Bioaffinity Fishing Procedure Using Secretory Phospholipase A2 for Screening for Bioactive Components: Modulation of Pharmacological Effect Induced by sPLA2 from Crotalus durissus terrificus by Hispidulin from Moquiniastrum floribundum

Bioaffinity capturing of molecules allows the discovery of bioactive compounds and decreases the need for various stages in the natural compound isolation process. Despite the high selectivity of this technique, the screening and identification methodology depends on the presence of a protein to capture potential ligands. However, some proteins, such as snake secretory phospholipase A2 (sPLA2), have never been investigated using this approach. The purpose of this study was to evaluate the use of a new method for screening natural compounds using a bioaffinity-guided ultrafiltration method on Crotalus durissus terrificus sPLA2 followed by HPLC-MS to identify the compounds, and this method could be used to discover new anti-inflammatory compounds from the various organisms originating from biodiversity. Different extracts were selected to evaluate their ability to inhibit sPLA2 activity. The extracts were incubated with sPLA2 and the resulting mixture was ultrafiltrated to elute unbound components. The resulting compounds were identified by HPLC-MS. We identified hispidulin as one of the components present in the Moquiniastrum floribundum leaf and evaluated the ability of this isolated compound to neutralize the inflammatory activity of sPLA2 from Crotalus durissus terrificus.

Effect of cooling on lung secretory phospholipase A2 activity in vitro, ex vivo, and in vivo

Hypothermia can modify surfactant composition and function. Secretory phospholipase A2 (sPLA2) hydrolyses surfactant phospholipids and is important in the pathobiology of several critical respiratory disorders. We hypothesize that sPLA2 activity might be influenced by the temperature partially explaining surfactant changes. This study aims to evaluate comprehensively the effect of hypothermia on sPLA2 activity. We measured sPLA2 activity at different temperatures, alone or combined with bile acids, in vitro (incubating human recombinant sPLA2-IIA and porcine sPLA2-IB), ex vivo (by cooling bronchoalveolar lavage samples from neonates with respiratory distress syndrome or no lung disease), and in vivo (using lavage samples obtained before and after 72 h of whole body cooling in neonates with hypoxic-ischemic encephalopathy). We also measured concentrations of various sPLA2 subtypes and natural sPLA2 inhibitors in in vivo cooled samples. Results were corrected for protein content and dilution. In vitro cooling did not show any effect of hypothermia on sPLA2. Ex vivo cooling did not alter total sPLA2 activity, and the addition of bile acids increased sPLA2 activity irrespective of the temperature and the type of sampled patient. In vivo hypothermia reduced median sPLA2 activity from 16.6 [15.2–106.7] IU/mg to 3.3 [2.7–8.5] IU/mg ( P = 0.026) and mean sPLA2-IIA from 1.1 (0.8) pg/μg to 0.6 (0.4) pg/μg ( P = 0.047), whereas dioleylphosphatidylglycerol increased from 8.3 (3.9)% to 12.8 (5.1)% ( P = 0.02). Whole body hypothermia decreases in vivo global sPLA2 activity in bronchoalveolar lavage fluids through the reduction of sPLA2-IIA and increment of dioleylphosphatidylglycerol. This effect is absent during in vitro or ex vivo hypothermia.

