Pitfalls in preparation of (3)H-unconjugated bilirubin by biosynthetic labeling from precursor (3)H-5-aminolevulinic acid in the dog.

We report problems encountered during preparation of tritium-labeled unconjugated bilirubin ((3)H-UCB) from precursor (3)H-5-aminolevulinic acid ((3)H-ALA) in 2 dogs with external biliary drainage installed into the animals under general anesthesia. Under prolonged sedation, 12.9 or 14.0 mCi of (3)H-ALA was administered intravenously in two divided doses, and bile was collected for 9 hours. In one animal, taurocholate (TC) infusion was needed to maintain bile flow. (3)H-UCB was isolated from the bile and recrystallized with the improved method of Webster et al (Webster CC, Tiribelli C, Ostrow JD. J Lab Clin Med 2001;137:370-3). Based on radioactivity and pigment content, hourly bile collections were pooled to optimize specific activities. Surprisingly, in the first dog, only 2.9% of injected radioactivity was recovered in bile and only 14.1% in urine, and the specific activities of the crystalline (3)H-UCB from the two pools were only 39.5 and 30.0 x 10(3) dpm/microg. High-performance liquid chromatography analysis revealed that only 4% of ALA degraded during 5 minutes in injection solution at pH 6.8. The low incorporation of (3)H-ALA and low specific activity of (3)H-UCB was apparently caused mainly by prior degradation and exchange of labile tritium of the (3)H-ALA and probably by enhanced endogenous ALA synthesis caused by the anesthetic/sedative agents. Revised procedures in the second dog improved the incorporation of (3)H-ALA to 11.9% excreted in bile and the specific activity of the crystalline (3)H-UCB to 122.0 and 50.8 x 10(3) dpm/microg, while urinary excretion of tritium increased to 28.5%. These experiences emphasize possible pitfalls in preparing (3)H-UCB by biosynthetic labeling from (3)H-ALA administered to dogs.

Identification of oxysterols in human bile and pigment gallstones.

BACKGROUND AND AIMS: Although cholesterol is the most abundant sterol in animal tissues, oxidized products of cholesterol (oxysterols) also occur in mammalian organs and blood and are cytotoxic, atherogenic, and carcinogenic. However, the presence of oxysterols in bile or gallstones has never been reported. METHODS: Fresh human bile and gallstones were collected. Sterol content and structure were analyzed using gas chromatography/mass spectrometry (GC/MS). Bacterial DNA was extracted from human gallstones. RESULTS: GC/MS identified cholesta-4,6-diene-3-one and cholest-4-ene-3-one, with several as yet unidentified oxysterols in bile and stone samples. Several plant and fungal sterols were also present in gallstones. When 102 human gallstones were analyzed for oxysterols, they were markedly higher (as percent of total sterols) in pigment gallstones, where bacterial DNA is most abundant. CONCLUSIONS: These observations suggest biliary oxysterols are associated with the presence of bacteria and may play a role in the pathogenesis of gallstones and biliary tract cancers.

Bacterial DNA in mixed cholesterol gallstones.

OBJECTIVE: Numerous investigators have proposed a role for bacteria in biliary lithogenesis. We hypothesized that bacterial DNA is present in gallstones, and that categorical differences exist between gallstone type and the frequency of bacterial sequences. METHODS: Polymerase chain reaction (PCR) was used to amplify bacterial 16S rRNA and uidA (encoding Escherichia coli [E. coli] beta-glucuronidase) genes in different types of gallstones. PCR products were sequenced. RESULTS: Bacterial 16S rRNA and uidA DNA sequences in E. coli were detected in all brown pigment, common bile duct, and mixed cholesterol gallstones (n = 14). In contrast, only one (14%) of seven pure cholesterol gallstones yielded a PCR product. Most (88%) mixed cholesterol gallstones yielded PCR amplification products from their central, as well as their outer, portions. Sequenced products possessed 88-98% identity to 16S rRNA genes of E. coli and Pseudomonas species. CONCLUSIONS: Bacterial DNA sequences are usually present in mixed cholesterol (to 95% cholesterol content), brown pigment, and common bile duct, but rarely in pure cholesterol gallstones. The presence of bacterial beta-glucuronidase is also suggested. The role of bacteria and their products in the formation of mixed cholesterol gallstones, which comprise the majority of cholesterol gallstones, warrants further study.

