Methods. 336 ARTERIOSCLEROSIS VOL 2, No 4, JULY/AUGUST 1982

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2 3 ARTERIOSCLEROSIS VOL 2, No 4, JULY/AUGUST 82 lipoprotein abnormalities, Fredrickson and coworkers 4 and later the World Health Organization (WHO) 5 recognized six types of hyperlipoproteinemias that are generally based on the concentrations of chylomicrons, VLDL, and LDL. Although an inverse relationship between HDL and coronary heart disease was reported by Barr et al. 6 in 51, HDL was not considered by the WHO, probably to keep the classification system simple enough for clinical purposes. Elevations in serum levels of LDL and VLDL, either individually or combined, were frequently found in survivors of myocardial infarction. 7 ' 8 To detect subtle lipoprotein abnormalities related to a particular form of hyperlipoproteinemia, it is essential, as a first step, to measure the concentration of cholesterol and triglycerides in the three major lipoprotein classes in a normal free-living population. Although extensive data are available in phenotypically abnormal individuals, to date there are only a few studies describing cholesterol and triglyceride composition of lipoprotein fractions of populations. " These studies have been limited to adults. The pathologic precursors of coronary artery disease are now recognized as originating during childhood. 12 Consequently, there has been a surge of interest in identifying problems associated with hyperlipoproteinemia in children. 1 *~ Recently serum lipid and lipoprotein distributions in U.S. children have been reported from major population-based studies. " 20 Of these, the Bogalusa Heart Study provides lipoprotein profiles of a biracial pediatric population from birth to 18 years of age. Furthermore, our observations on temporal relationships of risk factor variables indicate a high degree of serum lipoprotein level tracking in children and even in infants. 2 In this report we examine interrelationships of lipoprotein cholesterol and triglycerides within individual lipoprotein fractions and with serum total cholesterol and triglyceride levels in subgroups of children who belonged previously to extreme percentiles of distributions for p-lipoprotein cholesterol and/or pre-plipoprotein cholesterol. This report is a part of a Special Lipid Study designed to explore the anthropometric and biochemical determinants of serum lipoprotein levels in children. Methods Population Sample A detailed description of the sample selection and considerations of study design are reported elsewhere. Briefly, all children between the ages of 2 1 /2 and 14 years who fasted and were examined during both the and cross-sectional studies were entered into the sampling frame (n = 30). The B- and pre-3-lipoprotein cholesterol values from two major cross-sectional examinations were averaged and ranked separately. Four subgroups were selected based on age-, race-, and sex-specific distributions. In, both p- and pre-p-lipoprotein cholesterol levels were in the lowest quintile; in, p- and pre-p-lipoprotein cholesterol levels were in the highest and lowest quartiles respectively; in, both p- and pre-p-lipoprotein cholesterol levels were in the highest quintiles; in, p- and pre-p-lipoprotein cholesterol levels were in the lowest and highest quartiles respectively. Thus, phenotypically, our Groups 2, 3, and 4 were somewhat analogous to WHO'S classification system of Types Ha, lib, and IV, respectively. To increase the sample size of Groups 2 and 4, quartiles rather than quintiles were chosen. A total of 45 children were thus selected. Of the 7 children residing in Bogalusa in 78, 388 were actually seen between January and May of that year. Only fasting children with cholesterol and triglyceride values available on all three lipoprotein fractions following ultracentrifugation