Serum dithiothreitol-oxidizing capacity (DOC) is a promising biomarker for excluding significant liver fibrosis: a proof-of-concept study.

BACKGROUND: APRI and FIB-4 scores are used to exclude clinically significant fibrosis (defined as stage ≥ F2) in patients with chronic viral hepatitis. However, the cut-offs for these scores (generated by Youden indices) vary between different patient cohorts. This study aimed to evaluate whether serum dithiothreitol-oxidizing capacity (DOC), i.e., a surrogate test of quiescin sulfhydryl oxidase-1, which is a matrix remodeling enzyme, could be used to non-invasively identify significant fibrosis in patients with various chronic liver diseases (CLDs). METHODS: Diagnostic performance of DOC was compared with APRI and FIB-4 for identifying significant fibrosis. ROC curve analyses were undertaken in: a) two chronic hepatitis B (CHB) cohorts, independently established from hospitals in Wenzhou (n = 208) and Hefei (n = 120); b) a MASLD cohort from Wenzhou hospital (n = 122); and c) a cohort with multiple CLD etiologies (except CHB and MASLD; n = 102), which was identified from patients in both hospitals. Cut-offs were calculated using the Youden index. All CLD patients (n = 552) were then stratified by age for ROC curve analyses and cut-off calculations. RESULTS: Stratified by CLD etiology or age, ROC curve analyses consistently showed that the DOC test was superior to APRI and FIB-4 for discriminating between clinically significant fibrosis and no fibrosis, when APRI and FIB-4 showed poor/modest diagnostic performance (P < 0.05, P < 0.01 and P < 0.001 in 3, 1 and 3 cohort comparisons, respectively). Conversely, the DOC test was equivalent to APRI and FIB-4 when all tests showed moderate/adequate diagnostic performances (P > 0.05 in 11 cohort comparisons). DOC had a significant advantage over APRI or FIB-4 scores for establishing a uniform cut-off independently of age and CLD etiology (coefficients of variation of DOC, APRI and FIB-4 cut-offs were 1.7%, 22.9% and 47.6% in cohorts stratified by CLD etiology, 2.0%, 26.7% and 29.5% in cohorts stratified by age, respectively). The uniform cut-off was 2.13, yielded from all patients examined. Surprisingly, the uniform cut-off was the same as the DOC upper limit of normal with a specificity of 99%, estimated from 275 healthy control individuals. Hence, the uniform cut-off should possess a high negative predictive value for excluding significant fibrosis in primary care settings. A high DOC cut-off with 97.5% specificity could be used for detecting significant fibrosis (≥ F2) with an acceptable positive predictive value (87.1%). CONCLUSIONS: This proof-of-concept study suggests that the DOC test may efficiently rule out and rule in significant liver fibrosis, thereby reducing the numbers of unnecessary liver biopsies. Moreover, the DOC test may be helpful for clinicians to exclude significant liver fibrosis in the general population.

Putative Biomarkers for Malignant Pleural Mesothelioma Suggested by Proteomic Analysis of Cell Secretome.

BACKGROUND: Malignant pleural mesothelioma (MPM) a rare neoplasm linked to asbestos exposure is characterized by a poor prognosis. Soluble mesothelin is currently considered the most specific diagnostic biomarker. The aim of the study was to identify novel biomarkers by proteomic analysis of two MPM cell lines secretome. MATERIALS AND METHODS: The protein patterns of MPM cells secretome were examined and compared to a non-malignant mesothelial cell line using two-dimensional gel electrophoresis coupled to mass spectrometry. Serum levels of candidate biomarkers were determined in MPM patients and control subjects. RESULTS: Two up-regulated proteins involved in cancer biology, prosaposin and quiescin Q6 sulfhydryl oxidase 1, were considered candidate biomarkers. Serum levels of both proteins were significantly higher in MPM patients than control subjects. Combining the data of each receiver-operating characteristic analysis predicted a good diagnostic accuracy. CONCLUSION: A panel of the putative biomarkers represents a promising tool for MPM diagnosis.

Hepatic Deficiency of Augmenter of Liver Regeneration Predisposes to Nonalcoholic Steatohepatitis and Fibrosis.

