A novel method for the analysis of 3-mercaptopyruvate using high-performance liquid chromatography with fluorescence detection.

3-Mercaptopyruvate (3-MP) is a metabolite of cysteine present in mammalian tissues and is known to be a substrate of 3-mercaptopyruvate sulfurtransferase (3MST, EC.3.4.1.2). The physiological relevance of the 3-MP pathway has not been fully recognized because the metabolic behavior of 3-MP remains unclear. Here, we describe a novel method using high-performance liquid chromatography with fluorescence detection to measure 3-MP formation from cysteine. To demonstrate the practical value of the present method, we applied it to analyze the 3-MP produced in biological samples from mouse tissue.

[Development of assay methods for endogenous inorganic sulfur compounds and sulfurtransferases and evaluation of the physiological functions of bound sulfur].

Inorganic sulfur compounds, such as S(2-), SO(3)(2-) and S(2)O(3)(2-), are produced from sulfur- containing amino acids as intermediary metabolites in mammalian tissues through complex pathways and are ultimately incorporated into sulfate. Reduced sulfur is also produced via the desulfuration of cysteine by several sulfurtransferases present in mammalian tissues; these enzymes include gamma-cystathionase (gamma-CST), and 3-mercaptopyruvate sulfurtransferase (3-MST). This reduced sulfur is then incorporated into pools of active reduced sulfur (sulfane sulfur; polysulfides, polythionates, thiosulfate, thiosulfonates and elemental sulfur) that are involved in the detoxication of cyanide and in the biosynthesis of iron-sulfur cluster. Sulfane sulfur is labile and is reduced to H(2)S by reducing agents. The physiological function of these sulfur species is less clear. We have found that a reduced sulfur species is commonly present in mammalian sera and tissues as a high molecular weight material and as both a high and a low molecular weight material, respectively; we designated this sulfur species as "bound sulfur." Bound sulfur can be easily liberated as sulfide by reduction with DTT. This review describes sensitive and specific assay method for determining the presence of inorganic sulfur compounds as well as bound sulfur and related sulfurtransferases in biological samples. The physiological functions of bound sulfur in rat tissues were also evaluated using these assay methods. Bound sulfur was found to be located primarily in the rat liver cytosolic fraction in the form of high molecular weight components. The capacity of bound sulfur production was enriched in the cytosol fraction and depended on gamma-CST. Bound sulfur also affected redox regulation by modifying active thiol residues in some liver cytosol enzymes and effectively inhibited cytochrome P-450-dependent lipid peroxidation induced by CCl(4) and t-BuOOH.

Arsenic accumulation decreased in metallothionein null Cisplatin-resistant cell lines.

Metallothionein (MT) is known to play an important role in the resistance of tumor cells to cis-diamminedichloroplatinum (II) (cisplatin). To identify non-MT factors of cisplatin resistance, we characterized cisplatin-resistant cell lines derived from MT-null cells. All of the cisplatin-resistant MT- null cell lines, namely MKCr-1, -3, -4, -12, and -13, showed strong cisplatin resistance and decreased platinum accumulation. Some multidrug resistance proteins (MRPs) have been reported to contribute to cisplatin resistance. However, no significant difference of the MRPs was observed in any of these cell lines. The MKCrs showed cross-resistance to other metals such as arsenite, arsenate, cadmium and antimony. The arsenate and arsenite sensitivities were highly correlated with sensitivity to cisplatin. In addition, the degree of arsenite accumulation was correlated with the degree of cisplatin accumulation. These results suggest that the cisplatin resistance was strongly correlated with the arsenite transport mechanism in these cells.

Determination of plasma thiol bound to albumin using affinity chromatography and high-performance liquid chromatography with fluorescence detection: ratio of cysteinyl albumin as a possible biomarker of oxidative stress.

We examined the influence of oxidative stress on the relative amounts of various albumin-bound thiols in human plasma. To determine the ratio of thiols existing as mixed disulfides following oxidation, we developed a method combining fast purification of albumin using affinity columns and high-performance liquid chromatography (HPLC) with fluorescence detection for low molecular weight thiols which were labeled after reduction. When the effect of exposure of plasma to radical oxygen species on binding of thiols to albumin was determined by the present method, significant increases in the ratio of cysteine bound to albumin (Alb-Cys) to total cysteine were clearly demonstrated.

Aluminum inhibits proteolytic degradation of amyloid beta peptide by cathepsin D: a potential link between aluminum accumulation and neuritic plaque deposition.

