Differential regulation of γ-glutamyltransferase and glutamate cysteine ligase expression after mitochondrial uncoupling: γ-glutamyltransferase is regulated in an Nrf2- and NFκB-independent manner.

The enzymes γ-glutamyltransferase (GGT) and glutamate cysteine ligase (GCL) have important roles in glutathione (GSH) homeostasis, and both are frequently upregulated after acute oxidative stress. Mitochondria are major producers of ROS, and incubating the colorectal adenocarcinoma cell line HT-29 cells with mitochondrial uncouplers significantly increased endogenous ROS as well as mRNA for both GGT and GCLC (the catalytic subunit of GCL). However, no elevation in GGT protein or activity was detected, in contrast to the increased levels of GCLC protein found. The uncouplers initiated endoplasmic reticulum (ER) stress, as demonstrated by highly increased levels of CHOP and GRP78 mRNA. Using inhibitors of proteasomes and ER-associated degradation (ERAD) together with a mitochondrial uncoupler, increased GGT protein and activity levels were obtained indicating that GGT may be a substrate for ERAD. Uncoupling increased the mRNA levels of the two redox-regulated transcription factors Nrf2 and NFκB. Using siRNA to suppress Nrf2 and NFκB expression, downregulation of GCLC expression both at the basal level and after mitochondrial uncoupling was achieved. In contrast, the expression level of GGT was not affected by this treatment. These data strongly indicate a discrepancy between the regulation of GCLC and of GGT following the oxidative stress situation due to mitochondrial uncoupling. Both the enzymes are considered to be part of the cellular antioxidant system; however, the role of GGT as a consistent oxidative response parameter needs to be reevaluated.

Differential regulation of gamma-glutamyltransferase mRNAs in four human tumour cell lines.

Human gamma-glutamyltransferase (GGT) belongs to a multigenic family and at least three mRNAs are transcribed from the gene that codes for an active enzyme. Four human tumour cell lines (HepG2, LNCap, HeLa and U937) with different GGT levels were used to investigate how GGT activity, total GGT mRNA and each individual GGT mRNA subtype responded to tumour necrosis factor-alpha (TNF-alpha), 12-O-tetradecanoylphorbol 13-acetate (TPA) or sodium butyrate treatment. Butyrate reduced the GGT activity in HepG2 cells, and the level of total GGT mRNA accordingly, whereas TNF-alpha and TPA did not alter these parameters. In LNCap cells, TNF-alpha, TPA, and butyrate reduced the activity as well as the level of GGT total mRNA. In HeLa cells no significant changes were observed either in activity or in mRNA level whereas TPA induced both GGT activity and mRNA levels in U937 cells. The distribution of each GGT mRNA subtype (A, B and C) was found to be cell specific: type B mRNA was the major form in HepG2 cells, while type A was the major form in LNCap and HeLa, type A and type C were expressed almost at the same level in U937 cells. The GGT mRNA subtypes were also differently modulated in these cells after TNF-alpha, TPA or butyrate treatment, suggesting that they are regulated by distinct and cell type specific mechanisms.

Regulation of gamma-glutamyltransferase in cisplatin-resistant and -sensitive colon carcinoma cells after acute cisplatin and oxidative stress exposures.

Glutathione plays an important role in drug resistance of tumor cells and in their ability to resist oxidative stress. Improved salvage of glutathione can be obtained through increased activity of gamma-glutamyltransferase (GGT), which is of importance in the maintenance of cellular glutathione homeostasis. We investigated the regulation of GGT in 2 cisplatin-resistant and 1 cisplatin-sensitive colon carcinoma cell lines. Enzyme activity was induced in all 3 cell lines after acute exposure to cisplatin. The elevation was significantly higher in sensitive cells (3.3-fold) than in resistant (1.6- to 1.7-fold) cells. Exposure of cells to oxidative stress generated by menadione also resulted in enzyme induction but only in cisplatin-sensitive cells. Addition of anti-oxidants had different effects on the 2 inductions: N-acetylcysteine blocked the induction of both cisplatin and menadione, whereas catalase and glutathione-ester blocked only the menadione induction. Glutathione depletion alone was not sufficient to induce GGT in these cells. The data show that GGT is regulated by multiple mechanisms during anti-tumor drug treatment and oxidative stress and that reactive oxygen species were involved in the menadione, but not cisplatin, induction of the enzyme.

