Biomedical subjects
C Hirayama
Publications and source records attributed to C Hirayama.
[Pericecal abscess].
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Reduction of endotoxin contamination of various crude vaccine materials by gram-negative bacteria using aminated poly(gamma-methyl L-glutamate) spherical particles.
We describe a method for the removal of endotoxins from various crude antigen solutions originating from gram-negative bacteria using aminated poly(gamma-methyl L-glutamate) (PMLG) spherical particles. The aminated PMLG adsorbents showed high affinity for various purified endotoxins at an ionic strength of mu = 0.1. The endotoxin-adsorbing capacity of the adsorbent increased with increase in the amino-group content of the adsorbent. The adsorbent (3.2 meq/g amino-group content) showed the highest affinity for endotoxin at ionic strengths ranging from mu = 0.025-0.8. The adsorption of Bordetella pertussis antigen to the adsorbent decreased with increasing amino-group content of the adsorbent at an ionic strength of mu = 0.2. The adsorption of B. bronchiseptica protein to the adsorbent increased with increasing amino-group content of the adsorbent, but decreased with increasing ionic strength. The adsorbent (3.2 meq/g of amino-group content) selectively reduced endotoxin in crude antigen solutions originating from gram-negative bacteria, B. pertussis, B. bronchiseptica and Pasteurella multocida, even at a high ionic strength (mu = 0.2-0.4) without affecting the recovery of the protective antigens.
Metabolism of 15N-ammonia in patients with cirrhosis: a three-compartmental analysis.
Urinary 15N-ammonia and 15N-urea were measured by gas chromatography-mass spectrometry after the intravenous administration of 15N-ammonia (0.2 mumol/kg/hr) to 6 volunteers and 11 patients with cirrhosis. Urinary 15N-nitrogen excretion as ammonia and urea was measured during the 210-min infusion period, and urea synthesis and ammonia conversion to amino acids were analyzed with a three-compartment model using the nonlinear least-squares method. The rate of urea synthesis in control subjects was 14.1 +/- 1.2 mg/kg/hr (mean +/- S.E.M.), and in cirrhotic patients it was 11.0 +/- 3.2 mg/kg/hr. The cirrhotic group was divided into those with compensated cirrhosis (Child class A patients) and those with decompensated cirrhosis (Child classes B and C patients), and the rates of urea synthesis for these groups were 14.5 +/- 1.5 and 8.9 +/- 1.6 mg/kg/hr, respectively. The difference between decompensated cirrhotic patients and control subjects was statistically significant (p less than 0.001). The percentage of ammonia reutilization of a given dose of 15N-ammonia was 75.9% +/- 2.4% in compensated cirrhotic patients and 82.9% +/- 3.6% in decompensated cirrhotic patients (p less than 0.05). Fasting venous ammonia levels correlated inversely with urea synthesis (p less than 0.001) and correlated positively with ammonia reutilization (p less than 0.05). These results are consistent with a decreased capacity to synthesize urea and an increased capacity to convert ammonia to amino acids in chronic liver failure.
Removal of endotoxin from culture supernatant of Bordetella pertussis with aminated poly(gamma-methyl L-glutamate) spherical beads.
Attempts were made to prepare adsorbents having a high affinity for endotoxin in the culture supernatant of Bordetella pertussis. When poly(gamma-methyl L-glutamate) (PMLG) was used as a matrix and amino groups as the ligand, the highest affinity for endotoxin was attained even at a high ionic strength (mu = 0.2-0.4). PMLG beads containing amino groups of about 3.2 meq/g selectively removed endotoxin from the culture supernatant of B. pertussis without affecting the protective antigens. It was demonstrated that 1 ml of the wet adsorbent adsorbed 4.5 mg of endotoxin. The beads of PMLG derivatives, therefore, are considered to be a useful adsorbent for the removal of endotoxin from the pertussis vaccine, affecting neither filamentous hemagglutinin nor pertussis toxin.
[Tracer kinetics of amino acid].
In vivo turnover of an amino acid can be determined either by analysing the disappearance curve of a labeled amino acid after a single dose of labeled amino acid or by measuring the dilution of a continuously infused labeled amino acid at a steady state. From ethical concerns stable isotopically labeled amino acids, such as 13C-leucine and 15N-glycine have been routinely used to study amino acid kinetics with subsequent estimation of whole body protein turnover. Recently, instead of these 2 methods, pulse labeling by taking a single oral dose of labeled amino acid is more practical for the study of whole body protein turnover. We observed plasma 15N-glycine kinetics and urinary 15N excretion by a single oral dose of 15N-glycine simultaneously in healthy subjects and cirrhosis patients, and the results are discussed.
A simplified gelatin tolerance test to evaluate gastric and pancreatic proteolytic activities.
