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Of 987 cases of gastric adenocarcinoma seen at Nagoya University School of Medicine, we found 6 rare, extremely well-differentiated advanced gastric adenocarcinomas that could not be diagnosed as malignant tumors with only H&E staining, even with repeated biopsies under preoperative endoscopy. The aim of this study was to determine whether an immunohistochemical method using p53 and Ki-67 antibody would be helpful for preoperative pathologic diagnosis. The cancer control cases were 16 cases of ordinary well-differentiated advanced gastric adenocarcinoma, while the gastritis control cases were 22 cases of Helicobacter pylori–positive chronic gastritis. The p53 labeling index and the localization of Ki-67+ cells showed that the special adenocarcinomas in biopsy specimens were distinct from the surrounding normal mucosa and chronic gastritis, but not from the cancer control cases. These methods are useful markers for preoperative pathologic diagnosis of extremely welldifferentiated gastric adenocarcinoma, which sometimes is confused with regenerative atypical glands before operation. Diagnosis of gastric adenocarcinoma has not been difficult owing to technical advances in radiography, endoscopy

Background: Many different methods have been used to assay amylase activity, using nitrophenylated oligosaccharides as substrate; however, the hydrolysis steps in these methods are complex.Methods: We developed a new continuously monitoring assay for amylase activity in biological fluids, using 2-chloro-4-nitrophenyl-4-O-β-d-galactopyranosylmaltoside (GalG2CNP) as the substrate; this assay was used with anti-human salivary amylase monoclonal antibodies for specific determination of the pancreatic isoenzyme. Amylase converted GalG2CNP into β-d-galactopyranosylmaltose and 2-chloro-4-nitrophenol, which was measured at 405 nm.Results: GalG2CNP was cleaved between 2-chloro-4-nitrophenol and β-d-galactopyranosylmaltose and did not undergo transfer reactions. The within-assay CVs (n = 20) for total amylase (T-AMY) and pancreatic amylase (P-AMY) were 0.6–1.6% and 0.5–2.5%, respectively; and day-to-day CVs (n = 10) for T-AMY and P-AMY were 0.8–3.7% and 0.6–4.1%, respectively. T-AMY and P-AMY activities in serum or urine obtained by the proposed method correlated well with those determined by the 2-chloro-4-nitrophenyl 4-O-β-d-galactopyranosyl-β-maltotetraoside method or the modified IFCC method.Conclusions: This novel assay for T-AMY and P-AMY measures both activities stoichiometrically, directly, and easily, and may be suitable for routine procedures.

Various methods for determining calcium in body fluids have been reported. Atomic absorption spectrophotometry (AAS) (1) is a most reliable method; however, because the AAS instrument is expensive and maintenance is difficult, it is unsuitable for routine assay. Spectrophotometric methods by use of o -cresolphthalein complexone (CPC) (2)(3)(4)(5)(6), which is based on chelation, is now widely available to assay calcium in clinical laboratories. Several enzymatic methods that use porcine pancreatic α-amylase (EC 3.2.1.1) (7) or phospholipase D (PLD; EC 3.1.4.4) (8) to measure calcium have also been developed. These methods are based on the activation of the enzymes by calcium. On the other hand, Kimura et al. (9) reported a kinetic assay for calcium in serum that uses urea amidolyase (EC 6.3.4.6) based on the inhibition of the enzyme by calcium. Previously reported method for determining free calcium ions in serum using PLD required appropriate ionic strength for its reaction (8). We have established a new and stable enzymatic method for determining calcium in serum that uses PLD, stabilizing calcium status by adding a large excess of bovine albumin to the reaction system. The proposed enzymatic method for determining calcium in serum is based on the following sequence of reactions: \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{Diacetylphosphatidylcholine{+}\ H}_{\mathrm{2}}\mathrm{O}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\ \begin{array}{l}\mathrm{Phospholipase\ D}\\{\rightarrow}\\\mathrm{Calcium}\end{array}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{Choline\ {+}\ diacetylphosphatidic\ acid}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{Choline\ {+}\ 2\ O}\_{\mathrm{2}}\mathrm{\ {+}\ H}\_{\mathrm{2}}\mathrm{O\ }\ \begin{array}{l}\mathrm{Choline\ oxidase}\\{\rightarrow}\end{array}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{\ Betaine\ {+}\ 2\ H}\_{\mathrm{2}}\mathrm{O}\_{\mathrm{2}}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{2\ H}\_{\mathrm{2}}\mathrm{O}\_{\mathrm{2}}\mathrm{\ 4-aminoantipyrine\ {+}\ EMSE}\ \begin{array}{l}\mathrm{Peroxidase}\\{\rightarrow}\end{array}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{\ Quinone\ dye\ (purple)}\] \end{document} Diacetylphosphatidylcholine (DAPC) is used for the PLD reaction substrate. The reaction rate at which PLD hydrolyzes DAPC into choline and diacetylphosphatidic acid depends on the amount of calcium in serum. Choline oxidase (COD) produces betaine and H2O2 from choline and O2. Peroxidase (POD) produces quinone from H2O2 with 4-aminoantipyrine (4-AA) and N -ethyl- N -(3-methylphenyl)- N ′-succinyl-ethylenediamine (EMSE). The absorbance at 546 nm for the quinone dye is measured. This proposed method and the conventional methods were performed with the Hitachi Model 7170 automated …

