Masakazu Miura

Urinary pyridinoline and deoxypyridinoline as potential markers of bone metastasis in patients with prostate cancer

PURPOSE The levels of probable markers of bony metastatic disease were measured to evaluate their efficacy as predictors of disease and therapeutic outcome. MATERIALS AND METHODS Urinary pyridinoline, urinary deoxypyridinoline, serum alkaline phosphatase and serum osteocalcin were measured in patients with benign prostatic hyperplasia, clinically localized prostate cancer and prostate cancer with bone metastases. Also, urinary pyridinoline and deoxypyridinoline were compared in 2 groups of patients with metastatic prostate cancer of the bone who demonstrated progression or positive response to treatment. Urinary pyridinoline and deoxypyridinoline were determined by high performance liquid chromatography and were normalized to urinary creatinine. RESULTS Levels of pyridinoline and deoxypyridinoline in urine, and the level of alkaline phosphatase in serum from patients with bone metastatic prostate cancer were significantly greater than levels in patients with benign prostatic hyperplasia or localized prostate cancer. Serum osteocalcin levels failed to separate the 3 groups. Serial measurement of urinary pyridinoline and deoxypyridinoline was correlated with a positive response to treatment (decreased) and with clinical progression of disease (increased) before detection of new bone lesions by bone scintigraphy. CONCLUSIONS Measurement of urinary pyridinoline and deoxypyridinoline may provide a useful marker of prostate cancer metastatic to bone and may be useful in monitoring the response to treatment.

Sugar-chain heterogeneity of human alkaline phosphatases: differences between normal and tumour-associated isozymes

The sugar-chain heterogeneity of alkaline phosphatases (ALPs) from various human organs was investigated by using the serial lectin affinity technique. This technique revealed a possible structure of the sugar chain(s) of ALP isozymes and clarified a difference in affinity on the lectin column not only among three genetically different isozymes (liver/bone/kidney, intestinal and placental types) but also among liver, bone, and kidney ALPs. Lectin-binding profiles of ALPs in these human organs closely resembled those in the corresponding organs of the rat, as reported previously, suggesting that heterogeneities in sugar chains of ALPs have a specificity for the respective organs rather than being species-specific. Lectin-binding profiles of tumour-produced placental and liver ALPs were significantly different from those of ALPs in the respective normal organs. However, the two altered ALPs exhibited similar lectin-binding affinities. Isoelectric focusing analysis showed essentially no difference in protein charge between the normal and tumor-produced ALPs. Moreover, tumour-produced ALPs had the same N-terminal amino acid sequence and peptide mapping as normal ALPs. From these results, it is possible to suggest that organ-specific sugar chains in ALP isozymes are changed into those peculiar to tumours in association with malignant transformation.

Measurement of Bone-Specific Alkaline Phosphatase by an Immunoselective Enzyme Assay Method

We evaluated a new immunoselective enzyme assay of bone-specific alkaline phosphatase (ALP). The monoclonal antibody used in this assay was raised against purified bone-specific ALP obtained from SAOS-2 human osteosarcoma cell line. Calibration was based on the enzymes own activity. The relative activity of the antibody was 100% with bone ALP, 8·7% with liver ALP, and 0% with placental and intestinal ALPs. Intra- and inter-assay coefficients of variation were less than 4%. The sensitivity of the assay was 0·7 U/L, and the linearity extended from 2 to 140 U/L. The recovery of bone-specific ALP standard added to serum was 94–106%. The correlation coefficient between this method and the polyacrylamide gel (PAG) electrophoretic method was 0·94. The mean value of bone-specific ALP in 89 healthy adults (mean age 29 years, SD 5 years) was 18·5 U/L (SD 4·1 U/L). Interestingly, mean bone-specific ALP activities in 60 premenopausal women (mean age 39 years, SD 8 years) and 70 postmenopausal women (mean age 57 years, SD 5 years) were 20·3 U/L (SD 6·5 U/L) and 31·1 U/L (SD 11·1 U/L), respectively. The age-related increase in bone-specific ALP was significant and more pronounced in women (P < 0·01). We conclude that this new immunoassay of bone-specific ALP would be useful for clinical investigation of patients with osteoporosis or other metabolic diseases of bone.

Organ specific properties for human urinary alkaline phosphatases

We have re-evaluated the isolation and characteristics of human urinary alkaline phosphatases (ALPs). From the results of physicochemical properties and immunological identification, the urinary ALPs from healthy subjects and patients with hepatoma were found to be similar in nature to liver and/or bone-like ALP. In patients with chronic or acute nephritis, the ALPs contained a major band of kidney-like ALP with a minor band of bone and intestinal ALPs. However, the ALPs in pregnant women had not only liver and bone ALPs but also placental-like ALP. It is interesting that only bone-like ALP was detected in psychiatric patients administered chlorpromazine. In the conditions we investigated, the molecular sizes of the urinary ALPs were similar as those of original ALPs, except for the enzyme from renal failure. Moreover, the total activity of urinary ALP was closely related to the level of serum ALP, being in a ratio of 1/40. In general, urinary ALP may be derived from serum ALP by minor modification, suggesting that the identification of excreted ALP in urine is a good marker for disturbed organs in respective diseases.

