Improvement of sample throughput using fast gas chromatography mass-spectrometry for biochemical diagnosis of organic acid disorders

BACKGROUND: Gas chromatograph mass-spectrometric (GC/MS) method of analysis for urinary organic acids is used for the diagnosis of a variety of metabolic disorders. The method is time-consuming and does not allow for improvements in sample throughput. Although the sample preparation and the data processing have been improved, the long GC/MS analysis time still remains to be problematic. METHODS: The fast-GC/MS method, which utilizes a short microbore capillary GC column and fast temperature programming, was applied to the analysis for urinary organic acids. Urine samples obtained from 15 patients with 9 different disorders and 16 healthy controls were analyzed using conventional GC/MS and fast-GC/MS. RESULTS: Analysis cycle time was shortened from 1 h to 15 min. The automated data system uses retention indices determined by conventional-GC/MS for the identification of 134 organic acids. These retention indices can also be used in data obtained by fast-GC/MS. New fast-GC/MS method with the automated data system gave the same diagnostic results as conventional-GC/MS except for 1 healthy control. CONCLUSIONS: The combined system of fast-GC/MS and the automated data system will be powerful tools in clinical laboratories due to increased sample throughput and reduced analysis costs.     

Relationship between spontaneous echo contrast in the thoracic aorta and plasma von Willebrand factor

Purpose To clarify the relationships between spontaneous echo contrast (SEC) detected by transesophageal echocardiography (TEE) and coagulopathy, ultrasonographic findings that may correlate to biochemical coagulation markers were examined. Methods TEE was performed on 49 consecutive patients (mean age 64 ± 14 years; 28 men, 21 women). Blood samples were taken at the same time as TEE was carried out. Aortic SEC (Ao-SEC) and left atrial SEC (LA-SEC) were classified into three grades: absent, mild and marked. Levels of von Willebrand factor (vWF), thrombin antithrombin III complex (TAT), prothrombin fragments 1+2 (F1+2) and fibrinopeptide A (FPA) were measured. Results Mean plasma vWF levels by Ao-SEC grade were 144 ± 39% for absent, 177 ± 55% for mild and 210 ± 73% for marked, with significantly higher levels in the Ao-SEC marked group than in the Ao-SEC absent group (P < 0.05). Mean plasma vWF levels by LA-SEC were 185 ± 73% for absent, 180 ± 49% for mild and 201 ± 62% for marked, with no significant differences apparent between groups. Moreover, no relationships were identified between Ao-SEC grade and plasma levels of coagulation indicators TAT, F1+2 and FPA. Conclusion Plasma vWF levels correlated to grade of aortic SEC. Characteristics of the coagulation system differ between Ao-SEC and LA-SEC. Ao-SEC offers a clinical indicator of platelet thrombus formation.     

Responsiveness to DDAVP in Cushing’s disease is associated with USP8 mutations through enhancing AVPR1B promoter activity

Purpose

To clarify the characteristics of Cushing’s disease (CD) patients who respond to the desmopressin (DDAVP) test and its underlying mechanisms.

Methods

Forty-seven patients with CD who underwent DDAVP testing were included. Patients were divided into two groups: DDAVP test (+) (adrenocorticotropic hormone [ACTH] levels increased by?≥?1.5-fold during the DDAVP test) and DDAVP test (?) (ACTH levels increased by?<?1.5-fold). AVP receptor expression levels in these tumors were quantified using quantitative RT-PCR and immunohistochemistry. AVP receptor promoter activity was analyzed using a dual-luciferase reporter assay system.

Results

Females (96.9%) and USP8 mutants (85.7%) were more prevalent in the DDAVP test (+) than in the DDAVP test (?). Indeed, the ACTH and cortisol responsiveness to DDAVP was greater in USP8 mutation positive tumors than that in USP8 wild type tumors (3.0-fold vs. 1.3-fold, 1.6-fold vs. 1.1-fold, respectively). Responsiveness to DDAVP was correlated with the expression levels of AVPR1B, but not with those of AVPR2. Comparably, Avpr1b promoter activity was enhanced by the overexpression of mutant USP8 compared to the wild type.

Conclusions

We found that the responsiveness of ACTH to DDAVP in CD was greater in tumors with USP8 mutations. The present data suggest that USP8 mutations upregulate the AVPR1B promoter activity. Additionally, we showed that the DDAVP test can predict the presence of USP8 mutations.

     

Effects of candesartan and cilazapril on rats with myocardial infarction assessed by echocardiography

The purpose of this study was to compare the angiotensin II type 1 receptor antagonist candesartan cilexitil (candesartan) and the angiotensin-converting enzyme inhibitor cilazapril on cardiac function, assessed by Doppler echocardiography and cardiac gene expression associated with cardiac remodeling, in rats with myocardial infarction. Candesartan or cilazapril was administered after myocardial infarction. At 1 and 4 weeks after myocardial infarction, cardiac function and mRNA expression in noninfarcted myocardium were analyzed. Candesartan and cilazapril equally prevented increases in hypertrophy in noninfarcted myocardium, left ventricular dilatation, and ejection fraction at 4 weeks. The E-wave/A-wave velocity ratio and the rate of E-wave deceleration, measures of diastolic function, increased to 9.2+/-0.6 and 26.3+/-2. 6 m/s2 at 1 week after myocardial infarction. Candesartan and cilazapril, administered at a dose of 1 mg/kg per day, prevented increases in E-wave/A-wave velocity ratio and E-wave deceleration at 1 and 4 weeks. Candesartan and cilazapril significantly suppressed increased mRNA expression of beta-myosin heavy chain, alpha-skeletal actin, and atrial natriuretic peptide in noninfarcted ventricle at 1 and 4 weeks and expression of collagen I and III at 4 weeks to a similar extent. When given at a dose of 10 mg/kg per day, both candesartan and cilazapril prevented cardiac dysfunction and gene expression to the same extent as when given at 1 mg/kg per day. In conclusion, Doppler echocardiography showed that candesartan and cilazapril equally improved systolic and diastolic function and that ventricular remodeling accompanied modulation of cardiac gene expression.     

