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M Luzzana

Publications and source records attributed to M Luzzana.

At least 19 recordsLinked to original sources
PubMed1996

Simultaneous automated determination of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in whole blood.

We report a potentiometric fully automated method for determining red cell glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase activities and the glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index using 25 microliters of whole blood. No sample pre-treatment (e.g., preparation of the haemolysate) is needed and the measurements are performed at pH 8.0 and 37 degrees C under the conditions recommended by the ICSH committee. The reproducibility was constantly good, with within-run CV of 1.0% (glucose 6-phosphate dehydrogenase) and 5.9% (glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase) for activities in glucose 6-phosphate dehydrogenase non-deficient adults, and of 2.3% (glucose 6-phosphate dehydrogenase, G6PD) and 2.5% (glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase) for G6PDMediterranean heterozygotes. Linearity was observed up to an activity of 2800 U/l of glucose 6-phosphate dehydrogenase. Results of glucose 6-phosphate dehydrogenase activity (U/l) in whole blood (y) correlated well with those obtained with the previously described monostarter assay, performed at pH 9.2 (y = 0.60x + 37; n = 80; r = 0.991). Results of 6-phosphogluconate dehydrogenase (U/l) in whole blood (y) correlated well with those obtained by the ICSH recommended method (x) (y = 0.779x - 44; n = 23; r = 0.991). Reference intervals are reported for glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index relatively to normal, beta- and alpha-thalassaemic glucose 6-phosphate dehydrogenase non-deficient adults, to glucose 6-phosphate dehydrogenase deficient adult males and to G6PDMediterranean non-thalassaemic heterozygotes. We demonstrate that the diagnostic sensitivity of the glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index in detecting the G6PDMediterranean heterozygotes is superior to that of the glucose 6-phosphate dehydrogenase activity alone.

Adolescent↗
PubMed1995

A patient-side technique for real-time measurement of acetylcholinesterase activity during monitoring of eptastigmine treatment.

Rapid and reliable measurement of acetylcholinesterase (AChE) activity is of crucial importance to the pharmacodynamic monitoring of anticholinesterase drugs. A new assay has been developed to measure AChE from 10 microliter samples of capillary blood. AChE activity was calculated from the change in pH of the reaction medium caused by the hydrolysis of acetylcholine and measured with a highly sensitive differential pH apparatus (CL-10, Eurochem, Rome, Italy). Interference by butyrylcholinesterase was eliminated by a specific inhibitor, quinidine sulfate. The assay lasts 1 min. The coefficient of variation (CV) for replicated measurements was 2.8% (3267 U/L, n = 33). Linearity ranged from 0 to 10,000 U/L. The correlation coefficient between the new technique and Ellman's colorimetric method on washed erythrocytes was r = 0.987 (y = 1.299x - 63, n = 29). The correlation coefficient between assays on capillary and venous samples was r = 0.979 (y = 0.974x + 174, n = 47). A cross-laboratory validation study was performed in 10 centers using glycerol-stabilized hemolysates with normal and reduced AChE activity. Samples were assayed in triplicate. The within- and between-laboratory CVs for samples with normal AChE activity (6,018 U/L) were 2.2 and 8.1%, respectively. The new method was applied to a double-blind, placebo-controlled multicenter study of eptastigmine in Alzheimer patients and proved to be a simple, noninvasive, rapid, and reliable method for pharmacodynamic monitoring of this drug.

Acetylcholinesterase↗
PubMed1995

Fluorescence-based automated DNA sequencing by limited primer labeling.

The applicability of automated DNA sequencing systems to sequencing strategies that require a large number of primers is limited by the necessity for expensive fluorescence-labeled oligonucleotides. Here we present a simple procedure that allows the use of unlabeled oligonucleotides to perform fluorescence-based DNA sequencing. This method is based on a limited primer extension that incorporates three deoxynucleotides, one of which carries a fluorescent moiety. The elongated fluorescent primer is then used in a standard T7 sequencing reaction. This labeling procedure is both economical and straightforward and offers a valid alternative to current fluorescence-labeling protocols. Results of this method with different DNA templates demonstrate the reliability of the protocol.

DNA Primers↗
PubMed1995

Anion-exchange HPLC analysis of biotinylated oligonucleotides.

Biotinylated oligonucleotides combined with streptavidin-coated magnetic beads are commonly used in current molecular biology. Their quality and the level of incorporated biotin are essential for yielding good results in either solid-phase DNA sequencing or solid-phase purification procedures. This paper presents a very simple analytical test using anion-exchange HPLC and avidin to ascertain the quality of biotinylated oligonucleotides and to predetermine their ability to bind to avidin, which is a prerequisite for functionality in some solid-phase methods.

