Determination of plasma dibasic amino acids following trimethylsilyl–trifluoroacyl derivatization using gas chromatography–mass spectrometry

A rapid analytical method was developed to quantify dibasic amino acids (ornithine, lysine and arginine) after two-step derivatization procedure with good sensitivity and specificity on human plasma. If early diagnosis has not been made, patients with inborn metabolic disorders such as HHH syndrome, Hyperornithinemia and dibasic aminoaciduria rapidly progress to sudden death, physical defect or mental retardation resulting in storage of the toxic material into the brain. Therefore, it is necessary to develop the analytical method for rapid screening and/or correct confirmation diagnosis. The formation of trimethylsilyl derivative of the carboxylic (COO–) functional group was performed by adding MSTFA. Five μL of methyl orange was added to the residue until the color changed into yellow. Consecutively, the trifluoroacyl derivative of the amino (–NH₂) functional group was produced by adding MBTFA. Specific ions was chosen for quantification with following ions; m/z 166 and m/z 212 for ornithine, m/z 180 and m/z 395 for lysine, and m/z 292 and, m/z 519 for arginine. A calibration curve showed a linear relationship for the dibasic amino acids spiked to pooled normal plasma showing R² of 0.9955–0.9979 in the range of 0.1–600 ng investigated. The utility of the method for screening and diagnosis was demonstrated by recovery 80–125 % and reproducibility with RSD (9–17 %) at low, medium and high concentration fortified to pooled plasma. Collectively, the present study suggest that this method could be useful for diagnosis, screening, therapeutic monitoring of metabolic disorders on dietary therapy with excellent sensitivity and rapidity.     

RND-Type Efflux Pumps in Multidrug-Resistant Clinical Isolates of Acinetobacter baumannii: Major Role for AdeABC Overexpression and AdeRS Mutations

Increased expression of chromosomal genes for resistance-nodulation-cell division (RND)-type efflux systems plays a major role in the multidrug resistance (MDR) of Acinetobacter baumannii. However, the relative contributions of the three most prevalent pumps, AdeABC, AdeFGH, and AdeIJK, have not been evaluated in clinical settings. We have screened 14 MDR clinical isolates shown to be distinct on the basis of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) for the presence and overexpression of the three Ade efflux systems and analyzed the sequences of the regulators AdeRS, a two-component system, for AdeABC and AdeL, a LysR-type regulator, for AdeFGH. Gene adeB was detected in 13 of 14 isolates, and adeG and the intrinsic adeJ gene were detected in all strains. Significant overexpression of adeB was observed in 10 strains, whereas only 7 had moderately increased levels of expression of AdeFGH, and none overexpressed AdeIJK. Thirteen strains had reduced susceptibility to tigecycline, but there was no correlation between tigecycline MICs and the levels of AdeABC expression, suggesting the presence of other mechanisms for tigecycline resistance. No mutations were found in the highly conserved LysR regulator of the nine strains expressing AdeFGH. In contrast, functional mutations were found in conserved domains of AdeRS in all the strains that overexpressed AdeABC with two mutational hot spots, one in AdeS near histidine 149 suggesting convergent evolution and the other in the DNA binding domain of AdeR compatible with horizontal gene transfer. This report outlines the high incidence of AdeABC efflux pump overexpression in MDR A. baumannii as a result of a variety of single mutations in the corresponding two-component regulatory system.     

Therapeutic effects of strengthening exercise on gait function of cerebral palsy

Purpose. To assess the effectiveness of strengthening exercises of the lower limbs on improvement of muscle strength and gait function.

Method. Those included were diagnosed as spastic diplegic or hemiplegic type of cerebral palsy (CP) and verified as grade 2 or 3 of the Gross Motor Function Classification System (GMFCS). Participants were divided into an experimental group (n = 9) or a control group (n = 8). The experimental group completed a 5-week strengthening program and the control group took part in conventional physical therapy. Muscle tone and strength of lower limb, Gross Motor Function Measure, lateral step up, squat to stand, and three dimensional gait analysis were tested at pre-training, post-training, and 6 week follow up.

Results. Maximal hip extensor strength, and number of squat to stand were significantly increased at post-training and 6 weeks follow up in the experimental group compared with the control group. GMFM score D and E significantly improved in the experimental group at post-training. The experimental group demonstrated significant increase of gait speed and stride length and decrease of double support phase at post-training and 6 weeks follow up.

Conclusions. Strengthening exercises could be a useful method to improve gait function of patients with spastic CP.     

Protection of canine cardiac mitochondrial function by verapamil-cardioplegia during ischemic arrest

