N-acetyl Taurine Research Articles

Simultaneous determination of four ethanol biomarkers in blood: avalidated method for use in forensic toxicology in 257 postmortem samples

Purpose: Ethyl alcohol is the most widely and legally available intoxicating substance. However, excessive consumption is associated with numerous negative social consequences, including the potential for significant health risks. Rapid and simple diagnosis of alcohol use is necessary to initiate appropriate and effective treatment and is critical in forensic toxicological analysis. Ethanol biomarkers have clinical utility in the detection, diagnosis, and treatment of alcohol use disorders. Materials and Methods: An analytical method for the simultaneous determination of four different alcohol biomarkers ethyl glucuronide (EtG), ethyl sulphate (EtS), N-acetyltaurine (NAcT), and 16:0/18:1-phosphatidyl ethanol (PEth) in blood samples from forensic cases was developed, validated and verified for the accurate monitoring of alcohol abuse and dependence. Analyses were performed using a liquid chromatography-mass spectrometry system. Results: 257 blood and vitreous samples were collected from 255 male and 2 female subjects aged 16-80 years (average 44±14.82) from the Forensic Medicine Council of Turkey and analyzed for ethanol biomarkers and calculated ethanol concentrations. A total of 257 blood samples were found to contain ethanol, with concentrations ranging from 12.0 to 444.0 mg/dL. Vitreous concentrations ranged from 23 mg/dL to 597 mg/dL. The limit of detection (LOD) for EtG, EtS, NAcT, and PEth were 3.1, 3.9, 7.3, and 5.7 ng/mL respectively in blood samples. The limit of quantification (LOQ) for EtG, EtS, NAcT, and PEth were 7.8, 8.4, 18.3, and 13.1 ng/mL, respectively in blood samples. Conclusion: The method has potential in forensic toxicology as an invaluable tool for the accurate and simultaneous identification of biomarkers of alcohol use and dependence in different biological samples.

Characterization of Urinary N-Acetyltaurine as a Biomarker of Hyperacetatemia in Mice.

Acetate is an important metabolite in metabolic fluxes. Its presence in biological entities originates from both exogenous inputs and endogenous metabolism. Because the change in blood acetate level has been associated with both beneficial and adverse health outcomes, blood acetate analysis has been used to monitor the systemic status of acetate turnover. The present study examined the use of urinary N-acetyltaurine (NAT) as a marker to reflect the hyperacetatemic status of mice from exogenous inputs and endogenous metabolism, including triacetin dosing, ethanol dosing, and streptozotocin-induced diabetes. The results showed that triacetin dosing increased serum acetate and urinary NAT but not other N-acetylated amino acids in urine. The co-occurrences of increased serum acetate and elevated urinary NAT were also observed in both ethanol dosing and streptozotocin-induced diabetes. Furthermore, the renal cortex was determined as an active site for NAT synthesis. Overall, urinary NAT behaved as an effective marker of hyperacetatemia in three experimental mouse models, warranting further investigation into its application in humans.

Characterization of Urinary N-acetyltaurine as a Biomarker of Serum Acetate in Experimental Animal Models of Hyperacetatemia

Abstract Objectives Acetate is an intermediate metabolite originated from multiple important metabolic pathways. Even though blood acetate level has been associated with many health events, it is not commonly monitored in clinical practice, partially due to the needs for invasive blood collection and the challenges in acetate analysis. N-acetyltaurine (NAT) was earlier identified as a novel urinary metabolite of ethanol from the reaction between taurine and ethanol-derived acetate. As a direct metabolite of acetate, NAT has the potential to function as a urinary biomarker that reflects the acetate level inside the body. To test this hypothesis, this study examined the correlations between serum acetate level and urinary NAT level in three experimental animal models of hyperacetatemia. Methods Glycerol-triacetate (GTA) dosing, ethanol dosing, and streptozotocin (STZ)-induced Type 1 diabetes were used to achieve hyperacetatemia in mice. In GTA model, serum samples were collected at 2 h and urine samples were collected for 24 h after dosing the mice with 5.8 g/kg GTA. In ethanol dosing, serum were collected at 2 h after intraperitoneal injection of 4 g/kg ethanol, while 24 h urine samples were collected before and after 14-day feeding of modified semi-solid diet containing 2.2%–6.7% (v/v) ethanol. In the Type I diabetes model, urine and serum samples were collected before and 5 days after the intraperitoneal injection of 180 mg/kg STZ. The concentrations of NAT and creatinine in urine, as well as acetate in serum, were measured using their respective liquid chromatography-mass spectrometry (LC-MS) methods. Results The occurrence of hyperacetatemia in three animal models was confirmed by the clear elevation of serum acetate concentrations. The concentrations of urinary NAT were also dramatically increased in three animal models, suggesting the correlations between serum acetate and urinary NAT. Conclusions Urinary NAT is an effective metabolic marker of hyperacetatemia in three experimental models. The results warrant further investigation on its application in other pathophysiological conditions and in humans. Funding Sources This research was partially supported by the Agricultural Experiment Station project MIN-18–125 (C. C.) from the United States Department of Agriculture (USDA).

