In 1963, Cohn and Lyle (1) reported in an abstract that reduced glutathione (GSH) reacts with o-phthalaldehyde (OPT) at pH 8 to give a highly fluorescent compound under conditions such that none of a wide variety of other compounds (histamine, histidine, and many sulfur-and nonsulfurcontaining amino acids and peptides) gives appreciable fluorescence. In 1966, they published a more detailed description of a method of using this reaction for quantitative estimation of GSH in tissue extracts (2). The present paper is a report of investigations in this laboratory, based on Cohn and Lyle's original abstract, which partially duplicates their recently published findings and indicates some special precautions required in analyzing GSH in tissue extracts by a method similar to theirs.
Methods
Measurements of fluorescence were made with an Aminco-Bowman spectrophotofluorometer, model 4-8202, or with the same instrument equipped with an off-axis ellipsoidal mirror, providing increased sensitivity. All fluorescence wavelengths referred to herein are stated in terms of the original factory calibration of this instrument, not corrected by us to any wavelength standard. A Beckman DB speetrophotometer was employed for all extinction measurements.
Glass-distilled water was used in all aqueous solutions. GSH standards were prepared from crystalline reduced glutathione obtained from Sigma Chemical Company. OPT from Mann Research Laboratories and from K&K Laboratories gave satisfactory results without further purification. Aldrich Chemical Company was the source of 5,5'-dithiobis-2nitrobenzoic acid (BIS) for use in Elhnan's method for nonprotein sulfhydryl (NPSH) (3). All other chemicals were reagent grade, selected, when possible, from lots with minimal heavy metal contamination. Disodium EDTA was used at 0.001 M concentration in all buffers, tissue precipitants, and GSH solu-1 Supported by Public Health Service Grants 05981-05 and 05981-06.