Metabolomics in the developmental origins of obesity and its cardiometabolic consequences.

In this review, we discuss the potential role of metabolomics to enhance understanding of obesity-related developmental origins of health and disease (DOHaD). We first provide an overview of common techniques and analytical approaches to help interested investigators dive into this relatively novel field. Next, we describe how metabolomics may capture exposures that are notoriously difficult to quantify, and help to further refine phenotypes associated with excess adiposity and related metabolic sequelae over the life course. Together, these data can ultimately help to elucidate mechanisms that underlie fetal metabolic programming. Finally, we review current gaps in knowledge and identify areas where the field of metabolomics is likely to provide insights into mechanisms linked to DOHaD in human populations.

Clinical cancer trials in a rural community cancer center in Southwest Oklahoma from 2000-2009.

PURPOSE: The goal of this retrospective study is to describe the clinical trial participant population served at a rural community cancer center in Southwest Oklahoma from 2000-2009. METHODS: The medical records of clinical research participants from 2000-2009 were retrospectively reviewed. RESULTS: A total of 342 clinical trial registrations had occurred with 276 unique registrations. The demographics of the research participants showed the majority with a high school diploma/GED or higher education level (92.0%), female (62%), and White/Caucasian (77.2%). Enrolled patients were residents from 13 surrounding counties. CONCLUSION: The actualization of population-specific knowledge applied to clinical trials may allow the community oncologist to participate in and seek out protocols which are most relevant to his patients which potentially provides them with a more comprehensive cancer care.

Heparin cryoprecipitation reduces plasma levels of non-traditional risk factors for atherosclerosis in vitro.

AIMS: To show that heparin cryoprecipitation (HCP), an in vitro method of plasma purification, reduces the levels of in vivo modified proteins and non-traditional risk factors from plasma of atherosclerotic hemodialysis (HD) patients. METHODS: HCP was applied to plasma obtained from HD patients and controls, forming a precipitate--cryogel. Levels of fibrinogen, albumin, CRP, TNF-alpha, IL-6, advanced oxidation protein products, carbonylated fibrinogen and carbonylated albumin were determined in plasma before and after applying HCP and in the cryogel. RESULTS: Treatment of HD plasma with HCP, beyond the significant reduction of the increased levels of all the above-mentioned molecules, reduced fibrinogen, TNF-alpha, carbonylated fibrinogen and carbonylated albumin to control levels which were simultaneously found in the cryogel. CONCLUSIONS: HCP applied to plasma enables the simultaneous precipitation of modified molecules and circulating non-traditional risk factors for atherosclerosis. This study may serve as a base for the future development of a clinical purification technique.

Light chain muscle deposition caused rhabdomyolysis and acute renal failure in patient with multiple myeloma.

Case report of a 70-year-old woman with the diagnosis of multiple myeloma and acute renal failure due to rhabdomyolysis (RDM) that was caused from the deposition of kappa light chain in muscle fibers. In addition, the deposition was found in the liver.

The presence of erythropoietin receptors in the human peripheral nervous system.

Erythropoietin (EPO) is a well-known hematopoietic factor and a major determinant of tissue oxygenation. EPO receptors have been identified on a wide variety of non-erythroid cell types including human central nervous system and peripheral nervous system of animal models. The presence or function of EPO receptors in human peripheral nervous system is unknown. By examining nerve segments from radicular and autonomic nerves using immunohistochemical methods, we demonstrated the presence of EPO receptors on myelin sheath of radicular nerves in the human peripheral nervous system.

Effect of the antioxidant Mesna (2-mercaptoethane sulfonate) on experimental colitis.

