Ryuya Horiuchi

Two erbA homologs encoding proteins with different T3 binding capacities are transcribed from opposite DNA strands of the same genetic locus

Two erbA homologs, termed ear-1 and ear-7, are present in the human genome on chromosome 17. The two genes reside in the same genetic locus with overlapping exons and are transcribed from opposite DNA strands. In addition, the ear-7 mRNA is alternatively spliced to generate two protein isoforms, namely the ear71 and ear72 proteins. Nucleotide sequence analysis predicts that the ear71 protein is a human counterpart of the chicken c-erbA protein, a molecule closely related or identical to thyroid hormone receptor. Indeed, Scatchard analysis of proteins synthesized in vitro indicated very high affinity binding of T3 to the ear71 protein but not to the ear72 protein. Interestingly, the ear-1 gene product showed low, but appreciable, binding to T3, although its authentic ligand remains to be clarified.

VKORC1 gene variations are the major contributors of variation in warfarin dose in Japanese patients

To compare the genetic and clinical factors that cause large interpatient variability and ethnic differences in warfarin efficacy, we investigated variations of the VKORC1, CYP2C9, and CYP2C19 genes in Japanese subjects. Furthermore, we evaluated the genetic variations and clinical data as contributors of variation in warfarin maintenance dose.

Changes in Drug Plasma Concentrations of an Extensively Bound and Highly Extracted Drug, Propofol, in Response to Altered Plasma Binding

Cardiopulmonary bypass is known to result in a reduction in the plasma binding of drugs. The resulting effect on the hepatic clearance of drugs with low extraction is well understood. However, the situation with those that are highly extracted is less clear. Studies were, therefore, undertaken with one such drug, propofol, for which plasma binding was changed during cardiac surgery with cardiopulmonary bypass.

Genetic polymorphism of CYP2C9 and CYP2C19 in a Bolivian population: an investigative and comparative study

ObjectiveSeveral reports of CYP2C genetic polymorphism demonstrate its potential clinical role in determining both inter-individual and inter-ethnic differences in drug efficacy. We estimated the distribution of CYP2C9 and CYP2C19 common variants in the Bolivian population (a South American population), and compared these data with those from Asian, African, Caucasian and Oceanian populations.MethodsGenomic DNA was obtained from 778 unrelated healthy volunteers from Bolivia. The genotypic status of CYP2C9 and CYP2C19 was determined by means of polymerase chain reaction–restriction fragment length polymorphism.ResultsAllelic and genotypic frequencies of CYP2C9 and CYP2C19 were determined for the Bolivian population, and comparison of the data with other ethnic groups revealed a lower CYP2C9*2 frequency (4.8%) than in Caucasians, but a higher frequency than in Asians; frequencies of CYP2C9*3 (3.0%) and CYP2C9 (0.4%) poor metabolizers (PMs) were similar to those seen in Asian populations. Frequencies of CYP2C19*2 (7.8%), CYP2C19*3 (0.1%), and CYP2C19 PMs (1.0%) in the Bolivian population were for the most part lower than in Caucasian, Asian, Oceanian and African populations.ConclusionThis is the first study to investigate a South American population for genetic polymorphism in the CYP2C subfamily. The Bolivian population differs from most other ethnic groups in the incidence of CYP2C9 and CYP2C19 common variants that might be influenced by its admixture characteristics.

Human Kidneys Play an Important Role in the Elimination of Propofol

Background:Extrahepatic clearance of propofol has been suggested because its total body clearance exceeds hepatic blood flow. However, it remains uncertain which organs are involved in the extrahepatic clearance of propofol. In vitro studies suggest that the kidneys contribute to the clearance of this drug. The purpose of this study was to confirm whether human kidneys participate in propofol disposition in vivo. Methods:Ten patients scheduled to undergo nephrectomy were enrolled in this study. Renal blood flow was measured using para-aminohippurate. Anesthesia was induced with vecuronium (0.1 mg/kg) and propofol (2 mg/kg) and then maintained with nitrous oxide (60%), sevoflurane (1∼2%) in oxygen, and an infusion of propofol (2 mg · kg−1 · h−1). Radial arterial blood for propofol and para-aminohippurate analysis was collected from a cannula inserted in the radial artery. The renal venous sample and the radial arterial sample were obtained at the same time after the steady state of propofol was established. Results:The renal extraction ratio of propofol was 0.58 ± 0.15 (mean ± SD). The renal clearance of propofol was 0.41 ± 0.15 l/min (mean ± SD), or 27 ± 9.9% (mean ± SD) of total body clearance. Conclusion:Human kidneys play an important role in the elimination of propofol.

Rapid Single-Nucleotide Polymorphism Detection of Cytochrome P450 (CYP2C9) and Vitamin K Epoxide Reductase (VKORC1) Genes for the Warfarin Dose Adjustment by the SMart-Amplification Process Version 2

BACKGROUND Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2, CYP2C9*3) and the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (-1639G>A) greatly impact the maintenance dose for the drug warfarin. Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes. With current methodologies, therapy can be delayed by several hours to 1 day if genotyping is to determine the loading dose. A simpler and more rapid genotyping method is required. METHODS We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C9*2, CYP2C9*3, and VKORC1 -1639G>A polymorphisms. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 1 h after blood collection. RESULTS We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance. CONCLUSIONS The results validate the accuracy of the SMAP 2 for determination of SNPs critical to personalized warfarin therapy. SMAP 2 offers speed, simplicity of sample preparation, the convenience of isothermal amplification, and assay-design flexibility, which are significant advantages over conventional genotyping technologies. In this example and other clinical scenarios in which genetic testing is required for immediate and better-informed therapeutic decisions, SMAP 2-based diagnostics have key advantages.