Increased N-Acetyltaurine in the Skeletal Muscle After Endurance Exercise in Rat.

Taurine is metabolized to a novel metabolite, N-acetyltaurine (NAT), through N-acetylation with acetate. Furthermore, NAT production increases when the endogenous production of acetate is elevated in some situations, such as alcohol catabolism and endurance exercise. We have previously reported that both the serum concentration and urinary excretion of NAT from humans were increased after endurance exercise, and that NAT was secreted by cultured skeletal muscle cells exposed to both acetate and taurine. The present study evaluated the hypothesis that NAT is synthesized in the skeletal muscle after endurance exercise. Normal rats were loaded to a transient treadmill running until exhaustion. Serum, skeletal muscle, and liver were collected immediately after the exercise. The NAT concentration in the plasma and in the soleus muscle from the exercised rats was significantly increased compared to that in the samples from the sedentary control rats. There was a significant positive correlation in the NAT concentration between the plasma and soleus muscle. The NAT concentration in the liver was unchanged after the endurance exercise. These results confirm that the significantly increased NAT in both the serum and urine after endurance exercise is derived from NAT synthesis in the skeletal muscle.

Simultaneous Determination of N-Acetyl Cysteine and Taurine by HPTLC Method in Active Pharmaceutical Ingredient and Pharmaceutical Dosage Form

Specific, precise and sensitive TLC-Densitometric method was developed and validated for the simultaneous estimation of N-Acetyl cysteine and Taurine in active pharmaceutical ingredient and pharmaceutical dosage form. An effective separation was achieved on pre-coated silica gel HPTLC plates by using n-butanol:acetic acid:water (8:0.5:1.5 v/v/v). The spots were scanned densitometrically at 295 nm. The RF values of N-Acetyl cysteine and Taurine were found to be 0.29 and 0.52, respectively. Calibration curves were linear in the range of 30 - 180 and 100 - 600 ng/band for N-Acetyl cysteine and Taurine, correspondingly with correlation coefficients of 0.999. The developed method was validated as per ICH guidelines. The limits of detection were 11.24 and 63.40 ng/spot for NAC and TAU respectively. The method developed was found to be precise and specific for the simultaneous analysis of N-Acetyl cysteine and Taurine in pure and tablet dosage form.

N-Acetyltaurine as a novel urinary ethanol marker in a drinking study.

The forensic utility of N-acetyltaurine (NAcT) in urine as a marker for ethanol intake was examined. A HILIC-based liquid chromatography method for the mass spectrometric determination of NAcT, taurine, and creatinine in urine was developed and validated to investigate NAcT formation and elimination in a drinking study. Thereby, eight subjects ingested 0.66 to 0.84g/kg alcohol to reach a blood alcohol concentration (BAC) of 0.8g/kg. Blood and urine were taken every 1.5-2h, during the first 8h. NAcT and taurine levels were measured and corrected for the urine's dilution by normalization to a creatinine concentration of 1mg/mL. For the determination of NAcT and taurine, uncorrected lower limits of quantitation (LLOQs) were at 0.05μg/mL of urine. In the drinking study, NAcT proved to be an endogenous compound, which is present at a range of 1.0 to 2.3μg/mL in urine of alcohol-abstinent subjects. Maximum NAcT concentrations were reached in samples taken 3 to 6h after the start of drinking, whereby an upregulation in N-acetyltaurine could be found for all the subjects. The mean peak concentrations (c̅ max) of 14 ± 2.6μg/mL (range 9-17.5μg/mL) were reached. Within 24h, the NAcT levels declined to endogenous concentrations. The detectability of NAcT was found to be slightly shifted compared to BAC: When BAC was not detectable anymore, NAcT levels were still elevated. After 24h, when ethyl glucuronide (EtG) and ethyl sulfate (EtS) were still detectable, NAcT concentrations showed endogenous levels again. Positive NAcT results can be used as an indicator for recent alcohol consumption. A direct relationship between NAcT and taurine concentrations could not be found. Graphical abstract N-acetyltaurine concentrations for eight subjects during the first 24 h after an alcohol consumption of 0.8 g/kg.

