Jonatan Vukovic

Red wine induced modulation of vascular function: separating the role of polyphenols, ethanol, and urates

By using red wine (RW), dealcoholized red wine (DARW), polyphenols-stripped red wine (PSRW), ethanol-water solution (ET), and water (W), the role of wine polyphenols, ethanol, and urate on vascular function was examined in humans (n=9 per beverage) and on isolated rat aortic rings (n=9). Healthy males randomly consumed each beverage in a cross-over design. Plasma ethanol, catechin, and urate concentrations were measured before and 30, 60 and 120 minutes after beverage intake. Endothelial function was assessed before and 60 minutes after beverage consumption by normalized flow-mediated dilation (FMD). RW and DARW induced similar vasodilatation in the isolated vessels whereas PSRW, ET, and W did not. All ethanol-containing beverages induced similar basal vasodilatation of brachial artery. Only intake of RW resulted in enhancement of endothelial response, despite similar plasma catechin concentration after DARW. The borderline effect of RW on FMD (P=0.0531) became significant after FMD normalization (P=0.0043) that neutralized blunting effect of ethanol-induced basal vasodilatation. Effects of PSRW and ET did not differ although plasma urate increased after PSRW and not after ET, indicating lack of urate influence on endothelial response. Acute vascular effects of RW, mediated by polyphenols, cannot be predicted by plasma catechin concentration only.

Acute, food-induced moderate elevation of plasma uric acid protects against hyperoxia-induced oxidative stress and increase in arterial stiffness in healthy humans.

We examined the effects of acute, food-induced moderate increase of plasma uric acid (UA) on arterial stiffness and markers of oxidative damage in plasma in healthy males exposed to 100% normobaric oxygen. Acute elevation of plasma UA was induced by consumption of red wine, combination of ethanol and glycerol, or fructose. By using these beverages we were able to separate the effects of UA, wine polyphenols and ethanol. Water was used as a control beverage. Ten males randomly consumed test beverages in a cross-over design over the period of 4 weeks, one beverage per week. They breathed 100% O(2) between 60(th) and 90(th)min of the 4-h study protocol. Pulse wave augmentation index (AIx) at brachial and radial arteries, plasma antioxidant capacity (AOC), thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxides (LOOH) assessed by xylenol orange method, UA and blood ethanol concentrations were determined before and 60, 90, 120, 150 and 240 min after beverage consumption. Consumption of the beverages did not affect the AIx, TBARS or LOOH values during 60 min before exposure to hyperoxia, while AOC and plasma UA increased except in the water group. Significant increase of AIx, plasma TBARS and LOOH, which occurred during 30 min of hyperoxia in the water group, was largely prevented in the groups that consumed red wine, glycerol+ethanol or fructose. In contrast to chronic hyperuricemia, generally considered as a risk factor for cardiovascular diseases and metabolic syndrome, acute increase of UA acts protectively against hyperoxia-induced oxidative stress and related increase of arterial stiffness in large peripheral arteries.

Comparison of acute effects of red wine, beer and vodka against hyperoxia-induced oxidative stress and increase in arterial stiffness in healthy humans

OBJECTIVE We determined and compared acute effects of different alcoholic beverages on oxygen-induced increase in oxidative stress plasma marker and arterial stiffness in healthy humans. METHODS Ten males randomly consumed one of four tested beverages: red wine (RW), vodka, beer (0.32 g ethanol/kg body wt) and water as control. Every beverage was consumed once, a week apart, in a cross-over design. The volunteers breathed 100% normobaric O(2) between 60th and 90th min of 3h study protocol. Plasma lipid peroxides (LOOH) and uric acid (UA) concentration, blood alcohol concentration (BAC) and arterial stiffness (indicated by augmentation index, AIx) were measured before and 30, 60, 90, 120 and 180 min after beverage consumption. RESULTS Intake of all alcoholic beverages caused a similar increase of BAC. The oxygen-induced elevation in AIx was similarly reduced in all three groups relative to the control (3.4 ± 1.3%, 5.4 ± 2.2% and 0.2 ± 1.6% vs. 13.7 ± 2.6% for red wine, vodka, beer and control, respectively, 60 min after intake). Exposure to oxygen resulted in increased plasma LOOH in all groups. However, in RW group this increase was lowest (1.1 ± 0.5) in comparison to the vodka (2.1 ± 0.5), beer (1.6±0.3) and control (2.5 ± 0.4μM/L H(2)O(2)). 60 min after intake of RW and beer plasma UA significantly increased (34 ± 4 and 15 ± 3) in contrast to vodka and control (-6 ± 2 and -8 ± 2μmol/L). CONCLUSION All three alcoholic beverages provided similar protection against oxygen-induced increase in arterial stiffness, probably due to central vasodilatatory effect of alcohol itself, but only RW provided protection against oxygen-induced oxidative stress.

