Cathepsin B and D activity in stimulated peritoneal macrophages

Summary Highly sensitive and specific synthetic substrates were used to quantitate cathepsin B and D activity in peritoneal macrophages in response to stimulation in vivo with mineral oil and thioglycollate. After intraperitoneal instillation of mineral oil the activity of cathepsin B increased significantly (to 15 300 units/mg protein versus 7 340 in saline controls), reaching values approaching those found in alveolar macrophages (18 400 units/mg protein). Significantly greater stimulation of enzyme activity was obtained after intraperitoneal instillation of thioglycollate (23 600 units/mg protein). Cathepsin D activity also increased significantly after both mineral oil and thioglycollate. However, the increase was moderate (from 806 to about 1 200 units/mg protein), remaining still more than six times lower-than in alveolar macrophages. The data are the first to demonstrate that cathepsin B activity can be stimulated in vivo in peritoneal macrophages by instillation of agents that induce acute inflammation. They also point to a differential control of expression of cathepsin B and D activity in both peritoneal and alveolar macrophages in spite of the common lysosomal origin of the two enzymes.Abbreviations Cbz -N-benxyloxycarbonyl - 2NA 2-naphthylamine - EDTA ethylenediamine tetraacetate - DMSO dimethylsulfoxide - PBS phosphate-buffered saline - PM peritoneal macrophage - AM alveolar macrophage     

Porcine spleen cathepsin B is an exopeptidase

The major cathepsin B isozyme CB-I purified from porcine spleens was studied for its specificity against various peptide and denatured protein substrates. The enzyme degraded all the peptide substrates by an exopeptidase activity. The substrates were degraded mainly by a dipeptidyl carboxypeptidase activity of the enzyme except for angiotensin I, from which a COOH-terminal leucine residue was released. The enzyme failed to hydrolyze peptides having a proline or cysteic acid in the COOH-terminal, penultimate, and antepenultimate positions. Reduced and carboxymethylated soybean trypsin inhibitor was degraded by the same dipeptidyl carboxypeptidase action of cathepsin B. No significant endopeptidase activity was observed. These results do not support the general assumption that cathepsin B has both endo- and exopeptidase activities, neither do these observations support the postulation that cathepsin B might be involved in the in vivo proteolytic processing of protein precursors. We propose that the biological role of this enzyme is mainly the degradation of tissue proteins in lysosomes.     

A functional proteomics screen of proteases in colorectal carcinoma

BACKGROUND: Proteases facilitate several steps in cancer progression. To identify proteases most suitable for drug targeting, actual enzyme activity and not messenger RNA levels or immunoassay of protein is the ideal assay readout. MATERIALS AND METHODS: An automated microtiter plate assay format was modified to allow detection of all four major classes of proteases in tissue samples. Fifteen sets of colorectal carcinoma biopsies representing primary tumor, adjacent normal colon, and liver metastases were screened for protease activity. RESULTS: The major proteases detected were matrix metalloproteases (MMP9, MMP2, and MMP1), cathepsin B, cathepsin D, and the mast cell serine proteases, tryptase and chymase. Matrix metalloproteases were expressed at higher levels in the primary tumor than in adjacent normal tissue. The mast cell proteases, in contrast, were at very high levels in adjacent normal tissue, and not detectable in the metastases. Cathepsin B activity was significantly higher in the primary tumor, and highest in the metastases. The major proteases detected by activity assays were then localized in biopsy sections by immunohistochemistry. Mast cell proteases were abundant in adjacent normal tissue, because of infiltration of the lamina propria by mast cells. Matrix metalloproteases were localized to the tumor cells themselves; whereas, cathepsin B was predominantly expressed by macrophages at the leading edge of invading tumors. Although only low levels of urinary plasminogen activator were detected by direct enzyme assay, immunohistochemistry showed abundant protein within the tumor. CONCLUSIONS: This analysis, surveying all major classes of proteases by assays of activity rather than immunolocalization or in situ hybridization alone, serves to identify proteases whose activity is not completely balanced by endogenous inhibitors and which may be essential for tumor progression. These proteases are logical targets for initial efforts to produce low molecular weight protease inhibitors as potential chemotherapy.     

