Colorimetric enzyme-coupled assay for hyaluronic acid determination in complex samples
[Display omitted] •A highly sensitive colorimetric assay for hyaluronic acid determination is proposed.•Assay is based on 3-methyl-2-benothiazolinonehydrazone reaction with reducing ends.•S. pneumoniae lyase is used for hyaluronic acid depolymerization to disaccharides.•The sensitivity of new assay...
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| Veröffentlicht in: | European polymer journal 2017-09, Vol.94, p.460-470 |
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| creator | Pepeliaev, Stanislav Hrudíková, Radka Jílková, Jana Pavlík, Jaroslav Smirnou, Dzianis Černý, Zbyněk Franke, Lukáš |
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•A highly sensitive colorimetric assay for hyaluronic acid determination is proposed.•Assay is based on 3-methyl-2-benothiazolinonehydrazone reaction with reducing ends.•S. pneumoniae lyase is used for hyaluronic acid depolymerization to disaccharides.•The sensitivity of new assay is 1.5–2 times higher than that of Elson-Morgan assay.•The assay could be performed in 25–60min depending on sample complexity.
The present study describes the development of a fast, affordable and reliable method for hyaluronic acid detection in complex samples.
The method involves three principle steps. The first is the separation of hyaluronic acid (HA) from interfering glycosaminoglycans as well as mono- and oligosaccharides by cetyltrimethylammonium bromide fractioning. The second is subsequent digestion of HA with Streptococcus pneumoniae hyaluronate lyase to 4,5-unsaturated disaccharides (ΔHA2). The third is the reaction of ΔHA2 with 3-methyl-2-benothiazolinonehydrazone (MBTH) resulting in an intense blue-colored product. The extinction coefficient of ΔHA2-MBTH product is 34,735mol−1 at 654nm. The theoretical sensitivity of the assay is 0.07–0.09mg/l HA. The practical sensitivity is 0.3mg/l; the highest repeatability was achieved in the range of 3–2000mg/l HA (r2=0.9994). The analysis took 25–60min depending on sample complexity.
The described method was evaluated in an industrial setting for online monitoring of HA losses during downstream processes and for HA determination in veterinary products. It was positively rated by users and was introduced to routine laboratory practices. |
| doi_str_mv | 10.1016/j.eurpolymj.2017.07.036 |
| format | Article |
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•A highly sensitive colorimetric assay for hyaluronic acid determination is proposed.•Assay is based on 3-methyl-2-benothiazolinonehydrazone reaction with reducing ends.•S. pneumoniae lyase is used for hyaluronic acid depolymerization to disaccharides.•The sensitivity of new assay is 1.5–2 times higher than that of Elson-Morgan assay.•The assay could be performed in 25–60min depending on sample complexity.
The present study describes the development of a fast, affordable and reliable method for hyaluronic acid detection in complex samples.
The method involves three principle steps. The first is the separation of hyaluronic acid (HA) from interfering glycosaminoglycans as well as mono- and oligosaccharides by cetyltrimethylammonium bromide fractioning. The second is subsequent digestion of HA with Streptococcus pneumoniae hyaluronate lyase to 4,5-unsaturated disaccharides (ΔHA2). The third is the reaction of ΔHA2 with 3-methyl-2-benothiazolinonehydrazone (MBTH) resulting in an intense blue-colored product. The extinction coefficient of ΔHA2-MBTH product is 34,735mol−1 at 654nm. The theoretical sensitivity of the assay is 0.07–0.09mg/l HA. The practical sensitivity is 0.3mg/l; the highest repeatability was achieved in the range of 3–2000mg/l HA (r2=0.9994). The analysis took 25–60min depending on sample complexity.