Plasma nitrite concentration decreases after hyperoxia-induced oxidative stress in healthy humans.

The aim of this study was to measure plasma nitrite, the biochemical marker of endothelial nitric oxide (•NO) synthesis, before and after hyperoxia, in order to test the hypothesis that hyperoxia‐induced vasoconstriction is a consequence of reduced bioavailability of •NO caused by elevated oxidative stress. Ten healthy men breathed 100% normobaric O2 for 30 min between 15th and 45th min of the 1‐h study protocol. Plasma nitrite and malondialdehyde (MDA), arterial stiffness (indicated by augmentation index, AIx) and arterial oxygen (PtcO2) pressure were measured at 1st, 15th, 45th and 60th minute of the study. Breathing of normobaric 100% oxygen during 30 min caused an increase in PtcO2 (from 75 ± 2 to 412 ± 25 mm Hg), AIx (from −63 ± 4 to −51 ± 3%) and MDA (from 152 ± 13 to 218 ± 15 nm) values and a decrease in plasma nitrite (from 918 ± 58 to 773 ± 55 nm). During the 15‐min recovery phase, plasma nitrite, AIx and MDA values remained altered. This study suggests that the underlying mechanism of hyperoxia‐induced vasoconstriction may involve reduced •NO bioavailability caused by elevated and sustained oxidative stress.

Differences in Vasodilatory Response to Red Wine in Rat and Guinea Pig Aorta

We examined and compared mechanisms of the red wine (RW)-induced vasorelaxation in guinea pig (GP) and rat aorta. Acetylcholine-induced relaxation of norepinephrine-precontracted aortic rings was stronger in rat aorta than in GP aorta, whereas RW-induced vasorelaxation was stronger in GP aorta. l-nitro-arginine methyl ester (l-NAME) abolished RW-induced vasorelaxation in rat aorta, whereas in GP aorta, it was only reduced by 50%. To examine mechanisms of the l-NAME-resistant relaxation, GP aortic rings were additionally exposed to indomethacin, clotrimazole, and their combination. Indomethacin insignificantly reduced RW-induced relaxation, but in combination with l-NAME, the relaxation was synergistically decreased (80%). After clotrimazole exposure, the relaxation was reduced by 25%, and addition of indomethacin caused no further reduction. Only the combination of l-NAME, indomethacin, and clotrimazole prevented RW-induced vasorelaxation. RW-induced vasorelaxation in KCl-precontracted GP rings was significantly smaller (Emax 78.31% ± 6.09%) than the RW-induced relaxation in norepinephrine-precontracted rings (Emax 126.01% ± 2.11%). l-NAME in KCl-precontracted GP rings prevented RW-induced vasorelaxation. In conclusion, different pathways are involved in the RW-induced vasorelaxation in GP aorta, in contrast to rat aorta, in which NO plays main role. Therefore, the uncritical extrapolation of the results from one species to another could be misleading.

LC-MS/MS and GC-MS/MS measurement of plasma and urine di-paracetamol and 3-nitro-paracetamol: proof-of-concept studies on a novel human model of oxidative stress based on oral paracetamol administration.

Paracetamol (acetaminophen) is a widely used safe analgesic drug when administered at therapeutic doses. Given the chemical reactivity of its phenolic group towards electrophilic species, we assumed that detection of paracetamol metabolites distinctly different from its known phase I metabolite N-acetyl-p-benzoquinone imine (NAPQI) and the phase II glucuronic, sulfuric and mercapturic acids in biological samples upon oral administration of paracetamol (e.g., a 500-mg tablet) may represent a novel model of oxidative stress in humans. Such potential paracetamol metabolites are di-paracetamol and 3-nitro-paracetamol, in analogy to the well-investigated endogenous biomarkers di-tyrosine and 3-nitro-tyrosine. Di-paracetamol and 3-nitro-paracetamol are known to be formed both by enzymatic and non-enzymatic routes. In the present work we report on mouse and human pilot studies on the formation and appearance of di-paracetamol and 3-nitro-paracetamol in blood of mice intraperitoneally administered paracetamol, as well as in plasma and urine samples of healthy subjects who received a 500-mg paracetamol tablet or placebo. For the analysis of di-paracetamol and 3-nitro-paracetamol in plasma and urine samples, analytes were extracted by solvent extraction with ethyl acetate and subsequently analyzed by LC-MS/MS without and with derivatization with pentafluorobenzyl bromide. GC-MS/MS was used to detect 3-nitro-paracetamol and quantify paracetamol as pentafluorobenzyl derivatives. Our studies indicate that di-paracetamol and 3-nitro-paracetamol appear in plasma and urine when paracetamol is given orally to healthy humans at the therapeutic dosage of 5-7 mg/kg. The molar ratio of di-paracetamol to paracetamol in urine was determined to be 1:535 in the paracetamol group and 1:6844 in the placebo group; the molar ratio of 3-nitro-paracetamol to paracetamol in urine was determined to be 1:199 in the paracetamol group and 1:8657 in the placebo group. Our studies suggest that a fraction of circulating and excretory di-paracetamol and 3-nitro-paracetamol may be formed artefactually during sample workup including derivatization. Further studies based on the quantitative determination of di-paracetamol and 3-nitro-paracetamol in biological samples by LC-MS/MS and/or GC-MS/MS using stable-isotope labeled analogues as internal standards are warranted to test the utility of paracetamol as a probe of oxidative stress in animals and in humans in health and disease.

