A biomimetic enzyme-linked immunosorbent assay (BELISA) for the analysis of gonadorelin by using molecularly imprinted polymer-coated microplates.

An original biomimetic enzyme-linked immunoassay (BELISA) to target the small peptide hormone gonadorelin is presented. This peptide has been recently listed among the substances banned in sports by the World Antidoping Agency (WADA) since its misuse by male athletes triggers testosterone increase. Hence, in response to this emerging issue in anti-doping controls, we proposed BELISA which involves the growth of a polynorepinephrine (PNE)-based molecularly imprinted polymer (MIP) directly on microwells. PNE, a polydopamine (PDA) analog, has recently displayed impressive performances when it was exploited for MIP preparation, giving even better results than PDA. Gonadorelin quantification was accomplished via a colorimetric indirect competitive bioassay involving the competition between biotinylated gonadorelin linked to the signal reporter and the unlabeled analyte. These compete for the same MIP binding sites resulting in an inverse correlation between gonadorelin concentration and the output color signal (λ = 450 nm). A detection limit of 277 pmol L-1 was achieved with very good reproducibility in standard solutions (avCV% = 4.07%) and in urine samples (avCV% = 5.24%). The selectivity of the assay resulted adequate for biological specimens and non-specific control peptides. In addition, the analytical figures of merit were successfully validated by mass spectrometry, the reference anti-doping benchtop platform for the analyte. BELISA was aimed to open real perspectives for PNE-based MIPs as alternatives to antibodies, especially when the target analyte is a poorly or non-immunogenic small molecule, such as gonadorelin. Biomimetic enzyme-linked immunosorbent assay (BELISA).

Sensitivity and reproducibility enhancement in enzyme immunosorbent assays based on half fragment antibodies.

The free sulfhydryl groups of the hinge region of monovalent antibody fragments (rIgG) allow the orientation of rIgG on functionalized surfaces in immunosensors. To evaluate the contribution of reduction and orientation on signal enhancement we compared the performance of whole antibodies and their rIgG in ELISA performed on polystyrene or maleimide-functionalized microplates. Monoclonal anti-horseradish peroxidase (anti-HRP) and monoclonal anti-fPSA antibodies (1 mg/mL) were reduced with 2-mercaptoethylamine (53 mM). Western blot confirmed the presence of rIgG as a band at 75 kDa, detectable only by anti-heavy chain but not by anti-light chain antibodies, suggesting a possible folding rearrangement. Using anti-HRP we confirmed the retention of the antigen binding capacity of rIgG. Moreover, we observed a signal enhancement for rIgG even if randomly absorbed on polystyrene [linear regression slope (95%CI): rIgG 0.524 (0.434-0.614), IgG 0.370 (0.430-0.399); P = 0.0016] suggesting that chemical reduction might affect the antigen binding capacity of antibodies. ELISA with anti-fPSA rIgG coated on polystyrene confirmed these observations. Oriented anti-fPSA rIgG on a maleimide surface showed comparable signals to the assay performed on polystyrene for each analyzed concentration of antigen (PANOVA = 0.1980), anyway, with a significant improvement of the repeatability likely providing a more homogeneous capturing surface (SD rIgGmaleimide-rIgGpolystirene: fPSA 0.725 ng/mL:0.74-2.89; 1.45 ng/mL:1.56-8.69; 3.625 ng/mL:3.52-15.03; 7.25 ng/mL:7.78-18.44).

Inter-assay variability in automated serum free light chain assays and their use in the clinical laboratory.