BACKGROUND AND AIMS: The augmenter of liver regeneration (ALR) protein is critical for lipid homeostasis and mitochondrial function. We investigated high-fat/high-carbohydrate (HF/HC) diet-induced nonalcoholic fatty liver disease (NAFLD) in wild-type (WT), hepatocyte-specific ALR-knockout (ALR-H-KO), and ALR-heterozygous (ALR-H-HET) mice. ALR was measured in serum of human nonalcoholic steatohepatitis (NASH) and NASH-induced cirrhosis (serum and liver). APPROACH AND RESULTS: HF/HC feeding decreased ALR expression in all groups of mice. The otherwise normal ALR-H-HET mice gained more weight and steatosis than WT mice when challenged metabolically with the HF/HC diet; ALR-H-KO mice gained the least weight and had the least steatosis. These findings were consistent with correspondingly increased triglycerides and cholesterol and altered expression of carnitine palmitoyltransferase 1a, sterol regulatory element-binding protein, acetyl coenzyme A carboxylase, and fatty acid synthase. All HF/HC-fed mice developed insulin resistance, the magnitude being lower in ALR-H-KO mice. HF/HC-fed ALR-H-HET mice were more resistant to glucose challenge than WT or ALR-H-KO mice. The frequency of tumor necrosis factor alpha-producing, interleukin 6 (IL6)-producing, and IL17-producing cells was greater in ALR-H-KO than ALR-H-HET and lowest in WT mice. HF/HC feeding did not increase their number in ALR-H-KO mice, and the increase in ALR-H-HET was greater than that in WT mice except for IL17 cells. Cluster of differentiation 25-positive (CD25+ ) forkhead box P3-positive CD4+ regulatory T-cell frequency was lower in ALR-H-HET than WT mice and further reduced in ALR-H-KO mice; HF/HC reduced regulatory T-cell frequency only in WT mice. HF/HC-fed ALR-H-HET, but not WT, mice developed fibrosis; and ALR-H-KO mice progressed to cirrhosis. White adipose tissue of HF/HC-fed ALR-deficient mice developed strong inflammation, indicating bidirectional interactions with the liver. Hepatic and serum ALR levels were significantly reduced in patients with NASH-cirrhosis. Serum ALR was also significantly lower in patients with NASH. CONCLUSIONS: Hepatic ALR deficiency may be a critical predisposing factor for aggressive NAFLD progression.

Peripheral Blood RNA Levels of QSOX1 and PLBD1 Are New Independent Predictors of Left Ventricular Dysfunction After Acute Myocardial Infarction.

BACKGROUND: The identification of patients with acute myocardial infarction (MI) at risk of subsequent left ventricular (LV) dysfunction remains challenging, but it is important to optimize therapies. The aim of this study was to determine the unbiased RNA profile in peripheral blood of patients with acute MI and to identify and validate new prognostic markers of LV dysfunction. METHODS: We prospectively enrolled a discovery cohort with acute MI (n=143) and performed whole-blood RNA profiling at different time points. We then selected transcripts on admission that related to LV dysfunction at follow-up and validated them by quantitative polymerase chain reaction in the discovery cohort, in an external validation cohort (n=449), and in a representative porcine MI model with cardiac magnetic resonance-based measurements of infarct size and postmortem myocardial pathology (n=33). RESULTS: RNA profiling in the discovery cohort showed upregulation of genes involved in chemotaxis, IL (interleukin)-6, and NF-κB (nuclear factor-κB) signaling in the acute phase of MI. Expression levels of the majority of these transcripts paralleled the rise in cardiac troponin T and decayed at 30 days. RNA levels of QSOX1, PLBD1, and S100A8 on admission with MI correlated with LV dysfunction at follow-up. Using quantitative polymerase chain reaction, we confirmed that QSOX1 and PLBD1 predicted LV dysfunction (odds ratio, 2.6 [95% CI, 1.1-6.1] and 3.2 [95% CI, 1.4-7.4]), whereas S100A8 did not. In the external validation cohort, we confirmed QSOX1 and PLBD1 as new independent markers of LV dysfunction (odds ratio, 1.41 [95% CI, 1.06-1.88] and 1.43 [95% CI, 1.08-1.89]). QSOX1 had an incremental predictive value in a model consisting of clinical variables and cardiac biomarkers (including NT-proBNP [N-terminal pro-B-type natriuretic peptide]). In the porcine MI model, whole-blood levels of QSOX1 and PLBD1 related to neutrophil infiltration in the ischemic myocardium in an infarct size-independent manner. CONCLUSIONS: Peripheral blood QSOX1 and PLBD1 in acute MI are new independent markers of LV dysfunction post-MI.

