Metabolism of chylomicron-like emulsions in carriers of the S447X lipoprotein lipase polymorphism

Clinica Chimica Acta 335 (2003) 157 – 163 www.elsevier.com/locate/clinchim

Metabolism of chylomicron-like emulsions in carriers of the S447X lipoprotein lipase polymorphism Katia A. Almeida a, Roberto Schreiber b, Rosaˆngela F. Amaˆncio b, Se´rgio P. Bydlowski c, Adriana Debes-Bravo c, Jacqueline S. Issa b, Ce´lia M.C. Strunz b, Raul C. Maranha˜o a,* a

Faculty of Pharmaceutical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil b The Lipid Metabolism Laboratory, Heart Institute (InCor), Sa˜o Paulo, SP, Brazil c Department of Hematology of the Medical School Hospital, University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil Received 4 February 2003; received in revised form 9 June 2003; accepted 10 June 2003

Abstract Background: Lipoprotein lipase catalyzes the hydrolysis of the triglycerides contained in both very-low-density lipoproteins and chylomicrons for storage in the adipose tissue and muscle of fats of both hepatic and dietary origin. The S447X-Stop lipoprotein lipase is the most common polymorphism of the enzyme, affecting roughly 20% of the population and is accompanied by normal or diminished fasting triglycerides and perhaps lower incidence of coronary artery disease (CAD). Delay in the removal of chylomicron and remnant is now an established risk factor for CAD. Methods: Currently, the chylomicron metabolism has been evaluated in 12 normolipidemic subjects with the S447X-Stop and in 13 age- and sex-paired control subjects with no mutation. The doubly labeled chylomicron-like emulsion method was used to evaluate chylomicron metabolism. The emulsions labeled with cholesteryl-oleate (14C-CE) and tri[9,10-3H]oleate (3H-Tg) were injected intravenously and the decay curves of the labels were determined by blood sampling over 60 min followed by radioactive counting. Results: The fractional clearance rate (FCR, min 1) of the labels was not different in the S447X carriers compared with the noncarriers (FCR 3H-Tg 0.035 F 0.019 and 0.030 F 0.009 ; FCR 14C-CE 0.008 F 0.007 and 0.009 F 0.007, respectively). Conclusions: The chylomicron intravascular lipolysis monitored by the 3H-Tg emulsion and the remnant removal monitored by the 14C-CE emulsion were not altered by the presence of this polymorphism of great populational impact. D 2003 Elsevier B.V. All rights reserved. Keywords: Enzyme mutations; Triglycerides; Chylomicrons; Cholesterol; Emulsions; Lipoprotein lipase; Coronary artery disease

1. Introduction

* Corresponding author. Laborato´rio de Metabolismo de Lı´pides, Instituto do Coracß a˜o (InCor) do Hospital das Clı´nicas da FMUSP, Av. Ene´as de Carvalho Aguiar, 44-CEP 05403-000 Sa˜o Paulo, SP, Brazil. Fax: +55-11-3069-5574. E-mail address: [email protected] (R.C. Maranha˜o).

After entering the circulation, both triglyceride-rich lipoproteins—VLDL produced by the liver, and chylomicrons originated from the dietary lipids absorbed by the intestine and secreted into lymph—undergo the action of lipoprotein lipase on the capillary wall. Lipoprotein lipase is a key enzyme in lipoprotein

0009-8981/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0009-8981(03)00289-4

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metabolism [1]. Anchored to the luminal surface of the vascular endothelium, it promotes the hydrolysis of the triglyceride core of circulating VLDL and chylomicrons. This process generates free fatty acids and glycerol that are absorbed by body tissues, specifically by adipose tissue and muscle, where they are reesterified and stored to make up the main energy reservoir of the organism. The resulting catabolic products of VLDL and of chylomicrons such as, respectively, LDL and chylomicron remnants, are cleared from the circulation by LDL receptors and other receptor mechanisms [2,3]. Despite the common catabolic pathway and the resemblance in composition and structure, the half-lives of VLDL and chylomicrons in the plasma are remarkably different, the former being in the order of 2– 3 days, whereas the chylomicron half-life is about 15 min. An efficient lipolytic process is a prerequisite for the interaction of the lipoprotein particles with the receptors that remove chylomicron remnants and is determinant of the speed of removal of those particles from the plasma. The VLDL metabolism status is evaluated by the triglyceride concentration in the plasma after a 12 h fast. Being a non-steady-state lipoprotein, the assessment of the chylomicron metabolism status is much more complex and laborious and relies on plasma kinetic methodological approaches. Delayed chylomicron intravascular catabolism is now an established independent risk factor for coronary artery disease [4 –9]. Recently, a number of polymorphisms in the lipoprotein lipase gene have been described, such as the D9N and N291S polymorphisms that have been reported to increase plasma triglycerides and decrease HDL cholesterol, effects that are considered as proatherogenic. One of the most interesting lipoprotein lipase gene polymorphisms, and the one with the highest populational frequency so far identified, is the S447X [10,11]. The S447X creates a premature stop codon and loss of the terminal serine and glycine residues from the carboxy end of the protein [12]. The frequency of the S447X polymorphism in the population is in the range of 17 – 22% [11]. Interestingly, the S447X polymorphism appears to favorably affect the VLDL metabolism because carriers may tend to lower fasting triglyceride concentration values compared with noncarriers [13 –16]. HDL may also be found increased in carriers of this polymorphism. This is most likely accounted for the negative corre-