Serum κ and λ free light chain levels are markers of plasma cell proliferation, and their measurements have been included in recent guidelines by the International Myeloma Working Group for the management of patients with plasma cellular dyscrasias. Five in vitro diagnostic methods for the immunochemical quantification of serum free light chains (FLC) are available, three based on polyclonal antibodies (Freelite®, The Binding Site; FLC ELISA κ and λ, Sebia; human κ and λ FLC, Diazyme Laboratories) and two on monoclonal antibodies (N Latex FLC, Siemens Healthineers; Seralite®, Sebia). Several studies have shown that these methods cannot be used interchangeably for the follow-up of patients because measured κ and λ FLC concentrations may differ significantly, especially at high levels. Because no international reference material for the measurement of FLC is available, it is not possible to establish which method is the most accurate. For this reason, knowledge about the analytical and diagnostic performances of the assays used is important. The aim of this review is to describe the main analytical features of the κ and λ FLC assays and how they may influence the clinical use of these parameters.

Sweat chloride assay by inductively coupled plasma mass spectrometry: a confirmation test for cystic fibrosis diagnosis.

The current guidelines for sweat chloride analysis identify the procedures for sweat collection, but not for chloride assay, which is usually performed by methods originally not aiming at the low concentrations of chloride found in sweat. To overcome this limitation, we set up, characterized, and adopted an original inductively coupled plasma mass spectrometry (ICP-MS) method for sweat chloride determination, which was designed for its easy use in a clinical laboratory. The method was linear in the range 8.5E-3 to 272.0E-3 mM, precision exhibited a relative standard deviation < 6%, and accuracy was in the range 99.7-103.8%. Limit of blank, limit of detection, and limit of quantitation were 2.1 mM, 3.2 mM, and 7.0 mM, respectively, which correspond to real concentrations injected into the mass spectrometer of 3.9E-3 mM for LOD and 8.5E-3 mM for LOQ. At first, the method was tested on 50 healthy volunteers who exhibited a mean chloride concentration of 15.7 mM (25-75th percentile 10.1-19.3 mM, range 2.8-37.4 mM); then, it was used to investigate two patients with suspected cystic fibrosis, who exhibited sweat chloride values of 65.6 mM and 81.2 mM, respectively. Moreover, the method was cross-validated by assaying 50 samples with chloride concentration values in the range 10-131 mM, by both ICP-MS and coulometric titration, which is the technology officially used in Tuscany for cystic fibrosis newborn screening. The reference analytical performances and the relatively low cost of ICP-MS, accompanied by the advantageous cost of a single sweat chloride assay, make this technology the best candidate to provide a top reference method for the quantification of chloride in sweat. The method that we propose was optimized and validated for sweat samples ≥ 75 mg, which is the minimum amount requested by the international protocols. However, the method sensitivity and, in addition, the possibility to reduce the sample dilution factor, make possible the quantification of chloride even in samples weighting < 75 mg that are discarded according to the current guidelines. Graphical abstract.

Pilot, Open, Randomized, Prospective Trial for Normothermic Machine Perfusion Evaluation in Liver Transplantation From Older Donors.

Ex situ normothermic machine perfusion (NMP) might minimize ischemia/reperfusion injury (IRI) of liver grafts. In this study, 20 primary liver transplantation recipients of older grafts (≥70 years) were randomized 1:1 to NMP or cold storage (CS) groups. The primary study endpoint was to evaluate graft and patient survival at 6 months posttransplantation. The secondary endpoint was to evaluate liver and bile duct biopsies; IRI by means of peak transaminases within 7 days after surgery; and incidence of biliary complications at month 6. Liver and bile duct biopsies were collected at bench surgery, end of ex situ NMP, and end of transplant surgery. Interleukin (IL) 6, IL10, and tumor necrosis factor α (TNF-α) perfusate concentrations were tested during NMP. All grafts were successfully transplanted. Median (interquartile range) posttransplant aspartate aminotransferase peak was 709 (371-1575) IU/L for NMP and 574 (377-1162) IU/L for CS (P = 0.597). There was 1 hepatic artery thrombosis in the NMP group and 1 death in the CS group. In NMP, we observed high TNF-α perfusate levels, and these were inversely correlated with lactate (P < 0.001). Electron microscopy showed decreased mitochondrial volume density and steatosis and an increased volume density of autophagic vacuoles at the end of transplantation in NMP versus CS patients (P < 0.001). Use of NMP with older liver grafts is associated with histological evidence of reduced IRI, although the clinical benefit remains to be demonstrated.

