Chunxiu Hu

Analytical strategies in lipidomics and applications in disease biomarker discovery

Lipidomics is a lipid-targeted metabolomics approach aiming at comprehensive analysis of lipids in biological systems. Recently, lipid profiling, or so-called lipidomics research, has captured increased attention due to the well-recognized roles of lipids in numerous human diseases to which lipid-associated disorders contribute, such as diabetes, obesity, atherosclerosis and Alzheimers disease. Investigating lipid biochemistry using a lipidomics approach will not only provide insights into the specific roles of lipid molecular species in health and disease, but will also assist in identifying potential biomarkers for establishing preventive or therapeutic approaches for human health. Recent technological advancements in mass spectrometry and rapid improvements in chromatographic techniques have led to the rapid expansion of the lipidomics research field. In this review, emphasis is given to the recent advances in lipidomics technologies and their applications in disease biomarker discovery.

RPLC-ion-trap-FTMS method for lipid profiling of plasma : method validation and application to p53 mutant mouse model

A reversed-phase liquid chromatography-linear ion trap-Fourier transform ion cyclotron resonance-mass spectrometric method was developed for the profiling of lipids in human and mouse plasma. With the use of a fused-core C 8 column and a binary gradient, more than 160 lipids belonging to eight different classes were detected in a single LC-MS run. The method was fully validated and the analytical characteristics such as linearity ( R (2), 0.994-1.000), limit of detection (0.08-1.28 microg/mL plasma), repeatability (RSD, 2.7-7.9%) and intermediate precision (RSD, 2.7-15.6%) were satisfactory. The method was successfully applied to p53 mutant mice plasma for studying some phenotypic effects of p53 expression.

Discovery and Validation of Plasma Biomarkers for Major Depressive Disorder Classification Based on Liquid Chromatography–Mass Spectrometry

Major depressive disorder (MDD) is a debilitating mental disease with a pronounced impact on the quality of life of many people; however, it is still difficult to diagnose MDD accurately. In this study, a nontargeted metabolomics approach based on ultra-high-performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was used to find the differential metabolites in plasma samples from patients with MDD and healthy controls. Furthermore, a validation analysis focusing on the differential metabolites was performed in another batch of samples using a targeted approach based on the dynamic multiple reactions monitoring method. Levels of acyl carnitines, ether lipids, and tryptophan pronouncedly decreased, whereas LPCs, LPEs, and PEs markedly increased in MDD subjects as compared with the healthy controls. Disturbed pathways, mainly located in acyl carnitine metabolism, lipid metabolism, and tryptophan metabolism, were clearly brought to light in MDD subjects. The binary logistic regression result showed that carnitine C10:1, PE-O 36:5, LPE 18:1 sn-2, and tryptophan can be used as a combinational biomarker to distinguish not only moderate but also severe MDD from healthy control with good sensitivity and specificity. Our findings, on one hand, provide critical insight into the pathological mechanism of MDD and, on the other hand, supply a combinational biomarker to aid the diagnosis of MDD in clinical usage.

Application of plasma lipidomics in studying the response of patients with essential hypertension to antihypertensive drug therapy

Hypertension is a key risk factor in the progression of cardiovascular disease (CVD). Dyslipidemia, a strong predictor of CVD, frequently coexists with hypertension. Therefore, the control of hypertension and dyslipidemia may help reduce CVD morbidity and mortality. In the present study, the therapeutic effects of antihypertensive agents on blood pressure control and plasma lipid metabolism were evaluated. The plasma lipid profiles of patients with treated (n = 25) or untreated (n = 30) essential hypertension as well as of subjects with normotension (n = 28) were analyzed using liquid chromatography mass spectrometry. Principal component analysis of the lipidomics data revealed distinct clusters among studied subjects across three human populations. Phosphatidylcholines and triacylglycerols (TG) dominated the pattern of hypertension-influenced plasma lipid metabolism. Discriminatory lipid metabolites were analyzed using one-way analysis of variance followed by a post hoc multiple comparison correction. TG lipid class was significantly increased by 49.0% (p < 0.001) in hypertensive vs. normotensive groups while tended to decrease (-21.2%, p = 0.054) in hypertensive patients after treatment. Total cholesteryl esters were significantly decreased by -16.9% (p < 0.001) in hypertensive patients after treatment. In particular, a large number of individual neutral lipid species were significantly elevated in hypertensive subjects but significantly decreased after treatment with antihypertensive agents. The present study applied, for the first time, a systems biology based lipidomics approach to investigate differentiation among plasma lipid metabolism of patients with treated/untreated essential hypertension and subjects with normotension. Our results demonstrate that antihypertensive medications to lower blood pressure of hypertensive patients to target levels produced moderate plasma lipid metabolism improvement of patients with hypertension.

