Ohvanesse G. Ekindjian

Is α-ketoisocaproyl-glutamine a suitable glutamine precursor to sustain fibroblast growth?

Abstract Background: Glutamine is considered an essential nutrient for cellular growth. Aim: To test the suitability of α-ketoisocaproyl-Gln (Kic-Gin) as a new glutamine (Gin) precursor to sustain human fibroblast growth. Methods: [3H] thymidine uptake into cellular DNA of human fibroblasts. Extracellular and intracellular aminoacid patterns were determined with peptides and acylated compounds. Results: L-alanyl-L-glutamine (used here as a recognized Gin precursor) promoted DNA synthesis, whileN-acetyl-L-glutamine (used here as a negative control since it is known to be a poor Gin precursor) and α-ketoisocaproyl-glutamine had no effect. Alanyl-glutamine progressively gave rise to free glutamine in the growth medium. In contrast, glutaminesupplied in acylated form was poorly available and did not appear in free form in the medium. In addition, only alanyl-glutamine increased intracellular glutamine and glutamate levels. In contrast,Kic-Gln was able to sustain net protein synthesis as judged by total protein content and reduced intracellular levels of most essential amino acids. Conclusion: Kic-Gln appears to be a poor extra-cellular precursor of Gin to sustain cell growth.

Nifedipine decreases sVCAM-1 concentrations and oxidative stress in systemic sclerosis but does not affect the concentrations of vascular endothelial growth factor or its soluble receptor 1

Microvascular injury, oxidative stress, and impaired angiogenesis are prominent features of systemic sclerosis (SSc). We compared serum markers of these phenomena at baseline and after treatment with nifedipine in SSc patients. Forty successive SSc patients were compared with 20 matched healthy subjects. All SSc patients stopped taking calcium-channel blockers 72 hours before measurements. Twenty SSc patients were also examined after 14 days of treatment with nifedipine (60 mg/day). Quantitative ELISA was used to measure the serum concentrations of vascular endothelial growth factor (VEGF), soluble VEGF receptor 1 (sVEGFR-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), carbonyl residues, and advanced oxidation protein products (AOPP). The median concentrations of VEGF, sVEGFR-1, sVCAM-1, carbonyl residues, and AOPP were significantly higher in SSc patients than in healthy subjects at baseline. A correlation was found between VEGF concentration and carbonyl residue concentration (r = 0.43; P = 0.007). Nifedipine treatment led to a significant decrease in concentrations of sVCAM-1, carbonyl residues, and AOPP but did not affect concentrations of VEGF and sVEGFR-1. Nifedipine treatment ameliorated endothelium injury in patients with SSc, as shown by the concentrations of adhesion molecules and oxidative damage markers. The fact that VEGF and sVEGFR-1 concentrations were not changed whereas oxidative stress was ameliorated by nifedipine is consistent with the hypothesis that VEGF signalling is impaired in SSc. However, more experimental evidence is needed to determine whether the VEGF pathway is intrinsically defective in SSc.

Increased Plasma S-Nitrosothiol Concentrations Predict Cardiovascular Outcomes among Patients with End-Stage Renal Disease: A Prospective Study

The plasma concentrations of S-nitrosothiols, which are circulating nitric oxide metabolites with potential biologic activity, are increased among patients undergoing chronic hemodialysis (HD). However, the ability of S-nitrosothiols to release nitric oxide at physiologically relevant sites may be reduced among HD patients, because of impaired availability and/or activity of factors involved in S-nitrosothiol breakdown. The resultant lack of S-nitrosothiol bioavailability could contribute to the high cardiovascular risk for such patients. A possible relationship between plasma S-nitrosothiol levels and cardiac outcomes, as well as all-cause mortality rates, was investigated in a cohort of 250 chronic HD patients and who were undergoing regular dialysis three times per week were monitored for 1 yr. During that follow-up period, major cardiac events and all-cause deaths were prospectively recorded. At baseline, high plasma S-nitrosothiol levels (>2 micro M, corresponding to the top quartile of all measured values) were independently associated with pulse pressure in an adjusted multivariate analysis (odds ratio, 1.03; 95% confidence interval, 1.01 to 1.05; P = 0.007). During the follow-up period, 36 patients died (16 as a result of cardiac causes) and 33 patients experienced major adverse cardiac events. In an adjusted Cox proportional-hazards model, high plasma S-nitrosothiol concentrations (i.e., the top quartile versus the three other quartiles) were an independent predictor of cardiac events (hazard ratio, 3.30; 95% confidence interval, 1.61 to 6.76; P = 0.001) but not of all-cause death. Therefore, among chronic HD patients, markedly elevated plasma S-nitrosothiol levels are associated with pulse pressure and predict cardiovascular outcomes. These findings support the hypothesis that impaired S-nitrosothiol bioavailability in uremia is an important factor for the excessive cardiovascular risk among HD patients.

Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis

We have reported previously that dihydropyridine-type calcium-channel antagonists (DTCCA) such as nifedipine decrease plasma markers of oxidative stress damage in systemic sclerosis (SSc). To clarify the cellular basis of these beneficial effects, we investigated the effects in vivo and in vitro of nifedipine on superoxide anion (O2•-) production by peripheral blood monocytes. We compared 10 healthy controls with 12 patients with SSc, first after interruption of treatment with DTCCA and second after 2 weeks of treatment with nifedipine (60 mg/day). O2•- production by monocytes stimulated with phorbol myristate acetate (PMA) was quantified by the cytochrome c reduction method. We also investigated the effects in vitro of DTCCA on O2•- production and protein phosphorylation in healthy monocytes and on protein kinase C (PKC) activity using recombinant PKC. After DTCCA had been washed out, monocytes from patients with SSc produced more O2•- than those from controls. Nifedipine treatment considerably decreased O2•- production by PMA-stimulated monocytes. Treatment of healthy monocytes with nifedipine in vitro inhibited PMA-induced O2•- production and protein phosphorylation in a dose-dependent manner. Finally, nifedipine strongly inhibited the activity of recombinant PKC in vitro. Thus, the oxidative stress damage observed in SSc is consistent with O2•- overproduction by primed monocytes. This was decreased by nifedipine treatment both in vivo and in vitro. This beneficial property of nifedipine seems to be mediated by its cellular action and by the inhibition of PKC activity. This supports the hypothesis that this drug could be useful for the treatment of diseases associated with oxidative stress.

Action of ornithine α ketoglutarate on DNA synthesis by human fibroblasts

SummaryOrnithine α ketoglutarate (OKG) is largely used in clinical nutrition for its anabolic effects. However, the mechanism of its action remains questionable. We investigated the effect of OKG on the rate of DNA synthesis in human fibroblasts. The in vitro experimental procedure required to demonstrate in cell culture the anabolic effects of OKG observed in vivo was found to be glutamine-free and serum-poor medium with sparse cells. In these conditions, OKG induced a significant increase in [3H]thymidine incorporation compared to untreated control cells. This effect was dose-dependent and was observed in all the cultures tested. Taken individually, the two constituents of OKG, i.e. αKG and Orn, also showed a stimulatory effect, but did not demonstrate a dose-dependent response. Concomitant analysis of extracellular aminoacids showed in αKG-treated cultures an increase in glutamate and a decrease in aspartate, suggesting a cellular transamination of αKG. Glutamine, which is the preferential energetic substrate of fibroblasts, can be produced from glutamate and might play a role in the action of OKG. Moreover, OKG induced a rise in the cellular polyamine content. This, in association with the inhibitory effect on OKG action of difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, suggests a link between the polyamine biosynthesis pathway and the anabolic effect of OKG.

Metabolism and kinetics of parenterally administered ornithine and α-ketoglutarate in healthy and burned animals

Abstract To improve our understanding of the metabolism of ornithine α-ketoglutarate (OKG), used as an adjuvant of parenteral nutrition, we studied the plasma kinetics, localisation of target tissues and metabolism of α ketoglutarate (αKG) and ornithine (Orn) in healthy and burned animals. After parenteral administration, the kinetics of plasma disappearance of the two labelled compounds showed a biphasic decrease reflecting an open two-compartmental model of elimination, with the exception of Orn in burned rats. The appearance of metabolites in the plasma was rapid, particularly with regard to glutamate, proline and, following ornithine administration, glutamine. This witnessed the engagement of the substrates in multiple metabolic pathways. The study of tissular distribution by autoradiography demonstrated certain target tissues in common for αKG and Orn such as the liver, intestinal mucosa, salivary glands, kidney and muscle. This is consistent with a possible synergic action of these two compounds. The identification of labelled amino-acids and aliphatic polyamines was also performed in the tissues. Two major observations were made, which could be of interest in further work concerning cellular mechanisms of OKG action. Firstly, after αKG administration, labelled Glu and αKG were detected simultaneously in muscle; this would render possible the local biosynthesis of the anticatabolic compound α ketoisocaproate. Secondly, the injection of 14 C-ornithine was followed by the appearance in the intestinal mucosa and salivary glands of labelled aliphatic polyamines, which possess an anabolic effect.

B-type natriuretic peptides for the diagnosis of congestive heart failure in dyspneic oldest-old patients.

