Effects of albumin and other proteins during assay of amylase activity.

Addition of albumin, gamma-globulin, alpha-casein or submaxillary mucin to the assay system for chromogenic measurement of human or rat amylase with blue starch increased the amylase activity, albumin having the most effect. These proteins seemed to increase the activity by protecting amylase from inactivation. Amylase activity was higher in urine samples showing proteinuria than in urine samples without detectable protein. It is concluded that amylase assay is more reliable when a final concentration of albumin of 1 mg per ml is added at the dilution step and at the incubation step.     

A transient variant of serum lactate dehydrogenase isoenzymes.

An unusual variant of the serum lactate dehydrogenase (LDH) isoenzyme pattern is described in an apparently healthy young woman. The abnormal pattern consisted of a single zone of LDH activity, having the same mobility as, but more diffuse than, normal LDH-4. The molecular weight of the abnormal LDH complex is approximately 280 000, but the nature of the additional component remains unknown, as the isoenzyme pattern spontaneously reverted to normal six weeks after it was first noticed.     

Distribution of serum concentrations of creatine kinase MM and BB isoenzymes measured by radioimmunoassay.

Because of the recent interest in measuring serum enzyme concentration as opposed to catalytic activity, we measured the serum concentration of creatine kinase isoenzymes BB and MM by radioimmunoassay and the total creatine kinase enzymatic activity in healthy adults. For sex-race subgroups we report mean values and the 2.5, 5, 25, 50, 75, 90, 95 and 97.5 percentiles. Differences among average values of the subgroups were highly significant. Results within subgroups frequently departed from a gaussian distribution.     

Studies on alkaline phosphatase isoenzymes in hepatic diseases. Relation to gamma-glutamyltransferase.

Isoenzymes of alkaline phosphatase (ALP) and total gamma-glutamyltransferase (gamma GT) have been studied in patients with increased total ALP. Fractionation of alkaline phosphatase yielded clinical information which could not be obtained by determination ALP and gamma GT alone. 1. There was a high degree of correlation between isoALP 1 (biliary band) and total gamma GT. 2. The ALP2 fraction increases after cytolysis in acute and chronic hepatitis. 3. A new ALP4 fraction appears, probably due to fibroblastic activity, in some histological types of cirrhosis.     

Isolation and characterization of isoenzymes of human salivary and pancreatic alpha-amylase.

Human salivary and pancreatic alpha-amylase (1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) were separated by electrofocusing. In the first case we obtained six isoenzymes with isoelectric points of pH 5.70, 5.72, 6.23, 6.32, 6.73 and 6.88. Human pancreatic alpha-amylase has been separated into eight isoenzymes with isoelectric points of pH 5.72, 5.77, 5.88, 6.05, 6.23, 6.69, 6.72 and 6.95. Some of the isoenzymes were shown to be sialoproteins; others representing about 80% of the total activity did not contain neuraminic acid. The molecular weight of the non-sialoproteinic isoenzymes was found to be about 47 000 in all cases.     

Isoelectric focusing of serum alkaline phosphatase isoenzymes

We present a new method for the separation of alkaline phosphatase isoenzymes based on isoelectric focusing of serum proteins. Sera of healthy subjects studied by this method present seven groups of bands within a pH range of 3-9. To date, the organ source of six out of seven groups has been identified directly or indirectly. Serum of patients with histologically proven malignant neoplasms showed an additional group in the pH range 3.6-4.2. This fraction seems to be heterogeneous with respect to the pattern and stability to heat, and to L-phenylalanine and L-homoarginine inhibition. It was present in all but two of the proven malignant tumour patients but also in 26 out of 70 with benign disorders.     

Separation of acid and neutral alpha-glucosidase isoenzymes from fetal and adult tissues, cultivated fibroblasts and amniotic fluid cells by DEAE-cellulose and sephadex G-100 column chromatography.

