The amounts of total and cytochrome P450 proteins in circulating EVs were increased in human patients with alcoholism. (A) Analysis of the size of circulating EVs isolated from the sera of healthy controls (n = 9) who never drank alcohol due to religious reasons and patients with alcoholism (n = 14) with hepatitis. The inset figures represent transmission electron microscopy images. (B) EV size was determined by a Nanoparticle Tracking analysis (Nanosight).18 (C) Total number of EVs in the sera of healthy controls or patients with alcoholism was measured by Nanosight. (D) Analysis of the total protein amounts in circulating EVs isolated from the sera of healthy controls or patients with alcoholism. (E) Detection of the cytochrome P450 proteins (i.e., CYP2E1, CYP2A6, CYP1A1/2, CYP4A, and CYP4B) in circulating EVs from the different groups by immunoblot analyses, as indicated. CD63 was used as a loading control for the same amounts of total EV proteins in all samples. Densitometric quantitation of the immunoblots for CYP2E1, CYP2A6, CYP1A1/2, CYP4A, and CYP4 relative to CD63 (n = 4/group) is shown. *P < 0.05, **P < 0.01. Data are shown as means ± SD.