Silencing of chromosomal domains has been described in diverse systems such as position effect variegation in insects, silencing near yeast telomeres, and mammalian X chromosome inactivation. In mammals, silencing is associated with methylation at CpG dinucleotides, but little is known about how methylation patterns are established or altered during development. We previously described a strain-specific modifier locus, Ssm1, that controls the methylation of a complex transgene. In this study we address the questions of the nature of Ssm1's targets and whether its effect extends into adjacent sequences. By examining the inheritance of methylation patterns in a series of mice harboring deletion derivatives of the original transgene, we have identified a discrete segment, derived from the gpt gene of Escherichia coli, that is a major determinant for Ssm1-mediated methylation. Methylation analysis of sequences adjacent to a transgenic target indicates that the influence of this modifier extends into the surrounding chromosome in a strain-dependent fashion. Implications for the mechanism of Ssm1 action are discussed.

Elements of the mouse Immunoglobulin gamma 2a gene, near the membrane-specific poly(A) addition site, were inserted into a heterologous location in either a synthetic mouse gamma 2b gene or a gpt/SV40 chimeric gene and then assayed for their ability to terminate RNA polymerase II transcription in isolated nuclei of transfected myeloma cells. The intact gamma 2a membrane-specific 3'-untranslated region, with its potential stem loop forming sequences and poly(A) site, is able to efficiently terminate transcription in the absence of the downstream region in which transcription normally terminates (term). Termination efficiency in the presence of the termination fragment decreases either when sequences specifying a potential stem/loop, upstream of the poly(A) region, are interrupted or when the stronger membrane poly(A) site is substituted with a weaker, secretory-specific poly(A) site. We therefore conclude that the gamma 2a membrane-specific untranslated region plays a major role in specifying downstream termination. We further conclude that the immunoglobulin gamma 2a, membrane-specific, 3'-untranslated region can function in the context of the gpt gene, driven by an SV40 promoter, to terminate transcription in a poly(A) site dependent fashion.

A general method of relating molecular function and structure is to examine the biological and chemical effects of defined mutations. In many cases, particularly those concerned with the rate or efficiency of gene expression, it is important to assess mutations in the normal chromosomal context. There are two methods of obtaining such mutants: (i) site-directed mutagenesis of the chromosomal locus, using homologous recombination to target defined mutations to the gene of interest, and (ii) phenotypic selection of mutant organisms. For most mammalian genes the rarity of targeted recombinants and phenotypically evident mutants impede the use of either of these approaches. However, various genetic and biochemical features render the Ig heavy chain locus in B cell lines amenable to both gene targeting and phenotypic selection of mutants. We describe here a replacement-type vector in which the selectable marker is an enhancerless gpt gene which is particularly suitable for targeting the IgH locus. Deletion of the enhancer greatly decreased the frequency of gpt+ random transformants while still allowing properly targeted transformants to be gpt+, such that transformants with the predicted recombinant structure comprised 25% of the gpt+ population. Thus, the labor involved in mutagenizing the chromosomal locus using this method is comparable to the usual method of isolating randomly inserted transformants, but offers the important advantages that the copy number and integration site are the same in independent transformants. In the hybridoma cell lines which we have tested, the consistent copy number and integration site are sufficient to yield a uniform level of recombinant gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Damage to the Kupffer and endothelial cells of the liver sinusoids induced by the administration of sublethal doses of frog virus 3 (FV 3) renders A/J mice which are genetically resistant to mouse hepatitis virus type 3 (MHV 3) highly susceptible to this virus. Liver histopathology of these animals revealed typical necrotic foci containing MHV 3-specific antigens. FV 3-pretreated mice, after MHV 3 infection, showed higher levels of serum transaminase (GPT) than controls, and MHV 3 replicated more rapidly and to higher titres. Our results bear out the important role of the liver sinusoidal lining in protecting against hepatocyte infection and its direct involvement in the resistance of A/J mice to MHV 3 infection.

Objectives. To test the relation between white matter lesions (WML) location and physical performance, in aged patients. Methods.

METHODS

29 patients (17 males), aged >65 (mean age 72.6 ± 5.2), with leukoaraiosis. WML was quantified with a visual scale; Apparent Diffusion Coefficient (ADC) was measured bilaterally in frontal periventricular lesioned white matter and frontal and parieto-occipital normal appearing white matter (NAWM). Motor performance was studied using the Short Physical Performance Battery (SPPB), single leg stand time, finger tapping and grooved pegboard tests (GPT). Results. There were significant correlations between the frontal region visual scale scores and SPPB chair stands (r = -0.379; P = .039) and Grooved Pegboard (r = 0.393; P = .032); frontal NAWM ADC values and SPPB standing balance (r = -0.450; P = .014) and SPPB 4 meter walk (r = -0.379; P = .043). Conclusion. Frontal WML are negatively related to motor performance in patients with leukoaraiosis. DWI results suggest that this may be true even for NAWM.

