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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1082-1090.)
© 1997 American Heart Association, Inc.

Articles

Associations of Age, Adiposity, Menopause, and Alcohol Intake With Low-Density Lipoprotein Subclasses

Paul T. Williams; ; Ronald M. Krauss

From the Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, Calif.

	Abstract

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Abstract We used nondenaturing polyacrylamide gradient gel electrophoresis to examine the associations of age, adiposity, menopause, and alcohol intake with LDL subclasses in 355 individuals. The absorbency of protein stain was used as an index of mass concentrations at intervals of 0.05 nm within seven LDL subclasses: LDL-IVB (22.0 to 23.2 nm), LDL-IVA (23.3 to 24.1 nm), LDL-IIIB (24.2 to 24.6 nm), LDL-IIIA (24.7 to 25.5 nm), LDL-II (25.5 to 26.4 nm), LDL-I (26.0 to 28.5 nm), and intermediate-size lipoproteins (ISL, 28.0 to 32.0 nm). Age and alcohol intake were obtained from questionnaires, and body mass index was computed from clinic measurements of weight and height. In adult men, body mass index correlated positively with LDL-III, and alcohol intake correlated positively with larger LDL-I. Age was positively correlated with LDL-IIIA and ISL in both men and women and with LDL-IIIB and LDL-II in women. Postmenopausal women had higher LDL-IIIA, LDL-II, and ISL than both premenopausal and premenarchal females. Adult males, 18 years old, had higher levels of LDL-IIIA and LDL-II than younger males. Adjustment for fasting plasma triglyceride levels eliminated the significant associations between age and LDL-IIIA in both men and women and between age and LDL-II in women. Partial correlation analyses showed that reductions in the LDL peak diameter associated with increasing age, male sexual maturation, menopause, and adiposity are attributable to increases in the LDL-IIIA subclass. Thus, densitometric measurements of protein-stained gradient gels reveal specific relationships between LDL subclasses and age, adiposity, and alcohol intake beyond those identified by the LDL peak or average diameter.

Key Words: age • puberty • alcohol • adiposity • menopause • low-density lipoproteins

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Coronary heart disease risk increases with age and adiposity and decreases with alcohol intake.^{1 2 3} These relationships could be attributable in part to changes in LDL subclasses, as distinguished by size on nondenaturing polyacrylamide gradient gels.^{4 5} At least seven LDL subclasses have been identified: LDL-IVB (22.0 to 23.2 nm), LDL-IVA (23.3 to 24.1 nm), LDL-IIIB (24.2 to 24.6 nm), LDL-IIIA (24.7 to 25.5 nm), LDL-II (25.5 to 26.4 nm), LDL-I (26.0 to 28.5 nm), and ISL (28.0 to 32.0 nm).^{4 6} Several case-control studies suggest that the predominance of smaller LDL particles (ie, LDL-III or LDL-IV subclasses) is associated with increased CHD risk.^{7 8 9}

The principal methods for studying the distribution of LDL particles are based on the LDL major and minor peaks.^{4 5} The position of the predominant (major) LDL peak and the mean particle size based on the integration of all peaks have shown that adiposity, ^{8 10} male sex,^{5 11} and menopause¹² are all associated with a shift in the predominant LDL peak toward smaller particles. With respect to the plasma concentrations of the individual LDL subclasses, the average diameter and predominant LDL peak diameter are qualitative descriptions of the LDL particle distribution (ie, the diameter of the predominant LDL) rather than their quantitative description (ie, the concentration of individual subclasses, as provided, for example, by analytic ultracentrifugation¹³ ). The shift toward a smaller LDL peak diameter could occur if the percentage of smaller LDL (eg, LDL-III or LDL-IV subclasses) is increased relative to larger LDL (ie, LDL-II or LDL-I). This increase could be accomplished in different ways: (1) small and large LDL could both increase provided the increase is greater for small LDL, (2) small and large LDL could both decrease provided the decrease is less for small LDL, (3) small LDL could increase while large LDL decreases or is unchanged, or (4) large LDL could decrease while small LDL is unchanged.