Chlorpromazine alters bone metabolism of ratsin vivo

SummaryThe acute effect of chloropromazine (CPZ) on metabolic changes in rat was investigated. CPZ was found to markedly suppress45Ca incorporation into the calvarium and ileumin vitro. According to the serum and/or urinary levels of certain markers for bone metabolism, the increases of Ca and P in the serum and Ca, P, and γ-carboxyglutamate (Gla) in the urine were observed in rats given 10 mg CPZ/kg of their body weight, whereas the amount of alkaline phosphatase (ALP) activity, ionized Ca, calcitonin, 25-hydroxyvitamin D3 (25(OH)D3), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) as clearly or slightly reduced, suggesting the inhibitory effect of their bone formation. This was well supported since only bone type ALP was detected in the urine and loss of the vertebral bone density from rats given daily CPZ administration for a week. Moreover, in this case, there is little if any difference in the levels of mid-molecule parathyroid hormone (mid-PTH), osteocalcin, and urinary cAMP for the nontreated and CPZ-treated animals, resulting in the fact that CPZ may mainly inhibit the hydroxylase for active vitamin D3.

Partial characterization of human ileal alkaline phosphatase: differences between human ileal and duodenal enzymes

Properties of human ileal and duodenal alkaline phosphatase (ALP) were compared. The pH optimum, Km values, heat stability, inhibition of activity by amino acids, and antigenicity of ileal and duodenal ALPs were similar. Affinity for DEAE and Tyraminyl derivatives/Sepharose chromatographies, substrate specificity, molecular mass, isoelectric point, and sugar chain structure differed, suggesting two forms of intestinal enzyme. The N-terminal amino acid sequence, or peptide mapping or both suggest that the two major intestinal ALPs are identical, but the minor ALP may be differed from the sequence of major one.

An immunoglobulin g inhibiting lactate dehydrogenase activity

We found extremely low (6 U/l) serum lactate dehydrogenase (LDH, EC 1.1.1.27) activity in a patient with uterine myoma. The patients serum inhibited purified LDH isoenzymes. One milliliter serum neutralized 73 U purified LDH-1 isoenzyme, equivalent to 300-500-fold the amount of LDH in 1 ml of normal serum. The serum inhibitor was purified by ordinary procedures using chromatographies and identified as IgG which contains both kappa- and lambda-chains. The IgG is very unique in showing higher affinity to the H- than M-subunit of the LDH tetramer, in contrast with the IgGs already reported. After removal of the myoma, the anti-LDH activity gradually decreased with a half-life of 20 days corresponding to that of IgG and finally almost disappeared. This indicates a possibility that the myoma cells produce some factors such as B-cell growth and differentiation factors.

Similarity of the sugar moiety of human alkaline phosphatases between the kidney cortex and duodenum, or medulla and ileum.

Characteristics of human renal cortex and medulla alkaline phosphatase (ALP) were compared. Enzymatic and hydrophobic properties of both ALPs were almost similar. However, the results of concanavalin A affinity chromatography and wheat germ agglutinin affinity electrophoresis, exhibited that sugar chain structure(s) might be different between the cortex and medulla ALPs. In addition, the molecular mass and substrate specificity differed from each other, and these results of cortex and medulla ALPs were well accordant with those of human duodenal and ileal ALPs, respectively, as described previously (Clin Chim Acta 1987;163:279-287).

Sandwich Assay for Carcinoembryonic Antigen with Immobilized Lectins and a Monoclonal Antibody

A lectin-linked immunoradiometric assay (L-IRMA) using 7 different lectins and a monoclonal antibody (MoAb) directed against the protein moiety-specific epitope was developed to detect carcinoembryonic antigen (CEA). The method used for L-IRMA was as follows: certain CEAs that reacted with lectin agarose beads were allowed to bind further to an 125I-labeled anti-CEA MoAb, and the resulting trapped 125I-MoAb was counted in a gamma counter. From the results, CEAs which interacted with Phaseolus vulgaris erythroagglutinin and leukoagglutinin were presumed to contain the complex type of sugar chains. Furthermore, CEAs interacting specifically with wheat germ agglutinin lectin were found in the tested samples, suggesting that the CEA had a hybrid or a complex type of sugar chain as the core structure of the sugar chain, except for that in seminal plasma. These results obtained by L-IRMA were in good accord with the data obtained from serial lectin affinity chromatography. L-IRMA may therefore be a simple method to study the glycoprotein heterogeneities in tumors and in normal subjects.

Carbohydrate-mediated recognition of a circulating placental alkaline phosphatase-immunoglobulin M complex

We detected an abnormal alkaline phosphatase (AP) electrophoretically in the serum of a patient with rheumatoid arthritis, who had a macromolecular AP linked with immunoglobulin M (IgM) bearing a kappa light chain. The IgM isolated from the AP-IgM complex in the patients serum reacted apparently with all of the AP isozymes tested, i.e. those originating in the liver, bone, intestine and placenta, but the alpha-mannosidase-treated IgM from the patients serum bound to placental AP (PAP) alone. This suggests that untreated IgM recognizes multivalent epitopes of the AP and that the complex of AP with alpha-mannosidase-treated IgM is a specific antibody-antigen complex. In order to investigate further the multivalent binding capacity for the PAP-untreated IgM complex, we prepared a monoclonal antibody (MoAb) against PAP and identified it as an IgM with a kappa light chain. The binding affinities and their circulating half-lives of the synthetic complexes of PAP and respective MoAbs were examined with and without treatment with several glycosidases. The untreated MoAb bearing IgM had binding affinity for all of the AP isozymes tested, while alpha-mannosidase-treated IgM attached only to PAP, the same as the IgM isolated from the PAP-IgM complex in the patients serum. The circulating clearance of the PAP-IgM complex in rabbits was faster than either component alone. In addition, the PAP-IgM complex treated with alpha-mannosidase was found to have the shortest half-life of all the complexes of PAP and Igs treated with the several glycosidases tested. These results suggest that the formation of the PAP-IgM complex as an enzyme-linked antibody and the clearance of the complex in vivo are dependent on the sugar moieties of the Igs.