Repair of amino acid radicals by a vitamin E analogue

Free radicals derived from one-electron oxidation of the amino acids tryptophan, tyrosine, methionine and histidine have been found to be rapidly (k = 10(7) -10(9) dm3 mol-1 s-1) and efficiently repaired by Trolox C, a vitamin E analogue. The reactions form a relatively stable phenoxyl radical of Trolox C (lambda max = 440 nm; epsilon = 5.4 X 10(3) mol dm-3 cm-1). The radical cation of tryptophan is more rapidly repaired than the neutral tryptophan radical. Repair of tryptophanyl radicals in the enzyme lysozyme has also been observed. The results suggest that a function of alpha-tocopherol in membranes may be the repair of radicals of integral membrane proteins.     

Pulse radiolytic studies of radicals produced from methylated uracils via oxidation by SO4.

Using a pulse radiolysis technique, some nucleic base radicals were produced by the reactions of sulfate radical, SO4-, with 1-, 3-, 5-, and 6-methyluracils, and their optical and kinetic natures were observed. All of their absorption spectra showed main peaks at approximately 400 nm with absorption constants ranging from 1020 to approximately 1560 dm3 mol-1 cm-1. The rate constants of their formation were 1.6 to approximately 3.3 X 10(9) dm3 mol-1 s-1. For thymine and 6-methyluracil, the absorption coefficients of their radicals at approximately 500 nm changed according to pH, giving pK values of approximately 9. For N(3)-methylated uracil, on the other hand, no such acid-base equilibrium was found. When the N(1) position was methylated, another type of pH effect was found. From these spectral observations and the comparative discussions, it was shown that methylation at the N(1) position gives OH-adduct radicals and at other positions proton-released radicals. For 3- and 6-methyluracils, second intermediates were formed concomitantly with the disappearance of the initial radicals. They are tentatively assigned to their ring-opened radicals, presumably by the reaction of the initial radicals with S2O8(2-).     

Sensitive quantification of atomoxetine in human plasma by HPLC with fluorescence detection using 4-(4,5-diphenyl-1H-imidazole-2-yl) benzoyl chloride derivatization

The first HPLC-fluorescence method for the determination of atomoxetine in human plasma was developed and validated. Atomoxetine was derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) under mild conditions, and separated isocratically on a C18 column using a HPLC system with fluorescence detection (lambdaex: 318 nm, lambdaem: 448 nm). A linear calibration curve was obtained over the concentration range 1-1000 ng/mL (r=0.999). The limit of detection (S/N=3) was 0.3 ng/mL. The relative standard deviations of intra-day and inter-day variations were < or =8.30% and 7.47%, respectively. This method is rapid, sensitive, and suitable for both basic and clinical studies of atomoxetine.     

Determination of 1-hydroxypyrene in children urine using column-switching liquid chromatography and fluorescence detection

This study developed an acid hydrolysis method instead of using enzyme extraction, equipped with column-switching system for the pretreatment of samples, in the determination of 1-hydroxypyrene in the urine from children and pyrene in airborne particulates. We collected both types of samples from areas near a petrochemical industry and rural areas as reference. Samples were first treated with acid hydrolysis and followed by solvent extraction prior to being injected into the separation system for the determination with high performance liquid chromatography and fluorescence. A column-switching system was on-line with a C18 separation column to remove matrix interference and obtain a stable baseline of the chromatogram. The eluent used to separate the 1-hydroxypyrene was 60% (v/v) aqueous acetonitrile solution. A fluorescence detector was used to monitor 1-hydroxypyrene at lambdaex = 348 nm and lambdaem = 388 nm, and pyrene at lambdaex = 331 nm and lambdaem = 390 nm. Both calibration graphs were linear with very good correlation coefficients (r > 0.999) and the detection limits were ca. 2pg (5ng/l). Results showed that there was a significant association between 1-hydroxypyrene levels in urine specimens and pyrene levels in airborne particulate samples (r = 0.68, P < 0.05). The average levels of pyrene in the particulates (0.18 versus 0.09ng/m3) and of 1-hydroxypyrene in urine specimens (155.9 versus 110.2ng/g creatinine) were higher for the petrochemical area than for the rural area. This method is stable and sensitive for measuring polycyclic aromatic hydrocarbons in environmental samples.     

Oxidation of polyunsaturated fatty acids and lipids through thiyl and sulfonyl radicals: reaction kinetics, and influence of oxygen and structure of thiyl radicals.

