Apolipoprotein A-I and A-II Kinetic Parameters as Assessed by Endogenous Labeling With [2H3]Leucine in Middle-Aged and Elderly Men and Women
Abstract—The purpose of our study was to investigate high density lipoprotein (HDL) apolipoprotein (apo) A-I and apoA-II kinetics in a state of constant feeding after a primed-constant infusion of [5,5,5-2H3]l-leucine in 32 normolipidemic older men and postmenopausal women (aged 41 to 79 years). ApoA-I and apoA-II were isolated from plasma HDL, and enrichment was determined by gas chromatography/mass spectrometry. The fractional secretion rate was obtained by using a monoexponential equation calculated with the SAAM II program (Department of Bioengineering, University of Washington, Seattle). Mean HDL cholesterol (HDL-C) and total triglyceride levels were 23% higher and 27% lower, respectively, in women than in men. Mean plasma apoA-I levels were 10% greater in women than in men, whereas mean apoA-II levels were similar. HDL size, estimated by gradient-sizing gels and by the HDL-C/apoA-I+apoA-II ratio, was significantly higher in women than in men. Mean apoA-I secretion rates (SRs) were similar in men and women (12.28±3.64 versus 11.96±2.92 mg/kg per day), whereas there was a trend toward a lower (−13%) apoA-I fractional catabolic rate (FCR) in women compared with men (0.199±0.037 versus 0.225±0.062 pools per day, P=0.11). Mean apoA-II SRs (2.21±0.57 versus 2.27±0.91 mg/kg per day) and FCRs (0.179±0.034 versus 0.181±0.068 pools per day) were similar in men and women. For the group as a whole, there was an inverse association between the HDL-C/apoA-I+apoA-II ratio and apoA-I FCR and between the ratio and triglyceride levels. Plasma levels of apoA-I and apoA-II were correlated with their respective SRs but not FCRs. These data suggest a major role for apoA-I and apoA-II SRs in regulating the plasma levels of these proteins, whereas apoA-I FCR might be an important factor contributing to the differences in apoA-I levels between men and postmenopausal women. Moreover, plasma triglyceride levels are important determinants of HDL size and apoA-I catabolism.
- Received October 12, 1998.
- Accepted September 17, 1999.
High density lipoprotein cholesterol (HDL-C) levels are inversely correlated with the incidence of coronary heart disease (CHD) in population studies.1 2 3 Moreover, intervention trials aimed at lowering triglycerides and LDL cholesterol (LDL-C) levels suggest that increasing HDL-C levels with gemfibrozil may be beneficial in reducing CHD morbidity.4 Over the last 2 decades, researchers have examined the kinetics of HDL apoA-I and apoA-II to understand how plasma HDL levels are regulated and to determine whether apolipoprotein secretion and/or catabolism influences the levels of HDL in plasma.
The metabolism of apoA-I and apoA-II has been studied mostly by exogenous labeling of either HDL or apolipoproteins directly. Shepherd et al5 compared HDL apolipoprotein turnover in 10 normolipidemic young men and women and observed no significant differences in the fractional catabolic rate (FCR) or secretion rate (SR) of apoA-I and apoA-II between the 2 groups. In addition, Fidge et al6 reported that the SR was an important determinant of both apoA-I and apoA-II pool size in normolipidemic and hyperlipidemic subjects. Schaefer et al7 noted that premenopausal women had higher apoA-I and apoA-II SRs than did men. The major determinant of plasma apoA-I concentration for the whole group was the apoA-I FCR and not the SR. Furthermore, Gylling et al8 showed that in men apoA-I levels were correlated with SR but not FCR. The authors also noted that low HDL-C in obese subjects and smokers was associated with enhanced apoA-I FCR. In a series of studies, Brinton and colleagues9 10 have demonstrated the importance of HDL apoA-I and apoA-II FCR in regulating HDL-C levels. The authors reported that in women HDL-C levels are inversely correlated with the FCR but not with the SR of both apoA-I and apoA-II. Furthermore, low HDL-C levels were associated with high FCR of apoA-I and apoA-II in men and women regardless of plasma triglyceride levels.
In the present study, we have reexamined these issues by using a stable isotope tracer to measure the endogenous incorporation of the tracer into apoA-I and apoA-II. We hypothesized that postmenopausal women have higher plasma levels of apoA-I that are due to a higher apoA-I secretion. We studied middle-aged and older men and postmenopausal women not taking exogenous estrogen. Older people are the most rapidly growing segment of society, and the group commonly targeted for heart disease risk reduction.