Reactive oxygen species play a key role in intestinal inflammation, although interventional studies using antioxidants have shown only weak beneficial effects both in humans and animals. Hence, our aim was to examine the possible beneficial effect of the antioxidant 2-mercaptoethane sulfonate (Mesna) on experimental colitis. Colitis was induced in rats by intrarectal instillation of trinitrobenzene sulfonic acid (TNB) followed immediately by intrarectal Mesna or saline, administered for 14 days, twice daily. A beneficial effect of Mesna was observed, resulting in a significant reduction in inflammation followed by almost full recovery. iNOS mRNA expression and myeloperoxidase (MPO) activity were significantly increased in the TNB-Mesna group. These results suggest that the induction of iNOS in the presence of Mesna reduced intestinal inflammation. Mesna probably resolved this inflammation by scavenging reactive oxygen species generated by the augmented infiltration of polymorphonuclear leukocytes.

The polymorphonuclear leukocyte--a new target for erythropoietin.

A previous study from our laboratory has shown that erythropoietin (EPO), beside its traditional role in erythropoiesis, acts as an alleviator of oxidative stress and inflammation in chronic hemodialysis (HD) patients, conferred in part by activated polymorphonuclear leukocytes (PMNLs). To substantiate this phenomenon, the existence of EPO receptors (EPO-Rs) on PMNL membrane was examined at the transcriptional and translational levels. mRNA for EPO-R was detected in PMNLs using specific primers directed towards the extracellular region of human EPO-R cDNA. The predicted 300-bp fragment was amplified by reverse transcriptase-polymerase chain reaction. Subcloning and sequence analysis revealed 100% homology of this fragment with human EPO-R. The receptor protein was detected on the surface of intact PMNLs using (125)I-EPO. The protein was further demonstrated by flow cytometric analysis using a fluorescent monoclonal anti-EPO-R. The percentage of PMNLs expressing EPO-R showed a strong correlation with the level of EPO in the serum, suggesting an upregulation of the receptor by the hormone. Taken together with our recent findings that EPO attenuates the oxidative stress and inflammation contributed by PMNLs in HD patients, the detection of functional EPO-R expression in PMNLs places these cells among the nonerythroid, EPO-responsive target populations.

Selective dopaminergic vulnerability: 3,4-dihydroxyphenylacetaldehyde targets mitochondria.

Parkinson's disease (PD) is a major cause of age-related morbidity and mortality, present in nearly 1% of individuals at ages 70-79 and approximately 2.5% of individuals at age 85. L-DOPA (L-dihydroxyphenylalanine), which is metabolized to dopamine by dopa decarboxylase, is the primary therapy for PD, but may also contribute to disease progression. Association between mitochondrial dysfunction, monoamine oxidase (MAO) activity, and dopaminergic neurotoxicity has been repeatedly observed, but the mechanisms underlying selective dopaminergic neuron depletion in aging and neurodegenerative disorders remain unclear. We now report that 3,4-dihydroxyphenylacetaldehyde (DOPAL), the MAO metabolite of dopamine, is more cytotoxic in neuronally differentiated PC12 cells than dopamine and several of its metabolites. In isolated, energetically compromised mitochondria, physiological concentrations of DOPAL induced the permeability transition (PT), a trigger for cell death. Dopamine was > 1000-fold less potent. PT inhibitors protected both mitochondria and cells against DOPAL. Sensitivity to DOPAL was reduced > or = 30-fold in fully energized mitochondria, suggesting that mitochondrial respiration may increase resistance to PT induction by the endogenous DOPAL in the substantia nigra. These data provide a potential mechanism of action for L-DOPA-mediated neurotoxicity and suggest two potentially interactive mechanisms for the selective vulnerability of neurons exposed to dopamine.

Mesna: a novel renoprotective antioxidant in ischaemic acute renal failure.