Thiyl free radicals have been shown to react with polyunsaturated fatty acids via abstraction of bisallylic hydrogen, forming pentadienyl radicals, and via addition to the double bonds. In the absence of oxygen, the latter pathway leads to regeneration of thiyl radicals through beta-elimination or "repair" of the adduct radicals by thiols. In the presence of oxygen, fixation of thiyl-induced damage occurs through reaction of O2 with the pentadienyl radical (yielding conjugated dienyl peroxyl radicals) and also with the thiyl-to-double bond adduct radical. A quantitative reaction scheme evaluated from these data considers abstraction, addition, rearrangement, and repair reactions, and the evaluation of rate constants for the individual steps. Absolute rate constants have been measured, in particular, for reactions of thiyl free radicals from glutathione, cysteine, homocysteine, N-acetylcysteine, cysteine ethyl ester, penicillamine, captopril, mercaptoethanol, and dithiothreitol with polyunsaturated fatty acids (PUFAs) ranging from 18:2 to 22:6, and the lipids trilinolein and trilinolenin. The rate constants for hydrogen abstraction were found to be typically of the order of 10(7) mol-1 dm3 s-1 and to increase with increasing lipophilicity of the attacking thiyl radical. Thioperoxyl radicals, RSOO., were found to be rather unreactive toward PUFAs, in contrast to the isomer sulfonyl radicals, RSO2., which not only abstract hydrogen from the bisallylic methylene groups of the PUFAs (although only at relatively small yield) but also readily add to the PUFA double bonds (major pathway). Specific information was obtained on the optical properties of the thiyl radical derived from the ACE inhibitor captopril, CpS. (lambda max = 340 nm, epsilon = 460 +/- 50 mol-1 dm3 cm-1), and its conjugate disulfide radical anion (CpS:.SCp) (lambda max = 420 nm).     

Reaction of azide radicals with amino acids and proteins.

The azide radical N3 reacts selectively with amino acids, in neutral solution preferentially with tryptophan (k (N3 + TrpH) = 4.1 X 10(9) dm3 mol(-1s-1) and in alkaline solution also with cysteine and tyrosine (k(N3 + CyS-) = 2.7 X 10(9) dm3 mol-1s-1) and k(N3 + TyrO-) equals 03.6 X 10(9) dm3 mol-1s-1). Oxidation of the enzyme yADH by N3 involves primary attacks, mainly at tryptophan residues, and subsequent slow secondary reactions. N3-induced inactivation of yADH is likely to occur upon oxidation of tryptophan residues in the substrate binding pocket (58-TrpH and 93-TrpH) since the substrate ethanol although unreactive with N3, protects yADH and since elADH, which does not contain tryptophan in the substrate pocket, is comparatively resistant against N3-attack. N3 exhibits low reactivity with nucleic acid derivatives and it is inert towards aliphatic compounds such as methanol and sodium dodecylsulphate.     

Interactions of thiyl free radicals with oxygen: a pulse radiolysis study

A pulse radiolysis study of glutathione in aqueous solution at pH 5.5 containing N2O/O2 mixtures at various ratios indicates that oxygen rapidly adds to the thiyl glutathione radical yielding a transient absorption, with a maximum at 540 nm, whose characteristics appear to be compatible with assignment to the GSOO. radical. The reaction (Formula: see text) appears to be an equilibrium whose kinetic constants have been estimated (kf = 2.0 X 10(9) dm3 mol-1, kb = 6.2 X 10(5) s-1). Evidence for electron transfer from ascorbate to the GSOO. radical has been obtained and the respective rate constant has been determined to be 1.75 +/- 0.15 X 10(8) dm3 mol-1 s-1.     

Exciton coupling effects and conformational change of perhexyloligosilanes with optically active methyl(1-naphthyl)phenylsilyl terminals

Perhexyloligosilanes (R,R)-(+)-MeNpPhSi*(Hex(2)Si)(n)Si*PhNpMe (n = 2; (R,R)-(+)-4a, n = 4; (R,R)-(+)-6a, n = 6; (R,R)-(+)-8a) with chiral methyl(1-naphthyl)phenylsilyl terminals were synthesized and characterized. The absorption wavelengths lambda(max) by (1)L(a,Ph) transition of phenyl chromophore conjugated with oligosilane units in (R,R)-(+)-4a - (R,R)-(+)-8a show bathochromic shift of about 3-4 nm compared with those of the alpha,omega-phenyl substituted perhexyloligosilanes Ph(Hex(2)Si)(m)Ph (m = 4; 4b, m = 6; 6b, m = 8; 8b) having the same silicon chain length. Longer chain length induces the separated lambda(max) of (1)L(a,Ph) from (1)B(b,Np) of naphthyl chromophore with positive exciton chiralities. In (R,R)-(+)-8a, although the extremum wavelengths lambda(ext) of exciton coupling between (1)B(b,Np) and (1)L(a,Ph) are separated by about 80 nm, the compound retains the positive exciton chirality, which provides definite information on the absolute configuration of terminal chiral silicon atoms. Bulky terminal substituents and lowering the temperature affect the conformation of the main chain, inducing extended silicon backbone structure.     

