RESUMO
The assay in serum of non-caeruloplasmin copper, as exchangeable copper after complexation with EDTA (ExCu) and total copper has been evaluated and compared in patients with varying c-reactive protein(CRP). Measurement of ExCu and total copper, range 0.2-47.2 µmol/L, was developed using ICP-MS. The chelating agents EDTA and TEPA were compared over 0.0-10 g/L after incubation with serum for 60 mins followed by ultrafiltration with Amicon 10 kDa filter. The assay for ExCu was optimised with EDTA 3 g/L (8.1 mmol/L) maintained at pH 7.0-8.0 before ultrafiltration. TEPA was not as selective in chelation of copper. Patients n = 82 were studied in relation to changes in inflammatory marker CRP and a group of patients n = 37 with normal CRP. The ExCu assay gave excellent recoveries (94-102 % but poor recovery for free uncomplexed copper), good repeatability, limit of quantitation 0.19 µmol/l with a provisional reference range 0.48 to 1.63 µmol/L (n = 37 patients). The range for relative exchangeable copper (exchangeable copper divided by total serum copper) was 2.49 to 9.96 %. ExCu was elevated in conditions with increased CRP greater than 100 mg/L suggesting an effect of inflammation on the free copper fraction. A reliable and reproducible assay for ExCu and total copper has been developed. The upregulated inflammatory state increases the ExCu suggesting excess free copper.
Assuntos
Bioensaio , Cobre/sangue , Ácido Edético/química , Degeneração Hepatolenticular/diagnóstico , Espectrofotometria Atômica/normas , Adulto , Idoso , Proteína C-Reativa/metabolismo , Ceruloplasmina/metabolismo , Quelantes/química , Etilenodiaminas/química , Feminino , Degeneração Hepatolenticular/sangue , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Valores de Referência , Reprodutibilidade dos Testes , Espectrofotometria Atômica/métodos , Ultrafiltração/métodosRESUMO
Alkaptonuria (AKU) is a rare disease characterized by high levels of homogentisic acid (HGA); patients suffer from tissue ochronosis: dark brown pigmentation, especially of joint cartilage, leading to severe early osteoarthropathy. No molecular mechanism links elevated HGA to ochronosis; the pigment's chemical identity is still not known, nor how it induces joint cartilage degradation. Here we give key insight on HGA-derived pigment composition and collagen disruption in AKU cartilage. Synthetic pigment and pigmented human cartilage tissue both showed hydroquinone-resembling NMR signals. EPR spectroscopy showed that the synthetic pigment contains radicals. Moreover, we observed intrastrand disruption of collagen triple helix in pigmented AKU human cartilage, and in cartilage from patients with osteoarthritis. We propose that collagen degradation can occur via transient glycyl radicals, the formation of which is enhanced in AKU due to the redox environment generated by pigmentation.
Assuntos
Alcaptonúria/metabolismo , Cartilagem Articular/metabolismo , Osteoartrite/metabolismo , Pigmentação , Espectroscopia de Ressonância de Spin Eletrônica , Ácido Homogentísico/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Oxirredução , Pigmentos Biológicos/químicaRESUMO
BACKGROUND: Identification of unknown chemical entities is a major challenge in metabolomics. To address this challenge, we developed a comprehensive targeted profiling strategy, combining 3 complementary liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) techniques and in-house accurate mass retention time (AMRT) databases established from commercial standards. This strategy was used to evaluate the effect of nitisinone on the urinary metabolome of patients and mice with alkaptonuria (AKU). Because hypertyrosinemia is a known consequence of nitisinone therapy, we investigated the wider metabolic consequences beyond hypertyrosinemia. METHODS: A total of 619 standards (molecular weight, 45-1354 Da) covering a range of primary metabolic pathways were analyzed using 3 liquid chromatography methods-2 reversed phase and 1 normal phase-coupled to QTOF-MS. Separate AMRT databases were generated for the 3 methods, comprising chemical name, formula, theoretical accurate mass, and measured retention time. Databases were used to identify chemical entities acquired from nontargeted analysis of AKU urine: match window theoretical accurate mass ±10 ppm and retention time ±0.3 min. RESULTS: Application of the AMRT databases to data acquired from analysis of urine from 25 patients with AKU (pretreatment and after 3, 12, and 24 months on nitisinone) and 18 HGD -/- mice (pretreatment and after 1 week on nitisinone) revealed 31 previously unreported statistically significant changes in metabolite patterns and abundance, indicating alterations to tyrosine, tryptophan, and purine metabolism after nitisinone administration. CONCLUSIONS: The comprehensive targeted profiling strategy described here has the potential of enabling discovery of novel pathways associated with pathogenesis and management of AKU.