This report uses the densitometric measurements of absorbency on protein-stained gradient gels to assess the associations of LDL subclasses with age, adiposity, and alcohol intake in a population sample of men and women. Mean differences and correlation coefficients are computed for LDL levels (absorbency) at each LDL diameter value at intervals of 0.05 nm. This approach previously has been employed to assess gender differences and the relationships of LDL subclasses to other lipoproteins. The objectives of the current report are (1) to assess the associations of age, adiposity, and alcohol intake with protein-stained LDL subclasses; (2) to determine which LDL subclasses are responsible for the shift toward smaller LDL with age, menopause, and adiposity; and (3) to assess whether the relationships between LDL subclasses and age, adiposity, and alcohol intake can be attributed to metabolic factors associated with plasma triglyceride, HDL, or LDL cholesterol concentrations.

	Methods

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Subjects
The subjects were originally sampled as part of a family study of the inheritance of LDL subclass patterns.¹⁴ The selection of kindreds took place primarily among the Mormon community in the San Francisco Bay Area.¹⁴ Following the same protocol, several additional kindreds were recruited after the first report from this study. The kindreds were not selected for lipid disorders or family history of cardiovascular disease, but sequential sampling of kindreds for an LDL subclass phenotype expression was used,¹⁴ ie, the members of the pedigree were sampled if they had the potential to provide information on LDL subclass pattern A and B inheritance.¹⁵ Family members who were not pregnant, had no serious disease, and were at least 6 years of age were eligible to participate. Blood samples were obtained from each subject after an overnight fast. For nonlocal relatives, samples were delivered to Donner Laboratory by overnight mail at 4°C. Persons were excluded from this analysis if they reported taking drugs known to affect lipoproteins. All participants signed statements of informed consent approved by the institutional human use review board.

Of the 355 subjects with complete LDL and apoB measurements, 15 were excluded for missing data on cigarette and alcohol usage. Few subjects reported using cigarettes, oral contraceptives, or postmenopausal estrogen replacement. We did not test the relationships of these variables to LDL subclasses because of their low statistical power. The 50 subjects who used cigarettes, oral contraceptives, or estrogen were excluded in the analyses to follow because these factors could potentially confound relationships involving LDL subclasses. Four women were also excluded for not falling within one of three prescribed age categories: premenarchal girls (14 years old and not having periods), premenopausal women (12 to 50 years old and having periods), and postmenopausal women (40 years old and not having periods). The descriptive characteristics of the sample are presented in Table 1.

View this table: [in this window] [in a new window]   Table 1. Descriptive Characteristics of Subjects

Clinical Measurements and Questionnaires
Participants completed medical history questionnaires during their clinic visits, including questions on date of birth; drug and medication use; current and recent pregnancy, lactation, and hormone use in women; cigarette use; and usual intake of alcoholic beverages. BMI was calculated as the weight in kilograms divided by height in meters squared. Ounces of alcohol consumed per week were calculated on the basis of 0.48 oz per 12-oz bottle of beer, 0.48 oz per 4-oz glass of wine, 0.60 oz per drink of hard liquor (including cocktails and mixed drinks), and 0.4 oz per after-dinner drink.¹⁶ We have validated alcohol intake from reported usual drinks per week on questionnaires with recorded intakes from 4-day food records in a separate sample of 110 men. The correlation was r=.65 (data not shown). In our sample of Mormon men and women, the accuracy of the reported alcohol intake is supported by the positive correlations we have reported between their alcohol intake and HDL cholesterol (men: r=.38, P<.001; women: r=.26, P<.001) and apoA-I (men: r=.29, P<.001; women: r=.22, P<.01).¹⁷