Intermolecular charge transfer involving tryptophan, tyrosine and three electron-bonded intermediates derived from methionine

Oxidation processes of radiation-generated three-electron-bonded intermediates derived from methionine Met2[S+...S] and Met[S...X] (X=Cl,Br) were investigated through reaction with tryptophan and tyrosine, using the optical pulse radiolysis method. Bimolecular rate constants have been measured for the reactions Met2[S+...S] with TrpH(k=3.8 x 10(8) dm3 mol-1 s-1 and k = 4.9 X 10(8) dm3 mol-1 s-1 at at ph 1 and 4.3, respectively) and Met2[S+...S] with tyrosine, k=3.8 x 10(7) dm3 mol-1 s-1. Rate constants for intermolecular transformation of Met[S...Br] and Met[S...Cl] into TrpH+. or Trp. were also estimated. They varied from 4.7 X 10(8) dm3 mol-1 s-1 (bromide species) to 1.0 X 10(9)dm3 mol-1 s-1 (chloride species). It has also been established azide radicals N-6, N.3 in contrast to dihalide radicals (X-2) do not form transients of Met[S...X] (X = N3)-type. However, oxidation of N-3 by Met2[S+...S] occurs with a bimolecular rate constant of 2.8 X 10(8) dm3 mol-1 s-1. These results are discussed in the light of some equilibria which have been proposed earlier for methionine-halide systems.     

Reactions of linoleic acid peroxyl radicals with phenolic antioxidants: a pulse radiolysis study

Linoleic acid peroxyl radicals (LOO.) can be viewed as model intermediates occurring during lipid peroxidation processes. Formation and reactions of these species were investigated in aqueous alkaline solution using the technique of pulse radiolysis combined with kinetic spectroscopy. Irradiation of linoleic acid in N2O/O2-saturated solutions leads to a mixture of peroxyl radical isomers, whereas reaction of 13-hydroperoxylinoleic acid (13-LOOH) with azide radicals in N2O-saturated solution produces 13-LOO. radicals specifically. These peroxyl radicals cannot be observed directly, but their reactions with the two flavonols, kaempferol and quercetin, acting as radical-scavenging antioxidants, produced strongly absorbing aroxyl radicals (ArO.). The same aroxyl radicals were generated by .OH and N3. with rate constants exceeding 10(9) dm3 mol-1 s-1. Applying a reaction scheme that includes competing generation and decay reactions of both LOO. and ArO. radicals, we derived individual rate constants for LOO. reactions with the phenols (greater than 10(7) dm3 mol-1 s-1), with the aroxyl radicals to form covalent adducts (greater than 10(8) dm3 mol-1 s-1), as well as for their bimilecular decay (3.0 X 10(8) dm3 mol-1 s-1). These results demonstrate the high reactivity of both fatty acid peroxyl radicals and the flavone antioxidants in aqueous solution.     

Reactivity of halides with triplets of 6-chloro and 6-bromopicolinic acids: contrasting effects of bromide and chloride.

The reactivity of Br(-) and Cl(-) with triplet of anionic 6-chloropicolinic acid (pH = 5.4) and with triplets of 6-chloro and 6-bromopicolinic acids in zwitterionic forms (pH = 0.9) was studied by laser flash photolysis and steady-state irradiation. Br(-) was found to trap the three triplets. Triplet lifetime measurements gave quenching rate constants of 8 x 10(8) mol(-1) dm(3) s(-1) for the zwitterion of 6-chloropicolinic acid and of 3.4 x 10(5) mol(-1) dm(3) s(-1) for the anionic counterpart. No secondary transient species were observed indicating that the charge transfer intermediates are subject to dissipative processes. Cl(-) trapped triplet of zwitterions only, and reactions were found to be associated with a high quantum yield of radicals. The photolysis of 6-bromopicolinic acid photolysis was drastically enhanced by Cl(-), 6-chloropicolinic acid being produced with a chemical yield of about 90%. The 6-bromo-2-carboxypyridinyl radical could be characterized (lambda(max)/nm = 318 with shoulder at 370 nm and epsilon/mol(-1) dm(3) cm(-1) = 8100).     

