RESUMO
Choline plays a critical role in systemic lipid metabolism and hepatic function. Here we conducted a series of experiments to investigate the effect of choline supplementation on metabolically altered Pcyt2(+/-) mice. In Pcyt2(+/-) mice, the membrane phosphatidylethanolamine (PE) turnover is reduced and the formation of fatty acids (FA) and triglycerides (TAG) increased, resulting in hypertriglyceridemia, liver steatosis and obesity. One month of choline supplementation reduced the incorporation of FA into TAG and facilitated TAG degradation in Pcyt2(+/-) adipocytes, plasma and liver. Choline particularly stimulated adipocyte and liver TAG lipolysis by specific lipases (ATGL, LPL and HSL) and inhibited TAG formation by DGAT1 and DGAT2. Choline also activated the liver AMPK and mitochondrial FA oxidation gene PPARα and reduced the FA synthesis genes SREBP1, SCD1 and FAS. Liver (HPLC) and plasma (tandem mass spectroscopy and (1)H-NMR) metabolite profiling established that Pcyt2(+/-) mice have reduced membrane cholesterol/sphingomyelin ratio and the homocysteine/methionine cycle that were improved by choline supplementation. These data suggest that supplementary choline is beneficial for restoring FA and TAG homeostasis under conditions of obesity caused by impaired PE synthesis.
Assuntos
Colina/farmacologia , Fígado/efeitos dos fármacos , RNA Nucleotidiltransferases/deficiência , Proteínas Quinases Ativadas por AMP/metabolismo , Acilação , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Carnitina/análogos & derivados , Suplementos Nutricionais , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Camundongos Mutantes , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , RNA Nucleotidiltransferases/genética , Triglicerídeos/sangue , Triglicerídeos/metabolismo , Aumento de Peso/efeitos dos fármacosRESUMO
BACKGROUND: Propofol is a short-acting intravenous anesthetic agent. In rare conditions, a life-threatening complication known as propofol infusion syndrome can occur. The pathophysiologic mechanism is still unknown. Some studies suggested that propofol acts as uncoupling agent, others suggested that it inhibits complex I or complex IV, or causes increased oxidation of cytochrome c and cytochrome aa3, or inhibits mitochondrial fatty acid metabolism. Although the exact site of interaction is not known, most hypotheses point to the direction of the mitochondria. METHODS: Eight rats were ventilated and sedated with propofol up to 20 h. Sequential biopsy specimens were taken from liver and skeletal muscle and used for determination of respiratory chain activities and propofol concentration. Activities were also measured in skeletal muscle from a patient who died of propofol infusion syndrome. RESULTS: In rats, authors detected a decrease in complex II+III activity starting at low tissue concentration of propofol (20 to 25 µM), further declining at higher concentrations. Before starting anesthesia, the complex II+III/citrate synthase activity ratio in liver was 0.46 (0.25) and in skeletal muscle 0.23 (0.05) (mean [SD]). After 20 h of anesthesia, the ratios declined to 0.17 (0.03) and 0.12 (0.02), respectively. When measured individually, the activities of complexes II and III remained normal. Skeletal muscle from one patient taken in the acute phase of propofol infusion syndrome also shows a selective decrease in complex II+III activity (z-score: -2.96). CONCLUSION: Propofol impedes the electron flow through the respiratory chain and coenzyme Q is the main site of interaction with propofol.