Isoenzymes of alpha-glucosidases (EC 3.2.1.20) from various human organs and body fluids from fetuses and adults were separated by DEAE-cellulose column chromatography and gel filtration using Sephadex G-100. A minicolumn (0.35 X 2.5 cm) was used for the DEAE-cellulose column chromatography of extracts from tissues as well as cultivated cells of skin fibroblasts and amniotic fluid. The enzyme activity in the eluates was measured by the use of a methylumbelliferyl derivative as substrate and a very sensitive Microscope fluorimeter. In most tissue samples alpha-glucosidase was eluted mainly as a single peak when monitored at acid pH and as two peaks when the activity was measured at neutral pH in both columns. Another small peak representing alpha-glucosidase was found in fresh extracts of cultured cells on DEAE-cellulose columns. Neutral alpha-glucosidase especially in fibroblasts was extremely sensitive to storage at -20 degrees C. DEAE-cellulose column chromatography of plasma and amniotic fluid showed similar elution patterns of alpha-glucosidase. Differences were noticed in the elution pattern of urine from infants and adults. The tissue distribution and the different characteristics of the enzyme in samples of various origins and ages were discussed.     

Automated quantification of bone and liver alkaline phosphatase isoenzymes of human serum

Coupling two Technicon AAII samplers synchronised at 50 per hour with a 2 : 1 sample to wash ratio, sera are denatured and collected automatically. The incubation is done in continuous flow by passage through a U device made of large metallic needles soaked in a water bath at 60 ± 0.1°C. This allows a very quick temperature equilibration and a very reproducible incubation time of 35 sec. Initial and residual activities of alkaline phosphatase (ALP: EC 3.1.3.1) are measured on a Rotochem II (Aminco) with the procedure recommended by the Société Franlaise de Biologie Clinique (SFBC). For a mixture of bone and liver ALP, the initial rate constant of heat denaturation K_app = (A × K_b) + (B × K₁), where A and B are the fractions of each isoenzyme in the mixture, and K_b and K₁ the rate constants for bone (b) and liver (1) experimentally determined as 1.8 min^?1 and 0.45 min^?1 respectively. An equation was derived which converts the percent residual activity to a percentage of bone and liver isoenzyme: % bone ALP = 183 ? 2.38 ×% residual activity. This automated method was applied to 2700 people of both sexes from 4 to 100 years old.     

Modification of human pancreatic amylase isozymes by peptidoglutaminase I and II

The effects of peptidoglutaminase (PGln-ase) I and II on human pancreatic juice amylase purified as an isozyme were investigated. Several amylase isozymes were formed which corresponded to minor components of pancreatic amylase isozymes, indicating that appearance of amylase isozymes are due to enzymic deamidation. A similar result was observed when the purified amylase isozyme was incubated with the supernatant of human pancreatic juice whose amylase was previously removed by adsorption onto raw corn starch. These findings are discussed in connection with amylase isozymes in the sera of the patients suffering from pancreatic inflammation.     

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.     

Effect of albumin on amylase assay using blue starch polymer

The nature of the stimulation by albumin of amylase activity determined by use of blue starch polymer was studied. The binding of albumin to the starch polymer was responsible for the stimulation of amylase activity, since the stimulation was observed with the albumin-bound blue starch polymer as substrate irrespective of the presence or absence of free albumin in the reaction mixture. When the albumin-bound blue starch polymer was hydrolysed by amylase, the blue oligosaccharide fragments were released with the albumin attached. These oligosaccharide fragments were about 1.3 times larger in average molecular size than the fragments released in the absence of albumin, suggesting that the apparent stimulation of amylase by albumin, (about 1.3 times) is due to the liberation of the larger oligosaccharide fragments.     

Serum vitamin E,lipid peroxide and glutathione peroxidase in patients with chronic pancreatitis

It has been reported that lipid peroxidation increases in patients with antioxidant deficiencies, such as vitamin E and glutathione peroxidase. The relationships between serum lipid peroxide and vitamin E on the one hand and glutathione peroxidase on the other were examined in 22 patients with chronic pancreatitis, often accompanied by malabsorption of fats and fat-soluble vitamins due to the impaired exocrine pancreatic function.Both serum vitamin E concentrations and glutathione peroxidase activities were depressed, especially in patients with chronic calcifying pancreatitis. On the other hand, serum lipid peroxide levels were elevated. A significant negative correlation was found between the serum lipid peroxide levels and vitamin E concentration.These findings suggest that an elevation of the serum lipid peroxide level may be due to the lack of an antioxidative defense mechanism, such as vitamin E, against lipid peroxide.     

Determination of aspartate aminotransferase isoenzymes in hepatic diseases — preliminary findings

The study sought to establish a relationship between the AST isoenzyme levels in serum and degree of hepatic damage, by using a new and simple immunochemical method for the differential determination of the isoenzymes. Sixty-nine patients with various hepatic diseases were studied.During hepatic damage, cytoplasmic isoenzyme (s-AST) is found in greater quantities than mitochondria! isoenzyme (m-AST), but the m-AST level increases to a greater extent in acute liver diseases. However, m-AST in alcoholic hepatitis is higher than expected from the total AST (t-AST) values. The ratio of m-AST to t-AST seems to discriminate alcoholic hepatitis from other liver diseases.     

