H.Y. 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.

Male endocrine functions in workers with moderate exposure to lead

Evidence for the effect of occupational exposure to lead on the male endocrine system is conflicting. This study evaluated the primary (testicular) and secondary (hypothalamo pituitary testicular) effects of exposure to lead in 122 current lead workers and 49 non-exposed workers. The mean current blood lead concentration was 35.2 (range 9.6-77.4) micrograms/dl in the exposed workers, and 8.3 (range 2.6-14.8) micrograms/dl in the non-exposed workers. Concentrations of plasma luteinising hormone (LH) and follicle stimulating hormone (FSH) were both significantly higher in the exposed workers, but testosterone (T) was not significantly different between the two groups. In older exposed workers, however (greater than or equal to 40 years), plasma T concentrations were significantly lower, but LH and FSH concentrations were not significantly different. Compared with non-exposed workers, those exposed for less than 10 years had significantly raised LH and FSH and normal T concentrations whereas those exposed for 10 or more years had significantly lower T, and normal LH and FSH concentrations. The concentrations of LH and FSH showed a moderate increase in relation to blood lead concentrations in the range of 10 micrograms/dl to 40 micrograms/dl and thereafter reached a plateau or declined. No apparent trend for plasma T concentrations occurred. No significant difference in prolactin (PRL) concentration was noted. It is concluded that moderate exposure to lead was associated in dose related fashion with small but measurable changes in male endocrine functions that reflected both primary and secondary effects of lead on the testes and the hypothalamo pituitary testicular axis.

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.

Are salivary immunoglobulin A and lysozyme biomarkers of stress among nurses

Salivary immunoglobulin A (IgA) and lysozyme have been studied as possible biomarkers of stress. This study examined the stress levels among female nurses in various units and the relationship between these stress levels and salivary IgA and lysozyme secretion. One hundred ninety-five (43%) of 457 eligible female nurses from surgical wards/operating theaters (SURG), medical wards (MED), and outpatient clinics/day-surgery theaters (OPD) completed a self-administered questionnaire. From this group of 195 nurses, 124 provided a salivary sample accumulated over 5 minutes. Stress levels were assessed with a ten-point Stress Assessment Score (SAS) for Asians and a direct question on perceived life stress. Enzyme-linked immunosorbent assay and lyso-plate methods were used to determine salivary IgA and lysozyme levels. Forty-five percent of SURG, 35% of MED, and 17% of OPD nurses scored at least four points on the SAS. SURG nurses had the lowest IgA secretion (geometric mean; 95% confidence interval [CI]) rates (43 micrograms/min; 36 to 51 micrograms/min). The other groups had significantly higher salivary IgA secretion rates: MED (96 micrograms/min; 80 to 116 micrograms/min) and OPD (77 micrograms/min; 60 to 98 micrograms/min) Findings for salivary lysozyme (microgram/min) were similar; SURG (9 micrograms/min; 6 to 13 micrograms/min) MED (19 micrograms/min; 12 to 28 micrograms/min) and OPD (16 micrograms/min; 9 to 28 micrograms/min). The salivary IgA (Spearmans r = -0.22, P = 0.01) but not the lysozyme (Spearmans r = -0.01, P = 0.9) secretion rate correlated negatively with SAS. Nurses working in various units under different conditions experienced dissimilar levels of stress. Salivary IgA, but not lysozyme, correlated inversely with self-reported levels of stress. It may thus be a potential biomarker in future studies on stress.

Can salivary lead be used for biological monitoring of lead exposed individuals

Background: Measurement of blood lead (BPb) is the usual method for biomonitoring of persons exposed to inorganic lead. Aim: To explore the use of salivary lead (SPb) as an alternative. Methods: BPb and SPb levels were measured in a group of 82 lead exposed adults. Results: The mean BPb of the workers was 26.6 μg/dl (SD 8.6, range 10–48) and the mean SPb level 0.77 μg/dl, or 3% of the BPb level. As the SPb distribution was skewed, logarithmic transformation was performed to normalise the distribution. A bivariate scattergram of BPb and logSPb (r = 0.41, p = 0.00) had a line of best fit expressed as BPb = 29.7 + 8.95logSPb. The relation of logSPb and BPb was stronger among non-smokers (r = 0.42) compared to smokers (r = 0.3); and among those without a medical condition (r = 0.44). Multiple linear regression analysis (fitting smoking and medical condition into the model) yielded an R of 0.54, and an adjusted R2 of 0.26. Conclusion: The study findings do not support the use of SPb for biomonitoring at BPb levels ranging from 10 to 50 μg/dl.

Urinary N-acetyl-beta-D-glucosaminidase activity in workers exposed to inorganic lead.

