Article pubs.acs.org/jpr
Serum Metabolomics Study of Polycystic Ovary Syndrome Based on Liquid Chromatography−Mass Spectrometry Xinjie Zhao,†,⊥ Fang Xu,‡,⊥ Bing Qi,§,⊥ Songli Hao,‡ Yanjie Li,† Yan Li,‡ Lihong Zou,∥ Caixia Lu,‡ Guowang Xu,*,† and Lihui Hou*,‡ †
Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China ‡ Gynaecological Clinic of First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin 150040, China § Subsidiary Dongfang Hospital, Beijing University of Chinese Medicine, Beijing 100078, China ∥ The First Affiliated Hospital of Medical University, Harbin 150001, China S Supporting Information *
ABSTRACT: Polycystic ovary syndrome (PCOS) is a complex, heterogeneous disorder, which produces in 5−10% reproductive age women. In this study, a nontargeted metabolomics approach based on ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry is used to investigate serum metabolic characteristics of PCOS. PCOS women and healthy control can be clustered into two distinct groups based on multivariate statistical analysis. Significant increase in the levels of unsaturated free fatty acids, fatty acid amides, sulfated steroids, glycated amino acid and the decrease in levels of lysophosphatidylcholines, lysophosphatidylethanolamines, etc., were found. These metabolites showed abnormalities of lipid- and androgen-metabolism, increase of stearoyl-CoA desaturase (SCD) activity and accumulation of advanced glycation end-products in PCOS patients. On the basis of the binary logistic regression model, free fatty acid (FFA) 18:1/FFA 18:0, FFA 20:3, dihydrotestosterone sulfate, glycated phenylalanine, and uridine were combined as a diagnostic biomarker. The area under the curve (AUC) of combinational biomarker was 0.839 in 131 discovery phase samples and 0.874 in 109 validation phase samples. The findings of our study offer a new insight to understand the pathogenesis mechanism, and the discriminating metabolites may provide a prospect for PCOS diagnosis. KEYWORDS: Metabolomics, LC−MS, polycystic ovary syndrome, biomarkers
1. INTRODUCTION
Metabolomics is a power tool to study disease pathogenesis and to scan diagnostic biomarkers;17−19 it has been successfully applied in the study of metabolic diseases, such as insulin resistance,20 type 2 diabetes,21 and cardiovascular disease.18 Metabolomics studies of PCOS were also performed using nuclear magnetic resonance (NMR)22−25 and gas chromatography−mass spectrometry (GC−MS),23 and metabolic characteristics between different phenotypes were involved. Amino acids, creatinine, lactate, lipids, etc., were considered as the potential biomarkers of PCOS.23−25 Compared with other analytical techniques, main advantages of liquid chromatography−mass spectrometry (LC−MS) are wide dynamic range and coverage of a wide chemical diversity; it is a complementary analytical technique to NMR and GC−MS in metabolomics studies.26,27 In this work, LC−MS-based metabolomics approach was used to investigate the metabolic changes of PCOS patients. Furthermore, discriminative metabolites were evaluated on the
Polycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders; about 5−10% reproductive age women produce PCOS symptoms.1,2 PCOS leads to female subfertility and increases risk to type 2 diabetes and cardiovascular disease.3,4 Recent evidence suggests that women with PCOS have higher risk of developing endometrial cancer and ovarian cancer.5−7 PCOS is a complex disease, pathogenesis is not clear, but it is believed to be the result from genetic, environmental, and lifestyle interaction.8−10 It is relatively clear that PCOS is closely related with hormone abnormality.9,11,12 More and more evidence displayed that insulin signaling pathways are closely related to PCOS.3,12 PCOS may also be associated with chronic inflammation13,14 and increased level of oxidative stress.15,16 Diagnosis of PCOS is quite difficult since the symptoms of PCOS patients are not unified, including menstrual disturbances, elevated levels of male hormones, and polycystic ovaries.4 Rotterdam criteria are the most widely used diagnostic standard, which can be associated with a wide range of symptoms. © 2014 American Chemical Society
Received: November 16, 2013 Published: January 16, 2014 1101
dx.doi.org/10.1021/pr401130w | J. Proteome Res. 2014, 13, 1101−1111
Journal of Proteome Research
Article
Figure 1. Flowchart of study strategy in this study.