Different immunoreactivity of monomers and dimers makes automated free light chains assays not equivalent.

Background The automated immunochemical serum free light chains (FLC) assays, Freelite (a polyclonal antiserum) and N Latex FLC (a mixture of monoclonal antibodies), are not interchangeable, as they may provide different results on a same sample. This study was aimed to establish if the calibrators contain FLC oligomers, and if different reactivity against monomers and dimers contributes to the discrepancy. Methods Gel filtration chromatography fractions of the calibrators were subjected to a Western blot (WB) and analyzed by each reagent. The procedure was repeated after pretreating the N Latex FLC calibrator with the reducing agent dithiothreitol (DTT). Results Both calibrators contain FLC dimers and monomers. Both reagents detect (with different sensitivity) FLC kappa monomers and dimers; instead, Freelite detects only FLC lambda dimers, while N Latex FLC detects only FLC monomers. After DTT treatment, only the N Latex lambda still detects FLC with reduced protein thiols, while the reactivity of all other reagents is abolished. Conclusions Due to their different reactivity against FLC monomers and oligomers, the Freelite and N Latex FLC are calibrated against different components of their own calibrators, making the two reagents not equivalent. The redox status of FLC determines the immunoreactivity not only of FLC dimers, but also of the monomers.

Quantification of dehydroepiandrosterone in human serum on a routine basis: development and validation of a tandem mass spectrometry method based on a surrogate analyte.

In the clinical laboratories, dehydroepiandrostenedione (DHEA) is usually quantified by immunoassay-based methods, which are often affected by cross-reactivity with endogenous interferences, such as 4-androsten-3ß-ol-17-one. The interfering compounds lead to a poor accuracy of the measurements, mainly at a low concentration level. The present paper describes a validated method based on tandem mass spectrometry coupled to liquid chromatography, for the accurate quantification of DHEA in serum. The peculiarity of this method is the use of calibrators and quality controls prepared by adding measured amounts of DHEA-D5, a stable isotope-labeled analogue of DHEA, to real serum from healthy subjects. DHEA-D5 is used in place of DHEA, which is usually present in unstripped serum at physiological levels, as it has the same basic structure, provides an equivalent instrumental response, and can be easily distinguish by DHEA by mass spectrometry due to its different m/z value. The method proved to be sensitive, with a LLOD of 0.09 ng/mL and a LLOQ of 0.23 ng/mL, and selective, with overall performances that allow its use on a routine basis.

ß-trace protein is highly removed during haemodialysis with high-flux and super high-flux membranes.

BACKGROUND: Serum ß-trace protein (ßTP, MW 23-29 kDa) is a marker of GFR impairment in renal patients. Recent papers propose to predict residual renal function (RRF) in maintenance haemodialysis (MHD) patients from serum concentrations of ßTP and other small proteins, avoiding the collection of urine. Few data are available on the removal of ßTP in patients treated with dialysis membranes with different flux characteristics. The aim of this study was to evaluate the effects of haemodialysis with low-flux, high-flux and super high-flux membranes on serum concentrations of ßTP in MHD patients with null RRF. METHODS: Serum ßTP concentrations were measured before and after the first dialysis of the week in 51 MDH patients treated by low-flux (n = 24), high-flux (n = 17), or super high-flux (n = 10) membranes. The removal of ß2-microglobulin (ß2M, MW 11.8), cystatin C (Cys, MW 13.3), urea and creatinine was also analyzed. RESULTS: Low-flux membranes did not remove ßTP, ß2M and Cys whose concentration increased at the end of dialysis. High-flux membrane removed more efficiently ß2M and Cys than ßTP. Super high-flux membrane had the highest efficiency to remove ßTP: mean reduction ratio (RR) 53.4%, similar to ß2M (59.5%), and Cys (62.0%). CONCLUSIONS: In conclusion, the plasma clearance of small proteins and particularly of ßTP is dependent from the permeability of the dialysis membranes Therefore, the reliability of the formulas proposed to predict RRF from serum ßTP and other LMWP may be affected by the different permeability of the dialysis membranes.