Urinary N-acetyl-beta-D-glucosaminidase (NAG) had been shown to be a useful early marker of renal injury. In workers exposed to lead it seems to be the only early marker but the dose response and dose effect relations are weak. Furthermore, the significance and underlying mechanism of increased urinary NAG activity is far from clear. By studying the isoenzyme profiles of urinary NAG, the significance and underlying mechanism may be further clarified. The heat labile (NAG-A) and heat stable (NAG-B) isoenzyme profiles of 128 workers exposed to lead from a lead stabiliser factory were analysed. NAG activity was expressed as total NAG, NAG-A, and NAG-B activity as well as ratios (NAG-B/total NAG and NAG-B/NAG-A). Exposure indices included the recent concentration of blood lead (BPb), a cumulative blood lead index (TBPb), and the recent change in concentration of blood lead (CBPb). The NAG indices correlated best with CBPb. Nearly 50% of the variation in NAG-B activity could be explained by the combination of all three exposure indices but only the CBPb was highly significant. When these exposure indices were entered separately into the regression equation, CBPb accounted for 36.3% of the variation in NAG-B activity, 5.7% was accounted for by TBPb and 2.7% by BPb. There was also no dose-effect or dose-response relation between the NAG variables and BPb or TBPb groups. With CBPb, there were dose-effect and dose-response relations. With CBPb, there was an increase in NAG variables in the group with more than 25% increase in blood lead over the past six months. The increase in NAG activity in this study is likely to be due to a recent increase in concentration of blood lead and hence presumably a recent rise in renal burden of inorganic lead. This suggests that the increase in urinary NAG activity is a form of acute response to a sharp increase in renal burden of lead, rather than to a cumulative dose. Heat stable NAG is part of the lysosomal membrane and is present in the urine when there is breakdown of lysosomes. Our data therefore contradict suggestions that the increase in urinary NAG activity is due to exocytosis.

Renal tubular function of workers exposed to low levels of cadmium.

Cadmium induced renal tubular effects were examined in 65 female workers in a factory manufacturing nickel cadmium batteries. Urinary beta 2-microglobulin (beta 2m), urinary N-acetyl-D-glucosaminidase activity (NAG), and serum creatinine and serum urea concentrations were used to assess the renal effects. Of the four measures, only urinary NAG and urinary beta 2m showed a strong positive correlation with blood cadmium concentrations (r = 0.49 and 0.43 respectively); NAG showed a weaker correlation with urinary cadmium concentrations (r = 0.35). Urinary beta 2m has weak correlation with urinary cadmium (r = 0.04). Only urinary NAG showed a significant deterioration in renal function among the exposed group. NAG detects the largest proportion of abnormalities among the exposed group. Abnormal urinary beta 2m is detected in only 15.4% of the workers, half of whom have blood cadmium above 10 micrograms/l. The proportion of abnormalities detected by urinary NAG differs significantly from the proportion of abnormalities detected by urinary beta 2m (p less than 0.01). The age adjusted mean urinary NAG excretion showed a significant rise with urinary cadmium of above 3 micrograms/g creatinine. Urinary beta 2m failed to show any significant rise. With blood cadmium concentrations, the age adjusted mean urinary NAG excretion showed a rise from 1 microgram/l of blood cadmium followed by a plateau between blood cadmium concentrations of 3-10 micrograms/l. No significant rise in mean urinary excretion in beta 2m was seen until blood cadmium concentrations exceeded 10 micrograms/l.

Biological monitoring of occupational exposure to methyl ethyl ketone

SummaryThis study was conducted to evaluate the usefulness of three commonly used methods of biological monitoring for worker exposed to methyl ethyl ketone (MEK) under field conditions using blood, breath and urine. Environmental MEK exposures were measured by personal sampling with carbon-felt dosimeters. The correlation coefficient (r) between the time-weighted average (TWA) MEK concentration in air and the MEK concentration in blood collected at the end of the work shift was 0.85. The correlation coefficient between the TWA MEK level in air and the concentration exhaled in the breath of workers at the end of the work shift was 0.71. The end-of-shift urinary MEK excretion correlated best with the environmental concentration (r = 0.89). Correlations became lower after urine samples had been corrected for urinary creatinine (r = 0.83) or specific gravity (r = 0.73). After 8 h exposure to 200 ppm MEK, the corresponding end-of-shift urinary excretion was 5.11μol/l or 4.11 mg/g creatinine. This value is higher than that previously found in some studies, the difference probably being due to the physical acitivites of the present workers and their extensive skin contact with the solvent. The kinetics of inhaled MEK was also studied in eight subjects. Breath and urine samples were collected during the 8-h work shift on 2 consecutive Mondays. The results showed that urinary MEK excretion rose steadily until the end of exposure, whereas the MEK concentration in exhaled air varied markedly throughout the day. These findings suggest that the determination of MEK levels in end-of-shift urine samples appears to be the most reliable biological indicator of occupational exposure.