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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">An LC–MS/MS-Based Method for the Quantification
of Pyridox(am)ine 5′-Phosphate Oxidase Activity in Dried Blood
Spots from Patients with Epilepsy</title><author><name sortKey="Wilson, Matthew X0a P" sort="Wilson, Matthew X0a P" uniqKey="Wilson M" first="Matthew X0a P." last="Wilson">Matthew X0a P. Wilson</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Footitt, Emma J" sort="Footitt, Emma J" uniqKey="Footitt E" first="Emma J." last="Footitt">Emma J. Footitt</name><affiliation><nlm:aff id="aff2">Metabolic Medicine Unit,</nlm:aff></affiliation></author><author><name sortKey="Papandreou, Apostolos" sort="Papandreou, Apostolos" uniqKey="Papandreou A" first="Apostolos" last="Papandreou">Apostolos Papandreou</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Uudelepp, Mari Liis" sort="Uudelepp, Mari Liis" uniqKey="Uudelepp M" first="Mari-Liis" last="Uudelepp">Mari-Liis Uudelepp</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Pressler, Ronit" sort="Pressler, Ronit" uniqKey="Pressler R" first="Ronit" last="Pressler">Ronit Pressler</name><affiliation><nlm:aff wicri:cut=", and" id="aff2">Neurology Department</nlm:aff></affiliation></author><author><name sortKey="Stevenson, Danielle C" sort="Stevenson, Danielle C" uniqKey="Stevenson D" first="Danielle C." last="Stevenson">Danielle C. Stevenson</name><affiliation><nlm:aff id="aff2">Metabolic Medicine Unit,</nlm:aff></affiliation></author><author><name sortKey="Gabriel, Camila" sort="Gabriel, Camila" uniqKey="Gabriel C" first="Camila" last="Gabriel">Camila Gabriel</name><affiliation><nlm:aff id="aff2">Metabolic Medicine Unit,</nlm:aff></affiliation></author><author><name sortKey="Mcsweeney, Mel" sort="Mcsweeney, Mel" uniqKey="Mcsweeney M" first="Mel" last="Mcsweeney">Mel Mcsweeney</name><affiliation><nlm:aff id="aff2">Metabolic Medicine Unit,</nlm:aff></affiliation></author><author><name sortKey="Baggot, Matthew" sort="Baggot, Matthew" uniqKey="Baggot M" first="Matthew" last="Baggot">Matthew Baggot</name><affiliation><nlm:aff id="aff2">Chemical Pathology,<institution>Great Ormond Street Hospital for Children NHS Foundation Trust</institution>, London WC1N 3JH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Burke, Derek" sort="Burke, Derek" uniqKey="Burke D" first="Derek" last="Burke">Derek Burke</name><affiliation><nlm:aff id="aff2">Chemical Pathology,<institution>Great Ormond Street Hospital for Children NHS Foundation Trust</institution>, London WC1N 3JH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Stodberg, Tommy" sort="Stodberg, Tommy" uniqKey="Stodberg T" first="Tommy" last="Stödberg">Tommy Stödberg</name><affiliation><nlm:aff id="aff5">Neuropediatric Unit,<institution>Karolinska University Hospital</institution>, Stockholm SE-171 76,<country>Sweden</country></nlm:aff></affiliation></author><author><name sortKey="Riney, Kate" sort="Riney, Kate" uniqKey="Riney K" first="Kate" last="Riney">Kate Riney</name><affiliation><nlm:aff id="aff6">Neurosciences Unit,<institution>The Lady Cilento Children’s Hospital</institution>, 501 Stanley Street, South Brisbane, Queensland 4101,<country>Australia</country></nlm:aff></affiliation></author><author><name sortKey="Schiff, Manuel" sort="Schiff, Manuel" uniqKey="Schiff M" first="Manuel" last="Schiff">Manuel Schiff</name><affiliation><nlm:aff id="aff7">Reference Center for Inborn Errors of Metabolism,<institution>Robert Debré University Hospital</institution>, APHP, Paris 75019,<country>France</country></nlm:aff></affiliation></author><author><name sortKey="Heales, Simon J R" sort="Heales, Simon J R" uniqKey="Heales S" first="Simon J. R." last="Heales">Simon J. R. Heales</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Chemical Pathology,<institution>Great Ormond Street Hospital for Children NHS Foundation Trust</institution>, London WC1N 3JH,<country>United Kingdom</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff8">Neurometabolic Unit,<institution>National Hospital for Neurology and Neurosurgery</institution>, Queen Square, London WC1N 3BG,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Mills, Kevin A" sort="Mills, Kevin A" uniqKey="Mills K" first="Kevin A." last="Mills">Kevin A. Mills</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Gissen, Paul" sort="Gissen, Paul" uniqKey="Gissen P" first="Paul" last="Gissen">Paul Gissen</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Metabolic Medicine Unit,</nlm:aff></affiliation></author><author><name sortKey="Clayton, Peter T" sort="Clayton, Peter T" uniqKey="Clayton P" first="Peter T." last="Clayton">Peter T. Clayton</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Mills, Philippa B" sort="Mills, Philippa B" uniqKey="Mills P" first="Philippa B." last="Mills">Philippa B. Mills</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">28782931</idno><idno type="pmc">5588098</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5588098</idno><idno type="RBID">PMC:5588098</idno><idno type="doi">10.1021/acs.analchem.7b01358</idno><date when="2017">2017</date><idno type="wicri:Area/Pmc/Corpus">000689</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000689</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">An LC–MS/MS-Based Method for the Quantification
of Pyridox(am)ine 5′-Phosphate Oxidase Activity in Dried Blood
Spots from Patients with Epilepsy</title><author><name sortKey="Wilson, Matthew X0a P" sort="Wilson, Matthew X0a P" uniqKey="Wilson M" first="Matthew X0a P." last="Wilson">Matthew X0a P. Wilson</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Footitt, Emma J" sort="Footitt, Emma J" uniqKey="Footitt E" first="Emma J." last="Footitt">Emma J. Footitt</name><affiliation><nlm:aff id="aff2">Metabolic Medicine Unit,</nlm:aff></affiliation></author><author><name sortKey="Papandreou, Apostolos" sort="Papandreou, Apostolos" uniqKey="Papandreou A" first="Apostolos" last="Papandreou">Apostolos Papandreou</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Uudelepp, Mari Liis" sort="Uudelepp, Mari Liis" uniqKey="Uudelepp M" first="Mari-Liis" last="Uudelepp">Mari-Liis Uudelepp</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Pressler, Ronit" sort="Pressler, Ronit" uniqKey="Pressler R" first="Ronit" last="Pressler">Ronit Pressler</name><affiliation><nlm:aff wicri:cut=", and" id="aff2">Neurology Department</nlm:aff></affiliation></author><author><name sortKey="Stevenson, Danielle C" sort="Stevenson, Danielle C" uniqKey="Stevenson D" first="Danielle C." last="Stevenson">Danielle C. Stevenson</name><affiliation><nlm:aff id="aff2">Metabolic Medicine Unit,</nlm:aff></affiliation></author><author><name sortKey="Gabriel, Camila" sort="Gabriel, Camila" uniqKey="Gabriel C" first="Camila" last="Gabriel">Camila Gabriel</name><affiliation><nlm:aff id="aff2">Metabolic Medicine Unit,</nlm:aff></affiliation></author><author><name sortKey="Mcsweeney, Mel" sort="Mcsweeney, Mel" uniqKey="Mcsweeney M" first="Mel" last="Mcsweeney">Mel Mcsweeney</name><affiliation><nlm:aff id="aff2">Metabolic Medicine Unit,</nlm:aff></affiliation></author><author><name sortKey="Baggot, Matthew" sort="Baggot, Matthew" uniqKey="Baggot M" first="Matthew" last="Baggot">Matthew Baggot</name><affiliation><nlm:aff id="aff2">Chemical Pathology,<institution>Great Ormond Street Hospital for Children NHS Foundation Trust</institution>, London WC1N 3JH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Burke, Derek" sort="Burke, Derek" uniqKey="Burke D" first="Derek" last="Burke">Derek Burke</name><affiliation><nlm:aff id="aff2">Chemical Pathology,<institution>Great Ormond Street Hospital for Children NHS Foundation Trust</institution>, London WC1N 3JH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Stodberg, Tommy" sort="Stodberg, Tommy" uniqKey="Stodberg T" first="Tommy" last="Stödberg">Tommy Stödberg</name><affiliation><nlm:aff id="aff5">Neuropediatric Unit,<institution>Karolinska University Hospital</institution>, Stockholm SE-171 76,<country>Sweden</country></nlm:aff></affiliation></author><author><name sortKey="Riney, Kate" sort="Riney, Kate" uniqKey="Riney K" first="Kate" last="Riney">Kate Riney</name><affiliation><nlm:aff id="aff6">Neurosciences Unit,<institution>The Lady Cilento Children’s Hospital</institution>, 501 Stanley Street, South Brisbane, Queensland 4101,<country>Australia</country></nlm:aff></affiliation></author><author><name sortKey="Schiff, Manuel" sort="Schiff, Manuel" uniqKey="Schiff M" first="Manuel" last="Schiff">Manuel Schiff</name><affiliation><nlm:aff id="aff7">Reference Center for Inborn Errors of Metabolism,<institution>Robert Debré University Hospital</institution>, APHP, Paris 75019,<country>France</country></nlm:aff></affiliation></author><author><name sortKey="Heales, Simon J R" sort="Heales, Simon J R" uniqKey="Heales S" first="Simon J. R." last="Heales">Simon J. R. Heales</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Chemical Pathology,<institution>Great Ormond Street Hospital for Children NHS Foundation Trust</institution>, London WC1N 3JH,<country>United Kingdom</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff8">Neurometabolic Unit,<institution>National Hospital for Neurology and Neurosurgery</institution>, Queen Square, London WC1N 3BG,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Mills, Kevin A" sort="Mills, Kevin A" uniqKey="Mills K" first="Kevin A." last="Mills">Kevin A. Mills</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Gissen, Paul" sort="Gissen, Paul" uniqKey="Gissen P" first="Paul" last="Gissen">Paul Gissen</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Metabolic Medicine Unit,</nlm:aff></affiliation></author><author><name sortKey="Clayton, Peter T" sort="Clayton, Peter T" uniqKey="Clayton P" first="Peter T." last="Clayton">Peter T. Clayton</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation></author><author><name sortKey="Mills, Philippa B" sort="Mills, Philippa B" uniqKey="Mills P" first="Philippa B." last="Mills">Philippa B. Mills</name><affiliation><nlm:aff id="aff1">Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></nlm:aff></affiliation></author></analytic><series><title level="j">Analytical Chemistry</title><idno type="ISSN">0003-2700</idno><idno type="eISSN">1520-6882</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="ac-2017-01358c_0005" id="ab-tgr1"></graphic></p><p>We
report the development of a rapid, simple, and robust LC–MS/MS-based
enzyme assay using dried blood spots (DBS) for the diagnosis of pyridox(am)ine
5′-phosphate oxidase (PNPO) deficiency (OMIM 610090). PNPO
deficiency leads to potentially fatal early infantile epileptic encephalopathy,
severe developmental delay, and other features of neurological dysfunction.
