Validation of an HPLC method for the determination of urinary and plasma levels of N1-methylnicotinamide, an endogenous marker of renal cationic transport and plasma flow

N1-Methylnicotinamide (NMN) is an endogenous cationic metabolite of nicotinamide (niacine, vitamine PP) whose renal clearance reflects both the capacity of the renal tubular transport system to secrete organic cations and renal plasma flow. NMN is present in human plasma and urine at the 1-117-ng ml(-1) and 0.5-25-microg ml(-1) concentration range, respectively, and its level depends notably on pathophysiological (age, renal or hepatic diseases) conditions. We report the optimization and validation of an HPLC method for the measurement of endogenous NMN in biological fluids after derivatization into a fluorescent compound. Plasma is first deproteinized with TCA 20% and the urine diluted 1:10 with HCI 10(-4) M prior to the derivatization procedure, which includes a condensation reaction of NMN with acetophenone in NaOH at 0 degrees C, followed by dehydration in formic acid and subsequent formation of the fluorescent 1,6-naphthyridine derivatives after heating samples in a boiling water bath. The synthetic homologous derivative N1-ethylnicotinamide (NEN) reacts similarly and is added as internal standard into the biological fluid. The reaction mixture is subjected to reverse phase high performance liquid chromatography on a Nucleosil 100-C18 column using a mobile phase (acetonitrile 22%, triethylamine 0.5%, 0.01 M sodium heptanesulfonate adjusted to pH 3.2), delivered isocratically at a flow rate of 1 ml min(-1), NMN and NEN are detected at 7.8 and 10 min by spectrofluorimetry with excitation and emission wavelengths set at 366 and 418 nm, respectively. The addition-calibration method is used with plasma and urine pools. Calibration curves (using the internal standard method) are linear (r2 > 0.997) at concentrations up to 109 ng ml(-1) and 15.7 microg ml(-1) in plasma and urine, respectively. Both intra- and inter-assay precision of plasma control samples at 10, 50 and 90 ng ml(-1) were lower than 3.3% and concentrations not deviating more than 2.7% from their nominal values. In urine intra- and inter-assay CVs of control samples at 1, 5 and 9 microg ml(-1) are lower than 8.3%, with concentrations not deviating more than -9.0 to +11.8% from their nominal values. This analytical method has therefore the required sensitivity and selectivity to measure NMN in plasma and urine, enabling the non-invasive determination of the tubular secretory capacity of the kidney and the renal plasma flow

Specific determination of PAH and its N-acetyl metabolite by HPLC increases the accuracy and precision of 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. Although straightforward, the measurements must be corrected for the nonspecific residual response observed in blank plasma. We have therefore developed an HPLC method for the specific determination of PAH and its metabolite NAc-PAH using a gradient elution ion-pair reverse-phase chromatography with UV detection. The Nacetyltransferase (NAT-1 or NAT-2 dependent) activity does not seem clinically relevant nor does it affect notably PAH clearances, although NAc-PAH represents 10.2 +/- 2.7% of the PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC technique has been compared with the colorimetric method using urine and plasma samples collected from 12 healthy volunteers following a priming dose of PAH followed by a constant rate infusion. 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 while the concentrations in plasma are lower than those determined by HPLC. Hence, both renal (CLR = U x V/P) and systemic (CLS = Rinf/Css) clearances are systematically higher (35.1%, resp. 17.8%) 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 unphysio-logical values (> 1) in some cases. 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

Continuous infusion of ceftazidime with a portable pump is as effective as thrice-a-day bolus in cystic fibrosis children

Continuous infusion (CI) of beta-lactam antibiotics provides a stable concentration which may result in a better activity against gram-negative bacteria if exceeding the minimum inhibitory concentration (MIC). Treatment outcome after 24 h CI of ceftazidime (CAZ) in cystic fibrosis (CF) children was compared with the bolus administration regimen. Fourteen CF children with chronic Pseudomonas aeruginosa pulmonary infection were treated during 14 days with the conventional CAZ thrice-a-day bolus infusion (regimen A), and few months later with 24 h Cl of CAZ (regimen B) using a portable pump. Amikacin was added to both regimens. Clinical efficacy of treatment was assessed using pulmonary, inflammatory and nutritional variables. Bacteriological analyses and CAZ concentrations in serum and sputum were also measured. All patients improved clinically with both regimens. Among the parameters used to compare both regimens, only prealbumin values improved (regimen A: + 0.08 g/l versus regimen B: +0.11 g/l P = 0.015). No clinically significant side-effects were noted. In regimen A, the mean predose (trough level) CAZ concentration in serum was highly variable (range 2.2-45.4 gg/ml) with some values (32% of samples) below the MIC of P. aeruginosa isolates found in the sputum of the patients. In regimen B, the serum CAZ level achieved was 28.5+/-8.4 microg/ml without any value below the MIC. The mean sputum levels were comparable in both regimens. No CAZ resistant strains of P. aerugino.sa appeared between and directly after the treatments. CONCLUSION: The clinical outcome of children with cystic fibrosis treated with 24 h continuous infusion of ceftazidime was no different from that achieved with the conventional bolus infusion regimen. Continuous infusion provided a sustained serum ceftazidime level well above the P. aeruginosa minimum inhibitory concentration. Continuous infusion was well tolerated and appreciated by the children and this may promote home therapy for cystic fibrosis children

