JOURNAL OF APPLIED TOXICOLOGY, VOL. 10( l), 65-68 (1990)

Biological Monitoring of Workers Exposed to Lead Stearate C. N. Ong, L. H. Chua and K. Teramoto*t Department of Community, Occupational and Family Medicine, National University of Singapore, Kent Ridge 0511. Singapore and *Department of Environmental and Preventive Medicine, Osaka City University Medical School. Osaka 545, Japan

Key words: lead stearate; plasma lead; blood lead; ALAD.

This study was conducted to examine the usefulness of some of the commonly used biological parameters for monitoring of workers exposed to lead stearate. Forty-two lead stearate workers from a lead stabilizer factory and 26 workers exposed to inorganic lead compounds were involved in this study. Although the workers had similar blood lead values (PbB), subjects exposed to lead stearate were found to have a significantly higher concentration of lead in plasma (PbP), (1.0 0.57 pg dl-I) than workers exposed to inorganic lead compounds (0.42 ? 0.3). The ratio of PbP to PbB was ca. 2.5 times higher for lead stearate workers (0.38) than the inorganic lead workers (0.15). These data suggest that the different chemical properties of lead stearate may result in different distributional patterns of the metal in different blood components. On the other hand, the activity of 6-aminolevulinic acid dehydratase (ALAD), an enzyme highly sensitive to lead, was not so much depressed among the lead stearate workers as that of workers handling inorganic lead. A poor correlation was also observed between PbB and ALAD activity of the stearate workers. These findings indicate that PbB and ALAD are not good biological indicators for evaluating the toxicological effect of lead stearate exposure.

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INTRODUCTION

The biological monitorings of subjects exposed to lead is usually performed by measuring the concentration of lead in whole blood (PbB). This parameter is considered to be the best indicator of current exposure and reflects most closely a person’s ‘lead status’. However, it is generally agreed that blood contains lead in two forms, a non-diffusible form bound to erythrocyte and a diffusible form in plasma.’, * The diffusible form, because of its greater bioavailability, is more likely to produce effects in various organ^.^ There is, so far, little information relating to lead in plasma; this may be attributable either to the technical difficulties in their measurements4 or to the lack of correlation with whole blood c ~ n c e n t r a t i o n . ~ Recently, Cavalleri and Minoia6 reported that the mean lead concentration in plasma for workers exposed to lead stearate was higher than those exposed to inorganic lead compounds. This finding suggests that lead stearate may have different toxicokinetic and toxicodynamic properties as compared to inorganic compounds. Lead stearate (Pb(C,7H35C00)2) accounts for ca. 10% of the total stabilizer consumption in polyvinyl chloride (PVC) production. The manufacturing process of lead stearate is usually complex and this could result in the workers being exposed to a high concentration at the workplace.’ In comparison with other lead processes, the association between the toxicological effect and lead stearate is not noted as readily. Exposure to this compound I Author to

whom correspondence should be addressed.

0260437x/9o/01006SO4$05.~ Wiley & Sons, Ltd.

0 1990 by John

causes non-specific psychological complaints; such as memory loss and lack of concentration.x The conduction velocities of various nerves were also significantly lower than non-exposed subject^.^ The objectives of this study are: to examine the distribution of lead in the blood of workers exposed to lead stearate and other inorganic lead compounds; and to examine the relationships of lead in whole blood (PbB), lead in plasma (PbP) and 6-aminolevulinic acid dehydratase (ALAD) of lead stearate workers.

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MATERIALS AND METHODS Subjects

This study was carried out in a plant manufacturing lead stearate for PVC production and two automobile battery factories in Singapore. A total of 42 lead stearate workers and 24 inorganic lead workers were studied. Reagents and glassware

Triton-X-100 was purchased from Eastman Kodak (scintillation grade); all water used for dilution and rinsing was distilled and polished with a Milli-Q water purification system. Lead nitrate was purchased from BDH Chemicals Ltd. (spectro grade). Plastic ware and glassware were cleaned by soaking for 24 h in concentrated detergent, followed by soaking for 72 h in 25% (v/v) nitric acid. After cleaning, all containers were thoroughly rinsed with distilled-MilliQ-purified water and dried in a dust-free environment. Received 3 January 1989 Accepted 2 August 1989

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Samples were diluted with an Eppendorf automatic pipettor (mode 5203). Analysis of lead in blood and plasma

