707 Human HDL Cholesterol Levels Are Determined by ApoA-I Fractional Catabolic Rate, Which Correlates Inversely With Estimates of HDL Particle Size Effects of Gender, Hepatic and Lipoprotein Lipases, Triglyceride and Insulin Levels, and Body Fat Distribution Eliot A. Brinton, Shlomo Eisenberg, Jan L. Breslow D ow Abstract High-density lipoprotein (HDL) cholesterol stepwise multiple linear regression analysis, apoA-I FCR n (HDL-C) levels are a strong inverse predictor of atheroscle- alone accounted for 66% of the variability in HDL-C; two lo a rosis risk, but the physiological determinants of HDL-C levels other variables accounted for an additional 7%. Due to the d ed are poorly understood. We selected 57 human subjects (30 importance of apoA-I FCR, its determinants were sought fro women and 27 men) with a broad range of HDL-C levels and among the remaining variables. Two estimates of HDL size or m performed turnover studies of apolipoprotein (apo)A-I and density, the HDL-C/apoA-I+apoA-II ratio and the percent of http apoA-II, the two major apolipoproteins of HDL, to measure the apoA-I tracer found in the d>\2\ g/mL fraction, corre- ://atv pthoer t frraacttei o(nTalR c) aotaf btohleisce raptreo t(eFinCsR. )W aen dal spor omduecatsiuorne do rs etrvaenras-l (lart=ed-. 8s1tr onagnldy .w6i2th, reasppoeAct-iIv eFlyC; RP 2). The FCR of apoA-I insulin together predicted 46% of the variability of triglyceride y g tended to be lower in the women (0.248±0.077 versus levels and waist-to-hip ratio alone predicted 26% of the ue 0.277±0.069 pools/d, P=.l), although the difference was not variability in the ratio of lipoprotein lipase to hepatic lipase. s t o statistically significant. The FCR of apoA-II was also lower Based on these correlations, we hypothesize that abdominal n D (0.184±0.043 versus 0.216±0.056 pools/d, />=.O2). In contrast, fat, insulin sensitivity, lipase activity, and plasma triglyceride ec the apoA-I TR was equal in women and men (12.0±1.6 versus levels may regulate HDL size, which appears to be the primary em 12.1 ±2.8 mg/kg per day, P>2), and there was a trend toward determinant of apoA-I FCR, which may, in turn, be the major be lower apoA-II TR in women (2.19±.62 versus 2.61±1.06 determinant of HDL-C levels. These factors appear to play r 2 mg/kg per day, P=.O7). Linear regression analysis revealed a crucial roles in the regulation of HDL-C levels and may be 2 , 2 strong inverse correlation between HDL-C levels and the important in determining susceptibility to atherosclerosis. 01 FCRs of apoA-I and apoA-II (r=-.81 and -.76, respectively; (Arterioscler Thromb. 1994;14:707-720.) 7 /'<.0001 for both). In contrast, there was little or no association between HDL-C and the TRs of apoA-I and apoA-II Key Words • HDL • apoA-I metabolism • apoA-II (r=.O6 and —.35, /*=not significant and .01, respectively). In metabolism • triglycerides • lipoprotein lipase • hepatic lipase • insulin • body fat distribution H igh-density lipoprotein (HDL) cholesterol els.8 This inconsistency, as well as the small number of (HDL-C) levels are well established as an in- subjects and the small range of HDL-C levels in most of verse risk factor for atherosclerosis,12 but the these studies, has caused uncertainty in the interpretation regulation of HDL-C levels is only beginning to be under- of these data. Studies of subjects with high9 and low10 stood. Earlier HDL turnover studies suggested that the HDL-C levels and employing both high and low fat fractional catabolic rate (FCR) of apolipoprotein intake11 have established that apoA-I FCR is the primary (apo)A-I is elevated in subjects with low HDL-C34 and metabolic predictor of intersubject variability in HDL-C that apoA-I FCR correlates with HDL-C,56 but other levels and have suggested that HDL size or density, as investigators reported that apoA-I transport rate (TR) possibly influenced by fasting triglyceride (TG) levels and may better explain variability in HDL-C7 or apoA-I lev- endothelial lipase activity, may be a key determinant of Received June 19, 1992; revision accepted February 8, 1994. apoA-I FCR and hence of HDL-C levels. Nevertheless, From the Laboratory of Biochemical Genetics and Metabolism, knowledge of these relations remains preliminary. The Rockefeller University, New York, NY, and Hadassah Uni- Despite the evident importance of the gender differ- versity Hospital (S.E.), Jerusalem, Israel. ences in HDL-C and apoA-I levels, the metabolic basis Correspondence to Eliot A. Brinton, MD, Section on Endocri- of this difference has been incompletely explored. One nology and Metabolism, Bowman Gray School of Medicine, study reported a significantly higher apoA-I TR in Medical Center Boulevard, Winston-Salem, NC 27157-1047. women with no difference in apoA-I FCR,6 and another 708 Arteriosclerosis and Thrombosis Vol 14, No 5 May 1994 smaller study found nonsignificant trends toward lower eters. One subject (No. 37) had a history of hypertension and TR and FCR of apoA-I in women.12 Many studies have had been treated with metoprolol, a /3-blocker, and verapamil. shown an inverse correlation between abdominal adi- The /3-blocker was discontinued 2 weeks before the turnover study, and the subject's blood pressure remained normal posity (most commonly measured by the waist-to-hip throughout the study period. None of the other subjects took ratio) and HDL-C levels (see Reference 13 for review), medications known to alter glucose or lipid levels. but the relationship between body fat distribution and The clinical aspects of these studies were approved by the HDL metabolism, as measured by apolipoprotein turn- Institutional Review Board