High-performance liquid chromatography of the renal blood flow marker p-aminohippuric acid (PAH) and its metabolite N-acetyl PAH improves PAH clearance measurements.

PAH (N-(4-aminobenzoyl)glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction but the measurements must be corrected for the unspecific residual response observed in blank plasma. We have developed a HPLC method to specifically determine PAH and its metabolite NAc-PAH using a gradient elution ion-pair reversed-phase chromatography with UV detection at 273 and 265 nm, respectively. The separations were performed at room temperature on a ChromCart (125 mmx4 mm I.D.) Nucleosil 100-5 microm C18AB cartridge column, using a gradient elution of MeOH-buffer pH 3.9 1:99-->15:85 over 15 min. The pH 3.9 buffered aqueous solution consisted in a mixture of 375 ml sodium citrate-citric acid solution (21.01 g citric acid and 8.0 g NaOH per liter), added up with 2.7 ml H3PO4 85%, 1.0 g of sodium heptanesulfonate and completed ad 1000 ml with ultrapure water. The N-acetyltransferase activity does not seem to notably affect PAH clearances, although NAc-PAH represents 10.2+/-2.7% of PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC and the colorimetric method have been compared using urine and plasma samples collected from healthy volunteers. Good correlations (r=0.94 and 0.97, for plasma and urine, respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine and lower concentrations in plasma than those determined by HPLC. Hence, both renal (ClR) and systemic (Cls) clearances are systematically higher (35.1 and 17.8%, respectively) with the colorimetric method. The fraction of PAH excreted by the kidney ClR/ClS calculated from HPLC data (n=143) is, as expected, always <1 (mean=0.73+/-0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87+/-0.13 implying some unphysiological values (>1). In conclusion, HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.

Metabolic and structural rearrangement during dark-induced autophagy in soybean (Glycine max L.) nodules: An electron microscopy and P-31 and C-13 nuclear magnetic resonance study

The effects of dark-induced stress on the evolution of the soluble metabolites present in senescent soybean (Glycine max L.) nodules were analysed in vitro using C-13- and P-31-NMR spectroscopy. Sucrose and trehalose were the predominant soluble storage carbons. During dark-induced stress, a decline in sugars and some key glycolytic metabolites was observed. Whereas 84% of the sucrose disappeared, only one-half of the trehalose was utilised. This decline coincides with the depletion of Gln, Asn, Ala and with an accumulation of ureides, which reflect a huge reduction of the N-2 fixation. Concomitantly, phosphodiesters and compounds like P-choline, a good marker of membrane phospholipids hydrolysis and cell autophagy, accumulated in the nodules. An autophagic process was confirmed by the decrease in cell fatty acid content. In addition, a slight increase in unsaturated fatty acids (oleic and linoleic acids) was observed, probably as a response to peroxidation reactions. Electron microscopy analysis revealed that, despite membranes dismantling, most of the bacteroids seem to be structurally intact. Taken together, our results show that the carbohydrate starvation induced in soybean by dark stress triggers a profound metabolic and structural rearrangement in the infected cells of soybean nodule which is representative of symbiotic cessation.

Direct noninvasive estimation of myocardial tricarboxylic acid cycle flux in vivo using hyperpolarized (13)C magnetic resonance.

BACKGROUND: The heart relies on continuous energy production and imbalances herein impair cardiac function directly. The tricarboxylic acid (TCA) cycle is the primary means of energy generation in the healthy myocardium, but direct noninvasive quantification of metabolic fluxes is challenging due to the low concentration of most metabolites. Hyperpolarized (13)C magnetic resonance spectroscopy (MRS) provides the opportunity to measure cellular metabolism in real time in vivo. The aim of this work was to noninvasively measure myocardial TCA cycle flux (VTCA) in vivo within a single minute. METHODS AND RESULTS: Hyperpolarized [1-(13)C]acetate was administered at different concentrations in healthy rats. (13)C incorporation into [1-(13)C]acetylcarnitine and the TCA cycle intermediate [5-(13)C]citrate was dynamically detected in vivo with a time resolution of 3s. Different kinetic models were established and evaluated to determine the metabolic fluxes by simultaneously fitting the evolution of the (13)C labeling in acetate, acetylcarnitine, and citrate. VTCA was estimated to be 6.7±1.7μmol·g(-1)·min(-1) (dry weight), and was best estimated with a model using only the labeling in citrate and acetylcarnitine, independent of the precursor. The TCA cycle rate was not linear with the citrate-to-acetate metabolite ratio, and could thus not be quantified using a ratiometric approach. The (13)C signal evolution of citrate, i.e. citrate formation was independent of the amount of injected acetate, while the (13)C signal evolution of acetylcarnitine revealed a dose dependency with the injected acetate. The (13)C labeling of citrate did not correlate to that of acetylcarnitine, leading to the hypothesis that acetylcarnitine formation is not an indication of mitochondrial TCA cycle activity in the heart. CONCLUSIONS: Hyperpolarized [1-(13)C]acetate is a metabolic probe independent of pyruvate dehydrogenase (PDH) activity. It allows the direct estimation of VTCA in vivo, which was shown to be neither dependent on the administered acetate dose nor on the (13)C labeling of acetylcarnitine. Dynamic (13)C MRS coupled to the injection of hyperpolarized [1-(13)C]acetate can enable the measurement of metabolic changes during impaired heart function.

