Nozomu Takeuchi

Stabilities of N-acetyl-β-d-glucosaminidase (NAG) isoenzymes in urine: advantage of NAG isoenzyme B measurement in clinical applications

N-Acetyl-beta-D-glucosaminidase (NAG) is a widely used urinary enzyme for the assessment of renal diseases. We studied the stabilities of NAG isoenzymes in urine at 37 degrees C by enzyme assay and ELISA using a model simulating in vivo conditions. The stabilities were found to be affected by the pH. Under mild acidic condition (about pH 6), there was no significant loss of enzymatic activity of NAG isoenzyme A, enzymatic activity of NAG isoenzyme B and immunological activity of NAG isoenzyme B even after 8 h incubation. In contrast, under alkaline condition (about pH 8), the enzymatic activity of NAG isoenzyme A was rapidly lost, whereas both enzymatic and immunological activities of NAG isoenzyme B were maintained at more than 80% of their initial values. Also, we found that the ratios of endogenous NAG isoenzyme B to total NAG were elevated in alkaline urine samples. These results indicate that NAG isoenzyme A, which is a major isoenzyme in normal urine (pH 5-7), seems to be inactivated in alkaline urine. Our results suggest that for alkaline urine, NAG isoenzyme B should be measured to avoid misinterpretation of total NAG enzymatic activities.

Effects of β-lactam antibiotics on intestinal microflora and bile acid metabolism in rats

Wistar male rats were treated for six days with broad spectrum β-lactam antibiotics, latamoxef, and cefotaxime. On the seventh day, the number of fecal anaerobic microbes decreased, total fecal bile acids decreased, and bile acid pools increased. Secondary bile acids such as β-hyocholic, hyodeoxycholic, lithocholic, and deoxycholic acids decreased in the feces while the primary bile acids, cholic, β-muricholic, and chenodeoxycholic acids, became predominant. Coprostanol, a microbial metabolite of cholesterol, also disappeared from the feces during the treatment. The cecum enlarged to almost twice the size of that in control rats, whereas the liver weight was not significantly changed. After treatment was stopped, the number of fecal microbes returned to the initial counts within a week, but restoration of bile acid and cholesterol metabolism required at least three weeks.

Age-related changes of bile acid metabolism in rats

Cholesterol and bile acid leves were examined in young (8 weeks), middle-aged (12 months) and old (24 months) germ-free male rats, and young (8 weeks) and middle-aged (12 months) conventional male rats. The plasma cholesterol levels were higher in the aged rats, being more marked in the conventional rats. The liver cholesterol levels also increased with age and the increases were almost identical for both groups. No age-related changes were found in the biliary bile acid secretion, the pool size and distribution of bile acids in the bile, small intestine and large intestine, nor in the turnover frequency of bile acids, but the pool size in the young and middle-aged germ-free rats was much larger than that in the conventional rats. The turnover frequency was less in the germ-free rats. The bile acid synthesis presumed from the fecal bile acid excretion decreased in the aged germ-free rats but not in the conventional rats. A most remarkable age-related change was found in the bile acid composition; cholic acid increased and beta-muricholic acid derived from chenodeoxycholic acid in the rat decreased by aging, resulting in an increase of the CA/CDCA ratio (bile acids belonging to the cholic acid group/bile acids to the chenodeoxycholic acid group) in the bile, feces and pool. These results suggest that cholic acid synthesis increases while chenodeoxycholic acid synthesis is impaired by aging in rats.

Composition of very low density lipoproteins and in vitro effect of lipoprotein lipase

In order to clarify the relationship between composition and lipolytic responses to lipoprotein lipase (LPL), very low density lipoproteins (VLDL) from rats or humans were incubated with a commercially available LPL or with a partially purified LPL from postheparin human plasma and fatty acids released from VLDL were determined in vitro. VLDL from rats fed a diet containing 0.25% cholesterol for 6 months were rich in cholesterol and poor in triglycerides, and released less fatty acids from incubation with LPL than those from control rats. VLDL from normo-and hypertriglyceridemic human subjects were incubated with LPL. The fatty acid release poorly correlated with the apoprotein ratios of VLDL, apo C-III/C-II, B/E, and C/E with the exception of apo B/C, but it correlated well with the ratio of triglyceride/either one of the surface components including total apoproteins, free cholesterol and phospholipids in VLDL or the ratio of the triglyceride/total sum of the surface components. The correlation coefficients between fatty acid release and a ratio of triglyceride/total surface components were 0.774 (using the commercially available LPL) and 0.786 (using the partially purified human LPL). The fatty acid release increased after pretreatment of VLDL with phospholipase A2. The phospholipid content of VLDL was reduced without significant changes in other VLDL components. Thus, the responses of VLDL to LPL treatment may depend mainly upon the surface: core relationship of VLDL rather than its apoprotein composition except in rare clinical cases such as apo C-II deficiency.

In vitro degradation of very low density lipoprotein from diabetic patients by lipoprotein lipase

Fatty acid release by incubation with lipoprotein lipase (LPL) in vitro from very low density lipoproteins (VLDL) obtained from diabetic patients was low compared with that from healthy subjects, though the compositions were similar in both VLDL. Percentages of the large size VLDL decreased and those of the small size VLDL increased after the incubation with LPL. At the same time, on polyacrylamide gel disk electrophoresis, the smaller catabolic products from these VLDL appeared at a similar position to that of low density lipoproteins (LDL) and at the running front where high density lipoproteins (HDL) had migrated. The amount of the small size VLDL and the LDL-like lipoproteins produced from diabetic VLDL were less than those from normal VLDL and inversely correlated with the percent decrease of the large original size VLDL. This fact suggests that VLDL from diabetic patients are poor substrates for LPL compared with normal VLDL.

