František Duška

Frequent intravenous pulses of growth hormone together with glutamine supplementation in prolonged critical illness after multiple trauma: Effects on nitrogen balance, insulin resistance, and substrate oxidation*

Objectives:To estimate the efficacy and metabolic effects of growth hormone substitution as intravenous pulses together with alanyl-glutamine supplementation and tight blood glucose control in prolonged critical illness. Design:Prospective double-blind, randomized trial with open-label control arm. Setting:Intensive care unit of tertiary level hospital. Patients:Thirty multiple trauma patients (median Injury Severity Score 34). Interventions:Patients were randomized, at day 4 after trauma, to receive intravenous alanyl-glutamine supplementation (0.3 g/kg·day−1 from day 4 until day 17) and intravenous growth hormone (administered days 7–17, full dose 50 &mgr;g/kg·day−1 from day 10 onward) (group 1, n = 10) or alanyl-glutamine and placebo (group 2, n = 10). Group 3 (n = 10) received isocaloric isonitrogenous nutrition (proteins 1.5 g/kg·day−1) without alanyl-glutamine. Measurements and Main Results:Cumulative nitrogen balance for the whole study period was −97 ± 38 g of nitrogen for group 1, −193 ± 50 g of nitrogen for group 2, and −198 ± 77 g of nitrogen for group 3 (p < .001). This represents a daily saving of 300 g of lean body mass in group 1. Insulin-mediated glucose disposal, during euglycemic clamp, as a measure of insulin sensitivity, significantly worsened between days 4 and 17 in group 1 but improved in groups 2 and 3. Group 1 required significantly more insulin to control blood glucose, resulting in higher insulinemia (∼70 mIU in group 1 vs. ∼25 mIU in groups 2 and 3). Despite this, growth hormone treatment caused an increase in plasma nonesterified fatty acid (∼0.5–0.6 mM in group 1 in comparison with ∼0.2–0.3 mM in groups 2 and 3) but did not influence lipid oxidation. There were no differences in morbidity, mortality, or 6-month outcome among the groups. Conclusions:Treatment with frequent intravenous pulses of low-dose growth hormone together with alanyl-glutamine supplementation improves nitrogen economy in patients with prolonged critical illness after multiple trauma but worsens insulin sensitivity. Tight blood glucose control is possible but requires higher doses of insulin.

Determination of ammonia, creatinine and inorganic cations in urine using CE with contactless conductivity detection

CE with capacitively coupled contactless conductivity detection (C(4)D) was used to determine waste products of the nitrogen metabolism (ammonia and creatinine) and of biogenic inorganic cations in samples of human urine. The CE separation was performed in two BGEs, consisting of 2 M acetic acid + 1.5 mM crown ether 18-crown-6 (BGE I) and 2 M acetic acid + 2% w/v PEG (BGE II). Only BGE II permitted complete separation of all the analytes in a model sample and in real urine samples. The LOD values for the optimized procedure ranged from 0.8 microM for Ca(2+) and Mg(2+) to 2.9 microM for NH(4)(+) (in terms of mass concentration units, from 7 microg/L for Li(+) to 102 microg/L for creatinine). These values are adequate for determination of NH(4)(+), creatinine, Na(+), K(+), Ca(2+) and Mg(2+) in real urine samples.

Mechanism of paracetamol-induced hypotension in critically ill patients: A prospective observational cross-over study