Laboratory Measurements
All participants provided blood samples after an overnight fast. The cholesterol content of LDL was obtained by the application of the Friedewald et al¹⁸ algorithm to enzymatic end-point measurements of cholesterol from enzyme reagent kits (Ciba-Corning Diagnostics Corp) and a Ciba-Corning automated analyzer.^{18 19} Plasma apoB concentrations were determined by enzyme-linked immunosorbent assay using an antigen capture–antigen competition assay.^{20 21} Electrophoresis of LDL in the d<1.063-g/mL fraction was performed on a Pharmacia electrophoresis apparatus (GE 4-II Pharmacia) using slab gradient gels (PAA 2/16, Pharmacia).^{4 6 22} The protein-stained gels were obtained by agitating the gels in a 50- to 75-mL solution of 0.04% Coomassie G-250 and 3.5% perchloric acid after fixing the protein in 10% sulfosalicylic acid for 1 hour. The gels were scanned with a model RFT densitometer (Transidyne Corp) at a wavelength of 565 nm.⁶ A mixture of four globular proteins (HMW calibration kit, Pharmacia) was run on the central lane to calibrate for particle size. Latex beads were added to the high-molecular-weight standard to determine particle diameter. The migration distances (Rf) were measured from the beginning of the gel. A computer file of absorbency versus migration distance was obtained at 1000 points along the gradient gel. Calculus (transformation of variables) was then used to transform the LDL distribution from the migration-distance scale to the particle-diameter scale.²³ The height of the distribution as measured by absorbency at each diameter value was then determined by interpolation for each 0.05-nm value between 21.80 and 32.00 nm. The coefficient of variation for LDL absorbance, measured at the peak, was 17.6% when stained for protein. Lipoprotein(a) was not measured in any of the women studied. The protein-stained ISL could include lipoprotein(a), and some of the observed associations may reflect properties of lipoprotein(a) as well as IDL.

The total area of the protein-stained LDL was set equal to the total plasma concentration of apoB. Standardizing the absorbency of protein-stained LDL for apoB concentrations reduced the variability in LDL absorbance across individuals. This suggests that the reductions in errors associated with measuring absorbance (eg, sample application, staining, destaining, and measurement by the densitometer) were greater than the imprecision introduced by standardization (eg, interindividual differences in the proportion of apoB on particles outside the 22- to 32-nm-diameter range and interindividual differences in non-apoB protein within the 22- to 32-nm-diameter range).

Statistical Analysis
Student's t tests were used to evaluate differences in mean LDL levels between age groups. Pearson correlation coefficients were used to evaluate relationships between LDL levels and age, BMI, and alcohol intake. The correlations and differences were computed at each 0.05 nm between 21.8 and 32.0 nm. We also identified the LDL peak diameter by the maximum absorbency (highest peak) and calculated average LDL diameter by integration between 21.8 and 28.0 nm.

When assessing the effects of age, we excluded persons who drank to ensure that alcohol did not confound the differences between age groups. The correlations between BMI and LDL subclasses also excluded drinkers.

Analyses are presented using more than one person per nuclear family (ie, parents and children) to enhance statistical power. Because LDL levels may be positively correlated within families, the statistical significance may be overestimated when multiple family members are included. We therefore repeated the analyses using only one individual per nuclear family (more distant relations could be included in the same analyses). We also repeated the analyses with the proband excluded. This was done to assess the extent to which the ascertainment of pedigrees through an LDL pattern B proband might influence the results. All significance levels are two-tailed.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Correlations With Age, BMI, and Alcohol Intake
Fig 1 displays the correlations of protein-stained LDL with age, BMI, and alcohol intake by particle diameter. Significant associations (P<.05) in men and women are designated by the solid portions of the bars at the bottom of each graph. The approximate subclass intervals described by Krauss and Burke⁴ are provided for reference. The following results were observed.

View larger version (74K): [in this window] [in a new window]   Figure 1. Pearson correlation coefficients between levels of LDL protein and age (males, n=137; females, n=129), BMI (adult males, n=95; menstruating women, n=78), and amount of alcohol consumed in males (n=133). The solid portions of the bars at the bottom of the graphs designate the range of diameter values that correlate significantly at P.05. The correlations for age and BMI exclude drinkers. Correlations for alcohol intake involved 110 nondrinking adult men and 23 drinkers who consumed an average (±SD) of 3.7±3.1 oz of alcohol per week.

Age
Plasma ISL levels correlated positively with age in both nondrinking males (between 28.9 and 29.9 nm) and nondrinking females (between 27.8 and 30.0 nm). Age also correlated positively with smaller LDL in both sexes, involving LDL-IIIB, LDL-IIIA, and LDL-II in females (between 24.2 and 25.95 nm) and principally LDL-IIIA in males (between 24.4 and 25.45 nm). Plasma LDL-IVB levels were negatively correlated with age in men only (22.3 to 22.9 nm).