Thirty-two healthy volunteers (18 men and 14 women) underwent a complete physical examination and medical history before admission into the study. None of the subjects had any evidence of hepatic, cardiac, renal, or endocrine dysfunction or a family history of these illnesses. The subjects did not smoke or consume alcohol regularly and were not taking medications known to alter plasma lipid levels. All subjects were >40 years of age, and all women were postmenopausal. None of the women participants was on estrogen replacement therapy. The experimental protocol was approved by the Human Investigation Review Committee of the New England Medical Center and Tufts University. All subjects gave informed consent to participate in the study.
The subjects were placed on a baseline average US diet for 6 weeks. The diet consisted of 49% carbohydrate, 15% protein, 35% fat (14% saturated, 14% monounsaturated, and 7% polyunsaturated), and 147 mg of cholesterol/1000 kcal. All food and drink during the study periods were prepared and provided to the subjects by the Metabolic Research Unit of the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University. Energy intake was adjusted to keep body weight constant (±1 kg) throughout the 6-week period.
At the end of the experimental diet period, a primed-constant infusion of [5,5,5-2H3]l-leucine was carried out for 15 hours. After fasting for 12 hours, subjects were fed hourly for 20 hours starting at 6:00 am, and each meal consisted of 1/20th their daily caloric intake specific for the dietary period. Five hours after their first meal, subjects received an intravenous bolus dose (10 μmol/kg) followed by a constant infusion of [5,5,5-2H3]l-leucine at 10 μmol/kg per hour. After 15 hours, the primed-constant infusion and hourly feeding were terminated. Blood samples (20 mL), via a second intravenous line, were collected at hours 0, 1, 2, 3, 4, 6, 8, 10, 12, and 15.
Plasma Lipid and Lipoprotein Determinations
Blood samples were collected in tubes containing 0.15% EDTA and centrifuged at 2500 rpm for 20 minutes at 4°C to separate plasma. HDL-C was measured in plasma after precipitation of apoB-containing lipoproteins with dextran sulfate–MgCl2.11 HDL3-C was also measured from plasma by a modification of the dextran sulfate–MgCl2 method.12 HDL2-C concentration represents the difference between HDL-C and HDL3-C. Triglyceride-rich lipoproteins (d<l.006 g/mL) and HDL (1.063 to 1.21 g/mL) fractions were obtained by sequential ultracentrifugation (Beckman Instruments Inc) as previously described.13 Plasma lipids and the 1.006 g/mL supernatant-fraction cholesterol were determined by enzymatic methods with the use of an automated analyzer (Abbott Diagnostics Spectrum CCX analyzer) and Abbott enzymatic reagents.14 LDL-C was calculated as follows: total cholesterol−(1.006 g/mL supernatant-fraction cholesterol+HDL-C). All lipid and protein assays were performed in duplicate, and the coefficients of variation within and between runs were 2% to 5%.
HDL particle size was estimated according to the method of Li and colleagues15 16 and by calculating the HDL-C/apoA-I+apoA-II ratio.17 In the first method, plasma samples were loaded onto nondenaturing polyacrylamide gradient gels (4% to 30%) obtained from Pharmacia, electrophoresed, and stained with Sudan black B stain (Fisher). The migration distance of each band was compared with the migration of albumin by using an LKB Ultrascan XL laser densitometer (LKB Instruments Inc). A standard curve was obtained by using proteins of known sizes, and the HDL particle size was calculated as the mean diameter of each band multiplied by the fraction of the total area for that particular band, expressed in nanometers. In the second method, an estimate of the HDL core-to-surface ratio was defined as the molar ratio of HDL-C divided by apoA-I plus apoA-II.
Quantification and Isolation of Apolipoproteins
Plasma apoA-I concentration was measured by an immunoturbidimetric assay18 with the use of a Spectrum CCx analyzer (Abbott Diagnostics), with reagents and calibrators from Incstar Corp. Plasma apoA-II levels were assessed by an electroimmunodiffusion technique that used commercially available agarose gels with polyclonal anti–apoA-II incorporated into the gels (Laboratoires Sebia).19 Briefly, an excess of anti–apoA-II antibody is used to retain apoA-II–containing particles, whereas apoA-I particles without apoA-II continue to migrate until they react with anti–A-I antibody. A calibration curve was constructed with standards to determine the concentration of apoA-II. The coefficients of variation between and within runs were 10% and 7%, respectively.