About 3 ml of blood were collected from each subject by venepuncture. Precautions were taken during sample collection to prevent contamination as far as possible; the blood samples were collected heparinized, and in lead-free polypropylene tubes, which were sealed immediately and then transported to the laboratory. A 1-ml aliquot of this blood was used for ALAD and lead analysis. The remaining 2 ml were centrifuged for 20 min at 3500 rpm. A 1-ml aliquot of plasma was then pipetted into a lead-free polypropylene tube. Samples were analysed within 48 h of collection. Blood lead and plasma lead analysis were carried out using a Varian Tectron Spectra AA-30 graphite furnace atomic absorption spectrophotometer with an autosampler. Triton X-100 and NH4N03 were used as matrix modifiers. The matrix modification method was used throughout this study. In this procedure, the atomization rate of the analyte is retarded sufficiently to resolve the atomic absorption signal of the analyte from the non-atomic absorption signal of the matrix."' The detailed analytical procedures for PbB are as reported earlier.'l Ten microlitres of sample were used for the PbB determination, with background correction in a Spectra AA-30 graphite furnace. External quality control was carried out in collaboration with the National External Quality Assessment Scheme (NEQAS) in the UK. The mean running variance index score (MRVIS) during the time of analyses was 22-26. The analysis of lead was normally carried out in triplicate. The standard deviations (SD) for within-run precision (based on 15 repetitive measurements) for a blood lead concentration of 8 pg 100 ml-' was 0.34 and for a blood lead concentration of 84 pg 100 ml-l was 0.67. The coefficient of variation (C.V.) seldom exceeded 6%. Internal quality control for accuracy was checked by an analysis of a bovine liver standard from the US National Bureau of Standards. The value obtained from this laboratory was 0.35 +- 0.04 pg ggl for a sample certified to contain 0.34 ? 0.01 pg g-l. For PbP determination, 50 p1 of 10% NH4N03 was added to 0.5 ml of plasma and the specimens were further diluted six-fold with 0.1% Triton X-100. The atomization conditions for the graphite furnace were similar to that used earlier.4 Twenty microlitres of sample were used for each injection. With the use of the standard addition method, a mean of > 85% was obtained. Calibration curves were performed for every batch of about 15 analyses. The slope of the calibration graph varied from day to day, but the curve was linear for concentration ranges of 0.2-10 pg dl-'. The analysis was carried out in triplicate. The concentration of PbP was read directly off the calibration curve, and data were analysed using an IBM-XT microcomputer with statistical software. The detection limit (three SDs of blank) was 0.81 pg dl-I. The within-run reproducibility (based on 16 repetitive determinations) gave a C.V. of 5.8%. The sensitivity (= concentration for 0.0044 absorbence unit) was 0.08 pg d1-I. Precision was also determined

by measuring the daily percentage changes of a pooled plasma sample. The C.V. seldom exceeded 6%. The activity of the erythrocyte enzyme ALAD (E.C. 4.2.1.24) was measuring according to the European standardized method. l 2 RESULTS The mean PbB level of the 42 lead stearate workers was 29.38 2 9.67 pg dl-' and the mean PbB concentration for the reference group was 28.53 ? 8.94 pgdl-'. As shown in Fig. 1, the concentration of lead in plasma in the group exposed to lead stearate was much higher than the mean of the PbP level determined for the reference group. The PbP level was 1.00 ? 0.57 as compared to 0.42 ? 0.33 ( P < 0.001). The mean value of lead in urine for lead stearate workers (1.70 ? 0.57) was also found to be slightly higher than that of the reference group (1.58 0.90). It is interesting to note that the erythrocyte enzyme ALAD for workers exposed to lead stearate (0.72 2 0.13) was significantly higher ( P < 0.05) than that of workers handling other inorganic lead compounds (0.59 2 0.23). Differences were also noted for the PbP:PbB ratio x 100, which measures the fraction of lead in plasma to that in whole blood. The values were 3.76% (SD 2.0) and 1.47% (SD 1.1) for lead stearate workers and the reference group, respectively (Table 1). In order to rule out a possible influence of the difference in number of red blood cells between the two groups, we also measured the packed cell volume. No significant difference was observed for the number of erythrocytes between the two groups. The mean haematocrit values were 46.9 ? 4.1 and

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Pb P

Pb B

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Figure 1. Individual values of lead in whole blood (PbB), lead in plasma (PbP) and lead in urine (PbU) of workers exposed to lead stearate (ST) and exposed to inorganic lead compounds (IP) [-, mean value; - - - -, SD). All units in kg dl-'.

