Evaluation of anatomical references for locating the course of the posterior superior alveolar artery for dental implant surgery.

This retrospective cohort study aimed to identify the best anatomical reference for predicting the posterior superior alveolar artery (PSAA) location. Computed tomographic images of 90 maxillary sinuses were evaluated. We studied five references, including the alveolar crest, maxillary sinus floor, zygomatoalveolar crest, hard palate and soft palate, and measured the distances between them and the PSAA. Variations in the distance were evaluated by the standard deviation and coefficient of variation (CV). The zygomatoalveolar crest was an unstable reference, owing to its high standard deviation and CV. The smallest CV was for the distance between the alveolar crest and PSAA, although the distance was smaller in edentulous jaws than dentulous jaws. The distance between the sinus floor and PSAA was larger in male and edentulous patients. The PSAA was detected in 40.0%, 44.4%, 54.4% and 56.7% of the sinus walls at the first and second premolar and the first and second molar positions, respectively. At these tooth positions, the respective heights above the hard palate were 11.2 ± 4.9, 8.2 ± 4.9, 6.2 ± 2.8 and 8.1 ± 2.9 mm. The hard palate was the most stable reference for predicting the location of the PSAA, irrespective of sex, age and dentition.

He/Ar-atom scattering from molecular monolayers: C60/Pt(111) and graphene/Pt(111).

Supersonic He and Ar atomic beam scattering from C(60) and graphene monolayers adsorbed on a Pt(111) surface are demonstrated in order to obtain detailed insight into a gas-molecule collision that has not been studied in detail so far. The effective masses and phonon spectral densities of the monolayers seen by different projectiles are discussed based on classical models such as the hard cube model and the recently developed smooth surface model. Large effective masses are deduced for both the monolayers, suggesting collective effects of surface atoms in the single collision event. The effective Debye temperature of graphene was found to be similar to that reported in highly oriented pyrolytic graphite (HOPG), indicating that the graphene is decoupled well from the Pt substrate. A much smaller Debye-Waller factor was found for the C(60) layer, probably reflecting the strong C(60)-Pt(111) interaction.

Molecular heterogeneity of heat-stable toxin-producing and non-producing Escherichia coli O25 strains isolated in Japan.

Escherichia coli O25 strains that produce heat-stable toxin (ST) have been recently isolated in Japan, and epidemiological study of this type of enterotoxigenic E. coli is required. In this study the heterogeneity of 16 ST-producing and non-producing strains of E. coli O25 was investigated. All eight ST-producing strains were shown to have STIb gene, and seven of them had similar profiles of plasmids, ladder-banding of LPS in SDS-polyacrylamide gel electrophoresis, and chromosomal DNA digestions in pulsed-field gel electrophoresis (PFGE). In contrast, ST-non-producing strains were more heterogeneous in all parameters examined. PFGE of the digested chromosomal DNA with several restriction enzymes was proved to be an effective procedure to compare the closely related strains of E. coli O25.

Competition between copper and silver in Fischer rats with a normal copper metabolism and in Long-Evans Cinnamon rats with an abnormal copper metabolism.

Long-Evans Cinnamon (LEC) rats inherently lacking in serum ceruloplasmin (CP) activity and biliary Cu excretion were established from a closed colony of Long-Evans rats. These deficiencies, linked to a dysfunction of P-type ATPase, stimulate deposition of Cu and then of Cu metallothionein (MT) in the liver. Male LEC and Fischer rats were injected subcutaneously with Ag (AgNO3), which is an antagonist to Cu. They were operated on 24 h after the injection while under anesthesia. Total uptake of Ag into the liver was not stimulated, but its uptake into the MT fraction increased significantly in the LEC rats. Ag injection notably decreased the activity of serum CP in the Fischer rats, but not in the LEC rats. The decrease was accompanied by a reduction of serum Cu. In Fischer rat serum treated with Ag, Ag was detected mainly in the albumin region and partly in the CP fraction. In LEC rat serum, however, the Ag concentration was about 1/20 of that in the Fischer rats, and Ag was not detected in the CP fraction. Ag injection decreased the biliary excretion of Cu in the Fischer rats (0.183-0.052 microg Cu/20 min sampling), but not in the LEC rats (0.014-0.014 microg Cu/20 min sampling). On the other hand, biliary excretion of Ag was much greater in the Fischer rats (1.25 microg Ag/20 min) than in the LEC rats (0.04 microg Ag/20 min). Our results suggest that uptake of Ag into the liver is not dependent on the hepatic Cu content and status, but that biliary excretion of Ag from the liver is affected by these. Hepatic MT is not a transporter of hepatobiliary excretion of Cu and Ag. It seems likely that, unlike Cu excretion, Ag is excreted by not only the CP route but also by another route into the serum. Ag may compete with Cu in the uptake into CP (conversion of apo-CP to holo-CP).