Neuritic plaques are the key pathological feature of Alzheimer's disease, and amyloid beta (Abeta) peptides are major component of these plaques. In this study, we demonstrated the influence of aluminum (Al) on the Abeta peptide degradation by cathepsin D. Al did not directly affect the cathepsin D activity using small synthetic substrate. However, when Abeta peptides were used as substrate, the apparent inhibitory effect of Al on cathepsin D activity was observed. This inhibitory effect disappeared by treatment of desferrioxamine. These results indicate that Al has the potential to interact and disrupt Abeta peptide catabolism via the inhibition of proteolytic degradation.

Measurement of homocysteine thiolactone hydrolase activity using high-performance liquid chromatography with fluorescence detection and polymorphisms of paraoxonase in normal human serum.

We developed a non-radioactive and sensitive assay method for measurement of the HTL hydrolase (HTLase) activity in biological samples, using OPA as a fluorescent post-labeling agent, l-homocysteine thiolactone (L-HTL) as the substrate, and HPLC to achieve rapid and selective separation of the substrate and product. The method was applied to measure the activity of HTLase in human, rabbit, rat and mouse serum samples. In addition, the correlation between the serum HTLase activity and PON1 polymorphisms in Japanese subjects was also investigated. The serum HTLase activity in humans, as determined by measurement of the enzyme activity in 22 subjects, was found to be in the range of 0.89-2.06 nmol/min mg protein, with a mean activity of 1.44 nmol/min mg protein.

Accumulation of aluminum in ferritin isolated from rat brain.

The neurotoxic effects of aluminum have been widely reported but the mechanism of action and detoxification is poorly understood. To investigate the toxic potential of aluminum, we found it necessary to detail the behavior of absorbed aluminum in brain. The aim of this study was to clarify the distribution of aluminum in the brain. Rats were exposed to aluminum lactate intraperitoneally for 7 weeks. Although no marked differences in aluminum content was observed in brain regions, aluminum was eluted by gel filtration chromatography of the ferritin fraction from aluminum-loaded brain extracts; 5.9% of the total brain aluminum was recovered in purified ferritin from aluminum-loaded rat brains. These results suggest that ferritin may function as an aluminum detoxicant in the cell.

Novel camptothecin analogues that circumvent ABCG2-associated drug resistance in human tumor cells.

Irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin; CPT-11) is a widely used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I); however, overexpression of ABCG2 (BCRP/MXR/ABCP) can confer cancer cell resistance to SN-38, the active form of CPT-11. We have recently demonstrated that plasma membrane vesicles prepared from ABCG2-overexpressing PC-6/SN2-5H cells transported SN-38 and its glucuronide conjugate in an ATP-dependent manner (Nakatomi et al., Biochem Biophys Res Commun 2001;288:827-32). In the present study, we have characterized a total of 14 new camptothecin (CPT) analogues with respect to both the inhibition of Topo I and the substrate specificity of ABCG2. All of the tested CPT analogues, which have different substitutions at positions 10 and 11, strongly inhibited the Topo I activity in a cell-free system, as did SN-38. Their antitumor activities in the SN-38-resistant PC-6/SN2-5H2 cell line greatly varied, however, being correlated with intracellular accumulation levels. We have examined ATP-dependent transport of those CPT analogues by using plasma membrane vesicles prepared from both PC-6/SN2-5H2 cells and ABCG2-transfected HEK-293 cells. Based on the substrate specificity of ABCG2 thus evaluated, it is strongly suggested that CPT analogues with high polarity are good substrates for ABCG2 and are therefore effectively extruded from cancer cells. In this context, to circumvent ABCG2-associated drug resistance, low-polarity CPT analogues are considered to be potent lead compounds. The present study provides a practical approach to discover new CPT-based drugs for the chemotherapy of drug-resistant human cancer.

Simple non-ion-paired high-performance liquid chromatographic method for simultaneous quantitation of carboxylate and lactone forms of 14 new camptothecin derivatives.

SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite derived from the semi-synthetic compound camptothecin (CPT) named Irinotecan (CPT-11). The antitumor activity of SN-38 is 1000-fold more potent than the parent CPT-11. Fourteen new derivatives of camptothecin have recently been developed by Yakult Honsha (Tokyo, Japan). Here we describe a simple and cost-effective high-performance liquid chromatography (HPLC) method without an ion-pairing agent, which allows the simultaneous determination of both lactone and carboxylate forms of SN-38 and other camptothecin derivatives. A weak linear relationship between the HPLC retention factors (ln k') and the cellular concentrations of these compounds was observed. These results suggest that low-polarity compounds easily accumulate in cancer cells and may circumvent drug resistance. The HPLC analysis herein described is expected to greatly assist in derivative synthesis and chemical modification of camptothecin-based antitumor drugs.

