Comments on "Why not use it all?".
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Data Collection↗
Biomedical subjects
D Acosta
Publications and source records attributed to D Acosta.
[Intraesophageal stent in the prevention of stenosis caused by caustic ingestion].
Esophageal stenosis is a frequent consequence of lye ingestion, and their treatment is of longstanding and complex matter. The search of new solutions for this problem is challenging and always attractive if its associated with less aggressiveness to the patient. The use of intraesophageal stents is not new in the treatment of stenosis, but it was anecdotal, until histocompatible material came out in the market. The stent is designed in the OR, having the same length as the stenosis, previously observed by endoscopy, its made out of a silicone tube 30 or 36 French, mounted over an ng tube 16, all this is fixed in the ends of the silicone tube, that way won't slip over the ng tube. It is placed with a laryngoscope assistant, the proximal end of the ng tube comes out the nasal nares. Once the location of the tube is checked, the stent is left in place for six weeks. Our experience, with seven patients, has shown excellent tolerance to the stent; at the third post procedure day the patients were eating soft diet by mouth, we believe that the esophageal spasm had resolved by this day. Once the stent was withdraw, five cases were free of esophageal lesions, in the other two small areas of bleeding were visualized. In the follow-up the five cases that were free of lesions, had a normal esophageal diameter, in the other two, one had a moderate restenosis and the other case was severe.
Age Factors↗
High resolution electron microscopy of bis-(-2-hydroxyethyl)terephthalate crystalline polymers.
High resolution transmission electron microscopy (HRTEM) studies of Bis-(2-hydroxyethyl)terephthalate (BHET) crystals, prepared by a novel method which allows one to obtain large crystallites (of the order of several centimeters) are reported. The details of the crystallography, including polymorphism, as determined by X-ray diffractometry and infrared spectroscopy and their relation to the micrographs and electron diffraction results are discussed.
Crystallization↗
Adding metformin versus insulin dose increase in insulin-treated but poorly controlled Type 2 diabetes mellitus: an open-label randomized trial.
To compare the effect of adding metformin to insulin therapy with a moderate increase in insulin dose alone in insulin-treated, poorly controlled Type 2 diabetic patients, 47 consecutive such patients (baseline daily dose >0.5 IU kg(-1) and HbA1c >8%) were openly randomized either to a combination of their previous insulin schedule plus metformin (2.55 g daily in three divided doses, n = 24) or to a moderate insulin dose increase (20% of baseline, n = 23). The patient status/biochemical profile was assessed at entry and at 4 months. Among those assigned to insulin + metformin, 18 took the drug. Upon an intention-to-treat basis, patients assigned to insulin dose increase had a statistically significant weight gain (1.16+/-1.9 vs 0.3+/-4.5 kg, p < 0.05). Patients assigned to the insulin + metformin regimen experienced a significantly greater fall in HbA1c (-1.87+/-2.16 vs 0.03+/-1.68%, p < 0.01), total cholesterol (-0.56+/-0.89 vs 0.14+/-0.72 mmol l(-1), p < 0.05) and LDL-cholesterol (-0.51+/-0.73 vs 0.19+/-0.6 mmol l(-1), p < 0.01). These data suggest that adding metformin to insulin in poorly controlled Type 2 DM patients offers an advantage in terms of glycaemic control and lipid plasma profile.
Aged↗
Predictive value of in vitro model systems in toxicology.
The application of in vitro model systems to evaluate the toxicity of xenobiotics has significantly enhanced our understanding of drug- and chemical-induced target toxicity. From a scientific perspective, there are several reasons for the popularity of in vitro model systems. From the public perspective, in vitro model systems enjoy increasing popularity because their application may allow a reduction in the number of live animals employed in toxicity testing. In this review, we present an overview of the use of in vitro model systems to investigate target organ toxicity of drugs and chemicals, and provide selective examples of these model systems to better understand cutaneous and ocular toxicity and the role of drug metabolism in the hepatotoxicity of selected agents. We conclude by examining the value and use of in vitro model systems in industrial development of new pharmaceutical agents.
Animal Testing Alternatives↗
Characterization and partial purification of a leucine aminopeptidase from Fasciola hepatica.