were included in this report (n = 0). Since electrophoretic nomenclature was originally used in describing the serum lipoprotein profiles of the children in the current study, both electrophoretic and ultracentrifugal nomenclatures are used to describe earlier lipoprotein profiles of the four subgroups of children and their current lipoprotein cholesterol and triglyceride levels determined by ultracentrifugation. Collection of Blood Specimens Venous blood was collected in vacutainer tubes and allowed to clot at room temperature for about 1V2 hours. After centrifugation, sera were collected in tubes containing thimerosal (Aldrich Chemical Company, Milwaukee, Wisconsin) and sent by bus to New Orleans in a box cooled with frozen packs. Specimens arrived at the laboratory the same day and were immediately refrigerated at 4 C until analysis the following day. Serum Cholesterol and Triglyceride Determinations Serum total and individual lipoprotein cholesterol and triglycerides were determined in a Technicon AutoAnalyzer II according to the Laboratory Manual of the Lipid Research Clinics Program. 2 * The laboratory has been designated as standardized by the Center for Disease Control, Atlanta, Georgia. Errors in measurements of serum total cholesterol and triglycerides were evaluated from an approximate % random subsample (n = 33) of blind duplicate determinations. During the 5-month period of this study, coefficients of variation for serum total cholesterol and triglyceride were 1.8% and 2.8% respectively. Lipoprotein Separation Lipoprotein fractions were separated by ultracentrifugation, using a Type 35 rotor fitted with sevenhole adaptors. 18 Aliquots of serum (2.0 ml) were transferred to polycarbonate centrifuge tubes without caps ( x 7 mm) and diluted to 3.0 ml with a solution of d = The contents were centrifuged

3 LIPOPROTEIN COMPOSITION IN CHILDREN Srinivasan et al. 337 in a Model L2-65B preparative ultracentrifuge (Beckman Instruments Corporation, Palo Alto, California) at 33,000 rpm for hours at 17 C to separate VLDL (top 1.0 ml). The bottom fraction was then centrifugedatd = for 20 hours. At the end of the run, the top (1.0 ml) and bottom (2.0 ml) fractions, LDL and HDL respectively, were obtained. The densities of the solutions were adjusted with NaCI-NaBr according to the procedure of Havel et al. No attempt has been made to ascertain the presence of p-vldl (floating-p) and sinking pre-p-lipoprotein (Lp-a). The analytical recovery of serum total cholesterol in the lipoprotein fractions isolated by ultracentrifugation was 8.7 ± 6.6% (mean ± SD; n = 0). On the other hand, the recovery in terms of triglycerides ranged from 5.2% ±.3% in to 0.8% ± 17.3% in. Statistical Analysis A four-factor analysis of variance model was used to test for differences in the lipoprotein levels among the four groups of children. The four factors were race (white, ), sex (boy, girl), p-lipoprotein selection level (low, high), and pre-p-lipoprotein level (low, high). Both untransformed and log-transformed analyses were conducted for triglycerides, VLDL-T, and VLDL-C. The results were essentially the same. No three-factor or four-factor interactions on the untransformed data were noted. If a two-factor interaction was noted, then only simple effects rather than main effects were presented for those factors. 