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Biomedical subjects

J D Stuart

Publications and source records attributed to J D Stuart.

At least 19 recordsLinked to original sources
PubMed2001

Pharmacokinetic parameters and mechanisms of inhibition of rat type 1 and 2 steroid 5alpha-reductases: determinants for different in vivo activities of GI198745 and finasteride in the rat.

The interaction of baculovirus expressed rat steroid 5alpha-reductase types 1 and 2 (r5AR1 and r5AR2) with 17beta-N-(2,5-bis(trifluoromethyl)phenyl)carbamoyl-4-aza-5alpha-androst-1-en-3-one (GI198745) was investigated at pH 7 and 37 degrees. This 5alpha-reductase inhibitor was found previously to be a time-dependent inhibitor of the two human 5alpha-reductase isozymes. In contrast, we demonstrate in the present study that although GI198745 is a potent time-dependent inhibitor of r5AR2, it is a classical rapid-equilibrium inhibitor of r5AR1. This type of behavior with human and rat 5alpha-reductases has been shown for the inhibitor 17beta-(N-tert-butylcarbamoyl)-4-aza-5alpha-androst-1-en-3-one (finasteride), a current therapy for benign prostatic hyperplasia. Inhibition of r5AR1 by GI198745 was competitive with testosterone and followed Michaelis-Menten kinetics with a K(i) value of 0.3 +/- 0.02 nM. Data for the inhibition of r5AR2 by GI198745 were consistent with a two-step mechanism, where K(i) is the dissociation constant for an initial enzyme-inhibitor complex and k(3) is the rate constant for the second slow step. The pseudo-bimolecular rate constant (k(3)/K(i)) for the association of GI198745 with r5AR2 was (2.0 +/- 0.4) x 10(7) M(-1) sec(-1). The high affinity of this inhibitor for r5AR2 was further demonstrated by the inability of the enzyme-inhibitor complex to dissociate after approximately 7 days of dialysis at 4 degrees. Both GI198745 and finasteride appear to inactivate r5AR2 by apparent irreversible modification, but are classical, reversible inhibitors of r5AR1. Therefore, we hypothesize that because of its pharmacokinetic parameters and increased potency against r5AR1, GI198745 is more effective than finasteride in preventing the growth of the rat prostate.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed2001

A water extraction, static headspace sampling, gas chromatographic method to determine MTBE in heating oil and diesel fuel.

A method was developed to determine the fuel/water partition coefficient (KMTBE) of methyl tert-butyl ether (MTBE) and then used to determine low parts per million concentrations of MTBE in samples of heating oil and diesel fuel. A special capillary column designed for the separation of MTBE and to prevent coelution and a gas chromatograph equipped with a photoionization detector (PID) were used. MTBE was partitioned from fuel samples into water during an equilibration step. The water samples were then analyzed for MTBE using static headspace sampling followed by GC/PID. A mathematical relationship was derived that allowed a KMTBE value to be calculated by utilizing the fuel/water volume ratios and the corresponding PID signal. KMTBE values were found to range linearly from 3.8 to 10.9 over a temperature range of 5-40 degrees C. This analysis method gave a MDL of 0.7 ppm MTBE in the fuel and a relative average accuracy of +/-15% by comparison with an independent laboratory using purge and trap GC/ MS analysis. MTBE was found in home heating oil in residential tanks and in diesel fuel at service stations throughout the state of Connecticut. The levels of MTBE were found to vary significantly with time. Heating oil and diesel fuel from terminals were also found to contain MTBE. This research suggests thatthe reported widespread contamination of groundwater with MTBE may also be due to heating oil and diesel fuel releases to the environment. used extensively for the past 20 years as a gasoline additive (up to 15 wt %) to reduce automobile carbon monoxide and hydrocarbon emissions. The fact that MTBE is highly soluble in water (approximately 5 wt %) (3) and chemically inert when compared to other fuel constituents causes it to be often detected at high concentrations in groundwater in the vicinity of gasoline spills. The EPA has reported that low levels of MTBE in drinking water (above 40 microg/L) may cause unpleasant taste and odors and has designated MTBE as a possible human carcinogen (4). Past studies have concentrated on the reporting of MTBE levels in groundwater near gasoline spills. Happel et al. reported an MTBE occurrence rate of approximately 78% at locations where hydrocarbons have impacted groundwater (5). Johnson et al. estimate that 9,000 leaking underground fuel tanks have caused MTBE contamination at community water supplies in the 31 states surveyed (excluding California and Texas) (6). Robbins et al. reported finding a significant number of MTBE detections in groundwater samples taken at sites in Connecticut known to be contaminated by heating oil spills (7). Later, this same research group reported finding MTBE contamination to range from 9.7 to 906 mg/L in heating oil and from 74 to 120 mg/L in diesel fuel in samples collected from storage tanks in Connecticut (8). The method used to analyze these samples was based on fuel-water partitioning and GC analysis. This present study provides the detailed basis for that analytical method. MTBE fuel-water partition coefficients as a function of temperature, which are critical to the method, are also presented. This study also reports on variations in MTBE levels as a function of time observed at several residences and a service station. Analytical results are reported for samples taken from terminals as part of an effort to assess the sources of MTBE in heating oil and diesel fuel.