AKTIVITAS FOSFOLIPASE-A2 SEKRETORIS PLASMA TROMBOSITOPENIA DEMAM BERDARAH DENGUE

Infected macrophages by dengue virus will produce phospholipase-A2 (PLA2) enzyme, that can promote arachidonic acidmetabolism that produce inflammatory mediators, causing endhothelial damage and severe plasma leakage. Capillary endothelialdamage can cause platelet adhesion and aggregation, so that many platelets will be consumed. The role of sPLA2 (secretoryphospholipase-A2), which is a part of PLA2 in dengue and thrombocytopenia up to now has not been widely studied. The objective ofthe study is to analyze the association between the activity of plasma secretory phospholipase-A2 and the degree of thrombocytopeniain DHF adult patients. the study is carried out by a cross sectional, observational analytical study on 45 hospitalized adult patientssuffering dengue hemorrhagic fever in the Tropical Infection Ward, Department of Internal Medicine, Dr. Soetomo Hospital Surabaya,which has been conducted from February–December 2009. The diagnosis of Dengue Haemorrhagic Fever (DHF) was based on the1997 World Health Organization (WHO) criteria, that minimally had one positive serology marker of dengue. Venous blood wastaken from the patient for examining the activity of secretory phospholipase-A2 by correlated enzyme assay method, and plateletcount using automated hematology analyzer. The results of the secretory phospholipase-A2 activity and degree of thrombocytopeniawere analyzed by Pearson correlation test to determine the correlation between the two variables. In this study so far was foundthat the secretory phospholipase-A2 activity in DHF patients was 36.9–195.6 unit/mL (mean 97.49 unit/mL, SD 30.06 unit/mL).The mean of secretory phospholipase-A2 activity was increased according to the degree of thrombocytopenia severity. The mean ofsecretory phospholipase-A2 activities were 91.65 unit/mL, 98.94 unit/mL, and 110.47 unit/mL. The degree of thrombocytopeniawas divided into mild, moderate, and severe. Most of the patients showed mild thrombocytopenia. The sPLA2 activity in this studywas increased in DHF patients with second day of fever, and then decreased at the third and forth day of fever, and increased inDBD patients suffering fifth day of fever. The statistical analyzes show a non significant correlation between secretory phospholipaseA2 activity and degree of thrombocytopenia (p = 0.579). This result may be caused by several factors which influencing thethrombocytopenia in DHF, such as bone marrow suppression, dengue viral serotype, influence of cytosolic phospholipase-A2 (cPLA2)activity, and other proinflammatory cytokines which in this study could not be controlled. Statistical analyzes show a significantcorrelation between sPLA2 activity and the day of fever (p = 0.04). Further studies should have to be carried out in order to knowthe pattern of sPLA2 activity in DHF grade I, II, III, and IV, and to know the influence of other proinflammatory cytokines and viralserotypes in sPLA2 activities.

ANTI-INFLAMMATORY ACTIVITY OF BACTERIA ASSOCIATED WITH MARINE SPONGE (HALICLONA AMBOINENSIS) VIA REDUCTING NO PRODUCTION AND INHIBITING CYCLOOXYGENASE-1, CYCLOOXYGENASE-2, AND SECRETORY PHOSPHOLIPASE A2 ACTIVITIES

Objective: This study aimed to investigate the anti-inflammatory activity of methanol extract and fractions of bacteria associated with sponge (Haliclona amboinensis) and to evaluate their effect in reducing NO production and inhibiting cyclooxygenase-1 (COX-1), cyclooxgenase-2 (COX-2) and secretory phospholipase A2 (sPLA2) activity.Methods: All bacterial isolates were cultured and supernatants were collected for the extraction of secondary metabolites using diaion HP-20 to obtain methanol extracts. Evaluation of cytotoxicity property was carried out on macrophage cell lines (RAW264.7) by 3-(4,5-dimethylthiazol- 2-yl) 2,5-diphenyl tetrazoliumbromide assay. Anti-inflammatory screening was done by inducible nitric oxide assay on RAW264.7 cell lines with lipopolysaccharide (LPS) stimulation. Dianion HP-20 was used to remove salt content. A selected methanol extract was subjected to further fractionations by C-18 reverse phase and their anti-inflammatory potential was evaluated by COX-1 and COX-2, and sPLA2 enzymatic assay.Results: Seven methanol extracts showed no cytotoxic property against RAW 264.7 cell line (inhibitory concentration 50% > 30 μg/ml) and selected for anti-inflammatory screening assay. Result showed methanol extract HM 1.2 reduced NO production >80% and it has been selected for phytochemical screening, further fractionations and assay. Phytochemical screening showed alkaloids and terpenoids present in the HM 1.2. The HM 1.2 and its fractions (F1, F2, F1C1, F1C2, F1C3, and F1C4) were proven to inhibit COX-1, COX-2, and sPLA2 activity in the range of 60.516-116.886%, 20.554- 116.457%, and 70.2667-114.8148%, respectively.Conclusions: This study revealed that bacteria associated with H. amboinensis have produced anti-inflammatory activity via reducing NO production and inhibiting COX-1, COX-2, and sPLA2 activity.

