NMR-based metabonomic study of transgenic maize

The aim of this research was to verify the possibility of identifying and classifying maize seeds obtained from transgenic plants, in different classes according to the modification, on the basis of the concerted variation in metabolite levels detected by NMR spectra. It was possible to recognise the discriminant metabolites of transgenic samples as well as to classify non-a priori defined samples of maize. It is important to underline that the obtained results are useful to point out the metabolic consequences of a specific genic modification on a plant, without using a targeted analysis of the different metabolites, in fact it was possible to classify the seeds also without the complete assignment of the spectra. The analysis was performed by applying multivariate techniques (principal component analysis and partial least squares-discriminant analysis) to NMR data.     

NMR-based metabonomic study of Chinese medicine Gegen Qinlian Decoction as an effective treatment for type 2 diabetes in rats

Metabonomic profiles of the type 2 diabetic rats induced by streptozotocin and high-sugar, high fat diet on the treatment of Gegen Qinlian Decoction (GQD) for 9 weeks were investigated. Rats were randomly divided into five groups: normal control (NC), type 2 diabetes (DM), metformin hydrochloric, GQD in high and low dosages. Plasma samples for ¹H NMR-based metabolomic research, serum samples for clinical biochemistry, and liver and pancreas tissues for histopathology test were collected. Compared with NC rats, metabolic pathways of DM rats were revealed to be altered by pattern analyses of plasma NMR data, which was further correlated with serum biochemistry. Cross-validated scores mean trajectory derived from PLS-DA of NMR spectra demonstrated that GQD significantly restored the abnormal metabolic state in the long run, more potent than metformin hydrochloric. Detailed analysis of the altered metabolite levels indicated that GQD significantly ameliorated the disturbance in glucose metabolism, tricarboxylic acid cycle, lipid metabolism, amino acid metabolism and gut microbial metabolism and N-acetyl group metabolism. The results confirmed the hypoglycemic efficacy of GQD and its ability to ameliorate the diabetic symptoms in a global scale. NMR-based metabonomics approach is helpful for the further understanding of diabetes-related mechanisms.     

An NMR-based metabonomic investigation of the subacute effects of melamine in rats

The subacute toxic effects of 28 days of exposure to three dosages (250, 500, 1000 mg/kg/day) of melamine on Wistar rats were investigated using nuclear magnetic resonance spectra, histopathological examination, and biochemical analysis. Rats treated with melamine developed adverse health effects compared to the controls, including decrease in body weight and kidney damage. Blood biochemical analysis showed that the blood urea nitrogen and creatinine increased distinctly compared to the control group. Urinary metabonomic analysis indicated that melamine caused an increase in succinate and citrate. Serum metabonomic analysis showed that the lowest dose led to an increase in dimethylglycine, N-acetylglycoprotein (NAC), accompanied by a decrease in taurine and glucose. Rats treated with the highest dose developed high levels of serum choline and 3-hydroxybutyrate (3-HB) together with low lactate levels. Metabonomic analysis of liver tissue indicated that melamine caused an increase in NAC, choline, and creatine, accompanied by a decrease in lactate, trimethylamine-N-oxide, glutamate, and glucose. All three dosages resulted in an increase in glutamate, lactate, choline, glucose, and animo acids and a decrease in 3-HB and pyruvate in aqueous kidney extract. These results indicate that melamine not only caused renal disfunction but also disturbed the liver's glucose, protein, and nitrogen metabolism.     

Comparison of serum metabolite compositions between obese and lean growing pigs using an NMR-based metabonomic approach

Childhood obesity has become a prevalent risk to health of children and teenagers. To develop biomarkers in serum for altered lipid metabolism, genetically obese (Ningxiang strain) and lean (Duroc×Landrace×Large Yorkshire strain) growing pigs were used as models to identify potential differences in the serum metabonome between the two strains of pigs after consuming the same diet for 46 days. At the end of the study, pigs were euthanized for analysis of the serum metabonome and determination of body composition. Obese pigs had higher fat mass (42.3±8.8% vs. 21.9±4.5%) and lower muscle mass (35.4±4.5% vs. 58.9±2.5%) than lean pigs (P<.01). Serum concentrations of insulin and glucagon were higher (P<.02) in obese than in lean pigs. With the use of an NMR-based metabonomic technology, orthogonal projection to latent structure with discriminant analysis showed that serum HDL, VLDL, lipids, unsaturated lipids, glycoprotein, myo-inositol, pyruvate, threonine, tyrosine and creatine were higher in obese than in lean pigs (P<.05), while serum glucose and urea were lower in obese pigs (P<.05). In addition, changes in gut microbiota-related metabolites, including trimethylamine-N-oxide and choline, were observed in sera of obese pigs relatively to lean pigs (P<.05). These novel findings indicate that obese pigs have distinct metabolism, including lipogenesis, lipid oxidation, energy utilization and partition, protein and amino acid metabolism, and fermentation of gastrointestinal microbes, compared with lean pigs. The obese Ningxiang pig may be a useful model for childhood obesity research.     

