Original Articles
Clinical Evidence of Peroxynitrite Formation in Chronic Renal Failure Patients with Septic Shock

https://doi.org/10.1016/S0891-5849(96)00401-7Get rights and content

Abstract

The production of both nitric oxide (NO) and superoxide increases in septic shock. The cogeneration of these molecules is known to yield peroxynitrite, which preferentially nitrates tyrosine residues of protein and nonprotein origins. We present evidence of peroxynitrite production in septic shock by measuring plasma nitrotyrosine. The nitrotyrosine was measured by an HPLC C-18 reverse-phase column and ultraviolet detector in chronic renal failure patients with or without septic shock, and in healthy volunteers. Plasma nitrite+nitrate (NOx) was also measured to evaluate NO production. Nitrotyrosine was selected as an index for production of peroxynitrite because the direct measurement of peroxynitrite in vivo is difficult. Patients with renal failure were selected in order to minimize nitrotyrosine excretion through the kidney. Plasma nitrotyrosine levels were not detectable in volunteers, 28.0 ± 12.3 μM (1.6 ± 1.1% of total tyrosine) in renal failure patients without septic shock, and 118.2 ± 22.0 μM (5.5 ± 1.2% of total tyrosine) in patients with septic shock. NOx levels were also higher in patients with septic shock than in patients without septic shock (173.9 ± 104.7 vs. 75.6 ± 19.1 μM). Although renal failure itself increases plasma concentrations of both molecules, the higher levels in patients with septic shock suggest that peroxynitrite is generated and the nitration of tyrosine residues is increased in this disease. Copyright © 1997 Elsevier Science Inc.

Introduction

There is an increasing body of evidence showing that in septic shock nitric oxide synthase is rapidly induced, leading to the excessive production of nitric oxide (NO), a potent vasodilator. Thus, NO is proposed as a major molecule responsible for circulatory shock.1, 2, 3On the other hand, abundant evidence supports the notion that in septic condition superoxide (O2radical dot) production is augmented.[4]Under circumstances where both NO and O2radical dot are produced, they react with each other at a diffusion limited rate to form peroxynitrite (ONOO).[5]ONOO is a potent oxidant which by nitrating cellular constituents[6]may play an important role in tissue damage of rheumatic arthritis,[7]acute lung injury,[8]and myocyte injury.[9]Although it is plausible that enhanced production of both NO and O2radical dot in sepsis may lead to the formation of ONOO, investigations that relate ONOO with septic shock have not been undertaken. Here we present evidence of ONOO production in septic shock by measuring plasma nitrotyrosine, which is a nitrated product of tyrosine in blood or tissue and whose presence infers ONOO attack on tyrosine residues of protein and nonprotein origins. The reason to select nitrotyrosine as an index for production of ONOO is based on the fact that direct measurement of ONOO in vivo is difficult because of its very short half-life.[5]The investigation was focused on patients with chronic renal failure who were complicated by septic shock, because nitrotyrosine was shown to be rapidly excreted in the urine.[10]Patients with chronic renal failure without septic shock and healthy volunteers served as comparison. Plasma nitrite+nitrate (NOx), an end product of NO synthesis, was also measured to provide actual evidence for the increased production of NO.

Section snippets

Patients and Methods

Ten healthy volunteers were members of the hospital staff (6 men and 4 women, mean age 36.7 ± 10.0 years). Ten patients with chronic renal failure without sepsis (8 men and 2 women, mean age 52.9 ± 17.2 years) were consecutively selected from routine hemodialysis sessions. They gave their informed consent. Three patients with chronic renal failure with septic shock were a 72-year-old woman with sepsis induced by diverticulitis, a 68-year-old woman who developed sepsis during a course of

Results

Fig. 1A demonstrates the chromatogram of plasma from the patient with chronic renal failure and septic shock. Well-defined peaks at the retention time of tyrosine and nitrotyrosine were observed. The peak assigned to nitrotyrosine was eliminated with Na2S2O4 treatment, which confirmed that the peak is attributable to nitrotyrosine (Fig. 1B). In plasma of healthy volunteers, nitrotyrosine was not detectable and NOx was 28.7 ± 11.6 μM (Fig. 2). Renal failure patients without septic shock

Discussion

Our results show that plasma nitrotyrosine and NOx were increased in renal failure patients and the increases were more marked in patients complicated by septic shock. The rise in concentrations of both nitrotyrosine and NOx implies either an increased production of these molecules and/or their increased accumulation due to impaired excretion because both molecules are mainly eliminated through the kidney.[10]Although the dietary nitrate is known to influence the plasma NOx level, it is less

Acknowledgements

We thank Dr. Dennis J. Stuehr, Cleveland Clinic Foundation Research Institute for helpful discussion and peer review. This work was supported in part by Grand-in-Aid for International Scientific Research Program (No. 05045052) and Developmental Scientific Research (No. 07557006) from Ministry of Education, Science and Culture of Japan.

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