However, upon prompt treatment with high doses of vitamin B<sub>6</sub>, affected patients can have a normal developmental outcome. Prognosis
of these patients is therefore reliant upon a rapid diagnosis. PNPO
activity was quantified by measuring pyridoxal 5′-phosphate
(PLP) concentrations in a DBS before and after a 30 min incubation
with pyridoxine 5′-phosphate (PNP). Samples from 18 PNPO deficient
patients (1 day–25 years), 13 children with other seizure disorders
receiving B<sub>6</sub> supplementation (1 month–16 years),
and 37 child hospital controls (5 days–15 years) were analyzed.
DBS from the PNPO-deficient samples showed enzyme activity levels
lower than all samples from these two other groups as well as seven
adult controls; no false positives or negatives were identified. The
method was fully validated and is suitable for translation into the
clinical diagnostic arena.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Clayton, P T" uniqKey="Clayton P">P. T. Clayton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mills, P B" uniqKey="Mills P">P. B. Mills</name></author><author><name sortKey="Camuzeaux, S S" uniqKey="Camuzeaux S">S. S. Camuzeaux</name></author><author><name sortKey="Footitt, E J" uniqKey="Footitt E">E. J. Footitt</name></author><author><name sortKey="Mills, K A" uniqKey="Mills K">K. A. Mills</name></author><author><name sortKey="Gissen, P" uniqKey="Gissen P">P. Gissen</name></author><author><name sortKey="Fisher, L" uniqKey="Fisher L">L. Fisher</name></author><author><name sortKey="Das, K B" uniqKey="Das K">K. B. Das</name></author><author><name sortKey="Varadkar, S M" uniqKey="Varadkar S">S. M. Varadkar</name></author><author><name sortKey="Zuberi, S" uniqKey="Zuberi S">S. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Anal Chem</journal-id><journal-id journal-id-type="iso-abbrev">Anal. Chem</journal-id><journal-id journal-id-type="publisher-id">ac</journal-id><journal-id journal-id-type="coden">ancham</journal-id><journal-title-group><journal-title>Analytical Chemistry</journal-title></journal-title-group><issn pub-type="ppub">0003-2700</issn><issn pub-type="epub">1520-6882</issn><publisher><publisher-name>American
Chemical
Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">28782931</article-id><article-id pub-id-type="pmc">5588098</article-id><article-id pub-id-type="doi">10.1021/acs.analchem.7b01358</article-id><article-categories><subj-group><subject>Article</subject></subj-group></article-categories><title-group><article-title>An LC–MS/MS-Based Method for the Quantification
of Pyridox(am)ine 5′-Phosphate Oxidase Activity in Dried Blood
Spots from Patients with Epilepsy</article-title></title-group><contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Wilson</surname><given-names>Matthew
P.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath2"><name><surname>Footitt</surname><given-names>Emma J.</given-names></name><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" id="ath3"><name><surname>Papandreou</surname><given-names>Apostolos</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath4"><name><surname>Uudelepp</surname><given-names>Mari-Liis</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath5"><name><surname>Pressler</surname><given-names>Ronit</given-names></name><xref rid="aff2" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="ath6"><name><surname>Stevenson</surname><given-names>Danielle C.</given-names></name><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" id="ath7"><name><surname>Gabriel</surname><given-names>Camila</given-names></name><xref rid="aff2" ref-type="aff">‡</xref><xref rid="notes-2" ref-type="notes">○</xref></contrib><contrib contrib-type="author" id="ath8"><name><surname>McSweeney</surname><given-names>Mel</given-names></name><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" id="ath9"><name><surname>Baggot</surname><given-names>Matthew</given-names></name><xref rid="aff2" ref-type="aff">∥</xref></contrib><contrib contrib-type="author" id="ath10"><name><surname>Burke</surname><given-names>Derek</given-names></name><xref rid="aff2" ref-type="aff">∥</xref></contrib><contrib contrib-type="author" id="ath11"><name><surname>Stödberg</surname><given-names>Tommy</given-names></name><xref rid="aff5" ref-type="aff">⊥</xref></contrib><contrib contrib-type="author" id="ath12"><name><surname>Riney</surname><given-names>Kate</given-names></name><xref rid="aff6" ref-type="aff">#</xref></contrib><contrib contrib-type="author" id="ath13"><name><surname>Schiff</surname><given-names>Manuel</given-names></name><xref rid="aff7" ref-type="aff">∇</xref></contrib><contrib contrib-type="author" id="ath14"><name><surname>Heales</surname><given-names>Simon J. R.</given-names></name><xref rid="aff1" ref-type="aff">†</xref><xref rid="aff2" ref-type="aff">∥</xref><xref rid="aff8" ref-type="aff">■</xref></contrib><contrib contrib-type="author" id="ath15"><name><surname>Mills</surname><given-names>Kevin A.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath16"><name><surname>Gissen</surname><given-names>Paul</given-names></name><xref rid="aff1" ref-type="aff">†</xref><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath17"><name><surname>Clayton</surname><given-names>Peter T.</given-names></name><xref rid="cor1" ref-type="other">*</xref><xref rid="aff1" ref-type="aff">†</xref><xref rid="notes-50" ref-type="notes">△</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath18"><name><surname>Mills</surname><given-names>Philippa B.</given-names></name><xref rid="cor2" ref-type="other">*</xref><xref rid="aff1" ref-type="aff">†</xref><xref rid="notes-50" ref-type="notes">△</xref></contrib><aff id="aff1"><label>†</label>Genetics and Genomic Medicine,<institution>UCL GOS Institute of Child Health</institution>, 30 Guilford Street, London WC1N 1EH,<country>United Kingdom</country></aff><aff id="aff2"><sup>‡</sup>Metabolic Medicine Unit,<sup>§</sup>Neurology Department, and<sup>∥</sup>Chemical Pathology,<institution>Great Ormond Street Hospital for Children NHS Foundation Trust</institution>, London WC1N 3JH,<country>United Kingdom</country></aff><aff id="aff5"><label>⊥</label>Neuropediatric Unit,<institution>Karolinska University Hospital</institution>, Stockholm SE-171 76,<country>Sweden</country></aff><aff id="aff6"><label>#</label>Neurosciences Unit,<institution>The Lady Cilento Children’s Hospital</institution>, 501 Stanley Street, South Brisbane, Queensland 4101,<country>Australia</country></aff><aff id="aff7"><label>∇</label>Reference Center for Inborn Errors of Metabolism,<institution>Robert Debré University Hospital</institution>, APHP, Paris 75019,<country>France</country></aff><aff id="aff8"><label>■</label>Neurometabolic Unit,<institution>National Hospital for Neurology and Neurosurgery</institution>, Queen Square, London WC1N 3BG,<country>United Kingdom</country></aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>E-mail: <email>peter.clayton@ucl.ac.uk</email>.</corresp><corresp id="cor2"><label>*</label>E-mail: <email>p.mills@ucl.ac.uk</email>.</corresp></author-notes><pub-date pub-type="epub"><day>07</day><month>08</month><year>2017</year></pub-date><pub-date pub-type="ppub"><day>05</day><month>09</month><year>2017</year></pub-date><volume>89</volume><issue>17</issue><fpage>8892</fpage><lpage>8900</lpage><history><date date-type="received"><day>11</day><month>04</month><year>2017</year></date><date date-type="accepted"><day>07</day><month>08</month><year>2017</year></date></history><permissions><copyright-statement>Copyright © 2017 American Chemical Society</copyright-statement><copyright-year>2017</copyright-year><copyright-holder>American Chemical Society</copyright-holder><license><license-p>This is an open access article published under a Creative Commons Attribution (CC-BY) <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html">License</ext-link>, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.</license-p></license></permissions><abstract><p content-type="toc-graphic"><graphic xlink:href="ac-2017-01358c_0005" id="ab-tgr1"></graphic></p><p>We
report the development of a rapid, simple, and robust LC–MS/MS-based
enzyme assay using dried blood spots (DBS) for the diagnosis of pyridox(am)ine
5′-phosphate oxidase (PNPO) deficiency (OMIM 610090). PNPO
deficiency leads to potentially fatal early infantile epileptic encephalopathy,
severe developmental delay, and other features of neurological dysfunction.
