Estimation of IgM in cerebrospinal fluid by fluoroimmunoassay.

Acetylcholinesterase (AChE) activity and its molecular forms in the rectal tissue in Hirsprung's disease were studied. Total AChE activity was increased in Hirsprung's disease; the activity was separated by polyacrylamide gel electrophoresis into two molecular forms. The AChE form with higher electrophoretic mobility was not observed in healthy children, and it might be of diagnostic importance in this disease.     

A solid phase enzyme immunoassay for the quantitation of serum ferritin

A non-competitive solid phase enzyme immunoassay for the measurement of ferritin in human serum is described. This procedure involves the use of specific antibody covalently attached to derivatized hydrophilic microparticles and enzyme labeled specific antibody. Reproducible results were achieved within 4 h for ferritin in serum in the range of 4 ng/ml to 250 ng/ml. Ferritin levels as low as 0.4 ng/ml can be measured. The enzyme immunoassay and three commercially available radioimmunoassay (RIA) kits were used to determine serum ferritin levels in healthy adults. Good agreement was found between the enzyme immunoassay and the RIA methods.     

Platelet aggregation in six families with Bartter''s syndrome

Bartter's syndrome has so far been considered to be an autosomal recessive inherited disease, because of the occurrence of the syndrome in siblings. Both sexes are equally involved. Until now no family has been described in which parents as well as children suffered from the disease.

It has recently been found that ‘obligatory carriers’, i.e. parents of affected children, showed the same pattern of platelet aggregation inhibition as their affected children. We investigated eight patients as well as their parents and siblings, and found that all persons included in the study showed impaired thrombocyte aggregation, especially after low salt intake.

We conclude that the restriction of dietary sodium facilitates the measurement of the platelet abnormality.     

An ion chromatographie method for the simultaneous determination of inorganic phosphate,bromide, nitrate and sulphate in human serum

A direct method for the determination of inorganic phosphate, bromide, nitrate and sulphate in 100 μl serum is described. After a 10-fold dilution, the sample is directly injected into an ion Chromatograph without further pretreatment. The method described is highly accurate, and reproducible over longer periods. The serum concentrations of the four above-mentioned anions have been determined in 20 male and 20 female normal persons and found to be in agreement with the results of earlier studies.     

Plasma lysozyme level and reticuloendothelial system function in human liver disease

Plasma lysozyme levels have been reported to reflect the functional status of the reticuloendothelial system (RES). We measured plasma lysozyme levels in 22 patients with acute hepatitis and 21 patients with cirrhosis and a mesocaval shunt. In 17 of these patients RES function was assessed by measuring the disappearance rate (t/2) of radio-labelled sulphur colloid. In acute hepatitis plasma lysozyme levels and colloid t/2 were significantly lower than in healthy controls and cirrhotics. In the acute hepatitis patients, the plasma lysozyme levels rose significantly two weeks after admission as the hepatitis improved. The colloid t/2 of the 17 patients with liver disease was significantly correlated with the plasma lysozyme level (r = +0.66, p = 0.005).These results suggest that in human liver disease, in comparison with animal experiments, plasma lysozyme is dependent on RES functional status in the sense that a more active RES will result in a lower lysozyme level.     

Predictive value of serum enzyme determinations in acute myocardial infarction

The prognostic significance of serum enzyme measurements in acute myocardial infarction was studied in 146 patients hospitalized shortly after the attack. Creatine kinase (CK), CK-MB, aspartate aminotransferase (ASAT) and lactate dehydrogenase (LDH) were serially determined every four hours during the first three days following admission.Peak enzyme levels correlated well with the cumulated CK release (r = 0.95, 0.74, 0.70 for CK, ASAT and LDH respectively). Among all enzyme measurements, LDH levels determined when CK reached its peak value provided the best discrimination between acute phase survivors (15 days) and non-survivors. LDH was also the best measurement for identifying patients with ventricular impairment. LDH and ASAT peak levels were more powerful predictors of the patient's risk than CK peak levels. CK levels determined later in the course of myocardial infarction were more discriminant, indicating prolonged CK elevation in non-survivors. There was no significant difference in CK-MB levels, nor in cumulated CK-MB amounts for survivors and non-survivors.It is concluded that serum LDH activity is a better predictor of the short term evolution of myocardial infarction than CK levels or infarct size estimations from serial CK determinations.     

Initial activity and inactivation of alkaline phosphatase in different lots of buffer

Alkaline phosphatase activities were determined in six lots of 2-amino-2-methyl-l-propanol AMP) and in six lots of diethanolamine (DEA) buffers without preincubation of the sample.There appeared to be differences between the lot numbers in both cases, resulting in a variation in initial activity.When serum samples are preincubated with buffer a loss of activity was observed in 4 out of the 6 AMP buffers. Four human isoenzymes showed varying inactivation during preincubation with AMP buffer. No loss of activity was observed when the preincubation was done with the six DEA buffers. These results indicate that the purity of the commercially-available buffers is quite unsatisfactory.     