Effect of vitamins A, C and E on normal and HPV-immortalized human oral epithelial cells in culture.

There is experimental and epidemiological evidence that antioxidant vitamins can inhibit carcinogenesis. Since immortalization by Human Papilloma Virus (HPV) is one possible early step towards carcinogenesis in oral epithelia, we studied the differential effect of vitamins A, C and E on HPV-immortalized oral epithelial cells (IHGK) as compared to the normal counterpart. The dose response was determined by morphology, cell cycle by flow cytometry, and growth curve by cell number. The optimum dose in terms of inhibitory effect vs. toxicity was determined for each vitamin by morphology. Optimum doses were: vitamin A--1.4 x 10(-5) M, vitamin C--10(-3) M, and vitamin E--10(-6) M for both HPV-immortalized and normal cells. Growth curve showed reduction of proliferation by all three vitamins, with vitamins A and E more effective than C for both cell types. Flow cytometry showed that vitamins A and E reduced the percentage of cells at G2 phase of cell cycle and indicated arrest in the S phase. This effect was greatest in the immortalized cells with a 50% and 35% decrease of G2 for vitamins A and E respectively, whereas the normal counterpart showed a 48% decrease for A and a 12% increase for E. By organotypic culture, the morphology was not markedly different between the vitamin-treated and the control cells, except for a slight increase in the keratinization of normal cells with vitamin A. Also noted was a reduction in number of cell layers from five layers or more for controls to only one or two for vitamin E. In conclusion, we have demonstrated that the antioxidant vitamins inhibit proliferation, and show a preferential effect on IHGK cells.

Caveat: mycoplasma arginine deiminase masquerading as nitric oxide synthase in cell cultures.

We used confluent cultures of dog gallbladder epithelial cells, stimulated by conditioned medium from a culture of human neonatal foreskin fibroblasts, to establish the presence of inducible nitric oxide synthase (NOS, EC 1.14.13.39). Assay was by conversion of radiolabeled arginine to citrulline. By 4 days after addition of the conditioned medium, a relatively high level of activity was observed. However, further study showed that the enzyme did not require addition of the usual cofactors for maximal activity (NADPH, FAD, FMN and tetrahydrobiopterin) and was stable in the absence of anti-proteolytic agents. Our suspicion that this enzyme might not be NOS but arginine deiminase (EC 3.5.3.6) was confirmed by enzyme purification and by the liberation of ammonia during enzyme reaction. This enzyme, which is absent from primates and virtually confined to single-cell organisms, suggested the presence of Mycoplasma, a common contaminant of cell cultures, and it was subsequently confirmed that the fibroblast culture was a source of Mycoplasma. With the widespread interest in nitric oxide and NOS, and common use of the convenient [3H]arginine assay, there is a considerable danger of the two enzymes being confused. At the very least, it is necessary to check for activity in the absence of added cofactors.

Biliary lipid composition after liver transplantation: effect of allograft function and cyclosporine.

Biliary lipid composition and bile flow are altered after orthotopic liver transplantation. Cyclosporine may have additional effects on biliary lipid composition and secretion. We studied the effects of liver transplantation, allograft function, and cyclosporine on biliary lipids in humans. Changes in lipid composition and secretion were correlated with serum cyclosporine levels, clinical events, and allograft function. Bile samples were withdrawn via a T-tube at interval time points in 17 patients during the first 3 months posttransplantation. Total and individual bile acid, cholesterol, and phospholipid were determined using high-performance liquid chromatography. Biliary lipid profiles were then correlated with clinical events, serum cyclosporine levels, and other clinical laboratory values. Biliary lipid concentrations decreased in 3 patients during periods of graft dysfunction (acute cellular rejection, drug-induced hepatitis, and inferior vena caval thrombosis) and increased with resolution of the graft injury. Serum cyclosporine levels were positively correlated with total bile acid, cholesterol, and phospholipid concentrations in bile. There was no relationship between the composition of secreted bile acids and serum cyclosporine levels. Bile acid, cholesterol, and phospholipid secretion were not uncoupled in the presence of cyclosporine. We concluded that (1) a decrease in biliary lipid concentrations may be an indicator of worsened graft function in some allografts; (2) biliary lipid concentrations are correlated with increasing cyclosporine levels; and (3) bile acid composition is unchanged, and uncoupling of secretion of other biliary lipids is not observed in the presence of cyclosporine.

