numerous prospective epidemiological studies have firmly established that high - density 
 lipoprotein ( hdl ) , as assessed by its cholesterol content , is inversely correlated with the 
 incidence and prevalence of coronary heart disease due to atherosclerosis ( 1 ) . the anti - atherogenic action of hdl is probably related 
 to reverse cholesterol transport by which excess peripheral cholesterol is returned to the 
 liver ( 2 ) . however , hdl is heterogeneous , encompassing 
 several subclasses differing in size , density and apolipoprotein content . several 
 approaches , such as ultracentrifugation , precipitation , immunoaffinity chromatography , and 
 various types of electrophoresis , have been used to isolate and characterize hdl 
 subpopulations . as previous studies have shown , in agarose gel electrophoresis , most hdl has 
 -mobility , while some shown pre- mobility . by gradient gel 
 electrophoresis , pre -hdl can be further separated into pre 
 1-hdl , pre 2-hdl and pre 
 3-hdl , while -hdl can be separated into five distinct 
 subpopulations , hdl3c , hdl3b , hdl3a , hdl2a and 
 hdl2b , according to increasing particle size ( 3 , 4 ) . during the past few years , several reports have indicated that patients with 
 hyperlipidemia / dyslipidemia often have altered hdl subclass distributions . recent studies 
 using this classification have found relationships between hdl subclasses and different 
 metabolic and anthropometric parameters . that is , it has been demonstrated that patients 
 with hyperlipidemia have increased concentrations of small hdl particles ( hdl3b 
 and hdl3c ) and decreased concentrations of large hdl particles ( hdl2b 
 and hdl2a ) ( 5 ) . furthermore , it has been 
 demonstrated that there are positive correlations between increased body mass index ( bmi ) 
 and higher levels of hdl3b and lower levels of hdl2b , and inverse 
 correlerations between fasting plasma insulin concentrations and plasma hdl3a , 
 hdl2a , and hdl2b ( 6 ) . a very 
 recent report proposed that lipoprotein subclass parameters can be used to manage risk more 
 effectively and prevent cardiovascular disease in patients with insulin resistance ( 7 ) . it is widely recognized that the insulin resistant 
 state is associated with dyslipidemia and increased cardiovascular disease risk ( 8 , 9 ) . the childhood origin of atherosclerosis is well recognized , as is the expression in 
 childhood of characteristics that place individuals at increased risk for atherosclesois 
 later in life . although the relationships of 
 low - density lipoprotein ( ldl ) particle subclass and particle size to the insulin resistant 
 state or atherogenic metabolic state have received considerable attention even in children 
 ( 12,13,14 ) , there have been relatively few studies regarding hdl 
 subclass or particle size in children ( 15,16,17,18,19 ) . this study was carried out to document the relevance of assessing hdl particle size as 
 another feature of atherogenic dyslipidemia found in children , using a recently described 
 method ( 20 ) . findings obtained in this study could provide 
 additional information for understanding the pathogenesis of atherosclerosis in 
 children . the study subjects were 268 school children ( 137 boys and 131 girls ) 7 to 13 yr of age , 
 residing in s - cho , chiba , japan . parents or guardians of all the children mellitus , thyroid disease , 
 chronic renal disease , or hepatobilliary disease were excluded from this study . after an overnight fast , morning venous blood samples were collected from the children at 
 school . the obesity index was calculated as follows : obesity index = [ ( body weight  ideal 
 weight)/ideal weight ) ]  100 . children whose obesity index was over 20% were considered to 
 be obese . the body mass index ( bmi ) was also calculated from the weight and height . in regard to individual plasma lipid parameters , total cholesterol ( tc ) and triglycerides 
 ( tg ) were determined enzymaticaly . hdl - c was measured by precipitation of other 
 lipoproteins using the phosphowolframic acid method . apolipoproteins ( apoa1 
 and apob ) were quantified by turbidimetric immunoassay . the atherogenic index ( ai ) was 
 calculated as follows : ai = [ tc  hdl - c ]  hdl - c . an ai value below 3 is considered normal 
 ( 22 ) . the hdl particle diameter was determined according to the method described by perusse et 
 al ( 20 ) , by gradient gel electrophoresis using 4 to 
 25% polyacrylamide . after the gels had been equilibrated at 120 v for 60 min , 
 electrophoresis was performed for each gel containing the serum sample diluted 1:2 with 
 sample buffer ( consisting of 40% sucrose , 0.06% edta-2na , and 0.01% bpp ) , to a volume of 
 20 l . each gel also contained thyroglobulin , ferritin , catalase , and 
 lactate dehydrogenase as reference standards of known diameter . the gels were then 
 electrophoresed at 15 v for 15 min , followed by migration at 70 v for 20 min , at 75 v for 
 16 h , and finally at 125 v for 8 h. the gels were then stained for lipid overnight with 
 sudan black , and for reference standards with coomassie brilliant blue for 45 min . the decolorified 
 gels were then scanned with an image scanner ( epson gt-6500 : seiko epson corporation , 
 nagano , japan ) and analyzed using an image processing and analysis program for macintosh 