McArdle's disease: a study on the molecular basis of two different etiologies of myophosphorylase deficiency.

Three patients with myophosphorylase deficiency were investigated. Two had no detectable activity, while one had 1% residual activity. The patient with 1% residual activity had 40% of the normal amount of myophosphorylase protein. No myophosphorylase protein could be detected in the other two cases. A precipitin band in the Ouchterlony double immunodiffusion test was not present in any case. This study showed that modifying the normal enzyme (without changing the molecular weight) changed the immunoprecipitin activity of the phosphorylase protein. Therefore, immunoprecipitation is not a valid technique for differentiation of the variants of myophosphorylase deficiency, and another method, for example SDS-electrophoresis, should be applied.     

Dynamics and composition of serum lipids in hyperlipoproteinemias.

The microviscosity of serum lipids in patients with hyperlipoproteinemia and normal donors was determined by monitoring the degree of fluorescence polarization of the fluorescent probe 1,6-diphenyl-1,3,5-hexatrine (DPH). Determination of serum lipids--serum total cholesterol, serum triglycerides, and serum phospholipids--revealed that an increase in the ratio of cholesterol/triglyceride + phospholipid is accompanied by an increase in the degree of fluorescence polarization and an increase in the microviscosity values. Similar results were obtained with sonicated liposomes prepared from serum lipids extracts. This correlation between dynamics and composition of serum lipids was further supported by results obtained with an artificial model system of sonicated lipids dispersions. The results have shown that high microviscosity values are characteristic in patients with high levels of serum cholesterol, and that low microviscosity values are characteristic in patients with high levels of serum triglycerides. It is suggested therefore that this technique may serve as a basis for a rapid screening test for hyperlipoproteinemias.     

Renal hypouricaemia in insulin treated diabetes mellitus

Uric acid metabolism was investigated in 69 insulin-treated male diabetic outpatients and in 23 healthy male subjects, because of a reported coincidence between diabetes and gout. All subjects had normal serum creatinine concentrations and none received diuretic treatments.

Compared with normal, the diabetics had significantly lower mean serum uric acid concentrations (0.34 ± 0.08 (SD) mmol/l versus 0.23 ± 0.06 mmol/1, p < 0.001). 17% of the diabetic patients had serum concentrations below the normal mean—2 SD. In contrast, the diabetic patients had a 42% increase in renal uric acid excretion rate (p <0.01), and an 83% increase in the ratio of uric acid clearance/creatinine clearance (p < 0.001). These indices of renal uric acid excretion were both positively correlated to fasting blood glucose levels (r = 0.57, p < 0.001, and r = 0.50, p < 0.001, respectively), to the degree of glycosuria (r = 0.73, p < 0.001, and r = 0.63, p < 0.001, respectively), and to the magnitude of water diuresis (r = 0.60, p < 0.001, and r = 0.39, p < 0.01, respectively).

The hypouricaemia observed in these insulin-dependent diabetic male subjects may probably be caused by the increased renal excretion of uric acid in the presence of hyperglycaemia. The study gave no evidence of increased serum uric acid concentrations in insulin-dependent diabetics. It is therefore likely that any coincidence between gout and diabetes derives from other coexisting serum uric acid raising factors.     

The contribution of alpha1-antitrypsin to the total antitrypsin activity of human serum. A study of two phenotype PI OO individuals.

Comparison of the levels of alpha1-AT, alpha2-M, inter alpha-AT, C1 inactivator and antiplasmin and global antitrypsin activity in a group of normal phenotype PI MM individuals, a group of normal individuals with phenotypes with intermediate alpha1 AT activities and alpha2-AT-deficient persons show that alpha1-AT contributes more than 90 percent of the total antitrypsin activity of normal plasma. AT III and fast reacting antiplasmin are shown to contribute to the remaining activity. It can be assumed that due to test conditions the antitrypsin activity of alpha2-M is not assessed. C1 inactivator and inter alpha1-AT do not contribute to a perceptible extent to the overall antitrypsin activity estimated according to the method of Eriksson (Eriksson, S. (1965) Acta Med. Scand. 177, 1).     

Measurement of fibrinogen degradation products by a sandwich enzyme immunoassay technique

We describe a highly sensitive enzyme immunoassay technique using beta-D-galactosidase from Escherichia coli as a label to quantify fibrinogen and its degradation products. The minimum detectable concentrations of fibrinogen, fragment D and fragment E, were 0.6, 0.06 and 0.2 ng/ml of reaction mixture, respectively. The cross-reactions of the assay-system for fragment E with fibrinogen and fragments X, Y and D were 5, 8, 56 and 0% respectively. The assay system for fragment D did not cross-react with fragment E, but did cross-react with fragments X, Y and fibrinogen (38, 10 and 15% respectively). Fractionation of the serum sample on Sephadex G-200 column allowed specific quantification of fragments D and E in serum samples.     

