The effects of physical exercise on plasma prebeta-1 high-density lipoprotein

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this process involves cholesterol efflux from peripheral cells. 7,8upta et al 9 have shown that the net mass of free cholesterol transport out of cultured human fibroblasts into athlete's serum was greater than that of sedentary controls.This indicates that physical exercise may promote RCT by increasing cholesterol efflux from peripheral tissues.Because specific subfractions of HDL, such as the prebeta-1 HDL fraction, 10 may be especially efficient at mediating cholesterol removal from peripheral cells, physical exercise would be anticipated to increase plasma prebeta-1 levels yet this has not been previously reported.
Prebeta-1 HDL is a molecular species of HDL that was not recognized until 1985. 11Because of its high modal density, it was not included in HDL recovered by serum by ultracentrifugation in the traditional density interval of 1.063 to 1.21 g/mL.In addition, its rapid conversion to HDL species of larger diameter, ex vivo, by lecithin:cholesterol acyltransferase (LCAT), impeded its recognition by other techniques.The advent of a minimally perturbing chromatographic technique, selected affinity immunosorption 12 that recovers apolipoprotein A-I (Apo A-I)-containing particles quantitatively from human plasma, has permitted the subsequent recognition of HDL species of prebeta mobility, distinguishing them from the predominant mass of HDL that has alpha mobility (ie, alpha HDL). 11rebeta-1 HDL is a 67-kd species of plasma HDL that contains 2 copies of Apo A-I.Prebeta-1 HDL appears to be generated from both alpha HDL lipoproteins (A-I ϩ A-II) (Lp [A-I ϩ A-II]) and (A-I Ϫ A-II) (Lp [A-I Ϫ A-II]). 13The measurement of prebeta-1 HDL in human plasma by an ultrafiltration-isotope dilution technique permits the methodology to examine the metabolic transitions among the native molecular species of HDL. 13,14The evaluation of prebeta-1 HDL metabolism is may be of value in identifying mechanisms by which physical exercise-induced effects on HDL particles exert a protective action against coronary artery disease (CAD). 5he purpose of this study was to evaluate the effect of a single bout of protracted aerobic exercise on plasma prebeta-1 HDL levels.

Design
The study was designed to examine the effects of a bout of protracted physical exercise on fasting plasma lipids, lipoproteins, Apo A-I, and prebeta-1 HDL levels.

Subjects
Nineteen nonsmoking men and women volunteers in self-reported good health and not taking medications that would effect their plasma

Methods
Subjects refrained from participation in physical exercise and consumption of ethanol-containing beverages 3 days prior to the testing session.They reported following a 12-hour (overnight) fast to the exercise laboratory.A history and physical examination were performed to confirm each subject's health status (D.A.L.) and pre-exercise laboratory collection included the following measurements: (1) fasting plasma lipids, lipoproteins, Apo A-I, and prebeta-1 HDL; (2) percent body fat; and (3) dietary fat and cholesterol intake.Subjects were classified as exercise-trained (ET) if they reported regular participation in physical exercise of at least 45 minutes of vigorous, aerobic physical exercise training 3 times per week, or sedentary (SED) if they reported no regular physical exercise behavior.Subjects then underwent a bout of protracted physical exercise consisting of maximal cardiopulmonary exercise (CPX) stress testing followed by a 4-km run-jog (total exercise time, 45 to 60 minutes).Following the exercise bout measurements of fasting plasma lipids, lipoproteins, Apo A-I, and prebeta-1 HDL were repeated.

