PubMed Health⌕ Search

Biomedical subjects

M Luzzana

Publications and source records attributed to M Luzzana.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1984

The determination of ethanol in whole blood by differential pH measurements.

The application of a new technique based on the differential measurement of pH between two solutions to determine ethanol concentration in whole blood is reported. The ethanol is determined by measuring the change in pH following its enzymatic oxidation to acetaldehyde. The procedure correlates with the head space gas chromatographic method in the 0-108 mmol/l (0-5 g/l) ethanol whole blood concentration range according to the equation y = 1.344 + 1.013x (r = 0.997).

Acetaldehyde↗
PubMed1984

Urea, creatinine, and glucose determined in plasma and whole blood by a differential pH technique.

We report the conditions (buffer composition and enzyme activity) required for estimating three frequently determined analytes--urea, glucose, and creatinine--by use of an improved version of the differential pH apparatus previously described (Clin Chem 29: 80-85, 1983). For each analyte, we used only one specific enzyme, thus avoiding a chain of auxiliary and indicator reactions. The method requires about a minute for each determination in undiluted plasma or whole blood.

Blood Glucose↗
PubMed1983

Measurement of glucose in plasma by a differential pH technique.

A new automatic apparatus based on the differential measurement of pH between two solutions has been developed. Two 25-microL (internal volume) glass capillary electrodes are used to measure the results of automated (under microcomputer control) chemical reactions that lead to the liberation or the uptake of hydrogen ions. The sensitivity of the differential pH measurements is better than +/- 0.0001 pH unit, and the change in H+ concentration that can be detected by such an apparatus is 1 mumol/L for plasma and 3 mumol/L for whole blood. The technique has been applied to the measurement of glucose in plasma, giving results in agreement with the specifications of the Food and Drug Administration reference method for quantitative determination of glucose (hexokinase/glucose-6-phosphate dehydrogenase method).

Blood Glucose↗
PubMed1981

Equations and nomogram for the relationship of human blood p50 to 2,3-diphosphoglycerate, CO2, and H+.

We describe a new method for tonometry of small amounts of blood (up to 0.25 mL) at known pO2, PCO2, and temperature, in small, reusable, closed Pyrex flasks. Equilibrated blood is analyzed for oxygen saturation, pH, and organic phosphate concentration with standard techniques, and its p50 (the pO2 at which hemoglobin is half-saturated with oxygen) is determined with full control of all the variables known to affect it. The SD in the measurement of p50 is 0.044 kPa (0.33 mmHg). We made 63 determinations of p50 on normal human blood under different conditions of pH and pCO2, and with different concentrations of 2,3-diphosphoglycerate and ATP. Empirical equations and a nomogram were derived, which allow the calculation of p50 from known values of pCO2, pH, and [2,3-DPG]/[Hb4] molar ratio with a SD of 97 and 114 Pa (0.73 and 0.86 mmHg), respectively.

2,3-Diphosphoglycerate↗
PubMed1977

Oxygen equilibrium curve of normal human blood and its evaluation by Adair's equation.

Oxygen equilibrium curves of fresh, normal human blood have been measured by new methods which allow the control of pH, pCO2, and 2,3-diphosphoglycerate and which yield higher accuracy at the extremes of saturation than was possible previously. The curve determined by these techniques lies slightly to the right of the standard curve of Roughton et al. (Roughton, F.J.W., Deland, E.C., Kernohan, J.C., and Severinghaus, J.W. (1972) in Oxygen Affinity of Hemoglobin and Red Cell Acid Base Status (Astrup, P., and Rørth, M., eds) pp. 73-83, Academic Press, New York). The greatest difference is at low oxygen saturation, probably owing to the fact that the latter data were obtained under conditions which would lead to depletion of cellular 2,3-diphosphoglycerate. The range of p50 (oxygen pressure at half-saturation) values for four normal subjects was 28.3 mm Hg to 29.0 mm Hg. Adair's stepwise oxygenation scheme has been used to analyze the curves with the result that a1 = 0.1514 X 10(-1) (+/- 10%) mm-1; a2 = 0.9723 X 10(-3) (+/- 8%) mm-2; a3 = 0.1703 X 10(-3) (+/- 50%) mm-3; a4 = 0.1671 X 10(-5) (+/- 2%) mm-4 for the best of four data sets. Because these constants are very sensitive to changes in the shape of the oxygenation curve, this analysis is much more useful than p50 measurements in the investigation of the various allosteric effectors of the function of hemoglobin within the red cell.

Diphosphoglyceric Acids↗
PubMed1977

Simultaneous determination of hemoglobin derivatives, oxygen content, oxygen capacity, and oxygen saturation in 10 microliters of whole blood.

We describe a new method for simultaneous determination of four hemoglobin derivatives (deoxyhemoglobin, Hb; oxyhemoglobin, HbO2; methemoglobin, Hb+; and carbon monoxide hemoglobin, HbCO) and total oxygen content in 10 microliters of whole blood. Percentage HbO2, HbCO, Hb+, total hemoglobin (Hbt), and oxygen capacity can also be obtained from the experimental data by simple calculations. Total analysis time is 1 min. Blood is diluted 100-fold with a buffer contained in a quasi-anaerobic cuvette, where simultaneous measurements of oxygen pressure (by a po2 electrode) and adsorbance (at 497, 565, and 620 nm) are made. The decrease of oxygen pressure, as recorded by the oxygen electrode, is proportional to the amount of deoxyhemoglobin. The concentrations of HbO2, HbCO, and Hb+ can be obtained from absorbance measurements at the specified wavelengths. The new method eliminates the use of short-path optical cells and, due to the low sample volume requirement, makes possible the automated measurement of hemoglobin derivatives and oxygen saturation in arterialized capillary blood.

Carboxyhemoglobin↗
PubMed1976

Sherpas living permanently at high altitutde: a new pattern of adaptation.

Adaptation of Sherpas to high altitude has been studied and compared with that of Caucasians acclimatized to high altitude. Sherpas living permanently at 4000 m above sea level do not have increased hematological parameters (i.e., red cell number, hematocrit, hemoglobin content, and 2,3-diphosphoglycerate/hemoglobin ratio) and have a higher affinity of blood for oxygen as compared with acclimatized Caucasians. Sherpas permanently living at low altitude, on the contrary, have lower affinity of blood for oxygen than do Caucasians living at comparable altitude and are mildly "anemic,". Various other red cell biochemical parameters (possibly related to adaptation to altitude) have also been studied in the same population. We suggest that Sherpas are genetically better adapted to high altitude than are Amerindians living on the Peruvian highlands, possibly as a consequence of a much more prolonged exposure to such an ecological factor of selection as high altitude.

Adaptation, Physiological↗