Lipidomics Analysis Reveals Efficient Storage of Hepatic Triacylglycerides Enriched in Unsaturated Fatty Acids after One Bout of Exercise in Mice

Background Endurance exercise induces lipolysis, increases circulating concentrations of free fatty acids (FFA) and the uptake and oxidation of fatty acids in the working muscle. Less is known about the regulation of lipid metabolism in the liver during and post-exercise. Methodology/Principal Findings We performed an ultra fast liquid chromatography-mass spectrometry (UFLC-MS) based lipidomics analysis of liver tissue samples obtained from C57Bl/6J mice immediately after a 60 min treadmill run of moderate intensity, and after 3 h of recovery. The PLS-DA scores plot for 115 quantified lipid molecular species revealed a clear separation of the hepatic lipid profile of sedentary from recovering mice, but not from mice immediately after running. 21 lipid species were considered to be most responsible for the difference in the hepatic lipid profiles, including 17 triacylglycerides (TG), one lysophosphatidylcholine (LPC) and three phosphatidylcholines (PC). TG species were found to be more abundant in the recovery phase, while PC species were decreased. The degree of accumulation of individual TG species correlated well with the amount of theoretical energy stored whereas no increase was found for TG species containing only saturated or one monounsaturated fatty acid. Total liver TG content as assayed by an enzymatic method was increased to 163% in the recovery phase, while it was significantly decreased in skeletal muscle by the exercise bout and remained less in the recovery phase. Results from fasted and refed mice indicate that fasting-induced lipolysis was associated with a pronounced accumulation of hepatic TG, which is reversed by refeeding for 5 h. Thus food intake per se did not elevate hepatic TG. Conclusion These data indicate that high availability of FFA induced by endurance exercise or fasting resulted in a transient hepatic TG accumulation, while muscle TG content was decreased during exercise presumably due to increased muscle fatty acid oxidation.

Effect of Bisphenol A on Rat Metabolic Profiling Studied by Using Capillary Electrophoresis Time-of-Flight Mass Spectrometry

Bisphenol A (BPA), a chemical widely used in the manufacture of polycarbonate plastics, has raised considerable concern in recent decades because of its hormone-like properties. Whether BPA exposure is a health risk remains controversial in many countries. A metabolomics study based on capillary electrophoresis time-of-flight mass spectrometry (CE-TOF/MS) was performed to study the urine metabolic profiles of Sprague-Dawley rats fed with four dose levels of BPA (0, 1, 10, and 100 μg/kg body weight) for 45 days. Multivariate pattern recognition directly reflected the metabolic perturbations caused by BPA. On the basis of univariate analysis, 42 metabolites including amino acids, polyamines, nucleosides, organic acids, carbohydrates, pterins, polyphenols, and sugar phosphates were found as the most significantly differential metabolites. The marked perturbations were related with valine, leucine and isoleucine biosynthesis, D-glutamine and D-glutamate metabolism, etc. Significant alterations of neurotransmitters (glutamate, gamma-aminobutyric acid, and noradrenaline) and neurotransmitter-related metabolites (tyrosine, histamine, valine, and taurine) suggested that the toxic effects of small-dose BPA (below 50 mg/kg/day) may contribute to its interactions with the neuromediating system. Our study demonstrated that metabolomics may offer more specific insights into the molecular changes underlying the physiological effects of BPA.

Serum lipid profiling of patients with chronic hepatitis B, cirrhosis, and hepatocellular carcinoma by ultra fast LC/IT-TOF MS

In this study, an ultra fast LC/IT‐TOF MS (UFLC/IT‐TOF MS)‐based serum lipidomics method was employed to characterize the serum lipid profile of patients with chronic hepatitis B, cirrhosis, and hepatocellular carcinoma (HCC). After data collection and processing, 96 lipids including lysophosphatidylcholines, phosphatidylcholines, sphingomyelins, triacylglycerides, and cholesterol esters were identified and used for subsequent data analysis. Partial least squares‐discriminant analysis revealed that patients with liver diseases had distinctly different serum lipid profile from that of healthy controls; while cirrhosis and HCC patients had a similar serum lipid profile, but different from that of hepatitis patients. The ANOVA analysis found 75 of the 96 identified lipids to be abnormally regulated, among which most of these lipids were downregulated in cirrhosis and HCC patients compared with those of healthy controls and hepatitis patients, while hepatitis patients induced several lipids downregulated and others upregulated compared with those of healthy controls, indicating the aberrant lipid metabolism in patients with liver diseases. This work demonstrated the utility of UFLC/IT‐TOF MS‐based serum lipidomics as a powerful tool to investigate the lipid metabolism of liver diseases.

