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R Palombari

Publications and source records attributed to R Palombari.

7 recordsLinked to original sources
PubMed2003

Electrochemical assay of human haemoglobin S-nitrosylation by nitrosocysteine.

Oxyhaemoglobin (oxyHb) and methaemoglobin (metHb) react with S-nitrosocysteine (CysNO) to form nitroso derivatives. We test this reaction with a new method for evaluating transnitrosation reaction. The assay exploits an amperometric sensor developed in our laboratory. The results we obtain are in good agreement with those reported by others, although at much higher sensitivity, indicating the suitability of the method for examining high-mass nitroso compounds. The S-nitrosylation of oxyHb at a CysNO/haem ratio of 1 : 1 is about 5% in 60 min. In the same experimental conditions, the nitrosylation of met-Hb reaches 25%. OxyHb and metHb derivatize by 50% in 60 min upon using a CysNO/haem ratio of 10 : 1. The oxidation of haem iron occurs at ratios of haem/CysNO of 1 : 5 or higher. We conclude that CysNO transfers NO(+) both to metHb and oxyHb. We propose that NO transfer in RBC may occur through transnitrosation reactions between high and low-mass nitrosothiols.

Cysteine↗
PubMed2003

NO synthesis in human saliva.

Human saliva contains nitrate that is converted into nitrite by the activity of facultative, anaerobic bacteria of the oral cavity. Nitrite can be reduced to NO in the acidic gastric milieu; some NO may also form in the mouth at acidic pH values. In this paper, we show that bacteria (S. salivarius, S. mitis and S. bovis) isolated from saliva, may contribute to NO production in human saliva. NO formation by bacteria occurs at neutral pH values and may contribute to the antibacterial activity of saliva.

Adult↗
PubMed2001

Reaction mechanism between nitric oxide and glutathione mediated by Fe(III) myoglobin.

Ferrimyoglobin at pH 7.4 binds nitric oxide to yield nitric oxide adducts. In the presence of glutathione (GSH), nitrosoadducts of Mb(III) react with it to give nitrosoglutathione, whose concentration has been determined with an apparatus based on a specific and sensitive solid-state amperometric gas sensor. The reaction constant between the adduct and glutathione, kGSH = (47 +/- 1) M(-1) x s(-1), obtained by UV-Vis spectroscopy kinetic measurements, is about one-eighth of the constant with OH- determined by other authors. We can explain this fact with the higher nucleophilicity of OH- compared to GSH, due to the bulkiness and charge of the species. It is known that the formation of nitrosothiols starting from nitrite or NO (nitrogen monoxide) and glutathione, in the absence of oxygen, is impossible. Thus, from a biological point of view, it is important to point out that GSH reacts with NO in the presence of ferrimyoglobin, even at physiological pH, to form nitrosoglutathione.

Glutathione↗
PubMed2001

[The nitric oxide metabolism in the hypoxic, ischemic and reperfused human skeletal muscle cell: clinical and therapeutical observations].

BACKGROUND: The biochemical and metabolic role played by nitric oxide (NO) in course of oxidative stress due to cell hypoxia, ischemia and reperfusion has a determinant relevance in the mitochondrial adaptive changes which antagonize the irreversible morpho-functional damage. In particular conditions, such as in prolonged ischemia and/or exogenous NO supplementation, this element is present in the radical form (NOO*) concurring to peroxidative cell injury. Aim of this study was to investigate these opposite NO aspects in hypoxic, ischemic and reperfused human skeletal muscle tissue. METHODS: Skeletal muscle samples were taken during elective knee orthopedic surgery in 10 consecutive patients. The biopsies were obtained before, after 5+/-1 min and 58+/-2 min from tourniquet application and then after 18+/-3 min following muscle reperfusion. The samples, immediately frozen in liquid nitrogen, were assayed for endocellular free NO following the gas-amperometric method described by Palmerini C. RESULTS: When compared with normoxic tissues, a significant decrease in free NO content was observed in hypoxic samples. After about 60 min of prolonged ischemia the NO levels show an evident increase, while the tissue reperfusion leads to a progressive restoration of physiological content in the cellular free nitric oxide. CONCLUSIONS: The obtained data in hypoxic muscle cell seem to underline the pivotal role played by NO in adapting the cytochrome c oxidase oxidative activity to lower O2 bio-availability. On the other hand the prolonged ischemia leads to a consistent NOO* generation triggered by oxyradical generation and Ca2+ intracellular over load. Even if the tissue reoxygenation restores the normal NO levels it is arguable that the pre-treatment of ischemic cell with antioxidants, Ca-antagonist and Dexamethasone supplementation could represent a crucial and specific therapeutic approach to critically ill patient.

Adult↗
PubMed2000

Determination of S-nitrosohemoglobin using a solid-state amperometric sensor.

Nitric oxide (NO, nitrogen monoxide), generated in biological systems, plays important roles as a regulatory molecule. Its ability to bind to hemoglobin (Hb) iron is well known. Moreover, it may lose an electron, forming the nitrosonium ion, involved in the synthesis of nitrosothiols (RSNO). It has been suggested that S-nitrosohemoglobin (SNO-Hb) may act as a reservoir of NO. The S-nitrosylation of Hb can be detected after the incubation of CysNO and Hb for 60 min with a molecular ratio (CysNO/hem) of 1:1. Upon increasing the ratio to 10:1, about 50% of total Hb (100% of beta-chain -SH 93) was derivatized in 60 min. In this paper, we describe a new method for the quantitative assay of SNO-Hb, after the liberation of NO by Cu(2+)/Cu(+) and the simultaneous assessment of NO by solid-state amperometric sensor. The assay described by us is sensitive, rapid, easy to perform, and inexpensive. For this reason, we believe that it may represent an important analytical improvement for the study of the S-transnitrosylation reactions between RSNO and the Hb Cys-beta 93 and SNO-Hb and glutathione.

Biosensing Techniques↗
PubMed1998

A new assay for the determination of low-molecular-weight nitrosothiols (nitrosoglutathione), NO, and nitrites by using a specific and sensitive solid-state amperometric gas sensor.

Nitric oxide (NO) is generated in biological systems and plays an important role as a bioregulatory molecule. Its ability to bind hemoglobin and myoglobin is well known. Moreover, it may lose an electron forming the nitrosyl group involved in the formation of S-nitrosothiols. The main problem in analyzing NO is its extreme reactivity. We have tackled this task by using an amperometric sensor to determine free NO, S-nitrosothiols (such as S-nitrosoglutathione), and nitrite in cell-free systems and murine microglial cell cultures. The determination of nitrosothiols is of biochemical relevance and a difficult task particularly at low concentration values. In this article we describe a new method based on the reductive cleavage of the S-NO bond by cuprous ions followed by a solid-state amperometric determination. The system described by us is sensitive, rapid, does not require previous purification steps, is easy to perform, and is inexpensive. For this reason, we think that it may represent an important analytical improvement. It has been suggested that nitrosothiols may exert biological activity by acting as a reservoir of NO. We tested the production of nitrite and of RSNO in stimulated, cultured murine microglial cells and we have shown that nitrite accumulates in these conditions. GSNO also accumulates, provided that GSH is present in the medium.

Animals↗