C.N. Ong

High-performance liquid chromatographic method for routine determination of vitamins A and E and β-carotene in plasma

A simple and reliable reversed-phase high-performance liquid chromatographic (HPLC) method for the routine determination of vitamins A and E and beta-carotene in plasma (or serum) with wavelength-programmed ultraviolet-visible absorbance detection is described. A 200-microliters aliquot of serum or plasma sample, after deproteinization with ethanol, and containing tocopherol acetate as internal standard, was extracted with butanol-ethyl acetate. Sodium sulphate was added for dehydration. Analytes of extracted samples were found to be stable for at least four days. A 10-microliters aliquot of this organic extract was used for HPLC analysis. The mobile phase was methanol-butanol-water (89.5:5:5.5, v/v) and the flow-rate was set at 1.5 ml/min. The analytes of interest were well separated from other plasma constituents within 22 min at 45 degrees C. The lowest detection limits of vitamins A and E and beta-carotene were 0.02, 0.5 and 0.1 microgram/ml, respectively. The recovery and reproducibility of the present method were around 90%. The method is sensitive, specific and can be used for epidemiological studies and for routine determination of vitamin deficiency. Several important factors that may affect the analysis are also discussed in this paper.

Biomarkers of exposure to low concentrations of benzene: a field assessment.

OBJECTIVE: To carry out a comprehensive field investigation to evaluate various conventional and recently developed biomarkers for exposure to low concentrations of benzene. METHODS: Analyses were carried out on environmental air, unmetabolised benzene in blood and urine, urinary trans, transmuconic acid, and three major phenolic metabolites of benzene: phenol, catechol, and hydroquinone. Validations of these biomarkers were performed on 131 never smokers occupationally exposed to the time weighed average benzene concentration of 0.25 ppm (range, 0.01 to 3.5 ppm). RESULTS: Among the six biomarkers studied, unmetabolised benzene in urine correlated best with environmental benzene concentration (correlation coefficient, r = 0.76), followed by benzene in blood (r = 0.64). When urinary metabolites were compared with environmental benzene, trans, trans-muconic acid showed a close correlation (r = 0.53) followed by hydroquinone (r = 0.44), and to a lesser extent with urinary phenol (r = 0.38). No correlation was found between catechol and environmental benzene concentrations. Although unmetabolised benzene in urine correlates best with benzene exposure, owing to serious technical drawbacks, its use is limited. Among the metabolites, trans, trans-muconic acid seems to be more reliable than other phenolic compounds. Nevertheless, detailed analyses failed to show that it is specific for monitoring benzene exposures below 0.25 ppm. CONCLUSION: The overall results suggest that most of the currently available biomarkers are unable to provide sufficient specificity for monitoring of low concentrations of benzene exposure. If a lower occupational exposure limit for benzene is to be considered, the reliability of the biomarker and the technical limitations of measurements have to be carefully validated.

Determination of plasma ascorbic acid by high-performance liquid chromatography with ultraviolet and electrochemical detection

A convenient and reliable reversed-phase liquid chromatographic method for the routine determination of ascorbic acid with ultraviolet detection is described. This system avoids the use of modifier and ion-pairing reagent. The mobile phase consists of 20 mM ammonium dihydrogenphosphate with 0.015% metaphosphoric acid. This method enables the detection of plasma ascorbic acid at a concentration of 120 ng/ml within 5 min. The recovery and reproducibility were above 95%. A comparative study was also performed using ultraviolet and electrochemical detectors. Excellent agreement was observed between the two detection modes, with a correlation coefficient of 0.99. In addition, the storage conditions and stability of ascorbic acid in plasma and whole blood were investigated. The results showed that ascorbic acid was more stable in whole blood when stored below 4 degrees C.

Andrographolide sensitizes cancer cells to TRAIL-induced apoptosis via p53-mediated death receptor 4 up-regulation

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is an important member of the tumor necrosis factor subfamily with great potential in cancer therapy. Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess potent anti-inflammatory and anticancer activities. Here, we showed that pretreatment with Andro significantly enhances TRAIL-induced apoptosis in various human cancer cell lines, including those TRAIL-resistant cells. Such sensitization is achieved through transcriptional up-regulation of death receptor 4 (DR4), a death receptor of TRAIL. In search of the molecular mechanisms responsible for DR4 up-regulation, we found that the tumor suppressor p53 plays an essential role in DR4 transcriptional activation. Andro is capable of activating p53 via increased p53 phosphorylation and protein stabilization, a process mediated by enhanced reactive oxygen species production and subsequent c-Jun NH2-terminal kinase activation. Pretreatment with an antioxidant (N-acetylcysteine) or a c-Jun NH2-terminal kinase inhibitor (SP600125) effectively prevented Andro-induced p53 activation and DR4 up-regulation and eventually blocked the Andro-induced sensitization on TRAIL-induced apoptosis. Taken together, these results present a novel anticancer effect of Andro and support its potential application in cancer therapy to overcome TRAIL resistance. [Mol Cancer Ther 2008;7(7):2170–80]

