Determination of inorganic sulfate in human saliva and sweat by controlled-flow anion chromatography. Normal values in adult humans

Following the previous demonstration that low concentrations of inorganic sulfate (SO4) in human serum and cerebrospinal fluid can be accurately determined by controlled-flow anion chromatography, the assay has been extended to the quantitation of free SO4 in saliva and sweat by modification of the established methods of sample collection and preparation. Salivary secretions were ultrafiltered to remove macromolecular polyanions that bind irreversibly to the anion-exchange separator column and reduce resolution. Sweat was collected from 22 fasted adult volunteers using a method which utilizes absorbent filter pads applied to the forearm after secretion had been stimulated by pilocarpine iontophoresis. It was necessary to acid wash the filter pads to reduce sulfate contamination. Saliva ultrafiltrate or sweat was diluted and injected onto a Dionex D-10 Ion Analyzer using the standard anion column system. The mean inorganic SO4 concentration in saliva from seventeen adult fasting volunteers was 72 +/- 4 mumol/l (+/- S.E.); the mean SO4 concentration in sweat was 83 +/- 3 mumol/l. Both are significantly less than in matching serum, suggesting that SO4 is actively removed during formation of these glandular secretions. The ion chromatographic assay is shown to be capable of measuring SO4 in biological fluids at concentrations that are otherwise undetectable by conventional assay techniques.     

Determination of ultrafiltrable zinc in human milk by electrothermal atomic absorption spectrometry.

Percentages of non-protein-bound zinc in human milk have been reported by different workers, but ultrafiltration and zinc determination in human milk have not been comprehensively examined. However, zinc contamination and zinc membrane binding have been described for the determination of non-protein-bound zinc in serum. In this work, ultrafiltration was studied in terms of zinc contamination and zinc membrane binding. An MPS-1 micropartition system fitted with a YMT membrane was used. Zinc contamination was found to be less than 276 nmol dm-3 and the zinc recovery was 85 +/- 4%. The conditions for electrothermal atomic absorption spectrometry were also studied. The detection limit was found to be 26.4 nmol dm-3 and the upper linear range was 4 mumol dm-3. The precision varied from 3% (within-run) to 17% (between-run). The recovery of standard additions was 95 +/- 7% (n = 30, different human milk ultrafiltrate samples). Physiological values varied from 0.46 to 84 mumol dm-3 (4-56% of zinc in whole human milk). Expressed in mumol dm-3, zinc in human milk ultrafiltrate decreased slightly through the lactation period, whereas expressed as a percentage of the total zinc in milk, zinc in human milk ultrafiltrate remained constant from day 2 to day 69 post partum.     

Gas chromatographic-mass spectrometric study of deacetylation and oxidation of 2-acetylaminofluorene by rat liver epithelial cell lines upon cocarcinogen induction

Cell line cultures from postnatal and adult rats were incubated with 5-100 mumol/l [9-14C]-2-acetylaminofluorene. On incubation of 10 mumol/l, ring-hydroxylated metabolites, expressed as nmol hydroxy-2-acetylaminofluorene (OH-2-AAF)/mg cell protein/24 h, were 9-OH- 1.28 +/- 0.37, 7-OH- 1.08 +/- 0.28 and 5-OH- 0.30 +/- 0.08, and deacetylated 2-AAF (2-AF) 1.20 +/- 0.18. For 5, 10, 50 and 100 mumol/l 2-AAF, the total production of OH-2-AAF (same units) and 2-AF (%) were, respectively, 0.86 (0%), 3.86 (35%), 17.8 (60%) and 35.03 (89%). On preincubation with phenobarbital (BP) or 3-methylcholanthrene (3-MC) and then incubation of 10 mumol/l 2-AAF, the total synthesis of OH-2-AAF increased 1.9-fold (PB) and 2.5-fold (3-MC). In addition, four other OH-2-AAF (1-OH-, 3-OH- and two unknown OH-2-AAF) were produced and glucuronidation of all metabolites was induced and amounted to 57% of the total after PB and 75% after 3-MC preincubation. Metyrapone or alpha-naphthoflavone inhibition of BP or 3-MC, respectively, markedly affected the production of free and conjugated metabolites and, almost completely, the deacetylation of 2-AAF.     

