Mechanized assay of plasma prekallikrein by activation with Pseudomonas aeruginosa elastase and amidolysis of chromogenic substrate.

An automated assay of plasma prekallikrein is described. Prekallikrein was converted to kallikrein with Pseudomonas aeruginosa elastase, and the hydrolytic activity of kallikrein to H-D-Pro-Phe-Arg-paranitroanilide subsequently measured.

The conversion was complete within 8 minutes and the amidolytic activity remained stable at least another 10 min at 37 ° C. This method worked in plasma deficient in Hageman factor (blood coagulation factor XII). Using anti-prekallikrein antibody and plasma deficient in prekallikrein, the amidolytic activity generated in normal plasma was identified as due to kallikrein. With plasma samples, the coefficients of variation (CV) for multiple measurements within run (n = 10) and between run (n = 10) were as low as 5.0% and 6.6%, respectively, and the minimum measurable concentration of prekallikrein in plasma was 10% of the normal level.     

Polyphenols in lahpet-so and two new catechin metabolites produced by anaerobic microbial fermentation of green tea

The phenolic constituents of lahpet-so, a traditional postfermented tea of Myanmar produced under anaerobic conditions, were examined. The major polyphenols were identified to be pyrogallol and 4′-hydroxyphenyl-3-(2′′,4′′,6′′-trihydroxyphenyl)-propan-2-ol, 3′,4′-dihydroxyphenyl-3-(2′′,4′′,6′′-trihydroxyphenyl)-propan-2-ol, and 3′,4′,5′-trihydroxyphenyl-3-(2′′,4′′,6′′-trihydroxyphenyl)-propan-2-ol. The hydroxydiphenylpropan-2-ols were identical to the initial metabolites produced from green tea catechins by mammalian intestinal bacteria. In addition, an anaerobic mixed-fermentation experiment using lahpet-so and Japanese commercial green tea afforded two new catechin degradation products together with known compound bruguierol B and the above-mentioned catechin metabolites. Based on spectroscopic evidence, the structures of the new compounds were concluded to be 4-(2,5-dihydroxyhexyl)benzene-1,2-diol and (5S,8R)-6,7,8,9-tetrahydro-5-methyl-5·8-epoxy-5H-benzocycloheptene-2,3,4-triol. Interestingly, the production mechanism was deduced to be the inverse of the biosynthesis of the flavan-3-ol A ring.     

Application of a quantitative 1H-NMR method for the determination of amygdalin in Persicae semen, Armeniacae semen, and Mume fructus

A quantitative ¹H-NMR method (qHNMR) was used to measure the amygdalin content of Persicae semen, Armeniacae semen, and Mume fructus, in each of which amygdalin constitutes a major component. The purity of amygdalin was calculated from the ratio of the intensity of the amygdalin H-2 signal at δ 6.50 ppm in pyridine-d ₅ to that of the hexamethyldisilane (HMD) signal at 0 ppm. The HMD concentration was corrected by the International System of Units (SI) traceability with certified reference material (CRM)-grade bisphenol A. qHNMR revealed the amygdalin contents to be 2.72 and 3.13 % in 2 lots of Persicae semen, 3.62 and 5.19 % in 2 lots of Armeniacae semen, and 0.23 % in Mume fructus. Thus, we demonstrated the utility of this method for the quantitative analysis of crude drugs.     

Mechanism of the toxicity induced by natural humic acid on human vascular endothelial cells

Humic acid (HA), a group of high‐molecular weight organic compounds characterized by an ability to bind heavy metals, is normally found in natural water. Although the impairment of vascular endothelial cells in the presence of humic substances has been reported to be involved in some diseases, the mechanisms responsible for this involvement remain unclear. In this study, we examined the cytotoxicity of HA obtained from peatland in Central Kalimantan, Indonesia, to human vascular endothelial cells, as well as the mechanisms behind these effects. It was found that 50 mg/L HA showed cytotoxicity, which we considered to be mediated by apoptosis through the mitochondrial pathway because of an increase in the expression of caspases 6 and 9 in response to HA administration. In addition, this cytotoxicity was enhanced when cells in this experimental system were exposed to oxidative stress, while it was decreased by the addition of vitamin C. Thus, we conclude that the apoptosis induced by HA depends upon oxidative stress. Furthermore, an iron chelator, DFO, showed a tendency to decrease HA‐induced cytotoxicity, suggesting that iron may potentially mediate HA‐induced oxidative stress. In conclusion, long‐term consumption of HA‐rich water obtained from our study area may cause damage to endothelial cells and subsequent chronic health problems. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 916–925, 2014.     

