The nuclear receptors NUR77, NURR1 and NOR1 in obesity and during fat loss.

BACKGROUND: Adipose tissue is critical for systemic metabolic health. Identifying key factors regulating adipose tissue function is a research priority. The NR4A subfamily of nuclear receptors (NRs) (NR4A1/NUR77, NR4A2/NURR1 and NR4A3/NOR1) has emerged as important proteins in different disease states and in the regulation of metabolic tissues, particularly in liver and muscle. However, the expression of the NR4A members in human adipose tissue has not previously been described, and their target genes are largely unknown. OBJECTIVE: To determine whether the NR4As are differentially expressed in human adipose tissue in obesity, and identify potential NR4A target genes. DESIGN: Prospective analysis of s.c. adipose tissue before and 1 year after fat loss, and during in vitro differentiation of primary human preadipocytes. Case-control comparison of omental (OM) adipose tissue. SUBJECTS: A total of 13 extremely obese patients undergoing biliopancreatic diversion with duodenal switch for fat loss, 12 extremely obese patients undergoing laparoscopic sleeve gastrectomy and 37 lean individuals undergoing hernia repair or laparotomy were included in the study. Measurements were done by quantitative PCR gene expression analysis of the NR4A members and in silico promoter analysis based on microarray data. RESULTS: There was a strong upregulation of the NR4As in extreme obesity and normalization after fat loss. The NR4As were expressed at the highest level in stromal-vascular fraction compared with adipocytes, but were downregulated in both fractions after fat loss. Their expression levels were also significantly higher in OM compared with s.c. adipocytes in obesity. The NR4As were downregulated during differentiation of primary human preadipocytes. Moreover, the NR4As were strongly induced within 30 min of tissue incubation. Finally, promoter analysis revealed potential NR4A target genes involved in stress response, immune response, development and other functions. Our data show altered adipose tissue expression of the NR4As in obesity, suggesting that these stress responsive nuclear receptors may modulate pathogenic potential in humans.

Efficacy of tamoxifen based on cytochrome P450 2D6, CYP2C19 and SULT1A1 genotype in the Italian Tamoxifen Prevention Trial.

The role of pharmacogenomics and tamoxifen was investigated by analyzing several polymorphisms of cytochrome P450 and SULT1A1 gene in a nested case control study from the Italian Tamoxifen Prevention Trial. This study included 182 Caucasian subjects, 47 breast cancer (BC) cases and 135 matched controls. We used the AmpliChip CYP450 Test to screen 33 alleles of CYP2D6 and 3 of CYP2C19. One more variant for CYP2C19*17 and two single-nucleotide polymorphisms for the gene SULT1A1 were also performed. By using the AmpliChip CYP450 Test, out of 182 subjects, we identified 8 poor metabolizer (PM), 17 intermediate metabolizer (IM), 151 extensive metabolizer (EM) and 3 ultrarapid metabolizer (UM). PM women allocated to the tamoxifen arm showed a higher risk of developing BC compared to the remaining phenotypes (P=0.035). In an exploratory analysis, among 58 women with a CYP2D6*2A allele, 9 BCs were diagnosed in the placebo arm and only 1 in the tamoxifen arm (P=0.0001). CYP2C19 and SULT1A1 polymorphisms did not show any correlation with tamoxifen efficacy. Tamoxifen showed reduced efficacy in CYP2D6 PMs in the chemoprevention setting. Conversely, the CYP2D6*2A allele may be associated with increased efficacy of tamoxifen. These findings support the relevance of pharmaco-genomics in tailoring tamoxifen treatment.

Effects of CYP2D6 and SULT1A1 genotypes including SULT1A1 gene copy number on tamoxifen metabolism.

BACKGROUND: Tamoxifen is hydroxylated by cytochrome P450 (CYP) 2D6 to the potent metabolites 4-hydroxytamoxifen (4OHtam) and 4-hydroxy-N-demethyltamoxifen (4OHNDtam), which are both conjugated by sulphotransferase (SULT)1A1. Clinical studies indicate that CYP2D6 and SULT1A1 genotypes are predictors for treatment response to tamoxifen. Therefore, we examined the relationship between CYP2D6 genotype, SULT1A1 genotype, SULT1A1 copy number and the pharmacokinetics of tamoxifen. PATIENTS AND METHODS: The serum levels of tamoxifen and metabolites of 151 breast cancer patients were measured by high-pressure liquid chromatography-tandem mass spectrometry. The CYP2D6 and SULT1A1 polymorphisms and SULT1A1 copy number were determined by long PCR, PCR-based restriction fragment length polymorphism, DNA sequencing and fluorescence-based PCR. RESULTS: The levels of 4OHtam, 4OHNDtam and N-demethyltamoxifen were associated with CYP2D6 predicted enzymatic activity (P < 0.05). The SULT1A1 genotype or copy number did not influence the levels of tamoxifen and its metabolites. However, the ratios of N-demethyltamoxifen/tamoxifen and N-dedimethyltamoxifen/N-demethyltamoxifen were related to SULT1A1 genotype. CONCLUSION: CYP2D6 and SULT1A1 genotypes may partly explain the wide inter-individual variations in the serum levels of tamoxifen and its metabolites. We propose that therapeutic drug monitoring should be included in studies linking CYP2D6 and SULT1A1 genotypes to clinical outcome.