Adiposity
In nondrinking adult men, increasing levels of BMI were associated with higher levels of LDL-IIIA (between 24.5 and 25.3 nm).

Alcohol Intake
In men, alcohol intake was positively correlated with LDL-II (between 25.8 and 26.1 nm) and larger LDL-I (between 27.3 and 28.0 nm).

Mean Differences in LDL Absorbency by Age, Sex, and Drinking Status
The associations of age with LDL subclasses are further examined in Figs 2 and 3 by displaying the average distribution of LDL protein by age categories in men and by menstrual status in women. The curves were computed by averaging the heights of the individual LDL distributions at each diameter value. The mean differences between age groups are displayed in the lower panel, along with significance from two-sample t tests (indicated by the solid portions of the bar at the bottom of the graph). In nondrinking males, LDL-IIIA/LDL-II levels were significantly higher in adults 18 years and older than in boys 5 through 11 years old (specifically between 25.25 and 25.65 nm) or adolescents 12 through 17 years old (between 25.10 and 25.80 nm). Men also had higher plasma levels of ISL than adolescents (28.60 to 31.20 nm), higher levels of smaller LDL-I than boys (between 26.25 and 26.55 nm), and slightly lower LDL-IVB than boys (22.26 to 23.35 nm). Nondrinking postmenopausal females had higher LDL-IIIA, LDL-II, and ISL than females who were premenarchal (between 24.55 and 25.8 and between 27.85 and 32.0 nm) or premenopausal (between 24.20 and 25.85 and between 28.4 and 32.0 nm). Premenopausal women had higher ISL than premenarchal girls (between 28.10 and 30.75 nm) and lower LDL-IVB than both postmenopausal women (between 22.65 and 23.10 nm) and premenarchal girls (between 22.60 and 23.25 nm).

View larger version (65K): [in this window] [in a new window]   Figure 2. Mean absorbency of protein-stained LDL by particle size in boys (n=13), adolescents (n=14), and adult males (n=110, upper panel) and mean differences between age groups (lower panel). The solid portions of the bar at the bottom of the figure designate the diameter values that achieve statistical significance between age groups (P<.05) for two-sample t tests. Men who drank alcohol were excluded.

View larger version (61K): [in this window] [in a new window]   Figure 3. Mean absorbency of protein-stained LDL by particle size in premenarchal girls (n=14), menstruating women (n=81), and postmenopausal women (n=34, upper panel) and mean differences between age groups (lower panel). The solid portions of the bar at the bottom of the figure designate the diameter values that achieve statistical significance (P .05) for two-sample t tests. Women who smoked or drank alcohol or took birth control pills or postmenopausal estrogen replacement were excluded.

Fig 4 compares the LDL levels of males with the combined sample of premenarchal and menstruating females. There were only minor differences between boys and these females (the boys had less of the smaller LDL-I and slightly more LDL-IVB), and there was no significant difference between male adolescents and these females within the LDL range (ie, <28 nm). Compared with premenarchal and menstruating females, males had higher levels of ISL (29.3 to 30.3 nm) during both adolescence and adulthood. Adult males also had higher LDL-III and LDL-II than these females. Other analyses (not displayed) reveal that adult males have higher ISL (27.3 to 32.0 nm) and slightly lower LDL-IVB (22.2 to 22.3 nm) than postmenopausal women.

View larger version (72K): [in this window] [in a new window]   Figure 4. Mean differences in absorbency of protein-stained LDL by particle size between females (premenarchal girls and menstruating women combined, n=95) and boys (n=13), adolescents (n=14), and adult males (n=110). A positive difference indicates that the males have a higher LDL value than the females. The solid portions of the bar at the bottom of the figure designate the diameter values that achieve statistical significance (P<.05) for two-sample t tests. Males and females who drank alcohol and females who took birth control pills or postmenopausal estrogen replacement were excluded.

