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Effects of anabolic androgenic steroids on chylomicron metabolism
Steroids 77 (2012) 1321–1326
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Effects of anabolic androgenic steroids on chylomicron metabolism Aleksandra T. Morikawa a, Raul C. Maranhão a,b,⇑, Maria-Janieire N.N. Alves a, Carlos E. Negrão a, Jeferson L. da Silva a, Carmen G.C. Vinagre a a b
Heart Institute (InCor) of the Medical School Hospital, University of São Paulo, São Paulo, Brazil Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 28 September 2011 Received in revised form 15 June 2012 Accepted 7 August 2012 Available online 23 August 2012 Keywords: Exercise training Chylomicron metabolism Intravenous fat emulsions Lipoprotein lipase Hepatic lipase and cholesterol
a b s t r a c t Objective: To evaluate the effects of anabolic androgenic steroids (AAS) on chylomicron metabolism. Methods: An artificial lipid emulsion labeled with radioactive cholesteryl ester (CE) and triglycerides (TG) mimicking chylomicrons was intravenously injected into individuals who regularly weight trained and made regular use of AAS (WT + AAS group), normolipidemic sedentary individuals (SDT group) and individuals who also regularly weight trained but did not use AAS (WT group). Fractional clearance rates (FCR) were determined by compartmental analysis for emulsion plasma decay curves. Results: FCR-CE for the WT + AAS group was reduced (0.0073 ± 0.0079 min 1, 0.0155 ± 0.0100 min 1, 0.0149 ± 0.0160 min 1, respectively; p < 0.05), FCR-TG was similar for both the WT and SDT groups. HDL-C plasma concentrations were lower in the WT + AAS group when compared to the WT and SDT groups (22 ± 13; 41 ± 7; 38 ± 13 mg/dL, respectively; p < 0.001). Hepatic triglyceride lipase activity was greater in the WT + AAS group when compared to the WT and SDT groups (7243 ± 1822; 3898 ± 1232; 2058 ± 749, respectively; p < 0.001). However, no difference was observed for lipoprotein lipase activity. Conclusions: Data strongly suggest that AAS may reduce the removal from the plasma of chylomicron remnants, which are known atherogenic factors. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone and have been used since the 1930s to enhance athletic performance [1]. AAS may adversely affect the serum lipid profile by increasing the serum concentration of atherogenic lowdensity lipoprotein (LDL) cholesterol and apolipoprotein (apo) B [2]. On the other hand, AAS diminishes antiatherogenic highdensity lipoprotein (HDL) cholesterol and apo A1, the main apolipoprotein present in HDL [3–5]. These changes increase the risk of developing coronary artery disease (CAD) in users of AAS [1,3,5]. In respect to serum concentrations of fasting triglycerides that mainly reflect the levels of very-low density lipoprotein (VLDL), the triglyceride-rich lipoprotein produced by the liver, there are studies that show that the use of AAS either elevates triglyceride levels or does not, in fact, alter the levels [2,4]. Intravascular metabolism of the triglyceride-rich lipoproteins, such as VLDL, synthesized in the liver, and chylomicrons, synthetized in the intestine, from the absorbed dietary fats occurs in a two-step process: hydrolysis of the lipoprotein triglycerides by ⇑ Corresponding author. Address: Laboratory of Lipid Metabolism, Heart Institute, Medical School Hospital, University of São Paulo, Eneas de Carvalho Aguiar, 44 São Paulo, Brazil. Fax: +55 11 2661 5574. E-mail address: [email protected] (R.C. Maranhão). 0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.steroids.2012.08.004
lipoprotein lipase (LPL) on the endothelial surface of capillaries and uptake of the remaining remnants by lipoprotein receptors primarily in the liver. Lipolysis is triggered by apolipoprotein CII and the resulting remnants are taken-up by the liver. Hepatic lipase (HL) is also involved in the metabolism of chylomicron remnants, but appears to have little effect on triglyceride hydrolysis. HL is mainly responsible for phospholipase activity and facilitates remnant removal by B, E receptors [6]. A high content of phospholipids on surface of remnant particles leads to a non-exposure of the binding domain to the receptor. Thus, with the decrease of phospholipids, through the action of HL, the access to the receptor is improved which in turn increases binding affinity. HL also plays a role in remnant removal in conjunction with the related LDL receptor protein (LRP), a receptor which appears to be specific for apo E [7,8]. Defects in lipolysis or remnant removal are related to CAD [9]. One of the methods used to assess chylomicron metabolism in subjects is by way of chylomicron like-emulsions doubly labeled with radioactive triglycerides and cholesteryl esters. In this approach, emulsions are injected into the blood stream and labeled triglyceride plasma kinetics is used to trace the lipolysis process. Cholesteryl ester kinetics which is not independently removed from the emulsion particles traces the remnant removal. The kinetics of those emulsions are similar to that of chylomicrons rather than
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the kinetics of VLDL [10,11] since the metabolic pathway is common to both lipoproteins. Thus the method can also be used to trace VLDL metabolism. The current study was designed to verify how the use of AAS interferes with the metabolic pathway of triglyceride-rich lipoproteins described above, which can lead to lipid disorders associated with CAD. Using the doubly-labeled chylomicron-like emulsion method to evaluate this metabolism, weight training practitioners that were addicted to AAS were compared to both non-addicted AAS weight training practitioners and to sedentary subjects. 2. Experimental Twelve male individuals who weight trained, 4–5 times/week for more than three years and used AAS for at least two years (WT + AAS group) participated in the study. They were compared to 7 males who also practiced weight training 4–5 times/week for at least 1 year but were not under AAS use (WT group) and to 12 sedentary non-obese male subjects who made no use of AAS (SDT group). Use of AAS by the WT-AAS group and not used by the WT group was confirmed by the presence or absence of AAS and catabolic products by the method described below [12] in urine samples. Exclusion criteria for all study groups were: diabetes mellitus, cardiac, liver or thyroid disease, previous or current alcohol use and use of hypolipidemic drugs. The study protocol was approved by the Ethics Committee of the University of São Paulo Medical School Hospital. An informed-signed consent was obtained from each participant. 2.1. Urinary AAS AAS detection in urine samples was performed by gas chromatography associated to mass spectrometry Hewlett Packard, models 5972 e 6890, respectively, with Chemstation software for chromatogram and spectrum analysis. Qualitative analysis were performed using a chromatographic system with a Hewlett Packard ultra 1-metilsilicone column (17 m 200 lm 0.11 lm). The detection limit, the less detectable concentration with acceptable precision, was determined by the empiric method of successive dilutions with AAS-containing urine [12]. 2.2. Serum biochemical analysis Total cholesterol and triglycerides were determined using enzymatic test kits (Labtest, São Paulo, Brazil). HDL cholesterol and HDL triglycerides were determined after the precipitation of apo B-containing lipoproteins with MgCl2 and phosphotungstic acid. LDL cholesterol was calculated using the Friedewald equation [13]. Apo A1 and apo B levels were determined by an imunoturbidimetric method using automatic analyzers (COBASINTEGRA, Roche, Basel, Switzerland). Plasma concentration of apo B48 was determined by ELISA (Uscn Life Science, Wuhan, P.R. China). Non-esterified fatty acid (NEFA) plasma concentrations were determined by a colorimetric enzymatic method (NEFAC ACS-ACOD, Wako, Neuss, Germany). Alanine amino transferase (ALT) and aspartate aminotransferase (AST) were determined using kinetic test kit (Labtest, São Paulo, Brazil) and testosterone was determined using radioimmunoassay kits (DPC, Los Angeles, CA). All determinations described above were determined using blood samples taken after a 12 h fast. Blood pressure was monitored from an automatic cuff (DX2710, Dixtal, Manaus, Brazil).