Assuntos
Anestésicos Intravenosos/toxicidade , Propofol/toxicidade , Ubiquinona/metabolismo , Animais , Ciclo do Ácido Cítrico/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Ratos , Ratos Wistar , Respiração Artificial , SíndromeRESUMO
In strict carnivorous domestic cats, a metabolic competition arises between the need to use amino acids for gluconeogenesis and for protein synthesis both in health and disease. The present study investigated the amino acid-sparing potential of propionic acid in cats using dietary propionylated starch (HAMSP) supplementation. A total of thirty cats were fed a homemade diet, supplemented with either HAMSP, acetylated starch (HAMSA) or celite (Control) for three adaptation weeks. Propionylated starch was hypothesised to provide propionic acid as an alternative gluconeogenic substrate to amino acids, whereas acetic acid from HAMSA would not provide any gluconeogenic benefit. Post-adaptation, a 5-d total faecal collection was carried out to calculate apparent protein digestibility coefficients. Fresh faecal and blood samples were collected to analyse fermentation endproducts and metabolites. The apparent protein digestibility coefficients did not differ between supplements (P = 0·372) and were not affected by the protein intake level (P = 0·808). Faecal propionic acid concentrations were higher in HAMSP than in HAMSA (P = 0·018) and Control (P = 0·003) groups, whereas concentrations of ammonia (P = 0·007) were higher in HAMSA than in HAMSP cats. Tendencies for or higher propionylcarnitine concentrations were observed in HAMSP compared with HAMSA (P = 0·090) and Control (P = 0·037) groups, and for tiglyl- + 3-methylcrotonylcarnitine concentrations in HAMSP as compared with Control (P = 0·028) cats. Methylmalonylcarnitine concentrations did not differ between groups (P = 0·740), but were negatively correlated with the protein intake level (r -0·459, P = 0·016). These results suggest that HAMSP cats showed more saccharolytic fermentation patterns than those supplemented with HAMSA, as well as signs of sparing of valine in cats with a sufficient protein intake.
RESUMO
BACKGROUND: Carbohydrate-deficient transferrin (CDT) is one of the best indicators for chronic alcohol abuse and detection of relapse. In this study, we explore the microheterogeneity of ß-hexosaminidase (ß-HEX) in chronic alcohol abusers in the framework of a driver's license regranting program. Studies have shown that increased serum activity of ß-HEX B (isoforms P, I, and B) may be a sensitive marker for chronic alcohol abuse. Here, we describe methodology, limitations, and correlation of ß-HEX isoforms with CDT. METHODS: CDT was assayed at the central laboratory of the Ghent University Hospital by capillary zone electrophoresis, measured on the Capillarys 2™ system and was expressed as a percentage of total serum transferrin (%CDT). Serum of chronic alcohol abusers was compared to nonheavy drinkers using agarose gel isoelectric focusing (IEF). Total ß-HEX activity was assayed fluorimetrically following preparative IEF in 81 subjects. ß-HEX isoforms were investigated and compared between nonheavy drinkers and heavy drinkers. RESULTS: Agarose gel IEF shows additional cathodal bands in serum of chronic alcohol abusers. Mean total ß-HEX activity between pH 6.8 and 7.7, designated as HEX-7, showed the highest correlation with %CDT (r = 0.70, p < 0.0001, n = 68). In a selected subgroup, where CDT could not be quantified (n = 13) because of an atypical electropherogram, HEX-7 was in concordance with either estimated %CDT value or liver enzyme activities. CONCLUSIONS: In this proof-of-concept study, we introduce a novel approach to quantify ß-HEX isoforms using preparative IEF and fluorimetry. A highly significant correlation of HEX-7 and %CDT has been found. Because of exclusion of the P isoform, HEX-7 could be a useful supplementary marker for detecting chronic alcohol abuse.