OBJECTIVE To elucidate the mechanism of hypotension following intravenous administration of paracetamol (acetaminophen) to patients on the Intensive Care Unit. DESIGN Prospective observational cross-over study. SETTING Intensive Care Unit, University Hospital Královské Vinohrady, Prague, Czech Republic. METHODS Ventilated critically ill patients monitored by PiCCO and administered intravenous paracetamol at the same time were eligible for the study. We recorded haemodynamic indices, as well as core and peripheral temperatures, continuously for 3 h after the dose of paracetamol. Ranitidine was then used as a control drug known not to influence haemodynamics. RESULTS We included 6 subjects, and recorded 48 cycles of observations after administration of paracetamol, and 35 cycles after administration of the control drug. Haemodynamic parameters were not different at the baseline and administration of control drug did not result in any change in haemodynamics. After intravenous paracetamol, mean arterial pressure (MAP) dropped by 7% (p<0.001) with a nadir at the 19th minute. In 22 measurement cycles (45%) we noted >15% reduction in MAP with paracetamol. Analysis of these cycles suggests that hypotension with paracetamol can be caused by reduction of both cardiac index and systemic vascular resistance. In febrile cycles paracetamol caused narrowing of the gradient between central and peripheral temperatures suggesting skin vasodilation. These changes were not correlated to a change of systemic vascular resistance at any time point. CONCLUSION Hypotension with intravenous paracetamol in critically ill patients is caused by a reduction of both cardiac output and systemic vascular resistance. We did not demonstrate any relation between haemodynamic changes and antipyretic action of paracetamol. A possibility that cardiac output is reduced with paracetamol might be clinically important.

Intensive blood glucose control in acute and prolonged critical illness: endogenous secretion contributes more to plasma insulin than exogenous insulin infusion.

We investigated the contribution of impaired insulin secretion (observed as dynamics of C-peptide) and insulin resistance (measured by euglycemic clamps) to glucose dysregulation in 20 critically ill patients after severe trauma during feeding and intensive glucose control with intravenous insulin. Between the fourth and seventh day when insulin sensitivity is lowest, insulin secretion is highest and supranormal despite tight control of blood glucose by exogenous insulin. Afterward, plasma C-peptide decreases together with an improvement in insulin sensitivity. Multiple regression analysis revealed that plasma insulin is determined more by endogenous secretion than insulin infusion, even during the acute phase when exogenous insulin requirements are high.

Normalizing Glutamine Concentration Causes Mitochondrial Uncoupling in an In Vitro Model of Human Skeletal Muscle

BACKGROUND Glutamine has been considered essential for rapidly dividing cells, but its effect on mitochondrial function is unknown. MATERIALS AND METHODS Human myoblasts were isolated from skeletal muscle biopsy samples (n = 9) and exposed for 20 days to 6 different glutamine concentrations (0, 100, 200, 300, 500, and 5000 µM). Cells were trypsinized and manually counted every 5 days. Seven days before the end of exposure, half of these cells were allowed to differentiate to myotubes. Afterward, energy metabolism in both myotubes and myoblasts was assessed by extracellular flux analysis (Seahorse Biosciences, Billerica, MA). The protocol for myoblasts was optimized in preliminary experiments. To account for different mitochondrial density or cell count, data were normalized to citrate synthase activity. RESULTS Fastest myoblast proliferation was observed at 300 µM glutamine, with a significant reduction at 0 and 100 µM. Glutamine did not influence basal oxygen consumption, anaerobic glycolysis or respiratory chain capacity. Glutamine significantly (P = .015) influenced the leak through the inner mitochondrial membrane. Efficiency of respiratory chain was highest at 200-300 µM glutamine (~90% of oxygen used for adenosine triphosphate synthesis). Increased glutamine concentration to 500 or 5000 µM caused mitochondrial uncoupling in myoblasts and myotubes, decreasing the efficiency of the respiratory chain to ~70%. CONCLUSION Glutamine concentrations, consistent with moderate clinical hypoglutaminemia (300 µM), bring about an optimal condition of myoblast proliferation and for efficiency of aerobic phosphorylation in an in vitro model of human skeletal muscle. These data support the hypothesis of hypoglutaminemia as an adaptive phenomenon in conditions leading to bioenergetic failure (eg, critical illness).

