Interference of nicotine metabolites in cotinine determination by RIA.
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Antibody Specificity↗
Biomedical subjects
M Rosa
Publications and source records attributed to M Rosa.
Mutation analysis in Duchenne and Becker muscular dystrophy patients from Bulgaria shows a peculiar distribution of breakpoints by intron.
For the first time in Bulgaria, a deletion/duplication screening was performed on a group of 84 unrelated Duchenne/Becker muscular dystrophy patients, and the breakpoint distribution in the dystrophin gene was analyzed. Intragenic deletions were detected in 67.8% of patients, and intragenic duplications in 2.4%. A peculiar distribution of deletion breakpoints was found. Only 13.2% of the deletion breakpoints fell in the "classical" hot spot in intron 44, whereas the majority (> 54%) were located within the segment encompassing introns 45-51, which includes intron 50, the richest in breakpoints (16%) in the Bulgarian sample. Comparison with data from Greece and Turkey points at the probable existence of a deletion hot spot within intron 50, which might be a characteristic of populations of the Balkan region.
Bulgaria↗
Prognostic factors in mild dystrophinopathies.
One hundred twenty five patients from 105 families were considered, showing in-frame intragenic deletion or duplication of the dystrophin gene and/or abnormal dystrophin on muscle biopsy. According to clinical status of patients, the affection was classified as subclinical, benign, moderate or severe. Significant decrease of dystrophin abundance was observed with increasing clinical severity (p < 0.05). Detailed clinical data were available in 68 patients in whom a long-term follow-up (6-39 years) was obtained. Functional performance at different ages and disease endpoints were recorded in order to analyze the rate of disease progression. We identified three different disease courses: stable, slow and rapid progression. We observed a significantly lower level of dystrophin and immunohistochemical score (p < 0.05 vs. the other courses) in patients with rapid course. Deletion or duplication in the 5' end of the gene was associated with poor prognosis. Prognosis was substantially better, showing a stable course, in patients with large deletions or duplications in the proximal rod region. These subjects often suffered from a cramps/myalgia syndrome or experienced rhabdomyolisis. Cardiac involvement was detected in 65% of cases. A significant increase of right ventricular volume was seen in all clinical groups (p < 0.05). A left ventricular dilation was observed in 25% and a decreased ejection fraction in 29% of our patients. The reduction of ejection fraction and the increase of left ventricular volume were age-related. Since sudden death may occur as a consequence of cardiomyopathy, severe left ventricular dysfunction in dystrophinopathic patients is another important adverse prognostic factor, although not always directly correlated with skeletal muscle impairment.
Adolescent↗
Does the presence of ANCA in patients with ulcerative colitis necessarily imply renal involvement?
BACKGROUND: ANCA are thought to play a pathogenic role in renal vasculitis. ANCA may also be detected in patients with diseases not usually associated with renal pathology, such as ulcerative colitis. Our study was conducted to determine if the presence of ANCA in patients with ulcerative colitis is associated with renal pathology. METHODS: Eight ANCA-positive and five ANCA-negative patients with a histological and endoscopic diagnosis of active ulcerative colitis were investigated. Repeated complete urinalyses and determination of microalbuminuria and creatinine clearance were performed. Serum IgG and IgA ANCA were evaluated in all patients by indirect immunofluorescence and ELISA, and when detected the antibodies were further characterized by alpha granules preparation, myeloperoxidase, lactoferrin, and cathepsin G. RESULTS: In both ANCA-positive and ANCA-negative patients renal function was normal or near normal and urinalyses (including microalbuminuria) failed to disclose any abnormalities. ANCA exhibited a perinuclear pattern in all ANCA-positive patients. Interestingly, none of the ANCA-positive patients had antibodies to myeloperoxidase or to alpha granules which are usually found in the sera of patients with ANCA-associated vasculitis, and only one had antibodies to lactoferrin. The ANCA specificity remained undetermined in the remaining seven patients. At the end of the 1-year observation period, all ANCA-positive patients remained ANCA-positive without developing symptoms, signs or laboratory abnormalities consistent with renal involvement. CONCLUSIONS: Renal damage was not observed in ANCA-positive patients with ulcerative colitis even after 1 year of follow-up, suggesting that the ANCA found in these patients do not share the antigenic targets with the ANCA commonly found in renal vasculitis. Therefore the potential of ANCA of inducing renal lesions (if any) is dependent on their own antigenic specificity.
Adolescent↗
Effects of hypercholesterolemia of renal hemodynamics: study in patients with nephrotic syndrome.