Assuntos
Alcoolismo/sangue , beta-N-Acetil-Hexosaminidases/sangue , Exame para Habilitação de Motoristas , Biomarcadores/sangue , Feminino , Humanos , Focalização Isoelétrica , Isoenzimas/sangue , Masculino , Pessoa de Meia-Idade , Transferrina/análogos & derivados , Transferrina/metabolismoRESUMO
The present study evaluated the potential of affecting amino acid metabolism through intestinal fermentation in domestic cats, using dietary guar gum as a model. Apparent protein digestibility, plasma fermentation metabolites, faecal fermentation end products and fermentation kinetics (exhaled breath hydrogen concentrations) were evaluated. Ten cats were randomly assigned to either guar gum- or cellulose-supplemented diets, that were fed in two periods of 5 weeks in a crossover design. No treatment effect was seen on fermentation kinetics. The apparent protein digestibility (P= 0.07) tended to be lower in guar gum-supplemented cats. As a consequence of impaired small-intestinal protein digestion and amino acid absorption, fermentation of these molecules in the large intestine was stimulated. Amino acid fermentation has been shown to produce high concentrations of acetic and butyric acids. Therefore, no treatment effect on faecal propionic acid or plasma propionylcarnitine was observed in the present study. The ratio of faecal butyric acid:total SCFA tended to be higher in guar gum-supplemented cats (P= 0.05). The majority of large-intestinal butyric acid is absorbed by colonocytes and metabolised to 3-hydroxy-butyrylcoenzyme A, which is then absorbed into the bloodstream. This metabolite was analysed in plasma as 3-hydroxy-butyrylcarnitine, which was higher (P= 0.02) in guar gum-supplemented cats. In all probability, the high viscosity of the guar gum supplement was responsible for the impaired protein digestion and amino acid absorption. Further research is warranted to investigate whether partially hydrolysed guar gum is useful to potentiate the desirable in vivo effects of this fibre supplement.
Assuntos
Aminoácidos/metabolismo , Gatos/metabolismo , Dieta/veterinária , Fibras na Dieta/administração & dosagem , Fermentação/efeitos dos fármacos , Galactanos/administração & dosagem , Mananas/administração & dosagem , Gomas Vegetais/administração & dosagem , Animais , Peso Corporal , Ácido Butírico/análise , Ácido Butírico/metabolismo , Carnitina/análogos & derivados , Carnitina/sangue , Celulose/administração & dosagem , Estudos Cross-Over , Proteínas Alimentares/metabolismo , Digestão/efeitos dos fármacos , Ingestão de Energia , Ácidos Graxos Voláteis/análise , Fezes/química , Feminino , Galactanos/química , Mucosa Intestinal/metabolismo , Masculino , Mananas/química , Gomas Vegetais/química , ViscosidadeRESUMO
BACKGROUND: The Schwartz 2009 creatinine-based revised formula is the only pediatric GFR estimating formula, which is compatible with the recent global creatinine standardization. This formula is only applicable if enzymatic creatinine methods are used. We propose an equation, taking into account the relative bias caused by serum proteins to use Jaffe based creatinine data for GFR estimation. METHODS: In a cohort study of 100 pediatric patients, serum creatinine was measured using a kinetic rate-blanked Jaffe assay (modified kinetic alkaline picrate method), a kinetic rate-blanked Jaffe compensated assay for reactive proteins and an enzymatic assay (creatinine plus method). Serum total protein, albumin, urea, uric acid and total bilirubin were measured with the use of commercial agents. RESULTS: The difference in serum creatinine between the enzymatic method and the compensated Jaffe method was mainly dependent on the total protein concentration in serum (r(2)=0.61, p<0.001). After applying the proposed protein correction, corrected compensated Jaffe results and creatinine clearance values became interchangeable with enzymatic serum creatinine results (r(2)=0.99, p<0.001; Deming regression: slope: 0.9787, intercept: -0.351) and with the newly proposed Schwartz formula, respectively (r(2)=0.99, p<0.001; Deming regression: slope 1.004, intercept: 2.16). CONCLUSIONS: In this study, we demonstrated the usability of the alkaline picrate method in the Schwartz formula, taking into account the relative bias caused by serum proteins.
