Serum lysophosphatidylcholine level is not altered in coronary artery disease

Clinical Biochemistry 45 (2012) 793–797

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Serum lysophosphatidylcholine level is not altered in coronary artery disease Sang Hoon Song a, b, Yeomin Yoon c, Kyoung Un Park a, b, Junghan Song a, b, Jin Q Kim a, d,⁎ a

Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea Department of Laboratory Medicine, Seoul National University Bundang Hospital, Gyeonggi-do, Republic of Korea c Department of Laboratory Medicine, Konkuk University Hospital, Seoul, Republic of Korea d Department of Laboratory Medicine, Seoul National University Hospital, Seoul, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 6 July 2011 Received in revised form 12 March 2012 Accepted 24 March 2012 Available online 5 April 2012 Keywords: Lysophosphatidylcholine Coronary artery disease Atherosclerosis Phospholipase A2

a b s t r a c t Objectives: Lysophosphatidylcholine (LPC) is a promising biomarker for atherosclerosis and phospholipase activity. Serum LPC level in patients with coronary artery disease (CAD) was compared with controls. Design and methods: Eighty five CAD patients and 105 controls were enrolled. For sera from both groups of patients, twelve molecular species of LPC and lipid profile were measured. Associations with CAD were investigated and factors affecting serum LPC level were analyzed. Results: Individual LPC species, inter-species ratio, and the ratio to serum lipids were not associated with CAD. Diabetes was associated with decreased level of LPC 16:1. The ratios of LPC 16:0 to LPC 18:1, LPC 16:0 to 18:2, LPC 18:0 to LPC 18:1, and LPC 18:0 to LPC 18:2 were significantly affected by sex. Current smokers had lower LPC 18:0 to LPC 18:1 ratio. Conclusion: Serum LPC level is not altered in patients with CAD proven by coronary angiography. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc.

1. Introduction Lysophosphatidylcholine (LPC) is a major component of oxidized low-density lipoprotein (Ox-LDL), and it constitutes a group of lysophospholipids which play various roles in many important biological processes. In an atherosclerotic plaque, LPC exists in higher concentrations than in those of healthy individuals [1], and it has been suggested to be associated with many diseases, including systemic lupus erythematosus [2], ovarian cancer [3], and sepsis [4]. In vitro, LPC induces the expression of the intercellular adhesion molecule-1, vascular cell adhesion molecule-1 [5], cyclooxygenase-2 [6], and endothelial nitric oxide synthase [7], and induces the release of platelet-derived growth factor [8], arachidonic acid [9], and IL-8 [10] from endothelial cells. Recently, G proteincoupled receptors were identified as positive effectors of LPC [11], which suggests LPC has a role in a variety of biological processes, including cell proliferation, migration, inflammation, and apoptosis. LPC is produced from the hydrolysis of oxidized phosphatidylcholine in LDL by phospholipase A2 [12], which is itself abundant in atherosclerotic lesions [13]. Recently, this enzyme has been regarded as a potential therapeutic target in atherosclerosis [14]. Since it is the product of this enzyme, LPC could be used as a marker for atherosclerosis or other inflammatory diseases. We hypothesized that patients with coronary artery disease (CAD) might have higher level of LPC or altered ratio of its molecular species. ⁎ Corresponding author at: Seoul National University Hospital, 28 Yeongeon-dong, Jongno-gu, Seoul 110-744, Republic of Korea. Fax: +82 2 747 0359. E-mail address: [email protected] (J.Q. Kim).