Hemodynamic and mitochondrial function recover following 60 minutes of ischemic arrest and reperfusion in hearts pretreated with verapamil. The present study was carried out to determine whether verapamil prevents the onset of mitochondrial oxidative impairment after 60 minutes of ischemic arrest without reperfusion. Two preparations of mitochondria isolated following Polytron homogenization and subsequent treatment of the myofibrillar pellet with Nagarse were examined for phosphorylating respiration. The Polytron mitochondria were more sensitive to ischemic arrest than were the Nagarse mitochondria with either glutamate-malate (57% vs. 22% inhibition), succinate (+ rotenone) (41% vs. 14% inhibition), or palmitoylcarnitine (57% vs. 27% inhibition) as respiratory substrates. Verapamil pretreatment significantly increased oxidation of all substrates by the subsequently isolated Polytron mitochondria, but only succinate-supported respiration returned to control levels. In contrast, the small amount of respiratory inhibition exhibited by the Nagarse mitochondria after ischemic arrest was insensitive to verapamil pretreatment. We conclude that the Polytron preparation of mitochondria is more susceptible to ischemia than the Nagarse mitochondria, and this susceptibility correlates with a striking sensitivity to verapamil protection. In general, oxidation of NADH-linked substrates, including palmitoylcarnitine, is more affected by ischemic arrest than succinate, and only oxidation of the latter substrate is totally protected by verapamil. The beneficial action of verapamil on mitochondrial function occurs prior to reperfusion. The data suggest that alterations in calcium homeostasis occur during the ischemic period, as well as in the subsequent reperfusion period.     

Dialysis Access Graft Thrombolysis: Randomized Study of Pulse-Spray Versus Continuous Urokinase Infusion

Purpose: To compare pulse-spray to continuous-infusion thrombolysis with high-dose urokinase in thrombosed dialysis access grafts. Methods: A prospective randomized controlled trial was performed. From August 1992 to September 1993, 30 thrombosed polytetrafluoroethylene (PTFE) grafts in 24 patients were included, 15 grafts in each group. The success of thrombolysis, mean time to thrombolysis, mean urokinase dose, and 60-day patency rate were evaluated. Results: In the pulse-spray group, the mean time to thrombolysis was 72 min with a mean urokinase dose of 560,000 U. The 60-day patency rate was 71%. In the continuous-infusion group, the mean infusion time to thrombolysis was 55 min with a mean dose of 479,000 U. The 60-day patency rate was 73%. Conclusion: No statistically significant difference was found between the two techniques in the mean time to thrombolysis, the mean urokinase dose used, or the 60-day patency rate.     

The Clinical Utility of a Adding Lateral Approach to Conventional Vertical Approach for Prone Stereotactic Vacuum-Assisted Breast Biopsy

Objective

The purpose of this study is to evaluate the clinical utility of adding lateral approach to conventional vertical approach for prone stereotactic vacuum-assisted breast biopsies.

Materials and Methods

From April 2010 to May 2012, 130 vacuum-assisted stereotactic biopsies were attempted in 127 patients. While a vertical approach was preferred, a lateral approach was used if the vertical approach failed. The success rate of biopsies utilizing only a vertical approach was compared with that using both vertical and lateral approaches and the breast thickness for both procedures was measured and compared with that for vertical approach. In addition, pathology results were evaluated and the causes of the failed biopsies were analyzed.

Results

Of the 130 cases, 127 biopsies were performed and 3 biopsies failed. The success rate of the vertical approach was 83.8% (109/130); however, when the lateral approach was also used, the success rate increased to 97.7% (127/130) (p = 0.0004). The mean breast thickness was 2.7 ± 1 cm for the lateral approach and 4 ± 1.2 cm for the vertical approach (p < 0.0001). The histopathologic results in 76 (59.8%) of the biopsies were benign, 23 (18.1%) were high-risk lesions, and 28 (22.0%) were malignant. The causes of biopsy failure were thin breasts (n = 2) and undetected difficult lesion location (n = 1).

Conclusion

The addition of lateral approach to conventional vertical approach in prone stereotactic vacuum-assisted breast biopsy improved the success rate of stereotactic biopsy, especially in patients with thin breasts.     

MR Imaging in Patients with Suspected Liver Metastases: Value of Liver-Specific Contrast Agent Gadoxetic Acid

Objective

To compare the diagnostic performance of gadoxetic acid-enhanced magnetic resonance (MR) imaging with that of triple-phase multidetector-row computed tomography (MDCT) in the detection of liver metastasis.

Materials and Methods

Our institutional review board approved this retrospective study and waived informed consent. The study population consisted of 51 patients with hepatic metastases and 62 patients with benign hepatic lesions, who underwent triple-phase MDCT and gadoxetic acid-enhanced MRI within one month. Two radiologists independently and randomly reviewed MDCT and MRI images regarding the presence and probability of liver metastasis. In order to determine additional value of hepatobiliary-phase (HBP), the dynamic-MRI set alone and combined dynamic-and-HBP set were evaluated, respectively. The standard of reference was a combination of pathology diagnosis and follow-up imaging. For each reader, diagnostic accuracy was compared using the jackknife alternative free-response receiver-operating-characteristic (JAFROC).

Results

For both readers, average JAFROC figure-of-merit (FOM) was significantly higher on the MR image sets than on the MDCT images: average FOM was 0.582 on the MDCT, 0.788 on the dynamic-MRI set and 0.847 on the combined HBP set, respectively (p < 0.0001). The differences were more prominent for small (≤ 1 cm) lesions: average FOM values were 0.433 on MDCT, 0.711 on the dynamic-MRI set and 0.828 on the combined HBP set, respectively (p < 0.0001). Sensitivity increased significantly with the addition of HBP in gadoxetic acid-enhanced MR imaging (p < 0.0001).

Conclusion

Gadoxetic acid-enhanced MRI shows a better performance than triple-phase MDCT for the detection of hepatic metastasis, especially for small (≤ 1 cm) lesions.