A model of aluminum exposure associated with lipid peroxidation in rat brain.

We have developed a rat model to investigate the relationship between aluminum exposure and aluminum accumulation, and with oxidative damage in brain tissues. Intraperitoneal injections of aluminum lactate for 7 wk (the total aluminum dosage per rat was approx 100 mg) significantly increased aluminum levels in the brain. The concentration of lipid peroxidation products (thiobarbituric acid-reactive substances [TBARS]) also increased in the brain following aluminum lactate injections. No significant correlations between the concentrations of aluminum and of TBARS were found in the whole brain. Subcellular analysis revealed that aluminum lactate injections led to a significant increase in the concentration of aluminum in the mitochondrial fraction but had no significant effect on the concentration of peroxides in any subcellular fraction. These results suggest that aluminum accumulation induced by the aluminum lactate administration associates with the acceleration of lipid peroxidation in rat brain. Furthermore, these data indicate that the pro-oxidant effect of aluminum may be indirect and concentration independent. The experimental conditions used here provide an animal model of aluminum accumulation in the brain that should prove useful for further investigations of the mechanisms of aluminum neurotoxicity.

Enzymatic assay of gamma-cystathionase activity using pyruvate oxidase-peroxidase sequential reaction.

A highly sensitive method has been developed for the determination of gamma-cystathionase (EC. 4.4.1.1.) activity in rat tissues using beta-chloro-L-alanine as a substrate. This method is based on colorimetry for the determination of pyruvate produced from beta-chloro-L-alanine with the beta-elimination catalyzed by gamma-cystathionase, coupling a color enzymatic reaction with pyruvate oxidase and peroxidase. The absorbance increases with the oxidized color of a leuco dye, N-(carboxymethylamino)-4,4'-bis (dimethylamino)-diphenylamine at 727 nm is proportional to the gamma-cystathionase activity. The present method is more sensitive and more rapid than the usual methods and does not require troublesome steps such as centrifugation. The calibration curve is linear up to 1.6 microg of partially purified enzyme (100 U/l). Comparison with the usual method with L-homoserine as a substrate gave good correlation (r=0.990). The present method was applied to the determination of gamma-cystathionase activity in adult male rat tissues. The mean activities in liver and kidney were 8.03 and 3.91 U/g wet weight (n=10), respectively.

Effects of the administration routes and chemical forms of aluminum on aluminum accumulation in rat brain.

An experimental rat model of aluminum accumulation in the brain was developed to aid in determining neurotoxity of aluminum (Al). Al was administered orally, intravenously, and intraperitoneally, in the absence or presence of citric acid or maltol. Oral administration of Al hydroxide [Al (OH)3] or aluminum chloride (AlC13) with citric acid for 7 wk was not found to increase brain Al levels. Similarly, a single intravenous injection of AlCl3 in the presence or absence of either citric acid or maltol did not alter brain Al levels after 48 h. Only daily intraperitoneal injections of AlC13 (8 mg Al/kg body weight) and an equimolar amount of maltol over a 14-d period enhanced accumulation of Al in rat brain. No significant increases were observed for the experimental groups receiving intraperitoneal AlCl3 alone or with citric acid. This result suggests that the chemical form of Al strongly influences its bioavailability and that intraperitoneal administration of the Al-maltol complex appears to be useful in creating subacute model of Al accumulation in brain tissue.

Determination of homocysteine thiolactone and homocysteine in cell cultures using high-performance liquid chromatography with fluorescence detection.

A sensitive and simple method utilising fluorometric detection for the simultaneous routine monitoring of homocysteine thiolactone (HTL) and homocysteine (Hcy) in biological samples has been developed. Separation relies on isocratic ion-pairing and reversed-phase chromatography while the principle of the detection is that the lactone ring in HTL molecule is cleaved with an alkali to produce Hcy, which reacts with ortho-phthalaldehyde (OPA) in the absence of an added thiol reagent to form a stable fluorescent derivative. The method has a sensitivity of 200 fmol of HTL and 100 fmol for Hcy in the sample. The present method was applied to the determination of HTL and Hcy in Hep G2 cell.