Atherosclerosis, 91 (1992)31-51 0 1992 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved. 0021-9150/92/$05.00
31
Printed and Published in Ireland
ATHERO 049 I7
Acute physical exercise alters apolipoprotein E and C-III concentrations of apo E-rich very low density lipoprotein fraction Lavy
Klein”, Todd D. Millerb, Teresa E. Radama, Timothy O’Brien”, Tu T. Nguyena and Bruce A. Kottke”
UAtherosclerosis Research Unit and hthe Cardiovascular Health Clinic, Mayo Clinic and Foundation, Rochester, MN 55905 (USA)
(Received 21 February, 1992) (Revised, received 10 June, 1992) (Accepted 29 July, 1992)
Summary
To evaluate the influence of exercise on the apolipoprotein (apo) composition of very low density lipoprotein (VLDL) subfractions, we exercised 6 sedentary men for 30 min, 1 h after a fatty meal. VLDL fractions from samples drawn 4,6 and 8 h post-prandially were separated from pre-stained plasma by high performance liquid chromatography and fractionated to apo E-poor (heparin-unbound) and apo E-rich (heparin-bound) fractions. The postprandial peak area (volts) and apo E, C-II and C-III concentrations (mg/dl) of post-exercise VLDL fractions were compared with corresponding postprandial values obtained at rest. Plasma triglycerides (TG) levels (mg/dl) were significantly lower 4 (P < 0.05), 6 (P c 0.02) and 8 h (p < 0.05) postprandially; the apo E-poor VLDL fraction was not modified by exercise and its apo concentrations were in the low range of detection; the apo E-rich VLDL peak area significantly decreased 4 (P < O.Ol), 6 (P < 0.01) and 8 h (P < 0.05) postprandially; the apo E concentration of apo E-rich VLDL was significantly lower 4 (P c 0.02) and 6 h (P < 0.05) postprandially; the apo C-III concentration of apo E-rich VLDL significantly increased, 4 and 6 h postprandially (P < 0.05). Apo E-rich VLDL is, presumably, the metabolically active fraction of the particle and may regulate plasma TG level following exercise. The metabolic role of apo E-poor VLDL remains to be defined.
Key words: Apo E-poor VLDL fraction; Apo E-rich VLDL fraction; High performance liquid chromatography (HPLC); Size-exclusion separation column; Heparin-affinity column; Sudan Black B
Introduction
Very low density lipoproteins (VLDL) are triglyceride-rich particles that enter the circulation Correspondence to: Lavy Klein, International Medicine C, Rambam Medical Center, Haifa 31096, Israel. Tel.: 972-4-525473; Fax: 972-4-515710.
following synthesis and secretion from the liver. VLDL contains apolipoproteins B, E, C-I, C-II and C-III. Apolipoprotein (apo) B is required for particle formation and is considered to be the major source of apo B in low density lipoprotein (LDL). Apo E comprises about lo-20% of VLDL protein and has an important role in the
38
catabolism of VLDL, through a receptor mediated mechanism [1,2]. Apo C-II comprises 10% of VLDL proteins. Purified apo C-II has cofactor activity for the enzyme lipoprotein lipase, which catalyzes the hydrolysis of triglycerides (TG) in VLDL [3]. Apo C-III comprises about 50% of VLDL protein. Apo C-III has been shown in vitro to inhibit the activity of lipoprotein lipase [4,5]. Thus, degradation of triglyceride-rich VLDL is largely dependent on its apo E, C-II and C-III concentrations. The compositional heterogeneity of VLDL was assessed in several studies, by use of a heparin-affinity column [6-81. VLDL can be separated by heparin-affinity column chromatography into two discrete subfractions, which differ in their apo E content. Heparin-Sepharose chromatography studies of VLDL have shown that the apo E-poor fraction comprises about 40% of total VLDL in untreated type IV and type IIb hypertriglyceridemic patients, but only 25% of VLDL in normal subjects 191. It has been suggested that the apo E-rich subfraction contains metabolically active apo E. Injection of the subfractions to miniature pigs demonstrated that the apo E-poor subfraction was metabolized at onehalf the rate of the apo E-rich subfraction [8]. Physical activity, prolonged or acute, affects plasma triglyceride-rich lipoprotein metabolism [lo- 121. Although most of the studies have shown a reduction of the plasma level of triglyceride-rich lipoproteins after prolonged exercise, there is evidence that a lower level of TG in athletes and
in sedentary subjects is due to an acute exercise effect [ 131. It is believed that the decreased level of TG-rich lipoproteins after prolonged exercise is due to increased plasma lipoprotein lipase activity [14,15], but other studies have failed to detect a change in the activity of the enzyme after shortduration exercise [ 16,171. The purpose of the present study was to evaluate the effect of exercise on apolipoprotein composition of postprandial heparin-affinity VLDL subfractions. In order to avoid any long term influence of physical activity on VLDL composition, we selected sedentary untrained men for the study. Their VLDL fractions were separated from plasma by high performance size-exclusion liquid chromatography and were subsequently fractionated to apo E-poor and apo E-rich fractions on a heparin-affinity column. Material and Methods Subjects
Six healthy consenting adult men were recruited and admitted to the Clinical Research Center at Saint Marys Hospital, Rochester, MN. A sedentary patient was defined as a person who engaged in less than 30 rnin of formal exercise per week during the 6 months prior to the study. Patients with diabetes mellitus, gastrointestinal and liver disease, alcohol consumption, or those using medications known to affect lipoprotein metabolism were excluded. Table 1 summarizes
TABLE 1 SUBJECT CHARACTERISTICS BMI, body mass index. Subject
Age (yrs)
Weight (kg)
Height (cm)
BMI (ks/m2)
% Fata
Apo E phenotype
1
26 32 29 29 31 34
56.1
2 3 4 5 6
72.5 79.6 76.5 14.2 72.0
162 175 180 170 175 180
25.5 23.7 24.6 26.5 24.2 22.2
16.5 21.3 19.8 16.1 19.5 10.8
414 313 313 313 413 313
Mean S.D.
30.2 2.8
71.9 8
173.7 6.8
24.5 1.5
17.3 3.8
aPercentage of fat was estimated from axilla, triceps and suprailium skinfolds, using the skin caliper technique.
39
the subjects’ characteristics upon recruitment into the study, including: age, weight, height, body mass index (BMI, kg/m2), percentage of fat (% fat, the mean value of three different skinfold measurements by the skin caliper technique [ 181) and apo E phenotype. Fasting plasma lipids and apolipoproteins level are shown in Table 2. Study design
On the day of the interview, each subject performed a maximal exercise test on a motorized treadmill (Marquette Electronics, Milwaukee, WI). An incremental exercise protocol was designed to increase energy requirements by 3.5 ml 02/min per kg every 2 min. Continuous electrocardiographic monitoring allowed determination of the exercise heart rate. The participants spent two days at the Clinical Research Center. On the first morning (Day l), each participant consumed a fatty breakfast after 12-14 h of fasting. The fatty meal contained 55 g fat/m2 body surface, consisting of 60% of calories as fat, 25% as carbohydrates and 15% as protein. The breakfast was given as scrambled eggs, cheese, bread and milkshake and was eaten in 10 min. The subjects remain sedentary for the rest of the day. Blood samples were drawn at 0 h (before breakfast), and 4, 6 and 8 h after the meal. The participants did not eat any additional food, until the last blood sample had been drawn. Drinking of tap water was allowed. To reduce inconvenience,
TABLE
2
FASTING PLASMA OF SUBJECTS
LIPIDS
AND APOPROTEINS
TC, total cholesterol;
TG, triglycerides.
(mgidl)
Subject
TC
TG
APO E
APO C-II
APO C-III
1
2 3 4 5 6
176 193 174 154 166 170
62 88 67 75 96 87
4.4 5.4 5.2 4.3 3.1 4.4
2.3 2.3 1.6 1.9 1.1 1.9
17.2 13.6 13.9 14.3 13.2 15.7
Mean SD.
