24-hour Arterial Stiffness Monitoring in Kidney Transplant Recipients in the Early Postoperative Period.

INTRODUCTION: Laboratory tests and anthropometric assessments are essential in determining the risk for cardiovascular disease in patients after kidney transplantation (KTx). Patients with hypertension and elevated pulse wave velocity (PWV) are at a higher risk of cardiovascular mortality. The purpose of this study was to determine the role of blood pressure, arterial stiffness, and selected laboratory and anthropometric parameters in estimating the risk of cardiovascular disease in KTx patients. METHODS: A total of 17 KTx patients of the Clinical Department of Gastroenterological Surgery and Transplantation at Central Clinical Hospital of Ministry of the Interior and Administration (MSWiA Hospital) in Warsaw, Poland, were enrolled in this study between 3 to 7 days after undergoing kidney transplantation. Medical records of these patients were reviewed for the selected laboratory parameters. The patients' blood pressure and PWV values were monitored for 24 hours and their body mass index (BMI) values were calculated (BMI ≥ 25.0 is considered overweight). RESULTS: Hemoglobin concentration showed a negative correlation with PWV (r = -0.6), whereas red blood cell distribution width (RDW) showed a positive correlation with the PWV value (r = 0.29). There was a significant correlation (r = 0.21) between overweight measured via BMI and the PWV values. For results of kidney function blood tests, the estimated glomerular filtration rate (GFR) and creatinine levels showed no significant correlation with 24-hour PWV values (GFR r = -0.03; creatinine r = 0.03). CONCLUSIONS: The following were shown to be important indices of cardiovascular risk in the evaluated population of KTx patients: age, BMI, blood pressure, PWV, hemoglobin levels, red blood cells, and RDW%.

Comparative Analysis of Arterial Stiffness and Body Composition in Early and Late Periods After Kidney Transplantation.

INTRODUCTION: Diseases of the cardiovascular system are the most common cause of death in patients after kidney transplantation (KTx). Pulse wave velocity (PWV) measurement is a simple, noninvasive, and increasingly popular method to assess arterial stiffness, and thus to assess cardiovascular risk. The aim of the study was to compare arterial stiffness and body composition in patients after KTx in the early and late postoperative periods. METHODS: This research was carried out from January to November 2017 at two locations: (1) Department and Clinic of General and Transplant Surgery and (2) Nephrology and Transplantology Clinic Medical University of Warsaw, the Infant Jesus Teaching Hospital, Warsaw, Poland. The study group consisted of 30 patients in the early postoperative period (2-7 postoperative days) and 151 patients in the late period (6 months to 27 years) after KTx. A single blood pressure measurement, PWV, was performed using a Schiller BR-102 plus PWV. Body composition analysis was performed using a Tanita MC-780 device. RESULTS: The average PWV for patients in the early period after KTx was 8.02 ± 2.21 m/s and in the late period 8.09 ± 1.68 m/s. Positive correlations were found between adipose tissue in the abdominal cavity (R = 0.444, P = .033) and PWV value. There was no correlation between the values of PWV and time after transplantation (R = 0.034, P = .777). Upon analyzing patients after transplantation and taking into account the type of dialysis therapy, lower systolic blood pressure (142 ± 21 mm Hg vs 156 ± 24 mm Hg) and diastolic blood pressure (84 ± 13 mm Hg vs 98 ± 11 mm Hg) values were observed in patients treated with hemodialysis compared with those treated with peritoneal dialysis. CONCLUSION: Using PWV measurement, we found that arterial stiffness levels were similar for early and late periods after transplantation.

A novel real-time genotyping assay for detection of the E6-350G HPV 16 variant.

It has been suggested that some E6 human papillomavirus (HPV) type 16 variants could be involved in viral persistence and progression of HPV infection. A novel one-step allelic discrimination real-time PCR was evaluated for E6-350G variant detection in 102 endocervical HPV 16 positive samples. This assay was also used to assess the distribution of this variant in Spanish women with cervical cancer related to HPV 16. The detection limit for the allelic discrimination assay was 50 copies per reaction, even where the E6-350G variant represents only 20% of the variants in the sample. Complete concordance was observed between DNA sequencing and the novel AD RT-PCR assay. Fourteen E6-350T reference strains and 18 E6-350G variants were detected out of 32 endocervical samples from women with cervical cancer. The average age of women who were infected by the E6-350G HPV 16 variant was 10 years lower in these samples than in women who were infected by the reference strain. This novel allelic discrimination assay is a fast, sensitive and specific method for detection of the E6-350G HPV 16 variant.