Differential effect of unfractionated heparin and low molecular weight heparin on intravascular tissue factor pathway inhibitor: evidence for a difference in antithrombotic action.

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor (TF)-induced blood coagulation, which is increased several-fold in post-heparin plasma and thought to contribute significantly to the antithrombotic action of heparin. In the present study we investigated whether subcutaneous (s.c.) administration of a low molecular weight heparin (LMWH), enoxaparin, had a different effect on intravascular pools of TFPI compared with continuous i.v. infusion of unfractionated heparin (UFH). 18 healthy male volunteers were randomly assigned to continuous i.v. infusion with UFH (initially 450 U/kg/24 h, n = 6) or to s.c. treatment with LMWH once daily (enoxaparin, 1.5 mg/kg, n = 12) for 72 h. A bolus injection of 5000 IU UFH i.v. caused an 8-13-fold increase in plasma-free TFPI antigen (TFPI Ag), followed by a progressive decrease (81 +/- 4%, P<0.001) during the 72 h infusion with UFH. 4 h after discontinuation of the infusion, basal free TFPI Ag and heparin-releasable TFPI were significantly decreased compared with the concentrations before the infusion (30 +/- 9%, and 27 +/- 7%, respectively). In contrast, LMWH treatment did not reduce either basal or heparin-releasable TFPI Ag. The changes in plasma TFPI Ag by UFH and LMWH were statistically different between groups both in pre- (P<0.001) and post-heparin (P<0.0001) plasma. The differential effect of UFH and LMWH on intravascular pools of TFPI may contribute to the understanding of the apparent superior efficacy of LMWHs in the treatment of both arterial and venous thrombosis.

Elimination of alkaline phosphatases from circulation by the galactose receptor. Different isoforms are cleared at various rates.

Three isoforms of human alkaline phosphatase (liver, bone and placental ALP) were purified and their elimination studied after intravenous injection in rats. The rates of elimination were significantly inhibited by prior injection of asialofetuin, indicating that the uptake was mediated by the galactose receptor in liver. Their relative clearance rates differed, being rapid for the bone ALP, significantly slower for the liver isoform and very slow for the placental ALP. The bone ALP showed a rapid initial clearance, apparently related to its large glycan heterogeneity and to the presence of molecules with a low sialic acid content. When isolated from serum the liver and bone ALP isoforms showed clearance rates differing slightly from those of the organ derived forms. We conclude that differences in carbohydrate structure and amount of sialic acid of the three isoforms result in various clearance rates. These differences will also affect their serum concentrations as well as the composition and heterogeneity of the individual isoforms in serum.

Serum gamma-glutamyltransferase and alkaline phosphatase during experimental liver metastases. Detection of tumour-specific isoforms and factors affecting their serum levels.

Tumour-specific isoenzymes and tumour markers in serum are potentially useful in the detection and monitoring of liver metastases. An experimental rat model was used in the search for such isoenzymes and to study factors affecting their serum levels. Splenic injection of CC531 colon carcinoma cells in syngeneic WagRij rats caused liver metastases after 3 weeks with concomitant and significant increases in serum levels of gamma-glutamyltransferase (GT) and alkaline phosphatase (ALP). The presence of tumour-specific isoforms of both enzymes, as well as increased amounts of the liver isoform of ALP, were demonstrated in serum. The serum levels of the tumour variants were clearly related to their elimination rates from the circulation. Thus, the slow clearance of the tumour ALP resulted in high serum levels of this isoform, compared with the more rapid elimination of tumour GT and its lower serum level. When using another colon carcinoma cell line (DHD/K12), metastatic to liver in BD IX rats, no increases in serum GT were detected. This was related to the rapid elimination from the circulation of the GT variant from the DHD/K12 metastatic tissue. The relatively high amount of the tumour ALP isoform detected in serum during growth of the CC531 liver metastases indicated that this isoform could be useful as a marker of tumour growth.

Carbohydrate-deficient transferrin: marker of actual alcohol consumption or chronic alcohol misuse?

