Microheterogeneity of serum alkaline phosphatase isoenzymes as revealed by isoelectric focusing

The microheterogeneity of human serum alkaline phosphatase (ALP) was investigated by means of isoelectric focusing. Liver and bone isoenzymes focused in a similar pattern, with about 10 bands located between pH 3.7 and 4.9, but differed in the relative intensity of the various bands. Intestinal ALP exhibited 7 to 8 bands at pH 4.9-5.1, and the placental enzyme showed 2 to 3 bands at pH 4.9. Mild digestion with neuraminidase revealed that the banding of liver and bone isoenzymes was at least partly due to differences in the sialic acid content of the various fractions. Extensively desialylated liver and bone isoenzymes showed apparently identical patterns with 6 to 7 bands focused at pH 6.2-6.7. Isoelectric focusing is a useful method for characterizing the microheterogeneity of alkaline phosphatase isoenzymes. The clinical value of this method seems to be limited, however, since it did not distinguish between liver and bone isoenzymes and failed to detect 'specific' isoelectric fractions correlated to various diseases.     

Resolution of alkaline phosphatase isoenzymes in serum by isoelectric focusing in immobilized pH gradients

This new method for fractionation of serum alkaline phosphatase isoenzymes is based on isoelectric focusing on a mixed-type polyacrylamide support containing an immobilized pH gradient with a superimposed carrier-ampholyte gradient. All known forms of alkaline phosphatase are separated in an Immobiline pH 3.5-6.0 gradient, the sample being applied into pockets cast on a pH 8.0 plateau. Sharp zymogram bands are obtained by substituting alkaline-stable 5-bromo-4-chloro-3-indoxyl phosphate and tetrazolium salts for the standard 1- and 2-naphthyl phosphate-diazonium salt combinations. After hydrolysis of the phosphate group by the alkaline phosphatase the indoxyl moieties reduce tetrazolium salts to nearly insoluble and nondiffusible formazan precipitates. Normal sera show an array of about 10 isobands isoelectric between pH 3.9 and pH 4.79. In Paget's disease, two sharp isobands with pls of 4.97 and 5.09 are seen. Placental alkaline phosphatase overlaps with the higher pl bands of normal serum; however, upon heat destruction of the latter, it shows four sharp bands with the following pl's: 4.59, 4.62, 4.67 and 4.73.     

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.     

Analysis of human bone alkaline phosphatase isoforms: comparison of isoelectric focusing and ion-exchange high-performance liquid chromatography

BACKGROUND: Several isoforms of alkaline phosphatase (ALP) can be identified in human tissues and serum after separation by anion-exchange HPLC and isoelectric focusing (IEF). METHODS: We purified four soluble bone ALP (BALP) isoforms (B/I, B1x, B1 and B2) from human SaOS-2 cells, determined their specific pI values by broad range IEF (pH 3.5-9.5), compared these with commercial preparations of bone, intestinal and liver ALPs and established the effects of neuraminidase and wheat germ lectin (WGA) on enzyme activity. RESULTS: Whilst the isoforms B1x (pI=4.48), B1 (pI=4.32) and B2 (pI=4.12) resolved as well-defined bands, B/I resolved as a complex (pI=4.85-6.84). Neuraminidase altered the migration of all BALP isoforms to pI=6.84 and abolished their binding to the anion-exchange matrix, but increased their enzymatic activities by 11-20%. WGA precipitated the BALP isoforms in IEF gels and the HPLC column and attenuated their enzymatic activities by 54-73%. IEF resolved the commercial BALP into 2 major bands (pI=4.41 and 4.55). CONCLUSIONS: Migration of BALP isoforms is similar in IEF and anion-exchange HPLC and dependent on sialic acid content. HPLC is preferable in smaller scale research applications where samples containing mixtures of BALP isoforms are analysed. Circulating liver ALP (pI=3.85) can be resolved from BALP by either method. IEF represents a simpler approach for routine purposes even though some overlapping of the isoforms may occur.     

Isoelectric focusing of neuraminidase-treated alkaline phosphatase isoenzymes on agarose gel

We attempted to separate bone and liver alkaline phosphatase (EC 3.1.3.1) isoenzymes in human serum by isoelectric focusing on agarose gel. We found that in a pH 3-10 gradient the liver and bone isoenzymes focused into so many bands over a narrow pH range such that the information could not be quantified. However, when the bone isoenzyme in serum was first desialylated at 37 degrees C for a minimum of 6 h, catalyzed by neuraminidase (EC 3.2.1.18) at pH 5.8-6.0, we could detect four distinct bands with pls of 6.7, 6.8, 6.9, and 7.0. Under the same conditions, the liver isoenzyme in human serum focused into one band at pH 7.0. The multiple banding we observed for the desialylated bone isoenzyme has not been previously reported. The method is suited as a qualitative technique for detecting the bone alkaline phosphatase isoenzyme in serum.     

