Interconversion Of Metabolites Research Articles

Age-Specific Differences in Whole-Body Amino Acid Kinetics

Abstract Objectives Aging is associated with changes in body composition (eg. sarcopenia) but the overall effects of aging on systemic amino acid kinetics need further exploration. We previously reported metabolic differences in certain amino acids between young and older adults using comprehensive metabolic flux analysis. We expanded this novel single stable tracer pulse approach by the addition of several other isotopically-labeled amino acids to confirm and extend our findings in a new cohort of young and older adults. Methods We studied 18 healthy young (∼23 y, 9 females and 9 males) and 16 older adults (∼67 y, 8 females and 8 males) by administering a single dose of a mixture of stable amino acid tracers related to arginine-citrulline, glutamate, branched-chain amino acid (BCAA: leucine, isoleucine, valine), and protein-related metabolism. A baseline blood sample was collected before administration of the pulse tracer followed by 1.5 hours blood sampling protocol. We measured plasma enrichments by LC-MS/MS to calculate their whole body production (WBP) rates and metabolite interconversions. In addition, body composition by dual-energy X-ray absorptiometry was measured. Statistics were performed by unpaired student t-test. Results Older adults had a 13% higher Body Mass Index (P = 0.005) and 13% lower appendicular skeletal muscle index than the younger group (P = 0.04). WBP of glutamate was 26% lower (P < 0.05) in older adults whereas WBP of tau-methylhistidine was higher (31%, P = 0.045), in line with our previously reported data. In addition, older adults were characterized by lower WBP of all 3 BCAAs (P = 0.007), histidine (P = 0.001) and tryptophan (P = 0.003) by 17%, 16%, and 15%, respectively. However, higher whole-body production rates were observed for citrulline (24%, P = 0.036) and de novo arginine synthesis (21%, P = 0.027) in older adults. Conclusions Metabolic flux analysis reveals that the kinetics of a large set of amino acids differ between younger and older adults which indicates that amino acid metabolism is age-related. The clinical relevance of those changes needs further investigation. Funding Sources CTRAL Internal Funds.

Identification and classification of differentially expressed genes reveal potential molecular signature associated with SARS-CoV-2 infection in lung adenocarcinomal cells.

Genomic techniques such as next-generation sequencing and microarrays have facilitated the identification and classification of molecular signatures inherent in cells upon viral infection, for possible therapeutic targets. Therefore, in this study, we performed a differential gene expression analysis, pathway enrichment analysis, and gene ontology on RNAseq data obtained from SARS-CoV-2 infected A549 cells. Differential expression analysis revealed that 753 genes were up-regulated while 746 down-regulated. SNORA81, OAS2, SYCP2, LOC100506985, and SNORD35B are the top 5 upregulated genes upon SARS-Cov-2 infection. Expectedly, these genes have been implicated in the immune response to viral assaults. In the Ontology of protein classification, a high percentage of the genes are classified as Gene-specific transcriptional regulator, metabolite interconversion enzyme, and Protein modifying enzymes. Twenty pathways with P-value lower than 0.05 were enriched in the up-regulated genes while 18 pathways are enriched in the down-regulated DEGs. The toll-like receptor signalling pathway is one of the major pathways enriched. This pathway plays an important role in the innate immune system by identifying the pathogen-associated molecular signature emanating from various microorganisms. Taken together, our results present a novel understanding of genes and corresponding pathways upon SARS-Cov-2 infection, and could facilitate the identification of novel therapeutic targets and biomarkers in the treatment of COVID-19.

Purine Homeostasis Is Necessary for Developmental Timing, Germline Maintenance and Muscle Integrity in Caenorhabditis elegans.

