Gamma-glutamyltransferase fractions in human plasma and bile: characteristic and biogenesis

Abstract

Total plasma gamma-glutamyltransferase (GGT) activity is a sensitive, non-specific marker of liver dysfunction. Four GGT fractions (b-, m-, s-, f-GGT) were described in plasma and their differential specificity in the diagnosis of liver diseases was suggested. Nevertheless fractional GGT properties have not been investigated yet. The aim of this study was to characterize the molecular nature of fractional GGT in both human plasma and bile. Plasma was obtained from healthy volunteers; whereas bile was collected from patients undergoing liver transplantation. Molecular weight (MW), density, distribution by centrifugal sedimentation and sensitivity to both detergent (deoxycholic acid) and protease (papain) were evaluated. A partial purification of b-GGT was obtained by ultracentrifugation. Plasma b-GGT fraction showed a MW of 2000 kDa and a density between 1.063-1.210 g/ml. Detergent converted b-GGT into s-GGT, whereas papain alone did not produce any effect. Plasma m-GGT and s-GGT showed a MW of 1,000 and 200 kDa, and densities between 1.006-1.063 g/ml and 1.063-1.210 g/ml respectively. Both fractions were unaffected by deoxycholic acid, while GGT activity was recovered into f-GGT peak after papain treatment. Plasma f-GGT showed a MW of 70 kDa and a density higher than 1.21 g/ml. We identified only two chromatographic peaks, in bile, showing similar characteristics as plasma b- and f-GGT fractions. These evidences, together with centrifugal sedimentation properties and immunogold electronic microscopy data, indicate that b-GGT is constituted of membrane microvesicles in both bile and plasma, m-GGT and s-GGT might be constituted of bile-acid micelles, while f-GGT represents the free-soluble form of the enzyme.

Figure 1. GGT-specific (continuous line) and total cholesterol (dashed line) elution profiles in a plasma pool (pool-1) representative of four experiments, and in the lipoprotein fractions isolated from it.

A) Total plasma; B) VLDL (elution volume: 11.3 ml; density 0.950–1.006 g/ml; Stokes radius: 30–80 nm); C) LDL (15.0 ml; 1.006–1.063 g/ml; 18–25 nm); D) HDL (20.0 ml; 1.063–1.21 g/ml; 8–11 nm). Lipoproteins were separated by ultracentrifugation on a NaBr discontinuous density gradient. Elution profiles were obtained by size-exclusion chromatography associated with a post-column reaction specific for GGT or total cholesterol.

Figure 2. Papain and DOC effects on plasma samples.

Effect of papain (25 mmol/l, 24 h, RT) and DOC (1% w/v, 1 h, 37°C) treatments on GGT fractions in a plasma sample representative of four independent experiments. A) GGT-specific elution profile in the untreated control [continuous line; GGT activity (U/L): total = 38.8; b-GGT = 8.9; m-GGT = 3.1; s-GGT = 14.1; f-GGT = 12.6] and after papain treatment [dotted line; GGT activity (U/L): total = 49.8; b-GGT = 7.5; m-GGT = n.d.; s-GGT = n.d.; f-GGT = 42.3]. B) GGT-specific elution profile after DOC treatment [dashed line; GGT activity (U/L): total = 45.0; b-GGT = 1.3; m-GGT = 2.4; s-GGT = 31.2; f-GGT = 10.1] and after DOC and papain treatments [dotted line; GGT activity (U/L): total = 57.0; b-GGT = n.d.; m-GGT = n.d.; s-GGT = n.d.; f-GGT = 57.0]. n.d.: not detectable.

Figure 3. Papain and DOC effects on bile samples.

Effect of papain (25 mmol/l, 24 h, RT) and DOC (1% w/v, 1 h, 37°C) treatments on GGT fractions in sample of human bile representative of four independent experiments. Untreated control [continuous line; GGT activity (U/L): total = 127.5; b-GGT = 106.2; f-GGT = 21.3], after papain treatment [dashed line; GGT activity (U/L): total = 171.22; b-GGT = 62.1; f-GGT = 109.1] and after DOC and papain treatments [dotted line; GGT activity (U/L): total = 217.6; b-GGT = 19.6; f-GGT = 198.0].

Figure 4. GGT activity of isolated microparticles and exosomes.

GGT-specific elution profiles of isolated microparticles (continuous line), exosomes (dashed line) and microparticles/exosomes-free plasma (dotted line) samples. Microparticles were obtained from plasma by centrifugation (10,000 g, 45 min, 4°C) whereas exosomes were recovered by ultracentrifugation (100,000 g, 2 h, 4°C) of the corresponding microvesicles-free supernatant. Representative chromatograms of five independent experiments are reported.

Figure 5. GGT localization on exosomes by transmission electron microscopy.

Exosomes from A) plasma and B) bile were incubated with a primary antibody directed against GGT and with a secondary 15 nm gold particle-conjugated antibody. After fixation with glutaraldehyde, samples were negatively stained with uranyl acetate and examined under a transmission electron microscope.