2.3. Emulsion preparation The chylomicron like-emulsion was prepared from emulsified lipid mixtures by ultrasonic irradiation and purified by a two-step ultra-centrifugation in density gradients as described previously [10,11,14]. 14C-cholesteryl ester (cholesteryl[1-14C]oleate) and 3 H-triglycerides (glyceroltri[3H]oleate) (Perkin-Elmer, Waltham, MA) were added to the lipid mixtures for the determination of plasma kinetics. The emulsion was then sterilized by passage through a 0.2 lm filter (Millipore, São Paulo, Brazil) and evaluated for sterility and pyrogenicity prior to injection into the study participants. The final emulsion composition was 76.5% triolein, 1.9% free cholesterol, 11.2% cholesteryl oleate and 10.4% phosphatidylcholine. 2.3.1. Emulsion plasma kinetics After a 12 h overnight fast, blood samples were collected for biochemical analysis. The radiolabeled emulsion was injected in a bolus, (volume of about 100 lL) containing 74 kBq (2 lCi) of the 14C and 148 kBq (4 lCi) of the 3H label into the antecubital vein. Blood samples were collected from the antecubital vein of the contralateral arm, at pre-established intervals of 2, 4, 6, 10, 15, 20, 30, 45 and 60 min after the injection of the emulsion. Radioactivity in aliquots (1 mL) of plasma was measured in a scintillation liquid Ultima Gold™ XR (Packard, Groningen, Holland) using a Packard 1600TR spectrometer (Packard, Meridien, CT). The removal of the emulsion labeled lipids from the plasma is expressed as fractional clearance rate (FCR, in min 1), calculated by compartmental analysis of the plasma decay curves, according to a modification of the model proposed by Redgrave and Zech [15] (Fig. 1). The radiological dose of the labeled emulsion was evaluated according to the International Commission on Radiological Protection guidelines. The dose was well below the Annual Limit for Intake on Radionuclide (20 mSv) as previously discussed [10,11,15]. For each chylomicron-like emulsion kinetic study, the radioactive dose for 14C-cholesteryl ester and 3H-triglycerides was 0.02 and 0.0012 mSv, respectively. 2.4. Area under the curve (AUC) of NEFA generated from emulsion 3Htriglycerides To determine the NEFA originating from the lipolysis of the emulsion labeled triglycerides, plasma aliquots (1.5 mL) obtained in the same pre-determined intervals of the kinetic studies were submitted to lipid extraction [16] and to thin layer chromatography for lipid fraction separation. After separation, the radioactivity in the NEFA band was counted to determine the labeled NEFA curve and to calculate the AUC. 2.5. In vitro post-heparin LPL and HL activities Post-heparin LPL and HL activities in the plasma were determined the day after the kinetic studies, according to Ehnholm and Kuusi’s method [17]. After a 12 h fast, blood samples were collected from the participants 10 min after the intravenous injection of 100 IU heparin/kg body weight (Liquemine, Roche, São Paulo, Brazil). Assays were performed by using the chylomicronlike emulsion labeled with glycerol tri[3H]oleate (Amersham, UK) as the substrate. Plasma samples and the emulsion were incubated at 37 °C and the analysis performed after 5, 10, 15, 30, 45, 60, 120 and 180 min of incubation. Once incubated, the lipids were then extracted and separated by thin layer chromatography. The amount of radioactivity in the band corresponding to the NEFA fraction was measured in a scintillation liquid Ultima GoldTM XR (Packard, Groningen, Holland) using a Packard 1660 TR
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Fig. 1. Compartmental model used to analyze plasma kinetics of a labeled lipid emulsion. In short, five compartments were used to estimate the kinetic parameters for both 3 H-triglycerides (3H-TG) and 14C-cholesteryl ester (14C-CE) tracers. The kx,y constants represent the transfer or fractional catabolic rates from compartment x to compartment y. Compartments 1 and 3 represent the emulsion labeled with 3H-TG and 14C-CE introduced into the intravascular space, respectively. K1,0 and k3,0 represent the extracted fraction of the emulsion from the plasma compartment through a nonspecific pathway; k1,2 and k3,4 represent the emulsion that undergoes lipoprotein lipase activity, transforming into chylomicron remnants which is represented by compartments 2 and 4; k2,0 and k4,0 represent the chylmicron remnant fraction extracted from the plasma compartment, mainly through hepatic uptake; k3,5 and k4,5 represent the free fatty acid fraction generated from triglyceride lipolysis by LLP (compartment 5).
