Serum Alkaline Phosphatase Isoenzymes Research Articles

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.

Nitrogen utilization, enzyme activity, glucose intolerance and leukocyte chemotaxis in human experimental zinc depletion

We previously reported significant decreases in plasma, whole blood, urinary, seminal and fecal zinc in six young men consuming a semipurified formula diet providing 0.28 mg zinc and 0.8/kg protein per day for 4-9 weeks. During a one-week baseline period, 15.7 mg of zinc (as ZnSO4) were fed; three of the men were repleted with 6.0, 23.2 or 46.3 mg zinc for 2-5 weeks. Biochemical and functional measures of zinc status other than tissue zinc levels were also monitored. No one parameter appeared to parallel dietary zinc status in all subjects, although significant mean changes were seen in serum and leukocyte alkaline phosphatases. Inconsistent changes were noted in erythrocyte delta-amino levulinic acid dehydratase, plasma alkaline ribonuclease and the serum alkaline phosphatase isoenzymes. Nitrogen balance was unaffected by zinc nutritional status. However, alterations in hair root growth phase and morphology, decreases in lymphocyte counts and in transferrin levels during depletion suggest impairment in protein synthesis. Impaired leukocyte chemotaxis and clinical signs indicative of decreased resistance to infection were also noted.

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.

Reaction rate retardation as a method for serum alkaline phosphatase isoenzyme measurement.

A method for serum alkaline phosphatase isoenzymes using an enzyme reaction rate analyser is described. The complete urea-induced degradation of enzyme activity is monitored, from which individual isoenzyme activities are obtained by calculating the constituent exponential components of the degradation curve. Activities have been measured with adequate sensitivity and selectivity for up to four isoenzyme components in normal and in pathological sera. The identity of each isoenzyme present is assigned from its characteristic degradation half-life, and by this method bone and liver alkaline phosphatase are clearly distinguished and quantitated, and a composite value for placental-intestinal alkaline phosphatase activity is obtained. The approach promises to be applicable to a wide range of isoenzymes, and in analogy with 'reaction rate' the term 'reaction rate retardation' is suggested for the procedure.

Hypophosphatasia (adult form): quantitation of serum alkaline phosphatase isoenzyme activity in a large kindred.

We used heat inactivation, L-phenylalanine inhibition, and electrophoresis on polyacrylamide gel and cellulose acetate membranes--with and without use of specific antisera against the liver-bone, intestinal, and placental isoenzymes--to distinguish and quantitate the different alkaline phosphatase isoenzymes in sera from 23 adult members of a kindred affected by the adult form of hypophosphatasia. Nine subjects had values for total activity more than two standard deviations below the mean values for age- and sex-matched normal persons. Bone isoenzyme was diminished in all nine, whereas liver isoenzyme was subnormal in only four. Phosphoethanolamine and phosphoserine in the urine of eight hypophosphatasemic individuals correlated inversely with both total and liver alkaline phosphatase activity in their serum, but not with the activity of the bone isoenzyme. Total activity in the serum of adult kindred members correlated best with the circulating liver isoenzyme activity. The findings suggest that altered hepatic metabolism is responsible for the increased urinary excretion of phosphoethanolamine, and perhaps phosphoserine, in hypophosphatasia.

Hypophosphatasia (adult form): quantitation of serum alkaline phosphatase isoenzyme activity in a large kindred.

Abstract We used heat inactivation, L-phenylalanine inhibition, and electrophoresis on polyacrylamide gel and cellulose acetate membranes--with and without use of specific antisera against the liver-bone, intestinal, and placental isoenzymes--to distinguish and quantitate the different alkaline phosphatase isoenzymes in sera from 23 adult members of a kindred affected by the adult form of hypophosphatasia. Nine subjects had values for total activity more than two standard deviations below the mean values for age- and sex-matched normal persons. Bone isoenzyme was diminished in all nine, whereas liver isoenzyme was subnormal in only four. Phosphoethanolamine and phosphoserine in the urine of eight hypophosphatasemic individuals correlated inversely with both total and liver alkaline phosphatase activity in their serum, but not with the activity of the bone isoenzyme. Total activity in the serum of adult kindred members correlated best with the circulating liver isoenzyme activity. The findings suggest that altered hepatic metabolism is responsible for the increased urinary excretion of phosphoethanolamine, and perhaps phosphoserine, in hypophosphatasia.