subgroups (BMI ≥ 23) according to the BMI cutoff in the Chinese population.28 Obese subjects were included in the overweight subgroup. Fasting blood samples were collected from the study subjects: during the early follicular phase of the menstrual cycle (the days 3−5) in the female control groups and PCOS subjects who had spontaneous menses. For those PCOS subjects who did not have spontaneous menses, 10 mg of dydrogesterone was prescribed for 7 days to induce withdrawal bleeding, and blood sample will be drawn during days 3−5 after withdrawal bleeding. Sera were stored at −80 °C until analysis.
capability to diagnose PCOS. The flowchart of study strategy is shown in Figure 1. The aim is to offer a new insight in the complex dysregulation of the metabolism in PCOS patients and to provide a new way for PCOS diagnosis.
2. MATERIALS AND METHODS 2.1. Human Samples
All of the PCOS patients were recruited from the Gynaecological Clinic of First Affiliated Hospital, Heilongjiang University of Chinese Medicine. Healthy women (the control group) were recruited from the Physical Examination Center of the First Affiliated Hospital, Heilongjiang University of Chinese Medicine and Physical Examination Center of the First Affiliated Hospital of Harbin Medical University. The study was approved by the Institutional Review Board of the First Affiliated Hospital, Heilongjiang University of Chinese Medicine (2010HZYLL-014) in accordance with the Declaration of Helsinki. All study participants signed an informed consent form before the start of the study. According to the diagnostic criteria revised by the European Society of Human Reproduction and Embryology and the American Society for Reproductive Medicine at the Rotterdam in 2003,4 PCOS patients can be diagnosed if two of the three criteria are present after excluding congenial adrenal hyperplasia, Cushing’s syndrome, androgen secreting tumors, or other related disorders. The three criteria are (1) oligo- and/or anovulation; (2) clinical and/or biochemical signs of hyperandrogenism (clinical manifestations of hyperandrogenism include presence of acne, hirsutism, and androgenic alopecia); (3) polycystic ovaries by ultrasound examination: presence of 12 or more follicles in each ovary measuring 2−9 mm in diameter and/or ovarian volume >10 cm3. All subjects had the age between 15 and 35 years and had at least 2 years of menstrual history. Control subjects and PCOS subjects were further divided into lean subgroups (BMI < 23) and overweight
2.2. Clinical Biochemical Data
Radioimmunoassay was performed by automated chemiluminescence analyzer (Abbott Laboratories, USA) to assess the serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), prolactin (PRL), testosterone (T), dehydroepiandrosterone sulfate (DHEAS), androstenedione (AND), and sex hormone binding globulin (SHBG). Chemiluminescence was used to analyze fasting insulin (FINS), fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), and low density lipoprotein (LDL) by automated biochemical analyzer (Hitachi, Ltd. Japan). 2.3. Metabolomics Analysis
