Abstract
Concurrent Session
1:30 PM
Friday, January 29, 1999
Evaluation Of Disease Progression In Adpkd I Patients Using Four Common Diagnostic Assessments.
JS Swissman, AK Saggar-Malik. University of Nevada School of Medicne, Reno, NV, and St. George's Hospital Medical School, London, UK.
Autosomal Dominant Polycystic Kidney Disease I (ADPKD I) is an inherited disease caused by a mutation on chromosome 16p13.3. The disease manifests itself as several, small, fluid filled cysts in the kidney and may ultimately lead to renal failure and all the associated complications. The purpose of this study was to determine the progression of ADPKD I as it develops within a patient and to compare the current clinical assessments of renal function. The study population consisted of 39 Caucasian subjects diagnosed with ADPKD I. All diagnoses were initially made using ultrasound to identify renal cysts which were followed by karyotypes to establish chromosome mutation location. All patients were evaluated by the same physician to insure consistent diagnosis and treatment. Disease progression was examined using four different clinical assessments: chromium glomerular filtration rate (GFR), creatinine clearance from 24 hour urine collection (CrCI Ur), creatinine clerarance from serum creatinine using the Cockroft-Gault formula (CrCl CG), and the inverse serum creatinine (1/Cr). The GFR showed a mean±SD decrease in glomerular filtration rate of 0.01194±0.02398 mL/min/day. CrCl Ur yielded a decreased rate of 0.01030±0.03474 mL/min/day. CrCl CG also produced a decrease of 0.01184±0.01299 mL/min/day. Finally, 1/Cr yielded a decrease of 1.277±1.237 mL/min/day. There was not significant difference between the GFR, CrCl Ur, and CrCl CG methods of renal failure assessment (p=0.963 by ANOVA). The 1/Cr assessment was clearly the worst, producing significant differences with all three remaining assessments (p<0.0001). In conclusion, the GFR, CrCl Ur, and CrCl CG methods were superior in evaluating average renal function decline in the ADPKD I study patient population. The 1/Cr method was only suitable for evaluating disease progression in the individual patient.
An Arginine Provocative Test To Unmask Latent Diabetic Microalbuminuria.
Minimal microalbuminuria (MA) is most representative of tubular albumin reabsorptive fatigue on a background of preexisting hyperfiltration of albumin. Hence, even an early finding of MA will miss an undetermined number of patients with diabetic glomerulopathy. Arginine is a competitive inhibitor of tubular albumin reabsorption, and was used in an effort to unmask latent MA in diabetic subjects. Ten type II diabetics were studied, 4 with 80 to 400 mg daily albuminuria who were 100% positive for MA on eight screening tests (POS). Six had 1 to 135 mg of daily albuminuria with negative or equivocal readings on 75% of screening tests for albumin on spot and first a.m. urines, albumin/creatinine ratios and Micral® dipstick tests (BORDERLINE). Studies were done during water diuresis after breakfast with two 30 minute collections and midpoint plasmas. Thereafter, 30 g arginine was infused over 30 minutes as a 10% solution of Arg HCl, with urine and plasma collected for three more periods. Computations were made of albumin excretion rates (UaV) and apparent sieving coefficients (SC). In the POS group, UaV and SC, which were elevated at the beginning, did not rise. In the BORDERLINE group, UaV rose 30 ± 7.2 (sem) mg/24 hrs (scaled from a 30 minute colletion) (2P = .009), and apparent SC rose from 0.98 to 1.37 x 10⁵ (2P = .02).
It appears that an arginine provocative test may unmask latent diabetic microalbuminuria and render an as yet undiscovered and uncounted nephropathic population accessible for treatment. Control observations have yet to be made, and the route of arginine administration must be rendered into a convenient form.
Effects Of Dietary Supplements, 19-Norandrostenedione, Androstenediol And Androstenedione On The Profile Of Urine Steroids.
