A continuous fluorescence assay for the determination of calcium-dependent secretory phospholipase A2 activity in serum
Calcium-dependent secretory phospholipase A(2)-IIA (sPLA(2)-IIA) in the circulation is a marker of inflammation, associated with acute and chronic disease processes. We describe a quick, sensitive and reliable microplate continuous fluorescence assay for determining sPLA(2) activity in serum. Liposo...
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| Veröffentlicht in: | Clinica chimica acta 2007-04, Vol.379 (1-2), p.119-126 |
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| container_title | Clinica chimica acta |
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| creator | Tsao, Francis H C Shanmuganayagam, Dhanansayan Zachman, Derek K Khosravi, Mehdi Folts, John D Meyer, Keith C |
| description | Calcium-dependent secretory phospholipase A(2)-IIA (sPLA(2)-IIA) in the circulation is a marker of inflammation, associated with acute and chronic disease processes. We describe a quick, sensitive and reliable microplate continuous fluorescence assay for determining sPLA(2) activity in serum.
Liposomes composed of a fluorescent probe and varying amounts of L-alpha-phosphatidylglycerol (PG) and 1,2-dioleoyl-L-alpha-phosphatidylcholine (DOPC) were used as substrates to determine the optimal protocol for sPLA(2) activity determination without interference from serum albumin and lipoproteins.
Hydrolysis of the labeled substrate by sPLA(2)-IIA, characterized by increase in fluorescence intensity (FI) and confirmed by end-product analysis, occurred in a time-, calcium-, and protein-dependent manner. Liposomes containing 100% PG were most suitable for measurement of sPLA(2) activity without interference from serum components; LDL produced a Ca(2+)-independent increase in FI when liposomes containing DOPC were used. The assay determined that sPLA(2) activity in serum spiked with sPLA(2)-IIA and illustrated that endogenous sPLA(2) activity was markedly higher in sera from patients with sepsis than in healthy subjects. Intra-assay and inter-assay CVs were in the ranges of 1.6-8.8% and 3.0-11.5%, respectively.
The described method has potential for rapid and sensitive screening of sPLA(2) activity in both clinical and research settings. |
| doi_str_mv | 10.1016/j.cca.2006.12.023 |
| format | Article |
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Liposomes composed of a fluorescent probe and varying amounts of L-alpha-phosphatidylglycerol (PG) and 1,2-dioleoyl-L-alpha-phosphatidylcholine (DOPC) were used as substrates to determine the optimal protocol for sPLA(2) activity determination without interference from serum albumin and lipoproteins.
Hydrolysis of the labeled substrate by sPLA(2)-IIA, characterized by increase in fluorescence intensity (FI) and confirmed by end-product analysis, occurred in a time-, calcium-, and protein-dependent manner. Liposomes containing 100% PG were most suitable for measurement of sPLA(2) activity without interference from serum components; LDL produced a Ca(2+)-independent increase in FI when liposomes containing DOPC were used. The assay determined that sPLA(2) activity in serum spiked with sPLA(2)-IIA and illustrated that endogenous sPLA(2) activity was markedly higher in sera from patients with sepsis than in healthy subjects. Intra-assay and inter-assay CVs were in the ranges of 1.6-8.8% and 3.0-11.5%, respectively.