Characterization of an analog enzyme to cathepsin L produced by tumor cells

The activity of cathepsin L is examined in the culture supernatants of 38 human, murine and hamster tumor cell lines. It is found that all cell lines secrete the enzyme possessing cathepsin L activity. The supernatant of HPC-YP cell cultures is purified and characterized as the enzyme preparation, because this supernatant shows the highest cathepsin L activity. The results indicate that the enzyme produced in HPC-YP cells is different from cathepsin L of normal liver in the several points. The molecular weight of the enzyme is 68 kd, whereas it is 34 kd for the liver cathepsin L. The enzyme is more stable to heat treatment and at the various pH than the liver cathepsin L. Furthermore, the inhibitors, which inhibit the liver cathepsin L activity, do not inhibit the activity of this enzyme. It is concluded that the enzyme showing cathepsin L activity in the culture supernatants of human tumor cells is different from human normal liver cathepsin L.     

Chymase and tryptase in dog mastocytoma cells: asynchronous expression as revealed by enzyme cytochemical staining

Mast cell populations can be distinguished by differences in the content and substrate specificity of their two major cytoplasmic granule proteases, the chymases and the tryptases. To explore the origins of differences in the types of proteases present in mast cells, we used a double cytochemical staining technique to reveal both chymase and tryptase in cells from four lines of dog mast cell tumors containing both enzymes. We expected that if chymase and tryptase were expressed together during cell development the relative staining intensity of chymase compared to tryptase would be constant among different cells of each tumor. Instead, we found substantial variation in the relative intensity of chymase and tryptase staining among cells of a given mastocytoma line, each of which contained cells presumed to be monoclonal in origin but heterogeneous with respect to cell development. The overall staining intensity for chymase or tryptase correlated with the amount of protease activity in extracts of tumor homogenates. Staining specificity was established by use of selective inhibitors and competitive substrates and was tested on various types of dog cells obtained by bronchoalveolar lavage. The results suggest that active chymase and tryptase may be expressed differently during mast cell differentiation and support the possibility of a close developmental relationship between mast cells differing in protease phenotype. Moreover, the success of the staining procedures applied to mastocytoma cells suggests that they may be of general utility in phenotyping of mast cells according to the protease activities present in their granules.     

New assay using fluorogenic substrates and immunofluorescence staining to measure cysteine cathepsin activity in live cell subpopulations.

BACKGROUND: Cathepsins are endosomal/lysosomal proteases that play important roles in regulating cell physiological processes in cardiovascular, neurological, musculoskeletal, and immunological systems. Pathophysiological processes are often associated with a change in cathepsin expression and activity, leading to the possibility of using cathepsins as disease markers for diagnosis and prognosis. METHODS: We describe a new assay utilizing an argon laser flow cytometer to measure activities of cysteine cathepsins B, L, and S in live cells using cell permeable fluorogenic cresyl violet-conjugated peptides as selective substrates. Substrate concentration dependency and time kinetics studies were performed. The activity assay was combined with immunofluorescence staining to detect cell lineage-specific molecules and assess cathepsin activities in a heterogeneous cell population. RESULTS: Substrate concentrations utilized were not limiting, because MFI significantly increased in a macrophage cell line stimulated with bacterial lipopolysaccharide. Selective cathepsin inhibitors demonstrated the selectivity of substrate cleavage. Cells fixed and stored before analysis had no loss of fluorescence product. Activities of cathepsins B, L and S in splenic B cells, T cells and macrophages identified by immunofluorescence staining were analyzed. CONCLUSION: This novel technique determines cathepsin activities on a per cell basis without requiring purification of different cell types from a heterogeneous cell population.     

Purification and characterization of dog mastocytoma chymase: identification of an octapeptide conserved in chymotryptic leukocyte proteinases

