Living in an oxygenated environment has required the evolution of effective cellular strategies to detect and detoxify metabolites of molecular oxygen known as reactive oxygen species. Here we review evidence that the appropriate and inappropriate production of oxidants, together with the ability of organisms to respond to oxidative stress, is intricately connected to ageing and life span.

Oxidants, oxidative stress and the biology of ageing.

http://www.healthcare.uiowa.edu/corefacilities/esr/publications/buettnerpubs/pdf/FRBM-2001-30-1191-FQS-Redox.pdf

Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple.

The present article \"is a progress report rather than a review and in large part summarizes studies from a single laboratory\" on the minimal essential medium for cultivation of mammalian cells in either monolayer or suspension. Every cell culture examined, whether human or animal in origin, required at least 13 amino acids for survival and growth. All the cultured human cells examined were found to contain large amounts of glutathione, taurine, glutamine, ammonia, and glutamic acid. [The SCI® indicates that this paper was cited 2,255 times in the period 1961-1975.]

https://science.sciencemag.org/content/sci/130/3373/432.full.pdf

Amino Acid Metabolism in Mammalian Cell Cultures

Radicals are species containing one or more unpaired electrons. The oxygen radical superoxide (O 2 - ) and the non-radical oxidants hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) are produced during normal metabolism and perform several useful functions. Excessive production of O 2 - and H2O2 can result in tissue damage, which often involves generation of highly reactive hydroxy 1 radical (· OH) and other oxidants in the presence of “catalytic” iron or copper ions. A major form of antioxidant defence is the storage and transport of iron or copper ions in forms that will not catalyze formation of reactive radicals. Tissue injury, e. g., by ischaemia or trauma, can cause increased iron availability and accelerate free radical reactions. This may be especially important in the brain, since areas of this organ are rich in iron and cerebrospinal fluid cannot bind released iron ions. Oxidative stress upon nervous tissue can produce damage by several interacting mechanisms, including rises in intracellular free Ca2+ and, possibly, release of excitatory amino acids. Recent suggestions that free radical reactions are involved in the neurotoxicity of aluminium and in damage to the substantia nigra in Parkinson’s disease are reviewed. Finally, the nature of antioxidants is discussed, with a suggestion that antioxidant enzymes and chelators of iron ions may be more generally useful protective agents than chain-breaking antioxidants. Careful precautions must be taken in the design of antioxidants for therapeutic use.

Reactive Oxygen Species and the Central Nervous System

The human diet contains a great variety of natural mutagens and carcinogens, as well as many natural antimutagens and anticarcinogens. Many of these mutagens and carcinogens may act through the generation of oxygen radicals. Oxygen radicals may also play a major role as endogenous initiators of degenerative processes, such as DNA damage and mutation (and promotion), that may be related to cancer, heart disease, and aging. Dietary intake of natural antioxidants could be an important aspect of the body’s defense mechanism against these agents. Many antioxidants are being identified as anticarcinogens. Characterizing and optimizing such defense systems may be an important part of a strategy of minimizing cancer and other age-related diseases.

https://science.sciencemag.org/content/sci/221/4617/1256.full.pdf

Dietary carcinogens and anticarcinogens Oxygen radicals and degenerative diseases

Biochemistry of the amino acids

Glutathione is translocated out of cells; cells that have membrane-bound gamma-glutamyl transpeptidase can utilize translocated glutathione, whereas glutathione exported from cells that do not have appreciable transpeptidase enters the blood plasma. Glutathione is removed from the plasma by the kidney and other organs that have transpeptidase. Studies in which mice and rats were treated with buthionine sulfoximine, a selective and potent inhibitor of gamma-glutamylcysteine synthetase and therefore of glutathione synthesis, show that glutathione turns over at a significant rate in many tissues, especially kidney, liver, and pancreas; the rate of turnover in mouse skeletal muscle is about 60% of that in the kidney. Experiments on rats surgically deprived of one or both kidneys and treated with the gamma-glutamyl transpeptidase inhibitor D-gamma-glutamyl-(o-carboxy)phenylhydrazide establish that extrarenal gamma-glutamyl transpeptidase activity accounts for the utilization of about one-third of the total blood plasma glutathione. Normal animals treated with the transpeptidase inhibitor excrete large amounts of glutathione in their urine. They also excrete gamma-glutamylcysteine, suggesting that cleavage of glutathione at the cysteinylglycine bond may be of metabolic significance. The present and earlier findings lead to a tentative scheme (presented here) for the metabolism and translocation of glutathione, gamma-glutamyl amino acids, and related compounds.

https://www.pnas.org/content/pnas/76/11/5606.full.pdf

Glutathione: interorgan translocation, turnover, and metabolism.