Carbohydrate-deficient transferrin (CDT) is a useful indicator of excessive alcohol consumption with higher sensitivity and specificity than other markers that are used. In the present study, CDT was analysed in 161 patients hospitalized in a surgical ward to evaluate whether history of drinking and chronic alcohol misuse are important determinants of CDT elevations. Fifty-one of the patients were diagnosed as alcohol-dependent and they all reported a long history of alcohol abuse. Several of these, as well as many of the non-dependent patients, reported a high, recent alcohol consumption (> or = 60 g/day for the previous 2 weeks). CDT performed better in detecting patients with alcohol dependency than in detecting patients with high alcohol consumption irrespective of dependency, showing higher sensitivity (47 vs 37%), likelihood ratio (4.7 vs 3.4), and a statistically significant difference in the receiver-operating characteristic curve areas (P = 0.04 in a two-tailed comparison test). In two subgroups, one with alcohol-dependent and one with non-dependent patients, consuming similar amounts of alcohol (range: 60-170 g/day), the sensitivity of CDT was 52 and 5%, respectively. We conclude that CDT is a better marker for patients with chronic alcohol misuse than as a marker for high actual alcohol consumption alone.

Utility of biological markers during outpatient treatment of alcohol-dependent subjects: carbohydrate-deficient transferrin responds to moderate changes in alcohol consumption.

A group of 25 alcohol-dependent subjects in outpatient treatment were monitored for a period of 4 weeks. They were weekly interviewed for their alcohol consumption and their serum levels of carbohydrate-deficient transferrin (CDT) and gamma-glutamyltransferase (GT) were analyzed. The majority of the patients reported an excessive and fairly constant alcohol intake during the observation period. When selecting those patients that reported periods of 1 or 2 weeks with moderate changes in alcohol consumption, corresponding changes in CDT were demonstrated. Thus, of 14 patients reporting an increased alcohol consumption for 2 weeks (mean values increased from 57 to 101 g/day), 11 showed an increase in CDT at the end of the period. The mean CDT value of all 14 increased from 5.5 to 6.7% (p < 0.05). Slight, but not significant, increases were noted in GT, indicating that CDT is more sensitive than GT in detecting increased alcohol consumption. Furthermore, of 17 patients that reported decreased alcohol consumption for one or several weeks, 14 showed decreased CDT and GT values. The mean values of all 17 were reduced from 5.1% to 4.5% (CDT) and from 126 units/liter to 97 units/liter (GT) (p < 0.05 for both parameters). The results indicate that CDT responds to moderate changes in alcohol consumption in alcohol-dependent patients and may thus be useful as a corrective tool to self-reports of alcohol consumption during outpatient treatments.

Carbohydrate-deficient transferrin and alcohol dependency: variation in response to alcohol intake among different groups of patients.

Carbohydrate-deficient transferrin (CDT) and gamma-glutamyltransferase (GT) were evaluated as markers of alcohol dependency in two different groups of patients. Sensitivity of CDT was nearly 75% for patients hospitalized for detoxification, but lower than 50% for alcohol-dependent patients admitted to acute surgery. CDT correlated with self-reported alcohol consumption in both groups, and sensitivity increased with higher alcohol intake. Sensitivity of CDT for females in both groups was considerably lower than for males, although their alcohol consumption was not significantly different. Serum activity of GT showed almost identical performance as CDT when evaluated by receiver-operating characteristics curve analysis (ROC-analysis), but sensitivity and specificity of the two markers varied differently with both alcohol consumption and age. Among the surgical patients, the highest sensitivity of CDT was found for the middle-aged patients (36 to 50 years), whereas the highest sensitivity of GT was found for the eldest. A tendency for similar age-related differences were also observed among the patients warded for detoxification, but these differences were not statistically significant. A particular difference between the two groups was noted among the youngest patients (21 to 35 years), with a very low sensitivity of CDT (< 20%) for the surgical patients and a high sensitivity (77%) for the detoxification group. This difference was not only caused by differences in the present alcohol consumption, but would also be related to differences in drinking pattern or duration. Two commercial kits analyzing CDT were compared and ROC-analysis indicated identical performance of the two. However, a kit determining CDT as percentage of total transferrin showed a somewhat higher sensitivity among patients with low serum transferrin. We conclude that CDT and GT show variant responses to alcohol consumption in different groups of patients. The level of the two markers are related to sex, age, and alcohol consumption. Furthermore, the performance of both markers depend on the patients' history of alcohol abuse. CDT and GT are statistically independent markers and may therefore supplement each other.

Tissue factor pathway inhibitor in complex with low density lipoprotein isolated from human plasma does not possess anticoagulant function in tissue factor-induced coagulation in vitro.