of The Rockefeller University, and over, is just beginning to be explored. The FCRs of informed consent was obtained from all subjects. apoA-I and apoA-II have correlated positively with chest skinfold thickness14; however, abdominal fat, as Apolipoprotein Preparation and Labeling measured by the waist-to-hip ratio, may better predict ApoA-I and apoA-II were prepared from healthy donor lipoprotein metabolism and atherosclerosis risk (see plasma by ultracentrifugation, delipidation, and column chroma- Reference 15 for review), and the relation of this tography using standard methods.9 Apolipoprotein purity was measure to HDL metabolism remains unknown. Fasting verified by sodium dodecyl sulfate-polyacrylamide gel electro- insulin levels have been associated with increased phoresis followed by silver staining. The purified apoA-I and apoA-I FCR in diabetic16 and hypertensive17 subjects, apoA-II were dissolved in 6 mol/L urea and 0.05 mol/L tris(hy- but the relevance of insulin levels to HDL metabolism droxymethyl)aminomethane, pH 8.5, at a concentration of 0.5 to in a normoglycemic and normotensive population has 1.0 mg/mL for radioiodination by the iodine monochloride method and were tested for pyrogenicity and sterility.9 not been reported, and the potential mechanisms of an insulin effect on HDL metabolism are unclear. Kinetic Studies Do This study was designed to explore the metabolic The subjects were admitted to The Rockefeller University wn regulation of HDL-C levels by analyzing HDL apolipo- Hospital inpatient ward and kept on a metabolic diet consist- loa protein turnover data and related parameters in a ing of commonly available foods for 4 weeks. The diet had a d e sufficient number of subjects to allow multivariate sta- caloric distribution of 42% fat, 43% carbohydrate, and 15% d fro tistical analysis. The larger sample size also allowed protein. The total fat content corresponded to the 50th m comparisons between men and women as well as same- percentile of previous American intake according to the Lipid http://atvb gcgureonrrudepensrt oafns tasuluydbsyji esc.w tTsa hsfe ro oembx ttaepnirneseivvdieo usbsuy bs mjeccoatlm lepbroi npsitunulgad tiieonsna9 1ro1r ofw wtihteher ctIRiIuoe)rn.sr1ea9e lan rOtHc hfAe atmColttlehiarn li icaccansan dl( o LcNroRiuenCstsr,)u i tmpsiootpunltyd ioyuEnn1x8 s aaaamtcnucidrnoa arttdtoeiid ont nhg fe aSt t7ou 5 rctvtohhen eyps tsei(ertNuccoetHenndAtdi lN2Ne% EoaSf-, .aha many new subjects and by including new data on all monounsaturated fat 14%, and saturated fat 26%. Subjects jo subjects. We found that women and men differ more consumed 215 mg cholesterol per 1000 calories. Caloric need u rn clearly in FCR than in TR of HDL apolipoproteins. was estimated by the Harris-Benedict20 equation with adjust- a ls Relative amounts of abdominal fat by waist-to-hip ratio ment for physical activity,21 and caloric intake was not altered .o rg correlated directly with apolipoprotein HDL FCR. We during the study. After the first 2 days body weight remained b/ hypothesized an ordered hierarchy of factors that might constant. Daily weights during the final 2 weeks were averaged, y g regulate HDL-C levels. Multiple linear regression analy- and the body mass index was calculated as weight in kilograms u e divided by the square of height in meters. No alcohol intake s sis of the extended data set in this study was consistent t o was allowed, and subjects were instructed to maintain their n with this hypothetical hierarchy, suggesting that these D usual level of physical activity. e interacting factors may constitute a major mechanism of cem antiatherogenesis. After 2 weeks of equilibration on the metabolic diet, the b subjects received radioiodinated apoA-I and apoA-II (10 to 25 er 2 Methods /i,Ci 125I and 25 /iCi 131I, respectively, or vice versa) by intrave- 2 nous bolus injection. Subject No. 50 received only 25 fxCi , 20 Subjects 125I-labeled apoA-I. Although one study22 using labeling meth- 1 7 Thirty female and 27 male subjects were recruited for HDL ods somewhat harsher than ours reported that radiolabeling of metabolic studies from three sources: (1) patients from the purified HDL apolipoproteins results in faster plasma removal clinic of the Laboratory of Biochemical Genetics and Metab- than that obtained by protein labeling of intact HDL, subse- olism; (2) healthy adult volunteers working on the staff at The quent studies in different laboratories6'23'24 found that these Rockefeller University or at neighboring institutions; and (3) two methods give comparable results. This similarity is due to undergraduate students in a work-study program. The subjects the rapid association of free HDL apolipoproteins with plasma were recruited to obtain as broad a distribution of HDL-C and HDL in vivo. After bolus intravenous injection of tracer, blood TG levels as possible. All subjects were free from hepatic, was sampled at 10 minutes; 4, 12, 24, and 36 hours; and then renal, thyroid, and immunologic disorders by history and daily until 14 days. Each sample was handled and counted as laboratory screening and had normal brachial diastolic blood outlined.9 Since all subjects had steady-state apoA-I and pressure by examination. Studies on 21 subjects (Nos. 6, 9, 17, apoA-II levels, the radioactive decay curve was plotted directly 19, 20, 26, 30 through 33, 35 through 37, 41, 42, 44, 45, 47, 48, from the die-away of plasma counts. FCR was calculated from 54, and 55) are presented for the first time in this article, and this curve by the Matthews method.25 Absolute TR was individual data are given for the first time for an additional two calculated by