NMR-based metabonomic evaluation of livers from rats chronically treated with tamoxifen,mestranol, and phenobarbital

In this study, we look at the metabolic effects of long-term dosing with tamoxifen, mestranol or phenobarbital on the liver. Tamoxifen, mestranol and phenobarbital have all been reported to act as promoters of hepatic tumors. While tamoxifen and mestranol are known to have estrogenic activity, in the liver phenobarbital is a non-estrogenic compound. Aqueous and lipophilic liver extracts from control and chronically treated Fisher 344 rats were evaluated by nuclear magnetic resonance spectroscopy (NMR). In both the aqueous and lipophilic sample sets, the estrogenic action of mestranol appears to be responsible for the clustering of these samples with those animals treated with tamoxifen. Phenobarbital does not have estrogenic activity and, therefore, clusters away from the estrogenic and control groups. In the lipophilic samples, the fatty acid peak (CH₂)_n was higher in tamoxifen-treated rats than in control, phenobarbital- or mestranol-treated rats. In the aqueous samples, serine and choline levels were higher in phenobarbital-treated rats than controls, which may be an indication that the folate–homocysteine metabolic pathways were altered.     

Dynamic 1H NMR-based extracellular metabonomic analysis of oligodendroglia cells infected with herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) is a large, neurotropic, double-stranded DNA virus that establishes a lifelong latent infection in neurons and glial cells. Previous studies reveal that several metabolic perturbations are associated with HSV-1 infection. However, the extracellular metabolic alterations associated with HSV-1 infection have not been systematically profiled in human cells. Here, a proton nuclear magnetic resonance-based metabonomic approach was applied to differentiate the extracellular metabonomic profiles of HSV-1 infected human oligodendroglia cells (n = 18) and matched control cells (n = 18) at three time points (12, 24, and 36 h post-infection). Resulting spectra were analyzed by chemometric and statistical methods. Metabonomic profiling revealed perturbations in 21 extracellular metabolites. Partial least squares discriminant analysis demonstrated that the whole metabolic patterns enabled statistical discrimination between HSV-1 infected human oligodendroglia cells and control cells. Eight extracellular metabolites, seven of which were amino acids, were primarily responsible for score plot discrimination between HSV-1 infected human oligodendroglia cells and control cells at 36 h post-infection: alanine, glycine, isoleucine, leucine, glutamate, glutamine, histidine, and lactate. HSV-1 infection alters amino acid metabolism in human oligodendroglia cells cultured in vitro. HSV-1 infection may disturb these host cellular pathways to support viral replication. Through elucidating the extracellular metabolic changes incident to HSV-1 infection, this study also provides future directions for investigation into the pathogenic mechanism of HSV-1.     

Biofluid 1H NMR-based metabonomic techniques in nutrition research - metabolic effects of dietary isoflavones in humans

A metabonomic approach to nutrition research may provide an insight into in vivo mechanisms of action following nutritional intervention. This approach was applied to investigate changes in the (1)H NMR spectral profile of urine collected from controlled dietary intervention studies conducted in premenopausal women before and following soy or miso consumption. The aim of the study was to identify the biochemical effects of a diet rich in soy isoflavones, phytochemicals which are receiving significant attention because of their potential importance to human health and wide bioactivity in vitro. By applying various chemometric techniques to the data the biochemical effects of conjugated and unconjugated isoflavones were determined. The biochemical changes observed suggest that soy isoflavone ingestion had significant effects on several metabolic pathways associated with osmolyte fluctuation and energy metabolism. These biochemical changes were more significant following ingestion of the unconjugated soy isoflavone (miso) diet suggesting that the chemical composition of the isoflavones present in soy-based foods may have an effect on their biological efficacy in vivo. This study describes a novel application for (1)H NMR analysis by determining subtle differences in biochemical profiles following dietary intervention and providing further insight into the mechanisms of action of phytochemicals in vivo.     