However, upon prompt treatment with high doses of vitamin B<sub>6</sub>, affected patients can have a normal developmental outcome. Prognosis
of these patients is therefore reliant upon a rapid diagnosis. PNPO
activity was quantified by measuring pyridoxal 5′-phosphate
(PLP) concentrations in a DBS before and after a 30 min incubation
with pyridoxine 5′-phosphate (PNP). Samples from 18 PNPO deficient
patients (1 day–25 years), 13 children with other seizure disorders
receiving B<sub>6</sub> supplementation (1 month–16 years),
and 37 child hospital controls (5 days–15 years) were analyzed.
DBS from the PNPO-deficient samples showed enzyme activity levels
lower than all samples from these two other groups as well as seven
adult controls; no false positives or negatives were identified. The
method was fully validated and is suitable for translation into the
clinical diagnostic arena.</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>ac7b01358</meta-value></custom-meta><custom-meta><meta-name>document-id-new-14</meta-name><meta-value>ac-2017-01358c</meta-value></custom-meta><custom-meta><meta-name>ccc-price</meta-name><meta-value></meta-value></custom-meta></custom-meta-group></article-meta></front><body><p id="sec1">Pyridox(am)ine
5′-phosphate
oxidase (PNPO) deficiency (OMIM 610090) is an autosomal recessive
seizure disorder caused by impaired activity of the PNPO enzyme, which
catalyzes the oxidation of pyridoxamine 5′-phosphate (PMP)
and pyridoxine 5′-phosphate (PNP) to pyridoxal 5′-phosphate
(PLP), the active form of vitamin B<sub>6</sub>.</p><p>Dietary vitamin
B<sub>6</sub> is consumed as pyridoxine, pyridoxamine,
and pyridoxal (PN, PM, and PL), their phosphorylated analogues, and
the glucoside of PN.<sup><xref ref-type="bibr" rid="ref1">1</xref>−<xref ref-type="bibr" rid="ref4">4</xref></sup> The hydrolyzed vitamers are absorbed, and PN and PM undergo phosphorylation
prior to their conversion to PLP by PNPO (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>). The Enzyme Commission (<uri xlink:href="http://www.chem.qmul.ac.uk/iubmb/enzyme/">http://www.chem.qmul.ac.uk/iubmb/enzyme/</uri>) catalogues over 140 PLP-dependent enzymatic activities. In humans,
PLP-dependent enzymes include several involved in the synthesis or
degradation of amino acids or amines that serve as neurotransmitters
or neuromodulators in the brain.</p><fig id="fig1" position="float"><label>Figure 1</label><caption><p>Vitamin B<sub>6</sub> metabolism. (a)
Pyridoxine, (b) pyridoxal,
(c) pyridoxamine, (d) pyridoxine 5′-phosphate, (e) pyridoxal
5′-phosphate, and (f) pyridoxamine 5′-phosphate. PK
= pyridoxal kinase; PNPO = pyridox(am)ine 5′-phosphate oxidase.</p></caption><graphic xlink:href="ac-2017-01358c_0001" id="gr1" position="float"></graphic></fig><p>Classical PNPO deficiency has
been described as epileptic encephalopathy
refractive to conventional anticonvulsants and pyridoxine but responsive
to high doses (30–40 mg kg<sup>–1</sup> day<sup>–1</sup>) of PLP.<sup><xref ref-type="bibr" rid="ref5">5</xref></sup> In recent years, however, considerable
heterogeneity has been identified in the phenotype of affected patients
with approximately 40%<sup><xref ref-type="bibr" rid="ref6">6</xref></sup> of PNPO-deficient
patients showing some response to pyridoxine. Some of these individuals
have not tolerated a switch in treatment to PLP with their seizure
control worsening. In addition, patients have presented with seizures
after the neonatal period.<sup><xref ref-type="bibr" rid="ref2">2</xref>,<xref ref-type="bibr" rid="ref3">3</xref></sup> Patients that have severe
neonatal epileptic encephalopathy but survive into infancy have a
high disease burden with severe developmental delay, progressive seizures,
and microcephaly.<sup><xref ref-type="bibr" rid="ref2">2</xref>,<xref ref-type="bibr" rid="ref3">3</xref>,<xref ref-type="bibr" rid="ref7">7</xref></sup> However,
when diagnosed and treated early, patients can have a normal neurodevelopmental
outcome.<sup><xref ref-type="bibr" rid="ref8">8</xref></sup></p><p>Diagnosing patients with
PNPO deficiency can be challenging. There
are many practical difficulties in relying upon a clinical response
to PLP or PN as often neonates and infants can have multisystem pathology
and the response to treatment may not be immediate and total. Additionally,
there are other seizure disorders that respond to treatment with PLP
and/or PN, which cannot be differentiated from PNPO deficiency using
this approach.<sup><xref ref-type="bibr" rid="ref9">9</xref></sup> Seizures caused by pyridoxine-dependent
epilepsy (PDE) can be indistinguishable from those caused by a deficiency
of PNPO. Hyperprolinaemia type 2 (HPII) or hypophosphatasia can also
cause perinatal B<sub>6</sub>-responsive seizures.<sup><xref ref-type="bibr" rid="ref1">1</xref></sup> Other neurological disorders known to benefit from PLP/pyridoxine
supplementation include KCNQ2 deficiency and AADC deficiency.</p><p>Although biochemical findings that were initially thought to be
characteristic of PNPO deficiency can be indicative,<sup><xref ref-type="bibr" rid="ref5">5</xref></sup> for some individuals these may be present only transiently
or absent altogether. A more recent study has suggested that a high
plasma PM concentration can be indicative of patients with PNPO deficiency
irrespective of vitamin B<sub>6</sub> supplementation and that this
in conjunction with an elevated PM/pyridoxic acid (PA) ratio is a
biomarker for the selective screening of PNPO deficiency.<sup><xref ref-type="bibr" rid="ref10">10</xref></sup> However, this study used a small sample size
(<italic>n</italic> = 6), and it is currently still necessary to confirm
these findings genetically. Current practice relies on the detection
of mutations in the <italic>PNPO</italic> gene and subsequent expression
studies to confirm pathogenicity of any variants detected.<sup><xref ref-type="bibr" rid="ref11">11</xref>,<xref ref-type="bibr" rid="ref12">12</xref></sup> This is often carried out after the exclusion of other B<sub>6</sub>-responsive disorders such as pyridoxine-dependent epilepsy due to
mutations in <italic>ALDH7A1</italic> (PDE) and can cause a considerable
delay in diagnosis.</p><p>Published assays for the determination of
PNPO activity have shown
that human erythrocytes convert pyridoxamine 5′-phosphate (PMP),
a substrate of PNPO, to PLP. However, these assays are laborious,
not suitable for routine clinical diagnosis, require large sample
volumes, or use radiolabeled substrates.<sup><xref ref-type="bibr" rid="ref13">13</xref>−<xref ref-type="bibr" rid="ref18">18</xref></sup> The use of dried blood spots (DBS) from a heel or finger-prick is
an established method for sample collection, which was developed initially
for the measurement of phenylalanine levels in the diagnosis of phenylketonuria
in the 1960s.<sup><xref ref-type="bibr" rid="ref19">19</xref></sup> Subsequently, the use of
DBS to assay enzymes present in the circulating blood has been reported
for various metabolic disorders.<sup><xref ref-type="bibr" rid="ref20">20</xref></sup> The
noninvasive collection and simple transport/storage are a major advantage
of DBS.</p><p>We have recently established a mass spectrometry-based
method for
quantification of the individual forms of vitamin B<sub>6</sub><sup><xref ref-type="bibr" rid="ref21">21</xref></sup> and have further developed this to enable us
to assay PNPO activity in DBS. This highly sensitive method is more
specific than published methods and allows quantification of PNPO
activity without using large sample volumes or radiolabeled substrates.