Purine homeostasis is ensured through a metabolic network widely conserved from prokaryotes to humans. Purines can either be synthesized de novo, reused, or produced by interconversion of extant metabolites using the so-called recycling pathway. Although thoroughly characterized in microorganisms, such as yeast or bacteria, little is known about regulation of the purine biosynthesis network in metazoans. In humans, several diseases are linked to purine metabolism through as yet poorly understood etiologies. Particularly, the deficiency in adenylosuccinate lyase (ADSL)-an enzyme involved both in the purine de novo and recycling pathways-causes severe muscular and neuronal symptoms. In order to address the mechanisms underlying this deficiency, we established Caenorhabditis elegans as a metazoan model organism to study purine metabolism, while focusing on ADSL. We show that the purine biosynthesis network is functionally conserved in C. elegans Moreover, adsl-1 (the gene encoding ADSL in C. elegans) is required for developmental timing, germline stem cell maintenance and muscle integrity. Importantly, these traits are not affected when solely the de novo pathway is abolished, and we present evidence that germline maintenance is linked specifically to ADSL activity in the recycling pathway. Hence, our results allow developmental and tissue specific phenotypes to be ascribed to separable steps of the purine metabolic network in an animal model.

Measurement of Subcellular Metabolite Concentrations in Relation to Phloem Loading.

The key step of carbon export from green leaves is the loading of sugars into the phloem. To fully understand and quantify this process, it is essential to know the concentration of sugars in the different compartments of the cells along the phloem loading pathway. However, determining subcellular metabolite concentrations has been technically challenging. This paper describes a technique to measure metabolite levels in the chloroplast, the cytosol, and the vacuole of mesophyll cells with high accuracy. The nonaqueous fractionation (NAF) technique is arguably the method of choice to analyze the subcellular metabolite distributions as it minimizes the risk of metabolite interconversions or redistribution during the process. The principle of NAF is the separation of small subcellular particles, which are obtained by homogenization, lyophilization, and sonication, in a nonaqueous density gradient. Due to the varying composition-dependent density of the fragments, their segregation reflects compartmental distributions throughout the gradient. By determining marker enzymes for chloroplast stroma, cytosol, and vacuole in gradient fractions the proportions of each subcellular compartment in each gradient fraction can be analyzed. The measured distribution of marker enzymes and of metabolites in each fraction of the gradient can be used to calculate the subcellular distribution of the metabolites.

High-Throughput Solid-Phase Microextraction-Liquid Chromatography-Mass Spectrometry for Microbial Untargeted Metabolomics.

Nowadays, metabolomics data, when combined with other "omics" data, can provide important information regarding systems biology. Acquiring a comprehensive untargeted metabolome snapshot of complex sample matrices requires proper sample preparation, and access to sophisticated analytical instrumentation such as mass spectrometry. In metabolomics, sample preparation has substantial influence on the quality of the obtained metabolome profile. To achieve a real snapshot of the metabolome, the analysis method must be capable of inhibiting metabolite interconversion by immediately quenching all metabolome activity. Application of solid-phase microextraction (SPME), particularly in its in vivo set up, when undertaken in conjunction with a conscious selection of coating type based on the chosen sample matrix and the physicochemical properties of the analytes under study, is capable of providing extraction of representative metabolomes for many biological matrices. Metabolomes identified by SPME include low-abundance species and short-lived or unstable metabolites hardly captured by traditional extraction techniques. SPME coupled to liquid chromatography-high-resolution mass spectrometry has recently been introduced as an innovative alternative technique that integrates sampling, sample preparation, and extraction for metabolic profiling and isolation of candidate biomarkers. This chapter presents a detailed protocol for microbial metabolome analysis of Escherichia coli as a model organism, applying the high-throughput SPME-LC-MS workflow.

Metabolic phenotyping using kinetic measurements in young and older healthy adults