Beta-Spectrometer (Packard, Meridien, CT). The AUC of the NEFA was calculated. 2.6. HDL diameter The HDL size was measured by use of a ZetaPALS Zeta Potential Analyzer (Brookhaven Instruments, Holtsville, USA), as described elsewhere [18]. 2.7. Statistical analysis Parameters of all three groups studied at baseline were tested by One-way ANOVA for multiple comparisons. When differences showed statistical significance (p < 0.05), they were tested with the Bonferroni t test for Gaussian data and Kruskal Wallis for non-Gaussian data [19]. 3. Results As can be seen in Table 1, LDL cholesterol was higher in WT + AAS than in SDT (p = 0.047). However, this difference was not significant when compared to WT. On the other hand, HDL
cholesterol was lower in WT + AAS than in SDT and WT (p < 0.001). Total triglyceride, HDL triglyceride and NEFA concentrations were the same in the all three groups. Differences in apo B concentration among the three groups were not significant. However, apo A1 was lower in WT + AAS group than in SDT and WT groups. Apo B48 concentration was higher in WT + AAS than in SDT and WT groups (p < 0.001). WT + AAS group showed serum values of ALT and AST within the normal range (21 ± 7.7; 22 ± 11.3 U/I). Serum testosterone was also normal in all WT + AAS subjects (524 ± 391 ng/dL).Blood pressure measurements were elevated in WT + AAS group (140/ 85 ± 17/11 mmHg). HDL size was also the same for the WT + AAS, SDT and WT groups (8.9 ± 1.0; 8.6 ± 0.9; 8.7 ± 0.5 nm, respectively, p = 0,335). Fig. 2 shows the plasma decay curve of the 3H-triglyceride emulsion obtained for the WT + ASS group. Apparently there is no different between the SDT and WT group curves. In contrast, the WT + AAS group curve of the 14C-cholesteryl ester emulsion is clearly slower than those of the SDT and WT groups. While there was no difference of the 3H-triglyceride FCR in the three groups, 14 C-cholesteryl ester FCR for the WT + AAS group was about 50% less than that for the SDT and WT groups (Table 2).
Table 1 Physical characteristics and biochemical parameters in weight training AAS users (WT + AAS), sedentary (SDT) and weight training (WT) groups. Parameters
WT + AAS (n = 12)
Age (years) BMI (kg/m2) Cholesterol (mg/dL) Total LDL-C HDL-C Triglycerides (mg/dL) Glucose (mg/dL) Apo A1 (mg/dL) Apo B (mg/dL) Apo B48 (lg/mL) NEFA (mg/dL) HDL-triglycerides (mg/dL)
29 ± 5 28 ± 3a 184 ± 53 143 ± 54b 22 ± 13a 93 ± 71 83 ± 16 88 ± 50a 111 ± 4710 50 ± 2.89a 0.53 ± 0.25 5±4
Data expressed in mean ± SD. BMI = body mass index; NEFA = non-esterified fat acid. a WT + AAS group versus sedentary and WT groups. b WT + AAS group versus sedentary group.
Non-Users
p
SDT (n = 12)
WT (n = 7)
26 ± 5 25 ± 3 160 ± 36 101 ± 31 41 ± 7 96 ± 51 85 ± 11 156 ± 35 86 ± 200 94 ± 0.05 0.39 ± 0.21 5±3
26 ± 4 25 ± 2 168 ± 52 110 ± 52 38 ± 13 104 ± 37 92 ± 21 140 ± 24 86 ± 18 0.98 ± 0.05 0.40 ± 0.24 5±3
0.165 0.004 0.393 0.047 <0.001 0.604 0.372 <0.001 0.161 <0.0001 0.158 0.967
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Fig. 2. Chylomicron-like emulsion 3H-triglycerides (A) and weight training (WT) groups.
14
C-cholesteryl ester (B) plasma decay curves in weight training AAS users (WT + AAS), sedentary (SDT) and
Table 2 Emulsion 3H-triglycerides and 14C-cholesteryl ester fractional clearance rates (FCR) in weight training AAS users (WT + AAS), sedentary (SDT) and weight training (WT) groups. FCR (min
1
)
FCR 3H-TG FCR 14C-CE
WT + AAS (n = 12)
Non-users
p
SDT (n = 12)
WT (n = 7)
0.0254 ± 0.0152 0.0073 ± 0.0080*
0.0335 ± 0.0231 0.0155 ± 0.0100
0.0302 ± 0.0234 0.0164 ± 0.0159
NS <0.03
Data expressed in mean ± SD. NS, no significant. p < 0,03 WT + AAS group versus SDT and WT groups.
groups during increased time periods. Whereas the LPL activity was similar in the three groups, the HL activity was higher in the WT + AAS group than in the SDT and WT groups. AUC’s of the NEFA resulting from 3H-triglyceride hydrolysis in the LPL assay were 9951 ± 4888, 10706 ± 2782, 9495 ± 5723 (p = 0.997). AUC’s of the NEFA resulting from 3H-triglyceride hydrolysis in the HL assay were 7243 ± 1822, 3898 ± 1232, 2058 ± 749 (p < 0.001). 4. Discussion
*
Fig. 3. Non-esterified fatty acids (NEFA) generated from chylomicron-like emulsion triglycerides after an intravenously injected emulsion in a weight training AAS user group (WT + AAS), a sedentary (SDT) group and a weight training (WT) group.