 ( nih image 1.61 : national institutes of health , united states ) . the hdl particle diameter was then calculated by comparing the mobility of the 
 sample with that of the three calibrated standards on each gel . a higher integrated hdl 
 size indicated a greater proportion of  large  hdl particles , whereas a low hdl size 
 suggested an increased prevalence of  small  hdl particles . inter- and intra - assay 
 coefficients of variation for the integrated hdl size assessed by this method were < 3% 
 ( n=10 ) and < 2% ( n=9 ) , respectively . low - density lipoprotein ( ldl ) particle size was also measured , using the present blood 
 samples , according to the gradient gel electrophoresis method reported by krauss ( 23 ) and arisaka ( 11 ) ; type of solutions , duration time of electrophoresis , electric voltage , type 
 of lipid staining , and reference standard substances differed from the present hdl 
 electrophoresis . an 
 unpaired student s t - test was done to compare boys and girls for various parameters . relationships between hdl particle size and the individual parameters of the lipid profile 
 were analyzed using spearman s non parametric rank correlation . the study subjects were 268 school children ( 137 boys and 131 girls ) 7 to 13 yr of age , 
 residing in s - cho , chiba , japan . parents or guardians of all the children mellitus , thyroid disease , 
 chronic renal disease , or hepatobilliary disease were excluded from this study . after an overnight fast , morning venous blood samples were collected from the children at 
 school . the obesity index was calculated as follows : obesity index = [ ( body weight  ideal 
 weight)/ideal weight ) ]  100 . children whose obesity index was over 20% were considered to 
 be obese . the body mass index ( bmi ) in regard to individual plasma lipid parameters , total cholesterol ( tc ) and triglycerides 
 ( tg ) were determined enzymaticaly . hdl - c was measured by precipitation of other 
 lipoproteins using the phosphowolframic acid method . the atherogenic index ( ai ) was 
 calculated as follows : ai = [ tc  hdl - c ]  hdl - c . an ai value below 3 is considered normal 
 ( 22 ) . the hdl particle diameter was determined according to the method described by perusse et 
 al ( 20 ) , by gradient gel electrophoresis using 4 to 
 25% polyacrylamide . after the gels had been equilibrated at 120 v for 60 min , 
 electrophoresis was performed for each gel containing the serum sample diluted 1:2 with 
 sample buffer ( consisting of 40% sucrose , 0.06% edta-2na , and 0.01% bpp ) , to a volume of 
 20 l . each gel also contained thyroglobulin , ferritin , catalase , and 
 lactate dehydrogenase as reference standards of known diameter . the gels were then 
 electrophoresed at 15 v for 15 min , followed by migration at 70 v for 20 min , at 75 v for 
 16 h , and finally at 125 v for 8 h. the gels were then stained for lipid overnight with 
 sudan black , and for reference standards with coomassie brilliant blue for 45 min . the decolorified 
 gels were then scanned with an image scanner ( epson gt-6500 : seiko epson corporation , 
 nagano , japan ) and analyzed using an image processing and analysis program for macintosh 
 ( nih image 1.61 : national institutes of health , united states ) . the hdl particle diameter was then calculated by comparing the mobility of the 
 sample with that of the three calibrated standards on each gel . a higher integrated hdl 
 size indicated a greater proportion of  large  hdl particles , whereas a low hdl size 
 suggested an increased prevalence of  small  hdl particles . inter- and intra - assay 
 coefficients of variation for the integrated hdl size assessed by this method were < 3% 
 ( n=10 ) and < 2% ( n=9 ) , respectively . low - density lipoprotein ( ldl ) particle size was also measured , using the present blood 
 samples , according to the gradient gel electrophoresis method reported by krauss ( 23 ) and arisaka ( 11 ) ; type of solutions , duration time of electrophoresis , electric voltage , type 
 of lipid staining , and reference standard substances differed from the present hdl 
 electrophoresis . an 
 unpaired student s t - test was done to compare boys and girls for various parameters . relationships between hdl particle size and the individual parameters of the lipid profile 
 were analyzed using spearman s non parametric rank correlation . 1 ) table 1table 
 1 characteristics of subjects by sex 
 shows the characteristics of subjects profiles by sex . there was no statistically 
 significant difference regarding obesity index and various lipid levels between boys and 
 girls . 2 ) the hdl particle diameter was 11.4 ( mean )  0.61 ( sd ) nm in boys and 11.4  0.50 nm in 
 girls , not a statistically significant difference . even though particle size was analyzed by 
 dividing children into two age groups , those above 10 yr old ( 11.8  0.55 nm , n=162 ) and 
 those below 10 yr ( 11.5  0.62 , n=122 ) , there was no difference between boys and girls in 
 either age group . 3 ) relationships between hdl particle diameter and known individual risk factors for 
 atherosclerosis were evaluated for boys and girls combined , boys alone , and girls alone . therefore , we analyzed the data from boys and 
 girls together ( table 2table 2 relationship between hdl particle size and 
 individual lipid levels , anthropometric indices and blood pressure in 268 
 children , figs . . 