Nineteen men 18 years old reported drinking more than 1 oz of alcohol per week (average intake [±SD], 4.4±2.91 oz per week). Fig 5 shows that they had higher plasma levels of larger LDL-I (between 27.1 and 28.3 nm) than the 110 men who reported no alcohol consumption.

View larger version (75K): [in this window] [in a new window]   Figure 5. Mean absorbency of protein-stained LDL by particle size in male drinkers (>1 oz/wk, n=19) and nondrinkers (n=110, top panel) and mean differences between age groups (bottom panel). The solid portions of the bar at the bottom of the figure designate the diameter values that achieve statistical significance (P<.05) for two-sample t tests.

Statistical Adjustment by Partial Correlation and ANCOVA
Fig 6 (right column) displays the significance levels for the correlations of protein-stained LDL levels with age, BMI, and alcohol intake, adjusted for plasma concentrations of LDL cholesterol, triglycerides, and HDL-cholesterol. Adjustment for plasma triglycerides eliminated the positive correlation of LDL-III with age in both men and women but not the positive correlation of LDL-III with BMI. The correlation between alcohol intake and LDL-I was eliminated by adjustment for HDL cholesterol but was unaffected by the statistical adjustment for plasma triglyceride levels. Fig 6 (left column) shows that these adjustments had less effect on the mean LDL differences between men and premenopausal women, adult and younger males, postmenopausal and premenopausal females, and drinking and nondrinking subjects. The figure also shows that excluding first-degree relatives had little effect on the correlations and mean differences. Excluding the probands also had negligible influence on the domain of significant correlations and significant mean differences. Thus, the relations observed do not appear to be artifacts of the method of sampling.

View larger version (65K): [in this window] [in a new window]   Figure 6. Domain of significant mean differences and significant correlations when adjusted for plasma LDL cholesterol, triglyceride, and HDL cholesterol concentrations by ANCOVA and partial correlation analyses. The wide solid and dashed portions of the bars designate the domain of diameter values that are significantly different or correlated at P<.05 when adjusted. The significance levels for the unadjusted mean differences and correlations are also presented when the analyses are restricted to one person per nuclear family.

Correlational Analyses of LDL Peak Diameter and Average LDL Diameter
Table 2 shows that LDL peak diameter was negatively correlated with age in females (r=-.29, P<.01 for all ages combined) and with BMI in adult nondrinking males (r=-.27, P<.01). Partial correlation coefficients (not displayed) were computed to assess whether the relationships could be attributed to specific subclass intervals. The correlation with age in females is eliminated when adjusted for every individual diameter value between 24.5 and 25.6 nm, and the correlation with BMI in males is eliminated when adjusted for every individual diameter value between 24.45 and 25.25 nm (eg, the correlation between age and LDL peak diameter became nonsignificant when adjusted for LDL absorbance at 24.5 nm, 24.55 nm, 24.60 nm, and continuing up to 25.60 nm). Thus, the positive correlations between LDL peak diameter and age in women and adiposity in men appear to be due to higher levels of LDL-IIIA in postmenopausal women and more obese men.

View this table: [in this window] [in a new window]   Table 2. Correlations of Average LDL Diameter and LDL Peak Diameter With Age, BMI, and Alcohol Intake

Mean Differences in LDL Peak Diameter and LDL Average Diameter
Table 3 compares the LDL average diameter and LDL peak diameter by age groups. Adult men (18 years old) had significantly smaller LDL peak diameter than boys, male adolescents, and premenopausal women. Postmenopausal women had a significantly smaller LDL peak diameter than premenarchal girls and premenopausal women. ANCOVA was used to assess whether these differences could be attributed to specific subclass intervals, ie, whether they remained significant when adjusted for LDL levels at each diameter value. Adjustment for individual diameters within LDL-IIIA eliminated the significant difference in LDL peak diameter between all age groups (analysis not displayed).