Human Wharton's jelly-derived mesenchymal stromal cells engineered to secrete Epstein-Barr virus interleukin-10 show enhanced immunosuppressive properties.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) modulate the immune response and represent a potential treatment for inflammatory and autoimmune diseases. We hypothesized that this feature could be potentiated by co-administering anti-inflammatory cytokines. In this article, we asked whether engineering of Wharton Jelly-derived human MSCs (WJ-hMSCs) to express an anti-inflammatory cytokine increases cell immunomodulatory properties without altering their native features. METHODS: We used Epstein-Barr virus-derived interleukin-10 (vIL-10), which shares some immunosuppressive properties with human IL-10 but lacks immunostimulatory activity. Engineering was accomplished by transducing WJ-hMSCs with a self-inactivating feline immunodeficiency virus-derived vector co-expressing vIL-10 and herpes simplex virus type-1 thymidine kinase (TK). TK was added to allow future tracking of WJ-hMSC in vivo by positron electron tomography (PET). RESULTS: The results show that (i) expression of TK and/or vIL-10 does not change WJ-hMSC phenotypic and functional properties; (ii) vIL-10 is secreted, biologically active and enhances the immunosuppressing functions of WJ-hMSCs; (iii) v-IL10 and TK can be produced simultaneously by the same cells and do not interfere with each other. DISCUSSION: WJ-hMSCs engineered to secrete vIL-10 could be a powerful tool for adoptive cell therapy of immune-mediated diseases, and therefore, additional studies are warranted to confirm their efficacy in suitable animal disease models.

Effect of the three-dimensional organization of liver cells on the biogenesis of the γ-glutamyltransferase fraction pattern.

Context Four gamma-glutamyltransferase (GGT) fractions with different molecular weights (big-, medium-, small- and free-GGT) are detectable in human plasma. Objective Verify if liver cells can release all four GGT fractions and if the spatial cell organization influences their release. Methods Hepatoma (HepG2) and melanoma (Me665/2/60) cells were cultured as monolayers or spheroids. GGT released in culture media was analysed by gel-filtration chromatography. Results HepG2 and Me665/2/60 monolayers released the b-GGT fraction, while significative levels of s-GGT and f-GGT were detectable only in media of HepG2-spheroids. Bile acids alone or in combination with papain promoted the conversion of b-GGT in s-GGT or f-GGT, respectively. Conclusions GGT is usually released as b-GGT, while s-GGT and f-GGT are likely to be produced in the liver extracellular environment by the combined action of bile acids and proteases.

Non enzymatic upregulation of tissue factor expression by gamma-glutamyl transferase in human peripheral blood mononuclear cells.