Fig. 3 shows the plasma kinetics of the 3H-NEFA generated from the intravascular lipolysis of the 3H-triglyceride component of the emulsion injected into the subjects of the three study groups. In all three groups, the content of 3H-NEFA increased 20 min after the injection the time it apparently took to reach a plateau. The AUC of the 3H-NEFA found in the plasma and generated from the emulsion showed no difference in the WT + AAS, SDT and WT groups (2366 ± 670; 2283 ± 375; 2741 ± 980, respectively = 0.807). Fig. 4 shows the curves of the assays for LPL and HL activities obtained upon incubation of the chylomicron-like emulsion labeled with 3H-triglycerides with plasma samples of the three study
Hepatic damage, with altered ALT and AST is more frequent in users of per os AAS but the participants of this study used AAS intramuscular route, so that normal hepatic function found here in all WT + AAS subjects was as expected [20]. Due to the feed-back mechanisms in othalamic-pituitary axis, normal levels of total testosterone, as we found here, can also be expected [21,22]. The AAS group also showed elevated blood pressure; this is typical of AAS abusers and is known to be a dose-dependent feature [1]. AAS use resulted in increased LDL cholesterol and decreased HDL-cholesterol concentrations in the plasma which is in agreement with other published studies [1,3,5]. Increased LDL cholesterol by AAS use was ascribed, at least in part, to induction of hepatic triglyceride lipase. This increases VLDL catabolism leading to increased LDL generation [23]. The fact that apo B did not increase together with LDL cholesterol conforms to this assumption, i.e., while LDL apo B increases, apo B of the precursor VLDL decreases. Low levels of apo A1, the main apolipoprotein of HDL reflects the HDL cholesterol decrease in the WT + AAS group. Diminished HDL-cholesterol and apo A1 has also been pointed as a consequence to increased apo A1 catabolism due to induction of hepatic triglyceride lipase [23]. Nonetheless, as the apo A1 catabolism is largely independent from HDL catabolism, with involvement of renal recycling and excretion pathways, low apo1 low levels in AAS users can also be related to factors other than hepatic triglyceride lipase activation. Compared to fat-load tests, in which chylomicron remnant removal is estimated from the plasma kinetics of apo B48 or retinyl palmitate determined in plasma samples collected over several hours, the 1 h emulsion method used here is more practical and comfortable for the participant subjects. This method also has the advantage of bypassing the gastrointestinal tract transit and absorption component which varies largely among individuals. Furthermore, chylomicron competes with VLDL, which accumulates in the post-prandial period and adds-up for difficulties in
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Fig. 4. Activity of LPL (A) and HL (B) indicated by non-esterified fat acids (NEFA) generation curves from emulsion triglycerides hydrolysis in weight training AAS (WT + AAS), weight training (WT) and sedentary (SDT) groups.
interpretation of fat load tests. Straightforward interpretation of the decay curves generated by the labeled emulsion injection conceivably offers a more discriminative device to detect alterations of this metabolism, as shown in several clinical studies [9–11,14]. The results of this study show that AAS use impairs the removal from the plasma of the cholesteryl ester emulsion which in turn indicates that chylomicron remnants accumulate in the circulation of AAS users. This effect was so pronounced that the cholesteryl ester FCR was twice as less in the WT + AAS group when compared to both the SDT or WT groups. In the WT + AAS group the concentration in the plasma of fasting apo B48 was roughly tenfold higher than in SDT or WT, which confirms their pronounced retention of chylomicron remnants in the circulation even after the post-prandial period. Several transversal and prospective studies performed with different methodological approaches have shown that the accumulation of chylomicron remnants in the circulation is associated with the development of CAD [24–26]. With the kinetics emulsion method, we have shown that delayed removal of cholesteryl esters, that stands for remnant accumulation in the plasma, was an independent predictor of CAD and a marker of disease severity and, in a prospective study, also of disease evolution [9–11]. Therefore, remnant accumulation in the WT + AAS group indicates that AAS users bear a risk factor for CAD that had not yet been reported in other studies. The fact that the triglyceride FCR was unchanged in the WT + AAS group indicates that the lipolysis process that chylomicrons undergo in the circulation is not altered in that group. Indeed, in AAS users that practiced exercise training, triglyceridemia was not different from non-AAS users after the ingestion of a fat load as reported by Hislop et al. [27]. As occurred in this study, the WT + AAS group had normal fasting triglyceridemia. Here, the results of the in vitro test for LPL activity were again no different in the WT + AAS and WT groups. Another confirmatory finding was obtained in vivo from an experiment in which the generation of radioactive NEFA from the breakdown of the triglyceride emulsion after an intravenous injection. The fact that the curves of the appearance of labeled fatty acids in the circulation were the same for the three groups, WT + AAS, SDT and WT is consistent with normal triglyceride plasma emulsion decay curves, the normal LPL activity in the in vitro assay and finally, normal triglyceridemia in a demonstration that the lipolytic mechanisms are not altered by AAS use. In contrast with normal LPL activity was the activity of HL which was pronouncedly increased in the WT + AAS group. Increase in HL in administration of high stanozolol doses has been
reported in the literature [28,29]. HL has a phospholipase activity that facilitates lipoprotein uptake by the liver. The increase in the activity of HL accounts for the remarkable 50% decrease in HDL-cholesterol and apo A1 found in the WT + AAS group. Increase in HDL phospholipids degradation leads to increased of HDL clearance and consequently to decreased HDL-cholesterol. The fact that in the WT + AAS group the increase in HL activity did not elicit increased FCR of the cholesteryl ester emulsion is a novel finding. This is a clear-cut demonstration that the excess phospholipase activity of the enzyme that affects HDL catabolism so great that it has no effect on remnant catabolism. As the lipolysis process was unchanged, the decrease in chylomicron remnant removal can be ascribed to effects of AAS use on the mechanisms of remnant uptake. Nevertheless, it is also possible that the alteration in remnant structure produced by excess HL activity impairs the uptake by liver cells. It is interesting to note that LDL-cholesterol levels were increased in AAS users. As chylomicron remnants are also cleared, at least in part, by LDL receptors, it is possible that impairment of the same mechanisms that removes remnants also affects LDL removal from the plasma. In this respect, we showed in previous studies that the LDL cholesterol levels correlated with the FCR of the cholesteryl ester emulsions [9,10]. In fact, chylomicrons, chylomicron remnants, VLDL and LDL share the same catabolic cascade, mediated by LPL, lipoprotein receptors and apolipoproteins. The small number of study participants, due to recruitment difficulties, is a limitation of the study. In conclusion, this study shows that lipid metabolism alterations that contribute to the high incidence of CAD in AAS users are not restricted to increased HL and very low HDL cholesterol levels. An independent novel mechanism was shown here in AAS users, consisting of the delayed removal of chylomicron remnants. This defect may lead to an accumulation in the circulation of those atherogenic lipoproteins in the post-prandial state and which can be a predisposition for the development of CAD. 5. Conflict of interest None declared. Acknowledgments This study was supported by Fundação do Amparo à Pesquisa do Estado de São Paulo (FAPESP, Grant no. 06/58917-3), São Paulo, Brazil. Dr. Maranhão has a Research Award from Conselho Nacional