Intervention effects of puerarin on blood stasis in rats revealed by a 1H NMR-based metabonomic approach

Puerarin possesses a wide spectrum of biological activities including ameliorating effects on blood stasis, but the definite mechanism of this effect is still not known. In this study, a ¹H NMR-based plasma and urinary metabonomic approach was applied to comprehensively and holistically investigate the therapeutic effects of puerarin on blood stasis and its underlying mechanisms. Puerarin was injected intraperitoneally once daily for consecutive 7 days. The blood stasis rat model was established by placing the rats in ice-cold water during the time interval between two injections of adrenaline. With pattern recognition analysis, a clear separation of blood stasis model group and healthy control group was achieved and puerarin pretreatment group was located much closer to the control group than the model group, which was consistent with results of hemorheology studies. 15 and 10 potential biomarkers associated with blood stasis in plasma and urine, respectively, which were mainly involved in energy metabolism, lipid and membrane metabolisms, amino acid metabolism and gut microbiota metabolism, were identified. Puerarin could prevent blood stasis through partially regulating the disturbed metabolic pathways. This work highlights that metabonomics is a valuable tool for studying the essence of blood stasis as well as evaluating the efficacy of the corresponding drug treatment.     

Distinct T cell/renal tubular epithelial cell interactions define differential chemokine production: implications for tubulointerstitial injury in chronic glomerulonephritides

Chemokines can promote interstitial fibrosis that is, in turn, a strong predictor of renal failure in chronic glomerulonephritides (GN). Resident renal cells, including renal tubular epithelial cells (RTEC), represent a prominent source of chemokine expression. Evaluating those factors responsible for sustained chemokine production by RTEC during GN is therefore crucial. The contribution of interstitial T cells to such expression, and in particular the precise nature of their interactions with RTEC, are poorly understood. Activated T cell/RTEC coculture induced production of high levels of monocyte chemoattractant protein-1 (MCP-1), RANTES, and IFN-inducible protein-10 from RTEC. Using double-chamber cultures and activated T cell plasma membrane preparations we demonstrated that both cell contact and soluble factors contributed to RTEC chemokine production. Importantly, different chemokines exhibited distinct activation requirements. Thus, for RANTES cell contact was essential, but not sufficient. In contrast, either soluble factors or cell contact induced MCP-1 and IFN-inducible protein-10 production, although both pathways were required for a maximal response. Neutralization experiments identified critical roles in this process for proinflammatory cytokines such as TNF-alpha, IL-1beta, and IFN-gamma as well as membrane molecules such as LFA-1, CD40 ligand, and membrane bound TNF-alpha. Finally, chemotactic bioassays of T cell/RTEC coculture supernatants demonstrated 80% reduction of monocyte migration following MCP-1 neutralization, indicating a dominant role for this chemokine. In summary, activation of renal tubular cells by infiltrating T cells can amplify and perpetuate local inflammatory responses through chemokine production differentially mediated by soluble and cell contact-dependent factors. Recognition of this regulatory diversity has important implications in the choice of potential therapeutic targets in GN.     

Androgen receptor activation results in metabolite signatures of an aggressive prostate cancer phenotype: an NMR-based metabonomics study

Metabolomic studies have proven to provide a unique perspective of the cellular dysfunction developing as a result of prostate cancer (PCa) onset and progression, facilitated primarily by mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques. PCa develops as an androgen-dependent disease with the expression of the androgen receptor (AR), where patient treatment typically involves androgen ablation therapy. In response, it is theorized that PCa transforms to an androgen-hypersensitive or androgen-independent state, where treatment options are severely reduced. Under the hypothesis that AR stimulation increases the aggressivity of pre-existing PCa, NMR spectroscopy was utilized in the delineation of the metabonomic response of an androgen-dependent PCa cell line (LnCAP) as a result of AR activation. Metabolite profiles were determined after 12, 24, and 48?h of exposure to methyltrienolone (R1881), an AR agonist. Principal components analysis revealed the relative myo-inositol and phosphocholine levels were severely altered after androgen treatment. Furthermore, univariate analysis revealed multiple metabolic perturbations in response to R1881 exposure, including amino acid, choline, the phosphocholine/glycerophosphocholine ratio, and UDP-coupled sugar metabolism, which are consistent with reported measurements between normal and PCa samples. These results suggest that androgen-sensitive PCa may transform to an aggressive phenotype upon AR activation.     

