Blood oxidative stress biomarkers in women: influence of oral contraception, exercise, and N-acetylcysteine.

PURPOSE: To compare physiological responses to submaximal cycling and sprint cycling performance in women using oral contraceptives (WomenOC) and naturally cycling women (WomenNC) and to determine whether N-acetylcysteine (NAC) supplementation mediates these responses. METHODS: Twenty recreationally trained women completed five exercise trials (i.e., an incremental cycling test, a familiarisation trial, a baseline performance trial and two double-blind crossover intervention trials). During the intervention trials participants supplemented with NAC or a placebo 1 h before exercise. Cardiopulmonary parameters and blood biochemistry were assessed during 40 min of fixed-intensity cycling at 105% of gas-exchange threshold and after 1-km cycling time-trial. RESULTS: WomenOC had higher ventilation (ß [95% CI] = 0.07 L·min-1 [0.01, 0.14]), malondialdehydes (ß = 12.00 mmol·L-1 [6.82, 17.17]) and C-reactive protein (1.53 mg·L-1 [0.76, 2.30]), whereas glutathione peroxidase was lower (ß =  22.62 mU·mL-1 [- 41.32, - 3.91]) compared to WomenNC during fixed-intensity cycling. Plasma thiols were higher at all timepoints after NAC ingestion compared to placebo, irrespective of group (all p < 0.001; d = 1.45 to 2.34). For WomenNC but not WomenOC, the exercise-induced increase in malondialdehyde observed in the placebo trial was blunted after NAC ingestion, with lower values at 40 min (p = 0.018; d = 0.73). NAC did not affect cycling time-trial performance. CONCLUSIONS: Blood biomarkers relating to oxidative stress and inflammation are elevated in WomenOC during exercise. There may be an increased strain on the endogenous antioxidant system during exercise, since NAC supplementation in WomenOC did not dampen the exercise-induced increase in malondialdehyde. Future investigations should explore the impact of elevated oxidative stress on exercise adaptations or recovery from exercise in WomenOC.

Sexual dimorphism: increased sterol excretion leads to hypocholesterolaemia in female hyperbilirubinaemic Gunn rats.

Circulating bilirubin is associated with reduced serum cholesterol concentrations in humans and in hyperbilirubinaemic Gunn rats. However, mechanisms contributing to hypocholesterolaemia remain unknown. Therefore, this study aimed to investigate cholesterol synthesis, transport and excretion in mutant Gunn rats. Adult Gunn and control rats were assessed for daily faecal sterol excretion using metabolic cages, and water was supplemented with [1-13 C]-acetate to determine cholesterol synthesis. Bile was collected to measure biliary lipid secretion. Serum and liver were collected for biochemical analysis and for gene/protein expression using RT-qPCR and western blot, respectively. Additionally, serum was collected and analysed from juvenile rats. A significant interaction of sex, age and phenotype on circulating lipids was found with adult female Gunn rats reporting significantly lower cholesterol and phospholipids. Female Gunn rats also demonstrated elevated cholesterol synthesis, greater biliary lipid secretion and increased total faecal cholesterol and bile acid excretion. Furthermore, they possessed increased hepatic low-density lipoprotein (LDL) receptor and SREBP2 expression. In contrast, there were no changes to sterol metabolism in adult male Gunn rats. This is the first study to demonstrate elevated faecal sterol excretion in female hyperbilirubinaemic Gunn rats. Increased sterol excretion creates a negative intestinal sterol balance that is compensated for by increased cholesterol synthesis and LDL receptor expression. Therefore, reduced circulating cholesterol is potentially caused by increased hepatic uptake via the LDL receptor. Future studies are required to further evaluate the sexual dimorphism of this response and whether similar findings occur in females with benign unconjugated hyperbilirubinaemia (Gilbert's syndrome). KEY POINTS: Female adult hyperbilirubinaemic (Gunn) rats demonstrated lower circulating cholesterol, corroborating human studies that report a negative association between bilirubin and cholesterol concentrations. Furthermore, female Gunn rats had elevated sterol excretion creating a negative intestinal sterol balance that was compensated for by elevated cholesterol synthesis and increased hepatic low-density lipoprotein (LDL) receptor expression. Therefore, elevated LDL receptor expression potentially leads to reduced circulating cholesterol levels in female Gunn rats providing an explanation for the hypocholesterolaemia observed in humans with elevated bilirubin levels. This study also reports a novel interaction of sex with the hyperbilirubinaemic phenotype on sterol metabolism because changes were only reported in females and not in male Gunn rats. Future studies are required to further evaluate the sexual dimorphism of this response and whether similar findings occur in females with benign unconjugated hyperbilirubinaemia (Gilbert's syndrome).