Determination of trace lithium in human erythrocytes by electrothermal atomic-absorption spectrometry with pyrocoated graphite tubes and integrated platform

Electrothermal graphite-furnace atomic-absorption spectroscopy with pyrocoated graphite tubes, integrated platform and matrix modification was used to determine submicromolar concentrations of trace lithium in human red blood cells. Matrix-matched samples were used to establish calibration curves for concentrations up to 0.58 microM (addition-calibration method) with satisfactory linearity (r2 > 0.99) and intra- and inter-day variability (CV < 11.4%). The median concentration of trace lithium in the cells of 40 healthy Caucasian volunteers devoid of medical or psychiatric history was 0.23 microM (inter-quartile range 0.20-0.30). The levels of trace lithium in the red blood cells correlated (r2 = 0.83) with plasma concentrations (median 0.13 microM, inter-quartile range 0.11-0.19) measured in the same blood sample. Dietary factors (e.g. consumption of lithium-containing mineral water) affected both levels. The red blood cell/plasma lithium ratio had a median value of 1.57 (inter-quartile range 1.16-2.07), implying that trace lithium is accumulated in erythrocytes. This contrasts with most reports of red blood cell/plasma ratio, measured during therapeutic treatment with lithium, for which the average value is 0.5-0.8, albeit for much higher concentrations of lithium (approx. 500-800 microM). The proposed analytical method has the required sensitivity and accuracy for determination of trace lithium in red blood cells and makes it possible to perform epidemiological studies to assess human exposure to environmental lithium in diet and beverages, and inter-individual variations in trans-membrane and renal lithium kinetics at the submicromolar level

An EM-type approach for classification of bivariate MALDI-MS data and identification of high fertility markers

Dairy cows are responsible for a fair amount of gas emissions in the atmosphere (mainly methane, ammonia, and carbon dioxide), as well as waste outputs. Therefore, identifying high-fertility breeding cows and increasing fertility rates can diminish pollution and help minimize the effect of global warming and improve the environmental impact of the farming system. As a step to achieve this goal, changes in the lipid composition of the bovine uterus exposed to greater (LF-LCL group) or lower (SF-SCL group) concentrations of progesterone during postovulation were investigated by matrix-assisted laser desorption ionization mass spectrometry. Two measurements were made for each cow, and after preprocessing the data, the measurements available to analysis consist of relative intensities at significant 76 mass-to-charge ratio (m/z) values identifying specific ions in the spectra. Due to the small sample size, seven cows in the LF-LCL group and 10 cows in the SF-SCL group, the usual methods could not discriminate between groups. A model-based approach was therefore proposed, and due to the discrete nature of the data, a truncated mixture of bivariate beta distributions was fitted to the data using an expectation-maximization algorithm. However, unlike the usual approach for mixture density estimation problems, to each 76 m/z value, we assign an unobserved label shared by all cows in the same group. The role of these labels is similar to the frailty effect in survival models in which all cows in a given group would share some random effect due to group effect. These labels will be used to identify m/z values, which could potentially account for different fertility rates.303CNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo4420122014-4; 3025982014-62011/0326-4; 2011/06191-7; 2012/07206-

Estimation of glomerular filtration rate by sinistrin clearance using various approaches.

Two protocols for the determination of glomerular filtration rate (GFR) from sinistrin clearance are considered: a bolus injection and a bolus followed by infusion. On both occasions, serial blood and urine samplings are scheduled up to 6 h. Four calculation methods are compared for estimating GFR from the data obtained during each protocol: classical UV/P (ratio of urinary excretion rate over plasma concentration) after bolus or bolus plus infusion; 2-point (log-linear slope multiplied by apparent volume of distribution); D/AUC (ratio of dose over area under the curve) after bolus; and Rin/P (ratio of infusion rate over steady-state concentration) during infusion. Some refinements of the calculations are devised. Data are simulated by running a bicompartmental pharmacokinetic model with renal elimination, and contaminating the values with an array of random errors. The statistical performance of the respective calculation methods is assessed by graphical means. The UV/P method performs poorly during 2 hours following the bolus; on both bolus and infusion data, it suffers from imprecision on the urinary volume. The 2-point method is acceptable between 1 and 4 h after bolus; later, the estimates become much less precise. The D/AUC method appears highly reliable when integrating the concentrations up to 3 h after bolus; it requires extrapolation towards infinity. The Rin/P method is satisfactory if applied later than 3 to 4 h after the loading dose. The advantages and drawbacks of each methods must be evaluated in relation with the particular clinical setting in which GFR is to be estimated. D/AUC represents the most advisable approach for snapshot renal testing in subjects or patients without important renal impairment

