Determination of the relative contribution of uric acid level increases to the total measured antioxidative activity could be very useful for testing antioxidative products and their effect on human health. The aim of this report is to present a simple spectrophotometric method that combines the measurement of total antioxidative capacity of a sample by ferric reducing/antioxidative power (FRAP) assay, with the uricase-reaction (specific elimination of uric acid), in order to establish and correct for the contribution of uric acid in FRAP values. We measured FRAP values, with (uric acid-independent antioxidant capacity, TAC-UA) and without (total antioxidant capacity, TAC) uricase treatment, and expressed it as μmol/L of uric acid equivalents. In such way, it was possible to determine both total and uric acid-independent antioxidant capacity, plasma uric acid (UA, as the difference between TAC and TAC-UA), and the ratio of the uric acid in total antioxidant capacity (UA/TAC).
Surprisingly, to the authors' knowledge, there are only two published kinetic spectrophotometric methods for the determination of NAC. 15,16 Also, none of the cited methods for the determination of NAC has used Fe 3+ and 2,4,6-trypyridyl-s-triazine (TPTZ) as a reagent solution. In this report, a novel, simple and sensitive kinetic spectrophotometric method with TPTZ as the chromogenic reagent for the determination of NAC is described and validated. Ease of application, sensitivity, short analysis time, low cost and reliability are its main advantages. The initial rate and fixed-time methods, after their optimization and validation, are adopted for the determination of NAC in its pharmaceutical formulations. Experimental ReagentsAll chemicals were of analytical-reagent grade, and solutions were prepared in Milli-Q deionized water.A stock solution of NAC, 1.0 × 10 -2 mol L -1 , was prepared by dissolving 163.2 mg of NAC (Merck, Darmstadt, Germany) in deionized water up to 100.0 mL in volume. The stock solution was stable for at least 30 days when it was stored in a dark bottle at 4 C. Working solutions of lower concentration were prepared daily by appropriate dilution of the stock solution with deionized water.A stock solution of Fe 3+ , 1.0 × 10 -2 mol L -1 , was prepared by dissolving 270.3 mg of FeCl3·6H2O (Kemika, Zagreb, Croatia) in a portion of deionized water. Concentrated hydrochloric acid (0.5 mL) was added to prevent the hydrolysis of iron, before making up to a volume of 100.0 mL.A stock solution of TPTZ (Merck, Germany), 1.0 × 10 -2 mol L -1 , was prepared by dissolving 312.3 mg in 2.0 mL of HCl (c (HCl) = 6.0 mol L -1 ), and diluted to 100.0 mL with deionized water. A stock solution of TPTZ was stored in a dark bottle at 4 C. A novel simple kinetic spectrophotometric method for the determination of N-acetyl-L-cysteine (NAC) has been developed and validated. The proposed method is based on a coupled redox-complexation reaction, the first step of which is the reduction of Fe 3+ by NAC; the second one includes the complexation of Fe 2+ , resulting from the preceding redox reaction, with 2,4,6-trypyridyl-s-triazine (TPTZ). The stable Fe(TPTZ)2 2+ complex exhibits an absorption maximum at λ = 593 nm. The initial rate and fixed-time (at 5 min) methods were utilized for constructing calibration graphs. The graphs were linear in concentration ranges from 4.0 × 10 -6 to 1.0 × 10 -4 mol L -1 for the initial rate method and 1.0 × 10 -6 to 1.0 × 10 -4 mol L -1 for the fixed-time method, with detection limits of 1.0 × 10 -6 and 1.7 × 10 -7 mol L -1 , respectively. The proposed methods were successfully applied for the determination of NAC in its commercial pharmaceutical formulations.
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 O(2) 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 (P(tc)O(2)) 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 P(tc)O(2) (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.
S-Nitrosothiols or thionitrites with the general formula RSNO are formally composed of the nitrosyl cation (NO+) and a thiolate (RS−), the base of the corresponding acids RSH. The smallest S-nitrosothiol is HSNO and derives from hydrogen sulfide (HSH, H2S). The most common physiological S-nitrosothiols are derived from the amino acid L-cysteine (CysSH). Thus, the simplest S-nitrosothiol is S-nitroso-L-cysteine (CysSNO). CysSNO is a spontaneous potent donor of nitric oxide (NO) which activates soluble guanylyl cyclase to form cyclic guanosine monophosphate (cGMP). This activation is associated with multiple biological actions that include relaxation of smooth muscle cells and inhibition of platelet aggregation. Like NO, CysSNO is a short-lived species and occurs physiologically at concentrations around 1 nM in human blood. CysSNO can be formed from CysSH and higher oxides of NO including nitrous acid (HONO) and its anhydride (N2O3). The most characteristic feature of RSNO is the S-transnitrosation reaction by which the NO+ group is reversibly transferred to another thiolate. By this way numerous RSNO can be formed such as the low-molecular-mass S-nitroso-N-acetyl-L-cysteine (SNAC) and S-nitroso-glutathione (GSNO), and the high-molecular-mass S-nitrosol-L-cysteine hemoglobin (HbCysSNO) present in erythrocytes and S-nitrosol-L-cysteine albumin (AlbCysSNO) present in plasma at concentrations of the order of 200 nM. All above mentioned RSNO exert NO-related biological activity, but they must be administered intravenously. This important drawback can be overcome by lipophilic charge-free RSNO. Thus, we prepared the ethyl ester of SNAC, the S-nitroso-N-acetyl-L-cysteine ethyl ester (SNACET), from synthetic N-acetyl-L-cysteine ethyl ester (NACET). Both NACET and SNACET have improved pharmacological features over N-acetyl-L-cysteine (NAC) and S-nitroso-N-acetyl-L-cysteine (SNAC), respectively, including higher oral bioavailability. SNACET exerts NO-related activities which can be utilized in the urogenital tract and in the cardiovascular system. NACET, with high oral bioavailability, is a strong antioxidant and abundant precursor of GSH, unlike its free acid N-acetyl-L-cysteine (NAC). Here, we review the chemical and pharmacological properties of SNACET and NACET as well as their analytical chemistry. We also report new results from the ingestion of S-[15N]nitroso-N-acetyl-L-cysteine ethyl ester (S15NACET) demonstrating the favorable pharmacological profile of SNACET.
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