View this table: [in this window] [in a new window]   Table 3. Average LDL Diameter and LDL Peak Diameter in Men and Women by Age Group

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The LDL peak diameter and LDL average diameter provide perspectives on CHD risk and genetic inheritance that are independent of LDL cholesterol. The peak diameter has been used to identify a genetically influenced LDL subclass pattern (pattern B) characterized by a predominance of small LDL particles and associated with increased CHD risk.^{7 8} Family studies have indicated that this pattern is influenced by one or more major genes exhibiting autosomal dominant inheritance.¹⁴

We believe measurements of absorbency may have several advantages over measurements of peak or average diameter of LDL: (1) Absorbency provides a quantitative measurement of LDL levels by size.²³ LDL peak diameter and LDL average diameter are descriptive characteristics of the LDL particle distribution. Although the conversion from absorbency to plasma concentration is not known, the statistical test used in this report will be identical to those based on unknown plasma concentrations if the conversion is linear.^{24 25} (2) Absorbency describes the individual subclasses in absolute, as opposed to relative, amounts. The LDL peak diameter and average LDL diameter provide information on the properties of the individual subclasses relative to other subclasses, ie, a shift toward smaller LDL does not necessarily imply that smaller LDL levels have increased. (3) Absorbency may reveal properties of the individual subclasses that do not necessarily affect the average or mode of the total distribution. For example alcohol intake was associated with higher levels of LDL-I (Figs 1 and 5) without affecting the peak or average diameter (Table 2).

Alcohol Intake
Consistent with the observation by Campos et al,¹¹ we found that reported alcohol intake was unrelated to LDL peak diameter (Table 2). However, plasma levels of LDL-I were significantly higher in drinkers than nondrinkers (Fig 5), and LDL-I levels correlated positively with alcohol intake (Fig 1). We have reported elsewhere that these drinkers also had higher plasma levels of HDL_2a and less pronounced increases in HDL_2b than nondrinkers.¹⁷ Increases in more buoyant LDL mass²⁶ and LDL particle size²⁷ have been reported in alcohol-fed monkeys. Other investigators report larger LDL in alcoholic cirrhosis,²⁸ heterogeneous LDL in alcoholic hepatitis,²⁹ and polydisperse LDL in chronic alcoholism.³⁰

The increases in LDL-I and HDL_2a could both arise from the uptake of redundant surface material during accelerated turnover of VLDL. Sane et al³¹ found that chronic alcohol users have high fractional catabolic rates and high total synthesis (turnover) of VLDL triglycerides. Others have shown that the incorporation of free fatty acids into plasma VLDL triglycerides is increased after ethanol consumption.^{32 33} Chronic alcohol intake appears to increase lipoprotein lipase activity, which probably accounts for the increased fractional catabolic rate of VLDL triglycerides.^{34 35} Levels of both larger LDL^{36 37} and HDL₂³⁸ are associated with postheparin plasma lipoprotein lipase activity. It is also possible that higher LDL-I is the product of increased lipoprotein lipase–mediated catabolism of triglyceride-rich precursors, since, as seen in monkeys, conversion of VLDL to LDL is completely inhibited when lipoprotein lipase activity is blocked.³⁹ Reduced hepatic lipase activity may also lead to the accumulation of large LDL^{40 41} and HDL,⁴² but whether this phenomenon contributes to higher LDL-I and HDL concentrations in drinkers remains to be determined. The enzyme's activity is reported to be lower⁴³ or unchanged⁴⁴ after acute alcohol intake, unchanged by short-term moderate alcohol intake,^{45 46} and higher in chronic heavy drinkers.^{34 47}

Male Sexual Maturation and Female Menopause
ANCOVA and the partial correlation analyses suggest that increases in LDL-IIIA appear to explain the decreases in LDL particle size associated with male sexual maturation and female menopause. In women, the increase in small LDL during aging or menopause also encompassed LDL-II and LDL-IIIB increases (Figs 1 and 3). The higher ISL in women after menopause could reflect in part increases in lipoprotein(a).⁴⁸ Some caution is warranted in extrapolating these results to other populations, since the proportion of pattern B among postmenopausal women is reported to be less in other samples.^{11 49} In our sample, the proportion of pattern B individuals among postmenopausal women (44%) remained high (47%) when those who were first-degree relatives of the pattern B probands were excluded. Elsewhere we report that the HDL_3b subclass is also increased in association with male sexual maturation and female menopause and that HDL_2b is lower after male puberty.¹⁷ These concomitant changes in the HDL and LDL subclasses correspond to their apparent coordinate relationships as observed cross-sectionally in adult populations.²⁵