Diethyldithiocarbamate (DTC) and carbon disulfide (CS2), at nearly equimolar oral dose levels, protected mice against liver damage induced by carbon tetrachloride, chloroform, bromotrichloromethane, thioacetamide, bromobenzene, furosemide, acetaminophen, dimethylnitrosamine and trichloroethylene, as evidenced by the suppression of elevations in plasma GPT activity and liver calcium content, and of histopathological alterations. Both agents also prolonged hexobarbital sleeping time and zoxazolamine paralysis time in mice. DTC and SC, alone, given orally, decreased microsomal metabolism of several substrates (aniline, p-nitroanisole, hexobarbital, zoxazolamine, aminopyrine and 3,4-benzopyrene), CC14-induced lipid peroxidation, and cytochrome P-450 content. The loss of microsomal drug-metabolizing enzyme activity was also observed in the experiments in vitro using liver slices and isolated microsomes. Since a characteristic common to such diverse hepatotoxins is that they require metabolic activation before exhibiting hepatotoxicity, the protective mechanisms of DTC and CS2 may involve their interference with the process of metabolic activation of these hepatotoxins. The protective action of DTC may be mediated almost entirely through CS2 when administered orally and at least partly with parenteral administration, since, in CCl4-induced liver injury, DTC was most effective when given orally, while the action of CS2 was less dependent on the route of administration. Thus CS2 and CS2-producing agents in vivo such as dithiocarbamate derivatives and disulfiram may modify toxicological and pharmacological effects of foreign compounds by inhibiting microsomal drug-metabolizing enzyme activity in the liver.

To detect low-level DNA platination, a sensitive immunocyto- and histochemical technique was developed using a polyclonal antibody. The antibody GPt, derived after immunization of rabbits with highly platinated DNA and purified with affinity chromatography, detected the main platinum (Pt)-containing intrastrand and interstrand adducts. Double-fluorescence microscopy image analysis was used to quantify Pt-DNA adducts with Hoechst 33258 fluorescence to locate the nuclei and with fluorescein isothiocyanate fluorescence to measure the immunosignal. A two- to five-fold dose-dependent difference in the level of cisplatin (CDDP)-induced Pt-DNA adducts between a CDDP-sensitive and -resistant human tumour cell line was detected. Large differences in Pt-DNA adduct levels after in vitro CDDP incubation between human buccal cells, lymphocytes and biopsies of different tumour types were observed. Pt-DNA adduct levels were fivefold higher in human testicular tumours than in colon tumours, representing CDDP-sensitive and -resistant tumours, respectively, in the clinic. These data suggest the possibility of predictive testing by measuring Pt-DNA adduct levels. Pt-DNA adducts in patients after treatment with CDDP were shown in normal buccal cells and in imprints of fresh tumour biopsies as well as in paraffin-embedded tumour cells. The analysis of Pt-DNA adducts at a single-cell level in small samples of normal and tumour cells during and/or after treatment is feasible with GPt and will hopefully enable more selective treatment of patients.

Two patients with anorexia nervosa with evidence of pancreas and liver injury are reported. Both showed evidence of organ dysfunction in the presence of cachexia. Serum amylase, ASAT (GOT), and ALAT (GPT) became normal parallel with body weight. On the basis of reports on the effects of malnutrition on pancreas and liver function, it is suggested that nutritional pancreas and liver dysfunction might represent hitherto unrecognized complications of anorexia nervosa.

OBJECTIVE

A continuation of liver fibrosis after undergoing successful Kasai operation has become the important clinical issue in the long-term follow-up of patients with biliary atresia (BA). The aim of this study is to evaluate the efficacy of the herbal medicine Inchinko-to (TJ-135) on the treatment of liver fibrosis in patients with BA without jaundice, especially from the viewpoint of the long-term effects of TJ-135.