ApoA-I and apoA-II were isolated from the HDL fraction by SDS-PAGE with a 6% to 30% acrylamide linear gradient.20 The amount of protein loaded was ≈100 μg per sample. Protein bands were identified by comparing their migration bands with those of known molecular weight proteins (low molecular weight proteins, Sigma Chemical Co).
Isotopic Enrichment Determinations
Polyacrylamide gel bands for apoA-I and apoA-II were cut and hydrolyzed with 12N HCl at 110°C for 24 hours.20 The amino acids were derivatized by adding propanol/acetyl chloride and heptafluorobutyric anhydride reagents.21 The derivatized amino acids were extracted into ethyl acetate for analysis by using a 5890/5988 gas chromatograph/mass spectrometer (Hewlett-Packard Co). To identify labeled and unlabeled leucine, the amino acids were ionized by methane-negative chemical ionization. Selected ion monitoring at mass-to-charge ratios of 349, 350, and 352 was used to determined the isotopic ratio (labeled/unlabeled) of each sample. Enrichment was calculated from the isotopic ratio and then converted to tracer/tracee ratios22 according to the following formula: e(t)/e(I)−e(t), where e(t) is the enrichment at time t, and e(I) represents the isotopic abundance of the infusate, which for this study was 99.8%.
Fractional SRs (FSRs) of HDL apoA-I and apoA-II were determined by fitting the tracer/tracee ratios to a monoexponential function using the SAAM II program (Department of Bioengineering, University of Washington, Seattle). The data were fit to the function A(t)=Ap(l−e−k(t−d)), where A(t) is the tracer/tracee ratio at time t, Ap is the tracer/tracee ratio of the plateau of the curve representing the precursor pool, d is the delay time, and k is the FSR. Under steady-state conditions, the FSR equals the FCR. The delay was fixed at 30 minutes, representing the time of labeled leucine incorporation into the protein of interest.23 VLDL apoB-100 plateau enrichment, also determined by using a monoexponential function, was used to estimate the apoA-I and apoA-II precursor pool.20 In the present study, the VLDL apoB-100 plateau represents ≈75% of the plasma leucine enrichment plateau.
To analyze the kinetic data, we assumed that a primed-constant infusion provides a constant enrichment of plasma leucine, which is the immediate precursor of the leucine incorporated into apoA-I and apoA-II. Previous studies from our laboratory have reported that under these conditions, plasma leucine reaches a plateau within 1 hour and remains constant throughout the infusion.24 We also assumed that each subject was studied under steady-state conditions with regard to their lipid and apolipoprotein concentrations. The caloric content and composition of each hourly meal remained constant and was designed to achieve a steady state.25
The SRs of apoA-I and apoA-II were calculated as follows: FSR (pools per day)×apolipoprotein pool size (mg)/body weight (kg); the results are expressed as milligram per kilogram per day. Pool sizes were defined as follows: apolipoprotein plasma concentration (mg/dL)×plasma volume (0.045 1/kg body wt). The residence time represents the inverse of the FCR.
The SYSTAT 7.1 software program (SPSS, Inc) was used for all statistical analyses. The nonparametric Mann-Whitney U test statistic was used to test for the statistical significance (P<0.05) of differences between mean values for men and women and for the entire group. Pearson correlation coefficients were determined for the entire group. In addition, partial correlation coefficients were obtained after controlling for age and sex. Triglyceride levels were log-transformed prior to analysis to achieve normal distribution.
Characteristics of the study subjects are shown in Table 1⇓. On average, the women in the study were somewhat older than the men. Mean body mass index was similar for both groups. All plasma lipid, lipoprotein, and apolipoprotein concentrations were measured during the infusion period under constant-feeding conditions. There were no significant differences with regard to the mean plasma total cholesterol, VLDL-C, and LDL-C levels between men and women. Mean plasma triglyceride levels were significantly lower in women than in men (−27%, P=0.025). As expected, women had significantly higher mean HDL-C (23%, P=0.004), HDL2-C (50%, P=0.004), and HDL3-C (13%, P=0.024) levels than did men.