Avidin↗
PubMed1994

Mixed-mode fluorescent DNA sequencing.

Fluorescence-based, automated DNA sequences represent one of the major advances in recent molecular biology. Two main technologies have been developed in this field: the single-label/four-lane system and the four-label/one-lane system. The following present the use of single-label-sequencing chemistry, which resembles traditional radioactive DNA sequencing, using the four-label system ABI 373A that expands its flexibility and obtains data that are immediately interpretable without software manipulation. This method has been named mixed-mode fluorescent DNA sequencing. Here we show one of its possible applications in molecular genetic analysis.

Autoanalysis↗
PubMed1993

Pharmacodynamics and pharmacokinetics of eptastigmine in elderly subjects.

Eptastigmine is a new cholinesterase inhibitor, which may be potentially useful for the symptomatic treatment of Alzheimer's disease. A preliminary evaluation of its pharmacodynamic and pharmacokinetic profiles in the elderly has now been made in 6 healthy subjects (63-84 years of age) given 30 mg eptastigmine as a single oral dose. Blood was collected prior to and 1, 2, 3, 4, 6, 8, and 12 h after eptastigmine administration for measurement of cholinesterase inhibition in plasma and red blood cells and the plasma drug concentrations. The maximum plasma cholinesterase inhibition was 17%, which was reached 2.7 h after treatment. In red cells the maximum inhibition of the enzyme was 29% after 3.8 h. The estimated half-time of cholinesterase recovery was 12.4 h in plasma and 13.6 h in red blood cells. The peak plasma concentration of eptastigmine of 0.86 ng.ml-1 was reached after 1.4 h. Following absorption the drug was rapidly distributed into tissues (t1/2 alpha = 0.44 h) and then eliminated with a half-life of 12.1 h. The drug was well tolerated in all but one subject, who showed bradycardia with hypertension and nausea for about 2 h after the dose. The results indicate that oral administration of eptastigmine to elderly subjects produces long lasting inhibition of cholinesterase activity in plasma and in red blood cells.

Administration, Oral↗
PubMed1993

Rapid potentiometric determination of cholinesterases in plasma and red cells: application to eptastigmine monitoring.

Eptastigmine (MF 201) is a new physostigmine derivative with potent inhibitory activity on cholinesterases. Here we present a new potentiometric cholinesterase activity assay suitable for MF 201 monitoring. The analysis is performed on a differential pH system and has the following characteristics: (a) within-run precision: C.V. 2.0% (plasma cholinesterase), 1.8% (red cell cholinesterase); (b) between-run precision: C.V. 4.0% (plasma cholinesterase); (c) linearity: 1-10 kU/l (plasma cholinesterase), 6-70 U/g Hb (red cell cholinesterase); (d) comparison with a reference method (x, HITACHI 737 Boerhinger Mannheim, Italy): y = 0.785x - 0.07; n = 37; r = 0.998. The assay has been applied to the determination of plasma and red cell cholinesterase activity in volunteers over 60 years of age treated with a single oral dose of 30 mg eptastigmine. We found that red cell cholinesterase is selectively inhibited after MF 201 administration with the following kinetics (time, % of inhibition, mean +/- S.E., n = 6): 0 h, 0; 1 h, 17 +/- 4.6; 2 h, 24 +/- 4; 4 h, 23 +/- 4.4; 12 h, 14 +/- 3. Eptastigmine plasma levels were also determined by a HPLC method: maximum concentration was found one hour after drug administration.

Administration, Oral↗
PubMed1993

Introduction of magnetic beads in diagnosis: a simple and rapid method to detect mutations of beta globin gene, directly amplified from blood.

In this communication we present the results obtained by the use of magnetic beads in diagnosis, for the identification of genetic variants at the molecular level by sequencing, in comparison with the more laborious method of the production of ssDNA with asymmetric PCR. We compared the two techniques studying variants of beta globin gene: Hb Abruzzo [beta 143 (H21) His -> Arg] and Hb D Los Angeles [beta 121 (GH4) Glu -> Gln].

Base Sequence↗
PubMed1992

Dideoxy linear PCR on a commercial fluorescent automated DNA sequencer.

The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods play an important role in the actual effort to improve the efficiency of large-scale DNA analysis. Here we show the application of the linear PCR using a single fluorescent primer and dideoxynucleotide terminators in four separate sequencing reactions on the EMBL/Pharmacia's fluorescent automated DNA sequencer. We have used dideoxy/deoxynucleoside triphosphate ratios and linear amplification cycle conditions to obtain an accurate sequencing response of up to, and over, 500 bases from just 400 ng of double-stranded DNA template without chemical denaturation. The sequencing protocol described in this paper is effectively suited for enhancement of sensitivity and performance of the automated DNA sequencing system.