A multicentric study to evaluate efficacy and safety of cefetamet pivoxyl in lower respiratory tract infections in Indian patients.

This multicentric, open label, non-comparative study was designed to evaluate the extended spectrum of third generation oral cephalosporin, cefetamet pivoxyl in the treatment of patients with lower respiratory tract infections. This study was conducted among 111 patients with clinical, radiological and bacteriological findings consistent with the diagnosis. After obtaining written informed consent, patients were given cefetamet 500 mg tablet twice a day for 7 days. Cefetamet consistently decreased all clinical signs and symptoms at post-therapy visit. All the treated patients were either cured or improved. Cefetamet was well tolerated with a low incidence of drug related adverse events. The findings of this study indicate that cefetamet pivoxyl was well tolerated and is suitable option for the treatment of patients with lower respiratory tract infection.

Target cell-derived superoxide anions cause efficiency and selectivity of intercellular induction of apoptosis.

Transformed fibroblasts are specifically eliminated by their nontransformed neighbors through intercellular induction of apoptosis. This process depends on the number of nontransformed effector cells and on the local density of transformed target cells. Intercellular signalling is inhibited by SOD (a scavenger of superoxide anions), taurine (a scavenger of HOCl), 4-aminobenzoyl hydrazide (a mechanism-based inhibitor of peroxidase), DMSO (a hydroxyl radical scavenger), and two inhibitors of NO synthase. Therefore, selective apoptosis induction seems to be based on superoxide anion production by transformed cells, their spontaneous dismutation to hydrogen peroxide, and HOCl generation by a novel effector cell-derived peroxidase. HOCl then interacts with target cell-derived superoxide anions to yield hydroxyl radicals. Due to the short diffusion pathway of superoxide anions, hydroxyl radical generation is confined to the intimate vicinity of transformed cells. In parallel, NO derived from effector cells interacts with superoxide anions of target cells to yield the apoptosis inducer peroxynitrite. Reconstitution experiments using transformed or nontransformed cells in conjunction with myeloperoxidase, HOCl, or an NO donor demonstrated that superoxide anions generated extracellularly by transformed cells participate in intercellular signalling and at the same time determine transformed cells as selective targets for intercellular induction of apoptosis.

Transformed target cell-derived superoxide anions drive apoptosis induction by myeloperoxidase.

Myeloperoxidase induces apoptosis in src- or raxs-transformed fibroblasts, but not in parental nontransformed fibroblasts. This selectivity seems to be based on superoxide anion production by transformed cells, a recently described characteristic feature of transformed cells. Myeloperoxidase-mediated apoptosis induction is inhibited by SOD, catalase, 4-aminobenzoyl hydrazide, taurine and DMSO. This pattern of inhibition allows us to conclude that transformed cell derived superoxide anions dismutate to hydrogen peroxide, which fosters HOCl formation by myeloperoxidase. Hydrogen peroxide formation thereby is the rate-limiting step and depends on the cell density. In a second step, HOCl interacts with superoxide anions to yield the highly reactive apoptosis inducing hydroxyl radical. This conclusion was verified through selective apoptosis induction in transformed cells by direct addition of HOCl, which was also inhibited by SOD and DMSO. Our findings demonstrate a specific interplay between target cell derived superoxide anions and MPO during selective apoptosis induction.

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions.

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand - especially in the context of so-called "iron switches" - it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide. Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M(-1)s(-1) for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes. As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called "peroxide tone". Furthermore, it is proposed that - in the low (subtoxic) concentration range - hydroxylated compounds derived from reactions of "non-free" (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of "free" hydroxyl radicals.

Assaying for hydroxyl radicals: hydroxylated terephthalate is a superior fluorescence marker than hydroxylated benzoate.