172 13
79 13
4.4 0.8
1.9 0.4
14.6 1.5
blood was drawn from an indwelling plastic catheter, placed in the antecubital vein before breakfast. The catheter was flushed periodically with sterile saline solution. On the morning of the second study day (Day 2), a fasting blood sample was drawn, after which the participants consumed the same fatty breakfast served on Day 1. One hour later each subject exercised for 30 min on a motorized treadmill, at a heart rate corresponding to 75% of his previous peak oxygen consumption. Each exercise session consisted of a 5-min warm-up period, 30 min of exercise and a 5-min cool-down period. The heart rate was monitored during the session by a pulsimeter. After the exercise, a plastic catheter was placed in the antecubital vein of the participant and blood samples were drawn 4, 6 and 8 h after the meal. Analysis of samples Plasma samples. Blood samples were drawn into
tubes containing EDTA. Plasma was separated by centrifugation at 2700 rev./min for 10 min and the protease-inhibitor Aprotinin (Sigma Chemical Co., St. Louis, MO) was added at a concentration of 5&ml. Prior to HPLC analysis, another centrifugation of the samples was done for 10 min at 3000 rev./min, to eliminate residual cellular debris. The floating chylomicrons layer was not collected. The samples were stored at 4°C and subjected to HPLC analysis within 3 days, as recommended by Hughes et al. [19]. Samples staining. A stock solution of the lipophilic dye Sudan Black B (Color index 26150, Fisher Scientific Company, NJ) was prepared following Noble’s method [20], with modification [21]. The dye (0.1 g) was added to 100 ml of 60% ethanol, the mixture was shaken vigorously and allowed to stand for 12 h at 37°C. The undissolved dye was filtered off and the solution was stored in a dark bottle at 4°C and was stable for 2 months. Staining of the plasma was done immediately prior to HPLC analysis, by gently mixing 1 ~1 of the dye with 0.5 ml of plasma. HPLC studies. VLDL was obtained, following a method of separation of lipoprotein fractions from pre-stained plasma using high pressure liquid chromatography (Buithieu et al., unpublished). We used a Shimadzu Bio-Liquid Chromatograph
40
model LC-7A (Shimadzu Scientific Instrument, Columbia, MD), equipped with a system controller (model SCL-6B), an automatic loop injector (model SILdB), a low pressure mixing proportioning valves (model LPMdOO), a US-VIS spectrophotometric detector (model SPD-6AV) and an automatic fraction collector (model SF2120). Plasma separation was accomplished on two 10 X 300 mm size-exclusion separation columns (Superose 6 HR 10/30, Pharmacia LKB Biotechnology, Uppsala, Sweden), connected in series, at a flow rate of 0.67 ml/min. Superose 6 HR has a separation range for proteins from 5000 to 5 000 000 MW, with an average particle size of 13 f 2 pm. The columns were equilibrated with two column volumes (24 ml for each column) of the eluent buffer. Volumes of 0.5 ml from each plasma sample were injected into the HPLC columns and eluted with 0.15 M sodium chloride solution, containing 0.005 M Tris, 0.0005 M EDTA and 0.005% NaNs (buffer A). The buffer was previously filtered using 0.45 pm Millipore filter paper, degassed and aerated continuously with helium. The effluent was monitored at 600 nm, and VLDL, LDL and HDL fractions were detected, separated and automatically collected into plastic tubes containing Aprotinin (5 @ml). Each chromatogram was displayed and stored using a Shimadzu Chromatopac (model C-R4A). Heparin-affinity column studies. The separated VLDL fractions were concentrated from an initial elution volume of 4 ml to a concentrate volume of 50-100 ~1 using centrifugal concentrator/filtrator tubes (RCF-ConFiltiHollow Fiber Bundles, BioMolecular Dynamics, Beaverton, OR). The tubes were centrifuged at 1000 x g in a swinging bucket rotor for 25 min. During recovery, we washed the concentration cap with buffer A, ending with a final volume of 0.5 ml of concentrated VLDL. The samples were restained with Sudan Black B, as the dye, which is dissolved but not bound to the fraction, may be detached from the -particle during centrifugation. The fasting VLDL fractions contained 1.5-2.5 mg of protein and the postprandial VLDL fractions contained 2.5-3.5 mg protein, as determined by Lowry’s method of protein determination [22]. VLDL was fractionated on a ProgelTSK heparin-affinity column (Supelco, Bellefonte, PA) at a flow rate of 1.0 ml/min. The column was
monitored at 600 nm and the fractions were detected and automatically collected into plastic tubes containing Aprotinin (5 @ml). Apo E-poor (heparin-unbound) fraction was eluted first with buffer A, followed by elution of the free dye. When the absorbance of the eluate at 600 nm had decreased to baseline, the column was automatically flushed with 0.8 M NaCl solution containing 0.005 M Tris, 0.0005 M EDTA and 0.005% NaNs (buffer B). This resulted in the elution of the apo E-rich (heparin-bound) fraction. The fractions were concentrated from an initial elution volume of 4.0 ml to a volume of 75 ~1, using the Hollow-Fiber concentrator/filtrator tubes, in a method described previously. The filtration left the concentrated fractions free from the high-salt buffer and from the dye. The fractions were then brought to a final volume of 0.5 ml with buffer A and subjected to apolipoprotein analysis. Lipid and apolipoprotein analysis. Cholesterol and TG were measured in the plasma fractions using enzymatic-spectrophotometric kits (DriSTAT reagent, Cholesterol-ES and Triglycerides GPO, Beckman Instrument, Inc., Carsbad, CA). Apo C-II, C-III and E concentrations were determined using solid-phase polyclonal radioimmunoassays. To decrease interassay variation in radioimmunoassay of the relevant apolipoproteins, we used a quality control plasma sample as a secondary standard [23]. Statistical analysis
The differences between Day 1 and Day 2 results were analyzed for significance with a two-tailed ttest. A P-value < 0.05 was considered significant. Results
The mean f SD. values for fasting plasma level of cholesterol and TG were 172 f 13 and 79 f 13 mg/dl, respectively. The low fasting mean TG level is appropriately correlated with the low mean body weight (mean BMI = 24.5 f 1.5 kg/m2) and low percentage of body fat (mean % fat = 17.3 f 3.8). Postparandial level of cholesterol and TG are summarized in Table 3. Mean plasma TG concentration increased significantly 4, 6 and 8 h postprandialy on Day 1 and to a lesser but still significantly higher level on
41 TABLE
3
FASTING
AND POSTPRANDIAL
PLASMA
LIPID
The values are the mean f S.D. for six subjects. Fasting TC, TC, TG, TG,
Day Day Day Day
1 2 1 2
172 167 79 82
f f zt f
LEVELS,
PP, postprandial;
4 h PP 13 12 13 14
178 168 233 173
zt f f f
DAYS
1 AND 2
TC, total cholesterol;
6 h PP 16 10 23** 4Ott
*P < 0.05, **P < 0.01, fasting vs. postprandial
165 171 199 139 values,
zt f f f
TG, triglycerides.
8 h PP 11 12 21** 20t
166 174 114 84
f f f +
11 II 15; 28tt
Day 1. tP < 0.02, ttP < 0.05, Day 1 vs. Day 2 postprandial
values.
matogram overlay of plasma samples drawn from one participant on Day 1 of the study. The peak areas of the VLDL fractions (in volts) became larger at 4 and 6 h postprandially, compared to their areas at 0 h (fasting sample). The VLDL fraction peak-area enlargement is most probably due to enrichment of the particle with the endogenous load of TG and correlates with the increment of the postprandial plasma TG level. Figure 3 shows the chromatograms obtained on Day 1 and Day 2 of the study from one participant. The VLDL fraction peak area is observed to decrease following the exercise at 4, 6 and 8 h postprandially. This decrease in VLDL fraction peak area is correlated with reduced post-exercise plasma TG levels. A heparin-affinity column chromatogram of
Day 2 (post-exercise). Plasma cholesterol level did not change postprandially, neither was it modified by exercise. The postprandial and post-exercise levels of plasma TG are illustrated in Fig. 4. We have measured the plasma concentrations of apo C-II, C-III and E in the samples drawn from the subjects during the study and did not find a significant change between Day 1 and Day 2 measurements of the apolipoprotein levels (data are not shown). A Superose-HPLC chromatogram of fasting plasma sample is shown in Fig. 1. The lipoprotein fractions VLDL, LDL and HDL + albumin were eluted as peak Nos. 1, 2 and 3, respectively. Peak No. 4 represents free (undissolved) dye and did not contain lipids and apolipoproteins. Figure 2 displays a Superose-HPLC multichro3
0
10
20
30
40 Elution
50 time
60
70
80
90
loo
(min)
Fig. 1. A chromatogram of lipoprotein fractions, separated by HPLC from 0.5 ml plasma drawn from a patient after 14 h of fasting. Peak 1, VLDL; peak 2, LDL; peak 3, HDL + albumin; peak 4, free Sudan Black. Flow rate, 0.67 ml/mm.
42
D
' 1
Day 2 Day 12
0
10
20 30 40 50 Elution time (min)
60
Fig. 2. A multichromatogram overlay of lipoprotein fractions, separated by HPLC from plasma samples drawn from a patient after 14 h fasting (chromatog. A) and 4 h (chromatog. B), 6 h (chromatog. C) and 8 h (chromatog. D) postprandially. Peaks Al, Bl, Cl and Dl, VLDL fractions. Sample volume, 0.5 ml; flow rate, 0.67 ml/mitt.
VLDL, separated from a fasting plasma sample, is shown in Fig. 5. The apo E-poor peak is eluted first, followed by elution of free (undissolved) dye. The third peak represents the apo E-rich fraction, eluted after flushing the column with buffer B. The influence of exogenous TG loading on VLDL subfraction peak-area is shown in Fig. 6. Postprandial hypertriglyceridemia had no predictable influence on the apo E-poor fraction peakarea. In fact, overloading the column with triglyceride-rich VLDL did not change apo E-poor fraction peak-area. On the other hand, apo E-rich fraction peak-area increased during the postprandial period on Day 1 and was correlated with the increment of the postprandial plasma TG level. The exercise session significantly decreased the apo E-rich fraction peak area postprandially on Day 2 of the study (data in Table 3). Figure 7 compares heparin-affinity column studies of VLDL, separated from plasma samples of one participant, on Days 1 and 2. The difference between the mean values (six subjects), of apo E-rich fraction peakarea, measured on Day 1 and on Day 2, is illustrated by Fig. 8.