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Steroids journal homepage: www.elsevier.com/locate/steroids
Effects of anabolic androgenic steroids on chylomicron metabolism Aleksandra T. Morikawa a, Raul C. Maranhão a,b,⇑, Maria-Janieire N.N. Alves a, Carlos E. Negrão a, Jeferson L. da Silva a, Carmen G.C. Vinagre a a b
Heart Institute (InCor) of the Medical School Hospital, University of São Paulo, São Paulo, Brazil Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil
a r t i c l e
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Article history: Received 28 September 2011 Received in revised form 15 June 2012 Accepted 7 August 2012 Available online 23 August 2012 Keywords: Exercise training Chylomicron metabolism Intravenous fat emulsions Lipoprotein lipase Hepatic lipase and cholesterol
a b s t r a c t Objective: To evaluate the effects of anabolic androgenic steroids (AAS) on chylomicron metabolism. Methods: An artificial lipid emulsion labeled with radioactive cholesteryl ester (CE) and triglycerides (TG) mimicking chylomicrons was intravenously injected into individuals who regularly weight trained and made regular use of AAS (WT + AAS group), normolipidemic sedentary individuals (SDT group) and individuals who also regularly weight trained but did not use AAS (WT group). Fractional clearance rates (FCR) were determined by compartmental analysis for emulsion plasma decay curves. Results: FCR-CE for the WT + AAS group was reduced (0.0073 ± 0.0079 min 1, 0.0155 ± 0.0100 min 1, 0.0149 ± 0.0160 min 1, respectively; p < 0.05), FCR-TG was similar for both the WT and SDT groups. HDL-C plasma concentrations were lower in the WT + AAS group when compared to the WT and SDT groups (22 ± 13; 41 ± 7; 38 ± 13 mg/dL, respectively; p < 0.001). Hepatic triglyceride lipase activity was greater in the WT + AAS group when compared to the WT and SDT groups (7243 ± 1822; 3898 ± 1232; 2058 ± 749, respectively; p < 0.001). However, no difference was observed for lipoprotein lipase activity. Conclusions: Data strongly suggest that AAS may reduce the removal from the plasma of chylomicron remnants, which are known atherogenic factors. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone and have been used since the 1930s to enhance athletic performance [1]. AAS may adversely affect the serum lipid profile by increasing the serum concentration of atherogenic lowdensity lipoprotein (LDL) cholesterol and apolipoprotein (apo) B [2]. On the other hand, AAS diminishes antiatherogenic highdensity lipoprotein (HDL) cholesterol and apo A1, the main apolipoprotein present in HDL [3–5]. These changes increase the risk of developing coronary artery disease (CAD) in users of AAS [1,3,5]. In respect to serum concentrations of fasting triglycerides that mainly reflect the levels of very-low density lipoprotein (VLDL), the triglyceride-rich lipoprotein produced by the liver, there are studies that show that the use of AAS either elevates triglyceride levels or does not, in fact, alter the levels [2,4]. Intravascular metabolism of the triglyceride-rich lipoproteins, such as VLDL, synthesized in the liver, and chylomicrons, synthetized in the intestine, from the absorbed dietary fats occurs in a two-step process: hydrolysis of the lipoprotein triglycerides by ⇑ Corresponding author. Address: Laboratory of Lipid Metabolism, Heart Institute, Medical School Hospital, University of São Paulo, Eneas de Carvalho Aguiar, 44 São Paulo, Brazil. Fax: +55 11 2661 5574. E-mail address: [email protected] (R.C. Maranhão). 0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.steroids.2012.08.004
lipoprotein lipase (LPL) on the endothelial surface of capillaries and uptake of the remaining remnants by lipoprotein receptors primarily in the liver. Lipolysis is triggered by apolipoprotein CII and the resulting remnants are taken-up by the liver. Hepatic lipase (HL) is also involved in the metabolism of chylomicron remnants, but appears to have little effect on triglyceride hydrolysis. HL is mainly responsible for phospholipase activity and facilitates remnant removal by B, E receptors [6]. A high content of phospholipids on surface of remnant particles leads to a non-exposure of the binding domain to the receptor. Thus, with the decrease of phospholipids, through the action of HL, the access to the receptor is improved which in turn increases binding affinity. HL also plays a role in remnant removal in conjunction with the related LDL receptor protein (LRP), a receptor which appears to be specific for apo E [7,8]. Defects in lipolysis or remnant removal are related to CAD [9]. One of the methods used to assess chylomicron metabolism in subjects is by way of chylomicron like-emulsions doubly labeled with radioactive triglycerides and cholesteryl esters. In this approach, emulsions are injected into the blood stream and labeled triglyceride plasma kinetics is used to trace the lipolysis process. Cholesteryl ester kinetics which is not independently removed from the emulsion particles traces the remnant removal. The kinetics of those emulsions are similar to that of chylomicrons rather than
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the kinetics of VLDL [10,11] since the metabolic pathway is common to both lipoproteins. Thus the method can also be used to trace VLDL metabolism. The current study was designed to verify how the use of AAS interferes with the metabolic pathway of triglyceride-rich lipoproteins described above, which can lead to lipid disorders associated with CAD. Using the doubly-labeled chylomicron-like emulsion method to evaluate this metabolism, weight training practitioners that were addicted to AAS were compared to both non-addicted AAS weight training practitioners and to sedentary subjects. 2. Experimental Twelve male individuals who weight trained, 4–5 times/week for more than three years and used AAS for at least two years (WT + AAS group) participated in the study. They were compared to 7 males who also practiced weight training 4–5 times/week for at least 1 year but were not under AAS use (WT group) and to 12 sedentary non-obese male subjects who made no use of AAS (SDT group). Use of AAS by the WT-AAS group and not used by the WT group was confirmed by the presence or absence of AAS and catabolic products by the method described below [12] in urine samples. Exclusion criteria for all study groups were: diabetes mellitus, cardiac, liver or thyroid disease, previous or current alcohol use and use of hypolipidemic drugs. The study protocol was approved by the Ethics Committee of the University of São Paulo Medical School Hospital. An informed-signed consent was obtained from each participant. 2.1. Urinary AAS AAS detection in urine samples was performed by gas chromatography associated to mass spectrometry Hewlett Packard, models 5972 e 6890, respectively, with Chemstation software for chromatogram and spectrum analysis. Qualitative analysis were performed using a chromatographic system with a Hewlett Packard ultra 1-metilsilicone column (17 m 200 lm 0.11 lm). The detection limit, the less detectable concentration with acceptable precision, was determined by the empiric method of successive dilutions with AAS-containing urine [12]. 2.2. Serum biochemical analysis Total cholesterol and triglycerides were determined using enzymatic test kits (Labtest, São Paulo, Brazil). HDL cholesterol and HDL triglycerides were determined after the precipitation of apo B-containing lipoproteins with MgCl2 and phosphotungstic acid. LDL cholesterol was calculated using the Friedewald equation [13]. Apo A1 and apo B levels were determined by an imunoturbidimetric method using automatic analyzers (COBASINTEGRA, Roche, Basel, Switzerland). Plasma concentration of apo B48 was determined by ELISA (Uscn Life Science, Wuhan, P.R. China). Non-esterified fatty acid (NEFA) plasma concentrations were determined by a colorimetric enzymatic method (NEFAC ACS-ACOD, Wako, Neuss, Germany). Alanine amino transferase (ALT) and aspartate aminotransferase (AST) were determined using kinetic test kit (Labtest, São Paulo, Brazil) and testosterone was determined using radioimmunoassay kits (DPC, Los Angeles, CA). All determinations described above were determined using blood samples taken after a 12 h fast. Blood pressure was monitored from an automatic cuff (DX2710, Dixtal, Manaus, Brazil).