We measured serum LPC level in patients with CAD and investigated its association with CAD. 2. Materials and methods 2.1. Study subjects This study was reviewed and approved by the Institutional Review Board of Seoul National University Hospital. One hundred and ninety patients who underwent the coronary angiography were enrolled in this study. Peripheral blood was collected in serum separation tube. Serum was separated and frozen at −70 °C until analysis. The patients were divided into two groups based on the findings of coronary angiography, electrocardiogram, and serum levels of cardiac markers. CAD groups were defined as patients with more than 50% stenosis of at least one major coronary arteries without ST elevation on electrocardiogram or any evidence of significant increase of cardiac markers. Patients without significant coronary artery stenosis or elevation of cardiac markers were defined as controls. 2.2. Analysis of serum lysophosphatidylcholine The methanol (Burdick & Jackson, Muskegon, MI) and chloroform (Mallinckrodt Chemicals, Phillipsburg, NJ) were of HPLC grade. Ammonium acetate and 2,6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene, BHT) were purchased from Sigma, and KH2PO4 from Duksan Pure Chemical. An internal standard (LPC 19:0) and calibrators (LPC 16:0, 18:0, and 22:0) were all 1-acyl-2-hydroxyl-sn-glycero-3-phosphocholines (Avanti Polar Lipids, Alabaster, AL).

0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. doi:10.1016/j.clinbiochem.2012.03.031

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Serum lysophosphotidylcholine (LPC) level was measured using liquid chromatography–tandem mass spectrometry (Waters, Manchester, UK). Forty microliters of serum or calibrators was placed into a glass tube containing 100 μL of methanol, 60 μL of 0.5 mol/L KH2PO4 in water, and 300 μL of 1 μg/mL LPC 19:0 solution in chloroform. After mixing for 5 min, the mixture was centrifuged for 5 min at 3000 rpm. Two hundred microliters of the chloroform layer were extracted and transferred to a 96-well microplate. The separated chloroform layer was dried under nitrogen gas at 40 °C and dissolved in 100 μL of 10 mmol/L ammonium acetate in methanol-chloroform (3:1 by volume). The plate was then loaded into the autosampler of the HPLC system with a C18 guard cartridge (4.0 × 3.0 mm; Phenomenex, Torrance, CA) and the sample was eluted with 10 mmol/L of ammonium acetate in methanol-chloroform (3:1 by volume) at a constant flow rate of 50 μL/min. Ten microliters of each sample were injected and the run time was 2 min. The control samples were prepared in pooled plasma using a low and high level of LPC. The control samples were analyzed on each analytical run along with the patient's samples. Concentrations of the LPC species were calculated using the calibration curve of the calibrator with the closest plasma concentration. After preliminary evaluation of the concentration of each LPC species in serum samples, we divided the LPC species into three groups and assigned three different calibrators. For the calculation of LPC 16:0 concentration, we used the slope of the LPC 16:0 calibration curve; for LPC 14:0, 15:0, 22:5, and 22:0, we used the slope of the LPC 22:0 curve; and for LPC 16:1, 17:0, 18:2, 18:1, 18:0, 20:4, 20:3, and 22:6, we used the slope of the LPC 18:0 calibration curve. Quantification was achieved by using multiple reaction monitoring specific for phosphocholine-containing lipids in a positive ion mode with daughter's m/z 183.9. Data analysis was performed with QuanLynx 4.0 software and Excel spreadsheets.

2.3. Analysis of serum markers For all the patients enrolled, serum levels of total cholesterol, HDL cholesterol, triglyceride, total protein, and albumin were Table 1 Baseline characteristics and laboratory findings of study subjects. Variables

CAD (n = 85)

Control (n = 105)

p value

Age, years Male, % BMI, kg/m2 Hypertension, % Diabetes, % Smoking, % Statin medication, % Total cholesterol, mg/dL Triglyceride, mg/dL HDL cholesterol, mg/dL LDL cholesterol, mg/dL Albumin, mg/dL C-reactive protein, mg/dL LPC 16:0, μg/mL LPC 18:0, μg/mL LPC 18:2, μg/mL LPC 18:1, μg/mL LPC 14:0, μg/mL LPC 15:0, μg/mL LPC 16:1, μg/mL LPC 17:0, μg/mL LPC 20:4, μg/mL LPC 20:3, μg/mL LPC 22:6, μg/mL LPC 22:5, μg/mL Total LPCs, μg/mL Saturated LPCs, μg/mL Unsaturated LPCs, μg/mL Saturated/unsaturated LPCs