BackgroundThe aging process is often associated with the presence of sarcopenia. Although changes in the plasma concentration of several amino acids have been observed in older adults, it remains unclear whether these changes are related to disturbances in whole body production and/or interconversions. MethodsWe studied 10 healthy young (~22.7y) and 17 older adults (~64.8y) by administering a mixture of stable amino acid tracers in a pulse and in a primed constant infusion. We calculated whole body production (WBP) and metabolite to metabolite interconversions. In addition, we measured body composition, muscle function, and provided questionnaires to assess daily dietary intake, physical activity, mood (anxiety, depression) and markers of cognitive function. Plasma enrichments and metabolite concentrations were measured by GC- and LC-MS/MS and statistics were performed by student t-test. ResultsOlder adults had a 11% higher body mass index (p=0.04) and 27% reduced peak leg extension force (p=0.02) than the younger group, but comparable values for muscle mass, mood and cognitive function. Although small differences in several plasma amino acid concentrations were observed, we found older adults had about 40% higher values of WBP for glutamine (221±27 vs. 305±21μmol/kgffm/h, p=0.03) and tau-methylhistidine (0.15±0.01 vs. 0.21±0.02μmol/kgffm/h, p=0.04), 26% lower WBP value for arginine (59±4 vs. 44±4μmol/kgffm/h, p=0.02) and a reduction in WBP (50%; 1.23±0.15 vs. 0.69±0.06μmol/kgffm/h, p=0.001) and concentration (25%; 3.5±0.3μmol/l vs. 2.6±0.2μmol/l, p=0.01) for β-Hydroxy β-Methylbutyrate. No differences were observed in protein catabolism. Clearance of arginine was decreased (27%, p=0.03) and clearance of glutamine (58%, p=0.01), leucine (67%, p=0.001) and KIC (76%, p=0.004) were increased in older adults. ConclusionsSpecific differences exist between young and older adults in amino acid metabolism.

Extraction and Quantitation of Nicotinamide Adenine Dinucleotide Redox Cofactors.

Accurate analysis of dinucleotide redox cofactors nicotinamide adenine dinucleotide phosphate reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide reduced (NADH), and nicotinamide adenine dinucleotide (NAD+) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms. We compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues: a cold aqueous buffer commonly used in enzyme assays with and without detergent, hot aqueous buffer, and cold organic mixtures (80% methanol, buffered 75% acetonitrile, and acidic 40:40:20 acetonitrile:methanol:water with either 0.02 M or 0.1 M formic acid). Extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). To monitor the metabolite interconversion, cells were grown in 13C6-glucose medium, and unlabeled standards were spiked into the extraction solvents. Interconversion between the oxidized and reduced forms was substantial except for the enzyme assay buffer with detergent, 80% methanol and 40:40:20 acetonitrile:methanol:water, with the 0.1 M formic acid mix giving the least interconversion and best recoveries. Absolute NAD+, NADH, NADP+, and NADPH concentrations in cells and mouse tissues were measured with this approach. We found that the interconversion between the reduced and oxidized forms during extraction is a major barrier to accurately measuring NADPH/NADP+ and NADH/NAD+ ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards. Extraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.

The Mitochondrial Amidoxime Reducing Component (mARC): Involvement in Metabolic Reduction of N‐Oxides, Oximes and N‐Hydroxyamidinohydrazones

The mitochondrial amidoxime reducing component (mARC) is a molybdenum-containing enzyme and capable of reducing N-hydroxylated structures such as amidoxime prodrugs. In this study, we tested the involvement of mARC in the reduction of N-oxides (amitriptyline-N-oxide, nicotinamide-N-oxide), oximes ((E)-/(Z)-2,4,6-trimethylacetophenonoxime) and a N-hydroxyamidinohydrazone (guanoxabenz). All groups are reduced by mARC proteins, and the enzymes are therefore involved in the interconversion of N-oxygenated metabolites originating from cytochrome P450s and flavin-containing monooxygenases. In addition, these structures open up further options for serving as prodrugs. Thus, with respect to these reactions, testing of candidates with N-oxygenated structures should not solely be carried out in microsomal enzyme sources but as well in mitochondria. However, differences in the reduction of oximes and N-oxides between the two isoforms, namely mARC1 and mARC2, were detectable; N-oxides are exclusively reduced by mARC1. We therefore assume differences between the so far unknown 3D structures of the two proteins.

Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in a plausible Archean ocean.