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of the extrinsic coagulation system. In human plasma 70-85% is associated with apoB-containing lipoproteins whereas 10-20% exists in a carrier free form. The purpose of the present study was to assess the anticoagulant function of TFPI in complex with low density lipoproteins (LDL) on tissue factor (TF)-induced coagulation in vitro. LDL-TFPI complexes were isolated by preparative density gradient ultracentrifugation, LDL-free TFPI by preparative gel filtration and the anticoagulant properties were assessed by a diluted prothrombin time assay (dPT). LDL-free TFPI (0-0.46 U/ml) added to the dPT mixture, caused a prominent dose-dependent prolongation of dPT (0-42.2 sec.) which could be abolished by the addition of blocking anti-TFPI IgG. Contrary, increasing amounts of LDL-bound TFPI (0-4.0 U/ml) shortened dPT by 11.4 sec at the highest concentration. LDL-bound TFPI was not immunodetected by anti-TFPI IgG directed against the distal portion of the C-terminus, and appeared on Western blotting with a major band at 67 kDa and a weak band at 34 kDa which suggest that LDL-bound TFPI lack anticoagulant function due to carboxy terminal truncation. Our data provide evidence for the hypothesis that the anticoagulant function of TFPI is restricted to its carrier free form in human plasma.

Clearance of circulating gamma-glutamyltransferase by the asialoglycoprotein receptor. Enzyme forms with different sialic acid content are eliminated at different clearance rates and without apparent desialylation.

gamma-Glutamyltransferase is eliminated from the circulation via the asialoglycoprotein receptor in liver. After purifying the enzyme from human liver, a subfractionation into differently sialylated forms was obtained using MonoQ ion exchange chromatography. The uptake of such forms from rat circulation was studied and the slowest rate was measured for the most sialylated form. To test if the uptake of the sialylated enzymes was dependent on prior desialylation in the circulation the enzyme was recovered from liver after uptake and from serum after inhibiting the uptake with asialofetuin. Analysis of these recovered forms showed no apparent alteration in charge. The enzyme is apparently eliminated without prior desialylation through available galactose units which bind with low affinity to the receptor.

Evaluation of two biological markers combined as a parameter of alcohol dependency.

A test was constructed by combining carbohydrate-deficient transferrin (%CDT) and gamma-glutamyltransferase (GT) and was evaluated in detecting alcohol-dependent patients in a surgical ward. The performance of the combined test was significantly better than that of either %CDT alone or GT alone, as evaluated by calculating sensitivity and likelihood ratio at a specificity of 0.85 and by comparing areas under receiver-operating characteristic curves statistically. Improved performance was found for the whole group of patients, for men, for patients older than 35 years of age, and for those patients consuming more than 60 g alcohol per day. The performance of %CDT alone was similar to that of GT, %CDT being better in the age group 35-50 years, whereas GT was better for those drinking less than 60 g per day and for the oldest patients (> 50 years). Neither of the two tests or their combination performed well for patients younger than 36 years of age.

Depletion of intravascular pools of tissue factor pathway inhibitor (TFPI) during repeated or continuous intravenous infusion of heparin in man.

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of the extrinsic coagulation system. TFPI is increased several-fold in postheparin plasma and thereby thought to contribute significantly to the antithrombotic action of heparin. The present study was conducted to investigate how repeated (n = 8) and continuous (n = 6) administration of heparin affect plasma TFPI and the inhibition of tissue factor (TF)-induced coagulation ex vivo in humans. Free TFPI antigen (TFPI Ag) increased from 19.2 +/- 4.0 ng/ml to 204.7 +/- 31.7 ng/ml after intravenous injection of 5000 IU of unfractionated heparin. Five repeated injections of 5000 IU of heparin at 4 h intervals caused a progressive decrease (-45 +/- 8%, p < 0.0001 for time effect) in heparin-releasable TFPI and a progressive shortening of the clotting time as determined in a dilute prothrombin time assay (dPT) (-8.7 +/- 6.1 s, p < 0.0001). The basal concentration of TFPI Ag in plasma collected immediately before each heparin injection was decreased by 29 +/- 15% (p < 0.0001), whereas the dPT was decreased by 6.9 +/- 3.5 s (p < 0.0001). During a 24 h continuous infusion of heparin TFPI Ag decreased from 161.5 +/- 26.0 ng/ml to 35.6 +/- 4.7 ng/ml (-77.3 +/- 5.1%) (p < 0.0001). The contribution of TFPI to the inhibition of TF-induced coagulation during heparin infusion was estimated to decrease from 60 +/- 15% to 20 +/- 10% (p < 0.0001). The present data indicate partial depletion of intravascular pools of TFPI by repeated and continuous heparin administration and thereby attenuation of its contribution to the antithrombotic action of heparin.

