A Metabolomic Analysis of Omega-3 Fatty Acid-Mediated Attenuation of Western Diet-Induced Nonalcoholic Steatohepatitis in LDLR -/- Mice

Background

Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease and a risk factor for cirrhosis, hepatocellular carcinoma and liver failure. Previously, we reported that dietary docosahexaenoic acid (DHA, 22:6,n-3) was more effective than eicosapentaenoic acid (EPA, 20:5,n-3) at reversing western diet (WD) induced NASH in LDLR^-/- mice.

Methods

Using livers from our previous study, we carried out a global non-targeted metabolomic approach to quantify diet-induced changes in hepatic metabolism.

Results

Livers from WD + olive oil (WD + O)-fed mice displayed histological and gene expression features consistent with NASH. The metabolomic analysis of 320 metabolites established that the WD and n-3 polyunsaturated fatty acid (PUFA) supplementation had broad effects on all major metabolic pathways. Livers from WD + O-fed mice were enriched in saturated (SFA) and monounsaturated fatty acids (MUFA), palmitoyl-sphingomyelin, cholesterol, n-6 PUFA, n-6 PUFA-containing phosphoglycerolipids, n-6 PUFA-derived oxidized lipids (12-HETE) and depleted of C_20-22 n-3 PUFA-containing phosphoglycerolipids, C_20-22 n-3 PUFA-derived oxidized lipids (18-HEPE, 17,18-DiHETE) and S-lactoylglutathione, a methylglyoxal detoxification product. WD + DHA was more effective than WD + EPA at attenuating WD + O-induced changes in NASH gene expression markers, n-6 PUFA and oxidized lipids, citrate and S-lactosyl glutathione. Diet-induced changes in hepatic MUFA and sphingolipid content were associated with changes in expression of enzymes involved in MUFA and sphingolipid synthesis. Changes in hepatic oxidized fatty acids and S-lactoylglutathione, however, correlated with hepatic n-3 and n-6 C_20-22 PUFA content. Hepatic C_20-22 n-3 PUFA content was inversely associated with hepatic α-tocopherol and ascorbate content and positively associated with urinary F2- and F3-isoprostanes, revealing diet effects on whole body oxidative stress.

Conclusion

DHA regulation of hepatic SFA, MUFA, PUFA, sphingomyelin, PUFA-derived oxidized lipids and S-lactoylglutathione may explain the protective effects of DHA against WD-induced NASH in LDLR^-/- mice.     

Mechanisms for the prevention of vitamin E excess

The liver is at the nexus of the regulation of lipoprotein uptake, synthesis, and secretion, and it is the site of xenobiotic detoxification by cytochrome P450 oxidation systems (phase I), conjugation systems (phase II), and transporters (phase III). These two major liver systems control vitamin E status. The mechanisms for the preference for α-tocopherol relative to the eight naturally occurring vitamin E forms largely depend upon the liver and include both a preferential secretion of α-tocopherol from the liver into the plasma for its transport in circulating lipoproteins for subsequent uptake by tissues, as well as the preferential hepatic metabolism of non-α-tocopherol forms. These mechanisms are the focus of this review.     

Is there a vitamin E paradox?

In addition to epidemiologic studies that suggest a benefit for high intakes of alpha-tocopherol, studies of supplementation in humans have clearly shown that alpha-tocopherol decreases lipid peroxidation, platelet aggregation, and functions as a potent anti-inflammatory agent. In the five large prospective clinical trials with alpha-tocopherol therapy, four have shown a beneficial effect on cardiovascular end-points (two studies on a primary end-point and two studies on other cardiovascular end-points). Thus, the totality of evidence based on the epidemiologic data, in-vitro studies and animal models, and the clinical trials appears to support a benefit for alpha-tocopherol supplementation in patients with pre-existing cardiovascular disease. However, definitive recommendations must await ongoing clinical trials.     

