Evaluation of the T-KB-H and 3-HB Kits for the Measurement of Serum Ketone and beta-Hydroxybutyric Acid

BACKGROUND: Diabetes mellitus and alcohol consumption are the most common causes of ketoacidosis in adults. Recently, beta-hydroxybutyric acid (betaHBA) was reported to be a potential serum biomarker in the diagnosis and monitoring of ketoacidosis. We evaluated the performance of T-KB-H and 3-HB kits for the measurement of ketone bodies [acetoacetate (AcAc)+betaHBA] and betaHBA, respectively. METHODS: Quantitative enzymatic assays were performed using the T-KB-H and 3-HB kits (Nittobo Medical Co., Japan) and the Architect ci16200 Integrated System (Abbott Laboratories, USA). Simultaneously, the ketone body levels in these serum samples were determined by gas chromatography-mas spectrometry (GC-MS). We evaluated precision and linearity of these kits and correlation with GC-MS, and established reference intervals in children and adults. RESULTS: The coefficients of variation for the T-KB-H and 3-HB kits were less than 4.0% at analyte levels of 50, 100, and 400 micromol/L. Linearity was observed for AcAc and betaHBA over a 0-1,000 micromol/L range (R2<0.99). Results from the T-KB-H and 3-HB kits were in good agreement with those from the GC-MS analysis, with correlation coefficients of 0.94 for AcAc and 0.96 for betaHBA. Reference intervals determined for the T-KB-H kit were 9.8-270.1 micromol/L and 18.5-531.8 micromol/L in children and adults, respectively. For the 3-HB kit, the reference intervals were 6.4-234.0 micromol/L and 16.0-437.2 micromol/L in children and adults, respectively. CONCLUSIONS: The T-KB-H and 3-HB kits displayed good precision, clinically acceptable linearity, and reliable correlation with an established assay. This indicates that the kits can be used clinically for measuring serum ketone bodies.

Accuracy of capillary blood 3-beta-hydroxybutyrate determination for the detection and treatment of canine diabetic ketoacidosis

In human medicine, diagnosis of diabetic ketoacidosis (DKA) is usually based on measurement of capillary 3-beta-hydroxybutyrate (3-HB) with a hand held ketone sensor. This study was conducted to determine if measurement of capillary 3-HB could be useful for the diagnosis and monitoring of canine DKA. Fifteen dogs with diabetic ketosis and 10 with DKA were evaluated. Paired measurements of 3-HB of capillary and venous blood samples were analysed by the electrochemical sensor and reference method. Use of capillary 3-HB measurement during DKA management was then evaluated through simultaneous measurements of capillary 3-HB, urinary AcAc and venous blood gas analysis. Good agreement between capillary and venous 3-HB measurement was detected by the electrochemical sensor and reference method. Monitoring treatment of DKA revealed a significant correlation between capillary 3-HB and acidosis markers, while no significant correlation was observed between AcAc and acidosis markers. A cut-off value of capillary blood 3-HB >3.8 mmol/L for diagnosis of DKA resulted in 70% and 92% sensitivity and specificity. The electrochemical sensor accurately measures 3-HB concentration in both capillary and venous blood samples, is accurate in diagnosing canine DKA, and appears to reflect the patient's metabolic status during DKA treatment.

Study on the acetoacetate/β hydroxybutyrate determination in classification of type 1 and type 2 diabetes mellitus / 中华内分泌代谢杂志

The clinical values of acetoacetate ( AcAA ) and β hydroxybutyrate ( βHBA ) determination in classification of type 1 and2 diabetes were explored. 102 normal control subjects,33 cases of type 1 diabetes, and 104cases of type 2 diabetes were enrolled. Serum AcAA, βHBA, fasting plasma glucose ( FPG), C-peptide, and insulin levels were measured. The results showed that serum AcAA, βHBA, total ketone tody (TKB) levels in the diabetic groups were significantly higher than those of the normal group( P＜0. 01 ). AcAA, βHBA, TKB levels in type 1diabetes were higher as compared with those of type 2 diabetes( P＜0.01 ). The AcAA, βHBA, and TKB levels were negatively related with C-peptide and insulin in diabetic patients( P＜0. 01 ). All the type 1 diabetic patient were found to have TKB and lower C-peptide levels. TKB positive and lower C-peptide in type 2 diabetes were found in 47% and 26% respectively. Receiver operating characteristic (ROC) curve suggested that the area under the ROC curve of type 1 and type 2 diabetes was 0.926. The optimal operating point of the total ketone body was 0. 532 mmol/L with higher sensitivity and specificity. Enzymatic determination of acetoacetate and β hydroxybutyrate seems to have important clinical values for classification of type 1 and 2 diabetes.