Defective Activity of Recombinant Cytochromes P450 3A4.2 and 3A4.16 in Oxidation of Midazolam, Nifedipine, and Testosterone

Cytochrome P4503A4 (CYP3A4) is the most abundant cytochrome P450 in adult human liver and small intestine and oxidizes numerous clinically, physiologically, and toxicologically important compounds. The metabolic activity of CYP3A4 in patients varies at least 10-fold in vivo, and CYP3A4 genetic variants are considered one of the causes of individual differences. The cDNAs for the CYP3A4*2 (S222P), *7 (G56D), *16 (T185S), and *18 (L293P) mutant alleles, found in high frequencies in Caucasians or Asians, were constructed by site-directed mutagenesis and expressed in an Escherichia coli expression system. Midazolam (MDZ), testosterone (TST), and nifedipine (NIF) were used to assess the catalytic activities of the CYP3A4 wild type (CYP3A4.1) and its variants. The catalytic activities of CYP3A4.2 and CYP3A4.16 were reduced (lower Vmax and increased Km relative to CYP3A4.1) for all substrates. The CYP3A4.7 showed lower Vmax values for MDZ and NIF (60 and 84%, respectively) and a higher Km (2-fold) for TST but not for MDZ or NIF. Although CYP3A4.18 showed low Vmax values for MDZ, NIF, and TST (88, 72, and 80% of CYP3A4.1, respectively), no significant differences were identified in the ratio Vmax/Km. In summary, CYP3A4.2 and CYP3A4.16 exhibited significantly lower activity for MDZ, TST, and NIF oxidations than CYP3A4.1. Therefore, drugs metabolized by only CYP3A should be carefully administered to patients with these alleles.

Prediction of total propofol clearance based on enzyme activities in microsomes from human kidney and liver

ObjectivePropofol is commonly used for anesthesia and sedation in intensive care units. Approximately 53% of injected propofol is excreted in the urine as the glucuronide and 38% as hydroxylated metabolites. Liver, kidneys and intestine are suspected as clearance tissues. We investigated the contribution of the liver and kidneys to propofol metabolism in humans using an in vitro–in vivo scale up approach.MethodsRenal tissue was obtained from five patients who received nephrectomies. Each renal hydroxylation and glucuronidation enzymatic activities in microsomal fractions from patients were performed discretely and their estimation based on the decrease of propofol concentration. Hepatic hydroxylation and glucuronidation activities were also performed separately using human liver microsomes. This estimation is based on the decrease of propofol concentration, assuming that the contribution of hydroxylation activity without NADPH-generating system and glucuronidation activity without UDPGA in each microsomal fraction are negligible. Both renal and hepatic clearances were estimated assuming a well-stirred model.ResultsEnzymatic activity of propofol oxidation in renal microsomes was negligible. Although glucuronidation activity in microsomes from kidneys was comparable to that from liver, the hepatic intrinsic clearance predicted from in vitro study was higher than that in kidneys due to the larger tissue volume and higher protein concentration. However, glucuronidation clearance in kidney is relatively similar to that in liver because of blood flow limitation of clearance in both tissues.ConclusionThe high degree of hydroxylation activity in liver microsomes is consistent with the blood flow-limited hepatic clearance of propofol. Although the activity of propofol glucuronidation in liver is higher, glucuronidation in kidney may be a substantial contributor.

Systemic hyalinosis or juvenile hyaline fibromatosis

SummaryBiochemical examinations of a hyalinized skin tumor of systemic hyalinosis (juvenile hyaline fibromatosis) revealed an increase in chondroitin sulfate synthesis by skin fibroblasts with an enlarged, rough surfaced endoplasmic reticulum as well as an increase in chondroitin sulfate content. These results indicate that systemic hyalinosis is a connective tissue disorder characterized by abnormal GAG synthesis of fibroblasts.ZusammenfassungDurch biochemische Untersuchungen von hyalinisierten Hauttumoren der systematisierten Hyalinose (juvenile hyaline Fibromatose) zeigte sich eine gesteigerte Chondroitinsulfatsynthese von dermalen Fibroblasten, die sich in einem erweiterten rauhen endoplasmatischen Reticulum darstellt, sowie eine Steigerung des Chondroitinsulfat-Gehaltes im befallenen Hautbereich. Diese Ergebnisse weisen auf, daß es sich bei der systematisierten Hyalinose um eine Bindegewebserkrankung handelt, die durch eine abnorme Glykosaminoglykan-Synthese der Fibroblasten charakterisiert ist.

Identification of protein disulfide isomerase and calreticulin as autoimmune antigens in LEC strain of rats.

Long Evans Cinnamon (LEC) rats, showing spontaneous hereditary hepatitis and hepatic carcinoma, were found to possess autoimmune antibodies to liver microsomal proteins, particularly to proteins with the molecular weight of 56kD and 55kD. The antibodies occurred in association with acute lethal hepatitis in the LEC rats in our previous study. Two-dimensional immunoblot analysis of the antigenic proteins revealed that the 56kDa and 55kDa proteins showed 4.2 and 4.0 pI values and were estimated to be protein disulfide isomerase (PDI) and calreticulin, respectively, from NH2-terminal amino acid sequence analysis. These proteins were further identified by immunoblot analyses using purified proteins and specific antibodies. PDI was a major autoimmune antigenic protein.