Two regions of homozygous deletion clusters at chromosome band 9p21 in human lung cancer.

We examined 149 lung cancer cell lines for homozygous deletions using 24 DNA markers, which were mapped and ordered in chromosome band 9p21, to define the target regions for 9p21 deletions in human lung cancer. Homozygous deletions were detected in 39 (26%) cell lines and clustered at 2 independent regions. One was the region containing the p16/CDKN2A tumor suppressor gene, and this region was deleted in 32 (21%) cell lines. The other was the region containing D9S171, which is the locus approximately 3 Mb proximal to the CDKN2A locus. This region, designated as the D9S171 region, was deleted in 18 (12%) cell lines. Seven of the 18 cell lines had identical minimum deletions of a 17,036 bp sequence located 20 kb distal to the D9S171 locus. However, such a deletion was also observed in the corresponding B-lymphoblastoid cell line from 1 of the 7 cell lines and in 5 (16%) of 32 noncancerous tissues, suggesting that the deletion was a genetic polymorphism. By considering this polymorphism, 11 (7%) cell lines still had deletions at the D9S171 region. Two NSCLC cell lines showed deletions at the D9S171 region and retentions of the CDKN2A locus. Furthermore, an NSCLC cell line showed discontinuous deletions including either the CDKN2A or D9S171 locus. Therefore, the region surrounding the D9S171 locus was defined as another target region for the 9p21 deletions. It is possible that unknown tumor suppressor gene(s) are present in this chromosomal region. Genes Chromosomes Cancer 27:308-318, 2000.

Molecular dynamics of MHC genesis unraveled by sequence analysis of the 1,796,938-bp HLA class I region.

The intensely studied MHC has become the paradigm for understanding the architectural evolution of vertebrate multigene families. The 4-Mb human MHC (also known as the HLA complex) encodes genes critically involved in the immune response, graft rejection, and disease susceptibility. Here we report the continuous 1,796,938-bp genomic sequence of the HLA class I region, linking genes between MICB and HLA-F. A total of 127 genes or potentially coding sequences were recognized within the analyzed sequence, establishing a high gene density of one per every 14.1 kb. The identification of 758 microsatellite provides tools for high-resolution mapping of HLA class I-associated disease genes. Most importantly, we establish that the repeated duplication and subsequent diversification of a minimal building block, MIC-HCGIX-3.8-1-P5-HCGIV-HLA class I-HCGII, engendered the present-day MHC. That the currently nonessential HLA-F and MICE genes have acted as progenitors to today's immune-competent HLA-ABC and MICA/B genes provides experimental evidence for evolution by "birth and death," which has general relevance to our understanding of the evolutionary forces driving vertebrate multigene families.

An iron-deficient diet stimulates the onset of the hepatitis due to hepatic copper deposition in the Long-Evans Cinnamon (LEC) rat.