Effect of Silymarin Treatment on Circulating Bilirubin and Cardiovascular Disease Risk Factors in Healthy Men: A Single-Blind, Randomized Crossover Trial.

This clinical trial (ACTRN12619001296123) investigated the impact of silymarin (Legalon®) on circulating bilirubin concentration, lipid status, systemic inflammation, and antioxidant status. The study design was a randomized, placebo-controlled, single-blind crossover trial of healthy men (18-65 years), conducted at Griffith University, Gold Coast, Australia. Participants were recruited from Griffith University and were randomized to silymarin (140 mg silymarin capsules thrice daily) or placebo (3 capsules containing mannitol taken daily) for 14 days followed by a ≥4-week washout and crossover to the other arm. The main outcomes were whether silymarin treatment would increase serum bilirubin concentration by >0.29 mg/dL, change serum lipid status (cholesterol and triglycerides), inflammation (c-reactive protein), and antioxidant capacity (ferric reducing ability of plasma) compared with baseline. Silymarin consumption (n = 17) did not affect serum concentrations of unconjugated bilirubin (0.73 versus 0.67 mg/dL, P = .79), cholesterol (185 versus 189 mg/dL, P = .19), triglycerides (94.2 versus 92.3 mg/dL, P = .79), c-reactive protein (0.17 versus 0.09 mg/dL, P = .23), or antioxidant status (6.61 versus 6.67 mg Fe2+ /dL, P = .40). These findings challenge previous reports and manufacturer claims of hyperbilirubinemia following silymarin treatment and are critical to guiding researchers toward an effective means to mildly elevate bilirubin, which evidence suggests could protect from cardiovascular disease.

Temporal changes in blood oxidative stress biomarkers across the menstrual cycle and with oral contraceptive use in active women.

PURPOSE: To examine the temporal changes in blood oxidative stress biomarkers in recreationally-trained women that were naturally-cycling (WomenNC) or using oral contraceptives (WomenOC) across one month. METHODS: Blood samples were acquired at three timepoints of the menstrual cycle (1: early-follicular, 2: late-follicular and 3: mid-luteal) and oral contraceptive packet (1: InactiveOC, 2: Mid-activeOC and 3: Late-activeOC) for determination of estradiol, progesterone, oxidative stress, C-reactive protein (CRP) and other cardiometabolic biomarkers in plasma and serum. RESULTS: There was a Group by Time effect on estradiol (p < 0.001, partial η2 = 0.64) and progesterone (p < 0.001, partial η2 = 0.77). Malondialdehyde, lipid hydroperoxides and CRP concentrations were higher in WomenOC during Late-activeOC compared to InactiveOC (+ 96%, + 23% and + 104%, respectively, p < 0.05). However, there were no changes in these biomarkers across the menstrual cycle in WomenNC (p > 0.05). At all timepoints (i.e., 1, 2 and 3), WomenOC had elevated lipid hydroperoxides (+ 28, + 48% and + 50%) and CRP (+ 71%, + 117% and + 130%) compared to WomenNC (p < 0.05, partial η2 > 0.25). There was no Group by Time effect on non-enzymatic antioxidants or glutathione peroxidase; however, glutathione peroxidase was lower in WomenOC, i.e., main effect of group (p < 0.05, partial η2 > 0.20). CONCLUSION: These findings demonstrate that WomenOC not only have higher oxidative stress and CRP than WomenNC, but also a transient increase across one month of habitual oral contraceptive use. Since changes in oxidative stress and CRP often relate to training stress and recovery, these outcomes may have implications to workload monitoring practices in female athletes.

Biliverdin and bilirubin sulfonate inhibit monosodium urate induced sterile inflammation in the rat.