multiplying the FCR by the plasma pool (apo- subjects, Nos. 39 and 40. Turnover data from the other subjects lipoprotein level times plasma volume) and dividing by the have been reported in studies of elevated HDL-C in women,9 subject's body weight. Plasma volume was determined by of dietary effects on HDL turnover,11 and of low HDL-C in isotope dilution at the 0-minute point as calculated by log- men and women.10 linear backward extrapolation from the plasma content of Subject No. 23 took 5 mg glyburide, an oral hypoglycemic, tracer at the earliest sampling times. each morning. Subject No. 6 used insulin, 2 U regular and 8 U To show that the iodinated apoA-I and apoA-II associated NPH, subcutaneously each morning. Both were on stable with HDL, in each study aliquots of plasma were taken 10 dosage regimens throughout the study and had been taking minutes and 7 and 14 days after injection. Three fractions their respective medications for several years. These two (d< 1.063,1.063 1.21 g/mL) were prepared by subjects were omitted from the statistical analysis of correla- ultracentrifugation. The density distribution of radiolabel in tions between fasting insulin levels and the other study param- these samples was calculated and averaged, providing a mea- Brinton et al Determinants of HDL Metabolism 709 TABLE 1. Gender Differences in Selected Study Parameters Plasma Levels, mg/dL n Age, y BMI HDL-C ApoA-l ApoA-ll Women 30 41±19 25.1 ±5.7 56.7±21.4 147±32 34.5±7.4 Men 27 44+15 25.7±3.0 45.1 ±16.3 126±29 33.3±7.5 P >.2 >2 .02 .01 >.2 FCR, pools/d TR, mg/kg per day ApoA-l ApoA-ll ApoA-l ApoA-ll Women 0.248±0.077 0.184+0.043 12.0±1.6 2.19+0.62 Men 0.277±0.069 0.216±0.056 12.1 ±2.8 2.61+1.06 P .1 .02 >2 .07 Postheparin Llpolytic Activity, /unol/mL per hour Waist-to-HIp Ratio LPL HL MHR UHR D o Women 15.0±4.7 13.7±5.9 0.83±0.10 0.90+0.12 w n lo Men 15.2±5.8 21.2+8.7 0.91 ±0.06 0.95+0.06 a d ed P >.2 .0003 .0004 .04 fro BMI indicates body mass index; HDL-C, high-density lipoprotein cholesterol; Apo, apolipoprotein; FCR, fractional catabolic rate; TR, m h transport rate; LPL, lipoprotein lipase; HL, hepatic lipase; MHR, minimum-to-hip ratio; and UHR, umbilicus-to-hip ratio. Values are ttp mean±SD. ://a tv b sure of the density distribution of the subjects' own apolipo- major surface constituents of HDL are apoA-I and apoA-II. .ah proteins.6'9-2223 Since the percent tracer in the 1.21 g/mL fraction indicates lates inversely with the percent of plasma apoA-I both in the a ls the relative distribution between the two. Tracer in the d>\2\ d>1.21 g/mL fraction after ultracentrifugation (r=-.83, .org g/mL fraction was predominantly within the 1.21 <.0001) and in particles smaller than albumin by gel filtration b/ g/mL portion (very-high-density lipoprotein) in several repre- chromatography (r=-.61, P=.OO5) in human plasma samples y g sentative subjects; therefore, the percent of tracer in the (n=20) (E.A. Brinton and M.N. Nanjee, unpublished data, ue d>1.21 g/mL fraction was used as an indicator of the amount September, 1993). s t o of lipid-poor very-high-density lipoprotein relative to more n D lipid-rich HDL. Postheparin Lipase Activities ec After 11 days on the test diet and 3 days before isotope em Lipid, Lipoprotein, and injection, a postheparin lipase test was performed. After an ber 22 ApOonl idpaoyps r1o,t 3e,i n7 , D10e,t aenrdm 1in4 aotfi othnes turnover period, plasma odvoesern oigf hht epfaasrti,n ,t h6e0 sUu/bkjgec btso dwy ewree iggihvte. nF iaftne einn tmraivneuntoeus sl abteorl,u as , 20 was obtained after a 12-hour overnight fast for measurement blood sample was taken and immediately chilled to 4°C. The 17 of lipid, lipoprotein, and apolipoprotein levels. Lipid and plasma was promptly separated and stored at -70°C until lipoprotein measurements were done on fresh specimens by assay. Lipase activity was measured with [3H]triolein in a enzymatic methods.9 Aliquots of plasma were stored at -70°C sonicated Triton X-100 emulsion in the presence or absence of for subsequent apolipoprotein determinations. ApoA-I levels rabbit anti-human lipoprotein lipase (LPL) serum.9 Lipase were measured by a sandwich enzyme-linked immunosorbent activity was expressed as micromoles of free fatty acid liber- assay using a polyclonal goat antibody to human apoA-I9 ated per milliliter of plasma per hour. LPL activity was the generously supplied by Dr Peter Herbert of Yale University. difference between total activity and that remaining after ApoA-II levels were kindly determined by Dr John J. Albers in antibody inhibition, whereas the latter was the hepatic lipase the Northwest LRC laboratories and by Dr Linda Bausserman (HL) activity. Because LPL and HL have opposing effects on at Brown University. Both laboratories used comparable ra- HDL-C levels27 (see Reference 28 for an earlier review) and dioimmunoassays based on a radial immunodiffusion assay.26 because prior experience10 with a subset of the subjects of the Results from Brown correlated well with those from the LRC present study suggested the utility of combining LPL and HL using split aliquots (r=.8, P<.01, slope=0.9, y intercept=-4 data, the LPL-to-HL activity ratio was calculated and used in mg/dL). The coefficient of variation was approximately 8% the statistical analyses. within runs and 15% between runs for both the apoA-I and Other Study Parameters apoA-II assays. Over the 2-week turnover period, no temporal trends were observed in the lipid, lipoprotein, and apolipopro- Truncal girth was measured with a narrow steel tape with tein levels, and the means of all five determinations were used the subject standing. The subject was