To study effects of dietary Cu and Fe levels on the onset of hepatitis in Long-Evans Cinnamon (LEC) rats, female rats (40 days old) were fed a semipurified diet containing 0.1 or 10 mg Cu/kg and 1.5 or 150 mg Fe/kg in a 2 x 2 factorial arrangement for 35 days. At 75 days after birth, LEC rats (+Cu-Fe) fed a Cu-sufficient but Fe-deficient diet (Cu, 10 mg/kg; Fe, 1.5 mg/kg) showed jaundice, with lethargy, anorexia, and malaise. The biochemical variables relating to liver function were significantly increased compared to three other groups, a Cu- and Fe-deficient (-Cu-Fe) group, a Cu-deficient but Fe-sufficient (-Cu+Fe) group, and a Cu and Fe sufficient (+Cu+Fe) group. Furthermore, the +Cu-Fe rat liver showed massive necrosis with huge nuclei. The other three groups presented no biochemical and histological findings of hepatitis. Hepatic Cu and metallothionein concentrations were 289 +/- 87 (mean +/- SD) microg/g liver and 8.7 +/- 1.8 mg/g liver, respectively, in the +Cu-Fe rats. However, in the +Cu+Fe group the values were 196 +/- 28 microg Cu/g liver and 10.8 +/- 1.0 mg/g liver. Hepatic Fe deposition was not influenced significantly by the dietary Cu level. The +Cu-Fe group with jaundice showed the highest free Cu concentration in the liver among the four groups, but the hepatic free Fe concentration was similar to those in the -Cu+Fe and +Cu+Fe groups. Our results indicate that an Fe-deficient diet enhances the deposition of hepatic Cu due to increased absorption of Cu from the gastrointestinal tract. This deposition stimulated the onset of hepatitis.

Iron depletion prevents adenine nucleotide decomposition and an increase of xanthine oxidase activity in the liver of the Long Evans Cinnamon (LEC) rat, an animal model of Wilson's disease.

The Long Evans Cinnamon (LEC) rat, which accumulates excess Cu in the liver as in patients with Wilson's disease, is a mutant strain displaying spontaneous hepatitis. It was reported that Fe, like Cu, increases in the liver and that the severity of hepatitis is modified by Fe in the diet. In this experiment, oxidative stress increased by Fe was investigated before the onset of hepatitis. To examine the effect of Fe on the progress into hepatitis, LEC female rats were fed an Fe-regular (Fe 214 microg/g; Fe(+) group) or an Fe-restricted (Fe 14 microg/g; Fe(-) group) diet from 53 days of age for 35 days. Fischer rats were also fed as control animals. Adenine nucleotide decomposition was determined as an index of oxidative stress based on xanthine oxidase activity. The size of the hepatic pool of adenine nucleotides (ATP+ADP+AMP) was significantly smaller in LEC rats than Fischer rats. The energy charge (ATP+0.5ADP)/(ATP+ADP+AMP) was smaller in Fe(+) groups than in Fe(-) groups. In the LEC rat liver, the Fe concentration in the Fe(+) group was 160% of that in Fe(-) group and the correlation coefficient between the hepatic Fe concentration and the energy charge was significant. In this strain, an increase of xanthine oxidase activity resulted in an increase of xanthine, an oxidized metabolite of hypoxanthine in the liver. The results suggest the involvement of the Fe in the progression into hepatitis in the LEC rat, even if the dietary Fe concentration is similar to that of commercial diet.

Preventive effect of zinc compounds, polaprezinc and zinc acetate against the onset of hepatitis in Long-Evans Cinnamon rat.

It is known that Long-Evans Cinnamon (LEC) rats are characterized by the fulminant hepatitis occurring as a result of an abnormal hepatic deposition of Cu due to the lack of the Cu-transporter p-type ATPase. To prevent the hepatitis, two Zn compounds, Zn acetate and polaprezinc were given orally to LEC rats aged 30 days. At 100 days after birth, the control group composed of LEC rats fed a basal diet (Cu, 17 ppm; Zn, 50 ppm; Fe, 150 ppm) exhibited slight jaundice and showed high activities of serum enzymes related to hepatic function. The groups fed the diet fortified (1000 ppm as Zn) with Zn acetate or polaprezinc did not have jaundice. The hepatic Cu concentrations were 174 +/- 34 micrograms/g and 156 +/- 23 micrograms/g in the polaplezinc group and Zn acetate group, respectively. The control group showed 267 +/- 17 micrograms Cu/g and 298 +/- 62 micrograms Fe/g in the liver. The Fe concentration was about 1.7 times the concentration in the two Zn groups. Hepatic free Cu and Fe concentrations were 2.6 +/- 0.3 and 21.4 +/- 5.8 micrograms/g, 1.7 +/- 0.7 and 6.8 +/- 1.1 micrograms/g, and 1.3 +/- 0.1 and 6.2 +/- 0.8 micrograms/g in the control, polaprezinc and zinc acetate groups, respectively. Intestinal metallothionein (MT) concentrations were not increased significantly by the Zn diets. The two Zn compounds inhibit Cu absorption from the intestinal tract, resulting in a decrease of hepatic Cu deposition. The new Zn compound as well as Zn acetate is categorized as a therapeutic drug for Cu poisoning, including Wilson's disease.

