David James Maguire

Metabolism of prednisolone by the isolated perfused human placental lobule

Previous studies of the metabolism of 11 beta-hydroxy corticosteroids by placental tissue have indicated that the only product is the C11-oxidized metabolite. In the present study we have re-examined the metabolism of prednisolone in the isolated, perfused, dual recirculating human placental lobule, using a perfusate based on tissue culture medium 199. Four metabolites were identified in both the maternal and fetal compartments in 6 h perfusions by comparison of relative retention times measured by HPLC and capillary gas chromatography (GC) and of mass spectra recorded by capillary gas chromatography-mass spectrometry (GC-MS) with those of authentic reference standards. The steroids were derivatized as the MO-TMS ethers for mass spectral measurements. Analysis of samples from five perfusion experiments resulted in the following percentage conversions after 6 h perfusion (mean +/- SD, maternal and fetal perfusate, respectively): prednisone (49.1 +/- 7.8, 49.1 +/- 6.6), 20 alpha-dihydroprednisone (0.84 +/- 0.29, 0.81 +/- 0.35), 20 beta-dihydroprednisone (39.1 +/- 6.7, 39.2 +/- 5.9), 20 beta-dihydroprednisolone (6.8 +/- 2.7, 6.3 +/- 1.6) and unmetabolized prednisolone (4.1 +/- 1.8, 4.6 +/- 2.1). No evidence was found for metabolites formed by 6 beta-hydroxylation or cleavage of the C17-C20 bond.

Replication of Murine Mitochondrial DNA Following Irradiation

The effect of radiation on the mitochondrial genome in vivo is largely unknown. Though mitochondrial DNA (mtDNA) is vital for cellular survival and proliferation, it has little DNA repair machinery compared with nuclear DNA (nDNA). A better understanding of how radiation affects mtDNA should lead to new approaches for radiation protection. We have developed a new system using real-time PCR that sensitively detects the change in copy number of mtDNA compared with nDNA. In each sample, the DNA sequence coding 18S rRNA served as the nDNA reference in a run simultaneously with a mtDNA sequence. Small bowel collected 24 hours after 2 Gy or 4 Gy total body irradiation (TBI) exhibited increased levels of mtDNA compared with control mice. A 4 Gy dose produced a greater effect than 2 Gy. Similarly, in bone marrow collected 24 hours after 4 Gy or 7 Gy TBI, 7 Gy produced a greater response than 4 Gy. As a function of time, a greater effect was seen at 48 hours compared with 24 hours. In conclusion, we found that radiation increased the ratio of mtDNA:nDNA and that this effect seems to be tissue independent and seems to increase with radiation dose and duration following radiation exposure.

Oxygen transport to tissue XIII

Oxygen Transport Models: Modeling of Oxygen Transport to Skeletal Muscle J. Piiper The Haldane Effect of Rabbit Blood Under Different Acid-Base Conditions H. Kiwull-Schoone, et al. Methods and Instrumentation: A New Catheter for QuasiContinuous Measurement of Arterial Partial Oxygen Pressure E.P. Eijking, et al. Free Radicals: Cytochrome P450 Under Conditions of Oxidative Stress E. Serbinova, et al. Cardiovascular and Respiratory Systems: Estimation of Respiratory Mechanics in Dogs with Acute Lung Injury L.F. Zhang, et al. Tumors: Oxygenation of Mammary Tumors P. Vaupel, et al. Shock and Wound Healing: Impact of Ischemia on Tissue Oxygenation and Wound Healing M. Kamler, et al. Other Organs and Tissues: Human Placental Oxygen Metabolism D.J. Maguire, et al. 46 additional articles. Index.