Generation of hydroxyl radicals in terephthalate (benzene-1,4-dicarboxylic acid) solution yields fluorescent 2-hydroxy-terephthalate. The reaction product is stable for hours and can readily be assessed using standard fluorimeters. The efficiency, i.e. the relative increase of fluorescence per *OH radical, is about three times higher than that of the formation of salicylate (2-hydroxy-benzoate) from benzoic acid and approximately hundred-fold higher than that of the hydroxylation of phenylalanine. As the terephthalate molecule is symmetric with respect to ring-hydroxylation, only one isomer is formed; hence, mechanistic interpretation of the hydroxylation reaction is facilitated. The scavenging rate constant of terephthalate for *OH yielding the hydroxycyclohexadienyl adduct as first intermediate is close to the diffusion controlled limit (k = 3.3 x 10(9) M(-1) s(-1)). Therefore, competition of the detector molecule with biomolecules being present under physiological conditions is expected to be efficient. The assay can be used to detect 'free' *OH radicals produced by the radiolysis of water as well as 'hydroxyl analogous species' that have been suggested to arise from the interaction of complex-bound reduced metal with either oxygen or hydrogen peroxide, e.g. from Fenton reactions. Based on calibration with radiolytically generated hydroxyl radicals the detection limit of the method is estimated to be around 50 nmol/dm3. Terephthalate is classified non-toxic and hence may also prove useful for microdialysis and continuous flow experiments as observation of fluorescence is 'non-destructive' and the reporter substance does not necessarily have to be subjected to HPLC.

Phagocytic killing of microorganisms by radical processes: consequences of the reaction of hydroxyl radicals with chloride yielding chlorine atoms.

Chloride anions and hydrogen peroxide serve as substrates for myeloperoxidase (MPO) in order to produce hypochlorous acid (HOCl) as one of the major killing agents of phagocytic leukocytes. Apart from this role of being a substrate for the MPO-reaction the chloride anion has been considered as unreactive and has not been implicated in radical reactions which contribute to the killing process. From the inherent reactivities of the pertinent radicals (as determined by pulse radiolysis experiments), the great abundance of chloride, and the most probable distribution of reactants within the phagosome, we deduce estimates for the average life-time and free diffusion path-length in this milieu and arrive at a model according to which chloride ions enter into radical chains and influence the killing of ingested bacteria to an extraordinarily high extent. We propose that hydroxyl radicals--despite some controversial arguments in the literature--may still be considered as important contributors to cell killing especially since we show that their reactions are made more effective by producing chlorine radicals in a cyclic process. We furthermore present arguments how the phagocyte may protect itself from harmful actions of HOCl and H2O2 after the superoxide-generating activity of NADPH oxidase is turned off.

Radical functions in vivo: a critical review of current concepts and hypotheses.

Most of the basic knowledge about radical reactions comes from radiation chemical studies in vitro. In view of the rapidly increasing knowledge on radical reaction in vivo, it is important to reconcile the fundamental physico-chemical reaction characteristics of radicals with the need to explain their alleged biological effects. Severe problems in the understanding of their in vivo action remain unsolved. An example is phagocytosis, which seems to be a paradigm of a 'deleterious' radical process. The exact mechanism is not clear; so it is an open question whether the intruder is eventually killed by radicals (like OH) or by endproducts of radical reactions (like H2O2 and/or HOCl). It is even more difficult to understand signalling by radicals: owing to their chemical nature they are 'unspecifically' reacting species--they withdraw or add electrons--and thus their reactions are governed by redox-properties. Since all radicals have different redox characteristics and different molecular shapes, the usual key-and-keyhole picture for molecular interaction does not apply, as there, is no reactive site conceivable which has the property of reacting with radicals 'specifically. Our intent in this article is: (i) to briefly review some fundamental characteristics of in vitro radical reactions, (ii) to extrapolate from this to the conditions in vivo, and (iii) to discuss current hypotheses concerning the redox-regulation of cellular signalling. This leads us to the tentative conclusion that radicals per se must be tolerated by the cell and do not threaten its life, if they stay below a certain concentration limit. The main biological implication of radical-reactions seems to be that the cell derives signals from the balance of oxidative versus reductive processes and that radicals may interact with pathways of intra- and intercellular communication.

Radiation chemistry of physiological saline reinvestigated: evidence that chloride-derived intermediates play a key role in cytotoxicity.

