Metabolic Profiling of Plasma in Overweight/Obese and Lean Men using Ultra Performance Liquid Chromatography and Q-TOF Mass Spectrometry (UPLC-Q-TOF MS) Ji Young Kim,†,‡,§ Ju Yeon Park,‡,§,| Oh Yoen Kim,†,‡ Bo Mi Ham,‡ Hyun-Jin Kim,⊥ Dae Young Kwon,⊥ Yangsoo Jang,†,#,¶ and Jong Ho Lee*,†,‡,| Yonsei University Research Institute of Science for Aging, Yonsei University, Seoul, Korea, Laboratory of Clinical Nutrigenetics/Nutrigenomics, Yonsei University, Seoul, Korea, Department of Food and Nutrition, Brain Korea 21 Project, College of Human Ecology, Yonsei University, Seoul, Korea, Emerging Innovative Technology Research Division, Korean Food Research Institutes, Korea, Cardiology Division, Yonsei University College of Medicine, Yonsei Cardiovascular Center, Seoul, Korea, and Cardiovascular Genome Center, Yonsei Medical Institute, Yonsei University, Seoul, Korea Received February 3, 2010
Obesity is currently epidemic in many countries worldwide and is strongly related to diabetes and cardiovascular disease. This study investigated the differences in metabolomic profiling between overweight/obese and normal-weight men. Overweight/obese (n ) 30) and age-matched, normal-weight men (n ) 30) were included. Anthropometric parameters, conventional metabolites, and biomarkers were measured. Metabolomic profiling was analyzed with UPLC-Q-TOF MS. Overweight/obese men showed higher levels of HOMA-IR, triglycerides, total cholesterol, and LDL-cholesterol, and lower levels of HDL-cholesterol and adiponectin than lean men. Overweight/obese men showed higher proportion of stearic acid and lower proportion of oleic acid in serum phospholipids. Additionally, overweight/ obese individuals showed higher fat intake and lower ratio of polyunsaturated fatty acids to saturated fatty acids. We identified three lyso-phosphatidylcholine (lysoPC) as potential plasma markers and confirmed eight known metabolites for overweight/obesity men. Especially, overweight/obese subjects showed higher levels of lysoPC C14:0 and lysoPC C18:0 and lower levels of lysoPC C18:1 than lean subjects. Results confirmed abnormal metabolism of two branched-chain amino acids, two aromatic amino acids, and fatty acid synthesis and oxidation in overweight/obese men. Additionally, the amount of dietary saturated fat may influence the proportion of saturated fatty acids in serum phospholipids and the degree of saturation of the constituent acyl group of plasma lysoPC. Keywords: metabolic profiles • obesity • UPLC-Q-TOF MS • lyso-phosphatidylcholine • saturated fat
Introduction Obesity has reached epidemic proportions in many countries around the world and is strongly related to diabetes and cardiovascular disease.1 Whereas many comparisons of obese and lean subjects exist in the literature, they often focus on a small group of experimental variables. Obesity cosegregates with metabolic abnormalities, including dyslipidemia and glucose intolerance;2 however, obesity-induced perturbations * To whom correspondence should be addressed. Jong Ho Lee, PhD Department of Food & Nutrition, College of Human Ecology, Yonsei University, 134 Shinchon-Dong, Sudaemun-Gu, Seoul, 120-749, Korea. Fax: +82-2-364-9605. Tel: +82-2-2123-3122. E-mail:
[email protected]. † Yonsei University Research Institute of Science for Aging, Yonsei University. ‡ Laboratory of Clinical Nutrigenetics/Nutrigenomics, Yonsei University. § These authors contributed equally to this work. | Department of Food & Nutrition, Brain Korea 21 Project, College of Human Ecology, Yonsei University. ⊥ Korean Food Research Institutes. # Yonsei Cardiovascular Center. ¶ Yonsei Medical Institute, Yonsei University.
4368 Journal of Proteome Research 2010, 9, 4368–4375 Published on Web 06/18/2010
in metabolism have not been clearly established. Complex etiologies interacting with environmental factors highlight the need to understand how metabolite profiles are altered in this state. Metabolomics is an important technological discipline that focuses on the measurement of the relative concentrations of endogenous small molecules in biofluids, which characterizes changes in metabolism3 and helps unravel the metabolic state of biological systems.4 Metabolomics involves establishing relationships between phenotype and metabolism, which are key aspects of biological function. These approaches have been applied to identify serum/plasma metabolic markers involved in obesity,5-7 diabetes,8-10 and coronary artery disease11 through animal models or in humans. A new method of ultraperformance liquid chromatography coupled with Q-TOF mass spectrometry (UPLC-Q-TOF MS) was developed for sample analysis to obtain better quality and throughput for the analysis of complex mixtures. This sensitive, high-resolution system can acquire multiparametric metabolite profiles from biofluids rapidly and effectively as a powerful 10.1021/pr100101p
2010 American Chemical Society
Metabolic Profiling of Overweight/Obese and Lean Men 12,13
metabolomics tool. Here, we employed metabolomics analyses based on UPLC-Q-TOF MS to gain a broader understanding of metabolic differences between overweight/obese and lean men.
