The evaluation of the repeatability of the 13C-ketoisocaproate breath test for assessing hepatic mitochondrial function.

The 13C-ketoisocaproate (13C-KICA) breath test (BT) has been recently proposed as a non-invasive test for assessing hepatic mitochondrial function. Results of the 13C-KICA BT can be expressed as different parameters. However, the best parameter for expressing the 13C-KICA BT result is uncertain which hinders use of the BT in routine clinical practice. We have investigated the repeatability of different parameters of 13C-KICA BT. Thirteen healthy adult subjects (5 men and 8 women) underwent a 13C-KICA BT on two occasions separated by a gap of approximately 30 days. There were no significant differences between the repeated measurements for all the test parameters over 30 days. Furthermore, the Bland Altman statistics showed no fixed or proportional bias for any of the test parameters. The cumulative 13C-dose enrichment over 60 min had the lowest within-subject variability of 12% compared to all other test parameters. The cumulative 13C-dose enrichment over 60 min could be a very useful parameter for the 13C-KICA BT to detect impaired hepatic mitochondrial function in patients with chronic liver diseases.

Identification and quantification of oxo-bile acids in human faeces with liquid chromatography-mass spectrometry: A potent tool for human gut acidic sterolbiome studies.

Bile acids (BAs) are endogenous steroids involved in the transport of lipids in bile, acting also as molecular signaling hormones. Primary BAs synthesized in the liver undergo several metabolic pathways in the intestine by gut microbiota to produce secondary BAs. Together with secondary BAs, other metabolites have been recovered from human faeces, including many oxo-BA analogues produced in the colon through oxidation of BA hydroxy groups. However, the complete oxo-BA characterization in biospecimens (particularly intestinal content and faeces) has not been reported yet, hampering the assessment of their potential physiological role. Herein, we have developed and validated a new RP-HPLC-ESI-MS/MS method in negative ionization mode for the simultaneous analysis of 21 oxo-BAs and their 7 metabolic BAs precursors in human faeces. The elution was performed in gradient mode and 28 compounds, including primary, secondary BAs, and their oxo-derivatives, were separated within 50 min at 40 °C column temperature. The method is accurate (bias% <13%), precise (CV% <10%), with limits of quantification (LOQ <30 ng/mLextract samples), similar for all the studied compounds. The matrix effect does not significantly affect the analysis accuracy, allowing the use of standard solutions for the quantifications, without matrix-matched protocols. Thanks to the high detectability and the relatively high concentration of oxo-BAs (about µg/gwet faeces), the method does not require a pre-analytical clean-up step. This method was used to identify and quantify oxo-BAs in human faecal samples from healthy subjects, serving as a proof of concept for application in patients with hepatobiliary disease and bacteria overgrowth.

Scrutiny of 13C-phenylalanine breath test reproducibility.

We evaluated the reproducibility of the 13C-phenylalanine breath test (13C-PheBT). On three separate days, 21 healthy volunteers (11 F and 10 M) underwent 13C-PheBT with 100 mg l-[1-13C]phenylalanine taken orally. Short-term reproducibility was evaluated with paired examinations taken 3 days apart; paired examinations separated by 23 days (median) served for the medium-term reproducibility assessment. Expiratory air was sampled at 19 points throughout 3 h. Determined limited reproducibility of the 13C-PheBT must be taken into consideration while interpreting the results of this diagnostic tool. The results of this study imply the following conclusions: (i) From among the three parameters examined, the cumulative 13C recovery area under the curve (AUC) offers much better reproducibility than the maximum momentary 13C recovery in the expiratory air (Dmax) or the time to reach the maximum momentary 13C recovery (Tmax) (ii) Collection of the breath air samples for 2 h results in a much better reproducibility of AUC, than for 1 h only; (iii) Reproducibility of 13C-PheBT is affected neither by the duration of the time gap between repeated tests nor by gender; (iv) Comparison with data obtained formerly reveals that reproducibility of the 13C-PheBT is worse than either that of of the 13C-methacetin (13C-MBT) or the 13C-alpha-ketoisocaproic acic (13C-KICA-BT) breath tests. This finding will have to be taken into consideration while interpreting the results of this diagnostic tool.