Prognostic Utility of Secretory Phospholipase A2 in Patients with Stable Coronary Artery Disease

BACKGROUND Secretory phospholipase A2 (sPLA2) may contribute to atherogenesis. To date, few prospective studies have examined the utility of sPLA2 for risk stratification in coronary artery disease (CAD). METHODS We measured plasma sPLA2 activity at baseline in 3708 subjects in the PEACE randomized trial of trandolapril vs placebo in stable CAD. Median follow-up was 4.8 years. We used Cox regression to adjust for demographics, clinical risk factors, apolipoprotein B, apolipoprotein A1, and medications. RESULTS After multivariable adjustment, sPLA2 was associated with an increased risk of cardiovascular death, myocardial infarction, or stroke (adjusted hazard ratio Q4:Q1 1.55, 95% CI 1.13–2.14) and cardiovascular death or heart failure (1.91, 1.20–3.03). In further multivariable assessment, increased activity levels of sPLA2 were associated with the risk of cardiovascular death, myocardial infarction, or stroke (adjusted hazard ratio 1.47, 95% CI 1.06–2.04), independent of lipoprotein-associated phospholipase A2 mass and C-reactive protein, and modestly improved the area under the curve (AUC) beyond established clinical risk factors (AUC 0.668–0.675, P = 0.01). sPLA2, N-terminal pro-B-type natriuretic peptide, and high-sensitivity cardiac troponin T all were independently associated with cardiovascular death or heart failure, and each improved risk discrimination (P = 0.02, P < 0.001, P < 0.001, respectively). CONCLUSIONS sPLA2 activity provides independent prognostic information beyond established risk markers in patients with stable CAD. These data are encouraging for studies designed to evaluate the role of sPLA2 as a therapeutic target.

Cerebrospinal Fluid Secretory Ca2+-Dependent Phospholipase A2 Activity Is Increased in Alzheimer Disease

Background: The phospholipase A2 (PLA2) family comprises multiple isoenzymes that vary in their physicochemical properties, cellular localizations, calcium sensitivities, and substrate specificities. Despite these differences, PLA2s share the ability to catalyze the synthesis of the precursors of the proinflammatory mediators. To investigate the potential of PLA2 as a biomarker in screening neuroinflammatory disorders in both clinical and research settings, we developed a PLA2 assay and determined the predominant types of PLA2 activity in cerebrospinal fluid (CSF). Methods: We used liposomes composed of a fluorescent probe (bis-Bodipy® FL C11-PC [1,2-bis-(4,4- difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine]) and 1,2-dioleoyl-l-α-phosphatidylcholine as a substrate to measure CSF PLA2 activity in a 96-well microtiter plate format. We established the type of CSF PLA2 activity using type-specific inhibitors of PLA2. Results: Using 5 μL CSF per assay, our PLA2 activity assay was reproducible with CVs <15% in 2 CSF samples and for recombinant secretory Ca2+-dependent PLA2 (sPLA2) in concentrations ranging from 0.25 to 1 μmol/L. This PLA2 assay allowed identification of sPLA2 activity in lumbar CSF from healthy individuals 20–77 years old that did not depend on either sex or age. Additionally, CSF sPLA2 activity was found to be increased (P = 0.0008) in patients with Alzheimer disease. Conclusions: Adult human CSF has sPLA2 activity that can be measured reliably with the assay described. This enzyme activity in the CSF is independent of both sex and age and might serve as a valuable biomarker of neuroinflammation, as we demonstrated in Alzheimer disease.