The reaction sequences of central metabolism, glycolysis and the pentose phosphate pathway provide essential precursors for nucleic acids, amino acids and lipids. However, their evolutionary origins are not yet understood. Here, we provide evidence that their structure could have been fundamentally shaped by the general chemical environments in earth's earliest oceans. We reconstructed potential scenarios for oceans of the prebiotic Archean based on the composition of early sediments. We report that the resultant reaction milieu catalyses the interconversion of metabolites that in modern organisms constitute glycolysis and the pentose phosphate pathway. The 29 observed reactions include the formation and/or interconversion of glucose, pyruvate, the nucleic acid precursor ribose-5-phosphate and the amino acid precursor erythrose-4-phosphate, antedating reactions sequences similar to that used by the metabolic pathways. Moreover, the Archean ocean mimetic increased the stability of the phosphorylated intermediates and accelerated the rate of intermediate reactions and pyruvate production. The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. The reactions were particularly sensitive to ferrous iron Fe(II), which is understood to have had high concentrations in the Archean oceans. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron-rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.

Reduced Levels of Circulating 7α-Hydroxy-Dehydroepiandrosterone in Treated Adolescent Obese Patients

Elevated levels of glucocorticoids lead to the development of obesity and metabolic syndrome. Local glucocorticoid levels are regulated through the enzyme 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD 1), an enzyme that regenerates active cortisol from inert cortisone. Increased expression of 11beta-HSD 1 in adipose tissue promotes higher body mass index (BMI), insulin resistance, hypertension, and dyslipidemia. Human 11beta-HSD 1 is also responsible for inter-conversion of 7-hydroxylate metabolites of dehydroepiandrosterone (7-OH-DHEA) to their 7-oxo-form. To better understanding the mechanism of the action, we focused on 7-OH- and 7-oxo-DHEA, and their circulating levels during the reductive treatment in adolescent obese patients. We determined plasma levels of 7alpha-OH-DHEA, 7beta-OH-DHEA, and 7-oxo-DHEA in 55 adolescent patients aged 13.04-15.67 years, BMI greater than 90th percentile. Samples were collected before and after one month of reductive therapy. Circulating levels of 7alpha-OH-DHEA decreased during the reductive therapy from 1.727 (1.614; 1.854, transformed mean with 95 % confidence interval) to 1.530 nmol/l (1.435; 1.637, p<0.05) in girls and from 1.704 (1.583; 1.842) to 1.540 nmol/l (1.435; 1.659, p<0.05) in boys. With regard to the level of 7-oxo-DHEA, a significant reduction from 1.132 (1.044; 1.231) to 0.918 nmol/l (0.844; 1.000, p<0.05) was found after the treatment, but only in boys. No significant difference in 7beta-OH-DHEA levels was observed. In conclusions, diminished levels of 7alpha-OH-DHEA indicate its possible effect on activity of 11beta-HSD 1. Further studies are necessary to clarify whether competitive substrates for 11beta-HSD 1 such as 7alpha-OH-DHEA could inhibit production of glucocorticoids and may be involved in metabolic processes leading to reduction of obesity.

Inhibition of bladder cancer by broccoli isothiocyanates sulforaphane and erucin: Characterization, metabolism, and interconversion

Epidemiologic evidence suggests diets rich in cruciferous vegetables, particularly broccoli, are associated with lower bladder cancer risk. Our objectives are to investigate these observations and determine the role of isothiocyanates in primary or secondary bladder cancer prevention. We initially investigate the mechanisms whereby broccoli and broccoli sprout extracts and pure isothiocyanates inhibit normal, noninvasive (RT4), and invasive (J82, UMUC3) human urothelial cell viability. Sulforaphane (IC(50) = 5.66 ± 1.2 μM) and erucin (IC(50) = 8.79 ± 1.3 μM) are found to be the most potent inhibitors and normal cells are least sensitive. This observation is associated with downregulation of survivin, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2/neu), G(2) /M cell cycle accumulation, and apoptosis. In a murine UMUC3 xenograft model, we fed semipurified diets containing 4% broccoli sprouts, or 2% broccoli sprout isothiocyanate extract; or gavaged pure sulforaphane or erucin (each at 295 μmol/kg, similar to dietary exposure); and report tumor weight reduction of 42% (p = 0.02), 42% (p = 0.04), 33% (p = 0.04), and 58% (p < 0.0001), respectively. Sulforaphane and erucin metabolites are present in mouse plasma (micromolar range) and tumor tissue, with N-acetylcysteine conjugates as the most abundant. Interconversion of sulforaphane and erucin metabolites was observed. This work supports development of fully characterized, novel food products containing broccoli components for phase I/II human studies targeting bladder cancer prevention.