The association of an insertion/deletion (I/D) polymorphism in the angiotensin converting enzyme (ACE) gene or the serum activity of ACE with coronary artery disease (CAD) was investigated in Japanese men and women. The ACE genotype of 947 CAD subjects who underwent coronary angiography and of 893 control subjects was determined by polymerase chain reaction analysis. No association of the DD genotype or the D allele with CAD was observed in men or women. In a low risk group (defined by a body mass index below the median value and the absence of a history of hypertension, diabetes mellitus, and hypercholesterolemia), there was also no association between the ACE gene polymorphism and CAD. No significant difference in serum ACE activity was detected between CAD subjects and controls of all genotypes or of the same genotype, whereas a significant association was apparent between serum ACE activity and ACE genotype for both CAD subjects and controls among both men and women. These results indicate that the ACE I/D polymorphism and genotype associated variation in serum ACE activity are not risk factors for CAD in Japanese men or women.

We describe a new kinetic assay for determining urea in serum or urine with use of urease (EC 3.5.1.5) and leucine dehydrogenase (EC 1.4.1.9). The latter enzyme is suitable for the kinetic assay of NH4+ because itsKmvalue for NH4+ at pH 8.75 is large (∼500 mmol/L). Interference from endogenous NH4+ in serum or urine is obviated by subtraction of the assayed endogenous NH4+value in a sample blank. For serum, within-assay CVs (n = 10) were 0.39–0.58%; day-to-day CVs (n = 10) were 1.56–2.30%. In urine, within-assay CVs (n = 10) were 0.86–1.15%. Analytical recovery of urea (0.893–71.4 mmol/L) added to patients’ sera (urea 6.14 mmol/L) was 99.2–105.2%. The calibration curve for serum was linear through zero for urea concentrations up to 142.9 mmol/L and for urine up to 714.3 mmol/L. No influences of added ammonium ion, bilirubin, hemoglobin, ascorbic acid, or Intralipid were observed. The regression equations for this method (y) and conventional methods (x = Determiner-LUN for serum assays, Serotec UUR-R for urine) were: y = 1.016x − 0.12 mmol/L (r = 0.999, Sy|x = 0.34 mmol/L, n = 100) for sera, and y = 1.070x − 12.6 mmol/L (r = 0.998, Sy|x = 7.41 mmol/L, n = 100) for urine.

To clarify whether apoptosis is involved in endometriosis, we obtained eutopic endometrial tissues along with endometriotic tissues from the uterus (adenomyosis) (n = 12) and from the ovary (n = 12) from patients undergoing gynaecological surgery. Apoptosis-induced DNA fragmentation was detected by the TdT-mediated dUTP-biotin nick-end labelling method, and immunostaining with a monoclonal antibody against the Fas, Le(y) or B-cell leukaemia/lymphoma-2 (bcl-2) was also performed using the same tissue section. Analysis showed that apoptosis was occurring in all the samples of ovarian endometriotic tissue but in only two of the 12 adenomyotic and in five of the 24 eutopic endometrial tissue samples. In none of these cases was apoptosis correlated with phases of the menstrual cycle. The expression of bcl-2 in the eutopic endometrial and adenomyotic tissues was limited to the proliferative phase, and was observed in only one of the 12 cases of ovarian endometriosis. Fas and Ley were expressed randomly across a wide range in both the eutopic and ectopic endometrial tissues. These results suggest that the features of ovarian endometriosis are different from those of adenomyosis and eutopic endometrium in terms of the involvement of apoptosis. In addition, the regulatory mechanism involved in ovarian endometriosis may differ from that in other endometrial cells.