Changes with aging in serum lipoproteins and apolipoprotein C subclasses

The effects of aging on serum lipids, lipoproteins and apolipoprotein C subclasses in very low density lipoprotein (VLDL) were investigated in healthy male subjects aged from the 1st to the 9th decade. The serum cholesterol, phospholipid and triglyceride concentrations, the serum beta-lipoprotein concentration determined immunologically, and the beta-lipoprotein percentage determined by electrophoresis showed the lowest levels in the 2nd decade, increased gradually with age, attained the highest level in the 6th to 7th decade and slightly declined in the 9th decade. The VLDL-low density lipoprotein (LDL) cholesterol level changed almost in parallel with the serum total cholesterol level, but the HDL cholesterol level and the apolipoprotein A concentration remained almost constant showing no age-related change. The free cholesterol percentages in every lipoprotein fraction and the apolipoprotein content in LDL were higher in the subjects in the 6th and 7th decade than those in the 2nd to 3rd decade. The apo C II/C III ratio in VLDL increased with age. These data suggest that the ability to active lipoprotein lipase may not be impaired but the lecithin:cholesterol acyltransferase (LCAT) activity declines with age.

Role of membrane-associated lymphotoxin (mLT) in the killing activity of lymphokine-activated killer (LAK) cells towards various tumour cell lines.

Human lymphokine‐activated killer (LAK) cells developed by an incubation of peripheral mononuclear cells with IL‐2 express the membrane‐associated lymphotoxin (LT)‐related molecule (mLT). By a further cultivation of mLT expressing (mLT‐positive) LAK ceils for 24 h without IL‐2. mLT disappears (mLT‐negative LAK cells). Cytotoxicities of various tumour cell lines by either mLT‐positive or ‐negative LAK cells were compared. Eight out of 12 tumour cell lines, less susceptible to mLT‐negative LAK cells than mLT‐positive LAK cells, were categorized as group A, Two tumour ceils (K562 and Moit‐4) had the same susceptibility to both kinds of LAK cells. The others (Daudi and Jurkat) had less susceptibilities only when they were assessed at E:T ratios of less than 5. The four tumour cell lines in the latter two cases, containing K562. Moit‐4. Daudi and Jurkat cells, were categorized as group B. The cytotoxicities of group A tumour cells, but not group B tumourceils, by LAK cells were significantly suppressed by the presence of anti‐LT antibody. Group A tumour ceils had higher LT‐binding ability (2‐82‐16‐44 fmol/106 cells) than group B tumour cells (less than 1 46 fmol/106 cells). Both mLT‐positive and ‐negative LAK cells had similar perform activities and tumour cell‐binding capacities. These results suggest that the mLT‐mediated killing mechanism is involved in tumour ceil killing by LAK cells. Further, various tumour cell lines can be classified into two large groups according to their susceptibilities to the mLT‐mediated killing by LAK cells.

Enhancement of CD3‐mediated thymocyte apoptosis by the cross‐linkage of heat‐stable antigen

Heat‐stable antigen (HSA) is a murine differentiating antigen that is expressed on both CD4−CD8− double‐negative and CD4+CD8+ double‐positive thymocytes but not CD4+ or CD8+ single‐positive thymocytes. Effects of anti‐HSA monoclonal antibody, R13, on thymocyte apoptosis induced by various stimulations were investigated by a single‐cell suspension culture system. Immobilized R13 enhanced the CD3‐mediated DNA fragmentation and killing of thymocytes but not the dexamethasone‐induced or phorbol myristate acetate‐induced killing of thymocytes. Immobilized R13 by itself could not induce thymocyte apoptosis. Soluble R13 enhanced CD3‐mediated apoptosis when HSA and T‐cell receptor (TCR)/CD3 were co‐cross‐linked by a cross‐reactive secondary antibody. Even without the cross‐reactive secondary antibody, soluble R13 enhanced CD3‐mediated apoptosis, although a greater than 100‐fold increase in the amount of R13 was needed to give a similar enhancement compared with immobilized R13. Neither R13 by itself nor R13 plus secondary antibody induced cytosolic calcium influx, whereas R13 enhanced CD3‐mediated cytosolic calcium increase. These results suggest a functional role of HSA in promoting the activation‐induced apoptosis of thymocytes and the involvement of HSA in negative selection.

Effects of triton WR 1339 and orotic acid on lipid metabolism in rats

In order to investigate the effect of hepatic cholesterol flux on biliary bile acids, Triton WR 1339 and orotic acid were administered to rats, and the biliary cholesterol, phospholipids and bile acids were analyzed together with serum lipoproteins and hepatic lipids. Triton, which raised serum very low density lipoprotein and lipid levels and decreased serum high density lipoprotein liver lipid levels, increase the biliary cholic acid group/chenodeoxycholic acid group ratio (CA/CDCA) in the bile without affecting the total amount of bile acids and the other biliary lipids. Orotic acid, which decreased serum lipid and lipoprotein concentrations and increased liver lipid levels, increased the biliary excretion of cholesterol and phospholipids, but produced no significant change in the total amount of bile acids and in the CA/CDCA ratio in bile.

Aging changes of riboflavin concentration and glutathione reductase activity in erythrocytes

Aged erythrocytes obtained by fractionation using gradient centrifugation with Dextran 40, showed lower glutathione reductase activity and riboflavin content than young cells. However, both young and old cells displayed almost the same increase in enzyme activity upon addition of flavin adenine dinucleotide to the test hemolysate in vitro. The lipid peroxide content in the cell membranes showed no consistent changes with aging.