Current problems in clinical biochemistry最新文献

Ligandinuria is a useful index of acute tubular injury. Ligandin probably enters the urine at the time of initial necrosis and should be looked for soon after the toxic or ischemic event. Periodic examination of perfusates for this substance might yield useful information about techniques for storage of cadaver kidneys.

The excretion of antigens and enzymes derived from the brush border region was studied in patients with kidney diseases, after kidney transplantation, during administration of potential nephrotoxic drugs, before and after operations etc. The main portion of membrane constituents was excreted in the urine at an increased rate, compared to healthy persons, and was identical with glycoproteins artificially released from the brush border membrane surface. Antisera against brush border antigens, which had been isolated from urine by affinity chromatography, were used to localise the origin of urinary kidney tissue-proteins applying immunofluorescence microscopy.

This study compares the usefulness of serum and urine fibrin split products and the urinary enzyme, beta-glucuronidase, in the diagnosis and management of renal transplant rejection. Fibrin split products, determined by a tanned human red cell agglutination inhibition immunoassay, were measured as a reflection of the secondary fibrinolysis from fibrin deposited in the renal microvasculature as a result of rejection. Urinary beta-glucuronidase, expressed as the ratio of enzyme activity to creatinine concentration, was determined by a colorimetric technique following dialysis of urine to remove endogenous activators and inhibitors. Activity of this lysosomal enzyme is thought to reflect tubular injury. Twenty-nine renal transplant recipients (15 from living donors and 14 from cadaver donors) were evaluated. Both serum and urinary fibrin split products and urinary beta-glucuronidase were markedly elevated in the immediate postoperative period, probably reflecting ischemic trauma. Acute rejection occurring within the first three months was associated with elevations of fibrin split products (particularly urine) and beta-glucuronidase. Elevated values returned to normal following successful treatment with steroids and/or heparin, but remained high in the presence of continued rejection. After the first 48 hours post-transplant, in the absence of rejection, values for fibrin split products were within the normal range. Urinary beta-glucuronidase remained elevated if the transplanted kidney was recovering from acute tubular necrosis. Fibrin split products and urinary beta-glucuronidase were usually normal in chronic rejection.

The activities of several enzymes in urine are masked by the presence of interfering substances in native urine. From several methods proposed for the removal of low molecular mass interferences dilution, dialysis, gel filtration, and ultrafiltration have been successfully applied. Gel filtration seems to be of these most suitable. I is effective, accurate, precise and economical. Scale-down procedures provide for acceptable speed. By this method the complete separation of lactate dehydrogenase, gamma-glutamyltransferase, alkaline phosphatase, arylsulphatase A, alpha-glucosidase, beta-galactosidase, trehalase, N-acetyl-beta-glucosaminidase, beta-glucuronidase and leucine arylamidase from low molecular mass substances, e.g. a heat-stable, competitive inhibitor of N-acetyl-beta-glucosaminidase was possible. The preparation and determination of urinary enzymes should be thoroughly standardized and controlled. Acceptable precision (coefficient of variation less than 10% between-day) can be achieved with manual spectrophotometric methods.

The relative sensitivity of urinary enzyme measurements for detecting renal damage was determined for two nephrotoxins. Injection of a single dose of sodium phosphate (10 mmoles/kg) caused damage to the proximal tubules and led to a 15 fold increase in lactate dehydrogenase (LDH) activity excreted into the urine. In contrast to this change the serum LDH remained normal. Similar results were obtained following the injection of cephaloridine (2 g/kg) with an 18 fold increase in urinary LDH and a marginal increase in urinary glutamate dehydrogenase (GDH). By contrast the serum LDH was unchanged. Urinary enzymes are therefore more sensitive for detecting renal injury than enzymes. The four enzymes investigated are located in specific regions of the cell so that the involvement of the organelles and regions of the cell can be followed. Damage to the organelles does not appear to occur as the excretion of the lysosomal enzymes remained normal and only in the case of cephaloridine were marginal changes in the mitochondrial GDH excretion seen. The average alkaline phosphatase was also normal suggesting no gross damage to the plasma membrane although a few individual rats excreted abnormal activities of alkaline phosphatase. These rats however, also excreted high activities of LDH. This suggests that damage to the membrane causes leakage of LDH and in severe cases release of the plasma membrane enzyme alkaline phosphatase. The administration of cephaloridine at various doses showed that urinary enzyme measurements were as sensitive as histology for demonstrating renal damage and that of these enzymes, LDH was by far the most useful.