BACKGROUND: Reactive oxygen species (ROS) play a key role in renal ischaemia-reperfusion injury. After establishing the in vitro anti-oxidative potential of mesna, a sulfhydryl-containing compound, its effect on kidney function and morphology in a rat model of ischaemic acute renal failure (ARF) was examined. METHODS: Mesna (180 mg/kg) was administered at different time points relative to ischaemia and/or reperfusion onset. Kidney function was assessed by glomerular filtration rate (GFR) and fractional sodium excretion (FE(Na)) before a 45-min period of unilateral renal artery clamping and following 90 min of reperfusion. Mesna was administered by bolus, 30 min before the induction of ischaemia, 5 min before ischaemia, 5 min before reperfusion, and 5 min after the onset of reperfusion. RESULTS: Mesna improved function of the ischaemic kidney at each administration. When mesna was administered 5 min before the onset of reperfusion, GFR reached 90-100% of its pre ischaemic value and FE(Na) was improved by 75%. The beneficial effect of mesna was also demonstrated by light and electron microscopy. Kidneys treated with mesna 5 min before reperfusion resembled ischaemic non-reperfused kidneys and showed subtle morphological and ultrastructural changes compared with ischaemic-reperfused kidneys. Mesna had no haemodynamic effect on renal blood flow and did not induce any osmotic diuresis. CONCLUSIONS: We suggest that mesna acts as an antioxidant. Its antioxidant potential together with optimal protection achieved when administered 5 min before reperfusion, supports the conclusion that mesna scavenges ROS generated at the onset of reperfusion, thus diminishing reperfusion injury and organ damage.

Characterization of diet-dependent metabolic serotypes: analytical and biological variability issues in rats.

This report, the first in a series on diet-dependent changes in the serum metabolome (metabolic serotype), describes validation of the use of high performance liquid chromatography (HPLC) separations coupled with Coulometric array detectors to characterize changes in the metabolome. The long-term aim of these studies is to improve understanding of the effects of significant variation in nutritive status on physiology and on disease processes. Initial studies focus on identifying the effects of dietary (or caloric) restriction on the redox-active components of rat serum. Identification of compounds of interest is being carried out using HPLC separations coupled with coulometric array analysis, an approach allowing simultaneous examination of nearly 1200 serum compounds. The technical and practical issues discussed in this report are related to both analytical validity (HPLC running conditions, computer-automated peak identification, mathematical compensation for chromatographic drift, etc.) and biological variability (individual variability, cohort-cohort variability, outliers). Attention to these issues suggests approximately 250 compounds in serum are sufficiently reliable, both analytically and biologically, for potential use in building mathematical models of serotype.

Successful pregnancy in a patient with polycystic kidney disease and advanced renal failure without prophylactic dialysis.

Pregnancies in women suffering from advanced chronic renal failure are frequently associated with deterioration of maternal renal function, premature births and low birth weights. Prophylactic dialysis is sometimes instituted since this intervention ameliorates the uremic milieu and improves maternal status and fetal uterine environment. This report describes a successful pregnancy and delivery in a hypertensive woman with advanced chronic renal failure due to polycystic kidney disease without accelerating the natural deterioration of renal function and without instituting prophylactic dialysis. The infant was delivered at full term with a normal birth weight. Thirty months after delivery, growth and development of the child were normal and the rate of deterioration of maternal renal function, assessed by 1/creatinine, was unaffected by pregnancy. Conservative management and effective control of blood pressure may be sufficient to achieve successful pregnancy outcome when women with advanced chronic renal failure become pregnant.

Involvement of peripheral polymorphonuclear leukocytes in oxidative stress and inflammation in type 2 diabetic patients.