We isolated and characterized a chymotryptic serine proteinase from dog mastocytomas. Chymotryptic activity extracted at high ionic strength from mastocytomas propagated in nude mice was separated from tryptic activity by gel filtration and rapidly purified by sequential high-performance hydrophobic interaction and cation-exchange chromatography. The purified enzyme had an Mr of 27,000-30,000 by both analytical gel filtration and SDS-polyacrylamide gel electrophoresis, and a single amino-terminal sequence by automated Edman degradation. Like chymases from rat and human mast cells, the mastocytoma enzyme exhibited a high kcat/Km (1.1.10(5) M-1.s-1) employing succinyl-L-Val-Pro-Phe-p-nitroanilide, the best of several p-nitroanilide substrates screened. It was inhibited by diisopropyl fluorophosphate and soybean trypsin inhibitor, but not by aprotinin, distinguishing it from the otherwise closely related neutrophil enzyme, cathepsin G. The amino-terminal 25 residues of mastocytoma chymase were found to be 72 and 68% identical to the corresponding sequences of chymases from rat peritoneal and mucosal mast cells, respectively; they were also closely related to human cathepsin G and to proteinase sequences from mouse cytotoxic T-lymphocytes. The mastocytoma chymotryptic enzyme contained an octapeptide sequence which is common to all chymotryptic leukocyte proteinases sequenced to date from four mammalian species; this feature distinguishes chymases and other chymotryptic leukocyte proteinases from serine proteinases of coagulation and digestion.     

Cathepsin B in osteoblasts

Active cathepsin B has been found in cell extract and medium of human osteoblast-like cells and MG-63 cells. The released form is stable at neutral and alkaline pH and, in both cell types, intracellular and extracellular cathepsin B activities are increased by interleukin-1 beta (IL-1beta) and parathyroid hormone (PTH). To evaluate the physiological role of cathepsin B in osteoblasts, we investigated the production and secretion of this enzyme in normal human synovial fibroblasts and modulation by IL-1beta and PTH. Lactate secretion concurrent with release of cathepsin B and comparable responses in osteoblasts were also examined. Our data show that synovial fibroblasts respond differently to treatment with the two agents, suggesting a cell-specific regulation of cathepsin B and possible involvement in osteoblast physiology. Cathepsin B involvement was then evaluated in the activation of plasminogen activator (PA) in MG-63 cells using two specific inhibitors of cathepsin B, CA074 and CA074-Me, in constitutive conditions and after treatment with IL-1beta. As results of PA activity obtained in the presence of IL-beta were in contrast with previous reports, we examined the activities of PA, pro-PA activated with trypsin, and plasmin in cell extract and media of MG-63 cells after 24-h treatment with IL-1beta. Results show that in normal conditions and in the presence of IL-1beta, cathepsin B is involved in the activation of PA. Moreover, IL-1beta stimulates PA, pro-PA activated by trypsin, and plasmin activity in medium, whereas in cell extract it stimulates pro-PA activated by trypsin and plasmin activity. IL-1beta has no effect on cell extract-associated PA.     

Chymotrypsin- and trypsin-type serine proteases in rat mast cells: properties and functions

Two of the major enzymes present in and released from rat mast cells are chymotrypsin-type serine protease (chymase) and trypsin-type serine protease (tryptase), and these have been postulated to be important in the inflammatory reactions. There have been no clear data regarding the trypsin-type protease in rat mast cells. Tryptase was recently purified from rat peritoneal mast cells with an associated protein (trypstatin) that inhibited the protease activity above pH 7.5. Chymase was also purified from rat peritoneal cells by employing a one-step method involving hydrophobic chromatography on octyl-Sepharose 4B or arginine-Sepharose 4B. The properties of chymase and tryptase were described in relation to substrate specificity and their relative sensitivity to inhibitors. It was found that proteolytic activities of these enzymes were modulated by naturally occurring substances, such as phosphoglycerides, long-chain fatty acids, and trypstatin. There is as yet little evidence for the physiological roles of these enzymes in the inflammatory reaction. It has been found that the specific, low-molecular-weight inhibitor of chymase, chymostatin, and that of tryptase, leupeptin, inhibit histamine release induced by addition of anti-rat IgE to mast cells. However, the inhibitors with molecular weights of more than 6000 were found to have no effect in this process. The data suggest that chymase and tryptase in mast cell granules play a crucial or significant role in the process of degranulation.     

