Phase partitioning detects differences between phospholipase-released forms of alkaline phosphatase--a GPI-linked protein.

A number of enzymes are known to release alkaline phosphatase and other glycan phosphatidylinositol-anchored proteins from membrane surfaces. We describe a novel approach to detect and measure these activities by partitioning in aqueous phase systems. The procedures avoid the complications of micelle-formation involving hydrophobic molecules that may arise with detergent-based partition systems and can clearly distinguish between inositol-specific phospholipase C and D activities.

Malabsorption and macroamylasemia. Response to gluten withdrawal.

A 36 year old woman presented with malabsorption and macroamylasemia. The macroamylase was characterized and shown to be a complex of pancreatic amylase and immunoglobulin A(IgA). The patient had the clinical and histologic features of adult celiac disease, and responded to a gluten-free diet. The macroamylase complex disappeared from the serum after gluten withdrawal, a hitherto unreported finding in the syndrome of malabsorption and hyperamylasemia.

Bone scan flare predicts successful systemic therapy for bone metastases.

Changes in osteoblast function, assessed by serial bone scans and serum alkaline phosphatase bone isoenzyme (ALP-Bl) and osteocalcin, have been studied in 53 patients receiving systemic therapy for bone metastases from advanced breast cancer. In 12/16 patients with healing of lytic disease on x-ray a paradoxical deterioration in the bone scan appearances after 3 mo treatment was seen. This was characterized by increased activity in baseline lesions and the appearance of new foci of tracer uptake; changes which are indistinguishable from progressive disease. After 6 mo successful treatment the bone scan improved with reduced tracer uptake and no new lesions since the 3-mo scan. New lesions appearing after 6 mo indicated progressive disease. These changes are attributed to a flare in osteoblast activity induced by successful systemic therapy and confirmed by a transient rise in osteocalcin and ALP-Bl. After 1 mo of treatment 15/16 responders showed a rise in both parameters compared with only 5/23 nonresponders (p = less than 0.001). The flare response is the rule rather than the exception after successful systemic therapy for bone metastases. The appearance of new lesions or increasing activity in known lesions during the first 3 mo is as likely to herald radiological response as disease progression.

Effect of nitric oxide gas on the generation of nitric oxide by isolated blood vessels: implications for inhalation therapy.

1. We have investigated, using rat aortic rings, whether exogenous nitric oxide (NO) gas affects the activity or expression of the inducible, Ca(2+)-independent NO synthase. 2. Incubation of rings with lipopolysaccharide (LPS, S. typhosa) for 6 h resulted in a gradual loss of tissue tone, a time-dependent reduction in constrictor response to phenylephrine and significant expression and activity of Ca(2+)-independent NO synthase. 3. Following incubation of LPS-treated rings with NO gas, the expression of inducible NO synthase mRNA was still observed, although the enzyme activity was significantly reduced and there was no reduction in the response to phenylephrine. 4. Therefore, NO gas can inhibit the action but not the induction of an NO synthase likely to play a role in inflammatory states such as adult respiratory distress syndrome (ARDS). 5. These observations may explain the rebound phenomenon observed in some ARDS patients following inhalation therapy with NO gas.

Effect of hydrazine upon vitamin B12-dependent methionine synthase activity and the sulphur amino acid pathway in isolated rat hepatocytes.

The effect of the industrial chemical, hydrazine (4-12 mM), on methionine synthase (EC 2.1.1.13) activity and levels of the sulphur amino acids homocysteine, cysteine, and taurine as well as GSH were investigated in vitro in isolated rat hepatocyte suspensions and monolayers in order to explain some of the adverse in vivo effects of hydrazine. None of the concentrations of hydrazine were overtly cytotoxic in hepatocyte suspensions (measured as lactate dehydrogenase [LDH] leakage) after 3 hr. However, after 24 hr in culture cells treated with 12 mM, hydrazine showed a significant increase in LDH leakage. Methionine synthase activity was reduced by hydrazine (8 and 12 mM) in suspensions (by 45 and 55%, after 3 hr) and monolayers (12 mM; 65-80% after 24 hr). This was not due to nitric oxide production and the inhibitor of nitric oxide synthase, Nomega-nitro-L-arginine, failed to protect against the hydrazine-induced loss of ATP and GSH and the reduction in urea synthesis at 24 hr. Homocysteine export was increased by 6 mM hydrazine, and total taurine content of treated cells was increased by 12 mM hydrazine. Thus, hydrazine was found to have several important and possibly deleterious effects on some parts of the sulphur amino acid pathway.

Changes in serum enzyme levels accompanying cardiac surgery with extracorporeal circulation.

Serum lactic dehydrogenase, alpha-hydroxy-butyrate dehydrogenase, iso-citric dehydrogenase, and glutamic oxaloacetic transaminase activities were measured daily for two weeks postoperatively in the serum of 22 patients undergoing cardiac surgery with extracorporeal circulation.The length of perfusion was found to be a major factor affecting the extent of increased post-operative enzyme activities. Significantly higher levels were demonstrated in patients perfused for over one hour compared with those perfused for under one hour. Hepatocellular damage, age, and type of operation were not considered to be major factors in determining the extent of this increased activity.A considerable increase in enzyme activity was found to occur during perfusion when the dilution introduced by mixing the patient's circulation with the priming fluid of the heart-lung machine was taken into account. This dilution, when accounted for, increased the observed enzyme activity by 30 to 50%.

