Metabolic control analysis (MCA) provides a quantitative description of substrate flux in response to changes in system parameters of complex enzyme systems. Medical applications of the approach include the following: understanding the threshold effect in the manifestation of metabolic diseases; investigating the gene dose effect of aneuploidy in inducing phenotypic transformation in cancer; correlating the contributions of individual genes and phenotypic characteristics in metabolic disease (e.g., diabetes); identifying candidate enzymes in pathways suitable as targets for cancer therapy; and elucidating the function of "silent" genes by identifying metabolic features shared with genes of known pathways. MCA complements current studies of genomics and proteomics, providing a link between biochemistry and functional genomics that relates the expression of genes and gene products to cellular biochemical and physiological events. Thus, it is an important tool for the study of genotype-phenotype correlations. It allows genes to be ranked according to their importance in controlling and regulating cellular metabolic networks. We can expect that MCA will have an increasing impact on the choice of targets for intervention in drug discovery.

To determine mechanisms underlying the transgenerational presence of metabolic perturbations in the intrauterine growth-restricted secondgeneration adult females (F2 IUGR) despite normalizing the in utero metabolic environment, we examined in vivo glucose kinetics and in vitro skeletal muscle postinsulin receptor signaling after embryo transfer of first generation (F1 IUGR) to control maternal environment. Female F2 rats, procreated by F1 pre-and postnatally nutrientand growth-restricted (IUGR) mothers but embryo transferred to gestate in control mothers, were compared with similarly gestating age-and sex-matched control (CON) F2 progeny. Although there were no differences in birth weight or postnatal growth patterns, the F2 IUGR had increased hepatic weight, fasting hyperglycemia, hyperinsulinemia, and unsuppressed hepatic glucose production, with no change in glucose futile cycling or clearance, compared with F2 CON. These hormonal and metabolic aberrations were associated with increased skeletal muscle total GLUT4 and pAkt concentrations but decreased plasma membrane-associated GLUT4, total pPKC, and PKC enzyme activity, with no change in total SHP2 and PTP1B concentrations in IUGR F2 compared with F2 CON. We conclude that transgenerational presence of aberrant glucose/insulin metabolism and skeletal muscle insulin signaling of the adult F2 IUGR female offspring is independent of the immediate intrauterine environment, supporting nutritionally induced heritable mechanisms contributing to the epidemic of type 2 diabetes mellitus. glucose transporter; metabolic imprinting; epigenetic inheritance EPIDEMIOLOGICAL INVESTIGATIONS have linked pre-and postnatal nutrient restriction to adult-onset insulin resistance/type 2 diabetes mellitus, obesity, hypertension, and coronary artery disease (1, 2). Mimicking these conditions, animal models exposing the fetus or newborn to malnutrition in the form of either global (8,24,32) or selective nutrient restriction (6) with concomitant growth restriction predispose the adult offspring toward developing glucose intolerance (8, 24) and insulin resistance of postreceptor insulin-signaling pathways in skeletal muscle (21) and adipose tissue (6). This phenotype of aberrant glucose/insulin homeostasis persists transgenerationally from a gestationally diabetic adult intrauterine growthrestricted (IUGR) mother to the offspring (4). Various investigations have demonstrated a role for diminished pancreatic ␤-cells in type 2 diabetes mellitus as well, an aberration that is passed on transgenerationally (3, 17, 27). Although mutations of genetic loci responsible for insulin production are inherited (33, 34), emerging information suggests epigenetic regulation underlying this transgenerational inheritance pattern (7, 13, 18).In the first-generation (F1) adult female IUGR offspring with pre-and postnatal nutrient restriction, metabolic adaptations concerning glucose/insulin homeostasis consist of a diminution in glucose-induced insulin response with emerging hepatic insulin resistance (8) ...

BACKGROUND: Recent reports have described inherent problems with androgen immunoassays compared with mass spectrometry analyses.

Testosterone (T) and its metabolite dihydrotestosterone (DHT) are androgens with different biologic profiles. T and DHT measurements are required for assessment of patients with ambiguous genitalia, hirsutism, during 5 alpha reductase treatment of prostate disorders, and new androgen formulations. Our laboratory has developed and validated a method to simultaneously measure serum T and DHT with liquid chromatography tandem mass spectrometry (LC-MS/MS) for use in a clinical chemistry laboratory. Analysis of sera from blood collected in tubes containing clot activator gave results of T that were fourfold higher than blood collected in plain tubes. Changing the ion pair selected for monitoring eliminated this interference by clot activators. Blood collected in fluoride-coated tubes gave serum T and DHT levels that were 20 and 15% lower, respectively than levels measured in blood collected in plain tubes (no additives). Addition of T enanthate to blood collected in plain tubes caused a dose related increase serum T levels due to the action of non-specific esterases in the red cells. This esterase activity could be avoided by using fluoride tubes for blood collection. Serum DHT levels were consistently lower when measured by LC-MS/MS versus radioimmunoassay. The differences were concentration dependent and the variance for the difference was large when serum DHT concentration was low. Celite chromatograph prior to radioimmunoassay reduced the differences between the two methods, thus confirming that higher levels of DHT obtained by immunoassays were probably due to interfering substances which were partially removed by Celite chromatography.