BENZENE METABOLITES ENHANCE REACTIVE OXYGEN SPECIES GENERATION IN HL60 HUMAN LEUKEMIA CELLS

Benzene is myelotoxic and leukemogenic in humans. The mechanisms leading to these effects, however have not been fully elucidated. One of the underlying mechanisms is believed to be the oxidative damage caused by its metabolites. A comparative study was undertaken to examine the relationships between reactive oxygen species (ROS) production, lipid peroxidation and subse quent cytotoxicity induced by five major benzene meta bolites. The generation of ROS by benzene metabolites was demonstrated by the significant and dose-dependent increase of intracellular ROS formation in HL60 human promyelocytic leukemia cells in vitro. 1,4-Benzoquinone (BQ) was found to be the most potent metabolite in induction of ROS formation, followed by 1,2,4-benzene triol (BT) and to a lesser extent, phenol (PH) and trans,trans-muconaldehyde (MD). No significant effect was observed when the cells were treated with trans, trans-muconic acid (MA). The enhancement of ROS production by BQ was effectively inhibited by the addition of catalase, deferoxamine (DFO) and dimethyl sulfoxide (DMSO), but unchanged by superoxide dismutase (SOD), suggest that hydrogen peroxide (H2O2) and hydroxyl radicals (OH.) are the two major forms of ROS involved. The results also demonstrate that the ability of benzene metabolites in enhancing ROS generation is closely correlated to their capacity in causing lipid peroxidation and subsequent cytotoxicity. These findings together with earlier parallel observations on DNA damage suggest that ROS play an important role in the mechanism of carcinogenesis induced by benzene metabolites.

Simultaneous determination of hydroquinone, catechol and phenol in urine using high-performance liquid chromatography with fluorimetric detection

A method was developed for simultaneous determination of urinary hydroquinone, catechol and phenol using high-performance liquid chromatography (HPLC) with variable-wavelength fluorimetric detection. Urine samples, after acid hydrolysis, were saturated with sodium sulphate and extracted by diethyl ether. The two buffers used for gradient elution were (A) 10 mM sodium acetate containing 0.5% (v/v) acetic acid and (B) the same as buffer A but containing an additional 20% (v/v) acetonitrile. Hydroquinone, catechol and phenol were separated in a C18 column and detected at 2.9, 6.8 and 13.6 min, respectively. The recovery and reproducibility were generally over 90%. Over 300 extracted samples were analysed and no change in column efficiency was noted. Comparisons were also made with HPLC using ultraviolet (UV) detection and with gas chromatography (GC). The proposed method appears to be more sensitive and reliable than other existing methods. This new method was also validated with urine samples collected from cigarette smokers and from refinery workers exposed to low concentrations of benzene.

Evaluation of biomarkers for occupational exposure to benzene.

OBJECTIVE--To evaluate the relations between environmental benzene concentrations and various biomarkers of exposure to benzene. METHODS--Analyses were carried out on environmental air, unmetabolised benzene in urine, trans, trans-muconic acid (ttMA), and three major phenolic metabolites of benzene; catechol, hydroquinone, and phenol, in two field studies on 64 workers exposed to benzene concentrations from 0.12 to 68 ppm, the time weighted average (TWA). Forty nonexposed subjects were also investigated. RESULTS--Among the five urinary biomarkers studied, ttMA correlated best with environmental benzene concentration (correlation coefficient, r = 0.87). When urinary phenolic metabolites were compared with environmental benzene, hydroquinone correlated best with benzene in air. No correlation was found between unmetabolised benzene in urine and environmental benzene concentrations. The correlation coefficients for environmental benzene and end of shift catechol, hydroquinone, and phenol were 0.30, 0.70, and 0.66, respectively. Detailed analysis, however, suggests that urinary phenol was not a specific biomarker for exposure below 5 ppm. In contrast, ttMA and hydroquinone seemed to be specific and sensitive even at concentrations of below 1 ppm. Although unmetabolised benzene in urine showed good correlation with atmospheric benzene (r = 0.50, P < 0.05), data were insufficient to suggest that it is a useful biomarker for exposure to low concentrations of benzene. The results from the present study also showed that both ttMA and hydroquinone were able to differentiate the background level found in subjects not occupationally exposed and those exposed to less than 1 ppm of benzene. This suggests that these two biomarkers are useful indices for monitoring low concentrations of benzene. Furthermore, these two metabolites are known to be involved in bone marrow leukaemogenesis, their applications in biological monitoring could thus be important in risk assessment. CONCLUSION--The good correlations between ttMA, hydroquinone, and atmospheric benzene, even at concentrations of less than 1 ppm, suggest that they are sensitive and specific biomarkers for benzene exposure.