Isoelectric focusing of human tissue alkaline phosphatase isoenzymes in agarose gel

Human tissue alkaline phosphatases obtained by butanol treatment of tissue homogenates were subjected to isoelectric focusing in agarose gel. Inactivation of alkaline phosphatases by ampholytes was prevented by incorporating zinc into the gel prior to electrofocusing and to the authors' knowledge this has not been reported before. The addition of zinc did not alter the pH gradient and the method was reproducible. Ampholytes from two different manufacturers differed in their requirements for zinc in preventing inhibition of alkaline phosphatase. The method is more rapid than previously described methods and is very sensitive.     

Isoelectric focusing of human neutrophil alkaline phosphatase isoenzymes in agarose gel

An isoelectric focusing technique for human neutrophil alkaline phosphatase isoenzymes is described. After sonication with Zwittergent 3-12, butanol extraction and ultracentrifugation, dialysis of cytosols precedes focusing. Focusing patterns show a heterogeneity with two enzymatic activity areas: a main component at pI 6.7-6.8 with a minor component at pI 4.8-5.0 which is difficult to visualize due to its sensitivity to experimental conditions. The addition of 3 mmol/l ZnCl2 to the agarose gel improved the staining of focused bands and in particular the anodic component.     

A relative heat stability test for the identification of serum alkaline phosphatase isoenzymes

A new approach is presented for the identification of increased levels of serum alkaline phosphatase isoenzyme activity by heat inactivation. The pattern of enzyme stability at 56° over a 20-min period is compared with simultaneously determined patterns for control sera from patients with diagnosed liver and bone disease. This assessment of relative heat stability patterns has allowed the differentiation between liver and bone as the tissue source of increased serum alkaline phosphatase in 93% of the cases.     

Characterization of alkaline phosphatase isoenzymes in serum by agar gel electrophoresis

A simple method of characterizing alkaline phosphatase isoenzymes in serum by agar gel electrophoresis is described. By using sera from cases of obstructive jaundice as markers in the electrophoresis reproducible β₂-phosphatase could be obtained. Combining the electrophoretic tests with heat stability tests at 56° bile, liver, bone, intestinal, and placental isoenzymes could be assessed for routine clinical purposes. Results obtained with 221 normal subjects are given and the clinical use of the method is briefly discussed.     

Analysis for alpha-amylase isoenzymes by automated isoelectric focusing

We describe the fractionation of alpha-amylase (EC 3.2.1.1) isoenzymes by use of the Pharmacia "Phastsystem" electrophoresis apparatus. The separation is done by isoelectric focusing on "Phastgel IEF 5-8." A complete run, including staining, takes 4 h and can easily resolve eight isoenzymes. Samples can be analyzed in a routine laboratory with use of conventional reagents.     

Liquid-chromatographic determination of isoenzymes of alkaline phosphatase in serum and tissue homogenates

We describe a new method for separating alkaline phosphatase (AP) isoenzymes by means of "high-performance" liquid chromatography. Isoenzymes are eluted from the column (Mono Q HR 5/5, a strong anion-exchanger) with a stepwise gradient of LiCl. The isoenzymes originating from small intestine, bone, liver, and bile were identified by use of tissue homogenates, pathological sera, and heat inactivation.     

Abnormal alkaline phosphatase isoenzymes detected in the serum of elderly patients

Alkaline phosphatase (ALP) isoenzyme analysis of 101,832 serum samples was performed by electrophoresis using cellulose acetate membrane, and abnormal bands at the alpha(1) and alpha(2) globulin positions were detected in 23 samples. The physicochemical properties of these abnormal ALP isoenzyme fractions were examined. In brief, the abnormal fractions were heat-sensitive and inhibited by L-phenylalanine, and neither sialic acid in their polysaccharide chains nor the glycosyl-phosphatidylinositol anchor was detected by the enzyme treatment. The physicochemical properties of abnormal ALP isoenzyme fractions detected in Patients 1 - 22 were similar to those of the adult small intestine type. However, the molecular weight of the adult small intestine type abnormal fractions was smaller than that of the normal fractions. These adult small intestine type abnormal bands at the alpha(1)- to alpha(2)-globulin positions, which were identified in the serum of Patients 1 - 22, were detected in elderly patients. Most of them had various basal diseases such as renal insufficiency, fracture, interstitial pneumonia, and chronic pancreatitis. Some of them had severe diseases such as rectal cancer, descending colon cancer, and septic shock. In Patient 23, the polysaccharide chain had sialic acid and was heat sensitive physicochemical properties that were similar to those of the Kasahara ALP variant.