Enzymatic characterisation of recombinant murine inducible nitric oxide synthase.

A complementary DNA (cDNA) encoding murine inducible nitric oxide synthase was cloned from activated J774 macrophages. Expression of this cDNA in a baculovirus-insect cell system allowed comparison of the recombinant enzyme with the native homologue. Western blot analysis of activated J774 and baculovirus-infected insect cell cytosols demonstrated reactivity against a protein of 135 kDa. Kinetic studies on the recombinant and native enzymes revealed an absolute requirement for L-arginine and NADPH in order to achieve full activity. In addition, both enzymes were found to have similar maximum velocities and Km values for these two substrates. The nitric oxide synthase antagonists N-guanidino monomethyl L-arginine and N-iminoethyl L-ornithine inhibited both enzymes at a similar rate. Furthermore, comparable concentrations of inhibitor were required to achieve half maximal enzyme inhibition. These results indicate that recombinant inducible NO synthase appears to be pharmacologically indistinguishable from the native enzyme.

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.

The physical characteristics and enzymatic modification of fetal intestinal alkaline phosphatase in amniotic fluid.

The predominant form of alkaline phosphatase in amniotic fluid at 16 to 18 weeks gestation has the inhibition characteristics of the isoenzymes from adult and fetal small intestine. These characteristics are shared by an alkaline phosphatase of presumed intestinal origin occasionally found in the sera of premature neonates. However, in contrast to the rapid anodal migration of the latter enzyme, amniotic fluid phosphatase is of low electrophoretic mobility in polyacrylamide gel, and it has a high molecular weight. Digestion of amniotic fluid with bromelain produces a rapidly-migrating active enzyme form, with a molecular weight of about 135,000, compared with 145,000 for the fetal intestinal phosphatase in serum. The bromelain-treated amniotic fluid phosphatase is similar in size to a Kasahara isoenzyme in the serum of a cancer patient, which is itself thought to result from re-expression of a fetal intestinal phosphatase gene.

Interassay calibration as a major contribution to the comparability of results in clinical enzymology.

OBJECTIVE: Factors contributing to the applicability of interassay calibration of methods measuring enzyme catalytic activities are described. Also discussed are the properties essential for such a material. Similarity of specificity for the methods to be calibrated as well as commutability between the material(s) intended to be used as calibrator are the main criteria to be satisfied. RESULT: Several examples demonstrated that interassay calibration is feasible but a multi-enzyme calibrator with a wide commutability for the most popular methods remains to be developed. This is the project of the IFCC Working Group on Calibrators in Clinical Enzymology (WG-CCE). Several experimental data are also presented that indicate that the temperature at which the reaction is carried out is not a limiting factor in the implementation of interassay calibration in clinical enzymology.

Validation of an enzyme calibrator--an IFCC guideline. International Federation of Clinical Chemistry.

OBJECTIVES: The objective of this guideline is to improve standardization in clinical enzymology in order to improve intermethod comparability of patients' results. DESIGN AND METHODS: The reference system, combination of the reference method and the reference material, is used to produce a reference value for a given catalytic activity. Sets of methods are formed of methods exhibiting the same analytical specificity. Materials intended to be used as enzyme calibrators are experimentally checked for their commutability. RESULTS: The transfer of accuracy from the reference value to patients' results is dependent on methods (analytical specificity) and on materials (experimentally assessed commutability). The feasibility of this approach was demonstrated with materials of high level for several enzymes and for each of them for several routine methods. CONCLUSION: Expected advantages of this approach in clinical enzymology are presented.

Release of membrane-bound enzymes from cells and the generation of isoforms.

Enzymes bound to the surfaces of cells may be retained by a hydrophobic amino acid sequence (e.g. gamma-glutamyltransferase) or by a specific glycan phosphatidylinositol (GPI) anchor (e.g. alkaline phosphatase). In either case the attachment is by means of non-covalent hydrophobic interactions between the anchoring domain of the enzyme and lipid components of the cell membrane. Enzyme molecules released into the plasma or bile, complete with their hydrophobic domains, can undergo aggregation and complexation to give rise to high molecular weight isoforms of gamma-glutamyltransferase or alkaline phosphatase. However, the GPI domain of alkaline phosphatase can be degraded by an inositol-specific phospholipase in plasma, but not in bile, with production of the hydrophobic, non-aggregating isoform of alkaline phosphatase that predominates in plasma.

Methodological principles in the enzymatic determination of substrates illustrated by the measurement of uric acid.

