A normal phase high-performance liquid chromatography system for steroid 17α-hydroxylase/C17-20 lyase (cytochrome P-45021scc) assays
A normal phase HPLC system has been developed which is applicable to all of the steroid separations involved in the assay of steroid 17 alpha-hydroxylase (EC 1.14.99.9) and C17-20 lyase activities, in both the delta 4 (progesterone) and delta 5 (pregnenolone) pathways. A hexane-tetrahydrofuran (THF)...
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| Veröffentlicht in: | Analytical biochemistry 1988-11, Vol.175 (1), p.219-226 |
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| container_title | Analytical biochemistry |
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| creator | SCHATZMAN, G. L LAUGHLIN, M. E BLOHM, T. R |
| description | A normal phase HPLC system has been developed which is applicable to all of the steroid separations involved in the assay of steroid 17 alpha-hydroxylase (EC 1.14.99.9) and C17-20 lyase activities, in both the delta 4 (progesterone) and delta 5 (pregnenolone) pathways. A hexane-tetrahydrofuran (THF) gradient system is used with silica stationary phase and a flow cell radioactivity detector having a high efficiency for tritium. Folch extraction produces uniform extraction of substrates and products from the microsomal incubates, and this uniformity is maintained through HPLC separation and measurement. The hexane-THF mobile phase is convenient for product isolation and substrate purification and should be adaptable to other steroid separations. The system is especially useful for steroid enzyme assays utilizing radiolabeled substrates, since internal standards are not required for measuring recoveries of substrates and products. |
| doi_str_mv | 10.1016/0003-2697(88)90381-8 |
| format | Article |
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The system is especially useful for steroid enzyme assays utilizing radiolabeled substrates, since internal standards are not required for measuring recoveries of substrates and products.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1016/0003-2697(88)90381-8</identifier><identifier>PMID: 3266718</identifier><identifier>CODEN: ANBCA2</identifier><language>eng</language><publisher>San Diego, CA: Elsevier</publisher><subject>Aldehyde-Lyases - analysis ; Analytical, structural and metabolic biochemistry ; Animals ; Biological and medical sciences ; Cholesterol Side-Chain Cleavage Enzyme - analysis ; Chromatography, High Pressure Liquid - methods ; Cytochrome P-450 Enzyme System - analysis ; Enzymes and enzyme inhibitors ; Fundamental and applied biological sciences. Psychology ; Furans ; Hexanes ; Humans ; Kinetics ; Male ; Oxidoreductases ; Pregnenolone - metabolism ; Rats ; Steroid 17-alpha-Hydroxylase - analysis ; Steroid Hydroxylases - analysis ; Testis - enzymology</subject><ispartof>Analytical biochemistry, 1988-11, Vol.175 (1), p.219-226</ispartof><rights>1989 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=7166524$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3266718$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>SCHATZMAN, G. L</creatorcontrib><creatorcontrib>LAUGHLIN, M. E</creatorcontrib><creatorcontrib>BLOHM, T. R</creatorcontrib><title>A normal phase high-performance liquid chromatography system for steroid 17α-hydroxylase/C17-20 lyase (cytochrome P-45021scc) assays</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>A normal phase HPLC system has been developed which is applicable to all of the steroid separations involved in the assay of steroid 17 alpha-hydroxylase (EC 1.14.99.9) and C17-20 lyase activities, in both the delta 4 (progesterone) and delta 5 (pregnenolone) pathways. A hexane-tetrahydrofuran (THF) gradient system is used with silica stationary phase and a flow cell radioactivity detector having a high efficiency for tritium. Folch extraction produces uniform extraction of substrates and products from the microsomal incubates, and this uniformity is maintained through HPLC separation and measurement. The hexane-THF mobile phase is convenient for product isolation and substrate purification and should be adaptable to other steroid separations. The system is especially useful for steroid enzyme assays utilizing radiolabeled substrates, since internal standards are not required for measuring recoveries of substrates and products.