β nicotinamide adenine dinucleotide lithium salt from saccharomyces cerevisiae

An enzymatic assay using a standard curve of ethanol concentration was utilized to determine BECs (Jung and Ferard, 1978 (link)). Briefly, ethanol standard samples were prepared by mixing absolute ethanol and ultrapure water to yield a 1000 mg/dL ethanol stock solution. This solution was serially diluted to yield a set of eight standard samples in the range of 7.8–1000 mg/dL ethanol. Forty microliters of 3.75% perchloric acid was added to 10 μL of samples (serum) and standards and centrifuged for 6 minutes at 2,000 RPM. The supernatant was retained. β-Nicotinamide adenine dinucleotide lithium salt from Saccharomyces cerevisiae (Sigma #N7132) was added at a final concentration of 2.5 mM, and Alcohol Dehydrogenase from Saccharomyces cerevisiae (Sigma #A7011) was added at a final concentration of 5 μM to samples and standards, and incubated at 35 °C for 40 minutes. Samples and standards were read on a plate reader at 340 nm. In a representative DID experiment, BEC for the EtOH+Veh group was 90±20.5 mg/dL, while the BEC for the EtOH+ABT group was 37±21.9 mg/dL.