The reliability of urinary albumin assay by immunonephelometry in the clinical practice. The critical role of medical surveillance on laboratory routine.

Measurement of the urinary albumin excretion rate (UAER) is essential for the early diagnosis and monitoring of diabetic nephropathy; immunonephelometry is a procedure used worldwide for routine screening of diabetic patients. Since we have met with occasional inconsistent values of UAER in serial urine collections, we searched for possible sources of analytic error. To assess the best working conditions of the instrument in use, the stability of urine samples during storage and the need for previous urine centrifugation, we assayed repeatedly the six automatically diluted points of the standard curve (55.6 to 1.7 mg/l), four control samples of human albumin in saline (100 to 1 mg/l) and 24-h urine collections from outpatient diabetic subjects. The last were also assayed with and without previous centrifugation, and both immediately after collection as well as after storage at -20 degrees C for 7, 42, 79, 97, 128 and 161 days. We concluded that: (1) pre-analytic centrifugation of urine samples in unnecessary; (2) the intra-assay coefficient of variation (CV) of the standard curve changed from 2.4% to 9.3% when moving from the highest to the lowest concentration; the inter-assay CV changed from 4.1% to 14.4%, respectively; (3) the intra-assay CV of the control samples (manually prepared) changed from 5.7% to 10.2% and the inter-assay CV from 7.7% to 22.9%; there was a constant and significant (P < 0.01) underestimation (from -9% to -30%) of the obtained values compared with the expected concentrations; (4) a progressive decrease in recovered albumin by multiple freezing and thawing of urine samples did occur, which became significant after 161 days of storage. In the BNA workbook (menu 7.1, assay protocols), a 7-day validity of the reference curve is reported. Moreover, to economize, pre-dilution cuvettes were often recycled in our hospital central laboratory. We observed that: the intra-assay CV for urine samples was 79.4% with recycled cuvettes and stored standard curve, 11.3% with new cuvettes and stored standard curve, 4.9% with both new cuvettes and newly performed standard curve; the inter-assay CV was 32.6%, 10.5% and 6.4%, respectively. These data emphasize, from the laboratory viewpoint, the need for both accurate calibration of BNA and use of native urines; in addition, they stress the importance of careful supervision of laboratory routine and interpreting analytic results in the clinical setting.

Effects of CDP-choline on platelet aggregation and the antiaggregatory activity of arterial wall in the rat.

The effects of the acute (250 mg/kg) and chronic (250 mg/kg for two weeks) treatments with CDP-choline on platelet aggregation and thromboxane formation and on the platelet antiaggregatory activity of thoracic aorta, have been studied in the rat. The acute administration resulted mainly in reduced platelet reactivity to aggregating agents, with no change of platelet thromboxane formation. The antiaggregatory activity of aortic walls was also concomitantly reduced. After chronic treatment, the major effect was a greater antiaggregatory activity of the vessel wall in respect of the control values, whereas platelet function was not affected. CDP-choline treatment, thus exerts favourable effects especially in the acute treatment, by reducing platelet reactivity.

Diets rich in saturated, monounsaturated and polyunsaturated fatty acids differently affect plasma lipids, platelet and arterial wall eicosanoids in rabbits.

New Zealand male rabbits, on a moderate dietary fat intake (10.2% w/w) received, as the major dietary lipid, butter, olive oil and corn oil, respectively, for a period of 8 weeks. At the end of the dietary treatment, plasma total cholesterol was significantly decreased in the corn oil group, compared to butter, whereas the olive-oil-consuming rabbits had an intermediate cholesterolemia; the corn oil and olive oil groups had significantly elevated high-density lipoprotein (HDL) cholesterolemia, compared to the butter group. Maximal platelet aggregability, with collagen and arachidonic acid, did not appear to differ in the three treatment groups. Thromboxane B2 release in the sera of treated rabbits was slightly higher after corn oil administration. The arterial release of prostacyclin (PGI2), tested by perfusing platelet-rich plasma through the aorta of donor rabbits, was lowest in the corn oil group. Corn oil is the most effective dietary fat in reducing cholesterolemia, but it may also reduce PGI2 release from arteries. Butter has the most unfavorable effect on lipidemia and HDL-cholesterol, whereas dietary olive oil shows an intermediate lipid-lowering activity but preserves arterial PGI2 production.

Platelets from aged rats aggregate more, but are more sensitive to prostacyclin.

