[D-dimer: how about it?] / D-dimeerbepaling: hoe zit het ook alweer?

This lab quiz presents some practical case studies on the correct use of D-dimer tests in patients suspected of venous thromboembolism. Elevated D-dimer levels are associated with clotting activation and fibrinolysis and can be used as indirect biomarkers of thrombosis. The D-dimer test is highly sensitive and is used to rule out the presence of venous thromboembolism (VTE) when clinical probability is low, based on Wells scores. However, sensitivity and specificity of cut-off values for D-dimer for ruling out VTE are strongly assay-dependent due to lack of standardisation. Because of low specificity, use of these cut-off values is problematic in cases of sepsis and inflammation, after recent surgery and in cases of trauma and active malignancy as well as during anticoagulant therapy and pregnancy. Agedependent cut-off values for patients > 50 years old might improve specificity and could be safely used if clinically validated assays (latex-agglutination assays) are used as described for the ADJUST-study.

Novel CSF biomarkers to discriminate FTLD and its pathological subtypes.

OBJECTIVE: Frontotemporal lobar degeneration (FTLD) is the second most prevalent dementia in young patients and is characterized by the presence of two main protein aggregates in the brain, tau (FTLD-Tau) or TDP43 (FTLD-TDP), which likely require distinct pharmacological therapy. However, specific diagnosis of FTLD and its subtypes remains challenging due to largely overlapping clinical phenotypes. Here, we aimed to assess the clinical performance of novel cerebrospinal fluid (CSF) biomarkers for discrimination of FTLD and its pathological subtypes. METHODS: YKL40, FABP4, MFG-E8, and the activities of catalase and specific lysosomal enzymes were analyzed in patients with FTLD-TDP (n = 30), FTLD-Tau (n = 20), AD (n = 30), DLB (n = 29), and nondemented controls (n = 29) obtained from two different centers. Models were validated in an independent CSF cohort (n = 188). RESULTS: YKL40 and catalase activity were increased in FTLD-TDP cases compared to controls. YKL40 levels were also higher in FTLD-TDP compared to FTLD-Tau. We identified biomarker models able to discriminate FTLD from nondemented controls (MFG-E8, tTau, and Aß 42; 78% sensitivity and 83% specificity) and non-FTLD dementia (YKL40, pTau, p/tTau ratio, and age; 90% sensitivity, 78% specificity), which were validated in an independent cohort. In addition, we identified a biomarker model differentiating FTLD-TDP from FTLD-Tau (YKL40, MFGE-8, ßHexA together with ßHexA/tHex and p/tTau ratios and age) with 80% sensitivity and 82% specificity. INTERPRETATION: This study identifies CSF protein signatures distinguishing FTLD and the two main pathological subtypes with optimal accuracy (specificity/sensitivity > 80%). Validation of these models may allow appropriate selection of cases for clinical trials targeting the accumulation of Tau or TDP43, thereby increasing their efficiency and facilitating the development of successful therapies.

Novel diagnostic cerebrospinal fluid biomarkers for pathologic subtypes of frontotemporal dementia identified by proteomics.

INTRODUCTION: Reliable cerebrospinal fluid (CSF) biomarkers enabling identification of frontotemporal dementia (FTD) and its pathologic subtypes are lacking. METHODS: Unbiased high-resolution mass spectrometry-based proteomics was applied on CSF of FTD patients with TAR DNA-binding protein 43 (TDP-43, FTD-TDP, n = 12) or tau pathology (FTD-tau, n = 8), and individuals with subjective memory complaints (SMC, n = 10). Validation was performed by applying enzyme-linked immunosorbent assay (ELISA) or enzymatic assays, when available, in a larger cohort (FTLD-TDP, n = 21, FTLD-tau, n = 10, SMC, n = 23) and in Alzheimer's disease (n = 20), dementia with Lewy bodies (DLB, n = 20), and vascular dementia (VaD, n = 18). RESULTS: Of 1914 identified CSF proteins, 56 proteins were differentially regulated (fold change >1.2, P < .05) between the different patient groups: either between the two pathologic subtypes (10 proteins), or between at least one of these FTD subtypes and SMC (47 proteins). We confirmed the differential expression of YKL-40 by ELISA in a partly independent cohort. Furthermore, enzyme activity of catalase was decreased in FTD subtypes compared with SMC. Further validation in a larger cohort showed that the level of YKL-40 was twofold increased in both FTD pathologic subtypes compared with SMC and that the levels in FTLD-tau were higher compared to Alzheimer's dementia (AD), DLB, and VaD patients. Clinical validation furthermore showed that the catalase enzyme activity was decreased in the FTD subtypes compared to SMC, AD and DLB. DISCUSSION: We identified promising CSF biomarkers for both FTD differential diagnosis and pathologic subtyping. YKL-40 and catalase enzyme activity should be validated further in similar pathology defined patient cohorts for their use for FTD diagnosis or treatment development.