Assuntos
Proteínas Sanguíneas/química , Compostos Organometálicos/química , Adolescente , Proteínas Sanguíneas/metabolismo , Criança , Pré-Escolar , Estudos de Coortes , Creatinina/sangue , Feminino , Taxa de Filtração Glomerular/fisiologia , Humanos , MasculinoRESUMO
BACKGROUND: Citrulline is considered to be a marker of absorptive enterocyte mass. Citrulline levels can be measured in plasma or dried blood spot (DBS) samples. The purpose of this study is to calculate reference intervals for plasma and DBS citrulline concentrations in children and to examine the effect of age and gender. METHODS: In 151 healthy subjects ranging from 1 month to 20 years of age, plasma and DBS citrulline concentration were determined by using Liquid Chromatography-tandem Mass Spectrometry. Citrulline concentrations were examined in relation to age and gender. Reference values were calculated according to the guidelines of the International Federation of Clinical Chemistry and the National Committee on Clinical Laboratory Standards. RESULTS: No significant influence of age and gender could be discerned on plasma or DBS citrulline concentration. In children, the reference intervals for citrulline bounded by the 2.5 and 97.5 percentiles are 13.31-69.05 µmol/L and 23.70-49.04 µmol/L for plasma and DBS samples respectively. CONCLUSIONS: The reference intervals for citrulline levels in healthy children are widely dispersed. Measuring citrulline concentrations in dried blood spots delivers no additional value to plasma measurements for the calculation of reference intervals in children.
Assuntos
Citrulina/sangue , Adolescente , Adulto , Criança , Pré-Escolar , Cromatografia Líquida , Feminino , Humanos , Lactente , Masculino , Manejo de Espécimes , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
OBJECTIVE: To evaluate metabolic effects of sex steroids in nonfasting and fasting conditions, independent from changes in body composition. RESEARCH DESIGN AND METHODS: A randomized clinical trial was performed to create contrasting sex steroid levels in healthy young men: by letrozole (aromatase inhibitor) to lower estradiol (E(2)) and increase testosterone (group T, n = 10) versus letrozole plus E(2) patches to lower T and raise E(2) (group E, n = 10). Mixed meals and hyperinsulinemic-euglycemic clamps were performed before and after a 1-week treatment period. RESULTS: Following intervention, the postprandial triglyceride response displayed a diverging response with a decline in group T and an increase in group E; the postprandial glucose-dependent insulinotropic polypeptide (GIP) response increased in group T. Insulin sensitivity increased in group T but remained unaltered in group E. CONCLUSIONS: In healthy young men, short-term changes in sex steroids affect postprandial triglyceride and GIP response and insulin sensitivity.
Assuntos
Estradiol/sangue , Polipeptídeo Inibidor Gástrico/sangue , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Nitrilas/farmacologia , Testosterona/sangue , Triazóis/farmacologia , Triglicerídeos/sangue , Adulto , Inibidores da Aromatase/farmacologia , Jejum/sangue , Humanos , Letrozol , Masculino , Período Pós-Prandial , Triglicerídeos/metabolismo , Adulto JovemRESUMO
N balance and postprandial acylcarnitine profile following intestinal fermentation of oligofructose and inulin were investigated in healthy cats. Two diets were tested in a crossover design: a commercial high-protein cat food supplemented with 4 % DM oligofructose and inulin (spectrum: degree of polymerisation (DP) 2-10: 60 (SE 5) % DM; DP>10: 28 (SE 5) % DM) as high-fermentable fibre (HFF) diet, and the same commercial diet supplemented with 4 % DM cellulose as low-fermentable fibre diet. Eight adult cats were randomly allotted to each of the two diets at intervals of 4 weeks. At the end of each testing period, faeces and urine were collected over a 5-d period, and blood samples were obtained before and at the selected time points postprandially. No differences were found for N intake, N digestibility and faecal N excretion, whereas urinary N excretion was lower when the HFF diet was fed (P = 0.044). N balance was positive in all the cats, and tended to be increased when the HFF diet was fed (P = 0.079). Propionylcarnitine concentrations (P = 0.015) and their area under the curve (AUC) (P = 0.013) were increased when the HFF diet was fed, revealing a more pronounced production and absorption of propionate. Yet, methylmalonylcarnitine concentrations and concurrent AUC were not elevated when the HFF diet was fed, indicating reduced amino acid catabolism. 3-Hydroxy-3-methylglutarylcarnitine concentrations (P = 0.026) and their AUC (P = 0.028) were also reduced when the HFF diet was fed, implying diminished use of branched-chain amino acids as well. In healthy cats, oligofructose and inulin added to a high-protein diet were suggested to reduce postprandial amino acid-induced gluconeogenesis by substitution with propionate.