Metabolism of the vitamin D3 derivative (24R)-hydroxycalcidiol by human promyelocytic leukemia cells (HL-60). Isolation and identification of (5Z) and (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol

Human promyelocytic leukemia cells (HL-60 cells) incubated with (24R)-hydroxy[26,27-methyl-3H]calcidiol (0.2 microCi) or non-radioactive (24R)-hydroxycalcidiol (370 micrograms) produced significant quantities of two new vitamin D3 (calciol) metabolites. The metabolites were isolated from HL-60 cell culture media by methanol/chloroform extraction and a series of chromatographic procedures. The two new metabolites were identified as (5Z)- and (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol by HPLC analysis, ultraviolet absorption spectrophotometry, mass spectrometry and Fourier-transform infrared spectrophotometry. According to the isolation and purification procedures, the total amounts of 3.04 micrograms (5Z)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol (lambda max = 310 nm, epsilon = 17070 M-1 cm-1) and 8.89 micrograms (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol (lambda max = 312 nm, e = 24,500 M-1 cm-1) were calculated, assuming an Mr of 418. The activity of 19-nor-10-oxo-(24R)-hydroxycalcidiol to promote HL-60 cell differentiation was higher than the activity of the precursor (24R)-hydroxycalcidiol suggesting a possible biological action of this metabolite in HL-60 cells.     

Design of fluorogenic substrates for continuous assay of sialyltransferase by resonance energy transfer

Glycosyltransferases are important synthetic enzymes for the construction of naturally occurring glycoconjugates as well as for the design of neoglycoconjugates. The assay methods currently available for these enzymes require tedious and time-consuming procedures for separation of products and do not permit continual assay of enzyme activities. As a set of convenient fluorogenic substrates for continuous monitoring of sialyltransferase activities, we designed and synthesized a novel CMP-Neu5Ac derivative with a naphthylmethyl group at the C-9 position and N-acetyllactosamine derivative containing a dansyl group at the terminal position of aglycon. In such substrates, the emission peak of the naphthylmethyl group (lambdaem = 340 nm) of the glycosyl donor is successfully overlapped with the excitation peak due to the dansyl group (lambdaex = 335 nm) of the glycosyl acceptor. A coupling reaction of these two substrates catalyzed by rat liver 2,6-sialyltransferase caused an increase of dansyl fluorescence (lambdaem = 525 nm) and a decrease of naphthylmethyl fluorescence on the basis of resonance energy transfer between two fluorescence probes. The substrates presented here permit continuous fluorescent monitoring of enzymatic sugar combining reactions. Actually, using this time course of enzymatic reactions, kinetic constants of rat liver 2,6-sialyltransferase against glycosyl donor substrates were estimated to be Km = 4.85 microM and Vmax. = 0.119 micromol/min, respectively. This strategy allows precise and efficient analyses of enzyme kinetics not possible with the conventional assay methods for the glycosyltransferases that usually require separation of products from the reaction mixture.     

Chirality inversion in the bilirubin molecular exciton

The bichromophoric pigment bilirubin acts as a molecular exciton in its UV-visible and circular dichroism (CD) spectroscopy. In both polar and nonpolar solvents, an optically active analog, (beta R,beta 'R)-dimethylmesobilirubin-XIII alpha (1), exhibits intense bisignate CD Cotton effects in the region of its long wavelength UV-vis absorption near 400 nm: Delta epsilon(434)(max) + 337, Delta epsilon(389)(max) - 186 (CHCl(3)), and Delta epsilon(431)(max) + 285, Delta epsilon(386)(max) - 177 (CH(3)OH). However, introduction of an amine into a CHCl(3) solution of 1 causes the Cotton effect signs to become inverted, e.g., after addition of NH(3), Delta epsilon(433)(max) - 345, Delta epsilon(389)(max) + 243, and after addition of ethylene diamine, Delta epsilon(435)(max) - 420, Delta epsilon(390)(max) + 299. The sign inversions imply inversion of molecular chirality of the bilirubin and the phenomenon appears to be general for amines, including alpha,omega-diamines. 1,8-Diaminooctane was found to be more effective than longer or shorter chain analogs in producing CD sign inversion.     

