Bile acid metabolism in young-old parabiotic rats.

Serum cholesterol, triglyceride and phospholipid levels, liver cholesterol concentration, bile flow, biliary cholesterol, phospholipid and bile acid secretion rates, fecal sterol and bile acid levels and their bile acid compositions were examined in young-old parabiotic rats and compared with those in young and old control rats and young-young parabiotic rats. Bile acid composition was expressed in terms of the cholic acid group/chenodeoxycholic acid group (CA/CDCA) ratio. Body weight (BW) gain decreased after parabiosis especially in old rats, but the liver weight (g/100 g BW), diet-intake, feces dry weight, liver cholesterol concentration and fecal sterol level were almost the same in all the groups. The biliary bile acid secretion rate was higher and the fecal bile acid level was lower in old rats than those in young rats but both the levels became comparable with those in young rats after parabiosis of old rats with young rats. Young rats, however, showed no changes in these levels after parabiosis. The serum cholesterol level and the biliary and fecal CA/CDCA ratios in old rats were higher than those in young rats but decreased after parabiosis with young rats, although they were still higher than those in young rats. The serum cholesterol level in young rats increased after parabiosis with old rats, but not after parabiosis with young rats, and the fecal bile acid level and the CA/CDCA ratio were not changed in either case. It is concluded from these findings that the serum cholesterol level and the CA/CDCA ratio increased with age and that these increases were prevented after parabiosis with young rats while young rats, although their serum cholesterol level was increased, showed no increase in the CA/CDCA ratio after parabiosis with old rats.

[The taxonomic distribution, characteristic and susceptibility against antimicrobial agents of methicillin-resistant staphylococci isolated from blood].

The species, mecA gene, beta-lactamase activity, the ability of slime formation and drug susceptibilities of 386 strains of staphylococci which were isolated from blood in our laboratory were studied. The coagulase typing of each strains identified as S. aureus was also determined. These 386 strains consisted of various species, e.g., S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, S. simulans, S. haemolyticus, and S. lugdunensis. mecA (methicillin-resistant gene) was detected in 84 (67.7%) of 124 S. aureus and 195 (75.3%) of 259 CNS, but there was no statistical difference. However mecA positive rate was higher in S. epidermidis and S. caprae, lower in S. hominis compared with S. aureus, S. lugdunensis having mecA has not been reported, but one of two our S. lugdunensis strains had mecA. The positive rate (77.4%: 65/84) of beta-lactamase of methicillin-resistant Staphylococcus aureus (MRSA) was lower than that (95.5%: 187/195) of methicillin-resistant coagulase-negative staphylococci (MRCNS). Concerning the ability of slime formation, CNS had higher positive rate, especially in case of MRCNS (46.2%: 84/182) than S. aureus. On the other hand, MRCNS showed a tendency to be less resistant to some antimicrobials than MRSA. Especially against cephalothin (CET), the resistant rate of MRSA and MRCNS were 86.9% and 5.6%, respectively. Among the main species of MRCNS, S. capitis and S. caprae were more resistant than S. epidermidis and S. simulans to cefaclor (CCL), cefmetazole (CMZ), flomoxef (FMOX) and fosfomycin (FOM). Hereafter, one should be careful about infectious disease caused by CNS especially in immuno-compromised host, because many species of CNS have higher positive rate of beta-lactamase and slime formation than S. aureus beside mecA.

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

Wistar male rats were treated for six days with broad spectrum beta-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 beta-hyocholic, hyodeoxycholic, lithocholic, and deoxycholic acids decreased in the feces while the primary bile acids, cholic, beta-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.

Glycation index of hair for non-invasive estimation of diabetic control.

We propose a new indicator for diabetic control that shows the extent of glycation of hair protein (keratin), the glycation index (A(390)/A(412)) which is based on the ratio of glycated protein- to cystine-induced coloration, where A(390) and A(412) represent each absorbance in the color reactions of glycated protein and cystine in the hair protein. Samples can be quickly and non-invasively collected and easily stored. This index for the back and scalp hairs from hypercholesterolemic mice with hyperglycemia, diabetic rats and diabetic patients gave significantly higher values (2.0-6.0-fold) than those of normal subjects (p<0.01). The glycation indices (mean + or - S.D.) of hairs from diabetic and non-diabetic subjects were 3.00 + or - 0.96 (n = 21) and 1.51 + or - 0.45 (n = 30), respectively. These indices (y) correlated well with the levels of glycohemoglobin (HbA(1c), chi) in diabetic and non-diabetic subjects: y = 0.69 chi- 2.03 (r = 0.82, n = 31, p<0.01). Within-run precision (reproducibility, CV) for the assay of the glycation indices of hairs from the three groups was 6.7-9.4% (n = 10 each). The proposed glycation index of hair gave reasonable results for animals and humans with normo- and hyperglycemia, suggesting that it is reliable and can be diagnostically useful.