OBJECTIVE: To determine the extent to which peripheral polymorphonuclear leukocytes (PMNs) contributed to oxidative stress (OS) and inflammation in type 2 diabetic patients. RESEARCH DESIGN AND METHODS: PMNs and plasma were separated from blood withdrawn from 18 type 2 diabetic patients and 16 age- and sex-matched normal control subjects. The rate of superoxide release from phorbol 12-myristate 13-acetate (PMA)-stimulated PMNs and the plasma glutathione (GSH) levels served as measures of OS. Inflammation was assessed by PMN recruitment, expressed by peripheral blood PMN counts, and the in vitro survival of PMNs, which reflects cell necrosis. RESULTS: PMA-stimulated PMNs from diabetes released superoxide significantly faster, and plasma-reduced GSH was lower in diabetic patients than in normal control subjects. The rate of superoxide release from diabetic PMNs showed no correlation with the plasma glucose concentrations, whereas a positive linear correlation with HbA1c was found. The in vitro survival of diabetic PMNs was lower than normal control PMNs when each was incubated in its own serum. The in vitro survival of normal control PMNs was reduced when incubated with diabetic serum, whereas normal control sera promoted the survival of diabetic PMNs. Peripheral PMN counts were higher in diabetic patients than in normal control patients. CONCLUSIONS: Type 2 diabetes is accompanied by a priming of PMNs, resulting in OS and increased self-necrosis. Necrosis starts a chain of inflammatory reactions that result in cell recruitment and in the long run, with OS, may result in endothelial dysfunction. Understanding the contribution of PMNs to OS and inflammation in diabetes may illuminate new mechanisms through which endothelial dysfunction evolves and causes angiopathy and atherosclerosis.

Oxidative stress during hemodialysis: effect of heparin.

BACKGROUND: Patients on chronic hemodialysis (HD) are exposed to oxidative stress. An HD session is used in this study as an in vivo model for studying the influence of heparin on oxidative stress caused partially by activated peripheral blood polymorphonuclear leukocytes (PMNLs) during a HD session. METHODS: Each patient underwent HD once with and once without heparin. Oxidative stress was determined by evaluating both the rate of superoxide release from phorbol 12-myristate 13-acetate (PMA)-stimulated PMNLs and plasma levels of oxidized glutathione (GSSG), both measured before and after the dialysis session. RESULTS: In vitro, heparin reduced the rate of superoxide release from separated PMA-stimulated PMNLs. In vivo, the rate of superoxide release from PMNLs was always increased after the dialysis session, regardless of the presence of heparin. However, in the presence of heparin, this increase was significantly smaller. The augmentation in the rate of superoxide release after the dialysis session without heparin was accompanied by a significant elevation of GSSG, reflecting a preceding oxidation of plasma glutathione. CONCLUSIONS: The increase in both parameters, the rate of superoxide release, and the plasma GSSG concentration after HD treatment suggest that heparin in vivo alleviates the oxidative stress induced by the dialysis process. Based on our results, heparin should be the anticoagulant of choice because of its suppressant action on HD-induced oxidative stress.

Biochemical characterization of the mitochondrial permeability transition in isolated forebrain mitochondria.

Induction of the mitochondrial permeability transition (PT) has been proposed to contribute to neuronal cell death. Nearly all studies of the biochemistry of PT induction, however, have been conducted in isolated liver mitochondria. To better understand PT induction in brain mitochondria, we used Ficoll gradients to purify nonsynaptosomal mitochondria from the forebrains of male Fischer 344 rats. Incubation of these mitochondria with Ca(2+) was associated with a loss of absorbance. Inorganic phosphate enhanced this loss of absorbance, and the PT inhibitor cyclosporin A reduced it, especially in conjunction with ADP. These findings suggest that Ca(2+)-mediated loss of absorbance resulted from PT induction. Na(+), which enhances mitochondrial Ca(2+) efflux, but stimulates mitochondrial free radical production, had no effect on PT induction. These data confirm the existence of tissue-specific differences in the nature of PT induction.

High-throughput profiling of the mitochondrial proteome using affinity fractionation and automation.

Recent studies have demonstrated the need for complementing cellular genomic information with specific information on expressed proteins, or proteomics, since the correlation between the two is poor. Typically, proteomic information is gathered by analyzing samples on two-dimensional gels with the subsequent identification of specific proteins of interest by using trypsin digestion and mass spectrometry in a process termed peptide mass fingerprinting. These procedures have, as a rule, been labor-intensive and manual, and therefore of low throughput. The development of automated proteomic technology for processing large numbers of samples simultaneously has made the concept of profiling entire proteomes feasible at last. In this study, we report the initiation of the (eventual) complete profile of the rat mitochondrial proteome by using high-throughput automated equipment in combination with a novel fractionation technique using minispin affinity columns. Using these technologies, approximately one hundred proteins could be identified in several days. In addition, separate profiles of calcium binding proteins, glycoproteins, and hydrophobic or membrane proteins could be generated. Because mitochondrial dysfunction has been implicated in numerous diseases, such as cancer, Alzheimer's disease and diabetes, it is probable that the identification of the majority of mitochondrial proteins will be a beneficial tool for developing drug and diagnostic targets for associated diseases.