Assuntos
Alcaptonúria/metabolismo , Cicloexanonas/farmacologia , Metaboloma/efeitos dos fármacos , Nitrobenzoatos/farmacologia , Idoso , Alcaptonúria/tratamento farmacológico , Animais , Cromatografia Líquida/métodos , Cromatografia Líquida/estatística & dados numéricos , Bases de Dados de Compostos Químicos , Feminino , Técnicas de Silenciamento de Genes , Homogentisato 1,2-Dioxigenase/genética , Humanos , Masculino , Espectrometria de Massas/métodos , Espectrometria de Massas/estatística & dados numéricos , Metabolômica/métodos , Camundongos , Pessoa de Meia-Idade , FenótipoRESUMO
Metabolomic analyses in alkaptonuria (AKU) have recently revealed alternative pathways in phenylalanine-tyrosine (phe-tyr) metabolism from biotransformation of homogentisic acid (HGA), the active molecule in this disease. The aim of this research was to study the phe-tyr metabolic pathway and whether the metabolites upstream of HGA, increased in nitisinone-treated patients, also undergo phase 1 and 2 biotransformation reactions. Metabolomic analyses were performed on serum and urine from patients partaking in the SONIA 2 phase 3 international randomised-controlled trial of nitisinone in AKU (EudraCT no. 2013-001633-41). Serum and urine samples were taken from the same patients at baseline (pre-nitisinone) then at 24 and 48 months on nitisinone treatment (patients N = 47 serum; 53 urine) or no treatment (patients N = 45 serum; 50 urine). Targeted feature extraction was performed to specifically mine data for the entire complement of theoretically predicted phase 1 and 2 biotransformation products derived from phenylalanine, tyrosine, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenyllactic acid, in addition to phenylalanine-derived metabolites with known increases in phenylketonuria. In total, we observed 13 phase 1 and 2 biotransformation products from phenylalanine through to HGA. Each of these products were observed in urine and two were detected in serum. The derivatives of the metabolites upstream of HGA were markedly increased in urine of nitisinone-treated patients (fold change 1.2-16.2) and increases in 12 of these compounds were directly proportional to the degree of nitisinone-induced hypertyrosinaemia (correlation coefficient with serum tyrosine = 0.2-0.7). Increases in the urinary phenylalanine metabolites were also observed across consecutive visits in the treated group. Nitisinone treatment results in marked increases in a wider network of phe-tyr metabolites than shown before. This network comprises alternative biotransformation products from the major metabolites of this pathway, produced by reactions including hydration (phase 1) and bioconjugation (phase 2) of acetyl, methyl, acetylcysteine, glucuronide, glycine and sulfate groups. We propose that these alternative routes of phe-tyr metabolism, predominantly in urine, minimise tyrosinaemia as well as phenylalanaemia.
RESUMO
Alkaptonuria (AKU) is an inherited disorder of tyrosine metabolism caused by lack of active enzyme homogentisate 1,2-dioxygenase (HGD). The primary consequence of HGD deficiency is increased circulating homogentisic acid (HGA), the main agent in the pathology of AKU disease. Here we report the first metabolomic analysis of AKU homozygous Hgd knockout (Hgd -/-) mice to model the wider metabolic effects of Hgd deletion and the implication for AKU in humans. Untargeted metabolic profiling was performed on urine from Hgd -/- AKU (n = 15) and Hgd +/- non-AKU control (n = 14) mice by liquid chromatography high-resolution time-of-flight mass spectrometry (Experiment 1). The metabolites showing alteration in Hgd -/- were further investigated in AKU mice (n = 18) and patients from the UK National AKU Centre (n = 25) at baseline and after treatment with the HGA-lowering agent nitisinone (Experiment 2). A metabolic flux experiment was carried out after administration of 13C-labelled HGA to Hgd -/-(n = 4) and Hgd +/-(n = 4) mice (Experiment 3) to confirm direct association with HGA. Hgd -/- mice showed the expected increase in HGA, together with unexpected alterations in tyrosine, purine and TCA-cycle pathways. Metabolites with the greatest abundance increases in Hgd -/- were HGA and previously unreported sulfate and glucuronide HGA conjugates, these were decreased in mice and patients on nitisinone and shown to be products from HGA by the 13C-labelled HGA tracer. Our findings reveal that increased HGA in AKU undergoes further metabolism by mainly phase II biotransformations. The data advance our understanding of overall tyrosine metabolism, demonstrating how specific metabolic conditions can elucidate hitherto undiscovered pathways in biochemistry and metabolism.
