Determination of acetoacetate in urine by solid-phase spectrophotometry

A method for the determination of acetoacetate has been developed based on solid-phase spectrophotometry (SPS). The acetoacetate reacts with nitroprusside and glycine and the reaction product is sorbed on Dowex 1-X8 resin. The absorbance of the resin phase at 590 and 720 nm is measured directly. The calibration graph is linear up to 3.3 mg l-1 and the RSD is 1.9%. The detection limit is 7.6 micrograms l-1. The method has been applied to the determination of acetoacetate in normal and diabetic subjects' urine without pretreatment of the samples, and the results compared with those of 1H-NMR and homogeneous nitroprusside methods.     

Plasma leptin concentrations in Pima Indians living in drastically different environments

OBJECTIVE: Plasma leptin, an important signal for the regulation of energy stores, is known to be influenced by many hormonal factors, but may also be affected by behavioral and environmental factors. The purpose of this study was to investigate the impact of lifestyle (diet composition, level of physical activity) on plasma leptin concentrations among Pima Indians living in drastically different environments. RESEARCH DESIGN AND METHODS: We studied 224 Mexican Pima Indians (115 women, 109 men) living a traditional lifestyle in a remote, mountainous area of northwest Mexico and 418 U.S. Pima Indians (281 women, 137 men) living a North American lifestyle on the Gila River Indian Reservation in Arizona. We hypothesized that the absolute value of leptin would be lower in Mexican Pima Indians because of their lower percent body fat, but could be further influenced by their lifestyle, independent of body composition. RESULTS: Leptin concentration (enzyme-linked immunosorbent assay [ELISA]) was strongly correlated with percent fat (bioimpedance) in Mexican Pima Indians (r = 0.83, P < 0.0001) and U.S. Pima Indians (r = 0.86, P < 0.0001). Among U.S. Pima Indians, independent of percent fat, subjects with type 2 diabetes had lower leptin than nondiabetic subjects (difference = 6.9 +/- 1.0 ng/ml, P < 0.002). Among nondiabetic subjects, Mexican Pima Indians had lower absolute leptin concentrations than U.S. Pima Indians, but higher after adjustment for percent body fat, waist circumference, age, and sex. In a subset of 70 pairs of subjects matched for sex and percent body fat, leptin concentration was 4.4 +/- 1.0 ng/ml (P < 0.0001) higher in Mexican Pima Indians versus U.S. Pima Indians. CONCLUSIONS: These results suggest that independent of body composition, leptin concentration may be increased by environmental factors, such as a high-carbohydrate diet and a high level of physical activity.     

A highly sensitive and specific chemiluminescent enzyme-linked immunosorbent assay for diagnosis of active Trypanosoma cruzi infection

BACKGROUND: Chagas' disease is transmitted to man either by the bite of insects harboring Trypanosoma cruzi or by the transfusion of blood from infected donors. The conventional serologic testing as presently used in blood banks in South America is unsatisfactory, because of a high number of inconclusive and false-positive results. Other methods such as polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens have been proposed, but inherent difficulties have so far precluded their adoption in the large-scale screening required by blood banks. STUDY DESIGN AND METHODS: A highly sensitive and specific chemiluminescent ELISA using a purified trypomastigote glycoconjugate antigen and a complex epimastigote antigen was devised for the diagnosis of active T. cruzi infection. RESULTS: Chemiluminescent ELISA was 100-percent sensitive in the diagnosis of 100 cases of confirmed Chagas' disease. Inconclusive results and false-positive reactions were eliminated in a panel of 115 sera.The specificity of the chemiluminescent ELISA was 100 percent with a purified trypomastigote glycoconjugate antigen and 99.7 percent with a complex epimastigote antigen when applied to 1000 normal human sera and 288 heterologous sera from patients with other infections, including leishmaniasis, and vaccinated individuals. CONCLUSION: The chemiluminescent ELISAs provide a test that is highly sensitive (purified trypomastigote glycoconjugate and complex epimastigote antigens) and specific (purified trypomastigote glycoconjugate antigen) for Chagas' disease diagnosis. It can be used in blood bank screening and to monitor the treatment of patients undergoing chemotherapy.     

New Regenerative Vascular Grafts for Hemodialysis Access: Evaluation of a Preclinical Animal Model

The purpose of this study was to evaluate a suitable animal model for the in vivo evaluation of patency and vascular tissue regeneration in small intestinal submucosa (SIS) vascular grafts for hemodialysis access. First, a 4-mm U-shaped SIS vascular graft was implanted between the internal carotid artery (CA) and the external jugular vein (JV) in five sheep and six swine. The U-shape grafts remained functional for 53 ± 4 days in sheep and 32 ± 2 days in swine. The sheep model presented exaggerated inflammation, so the swine model was selected for the in vivo study. Based on these initial results, a 4-mm C-shape SIS vascular graft with SIS circumferential reinforcement was developed to mechanically improve the vascular graft and manage complications identified during surgery in both sheep and swine. The C-shape vascular graft was implanted in a swine model (n = 3) between the CA and JV. GORE-TEX® vascular grafts were used as controls in the contralateral side of the neck. C-shape grafts remained patent for 47 ± 4 days, whereas the GORE-TEX® grafts were patent for 30 ± 15 days. The C-shape vascular graft was easier to handle during surgery, and its circumferential reinforcement improved in vivo patency, avoiding kinks in the graft after implantation. Histological results showed neovascularization and some regeneration with the alignment of endothelial cells in the vascular wall of the grafts. The model developed may be helpful in other research involving in vivo studies of vascular grafts for hemodialysis access.     

