Increase of LDL susceptibility to oxidation occurring after intense, long duration aerobic exercise

ATHEROSCLEROSIS Atherosclerosis

118 (1995) 297-305

Increase of LDL susceptibility to oxidation occurring after intense, long duration aerobic exercise J.L. Sgnchez-Quesada”,“, R. Horns- Serradesanferm”, J. Serrat-Serrat”, J.R. Serra-Grimab, F. Gonz61ez-Sastre”,c, J. Ordbiiez-Llanos”“,’ “Servei de Bioyuimica, Hospiial de la Sanla Creu i Sunt Pau. Avinguda Sam Anioni Ma Claret, 167 08025 Barcelow, Spam bDcparramcw de Cardiologiu. Hospital de la Santa Crew i Sant Pau, AGguda Sam Antoui Ma Claret. 167 08025 Barcelona, Spain ‘Departament de Bioyuimica i Biologiu Mokular, Unicersirat Aurcinomcl de Barcelona. Barcelona. Spain

Received 21 March

1995; revision received 18 May 1995; accepted 31 May 1995

Abstract The elect of heavy, long duration aerobic exercise on low density lipoprotein (LDL) susceptibility to oxidation and on distribution of LDL subfractions was studied. Six well-trained runners, previously fasted, ran continuously for 4 h. Controlled intake of liquid and food was permitted during exercise. Total plasma and LDL triglyceride increased significantly. LDL susceptibility to oxidation, measured as conjugated dienes formation, was modified significantly (P < 0.05) after running (14X reduction in lag phase time, and 8X increase in maximal curve slope). The percentage of electronegative LDL form (named LDLB) also increased significantly (P I 0.05) after exercise both basally (from 7.3%)to 11X) and after 211of induced oxidation (from 40.6’%,to 52.3%)).Neither LDL susceptibility to oxidation nor increase of LDLB was statistically associated with food consumed during the race or modifications of triglycerides suggesting that this effect was due to exercise rather than food-related. The pattern of LDL subfractions was type A in all athletes before and after running. The oxidative LDL changes, seen in exercise conditions similar to those of hard training or competition, demonstrated an unfavourable effect of very intense exercise on lipoprotein metabolism. Krvrcord.s:

LDL susceptibility to oxidation: LDL subfractions: Acute aerobic exercise

major effects of exercise, whatever its intensity, in

1. Introduction

lipoprotein metabolism [2,3]. Slight decreases in It is well established that regular aerobic exercise has some protective effects against atherosclerosis development [l]. A decrease in plasma triglyceride levels, as well as an increase in high density lipoprotein (HDL) cholesterol, are the * Corresponding

author. Fax.: + 34-3-2919196.

LDL

aerobically-trained

OO21-9150’95/$09.50 0 1995ElsevierScience Ireland Ltd. All rights reserved SSDlOO21-9150(95)05617-6

cholesterol

concentrations

(5%- 10%) are

seen as exercise adaptation, but higher diminutions are only observed after repeated, intense exercises[4]. Like others, we have demonstrated a decrease of activity and mass concentration [5,6] of cholesterol ester transfer protein (CETP) in people;

an

increase

of

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1ecitin:cholesterol acyl transferase (LCAT) activity has also been described in these individuals [7]. Recently, predominance of large, light LDL particles has been identified as an adaptative phenomenon related to exercise [8], whereas acute responses of LDL subfraction pattern to exercise appear to be dependent on variations in plasma levels of triglycerides [9,10], which are directly related to the fasting state and intensity of exercise. Large, lighter LDL particles are less susceptible to in vitro oxidation, in opposition to small, denser ones that show an increased susceptibility to oxidative processes[ 11,121.Exercise therefore, seemsto represent a protective mechanism against LDL oxidation. However, during heavy aerobic exercise, 0, consumption rises up to 20-fold, and free radical production is subsequently increased. The natural antioxidant mechanisms [13] may be overcome, and as a result, LDL particles could become oxidized. Oxidized LDL can be atherogenie in several ways, one of which is its uptake by the scavenger receptor, leading to stimulation of cholesterol esterification and foam cell formation [14]. This is apparently contradictory to the well-known protective effects of exercise against atheromatous disease, especially in highly-trained people. To our knowledge there is no data in the literature about the acute effects of intense, aerobic exercise on LDL susceptibility to oxidation. Our aim was to study the susceptibility to in vitro oxidation and the subfraction pattern of LDL particles isolated in well-trained runners after an intense exercise. The exercise was done under conditions of energy and food allowance similar to those during hard training or competitive races. 2. Materials

and methods

2.1. Design of the rue Experimental data were acquired immediately before and after the ‘Vth Scientific Race of the Sant Pau Hospital’ which is a competitive race designed to obtain medical data on participants. Athletes run continuously for 4 h in a 1100 m circuit located inside the hospital, and some voluntarily participate in medical studies. Scientific results obtained in previous editions of the race have been published elsewhere [ 15- 171.

