Origin of an elevated plasma alkaline phosphatase activity in non-jaundiced patients.

Samples from 260 non-jaundiced patients with elevated plasma alkaline phosphatase activities were analysed for gamma-glutamyltransferase and 5'-nucleotidase activity, and for alkaline phosphatase isoenzyme pattern. The plasma gamma-glutamyltransferase activity was found to be a more sensitive index than that of plasma 5'-nucleotidase in confirming the presence of a liver component of the elevated plasma alkaline phosphatase. If the gamma-glutamyltransferase level is normal it is probable that the increase in plasma alkaline phosphatase activity is of bone origin. However, and elevated gamma-glutamyltransferase result does not exclude a bone component; in this situation plasma alkaline phosphatase isoenzymes should be estimated. The causes of elevated activities of plasma alkaline phosphatase, 5'-nucleotidase and gamma-glutamyltransferase, found in this investigation were generally the same as those found by other worker. The effect of treatment by drugs on gamma-glutamyltransferase, an inducible enzyme, needs more investigation.     

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.     

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.     

Urinary hydroxyproline and serum alkaline phosphatase in sickle cell disease.

Serum alkaline phosphatase, alkaline phosphatase isoenzymes, and urinary hydroxyproline excretion were studied in 20 young adult sickle cell patients and 58 matching normal controls. Total alkaline phosphatase was significantly higher in the sickle cell patients than in controls. Heat inactivation test and isoenzyme electrophoresis indicated that bone is the predominant isoenzyme in patients. Hydroxyproline excretion was significantly higher in the sickle cell patients than in controls. Serum total alkaline phosphatase correlated well with urinary hydroxyproline excretion in sickle cell patients (r = 0.73). Both alkaline phosphatase and hydroxyproline increased with age in the sickle cell patients. This study suggests that delayed growth and/or bone destruction may contribute to the elevated levels of alkaline phosphatase and urinary hydroxyproline.     

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.     

Reference limits of bone and liver alkaline phosphatase isoenzymes in the serum of healthy subjects according to age and sex as determined by wheat germ lectin affinity electrophoresis

Wheat germ lectin affinity electrophoresis was employed for quantitating the bone and liver isoenzymes of alkaline phosphatase (EC 3.1.3.1) in serum and for determining the reference limits of each isoenzyme activity in 488 healthy individuals. Bone phosphatase activity was detected even after bone growth, accounting for 60-70% of the total activity. An increase in bone phosphatase activity occurred in older females, but there was a decrease in older males. Liver phosphatase activity gradually increased with age in both sexes, males showing higher activity than females at all ages. Wheat germ lectin affinity electrophoresis of serum alkaline phosphatase is a simple and useful method for quantitating bone and liver alkaline phosphatase activities.     

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.     

Alterations in human serum alkaline phosphatase and its bone isoenzyme by chronic administration of lithium

There are conflicting data on the effects of lithium on bone metabolism. Bone formation is known to be reflected by the activity of alkaline phosphatase of the bone tissue. We have found significant differences in mean serum alkaline phosphatase and its bone isoenzyme levels between a group receiving lithium and a control group. In this study increased bone isoenzyme of alkaline phosphatase activity above the normal range was found in 27 of 41 patients treated with lithium carbonate. In 19 of 41 patients treated with lithium the activity of bone isoenzyme was increased above the normal range even in the absence of increased activity of serum total alkaline phosphatase.     

Changes in the bone and liver isoenzymes of alkaline phosphatase in postmenopausal women being treated with norethisterone

The effects of norethisterone therapy on alkaline phosphatase isoenzyme activities were studied in a group of postmenopausal women. There was a significant fall in total alkaline phosphatase activity after 8 wk which was still in evidence after 24 wk. Both bone and liver alkaline phosphatase isoenzyme activities were decreased during the first 16 wk on treatment, but after 24 wk only the bone phosphatase activity was significantly lower than the pretreatment level. The other biochemical indices of bone metabolism and liver function were also measured during the study. The results indicate that bone specific alkaline phosphatase activity is a more sensitive index of bone activity than total alkaline phosphatase and that monitoring of total activity may in some instances be misleading.     

Alkaline phosphatase isoenzymes in the plasma of preterm and term infants: serial measurements and clinical correlations

Serial measurements of the bone and fetal intestinal isoenzymes of alkaline phosphatase (EC 3.1.3.1) in the plasma of 43 term and 43 preterm infants, from birth to six weeks later, indicate that the bone isoenzyme gradually increases over this period in both preterm and term infants fed with unsupplemented commercial formulas. Preterm babies given formula supplemented with calcium (with or without additional phosphate) had significantly lower bone isoenzyme activities for most of the study period. The concentrations of fetal intestinal isoenzyme increased, under the stimulation of milk feeding, from generally undetectable at birth to a peak during the first two weeks postpartum, and then declined. This increase was highly significantly negatively correlated with gestational age, the preterm infants having a much higher and more prolonged increase in this isoenzyme than did term infants. Unlike the adult isoenzyme, fetal intestinal alkaline phosphatase in plasma showed no relationship with blood group status.     

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 isoenzyme patterns in malignant disease.

