there is a strong inverse relationship between plasma high density lipoprotein ( hdl ) level and the risk of developing coronary artery disease ( cad ) .
the major role of hdl is the reverse cholesterol transport , which leads to cholesterol movement from peripheral tissue and vessel wall to the liver .
anti - inflammatory , antioxidant , anticoagulant , and profibrinolytic actions of hdl are among other functions that may contribute further to its ability to protect against cad .
hdl particles are heterogeneous in their composition , structure , and size . several methods such as immunoaffinity chromatography , ultracentrifugation .
electron microscopy . and nuclear magnetic resonance ( nmr ) have been used to isolate and characterize hdl subfractions and at least five subfractions of hdl with sizes of 7.3 - 13 nm have been identified .
one of the best known anti - inflammatory and antioxidant functions of hdl is its ability to inhibit the oxidation of low - density lipoprotein ( ldl ) .
hdl exerts its inhibitory effects on oxidative modification of ldl , in part , by its related enzyme paraoxonase-1 ( pon1 ) which can hydrolyze lactones , and several non - physiological substrates , such as aryl esters and organophosphates .
pon1 is transported in plasma as a component of hdl , and many studies have also shown that pon1 inhibits ldl oxidation in vitro .
hdl facilitates the secretion of the pon1 by the liver , stabilizes the enzyme , and provides a hydrophobic environment which is needed for pon1 function . in return
there is a wide inter - individual variation in pon1 activity and concentrations due to variation in pon1 gene , and also , because of the influences of lifestyle factors such as smoking , alcohol consumption , etc .
a study of populations with high prevalence of atherosclerosis suggested that pon1 could influence hdl concentrations in patients with familial hypercholesterolemia .
it has been demonstrated that pon1 is present in small dense subclasses of hdl particles including apolipoprotein a - i ( apo a - i ) and apolipoprotein j. investigations have also indicated that there is a relationship between pon1 activity and hdl size with cad risk .
, the protective role of hdl against cad seems to be a complicated and multi - factorial procedure that depends on the hdl particle size , number , composition , distribution , and its related enzymes such as pon1 .
the aim of this study was to investigate the associations between hdl mean particle size and serum pon1 activity in relation to serum hdl - c levels in healthy subjects .
for this study , 140 healthy men between 20 and 50 years of age from isfahan area were recruited to isfahan cardiovascular research center between june and september 2010 .
all subjects were healthy , non - smoking , non - obese [ body mass index ( bmi ) 30 ] , and non - diabetic [ fasting blood sugar ( fbs ) 110 mg / dl ] volunteers , without familiar hypercholesterolemia and any sign of cardiovascular or inflammatory diseases , and no one was taking lipid - modifying medication .
all participants were interviewed and a checklist relating to their age , medical history , and lifestyle information was completed .
all participants signed an informed consent document approved by the isfahan university of medical sciences medical ethics committee .
this study was funded by grant number 188140 from isfahan university of medical sciences , isfahan , iran .
weight was measured while subjects were minimally clothed without shoes and height was measured in a standing position .
waist circumference ( wc ) was measured at the narrowest level and that of hip at the maximal level over light clothing , using a tape measure .
waist hip ratio ( whr ) was calculated as wc divided by hip circumference . to avoid subjective error ,
blood samples ( 20 ml ) were collected by venopuncture after 12 - 14 h fasting .
a 100 l aliquot of serum was stored at 80c until the measurement of serum pon1 activity .
hdl - c and ldl - c were determined by direct enzymatic procedures ; fbs , total cholesterol ( tc ) , and triglyceride ( tg ) were measured by enzymatic methods ; and apo a - i and apolipoprotein b-100 ( apo b-100 ) were measured by immunoturbidometric methods using an automated analyzer ( hitachi 902 , kyoto , japan ) and commercial kits ( diasys diagnosis , holzheim , germany ) .