Zn2+ inhibits alpha-ketoglutarate-stimulated mitochondrial respiration and the isolated alpha-ketoglutarate dehydrogenase complex.

Intracellular free Zn(2+) is elevated in a variety of pathological conditions, including ischemia-reperfusion injury and Alzheimer's disease. Impairment of mitochondrial respiration is also associated with these pathological conditions. To test whether elevated Zn(2+) and impaired respiration might be linked, respiration of isolated rat liver mitochondria was measured after addition of Zn(2+). Zn(2+) inhibition (K(i)(app) = approximately 1 micrometer) was observed for respiration stimulated by alpha-ketoglutarate at concentrations well within the range of intracellular Zn(2+) reported for cultured hepatocytes. The bc(1) complex is inhibited by Zn(2+) (Link, T. A., and von Jagow, G. (1995) J. Biol. Chem. 270, 25001-25006). However, respiration stimulated by succinate (K(i)(app) = approximately 6 micrometer) was less sensitive to Zn(2+), indicating the existence of a mitochondrial target for Zn(2+) upstream from bc(1) complex. Purified pig heart alpha-ketoglutarate dehydrogenase complex was strongly inhibited by Zn(2+) (K(i)(app) = 0.37 +/- 0.05 micrometer). Glutamate dehydrogenase was more resistant (K(i)(app) = 6 micrometer), malate dehydrogenase was unaffected, and succinate dehydrogenase was stimulated by Zn(2+). Zn(2+) inhibition of alpha-ketoglutarate dehydrogenase complex required enzyme cycling and was reversed by EDTA. Reversibility was inversely related to the duration of exposure and the concentration of Zn(2+). Physiological free Zn(2+) may modulate hepatic mitochondrial respiration by reversible inhibition of the alpha-ketoglutarate dehydrogenase complex. In contrast, extreme or chronic elevation of intracellular Zn(2+) could contribute to persistent reductions in mitochondrial respiration that have been observed in Zn(2+)-rich diseased tissues.

Purine catabolism: links to mitochondrial respiration and antioxidant defenses?

Type I diabetes in rodents is associated with a spectrum of liver mitochondrial abnormalities ranging from evidence of oxidative stress and altered antioxidant defenses to frank defects in respiration rates and respiratory control ratios. To better address the myriad changes in redox metabolism in these mitochondria, we have applied new chromatographic techniques that enable simultaneous analysis of multiple components of pathways of interest (e.g., purine catabolites and oxidation by-products). We report here a portion of these results, which, in conjunction with other reported data, suggest that purine catabolism may contribute to mitochondrial antioxidant defenses by producing the antioxidant urate. In liver mitochondria from diabetic rats, increases in uric acid (threefold) and its direct precursor xanthine (sixfold) were observed in moderate diabetes, but levels fell essentially to normal in severe disease. Failure to maintain elevated xanthine and uric acid occurred contemporaneously with progressive mitochondrial dysfunction. Regression analysis revealed altered precursor-product relationships between xanthine, its precursors, and uric acid. An independent set of studies in isolated rat liver mitochondria showed that mitochondrial respiration was associated with essentially uniform decreases (approximately 30%) in all purine catabolites measured (urate, xanthine, hypoxanthine, guanine, guanosine, and xanthosine). That result suggests the potential for steady production of urate. Taken together, the two studies raise the possibility that purine catabolism may be a previously unappreciated component of the homeostatic response of mitochondria to oxidant stress and may play a critical role in slowing progressive mitochondrial dysfunction in certain disease states.