118 (1995) 297-305

2.2. Runners A subgroup of 6 well-trained male aerobic runners, selected from among all participants in the race for their homogeneity in anthropometric measures and endurance level, constituted the experimental group. Mean age was 41.5 f 4.9 years, body mass index (BMI) was 21.3 f 1.2 kg/m2, and maximal 0, consumption (VOZmax) was 556 ml/kg/min. A mean of 47.8 &- 8.5 km was run by athletes (range 38-56 km) during the 4-h period at an average speed of 11.95 km/h. In these conditions, the mean energy expenditure of the race was calculated as 3400 Kcal. All subjects were active competitors in marathon and ultramarathon events and trained between 75 and 135 km/week. Subjects were fasting for at least 10 h prior to the race and were free of any medication. In order to avoid dehydration, orange juice and/ or isotonic carbohydrate containing drinks (100% carbohydrates) were allowed during the race and consumption was recorded. Biscuits containing 65% carbohydrates and 30% fat (10% saturated fat) were also allowed and intake was recorded. Total energy allowance for each participant, as well as food composition, were calculated by the nutritional software package ‘Nutridiet Pro3.0@‘. 2.3. Biochemical and hematological analysis Elementary analysis, including current biochemical and hematological constituents, was conducted in all subjects using automated standard methods. 2.4. Analysis of lipids Serum was obtained from venous blood in Vacutainer tubes. Lipoproteins were isolated by the combined ultracentrifugation-precipitation method, as recommended by the Lipid Research Clinics Laboratory [18]. Cholesterol and triglyceride concentrations from serum and lipoprotein fractions were determined by enzymatic methods (Boehringer Mannheim, Germany) adapted to an RA-XT autoanalyzer (Technicon Instruments, Tarrytown, NY, USA). LDL composition was calculated from plasma-isolated LDL (densities 1025- 1050 g/I), and cholesterol, triglyceride, method, (enzymocolorimetric phospholipid Wako, Japan) and protein content [19] were expressed as % of total LDL mass.

J.L. Srincher-Quesnr

rt al. ! Arllrrosclrrosis

2.5. LDL oxidution studies Isolution of nutive LDL particles. Plasma aliquots were isolated by centrifugation at 1500 x g for 15 min at 4°C from venous blood drawn in 7.5% ethylenediamine tetraacetic acid (EDTA)containing Vacutainer tubes. Plasma was readily supplemented with preserving solution [20], and native LDL (nLDL), free of lipoprotein(a) (Lp(a)) and intermediate density lipoprotein (IDL) (densities 1025- 1050 g/l), was immediately isolated by sequential flotation ultracentrifugation [21]. To avoid oxidative modifications of lipoproteins, ultracentritugation was performed at 4°C with KBr solutions containing 1 mM EDTA. nLDL was dialyzed extensively against degassed 10 mM Tris-HCl buffer pH 7.4, containing 1 mM EDTA (buffer A). and stored at 4°C in the dark. Ident$ication of electronegative subfi)rnu oj nLDL. Electronegative subforms of LDL were isolated from nLDL by chromatography in an anion exchange column (Mono Q 515) with a fast protein liquid chromatography (FPLC) system (Pharmacia Sweden), as described by Vedie et al. [22]. Two LDL forms named LDLA and LDLB, differing in their electronegativity, were identified at 280 nm, and their relative proportion quantified by peak integration. In our laboratory, we have established a within-day imprecision of 1.9% and 3.2% (coefficient of variation of 10 replicates) for LDLA and LDLB separation respectively. Between-day imprecision was 2.4% and 10.2% for LDLA and LDLB, respectively (coefficient of variation of 6 replicates). ‘In vitro’ susceptibility of nLDL to oxidution. nLDL was dialyzed against phosphate buffered saline (PBS) pH 7.4 by gel filtration chromatography in a G-25 Sephadex column (Pharmacia, Sweden), diluted to a concentration of 70 mg of protein/l, and incubated with 1.6 PM CuSO, at 37°C. Conjugated dienes formation of an aliquot of nLDL was determined by continuous monitoring at 234 nm for 4 h according to Esterbauer [23] in a Biochrom 4060 spectrophotometer equipped with a seven-position cell changer (Pharmacia LKB, Sweden). In a parallel aliquot, the oxidative process was followed at fixed time intervals of 2, 4, 8 and 24 h, being stopped by dialysis against buIIer A containing 2 /IM butylated hydroxy-