Determination of pKa and Hydration Constants for a Series of α-Keto-Carboxylic Acids Using Nuclear Magnetic Resonance Spectrometry.

The determination of the acid-base dissociation constants, and thus the pKa values, of α-keto acids such as pyruvic acid is complex because of the existence of these acids in their hydrated and nonhydrated or oxo state. Equilibria involved in the hydration and dehydration of the α-keto group of pyruvic acid and three other α-keto acids, 3-methyl-2-oxobutanoic acid, 4-methyl-2-oxopentanoic acid, and 2-oxo-2-phenylacetic acid, were investigated by proton and carbon nuclear magnetic resonance spectrometry, at constant ionic strength, 0.15, and 25 °C. Dissociation constants for the oxo (pKa(oxo)) and hydrated (pKa(hyd)) acids of each compound were estimated from the change in the degree of hydration with changes in pH and directly from the changes in chemical shifts of various hydrogen and carbons nuclei with pH. α-Keto acids showed greater hydration in their acidic forms than their carboxylate forms. The degree of hydration was sensitive to steric and electronic/resonance factors. As expected, the oxo forms of the acids were stronger acids compared with their hydrated analogs, and their dissociation constants were also sensitive to steric and electronic factors.

[Simultaneous determination of five active components of compound α-ketoacid tablet in human urine by ion-pair reversed-phase high performance liquid chromatography].

A simple and sensitive method for the simultaneous determination of five active components, D, L-α-hydroxymethionine calcium (HMACa), α-ketovaline calcium (KVCa), D, L-α-ketoisoleucine calcium (KILCa), α-ketoleucine calcium (KLCa) and α-ketophenylalanine calcium (KPACa) of compound α-ketoacid tablet in human urine by ion-pair reversed-phase high performance liquid chromatography (RP-HPLC) was developed and validated. The separation conditions, such as the concentration of ion-pair reagent, the pH value of the mobile phase and the concentration of the buffer were optimized. All the five analytes were separated well on a C18 column (250 mm x 4.6 mm, 5 µm) with diode array detection at 210 nm and the column temperature of 35 °C. The mobile phases were acetonitrile and 20 mmol/L phosphate buffer (containing 15 mmol/L tetrabutylammonium hydroxide; pH 7) at the flow rate of 1. 0 mL/min with gradient elution. The calibration curves for the five components were linear in the range from 20 to 200 mg/L (r≥ 0. 9990). The limits of detection (LODs, S/N= 3) were 3.0, 5. 0, 3. 6, 5.7 and 2. 5 mg/L, and the limits of quantification (LOQs, S/N= 10) were 9. 6, 16.7, 12.0, 19.0 and 8.3 mg/L for HMACa, KVCa, KILCa, KLCa and KPACa, respectively. The intra-day and inter-day precisions were less than 7%, and the average recoveries were between 86.79% and 112. 00% in the human urine with RSDs lower than 9% (n= 5). The method proved precise, specific and reproducible, and can be used for the determination of the five components in urine.

Determination of α-keto acids in pork meat and Iberian ham via tandem mass spectrometry.

An analytical method which offers accurate determination and identification of eight α-keto acids (α-ketoglutaric acid, pyruvic acid, 4-hydroxyphenylpyruvic acid, 3-methyl-2-oxobutyric acid, α-keto-γ-methylthiobutyric acid, 4-methyl-2-oxovaleric acid, 3-methyl-2-oxovaleric acid, and phenylpyruvic acid) in pork meat and Iberian ham samples is reported. The method utilises a highly selective and sensitive method of multiple reaction monitoring (MRM) by mass spectrometry. The analytical method is simple (although the chemical derivatisation of the α-keto acids with dansylhydrazine is required), precise (<18% RSD), accurate (90-110%), sensitive (0.01-0.34 mg/kg of defatted and freeze-dried meat depending on the α-keto acid) and linear (R>0.99) over several orders of magnitude (until 0.01-146.1 mg/kg of defatted and freeze-dried meat depending on the α-keto acid). Using this methodology, α-keto acids were found to be present in pork meat to a low extent, and their concentration increased when they were determined in Iberian ham. This is the first report of the presence of α-keto acids in both pork meats and Iberian hams.