Autoanalysis↗
PubMed1987

A horizontal apparatus for isoelectric protein purification in a segmented immobilized pH gradient.

A modification of the previously described apparatus (Faupel et al. (1987) J. Biochem. Biophys. Methods 15, 147-162), for recycling isoelectric focusing in a segmented immobilized pH gradient, is here reported. The most important improvements are: (1) a horizontal, vs. the previously vertical assembly; (2) a reduction of the thickness of the central flow chamber to 6 mm, vs. the previous 3 cm length and (3) the introduction, at both gel extremities of each Immobiline segment, of polypropylene filters, thus efficiently blocking the gel in situ. The advantages are: (i) the spontaneous removal of air bubbles, which in the vertical apparatus tend to accumulate in the ceiling of the flow chamber and to obstruct the flow of electric current; (ii) a more efficient hydraulic flow with a reduced chance of heating the liquid stream in the flow chamber, due to its reduced length along the separation path and (iii) a reduced risk of gel detachment from the tube walls, due to osmotic swelling caused by focused protein zones in the gel phase and by the fixed Immobiline charges in the polyacrylamide matrix.

Adult↗
PubMed1987

Urea determination in dialysis, based on a differential pH technique.

The application of a new technique, based on differential measurements of pH, to determine urea concentration in patients of a dialysis center, is reported. Urea in plasma, whole blood or dialysis fluids is measured by an enzymatic reaction, with urease; the procedure, requiring 10 microL of sample, is simple, fast and correlates well with a reference spectrophotometric method, in the 0-300 mg/dL concentration range, according to the equation y = 1.0291 X -0.0777; r = 0.9991; n = 73.

Autoanalysis↗
PubMed1987

Erythrocytic glucose-6-phosphate dehydrogenase measured by a differential pH technique.

We propose a new quantitative electrochemical method for determining glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity in purified erythrocytes or in whole blood, based on measurement of the pH change caused by oxidation of glucose 6-phosphate to 6-phosphogluconic acid, with simultaneous reduction of NADP+ to NADPH + H+. No sample pretreatment (e.g., preparation of hemolysate) is needed, and the automatic correction for sample blanks obviates interference from 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The method is simple and fast, and the standard curve is linear to at least 2200 U/L at 37 degrees C. The within-day CV was 3.9% for activities in healthy individuals (mean value 1204 U/L), and 10% for deficient ones (classified as belonging to class II, mean value 407 U/L). Results (y) correlated well with those obtained with the WHO-recommended method (x): y = 1.13x + 0.02 (r = 0.971) for purified erythrocytes. Normal reference intervals are reported for the enzyme in purified erythrocytes and in whole blood.

Erythrocytes↗
PubMed1986

Measurement of erythrocyte acetylcholinesterase and plasma cholinesterase activity by a differential pH technique.

We describe a new electrochemical method for the determination of erythrocyte acetylcholinesterase activity (EC 3.1.1.7) and plasma cholinesterase (EC 3.1.1.8) activity, based on the measurements of pH variation due to release of acetic acid from acetylcholine. The major advantages of the differential pH procedure are simplicity, high reproducibility, no need for pre-treatment of samples, automatic correction of sample blanks, and speed and direct measurement of enzymatic reaction. The proposed methods are linear up to 7400 U/L at 30 degrees C and correlate well with the manual spectrophotometric method of Ellman for plasma cholinesterase and for washed erythrocytes. We adapted the same technique for the determination of erythrocyte cholinesterase using whole blood as sample and quinidine sulphate as inhibitor of pseudocholinesterase.

Acetylcholinesterase↗
PubMed1985

Measurement of lipase activity by a differential pH technique.

This is a new electrochemical method for determination of lipase activity in biological fluids, including serum, plasma, and duodenal juice. Advantages of turbidimetric methods--short reaction time, and small sample and reagent volumes--are combined with those of titrimetric methods: measurement of absolute activity (i.e., no standardization required), saturated substrate conditions, and direct measurement of reaction products. The proposed method is easy, inexpensive, and takes only 3 min. Precision is good: CV = 3.74% within day and 7.3% between days at the clinical-decision concentration, CV = 1.86% within day and 4.65% between days for above-normal lipase activities. The standard curve is linear up to 4500 U/L. Results (y) correlate well with those by turbidimetry (x): y = 0.9287x - 65.3 (r = 0.9719). Reference values are between 0 and 130 U/L.

Adult↗