Assuntos
Aminoácidos/metabolismo , Carnitina/metabolismo , Proteínas Alimentares/metabolismo , Mucosa Intestinal/metabolismo , Inulina/farmacologia , Nitrogênio/metabolismo , Oligossacarídeos/farmacologia , Animais , Área Sob a Curva , Bactérias/metabolismo , Carnitina/análogos & derivados , Gatos , Estudos Cross-Over , Proteínas Alimentares/administração & dosagem , Suplementos Nutricionais , Feminino , Fermentação , Gluconeogênese , Intestinos/microbiologia , Inulina/metabolismo , Masculino , Oligossacarídeos/metabolismo , Período Pós-Prandial , Prebióticos , Propionatos/metabolismoRESUMO
The effect of dietary oligofructose and inulin supplementation on glucose metabolism in obese and non-obese cats was assessed. Two diets were tested in a crossover design; a control diet high in protein (46 % on DM basis), moderate in fat (15 %), low in carbohydrates (27 %), but no soluble fibres added; and a prebiotic diet, with 2.5 % of a mixture of oligofructose and inulin added to the control diet. Eight non-obese and eight obese cats were allotted to each of two diets in random order at intervals of 4 weeks. At the end of each testing period, intravenous glucose tolerance tests were performed. Area under the glucose curve (AUCgluc) was increased (P = 0.022) and the second insulin peak was delayed (P = 0.009) in obese compared to non-obese cats. Diets did not affect fasting plasma glucose concentrations, blood glucose response at each glucose time-point after glucose administration, AUCgluc, fasting serum insulin concentrations, area under the insulin curve, and height and appearance time of insulin response. Yet, analysis of acylcarnitines revealed higher propionylcarnitine concentrations (P = 0.03) when fed the prebiotic diet, suggesting colonic fermentation and propionate absorption. Prebiotic supplementation reduced methylmalonylcarnitine (P = 0.072) and aspartate aminotransferase concentrations (P = 0.025), both indicating reduced gluconeogenesis from amino acids. This trial evidenced impaired glucose tolerance and altered insulin response to glucose administration in obese compared to non-obese cats, regardless of dietary intervention; yet modulation of glucose metabolism by enhancing gluconeogenesis from propionate and inhibition of amino acid catabolism can be suggested.
Assuntos
Aminoácidos/metabolismo , Inulina/farmacologia , Obesidade/veterinária , Oligossacarídeos/farmacologia , Propionatos/metabolismo , Animais , Área Sob a Curva , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Peso Corporal , Doenças do Gato/prevenção & controle , Gatos , Feminino , Insulina/sangue , Inulina/uso terapêutico , Masculino , Obesidade/prevenção & controle , Oligossacarídeos/uso terapêutico , Orquiectomia/veterinária , Ovariectomia/veterinária , PrebióticosRESUMO
BACKGROUND: Anderson-Fabry disease (AFD) is an X-linked condition originating from a deficiency in alpha-galactosidase, a lysosomal enzyme. Multi-organ involvement ensues in early adulthood and vital organs are affected: the kidneys, brain, heart. Several reports however suggest that AFD is underdiagnosed. METHODS: We screened a kidney transplant population using a two-tier approach. The first tier was the determination of alpha-galactosidase A (AGALA) activity using a dried blood spot on filter paper (DBFP); in the second tier, patients with the lowest alpha-galactosidase levels were further subjected to mutation analysis of the GLA gene. RESULTS: From the database of 2328 patients, 1233 subjects met the inclusion criteria. Finally, after informed consent, 673 patients were screened (54.5%-395 women and 278 men). DBFP analysis resulted in a mean AGALA of 2.63 +/- 2.48 micromol/L/h (2.5 and 97.5 percentile were 0.0001 and 5.07 micromol/L/h, respectively). Eleven patients were subjected to further genetic analysis. In a male patient a pathogenic missense mutation p.Ala143Thr (c.427A>G) was identified. CONCLUSIONS: Our results show that the proposed approach can detect AFD patients in a until now seldomly screened high-risk group: kidney transplant patients. We conclude that screening for AFD in high-risk populations is a cost-effective, technically feasible and clinically valuable objective.