Keto acid profiling analysis as ethoxime/tert-butyldimethylsilyl derivatives by gas chromatography-mass spectrometry.

Organic acids, including keto acids, are key intermediates of central pathways in cellular metabolism. In this study, a comprehensive and reliable method was developed and optimized for the simultaneous measurement of 17 keto acids in various biological samples. The keto acids were converted to solvent extractable forms by ethoximation followed by tert-butyldimethylsilylation for direct analysis by gas chromatography-mass spectrometry in selected ion monitoring mode. The proposed method was precise (0.05-8.3, % RSD) and accurate (-10.5 to 5.3, % RE) with low limit of detection (0.01-0.5ng/mL) and good linearity (r>0.995) in the range of 0.01-5.0µg/mL. This was suitable for profiling analysis of targeted keto acids in human plasma, urine and rat brain tissue.

Probing AGXT2 enzyme activity in mouse tissue by applying stable isotope-labeled asymmetric dimethyl arginine as substrate.

Asymmetric dimethylarginine (ADMA) is a metabolite of the amino acid L-arginine. It competitively inhibits the enzymatic production of the cell-signaling substance nitric oxide. Therefore, increased levels of ADMA are associated with a range of cardiovascular and other diseases. ADMA is biologically eliminated by direct renal excretion and hydrolysis by the enzyme DDAH. Recently, a further elimination pathway via the transamination by the enzyme AGXT2 to α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) has come into the focus of biological research. In this work, we describe an assay for the AGXT2 activity in mouse liver and kidney tissue. It is based on the transformation of isotope-labeled ADMA-d(6) to DMGV-d(6). The quantification of the DMGV-d(6) produced by this reaction in tissue homogenate samples was accomplished by chromatographic separation on a porous graphitic carbon column and tandem mass spectrometric detection. DMGV-d(6) with the deuterium labels in different molecular positions was used as internal standard. The overall production rates of DMGV-d(6) in mice were 195.37 pmol/min/mg total protein in liver and 85.21 pmol/min/mg total protein in kidney tissue, with coefficients of variation of 6.31% and 11.25%, respectively. This method can be applied as a tool for the characterization of the ADMA elimination by the AGXT2 pathway.

Detection and quantification of α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid: a metabolite of asymmetric dimethylarginine.

Nitric oxide is an ubiquitary cell signaling substance. Its enzymatic production rate by nitric oxide synthase is regulated by the concentrations of the substrate L-arginine and the competitive inhibitor asymmetric dimethylarginine (ADMA). A newly recognized elimination pathway for ADMA is the transamination to α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) by the enzyme alanine-glyoxylate aminotransferase 2 (AGXT2). This pathway has been proven to be relevant for nitric oxide regulation, but up to now no method exists for the determination of DMGV in biological fluids. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of DMGV. D(6)-DMGV was used as internal standard. Samples were purified online by column switching, and separation was achieved on a porous graphitic carbon column. The calibration was linear over ranges of 10 to 200 nmol/L for plasma and 0.1 to 20 µmol/L for urine. The intra- and interday accuracies and precisions in plasma and urine were better than 10%. In plasma samples, DMGV was present in concentrations between 19.1 and 77.5 nmol/L. In urine samples, concentrations between 0.0114 and 1.03 µmol/mmol creatinine were found. This method can be used as a tool for the scientific investigation of the ADMA conversion to DMGV via the enzyme AGXT2.

Simultaneous determination of D-amino acids by the coupling method of D-amino acid oxidase with high-performance liquid chromatography.