Acute application of antioxidants protects against hyperoxia-induced reduction of plasma nitrite concentration.

We investigated the effects of acute intake of antioxidants on hyperoxia‐induced oxidative stress, reduction of plasma nitrite and change in arterial stiffness. Twelve healthy males randomly consumed either placebo or an oral antioxidant cocktail (vitamin C, 1000 mg; vitamin E, 600 IU; alpha‐lipoic acid, 600 mg). Every therapy was consumed once, a week apart, in a cross‐over design, 30 min before the experiment. The volunteers breathed 100% normobaric oxygen between 30th and 60th min of 1‐h study protocol. Plasma levels of nitrite, lipid peroxides (LOOH) and vitamin C, arterial stiffness (indicated by augmentation index, AIx) and arterial oxygen (PtcO2) pressure were measured before and after hyperoxia. Exposure to oxygen caused a similar increase of PtcO2 in both placebo and antioxidants groups, confirming comparable exposure to hyperoxia (438 ± 100 versus 455 ± 83 mm Hg). Vitamin C was increased in the antioxidants group confirming successful application of antioxidants (69 ± 14 versus 57 ± 15 μm). Hyperoxia resulted in increased AIx and LOOH and decreased nitrite in placebo (−32 ± 11 versus −47 ± 13%, 72 ± 7 versus 62 ± 6 μm H2O2 and 758 ± 184 versus 920 ± 191 nm, respectively), but not in the antioxidants group (−42 ± 13 versus −50 ± 13%, 64 ± 9 versus 61 ± 8 μm H2O2 and 847 ± 156 versus 936 ± 201 nm, respectively). The acute intake of selected antioxidants was effective in preserving bioavailabity of ˙NO and vascular function, against hyperoxia‐induced oxidative stress.

Signet Ring Carcinoma of the Appendix Presenting as Crohn’s Disease in a Young Male

Primary signet ring cell carcinoma is a rare event in surgery. It looks like acute appendicitis and it is difficult to diagnose it on clinical grounds alone. The diagnosis is always confirmed by histopathology of a surgically removed appendix. A young man, 22 years old, presented with vomiting, diarrhea, and cramps in his abdomen without abdominal tenderness (mild abdominal discomfort in the right lower abdominal quadrant without signs of peritoneal irritation) during the previous month. The first endoscopic results showed only changes of mucosa that could be attributed to endoscopic and clinical representation of Crohn’s disease. A few days after the initiation of the therapy with aminosalicylates and corticosteroids, the patient went into ileus and was transferred to the Department of Surgery, where he underwent an emergency right-sided hemicolectomy with resection of the transversal colon and forming of an ileostoma. The first pathohistological diagnosis was pseudomembranous colitis. Because the patient’s condition was deteriorating, a revision of the pathohistological diagnosis was done. After careful revision and extensive sampling, a signet ring cell carcinoma arising in the appendix with infiltration of the ileocecal region was found. Immunohistochemically, tumor cells were positive for CDX-2 CK7, CK20, CK19, and carcinoembryonic antigen and negative for chromogranin A. Sixteen isolated lymph nodes were negative. Although the patient had a disease that was localized to the appendix and ileocecal region with no apparent distal metastasis, his clinical condition was worsening rapidly and he died after 2 months. This case shows the aggressive biological behavior of the appendix signet ring cell carcinoma. Scrupulous histopathological examination of the appendix is an obligatory procedure. Elimination of the signet ring cell carcinoma from other carcinoma subtypes is of special importance as it has an exceptionally poor prognosis and is generally diagnosed in its advanced stages.