An aminopeptidase activity capable of hydrolyzing different aminomethylcoumarin amino acids, but mainly leucine-7-amino-4-methylcoumarin (Leu-NHMc), was detected in deoxycholic soluble extracts from adult Fasciola hepatica. The enzyme (EC 3.4.11.1) was partially purified by gel filtration and EAH-Sepharose affinity chromatography using bestatin as a ligand. Results obtained by gel filtration, direct fluorogenic substrate analysis in polyacrylamide gel, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that in a native form the enzyme might be aggregated as a high molecular weight complex. By affinity chromatography on concanavalin A Sepharose, the enzyme did not bond to the column showing that it lacks mannose residues. The F. hepatica aminopeptidase was characterized as a metalloproteinase based on its activation by Mn2+ and Mg2+, and its inhibition by EDTA, 1,10-phenanthroline, and bestatin. It has an optimal activity at a pH between 8.0 and 8.5. Histochemical localization revealed strong leucine naphthylamidase activity at the cells lining the gut epithelium of the parasite.
Animals↗
N-deacetyl ketoconazole-induced hepatotoxicity in a primary culture system of rat hepatocytes.
Ketoconazole (KT) is an azole antifungal agent that has been associated with hepatotoxicity. The mechanism of its hepatotoxicity has not yet been resolved. It has been suggested that a reactive metabolite may be the cause of toxicity because the hepatic injury does not appear to be mediated through an immunoallergic mechanism. Several metabolites of KT have been reported in the literature of which the deacetylated metabolite, N-deacetyl ketoconazole (DAK), is the major metabolite which undergoes further metabolism by the flavin-containing monooxygenases (FMO) to form a potentially toxic dialdehyde. The objective of this study was to evaluate DAK's cytotoxicity and the role of FMO in a primary culture system of rat hepatocytes. Cytotoxicity was evaluated by measuring the leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), into the medium and by assessing mitochondrial reduction of 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT). The cultures were exposed to various concentrations of DAK (20-160 microM) for 0.5-4 h. There was a significant increase (P < 0.05) in LDH leakage and an immediate decrease in MTT reduction (P < 0.05) as early as 0.5 h. The MTT reduction assay appeared to be more sensitive than the LDH assay in that lower concentrations were needed to observe a 50% reduction of MTT (107, 90, 75, 58 microM DAK at 0.5, 1.0, 2.0 and 4.0 h, respectively). The concentrations to observe 50% LDH leakage from the hepatocytes were 155, 133, 100, 70 microM DAK at 0.5, 1.0, 2.0 and 4.0 h, respectively. Moreover, co-treatment with methimazole, a competitive substrate for FMO, produced a significant decrease (P < 0.05) in % LDH leakage as early as 0.5 h, when compared to cells treated solely with DAK. Also, the toxicity was significantly (P < 0.05) enhanced as early as 0.5 h by n-octylamine, a known positive effector for FMO. These results demonstrate that DAK is a more potent cytotoxicant than its parent compound, KT, as reported previously by our laboratory (Rodriguez and Acosta, Toxicology, 96: 83-92, 1995) and its toxicity was expressed in a dose- and time-dependent manner. Furthermore, DAK's cytotoxicity was enhanced with n-octylamine and suppressed with methimazole, suggesting a role for FMO in the toxicity of the metabolite.
Animals↗
Ratiometric measurement of intracellular pH of cultured cells with BCECF in a fluorescence multi-well plate reader.
