Kiyoshi Okuda

Changes in amounts of cytochrome P450 isozymes and levels of catalytic activities in hepatic and renal microsomes of rats with streptozocin-induced diabetes

Hepatic microsomal cytochrome P450s, which are involved in the metabolism of drugs, hormones, prostaglandins and fatty acids, change when animals develop diabetes. We studied changes in cytochrome P450 isozymes in both hepatic and renal microsomes of rats with diabetes caused by streptozocin, and compared the results with changes in catalytic activities in the microsomes. In hepatic microsomes of diabetic rats, the amount of cytochrome P450 2E1, an acetone-inducible isozyme, was two and a half times that of control rats, and that of P450 4A2, a major renal isozyme, was three times that in the controls. The amounts of cytochrome P450s 2A1, 2C6, 2C7, 3A2 and 4A3 increased in hepatic microsomes of diabetic rats, and P450 2C11 decreased. Treatment with insulin restored these to the levels in the controls. The catalytic activities of aniline hydroxylation, 7-ethoxycoumarin O-dealkylation, testosterone 2 beta, 6 beta, 7 alpha, and 16 beta-hydroxylation, and omega-, (omega-1)-hydroxylation of lauric acid were high in the hepatic microsomes of diabetic rats, and testosterone 2 alpha and 16 alpha-hydroxylation activities were low. In renal microsomes of diabetic rats, cytochrome P450s 2E1, 4A2 and K-4 were induced, and omega- and (omega-1)-hydroxylation activities were high. These changes were reversed by insulin treatment. The induction and suppression of cytochrome P450 isozymes in diabetic rats were consistent with the changes in the catalytic activities. In both hepatic and renal microsomes, P450s 2E1 and 4A2 were induced, altered metabolism of ketones and fatty acids in diabetes may contribute to these changes.

A new direction in automated laboratory testing in Japan: five years of experience with total laboratory automation system management.

The introduction of integrated laboratory systems has proceeded rapidly in Japan in these 15 years, but they require large initial investment for installation and do not always succeed in reducing laboratory cost. We also experienced three major events that taught us that total laboratory systems are not always effective: these were an earthquake, a nerve gas attack, and an outbreak of food poisoning. Political changes in the national health care system in Japan have forced the cutting of expenses for laboratory testing. In this context, cost-effective laboratory testing has been considered, and many hospitals have replaced total laboratory systems with small laboratory systems. Our University Hospital introduced a mini-lab system consisting of compact instruments to increase laboratory efficiency, and we have begun point-of-care testing education for medical students. This combination enables rapid and convenient testing, and is responsive to the political changes in the Japanese health care system.

Comparison of n-acetyl-β-d-glucosaminidase and alanine aminopeptidase activities for evaluation of microangiopathy in diabetes mellitus

The activities of urinary N-acetyl-beta-D-glucosaminidase (NAG) and alanine aminopeptidase (AAP) were measured in 207 diabetic patients and 57 healthy controls, and the relationship of these enzymes to different stages of diabetic microangiopathy was studied. Diabetics with clinical proteinuria had higher urinary NAG and AAP (17.7 +/- 1.9 and 42.8 +/- 4.9 U/g creatinine, mean +/- SE, respectively) than healthy controls (1.8 +/- 0.1 and 10.0 +/- 0.4) or diabetics without proteinuria. Among diabetics without proteinuria, NAG excretion in those with retinopathy was slightly higher than in those without (6.4 +/- 0.5 v 5.4 +/- 0.4), and AAP in those with retinopathy was significantly higher than in those without (23.0 +/- 1.5 v 17.4 +/- 0.8, P less than 0.01). Urinary albumin measured by radioimmunoassay and lysozyme in diabetics with retinopathy but without proteinuria was higher than those without retinopathy (P less than 0.001 and P less than 0.01). The increase in albumin was the greatest in diabetics with long duration of the disease (greater than or equal to 8 years); however, NAG and AAP increased more significantly in those with high hemoglobin A1c than in patients with long duration.(ABSTRACT TRUNCATED AT 250 WORDS)

A new method for screening for hyperammonemia.

A new method for the detection of hyperammonemia, using a kit based on the principle of microdiffusion of ammonia, is described. The method requires only one drop of blood and takes only 15 min to complete. Experiments for recovery and reproducibility were satisfactory, and good correlation was obtained when compared with an enzymatic method for blood ammonia determination. The new method is considered to be useful for routine, low-cost mass-screening of newborn infants for hyperammonemia. It will also be useful for monitoring blood ammonia levels at the bedside in cases with hepatic disease or receiving parenteral nutrition.

Automated measurement of trypsin inhibitor in urine with a centrifugal analyzer: comparison with other acute phase reactants

Automated measurement of trypsin inhibitor in urine was performed with good precision using the COBAS FARA. Elevated levels of both trypsin inhibitor in urine and acute phase proteins in serum were shown in most cases of major abdominal surgery. We suggest that the automated assay of urinary trypsin inhibitor might be useful for the clinical diagnosis of acute phase response.