NMR-based metabonomic analysis on effect of light on production of antioxidant phenolic compounds in submerged cultures of Inonotus obliquus

This study was designed to investigate the light effect on biosynthesis of antioxidant phenolic compounds by Inonotus obliquus grown in submerged cultures using ¹H NMR spectroscopy combining multivariate pattern recognition strategies. I. obliquus were exposed to a range of light conditions and resultant data were compared to those from field-grown sclerotia and the mycelia grown in daylight. Daylight illumination inhibited biosynthesis of davallialactone and phelligridins and other hispidin analogs. Continuous darkness enhanced the formation of phelligridins, davallialactone and inoscavins. Phelligridins and davallialactone also occurred in the mycelia grown in blue and red light with levels lower than those found in darkness. In addition, polyphenols synthesized under daylight conditions showed less potential antioxidant activity than those determined with other light regimes. These findings demonstrate that light regulates biosynthesis of polyphenols in I. obliquus and their subsequent antioxidant activities, and ¹H NMR-based metabolic profiling is a cost-effective approach for evaluating light effects on fungal metabolisms.     

A metabonomic approach to the investigation of drug-induced phospholipidosis: an NMR spectroscopy and pattern recognition study

¹H NMR spectroscopy of urine and pattern recognition analysis have been used to study the metabolic perturbations caused following dosing of five novel drug candidates, two of which (GWA, GWB) caused mild lung and liver phospholipidosis, whilst the rest (GWC-GWE) did not cause any detectable toxicity. Urine samples were collected predose, 0-8 h, 8-16 h, 16-24 h and 24-32 h after single, oral dosing with each compound to Han Wistar rats (n = 3 per group), and liver and lung samples for were taken at 48 h for histology. ¹H NMR spectra of whole urine were acquired, processed and subsequently analysed using principal component analysis. All animals administered the drug candidates showed a significant reduction in serum triglycerides and those animals administered either GWA or GWB were observed to have foamy alveolar macrophages and the presence of multilamellar bodies in hepatocytes by electron microscopy. In the plot of the first two principal components, urinary spectra of those animals dosed with GWA or GWB mapped separately to controls, all pre-dose samples and animals dosed with GWC-GWE. Inspection of the principal components loadings indicated an increase in urinary phenylacetylglycine with a concomitant decrease in urinary citrate and 2-oxoglutarate, possibly constituting a novel urinary biomarker set for phospholipidosis. This work exemplifies the use of NMR spectroscopy and pattern recognition methods for the detection of novel biomarker combinations for poorly understood toxicity types and the potential in screening novel drugs for toxicity.     

A metabonomic approach to the investigation of drug-induced phospholipidosis: an NMR spectroscopy and pattern recognition study

¹H NMR spectroscopy of urine and pattern recognition analysis have been used to study the metabolic perturbations caused following dosing of five novel drug candidates, two of which (GWA, GWB) caused mild lung and liver phospholipidosis, whilst the rest (GWC-GWE) did not cause any detectable toxicity. Urine samples were collected predose, 0-8 h, 8-16 h, 16-24 h and 24-32 h after single, oral dosing with each compound to Han Wistar rats (n = 3 per group), and liver and lung samples for were taken at 48 h for histology. ¹H NMR spectra of whole urine were acquired, processed and subsequently analysed using principal component analysis. All animals administered the drug candidates showed a significant reduction in serum triglycerides and those animals administered either GWA or GWB were observed to have foamy alveolar macrophages and the presence of multilamellar bodies in hepatocytes by electron microscopy. In the plot of the first two principal components, urinary spectra of those animals dosed with GWA or GWB mapped separately to controls, all pre-dose samples and animals dosed with GWC-GWE. Inspection of the principal components loadings indicated an increase in urinary phenylacetylglycine with a concomitant decrease in urinary citrate and 2-oxoglutarate, possibly constituting a novel urinary biomarker set for phospholipidosis. This work exemplifies the use of NMR spectroscopy and pattern recognition methods for the detection of novel biomarker combinations for poorly understood toxicity types and the potential in screening novel drugs for toxicity.     

