Bei Yan

Differences in metabolite profile between blood plasma and serum.

In metabolomic research, blood plasma and serum have been considered to possess similar compositions and properties. Their perceived equivalence has resulted in researchers choosing arbitrarily between serum and plasma for analysis. Here, routine serum and plasma were prepared and their low-molecular-weight compounds were determined using gas chromatography/time-of-flight mass spectrometry. Principal components analysis was applied to process the acquired data, and marked differences in metabolite profiles were observed between serum and plasma. Of the 72 identified compounds, 36 (50%) discriminate serum from plasma, with 29 and 7 metabolites showing a significantly higher abundance (t test, P<0.05) in serum and plasma, respectively. Incubation of blood had distinct effects on the analyte peak areas, with the effects being more pronounced for plasma than for serum and more pronounced for a shorter incubation than for a longer incubation. These results highlight the importance in choosing serum or plasma as the analytical sample and in stipulating the incubation time. Because incubation affected the analyte peak areas less in serum than in plasma, we recommend serum as the sample of choice in metabolomic studies.

Sensitive determination of 20(S)-protopanaxadiol in rat plasma using HPLC–APCI-MS: Application of pharmacokinetic study in rats

20(S)-Protopanaxadiol (PPD), the main metabolite of protopanoxadiol type ginsenosides (e.g. Rg3 and Rh2), is a very promising anti-cancer drug candidate. To evaluate the pharmacokinetic property of PPD, we reported a reliable, sensitive and simple method utilizing liquid chromatography (HPLC)-atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) to determine PPD. PPD and the internal standard, panoxadiol (PD) were extracted from plasma with acetic ether, separated on a C18 reverse column, and then analyzed by APCI-MS. Targeting fragment ion at m/z 425 for both PPD and PD was monitored in selected-ion monitoring (SIM) mode. PPD can be quantitatively determined at the concentration as low as 1 ng/mL using 200 microL plasma. And the sensitive method showed excellent linearity over a range from 1 to 1000 ng/mL, high recovery, accuracy and precision at the concentrations of 2.5, 100.0 and 1000.0 ng/mL, respectively. The method was successfully applied to pharmacokinetic study of PPD in rats. Pharmacokinetic parameters were calculated and absolute bioavailability of PPD was 36.8+/-12.4%, at least ten times higher than that of Rg3 and Rh2, indicating its good absorption in gastrointestinal tract. It was further suggested that PPD be a promising anti-cancer candidate and probably responsible for the observed pharmacological activity of Rg3 and Rh2.

Global analysis of metabolites in rat and human urine based on gas chromatography/time-of-flight mass spectrometry

Sediment in urine may contain low-molecular-weight compounds that should be included in the analysis. To date, no systematic investigation has addressed this issue. We investigated three primary factors that influence the extraction efficiency of metabolites during preparation of urine samples for metabolomic research: centrifugation, pH, and extraction solvents. Obtained with the use of gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) technique and principal component analysis (PCA), our results indicate that (1) conventional centrifugation causes an apparent loss of some metabolites, indicating that urine samples for metabolomic research should not be centrifuged before procedures are undertaken to recover the metabolites; (2) pH adjustment has a large impact on the recovery of metabolites and is therefore not encouraged; (3) with design of experiment analysis, methanol and water yield the optimal extraction efficiency. Differences between rat and human urine were observed and are discussed. Ninety-nine metabolites identified in rat and human urine are presented. An efficient protocol is proposed for the pretreatment of urine samples.

23-Hydroxybetulinic acid from Pulsatilla chinensis (Bunge) Regel synergizes the antitumor activities of doxorubicin in vitro and in vivo.

UNLABELLED ETHNOPHARMACOLOGICAL REVELANCE: Pulsatilla chinensis (Bunge)Regel has been used as adjuvant in chemotherapy in traditional Chinese medicine. 23-Hydroxybetulinic acid, an isolated pentacyclic triterpene, is the major active constituent of Pulsatilla chinensis (Bunge) Regel. AIM OF THIS STUDY To evaluate the combinational anticancer effect of 23-hydroxybetulinic acid and doxorubicin in vitro and in vivo. MATERIALS AND METHODS The effect of combination treatment with 23-hydroxybetulinic acid and doxorubicin was evaluated with a quantitative combination index method based on the median-effect analysis in various cancer cell lines. And in vivo efficacy of combination chemotherapy was also evaluated using ICR mice bearing sarcoma 180 carcinoma tumors. RESULTS 23-Hydroxybetulinic acid showed a synergistic cytotoxic effect on multiple cancer cell lines by combined use with doxorubicin. In vivo studies further demonstrated that co-administration of 23-HBA significantly improved the sensitivity of the tumor to doxorubicin through increasing intra-tumor doxorubicin concentration and inhibiting doxorubicin-induced up-regulation of P-gp in tumor. CONCLUSION These results suggest that the combined therapy with 23-hydroxybetulinic acid and doxorubicin may be a new promising strategy to promote the clinical chemotherapy, which needs further verification.

