The effect of ingested sulfite on active avoidance in normal and sulfite oxidase-deficient aged rats.

The aim of this study was to investigate the possible toxic effects of sulfite on neurons by measuring active avoidance learning in normal and sulfite oxidase (SOX)-deficient aged rats. Twenty-four months of age Wistar rats were divided into four groups: control (C), sulfite-treated group (S), SOX-deficient group (D) and SOX-deficient + sulfite-treated group (DS). SOX deficiency was established by feeding rats with a low molybdenum (Mo) diet and adding 200 ppm tungsten (W) to their drinking water. Sulfite in the form of sodium metabisulfite (25 mg/kg) was given by gavage for six weeks. Active avoidance responses were determined by using an automated shuttle box. Hepatic SOX activity was measured to confirm SOX deficiency. The hippocampus was used for determining the activity of cyclooxygenase (COX) and caspase-3 enzymes and the level of prostaglandin E2 (PGE2) and nitrate/nitrite. SOX-deficient rats had an approximately 10-fold decrease in hepatic SOX activity compared with normal rats. Sulfite did not induce impairment of active avoidance learning in SOX-deficient rats and in normal rats compared with their control groups. Sulfite had no effect on the activity of COX and caspase-3 in the hippocampus. Treatment with sulfite did not significantly increase the level of PGE2 and nitrate/nitrite in the hippocampus.

Determination of andrograpolide sodium bisulphite in Beagle dog plasma by LC-MS/MS and its application to pharmacokinetics.

A sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated for the determination of andrographolide sodium bisulphite (ASB) in dog plasma using dehydroandrographolide (DAG) as an internal standard. Chromatographic separation was achieved on a Hypersil Gold C(18) column (50 mm × 2.1 mm, 1.9 µm) with gradient elution that consisted of methanol and water at a flow rate of 0.2 mL/min. Quantification was done using selected reaction monitoring (SRM) mode to monitor precursor-product ion transitions of m/z 413.2→287.2 for ASB and 331.2→303.3 for DAG at negative ionization mode. Good linearity was obtained over the range of 10-1000 ng/mL and the correlation coefficient was better than 0.99. The intra- and inter-day accuracies ranged from 97.2% to 107.8% and precisions (RSD) were within 13.9%. ASB was found stable under three freeze-thaw cycles, short-term temperature, post-preparative and long-term temperature conditions. The method was successfully applied to a pharmacokinetic study of ASB intravenously administered to Beagle dogs.

Covalent protein binding and tissue distribution of houttuynin in rats after intravenous administration of sodium houttuyfonate.

AIM: To investigate the potential of houttuynin to covalently bind to proteins in vitro and in vivo and to identify the adduct structures. METHODS: Male Sprague-Dawley rats were intravenously injected with sodium houttuyfonate (10 mg/kg). The concentrations of houttuynin in blood, plasma and five tissues tested were determined using an LC/MS/MS method. The covalent binding values of houttuynin with hemoglobin, plasma and tissue proteins were measured in rats after intravenous injection of [1-(14)C]sodium houttuyfonate (10 mg/kg, 150 mCi/kg). Human serum albumin was used as model protein to identify the modification site(s) and structure(s) through enzymatic digestion and LC/MS(n) analysis. RESULTS: The drug was widely distributed 10 min after intravenous injection. The lungs were the preferred site for disposition, followed by the heart and kidneys with significantly higher concentrations than that in the plasma. The extent of covalent binding was correlated with the respective concentrations in the tissues, ranging from 1137 nmol/g protein in lung to 266 nmol/g protein in liver. Houttuynin reacted primarily with arginine residues in human serum albumin to form a pyrimidine adduct at 1:1 molar ratio. The same adduct was detected in rat lungs digested by pronase E. CONCLUSION: This study showed that the ß-keto aldehyde moiety in houttuynin is strongly electrophilic and readily confers covalent binding to tissue proteins, especially lung proteins, by a Schiff's base mechanism. The findings explain partially the idiosyncratic reactions of houttuyniae injection in clinical use.

Quantification of andrographolide sodium bisulphite in urine after intravenous injection to rats by LC-MS/MS.