Rapid method for the determination of organochlorine pesticides in milk

This method involved one step solvent extraction of milk with ethyl acetate-acetone-methanol by ultrasonication. The supernatants were further cleaned-up and enriched by solid-phase extraction using octadecyl (C18)-bonded silica cartridges, then assayed by capillary gas-liquid chromatography with electron capture detection. The recoveries of eleven organochlorine pesticides (OCPs) from raw milks were quantitative, ranging from 90-110% at 10 times the limit of detection (LOD). The LOD ranged from 0.5 micrograms/l whole milk for alpha-hexachlorocyclohexane to 2.5 micrograms/l whole milk for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane. The day-to-day variation of the method was evaluated over 7 days using 3 different pools of spiked cow milks (at the LOD, 5 and 10 times the LOD). The coefficient of variations (C.V.s) were 16 +/- 6, 10 +/- 2 and 9 +/- 3% (mean S.D.), respectively. The method showed no emulsion problems common with conventional non-polar solvent extraction, and the use of solid-phase extraction considerably reduced the sample clean-up process compared with the existing methods. The method also showed that OCPs in milk could be extracted quantitatively without extraction of total fat, and that OCPs spiked into cows milk could be used to construct calibration curves for human milk determinations.     

高效液相色谱法测定大鼠神经组织中的硝酸盐和亚硝酸盐

 建立了大鼠神经组织中亚硝酸盐和硝酸盐的高效液相色谱测定方法,以用于研究一氧化氮在糖尿病慢性神经病变中的作用。在1 μmol/L～25 μmol/L的浓度范围内,NO2-和NO3-的峰面积与浓度的线性相关系数>0.991;最低检测浓度分别为0.2 μmol/L和0.5 μmol/L;日内、日间相对标准偏差<14%。对各实验组大鼠的初步测定结果表明,糖尿病组及糖尿病胰岛素(IGF)治疗组的NO2-和NO3-水平均低于对照组。 关键词：高效液相色谱法;一氧化氮;硝酸盐;亚硝酸盐;糖尿病神经病变     

Fluorimetric determination of the levels of urinary neopterin and serum thiobarbituric acid reactive substances in the nonagenarians

Twelve self-sustaining nonagenarians, 10 women and two men, aged 94+/-3 years, and eight institutionalised nonagenarians, eight women, aged 91+/-1 year as well as 11 control subjects, seven women and four men, aged 84+/-5 years entered the study. Urinary neopterin, an indicator of systemic immune activation, and serum thiobarbituric acid reactive substances (TBARS), a marker of lipoperoxidation, were determined initially, and collection of the blood and urine samples was repeated at 3-month interval. Neopterin was measured in the urine specimens by reversed-phase high performance liquid chromatography. A C(18) reversed-phase column 3.3x150 mm, 5 mum-diameter packing Separon SGX was used. Potassium phosphate buffer (15 mmol l(-1), pH 6.4) at flow rate of 0.8 ml min(-1) was used as mobile phase. After centrifugation (5 min, 1300xg) and diluting 100 mul of urine specimens with 1.0 ml of mobile phase containing 2 g of disodium-EDTA per litre, a 20 mul sample was injected on a column. Neopterin was identified by its native fluorescence (353 nm excitation, 438 nm emission). Creatinine was determined by Jaffé kinetic reaction after dilution of sample 1:50 (v/v). The concentration of neopterin in urine was expressed as neopterin/creatinine ratio (mumol mol(-1) creatinine). TBARS were determined spectrofluorometrically using LS-5 spectrofluorimeter (excitation wavelength 528 nm, emission wavelength 558 nm) after extraction with n-butanol treatment with thiobarbituric acid. The significance of differences between nonagenarians and control group was examined by ANOVA-Kruskal-Wallis tests, using statistical software NCSS 6.0.21 (Kaysville, UT, 1996). The decision on significance was based on P=0.05. Urinary neopterin was significantly higher in institutionalised compared to self-sustaining subjects and controls (625+/-565 vs. 203+/-63 mumol mol(-1) creatinine, and 198+/-128 mumol mol(-1) creatinine, respectively, P=0.006). The serum TBARS were higher in both groups of nonagenarians (3.23+/-1.16 mumol l(-1) and 2.69+/-0.39 vs. 2.12+/-0.83 mumol l(-1) for the self-sustaining, institutionalised and controls, respectively, P=0.023). We conclude that the fluorimetric determinations of urinary neopterin and serum TBARS can be useful for the monitoring health status in the elderly patients.     

Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N(epsilon)-fructoselysine (FL), N(epsilon)-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34+/-3.81 nmol CML per micromol of free Lys (Lys(free)) and 81.5+/-87.8 nmol Pyr micromol(-1) Lys(free)(-1) vs. 3.72+/-1.29 nmol FL micromol(-1) Lys(free)(-1). In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47+/-0.08 nmol FL micromol(-1) of protein-bound Lys (Lys(p-b)), 0.04+/-0.03 nmol CML micromol(-1) Lys(p-b)(-1) and 0.06+/-0.02 nmol Pyr micromol(-1)Lys(p-b)(-1). It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.     

High-performance liquid chromatographic determination of formate as benzimidazole in biological samples

Formate was determined as benzimidazole by high-performance liquid chromatography after reaction with o-phenylenediamine at 130 degrees C for 2 h in 1 M perchloric acid. The useful concentration range was 1.6-40 mumol/l and the determination limit was 20 pmol. The recoveries from rat liver homogenate and human urine were 90.3 +/- 2.9 and 89.4 +/- 2.5%, respectively. Using this method, the activity of formaldehyde dehydrogenase in biological samples could be measured, and also the formate concentration in the liver and urine of rats to which methanol had been administered.     

Three component flow injection analysis with on-line dialysis. Simultaneous determination of free calcium, total calcium and total chloride in milk by flow injection analysis and on-line dialysis

Summary A fast and reliable fully automated three-component flow injection procedure for the simultaneous determination of free calcium, total calcium and total chloride in milk is described where the three components from a single sample injection (30 l milk samples) are determined at a sampling rate of 60 samples per hour. The samples are directed to three different channels by using two dialysers in series. Interferences in the determination of free calcium are eliminated by using a dialyser (first in the series) which also separates the total and free calcium. Free calcium is determined by UV/VIS spectrophotometry at 580 nm and total calcium by atomic absorption spectrometry (AAS) at 422.7 nm. Interferences from phosphates in milk in the determination of total calcium by AAS are overcome by using a nitrous oxide-acetylene flame with the necessary suppression with potassium ions. A second dialyser in series is used to eliminate interferences, especially the interference of casein, before the dialysed chloride is measured with a coated tubular chloride-selective electrode. The results obtained for the free calcium, total calcium and total chloride in milk at a sampling rate of 60 samples per hour compared well with data obtained by standard methods. With 30 l samples the relative standard deviation for milk samples having different concentrations of free calcium (110–170 mg/l free calcium), total calcium (1000–1500 mg/l total calcium) and total chloride (1000–1800 mg/l total chloride) were better than 0.37, 1.01 and 0.25, respectively.     

A simple flow injection system with bead injection for trace iron determination

A simple and low cost flow injection (FI) system with bead injection (BI) was developed for determination of low concentration (mumol l(-1)) of iron in water samples. Chelex-100 chelating resin beads, trapped in a jet ring cell, were employed. The intensity of red complex of 1,10-phenanthroline with Fe(2+) was monitored using colorimetric detector with a LED green light source. Amount of total Fe (Fe(2+) and Fe(3+)) and Fe(2+) can be evaluated by with and without reduction of Fe(3+) using ascorbic acid. Lowest detectable levels of Fe(2+) were 0.90 and 0.45 mumol l(-1) for sample loading time of 3 and 5 min, respectively. Working range was up to 3.90 mumol l(-1) using 0.3% w/v 1, 10-phenanthroline. Percent recoveries of spiked water samples (0.90-2.33 mumol l(-1) of Fe(2+)) were 100-110%.     

High-performance liquid chromatographic separation of malondialdehyde-thiobarbituric acid adduct in biological materials (plasma and human cells) using a commercially available reagent.