26 Both Pearson product moment correlation coefficients and Spearman rank correlation coefficients were used to examine interrelationships between serum lipids and lipoproteins. Both statistics gave similar coefficients; hence, only the Pearson coefficients are presented. Linear regression lines of lipoprotein triglyceride on cholesterol for, and approximate 5% confidence intervals for individual values, were computed. The lines were then superimposed onto the corresponding race- and sex-specific plots for Groups 2, 3, and 4. The relationship of the various lipoprotein fractions to age was examined by linear regression. Results Serum Lipoprotein Cholesterol and Triglycerlde Levels The mean ± 2 SE levels of serum total and individual lipoprotein (VLDL, LDL, and HDL) cholesterol (C) and triglyceride (T) levels in four groups of children are given by race and sex in tables 1-4. As expected, serum total cholesterol and triglycerides and VLDL- C and LDL-C levels followed the criteria of sample selection. Children (Groups 2 and 3) who were selected based on elevated p-lipoprotein cholesterol levels had higher total cholesterol, VLDL-C, and LDL-C levels than their counterparts (Groups 1 and 4) with earlier low p-iipoprotein cholesterol levels. Likewise, children (Groups 3 and 4) who had elevated pre-p-lipoprotein cholesterol levels before the study showed higher serum triglycerides, VLDL-C, VLDL-T, and LDL-T levels than children (Groups 1 and 2) with low pre-p-lipoprotein cholesterol. Of particular interest is the observations on HDL-C and HDL-T levels in these four lipoprotein groups, since a-lipoprotein levels were not taken into consideration in sample selection. Children with earlier low pre-plipoprotein cholesterol levels (Groups 1 and 2) had higher HDL-C levels and lower HDL-T levels than children with high pre-p-lipoprotein cholesterol Table 1. Serum Total Cholesterol and Triglycerlde Levels by Race and Sex In Children with Different Lipoprotein Profiles: Bogalusa Heart Study, 78 Total cholesterol* Triglycerides't ± 6.5 ± 7.0 ± 5.8 ±. ± 3.8 a 4.7 dt dt dt dt dt 3.5 e ± 22.1 ± 17.1 ±.6 ± 7. ± 7.6 b ±.8 ± 6.8 ± 21.2 ± 7.1 ± ± ± ± ± ± 4.= 8.2 ± ± ± ± ± ± 8.0 ± ± 0.0 ± ± 72.4 ±.8 ± 66.6 ± 62.1 ± 61.7 ± Race, p < tlog transformed before statistical analysis. Statistical differences: (b and c) > (a and d), p < ; (f and g) > (e and h), p < ; (g and h) > (e and1), p < < h

4 338 ARTERIOSCLEROSIS VOL 2, No 4, JULY/AUGUST 82 Table 2. Serum VLDL-Cholesterol and Triglyceride Levels by Race and Sex In Children with Different Lipoprotein Profiles: Bogalusa Heart Study, 78 VLDL cholesterol* t VLDL-triglyceride* t 8.7 ± ± ± ± ± 0.8 a 26.4 ± 4.2. ± 4..8 ± ± ± 2.7 a.6 ± ± ± ± ± 1.1" 33.5 ± 8..3 ± ± ± ± 4.5" 17.3 ± ± ± ± ± 1.5 C 61.5 ± ± ± ±.0.0 ± 6. C 12.2 ± ± ± ± ± ^. d 43.6 ± ±.3.3 ± ±.5.0 ± 6.4 d Race, p < tsignificant differences (log transformed data): (b and c) > (a and d), p < 0.001; (c and d) > (a and b), p < Table 3. Serum LDL-Cholesterol and Triglyceride Levels by Race and Sex in Children with Different Lipoprotein Profiles: Bogalusa Heart Study, 78 LDL-cholesterol LDL-triglyceride* 68.2 ± ± ± ± ± 2.6" 14.4 ± ± ± ± ± 0.7" 8.4 ±.2 4. ± ±.3 2. ± ± 5.8" 20. ± 2..3 ± ± ± ± 1.1' 7. ± ± ± ± ± ± ± ± ± ± 1.38 'Race, p = (b and c) > (a and d), p < ; (f and g) > (e and h), p < ; (g and h) > (e and f), p < ± ± ± ± ± 4.3 d. ± ± ± ± ± 1.4 h Table 4. Serum HDL-Cholesterol and Trigiycerlde Levels by Race and Sex In Children with Different Lipoprotein Profiles: Bogalusa Heart Study, 78 HDL-cholesterol* HDL-triglyceridef *Race, p < tsex, p < (a and b) > (c andd), P< 54.0 dt dt dt dt dt 2.6 a.6 dt dt dt dt dt 0.8«0.01; 58.4 ± ± ± ± ± 3.5".8 ± ± ± ± ± 0.8' 4.7 ± 57.2 ± 51.7 ± 5.4 ±.5 ±.4 ±.2 ±.4 ±.6 ±. ± 'f and g) > (e and h), p< 0.01; (g and h) > (e and f), P < ± ± ±.3.1 ± ± 3.1 d.4 ± ± 1.4. ± ± ± 1.1 h