Assessing the function of mitochondria in cytosolic context in human skeletal muscle: Adopting high-resolution respirometry to homogenate of needle biopsy tissue samples

Using skeletal muscle homogenates for respirometry has many advantages, but the main challenge is avoiding the damage to outer mitochondrial membrane (OMM) and complex I. By optimising the amount of muscle and careful titration of substrates and inhibitors we developed a new protocol and compared it to isolated mitochondria. We found acceptable damage to OMM (~10-15% increment of oxygen flux after addition of cytochrome c) and to complex I (~70% of electron flux). Homogenate retained ~90% of phosphorylation capacity of isolated mitochondria. The use of fresh homogenate was crucial as mitochondrial function declined rapidly after 2-3h of cold storage.

Effects of Propofol on Cellular Bioenergetics in Human Skeletal Muscle Cells

Objectives: Propofol may adversely affect the function of mitochondria and the clinical features of propofol infusion syndrome suggest that this may be linked to propofol-related bioenergetic failure. We aimed to assess the effect of therapeutic propofol concentrations on energy metabolism in human skeletal muscle cells. Design: In vitro study on human skeletal muscle cells. Settings: University research laboratories. Subjects: Patients undergoing hip surgery and healthy volunteers. Interventions: Vastus lateralis biopsies were processed to obtain cultured myotubes, which were exposed to a range of 1–10 &mgr;g/mL propofol for 96 hours. Measurements and Main Results: Extracellular flux analysis was used to measure global mitochondrial functional indices, glycolysis, fatty acid oxidation, and the functional capacities of individual complexes of electron transfer chain. In addition, we used [1-14C]palmitate to measure fatty acid oxidation and spectrophotometry to assess activities of individual electron transfer chain complexes II–IV. Although cell survival and basal oxygen consumption rate were only affected by 10 &mgr;g/mL of propofol, concentrations as low as 1 &mgr;g/mL reduced spare electron transfer chain capacity. Uncoupling effects of propofol were mild, and not dependent on concentration. There was no inhibition of any respiratory complexes with low dose propofol, but we found a profound inhibition of fatty acid oxidation. Addition of extra fatty acids into the media counteracted the propofol effects on electron transfer chain, suggesting inhibition of fatty acid oxidation as the causative mechanism of reduced spare electron transfer chain capacity. Whether these metabolic in vitro changes are observable in other organs and at the whole-body level remains to be investigated. Conclusions: Concentrations of propofol seen in plasma of sedated patients in ICU cause a significant inhibition of fatty acid oxidation in human skeletal muscle cells and reduce spare capacity of electron transfer chain in mitochondria.

Lactate production without hypoxia in skeletal muscle during electrical cycling: Crossover study of femoral venous-arterial differences in healthy volunteers

Background Aim of the study was to compare metabolic response of leg skeletal muscle during functional electrical stimulation-driven unloaded cycling (FES) to that seen during volitional supine cycling. Methods Fourteen healthy volunteers were exposed in random order to supine cycling, either volitional (10-25-50 W, 10 min) or FES assisted (unloaded, 10 min) in a crossover design. Whole body and leg muscle metabolism were assessed by indirect calorimetry with concomitant repeated measurements of femoral venous-arterial differences of blood gases, glucose, lactate and amino acids. Results Unloaded FES cycling, but not volitional exercise, led to a significant increase in across-leg lactate production (from −1.1±2.1 to 5.5±7.4 mmol/min, p<0.001) and mild elevation of arterial lactate (from 1.8±0.7 to 2.5±0.8 mM). This occurred without widening of across-leg VA O2 and CO2 gaps. Femoral SvO2 difference was directly proportional to VA difference of lactate (R2= 0.60, p=0.002). Across-leg glucose uptake did not change with either type of exercise. Systemic oxygen consumption increased with FES cycling to similarly to 25W volitional exercise (138±29% resp. 124±23% of baseline). There was a net uptake of branched-chain amino acids and net release of Alanine from skeletal muscle, which were unaltered by either type of exercise. Conclusions Unloaded FES cycling, but not volitional exercise causes significant lactate production without hypoxia in skeletal muscle. This phenomenon can be significant in vulnerable patients’ groups.

