Biomedical subjects
Publications and source records attributed to I Fermo.
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OBJECTIVE: To evaluate the prevalence of moderate hyperhomocysteinemia and inherited thrombophilia disorders (congenital defects of the natural anticoagulant or fibrinolytic mechanisms) in patients with early-onset venous or arterial thromboembolic disease. DESIGN: Cross-sectional 2-year evaluation of consecutive unrelated patients with a history of venous or arterial occlusive disease occurring before the age of 45 years or at unusual sites, in the absence of local predisposing factors. SETTING: Thrombosis research unit of a community hospital. PATIENTS: 107 patients with venous thromboembolism (mean age at event, 32.9 +/- 11.9 years) and 50 patients with arterial occlusive disease (mean age at event, 31.1 +/- 10 years) who did not have acquired coagulation defects, overt cancer, or acquired conditions affecting methionine metabolism. MEASUREMENTS: Total plasma homocysteine (fasting levels), antithrombin III, protein C, protein S, activated protein C resistance, plasminogen, and heparin cofactor II were measured at least 3 months after the event. In 87 patients, total plasma homocysteine levels were also measured 8 hours after an oral methionine load was administered (L-methionine, 0.1 g/kg body weight). Ninety-fifth percentiles of the distribution of these variables were established in 60 apparently healthy persons; sex-specific ranges were used for protein S and total plasma homocysteine. Relatives of patients with laboratory abnormalities were studied to confirm inheritance of the defects. RESULTS: Moderate hyperhomocysteinemia was detected in 13.1% (95% CI, 7.6% to 21.3%) and in 19.2% (CI, 9.0% to 31.9%) of patients with venous or arterial occlusive disease. The prevalence of hyperhomocysteinemia was almost twice as high when based on homocysteine measurements done after oral methionine load as when based on fasting levels. The remaining defects were detected only in patients with venous occlusive disease (activated protein C resistance in 11.2% of patients, protein S or C deficiency in 6.6%, and plasminogen deficiency in 0.9%), with an overall prevalence of 18.7% (CI, 12.1% to 27.6%). Inheritance of hyperhomocysteinemia and of the other defects was confirmed in 26 of the 30 families studied. Event-free survival analysis showed that the relative risk for occlusive disease in patients with moderate hyperhomocysteinemia and other defects was 1.70 times (CI, 1.19 to 2.42; P < 0.01) greater than in patients without defects. After adjustment for the presence of predisposing factors (for example, use of contraceptive drugs, pregnancy, surgery, prolonged bedrest, smoking, mild hypertension or dyslipidemia) and a family history of thrombosis, the age at first event of patients with moderate hyperhomocysteinemia was similar to that of patients with the other defects (26.4 +/- 11.2 years compared with 25.2 +/- 10.6 years), and the 43 patients with defects were significantly younger at first event than the 114 patients without defects (25.5 +/- 11.1 years compared with 31.0 +/- 12.3; P < 0.005). Patients with mild hyperhomocysteinemia had a higher rate of recurrence than those without defects (52% compared with 25%; P = 0.01); among the 56 patients who had their first event more than 1 year before observation, the recurrence rate was higher (80% [CI, 51% to 95%]) in patients with defects than in patients without defects (41% [CI, 26% to 57%] P = 0.01). CONCLUSIONS: Moderate hyperhomocysteinemia may have pathogenic significance in premature venous and arterial occlusive disease and should be included among the (inherited) disorders of venous and arterial thrombophilia.
The aim of the study was to evaluate the effects of an acute increase in triglyceride levels induced by Intralipid (Kabivitrum, Stockholm, Sweden) infusion on forearm glucose uptake, glucose oxidative metabolism, and hepatic glucose production independent of circulating free fatty acid (FFA) levels in man. Six normal subjects underwent three different tests in random order. Each test consisted of a control period of 120 minutes followed by a euglycemic, hyperinsulinemic clamp lasting 120 minutes. In test 1, a high-dose intravenous Intralipid infusion was performed to increase triglyceride and FFA levels. In test 2, heparin (30 U/min) plus low-dose Intralipid infusions were performed to maintain triglyceride at normal levels and increase only FFA levels. Test 3 was performed as a control study. During the 120-minute control period, forearm glucose uptake and hepatic glucose production were not affected by increasing only FFA levels (test 2) or FFA and triglyceride levels (test 1) as compared with the control study. On the contrary, glucose oxidation was significantly decreased as compared with the control study during tests 1 and 2, without a further significant decrease during simultaneously increased FFA and triglyceride levels. Concomitantly, lipid oxidation was similar in tests 1 and 2, at values significantly greater than in test 3. During the euglycemic clamp, forearm glucose uptake and glucose oxidation were significantly lower during tests 1 and 2 than test 3. At variance with the control period, the increase of triglyceride levels during test 1 caused a significant 30% to 40% decrease of both parameters as compared with test 2.(ABSTRACT TRUNCATED AT 250 WORDS)
This HPLC assay with o-phthalaldehyde precolumn derivatization is used to measure the total, oxidized, and protein-bound forms of glutathione in human blood, plasma, and rat tissue. Total glutathione (i.e., sum of reduced, oxidized, and protein-bound fractions) was determined after reduction with dithiothreitol and protein precipitation with perchloric acid (PCA). A preliminary selective blockage of free sulfhydryl groups with N-ethylmaleimide was necessary to evaluate the different oxidized forms. The assay showed high sensitivity (< 0.05 pmol injected) and good precision (within-day CVs of 5.5% to 6.4%), recovery (101% +/- 4%), and linearity (r > 0.999). Samples, after PCA acidification, were stable at room temperature and 4 degrees C for 3 days, and at -20 degrees C and -80 degrees C for > 1 month. The method (involving automated derivatization) not only is very rapid and simple but also allows immediate processing of many different biological samples.
