We report a robust, reliable, and comprehensive analytical method for the identification and quantification of the entire class of coenzyme A (CoA) activated substances, particularly short-, medium-, and long-chain acyl-CoAs derived from various biological tissues. This online SPE-LC/MS/MS-based method is characterized by a simple three-step sample preparation: (1) addition of buffer, organic solvents, and internal standards; (2) homogenization; and (3) centrifugation. The supernatant is injected directly into the SPE-LC/MS/MS system. Identification of CoA activated compounds is performed by accurate mass determination within the HPLC run. Method validation for short-, medium-, and long-chain acyl-CoA fatty acids revealed excellent quality. Accuracy was found to be between 87 and 107% and precision was between 0.1 and 12.8% in mouse skeletal muscle. The lower limit of quantification for all investigated compounds was well below 3.1% of estimated physiological levels in 200 mg of mouse tissue. Comparable results were obtained for mouse liver, mouse brown white adipose tissue and rat liver. For all investigated tissues, no matrix effect was observed.
Inflammatory cytokines released from adipose tissue play an important role in different pathological processes. In the present study, we investigated the inflammatory cytokine response of human subcutaneous adipose tissue (SAT) by applying the open-flow microperfusion technique. Four standard 18-gauge microperfusion catheters were inserted into periumbilical SAT of eight healthy male volunteers [29 Ϯ 3 yr, BMI 24.3 Ϯ 1.9 (mean Ϯ SD)]. SAT probe effluents were collected at 60-min intervals for 8 h after catheter insertion. Different perfusion fluids were used to measure the local effect of insulin and/or glucose on the cytokine response. SAT probe effluents were analyzed for IL-1, IL-6, CXCL8 (IL-8), and TNF-␣. SAT concentrations of IL-1 increased 100-fold from 1.0 Ϯ 0.2 pg/ml (mean Ϯ SE) to 101.5 Ϯ 23.2 pg/ml (P Ͻ 0.001) after 8 h. A 130-fold increase was observed for CXCL8, from 49 Ϯ 29 to 6,554 Ϯ 1,713 pg/ml (P Ͻ 0.001). Furthermore, a 20-fold increase of IL-6 was observed within the first 5 h (from 159 Ϯ 123 to 3,554 Ϯ 394 pg/ml; P Ͻ 0.001), and a significant decline to 2,154 Ϯ 216 pg/ml (P Ͻ 0.01) was seen thereafter. Finally, TNF-␣ increased from 1.4 Ϯ 0.6 to 2.5 Ϯ 0.5 pg/ml (P Ͻ 0.05) in hour 2 and remained stable thereafter. Local administration of insulin exerted a stimulatory effect on the inflammatory response of IL-6. In conclusion, SAT exerts a highly reproducible and consistent proinflammatory cytokine response after minimally invasive trauma caused by the insertion of a catheter in humans.
OBJECTIVE-Physiologically elevated insulin concentrations promote access of macromolecules to skeletal muscle in dogs. We investigated whether insulin has a stimulating effect on the access of macromolecules to insulin-sensitive tissues in humans as well.RESEARCH DESIGN AND METHODS-In a randomized, controlled trial, euglycemic-hyperinsulinemic clamp (1.2 mU ⅐ kg Ϫ1 ⅐ min Ϫ1 insulin) and saline control experiments were performed in 10 healthy volunteers (aged 27.5 Ϯ 4 years, BMI 22.6 Ϯ 1.6 kg/m 2 ). Distribution and clearance parameters of inulin were determined in a whole-body approach, combining primed intravenous infusion of inulin with compartment modeling. Inulin kinetics were measured in serum using open-flow microperfusion in interstitial fluid of femoral skeletal muscle and subcutaneous adipose tissue.RESULTS-Inulin kinetics in serum were best described using a three-compartment model incorporating a serum and a fast and a slow equilibrating compartment. Inulin kinetics in interstitial fluid of peripheral insulin-sensitive tissues were best represented by the slow equilibrating compartment. Serum and interstitial fluid inulin kinetics were comparable between the insulin and saline groups. Qualitative analysis of inulin kinetics was confirmed by model-derived distribution and clearance parameters of inulin. Physiological hyperinsulinemia (473 Ϯ 6 vs. 18 Ϯ 2 pmol/l for the insulin and saline group, respectively; P Ͻ 0.001) indicated no effect on distribution volume (98.2 Ϯ 6.2 vs. 102.5 Ϯ 5.7 ml/kg; NS) or exchange parameter (217.6 Ϯ 34.2 vs. 243.1 Ϯ 28.6 ml/min; NS) of inulin to peripheral insulin-sensitive tissues. All other parameters identified by the model were also comparable between the groups. CONCLUSIONS-Our data suggest that in contrast to studies performed in dogs, insulin at physiological concentrations does not augment recruitment of insulin-sensitive tissues in healthy humans. Diabetes
A highly sensitive alternative to established methods for measuring oxygen transmission rates of ultra-barrier membranes is presented. The key idea is the employment of an optochemical sensor (a luminescent dye immobilized in a matrix) which has vital advantages over electrochemical sensors, such as consumption-less detection and extraordinary sensitivity. The luminescent response to modulated light, which is altered in the presence of oxygen, is recorded by an opto-electronic instrument in a non-invasive manner. Provided with said sensing system, a reusable stand-alone permeation cell was developed. Unlike in conventional devices, the permeating oxygen is accumulated while being simultaneously monitored. The demonstrator unit achieves a limit of detection in the 10 -5 cm 3 m -2 day -1 bar -1 regime. It is therefore among the most sensitive O 2 permeability testers, outperforming commercial instruments by two orders of magnitude, yet offering reusability and similar convenience in operation.
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