Embryonic gene expression of Coregonus palaea (whitefish) under pathogen stress as analyzed by high-throughput RNA-sequencing.

Most fishes produce free-living embryos that are exposed to environmental stressors immediately following fertilization, including pathogenic microorganisms. Initial immune protection of embryos involves the chorion, as a protective barrier, and maternally-allocated antimicrobial compounds. At later developmental stages, host-genetic effects influence susceptibility and tolerance, suggesting a direct interaction between embryo genes and pathogens. So far, only a few host genes could be identified that correlate with embryonic survival under pathogen stress in salmonids. Here, we utilized high-throughput RNA-sequencing in order to describe the transcriptional response of a non-model fish, the Alpine whitefish Coregonus palaea, to infection, both in terms of host genes that are likely manipulated by the pathogen, and those involved in an early putative immune response. Embryos were produced in vitro, raised individually, and exposed at the late-eyed stage to a virulent strain of the opportunistic fish pathogen Pseudomonas fluorescens. The pseudomonad increased embryonic mortality and affected gene expression substantially. For example, essential, upregulated metabolic pathways in embryos under pathogen stress included ion binding pathways, aminoacyl-tRNA-biosynthesis, and the production of arginine and proline, most probably mediated by the pathogen for its proliferation. Most prominently downregulated transcripts comprised the biosynthesis of unsaturated fatty acids, the citrate cycle, and various isoforms of b-cell transcription factors. These factors have been shown to play a significant role in host blood cell differentiation and renewal. With regard to specific immune functions, differentially expressed transcripts mapped to the complement cascade, MHC class I and II, TNF-alpha, and T-cell differentiation proteins. The results of this study reveal insights into how P. fluorescens impairs the development of whitefish embryos and set a foundation for future studies investigating host pathogen interactions in fish embryos.

L'oxalate: un déchet organique peu soluble, avec conséquences [Oxalate: a poorly soluble organic waste with consequences].

Oxalate is a highly insoluble metabolic waste excreted by the kidneys. Disturbances of oxalate metabolism are encountered in enteric hyperoxaluria (secondary to malabsorption, gastric bypass or in case of insufficient Oxalobacter colonization), in hereditary hyperoxaluria and in intoxication (ethylene glycol, vitamin C). Hyperoxaluria causes a large spectrum of diseases, from isolated hyperoxaluria to kidney stones and nephrocalcinosis formation, eventually leading to kidney failure and systemic oxalosis with life-threatening deposits in vital organs. New causes of hyperoxaluria are arising recently, in particular after gastric bypass surgery, which requires regular and preemptive monitoring. The treatment of hyperoxaluria involves reduction in oxalate intake and increase in calcium intake. Optimal urine dilution and supplementation with inhibitors of kidney stone formation (citrate) are required. Some conditions may need vitamin B6 supplementation, and the addition of probiotics might be useful in the future. Primary care physicians should identify cases of recurrent calcium oxalate stones and severe hyperoxaluria. Further management of hyperoxaluria requires specialized care.

AMP-activated protein kinase plays an important evolutionary conserved role in the regulation of glucose metabolism in fish skeletal muscle cells

AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP:ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1α) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish.

Reference intervals for 24 laboratory parameters determined in 24-hour urine collections.

BACKGROUND: Reference intervals for many laboratory parameters determined in 24-h urine collections are either not publicly available or based on small numbers, not sex specific or not from a representative sample. METHODS: Osmolality and concentrations or enzymatic activities of sodium, potassium, chloride, glucose, creatinine, citrate, cortisol, pancreatic α-amylase, total protein, albumin, transferrin, immunoglobulin G, α1-microglobulin, α2-macroglobulin, as well as porphyrins and their precursors (δ-aminolevulinic acid and porphobilinogen) were determined in 241 24-h urine samples of a population-based cohort of asymptomatic adults (121 men and 120 women). For 16 of these 24 parameters creatinine-normalized ratios were calculated based on 24-h urine creatinine. The reference intervals for these parameters were calculated according to the CLSI C28-A3 statistical guidelines. RESULTS: By contrast to most published reference intervals, which do not stratify for sex, reference intervals of 12 of 24 laboratory parameters in 24-h urine collections and of eight of 16 parameters as creatinine-normalized ratios differed significantly between men and women. For six parameters calculated as 24-h urine excretion and four parameters calculated as creatinine-normalized ratios no reference intervals had been published before. For some parameters we found significant and relevant deviations from previously reported reference intervals, most notably for 24-h urine cortisol in women. Ten 24-h urine parameters showed weak or moderate sex-specific correlations with age. CONCLUSIONS: By applying up-to-date analytical methods and clinical chemistry analyzers to 24-h urine collections from a large population-based cohort we provide as yet the most comprehensive set of sex-specific reference intervals calculated according to CLSI guidelines for parameters determined in 24-h urine collections.

