Half-Life of Sulfonylureas in HNF1A and HNF4A Human MODY Patients is not Prolonged as Suggested by the Mouse Hnf1a(-/-) Model.

OBJECTIVES: Sulfonylurea derivatives are widely used for clinical treatment of human subjects with Maturity Onset Diabetes of the Young (MODY) caused by mutations in HNF-1α or HNF-4α despite the mechanism leading to their hypersensitivity is incompletely understood. In Hnf1a(-/-) mice, serum concentrations and half-life of sulfonylurea derivatives are strongly increased. We thus hypothesized that reduced sulfonylurea derivatives clearance stands behind their therapeutic potential in human HNF1A/HNF4A MODY subjects. DESIGN AND METHODS: Single doses of 3 mg glipizide and 5 mg glibenclamide/glyburide were administered sequentially to seven HNF1A/HNF4A MODY subjects and six control individuals matched for their age, BMI and CYP2C9 genotype. Pharmacokinetic (plasma concentration levels, Cmax, tmax, t1/2, AUC) and pharmacodynamic parameters (glycemia, C-peptide and insulin plasma levels) were followed for 24 hours after drug administration. RESULTS: We provide the first evidence on the pharmacokinetics and pharmacodynamics of sulfonylurea derivatives in human MODY subjects. The half-life of glipizide did not change, and reached 3.8±0.7 and 3.7±1.8 h in the MODY and control subjects, respectively. The half-life of glibenclamide was increased only in some MODY subjects (t1/2 9.5±6.7 and 5.0±1.4 h, respectively). Importantly, the intra- individual responses of MODY (but control) subjects to glipizide and glibenclamide treatment were highly correlated. With regards to pharmacodynamics, we observed a differential response of control but not MODY subjects to the doses of glipizide and glibenclamide applied. CONCLUSIONS: We rejected the hypothesis that all human MODY-associated mutations in HNF1A / HNF4A induce changes in the pharmacokinetics of sulfonylureas in humans analogically to the Hnf1a(-/-) mouse model.

Clinical manifestation and molecular genetic characterization of MYH9 disorders.

Currently, the May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndrome are considered to be distinct clinical manifestations of a single disease caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). Manifestations of these disorders include giant platelets, thrombocytopenia and combinations of the presence of granulocyte inclusions, deafness, cataracts and renal failure. We examined 15 patients from 10 unrelated families on whom we performed immunostaining of NMMHC-IIA in blood samples. Polymerase chain reaction (PCR) analysis of selected exons of the MYH9 gene revealed mutations in nine samples with one novel mutation. Results of fluorescence and mutational analysis were compared with clinical manifestations of the MYH9 disorder. We also determined the number of glycoprotein sites on the surface of platelets. Most patients had an increased number of glycoproteins, which could be due to platelet size.

High-resolution melting analysis for detection of MYH9 mutations.

May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndromes are rare autosomal dominant disorders with giant platelets and thrombocytopenia. Other manifestations of these disorders are combinations of the presence of granulocyte inclusions and deafness, cataracts and renal failure. Currently, MHA, SBS, FTNS and EPS are considered to be distinct clinical manifestation of a single illness caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). As the MYH9 gene has a high number of exons, it takes much time and material to use this method for the detection of MYH9 mutations. Recently, a new method has been introduced for scanning DNA mutations without the need for direct sequencing: high-resolution melting analysis (HRMA). Mutation detection with HRMA relies on the intercalation of the specific dye (LC Green plus) in double-strand DNA and fluorescence monitoring of PCR product melting profiles. In our study, we optimized the conditions and used HRMA for rapid screening of mutations in all MYH9 exons in seven affected individuals from four unrelated families with suspected MYH9 disorders. Samples identified by HRMA as positive for the mutation were analysed by direct sequencing. HRMA saved us over 85% of redundant sequencing.

Serum adiponectin and resistin concentrations in patients with restrictive and binge/purge form of anorexia nervosa and bulimia nervosa.

To study the role of adipose tissue-derived hormones in the pathophysiology of eating disorders, circulating levels of adiponectin, resistin, and other hormonal and metabolic parameters were measured in 16 females with the restrictive subtype of anorexia nervosa (R-AN), 10 females with the binge/purge subtype of anorexia nervosa (P-AN), 15 females with bulimia nervosa (BN), and 12 age-matched healthy females (C). Body mass index (BMI), body fat content, and serum leptin levels were severely decreased in R-AN and moderately decreased in P-AN patients, whereas the BN group did not differ from C in these parameters. Serum soluble leptin receptor levels were increased in R-AN and P-AN and unchanged in BN patients. Circulating adiponectin levels were inversely related to BMI and were unchanged in BN patients and increased by 53% in P-AN and by 96% in R-AN relative to C group, respectively. In contrast, resistin levels in malnourished R-AN and P-AN were not different from either C or BN groups and showed no significant relationship to BMI or body fat content. We suggest that increased adiponectin levels reflect decreased body fat content in AN patients. In contrast, circulating resistin levels do not appear to be closely related to the nutritional status.

Human cytosolic enzymes involved in the metabolic activation of carcinogenic aristolochic acid: evidence for reductive activation by human NAD(P)H:quinone oxidoreductase.

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Understanding which human enzymes are involved in AA metabolism is important in the assessment of an individual's susceptibility to this carcinogen. Using the 32P-postlabeling assay we examined the ability of enzymes of cytosolic samples from 10 different human livers and from one human kidney to activate the major component of the plant extract AA, 8-methoxy- 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI), to metabolites forming adducts in DNA. Cytosolic fractions of both organs generated AAI-DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(Deoxyadenosin-N6-yl)aristolactam I, 7-(deoxyguanosin-N2-yl)aristolactam I and 7-(deoxyadenosin-N6-yl)aristolactam II, indicating a possible demethoxylation reaction of AAI, were identified as AA-DNA adducts formed from AAI by all human hepatic and renal cytosols. To define the role of human cytosolic reductases in the activation of AAI, we investigated the modulation of AAI-DNA adduct formation by cofactors or selective inhibitors of the NAD(P)H:quinone oxidoreductase (NQO1), xanthine oxidase (XO) and aldehyde oxidase. We also determined whether the activities of NQO1 and XO in different human hepatic cytosolic samples correlated with the levels of AAI-DNA adducts formed by the same cytosolic samples. Based on these studies, we attribute most of the activation of AA in human cytosols to NQO1, although a role of cytosolic XO cannot be ruled out. With purified NQO1 from rat liver and kidney and XO from buttermilk, the major role of NQO1 in the formation of AAI-DNA adducts was confirmed. The orientation of AAI in the active site of human NQO1 was predicted from molecular modeling based on published X-ray structures. The results demonstrate for the first time the potential of human NQO1 to activate AAI by nitroreduction.