2.3. Emulsion preparation The chylomicron like-emulsion was prepared from emulsified lipid mixtures by ultrasonic irradiation and purified by a two-step ultra-centrifugation in density gradients as described previously [10,11,14]. 14C-cholesteryl ester (cholesteryl[1-14C]oleate) and 3 H-triglycerides (glyceroltri[3H]oleate) (Perkin-Elmer, Waltham, MA) were added to the lipid mixtures for the determination of plasma kinetics. The emulsion was then sterilized by passage through a 0.2 lm filter (Millipore, São Paulo, Brazil) and evaluated for sterility and pyrogenicity prior to injection into the study participants. The final emulsion composition was 76.5% triolein, 1.9% free cholesterol, 11.2% cholesteryl oleate and 10.4% phosphatidylcholine. 2.3.1. Emulsion plasma kinetics After a 12 h overnight fast, blood samples were collected for biochemical analysis. The radiolabeled emulsion was injected in a bolus, (volume of about 100 lL) containing 74 kBq (2 lCi) of the 14C and 148 kBq (4 lCi) of the 3H label into the antecubital vein. Blood samples were collected from the antecubital vein of the contralateral arm, at pre-established intervals of 2, 4, 6, 10, 15, 20, 30, 45 and 60 min after the injection of the emulsion. Radioactivity in aliquots (1 mL) of plasma was measured in a scintillation liquid Ultima Gold™ XR (Packard, Groningen, Holland) using a Packard 1600TR spectrometer (Packard, Meridien, CT). The removal of the emulsion labeled lipids from the plasma is expressed as fractional clearance rate (FCR, in min 1), calculated by compartmental analysis of the plasma decay curves, according to a modification of the model proposed by Redgrave and Zech [15] (Fig. 1). The radiological dose of the labeled emulsion was evaluated according to the International Commission on Radiological Protection guidelines. The dose was well below the Annual Limit for Intake on Radionuclide (20 mSv) as previously discussed [10,11,15]. For each chylomicron-like emulsion kinetic study, the radioactive dose for 14C-cholesteryl ester and 3H-triglycerides was 0.02 and 0.0012 mSv, respectively. 2.4. Area under the curve (AUC) of NEFA generated from emulsion 3Htriglycerides To determine the NEFA originating from the lipolysis of the emulsion labeled triglycerides, plasma aliquots (1.5 mL) obtained in the same pre-determined intervals of the kinetic studies were submitted to lipid extraction [16] and to thin layer chromatography for lipid fraction separation. After separation, the radioactivity in the NEFA band was counted to determine the labeled NEFA curve and to calculate the AUC. 2.5. In vitro post-heparin LPL and HL activities Post-heparin LPL and HL activities in the plasma were determined the day after the kinetic studies, according to Ehnholm and Kuusi’s method [17]. After a 12 h fast, blood samples were collected from the participants 10 min after the intravenous injection of 100 IU heparin/kg body weight (Liquemine, Roche, São Paulo, Brazil). Assays were performed by using the chylomicronlike emulsion labeled with glycerol tri[3H]oleate (Amersham, UK) as the substrate. Plasma samples and the emulsion were incubated at 37 °C and the analysis performed after 5, 10, 15, 30, 45, 60, 120 and 180 min of incubation. Once incubated, the lipids were then extracted and separated by thin layer chromatography. The amount of radioactivity in the band corresponding to the NEFA fraction was measured in a scintillation liquid Ultima GoldTM XR (Packard, Groningen, Holland) using a Packard 1660 TR
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Fig. 1. Compartmental model used to analyze plasma kinetics of a labeled lipid emulsion. In short, five compartments were used to estimate the kinetic parameters for both 3 H-triglycerides (3H-TG) and 14C-cholesteryl ester (14C-CE) tracers. The kx,y constants represent the transfer or fractional catabolic rates from compartment x to compartment y. Compartments 1 and 3 represent the emulsion labeled with 3H-TG and 14C-CE introduced into the intravascular space, respectively. K1,0 and k3,0 represent the extracted fraction of the emulsion from the plasma compartment through a nonspecific pathway; k1,2 and k3,4 represent the emulsion that undergoes lipoprotein lipase activity, transforming into chylomicron remnants which is represented by compartments 2 and 4; k2,0 and k4,0 represent the chylmicron remnant fraction extracted from the plasma compartment, mainly through hepatic uptake; k3,5 and k4,5 represent the free fatty acid fraction generated from triglyceride lipolysis by LLP (compartment 5).