Evaluation of coagulation activation after Rhinovirus infection in patients with asthma and healthy control subjects: an observational study

Background

Asthma exacerbations are frequently triggered by rhinovirus infections. Both asthma and respiratory tract infection can activate haemostasis. Therefore we hypothesized that experimental rhinovirus-16 infection and asthmatic airway inflammation act in synergy on the haemostatic balance.

Methods

28 patients (14 patients with mild allergic asthma and 14 healthy non-allergic controls) were infected with low-dose rhinovirus type 16. Venous plasma and bronchoalveolar lavage fluid (BAL fluid) were obtained before and 6 days after infection to evaluate markers of coagulation activation, thrombin-antithrombin complexes, von Willebrand factor, plasmin-antiplasmin complexes, plasminogen activator inhibitor type-1, endogenous thrombin potential and tissue factor-exposing microparticles by fibrin generation test, in plasma and/or BAL fluid. Data were analysed by nonparametric tests (Wilcoxon, Mann Whitney and Spearman correlation).

Results

13 patients with mild asthma (6 females, 19-29 y) and 11 healthy controls (10 females, 19-31 y) had a documented Rhinovirus-16 infection. Rhinovirus-16 challenge resulted in a shortening of the fibrin generation test in BAL fluid of asthma patients (t = -1: 706 s vs. t = 6: 498 s; p = 0.02), but not of controls (t = -1: 693 s vs. t = 6: 636 s; p = 0.65). The fold change in tissue factor-exposing microparticles in BAL fluid inversely correlated with the fold changes in eosinophil cationic protein and myeloperoxidase in BAL fluid after virus infection (r = -0.517 and -0.528 resp., both p = 0.01).Rhinovirus-16 challenge led to increased plasminogen activator inhibitor type-1 levels in plasma in patients with asthma (26.0 ng/mL vs. 11.5 ng/mL in healthy controls, p = 0.04). Rhinovirus-16 load in BAL showed a linear correlation with the fold change in endogenous thrombin potential, plasmin-antiplasmin complexes and plasminogen activator inhibitor type-1.

Conclusions

Experimental rhinovirus infection induces procoagulant changes in the airways of patients with asthma through increased activity of tissue factor-exposing microparticles. These microparticle-associated procoagulant changes are associated with both neutrophilic and eosinophilic inflammation. Systemic activation of haemostasis increases with Rhinoviral load.

Trial registration

This trial was registered at the Dutch trial registry (http://www.trialregister.nl): NTR1677.     

Cracking the molecular weight barrier: fragment screening of an aminotransferase using an NMR-based functional assay

NMR-based screening of protein targets has become a well established part of the drug discovery process especially with respect to fragments. However, as target size increases the two-dimensional spectra typically used for such screening become more crowded due to the increased number of signals, and the individual signals broaden due to the decreased rotational correlation time of the protein. Here we present an NMR-based functional assay for the branched-chain aminotransferase BCATc, a dimer with a total molecular weight of 88 kDa, which overcomes the limitations of the typical protein-based NMR screening method. BCATc is involved in glutamate production in the brain and is a therapeutic target for neuronal disorders involving a glutamatergic mechanism. Several fragments which inhibit BCATc were discovered using this assay and these may serve as novel cores for the development of potent BCATc inhibitors.     

Evaluation of extraction methods for use with NMR-based metabolomics in the marine polychaete ragworm, Hediste diversicolor

The sediment-dwelling polychaete, Hediste diversicolor, is commonly found in Northern temperate estuaries. Its limited mobility and tolerance to polluted conditions makes it a good candidate for biological monitoring. Moreover, its importance in the functioning of the sediment ecosystem has caused it to be described as a keystone species. Here we present the development of analytical methodology that will enable the use of H. diversicolor in environmental metabolomics studies for the biomonitoring of estuarine ecosystems. Polar and non-polar extraction solvents have been used to solubilise a wide range of metabolites. Extraction solvents assessed include: aqueous phosphate buffer solution, methanol:chloroform:water (1:1:0.9), methanol:water (1:1 and 2:1) and chloroform. The metabolites were analysed using 1-dimensional (1D) ¹H nuclear magnetic resonance (NMR) spectroscopy. Using the methanol:water (1:1) method, previous freezing to aid cell rupture did not result in an enhanced extraction. Removal of methanol with a speed vacuum resulted in reduction in yield. Methanol:water (1:1) and chloroform extractions proved to be the most appropriate techniques based on the sample yield and repeatability. NMR-based metabolomics in the ragworm can now be used to understand the ecophysiology of this important estuarine organism and has applications in biomonitoring, biomarker development and ecotoxicological studies.     