Assuntos
Doença de Fabry/diagnóstico , Transplante de Rim , Adulto , Idoso , Criança , Análise Mutacional de DNA , Doença de Fabry/complicações , Doença de Fabry/enzimologia , Doença de Fabry/genética , Feminino , Testes Genéticos , Humanos , Falência Renal Crônica/enzimologia , Falência Renal Crônica/etiologia , Falência Renal Crônica/genética , Masculino , Mutação de Sentido Incorreto , Linhagem , Adulto Jovem , alfa-Galactosidase/genéticaRESUMO
BACKGROUND: The European In Vitro Diagnostics (IVD) directive requires traceability to reference methods and materials of analytes. It is a task of the profession to verify the trueness of results and IVD compatibility. METHODS: The results of a trueness verification study by the European Communities Confederation of Clinical Chemistry (EC4) working group on creatinine standardization are described, in which 189 European laboratories analyzed serum creatinine in a commutable serum-based material, using analytical systems from seven companies. Values were targeted using isotope dilution gas chromatography/mass spectrometry. Results were tested on their compliance to a set of three criteria: trueness, i.e., no significant bias relative to the target value, between-laboratory variation and within-laboratory variation relative to the maximum allowable error. RESULTS: For the lower and intermediate level, values differed significantly from the target value in the Jaffe and the dry chemistry methods. At the high level, dry chemistry yielded higher results. Between-laboratory coefficients of variation ranged from 4.37% to 8.74%. Total error budget was mainly consumed by the bias. Non-compensated Jaffe methods largely exceeded the total error budget. Best results were obtained for the enzymatic method. The dry chemistry method consumed a large part of its error budget due to calibration bias. CONCLUSIONS: Despite the European IVD directive and the growing needs for creatinine standardization, an unacceptable inter-laboratory variation was observed, which was mainly due to calibration differences. The calibration variation has major clinical consequences, in particular in pediatrics, where reference ranges for serum and plasma creatinine are low, and in the estimation of glomerular filtration rate.
Assuntos
Análise Química do Sangue/métodos , Creatinina/sangue , Análise Química do Sangue/normas , Europa (Continente) , Cooperação Internacional , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
INTRODUCTION: Fabry's disease (AFD) is an X-linked lysosomal storage disease, resulting from a deficiency in alpha-galactosidase A (AGALA). Untreated, this leads to precocious failure of vital organ function and death. As enzyme replacement therapy is available, it is of vital importance that affected individuals can be traced. MATERIALS AND METHODS: We set up a screening in the Flemish haemodialysis population using a two-tier approach. The first tier was a determination of alpha-galactosidase A activity using a dried blood spot on filter paper, in the second tier, patients with the lowest alpha-galactosidase levels were further subjected to mutation analysis of the GLA gene. RESULTS: 1284 patients (1047 women, 237 men) were evaluated for inclusion, eliminating patients with definite renal diagnoses. Total 922 patients (71.8 %) were screened (742 women, 180 men). Fifty seven patients were subjected to further genetic analysis. Three GLA mutation carriers were identified: two apparently nonrelated female patients carry the missense mutation p.Ala143Thr (c.427G > A), a missense mutation p.Trp236Arg (c.706T > C) was identified in a man. While the male patient had been clinically diagnosed with AFD, the female patients had remained unrecognized. Additional family based screening resulted in the identification of nine mutation carriers (four males and five females). DISCUSSION: We demonstrated that the prevalence of GLA mutation carriers in our haemodialysis population is 0.3%. Our results show that the proposed approach accurately detects AFD patients. We conclude that screening for AFD in high risk populations is a cost-effective, technically feasible and clinically valuable objective.