118 (1995) 297- 305

299

toluene (BHT) in a G-25 Sephadex column. Five forms of LDL with increasing negative charge, named LDLA to LDLE, were identified by anion exchange FPLC after CuSO,-induced LDL oxidation [22]. The LDL forms were identified at 280 nm and quantified by peak integration. 2.4. LDL subfraction distribution LDL subfractions were isolated from plasmaEDTA aliquots according to Griffin et al. [24] with minor modifications. Six LDL subfractions, named LDLl (mean density 1024 g/l), LDL2 (d= 1028) LDL3 (d= 1033), LDL4 (d= 1039) LDLS ((II= 1047) and LDL6 (d= 1056) were isolated by aspiration of 0.8 ml aliquots. The density of each subfraction was measured by assessingits respective potassium concentration. Cholesterol content of subfractions was determined as above described and results expressed as ‘%Iof each LDL subfraction cholesterol with respect to total LDL cholesterol and a ratio ((LDLl + LDL2 + LDL3); (LDL4 + LDLS + LDL6)) was calculated. Data obtained in our laboratory have shown that normolipemic and hypercholesterolemic subjects present a ratio above 1.8, severe hypertriglyceridemic patients (total triglyceride above 5.0 mmol,i 1,) a ratio below 1.1, and mildly hypertriglyceridemic patients (total triglyceride between 2.5 and 5.0 mmoll) a ratio between 1.1 and 1.8 (unpublished observations). Using this ratio we classified an LDL subfraction pattern as A when the ratio exceeded 1.8, as B if the ratio was below 1.1. and as AB if the ratio was between 1.1 and 1.8. Intrassay imprecision was measured by assaying 6 replicates of two different sera presenting A and B LDL patterns. We obtained an intrassay coefficient of variation ranging from 1.2% to 10.6% for the different LDL subfractions. Preand post-race samples corresponding to the same individual were assayed in the same batch. 2.7. Stutistical methods Intraindividual differences were tested using Wilcoxon’s paired T-test. Association between parameters was tested by Spearman’s rank correlation coefficient (Rs). In both cases,a probability I 0.05 was accepted as significant.

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Table 1 Analysis of food intake composition consumed during the race for each runner

Athlete Athlete Athlete Athlete Athlete Athlete

I 2 3 4 5 6

Kcal

‘%,CH”

‘%,Prot”

‘%IFat”

Vit Cb

193 404 124 331 1104 261

76 78 69 74 72 100

6 4 6 6 5 0

18 18 25 20 23 0

100 61 3 106 46 52

I18 (1995)

297-305

ride appeared positively associated with the energy allowance during the race (Rs = 0.94, P = 0.035). No significant changes occurred in serum total, very low density lipoprotein (VLDL), LDL and HDL cholesterol or in the lipid/protein ratio. 3.4. LDL composition Triglyceride content of LDL particles increased significantly (P I 0.05) after the race from 7.6% & 2.9% of LDL mass before to 10.9% + 3.1% after the race, whereas a significant decrease (P I 0.05) in phospholipid content from 29.1% &1.0% to 27.6% f 1.1% also occured (Table 3). Observed modifications were not correlated with energy allowance during the race.

CH, carbohydrates; Prot, proteins; Vit C, vitamin C. “Expressed as ‘% of total Kcal; bExpressed as mg.

3. Results

3.1. Analysis of’ energy and food intake Table 1 shows total energy allowance and composition of liquid and food ingested by runners during the race. Energy intake differed notably among athletes from 124 to 1104 Kcal, mainly apported by carbohydrates (69% to lOO”/oof allowed energy). The only antioxidant food ingested was vitamin C, apported by flavours included in the carbohydrate-enriched beverage consumed during the race, and its intake varied from 3 to 106 mg.

