RESUMO
The main objective of this work was to evaluate a comprehensive two-dimensional gas chromatographic (GCxGC) coupled to quadrupole mass spectrometry (qMS) method in the field of biomarker candidates' discovery. To this purpose we developed a GCxGC-qMS method suitable for the separation of organic acids and other classes of compounds with silylable polar hydrogen such as sugars, amino-acids, and vitamins. As compared to those obtained by a widely used 1D-GC method, the urinary chromatographic profiles performed by the proposed 2D-GC method exhibit higher resolution and sensitivity, leading to the detection of up to 92 additional compounds in some urine samples including some well-known biomarkers. In order to validate the proposed method we focused on three metabolites of interest with various functional groups and polarities including CH3-malonic acid (MMA: biomarker of methylmalonic acidemia), 3-hydroxy-3-methyl-glutaric acid (3-OHMGA: biomarker of 3-hydroxy-3-methylglutaric acidemia), and phenylpiruvic acid (PhPA: marker of phenylketonuria). While these three metabolites can be considered as representative of organic acids classically determined by 1D-GC, they cannot be representative of new detected metabolites. Thus, we also focused on quinolic acid (QUIN), taken as an example of biomarker not detected at basal levels with the classical 1D GC-qMS method. In order to obtain sufficient recoveries for all tested compounds, we developed a sample preparation protocol including a step of urea removal followed by two extraction steps using two solvents of different polarity and selectivity. Recoveries with the proposed method reached more than 80% for all targeted compounds and the linearity was satisfactory up to 50µmol/L. The CVs of the within-run and within-laboratory precisions were less than 8% for all tested compounds. The limits of quantification (LOQs) were 0.6µmol/L for MMA, 0.4µmol/L for 3-OHMGA, 0.7µmol/L for PhPA, and 1µmol/L for QUIN. The LOQs of these metabolites obtained by a classical GC-MS method under the same chromatographic conditions were 5µmol/L for MMA, 4µmol/L for 3-OHMGA, 6µmol/L for PhPA while QUIN was below the limit of detection. As compared to 1D-GC, these results highlight the enhanced detectability of urine metabolites by the 2D-GC technique. Our results also show that for each new detected compound it is necessary to develop and validate an appropriate sample preparation procedure.
Assuntos
Cromatografia Gasosa/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Ácidos Quinolínicos/urina , Criança , HumanosRESUMO
A new and rapid method for the determination of the excitotoxic tryptophan metabolite quinolinic acid is based on its enzymatic conversion to nicotinic acid mononucleotide and, in a second step utilizing [3H]ATP, further to [3H] deamido-NAD. Specificity of the assay is assured by using a highly purified preparation of the specific quinolinic acid-catabolizing enzyme, quinolinic acid phosphoribosyltransferase, in the initial step. The limit of sensitivity was found to be 2.5 pmol of quinolinic acid, sufficient to conveniently determine quinolinic acid levels in small volumes of human urine and blood plasma.
Assuntos
Piridinas/análise , Ácidos Quinolínicos/análise , Adulto , Humanos , Masculino , NAD/análogos & derivados , Mononucleotídeo de Nicotinamida/análogos & derivados , Pentosiltransferases , Ácido Quinolínico , Ácidos Quinolínicos/sangue , Ácidos Quinolínicos/urina , TrítioRESUMO
We measured the excretion of the endogenous neurotoxin quinolinic acid in 14 patients with Huntington's disease and in 11 age matched control subjects. Huntingtonian patients excreted less quinolinic acid, than controls. When normalised to urea or creatinine output quinolinic acid excretion was normal. We conclude that Huntington's disease is not associated with a generalised disturbance of quinolinic acid metabolism, however, a local hyperproduction of quinolinic acid cannot be excluded from our results.
Assuntos
Doença de Huntington/urina , Piridinas/urina , Ácidos Quinolínicos/urina , Adolescente , Adulto , Creatinina/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ácido Quinolínico , Ureia/urinaRESUMO
Three human metabolic studies, each 35 days in length, were performed to investigate the relationship between tryptophan intake and the proportion of dietary tryptophan converted to niacin and the effect of supplements of L-leucine and vitamin B6 on this conversion. Nine college men consumed a basal diet that provided 8 mg of niacin, 1 mg of vitamin B6, and either 245, 548, or 845 mg of tryptophan from proteins per day. During each 35-day study, for one 15-day period basal diet alone was consumed, for another 15-day period basal diet plus 10 g of L-leucine per day was consumed, and for the last 5-day period, 20 mg of vitamin B6 per day was added to the diets of both groups. N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and quinolinic acid were measured in 24-hr urine samples. There were no significant or consistent effects of L-leucine or vitamin B6 supplements on the excretin of any of the metabolites measured. The proportion of tryptophan converted to niacin tended to increase as tryptophan consumption increased; however, this change was small and was probably not significant over the range of tryptophan intakes studied. The average conversion ration of tryptophan to niacin was approximately 72:1 in these subjects.