 2 relationship between hdl particle diameter ( range below 11 nm ) and 
 triglycerides level . relationship between hdl particle diameter and hdl - cholesterol 
 level .   relationship between hdl particle diameter ( range below 11 nm ) and 
 triglycerides level . the hdl particle diameter showed a significant positive correlation with hdl - c ( r=0.363 , 
 p<0.001 ) and apoa1 ( r=0.310 , p<0.05 ) , while it showed an inverse correlations with ai 
 ( r=0.316 , p<0.05 ) and bmi ( r=0.138 , p<0.05 ) . no statistically significant 
 correlation was noted between hdl particle size and tc , ldl - c , apob , tg , obesity index or 
 blood pressure . 4 ) there was no significant correlation between hdl particle size and tg levels in the 
 overall analysis ( n=268 ) , however , when this relation was analyzed in the limited hdl size 
 range below 11 nm , a significant inverse relation appeared between particle size and tg 
 levels ( r= 0.546 , p<0.01 , n=75 ) ( fig . 5 ) there was a statistically significant correlation between hdl particle size and ldl 
 particle size ( r=0.290 , p<0.05 ) . changes in hdl particle size , in association with changes in hdl subclasses distribution 
 are considered to reflect the metabolic process of uptake and transport of cholesterol by 
 circulating hdl . also , changes in hdl particle size may be related to altered lipid 
 metabolism caused by insulin resistance , which is the background of atherosclerosis ( 24,25,26 ) . lipoprotein lipase is the 
 rate - limiting enzyme in the clearance of tg - rich lipoproteins from plasma and is involved in 
 the formation of hdl , whereas hepatic lipase plays a role in the catabolism of hdl . therefore , abnormalities in the regulation of tg metabolism by lipoprotein lipase and 
 hepatic lipase may reduce hdl levels . furthermore , cholesteryl ester transfer protein ( cetp ) 
 and phospholipid transfer protein ( pltp ) are two other factors involved in the metabolism of 
 hdl ( 26 , 27 ) . pltp and cetp are released from adipose tissues and appear to directly modulate lipoprotein 
 metabolism : enhanced pltp activity results in a lower hdl - c fraction accompanied by a 
 relative increase in small pre -hdl . cetp also catalyzes the transfer of 
 cholesteryl ester from hdl to tg - rich lipoprotein in exchange for tg and leads to an 
 enrichment of hdl and ldl with tg . tg - enriched hdl and ldl particles can be subjected to 
 further lipolysis by hepatic lipase , leading to the formation of small , dense hdl and ldl 
 particles ( 28 , 29 ) . in obesity , increased synthesis of these lipid transfer proteins by the 
 enlarged mass of adipose tissue occurs and it has been demonstrated that weight reduction is 
 associated with a decrease in plasma cetp activity ( 30,31,32 ) . factors that promote the conversion of hdl and ldl into populations of 
 smaller particles is thought to be related to insulin resistance ( 19 , 32 ) . relatively few studies have examined hdl particle size in children ( 15,16,17,18,19 ) . in the present study , relationships among lipids , lipoproteins , 
 anthropometric measures and hdl particle sizes in children were investigated . it was found 
 that hdl particle size showed a general shift toward smaller size in association with 
 decreased hdl - c and apoa1 , and increased atherogenic index , tg and bmi , indicating the 
 features of atherogenic metabolic triad . although circulating hdl - c concentration is 
 recognized for its inverse association with the occurrence of cardiovascular disease , hdl - c 
 is only moderately predictive of disease ( 1 , 2 ) . this is exactly because circulating hdl , as assessed 
 by its cholesterol content ( hdl - c ) , does not reflect the heterogeneity of hdl particles ( hdl 
 subfractions ) . therefore , it is reasonable that hdl particle size and hdl - c level are not 
 strongly correlated , as shown in the present study . these results favor the idea that the 
 low cardiovascular disease risk may be due in part to their anti - atherogenic hdl pattern , 
 rather than the hdl - c concentration . in addition , no significant correlation was found 
 between hdl particle size and either tc or ldl - c , providing further support for the 
 previously published notion that the measurement of tc or ldl - c alone does not allow proper 
 identification of individuals who are carriers of atherogenic dyslipidemia . regarding the relationship between hdl particle size and plasma tg levels , a tendency for 
 an inverse relationship was recognized in the hdl size ranges below approximately 11 nm . 
