Vitamin E sequestration by liver fat in humans

Abstract

BACKGROUNDWe hypothesized that obesity-associated hepatosteatosis is a pathophysiological chemical depot for fat-soluble vitamins and altered normal physiology. Using α-tocopherol (vitamin E) as a model vitamin, pharmacokinetics and kinetics principles were used to determine whether excess liver fat sequestered α-tocopherol in women with obesity-associated hepatosteatosis versus healthy controls.METHODSCustom-synthesized deuterated α-tocopherols (d3- and d6-α-tocopherols) were administered to hospitalized healthy women and women with hepatosteatosis under investigational new drug guidelines. Fluorescently labeled α-tocopherol was custom-synthesized for cell studies.RESULTSIn healthy subjects, 85% of intravenous d6-α-tocopherol disappeared from the circulation within 20 minutes but reappeared within minutes and peaked at 3-4 hours; d3- and d6-α-tocopherols localized to lipoproteins. Lipoprotein redistribution occurred only in vivo within 1 hour, indicating a key role of the liver in uptake and re-release. Compared with healthy subjects who received 2 mg, subjects with hepatosteatosis had similar d6-α-tocopherol entry rates into liver but reduced initial release rates (P < 0.001). Similarly, pharmacokinetics parameters were reduced in hepatosteatosis subjects, indicating reduced hepatic d6-α-tocopherol output. Reductions in kinetics and pharmacokinetics parameters in hepatosteatosis subjects who received 2 mg were echoed by similar reductions in healthy subjects when comparing 5- and 2-mg doses. In vitro, fluorescent-labeled α-tocopherol localized to lipid in fat-loaded hepatocytes, indicating sequestration.CONCLUSIONSThe unique role of the liver in vitamin E physiology is dysregulated by excess liver fat. Obesity-associated hepatosteatosis may produce unrecognized hepatic vitamin E sequestration, which might subsequently drive liver disease. Our findings raise the possibility that hepatosteatosis may similarly alter hepatic physiology of other fat-soluble vitamins.TRIAL REGISTRATIONClinicalTrials.gov, NCT00862433.FUNDINGNational Institute of Diabetes and Digestive and Kidney Diseases and NIH grants DK053213-13, DK067494, and DK081761.

Keywords: Hepatology; Metabolism; Obesity.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Recruitment scheme.

The recruitment scheme is shown for this clinical intervention (NCT00862433), conducted at the NIH Clinical Research Center. Data from all participants are included in the analyses. Green indicates 6 healthy women of the 10 healthy women who received 2 mg α-tocopherol who also received, after a period of washout, 5 mg α-tocopherol. IV, intravenous; PO, per oral administration.

Figure 2. Plasma concentrations of d6- and d3-α-tocopherol and redistribution into lipoproteins in healthy women.

(A) Protocol sampling scheme. (B) At time 0 in 6 subjects, d6-α-tocopherol 5 mg (●) was administered IV and d3-α-tocopherol 5 mg (○) was administered orally, with plasma samples obtained over 72 hours. (C) Plasma samples in B, magnified time interval –2 to 8 hours. (D) Percentage enrichment of d6-α-tocopherol (▲, HDL; ■, LDL; ●, VLDL) in lipoproteins following administration of 5 mg at time 0 in 6 subjects. (E) Percentage enrichment of d3-α-tocopherol (Δ, HDL; □, LDL; ○, VLDL) in lipoproteins following administration of 5 mg at time 0 in 6 subjects. Because HDL and LDL points were superimposed, for clarity HDL and LDL curves were slightly offset on the y axis. (F) Percentage of recovery of d3-α-tocopherol (○) and d6-α-tocopherol (■) in all lipoproteins compared with plasma concentrations in 6 subjects. (G) Distribution of 7 μM of d6-α-tocopherol in lipoproteins over time ex vivo (Δ, HDL; □, LDL; ○, VLDL). (H) At time 0 in 6 subjects, d6-α-tocopherol 5 mg (●) and 2 mg (●) was administered IV, with plasma samples obtained over 72 hours; inset: initial rates of d6-α-tocopherol reappearance for these 2 doses. SL, slope: 0.195 μM/h for 5-mg dose and 0.083 μM/h for 2-mg dose. Data represented as mean ± SEM. n = 6 for HS and n = 10 for healthy cohorts.