Therapeutic effects of tetrathiomolybdate on hepatic dysfunction occurring naturally in Long-Evans Cinnamon (LEC) rats: a bona fide animal model for Wilson's disease.

Long-Evans Cinnamon (LEC) rats were fed a diet containing 7 ppm Cu and 30 ppm Cu from 60 days after birth. Fischer (Fischer group) and LEC (LEC-control group) rats fed a 7 ppm Cu diet showed normal growth throughout the whole period (60 to 125 days after birth). On the other hand, LEC rats fed the 30 ppm Cu diet had decreased body weight and showed slight jaundice at around 100 days after birth. Tetrathiomolybdate (TTM, 10 mg/kg bw) was injected sub-cutaneously at 101 and 105 days after birth into half of the LEC rats fed the 30 ppm Cu diet. LEC rats given TTM (LEC+TTM group) recovered their body weight and the jaundice rapidly disappeared. However, LEC rats without TTM (LEC-TTM group) had sharply decreased body weight and showed severe jaundice at 103 days after birth. The hepatic Cu concentration in LEC+TTM rats (460 micrograms/g) exceeded that of LEC-control rats (330 micrograms/g) at 125 days after birth. Our data suggest that TTM is effective for treatment of acute hepatic injury in the LEC rat.

The effect of subcutaneous tetrathiomolybdate administration on copper and iron metabolism, including their regional redistribution in the brain, in the Long-Evans Cinnamon rat, a bona fide animal model for Wilson's disease.

The present work was performed to examine the effect of tetrathiomolybdate on Cu and Fe metabolism, especially redistribution of Cu and Fe in the brains of Long-Evans Cinnamon rats, with inherently abnormal Cu deposition in the liver. The drug was injected subcutaneously at 5 mg/kg of body weight twice a week for 65 days (total dose of 20 mg) into 40-day-old Long-Evans Cinnamon rats. In Long-Evans Cinnamon rats treated with tetrathiomolybdate, the hepatic Cu concentration was 60 microg/g wet weight, compared to 170 microg/g in untreated rats. In seven brain regions (cerebellum, medulla oblongata, hypothalamus, striatum, midbrain, hippocampus and cortex) of the Long-Evans Cinnamon rats treated with tetrathiomolybdate. the Cu concentration (1.5 to 2.3 microg/g) was slightly lower (1.6 to 2.7 microg/g) than in untreated rats. A significant difference between the two groups was found only in the midbrain. Brain Fe concentrations in regions other than the striatum were not changed significantly by the tetrathiomolybdate injections. The hepatic Fe concentration was about 120 microg/g in Long-Evans Cinnamon rats without tetrathiomolybdate. Tetrathiomolybdate injection further increased the concentration to about 250 microg/g. Our results indicated that subcutaneous tetrathiomolybdate injection did not have an effect that stimulated redistribution of Cu and Fe in the seven brain regions examined, although hepatic Cu was markedly decreased and the removed Cu was deposited in kidneys, spleen and testes. The increased hepatic Fe level should be taken into account when considering side effects of the compound.

Mapping of a novel human carbonyl reductase, CBR3, and ribosomal pseudogenes to human chromosome 21q22.2.

To find the genes contributing to Down syndrome, we constructed a 4-Mb sequence-ready map spanning chromosome 21q22.2 with megabase-sized cosmid/P1-derived artificial chromosome (PAC) contigs. The restriction map with rare cutting enzymes, followed by sequencing from the clustering sites, has defined CpG islands and revealed the genes associated with CpG islands (Accession No. D85771). Of these, two human carbonyl reductases (CBR; EC1.1.1.184) were found in a PAC 25P16 clone. CBR catalyzes the reduction of a large number of biologically and pharmacologically active carbonyl compounds to their corresponding alcohols and has been mapped in 21q22.1. To confirm these results, we sequenced the PAC clone in shotgun strategies and identified a novel carbonyl reductase, designated CBR3, 62 kb downstream from the original CBR. In addition, three ribosomal pseudogenes, L23a, S9, and L3, and some cDNAs with ESTs were mapped in the sequence. In conclusion, the sequence analysis for CpG islands predicted from the megabase-sized contigs will reveal and identify the genes involved in Down syndrome.