Pathway and kinetics of prednisolone metabolism in the human placenta

Prednisolone is metabolized in the perfused human placental lobule to prednisone, 20 alpha-dihydroprednisone, 20 beta-dihydroprednisone and 20 beta-dihydroprednisolone. The pathway of metabolite formation was defined in perfusions of placental lobules using prednisone and 20 beta-dihydroprednisone separately as substrates and with prednisolone co-perfused with glycyrrhetinic acid, a potent inhibitor of the 11-oxidase component of the 11 beta-hydroxysteroid dehydrogenase enzyme system. The pattern of metabolites identified from 6 h samples indicated a reversible formation of prednisone from prednisolone, the production of the 20 alpha- and 20 beta-dihydro metabolites of prednisone from prednisone, the formation of 20 beta-dihydroprednisolone from 20 beta-dihydroprednisone only and no direct formation of 20 beta-dihydroprednisolone from prednisolone. Kinetic analysis at two substrate concentrations confirmed that the formation of three of the four steroid metabolites followed first order kinetics. In perfusions with an initial prednisolone concentration of 1 microgram/ml (n = 4) or 100 ng/ml (n = 3), the rate constants obtained were (mean +/- SD, maternal compartment, h-1): prednisone, 1.97 +/- 0.49 and 2.25 +/- 0.15, P > 0.1; 20 alpha-dihydroprednisone, 0.0006 +/- 0.0004 and 0.0017 +/- 0.0006, P < 0.1; 20 beta-dihydroprednisone, 0.15 +/- 0.022 and 0.15 +/- 0.0077, P > 0.1. In contrast, the rate constant for formation of 20 beta-dihydroprednisolone at an initial prednisolone concentration of 100 ng/ml (0.083 +/- 0.0095 h-1) was significantly (P < 0.01) greater than the corresponding rate constant at the higher initial prednisolone concentration (0.039 +/- 0.015 h-1). A significant increase (P < 0.05) was observed for the formation of 20 beta-dihydroprednisolone at the end of 6 h perfusions at the lower initial substrate concentration (11.2 +/- 1.9%) compared with the 1 microgram/ml concentration (6.0 +/- 2.5%).

Alanine in HI: A Silent Mutation Cries Out!

It is a widely held paradigm in molecular biology that a change in the third base of a codon is silent in terms of expression. In this investigation, results are presented that challenge that paradigm, at least in terms of one polymorphism in KCNJ11, which is one of five genes that have been implicated in the disorder Hyperinsulinism of Infancy. In two cohorts of Australian patients, an uneven distribution of KCNJ11 SNPs was observed. A silent polymorphism at codon 190 was over-represented in the patients who responded well to medical treatment and under-represented in those that required radical surgical intervention. In an attempt to investigate this polymorphism, it was expressed in vitro and western blot analysis showed that there were virtually no bands from the homozygous variant samples, while strong bands were seen in normal controls. The human genome is highly redundant in terms of tRNA species for each amino acids but enigmatically under-represents a number of specific codons. The polymorphism in question occurs within one such codon. We propose that the presence of a base change at the third position of codon that is not represented by a corresponding anti-codon within the human nuclear tRNA leads to a decreased rate of expression of the protein.

Liquid-Chromatographic Analysis of Prednisolone, Prednisone and Their 20-Reduced Metabolites in Perfusion Media

A reversed-phase liquid chromatographic assay was developed to quantitate prednisolone, prednisone and the 20 alpha-dihydro and 20 beta-dihydro reduced metabolites of both parent compounds in tissue culture media from in vitro perfusions of the human placental lobule. Steroids were extracted from perfusate, using reversed-phase cartridges, with average recoveries of 95.2% or greater. The internal standard for the analyses was 6 alpha-methylprednisolone. In this assay cortisol coelutes with prednisolone, however, no other significant interferences were found. Assay of each steroid was linear in the range 0-1 microgram/ml. Intra-assay coefficients of variation were measured at 10 and 750 ng/ml with ranges of 3.4% (20 alpha-dihydroprednisone) to 8.8% (20 beta-dihydroprednisolone) and 4.1% (20 beta-dihydroprednisone) to 8.8% (prednisone). The corresponding inter-assay coefficients of variation were 3.3% (20 alpha-dihydroprednisone) to 9.1% (20 beta-dihydroprednisolone) and 1.9% (prednisolone) to 3.5% (prednisone). The analyses utilized two C18 columns which were linked together and maintained at 40 degrees C.