Potential effect of cyclosporin A in formation of cholesterol gallstones in pediatric liver transplant recipients.

Recent advancements in liver transplantation have resulted in extended survival both for grafts and recipients. Such improvement, together with the shortage of donor organs has prompted expansion of the donor pool to include less than ideal donors, especially in life-threatening situations. The use of older liver donors has been associated with lower long-term survival. However, potential morbidity such as gallstone formation has not been explored. We analyzed bile composition in a child who developed cholesterol gallstones in the proximal bile duct two years after undergoing emergency liver transplantation with a liver from a 78-year-old donor. Oral administration of ursodeoxycholic acid (ursodiol) shifted the cholesterol composition of the bile from a supersaturated, potentially crystallized state to a liquid (micellar) state. Unlike cyclosporin A, FK506 showed an increase in the proportion of chenodeoxycholic acid and a decrease in the proportion of cholic acid, and thus may exhibit minimal or no hepatotoxic effect. Thus, in donor livers with factors known to be associated with cholesterol gallstone formation (such as age, sex, or obesity), one may consider analyzing the bile composition at the time of procurement. Depending on cholesterol and bile acid composition the use of FK506 with or without addition of ursodeoxycholic acid may be warranted.

Blood ionized magnesium concentrations during cardiopulmonary bypass and their correlation with other circulating cations.

BACKGROUND AND AIM: The recent introduction of new measurement technology (using ion specific electrodes) makes intraoperative evaluation of blood ionized magnesium (Mg2+, or iMg)--the bioactive fraction of circulation magnesium--possible. The goals of this study were: (1) to examine the longitudinal pattern(s) of change in blood iMg during cardiopulmonary bypass (CPB); and (2) to determine the relationship of iMg to Ca2+ (iCa), K, pH, Na, and hematocrit (Hct) during CPB. METHODS: Blood was collected serially before, during, and after CPB on 30 patients undergoing elective coronary artery bypass graft procedures and the iMg was measured with an AVL Scientific Corp., model 988-4 instrument. RESULTS: Overall, 73% of iMg results were abnormally low, 50% during CPB. Some cases had both hypo- and hyperionized magnesemic episodes. There were low iCa during CPB in 97% of cases. Using Spearman's rank order correlations and p < 0.05, iMg and K were directly correlated before, during, and after bypass, suggesting their parallel movement between tissue and blood. iMg and iCa were directly correlated before, and inversely correlated after, CPB, but unassociated during bypass. iMg and Na were inversely correlated after bypass in all cases. iMg was inversely correlated to pH and positively correlated to Hct during CPB only, and only in patients with concurrent association of iMg and iCa. CONCLUSIONS: Blood iMg depletion occurs frequently in CPB patients. iMg changes are not readily predictable. The association of intraoperative iMg depletion with postsurgical atrial fibrillation--reported to have a hypomagnesemic connection- should be investigated.

Loss of the adenomatous polyposis coli gene and human papillomavirus infection in oral carcinogenesis.

Recent evidence suggests that loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) gene plays a role in colorectal tumorigenesis and other cancers. However, little is known as to whether the APC gene contributes to the pathogenesis of oral squamous cell carcinoma. To assess involvement of both the APC gene and the human papillomavirus (HPV) in the development of oral pre-malignant and malignant lesions, we analysed DNA from 14 paired oral normal and pre-malignant or malignant paraffin-embedded biopsy specimens, and DNA from cultured normal and HPV 16-immortalised oral epithelial cells for the presence of LOH of APC and for HPV infection, using PCR based techniques. LOH of APC occurred in 80% of cases of oral epithelial dysplasia, 67% of carcinoma in situ, 50% of invasive squamous cell carcinoma cases, and in the HPV 16-immortalised oral epithelial cells. HPV was detected in half of the biopsy specimens, with HPV 16 as the dominant type. More than half of the carcinoma cases were found to contain both LOH of APC and HPV infection. These results suggest that LOH of APC is an early event during oral tumorigenesis. Our findings also suggest a strong correlation between HPV infection, particularly HPV 16, and LOH of the APC gene in oral squamous cell carcinomas.