Nucleotide sequencing analysis of the 146-kilobase segment around the IkBL and MICA genes at the centromeric end of the HLA class I region.

To elucidate the complete gene structure and to identify new genes involved in the development of HLA class I antigen-associated diseases in the class I region of the human major histocompatibility complex on chromosome 6, a YAC clone (745D12) covering the 146-kb segment around the IkBL and MICA loci was isolated from a YAC library constructed from the B-cell line, BOLETH. A physical map of this region was constructed by isolation of overlapping cosmid clones derived from 745D12. Of these, five contiguous cosmids were chosen for DNA sequencing by the shotgun strategy to give a single contig of 146,601 bp from 2.8 kb telomeric of the IkBL gene to exon 6 of MICA. This region was confirmed to contain five known genes, IkBL, BAT1, MICB, P5-1, and HLA-X (class I fragment), from centromere to telomere, and their exon-intron organizations were determined. The 3.8-1 homologue gene (3.8-1-hom) showing 99.7% identity with the 3.8-1 cDNA clone, which was originally isolated using the 3.8-kb EcoRI fragment between the HLA-54/H and the HLA-G genes, was detected between MICA and MICB and was suggested to represent the cognate 3.8-1 genomic sequence from which the cDNA clone was derived. No evidence for the presence of expressed new genes could be obtained in this region by homology and EST searches or coding and exon prediction analyses. One TA microsatellite repeat spanning 2545 bases with as many as 913 repetitions was found on the centromeric side of the MICA gene and was indicated to be a potential hot spot for genetic recombination. The two segments of approximately 35 kb upstream of the MICA and MICB genes showed high sequence homology (about 85%) to each other, suggesting that segmental genome duplication including the MICA and MICB genes must have occurred during the evolution of the human MHC.

Nucleotide sequence analysis of the HLA class I region spanning the 237-kb segment around the HLA-B and -C genes.

To elucidate the detailed gene organization of the human leukocyte antigen (HLA) class I region on chromosome 6, seven contiguous cosmid genomic clones covering the 237-kb segment around the HLA-B and -C loci were subjected to DNA sequencing by the shotgun strategy to give a single contig of 236,822 bp from the MICA gene (58.2 kb centromeric of HLA-B) to 90.8 kb telomeric of HLA-C. This region was confirmed to contain four known genes, MICA, HLA-17, HLA-B, and HLA-C, from centromere to telomere. Further, a new member of the P5 multicopy genes was found to be about 1.3 kb upstream of the HLA-17 gene and designated P5.8. Five novel genes designated NOB1-5 were identified by RT-PCR and Northern blot hybridization. In addition, two pseudogenes, dihydrofolate reductase pseudogene (DHFRP) and ribosomal protein L3 homologous gene (RPL3-Hom), were also found in the vicinity of the HLA-B and -C genes, respectively. The two segments (about 40 kb) downstream of the HLA-B and HLA-C genes showed high sequence homology to each other, suggesting that segmental genome duplication including the major histocompatibility complex (MHC) class I gene must have occurred during the evolution of the MHC.

Antigenicity of cell wall mannans of Candida albicans NIH B-792 (serotype B) strain cells cultured at high temperature in yeast extract-containing sabouraud liquid medium.

