Simultaneous free and glycosylated pyridinium crosslink determination in urine: Validation of an HPLC-fluorescence method using a deoxypyridinoline homologue as internal standard
Pyridinoline (Pyr), deoxypyridinoline (D-Pyr), galactosyl-pyridinoline (Gal-Pyr) and glucosyl–galactosyl pyridinoline (GluGal-Pyr) are enzymatic mature pyridinium crosslinks. Generally, only total Pyr and D-Pyr urinary amounts (free + bound forms) are evaluated by HPLC as indices of bone resorption....
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| Veröffentlicht in: | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2011-09, Vol.879 (26), p.2764-2771 |
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| creator | Monticelli, Elena Aman, Caroline Stéphanie Costa, Maria Letizia Rota, Paola Bogdan, Daniela Allevi, Pietro Cighetti, Giuliana |
| description | Pyridinoline (Pyr), deoxypyridinoline (D-Pyr), galactosyl-pyridinoline (Gal-Pyr) and glucosyl–galactosyl pyridinoline (GluGal-Pyr) are enzymatic mature pyridinium crosslinks. Generally, only total Pyr and D-Pyr urinary amounts (free
+
bound forms) are evaluated by HPLC as indices of bone resorption. This report describes the validation of an HPLC-fluorescence method for the simultaneous evaluation of free Pyr and D-Pyr, together with GluGal-Pyr and Gal-Pyr, in urine of healthy women (
n
=
20, aged 27–41) and girls (
n
=
20, aged 5–10). The use of an unnatural D-Pyr homologue, here proposed for the first time as internal standard, and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr synthesized to be used as primary calibrators, guarantees method specificity and correct crosslink quantification. Urine, spiked with IS, was solid-phase extracted prior to HPLC analysis. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis. The HPLC method was validated for selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Both free and total Pyr and D-Pyr as well as GluGal-Pyr and Gal-Pyr amounts were significantly higher in girls than in women (
p
<
0.0001), indicating an increased collagen turnover rather than only bone turnover. Gal-Pyr, for the first time evaluated in girls, was under its lower quantification limit ( |
| doi_str_mv | 10.1016/j.jchromb.2011.07.044 |
| format | Article |
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+
bound forms) are evaluated by HPLC as indices of bone resorption. This report describes the validation of an HPLC-fluorescence method for the simultaneous evaluation of free Pyr and D-Pyr, together with GluGal-Pyr and Gal-Pyr, in urine of healthy women (
n
=
20, aged 27–41) and girls (
n
=
20, aged 5–10). The use of an unnatural D-Pyr homologue, here proposed for the first time as internal standard, and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr synthesized to be used as primary calibrators, guarantees method specificity and correct crosslink quantification. Urine, spiked with IS, was solid-phase extracted prior to HPLC analysis. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis. The HPLC method was validated for selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Both free and total Pyr and D-Pyr as well as GluGal-Pyr and Gal-Pyr amounts were significantly higher in girls than in women (
p
<
0.0001), indicating an increased collagen turnover rather than only bone turnover. Gal-Pyr, for the first time evaluated in girls, was under its lower quantification limit (<LLOQ, <21.20
pmol/mL) in women. The measurement of free and glycosylated pyridinium crosslinks might provide more information on the degradation of various types of collagen than only that of total Pyr and D-Pyr. Moreover, this validated method could be a useful non-invasive technique for studying pathological conditions characterized by modified glycosylation enzyme activity and for more clinical investigation on bone fragility.