Early markers of cardiovascular disease are associated with occupational exposure to polycyclic aromatic hydrocarbons

Occupational exposure to soot, rich in polycyclic aromatic hydrocarbons (PAH), has been associated with increased risk of cardiovascular disease (CVD). However, our knowledge about PAH exposure and early markers of CVD remains limited. In this cross-sectional study of 151 chimney sweeps and 152 controls, we investigated occupational exposure to PAH and early markers of CVD. Blood pressure (BP) (chimney sweeps only), urinary PAH metabolites and serum biomarkers were measured (C-reactive protein, homocysteine, gamma-glutamyltransferase, cholesterol, HDL, LDL, and triglycerides). Chimney sweeps had up to 7 times higher concentrations of PAH metabolites in urine than controls (P < 0.001): median concentrations (adjusted for specific gravity) for 1-hydroxypyrene, 2-hydroxyphenanthrene, 3-hydroxybenzo[a]pyrene, and 3-hydroxybenzo[a]anthracene were 0.56 µg/L, 0.78 µg/L, 4.75 ng/L, and 6.28 ng/L, respectively. Compared with controls, chimney sweeps had increased homocysteine, cholesterol, and HDL (β = 3.4 µmol/L, 0.43 mmol/L, and 0.13 mmol/L, respectively, P ≤ 0.003, adjusted for age, BMI, and smoking). In chimney sweeps, PAH metabolites correlated positively with the percentage of soot sweeping (P < 0.001). 2-hydroxyphenanthrene, 3-hydroxybenzo[a]pyrene, and 3-hydroxybenzo[a]anthracene were positively associated with diastolic BP (P < 0.044, adjusted for age, BMI, and smoking). PAH exposure among chimney sweeps resulted in elevated levels of markers for CVD risk. These findings stress the need to reduce occupational exposure to PAH.

Urine samples for calibration and for quality control (QC) were obtained from healthy volunteers at our laboratory and PAH-exposed subjects. The QC samples for 1-OH-PYR had concentrations of 6 and 26 nmol/L and for 2-OH-PH 20 and 95 nmol/L.

Instrumentation
The quantitative analysis used a triple quadrupole linear ion trap mass spectrometer (MS) equipped with an electrospray ion source (QTRAP 5500; AB Sciex, Foster City, CA, USA) coupled to a liquid chromatograph with four pumps (LC-MS/MS; Shimadzu Corporation, Kyoto, Japan). Air was used as nebulizer and auxiliary gas, pure nitrogen as curtain and collision gas.
The MS analyses were carried out by selected reaction monitoring in the negative mode (Supplementary Table S6).

Instrumental Analysis
For analysis of 1-OH-PYR and 2-OH-PH, sample aliquots of 5 µL were injected onto a C18 column (Genesis Lightn, 2.1 mm i.d. x 100 mm, Genesis, Grace Vydac, Hesperia, CA, USA) kept at 60°C. A mobile phase gradient, with a flow rate of 0.3 mL/min, consisting of water (A) and methanol (B) was kept at 5% B for 1 min after injection, raised to 95% B in 6.7 min, kept there for 2.3 min; and for the next injection, the column was conditioned at 5% B for 3 min. A diverter valve was used to introduce the column effluent into the mass spectrometer between 5.8 and 6.9 min. The ion source temperature was 700°C; for other parameters see Supplementary   Table S6.
For analysis of 3-OH-BaA and 3-OH-BaP, a two-dimensional separation was carried out, using two analytical columns: column I: Genesis (C8, 4.6 × 20 mm, 4 µm) and column II: Genesis Lightn (C18, 4.6 × 100 mm, 4 µm,), and four LC pumps. The columns and LC pumps were connected through a diverter valve. The two mobile phases used consisted of water (A) and methanol (B). An aliquot of 20 µL of the sample was injected on column I and the separation was carried out by gradient elution, beginning with 55% mobile phase B for 1.55 min and a gradient to 70% B for 1.5 min. After 2.85 min, the diverter valve switched over and the effluent was diverted onto column II for 1.3 min. The second set of pumps continued the gradient from 70% B to 95% B for 2 minutes on column II. A valve on the MS diverted the column II effluent to the MS between 4.5-6.3 min. Column I was reconditioned with 95% mobile phase B for 1 min, followed by equilibration with 55% mobile phase A for 2.5 min and column II was reconditioned with 95% mobile phase B for 1 min in the end of the analytical run and then equilibrated with 70% mobile phase B during the beginning of the next analytical run. The columns were maintained at 60°C and the flow rate was 0.6 mL/min. The ion source temperature was 700°C, for other parameters see Supplementary Table S6.
Data were acquired and processed using the supplied software (Analyst 1.6.1, Multiquant 2.1, AB Sciex). Concentrations were determined by peak area ratios of the analytes versus the ISs.
Within each analytical batch, 80 urine samples, a calibration curve, 2 QC samples for 1-OH-PYR and 2-OH-PH and 2 chemical blanks were included. All samples were prepared in duplicate and analyzed by single injections. The average concentration of the duplicate samples was used.
A hand refractometer was used for specific gravity (SG) determinations of urine, the concentrations were adjusted for urinary SG according to C SG = C(observed) × (1.020 − 1)/(⍴-1), where C(observed) was the determined concentration in a urine sample, ⍴ the measured SG, and 1.020, the average SG of all urine samples in this study.

Validation
The limit of detection (LOD) was estimated from the blank samples and it was 50 pg/mL for 1-OH-PYR, and 2-OH-PH, and 2 pg/mL for 3-OH-BaP and 3-OH-BaA. Analytical reproducibility, expressed as coefficient of variation (CV) in n = 400 duplicate urine samples, was 14% for 1-OH-PYR at 0.5 ng/mL, and 9% for 2-OH-PH at 0.8 ng/mL.