Electrophoretic identification of serum immunoglobulins linked to amylase:macroamylase

The amylase-linked immunoglobulins in 16 cases with macroamylasemia were analyzed by employing immunoelectrophoresis on agar gel plates. The classes of the amylase-binding immunoglobulins were identified as follows: in the heavy chain classes, 10 cases were alpha, two cases gamma, one case both alpha and gamma and three cases could not be identified; in the light chain types, nine cases were kappa, two cases lambda and five cases unidentified.     

The measurement of serum free insulin by steady-state gel filtration

A method for the separation of bound and free insulin in the serum of insulin treated diabetics by steady state gel filtration is described. The method meets the criteria which have been suggested for the validation of a method of separating bound from free ligand. The precision of the method is satisfactory, and the results compare well with those obtained by the methods of ethanol precipitation and polyethylene glycol precipitation.     

Isoelectric focusing of serum in cystic fibrosis: Failure to distinguish between homozygote and heterozygote sera

Thin-layer isoelectric focusing was performed on samples of sera from patients with Cystic Fibrosis, siblings and obligate heterozygotes (parents), and children without cystic fibrosis (controls). The protein band with an isoelectric point of pH 5.48, previously reported to be absent in homozygote cystic fibrosis sera, was found to have a pI of 5.25. It was present in approximately one-half of the homozygote and heterozygote sera tested, and absent from 18 percent of control sera. The presence of this band is not therefore a reliable marker for the normal gene, and cannot be used to identify the heterozygous carrier for cystic fibrosis.     

2-Ethylhydracrylic acid: a newly described urinary organic acid

2-Ethylhydracrylic acid has been identified as a significant component in the organic acid fraction of normal human urine. Its excretion is increased after an isoleucine load in a normal subject and was elevated in the urine of a child with an apparent inborn error of metabolism of isoleucine at the 3-ketoacyl thiolase step.     

Radioimmunoassay and heat denaturation enzyme assay for the detection of Tay-sachs heterozygotes during pregnancy

Tay-Sachs disease results from a loss of activity of hexosaminidase A (HEXA) in body tissues and fluids. Heterozygotes for the disease are usually identified by their relatively low ratio of heat-labile HEX A to total hexosaminidase. During pregnancy an intermediate isoenzyme (HEX I) increases in activity in serum and obscures the heterozygote status. HEX I dose not increase in leucocytes, tears and other body tissues but because of technical difficulties in these assays we examined the feasibility of using a radioimmunoassay for HEX A. By univariate analysis, the heat denaturation assay gave a lower cost of misclassification for non-pregnant normals while RIA did so for pregnant normals. A combination of both tests led to reduced cost of misclassification compared to either alone. Bayesian analysis of bivariate gaussian density functions for heat denaturation and for radioimmunoassay of HEX isoenzymes was employed to calculate misclassification frequencies. Among the parameters examined, HEX A measured by RIA and % HEX A by heat-denaturation assay were the two having the best discriminatory power.     

Dicarboxylic aciduria: the response to fasting.

The urine of a child who presented with an episode of a disease resembling Reye's syndrome was found to contain large quantities of the dicarboxylic acids adipic and suberic acids, as well as the glycine conjugate of suberic acid, suberyl glycine. A variety of other dicarboxylic acids, both saturated and unsaturated, were also found in the urine at the time of the attack. It was found that the excretion of these unusual metabolites could be markedly increased by fasting for periods of greater than 10 h. These results indicate that the patient may have a defect in fatty acid oxidation which becomes clinically significant during periods of prolonged fasting.     

Interference of sodium azide with the quantitation of serum cholesterol: a comparative study.

Interference of sodium azide (employed widely as a preservative for serum) with six different cholesterol methods and two modified procedures is analyzed and compared. Sodium azide is shown to lower the serum cholesterol values of the iron-cholesterol methods from 5 to 50% at 0.1 g% azide, and from 60 to 95% at 1.0 g% azide concentration, depending on the method employed. This inhibition is independent of serum cholesterol concentration. Increased serum to reagent (v/v) ratio, i.e., increased azide concentration, caused increased depression of serum cholesterol values. Appropriate choice of low serum-to-reagent (v/v) ratio thus decreases the magnitude of the azide interference in the iron-cholesterol reaction to 3-10% for the precipitation procedures, and to 25% for the non-precipitation procedure at 0.1 g% azide concentration in serum.     