Different types of urinary protein excretion may be recognized by determination of the proteins molecular weight. Beside chromatography different electrophoretic procedures have been applied to urinary proteins to study the underlying renal disease. The various zone electrophoreses separate merely by surface charge, proteins however covered by sodium dodecyl sulfate (SDS) migrate according to their molecular radius. So by SDS-polyacrylamide electrophoresis (SDS-PAe) macromolecular proteinurias (Mr 60,000- greater than 300,000 daltons) due to glomerular damage may be distinguished from micromolecular forms (Mr 10,000-70,000 d) due to tubular dysfunction. By densitometric quantitation of the separated Ig and transferrin an index of the glomerular selectivity is obtained, i.e. the capacity of the glomerular system, to retain serum proteins of a Mr above 150,000 d. By this procedure proliferative and degenerative glomerulopathies may be distinguished from minimal change disease, focal glomerular sclerosis and early membranous nephropathy; serial determinations of this selectivity index in the latter two disease entities show a gradual deterioration of glomerular protein handling with time. A glomerular proteinuria of even "physiological" quantity has been proved as early sign of renal involvment in systemic diseases; it may be detected earlier as for example the retinopathy in juvenile diabetics. Micromolecular proteinurias also occur at least in two forms: the typical tubular proteinuria (MW 10,000-70,000 d) is associated with acute or chronic severe tubular dysfunction as in interstitial nephritis and acute kidney failure; rejection episodes of kidney transplants lead to transient tubular proteinurias, too. The second form of micromolecular proteinuria (Mr 40,000-70,000 d) has been found frequently in association with a glomerular in diabetic and hypertensive glomerulosclerosis. By measuring clearances of the microproteins, the proteinuria with this pattern could be established as form independant from glomerular and tubular proteinurias. The constancy of the two micromolecular proteinurias led to the hypothesis of at least two selective mechanism of tubular protein resorption. SDS-PAe additionally allows the differentiation of extrarenal proteinurias, as caused by overflow, paraproteins, postrenal Ig-secretion or bleeding etc. In comparing clinical and in part histological data of about 2,000 patients suffering from kidney diseases the analysis of urinary proteins by this method has been proved as valuable non-invasive tool for diagnosis and follow-up.

Isoenzyme patterns of alkaline phosphatase are determined quantitatively in extracts of human kidney as well as in human urine by means of immunotitration technique. Both media contain two types of AP isoenzymes: liver and intestinal like AP. Intestinal AP is located as a minor component of total AP activity (1-4%) in particle-free fraction of the kidney. Urinary AP activity is found after high speed centrifugation in supernatant (100,000 Xg) as well as in the 100,000 Xg sediment and can only be made soluble from the latter by n-butanol treatment. Intestinal AP in urine is concentrated in the supernatant while in sediment the isoenzyme pattern resembles to that of kidney. Urine of normal persons contains most of AP activity in the sediment and consists mainly of liver type AP. Urinary AP of patients with renal diseases or after application of cytotoxins contains little sedimentable activity, mainly intestinal AP.

Human urine and urine of various animals contains a powerful procoagulant which converts prothrombin in presence of factors V, VII, X, and phospholipids or thrombocytes into thrombin. In human beings its content of the urine is markedly reduced or totally absent in kidney diseases, but normal in hemophilic patients. Only 0.2 ml urine are required for its assessment. In experimental kidney diseases in rabbits and rats there is an inverse relationship between procoagulant and protein excretion. In the test tube 1 part of urine corrects the clotting of 5-10 parts of hemophilic plasma, even in the presence of very strong coagulation inhibitors.