5 LIPOPROTEIN COMPOSITION IN CHILDREN Srinivasan et al. 3 Table 5. Proportion of HDL-Cholesterol to Total Cholesterol, LDL-Cholesterol and VLDL + LDL- Cholesterol In Serum of Children with Different Lipoprotein Profiles: Bogalusa Heart Study, 78 HDL-C (%)*t LDL-C/HDL-C** VLDL + LDL-C/HDL-C*:}: (n = ) 42.7 ± 1.4 a 1. ± 0.0 e 1.41 ± 0. e (n = ) 32.8 ± 1.5 b 1.3 ± 0.20' 2. ± 0.22' (n = ) 2.3 ± ± ± 0. (n = ) 40.3 ± 1.7" 1.31 ± 0. h 1. ± 0. h Race, p < thdl-c/total cholesterol x 0; arc sine transformed prior to statistical analysis: (b and c) < (a and d), p < ; (c and d) < (a and b), p < $Log transformed prior to statistical analysis: (f and g) > (e and h), p < ; (g and h) > (e and f), p <0.01. (Groups 3 and 4). In general, white children had lower serum total cholesterol and HDL-C and higher serum triglycerides VLDL-C, VLDL-T, and LDL-T than children. Similar race differences were not noted for LDL-C and HDL-T. Among the variables, sex-related differences were noted only for HDL-T, with girls showing significantly higher values than boys. The status of HDL-C in proportion to other lipoproteins in the four groups were further evaluated in terms of percentage of total cholesterol (table 5). HDL-C constituted a smaller percentage of serum total cholesterol in children who had either elevated p-lipoprotein cholesterol levels (Groups 2 and 3) or elevated pre-p-lipoprotein cholesterol levels (Groups 3 and 4) than their counterparts. This was reflected in the relatively higher mean ratios of LDL- C/HDL-C and VLDL + LDL-C/HDL-C observed in children with high p-lipoprotein cholesterol levels (Groups 2 and 3) or pre-p-lipoprotein cholesterol levels (Groups 3 and 4). Irrespective of liproprotein groups, white children had significantly lower percentages of total cholesterol as HDL-C and higher ratios of LDL-C/HDL-C and VLDL + LDL-C/HDL-C than children. Liproprotein Composition Changes in lipoprotein composition among the four groups of children were evaluated in terms of the ratios of C/T in the VLDL, LDL, and HDL, as well as the relationship between cholesterol and triglycerides in each of the fractions. It can be seen from table 6 that, in general, children with low pre-p-lipoprotein cholesterol (Groups 1 and 2) had higher ratios of VLDL-C/T, LDL-C/T, and HDL-C/Tthan children with high pre-p-lipoprotein cholesterol (Groups 3 and 4). Among the four lipoprotein groups, the ratios of VLDL-C/T and HDL-CAT were lowest in children, while the ratio of LDL-CAT was highest in Group 2 children. Black children had significantly higher ratios of VLDL-C/T and LDL-C/T than white children. Correlation coefficients of the relative distribution of cholesterol and triglycerides in VLDL, LDL, and HDL of the four groups of children are given in table 7. For VLDL, there was an extremely strong positive correlation between cholesterol and triglycerides in Table 6. Cholesterol to Trlglycerlde Ratios of Serum Lipoprotein In Children with Different Lipoprotein Profiles: Bogalusa Heart Study, 78 Group VLDL-C/T*t LDL-C/T*f HDL-C/T*t 1 0. ± 0.05 a ± 0.06 b ± ± 0.03 d 5.07 ± ± ± ± 0. h 5.24 ± 0.61' 4.33 ± 0.30J 3.73 ± 0.30 k 4. ± 0.42' 'Significant differences (log transformed data): (a and b) > (c and d), p < ; (f and g) > (e and h), p < ; (e and f) > (g and h), p < 0.002; (i and I) > (j and k), p < 0.01; (i and j) > (k and I) p < trace difference, p < all four groups. There was also a significant positive correlation between cholesterol and triglycerides for LDL in Groups 1, 3, and 4, but the magnitude was of low order; in contrast no correlation was noted in children. A lack of correlation between cholesterol and triglycerides for HDL was noted in all four groups. The relationship between cholesterol and triglyceride content of VLDL in the four groups of children by sex is shown in figure 1 for white children and in figure 2 for children. The regression line and 5% confidence interval were constructed for Group 1, and these lines were superimposed on the corre- Table 7. Correlation Coefficients of Relative Distribution of Cholesterol and Triglycerides In Serum Llpoprotelns of Children with Different Lipoprotein Profiles: Bogalusa Heart Study, 78 Cholesterol vs triglycerides Group *p< tp < Lipoprotein fraction VLDL LDL HDL 0.80* 0.81* 0.3* 0.1* 0.37* * 0.30j

