Study On The Redox State Of Human Body

Human body was continuously attacked by endogenous and exogenous reactive oxygen species (ROS), but under the protective effect of the affluent antioxidant defense system, the balance of pro-oxidant and antioxidant was maintained in a stable redox state (REDST), to keep the homeostasis of the body. When the balance of pro-oxidant and antioxidant system was disrupted, the oxidative or reductive shift of the redox state was occurred. If the shift of redox state exceeded a certain threshold, it would produce oxidative or reductive damage on the body by redox sensitive mechanism, and induce various kinds of disease related. Currently, the oxidative damage was evaluated with the changes of content of antioxidant and the increase of the oxidative metabolic products of proteinum, lipid and DNA, and there was not any available method to evaluate the redox state of the human body. As an important endogenous antioxidant, GSH is the most important redox buffer. GSH participated in antioxidant defense, and composed a consummate redox network with reduced form of nicotinamide-adenine dinucleotide phosphate, ascorbic acid, tocopherol(s), thioredoxin and other antioxidant enzymes related. Therefore we did some works on these issues, especially emphasizedthe changes of redox state of GSH/GSSG and NADPH/NADP+ redox couplesin blood plasma in the normal and various morbid state.Firstly, based on the specific property of GSH molecule, which can combine with o-phthalaldehyde to form a fluorescent compound we established a new experiment condition to measure the free glutathione of both reduced and oxidized (i.e. GSH and GSSG) in plasma with fluorophotometry. The GSSG molecule can be reduced to GSH by the dithiothreitol, and the primary amines and thiol group contained in GSH molecule can combine with o-phthalaldehyde to form a fluorescent conjugation product at pH 8.0, and then the content of free GSH and GSSG in blood plasma can be measured with a spectrofluorophotometric detector. This is a simple method with great sensitivity and selectivity for rapid measuring GSH and GSSG in human blood plasma simultaneously.Secondly, we revised the spectrophotometry for measuring the NADPH and NADP+ in serum built in our laboratory. With the help of phenazine methosulfate (PMS), the methyl thiazolyl tetrazolium (MTT) can be reduced to form formazan by the NADPH, which was oxidized to form NADP+, and the NADP+ can be reduced to form NADPH with the reducing equivalent produced in the dehydrogenation reaction of glucose-6-phosphate (G6P) catalyzed by glucose-6-phosphate dehydrogenase (G6PD), the production rate of formazan is proportion to the content of NADP. As there is the maximal absorbance for formazan in 570nm, the content of NADPH and NADP+ can be measured the change rate of the absorbance for formazan with spectrophotometer. As NADP+ can be destructed in hot alkali conditions, but the NADPH can maintain intact, we can measure the content of NADPH andNADP+ in blood plasma with or without heating respectively. This is a simple method with great sensitivity and selectivity for rapid measuring NADPH and NADP+ in human blood plasma simultaneously.Finally, the content of free GSH, GSSG, NADPH and NADP+ in blood plasma of 120 healthy adults, 74 various kinds of cancer patients, 48 cases of chronic obstructive pulmonary disease (COPD) patients and 68 cases of acute pancreatitis (AP) patients was measured with methods mentioned above. In addition, the redox potential of GSH/GSSG redox couple and NADPH/NADP+ redox couple (i.e. E^gsh/gsso and Eh(NADPH/NADP+)) in the blood plasma were calculated with the Nernst equation. Results were showed as follow. No significant differences of the content of gluthione, glutathione disulfide, NADPH and NADP+ and the value of Eh(GSH/GssG) and Eh(nadph/nadp+) in blood plasma were found between male and female healthy of the same age group (P > 0.05). Before 40 years old, the content of gluthione, glutathione disulfide, NADPH and NADP+ and the value of Eh(GSH/GSSG) and Eh(nadph/nadp+) in blood plasma was kept in a stable level, without significant difference between 20-29 and 30-39 years group. There was no significant difference of the content of glutathione disulfide and NADP+ among all different age groups (P > 0:05). But 40 years later, the content of glutathione and NADPH decreased and the value of Eh(GSH/GSSG), Eh(NADPH/NADP+) increased significantly (P < 0.05), the older, the lower of the content of glutathione and NADPH, and