Here, we report the development and validation of this assay as well
as its utility for the analysis of control and PNPO-deficient patient
samples.</p><sec id="sec2"><title>Experimental Section</title><sec id="sec2.1"><title>Sample Collection and Storage of DBS</title><p>This study was
approved by the National Research Ethics Service (NRES) Committee
(London, Bloomsbury [REC ref no. 3/LO/0168]). The majority of control
DBS were obtained from patients attending Great Ormond Street
Hospital for Children, London, UK. Written informed consent was obtained
for all subjects, and inclusion/exclusion criteria are discussed below.
DBS samples were also received from patients with undiagnosed B<sub>6</sub>-responsive seizure disorders, after informed consent, from
centers across Europe. DBS for validation of the measurement of PNPO
activity were collected from healthy adults.</p><p>Whatman 903 filter
paper was used for sample collection. Blood, either venous or capillary,
was spotted in the middle of the preprinted circle of a Protein Saver
card (GE Healthcare, Little Chalfont, Bucks, UK). The spotted blood
was allowed to dry for 16–24 h at room temperature (22 °C)
prior to the card being placed in a sealed foil zip-lock bag with
desiccant (to protect from light and humidity) at room temperature
for a maximum of 7 days. DBS samples were then stored at −20
or −80 °C until analysis.</p><p>Cards were visually inspected
upon analysis and rejected if poor
sampling protocol was suspected. Care was taken to avoid sample collection
from recipients of blood transfusions within the preceding 100 days.</p></sec><sec id="sec2.2"><title>Materials</title><p>Trizma base, PLP, LC–MS/MS grade acetic
acid, heptafluorobutyric acid (HFBA), PMP, PL, PM, <italic>d</italic><sub>3</sub>-PM, PA, <italic>d</italic><sub>2</sub>-PA, and PN were
purchased from Sigma-Aldrich (Gillingham, Dorset, UK). Orthophosphoric
acid was purchased from BDH Chemicals (Poole, Dorset, UK). PNP was
purchased from Toronto Research Chemicals (Toronto, Canada). <italic>d</italic><sub>2</sub>-PN and <italic>d</italic><sub>3</sub>-PLP
were purchased from CDN Isotopes (Thaxted, Essex, UK) and Buchem BV
(Appeldoorn, The Netherlands), respectively. 6.1 N (=6.1 M) trichloroacetic
acid (TCA) was purchased from MP Biomedicals (Solon, U.S.). Whatman
desiccant was purchased from Scientific Laboratory Supplies (Nottingham,
UK). Flavin mononucleotide (FMN) was purchased from Applichem (Darmstadt,
Germany). All water used was purified by a Millipore Milli-Q Direct
8 system with a 0.22 μm filter.</p></sec><sec id="sec2.3"><title>Measurement of PNPO Activity</title><p>For each patient sample,
two 3 mm punches (T0 and T30) were taken from the spotted blood card.
These punches were rehydrated in a 1.5 mL polypropylene tube containing
60 μL of a 40 mmol/L Tris-phosphate buffer, prepared by adjusting
a 40 mmol/L Tris solution to pH 7.6 using 8.5% orthophosphoric acid.
The tubes were then sonicated in a Grant XUBA3 ultrasonic bath for
2 min. Subsequently, 60 μL of a reaction mix containing 800
nmol/L PNP substrate and 3 μmol/L FMN was added to each tube
to a final concentration of 400 nmol/L and 1.5 μmol/L, respectively.
120 μL of a reaction stop mix (0.3 M TCA containing 50 nmol/L
of the internal standard <italic>d</italic><sub>3</sub>-PLP to a final
concentration of 0.15 M and 25 nmol/L, respectively) was added immediately
to tube T0 prior to incubation on ice for 45 min. Tube T30 was incubated
for 30 min at 37 °C with shaking at 300 rpm in an Eppendorf Thermomixer
C prior to the addition of 120 μL of reaction stop mix and incubation
on ice for 45 min. After incubation with TCA, all samples were sonicated
for 5 min prior to centrifugation at 16 000<italic>g</italic> for 10 min at 4 °C. The resulting supernatant, containing the
B<sub>6</sub> vitamers, was removed and stored at −20 °C
until time of analysis. All samples were recentrifuged at 16 000<italic>g</italic> for 10 min at 4 °C after thawing, and the supernatants
were transferred to 300 μL amber insert vials (Fisher Scientific).
Because of the known photolability of PLP,<sup><xref ref-type="bibr" rid="ref22">22</xref>,<xref ref-type="bibr" rid="ref23">23</xref></sup> care was taken at all times to avoid light exposure, and all standards
were stored on ice during experimentation. For long-term storage,
B<sub>6</sub> vitamer stock solutions were kept in aqueous form at
−80 °C. TCA addition and subsequent incubation was necessary
for the precipitation of protein found in the DBS, release of protein-bound
PLP into solution, and cessation of enzymatic activity. Acidic conditions
are also known to stabilize the B<sub>6</sub> vitamers in aqueous
solution.<sup><xref ref-type="bibr" rid="ref24">24</xref></sup></p></sec><sec id="sec2.4"><title>Liquid Chromatography–Mass
Spectrometry</title><p>Quantification
of the B<sub>6</sub> vitamers and pyridoxic acid was performed essentially
as described in Footitt et al.<sup><xref ref-type="bibr" rid="ref21">21</xref></sup> with minor
modifications. UPLC–MS/MS was carried out using a Waters Acquity
H-Class UPLC system connected to a Waters Xevo TQ-S triple quadrupole
mass spectrometer using electrospray ionization and multiple reaction
monitoring (MRM). All compounds were detected in positive ion mode.