Comparison of Pulmonary Artery Pressure Measurements in the Supine and 60° Lateral Positions Leanne is continuing this research in her doctoral studies

Pulmonary artery pressure monitoring, with the patient in both the supine and lateral positions, is an essential element in the assessment of critically ill patients. Previous work offers conflicting results regarding the accuracy of measurements obtained with the patient in the lateral position. The purpose of this study was to determine if accurate pulmonary artery pressure measurements can be obtained in the cardiac surgical patient. Thirty-five patients underwent repositioning between the supine and both the left and right 60° lateral position while being mechanically ventilated and then breathing spontaneously. Pulmonary artery pressure measurements were recorded prior to, two minutes following and ten minutes following repositioning. Despite some variation in results the pulmonary capillary wedge pressure measurement was reliable ten minutes after repositioning in both the spontaneously breathing and mechanically ventilated patient. Other pulmonary artery pressure measurements were not so reliable in the lateral position. This study concludes that clinical practitioners can obtain accurate pulmonary capillary wedge pressure measurements in post-operative cardiac surgical patients positioned in either the left or right 60° lateral position. Further research is however required, with larger numbers from all sub-groups of the critical care population. Physiological and pathophysiological characteristics which preclude reliable pulmonary artery pressure measurements need to be identified.     

Trichinella infection and clinical disease

Trichinellosis is caused by ingestion of insufficiently cooked meat contaminated with infective larvae of <it>Trichinella</it> species. The clinical course is highly variable, ranging from no apparent infection to severe and even fatal disease. We report two illustrative cases of trichinellosis. Returning to Denmark a few days after having eaten roasted pork in the Republic of Serbia, a female patient suffered from severe vomiting, epigastric pain, diarrhoea, and later myalgia, generalized oedema, and prostration. A biopsy showed heavy infestation with <it>Trichinella spiralis</it>, 2000 larvae/g of muscle. Life-threatening cardiopulmonary, renal and central nervous system complications developed. The patient recovered after several months. Her husband, who also ate the pork, did not have clinical symptoms, but an increased eosinophil count and a single larva in a muscle biopsy confirmed infection. The epidemiology, clinical manifestations, diagnosis, treatment and prevention of trichinellosis are reviewed.      

Binding of receptor for advanced glycation end products (RAGE) ligands is not sufficient to induce inflammatory signals: lack of activity of endotoxin-free albumin-derived advanced glycation end products

Aims/hypothesis Activation of the receptor for advanced glycation end products (RAGE) reportedly triggers cellular responses implicated in the pathogenesis of diabetes, such as increasing vascular cell adhesion molecule-1 (VCAM-1) expression on vascular endothelial cells and inducing TNF- secretion by mononuclear cells. The objective of this study was to evaluate whether RAGE binding affinity of AGE-BSAs and cellular activation correlate.Methods To produce AGEs with varying glycation, bovine albumin AGEs were prepared with 500 mmol/l of glucose, fructose or ribose at times of incubation from 1 to 12 weeks. In addition, AGE-BSA was generated using either glyoxylic acid or glycolaldehyde. Cellular binding of the AGE-BSAs and the effect on endothelial cell VCAM-1 expression were studied in RAGE-expressing human microvascular endothelial cell line-4 cells. Induction of TNF- secretion was assessed using RAGE-expressing human peripheral blood mononuclear cells (PBMCs).Results Cellular binding of the different AGE preparations correlated well with RAGE affinity. Interestingly, we found that the AGE preparations, which were essentially endotoxin free (0.2 ng/mg protein), were incapable of inducing VCAM-1 or TNF- secretion regardless of RAGE binding affinity, AGE concentration or incubation time. In contrast, the reported RAGE ligand S100b was confirmed to induce VCAM-1 expression on endothelial cells and TNF- secretion by PBMCs after 24 h of treatment.Conclusions/interpretation The results of this study suggest that AGE modification and high RAGE binding affinity are not sufficient to generate pro-inflammatory signalling molecules. Thus, RAGE binding affinity of AGE-BSAs does not seem to correlate with cellular activation. Our findings using AGEs with strong RAGE-binding properties indicate that AGEs may not uniformly play a role in cellular activation.Abbreviations Ctrl-BSA BSA incubated in the absence of modifying agent - FruBSA BSA incubated with fructose - G50 BSA BSA incubated with 50 mmol/l glycolaldehyde - G70 BSA BSA incubated with 70 mmol/l glycolaldehyde - GA BSA BSA incubated with glyoxylic acid - GluBSA BSA incubated with glucose - HMEC-4 human microvascular endothelial cell line-4 - HRP horseradish peroxidase - hsRAGE human-soluble RAGE - IB- inhibitor of nuclear factor-B - LPS lipopolysaccharide/endotoxin - NF-B nuclear factor-B - PBMCs peripheral blood mononuclear cells - RAGE receptor for AGE - RibBSA BSA incubated with ribose - RT room temperature - SRB sulphorhodamine B - TBS-casein Tris-buffered saline plus casein - VCAM-1 vascular cell adhesion molecule-1