lation between triglycerides and HDL cholesterol determined by the metabolic relationships of the two lipoproteins. The effects of the polymorphism presence upon the chylomicron metabolism, however, have not yet been explored and this issue is of major importance due to the very high populational incidence of the polymorphism and the association between the speed of chylomicron catabolism and coronary artery disease (CAD). To address the question of whether or not the S447X polymorphism affects chylomicron lipolysis and remnant removal, we studied this metabolism in 12 carriers of the S447X polymorphism as compared with 13 noncarriers with similarly normal plasma fasting triglycerides and cholesterol. For this purpose, it was utilized the method of the determination of the plasma kinetics of doubly labeled chylomicron-like emulsions, which offers a straightforward and integrated view of the intravascular chylomicron metabolism.

2. Materials and methods 2.1. Subjects One hundred forty-nine healthy subjects with normal lipid profile that were employees of the Heart Institute had been previously genotyped and 24 (16%) were shown to be carriers of the S447X lipoprotein lipase polymorphism. Among this latter group of subjects, 12 (seven male and five female) volunteered to participate in the study. Selected from the same group of 149 healthy subjects, 13 (six male and seven female) volunteer noncarriers matched for age, sex and body mass index (BMI) were recruited allocated in the study as controls. All participants were of general Caucasian population, sedentary, were not smokers or alcohol abusers and none had arterial hypertension. They did not have apparent or reported diseases, amenorrhea, pregnancy or breast feeding. Their physical characteristics and plasma lipids and apolipoprotein data are displayed in Table 1. The design and the objective of the study were explained to each participant before the study, and an informed written consent was obtained from all. The study was approved by the Scientific and Ethics Committee of the Heart Institute of the Hospital of the University of Sa˜o Paulo School of Medicine.

K.A. Almeida et al. / Clinica Chimica Acta 335 (2003) 157–163 Table 1 Physical characteristics, plasma lipids, apolipoproteins, lipoprotein (a) [Lp(a)] and fractional clearance rates (FCR) of the emulsion radioactive triglycerides (3H-Tg) and cholesteryl esters (14C-CE) in the group of noncarriers and of carriers of the lipoprotein lipase S447X polymorphism

Number of subjects Male/female Age (years) BMI (kg/m2) Cholesterol (mg/dl) Total LDL HDL VLDL Triglycerides (mg/dl) Apo A1 (g/l) Apo B (g/l) Lipoprotein (a) (mg/dl) FCR—3H-Tg (min 1) FCR—14C-CE (min 1)

Noncarriers

Carriers

13 6/7 33 F 8 25 F 4

12 7/5 36 F 9 24 F 3

199 F 45 133 F 40 49 F 12 18 F 7 88 F 35 1.32 F 0.21 0.94 F 0.26 37.3 F 36.6 0.035 F 0.019 0.008 F 0.007

203 F 51 130 F 41 51 F 15 22 F 15 109 F 76 1.34 F 0.25 0.97 F 0.29 20.1 F 27.7 0.030 F 0.009 0.009 F 0.007

2.2. Genotyping The participants were selected for this study after a screening for the S447X lipoprotein lipase polymorphism. DNA was extracted from peripheral blood leukocytes. The target for amplification was the MnlI polymorphic sites in introns 8 and 9 of the lipoprotein lipase gene. All PCR reagents were obtained from Life Technologies. PCR amplification was carried out using the primers 5V-TACACTAGCAATGTCTAGGTGA-3V and 5V-TCAGCTTTAGCCCAGAATGC-3V as described [17]. Briefly, samples were amplified in a 50Al reaction mixture containing 0.1 Ag genomic DNA, 1.5 Al of 50 mmol/l MgCl2, 0.5 Ag BSA, 40 pmol of each primer, 4 pmol dNTP and 0.5 U Taq DNA polymerase. Amplification (30 cycles; 94 jC for 1 min, 60 jC for 30 s, 72 jC for 30 s) was performed in a Peltier thermal cycler PTC-200 (MJ Research, USA). The amplified product of 488 bp was digested with 10 U MnlI and polymorphisms were visualized after electrophoresis on 2% agarose gel stained with ethidium bromide. 2.3. Biochemical analysis Serum total cholesterol and triglyceride concentrations were determined by enzymatic test kits (CHOD-