Identification of a cathepsin G-like proteinase in the MCTC type of human mast cell

Human mast cells can be divided into two subsets based on serine proteinase composition: a subset that contains the serine proteinases tryptase and chymase (MCTC), and a subset that contains only tryptase (MCT). In this study we examined both types of mast cells for two additional proteinases, cathepsin G and elastase, which are the major serine proteinases of neutrophils. Because human mast cell chymase and cathepsin G are both chymotrypsin-like proteinases, the properties of these enzymes were further defined to confirm their distinctiveness. Comparison of their N-terminal sequences showed 30% nonidentity over the first 35 amino acids, and comparison of their amino acid compositions demonstrated a marked difference in their Arg/Lys ratios, which was approximately 1 for chymase and 10 for cathepsin G. Endoglycosidase F treatment increased the electrophoretic mobility of chymase on SDS gels, indicating significant N-linked carbohydrate on chymase; no effect was observed on cathepsin G. Immunoprecipitation and immunoblotting with specific antisera to each proteinase revealed little, if any, detectable cross-reactivity. Immunocytochemical studies showed selective labelling of MCTC type mast cells by cathepsin G antiserum in sections of human skin, lung, and bowel. No labeling of mast cells by elastase antiserum was detected in the same tissues, or in dispersed mast cells from lung and skin. A protein cross-reactive with cathepsin G was identified in extracts of human skin mast cells by immunoblot analysis. This protein had a slightly higher Mr (30,000) than the predominant form of neutrophil cathepsin G (Mr 28,000), and could not be separated from chymase (Mr 30,000) by SDS gel electrophoresis because of the size similarity. Using casein, a protein substrate hydrolyzed at comparable rates by chymase and cathepsin G, it was shown that about 30% of the caseinolytic activity in mast cell extracts was sensitive to inhibitors of cathepsin G that had no effect on chymase. Hydrolytic activity characteristic of elastase was not detected in these extracts. These studies indicate that human MCTC mast cells may contain two different chymotrypsin-like proteinases: chymase and a proteinase more closely related to cathepsin G, both of which are undetectable in MCT mast cells. Neutrophil elastase, on the other hand, was not detected in human mast cells by our procedures.     

Cell growth and substrate effects on characteristics of a lysosomal enzyme (cathepsin C) in Duchenne muscular dystrophy fibroblasts

Skin fibroblasts from normal males and males suffering from Duchenne muscular dystrophy were studied in culture over a 10-week period. The lysosomal enzyme cathepsin C (dipeptidyl aminopeptidase I; EC 3.4.14.1), defined by the chloride-dependent hydrolysis of dipeptide-beta-naphthylamide (dipeptide-beta-NA) substrates at pH 5.1, was significantly lower in Duchenne cell sonicates and cell lysosomal preparations. The apparent difference in activity tended to increase with in vitro cell culture age, with the Duchenne cells being found also to grow faster and yield a greater number of cells at confluence. An analysis of all 10 cell lines as a group indicated that cathepsin C activity was related to growth rate. In addition, while analyses of cell homogenization and fractionation showed that the yield of cathepsin C was not different in Duchenne lysosomal preparations, the enzyme showed significantly lower latent activity in the Duchenne lysosomes with Gly-Phe-NA used as substrate. However, despite significant differences in specific activity compared with normal lysosomal preparations, no latency difference was observed if three other substrates were used (Gly-Arg-, Pro-Arg-, and Pro-Phe-NAs). The expression of this enzyme can thus be differentially influenced by cell growth and its latency characteristics can be influenced by the substrate used in assays.     

Rodent alpha-chymases are elastase-like proteases.

Although the alpha-chymases of primates and dogs are known as chymotrypsin-like proteases, the enzymatic properties of rodent alpha-chymases (rat mast cell protease 5/rMCP-5 and mouse mast cell protease 5/mMCP-5) have not been fully understood. We report that recombinant rMCP-5 and mMCP-5 are elastase-like proteases, not chymotrypsin-like proteases. An enzyme assay using chromogenic peptidyl substrates showed that mast cell protease-5s (MCP-5s) have a clear preference for small aliphatic amino acids (e.g. alanine, isoleucine, valine) in the P1 site of substrates. We used site-directed mutagenesis and computer modeling approaches to define the determinant residue for the substrate specificity of mMCP-5, and found that the mutant possessing a Gly substitution of the Val at position 216 (V216G) lost elastase-like activity but acquired chymase activity, suggesting that the Val216 dominantly restricts the substrate specificity of mMCP-5. Structural models of mMCP-5 and the V216G mutant based on the crystal structures of serine proteases (rMCP-2, human cathepsin G, and human chymase) revealed the active site differences that can account for the marked differences in substrate specificity of the two enzymes between elastase and chymase. These findings suggest that rodent alpha-chymases have unique biological activity different from the chymases of other species.     