To evaluate gastric, pancreatic and intestinal proteolytic activities, a gelatin tolerance test was standardized, and its clinical significance was evaluated in 16 normal subjects and 25 patients who had undergone gastrectomy or had a pancreatic disease. A 15g gelatin dose given to normal subjects resulted in an increase of serum-free and peptide-bound hydroxyproline (HYP), with the peak values being observed at 1 hr and 2 hr, respectively. In patients who had malabsorption, both peak levels of serum-free and peptide-bound HYP were decreased, and the peak value of free HYP was more frequently lower than that of peptide-bound HYP. The determination of serum free HYP after oral administration og gelatin was significantly correlated with BT-PABA test. These results indicate that usefulness of determining serum-free HYP for the gelatin tolerance test as a simple reliable test for digestion and absorption of proteins.
Hepatic collagen synthesis in patients with alcoholic and nonalcoholic liver disease.
To examine the synthesis of hepatic collagen in patients with alcoholic and nonalcoholic liver disease, liver biopsy specimens were incubated in vitro with 14C-proline, and the radioactivity of the newly synthesized protein-bound 14C-hydroxyproline was measured. Mean hepatic collagen synthesis was 0.82 +/- 0.19 pmole of 14C-hydroxyproline/g liver/2 h in control subjects without histological liver fibrosis. Hepatic collagen synthesis was increased in patients with alcoholic and nonalcoholic liver diseases, especially in those with alcoholic fibrosis, alcoholic cirrhosis and chronic active hepatitis. The raised collagen synthesis in alcoholic liver disease rapidly decreased after withdrawal of alcohol. When alcoholic liver disease were compared with nonalcoholic liver disease, there was no significant difference in hepatic collagen synthesis.
Serum lysyl oxidase activity in chronic liver disease in comparison with serum levels of prolyl hydroxylase and laminin.
Lysyl oxidase was partially purified from serum by a diethylaminoethyl batch procedure in the presence of 6 mol/L urea and dialyzed against 3 mol/L KSCN. Using this method, we determined serum lysyl oxidase activity in 52 patients with liver disease and in 14 healthy controls, and we examined usefulness of serum lysyl oxidase in assessing liver fibrogenesis. For this purpose, serum lysyl oxidase activity in chronic liver disease was compared with serum levels of prolyl hydroxylase and laminin P1. As compared with controls, serum lysyl oxidase activity increased 1.6-fold in chronic persistent hepatitis, 4.4-fold in chronic active hepatitis and 11.8-fold in cirrhosis, indicating an increase in concert with the development of liver fibrosis. In hepatocellular carcinoma, the serum activity, although significantly increased, was lower than that in cirrhosis. Serum prolyl hydroxylase was significantly increased in chronic active hepatitis, in liver cirrhosis and in hepatocellular carcinoma. Serum laminin P1 was significantly increased in chronic active hepatitis, in cirrhosis and in hepatocellular carcinoma. Serum lysyl oxidase activity did not correlate significantly with serum levels of prolyl hydroxylase and laminin P1 in any subject or in any subgroup. The magnitude of the increase and the abnormal percentage of serum lysyl oxidase activity were larger than those for serum prolyl hydroxylase and laminin P1. These results suggest that serum lysyl oxidase activity is a more sensitive indicator of liver fibrosis than serum prolyl hydroxylase and laminin P1.
Lectin-reactive patterns of markedly elevated serum alpha-fetoprotein in patients with chronic active hepatitis.
Four cases of chronic hepatitis associated with high serum levels of alpha-fetoprotein (AFP) without hepatocellular carcinoma are reported. All showed transient elevations of serum AFP, with peak levels of 13,500, 8,000, 4,450, and 3,000 ng/ml shortly after aggravation resulting from liver function tests. Liver biopsies revealed severe parenchymal damage in all the cases with piece-meal necrosis, bridging necrosis or bridging fibrosis. In two of four cases, there was a lobular distortion. AFP stain by an immunoperoxidase method showed a positive result in surviving hepatocytes. Lectin affinity electrophoresis of AFP in the four cases, together with an additional 12 patients with chronic hepatitis and cirrhosis and 44 patients with hepatocellular carcinoma, all having AFP levels above 1,000 ng/ml, revealed that the chronic hepatitis patients had a benign pattern of AFP bands, in contrast with the pattern of hepatocellular carcinoma with increased proportions of lentil lectin-reactive AFP-L3 and/or erythroagglutinating phytohemagglutinin-reactive AFP-P4, indicating that the analysis of lectin reactivity of AFP has a great value in differentiating the benign and malignant conditions with increased serum levels of AFP above 1,000 ng/ml.
[Current management of amyloidosis].
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Increase in lipoperoxides and prolyl hydroxylase activity in rat liver following chronic ethanol feeding.
The effect of lipid peroxidation on hepatic collagen synthesis was investigated in male Wistar strain rats after 7 weeks of ethanol feeding. Compared with control rats, the ethanol-fed rats had a significantly higher lipoperoxide content and a significantly lower reduced glutathione content al all times following ethanol treatment. Except for the earliest time (2 days), hepatic prolyl hydroxylase activity was also significantly increased and finally reached up to 214% of the control level. Hepatic hydroxyproline content was slightly increased, but not statistically significant. The lipoperoxides content was significantly correlated with prolyl hydroxylase activity and inversely correlated with reduced glutathione content. These findings were also confirmed in ethanol-pyrazole-treated rats. These results suggest that elevated lipoperoxides mediate an acceleration of collagen synthesis, even at an early stage, in ethanol-induced hepatic injury.