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PAP and GPO-PAP respectively, both from HoffmanLa Roche, Basel, Switzerland). HDL cholesterol was measured by the same cholesterol method, after precipitation with MgCl2 and phosphotungstic acid. VLDL and LDL cholesterol was calculated using the Friedewald equation [18]. Plasma apolipoprotein A1 (apo A1), apolipoprotein B (apo B) and lipoprotein (a) were assayed by an immunoturbidimetric method (Hoffman-La Roche). 2.4. Emulsion preparation The chylomicron-like emulsion was prepared as previously described [9]. Briefly, dried lipid mixtures composed of 2% cholesterol, 23% lecithin,6% cholesteryl oleate and 69% triolein (Sigma, St. Louis, MO) were prepared with cholesteryl-oleate (14C-CE) and tri[9,10-3H]oleate (3H-Tg) (Amersham, Surrey, UK) and the mixtures were emulsified by ultrasonic irradiation in aqueous medium and purified by two-step ultracentrifugation, sterilized by passage through a 0.2-Am filter and tested for sterility and pyrogenicity prior to injection into the patients. 2.5. Kinetics of the emulsion The determination of the plasma kinetics of the chylomicron-like emulsion was performed after a 12-h fast. The cubital veins from both arms were cannulated and kept with a saline flush. On average, 100 ml of the emulsion containing 148 kBq (4 mCi) 3H-triolein and 74 kBq (2 mCi) 14C-cholesteryl-oleate were injected in a bolus followed by a 5-ml saline flush. Blood samples were collected from the contralateral arm vein at 2, 4, 6, 10, 15, 30, 45 and 60 min after injection. Blood was dispensed into tubes containing 50 ml of sodium heparin (Liquemine, Roche Laboratories, Sa˜o Paulo, Brazil). Blood was centrifuged and the radioactivity contained in 1.0 ml of plasma was measured by liquid scintillation counting (Packard 1.660 TR, Meridien, CT). The results were expressed as the fractional clearance rate (FCR) of both radioactive labels. The safety of the radioactive dose injected into the subjects was warranted according to radioprotection regulations as described elsewhere [9].

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2.6. Statistical analysis All recorded variables were expressed as means F S.D. The differences of data were evaluated by the Student’s t-test or Mann – Whitney’s test when applicable, with the level of significance set at p < 0.05 for all comparisons.

3. Results Table 1 shows the physical characteristics and the plasma lipid and apolipoprotein data of the carriers

and noncarriers of the S447X polymorphism. There was no difference between the two groups regarding age, BMI and plasma values: total, LDL, VLDL and HDL cholesterol, triglycerides, lipoprotein (a), apo B and apo A1. Fig. 1 shows the plasma decay curves of the emulsion radioactive lipids obtained in both groups, S447X polymorphism carriers and noncarriers. It is apparent that the decay curves of both the emulsion triglycerides and cholesteryl ester moieties were not different. In fact, both 3H-triglyceride and 14C-cholesteryl ester FCRs of the S447X polymorphism carriers were similar to those obtained from the noncarriers controls (Table 1).

4. Discussion

Fig. 1. Decay curves of the emulsion 3H-triglycerides (above) and 14 C-cholesteryl esters (below) in 12 carriers of the S447X polymorphism (black) and in 13 noncarriers (white). The doubly labeled emulsion was intravenously injected in a bolus and blood samples were collected in preestablished intervals and analyzed for radioactivity in a liquid scintillation counter.