Figure 3. Influence of liver fat on d6-α-tocopherol plasma concentrations: pharmacokinetics and kinetics in 10 healthy and 6 HS subjects.

(A) d6-α-Tocopherol 2 mg administered IV in healthy (●) or HS subjects (●) over 72 hours. (B–D) Initial rates of d6-α-tocopherol reappearance matched to patient status (C), to percentage liver fat by MRI (D). (E and F) Initial rates of d6-α-tocopherol reappearance to percentage body fat by DEXA in healthy (E) and HS (F) subjects. n = 6 for HS and n =10 for healthy cohorts. (A and B) Data represented as mean ± SEM. (C) Data represented as median.

Figure 4. Fat traps vitamin E in vitro and in vivo.

(A–C) Bodipy-α-tocopherol (BDP-α-tocopherol) quantification by flow cytometry 30 hours after incubation using Huh7.5, HepG2C2A, or immortalized human hepatocyte (IHH) cells without/with 100 μM oleic acid. n = 6, representative experiment of 3 independent repeats. (D) Confocal microscopy of McARH7777 cells without/with 100 μM oleic acid (Nile Red staining) incubated 2 hours with 10 μM BDP-α-tocopherol (shown in green). Outlined areas indicate individual cell contours. (E) Mouse liver α-tocopherol (HPLC analyses) using animals fed normal chow/high-fat normal vitamin E chow (HFNE); n = 5. **P < 0.01; and ***P < 0.001, Student’s t test (unpaired, 2 tailed). Data represented as mean ± SD.

Figure 5. Plasma concentrations of d6- and d3-α-tocopherol and redistribution into lipoproteins in 10 healthy versus 6 HS women: dual-isotope analyses.

(A) d3-α-Tocopherol 2 mg administered IV in healthy (○) or HS subjects (○) over 72 hours; data represented as mean ± SD. (B and C) AUC of (B) d3-α-tocopherol percentage enrichment and (C) d6-α-tocopherol percentage enrichment for VLDL, LDL, and HDL from 0 to 8 hours following administration of 2 mg d3- or d6-α-tocopherol in healthy and HS subjects. (D and E) Percentage enrichment of (D) d3-α-tocopherol and (E) d6-α-tocopherol in VLDL in healthy (●) and HS (●) at time 8 hours; data represented as median. *P < 0.05; **P < 0.01, Student’s t test (unpaired, 2-tailed). (F and G) AUC0–72h of d3-α-tocopherol matched to percentage body fat by DEXA for HS (F) and healthy (G) subjects. n = 6 for HS and n = 10 for healthy cohorts.

Figure 6. Proposed vitamin E (α-tocopherol) physiology in healthy subjects and pathophysiology in subjects with HS.

Left (healthy subjects): Following oral ingestion, α-tocopherol is transported (via chylomicrons to the thoracic duct and general circulation) to hepatocytes on lipoproteins. Internalized α-tocopherol is specifically recognized and transported through hepatocytes by tocopherol transfer protein (TTP), with release into the space of Disse and lipoprotein capture, shown as VLDL. Biologically available vitamin E quenches reactive oxygen species (ROS) generated by normal hepatocyte metabolism. Right (subjects with HS): Following oral ingestion, some α-tocopherol might be sequestered in intestinal fat (i.e., extrahepatic fat) with a reduction in availability. Remaining available vitamin E is transported to hepatocytes on lipoproteins. Internalized α-tocopherol is diverted into liver fat, acting as a chemical sink, resulting in decreased vitamin E availability within the hepatocyte and a functional local hepatocyte deficiency. Additionally, vitamin E local release into the space of Disse may be reduced. Less local vitamin E could lead to unquenched ROS that can damage hepatocytes directly and/or activate pericytes and Kupffer cells, over time producing inflammation, hepatitis, and fibrosis (cirrhosis).