instructed to push out in the data analysis. the abdomen as far as possible and then allow it to relax. Girth The molar ratio of HDL-C/apoA-I+apoA-II was calculated was measured transversely at three levels, the first being the as an approximation of the core-to-surface ratio of HDL. The "minimum" girth, measured at end expiration at the level of majority of cholesterol in HDL is esterified, and cholesteryl intersection of the anterior costal margin with the midclavic- ester is the major constituent of the HDL core (unesterified ular line. This usually constituted the minimum truncal girth. cholesterol, a minor fraction of total HDL-C, is divided The second measurement was at the umbilicus, also made at approximately evenly between the core and surface). The end expiration after pushing out and relaxing the abdominal 710 Arteriosclerosis and Thrombosis Vol 14, No 5 May 1994 A. B. 100- 1 60 O FIG 1. Scatterplots showing relations between high-density lipoprotein (HDL) choles- 1 40 terol levels and (A) apolipoprotein (Apo) A-l O and (B) Apo A-ll concentrations in plasma. Circles indicate women; triangles, men. r=.19 p=.15 100 200 30 40 50 Apo A-l Level (mg/dl) Apo A-ll Level (mg/dl) wall. The third measurement ("hip" girth) was made at the these 8 subjects from analysis of girth ratio correlations did not symphysis pubis, nearly always coinciding with the maximum alter the results. All but 12 subjects were measured by one of posterior curvature of the buttocks. Care was exercised to the authors (E.A. Brinton). The others were measured by maintain the tape completely horizontal (transverse), to main- other physicians or by the subjects themselves after careful tain continuous contact with the skin, and to avoid compress- instruction in the proper technique. Exclusion of the measure- D ing it inward. Girth was measured to the nearest millimeter, ments not made by the authors did not alter the statistical ow and two separate measurements were taken at each level by correlations with other parameters. nlo repositioning the tape in the opposite direction. If the first two Insulin concentration was measured in four fasting plasma ad measurements differed by more than 5 mm they were repeated samples taken during the final 2 weeks of the metabolic diet ed until two measurements differing by no more than 5 mm were period for each subject; the samples were prepared and stored fro obtained. The mean of the two measurements at each level was in the same way as for the apolipoprotein determinations. hm used for calculating the minimum-to-hip ratio (MHR) and Insulin levels were kindly measured by Dr William Bauman of ttp umbilicus-to-hip ratio (UHR), which were used for the statis- the Bronx Veterans Administration Medical Center by radio- ://a tical analyses. immunoassay.29 The results of duplicate assays on the four tv Measurements on 18 of the subjects were obtained during samples were averaged, and the mean value for each subject b .a the turnover study. The remaining 39 subjects were measured was used in statistical analyses. h ajo from 3 months to 4 years later (average, 1 year); however, all All parameters (with exception of the truncal girth in some u but 8 of these subjects (Nos. 2, 13, 27, 28, 38, 43, 46, and 56) subjects as noted above) were measured during the steady- rna were measured within 5 kg of the study weight. Exclusion of state metabolic diet period. ls .o rg by/ A. B. g ues o r = -.81 O r=.O6 t o 100- p<.0001 p=NS n o o D A e ce 1 60 A m b er 2 ! 40 A 2 2 , 2 o 0 1 7 20 10- FIG 2. Scatterplots showing relations 0.1 0.2 0.3 0.4 0.5 8 10 12 14 16 18 20 between high-density lipoprotein Apo A-l FCR (pools/day) Apo A-l TR (mg/kg*d) (HDL) cholesterol levels and (A) fractional catabolic rate (FCR) and (B) transport rate (TR) of apolipoprotein C. D. (Apo) A-l and (C) FCR and (D) TR of Apo A-ll. Circles indicate women; tri- 1 o r = -.76 angles, men. 100- X p<.0001 o 60 sti e 40 ol h C • L 20 ' D H 10- 0.1 0.2 0.3 0.4 2 3 4 5 Apo A-ll FCR (pools/day) Apo A-ll TR (mg/kg-d) Brinton et al Determinants of HDL Metabolism 711 TABLE 2. Stepwise Multiple Linear Regression Analysis TABLE 3. Stepwise Multiple Linear Regression Analysis of Correlates of HDL-C Levels of Correlates of ApoA-l FCR ApoA-l FCR LPL/HL TG r r2 HDL-C/ ApoA-l Tracer, ApoA-l+ApoA-ll d>1.21 g/mL UHR r r2 .0001 -.81 .66 .0001 -.81 .66 .0001 .84 .71 .005 .84 .70 .05 .85 .73 .04 .85 .72 HDL-C indicates high-density lipoprotein cholesterol; Apo, apolipoprotein; FCR, fractional catabolic rate; LPL, lipoprotein Apo indicates apolipoprotein; FCR, fractional catabolic rate; lipase; HL, hepatic lipase; and TG, triglyceride. The independent HDL-C, high-density lipoprotein cholesterol; and UHR, umbili- variables are listed in descending order of strength of correlation cus-to-hip ratio. The independent variables, probability values, from left to right. The probability value to add the variable is listed and the r and r2 values are given as noted for Table 2. underneath the variable name, and the r and r2 values for each stepwise combination of independent variables are given on the versus 0.286 pools/d, /J=.OO7). In contrast, adjustment for right. All independent variables adding at P<.05 are shown. other key parameters, including HDL-C/apoA-I+apoA- II, percent apoA-I in the d>1.21 g/mL fraction, TG Statistical Analysis LPL/HL, and UHR each eliminated the gender difference Linear regression analysis was performed by using the in apoA-I FCR (P=3 to .9), suggesting that they may least-squares method, and statistical significance was defined mediate that difference. as a probability value of less than .05. Parameters that