An enzymatic assay system of D-amino acids was established using the D-amino acid oxidase of Schizosaccharomyces pombe. In this method, the enzyme converts the D-amino acids to the corresponding α-keto acids, which are then reacted with 1,2-diamino-4,5-methylenedioxybenzene (DMB) in an organic solvent. The resultant fluorescent compounds are separated and quantified by high-performance liquid chromatography (HPLC). Use of an organic solvent following the α-keto acid modification with DMB prevents the non-enzymatic deamination of L-amino acids, which are generally present at much higher concentrations than D-amino acids in biological samples. With this method, D-Glu, D-Asn, D-Gln, D-Ala, D-Val, D-Leu, D-Phe, and D-Ile can be quantified in the order of micromolar, and other D-amino acids except D-Asp can be assayed within a sensitivity range of 50-100 µM. The established enzymatic method was used to analyze the d-amino acid contents in human urine. The concentration of D-Ser obtained using this enzymatic method (223 µM) was in good agreement with that obtained using the conventional HPLC method (198 µM). The enzymatic method also demonstrated that the human urine contained 5.45 µM of d-Ala and 0.91 µM of D-Asn. Both D-amino acids were difficult to be identified using the conventional method, because the large signals from L-amino acids masked those from d-amino acids. The enzymatic method that we have developed can circumvent this problem.

Computational screening of novel thiamine-catalyzed decarboxylation reactions of 2-keto acids.

A molecular modeling strategy to screen the capacity of known enzymes to catalyze the reactions of non-native substrates is presented. The binding of pyruvic acid and non-native ketoacids in the active site of pyruvate ferredoxin oxidoreductase was examined using docking analysis, and our results suggest that enzyme-non-native ketoacid-bound species are feasible. Quantum mechanics/molecular mechanics methods were then used to study the geometry of the covalent intermediate formed from the enzyme and the various ketoacids. Finally, quantum mechanical methods were used to study the decarboxylation reaction of 2-keto acids at the mechanistic level. This hierarchical screening ranked the substrates from those that cannot be accommodated by the enzyme (phenyl pyruvate) to those whose conversion rate would most closely approach that of the native substrate (2-ketobutanoic acid and 2-ketovaleric acid). Most notably, our investigation suggests that novel pathways generated using generalized enzyme actions may be screened using the hierarchical approach employed here.

Intracellular alpha-keto acid quantification by fluorescence-HPLC.

Procedures for the analysis of free alpha-keto acids in human fluids (i.e. plasma, cerebrospinal fluid, urine, etc.) as well as for studying the dynamic free alpha-keto acid pools in differentiated tissues and organ cells have been the subject of growing clinical interest in the study of metabolic regulatory and pathophysiological phenomena. Due to the high instability and polarity of the alpha-keto acids being examined, the development of a quantitative and reproducible analysis of metabolically relevant intracellular alpha-keto acids still presents a substantial methodological challenge. The aim of small sample size, rapid, non-damaging and "metabolism-neutral" cell isolation, careful sample preparation and stability, as well as reproducible analytics technology is not often achieved. Only few of the methods described can satisfy the rigorous demands for an ultra-sensitive, comprehensive and rapid intracellular alpha-keto acid analysis.

Quantitative determination of epoxy acids, keto acids and hydroxy acids formed in fats and oils at frying temperatures.

A method based on derivatization to fatty acid methyl esters and GC is proposed for the quantitative analysis of hydroxy acids, keto acids and epoxy acids in fats and oils. Isolation of the analytes by solid-phase extraction is proposed to prevent analytical interferences caused by non-altered fatty acids naturally occurring in oils. In addition, hydrogenation is required before the GC analysis to improve repeatability. The analytical method was applied to thermoxidized samples of high linoleic sunflower oil, high oleic sunflower oil and high palmitic sunflower oil. Results showed total levels of these compounds in the order of mg/g of oil in samples with contents of polar compounds ranging from 6.7 to 25.7%. The compounds analyzed constituted major fractions of the oxidized fatty acids.

