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.     

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.     

Two new methods for separating and quantifying bone and liver alkaline phosphatase isoenzymes in plasma

We describe two new methods for the separation and quantification of the bone and liver isoenzymes of alkaline phosphatase (EC 3.1.3.1) in plasma. In the first, we use wheat-germ lectin to precipitate the bone isoenzyme. About 80% of this, but minimal liver isoenzyme, is precipitated. The activity of the bone isoenzyme is calculated from measuring the alkaline phosphatase activity in the precipitate, that of liver alkaline phosphatase by subtracting the activity of the bone isoenzyme from total alkaline phosphatase activity. The liver fraction will also contain biliary, intestinal, and placental alkaline phosphatase if these are present in the original plasma, but correction for such activity is readily made. In the second method, samples are separated on cellulose acetate membranes that, before electrophoresis, have been soaked in buffer containing wheat-germ lectin. The bone isoenzyme is retarded and clearly separated from the liver fraction, allowing these isoenzymes to be quantified by densitometry. Both methods are rapid, reproducible, and suitable for use in the diagnostic laboratory.     

Affinity electrophoresis of alkaline phosphatase using polyacrylamide gels.

A method is described for the separation of liver and bone isoenzymes of alkaline phosphatase in serum using wheat germ lectin affinity electrophoresis in a polyacrylamide gel matrix. The electrophoretic mobilities of liver and intestinal isoenzyme are essentially not affected by lectin, but the bone enzyme is retarded and separated from the liver fraction. Affinity electrophoresis in polyacrylamide gel, combined with agarose gel electrophoresis, and a solid-phase linked antibody precipitation procedure for intestinal alkaline phosphatase allowed the various isoenzyme fractions, biliary, liver, bone and intestinal, to be quantitated.     

Characterization of serum alkaline phosphatase isoenzymes by affinity electrophoresis in agarose gel containing lectin combined with agar gel electrophoresis

The combined use of affinity electrophoresis in agarose gel containing lectin and of agar gel electrophoresis for the quantitation of liver, bone, biliary and intestinal alkaline phosphatase isoenzymes is described. Sera from patients with various diseases and from normal subjects (blood donors) have been analyzed. Data from normal subjects show that the bone isoenzyme is the predominant fraction (about 62%) in adults. The relative proportions of the alkaline phosphatase isoenzymes are similar in both sexes in adulthood (21-50 years). The higher alkaline phosphatase activity found in men than in women (ages 21-50 years) is due to higher values for both liver and bone isoenzymes. The difference between men and women tends to decrease after the age of 50 mainly due to an increase of the bone isoenzyme in women.     

Alkaline Phosphatase: Diagnostic Value of Isoenzyme Determination

Abstract. The isoenzyme pattern of the alkaline phosphatases was determined in the sera of 51 normal subjects, 28 patients with hepatobiliary diseases and 17 patients with bone diseases. Two quick and technically simple methods of differentiation were used for a semiquantitative determination: stereospecific sensitivity to L-phenylalanine, especially of the small intestine phosphatase, and the separation of the bone and liver/biliary tract phosphatases by a combination of heat inactivation and stereo-specific inhibition. The basic principles of these methods are described. The results, statistically evaluated, are discussed. The alkaline phosphatase activity in the serum of healthy adults stems from isoenzymes of the small intestine (about 20%), of bone and of hepatobiliary origin. In hepatobiliary diseases the proportion of bone to liver/biliary tract phosphatases changes significantly in favour of the latter. In diseases of the skeleton, however, which are accompanied by increased activity of serum alkaline phosphatase, there is a significant increase in the absolute fraction of bone phosphatase in the total activity. In addition to the demonstration of these qualitative and quantitative changes in the isoenzyme distribution patterns, limiting values were determined for a normal group; values outside these can be considered as pathognomonic for diseases of the liver and biliary tract and the skeleton respectively. Of particular importance for early diagnosis is the fact that changes in the isoenzyme distribution pattern are demonstrable not only when the total activity is increased, but at a time when the serum activity lies still within normal limits. The methods used are suitable for a rapid and reproducible semiquantitative determination of the isoenzymes of alkaline phosphatase, and for early differential diagnosis of diseases of the skeleton (especially metastatic tumours), the liver and biliary tract.     