3.5. LDL basal oxidation state The nLDL chromatographic profile presented two LDL forms, named A and B. The proportion of the electronegative form, LDLB increased significantly (P I 0.05) after the exercise from a basal value of 7.3% f 1.7% to 11.O% f 2.7% (Table 4). No significant correlation was observed between allowance of energy or vitamin C ingestion and increase in LDLB. 3.6. LDL susceptibility to oxidation CuSO,-induced in vitro LDL oxidation appeared accelerated at the early stages of the process in all post-race samples. A significant reduction (P I 0.05) of 14% in the lag phase times of conjugated dienes formation (Fig. la) was observed after the race (71.2 f 10.2 min before, 61.8 f 10.2 min after). In addition, all post-race samples showed a significant (P I 0.05) increase of 8% of maximal curve slope of conjugated dienes formation (from 0.028 f 0.005 to

3.2. Biochemical and hematological analysis No significative changes of hematocrit (2.1% decrease), glucose (4.1% decrease) or albumin (2.1% increase) (P > 0.4) were seen. 3.3, Analysis of serum lipids As indicated in Table 2, a significant increase (P I 0.05) of serum triglyceride concentrations from 0.92 _+ 0.35 to 1.25 + 0.28 mmol/l was observed after the exercise. The increase in serum triglyceTable 2 Lipoprotein concentrations of athletes before and after the race

Pre-race Post-race

Chol”

TG

cVLDL

cLDL

cHDL

5.03 f 0.76 5.23 + 0.78

0.92 f 0.35 I .25 & 0.28*

0.29 + 0. I5 0.34 & 0.16

3.18 + 0.75 3.29 i 0.71

1.56+ 0.27 1.60f 0.33

Chol, total serum cholesterol: TG, total serum triglycerides. “Results expressed as mean & SD. in mmol/l. *Significant differences (P I 0.05) as compared to pre-race values.

J. L. Scir~lw~-Qu~~da

et al.

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Athrro.sc~/et~osi.sI18 (I 995) ZW3O-C

Table 3 LDL composition (expressed as ‘%Iof total LDL mass) and LDL subfraction distribution pattern before and after the race

Pre-race Post-race

45.1 * 2.1 45.0 f 2. I

7.6 i 2.9 10.9f 3.1*

‘ii, PH”

‘%,PT

Lipid/PT

Patternh

29.1 * 1.0 27.6 i l.l*

17.7+ 2.9 17.2t 2.7

4.65 4.81

2.93 + 0.94 2.41 & 0.91

Chol. cholesterol; TG, triglyceride; PH. phospholipid: PT. protein. bExpressed as the ratio (LDLI + LDL2 + LDL3).‘(LDL4 + LDL5 + LDL6). *Significant ditferences (P I 0.05) as compared to pre-race values.

0.031 * 0.006) (Fig. lb). In agreement with these results, the proportion of LDL electronegative forms identified by anion exchange chromatography after 2 h of in vitro oxidation showed significant differences (P I 0.05) between pre (LDLA: 59.4%, LDLB: 40.6%) and post-race (LDLA: 47.7%, LDLB: 52.3%) samples (Table 4). Further steps of ‘in vitro’ oxidation (4, 8 and 24 h) showed a non-statistically significant shift towards the accelerated formation of more electronegative forms (C-E) of LDL. No significant association was observed between energy or vitamin C intake and changes in LDL susceptibility to oxidation. 3.7. LDL subfraction distribution

After exercise, a significant decrease(P 6 0.05) of LDLl (from 19.3% to 15.3%) and increase of LDL3 (from 24.5% to 27.3%) subclasseswas observed. However, the ratio ((LDLl + LDL2 + LDL3)/(LDL4 + LDL5 + LDL6)) was 2.93 + 0.94 before and 2.41 * 0.91 after the race, and no statistical differences or change in the LDL subclass pattern from type A to type AB or B was observed. 4. Discussion Results of this study were obtained in a small, but homogeneous group of well-trained runners, in similar conditions to competitive races. We analyzed the acute effect on lipoprotein metabolism of intense exercise. It is worthy to note that during heavy, long duration exercise, energy consumption is required. Marathon and ultramathon runners utilize carbohydrates as energy allowance during exercise in order to delay fatigue ca.usedby decrease in blood glucose and