Contrary to common belief, hydrogen peroxide (H2O2) and hypochlorite (HOCl) are not produced continuously and independently during the irradiation of buffer solution containing chloride. Different buildup and decay reactions are involved in a complex interaction of these substances during irradiation. Which of the species predominates is determined by the parameters of the solution. The amount of either compound detectable after irradiation depends on the dissolved gas (O2, N2O or N2), on the pH value and to some extent on the presence of catalytic metals: Under slightly acidic conditions, low oxygen content and high generation rates of OH radicals, the only detectable species is hypochlorite; at high oxygen content and at pH values in the physiological range, hydrogen peroxide is the main detectable product. However, H2O2 and HOCl react with each other in a pH-dependent way, yielding the stable products O2 and Cl-. This reaction limits the expected lifetime of both species in aqueous solution to some tens of seconds. Therefore, analysis of the sample solution after irradiation determines only the substance that was present in greater relative concentration at the termination of irradiation. Such analysis, however, does not allow conclusions about the processes that occurred during irradiation. We have investigated the decay and formation reactions of H2O2 and HOCl under all relevant irradiation conditions and found evidence that the formation and further reaction of HOCl-, the precursor of HOCl, is of central importance even in cases where no significant amounts of H2O2 or HOCl are detectable after irradiation. We discuss the consequences of these results for the cytotoxicity observed after irradiation of cells suspended in physiological saline and conclude that analogous processes must also be relevant for irradiations under in vivo conditions.

Hydrogen peroxide protects yeast cells from inactivation by ionizing radiation: a radiobiological paradox.

PURPOSE: To elucidate mechanisms of the interaction of hydrogen peroxide with chloride-derived cytotoxins under steady-state irradiation conditions and to determine the effects on cell viability. MATERIALS AND METHODS: Yeast cells were suspended in phosphate-buffered saline and exposed to 60Co gamma-irradiation under different conditions. The colony-forming ability was determined. RESULTS: Irradiation of PBS produces H2O2 and HOCl simultaneously. Under slightly acidic conditions and low oxygen tension the yield of HOCl exceeds that of H2O2 while at physiological pH and normoxic conditions H2O2 exceeds HOCl. Both substances react with each other rapidly in a pH-dependent way, even during an irradiation that lasts several seconds. As HOCl is about 1000-fold more toxic than H2O2 to the strain of Saccharomyces cerevisiae used in these experiments, it is evident that in an irradiation that produces more HOCl than H2O2 the radiation-induced damage will be large. If, in contrast, the cells are irradiated under conditions in which H2O2 production predominates, the damage will be small. One would therefore predict that addition of hydrogen peroxide to a cell suspension prior to irradiation should result in protection for suspended cells if H2O2 interferes with the generation of HOCl and thereby inactivates this more powerful toxin. Our data show that addition of H2O2 in sublethal concentration decreases radiation-induced cell death to the level that is found in chloride-free solution, i.e. depending on pH, reduces it by a factor of > or = 3.

Radiation-induced cell killing is highly dependent upon buffer treatment (filtration compared to autoclaving) due to metal-catalyzed formation of hypochlorite: a cautionary note.

Buffer solutions used in experiments in radiation biology may be sterilized by either autoclaving or filtration. We show here that for phosphate-buffered saline such differences in buffer treatment may result in widely differing dose-effect curves for cell killing. The temperature-dependent transformation of monophosphate ions into di- or polyphosphate evidently proceeds to an appreciable extent upon autoclaving the buffers at 120 degrees C for 10 to 20 min. This increases the capability of the buffer to chelate spurious metal contaminations and, as a consequence, to reduce the amount of cytotoxic hypochlorite being produced. Depending on conditions of buffer treatment we have observed dose modification factors for the colony-forming ability of yeast cells up to the order of 3. Thus effects due to buffer treatment might easily outweigh the effect which the experiment was originally designed to determine. We strongly advise, therefore, that results of parallel sets of experiments in which different methods of buffer sterilization have been used should not be compared directly.

Kinetic and spectroscopic studies on a superoxide dismutase from Propionibacterium shermanii that is active with iron or manganese: pH-dependence.

Kinetic studies were performed on the superoxide dismutases isolated from the anaerobic bacterium Propionibacterium shermanii as active enzymes with either iron or manganese, which were naturally incorporated into the same molecule depending on the metal supply. Both the Fe- and Mn- forms showed decreasing activity with increasing pH. This suggests the protonation of some groups near the metal, possibly a metal-bound water molecule. Thus the kinetic behaviour of this superoxide dismutase is much more dependent on the protein structure than on the metal incorporated into the active site. The secondary structures of both forms were not influenced by variations in pH, whereas the EPR spectra of the Fe-superoxide dismutase changed as a function of pH. The EPR spectra apparently consist of two overlapping species. Steady-state experiments proved that all iron-containing species show catalytic activity, but the species predominating in the alkaline pH range displays a lower reaction rate. The Michaelis constant and maximal turnover number for the Fe-superoxide dismutase were determined polarographically as Km = 0.54 mmol/l and Vmax. = 2000 mol.s-1 at pH 9.5. These data indicate that, in anaerobic bacteria under physiological conditions, the superoxide dismutase is not saturable with O2-. and the catalytic activity is similar to that of metal-specific Fe- or Mn-superoxide dismutases from aerobic organisms.