A number of methods have been developed to measure intracellular pH (pHi) because of its importance in intracellular events. A major advance in accurate pHi measurement was the development of the ratiometric fluorescent indicator dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). We have used a fluorescence multi-well plate reader and a ratiometric method for determining pHi in primary cultures of rabbit corneal epithelial (CE) cells with BCECF. Fluorescence was measured at excitation wavelengths of 485 +/- 11 nm and 395 +/- 12.5 nm, with emission detected at 530 +/- 15 nm. Cells grown in multi-well plates were loaded with 4 microM BCECF for 30 min at 37 degrees C. Resting pHi was 7.34 +/- 0.03 (2 cultures, N = 5 wells). Changes in pHi determined with the fluorescence multi-well plate reader after the addition and removal of NH4Cl or sodium lactate were comparable to changes in cells analyzed with a digitized fluorescence imaging system. A concentration-response relationship involving changes in pHi was easily demonstrated in CE cells after treatment with ionomycin, a calcium ionopore. Low doses of ionomycin (2.5-5 microM), produced a prolonged acidification; 7.5 microM ionomycin produced a transient acidification; and 10 microM ionomycin resulted in a slight alkalinization. We conclude that accurate pHi measurements can be obtained with a ratiometric method with BCECF in a multi-well plate reader. This technology may simplify screening studies evaluating effects of hormones, growth factors, or toxicants on pHi homeostasis.
Ammonium Chloride↗
Urinary albumin excretion rate and cardiovascular disease in Spaniard type 2 diabetic patients.
To assess the prevalence of urinary albumin excretion abnormalities and their associations with cardiovascular disease or its classical risk factors in type 2 diabetes mellitus, 1348 clinic-proceeding patients have been studied retrospectively. The overnight urinary albumin excretion rate, blood pressure, smoking, ophthalmic and cardiovascular status, current therapies, estimates of glycemic control, plasma lipids, serum creatinine and uric acid have been ascertained. 767 (56.8%) patients were found normoalbuminuric, 461 (34.1%) microalbuminuric and 120 (8.9%) macroalbuminuric. In bivariate analyses, the urinary albumin excretion rate had statistically significant (P < 0.05) relationships with age, duration of diabetes, male sex, waist-to-hip ratio, systolic and diastolic pressure, coronary heart disease, cerebrovascular disease, peripheral vascular disease, hypertension, antihypertensive therapy, laser-treated retinopathy, kind of treatment, smoking habit, fasting glycaemia, HbA1c, creatinine, uric acid, triglycerides, high density lipoprotein (HDL)-cholesterol and apolipoprotein B. Borderline statistically significant (P < 0.1) relationships were found with hypolipidaemic therapy, insulin dose, non-HDL-cholesterol, apolipoprotein A1 and lipoprotein (a). In a multivariate stepwise logistic regression model, HbA1c, hypertension, male sex, age, diastolic blood pressure, coronary heart disease and body-mass index were sequentially selected as variables independently associated with microalbuminuria. Serum creatinine, HbA1c, male sex and hypertension were sequentially selected as independently associated with macroalbuminuria. Micro and macroalbuminuria are frequent abnormalities associated with poorly controlled and complicated disease, with overt cardiovascular disease and its classical risk factors as well as with the male sex.
Aged↗
Relationships of C-peptide levels and the C-peptide/bloodsugar ratio with clinical/biochemical variables associated with insulin resistance in orally-treated, well-controlled type 2 diabetic patients.
The aim of the present study was to evaluate the relationship of C-peptide and the C-peptide/bloodsugar ratio with clinical/biochemical variables presenting a well-known association with insulin resistance in NIDDM patients in acceptable control, obtained without the use of exogenous insulin. A total of 118 non insulin dependent diabetes mellitus (NIDDM) patients treated with diet/oral drugs and having a HbA(1c) level < 7.5% have been studied. Non-stimulated C-peptide levels (RIA) and the C-peptide/bloodsugar ratio have been determined and their relationships with the blood pressure status, blood pressure figures, estimates of adiposity, age, known duration of diabetes, current therapies, plasma lipids, glycaemic control, urinary albumin excretion rate, uric acid and creatinine have been ascertained. C-peptide levels were significantly (P < 0.05) correlated with systolic (r = 0.21) and diastolic blood pressure (r = 0.19), BMI (r = 0.21), high density lipoprotein (HDL) (r = -0.22), non-HDL-cholesterol (r = 0.23), apolipoprotein B (r = 0.29), log of triglycerides (r = 0.39) and uric acid (r = 0.35). The C-peptide/bloodsugar ratio had statistically significant correlations with known duration of diabetes (r = -0.23), diastolic blood pressure (r = 0.21), body mass index (BMI) (r = 0.22), log of triglycerides (r = 0.23) and uric acid (r = 0.36). Hypertensives had higher C-peptide levels than normotensives (1.04 +/- 0.04 versus 0.88 +/- 0.04 nmol/ml, respectively (mean +/- S.E.), P < 0.05) and this statistically significant difference remained after adjustment for age and known duration of diabetes. In well-controlled NIDDM patients not receiving exogenous insulin, both C-peptide levels and the C-peptide/bloodsugar ratio have statistically significant relationships with clinical/biochemical variables presenting a well-known association with insulin resistance.
