Measurement of respiratory function in isolated cardiac mitochondria using Seahorse XFe24 Analyzer: applications for aging research

Mitochondria play a critical role in the cardiomyocyte physiology by generating majority of the ATP required for the contraction/relaxation through oxidative phosphorylation (OXPHOS). Aging is a major risk factor for cardiovascular diseases (CVD) and mitochondrial dysfunction has been proposed as potential cause of aging. Recent technological innovations in Seahorse XFe24 Analyzer enhanced the detection sensitivity of oxygen consumption rate and proton flux to advance our ability study mitochondrial function. Studies of the respiratory function tests in the isolated mitochondria have the advantages to detect specific defects in the mitochondrial protein function and evaluate the direct mitochondrial effects of therapeutic/pharmacological agents. Here, we provide the protocols for studying the respiratory function of isolated murine cardiac mitochondria by measuring oxygen consumption rate using Seahorse XFe24 Analyzer. In addition, we provide details about experimental design, measurement of various respiratory parameters along with interpretation and analysis of data.     

Randomized comparison of nasojejunal and nasogastric feeding in critically ill patients

OBJECTIVE: Critically ill patients often develop large gastric residual volumes during nasogastric feeding as a result of poor gastroduodenal motility. Nasojejunal feeding may decrease the severity of this complication. The aim of this study was to determine whether nasojejunal feeding improved tolerance of enteral nutrition by reducing gastric residual volumes. DESIGN: Randomized, prospective, clinical study. SETTING: Intensive care unit of a university-affiliated hospital. PATIENTS: Seventy-three intensive care unit patients expected to require nutritional support for at least 3 days. INTERVENTIONS: Patients were randomized to receive enteral nutrition via a nasojejunal tube (placed endoscopically) (34 patients) or a nasogastric tube (39 patients). A strict protocol was followed, which included regular gastric residual volume measurement (in both groups), the use of predetermined criteria for intolerance, and an attempt at nasojejunal feeding for those nasogastrically fed patients who were intolerant of enteral nutrition. MEASUREMENTS AND MAIN RESULTS: Endoscopic placement of nasojejunal tubes was successful in 98% with no complications of insertion. Patients fed via a nasojejunal tube had 1) a reduced total gastric residual volume in both the first 24 (197 vs. 491 mL, p = .02) and 48 hrs (517 vs. 975 mL, p = .02); 2) a reduced incidence of a single gastric residual volume >150 mL (32% vs. 74%, p = .001); and 3) a trend toward a reduced incidence of intolerance of enteral nutrition (13% vs. 31%, p = .09). Only 13% of those nasogastrically fed patients who were initially intolerant of enteral nutrition remained intolerant once fed via a nasojejunal tube, and only 1.4% of all patients met criteria for commencement of parenteral nutrition. CONCLUSIONS: Enteral nutrition delivered via a nasojejunal tube is associated with a significant reduction in gastric residual volume, a strong trend toward improved tolerance of enteral nutrition, and an extremely low requirement for parenteral nutrition.     

Chylothorax as a complication of coronary artery bypass grafting operation

The authors present the case of a 72-year-old woman who underwent coronary bypass grafting. Left sided chylothorax due to accidental dissection of a thoracic duct branch developed 2 months after sternotomy. As conservative therapy has failed, surgical pleurodesis was performed successfully. Chylothorax is a rare and underestimated complication of coronary bypass grafting. The worldwide increasing number of coronary artery bypass grafting surgeries makes it important to pay attention to this condition. Thus diagnosis of the chyle is relatively easy by its high chylomicron and triglyceride content, but identification of the etiology and its treatment is sometimes challenging for the physician. The treatment of chylothorax is usually conservative. The main goal is to keep the volume of the chyle under control. The number of surgical interventions because of chylothorax is increasing due to an increase of iatrogenic etiology.     

Hormonal evaluation and mutation screening for steroid 21-hydroxylase deficiency in patients with unilateral and bilateral adrenal incidentalomas

OBJECTIVE: The aims of the present study were (a) to examine the occurrence of 21-hydroxylase gene (CYP21) mutations in patients with unilateral and bilateral adrenal incidentalomas and (b) to correlate the results of mutation screening with hormonal parameters of 21-hydroxylase deficiency. DESIGN: The frequency of the eight commonly occurring CYP21 mutations in blood DNA samples of 19 patients with bilateral, as well as in blood and tumoral tissue DNA samples of 31 patients with unilateral adrenal incidentalomas, was determined. In all patients, hormonal evaluation for 21-hydroxylase deficiency was performed using measurements of basal and ACTH-stimulated plasma 17-hydroxyprogesterone (17-OHP) concentrations. METHODS: Blood and tumoral DNA samples were analyzed by allele-specific PCR for the detection of the eight commonly occurring CYP21 mutations (deletion/large gene conversion, intron 2 splicing, Ile172Asn, exon 6 cluster, Val281Leu, Leu307insT, Gln318Stop and Arg356Trp mutations). Plasma 17-OHP concentrations were measured by radioimmunoassay. RESULTS: Of the 19 patients with bilateral adrenal incidentalomas, one patient had homozygous (Val281Leu) and three patients had heterozygous germline CYP21 mutations (Val281Leu in two cases and Arg356Trp in one case). Heterozygous germline CYP21 mutations were also detected in five of the 31 patients with unilateral adrenal incidentalomas (Ile172Asn in three cases and Val281Leu in two cases). Mutation screening of tumoral DNA in unilateral incidentalomas showed the presence of corresponding germline mutations but no additional somatic mutations were found. ACTH-stimulated plasma 17-OHP concentrations were above 1500 ng/dl in all patients with bilateral incidentalomas who had homozygous and heterozygous CYP21 mutations, but heterozygous carriers with unilateral incidentalomas had highly variable ACTH-stimulated plasma 17-OHP levels (between 111 and 1705 ng/dl). CONCLUSIONS: These results suggest a similar frequency of germline CYP21 mutations in patients with bilateral and unilateral adrenal incidentalomas (21.1% and 16.1% respectively). Therefore, it cannot be ruled out that, in at least some patients, CYP21 mutations may play a role in the pathomechanism of bilateral and unilateral adrenal incidentalomas. However, the lack of clear association of CYP21 mutations with increased ACTH-stimulated plasma l7-OHP response, especially in patients with unilateral incidentalomas, suggests that the effect of CYP21 mutations on adrenocortical tumor formation may also involve mechanism(s) independent of ACTH-induced changes in 17-OHP secretion.     