Evidence is presented that rat kidney contains enzymes that catalyze the synthesis and utilization of glutathione; these reactions, which involve the uptake and release of amino acids from γ-glutamyl linkage, constitute a cyclical process which is termed „the γ-glutamyl cycle.” The γ-glutamyl cycle has properties that fulfill the requirements of an amino acid transport system. Thus, γ-glutamyl transpeptidase may function in translocation and γ-glutamylcysteine synthetase and glutathione synthetase may catalyze energy-requiring „recovery” steps in transport. These and other considerations suggest that glutathione serves a carrier function in amino acid transport.

The γ-Glutamyl Cycle: A Possible Transport System for Amino Acids

Glutathione deficiency induced in newborn rats by giving buthionine sulfoximine, a selective inhibitor of gamma-glutamylcysteine synthetase, led to markedly decreased cerebral cortex glutathione levels and striking enlargement and degeneration of the mitochondria. These effects were prevented by giving glutathione monoethyl ester, which relieved the glutathione deficiency, but such effects were not prevented by giving glutathione, indicating that glutathione is not appreciably taken up by the cerebral cortex. Some of the oxygen used by mitochondria is known to be converted to hydrogen peroxide. We suggest that in glutathione deficiency, hydrogen peroxide accumulates and damages mitochondria. Glutathione, thus, has an essential function in mitochondria under normal physiological conditions. Observations on turnover and utilization of brain glutathione in newborn, preweaning, and adult rats show that (i) some glutathione turns over rapidly (t 1/2, approximately 30 min in adults, approximately 8 min in newborns), (ii) several pools of glutathione probably exist, and (iii) brain utilizes plasma glutathione, probably by gamma-glutamyl transpeptidase-initiated pathways that account for some, but not all, of the turnover; thus, there is recovery or transport of cysteine moieties. These studies provide an animal model for the human diseases involving glutathione deficiency and are relevant to oxidative phenomena that occur in the newborn.

https://www.pnas.org/content/pnas/88/5/1913.full.pdf

Glutathione deficiency leads to mitochondrial damage in brain

Publisher Summary This chapter presents a procedure for the preparation of γ- glutamyl transpeptidase In the assay of γ- glutamyl transpeptidase the most widely used substrate is L-y-glutamyl-p-nitroanilide p -Nitroaniline, released during transpeptidation and hydrolysis, is readily determined from the increase in absorbance at 410 nm A variety of other procedures has been used to determine the activity of γ-glutamyl transpeptidase Other chromogenic substrates include L-γ- glutamylanilide and L-γ-glutamylnaphthylamides Assay procedures in which glutathione is used are complicated by its oxidation to glutathione disulfide; oxidation can be retarded by including ethylenediaminetetraacetic acid in the reaction mixtures Convenient spectrophotometric assays involving the use of S -substituted glutathione derivatives are also available Such derivatives include S -pyruvoylglutathione and S -acetophenoneglutathione  For purification procedures, various methods of solubilization are employed including the treatment of the particulate enzyme with detergents, organic solvents, and proteinases The rat kidney enzyme can be purified following its solubilization with either proteinases (eg, papain and bromelain) or detergents (eg, Triton X-100 and Lubrol WX)

Alton Meister

Papers

Biochemistry of the amino acids

Glutathione: interorgan translocation, turnover, and metabolism.

The γ-Glutamyl Cycle: A Possible Transport System for Amino Acids

Glutathione deficiency leads to mitochondrial damage in brain

Gamma-glutamyl transpeptidase.