Quiescin Sulfhydryl Oxidase 1 (QSOX1) Secreted by Lung Cancer Cells Promotes Cancer Metastasis.

As lung cancer shows the highest mortality in cancer-related death, serum biomarkers are demanded for lung cancer diagnosis and its treatment. To discover lung cancer protein biomarkers, secreted proteins from primary cultured lung cancer and adjacent normal tissues from patients were subjected to LC/MS⁻MS proteomic analysis. Quiescin sulfhydryl oxidase (QSOX1) was selected as a biomarker candidate from the enriched proteins in the secretion of lung cancer cells. QSOX1 levels were higher in 82% (51 of 62 tissues) of lung cancer tissues compared to adjacent normal tissues. Importantly, QSOX1 serum levels were significantly higher in cancer patients (p < 0.05, Area Under curve (AUC) = 0.89) when measured by multiple reaction monitoring (MRM). Higher levels of QSOX1 were also uniquely detected in lung cancer tissues, among several other solid cancers, by immunohistochemistry. QSOX1-knock-downed Lewis lung cancer (LLC) cells were less viable from oxidative stress and reduced migration and invasion. In addition, LLC mouse models with QSOX1 knock-down also proved that QSOX1 functions in promoting cancer metastasis. In conclusion, QSOX1 might be a lung cancer tissue-derived biomarker and be involved in the promotion of lung cancers, and thus can be a therapeutic target for lung cancers.

Serum thioredoxin reductase is highly increased in mice with hepatocellular carcinoma and its activity is restrained by several mechanisms.

Increased thioredoxin reductase (TrxR) levels in serum were recently identified as possible prognostic markers for human prostate cancer or hepatocellular carcinoma. We had earlier shown that serum levels of TrxR protein are very low in healthy mice, but can in close correlation to alanine aminotransferase (ALT) increase more than 200-fold upon chemically induced liver damage. We also found that enzymatic TrxR activity in serum is counteracted by a yet unidentified oxidase activity in serum. In the present study we found that mice carrying H22 hepatocellular carcinoma tumors present highly increased levels of TrxR in serum, similarly to that reported in human patients. In this case ALT levels did not parallel those of TrxR. We also discovered here that the TrxR-antagonistic oxidase activity in serum is due to the presence of quiescin Q6 sulfhydryl oxidase 1 (QSOX1). We furthermore found that the chemotherapeutic agents cisplatin or auranofin, when given systemically to H22 tumor bearing mice, can further inhibit TrxR activities in serum. The TrxR serum activity was also inhibited by endogenous electrophilic inhibitors, found to increase in tumor-bearing mice and to include protoporphyrin IX (PpIX) and 4-hydroxynonenal (HNE). Thus, hepatocellular carcinoma triggers high levels of serum TrxR that are not paralleled by ALT, and TrxR enzyme activity in serum is counteracted by several different mechanisms. The physiological role of TrxR in serum, if any, as well as its potential value as a prognostic marker for tumor progression, needs to be studied further.

Disulfide bond generation in mammalian blood serum: detection and purification of quiescin-sulfhydryl oxidase.