Lipase activities in post-heparin plasma and tissues, and susceptibilities of lipoproteins in experimental diabetic rats.

Heparin administration to diabetic rats caused no change in VLDL, an increase in IDL and a decrease in LDL on electrophoretic analysis of plasma lipoproteins, while the administration to control rats markedly decreased VLDL and increased IDL and LDL. Both hepatic triglyceride lipase (HTGL) and lipoprotein lipase (LPL) activities in the postheparin plasma were lower in the diabetic rats than in the controls, and the reduction of HTGL activity was greater than that of LPL activity in the diabetic rats. The LPL activity in the adipose tissue was lower in the diabetic rats than in the controls, but the activities in the cardiac and skeletal muscles were similar in the two rats. The HTGL-catalyzed fatty acid (FA) releases from the diabetic VLDL and IDL were lower than those from the normal rat VLDL and IDL, while the LPL-catalyzed FA release in the diabetic rats was not different from those in the controls. The decreases in LPL and HTGL activities and the markedly impaired susceptibility of IDL to HTGL coincide well with the postheparin changes in plasma lipoproteins in diabetic rats, an increase in IDL and a decrease in LDL.

A new genetic assay for rifampicin susceptibility of Mycobacterium tuberculosis.

A new method for testing rifampicin (RFP) susceptibility of Mycobacterium tuberculosis was developed. This method is based on detection of the internal sequence derived from 71-kDa heat shock protein mRNA in tubercle bacilli heat-treated in the presence of RFP. The target sequence was amplified by reverse transcription and PCR, followed by agarose gel electrophoretic analysis. No amplification occurred in one RFP-susceptible strain by exposure to 45 degrees C for 45 min in Middlebrook 7H9 broth containing RFP (10 micrograms/ml) after overnight incubation at 37 degrees C. On the other hand, an amplified 275-bp product was obtained from the RFP-resistant strain MY-129. In a subsequent trial using 65 clinical isolates, this method defined their RFP susceptibility levels as well as the verification of the MICs obtained by the conventional agar dilution method, with the exception of one RFP-susceptible strain. Thus, this method provides a rapid and practical system to determine RFP susceptibility in M. tuberculosis.

Hepatitis C viral complexity detected by single-strand conformation polymorphism and response to interferon therapy.

BACKGROUND/AIMS: Hepatitis C virus (HCV) genome heterogeneity by sequence analysis in association with interferon (IFN) inefficacy has been reported. This study was performed to establish a convenient method for detecting the HCV quasispecies complexity and to determine the correlation between the complexity and the responsiveness to IFN therapy in patients with chronic hepatitis C. METHODS: The quasispecies complexity of HCV hypervariable region 1 in patients treated with IFN-alpha was analyzed by polymerase chain reaction-mediated single-strand conformation polymorphism (SSCP). RESULTS: Seven of 25 patients (28%) with low complexity (SSCP band number of < or = 2) were HCV RNA negative after treatment, whereas in 24 patients with high complexity (SSCP band number of > or = 3), the response to IFN was almost insignificant because only 1 patient (4.5%) remained HCV RNA negative after treatment (P < 0.05). Among type 1b patients, IFN therapy was only effective for patients with low amounts of HCV RNA (< or = 10(7.5) copies/mL serum) and low complexity. In contrast, most type 2a patients tended to respond to the therapy with exceptions being those with high amounts of HCV RNA and high complexity. CONCLUSIONS: The complexity of the hypervariable region 1 quasispecies may be a factor for predicting IFN inefficacy in patients with chronic hepatitis C.

Evaluation of nested polymerase chain reaction for detecting mycobacterial DNA in pleural fluid.