Creatine phosphokinase isoenzymes: A comparison of a kinetic and an electrophoretic method in the diagnosis of myocardial infarction

A comparison has been made of the results of the levels of the MB isoenzyme of creatine phosphokinase by a kinetic and an electrophoretic method performed on patients suspected of having myocardial infarction. Reviewed in depth are those cases in which discrepant results have occurred. On the basis of the combined clinical and laboratory data, the kinetic method is a more sensitive but less specific indicator of myocardial necrosis. Thus, the kinetic method may be used as a screening test, with confirmation of positive results by electrophoresis.     

Urinary alpha-glucosidase analysis for the detection of the adult form of Pompe's disease.

Two alpha-glucosidases from human heart, liver, muscle, kidney and urine have been separated by means of Sephadex G-100 gel filtration. The first peak (Peak I) was neutral alpha-glucosidase and the second peak (Peak II) was lysosomal acid alpha-glucosidase. Peak II was absent in a patient with the adult form of Pompe's disease. KCl stimulated the activity of the Peak II enzyme but it strongly inhibited the activity of the Peak I enzyme measured at pH 4.0. Decreases in the urinary alpha-glucosidase activity measured at pH 4.0 with added KCl and the ratio of the activity at pH 4.0 with added KCl/the activity at pH 6.5 without KCl may aid in the detection of homozygotes or heterozygotes with the adult form of Pompe's disease.     

Improved electrophoretic resolution of LDH isoenzymes in agar-agarose mixtures by utilizing its endosmotic properties

A simple technique is described which gives a better separation of lactate dehydrogenase isoenzymes; by mixing a correct proportion of a gel with a high electroendosmotic property such as agar, and a gel with a low electroendosmotic property such as agarose.The different positions of the application slit in lactate dehydrogenase isoenzyme patterns and the effect on the positioning of the application slit after mixing gels with different electroendosmotic properties are discussed.     

Plasma pancreatic and salivary-type amylase and immunoreactive trypsin concentrations: variations with age and reference ranges for children

The differential alpha-amylase (EC 3.2.1.1) assay was applied to 166 control children in the age range 0.1--13 years. Circulating levels of both pancreatic and salivary-type amylase were very low (mean 20 U/l) in the first four months of life. Pancreatic levels increased gradually with age, reaching adult levels (mean 74 U/l) by the age of eight years. Salivary amylase levels showed a sharp rise in the 0.9--1.9 year period reaching maximum levels (mean 99 U/l) by age 5--6 years. While linear regression analysis showed significant correlation between age of subject and pancreatic and salivary amylase levels, no such correlation was evident between age and plasma immunoreactive trypsin levels over the age range studied. Plasma trypsin levels in children were lower than reported adult values. Reference ranges for pancreatic and salivary-type amylase and immunoreactive trypsin in children are presented. The importance of age-matching, when pancreatic amylase and plasma trypsin are being investigated in children, is emphasised.     

Erythrocyte glutathione synthetase in 5-oxoprolinuria: kinetic studies of the mutant enzyme and detection of heterozygotes.

The primary metabolic defect in 5-oxoprolinuria is a generalized deficiency of glutathione synthetase. The activity of this enzyme was determined in cell-free extracts of erythrocytes from patients with 5-oxoprolinuria, their parents and a sibling as well as from normal control individuals. The following activities (pkat/mg of hemoglobin) for glutathione synthetase were obtained: homozygotes mean 0.10 (range 0.07-0.12), heterozygotes mean 3.1 (range 2.8-3.7) and control individuals mean 6.1 (range 5.4-6.7). These results indicate that 5-oxoprolinuria, i.e. the defective gluthione synthetase gene(s), is transmitted by autosomal recessive inheritance. Studies of the kinetics of the low remaining activity of erythrocyte glutathione synthetase in patients with 5-oxoprolinuria failed to reveal defective affinity for glycine, gamma-glutamyl-alpha-aminobutyrate, ATP and Mg2+ ions. Furthermore, the pH optimum, time curves and temperature dependence for the mutant enzyme activity did not significantly differ from the corresponding parameters observed with normal enzyme.