BACKGROUND: Besides maintaining intracellular glutathione stores, gamma-glutamyltransferase(GGT) generates reactive oxygen species and activates NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the principal initiator of the clotting cascade. Thus, GGT might be involved in TF-mediated coagulation processes, an assumption untested insofar. METHODS: Experiments were run with either equine, enzymatically active GGT or human recombinant (hr) GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation. TF Procoagulant Activity (PCA, one-stage clotting assay), TF antigen(ELISA) and TFmRNA(real-time PCR) were assessed in unpooled human peripheral blood mononuclear cell(PBMC) suspensions obtained from healthy donors through discontinuous Ficoll/Hystopaque density gradient. RESULTS: Equine GGT increased PCA, an effect insensitive to GGT inhibition by acivicin suggesting mechanisms independent of its enzymatic activity, a possibility confirmed by the maintained stimulation in response to hrGGT, an enzymatically inactive molecule. Endotoxin(LPS) contamination of GGT preparations was excluded by heat inactivation studies and direct determination(LAL method) of LPS concentrations <0.1 ng/mL practically devoid of procoagulant effect. Inhibition by anti-GGT antibodies corroborated that conclusion. Upregulation by hrGGT of TF antigen and mRNA and its downregulation by BAY-11-7082, a NFkB inhibitor, and N-acetyl-L-cysteine, an antioxidant, was consistent with a NFkB-driven, redox-sensitive transcriptional site of action. CONCLUSIONS: GGT upregulates TF expression independent of its enzymatic activity, a cytokine-like behaviour mediated by NFκB activation, a mechanism contributing to promote acute thrombotic events, a possibility in need, however, of further evaluation.

Circulating gamma-glutamyltransferase fractions in cirrhosis.

BACKGROUND & AIMS: Four gamma-gultamyltransferases (GGT) fractions (b-, m-, s-, and f-GGT) have been identified in human plasma and their concentrations and ratios vary in different pathological conditions. To assess the behaviour of fractional GGT in cirrhotic patients evaluated for liver transplantation. METHODS: This was a single-centre, cross-sectional study; GGT fractions were determined by gel-filtration chromatography. RESULTS: 264 cirrhotic patients (215 males; median age 54.5 years) were included and compared against a group of 200 healthy individuals (100 males; median age 41.5). Median (25th-75th percentile) total and fractional GGT were higher in cirrhotics, with s-GGT showing the greatest increase [36.6 U/L (21.0-81.4) vs. 5.6 U/L (3.2-10.2), P<0.0001], while the median b-GGT/s-GGT ratio was lower in cirrhotics than in healthy controls [0.06 (0.04-0.10)] vs. 0.28 (0.20-0.40), P<0.0001]. The ratio showed higher diagnostic accuracy (ROC-AUC, 95% CI: 0.951, 0.927-0.969) then either s-GGT (0.924, 0.897-0.947; P<0.05) or total GGT (0.900, 0.869-0.925; P<0.001). The diagnostic accuracy of the ratio was maintained (0.940, 0.907-0.963) in cirrhotic patients (n=113) with total GGT values within the reference range. The s-GGT fraction consisted of two components, with one (s2-GGT) showing a significant positive correlation with serum aspartate aminotransferases, alanine aminotransferase, lactate dehydrogenases (LDH), alkaline phosphatases and bilirubin, and negative with albumin. The b-GGT fraction showed a positive correlation with albumin, fibrinogen, and platelet counts, and negative with international normalized ratio, bilirubin and LDH. CONCLUSIONS: The ratio performs as a sensitive biomarker of the liver parenchymal rearrangement, irrespective of aetiology of cirrhosis and presence of hepatocellular carcinoma, even in patients with total GGT values within the reference range.

Sequential Normothermic Regional Perfusion and End-ischemic Ex Situ Machine Perfusion Allow the Safe Use of Very Old DCD Donors in Liver Transplantation.

BACKGROUND: In Italy, 20 min of continuous, flat-line electrocardiogram are required for death declaration. Despite prolonged warm ischemia time, Italian centers reported good outcomes in controlled donation after circulatory death (cDCD) liver transplantation by combining normothermic regional and end-ischemic machine perfusion (MP). The aim of this study was to evaluate the safety and feasibility of the use of septuagenarian and octogenarian cDCD donors with this approach. METHODS: All cDCD older than 70 y were evaluated during normothermic regional perfusion and then randomly assigned to dual hypothermic or normothermic MP. RESULTS: In the period from April 2021 to December 2022, 17 cDCD older than 70 y were considered. In 6 cases (35%), the graft was not considered suitable for liver transplantation, whereas 11 (65%) were evaluated and eventually transplanted. The median donor age was 82 y, being 8 (73%) older than 80. Median functional warm ischemia and no-flow time were 36 and 28 min, respectively. Grafts were randomly assigned to ex situ dual hypothermic oxygenated MP in 6 cases (55%) and normothermic MP in 5 (45%). None was discarded during MP. There were no cases of primary nonfunction, 1 case of postreperfusion syndrome (9%) and 2 cases (18%) of early allograft dysfunction. At a median follow-up of 8 mo, no vascular complications or ischemic cholangiopathy were reported. No major differences were found in terms of postoperative hospitalization or complications based on the type of MP. CONCLUSIONS: The implementation of sequential normothermic regional and end-ischemic MP allows the safe use of very old donation after circulatory death donors.

