R.S. Addison

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.

Melphalan uptake, hyperthermic synergism and drug resistance in a human cell culture model for the isolated limb perfusion of melanoma

Isolated limb perfusion with melphalan Is a long-standing treatment for melanoma but the clinical conditions have not been subjected to a systematic evaluation. In order to establish optimal conditions for perfusion, three human melanoma cell lines were cultured with melphalan In vitro under conditions comparable to In vivo therapy. The most important findings were that: (a) 41.5°C was synerglstlc for melphalan killing of three human melanoma cell lines; (b) prolonging the treatment time beyond 1 h had little additional toxiclty; and (c) varying the Initial pH of the culture medium had no effect. After 1 h of treatment, cells accumulated more melphalan at 41.5°C than at 37°C, relative to the extracellular concentration. A cell line (MM418) derived from a primary tumour was the most resistant of the three lines; plgmented or non-plgmented sublines were equally resistant. The A2058 line showed the lowest level of synergism with hyperthermia, and displayed a marked plateau at 10% of controls in the dose-response for survival, yet no melphalan-reslstant subpopulation could be isolated. The implications of this work are that (a) enhanced cellular uptake of melphalan may account for hyperthermic synergism of melphalan; (b) varying conditions other than treatment time will be necessary to deal with the variation In resistance between tumours; and (c) repeated cycles of treatment may be needed for phenotypes such as A2058 where melphalan resistance appears to be based on an epigenetic mechanism.

High-performance liquid chromatographic assay for the measurement of melphalan and its hydrolysis products in perfusate and plasma and melphalan in tissues from human and rat isolated limb perfusions

A sensitive, specific and rapid reversed-phase high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of melphalan and its hydrolysis products in samples from the isolated perfusion of human and rat limbs. Samples of perfusate, plasma and tissue were analysed, following methanol precipitation, using a phenyl column and fluorescence detection. Dansyl-arginine (38 micrograms ml-1) was employed as the internal standard. Good resolution was observed allowing quantitation of melphalan, monohydroxymelphalan (MOH) and dihydroxymelphalan (DOH) in perfusate and plasma were all 100 +/- 10%. The recovery of melphalan in tissue was 93.5%. A linear response was demonstrated for melphalan in the concentration range 1.8 - 56.8 micrograms ml-1, for DOH in the concentration range 0.5 - 30.0 micrograms ml-1 and for MOH in the range 1.4-25.1 micrograms ml-1, in perfusate and plasma. The lower limits of quantitation of melphalan, MOH and DOH in perfusate and plasma were 1.4, 2.4 and 1.2 ng on column, respectively, and 7.2 ng of melphalan on column in tissue. Intra-assay coefficients of variation (C.V.) for melphalan, MOH and DOH, at low and high concentrations were all less than 5% and the inter-assay C.V.s were less than 9%. An ultra-filtration study to determine the protein binding of melphalan and the hydrolysis products showed that the unbound fractions (fu) of melphalan in buffer containing dextran and bovine serum albumin were 0.873 and 0.521, respectively. The assay was used to quantitate melphalan and its hydrolysis products in samples from isolated perfusions in the human limb and rat hindlimb.

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%).

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.

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...

Rapid Determination of Prazosin in Perfusion Media By HPLC With Solid Phase Extraction

Abstract A method is described for the quantitation of prazosin in tissue culture medium used for in vitro perfusion of human placental lobules. Prazosin was extracted using solid phase cartridges and the samples analysed by high performance liquid chromatography. The analysis utilised a C18 reversed-phase column maintained at 40°C with quantitation by fluorescence detection. The assay was linear to 100 ng/mL, intra-assay coefficients of variation measured at concentrations of 5 and 50 ng/mL were 5.8 and 6.2% respectively and inter-assay coefficients of variation were 4.9 and 2.7% for the same concentrations. The mean recovery of prazosin was 90.9 and 85.2% from solutions with concentrations of 5 and 50 ng/mL respectively. The minimum detectable limit was 0.1 ng/mL and the internal standard for this assay was propranolol. Analysis of a range of endogenous and exogenous compounds likely to be present in pregnancy plasma revealed only minor interference from lignocaine.

A Comparison of Parameters Used to Standardize Results From In Vitro Perfusions of Human Placentae

The placenta is a relatively short-lived organ which is required to mimic some of the complex functions of highly specialized organs. Some examples of this include; n n(i) n nTrans-placental gaseous exchanges (oxygen and carbon dioxide) which must occur at rates approaching those exhibited by alveolar tissue in lungs. n n n n n(ii) n nDetoxification of foetal waste-products, in particular various amines associated with developing tissues, thereby mimicking one of the important roles of hepatic tissue. n n n n n(iii) n nSynthesis of a number of pregnancy-specific peptide hormones, such as prolactin and gonadotrophin, and transformation of steroid hormones of maternal origin. These “endocrine-like” activities are essential to survival of the developing foetus and are, indeed, used clinically to monitor foetoplacental function and detect foetal distress. n n n n n(iv) n nTrans-placental solute exchange. Although this exchange does not occur against a concentration gradient, this activity is similar to some aspects of renal function.

An isolated perfused human placental lobule model for multiple indicator dilution studies

We describe a method for multiple indicator dilution studies in the isolated perfused human placental lobule developed to investigate the relationships between changes in pressure and flow and solute clearance. A peripheral lobule of a human placenta is perfused with a tissue culture-based medium and the perfusate oxygen tension, arterial and venous pressures, pH and perfusion temperature continuously monitored by a computerized system. Flow rates are readily changed. Bolus injections of vascular, extracellular and water space markers, and study compounds can be made into either maternal or fetal circulations, and precisely timed outflow fractions can be collected with computer-controlled fraction collectors, allowing simultaneous determination of concentration-time profiles of each marker.

Oxygen Supply and Placental Oxygen Metabolism

There have been numerous previous reports of investigations into the relationship between oxygen supply and oxygen metabolism in human tissues and organs and a variety of animal tissues and organs. Those studies have encompassed both in vivo and in vitro preparations and have included perfusions with whole blood and with artificial perfusion in the presence and absence of synthetic, natural and modified natural oxygen carriers.