Development and application of a simple radioimmunoassay for urinary aldosterone

A simple, economical and direct assay was developed to measure aldosterone in urine, using aldosterone antibody of high specificity and gamma labelled ligand. The assay allows the direct measurement of aldosterone in 100 μl aliquots of urine after acid hydrolysis. It does not require preliminary solvent extraction and purification steps and hence a large number of samples in a single batch can be assayed simultaneously. An excellent correlation was obtained between the results of the direct assay and the levels measured after extraction and paper chromatography (Y = 0.97X + 0.89, r = 0.99, p< 0.001) or after extraction alone (Y = 0.98X + 1.75, r = 0.99, p< 0.001). The coefficients of variation for inter-assay and intra-assay determinations of samples from normal and high urine pools were 4.2–6.5% and 5.6–9.8%, respectively. Total urinary aldosterone excretion in 21 normal subjects on unrestricted sodium diet ranged from 3.8–20.2 μg/24 h (10.5–55.0 nmol/24h) with a mean of 12.5 ± 4.6 (SD) μg/24 h (34.7 ± 12.8 (SD) nmol/24 h).     

Use of magnesium silicate before gas-liquid chromatography for determination of "total estrogens" in urine during pregnancy.

The authors propose a simple, rapid, reproducible and reliable method for determination of "total estrogens" in urine during the last three months of pregnancy. The procedure consists of separation of free urinary estrogens, obtained after rapid hydrolysis, on a column of magnesium silicate. The estrogens are adsorbed on the column at acid pH and eluted by 1 M potassium hydroxide. Following extraction of the eluate by diethyl ether and formation of trimethylsilyl ether derivatives, the steroids are analysed by gas-liquid chromatography. This new procedure is used routinely in our laboratory, one assay being carried out in less than three hours. The results appear to be comparable to those obtained with classic methods. We wish to report the elimaination curves of "total estrogens" during normal pregnancies and their allowable limits.     

Zero electrophoretic mobility of very low density lipoproteins in type III hyperlipidemic patients during treatment.

In some cases of type III hyperlipoproteinemia reduction of lipid levels by diet and/or drugs results in a complete loss of mobility of the VLDL fraction on paper electrophoresis.     

Polymorphism of human serum Zn-alpha2-glycoprotein and its behaviour during ontogenesis using quantitative crossed starch gel immunoelectrophoresis

Using crossed starch gel immunoelectrophoresis with rabbit anti-Zn-alpha₂-glycoprotein (Zn-alpha₂-GP) immune serum and planimetric evaluation of antigen-antibody precipitation peak area, human serum Zn-alpha₂-GP was found to consist of four major antigenically identical components (A, B, C, D), the mean concentrations of which were 8.3 ± 3.5 mg/l (A), 27.1 ± 3.0 mg/l (B), 38.7 ± 3.4 mg/l (C) and 11.1 ± 3.5 mg/l (D). The mean relative percentages of individual Zn-alpha₂-GP components were 9.7 ± 4.0% (A),31.8 ± 3.4% (B), 45.4 ± 5.2% (C) and 13.0 ± 4.0% (D). The concentrations of human serum Zn-alpha₂-GP components A, B and C increase from the lowest values in the fetal and early newborn period to the highest ones in children and adults. During ontogenesis, the mean relative percentages of the Zn-alpha₂-GP components A and B increase while those of the Zn-alpha₂-GP components C and D decrease simultaneously. The anodal mobility of the Zn-alpha₂-GP components decreases after treatment with neuraminidase. The pattern of the Zn-alpha₂-GP components in human serum was found to be very similar to that in human urine and amniotic fluid.     

The mass spectrometric identification of dipeptides in the urine of a patient suffering from chronic skin ulceration and oedema

The identification of a complex ninhydrin positive mixture present in the urine of a child suffering from chronic skin ulceration and oedema by direct chemical ionisation mass spectrometric analysis is described. The compounds were shown to be dipeptides, of which glycylproline was the major constituent. At least 15 dipeptides were identified in the urine, most of which contained proline or hydroxyproline in the carboxy terminal position. The results suggested that the patient suffered from a defect in collagen metabolism. This hypothesis was subsequently confirmed by a grossly diminished level of prolidase in cultured fibroblasts and erythrocytes.     

A gel filament as an analytical tool: the testing of an affinity chromatographic, product-immobilizing linear gel for determination of enzymatic activity.