Urinary di-(2-ethylhexyl) phthalate metabolites for detecting transfusion of autologous blood stored in plasticizer-free bags.

BACKGROUND: Autologous blood transfusion (ABT) efficiently increases sport performance and is the most challenging doping method to detect. Current methods for detecting this practice center on the plasticizer di(2-ethlyhexyl) phthalate (DEHP), which enters the stored blood from blood bags. Quantification of this plasticizer and its metabolites in urine can detect the transfusion of autologous blood stored in these bags. However, DEHP-free blood bags are available on the market, including n-butyryl-tri-(n-hexyl)-citrate (BTHC) blood bags. Athletes may shift to using such bags to avoid the detection of urinary DEHP metabolites. STUDY DESIGN AND METHODS: A clinical randomized double-blinded two-phase study was conducted of healthy male volunteers who underwent ABT using DEHP-containing or BTHC blood bags. All subjects received a saline injection for the control phase and a blood donation followed by ABT 36 days later. Kinetic excretion of five urinary DEHP metabolites was quantified with liquid chromatography coupled with tandem mass spectrometry. RESULTS: Surprisingly, considerable levels of urinary DEHP metabolites were observed up to 1 day after blood transfusion with BTHC blood bags. The long-term metabolites mono-(2-ethyl-5-carboxypentyl) phthalate and mono-(2-carboxymethylhexyl) phthalate were the most sensitive biomarkers to detect ABT with BTHC blood bags. Levels of DEHP were high in BTHC bags (6.6%), the tubing in the transfusion kit (25.2%), and the white blood cell filter (22.3%). CONCLUSIONS: The BTHC bag contained DEHP, despite being labeled DEHP-free. Urinary DEHP metabolite measurement is a cost-effective way to detect ABT in the antidoping field even when BTHC bags are used for blood storage.

Stabilization of the ionic form of weak acid cation exchange resins

Weak acid cation exchange (WAC) resins are used in the chromatographic separation of betaine from vinasse, a by-product of sugar industry. The ionic form of the resin determines the elution time of betaine. When a WAC-resin is in hydrogen form, the retention time of betaine is the longest and betaine elutes as the last component of vi-nasse from the chromatographic column. If the feed solution contains salts and its pH is not acidic enough to keep the resin undissociated, the ionic form of the hydrogen form resin starts to alter. Vinasse contains salts and its pH is around 5, it also contains weak acids. To keep the metal ion content (Na/H ratio) of the resin low enough to ensure successful separation of betaine, acid has to be added to either eluent (water) or vinasse. The aim of the present work was to examine by laboratory experiments which option requires less acid. Also the retention mechanism of betaine was investigated by measuring retention volumes of acetic acid and choline in different Na/H ratios of the resin. It was found that the resulting ionic form of the resin is the same regardless of whether the regeneration acid is added to the eluent or the feed solution (vinasse). Be-sides the salt concentration and the pH of vinasse, also the concentration of weak acids in the feed affects the resulting ionic form of the resin. The more buffering capacity vinasse has, the more acid is required to keep the ionic form of the resin desired. Vinasse was found to be quite strong buffer solution, which means relatively high amounts of acid are required to prevent the Na/H ratio from increasing too much. It is known that the retention volume of betaine decreases significantly, when the Na/H ratio increases. This is assumed to occur, because the amount of hydrogen bonds between the carboxylic groups of betaine and the resin decreases. Same behavior was not found with acetic acid. Choline has the same molecular structure as betaine, but instead of carboxylic group it has hydroxide group. The retention volume of choline increased as the Na/H ratio of the resin increased, because of the ion exchange reaction between choline cation and dissociated carboxylic group of the resin. Since the retention behavior of choline on the resin is opposite to the behavior of be-taine, the strong affinity of betaine towards hydrogen form WAC-resin has to be based on its carboxylic group. It is probable that the quaternary ammonium groups also affect the behavior of the carboxylic groups of betaine, causing them to form hydrogen bonds with the carboxylic groups of the resin.