Identification of N‐acetyltaurine as a novel metabolite of ethanol through metabolomics‐guided biochemical analysis

The influence of ethanol on small‐molecule metabolome and the role of CYP2E1 in ethanol‐induced hepatotoxicity were investigated using liquid chromatography‐mass spectrometry (LC‐MS) based metabolomics platform and Cyp2e1‐null mouse model. Histological and biochemical examinations of ethanol‐exposed mice indicated that the Cyp2e1‐null mice were more resistant to ethanol‐induced hepatic steatosis and transaminase activity than the wild‐type mice, suggesting CYP2E1 contributes to ethanol‐induced toxicity. Metabolomic analysis of urinary metabolites revealed time‐ and dose‐dependent changes in the chemical composition of urine. N‐acetyltaurine (NAT) was identified as a urinary metabolite that is highly responsive to ethanol exposure. Stable isotope labelling analysis determined that NAT, with its acetyl group originated from ethanol metabolites, is a novel metabolite of ethanol, and NAT biosynthesis during ethanol treatment is contributed by both endogenous metabolism and ethanol metabolism. In vitro incubation assays showed that both acetyl‐CoA and acetate can react with taurine to form NAT. However, the enzymatic reaction between taurine and acetate is much more relevant to the metabolite concentrations in vivo than the reaction between taurine and acetyl‐CoA, indicating the former reaction is the main route of NAT biosynthesis in vivo. Furthermore, the contribution of acetate to NAT biosynthesis was confirmed by the immediate presence and increase of deuterated NAT after feeding deuterated acetate. The incubations of tissues and cellular fractions with taurine and acetate indicated that the kidney has the highest NAT synthase activity among tested organs while the cytosol is the main site of NAT biosynthesis inside the cell. Overall, the combination of biochemical and metabolomic analysis revealed NAT is a novel metabolite of ethanol and a potential biomarker of hyperacetatemia.Grant Funding Source: UMN startup fund for Chi Chen

Identification of N-Acetyltaurine as a Novel Metabolite of Ethanol through Metabolomics-guided Biochemical Analysis

The influence of ethanol on the small molecule metabolome and the role of CYP2E1 in ethanol-induced hepatotoxicity were investigated using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics platform and Cyp2e1-null mouse model. Histological and biochemical examinations of ethanol-exposed mice indicated that the Cyp2e1-null mice were more resistant to ethanol-induced hepatic steatosis and transaminase leakage than the wild-type mice, suggesting CYP2E1 contributes to ethanol-induced toxicity. Metabolomic analysis of urinary metabolites revealed time- and dose-dependent changes in the chemical composition of urine. Along with ethyl glucuronide and ethyl sulfate, N-acetyltaurine (NAT) was identified as a urinary metabolite that is highly responsive to ethanol exposure and is correlated with the presence of CYP2E1. Subsequent stable isotope labeling analysis using deuterated ethanol determined that NAT is a novel metabolite of ethanol. Among three possible substrates of NAT biosynthesis (taurine, acetyl-CoA, and acetate), the level of taurine was significantly reduced, whereas the levels of acetyl-CoA and acetate were dramatically increased after ethanol exposure. In vitro incubation assays suggested that acetate is the main precursor of NAT, which was further confirmed by the stable isotope labeling analysis using deuterated acetate. The incubations of tissues and cellular fractions with taurine and acetate indicated that the kidney has the highest NAT synthase activity among the tested organs, whereas the cytosol is the main site of NAT biosynthesis inside the cell. Overall, the combination of biochemical and metabolomic analysis revealed NAT is a novel metabolite of ethanol and a potential biomarker of hyperacetatemia.