Separation Of Alkaline Phosphatase Isoenzymes Research Articles

Alkaline phosphatase isoenzymes in children with respiratory disease

Aim: To present the electrophoretic pattern of alkaline phosphatase (ALP) isoenzymes in serum of infants and children exhibiting increased total ALP catalytic activity in the course of acute respiratory disease. Subjects and methods: Results obtained in 21 children (17 of them infants), including 13 male and eight female children aged 2 months to 8 years, hospitalized for respiratory diseases are presented. Total ALP catalytic activity was determined and electrophoretic separation of ALP isoenzymes was performed in children's sera. Results: Increased total ALP catalytic activity (range, 528-5622 U/L) during hospital stay was recorded in eight (38.1%) children. A typical picture of be-nign transient hyperphosphatasemia (TH), which implies the occurrence of fast anodal fraction (faster than hepatic fraction) and near-cathode fraction (faster than bone fraction), was recorded in five children. The placental-like isoenzyme was detected in two children. Expression of bone fraction and placental-like fraction was recorded in a rachitic child. Prehepatic ALP was expressed in two children, and hepatic ALP isoenzyme in one child. Conclusion: Acute respiratory disease in infants and children may entail transient increase in the ALP catalytic activity with the occurrence of various isoenzyme bands such as fast anodal and near-cathode fraction (in TH), prehepatic fraction and placental-like fraction. TH is verified when total ALP activity has decreased and returned to reference intervals. In this case, no additional testing is required.

Comparison of two commercially available systems for the electrophoretic separation of alkaline phosphatase isoenzymes

Two commercially available electrophoretic methods for the separation of the human serum enzyme alkaline phosphatase (ALP, EC 3.1.3.1) were evaluated: the Isopal system (Beckman, Brea, CA, USA) was compared with the Iso-PAL system (Sebia, Issy-les-amoulineux, France). Both use agarose as supporting medium to separate ALP into its clinically relevant isoenzymes: bone, liver, high- M r (or “fast liver”), intestinal and placental ALP. With both methods, additional fractions for bone, liver and intestinal ALP were found, true isoforms that were called “variant” fractions. The migration pattern differed considerably between the systems, owing to the use of different detergents. Bone and liver ALP were partially separated with both methods. However, when bone ALP exceeded 50% of the total ALP activity, sample treatment was necessary, either with neuraminidase (Beckman) or by applying the sample on a second gel containing wheat-germ lectin to precipitate bone ALP (Sebia). The within-and between-gel reproducibilities of both systems were comparable and remained between 2 and 6% for normal isoenzyme activities. Both systems correlated well, except for high M r ALP. The Sebia system was more sensitive for detecting intestinal ALP, whereas higher liver and bone variant ALP activities were detected with the Beckman system. It is concluded that both methods are convenient for routine use in the clinical laboratory.

Electrophoretic separation of alkaline phosphatase isoenzymes in synovial fluid and serum from patients with rheumatoid arthritis.

The alkaline phosphatase enzyme in both serum and synovial fluid from 28 cases of rheumatoid arthritis and from the serum of 30 controls was measured. The enzyme was further studied by separating its isoenzymes to clarify their origin in both synovial fluid and serum of 10 patients with elevated level of the enzyme in their sera. The level of the enzyme in serum was elevated in 37% of patients confirming previous reports on that point. The most abundant isoenzyme in the synovial fluid (66.9%) was found to be bone in origin while in serum the most abundant isoenzyme was found to be hepatic (60.5%). This may be responsible for increased bone turn-over in rheumatoid joints whether in formation or resorption.

HPLC separation of alkaline phosphatase isoenzymes. Application to the amniotic fluid

Alkaline phosphatase (AP; orthophosphoric-monoester phosphohydrolase, alkaline optimum; EC 3.1.3.1) is a polymorph glycoprotein. Three gene loci encode two tissue-specific isoenzymes intestinal and placental and a third nonspecific isoenzyme found in liver, bone and kidney. These last isoforms differ by their carbohydrate content [l]. In a previous publication, we described the link existing between the lecithin/ sphingomyelin ratio [2] and the amniotic fluid (AF) AP activity expressed as the ratio; AP heat labile activity versus AP total activity. This ratio, independent of analytical conditions and amniotic fluid volume variations, allowed the prediction of foetal lung maturity and was considered to be helpful in assessing the risk of respiratory distress syndrome in the neonate [3]. In order to account for the relationship between amniotic fluid AP activity and foetal lung maturity, it is necessary to identify the AP isoenzyme responsible for the increasing activity demonstrated during the last term of gestation [3,4]. In this study we describe a rapid and reproducible HPLC method to separate and identify AP isoenzymes in low-activity biological medium such as amniotic fluid.