The described method has potential for rapid and sensitive screening of sPLA(2) activity in both clinical and research settings.</description><identifier>ISSN: 0009-8981</identifier><identifier>DOI: 10.1016/j.cca.2006.12.023</identifier><identifier>PMID: 17292873</identifier><language>eng</language><publisher>Netherlands</publisher><subject>Cholesterol, LDL - blood ; Group II Phospholipases A2 ; Humans ; Phospholipases A - blood ; Phospholipases A2 ; Sensitivity and Specificity ; Spectrometry, Fluorescence - methods ; Spectrometry, Fluorescence - standards ; Substrate Specificity</subject><ispartof>Clinica chimica acta, 2007-04, Vol.379 (1-2), p.119-126</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c214t-1f15cdc3c96e67ab4fbc6c01c912e5a86941d380ce759981f5d352ce54081f4c3</citedby><cites>FETCH-LOGICAL-c214t-1f15cdc3c96e67ab4fbc6c01c912e5a86941d380ce759981f5d352ce54081f4c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27923,27924</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17292873$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Tsao, Francis H C</creatorcontrib><creatorcontrib>Shanmuganayagam, Dhanansayan</creatorcontrib><creatorcontrib>Zachman, Derek K</creatorcontrib><creatorcontrib>Khosravi, Mehdi</creatorcontrib><creatorcontrib>Folts, John D</creatorcontrib><creatorcontrib>Meyer, Keith C</creatorcontrib><title>A continuous fluorescence assay for the determination of calcium-dependent secretory phospholipase A2 activity in serum</title><title>Clinica chimica acta</title><addtitle>Clin Chim Acta</addtitle><description>Calcium-dependent secretory phospholipase A(2)-IIA (sPLA(2)-IIA) in the circulation is a marker of inflammation, associated with acute and chronic disease processes. We describe a quick, sensitive and reliable microplate continuous fluorescence assay for determining sPLA(2) activity in serum.
Liposomes composed of a fluorescent probe and varying amounts of L-alpha-phosphatidylglycerol (PG) and 1,2-dioleoyl-L-alpha-phosphatidylcholine (DOPC) were used as substrates to determine the optimal protocol for sPLA(2) activity determination without interference from serum albumin and lipoproteins.
Hydrolysis of the labeled substrate by sPLA(2)-IIA, characterized by increase in fluorescence intensity (FI) and confirmed by end-product analysis, occurred in a time-, calcium-, and protein-dependent manner. Liposomes containing 100% PG were most suitable for measurement of sPLA(2) activity without interference from serum components; LDL produced a Ca(2+)-independent increase in FI when liposomes containing DOPC were used. The assay determined that sPLA(2) activity in serum spiked with sPLA(2)-IIA and illustrated that endogenous sPLA(2) activity was markedly higher in sera from patients with sepsis than in healthy subjects. Intra-assay and inter-assay CVs were in the ranges of 1.6-8.8% and 3.0-11.5%, respectively.
The described method has potential for rapid and sensitive screening of sPLA(2) activity in both clinical and research settings.</description><subject>Cholesterol, LDL - blood</subject><subject>Group II Phospholipases A2</subject><subject>Humans</subject><subject>Phospholipases A - blood</subject><subject>Phospholipases A2</subject><subject>Sensitivity and Specificity</subject><subject>Spectrometry, Fluorescence - methods</subject><subject>Spectrometry, Fluorescence - standards</subject><subject>Substrate Specificity</subject><issn>0009-8981</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpFkEtLBDEQhHNQXF39AV4kJ28zpjPv4yK-QPCi55Dt6cEsM8mYZJT992ZxwUPTFFQV1MfYNYgcBNR3uxxR51KIOgeZC1mcsHMhRJe1XQsrdhHCLslS1HDGVtDITrZNcc5-NhydjcYubgl8GBfnKSBZJK5D0Hs-OM_jJ_GeIvnJWB2Ns9wNHPWIZpmynmayPdnIA6Gn6Pyez58upBvNrAPxjeQao_k2cc-NTTa_TJfsdNBjoKvjX7OPx4f3--fs9e3p5X7zmqGEMmYwQIU9FtjVVDd6Ww5brFEAdiCp0m3dldAXrUBqqi4NHaq-qCRSVYokSizW7Pavd_bua6EQ1WTSvnHUltJi1QhZd6KBZIQ_I3oXgqdBzd5M2u8VCHUgrHYqEVYHwgqkSoRT5uZYvmwn6v8TR7zFLy2OfIQ</recordid><startdate>200704</startdate><enddate>200704</enddate><creator>Tsao, Francis H C</creator><creator>Shanmuganayagam, Dhanansayan</creator><creator>Zachman, Derek