The assay of malondialdehyde (MDA) is widely used in clinical chemistry laboratories to investigate lipid peroxidation in oxidative pathologies. In the present work, the thiobarbituric acid (TBA) reaction was carried out on plasma, human erythrocytes and fibroblasts. The reagents used were those of the fluorimetry MDA kit manufactured by Sobioda. We have defined the application of this kit to high-performance liquid chromatography. This adaptation satisfied the criteria of good analytical practice. The detection limit was 2.5 pmol per injection. The retention time of the MDA-TBA2 peak (4.96 +/- 0.07 min) led to excellent resolution of the complex. The within-assay (6-12%) and between-assay (11-12%) precisions were satisfactory. The analytical recovery of MDA after spiking samples of human plasma with tetraethoxypropane standards varied from 70 to 100%. The mean lipoperoxide concentration determined in 32 healthy adults (20-40 years) was 1.04 +/- 0.23 mumol l-1 in plasma. Applied to the erythrocytes of fifteen laboratory workers, the method furnished physiological values of 0.59 +/- 0.21 mumol l-1. Concentrations were significantly higher in chronic renal dialysis patients (4.15 +/- 2.35 mumol l-1. The MDA content of fibroblasts cultured in standard medium was 0.38 +/- 0.04 mumol per g of protein and increased (5.78 +/- 1.38 mumol per g of protein) if the cells were grown in an iron-enriched medium. This accurate high-performance liquid chromatographic method for detection of MDA is the first one which can be applied to plasma, red blood cells and cultured cells. This technique will prevent false positives and should make inter-laboratory comparisons possible.     

Simultaneous determination of myocardial creatine phosphate and adenine nucleotides by reversed-phase HPLC

An isocratic HPLC system has been developed which allows for the rapid (single run of 20 min) measurement of creatine phosphate (PCr) and adenine nucleotides (ATP, ADP and AMP) in extracts from freeze-clamped and freeze-dried myocardial tissues. The separation was achieved at room temperature by using a RP18 column and a dual variable wavelength spectrophotometer, set at 210 and 254 nm. The solvent was 30 mM potassium dihydrogen phosphate, 15 mM tetrabutylammonium hydrogen sulfate, pH 6.7, 19% (v/v) acetonitrile. A distinct separation (confirmed with the retention time of standard sample) of these high energy compounds was achieved. Standard curves were linear. In isolated rat hearts the following values were obtained (mumol/g dry wt, mean +/- SEM): ATP 21.5 +/- 1.3, ADP 4.6 +/- 0.2, AMP 1.5 +/- 1.1 and PCr 32.5 +/- 1.3; which are consistent with previously published values for high energy compounds in this tissue.     

Determination of 2-mercaptopropionylglycine in plasma and urine by high-performance liquid chromatography

Methods for quantitative analysis of total and non-protein-bound 2-mercaptopropionylglycine (2-MPG) in plasma, and total 2-MPG in urine, have been developed. By reduction of urine, plasma or deproteinized plasma samples with tributylphosphine, 2-MPG is liberated from its disulphides, and after clean-up of the sample, 2-MPG is derivatized with N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM). The 2-MPG-DACM derivative is then quantified by high-performance liquid chromatography (HPLC) with fluorimetric detection. Both ion-suppression and ion-pair HPLC gave satisfactory chromatograms. The precision of the methods was satisfactory (coefficient of variation 3.1-5.8%), analytical recovery was quantitative (85-99%) and the two HPLC techniques were well correlated (r = 0.99). Five healthy subjects receiving 500 mg of 2-MPG showed maximal total plasma concentration of 13.8-26.9 mumol/l at 3-5 h after intake, and their non-protein-bound 2-MPG was, at the same time, 62-77% of the total 2-MPG. The urinary excretion was 27.8 +/- 3.8% (mean +/- S.D.) of the given dose, most of it excreted within 12 h after intake.     

Analysis of Free Amino Acids and Protein Contents of Mature Human Milk from Turkish Mothers

Abstract

This study was designed to evaluate free amino acid (FAA) composition and total protein in mature human milk from Turkish mothers. Free amino acid concentrations in mature human milk were determined in all subjects using a high‐performance liquid chromatography (HPLC) with postcolumn derivatization system, with a fluorescence detector. Total protein content was determined by the classical biuret method. Total protein concentration was found to be 1.3±0.4 mg/dl. Glutamic asid plus glutamine is the most abundant amino acid (1275 µmol/L), followed by taurine (353 µmol/L) and alanine (261 µmol/L). Glutamic acid plus glutamine accounts for the most free amino acids in mature human milk and their sum represents 40% of total FAA. On the other hand, some amino acid derivatives such as citrulline, ethanolamine, ammonium, ornithine, ortophosphoserine, and phosphoethanolamine, not usually a part of protein, are determined and this fraction represented ～21% of the total FAA in mature human milk in the present study.     