Procedures
Body composition.Prior to undergoing CPX testing, body composition was determined via skinfold thickness measurements at specific sites (chest, abdomen, and thigh for men; tricept, suprailliac, and thigh for women) using standard procedures. 15Measurements at each site and on each subject were made by an experienced investigator using Harpenden skinfold calipers (British Indicators, Dover, England).Each skinfold site was measured using the thumb and index finger of the left hand to elevate a double fold of skin, with subcutaneous tissue approximately 1 cm proximal to the site being measured.Each skinfold was raised perpendicular to the surface of the body at the measurement site, with the long axis of the skinfold parallel to the natural cleavage lines of the skin.The skinfold was kept elevated until the measurement was completed, and the jaws of the caliper were placed so that the thicknesss of the skinfold was measured perpendicular to its long axis when the pressure of the caliper was released and the caliper jaws moved toward each other.A reading of the skinfold thickness, in millimeters, was made 2 seconds after the caliper was released at the skinfold site.All sites on each subject were measured once, and then each site was measured a second time.A third skinfold measurement was made if the second measurement at a particular site exceeded the first by more than 1 mm.The skinfold thickness at a particular site was taken as the average of the 2 closest measurements.Intrameasurer reliability coefficients for experienced investigators using this technique are 0.97 (tricept), 09.6 (chest), 0.98 (abdomen), 0.97 (suprailliac), and 0.98 (thigh).
CPX testing.CPX testing was conducted using a standard Bruce protocol. 16Heart rate was continuously monitored during CPX testing by telemetry (Polar Vantage XL, Polar USA, Hartford, CT), with heart rates recorded at one-minute intervals for 10 minutes following exercise.Metabolic measurements for determination of maximal aerobic capacity (VO 2max ) were made using a MedGraphics CardioO 2 system (Medical Graphics, St Paul, MN).Expired gas measurements were recorded at 30 second intervals, and VO 2max was determined by the following criteria: (1) Ͻ 100 mL increases in VO 2 in consecutive 30 second sampling periods prior to exhaustion, (2) respiratory exchange ratio (RER) in excess of 1.10, and (3) attainment of predicted maximal heart rate Ϯ15 bpm.
4-km exercise bout.Approximately 5 to 10 minutes after the CPX test, when subjects' heart rates had recovered to Ͻ 100 bpm, they engaged in a run-jog exercise bout covering 4 km on a running track adjacent to the laboratory, at a self-selected pace.This distance was selected such that, when combined with the CPX test, the subjects would expend approximately 300 to 350 kcal in aerobic exercise.Upon completion of the run-jog, the subjects underwent a final venipuncture.
Dietary surveys.A 3-day dietary record was dispensed to all subjects within 1 week prior to the run-jog exercise session. 17,18Subjects were instructed to list all daily food and drink intake for 2 days of the week and one day of the weekend.Portion sizes were estimated using household measures, and adherence to typical dietary intake was strongly recommended. 19Daily caloric intake, dietary cholesterol, and fat were calculated using food composition tables. 20lasma lipoproteins and apoproteins.Laboratory analysis was performed in a laboratory that participates in the standardization procedure for the National Heart Lung and blood Institute-Centers for Disease Control Lipid Standardization Program coordinated by the Centers for Disease Control, Atlanta, GA, and is certified.Two 20-mL fasting blood samples were obtained in EDTA-containing vacutainers from each subject before and immediately after exercise.The samples were stored immediately on wet ice for transport to the analytic laboratory.Each sample was cold centrifuged to collect the plasma and harvested on Ϫ60°C until assayed.Three major classes of plasma lipoproteins were separated by preparative ultracentrifugation, followed by measurement of the content of cholesterol and triglycerides in each. 21The content of Apo A-I was measured by enzyme-linked immunoassay (ELISA). 22The content of prebeta-1 HDL in plasma was measured by an ultrafiltration-isotope dilution technique, in which both the relative percentage of plasma Apo A-I that is present as prebeta-1 HDL and the absolute amount of prebeta-1 HDL in plasma are determined. 23The ELISA used to measure prebeta-1 HDL was demonstrated to react with Apo A-I equivalently in prebeta-1 HDL and in larger HDL species of alpha electrophoretic mobility.

Analysis
Data were manually entered and analyzed using EXCEL software (Microsoft, Seattle, WA) in an Intel Pentium computer (Intel, Santa Clara, CA).Means Ϯ SD were determined for the following entry characteristics: age, percent body fat, VO 2max , dietary caloric intake, dietary cholesterol intake, and percentage dietary fat intake.As an internal validity check, 1-way analysis of variance (ANOVA) was conducted between ET and SED subjects' VO 2max and pre-exercise plasma levels of plasma HDL-cholesterol, and between male and female subjects' pre-exercise plasma levels of HDL-cholesterol.Paired t tests were conducted for all subjects (N ϭ 19) between pre-and post-exercise plasma levels of fasting plasma lipids, lipoproteins, Apo A-I, and prebeta-1 HDL.