Hydrolysis of cyclosporin A (CsA) was studied in order to clarify the still undefined point of attack of the acidic degradation. Among ether extractable and water-soluble products formed from CsA in HCl, two open-chain peptides were isolated by high-performance liquid chromatography which were identified as the deca- and nonapeptides deriving from CsA through the hydrolytic cleavage of amino acid residue 11 and both residues 11 and 10, respectively. Identification was carried out by fast atom bombardment tandem mass spectrometry.
The aim of the study was to investigate the effects of two hypocaloric (800-kcal) diets on body weight reduction and composition, insulin sensitivity, and proteolysis in 25 normal glucose-tolerant obese women. The two diets had the following composition: 45% protein, 35% carbohydrate (CHO), and 20% fat (HP diet, 10 subjects), and 60% CHO, 20% protein, and 20% fat (HC diet, 15 subjects); both lasted 21 days. A euglycemic hyperinsulinemic (25 mU/kg/h) clamp lasting 150 minutes combined with indirect calorimetry was performed before and after the diet. Both diets induced a similar decrease in body weight and fat mass (FM), whereas fat-free mass (FFM) decreased only after the HC diet. 3-Methylhistidine (3-CH3-HIS) excretion was reduced by 48% after the HP diet and remained unchanged after the HC diet (P < .05). A significant correlation was found between the changes in FFM and in 3-CH3-HIS excretion after the diet (rs = .50, P < .02). Blood glucose remained unchanged, while insulin decreased in both diets. Free fatty acids (FFA) significantly increased only after the HC diet (P < .05). During the clamp period, glucose disposal and glucose oxidation significantly increased after the HP diet and significantly decreased after the HC diet. Opposite results were found when measuring lipid oxidation. In conclusion, our experience suggests that (1) a hypocaloric diet providing a high percentage of natural protein can improve insulin sensitivity; and (2) conversely, a hypocaloric high-polysaccharide-CHO diet decreases insulin sensitivity and is unable to spare muscle tissue.
The capillary zone electrophoresis (CZE) technique was evaluated for separation and quantification of human growth hormone (hGH), human insulin (hI), and proinsulin. Three different molecular forms of biosynthetic hGH (20K, 22K, 44K), methionyl-hGH, biosynthetic hI, and proinsulin, were studied. The hormones were separated with uncoated capillaries, and various analytical conditions were tested (different buffers, ionic strength, pH). The samples were introduced both at positive pressure and electrokinetically, and the voltage applied was varied in each case. Linearity, repeatibility, and limit of detection (0.8 microM for hGH and 1.3 microM for insulin) were determined. Different purification steps were tried in order to find a suitable preanalytical procedure for hGH and hI purification from plasma. Recovery rates from 65 to 80% were obtained.
A high-performance liquid chromatographic method for the determination of total plasma homocyst(e)ine [H(e)] after reduction with sodium tetrahydroborate and precolumn derivatization with o-phthaldialdehyde is described. The analyses, carried out on a reversed-phase C18 column, were based on spectrofluorimetric detection. The sensitivity was 1 pmol per injection and the intra- and inter-assay relative standard deviations were 1.8% and 5%, respectively. The plasma H(e) concentration determined in 40 healthy volunteers (20-60 years old) was 12.4 +/- 2.9 microM (mean +/- S.D.), in good agreement with reference values.