Experimental and clinical studies have demonstrated a positive relationship between hyperlipidemia and rate of progression of renal disease, suggesting that lipids can induce or aggravate glomerular injury mainly by interacting with mesangial cells. Nevertheless, recently has been demonstrated that increased cholesterol levels can also induce endothelial cell dysfunction. Thus, since endothelium is known to play a major role in modulating the vascular tone, we have tested the possibility that hypercholesterolemia impairs the renal hemodynamics in patients with active nephrotic syndrome and elevated serum cholesterol levels. In this single-blind, nonrandom study, 12 patients were treated with pravastatin (group T, treated, n = 12) and 8 with placebo (group C, controls, n = 8). The controls were studied after the pravastatin group had been completed. Before starting the treatment the patients underwent basal determinations including routine laboratory investigations and PAH and inulin clearances. The same determinations were repeated after 48 h, and 6 and 12 weeks from the beginning of the treatment. The study at 48 h was performed to see if pravastatin had a direct, cholesterol-independent effect on renal function. The following basal results were reported (mean +/- SEM; group T vs. group C): serum cholesterol (mmol/l) 9.7 +/- 0.4 vs. 9.1 +/- 0.3 (NS); proteinuria (g/24 h): 6.2 +/- 0.2 vs. 7.0 +/- 0.7 (NS); PAH clearance (ml/min): 353 +/- 21 vs. 385 +/- 31 (NS); inulin clearance (ml/min): 62.5 +/- 7.7 vs. 67 +/- 9.3 (NS). After 48 h, no changes were observed in both groups. Subsequently, in group T, the following percentage changes of basal levels were observed: serum cholesterol -21.4 +/- 3.2% at 6 weeks (p < 0.05) and -34.9 +/- 3.2% at 12 weeks (p < 0.01); inulin clearance +3 +/- 3.7% at 6 weeks (NS) and +9.3 +/- 2.9% at 12 weeks (p < 0.05); PAH clearance +7 +/- 3.1% at 6 weeks (p < 0.05) and +21.2 +/- 5.5% at 12 weeks (p < 0.01). By contrast, no significant changes of these parameters occurred in group C at any time, so that the percent changes of baseline values of CPAH were significantly greater in group T (at 6 weeks: p < 0.05; at 12 weeks p < 0.005). These results indicate that the reduction of cholesterol is associated with a significant increase in renal plasma flow, thus, suggesting that hypercholesterolemia may actually impair the renal hemodynamics. We speculate that this effect may contribute to increase the risk of ischemic acute renal failure in nephrotic patients and, along with changes induced in the mesangium by other mechanisms, to contribute to the progression of renal disease.
Adult↗
Household and community determinants of exposure to involuntary smoking: a study of urinary cotinine in children and adolescents.
The study examines the role of several potential predictors of urinary cotinine levels in a cross-sectional sample of 1,072 nonsmoking children and adolescents in Latium, Italy, during 1990-1991. As expected, there was a strong relation between passive exposure to smoking and the amount of maternal and paternal self-reported smoking. The urinary cotinine level increased with a decreasing level of paternal education and with an increasing index of household crowding; self-report of recent exposure to smoking outside the home was a strong predictor of the biologic marker. The analysis was then restricted to 346 subjects whose parents claimed that they were nonsmokers and that there were no smokers at home. In this group, however, 57 children reported some active smoking at home by their parents. Those with parents suspected to be "deceivers" had higher level of urinary cotinine than did those truly not exposed. In addition, urinary cotinine in this group was clearly associated with duration of exposure to smoking outside home. The study indicates that both factors related to family circumstances and exposure outside the household setting are strong determinants of urinary cotinine levels. The finding may be considered a direct confirmation that passive smoking among children should be viewed as a specific community responsibility.
Adolescent↗
Solid-phase extraction of nicotine and its metabolites for high-performance liquid chromatographic determination in urine.
A solid-phase extraction, using Extrelut-1 glass columns, has been applied to urine samples of both passive and active smokers for high-performance liquid chromatographic determination of nicotine and its metabolites cotinine and trans-3'-hydroxycotinine. Chromatography was performed using a reversed-phase LC8DB column and a mobile phase consisting of water-acetonitrile (80:9, v/v) containing 5 ml triethylamine, 670 mg/l sodium heptanesulphonate, and 0.034 M each of K2HPO4 and citric acid (pH 4.4), at a flow-rate of 1.6 ml/min. The results obtained indicate that solid-phase extraction is a reliable and quick procedure which can be applied also to other nicotine metabolites.