6 340 ARTERIOSCLEROSIS VOL 2, No 4, JULY/AUGUST 82 WHITE BOYS (n = ) (n = ) WHITE GIRLS (n = ) (n = ) * I p I I Q > 0 0 (n = ) (n = ) (n = ) (n = ) VLDL-Cholesterol Figure 1. Relationship between cholesterol and triglyceride contents of VLDL in white children who had different lipoprotein profiles. : low p- and pre-p-lipoprotein cholesterol; : high p- and low pre-p-lipoprotein cholesterol; Group 3: high p- and high pre-p-lipoprotein cholesterol; : low p- and high pre-p-lipoprotein cholesterol. The regression line and 5% confidence interval were constructed for and these lines were superimposed on corresponding sexspecific Groups 2, 3, and 4 plots. Open dots represent two observations. sponding race- and sex-specific Groups 2, 3, and 4 plots. Although the low levels of both p- and pre-plipoprotein cholesterol of may not be considered "normal" in a statistical sense, the regression data of this group were used for comparative purposes. A strong linear relation between cholesterol and triglycerides almost containing the origin can be seen in children of both races and sexes. A considerable number of children from, and to a certain extent, fell above the 5% confidence interval set for corresponding race- and sexspecific. Such outliers were highest in white girls from, indicating a high prevalence of triglyceride-rich particles in this group. Furthermore, for a given VLDL-T level, especially below 0 mg/dl, white girls from tended to have more triglyceride-rich particles (in relation to cholesterol) than the other race-sex groups. Similar regression plots

7 LIPOPROTEIN COMPOSITION IN CHILDREN Srinivasan et al. 341 BLACK BOYS (n = ), BLACK GIRLS (n = ) I I I i I I I VLDL-Cholesterol Figure 2. Relationship between VLDL-cholesterol and VLDL-triglycerides in children with different lipoprotein profiles. Lipoprotein groups and construction of regression line and 5% confidence interval are the same as in figure 1. were constructed for LDL and HDL (figures not shown). The prevalence of triglyceride-rich lipoprotein particles above the 5% confidence interval noted for individual lipoprotein cholesterol-triglyceride relationship in is summarized by sex and race in table 8. Like VLDL, the prevalence of triglyceride-rich particles was high in and children, especially in for LDL and HDL. Within or Groups 2, 3, and 4 combined, white girls had more triglyceride-rich LDL particles and and girls had more triglyceriderich HDL particles than corresponding race-sex groups. Interrelationship between Serum Llplds and Lipoprotein Llplds Correlation coefficients (t) between serum total cholesterol and triglyceride levels and lipoprotein cholesterol and triglyceride levels in the four groups of children are given in table. There was a significant negative correlation between serum total cholesterol and VLDL-C in children. Although serum total cholesterol correlated significantly with LDL-C and HDL-C in all four groups, the order of magnitude of correlation coefficients varied among the groups. Serum total cholesterol correlated very highly with LDL-C in and 3 children when compared to children from Groups 1 and 4. On the other hand, correlation between serum total cholesterol and HDL-C was highest in and lowest in children. The correlation coefficients between serum total cholesterol levels and VLDL-T, LDL-T, and HDL-T levels were of a low order of magnitude and reached a significant inverse relationship for VLDL only in and a positive relationship for LDL and HDL only in. Serum triglycerides correlated very highly with VLDL-C in all four groups. A significant inverse relationship was noted for LDL-C only in children and for HDL-C in all four groups. Serum triglyceride levels correlated very highly with VLDL-T in all four groups, as one would expect. Of particular interest is the high positive correlations obtained for LDL- T in all but children. Correlations were of a low order of magnitude for HDL-T.

