Evaluation of Serum and Urinary Neopterin Levels as a Biomarker for Occupational Exposure to Crystalline Silica

ABSTRACT

Background:

Crystalline silica is a commonly used mineral in various industries and construction activities, and it is so important introducing potential biomarkers to identify early indicators of biological effects in its high-risk occupational exposures.

Aim:

The present study was aimed to assess the blood and urinary neopterin as an early biomarker of exposure in the workers of an insulator manufacturing plant who are exposed to crystalline silica.

Subjects and Methods:

This analytical descriptive study was done among two groups of exposed workers (n = 55) and unexposed office workers (n = 38) of an insulator manufacturing plant. Statistical software R was used to determine sample size and select the participants by random sampling among nonsmoker workers. Sampling of airborne silica in breathing zone of participants was done based on the National Institute for Occupational Safety and Health method 7601. The urinary and blood samples were collected and prepared for analysis by high-performance liquid chromatography to determine the level of urinary and serum neopterin. All of the statistical analyses were carried out using SPSS 22.

Results:

The airborne silica concentration was significantly different between two exposed and unexposed groups (P < 0.001, 0.27 [0.11] vs. 0.0028 [0.0006] mg/m3, respectively). The urinary neopterin in exposed group is significantly higher than the unexposed one (P < 0.001, 97.67 [30.24] vs. 55.52 [2.18] μmol/mol creatinine, respectively). Neopterin level of serum in exposed group is higher than the unexposed group, and there is a significant difference between them (P < 0.001, 6.90 [2.70] vs. 2.20 [1.20] nmol/l, respectively). The positive significant correlations were found between silica exposure concentration with urinary and serum neopterin (P < 0.001, r = 0.36 and 0.59, respectively).

Conclusions:

Considering the sensitively and easily measurement of neopterin in biological fluid and also the statistically significant positive relationships which were found between the airborne silica concentration and neopterin levels in the present study, the serum and urinary neopterin levels can be considered the potential biomarkers of silica exposure for doing further comprehensive studies in this area.

SUBJECTS AND METHODS

Sample size determination

This analytical descriptive study was done among two groups of exposed and unexposed workers of an insulator manufacturing plant from June to August 2015. Statistical software R 3.2.2 (2015; R Development Core Team, Auckland University, New Zealand) was used to determine sample size and select the participants by random sampling among nonsmoker workers. A list of all workers (n = 181) involving their history exposure level to silica was provided. To have 90% power to detect 0.70 μg/ml difference between the each exposed group with the unexposed group, when the type I error assumed to be 0.05 and the standard deviation assumed to be 0.65 μg/ml, sample size was calculated to be 18 in each exposed and unexposed groups. As there were five exposed groups which compared with one unexposed group, it was decided to change the ratio to 4-1 in unexposed group compared with each exposed group. In this way, the final sample size has been determined 11 in each exposed group (total = 55) and 44 in unexposed group. Forty-four male healthy and nonexposed office employees to crystalline silica, who their age and work experience were in accordance with exposed participants, were selected through simple random sampling. There were some data missing including six participants from unexposed group.

A signed informed consent had been obtained from all participants. Workers who have suffered from any infectious disease, autoimmune diseases and other inflammatory diseases, and malignant diseases and were under any other special medical treatment and also have work experience <2-year-old were excluded from the study. Ethical approval was granted by the Research Ethics Committee of Tehran University of Medical Sciences.

Measurement of the concentration of airborne silica

National Institute of Occupational Safety and Health Method 7601 was used for sampling of airborne silica in breathing zone of all exposed and unexposed participants to evaluate the status of their respiratory exposure (sampling time: 6–8 h). The collected samples were carried to the laboratory for quantitative analysis. The stock and working solutions prepared according to method were used to plot the calibration curve. To prepare the samples for spectrophotometric analysis, silica standard solutions were filtered through the mixed cellulose ester filters (MCE-37 mm, 0.8 μm; SKC, Pennsylvania, USA). Finally, samples were analyzed by spectrophotometer (Unico SpectroQuest Model SQ2800 Single Beam UV/Visible Scanning Spectrophotometer, Ottawa, Canada).