DaY
1_
Day
Day 1 ElutiOn time (min) Fig. 3. The postprandial chromatograms of lipoprotein fractions, separated by HPLC from plasma samples drawn from a patient on Day 1 and Day 2 of the study. Plasma was drawn 4 h (a), 6 h (b) and 8 h (c) postprandially. Peak 1, Day 1 and Day 2, VLDL. Sample volume, 0.5 ml; flow rate, 0.67 ml/mm.
43
300,
I *Day1
250
*Day
2
is200 z. $50 k 0 $100 c 50
0’
J
0
2
1
3
5
4
Time (postprandial Fig. 4. Postprandial
levels of plasma
triglycerides (mg/dl) during mean f SD. of 6 subjects.
the study,
6
8
7
hour) Day
1 and Day 2 compared.
The values
are the
*P < 0.05, **P < 0.02.
postprandial values). On Day 2, the apo E concentration of apo E-rich fractions was significantly decreased following exercise, 4 h (P < 0.02) 6 h (P < 0.05) and 8 h (P < 0.01) postprandially. The difference between apo E concentrations during Day 1 and Day 2 of the study is shown on Fig. 9.
We measured the concentrations of apo E, C-II and C-III in the apo E-rich VLDL fractions. Table 4 summarizes the mean values f S.E.M. from six subjects. Apo E concentration of apo E-rich fractions significantly increased 4 and 6 h postprandially during Day 1 (P < 0.05, fasting vs. FSB
I
AER
I
0
5
10
15
20 Elution
25
30
time
(min)
35
40
45
50
Fig. 5. A heparin-affinity column chromatogram of VLDL, separated from a fasting plasma sample by HPLC. AEP, apo E-poor VLDL; FSB, free Sudan Black; AER, apo E-rich VLDL. Sample volume, 0.075 ml; flow rate, 1.0 mlimin.
44
0
5
10
15
20
25
30
Elution time (min) Fig. 6. An overlay of heparin-affinity column chromatograms of VLDL, separated by HPLC from plasma samples drawn from a patient after 14 h fasting (chromatog. A) and 4 h (chromatog. B), 6 h (chromatog. C) and 8 h (chromatog. D) postprandially. Al, Bl, Cl and Dl, apo E-poor VLDL; A2, B2, C2 and D2, apo E-rich VLDL. Sample volume, 0.075 ml; flow rate, 1.0 ml/mitt.
The concentrations of apo C-II and apo C-III of the apo E-rich fractions increased significantly 4 and 6 h postprandially, during Days 1 and 2 of the study (P < 0.01, fasting vs. postprandial values). Apo C-III concentration was still significantly increased 8 h postprandially (P < 0.02). Following exercise, significantly higher concentrations of apo C-III, but not of apo C-II, were measured in the apo E-rich fractions 4 and 6 h postprandially (P < 0.01). Apo C-II and C-III concentrations of apo Erich VLDL during the study are shown in Fig. 10. Discussion Shelburne and Quarfordt [24] originally described the separation triglyceride-rich VLDL into two distinct subfractions with different content of an ‘arginine-rich protein’ (later, apo E), using a heparin-column. This observation was made before the functional role of apo E was known. One of the major conclusions from that study was that the interaction of apo E with heparin in vitro may imply an in vivo interaction of apo E with lipoprotein lipase, as the enzyme and
heparin are intimately associated at the vascular endothelium [25]. It was hypothesized that the interaction of apo E with heparin mediates an enzyme (lipoprotein-lipase)-substrate (triglyceriderich VLDL) interaction. Thus, the observation that apo E binds to heparin suggested a function for the apolipoprotein in VLDL catabolism. The physiologic importance of apo E-heparin interaction was emphasized by Weisgraber et al. [26], who demonstrated that the heparin binding domain and the LDL receptor binding domain of apo E are in the same region of the apo E molecule. Injection of VLDL into a heparinaffinity column will result in binding of a VLDL subfraction containing metabolically active apo E. The metabolic significance of VLDL apo Epoor and apo E-rich subfractions is not entirely understood. Fractionation of VLDL, obtained from healthy subjects, on a heparin-Sepharose column, has initially resulted in elution of four subfractions. The subfractions were eluted at different molar concentration of haC1 and differed in chemical composition and apolipoprotein pattern [7]. In vitro studies have indicated that only apo E-
45
Elutfon
time
(min)
Fig. 7. Postprandial heparin-affinity column chromatograms of VLDL, separated by HPLC, from plasma samples drawn from a patient on Day 1 and Day 2 of the study. Plasma was drawn 4 h (a), 6 h (b) and 8 h (c) postprandially. Peak 1, Day I and Day 2. apo E-poor VLDL; peak 2, Day I and Day 2, apo E-rich VLDL. Sample volume, 0.075 ml; flow rate. 1.0 mlimin.
46
700 E c600
A Day1 ADay
0” Z_500 g e 400
“0
12
3
4
5
Time (postprandial
6
7
8
hour)
Fig. 8. The postprandial peak area (volts) of apo E-rich VLDL fractions during the study, Day 1 and Day 2 compared. AERV, apo E-rich VLDL; A, absorbance. The values are the mean of 6 subjects. The S.D. for each value is shown in Table 3. *P < 0.01, **P < 0.05.
rich fractions compete with LDL for LDL receptors. It was suggested that the various subfractions represent VLDL at different stages of catabolism. Huff and Telford [8] have shown that only one bound apo E-rich fraction was eluted with a gradient (0.05-0.8 M) NaCl and that it was metabolized faster than the apo E-poor fraction. They have suggested that each fraction is synthesized and catabolized independently. The presence of apo E in VLDL determines the particle metabolic fate: the apo E-rich fraction is rapidly removed by the liver, while the apo E-poor fraction will be converted to LDL [27]. The metabolic role of apo E-poor VLDL is not clear. This VLDL subfraction has been found to be metabolically inactive [28], and may represent a nascent particle. Apo E-poor VLDL accumulates abnormally in pathologic hypertriglyceridemia. Evans et al. [9] have shown that in type IV and IIb hyperlipidemic patients, there was a substantial contribution made by the accumulation of the apo E-poor fraction to the increased VLDL pool size. In normal individuals, the apo Epoor fraction comprised a significantly smaller proportion of total VLDL. Nestel et al. [6] have suggested that the apo E-poor fraction may be the precursor of the apo E-rich fraction and a conver-
sion of apo E-poor to apo E-rich must occur, prior to its degradation. A significant change in the apo E-poor VLDL peak area, following the exogenous load of fat, could not be chromatographically detected during heparin fractionation of VLDL in our study. This may be due to the fact that we studied normolipidemic volunteers. The concentrations of apo E, C-II and C-III of the apo E-poor fractions (when detected) were below the lower range of our assays. It has been suggested that the amount of VLDL protein injected into the heparin-affinity column determines the fractionation outcome [29]. In various VLDL preparations, separated from baboons’ plasma, 31-91% of the VLDL proteins were bound to the column. In human studies, underloading the column (injection of < 1.25 mg of VLDL protein) may result in binding of all the VLDL to the column and the apo E-poor fraction cannot be detected [9]. In our study, we were close to overloading the column by injecting > 3.0 mg of postprandial VLDL protein, yet most of the time we could not detect a substantial apo E-poor fraction. Therefore, in healthy subjects, apo E-poor VLDL may comprise only a small portion of the VLDL pool, being an intermediate particle in VLDL metabolism. The increase in apo E, C-II and C-III concentra-
AND EXERCISE
247 ?? 155 0.71 f 0.38 0.18 zt 0.08 0.22 f 0.09
238 f 140 0.67 f 0.32 0.2 f 0.09 0.19 ?? 0.09
601 1.18 0.38 0.48
f f f zt
380*** 0.57 0.14*** 0.14***
397 f 243ttt 0.94 ?? 0.4377 0.44 f 0.14 0.64 f 0.12ttt
*P < 0.05, **P < 0.02, ***P < 0.01, fasting vs. Day 1 postprandial values. tP < 0.05, ttP < 0.02, ittP < 0.01, Day 1 vs. Day 2 values.
AER peak area Apo E Apo C-II Apo C-III 584 0.92 0.4 0.56
Dl
Dl
D2
6h
4h
Dl
D2
Postprandial
Fasting
f zt zt f
351 f 175ttt 0.73 ?? 0.23t 0.36 zt 0.18 0.74 + 0.28ttt
D2
CONCENTRATION
370*** 0.55* 0.16*** 0.26***
EFFECT ON PEAK AREA (VOLTS), AND APOLIPOPROTEIN
The values are the mean f S.D. for six subjects. D, Day; AER, Apo E-rich VLDL
POSTPRANDIAL
TABLE 4
287 0.85 0.2 0.34
Dl
8h
zt zt zt f
151 0.6 0.08 0.16**
106 zt 557 0.54 ?? 0.3277 0.16 + 0.08 0.4 f 0.18
D2
(mg/dl) OF APO E-RICH VLDL
1
l*DW 1
0
I
I
1
2
3
4
5
Time (postprandial
6
7
8
hour)
Fig. 9. The postprandial concentration (mg/dl) of apo E, as measured in the apo E-rich VLDL fractions during the study, Day 1 and Day 2 compared. The values are the mean f S.D. of 6 subjects. *P < 0.02, **P < 0.05, ***P< 0.001.
tions of apo E-rich VLDL was significant 4 and 6 h postprandially. This increase of VLDLregulating proteins, following the load of exogenous TG, was most probably due to increased secretion of the apolipoproteins from the liver and
1.2
r
*C-Ill,
Dl
f
C-III,
D2
*
C-II,
*C-II,
redistribution of apo E between HDL and VLDL [30]. We have shown that the apo E-rich fraction of VLDL is the fraction in which the postprandial metabolic changes could be detected chromatographically and analytically, and in which the
Dl D2
0.2
I
0
1
2
3
4
Time (postprandial
5
6
7
8
hour)
Fig. 10. The postprandial concentration (mgdl) of apo C-II and apo C-III, as measured in the apo E-rich VLDL fractions during the study, Day 1 and Day 2 compared. C-II, apo C-II; C-III, apo C-III; Dl, Day 1; D2, Day 2. The values are the mean f SD. of 6 subjects. *P < 0.01.