Chromatography, High Pressure Liquid↗
Environmental tobacco smoke: nicotine and cotinine concentration in semen.
Forty-three nonsmokers attending an infertility clinic reported their degree of passive exposure to environmental tobacco smoke (ETS) and provided samples of blood and seminal plasma. Levels of nicotine and cotinine in biological fluid were evaluated by a radioimmunoassay. The data indicate that exposure to ETS results in measurable nicotine and cotinine levels in seminal plasma. Seminal plasma cotinine concentration also shows a significant positive correlation with degree of reported exposure.
Adult↗
Sequential slicing of lower deciduous teeth to resolve incisor crowding.
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Child↗
Determination of nicotine and four metabolites in the serum of smokers by high-performance liquid chromatography with ultraviolet detection.
A simple and reliable reversed-phase high-performance liquid chromatographic method with ultraviolet detection is described for the quantitation of nicotine and its metabolites cotinine, trans-3'-hydroxycotinine, norcotinine and cotinine N-oxide in human serum. The analytes and the internal standard, N-ethylnorcotinine, were extracted by solid-phase extraction before chromatography. Two different columns and mobile phases with gradient systems were used. The detection limit of the assay was 10 ng/ml for nicotine, 3 ng/ml for cotinine N-oxide and 5 ng/ml for cotinine, trans-3'-hydroxycotinine and norcotinine. The concentrations of nicotine and its metabolites in the serum of 12 cigarette smokers are reported.
Chromatography, High Pressure Liquid↗
Determination of nicotine and two major metabolites in serum by solid-phase extraction and high-performance liquid chromatography, and high-performance liquid chromatography-particle beam mass spectrometry.
A rapid and selective assay of nicotine, cotinine and trans-3'-hydroxycotinine in human serum, based on high-performance liquid chromatography with UV detection has been developed. The compounds were subjected to solid-phase extraction, using Extrelut 1 cartridges. Recoveries were ca. 95% for nicotine, 90% for cotinine and 50-55% for trans-3'-hydroxycotinine. The limit of quantitation observed with this method was 10 ng/ml for nicotine and 5 ng/ml for each of the metabolites. The compounds were also identified using high-performance liquid chromatography with particle beam mass spectrometry, to confirm their presence in human serum.
Chromatography, High Pressure Liquid↗
Nicotine, cotinine, and trans-3-hydroxycotinine levels in seminal plasma of smokers: effects on sperm parameters.
Sperm samples from 44 cigarette smokers and 50 nonsmokers attending an infertility clinic were examined by high-performance liquid chromatography (HPLC) assay and HPLC-mass spectrometry for the presence of nicotine (NIC), cotinine (COT), and trans-3'-hydroxycotinine (THOC) in seminal plasma. Smokers were found to have levels of COT and THOC in seminal plasma that were similar to those found in serum. The level of NIC was significantly increased in seminal plasma compared to serum. Total motility of spermatozoa was significantly and negatively correlated to COT and THOC levels in seminal plasma. Forward motility of spermatozoa was correlated only with cotinine semen levels. On the basis of these results, we suggest that the presence of tobacco smoke constituents in seminal plasma could provide a warning of the adverse effects of cigarette smoke on the physiology of reproduction.
Adolescent↗
Simultaneous determination of cotinine and trans-3'-hydroxycotinine in human serum by high-performance liquid chromatography.
A high-performance liquid chromatographic method with ultraviolet photometric detection has been developed for the quantitation of cotinine and trans-3'-hydroxycotinine in human serum. A solid-phase extraction procedure was performed for the analytes and the internal standard, N-ethylnorcotinine, before chromatography. The use of a 30-cm reversed-phase column and a mobile phase of water-methanol-0.1 M sodium acetate-acetonitrile (67:24.5:6.5:2, v/v), pH 4.3, prevented the co-elution of caffeine with cotinine. The limit of quantitation observed with this method was 5 ng/ml for both cotinine and trans-3'-hydroxycotinine. The present method proved useful for the determination of serum levels of these metabolites, correlating with nicotine daily intake.
Chromatography, High Pressure Liquid↗
How the steady-state cotinine concentration in cigarette smokers is directly related to nicotine intake.