Assaying ATP Synthase Rotor Activity

With the development of laser excitation and detection systems, it has become possible to consider analyses that have otherwise been beyond the capacity of experimentalists. In this investigation, a case is made for the development of a device to analyse ATP synthase activity in-vivo. Such analyses ultimately have applications in clinical practice, particularly in the field of mitochondrial disorders, of both nuclear and mitochondrial DNA origin.

High Performance Liquid Chromatographic Separation of Cortisol, Cortisone, and Their 20–Reduced Metabolites in Perfusion Media

Abstract A reversed-phase liquid chromatographic assay to quantitate cortisol, cortisone and their respective 20α- and 20β-dihydro reduced metabolites in tissue culture media from in vitro perfusions of the human placental lobule is described. The internal standard used in this assay was 6α-methyl-prednisolone. Steroids were extracted from the perfusion medium using Sep-Pak reversed-phase cartridges with the average recoveries of each steroid at 150 and 600 nmol/L ranging from 84.4 to 99.1% and 85.6 to 93.5% respectively. The separation was achieved by using two C18 columns linked in series at 40°C with a mobile phase of methanol/water (53/47 v/v) and a flow rate of 1.1 mL/min. The eluant was monitored by UV absorption at 242 nm. The assay was linear for each steroid to a concentration of 750 nmol/L with a lower detectable limit of 5 nmol/L. Intra-assay coefficients of variation were measured at 150 and 750 nmol/L with ranges of 4.0% (cortisone) to 5.5% (cortisol) and 2.8% (cortisol) to 4.0% (cortisone an...

The Anomalous Einstein-Stokes Behaviour of Oxygen and Other Low Molecular Weight Diffusants

Almost a century ago, Einstein and Sutherland independently derived equations that describe the relationship between diffusion of solutes and the molecular parameters of those solutes. In that time it has been recognized that, although the equations adequately describe the diffusion of large and medium-sized molecules, there is deviation from this relationship for small molecules. Many authors have attempted to redefine the equations for diffusion, with varying degrees of success, but generally have not attempted to consider the fundamental events that may be occurring at the molecular level during the diffusion of small molecules. In this presentation, we attempt to provide such an explanation, particularly with respect to the diffusion of oxygen through water. We consider the possibility of a random rotational model that complements the (slower) translational process of traditional diffusion and thereby provides accelerated diffusion of small molecules. It is hoped that our description of this model may provide a basis for the development of mathematical modelling of the process.

The role of atp sensitive channels in insulin secretion and the implications in persistent hyperinsulinemic hypoglycaemia of infancy (PHHI)

Persistent Hyperinsulinemic Hypoglycaemia of Infancy (PHHI) is a metabolic syndrome of unregulated insulin secretion. It is a heterogenous disease with causes linked to mutations of the ATP sensitive potassium channels of the beta cell, as well as to metabolism in the beta cell. 5 candidate genes--ABCC8, KCNJ11, GCK, GLUD1 and SCHAD have been implicated in the disease so far, however the aetiology of the disease remains unknown in up to 50% of all patients. We genotyped 43 subjects with PHHI (20 surgically treated and 23 medically treated) for disease associated mutations in the candidate genes. Mutations on ABCC8 were identified in 16 of the 20 (80%) of the surgically treated patients. One putative mutation was identified in the medically treated cohort. The polymorphism E23K on KCNJ11 that is associated with NIDDM was differentially distributed in the 2 cohorts. We discuss the mutations identified, emphasise the importance of the K-ATP channel in physiological processes and discuss the possibility that the disease is caused by mutations in other genes associated with insulin release, glucose metabolism in the beta cell or beta cell apoptosis and survival. We propose that these processes must be explored in order to further our understanding of PHHI.