A protocol based on the polymerase chain reaction (PCR) is the most sensitive method for detecting mycobacteria in clinical samples. However, few studies have assessed the usefulness of this method in the diagnosis of tuberculous effusion. We developed a highly sensitive and specific nested PCR method, that amplifies the bovine tuberculous MPB70 gene and the mycobacterial 16S rRNA gene for use in detecting Mycobacterium tuberculosis (M. tuberculosis) and mycobacteria, respectively, in clinical samples. We determined the sensitivity of this method for detecting mycobacteria in samples containing known amounts of mycobacterial DNA and in DNA extracted from pleural effusions obtained from 10 patients with pulmonary tuberculosis in whom standard microbiological techniques had detected mycobacteria in sputum but not in pleural effusion. The nested PCR method for the bovine tuberculous MPB70 gene and the mycobacterial 16S RNA gene was able to detect M. tuberculosis and mycobacterial genomes only if there were at least 2 copies per sample. Positive results for M. tuberculosis and the mycobacterial genomes were obtained by nested PCR in 2 of 10 and in 3 of 10 samples of pleural fluid, respectively but no mycobacteria were detected in malignant effusions obtained from 9 patients with lung cancer. The nested PCR method represents a rapid means for detecting mycobacteria in some pleural effusions previously found to be negative by culture. We speculate that the reaction of the host against mycobacteria is more important than the mycobacteria themselves in the pathogenesis of pleural effusion in which mycobacteria are not detected.

Molecular analysis of T cell receptor V beta chain to detect leukemia cell clonality in patients by adaptor ligation-mediated polymerase chain reaction.

We have developed a simple and rapid method to analyze the clonality of leukemia cells. After three rounds of amplification by adaptor-ligation polymerase chain reaction (PCR), the cDNA is cut with AluI, HaeIII, RsaI, and Sau3AI, and analyzed by polyacrylamide gel electrophoresis. The size of the restriction fragments is compared to that of the published restriction fragments size each TCR-beta subfamily V region. The sensitivity of adaptor-ligation PCR restriction enzyme analysis (AL-PCR-REA) was 10(-4) MOLT-4 T-ALL cell population in the normal peripheral blood lymphocytes (PBL). Application of AL-PCR-REA to PBL and bone marrow (BM) cells from eight clinical leukemia samples indicated that a detection sensitivity was rather low, but revealed the clonality of all eight clinical samples. This AL-PCR-REA method can detect clonality without the need for either radioisotopes or sequencing procedures.

[Determination of serotonin in whole blood and platelet rich plasma by high-performance liquid chromatography with fluorometric detection].

An improved high-performance liquid chromatographic method with fluorometric detection (Ex 285nm, Em 345nm) for the assay of serotonin in whole blood and platelet rich plasma was developed for the routine clinical examination. Clinical sample was successfully deproteinized with perchloric acid in the presence of a reducing agent (ascorbic acid). Precision (CV < 5.0%), accuracy (recovery > 95%), lower assay limit (20ng/ml) and assay time (15min/sample) were allowable for the routine clinical examination. The level of serotonin (ng/ml, mean +/- SD) in whole blood and platelet rich plasma from normal volunteers (n = 53) was 143 +/- 47 and 180 +/- 60, respectively.

A comparative study usefulness of magnetic resonance imaging in the diagnosis of acute cholecystitis.

The utility of magnetic resonance imaging (MRI) in the diagnosis of acute cholecystitis was evaluated in 72 consecutive individuals (5 healthy, 13 with chronic cholecystitis and silent gallbladder stones, 43 without biliary or diffuse liver disease, and 11 with acute cholecystitis and gallbladder stones) with a 0.5-T superconducting unit. On the T1-weighted (500/20) and less T1-weighted axial spin-echo images (620/25), the liver/gallbladder signal intensity ratio (mean +/- SD) was 2.5 +/- 0.51 (n = 5) and 1.8 +/- 0.29 (n = 8) in acute cholecystitis; 0.9 +/- 0.42 (n = 6) and 1.0 +/- 0.29 (n = 9) in chronic cholecystitis; and 0.9 +/- 0.14 (n = 5) and 0.8 +/- 0.19 (n = 43) in normal gallbladder, respectively. Our results indicate that the liver/gallbladder signal intensity ratio on the T1-weighted image may be a simple and reliable indicator for the diagnosis of acute cholecystitis; we suggest further investigation to confirm these results.

Delta 22-beta-muricholic acid in monoassociated rats and conventional rats.