A precolumn derivatization method was optimized for rapid and specific analysis of total urinary hydroxyproline by HPLC. After an overnight hydrolysis, urine samples dried and reconstituted with the internal standard cysteic acid (in sodium hydrogen carbonate, pH 9.3) were derivatized with N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA) at 100 degrees C for 20 min. The DNDEA-hydroxyproline adduct was separated on an Ultrasphere ODS column with a mobile phase of acetate buffer (containing triethylamine, 6 mL/L, pH 4.3) and acetonitrile (80/20, by vol), and was detected at 360 nm. A single run took 18 min with a hydroxyproline retention time of 7.3 min. The assay showed a linear response to hydroxyproline concentrations from 5 to 100 mg/L with a detection limit of 0.8 ng injected, corresponding to 2 mg/L in urine. Mean (SD) analytical recovery was 94.2 (13)% and 104 (9)% at 10 and 50 mg/L, respectively. Within-run and between-run CVs (n = 10) were 3.74% and 4.33%, respectively, for 25 mg/L. Results for samples (n = 50) analyzed by HPLC (y) vs ion-exchange chromatography with postcolumn ninhydrin reaction (x) correlated well: y = 0.98x + 1.02 (r = 0.985, Sxy = 3.13). In another comparison, involving 173 samples, a colorimetric procedure (Hypronosticon, x) gave slightly higher values than the HPLC method (y): y = 0.83x + 2.21 (r = 0.937, Sxy = 4.6).
We compared two pre-column derivatization methods, o-phthaldialdehyde (OPA) and N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA), for analysis of serum amino acids by reversed-phase high-performance liquid chromatography. Separations took 102 and 106 min for FDNDEA and OPA (reconditioning included), respectively, allowing a very good resolution of 30 amino acids by the former process and 38 by the latter. Linearity, within- and between-day variability and advantages in terms of accuracy and speed were studied for both methods. Twenty serum samples from healthy volunteers were assayed with OPA, FDNDEA and with the reference method of ion-exchange and post-column ninhydrin reaction (amino acid analyser), which took 170 min. The correlation between OPA and ninhydrin was good for all the amino acids (r = 0.959) except for the last-eluting lysine. Good agreement was found for FDNDEA (r = 0.987), which appeared in general to be a highly reproducible technique. Both pre-column methods were more sensitive than the post-column ninhydrin method.
We describe an HPLC ion-pair procedure for rapid and specific evaluation of creatinine in serum and urine. We used a 15 cm X 4.6 mm ODS column with a 50/50 (by vol) mixture of sodium decanesulfonic acid (10 mmol/L, pH 3.2) and methanol and measured absorbance at 236 nm. Serum (100 microL) or 30-fold-diluted urine (100 microL) was added to 400 microL of acetone. After centrifugation, the supernates (300 microL) were dried, reconstituted with the mobile phase, and injected into the HPLC. Assay precision was tested for concentrations of 10, 29, and 130 mg/L and yielded, respectively, 3.1%, 2.1%, and 1.1% for within-day CV and 2.8%, 2.1%, and 2.2% for total CV. Analytical recovery was 102 (+/- 6.7%). Linearity was demonstrated in the 0-200 mg/L range for serum and 0-3.5 g/L range for urine (r greater than or equal to 0.999). The detection limit for creatinine (signal-to-noise ratio = 3) was 0.5 mg/L. We used cimetidine for internal standardization. Correlation was good between this procedure and the Jaffé kinetic, the enzymatic (creatinine amidohydrolase), and the Fuller's earth alkaline picrate methods.
We compared the performance of two highly resolving methods, chromatofocusing (CRF) and isoelectric focusing in immobilized pH gradients (IPGF), for the separation of human hemoglobin variants. Lysates containing 13 different hemoglobins, including variants of clinical and geographical importance, and four electrophoretically "silent" variants (Hb Brockton, Hb Cheverly, Hb Köln, and Hb Waco) were analyzed. Both techniques showed a good intrarun precision (CV = 0.87% for CRF, 0.27% for IPGF) and high and similar resolving power (0.010 pH units, with the pH gradients used in this work). The use of an ultranarrow IPGF range (pH 7.15-7.35; pH gradient = 0.019 pH/cm) allowed the resolution between Hb Brockton, Hb Köln, and Hb A. In some cases (Hb D-Los Angeles, Hb F, Hb Waco), the variants were separated from Hb A in different orders, depending on which technique was used, probably because of the different analytical principles of the two methods. As a second-level test, both procedures are informative for characterization of human hemoglobin variants.
A new method for the separation and quantification of primary and secondary amino acids by reversed-phase high-performance liquid chromatography and spectrophotometric detection is described. The use of the novel derivatizing reagent N,N-diethyl-2,4-dinitro-5-fluoroaniline yields derivatives that are more stable to light and heat in solution than the traditional ones. A simple gradient between acidic acetate buffer and acetonitrile allows the complete separation of 21 amino acids in 80 min at room temperature. The good reproducibility of peak areas and retention times allows the application of this simple and low-priced method to the determination of amino acids in the range 50-500 pmol per residue.