Administration, Oral↗
Lack of association of lipoprotein (a) with coronary heart disease in Spaniard type 2 diabetic patients.
We tried to elucidate the possible relationship between lipoprotein (a) levels and coronary heart disease by assessing the presence of lipoprotein (a) covariates in NIDDM. We selected 41 type 2 diabetic patients with coronary heart disease and 82 type 2 diabetic patients free from cardiovascular disease. They were adjusted for age, sex and duration of diabetes. Routine chemical analysis was carried out using standard procedures, HbA1c by HPLC and lipoprotein (a) and urinary albumin excretion rate by immunonephelometry. No difference has been found in lipoprotein (a) levels between both groups of patients (18 [144.25] mg/dl in cases vs. 23 [197.25] mg/dl in controls (median [range]), Mann Whitney U-test, P > 0.1). No association has been found between coronary heart disease and lipoprotein (a) levels greater than 30 mg/dl (Pearson's chi 2, P > 0.1). Significant and independent linear relationships have been found between the square root of lipoprotein (a) levels, serum creatinine and total cholesterol (multiple r2: 0.15, P < 0.001). Patients treated with insulin had greater square root of lipoprotein (a) levels, even after adjusting for serum creatinine and total cholesterol (5.87 +/- 0.35 vs. 4.76 +/- 0.36 (mean +/- S.E.), ANCOVA, P < 0.05). These data do not show an association between symptomatic coronary heart disease and lipoprotein (a) in NIDDM. Significant and independent relationships have been found between this variable and serum creatinine, total cholesterol and insulin therapy.
Aged↗
IRAG working group 4. Cell cytotoxicity assays. Interagency Regulatory Alternatives Group.
Twenty-seven data sets from 12 cellular cytotoxicity assays, intended to predict ocular irritation, were submitted to the Interagency Regulatory Alternatives Group (IRAG) for review. These data consisted of paired in vivo (Draize) and in vitro responses to individual chemicals and formulations. In vivo data consisted of individual tissue scores so that the predictive value of the in vitro assay could be assessed for each tissue response normally measured in the standard Draize assay. Data were compiled and evaluated according to the IRAG Guidelines Document. The Pearson's linear correlation coefficient was used as the first step in assessing the relationship between the in vitro and in vivo responses. The majority of the data sets represented the study of surfactant-based materials. In many cases, there was good correlation between the in vitro scores and the in vivo tissue responses. Most pronounced were the particularly good correlations between the in vitro scores and conjunctival redness scores across most of the assays. Based on the data submitted, a number of the cell cytotoxicity assays show considerable promise as screens for ocular irritancy. None of the submitters recommended that their cell cytotoxicity assay be used as a sole replacement for in vivo assessment. For almost all of these assays, the materials being tested should be water-soluble/miscible. The toxicity of products with reserve acidity or alkalinity or with high reactivity may be underestimated. A given user may prefer certain assays depending on the types of materials to be tested, the expected range of toxicities and the resources available. The cell cytotoxicity assays can serve as a valuable component of a tiered or battery testing program. As with any assay, a sufficient number of replicate values, concurrent positive and negative controls, and a strict adherence to assay acceptance criteria are essential to produce credible data.
Animal Testing Alternatives↗
Proteinases secreted by Fasciola hepatica: time course of the inhibitory effect of serum from experimentally infected rabbits demonstrated by gelatin-substrate polyacrylamide gel electrophoresis.
