Determination of phosphate-activated glutaminase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry)

Phosphate-activated glutaminase (PAG) converts glutamine to glutamate as part of the glutaminolysis pathway in mitochondria. Two genes, GLS1 and GLS2, which encode for kidney-type PAG and liver-type PAG, respectively, differ in their tissue-specific activities and kinetics. Tissue-specific PAG activity and its kinetics were determined by metabolic mapping using a tetrazolium salt and glutamate dehydrogenase as an auxiliary enzyme in the presence of various glutamine concentrations. In kidney and brain, PAG showed Michaelis-Menten kinetics with a K(m) of 0.6 mM glutamine and a V(max) of 1.1 µmol/mL/min when using 5 mM glutamine. PAG activity was high in the kidney cortex and inner medulla but low in the outer medulla, papillary tip, glomeruli and the lis of Henle. In brain tissue sections, PAG was active in the grey matter and inactive in myelin-rich regions. Liver PAG showed allosteric regulation with a K(m) of 11.6 mM glutamine and a V(max) of 0.5 µmol/mL/min when using 20 mM glutamine. The specificity of the method was shown after incubation of brain tissue sections with the PAG inhibitor 6-diazo-5-oxo-L-norleucine. PAG activity was decreased to 22% in the presence of 2 mM 6-diazo-5-oxo-L-norleucine. At low glutamine concentrations, PAG activity was higher in periportal regions, indicating a lower K(m) for periportal PAG. When compared with liver, kidney and brain, other tissues showed 3-fold to 6-fold less PAG activity. In conclusion, PAG is mainly active in mouse kidney, brain and liver, and shows different kinetics depending on which type of PAG is expresse

Value of magnetic resonance imaging, angiography, and fractional flow reserve to evaluate the left main coronary artery after direct surgical angioplasty

Background: Direct surgical angioplasty of the left main coronary artery is aimed to restore a more physiologic blood flow through the left main coronary artery compared with conventional bypass surgery and allows subsequent percutaneous coronary interventions of more distal coronary lesions. Some data on anatomic evaluation with coronary angiography and magnetic resonance imaging (MRI) are known, and we conducted a study to report the physiologic evaluation. Methods: Coronary angiography, MRI, and fractional flow reserve measurements were performed in 18 patients 8 years after direct surgical angioplasty of the left main coronary artery. Results: At coronary angiography and MRI, a dilated funnel-shaped left main coronary artery was seen in all 18 patients, but both methods failed to demonstrate a flow-limiting lesion in the distal left main coronary artery in 1 patient. The functional severity was shown by fractional flow reserve measurement, and subsequently, this patient underwent repeated bypass grafting surgery. Conclusions: After long-term follow-up, 17 of 18 patients had an excellent result of direct surgical angioplasty of the left main coronary artery. MRI is a safe and noninvasive way to visualize the left main coronary artery after direct surgical angioplasty, but quantitative assessment of a lesion is not reliable. Fractional flow reserve measurements are mandatory to evaluate the hemodynamic properties of the left main coronary artery after direct surgical angioplasty

Determination of glutamate dehydrogenase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry)

Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)(+) to NAD(P)H or vice versa. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we assessed the effects of various glutamate concentrations in combination with either the coenzyme NAD(+) or NADP(+) on GDH activity in mouse liver cryostat sections using metabolic mapping. NAD(+)-dependent GDH V(max) was 2.5-fold higher than NADP(+)-dependent V(max), whereas the K(m) was similar, 1.92 mM versus 1.66 mM, when NAD(+) or NADP(+) was used, respectively. With either coenzyme, V(max) was determined at 10 mM glutamate and substrate inhibition was observed at higher glutamate concentrations with a K(i) of 12.2 and 3.95 for NAD(+) and NADP(+) used as coenzyme, respectively. NAD(+)- and NADP(+)-dependent GDH activities were examined in various mouse tissues. GDH activity was highest in liver and much lower in other tissues. In all tissues, the highest activity was found when NAD(+) was used as a coenzyme. In conclusion, GDH activity in mice is highest in the liver with NAD(+) as a coenzyme and highest GDH activity was determined at a glutamate concentration of 10 m

Long-term outcome after surgical left main coronary angioplasty

Background. Direct surgical angioplasty of the left main coronary artery is aimed to restore a more physiologic flow of the left coronary artery compared with conventional coronary artery bypass graft surgery and allows subsequent percutaneous coronary interventions of more distal coronary lesions if necessary. Relatively little data are known about long-term outcome in these patients. Methods. In 1996 and 1997, in 31 patients in our hospital, surgical angioplasty of the left main coronary artery was attempted. The left main coronary artery was approached in the anterior way. Follow-up was performed during 8 years and concluded by invasive anatomic and functional evaluation of the left main coronary artery. Results. In 4 of these patients, the procedure was converted to conventional coronary artery bypass graft surgery owing to calcification of the left main coronary artery. Of the remaining 27 patients, 3 patients died in the perioperative period and 4 other patients died during follow-up. In 18 of the 20 survivors, coronary angiography was performed after 8 years, and the left main coronary artery was also evaluated by intravascular ultrasound and coronary pressure-based fractional flow reserve measurement. At angiography and intravascular ultrasound, a dilated funnel-shaped left main coronary artery was seen in all of these patients. In 1 patient, a hemodynamically significant left main coronary artery stenosis was present (fractional flow reserve < 0.75), and in this patient coronary artery bypass graft surgery was performed. Conclusions. Although the total mortality of 23% was somewhat disappointing, the majority of the survivors had an excellent anatomic and physiologic result after direct surgical angioplasty of the left main coronary artery. Therefore, this technique deserves a place in the surgical armamentarium

Chronic vagal stimulation for the treatment of low ejection fraction heart failure : results of the NEural Cardiac TherApy foR Heart Failure (NECTAR-HF) randomized controlled trial

AIM: The neural cardiac therapy for heart failure (NECTAR-HF) was a randomized sham-controlled trial designed to evaluate whether a single dose of vagal nerve stimulation (VNS) would attenuate cardiac remodelling, improve cardiac function and increase exercise capacity in symptomatic heart failure patients with severe left ventricular (LV) systolic dysfunction despite guideline recommended medical therapy. METHODS: Patients were randomized in a 2 : 1 ratio to receive therapy (VNS ON) or control (VNS OFF) for a 6-month period. The primary endpoint was the change in LV end systolic diameter (LVESD) at 6 months for control vs. therapy, with secondary endpoints of other echocardiography measurements, exercise capacity, quality-of-life assessments, 24-h Holter, and circulating biomarkers. RESULTS: Of the 96 implanted patients, 87 had paired datasets for the primary endpoint. Change in LVESD from baseline to 6 months was -0.04 ± 0.25 cm in the therapy group compared with -0.08 ± 0.32 cm in the control group (P = 0.60). Additional echocardiographic parameters of LV end diastolic dimension, LV end systolic volume, left ventricular end diastolic volume, LV ejection fraction, peak V02, and N-terminal pro-hormone brain natriuretic peptide failed to show superiority compared to the control group. However, there were statistically significant improvements in quality of life for the Minnesota Living with Heart Failure Questionnaire (P = 0.049), New York Heart Association class (P = 0.032), and the SF-36 Physical Component (P = 0.016) in the therapy group. CONCLUSION: Vagal nerve stimulation as delivered in the NECTAR-HF trial failed to demonstrate a significant effect on primary and secondary endpoint measures of cardiac remodelling and functional capacity in symptomatic heart failure patients, but quality-of-life measures showed significant improvement