were log Women also had a lower apoA-II FCR both in the D normally distributed (HDL-C, TG, and LPL-to-HL ratio) o overall group (0.184±0.043 versus 0.216±0.056 pools/d, w were logarithmically transformed for t test and linear regres- nlo sion analysis. Calculations were performed on The Rockefeller P=.O2) and among the normolipemic subjects as well ade University Hospital CLINFO system. Results are reported as (0.171±0.031 versus 0.213±0.020 pools/d, P=.01). In con- d from mean±SD. mtraesats, uwreodm einn athned emnetinr eh agdr oeuqpu i(v1a2le.0n±t 1a.p6o Ave-rIs TusR 1w2.1h e±th2e.8r h Results mg/kg per day, P>.2) or among the subjects with normal ttp://atv apoLAev-eI lsa nodf aHpDoAL--ICI, awnedr eo mf eitass umreadjo, r alaopnogl iwpoitphr ootethinesr, HpeDr Ld-aCy ,a nPd> T2G). leTvheels (a1p1o.A8±-I1 .9T Rve rsouf s p1o1s.7tm±3e.n5o pmagu/skagl b .a factors reported to relate to HDL-C levels. Men and women differed neither from that of premenopausal h ajo women were compared to explore gender effects on HDL women nor from that of men (12.4±1.1 versus 11.8±1.8 urn metabolism (Table 1) and to evaluate the appropriateness and 12.1±2.8 mg/kg per day for postmenopausal and als of pooling male and female subjects for other statistical premenopausal women and for men, respectively; P>.2). .org analyses. Women had higher levels than men for HDL-C Women tended toward a lower apoA-II TR than men b/ (56.7±21.4 versus 45.1±16.3 mg/dL, />=.O3) and apoA-I (2.19±0.62 versus 2.61 + 1.06 mg/kg per day, P=.O7). Al- y g (147±32 versus 126±29 mg/dL, P=.til) but did not differ though there was no significant gender difference in HDL u es in apoA-II levels (34.5±7.4 versus 33.3±7.5 mg/dL, P>2). size or density as estimated either by HDL-C/apoA- t on None of these levels varied during the study period. I+apoA-II ratio (19.7±4.7 versus 17.8±3.9, P=.10) or by D Women tended to have lower apoA-I FCR than men percent apoA-I tracer in the d>1.21 fraction (12.5±5.9 e ce (0.248±0.077 versus 0.277±0.069 pools/d,/>=.l), although versus 14.5±7.9, P=3), both parameters tended to show m be the difference did not reach statistical significance in the smaller, denser HDL in men. Men and women did not r 2 overall group, possibly due to subject heterogeneity. The differ in postheparin LPL activity (15.0±4.7 versus 2 , 2 gender difference in apoA-I FCR reached statistical sig- 15.2±5.8 !u,inol/mL per hour, P>.2), but women had far 017 nificance only when subjects with normal HDL and TG lower HL activity (13.7±5.9 versus 21.2±8.7 ^mol/mL per levels (both between the 20th and 80th percentile for age hour, P=.0003) and thus had a higher LPL/HL ratio and sex) were analyzed (0.277±0.038 [n=8] versus (1.47±1.58 versus 0.92±0.70, P=.OO9 for log-transformed 0.295 ±0.059 [n=6] pools/d for women versus men, respec- data). Fasting insulin levels did not differ significantly tively; P=.Q2). Furthermore, by ANCOVA, apoA-I FCR between female and male subjects (14.6±8.2 versus did differ significantly between women and men in the 11.6±4.2 /xU/mL, P=.10), but the women had a far lower entire group when adjusted for plasma insulin (0.239 MHR (.83±.1O versus .91±.O6, />=.0004) and a moder- 0.5-, B. 0.4- FIG 3. Scatterplots showing relations between the apolipoprotein (Apo) A-l fractional catabolic rate (FCR) and high-density lipopro- 0.3 -| tein (HDL) size or density as measured by (A) the HDL cholesterol (HDL-C)/Apo A-l+Apo A-ll ratio and (B) percent Apo A-l tracer in the 0.2- d>L21 g/mL fraction. Circles indicate women; triangles, men. —I— —I— —I 10 20 30 0 10 20 30 40 HDL-C/Apo A-l + Apo A-ll (molar ratio) Apo A-l Tracer D>1.21 (percent) 712 Arteriosclerosis and Thrombosis Vol 14, No 5 May 1994 TABLE 4. Stepwise Multiple Linear Regression Analysis apoA-I FCR (r2=.66; Table 2), with no additional of Correlates of HDL-C/ApoA-l+ApoA-ll contribution by the second strongest univariate correlate, apoA-II FCR. There was a modest addition from TG LPL/HL the third, LPL/HL (increasing r2 to .71), and only a .0001 .74 .55 small addition by any other variable (r2 increasing to .73 .0001 .85 .72 with fasting TG added to apoA-I FCR and LPL/HL). Since apoA-II FCR failed to add to the prediction of HDL-C indicates high-density lipoprotein cholesterol; Apo, HDL-C by apoA-I FCR, correlations with apoA-II FCR apolipoprotein; TG, triglyceride; LPL, lipoprotein lipase; and HL, hepatic lipase. The independent variables, probability values, were not pursued further. and the r and r2 values are given as noted for Table 2. Based on known physiological relations and data on HDL apolipoprotein turnover in patients with normal ately lower UHR (.90±0.12 versus .95±0.06, P=M) than and low HDL-C,10 we have hypothesized a hierarchical the men. schema to interconnect various HDL-related parame- Relations among these study parameters were ex- ters.10 We sought to confirm and extend this model in plored by linear regression analysis. Despite the signif- the current study with a larger subject group, including icant differences between men and women in some of subjects with low through high HDL-C levels, and with these variables, the linear correlations among nearly all added study parameters. We studied each parameter variables were similar between the genders, so data for from HDL downward as the dependent variable, using men and women were pooled for regression analyses. the remaining lower factors as independent variables. There was a correlation between HDL-C and apoA-I ApoA-I FCR was the strongest correlate of HDL-C D levels (r=.87, P<.0001; Fig 1A), but there was no levels among all the measured parameters, both in o wn significant relationship between HDL-C and apoA-II univariate and multivariate analyses. We next explored loa (r=.19, P=.