63.3 ± 9.8 74.1 25.6 ± 3.1 70.6 28.2 25.9 17.6 178.3 ± 33.5 147.5 ± 87.9 46.3 ± 11.5 94.2 ± 25.7 4.0 ± 0.3 0.4 ± 0.8 83.2 ± 19.6 25.2 ± 6.6 14.2 ± 6.2 11.5 ± 3.7 0.29 ± 0.12 0.26 ± 0.10 1.30 ± 0.57 1.05 ± 0.47 2.98 ± 1.46 1.21 ± 0.86 2.21 ± 1.87 0.25 ± 0.15 143.7 ± 32.9 110.0 ± 25.1 33.7 ± 12.4 3.53 ± 1.10

60.2 ± 12.3 62.9 25.2 ± 3.5 53.3 25.7 17.1 35.2 168.0 ± 35.8 136.0 ± 65.2 47.9 ± 12.1 86.5 ± 27.3 4.1 ± 0.3 0.2 ± 0.4 80.1 ± 19.4 24.5 ± 7.3 13.2 ± 7.9 11.5 ± 5.7 0.26 ± 0.13 0.23 ± 0.09 1.31 ± 0.68 0.98 ± 0.44 3.20 ± 2.71 1.27 ± 2.12 2.36 ± 4.63 0.26 ± 0.40 139.4 ± 38.0 106.3 ± 26.1 33.1 ± 22.6 3.64 ± 1.10

0.052 0.098 0.354 0.015 0.697 0.142 0.007 0.044 0.304 0.354 0.051 0.039 0.034 0.271 0.673 0.358 0.922 0.198 0.054 0.935 0.304 0.517 0.818 0.778 0.767 0.414 0.326 0.829 0.488

analyzed using an automated chemistry analyzer Toshiba-200FR (Toshiba, Tokyo, Japan). Total cholesterol and triglyceride were determined with enzymatic method. LDL cholesterol and HDL cholesterol were measured by homogenous enzymatic assay. Albumin was measured with dye-binding method using bromcresol green. 2.4. Statistical analysis The difference of serum lysophosphatidylcholine concentration between CAD patients and controls were analyzed by Student's t-test. Chi-square test was performed to identify specific difference of categorical variables such as sex and presence of hypertension or diabetes. Correlations between LPC species and/or other laboratory markers were analyzed by Pearson's regression analysis. Influences of sex, presence of hypertension and diabetes, and current smoking on serum LPC level and ratios were analyzed using multiple logistic regression analysis. Continuous variables were expressed as mean ± SD, and P b 0.05 was considered as statistically significant. Only significant associations were shown in the results. All the statistical analyses were performed using SPSS for Windows 12.0 (SPSS Inc., Chicago, IL). 3. Results 3.1. Demographics and basic laboratory findings Baseline characteristics of the 190 patients who underwent the coronary angiography are presented in Table 1. Patients with CAD were more likely to be hypertensive and have higher serum level of total cholesterol, LDL cholesterol, and C-reactive protein. Statin medication was more prevalent in controls than in patients with CAD. Other risk factors for CAD such as age, sex, presence of diabetes, serum level of HDL cholesterol, and current smoking were not significantly different between the two groups. 3.2. Serum LPC species Each LPC species could be quantitated using the MRM scan with precursors' m/z N 183.9 (Fig. 1). The spectra of parent ion scan for a calibrator and a patient are presented in Fig. 2. For 12 LPC species, there were no molecular species of LPCs which showed significantly different serum levels between the CAD patients and controls. Total LPCs, saturated LPCs, unsaturated LPCs, and saturated LPCs to unsaturated LPCs ratio were not different between the two groups. LPC 16:0, LPC 18:2, LPC 18:1, LPC 18:0 were predominant species of LPC comprising about 93% of total LPCs. LPC species with saturated acyl group were about three to four times more abundant than unsaturated forms (Table 1). Strong correlations of serum concentrations between LPC species were observed depending on the species (Table 2). The most predominant species LPC 16:0 was the most correlated with LPC 18:0, and the strength of correlation weakened as the number of double bonds increased. Total cholesterol, triglyceride, and LDL cholesterol were correlated with saturated LPCs, while HDL cholesterol with unsaturated LPC species. Body mass index (BMI) was correlated with saturated LPC species such as LPC 16:0, LPC 18:0, LPC 14:0, and LPC 15:0, but not with unsaturated LPC species. 3.3. Factors associated with serum LPC species As serum LPC level was not associated with coronary artery disease, we investigated if there are other variables which affect it. LPC 16:0 to LPC 18:2, LPC 16: to LPC 18:1, LPC 18:0 to LPC 18:2, and LPC 18:0 to LPC 18:1 were independently associated with sex (Table 3). LPC 16:1 was associated with the presence of diabetes. Presence of