K</creator><creator>Khosravi, Mehdi</creator><creator>Folts, John D</creator><creator>Meyer, Keith C</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>200704</creationdate><title>A continuous fluorescence assay for the determination of calcium-dependent secretory phospholipase A2 activity in serum</title><author>Tsao, Francis H C ; Shanmuganayagam, Dhanansayan ; Zachman, Derek K ; Khosravi, Mehdi ; Folts, John D ; Meyer, Keith C</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c214t-1f15cdc3c96e67ab4fbc6c01c912e5a86941d380ce759981f5d352ce54081f4c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>Cholesterol, LDL - blood</topic><topic>Group II Phospholipases A2</topic><topic>Humans</topic><topic>Phospholipases A - blood</topic><topic>Phospholipases A2</topic><topic>Sensitivity and Specificity</topic><topic>Spectrometry, Fluorescence - methods</topic><topic>Spectrometry, Fluorescence - standards</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Tsao, Francis H C</creatorcontrib><creatorcontrib>Shanmuganayagam, Dhanansayan</creatorcontrib><creatorcontrib>Zachman, Derek K</creatorcontrib><creatorcontrib>Khosravi, Mehdi</creatorcontrib><creatorcontrib>Folts, John D</creatorcontrib><creatorcontrib>Meyer, Keith C</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Clinica chimica acta</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Tsao, Francis H C</au><au>Shanmuganayagam, Dhanansayan</au><au>Zachman, Derek K</au><au>Khosravi, Mehdi</au><au>Folts, John D</au><au>Meyer, Keith C</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A continuous fluorescence assay for the determination of calcium-dependent secretory phospholipase A2 activity in serum</atitle><jtitle>Clinica chimica acta</jtitle><addtitle>Clin Chim Acta</addtitle><date>2007-04</date><risdate>2007</risdate><volume>379</volume><issue>1-2</issue><spage>119</spage><epage>126</epage><pages>119-126</pages><issn>0009-8981</issn><abstract>Calcium-dependent secretory phospholipase A(2)-IIA (sPLA(2)-IIA) in the circulation is a marker of inflammation, associated with acute and chronic disease processes. We describe a quick, sensitive and reliable microplate continuous fluorescence assay for determining sPLA(2) activity in serum.
Liposomes composed of a fluorescent probe and varying amounts of L-alpha-phosphatidylglycerol (PG) and 1,2-dioleoyl-L-alpha-phosphatidylcholine (DOPC) were used as substrates to determine the optimal protocol for sPLA(2) activity determination without interference from serum albumin and lipoproteins.
Hydrolysis of the labeled substrate by sPLA(2)-IIA, characterized by increase in fluorescence intensity (FI) and confirmed by end-product analysis, occurred in a time-, calcium-, and protein-dependent manner. Liposomes containing 100% PG were most suitable for measurement of sPLA(2) activity without interference from serum components; LDL produced a Ca(2+)-independent increase in FI when liposomes containing DOPC were used. The assay determined that sPLA(2) activity in serum spiked with sPLA(2)-IIA and illustrated that endogenous sPLA(2) activity was markedly higher in sera from patients with sepsis than in healthy subjects. Intra-assay and inter-assay CVs were in the ranges of 1.6-8.8% and 3.0-11.5%, respectively.
The described method has potential for rapid and sensitive screening of sPLA(2) activity in both clinical and research settings.</abstract><cop>Netherlands</cop><pmid>17292873</pmid><doi>10.1016/j.cca.2006.12.023</doi><tpages>8</tpages></addata></record> |
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| language | eng |
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| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | Cholesterol, LDL - blood Group II Phospholipases A2 Humans Phospholipases A - blood Phospholipases A2 Sensitivity and Specificity Spectrometry, Fluorescence - methods Spectrometry, Fluorescence - standards Substrate Specificity |
| title | A continuous fluorescence assay for the determination of calcium-dependent secretory phospholipase A2 activity in serum |
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