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BACKGROUND: Manganese (Mn) toxicity has been reported in patients receiving total parenteral nutrition. To avoid unnecessary exposure it is recommended by NICE (National Institute for Clinical Excellence) that blood Mn concentrations are monitored. The aim of the study was to develop a method using inductively coupled plasma mass spectrometry (ICP-MS) for the reliable determination of Mn in plasma and whole blood, as indices of acute and chronic exposure. METHODS: Whole blood and plasma samples were prepared by appropriate dilution (diluent containing 0.005% Triton X-100, 0.2% propan-2-ol, 0.2% butan-1-ol and 1% nitric acid) addition of an internal standard gallium, followed by centrifugation to remove cell debris. Thermo Fisher Scientific ExCell and X Series ICP-MS instruments were used to define and correct for polyatomic interference on Mn assay. RESULTS: Mn was quantified at mass 55 using aqueous calibration and the polyatomic interference from FeH was successfully eliminated by modified (Xt) skimmer cones but not with the collision cell (collision gas 7% H2 in He, flow rate 4-7 mL/min). The assay was validated showing good precision, limit of detection and percentage recovery. Good agreement was observed with the All Laboratory Trimmed Mean of External Quality Assurance samples y (in house)=1.1 (ALTM)-45.0 between values of 250 and 750 nmol/L. CONCLUSIONS: A method has been developed using ICP-MS for the analysis of whole blood and plasma Mn incorporating a novel method of eliminating interference by utilizing the different geometries of the Xt interface cones. The procedure is simple and robust with good precision and recovery over a wide dynamic range.
Assuntos
Cromatografia por Troca Iônica , Manganês/sangue , Espectrometria de Massas , Plasma/química , Cromatografia por Troca Iônica/normas , Humanos , Limite de Detecção , Espectrometria de Massas/normas , Padrões de ReferênciaRESUMO
BACKGROUND: Pseudohyperkalaemia may result from delay in centrifugation and storage at 4°C. We investigated whether the stage of chronic kidney disease (CKD), its aetiology or medications influence this. METHODS: Seventy-seven patients with CKD were recruited. Lithium heparin plasma samples were analysed for sodium, potassium, urea and creatinine, chloride, bicarbonate, magnesium, calcium and inorganic phosphate at 0 h and after storage of whole blood at 4°C for 6 h and 20 h. K-EDTA and fluoride-EDTA samples were analysed for full blood count and glucose at 0 h. CKD stage was determined by standard criteria. RESULTS: K(+) increased on average by 1.0 and 3.6 mmol/L after 6 and 20 h storage of whole blood at 4°C, independent of cause or stage of CKD. K(+) increase at 6 h was correlated with haemoglobin but not with white blood cell count, platelet count or glucose. Patients taking ACE inhibitors and/or angiotensin receptor blockers (ARBs) had slightly higher K(+) at 0 h and increased K(+) after storage for 6 h. Na(+) decreased on average by 3.8 mmol/L at 20 h and was independent of CKD stage, and correlated with K(+) increase. CONCLUSIONS: K(+) increased significantly with time in samples stored at 4°C in all stages of CKD. This was greater in some patients on ACE inhibitors and ARBs, and increased with haemoglobin, but was not related to the stage of CKD, white blood cell count or platelet count for the samples used in this study.