Regulatory function of divalent cations in controlling the acidogenic biohydrogen production process

The functional role of divalent cations viz., iron (Fe2+), magnesium (Mg2+), nickel (Ni2+), zinc (Zn2+) and manganese (Mn2+) in regulating the biohydrogen (H2) production potential of a biocatalyst was evaluated under varying concentrations. The optimum conditions were evaluated in detail and compared with the principle conditions for enhanced conversion efficiency. Higher concentration of Fe2+ showed an enhanced H2 production efficiency of the biocatalyst (61.94 mmol) due to its role as a component of hydrogenase and ferredoxin. On the contrary, a higher concentration of Mg+2 showed a higher substrate utilization capability (76.46%) of the biocatalyst. The optimum metal concentration showed a higher H2 yield (18.23 mol kg−1 CODR; 3.5 mol mol−1 glucose) over principle operation conditions (14.76 mol kg−1 CODR; 2.84 mol mol−1 glucose). Higher concentrations of acetate (78 ± 6%) as the metabolic intermediate over butyrate (12 ± 2%) supported the observed higher H2 yields. Higher reductive catalytic current over oxidative current along with the enhanced dehydrogenase activity supported the higher H2 production efficiency. Shift in the redox Tafel slope was witnessed near zero in the case of Fe2+ and optimum conditions indicating the simultaneous oxidation and reduction reactions facilitating the availability of higher number of protons through metabolite inter-conversions that can make H2. The presence of divalent cations at an optimum concentration will enhance the H2 production capability of the biocatalyst. However, elevated levels of metal concentration showed a negative influence on the biocatalyst efficiency.

A systems biology approach to analyse leaf carbohydrate metabolism in Arabidopsis thaliana

Plant carbohydrate metabolism comprises numerous metabolite interconversions, some of which form cycles of metabolite degradation and re-synthesis and are thus referred to as futile cycles. In this study, we present a systems biology approach to analyse any possible regulatory principle that operates such futile cycles based on experimental data for sucrose (Scr) cycling in photosynthetically active leaves of the model plant Arabidopsis thaliana. Kinetic parameters of enzymatic steps in Scr cycling were identified by fitting model simulations to experimental data. A statistical analysis of the kinetic parameters and calculated flux rates allowed for estimation of the variability and supported the predictability of the model. A principal component analysis of the parameter results revealed the identifiability of the model parameters. We investigated the stability properties of Scr cycling and found that feedback inhibition of enzymes catalysing metabolite interconversions at different steps of the cycle have differential influence on stability. Applying this observation to futile cycling of Scr in leaf cells points to the enzyme hexokinase as an important regulator, while the step of Scr degradation by invertases appears subordinate.

Drug targeting of sphingolipid metabolism: sphingomyelinases and ceramidases

Sphingolipids represent a class of diverse bioactive lipid molecules that are increasingly appreciated as key modulators of diverse physiologic and pathophysiologic processes that include cell growth, cell death, autophagy, angiogenesis, and stress and inflammatory responses. Sphingomyelinases and ceramidases are key enzymes of sphingolipid metabolism that regulate the formation and degradation of ceramide, one of the most intensely studied classes of sphingolipids. Improved understanding of these enzymes that control not only the levels of ceramide but also the complex interconversion of sphingolipid metabolites has provided the foundation for the functional analysis of the roles of sphingolipids. Our current understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from loss-of-function/gain-of-function studies utilizing genetic deletion/downregulation/overexpression of enzymes of sphingolipid metabolism (e.g. knockout animals, RNA interference) and from the use of pharmacologic inhibitors of these same enzymes. While genetic approaches to evaluate the functional roles of sphingolipid enzymes have been instrumental in advancing the field, the use of pharmacologic inhibitors has been equally important in identifying new roles for sphingolipids in important cellular processes.The latter also promises the development of novel therapeutic targets with implications for cancer therapy, inflammation, diabetes, and neurodegeneration. In this review, we focus on the status and use of pharmacologic compounds that inhibit sphingomyelinases and ceramidases, and we will review the history, current uses and future directions for various small molecule inhibitors, and will highlight studies in which inhibitors of sphingolipid metabolizing enzymes have been used to effectively treat models of human disease.