BACKGROUND: Biliverdin, a by-product of haem catabolism, possesses potent endogenous antioxidant and anti-inflammatory properties. Bilirubin-C10-sulfonate (BRS), an active metabolite formed after enteral administration of BV in the rat, also possess antioxidant properties. Therefore, we investigated the anti-inflammatory and antioxidant activity of BV and BRS in an in vivo model of monosodium urate induced sterile inflammation. METHODS: Subcutaneous air pouches were created on the dorsal flanks of Wistar rats (10-12 weeks of age). Prior to stimulation of the 6-day old pouch with monosodium urate (25 mg), groups were pre-treated with intraperitoneal BRS (27 mg/kg) and BV (27 mg/kg). Total and differential leukocyte counts were determined in pouch fluid aspirate at 1, 6, 12, 24 and 48 h after monosodium urate stimulation. Biliverdin (BV), BRS and unconjugated bilirubin (UCB) concentrations in the serum and pouch fluid were quantified using liquid chromatography-mass spectrometry. Pouch fluid cytokine concentrations (IL-1ß, IL-1α, TNF-α, IL-17A, IL-12, GM-CSF, IL-33, IFN-γ, IL-18, IL-10, MCP-1, CXCL-1 and IL-6) were assessed after 6 h. In addition, 24 h protein carbonyl and chloramine concentrations were assessed in pouch fluid using ELISA and spectrophotometry, respectively. RESULTS: BRS and BV significantly (p < 0.05) inhibited leukocyte (total, neutrophil and macrophage) infiltration into the pouch fluid from 6 to 48 h. For example, after 6 h neutrophil counts decreased following BRS (0.32 ± 0.11 × 106 cells mL-1) and BV (0.17 ± 0.03 × 106 cells mL-1) compared to MSU only (3.51 ± 1.07 × 106 cells mL-1). Both BV and BRS significantly (p < 0.05) reduced pouch GM-CSF (BV: 5.8 ± 1.2 pg mL-1, BRS: 6.9 ± 1.5 pg mL-1 vs MSU only: 13.0 ± 1.9 pg mL-1) and MCP-1 concentrations at 6 h (BV: 1804 ± 269 pg mL-1, BRS: 7927 ± 2668 pg mL-1 vs MSU only: 17,290 ± 4503 pg ml-1), whilst BV additionally inhibited IL-6 (4354 ± 977 pg mL-1 vs MSU only: 25,070 ± 5178 pg mL-1) and IL-18 (17.6 ± 2.0 pg mL-1 vs MSU only: 81.5 ± 19.9 pg mL-1) concentrations at 6 h (p < 0.05). Despite these differences, no change in pouch chloramine or protein carbonyl concentrations occurred at 24 h (p > 0.05). Serum BV concentrations rapidly diminished over 6 h, however, BRS was readily detected in the serum over 48 h, and in pouch fluid over 12 h. CONCLUSIONS: This study is the first to elucidate anti-inflammatory activity of BRS and the efficacy of BV administration in a model of gouty inflammation. Reduced leukocyte infiltration and cytokine production in response to sterile inflammation further support the importance of these molecules in physiology and their therapeutic potential in sterile inflammation.

Muscle fiber typology is associated with the incidence of overreaching in response to overload training.