49
changes in the apolipoprotein concentrations corresponded with the changes in plasma TG level. The duration and mechanism of plasma TG response to physical exercise in sedentary men is a controversial issue, partially due to the heterogenous designs of the numerous studies that have been published in this area. Highly trained athletes have low plasma TG concentration when compared with sedentary controls or the general population [3 I-331. Endurance exercise training frequently induces a decrease in plasma TG [ 15,341, even when the base line levels are elevated [35,36]. In addition, endurance training is associated with a diminished postprandial lipemia in athletes compared with sedentary men [11,37,38]. The magnitude to which plasma TG level will decrease after an acute exercise session is dependent on the pre-exercise values and how long after the exercise the measurements are made. Baseline low plasma TG level remains relatively stable after a short exercise session [39]. In contrast, hypertriglyceridemic subjects have markedly decreased their plasma TG level with only moderate amount of exercise [35,40,41]. We report a significant reduction of plasma postprandial TG level after a short and nonvigorous exercise session. This is due, most likely, to the direct effect of exercise on the major components of the plasma TG removal system, i.e. plasma LPL activity. LPL, which is located at the endothelial surface of muscle and adipose tissue, is the key enzyme for catabolism of the TG-rich lipoproteins [42]. Both the acute and the chronic lowering of plasma TG level have frequently been attributed to an increase in skeletal muscle or adipose tissue LPL activity, or both [43]. In our postprandial exercise study, large amounts of substrate have come into contact with the capillary-bound LPL, as a result of an increased blood flow in the muscles. Following exercise, we have measured significantly lower levels of plasma TG 4, 6 and 8 h postprandially. Since most of the TG in plasma of normolipidemic subjects is carried as VLDLTG, changes of plasma TG reflect the changes in composition of its lipoprotein carrier. A decrease in VLDL-TG concentration has been reported following a prolonged period of vigorous exercise
[44], or repeated bouts of exercise on consecutive days [45]. Others have shown that a reduction in VLDL-TG concentration could be recorded following 1.5 h of exercise and was mainly due to a decrease in the concentration of TG in the VLDL fraction [lo]. Although the plasma levels of apo E, C-II and C-III had not changed after the exercise (a finding consistent with the results of Annuzzi et al. [lo]), we have shown, by fractionation of postprandial VLDL, that exercise resulted in a decreased concentration of apo E and increased concentration of apo C-III in the apo E-rich VLDL fraction, 4 and 6 h postprandially. Thus, the changes which occur in response to exercise could be detected only at the level of the VLDL subfractions. We assume that the decrease in apo E concentration of apo E-rich VLDL is due to increased uptake of the particle through the apo E/ape B/LDL receptor and to transfer of the protein components of VLDL to HDL during VLDL degradation. The increase in apo C-III concentration in the apo Erich VLDL fraction following exercise may result from increased secretion of the protein from the liver, or its redistribution between the two VLDL subfractions. We cannot explain why apo C-II concentration in apo E-rich VLDL was not modified following exercise. In conclusion, the following observations on VLDL metabolism have been noted: (1) The concentrations of apo C-II, C-III and E of the apo E-rich VLDL fraction were modified by an exogenous fat load. (2) Physical exercise altered apo E and C-III concentrations of postprandial apo E-rich VLDL. Apo E-rich VLDL is, most likely, the metabolically active fraction of VLDL. The role and the metabolic fate of the apo E-poor VLDL remain to be determined. Acknowledgment
This work has been supported by a grant from the Merck Foundation and by the Mayo Foundation. Lavy Klein is a recipient of a fellowship from the American Physicians Fellowship for Medicine in Israel. The authors gratefully acknowledge the assistance of Mrs. Nina D. Bren, Brenda J.
50
Hallaway and Mr. Mark A. Wentworth for their excellent technical assistance in lipoprotein assays, and Mrs. Susan Peterson for her secretarial assistance.
14
15 1
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9
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Bradley, W.A., Hwang, S.L.C., Karlin, J.B., Lin, A.H.Y., Prasad, SC., Gotto, A.M., Jr. and Gianturco, S.H., Lowdensity lipoprotein receptor binding determinants switch from apolipoprotein E to apolipoprotein B during conversion of hypertriglyceridemic very-low-density lipoprotein to low-density lipoproteins, J. Biol. Chem., 259 (1984) 14728. Breslow, J.L., Human apolipoprotein molecular biology and genetic variation, Annu. Rev. B&hem., 54 (1985) 699. Breckenridge, WC., Little, J.A., Steiner, G., Chow, A., and Poapst, M., Hypertriglyceridemia associated with deficiency of apolipoprotein C-II, N. Engl. J. Med., 298 (1978) 1265. Brown, W.V., Baginsky, M.L., Inhibition of lipoprotein lipase by an apoprotein of human very low density lipoprotein, Biochem. Biophys. Res. Commun., 46 (1972) 375. Krauss, R.M., Herbert, P.N., Levy, RI. and Fredrickson, D.S., Further observations on the activation and inhibition of lipoprotein lipase by apolipoproteins, Circ. Res., 33 (1973) 403. Nestel, P., Tada, N., Billington, T., Huff, M. and Fidge, N., Changes in very low density lipoproteins with cholesterol loading in man, Metabolism, 31 (1982) 398. Trezzi, E., Calvi, C. and Catapano, A.L., Subfractionation of human low density lipoproteins by heparinSepharose affinity chromatography, J. Lipid Res., 24 (1983) 790. Huff, M.W. and Telford, D.E., Characterization and metabolic fate of two very-low-density lipoprotein subfractions separated by heparin-Sepharose chromatography, B&him. Biophys. Acta, 796 (1984) 251. Evans, A.J., Huff, M.W. and Wolfe, B.M., Accumulation of an apo E-poor subfraction of very low density lipoprotein in hypertriglyceridemic men, J. Lipid Res. (1989) 30, 1691. Annuzzi, G., Jansson, L, Kaijser, L., Holmquist, L. and Carlson, L.A., Increased removal rate of exogenous triglycerides after prolonged exercise in man, time course and effect of exercise duration, Metabolism, 36 (1987) 438. Sady, P.S., Thompson, D.P., Cullinane M.E., Kantor, A.M., Domagala, E. and Herbert, N.P., Prolonged exercise augments plasma triglyceride clearance, J. Am. Med. Assoc., 256 (1986) 2552. Haskell, L.W., The influence of exercise on the concentrations of triglycerides and cholesterol in human plasma, Exercise Sport Sci. Rev., 12 (1984) 205. Cullinane, E., Siconolli, S., Saritelli, A. and Thompson,
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D.P., Acute decrease in serum triglycerides with exercise: is there a threshold for an exercise effect? Metabolism, 3 1 (1982) 844. Taskinen, M.R., Nikkila, E.A., Rehunen, S. and Gordin, A., Effect of acute vigorous exercise on lipoprotein lipase activity of adipose tissue and skeletal muscle in physically active men, Artery, 6 (1980) 471. Weintraub, M.S., Rosen, Y., Otto, R., Eisenberg, S. and Breslow, J.L., Physical exercise conditioning in the absence of weight loss reduces fasting and postprandial triglyceride-rich lipoprotein level, Circulation, 79 (1989) 1007. Mamiemi, J., Peltonen, P., Vuori, I. and Hietanen, E., Triglyceride lipase activities of human postheparin plasma in obese and non-obese subjects in physical exercise, Acta Physiol. Stand., 102 (1978) A38. Lithel, H., Hellsing, K., Lundqvist, G. and Malmberg, P., Lipoprotein-lipase activity of human skeletal muscle and adipose tissue after intensive physical exercise, Acta Physiol. Stand., 105 (1979) 312. Pollack, M.L., Schmidt, D.H. and Jackson, AS., Measurement of cardiorespiratory fitness and body composition in the clinical setting, Compr. Ther., 6 (1980) 12. Hughes, T.A., Moore, M.A., Neame, P., Medely, M.F. and Chung, B.H., Rapid quantitative apolipoprotein analysis by gradient ultracentrifugation and reversedphase high performance liquid chromatography, J. Lipid Res., 29 (1988) 363. Noble, R.P., Electrophoretic separation of plasma lipoproteins in agarose gel, J. Lipid Res., 9 (1968) 693. Mills, G.L., Lane, P.A., A guidebook to lipoprotein technique. In: Burdon, R.H. and van Knippenberg, P.H. (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, Elsvier Science publishers, 1984, pp. 474-475. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurements with the Folin Phenol reagent, J. Biol. Chem., 193 (1951) 265. Au, W.P.T., Hallaway, B.J. and Kottke, B.A., Use of quality-control plasma samples to decrease interassay variation in radioimmunoassay of apolipoprotein A-I, Clin. Chem., 32 (1986) 1394. Shelburne, F.A. and Quarfordt, S.H., The interaction of heparin with an apoprotein of human very low density lipoprotein, J. Clin. Invest., 60 (1977) 944. Kom, E.D., Assay of lipoprotein lipase in vivo and in vitro, Methods Biochem. Anal., 7 (1959) 145. Weisgraber, K.M., Rall, SC., Mahley, R.W., Milne, R.W., Marcel, Y.L. and Sparrow, J.T., Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein Es, J. Biol. Chem., 261 (1986) 2068. Yamada, N., Shames, D.M., Stoudemire, J.B. and Have], R.J., Metabolism of lipoproteins containing apolipoprotein B-100 in blood plasma of rabbits, Heterogeneity related to the presence of apolipoprotein E, Proc. Natl. Acad. Sci. USA, 85 (1986) 3479. Fielding, C.J., Reaven, G.M., Lui, G. and Fielding, P.E.,