The effect of non-insulin-dependent diabetes on serum concentrations of tumor-associated carbohydrate antigens of CA19-9, CA-50, and sialyl SSEA-1 in association with the Lewis blood phenotype

Serum concentrations of the tumor-associated carbohydrate antigens CA19-9, CA-50, and sialyl SSEA-1 were measured in non-insulin-dependent diabetic patients without diseases causing the elevation of those antigens, and the relationship to diabetic conditions was studied. The patients of the Lewis blood group phenotype of Lea (23%) had higher serum CA19-9, CA-50, and sialyl SSEA-1 than those of Leb (67%) and Le(-) (10%). Lea patients with high HbA1c (greater than 10%) had significantly higher serum CA19-9 and CA-50 than those with low HbA1c (less than or equal to 7%). Leb patients with high HbA1c also had elevated CA19-9 and sialyl SSEA-1. In Leb patients, diabetic nephropathy was associated with increased CA19-9 levels. Diabetic retinopathy was also accompanied by high carbohydrate antigens in Leb patients, but the difference was not significant. Leb patients treated with sulfonylurea or insulin had increased CA19-9 and CA-50. The changes in serum concentrations of these carbohydrate antigens might have some relationship not only to the Lewis blood phenotype, but also to diabetes.

Purification of calcium-sensitive regulatory protein of platelets which inhibits the gelation of actin

Abstract Ca2+-sensitive regulatory protein of human platelets which inhibits the gelation of actin was purified by DEAE-Sepharose and an affinity column using actin as a ligand. The protein was a single polypeptide chain with an average molecular weight of 90,000 and it bound to actin and inhibited its gelation at concentration from 10−6–10−7M of free calcium. Since the protein existed in the form of a complex with actin even though at concentration lower than 10−7M of free calcium, binding and dissociation of actin and the protein appeared to be dependent on the concentration of free calcium, and complete dissociation was not seen.

Introduction of phalloidin labelled with fluorescein isothiocyanate into living polymorphonuclear leukocytes by electroporation.

To examine the mechanism by which polymorphonuclear leukocytes (PMNs) move, phalloidin labelled with fluorescein isothiocyanate was introduced into freshly sampled cells by use of an electric-cell fusion system. The best conditions for treatment were three pulses of direct current at 100 V for a pulse duration of 3 microseconds. The treated cells retained their usual motility when observed under a microscope, so the method was suitable for the analysis of motile living cells. We used the method to study PMNs during locomotion, spreading and phagocytosis. In locomotion, fluorescence first appeared at the head of the cell and shifted gradually along the cell margin from head to tail. In spreading, diffuse fluorescence around the marginal part of the cytoplasm was strongest near both the attachment sites and the perinuclear area of the cell and spots of fluorescence appeared in the cytoplasm. In phagocytosis, fluorescence developed from the attachment sites, spread to the entire phagocytizing area of the cytoplasm and disappeared when phagocytosis ended. Cells treated with cytochalasin B were randomly spotted with fluorescence. Freshly sampled cells had diffuse and scattered fluorescence, without the lines observed in fixed cells.

Neutrophil chemiluminescence induced by platelet activating factor and suppressed by C-reactive protein

We studied the effects of two substances related to acute inflammation, platelet activating factor (PAF) and C-reactive protein (CRP), on the chemiluminescence reaction of human neutrophils. PAF caused chemiluminescence dosedependently when calcium ion was present. This luminescence was inhibited competitively by CV-3988, a structural analogue of PAF. However, when CRP, an indicator of inflammation, was added, the luminescence reaction was inhibited. The findings suggested that CRP neutralizes the action of PAF in acute inflammation.

Quantitative analysis of Lewis antigens on erythrocytes by flow cytometry

We have developed a method for the quantitative analysis of Lewis antigens on human red blood cells (RBC) using immunofluorescence labeling and flow cytometry. Initially, Lewis a and Lewis b (Le(a) and Le(b)) antigens were labeled with monoclonal anti-Le(a) or anti-Le(b) antibodies followed by labeling with the fluorescein isothiocyanate (FITC)-conjugated second antibody. This method was not sensitive enough to identify the Lewis antigens on RBC, although the FITC method is very commonly used for antigens on white blood cells. Next, we selected the enhanced labeling technique using the avidin-biotin procedure. Biotinylated anti-mouse IgM was used for the second label and the reaction with R-phycoerythrin (RPE)-conjugated streptavidin followed to produce the fluorescence. The method was found to be effective for our objectives. From the results analyzed by the enhanced labeling technique, differences were not found in either the levels of the antigen-positive percentage and the peak mean channel of Le(a) antigens on RBC in the groups of blood type O and A (in ABO system). On the other hand, both the levels of Le(b) antigens on RBC were higher in the groups of blood type O than in those of blood type A. We found both Le(a) and Le(b) antigens on RBC from a few blood type O subjects. We conclude that enhanced labeling and flow cytometry constitute a useful technique for the determination of Lewis antigens on RBC and that this method enables the precise quantification of such antigens.