Energy restriction in obese subjects impact differently two mitochondrial function markers

Excessive fat deposition is the key feature in obesity, which is empowered bycytokines overproduction and stimulation of cell oxidative stress processes, but littleis known about energy availability in the form of ATP and mitochondrial functionin the obese subjects. Thus, the aim of this study was to evaluate the possible changesin energy metabolism after a 8-weeks balanced-hypocaloric diet in obese subjects bymeasuring the ATP-content in leukocytes, by assessing 2-keto[1-13C]isocaproatebreath test (KICA-BT) parameters related to mitochondrial function and by analyzinginflammatory and oxidative stress biomarkers. All the recruited obese subjects(n=19) lost body weight after dieting (-5.55±2.88%). The hypocaloric treatmentinduced a decrease in leptin levels and lipid peroxidation markers. Interestingly, theATP content in blood leukocytes increased (49.9±32.5 vs 36.2±27.9 pmol/mg prot.;p<0.05), while KICA tracer mitochondrial oxidation decreased (30.9±5.9 vs. 33.1±4.5%13C; p<0.05) after weight loss. These results show that two minimally invasivemethods were able to detect changes in mitochondrial function as induced by ahypocaloric diet, which is of great interest in order to understand oxidative processesassociated with weight homeostasis as well as to establish newer anti-obesity therapeutictargets by using mitochondrial function markers in vivo (AU)

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Energy restriction in obese subjects impact differently two mitochondrial function markers.

Excessive fat deposition is the key feature in obesity, which is empowered by cytokines overproduction and stimulation of cell oxidative stress processes, but little is known about energy availability in the form of ATP and mitochondrial function in the obese subjects. Thus, the aim of this study was to evaluate the possible changes in energy metabolism after a 8-weeks balanced-hypocaloric diet in obese subjects by measuring the ATP-content in leukocytes, by assessing 2-keto[1-13C]isocaproate breath test (KICA-BT) parameters related to mitochondrial function and by analyzing inflammatory and oxidative stress biomarkers. All the recruited obese subjects (n = 19) lost body weight after dieting (-5.55 +/- 2.88%). The hypocaloric treatment induced a decrease in leptin levels and lipid peroxidation markers. Interestingly, the ATP content in blood leukocytes increased (49.9 +/- 32.5 vs 36.2 +/- 27.9 pmol/mg prot.; p < 0.05), while KICA tracer mitochondrial oxidation decreased (30.9 +/- 5.9 vs. 33.1 +/- 4.5 % 13C; p < 0.05) after weight loss. These results show that two minimally invasive methods were able to detect changes in mitochondrial function as induced by a hypocaloric diet, which is of great interest in order to understand oxidative processes associated with weight homeostasis as well as to establish newer anti-obesity therapeutic targets by using mitochondrial function markers in vivo.

Simultaneous determination of ketoacids and dicarbonyl compounds, key Maillard intermediates on the generation of aged wine aroma.

The aim of this work was the simultaneous determination of both ketoacids and dicarbonyl compounds in wine. To detect ketoacid compounds in wine, a method based on the quinoxaline derivatives by the reaction with diaminobenzene, currently employed to detect alpha-dicarbonyl compounds, was developed. The quinoxaline derivatives were detected by RP-HPLC with UV detection, which allows the determination of the major dicarbonyl compounds in wine: glyoxal, methylglyoxal, diacetyl and pentane-2,6-dione, and the quinoxaline/quinoxalinol derivatives of alpha-keto-gamma-(methylthio)butyric acid and beta-phenylpyruvic acid (intermediate ketoacid compounds of methional and phenylacetaldehyde) were simultaneously detected by a fluorescence detector. The identification was performed by comparison with standards and also by using LC-MSMS. The levels found in 15 wines analyzed (white wines, Madeira wines, and Port wines) diverge according to the type and the age of the wine. The ketoacid compounds ranged from 0.2 to 5.7 mg/L for alpha-keto-gamma-(methylthio)butyric acid and 0.1 to 9.6 mg/L for beta-phenylpyruvic acid. The quantities observed for dicarbonyl compounds were similar to those already reported.

[Aldehydes, ketones, and ketoacids produced during ozonation of NOM fractions isolated from filtrated water].