The women had significantly higher apoA-I mean levels (+10%, P=0.024) than the men, but apoA-II levels were similar between both groups. With regard to HDL particle size, the estimates obtained from the gradient sizing gels showed that women had a mean HDL particle size 5% larger than in men (P=0.025). However, the HDL-C/apoA-I+apoA-II ratio was 14% larger in women than in men (P=0.028).
ApoA-I and ApoA-II Kinetic Parameters
The Figure⇓ shows the mean apoA-I and apoA-II tracer/tracee ratios in women (top) and men (bottom). In women, the slope of the apoA-I and apoA-II curves was 0.060 and 0.058, with 95% CIs of 0.058 to 0.062 and 0.056 to 0.059, respectively. In men, the apoA-I and apoA-II slopes were 0.068 and 0.051 with 95% CIs of 0.066 to 0.070 and 0.047 to 0.054, respectively. Women had a lower apoA-I slope and a higher apoA-II slope than did men. In men, the apoA-II enrichment curve had a noticeable deviation at 4 hours. This was probably due to the greater variability observed at this particular time point.
Mean apoA-I pool size was similar between men and women (Table 2⇓). However, after adjusting the pool size by body weight, women had a significantly higher mean normalized apoA-I pool size. Although not statistically significant, women had a 13% lower mean apoA-I FCR than did men. This finding may account for the higher apoA-I levels observed in women in the present study, because the mean apoA-I SRs were similar in both groups.
No sex effect was observed with regard to apoA-II kinetic parameters, reflecting the lack of significant difference in apoA-II concentrations between women and men. Both groups had a similar apoA-II FCR and SR (Table 3⇓). The mean apoA-I FCR was significantly higher than the mean apoA-II FCR in women (0.199 versus 0.181 pools per day, P=0.007) and men (0.225 versus 0.179 pools per day, P=0.007), as well as in the entire group (0.213 versus 0.180 pools per day, P=0.002).
Data for men and women were pooled to evaluate the linear relation between variables (Table 4⇓). There was a significant correlation between apoA-I levels and apoA-I SR (r=0.400, P=0.023), but not apoA-I FCR. ApoA-II levels were significantly correlated with apoA-II SR (r=0.471, P=0.007), but not apoA-II FCR. Although not statistically significant, there was a tendency for HDL-C, in contrast to apoA-I, to be inversely correlated with apoA-I FCR (r=−0.307, P= 0.087). HDL-C levels were significantly correlated with apoA-II SR (r=0.386, P=0.029). The HDL-C/apoA-I+apoA-II ratio was inversely correlated with apoA-I FCR (r=−0.383, P=0.03). After adjustment for age and sex (data not shown), apoA-I levels remained correlated with apoA-I SR. ApoA-II levels also remained associated with apoA-II SR, and HDL-C remained associated with apoA-II SR.
Both estimates of HDL particle size (HDL-C/apoA-I+apoA-II ratio and HDL particle size by gradient gels) were inversely correlated with plasma triglyceride levels (r= −0.535, P=0.002 and r=−0.430, P=0.018, respectively). However, after adjusting for age and sex, only the HDL-C/apoA-I+apoA-II ratio remained correlated with triglyceride levels. Plasma triglyceride levels accounted for 36% of the variability in the HDL-C/apoA-I+apoA-II ratio after controlling for age and sex.
The purpose of the present study was to examine the kinetic behavior of plasma HDL apoA-I and apoA-II in a population of older men and postmenopausal women between the ages of 41 to 79 years with mean levels of HDL-C and apoA-I within the normal range.18 26 We used endogenous labeling, which circumvents the potential problem of altering the metabolic properties of apolipoproteins by isolation and radioiodination.27 The mean apoA-I FCR was significantly greater than the mean apoA-II FCR, which agrees with the results obtained with radioiodination studies.5 7
Although women had 23% higher mean HDL-C levels than did men, their mean apoA-I levels were only 10% higher. The mean HDL particle size, estimated by using gradient sizing gels, was 5% higher in women than in men. However, for HDL particle size on the basis of HDL composition (HDL-C/apoA-I+apoA-II ratio), women had 14% higher mean HDL size than did men. The discrepancy between the results obtained with the above-mentioned methods to estimate HDL size is likely due to differences in the characteristics of the particle assessed by the 2 measures.