Assuntos
Doença de Fabry/diagnóstico , Doença de Fabry/genética , Diálise Renal , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fatores SexuaisRESUMO
BACKGROUND: Plasma Citrulline concentration has been correlated to functional enterocyte mass. Dried blood spot (DBS) analysis using LC-MSMS reduces sample amount needed. We optimized DBS elution to increase precision and accuracy of DBS LC-MSMS analysis. METHOD: DBS control samples were eluted in varying pH (2.2-7.0) and for varying times (15-75 min) and Cit, Arg and Orn were analyzed using LC-MSMS, with and without derivatization. In 20 volunteers, the DBS LC-MSMS assay was correlated with a plasma ion exchange HPLC method. RESULTS: For Citrulline an extraction optimum was obtained at pH 2.6, whereas lower Arginine concentrations were found using low extraction pH. Increasing elution times lead to increased concentrations. Within-run CV was higher with, compared to without derivatization. No close association could be found between plasma HPLC and DBS LC-MSMS concentrations. CONCLUSION: Analysis of amino acids on DBS using LC-MSMS should be optimized regarding the purpose of the assay. In our study, most optimal results were obtained without derivatization and elution in pH 2.6 for 45 min. Cellular amino acids in DBS might influence the correlation of Cit with severity of enteral disorders. Therefore, further evaluation of DBS Cit as a marker for enteral disorders is warranted.
Assuntos
Aminoácidos/metabolismo , Membrana Celular/metabolismo , Citrulina/sangue , Monitorização Fisiológica/métodos , Aminoácidos/química , Arginina/química , Arginina/metabolismo , Membrana Celular/química , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Concentração de Íons de Hidrogênio , Espectrometria de Massas/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
BACKGROUND: Recently, automated urine test strip readers became available that can report quantitative data. We explored the possibility of measuring all ketone bodies (acetone, acetoacetate, 3-hydroxybutyrate) in urine with these test strips. Monitoring urinary ketone concentrations could offer the advantages of measuring higher values (due to the low renal thresholds) and being less sensitive to fluctuations. METHODS: We evaluated URISYS 2400 (Roche) quantitative reflectance data for the ketone reflectance field and compared it with biochemical data from urine samples. Using an easy sample pre-treatment with 3-hydroxybutyrate dehydrogenase, we were able to assay 3-hydroxybutyrate as well, which normally does not react on urine test strips. RESULTS: Within- and between-run reproducibility of the reflectance signal for high- and low-concentration urine pools was 11.0-3.6% and 11.0-5.8% for aceto-acetate, 8.2-9.2% and 10.4-16.1% for acetone, and 5.1-3.0% and 5.6-3.5% for 3-hydroxybutyrate, respectively. The lower limit of detection for acetoacetate was 0.13 mmol/L (CV=3.6%). Fair agreement was obtained between test strip data for ketones andcolorimetrically determined acetoacetate values (r=0.90). CONCLUSIONS: In urine test strip analysis, quantitative ketone reflectance data allow a simple and fast analysis, offering affordable screening for the detection of ketone body production in diabetes, especially in emergency settings.