Eight microliters of sample was flow-injected by the autosampler onto
a Waters Acquity UPLC HSS T3 column (1.8 μm, 2.1 × 50 mm)
protected by a 1.8 μm Acquity UPLC HSS T3 guard column. The
mobile phase components consisted of (A) 3.7% acetic acid in water
with 0.01% heptafluorobutyric acid (HFBA) and (B) 100% methanol. The
gradient profile used is as detailed in <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Table S-1</ext-link>, and the flow rate was 0.4 mL/min. A representative chromatogram
can be found in <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Figure S-1</ext-link>. The precursor
and product ions used for the detection of PLP, PNP, PMP, PL, PN,
PM, PA, <italic>d</italic><sub>3</sub>-PLP, <italic>d</italic><sub>2</sub>-PN, <italic>d</italic><sub>3</sub>-PM, and <italic>d</italic><sub>2</sub>-PA can be found in <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Table S-2</ext-link>. The retention times observed as well as the cone voltages and collision
energies used for the analysis of these compounds are also detailed
in <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Table S-2</ext-link>. The mass spectrometry settings
were as follows: capillary voltage, 2.50 kV; source temperature, 150
°C; desolvation temperature, 600 °C; cone gas flow rate,
150 L/h; desolvation gas flow rate, 1200 L/h; dwell time, 17 ms. Data
collection and analysis was performed using Waters MassLynx software.</p><p>PNPO activity was calculated by subtracting the endogenous PLP
measured at 0 min in the T0 DBS punch from the amount of PLP present
after 30 min of incubation of the DBS with PNP substrate in the T30
DBS punch. The enzyme activity was calculated and reported as pmol
PLP (3 mm DBS)<sup>−1</sup> h<sup>–1</sup>. To make
comparisons with published data, we made the assumption that a 3 mm
DBS punch contains 3.2 μL of blood,<sup><xref ref-type="bibr" rid="ref23">23</xref></sup> and a conversion factor of 0.3125 was used to convert this activity
to pmol PLP μL blood<sup>–1</sup> h<sup>–1</sup>. Although not used to quantify PNPO activity, concentrations of
PNP were routinely quantified. During assay optimization, PMP, PL,
PN, PM, and PA were also quantified. Despite having similar retention
times, all analytes were well differentiated, and there was no cross
talk between ion pairs except at high PNP concentrations as discussed
below. Calibration curves from 1.25 to 200 nmol/L were constructed
using the analyte:internal standard peak area ratios of PLP, PNP,
and PMP:<italic>d</italic><sub>3</sub>-PLP and were linear over this
range (<italic>r</italic><sup>2</sup> > 0.99). A calibration curve,
over the same range, was constructed for PM:<italic>d</italic><sub>3</sub>-PM, PA:<italic>d</italic><sub>2</sub>-PA, and PN:<italic>d</italic><sub>2</sub>-PN; these were also found to be linear (<italic>r</italic><sup>2</sup> > 0.99). Pyridoxal was not quantified using
a calibration curve or internal standard as measurement was only performed
in early stages of method development. The area under the curve (AUC)
upon LC–MS/MS elution was thus used to quantify pyridoxal levels.</p></sec><sec id="sec2.5"><title>Statistical Analysis</title><p>All analyses were carried out
using GraphPad Prism version 6.05 for Windows (GraphPad Software,
La Jolla, CA). Statistical analysis was performed using one-way ANOVA
followed by Tukey’s multiple comparisons test; ** = <italic>P</italic> < 0.01; **** = <italic>P</italic> < 0.0001.</p></sec></sec><sec id="sec3"><title>Results
and Discussion</title><sec id="sec3.1"><title>PNPO Assay Optimization</title><p>The DBS
assay was evaluated
and optimized with regards to pH, buffer, incubation time, and cofactor/substrate
concentrations.</p><p>PNPO enzyme activity in erythrocytes has been
reported to be optimal at pH’s between pH 7.1 and 8.0.<sup><xref ref-type="bibr" rid="ref13">13</xref>,<xref ref-type="bibr" rid="ref15">15</xref></sup> The pH dependence of the enzyme in the DBS was verified over this
range, and it was found that there was little difference in PNPO activity
when a 3 mm DBS from an adult control was incubated at pH’s
at or between pH 7–8 (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Table S-3</ext-link>).
Analysis of the chromatogram of samples incubated at pHs > 7.8,
however,
revealed peak-splitting of PLP. Therefore, all subsequent incubation
conditions were optimized while keeping the pH constant at 7.6.</p><p>The effects of different biological buffers on PNPO activity that
were capable of buffering at pH 7–8 were investigated. A Tris-HCl
buffer system, similar to that which we have used previously for analysis
of PNPO activity in Chinese Hamster ovary cells overexpressing human
PNPO,<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> was considered initially. PNPO is
regulated by feedback inhibition of its product, PLP, and because
Tris complexes with PLP it would therefore be expected to minimize
any such inhibition.<sup><xref ref-type="bibr" rid="ref25">25</xref></sup> However, it was
evident that under the conditions used, approximately 12% of the PLP
formed was subsequently hydrolyzed to PL (data not shown) presumably
by the activity of multiple endogenous phosphatases that are present
in blood, which hydrolyze the 5′ ester bond of PLP. Inorganic
phosphate is known to be a product inhibitor of phosphatase activity;<sup><xref ref-type="bibr" rid="ref15">15</xref>,<xref ref-type="bibr" rid="ref26">26</xref></sup> therefore, a custom 20 mmol/L Tris-phosphate buffer (pH 7.6) was
adopted, and at the concentration of phosphate present negligible
PLP hydrolysis was observed (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Figure S-2</ext-link>). There was additional concern, particularly for the analysis of
DBS from individuals on high-dose supplementation where the levels
of B<sub>6</sub> vitamers would be elevated, that any endogenous pyridoxal
kinase activity present in the DBS would act on endogenous PL levels
to form PLP and that PLP measured in the T30 DBS would not be solely
representative of PNPO activity but of combined PNPO and pyridoxal
kinase activity. Previous studies have shown, however, that pyridoxal
kinase requires the presence of ATP and a divalent cation (e.g., Mg<sup>2+</sup>) for activity,<sup><xref ref-type="bibr" rid="ref27">27</xref>,<xref ref-type="bibr" rid="ref28">28</xref></sup> cofactors that are
not added to the PNPO reaction buffer. We have confirmed these findings
in our DBS system. In the absence of exogenous ATP and Mg<sup>2+</sup>, PN phosphorylation to PNP was undetectable (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Figure S-3</ext-link>).</p><p>PNP was chosen as a substrate for the assay
rather than PMP because
oxidation of PNP to PLP, under the conditions employed, was much more
rapid than that observed when PMP was used as a substrate. The <italic>V</italic><sub>max</sub> of PNP conversion to PLP upon the incubation
of a 3 mm adult control DBS was found to be approximately twice that
of PMP to PLP (39.5 vs 19.8 pmol DBS<sup>–1</sup> h<sup>–1</sup>). Equally, the <italic>K</italic><sub>m</sub> for PNP was 0.32 μmol/L
as compared to 0.53 μmol/L for PMP (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Figure S-4</ext-link>). The conversion of PMP (400 nmol/L) to PLP, when incubated
for 30 min with an adult control DBS, was only 14.1% of that observed
when the same concentration of PNP was used as a substrate (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Figure S-5</ext-link>). In addition, the PMP standard utilized
was found to contain a not insignificant (1–2%) proportion
of PLP. This background of PLP could interfere with measurement of
endogenous PLP in the DBS and lead to feedback inhibition.</p><p>A
concentration of 400 nmol/L PNP was used for the assay for several
reasons: (i) This concentration of PNP permitted analysis of the resulting
supernatant by UPLC–MS/MS without further dilution steps. (ii)
Upon the investigation of higher PNP concentrations for the determination
of Michaelis–Menten kinetics, it was noted that there was a
small amount of cross talk between the MRM channels for PNP and <italic>d</italic><sub>3</sub>-PLP (<1%). Although at a concentration
of 200 nmol/L upon analysis (as in the T0 repeats of the assay in
its current form) the effect was negligible, at higher PNP concentrations
(>1 μmol/L) interference was unacceptable and could affect
results
through aberrant PLP quantification. (iii) Saturation of the enzyme
with substrate will not facilitate detection of PNPO deficiency in
patients with mutations that cause an alteration in the <italic>K</italic><sub>m</sub> of PNPO where the enzyme has residual activity and the <italic>V</italic><sub>max</sub> and <italic>K</italic><sub>cat</sub> are
relatively unaffected. By mildly limiting the substrate concentration
but still achieving easily measurable conversion in controls, it is
likely that the diagnosis of PNPO deficiency in patients with “milder”
mutations is enabled.</p><p>There were concerns that the concentration
of FMN, the cofactor
for PNPO, in DBS samples may be dependent upon the vitamin B<sub>2</sub> status of each subject. Indeed, activity in an adult control measured
in the presence of endogenous FMN was only 61% of that seen when 1.5
μmol/L FMN was added to the reaction buffer, a concentration
that we have used previously when studying PNPO activity in an overexpression
system.<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> To ensure that the concentration
of FMN used in the assay was not limiting the activity of PNPO, punches
from DBS collected from the same control subject were incubated with
varying concentrations of FMN. No significant difference in PNPO activity
was observed when 0.75–3 μmol/L FMN was included in the
reaction buffer (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Figure S-5</ext-link>). A cofactor
concentration of 1.5 μmol/L was used for all subsequent experimentation.</p><p>PNPO activity as a function of the amount of enzyme was established
by incubating the substrate with an increasing weight of dried blood
spot. Initial experiments showed that it was possible to accurately
quantify PNPO activity using a 3 mm DBS punch, the weight of which
is approximately 26 mg. PLP production was measured in the presence
of varying amounts of DBS (0–80 mg) prepared using blood from
a healthy adult volunteer. An approximately linear correlation was
observed to 44 mg (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Figure S-6</ext-link>). No PLP
formation was observed when a 3 mm punch from a blank Whatman 903
card was incubated for 30 min under the final assay conditions; hence
the enzyme activity in the DBS samples was not corrected.</p><p>The
linearity of PLP production from PNP, under the conditions
described above, was monitored in two healthy adult controls and one
child hospital control (age 7 months) over 120 min. Product formation
increased linearly for the first 60 min in all controls (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Figure S-7</ext-link>). It was decided that an incubation
time of 30 min would be used for all analyses as this provided adequate
PLP formation for accurate quantification by LC-MS/MS while keeping
the incubation time as short as possible to increase throughput in
a clinical diagnostic arena. It also ensured linearity of the assay
in subjects with particularly high PNPO activity.</p></sec><sec id="sec3.2"><title>Imprecision
and Limits of Detection and Quantification</title><p>To validate assay
precision, PNPO activity was measured in DBS from
one healthy adult control. The intra-assay precision was 7.9% and
was determined by measuring the PNPO activity in 10 different DBS
punches from the same individual on 1 day. Interassay variability
was determined by measuring enzyme activity in 10 different DBS punches
from the same individual 5 times over a 4 week period. The observed
interassay variability was 10.3%.</p><p>For quantification of PLP
and PNP, the lower and upper limits of quantification (LLOQ and ULOQ)
were set at the lower and upper bounds of the calibration curve created.