</description><identifier>ISSN: 1570-0232</identifier><identifier>EISSN: 1873-376X</identifier><identifier>DOI: 10.1016/j.jchromb.2011.07.044</identifier><identifier>PMID: 21855424</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Adult ; Amino Acids - analysis ; Amino Acids - chemistry ; Amino Acids - urine ; Analysis ; Analytical, structural and metabolic biochemistry ; Biological and medical sciences ; bone resorption ; Bone Resorption - urine ; Bones ; Calibration ; Child ; Child, Preschool ; Chromatography, High Pressure Liquid - methods ; collagen ; Collagens ; Crosslinking ; Deoxypyridinoline ; Drug Stability ; enzyme activity ; Female ; Fluorescence detection ; Fragility ; Fundamental and applied biological sciences. Psychology ; Galactosides - chemistry ; Galactosides - urine ; General pharmacology ; Girls ; Glycosylated pyridinoline ; Glycosylation ; high performance liquid chromatography ; HPLC ; Human urine ; Humans ; Hydrolysis ; Least-Squares Analysis ; Medical sciences ; Pharmacology. Drug treatments ; Pyridinoline ; Reproducibility of Results ; Sensitivity and Specificity ; Spectrometry, Fluorescence ; Urine ; women</subject><ispartof>Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 2011-09, Vol.879 (26), p.2764-2771</ispartof><rights>2011 Elsevier B.V.</rights><rights>2015 INIST-CNRS</rights><rights>Copyright © 2011 Elsevier B.V. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c451t-49a5d9f6123098334089bee8f3b56d5bff4abe2505d47800559697fb17988773</citedby><cites>FETCH-LOGICAL-c451t-49a5d9f6123098334089bee8f3b56d5bff4abe2505d47800559697fb17988773</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S1570023211005125$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=24512396$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21855424$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Monticelli, Elena</creatorcontrib><creatorcontrib>Aman, Caroline Stéphanie</creatorcontrib><creatorcontrib>Costa, Maria Letizia</creatorcontrib><creatorcontrib>Rota, Paola</creatorcontrib><creatorcontrib>Bogdan, Daniela</creatorcontrib><creatorcontrib>Allevi, Pietro</creatorcontrib><creatorcontrib>Cighetti, Giuliana</creatorcontrib><title>Simultaneous free and glycosylated pyridinium crosslink determination in urine: Validation of an HPLC-fluorescence method using a deoxypyridinoline homologue as internal standard</title><title>Journal of chromatography. B, Analytical technologies in the biomedical and life sciences</title><addtitle>J Chromatogr B Analyt Technol Biomed Life Sci</addtitle><description>Pyridinoline (Pyr), deoxypyridinoline (D-Pyr), galactosyl-pyridinoline (Gal-Pyr) and glucosyl–galactosyl pyridinoline (GluGal-Pyr) are enzymatic mature pyridinium crosslinks. Generally, only total Pyr and D-Pyr urinary amounts (free
+
bound forms) are evaluated by HPLC as indices of bone resorption. This report describes the validation of an HPLC-fluorescence method for the simultaneous evaluation of free Pyr and D-Pyr, together with GluGal-Pyr and Gal-Pyr, in urine of healthy women (
n
=
20, aged 27–41) and girls (
n
=
20, aged 5–10). The use of an unnatural D-Pyr homologue, here proposed for the first time as internal standard, and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr synthesized to be used as primary calibrators, guarantees method specificity and correct crosslink quantification. Urine, spiked with IS, was solid-phase extracted prior to HPLC analysis. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis. The HPLC method was validated for selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Both free and total Pyr and D-Pyr as well as GluGal-Pyr and Gal-Pyr amounts were significantly higher in girls than in women (
p
<
0.0001), indicating an increased collagen turnover rather than only bone turnover. Gal-Pyr, for the first time evaluated in girls, was under its lower quantification limit (<LLOQ, <21.20
pmol/mL) in women. The measurement of free and glycosylated pyridinium crosslinks might provide more information on the degradation of various types of collagen than only that of total Pyr and D-Pyr. Moreover, this validated method could be a useful non-invasive technique for studying pathological conditions characterized by modified glycosylation enzyme activity and for more clinical investigation on bone fragility.