Selective effects of clofibrate on alkaline phosphatase isoenzymes in serum

Administration of clofibrate to both hyperlipidaemic patients and normolipidaemic subjects produced a significant decrease, averaging 22%, in serum alkaline phosphatase activity. Quantitative isoenzyme analysis showed that this change was entirely attributable to an average reduction of 39% in the activity of liver alkaline phosphatase, and that no significant change in the bone isoenzyme occurred. An accompanying fall in serum γ-glutamyltransferase activity was seen in some subjects but this change was not statistically significant in the group as a whole.

Bone isoenzyme of serum alkaline phosphatase in acromegaly

In 37 patients with active acromegaly and in 15 patients with inactive acromegaly, activity of bone isoenzyme of serum alkaline phosphatase cor-related ( P< 0.001) with serum concentration of immunoreactive growth hormone. By using stepwise regression analysis, the prediction of serum growth hormone values based on serum levels of bone isoenzyme of serum alkaline phosphatase, gamma-glutamyl transferase and calcium in these patients with acromegaly was within 1 S.D. range in 37 patients and in only 2 patients was it out of 2 S.D. range. By using discriminant analysis, based on bone and liver isoenzymes of serum alkaline phosphatase and urinary hydroxyproline excretion, 87%, 60%, and 97% of the classification of patients with active and inactive acromegaly and healthy adults, respectively, was correct. The multivariate approach offers a quantitative appraisal of the biochemical parameters of peripheral growth hormone action used as an indicator of growth hormone concentration in patients with acromegaly.

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.

Clinical Significance of Serum Alkaline Phosphatase Isoenzyme Levels in Advanced Prostatic Carcinoma

The alkaline phosphatase isoenzymes in 105 patients with stage D carcinoma of the prostate who entered the National Prostatic Cancer Study were analyzed and these values were correlated to clinical response. Only patients with at least 3 measurements of alkaline phosphatase were evaluated. In 91 per cent of patients with metastatic bone disease, bone alkaline phosphatase was elevated. Those patients with higher pre-treatment levels of alkaline phosphatase generally showed a poorer response to therapy. The results of alkaline phosphatase isoenzyme estimation indicate that these biological markers may be used in the evaluation of patients with metastatic prostatic cancer to predict and monitor their response to chemotherapy. The evaluation of bone and liver alkaline phosphatase isoenzymes in earlier stages also may be valuable.

Alkaline phosphatase isoenzymes in liver cirrhosis.

The study of a group of 114 hepatic patients allowed us to demonstrate the presence of an isophosphatase band, called ALP4, which can be of value as an index of evolutive cirrhosis. A certain correlation between the presence of this alkaline phosphatase isoenzyme in serum and histological evidence of moderate or extreme inflammatory response in liver biopsies was shown.

Serum alkaline phosphatase isoenzyme patterns in patients with chronic renal failure

1. 1. Using acrylamide gel electrophoresis the serum alkaline phosphatase (ALP) isoenzyme patterns of 204 patients with chronic renal failure have been examined for periods up to 18 months in length. 2. 2. Of those with elevated serum ALP activity the bone isoenzyme was largely responsible. The presence of increasing amounts of the bone isoenzyme even if the total serum ALP activity remains within the normal reference range should also indicate bone pathology. 3. 3. Intestinal alkaline phosphatase was the major serum alkaline phosphatase in 15% of patients on regular haemodialysis and 10% of uraemic patients not on dialysis. The overall incidence of detectable intestinal alkaline phosphatase in those with normal serum ALP activity was 36%. 4. 4. With those patients whose serum ALP activity changed significantly during the investigation this usually reflected changes in the amount of the bone isoenzyme. Patients with abnormal amounts of the intestinal isoenzymes in their serum usually showed little variation in serum ALP activity over the period of the study.

Serum alkaline phosphatase isoenzymes in epileptic children receving anticonvulsant drugs.

