Luigi Cucchiarini

Simultaneous extraction and reverse-phase high-performance liquid chromatographic determination of adenine and pyridine nucleotides in human red blood cells☆

A simple and rapid method for the determination of ATP, ADP, AMP, NADP+, NAD+, NADPH, and NADH in human erythrocytes is described. A single-step extraction procedure employing alkaline medium and CF 50A Amicon ultrafiltration membranes allows a simultaneous and total recovery of the compounds of interest. Analysis is performed by reverse-phase high-performance liquid chromatography on a 5-micron Supelcosil LC-18 column and uv detection. Extraction and analysis require about 30 min. Levels of adenine and pyridine nucleotides in normal adults are also presented.

A very fast ion-pair reversed-phase HPLC method for the separation of the most significant nucleotides and their degradation products in human red blood cells

A simple and fast ion pair reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of ATP, ADP, AMP, GTP, GDP, IMP, NADP+, NADPH+, NAD+, NADH, ADP-ribose, inosine, adenosine, hypoxanthine, and xanthine. This method allows us to have a complete picture of the most important nucleotides present in fresh human erythrocytes. Furthermore it is particularly useful in the study of the erythrocyte adenine nucleotide catabolism allowing the detection of degradation products such as IMP, inosine, adenosine, hypoxanthine, and xanthine. The separation of the compounds under investigation is achieved in less than 15 min using a reversed-phase 3-micron Supelcosil LC-18 column and adding tetrabutylammonium, as ion-pair agent, to the buffers. The short time of analysis, the high reproducibility of the system, and the accurate evaluation of the compounds of interest make this method particularly suitable for routine analysis. Finally it is possible to use this assay as an alternative method of measuring activities of enzymes which catalyze reactions involving some of these compounds, as in the case of Na+-K+ ATPase, AMP deaminase, and adenosine deaminase.

Quercetin prevents glutathione depletion induced by dehydroascorbic acid in rabbit red blood cells

Exposure of rabbit red blood cells to dehydroascorbic acid (DHA) caused a significant decline in glutathione content which was largely prevented by quercetin, whereas it was insensitive to various antioxidants, iron chelators or scavengers of reactive oxygen species. This response was not mediated by chemical reduction of either extracellular DHA or intracellular glutathione disulfide. In addition, the flavonoid did not affect the uptake of DHA or its reduction to ascorbic acid. Rather, quercetin appeared to specifically stimulate downstream events promoting GSH formation.

Reversed-phase high-performance liquid chromatography separation of dimethylaminoazobenzene sulfonyl- and dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the picomole level

A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of cysteic acid, S-sulfocysteine, hydroxyproline, taurine, norleucine, cystine, and delta-hydroxylysine. The precolumn derivatization of amino acids with dimethylaminoazobenzene sulfonyl chloride (DABS-Cl) is simple and quick (10 min at 70 degrees C) and allows the complete reaction of primary and secondary amino acids. The separation of the compounds under investigation is achieved in 25 min using a reversed-phase 3-microns Supelcosil LC-18 column at room temperature. The versatility of the proposed method is documented by amino acid determination on protein samples obtained using different hydrolysis techniques (HCl, methane-sulfonic acid, and NaOH), with attention given to the detection of tryptophan in protein samples with high sugar concentration. Furthermore, we have reported the experimental conditions necessary to apply this method to the amino acid analysis of very low amount of proteins (1 to 5 micrograms) electroeluted from a stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stability of DABS-derivatives, the short time of analysis, the high reproducibility and sensitivity of the system, and the complete resolution of all compounds of interest make this method suitable for routine analysis. Furthermore, we have also developed a fast reversed-phase high-performance liquid chromatographic method for the complete separation of dimethylaminoazobenzene thiohydantoin (DABTH)-amino acids. The separation of the compounds under investigation is obtained, at room temperature, in less than 18 min using a reversed-phase Supelcosil LC-18 DB column, 3-micron particles, and also allows the complete separation of DABTH-Ile, DABTH-Leu, and DABTH-Norleu. The short time of analysis, together with the high reproducibility of the system and its sensitivity at picomole levels, make this method very suitable for the identification of DABTH-amino acids released during microsequencing studies of proteins and peptides with the dimethylaminoazobenzene isothiocyanate reagent. In addition, we have shown that it is possible to obtain complete separation of DABTH-amino acids also under isocratic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Rhodiola Rosea as antioxidant in red blood cells: ultrastructural and hemolytic behaviour

Rhodiola rosea L. (Crassulaceae) is a plant that lives at high altitude in Europe and Asia, widely used for its high capacity to increase the organism resistance to different stress conditions. Although a few international literature supports these effects, today R. rosea has become a common component of many dietary supplements also in the Western world. The aim of the present study was to investigate the effect of the R. rosea roots aqueous extract on in vitro human erythrocytes exposed to hypochlorous acid (HOCl)-oxidative stress. Several damages occur in human erythrocytes exposed in vitro to HOCl, among these membrane protein and lipid modifications, shifting from the discocyte shape to the echinocyte one, and determining lysis ultimately. Therefore, in the present work, the evaluation of the antioxidant capacity of the Rhodiola extract has been carried out by means of scanning electron microscopy and of hemolytic behaviour on human erythrocytes exposed to HOCl in the presence of increasing doses of the aqueous extract in different experimental environments (co-incubation and subsequent incubations). The results obtained are consistent with a significant protection of the extract in presence of the oxidative agent, but a cautionary note emerges from the analysis of the data related to the cell exposition to the plant extract in the absence of any induced oxidative stress. In fact, the addition to erythrocyte of high doses of R. rosea extract always determines severe alterations of the cell shape.