A micromethod for simultaneous estimation of blood levels of some commonly used antiepileptic drugs.

The analysis of serum samples for the antiepileptic drugs carbamazepine, phenobarbital and phenytoin by a simple extraction technique and chromatographic analysis by high-pressure liquid chromatography is described. The method is a micro one which correlates well with gas-liquid chromatographic procedures. Coefficients of variation are smaller than 3%.     

Amino acid composition of gliadin fractions which may be toxic to individuals with coeliac disease

Fraction 9, prepared by chromatography of a peptic-tryptic-pancreatinic digest of gliadin on S.P. Sephadex C-25, was re-chromatographed on Q.A.E. Sephadex A-25 and subfractions 9-1 and 9-2 further purified on S.P. Sephadex C-25. Sub-fractions 9-1 and 9-2 and the purified sub-fractions 9-1B and 9-2B appeared to be toxic to patients with coeliac disease on the basis of causing a reduction in d-xylose absorption.Amino acid analysis of undigested residues from sub-fractions 9-1B and 9-2B obtained after ‘in vitro’ digestion with remission coeliac mucosa contained mainly glutamine/glutamic acid and proline with some serine, leucine, phenylalanine and glycine.Another fraction (fraction 3) of wheat gliadin prepared by peptic-tryptic digestion and ion-exchange chromatography on S.P. Sephadex, previously shown to produce a skin-reaction in adults with coeliac disease, has been further purified by ion exchange chromatography, isoelectric focusing and gel filtration. The sub-fractions were submitted to amino acid analysis and the results compared with those from the undigested residues above.Isoelectric focusing of fraction 3 of the P.T. digest and its sub-fractions showed the presence of peptides of pI approximately 4.8 and 5.6 with only small amounts of peptides on either side of this region.Mucosal digestion of fractions of peptic-tryptic-pancreatinic gliadin digests ‘in vitro’ appears to be a promising method for the elucidation of the primary structure of that section of the gliadin which may be responsible for the lesion in coeliac disease. The evaluation of higher molecular mass peptic-tryptic digests by intradermal skin tests could also be useful for the preliminary screening of fractions for feeding tests, but this approach seems less likely to indicate the toxic region of the gliadin molecule.     

Physico-chemical and immunological properties of acid alpha-glucosidase from various human tissues in relation to glycogenosis type II (Pompe's disease).

The physico-chemical and immunological properties of acid alpha-glucosidase from various human tissues have been studied. Heat stability of acid alpha-glucosidase from heart, liver and skeletal muscle is identical, but for kidney some different results are obtained. Identical isoelectrofocussing patterns are found for heart, liver and skeletal muscle. Furthermore, the effect of antiserum against human liver acid alpha-glucosidase on the activity of acid alpha-glucosidase from various tissues is studied. The results are discussed in relation to glycogenosis type II (Pompe's disease).     

Urinary excretion of N-acetyl-beta-D-glucosaminidase and alanine aminopeptidase in patients receiving amikacin or cis-platinum

Urinary excretion of alanine aminopeptidase (EC 3.4.11.2) and N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) was determined for 25-70 days in five patients receiving cis-platinum and for 8-53 days in six patients receiving amikacin. This study was performed to investigate if the excretion of urinary enzymes represents a sensitive parameter for the early detection of toxic kidney damage. The determination of N-acetyl-beta-D-glucosaminidase was carried out by the method of Knoll et al. [13]. The procedure of Mondorf et al. [14] for the estimation of alanine aminopeptidase activity was adapted to the Gemsaec Fast-Analyzer. In both patient groups an increase in the excretion of the two enzyme activities could be demonstrated. In patients receiving amikacin, the excretion of alanine aminopeptidase was always higher than that of N-acetyl-beta-D-glucosaminidase, whereas in three patients receiving cis-platinum it was the opposite. In two cis-platinum patients the excretion of both enzymes was of the same size. The changes during amikacin therapy seem to be reversible, whereas in four cis-platinum patients these changes seemed to be partly irreversible. Serum creatinine concentration was less sensitive than the urinary enzyme excretion for detection of kidney damage.     

A solid phase fluorescent immunoassay for the quantitation of the C3 component of human complement.

A non-competitive method for the determination of the C3 component of human complement in serum is described. This procedure involves use of a specific antibody covalently attached to derivatized polyacrylamide beads and a fluorescently labeled specific antibody. Reproducible results were achieved for C3 in serum in the range of 20 mg/d1 to 195 mg/d1 within 2 h. C3 levels as low as 125 ng/m1 can also be measured. Fluorescent immunoassay and radial immunodiffusion were used to determine C3 levels in healthy adults. Good agreement was found between the two methods.