Steroids sold over-the-counter (OTC) as dietary supplements have become popular among those who seek to enhance athletic performance. We examined the effect of three OTC steroids: 19-norandrostenedione (19-nor-4-androsten-3, 17-dione); androstenediol (4-androsten-3, 17-diol); and androstenedione (4-androsten-3, 17-dione) on the profile of urine steroids. The urine samples were analyzed by gas chromatography-mass spectrometry (GC-MS). Urine samples from individuals who took 19-norandrostenedione contained the metabolites of 19-nortestosterone (19-norandrosterone, 19-noretiocholanolone). 19-nortestosterone is a potent androgen that is banned by the International Olympic Committee (IOC). IOC testing procedures determine the ratio of testosterone (T) to epitestosterone (T/E) and report cases with T/E >6 to the sport authorities. The T/E is considered a marker for T administration but it is not definitive, thus the sport authorities have the responsibility to conduct further studies to determine if the elevated T/E is due to T administration or some other cause. Urine samples from individuals who ingested androstenediol revealed T/Es >6. Urine obtained from individuals who ingested androstenedione showed large increases in metabolites of T. These findings indicate that individuals who ingest OTC steroids may result in changes in their urine steroid profile consistent with testosterone or 19-nortestosterone administration.
Mechanism Of Disulfiram Inhibition Of P450 2E1 In Human Liver Microsomes.
Numerous environmental contaminants and some therapeutic drugs can cause hepatotoxicity when metabolized by cytochrome P450 2E1. Disulfiram and its primary metabolite diethydithiocarbamate (DDTC) inhibit P450 2E1 in vitro and in vivo, and disulfiram may prevent metabolism-based toxicity. Disulfiram and DDTC are mechanism-based inhibitors requiring P450 2E1-mediated activation to the ultimate inhibitory species. Disulfiram is extensively metabolized in vivo, however, the identity of the most inhibitory disulfiram metabolite is unknown. This project tests the hypothesis that disulfiram metabolites is/are the ultimate species responsible for P450 2E1 inhibition.
Human liver microsomal P450 2E1 activity was determined by chlorzoxazone 6-hydroxylation, in the presence and absence (controls) of the disulfiram metabolites: DDTC, S-methyl-N, N-diethyldithio-carbamate (DDTC-Me), S-methyl-N, N-diethylthiocarbamate (DETC-Me), S-methyl-N, N-diethylthiocarbamate sulfoxide (DETC-MESO), S-methyl-N, N-diethyldithiocarbamate sulfoxide (DDTC-MeSO), S-methyl-N, N-diethylthiocarbamate sulfone (DETC-MeSO2), S-methyl-N, N-diethyldithiocarbamate sulfone (DDTC-MeSO2), and carbon disulfide (CS2). Initially, reaction mixtures contained human liver microsomes (0.15mg), chlorzoxazone (62µM), and inhibitor (0 or 0.3-1,000µM) in 100mM KPi buffer. NADPH (1mM final) was added to start the reaction (10 min., 37°C). 6-hydroxychlorzoxazone was extracted and concentrations determined by HPLC. To investigate if inhibitors required metabolic activation, experiments were repeated, however, inhibitor was incubated with microsomes and NADPH for 15 minutes before adding substrate. Data were analyzed by non-linear regression analysis of sigmoidal log concentration-rate curves. All the compounds showed inhibitory activity. Compounds con-taining a diethyldithiol or dithiol moiety were better inhibitors than those containing a diethylthiol group. The most effective inhibitors were the dithiol containing compounds. Furthermore, pre-incubation of the diethyldithiol compounds significantly lowered the IC50. In conclusion, diethyldithiol compounds showed evidence of metabolic. activation. Furthermore, the diethyldithiol metabolites of disulfiram are likely to be the actual inhibitors of P450 2E1. This knowledge could be useful to enhance the prevention of toxicity caused by an-esthetics, other drugs or carcinogens. Supported by NIH GM 4P712.
Mechanism Of Irritant Dermatitis In Man.
