Detection of Bence-Jones protein in serum by immunoblotting.

To detect Bence-Jones protein (BJP) in serum we precipitated intact immunoglobulins (Ig) using polyethylene glycol (PEG) and subjected the BJP in solution to electrophoresis in agarose gel, followed by transfer to a polyvinylidene difluoride membrane, and immunoenzymatic staining (successively using rabbit anti-human light/heavy chain of Ig, biotinylated swine anti-rabbit Ig, and alkaline phosphatase-conjugated streptavidin). Treatment with PEG effectively reduced background staining of polyclonal Ig in the immunoblots, although intact monoclonal Ig was not always completely removed. To compare the present method with immunoelectrophoresis (IEP), we selected samples from patients demonstrating BJP by IEP in both serum and urine (n = 40), serum only (n = 18), and urine only (n = 32); 21 of these patients had BJP alone and 69 had BJP in addition to intact monoclonal Ig. Efficiency of detection of BJP in serum was increased by the present method: serum BJP was detected in 70 patients by the present method versus 58 by IEP. The present method demonstrated single BJP bands in the samples from 16 patients (kappa, n = 7; lambda, n = 9) and multiple BJP bands (range: 2-9) in the samples from 54 patients (kappa, n = 31; lambda, n = 23). This method could be useful for detecting BJP in serum from patients suspected of having light chain gammopathy (without the need for urine testing) and may complement urine testing in patients excreting polyclonal free light chains of Ig.

A case of recurrent adrenocortical carcinoma, with observations on long-term o,p'-DDD therapy and complications.

This report describes a patient with a recurring, one stemline-aneuploid, adrenocortical carcinoma. The condition showed a number of unusual characteristics over a period of 22 yr. It changed from a biochemically functioning, low-grade malignant tumour into a non-functioning malignancy with pronounced mitotic activity, accompanied by an ovarian carcinosarcoma 1 yr before death. Quality of life was reasonable for many years despite chemotherapy, consisting of a total of almost 10 kg of o,p'-DDD administered over a period of 8 yr, and the subsequent side effects (e.g. low T4; increased bleeding time). A reduced mineralocorticoid activity, induced by o,p'-DDD, was reversed after discontinuation of o,p'-DDD treatment. During o,p'-DDD administration the substitution requirements for both hydrocortisone and fludrocortisone acetate increased, leading to periods of hypoadrenocorticism with prerenal uraemia.

Complexes of creatine kinase and immunoglobulins in serum identified by a nonimmune binding method.

We have developed a method for identifying IgG-complexed creatine kinase (CK; EC 2.7.3.2) (IgG-CK) and IgA-complexed CK (IgA-CK) in serum. We used immobilized Protein G to bind IgG-CK and immobilized jacalin to bind IgA-CK, leaving noncomplexed CK in solution. The noncomplexed CK and total CK were measured kinetically. The results are reported as CK bound to immobilized Protein G and CK bound to immobilized jacalin. We validated the method by using sera determined immunochemically to contain IgG-CK, IgA-CK, mitochondrial CK (CKmt), and free CK-BB. We demonstrated concomitant binding of CK and approximately 99% of IgG, and of CK and approximately 87% of IgA. For CK bound to immobilized Protein G and to immobilized jacalin, intra- and interassay precisions ranged from 2.5% to 9.6%, and detection limits were less than 9 318 U/L in 40 sera containing IgG-CK, and CK bound to immobilized jacalin ranged from 10 to 59 U/L in eight sera containing IgA-CK. These ranges represent the activities of immunoglobulin-bound CK in the sera. In 13 sera containing CKmt and in eight sera containing free CK-BB, the binding of CK was less than 9 U/L. Evidently, this method is useful for identifying IgG-CK and IgA-CK in serum.

Radial immunodiffusion and immunoelectrophoresis compared for identifying autoantibodies to lactate dehydrogenase in human serum.

Variant electrophoretic patterns of lactate dehydrogenase isoenzymes were studied. By radial immunodiffusion and immunoelectrophoresis, immunoglobulin and light chain class of autoantibodies to lactate dehydrogenase were identified in nine sera: seven of these sera demonstrated IgG (5 lambda, 2 kappa) autoantibodies to lactate dehydrogenase, the other two demonstrated IgA (both kappa) autoantibodies to lactate dehydrogenase, the other two demonstrated IgA (both kappa) autoantibodies to lactate dehydrogenase. We conclude that radial immunodiffusion and immunoelectrophoresis are equally effective for identifying auto-antibodies to lactate dehydrogenase in serum. Radial immunodiffusion, however, is easier to perform than immunoelectrophoresis.

[Misleadingly high amylase and pancreatic amylase activity in the plasma of patients with macroamylasemia]. / Misleidende hoge amylase- en pancreasamylase-activiteit in het serum van een patiënt met macro-amylasemie.

A man aged 68 years with choledocholithiasis and cholangitis, with no clinical signs suggestive of acute pancreatitis and with a low excretion of amylase in the urine, showed persistent hyperamylasaemia which appeared to be caused by macroamylasaemia. The macroamylase (an IgA-lambda-amylase complex) accounted for nearly all (90%) of the amylase activity in the serum. The activity of pancreatic amylase in serum, determined by an immunoinhibition test which selectively blocks salivary amylase activity, constituted 99% of the amylase activity in serum (normal reference range 19-71). We showed, however, that complexed salivary amylase is not inhibited in the test, resulting in a falsely-increased activity of pancreatic amylase in serum. We conclude that macroamylasaemia can lead to a clinically misleading increase in the activity of pancreatic amylase in serum.

Autoantibodies to lactate dehydrogenase in serum identified by use of immobilized protein G and immobilized jacalin, a jackfruit lectin.