Atherosclerotic plaque with ultrasonic attenuation affects coronary reflow and infarct size in patients with acute coronary syndrome: an intravascular ultrasound study.

BACKGROUND: No reflow following percutaneous coronary intervention (PCI) is a major concern in patients with acute coronary syndrome (ACS) and it may be influenced by the preexisting plaque type. METHODS AND RESULTS: To evaluate the impact of plaque characteristics on coronary reflow following PCI in patients with ACS, a total of 110 patients (89 acute myocardial infarction, 21 unstable angina) were assessed by intravascular ultrasound. Plaque type was categorized as either atherosclerotic plaque without ultrasonic attenuation (group 1) or atherosclerotic plaque with attenuation (group 2). External elastic membrane, plaque plus media, and lumen area were measured. Coronary flow was assessed by Thrombolysis in Myocardial Infarction (TIMI) grade and TIMI frame count. Although the final TIMI frame count was similar between the 2 groups, TIMI frame count immediately after the first balloon inflation was significantly higher in group 2 (p=0.03). Despite the similar final TIMI grade and TIMI frame count, peak creatine kinase level was significantly higher (3,035+/-2,553 vs 1,950+/-1,958 IU/L, p=0.04) and fatal arrhythmia more frequently observed (16.4% vs 2.7%, p=0.04) in group 2 than in group 1. CONCLUSIONS: Atherosclerotic plaque with ultrasonic attenuation may be related to a transient deterioration in coronary flow and as a result larger infarct size and higher incidence of fatal arrhythmia following PCI in patients with ACS. These results may help in selecting lesions suitable for distal protection devices.     

Risk stratification in patients with Brugada syndrome: analysis of daily fluctuations in 12-lead electrocardiogram (ECG) and signal-averaged electrocardiogram (SAECG)

Introduction: Risk stratification between symptomatic and asymptomatic patients with Brugada syndrome is not yet established. We compared daily fluctuations in 12-lead electrocardiogram (ECG) and signal-averaged ECG (SAECG) characteristics between symptomatic and asymptomatic patients with Brugada syndrome to identify new markers for distinguishing between high- and low-risk patients.
Methods and Results: Thirty-five patients with Brugada syndrome underwent ECG and SAECG simultaneously at least 4 times every 3 months. We evaluated daily fluctuations (differences between maximum and minimum values) in ECG and SAECG characteristics and compared them between symptomatic  (N = 11)  , and asymptomatic  (N = 24)  patients. On ECG, the daily fluctuations in r-J interval (interval from QRS onset to J point) in leads V1, V2, and V6 were significantly larger in symptomatic than in asymptomatic patients  (V1; 20 ± 6 vs 10 ± 8 msec, P < 0.01, V2; 22 ± 8 vs 11 ± 4 msec, P < 0.01, and V6; 24 ± 7 vs 14 ± 7 msec, P < 0.01)  . On SAECG, daily fluctuations in filtered QRS (f-QRS) duration and LAS40 were significantly larger in symptomatic than in asymptomatic patients (f-QRS;  15 ± 7 vs 9 ± 4 msec, P < 0.05  , and LAS40;  21 ± 7 vs 10 ± 6 msec, P < 0.05  ).
Conclusions: Instability of depolarization appears to be related to the risk of fatal ventricular arrhythmias in patients with Brugada syndrome. Daily fluctuations in ECG and SAECG characteristics could be useful for distinguishing between high- and low-risk patients with Brugada syndrome.     

Adipose tissue-derived stromal cells as a novel option for regenerative cell therapy

Adult stem cells hold great promise for use in tissue repair and regeneration, and the delivery of autologous progenitor cells into ischemic tissue is emerging as a novel therapeutic option. We and others have recently demonstrated the potential impact of adipose tissue-derived stromal cells (ADSC) on regenerative cell therapy for ischemic diseases. The main benefit of ADSC is that they can be easily harvested from patients by a simple, minimally invasive method and also easily cultured. Cultured ADSC can be induced to differentiate into not only adipocytes, but also bone, neurons or endothelial cells in certain conditions. Interestingly, they secrete a number of angiogenesis-related cytokines, such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), which might be suitable for regenerative cell therapy for ischemic diseases. In the ischemic mouse hindlimb, the angiogenic score was improved in the ADSC-treated group. Moreover, recent reports demonstrated that these ADSC can also be induced to differentiate into cardiac myocytes. These adipose tissue-derived cells have potential in angiogenic cell therapy for ischemic disease, and might be applied for regenerative cell therapy instead of bone marrow cells in the near future.