Isoelectric focusing on cellulose acetate membrane: a separation procedure for alkaline phosphatase isoenzymes

An isoelectric focusing technique for the separation of alkaline phosphatase isoenzymes on cellulose acetate membrane is described. Optimal conditions for isoelectric focusing were established by changing ampholine concentration and focusing conditions. Bone, liver, intestinal, and placental isoenzymes can be resolved into vacious sub-bands in a pH range of 4.1 to 5.2. These sub-bands were correlated with the findings of electrophoretic isoenzyme separation. The whole procedure proves very simple to perform and comparatively time saving (4 h). This procedure may help clarify the problems of ALP isoenzyme differention when electrophoretic patterns are unresolved.

Affinity electrophoresis of human serum alkaline phosphatase isoenzymes in agarose gel containing lectin

Separation of alkaline phosphatase isoenzymes using affinity electrophoresis in agarose gel containing lectin is described. The bone and biliary isoenzymes precipitate during electrophoresis and are clearly separated from the liver isoenzyme. The liver, intestinal and placental alkaline phosphatases are essentially not affected by the lectin. The migration distances of the precipitating bone and biliary fractions vary with their alkaline phosphatase activity. The bone isoenzyme is more heterogeneous than the biliary isoenzyme with respect to interaction with lectin forming both insoluble and soluble complexes. Affinity electrophoresis in agarose gel containing lectin can be used for quantitation by densitometry of liver and bone isoenzymes in sera containing only these two fractions but must be combined with conventional electrophoresis, preferably in agar gel, if biliary, intestinal, or placental isoenzymes are also present.

Clinical relevance of alkaline phosphatase isoenzyme determinations by high performance liquid chromatography.

A new method for the separation of alkaline phosphatase isoenzymes by means of high performance liquid chromatography (HPLC) is presented. One isoenzyme was identified in homogenate of small intestine, two were identified in bone, and two in liver, and fragment and biliary isoenzymes were identified in bile. Sera from 32 patients with different diseases of the skeletal system or the liver were analysed. High activities of the bone isoenzymes were detected in bone diseases, of the second liver isoenzyme in acute hepatitis and of the first liver and biliary isoenzymes in biliary obstruction. There are indications that the first liver isoenzyme is derived from the cell membrane and the second liver isoenzyme from the cytosol. The biliary isoenzyme is considered to be a highly sensitive and specific indicator for cholestasis.

Clinical assessment of the electrophoretic separation of alkaline phosphatase isoenzymes.

Over a five-month period, using data from patients in whom alkaline phosphatase (ALP) isoenzyme studies were requested routinely, we compared actual clinical diagnoses with the predicted diagnoses based on the results of electrophoretic separation of ALP isoenzymes on cellulose acetate before and after heat treatment and on elevated enzymatic activity of gamma glutamyl transferase (GGT) and alanine aminotransferase (ALT) activity. ALP isoenzymes were interpreted on a qualitative basis (presence or absence of liver, bone, or other isoenzyme) by individual clinical pathologists. Overall, the consistency of agreement in 61 patients was 66% for GGT, 51% for ALP isoenzymes, and 21% for ALT. In 44 patients with definite diagnoses, the sensitivity and specificity, respectively, of each laboratory test for patients with liver disease were 88 +/- 5.7% and 64 +/- 14.5% (ALP isoenzymes); 88 +/- 5.7% and 91 +/- 8.6% (GGT); and 6 +/- 4.1% and 91 +/- 8.6% (ALT). In patients with bone disease, the sensitivity and specificity of ALP isoenzymes was 75 +/- 10.8% and 86 +/- 6.6%, respectively. The results indicate that isoenzymes as currently performed need to be improved through standardization of the interpretation of ALP isoenzyme patterns to establish uniformity of comments.

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 alkaline phosphatase isoenzymes from human kidney and comparison with alkaline phosphatases from other human tissues, urine, and Escherichia coli.

Human kidney isoenzymes of alkaline phosphatase (EC 3.1.3.1) after extraction with butan-1-ol were separated by ammonium sulfate precipitation, gel filtration, and chromatofocusing fractionation methods. The separation at each fractionation step was monitored by starch gel and equilibrium-gradient-pore electrophoresis, the latter technique also being used to determine molecular mass. The determined molecular mass (daltons) of alkaline phosphatase from human placenta was 132 000, from urine 95 000, and three isoenzymes from kidney were 195 000, 140 000, and 95 000, respectively. The mass of Escherichia coli alkaline phosphatase was 80 000 daltons, and that of human liver alkaline phosphatase was assumed to be 160 000 daltons. The urinary isoenzyme and the electrophoretically fastest migrating kidney isoenzyme were similar with regard to pH optima, charge, and molecular mass as well as response to L-phenylalanine, L-homoarginine, heat, and urea. Bacterial alkaline phosphatase could be distinguished from the alkaline phosphatases in human tissues and urine by differences in the response to changes in pH and several other physicochemical properties.