Assuntos
Hiperpotassemia/sangue , Hiperpotassemia/diagnóstico , Nefropatias/sangue , Potássio/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença Crônica , Reações Falso-Positivas , Feminino , Humanos , Nefropatias/tratamento farmacológico , Nefropatias/etiologia , Nefropatias/patologia , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
BACKGROUND: Aluminium (Al) toxicity was frequent in the 1980s in patients ingesting Al containing phosphate binders (Alucaps) whilst having HD using water potentially contaminated with Al. The aim of this study was to determine the risk of Al toxicity in HD patients receiving Alucaps but never exposed to contaminated dialysate water. METHODS: HD patients only treated with Reverse Osmosis(RO) treated dialysis water with either current or past exposure to Alucaps were given standardised DFO tests. Post-DFO serum Al level > 3.0 µmol/L was defined to indicate toxic loads based on previous bone biopsy studies. RESULTS: 39 patients (34 anuric) were studied. Mean dose of Alucap was 3.5 capsules/d over 23.0 months. Pre-DFO Al levels were > 1.0 µmol/L in only 2 patients and none were > 3.0 µmol/L. No patients had a post DFO Al levels > 3.0 µmol/L. There were no correlations between the serum Al concentrations (pre-, post- or the incremental rise after DFO administration) and the total amount of Al ingested.No patients had unexplained EPO resistance or biochemical evidence of adynamic bone. CONCLUSIONS: Although this is a small study, oral aluminium exposure was considerable. Yet no patients undergoing HD with RO treated water had evidence of Al toxicity despite doses equivalent to 3.5 capsules of Alucap for 2 years. The relationship between the DFO-Al results and the total amount of Al ingested was weak (R² = 0.07) and not statistically significant. In an era of financial prudence, and in view of the recognised risk of excess calcium loading in dialysis patients, perhaps we should re-evaluate the risk of using Al-based phosphate binders in HD patients who remain uric.
Assuntos
Compostos de Alumínio/sangue , Alumínio/sangue , Alumínio/toxicidade , Hiperfosfatemia/tratamento farmacológico , Falência Renal Crônica/complicações , Fosfatos/sangue , Diálise Renal , Administração Oral , Alumínio/farmacocinética , Desferroxamina , Soluções para Diálise/administração & dosagem , Humanos , Hiperfosfatemia/sangue , Hiperfosfatemia/prevenção & controle , Falência Renal Crônica/terapia , Pessoa de Meia-Idade , Osmose , Medição de Risco/métodos , Sideróforos , Resultado do TratamentoRESUMO
BACKGROUND: The goal of our study was to test the relative stability of urine, unconjugated, free catecholamines and the methyl derivatives. We measured the change in concentrations in commercially available urines after storage at various pH values, temperatures and time, from days up to 10 weeks. METHODS: Samples of commercial control urines were adjusted to pH 2.0, 4.0, 6.0 and 8.0 and aliquots stored at ambient temperature (20-26 degrees C), 4 degrees C and -18(o)C. The free catecholamines (cats) and the free methyl derivatives (mets) were measured after 1, 2, 3 and 6 days and 1, 2, 3 and 10 weeks using the automated sample trace enrichment dialysis (ASTED) procedure with reversed phase ion pair high performance liquid chromatography (HPLC) and coulometric detection. RESULTS: Free catecholamines were relatively stable, with <15% loss of concentration, when stored at pH 6.0 or less for at least 4 days and up to 10 weeks at pH 2.0 at either 4(o)C or -18(o)C. At pH 8.0, the concentration fell to <60% after 48 h and at a pH of 6.0 or 8.0, up to 90% was lost within the first week at 4(o)C and 25(o)C. More than 40% of free normetadrenaline and metadrenaline were lost after 1-2 weeks when stored at 20-25(o)C and pH 8.0. After 10 weeks at pH 4.0, 6.0 and 8.0, up to 90% loss was observed at 25(o)C. Free cats were stable at pH 2.0 and 4.0 at -18(o)C and the free mets were stable at -18(o)C over the entire time period studied and at all pHs. CONCLUSIONS: In the analysis of free catecholamine and the free methyl derivatives, urine samples should be acidified to a pH range 2.0-3.0 to ensure stability and hence the correct analysis.