A systems biology approach for the analysis of carbohydrate dynamics during acclimation to low temperature in Arabidopsis thaliana

Low temperature is an important environmental factor affecting the performance and distribution of plants. During the so-called process of cold acclimation, many plants are able to develop low-temperature tolerance, associated with the reprogramming of a large part of their metabolism. In this study, we present a systems biology approach based on mathematical modelling to determine interactions between the reprogramming of central carbohydrate metabolism and the development of freezing tolerance in two accessions of Arabidopsis thaliana. Different regulation strategies were observed for (a) photosynthesis, (b) soluble carbohydrate metabolism and (c) enzyme activities of central metabolite interconversions. Metabolism of the storage compound starch was found to be independent of accession-specific reprogramming of soluble sugar metabolism in the cold. Mathematical modelling and simulation of cold-induced metabolic reprogramming indicated major differences in the rates of interconversion between the pools of hexoses and sucrose, as well as the rate of assimilate export to sink organs. A comprehensive overview of interconversion rates is presented, from which accession-specific regulation strategies during exposure to low temperature can be derived. We propose this concept as a tool for predicting metabolic engineering strategies to optimize plant freezing tolerance. We confirm that a significant improvement in freezing tolerance in plants involves multiple regulatory instances in sucrose metabolism, and provide evidence for a pivotal role of sucrose-hexose interconversion in increasing the cold acclimation output.

Comparison of the secondary metabolites in Penicillium chrysogenum between pilot and industrial penicillin G fermentations

The disparity of secondary metabolites in Penicillium chrysogenum between two scales of penicillin G fermentation (50 L as pilot process and 150,000 L as industrial one) was investigated by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry. In industrial process, the pools of intracellular L-α-aminoadipyl-L-cysteinyl-D-valine (LLD-ACV) and isopenicillin N (IPN) were remarkably less than that in the pilot one, which indicated that the productivity of penicillin G might be higher in the large scale of fermentation. This conclusion was supported by the higher intracellular penicillin G concentration as well as its higher yield per unit biomass in industrial cultivation. The different changing tendencies of IPN, 6-aminopenicillanic acid and 6-oxopiperide-2-carboxylic acid between two processes also suggested the same conclusion. The higher content of intracellular LLD-ACV in pilot process lead to a similarly higher concentration of bis-δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, which had an inhibitory effect on ACV synthetase and also subdued the activity of IPN synthetase. The interconversion of secondary metabolites and the influence they put on enzymes would intensify the discrepancy between two fermentations more largely. These findings provided new insight into the changes and regulation of secondary metabolites in P. chrysogenum under different fermentation sizes.

Peptide Lost and Found: Internal Standards and the Mass Spectrometric Quantification of Peptides

Think of all the tubes of blood that are drawn and transported to the clinical laboratory to be processed every day. After being logged in, they make their way to an instrument for analysis, wherever that might be: in the same room, down the hall, across town, or a plane ride away. During that time, as the samples sit at room temperature, refrigerated, or on dry ice, chemical processes continue, albeit at varied rates for different molecules. Indeed, even at subfreezing temperatures enzymatic lipolysis takes place (1). Oxidation, which could obscure endogenous oxidation events that may represent important novel biomarkers, can also occur (2). Clinical chemists are careful to evaluate these and other preanalytical variables that may affect assay results for their patients. Unfortunately, there is often no way to tell if something has gone unexpectedly wrong with sample collection, storage, or preparation until it is too late and a result is already in a medical record being interpreted by a care provider. It would be highly desirable to have an easy way to tell if a sample has been compromised in a way that would effectively eliminate the possibility of accurate protein quantification, but this option will never be possible for the traditional automated immunochemical platforms that serve as the mainstay in clinical laboratories. In addition to the preanalytical issues with clinical assays, a variety of analytical problems can also complicate the analysis of a biomarker in human samples. For example, lipemia and hyperproteinemia can interfere with the indirect potentiometric measurement of sodium (3). In addition, interconversion of folate metabolites from lysed erythrocytes occurs during the deconjugation reaction before certain chromatographic assays (4). In this issue of Clinical Chemistry , Bystrom et al. (5) describe taking …