Assuntos
Leucina/farmacologia , Ácidos Nicotínicos/metabolismo , Piridoxina/farmacologia , Triptofano/metabolismo , Adulto , Relação Dose-Resposta a Droga , Humanos , Masculino , Niacinamida/análogos & derivados , Niacinamida/urina , Ácidos Nicotínicos/biossíntese , Nitrogênio/metabolismo , Ácidos Quinolínicos/urina , Triptofano/farmacologiaAssuntos
Leucina/farmacologia , Ácidos Nicotínicos/metabolismo , Triptofano/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Dieta , Eritrócitos/análise , Feminino , Ácido Hidroxi-Indolacético/urina , Isoleucina/deficiência , NAD/sangue , NADP/sangue , Ácidos Nicotínicos/deficiência , Ácidos Nicotínicos/urina , Tamanho do Órgão/efeitos dos fármacos , Pelagra/etiologia , Ácidos Quinolínicos/urina , Ratos , Fatores de Tempo , Valina/deficiência , Deficiência de Vitamina B 6/metabolismoRESUMO
1. Six groups of rats were given diets containing protein at three levels (50, 100 and 200 g/kg), with and without nicotinic acid. After 4 weeks on these diets some key enzymes of the tryptophan and nicotinic acid-NAD pathway, liver nicotinamide nucleotide concentration, and urinary metabolites of tryptophan and nicotinic acid were studied. 2. Liver nicotinamide nucleotide levels were lower in rats given the diet with 50 g protein/kg as compared to those in rats given diets with 100 and 200 g protein/kg. The addition of nicotinic acid to the diet resulted in a significant increase in the levels of nicotinamide nucleotides only in rats given 50 g protein/kg diet but not in those given either 100 or 200 g protein/kg diet. 3. Liver tryptophan oxygenase (EC 1.13.1.12) activity increased with increasing dietary protein level. Niconitic acid in the diet had no effect on its activity. 4. Quinolinate phosphoribosyltransferase (EC 2.4.2.a) activity in liver was inversely related to dietary protein level, and nicotinic acid in the diet had no effect on its activity. 5. Liver nicotinate phosphoribosyltransferase (EC 2.4.2.11) activity and kidney picolinate carboxylase (EC 4.1.1.45) activity were not altered either by dietary protein level or nicotinic acid in the diet. 6. The addition of nicotinic acid to the diet resulted in increased excretion of N'-methylnicotinamide at all dietary protein levels. 7. The inverse relationship between protein level in the diet and liver quinolinate phosphoribosyltransferase activity, the rate-limiting enzyme of the tryptophan-NAD pathway suggests that the efficiency of conversion of tryptophan to NAD is related to protein level in the diet, the efficiency decreasing with an increase in the level of dietary protein.
Assuntos
Proteínas Alimentares/metabolismo , NAD/metabolismo , Ácidos Nicotínicos/metabolismo , Triptofano/metabolismo , Animais , Indolamina-Pirrol 2,3,-Dioxigenase , Rim/enzimologia , Rim/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Pentosiltransferases/metabolismo , Ácidos Quinolínicos/urina , Ratos , Triptofano Oxigenase/metabolismoRESUMO
Six South African White miners were studied with the 2-g L-tryptophan load test and tracer doses of L-tryptophan-7a-14C, L-kynurenine-keto-14C and hydroxy-L-kynurenine-keto-14C. The breath 14CO2 and 14 urinary metabolities were measured. When they were compared with a previous study of American women with scleroderma, similar 14CO2 and tryptophan metabolite excretion patterns were observed in the data from the miners. The labelled quinolinic acid excretion was more significantly elevated in the South African miners' urine than in the urine of the American women. The data from both studies suggest that some patients with scleroderma have an altered step in the tryptophan metabolic pathway after hydroxy-anthranilic acid. What relationship exists between the induction of pulmonary silicosis and the subsequent development of scleroderma, requires additional human studies.