Assuntos
Catecolaminas/urina , Catecolaminas/química , Cromatografia Líquida de Alta Pressão/métodos , Dopamina/urina , Epinefrina/urina , Humanos , Concentração de Íons de Hidrogênio , Metanefrina/urina , Norepinefrina/urina , Normetanefrina/urina , Manejo de Espécimes , TemperaturaRESUMO
Selected-ion flow-tube mass spectrometry (SIFT-MS) is particularly suited for the analysis of volatile low molecular weight compounds. We have evaluated this technique for the assay of different alcohols in aqueous solutions, including blood plasma, and in particular whether the osmolality or sample volume affected vapourisation. Solutions of three different alcohols (methanol, ethanol and isopropanol) ranging from 0.005 to 50 mmol/L were prepared in deionised water (0 milliosmol), phosphate-buffered saline (690 mOsm), isotonic saline (294 mOsm) and plasma (296 mOsm). The vapour above the sample (50 to 1000 microL) contained in air-tight tubes at 37 degrees C was aspirated into the instrument. The outputs for ethanol, methanol and isopropanol were linear over the concentration range and independent of the sample volume and relatively independent of the osmolar concentration. SIFT-MS can reliably and accurately measure common alcohols in the headspace above aqueous solutions, including serum/plasma. This novel application of SIFT-MS is easy to follow, requires no sample preparation and the wide dynamic range will facilitate measurement of alcohols present from normal metabolism as well as when taken in excess or in accidental poisoning.
Assuntos
2-Propanol/química , Etanol/química , Espectrometria de Massas/métodos , Metanol/química , Espectrometria de Massas/instrumentação , Concentração OsmolarAssuntos
Anticonvulsivantes/intoxicação , Ácido Valproico/intoxicação , Equilíbrio Ácido-Base , Acidose Láctica/induzido quimicamente , Adulto , Carnitina/uso terapêutico , Overdose de Drogas , Epilepsia/tratamento farmacológico , Humanos , Hiperamonemia/induzido quimicamente , Hipernatremia/induzido quimicamente , MasculinoRESUMO
Aluminium (Al)-induced injury to various cells including brain and bone is well described. We have previously shown that Al initiated damage to kidney cells in culture assessed by loss of cell viability, enzyme release and damage to cell brush borders. However, little is known about the mechanism(s) of these effects, we therefore investigated whether lipid peroxidation and/or sulphydryl depletion, i.e. cellular glutathione (GSH) depletion, could be part of this process(es). Monolayers of porcine kidney proximal tubular cells (PTC), LLC-PK1, were either exposed to 100 micromol/L Al as a citrate complex or cis-platin (cis-Pt) or mercury (Hg) as damage mediating controls (positive), or no added metals (negative control). Malondialdehyde (MDA), a marker of lipid peroxidation, cellular GSH as the intracellular sulphydryl compound, heat shock protein 70 (Hsp70), glutathione peroxidase (GPx) as a reactive oxygen species scavenger enzyme and cellular uptake of Al were assessed. MDA increased significantly (p = 0.03) more than two-fold in the cell lysate after PTCs were exposed to Al for 48 h. In Al-treated PTC, the GSH content of the cell lysate increased to 1.042 +/- 0.080 mmol/L compared with the 0.80 +/- 0.1.64 mmol/L control value. The Hsp70 content of the cells showed no significant change for Al (6.5% vs. 4.4% control value) and GPx activity increased slightly from 0.41 to 0.45 mU/mg protein. Treatment with cis-Pt and Hg also resulted in a significant increase in MDA and Hsp70 and reduction in cellular GSH and GPx activity. The low cellular Al uptake as well as some inherent insensitivity related to the cell type might be contributory factors for the small effect of Al on kidney cells and markers of oxidative damage.