Within-day reproducibility of an HPLC-MS-based method for metabonomic analysis: application to human urine

Self-evidently, research in areas supporting "systems biology" such as genomics, proteomics, and metabonomics are critically dependent on the generation of sound analytical data. Metabolic phenotyping using LC-MS-based methods is currently at a relatively early stage of development, and approaches to ensure data quality are still developing. As part of studies on the application of LC-MS in metabonomics, the within-day reproducibility of LC-MS, with both positive and negative electrospray ionization (ESI), has been investigated using a standard "quality control" (QC) sample. The results showed that the first few injections on the system were not representative, and should be discarded, and that reproducibility was critically dependent on signal intensity. On the basis of these findings, an analytical protocol for the metabonomic analysis of human urine has been developed with proposed acceptance criteria based on a step-by-step assessment of the data. Short-term sample stability for human urine was also assessed. Samples were stable for at least 20 h at 4 degrees C in the autosampler while queuing for analysis. Samples stored at either -20 or -80 degrees C for up to 1 month were indistinguishable on subsequent LC-MS analysis. Overall, by careful monitoring of the QC data, it is possible to demonstrate that the "within-day" reproducibility of LC-MS is sufficient to ensure data quality in global metabolic profiling applications.     

A metabonomic study of inhibition of GABA uptake in the cerebral cortex

GABAergic activity is regulated by rapid, high affinity uptake of GABA from the synapse. Perturbation of GABA reuptake has been implicated in neurological disease and inhibitors of GABA transporters (GAT) have been used therapeutically but little detail is known about the ramifications of GAT inhibition on brain neurochemistry. Here, we incubated Guinea pig cortical tissue slices with [3-¹³C]pyruvate and major, currently available GABA uptake inhibitors. Metabolic fingerprints were generated from these experiments using ¹³C/¹H NMR spectroscopy. These fingerprints were analyzed using multivariate statistical approaches and compared with an existing library of fingerprints of activity at GABA receptors. This approach identified five distinct clusters of metabolic activity induced by blocking GABA uptake. Inhibition of GABA uptake via GAT1 produced patterns similar to activity at mainstream GABAergic synapses in particular those containing α1-subunits but still statistically separable. This indicated that inhibition of GABA uptake, an indirect method of activating GABA receptors, produces different effects to direct receptor activation or to exogenous GABA. The mechanism of inhibitor function also produced different outcomes, with the channel blocker SKF 89976A yielding a unique metabolic response. Blocking GAT1 and GAT3 simultaneously induces a large metabolic response consistent with induction of tonic inhibition via high affinity GABA receptors. Blocking BGT produces patterns similar to activity at less common receptors such as those containing α5 subunits. This approach is useful for determining where in the spectrum of GABAergic responses a particular GABA transport inhibitor is effective.     

Altered fatty acid metabolism in long duration road transport: An NMR-based metabonomics study in sheep

The physical, endocrine, and metabolic responses of livestock to road transport have been evaluated by conventional hematological and biochemistry parameters for more than 20 years. However, these measures are relatively insensitive to subtle metabolic adaptations. We applied NMR-based metabonomics to assess system-wide metabolic responses as expressed in urine and serum of a large cohort of animals (n = 80) subjected to 12 and 48 h road transport. The profiling of (1)H NMR spectra revealed that the transported animals experienced altered gut and energy metabolism, muscle catabolism, and possibly a renal response. The animals transported for 48 h exhibited a deeper metabolic response to the transport event and a complex and expanded metabolic trajectory over the 72 h recovery period. Intriguingly, excretion of acyl glycines and a dicarboxylic acid was observed after transport and during recovery, implicating peroxisomal fatty acid oxidation as a metabolic response to transport-induced stress.