RESULTS
All subjects completed the exercise protocol.Group characteristics are shown in Table 1.It is notable that subjects no. 2 and 18 reported zero cholesterol and very low fat diets.These subjects were young women who where using commercial very-low-fat meal replacements for weight management.Most subjects (N ϭ 13) did not consume ethanol-containing beverages and the remainder consumed fewer than 5 ethanol-containing beverages per week.Subjects refrained from consuming ethanol-containing beverages during the 3 days preceding the exercise session.The comparisons made between ET and SED subjects as an internal validity check showed that VO 2max was greater in ET than in SED subjects (52.8 Ϯ 6.8 v 45.0 Ϯ 7.3 mL O 2 /kg/min, P ϭ .026),and HDL-cholesterol levels were greater in ET than in SED subjects (54.8 Ϯ 11.2 v 43.3 Ϯ 8.7 mg/dL, P ϭ .024).Pre-exercise plasma HDL-cholesterol levels were not significantly different between male (n ϭ12, 48.0 Ϯ 13.3 mg/dL) and female (n ϭ 7, 51.1 Ϯ 9.4 mg/dL) subjects.
Table 2 summarizes mean Ϯ SD values of fasting lipids, lipoproteins, Apo A-I, and prebeta HDL before and after exercise.Paired t tests conducted for all subjects (N ϭ 19) between pre-and post-exercise plasma levels of fasting lipids, lipoproteins, Apo A-I, and prebeta-1 HDL showed a significant decrease in plasma HDL-triglyceride levels (23.3 Ϯ 10.8 to 12.5 Ϯ 5.6 mg/dL, P Ͻ .012)and an increase in absolute prebeta-1 HDL (0.10 Ϯ 0.05 to 0.13 Ϯ 0.07, P ϭ .039)before and after exercise.Table 3 shows individual subjects' plasma  prebeta-1 HDL levels before and after exercise.The response was heterogeneous but favored increased plasma prebeta-1 HDL levels (n ϭ 11).Plasma prebeta-1 HDL levels were unchanged in 6 subjects and decreased slightly in 3 subjects.