Reactions of hydrogen peroxide with superoxide dismutase from Propionibacterium shermanii--an enzyme which is equally active with iron or manganese--are independent of the prosthetic metal.

Propionibacterium shermanii contains a single constitutive superoxide dismutase (SOD) which is active with either iron or manganese incorporated in the same protein moiety. Copper and cobalt can also be incorporated by the bacteria in the active center of the SOD under conditions of metal deficiency, but in this case the enzyme is enzymatically inactive. In contrast to other bacterial SODs, the Fe-SOD of P. shermanii remains highly resistant to inactivation by hydrogen peroxide, as does Mn-SOD. Both SOD types cannot be distinguished by their inactivation patterns. Incubation with hydrogen peroxide results in a concentration- and time-dependent decrease in tryptophan fluorescence, independent of the metal present in the active center. Moreover, the Fe-SOD shows a time-dependent decrease in spin concentration after addition of hydrogen peroxide, which reflects alterations in the environment of the metal rather than a reduction of Fe3+ to Fe2+. No obvious correlations exist, however, between these effects and the enzymatic activity of the enzyme. The resistance of the SODs from P. shermanii to inactivation by hydrogen peroxide seems to be caused by the fact that a tryptophan residue near the metal-chelating histidine-75--which is present in all Fe-SODs being rapidly inactivated by this agent--is exchanged for valine.

Signalling by O2-. and NO.: how far can either radical, or any specific reaction product, transmit a message under in vivo conditions?

With regard to the stability of the NO. radical as a chemical entity, it is without doubt able to serve as an intra- as well as an intercellular messenger. The radical O2-., in contrast, does not seem to be suited to far-range signalling in the vascular system. Its short chemical half-life, which is limited by the presence of various reactive blood constituents to below 50 ms, results in a free diffusion path length of less than 40 microns, i.e. only the distance between just a few cells. While accelerated 'downstream' transport by arterial blood may help to extend the action sphere, there is no possibility for O2-. to serve as a signal in an upstream direction. The estimates presented, however, do not invalidate arguments for a possible role of superoxide anions in intra- or pericellular signalling phenomena. Cross-talk between NO.- and O2-.-dependent signal routes, e.g. by peroxynitrite formation, is unlikely to be a relevant process under the conditions which prevail in the vascular system.

On the cytotoxicity of irradiated media. To what extent are stable products of radial chain reactions in physiological saline responsible for cell death?

In a previous publication (Czapski et al. 1992) we reported that HOCl accounts for the toxicity of irradiated phosphate-buffered saline towards Escherichia coli bacterial cells. We have now investigated the respective toxicities towards lambda phage and mammalian cells. For phage, as with bacteria, cytotoxicity of the irradiated media seems to derive from HOCl without detectable contribution of H2O2. Mammalian cells (V79 CHO), in contrast, are more sensitive to H2O2 than to HOCl. Both agents, however, are not able to account quantitatively for the toxicity of irradiated solutions towards V79 cells; a hitherto unidentified chlorine/oxygen derivative--being formed in the sub-micromolar concentration range--is suggested to be responsible for toxicity in the case of eukaryotes.

Radical intermediates during the oxidation of nitropropanes. The formation of NO2 from 2-nitropropane, its reactivity with nucleosides, and implications for the genotoxicity of 2-nitropropane.

The chemistry of the nonenzymatic oxidation of the rat liver carcinogen, 2-nitropropane, and its anionic form, propane-2-nitronate, was investigated using pulse radiolysis and EPR/spin trapping with 3,5-dibromo-4-nitrosobenzenesulfonic acid as the trapping agent. The results suggest that, following initial oxidation to a secondary alkyl radical, propane-2-nitronate is effectively degraded in a peroxidative chain reaction with the intermediary formation of peroxyl and NO2.radicals. The latter radical was shown to react appreciably fast with ribonucleosides, deoxyribonucleosides, and guanosine nucleotides. It is proposed that nonenzymatic formation of NO2.radicals after enzymatic oxidation of propane-2-nitronate to the corresponding secondary alkyl radical accounts for the induction of DNA damage observed after exposure of rats to 2-nitropropane.