Live Tissue Imaging Reveals Distinct Transcellular Pathways for Organic Cations and Anions at the Blood-Cerebrospinal Fluid Barrier.

Formed by the choroid plexus epithelial (CPE) cells, the blood-cerebrospinal fluid barrier (BCSFB) plays an active role in removing drugs, toxins, and metabolic wastes from the brain. Several organic cation and anion transporters are expressed in the CPE cells, but how they functionally mediate transepithelial transport of organic cations and anions remain unclear. In this study, we visualized the transcellular transport of fluorescent organic cation and organic anion probes using live tissue imaging in freshly isolated mouse choroid plexuses (CPs). The cationic probe, 4-[4-(dimethylamino)phenyl]-1-methylpyridinium iodide (IDT307) was transported into CPE cells at the apical membrane and highly accumulated in mitochondria. Consistent with the lack of expression of organic cation efflux transporters, there was little efflux of IDT307 into the blood capillary space. Furthermore, IDT307 uptake and intracellular accumulation was attenuated by approximately 70% in CP tissues from mice with targeted deletion of the plasma membrane monoamine transporter (Pmat). In contrast, the anionic probe fluorescein-methotrexate (FL-MTX) was rapidly transported across the CPE cells into the capillary space with little intracellular accumulation. Rifampicin, an inhibitor of organic anion transporting polypeptides (OATPs), completely blocked FL-MTX uptake into the CPE cells whereas MK-571, a pan-inhibitor of multidrug resistance associated proteins (MRPs), abolished basolateral efflux of FL-MTX. In summary, our results suggest distinct transcellular transport pathways for organic cations and anions at the BCSFB and reveal a pivotal role of PMAT, OATP and MRP transporters in organic cation and anion transport at the blood-cerebrospinal fluid interface. SIGNIFICANCE STATEMENT: Live tissue imaging revealed that while organic cations are transported from the cerebrospinal fluid (CSF) into the choroid plexus epithelial cells by plasma membrane monoamine transporter without efflux into the blood, amphipathic anions in the CSF are efficiently transported across the BCSFB through the collaborated function of apical organic anion transporting polypeptides and basolateral multidrug resistance associated proteins. These findings contribute to a mechanistic understanding of the molecular and cellular pathways for choroid plexus clearance of solutes from the brain.

Ginsenoside 20(S)-Rh2 promotes cellular pharmacokinetics and intracellular antibacterial activity of levofloxacin against Staphylococcus aureus through drug efflux inhibition and subcellular stabilization.

Intracellular Staphylococcus aureus (S. aureus) often causes clinical failure and relapse after antibiotic treatment. We previously found that 20(S)-ginsenoside Rh2 [20(S)-Rh2] enhanced the therapeutic effect of quinolones in a mouse model of peritonitis, which we attributed to the increased concentrations of quinolones within bacteria. In this study, we investigated the enhancing effect of 20(S)-Rh2 on levofloxacin (LVF) from a perspective of intracellular bacteria. In S. aureus 25923-infected mice, coadministration of LVF (1.5 mg/kg, i.v.) and 20(S)-Rh2 (25, 50 mg/kg, i.g.) markedly increased the survival rate, and decreased intracellular bacteria counts accompanied by increased accumulation of LVF in peritoneal macrophages. In addition, 20(S)-Rh2 (1, 5, 10 µM) dose-dependently increased the uptake and accumulation of LVF in peritoneal macrophages from infected mice without drug treatment. In a model of S. aureus 25923-infected THP-1 macrophages, we showed that 20(S)-Rh2 (1, 5, 10 µM) dose-dependently enhanced the intracellular antibacterial activity of LVF. At the cellular level, 20(S)-Rh2 increased the intracellular accumulation of LVF by inhibiting P-gp and BCRP. PK-PD modeling revealed that 20(S)-Rh2 altered the properties of the cell but not LVF. At the subcellular level, 20(S)-Rh2 did not increase the distribution of LVF in lysosomes but exhibited a stronger sensitizing effect in acidic environments. Molecular dynamics (MD) simulations showed that 20(S)-Rh2 improved the stability of the DNA gyrase-LVF complex in lysosome-like acidic conditions. In conclusion, 20(S)-Rh2 promotes the cellular pharmacokinetics and intracellular antibacterial activities of LVF against S. aureus through efflux transporter inhibition and subcellular stabilization, which is beneficial for infection treatment.