Materials and Methods Subjects. Study subjects were 60 healthy men between 30 and 50 years of age. They were recruited during routine checkups at a health promotion center at National Health Insurance Corporation Ilsan Hospital. Overweight/obese men were defined as those with a body mass index (BMI) greater than or equal to 25 kg/m2. Finally, 30 overweight/obese subjects (27 e BMI e 33) and 30 age-matched, lean subjects as controls (18 e BMI e 23) were included in this study. Subjects were excluded if they had significant cardiopulmonary, renal, or liver disease, or were taking a diabetes medication, systemic corticosteroids, or using weight loss mediation. The study protocol was approved by the Institutional Review Board of Yonsei University, and all participants provided written informed consent. Anthropometric Parameters, Blood Pressure, and Blood Collection. Body weights and heights were measured in unclothed subjects in the morning for the calculation of BMI (kg/m2). Waist and hip circumferences were measured for the calculation of waist/hip ratio (WHR). Blood pressures (BPs) were measured in the left arm of seated patients with an automatic BP monitor (TM-2654, A&D, Tokyo, Japan) after a 20-min rest. After a 12-h fasting period, venous blood specimens were collected in EDTA-treated and plain tubes, centrifuged to yield plasma or serum, and stored at -70 °C until analysis. Assessment of Dietary Intake. The dietary intake was assessed with a 24-h recall method and semiquantitative food frequency questionnaire. Dietary energy values and nutrient content were calculated using the Computer Aided Nutritional analysis program (CAN-pro 2.0. Korean Nutrition Society, Seoul, Korea). Total energy expenditure (TEE) was calculated from activity patterns including basal metabolic rate (BMR), physical activity for 24-h and specific dynamic action of food. BMR of each subject was calculated with the Harris-Benedict equation. Glucose, Insulin, and HOMA-IR. Fasting glucose levels were measured using a glucose oxidase method with a Beckman Glucose Analyzer (Beckman Instruments, Irvine, CA). Insulin levels were measured by radioimmunoassay using commercial kits from Immuno Nucleo Corporation (Stillwater, MN). Insulin resistance (IR) was calculated by the homeostasis model assessment (HOMA) using the following equation: IR ) [fasting insulin (µIU/mL) × fasting glucose (mmol/L)]/22.5. Serum Lipid Profile. Fasting total cholesterol and triglycerides were measured using commercially available kits on a Hitachi 7150 Autoanalyzer (Hitachi Ltd., Tokyo, Japan). After precipitation of serum chylomicrometers using dextran sulfate magnesium, the concentrations of LDL-cholesterol and HDLcholesterol in the supernatants were measured using an enzymatic method. LDL-cholesterol was indirectly estimated in subjects with serum triglyceride concentrations less than 400 mg/mL using the Friedewald formula {LDL-cholesterol ) totalcholesterol - [HDL-cholesterol + (triglycerides/5)]. Plasma Lp-PLA2 Activity, Oxidized LDL, and Adiponectin Concentrations. The activity of Lp-PLA2 (phospholipase A2), which is also known as platelet-activating factor acetylhydrolase (PAF-AH), was measured using a previously described modified method.14 Plasma oxidized LDL (ox-LDL) was measured using
research articles an enzyme immunoassay (Mercodia, Uppsala, Sweden), and the resulting color reaction was read at 450 nm on a Victor2 plate reader (Perkin-Elmer Life Sciences, Turku, Finland). Plasma adiponectin concentrations were measured using an enzyme immunoassay (Human Adiponectin ELISA kit, B-Bridge International Inc., CA, USA). Assays were read using a Victor2 plate reader at 450 nm, and wavelength correction was set to 540 nm. Lipid Peroxidation: Urinary 8-Epi-prostaglandin F2r and Plasma Malondialdehyde. Urine was collected in polyethylene bottles containing 1% butylated hydroxytoluene after 12 h of fasting. The tubes were immediately covered with aluminum foil and stored at -70 °C until analysis. The compound 8-epiprostaglandin F2R (8-epi-PGF2R) was measured using an enzyme immunoassay (BIOXYTECH urinary 8-epi-PGF2R Assay kit, OXIS International Inc., OR). The resulting color reaction from the enzyme immunoassay was read at 650 nm on a Victor2 plate reader. Urinary creatinine was determined by the alkaline picrated (Jeffe) reaction. Urinary 8-epi-PGF2R concentrations were expressed as pmol/mmol creatinine. Malondialdehyde (MDA) was measured from thiobarbituric acid-reactive substances (TBARS Assay Kit, Zepto-Metrix Co., Buffalo, NY). Assays were read using a Victor2 plate reader at 540 nm. Serum hs-CRP. Serum hs-CRP (C-reactive protein) concentrations were measured with an Express+ autoanalyzer (Chiron Diagnostics Co., Walpole, MA) using a commercially available, high-sensitivity CRP-Latex (II) X2 kit (Seiken Laboratories Ltd., Tokyo, Japan) that allowed detection of CRP concentrations as low as 0.001 mg/dL and as high as 32 mg/dL. Fatty Acid Composition in Serum Phospholipids. Total lipids were extracted with chloroform:methanol (2:1, v/v) as described by Folch et al.15 Phospholipids were methylated as described by Lepage and Roy after separation using thin-layer chromatography.16 Fatty acid methyl esters were analyzed by gas chromatography (GC) (HP 6890A, Agilent Technologies, Inc., Santa Clara, CA) using an Omegawax 320 fused-silica capillary column (30 m × 0.32 mm with 0.25-µm film; Supelco Inc., Bellefonte, PA). Fatty acids were identified by comparing their retention times with those of standard fatty acid methyl esters. Percentages of individual fatty acids were calculated according to the peak areas relative to the total area (total fatty acid area was set at 100%). Global (Nontargeted) Metabolic Profiling Analysis of Plasma by UPLC-Q-TOF MS. Sample Preparation and Analysis. Prior to analysis, 800 µL acetonitrile was added to 100 µL plasma. After shaking for 10 min at 4 °C, the mixture was centrifuged at 10 000 rpm for 5 min at 4 °C. The supernatant was freeze-dried at -70 °C and dissolved in 10% methanol. The supernatant was transferred into a vial, and a 7-µL sample was injected into the UPLC-Q-TOF MS (Waters, Milford, MA). The plasma extract was injected into an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm; Waters) in line with the UPLC system and equilibrated with water containing 0.1% formic acid. Samples were eluted by an acetonitrile gradient containing 0.1% formic acid at a flow rate of 0.35 mL/ min for 18 min, and metabolites were separated by UPLC, analyzed, and assigned by Q-TOF-MS (Waters). The Q-TOF was operated in ESI-positive mode. The capillary and sampling cone voltages were set at 2.78 kV and 26 V, respectively. The desolvation flow was set to 700 L/h at a temperature of 300 °C, and the source temperature was set to 110 °C. The TOF-MS data were collected in the range of m/z 50-1000 with a scan time of 0.2 s and interscan delay Journal of Proteome Research • Vol. 9, No. 9, 2010 4369
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Kim et al.
Table 1. Clinical Characteristics, Conventional Metabolites, and Inflammatory Markers in Normal-Weight and Overweight/Obese Mena normal-weight (n ) 30)
Age (yrs) Body mass index (kg/m2) Body fat (%) Waist-hip ratio Cigarette smoker, n (%) Alcohol drinker, n (%) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Glucose (mg/dl)b Insulin (µU/ml)b 1 HOMA-IRb Free fatty acid (uEq/l)b Triglyceride (mg/dl)b Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl)b Lp-PLA2 activity (nmol/mL/min) oxidized LDL (U/l)b Adiponectin (µg/mL)b hs-CRP (mg/dl)b Malondialdehyde (nmol/mL) b 8-epi-PGF2R(pg/mg creatinine)b
overweight/obese (n ) 30)
P value
9.5 ( 1.22 20.9 ( 0.14 15.3 ( 0.52 0.86 ( 0.01 15 (53.6) 26 (92.9) 115.1 ( 2.05 66.2 ( 1.45 91.4 ( 1.51 7.63 ( 0.48 1.72 ( 0.11 458.8 ( 48.8 85.5 ( 10.9 183.6 ( 4.72 114.2 ( 4.61 52.3 ( 2.22 33.9 ( 2.69
39.6 ( 1.24 28.9 ( 0.20 27.7 ( 0.55 0.93 ( 0.01 17 (58.6) 27 (93.1) 125.6 ( 2.00 76.7 ( 1.65 97.7 ( 1.85 13.7 ( 1.15 3.34 ( 0.30 618.2 ( 38.3 145.8 ( 11.1 196.9 ( 5.84 127.1 ( 5.02 40.7 ( 1.63 34.0 ( 2.07
0.994