METHODS

Six postoperative patients with BA ranging between 3 and 13 years of age with normal serum total bilirubin levels (total bilirubin < 1.0 mg/dL [17 micromol/L]) received TJ-135 from 2 to 4 years. The liver enzyme (glutamic oxaloacetic transaminase [GOT], glutamic pyruvic transaminase [GPT], gamma glutamyl transpeptidase[gamma-GTP]transpeptidase[gamma-GTP] levels and hyaluronic acid (HA) levels were compared before and after the administration of TJ-135. The monthly collected data were averaged on a 1-year basis. The record of one postoperative patient with BA and a normal serum total bilirubin level was incorporated as a control. This patient showed portal hypertension and did not receive TJ-135.

RESULTS

Five of the six patients who showed abnormal values for liver enzymes, exhibited a significant decrease in serum GOT, gamma-GTP, or GPT levels after a 1 to 3-year administration of TJ-135, and the improvement in these parameters persisted thereafter. Furthermore, one patient who had an abnormally high value of HA also showed a significant decrease in the serum level of HA. In the remaining patient with normal liver enzyme values, no significant change was observed during the administration of TJ-135. The control patient exhibited a chronological decrease in the serum GOT and GPT levels by 5 years of age, but the serum gamma-GTP and HA levels remained stable throughout the postoperative period.

CONCLUSIONS

The long-term effectiveness of TJ-135 was only found in those patients with abnormal liver enzyme levels and HA, thereby suggesting that TJ-135 has a protective and antifibrotic effect on the liver.

After 2 years of steroid therapy that had effectively controlled her systemic lupus erythematosus, a 37-year-old woman presented with fever, erythema (face, upper chest), and low CH50. Increased oral steroid (prednisolone from 15 mg to 40 mg) and intravenous methylprednisolone (mPSL) (80 mg for 3 days) alleviated these symptoms except for the fever. Subsequently, the patient's fever worsened and leukocytopenia, abnormal liver function, lymphadenopathy (neck, axilla), and salivary gland swelling developed. Lymph node histology revealed features characteristic of Kikuchi-Fujimoto's disease (KFD). Laboratory examinations showed WBC 600/microliter, Hb 9.5 g/dl, platelets 90,000/microliter, GOT 766 IU/l, GPT 646 IU/l, LDH 4,228 IU/l, TG 1,622 mg/dl, and ferritin 6,330 ng/ml. Serum interferon gamma was also elevated (673 U/ml). Because a bone marrow smear revealed hemophagocytosis, mPSL pulse therapy (1 g for 3 days) was started for treatment of hemophagocytic syndrome. The fever promptly disappeared, and the patient's clinical symptoms resolved within 2 weeks. The abnormal laboratory data related to KFD and hemophagocytosis returned to normal within 4 weeks after the initiation of mPSL pulse therapy. We speculated that the hemophagocytosis and salivary gland involvement in this patient were also symptoms of KFD. This case indicated that corticosteroid pulse therapy is effective for KFD with serious clinical symptoms.

The activity of ornithine carbamyl transferase (OCT) and glutamate pyruvate transaminase (GPT) in serum has been correlated with the extent of necrosis 24 h after different periods of ischaemia in rat liver. The extent of necrosis has been quantified as the volume density of necrosis in the total ischaemic liver lobes using tetranitro BT. The GPT-activity in serum is maximal between 1 and 5 h after different periods of ischaemia, whereas OCT reaches its maximum between 5 and 12 h after ischaemia. The total amount of leaked enzyme-activity as well as the peak value give a linear correlation with the extent of necrosis for OCT and GPT. There is a difference between the character of these two enzymes in that a small leakage of GPT does not indicate liver cell necrosis later on. However, the appearance of OCT in the blood, an enzyme localized in the mitochondrial matrix, has a predictive value for the extent of necrosis, likely to occur later on. GPT, an enzyme from the cytoplasm, can also occur in the blood during the reversible stage of liver cell damage.

Lead and mercury are toxic metals that are widely distributed in the atmosphere, soil, and groundwater. It is estimated that 2-4 x 10(4) tons of these metals are released annually into the environment by natural and industrial processes. Therefore, human exposure to low relatively nontoxic concentrations of these metals is unavoidable. However, the possible health effects of such exposure remain controversial. We have previously reported that low, subthreshold concentrations (0.1-1 microM) of these metals are mutagenic in the transgenic Chinese hamster ovary cell line AS52. The purpose of the present study is to determine the types of mutations induced in the gpt gene in AS52 cells. Using multiplex polymerase chain reaction and southern blot analyses, we characterized the 138 lead-induced, 192 mercury-induced, 29 reactive oxygen radical-induced, and 20 spontaneously arising mutants for point and deletion mutations in the gpt gene. Similar levels of point mutations were observed in the lead- and mercury-induced populations (47.8 and 53.6, respectively), which was significantly less than that occurring in the spontaneously arising and reactive oxygen intermediate-induced mutants. However, further examination of the data revealed that at concentrations of the metals of equal to or less than 0.4 microM, the majority of the mutations in the gpt gene were point mutations, while at higher concentrations, deletions (partial and complete) were the predominant type of mutation. These results are consistent with the hypothesis that lead and mercury induce mutations in eukaryotic cells by at least two distinct mechanisms.

