Investigating the In vitro Metabolism of Propylene Glycol in Human Liver Cytosol

Abstract

Background: Propylene glycol (PG; 1,2-propylene glycol) is widely used as a pharmaceutical excipient. High plasma levels of PG have been linked to saturable metabolism of PG and have been reported to result in adverse effects such as hypotension and metabolic acidosis. PG is metabolised by the alcohol dehydrogenase (ADH) pathway in the liver and is also renally excreted. In vitro characterisation of PG metabolism is essential in understanding and mitigating high plasma levels in vivo. Aims and Objectives: This project aims to investigate the efficiency and speed of PG metabolism in human liver cytosol (HLC) through the ADH pathway. The primary objective of this study is to determine critical kinetic parameters, such as Km and Vmax values, which will provide insights into the efficiency and capacity of PG metabolism via ADH in HLC. Method: The study involved preparing different concentrations of PG through serial dilutions from 1 M PG. We prepare a NAD+ stock solution by combining NAD+ powder with deionized water (DIW). We also developed a Reaction Mix (RM) with precise additives and meticulous temperature control. These solutions are employed in various tests, including spectrophotometry and gas chromatography-mass spectrometry (GC-MS) analysis. Spectrophotometry involves placing the samples in a 96-microplate, adding diluted PG GM in each well, and measuring the absorbance at 340 nm. We assess ADH enzyme activity using conventional methods. While for assessing its pharmacokinetic properties, we measure the rate of NADH reaction in cytosolic protein. We employ PG diluents and GM in GC-MS analysis, prepare samples at specific times, and collect aliquots for additional testing following incubation to determine PG pharmacokinetic properties by measuring the PG rate of reaction per cytosolic protein. We employ tools such as Microsoft Excel and GraphPad Prism and particular formulae to measure ADH activity in HLC for statistical analysis. Results: Spectral analysis of ADH activity unveiled an ADH activity of 0.16 U/mg of cytosolic protein, with a Vmax measurement of 2.34 µmole/min/mg and a Km measurement of 23.71 mM for cytoplasmic proteins. Furthermore, GC-MS analysis delved into the kinetics of PG disappearance rate, exposing a Vmax value of 67.62 µmole/min/mg of cytosolic protein of cytosolic protein and a Km value of 4.101 mM. This investigation illuminated the intricacies of enzymatic dynamics and the intricate interplay between ADH and PG.