Fat-free mass depletion in cystic fibrosis: Associated with lung disease severity but poorly detected by body mass index

ObjectiveMalnutrition in cystic fibrosis (CF) is associated with poorer survival, but the determinants of fat-free mass (FFM) depletion are not well-characterized. It is unknown whether routine nutritional indicators, including body mass index (BMI), are adequate for detecting FFM depletion. This study aimed to determine the prevalence of FFM depletion in adults with CF, to compare fat-free mass index (FFMI) with BMI, and to identify predictors of FFM depletion.MethodsThis was a prospective cross-sectional study of 86 adults with CF (19–59 y old). Body composition was assessed using dual-energy X-ray absorptiometry to determine FFMI and BMI. FFMI percentiles and Z-scores were derived from a reference population of 156 healthy adults. FFM depletion was defined as an FFMI below the fifth percentile for age and gender and low BMI as <18.5 kg/m². Univariate and multivariate analyses identified predictors of FFMI and FFMI Z-score.ResultsMean FFMIs were 18.3 ± 1.9 kg/m² in men with CF and 15.8 ± 1.1 kg/m² in women with CF (P < 0.0005). FFM depletion was found in 14% of adults with CF, and low BMI was found in 18.6%. The sensitivity of BMI for detecting FFM depletion was 42%. Forced expiratory volume in 1 s as a percentage of predicted was independently associated with FFMI in women (r = 0.62, P < 0.0001) and men (r = 0.28, P = 0.045) and FFMI Z-score (r = 0.41, P < 0.0001).ConclusionFFM depletion was found in 14% of adults with CF, but was undetectable by BMI in 58% of these patients. These findings, together with the association of FFMI with forced expiratory volume in 1 s predicted, suggest a role for body composition assessment in adult CF care.     

Cutoff values of midnight salivary cortisol for the diagnosis of overt hypercortisolism are highly influenced by methods

BackgroundMidnight salivary cortisol (MSC) concentration has been considered as a sensitive marker of overt hypercortisolism. Because no studies on commercially available automated, non-isotopic MSC assays have been reported, we determined and compared the diagnostic performance of an automated electrochemiluminescent immunoassay (ECLIA, Elecsys E170) and an in-house radioimmunoassay (RIA) for MSC measurement.MethodsThe study involved 126 consecutive patients referred for evaluation because of symptoms of Cushing's syndrome, obesity, or the presence of incidentally discovered adrenal adenoma. Using detailed clinical, hormonal and radiological evaluation overt endogenous hypercortisolism was confirmed in 9 patients and was excluded in 117 patients.ResultsROC analysis indicated that the best performance of MSC was obtained at cutoff value of 0.35 µg/dl using ECLIA (sensitivity, 100%; specificity, 88%) and 0.29 µg/dl (sensitivity, 100%; specificity, 71%) using RIA. When the findings were compared to those obtained from low dose dexamethasone test, both ECLIA and RIA of MSC showed a better diagnostic performance.ConclusionMSC measurement is useful for the diagnosis of overt hypercortisolism but the cutoff value is highly dependent on the method used. We recommend the use of automated ECLIA for MSC assay, and we propose further studies on other automated immunoassay analyzers potentially suitable for MSC measurements.     

Die Bedeutung der Phagozyten und des T-Lymphozyten-Systems bei der Abwehr von Candida albicans-Infektionen*(Übersicht)

Zusammenfassung: Die Immunität gegenüber Candida-Infektionen stellt ein komplexes Geschehen dar. Als Ergebnis der unterschiedlichen Schädigungsmöglichkeiten der verschiedenen Abwehrfaktoren kommt es zur Variabilität der klinischen Erscheinungsformen der Candidosen. Zu ausgedehnten und schweren Mykosen kann es kommen bei Mängeln der Barrierefunkrion der Haut, bei verminderterbiologischerAktivitätderSchleinihäute, oder bei spontanenbzw. artefiziellen strukturellen oder funktionellen Schädigungen der Granulozyten und Lymphozyten. Summary: Immunity to Candida infections is based on a number of complex mechanisms. The diversity of clinical features of candidoses is determined by a variety of modes of impairment of the defense factors. Extended and severe mycoses may develop if the barrier function of the skin is damaged, if the mucous membranes lose their biological defense activity and if structural or functional deficiencies of polymorphonuclear cells or lymphocytes occur.