Measurement of the blood and urinary neopterin levels

Blood and urine samples were taken from all participants in the early morning before work, and then they were transferred into 15 and 50 ml polyethylene tubes, respectively. Samples were stored immediately in an ice box and carried to the laboratory. All blood and urine samples were stored at −20°C until analysis. These biological samples were prepared for analysis by high-performance liquid chromatography (Merck Hitachi model L-7420 HPLC-UV, Midland, ON, Canada, equipped with RP18 column, absorbance wavelength of 353 nm). To analyze the biomarkers in serum and urine samples, calibration standard solutions were prepared, and the calibration curve was plotted based on the obtained data (R2 = 0.9992, linear dynamic range: 1–2000 ng/ml). Using the obtained model, limit of detection and limit of quantification were computed, 0.0089 and 0.029 ng/ml, respectively. Since the urinary neopterin is reported in terms of urinary creatinine, the creatinine level was also determined by Kinetic Jaffe methods.

Statistical analysis

Microsoft Excel version 2010 (Microsoft Inc., USA) was used to plot the calibration curves. All statistical analyses were carried out by SPSS version 22.00 (Chicago, IL, USA). Descriptive statistics including mean, standard deviation, median, range, frequency, and percentage were used to describe data, and analytical statistics including t-test, Mann–Whitney, and analysis of variance (ANOVA) were used to compare the results between the groups. Furthermore, Pearson correlation coefficient is used to measure the strength of a linear association between two variables. Normality of the study data was tested with a one-sample Kolmogorov–Smirnov test. The significance level was set to 0.05.

RESULTS

Table 1 shows some demographic characteristics of study participants. There is no significant difference between the exposed and unexposed groups regarding the age and work experience (t-test, P = 0.89 and 0.14, respectively).

The time-weighted average concentration of silica for exposed and unexposed participants can be seen in Table 2. The significant difference was found between two groups regarding the silica level exposure (P < 0.001). The highest silica concentration (0.36 [0.13] mg/m3) was related to glazing workers, and the lowest concentration (0.11 [0.04] mg/m3) was obtained for the workers of subordinate and small parts of the factory [Table 2]. According to results, the exposure concentration of silica was significant difference between workers of all parts of factory and unexposed ones(P < 0.001). The concentration of urinary neopterin (μmol/mol creatinine) in both groups is presented in Table 3. The urinary neopterin in exposed group is significantly higher than the unexposed one (Mann–Whitney, P < 0.001). According to findings, the highest urinary concentration of neopterin (139.77 [14.95] μmol/mol creatinine) was related to glazing workers, and the lowest one (55.52 [2.18] μmol/mol creatinine) was obtained for unexposed group. The urinary concentration of neopterin was significantly different between exposed and unexposed groups in all parts of factory (P < 0.001).

Table 4 indicates the serum level of neopterin (nmol/l) in both groups. Based on results, neopterin level of serum in exposed group is higher than the unexposed group, and there is a significant difference between them (ANOVA, P < 0.001). As it can be seen in Table 4, the highest and lowest serum concentrations of neopterin (7.80 [0.20] and 2.20 [1.20] nmol/l) were obtained for glazing workers and unexposed group, respectively. The concentration of neopterin in serum was significantly different between exposed and unexposed groups in all parts of factory (P < 0.001).

Table 5 illustrates the relationship of silica concentration, age, and work experience with serum and urinary neopterin. Findings demonstrate that there was no significant correlation between these two these demographic variables and urinary and serum neopterin (Pearson correlation test, P > 0.06). The positive significant correlations were found between silica exposure concentration and urinary and serum neopterin (P < 0.001, r = 0.36 and 0.59, respectively).
