Publications by Year: 2015

BACKGROUND: Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). RESULTS/METHODOLOGY: We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects.

CONCLUSION: Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease.

INTRODUCTION: We previously found decreased levels of Coenzyme Q10 (CoQ10) in patients with septic shock. The objective of the current study was to assess whether the provision of exogenous ubiquinol (the reduced form of CoQ10) could increase plasma CoQ10 levels and improve mitochondrial function.

METHODS: We performed a randomized, double-blind, pilot trial at a single, tertiary care hospital. Adults (age ≥18 years) with severe sepsis or septic shock between November 2012 and January 2014 were included. Patients received 200 mg enteral ubiquinol or placebo twice a day for up to seven days. Blood draws were obtained at baseline (0 h), 12, 24, 48, and 72 h. The primary outcome of the study was change in plasma CoQ10 parameters (total CoQ10 levels, CoQ10 levels relative to cholesterol levels, and levels of oxidized and reduced CoQ10). Secondary outcomes included assessment of: 1) vascular endothelial biomarkers, 2) inflammatory biomarkers, 3) biomarkers related to mitochondrial injury including cytochrome c levels, and 4) clinical outcomes. CoQ10 levels and biomarkers were compared between groups using repeated measures models.

RESULTS: We enrolled 38 patients: 19 in the CoQ10 group and 19 in the placebo group. The mean patient age was 62 ± 16 years and 47% were female. Baseline characteristics and CoQ10 levels were similar for both groups. There was a significant increase in total CoQ10 levels, CoQ10 levels relative to cholesterol levels, and levels of oxidized and reduced CoQ10 in the ubiquinol group compared to the placebo group. We found no difference between the two groups in any of the secondary outcomes.

CONCLUSIONS: In this pilot trial we showed that plasma CoQ10 levels could be increased in patients with severe sepsis or septic shock, with the administration of oral ubiquinol. Further research is needed to address whether ubiquinol administration can result in improved clinical outcomes in this patient population.

TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT01948063. Registered on 18 February 2013.

INTRODUCTION: Pyruvate dehydrogenase (PDH) is a key gatekeeper enzyme in aerobic metabolism. The main purpose of this study was to determine if PDH activity is affected by major stress in the form of coronary artery bypass grafting (CABG), which has previously been used as a model of critical illness.

METHODS: We conducted a prospective, observational study of patients undergoing CABG at an urban, tertiary care hospital. We included adult patients undergoing CABG with or without concomitant valve surgery. Measurements of PDH activity and quantity and thiamine were obtained prior to surgery, at the completion of surgery, and 6 h after surgery.

RESULTS: Fourteen patients were enrolled (aged 67 ± 10 years, 21% female). Study subjects had a mean 41.7% (SD, 27.7%) reduction in PDH activity after surgery and a mean 32.0% (SD, 31.4%) reduction 6h after surgery (P < 0.001). Eight patients were thiamine deficient (≤ 7 nmol/L) after surgery compared with none prior to surgery (P = 0.002). Thiamine level was significantly associated with PDH quantity at all time points (P = 0.01). Postsurgery lactate levels were inversely correlated with postsurgery thiamine levels (r = -0.58 and P = 0.04).

CONCLUSION: The stress of major surgery causes decreased PDH activity and quantity and depletion of thiamine levels.

RATIONALE: Rodent studies have shown that pyruvate dehydrogenase (PDH) levels are low in sepsis. This may cause cells to shift to anaerobic metabolism, resulting in increased lactate production. Alterations in PDH during sepsis have never been studied in humans.

OBJECTIVES: The objective of this pilot study was to measure PDH activity and quantity in patients with severe sepsis.

METHODS: We conducted a pilot case-control study at a single urban tertiary care center. We compared PDH activity and quantity between patients with severe sepsis admitted to the intensive care unit and healthy control subjects. PDH activity and quantity were measured in isolated peripheral blood mononuclear cells. We measured PDH activity and quantity in control subjects at baseline and in patients with sepsis at 0 (baseline), 24, 48, and 72 hours.

MEASUREMENTS AND MAIN RESULTS: We enrolled 56 patients with sepsis and 20 control subjects with at least one blood sample being drawn from each patient. PDH activity and quantity in the sepsis group were significantly lower than the control group (P < 0.001). In multivariable linear regression adjusting for age, race, sex, and assay plate, the difference remained significant. Patients with sepsis who died had significantly lower PDH activity compared with those who survived (P = 0.03).

CONCLUSIONS: PDH activity and quantity is decreased in peripheral blood mononuclear cells of humans with severe sepsis when compared with healthy control subjects, and may be associated with mortality. Whether decreased PDH activity plays a role in lactate metabolism or whether pharmacologic modification of PDH activity may improve outcomes remains unknown.

BACKGROUND/OBJECTIVES: Obesity is characterized by chronic inflammation and immune dysregulation, as well as insulin resistance, but the link between obesity and adaptive immunity remains to be fully studied.

METHODS: To elucidate the role of adaptive immunity on body composition, glucose homeostasis and inflammation, recombination-activating gene 1 knockout (Rag1-/-) mice, without mature T-lymphocytes or B-lymphocytes, were maintained on a low- or high-fat diet (LFD and HFD, respectively) for 11 weeks.

RESULTS: Rag1-/- mice fed HFD gained significantly more weight and had increased body fat compared with wild type. Downregulation of energy expenditure as well as brown fat uncoupling protein UCP-1 and UCP-3 gene expression were noticed in HFD-fed Rag1-/- mice compared with LFD. HFD mice had significantly decreased energy intake compared with LFD mice, consistent with decreased agouti-related protein and increased pro-opiomelanocortin gene expression levels in the hypothalamus. Moreover, compared with wild type, Rag1-/- mice had lower interleukin (IL)-4 levels, a cytokine recently found to induce browning in white adipocytes, and higher IL-12 levels in HFD-fed Rag1-/- mice. Despite that HFD Rag1-/- mice were more obese, they had similar glucose, insulin and adiponectin levels, while leptin was marginally increased.

CONCLUSIONS: Mice with deficiency in adaptive immunity are obese, partly owing to decreased energy expenditure, but are metabolically normal, suggesting that mature lymphocytes have necessary roles in the development of obesity-related metabolic dysregulation.