This corresponds to concentrations of 1.25 and 200 nmol/L, respectively.
At LLOQ, the signal/noise (S/N) ratio was greater than 10. The limit
of detection (S/N greater than 3) was therefore lower than 1.25 nmol/L.
It was ensured that, when back-calculated, the values at LLOQ and
ULOQ never deviated from predetermined criteria specified by the European
Medicines Agency analytical consideration guidelines (<20% imprecision
for LLOQ and <15% for ULOQ). QC standards of PLP and PNP were analyzed
alongside each LC–MS/MS run to determine precision of measurement.
The %CVs of five repeated injections of QC standards of 5, 10, 100,
and 175 nmol/L were 2.28%, 0.90%, 2.30%, and 3.45%, respectively.</p><p>While the concentration of endogenous PLP levels was below the
limits of quantification (<1.25 nmol/L in the reaction/stop mixture,
equivalent to 94 nmol/L whole blood) in controls not on B<sub>6</sub> supplementation, in individuals on supplementation the concentration
of endogenous PLP in the reaction/stop mixture ranged from 2.5–199
nmol/L. Accuracy of the extraction method of PLP from DBS was investigated
to ensure that a high endogenous concentration of PLP in the T0 punch
would not lead to discrepancies in measured PNPO activity. The intra
and interassay %CVs for the extraction of endogenous PLP in a DBS
card collected from a PNPO-deficient patient on supra-physiological
doses (42 mg/kg/day) of B<sub>6</sub> were 5.92 and 7.76, respectively.</p></sec><sec id="sec3.3"><title>Effect of the Presence of Supra-physiological Concentrations
of B<sub>6</sub> Vitamers on Pyridox(am)ine 5′-Phosphate Oxidase
Activity</title><p>While DBS were incubated in a Tris-based buffer
system to minimize any feedback inhibition that PLP may have on PNPO
activity, there was concern that PLP present in DBS from patients
on supra-physiological doses may inhibit PNPO activity. DBS from a
healthy adult not on supplementation were incubated with increasing
concentrations of PLP added to the reaction buffer (0–400 nmol/L)
corresponding to concentrations of 0–200 nmol/L in the reaction/stop
mixture after TCA precipitation (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Table S-4</ext-link>). The PLP concentrations present in the reaction/stop mixture observed
in controls and PNPO patients on B<sub>6</sub> supplementation were
2.5–199 nmol/L. No product inhibition was seen in the presence
of PLP concentrations ≤50 nmol/L. Product inhibition was observed
at concentrations higher than this with a decrease in PNPO activity
by 15–20% in the presence of 100–150 nmol/L PLP. The
majority of patients analyzed receiving B<sub>6</sub> supplements
(13/18 PNPO; 14/16 others) had concentrations of PLP < 100 nmol/L
in the T0 DBS reaction/stop mixture (data not shown).</p><p>PNPO-deficient
patients have been reported to have high concentrations of PMP, and
sometimes PNP, in plasma.<sup><xref ref-type="bibr" rid="ref21">21</xref></sup> Concentrations
of endogenous PNP and PMP were therefore measured in the T0 DBS punches
so that we could evaluate the effect of these vitamers on PNPO activity.
In control samples from individuals not receiving/on high dose B<sub>6</sub> supplementation (that were not PNPO deficient), the concentrations
of PNP and PMP in the reaction/stop mixture were below the LLOQ (1.25
nmol/L). As compared to the concentration of PNP added to the assay
(200 nmol/L), this is negligible. The concentration of PNP and PMP
in the reaction/stop mixture of PNPO-deficient patients ranged from
0–137 and 0–121 nmol/L, respectively. The total concentration
of substrate present in the reaction mixture (i.e., endogenous and
added) of some PNPO-deficient patients will be higher therefore than
that of control samples. However, as mentioned above, our data show
that the rate of oxidation of PMP using the assay conditions employed
was far lower than that of PNP. When a 3 mm DBS from an adult control
was incubated with 400 nmol/L PMP in addition to 400 nmol/L PNP (equivalent
to 200 nmol/L upon analysis of the reaction/stop mixture), a small
decrease in PLP formation was identified; however, this was not statistically
significant (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Figure S-5</ext-link>). PMP is not thought
to inhibit PNPO activity;<sup><xref ref-type="bibr" rid="ref25">25</xref></sup> therefore,
high endogenous PMP levels should not significantly affect results.</p></sec><sec id="sec3.4"><title>Pyridox(am)ine 5′-Phosphate Oxidase Activity in Dried
Blood Spots Following Storage</title><p>Stability studies were performed
using DBS from a healthy adult control. Venous blood was collected
and immediately spotted onto Whatman 903 Protein Saver cards. PNPO
stability was evaluated after storage of the card at various temperatures.
Previous studies have shown that ambient humidity can affect enzyme
stability;<sup><xref ref-type="bibr" rid="ref29">29</xref>−<xref ref-type="bibr" rid="ref31">31</xref></sup> hence, duplicate cards were stored, one without desiccant
and the other in a sealed foil bag with desiccant after drying for
16–24 h. Enzyme activities observed after storage were compared
to the PNPO activity present in a DBS after the 16–24 h drying
period.</p><p>DBS collected for short-term stability studies were
stored in the dark at 22 °C for 3, 5, 7, 14, and 28 days with/without
desiccant (<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>). All cards were then stored with desiccant
at −80 °C before being assayed on day 28. When stored
at room temperature without desiccant for 3 days, a residual activity
of 83% was seen. This decreased to 52% over the 28-day period studied.
As expected, the decrease in activity was mitigated when DBS were
stored with desiccant in sealed foil bags after the initial 16–24
h drying period. Residual activity under these conditions was 92%
and 74% after 3 and 28 days, respectively.</p><fig id="fig2" position="float"><label>Figure 2</label><caption><p>Effect of humidity on
the short-term stability of the PNPO enzyme
in dried blood spots. Error bars indicate SEM. DBS were stored in
sealed foil bags with desiccant (▲) or under ambient conditions
(■) at room temperature (22 °C). Data points represent
the mean percentage of activity as compared to that found after 1
day (<italic>n</italic> = 2).</p></caption><graphic xlink:href="ac-2017-01358c_0002" id="gr2" position="float"></graphic></fig><p>Long-term stability
of
the enzyme in DBS was determined after storage for 1, 4, 8, and 12
weeks at 4, −20, and −80 °C, again after an initial
16–24 h drying period at 22 °C (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>). After storage for 12 weeks, a reduction
in activity of 27% was measured in those spots stored at 4 °C.
In DBS stored at −20 and −80 °C, activity was 92.7%
and 87.0%, respectively, as compared to baseline levels after 12 weeks.
Under these conditions, minimal difference was seen in the activity
of the enzyme after storage of the DBS without desiccant.</p><fig id="fig3" position="float"><label>Figure 3</label><caption><p>Effect of temperature
(4 °C [●], −20 °C
[■], and −80 °C [▲]) on the long-term stability
of the PNPO enzyme in dried blood spots (DBS). Data points represent
the mean percentage of PNPO activity relative to measurement at time
zero. Error bars indicate SEM. No significant difference was seen
in activities of samples with/without desiccant; the data from DBS
stored both with (<italic>n</italic> = 2) and without (<italic>n</italic> = 2) desiccant at each temperature have therefore been combined
(<italic>n</italic> = 4).</p></caption><graphic xlink:href="ac-2017-01358c_0003" id="gr3" position="float"></graphic></fig></sec><sec id="sec3.5"><title>Analysis of Clinical Samples</title><p>PNPO enzyme activity was
measured in DBS from 37 child hospital controls (age range 5 days–15
years) and 7 healthy adult controls. These individuals did not have
seizures. Sixteen samples were also collected from children with other
seizure disorders receiving vitamin B<sub>6</sub> supplementation
in whom either PNPO deficiency had been excluded by Sanger sequencing
or another conclusive genetic diagnosis had been made; this included
2 individuals with mutations in <italic>ALDH7A1</italic>, 1 subject
with PROSC deficiency,<sup><xref ref-type="bibr" rid="ref32">32</xref></sup> 7 subjects with
other genetic seizure disorders, and 6 individuals in whom the etiological
basis of their seizures is unknown (age range 1 month–16 years).