Clinical irritant dermatitis refers to inflammation of the skin resulting from non immunologic damage by chemicals in contact with the skin immediately (acute irritant dermatitis) or repeatedly (cumulative irritant dermatitis). Past trials using the standard human assay for low grade irritants, known as the cumulative irritant assay, have proven unsuccessful in predicting development of chronic low grade dermatitis in patients exposed to irritants. This study used a subclinical approach in predicting clinical changes induced by low grade chemicals.
The study was conducted as a bilateral and paired comparison. Six sites were chosen on the volar aspect of both forearms of ten healthy Caucasian volunteers aged 35-55 with no past history of atopic skin disease. Baseline bioengineering measurements of transepidermal water loss (TEWL), skin hydration (Corneometer), and skin color (Chromameter) were taken before treatment as indicators of skin barrier function and integrity. In addition, baseline squamometry was performed to sample unirritated stratum corneum tape strippings for later characterization of intercorneocyte cohesion (via microscope) and toludine blue dye staining of stratum corneum proteins. Application of 50 µl of three dilute irritants (1% HCI, 0.1% KOH, 1.5% phenol) and three control sites of water, chamber, and no treatment were applied with chamber occlusion. After 24 hours, the chambers were removed and the same parameters were measured again, as well as squamometry tape strippings. Subsequently, subjective analysis of intercorneocyte cohesion of the tape strippings was done under microscope based on a standardized grading system. Dye intensity on the discs was also measured by the Chromameter.
Subclinical indications are important in predicting clinical contact dermatitis. This study attempted to characterize non-visible stratum corneum changes upon mild skin irritation through bioengineering methods and squamometry. Several parameters indicative of stratum corneum alterations upon irritation were measured. Baseline measurements at 0 hours were compared to those at 24 hours post treatment. Results showed generally increased transpepidermal water loss (as expected from decreased stratum corneum integrity), decreased skin hydration (as the stratum corneum's water retaining ability is compromised), and increased chromameter values (a measure of skin redness) after irritation. Despite work in previous studies, squamometry results were not as informative. This study has shed light on subclinical changes in the skin and their diagnostic value in clinical dermatitis. This can ultimately lead to more predictive and less damaging skin formulations as identified by these subclinical assays.
Cyclodextrins, But Not Other Cholesterol-Lowering Pharmacologics Or Genetic Manipulations, Delays The Onset Of Neurological Symptoms In Niemann Pick C Mice.
The gene for Niemann Pick C (Npc1 in mice) has recently been cloned and its predicted structure suggests that it could be involved in cholesterol movement - an hypothesis supported by a number of analyses. We have attempted to ameliorate the symptoms of NPC in a “perfect” mouse model. Our approach has included both pharmacological agents and introducing the low density lipoprotein receptor (LDLR) knockout. In regards to the latter, Npc1 homozygous mice, which were also homozygous for the the LDLR knockout, showed unaltered storage of unesterified cholesterol in the liver (normally about 20-fold elevated in Npc1/homozygous mice) but a slight delay in onset of neurological symptoms - by about one week. This may, however, be due to the genetic background on which the LDLR knockout was introduced since LDLR knockout heterozygous mice also showed a delay of about a week in onset of symptoms. Probucol and nifedipine had marked effects on the storage of unesterified cholesterol, reducing it to only about 3 times normal. They had a lesser effect on liver levels of esterified cholesterol which are elevated in Npc1 homozygotes. However, there was little or no effect on the age of onset of neurological symptoms. In contrast, cyclodextrins (2-hydroxy propyl substituted) lowered liver unesterified cholesterol to about 3 times normal and delayed the onset of neurological symptoms by nearly 2 weeks, a very significant delay. We are currently studying cyclodextrins substituted to different degrees for increased efficacy. We are also mating Npc1 mice to mdria knockout mice (mdria is a P-glycoprotein which wh is highly important for the function of the blood brain barrier) to determine whether or not the blood brain barrier is important in limiting the efficacy of these cyclodextrins.