Assuntos
Alumínio/toxicidade , Biomarcadores/metabolismo , Túbulos Renais Proximais , Animais , Antineoplásicos/toxicidade , Cisplatino/toxicidade , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Peroxidação de Lipídeos , Malondialdeído/metabolismo , Mercúrio/toxicidade , SuínosRESUMO
BACKGROUND: Patients with end-stage chronic renal failure (CRF) (uremia) have a high prevalence of inflammation, malnutrition, and oxidative stress. All of these features seem to be associated with the increased cardiovascular mortality observed in these patients. Nitric oxide (NO) is involved in the pathogenesis of CRF. The present study investigates the effects of nutritional status on L-arginine transport (NO precursor), plasma amino acid profile, and concentration of tumor necrosis factor (TNF)-alpha in uremic patients on hemodialysis (HD). METHODS: A total of 32 uremic patients on regular HD and 16 healthy controls were included in this study. Kinetic studies of L-arginine transport, mediated by cationic transport systems y(+) and y(+)L into red blood cells, plasma concentrations of amino acids (measured by high-performance liquid chromatography), and plasma TNF-alpha level (evaluated by enzyme-linked immunosorbent assay), were analyzed in malnourished and well-nourished patients (isolated by body mass index). RESULTS: L-arginine influx by system y(+) in red blood cells (micromol/L cells(-1)h(-1)) was increased in both malnourished (377 +/- 41) and well-nourished (461 +/- 63) patients with CRF compared with controls (287 +/- 28). Plasma levels of all cationic amino acids (L-arginine, L-ornithine, and L-lysine) were low in uremic patients compared with controls. Among the uremic population, the reduction in plasma cationic amino acids levels was greater in malnourished patients. L-cysteine and L-glutamate, precursors of glutathione, were dramatically increased in plasma from uremic patients, independently of nutritional status. In addition, TNF-alpha concentration in plasma was enhanced in malnourished uremic patients (3.4 +/- 0.7 pg/mL) compared with controls (1.2 +/- 0.1 pg/mL) and well-nourished patients (1.9 +/- 0.1 pg/mL). CONCLUSIONS: Our results suggest an increased catabolism of cationic amino acids, inflammatory markers, and oxidative stress in CRF, especially in malnourished patients. The reduced plasma concentration of plasma L-arginine is counterbalanced by enhanced rates of transport, resulting in an activation of NO synthesis in uremia.
Assuntos
Aminoácidos/sangue , Arginina/sangue , Eritrócitos/metabolismo , Falência Renal Crônica/sangue , Desnutrição/sangue , Adulto , Índice de Massa Corporal , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Falência Renal Crônica/complicações , Falência Renal Crônica/terapia , Lisina , Masculino , Desnutrição/complicações , Pessoa de Meia-Idade , Ornitina , Diálise Renal , Trítio , Fator de Necrose Tumoral alfa/sangueRESUMO
The well-described long-term effects of sustained exposure to aluminium in patients with end-stage renal failure (ESRF) are a result of uptake and storage of aluminium, leading to cellular toxicity. A case is presented suggesting that this aluminium may be mobilizable, and indicating the consequence of such release. A patient on haemodialysis (HD) presented acutely with infection, a raised CRP, decreased conscious level, impaired cognition and agitation. Subsequent neurological recovery over six to seven days appeared to follow the return of markedly elevated plasma aluminium concentrations to basal (i.e. from 25.2 micromol/L to 2.5 micromol/L; reference range < 0.5 micromol/L), coupled with a resolution of the infection. The patient was on long-term aludrox therapy 3 g/day, and showed relative resistance to the exogenous hormone erythropoietin, resulting in a refractory anaemia and suggesting aluminium toxicity. A series of HD patients (n = 5) presenting with bacteraemia, not on aludrox, showed no appreciable rise in the plasma aluminium mean of 1.3 micromol/L (SD 0.9; range 0.6-2.0 micromol/L). We suggest that infection can result in release of tissue aluminium, leading to acutely elevated plasma aluminium concentrations and signs of neurotoxicity. The amount of tissue storage and resultant aluminium release seemed to be related to the use of aluminium hydroxide as a phosphate binder.