Diabetes mellitus (DM) is a metabolic disease that has grown to pandemic proportions. Recent reports have highlighted the effect of DM on male reproductive function. Here, we hypothesize that testicular metabolism is altered in type 1 diabetic (T1D) men seeking fertility treatment. We propose to determine some metabolic fingerprints in testicular biopsies of diabetic patients. For that, testicular tissue from five normal and five type 1 diabetic men was analyzed by high-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy. mRNA and protein expression of glucose transporters and glycolysis-related enzymes were also evaluated. Our results show that testes from diabetic men presented decreased levels of lactate, alanine, citrate and creatine. The mRNA levels of glucose transporter 1 (GLUT1) and phosphofructokinase 1 (PFK1) were decreased in testes from diabetic men but only GLUT3 presented decreased mRNA and protein levels. Lactate dehydrogenase (LDH) and glutamate pyruvate transaminase (GPT) protein levels were also found to be decreased in testes from diabetic men. Overall, our results show that T1D alters glycolysis-related transporters and enzymes, compromising lactate content in the testes. Moreover, testicular creatine content was severely depressed in T1D men. Since lactate and creatine are essential for germ cells development and support, the data discussed here open new insights into the molecular mechanism by which DM promotes subfertility/infertility in human males.

UNASSIGNED

Tumor microenvironment plays an important role in the chemoresistance of oral squamous cell carcinoma (OSCC). Hypoxia in the microenvironment is one of the important factors that contributes to OSCC chemoresistance; therefore overcoming hypoxia-mediated chemoresistance is one of the great challenges in clinical practice.

UNASSIGNED

In this study, we developed a drug delivery system based on Pt-loaded, polyethylene glycol-modified graphene quantum dots via chemical oxidation and covalent reaction.

UNASSIGNED

Our results show that synthesized polyethylene glycol-graphene quantum dots-Pt (GPt) is about 5 nm in diameter. GPt sensitizes OSCC cells to its treatment in both normoxia and hypoxia conditions. Inductively coupled plasma-mass spectrometry assay shows that GPt enhances Pt accumulation in cells, which leads to a notable increase of S phase cell cycle arrest and apoptosis of OSCC cells in both normoxia and hypoxic conditions. Finally, compared with free cisplatin, GPt exhibits a strong inhibitory effect on the tumor growth with less systemic drug toxicity in an OSCC xenograft mouse tumor model.

UNASSIGNED

Taken together, our results show that GPt demonstrates superiority in combating hypoxia-induced chemoresistance. It might serve as a novel strategy for future microenvironment-targeted cancer therapy.

The developmental and hormonal regulation of UDP-GlcNA:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme which initiates the biosynthesis of asparagine-linked glycoproteins, was investigated in mouse mammary glands. An anti-peptide antibody raised against the carboxyl-terminal 11 amino acids of mouse GPT, immunoadsorbed GPT activity and recognized a protein of expected size (approximately 48 kDa) on Western blots. Mouse mammary glands at different stages of development were examined for GPT activity, immunoreactive protein, and GPT mRNA. All three parameters showed a similar trend, i.e. they were low in tissues from virgin and pregnant animals, increased steadily during lactation, reaching a peak around mid to late lactation, and declined thereafter in glands from post-lactating animals. At mid-lactation, the increase in GPT activity, immunoreactive protein, and GPT mRNA relative to the virgin stage was 9.5-, 3.3- and 5.4-fold respectively, on a per cell basis. These data suggest possible transcriptional and post-transcriptional modulation of GPT gene expression during development of mouse mammary gland. The results on hormonal regulation of GPT in mouse mammary explants and primary mouse mammary epithelial cells showed that all three parameters cited above were stimulated maximally by the combined presence of insulin, hydrocortisone, and prolactin, indicating that the hormonal regulation of GPT expression is also mediated at the level of RNA.