The described method was evaluated in an industrial setting for online monitoring of HA losses during downstream processes and for HA determination in veterinary products. It was positively rated by users and was introduced to routine laboratory practices.</description><identifier>ISSN: 0014-3057</identifier><identifier>EISSN: 1873-1945</identifier><identifier>DOI: 10.1016/j.eurpolymj.2017.07.036</identifier><language>eng</language><publisher>Oxford: Elsevier Ltd</publisher><subject>Acids ; Analytical chemistry ; Cetyltrimethylammonium bromide ; Colorimetry ; Complexity ; Glycosaminoglycan precipitation ; Glycosaminoglycans ; Hyaluronic acid ; Hyaluronic acid assay ; Oligosaccharides ; Sensitivity ; Streptococcus pneumoniae hyaluronan lyase ; Veterinary medicine ; Viscosity</subject><ispartof>European polymer journal, 2017-09, Vol.94, p.460-470</ispartof><rights>2017 Elsevier Ltd</rights><rights>Copyright Elsevier BV Sep 2017</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c380t-2a05730fb543735b8f0c57e8cbed9b258db2983e2726d20e02a92b5496db55ee3</citedby><cites>FETCH-LOGICAL-c380t-2a05730fb543735b8f0c57e8cbed9b258db2983e2726d20e02a92b5496db55ee3</cites><orcidid>0000-0001-5380-0878</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.eurpolymj.2017.07.036$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,777,781,3537,27905,27906,45976</link.rule.ids></links><search><creatorcontrib>Pepeliaev, Stanislav</creatorcontrib><creatorcontrib>Hrudíková, Radka</creatorcontrib><creatorcontrib>Jílková, Jana</creatorcontrib><creatorcontrib>Pavlík, Jaroslav</creatorcontrib><creatorcontrib>Smirnou, Dzianis</creatorcontrib><creatorcontrib>Černý, Zbyněk</creatorcontrib><creatorcontrib>Franke, Lukáš</creatorcontrib><title>Colorimetric enzyme-coupled assay for hyaluronic acid determination in complex samples</title><title>European polymer journal</title><description>[Display omitted]
•A highly sensitive colorimetric assay for hyaluronic acid determination is proposed.•Assay is based on 3-methyl-2-benothiazolinonehydrazone reaction with reducing ends.•S. pneumoniae lyase is used for hyaluronic acid depolymerization to disaccharides.•The sensitivity of new assay is 1.5–2 times higher than that of Elson-Morgan assay.•The assay could be performed in 25–60min depending on sample complexity.
The present study describes the development of a fast, affordable and reliable method for hyaluronic acid detection in complex samples.
The method involves three principle steps. The first is the separation of hyaluronic acid (HA) from interfering glycosaminoglycans as well as mono- and oligosaccharides by cetyltrimethylammonium bromide fractioning. The second is subsequent digestion of HA with Streptococcus pneumoniae hyaluronate lyase to 4,5-unsaturated disaccharides (ΔHA2). The third is the reaction of ΔHA2 with 3-methyl-2-benothiazolinonehydrazone (MBTH) resulting in an intense blue-colored product. The extinction coefficient of ΔHA2-MBTH product is 34,735mol−1 at 654nm. The theoretical sensitivity of the assay is 0.07–0.09mg/l HA. The practical sensitivity is 0.3mg/l; the highest repeatability was achieved in the range of 3–2000mg/l HA (r2=0.9994). The analysis took 25–60min depending on sample complexity.
The described method was evaluated in an industrial setting for online monitoring of HA losses during downstream processes and for HA determination in veterinary products. It was positively rated by users and was introduced to routine laboratory practices.</description><subject>Acids</subject><subject>Analytical chemistry</subject><subject>Cetyltrimethylammonium bromide</subject><subject>Colorimetry</subject><subject>Complexity</subject><subject>Glycosaminoglycan precipitation</subject><subject>Glycosaminoglycans</subject><subject>Hyaluronic acid</subject><subject>Hyaluronic acid assay</subject><subject>Oligosaccharides</subject><subject>Sensitivity</subject><subject>Streptococcus pneumoniae hyaluronan lyase</subject><subject>Veterinary medicine</subject><subject>Viscosity</subject><issn>0014-3057</issn><issn>1873-1945</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><recordid>eNqFUE1LxDAUDKLguvobLHju-pK0TXtcFr9gwYt6DWnyiiltU5NWrL_eLCtehYG5zMx7M4RcU9hQoMVtu8HZj65b-nbDgIoNRPDihKxoKXhKqyw_JSsAmqUccnFOLkJoAUDwgq_I2851ztseJ291gsP30mOq3Tx2aBIVglqSxvnkfVHd7N0QNUpbkxic0Pd2UJN1Q2KHRLs-Wr6SoA4cLslZo7qAV7-8Jq_3dy-7x3T__PC02-5TzUuYUqbiRxyaOs-44HldNqBzgaWu0VQ1y0tTs6rkyAQrDAMEpioWxVVh6jxH5Gtyc8wdvfuYMUyydbMf4klJq4JmDCooo0ocVdq7EDw2coyVlV8kBXkYUbbyb0R5GFFCRBxoTbZHJ8YSnxa9DNrioNFYj3qSxtl_M34A71aA-Q</recordid><startdate>20170901</startdate><enddate>20170901</enddate><creator>Pepeliaev, Stanislav</creator><creator>Hrudíková, Radka</creator><creator>Jílková, Jana</creator><creator>Pavlík, Jaroslav</creator><creator>Smirnou, Dzianis</creator><creator>Černý, Zbyněk</creator><creator>Franke, Lukáš</creator><general>Elsevier