Effect of cholesterol lowering on intravascular pools of TFPI and its anticoagulant potential in type II hyperlipoproteinemia.

Tissue factor pathway inhibitor (TFPI) inhibits the extrinsic coagulation system. A major pool of TFPI is associated with the vascular endothelium and can be mobilized into the circulation by heparin. In circulating blood, TFPI is mainly associated with LDL (80%), whereas 10% to 20% is carrier free. In this study, heparin administration caused a 2.2-fold and a 7.5-fold increase in TFPI activity and TFPI antigen, respectively, in 25 patients with phenotypes IIa and IIb hyperbetalipoproteinemia. Because the antigen determination of TFPI almost exclusively measures carrier-free TFPI, more than 90% of the heparin-induced increase in TFPI activity was caused by mobilization of carrier-free TFPI from the vascular endothelium. Therapeutic lowering of total cholesterol (a decrease of 31.1 +/- 11.6%, P < .001) by 40 mg/d lovastatin in 17 patients with hyperbetalipoproteinemia was accompanied by a parallel decrease in TFPI activity (of 27.7 +/- 24.2%, P < .001) because of a reduction in LDL-TFPI complexes. However, drug intervention did not affect carrier-free TFPI or the magnitude of the vascular pool of TFPI that could be mobilized into the circulation by heparin. Moreover, this reduction of LDL-TFPI complexes did not reduce the anticoagulant potency of TFPI in plasma or of the vascular endothelial pool. The results of this study may imply that the anticoagulant potency of TFPI is associated with its carrier-free form in plasma or on the endothelium and that downregulation of LDL affects neither the size nor the anticoagulant potency of the endothelial pool of TFPI.

Tissue-factor pathway inhibitor and lipoproteins. Evidence for association with and regulation by LDL in human plasma.

Tissue-factor pathway inhibitor (TFPI) is a potent inhibitor of extrinsic coagulation, which is mainly associated with lipoproteins in circulating blood. Gel filtration of human plasma confirmed the presence of three peaks in which approximately 10%, 70%, and 20% of total TFPI activity was retained. Precipitation of very-low-density lipoproteins and low-density lipoproteins (LDLs) in plasma by polyethylene glycol almost completely abolished peaks and I and II. LDL isolated by ultracentrifugation revealed two peaks of TFPI after gel filtration that coeluted with peaks I and II, respectively, from gel filtration of total plasma. TFPI activity in peaks I and II was also precipitated by anti-apolipoprotein B antibodies. Fourteen patients with familial hypercholesterolemia had higher plasma TFPI activity than did age- and sex-matched normolipemic control subjects (1.45 +/- 0.27 U/mL versus 0.80 +/- 0.09 U/mL, P < .001). Plasma TFPI was correlated with LDL cholesterol (r = .73, P < .001) and apolipoprotein B (r = .69, P < .001). No association was found with high-density lipoprotein cholesterol or apolipoprotein A-I. In a double-blind, placebo-controlled trial among the familial hypercholesterolemia patients, lovastatin alone or in combination with fish oil concentrate lowered plasma TFPI in parallel with LDL cholesterol. Gel filtration of plasma from these patients demonstrated a specific drop in apolipoprotein B-TFPI complexes, whereas TFPI not associated with lipoproteins was unchanged. This study demonstrated that plasma TFPI was associated with and regulated by LDL in plasma from healthy subjects and patients with familial hypercholesterolemia.

Renal and hepatic toxicity after high-dose 7-hydroxymethotrexate in the rat.

To examine directly the hepatic and renal toxicity of 7-hydroxymethotrexate (7-OH-MTX) without interference of the parent compound methotrexate (MTX), we purified and gave 100 mg/kg 7-OH-MTX to rats, a dose resulting in serum levels of 7-OH-MTX comparable with those achieved in the clinic after the administration of high-dose MTX (HD-MTX). After only 5 h, the 7-OH-MTX-treated rats demonstrated 2.6-fold increases in serum creatinine values and 2-fold elevations in serum aspartate aminotransferase (ASAT) levels as compared with the controls. Morphologic evidence of toxicity, however, was apparent only in the kidneys. Intraluminal cellular debris containing membranous material and deteriorated organelles was seen, but no precipitate of the delivered drug. The peak serum concentration of 7-OH was up to 939 microM, and concentrations of 7-OH-MTX declined triphasically, showing a t1/2 alpha value of 2.45 min, a t1/2 beta value of 30.5 min, and a terminal half-life (t1/2 gamma) of 240 min. The total clearance value was 14.5 ml min-1 kg, and the postdistributional volume of distribution (V beta) was 5070 ml/kg. Our results may indicate a direct toxic effect of 7-OH-MTX on kidney and liver cells.