pon1 activity was measured using 2 mm paraoxon ( diethyl p - nitrophenyl phosphate , sigma chemical co. , st .
louis , mo , usa ) as the substrate in 100 mm tris buffer ( ph 8.0 ) containing 2 mm of cacl2
. aliquots of 40 l of serum were added to 500 l of substrate medium and the initial rate of hydrolysis ( generation of p - nitrophenol ) was monitored at 412 nm , using a continuously recording spectrophotometer ( uv 3100 , shimadzu , kyoto , japan ) over a period of 2 min . a pon1 activity of 1 unit / l was defined as 1 mol of p - nitrophenol formed per minute .
the molar extinction coefficient of p - nitrophenol was taken at 17,000 m / cm .
all assays were performed in duplicate at 25c . the within - assay coefficient of variation ( cv ) for 10 separate measurements was 2.9% and between - assay cv for 10 separate measurements during 20 days was 3.6% . for hdl separation ( 1.063 g / cm <  < 1.21 g / cm ) ,
polyallomer ultracentrifuge tubes with 8.9-ml capacity ( optiseal , beckman / coulter , fullerton , ca , usa ) were first taken with 5.9 ml of serum sample and the sample was overlaid with 3 ml of solution a [ containing 0.195 mmol nacl , 0.62 mmol naoh , 0.01% ( w / v ) ethylenediaminetetraacetic acid disodium salt ( edta - na2 ) , d = 1.006 g / ml ] in order to make discontinuous density gradient .
the tubes were centrifuged in a beckman coulter optima l-100 xp ultracentrifuge equipped with a type 90ti fixed angle rotor at 60,000 rpm for 6 h at 16c , acceleration:5  and deceleration:7  .
after centrifugation , the very low density lipoprotein ( vldl ) fraction ( the white layer at the supernatant ) accompanied by 3 ml of the upper layer solution in tubes was removed and residual content of tubes was mixed with 3 ml of solution b ( containing 24.8 g nabr in 100 ml of solution a ) .
the tubes were centrifuged at 60,000 rpm for 12 h at 16c , acceleration:9  and deceleration:7  .
after centrifugation , the ldl fraction ( appearing as a yellow - orange band at the supernatant ) accompanied by 3 ml of the upper solution was removed .
the residual content of tubes was mixed with 3 ml of solution c ( containing 78.32 g of nabr in 100 ml of solution a ) and centrifuged for another 12 h at 16c , acceleration:9  and deceleration:7  .
after centrifugation , the hdl fraction appearing as a yellow band at the supernatant was collected carefully and stored at 80c until further analysis .
the protein concentration of the hdl fractions was determined according to a modified lowry method . a zetasizer nano zs instrument ( malvern , worcestershire , uk ) with a 532-nm green laser beam was used for the study .
the scattered light was collected by detector at an angle of 173 using non - invasive back - scatter ( nibs ) technology and directed to a correlator .
the data were analyzed by zetasizer software ( dts , nano series , version 5.02 , malvern ) and size information was reported as the z - average by intensity .
all measurements were performed at 25c in duplicate with automatic duration measurements . in order to determine the suitable solvent with the best reproducibility prior to commencing the measurements , six different solvents [ including distilled water , normal saline , and 0.2 m phosphate buffer containing 0.1% ( w / v ) edta - na2 at three different ph values of 5.6 , 7.4 , and 8.3 ]
hourly measurements of size were made over a period of 5 h and the results were compared . consequently
, 0.2 m phosphate buffer at ph of 7.4 containing 0.1% ( w / v ) edta - na2 was selected as the most repeatable dispersant with the lowest aggregation index . in order to measure hdl mean
particle size by dynamic light scattering ( dls ) methodology , isolated hdl samples were mixed gently with 1 ml of dispersant ( at a final protein concentration of 250 g / ml ) and passed through a syringe filter ( millipore cellulose acetate membrane , 30 mm , 0.2 m pore size ) prior to injection into a disposable polystyrene cell ( malvern ) in order to remove dust particles and were then subjected to size determination .
viscosity and refractive index ( ri ) of water as the dispersant were applied to standard operating protocol ( sop ) prior to size determination .
the accuracy of size measurements was examined using standard size nanoparticles [ gold nanoparticles , 20 nm , 0.01% ( w / v ) aqueous solution , nanocs inc .