Gas chromatography/time-of-flight mass spectrometry based metabonomic approach to differentiating hypertension-and age-related metabolic variation in spontaneously hypertensive rats

Metabonomics is a systematic approach to the study of in vivo metabolic profiles and therefore allows deep insight into and a better understanding of the pathogenesis of disease. To characterize the development of hypertension, a hypertensive animal model, the spontaneously hypertensive rat (SHR), and its normotensive control, the Wistar Kyoto (WKY) rat, were investigated and their blood plasma analyzed using the high-throughput metabolomic tool, gas chromatography/time-of-flight mass spectrometry (GC/TOFMS). A total of 187 peaks were quantitatively determined after deconvolution, and 78 of them were identified. Principal components analysis (PCA) and projection to latent structure partial least-squares discriminant analysis (PLS-DA) were used to process the GC/TOFMS data. The resulting mathematical models were further validated by cross-validation. Plasma compositional differences of many identified compounds showed hypertension-related variation between SHR and WKY rats, and age-related changes from 10 to 18 weeks for both the SHR and WKY rats. These compositional changes involved compounds such as hexadecanoic acid, linoleic acid, oleic acid, stearic acid, 3-hydroxybutyric acid, citric acid, threonic acid, tyrosine, tryptophan, threonine, phenylalanine, serine, ornithine, methionine, 3-hydroxyproline, creatinine, erythrose, myo-inositol, D-methylglucopyranoside, tocopherol, sitosterol, and nonesterified cholesterol. Significantly elevated free fatty acids (FFA) were observed in SHR relative to those in WKY rats, and their levels increased as the SHR aged from 10 to 18 weeks. The close correlation between FFA and hypertension suggests that FFA are potential biomarker candidates for hypertension and they may play an important role in the development of hypertension in SHR. It is also indicated that GC/TOFMS-based metabonomics is a powerful approach to identifying potential biomarkers and investigating the pathological processes of hypertension and the physiological developments of aging.

Metabonomic characterization of early atherosclerosis in hamsters with induced cholesterol

Atherosclerosis is a complicated and multifactorial disease, induced not only by genotype, but also, even more importantly, by environmental factors. Study on the metabolic perturbation of endogenous compounds may offer deeper insight into development of atherosclerosis. Gas chromatography/mass spectrometry (GC/MS)-based metabonomics was used to profile a metabolic fingerprint of serum obtained from hamsters with induced cholesterol. The deconvoluted GC/MS data were processed by multivariate statistical analysis tools, such as principal component analysis (PCA) and partial least squares projection to latent structure and discriminant analysis (PLS-DA). For the first time we showed a time-dependent development of the model animal from normal to hypercholesterolaemia, and further to early atherosclerosis. Twenty-one compounds were identified as markers involved in the development to atherosclerosis. Identification of the compounds suggests that amino acid metabolism and fatty acid oxidation are significantly perturbed following cholesterol overloading. The data provide novel information to approach the pathophysiological processes of the hypercholesterolaemia and atherosclerosis disease continuum.

Metabonomic profiling of liver metabolites by gas chromatography–mass spectrometry and its application to characterizing hyperlipidemia

The measurement of metabolites in tissues is of great importance in metabonomic research in the biomedical sciences, providing more relevant information than is available from systemic biofluids. The liver is the most important organ/tissue for most biochemical reactions, and the metabolites in the liver are of great interest to scientists. To develop an optimized extraction method and comprehensive profiling technique for liver metabolites, organic solvents of various compositions were designed using design of experiments to extract metabolites from the liver, and the metabolites were profiled by gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). The resolved peak areas were processed by principle components analysis, partial least-squares projections to latent structures, and discriminant analysis. The results suggest the highest extraction efficiency was for methanol-water, which maximized the majority of GC/TOF-MS responses. The optimal solvent was applied to extract metabolites in liver of hyperlipidemia hamster and the control. The GC/TOF-MS profiles of liver metabolites showed obvious differences between hyperlipidemic hamsters and controls. A comparison of liver and serum data from the same animals identified common biomarkers and presented complementary information. Our results suggest that liver metabonomics is a valuable technique and that the combined analysis of systematic biofluids and local tissues is meaningful and complementary, recovering more comprehensive metabonomic data than either analysis alone.