</description><subject>Adult</subject><subject>Amino Acids - analysis</subject><subject>Amino Acids - chemistry</subject><subject>Amino Acids - urine</subject><subject>Analysis</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Biological and medical sciences</subject><subject>bone resorption</subject><subject>Bone Resorption - urine</subject><subject>Bones</subject><subject>Calibration</subject><subject>Child</subject><subject>Child, Preschool</subject><subject>Chromatography, High Pressure Liquid - methods</subject><subject>collagen</subject><subject>Collagens</subject><subject>Crosslinking</subject><subject>Deoxypyridinoline</subject><subject>Drug Stability</subject><subject>enzyme activity</subject><subject>Female</subject><subject>Fluorescence detection</subject><subject>Fragility</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Galactosides - chemistry</subject><subject>Galactosides - urine</subject><subject>General pharmacology</subject><subject>Girls</subject><subject>Glycosylated pyridinoline</subject><subject>Glycosylation</subject><subject>high performance liquid chromatography</subject><subject>HPLC</subject><subject>Human urine</subject><subject>Humans</subject><subject>Hydrolysis</subject><subject>Least-Squares Analysis</subject><subject>Medical sciences</subject><subject>Pharmacology. Drug treatments</subject><subject>Pyridinoline</subject><subject>Reproducibility of Results</subject><subject>Sensitivity and Specificity</subject><subject>Spectrometry, Fluorescence</subject><subject>Urine</subject><subject>women</subject><issn>1570-0232</issn><issn>1873-376X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkcGu1CAYhRuj8V5HH0FlY3TTEQqU1o0xE_WaTKLJvRp3hJafGUYKI7TGvpZPKGNH3SkbCPk45-econhI8JpgUj8_rA_9PoahW1eYkDUWa8zYreKSNIKWVNSfb-czF7jEFa0uinspHTAmAgt6t7ioSMM5q9hl8ePaDpMblYcwJWQiAFJeo52b-5Bmp0bQ6DhHq62304D6GFJy1n9BGkaIg_VqtMEj69EUrYcX6JNyVi-XwWQtdPVhuymNm0KE1IPvAQ0w7oNGU7J-h1RWCt_ns0fI2oD2YQgu7KY8S8rS2cgrh1KeUquo7xd3jHIJHpz3VXHz5vXN5qrcvn_7bvNqW_aMk7FkreK6NTWpKG4bShlu2g6gMbTjteadMUx1UHHMNRMNxpy3dStMR0TbNELQVfF0kT3G8HWCNMrB5g84t2QlM9QyRvNaFc_-SZJaENpiRtuM8gX9FWQEI4_RDirOkmB56lUe5LlXeepVYiFzr_ndo7PF1A2g_7z6XWQGnpwBlXrlTFS-t-kvlyOpaFtn7vHCGRWk2sXMfLzOTjXGuOacNJl4uRCQs_1mIcrU21Nv2kboR6mD_c-wPwH-8tDi</recordid><startdate>20110915</startdate><enddate>20110915</enddate><creator>Monticelli, Elena</creator><creator>Aman, Caroline Stéphanie</creator><creator>Costa, Maria Letizia</creator><creator>Rota, Paola</creator><creator>Bogdan, Daniela</creator><creator>Allevi, Pietro</creator><creator>Cighetti, Giuliana</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>FBQ</scope><scope>IQODW</scope><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>7TB</scope><scope>8FD</scope><scope>FR3</scope><scope>7X8</scope></search><sort><creationdate>20110915</creationdate><title>Simultaneous free and glycosylated pyridinium crosslink determination in urine: Validation of an HPLC-fluorescence method using a deoxypyridinoline homologue as internal standard</title><author>Monticelli, Elena ; Aman, Caroline Stéphanie ; Costa, Maria Letizia ; Rota, Paola ; Bogdan, Daniela ; Allevi, Pietro ; Cighetti, Giuliana</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c451t-49a5d9f6123098334089bee8f3b56d5bff4abe2505d47800559697fb17988773</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Adult</topic><topic>Amino Acids - analysis</topic><topic>Amino Acids - chemistry</topic><topic>Amino Acids - urine</topic><topic>Analysis</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Biological and medical sciences</topic><topic>bone resorption</topic><topic>Bone Resorption - urine</topic><topic>Bones</topic><topic>Calibration</topic><topic>Child</topic><topic>Child, Preschool</topic><topic>Chromatography, High Pressure Liquid - methods</topic><topic>collagen</topic><topic>Collagens</topic><topic>Crosslinking</topic><topic>Deoxypyridinoline</topic><topic>Drug Stability</topic><topic>enzyme activity</topic><topic>Female</topic><topic>Fluorescence detection</topic><topic>Fragility</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Galactosides - chemistry</topic><topic>Galactosides - urine</topic><topic>General pharmacology</topic><topic>Girls</topic><topic>Glycosylated pyridinoline</topic><topic>Glycosylation</topic><topic>high performance liquid chromatography</topic><topic>HPLC</topic><topic>Human urine</topic><topic>Humans</topic><topic>Hydrolysis</topic><topic>Least-Squares Analysis</topic><topic>Medical sciences</topic><topic>Pharmacology. Drug treatments</topic><topic>Pyridinoline</topic><topic>Reproducibility of Results</topic><topic>Sensitivity and Specificity</topic><topic>Spectrometry, Fluorescence</topic><topic>Urine</topic><topic>women</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Monticelli, Elena</creatorcontrib><creatorcontrib>Aman, Caroline Stéphanie</creatorcontrib><creatorcontrib>Costa, Maria