Oral presentation of posttransplantation lymphoproliferative disorders. An unusual manifestation.

Cyclosporine, an immunosuppressive agent widely used in organ transplantation, has several undesirable side effects, including gingival hyperplasia, which occurs in up to 70% of patients. Another complication associated with use of cyclosporine and other immunosuppressants is an increased incidence of malignancies. Long-term use of cyclosporine also is associated with a spectrum of hyperproliferative disorders ranging from reactive lymphoid hyperplasia to aggressive malignant lymphomas. While cyclosporine-related lymphoproliferative disorders have been widely reported, they have not been described in the oral cavity as the first manifestation of this disease. We report on two cardiac transplantation patients with a history of cyclosporine use who presented initially with oral symptoms of lymphoproliferative disorder. Both had erythematous to cyanotic and hyperplastic gingiva. On gingivectomy, the fixed tissue was soft, glistening, and tan colored, in contrast to the usual firm, white, cyclosporine-associated, benign gingival fibrous hyperplasia. Histologically, a dense, diffuse infiltrate of lymphoplasmacytoid cells with vesicular nuclei, prominent nucleoli, a moderate amount of cytoplasm, and high mitotic activity was observed. Immunocytochemical studies confirmed that the cells were monoclonal for lambda light chains in one patient and kappa light chains in the other. The cells from one patient were positive for CD45, while both patients were negative for CD20 and all nonhematopoietic antigens tested. Both tissues were strongly positive for Epstein-Barr virus. Morphology and immunocytochemistry findings are consistent with a posttransplant lymphoproliferative disorder. These are the first two reported cases of cyclosporine-associated posttransplant lymphoproliferative disorders presenting as gingival hyperplasia.

The characterisation and cyclic production of a highly unsaturated homoserine lipid in Chlorella minutissima.

The marine alga Chlorella minutissima contains DGTS (diacylglyceryl-N,N,N-trimethylhomoserine) as a major component (up to 44% of total lipids). This lipid is absent from other members of the Chlorococcales, except for C. fusca, which contains DGTS as 1.3% of total lipids. Contrary to expectation, the DGTS is accompanied by PC (phosphatidylcholine) as the major phospholipid. DGTS is normally highly saturated in the C-1 position of glycerol, but in C. minutissima, both C-1 and C-2 are acylated with EPA (eicosapentaenoic acid, 20:5) in the major molecular species (over 90% of total). The DGTS level shows a marked rhythmic fluctuation with time which is inversely correlated with the level of MGDG (monogalactosyldiacylglycerol), the other major lipid. Improved NMR data and the first electrospray MS data on this lipid are presented.

Organotypic culture of human gallbladder epithelium.

We have modified methods of growing human gallbladder epithelial cells in monolayer and organotypic culture. These cells were grown in the presence of fetal bovine serum and with coculture of feeder layers of human gallbladder fibroblasts. Human gallbladders were obtained from cholecystectomy specimens, and the cells were dissociated with trypsin/EDTA. Cells, which were grown with feeder layer on collagen-coated plates in the presence of 10% FBS, grew rapidly and formed islands of cuboidal cells with morphology typical of epithelial cells in culture. They could be passaged up to four times. The cells were also successfully grown by organotypic technique producing a monolayer of tall, columnar, palisade, epithelial cells. These cells, both in monolayer and in organotypic culture, were positive to antibodies for simple epithelial keratin and negative to antibody for vimentin or any of the mesenchymal antibodies. These cells respond to agonists (prostaglandin E2, isoproterenol) by the intracellular generation of cAMP. Secreted mucin on the apical surface stained strongly with periodic acid-Schiff. Organotypic culture of human gallbladder epithelium may serve as a cell preparation for the study of pathobiology of columnar epithelial cells.