Fasciola hepatica secretes proteolytic enzymes to aid it to penetrate and migrate through the host tissues. Two of these proteinases have been purified and shown to be cathepsin L-like, and are termed, CL1 (27.5 kD) and CL2 (29 kD). The immunogenicity of these proteinases was investigated over the course of an experimental infection and following drug treatment. Four groups of rabbits were studied: group 1: orally infected with 50 metacercariae; group 2: infected and treated 8 weeks after infection; group 3: infected, treated at week 8 and reinfected at week 13 and group 4: non-infected control group. Sera were collected weekly from each group until week 20 postinfection. CL1 and CL2 were incubated with the different sera and then analysed by gelatin substrate polyacrylamide gel electrophoresis (GS-PAGE). Analysis of groups 1, 2 and 3 showed that CL1 and CL2 neutralizing antibodies appear at week 5 post-infection. In group 1, these remained throughout the 20 weeks of infection. In group 2, neutralizing antibodies disappeared at week 13, that is, 5 weeks after anti-Fasciola treatment. In group 3, CL1 and CL2 neutralizing antibodies disappeared at week 13 but reappeared by week 15, that is 2 weeks after reinfection. Pooled sera from group 4, showed no inhibitory capacity. ELISA results using CL1 and CL2 as antigen, correlate very well with the inhibitiory time course observed by GS-PAGE. These results suggest that purified cathepsin Ls are antigenic molecules recognized early in the infective process and capable of inducing a specific humoral response, strong enough to neutralize, at least partially, their enzymatic activity.
Animals↗
High lipoprotein(a) levels in type 1 and type 2 diabetic patients with macroalbuminuria.
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Albuminuria↗
Metabolism of ketoconazole and deacetylated ketoconazole by rat hepatic microsomes and flavin-containing monooxygenases.
Ketoconazole (KT) has been reported to cause hepatotoxicity, which is probably not mediated through an immunoallergic mechanism. Although KT is extensively metabolized by hepatic microsomal enzymes, the nature, route of formation, and toxicity of suspected metabolites are largely unknown. Recent reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice, which, like lipophilic 4-alkylpiperazines, is susceptible to successive oxidative attacks on the N-1 position producing ring-opened dialdehydes. The rate of formation of DAK from hepatic rat microsomal incubations of KT was determined by HPLC. The rate of disappearance for KT was almost equal to the rate of DAK formation: 5.96 and 5.88 microM/hr, respectively. Also, the potential bioactivation of DAK was evaluated by measuring substrate activity of DAK with purified pig liver flavin-containing monooxygenase (FMO) and rat liver microsomes. Activity was measured by following DAK-dependent oxygen uptake polarographically at 37 degrees C in pyrophosphate buffer (pH 8.8) containing the glucose-6-phosphate NADPH-generating system. The K(M)'s of DAK were 34.6 and 77.4 microM for the purified FMO and rat microsomal FMO, respectively. Lastly, DAK was found to be metabolized by an NADPH-dependent rat liver microsomal monooxygenases at pH 8.8 to two metabolites as determined by HPLC. Heat inactivation of rat liver microsomal FMO abolished the formation of these metabolites from DAK. SKF-525A and anti-rat NADPH cytochrome P450 reductase did not inhibit this reaction. These results suggest that deacetylation of KT yields a major product, DAK, for further metabolism by microsomal monooxygenases that seem to be FMO-related.
Animals↗
Evaluation of PAPNET testing as an ancillary tool to clarify the status of the "atypical" cervical smear.
To assess the utility of the PAPNET system (Neuromedical Systems Inc., Suffern, NY) in clarifying the status of cervical smears showing borderline abnormalities, we analyzed the results of five cytotechnologists who reclassified 200 "atypical" smears by evaluating PAPNET images only. The interobserver agreement (reliability) of the PAPNET reviewers was computed, and their readings were compared with three standards: the consensus diagnosis of five pathologists who used light microscopy, the detection of cancer-associated human papillomavirus DNA by Southern analysis, and the correlation with diagnoses of biopsy specimens obtained during passive follow-up. The PAPNET reviewers classified 18 to 65% of cases as normal, 25 to 42% as equivocal, and 10 to 55% as abnormal. Unanimous interobserver agreement was achieved in only 24 (13%) cases. Four of the five PAPNET reviewers agreed moderately well with the results of the pathology reference panel. In four of the five PAPNET reviews, classification of cases as abnormal was strongly correlated with the detection of cancer-associated types of human papillomavirus. Consensus PAPNET results of abnormal were predictive of abnormal histologic findings at follow-up. Theoretically, if colposcopy had been performed on all of the women with equivocal or abnormal PAPNET results (based on the consensus of the panel), as much as 95% of biopsy-confirmed lesions could have been detected, but 79% of women would have been referred. Restriction of colposcopy referral to women with definitely abnormal PAPNET readings would have reduced referrals to 31%, but the sensitivity of the triage would have dropped to 51% of biopsy-confirmed lesions.