</description><subject>Aldehyde-Lyases - analysis</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Cholesterol Side-Chain Cleavage Enzyme - analysis</subject><subject>Chromatography, High Pressure Liquid - methods</subject><subject>Cytochrome P-450 Enzyme System - analysis</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Furans</subject><subject>Hexanes</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Male</subject><subject>Oxidoreductases</subject><subject>Pregnenolone - metabolism</subject><subject>Rats</subject><subject>Steroid 17-alpha-Hydroxylase - analysis</subject><subject>Steroid Hydroxylases - analysis</subject><subject>Testis - enzymology</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1988</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo9kM9O3DAQh62qiC4Lb9BKPlQVHAwzdvwnR7SCttJKcGjPq1nHIamSTbCzEnmAPhAvwjNh2hWnGc3v06fRj7HPCJcIaK4AQAlpSnvu3EUJyqFwH9gCoTQCFJQf2eId-cROUvoDgFhoc8yOlTTGoluwv9d8N8SeOj42lAJv2odGjCHWb8edD7xrH_dtxX0Th56m4SHS2Mw8zWkKPc8Uz0scMoH25Vk0cxWHp7nLqqsVWiGBd_Ob99zP0_BPEvi9KDRITN5fcEqJ5nTKjmrqUjg7zCX7fXvza_VDrO--_1xdr8UolZ5EkIWqvQ5FqcB5rWm7LRArqq1ESRYqsITO1YVEssEY6T1UJh9oG7DAUi3Zt__eMQ6P-5CmTd8mH7qOdmHYp411VqNWNoNfDuB-24dqM8a2pzhvDr3l_Oshp-Spq2Puqk3vmEVjdH72Fa1AfZM</recordid><startdate>19881115</startdate><enddate>19881115</enddate><creator>SCHATZMAN, G. L</creator><creator>LAUGHLIN, M. E</creator><creator>BLOHM, T. R</creator><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7X8</scope></search><sort><creationdate>19881115</creationdate><title>A normal phase high-performance liquid chromatography system for steroid 17α-hydroxylase/C17-20 lyase (cytochrome P-45021scc) assays</title><author>SCHATZMAN, G. L ; LAUGHLIN, M. E ; BLOHM, T. R</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p235t-e243fc5e49308c55abb411daf7212a70d07a188f421a7e662cc0d688fabe14193</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1988</creationdate><topic>Aldehyde-Lyases - analysis</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Cholesterol Side-Chain Cleavage Enzyme - analysis</topic><topic>Chromatography, High Pressure Liquid - methods</topic><topic>Cytochrome P-450 Enzyme System - analysis</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Furans</topic><topic>Hexanes</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Male</topic><topic>Oxidoreductases</topic><topic>Pregnenolone - metabolism</topic><topic>Rats</topic><topic>Steroid 17-alpha-Hydroxylase - analysis</topic><topic>Steroid Hydroxylases - analysis</topic><topic>Testis - enzymology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>SCHATZMAN, G. L</creatorcontrib><creatorcontrib>LAUGHLIN, M. E</creatorcontrib><creatorcontrib>BLOHM, T. 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R</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A normal phase high-performance liquid chromatography system for steroid 17α-hydroxylase/C17-20 lyase (cytochrome P-45021scc) assays</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>1988-11-15</date><risdate>1988</risdate><volume>175</volume><issue>1</issue><spage>219</spage><epage>226</epage><pages>219-226</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><coden>ANBCA2</coden><abstract>A normal phase HPLC system has been developed which is applicable to all of the steroid separations involved in the assay of steroid 17 alpha-hydroxylase (EC 1.14.99.9) and C17-20 lyase activities, in both the delta 4 (progesterone) and delta 5 (pregnenolone) pathways. A hexane-tetrahydrofuran (THF) gradient system is used with silica stationary phase and a flow cell radioactivity detector having a high efficiency for tritium. Folch extraction produces uniform extraction of substrates and products from the microsomal incubates, and this uniformity is maintained through HPLC separation and measurement. The hexane-THF mobile phase is convenient for product isolation and substrate purification and should be adaptable to other steroid separations. The system is especially useful for steroid enzyme assays utilizing radiolabeled substrates, since internal standards are not required for measuring recoveries of substrates and products.</abstract><cop>San Diego, CA</cop><pub>Elsevier</pub><pmid>3266718</pmid><doi>10.1016/0003-2697(88)90381-8</doi><tpages>8</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0003-2697 |
| ispartof | Analytical biochemistry, 1988-11, Vol.175 (1), p.219-226 |
| issn | 0003-2697 1096-0309 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_78751537 |
| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Aldehyde-Lyases - analysis Analytical, structural and metabolic biochemistry Animals Biological and medical sciences Cholesterol Side-Chain Cleavage Enzyme - analysis Chromatography, High Pressure Liquid - methods Cytochrome P-450 Enzyme System - analysis Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Furans Hexanes Humans Kinetics Male Oxidoreductases Pregnenolone - metabolism Rats Steroid 17-alpha-Hydroxylase - analysis Steroid Hydroxylases - analysis Testis - enzymology |
| title | A normal phase high-performance liquid chromatography system for steroid 17α-hydroxylase/C17-20 lyase (cytochrome P-45021scc) assays |
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