Cultivation of Candida albicans NIH B-792 (serotype B) at high temperature (37 degrees C) for 48 h in yeast extract-containing Sabouraud liquid medium (YSLM) provided the following findings in comparison with the findings obtained after incubation at 27 degrees C. Growth of the blastoconidia of this strain was decreased, with a dry weight of 9%, and the cells were deficient in cytokinesis. The cells did not undergo agglutination with serum factor 5 from a commercially available serum factor kit (Candida Check). Mannan (B-37-M) obtained from the cells cultured at 37 degrees C had partially lost its reactivity against serum factor 4 and lost most of its reactivity against serum factor 5 in an enzyme-linked immunosorbent assay (ELISA) in contrast to that (B-27-M) at 27 degrees C. Both cells and mannan prepared by cultivation first at 37 degrees C and then at 27 degrees C entirely recovered their reactivities with serum factors 4 and 5. 1H-nuclear magnetic resonance analysis also revealed that B-37-M had lost a beta-1,2-linked mannopyranose unit and retained a phosphate group. Similar changes were observed in the three other serotype B strains used in the study. The beta-1,2-linked mannooligosaccharides longer than mannotetraose were not included among the products released from B-37-M by mild acid treatment. The results of the inhibition ELISA with a series of beta-1,2-linked mannooligosaccharides from biose to octaose (M2 to M8, respectively) showed that the reactivity against serum factor 4 was inhibited most strongly by the oligosaccharides M4 to M8 and that the reactivity against serum factor 5 was inhibited completely by relatively longer oligosaccharides, M5 to M8, indicating their participation as the antigenic factor 5 epitopes.

Accumulation of orally given cadmium in Long-Evans Cinnamon (LEC) rats with an inherently abnormal copper metabolism.

An inherent defect of biliary Cu excretion and subsequent Cu deposition in the liver have been found in Long-Evans Cinnamon (LEC) rats, which are promising models of Wilson disease. LEC and Fischer rats were given water containing Cd (CdCl2) at a level of 5 ppm for 30 days. Regardless of drinking Cd water, LEC rats showed a very high concentration of Cu (200 to 250 microgram/g) and Cu-metallothionein (Cu-MT) (18 mg/g) in the liver. There was no difference of Cd accumulation in the liver between the two strains exposed to Cd (2.6 and 2.7 microgram/g in the Fischer and LEC groups, respectively). However, the renal Cd concentration was slightly but significantly higher in LEC rats (3.5 microgram/g) than in Fischer rats (2.0 microgram/g). The ratio of renal Cd contents to the sum of renal and hepatic Cd contents was significantly higher in LEC rats (0.25) than in Fischer rats (0.15). The serum Cd concentration in Cd-treated LEC rats increased threefold compared to Cd-treated Fischer rats. It seems likely that Cd from the liver is transported into the kidney in the form of Cd, Cu-MT. There was no difference in uptake of Cd in the hepatic MT fraction between the two strains. Although biliary Cu excretion in LEC rats was significantly lower than that in Fischer rats, reduced excretion of Cd into bile was not found in LEC rats. The gross amounts of Cu and Cu-MT influenced the accumulation of Cd in the kidney rather than in the liver when Cd was given orally at a low level to LEC rats. Our results suggest tht Cu and Cd do not share the same sites of hepatobiliary excretion in rats, although the main route of their excretion is via bile.

Response of hepatic function to hepatic copper deposition in rats fed a diet containing copper.

Fischer rats were a fed diet supplied with copper chloride (150-600 ppm) for 60 d from weaning. Serum (glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activities were increased with the increase of Cu concentration in the diet. Biliary excretion of Cu was related to the dietary Cu level. Depositions of hepatic and renal Cu were also related to the dietary Cu level in a dose-dependent manner. In particular, hepatic (155.2 +/- 13.3 micrograms/g) and renal (44.9 +/- 4.4 micrograms/g) Cu concentrations increased abruptly in the Cu-600 ppm group. In the liver, about 60% of Cu was distributed in the soluble fraction (100,000 g supernatant). In the Cu-600 ppm group, 25% of cystosolic Cu was bound to metallothionein (MT). Our results suggest that chronic exposure to Cu appears to have a deleterious effect on the hepatic function, and further, that even in rats with normal biliary Cu excretion, clearance of Cu from the liver may be marginal when dietary Cu is near the 600-ppm level. Although Cu is an essential nutrient, an overload of Cu should be avoided.

Biliary excretion of copper, metallothionein, and glutathione into Long-Evans Cinnamon rats: a convincing animal model for Wilson disease.