Carcinogens↗
PubMed2000

Detection of chemically induced DNA damage by derivative square wave voltammetry.

Damage of DNA films after reaction with styrene oxide was detected using derivative square wave voltammetry. Double-stranded (ds) DNA films with initially low backgrounds developed oxidation peaks for DNA bases during incubation with styrene oxide. Films were prepared on pyrolytic graphite (PG) electrodes by casting mixtures of DNA with the poly(ester sulfonic acid) ionomer Eastman AQ38S or by covalent binding of DNA onto oxidized PG. While both types of films gave oxidation peaks in the region 0.6-1.1 V vs SCE after incubations with styrene oxide, DNA/AQ films gave the best signal-to-background ratios. Damage of DNA by reaction with styrene oxide under the electrode incubation conditions was confirmed by capillary electrophoresis. Total integrals of oxidation peaks increased with time of incubation with styrene oxide. Relative peak heights depended on the type of DNA in the order calf thymus ds DNA > salmon sperm ds DNA > supercoiled ds DNA > highly polymerized calf thymus ds DNA.

Animals↗
PubMed1996

Inhibition of human steroid 5alpha reductases type I and II by 6-aza-steroids: structural determinants of one-step vs two-step mechanism.

We have discovered that 17beta-[N,N-(diethyl)carbamoyl]-6-azaandrost-4-en-3-one is a time-dependent inhibitor of type II 5alpha-reductase, as is the drug finasteride. Unlike finasteride, the 6-aza-steroid is not a time-dependent inhibitor of type I 5 alpha-reductase. Finasteride inhibition of type II enzyme proceeds in a two-step mechanism. At pH 6 and 37 degrees C, an initial finasteride-reductase complex is formed with a K(i)(app) of 11.9 +/- 4.1 nM. In a second step, an irreversible complex is formed with a rate constant of inactivation of 0.09 +/- 0.01 s(-1). In contrast, the 6-aza-steroid is a reversible inhibitor. From the results of a simplified mathematical analysis, based on the rapid equilibrium approximation, the inhibitor and the enzyme form an initial complex with a K(i) of 6.8 +/- 0.2 nM. The reversible formation of a final complex, with an overall K(i) of 0.07 +/- 0.02 nM, is characterized by a first-order isomerization rate constant 0.0035 +/- 0.0001 s(-1) for the forward step and 0.00025 +/- 0.00006 s(-1) for the backward step. All rate constants for the two-step mechanism were obtained by using a general numerical integration method. The best fit values for the association and dissociation rate constants were 5.0 microM(-1) s(-1) and 0.033 +/- 0.008 s(-1), respectively, and the isomerization rate constants were 0.0035 +/- 0.007 s(-1) and 0.000076 +/- 0.000019 s(-1). These values correspond to an initial K(i) of 6.5 nM and an overall dissociation constant of 0.14 nM. The data presented here show that both finasteride and the 6-aza-steroid analogs are potent against type II 5alpha-reductase, although their mechanisms of inhibition are different.

