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Sensitive Enzymatic Cycling Assay for Glutathione: Measurements of Glutathione Content and Its Modulation by Buthionine Sulfoximine in Vivo and in Vitro in Human Colon Cancer¹

Departments of Medicine [S. J. B., D. G., E. Z., J. K. V. W., N. A. B.] and Biochemistry [S. J. B., N. A. B.] and Ireland Cancer Center, University Hospitals of Cleveland, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4937

We have measured glutathione content in small tissue samples derived from biopsies of primary and metastatic human colon tumors and from colon cancer cell lines in tissue culture and xenografts in athymic mice. Measurements were performed using an enzymatic cycling assay designed to quantitate extremely low levels of glutathione (GSH) (down to 10^-14 mol) from perchlorate extracts of tissue samples weighing less than 1 mg wet weight. Glutathione was stable in these acid extracts for at least 6 months when stored at -80°C. A survey of normal tissues in mice, rats, and some human tissues showed considerable variation in GSH content of different tissues but generally similar levels were identifiable for the same tissues from different species. The highest GSH level was 56.9 nmol/mg protein in rat liver and the lowest was 1.8 nmol/mg protein in rat skeletal muscle. High GSH levels were also determined in mouse and human liver, while low GSH levels were detected in mouse muscle. Human colon cancer cell lines showed slightly higher GSH levels than did colon cancer tumor samples obtained from biopsies. These studies revealed a marked inter-individual difference in tumor GSH content, as well as a difference in GSH content between tumor deposits at different metastatic sites in the same individual. These results indicate the importance of direct tumor measurements of GSH content in clinical trials designed to modulate tumor glutathione content to try to increase sensitivity to chemotherapy or radiation therapy. Buthionine sulfoximine, an inhibitor of -glutamyl cysteine synthetase, was shown to produce almost complete depletion of GSH in four different human colon cancer cell lines in 24 h. Buthionine sulfoximine was also shown to be capable of producing drastic depletion of GSH in human colon cancer grown as xenografts in athymic animals.

¹ This work was supported in part by National Cancer Institute Grants P01 CA51183 and P30 CA43703.

² To whom requests for reprints should be addressed, at Department of Medicine, Case Western Reserve University, Biomedical Research Building, 3 West, 10900 Euclid Avenue, Cleveland, OH 44106-4937.

Received 2/16/94. Accepted 6/ 1/94.

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