Bile acids were analyzed in the bile, small and large intestines, and feces of germ-free rats after a single inoculation with one of six intestinal bacteria that had been originally isolated from human feces. Bacteroides vulgatus and Bifidobacterium longum preferentially deconjugated tauro-beta-muricholic acid and taurocholic acid, respectively. Clostridium ramosum, Peptostreptococcus productus and Lactobacillus gasseri deconjugated both bile acids, but Escherichia coli did not deconjugate either one. Rats inoculated with bacteria that deconjugated tauro-beta-muricholic acid produced delta 22-beta-muricholic acid in the feces. In contrast, delta 22-cholic acid could not be detected in rats inoculated with bacteria that deconjugated taurocholic acid.

DMSO preparation as a direct solubilizer of calcium bilirubinate stones.

Despite considerable progress in operative techniques, biliary surgeons continue to encounter difficulties when treating patients with intrahepatic gallstones. We have experienced a significant number of patients in whom complete stone removal was impossible during an operation due to the anatomical complexity of intrahepatic bile ducts, and the variety in the size and hardness of the stones. Under such circumstances, the application of a direct solubilizer capable of dissolving intrahepatic calcium bilirubinate stones, might be a possible alternative. Up to now, however, only calcium chelating agents have been used for removing calcium from calcium bilirubinate stones, and they are not always effective. As a promising solution to this problem, we have now developed a direct solubilize for bilirubin complexes--a dimethyl sulfoxide (DMSO) preparation. DMSO is a well-known bipolar nonprotonic solvent. We prepared solutions of DMSO and performed toxicological studies by administering this preparation to rats and dogs. Biochemical indices of liver and renal function remained unchanged after 7-28 days of DMSO administration. Nor were there any histological changes in the DMSO-treated animals on systemic survey of various organs. On comparing the in vitro capability for solubilizing calcium bilirubinate stones, the DMSO preparation was consistently superior to a calcium chelating agent alone. The combination of a chelating agent with DMSO had the highest solubilizing capacity.

Development of atherosclerosis in alloxan diabetic rats.

Rats with alloxan-induced diabetes developed severe atherosclerotic lesions when they were maintained on a 0.25% cholesterol diet for one year. The atheromatous changes developed at the aortic arch, appeared as early as 3 months after the start of the experiment, and increased thereafter. The diabetic rats also developed atherosclerosis when they were fed standard rat chow, but the area of the atheromatous lesion was about one tenth of that in rats fed the high-cholesterol diet. Normal rats did not develop atherosclerosis even when fed the high-cholesterol diet for one year. The alloxan diabetic rats showed no increase in body weight, but developed serum glucose levels as high as 600-800 mg/dl as well as high serum cholesterol levels and lower serum HDL-cholesterol levels. The development of atherosclerosis in these rats was significantly related to an increase in the serum cholesterol/phospholipid ratio, the atherogenic index (TC-HDLC/HDLC), and the serum total cholesterol level, but was not related to the serum glucose, HDL-cholesterol, triglyceride, or lipid peroxide levels. These relationships were found as early as B-16 weeks after the start of the experiment. These data suggest that the serum cholesterol/phospholipid ratio, the atherogenic index, and the total cholesterol level are important risk factors for the development of atherosclerosis in rats with alloxan diabetes.

[Detection of Mycobacterium avium, M. intracellulare and M. kansasii in sputum by two-step PCR].

A DNA amplification assay using PCR, which consists of amplification of genus specific mycobacterial 16S rRNA gene as the 1st step and reamplification of the amplicon with species specific primers as the next step, could detect M. avium, M. intracellular and M. kansasii in the sputum. The results with type or standard strains showed that M. avium PCR and M. intracellular PCR were highly specific for identification of each species but M. kansasii PCR detected M. gastri besides M. kansasii. Among 22 clinical samples which were positive by PCR, the 17 results were confirmed by culture. The PCR detected 27 (7.5%) of nontuberculous mycobacteria from 360 sputum and showed that the 27 of NTM consists of 9 M. avium, 8 M. intracellulare, 5 M. kansasii, 1 reacted both M. avium and M. intracellulare and 4 unidentified.

[A healthy carrier of verotoxin-producing Escherichia coli, who was detected by periodic feces examinations].