A sensitive new plate-reader assay has been developed showing that adult mammalian blood serum contains circulating soluble sulfhydryl oxidase activity that can introduce disulfide bonds into reduced proteins with the reduction of oxygen to hydrogen peroxide. The activity was purified 5000-fold to >90% homogeneity from bovine serum and found by mass spectrometry to be consistent with the short isoform of quiescin-sulfhydryl oxidase 1 (QSOX1). This FAD-dependent enzyme is present at comparable activity levels in fetal and adult commercial bovine sera. Thus cell culture media that are routinely supplemented with either fetal or adult bovine sera will contain this facile catalyst of protein thiol oxidation. QSOX1 is present at approximately 25 nM in pooled normal adult human serum. Examination of the unusual kinetics of QSOX1 toward cysteine and glutathione at low micromolar concentrations suggests that circulating QSOX1 is unlikely to significantly contribute to the oxidation of these monothiols in plasma. However, the ability of QSOX1 to rapidly oxidize conformationally mobile protein thiols suggests a possible contribution to the redox status of exofacial and soluble proteins in blood plasma. Recent proteomic studies showing that plasma QSOX1 can be utilized in the diagnosis of pancreatic cancer and acute decompensated heart failure, together with the overexpression of this secreted enzyme in a number of solid tumors, suggest that the robust QSOX assay developed here may be useful in the quantitation of enzyme levels in a wide range of biological fluids.

Unbiased plasma proteomics for novel diagnostic biomarkers in cardiovascular disease: identification of quiescin Q6 as a candidate biomarker of acutely decompensated heart failure.

AIMS: Biochemical marker testing has improved the evaluation and management of patients with cardiovascular diseases over the past decade. Natriuretic peptides (NPs), used in clinical practice to assess cardiac dysfunction, exhibit many limitations, however. We used an unbiased proteomics approach for the discovery of novel diagnostic plasma biomarkers of heart failure (HF). METHODS AND RESULTS: A proteomics pipeline adapted for very low-abundant plasma proteins was applied to clinical samples from patients admitted with acute decompensated HF (ADHF). Quiescin Q6 (QSOX1), a protein involved in the formation of disulfide bridges, emerged as the best performing marker for ADHF (with an area under the receiver operator characteristic curve of 0.86, 95% confidence interval: 0.79-0.92), and novel isoforms of NPs were also identified. Diagnostic performance of QSOX1 for ADHF was confirmed in 267 prospectively collected subjects of whom 76 had ADHF. Combining QSOX1 to B-type NP (BNP) significantly improved diagnostic accuracy for ADHF by particularly improving specificity. Using thoracic aortic constriction in rats, QSOX1 was specifically induced within both left atria and ventricles at the time of HF onset. CONCLUSION: The novel biomarker QSOX1 accurately identifies ADHF, particularly when combined with BNP. Through both clinical and experimental studies we provide lines of evidence for a link between ADHF and cardiovascular production of QSOX1.

Alrp, a survival factor that controls the apoptotic process of regenerating liver after partial hepatectomy in rats.

Augmenter of Liver Regeneration (Alrp) enhances, through unknown mechanism/s, hepatocyte proliferation only when administered to partially hepatectomized (PH) rats. Liver resection, besides stimulating hepatocyte proliferation, induces reactive oxygen species (ROS), triggering apoptosis. To clarify the role of Alrp in the process of liver regeneration, hepatocyte proliferation, apoptosis, ROS-induced parameters and morphological findings of regenerating liver were studied from PH rats Alrp-treated for 72 h after the surgery. The same parameters, evaluated on regenerating liver from albumin-treated PH rats, were used as control. The results demonstrated that Alrp administration induces the anti-apoptotic gene expression, inhibits hepatocyte apoptosis and reduces ROS-induced cell damage. These and similar data from in vitro studies and the presence of 'Alrp homologous proteins' in viruses as well as in mammals (i) allow to hypothesize that Alrp activity/ies may not be exclusive for regenerating liver and (ii) suggest the use of Alrp in the treatment of oxidative stress-related diseases.

Innate immune recognition triggers secretion of lysosomal enzymes by macrophages.