A new technique for quantitative analysis is proposed. An underfined volume of the test solution is exposed to a reagent in the form of a gel filament of capillary dimensions. The linear progress in the gel of the compound in question is followed with the aid of the coloured reaction products deposited along its path. With the use of the enzyme gamma-glutamyl transpeptidase it is shown that the velocity in the system of the component to be analysed, as determined by measurement of its coloured track, is related to its activity in the test solution.     

Quality control in haemoglobinometry with special reference to the stability of haemiglobincyanide reference solutions

In haemoglobinometry grave errors are still being made even though an internationally accepted standardized method is available for the determination of the haemoglobin content of blood. Up to the present only haemiglobincyanide reference solutions have been available on a wide scale to check the measuring stage of the standardized haemiglobincyanide method. These reference solutions are shown to remain stable, under proper storage conditions, for more than 10 years.Concentrated haemoglobin solutions have become available recently, offering the possibility to control the dilution and conversion steps of the haemiglobincyanide method. Such a solution is shown to remain stable, under proper storage conditions, for at least a year.Using both haemiglobincyanide reference solutions and concentrated haemoglobin solutions, as well as having the possibility of checking the cyanide content of the reagent used, an acceptable intra-laboratory control program may now be set up.     

Subjective sensitization to tonic heat as an indicator of thermal pain

In 144 healthy subjects tonic heat stimuli were applied with a contact thermode and systematically varied with respect to 3 parameters: temperature T, rate of temperature change RTC, and duration D. In addition, the stimulus temperature at which the first sensation of pain occurred was produced by some subjects. In both types of experiments, subjects compared heat intensity felt at the beginning and the end of the stimulus and then set stimulus temperature to correspond with their initial sensation. The direction of this temperature change (delta T) indicates whether the subject senses an augmentation or a diminution of heat intensity. There was a parallel occurrence of pain and sensitization to sustained heat. The average skin temperature of the point of transition from adaptation to sensitization was equal to the average pain threshold temperature. The temperature change response maintained individual differences of thermal and pain sensitivity and was highly consistent for each subject. Potential applications of the procedure in clinical and experimental pain research are discussed.     

The metabolism of L-m-tyrosine: the use of a putative precursor to investigate the increased production of m-hydroxymandelic acid in phenylketonuria

Several urinary meta-substituted phenols appear to be of endogenous origin. However, the production of these compounds is reduced in phenylketonuria with the exception of m-hydroxymandelic acid whose excretion is approximately doubled. This phenomenon has been investigated in two patients with phenylketonuria using the putative precursor L-m-tyrosine labelled with deuterium. Metabolism of this compound in these patients was comparable to that in healthy adults although much less was converted to m-hydroxymandelic acid and the excretion pattern of this metabolite was different. This apparent anomaly is attributed to smaller metabolic compartments in phenylketonuria and a lower threshold for the metabolism of m-tyramine via beta-hydroxylation. Incorporation into the natural pathway was shown by depletion of endogenous m-hydroxymandelic acid. The results are further support for the ideas that the amine precursors of m-hydroxymandelic acid, m-tyramine and m-octopamine, have a functional role and may be important in the pathogenesis of phenylketonuria.     

The response to 1,25-dihydroxycholecalciferol and to dihydrotachysterol in adult-onset hypophosphataemic osteomalacia.

The biochemical changes observed in a patient with adult-onset hypophosphataemic osteomalacia after three weeks treatment with 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) followed by dihydrotachysterol (DHT) are reported. The treatment with 1,25-(OH)2D3 resulted in restoration of intestinal phosphate absorption to normal with a small rise in plasma phosphate concentration; there was no significant change in tubular reabsorption of phosphate. The tubular reabsorption of bicarbonate, which was initially low, returned almost into the normal range with normalisation of plasma bicarbonate concentration. Aminoaciduria decreased. There were no changes in plasma or urinary calcium but immunoreactive parathyroid hormone (i-PTH) which was initially elevated fell but still remained above the normal range. These changes were maintained after replacing the 1,25-(OH)2D3 treatment with dihydrotachysterol (DHT).     

Studies on the use of skin fibroblasts for the measurement of cystathionine synthase activity with respect to homocystinuria.

The levels of cystathionine synthase have been examined in cultured skin fibroblasts obtained from a patient suffering from pyridoxine-non-responsive homocystinuria and compared with the normal and heterozygous states. Levels of synthase activity were found to vary with time in culture and composition of culture media. The physical properties of normal and abnormal synthase activities were markedly different. It has been concluded that caution has to be exercised when using cell culture methods for the purpose of defining normal, heterozygous and homozygous states with reference to homocystinuria.