The Effect of Hypoxia and Metformin on Fatty Acid Uptake, Storage, and Oxidation in L6 Differentiated Myotubes

Metabolic impairments associated with obstructive sleep apnea syndrome (OSA) are linked to tissue hypoxia, however, the explanatory molecular and endocrine mechanisms remain unknown. Using gas-permeable cultureware, we studied the chronic effects of mild and severe hypoxia on free fatty acid (FFA) uptake, storage, and oxidation in L6 myotubes under 20, 4, or 1% O2. Additionally, the impact of metformin and the peroxisome proliferator-activated receptor (PPAR) β/δ agonist, called GW501516, were investigated. Exposure to mild and severe hypoxia reduced FFA uptake by 37 and 32%, respectively, while metformin treatment increased FFA uptake by 39% under mild hypoxia. GW501516 reduced FFA uptake under all conditions. Protein expressions of CD36 (cluster of differentiation 36) and SCL27A4 (solute carrier family 27 fatty acid transporter, member 4) were reduced by 17 and 23% under severe hypoxia. Gene expression of UCP2 (uncoupling protein 2) was reduced by severe hypoxia by 81%. Metformin increased CD36 protein levels by 28% under control conditions and SCL27A4 levels by 56% under mild hypoxia. Intracellular lipids were reduced by mild hypoxia by 18%, while in controls only, metformin administration further reduced intracellular lipids (20% O2) by 36%. Finally, palmitate oxidation was reduced by severe hypoxia, while metformin treatment reduced non-mitochondrial O2 consumption, palmitate oxidation, and proton leak at all O2 levels. Hypoxia directly reduced FFA uptake and intracellular lipids uptake in myotubes, at least partially, due to the reduction in CD36 transporters. Metformin, but not GW501516, can increase FFA uptake and SCL27A4 expression under mild hypoxia. Described effects might contribute to elevated plasma FFA levels and metabolic derangements in OSA.

Postoperative inflammation and insulin resistance in relation to body composition, adiposity and carbohydrate treatment: a randomised controlled study

Summary Background & aims The aims of this study were to identify whether differences in distribution of adipose tissue and skeletal muscle in obese and non-obese individuals contribute to the magnitude of the postoperative inflammatory response and insulin resistance, with and without preoperative treatment with carbohydrate drinks. Methods Thirty-two adults (16 obese/16 non-obese) undergoing elective major open abdominal surgery participated in this 2 × 2 factorial, randomised, double-blind, placebo-controlled study. Participants received Nutricia preOp® or placebo (800 ml on the night before surgery/400 ml 2–3 h preoperatively) after stratifying for obesity. Insulin sensitivity was measured using the hyperinsulinaemic-euglycaemic clamp preoperatively and on the 1st postoperative day. Vastus lateralis, omental and subcutaneous fat biopsies were taken pre- and postoperatively and analysed after RNA extraction. The primary endpoint was within subject differences in insulin sensitivity. Results Major abdominal surgery was associated with a 42% reduction in insulin sensitivity from mean(SD) M value of 37.3(11.8) μmol kg−1 fat free mass (FFM) to 21.7(7.4) μmol kg−1 FFM, but this was not influenced by obesity or preoperative carbohydrate treatment. Activation of the triggering receptor expressed on myeloid cells (TREM1) pathway was seen in response to surgery in omental fat samples. In postoperative muscle samples, gene expression differences indicated activation of the peroxisome proliferator-activated receptor (PPAR-α)/retinoid X-receptor (RXR-α) pathway in obese but not in non-obese participants. There were no significant changes in gene expression pathways associated with carbohydrate treatment. Conclusion The reduction in insulin sensitivity associated with major abdominal surgery was confirmed but there were no differences associated with preoperative carbohydrates or obesity.