Assuntos
Alumínio/sangue , Alumínio/toxicidade , Infecções/complicações , Falência Renal Crônica/complicações , Doenças do Sistema Nervoso/induzido quimicamente , Idoso , Hidróxido de Alumínio/efeitos adversos , Hidróxido de Alumínio/metabolismo , Combinação de Medicamentos , Humanos , Hidróxido de Magnésio/efeitos adversos , Hidróxido de Magnésio/metabolismo , Masculino , Diálise Renal/efeitos adversosRESUMO
Zinc (in relatively high concentrations) can be toxic to intestinal cells. The aim of the present study was to quanitfy cellular injury in preconfluent, colonic cancerous cells and in postconfluent, differentiating human intestinal Caco-2 cells. Cellular damage was measured by using cell proliferation, lactate dehydrogenase (LDH)-release, and apoptosis studies. Furthermore, the activities of the major antioxidative enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase] and differentiation markers (alkaline phosphatase and aminopeptidase-N) were determined after exposure of the cells to increasing amounts of zinc sulfate. Proliferation and viability decreased in a concentration-dependent manner. A noticeable increase of LDH-release correlated to cell rounding and detachment at relatively high zinc levels (200 muM) was observed in both groups of cells. Above 100 muM of zinc, significant apoptotic activity was found in the preconfluent cells. Zinc supplementation did not alter SOD activities. However, GPx and, in part, catalase activities tended to be higher in zinc-treated cells (nevertheless the results were not significant). Differentiation markers were noticeably induced by increasing amounts of zinc, especially in the preconfluent cells. In conclusion, we suggest that the susceptibility to zinc induced damage is equal in both confluentation groups of Caco-2 cells. Risk assessment for high concentrations seems recommendable.
Assuntos
Intestinos/efeitos dos fármacos , Zinco/metabolismo , Zinco/farmacologia , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Biomarcadores/análise , Antígenos CD13/efeitos dos fármacos , Antígenos CD13/metabolismo , Células CACO-2 , Catalase/efeitos dos fármacos , Catalase/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Glutationa Peroxidase/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Humanos , Mucosa Intestinal/metabolismo , L-Lactato Desidrogenase/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Superóxido Dismutase/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Zinco/toxicidadeRESUMO
UNLABELLED: This study was carried out to investigate whether zinc can potentiate renal toxicity using monolayer cultures of kidney proximal tubular cells and if so to establish the chemical species and the mechanism involved. METHODS: Zinc was prepared as the citrate complex at pH 7.4 in phosphate buffered saline. Monolayers of kidney proximal tubular cells under standard cell culture conditions were exposed to zinc concentrations of 0, 5 10, 20, 50 and 100 µmol/L. To assess cellular damage, thiazol blue (MTT) uptake, NAG and LDH release, DAPI staining and Tunel assay were used. Cytoprotective agents: trolox, cysteine, glutathione, ascorbic acid and sodium selenite were used to investigate if the damage was reversible. RESULTS: Incubation of kidney cells with zinc citrate showed a dose related reduction in cell viability (p<0.005) associated with cellular uptake of zinc ions. After 24 h incubation with 100 µmol/L Zn citrate, NAG release was not significantly different compared to the control whereas LDH increased 3 fold. DAPI staining showed apoptotic bodies within the cells confirmed by Tunel assay using flow cytometry. Electron microscopy showed significant morphological changes including loss of brush border, vacuolated cytoplasm and condensed nuclei. Trolox almost completely (>85±5%) and sodium selenite partially recovered (40±4%) the viability of cells exposed to Zn but no protection was observed with other cytoprotectants, e.g. glutathione, cysteine or ascorbic acid. In conclusion zinc can induce damage to kidney cells by a mechanism dependent on zinc ions entering the cell, binding to the cell organelles and disrupting cellular processes rather than damage initiated by free radical and ROS production.
Assuntos
Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Zinco/química , Zinco/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Túbulos Renais Proximais/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: We have investigated whether blood ammonia is increased with worsening CKD. METHODS: Fifty eight subjects with a range of CKD were recruited for analysis of plasma ammonia and other electrolytes. RESULTS: The concentrations of plasma ammonia were all within the normal reference range and there was no correlation between ammonia and CKD without any effect of dialysis. CONCLUSIONS: Blood ammonia is not elevated in or related to the severity of chronic kidney disease.