A liquid chromatography-tandem mass spectrometry method for the determination of andrographolide sodium bisulphite (ASB) in rat urine was established and validated. To our knowledge, the analytical method is the first developed assay for the determination of ASB in urine samples. Dehydroandrographolide (DAG) was used as an internal standard. ASB and DAG were separated on a C(18) column and detected at negative ion mode using the mass transitions of m/z 413.2→287.2 and m/z 331.2→303.3, respectively. Good linearity was obtained over the range of 50-5000 ng/mL and the correlation coefficient was better than 0.99. The intra- and inter-day accuracy at all levels fell in the ranges of 85.8-101.4% and 87.9-97.5%, and the intra- and inter-day precision (RSD) were in the ranges of 4.3-11.2% and 8.4-13.3%, respectively. The recovery ranged from 96.1% to 98.3% and the matrix effects from 96.2% to 98.1%. Good stability was found under tested conditions. The method was successfully applied to a urinary excretion study of ASB in rats following intravenous administration of 80 mg/kg ASB.

A novel administration route of edaravone--II: mucosal absorption of edaravone from edaravone/hydroxypropyl-beta-cyclodextrin complex solution including L-cysteine and sodium hydrogen sulfite.

We examined the pharmacokinetics of edaravone when edaravone/hydroxypropyl-beta-cyclodextrin (HPbetaCD) complex solution, including L-cysteine (L-Cys) and sodium hydrogen sulfite (SHS), was administered intravenously, rectally and via the oral mucosa. In oral mucosal administration, atomized edaravone/HPbetaCD complex solution that contained L-Cys and SHS was sprayed into the mouth of Wistar rats. Oral mucosal and rectal administration of edaravone/HPbetaCD complex solution that contained L-Cys and SHS was compared with that for edaravone/HPbetaCD complex solution without L-Cys and SHS. When edaravone 0.25-1.0 mg was administered intravenously, C(0) and AUC(0-60) were linear. In oral mucosal and rectal administration, C(max) and AUC(0-60) of edaravone/HPbetaCD with L-Cys and SHS were significantly higher than those of edaravone/HPbetaCD without L-Cys and SHS. On the other hand, bioavailability of oral mucosal, rectal and oral administration was about 100, 63.5 and 26.6%, respectively. This study suggested that L-Cys and SHS were useful for the oral mucosal and rectal administration of edaravone.

Spectral analysis of EEG in normal and sulfite oxidase deficient rats under sulfite administration.

This article investigated the possible neurotoxic effect of sulfite in normal and sulfite oxidase (SOX) deficients rats by evaluating EEG spectral analysis. Rats were divided into four groups: control (C), sulfite treated (25 mg/kg) (ST), SOX deficient (SD), and sulfite treated SOX deficient (STSD) groups. The qEEG spectral analyses of two spectral parameters including power and relative power were performed. The mean power of SD group was found to be increased compared to the all other groups and returned to control levels after sulfite administration. The power of the four frequency bands (delta, theta, alpha, beta) of the SD group corresponds to the mean power. EEG relative power increased in the delta band with concomitant decreases in power measured in the alpha frequency range. It was concluded that exogenous administration of sulfite affected the brain electrical activity in SOX deficiency, and improved neuroprotection.

Investigation of nail permeation enhancement by chemical modification using water as a probe.

Our objective was to screen molecules that could interact with keratin in the human nail and thereby improve the topical penetration of actives into and through the nail plate. We used specialized Franz-type diffusion cells for our permeation experiments and water as a marker molecule. Aqueous/hydroalcoholic gels containing the enhancers were spiked with tritiated water and compared with a control (without enhancer). We computed the normalized water flux (defined as a product of flux and nail thickness) for each gel. We defined an enhancement factor for water as the ratio of the normalized water flux from a gel containing enhancer to that of the control. Our results indicate that the chemical structure of the modifier is most important in determining its ability to enhance penetration. The best enhancement effect was obtained using N-(2-mercaptopropionyl) glycine, a mercaptan derivative of an amino acid, in combination with urea. The concentration of each chemical modifier was linearly related to normalized water flux and mercaptan levels were more important that urea levels in penetration enhancement. Barrier integrity of nails was compromised after treatment with effective chemical modifiers. Thus, we have developed a suitable technique to screen nail penetration enhancers using water as a probe.

Determination of total serum sulfite by HPLC with fluorescence detection.