Metabolic shift and electron discharge pattern of anaerobic consortia as a function of pretreatment method applied during fermentative hydrogen production

We have made an attempt to evaluate the variation in the electron discharge (ED) pattern of anaerobic consortia as a function of pretreatment viz., chemical, heat-shock, acid and oxygen-shock in comparison with untreated mixed consortia during fermentative hydrogen (H 2) production. Experiments were performed with dairy wastewater as substrate using anaerobic mixed consortia as biocatalyst (pretreated individually and in combination). Cyclic voltammetry (CV) elucidated significant variation in the ED pattern of mixed consortia along with H 2 production and substrate degradation (SD) as a function of pretreatment method applied. Higher ED was observed with all pretreated consortia which can be attributed to the stable proton (H +) shuttling due to the suppression of methanogenic activity. Oxygen-shock method and untreated consortia showed lower H 2 production and higher SD among the variations studied, while, combined pretreated consortia resulted higher H 2 production and lower SD. Lower ED observed with untreated consortia suggests the H + reduction during methanogenesis rather than the inter-conversion of metabolites, which is presumed to be necessary for H 2 production. ED observed with combined pretreated consortia corroborated well with the observed H 2 production. Redox pairs were visualized on the voltammograms with almost all the experimental variations studied except untreated consortia. The potentials ( E 0) of redox pairs observed were corresponding to intracellular electron carriers viz., NAD +/NADH ( E 0 −0.32 V) and FAD +/FADH 2 ( E 0 −0.24 V).

Insight into the dehydrogenase catalyzed redox reactions and electron discharge pattern during fermentative hydrogen production

Dehydrogenase (DH) activity associated with bio-electrochemical behavior was analyzed for the first time to understand the redox reactions involved in fermentative hydrogen (H 2) production process in concurrence with proton (H +) shuttling and electron (e −) discharge (ED) pattern. DH facilitates the availability of H + through redox reactions to make H 2. We have designed a comprehensive experimental study to evaluate the DH activity (H + shuttling) and ED to understand the biochemical process with the function of pH (5, 6, 7 and 8) and metabolic microenvironment [anaerobic, anoxic and aerobic (control)]. DH activity was observed to be higher during anaerobic operation suggesting the higher availability of H + and e − due to the inter-conversion of metabolites and the same was reflected in the voltammetry analysis. Higher H 2 production under anaerobic operation corroborated well with these findings. The DH activity associated with H + shuttling and ED was also correlated with the substrate degradation pattern.

Interactions between dehydroepiandrosterone and glucocorticoid metabolism in pig kidney: Nuclear and microsomal 11β-hydroxysteroid dehydrogenases

The 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) activates glucocorticoids (GC) by reversibly converting 11-keto-GC to 11-hydroxy-GC, while 11βHSD2 and 11βHSD3 only catalyzes the reverse reaction. Recently, rat and human 11βHSDs were shown to interconvert 7α- and 7β-hydroxy-dehydroepiandrosterone (7α- or 7β-OH-DHEA) with 7-oxo-DHEA. We report that pig kidney microsomes (PKMc) and nuclei (PKN) oxidize 7α-OH-DHEA to 7-oxo-DHEA at higher rates with NAD +, than with NADP +. Corticosterone (CS), dehydrocoticosterone (DHC), 11α- and 11β-hydroxyprogesterone, and carbenoxolone completely inhibited these reactions, while 7-oxo-DHEA only inhibited the NAD +-dependent reaction. Conversely, CS oxidation was not inhibited by 7α-OH-DHEA or 7-oxo-DHEA. PKMc and PKN did not convert 7-oxo-DHEA to 7-OH-DHEA with either NADPH or NADH. Finally, PKN contained a high affinity, NADPH-dependent 11βHSD that reduces DHC to CS. The GC effects on interconversion of DHEA metabolites may have clinical significance, since DHEA and its 7-oxidized derivatives have been proposed for treatment of human autoimmune and inflammatory disorders.