The relationship between nicotine intake and steady-state cotinine concentration was studied in a sample of 125 subjects who smoked their usual brands of cigarettes. Nicotine and tar yield of cigarettes was determined with a smoking machine, under standardized conditions. Blood was drawn about 8 hours after the last cigarette was smoked and serum cotinine was measured by high performance liquid chromatography. Cotinine levels ranged from 11 to 400 ng/ml, and nicotine daily intake ranged from 1 to 33 mg/day. Regression analysis and the correlation coefficient, r = 0.919, significant at p less than 0.0001, showed that steady-state cotinine level was linearly and directly related to daily available nicotine, with an increase in correlation coefficient directly related to the increase in tar and nicotine yield. From the findings we also conclude that smokers of low-tar cigarettes do not tend to compensate for lower yields of nicotine.
Adolescent↗
L-carnitine uptake in differentiating human cultured muscle.
We studied carnitine uptake in human skeletal muscle growing in culture for up to 30 days, and correlated it to the degree of muscle differentiation revealed by myotube formation and muscle-specific creatine-kinase isozyme accumulation. In our study carnitine uptake was a saturable specific process with two distinct components: a high affinity uptake at carnitine concentration between 0.5 and 10 microM and a low affinity uptake at carnitine concentration between 25 and 200 microM. High affinity uptake (Km 4.17-5.50 microM, Vmax 11.78-19.6 pmol/h per mg protein) did not change during muscle maturation in culture. Low affinity uptake showed significant changes in Km and Vmax in the various stages of muscle differentiation. Our studies suggest the existence of a muscle-specific system, operating at physiological carnitine concentration, which gradually develops during muscle maturation in culture. We hypothesize that a defect of the low affinity muscle-specific uptake might be the cause of the primary muscle carnitine deficiency syndrome.
Adult↗
Expression and functional characterization of a soluble form of vascular cell adhesion molecule 1.
Vascular cell adhesion molecule 1 (VCAM1) is a leukocyte adhesion molecule induced on human endothelium in vitro and in vivo by inflammatory stimuli. A truncated cDNA for VCAM1 was constructed, stably expressed in Chinese Hamster Ovary (CHO) cells, and the secreted recombinant soluble form of VCAM1 (rsVCAM1) purified to homogeneity by immunoaffinity chromatography. Immobilized rsVCAM1 is a functional adhesion protein, and selectively binds only VLA4-expressing cells, including human B and T lymphocytes, NK cells, and certain lymphoblastoid cell lines. T cell subset analyses indicate preferential binding of CD8+ memory cells. rsVCAM1 should prove valuable for the further study of the role of VCAM1 during inflammatory and immune responses in vivo.
Amino Acid Sequence↗
Elimination of caffeine interference in high-performance liquid chromatographic determination of cotinine in human plasma.
A high-performance liquid chromatographic method with ultraviolet photometric detection for the determination of cotinine in human plasma was described. The use of a 30-cm reversed-phase column and of a mobile phase consisting of water-methanol-0.1 M sodium acetate-acetonitrile (72:21:5.6:1.4, v/v), pH 4.1, eliminated caffeine interference. A simplified solid-phase extraction procedure was also performed for plasma samples.
Caffeine↗
Endothelial-leukocyte adhesion molecule 1 stimulates the adhesive activity of leukocyte integrin CR3 (CD11b/CD18, Mac-1, alpha m beta 2) on human neutrophils.
Two classes of adhesion molecules have well-defined roles in the attachment of unstimulated polymorphonuclear leukocytes (PMN) to cytokine-treated endothelial cells. Endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial cells interacts with specific carbohydrate residues on the PMN, and the leukocyte integrins (CD18 antigens) on PMN interact with intracellular adhesion molecule 1 and other structures on endothelium. Here we show that these two classes of molecules can act sequentially in an "adhesion cascade". Interaction of PMN with ELAM-1-bearing endothelial cells causes PMN to express enhanced adhesive activity of the integrin CR3 (CD11b/CD18). Expression of ELAM-1 on the cytokine-treated endothelium appears both necessary and sufficient for the stimulation of CR3 activity since blockade of ELAM-1 with mAbs prevents the activation of CR3 by cytokine-treated endothelium, and immobilized recombinant ELAM-1 activates CR3. The ability to activate CR3 is shared by chemattractants, suggesting that ELAM-1 may serve as a "tethered chemattractant." This hypothesis is strengthened by the observation that recombinant soluble ELAM-1 directs movement of PMN in chemotaxis chambers. These results suggest a mechanism by which multiple adhesive molecules may function together in diapedesis. ELAM-1 serves both as an adhesin and as a trigger that recruits the participation of additional adhesion molecules. Our results also suggest that ligands for adhesion molecules may also be "receptors" capable of generating intracellular signals.
Antibodies, Monoclonal↗