Apoptogenic ganglioside GD3 directly induces the mitochondrial permeability transition.

Early events in apoptotic cascades initiated by ceramides or by activation of the surface receptor CD95 (Fas/APO-1) include the formation of ganglioside GD3. GD3 appears to be both necessary and sufficient to propagate this lipid-mediated apoptotic pathway. Later events common to many apoptotic pathways include induction of the mitochondrial permeability transition (PT) and cytochrome c release, which in turn triggers downstream caspases and cell death. The links between GD3 formation and downstream stages of apoptosis are unknown. We report that ganglioside GD3 directly induces the PT in isolated rat liver mitochondria at 30-100 microM in the presence of exogenous substrate (succinate) and at approximately 3 microM in the absence of exogenous substrate. In contrast, other gangliosides tested (e.g. GM1) have only weak stimulatory effects in the presence of succinate and protect against PT induction in the absence of respiratory substrates. GD3-mediated induction of PT was antagonized by known PT inhibitors, namely cyclosporin A, ADP, trifluoperazine, and Mg(2+). GD3 induced PT even in the presence of submicromolar Ca(2+); GD3 is therefore the first biological PT inducer identified that does not require elevated Ca(2+). Exposure to GD3 also led to mitochondrial cytochrome c release. In contrast, C(2)-ceramide, which can initiate the lipid-mediated apoptotic cascade in susceptible cells, failed to either induce PT or release cytochrome c. These observations suggest that GD3 propagates apoptosis by inducing the PT and cytochrome c release. This model provides a mechanistic link between the earlier and later stages of CD95-induced/ceramide-mediated apoptosis.

A DLST genotype associated with reduced risk for Alzheimer's disease.

Recent studies suggest that variants of the DLST gene alter the risk of AD. DLST encodes the core subunit of the mitochondrial alpha-ketoglutarate dehydrogenase complex, which is deficient in AD. The authors report that in 247 US white subjects, homozygosity for DLST A19,117, T19,183 was associated with a reduced risk of AD (odds ratio [OR] = 0.35, p = 0.018). The reduced risk was marked in subjects who did not carry the apolipoprotein (APOE)-4 allele (OR = 0.16, p = 0.014). Further study of DLST in AD appears warranted.

Metabolic impairment induces oxidative stress, compromises inflammatory responses, and inactivates a key mitochondrial enzyme in microglia.

Microglial activation, oxidative stress, and dysfunctions in mitochondria, including the reduction of cytochrome oxidase activity, have been implicated in neurodegeneration. The current experiments tested the effects of reducing cytochrome oxidase activity on the ability of microglia to respond to inflammatory insults. Inhibition of cytochrome oxidase by azide reduced oxygen consumption and increased reactive oxygen species (ROS) production but did not affect cell viability. Azide also attenuated microglial activation, as measured by nitric oxide (NO.) production in response to lipopolysaccharide (LPS). It is surprising that the inhibition of cytochrome oxidase also diminished the activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a Krebs cycle enzyme. This reduction was exaggerated when the azide-treated microglia were also treated with LPS. The combination of the azide-stimulated ROS and LPS-induced NO. would likely cause peroxynitrite formation in microglia. Thus, the possibility that KGDHC was inactivated by peroxynitrite was tested. Peroxynitrite inhibited the activity of isolated KGDHC, nitrated tyrosine residues of all three KGDHC subunits, and reduced immunoreactivity to antibodies against two KGDHC components. Thus, our data suggest that inhibition of the mitochondrial respiratory chain diminishes aerobic energy metabolism, interferes with microglial inflammatory responses, and compromises mitochondrial function, including KGDHC activity, which is vulnerable to NO. and peroxynitrite that result from microglial activation. Thus, activation of metabolically compromised microglia can further diminish their oxidative capacity, creating a deleterious spiral that may contribute to neurodegeneration.