higher rates of utilization of fat as exercise fuel [25]. For this reason, in a long distance race as studied in this paper, fasting maintenance induces precocious (3 or fewer hours after beginning exercise) fatigue [26]. Results observed in our conditions can therefore differ substantially from those observed in fasting conditions generally used in lipoprotein metabolism studies. However, our results represent the real metabolic status of athletes during long duration exercise. The rise in OZ consumption occurring with exercise promotes an enhancement of free radical production and, hence could induce lipid peroxidation in the cellular membranes and in the lipid moiety of lipoproteins. This latter effect could be involved in the oxidative modification of LDL. Physical exercise acutely promoted changes in circulating LDL particles. A significant increase in the proportion of the LDLB form was measured in nLDL of all runners after exercise. This electronegative form has been described [21,27,28] as mildly oxidized, while LDLA is described as the major form in readily isolated nLDL and considered as the non-modified LDL form. Vedie et al. [22] reported that in vitro-induced oxidation of nLDL increases LDLB and raises another three identifiable forms of oxLDL (named LDLC to LDLE) with increasing negative charge. They showed that these oxLDL forms (LDLB to LDLE) promoted the intracellular accumulation of cholesteryl esters in macrophage cultures, leading to foam cell formation. In our work we observed that LDLB constituted a minor part of readily isolated nLDL, but its proportion in relation to LDLA increased significantly after exercise. A possible explanation for this finding is that increased free radical production associated with

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I18 (1995) 297-305

Table 4 Fast protein liquid chromatographic profiles of native and CuSO,-induced oxidized LDL of six athletes before and after exercise LDL subform

nLDL (‘A,)

Ox LDL (‘X)

Basal”

2h

92.1 (88-95) 7.3 (5-12)

59.4 (O-90) 40.6 (10-100)

4h

8h

97.2 (93-100) 2.8 (O-9)

8.7 (O-52) 76.3 (48-95) 15.0 (0.22)

87.2 (5% 100) 12.8 (O-42)

2.8 (O- 17) 72.2 (28-89) 25.0 (O-71)

24h

Pre-race values

A B C D E

2.3 (0.14) 89.8 (77p 100) 7.9 (O-l 1)

Post-race values

A B C D E

89.0 (87792)* 11.0 (8-13)*

47.7 (o-77)* 52.3 (23- lOO)*

89.6 (63398) 10.4 (I-37)

“Results are expressed as the mean ‘%Iof each isolated LDL subform. The range of the ‘Xl is expressed in parenthesis. *Significant differences (P 5 0.05) as compared to pre-race samples.

aerobic exercise [13] can affect the oxidative state of circulating LDL particles, leading to an increase in LDLB. To our knowledge, this is the first time that such oxidative modifications of LDL particles are observed in association with exercise. In agreement with the increase of circulating LDLB form, LDL susceptibility to CuSO,-induced oxidation appeared enhanced after exercise. Both the lag phase time (14% of decrease) and maximal curve slope of conjugated diene production (8% increase) modified significantly after exercise in all our athletes. In addition, when CuSO,-induced LDL oxidation was stopped in earlier stages (2 h), statistically significant differences in the formation of electronegative LDL forms were observed between pre- and post-race samples, whereas such significant differences were not seen when in vitro oxidation was halted at later stages (4 h or more). These findings indicate that during the heavy, aerobic exercise studied herein, LDL particles increased their susceptibility to oxidation. As the decreasein lag phase time of conjugated diene formation was the most significative change observed, it could be assumed that depletion of antioxidant content is implicated in oxidative modifications of LDL observed postrace. In opposition to the well-known benefical

effects of light-moderate aerobic exercise on lipoprotein metabolism, this increased susceptibility to oxidation of LDL particles seen in our athletes appears to be an undesirable effect of heavy exercise. Consequently, such intense exercise should be performed with caution. In agreement with [29], we also demonstrated some undesirable effects of strenuous exercise on cardiac function and morphology, even in healthy subjects [17]. If these deleterious effects extend to metabolism, such heavy exercise should be discouraged. Secondly, in view of the results of LDL oxidation ability, further experiments must be done to measure the consumption of antioxidants contained on LDL particles during heavy exercises and to assay the possible protective effect of ingested antioxidants (e.g., those contained in some carbohydrate-enriched beverages or in fruit juices) on LDL susceptibility to oxidation. In agreement with results obtained by Goodyear et al. [30] in non-fasted runners after a marathon race, we observed a significant increase in total plasma triglyceride levels after running. A similar increase of triglyceride LDL content after a race was also noted. These data could indicate that changes in oxidation parameters of LDL seen after a race are related to a postprandial situation, rather than to exercise. However, two findings