Fig 6 shows that the correlation between age and LDL subclasses was eliminated when adjusted for plasma triglyceride levels. Cross-sectional studies show a positive correlation between the preponderance of small LDL and elevated triglyceride-rich lipoproteins in fasting and postprandial plasma.⁵⁰ Excessive levels of triglyceride-rich lipoproteins may promote small LDL formation by (1) facilitating the exchange of LDL cholesterol for triglycerides, which can then be hydrolyzed by lipases to form smaller LDL particles,⁵¹ and (2) promoting the synthesis of triglyceride-enriched IDL precursors of small LDL.⁵² Fasting plasma triglyceride levels are closely related to the degree of postprandial lipemia.⁵³ Postprandial lipemia may promote small LDL and restrict large LDL formation by enhancing cholesteryl ester–triglyceride exchange.⁵⁴

Adiposity
Partial correlation analyses suggest that increases in LDL-IIIA also appeared to explain the decrease in the LDL peak diameter associated with male adiposity. Fig 1 shows that LDL-IIIA and larger LDL-IIIB were positively correlated with BMI. Elsewhere we have shown that adiposity was also associated with higher HDL_3b and lower HDL_2b in these men.¹⁷ These observations are in accordance with the changes in HDL and LDL subfractions observed during weight loss.^{55 56} Adiposity is associated with increased synthesis of VLDL triglycerides, VLDL apoB, and LDL apoB, increased IDL and LDL apoB catabolism, and increased VLDL clearance before conversion to LDL.^{57 58 59 60} The excess of triglyceride-rich lipoproteins in fasting and postprandial plasma⁶¹ and elevated plasma free fatty acid concentrations^{62 63} in overweight individuals may enhance their cholesteryl ester–triglyceride exchange,⁶⁴ thereby preventing the accumulation of HDL_2b cholesterol and increasing LDL-III. Adiposity is also associated with higher hepatic lipase activity,⁴² which may enhance IDL and LDL lipid hydrolysis^{41 65} and the formation of small LDL.³⁷

In conclusion, we have shown that LDL subclasses exhibit specific relationships to age, adiposity, and alcohol intake when densitometric measurements of absorbency on protein-stained gradient gels are analyzed in a population sample. The measurement error associated with protein-stained LDL (see "Methods") does not bias the estimated mean LDL levels within age, sex, or drinking categories but attenuates the correlation of LDL with other variables. The added error increases the probability of a type II statistical error (ie, false negative result) rather than a type I error (ie, false positive result) and is therefore conservative. Nevertheless, our analyses suggest that plasma levels of LDL-I are higher in male drinkers than nondrinkers. Plasma levels of LDL-III are higher in adult men than younger males, higher in overweight than leaner men, and higher in postmenopausal than premenopausal women. Reductions in the LDL peak diameter associated with increasing age, male sexual maturation, menopause, and adiposity can be related to increases in the LDL-IIIA subclass. These analyses attribute the effects of alcohol, age, and adiposity to specific LDL subfractions, thereby providing a more detailed assessment than the analysis of the LDL peak diameter or LDL average diameter.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein BMI = body mass index CHD = coronary heart disease IDL = intermediate density lipoprotein ISL = intermediate-size lipoprotein

	Acknowledgments

 
This study was supported in part by grants HL-49857, HL-45652, HL-24462, and HL-18574 from the National Heart, Lung, and Blood Institute and was conducted at the Lawrence Berkeley Laboratory (Department of Energy DE-AC03-76SF00098 to the University of California). We wish to thank Melissa A. Austin for her role in collecting the data and Laura Holl, Charlotte Brown, and Bahareh Sahami for laboratory analysis of gradient gel electrophoresis.

	Footnotes

 
Reprint requests to Paul T. Williams, PhD, Life Sciences Division, Lawrence Berkeley National Laboratory, Bldg 934, Berkeley, CA 94720.

Received March 31, 1996; accepted October 21, 1996.

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