, new york , ny , usa ] under the same experimental conditions and the results were matched with the diameter quoted by the manufacturer .
the within - assay cv for 10 measurements was 1.2% and between - assay cv for 8 measurements was 3.1% .
the detection limit of the assay for zetasizer instrument used in the present study was 0.3 nm to 10 m . in order to calculate the ratio of hdl2 to hdl3
, the accumulative percentage of particles with sizes larger than 8.2 nm was divided to the accumulative percentage of particles with sizes smaller than 8.2 nm .
statistical analyses were performed using spss 16.0 ( spss , chicago , il , usa ) . before analyses
, subjects were divided into three groups based on their serum hdl - c levels [ group 1 ( n = 49 ) : hdl 39 mg / dl ; group 2 ( n = 46 ) : 39 > hdl 49 mg / dl ; group 3 ( n = 45 ) : hdl > 49 mg / dl ] , and variables that were not normally distributed ( i.e. tg , apo a - i and apo b-100 ) were subjected to natural log transformation .
quantitative parameters were compared between groups by analysis of variance ( anova ) except for pon1 and hdl2/hdl3 that were analyzed by a nonparametric kruskal - wallis test and data were expressed as means  sd .
spearman 's rho correlation coefficient analysis was used to calculate the correlation between variables that were not normally distributed ( i.e. serum pon1 activity ) .
for this study , 140 healthy men between 20 and 50 years of age from isfahan area were recruited to isfahan cardiovascular research center between june and september 2010 .
all subjects were healthy , non - smoking , non - obese [ body mass index ( bmi ) 30 ] , and non - diabetic [ fasting blood sugar ( fbs ) 110 mg / dl ] volunteers , without familiar hypercholesterolemia and any sign of cardiovascular or inflammatory diseases , and no one was taking lipid - modifying medication .
all participants were interviewed and a checklist relating to their age , medical history , and lifestyle information was completed .
all participants signed an informed consent document approved by the isfahan university of medical sciences medical ethics committee .
this study was funded by grant number 188140 from isfahan university of medical sciences , isfahan , iran .
weight was measured while subjects were minimally clothed without shoes and height was measured in a standing position .
waist circumference ( wc ) was measured at the narrowest level and that of hip at the maximal level over light clothing , using a tape measure .
waist hip ratio ( whr ) was calculated as wc divided by hip circumference . to avoid subjective error ,
blood samples ( 20 ml ) were collected by venopuncture after 12 - 14 h fasting .
a 100 l aliquot of serum was stored at 80c until the measurement of serum pon1 activity .
hdl - c and ldl - c were determined by direct enzymatic procedures ; fbs , total cholesterol ( tc ) , and triglyceride ( tg ) were measured by enzymatic methods ; and apo a - i and apolipoprotein b-100 ( apo b-100 ) were measured by immunoturbidometric methods using an automated analyzer ( hitachi 902 , kyoto , japan ) and commercial kits ( diasys diagnosis , holzheim , germany ) .
pon1 activity was measured using 2 mm paraoxon ( diethyl p - nitrophenyl phosphate , sigma chemical co. , st .
louis , mo , usa ) as the substrate in 100 mm tris buffer ( ph 8.0 ) containing 2 mm of cacl2 .
aliquots of 40 l of serum were added to 500 l of substrate medium and the initial rate of hydrolysis ( generation of p - nitrophenol ) was monitored at 412 nm , using a continuously recording spectrophotometer ( uv 3100 , shimadzu , kyoto , japan ) over a period of 2 min . a pon1 activity of 1 unit / l was defined as 1 mol of p - nitrophenol formed per minute .