Sera were thawed at 4 °C. A 100 μL serum was deproteinized with 4 volume of acetonitrile including 12 internal standards, centrifuged at 14 000g for 12 min, and the supernatant was dried in a vacuum centrifuge. The 12 internal standards were carnitine C2:0-d3, carnitine C10:0-d3, carnitine C16:0-d3, cholic acid-d4, LPC 12:0, LPC 19:0, leucine-d3, tryptophan-d5, FFA 16:0-d3, chenodeoxycholic acid-d4, leucine-enkephalin, and lansoprazole. For the analysis, the samples were redissolved in 150 μL of acetonitrile/water (2:8) and analyzed by ultra high-performance liquid chromatography (UHPLC) (Agilent 1290 Infinity, USA), coupled to electrospray ionization (ESI) 1102
dx.doi.org/10.1021/pr401130w | J. Proteome Res. 2014, 13, 1101−1111
Journal of Proteome Research
Article
Table 1. Clinical Biochemical Data of the Study Groups
age (years) BMI (kg/m2) waist−hip ratio age of menarche (years) systolic blood pressure (mmHg) diastolic blood pressure (mmHg) F−G score acanthosis nigricans incidence (%) acne incidence (%) resident skin oil incidence (%) FINS (mmol/L) FPG (μIU/ml) HOMA-IR TG (mmol/L) TC (mmol/L) HDL (mmol/L) LDL (mmol/L) FSH (mIU/ml) LH (mIU/ml) LH/FSH E2 (pg/mL) PRL (ng/mL) T (ng/dl) DHEAS (ug/Dl) AND (ng/mL) SHBG (nmol/L)
lean control (n = 32)
overweight control (n = 22)
lean PCOS (n = 40)
overweight PCOS (n = 37)
24.00 ± 2.57 20.23 ± 1.70 0.81 ± 0.06 13.31 ± 1.56 111.13 ± 10.80 71.92 ± 8.05 2.13 ± 2.09 0 48.7 51.3 8.08 ± 3.40 4.38 ± 0.45 1.60 ± 0.74 0.74 ± 0.25 3.82 ± 0.48 1.49 ± 0.32 2.16 ± 0.51 5.63 ± 1.46 6.62 ± 4.69 1.16 ± 0.66 38.56 ± 17.57 14.76 ± 5.89 26.35 ± 12.88 209.31 ± 84.94 2.93 ± 1.40 52.13 ± 19.87
24.59 ± 3.42 26.57 ± 1.93a 0.86 ± 0.04a 13.09 ± 0.97 113.5 ± 7.73a 69.27 ± 7.02 1.50 ± 1.47 0 54.5 63.6 12.50 ± 4.48a 4.64 ± 0.39a 2.61 ± 1.04a 1.15 ± 0.54a 4.23 ± 0.94 1.31 ± 0.41 2.73 ± 1.03a 4.55 ± 0.96a 5.26 ± 2.83 1.17 ± 0.58 35.05 ± 10.99 12.34 ± 5.07 25.67 ± 12.42 240.52 ± 103.14 3.21 ± 1.33 38.59 ± 21.73a
24.13 ± 3.97 20.16 ± 1.35 0.82 ± 0.05 14.20 ± 1.95c 107.30 ± 10.34 71.43 ± 7.28 3.70 ± 3.73c 10c 75c 65 7.37 ± 5.22 4.81 ± 0.4c 1.38 ± 1.18 0.97 ± 0.54c 4.33 ± 0.60c 1.25 ± 0.25c 2.60 ± 1.05c 5.89 ± 1.76 12.65 ± 7.67c 2.19 ± 1.19c 51.01 ± 23.67c 16.47 ± 22.47 54.75 ± 24.89c 249.36 ± 105.36c 4.70 ± 2.18c 45.61 ± 38.57
24.38 ± 3.04 27.99 ± 3.69b 0.90 ± 0.07b,d 13.28 ± 1.52d 116.69 ± 15.65b,d 76.33 ± 9.62d 3.36 ± 2.79d 61.5b,d 41d 92.3b,d 17.65 ± 18.96b 5.14 ± 0.71b,d 4.34 ± 5.66b 1.32 ± 0.61d 4.56 ± 1.01 1.00 ± 0.18b,d 3.08 ± 1.08 5.43 ± 1.57d 9.14 ± 4.45b,d 1.70 ± 0.74b,d 50.55 ± 21.28d 12.77 ± 6.24 57.91 ± 26.07d 234.93 ± 117.27 4.34 ± 1.71d 30.16 ± 30.22
PCOS and control p-value
0.003 0.014 0.019 0.0005