Removal of endotoxins using macroporous spherical poly(gamma-methyl L-glutamate) beads and their derivatives.
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Free and small peptide-bound [14C]hydroxyproline synthesis in vitro in ethanol-induced hepatic injury in the rat liver.
To clarify the significance of free and small peptide-bound hydroxyproline synthesis in ethanol-induced liver injury, we measured the in vitro synthesis of [14C]hydroxyproline in the 67% ethanol-soluble fraction in rat liver slices, together with hepatic protein-bound [14C]hydroxyproline synthesis. The synthesis of free and small peptide-bound [14C]hydroxyproline was 11.1 +/- 2.0 dpm x 10(-4)/g liver/3 hr and the synthesis of protein-bound [14C]hydroxyproline was 10.1 +/- 3.3 dpm x 10(-4)/g liver/3 hr in control rat liver. In the ethanol-fed rat liver, the synthesis of free and small peptide-bound [14C]hydroxyproline significantly increased 1.5-fold and the synthesis of protein-bound [14C]hydroxyproline significantly increased 1.6-fold, while the hepatic collagen content did not change. There was a significant correlation between free and small peptide-bound [14C]hydroxyproline synthesis and protein-bound [14C]hydroxyproline synthesis. These results suggest that free and small peptide-bound hydroxyproline synthesis plays an important role in regulating the content of hepatic collagens.
Nonoxidative metabolism of ethanol in the pancreas; implication in alcoholic pancreatic damage.
Alcohol dehydrogenase activity and fatty acid ethyl ester synthase activity were measured in various organs of male Wistar strain rats. The mean (+/- SE) values of alcohol dehydrogenase activity in liver, testis, pancreas and brain were 223 +/- 34, 35 +/- 13, 27 +/- 17 and 24 +/- 15 nmol/hr/mg protein, respectively, but not detectable in heart and skeletal muscle. Fatty acid ethyl ester synthase activity in pancreas, liver, testis and heart were 1348 +/- 263, 23 +/- 14, 17 +/- 3 and 2 +/- 1 nmol/hr/mg protein, respectively, but not detectable in brain and skeletal muscle. Alcohol dehydrogenase activity, fatty acid ethyl ester synthase activity, fatty acid ethyl ester content and amylase activity were measured in pancreas of rat after 7 weeks of ethanol feeding. Compared with control rats, ethanol-fed rats had normal fatty acid ethyl ester synthase activity and alcohol dehydrogenase activity. However, fatty acid ethyl ester content increased five-fold and amylase activity decreased up to 20% of the control group. Fatty acid ethyl ester content was inversely correlated with amylase activity. These results suggest that fatty acid ethyl ester may be responsible for the development of pancreatic damage by ethanol.
Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells.
Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.
An association between alpha 1-antitrypsin phenotype and chronic liver disease.
The phenotypes of alpha-1-antitrypsin have been analyzed by isoelectric focusing on polyacrylamide gels in 232 healthy Japanese blood donors and in 240 Japanese patients with chronic liver diseases: 69 with chronic active hepatitis, 122 with liver cirrhosis, 41 with hepatocellular carcinoma and 8 with primary biliary cirrhosis. The liver cirrhosis patients had a gene frequency of 0.07 for P1*M3, which was significantly higher (P less than 0.01) than that (0.03) in blood donors. The gene frequency of P1*M3 was significantly increased in cryptogenic liver cirrhosis (P less than 0.05), and there was a tendency toward an increased frequency of P1*M3 in post-transfusion groups, and in primary biliary cirrhosis. There were also tendencies toward increased frequencies of P1*M3 in cryptogenic and post-transfusion groups of patients with chronic active hepatitis. The present study indicates that P1*M3 is a genetic or predisposing factor for chronic liver diseases, especially for cryptogenic and/or non A-non B viral chronic liver disease and also for primary biliary cirrhosis.
[15N]glycine metabolism in normal and cirrhotic subjects.
Following a single oral dose of 10 mg/kg of [15N]glycine, plasma [15N]glycine kinetics and urinary 15N excretion were measured in 12 cirrhosis patients and in 6 control subjects. Cirrhosis patients were divided into two groups of 6 patients with and without a history of hepatic encephalopathy designated as group II and group I, respectively. Thirty minutes after oral administration of labeled glycine, the plasma concentration of [15N]glycine was significantly higher in both cirrhosis groups than that in the control group (P less than 0.05 and P less than 0.01). The elimination constant of plasma [15N]glycine slightly decreased in group II, but not significantly. Urinary 15N excretion did not differ among the three groups, but the rate of urinary ammonia 15N in urinary 15N was significantly increased in group II (P less than 0.05). The whole-body protein flux did not differ among the three groups, but whole-body protein breakdown was significantly increased in group II cirrhosis patients (P less than 0.05). These findings indicated that the kinetics of glycine were substantially altered in severe cirrhosis patients. Because hepatic uptake and oxidation of glycine was well maintained even in group II, increased endogenous protein breakdown seemed to be responsible for hyperglycinemia and also for the negative nitrogen balance seen in this group.