Effect of Pregnancy on Paroxetine-Induced Adiposity and Glucose Intolerance in Mice.

Long-term use of selective serotonin reuptake inhibitors (SSRIs) targeting the serotonin transporter (SERT) has been suggested to be associated with an increased risk for obesity and type 2 diabetes. Previously, using a murine knockout model of SERT, we showed that estrogen suppression is involved in SERT deficiency-induced obesity and glucose intolerance in nonpregnant mice. The present study investigated the effects of chronic paroxetine treatment on adiposity and glucose tolerance in mice before and during pregnancy. Chronic paroxetine treatment in nonpregnant mice resulted in visceral adiposity and glucose intolerance accompanied by reduced circulating 17ß-estradiol levels and ovarian expression of the aromatase (CYP19a1). Remarkably, pregnancy significantly reduced adiposity and improved glucose tolerance in paroxetine-treated mice by rebooting ovarian CYP19a1 expression and 17ß-estradiol production. These effects appear to be reversible as ovarian CYP19a1 expression and circulating 17ß-estradiol returned to prepregnancy levels soon after parturition. As in pregnant mice, 17ß-estradiol replacement treatment in nonpregnant mice reduced paroxetine-induced adiposity. Our findings further suggested that modulation of estrogen synthesis underlies the observed metabolic adverse effects of SSRIs. Although our data revealed a transient reversal effect of pregnancy on SSRI-induced metabolic abnormalities, these observations are experimental and limited to mice. The use of SSRIs during human pregnancy should be cautioned because of potential adverse effects to the fetuses.

Transporter-mediated natural product-drug interactions for the treatment of cardiovascular diseases.

The growing use of natural products in cardiovascular (CV) patients has been greatly raising the concerns about potential natural product-CV drug interactions. Some of these may lead to unexpected cardiovascular adverse effects and it is, therefore, essential to identify or predict potential natural product-CV drug interactions, and to understand the underlying mechanisms. Drug transporters are important determinants for the pharmacokinetics of drugs and alterations of drug transport has been recognized as one of the major causes of natural product-drug interactions. In last two decades, many CV drugs (e.g., angiotensin II receptor blockers, beta-blockers and statins) have been identified to be substrates and inhibitors of the solute carrier (SLC) transporters and the ATP-binding cassette (ABC) transporters, which are two major transporter superfamilies. Meanwhile, in vitro and in vivo studies indicate that a growing number of natural products showed cardioprotective effects (e.g., gingko biloba, danshen and their active ingredients) are also substrates and inhibitors of drug transporters. Thus, to understand transporter-mediated natural product-CV drug interactions is important and some transporter-mediated interactions have already shown to have clinical relevance. In this review, we review the current knowledge on the role of ABC and SLC transporters in CV therapy, as well as transporter modulation by natural products used in CV diseases and their induced natural product-CV drug interactions through alterations of drug transport. We hope our review will aid in a comprehensive summary of transporter-mediated natural product-CV drug interactions and help public and physicians understand these type of interactions.

Cerebral infarction due to malposition of cervical pedicle screw: A case report.