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Increased free cholesterol in plasma low and very low density lipoproteins in non-insulin-dependent diabetes mellitus, Its role in the inhibition of cholesteryl ester transfer, Proc. Natl. Acad. Sci. USA, 81 (1984) 2512. Rainwater, D.L. and Kushwaha, R.S., Characterization of very-low-density lipoproteins isolated from baboons and fractionation using heparin-Sepharose chromatography, B&him. Biophys. Acta, 958 (1988) 416. Falko, J.M., Schonfeld, G., Witztum, J.L., Kolar, J.B., Weidman, SW. and Steelman, R., Effects of diet on apo E levels and on the apo E subspecies in human plasma lipoproteins, J. Clin. Endocrinol. Metab., 50 (1980) 521. Martin, R.P., Haskell, W.L. and Wood, P.D., Blood chemistry and lipid profiles of elite distance runners, Ann. N.Y. Acad. Sci., 301 (1977) 346. Wood, P.D., Haskell, W.L., Klein, H., Lewis, S., Stern, M.P. and Farquhar, J.W., The distribution of plasma lipoprotein in middle-aged male runners, Metabolism, I I (1976) 1249. Lehtonen, A. and Viikari, J., Serum triglycerides and cholesterol and serum high density lipoprotein cholesterol in highly physical active men, Acta Med. Stand., 204 (1978) 111. Huttunen, J.K.,, Effect of moderate exercise on serum lipoproteins - Controlled clinical trial with special reference to serum high-density lipoproteins, Circulation, 60 (1979) 1220. Goode, R., Firstbrook, J. and Shepard, R., Effects of exercise and a cholesterol free diet on human serum lipids, Can. J. Physiolol. Pharmacol., 44 (1966) 575. Gyntelberg, F., Brennan, R., Holloszy, J., Schonfeld, G., Rennie, M. and Weidman, S., Plasma triglyceride lowering by exercise despite increased food intake in patients with Type-IV hyperlipoproteinemia, Am. J. Clin. Nutr., 30 (1977) 716.
Cohen, J.C., Noakes, T.D. and Benade, A.J., Postprandial lipemia and chylomicrons clearance in athletes and in sedentary men, Am. J. Clin. Nutr., 49 (1989) 443. 38 Merrill, J.R., Holly, R.J., Anderson, R.L., Rifai, N., King, M.E. and Demeersman, R., Hyperlipidemic response of young trained and untrained men after a high fat meal, Arteriosclerosis, 9 (1989) 217. 39 Hurter, R., Swale, J., Peyman, M.A. and Barnett, C.W.H., Some immediate and long-term effects of exercise on the plasma lipids, Lance& 2 (1972) 671. 40 Holloszy. J.O., Skinner, J.S., Toro, G. and Cureton, T.K., Effects of a six-months program for endurance exercise on serum lipids of middle-aged men, Am. J. Cardiol., 14 (1964) 753. 41 Oscai, L.B., Patterson, J.A., Bogard, D.L., Beck, R.J., and Rothermell, B.L., Normalization of serum triglycerides and lipoprotein electrophoretic patterns by exercise, Am. J. Cardiol., 30 (1972) 755. 42 Eckel, R.H., Lipoprotein lipase - A multifactorial enzyme relevant to common metabolic disease, N. Engl. J. Med., 320 (1989) 1060. 43 Nikkill, E.A., Taskinen, M.R., Rehunen, S. and Hlrkonen, M., Lipoprotein lipase activity in adipose tissue and skeletal muscles of runners, Relation to serum lipoproteins, Metabolism, 27 (1978) 1661. 44 Carlson, L.A. and Mossfeldt, F, Acute effects of prolonged, heavy exercise on the concentrations of plasma lipids and lipoprotein in men, Acta Physiol. Stand., 62 (1964) 51. 45 Carbon, L.A. and Froberg, SO., Blood lipid and glucose levels during a ten day of low-caloric intake and exercise in man, Metab. Clin. Exp., 16 (1971) 624. 37
31
Printed and Published in Ireland
ATHERO 049 I7
Acute physical exercise alters apolipoprotein E and C-III concentrations of apo E-rich very low density lipoprotein fraction Lavy
Klein”, Todd D. Millerb, Teresa E. Radama, Timothy O’Brien”, Tu T. Nguyena and Bruce A. Kottke”
UAtherosclerosis Research Unit and hthe Cardiovascular Health Clinic, Mayo Clinic and Foundation, Rochester, MN 55905 (USA)
(Received 21 February, 1992) (Revised, received 10 June, 1992) (Accepted 29 July, 1992)
Summary
To evaluate the influence of exercise on the apolipoprotein (apo) composition of very low density lipoprotein (VLDL) subfractions, we exercised 6 sedentary men for 30 min, 1 h after a fatty meal. VLDL fractions from samples drawn 4,6 and 8 h post-prandially were separated from pre-stained plasma by high performance liquid chromatography and fractionated to apo E-poor (heparin-unbound) and apo E-rich (heparin-bound) fractions. The postprandial peak area (volts) and apo E, C-II and C-III concentrations (mg/dl) of post-exercise VLDL fractions were compared with corresponding postprandial values obtained at rest. Plasma triglycerides (TG) levels (mg/dl) were significantly lower 4 (P < 0.05), 6 (P c 0.02) and 8 h (p < 0.05) postprandially; the apo E-poor VLDL fraction was not modified by exercise and its apo concentrations were in the low range of detection; the apo E-rich VLDL peak area significantly decreased 4 (P < O.Ol), 6 (P < 0.01) and 8 h (P < 0.05) postprandially; the apo E concentration of apo E-rich VLDL was significantly lower 4 (P c 0.02) and 6 h (P < 0.05) postprandially; the apo C-III concentration of apo E-rich VLDL significantly increased, 4 and 6 h postprandially (P < 0.05). Apo E-rich VLDL is, presumably, the metabolically active fraction of the particle and may regulate plasma TG level following exercise. The metabolic role of apo E-poor VLDL remains to be defined.
Key words: Apo E-poor VLDL fraction; Apo E-rich VLDL fraction; High performance liquid chromatography (HPLC); Size-exclusion separation column; Heparin-affinity column; Sudan Black B
Introduction
Very low density lipoproteins (VLDL) are triglyceride-rich particles that enter the circulation Correspondence to: Lavy Klein, International Medicine C, Rambam Medical Center, Haifa 31096, Israel. Tel.: 972-4-525473; Fax: 972-4-515710.
following synthesis and secretion from the liver. VLDL contains apolipoproteins B, E, C-I, C-II and C-III. Apolipoprotein (apo) B is required for particle formation and is considered to be the major source of apo B in low density lipoprotein (LDL). Apo E comprises about lo-20% of VLDL protein and has an important role in the
38
catabolism of VLDL, through a receptor mediated mechanism [1,2]. Apo C-II comprises 10% of VLDL proteins. Purified apo C-II has cofactor activity for the enzyme lipoprotein lipase, which catalyzes the hydrolysis of triglycerides (TG) in VLDL [3]. Apo C-III comprises about 50% of VLDL protein. Apo C-III has been shown in vitro to inhibit the activity of lipoprotein lipase [4,5]. Thus, degradation of triglyceride-rich VLDL is largely dependent on its apo E, C-II and C-III concentrations. The compositional heterogeneity of VLDL was assessed in several studies, by use of a heparin-affinity column [6-81. VLDL can be separated by heparin-affinity column chromatography into two discrete subfractions, which differ in their apo E content. Heparin-Sepharose chromatography studies of VLDL have shown that the apo E-poor fraction comprises about 40% of total VLDL in untreated type IV and type IIb hypertriglyceridemic patients, but only 25% of VLDL in normal subjects 191. It has been suggested that the apo E-rich subfraction contains metabolically active apo E. Injection of the subfractions to miniature pigs demonstrated that the apo E-poor subfraction was metabolized at onehalf the rate of the apo E-rich subfraction [8]. Physical activity, prolonged or acute, affects plasma triglyceride-rich lipoprotein metabolism [lo- 121. Although most of the studies have shown a reduction of the plasma level of triglyceride-rich lipoproteins after prolonged exercise, there is evidence that a lower level of TG in athletes and
in sedentary subjects is due to an acute exercise effect [ 131. It is believed that the decreased level of TG-rich lipoproteins after prolonged exercise is due to increased plasma lipoprotein lipase activity [14,15], but other studies have failed to detect a change in the activity of the enzyme after shortduration exercise [ 16,171. The purpose of the present study was to evaluate the effect of exercise on apolipoprotein composition of postprandial heparin-affinity VLDL subfractions. In order to avoid any long term influence of physical activity on VLDL composition, we selected sedentary untrained men for the study. Their VLDL fractions were separated from plasma by high performance size-exclusion liquid chromatography and were subsequently fractionated to apo E-poor and apo E-rich fractions on a heparin-affinity column. Material and Methods Subjects
Six healthy consenting adult men were recruited and admitted to the Clinical Research Center at Saint Marys Hospital, Rochester, MN. A sedentary patient was defined as a person who engaged in less than 30 rnin of formal exercise per week during the 6 months prior to the study. Patients with diabetes mellitus, gastrointestinal and liver disease, alcohol consumption, or those using medications known to affect lipoprotein metabolism were excluded. Table 1 summarizes
TABLE 1 SUBJECT CHARACTERISTICS BMI, body mass index. Subject
Age (yrs)
Weight (kg)
Height (cm)
BMI (ks/m2)
% Fata
Apo E phenotype
1
26 32 29 29 31 34
56.1
2 3 4 5 6
72.5 79.6 76.5 14.2 72.0
162 175 180 170 175 180
25.5 23.7 24.6 26.5 24.2 22.2
16.5 21.3 19.8 16.1 19.5 10.8
414 313 313 313 413 313
Mean S.D.
