The PASAT performance among patients with multiple sclerosis: analyses of responding patterns using different scoring methods.

The Paced Auditory Serial Addition Test (PASAT) is widely used in the evaluation of multiple sclerosis (MS) patients' cognitive performance, and also used as the sole measure of cognition in a recently developed assessment tool for MS clinical trials, the Multiple Sclerosis Functional Composite (MSFC). We analysed if MS patients and healthy controls have different patterns of responding in the PASAT, and whether different scoring methods influence the PASAT's sensitivity and specificity in detecting disease-associated cognitive impairment. Forty-five relapsing-remitting MS patients and 48 healthy controls were evaluated using the PASAT and a comprehensive neuropsychological examination. Cognitively deteriorated MS patients compensated for their difficulties in PASAT by omitting rather than guessing answers. They skipped items intermittently, which reduces the difficulty of the task. Furthermore, towards the end of the PASAT's 60-item series MS patients' performance had a trend to fade whereas controls' performance was more even throughout the task. The dyad score or the percent dyad score did not essentially improve the sensitivity or the specificity, but the accuracy improved when the answers at the end of the PASAT series were specifically emphasized. Using the combined score, 73% of the patients were correctly classified as cognitively impaired or unimpaired.

Electrospray ionization mass spectrometry of synthetic oligonucleotides using 2-propanol and spermidine

Oligonucleotides have become widely used tools in molecular biology and molecular diagnostics. Their parallel synthesis in large numbers and the increasing interest in microarray technology has raised the requirement for fast and informative analytical tools for their quality control. A direct injection electrospray ionization mass spectrometry (ESI-MS) technique based on the use of aqueous 2-propanol as running eluent, and spermidine (or triethylamine) as DNA modifiers, has been applied to analyze a large set of samples (about 200 synthetic oligonucleotides) ranging from 5 to 15 kDa (17-51mers) with good results in terms of sensitivity, suppression of sodium adduct formation, and speed of analysis. Copyright 2000 John Wiley & Sons, Ltd.

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.

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.

IHP entrapment into human erythrocytes: comparison between hypotonic dialysis and DMSO osmotic pulse.

Three different blood units were treated separately by the hypotonic dialysis (HD) and the dimethylsulphoxide osmotic pulse (DMSO) method, in order to load the erythrocytes with inositol hexaphosphate. A detailed comparison between the two loading techniques was performed by monitoring the red cell distribution patterns on discontinuous Percoll density gradients, the RBC oxygen affinity and the amount of the main intracellular organic phosphates with the 31P-NMR. The results obtained showed that: (1) The HD loading produces a redistribution of the RBC fractions with a concomitant smoothing of the relative differences among distinct fractions (2) only a minor portion of erythrocytes (from 8.5 to 24.9% of total RBCs) are loaded with IHP after the DMSO treatment. All of these cells move to the lightest fraction (d = 1.080 g/ml). (3) Both HD and DMSO IHP-loaded cells show an increase in P50 (basal vs. after loading, means +/- SD: 25.8 +/- 3.0 vs. 52.5 +/- 3.2 mm Hg) correlated to the IHP incorporation (mean intracellular IHP concentration: 4.2 mmol/l RBC). (4) probably the IHP incorporation efficiency could be probably improved at least by increasing the IHP concentration during the treatment.