contradict this relationship. First, all six runners presented a clearly negative energy balance (600 Kcal of allowance, 3400 Kcal of expenditure). In such conditions, food intake will be mainly used to avoid hypoglycemia [26], not to modify lipid concentrations or LDL composition. Secondly, parameters measuring LDL susceptibility to oxidation were statistically unrelated to calories, carbohydrate, fat or vitamin C consumed during the race or plasma triglyceride increase after running.

4 0 LDL 1

LDLZ

LDL3 LDL

t-

a)

Pre-race

LDLd

LDL5

LDL6

subfract~om -*-

Post-race

Fig. 2. Changes in LDL subfractions distribution before (- + -) and after (-O-) the race. *Significant differences (P 5 0.05) as compared to pre-race values.

J Athlete Athlete Athlete Athlete Athlete Athlete mean

1 2 3 4 5 6 l

Pm mce

Post mce

77 79 62 55 79 75

61 77 66 47 66 66

71.2 * 10.2

61.6 * 10.2.

-+-*----SD

pm-race Athlete Athlete Athlete Athlete Athlete Athlete

1 2 3 4 6 6

+ -* --i+ * -

mean ? SD

0,036 0,027 0,031 0,026 0,019 0,026 0.026

f 0.005

Po(It-race 0,036 0.026 0,034 0,027 0,023 0,027 0.030

* 0.005*

Fig. 1. Conjugated dienes formation of pre- and post-race nLDL (0.07 g/l) induced by CuSO, (1.6 PM) incubation at 37°C. (a) Lag phase time (expressed as minutes). (b) Maximal curve slope (expressed as AAbs/minute). *Significant ditherences (P 2 0.05) as compared to pre-race values.

In accordance with these results, the rise in plasma and LDL triglyceride may be related to re-esterification of glycerol and fatty acids coming from tissue-mobilized triglycerides, not to a postprandial effect. So, modifications of LDL oxidability could be attributable to the effect of intense exercise on such particles. The relative abundance of the LDL subclasses defines two well-described patterns, named A and B [31,32]. Pattern B characterized by the existence of smaller, denser LDL particles is associated with an increased risk of arteriosclerosis development. The six runners presented a pre-race LDL pattern A. In spite of the increase of plasma triglyceride after exercise observed changes on LDL subfractions pattern were not significant. The slight shift in LDLl and LDL3 subfractions could be a result of the increase of triglyceride content on LDL particles. In contrast with our data, Lamon-Fava et al. [9] described a dramatic decrease in serum triglyceride concentration after exercise and an acute reduction in dense LDL particles in seven of the athletes analyzed, who presented smaller LDL particles pre-exercise. In our work, all the athletes clearly showed a LDL pattern A. Moreover, the energy expenditure of the exercise in our athletes (that necessary to run 48 km) and those of Lanon-Fava (the energy required to swim 4 km, a bicycle ride of 180 km plus a marathon race of

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J.L. Scinchez-Quesada et al. 1 Atherosclerosis

42 km) differed dramatically. Differences in energy expenditure of exercise and specially in basal LDL pattern of subjects can explain these discrepancies in the effect of heavy exercise on LDL subfraction pattern. In addition to the well described effects on lipid metabolism, light-moderate aerobic exercise is known to develop antioxidant systems in order to prevent oxidative damage. An increase in the concentration of antioxidants and antioxidant activities [33] and in the activity of muscle superoxide dismutase [34], as well as a reduction of the production of lipid peroxide products [35], have been described in such athletes. However, our results, representative of the real metabolic status in some athletes during long-distance races indicate that this type of heavy exercise acutely ensusceptibility to oxidation, hances LDL suggesting caution regarding the supposedly benefical effects of such exercise on lipid metabolism. Further studies are needed to elucidate mechanisms responsible for such oxidative modifications and to ascertain if these can be attenuated or prevented by antioxidant agents. Acknowledgments This work was supported by grants to J.O.L. from the Direction General de Ciencia y Tecnologia (DGICYT DEP 91/0773 and SAF93/03 13) of the Ministerio de Education y Ciencia and to J.O.L. and J.S.S. from the Direccio General de 1’Esport of the Generalitat de Catalunya. We are indebted to Agustina Castellvi-Griso and Carme Burg&-Mauri for their helpful nursing assistance. References [I] Berlin JA, Colditz GA. A meta-analysis of physical activity in the prevention of coronary heart disease, Am J Epidemiol 1990;132612. [2] Naklmiura N, Uzawa H, Haeda H, Inomoto T. Physical fitness, its contribution to serum high density lipoprotein. Atherosclerosis 1983;48:173. [3] Weintraub MS, Rosen Y, Otto R, Eisenberg S, Breslow JL. Physical exercise conditioning in the absence of weight loss reduces fasting and post-prandial triglyceride rich lipoprotein levels. Circulation 1989;79:1007.