8 342 ARTERIOSCLEROSIS VOL 2, No 4, JULY/AUGUST 82 Table 8. Prevelance of Trlglycerlde-Rich Lipoprotein Particles* In Major Serum Lipoprotein Fractions of Children with Different Lipoprotein Profiles: Bogalusa Heart Study, 78 Lipoprotein VLDL LDL HDL (%) 1() 1() (%) 7(18) 5(22) 17() () 3() 14(32) 5(22) 6() 2() 3(7) 4(17) (%) 1(6) 2(22) 2() 3(33) 3(20) 1(6) 3(20) Combined ( ) (%) 8(12) 5() (30) 12(18) 6(12) (24) 8(14) 7() 2(4) 3(5) 7(12) "Above the 5% confidence interval noted for lipoprotein triglycerides-cholesterol relationship in. Dashes indicate no particles present. Effect of Age To assess age effects (6- to 18-year age interval) on serum lipoprotein cholesterol and triglyceride levels among the four groups of children, regression analyses of the lipoprotein fractions on age were made by race and sex. The summary of statistically significant results only is provided in table. While VLDL-C showed positive association with age only in boys from both races, VLDL-T showed associations in and boys from both races and in. Associations with age were not significant for LDL-C in all four groups, whereas LDL-T associated positively with age only in girls from. Of particular interest is the significant inverse relationship of HDL-C with age, seen only in from Groups 2, 3, and 4, but not in their counterparts from. Only children from showed a positive association with age for HDL-T. Discussion The present study provides lipid (cholesterol and triglycerides) composition and interrelationships of major lipoprotein fractions in subgroups of children from a total biracial population whose p- and/or prep-lipoprotein cholesterol levels were in the extremes of the general distribution. The problem of regression to the mean raises the question of whether the subgroups of children remained in their respective rankings. As we have observed previously, persistence in ranks (tracking) was stronger for p- than for pre-plipoprotein cholesterol. Because of inherent Table. Correlation Coefficients Between Serum Total Cholesterol and Triglycerlde Levels and Lipoprotein Cholesterol and Triglyceride Levels In Children with Different Lipoprotein Profiles: Bogalusa Heart Study, 78 Lipoprotein fraction VLDL-C* LDL-C HDL-C VLDL-T* LDL-T HDL-T *Log transformed. +p < tp< p< (n = ) Serum total cholesterol (n = ) (n = ) -0.28t t t (n = ) (n = ) Serum triglycerides* (n = ) t -O.32t (n = ) (n = ) *

9 LIPOPROTEIN COMPOSITION IN CHILDREN Srinivasan et al. 343 Table. Regression Analyses with Age of Serum LIpoproteIn Cholesterol and Triglycerides In Children with Different LIpoproteIn Profiles. Bogalusa Heart Study, 78 Variable VLDL-C HDL-C VLDL-T LDL-T HDL-T LJpoprotein group Only statistically significant data are included. *p < tp< Race, sex boys boys boys girls boys girls Slope 0.87t 1.* -2.55* -1.* -1.65* 2.48* 1.73* 4.* 3.77f 3.78* 0.85* 0.64f 0.58* Intercept (Y-axis) biologic variability and the measurement error of a single sample, 12 it is to be expected that upon reexamination some subjects will no longer represent the originally selected groups. However, the mean values of p- and pre-p-lipoprotein cholesterol upon reexamination showed remarkable consistency in maintaining the group differences. The lipoprotein lipid values represent actual direct analysis of individual fractions isolated by ultracentrifugation. Although children were grouped according to percentile levels, it should be emphasized that these children are free-living healthy individuals. Here, the definition of normality was made from a statistical, rather than a clinical or biological viewpoint. These lipoprotein compositional data, however, provide useful baseline values for children with defined lipoprotein categories within a population. Although race- and sex-related differences among children are well established for serum total cholesterol, triglycerides, VLDL-C, LDL-C, and HDL-C, - 20 the current o!,servations that white children generally had significantly higher VLDL-T and LDL-T than children have not been noted before. While HDL-C differed between and white children as expected, HDL-T showed no difference. On the other hand, HDL-T levels were significantly higher in girls than in boys from both races. The