Browsing by Author "Hughes, Michael Douglas Jr."

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    Investigating the Influence of Fresh and Aged Garlic Extracts on the Biosynthesis of Trimethylamine N-Oxide
    Hughes, Michael Douglas Jr. (Virginia Tech, 2021-01-07)
    Introduction: Garlic-derived organosulfur compounds are associated with physiological benefits, including the reduction of cardiovascular disease (CVD) risk, possibly by reducing the risk marker trimethylamine-N-oxide (TMAO). TMAO production in humans is largely influenced by the metabolic activity of the intestinal bacteria on dietary precursors including L-carnitine. Dietary supplementation of bioactive garlic phytochemical allicin has recently been suggested to reduce the formation of TMAO precursor molecule trimethylamine (TMA) from L-carnitine through impact on the intestinal bacteria, thereby limiting the formation of TMAO by the host. Purpose: The objective of this research was to evaluate and compare the efficacy of fresh and aged garlic extracts (rich in alliin and allicin, respectively) in the reduction of circulating TMAO levels produced from L-carnitine metabolism and identify shifts in the abundances of gastrointestinal bacterial genes that may contribute to reduction in circulating TMA levels, which may, in turn, influence the levels of circulating TMAO. Methods: Five-week old female C57BL/6 mice (n = 12) were challenged with L-carnitine to assess the animal's capacity for TMAO production. Animals were gavaged daily with fresh or aged garlic extract dissolved in L-carnitine for 13 days, then challenged with L-carnitine post-treatment to evaluate changes in TMAO production. Whole blood samples were evaluated for TMAO content using UPLC-MS/MS and compared to non-extract consuming control groups. Post-mortem hepatic tissues were collected and analyzed for TMA-oxidizing flavin monooxygenase 3 (Fmo3) gene abundance and protein expression using quantitative real-time PCR (qPCR) and ELISA. Fecal samples collected prior to and following treatment were analyzed using qPCR to quantify shifts in the abundance of L-carnitine metabolizing genes cntAB and grdH. Results: Postprandial and circulating TMAO levels were not significantly affected (p < 0.05) by inclusion of garlic extract in the diet. Dietary intervention with extracts significantly increased L-carnitine-derived proatherogenic CVD risk marker γ-butyrobetaine levels ~28% higher than the increased levels observed in the positive control group supplemented with L-carnitine only. Mice administered garlic extracts had significant increases of, γ-butyrobetaine, relative to negative control mice and mice supplemented with broad-spectrum antibiotics. Mice supplemented fresh garlic extract saw a 25-fold increase in circulating γ-butyrobetaine levels after intervention; mice supplemented aged garlic extract saw a 23-fold increase in circulating γ-butyrobetaine levels after intervention. Furthermore, FMO3 protein expression levels in either extract treatment group were not significantly different (p < 0.05) from controls. Abundances of L-carnitine metabolizing genes in fecal samples of mice fed either garlic extract were not significantly higher than levels observed in positive or negative controls. Interestingly, treatment with broad-spectrum antibiotics significantly increased abundances of L-carnitine metabolizing genes cntAB and grdH when compared with controls. Abundances of hepatic Fmo3 mRNA transcript in mice supplemented garlic extracts were not significantly different from the positive control group when data were normalized to mg of liver used. Mice supplemented aged garlic extracts significantly lowered Fmo3 mRNA transcript levels relative to the negative control. Significance: This research suggests that garlic extract supplementation in conjunction with excess L-carnitine consumption may not be an appropriate dietary intervention strategy to reduce CVD risk. As it stands, garlic extract supplementation may increase CVD risk by promoting the biosynthesis of proatherogenic γ-butyrobetaine. The impact of garlic extract mediated increases in γ-butyrobetaine should be further investigated in tandem with CVD outcomes to confirm the findings presented in this study.