RATIONALE: Malposition of cervical pedicle screw (CPS) has a risk of vertebral artery (VA) injury which sometimes may cause unexpected and catastrophic outcome. A rare case of delayed onset of cerebral infarction caused by malposition of CPS was reported. PATIENT CONCERNS: A 23-year-old man who underwent a posterior cervical reduction and fusion of C4-5 using CPS fixation and allograft for cervical spine injury is presented. The patient suffered progressively weakness and numbness for both of upper and lower extremities 1 day after the operation. Computed tomography scans revealed bilateral occupation of the pedicle screws in the foramen of C4 and C5 and the magnetic resonance imaging (MRI) displayed several areas of infarction in the brainstem and cerebellum. DIAGNOSES: Plain radiographs of the cervical spine revealed the C4 vertebral body and MRI displayed a disruption of the anterior longitudinal ligament on the level of C4-5 and severe injury to the soft tissues of the cervical spine at admission. Brainstem and cerebellum infarction was diagnosed at postoperative. INTERVENTION: A revision surgery was decided to remove all of the pedicle screws and place lateral mass screws instead. OUTCOMES: The patient felt better on his all of 4 extremities following revision surgery. Fortunately, he was neurologically close to normal at a 3-month follow-up. LESSONS: Delayed onset of cerebral infarction is rarely reported complication caused by malposition of CPS. When a CPS perforates the transverse foramen and causes symptom of cerebral infarction, a revision surgery in time is strongly recommended to prevent further sequelae.

Serotonin transporter deficiency drives estrogen-dependent obesity and glucose intolerance.

Depression and use of antidepressant medications are both associated with increased risk of obesity, potentially attributed to a reduced serotonin transporter (SERT) function. However, how SERT deficiency promotes obesity is unknown. Here, we demonstrated that SERT -/- mice display abnormal fat accumulation in both white and brown adipose tissues, glucose intolerance and insulin resistance while exhibiting suppressed aromatase (Cyp19a1) expression and reduced circulating 17ß-estradiol levels. 17ß-estradiol replacement in SERT -/- mice reversed the obesity and glucose intolerance, supporting a role for estrogen in SERT deficiency-associated obesity and glucose intolerance. Treatment of wild type mice with paroxetine, a chemical inhibitor of SERT, also resulted in Cyp19a1 suppression, decreased circulating 17ß-estradiol levels, abnormal fat accumulation, and glucose intolerance. Such effects were not observed in paroxetine-treated SERT -/- mice. Conversely, pregnant SERT -/- mice displayed normalized estrogen levels, markedly reduced fat accumulation, and improved glucose tolerance, which can be eliminated by an antagonist of estrogen receptor α (ERα). Together, these findings support that estrogen suppression is involved in SERT deficiency-induced obesity and glucose intolerance, and suggest approaches to restore 17ß-estradiol levels as a novel treatment option for SERT deficiency associated obesity and metabolic abnormalities.

Involvement of organic cation transporter 3 (Oct3/Slc22a3) in the bioavailability and pharmacokinetics of antidiabetic metformin in mice.

Metformin is widely used for the treatment of type II diabetes mellitus. It was reported to be substrate of OCT3/Oct3, which is expressed in the brush boarder membrane of the enterocytes. However, the role of OCT3/Oct3 in the intestinal absorption process of metformin remains obscure. In the present study, we aimed to clarify the impact of Oct3 on the oral bioavailability and pharmacokinetics of metformin in mice, by means of in vivo pharmacokinetic study using wild-type (Oct3+/+) and Oct3-knockout (Oct3-/-) mice. When metformin (8.0 mg/kg) was intravenously administered to male Oct3+/+ and Oct3-/- mice, AUC0-∞ of metformin was evaluated to be 659 ± 133 µg h/mL and 734 ± 213 µg h/mL, respectively. In the case of orally administered metformin (15 mg/kg), AUC0-∞ was 578 ± 158 µg h/mL and 449 ± 101 µg h/mL in Oct3+/+ and Oct3-/- mice, respectively. Based on these pharmacokinetic parameters, absolute bioavailability (F) of metformin in Oct3+/+ mice was evaluated as 46.8%, and it was significantly decreased to 32.6% in Oct3-/- mice. Taking into account the fact that metformin undergoes negligible metabolism, these results imply that intestinal absorption of metformin is mediated at least in part by Oct3 in mice.