Mini-dialysis tubes as tools to prepare drug-protein adducts of P450-dependent reactive drug metabolites.

The modification of critical cellular proteins by reactive metabolites (RMs) resulting from P450-dependent drug bioactivation is considered essential to the onset of many idiosyncratic drug reactions. In this study, we report a novel method that can be used to prepare and study drug-protein adducts. Drug bioactivation by P450s was performed in a small container containing a mini-dialysis tube with the model target protein human glutathione-S-transferase P1-1 (hGST P1-1), allowing RMs to translocate from P450 to hGST P1-1 via a semi-permeable membrane (6-8kDa). GST P1-1 modification was evaluated by LC-MS analysis of intact protein adducts and following digestion of protein with trypsin. As proof of principle, the described methodology was first applied to the direct electrophile monochlorobimane. A highly active P450 BM3 mutant (CYP102A1M11H) was subsequently used for bioactivation of acetaminophen, clozapine, diclofenac (DF) and mefenamic acid (MFA), but hGST P1-1 adducts were only observed for the latter two drugs. CYP2C9 and CYP3A4, which metabolize DF to p-benzoquinone imines, were tested to investigate the applicability of human P450s. Finally, it was evaluated whether bioactivation of MFA by human and rat liver microsomes resulted in modification of hGST P1-1. The results show that our adduct preparation method can also be used in combination with membrane-bound P450 bioactivation systems, as long as formed RMs have sufficient life-time to reach hGST P1-1 inside the dialysis tube.

Human NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated inactivation of reactive quinoneimine metabolites of diclofenac and mefenamic acid.

NAD(P)H: quinone oxidoreductase 1 (NQO1) is an enzyme capable of reducing a broad range of chemically reactive quinones and quinoneimines (QIs) and can be strongly upregulated by Nrf2/Keap1-mediated stress responses. Several commonly used drugs implicated in adverse drug reactions (ADRs) are known to form reactive QI metabolites upon bioactivation by P450, such as acetaminophen (APAP), diclofenac (DF), and mefenamic acid (MFA). In the present study, the reductive activity of human NQO1 toward the QI metabolites derived from APAP and hydroxy-metabolites of DF and MFA was studied, using purified bacterial P450 BM3 (CYP102A1) mutant M11 as a bioactivation system. The NQO1-catalyzed reduction of the QI metabolites was quantified relative to spontaneous glutathione (GSH) conjugation. Addition of NQO1 to the incubations strongly reduced the formation of all corresponding GSH conjugates, and this activity could be prevented by dicoumarol, a selective NQO1 inhibitor. The GSH conjugation was strongly increased by adding human GSTP1-1 in a wide range of GSH concentrations. Still, NQO1 could effectively compete with the GST catalyzed GSH conjugation by reducing the QIs. In conclusion, we identified the QI metabolites of the 4'- and 5-hydroxy-metabolites of DF and MFA as novel substrates for human NQO1. NQO1-mediated reduction proves to be an effective pathway to detoxify these QI metabolites in addition to GSH conjugation. Genetically determined deficiency of NQO1 therefore might be a risk factor for ADRs induced by reactive QI drug metabolites.