OBJECTIVES To evaluate the accuracy of B-type natriuretic peptide (BNP) and amino-terminal pro-brain natriuretic peptide (NT-proBNP) for the diagnosis of congestive heart failure (CHF) in dyspneic patients aged >or=85 years admitted to the Emergency Department (ED), and to define threshold values in this oldest-old population. DESIGN AND METHODS This study involved 210 oldest-old patients, and 360 patients aged from 65 to 84 years (<85 years), admitted to the ED for dyspnea. RESULTS Median BNP and NT-proBNP levels were significantly higher in CHF oldest-old patients (p<0.001). BNP and NT-proBNP threshold values were higher in oldest-old patients (290 and 2800 pg/mL, respectively) compared to that of patients <85 years (270 and 1700 pg/mL, respectively). In a multivariate analysis, both BNP and NT-proBNP were the strongest variables associated with CHF in oldest-old patients. Neither renal function nor gender had impact on the diagnostic utility of the two tests. CONCLUSION Both BNP and NT-proBNP could potentially be reliable biomarkers for the diagnosis of CHF in oldest-old patients admitted with acute dyspnea to the ED.

Variability in urinary excretion of bone resorption markers: limitations of a single determination in clinical practice

In this study we assessed the within and between-subject variability of the concentrations of two urinary markers, free deoxypyridinoline (DPD) and C telopeptide (CTX-I), in healthy patients with the aim of setting reliable thresholds to enable physicians to take decisions about individual patients with confidence.Between-subject variability for the women was 25.4% for DPD and 38.2% for CTX-I, and for the men was 12.9% for DPD and 23.8% for CTX-I. The coefficients of variation were similar for daily, weekly and monthly determinations, giving means of 13.8 and 28.1% for DPD and CTX-I respectively. Critical difference (CD) was lower for DPD than for CTX-I (about 44 and 80% respectively). The number of samples required to determine the true mean with a CD at the 5% level was 29 for DPD and more than 113 for CTX-I.DPD was the least biologically variable. One determination was not sufficient to determine bone resorption status and a 44% decrease in DPD levels and an 80% decrease in CTX-I levels were required to demonstrate the efficacy of antiresorptive therapy in individual patients.

Ornithine α-Ketoglutarate and Glutamine Supplementation During Refeeding of Food-Deprived Rats

The aim of this study was to compare the efficiency of ornithine alpha-ketoglutarate (OKG) and glutamine supplementation in an experimental model of denutrition that provides well-characterized disturbances of amino acid patterns. Male Wistar rats (187 +/- 11 g; five in each group) were starved for 3 days and then refed for 7 days with an oral diet (192 kcal kg-1.day-1 and 2.25 g of nitrogen kg-1.day-1), supplemented with 0.19 g of nitrogen kg-1.day-1 in the form of OKG, glutamine, or casein (control group). Food deprivation induced a fall in most tissue amino acids, with the notable exception of muscle leucine and liver glutamate, which increased by 43% (p < .01), and 11% (p < .05), respectively. The main effect of OKG was seen in the viscera, with a normalization of most amino acid pools (including proline and branched-chain amino acids) in the small bowel and liver. The main effect of glutamine was observed in the muscle, with a normalization of the glutamine and leucine pools. We conclude that, in this model and with the doses used, OKG and glutamine act in different target tissues, ie, splanchnic areas and muscle, respectively.

Interleukin-1β-mediated glucose uptake by chondrocytes. Inhibition by cortisol

Summary The aim of this study was to investigate the in vitro effects of interleukin-1 β (IL-1 β ) on cultured human articular chondrocytes from patients with osteoarthritis, by the evaluation of glucose uptake. We also investigated the inhibitory effect of cortisol on IL-1 β -mediated glucose uptake. Experiments were performed by using 2-deoxy-D-[1- 3 H]glucose (2-DOG) and confluent monolayer cells at first passage. Confluent cells were also treated for 24 h with different concentrations of cortisol (10 −5 , 10 −6 and 10 −7 mol/l). IL-1 β (100 pg/ml) was added 6 h before glucose uptake studies. Glucose uptake stimulation was observed 3 h after the addition of 100 pg/ml IL-1 β (+70%) and increased up to 24 h (+145%). The sensitivity and responsiveness of chondrocytes to IL-1 β , studied after a 6 h association time, appeared to be dose-dependent from 0.1 pg/ml IL-1 β (+50%) to 100 pg/ml (+130%) over basal values. The effect of the cytokine was protein synthesis-dependent, as demonstrated by using cycloheximide. Cortisol inhibited the action of IL-1 β on glucose uptake because it reduced stimulating effects by 28% at concentrations as weak as 10 −6 mol/l. Results appeared similar when IL-1 β and cortisol were added simultaneously 6 h before 2-DOG uptake. The rapid effect of cortisol was protein-synthesis dependent, as indicated by inhibition by cycloheximide. These results suggest that IL-1 β stimulates chondrocyte metabolic activity. The inhibition of IL-1 β -mediated glucose uptake is suggested for studying the anti-IL-1 effect of other anti-rheumatic drugs.