Clinical usefulness of the simultaneous localisation of the isoenzymes of alkaline phosphate and gamma-glutamyl transferase

Simultaneous localisation of the isoenzymes of alkaline phosphatase and gamma-glutamyl transferase was carried out on 173 serum samples from patients with hepatobiliary and bone disease to determine (i) if a better discrimination of the underlying pathology could be obtained by such an approach, and (ii) if the pathology suggested by the alkaline phosphatase isoenzyme separation could help in determining the cellular location of the gamma-glutamyl transferase isoenzymes. It was found that the localisation of the gamma-glutamyl transferase isoenzymes had, with a few exceptions, little advantage over the measurement of the total activity but that some evidence concerning the cellular location of the gamma-glutamyl transferase isoenzymes could be gained by such an approach. There was support for the view that the alpha 2-migrating isoenzyme is derived from the liver parenchymal cytoplasm, but little support for the view that the alpha 1-migrating isoenzyme is predominantly the induced microsomal enzyme or that the beta-migrating isoenzyme is the result of impaired biliary secretion.     

碱性磷酸酶同工酶对原发性胆汁性肝硬化的诊断价值初探

目的　初步探讨血清碱性磷酸酶(ALP)同工酶对原发性胆汁性肝硬化(PBC)的辅助诊断价值。方法 　用Sebia琼脂糖凝胶电泳分离30名正常人、23例PBC患者和25例其他肝胆疾病患者的血清ALP同工酶,经 光密度扫描仪扫描后,分析各型同工酶的相对含量。结果　血清ALP同工酶经电泳后可分为肝型、胆汁型、肠 型和骨型4个区带。PBC患者的肝型ALP的相对含量最高,约为56%～70%,显著高于正常对照及乙肝后肝硬 化患者(P<0.05),而其胆汁型ALP的相对含量显著低于肝外阻塞性黄疸患者(P<0.05)。结论　ALP同工酶 分析可辅助诊断PBC。     

Quantitative fractionation of alkaline phosphatase isoenzymes according to their thermostability.

Continuous monitoring of heat denaturation of a mixture of alkaline phosphatase isoenzymes at 60 degrees C and pH 7.5 permits the simultaneous direct identification and quantitation of three isoenzymes: the placental isoenzyme, the L-phenylalanine-sensitive intestinal isoenzyme, and the liver isoenzyme (hepatocytic). The isoenzyme that is principally of bone origin cannot be identified as such without the help of other diagnostic aids and the patient's medical history. All human tissues contain alkaline phosphatase, many organs more than one of the isoenzymes. Liver alkaline phosphatase, which constitutes 40-50% of normal serum alkaline phosphatase activity, was measured in the serum of persons with various liver diseases. Its activity exceeded normal in all types of liver disease; in 80% of cases this increase was accompanied by increased gamma-glutamyl-transferase activity, but the quantitative correlationship (r = 0.54) was not as good as expected if both enzymes come from the same source and are indices of liver dieases. Liver alkaline phosphatase activity increases in the blood early in liver disease, before most liver tests show abnormalities. The other major isoenzyme of normal serum probably represents a mixture of isoenzymes from bone and reticulo-endothelial and vascular tissues, which all contain the same "very heat-labile" alkaline phosphatase. Cord blood and children's sera contain mostly this very heat-labile isoenzyme.     

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.     

Alkaline phosphatase isoenzymes in serum determined by high-performance anion-exchange liquid chromatography with detection by enzyme reaction

This rapid, reproducible method for separating and determining individual alkaline phosphatase (EC 3.1.3.1) isoenzymes in serum is based on high-performance liquid chromatography with a weak anion-exchange column (SynChropak AX 300). The isoenzymes so resolved are detected by using an on-line enzyme reaction followed by spectrophotometric monitoring at 405 nm of the 4-nitrophenol formed. Complete diagnostic profiles of the various isoenzymes present in normal and pathological sera are obtained within 20 min. The mean (and SD) normal concentrations of the bone B1 and intestinal isoenzymes in serum of adults were 3.7 (4.3) and 4.5 (3.9) U/L, respectively (n = 14), and of the bone isoenzyme B2 and liver isoenzymes L1 and L2, 5.8 (8.6), 33.0 (10.6), and 12.0 (4.8) U/L, respectively (n = 17). Concentrations of the B2 and L1 isoenzymes in adults over age 40 years differed significantly from those in adults younger than 40 years, that of bone isoenzyme being lower (P less than 0.05) and that of the liver isoenzyme being higher (P less than 0.001) in the younger adults.     