Fluorescence Detection of Malignant Liver Tumors using 5-Aminolevulinic Acid-Mediated Photodynamic Diagnosis: Principles,Technique, and Clinical Experience

Background

Photoactive drugs selectively accumulate in malignant tissue specimens and cause drug-induced fluorescence. Photodynamic diagnosis (PDD) and fluorescence can distinguish normal from malignant tissue.

Objective

Methods

From May 2012 to September 2013, a total of 70 patients underwent hepatic resections using 5-ALA-mediated PDD for liver tumors at our hospital.

Results

5-ALA fluorescence was detected in all hepatocellular carcinoma cases with serosa invasion. In liver metastasis from colorectal cancer cases with serosa invasion, 18 patients (85.7 %) were detected, and three patients (14.2 %) whose tumors showed complete response to neoadjuvant chemotherapy showed no fluorescence. Both superficial and deep malignant liver tumors were detected with 92.5 % sensitivity. Using 5-ALA-mediated PDD, tumors remaining at the cut surface and postoperative bile leakage were less frequent than in our previous hepatic resections using conventional white-light observation. Moreover, all malignant liver tumors were completely removed with a clear microscopic margin using 5-ALA, with a significant difference in resection margin width between 5-ALA-mediated PDD (6.7 ± 6.9 mm) and white-light observation (9.2 ± 7.0 mm; p = 0.0083).

Conclusions

With the detection of malignant liver tumors, residual tumor and bile leakage at the cut surface of the remnant liver were improved by PDD with 5-ALA. This procedure may provide greater sensitivity than the conventional procedure. Furthermore, 5-ALA-mediated PDD can ensure histological clearance regardless of the resection margin and preserve as much liver parenchyma as possible in patients with impaired liver function.     

Short-Term and Long-Term Outcomes After Gastrectomy for Gastric Cancer in Patients With Chronic Kidney Disease

Background

The incidence of chronic kidney disease (CKD) is increasing, which might be an obstacle to various aspects of gastric cancer treatment, such as perisurgical management and postsurgical follow-up. The present study aimed to evaluate the short- and long-term surgical outcomes following gastrectomy in patients with CKD.

Methods

We retrospectively analyzed surgical complications and prognosis of patients with gastric cancer. These patients were divided into three groups according to the glomerular filtration rate (GFR): 49 patients with severe CKD (GFR < 29 ml/min/1.73 m²), 128 with mild CKD (GFR 30–59), and 798 in the controls (GFR ≥ 60).

Results

The incidences of anastomotic leakage and intraabdominal abscess in the mild and severe CKD groups were higher than that in the control group. The incidences of wound infection, cardiovascular and pulmonary events, and in-hospital mortality in the severe CKD group were higher than those in the other two groups. Severe surgical complications were associated with co-morbidities other than CKD, serum albumin level, estimated blood loss, surgery duration in the mild and severe CKD group. The 3-year overall survival rates for the severe CKD, mild CKD, and control groups were 48.6, 80.9, and 85.0 %, respectively, indicating significant differences between the severe CKD group and other two groups.

Conclusions

Patients with severe CKD show an increased risk of morbidity and mortality following gastrectomy, and their prognosis is usually poor. Studies with a large cohort are essential to refine the risk stratification for gastrectomy in this high-risk population.     

Clinical significance of definite muscle layer in TUR specimen for evaluating progression rate in T1G3 bladder cancer: multicenter retrospective study by the Sapporo Medical University Urologic Oncology Consortium (SUOC)

Purpose

To evaluate the clinical impact on progression and recurrence according to presence and absence of a muscle layer, we conducted a retrospective, multicenter study.

Methods

We retrospectively reviewed 247 patients who received transurethral resection (TUR) of bladder tumors and were pathologically diagnosed as having T1G3 bladder cancer from 1990 to 2009. We ruled out 8 patients who received immediate cystectomy and analyzed the remaining 239 T1G3 patients. Patients who had invasion to the prostatic urethra and patients who underwent a second TUR were not included.

Results

TUR specimens from 194 patients were confirmed to have a definite muscle layer and those from 45 did not. The median follow-up period was 53 months, ranging from 3 to 181 months. The progression-free survival rates at 5 years after TUR were 91.1 % for patients who had a muscle layer in their specimen and 77.3 % for those who did not (p = 0.005, log-rank test). Multivariate analysis indicated that the absence of a muscle layer was a risk factor for progression (p = 0.006, Cox proportional hazards analysis).

Conclusions

Patients without a muscle layer in the specimen had high risk for progression. The initial TUR must have a muscle layer in the specimen. Variations of progression rates in previous studies might be due to different proportions of patients who had a muscle layer in TUR specimens.