Standard kit

Blood samples and 24 h urine were drawn after an overnight fast for biochemistry measurements. The Roche electrochemiluminescence system measured beta-C-telopeptides of type I collagen (β-CTX) (E170, Roche Diagnostics, Basel, Switzerland). Serum-free soluble receptor activator of nuclear factor-kappaB ligand (sRANK), osteoprotegerin (OPG), and DKK1 were measured by an enzyme-linked immunosorbent assay (ELISA) using standard kits (Human sRANKL (Total), Human osteoprotegerin, Biovendor, Brno, Czech Republic; Human DKK-1 Quantikine, R&D Systems). Urinary PGE2 and PGEM levels were measured by competitive ELISA using standard kits (Cayman Chemicals, Ann Arbor, Michigan, USA). All biochemical parameters were evaluated in PHO patients. Simultaneously, DKK1, sRANKL, and OPG were assessed in HCs.

Catalase and superoxide dismutase (SOD) activity of tumor and liver lysates (of subset III mice) was estimated using standard kits (Cayman Chemicals, Ann Arbor, MI, USA) following manufacturer’s instructions. Statistical significance was tested using unpaired Student’s t-test where P<0.05 was considered as the minimal level of significance.

Plasma TAC was determined using a standard kit (Cayman Chemicals, Ann Arbor, MI), which relies on the ability of antioxidants to inhibit of the oxidation of ABTS (2 ,2'‐azino‐di‐[3‐ethylbenzthiazoline sulphonate]) to ABTS^•+ in the presence of metmyoglobin.¹⁵ We monitored the formation of ABTS^•+ by measuring the absorbance at 405 nm in a 96‐well clear bottom plate using a SynergyMx Multi‐mode Microplate Reader (BioTek Instruments Inc., Vermont). The assay was measured before and after e‐hookah vaping sessions with and without ascorbic acid infusion and was calibrated with vitamin E analogue trolox and the results were expressed in millimolar trolox equivalent.