BIOLOGICAL MONITORING OF LEAD STEARATE EXPOSURE

l’able 1. Mean values and standard deviations of various parameters

Parameter

Workers exposed to lead stearate

Workers exposed to inorganic lead

PbB (pg dlk’) PbP (pg dl ’1 PbU (pg dl ‘1 ALAD (pmol h - ’ ml-I RBC) PbP: PbB (%I

29.38 2 9.67 1.00 2 0.57 1.70 2 1.91 0.72 2 0.13 3 . 7 6 2 2.22

28.53 0.42 1.58 0.59 1.47

2 8.94 2 0.33 5

0.90

2 0.23 5

1.15

37.4 2 3.5 for lead stearate workers and the reference group, respectively. The relationships between blood lead, plasma lead, urine lead (PbU) and ALAD are summarized in Table 2. I t is important to note that for workers exposed to lead stearate the blood lead concentration did not appear to correlate very well with lead in plasma ( Y = 0.12). Whereas, for workers exposed to other lead compounds a strong association was observed between PbB and PbP (Y = 0.57). It was also noted that the PbP and PbU showed a very poor association for the lead stearate workers (Y = 0.091). On the other hand, a significant correlation for PbP and PbU was observed for the reference group. Although the erythrocyte enzyme ALAD showed significant correlations with PbB and PbP for both groups of lead workers, the associations were generally lower for the lead stearate workers (Table 2).

DISCUSSION

Biological monitoring of subjects exposed to lead is usually performed by measuring the concentration of lead in whole blood (PbB). It is believed that PbB reflects best the current exposure status of a person. However, it is generally agreed that blood contains lead in two forms: a non-diffusible form associated with the erythrocytes and a more biologically active form associated with the plasma. Thus, the biological significance and toxico-kinetics of lead in different blood components are not the same. Since the plasma leiid has a higher bioavailability, the lead concentration

Table 2. Relative agreements between biological indicators Correlation coefficient, r

Relationship

Workers exposed to lead stearate

Workers exposed to inorganic lead

PbB PbB PbP PbB PbP

0.12 0.36 0.09 -0.66 -0.32

0.57 0.32 0.41 -0.81 -0.56

vs. PbP vs. PbU vs. PbU vs. ALAD vs. ALAD

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in plasma should not be overlooked in an evaluation of the potential health effect of lead in the body. A significant relationship has been demonstrated between lead in plasma and the concentration of lead in the cerebrospinal fluid, but not between PbB and the cerebrospinal fluid lead level^.^ In the present study, although similar concentrations of lead in whole blood were observed for lead stearate workers and the reference group, a significantly higher concentration of lead in plasma was observed in the former group. The PbP:PbB ratio was 3.76 for the lead stearate workers and 1.47 in the reference group. This finding suggests that the different chemical properties of lead stearate may result in a different distribution pattern of the metal in different compartments. In other words, with a similar absorbed dose, the plasma fraction of lead stearate workers had twice the bioavailable lead than workers exposed to other inorganic lead compounds. These data confirmed and extend a recent report by Cavalleri and Minoia6 that lead stearate workers have a higher fraction of lead in the plasma. They believed that this shift of lead from erythrocyte to plasma may produce more severe toxic effects than those caused by other lead compounds at a similar absorbed dose, as a larger proportion of absorbed lead is available for delivering its toxic effect. The depression of 6-aminolevulinic acid dehydratase (ALAD) activity has been considered as the most sensitive indicator of the effect of lead.13, l4 The activity of ALAD was used in the present study to evaluate the biological effect of exposure to lead stearate. It is interesting to note that the results here showed that with a similar absorbed dose the lead stearate workers’ ALAD activity was not inhibited as much (Table 1) as that of the reference group (I‘ > 0.05). Furthermore, the correlation of ALAD with PbB or PbP for stearateexposed subjects was not as good as that of workers exposed to other lead compounds (Table 2). This may be explained by the fact that ALAD is an erythrocyte enzyme and its suppression is caused by lead within the erythrocyte^.'^ Although, with a similar dose in the blood the lead stearate workers have a smaller fraction of lead in the erythrocytes and thus the biological effect on ALAD would not be as significant as that of other lead workers who have a larger fraction of absorbed lead with the erythrocytes. These findings tend to suggest that ALAD activity is not a suitable biological indicator for evaluating the toxic effect of lead stearate. In an earlier study it was shown that the solubility of lead stearate was much higher than lead oxide;’ it is, however, not known that this difference in physical property may lead to different affinities of lead to different blood constituents. Two conclusions that can be drawn from the present study are: (i) lead stearate workers had a higher proportion of lead in the plasma than that of other inorganic lead workers; (ii) the biological effect of lead stearate is likely to be different from that of other inorganic lead compounds. The results presented here clearly indicated that for lead stearate workers the ratio of lead in plasma to that of lead in erythrocytes might be a better estimate