Gamma interferon-induced lysosomal thiolreductase (GILT) is expressed constitutively in antigen-presenting cells, where it reduces disulfide bonds to facilitate antigen presentation. GILT is synthesized as an enzymatically active precursor protein and is processed in early endosomes to yield the mature enzyme. The exposure of the promonocytic cell line THP-1 to Escherichia coli causes a differentiation-dependent induction of GILT expression in which the majority of precursor GILT is secreted as active enzyme. We confirm this result in cultured primary monocytes and macrophages, and demonstrate, as an in vivo correlate of the phenomenon, upregulation of precursor GILT levels in the serum of mice injected with lipopolysaccharide. We show that macrophage differentiation is accompanied by a transcriptional downregulation of mannose-6-phosphorylation, which likely prevents the recognition and proper sorting of soluble lysosomal enzymes by the mannose-6-phosphate receptors. We provide evidence for a mechanism of generalized soluble lysosomal enzyme secretion through the constitutive secretory pathway.

New approaches towards laboratory diagnosis of isolated sulphite oxidase deficiency.

BACKGROUND: Molybdenum cofactor deficiency (resulting in combined deficiencies of the enzymes sulphite oxidase, xanthine dehydrogenase and aldehyde dehydrogenase) and isolated sulphite oxidase deficiency are inherited metabolic diseases which follow an autosomal recessive trait of inheritance. Detection of these diseases in selective screening for inborn errors of metabolism is not easy because relevant metabolites are either not routinely determined or are unstable. METHODS: We have searched for additional markers for these diseases and studied plasma total homocysteine (determined by enzyme immunoassay) and S-sulphonation of transthyretin (assessed by electrospray ionization mass spectrometry). RESULTS AND CONCLUSION: We found total homocysteine concentrations below the limit of quantification (<1 micromol/L) in all samples of patients with sulphite oxidase deficiency studied in this regard and that the proportion of S-sulphonated transthyretin is clearly increased in such samples. Our observations suggest additional tools for selective screening and diagnostic work-up of patients suspected of having sulphite oxidase deficiency.

Alternative pathways of sulfite oxidation in human polymorphonuclear leukocytes.

Sodium sulfite is metabolized by human polymorphonuclear leukocytes by two alternative pathways, one enzymatic route dependent on sulfite oxidase and one non-enzymatic which involves intermediate formation of sulfur trioxide anion radicals. Initiation of the oxidative burst by phorbol myristate acetate significantly stimulates sulfate formation through the second pathway. The activity of sulfite oxidase in polymorphonuclear leukocytes varies greatly among individuals, a variation consistent with the suggested polymorphic distribution of sulfite oxidase in the human population.

M4 and M9 antibodies in the overlap syndrome of primary biliary cirrhosis and chronic active hepatitis: epitopes or epiphenomena?

Before the identification of the major mitochondrial antigens of primary biliary cirrhosis as components of the 2-oxo-acid dehydrogenase enzyme family, mitochondrial autoantigens were believed to be extremely heterogeneous and were divided into nine subtypes termed M1 to M9. This classification was based on the data derived from the relatively nonspecific biochemical and immunological techniques that were available. After the cloning and definition of the major autoantigens, more than 95% of the sera of patients with primary biliary cirrhosis were found to react with components of the 2-oxo-dehydrogenase enzymes; these enzymes correspond to the old M2 classification. Two other "M" species, dubbed M4 and M9, have attracted significant attention because they have been postulated to be prognostic indicators and more recently have been tentatively identified respectively as sulfite oxidase (EC 1.8.3.1) and glycogen phosphorylase (EC 2.4.1.1). Indeed, patients with the "overlap syndrome" are reported to have antibodies to M4 and a poor prognosis, whereas patients with antibodies to M9 have a favorable prognosis. To address the significance and definition of M4 and M9, we performed in-depth studies of sera from 11 patients with the overlap syndrome, 75 patients with primary biliary cirrhosis, 19 chronic active hepatitis patients, 13 patients with primary sclerosing cholangitis, 10 patients with cholangiocarcinoma, 20 patients with systemic lupus erythematosus, 20 patients with alcoholic cirrhosis, 17 patients with scleroderma and 30 normal individuals, using techniques of ELISA, complement fixation, immunoblotting and enzyme inhibition. We report herein that we were unable to show any disease-specific reactivity toward the proposed M4 and M9 antigens.(ABSTRACT TRUNCATED AT 250 WORDS)