Characterization of the Cytochrome P450 epoxyeicosanoid pathway in non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is an emerging public health problem without effective therapies. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into bioactive epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory and protective effects. However, the functional relevance of the CYP epoxyeicosanoid metabolism pathway in the pathogenesis of NASH remains poorly understood. Our studies demonstrate that both mice with methionine-choline deficient (MCD) diet-induced NASH and humans with biopsy-confirmed NASH exhibited significantly higher free EET concentrations compared to healthy controls. Targeted disruption of Ephx2 (the gene encoding for soluble epoxide hydrolase) in mice further increased EET levels and significantly attenuated MCD diet-induced hepatic steatosis, inflammation and injury, as well as high fat diet-induced adipose tissue inflammation, systemic glucose intolerance and hepatic steatosis. Collectively, these findings suggest that dysregulation of the CYP epoxyeicosanoid pathway is a key pathological consequence of NASH in vivo, and promoting the anti-inflammatory and protective effects of EETs warrants further investigation as a novel therapeutic strategy for NASH.

Urinary metabolomic study of systemic lupus erythematosus based on gas chromatography/mass spectrometry.

Systemic lupus erythematosus (SLE) is an autoimmune disease with heterogeneous organ and system manifestations. In this study, urinary metabolic alterations related to SLE were investigated by performing gas chromatography/mass spectrometry (GC/MS) based metabolomics and multivariate statistical analysis. Patients with SLE and healthy controls could be clearly differentiated in view of the metabolic abnormity in urine. Among 70 identified endogenous metabolites, 23 metabolites were dramatically increased in SLE patients, which involved in several key metabolic pathways including energy metabolism, nucleotide metabolism, oxidative stress and gut-microbiome-derived metabolism. This noninvasive and GC/MS-based metabolomic technique is a promising and potent strategy for identifying novel biomarkers and understanding pathogenesis of SLE. Copyright © 2016 John Wiley & Sons, Ltd.

Serum metabolomic profiling in patients with systemic lupus erythematosus by GC/MS.

OBJECTIVES: The aim of this study is to characterize the serum metabolic profiles of patients with systemic lupus erythematosus (SLE) using metabolomics. METHODS: Serum samples were collected from patients with SLE (n = 80) and gender- and age-matched healthy controls (n = 57). Metabolite profiles were performed with gas chromatography-mass spectrometry in conjunction with multivariate statistical analysis, and possible biomarker metabolites were identified. RESULTS: SLE and disease severity-related metabolic phenotypes were identified in sera. Parameters of the metabolomic model were correlated with SLEDAI (SLE disease activity index) scores in SLE. The metabolic signature of SLE patients comprised metabolite changes associated with amino acid turnover or protein biosynthesis, saccharometabolism, lipid metabolism, and gut microbial metabolism. Disease activity-related alterations included glutamate, 2-hydroxyisobutyrate, citrate, glycerol, linoleic acid, and propylparaben metabolites. Parts of endogenous metabolites related to SLE had the relationship with serum immunological parameters and organ manifestations. Moreover, receiver operating characteristic curve analysis revealed a higher diagnosis accuracy of endogenous metabolites. CONCLUSIONS: Our study distinguished serum metabotypes associated with SLE and disease activities. The implementation of this metabolomic strategy may help to develop biochemical insight into the metabolic alterations in SLE.

A metabolomic and pharmacokinetic study on the mechanism underlying the lipid-lowering effect of orally administered berberine.