Eighteen samples were collected from subjects (age range 1 day–25
years) with known variants in the <italic>PNPO</italic> gene (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Table S-5</ext-link>). Three of these samples were from
individuals homozygous for the PNPO variant p.R116Q. We were interested
in looking at the effect of p.R116Q as it is predicted to affect FMN
binding and binding of pyridox(am)ine 5′-phosphate oxidase
monomers to form the dimeric form of the enzyme; however, previous
in vitro studies have suggested that this variant has high residual
activity and indeed it is present in the ExAC database with an allele
frequency of 0.0558, suggesting it could be a polymorphism.<sup><xref ref-type="bibr" rid="ref33">33</xref></sup> We have concluded previously,<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> because of its higher incidence in B<sub>6</sub>-responsive
epilepsy patients relative to the general population, that it contributes
to the pathogenesis of epilepsy and that it may be a common mutation,
perhaps responsible for the susceptibility locus for genetic generalized
epilepsy on 17q21.32 (close to rs72823592).<sup><xref ref-type="bibr" rid="ref34">34</xref></sup> Two of the individuals homozygous for p.R116Q (subjects 3 and 13)
receive B<sub>6</sub> for the treatment of their epilepsy; however,
subject 4, the sibling of subject 3, has never had a seizure and is
not on B<sub>6</sub> supplementation. DBS from the parents of the
two siblings homozygous for p.R116Q were also analyzed. All DBS samples
were transferred into sealed foil bags after drying at room temperature
for 16–24 h after collection. They were kept for a maximum
period of 1 week at room temperature prior to storing at −20
°C or temperatures lower than this.</p><p>The mean activity of
PNPO in DBS samples from hospital controls was 41.7 pmol DBS<sup>–1</sup> h<sup>–1</sup> (range: 10.0–95.0 pmol DBS<sup>–1</sup> h<sup>–1</sup>). The activity of PNPO-deficient patients
(range: 0.0–4.6 pmol DBS<sup>–1</sup> h<sup>–1</sup>; mean 1.1 pmol DBS<sup>–1</sup> h<sup>–1</sup>) was
significantly lower than the controls (<italic>p</italic> < 0.0001).
This PNPO-deficient cohort includes the 3 samples from the individuals
homozygous for the p.R116Q variant in PNPO. The range for patients
with other seizure disorders receiving vitamin B<sub>6</sub> supplementation,
similar to that of the hospital controls, was significantly higher
than that of the PNPO-deficient patients (23.0–85.9 pmol DBS<sup>–1</sup> h<sup>–1</sup>; mean = 56.0 pmol DBS<sup>–1</sup> h<sup>–1</sup>). Seven healthy adult controls displayed activity
from 13.8–44.0 pmol DBS<sup>–1</sup> h<sup>–1</sup> with a mean of 28.3 pmol DBS<sup>–1</sup> h<sup>–1</sup>. The p.R116Q heterozygous parents of subjects 3 and 4 had intermediate
PNPO activities of 7.1 and 10.8 pmol DBS<sup>–1</sup> h<sup>–1</sup> (<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>). Patients with mutations in <italic>PNPO</italic> could
be clearly delineated from all of the control groups with the lowest
control having a PNPO activity more than 2-fold greater than that
of the PNPO patient with highest residual activity. Differences between
the groups were shown to be statistically significant (<italic>P</italic> values shown in <xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>). No false positives were identified. The large range of
PNPO activity seen in controls could be representative of population
differences reported previously.<sup><xref ref-type="bibr" rid="ref13">13</xref>,<xref ref-type="bibr" rid="ref14">14</xref></sup> There was
no direct correlation of PNPO activity with age in the samples from
control individuals not receiving B<sub>6</sub> supplementation (age
range 5 days–15 years and adult samples). In the future, it
will be important to expand this age range and measure PNPO activity
in DBS of babies born prematurely as Kang et al.<sup><xref ref-type="bibr" rid="ref35">35</xref></sup> have reported that fetal expression of PNPO is relatively
low as compared to that of adults. The mean PNPO activity in 5 control
neonates (<1 month of age) not receiving B<sub>6</sub> supplementation
measured in this study was 52.7 pmol DBS<sup>–1</sup> h<sup>–1</sup> (range 41.3–66.3 pmol DBS<sup>–1</sup> h<sup>–1</sup>). This is not significantly different from
the PNPO activity seen in controls (>1 month of age) irrespective
of whether they are receiving B<sub>6</sub> supplementation or not.
The youngest neonate (5 days of age) had PNPO activity of 64.3 pmol
DBS<sup>–1</sup> h<sup>–1</sup>.</p><fig id="fig4" position="float"><label>Figure 4</label><caption><p>DBS PNPO enzyme activity
in PNPO-deficient patients and control
samples. Box plots indicate range, interquartile range, and median.
Statistical analysis was performed using one-way ANOVA followed by
Tukey’s multiple comparisons test; ** = <italic>P</italic> <
0.01; **** = <italic>P</italic> < 0.0001.</p></caption><graphic xlink:href="ac-2017-01358c_0004" id="gr4" position="float"></graphic></fig><p>The results obtained using the DBS assay were compared to
those
from previous assays of erythrocyte PNPO activity described in the
literature. Anderson et al.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> reported
mean PNPO activity levels of approximately 10 nmol PLP gHb<sup>–1</sup> h<sup>–1</sup>, and similar activity levels were reported
more recently by Mushtaq et al.<sup><xref ref-type="bibr" rid="ref36">36</xref></sup> To compare
our results directly, two assumptions had to be made: (1) a 3 mm punch
from a DBS contains 3.2 μL of whole blood, and (2) the mean
hemoglobin level of our sample cohort was 14 gHb/dL (hemoglobin levels
were not available for our subjects). If these assumptions are accepted,
a conversion factor of 2.2 can be used to convert pmol PLP DBS<sup>–1</sup> h<sup>–1</sup> to nmol PLP gHb<sup>–1</sup> h<sup>–1</sup>. This gave mean PNPO activity levels of 62.2
and 91.8 nmol gHb<sup>–1</sup> h<sup>–1</sup> in our
adult and child control cohorts, respectively (those not receiving
B<sub>6</sub> supplementation). Although these values are significantly
higher than those reported previously, there are major differences
in methodology that can account for this difference. First, we have
used PNP rather than PMP as a substrate. As mentioned previously,
the oxidation of PMP to PLP under the conditions used is only 14.1%
of that observed when PNP is used as a substrate, that is, 7-fold
less. Equally, we have shown that upon use of PNP as substrate, a
lower <italic>K</italic><sub>m</sub> and higher <italic>V</italic><sub>max</sub> are identified, when compared to PMP. Second, methods
previously published have not added FMN to their assay and rely on
endogenous FMN present in the samples, and both our results and previous
work<sup><xref ref-type="bibr" rid="ref13">13</xref></sup> have shown that addition of FMN
increases PNPO activity in vitro.</p><p>Recently, Mathis et al.<sup><xref ref-type="bibr" rid="ref10">10</xref></sup> have shown
that a raised plasma PM/PA ratio can be used as a diagnostic marker
of PNPO deficiency. PM/PA ratios in dried blood spots of 18 PNPO deficient
subjects and 13 individuals from the cohort of controls receiving
B<sub>6</sub> supplementation were measured, although it was not possible
to ascertain recovery of these vitamers due to interconversion in
spiked whole blood by endogenous enzymes. Ratios were found to range
from 0.00–0.95 (mean 0.28) and from 0.00–0.74 (mean
0.09) in subjects with mutations in PNPO and controls receiving B<sub>6</sub> supplementation, respectively (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Figure S-8</ext-link>). Although the PM/PA ratios in patients with mutations
in PNPO were significantly higher (<italic>p</italic> = 0.0059), it
was not possible to use the PM/PA ratio in dried blood spots to differentiate
PNPO-deficient individuals from controls receiving B<sub>6</sub> for
seizure control with certainty.</p><p>There are several factors that
may explain the different findings
between the two studies. Our study (<italic>n</italic> = 18 PNPO patients)
has investigated the ratio in DBS, while that of Mathis et al.<sup><xref ref-type="bibr" rid="ref10">10</xref></sup> (<italic>n</italic> = 6) studied plasma. Plasma
samples were not available in our study. Comparison of whole blood
and plasma B<sub>6</sub> vitamer concentrations will be important
in the future. The <italic>PNPO</italic> genotype may also be influential,
as PNPO-deficient patients investigated previously were homozygous
for either the c.674G > A (<italic>n</italic> = 4), c.263 + 2T
> C
(<italic>n</italic> = 1), and c.416A > C (<italic>n</italic> =
1).