Metabonomic phenotype and identification of "heart blood stasis obstruction pattern" and "qi and yin deficiency pattern" of myocardial ischemia rat models.

The traditional Chinese medicine concepts of “Xinxueyuzuzheng (heart blood stasis obstruction pattern)” and “Qiyinliangxuzheng (qi and yin deficiency pattern)” for myocardial ischemia rat models were constructed in the present study. Endogenous metabolites in rat plasma were analyzed using the GC/TOF-MS-based metabonomic method. Significant metabolic differences were observed between the control and two model groups, and the three groups were distinguished clearly by pattern recognition. Compared with those of the control, the levels of hydroxyproline, threonic acid, glutamine and citric acid were strikingly up- or down-regulated in model rats. The metabolites contributing most to the classification between the two “pattern” rats were identified, such as valine, serine, threonine, ornithine, hydroxyproline, lysine, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, galactofuranose and inositol. These compounds were indicated as the potential biomarkers. The results suggested that the two “patterns” are involved in dysfunction in oxidative stress, energy metabolism and amino acid metabolism. These findings also provided the substantial foundation for exploring the scientific connotation of these two “Zhengxing (pattern types)” of myocardial ischemia, and “Bianzheng (pattern identification)”.

Simultaneous determination of GFA and its active metabolites in human plasma by liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic studies

A sensitive and rapid liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for simultaneous quantification of guanfu base A (GFA) and its metabolites guanfu base I (GFI) and guanfu alcohol-amine (AA) in human plasma with phenoprolamine hydrochloride (DDPH) as the internal standard. The analytes were extracted from human plasma by using liquid-liquid extraction with ethyl acetate and the LC separation was performed on a Diamonsil C(18) analytical column (150 mm x 2.1 mm i.d., 5 microm). The MS acquisition was performed in selected ion monitoring (SIM) mode of positive ions. Analysis was carried out in SIM mode at m/z 430.25 for GFA [M+H](+), m/z 388.25 for GFI [M+H](+), m/z 346.25 for AA [M+H](+) and m/z 344.20 for the IS DDPH [M+H](+). The calibration curves were linear over the range of 50-5000 ng/mL for GFA and 5-1000 ng/mL for GFI and AA, with coefficients of correlation above 0.999. The lower limit of quantification for GFA was 1 ng/mL, while for GFI and AA were both 5 ng/mL. The intra- and inter-day precisions (CV) of analysis were within 9%, and the accuracy ranged from 91% to 108%. The overall recoveries for GFA, GFI and AA were about 94.2%, 87.8% and 80.6%, respectively. The total LC-MS run-time was only 5.5 min. This quantitation method was successfully applied to the simultaneous determination of GFA and its metabolites in human plasma for the metabolic study and pharmacokinetic evaluation.

Metabonomic Approach to Evaluating Pharmacodynamics of Ginkgo biloba Extract on the Perturbed Metabolism in Hamsters with Atherosclerosis by High Fat Diet

Abstract Aim To investigate the effects of Ginkgo biloba extract (GBE) on the perturbed metabolism of a diet-induced atherosclerosis hamster model. Methods Gas chromatography coupled with time-of-flight mass spectrometry (GC/TOF-MS) spectroscopy was used to profile serum samples of hamsters induced by high fat diet for 0, 3, 6, 12 weeks, and hamsters administered with GBE in the meanwhile. Multivariate analyses were employed to identify treatment-related fingerprint and potential biomarkers regarding the anti-atherosclerosis effect and mechanisms of GBE. Results For the model animals, high fat diet (HFD) resulted in a gradual elevation of serum cholesterol and triglyceride, low-density lipoprotein cholesterol (LDL-C), lesion in aorta arch, and a dynamic trajectory of the metabonomic profile. GBE treatment did not only lead to a marked reduction in total cholesterol and LDL-C levels in hamster serum, but also showed regulatory effects on serum metabolome and restored their scores plot close to normal. Amongst the discriminatory metabolites in serum that characterize atherosclerosis, 8 metabolites restored to normal after treatment with GBE, including succinic acid, glyceric acid, linoleic acid, arachidonic acid, 1-monooleoylglycerol, β-tocopherol, cholest-5-ene, lysine, while 6 metabolites were regulated towards normal, including tyrosine, oleic acid, 2-monooleoylglycerol, γ-tocopherol, α-tocopherol and deoxycholic acid. Conclusion These results indicate that the effect of GBE on the metabolites is involved in lipid metabolism, bile acid synthesis and amino acids turn-over, and is indirect in anti-atherosclerosis effect of GBE. GC/MS-based metabonomics is a potential approach to exploring pharmacological mechanism of GBE effects and to the assessment of pharmacodynamics.