Verotoxin-producing Escherichia coli (VTEC) was rapidly detected by the PCR method in one of the 9 feces samples. They were collected from the people who had been cooking meals for patients on a periodic feces examination on November 25, 1992. It was confirmed by PCR that the isolate had both VT1 and VT2vh toxic genes and showed cytotoxicity on Verocells. In this case, the isolate was highly susceptible to common antibiotic agents, it was removed by the administration of tosfulaxacin. Some isolates which had the same properties as those of the strain described in "VITEK GNI card" and the antimicrobial susceptibility and VT toxin types, were detected in 2 of the 3 members of her family, they were healthy carriers and did not have any subjective symptoms such as diarrhea or abdominal pain. In order to detect Verotoxic genes, the sample of feces preincubated for 3 h in trypticase broth was subjected to PCR. We recommend that this method is much more useful because of rapid detection and identification of VTEC compared with the classical culture method.

Ursodeoxycholic acid in the Ursidae: biliary bile acids of bears, pandas, and related carnivores.

The biliary bile acid composition of gallbladder bile obtained from six species of bears (Ursidae), the Giant panda, the Red panda, and 11 related carnivores were determined by reversed phase liquid chromatography and gas chromatography-mass spectrometry. Bile acids were conjugated solely with taurine (in N-acyl linkage) in all species. Ursodeoxycholic acid (3 alpha, 7 beta-dihydroxy-5 beta-cholan-24-oic acid) was present in all Ursidae, averaging 1-39% of biliary bile acids depending on the species; it was not detected or present as a trace constituent (< 0.5%) in all other species, including the Giant panda. Ursodeoxycholic acid was present in 73 of 75 American Black bears, and its proportion averaged 34% (range 0-62%). Ursodeoxycholic acid averaged 17% of biliary bile acids in the Polar bear (n = 4) and 18% in the Brown bear (n = 6). Lower proportions (1-8%) were present in the Sun bear (n = 2), Ceylon Sloth bear (n = 1), and the Spectacled bear (n = 1). Bile of all species contained taurine-conjugated chenodeoxycholic acid and cholic acid. In some related carnivores, deoxycholic acid, the 7-dehydroxylation product of cholic acid, was also present. To determine whether the 7 beta hydroxy group of ursodeoxycholic acid was formed by hepatic or bacterial enzymes, bile acids were determined in hepatic bile obtained from bears with chronic biliary fistulae. Fistula bile samples contained ursodeoxycholic acid, chenodeoxycholic acid, and a trace amount of cholic acid, all as taurine conjugates, indicating that ursodeoxycholic acid is a primary bile acid formed in the liver in Ursidae.(ABSTRACT TRUNCATED AT 250 WORDS)

[Direct gallstone dissolution therapy with GS-100].

We have developed GS-100, a new direct dissolving drug with strong dissolution property for cholesterol stone by supplementing d-limonene, a dissolvent used since 1974, with 30% medium-chain monoglyceride (MCM). The new drug was applied in 23 patients with gallbladder stones and three with bile duct stone. The presumptive analysis of the composition of the stones was drawn from the CT recordings. As non-invasive therapies, methyl tert-butyl ether (MTBE) dissolution and extracorporeal shock wave lithotripsy (ESWL), which are commonly used in Europe and America, have been reported as being favourable; however, GS-100 is superior in respect to safety and applicative dimension, suggesting of the possibility of using GS-100 as an important drug in the non-invasive therapy for gallstone in the near future.

Isolation of a human biliary glycoprotein inhibitor of cholesterol crystallization.

BACKGROUND: About 50% of populations in developed countries have bile supersaturated with cholesterol, which is a major risk factor for cholesterol gallstone formation. Despite the prevalence of supersaturated bile, only about 10% of these populations develop gallstones. The existence of a biliary protein that inhibits cholesterol crystallization was hypothesized to explain this discrepancy. This report outlines the purification and characterization of such a human biliary glycoprotein. METHODS: Chromatographic methods were used for separation and characterization. Additional steps included activity analysis by crystal growth assay, electrophoresis, and deglycosylation. RESULTS: The glycoprotein consists of a heterodimer, M(r) of 120 kilodalton, with subunits of M(r) of 63 kilodalton and 58 kilodalton. Each of the subunits is characterized by an isoelectric point of 6.6 and shows comparable inhibitory activity. Deglycosylation of the subunits show that they share a similar polypeptide backbone (M(r) of 35 kilodalton) based upon a highly similar amino acid profile. This suggests that differential subunit glycosylation alone may account for the apparent heterodimeric structure. CONCLUSIONS: No other human biliary glycoprotein has been found thus far that shows cholesterol crystal growth-inhibiting activity. Thus, it may be of importance in preventing gallstone formation in healthy populations.