Metalloinitiation routes to biocompatible poly(lactic acid) and poly(acrylic acid) stars with luminescent ruthenium tris(bipyridine) cores

Poly(lactic acid) (PLA) and poly(acrylic acid) (PAA) biomaterials with luminescent ruthenium tris(bipyridine) centers couple drug delivery and imaging functions. Hydrophobic [Ru(bpyPLA2)3](PF6)2 (1) was generated from [Ru[bpy(CH2OH)2]3](PF6)2 in bulk monomer using 4-(dimethylamino)pyridine as the catalyst. The bromoesters, [Ru[bpy(CH2OR)2]3](PF6)2, [Ru[bpy(C13H27)2][bpy(CH2OR]2](PF6)2 (4), and [Ru[bpy(PLAOR)2]3]2+ (9) (R=COCBr(CH3)2), served as initiators for tert-butyl acrylate (tBA) polymerization. Conversion of PtBA to PAA via hydrolysis affords water soluble materials, [Ru(bpyPAA2)3]2+ (7) and [Ru[bpy(C13H27)2](bpyPAA2)2]2+ (8) and the amphiphilic star polymer [Ru[bpy(PLA-PAA)2]3)](PF6)2 (11), which is soluble in a H2O/CH3CN (1:1) mixture. Luminescence excitation and emission spectra of the Ru polymers were in agreement with the parent [Ru(bpy)3]2+ chromophore (lambdaex=468, lambdaem=621 nm). Lifetimes of tau approximately 700 ns in both air and nitrogen atmospheres are typical for most materials; however, the amphiphilic star block copolymer 11 is quenched by oxygen to some degree. Thermal analysis shows the expected glass transitions for the polymeric ruthenium complex materials.     

Resonance Raman spectroscopy of pyridoxal Schiff bases

Resonance Raman (RR) spectra are reported for amino acid and amine adducts of pyridoxal 5'-phosphate (PLP) and 5'-deoxypyridoxal (5'-dPL) in aqueous solution. For the valine adducts, a detailed study has been carried out on solutions at pH and pD 5, 9, and 13, values at which the pyridine and imine protons are successively ionized, and on the adducts formed from 15N-valine, alpha-deuterovaline, and N-methyl-PLP. Good quality spectra were obtained, despite the strong fluorescence of pyridoxal Schiff bases, by adding KI as a quencher, and by exciting the molecules on the blue side of their absorption bands: 406.7 nm (cw Kr+ laser) for the pH 5 and 9 species (lambda max = 409 and 414 nm), and 354.7 nm (pulsed YAG laser, third harmonic) for the pH 13 species (lambda max = 360 nm). A prominent band at 1646 cm-1 is assigned to the imine C=N stretch via its 13 cm-1 15N shift. A 12 cm-1 down-shift of the band in D2O confirms that the Schiff base linkage is protonated at pH 9. Deprotonation at pH 13 shifts VC = N from 1646 to 1629 cm-1, values typical of conjugated Schiff bases. The strongest band in the spectrum, at 1338 cm-1, shifts to 1347 cm-1 upon pyridine protonation at pH 5, and is assigned to a ring mode with a large component of phenolate C-O stretch. A shoulder on its low-frequency side is assigned to the C4-C4' stretch. Large enhancements of these modes can be understood qualitatively in terms of the dominant resonance structures contributing to the ground and resonant excited states. A number of weaker bands are observed, and assigned to pyridine ring modes. These modes gain significantly in intensity, while the exocyclic modes diminish, when the spectra are excited at 266 nm (YAG laser, fourth harmonic) in resonance with ring-localized electronic transitions.     

On the reaction of molecular oxygen with thiyl radicals: a re-examination

Absolute rate constants for the addition of oxygen to thiyl radicals, i.e. RS. + O2----RSOO., have been determined by applying a new competition method based on RS. formation via one-electron reduction of the corresponding disulphides, and the competition between RS. reacting with O2 and an electron donor such as ascorbate. Bimolecular rate constants have been obtained for the thiyl radicals derived from cysteine (6.1 X 10(7) mol-1 dm3 s-1), penicillamine (2.5 X 10(7) mol-1 dm3 s-1), homocysteine (8.0 X 10(7) mol-1 dm3 s-1), cysteamine (2.8 X 10(7) mol-1 dm3 s-1), 3-thiopropionic acid (2.2 X 10(8) mol-1 dm3 s-1) and glutathione (3.0 X 10(7) mol-1 dm3 s-1), respectively. The values obtained for the O2 addition to the thiyl radicals from glutathione and cysteine are considerable lower (by about two orders of magnitude) than those previously published. This indicates that the RS. + O2 reaction may be of complex nature and is generally a process which is not solely controlled by the diffusion of the reactants.     