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Fig. 1. MRM chromatography of 13 LPC species and LPC 19:0 (internal standard). Total analysis time for each sample was 2 min. Each number above the peak is the retention time of corresponding LPC species.

hypertension was not associated with any LPC species or interspecies ratio. Current smoking significantly lowered LPC 18:0 to LPC 18:1 ratio. Other combinations of serum markers with LPC species, such as LPC species to LDL cholesterol and LPC species to albumin, were not related to the presence of CAD or other status.

4. Discussion It has been suggested that lysophosphatidylcholine may be involved in many pathologic conditions. As the major component of oxidized LDL, it has been advocated to be involved in the process of

Fig. 2. Spectra of parent ion scans of a calibrator (upper panel) and a patient (lower panel).

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Table 2 Pearson's correlation coefficients between LPC species and serum markers.

LPC 16:0 LPC 18:0 LPC 18:1 LPC 18:2 TC TG HDL-C LDL-C BMI

LPC 16:0

LPC 18:0

1 0.799⁎⁎ 0.473⁎⁎ 0.225⁎⁎ 0.391⁎⁎ 0.348⁎⁎

0.799⁎⁎ 1 0.444⁎⁎ 0.231⁎⁎ 0.281⁎⁎ 0.248⁎⁎ 0.122 0.171⁎⁎ 0.197⁎⁎

0.108 0.260⁎⁎ 0.180⁎

LPC 18:1 0.473⁎⁎ 0.444⁎⁎ 1 0.806⁎⁎ 0.172⁎ 0.070 0.214⁎⁎ 0.056 − 0.062

LPC 18:2 0.225⁎⁎ 0.231⁎⁎ 0.806⁎⁎ 1 0.091 − 0.004 0.220⁎⁎ − 0.003 − 0.067

TC

TG

HDL-C

0.391⁎⁎ 0.281⁎⁎ 0.172⁎ 0.091 1 0.231⁎⁎ 0.266⁎⁎ 0.909⁎⁎

0.348⁎⁎ 0.248⁎⁎ 0.070 − 0.004 0.231⁎⁎ 1 − 0.304⁎⁎

0.108 0.122 0.214⁎⁎ 0.220⁎⁎ 0.266⁎⁎ − 0.304⁎⁎ 1 0.075 − 0.048

0.077

0.039 0.304⁎⁎

LDL-C

BMI

0.260⁎⁎ 0.171⁎⁎

0.180⁎ 0.197⁎⁎

0.056 − 0.003 0.909⁎⁎ 0.039 0.075 1 − 0.019

− 0.062 − 0.067 0.077 0.304⁎⁎ − 0.048 − 0.019 1

TC, total cholesterol; TG, triglyceride, HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; and BMI, body mass index. ⁎⁎ p b 0.01. ⁎ p b 0.05.

