What is oxidative damage? Please, 3Q.

Glutathione is a small molecular tripeptide compound widely existing in cells. Participating in the regulation of intracellular amino acid transport, glucose metabolism and DNA synthesis plays an important role in antagonizing exogenous poisons and oxygen free radical damage, regulating immune function, maintaining the structure and function of protein cells, and inhibiting apoptosis [1]. Over the years, people have tried to evaluate the damage of oxygen free radical lipid peroxidation according to the degree of GSH redox, and made a lot of progress. 1. General physiological characteristics of glutathione Glutathione is composed of glutamic acid, cysteine and glycine, which is a kind of compound with the richest sulfhydryl group in cells, and -SH on the α amino group of cysteine is the active center of this kind of molecular compound. The concentration of GSH in different organs is different, the liver is the highest, followed by spleen, kidney, lung, brain, heart, pancreas and bone marrow, and the blood is the lowest [2]; The GSH concentration in different parts of the same organ is also different, and the GSH content in different organelles in the same cell is also different. Glutathione oxidized form (GSSG) is an oxidized form of glutathione, which is oxidized to GSSG by glutathione peroxidase (GSH-Px) under the action of oxidant. The latter is the hydrogen provided by NADPH, which is reduced to GSH under the action of GSH-Rx, forming a dynamic balance, keeping GSSG at the level of 65,438+0% ~ 65,438+00% of the total GSH [3], forming an effective antioxidant system. Under physiological conditions, the ratio of GSH/GSSG is maintained at a high level, but under oxidative stress, GSH is oxidized to GSSG, and the ratio of GSH/GSSG decreases, so it can be used to evaluate lipid peroxidation damage. GSH is mainly synthesized in the liver, and its synthesis is not only related to the contents of cysteine and NADPH, but also plays an important role in the synthesis of rate-limiting enzyme glutathione synthase (GCS). GCS has two subunits, GCLR and GCLS, in which GCLR is the main unit and GCLS plays a regulatory role. In the oxidation state, GCLR mRNA is highly expressed in a dose-dependent manner, but the effect of low-level glutathione on GCLR mRNA is not obvious. On the contrary, after GSH-Rx is inhibited, the mRNA of GCLR is highly expressed, suggesting that GSSG can regulate GCLR and then feed back GSH level [4]. The clearance of GSH is mainly in the kidney, accounting for 50% ~ 65% of the total blood circulation. 80% GSH and GSSG in bilateral renal arteries are filtered after one renal cycle, but can be reabsorbed by Na+-dependent GSH enzyme transport system. Second, GSH antagonizes the mechanism of free radical lipid peroxidation damage. GSH plays an important role in the fight against antioxidant poisons. On the one hand, it can react with toxic molecules and their metabolites to reduce the toxicity of poisons; On the other hand, redox reaction can reduce the peroxide ability of poisons, so that sulfhydryl-containing enzymes are not activated by heavy metals and oxidants, or the oxidized sulfhydryl-containing enzymes are reduced to restore their activities. When a large number of free radicals are produced, unsaturated fatty acids in cell membranes are oxidized into lipid peroxides, causing a series of secondary injuries. GSH can directly antagonize the toxicity of oxygen free radicals by providing H+, terminate the chain reaction, and be oxidized into GSSG. Similarly, GSH also plays an important role in resisting oxygen free radical peroxidation and inhibiting cell apoptosis, necrosis and homeostasis caused by it. GSH/GSSG has many metabolic pathways in the body. In the oxidation state, GSH is oxidized to GSSG on the one hand, which shows that GSH/GSSG decreases correspondingly; On the other hand, GSH reacts with exogenous poisons and their metabolites, and finally generates thiosemicarbazone, which is excreted with urine. At this time, only the corresponding GSH decreased, but the change of GSSG may not be great, and even GSSG decreased due to GSH consumption [5]. GSSG can be reduced to GSH or combined with GST (glutathione -S- transferase). 3. Factors affecting GSH level in vivo Because GSH in blood mainly comes from organs such as liver, GSH level in plasma is undoubtedly an ideal index to indirectly reflect GSH level in liver, kidney and other related organs. At present, the determination is still in the stage of animal experiments, and there are few reports about GSH concentration in human blood. Recently, it was reported that the concentration of GSH in human blood was1.0000 0.167 (715 cases) [1]. In many pathological conditions, such as diabetes, alcoholic liver disease, liver cirrhosis and peroxidation caused by exogenous poisons, GSH levels decrease. Recently, it was found that GSH in patients with AIDS, Parkinson's disease, aging and hypoxemia also decreased, and the health status of the elderly with low GSH was worse than that of the elderly with high GSH. Hagg et al. reported that the GSH level of men was higher than that of women, that of vegetarians was higher than that of non-vegetarians, that of the elderly decreased significantly, and that of blacks was higher than that of whites [1, 6]. In addition, people also found that GSH level is related to physical activity and nutritional level. Interestingly, the GSH of smokers is higher than that of non-smokers, which may be the adaptive adjustment response of the body to the chronic oxidation state caused by long-term smoking, further suggesting that GSH plays an important role in the anti-oxidation process in the body. 4. Changes of GSH/GSSG under lipid peroxidation induced by different chemical poisons. In vitro experiments showed that GSH did not decrease but increased when cells were exposed to toxic substances with sub-toxic concentration. Cookson et al. [7] cultured glial cells with sub-toxic concentrations of trimethyltin and triethyltin for 24 hours, and the GSH in cells increased significantly; When Ochi [8] China hamster V79 cells were incubated with sub-toxic concentration of arsenic * * *, it was found that GSH increased the most at 8 hours, and then decreased. In addition, a large number of experiments show that exposure to toxic concentrations of lead, mercury, methylmercury and other compounds also increases GSH [9], suggesting that this may be a protective mechanism adopted by cells