5-alpha Reductase Inhibitors↗
PubMed1996

Field examination of ground water quality as an indicator of microbiological activity at gasoline contaminated sites.

Various portable electrodes and an on-line colorimetric test kit were used in the field to examine ground water quality as an indicator of natural bioremediation across two sites in Connecticut having subsurface gasoline contamination. The parameters examined included dissolved oxygen, dissolved carbon dioxide, direct redox potential (Eh), nitrate, ammonia and pH. These parameters permitted delineating regions of aerobic and anaerobic microbiological activity. Variations in these parameters over an eighteen month period along with gas chromatographic analyses of certain gasoline components in the ground water indicated that in-situ bioremediation was effective at containing the petroleum contamination at both sites. It was found that a new on-line colorimetric test kit for the determination of oxygen was more accurate than a commonly used dissolved oxygen electrode.

Ammonia↗
PubMed1996

Synthesis of 4,17-diazasteroid inhibitors of human 5 alpha-reductase.

The synthesis of the 17-aza isomer of finasteride is described. With the side chain amide group of the compound existing in the Z configuration the structure is similar to one of the two favored conformations of finasteride. A series of 4,17-diazasteroids was assayed against the isoenzymes of human 5 alpha-reductase.

5-alpha Reductase Inhibitors↗
PubMed1995

Structure-activity relationships for inhibition of type 1 and 2 human 5 alpha-reductase and human adrenal 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase by 6-azaandrost-4-en-3-ones: optimization of the C17 substituent.

A variety of C17 amide-substituted 6-azaandrost-4-en-3-ones were prepared and tested versus human type 1 and 2 steroid 5 alpha-reductase (5AR) and human adrenal 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase (3BHSD) in order to optimize potency versus both isozymes of 5AR and selectivity versus 3BHSD. Two series of potent and selective C17 amides were discovered, 2,5-disubstituted anilides and (arylcycloalkyl)amides. Compounds from each series with picomolar IC50's versus human type 2 5AR and low nanomolar to picomolar IC50's versus human type 1 5AR possessing 100-500-fold selectivity versus 3BHSD were identified. A conformational model to predict 3BHSD potency was developed which could rationalize 3BHSD potency within three different series of compounds. Evaluation of some optimal compounds from this series in a chronic castrated rat model of 5AR inhibitor induced prostate involution, and pharmacokinetic measurements identified compounds (9, 12, 16, and 29) with good in vivo efficacy and half-life in the dog. An intact rat model of in vivo selectivity for 5AR versus 3BHSD inhibition was also developed. Dual inhibitors of both human 5AR's may show advantages over type 2 selective 5AR inhibitors, such as finasteride (1), in the treatment of disease states which depend upon dihydrotestosterone.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

17 beta-(N-tert-butylcarbamoyl)-4-aza-5 alpha-androstan-1-en-3-one is an active site-directed slow time-dependent inhibitor of human steroid 5 alpha-reductase 1.

17 beta-(N-tert-butylcarbamoyl)-4-aza-5 alpha-androstan-1-en-3-one (finasteride), which has been approved for treatment of benign prostatic hyperplasia, is shown here to be a slow time-dependent inhibitor of human steroid 5 alpha-reductase isozyme 1. This inhibition is characterized by an initial, fast step where the inhibitor binds to the enzyme followed by a slow step that leads to a final enzyme-inhibitor complex (EI*). No recovery of activity from this EI* complex was observed after dialysis for 3 days. The formation of EI* is diminished in the presence of a competitive, reversible inhibitor, indicating that the inhibition is active site-directed. At 37 degrees C and pH 7.0, the rate constant for the second, slow inhibition step, k3, is (1.40 +/- 0.04) x 10(-3) s-1 and the pseudo-bimolecular rate constant, k3/Ki, is (4.0 +/- 0.3) x 10(3) M-1 s-1. This latter rate constant is less than the value of 2.7 x 10(5) M-1 s-1 determined for the inhibition of 5 alpha-reductase 2 by finasteride [Faller, B., Farley, D., & Nick, H. (1993) Biochemistry 32, 5705-5710].(ABSTRACT TRUNCATED AT 250 WORDS)