the molar extinction coefficient of p - nitrophenol was taken at 17,000 m / cm .
all assays were performed in duplicate at 25c . the within - assay coefficient of variation ( cv ) for 10 separate measurements was 2.9% and between - assay cv for 10 separate measurements during 20 days was 3.6% .
for hdl separation ( 1.063 g / cm <  < 1.21 g / cm ) , polyallomer ultracentrifuge tubes with 8.9-ml capacity ( optiseal , beckman / coulter , fullerton , ca , usa ) were first taken with 5.9 ml of serum sample and the sample was overlaid with 3 ml of solution a [ containing 0.195 mmol nacl , 0.62 mmol naoh , 0.01% ( w / v ) ethylenediaminetetraacetic acid disodium salt ( edta - na2 ) , d = 1.006 g / ml ] in order to make discontinuous density gradient .
the tubes were centrifuged in a beckman coulter optima l-100 xp ultracentrifuge equipped with a type 90ti fixed angle rotor at 60,000 rpm for 6 h at 16c , acceleration:5  and deceleration:7  .
after centrifugation , the very low density lipoprotein ( vldl ) fraction ( the white layer at the supernatant ) accompanied by 3 ml of the upper layer solution in tubes was removed and residual content of tubes was mixed with 3 ml of solution b ( containing 24.8 g nabr in 100 ml of solution a ) .
the tubes were centrifuged at 60,000 rpm for 12 h at 16c , acceleration:9  and deceleration:7  .
after centrifugation , the ldl fraction ( appearing as a yellow - orange band at the supernatant ) accompanied by 3 ml of the upper solution was removed .
the residual content of tubes was mixed with 3 ml of solution c ( containing 78.32 g of nabr in 100 ml of solution a ) and centrifuged for another 12 h at 16c , acceleration:9  and deceleration:7  .
after centrifugation , the hdl fraction appearing as a yellow band at the supernatant was collected carefully and stored at 80c until further analysis .
the protein concentration of the hdl fractions was determined according to a modified lowry method .
a zetasizer nano zs instrument ( malvern , worcestershire , uk ) with a 532-nm green laser beam was used for the study .
the scattered light was collected by detector at an angle of 173 using non - invasive back - scatter ( nibs ) technology and directed to a correlator .
the data were analyzed by zetasizer software ( dts , nano series , version 5.02 , malvern ) and size information was reported as the z - average by intensity .
all measurements were performed at 25c in duplicate with automatic duration measurements . in order to determine the suitable solvent with the best reproducibility prior to commencing the measurements , six different solvents [ including distilled water , normal saline , and 0.2 m phosphate buffer containing 0.1% ( w / v ) edta - na2 at three different ph values of 5.6 , 7.4 , and 8.3 ] were used as the hdl dispersant .
hourly measurements of size were made over a period of 5 h and the results were compared .
consequently , 0.2 m phosphate buffer at ph of 7.4 containing 0.1% ( w / v ) edta - na2 was selected as the most repeatable dispersant with the lowest aggregation index . in order to measure hdl mean particle size by dynamic light scattering ( dls ) methodology ,
isolated hdl samples were mixed gently with 1 ml of dispersant ( at a final protein concentration of 250 g / ml ) and passed through a syringe filter ( millipore cellulose acetate membrane , 30 mm , 0.2 m pore size ) prior to injection into a disposable polystyrene cell ( malvern ) in order to remove dust particles and were then subjected to size determination .
viscosity and refractive index ( ri ) of water as the dispersant were applied to standard operating protocol ( sop ) prior to size determination .
the accuracy of size measurements was examined using standard size nanoparticles [ gold nanoparticles , 20 nm , 0.01% ( w / v ) aqueous solution , nanocs inc .