Effects of ssDNA sequences on non-sequence-specific protein binding

The circular dichroism (CD) spectra of single-stranded DNAs (ssDNAs) are significantly perturbed by the binding of single-stranded DNA binding proteins such as the Ff bacteriophage gene 5 protein (g5p) and the A domain of the 70 kDa subunit of human replication protein A (RPA70-A). These two proteins have similar OB-fold secondary structures, although their CD spectra at wavelengths below 250 nm differ greatly. The spectrum of g5p is dominated by a tyrosyl L(a) band at 229 nm, while that of RPA70-A is dominated by its beta secondary structure. Despite differences in their inherent spectral properties, these two proteins similarly perturb the spectra of bound nucleic acid oligomers. CD spectra of free, non-protein-bound ssDNAs are dependent on interactions of the nearest-neighboring nucleotides in the sequence. The CD spectra (per mol of nucleotide) of simple repetitive sequences 48 nucleotides in length and containing simple combinations of A and C are related by nearest-neighbor equations. For example, 3 x Deltaepsilon[d(AAC)(16)] = 3 x Deltaepsilon[d(ACC)(16)] + Deltaepsilon[d(A)(48)] - Deltaepsilon[d(C)(48)]. Moreover, nearest-neighbor equations relate the spectra of ssDNAs when they are bound by g5p, indicating that each type of perturbed nearest neighbor has a similar average structure within the binding site of the protein.     

Antioxidant properties of melatonin: a pulse radiolysis study

Various one-electron oxidants such as OH*, tert-BuO*, CCl3OO*, Br2*- and N3*, generated pulse radiolytically in aqueous solutions at pH 7, were scavenged by melatonin to form two main absorption bands with lambda(max) = 335 nm and 500 nm. The assignment of the spectra and determination of extinction coefficients of the transients have been reported. Rate constants for the formation of these species ranged from 0.6-12.5x10(9) dm3 mol(-1) s(-1). These transients decayed by second order, as observed in the case of Br2*- and N3* radical reactions. Both the NO2* and NO* radicals react with the substrate with k = 0.37x10(7) and 3x10(7) dm3 mol(-1) s(-1), respectively. At pH approximately 2.5, the protonated form of the transient is formed due to the reaction of Br2*- radical with melatonin, pKa ( MelH* <=> Mel* + H+) = 4.7+/-0.1. Reduction potential of the couple (Mel*/MelH), determined both by cyclic voltammetric and pulse-radiolytic techniques, gave a value E(1)7 = 0.95+/-0.02 V vs. NHE. Repair of guanosine radical and regeneration of melatonin radicals by ascorbate and urate ions at pH 7 have been reported. Reactions of the reducing radicals e(aq)- and H* atoms with melatonin have been shown to occur at near diffusion rates.     

Determination of thiocyl in biological samples by liquid chromatography with ThioGlo 3 derivatization

Thiocyl (sodium thiosalicylate) belongs to a salicylate group of drugs, thus it has analgesic, antipyretic and anti-inflammatory effects. It possesses metal chelating function because it also belongs to a thiol-containing group of compounds which are well-known chelators. The studies of our research group showed that thiocyl is a promising chelator of lead poisoning due to its antioxidant and metal-chelating abilities. To the best of our knowledge, no methods were currently available for measuring thiocyl in biological samples. Therefore, we developed a reversed-phase HPLC method using fluorescence detection (lambdaex = 365 nm, lambdaem = 445 nm) with a one-step derivatizing reaction between thiocyl and a derivatizing agent-ThioGlo 3 (9-acetoxy-2-(4-(2, 5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)pyenyl)-3-oxo-3H-naphtho[2,1-b]pyran). Most biological thiols (such as N-acetylcysteine (NAC), cysteine (CYS), glutathione (GSH) and homocysteine (HCYS)) do not interfere with the detection of thiocyl by using this technique. The linear range of its calibration curve was determined to be 25-2500 nM, and the detection limit of thiocyl was found to be 3 nM with 20 microL injection volume. The coefficients of variation (CV) for within-run precision and between-run precision ranged from 0.93 to 7.21%. This assay proved to be a rapid, sensitive and simple method for determining thiocyl in biological samples.