Assuntos
Diabetes Mellitus/urina , Corpos Cetônicos/urina , Urinálise/métodos , Ácido 3-Hidroxibutírico/urina , Acetoacetatos/urina , Acetona/urina , Adulto , Diabetes Mellitus/tratamento farmacológico , Feminino , Humanos , Sistemas de Infusão de Insulina , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Urinálise/estatística & dados numéricosRESUMO
The quantification of monoclonal immunoglobulins by protein electrophoresis has been helpful in determining the prognosis for the patient. In a 65-year old man with documented IgG lambda monoclonal gammopathy, a discrepancy in the detection and quantification of this M-protein was found when studied by four different electrophoretic methods. On a high-resolution agarose method with acid violet staining a prominent peak was seen in the fast gamma-region. A lower resolution agarose method using an amido black staining showed a small mid-gamma restriction, and no spike was detected by high-resolution or lower resolution capillary zone electrophoresis. The discrepancy was not attributable to a migration in the beta-region leading to masking by transferrin or C3 peak, incorrect separation on CZE due to a high isoelectric point (pI > 8.5) or pentamerization of IgM M-proteins. Therefore, other causes for discrepancies should be investigated.
Assuntos
Eletroforese das Proteínas Sanguíneas , Eletroforese Capilar , Imunoglobulina M/análise , Cadeias lambda de Imunoglobulina/análise , Paraproteinemias/imunologia , Idoso , Eletroforese em Gel de Ágar , Humanos , Imunoglobulina M/sangue , Cadeias lambda de Imunoglobulina/sangue , Masculino , Reprodutibilidade dos TestesRESUMO
BACKGROUND: The understanding of iron metabolism has increased substantially during the last decade. Several new transporters and iron regulating molecules have been described. Hepcidin, a small hepatic peptide has recently been proposed as a central mediator of dietary iron absorption. We have investigated the relationship between prohepcidin, the prohormone of hepcidin, and renal function and iron status. METHODS: Forty six patients, referred for 51Cr-EDTA clearance were included in this study. Renal function was assessed by determination of serum creatinine, creatinine clearance, serum cystatin C and serum beta-trace protein. Iron status was evaluated by determination of serum iron, transferrin, transferrin saturation and serum ferritin. All determinations were performed using commercial reagents (Roche Diagnostics, Dade Behring). Serum prohepcidin was determined using an ELISA kit. RESULTS: Serum prohepcidin was found to correlate with 51Cr-EDTA clearance (r = -0.44; p = 0.005), creatinine clearance, serum creatinine, beta-trace protein and cystatin C. No significant relationship was observed between serum prohepcidin concentrations and red cell count, hemoglobin concentration or hematocrit. No significant correlation was found in this population between prohepcidin concentrations and iron status. CONCLUSION: Increased serum prohepcidin concentrations were observed with declining kidney function. We observed no relationship between red cell indices or iron status and serum prohepcidin concentrations.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Ferro/metabolismo , Insuficiência Renal/sangue , Idoso , Creatinina/sangue , Cistatina C , Cistatinas/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Ferritinas/sangue , Hepcidinas , Humanos , Oxirredutases Intramoleculares/sangue , Lipocalinas , Masculino , Taxa de Depuração Metabólica , Precursores de Proteínas , Transferrina/metabolismoRESUMO
Carbohydrate-deficient transferrin (CDT) is currently considered to be the best available marker for the diagnosis of chronic alcoholism. A large variety of methods have been developed, demonstrating the need for standardisation. Commercially available anion-exchange chromatographic-based assays are easy to use and require no specialised, expensive instruments. However, these methods cannot identify genetic transferrin variants or the carbohydrate-deficient glycoprotein syndrome. In 1989, a capillary isoelectric focusing method was developed for quantitative measurement of CDT. Despite the optimal resolution, this method is not easily applied in a clinical routine environment due to the complexity of analysis. Capillary electrophoresis in a polymer network using coated capillaries allowed full resolution of the sialoforms of human transferrin. The drawbacks due to an expensive and time-consuming sample preparation were eliminated when a method in neat serum was developed. Capillary zone electrophoresis allowed full resolution of the transferrin isoforms with a high analytical performance in a short analysis time thanks to a strong electroosmotic flow. Genetic transferrin variants were easily detected, avoiding false-positive results. Also, using capillary zone electrophoresis, it was shown that CDT is a suitable marker of chronic alcohol abuse detection in transferrin CD (common/cathodal) variants.