Beta-Spectrometer (Packard, Meridien, CT). The AUC of the NEFA was calculated. 2.6. HDL diameter The HDL size was measured by use of a ZetaPALS Zeta Potential Analyzer (Brookhaven Instruments, Holtsville, USA), as described elsewhere [18]. 2.7. Statistical analysis Parameters of all three groups studied at baseline were tested by One-way ANOVA for multiple comparisons. When differences showed statistical significance (p < 0.05), they were tested with the Bonferroni t test for Gaussian data and Kruskal Wallis for non-Gaussian data [19]. 3. Results As can be seen in Table 1, LDL cholesterol was higher in WT + AAS than in SDT (p = 0.047). However, this difference was not significant when compared to WT. On the other hand, HDL
cholesterol was lower in WT + AAS than in SDT and WT (p < 0.001). Total triglyceride, HDL triglyceride and NEFA concentrations were the same in the all three groups. Differences in apo B concentration among the three groups were not significant. However, apo A1 was lower in WT + AAS group than in SDT and WT groups. Apo B48 concentration was higher in WT + AAS than in SDT and WT groups (p < 0.001). WT + AAS group showed serum values of ALT and AST within the normal range (21 ± 7.7; 22 ± 11.3 U/I). Serum testosterone was also normal in all WT + AAS subjects (524 ± 391 ng/dL).Blood pressure measurements were elevated in WT + AAS group (140/ 85 ± 17/11 mmHg). HDL size was also the same for the WT + AAS, SDT and WT groups (8.9 ± 1.0; 8.6 ± 0.9; 8.7 ± 0.5 nm, respectively, p = 0,335). Fig. 2 shows the plasma decay curve of the 3H-triglyceride emulsion obtained for the WT + ASS group. Apparently there is no different between the SDT and WT group curves. In contrast, the WT + AAS group curve of the 14C-cholesteryl ester emulsion is clearly slower than those of the SDT and WT groups. While there was no difference of the 3H-triglyceride FCR in the three groups, 14 C-cholesteryl ester FCR for the WT + AAS group was about 50% less than that for the SDT and WT groups (Table 2).
Table 1 Physical characteristics and biochemical parameters in weight training AAS users (WT + AAS), sedentary (SDT) and weight training (WT) groups. Parameters
WT + AAS (n = 12)
Age (years) BMI (kg/m2) Cholesterol (mg/dL) Total LDL-C HDL-C Triglycerides (mg/dL) Glucose (mg/dL) Apo A1 (mg/dL) Apo B (mg/dL) Apo B48 (lg/mL) NEFA (mg/dL) HDL-triglycerides (mg/dL)
29 ± 5 28 ± 3a 184 ± 53 143 ± 54b 22 ± 13a 93 ± 71 83 ± 16 88 ± 50a 111 ± 4710 50 ± 2.89a 0.53 ± 0.25 5±4
Data expressed in mean ± SD. BMI = body mass index; NEFA = non-esterified fat acid. a WT + AAS group versus sedentary and WT groups. b WT + AAS group versus sedentary group.
Non-Users
p
SDT (n = 12)
WT (n = 7)
26 ± 5 25 ± 3 160 ± 36 101 ± 31 41 ± 7 96 ± 51 85 ± 11 156 ± 35 86 ± 200 94 ± 0.05 0.39 ± 0.21 5±3
26 ± 4 25 ± 2 168 ± 52 110 ± 52 38 ± 13 104 ± 37 92 ± 21 140 ± 24 86 ± 18 0.98 ± 0.05 0.40 ± 0.24 5±3
0.165 0.004 0.393 0.047 <0.001 0.604 0.372 <0.001 0.161 <0.0001 0.158 0.967
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Fig. 2. Chylomicron-like emulsion 3H-triglycerides (A) and weight training (WT) groups.
14
C-cholesteryl ester (B) plasma decay curves in weight training AAS users (WT + AAS), sedentary (SDT) and
Table 2 Emulsion 3H-triglycerides and 14C-cholesteryl ester fractional clearance rates (FCR) in weight training AAS users (WT + AAS), sedentary (SDT) and weight training (WT) groups. FCR (min
1
)
FCR 3H-TG FCR 14C-CE
WT + AAS (n = 12)
Non-users
p
SDT (n = 12)
WT (n = 7)
0.0254 ± 0.0152 0.0073 ± 0.0080*
0.0335 ± 0.0231 0.0155 ± 0.0100
0.0302 ± 0.0234 0.0164 ± 0.0159
NS <0.03
Data expressed in mean ± SD. NS, no significant. p < 0,03 WT + AAS group versus SDT and WT groups.
groups during increased time periods. Whereas the LPL activity was similar in the three groups, the HL activity was higher in the WT + AAS group than in the SDT and WT groups. AUC’s of the NEFA resulting from 3H-triglyceride hydrolysis in the LPL assay were 9951 ± 4888, 10706 ± 2782, 9495 ± 5723 (p = 0.997). AUC’s of the NEFA resulting from 3H-triglyceride hydrolysis in the HL assay were 7243 ± 1822, 3898 ± 1232, 2058 ± 749 (p < 0.001). 4. Discussion
*
Fig. 3. Non-esterified fatty acids (NEFA) generated from chylomicron-like emulsion triglycerides after an intravenously injected emulsion in a weight training AAS user group (WT + AAS), a sedentary (SDT) group and a weight training (WT) group.