Correlation of free radical yields with strand break yields produced in plasmid DNA by the direct effect of ionizing radiation

The purpose of this study was to determine how free radical formation (fr) correlates with single strand break (ssb) and double strand break (dsb) formation in DNA exposed to the direct effects of ionizing radiation. Chemical yields have been determined of (i) total radicals trapped on DNA at 4 K, G(Sigmafr), (ii) radicals trapped on the DNA sugar, Gsugar(fr), (iii) prompt single strand breaks, Gprompt(ssb), (iv) total single strand breaks, Gtotal(ssb), and (v) double strand breaks, G(dsb). These measurements make it possible, for the first time, to quantitatively test the premise that free radicals are the primary precursors to strand breaks. G(fr) were measured by EPR applied to films of pEC (10,810 bp) and pUC18 (2686 bp) plasmids hydrated to Gamma = 22 mol of water/nucleotide and X-irradiated at 4 K. Using these same samples warmed to room temperature, strand breaks were measured by gel electrophoresis. The respective values for pEC and pUC18 were G(fr) = 0.71 +/- 0.02 and 0.61 +/- 0.01 micromol/J, Gtotal(ssb) = 0.09 +/- 0.01 and 0.14 +/- 0.01 micromol/J, G(dsb) = 0.010 +/- 0.001 and 0.006 +/- 0.001 micromol/J, and Gtota)(ssb)/G(dsb) approximately 9 and approximately 20. Surprisingly, Gsugar(fr) approximately 0.06 mumol/J for pUC18 films, less than half of Gtotal(ssb). This indicates that a significant fraction of strand breaks are derived from precursors other than trapped DNA radicals. To explain this disparity, various mechanisms were considered, including one that entails two one-electron oxidations of a single deoxyribose carbon.     

Determination of total sulfite in wine. Zone electrophoresis-isotachophoresis quantitation of sulfate on a chip after an in-sample oxidation of total sulfite

This work deals with the determination of total sulfite in wine. The determination combines an in-sample hydrogen peroxide oxidation of total sulfite in alkalized wine to sulfate with the separation and quantitation of the latter anion by zone electrophoresis (ZE) on-line coupled with isotachophoresis (ITP) on a column-coupling chip. Sample clean up, integrated into the ITP-ZE separation, eliminated wine matrix in an extent comparable to that provided by a highly selective distillation isolation of sulfite. At the same time, conductivity detection, employed to the detection of sulfate in the ZE stage of the ITP-ZE combination, provided for sulfate the concentration limit of detection corresponding to a 90 microg/l concentration of sulfite in the loaded sample (0.9 microl). Such a detectability allowed a reproducible quantitation of total sulfite when its concentration in wine was 15 mg/l. Formaldehyde binding of free sulfite in wine, included into the pre-column sample preparation, prevented an uncontrolled oxidation of this sulfite form. This step contributed to an unbiased determination of sulfate present in the original wine sample (this determination corrected for the concentration of sulfate determined in the sample after the peroxide oxidation of sulfite to the value equivalent to the total sulfite). The 99-101% recoveries of sulfite, determined for appropriately spiked wine samples, indicate a very good accuracy of the present method. Such a statement also supports excellent agreements of the results of quantitation based on the in-sample peroxide oxidation of the total sulfite (bound sulfite released at a high pH) with those in which this analyte was isolated from wine by distillation (bound sulfite released at a very low pH).     

Quantitative analysis of desmosterol, cholesterol and cholesterol sulfate in semen by high-performance liquid chromatography.