Biopsy↗
Cocaine-induced mitochondrial dysfunction in primary cultures of rat cardiomyocytes.
Morphological alterations of cardiac mitochondria have been observed in rats chronically treated with cocaine. Whether cocaine directly causes heart mitochondrial dysfunction remains unclear. The present study was undertaken to investigate the effect of cocaine on mitochondrial function in cultured myocardial cells. Cells were incubated with cocaine (10(-5)-10(-3) M) for 3-72 h, using either a repeated or single exposure protocol. Cocaine (10(-3) M) produced severe cytotoxicity after repeated exposure (24-72 h), as elevated by leakage of lactate dehydrogenase. Treatment of the cultures with a single exposure protocol (10(-5)-10(-3) M for 24 h or less) produced a very modest cytotoxic effect, as shown by a small increase in LDH leakage. However, cellular ATP levels showed a time-dependent decline in cultures treated with the single exposure protocol. Experiments using a digitized fluorescence imaging system revealed that cocaine (single exposure protocol) caused a dose- and time-dependent decrease in mitochondrial membrane potential, and the decline in membrane potential occurred prior to manifestation of cytotoxicity shown with the repeated exposure protocol. Cytosolic and mitochondrial calcium levels, as determined by fura-2, were not affected during treatment with cocaine. Our results suggest that cocaine may compromise cardiac mitochondrial function and may lead to cardiotoxicity.
Adenosine Triphosphate↗
Inhibition of mitochondrial function in isolated rate liver mitochondria by azole antifungals.
Ketoconazole is an imidazole oral antifungal agent with a broad spectrum of activity. Ketoconazole has been reported to cause liver damage, but the mechanism is unknown. However, ketoconazole and a related rug, miconazole, have been shown to have inhibitory effects on oxidative phosphorylation in fungi. Fluconazole, another orally administered antifungal azole, has also been reported to cause liver damage despite its supposedly low toxicity profile. The primary objective of this study was to evaluate the metabolic integrity of adult rat liver mitochondria after exposure to ketoconazole, miconazole, fluconazole, and the deacetylated metabolite of ketoconazole by measuring ADP-dependent oxygen uptake polarographically and succinate dehydrogenase activity spectrophotometrically. Ketoconazole, N-deacetyl ketoconazole, and miconazole inhibited glutamate-malate oxidation in a dose-dependent manner such that the 50% inhibitory concentration (I50) was 32,300, and 110 microM, respectively. In addition, the effect of ketoconazole, miconazole, and fluconazole on phosphorylation coupled to the oxidation of pyruvate/malate, ornithine/malate, arginine/malate, and succinate was evaluated. The results demonstrated that ketoconazole and miconazole produced a dose-dependent inhibition of NADH oxidase in which ketoconazole was the most potent inhibitor. Fluconazole had minimal inhibitory effects on NADH oxidase and succinate dehydrogenase, whereas higher concentrations of ketoconazole were required to inhibit the activity of succinate dehydrogenase. N-deacetylated ketoconazole inhibited succinate dehydrogenase with an I50 of 350 microM. In addition, the reduction of ferricyanide by succinate catalyzed by succinate dehydrogenase demonstrated that ketoconazole caused a dose-dependent inhibition of succinate activity (I50 of 74 microM). In summary, ketoconazole appears to be the more potent mitochondrial inhibitor of the azoles studied; complex I of the respiratory chain is the apparent target of the drug's action.
Animals↗