Identification and quantification of tamoxifen and four metabolites in serum by liquid chromatography-tandem mass spectrometry.

We have developed a method for the determination of tamoxifen (tam) and its metabolites 4-hydroxytamoxifen (4OHtam), N-demethyltamoxifen (NDtam), N-dedimethyltamoxifen (NDDtam), tamoxifen-N-oxide (tamNox), and 4-hydroxy-N-demethyltamoxifen (4OHNDtam) in 50 microl human serum. Serum proteins were precipitated with acetonitrile. Deuterated-tamoxifen (D5 tam) was added as internal standard. Sample supernatant was injected into an on-line reversed-phase extraction column coupled with a C18 analytical column and analytes were detected by tandem mass spectrometry. The lower limits of quantification were 0.25 ng/mL for 4OHtam, NDtam and tam, 1.0 ng/mL for NDDtam and tamNox. Ranges of within- and between-day variation were 2.9-15.4% and 4.4-12.9%, respectively.

Tamoxifen administration and metabolism in nude mice and nude rats.

We investigated the kinetics of tamoxifen (tam) in immunodeficient mice and rats after oral treatment and compared drug and metabolite profile in nude rat serum and tissues after oral and subcutaneous (s.c.) routes of administration. The serum levels were compared to those observed in man. After oral dosing in mice, tam and the potent metabolite 4-hydroxytamoxifen (4-hydroxytam), were detectable in liver and lung tissue, but not in serum. The levels of 4-hydroxytam in these tissues were significantly higher than those of tam, a profile opposite to that observed in rat and man. In rats and man, the 4-hydroxytam/tam serum concentration ratios were 0.16 and 0.02, respectively. Compared to oral route, the s.c. pellets yielded only trace amounts of the demethylated derivatives of tam in rats. Thus, the kinetics of tam observed in the present study suggest that the nude rat may represent a preferable animal model in studying the pharmacokinetics of tam and that, the oral route yielded higher serum and tissue levels of tam and metabolites than equivalent s.c. pellet implants.

Thin layer iso-electro focusing of intracellular bacterial protein in classification of some representatives of the families Enterobacteriaceae and Vibrionaceae.

Intracellular bacterial protein from some representatives of the families Enterobacteriaceae and Vibrionaceae has been investigated using thin layer iso-electric focusing in polyacrylamide gel. A marked difference between the protein patterns was observed between species within the same family. Carrying out the iso-electric focusing at pH range 3.5-9 gave a large number of protein bands. Increased resolution of the protein bands was obtained using iso-electric focusing at the pH range 4-6.5. Careful standardization of sample preparation, application and running conditions gave good reproducibility of intracellular proteins from bacterial subcultures.

Fatty acid patterns in the classification of some representatives of the families Enterobacteriaceae and Vibrionaceae.

Twenty-three strains representing the families Enterobacteriaceae and Vibrionaceae were analysed for fatty acid composition of whole cells by means of glass capillary column gas chromatography. Among the several alternatives tested, cluster analysis based on data normalized to hexadecanoate and logarithmically transformed provided good separations of species, genera and families. Strains from the genera Salmonella, Escherichia, Proteus, Enterobacter, Klebsiella, Vibrio and Aeromonas were studied.

[A bacteriological investigation of Norwegian cod liver oil (author's transl)]. / En bakteriologisk undersøkelse av norsk medisintran

Samples of medicinal cod liver oil collected from four of the main producers in Norway have been investigated for content of microorganism. Viable counts for bacteria and fungi were found to be low. No coliform bacteria, coagulase positive Staphylococcus or Pseudomonas could be detected. The findings suggest that the main part of the flora found in cod liver oil belongs to the family Micrococcaceae. Several of the isolated colonies showed lipolytic activity.