Early treatment of patients with malignant disease and liver or bone metastasis may increase their survival time. We have used the activity patterns of liver and bone isoenzymes of alkaline phosphatase (ALP), separated by agarose gel electrophoresis, to detect early metastasis. We studied ALP isoenzyme patterns in a background population of 101 patients with no evidence of any disease that might influence this pattern; a healthy reference population (n = 330); and the following three groups of patients: 143 with malignant disease, 47 with nonmalignant liver disease, and 22 with nonmalignant bone disease. Cutoff and predictive values of liver ALP, high-molecular-mass (high-M(r)) ALP, and bone ALP were established for detecting liver and bone metastasis. The positive predictive value of liver and high-M(r) ALP was higher than that of total ALP in detecting liver metastasis, but liver and high-M(r) ALP did not enable us to differentiate between malignant and nonmalignant liver disease. Total ALP activity was of slightly more value than liver and high-M(r) ALP in enabling us to rule out liver metastasis. From bone ALP activity we could not distinguish between nonmalignant bone disease and bone metastasis. The negative predictive value of bone ALP in the diagnosis of bone metastasis was low, but its positive predictive value was high and superior to that of total ALP.     

Activities of bone and liver alkaline phosphatases in serum in health and disease

A heat-inactivation method for determining absolute activities of liver and bone alkaline phosphatases in serum has been applied extensively in routine diagnosis. Values for each isoenzyme in healthy individuals of different ages are reported together with results obtained in various diseases. Data from normal subjects show that bone alkaline phosphatase contributes about half the total alkaline phosphatase activity in adults. Liver phosphatase shows a slight increase with age. The method is also able to detect reliably the presence of carcinoplacental isoenzymes.     

Wheat-germ agglutinin method for measuring bone and liver isoenzymes of alkaline phosphatase assessed in postmenopausal osteoporosis

In the method of Rosalki and Foo (Clin Chem 1984;30:1182-6) bone and liver isoenzymes of alkaline phosphatase (EC 3.1.3.1) are quantified by using wheat-germ agglutinin (WGA). I suggest standardizing the procedure by using a WGA concentration that precipitates half of the alkaline phosphatase activity of serum pooled from an equal number of healthy women and men. By applying knowledge of the precipitation pattern in serum samples containing predominantly or exclusively bone or liver sources of alkaline phosphatase, I obtained results for the isoenzymes in healthy subjects that agreed with those by the heat-inactivation methods, as reported earlier in the literature. I then assessed the utility of the standardized procedure in a clinical study of prevention of postmenopausal bone loss. In patients receiving hormone replacement therapy, which is known to decrease bone turnover, the decrease in total alkaline phosphatase activity in serum was entirely ascribable to decreases in the bone isoenzyme activity, probably reflecting reduced bone formation, whereas the activity concentration of liver alkaline phosphatase remained unchanged.     

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.     

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.     

Isoenzyme pattern of alkaline phosphatase in the serum of children with leukaemia after long-term chemotherapy

Chemotherapy was discontinued in thirteen children with acute lymphoblastic leukaemia after 2-3 years without relapse. In the following months, a continuous increase was observed in the serum alkaline phosphatase. A maximum was reached at different times but all patients had normal values again after 19 months. The rise in the total activity is due to an isolated increase of the bone isoenzyme. Increased enzyme activity in this post-therapy phase of the disease does not signify liver damage from the preceding chemotherapy, but rather is due to an increased osteoblast activity associated with a temporary increase in growth rate.     

High-molecular-mass ("biliary") isoenzyme of alkaline phosphatase and the diagnosis of liver dysfunction in cystic fibrosis

The high-Mr isoenzyme of alkaline phosphatase (AP, EC 3.1.3.1), a highly sensitive index to cholestasis, was measured by liquid chromatography in 45 patients with cystic fibrosis. Results of serum tests for liver dysfunction--including gamma-glutamyltransferase, aspartate aminotransferase, alanine aminotransferase, total AP, bilirubin, and bile acids--were compared with those for high-Mr AP. Values for high-Mr AP were increased in 44.4% of our patient population, with activities ranging from 0.4 to 17.3 U/L. The upper limit in the control group was 2.5 U/L. We find increased high-Mr AP to be a more sensitive indicator of liver dysfunction in patients with cystic fibrosis than are other tests.     

Measurement of serum alcohol dehydrogenase activity at different pH-values during the course of viral hepatitis in children

Serum alcohol dehydrogenase activity was estimated at pH 10.4 (optimum for the typical liver isoenzyme), 8.8 (optimum for atypical liver isoenzyme), at the physiological serum pH of 7.4, and at pH 9.2, with a view to obtaining the greatest possible difference between patients and controls. Measurements were performed on the sera of 39 children aged from 2 to 13 years, using the Technicon analyzer RA-1000 with the continuously measuring method of Bonnichsen & Brink. Blood sera were tested at the onset of viral hepatitis, in the first week of hospitalization, and three times thereafter at intervals of 7 to 9 days. During the illness, the activity of serum alcohol dehydrogenase, measured at different pH-values, was higher than that of controls. The ratio of activity at pH 10.4 to activity at pH 8.8 in the sera differed from that previously reported for liver cells. The highest increase in alcohol dehydrogenase activity was at pH 9.2. The diagnostic sensitivity of alcohol dehydrogenase determination at this pH is lower than that of alanine aminotransferase, gamma-glutamyltransferase and aspartate amino-transferase, but higher than that of lactate dehydrogenase, alkaline phosphatase and bilirubin; alcohol dehydrogenase activity also shows the best correlation with the activity of gamma-glutamyltransferase.     

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 correlated (P less than 0.001) with serum concentration of immunoreactive growth hormone. By using stepwise regression analysis, the predication 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 alaline 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.