showed that the amounts of LPC 16:0 and LPC 18:0 per gram protein in LDL were increased in diabetic patients, when compared with non-diabetics. This is the only case control study which has noted the importance of LPC species in atherosclerosis. In our study, serum LPC 16:1 was decreased in patients with diabetes. Neither LPC 16:0 to LDL nor LPC 18:0 to LDL ratio was significantly associated with diabetes. The main difference between the previous study and this study is that they measured LPC in LDL and we measured it in the whole serum. From both studies, diabetic patients may have altered oxidized LDL or phospholipid metabolism. LPC is also known to be associated with some cancers. Okita et al. found that LPC 18:2 to LPC 16:0 ratio in the plasma of ovarian cancer patients is altered [3]. Recently, plasma levels of several LPC species including LPC 16:0, LPC 18:0, LPC 18:1, and LPC 18:2 were shown to be significantly decreased in patient with colorectal cancer, and suggested as potential biomarkers [24]. In our study, saturated LPCs were significantly correlated with total cholesterol, triglyceride, and LDL cholesterol that are known as risk factors for CAD or metabolic syndrome. BMI was also correlated with saturated LPC species. In contrast, unsaturated LPCs were significantly correlated with HDL cholesterol. This suggests that LPCs in the form of saturation or unsaturation might contribute to the pathogenesis of CAD or metabolic syndrome to some degree. Plasma total LPC level was also suggested to be an indicator of severity of malignant disease, as it decreases in proportion to the severity of weight loss and inflammatory parameters [25]. From above and our study, it could be suggested that serum LPC level is affected by nutritional or metabolic status of patients. Furthermore, CAD may not alter the metabolism involved in the generation of LPC. This is quite opposite to the current belief that patients with CAD have increased phospholipase A2 activity [26,27]. That might be explained by the diversity and different substrate specificity of phospholipase A2 enzyme. Serum LPC level reflects the activity of both secretory and lipoprotein-associated phospholipase A2. There are several isoenzymes and they show different substrate specificity for phospholipids [28,29]. However, to our best knowledge, there is no study on the specificity of each enzyme for LPC species. Further study that includes the measurement of phospholipase A2 activity may be required for validation of our study.

atherosclerosis. Currently, both secretory phospholipase A2 and lipoprotein associated phospholipase A2 are suggested to be involved in the pathogenesis of CAD [13] and are emerging as therapeutic targets [15]. Thus, studies about appropriate biomarker that can reflect their activities are mandatory. LPC is one of the promising markers as it is a metabolite by phospholipase A2 through hydrolysis of phosphatidylcholine [12]. In our study, however, serum LPC level was not different between patients with CAD and controls. A few explanations for our results could be suggested. We measured LPC from total lipid fractions of serum. Serum LPC exits in LDL cholesterol, HDL cholesterol, free form, and protein-bond form [16]. Most studies on the association between oxidized LDL and CAD have measured oxidized LDL using monoclonal antibodies to oxidized LDL [17–19] or ultracentrifugation [20]. Though we did not measure LPC in LDL or oxidized LDL fractions, we can hypothesize that even if oxidized LDL increase in CAD, serum LPC does not reflect its change. One possible reason of the discrepancy is that oxidized LDL measured using monoclonal antibodies and LPC using tandem mass spectrometry may be a different target measured. There is a recent report that LPC contents measured with mass spectrometry in LDL cholesterol from diabetic patients are higher than those from healthy controls [21]. Control and patients in our study were those who were diagnosed as or suspicious of coronary artery disease and underwent the coronary angiography. Another reason may be that other fractions in serum, such as HDL cholesterol and protein-bound form compensates for the change in LPC level, in which case LPC might be the source of native phosphatidylcholine and should be further elucidated. Our small sample size may not be large enough to reflect the change of serum LPC level. However, we studied three times with different pools of patients and controls with the same inclusion and exclusion criteria. Our conclusion is that there is no change in serum LPC level that reflects the progress of atherosclerosis of coronary artery. There is a recent study that reported higher LPC level in the symptomatic carotid artery plaques than that in the asymptomatic [22]. Studies in coronary artery plaque and of its association with stages of coronary artery disease and serum LPC level are mandatory. A few studies on LPC species in biological fluids have proven its significance in some pathologic conditions. Shi et al. demonstrated the importance of LPC in patients with type 2 diabetes [23]. They

Table 3 Factors associated with serum LPC species or inter-species ratios. Sex

LPC LPC LPC LPC LPC

16:0/LPC 18:2 16:0/LPC 18:1 18:0/LPC 18:1 18:0/LPC 18:2 16:1, μg/mL

Diabetes

Male

Female

6.45 ± 2.26 7.33 ± 1.55 2.21 ± 0.51 1.96 ± 0.79

7.99 ± 3.63 7.88 ± 2.26 2.44 ± 0.61 2.42 ± 0.90

Variables with p b 0.05 after multivariate analysis are presented.

