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.     

Intestinal variant alkaline phosphatase in plasma in disease.

We investigated by enzyme electrophoresis after prolonged neuraminidase treatment the activity of "intestinal variant" (alpha 2-globulin mobility) alkaline phosphatase (EC 3.1.3.1; ALP) in the plasma of 189 patients selected for disorders (diabetes mellitus, liver cirrhosis, and chronic renal failure) with a known high frequency of increased plasma intestinal (beta-globulin mobility) ALP activity. The overall frequency of the variant ALP was 23.8%, whereas in the samples showing intestinal ALP it was 45.0%. The variant ALP was not observed in the absence of intestinal ALP, nor in patients of blood group A. Its frequency did not differ significantly between the different patient groups. Quantification of the variant ALP by densitometry was unsatisfactory but the quantity could be estimated by subtracting the intestinal ALP activity measured by electrophoresis from the activity determined by immunoassay with monoclonal antibody that reacts with both the intestinal and the variant forms. This indicated median activity of 12 U/L for the variant, approximately equal to that of the concomitant intestinal ALP. From the effects of papain and bromelain treatments, we suggest that "intestinal variant" represents intestinal ALP with attached membrane-binding domain.     

Value of serum alkaline phosphatase in evaluating hyperplasia of parathyroid glands in chronic hemodialysis patients

To investigate the value of serum alkaline phosphatase in evaluating hyperplasia of parathyroid glands in hemodialysis patients, 28 hemodialysis patients who had parathyroid sonography examinations for secondary hyperparathyroidism were studied retrospectively. There were significant elevations of serum alkaline phosphatase, parathyroid hormone (PTH), serum total calcium, and dialysis duration in patients with sonography-detectable parathyroid (N = 17) as compared with those of sonography-undetectable (N = 11) parathyroid. Hemodialysis patients who have both higher serum alkaline phosphatase (>94 IU/ L) and considerably elevated serum PTH (9× or higher) are likely to have sonography-detectable parathyroids (positive predictive value of 93%). Patients with mildly or moderately elevated serum PTH but normal serum alkaline phosphatase are less likely to have sonography-detectable parathyroids (negative predictive value is 100%). These findings suggest that the elevation of serum alkaline phosphatase could be a valuable parameter in addition to the high serum PTH level in predicting hyperplasia of parathyroid glands in chronic hemodialysis patients. © 1994 John Wiley & Sons, Inc.     

Various forms of plasma cysteine and its metabolites in patients undergoing hemodialysis

The thiol tripeptide glutathione (GSH) is particularly important as an antioxidative protector in the cells. GSH is also a form of storage and transport of cysteine. Under physiological conditions, the kidney plays an essential role in GSH biodegradation to free cysteine via gamma-glutamyl cycle and subsequently in further metabolism of this sulfur amino acid. Our aim was to assess to what degree renal insufficiency affects the level of various cysteine forms and its metabolites (sulfates and sulfane sulfur compounds) in the plasma, and whole blood GSH levels. The concentrations of all the above mentioned sulfur compounds were measured in plasma of patients with end stage renal failure (ESRF) before and after dialysis and in a group of healthy controls. In plasma of patients with ESRF before dialysis tendency towards significant elevation of cystine, protein-bound cysteine and sulfates levels was evident. Simultaneously, a decrease of plasma level of sulfane sulfur compounds, products of anaerobic sulfur metabolism, and whole blood GSH concentration was found. As a consequence, the ratio between the reduced cysteine and the total cysteine concentration was markedly decreased. The dialysis session restore this ratio to the value observed in plasma of control individuals. These findings indicate disturbances in the thiol-disulfide equilibrium and show a higher oxidation status in plasma of patients with ESRF than in healthy controls.     

Multiple forms of human plasma renin substrate.

The objective of this investigation was to determine whether heterogeneity of plasma renin substrate could be observed in states of steroid excess and various forms of hypertensive disease. In states of stimulated renin substrate production by estrogens or glucocorticoids, multiple forms of renin substrate were apparent when stimulation was excessive. Stimulation of substrate production caused by uremia associated with hypertension showed similar results. None, or only trace quantities of the additional forms of renin substrate were evident in subjects with normal or suppressed levels of plasma renin substrate. The additional forms of renin substrate could be distinguished from the normal form on the basis of cross-reactivity with a specific antiserum to the normal form, electrophoretic mobility, and kinetic rate constants. Differences in rate constants of the various forms of plasma renin substrate may account for the altered rate of the renin reaction associated with several states of hypertension. In plasma of patients with renovascular hypertension, significant quantities of a protein which cross-reacted with the antiserum but could not generate angiotensin I were observed.     

Endoprotease in human liver transforming multiple forms of alkaline phosphatase

Two forms of alkaline phosphatase, extracted from human liver and named API1 and API3, are of high molecular mass, but API3 is the larger molecule and is membrane-bound while API1 is smaller and soluble. Enzyme kinetics are identical. It is suggested that API1 is produced from API3 by an endoprotease. We demonstrated the action of an endoprotease in human liver homogenate converting API3 into API1. In the absence of this enzyme no conversion occurred. This enzyme is active at an acidic pH (less than 6.5) in the presence of Ca.. or Mg.. -ions. It is inhibited by traces of EDTA. It is insensitive to diisopropyl fluoro-phosphate, to leupeptin and to reducing or oxidizing chemicals. At alkaline pH (8.6) its activity is rapidly destroyed. The enzyme is stable in acidic buffer. We conclude that API1 is indeed formed from API3 in the living cell by enzymatic conversion.     