Long-Evans Cinnamon (LEC) rats, characterized by a gross accumulation of hepatic Cu and the spontaneous onset of hepatitis, have been established to be an animal model for Wilson disease. They were used to estimate the relationships among copper (Cu), metallothionein (MT), and reduced glutathione (GSH) in biliary excretion in this study. Even though a huge amount of MT existed in the LEC rat liver (5016 micrograms/g liver) compared to that (63 micrograms/g liver) of controls (Fischer rats), the biliary excretion of MT (65 ng/ml bile) did not reflect the accumulated MT level in LEC rats. It seems likely that MT does not excrete intrinsically into the bile. Biliary excretion of Cu (0.17 microgram/ml) in LEC rats was significantly lower than that (0.57 microgram/ml) in Fischer rats. The difference in biliary excretion of GSH between the two groups was significant but slight. The reduced excretion of GSH into bile in LEC rats may be due to increased hepatic gamma-glutamyltransferase but not to hepatic GSH levels. There were no differences in biliary potassium and inorganic phosphorous between the two groups. On the other hand, excretion of lysosomal enzymes such as beta-N-acetylglucosaminidase into bile was much lower in LEC rats (15.6 units/liter) than in controls (42.5 units/liter). The defective biliary excretion of Cu may be due to impaired lysosomal exocytosis, rather than canalicular membrane impairment. The LEC rat is very useful for research into the dynamics of metal excretion via the hepatobiliary system.

Biliary excretion of copper in Fischer rats treated with copper salt and in Long-Evans cinnamon (LEC) rats with an inherently abnormal copper metabolism.

Increased biliary Cu excretion was found in Fischer rats injected with Cu. The biliary Cu was located at the void (large-molecule region) and total (small-molecule region) volume of a Sephadex G-75 column. The most Cu was found in the total volume. The two Cu peaks comigrated with absorbance at 280 nm. Although the bile from Cu-untreated Fischer rats did not show Cu absorbance in the total volume, absorbance at 280 nm was also found in this region. Even though Long-Evans Cinnamon (LEC) rats deposited a gross amount of Cu (194.0 +/- 27.8 micrograms/g liver) in the liver, they conversely showed reduced Cu excretion into the bile. LEC bile did not show Cu absorbance but rather absorbance at 280 nm in the total volume. Therefore, it seems unlikely that the small molecules found in the Sephadex G-75 regulate biliary Cu excretion in Cu-loaded rats, although the molecules bind to Cu. When the bile from Cu-untreated Fischer and LEC rats was incubated with CuCl2 solution, the most Cu was recovered in the total volume of this column. Our results suggest that reduced biliary Cu excretion in LEC rats is not related to the small molecules, and that Cu cannot be excreted in the form of macromolecules in rats to decrease Cu from the Cu-loaded liver.

Removal of copper from the liver of Long-Evans Cinnamon (LEC) rats by tetrathiomolybdate (TTM) injection: the main excretion route is via blood, not bile.

Tetrathiomolybdate (TTM) was injected at a dose of 10 mg/kg bw daily for eight consecutive days into Long-Evans Cinnamon (LEC) rats, which inherently abnormally deposit Cu (260 micrograms/g) in the liver. The hepatic Cu (100 micrograms/g) and metallothionein (MT) bound Cu (from 2,600 to 540 micrograms/g protein) concentrations were decreased greatly by the injection. On the other hand, the renal Cu concentration increased significantly, but the brain Cu concentration only very slightly. The reduction of the hepatic Cu concentration was accompanied by reductions of Zn and Fe concentrations in the liver, kidney and brain. The TTM compound slightly stimulated excretion (about 3-fold) of Cu into the bile, but greatly (about 40-fold) into the blood. In rats not treated with TTM, most biliary (100%) and serum (78%) Cu was recovered in the trichloroacetic acid (TCA) soluble fraction. On the other hand, in rats treated with TTM, bile and serum Cu were recovered overwhelmingly in the TCA insoluble fraction, probably in the form of a Cu-TTM-albumin complex. Our results suggest that although there is an inherent failure in the intrinsic secretory process of Cu from the liver in LEC rats, the TTM compound can remove Cu from Cu-MT, resulting in a decrease of hepatic Cu.