Extração de ferro de esmectita brasileira com emprego do método ditionito-citrato-bicarbonato

A natural clay from Campina Grande region (Paraíba, Brazil), with 8.57% of Fe2O3, was used to study the most appropriate condition to carry out the iron extraction, without altering the clay structure in a significant way. Samples were treated with the Dithionite-Citrate-Bicarbonate method (DCB) for 30 and 120 minutes (pH=9.1), and also with citric acid (pH=1.8; time=15min), at 75°C. Conductivity measurements, X-ray fluorescence, X-ray diffraction, energy-dispersive spectrometry, electron-diffraction with transmission electron microscopy and textural evaluation by nitrogen adsorption were done. The treatment in a basic medium was more selective for iron removal than in acid condition. The time of 30 minutes, with 1.6 g Na2S2O4/10 g clay, was the best condition for the iron extraction.

Estudo polarográfico sobre a determinação de Fe(III) utilizando-se a técnica da polarografia de pulso diferencial

A differential pulse polarographic study with the objective to determine iron (III) in presense of copper (II) in a supporting electrolyte based on citrate - EDTA was made. The best experimental conditions found were a supporting electrolyte of citrate 0.25 mol L-1, EDTA 0.050 mol L-1 and KNO3 0.50 mol L-1, pH 5.00. In this media iron (III) showed a polarographic peak in -0.08 V and the copper (II) in -0.34 V, both vs. Ag/AgCl (saturated KCl). Thus, a analytical method was developed and applied to determine iron (III) in brass alloy, a matrix were copper is in large excess over iron. The results obtained showed no interference of copper in the iron determination. The value of 0.21% of iron in the sample alloy composition was obtained and the method was validated by atomic absortion and recovery test, and the results exhibited a good agreement with the proposed method.

Mechanism of 3,4-dihydroxybenzaldehyde electropolymerization at carbon paste electrodes: catalytic detection of NADH

Cyclic voltammetry was used to study 3,4-dihydroxybenzaldehyde (3,4-DHB) electropolymerization processes on carbon paste electrodes. The characteristics of the electropolymerized films were highly dependent on pH, anodic switching potential, scan rate, 3,4-DHB concentrations and number of cycles. Film stability was determined in citrate/phosphate buffer solutions at the same pH used during the electropolymerization process. The best conditions to prepare carbon paste modified electrodes were pH 7.8; 0.0 <= Eapl <= 0.25 V; 10 mV s-1; 0.25 mmol L-1 3,4-DHB and 10 scans. These carbon paste modified electrodes were used for NADH catalytic detection at 0.23 V in the range 0.015 <= [NADH] <= 0.21 mmol L-1. Experimental data were used to propose a mechanism for the 3,4--DHB electropolymerization processes, which involves initial phenoxyl radical formation.

Influência da calcinação sobre a remoção de ferro da caulinita e ilita e seus efeitos sobre a acidez

Samples of natural clay composed by kaolinite, illite, goethite and quartz, were calcinated and submitted to lixiviation with citrate and chloridric acid in order to remove iron. Investigation due to extraction consequences was carried cut by analyzing its acid properties using ammonia gas as probe in infrared spectrophotometry analysis. The sample that were treated with citrate followed by acid lixiviation yield materials twice more acid than samples treated with acid only.

Determinação potenciométrica em fluxo de cloreto de cetilpiridinio em desinfectantes bucais

The work describes a new procedure for cetylpyridinium chloride determination in oral disinfectants, based on a flow-injection system with potentiometric detection. The determination was based on the measurement of picrate concentration decrease as result of ion-pair reaction with the analyte present in the injected sample. In the optimised set-up the sample injection volume was kept at 400 µL and merged downstream with the reagent solution containing 1,0 x10-5 mol/L of picrate adjusted to pH 5.0 with citrate/citric acid buffer. The flow rate was fixed at 8 mL/min and the reactor length at 40 cm. The proposed procedure enables the determination of cetylpyridinium in the analytical range of 5,0x10-6 - 7,5x10-5 mol/L at a sampling rate of 60/h. The results for real samples had a precision better than 3% and were comparable to the labelled values.

Validation and statistical analysis of two high performance liquid chromatography methods for the determination of indinavir sulfate raw material and capsules

Two high performance liquid chromatography (HPLC) methods for the quantitative determination of indinavir sulfate were tested, validated and statistically compared. Assays were carried out using as mobile phases mixtures of dibutylammonium phosphate buffer pH 6.5 and acetonitrile (55:45) at 1 mL/min or citrate buffer pH 5 and acetonitrile (60:40) at 1 mL/min, an octylsilane column (RP-8) and a UV spectrophotometric detector at 260 nm. Both methods showed good sensitivity, linearity, precision and accuracy. The statistical analysis using the t-student test for the determination of indinavir sulfate raw material and capsules indicated no statistically significant difference between the two methods.