Multiple forms of alkaline phosphatases in human liver tissue

Alkaline phosphatases (AP) extracted in the presence of n-butanol from human liver are separated by affinity chromatography on phenylsepharose Cl-4B into two fractions named APII and APIIII. By repeated chromatography, APII was purified to a single enzyme entity with a specific activity of 1,684 kU/g protein. APIIII was purified to a specific activity of 535 kU/g protein. It consisted of only APIIII enzyme activity, but still contained gamma-glutamyltransferase activity. These two forms of AP are different in chromatographic and electrophoretic behaviour, APIIII being a larger molecule than APII. APII and APIIII are very similar in enzyme kinetic behavior, such as substrate activity, thermolability and sensitivity to different inhibitors. It is concluded from these experiments that multiple forms of AP in liver bear identical active centres, the difference is due to a modification of protein residue. It is possible that both are modified forms of one enzyme. Both are different from the AP isoenzyme that appears in serum in cholestatic patients.     

Multiple forms of alanine aminopeptidase, alkaline phosphatase and gamma-glutamyltransferase in urine of healthy persons, patients suffering from kidney diseases and patients with kidney transplants

The catalytic activities of alanine aminopeptidase, alkaline phosphatase and gamma-glutamyltransferase were determined in the soluble and particulate fractions of urine, after ultracentrifugation. In healthy adults the fractional catalytic activities in the supernatants were 0.53, 0.56 and 0.24, respectively. Nearly the same proportions were found in children. In patients suffering from chronic kidney diseases there was a tendency for the proportion of catalytic activity in the soluble fraction to increase. However, the separation into the multiple forms gave no higher diagnostic reliability than the determination of total catalytic activity of the respective enzymes. The determination of multiple forms has no clinical significance in the detection of rejection episodes in renal transplant recipients.     

The properties and clinical significance of some electrophoretically slow forms of alkaline phosphatase.

Sera from 8 patients with a marked slow-moving alkaline phosphatase band on electrophoresis were investigated. Inhibitor studies and treatment with neuraminidase showed that all the patients had slow bands with alkaline phosphatase properties resembling those of the liver or bone isoenzyme. In no case did the slow band resemble the intestinal isoenzyme. Immunoelectrophoretic and molecular weight studies indicated that the slow band consisted of an IgG-alkaline phosphatase complex of molecular weight 540 000. Serum from a patient with the slow band was able to bind liver or bone, but not intestinal, alkaline phosphatase from other patients to form the slow band. Serum from patients with the slow band probably contains an abnormal IgG molecule which can bind alkaline phosphatase in the ratio 2:1. No clinical condition was common to all 8 patients although most of them had either intestinal or lung disease.     

Multiple form of L-phenylalanine sensitive alkaline phosphatase in rat fecal extracts

Alkaline phosphatase activity was detected in fecal extracts of male rats. 58% of the total enzyme activity was inhibited by 30 mM L-phenylalanine, which is an inhibitor specific for intestinal alkaline phosphatase. L-Phenylalanine sensitive alkaline phosphatase in the fecal extracts revealed multiple peaks on DEAE-Sephadex A-25 ion exchange column with a linear gradient of NaCl. It is discussed that a part of the fecal alkaline phosphatase might be a set of catabolites of the intestinal enzyme.     

Multiple forms of renin substrate in human plasma

When adapting the immediate bromcresol green method for urinary albumin determination a correlation study with the Mancini single radial immunodiffuon (SRID) method was performed. This study showed large disparities between the two methods, SRID giving the higher results. The unsuitability of the currently used SRID methods is demonstrated and improvements to the method are suggested.

Known amounts of albumin were added to urine samples as well as to a “synthetic urine”, both giving falsely elevated results with the SRID method. On investigating the different components of the “synthetic urine”, it was found that the disparities were due to the influence of citrate and phosphate.

On addition of citric acid or phosphate to the dilution buffer and/or the gel buffer, the results of the SRID method agreed with those of other methods and with the expected values.

The findings presented in this paper can probably be extended to other immunological methods too since it seems to be the antigen-antibody reaction which is affected.     

Perspectives in alkaline phosphatase research.

Gene cloning and site-directed mutagenesis have had a profound effect on alkaline phosphatase research. Four distinct structural genes encoding placental, intestinal, and tissue-nonspecific isoenzymes have been cloned, sequenced, and mapped to human chromosomes. Differences in properties between the respective gene products are due to variations in primary structure involving only one, or a few, key amino acid residues. Recognition that alkaline phosphatase belongs to the category of molecules that are localized to cell membranes through a COOH-terminal glycan-phosphatidylinositol anchor provides a basis for understanding the generation of isoforms observed in plasma in disease. Isoforms produced by differential cleavage or preservation of the glycan-phosphatidylinositol anchor may offer new correlations with disease that are of diagnostic value. However, a more important contribution of alkaline phosphatase research to clinical chemistry may prove to be an increased understanding of disease processes at the molecular level.     

Changes in alkaline phosphatase and 5'-nucleotidase multiple forms after surgical management of biliary obstruction.

Serial measurements of alkaline phosphatase and 5'-nucleotidase multiple forms in two patients undergoing surgical procedures to release biliary obstruction suggested an inverse relationship between high-M(r) isoforms and serum bile acids concentrations. Furthermore, the study of several groups of patients with cholestatic disorders confirmed this inverse correlation. Mechanisms responsible for these observations are discussed.