In this study, we have studied the ratios of subjects with 14 different
genotypes (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">Table S-5</ext-link>), only one of which
was studied previously (c.263 + 2T > C; subject 18). The individual
with this genotype was found to have a raised PM/PA ratio in both
studies. Equally, in those patients for whom supplementation type
was known, a larger proportion of our subjects were receiving PLP
monotherapy for the treatment of their seizures as opposed to PN:
19/23 overall, 14/17 PNPO deficient versus 2/37 overall, and 2/6 PNPO
deficient in Mathis et al.<sup><xref ref-type="bibr" rid="ref10">10</xref></sup> Type of B<sub>6</sub> supplementation and dosage could be important if using PM/PA
ratios for diagnosis of PNPO deficiency.</p><p>The three subjects
(3, 4, and 13) homozygous for the p.R116Q variant
had extremely low PNPO activities (<0.2 pmol DBS<sup>–1</sup> h<sup>–1</sup>), while the two parents who were heterozygous
for the p.R116Q PNPO variant had intermediate PNPO activities of 7.1
and 10.8 pmol DBS<sup>–1</sup> h<sup>–1</sup>. This
contrasts to our previous findings where variants had been investigated
in a human in vitro HeLa cell lysate expression system, in which p.R116Q
was shown to result in 83% activity as compared to wild-type PNPO
activity.<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> We have previously suggested
that p.R116Q contributes to the pathogenesis of epilepsy. The results
of the DBS assay confirm this. The difference observed between the
two systems with regard to this variant may be due to how PNPO utilizes
PNP as a substrate, as we have used in this DBS assay, rather than
PMP, as used in the in vitro overexpressed system. Indeed, a patient
homozygous for p.R116Q has previously been reported to have a normal
plasma PM/PA ratio.<sup><xref ref-type="bibr" rid="ref10">10</xref></sup> In this study, 2/3
subjects homozygous for p.R116Q also had low DBS PM/PA ratios. Why
subject 4, the sibling homozygous for this variant, exhibits negligible
activity and has not presented with seizures has yet to be explained.
It may be that there are other environmental or genetic factors that
are implicated. The mother of this subject received multivitamin tablets
containing PN during pregnancy, and it is possible that this increased
the PLP and PL in her plasma/breast milk to concentrations high enough
to provide adequate intracranial PLP concentrations in the subject
as a fetus/neonate. Accordingly, it is also possible that a “second
hit” is required at another locus for an epileptic phenotype
to become apparent. Further work will be needed to clarify this and
to investigate the effects of p.R116Q on PNPO activity.</p><p>There
have been reports of liver dysfunction in individuals receiving
high doses (>30 mg kg<sup>–1</sup> d<sup>–1</sup>) of
PLP for the treatment of seizures. In particular, this has been noted
in PNPO-deficient patients.<sup><xref ref-type="bibr" rid="ref37">37</xref>,<xref ref-type="bibr" rid="ref38">38</xref></sup> Transiently raised
liver function tests have also been reported in 14/28 patients with
infantile spasms when receiving 30–50 mg kg<sup>–1</sup> d<sup>–1</sup> PLP.<sup><xref ref-type="bibr" rid="ref39">39</xref></sup> A clear
mechanism behind this pathology has yet to be elucidated, but it would
be prudent to minimize patient exposure to high PLP doses where possible.<sup><xref ref-type="bibr" rid="ref40">40</xref></sup> This DBS assay can identify individuals receiving
PLP supplementation in whom PNPO activity is normal and could therefore
help to assess which patients would be more likely to tolerate a switch
to PN supplementation while maintaining seizure control. This would
help to eliminate potential liver damage in these individuals. An
alternative treatment approach could be pyridoxal supplementation;
however, this has not been tested clinically.</p><p>This DBS assay
will also be useful for the diagnosis of patients
in whom only one heterozygous pathogenic variant has been identified
in the <italic>PNPO</italic> gene upon molecular genetic investigation.
Subject 6 is heterozygous for the <italic>PNPO</italic> variant c.[641dupA];
however, no other pathogenic variant has been identified. They had
undetectable PNPO activity using our DBS assay, confirming subject
6 is PNPO deficient.</p><p>Currently, in patients with neonatal/infantile
seizures, if CSF
analysis shows alterations in neurotransmitter amine metabolites (e.g.,
raised 3-<italic>O</italic>-methyldopa; low homovanillic acid, 5-hydroxyindoleacetic
acid) or amino acids (e.g., raised glycine, threonine) indicative
of PLP deficiency or low CSF PLP, urinary α-aminoadipic semialdehyde
(α-AASA) is measured to exclude ALDH7A1 deficiency. This DBS
assay could, in combination with the clinical presentation of these
individuals, be used for the simultaneous assessment of PNPO activity
alongside urinary α-AASA measurement. This will aid the development
of protocols for the rapid diagnosis and treatment of neonatal seizures.</p></sec></sec><sec id="sec4"><title>Conclusions</title><p>We have developed a novel LC–MS/MS-based
assay for the determination
of PNPO activity, which would be suitable for translation into the
clinical diagnostic setting. The method is simple, robust, and clearly
separates PNPO-deficient patients from individuals with normal PNPO
activity. It does not require prior enzyme extraction from the DBS
nor derivatization, requires minimal sample preparation, and can be
performed in 1.5 h, followed by a 6.5 min LC–MS/MS analytical
run. All previously published assays for measuring PNPO activity have
not been suitable for the clinical setting and often require large
sample volumes not possible to obtain from severely ill neonates.
This new rapid DBS PNPO assay will now allow clinicians to distinguish
PNPO deficiency from other disorders, potentially in combination with
the plasma or DBS PM/PA ratio. This will be a powerful tool for clinicians
to help diagnose these patients.</p></sec></body><back><notes id="notes-1" notes-type="si"><title>Supporting Information Available</title><p>The Supporting Information
is available free of charge on the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org">ACS Publications website</ext-link> at DOI: <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/abs/10.1021/acs.analchem.7b01358">10.1021/acs.analchem.7b01358</ext-link>.<list id="silist" list-type="simple"><list-item><p>Tables and figures
showing experimental data collected
during optimization of enzyme assay conditions as well as more detailed
information on the PNPO-deficient subjects analyzed during method
development (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01358/suppl_file/ac7b01358_si_001.pdf">PDF</ext-link>)</p></list-item></list></p></notes><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="sifile1"><media xlink:href="ac7b01358_si_001.pdf"><caption><p>ac7b01358_si_001.pdf</p></caption></media></supplementary-material></sec><notes id="notes-2"><title>Author Present Address</title><p><sup>○</sup> C.G.: Dana Farber Cancer Institute, 440
Brookline Avenue, Boston,
Massachusetts 02215, United States.</p></notes><notes id="notes-50"><title>Author Contributions</title><p><sup>△</sup> P.T.C.
and P.B.M. contributed equally. M.P.W. undertook all laboratory
analyses supervised principally by P.B.M. P.T.C., S.J.R.H., K.A.M.,
and P.G. contributed valuable intellectual support and discussion
and were involved in experimental design. E.J.F., A.P., M.-L.U., R.P.,
D.C.S., C.G., T.S., K.R., M.S., M.B., and D.B. obtained patient samples
and supplied clinical details. The manuscript was written by M.P.W.,
P.T.C., and P.B.M. All authors have given approval to the final version
of the manuscript.</p></notes><notes id="notes-4" notes-type="COI-statement"><p>The authors
declare no competing financial interest.</p></notes><ack><title>Acknowledgments</title><p>We would
like to thank Dr. F. Feillet, Dr. O. Maier, Dr. N.
Gayatri, Dr. M. Taylor, Dr. H. Mundy, Dr. E. Del Giudice, Dr. L. Delfiner,
Dr. C. Freihuber, Dr. P. Bala, Dr. Livingston, and Dr. I. Kern for
their involvement in the study and Dr. E. S. Reid for her technical
help and support. M.P.W., P.T.C., and P.B.M. are supported by Great
Ormond Street Children’s Charity (GOSHCC). This project was
funded by GOSHCC. P.B.M., P.T.C., P.G., and S.J.R.H. are supported
by the National Institute for Health Research Biomedical Research
Centre at GOSH for Children NHS Foundation Trust and University College
London. P.G. was supported by a Wellcome Trust Senior Clinical Fellowship
(WT095662MA). We are indebted to the patients and their families for
participating in this study.</p></ack><ref-list><title>References</title><ref id="ref1"><mixed-citation publication-type="journal" id="cit1"><name><surname>Clayton</surname><given-names>P. T.</given-names></name><source>J. Inherited Metab.
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