30.2 2.8
71.9 8
173.7 6.8
24.5 1.5
17.3 3.8
aPercentage of fat was estimated from axilla, triceps and suprailium skinfolds, using the skin caliper technique.
39
the subjects’ characteristics upon recruitment into the study, including: age, weight, height, body mass index (BMI, kg/m2), percentage of fat (% fat, the mean value of three different skinfold measurements by the skin caliper technique [ 181) and apo E phenotype. Fasting plasma lipids and apolipoproteins level are shown in Table 2. Study design
On the day of the interview, each subject performed a maximal exercise test on a motorized treadmill (Marquette Electronics, Milwaukee, WI). An incremental exercise protocol was designed to increase energy requirements by 3.5 ml 02/min per kg every 2 min. Continuous electrocardiographic monitoring allowed determination of the exercise heart rate. The participants spent two days at the Clinical Research Center. On the first morning (Day l), each participant consumed a fatty breakfast after 12-14 h of fasting. The fatty meal contained 55 g fat/m2 body surface, consisting of 60% of calories as fat, 25% as carbohydrates and 15% as protein. The breakfast was given as scrambled eggs, cheese, bread and milkshake and was eaten in 10 min. The subjects remain sedentary for the rest of the day. Blood samples were drawn at 0 h (before breakfast), and 4, 6 and 8 h after the meal. The participants did not eat any additional food, until the last blood sample had been drawn. Drinking of tap water was allowed. To reduce inconvenience,
TABLE
2
FASTING PLASMA OF SUBJECTS
LIPIDS
AND APOPROTEINS
TC, total cholesterol;
TG, triglycerides.
(mgidl)
Subject
TC
TG
APO E
APO C-II
APO C-III
1
2 3 4 5 6
176 193 174 154 166 170
62 88 67 75 96 87
4.4 5.4 5.2 4.3 3.1 4.4
2.3 2.3 1.6 1.9 1.1 1.9
17.2 13.6 13.9 14.3 13.2 15.7
Mean SD.
172 13
79 13
4.4 0.8
1.9 0.4
14.6 1.5
blood was drawn from an indwelling plastic catheter, placed in the antecubital vein before breakfast. The catheter was flushed periodically with sterile saline solution. On the morning of the second study day (Day 2), a fasting blood sample was drawn, after which the participants consumed the same fatty breakfast served on Day 1. One hour later each subject exercised for 30 min on a motorized treadmill, at a heart rate corresponding to 75% of his previous peak oxygen consumption. Each exercise session consisted of a 5-min warm-up period, 30 min of exercise and a 5-min cool-down period. The heart rate was monitored during the session by a pulsimeter. After the exercise, a plastic catheter was placed in the antecubital vein of the participant and blood samples were drawn 4, 6 and 8 h after the meal. Analysis of samples Plasma samples. Blood samples were drawn into
tubes containing EDTA. Plasma was separated by centrifugation at 2700 rev./min for 10 min and the protease-inhibitor Aprotinin (Sigma Chemical Co., St. Louis, MO) was added at a concentration of 5&ml. Prior to HPLC analysis, another centrifugation of the samples was done for 10 min at 3000 rev./min, to eliminate residual cellular debris. The floating chylomicrons layer was not collected. The samples were stored at 4°C and subjected to HPLC analysis within 3 days, as recommended by Hughes et al. [19]. Samples staining. A stock solution of the lipophilic dye Sudan Black B (Color index 26150, Fisher Scientific Company, NJ) was prepared following Noble’s method [20], with modification [21]. The dye (0.1 g) was added to 100 ml of 60% ethanol, the mixture was shaken vigorously and allowed to stand for 12 h at 37°C. The undissolved dye was filtered off and the solution was stored in a dark bottle at 4°C and was stable for 2 months. Staining of the plasma was done immediately prior to HPLC analysis, by gently mixing 1 ~1 of the dye with 0.5 ml of plasma. HPLC studies. VLDL was obtained, following a method of separation of lipoprotein fractions from pre-stained plasma using high pressure liquid chromatography (Buithieu et al., unpublished). We used a Shimadzu Bio-Liquid Chromatograph
40
model LC-7A (Shimadzu Scientific Instrument, Columbia, MD), equipped with a system controller (model SCL-6B), an automatic loop injector (model SILdB), a low pressure mixing proportioning valves (model LPMdOO), a US-VIS spectrophotometric detector (model SPD-6AV) and an automatic fraction collector (model SF2120). Plasma separation was accomplished on two 10 X 300 mm size-exclusion separation columns (Superose 6 HR 10/30, Pharmacia LKB Biotechnology, Uppsala, Sweden), connected in series, at a flow rate of 0.67 ml/min. Superose 6 HR has a separation range for proteins from 5000 to 5 000 000 MW, with an average particle size of 13 f 2 pm. The columns were equilibrated with two column volumes (24 ml for each column) of the eluent buffer. Volumes of 0.5 ml from each plasma sample were injected into the HPLC columns and eluted with 0.15 M sodium chloride solution, containing 0.005 M Tris, 0.0005 M EDTA and 0.005% NaNs (buffer A). The buffer was previously filtered using 0.45 pm Millipore filter paper, degassed and aerated continuously with helium. The effluent was monitored at 600 nm, and VLDL, LDL and HDL fractions were detected, separated and automatically collected into plastic tubes containing Aprotinin (5 @ml). Each chromatogram was displayed and stored using a Shimadzu Chromatopac (model C-R4A). Heparin-affinity column studies. The separated VLDL fractions were concentrated from an initial elution volume of 4 ml to a concentrate volume of 50-100 ~1 using centrifugal concentrator/filtrator tubes (RCF-ConFiltiHollow Fiber Bundles, BioMolecular Dynamics, Beaverton, OR). The tubes were centrifuged at 1000 x g in a swinging bucket rotor for 25 min. During recovery, we washed the concentration cap with buffer A, ending with a final volume of 0.5 ml of concentrated VLDL. The samples were restained with Sudan Black B, as the dye, which is dissolved but not bound to the fraction, may be detached from the -particle during centrifugation. The fasting VLDL fractions contained 1.5-2.5 mg of protein and the postprandial VLDL fractions contained 2.5-3.5 mg protein, as determined by Lowry’s method of protein determination [22]. VLDL was fractionated on a ProgelTSK heparin-affinity column (Supelco, Bellefonte, PA) at a flow rate of 1.0 ml/min. The column was
monitored at 600 nm and the fractions were detected and automatically collected into plastic tubes containing Aprotinin (5 @ml). Apo E-poor (heparin-unbound) fraction was eluted first with buffer A, followed by elution of the free dye. When the absorbance of the eluate at 600 nm had decreased to baseline, the column was automatically flushed with 0.8 M NaCl solution containing 0.005 M Tris, 0.0005 M EDTA and 0.005% NaNs (buffer B). This resulted in the elution of the apo E-rich (heparin-bound) fraction. The fractions were concentrated from an initial elution volume of 4.0 ml to a volume of 75 ~1, using the Hollow-Fiber concentrator/filtrator tubes, in a method described previously. The filtration left the concentrated fractions free from the high-salt buffer and from the dye. The fractions were then brought to a final volume of 0.5 ml with buffer A and subjected to apolipoprotein analysis. Lipid and apolipoprotein analysis. Cholesterol and TG were measured in the plasma fractions using enzymatic-spectrophotometric kits (DriSTAT reagent, Cholesterol-ES and Triglycerides GPO, Beckman Instrument, Inc., Carsbad, CA). Apo C-II, C-III and E concentrations were determined using solid-phase polyclonal radioimmunoassays. To decrease interassay variation in radioimmunoassay of the relevant apolipoproteins, we used a quality control plasma sample as a secondary standard [23]. Statistical analysis
The differences between Day 1 and Day 2 results were analyzed for significance with a two-tailed ttest. A P-value < 0.05 was considered significant. Results
The mean f SD. values for fasting plasma level of cholesterol and TG were 172 f 13 and 79 f 13 mg/dl, respectively. The low fasting mean TG level is appropriately correlated with the low mean body weight (mean BMI = 24.5 f 1.5 kg/m2) and low percentage of body fat (mean % fat = 17.3 f 3.8). Postparandial level of cholesterol and TG are summarized in Table 3. Mean plasma TG concentration increased significantly 4, 6 and 8 h postprandialy on Day 1 and to a lesser but still significantly higher level on
41 TABLE
3
FASTING
AND POSTPRANDIAL
PLASMA
LIPID
The values are the mean f S.D. for six subjects. Fasting TC, TC, TG, TG,
Day Day Day Day
1 2 1 2
172 167 79 82
f f zt f
LEVELS,
PP, postprandial;
4 h PP 13 12 13 14
178 168 233 173
zt f f f
DAYS
1 AND 2
TC, total cholesterol;
6 h PP 16 10 23** 4Ott
*P < 0.05, **P < 0.01, fasting vs. postprandial
165 171 199 139 values,
zt f f f
TG, triglycerides.