Orientation of capture antibodies on gold nanoparticles to improve the sensitivity of ELISA-based medical devices.

The sensitivity of ELISA-based devices strongly depends on the right orientation of antibodies on the sensor surface. The aim of this work was to increase the analytical performance of a commercial ELISA-based medical device (VIDAS®), thanks to the specific orientation of antibodies on gold nanostructured disposables. For this purpose, fPSA VIDAS® assay was used as model and the disposable providing the antigen binding surface (SPR®) was functionalized with gold nanostructures coated with monovalent half-fragment antibodies (reduced IgG, rIgG). The functionalization of polystyrene SPRs® with gold nanostructures was achieved through a one-step incubation of gold dispersions in a mixture of non-toxic solvents. Five different concentrations of gold nanoparticles (NPs) were tested with a maximum fluorescence enhancement for NPs density around 3-8 *103 NPs/µm2 (752 ± 11 RFV vs 316 ± 5 RFV of bare SPRs®). The comparison of the dose-response curve obtained with commercial and gold coated-SPRs® revealed a significant improvement (p < 0.0001) of the analytical sensitivity of the VIDAS® system using nanostructured disposables. This improved version of SPRs® allows to distinguish small variations of fPSA concentrations opening the way to the application of this biomarker to other kinds of cancer as recently described in the literature.

Accuracy of b-GGT fraction for the diagnosis of non-alcoholic fatty liver disease.

BACKGROUND: Serum gamma-glutamyltransferase (GGT) activity is a sensitive but non-specific marker of non-alcoholic fatty liver disease (NAFLD). Recently, four GGT fractions (big-, medium-, small-, free-GGT) were described in humans. AIM: We aimed to investigate whether a specific GGT fraction pattern is associated with NAFLD. METHODS: Gamma-glutamyltransferase fractions were determined in patients with NAFLD (n = 90), and compared with those in control subjects (n = 70), and chronic hepatitis C (CHC, n = 45) age and gender matched. RESULTS: Total GGT was elevated in NAFLD as compared to controls (median, 25°-75° percentile: 39.4, 20.0-82.0 U/L vs. 18.4, 13.2-24.9 U/L respectively, P < 0.001). All fractions were higher in NAFLD than in controls (P < 0.001). The b-GGT showed the highest diagnostic accuracy for NAFLD diagnosis [area under ROC curve (ROC-AUC): 0.85; cut-off 2.6 U/L, sensitivity 74%, specificity 81%]. Also subjects with CHC showed increased GGT (41.5, 21.9-84.5 U/L, P < 0.001 vs. controls, P = n.s. vs. NAFLD), as well as m-, s-, and f-GGT, while b-GGT did not show any significant increase (P = n.s. vs. HS, P < 0.001 vs. NAFLD). In subjects with CHC, s-GGT showed the best diagnostic value (ROC-AUC: 0.853; cut-off 14.1 U/L, sensitivity 73%, specificity 90%). Serum GGT did not show any value in the differential diagnosis between NAFLD and CHC (ROC-AUC 0.507, P = n.s.), while b-GGT/s-GGT ratio showed the highest diagnostic accuracy for distinguishing NAFLD and CHC (ROC-AUC: 0.93; cut-off value 0.16, sensitivity 82%, specificity 90%). CONCLUSIONS: b-GGT increases in NAFLD, but not in CHC. GGT fraction analysis might help in improving the sensitivity and specificity of the diagnosis of NAFLD and other liver dysfunctions.