An estimated 500,000 individuals in the US, mostly steroid-dependent asthmatics, suffer severe adverse reactions to sulfites in foods, beverages, and pharmaceutical products. In an attempt to understand the pathogenesis of sulfite hypersensitivity, we have developed an assay for the determination of total serum sulfite by utilizing: (a) reductive release of serum protein-bound sulfite; (b) derivatization of free sulfite with monobromobimane; (c) separation of sulfite-bimane from thiol-bimanes by reversed-phase HPLC; and (d) quantitation of sulfite-bimane by fluorescence detection. The detection limit of this assay was 0.44 mumol/L serum sulfite. The intra- and interassay CVs for total serum sulfite at 5.4 mumol/L were 8.1% and 22.0%, respectively. The standard addition method was used to determine total serum sulfite in normal subjects. More than 70 samples were prepared in 2-3 h, followed by automated overnight analysis. The mean concentrations (+/- SD) of total serum sulfite in female (n = 41) and male (n = 35) donors were 4.63 +/- 2.33 and 5.16 +/- 2.68 mumol/L, respectively (not statistically significant: P = 0.368). The combined mean concentration of total sulfite in both sexes was 4.87 +/- 2.49 mumol/L. There was no correlation between total serum sulfite and total serum cysteine, cysteinylglycine, homocysteine, subject age, serum cobalamin, or serum folic acid. The reference range (mean +/- 2 SD) for total serum sulfite in normal subjects is 0-9.85 mumol/L.

Nitritocobalamin and nitrosocobalamin may be confused with sulfitocobalamin using cation-exchange chromatography.

The previously reported ability of SP-Sephadex C25 column chromatography for partitioning biologically important cobalamins has been modified to include analytical separation of nitritocobalamin (NO2-Cbl) and nitrosocobalamin (NO-Cbl). Gel column dimensions (1.5 x 11.0 cm), a low eluent flow-rate (125 microliters/min), collection of small eluate fractions (160 microliters) plus maintenance of He saturated mobile and gel phases all combined to eliminate ordinarily confusing proximal elution of NO2-Cbl and NO-Cb1 with sulfitocobalamin (SO3-Cbl) and cyanocobalamin. Cobalamin elution profiles from the gel column were monitored by direct radiometric analysis of 57Co-labelled cobalamin standards or competitive intrinsic factor radioassays for cobalamin sample sizes up to 10.0 ng. Failure to implement the chromatographic conditions detailed here totally obscured analysis of NO2-Cbl coexisting with SO3-Cbl in brain tissues for chicks exposed to dietary sulfites and caused oversight of NO-Cb1 normally coexisting in prepared NO2-Cbl standards.

Effect of sodium metabisulphite on bronchial blood flow in conscious sheep: pharmacological modulation.

1. Sodium metabisulphite (MBS) can induce bronchoconstriction in patients with asthma. We investigated the effects of MBS aerosol on bronchial blood velocity (Vbr) and pulmonary resistance in intubated conscious sheep. 2. Bronchial blood velocity was measured by implanting a 20 MHz ultrasonic Doppler flow probe on the common bronchial branch of the bronchoesophageal artery. 3. Inhaled MBS induced a dose-dependent, transient increase in Vbr lasting for a few minutes without any changes in aortic and pulmonary artery pressures. There was some tachyphylaxis of the Vbr response to successive inhalations of MBS. 4. The cholinoceptor antagonist, ipratropium bromide and the H1 and H2 histamine antagonists, chlorpheniramine and cimetidine, had no significant effect on MBS-induced increase on Vbr. The loop diuretic, frusemide, and the anti-inflammatory drug, nedocromil sodium, which both inhibit MBS-induced bronchoconstriction in patients with asthma, were also without effect. 5. We conclude that MBS induces bronchial vasodilatation in conscious sheep, and that this effect is not dependent on the release of histamine or other mediators, or an activation of cholinergic pathways.

Comparison of sulphur dioxide and metabisulphite airway reactivity in subjects with asthma.

BACKGROUND: In asthmatic subjects bronchoconstriction is induced by inhalation of the common food preservatives sulphur dioxide (SO2) and metabisulphite (MBS). SO2 and MBS challenges share many similarities, but it is not known whether they are equivalent. In this study of subjects with mild clinical asthma equivalence was assessed by comparing SO2 and MBS reactivity by estimating the total dose of SO2 inhaled during SO2 and MBS challenges, and by calculating SO2 uptake during both challenges. In addition, as the MBS solutions inhaled were acidic and hyperosmolar, the effect of these factors on MBS responsiveness was investigated. METHODS: Fifteen subjects were challenged on separate days with doubling (0.5 to 8.0 ppm) concentrations of SO2 gas inhaled during three minute periods of isocapnic hyperventilation and MBS administered in doses ranging from 0.1 to 12.8 mumol using the Wright protocol. On two other days SO2 and MBS challenges were preceded by a challenge with phosphate buffered saline (PBS) solutions of pH and osmolarity similar to MBS solutions. Response was measured as the dose or concentration causing a 20% fall in FEV1 (PD20 or PC20). RESULTS: All subjects reacted to MBS and 14 responded to SO2. Geometric mean histamine PD20 was 1.61 mumol (95% confidence interval 0.72 to 3.60). MBS and SO2 airway responsiveness were not significantly related. Estimates of the mean concentration of SO2 inhaled during SO2 and MBS challenges differed, as did estimates of the mean SO2 uptake during both challenges. MBS and SO2 reactivity were not affected by prior challenge with PBS solutions. CONCLUSIONS: SO2 and MBS challenges are not comparable. MBS reactivity was not affected by the hyperosmolar, acidic nature of its solutions.