Luteolin induces G1 arrest in human nasopharyngeal carcinoma cells via the Akt-GSK-3β-Cyclin D1 pathway.

Luteolin, a plant flavonoid is known to possess multiple biological activities such as anti-inflammation, anti-allergy, anti-oxidant as well as anti-cancer. At present, the anti-proliferative potential of luteolin has not been fully understood. In this study, we focused on the effect of luteolin on cell cycle regulation in human nasopharyngeal carcinoma (NPC) cells. First, we found that luteolin inhibited cell cycle progression at G1 phase and prevented entry into S phase in a dose- and time-dependent manner. Next, it was found that luteolin treatment led to down-regulation of cyclin D1 via enhanced protein phosphorylation and proteasomal degradation, leading to reduced CDK4/6 activity and suppression of retinoblastoma protein (Rb) phosphorylation, and subsequently inhibition of the transcription factor E2F-1. In search of the molecular mechanisms underlying luteolin-mediated cyclin D1 down-regulation, it was found that luteolin was capable of suppressing Akt phosphorylation and activation, resulting in de-phosphorylation and activation of glycogen synthase kinase-3β (GSK-3β). Activated GSK-3β then targeted cyclin D1, causing phosphorylation of cyclin D1 at Thr(286) and subsequent proteasomal degradation. The above findings were reinforced by the fact that luteolin was able to abrogate the effect of insulin on the Akt/GSK-3β/Cyclin D1 pathway, resulting in suppression of insulin-induced cell proliferation. Since Akt is often over-activated in many human cancers including NPC, it is thus believed that data from this study support the potential application of luteolin as a chemotherapeutic or chemopreventive agent in human cancer.

Automated high-performance liquid chromatographic method with precolumn reduction for the determination of ubiquinol and ubiquinone in human plasma

We developed a gradient HPLC method with automated precolumn reduction for direct electrochemical detection of ubiquinol-10 (CoQ10H2) and total coenzyme Q10 (TQ10) in human plasma. The concentration of ubiquinone-10 (CoQ10) was calculated by subtraction of CoQ10H2 from TQ10. Preparation of reducing agent and precolumn reduction was performed by a programmable auto-injector. The two mobile phases used were: A, 100% of methanol containing 50 mM sodium perchlorate and 10 mM perchloric acid; and B, a mixture of ethanol and tert.-butanol (80:20, v/v). Sample preparation was simply a deproteinisation process with 10-fold ethanol. A good linear relationship was obtained for CoQ10H2 concentration from 0.1 to 3 micromol/l. The detection limit was 2.5 nmol/l with an injection volume of 20 microl. The analytical recovery and reproduciblity were generally >90%. To validate the method, 18 freshly collected plasma samples of normal healthy subjects were analysed. The mean ratio of CoQ10H2/CoQ10 was 93:7. The proposed method is sensitive, reliable and can be used for clinical investigation.

Biological monitoring of kidney function among workers occupationally exposed to trichloroethylene

Aims: To investigate the nephrotoxic potential of trichloroethylene in a currently exposed population using sensitive urinary markers of kidney toxicity. Methods: Renal dysfunction was monitored in a cross-sectional study of 70 workers currently exposed to trichloroethylene. An age and sex matched control population of 54 individuals was drawn from hospital and administrative staff. Results: The mean exposure to trichloroethylene, estimated from urinary trichloroacetic acid concentrations, was 32 ppm (range 0.5–252 ppm) with an average duration of exposure of 4.1 years (range 1–20 years). Significant differences between the exposed and control populations were found for nephrotoxicity markers N-acetylglucosaminidase (NAG) and albumin, and for the mode of action marker, formic acid. However, neither NAG nor albumin showed a significant correlation with either the magnitude or duration of exposure to trichloroethylene. There was a significant correlation between urinary formic acid and trichloroacetic acid concentrations. Within the exposed population there were dose dependent increases in urinary methylmalonic acid concentrations and urinary glutathione S-transferase α activity. Although still within the control range, these changes were clearly dose dependent and consistent with one of the proposed mechanisms of trichloroethylene induced kidney toxicity. Conclusion: Although there was no evidence of kidney toxicity within the population studied, the results suggest that kidney damage could occur at exposure concentrations higher (>250 ppm) than those encountered in this study.