Developmental variations of plasma gamma-glutamyltransferase fractions in humans and in laboratory mammalians.

Plasma samples from human cord blood, and fetuses, newborns, and adults of different mammalians species were analyzed by gel-filtration chromatography, to ascertain whether gamma-glutamyltransferase (GGT) fractions reflect liver maturation. Human cord blood plasma showed higher b-, m-, and s-GGT fraction as compared to adult women. In rat and mouse fetuses and in newborns, b-GGT was the most abundant fraction. As in adult humans, in adult rats, mice, rabbits, sheep, and mini pigs, f-GGT was the most abundant fraction. GGT fractions are a common feature of all mammalian species tested. Their pattern changes seem to reflect liver postnatal maturation, function.

A method to obtain purified free light chain monomers and dimers from urine samples of patients with multiple myeloma.

Antibody light chains are synthesized in excess by plasma cells, and this excess can be secreted into biological fluids as dimers or monomers in various proportions. Structural differences between monomers or dimers of free light chains (FLC) can affect their biological functions and possibly their pathogenicity. They also may exhibit differential immune reactivity, perhaps explaining discrepant quantifications when measured by different immunoreagents. Having purified FLC monomers and dimers available can be useful for studying their properties. Here we propose a simple preparatory procedure to purify FLC monomers and dimers from urine samples of patients with plasma cell disorders. Two representative urine samples containing lambda or kappa FLC were loaded into a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel strips containing separate monomers and dimers were excised, electroeluted, and the FLC recovered. The FLC were recovered from SDS-PAGE gel in sufficient amounts to be quantified by UV and two automated nephelometric assays immunochemical. The procedure was found to be simple, reproducible, and with a high yield, thus offering the opportunity to compare different assays. Not all urine samples are suitable for this procedure, but this approach allows for the purification of FLC monomers and dimers from many selected urine samples which maintain their oligomeric organization.

Big and Free Fractions of Gamma-Glutamyltransferase: New Diagnostic Biomarkers for Malignant Mesothelioma?

Malignant pleural mesothelioma (MPM) is a cancer mainly caused by asbestos fiber inhalation, characterized by an extremely long latency and poor prognosis. Recently, researchers have focused on testing the diagnostic ability of several biomarkers. Gamma-Glutamyltransferase (GGT) has been demonstrated to be the sum of several GGT sub-fractions activity, classified based on their molecular weight in big-GGT, medium-GGT, small-GGT, and free-GGT. This work aims to evaluate whether specific GGT fractional enzymatic activity patterns could be helpful in MPM diagnosis. We analyzed blood samples from 175 workers previously exposed to asbestos, 157 non-exposed healthy subjects, and 37 MPM patients through a molecular exclusion chromatographic method. We found a specific profile of GGT fractions activity, significantly associated with MPM, resulting in an increase in b-, m- activity, along with an evident, yet not significant, decrease in f-activity. Receiver-operating characteristic (ROC) analysis showed that the best Area Under Curve (AUC) value resulted from the combined index b/f (0.679, 95% CI: 0.582-0.777). Combining the b-/f-GGT activity with the levels of serum mesothelin-related protein (SMRP; another promising MPM biomarker) improved the diagnostic accuracy, increasing the AUC value to 0.875 (95% CI: 0.807-0.943, p = <0.0001). Since MPM has a specific pattern of GGT enzymatic activity, we could hypothesize that GGT fractions play different specific biochemical roles. The improvement in the diagnostic power given by the combination of these two biomarkers confirms that the strategy of biomarkers combination might be a better approach for MPM diagnosis.