\5; Fig IB). We found a strong inverse its possible determinants among the other study param- ded correlation between HDL-C and apoA-I FCR (r= -.81, eters. The best correlate of apoA-I FCR by single linear fro P<.0001; Fig 2A) and a similar relationship between regression was the HDL-C/apoA-I+apoA-II ratio, an m HDL-C and apoA-II FCR (r=-.76, /><.0001; Fig 2C). approximation of the core-to-surface ratio of HDL (see h ttp In contrast, there was no correlation between HDL-C "Methods"), which had a strong inverse relation ://a and apoA-I TR (r=.O6, P>.2; Fig 2B), and the moder- (r=-.81, P<.0001; Fig 3A). The second best univariate tvb ate inverse correlation between HDL-C and apoA-II correlate of apoA-I FCR was another parameter of .ah TR (r=-35, P=.01; Fig 2D) was diminished on exclu- HDL density or size, the percentage of the apoA-I a jo sion of two outliers with extremely high apoA-II TRs tracer present in the d>\2\ g/mL fraction (r=.62, u rn (after exclusion, r=-.3O, P=.O3). P<.0001; Fig 3B). Although all of the other study a ls.o The study parameters were selected for reported parameters correlated with apoA-I FCR in univariate brg/ relations with HDL-C levels, and such correlations were afinrastl ytswiso, pianr asmteeptweriss ea cmcouultnipteled floinr e7a0r %re ogfr etshsei ovna rtiahbeisle- y confirmed between HDL-C and the percent of the g ity in apoA-I FCR (r2=.7O; Table 3), and only a small u apoA-I and apoA-II tracers in the d> 1.21 g/mL fraction es increment in correlation was obtained by the addition of t on D f(ars=ti-n.g4 6T aGn d( r-=.3-.65, 9P, 0<0.0010 0a1n)d a n<.0001; Fig 4B). In stepwise multivariate linear dependent variables (excluding apoA-I and apoA-II regression these both added to predict nearly three levels). Dependent variables were added in descending fourths of the variability in HDL-C/apoA-I+apoA-H order of univariate correlation with HDL-C. Two thirds (r2=.72; Table 4); the other remaining study parameters of the variability in HDL-C was accounted for by failed to add to its prediction. LPL and HL activities 30- A. o B. ! r = -.74 25- e*o fx.0001 a"V o oo o < owv o FIG 4. Scatterplots showing relations be- 20- °\° tween the high-density lipoprotein cholesterol + (HDL-C)/apolipoprotein (Apo) A-I+Apo A-ll ra- 15- * * V 0 tio and (A) fasting plasma triglyceride level and (B) the lipoprotein lipase/hepatic lipase < •v (LPL/HL) activity ratio in postheparin plasma. 10- o N r =.62 Circles indicate women; triangles, men. [X.0001 E A 5- 10 100 1000 10000 01 Plasma Triglyceride Level (mg/dl) LPL/HL Activity Ratio Brinton et al Determinants of HDL Metabolism 713 A. B. 1000- 500 FIG 5. Scatterplots showing relations between fasting plasma triglyceride and (A) fast- 200 ing insulin levels and (B) the umbilicus-to-hip girth ratio. Circles indicate women; triangles, men. —1— —I— 10 20 30 08 09 1 11 Fasting Insulin (MU/ITII) Umbilicus/Hip Girth Ratio correlated, directly and inversely, respectively, with the from plasma. UHR also correlated with the HDL-C/ HDL-C/apoA-I+apoA-II ratio. LPL and HL taken simul- apoA-I+apoA-II ratio (r=-.59, P<.0001), the percent taneously as two separate variables correlated approxi- apoA-I tracer in the d>1.21 g/mL fraction (r=.36, mately as well as did the LPL/HL ratio as a single variable. P= .006), and fasting insulin (r= .37, P=.006) in addition to D Since the correlations of LPL and HL were consistently the previously mentioned relations with TG and lipase ow opposite each other, and because of their known opposing activity. Thus, the correlation of body fat distribution with n lo actions on HDL, to simplify our statistical analyses the HDL-C appears to be mediated by its proximate relations a de LPL/HL ratio was used instead of the two separate with TG, lipase, HDL size, and apoA-I FCR. d fro variables. In summary, our earlier schema10 was confirmed in a m By univariate linear regression, fasting plasma TG much larger and more diverse group of subjects. Further- http was predicted both by fasting plasma insulin (r=.53, more, two new parameters, fasting insulin levels and body ://a /><.0001; Fig 5A) and by UHR (r=.52, P<.0001; Fig fat distribution, correlated with the other factors in a way tv 5B), and in multivariate linear regression analysis these confirmatory of their hypothesized contribution to lipo- b .ah two together predicted nearly one half of the variability protein metabolism.13 The data in the current study are ajo in TG levels (r2=.46; Table 5). The LPL/HL activity consistent with a new expanded schema (Fig 7). u rn ratio correlated with UHR (r= -.51, /><.0001; Fig 6). In Individual data from each subject regarding apoA-I als.org cthoen troatshte tro tphaer aomtheetre rcso, rrienl atwiohnicsh b eLtwPLee na nLdP LH/HLL baontdh aarned parpeosAen-ItIe dt urinn ovTearb laensd 6s etvherroaul grhe la9t.e dS upbajreacmts etaerres by/ contributed to the relations seen with the LPL/HL ratio, divided by gender and ranked in descending order of gu the relationship between UHR and LPL/HL was due to HDL-C level. Table 6 presents subject age and lipopro- est on D UHLH Ral,o rn=e -(.H3L, Pve=r.s1u5s )U. IHn Ra,l lr =ot h.5e2r, Pca.\). Discussion , 20 Another goal of this study was to explore relationships 1 The object of this study was to examine associations 7 between body fat distribution and HDL metabolism. Both among parameters relating to HDL metabolism and to UHR and MHR correlated well with HDL-C levels postulate mechanisms that might explain these associa- (/•= -.52, P<.0001 and r= -.46, /><.001, respectively), but since UHR appeared to correlate more strongly in this and other analyses, UHR alone was used as the parameter 10-1 of body fat distribution. UHR also correlated well with the FCR of apoA-I (r=.61, P<.0001) and apoA-II (r=0.55, r = -.51 P<.0001) but only moderately or not at all with the TR of p<.0001 3 apoA-I or apoA-II (r=. 