Comparison of alkaline phosphatase isoenzymes determined by an inhibition method and by electrophoresis

A chemical inhibition procedure suitable for the routine determination of alkaline phosphatase (AP) isoenzymes in serum has been adapted for use with a fast kinetic analyzer, System Olli 3000. The results of this procedure are compared with the electrophoretic separation of alkaline phosphatase isoenzymes. The comparison of the results obtained indicates that the AP-urea/AP ratio can be used to differentiate between patients with bone and liver disease and that it is possible to estimate the relative bone and liver isoenzyme activities from this ratio quickly using two simple equations.

Serum alkaline phosphatase isoenzymes in childhood.

Using a combination of L-phenylalanine inhibition and heat inactivation, the serum alkaline phosphatase (AP) in 2 to 13 year old children without evidence of hepatobiliary, osseous, or intestinal disease was separated in three fractions; i.e. L-phenylalanine sensitive AP (LPSAP), heat-stable non-L-phenylalanine sensitive AP (heat-stable non-LPSAP) and heat sensitive non-L-phenylalanine sensitive AP (heat-sensitive non-LPSAP). The activities of total AP and the different fractions were measured using optimized test conditions. LPSAP, (mainly intestinal AP), accounts for approximately 12% of the total serum AP activity, heat-stable non-LPSAP (mainly hepatobiliary AP) for approximately 9%, and heat-sensitive non-LPSAP (mainly bone AP) for approximately 77%. To give a better differentiation between bone and liver AP, the percentage ratios of heat-stable non-LPSAP/non-LPSAP (Q value), and heat-stable non-LPSAP/total AP, were determined. Both quotients showed a significant negative correlation with total AP, which has to be taken into account in the interpretation of the results of isoenzyme determinations of serum AP activity. The above semiquantitative separation of AP isoenzymes can be readily done in a routine clinical laboratory.

Separation of alkaline phosphatase isoenzymes and evaluation of the clinical usefulness of this determination.

A simple technic for separation of alkaline phosphatase isoenzymes on agarose gel, using thymolphthalein monophosphate as the isoenzyme indicator substrate, is described. Results demonstrate that separation of isoenzymes of alkaline phosphatase in serum do not always directly identify diseased organs, except in cases in which liver or bone is the organ primarily involved. In most cases physicochemical tests such as heat treatment of serum and assay of γ -glutamyl transpeptidase activity provide as much information as separation of alkaline phosphatase isoenzymes. When both liver and bone diseases are suspected, separation of alkaline phosphatase isoenzymes can provide additional information.

Further improvements on the separation of alkaline phosphatase isoenzymes by gradient pore electrophoresis

Gradient pore electrophoresis is an excellent method for the separation of alkaline phosphatase (AP) isoenzymes. There are problems associated with brittleness of the gel at high acrylamide concentrations, when 5% of total acrylamide is the crosslinking agent N, N'-methylene bisacrylamide (BIS), as is generally used. If the concentration of BIS is reduced too much the gels undergo excessive swelling on subsequent treatment. The use of a gradient gel with a constant BIS concentration of 2 g/l overcomes these problems. Gradients with this concentration of BIS also show improved separation of AP isoenzymes, with all isoenzymes so far investigated giving bands of different mobility. The use of thin layer slabs with linear gradients of 100 g/l to 200 g/l and a constant BIS concentration of 2 g/l offer an excellent method for separating AP isoenzymes. Gradient pore electrophoresis is an excellent method for the separation of alkaline phosphatase (AP) isoenzymes. There are problems associated with brittleness of the gel at high acrylamide concentrations, when 5% of total acrylamide is the crosslinking agent N, N'-methylene bisacrylamide (BIS), as is generally used. If the concentration of BIS is reduced too much the gels undergo excessive swelling on subsequent treatment. The use of a gradient gel with a constant BIS concentration of 2 g/l overcomes these problems. Gradients with this concentration of BIS also show improved separation of AP isoenzymes, with all isoenzymes so far investigated giving bands of different mobility. The use of thin layer slabs with linear gradients of 100 g/l to 200 g/l and a constant BIS concentration of 2 g/l offer an excellent method for separating AP isoenzymes.