The aim of this study was to identify markers of training stress and characteristics of middle-distance runners related to the incidence of overreaching following overload training. Twenty-four highly trained middle-distance runners [n = 16 male, peak oxygen uptake (V̇o2peak) = 73.3 (4.3) mL·kg·min-1; n = 8 female, V̇o2peak = 63.2 (3.4) mL·kg·min-1] completed 3 wk of normal training (NormTr), 3 wk of high-volume training (HVTr; a 10%, 20%, and 30% increase in training volume each successive week from NormTr), and 1 wk of taper (TapTr; 55% exponential reduction in training volume from HVTr week 3). Before and immediately after each training period, an incremental treadmill-running test was performed, while resting metabolic rate (RMR), subjective fatigue responses, and various resting blood biomarkers were assessed. Muscle fiber typology of the gastrocnemius was estimated by quantification of muscle carnosine using proton magnetic resonance spectroscopy and expressed as a z-score relative to a nonathlete control group. Twelve runners were classified as functionally overreached (FOR) following HVTr [decreased running time to exhaustion (TTE)], whereas the other 12 were classified as acutely fatigued (AF; no decrease in running TTE). The FOR group did not demonstrate systematic alterations in RMR, resting blood biomarkers, or submaximal exercise responses, compared with the AF group. The gastrocnemius carnosine z-score was significantly higher in the FOR group (-0.44 ± 0.57) than in the AF group (-1.25 ± 0.49, P = 0.004, d = 1.53) and was also negatively correlated with changes in running TTE from pre- to post-HVTr (r = -0.55, P = 0.005) and from pre-HVTr to post-TapTr (r = -0.64, P = 0.008). Muscle fiber typology is related to the incidence of overreaching and performance supercompensation following increased training volume and a taper.NEW & NOTEWORTHY Variability in the performance responses following an overload training period and subsequent taper was associated with the variation in the muscle fiber typology of the gastrocnemius. Runners with an estimated higher proportion of type I fibers (i.e., lower carnosine z-score) were able to maintain performance in response to an overload training period and subsequently achieve a superior performance supercompensation. These findings show that muscle fiber typology contributes to the variability in performance responses following training.

The impact of bilirubin ditaurate on platelet quality during storage.

Bilirubin ditaurate (BRT), a conjugated bilirubin analogue, has demonstrated anti-platelet characteristics following acute ex vivo exposure. Scavenging of mitochondrial superoxide and attenuation of granule exocytosis suggested a potential benefit for including BRT for storage. With no reports of cytotoxicity following acute exposure, the impact of 35µM BRT on platelet function was investigated, in clinically suppled units, for up to seven days. Exposure to 35µM BRT significantly reduced mitochondrial membrane potential and increased glucose consumption until exhaustion after 72 hours. Platelet aggregation and activation was significantly impaired by BRT. Mitochondrial superoxide production and phosphatidylserine expression were significantly elevated following glucose exhaustion, with decreased viability observed from day five onwards. Lactate accumulation and loss of bicarbonate, support a metabolic disturbance, leading to a decline of quality following BRT inclusion. Although acute ex vivo BRT exposure reported potentially beneficial effects, translation from acute to chronic exposure failed to combat declining platelet function during storage. BRT exposure resulted in perturbations of platelet quality, with the utility of BRT during storage therefore limited. However, these are the first data of prolonged platelet exposure to analogues of conjugated bilirubin and may improve our understanding of platelet function in the context of conjugated hyperbilirubinemia.

Acute bilirubin ditaurate exposure attenuates ex vivo platelet reactive oxygen species production, granule exocytosis and activation.

BACKGROUND: Bilirubin, a by-product of haem catabolism, possesses potent endogenous antioxidant and platelet inhibitory properties. These properties may be useful in inhibiting inappropriate platelet activation and ROS production; for example, during storage for transfusion. Given the hydrophobicity of unconjugated bilirubin (UCB), we investigated the acute platelet inhibitory and ROS scavenging ability of a water-soluble bilirubin analogue, bilirubin ditaurate (BRT) on ex vivo platelet function to ascertain its potential suitability for inclusion during platelet storage. METHODS: The inhibitory potential of BRT (10-100 µM) was assessed using agonist induced platelet aggregation, dense granule exocytosis and flow cytometric analysis of P-selectin and GPIIb/IIIa expression. ROS production was investigated by analysis of H2DCFDA fluorescence following agonist simulation while mitochondrial ROS production investigated using MitoSOX™ Red. Platelet mitochondrial membrane potential and viability was assessed using TMRE and Zombie Green™ respectively. RESULTS: Our data shows ≤35 µM BRT significantly inhibits both dense and alpha granule exocytosis as measured by ATP release and P-selectin surface expression, respectively. Significant inhibition of GPIIb/IIIa expression was also reported upon ≤35 µM BRT exposure. Furthermore, platelet exposure to ≤10 µM BRT significantly reduces platelet mitochondrial ROS production. Despite the inhibitory effect of BRT, platelet viability, mitochondrial membrane potential and agonist induced aggregation were not perturbed. CONCLUSIONS: These data indicate, for the first time, that BRT, a water-soluble bilirubin analogue, inhibits platelet activation and reduces platelet ROS production ex vivo and may, therefore, may be of use in preserving platelet function during storage.