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.     

Atypical alkaline phosphatase isoenzyme in serum from a patient with Hodgkin's disease

An alkaline phosphatase isoenzyme that did not move from the origin in agarose gel electrophoresis was detected in serum from a 51-year-old woman with Hodgkin's disease. Inhibitor and heat-inactivation studies of the patient's serum alkaline phosphatase showed properties resembling those of both liver and bone isoenzymes. No immunoglobulin or high-molecular-mass complexes with the alkaline phosphatase isoenzyme were detected. The relative molecular mass (Mr) of the atypical alkaline phosphatase isoenzyme was 182 000, that of the liver alkaline phosphatase isoenzyme control 170 000. Treatment of both of these isoenzymes with neuraminidase gave a product with an Mr of 140 000. We propose that a post-translational modification increased the carbohydrate content of the liver alkaline phosphatase isoenzyme, thus changing the charge characteristics of the enzyme and decreasing its electrophoretic mobility. We believe this to be the first report of a post-translational modification in a heat-sensitive isoenzyme of alkaline phosphatase.     

Diagnostic aspects of alkaline phosphatase and its isoenzymes

The changes in serum alkaline phosphatase that are of main diagnostic importance result from increased entry of enzyme into the circulation. This results from increased osteoblastic activity in bone disease, and increased synthesis of alkaline phosphatase by hepatocytes in hepatobiliary disease. The liver and bone forms of alkaline phosphatase are differently-glycosylated forms of a single gene product. The main value of their specific estimation is found in patients in whom bone and liver diseases co-exist, for example, as a result of cancer. Abnormal expression of genetically-distinct alkaline phosphatase isoenzymes is valuable in monitoring cancers, particularly germ-cell tumors. These isoenzymes include Regan and Nagao isoenzymes, which correspond respectively to normal placental and placental-like alkaline phosphatases, and the Kasahara isoenzyme which appears to result from re-expression of a fetal intestinal alkaline phosphatase gene.     

Isoenzymes of alanine arylamidase (AAP, EC 3.4.11.2) and gamma-glutamyltranspeptidase (GGTP, EC 2.3.2.2) in chronic pancreatitis and pancreas neoplasm (author's transl)]

In 22 patients with chronic pancreatitis and 16 patients with pancreas neoplasms gamma-glutamyltranspeptidase (GGTP) and alanine arylamidase (AAP) in serum were measured and the isoenzymes were determined by gel electrophoresis on Agar. No specific isoenzyme pattern was found for chronic pancreatic diseases in a comparative investigation with a group of 19 patients suffering from hepatobiliary diseases. Two fractions of AAP and GGTP isoenzymes were found on agar gel electrophoresis: alpha-1 and alpha-2 (GGTP between alpha-2 and beta-globulin). The alpha-1 fraction of AAP and GGTP seems to be a specific liver isoenzyme. The slower fraction of both enzymes was also found in chronic pancreatic diseases and cholestatic diseases as in neoplasms of liver, pancreas and biliary tract. Practical importance of the findings is diminished by large variation coefficients of the results. A significantly low ratio of alpha-1 to alpha-2 fraction (or beta-globulin) on electrophoresis of the isoenzymes of AAP and GGTP was found in the group with neoplasm of pancreas (especially neoplasm of the pancreas head) as compared to the group with intrahepatic cholestasis. The possible causes and diagnostic importance of the findings are discussed.     