Investigations into the properties of haemoglobin often require the isolation of the valence intermediates (alpha o2 beta+)2 and (alpha+ beta o2)2. Chromatofocusing with an anion-exchange gel (Mono PTM; Pharmacia, particle size 10 micron) in an HR5/20 column at various temperatures (10-25 degrees C) provides an excellent method for this task. A linearly decreasing pH gradient (8 to 7, generated by Polybuffer 96, Pharmacia) eluted sequentially the species methaemoglobin, (alpha o2 beta+)2, (alpha+ beta o2)2 and oxygenated haemoglobin. Calibration graphs help in quantitative analyses. This method is simpler and less time consuming and provides a similar or even better resolution than the traditional ion-exchange or isoelectric focusing methods.
We describe here conditions for the in vitro preparation of the main metabolite of cyclosporin A, namely AM1, with rabbit liver enzymes. The compound was purified and was identified by comparison with the metabolite isolated from urine of rats treated with [3H]CsA and by its Mass-Spectrometry Fast Ion Bombardment (MS-FIB) and 1HNMR spectra. The procedure is simple and with the enzyme fraction derived from one rabbit liver 3-4 mg of the pure metabolite can be obtained.
BACKGROUND: The plasma concentration of asymmetrical dimethylarginine (ADMA), an inhibitor of nitric-oxide synthase, which has been linked to endothelial dysfunction and atherosclerosis in the general population, is raised in patients with end-stage renal disease and could contribute to the high cardiovascular risk in patients with chronic renal failure. We investigated the relation between cardiovascular risk factors and plasma ADMA concentration in a cohort of haemodialysis patients (n=225), and tested the predictive power of ADMA for mortality and cardiovascular outcomes. METHODS: Patients had standard dialysis three times a week. We accurately recorded cardiovascular events over a mean follow-up of 33.4 months (SD 14.6); these events were reviewed by a panel of physicians. We identified correlates of plasma ADMA by univariate and multivariate analyses. FINDINGS: On univariate analysis, ADMA concentration in plasma was directly related to concentrations of fibrinogen and L-arginine in plasma, duration of dialysis treatment, and serum cholesterol concentration, and was inversely related to serum albumin concentration. On multivariate analysis, only plasma fibrinogen (p=0.0001) and serum albumin (p=0.04) concentrations were independently related to plasma ADMA concentration (multiple r=0.44, p=0.0001). 83 patients died, 53 (64%) by cardiovascular causes. In a Cox's proportional-hazards model, plasma ADMA ranked as the second factor predicting overall mortality (hazard ratio 1.26, 95% Cl 1.11-1.41, p=0.0001) and cardiovascular events (1.17, 1.04-1.33, p=0.008). INTERPRETATION: In haemodialysis patients, plasma ADMA is a strong and independent predictor of overall mortality and cardiovascular outcome. These findings lend support to the hypothesis that accumulation of ADMA is an important risk factor for cardiovascular disease in chronic renal failure.
BACKGROUND AND OBJECTIVE: In spite of the large number of reports showing that hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis and arterial occlusive disease, this metabolite of the methionine pathway is measured in relatively few laboratories and its importance is not fully appreciated. Recent data strongly suggest that mild HHcy is also involved in the pathogenesis of venous thromboembolic disease. The aim of this paper is to analyze the most recent advances in this field. EVIDENCE AND INFORMATION SOURCES: The material examined in the present review includes articles and abstracts published in journals covered by the Science Citation Index and Medline. In addition the authors of the present article have been working in the field of mild HHcy as cause of venous thromboembolic disease. STATE OF ART AND PERSPECTIVES: The studies examined provide very strong evidence supporting the role of moderate HHcy in the development of premature and/or recurrent venous thromboembolic disease. High plasma homocysteine levels are also a risk factor for deep vein thrombosis in the general population. Folic acid fortification of food has been proposed as a major tool for reducing coronary artery disease mortality in the United States. Vitamin supplementation may also reduce recurrence of venous thromboembolic disease in patients with HHcy. At the present time, however, the clinical efficacy of this approach has not been tested. In addition, the bulk of evidence indicates that fasting total homocysteine determinations can identify up to 50% of the total population of hyperhomocysteinemic subjects. Patients with isolated methionine intolerance may benefit from vitamin B6 supplementation. Homocysteine-lowering vascular disease prevention trials are urgently needed. Such controlled studies, however, should not focus exclusively on fasting homocysteine determinations and folic acid monotherapy.
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