Clearance of circulating gamma-glutamyltransferase by the hepatic galactose receptor. Variability in clearance rate due to carbohydrate heterogeneity of the enzyme.

The clearance and organ uptake of gamma-glutamyltransferase was studied by injecting the purified human liver enzyme intravenously in the rat. The enzyme was almost exclusively taken up by liver hepatocytes with a rapid initial uptake. The clearance was significantly inhibited by asialofetuin as well as by galactose and fucose. The uptake of neuraminidase-treated enzyme was much more rapid than that of the native enzyme. Subfractions of gamma-glutamyltransferase obtained by lectin affinity chromatography revealed significant differences in clearance rates. The data strongly indicates that the uptake of circulating gamma-glutamyltransferase involves the galactose (asialo-glycoprotein) receptor of the parenchymal cells, and that the heterogeneity of gamma-glutamyltransferase results in varying clearance rates.

Phosphoinositide metabolism in a polyoma-BK-virus-transformed pancreatic islet cell line: evidence for constitutively activated phospholipase C.

We have characterized the phosphoinositide metabolism in a polyoma-BK-virus-transformed rat pancreatic islet cell line which has highly malignant characteristics, expresses viral T-antigen and has lost insulin-secreting capacity. After incorporation with [3H]inositol to isotopic equilibrium, all inositol metabolites were analyzed. When compared with normal pancreatic islets, increased levels of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), inositol 1,3,4-trisphosphates and inositol tetrakisphosphate (Ins-P4), and decreased levels of phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) were found. The Ins-1,4,5-P3/PIP2 ratio increased, whereas the PIP2/PIP ratio was not altered after the transformation. In the pancreatic islet cell line there was a stable accumulation of inositol phosphates at 3.3 mM glucose. Glucose, KCl, cholecystokinin (CCK) and carbachol with and without LiCl were all without effect on the accumulation of inositol phosphates. Somatostatin inhibited the accumulation of inositol phosphates but a Ca(2+)-free/EDTA solution did not. Preincubation with cholera toxin or pertussis toxin inhibited the accumulation of inositol phosphates at 3.3 mM glucose except for Ins-P4, whereas no effect was observed on the phosphoinositides. NaF stimulated the accumulation of inositol phosphates, with a concomitant decrease in the phosphoinositides, whereas neomycin was without effect on the inositol phosphates. In normal pancreatic islets, pertussis toxin inhibited the CCK-induced increase in Ins-1,4,5-P3, whereas no effect was seen at 3.3 mM glucose. Finally, pertussis toxin inhibited the CCK-induced increase in the Ins-1,4,5-P3/PIP2 ratio in normal pancreatic islets. The same inhibition was also found in the pancreatic islet cell line at 3.3 mM glucose. We conclude that in the transformed pancreatic islet cell line the phosphoinositide hydrolysis is constitutively activated at the level of phospholipase C, with a substantial loss of regulatory control.

The level of gamma-glutamyltransferase in serum, effect of carbohydrate heterogeneity on clearance rate.

The measurement of serum gamma-glutamyltransferase (GT) is a frequently used parameter of liver diseases. The serum enzyme originates from liver and is cleared from the circulation by the galactose receptor in liver. The rate of uptake will thus vary with the amount of terminal galactose residues on the enzymes' carbohydrate moiety. Using an experimental rat model we have studied the relative clearance rates of variant forms of GT with different carbohydrate composition. GT purified from pancreas and kidney contains less sialic acid and showed considerably higher clearance rates than the enzyme from liver. The rapid uptake of the kidney and pancreas enzymes indicates that these enzymes may not reach detectable levels if released from these organs to the circulation. On the other hand, GT in serum of alcoholics contains increased amount of sialic acid and this enzyme variant showed a slightly decreased clearance rate compared to the normal liver enzyme. Increased sialylation of GT may thus contribute to the increased level of the enzyme in serum after alcohol abuse.