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than the level of lead in whole blood. ALAD is not a good biological indicator for evaluating the health effect of lead stearate exposure. The use of lead stearate in PVC production is increasing and we have recently shown that exposure to lead stearate resulted in subclinical changes in both the neurophysiologicalX and neuropsychological function^.^ These findings, together with the present data, suggest that there is a need for more stringent monitoring of workers exposed to lead stearate. Due to its greater bioavailability in the blood and the fact that the effect of lead stearate o n biological systems

may be very different to that of other inorganic lead compounds, more studies on the toxicological effect for this compound are obviously needed.

Acknowledgements The authors wish to thank H. Y. Ong, B. L. Lee and S. L. Yeong for their assistance in A L A D analysis and preparation of the manuscript. We are also grateful to the management and staff of Sunance (S), Chloride (South East Asia) and Yuasa Battery for their cooperation.

REFERENCES 1. A. Cavarelli, C. Minoia, L. Pozzoli and A. Baruffini, Determination of plasma lead levels in normal subjects and in lead exposed workers. Br. J. Ind. Med. 35, 21-26 (1978). 2. C. N. Ong and W. R. Lee, Distribution of lead-203 in human peripheral blood in vitro. Br. J. lnd. Med. 37, 78-84 (1980). 3. A. Cavalleri, C. Minoia, M. Ceroni and M. Poloni, Lead in cerebrospinal fluid and its relationship to plasma lead in humans. J. Appl. Toxicol. 4, 63-65 (1984). 4. C. N. Ong, W. 0. Phoon, B. L. Lee, L. E. Lim and L. H. Chua, Lead in plasma and its relationships to other biological indicators. Ann. Occup. Hyg. 30, 219-228 (1986). 5. J. F. Rosen, Plasma lead levels in normal and lead intoxicated children. J. Pediatr. 84, 45-48 (1974). 6. A. Cavalleri and C. Minoia, Lead level of whole blood and plasma in workers exposed to lead stearate. Scand. J. Work Environ. Health 13, 218-220 (1987). 7. C. N. Ong, H. Y. Ong and N. Y. Khoo, Lead exposure in PVC stabilizer production. Appl. Ind. Hyg. (in print). 8. J. Jeyaratnam, K. W. Boey, C. N. Ong, C. B. Chia and W. 0. Phoon, Neuropsychological studies on lead workers in Singapore. Br. J. Ind. Med. 43, 626-630 (1986). 9. J. Jeyaratnam, G. Devathasan, C. N. Ong, W. 0. Phoon and P. K. Wong, Neurophysiological studies on workers exposed

to lead. Br. J. Ind. Med. 42, 173-178 (1985). 10. K. S. Subramaniam and J. C. Meranger, A rapid electrothermal AAS method for lead in human whole blood. Clin. Chem. 27, 1866-1871. 11. C. N. Ong, W. 0. Phoon, H. Y. Law, C. Y. Tye and H. H. Lim, Concentration of lead in material blood, cord blood and breast milk. Arch. Dis. Child. 60, 756-760 (1985). 12. A. Berlin and K. H. Schaller, European standardized method for the determination of ALAD activity in blood Z. Klin. Chem. Klin. Biochern. 12, 389-390 (1974). 13. S. Hernberg, Biochemical, subclinical and clinical responses of lead and their relation to different exposure levels. In Effects and Dose-Response Relationships of Toxic Metals, ed. by G . F. Norberg, pp404-415. Elsevier, Amsterdam (1980). 14. S. Telisman, A. Kerang and D. Pripic-Mazic, The relevance of arguments for excluding ALAD from the recommended biological limit values in occupational exposure to inorganic lead. Int. Arch. Occup. Environ. Health 50, 399-342. 15. T. Sakai, S. Yanagihara, Y. Kunugi and K. Ushio, Relationships between distribution of lead in erythrocytes in vivo and in vitro and inhibition of ALA-D. Br. J. Ind. Med. 39, 283-387.

Biological monitoring of workers exposed to lead stearate.

This study was conducted to examine the usefulness of some of the commonly used biological parameters for monitoring of workers exposed to lead steara...
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