Pathophysiological analysis of combined burn and smoke inhalation injuries in sheep

We investigated the pathophysiological alterations seen with combined burn and smoke inhalation injuries by focusing on pulmonary vascular permeability and cardiopulmonary function compared with those seen with either burn or smoke inhalation injury alone. To estimate the effect of factors other than injury, the experiments were also performed with no injury in the same experimental setting. Lung edema was most severe in the combined injury group. Our study revealed that burn injury does not affect protein leakage from the pulmonary microvasculature, even when burn is associated with smoke inhalation injury. The severity of lung edema seen with the combined injury is mainly due to augmentation of pulmonary microvascular permeability to fluid, not to protein. Cardiac dysfunction after the combined injury consisted of at least two phases. An initial depression was mostly related to hypovolemia due to burn injury. It was improved by a large amount of fluid resuscitation. The later phase, which was indicated to be a myocardial contractile dysfunction independent of the Starling equation, seemed to be correlated with smoke inhalation injury.     

Acute effects of combined burn and smoke inhalation injury on carboxyhemoglobin formation, tissue oxygenation, and cardiac performance

The objective of this study was to determine the relationship between carboxyhemoglobin (COHb) formation, global oxygen transport, and cardiac performance in the acute phase of combined burn and smoke inhalation injury. Following a third degree burn of 20% of the total body surface area, adult sheep were subjected to cotton smoke (4x12 breaths) according to an established protocol. Compared with baseline (BL), the burn injury led to an immediate and sustained COHb-independent depression in myocardial contractility. Despite a progressive increase in COHb formation, up to a maximum of 78+/-3% (P < 0.001 vs BL), smoke inhalation did not further impair these hemodynamic changes. This study demonstrated that in the early stage of combined burn and smoke inhalation injury, the depression in cardiac function is basically triggered by the burn injury, whereas COHb generation secondary to cotton smoke exposure primarily contributes to pulmonary shunting.     

Vitamin E delivery to human skin: studies using deuterated alpha-tocopherol measured by APCI LC-MS

Enrichment of skin surface lipids with deuterium-labeled alpha-tocopherol was compared with plasma enrichment to evaluate kinetics of the delivery of vitamin E to skin surface lipids. For 7 d, subjects consumed 75 mg each of RRR-alpha-[5-(C2H3)]- (d3) and all rac-alpha-[5,7-(C2H3)2]- (d6) tocopheryl acetates with breakfast. Blood was drawn and skin lipids were collected daily for 2 weeks, then every other day for 2 weeks. A liquid chromatography-mass spectrometry atmospheric pressure chemical ionization method for quantification of deuterium labeled (d3, d6, d9-alpha-tocopherols) and unlabeled (d0-) alpha- and gamma-tocopherols was developed. Tocopherols were quantified at their m/z [M-1] using single ion recording. alpha-Tocopherol detection was linear from 1 to 100 pmol with a detection limit of 40 pg (93 fmol). Detection of gamma-tocopherol was twice as sensitive due to greater ionization efficiency. Though d3- and d6-alpha-tocopherols appeared in plasma within 24 h of the first dose, d3-alpha-tocopherol was not detected in skin surface lipids until approximately 1 week. Plasma percentage d3 peaked at day 8, while skin surface lipid percentage d3 increased on average until day 19. Apparently skin employs a mechanism to deliver alpha-tocopherol into skin via lipid secretions.     

Enrichment of rat hepatic organelles by vitamin E administered subcutaneously

Novel modes of administering antioxidants to improve delivery to targeted tissues or cells may be advantageous in preventing oxidant-induced pathologies. Vitamin E (alpha-tocopherol) has been shown to be protective in several models of liver injury. The objectives of this study were: (1) to determine if subcutaneously (s.q.) administered emulsified vitamin E enriched liver and hepatic subcellular fractions with the antioxidant and (2) to carry out a time-dependent analysis of serum and tissue vitamin E in rats receiving daily s.q. vitamin E. In the first experiment rats injected daily s.q. with emulsified vitamin E for 9 d increased serum, total liver, liver mitochondria, and liver microsomes by 8-, 16-, 30-, and 29-fold, respectively, compared with placebo injections. Similar enrichment was observed after intramuscular injections. In the second experiment, daily doses of s.q. vitamin E increased liver concentrations 40-fold by 9 d, which decreased to 22-fold by 18 d, whereas serum adjusted vitamin E levels maximized with a 24-fold increase by day 3 and plateaued thereafter. In conclusion, s.q. administration of emulsified vitamin E to rats resulted in substantially elevated serum and liver concentrations of alpha-tocopherol compared with levels achievable by dietary supplementation. The s.q. route of administration is a potentially effective parenteral mode of delivery of vitamin E for conditions in which hepatic oxidative stress is present.