A cloned DNA fragment, carrying the gene for peptidase D (pepD) of Escherichia coli, was partially sequenced. By purification of peptidase D and sequence determination of an amino-terminal oligopeptide the reading frame of the pepD gene, starting with a GTG initiator codon, was unambiguously identified. An overlap of the established nucleotide sequence with the previously sequenced 5' flanking region of the gpt gene allowed the exact distance between pepD and gpt to be calculated. The two genes are pointing towards each other and are separated by 260 bp. A search for open reading frames (ORFs) and the analysis of possible codon usage in the intercistronic region indicate the absence of an additional gene (lpcA) between pepD and gpt.

Two recombinant retroviral systems are described that can be used to analyze antiretroviral drug activity and HIV breakthrough (replication in the presence of the drug). The first system utilizes a recombinant HIV encoding beta-galactosidase as a reporter gene (HIV-LacZ). The defective HIV-LacZ virus is produced in COS cells after co-transfection of a plasmid encoding the HIV-LacZ genome with a plasmid encoding HIV proteins necessary for packaging and infectivity. Subsequent infection of CD4+ target cells, followed by assay for LacZ expression, permits the rapid identification of individual virus-infected cells. This system can be used to quantitate the inhibition of early events in the HIV replicative cycle and is suitable for the screening of compounds for anti-HIV activity. However, this system cannot be used to analyze HIV drug resistance because of the limited genetic heterogeneity of the virus that is produced in COS cells. To circumvent this problem, a second system has been developed in which heterogenous recombinant HIV is produced by rescue with replication-competent 'helper' HIV. This system required the production of CD4+ cell lines containing defective proviruses encoding either LacZ or guanosine phosphoribosyl transferase (gpt). The defective proviruses are rescued by infection of the cell lines with 'helper' HIV and used to infect target cells in the presence of antiretroviral agents. Subsequent reporter gene assay is used to identify virus-infected cells. This system has been used to detect rare HIV breakthrough infection of cells in the presence of the non-nucleoside reverse transcriptase inhibitor TIBO R82150. Similar analyses with other antiretroviral agents, alone and in combination, may help identify therapeutic strategies that minimize breakthrough replication of HIV.

The animals used in liver fibrosis studies must usually be sacrificed. Ultrasound has been demonstrated to have the ability to diagnose hepatic fibrosis and cirrhosis in experimental small-animal models. However, few studies have used high-frequency ultrasound (HFU, 40 MHz) to monitor changes in the rat liver and other hollow organs longitudinally. In this study, liver fibrosis was induced by administering dimethylnitrosamine (DMN) in SD rats, aged 8 weeks, for three consecutive days per week for up to 4 weeks. A Chinese herbal medicine Yi Guan Jian (YGJ) was orally administered (1.8 g/kg daily) to DMN-induced liver fibrosis rats for 2 weeks. Compared with the normal control rats, rats treated with DMN for either 2 weeks or 4 weeks had significantly lower body weights, liver indexes and elevation of hydroxyproline, GOT, and GPT contents. YGJ herbal treatment remarkably prevented rats from DMN-induced liver fibrosis. The HFU scoring results among the normal controls, 2-week DMN-treated rats, 4-week DMN-treated rats, and combined 2-week YGJ therapy with 4-week DMN-treated rats also reached statistical significance. Thus, HFU is an accurate tool for the longitudinal analysis of liver fibrosis progression in small-animal models, and the YGJ may be useful in reversing the development of hepatic fibrosis.

Many eukaryotic DNA sequences, especially lenti-retrovirus proviral genomes and their env genes, are unstable when cloned in high-copy-number plasmids in Escherichia coli. Stability can be increased by the use of low-copy-number vectors, although plasmid yields are low. Vectors are described here that contain the intermediate-copy-number P15A ori for cloning, stable propagation and higher-yield production of plasmid DNA in E. coli, and the f1 ori for propagation as single-stranded phage. These vectors also have the capacity to direct high-yield production of protein in mammalian cells, and the option of incorporation into and expression via a T7 promoter in vaccinia virus. The SR alpha promoter, encephalomyocarditis (EMC) virus untranslated leader sequence, and poly(A) signal sequence serve as a high-yield mammalian cell expression cassette without the requirement for mRNA capping. A polyhistidine sequence is available at the 3' end of the cassette to facilitate chromatographic purification of protein. neo and gpt genes were included in some vectors to serve as selectable markers, and the dhfr gene was included in one to achieve gene amplification in mammalian cells. Dicistronic mRNAs can be generated by insertion of coding sequences up and downstream from the EMC leader. The utility of these vectors was shown through expression of feline immunodeficiency virus (FIV) Env protein, in conjunction with the tissue plasminogen activator (tPA) leader sequence.

Hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT), which initiates N-linked glycosylation by catalyzing the synthesis of GlcNAc-P-P-dolichol, has multiple transmembrane spans and a catalytic site that probably exists on the cytosolic face of the endoplasmic reticulum membrane (Dan, N., Middleton, R. M., and Lehrman, M. A. (1996) J. Biol. Chem. 271, 30717-30725). In this report, we demonstrate that GPT forms functional oligomers, probably dimers. Oligomers were detected by chemical cross-linking of GPT and by a dominant-negative effect caused by co-expression of enzymatically inactive (but properly folded) GPT mutants. The GPT mutants had no effect on two other dolichol-P-dependent endoplasmic reticulum enzymes. Mixing experiments indicated that mature GPT was competent for oligomerization. Oligomerization appeared to be favored in detergent extracts compared with intact microsomes. Detergent treatments were found to prevent, rather than promote, nonspecific aggregation of GPT. These results demonstrate that GPT subunits can physically interact and influence each other. The implications of oligomerization for enzyme function are discussed. From these results, we conclude that GPT is one of a very small number of multitransmembrane span enzymes that can form multimers.

Estragole (ES) is a natural organic compound used frequently as a flavoring food additive. Although it has been reported to be tumorigenic and induce DNA adducts in the mouse liver, there have been no reports regarding ES hepatocarcinogenicity in rats. In the current study, we therefore examined potent carcinogenicity, DNA adduct formation and in vivo genotoxicity of ES in the livers of wild and reporter gene-carrying F344 rats. Males were administered 600 mg/kg bw ES by gavage and sequentially sacrificed at weeks 4, 8 and 16 for GST-P and PCNA immunohistochemistry and measurement of ES-specific DNA adducts by LC-MS/MS in the livers. GST-P-positive foci increased with time in ES-treated rats from week 4, PCNA-labeling indices being similarly elevated at both weeks 4 and 8. ES-specific DNA adducts such as ES-3'-N(2)-dG, 3'-8-dG and 3'-N(6)-dA were consistently detected, particularly at week 4. In a second study, male F344 gpt delta rats were administered 0, 22, 66, 200 or 600 mg/kg bw ES for 4 weeks. Gpt mutant frequency in the liver was increased in a dose-dependent manner, with significance at 200 and 600 mg/kg bw in good correlation with PCNA-labeling indices. Mutation spectra analysis showed A:T to G:C transitions to be predominantly increased in line with the formation of ES-3'-N(6)-dA or 3'-8-dG. These results indicate that ES could be a possible genotoxic hepatocarcinogen in the rat, at least when given at high doses.

Elevated blood vitamin B(12) (VitB(12)) level has recently been identified as a prognostic indicator for advanced cancer patients. The predictive value of blood VitB(12) for survival of patients with hepatocellular carcinoma (HCC) remains unclear. Our objective was to examine the determinants of elevated serum VitB(12) levels and their associations with prognosis of patients with HCC. The cohort study included 90 HCC patients who were consecutively admitted to the Chi-Mei Hospital, Taiwan, from April 2005 to December 2006. Nutrition and clinical pathological data were collected. Serum VitB(12) levels were determined by radioimmunoassay. Survival curves were calculated by the Kaplan-Meier method. Multivariate analysis of outcome predictors was assessed by Cox regression. Elevated serum VitB(12) levels of HCC patients were associated with reduced levels of albumin, hemoglobin, red blood cells count, and glutamate-pyruvate transaminase (GPT) (P < 0.05). Serum VitB(12) levels were positively correlated with alpha-fetal protein (AFP) levels (r = 0.623, P = 0.001) and tumor size (r = 0.630, P = 0.001; Table 3). By univariate analysis, survival was significantly worse in patients with elevated serum AFP >> 200 mu g/l) and VitB(12) levels >> 1,500 ng/l; P < 0.05). In multivariate analysis, both elevated AFP >> 200 vs. < 20; HR 4.4; CI = 1.9-10.3, P = 0.001) and VitB(12) levels >> 699 vs. < or = 699; HR = 2.88; CI = 1.26-6.6, P = 0.012) were found to be favorable predictive factors for HCC survival. This study shows that the determinants of elevated serum VitB(12) levels in HCC patients were in association with malnourishment, liver injuries, and tumor progression. Elevated VitB(12) levels in concurrence with AFP levels serve as the prognostic factors predictive for poor survival of HCC patients.