The metabolism of sulfite in liver. Stimulation of sulfate conjugation and effects on paracetamol and allyl alcohol toxicity.

Sulfite is rapidly oxidized to sulfate in the liver. This was shown both in isolated rat hepatocytes and isolated perfused liver. In addition sulfite treatment resulted in release of GSH originating probably from low molecular disulfides such as GSSG and/or mixed disulfides between GSH and protein sulfhydryl groups. Sulfite was demonstrated to be an efficient precursor for sulfate conjugation. This was demonstrated using paracetamol as a substrate. Sulfite was even more efficient in supplying sulfate for sulfate conjugation than inorganic sulfate. Sulfite was furthermore shown to be protective against the toxicity of both N-acetyl-p-benzoquinone imine (NAPQI), the reactive paracetamol metabolite, and acrolein, a reactive aldehyde which is a metabolite of allyl alcohol. This protection is most likely due to direct reaction between sulfite and these reactive metabolites in a manner similar to that occurring with GSH and other thiols. When NAPQI and acrolein were generated intracellularly in isolated hepatocytes from paracetamol and allyl alcohol, respectively, toxicity was also expressed. In this case sulfite only protected against allyl alcohol induced toxicity and not against paracetamol induced toxicity. The reason for this discrepancy is not clear but may depend on factors such as site of generation of the reactive metabolite or the reactivity of the reactive metabolite.

Accumulation of sulphite by Saccharomyces cerevisiae and Zygosaccharomyces bailii as affected by phospholipid fatty-acyl unsaturation and chain length.

Analyses were made of the fatty-acyl composition of phospholipids from each of two strains of Saccharomyces cerevisiae and Zygosaccharomyces bailii grown aerobically. Residues of C16:0, C16:1 and C18:1 predominated in phospholipids from strains of the first yeast, while phospholipids from Z. bailii contained mainly C16:0, C18:1 and C18:2 residues. S. cerevisiae NCYC 431, grown anaerobically in media supplemented with ergosterol and C14:1, C16:1, C18:1, C18:2, C18:3 or C20:1 fatty acids, contained phospholipids enriched with residues of the exogenously provided acid, to a greater extent with shorter chain than longer chain acids. A plot of the permeability coefficient for sulphite, derived from Woolf-Eadie plots, against the degree of unsaturation in phospholipids (expressed as delta mol-1 value) showed that the coefficient was greater the lower the degree of unsaturation in the phospholipids. A plot of the permeability coefficient against values for the mean fatty-acyl chain length divided by the delta mol-1 value, which is an approximation of the cross-section surface area of a phospholipid molecule, showed that the permeability coefficient tended to increase the greater the surface-area value.

Reactions of Saccharomyces cerevisiae and Zygosaccharomyces bailii to sulphite.

Sulphite inhibited growth of all four yeasts studied, Zygosaccharomyces bailii NCYC 563 being most sensitive and Saccharomyces cerevisiae NCYC 431 the least. Vertical Woolf-Eadie plots were obtained for initial velocities of 35S accumulation by all four yeasts suspended in high concentrations of sulphite. Equilibrium levels of 35S accumulation were reached somewhat faster with strains of S. cerevisiae than with those of Z. bailii. With all four yeasts, the greater the extent of 35S accumulation, the larger was the decline in internal pH value. Growth of S. cerevisiae TC8 and Z. bailii NCYC 563, but to a lesser extent of S. cerevisiae NCYC 431 and Z. bailii NCYC 1427, was inhibited when mid exponential-phase cultures were supplemented with 1.0 or 2.0 mM-sulphite, the decrease in growth being accompanied by a decline in ethanol production. Unless growth was completely inhibited, the sulphite-induced decline in growth was accompanied by production of acetaldehyde and additional glycerol.