Nils-Erik Huseby

The Tromsø Heart Study: distribution of, and determinants for, gamma-glutamyltransferase in a free-living population.

Gamma-glutamyltransferase (GGT) was measured in 1579 men, aged 20-54 years, and 1654 women, aged 20-49 years, screened for coronary risk factors. The distribution was right-skewed with medians 15 and 10 U/l for men and women, respectively. Less than 3.8% of the men and 0.8% of the women had GGT greater than or equal to 50 U/l. The low level of GGT matched well with the low mortality of cirrhosis and the modest use of alcohol in Norway. Multiple regression analysis for each sex showed a strong positive association with body mass index, use of alcohol and, unexpectedly, a negative association with coffee consumption, whereas serum triglycerides and the time since the last meal showed a weaker positive association. In women, use of oral contraceptives was positively associated with GGT.

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.

Practical Points Regarding Routine Determination of γ -Glutamyl Transferase (γ-GT) in Serum with a Kinetic Method at 37°C

Kinetic data at 37°C on γ-GT activity in serum with γ-glutamyl-4-nitroanilide and glycylglycine as substrates are reported. The apparent Km for the two substrates were 0.95 and 9.5 mmol/l, respectively. Without glycylglycine the enzymatic formation of 4-nitroaniline was only 5 per cent of that with both substrates. 4-nitroaniline showed a slight inhibitory effect on the γ-GT activity. On the basis of the results an optimized modification at 37°C sof the traditional method of Szasz (17) is described. The method is well suited for automation, e.g. in LKB 8600 Reaction Rate Analyzer. Somewhat higher reference values for men (9-35 U/l) than women (6-25 U/l) were found. Data on purity, solubility, and stability of the reagents, and on the precision of the method are given.

The Role of Reactive Oxygen Species in Integrin and Matrix Metalloproteinase Expression and Function

Cell adhesion and migration is largely dependent on integrin binding to extracellular matrix, and several signalling pathways involved in these processes have been shown to be modified by reactive oxygen species (ROS). In fact, integrin activation is linked to increased ROS production by NADPH-oxidases, 5-lipoxygenase, and release from mitochondria. Cell migration is intimately linked to degradation of the extracellular matrix, and activated matrix metalloproteinases (MMPs) are a prerequisite for cancer cell invasion and metastasis. In this minireview, we focus on the interplay between integrin-mediated ROS production and MMP expression as well as its biological and pathobiological significance.

Multiple forms of serum γ-glutamyltransferase. Association of the enzyme with lipoproteins

Abstract The association of γ-glutamyltransferase activity with lipoproteins was investigated in serum from hepatobiliary diseased patients. From 60 to 80% of the total activity in such sera was bound in lipoprotein complexes. These complexes or multiple forms of γ-glutamyltransferase could be separated into two main fractions by gel filtration or agarose gel electrophoresis. One fraction was characterized by γ-glutamyltransferase of high molecular mass ( M r > 600 000) and β-mobility and was detected in increased amounts in serum from patients with cholestasis. The main part of this activity was associated with lipoprotein-X and this complex could amount to 50% in some sera. The other fraction contained γ-glutamyltransferase eluting in the molecular mass range of 250 000 to 450 000 and migrating with α 1 α 2 -mobility. In this fraction complexes of γ-glutamyltransferase and high-density lipoprotein were detected which could amount to 70% in non-icteric sera. However, heterogeneity in size, charge and density could be demonstrated in both fractions.

Multiple forms of γ-glutamyltransferase in normal human liver, bile and serum

Abstract A study of the multiple forms of γ-glutamyltransferase ((γ-glutamyl)-peptide:amino acid γ-glutamyltransferase, EC 2.3.2.2) in normal human liver, bile and serum are reported. An amphiphilic form of the enzyme was demonstrated in all three samples. When solubilized with detergent, estimated values for Stokes radius of 48 A and sedimentation coefficient of 5 S were obtained for this form. A hydrophilic form was also present in serum and bile, which showed identical properties to the enzyme form obtained after papain-treatment of the three samples. The Stokes radius was found to be 37 A, and the sedimentation coefficient 5 S. It was concluded that the heterogeneity of enzyme activity found both on gel filtration and on electrophoresis was due to aggregates of the amphiphilic form with lipids and other proteins, and could not be ascribed to the presence of isoenzymes.

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.

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.

Purification and some properties of γ-glutamyltransferase from human liver

Abstract The purification of γ-glutamyltransferase ((γ-glutamyl)-peptide: amino acid γ-glutamyltransferase, EC 2.3.2.2) from normal human liver is described. The procedure includes solubilization of the enzyme from membranes using deoxycholate and Lubrol W, treatment with acetone and butanol, and affinity chromatography on immobilized concanavalin A. Treatment with papain was used to release the enzyme from aggregates of lipid and protein, prior to further purification. An overall purification of 9400 was achieved and analytical polyacrylamide gel electrophoresis indicated that the final product was homogeneous, and had a molecular weight of 90 000. Gel filtration, however, indicated a molecular weight of 110 000. Two subunits were identified on dodecyl sulfate gel electrophoresis with estimated molecular weights of 47 000 and 22 000. The kinetic properties studied for the purified enzyme were similar to those found for partially purified (not papain-treated) enzyme, and resembled those of serum γ-glutamyltransferase. The true Km values for the liver enzyme were estimated to 0.81 mM for γ-glutamyl-p-nitroanilide and to 12.4 mM for glycylglycine.

γ-Glutamyltransferase is upregulated after oxidative stress through the Ras signal transduction pathway in rat colon carcinoma cells

γ-Glutamyltransferase (GGT) plays a central role in the homeostasis of the antioxidant glutathione (GSH). The expression of GGT has been shown to be upregulated after oxidative stress, but the signalling pathways implicated remain poorly characterized. The results here show that acute exposure of CC531 cells to oxidative stress resulted in activation of Ras and augmented GGT enzyme activity, both at the transcriptional and at the translation level. Moreover, an involvement of the GGT promoter II was detected after RT-PCR and transient transfection studies. Ectopic expression of activated Ras, but not dominant negative Ras, also resulted in increased GGT promoter II transcriptional activity, an effect that was attenuated by over-expression of dominant negative mutants of Akt, p38 MAPK and MEK1. Addition of specific inhibitors of these kinases during oxidative stress diminished the activation of GGT. In conclusion, oxidative stress-induced activation of GGT involves Ras and several downstream signalling pathways.