5-alpha Reductase Inhibitors↗
PubMed1994

Effect of the plant compound indole-3-carbinol on hepatic cholesterol homoeostasis.

The aim of this study was to elucidate the effects of the compound indole-3-carbinol (I3C), which is found in cruciferous vegetables, on hepatic cholesterol homoeostasis and metabolism in male CD-1 mice. Oral administration of 500 and 750 mg I3C/kg/day to mice for 1 wk resulted in increased liver mass and microsomal protein content. Hepatic microsomal cholesterol levels were not significantly altered following treatment with 100 and 250 mg I3C/kg/day, but were significantly decreased following treatment with 500 and 750 mg/kg/day. Conversely, the lower doses of I3C administered decreased serum cholesterol levels whereas the higher doses of I3C had no effect on this parameter. Alterations in cholesterol homoeostasis by I3C were not related to liver hypertrophy, since administration of phenobarbital to mice increased liver size, but had no significant effect on hepatic microsomal or serum cholesterol levels. Activities of the hepatic enzymes cholesterol ester hydrolase and cholesterol 7 alpha-hydroxylase were not altered by I3C. However, 500 and 750 mg I3C/kg/day elevated the activity of hepatic acyl-CoA:cholesterol acyltransferase (ACAT), the enzyme responsible for the formation of hepatic cholesteryl esters. These results demonstrate that (a) I3C lowers serum cholesterol levels at concentrations that have no discernible effect on hepatic cholesterol homoeostasis, and (b) at higher doses of I3C, hepatic microsomal cholesterol levels are significantly lowered and ACAT activity is significantly elevated. These latter effects are not accompanied by changes in serum cholesterol levels and may represent compensatory mechanisms to restore cholesterol homoeostasis in the body. Mechanisms responsible for the effects of I3C on cholesterol homoeostasis are proposed.

Animals↗
PubMed1992

Effects of neural transplantation on seizures in the immature genetically epilepsy-prone rat.

To study the hypothesis that neural transplantations can alter seizure susceptibility in a genetic animal model of epilepsy, 93 pubescent genetically epilepsy-prone rats with stage 9 seizures received either bilateral inferior colliculi (N = 21) or lateral ventricle (N = 42) transplants or sham transplants (N = 30). The grafts consisted of embryonic locus ceruleus, neocortical, or cerebellar tissue. Starting 2 days after the transplantation the rats were subjected to audiogenic stimulations every other day for 61 days. Latency to the running and tonic phase, seizure severity score, and duration of the tonic and clonic phase were compared in the neural transplant and sham-operated controls. Rats that received transplants had a longer latency to the tonic phase and a shorter duration of the clonic phase than the controls. At age 110 days the rats had electrodes implanted bilaterally into the angular bundle and were kindled. No difference in kindling rate was found between the rats that received neural grafts and the sham-operated controls. Cerebrospinal fluid concentration of norepinephrine was not altered by the transplants. This study demonstrates that the anticonvulsant effects of neural transplants, using the genetically epilepsy-prone model of epilepsy, are mild.

Acoustic Stimulation↗
PubMed1990

Single-donor fibrin glue for hand burns.