Letizia</creatorcontrib><creatorcontrib>Rota, Paola</creatorcontrib><creatorcontrib>Bogdan, Daniela</creatorcontrib><creatorcontrib>Allevi, Pietro</creatorcontrib><creatorcontrib>Cighetti, Giuliana</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of chromatography. B, Analytical technologies in the biomedical and life sciences</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Monticelli, Elena</au><au>Aman, Caroline Stéphanie</au><au>Costa, Maria Letizia</au><au>Rota, Paola</au><au>Bogdan, Daniela</au><au>Allevi, Pietro</au><au>Cighetti, Giuliana</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Simultaneous free and glycosylated pyridinium crosslink determination in urine: Validation of an HPLC-fluorescence method using a deoxypyridinoline homologue as internal standard</atitle><jtitle>Journal of chromatography. B, Analytical technologies in the biomedical and life sciences</jtitle><addtitle>J Chromatogr B Analyt Technol Biomed Life Sci</addtitle><date>2011-09-15</date><risdate>2011</risdate><volume>879</volume><issue>26</issue><spage>2764</spage><epage>2771</epage><pages>2764-2771</pages><issn>1570-0232</issn><eissn>1873-376X</eissn><abstract>Pyridinoline (Pyr), deoxypyridinoline (D-Pyr), galactosyl-pyridinoline (Gal-Pyr) and glucosyl–galactosyl pyridinoline (GluGal-Pyr) are enzymatic mature pyridinium crosslinks. Generally, only total Pyr and D-Pyr urinary amounts (free
+
bound forms) are evaluated by HPLC as indices of bone resorption. This report describes the validation of an HPLC-fluorescence method for the simultaneous evaluation of free Pyr and D-Pyr, together with GluGal-Pyr and Gal-Pyr, in urine of healthy women (
n
=
20, aged 27–41) and girls (
n
=
20, aged 5–10). The use of an unnatural D-Pyr homologue, here proposed for the first time as internal standard, and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr synthesized to be used as primary calibrators, guarantees method specificity and correct crosslink quantification. Urine, spiked with IS, was solid-phase extracted prior to HPLC analysis. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis. The HPLC method was validated for selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Both free and total Pyr and D-Pyr as well as GluGal-Pyr and Gal-Pyr amounts were significantly higher in girls than in women (
p
<
0.0001), indicating an increased collagen turnover rather than only bone turnover. Gal-Pyr, for the first time evaluated in girls, was under its lower quantification limit (<LLOQ, <21.20
pmol/mL) in women. The measurement of free and glycosylated pyridinium crosslinks might provide more information on the degradation of various types of collagen than only that of total Pyr and D-Pyr. Moreover, this validated method could be a useful non-invasive technique for studying pathological conditions characterized by modified glycosylation enzyme activity and for more clinical investigation on bone fragility.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>21855424</pmid><doi>10.1016/j.jchromb.2011.07.044</doi><tpages>8</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1570-0232 |
| ispartof | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 2011-09, Vol.879 (26), p.2764-2771 |
| issn | 1570-0232 1873-376X |
| language | eng |
| recordid | cdi_proquest_miscellaneous_887944333 |
| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Adult Amino Acids - analysis Amino Acids - chemistry Amino Acids - urine Analysis Analytical, structural and metabolic biochemistry Biological and medical sciences bone resorption Bone Resorption - urine Bones Calibration Child Child, Preschool Chromatography, High Pressure Liquid - methods collagen Collagens Crosslinking Deoxypyridinoline Drug Stability enzyme activity Female Fluorescence detection Fragility Fundamental and applied biological sciences. Psychology Galactosides - chemistry Galactosides - urine General pharmacology Girls Glycosylated pyridinoline Glycosylation high performance liquid chromatography HPLC Human urine Humans Hydrolysis Least-Squares Analysis Medical sciences Pharmacology. Drug treatments Pyridinoline Reproducibility of Results Sensitivity and Specificity Spectrometry, Fluorescence Urine women |
| title | Simultaneous free and glycosylated pyridinium crosslink determination in urine: Validation of an HPLC-fluorescence method using a deoxypyridinoline homologue as internal standard |
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