Mecanismo de oxidação aeróbica de acetoacetato e 2-metilacetoacetato catalisada por mioglobina: implicações em desordens cetogênicas / Mechanism of the aerobic oxidation of acetoacetate and 2- methylacetoacetate catalyzed by Mb: implications for ketogenic disorders

Acetoacetato (AA) e 2-metilacetoacetato (MAA) são compostos β-cetoácidos acumulados em diversas desordens metabólicas como no diabetes e na isoleucinemia, respectivamente. Examinamos o mecanismo de oxidação aeróbica de AA e MAA iniciada por intermediários reativos de mioglobina de coração de cavalo (Mb) gerados pela adição de H2O2. Uma rota quimioluminescente que envolve um intermediário dioxetânico cuja termólise gera espécies α-dicarbonílicas (metilglioxal e biacetilo) foi proposta e estudada. Emissão de luz ultra fraca acompanha a reação, e sua intensidade aumenta linearmente pelo aumento da concentração tanto de Mb (10-500 µM) quando AA (10-100 mM). Estudos de consumo de oxigênio mostraram que MAA é, como esperado, quase uma ordem de grandeza mais reativo que AA. Estudos de EPR com captação de spin, utilizando MNP, possibilitaram detectar adutos de MAA atribuíveis a um radical centrado no Cα (aN = 1.55 mT) e ao radical acetila (aN = 0.83 mT). O sinal do radical acetila é totalmente suprimido por sorbato, um conhecido e eficiente supressor de espécies tripletes, o que é consistente com uma rota reacional envolvendo um intermediário dioxetânico. Clivagem-α da ligação carbonila-carbonila do produto biacetilo triplete produziria, de fato, radicais acetila. Além disso, utilizando AA como substrato para Mb/H2O2, um sinal de EPR atribuível ao aduto MNP-AA• (aN = 1.46 mT e aH = 0.34 mT) foi observado e confirmado por efeito isotópico. O consumo de oxigênio e o rendimento de compostos α-dicarbonílicos foram dose-dependentes à concentração de AA ou MAA (1-50 mM) bem como à concentração de H2O2 adicionado às misturas de reação contendo Mb (até 1:10 quando medido o consumo de oxigênio, e até 1:25 quando medido o rendimento de compostos α-dicarbonílicos) e tert-butilhidroperóxido (até 1:200). Os perfis de pH (5,8-7,8) para consumo de oxigênio e rendimento de compostos α-dicarbonílicos mostraram maiores rendimentos para baixos valores de pH, indicativo de ferrilMb...

Acetoacetate (AA) and 2-methylacetoacetate (MAA) are β-ketoacids accumulated in several metabolic disorders such as diabetes and isoleucinemia, respectively. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by the reactive enzyme intermediates formed by the reaction of muscle horse myoglobin (Mb) with H2O2. A chemiluminescent route involving a dioxetane intermediate whose thermolysis yields triplet α-dicarbonyl species (methylglyoxal and diacetyl) is envisaged. Accordingly, the ultraweak light emission that accompanies the reaction increases linearly by raising the concentration of both Mb (10-500 µM) and AA (10- 100 mM). Oxygen uptake studies revealed that MAA is, expectedly, almost one order of magnitude more reactive than AA. EPR spin-trapping studies with MNP detected spin adducts from MAA attributable to an α-carbon-centered radical (aN = 1.55 mT) and to an acetyl radical (aN = 0.83 mT). As the acetyl radical signal is totally suppressed by sorbate, a well-known efficient triplet species quencher, the dioxetane hypothesis seems to be reliable. The α-cleavage of the carbonyl-carbonyl bond of a putative excited triplet diacetyl product would, in fact, leads to an acetyl radical. Furthermore, using AA as substrate for Mb/H2O2, an EPR signal assignable to a MNP-AA• adduct (aN = 1.46 mT and aH = 0.34 mT) was observed and confirmed by isotope effect. Oxygen consumption and α-dicarbonyl yield were also dependent on AA or MAA concentrations (1-50 mM) as well as on the concentration of peroxide added to the Mb-containing reaction mixtures: H2O2 (up to 1:10 when measuring oxygen uptake and up to 1:25 when measuring the α-dicarbonyl yield) and t-butOOH (up to 1:200). The pH profiles (5.8-7.8) of oxygen consumption and α-dicarbonyl yield show higher reaction rates at lower pHs, indicative of a ferrylMb intermediate. Evaluating Mb lesion, both β-ketoacids reduced disorganization of the secondary and tertiary protein structure elicited by H2O2...

Lipoperoxidação, perda de viabilidade celular e diminuição da liberação de IL-8 de neutrófilos humanos na presença de corpos cetônicos / Lipoperoxidation, loss of cell viability and inhibition of release of IL-8 BY human neutrophils in the presence of ketone bodies