DISCUSSION
Our findings confirm the expected result that young adults' level of regular habitual exercise status is related to increased VO 2max and plasma HDL-cholesterol levels in ET subjects compared with SED counterparts.To our knowledge the effect of physical exercise on plasma prebeta-1 HDL levels has not been previously reported.Plasma prebeta-1 HDL levels increased and plasma HDL-triglyceride levels were lowered following physical exercise.
The study was conducted in young, healthy adults, and hence the findings may not extend to older persons.Although the study did not include an nonexercising control group, we do not regard this as a major design flaw because the substantial physiological impact from the protracted intervention resulted in changes in plasma lipoprotein levels that we cannot ascribe as artifact.In addition, we did not measure specific enzymes involved in prebeta-1 HDL metabolism.Acute exercise has generally been shown to increase plasma HDL-cholesterol levels by 4% to 43%. 24The lack of acute exercise-induced increase in plasma HDL-cholesterol noted in our study may reflect a "masking effect" of exercise-induced offsetting reciprocal changes in plasma HDL 2 -cholesterol (increased) and HDL 3 -cholesterol (decreased) subparticle levels. 25Our study did not measure HDL subparticle composition.
Physical exercise is recognized to have a triglyceride-lowering effect 26 that is prominent following acute 27 and chronic exercise in hypertriglyceridemics. 28The lack of an acute exercise response on total plasma triglyceride lowering may be reconciled by the relatively low pre-exercise triglyceride levels among these subjects as exercise-induced triglyceride lowering occurs more prominently among those with increasing triglyceride levels. 29rebeta-1 HDL behaves as the quantum HDL particle in the initial phase of the pathway for cholesterol retrieval from peripheral cells to the liver. 6It is the first acceptor of unesterified cholesterol, becoming a substrate for LCAT.As the cholesterol is esterified, the prebeta particles are ultimately subsumed into larger, spherical alpha HDL particles. 7As cholesteryl esters are then transferred, under the influence of cholesteryl ester transfer protein (CETP), to acceptor lipoproteins that contain the B apolipoproteins, prebeta-1 particles are regenerated.Thus the circulating level of prebeta-1 HDL reflects the relative activities of LCAT and CETP, in what is now recognized as a cyclic process.Increased LCAT activity increases alpha HDL formation and has been shown to be greater in athletes than sedentary counterparts, 9,30,31 and is increased immediately after acute exercise. 32,33Decreased CETP activity increases alpha HDL levels by increasing cholesterol ester content, and concomittently reduces HDL-triglyceride content by triglyceride transfer to non-HDL lipoprotein species.On the other hand, increased CEPT activity could facilitate alpha HDL turnover and promote prebeta HDL formation.In some studies CETP activity is greater among athletes than sedentary indi-viduals, 9 while it has also been shown to decrease following physical exercise training. 34Following acute exercise CEPT activity is shown to decrease 35 or be unchanged. 36ur findings showing that acute exercise increases prebeta-1 HDL without increasing plasma Apo A1 levels are consistent with studies showing that prebeta-1 HDL is derived from alpha HDL particles rather from de novo Apo A-I synthesis. 13Both HDL particle species Lp (A-I Ϫ A-II) and Lp (A-I ϩ A-II) contribute Apo A-I that is dissociated from these particles in the formation of prebeta HDL. 13 Prebeta HDL particles are generated from alpha HDL during cholesterol clearance by several pathways, including: (1) CETP-mediated exchange of cholesterol esters from HDL to the very-low-density lipoprotein (VLDL)/low-density lipoprotein (LDL) particle series which then provides cholesterol removal by the LDL receptor pathway 6,37 ; and (2) direct HDL-mediated receptor pathways that include (a) cubulin 38,39,40 that mediates HDL holoparticle endocytosis, and (b) the scavenger receptor, type BI (SR-BI) that binds HDL and selectively mediates the uptake or removal of HDL cholesterol esters without the uptake/degradation of HDL-Apo A-I. 38,41The SR-BI receptor appears to act as a "docking" site where cholesterol esters can be removed and subsequently the HDL particle (depleted of cholesterol esters) "undocks" and recirculates to pick up more cholesterol esters from peripheral tissues.Exercise-induced modulation of these HDL catalytic pathways could potentially play an important role in regulating plasma prebeta-1 HDL levels.Hepatic lipase has been shown to play a major role in HDL catabolism by hydrolyzing both triglycerides and phospholipids within HDL particles. 42,43P A-I particles appear to be the most efficient in facilitating cholesterol ester uptake by SR-BI receptors: Hep G 2 cell uptake of [ 3 H] cholesterol ester is approximately 75% greater from LP A-I than LP A-I ϩ A-II particles.44 Niacin has been shown to selectively inhibit plasma LP A-I removal without affecting cholesterol ester uptake.45 Niacin inhibition of plasma LP A-I removal may promote prebeta-1 HDL regeneration.It is conceivable that physical exercise could exert a similar effect to increase plasma LP A-I levels by reducing LP A-I removal.Indeed Herbert et al 46 found that the mean biological half-life of HDL proteins was longer in runners (6.2 days) than in sedentary controls (3.8 days).Tracer studies of radioiodinated autologous HDL demonstrated that runners did not produce more HDL protein but rather catabolized less.7 who showed that participation in 8 to 11 months of aerobicbased PET increased the biological half-life of Apo A-I by 10% (P ϭ .07)by decreasing the fractional catabolic rate by a corresponding amount and not affecting Apo A-I synthetic rate.A similar finding was noted for Apo A-II. Soe 48,49,50 but not other 51,52 studies indicate that physical exercise training raises plasma Apo A-I levels.We found no difference in plasma Apo A-I levels between entry and Apo A-I levels following acute exercise.These findings are consistent with the possibility that physical exercise promotes alpha HDL recycling and regeneration of HDL.
It is likely that intravascular lipolysis is a further contributor to the levels of prebeta-1 HDL in plasma.As lipoprotein lipase-mediated lipolysis proceeds, phospholipid, and some small molecular weight apolipoproteins move from the surface monolayers of triglyceride-rich lipoproteins to HDL.Exerciseinduced lipoprotein lipase activity may account for exerciserelated reductions in plasma HDL-triglyceride levels. 53,54Lipoprotein lipases are a family of tissue-specific hydrolytic enzymes that are rate-limiting for the removal of circulating lipoprotein triglycerides and have been implicated in atherogenesis. 55,56Adipose lipoprotein lipase activity, involved in triglyceride storage of acyl groups derived primarily from the hydrolysis of plasma lipoproteins, increases with physical exercise training. 57Acute exercise also increases lipoprotein lipase activity and fat tolerance. 58,59This has been most widely shown in fit individuals performing at high levels of protracted physical exertion, but also has been shown to occur in sedentary individuals exercising for as little as 1 hour at 80% of maximal heart rate. 60These observations have lead to the hypothesis that exercise acutely depletes intramuscular triglycerides, a finding that has recently been confirmed. 61This stimulates the synthesis or translocation of lipoprotein lipase, which hydrolyzes triglycerides from lower-density lipoproteins with transfer of the excess surface cholesterol to the HDL particle. 62Skeletal muscle lipoprotein lipase has become the recent focus of exercise-related effects on plasma lipids and lipoproteins. 63Exercise raises skeletal lipoprotein lipase activity in the capillary beds of skeletal muscles where fatty acids are hydrolyzed from circulating lipoproteins for energy utilization.This could influence plasma HDL-triglyceride metabolism as we have previously shown acute exercise to reduce plasma HDL-triglycerides by 30% in hypertriglyceridemics. 27n conclusion, both acute physical exercise and regular physical exercise training are recognized for their effects of HDL metabolism that can mitigate coronary artery disease risk.Our findings indicate that the effect of acute physical exercise on plasma HDL particles involves increasing prebeta-1 HDL particles.Additional studies are warranted to further evaluate the role of exercise-related effects upon plasma prebeta HDL relating to coronary artery disease risk reduction.
lipid-lowering agents and oral contraceptives), maintaining their usual diets, and able to complete the required exercise protocol signed informed consent that was approved by the Pepperdine University Institutional Review Board.