Incubation with neuraminidase and affinity electrophoresis with wheat-germ lectin compared for separating and quantifying alkaline phosphatase isoenzymes in plasma

Of 98 patients' specimens examined for alkaline phosphatase (EC 3.1.3.1) isoenzymes by electrophoresis on cellulose acetate membrane after incubation with neuraminidase, 50 showed only a single liver or bone isoenzyme staining band; in 15 of these, the tissue origin of the fraction could not be accurately identified from its electrophoretic location. In the remaining 48 specimens, both liver and bone fractions were identifiable, but in only 25 of these was the electrophoretic resolution sufficient to yield separate peaks on densitometry. In contrast, both liver and bone alkaline phosphatase isoenzymes were identified in 95 of the 98 specimens by affinity electrophoresis involving wheat-germ lectin, the detection of both fractions being in agreement with the results of sequential heat inactivation. The tissue origin of the enzyme bands was readily ascertainable from their consistent electrophoretic location in this medium, and in 89 of the specimens the isoenzyme fractions could be resolved into separate peaks on densitometry. We conclude that resolution of liver and bone alkaline phosphatase by incubation with neuraminidase followed by cellulose acetate electrophoresis is greatly inferior to that obtained by wheat-germ lectin affinity electrophoresis.     

Electrophoretic method for assessing the normal and pathological distribution of alkaline phosphatase isoenzymes in serum.

We describe a simple, reproducible, discontinuous system for polyacrylamide disc gel-electrophoresis, with which the alkaline phosphatase isoenzymes in human serum can be fractionated. No sample preparation is needed. The isoenzymes are classified according to their electrophoretic mobilities (R-F values) and quantitated by peak area measurements from spectrophotometric scans. The four alkaline phosphatase isoenzymes usually present in normal sera, in order of descending mobilities (and designated according to principal tissue of origin) are: "fast" liver, "slow" liver, bone, and intestine. Sera of diseased patients show a greater variety of isoenzyme distribution patterns, but the most frequently observed patterns are the same as normal patterns. We conclude that the finding of "fast" liver only is not pathognomonic, as previously reported by others, and that information on relative distributions per se is not diagnostically useful, although information on specific increases in activity is useful. With this system, hepatobiliary disorders can be differentiated from other forms of liver and bone diseases.     

胆总管囊肿患儿血清肝型碱性磷酸酶同工酶的分析

目的分析胆总管囊肿患儿血清碱性磷酸酶（ALP）同工酶[肝型ALP（L-ALP），特别是L2-ALP]的临床价值。方法用Sebia琼脂糖凝胶电泳分离25名胆总管囊肿患儿和48名对照儿童的血清ALP同工酶，经光密度扫描仪扫描后，分析各型同工酶的相对含量。结果血清ALP同工酶经电泳后可分为肝型（L1和L2）、胎盘型（P1和P2）、肠型（I1、I2、I3）和骨型（B）4个部分。通过这种方法，L-ALP和B-ALP得到了很好的分离。儿童血清中L2-ALP的水平与胆总管囊肿的发生具有明显的相关性（P〈0．01）；胆总管囊肿患儿术后L2-ALP水平显著降低。结论胆总管囊肿患儿血清L-ALP同工酶的测定具有一定的临床价值。     

Total bone and liver alkaline phosphatases in plasma: biological variations and reference limits

We have studied factors affecting biological variation in total plasma alkaline phosphatase in a population of 32 329 apparently healthy subjects four years old or older. Quantification of the bone and liver isoenzymes after thermal denaturation made it possible to specify the contributions of each isoenzyme to variations in the total activities. The main factors that modify plasma alkaline phosphatase activity are age, sex, hormonal state (puberty or menopause), and morphometric parameters (height, body weight, or degree of overweight). The bone isoenzyme is mainly responsible for the variations associated with age, sex, and puberty and to some extent with the menopause. Activity of the liver isoenzyme was also altered at the menopause and by certain drugs, such as oral contraceptives and blood-lipid-lowering agents. These data allow us to propose reference limits for total plasma, bone, and liver alkaline phosphatases according to age and sex.     

"Fragmented" isoenzymes of alkaline phosphatase in the diagnosis of transient hyperphosphatasemia

We describe the appearance of "fragmented" isoenzymes of serum alkaline phosphatase (EC 3.1.3.1) in two cases of transient hyperphosphatasemia. We determined the isoenzymes by liquid chromatography, then characterized them by heat inactivation, inhibition with 5 mmol/L L-phenylalanine solution, and electrophoresis on cellulose acetate membranes. We suspect that a virus-induced decrease in clearance of the enzyme from serum is responsible for a similar increase of bone and liver isoenzyme activities and for the presence of these fragmented isoenzymes in transient hyperphosphatasemia.