Detection of Exogenous γ-Hydroxybutyric Acid in Rat Blood Exosomes / 法医学杂志

OBJECTIVES@#To find a method to distinguish exogenous gamma-hydroxybutyrate (GHB) from endogenous GHB by establishing ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) based on exosome for quantitative detection of GHB in the rat blood.@*METHODS@#Adult male SD rats were divided into 1 h, 5 h, 10 h administration group and control group. After 1 h, 5 h and 10 h of single precursor of GHB gamma-butyrolactone (GBL) intraperitoneal injection in administration groups, 5 mL blood was collected from the abdominal aorta. Meanwhile, the control group was given a same dose of normal saline, and 5 mL blood was collected at 1 h. Among the 5 mL blood, 0.5 mL was directly detected by HPLC-MS after pretreatment, and exosomes were extracted from the remaining blood by differential centrifugation and detected.@*RESULTS@#The concentration of GHB in the control group was (87.36±33.48) ng/mL, and the concentration with administration at 1 h, 5 h and 10 h was (110 400.00±1 766.35) ng/mL, (1 479.00±687.01) ng/mL and (133.60±12.17) ng/mL, respectively. The results of exosome detection showed that no peak GHB signal was detected in the control group and the 10 h administration group, and the concentrations of GHB at 1 h and 5 h administration groups were (91.47±33.44) ng/mL and (49.43±7.05) ng/mL, respectively.@*CONCLUSIONS@#GHB was detected in blood exosome by UPLC-MS, which indicated that exogenous GHB could be detected in plasma exosomes, while endogenous GHB could not be detected, suggesting that this method may be used as a basis to determine whether there is exogenous drug intake.

Isolation and identification of poly beta hydroxybutyric acid accumulating bacteria of Staphylococcal sp. and characterization of biodegradable polyester.

Staphylococcus sp. strain BP/SU1, capable of degrading the biopolymer and utilize it as a source of carbon and energy, was isolated from activated sludge using METABOLIX (MBX D411G). It was found that this strain was capable of accumulating poly(3-hydroxybutyric acid) P(3-HB), as granule poly (3-hydroxybutyric acid), p(3-HB), inclusion bodies when grown under suitable nutrient conditions. These strains could sustain cell growth up to a dry mass of 9.24 g/l with a doubling time of 8 to 10 hr and could accumulate P(3-HB) as granular inclusion bodies to a cell dry weight of more than 12%. P(3-HB) accumulated by this organism was isolated and characterized through NMR, FT-IR spectroscopy, UV Spectroscopy, Mass spectroscopy and Differential Scanning Calorimetry. P(3-HB) granules so isolated showed physical and chemical properties that should be possessed by a superior quality thermoplastic biopolymer.

Inhibition of in vitro CO2 production and lipid synthesis by 2-hydroxybutyric acid in rat brain

2-Hydroxybutyric acid appears at high concentrations in situations related to deficient energy metabolism (e.g., birth asphyxia) and also in inherited metabolic diseases affecting the central nervous system during neonatal development, such as "cerebral" lactic acidosis, glutaric aciduria type II, dihydrolipoyl dehydrogenase (E3) deficiency, and propionic acidemia. The present study was carried out to determine the effect of 2-hydroxybutyric acid at various concentrations (1-10 mM) on CO2 production and lipid synthesis from labeled substrates in cerebral cortex of 30-day-old Wistar rats in vitro. CO2 production was significantly inhibited (30-70 percent) by 2-hydroxybutyric acid in cerebral cortex prisms, in total homogenates and in the mitochondrial fraction. We also demonstrated a significant inhibition of lipid synthesis (20-45 percent) in cerebral cortex prisms and total homogenates in the presence of 2-hydroxybutyric acid. However, no inhibition of lipid synthesis occurred in homogenates free of nuclei and mitochondria. The results indicate an impairment of mitochondrial energy metabolism caused by 2-hydroxybutyric acid, a fact that may secondarily lead to reduction of lipid synthesis. It is possible that these findings may be associated with the neuropathophysiology of the situations where 2-hydroxybutyric acid is accumulated

A simple chemical method for the oxidation of beta-hydroxybutyrate; application of the method for the estimation of ketone bodies.

A simple one-step method employing potassium persulphate as an oxidising agent in presence of catalyst, Ag2+, for the oxidation of beta-hydroxybutyrate (beta-OHB) to acetoacetate (AcAc) has been developed and standardized. Under the condition of assay, beta-OHB (0.079-0.395 microM) was quantitatively transformed to AcAc. The reaction linearity was observed from 0.079 to 0.634 microM. Optimum conditions were: pH, 6.2; temp., 40 degrees C; persulphate saturation, 40% and catalyst, 1.82 mM. Under the experimental condition, no reversal of inhibition caused by chloride (22.96 mM) was observed at Ag+ concentrations (9.09 and 18.18 mM), while higher conc. of Ag+ (27.27 mM) caused significant reversal of inhibition (about 60%). The maximum reversal of inhibition was achieved at Ag+ (36.36 mM). The level of ketone bodies, when estimated by the present method, was greatly enhanced during starvation period and about 2- and 12-fold higher level of ketone bodies was observed (compared to control) in rats fasted for 24 and 48 hr respectively.

Spectral evidence for the oxidation of beta-hydroxybutyrate to acetoacetate: diminution of spectra by glucose and chloride.

Chemical oxidation of beta-hydroxybutyrate (beta-OHB) to acetoacetate (AcAc) has been carried out by a simple and new method employing potassium persulphate as an oxidising agent. Under the conditions of assay, beta-OHB (0.079-0.395 microM) was instantaneously oxidised to AcAc and the authenticity of the oxidised product was proved by absorption spectroscopy. A common absorption maxima at about 446 nm was observed in all the spectra recorded for the product (AcAc-complex) obtained after the oxidation of beta-OHB (0.079-0.395 microM) to AcAc followed by coupling with diazotized p-nitroaniline. This absorption maxima was almost equal to that obtained for AcAc-complex using AcAc as reference standard. It implies that AcAc formed by the chemical oxidation of beta-OHB is identically similar to the AcAc used as reference standard for the study. This fact was further strengthened when absorption spectra, recorded either individually or in combination (mixed-type), exhibited a single peak with a common absorption maxima at about 446 nm. Absorption spectra was found to be partially diminished by glucose (1.77 microM) and chloride (17.1 microM), while almost complete diminution of absorption spectra was observed at higher concentration of glucose (8.88 microM) and chloride (51.3 microM).