, new york , ny , usa ] under the same experimental conditions and the results were matched with the diameter quoted by the manufacturer .
the within - assay cv for 10 measurements was 1.2% and between - assay cv for 8 measurements was 3.1% .
the detection limit of the assay for zetasizer instrument used in the present study was 0.3 nm to 10 m .
in order to calculate the ratio of hdl2 to hdl3 , the accumulative percentage of particles with sizes larger than 8.2 nm was divided to the accumulative percentage of particles with sizes smaller than 8.2 nm .
statistical analyses were performed using spss 16.0 ( spss , chicago , il , usa ) . before analyses , subjects were divided into three groups based on their serum hdl - c levels [ group 1 ( n = 49 ) : hdl 39 mg / dl ; group 2 ( n = 46 ) : 39 > hdl 49 mg / dl ; group 3 ( n = 45 ) : hdl > 49 mg / dl ] , and variables that were not normally distributed ( i.e. tg , apo a - i and apo b-100 ) were subjected to natural log transformation .
quantitative parameters were compared between groups by analysis of variance ( anova ) except for pon1 and hdl2/hdl3 that were analyzed by a nonparametric kruskal - wallis test and data were expressed as means  sd .
spearman 's rho correlation coefficient analysis was used to calculate the correlation between variables that were not normally distributed ( i.e. serum pon1 activity ) .
all subjects in the three groups were age matched . as seen in table 1 ,
the mean bmi value in group 3 subjects was lower compared with that in group 1 ( p = 0.002 ) .
whr in groups 2 and 3 was lower compared with that in group 1 ( p < 0.05 and p = 0.001 , respectively ) .
there was a significant difference in the mean tg values among the three groups ( groups 1 and 2 , p < 0.001 ; groups 2 and 3 , p = 0.002 ; and groups 1 and 3 , p < 0.001 ) , with group 3 having the lowest values .
tc was higher in group 3 compared with those in groups 1 and 2 ( p = 0.003 and p = 0.002 , respectively ) .
apo a - i was also significantly higher in groups 2 and 3 in parallel with the increasing serum hdl - c levels . the serum pon1 activity level was lower in group 3 compared with those in groups 2 and 1 ( p = 0.023 and p = 0.004 , respectively ) , whereas it was comparable in groups 1 and 2 .
pon1 activity adjusted for the hdl level ( pon1/hdl - c ) was also significantly lower in group 3 compared with those in groups 1 and 2 of our study ( p < 0.001 ) and lower in group 2 compared with that in group 1 ( p = 0.01 ) .
the mean hdl particle size and hdl2/hdl3 ratio was higher in the high hdl - c group compared with those in group 2 ( p = 0.003 and p = 0.001 , respectively ) and group 1 ( p < 0.001 and p < 0.001 , respectively )
. the hdl2/hdl3 ratio also correlated with hdl - c levels in groups 1 and 2 .
there were no significant differences in blood pressure , fbs , ldl - c , and apo b-100 levels among the three groups .
clinical characteristics of the study participants correlations between hdl - c , hdl mean particle size , hdl2/hdl3 ratio , and serum pon1 activity with anthropometric indices and clinical biomarkers are shown in table 2 .
serum hdl - c levels were positively correlated with tc , apo a - i , and ldl - c levels , and were negatively correlated with bmi , whr , and tg ( p < 0.01 ) .
hdl mean particle size and hdl2/hdl3 ratio were negatively correlated with bmi ( p < 0.01 ) , whr , and serum tg levels ( p < 0.05 and p < 0.01 , respectively ) , and were positively correlated with serum hdl - c levels ( p < 0.01 ) .
