Influence of protein binding on acrolein turnover in vitro by oxazaphosphorines and liver microsomes.
For a correct determination of acrolein amounts generated in in vitro turnover experiments with oxazaphosphorines, it is necessary to characterize the interaction of acrolein with liver microsomal proteins. Acrolein, a highly reactive metabolite of oxazaphosphorines, readily forms covalent adducts with proteins by electrophilic attack on nucleophiles, such as the sulfhydryl group of cysteine, imidazole group of histidine, and amino group of lysine. The current investigations were mainly directed toward determination of the degree of acrolein-protein binding under conditions of in vitro experiments with liver microsome preparations. The acrolein concentration in protein dilution was determined by a fluorescence method. Moreover, the influence of sucrose and glycerine on the extent of acrolein-protein binding commonly used for the stabilization of microsomal preparations during storage was investigated. The current investigations show evidence that the chemical reaction of acrolein with liver microsomal proteins strictly follows first order kinetics. The main part of the formed acrolein in the in vitro attempts is available as bound part. Results of these investigations indicate that the calibration should be carried out with mixtures from liver microsome preparations and known amounts of acrolein under the same conditions as the in vitro experiments to record the entirely formed acrolein part (free and bound) in oxazaphosphorine turnover experiments. Glycerine is recommended as a preservative to store liver microsomes instead of sucrose because the latter reacts with acrolein.
Baumann
Frank
F
Institute of Clinical Pharmacology, University Leipzig, Germany. baumf@medizin.uni-leipzig.de
Schmidt
Renate
R
Teichert
Jens
J
Preiss
Rainer
R
D000171:Acrolein / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism; D001711:Biotransformation; D003520:Cyclophosphamide / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry; D008862:Microsomes, Liver / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013395:Sucrose / Q000737:chemistry
D018906:Antineoplastic Agents, Alkylating; D011506:Proteins; D013395:Sucrose; D000171:Acrolein; D003520:Cyclophosphamide; D005990:Glycerol