Eosinophils and immune mechanisms: V. Demonstration of mouse spleen cell-derived chemotactic activities for eosinophils and mononuclear cells and comparisons with eosinophil stimulation promoter

The chemotactic response of mouse eosinophil-rich peritoneal exudative cells to the lymphokine eosinophil stimulation promoter (ESP) was examined. Both eosinophils and monocytes are chemotactically attracted across a 3-μm-pore size polycarbonate filter toward a concentration gradient of ESP-containing culture supernatant fluids. Deactivation of both cell types occurs following preincubation of the responding cells in culture supernates containing ESP activity. The chemotactic activity for both eosinophils and mononuclear cells is stable when incubated at 60 °C for 30 min but is labile at 80 °C, is nondialyzable, and at peak activity exhibits an apparent molecular weight of approximately 25,700 daltons, based on Sephadex G-100 gel chromatography. Production conditions required for the generation of chemotactic and ESP activities are identical, and fractions of culture supernatant fluids possessing one activity are also positive for the other. Preliminary results therefore indicate that the lymphokine ESP attracts both eosinophils and monocytes in a gradient-induced chemotaxis assay.     

Activation of paracrine TGF-beta1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase.

As a source of transforming growth factor beta1 (TGF-beta1), mast cells have been implicated as potential effector cells in many pathological processes. However, the mechanisms by which mast cells express, secrete, and activate TGF-beta1 have remained vague. We show here by means of RT-PCR, immunoblotting, and immunocytochemistry that isolated rat peritoneal mast cells synthesize and store large latent TGF-beta1 in their chymase 1-containing secretory granules. Mast cell stimulation and degranulation results in rapid secretion of the latent TGF-beta1, which is converted by chymase 1 into an active form recognized by the type II TGF-beta serine/threonine kinase receptor (TbetaRII). Thus, mast cells secrete active TGF-beta1 by a unique secretory mechanism in which latent TGF-beta1 and the activating enzyme chymase 1 are coreleased. The activation of latent TGF-beta1 specifically by chymase was verified using recombinant human latent TGF-beta1 and recombinant human chymase. In isolated TbetaRI- and TbetaRII-expressing peritoneal macrophages, the activated TGF-beta1 induces the expression of the plasminogen activator inhibitor 1 (PAI-1), whereas in the mast cells, the levels of TbetaRI, TbetaRII, and PAI-1 expression were below detection. Selective stimulation of mast cells in vivo in the rat peritoneal cavity leads to rapid overexpression of TGF-beta1 in peritoneal mast cells and of TbetaRs in peritoneal macrophages. These data strongly suggest that mast cells can act as potent paracrine effector cells both by secreting active TGF-beta1 and by enhancing its response in target cells.     

Cathepsin B synthesis by the HL60 promyelocytic cell line: effects of stimulating agents and anti-inflammatory compounds

Cathepsin B synthesis by the human HL60 promyelocyte cell line was investigated by immunohistochemistry and by the assay of the enzyme in cell lysates using a fluorimetric substrate. HL60 cells were shown to produce cathepsin B in response to treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Intracellular levels of cathepsin B and immunohistochemical staining of the enzyme were related to time in culture with increasing concentrations of TPA from 1 nmol/1 to 8.0 nmol/1. Synthesis of cathepsin B was associated with TPA-induced phagocytic activity of cells in culture, expression of alpha-naphthyl acetate esterase and reduced cell division. Cathepsin B production was, therefore, related to differentiation of the HL60 promyelocytes into mature macrophage-like cells. Cathepsin B activity in HL60 cell lysates was significantly increased by incubation of the cells with 10 micrograms/ml endotoxin (lipopolysaccharide) from Escherichia coli, but not carrageenan. The production of cathepsin B by TPA-induced HL60 cells was significantly reduced by 0.25 mumol/1 dexamethasone and the non-steroidal anti-inflammatory compound 4-(6-methoxy-2-naphthyl)-butan-2-one but not by indomethacin. The HL60 promyelocytic cell line is a useful model for the study of factors affecting proteinase synthesis by human mononuclear phagocytes.     

Elevated proteinase activities in mouse lung tumors quantitated by synthetic fluorogenic substrates.