No

1.37 ± 0.68

Current smoking Yes

1.10 ± 0.45

No

Yes

2.33 ± 0.55

2.13 ± 0.54

S.H. Song et al. / Clinical Biochemistry 45 (2012) 793–797

We used LPC 16:0, 18:0, and 22:0 for calibration and quantitation of other different species of LPC. It is ideal to use the same calibrator for the targeting molecule. However, the aim of this study was not to get the absolute amount of each LPC species but to compare the level in the CAD patients and controls. We supposed that the variation in the ionization efficiency could be compensated in the comparison process. There are also other studies that used two or three calibrators for quantification of multiple LPC species [30,31]. Profiling of multiple analytes and/or metabolites in the clinical samples using mass spectrometers are getting more attention [32] and experiences from other analytes [33,34] could be considered for further investigation. In summary, serum LPC level was not associated with CAD. Further studies on enzyme diversity and substrate specificity are needed to elucidate its role in atherosclerosis. Acknowledgments This study was supported by grant no 04-2004-27 from the SNUH Research Fund. References [1] Portman OW, Alexander M. Lysophosphatidylcholine concentrations and metabolism in aortic intima plus inner media: effect of nutritionally induced atherosclerosis. J Lipid Res 1969;10:158–65. [2] Wu R, Svenungsson E, Gunnarsson I, Andersson B, Lundberg I, Schafer Elinder L, et al. Antibodies against lysophosphatidylcholine and oxidized LDL in patients with SLE. Lupus 1999;8:142–50. [3] Okita M, Gaudette DC, Mills GB, Holub BJ. Elevated levels and altered fatty acid composition of plasma lysophosphatidylcholine(lysoPC) in ovarian cancer patients. Int J Cancer 1997;71:31–4. [4] Chen G, Li J, Qiang X, Czura CJ, Ochani M, Ochani K, et al. Suppression of HMGB1 release by stearoyl lysophosphatidylcholine:an additional mechanism for its therapeutic effects in experimental sepsis. J Lipid Res 2005;46:623–7. [5] Kume N, Cybulsky Jr MI, Gimbrone MA. Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. J Clin Invest 1992;90:1138–44. [6] Rikitake Y, Hirata K, Kawashima S, Takeuchi S, Shimokawa Y, Kojima Y, et al. Signaling mechanism underlying COX-2 induction by lysophosphatidylcholine. Biochem Biophys Res Commun 2001;281:1291–7. [7] Cieslik K, Zembowicz A, Tang JL, Wu KK. Transcriptional regulation of endothelial nitric-oxide synthase by lysophosphatidylcholine. J Biol Chem 1998;273:14885–90. [8] Kume N, Gimbrone Jr MA. Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human endothelial cells. J Clin Invest 1994;93:907–11. [9] Wong JT, Tran K, Pierce GN, Chan AC, O K, Choy PC. Lysophosphatidylcholine stimulates the release of arachidonic acid in human endothelial cells. J Biol Chem 1998;273:6830–6. [10] Riederer M, Lechleitner M, Hrzenjak A, Koefeler H, Desoye G, Heinemann A, et al. Endothelial lipase (EL) and EL-generated lysophosphatidylcholines promote IL8 expression in endothelial cells. Atherosclerosis 2011;214:338–44. [11] Radu CG, Yang LV, Riedinger M, Au M, Witte ON. T cell chemotaxis to lysophosphatidylcholine through the G2A receptor. Proc Natl Acad Sci U S A 2004;101: 245–50. [12] Itabe H. Oxidized phospholipids as a new landmark in atherosclerosis. Prog Lipid Res 1998;37:181–207. [13] Romano M, Romano E, Bjorkerud S, Hurt-Camejo E. Ultrastructural localization of secretory type II phospholipase A2 in atherosclerotic and nonatherosclerotic regions of human arteries. Arterioscler Thromb Vasc Biol 1998;18:519–25. [14] Macphee CH, Nelson JJ, Zalewski A. Lipoprotein-associated phospholipase A2 as a target of therapy. Curr Opin Lipidol 2005;16:442–6.

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