Clinical and animal studies demonstrated that orally administered berberine had a distinct lipid-lowering effect. However, pharmacokinetic studies showed that berberine was poorly absorbed into the body so the levels of berberine in the blood and target tissues were far below the effective concentrations revealed. To probe the underlying mechanism, the effect of berberine on the biological system was studied on a high-fat-diet-induced hamster hyperlipidemia model. Our results showed that intragastrically-administered berberine was poorly absorbed into circulation and most berberine accumulated in gut content. Although the bioavailability of intragastrically administered berberine was much lower than that of intraperitoneally administered berberine, it had a stronger lipid-lowing effect, indicating that the gastrointestinal tract is a potential target for the hypolipidemic effect of berberine. A metabolomic study on both serum and gut content showed that orally administered berberine significantly regulated molecules involved in lipid metabolism, and increased the generation of bile acids in the hyperlipidemic model. DNA analysis revealed that the orally administered berberine modulated the gut microbiota, and berberine showed a significant inhibition of the 7α-dehydroxylation conversion of cholic acid to deoxycholic acid, indicating a decreased elimination of bile acids in the gut. However, in model hamsters, elevated bile acids failed to downregulate the expression and function of CYP7A1 in a negative feedback loop. It was suggested that the hypocholesterolemic effect of orally administered berberine involves modulating the turnover of bile acids and the farnesoid X receptor signal pathway.

Correction: A metabolomic and pharmacokinetic study on the mechanism underlying the lipid-lowering effect of orally administered berberine.

Correction for 'A metabolomic and pharmacokinetic study on the mechanism underlying the lipid-lowering effect of orally administered berberine' by Shenghua Gu et al., Mol. BioSyst., 2015, DOI: .

The cytochrome P450 epoxygenase pathway regulates the hepatic inflammatory response in fatty liver disease.

Fatty liver disease is an emerging public health problem without effective therapies, and chronic hepatic inflammation is a key pathologic mediator in its progression. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory effects. Although promoting the effects of EETs elicits anti-inflammatory and protective effects in the cardiovascular system, the contribution of CYP-derived EETs to the regulation of fatty liver disease-associated inflammation and injury is unknown. Using the atherogenic diet model of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), our studies demonstrated that induction of fatty liver disease significantly and preferentially suppresses hepatic CYP epoxygenase expression and activity, and both hepatic and circulating levels of EETs in mice. Furthermore, mice with targeted disruption of Ephx2 (the gene encoding soluble epoxide hydrolase) exhibited restored hepatic and circulating EET levels and a significantly attenuated induction of hepatic inflammation and injury. Collectively, these data suggest that suppression of hepatic CYP-mediated EET biosynthesis is an important pathological consequence of fatty liver disease-associated inflammation, and that the CYP epoxygenase pathway is a central regulator of the hepatic inflammatory response in NAFLD/NASH. Future studies investigating the utility of therapeutic strategies that promote the effects of CYP-derived EETs in NAFLD/NASH are warranted.

Functional characterization of cytochrome P450-derived epoxyeicosatrienoic acids in adipogenesis and obesity.

Adipogenesis plays a critical role in the initiation and progression of obesity. Although cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) have emerged as a potential therapeutic target for cardiometabolic disease, the functional contribution of EETs to adipogenesis and the pathogenesis of obesity remain poorly understood. Our studies demonstrated that induction of adipogenesis in differentiated 3T3-L1 cells (in vitro) and obesity-associated adipose expansion in high-fat diet (HFD)-fed mice (in vivo) significantly dysregulate the CYP epoxygenase pathway and evoke a marked suppression of adipose-derived EET levels. Subsequent in vitro experiments demonstrated that exogenous EET analog administration elicits potent anti-adipogenic effects via inhibition of the early phase of adipogenesis. Furthermore, EET analog administration to mice significantly mitigated HFD-induced weight gain, adipose tissue expansion, pro-adipogenic gene expression, and glucose intolerance. Collectively, these findings suggest that suppression of EET bioavailability in adipose tissue is a key pathological consequence of obesity, and strategies that promote the protective effects of EETs in adipose tissue offer enormous therapeutic potential for obesity and its downstream pathological consequences.

An in vitro metabolic system of gut flora and the metabolism of ginsenoside Rg3 and cholic acid.