Effect of age on some properties of mice erythrocytes

The hematological parameters of young (2-month-old) and old (2-year-old) mice were compared. No differences could be detected with the exception of an increased percentage of reticulocytes in the old animals suggesting that anemia in senescent mice does not occur. Red blood cell mean half-life in old mice was 8 +/- 0.8 days compared to 12 +/- 1 days in young mice. This reduced survival of red blood cell is not due to a different rate of cell phagocytosis in the reticulohistiocytic system of young and old animals since erythrocytes from young mice have the same mean half-life when injected both in young and old animals and vice versa. Thus, the old mice have a reduced red cell life-span but the same hematocrit of the young, suggesting that old animals possess a chronologically younger population of erythrocytes than do young animals. This has been confirmed by measuring the specific activities of some red blood cell age-dependent enzymes (hexokinase, glucose-6-phosphate dehydrogenase, pyruvate kinase) that were found to be higher in the older animals, and by the separation of erythrocytes into different density (age) groups by Percoll/albumin density gradient centrifugation. However, the erythrocytes osmotic fragility, and the cellular contents of adenine and pyridine nucleotides, as well as the content of 2,3-diphosphoglycerate and reduced glutathione, show that circulating erythrocytes in old animals constitute an heterogeneous cell population whose properties cannot be explained on the basis of a chronologically younger erythrocyte population. Furthermore, evaluation of cell components in hemopoietic tissues have shown an increased porportion of erythroid precursor cells in old animals confirming that old mice compensate for reduced red cell survival with an increased erythropoiesis.

Complete high-performance liquid chromatographic separation of 4-N,N-dimethylaminoazobenzene-4′-thiohydantoin and 4-dimethylaminoazobenzene-4′-sulphonyl chloride amino acids utilizing the same reversed-phase column at room temperature

Abstract Reversed-phase high-performance liquid chromatographic methods for the complete separation of all 4-dimethylaminoazobenzene-4′-sulphonyl chloride and 4-N,N-dimethylaminoazobenzene-4′-thiohydantoin amino acids on the same Supelcosil LC-18 column at room temperature are described. The procedures are simple and reproducible, and the systems are easily interconvertible. The use of a fixed-wavelength detector at 436 nm permits amino acid analysis at levels lower than 1 pmol with a stable baseline.

Rhodiola rosea ability to enrich cellular antioxidant defences of cultured human keratinocytes

Keratinocytes are cells strongly exposed to oxidative stress, but normally good equipped for antioxidant responses. However, it has long been suggested that exogenous antioxidants could play a useful role in minimizing the adverse skin responses associated with such oxidant species. In this work it was paid attention to the extract of Rhodiola rosea L. roots by using the phytocomplex as a whole because of the important activity of its composition and mutual distribution of its components. We have measured the protection afforded by the extract to reduced glutathione levels, glyceraldehyde-3-phosphate dehydrogenase activity, and thiobarbituric acid reactive substances levels in cultured human keratinocytes (NCTC 2544) exposed to different oxidative insults: Fe(II)/ascorbate, Fe(II)/H2O2, and tert-butyl-hydroperoxide. We also have investigated the influence of the R. rosea extract on the production of intracellular reactive oxygen species and on the activity of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase). Furthermore, we have demonstrated that R. rosea extract was able to increase in a time- and dose-dependent manner the activity of the trans plasma membrane oxido reductase activity as an indirect evaluation of the intracellular redox status and this effect was already evident with small concentration of the extract and in a long time. As a result, NCTC 2544 are able to better counteract to several oxidative insults if incubated with R. rosea extract demonstrating a very good antioxidant activity of this phytocomplex.

Redox and Energetic State of Red Blood Cells in G6PD Deficiency, Heterozygous β-Thalassemia and the Combination of Both

The levels of ATP, ADP, AMP, NADP, NADPH, NAD, NADH and reduced glutathione were determined in the red blood cells of individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, beta-thalassemia (beta-thal) heterozygotes and in a boy carrying both mutations. The results obtained confirmed a reduced concentration of NADPH in G6PD deficiency and showed that with the combination of both diseases, the red blood cell contained practically undetectable levels of NADPH. Assays of some red blood cell enzyme activities known to be markedly influenced by cell age suggested that a younger mean red cell population is present in beta-thal/G6PD deficiency. Thus, the marked oxidative stress caused by beta-thal, that is apparently incompatible with G6PD deficiency, in fact exists, probably because of the residual activity of this enzyme in the younger red cells.

High resolution of multiple forms of rabbit reticulocyte hexokinase type I by hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) has been employed extensively in the separation of proteins by elution using a descending salt gradient, with and without the use of detergents or denaturing agents. In this study, a new hydrophobic interaction chromatographic support, Toyopearl Phenyl 650 S, was investigated in order to examine the distribution of multiple forms of rabbit reticulocyte hexokinase type I. These distinct forms of the enzyme, designated hexokinase Ia, Ia* and Ib, show similar kinetic and physical properties, similar molecular masses (ca. 100,000) and a different intracellular distribution. The results obtained using Toyopearl Phenyl 650 S of 20-50-microns particle diameter show that this HIC support allows very high resolution, comparable to that obtainable with HIC-HPLC columns but with the advantage of charging a higher amount of starting material even with a high protein concentration. These characteristics render Toyopearl Phenyl 650 S suitable for analytical and preparative purposes. Further, in the separation of multiple forms of rabbit reticulocyte hexokinase, the HIC method was shown to be superior to RP-HPLC, making possible the efficient separation of proteins with high molecular mass and their recovery in active forms. The Toyopearl Phenyl 650 S column was also shown to be more efficient than the ion-exchange chromatographic media previously used, allowing a quicker analysis of the multiple forms of rabbit reticulocyte hexokinase under different biological conditions.