8 h PP 11 12 21** 20t
166 174 114 84
f f f +
11 II 15; 28tt
Day 1. tP < 0.02, ttP < 0.05, Day 1 vs. Day 2 postprandial
values.
matogram overlay of plasma samples drawn from one participant on Day 1 of the study. The peak areas of the VLDL fractions (in volts) became larger at 4 and 6 h postprandially, compared to their areas at 0 h (fasting sample). The VLDL fraction peak-area enlargement is most probably due to enrichment of the particle with the endogenous load of TG and correlates with the increment of the postprandial plasma TG level. Figure 3 shows the chromatograms obtained on Day 1 and Day 2 of the study from one participant. The VLDL fraction peak area is observed to decrease following the exercise at 4, 6 and 8 h postprandially. This decrease in VLDL fraction peak area is correlated with reduced post-exercise plasma TG levels. A heparin-affinity column chromatogram of
Day 2 (post-exercise). Plasma cholesterol level did not change postprandially, neither was it modified by exercise. The postprandial and post-exercise levels of plasma TG are illustrated in Fig. 4. We have measured the plasma concentrations of apo C-II, C-III and E in the samples drawn from the subjects during the study and did not find a significant change between Day 1 and Day 2 measurements of the apolipoprotein levels (data are not shown). A Superose-HPLC chromatogram of fasting plasma sample is shown in Fig. 1. The lipoprotein fractions VLDL, LDL and HDL + albumin were eluted as peak Nos. 1, 2 and 3, respectively. Peak No. 4 represents free (undissolved) dye and did not contain lipids and apolipoproteins. Figure 2 displays a Superose-HPLC multichro3
0
10
20
30
40 Elution
50 time
60
70
80
90
loo
(min)
Fig. 1. A chromatogram of lipoprotein fractions, separated by HPLC from 0.5 ml plasma drawn from a patient after 14 h of fasting. Peak 1, VLDL; peak 2, LDL; peak 3, HDL + albumin; peak 4, free Sudan Black. Flow rate, 0.67 ml/mm.
42
D
' 1
Day 2 Day 12
0
10
20 30 40 50 Elution time (min)
60
Fig. 2. A multichromatogram overlay of lipoprotein fractions, separated by HPLC from plasma samples drawn from a patient after 14 h fasting (chromatog. A) and 4 h (chromatog. B), 6 h (chromatog. C) and 8 h (chromatog. D) postprandially. Peaks Al, Bl, Cl and Dl, VLDL fractions. Sample volume, 0.5 ml; flow rate, 0.67 ml/mitt.
VLDL, separated from a fasting plasma sample, is shown in Fig. 5. The apo E-poor peak is eluted first, followed by elution of free (undissolved) dye. The third peak represents the apo E-rich fraction, eluted after flushing the column with buffer B. The influence of exogenous TG loading on VLDL subfraction peak-area is shown in Fig. 6. Postprandial hypertriglyceridemia had no predictable influence on the apo E-poor fraction peakarea. In fact, overloading the column with triglyceride-rich VLDL did not change apo E-poor fraction peak-area. On the other hand, apo E-rich fraction peak-area increased during the postprandial period on Day 1 and was correlated with the increment of the postprandial plasma TG level. The exercise session significantly decreased the apo E-rich fraction peak area postprandially on Day 2 of the study (data in Table 3). Figure 7 compares heparin-affinity column studies of VLDL, separated from plasma samples of one participant, on Days 1 and 2. The difference between the mean values (six subjects), of apo E-rich fraction peakarea, measured on Day 1 and on Day 2, is illustrated by Fig. 8.
DaY
1_
Day
Day 1 ElutiOn time (min) Fig. 3. The postprandial chromatograms of lipoprotein fractions, separated by HPLC from plasma samples drawn from a patient on Day 1 and Day 2 of the study. Plasma was drawn 4 h (a), 6 h (b) and 8 h (c) postprandially. Peak 1, Day 1 and Day 2, VLDL. Sample volume, 0.5 ml; flow rate, 0.67 ml/mm.
43
300,
I *Day1
250
*Day
2
is200 z. $50 k 0 $100 c 50
0’
J
0
2
1
3
5
4
Time (postprandial Fig. 4. Postprandial
levels of plasma
triglycerides (mg/dl) during mean f SD. of 6 subjects.
the study,
6
8
7
hour) Day
1 and Day 2 compared.
The values
are the
*P < 0.05, **P < 0.02.
postprandial values). On Day 2, the apo E concentration of apo E-rich fractions was significantly decreased following exercise, 4 h (P < 0.02) 6 h (P < 0.05) and 8 h (P < 0.01) postprandially. The difference between apo E concentrations during Day 1 and Day 2 of the study is shown on Fig. 9.
We measured the concentrations of apo E, C-II and C-III in the apo E-rich VLDL fractions. Table 4 summarizes the mean values f S.E.M. from six subjects. Apo E concentration of apo E-rich fractions significantly increased 4 and 6 h postprandially during Day 1 (P < 0.05, fasting vs. FSB
I
AER
I
0
5
10
15
20 Elution
25
30
time
(min)
35
40
45
50
Fig. 5. A heparin-affinity column chromatogram of VLDL, separated from a fasting plasma sample by HPLC. AEP, apo E-poor VLDL; FSB, free Sudan Black; AER, apo E-rich VLDL. Sample volume, 0.075 ml; flow rate, 1.0 mlimin.
44
0
5
10
15
20
25
30
Elution time (min) Fig. 6. An overlay of heparin-affinity column chromatograms of VLDL, separated by HPLC from plasma samples drawn from a patient after 14 h fasting (chromatog. A) and 4 h (chromatog. B), 6 h (chromatog. C) and 8 h (chromatog. D) postprandially. Al, Bl, Cl and Dl, apo E-poor VLDL; A2, B2, C2 and D2, apo E-rich VLDL. Sample volume, 0.075 ml; flow rate, 1.0 ml/mitt.
The concentrations of apo C-II and apo C-III of the apo E-rich fractions increased significantly 4 and 6 h postprandially, during Days 1 and 2 of the study (P < 0.01, fasting vs. postprandial values). Apo C-III concentration was still significantly increased 8 h postprandially (P < 0.02). Following exercise, significantly higher concentrations of apo C-III, but not of apo C-II, were measured in the apo E-rich fractions 4 and 6 h postprandially (P < 0.01). Apo C-II and C-III concentrations of apo Erich VLDL during the study are shown in Fig. 10. Discussion Shelburne and Quarfordt [24] originally described the separation triglyceride-rich VLDL into two distinct subfractions with different content of an ‘arginine-rich protein’ (later, apo E), using a heparin-column. This observation was made before the functional role of apo E was known. One of the major conclusions from that study was that the interaction of apo E with heparin in vitro may imply an in vivo interaction of apo E with lipoprotein lipase, as the enzyme and
heparin are intimately associated at the vascular endothelium [25]. It was hypothesized that the interaction of apo E with heparin mediates an enzyme (lipoprotein-lipase)-substrate (triglyceriderich VLDL) interaction. Thus, the observation that apo E binds to heparin suggested a function for the apolipoprotein in VLDL catabolism. The physiologic importance of apo E-heparin interaction was emphasized by Weisgraber et al. [26], who demonstrated that the heparin binding domain and the LDL receptor binding domain of apo E are in the same region of the apo E molecule. Injection of VLDL into a heparinaffinity column will result in binding of a VLDL subfraction containing metabolically active apo E. The metabolic significance of VLDL apo Epoor and apo E-rich subfractions is not entirely understood. Fractionation of VLDL, obtained from healthy subjects, on a heparin-Sepharose column, has initially resulted in elution of four subfractions. The subfractions were eluted at different molar concentration of haC1 and differed in chemical composition and apolipoprotein pattern [7]. In vitro studies have indicated that only apo E-
45
Elutfon
time
(min)
Fig. 7. Postprandial heparin-affinity column chromatograms of VLDL, separated by HPLC, from plasma samples drawn from a patient on Day 1 and Day 2 of the study. Plasma was drawn 4 h (a), 6 h (b) and 8 h (c) postprandially. Peak 1, Day I and Day 2. apo E-poor VLDL; peak 2, Day I and Day 2, apo E-rich VLDL. Sample volume, 0.075 ml; flow rate. 1.0 mlimin.