Proteinase activities in malignant and normal lung tissues were measured using two synthetic substrates that consist of a fluorophor coupled to a peptide moiety. The hydrolysis of CBZ-Val-Lys-Lys-Arg-4-methoxy-2-naphthylamide and BZ-Gly-Gly-Arg-4-methoxy-2-naphthylamide were studied in homogenates of two types of mouse lung tumors, the Lewis lung tumor of the C57 black mouse and the KHT tumor of the C3H mouse. The activity of CBZ-Val-Lys-Lys-Arg-4-methoxy-2-naphthylamide hydrolysis had a pH optimum of 6.3 and a Km of 2.1 x 10(-4) M, required a thiol activator, and was inhibited by leupeptin suggesting the activity of a cathepsin B-like enzyme. The activity of BZ-Gly-Gly-Arg-4-methoxy-2-naphthylamide hydrolysis had a pH optimum of 6.7 and a Km of 3 x 10(-5) M. Lung tumor homogenates contained higher hydrolytic activities for both substrates than normal lung homogenates.     

The Human Mast Cell Chymase Gene (CMA1): Mapping to the Cathepsin G/Granzyme Gene Cluster and Lineage-Restricted Expression

Genes encoding T-cell-receptor α/δ chains, neutrophil cathepsin G, and lymphocyte CGL/granzymes are closely linked on chromosomal band 14q11.2. The current work identifies the human mast cell chymase gene (CMA1) as the fourth protease in this cluster and maps the gene to within 150 kb of the cathepsin G gene. The gene order is centromere-T cell receptor α/δ-CGL-1/granzyme B-CGL-2/granzyme H-cathepsin G-chymase. Chymase and cathepsin G genes are shown to be cotranscribed in the human mast cell line HMC-1 and in U-937 cells. Other cells transcribe cathepsin G or CGL/granzyme genes, but not chymase genes, suggesting a capacity for independent regulation. Comparison of the 5′ flank of the chymase gene with those of cathepsin G and CGL/granzymes reveals little overall homology. Only short regions of the 5′ flanks of the human and murine chymase genes sequenced to date are similar, suggesting that they are more distantly related than human and rodent CGL-1/granzyme B, the flanks of which are highly homologous. The expression patterns and clustering of genes provide possible clues to the presence of locus control regions that orchestrate lineage-restricted expression of leukocyte and mast cell proteases.     

Localization of cathepsin B in two human lung cancer cell lines

We demonstrated the cysteine proteinase cathepsin B in two human lung tumor cell lines by cytochemical and immunocytochemical methods. The cell lines were derived from a squamous cell carcinoma of the lung (HS-24) and a metastasis to the adrenal gland from an adenocarcinoma of the lung (SB-3). For comparison and control, normal human lung fibroblasts cells (Wi-38) were also investigated. Intracellular cathepsin B activity was detected in all three cell lines. SB-3 and the normal fibroblast cells showed almost equal cathepsin B activity, which was considerably stronger than that in the HS-24 cells. Specific inhibitors for cathepsin B (E64, leupeptin, antipain) suppressed its activity completely. Stefin A, the physiological cathepsin B inhibitor, was less effective; this might depend on its limited penetrability into living cells. Localization of the cathepsin B was performed by conventional immunofluorescence microscopy and laser scanning microscopy. With specific anti-cathepsin B antibodies, the enzyme was localized in HS-24, SB-3, and Wi-38 fibroblast cells within perinuclear granules representing the lysosomal compartment. In the SB-3 cells, we additionally localized a minor fraction of the enzyme bound to the plasma membrane in a speckled distribution, accessible to the antibodies from the outside. This direct demonstration of cathepsin B distribution supports biochemical data about the dual localization of the enzyme in tumor cells. It also supports the possibility of a direct involvement of cathepsin B in the degradation of the extracellular matrix, and thus a contribution of the enzyme in invasion and metastasis.     

Comparative study of alanine-amino-peptidase and gamma-glutamyl-transferase activity in normal Wistar rat urine

24 h urinary alanine-amino-peptidase (AAP) and gamma-glutamyl transferase (GGT) activities were studied from the 3rd-7th month of life in male Wistar rats. A close relationship was found between AAP and GGT activity, except at the beginning and at the end of this period. At the end there appear 2 subgroups, the larger (70%) showing a strong correlation between AAP and GGT activity and the other (30%), demonstrating no correlation at all. A good correlation between AAP and GGT activities, creatinine, 24 h urine volume and 24 h creatinine output was found.