19, P= .2 and r= .29, P= .03, respectively). Thus, the amount of central (abdominal) fat rela- 2 tive to lower body (gluteal) fat directly relates to the fractional removal of the major HDL apolipoproteins 1 .7 TABLE 5. Stepwise Multiple Linear Regression Analysis .5 of Correlates of Fasting Plasma TG Levels .3 Fasting Insulin UHR r r2 .0001 .58 .33 0 7 0.8 0.9 1.0 1.1 1.2 .0001 .68 .46 Umbilicus/Hip Girth Ratio TG indicates triglyceride; UHR, umbilicus-to-hip ratio. The FIG 6. Scatterplot showing relation between the lipoprotein independent variables, probability values, and the r and r2 values lipase/hepatic lipase (LPL/HL) activity ratio and the umbilicus-to- are given as noted for Table 2. hip girth ratio. Circles indicate women; triangles, men. 714 Arteriosclerosis and Thrombosis Vol 14, No 5 May 1994 variable. In other words, by ANCOVA we asked Atherosclerosis whether women and men would differ in apoA-I FCR if they had equal values of a given covariate. With plasma insulin as the covariate, apoA-I FCR was a highly HDL-C Level significant 16% lower in women (P=.OO7), strengthen- ing our impression that women differ from men in apoA-I FCR. Interestingly, when we used any of the Apo A-l FCR other key parameters (HDL-C/apoA-I+apoA-II, percent apoA-I in the d>\2\ g/mL fraction, TG, LPL/HL, or UHR) as a covariate, ANCOVA showed no gender HDL Size or Density difference in apoA-I FCR (P=3 to .9). However, rather (HDL-C/Apo A-l + Apo A-ll Ratio) than negating a gender difference in apoA-I FCR, these findings suggest possible mechanisms for that differ- / \ ence. Women and men differed (significantly or nearly Core Lipid Exchange Lipase Activity so) in every one of these other key parameters in ways (Plasma TG Level) (LPL/HL Ratio) that should result in a lower FCR in women. This is suggestive evidence that one or more of these factors \ may cause apoA-I FCR to be reduced in women and Insulin Responsiveness (Insulin Level) may thus cause their HDL-C to be higher as well. Our findings of a lower apoA-I FCR and the lack of D an increase in apoA-I TR in women concur with those o w of Shepherd and coworkers,12 who reported nonsignifi- n lo Abdominal Fat cant trends in women toward reduced FCR of apoA-I a (Waist/Hip Ratio) de and apoA-II (13% and 10% lower, respectively) and d from tFaIGb o7li.s mH aynpdo tlheevteiclsa lo sf chhigehm-dae onfs itthye lifpaocptororste iinnf lu(HeDncLi)n ge xtphaen mdeed- creodnutrcaesdt , TSRc hoafe afepro Aan-Id acnodl laepaogAue-sI6I (rbepotohr t6e%d sloigwneifri)c.a Innt h from Reference 9 to show the proposed interaction of the ttp additional parameters reported in this study. HDL-C indicates elevations of apoA-I and apoA-II TRs in women (22% ://a HDL cholesterol; Apo, apolipoprotein; FCR, fractional catabolic and 20%, respectively) along with higher FCRs (a tvb rate; TG, triglyceride; LPL, lipoprotein lipase; and HL, hepatic nonsignificant 8% increase for apoA-I and a significant .a lipase. 17% increase for apoA-II). Reasons for the major h ajo differences among these studies are unclear. Schaefer et u rn tions. We studied human subjects whose HDL-C levels al included only young adult subjects and had just over a ls varied across a broad range despite their consumption one third as many subjects as our study. However, we .o rg of identical metabolic diets. We measured several pa- found no gender difference in apoA-I TR among sub- b/ rameters in these subjects and tested for gender differ- jects who were less than 49 years old (women, 11.8±1.8 y g ences and for intercorrelation among the parameters. versus men, 12.1±2.8 mg/kg per day, P>2), nor did we u est on thaWn ommeenn, tahree awveelrla kgneo bwenin gto a hbaovuet h5i5g mhegr/ dHLD fLor- Cw loemveelns fminedn oap daiufsfearle nsucbe jebcettsw (e1e2n. 4o±u1r .1p rveemrseunso p1a1u.s8a±l1 a.8n dm pgo/kstg- De and about 45 mg/dL for men in Westernized societies.30 per day, P> .2). Furthermore, the lack of difference in c em We found similar mean values in the small, selected apoA-I TR by gender was not due to our inclusion of be subject population of the current study, suggesting that subjects with a broad range of lipid levels because it did r 2 our subjects may be representative of the general pop- not differ by gender among our normolipemic subjects 2 , 2 ulation. Nevertheless, because we selected for overrep- (women, 11.8+1.9 versus men, 11.7±3.5 mg/kg per day, 0 1 resentation of extreme levels, the variability of HDL-C P>2). 7 levels was much higher among our subjects than in Superficially, the elevated TR of apoA-I and apoA-II unselected populations. We explored possible metabolic in women reported by Schaefer and colleagues6 is bases of the gender difference in HDL-C levels by confirmed by their later study,31 which showed an unpaired t test. We found 17% higher apoA-I levels in increase in apoA-I TR during oral estrogen therapy. women but a nonsignificant 3% difference in apoA-II However, since endogenous estrogens enter the plasma levels, similar to previous reports.6 The 17% elevation in outside of the portal circulation, the oral route is apoA-I was associated with a nonsignificant trend to- nonphysiological for estrogen replacement. Due to inward a 10% reduction in its FCR in women, while its testinal contact during enteral absorption and due to TR did not