46
700 E c600
A Day1 ADay
0” Z_500 g e 400
“0
12
3
4
5
Time (postprandial
6
7
8
hour)
Fig. 8. The postprandial peak area (volts) of apo E-rich VLDL fractions during the study, Day 1 and Day 2 compared. AERV, apo E-rich VLDL; A, absorbance. The values are the mean of 6 subjects. The S.D. for each value is shown in Table 3. *P < 0.01, **P < 0.05.
rich fractions compete with LDL for LDL receptors. It was suggested that the various subfractions represent VLDL at different stages of catabolism. Huff and Telford [8] have shown that only one bound apo E-rich fraction was eluted with a gradient (0.05-0.8 M) NaCl and that it was metabolized faster than the apo E-poor fraction. They have suggested that each fraction is synthesized and catabolized independently. The presence of apo E in VLDL determines the particle metabolic fate: the apo E-rich fraction is rapidly removed by the liver, while the apo E-poor fraction will be converted to LDL [27]. The metabolic role of apo E-poor VLDL is not clear. This VLDL subfraction has been found to be metabolically inactive [28], and may represent a nascent particle. Apo E-poor VLDL accumulates abnormally in pathologic hypertriglyceridemia. Evans et al. [9] have shown that in type IV and IIb hyperlipidemic patients, there was a substantial contribution made by the accumulation of the apo E-poor fraction to the increased VLDL pool size. In normal individuals, the apo Epoor fraction comprised a significantly smaller proportion of total VLDL. Nestel et al. [6] have suggested that the apo E-poor fraction may be the precursor of the apo E-rich fraction and a conver-
sion of apo E-poor to apo E-rich must occur, prior to its degradation. A significant change in the apo E-poor VLDL peak area, following the exogenous load of fat, could not be chromatographically detected during heparin fractionation of VLDL in our study. This may be due to the fact that we studied normolipidemic volunteers. The concentrations of apo E, C-II and C-III of the apo E-poor fractions (when detected) were below the lower range of our assays. It has been suggested that the amount of VLDL protein injected into the heparin-affinity column determines the fractionation outcome [29]. In various VLDL preparations, separated from baboons’ plasma, 31-91% of the VLDL proteins were bound to the column. In human studies, underloading the column (injection of < 1.25 mg of VLDL protein) may result in binding of all the VLDL to the column and the apo E-poor fraction cannot be detected [9]. In our study, we were close to overloading the column by injecting > 3.0 mg of postprandial VLDL protein, yet most of the time we could not detect a substantial apo E-poor fraction. Therefore, in healthy subjects, apo E-poor VLDL may comprise only a small portion of the VLDL pool, being an intermediate particle in VLDL metabolism. The increase in apo E, C-II and C-III concentra-
AND EXERCISE
247 ?? 155 0.71 f 0.38 0.18 zt 0.08 0.22 f 0.09
238 f 140 0.67 f 0.32 0.2 f 0.09 0.19 ?? 0.09
601 1.18 0.38 0.48
f f f zt
380*** 0.57 0.14*** 0.14***
397 f 243ttt 0.94 ?? 0.4377 0.44 f 0.14 0.64 f 0.12ttt
*P < 0.05, **P < 0.02, ***P < 0.01, fasting vs. Day 1 postprandial values. tP < 0.05, ttP < 0.02, ittP < 0.01, Day 1 vs. Day 2 values.
AER peak area Apo E Apo C-II Apo C-III 584 0.92 0.4 0.56
Dl
Dl
D2
6h
4h
Dl
D2
Postprandial
Fasting
f zt zt f
351 f 175ttt 0.73 ?? 0.23t 0.36 zt 0.18 0.74 + 0.28ttt
D2
CONCENTRATION
370*** 0.55* 0.16*** 0.26***
EFFECT ON PEAK AREA (VOLTS), AND APOLIPOPROTEIN
The values are the mean f S.D. for six subjects. D, Day; AER, Apo E-rich VLDL
POSTPRANDIAL
TABLE 4
287 0.85 0.2 0.34
Dl
8h
zt zt zt f
151 0.6 0.08 0.16**
106 zt 557 0.54 ?? 0.3277 0.16 + 0.08 0.4 f 0.18
D2
(mg/dl) OF APO E-RICH VLDL
1
l*DW 1
0
I
I
1
2
3
4
5
Time (postprandial
6
7
8
hour)
Fig. 9. The postprandial concentration (mg/dl) of apo E, as measured in the apo E-rich VLDL fractions during the study, Day 1 and Day 2 compared. The values are the mean f S.D. of 6 subjects. *P < 0.02, **P < 0.05, ***P< 0.001.
tions of apo E-rich VLDL was significant 4 and 6 h postprandially. This increase of VLDLregulating proteins, following the load of exogenous TG, was most probably due to increased secretion of the apolipoproteins from the liver and
1.2
r
*C-Ill,
Dl
f
C-III,
D2
*
C-II,
*C-II,
redistribution of apo E between HDL and VLDL [30]. We have shown that the apo E-rich fraction of VLDL is the fraction in which the postprandial metabolic changes could be detected chromatographically and analytically, and in which the
Dl D2
0.2
I
0
1
2
3
4
Time (postprandial
5
6
7
8
hour)
Fig. 10. The postprandial concentration (mgdl) of apo C-II and apo C-III, as measured in the apo E-rich VLDL fractions during the study, Day 1 and Day 2 compared. C-II, apo C-II; C-III, apo C-III; Dl, Day 1; D2, Day 2. The values are the mean f SD. of 6 subjects. *P < 0.01.
49
changes in the apolipoprotein concentrations corresponded with the changes in plasma TG level. The duration and mechanism of plasma TG response to physical exercise in sedentary men is a controversial issue, partially due to the heterogenous designs of the numerous studies that have been published in this area. Highly trained athletes have low plasma TG concentration when compared with sedentary controls or the general population [3 I-331. Endurance exercise training frequently induces a decrease in plasma TG [ 15,341, even when the base line levels are elevated [35,36]. In addition, endurance training is associated with a diminished postprandial lipemia in athletes compared with sedentary men [11,37,38]. The magnitude to which plasma TG level will decrease after an acute exercise session is dependent on the pre-exercise values and how long after the exercise the measurements are made. Baseline low plasma TG level remains relatively stable after a short exercise session [39]. In contrast, hypertriglyceridemic subjects have markedly decreased their plasma TG level with only moderate amount of exercise [35,40,41]. We report a significant reduction of plasma postprandial TG level after a short and nonvigorous exercise session. This is due, most likely, to the direct effect of exercise on the major components of the plasma TG removal system, i.e. plasma LPL activity. LPL, which is located at the endothelial surface of muscle and adipose tissue, is the key enzyme for catabolism of the TG-rich lipoproteins [42]. Both the acute and the chronic lowering of plasma TG level have frequently been attributed to an increase in skeletal muscle or adipose tissue LPL activity, or both [43]. In our postprandial exercise study, large amounts of substrate have come into contact with the capillary-bound LPL, as a result of an increased blood flow in the muscles. Following exercise, we have measured significantly lower levels of plasma TG 4, 6 and 8 h postprandially. Since most of the TG in plasma of normolipidemic subjects is carried as VLDLTG, changes of plasma TG reflect the changes in composition of its lipoprotein carrier. A decrease in VLDL-TG concentration has been reported following a prolonged period of vigorous exercise
[44], or repeated bouts of exercise on consecutive days [45]. Others have shown that a reduction in VLDL-TG concentration could be recorded following 1.5 h of exercise and was mainly due to a decrease in the concentration of TG in the VLDL fraction [lo]. Although the plasma levels of apo E, C-II and C-III had not changed after the exercise (a finding consistent with the results of Annuzzi et al. [lo]), we have shown, by fractionation of postprandial VLDL, that exercise resulted in a decreased concentration of apo E and increased concentration of apo C-III in the apo E-rich VLDL fraction, 4 and 6 h postprandially. Thus, the changes which occur in response to exercise could be detected only at the level of the VLDL subfractions. We assume that the decrease in apo E concentration of apo E-rich VLDL is due to increased uptake of the particle through the apo E/ape B/LDL receptor and to transfer of the protein components of VLDL to HDL during VLDL degradation. The increase in apo C-III concentration in the apo Erich VLDL fraction following exercise may result from increased secretion of the protein from the liver, or its redistribution between the two VLDL subfractions. We cannot explain why apo C-II concentration in apo E-rich VLDL was not modified following exercise. In conclusion, the following observations on VLDL metabolism have been noted: (1) The concentrations of apo C-II, C-III and E of the apo E-rich VLDL fraction were modified by an exogenous fat load. (2) Physical exercise altered apo E and C-III concentrations of postprandial apo E-rich VLDL. Apo E-rich VLDL is, most likely, the metabolically active fraction of VLDL. The role and the metabolic fate of the apo E-poor VLDL remain to be determined. Acknowledgment
This work has been supported by a grant from the Merck Foundation and by the Mayo Foundation. Lavy Klein is a recipient of a fellowship from the American Physicians Fellowship for Medicine in Israel. The authors gratefully acknowledge the assistance of Mrs. Nina D. Bren, Brenda J.
50
Hallaway and Mr. Mark A. Wentworth for their excellent technical assistance in lipoprotein assays, and Mrs. Susan Peterson for her secretarial assistance.
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8
9
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