In this method for identifying autoantibodies to lactate dehydrogenase (anti-LDs) in serum, we used immobilized Protein G to bind IgG-complexed LD and immobilized jacalin to bind IgA-complexed LD, leaving non-complexed LD in solution. The non-complexed LD and total LD were kinetically measured. We report results as LD bound to immobilized Protein G and LD bound to immobilized jacalin. Using sera demonstrating IgG and IgA anti-LDs by immunoelectrophoresis (IEP), respectively, we optimized the method for incubation time and concentration of binding agents. We demonstrated concomitant binding of LD and greater than or equal to 98% of IgG and of LD and greater than or equal to 92% of IgA. For LD bound to immunobilized Protein G the detection limit was 10 U/L, within- and between-run CVs ranged from 2.9% to 9.1%, and values for normal sera were less than or equal to 3% of total LD. Results for LD bound to immobilized jacalin were similar. We tested 10 sera displaying aberrant LD electrophoretograms: In seven, LD bound to immobilized Protein G was increased (range: 26-99% of total LD), indicating IgG-complexed LD. This was confirmed by IEP, demonstrating IgG1,2, or IgG3 anti-LDs in these sera. In the other three sera, LD bound to immobilized jacalin was increased (range: 38-72% of total LD), indicating IgA-complexed LD. This was confirmed by IEP, demonstrating IgA anti-LDs in these sera. Evidently this method is an alternative to IEP for identifying anti-LDs in serum.

Reversible loss of lactate dehydrogenase isoenzymes and lactate dehydrogenase activity in patient's serum.

An abnormal lactate dehydrogenase (LD; EC 1.1.1.27) electrophoretogram (only one band, at the application site) and a low LD activity (7 U/L) was seen for a patient's serum during storage at 22 and 4 degrees C. Both reverted to normal when the serum was incubated at 37 degrees C.

Evaluation of the Hitachi 704 automatic analyzer.

We evaluated the analytical performance of the Hitachi 704 automatic analyzer. The spectrophotometer showed a linearity of response at 340 nm up to 2.8 A. Photometric imprecision measured bichromatically at 340 and 376 nm was 0.49% at 0.16 A, 0.14% at 0.46 A, and 0.17% at 0.76 A. Imprecision of the sample probe was 0.4% for 5, 10, and 20 microL, and the volume delivered deviated -2.4%, -4.4%, and -4.2% from these preset volumes, respectively. Imprecision of the reagent probe over the range 50 to 500 microL ranged from 0.14% to 0.29%; volume delivered deviated from +1.7% to +4.4%). At equilibrium, the temperature in the cuvets was 29.8 (SD 0.05) degree C as measured by cresol red spectrophotometry. No sample carryover was detected. Reagent carryover was detected when a bilirubin assay was preceded by a total protein assay and when lactate dehydrogenase was measured after alanine aminotransferase. Imprecision for nine tests at three concentrations ranged from 1.1% to 4.4%. Comparison of methods with the SMAC II as reference method showed good results. Precision was better than reported for the Hitachi 705 automatic analyzer.

Increased serum lactate dehydrogenase isoenzyme 1 and macro creatine kinase type 2 in a patient with lung cancer.

A 72-yr-old man, having a lung tumor with metastatic spread to liver and bone, was hospitalized with a lactate dehydrogenase (LD; EC 1.1.1.27) activity and a creatine kinase (CK; EC 2.7.3.2) activity in serum of 30 and 2 times the upper reference limit (URL), respectively. The LD isoenzyme-1/LD activity ratio was 62% (2 times URL). This ratio was maintained throughout hospitalization, during which LD activity increased up to 90 times URL. Macro CK type 2 activity represented almost all of the CK activity, which increased up to 4 times URL during hospitalization. The patient died the 29th day after admission. The enzyme abnormalities were thought to stem from tumor tissue.

Determination of urinary cystine for monitoring anticystinuric therapy.

Cystine is assayed in urine by a method adapted from the procedure of Haux and Natelson (Clin Chem 1970;16:366-369) and modifications of the method, which improved its specificity, are presented. Assay imprecision (CV) at four different cystine concentrations (range: 0.042-1.658 mmol/l) varied from 23.1% to 3.7%. Analytical recovery was 94.6 +/- 6.0%. D-Penicillamine, alpha-mercaptopropionylglycine, L-glutamine and metabolites, added to urine, had negligible effect on the results of the cystine assay. Samples were stable for 3 wk when stored at 4 degrees C or frozen. Cystine excretion (mmol/24 h) in apparently healthy persons ranged from 0.041 to 0.170 (mean +/- SD: 0.099 +/- 0.039). Cystine excretions are presented in patients treated with anticystinuric drugs.

Adrenocortical tumour in untreated congenital adrenocortical hyperplasia associated with inadequate ACTH suppressibility.

Data are presented concerning a 60-year-old woman with untreated congenital adrenocortical hyperplasia due to 21-hydroxylase deficiency, who presented with a tumour of the left adrenal gland. Steroid excretion was partly suppressed with dexamethasone. After removal of the tumour, the excretion of several steroid fractions decreased substantially, but suppression by dexamethasone remained inadequate. Preoperatively, plasma ACTh was elevated in the afternoon and decreased only slightly after dexamethasone administration. After surgery, cortisol secretion decreased markedly, whereas ACTH dysregulation became more prominent. Negative feedback failure precluded the use of normal suppressive therapy with low doses of glucocorticosteroids and led to the therapeutic removal of the right adrenal gland, which showed histological signs of nodular hyperplasia.

Determination of serum 5'-nucleotidase with a centrifugal analyzer.

We describe a multi-point method for the determination of the serum enzyme activity 5'-nucleotidase by means of an NADH sensor reaction. Studies show that the assay may be carried out in one step, without preincubation, by using a GEMSAEC Fast Analyzer.