during stress, similar to the increase of GSH caused by long-term smoking. Palmeira et al. [10] cultured male Wistar rat hepatocytes in vitro with different toxic concentrations of herbicide paraquat and 2,4-d * *, and detected GSH/GSSG by Hissin enzyme chemistry method. The results showed that GSH concentration decreased with the extension of contact time, and reached the lowest level after incubation for 2 ~ 3h hours, and there was a dose-response relationship, while GSSG increased proportionally. Lora et al. incubated male Fischer344 rats with different concentrations of dichloroethyl nitrosourea, and detected GSH/GSSG level by RP-HPLC, and got the same conclusion [1 1]. In vivo experiments, the situation is much more complicated. Stone et al. [12] exposed mice to different concentrations of vitamin K3(0, 30, 60 and 100 μmol/L), and determined GSH/GSSG by Hissin-Hilf enzyme chemistry method. The results showed that the decrease of reduced glutathione and the increase of GSSG were dose-dependent, but the decrease of reduced glutathione was disproportionate to the increase of GSSG. When GSH value (μmol/g) decreased from1.45 0.28 to 0.57 0.07 (61%), the increase of GSSG was only 10% of GSH loss. Male rats were exposed to methanol (3.0 g/kg and 6.0 g/kg), and GSH/GSSG of hepatocytes, erythrocytes and plasma were determined at different times. The results showed that GSH reached the lowest level at +0.2 hours, decreased from 4.4 μmol/g to 3.4 μ mol/g (P < 0.05), and then slowly recovered. The activities of GSH-Px, GSH-Rx and GSH also increased and decreased synchronously, but the change of GSSG was not obvious, which may be due to the direct combination of GSH and newly generated formaldehyde without generating GSSG [13]. Another experiment showed that due to the combination of GSH and poisons, the production of GSSG also decreased, and the ratio of GSH/GSSG increased [5, 13]. Some experiments show that the change of GSSG in peripheral blood is more sensitive than GSH, which may be related to the different levels of GSH and GSSG released to the extracellular after cell injury. Navarro et al. [14] irradiated 1 mice with high-energy X-rays, and measured GSH/GSSG in their blood at different times. In addition, when patients with breast cancer and lung cancer receive different doses of radiotherapy, the above indexes are also determined by HPLC. The results showed that the change of GSH concentration in blood of mice was not significant, GSSG increased and GSH/GSSG decreased in a dose-and time-dependent manner, especially within 2 hours. The changes of liver, heart and pancreas in rats were also obvious, and GSH decreased statistically. The results of tumor radiotherapy patients also showed that the level of GSSG in peripheral blood increased with the increase of radiotherapy accumulation, which was statistically significant, but the change of GSH was not significant. However, it is necessary to pay attention to the participation of GSH/GSSG system when evaluating lipid peroxidation damage with this index. Lii et al [15] used male SD rats, which were exposed to paraquat (20 and 40 g/kg) and diquat (85 and 190 mg/kg) respectively. The results showed that the changes of GSH and GSSG in hepatocytes and their ratio were not statistically significant, but at this time, the detection of thiobarbituric acid, the product of oxygen free radical lipid peroxidation, showed that hepatocytes were seriously damaged by oxygen free radical lipid peroxidation. Using GSH/GSSG as an index to evaluate lipid peroxidation damage, many experimental results are quite different, which may be related to the following factors: 1. Determination method: There are many methods to detect GSH/GSSG, which can be summarized into two main categories: enzyme chemistry method and HPLC method. The former mainly reflects the level of GSH/GSSG indirectly according to the change of enzyme metabolism kinetics during GSH redox process, such as the degree to which GSH is oxidized by dithio-p-dinitrobenzene to nitrosobenzoic acid. HPLC method can directly determine GSH/GSSG according to the experimental spectrum, which has high sensitivity and specificity, but the operation is complicated. The results of the two methods are mostly consistent [1, 15], while Floreani and others [16] think that HPLC is more sensitive than chemical methods 10 times. The results of the two methods are significantly different, but they all face the same problems, such as the intracellular separation and purification of GSH/GSSG. 2. Quality control: GSH is in contact with air and can be rapidly oxidized and consumed without stabilizer; Its stability is different at different temperatures. For example, GSH can be stored for at least 3 weeks by freezing at -70℃ immediately after blood samples are obtained, but it gradually degrades at -20 ~ 4℃. After adding stabilizer, it can be stored at -20℃ for 1 year and at room temperature for 1 day. Therefore, the pretreatment of samples has a direct and very important influence on the measurement results. 3. Multi-antioxidant system of the body and multi-pathways of GSH/GSSG metabolism: For example, the body protects cells through protein -S- glutathionylation and carbonic anhydrase III, and GSH/GSSG system does not participate in antioxidant metabolism at this time; GSH/GSSG defense system is restricted by many enzyme activities in physiological state, and the changes of enzyme activities in pathological state will also affect GSH/GSSG level. Glutathione and GSSG have many metabolic pathways. If the GSSG does not increase, it can reflect the low level of lipid peroxidation or the low content of GSH, but it may also be because GSH directly combines with poisons without forming GSSG, or the newly generated GSSG has a new binding reaction. To sum up, GSH/GSSG can be used as a sensitive index to evaluate the lipid peroxidation damage caused by oxygen free radicals, and can be used to explore the toxic mechanism of poisons, but it is still necessary to comprehensively analyze GSH/GSSG according to the types, concentrations and action time of poisons. In addition, the study of GSH/GSSG as an index of oxygen free radical damage is still in the stage of animal experiments, and there are few studies on people, so the improvement of its specificity is still a problem to be solved. There is still a long way to go before it can be used to monitor people or evaluate patients with clinical poisoning. 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