Assuntos
Cinurenina/metabolismo , Mineração , Doenças Profissionais/metabolismo , Escleroderma Sistêmico/metabolismo , Triptofano/metabolismo , Dióxido de Carbono/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Ácidos Nicotínicos/urina , Doenças Profissionais/urina , Ácidos Quinolínicos/urina , Escleroderma Sistêmico/urinaRESUMO
The effect of vitamin B-6 deficiency on excretion of tryptophan metabolites was compared in rats, guinea pigs, hamsters, and humans. With adequate vitamin B-6 intake, a high percentage of the tryptophan administered was excreted as kynurenic acid and quinolinic acid in rat urine, and as acetylkynurenine and kynurenine in hamster urine. None of the tryptophan metabolites measured in normal guinea pig urine or human urine accounted for more than 1% to 2% of the tryptophan administered. During vitamin B-6 deficiency, the percentages of the tryptophan load excreted as xanthurenic acid, kynurenine, and o-hydroxykynurenine, (which precede the 3-hydroxykynureninase step in the kynurenine pathway) were increased in all four species. However, the percentages excreted as 3-hydroxyanthranilic acid and quinolinic acid, which are beyond the 3-hydroxy-kynureninase step, responded differently. The 3-hydroxyanthranilic acid percentage was not changed in rat urine, but was increased in human and guinea pig urines. The quinolinic acid percentage was decreased in rat urine, unchanged in guinea pig and hamster urine, and increased in human urine. In rats, depression of 3-hydroxykynureninase activity was apparently the major factor causing a change in the pattern. However, in hamsters, kynurenine hydroxylase and o-hydroxykynureninanse activities apparently were depressed. In humans, 3-hydroxykynureninase activity also was apparently depressed and the total amount of administered tryptophan accounted fro in the urine as metabolites of the kynurenine pathway was increased. Levels of urinary metabolites reached a maximum in guinea pigs after only 1 week of consuming the vitamin B-6 deficient diet, suggesting that the vitamin deficiency developed very rapidly in this species.
Assuntos
Triptofano/metabolismo , Deficiência de Vitamina B 6/urina , Adulto , Animais , Cricetinae , Dieta , Feminino , Cobaias , Humanos , Ácido Cinurênico/urina , Cinurenina/urina , Piridoxina/metabolismo , Ácidos Quinolínicos/urina , Ratos , Xanturenatos/urinaRESUMO
Distrubances in the tryptophan-niacin pathway seen in endemic pellagra among sorghum eaters have been ascribed to high dietary intake of leucine. Vitamin B6 plays an important role in several steps of this pathway. Therefore, studies on possible metabolic interrelations between excess dietary leucine and vitamin B6 were undertaken in normal healthy human subjects. The results indicated that vitamin B6 could successfully counteract the effects of leucine on quinolinic acid excretion in urine, and on in vitro nicotinamide nucleotide synthesis by erythrocytes, and also could correct the abnormalities of 5-hydroxytryptamine metabolism induced by excess leucine. These observations suggest that vitamin B6 nutritional status may have a contributory role in the pathogenesis of endemic pellagra.
Assuntos
Leucina/farmacologia , Pelagra/etiologia , Piridoxina/farmacologia , Adulto , Plaquetas/metabolismo , Sinergismo Farmacológico , Grão Comestível , Eritrócitos/metabolismo , Comportamento Alimentar , Humanos , Ácido Hidroxi-Indolacético/urina , Índia , Masculino , Ácidos Nicotínicos/metabolismo , Ácidos Quinolínicos/urina , Ribonucleotídeos/sangue , Serotonina/sangue , Triptofano/metabolismoRESUMO
Administration of leucine at 3% level in the diet has been shown to increase tryptophan oxygenase activity and to decrease kynureninase activity in the liver and to increase quinolinic acid excretion in the urine of rats fed low amounts of vitamin B6 (0.5 mug/g diet). Vitamin B6 at 3 mug/g of diet was able to reverse the effects of leucine on enzyme activities, but not on quinolinic acid excretion. Isoleucine at 0.2% level could counteract the leucine effect on kynureninase but not on tryptophan oxygenase. Administration of leucine also decreased the 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the brain. Simultaneous administration of isoleucine could counteract the effects of leucine on brain 5-HT and 5-HIAA partially at low amount of vitamin B6 but completely at higher levels of vitamin B6 in the diet.