A simple, rapid and accurate method to separate and quantify cholesterol, desmosterol and cholesterol sulfate in human spermatozoa and seminal plasma (SP) is described. This high-performance liquid chromatographic procedure is based on reversed-phase chromatography on a Inertsil ODS2 5 microm silica column with a binary gradient of mixtures of chloroform-methanol and chloroform-methanol-water as the mobile phase at a flow-rate of 0.25 ml/min. Sterols are separated with good resolution and high reproducibility. The eluted sterols are quantified using a light-scattering (mass) detector. As little as 64, 64 and 68 pmol of cholesterol, desmosterol and cholesterol sulfate, respectively, can be quantified under these conditions. Cholesterol is the predominant sterol both in spermatozoa (107+/-7 nmol/10(8) spermatozoa) and SP (0.83+/-0.10 micromol/ml) whereas the concentrations of desmosterol were 38+/-6 nmol/10(8) in spermatozoa and 0.18+/-0.02 micromol/ml in SP. Cholesterol sulfate represents about 6% of total cholesterol in the spermatozoa and SP. In conclusion, this method offers interesting perspectives for the quantitative analysis of these sterols not only in semen, but also in other biological samples.     

HPLC analysis of methylxanthines in human breast milk

A sensitive and specific high-performance liquid chromatographic (HPLC) procedure is developed for simultaneously quantitating the levels of caffeine, theophylline, theobromine and paraxanthine in breast milk. The method involved the precipitation of proteins present in the milk samples with a 6% v/v perchloric acid solution containing the internal standard, proxyphylline, followed by centrifugation at 12,800 Xg for 10 minutes. The clear supernatant was then chromatographed on a C18 reversed-phase analytical column at ambient temperature using a wavelength of 272 nm. Samples were eluted from the column at a constant flow rate of 1.5 mL/min using a gradient program in which the concentration of methanol in the mobile phase varied from 0 to 16%. The mean recoveries of the methylxanthines averaged over all the concentrations examined were generally excellent and ranged from 96.3 +/- 5.4% for caffeine to 102.3 +/- 8.9% for paraxanthine. The assay precision was very good and the peaks of interest were extremely well resolved. The method is recommended for assessing the total caffeine and dimethylxanthine load to which the nursing infant is exposed in mothers ingesting typical amounts of caffeine.     

Determination by high-performance liquid chromatography with electrochemical detection of free and conjugated N-acetyldopamine excretion in urine of children with neuroblastoma and nephroblastoma.

A simple method for the determination of N-acetyldopamine (NADA) (both free and conjugated) in children's urine by high-performance liquid chromatography with electrochemical detection has been developed. Conjugated NADA was measured as the free compound after enzymatic hydrolysis and purification on alumina. The total analysis time is 25 min. The results show a linearity of the whole assay from 0.005 to 20 mumol/l NADA; the sensitivity is 0.2 pmol per 20 microliters injected sample. Mean recoveries of 96.7 and 86.6% were obtained for free and total NADA, respectively. Modifications of the retention times (between 2 and 50 min) induced by changes in the eluent were determined. Conjugated NADA accounted for about 90% of the total excretion of NADA. These results suggest that this compound could play an important part in neuroblastoma; its concentration is thirteen times higher in children with neuroblastoma than in normal subjects.     

Determination of thiols and disulfides in normal rat tissues and hamster pancreas treated with N-nitrosobis(2-oxopropyl)amine using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate

Biological thiols and disulfides in rat and hamster tissues were simultaneously determined by HPLC-fluorescence detection using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). The coefficients of variation (CV) of the method for reduced glutathione (GSH) and oxidized glutathione (GSSG) in liver and for cysteine (CySH) and cystine (CySSCy) in kidney were less than 3.1%. In 11 tissues of Wistar rats (liver, spleen, heart, lung, stomach, bladder, ovary, uterus, adrenal, kidney and pancreas), only CySH, CySSCy, GSH and/or GSSG were detected. Other thiols and disulfides were at extremely low levels in all samples. Both concentrations of CySH and CySSCy in the livers of old rats (111 weeks old, F344) were significantly higher than those of young rats (8 weeks old) (CySH, 0.246 +/- 0.099 vs 0.130 +/- 0.020 mumol/g; CySSCy, 0.051 +/- 0.027 vs 0.013 +/- 0.002 mumol/g). Administration of N-nitrosobis(2-oxopropyl)amine (BOP), a selective carcinogen of hamster pancreatic cancer, to Syrian golden hamsters (38 weeks old) resulted in the increase in the pancreas of GSH to a level 19 times as high and of GSSG to a level 14 times as high as those in untreated hamsters (GSH, 1.173 +/- 0.272 vs 0.062 +/- 0.017 mumol/g; GSSG, 0.155 +/- 0.063 vs 0.011 +/- 0.001 mumol/g).