results in table 2 also indicate a significant negative correlation between serum pon1 activity and serum hdl - c levels ( p < 0.01 ) and apo a - i ( p < 0.05 ) , and a significant positive correlation with whr ( p < 0.05 ) . bivariate correlations between clinical biomarkers and hdl mean size , hdl2/hdl2 ratio , and pon1 activity in all subject figure 1 shows individual correlations of serum pon1 activity with serum hdl - c level , hdl mean particle size , and hdl2/hdl3 ratio . as indicated , serum pon1 activity was negatively correlated with serum hdl - c level ( r = 0.25 , p = 0.003 ) , hdl mean particle size ( r = 0.22 , p 0.008 ) , and hdl2/hdl3 ratio ( r = 0.23 , p = 0.007 ) .
there was also a strong positive correlation between serum hdl - c level and hdl mean particle size ( r = 0.45 , p < 0.001 ) [ figure 2 ] .
correlations between serum pon1 activities with serum hdl - c levels , hdl mean particle size , and hdl2/hdl3 ratio correlations between hdl mean particle size and serum hdl - c levels
the present study describes the correlation between hdl mean particle size and serum pon1 activity and their relationship with anthropometric indices and lipid profile in healthy male subjects .
we found a significant negative correlation between serum pon1 activity and hdl mean particle size in all subjects .
these findings are in accordance with kontush 's investigation which demonstrated that small hdl3 particles posse higher pon1 activity than large hdl2 particles .
nishio 's investigation on the susceptibility of hdl subfractions to copper - mediated oxidation indicated that hdl2 is more susceptible to oxidation than hdl3 .  
it is conceivable that this protective effect is mediated by pon1 activity being higher in small hdl3 particles compared with large hdl2 particles .
there is increasing evidence suggesting that pon1 makes a central contribution to the antioxidant capacity of hdl .
however , hasselwander 's investigation has indicated that reduction in arylesterase activity of paraoxonase had no effect on the hdl ability to inhibit ldl oxidation in patients on regular hemodialysis .
moreover , it has been demonstrated that hdl also exerts inhibitory effects on ldl oxidation , which are independent of paraoxonase activity .
several independent studies have indicated that the concentration and activity of pon1 are decreased in cad patients .
mackness et al . in a prospective study reported a reverse relationship between pon1 activity and cad risk in men , whereas in another prospective investigation on healthy middle - aged men , troughton et al . did not find any effect of pon1 paraoxonase activity on coronary heart disease ( chd ) outcomes . furthermore , van himbergen et al .
have reported that in a general population of dutch women , increased pon1 paraoxonase activity was a risk factor for cardiovascular outcomes instead of being protective .
our findings have indicated that serum pon1 activity in subjects with high hdl - c levels ( group 3 of our study ) is significantly decreased in comparison with subjects with medium and low hdl - c levels .
this finding is in contradiction with a majority of investigations demonstrating that high pon1 levels and high pon1 activity contribute to increased levels of hdl - c . in many examples of hdl deficiency , such as tangier disease and fish eye disease ,
serum pon1 is significantly diminished , but in other hdl deficiencies , this is not always the case .
blatter et al . have reported a positive correlation between pon1 activity and hdl - c levels in control subjects and cad patients .
they also demonstrated a significant decrease in pon1 concentration and its arylesterase and paraoxonase activities in cad patients in comparison with control subjects .
in addition , pon1 levels and activity could be modified by lifestyle determinants such as smoking , vitamin c and e consumption , and alcohol intake .
the contribution of pon1 genotypes to hdl - c levels was described by van himbergen et al . who investigated on a population with familial hypercholesterolemia .
they described that 55 l , 192 r , 107c , and 907 g variants of the l55 m , q192r , 107c / t , and 907 g / c polymorphisms predicted increased hdl - c levels .
they also indicated that like the pon1 genotypes , pon1 levels and paraoxonase , diazoxonase , and arylesterase activities also contributed to increased hdl - c levels . in our study , a strong positive correlation was found between serum hdl - c levels with hdl mean particle size and hdl2/hdl3 ratio . a similar direct relationship between hdl particle size and serum hdl - c levels
has been reported by others . decreased serum pon1 activity parallel to increased serum hdl - c levels observed in the present study may be a result of increased hdl particle size and the ratio of hdl2/hdl3 , and consequently , decrease in the small dense hdl particles .