the higher of the value of Eh(GSH/GSSG) and Eh(NADPH/NADP+)- Compared with the healthy before 40 years old, the content of glutathione and NADPH in blood plasma of cancer patients decreased, the content of glutathione disulfide and NADP+ and the value of Eh(GSH/GssG) and Eh(NADPH/NADP+) of cancer patients increasedsignificantly (P < 0.05). Compared with the healthy 40 years later, there was no significant difference of the content of glutathione and NADPH and the value of Eh(NADPH/NADP+) of cancer patients, but the content of glutathione disulfide and NADP+ and the value of E^gsh/gssg) increased significantly (P < 0.05). The content of glutathione of digestive system cancer patients was lower and the value of E^gsh/gssg) was higher than that of respiration system cancer and other kinds of cancer patients, but there was no significant difference of NADPH content and Ei,(nadph/nadp+) value (P > 0.05). The content of glutathione and NADPH in blood plasma of COPD patients was significant lower than that of young healthy, and approximate to that of old healthy whose age was matched to the COPD patients, but the value of Eh(GSH/ossG) and Eh(NADPH/NADP+) was significant higher than that of all young and old healthy, became more oxidative. The content of glutathione and NADPH in blood plasma of acute exacerbated COPD patients was lower than that of stable COPD patients, but without significant difference. Compared with the stable COPD patients, the content of glutathione disulfide and NADP+ and the value of Eh(GSH/GSSG) and Eh(nadph/nadp+) was higher significantly, and became more oxidative. Compared with the healthy, the glutathione content of AP patients was lower, the glutathione disulfide and NADP+ content and the Eh(NADPH/NADP+) and Eh(GSH/GssG) value higher significantly, and become more oxidative, but without significant difference of NADP+ content. There was no significant difference of glutathione disulfide and NADPH between minor severe acute pancreatitis (MAP) and severe acute pancreatitis (SAP) patients, but compared with MAP patients, the glutathione content of SAP was lower, the NADP+ higher, and the value higher significantly, and became moreoxidative.Conclusions: 1. We established a new experiment condition to measure the free glutathione of both reduced and oxidized (i.e. GSH and GSSG) with fluorophotometry, which was high sensitive and selective in measuring GSH and GSSG in human blood plasma simultaneously. 2. We revised the the spectrophotometry for measuring the NADPH and NADP+ in serum built in our laboratory to measure the NADPH and NADP+ in blood plasma, which was more concise with high accurate and sensitive. 3. With the autogenous regulation, the balance of pro-oxidant and antioxidant system of the human body of different age grades was maintained in different relative stable levels. Before 40 years, the redox state of human body was kept stable, but 40 years later, the antioxidant capacity decreased and the redox state became more and more oxidized with the increase of age, which suggested that at the age of 40 years old more or less, it might be the reversal point of human body to begin aging continuously. 4. The antioxidant capacity decreased and the redox state of various kinds of cancer patients, COPD and AP patients became oxidized significantly than the healthy. Although there existed constant oxidative stress in stable COPD patients, the redox state of COPD patients were kept as the same as that of the age-matched healthy individuals. And the redox state of the acute exacerbated COPD patients became more oxidative than that of the stable COPD patients. The antioxidant capacity of severe acute pancreastitis patients was lower and the redox state became more oxidative than that of the minor acute pancreasstitis patients. These suggested that the extent of oxidative shift of redox state of patients was correlated with the severity of the pathogenetic condition.In a word, with the autogenous regulation, the redox state of human body was maintained in different stable state in different physiological and pathological condition. In all acute and chronic pathological processes and aging process, the antioxidant capacity decreased and oxidative shift of the redox state occurred, which was correlated with the severity of the pathogenetic condition. And the redox state might be judged with the integration of the redox state of GSH/GSSG couple and NADPH/NADP+ couple, which might be a useful method to measure the prooxidant-antioxidant imbalance in clinic.