Protein metabolism in leg muscle following an endotoxin injection in healthy volunteers

A B S T R A C T The human endotoxin model has been used to study the early phase of sepsis. The aim of the present study was to assess leg muscle protein kinetics after an endotoxin challenge given to healthy human volunteers. Six healthy male subjects were studied in the post-absorptive state before and during 4 h following an intravenous endotoxin bolus (4 ng/kg of body weight). Primed continuous infusion of [ 2 H 5 ]phenylalanine and [ 2 H 3 ]3-methylhistidine in combination with sampling from the radial artery, femoral vein and muscle tissue were used to assess leg muscle protein kinetics. Both two-and three-compartment models were used to calculate protein kinetics. In addition 26S proteasome activity and protein ubiquitination were assessed. An increase in the net release of phenylalanine from the leg following the endotoxin challenge was observed; however, this phenylalanine originates from the free intracellular pool and not from protein. Net protein balance was unchanged, whereas both protein synthesis and breakdown were decreased. Degradation rates of contractile proteins were not affected by endotoxin, as indicated by an unchanged rate of appearance of 3-methylhistidine from leg muscle. In addition, proteasome activity and protein ubiquitination were unaffected by endotoxaemia. In conclusion, intravenous endotoxin administration to healthy volunteers resulted in an increased release of free phenylalanine from skeletal muscle, whereas protein balance was unaffected. Both protein synthesis and breakdown were decreased to a similar extent

Critical evaluation of colon submucosal microdialysis in awake, mobile rats

<div><p>Sensors able to record large bowel physiology and biochemistry <i>in situ</i> in awake rodents are lacking. Microdialysis is a mini-invasive technique that may be utilized to continuously deliver or recover low-molecular substances from various tissues. In this experiment we evaluated the feasibility of <i>in vivo</i> microdialysis to monitor extracellular fluid chemistry in the descending colon submucosa of conscious, freely moving rodents. Following surgical implantation of a microdialysis probe, male Wistar rats were housed in metabolic cages where they were analgized and clinically followed for four days with free access to standard diet and water. To assess local microcirculation and probe function, glucose, lactate, glucose-to-lactate ratio and urea clearance were determined in the dialysates from the three postoperative days with focus on the final 24-h period. In an attempt to mitigate the expected tissue inflammatory response, one group of animals had the catheters perfused with 5-aminosalicylic acid-enriched medium with final concentration 1 μmol/L. For verification of probe position and the assessment of the surrounding foreign body reaction, standard histological and immunohistochemical methods were employed. Microdialysis of rat gut is associated with considerable technical challenges that may lead to the loss of probe function and high drop-out rate. In this setting, limited data did not allow to draw any firm conclusion regarding local anti-inflammatory effectiveness of 5-aminosalicylic acid perfusion. Although intestinal microdialysis may be suitable for larger anesthetized animals, low reproducibility of the presented method compromises its routine experimental use in awake and freely moving small-sized rodents.</p></div

Summary of experimental challenges.

<p>Summary of experimental challenges.</p

Experimental design.

<p>Sampling pattern reflected our interest in the fourth 24-h period (day 4). Information from preceding time periods was intentionally restricted to two solitary measurements.</p

Routine biochemistry and hematology.

<p>Routine biochemistry and hematology.</p

Microdialysis data.

<p>Panels a-d depict data from three eligible animals (one from group A with perfusate A and two from group B with anti-inflammatory perfusate B). Data are displayed as individual measurements per time interval. RD_urea (%) was expressed as median (min-max) of values obtained on day 4. Symbols denote: *p<0.05 and **p<0.01 <i>vs</i>. rat A. Statistical approach was restricted to the evaluation of inter-individual differences owing to low number of subjects. Possible effect of 5-ASA (perfusate B) could not be determined. Due to ethical reasons the attempts to harvest more data were discontinued.</p

Correlations between microdialysis parameters.

<p>Correlations between microdialysis parameters.</p

Animal physiology.

<p>Animal physiology.</p