Natural organic matter (NOM) in the filtrated surface water from a water treatment plant was isolated and fractionated into six types of fractions. These fractions were ozonated at typical ozone dose and reaction time. The yields of several small molecular ozonation by-products, such as, aldehydes, ketones and ketoacids, were monitored after ozonation of the individual NOM fractions. The results showed that formaldehyde and pyruvic acid had the maximal yields among the aldehydes and ketoacids, respectively. Hydrophobic neutral (HON) produced the most significant yield of the ozonation by-products with the specific yield of formaldehyde up to 70.58% of the specific yields and pyruvic acid being 103.2 microg/mg. The hydrophobic NOM fractions produced higher yields of these by-products than the hydrophilic fractions. For example, HON and hydrophobic acid (HOA) produced much higher yields of the ozonation by-products than other fractions. Aldehydes and ketones from HON and HOA and ketoacids from the two fractions accounted for 55.56% and 60% of the ozonation by-products from all the NOM, respectively. Lower production of these by-products was also observed for the basic fractions compared to the acidic and neutral ones. HON and HOA were more biodegradable after ozonation, because their ozonation by-products contributed much higher amount of DOC to the final DOC after ozonation.

Structural analysis of oligomeric molecules formed from the reaction products of oleic acid ozonolysis.

The products arising from the ozonolysis of oleic acid (cis-9-octadecenoic acid) in solution have been studied using negative ion mode electrospray ionization ion trap mass spectrometry. Oleic acid is an important component of atmospheric organic aerosol and is a key model species in predicting aerosol physical and chemical characteristics. The four predicted reaction products, 1-nonanal, nonanoic acid, 9-oxononanoic acid, and azelaic acid, were all observed in roughly equal yields. In addition to these products a large number of higher molecular weight compounds were detected with m/z ratios of up to 1000 Daltons. Tandem mass spectrometry of these larger ions revealed thatthey represented a complex mixture of linear alpha-acyloxyalkyl hydroperoxides, secondary ozonides, and cyclic diperoxides, formed by reactions between ozonolysis products and Criegee intermediates. These comprise the first directly elucidated structures of large oligomeric species from oleic acid ozonolysis. The degree of oligomerization and hence molecular weight distribution was observed to increase with reaction time in solution.

Low molecular weight dicarboxylic acids, ketoacids, and dicarbonyls in the fine particles from a roadway tunnel: possible secondary production from the precursors.

Low molecular weight dicarboxylic acids (DCAs), ketoacids, and alpha-dicarbonyls have been determined for the PM2.5 samples in a Hong Kong roadway tunnel, using a water extraction followed by a butyl ester and/or dibutyl acetal derivatization technique. For the most wintertime sampling runs, outlet and inlet concentrations of the quantified compounds were found to be quite similar (ca. 10% differences), leading to the conclusion that direct emissions of the organic compounds are insignificant from vehicles in hot-stabilized operations although vehicular emissions can provide important precursors to them. In contrast, a significant concentration increase of most compounds was observed at the outlet station compared to the inlet station in the summertime runs, which might be explained by the secondary production of aerosols in the tunnel. The organic compounds studied comprised a small fraction (<1%) of aerosol organic carbon (OC). In winter, their abundances relative to that of OC in outlet samples were found to be significantly less than those in inlet samples. On the basis of the summer data, apparent secondary production factors of the compounds were calculated, which indicate that adipic and m-phthalic acids can be favorably formed in the tunnel. However, like other DCAs, direct emissions of adipic, m-phthalic, and p-phthalic acids from automobiles are suggested to be insignificant.

Secondary organic aerosol production from terpene ozonolysis. 1. Effect of UV radiation.

We report secondary organic aerosol (SOA) yields from the ozonolysis of alpha-pinene under both dark and UV-illuminated conditions. Exposure to UV light reduces SOA yield by 20-40%, with a maximum reduction in yield coinciding with a minimum in the amount of terpene consumed (15-30 ppb). The data are consistent with a constant absolute reduction in the yield of approximately 0.03. Gas chromatography mass spectrometry analysis of filter samples indicates that the major products found in alpha-pinene SOA include organic acids (e.g., pinic acid), keto acids (e.g., pinonic acid), and hydroxy keto acids (e.g., 10-hydroxypinonic acid). Analysis of filter-based results suggests that yield reduction is a result of the formation of a more volatile product distribution when experiments are conducted in the presence of UV light. These results implythat previous "dark bag" experiments may overestimate SOA generation from monoterpenes and also that SOA generation in the atmosphere may depend significantly on actinic flux.