Fig. 3 shows the plasma kinetics of the 3H-NEFA generated from the intravascular lipolysis of the 3H-triglyceride component of the emulsion injected into the subjects of the three study groups. In all three groups, the content of 3H-NEFA increased 20 min after the injection the time it apparently took to reach a plateau. The AUC of the 3H-NEFA found in the plasma and generated from the emulsion showed no difference in the WT + AAS, SDT and WT groups (2366 ± 670; 2283 ± 375; 2741 ± 980, respectively = 0.807). Fig. 4 shows the curves of the assays for LPL and HL activities obtained upon incubation of the chylomicron-like emulsion labeled with 3H-triglycerides with plasma samples of the three study
Hepatic damage, with altered ALT and AST is more frequent in users of per os AAS but the participants of this study used AAS intramuscular route, so that normal hepatic function found here in all WT + AAS subjects was as expected [20]. Due to the feed-back mechanisms in othalamic-pituitary axis, normal levels of total testosterone, as we found here, can also be expected [21,22]. The AAS group also showed elevated blood pressure; this is typical of AAS abusers and is known to be a dose-dependent feature [1]. AAS use resulted in increased LDL cholesterol and decreased HDL-cholesterol concentrations in the plasma which is in agreement with other published studies [1,3,5]. Increased LDL cholesterol by AAS use was ascribed, at least in part, to induction of hepatic triglyceride lipase. This increases VLDL catabolism leading to increased LDL generation [23]. The fact that apo B did not increase together with LDL cholesterol conforms to this assumption, i.e., while LDL apo B increases, apo B of the precursor VLDL decreases. Low levels of apo A1, the main apolipoprotein of HDL reflects the HDL cholesterol decrease in the WT + AAS group. Diminished HDL-cholesterol and apo A1 has also been pointed as a consequence to increased apo A1 catabolism due to induction of hepatic triglyceride lipase [23]. Nonetheless, as the apo A1 catabolism is largely independent from HDL catabolism, with involvement of renal recycling and excretion pathways, low apo1 low levels in AAS users can also be related to factors other than hepatic triglyceride lipase activation. Compared to fat-load tests, in which chylomicron remnant removal is estimated from the plasma kinetics of apo B48 or retinyl palmitate determined in plasma samples collected over several hours, the 1 h emulsion method used here is more practical and comfortable for the participant subjects. This method also has the advantage of bypassing the gastrointestinal tract transit and absorption component which varies largely among individuals. Furthermore, chylomicron competes with VLDL, which accumulates in the post-prandial period and adds-up for difficulties in
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Fig. 4. Activity of LPL (A) and HL (B) indicated by non-esterified fat acids (NEFA) generation curves from emulsion triglycerides hydrolysis in weight training AAS (WT + AAS), weight training (WT) and sedentary (SDT) groups.
interpretation of fat load tests. Straightforward interpretation of the decay curves generated by the labeled emulsion injection conceivably offers a more discriminative device to detect alterations of this metabolism, as shown in several clinical studies [9–11,14]. The results of this study show that AAS use impairs the removal from the plasma of the cholesteryl ester emulsion which in turn indicates that chylomicron remnants accumulate in the circulation of AAS users. This effect was so pronounced that the cholesteryl ester FCR was twice as less in the WT + AAS group when compared to both the SDT or WT groups. In the WT + AAS group the concentration in the plasma of fasting apo B48 was roughly tenfold higher than in SDT or WT, which confirms their pronounced retention of chylomicron remnants in the circulation even after the post-prandial period. Several transversal and prospective studies performed with different methodological approaches have shown that the accumulation of chylomicron remnants in the circulation is associated with the development of CAD [24–26]. With the kinetics emulsion method, we have shown that delayed removal of cholesteryl esters, that stands for remnant accumulation in the plasma, was an independent predictor of CAD and a marker of disease severity and, in a prospective study, also of disease evolution [9–11]. Therefore, remnant accumulation in the WT + AAS group indicates that AAS users bear a risk factor for CAD that had not yet been reported in other studies. The fact that the triglyceride FCR was unchanged in the WT + AAS group indicates that the lipolysis process that chylomicrons undergo in the circulation is not altered in that group. Indeed, in AAS users that practiced exercise training, triglyceridemia was not different from non-AAS users after the ingestion of a fat load as reported by Hislop et al. [27]. As occurred in this study, the WT + AAS group had normal fasting triglyceridemia. Here, the results of the in vitro test for LPL activity were again no different in the WT + AAS and WT groups. Another confirmatory finding was obtained in vivo from an experiment in which the generation of radioactive NEFA from the breakdown of the triglyceride emulsion after an intravenous injection. The fact that the curves of the appearance of labeled fatty acids in the circulation were the same for the three groups, WT + AAS, SDT and WT is consistent with normal triglyceride plasma emulsion decay curves, the normal LPL activity in the in vitro assay and finally, normal triglyceridemia in a demonstration that the lipolytic mechanisms are not altered by AAS use. In contrast with normal LPL activity was the activity of HL which was pronouncedly increased in the WT + AAS group. Increase in HL in administration of high stanozolol doses has been
reported in the literature [28,29]. HL has a phospholipase activity that facilitates lipoprotein uptake by the liver. The increase in the activity of HL accounts for the remarkable 50% decrease in HDL-cholesterol and apo A1 found in the WT + AAS group. Increase in HDL phospholipids degradation leads to increased of HDL clearance and consequently to decreased HDL-cholesterol. The fact that in the WT + AAS group the increase in HL activity did not elicit increased FCR of the cholesteryl ester emulsion is a novel finding. This is a clear-cut demonstration that the excess phospholipase activity of the enzyme that affects HDL catabolism so great that it has no effect on remnant catabolism. As the lipolysis process was unchanged, the decrease in chylomicron remnant removal can be ascribed to effects of AAS use on the mechanisms of remnant uptake. Nevertheless, it is also possible that the alteration in remnant structure produced by excess HL activity impairs the uptake by liver cells. It is interesting to note that LDL-cholesterol levels were increased in AAS users. As chylomicron remnants are also cleared, at least in part, by LDL receptors, it is possible that impairment of the same mechanisms that removes remnants also affects LDL removal from the plasma. In this respect, we showed in previous studies that the LDL cholesterol levels correlated with the FCR of the cholesteryl ester emulsions [9,10]. In fact, chylomicrons, chylomicron remnants, VLDL and LDL share the same catabolic cascade, mediated by LPL, lipoprotein receptors and apolipoproteins. The small number of study participants, due to recruitment difficulties, is a limitation of the study. In conclusion, this study shows that lipid metabolism alterations that contribute to the high incidence of CAD in AAS users are not restricted to increased HL and very low HDL cholesterol levels. An independent novel mechanism was shown here in AAS users, consisting of the delayed removal of chylomicron remnants. This defect may lead to an accumulation in the circulation of those atherogenic lipoproteins in the post-prandial state and which can be a predisposition for the development of CAD. 5. Conflict of interest None declared. Acknowledgments This study was supported by Fundação do Amparo à Pesquisa do Estado de São Paulo (FAPESP, Grant no. 06/58917-3), São Paulo, Brazil. Dr. Maranhão has a Research Award from Conselho Nacional
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