Two-hour Sephadex column method for assay of unconjugated estriol in serum.

The column method of Christner and Fetter (Steroids 24: 327, 1974) has been modified to give a simple, rapid assay for unconjugated estriol in serum. Estriol is isolated from serum by being retained on a Sephadex column, while estriol conjugates and serum proteins are eluted. The sample and labeled estriol compete for antibody on the column. Antibody is eluted, removing proportional amounts of sample and labeled estriol. Our modifications include using stable serum-based standards, shortening the incubation to 10 min, complete removal of conjugates, and regeneration of the columns so that they can be used repeatedly. The assay, which can be completed in 2 h, has a mid-range interassay CV of 8.2%.

The structure of novel C35 pentacyclic terpenes from Acetobacter xylinum.

A novel C(35) terpene and its monounsaturated analogue were isolated from cultures of Acetobacter xylinum, together with traces of their C(36) homologues. These substances were found to be hopane derivatives substituted by a five-carbon chain bearing four vicinal hydroxyl groups. For the parent hydrocarbon the term bacteriohopane is proposed. The elucidation of the structures utilized high-resolution mass spectrometry of the terpenes, degradation to C(32) hydrocarbons and detailed mass-spectrometric comparison of these with C(32) hydrocarbons synthesized from known pentacyclic triterpenes. High-resolution mass-spectral data of the terpenes are presented. N.m.r. data are in agreement with the proposed structures, which are further supported by the isolation from the same organism of 22-hydroxyhopane and derivative hopene(s).

Induction of orientation of bacterial cellulose microfibrils by a novel terpenoid from Acetobacter xylinum.

1. The bacterium Acetobacter xylinum produces extracellular cellulose microfibrils that form a pellicle in the medium enmeshing the bacterial cells. These microfibrils may show some localized alignment, which can be seen as birefringence when the culture is viewed between crossed Polaroid sheets. 2. An increase in birefringence can be induced by the addition of small amounts of certain classes of lipids, particularly sterols, to the cultures. 3. A crude lipid extract from Acetobacter cells induced greatly increased birefringence when added to fresh cultures of this organism. 4. When the bacterial lipids were fractionated, most of the activity was recovered in a complex, polar lipid. The lipid is secreted into the medium during growth and is unstable. The non-saponifiable portion of this lipid is shown to be a 1:1 mixture of a saturated and a monounsaturated C(35) tetrahydroxy terpene with a hopane ring system in the accompanying paper by Förster et al. (1973). The saturated molecule is referred to as tetrahydroxybacteriohopane. 5. Tetrahydroxybacteriohopane is itself capable of inducing birefringence in cultures as is 22-hydroxyhopane, which was also isolated from the non-saponifiable fraction of the total lipids. 6. The mechanism of induction of birefringence (orientation of microfibrils) is not known. This is unlikely to be a specific effect, since all the above compounds are active (intact lipid, tetrahydroxybacteriohopane, 22-hydroxyhopane), as are other classes of lipid. It is suggested, however, that a common mechanism may be involved and that similar compounds may be concerned with control of microfibril alignment in the cells of higher plants.

Acetylenic acid biosynthesis inCrepis rubra.

Time studies of crepenynic acid synthesis inCrepis rubra show that this acid is not present in the seed for several days after, flowering commences but builds up rapidly between the 14th and 28th days to become the major fatty acid of the seed oil.Radioactive tracer studies clearly demonstrate that the acetylenic bond is introduced into the carbon chain of a preformed long-chain fatty acid rather than built in during formation of the carbon chain. The nearest precursor found is oleic acid. There is no conversion to crepenynic acid by seed preparations ofcis,cis-linoleic acid,cis,trans (trans,cis)-linoleic acid, orcis-12,13-epoxy-oleic acid. Possible biosynthetic pathways to explain these results are suggested.The crepenynic acid is chiefly, but not entirely, in triglycerides in the seed oil, and it has been shown to be esterified in the 2- and 3- positions of the triglyceride.