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[4] Cullinane E, Siconolfi S, Saritelli A, Thompson PD. Acute decrease in serum triglycerides with exercise: is there threshold for an exercise effect? Metabolism 1982;31:844. [5] Serrat-Serrat J, Ordoiiez-Llanos J, Serra-Grima JR Gomez-Gerique JA, Pelhcer-Thoma E, Payes-Romero A Gonzalez-Sastre F. Marathon runners presented lower serum cholesteryl ester transfer activity than sedentary people. Atherosclerosis 1993;101:43. [6] Seip RL, Moulin P, Cocke T, Tall A, Kohrt WM, Mdnkowitz K, Semenkovich CF, Ostlund R, Schonfeld G. Plasma lipid transfer proteins. Arterioscler Thromb 1993;13:1359. [71 Tsopanakis C, Kotsarellis D, Tsopanakis A. Plasma lecithin:cholesterol acyltransferase in elite athletes from selected sports. Eur J Appl Physiol 1988;58:262. PI Williams PT, Krauss RM, Wood PD, Lindgren FT, Giotas C Vranizan KM. Lipoprotein subfractions of runners and sedentary men. Metabolism 1986;35:45. [91 Lamon-Fava S, McNamara JR, Farber HW, Hill NS, Schaefer EJ. Acute changes in lipid, lipoprotein, apolipoprotein, and low density lipoprotein particle size after an endurance triathlon. Metabolism 1989;38:921. [lOI Baumstark MW, Frey I, Berg A. Acute and delayed etfects of prolonged exercise on serum lipoproteins. II. Concentration and composition of low density lipoproteins subfractions and very low density lipoproteins. Eur J Appl Physiol 1993;66:526. de Graaf J, Hak-Lemmers HLM, Hectors MPC, Demacker PNM Hendriks JCM, Stalenhoef AFH. Enhanced susceptibility to in vitro oxidation of the dense low density lipoprotein subfraction in healthy subjects. Arterioscler Thromb 1991;11:298. 1121Tribble DL, Ho11LG, Wood PD, Krauss RM. Variations in oxidative susceptibility among six low density lipoprotein subfractions of differing density and particle size. Atherosclerosis 1992;93:189. [I31 Li LJ. Antioxidant enzyme response to exercise and aging. Med Sci Sports Exert 1993;25:225. 1141 Steinberg D, Parthasarathy S, Carew TE, Khoo JC Witzturn JL. Beyond cholesterol. Modifications of low density lipoprotein that increase its atherogenicity. N Engl J Med 1989;320:915. 1151Ordoiiez-Llanos J, Serra-Grima JR, Merce-Muntaiiola J Gonzalez-Sastre F. Ratio of creatine kinase 2 mass concentration to total creatine kinase activity not altered by heavy physical exercise. Chn Chem 1992;38:2224. [I61 Wu AHB, Xue-Ming W, Gornet TG, Ordofiez-Llanos J. Creatine kinase MB isoforms in patients with skeletal muscle injury: Ramifications for early detection of acute myocardial infarction. Clin Chem 1992;38:2396. s71 Carrio I, Serra-Grima R, Berna L, Estorch M MartinezDuncker C, Ordofiez J. Transient alterations in cardiac performance after a six-hour race. Am J Cardiol 1990;65:1471. [I81 Lipid Research Clinic Program. Manual of laboratory operations, lipid and lipoprotein analysis. In: Hainline A

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