study of relative distributions of cholesterol and triglycerides within lipoprotein fractions offers a means by which compositional changes among children who had different p- and pre-p-lipoprotein cholesterol profiles can be evaluated. Such information may reveal the atherogenic potential of lipoproteins. In hyperlipoproteinemic subjects, a high VLDL-C/T ratio is associated with a highly atherogenic particle, P-VLDL 5 Likewise, LDL particles with a high C/T ratio are known to nave more ordered and less fluid lipid cores that are considered potentially atherogenic Furthermore, a decrease in LDL fluidity might also reduce its catabolism, as suggested from the studies of Shepherd et al. 2 In these apparently normal children, one can detect significant differences in C/T ratios of lipoproteins, depending upon the lipoprotein profile. In general, children (Groups 1 and 2) with earlier low serum triglycerides and pre-plipoprotein cholesterol had higher VLDL-C/T, LDL-C/ T, and HDL-C/T ratios than their counterparts (Groups 3 and 4). However, ratios alone without the absolute levels of the components involved are of limited use, at least for VLDL-C/T ratios. 18 The VLDL- C/T ratio of 0.42 has been considered the upper limit for separating individuals with normal VLDL from those with p-vldl. 5 M In adults this ratio seems to be influenced by the serum triglyceride level. 31 The high VLDL-C/T ratios in and 2 children may be related to extremely low serum triglyceride levels (table 1), since the occurrence of p-vldl as in Type III disorder is characterized by an elevation of both cholesterol and triglycerides in plasma. 0 On the other hand, it is noteworthy that for elevated LDL-C levels of similar magnitude (Groups 2 and 3), children with high p- and low pre-p-lipoprotein cholesterol () had the highest LDL-C/T ratio among the four groups. In addition, there was absolutely no relationship between cholesterol and triglycerides within LDL particles in (table 6). It is tempting to speculate that LDL particles in children may have less fluid lipid cores and may be potentially atherogenic. Although extremely high correlation coefficients for VLDL-C and VLDL-T were noted among the four groups, a close inspection of a linear relationship between cholesterol and triglyceride content of VLDL by sex and race indicates definite compositional differences in certain children within and (figures 1 and 2). The prevalence of triglyceride-rich (in relation to cholesterol) VLDL particles was highest in white girls from Groups 3 and 4

10 3 ARTERIOSCLEROSIS VOL 2, No 4, JULY/AUGUST 82 with no instance of unusually high enrichment noted in girls. Compositional changes noted may be either intrinsic or may be a reflection of serum cholesterol and triglyceride levels characterizing each group. Although the triglyceride content of lipoproteins is known to be dependent on serum triglyceride levels, 32 " 34 white girls from tended to have more outliers in terms of triglyceride-rich VLDL particle for a given VLDL-T level (especially below 0 mg/dl) than the rest of the race-sex groups. Furthermore, since serum triglyceride levels of boys and girls from were similar, there may be an intrinsic difference in VLDL-C/T ratio as a function of sex in white children. Since sex hormones significantly influence lipoprotein levels, it would be of considerable interest to be able to follow such changes through puberty as will be discussed below. The relationship of LDL-C and LDL-T, although positive and statistically significant, was not strong and did not contain origin. This may be due to the fact that LDL fraction between density and is heterogeneous and comprises at least two distinct lipoprotein classes, triglyceride-rich S, (d = ) fraction and cholesterol-rich S, 0-12 (d = ) fraction. 33 Among the four race-sex groups, white children tended to have more triglyceride-rich LDL. The strong correlation of LDL-T with serum triglyceride levels in Groups 1, 3, and 4 (table ) is in accordance with earlier reports that lipoproteins of greater density than VLDL also carry increased proportions of triglycerides at increased concentrations of serum triglycerides due to an exchange between lipoproteins. 