Early tangential excision sometimes results in considerable blood loss, prolonged operative time, and partial loss of the graft secondary to hematoma formation. Previous reports document positive hemostatic effects and improved skin fixation with fibrin "glue." The commercial preparation used in Europe, however, has not been approved by the United States Food and Drug Administration because of the high risk of hepatitis and human immunodeficiency virus transmission. Using a method developed at the University of Virginia, we applied single-donor fibrin glue as an adjunct in early excision and grafting in 16 patients (26 hands). The overall graft take was 99%. In all patients, better adherence of the split-thickness graft to the recipient bed, during and immediately after application, was noted. We have observed no negative effects with regard to infection or healing. We recommend the use of single-donor fibrin glue to reduce operative blood loss, improve survival and ease of graft application, and possibly to accelerate healing.

Adult↗
PubMed1989

Nerve compression syndromes of the lower extremity.

Nerve compression syndromes of the lower extremity present a challenge in differential diagnosis. Compression of the common peroneal nerve occurs relatively frequently; compression of the sciatic nerve occurs infrequently. The pattern of weakness helps distinguish lumbar root entrapment from peripheral compression syndromes. Compression syndromes must also be differentiated from diabetic, alcoholic or vasculitic neuropathy. Recovery correlates with the degree and duration of nerve injury. Thus, early diagnosis and treatment are important.

Diagnosis, Differential↗
PubMed1987

Improvement in the resolution of o-phthalaldehyde derivatized amino acids by applying gradient steepness optimization to five reversed-phase columns of different lengths and particle sizes.

Twenty-two o-phthalaldehyde (OPA) derivatized amino acids were separated on five different reversed-phase, octadecyl columns. Each column was packed with the same type of spherical silica on which matched bonding chemistry had been performed. Columns of 250, 150 or 50 mm x 4.5 mm I.D. were packed with 5 microns particles, and columns of 100 or 50 mm x 4.5 mm I.D. were packed with 3 microns particles. Snyder's linear solvent strength gradient optimization method was used to determine the optimum gradient steepness by maximizing the resolution of four pairs of adjacent OPA-derivatized amino acids. An asymptotic dependence of improvement in resolution with increase in gradient time was obtained for each pair of compounds on each column. For short (50 mm) columns, resolution of very closely eluting compounds required the use of gradient steepness parameters as low as 0.05 and 0.02, due to the low efficiencies intrinsic to these short column lengths.

Amino Acids↗
PubMed1987

Pediatric burns.

Burns are the second most common cause of death in childhood. More than half of pediatric burns are partial-thickness scald burns; the majority occur in the kitchen. Generally, minor burns may be treated on an outpatient basis, while moderate burns are treated in a community hospital. Children with major burns should be transferred to a regional burn unit as soon as possible after stabilization, provision of fluids and, if necessary, intubation.

Age Factors↗
PubMed1985

High-performance liquid chromatography of substituted p-benzoquinones and p-hydroquinones. II. Retention behavior, quantitative structure-retention relationships and octanol-water partition coefficients.

The retention behavior of methoxy-substituted p-benzoquinones and the corresponding hydroquinones in reversed-phase chromatography was examined on octylsilica and two octadecylsilica stationary phases and with five hydroorganic mobile phases containing acetonitrile, methanol or tetrahydrofuran and additionally in most cases (NH3OH)3PO4 used as a reducing and buffering agent. The retention order of benzoquinones and hydroquinones was the same on each stationary phase with either methanol or acetonitrile as the organic modifier. On the other hand, minor differences in the retention order were observed with the various stationary phases. In all cases, satisfactory quantitative structure-retention relationships (QSRRs) were found and the data suggest that the differences in the retention behaviour of octadecylsilicas used in this study are silanophilic interactions which, together with solvophobic interaction contribute to the retention of these eluites. Further analysis showed that QSRRs of sterically crowded molecules must take into account reduced surface area available for binding. The retention data obtained with use of aqueous tetrahydrofuran as mobile phase failed to give rise to satisfactory QSRRs. This was attributed to selective solvation of eluite by tetrahydrofuran and/or nearly equipotent binding of eluite and tetrahydrofuran to stationary phase.

Benzoquinones↗