Portadores de diabetes tipo-1 são acometidos por episódios freqüentes de acidose causada pelo aumento no metabolismo de ácido graxos com conseqüente acúmulo de acetoacetato (AcAc) e beta-hidroxibutirato (beta -OB). Neste trabalho estudou-se o efeito de concentrações patológicas destes metabólitos na lipoperoxidação, viabilidade e liberação da quimiocina CXCL8 (IL-8) por neutrófilos em cultura. Neutrófilos de indivíduos saudáveis foram isolados por gradiente de densidade (Histopaque 1077/1119) e incubados com os corpos cetônicos. A lipoperoxidação foi determinada pela presença de substâncias reagentes ao ácido tiobarbitúrico (TBARS). A viabilidade celular foi avaliada pela liberação da enzima lactato desidrogenase. A liberação de CXCL8 para o meio extracelular foi medida após cultura de 24 horas de neutrófilos estimulados por zymosan opsonizado por ensaio imunoenzimático (ELISA). O AcAc causou um aumento na lipoperoxidação dos neutrófilos e este efeito foi dependente da sua concentração (p < 0.05; r = 0.99146); não se observou efeito do beta-HOB. No estudo do efeito citotóxico, houve aumento dose-dependente da liberação da LDH até 40 mM de AcAc (p < 0.05); não se observou efeito do beta-HOB. A liberação de CXCL8 foi suprimida de modo dose-dependente por AcAc e beta-HOB. Estes resultados sugerem que o acúmulo de corpos cetônicos pode contribuir para aumentar o tempo de remissão de doenças e mesmo estar relacionado com a gravidade destas em indivíduos diabéticos.

Type-1 diabetes patients suffer from frequent episodes of acidosis caused by an increased fatty acid metabolism and consequently increased plasma level of acetoacetate (AcAc) and beta-hydroxybutyrate (beta-HOB). This article describes a study of the effects of pathological concentrations of AcAc and beta-HOB on lipoperoxidation, cell viability and the release of the CXCL8 (IL-8) cytokine by activated neutrophils. Neutrophils from healthy donors were isolated by density gradient (Histopaque® 1077/1119) and incubated with the ketone bodies. Lipoperoxidation was determined as thiobarbituric acid reactive substances (TBARS). The cell viability was evaluated by the release of intracellular lactate dehydrogenase. The release of CXCL8 was measured by ELISA in a 24-h culture of opsonized zymosanstimulated neutrophils. AcAc, but not beta-HOB, provoked a dose-dependent increase in the neutrophil membrane lipoperoxidation (p<0.05; r =0.9915). In the cytotoxicity assay, a dose-dependent release of LDH was observed when the neutrophils were incubated with AcAc in concentrations up to 40 mM (p<0.05). beta-HOB was devoid of effect. The release of CXCL8 was inhibited by AcAc and beta-HOB in a dose-dependent manner. In conclusion, these results suggest that the accumulation of ketone bodies in diabetic patients could be involved in their usually increased susceptibility to infection.

Effects of the extract from semen viticis negundo with acetoactate on human gastric carcinoma SGC-7901 cell in vitro and in vivo / 中国药理学通报

Aim To study the effects of EVn-50 on human gastric carcinoma SGC-7901 cells in vitro and invivo. Methods Human gastric carcinoma SGC-7901 cells were cultured in vitro. The inhibitory rate of cells was determined by cell counting and the cell growth curve was made. Plate clone formation assay was carried out to detect the phenotypes of colony formation. Trail of human gastric carcinoma xenografts in nude mouse model was used to draw the transplant tumor growth curve and test inhibition rate of EVn-50 on human gastric carcinoma. The histopathological changes were observed by lightmicroscopy and electronmicroscopy. Results In vitro,EVn-50 at 1,10,100 mg?L-1inhibited the growth and proliferation of SGC-7901 cells in a dose-dependent manner and a time-dependent manner; The colony-forming rate was reduced drastically compared with control group(P

Mode of action of lipoic acid in diabetes.

Metabolic aberrations in diabetes such as hyperglycemia, ketonemia, ketonuria, reduced glycogen in tissues and reduced rates of fatty acid synthesis in the liver are corrected by the administration of lipoic acid. Dithiol octanoic acid is formed from lipoic acid by reduction and substitutes for Coenzyme A in several enzymatic reactions such as pyruvate dehydrogenase, citrate synthase, acetyl Coenzyme A carboxylase, fatty acid synthetase, and triglyceride and phospholipid biosynthesis; but not in the oxidation of fatty acids because of the slow rates of thiolysis of ß-keto acyl dithioloctanoic acid. The overall effect of these changes in the key enzymic activities is seen in the increased rates of oxidation of glucose and a reduction in fatty acid oxidation in diabetes following lipoic acid administration.

Lipoic acid and diabetes: Effect of dihydrolipoic acid administration in diabetic rats and rabbits.

Relative α-lipoic acid content of diabetic livers was considerably less than that of normal livers as determined by gas chromatography. It was not possible to detect any dihydrolipoic acid in the livers. Biochemical abnormalities such as hyperglycaemia, ketonemia, reduction in liver glycogen and impaired incorporation of [2-14C] -acetate into fatty acids in alloxan diabetic rats were brought to near normal levels by the oral or intraperitoneal administration of dihydrolipoic acid. The effect of α-lipoic acid was comparable to that of dihydrolipoic acid in reducing the blood sugar levels of diabetic rabbits during a glucose tolerance test. The results suggest that the mode of action of lipoic acid was through stimulation of pyruvate dehydrogenase.