaired renal function in ckd [ 10 , 11 ] .
the similar association of ptx3 with dn is seen in turkish patients with t2 dm . in the present study , however ,
we demonstrate that decreased ptx3 levels are associated with dn in malay men with t2 dm .
dubin et al . have demonstrated that there are racial differences of ptx3 in term of association with kidney dysfunction .
second , the ages of turkish t2 dm subjects with dn ( at 42 years old )   are younger and their duration of diabetes are shorter compared to malay t2 dm patients with dn ( at the age of 55 years old ) in the present study .
furthermore , clinical observations have indicated that two main causes of ckd are diabetes and high blood pressure , which are responsible for up to two - thirds of the cases .
the progresses and mechanisms of dn and ckd may be different , while ptx3 may have different effects in these two diseases .
we have shown that plasma ptx3 levels were inversely correlated with bmi in males with ngt , which is consistent with previous reports [ 7 , 12 , 13 ] . in the present study
, there is a limitation with lack of lean subjects because all patients with t2 dm and female subjects with ngt had mean values of bmi at least 27.2  kg / m .
recently , a study has demonstrated that ptx3 is expressed in adipose tissue , and its tissue specific expression reflects endothelial dysfunction .
although we did not analyze plasma levels of adiponectin in this malay cohort , the accumulated documents have shown that adiponectin is inversely proportional to obesity and t2 dm in different populations including malaysians .
plasma / serum adiponectin levels in the patients with t2 dm and obese subjects are decreased compared to that in healthy control subjects [ 31 , 32 ] .
therefore , we hypothesize that ptx3 , as similar to adiponectin , may have protective effects in increased body weight . further investigation is needed to fully understand the cellular mechanism of ptx3 reduction in t2 dm and dn . in conclusion ,
the present study provides the first evidence that decreased plasma ptx3 levels are associated with t2 dm and dn in malay men and also suggests that ptx3 may have different effects in dn and ckd .
 recent reports have demonstrated that elevated plasma long pentraxin 3 ( ptx3 ) levels are associated with cardiovascular and chronic kidney diseases . in the current study , we investigated the plasma ptx3 levels in 296 malay subjects including the subjects with normal glucose tolerance ( ngt ) and type 2 diabetes ( t2 dm ) patients with or without dn by using an enzyme - linked immune - sorbent assay . 
 results showed that in males , plasma ptx3 levels in t2 dm patients without dn were lower than that in the subjects with ngt ( 2.78 versus 3.98  ng / ml ; p = 0.021 ) . 
 plasma ptx3 levels in t2 dm patients with dn were decreased compared to the patients without dn ( 1.63 versus 2.78  ng / ml ; p = 0.013 ) . in females , however , no significant alteration of plasma ptx3 levels among ngt subjects and t2 dm patients with and without dn was detected . 
 furthermore , an inverse correlation between ptx3 and body mass index was found in male subjects with ngt ( p = 0.012 ; r = 0.390 ) , but not in male t2 dm patients , neither in all females . 
 the current study provided the first evidence that decreased plasma ptx3 levels are associated with t2 dm and dn in malay men and also suggested that ptx3 may have different effects in dn and chronic kidney diseases . 