This cross-sectional comparative study was conducted at a non-ferrous smelter in Helwan, Greater Cairo, Egypt, between May and June 2024. Workers in this facility are exposed to lead (Pb) during various high-risk activities involved in processing non-ferrous metals such as aluminum and copper. Their tasks include loading metal ores or concentrates into furnaces, which release Pb fumes and dust into the air during the smelting process.

Additionally, workers are engaged in refining molten metals, where chemical treatments and high-temperature operations further vaporize Pb compounds, increasing the risk of inhalation exposure. Maintenance activities, such as cleaning furnaces, repairing equipment, and managing slag, often disturb accumulated Pb dust, leading to significant exposure through both inhalation and dermal contact. Furthermore, tasks such as sampling or quality testing of molten metal frequently expose workers to direct Pb fumes.

Based on the sample size calculation, the study included 48 smelter workers with chronic occupational exposure to Pb and a control group of 48 administrative personnel who were not exposed to Pb. To qualify for the study, each exposed worker had to have been actively involved in the production process within the industrial setting for a minimum of two years. Notably, all exposed workers reported proper use of personal protective equipment (PPE). The PPE utilized included N95 air-purifying respirators, safety goggles, face shields, heat-resistant and flame-retardant suits, overalls, gloves, and boots. Additionally, the local exhaust ventilation system appeared to function effectively.

Environmental monitoring of heavy metals, including lead (Pb), aluminum (Al), and copper (Cu), is conducted annually within the smelter to ensure compliance with national safety standards. Data from these monitoring efforts consistently show that the concentrations of these metals in the workplace air and surrounding environment remain within the permissible limits set by national regulatory bodies. The authors reviewed the results of this monitoring in collaboration with the smelter administration; however, the data were not made available for inclusion in this study. According to the Egyptian Environmental Affairs Agency (EEAA), the threshold limit value (TLV) for Pb in workplace air is set at 0.05 mg/m³ (50 µg/m³), averaged over an 8-hour workday. For Al and Cu, the EEAA has established TLVs of 5 mg/m³ and 1 mg/m³, respectively, for respirable dust fractions (EEAA 1995).

Sample size calculation: The Power and Sample Size Calculator tool (version 3.0.43) was used to determine the sample size. The calculation was based on the following inputs: a significance level of 0.05, a mean difference between groups (g1 and g2) of 0.58, and a total sample size of 96 participants, with 48 individuals in each group (Yadav et al. 2018).

Participants with endocrine or metabolic disorders affecting bone tissue, or those using medications known to influence calcium-phosphate metabolism and bone turnover, were excluded from the study.

To form the control group, 48 matched individuals without a history of occupational Pb exposure were selected from the administrative department of the investigated smelter. After applying the exclusion criteria, control participants were chosen to match the exposed group in terms of sex, age, socioeconomic status, and specific medically significant behaviours, such as smoking.

The Research Ethics Committee of the Faculty of Medicine, Cairo University, Egypt, approved the study (approval number: N-467-2023). The Chief Executive Officer (CEO) of the smelter also presented formal consent for the study.

After receiving a detailed explanation of the study’s purpose, each participant voluntarily provided written informed consent to participate. Strict confidentiality and adherence to good clinical practice principles were maintained throughout the processes of sample collection, testing, coding, and recording of results.

A team of experienced occupational doctors conducted interviews with the participants to gather personal and occupational information. The medical history and clinical examination focused on any complaints or signs indicating skeletal system involvement, including generalized bone aches, back pain, joint pain, limited joint movement, a history of fractures from minor impact or low-energy activities, and delayed healing of previous fractures.

Five millilitres of whole blood were drawn from each participant at the end of the work shift: 2 mL in heparinized tubes and 3 mL in plain tubes. The 2-mL sample was used to estimate blood lead levels (BLL). The 3-mL sample was centrifuged at 4 °C and 3000 RPM for 10 min to extract serum for measuring serum vitamin D3 and CTX-1. Laboratory assays were conducted at the Clinical and Chemical Pathology Department Laboratory, Faculty of Medicine, Cairo University, Egypt.

Until analysis, the 2-mL blood sample was kept at -20 °C. The sample was digested with 2 mL of nitric acid and 0.2 mL of hydrogen peroxide using ETHOS-D (Milestone Microwave Laboratory Systems, Italy) while keeping power, temperature, and process duration constant. Using distilled water, the digested samples were diluted to a volume of 5 mL and then centrifuged. Using an atomic absorption spectrophotometer (GBC Avanta P, Australia), the concentration of Pb was determined. For the sample with the lowest concentration, a standard solution containing 20 µg/dL of lead was generated using a stock solution. After three repetitions, the study revealed 100% recovery with % relative standard deviation (RSD) at < 0.5 (Kalahasthi et al. 2016).

The enzyme-linked immunosorbent assay (ELISA) was used to determine the amounts of serum vitamin D3 and CTX-1 (BT LAB Bioassay Technology Laboratory, Shanghai, China). Thermo Scientific Multiskan EX-reader (USA) was used to detect the absorbance of samples and standard specimens at 450 nm. The unknown sample’s concentration was determined through the use of a standard curve and linear regression. For vitamin D3 and CTX-1, the range of the technique was 7–500 pg/mL and 7–1500 ng/mL, respectively. For vitamin D3 and CTX-1, the method’s sensitivity was 3.14 pg/mL and 4.21 ng/mL, respectively (Ali et al. 2024).

The statistical software for the social sciences (SPSS) version 28 (IBM Corp., Armonk, NY, USA) was used to code and input the data. For quantitative variables, the mean, standard deviation, median, minimum, and maximum were used to summarize the data; for categorical variables, the frequencies (number of cases) and relative frequencies (percentages) were used. For normally distributed quantitative variables, group comparisons were carried out using the unpaired t-test; for non-normally distributed quantitative variables, the non-parametric Mann-Whitney test was employed (Chan 2003a). The normally distributed variables were the age, BMI, and levels of BLL, vitamin D3 and CTX-1 of the studied participants. The Chi-square (χ2) test was used to compare categorical data. When the predicted frequency is less than five, the Exact test was utilized in its place. A 95% confidence interval for the odds ratio (OR) was computed (Chan 2003b). The Spearman correlation coefficient was used to perform correlations between quantitative variables (Chan 2003c). Logistic regression was done to detect independent predictors of the clinical manifestations of skeletal system affection (Chan 2004a). For identifying independent predictors of vitamin D3 and CTX-1 levels, linear regression analysis was used (Chan 2004b). The significance level of 0.05 for a P-value was deemed statistically significant.