Ltd</general><general>Elsevier BV</general><scope>AAYXX</scope><scope>CITATION</scope><scope>7SR</scope><scope>8FD</scope><scope>JG9</scope><orcidid>https://orcid.org/0000-0001-5380-0878</orcidid></search><sort><creationdate>20170901</creationdate><title>Colorimetric enzyme-coupled assay for hyaluronic acid determination in complex samples</title><author>Pepeliaev, Stanislav ; Hrudíková, Radka ; Jílková, Jana ; Pavlík, Jaroslav ; Smirnou, Dzianis ; Černý, Zbyněk ; Franke, Lukáš</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c380t-2a05730fb543735b8f0c57e8cbed9b258db2983e2726d20e02a92b5496db55ee3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Acids</topic><topic>Analytical chemistry</topic><topic>Cetyltrimethylammonium bromide</topic><topic>Colorimetry</topic><topic>Complexity</topic><topic>Glycosaminoglycan precipitation</topic><topic>Glycosaminoglycans</topic><topic>Hyaluronic acid</topic><topic>Hyaluronic acid assay</topic><topic>Oligosaccharides</topic><topic>Sensitivity</topic><topic>Streptococcus pneumoniae hyaluronan lyase</topic><topic>Veterinary medicine</topic><topic>Viscosity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Pepeliaev, Stanislav</creatorcontrib><creatorcontrib>Hrudíková, Radka</creatorcontrib><creatorcontrib>Jílková, Jana</creatorcontrib><creatorcontrib>Pavlík, Jaroslav</creatorcontrib><creatorcontrib>Smirnou, Dzianis</creatorcontrib><creatorcontrib>Černý, Zbyněk</creatorcontrib><creatorcontrib>Franke, Lukáš</creatorcontrib><collection>CrossRef</collection><collection>Engineered Materials Abstracts</collection><collection>Technology Research Database</collection><collection>Materials Research Database</collection><jtitle>European polymer journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Pepeliaev, Stanislav</au><au>Hrudíková, Radka</au><au>Jílková, Jana</au><au>Pavlík, Jaroslav</au><au>Smirnou, Dzianis</au><au>Černý, Zbyněk</au><au>Franke, Lukáš</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Colorimetric enzyme-coupled assay for hyaluronic acid determination in complex samples</atitle><jtitle>European polymer journal</jtitle><date>2017-09-01</date><risdate>2017</risdate><volume>94</volume><spage>460</spage><epage>470</epage><pages>460-470</pages><issn>0014-3057</issn><eissn>1873-1945</eissn><abstract>[Display omitted]
•A highly sensitive colorimetric assay for hyaluronic acid determination is proposed.•Assay is based on 3-methyl-2-benothiazolinonehydrazone reaction with reducing ends.•S. pneumoniae lyase is used for hyaluronic acid depolymerization to disaccharides.•The sensitivity of new assay is 1.5–2 times higher than that of Elson-Morgan assay.•The assay could be performed in 25–60min depending on sample complexity.
The present study describes the development of a fast, affordable and reliable method for hyaluronic acid detection in complex samples.
The method involves three principle steps. The first is the separation of hyaluronic acid (HA) from interfering glycosaminoglycans as well as mono- and oligosaccharides by cetyltrimethylammonium bromide fractioning. The second is subsequent digestion of HA with Streptococcus pneumoniae hyaluronate lyase to 4,5-unsaturated disaccharides (ΔHA2). The third is the reaction of ΔHA2 with 3-methyl-2-benothiazolinonehydrazone (MBTH) resulting in an intense blue-colored product. The extinction coefficient of ΔHA2-MBTH product is 34,735mol−1 at 654nm. The theoretical sensitivity of the assay is 0.07–0.09mg/l HA. The practical sensitivity is 0.3mg/l; the highest repeatability was achieved in the range of 3–2000mg/l HA (r2=0.9994). The analysis took 25–60min depending on sample complexity.
The described method was evaluated in an industrial setting for online monitoring of HA losses during downstream processes and for HA determination in veterinary products. It was positively rated by users and was introduced to routine laboratory practices.</abstract><cop>Oxford</cop><pub>Elsevier Ltd</pub><doi>10.1016/j.eurpolymj.2017.07.036</doi><tpages>11</tpages><orcidid>https://orcid.org/0000-0001-5380-0878</orcidid></addata></record> |
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| subjects | Acids Analytical chemistry Cetyltrimethylammonium bromide Colorimetry Complexity Glycosaminoglycan precipitation Glycosaminoglycans Hyaluronic acid Hyaluronic acid assay Oligosaccharides Sensitivity Streptococcus pneumoniae hyaluronan lyase Veterinary medicine Viscosity |
| title | Colorimetric enzyme-coupled assay for hyaluronic acid determination in complex samples |
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