Large-scaled human serum sphingolipid profiling by using reversed-phase liquid chromatography coupled with dynamic multiple reaction monitoring of mass spectrometry: Method development and application in hepatocellular carcinoma

Sphingolipids are a family of bioactive molecules with high structural diversity and complexity. They not only serve as integral components of cellular membrane, but also play pivotal roles in signaling and other cellular events. It is desirable for the development of sensitive, robust and structural-specific analytical approaches enabling rapid determination of as many sphingolipid species as possible. Herein we present an analytical method for large-scaled profiling of sphigolipids in human serum, which consisted of an improved extraction protocol using tert-butyl methyl ether combined with mild alkaline hydrolysis, and an ultra high performance reversed-phase liquid chromatography-dynamic multiple reaction monitoring-mass spectrometric (RPLC-dynamic MRM-MS) method. In total 84 endogenous sphingolipid species covering six subcategories (i.e. free sphingoid base, dihydroceramide, ceramide, hexosylceramide, lactosylceramide, and sphingomyelin), were separated and quantified in a single run within 10min. A broad linear range over 2.5-4 orders of magnitude (r(2)>0.99), a limit of detection of 0.01-0.17pmol/mL, and a limit of quantitation of 0.02-0.42pmol/mL were obtained for each subcategory. Average recovery of each subcategory was within 85.6-95.6%. Median values of coefficient of variation (CV) of all detected 84 sphingolipids were 3.9% and 6.8% for intraday and interday precision, respectively. This method was exemplarily applied in a study regarding dysregulated sphingolipid homeostasis in hepatocellular carcinoma. The establishment of this method provides a useful tool for serum-based high throughput screening of sphingolipid biomarkers and mechanism investigation of sphingolipid metabolic regulation in human disease.

Metabolomics Identifies Biomarker Pattern for Early Diagnosis of Hepatocellular Carcinoma: from Diethylnitrosamine Treated Rats to Patients

Early diagnosis of hepatocellular carcinoma (HCC) remains challenging to date. Characteristic metabolic deregulations of HCC may enable novel biomarkers discovery for early diagnosis. A capillary electrophoresis-time of flight mass spectrometry (CE-TOF/MS)-based metabolomics approach was performed to discover and validate potential biomarkers for HCC from the diethylnitrosamine-induced rat hepatocarcinogenesis model to human subjects. Time series sera from the animal model were evaluated using multivariate and univariate analyses to reveal dynamic metabolic changes. Two independent human cohorts (populations I and II) containing 122 human serum specimens were enrolled for validations. A novel biomarker pattern of ratio creatine/betaine which reflects the balance of methylation was identified. This biomarker pattern achieved effective classification of pre-HCC and HCC stages in animal model. It was still effective in the diagnosis of HCC from high-risk patients with cirrhotic nodules, achieving AUC values of 0.865 and 0.905 for two validation cohorts, respectively. The diagnosis of small HCC from cirrhosis with an AUC of 0.928 highlighted the potential for early diagnosis. This ratio biomarker can also improve the diagnostic performance of α-fetoprotein (AFP). This study demonstrates the efficacy of present strategy for biomarker discovery, and the potential of metabolomics approach to provide novel insights for disease study.

Linking biological activity with herbal constituents by systems biology-based approaches: effects of Panax ginseng in type 2 diabetic Goto-Kakizaki rats

Although a number of animal experiments and clinical trials have investigated the effects of ginseng roots on diabetes, the relationship between their therapeutic effects on diabetes and the quality and the growth age of this herb have not yet been reported. This study systematically investigated the effects of 3- to 6-year-old ginseng roots on glycemic and plasma lipid control in a rat model of type 2 diabetes. Six groups of male Goto-Kakizaki (GK) rats received either metformin, 3- to 6-year-old ginseng roots, or no treatment. The treatments were administered twice daily for 9 weeks. A combined approach was used that involved applying liquid chromatography-mass spectrometry-based lipidomics, measuring biochemical parameters and profiling the components of ginseng roots of different ages. Compared to the untreated controls, treatment with 4- and 6-year-old ginseng roots significantly improved glucose disposal, and 5-year-old ginseng treatment significantly increased high density lipoprotein cholesterol. Treatment with 6-year-old ginseng significantly decreased total plasma triacylglyceride (TG) and very-low-density lipoprotein cholesterol and improved plasma glycated hemoglobin (HbA1c). In addition, treatment with 4- to 6-year-old ginseng influenced plasma lipidomics in diabetic GK rats by reducing TG lipid species. Metformin significantly reduced fasting blood glucose by 41% and reduced HbA1c by 11%, but showed no effects on the plasma lipid parameters. The present study demonstrates that ginseng roots show growth age-dependent therapeutic effects on hyperlipidemia and hyperglycemia in diabetic GK rats. These age-dependent effects may be linked with the variation in both the ratios and concentrations of specific bioactive ginsenosides in ginseng roots of different growth ages. This study introduced novel systems biology-based approaches for linking biological activities with potential active components in herbal mixtures.