33 ' 35 The complete lack of a relationship between the cholesterol and triglyceride content of HDL suggests a wide variability in the triglyceride content among HDL particles. Although serum triglyceride levels correlated positively with HDL-T as expected (table 8), no distinct pattern emerged among the four race-sex groups in terms of triglyceride-rich HDL. The proportion of HDL-C to total cholesterol, LDL- C, and VLDL + LDL-C in these groups of children (table 5) is of practical significance considering the antiatherogenic nature of HDL. Observations from the Framingham Study in an adult population, aged 50 to 80 years, indicate that the risk of coronary heart disease can be more accurately assessed by the relative proportion of HDL-C to serum total cholesterol, LDL-C, or LDL + VLDL-C. However, corresponding ratios for children with extreme percentiles of the distribution for p- and/or pre-s-lipoprotein cholesterol levels in the present study are well below the reported average risk ratios for adults. This shows that these ratios are subject to change to higher values during adulthood. Cross-sectional studies in children 38 indicate that these ratios begin to increase progressively, especially in, at the onset of puberty. The highest ratios of LDL-C/HDL-C and VLDL + LDL-C/HDL-C in children may be due to high serum triglyceride levels, a condition known to relate inversely to HDL-C It is noteworthy that the relationship between age and lipoprotein lipid levels is not the same in children with different lipoprotein profiles (table ). In general, cross-sectional studies, including ours, indicate a positive association with age for VLDL-C and a negative association with age for HDL-C and LDL-C around puberty. 38 ' M No age-related trends were noted in children who had low p- and pre-p-lipoprotein cholesterol levels (). Age-related trends in other groups who had elevated p- and/or pre-p-lipoprotein cholesterol were observed mainly in boys. Of particular interest is the significant inverse relationship with age for HDL-C noted only in from Groups 2, 3, and 4, emphasizing the atherogenic potential of lipoprotein changes occurring in these groups of children. Since serum lipoprotein levels persist, 2 the above observations may help identify subgroups of children at young ages who are potential targets for coronary artery disease and for initiation of appropriate preventive measures. The present studies demonstrate subtle but definite compositional differences in terms of cholesterol and triglyceride content of major lipoprotein classes among subgroups of free-living, healthy children. Since the lipid transport by lipoprotein is a highly integrated, dynamic, and complex metabolic process, serum lipoproteins are probably affected both quantitatively and qualitatively in several ways. 40 Both genetic and environmental factors obviously determine lipoprotein concentrations. The differences that exist in lipoprotein classes among subgroups of children who are exposed to the same environmental factors suggest that the metabolic process is altered by several determinants. Anthropometric and biochemical profiles such as carbohydrate tolerance, sex hormone levels, etc., are being evaluated in these children in an effort to identify possible associations or determinants. The results will be the subject of another report. Acknowledgments The authors thank Carl Whitaker and Mildred Caro for their work in the SCOR-A Core LJpid Laboratory. References 1. Kannel WB, Castelll WP, Gordon T, McNamara PM. Serum cholesterol, lipoproteins and risk of coronary heart disease: The Framingham Study. Ann Intern Med 71 ;74: Castelll WP, Cooper GR, Doyle JT, et al. Distribution of triglycerides and total, LDL, and HDL cholesterol in several populations: A cooperative lipoprotein phenotyping study. J Chronic Dis 77;30: Gofman JW, Young W, Tandy R. Ischemic heart disease, atherosclerosis and longevity. Circulation 66;34: Fredrickson DS, Levy Rl, Lees RS. Fat transport and lipoproteins an integrated approach to mechanisms and disorders. N Engl J Med 67;276:34-^4, 4-3,148-1, 2-2, Beaumont JL, Carlson JA, Cooper GR, Fejfar Z, Fredrick-

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