differ. ApoA-I TR did not differ between hepatic first-pass clearance, oral administration of es- women and men even when postmenopausal subjects trogen in doses sufficient to provide physiological con- were excluded (1% and 3% lower than men with and centrations in the systemic circulation exposes both the without postmenopausal subjects, respectively). The small intestine and the liver to superphysiological con- lack of gender difference in apoA-II levels (3% higher centrations of hormone. Since these two organs are the in women) resulted from a 15% reduction in its FCR in primary sites of apoA-I synthesis, it is plausible that women counterbalanced by a 16% reduction in its TR. such exposure could drive apoA-I production rates to We further explored the relation of gender to apoA-I artificially high levels. Other studies32 in postmeno- FCR by ANCOVA using key parameters from our pausal women confirmed that oral administration of linear regression analyses (Fig 7). ANCOVA refines the estrogen may raise apoA-I TR, whereas parenteral study of differences in a primary or dependent variable (percutaneous) administration may have no effect. De- (in this case, apoA-I FCR) between groups (women and spite the drawback that the parenteral estrogen was of men) by adjusting for covariates (the other key param- insufficient dose or duration to reverse the expected eters) that correlate or covary with the dependent postmenopausal reduction in HDL levels, the study Brinton et al Determinants of HDL Metabolism 715 TABLE 6. Age and Cholesterol and Triglyceride Levels of Study Subjects Subject Age, TC, VLDL-C, LDL-C, HOL-C, TG, No. y mg/dL mg/dL mg/dL mg/dL mg/dL Women 1 34 442 21 299 122 90 2 29 355 33 229 93 105 3 33 194 13 92 90 59 4 67 308 38 192 78 151 5 22 221 24 125 72 96 6 51 391 49 271 71 194 7 19 150 11 70 70 46 8 64 639 37 532 70 111 g 20 289 28 194 67 116 10 22 200 14 119 67 60 11 22 223 31 131 61 87 12 21 178 25 92 60 84 13 24 159 10 91 58 56 14 21 174 23 93 57 73 15 21 167 16 95 56 59 16 22 181 14 112 55 62 D o 17 68 446 56 337 53 248 w n 18 79 180 51 78 51 126 lo ad 19 27 252 49 153 50 128 e d from 2201 4391 710937 44466 211029 4453 1163287 h 22 47 203 19 144 40 87 ttp://atv 2234 6567 321517 5709 119658 3378 218329 b .a 25 67 250 38 175 36 147 h ajo 26 61 611 338 237 36 778 urn 27 52 475 229 211 36 783 a ls 28 41 254 60 160 34 208 .o rg 29 41 287 140 120 28 733 by/ 30 45 617 391 201 25 1156 gu Mean±SD 41±19 311±159 79±115 175±96 57±21 268+373 e s t o Men 31 59 335 21 231 83 82 n D 32 70 263 23 159 81 94 ec 33 53 267 27 172 69 83 e mb 34 24 206 22 119 65 85 e r 2 35 41 223 39 119 64 147 2, 2 36 46 272 29 179 63 109 01 37 67 390 37 292 61 292 7 38 20 196 21 127 48 100 39 28 213 38 130 44 140 40 56 290 51 196 43 133 41 48 313 55 215 43 209 42 64 361 144 175 42 590 43 45 268 35 191 42 135 44 67 433 222 169 41 410 45 48 572 320 212 40 907 46 23 166 15 112 38 71 47 47 289 161 92 36 311 48 20 128 45 47 36 150 49 48 274 129 110 35 777 50 45 196 60 102 35 260 51 46 411 105 274 33 175 52 38 391 31 328 32 112 53 37 353 127 194 31 479 54 23 204 38 135 31 167 55 39 181 76 75 30 164 56 34 145 30 85 30 105 57 58 264 187 56 22 1354 Mean±SD 44±15 282±101 77 ±75 159±70 45±16 283±304 716 Arteriosclerosis and Thrombosis Vol 14, No 5 May 1994 TABLE 7. ApoA-l and ApoA-ll Parameters in All Study Subjects ApoA-l ApoA-ll Subject No. Level, mg/dL FCR, pools/d TR, mg/kg per day Level, mg/dL FCR, pools/d TR, mg/kg per day Women 1 251 0.133 13.5 31 0.142 1.79 2 207 0.162 9.7 46 0.136 1.81 3 200 0.151 13.7 38 0.143 2.46 4 181 0.221 13.2 34 0.152 1.69 5 162 0.189 12.9 45 0.161 3.07 6 157 0.188 10.9 47 0.164 2.82 7 158 0.176 13.7 35 0.167 2.89 8 163 0.185 12.1 34 0.131 1.80 9 128 0.196 8.5 42 0.156 2.23 10 155 0.171 9.3 26 0.147 1.33 11 153 0.184 10.1 35 0.147 1.86 12 162 0.242 14.2 29 0.171 1.80 13 171 0.227 14.1 28 0.210 2.10 14 127 0.233 10.1 24 0.164 1.32 15 137 0.256 10.4 29 0.200 1.69 D 16 134 0.195 10.2 24 0.141 1.34 o w 17 144 0.257 10.2 28 0.158 1.21 n lo 18 134 0.291 13.4 27 0.205 1.88 a d e 19 157 0.208 11.9 44 0.163 2.60 d fro 20 134 0.305 12.4 49 0.200 2.98 m 21 138 0.234 10.9 35 0.178 2.12 h ttp 22 127 0.322 13.2 27 0.186 1.63 ://a 23 111 0.399 13.0 32 0.247 2.30 tvb 24 117 0.304 13.0 32 0.195 2.30 .a ha 25 114 0.249 13.1 28 0.193 2.48 jou 26 120 0.249 11.9 42 0.192 3.18 rn a 27 139 0.328 12.8 43 0.267 3.23 ls .o 28 113 0.415 12.7 29 0.255 1.99 rg b/ 29 128 0.377 12.3 39 0.246 2.43 y g 30 99 0.383 11.6 37 0.298 3.35 ue Mean±SD 147±32 0.248±0.077 12.0+1.6 35±7 0.184±0.043 2.19±0.62 s t on Men 31 160 0.196 12.3 48 0.143 2.70 D e 32 167 0.136 9.6 34 0.135 1.93 c em 33 175 0.216 12.5 32 0.150 1.56 b e 34 141 0.175 12.4 23 0.159 1.80 r 22 35 168 0.221 11.7 45 0.196 2.75 , 20 36 164 0.161 8.6 29 0.143 1.35 1 7 37 179 0.266 18.4 40 0.214 3.33 38 127 0.242 8.3 28 0.218 1.65 39 134 0.313 9.7 33 0.204 1.57 40 113 0.274 9.7 35 0.184 2.01 41 127 0.396 18.1 38 0.243 3.34 42 133 0.327 12.8 43 0.219 2.75 43 124 0.234 11.9 38 0.208 3.27 44 141 0.346 15.2 30 0.234 2.20 45 125 0.300 13.1 48 0.211 3.54 46 115 0.308 12.6 23 0.222 1.81 47 109 0.351 15.7 39 0.338 5.44 48 91 0.218 8.2 35 0.178 2.52 49 113 0.321 13.5 26 0.268 2.62 50 124 0.251 11.7 31 51 104 0.320 12.1 26 0.207 1.94 52 92 0.273 9.5 23 0.178 1.55 53 97 0.282 12.7 27 0.226 2.77 54 96 0.372 13.9 39 0.364 5.55 55 75 0.271 7.0 27 0.218 2.05 56 89 0.316 12.6 26 0.268 3.07 57 121 0.393 13.6 34 0.281 2.71 Mean±SD 126±29 0.277+0.069 12.1+2.8 33+8 0.216±0.056 2.61 ±1.06

Human HDL Cholesterol Levels Are Determined by ApoA-I Fractional Catabolic Rate, Which

Similar Human HDL Cholesterol Levels Are Determined by ApoA-I Fractional Catabolic Rate, Which