Inadequate concentrations of progesterone during gestation can result in impaired embryonic growth and losses. These losses may be attributed to an overactive mechanism of progesterone catabolism or improper luteal function, which results in low concentration of progesterone. Progesterone catabolism occurs to the greatest extent by the liver, which holds a vast supply of cytochrome P450 enzymes and aldo-keto reductases that are involved in steroid inactivation. Insulin is a hormone produced by the pancreas that is involved in glucose uptake and metabolism. Progesterone catabolism is decreased in the presence of elevated insulin levels. Lipoic acid is a naturally occurring antioxidant and multienzyme cofactor which has been shown to increase insulin sensitivity and enhance glucose uptake in a number of species. The objectives of the current experiments were to 1) determine if administering a racemic mixture of lipoic acid by gavage at a dose of 32 mg/kg BW would increase peripheral progesterone concentrations, decrease progesterone clearance rates, or modulate cytochrome P450 2C (CYP2C), cytochrome P450 3A (CYP3A), or aldo-keto reductase 1 C (AKRIC) hepatic enzyme activity, and 2) determine if dosing lipoic acid directly into the rumen at 32 mg/kg BW or 64 mg/kg BW would increase progesterone in the blood, decrease progesterone clearance rates, or modulate insulin. In the first trial, Katahdin cross ovariectomized ewes were randomly assigned to a control or a lipoic acid treatment group. In this experiment, a controlled internal drug release (CIDR) device was inserted in all ewes and serum samples were collected daily for five days to determine progesterone. Liver biopsies were performed on day 10 to measure CYP2C, CYP3A, and AKRI C activity. Following liver biopsies, CIDRs were removed and an intensive blood sampling was performed to measure progesterone decay from peripheral circulation. We found that while lipoic acid does not have an effect on peripheral progesterone concentrations or hepatic enzyme activity, lipoic acid supplemented ewes have decreased progesterone clearance rates compared to control ewes. In the second trial, ovariectomized Katahdin cross ewes were randomly assigned to a control, low lipoic acid (32 mg/kg BW), or a high lipoic acid (64 mg/kg BW) treatment group. A CIDR was inserted in all ewes and blood samples were taken daily for 4 days. Following CIDR removal on day 11, an intensive blood sampling was performed to measure progesterone decay from peripheral circulation. One week following CIDR removal, ewes underwent an intravenous glucose tolerance test. It was found that lipoic acid supplementation did not affect progesterone concentrations, progesterone clearance, or insulin area under the curve. There was a treatment effect such that high lipoic acid dosed ewes had higher area under the curve for glucose when compared to control and low lipoic acid dosed ewes. Although no differences in progesterone concentrations were seen in the second trial, we speculate that the administration method rather than the efficacy of lipoic acid may account for the lack of differences observed. This theory is based on evidence from our first trial that oral lipoic acid supplementation did in fact reduce progesterone catabolism, as well as published data demonstrating that ruminally dosed lipoic acid is less effective than the equivalent oral dose.

The vascular endothelium is a single cell layer that lines the lumen of the entire vasculature. It is the site of synthesis of nitric oxide (NO), a vasodilatory compound synthesized by endothelial nitric oxide synthase (eNOS). NO causes intracellular calcium sequestration of the vascular smooth muscle cells, relaxing and dilating the arteries. Age profoundly affects endothelium-dependent vasodilation, leading to specific losses of NO. We sought to determine what causes the age-specific loss of endothelial NO. This was accomplished by investigating whether there are differences in markers of eNOS post-translational regulation elements in the aortic endothelium of young (2-4 months; corresponding to an adolescent human adult) and old (32-34 months; corresponding to a 65-75 year-old human). F 344 x Brown Norway hybrid rats. Results show that maximal eNOS activity significantly declines with age (n=4;p���0.05) though there was no change in eNOS protein levels in the aortic endothelium. Endothelial NOS exists in two distinct subcellular fractions. No alterations were detected in the soluble, inactive fraction while significantly less eNOS protein is detected in the active, plasma membrane fraction of the endothelium (n=4;p���0.02). Endothelial NOS activation is also controlled by its phosphorylation state. In this work we demonstrate that free ceramides and ceramide-activated phosphatase (PP2A) activity are significantly elevated with age in the endothelium and correlate with specific alterations in eNOS phosphorylation status consistent with its inactivation. These changes were concomittent with an age-associated decline in endothelial glutathione (GSH) and increased sphingomyelinase activity which liberates ceramides from membrane sphingolipids. In previously published reports we demonstrated that the dithiol compound R-��-lipoic acid (LA) increased maximal NO synthesis in cultured endothelial cells and that LA improved age-associated loss of eNOS stimulatory phosphorylation in rats. Therefore, we administered pharmacologic doses of LA (40 mg/kg, i.p. over 24 h) to old rats to determine whether it restored NO-dependent vasomotor function. Results show that LA significantly increased endothelial GSH (p���0.05 compared to saline controls), decreased sphingomyelinase activity and reversed the age-related increase in ceramide (p���0.01) in old animals. Finally, LA significantly improved endothelium-dependent vasodilation, suggesting that it might be a good therapeutic agent for age-related vascular endothelial dysfunction. / Graduation date: 2005

Oxidative stress is the major driving force behind the aging process and many age-related diseases. However, direct experimental evidence of whether antioxidants, such as ascorbate (AA) and lipoic acid (LA) can slow the progression of aging process and/or reduce risks of developing degenerative disease is largely absent. This suggests a better understanding of the precise mechanism of how dietary micronutrient affect parameters of involved in cellular redox balance and aging are warranted. In this dissertation, young and old rats were used as our model to understand potential pro-oxidant events that contribute to increases in oxidative stress in various tissues and how antioxidants such as ascorbate and lipoic acid influence these events. Our major findings are that the age-related impairment of mitochondria and increased deposition of iron contribute significantly to heighten levels of oxidative stress, as evidenced by the resultant increases in the rates of oxidant appearance and in the levels of oxidative damage to DNA, lipids and proteins. We find that AA and LA strongly protected against transition metal-ion dependent increases in oxidative stress. AA effectively inhibited transition metal-mediated lipide peroxidation in human plasma. LA in its reduced form effectively binds iron and copper in a redox inactive manner and reversed chronically elevated levels of iron in the brain without removing enzyme bound transition metal ions. LA also significantly attenuated the age-related increase in oxidative stress associated with mitochondrial decay in the heart, as evidenced by the improvements in AA levels and glutathione redox status. The declines in tissue GSH levels in aged rats were strongly associated with the diminished γ-GCL activity (in parallel with decreased expression of the catalytic and modulatory subunits), and lowered Nrf2 expression and binding to ARE sequence in rat liver. Remarkably, all these events were effectively reversed by the administration of LA, modulating the parameters to return to the observed in young animals. The implications of this work open new avenues not only for further understanding of the aging process but also for possible strategies in its modulation by the micronutrients. / Graduation date: 2004