For orally administered drugs, the metabolism of a drug by the gut flora plays an important role in the bioavailability, activation and disposition of the drug in vivo. However, no in vitro system is currently available to evaluate the metabolism of a drug by the gut flora before the drug is absorbed into the body. This paper presents an in vitro metabolic system in an anaerobic environment that could be used to evaluate the metabolism of an endogenous compound, cholic acid, and a xenobiotic compound, ginsenoside Rg3. We showed that the proliferation of the anaerobic bacteria of the gut content of hamsters produced a similar composition of gut flora in a culture medium for yeast to that in vivo. Incubation of ginsenoside Rg3 and cholic acid in the anaerobic in vitro system efficiently produced the metabolites Rh2 and deoxycholic acid, respectively, similar to those seen in the gut content in vivo. In comparison with in vivo analysis, this anaerobic in vitro metabolic system is convenient, reproducible, economic and animal saving, and can easily be applied to assess the transformation and disposition of a drug before it enters into the circulatory system.

Metabolomic approach to evaluating adriamycin pharmacodynamics and resistance in breast cancer cells.

Continuous exposure of breast cancer cells to adriamycin induces high expression of P-gp and multiple drug resistance. However, the biochemical process and the underlying mechanisms for the gradually induced resistance are not clear. To explore the underlying mechanism and evaluate the anti-tumor effect and resistance of adriamycin, the drug-sensitive MCF-7S and the drug-resistant MCF-7Adr breast cancer cells were used and treated with adriamycin, and the intracellular metabolites were profiled using gas chromatography mass spectrometry. Principal components analysis of the data revealed that the two cell lines showed distinctly different metabolic responses to adriamycin. Adriamycin exposure significantly altered metabolic pattern of MCF-7S cells, which gradually became similar to the pattern of MCF-7Adr, indicating that metabolic shifts were involved in adriamycin resistance. Many intracellular metabolites involved in various metabolic pathways were significantly modulated by adriamycin treatment in the drug-sensitive MCF-7S cells, but were much less affected in the drug-resistant MCF-7Adr cells. Adriamycin treatment markedly depressed the biosynthesis of proteins, purines, pyrimidines and glutathione, and glycolysis, while it enhanced glycerol metabolism of MCF-7S cells. The elevated glycerol metabolism and down-regulated glutathione biosynthesis suggested an increased reactive oxygen species (ROS) generation and a weakened ability to balance ROS, respectively. Further studies revealed that adriamycin increased ROS and up-regulated P-gp in MCF-7S cells, which could be reversed by N-acetylcysteine treatment. It is suggested that adriamycin resistance is involved in slowed metabolism and aggravated oxidative stress. Assessment of cellular metabolomics and metabolic markers may be used to evaluate anti-tumor effects and to screen for candidate anti-tumor agents.

Pharmacokinetic interactions between 20(S)-ginsenoside Rh2 and the HIV protease inhibitor ritonavir in vitro and in vivo.

AIM: 20(S)-Ginsenoside Rh2 (Rh2) has shown potent inhibition on P-glycoprotein (P-gp), while most HIV protease inhibitors are both substrates and inhibitors of P-gp and CYP3A4. The aim of this study was to investigate the potential pharmacokinetic interactions between Rh2 and the HIV protease inhibitor ritonavir. METHODS: The effects of Rh2 on the cellular accumulation and transepithelial transport of ritonavir were studied in Caco-2 and MDCK-MDR1 cells. Male rats were administered Rh2 (25 or 60 mg/kg, po) or Rh2 (5 mg/kg, iv), followed by ritonavir (25 mg/kg, po). The P-gp inhibitors verapamil (20 mg/kg, po) or GF120918 (5 mg/kg, po) were used as positive controls. The concentrations of ritonavir in plasma, bile, urine, feces and tissue homogenates were analyzed using LC-MS. RESULTS: Rh2 (10 µmol/L) significantly increased the accumulation and inhibited the efflux of ritonavir in Caco-2 and MDCK-MDR1 cells, as verapamil did. But Rh2 did not significantly alter ritonavir accumulation or transport in MDCK-WT cells. Intravenous Rh2 significantly increased the plasma exposure of ritonavir while reducing its excretion in the bile, and oral verapamil or GF120918 also increased plasma exposure of ritonavir but without changing its excretion in the bile. Interestingly, oral Rh2 at both doses did not significantly change the plasma profile of ritonavir. Moreover, oral Rh2 (25 mg/kg) significantly elevated the ritonavir concentration in the hepatic portal vein, and markedly increased its urinary excretion and tissue distribution, which might counteract the elevated absorption of ritonavir. CONCLUSION: Rh2 inhibits the efflux of ritonavir through P-gp in vitro. The effects of Rh2 on ritonavir exposure in vivo depend on the administration route of Rh2: intravenous, but not oral, administration of Rh2 significantly increased the plasma exposure of ritonavir.