The enzymatic method of Haeckel for the determination of uric acid in serum has been used to compare three different kinetic approaches with the corresponding equilibrium (end-point) procedure. The kinetic methods were: measurement of the difference in absorbance between two pre-selected fixed instants during the course of the reaction under first-order conditions; measurement of the initial rate of reaction at substrate concentrations sufficiently low for first-order kinetics to apply, and determination of initial rate with substrate concentrations approximating to, or greater than, the Michaelis constant (pseudo-zero order kinetics). In the third method results were obtained by solving the Michaelis-Menten equation. The results emphasise the superiority of the two-point kinetic approach, which is apparent even when the timed absorbance values are obtained from an analogue signal displayed on a recorder chart. Conditions are described for the determination or uric acid in serum according to this principle.

Physicochemical and pathophysiological factors in the release of membrane-bound alkaline phosphatase from cells.

Alkaline phosphatase is bound to cell membranes by a glycan phosphatidylinositol anchoring domain. The structure of this domain and ways in which it may be cleaved by chemical and enzymatic means provide a basis for understanding the solubilization of alkaline phosphatase from tissues in vitro and in vivo and the generation of isoforms.

Reactivation of the apoenzyme of aspartate aminotransferase in serum.

Apoenzyme of aspartate aminotransferase in serum can be reactivated conveniently by addition of 100 mumoles/l pyridoxal phosphate to the reaction mixture, without extending the usual minimum pre-incubation period in the operation of the LKB 8600 reaction rate analyzer. Normal sera contain some apoenzyme, the amount of which, as well as that of holoenzyme, is greatly increased by damage to skeletal muscle. This may be due to direct injury or to the indirect effects of anoxia; e.g., following surgery with extracorporeal circulation. Myocardial infarction also increases the levels of both apo- and holoenzymes, but changes in the two levels follow similar time courses and apo- and holo-aminotransferases disappear from the circulation at similar rates.

The place of reference materials in clinical enzymology.

Standardization embraces two concepts: achievement of a uniform standard of practice, or comparison with a standard of known or defined properties. In clinical enzymology the former concept can be equated with the adoption of recommended standardized methodology, which has had noteworthy successes in improving comparability of results. The use of enzyme standards or calibrators has received relatively little attention so far, but is increasingly important with the introduction of analysers which cannot readily utilize recommended methods. The main factor in the use of calibrators is that of commutability of calibrators and patients' samples between methods, which must be the subject of thorough experimental tests.

Changes in enzyme expression related to differentiation and regulatory factors: the acid phosphatase of osteoclasts and other macrophages.

Human tartrate-resistant Type 5 acid phosphatase is a unique isoenzyme encoded by a gene located on chromosome 19. It is a member of a widely-distributed and structurally highly-conserved group of iron-containing proteins. It is normally expressed in certain tissue macrophages, notably osteoclasts and alveolar macrophages, but is virtually absent from the precursor monocytes. Factors which enhance or inhibit expression of this specific isoenzyme can be studied in monocytes and osteoclasts cultured in vitro. This provides opportunities to develop the use of Type 5 acid phosphatase as a reporter of pathophysiological events and an essential, though not sufficient, role in bone resorption by osteoclasts has been established by such studies.

Further observations on the differential precipitation of alkaline phosphatase isoenzymes by ethanol.

The proportions of the total activities of different isoenzymes of human alkaline phosphatase precipitated from serum by ethanol (20% v/v) were: liver phosphatase, 37%; placental phosphatase, 23%; bone phosphatase, 8.0%, and small-intestinal phosphatase, 3.7%. Treatment of the isoenzymes with neuraminidase reduced the percentages of non-intestinal phosphatases precipitated by ethanol to below 10%. Precipitation of intestinal alkaline phosphatase was unaffected by this treatment. The degree of solubility in ethanol therefore appears to be largely determined by the content of terminal sialic acid residues in the alkaline phosphatase molecules. In contrast the stabilities of the isoenzymes to heating at 56 degrees C were not significantly altered by neuraminidase digestion.

A fluorescent artefact resembling BB-creatine kinase in sera of patients with prostatic disease.

A fluorescent zone with mobility towards the anode almost equal to that of human BB-creatine kinase has been detected after electrophoresis on cellulose acetate of sera from each of 28 patients with prostatic carcinoma. The zone is not due to the BB isoenzyme and its appearance does not depend on the presence of substrates of creatine kinase. It therefore appears to be a further example of a fluorescent artefact resembling a creatine kinase ieoenzyme. These observations indicate a need for caution in assessing the possible value of BB-creatine kinase in patients with prostatic disease.

Analysis of heat inactivation curves of alkaline phosphatase isoenzymes in serum.

The course of the decline in alkaline phosphatase activity during exposureof serum samples to a temperature of 56 degree C can be resolved into two phases. These represent the exponential decay of an enzyme component with an average half-inactivation time of 112 seconds and of a second component with an average half-inactivation time of 456 seconds. The more rapid fall is due to inactivation of bone alkaline phosphatase and the slower to inactivation of liver and, when present, intestinal phosphatases. The half-inactivation times of the different enzyme species show considerable variation from one serum sample to another. The implications of this variation for methods of estimating the relative proportions of alkaline phosphatase isoenzymes by selective inactivation procedures are discussed.