 however , in the diameter ranges above 11 nm , this association was not found . the association 
 of reduced hdl particle size with elevated tg may be explained by the bidirectional exchange 
 of tg between hdl and tg - rich lipoprotein : hypertriglyceridemia appears to promote tg 
 transfer from tg - enriched lipoprotein to hdl particles under the action of cetp with a 
 subsequent hydrolysis of tg - enriched hdl by hepatic lipase or lipoprotein lipase , producing 
 small hdl particles ( 4 , 28 , 32 , 34 ) . although it is thought that tg is a critical component of 
 lipoprotein particles and plasma tg level affects lipoprotein size , both tg level -related 
 metabolic effect and tg level - independent genetic effect may be operated for determining hdl 
 particle size ( 4 , 35 ) . we also noted a positive relationship between hdl particle size and ldl particle size , 
 suggesting that the synergic reduction in the size of these two lipoproteins may be the 
 consequence of a common metabolic alteration , probably related to insulin resistance ( 8 , 9 , 23 , 24 ) . therefore , hdl size may represent another relevant marker of atherogenic dysmetabolism . in conclusion , although the mechanisms by which hdl size could affect cardiovascular 
 disease risk remain to be further investigated , the results of this study imply that a 
 general shift toward smaller hdl particle size may be related to the pathogenesis of 
 atherosclerosis in children . prospective studies are clearly warranted to quantify the 
 contribution of hdl particle size to the evaluation of the future risk of cardiovascular 
 disease development .
atherosclerosis begins in childhood . protection from atherosclerosis 
 is 
 provided by high - density lipoprotein ( hdl ) , a heterogeneous particle , which includes 
 several subclasses differing in size , density and apolipoprotein content . 
 the objective of 
 this study was to document the relevance of assessing hdl particle size as another feature 
 of dyslipidemia related to the develpment of atheosclerosis during childhood . 
 for that 
 purpose , hdl particle size in 268 community - based children ( 137 boys and 131 girls ) , 713 
 years old , was measured by gradient gel electrophoresis , and relationships of hdl particle 
 size to plasma lipids parameters and the anthropometric indices were analyzed . 
 there was 
 no gender difference in hdl particle diameter . 
 the results of analysis revealed 
 significant positive correlations between hdl particle diameter and hdl - cholesterol level 
 ( r=0.363 , p<0.01 ) , apolipoprotein ai level ( r=0.310 , r<0.05 ) and low - density 
 lipoprotein particle ( ldl ) size ( r=0.290 , p<0.05 ) , while there was an inverse 
 correlation with atherogenic index ( r=0.316 , p<0.05 ) . 
 there was no significant 
 correlation between hdl particle size and triglyceride levels in the overall analysis 
 ( n=268 ) , however , when this relation was analyzed in the limited hdl size range below 11 
 nm , a significant inverse relation appeared between particle size and tg levels ( r 
 = 0.546 , p<0.01 , n=75 ) . 
 these findings indicate that the general shift toward smaller 
 hdl particle size was associated with dyslipidemia characterized by higher atherogenic 
 index and triglyceride level , lower hdl - c level and smaller ldl particle size . 
 therefore , 
 hdl size may represent another relevant marker of atherogenic lipid metabolism .