We found that, on average, women had 13% lower apoA-I FCRs than did men. This difference, although not significant, might account for the 10% higher apoA-I levels observed in women compared with men. The coefficient of variation of the apoA-I measurement was 11% and 9%, whereas for apoA-I FCR, it was 19% and 27% in women and men, respectively. These data suggest that a greater number of subjects would be required to reach statistical significance because of the greater variation in the FCR measurement. In contrast, mean apoA-II FCR was similar in men and women.
In the present study, postmenopausal women had apoA-I SRs that were similar to those in older men. It has previously been reported that apoA-I SR is higher in premenopausal women than in men7 and that exogenous estrogen increases apoA-I SR.28 29 30 However, Brinton et al17 showed no apoA-I SR difference in women versus men. The authors reported a trend toward a lower apoA-I FCR and a significantly lower apoA-II FCR in women than in men. The study of Brinton et al17 was carried out in subjects with a very wide range of HDL-C, apoA-I, and triglyceride levels. In the above-mentioned study, there were 16 women (14 of whom were in the premenopausal age group) and 12 men. In these subjects, the mean apoA-I SRs were 12.14 mg/kg per day in women and 10.60 mg/kg per day in men (P=0.03), whereas the mean apoA-I FCR values were very similar in the 2 groups (0.233 pools per day in women and 0.246 pools per day in men, P=0.65). These analyses indicate that premenopausal women had a higher apoA-I SR than did men. The fact that in the present study women were postmenopausal and had lost both estrogen and progesterone may account for the difference observed.
It should also be noted that the women in the present study had mean triglyceride levels that were 27% lower than those in men (as assessed in the fed state). These high apoA-I levels in women could best be accounted for by decreased apoA-I FCR. Schaefer and Ordovas31 showed a markedly enhanced apoA-I FCR in 6 subjects with severe hypertriglyceridemia (type I and type V hyperlipoproteinemia) (Table 5⇓). Le and Ginsberg32 reported that subjects with low HDL-C and high triglyceride levels had significantly enhanced HDL apoA-I FCRs, whereas those with low HDL-C levels only had decreased apoA-I SRs. Moreover, in a study by Brinton et al,17 fasting triglyceride levels were inversely correlated with HDL size, which was estimated by the HDL-C/apoA-I+apoA-II ratio. The authors observed that HDL size was the best predictor of apoA-I FCR. In the present study, triglyceride levels were also inversely correlated with both estimates of HDL particle size. It has been proposed that elevated plasma triglyceride levels cause an increased exchange of HDL cholesteryl ester with triglyceride. As a result, triglyceride-enriched HDL particles become more susceptible to lipolysis, followed by an enhanced catabolism of apoA-I.33
For the entire group, we observed a significant correlation between apoA-I concentration and apoA-I SR but not apoA-I FCR. An association of apoA-I levels or pool size with apoA-I SR was reported in the studies of Fidge et al6 and Gylling et al.8 Other investigators have reported that apoA-I levels are inversely associated with apoA-I FCR.7 9 With regard to HDL-C, there was a trend toward an inverse correlation between HDL-C levels and apoA-I FCR. In addition, we observed an inverse association between apoA-I FCR and plasma triglyceride levels. However, this association was lost after adjusting for age and sex. Nevertheless, plasma triglyceride levels seem to play an important role in apoA-I catabolism, and this deserves future attention, perhaps in a larger group of normolipidemic men and women. For a summary of data to date involving apoA-I and apoA-II kinetic parameters, please refer to Table 5⇑. The overall data indicate that both SR and FCR can play an important role in affecting plasma apoA-I and apoA-II pool sizes.
In summary, sex differences in apoA-I levels in older individuals appear to relate to FCR and not SR, with postmenopausal women having delayed catabolism compared with men. Our data suggest that the lower mean triglyceride levels in women may have contributed to their larger HDL particles and lower apoA-I catabolism.
This study was supported by a grant from the National Institutes of Health (HL-39236) and a contract from the US Department of Agriculture Research Service (No. 53-3k06-5-10). The authors would like to thank Dr Gerard Dallal for assistance with the statistical analyses, Richard Lirio for his technical assistance, Dr Julian Marsh for his helpful comments and criticisms, the staff of the Metabolic Research Unit for the care and service provided to the subjects, and the volunteers for participating in the study.
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