In 40 epileptic children on long-term anticonvulsant treatment, serum alkaline phosphatase (AP) isoenzymes were separated semiquantitatively using a combination of L-phenylalanine inhibition and heat inactivation. Though mean total serum AP activity was significantly increased compared to age matched controls, only 4 individual values exceeded the upper limit (mean + 2SD) of the reference sample. In epileptics the mean activity of the heat-sensitive non L-phenylalanine sensitive AP fraction (non-LPSAP) was significantly increased (P less than 0.01) and the mean Q-value (i.e. percentage ratio of heat-stable non-LPSAP/non-LPSAP) was significantly decreased (P less than 0.05), thus indicating an enhancement of the bone isoenzymes during anticonvulsant treatment. In 4 patients the isoenzyme pattern was abnormal although total serum AP activity was normal and in 3 of them the deviation indicated enhanced bone isoenzyme. The data provide evidence that in anticonvulsant treated children the bone isoenzyme, rather than hepatobiliary isoenzyme fraction, may be increased even when total serum AP activity is normal. Thus, semiquantitative separation of serum AP isoenzymes may be a helpful guide as to whether or not an eplieptic child should be given vitamin D.

Alkaline phosphatase isoenzyme patterns in normal sera of Nigerians

Serum alkaline phosphatase isoenzymes have been examined in Nigerian subjects by cellulose acetate electrophoresis to determine the variations that may occur in the ethnic groups and to establish the normal patterns in health. The results have shown no differences in the isoenzyme distribution patterns in healthy Nigerian and Caucasian subjects.

A modified inactivation - inhibition method for determining the serum activity of alkaline phosphatase isoenzymes

A procedure using heat inactivation and L-phenylalanine inhibition to quantitate the activities of bone, liver and intestinal alkaline phosphatase isoenzymes in human serum was confirmed by alkaline phosphatase isoenzyme analysis using an electrophoretic procedure. The results of this assay were compared with the radionuclear 85Sr test, and gamma-glutamyl transpeptidase activity in a group of patients with hepatobiliary and bone diseases.

Improved Electrophoretic Resolution of Human Serum Alkaline Phosphatase Isoenzymes in Agarose Gel by Triton X-100

Improved Electrophoretic Resolution of Human Serum Alkaline Phosphatase Isoenzymes in Agarose Gel by Triton X-100

Practical aspects on the determination of serum alkaline phosphatase isoenzymes using l-phenylalanine and urea

Simple methods for estimation of the most frequently occurring alkaline phosphatase isoenzymes in serum are evaluated. Methods based on inhibition by l-phenylalanine or denaturation by heat or urea have been adopted for a reaction-rate instrument (LKB 8600) using conditions recommended by the Scandinavian Enzyme Committee, when applicable. The analytical results are directly evaluated for various groups of patients and also used in a mathematical “three-isoenzyme” model. These calculations did not improve the clinical value of the primarily obtained data. Thus, it was concluded that under certain precautions the results obtained in the presence of l-phenylalanine or urea should be used separately.

Isoenzymes of alkaline phosphatase in serum of newly born lambs

In the serum of lambs at birth most of the circulating alkaline phosphatase, identified by electrophoretic analysis, was of skeletal origin. Suckling was associated with a rapid increase in activity of intestinal alkaline phosphatase isoenzymes in serum. There was a coincidental increase in level of circulating gamma globulin. Activity of the intestinal isoenzymes in serum began to fall between 15 and 21 h after birth and this fall preceded a fall in serum gamma globulin concentration. The electrophoretic characteristics of the intestinal isoenzymes in serum changed as activity of these isoenzymes increased and then decreased.

Isoenzymes of alkaline phosphatase in serum of lambs and ewes

Electrophoresis in polyacrylamide gels was used to identify isoenzymes of serum alkaline phosphatase in sheep. Skeletal isoenzyme predominated in the serum of lambs and liver, skeletal and intestinal isoenzymes were found in the serum of adult ewes. In r ewes (R-r-i blood group system) intestinal isoenzyme activity was 67 per cent of total serum activity; in R ewes intestinal activity was only 36 per cent of total activity. Relative activities of the three isoenzymes differed greatly within individual ewes and between individual ewes during pregnancy and lactation.

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.