Lipoprotein lipase has a pivotal role in the intravascular lipid metabolism. By catalyzing the breaking down of VLDL and chylomicron triglycerides, it is directly responsible for the input of fat into the adipose tissue cells and muscle. Consequently, the enzyme is responsible for the formation of the energy stores of the organism. Mutations affecting the enzyme or its cofactors, namely apo CII and apo CIII, may potentially have a great impact on the lipid metabolism regulation. Therefore, a high-frequency polymorphism affecting roughly one out of each five subjects in the general population, such as the S447X polymorphism, should be carefully examined regarding its metabolic and pathophysiological consequences. Double-labeled chylomicron-like emulsions allow reproduction in man of the operative conditions in experimental animal models for studies of chylomicron metabolism, where chylomicrons obtained from the intragastric administration of fatty bolus with radioactively labeled cholesterol and triglycerides or fatty acids are recovered from the lymph and reinjected into the blood stream. The emulsions are injected intravenously in a bolus so that the gastrointestinal component is bypassed and the determination of the plasma decay curves of the emulsion radiolabels is performed in a straightforward manner to assess, by analogy, chylomicron degradation and removal from plasma. After a 12-h fast, injection of minimal amounts of the emulsion does not disturb VLDL metabolism as occurs in fat load tests. After

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injection into the circulation, these emulsions, which are devoid of proteins, adsorb Apo CII and Apo E and other low-molecular-weight apolipoproteins that modulate their metabolism [22]. Thus, in a similar process to that involved when lymph chylomicrons enter into the circulation, emulsion triglycerides are hydrolyzed by lipoprotein lipase and the resulting remnants bind to hepatic receptors resulting in their removal from plasma. Remnant removal is evaluated by the emulsion cholesteryl esters, since this label does not shift in appreciable amounts from the emulsion to particles as native plasma lipoproteins or other [7]. Thus, the chylomicron-like emulsion model is a useful tool that facilitates studies of this metabolic pathway in humans which has been validated in human studies [9,23]. In animal and human subjects, the half-lives of the emulsion corresponded to those of lymph chylomicrons [9,23,24]. The emulsion model has unraveled chylomicron metabolism defects in malignant hypertension [25], systemic lupus erythematosus [26], in patients submitted to heart transplantation [27] and in obesity [28]. It has also been used to investigate the effects of lipid-lowering drugs on chylomicron metabolism [29,30]. In this study, we showed that the S447X polymorphism occurring in subjects does not affect the chylomicron intravascular catabolism, as evaluated with the use of chylomicron-like emulsions. Awareness of chylomicron status as a CAD risk factor has been steadily increasing. In oral fat load tests, chylomicron remnant retention, as estimated by postprandial retinyl palmitate or apo B48, has been observed in patients with CAD [4 – 6]. Slower lipolysis and remnant removal from the plasma were also documented in CAD patients by using the chylomicron-like emulsion test [9]. Conflicting results have been described in the literature concerning the effects of the S447X polymorphism upon the activity of lipoprotein lipase. The activity of the enzyme, as determined in vitro in the post-heparin plasma of the carriers, has been reported as equal [19], lower [20] or higher [17,21] than in the noncarriers of the polymorphism. Nonetheless, it is worthwhile to point out that if this method may correctly evaluate the enzyme function it hardly reflects the physiological lipolysis process occurring in vivo in the vascular endothelial surface. The access of triglyceride-rich lipoproteins to lipo-

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protein lipase might be affected by several factors, e.g., by capillary blood flow. Consistent with this assumption, the injection of heparin causes a rapid and transitory reduction of plasma triglyceride levels in subjects, thereby indicating that the availability of LPL is significantly increased after heparin injection [31,32]. Moreover, there is a very weak correlation between post-heparin plasma lipoprotein lipase protein and activity and VLDL triglyceride concentration (r = 0.3, p < 0.05) [33]. In this context, we also observed a weak association between postheparin lipase activity and the emulsion triglyceride FCR (r = 0.34; p = 0.026), suggesting that modulation of intravascular lipolysis in vivo is not measured in the assay in vitro (Santos, R., unpublished data). On the other hand, kinetic analysis of the intravascular catabolism of chylomicron-like emulsions provides a high-resolution view of triglyceride-rich particles metabolism in the circulation. The kinetics of the emulsion 3H-triglycerides mirrors the overall intravascular lipolysis activity in vivo. Although chylomicrons and VLDL share the same catabolic pathway that is driven by lipoprotein lipase, slow chylomicron catabolism does not necessarily imply in increased plasma concentration of VLDL, as expressed by increased fasting triglycerides. In our previous study with chylomicron-like emulsions, slow chylomicron catabolism occurred in CAD patients despite there were no differences between patients and controls regarding fasting plasma triglycerides. Therefore, chylomicron-like emulsion plasma kinetics and the concentration of fasting triglycerides are independent parameters. In conclusion, our results show that both chylomicron lipolysis and remnant removal, as evaluated by emulsion models of chylomicrons, are unaffected by the presence of the S447X polymorphism.

Acknowledgements This study was supported by Fundacßa˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP, Grant 99/01299-2), Sa˜o Paulo, Brazil. R.C. Maranha˜o has a Research Award from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Brasilia, Brazil.

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