Lipidomic alterations of in vitro macrophage infection by L. infantum and L. amazonensis

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOParticular lipid profiles have been found in two different protozoa of the Leishmania genus. Leishmania infantum, a visceral leishmaniasis causative agent and Leishmania amazonensis, a cutaneous leishmaniasis, reveal distinctive lipid contents of phosphat131124012406FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2010/51677-22015/23767-02016/11517-22012/07206-02013/11100-6162191/2015-

Phospholipid profile and distribution in the receptive oviduct and uterus during early diestrus in cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Phospholipid metabolism and signaling influences on early pregnancy events in cattle are unknown. This study aimed to characterize global phospholipid composition of oviduct and uterus during early diestrus in a model of contrasting embryo receptivity. Beef cows were treated to ovulate a larger (LF-LCL group, associated with greater receptivity) or smaller (SF-SCL group) follicle and, consequently, to present greater or smaller plasma concentrations of estradiol during proestrusestrus, as well as progesterone during early diestrus. Oviduct and uterus (4 days after gonadotropin-releasing hormone-induced ovulation; D4) as well as the uterus (D7) were collected, and lipid profiles were monitored by matrix-assisted laser desorption/ionization mass spectrometry (MAL-DI-MS). This technique allowed the identification and tissue localization of sphingomyelins (SM), phosphatidylcholines (PC), ceramides (Cer), and phosphatidylethanolamines (PE). Multivariate statistics were used to separate samples into groups with distinctly different phospholipid profiles in the uterus at D4 and D7. Different abundance of ions corresponding to specific lipids were detected on D4 (Cer [42: 1], PC [31: 0], PC [32: 1], PC [34: 4], and PC [36: 4] greater for LF-LCL group; and PC [38: 7], PC [38: 5], PC [38: 4], PC [40: 7], and PC [40: 6] greater for SF-SCL group) and D7 (SM [34: 2], SM [34: 1], PC [32: 1], and PC [35: 2] greater for LF-LCL group). The MALDI-MS imaging showed the spatial distributions of major phospholipids. In conclusion, distinct phospholipid profiles were associated with animals treated to show contrasting receptivity to the embryo. Functional roles of the identified phospholipids on uterine function and preimplantation embryo development deserve further studies.Phospholipid metabolism and signaling influences on early pregnancy events in cattle are unknown. This study aimed to characterize global phospholipid composition of oviduct and uterus during early diestrus in a model of contrasting embryo receptivity. Be956111FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)2012/07206-0; 2011/06191-7; 2011/03226-4; 2013/19620-9sem informaçãosem informaçã

MALDI mass spectrometry reveals that cumulus cells modulate the lipid profile of in vitro-matured bovine oocytes

The influence of cumulus cells (CC) on the lipid profile of bovine oocytes matured in two different lipid sources was investigated. Cumulus-oocyte complexes (COC) or denuded oocytes (DO) were matured in tissue culture medium (TCM) supplemented with fetal bovine serum (FBS) or serum substitute supplement (SSS). Lipid profiles of TCM, serum supplements, immature CC and oocyte (IO), and in vitro-matured oocytes from COC and DO were then analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and submitted to partial least squaresdiscriminant analysis (PLS-DA). The developmental competence of such oocytes was also assessed. Differences in lipid composition were observed between two types of sera and distinctly influenced the lipid profile of CC. As revealed by PLS-DA, the abundance of specific ions corresponding to triacylglycerols (TAG) or phospholipids (PL) were higher in COC compared to DO both supplemented with FBS or SSS and to some extent affected the subsequent DO in vitro embryo development. DO exposed to SSS had however a marked diminished ability to develop to the blastocyst stage. These results indicate a modulation by CC of the oocyte TAG and PL profiles associated with a specific cell response to the serum supplement used for in vitro maturation.The influence of cumulus cells (CC) on the lipid profile of bovine oocytes matured in two different lipid sources was investigated. Cumulus-oocyte complexes (COC) or denuded oocytes (DO) were matured in tissue culture medium (TCM) supplemented with fetal6328699sem informaçãosem informaçã