in other words , changes in hdl subspecies distribution could explain the reduced activity of serum pon1 .
indicated that serum pon1 activity was decreased in chronic renal failure patients under maintenance hemodialysis .
they explained that reduction in pon1 activity was the consequence of the change in hdl subspecies distribution .
they found that the pon1 enzyme activity on hdl was almost totally expressed by the hdl3 subspecies fraction .
the levels of small hdl3 b and hdl3 c ( 7.3 - 8.2 nm ) were directly correlated with cad severity , whereas the levels of intermediate hdl3 a and hdl2 a ( 8.2 - 10 nm ) and large hdl2 b ( 10 - 13 nm ) were inversely correlated with cad severity .
small dense hdl3 has been shown to exert more powerful antioxidative , protective effects against ldl oxidation compared with the large hdl2 subfraction .
as ox - ldl is the most atherogenic form of modified ldl , this protective role of hdl3 particles is very important .
results of the anthropometric determinations in our study have indicated a reverse relationship between hdl mean particle size with bmi and whr and a direct relationship between pon1 activity and these anthropometric indices .
because of the effects of female sex hormones such as estrogen and estradiol on lipid profile and pon1 activity and alterations of these hormones during menstrual cycles in women , in the present study we investigated only healthy male subjects ; our results need to be verified in women .
it is also recommended to determine the relationship between hdl mean particle size and pon1 activity in cad patients in comparison with healthy subjects .
in conclusion , our results indicate that there is negative correlation between hdl mean particle size and serum pon1 activity in healthy iranian male subjects .
an inverse correlation also exists between hdl mean particle size and anthropometric indices , whereas plasma pon1 activity is directly correlated with them .
considering the complicated role of hdl in cad prevention and its heterogeneous nature , further characterization of hdl subclasses , including particle size and its associated enzymes such as pon1 , may be helpful for better understanding of cad risks and prognosis and also may help to design therapeutic protocols toward beneficial modifications of hdl characteristics .
 background : high density lipoprotein ( hdl ) particles are heterogeneous in composition , structure , size , and may differ in conferring protection against cardiovascular disease . 
 hdl associated enzyme , paraoxonase-1 ( pon1 ) , has an important role in attenuation of atherogenic low density lipoprotein ( ldl ) oxidation . 
 the aim of this study was to investigate the associations between hdl particle size and pon1 activity in relation to serum hdl cholesterol ( hdl - c ) levels.materials and methods : one hundred and forty healthy subjects contributed to this study . 
 hdl was separated by sequential ultracentrifugation and its size was estimated by dynamic light scattering . 
 paraoxonase activity was measured spectrophotometrically using paraoxon as substrate.results:results of this study showed that pon1 activity had negative correlations with hdl mean particle size ( r = 0.22 , p < 01 ) , hdl2/hdl3 ratio , and serum hdl - c levels ( r = 0.25 , p < 0.01 ) . 
 hdl mean particle size and hdl2/hdl3 ratio had negative correlation with body mass index ( bmi ) , waist hip ratio ( whr ) , and serum triglyceride ( tg ) levels , and positive correlation with serum hdl - c levels . 
 serum hdl - c levels had significant positive correlations with age , total cholesterol ( tc ) , and apolipoprotein a - i ( apo a - i ) and significant negative correlation with bmi , whr , and tg.conclusion:based on the results of this study , determination of hdl mean particle size beside the serum pon1 activity may help to better understand the cad risks , pathogenesis , and prognosis , and may also help to design therapeutic protocols toward beneficial modifications of hdl characteristics . 
INTRODUCTION
MATERIALS AND METHODS
Subjects
Anthropometric measurements
Biochemical analysis
Serum PON1 activity
HDL separation
HDL mean size determination
HDL
Statistical analysis
RESULTS
DISCUSSION
CONCLUSION