The balance between vascular eicosanoids has been explored in young (1 month of age) and aged (11 month of age) rats. PRP from mature rats was more reactive to collagen (lower threshold, greater amplitude of aggregation curve and higher TxB2 formation) than PRP from young animals. Release of 6-keto-PGF1 alpha from PRP-perfused isolated aortas (pg@ul) was higher and the inhibition of PRP aggregation after perfusion correspondingly greater, in mature rats. Platelets from aged rats were more sensitive to exogenous prostacyclin (higher inhibition of aggregation and greater accumulation of cAMP). The fatty acid compositions of plasma and platelet lipids were not different in the two age groups. Compensatory mechanisms were operating in the aged rats, counteracting the greater platelet aggregability with higher vascular prostacyclin production and greater sensitivity of platelets to this eicosanoid.

Heated fat, vitamin E and vascular eicosanoids.

A semisynthetic diet containing adequate amounts of vitamin E and 10% (w/w) of a mixture of polyunsaturated oils subjected to heating and characterized by elevated indexes of thermal alteration (polar component, dimer triglyceride, altered triglyceride contents and reduced alpha-tocopherol levels) was fed to growing male rats for a period of eight weeks. It resulted in a selective alteration of the production of vascular eicosanoids (elevation of platelet thromboxane formation and decrease of vascular prostacyclin release) compared to the values found in rats fed a diet containing a fresh mixture of polyunsaturated oils. Major nutritional parameters, plasma lipids and the fatty acid profiles of plasma, liver and heart lipids were not different in the two groups of animals. Supplementation of an excess vitamin E (300 mg/kg) to the diet containing heated fat neutralized the adverse effects of heated fat on vascular eicosanoid production.

Sex differences in platelet thromboxane and arterial prostacyclin production in control and n-6 fatty acid supplemented rats.

Sex differences in eicosanoid production in platelets and vessel walls have been studied in control and n-6 fatty acid supplemented rats. In platelet rich plasma (PRP) of control female rats, arachidonic acid (AA) levels in phospholipids (PL), thromboxane B2 (TxB2) formation following collagen stimulation and aggregatory responses to collagen were higher than in PRP of male rats. 6 keto PGF1 alpha release from PRP-perfused isolated aortas were the same for both sexes, but the antiaggregatory activity of the wall was higher in males than in females, in association with a greater sensitivity of male platelets to prostacyclin. The administration of n-6 fatty acid supplements increased AA level in PL, TxB2 production and aggregation only in male platelets. Production of 6 keto PGF1 alpha and the antiaggregatory activity of aortic walls were reduced after dietary treatment in males, but biochemical and functional parameters were not correlated in females. The results indicate complex sex-related differences in fatty acid metabolism and eicosanoid production, and in responses to n-6 dietary fatty acids in platelets and the vascular system in the rat.

Blood lipids in Italy. Regional differences.

Serum cholesterol and triglyceride levels have been evaluated in samples from fasting males aged 20--59 in Northern (Brisighella), Central (Rome) and Southern (Pozzuoli) Italy. Regularly performed quality controls between laboratories assured comparability of data. A statisitically significant difference of mean serum cholesterol and triglyceride levels was observed for most age-groups in the 3 different areas, lower values being found in the southern population as compared to the central and northern ones. These results support previous findings and the thesis that large differences in blood lipid levels may still exist even within the same country and that they at least in part may be culturally determined in connection with different dietary habits.

[Evaluation and standardization of radioimmunoassays]. / Valutazione e standardizzazione dei dosaggi radioimmunologici

The term "standardization" may be applied to RIA methods to indicate the "definition of quality characteristics" the level of which must be defined on the basis of the evaluation of the "analytical procedure/specific reagents" system. Each laboratory, by applying a suitable long-term quality control system, must assure the consistent maintenance of the "standardization status" established for all mehtods it uses. As a matter of fact, in the case of RIA, materials are used that have a limited stability, that cannot be produced in large quantities, and that possess a complex composition not always well defined chemically. However, a remarkable role in the standardization process belongs to the producers of special reagents intended for RIA. In order to help the standardization of RIA methods, some programs of inter-laborator cooperation have been worked out in recent years. Among them mention is specifically made of three studies regarding the dosage of insulin: the most recent of them is devised and coordinated by the Instituto Superiore di Santià in cooperation with the Laboratory of Clinical Physiology of the National Research Council of Pisa. The availability of standardized materials is estremely important: for antigens, some materials are already in existence, but it is desirable that the various manufacturers shall follow more uniform criteria in their standardization. As far as the application of the RIA methods in the diagnostic field is concerned, the need exists for the use of standardized basic schemes that would include and unambiguously express all the useful information available in order to define better the clinical significance of the analytical data obtained.