[Evaluation of myocardial ischemia rat model based on metabonomic method of small molecule metabolites of plasma and cardiac muscle].

Isoproterenol (ISO)-induced myocardial ischemia animal model has been widely applied to the study of myocardial ischemia and evaluation of drug efficacy. Metabolic profiling of endogenous compounds can make a deep insight into biochemical process of the ISO-induced myocardial ischemia rats. Herein, rats were treated with ISO (2 mg x kg(-1)) for 10 days. After the model was established by measuring myocardial histopathology and plasma creatine kinase, GC/TOF-MS was used to determine endogenous metabolites in plasma and cardiac muscle of rats, and pattern recognition was used to process the data. Results showed that the plasma metabolic profiling of ISO-induced myocardial ischemia rats was significantly different from that of the control, and it had the tendency to the normal state after the discontinue of ISO injection. Besides, the cardiac muscle of rats treated with ISO for 10 days and the normal cardiac muscle could also be separated clearly. The potential biomarkers in plasma and cardiac muscle of model rats had homogeneity and their own specialty. Biochemical metabolic pathway analysis indicated that this myocardial ischemia model was involved in the alternation of energy metabolism, saccharometabolism, lipid metabolism, nucleoside metabolism and amino acid metabolism, and in relationship with oxidative stress. These findings revealed that metabonomics may be a promising tool to evaluate myocardial ischemia rat model induced by ISO and could further extend the study of pharmacodynamic action of drugs at the molecular level.

GC/TOFMS analysis of metabolites in serum and urine reveals metabolic perturbation of TCA cycle in db/db mice involved in diabetic nephropathy.

Early diagnosis of diabetic nephropathy (DN) is difficult although it is of crucial importance to prevent its development. To probe potential markers and the underlying mechanism of DN, an animal model of DN, the db/db mice, was used and serum and urine metabolites were profiled using gas chromatography/time-of-flight mass spectrometry. Metabolic patterns were evaluated based on serum and urine data. Principal component analysis of the data revealed an obvious metabonomic difference between db/db mice and controls, and db/db mice showed distinctly different metabolic patterns during the progression from diabetes to early, medium, and later DN. The identified metabolites discriminating between db/db mice and controls suggested that db/db mice have perturbations in the tricarboxylic acid cycle (TCA, citrate, malate, succinate, and aconitate), lipid metabolism, glycolysis, and amino acid turnover. The db/db mice were characterized by acidic urine, high TCA intermediates in serum at week 6 and a sharp decline thereafter, and gradual elevation of free fatty acids in the serum. The sharp drop of serum TCA intermediates from week 6 to 8 indicated the downregulated glycolysis and insulin resistance. However, urinary TCA intermediates did not decrease in parallel with those in the serum from week 6 to 10, and an increased portion of TCA intermediates in the serum was excreted into the urine at 8, 10, and 12 wk than at 6 wk, indicating kidney dysfunction occurred. The relative abundances of TCA intermediates in urine relative to those in serum were suggested as an index of renal damage.