Assuntos
Amônia/sangue , Rim/fisiopatologia , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Bicarbonatos/sangue , Creatinina/sangue , Taxa de Filtração Glomerular , Humanos , Pessoa de Meia-Idade , Estudos Prospectivos , Diálise Renal , Insuficiência Renal Crônica/terapia , Índice de Gravidade de Doença , Ureia/sangue , Uremia/sangue , Uremia/fisiopatologia , Uremia/terapia , Adulto JovemRESUMO
Evidence shows that raised cobalt (Co), chromium (Cr), and nickel (Ni) whole blood concentrations correlate with poor device outcome in patients following metal-on-metal (MoM) hip arthroplasty. To understand the local and systemic pathological effects of these raised metal concentrations it is important to define their distribution between whole blood, plasma, and urine. The metals were measured by Inductively Coupled Plasma Mass Spectrometry (ICPMS). Two hundred and five plasma, 199 whole blood, and 24 sets of urine samples were analyzed from 202 patients with Co-Cr alloy MoM hip prostheses implanted between 8 months to 12 years (mean 6.0 years) prior to analysis. Plasma Co (median 39.1 nmol/L) showed significantly positive 1:1 correlation with whole blood Co (median 45.9 nmol/L; R(2) = 0.98, p < 0.001, slope = 1.0). Plasma Cr (median 53.8 nmol/L) and whole blood Cr (median 40.3 nmol/L) were also correlated; however, concentrations were significantly higher in plasma indicating relatively little blood cell uptake (R(2) = 0.96, p < 0.001, slope = 1.6). Urinary Co was up to threefold higher than Cr (median 334.0 vs. 97.3 nmol/L respectively). Nickel concentrations in whole blood, plasma, and urine were low relative to Co and Cr. The analysis shows fundamental differences in the physiological handling of these metals: Co is distributed approximately equally between blood cells and plasma, whereas Cr is mainly in plasma, despite which, Cr had far less renal excretion than Co.
Assuntos
Artroplastia de Quadril , Cromo/sangue , Cobalto/sangue , Prótese de Quadril/efeitos adversos , Níquel/sangue , Cromo/urina , Cobalto/urina , Humanos , Níquel/urinaRESUMO
OBJECTIVES: There is limited data regarding the phenomenon of seasonal pseudohypokalemia. We aimed to demonstrate the incidence of spurious hypokalemia during the summer months and to investigate the mechanism of cause. DESIGN AND METHODS: Potassium and glucose results from primary care and hospital patients were collected retrospectively for a period of 1 year to assess the incidence of pseudohypokalemia. Experiments were undertaken to confirm that this was a reversible in vitro phenomenon due to increased temperature mediated by sodium-potassium-exchanging-ATPase. RESULTS: Our data show an increased incidence of hypokalemia associated with increasing ambient temperature during June-August in samples from primary care but not in hospital samples. In a subset of patients, we showed that the repeat results were within or at the lower limit of the reference range. Experiments showed that this phenomenon was mediated by the sodium-potassium-exchanging-ATPase. CONCLUSIONS: There is an increased incidence of pseudohypokalemia during the summer (seasonal pseudohypokalemia) in samples from primary care and this is an in vitro pseudo-phenomenon mediated by sodium-potassium-exchanging-ATPase.
Assuntos
Glicemia/metabolismo , Hipopotassemia/sangue , Hipopotassemia/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Temperatura , Diazóxido/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Hipopotassemia/metabolismo , Moduladores de Transporte de Membrana/farmacologia , Pinacidil/farmacologia , Potássio/sangue , Tolbutamida/farmacologiaRESUMO
BACKGROUND: Electrospray ionization mass spectrometry (ESI-MS) can be used for the measurement of glycated adult hemoglobin. Here, we describe the evaluation of ESI-MS for measurement of glycated (GHbF) and acetylated (AcHbF) fetal hemoglobin and the identification by mass of different chains of fetal hemoglobin. METHODS: Blood samples were diluted in an acidic denaturing solvent, desalted with AG 50W-X8 resin and introduced directly into the mass spectrometer. Resulting mass spectra were processed to determine the percentage of GHbF and AcHbF and the gamma-chain masses. RESULTS: The procedure yielded reproducible quantitative assay of GHbF and AcHbF, with coefficients of variation <4.9%. Measurement of alpha-chain glycation was similarly reproducible and is suggested as an alternative marker of glycemic control. Marked increases in glycation occurred in dried spot blood samples, which were related to duration of storage, temperature and glucose concentration. Molecular masses of fetal hemoglobin chains were also determined and in 42 neonates studied, two types A and B were identified, two-thirds were type A with gamma-chain masses corresponding to (G)gamma and (A)gamma. In type B, the relative abundance of the (A)gamma-chain was less and the apparent intensity of the (G)gamma-chain was higher. CONCLUSIONS: ESI-MS can be used for the estimation of GHbF and AcHbF and the accurate measurement of fetal gamma-chain masses. The use of whole blood is preferred for analysis.