Dennis J. Dietzen

Recent work has demonstrated that p56lck, a member of the Src family of protein tyrosine kinases (PTKs), is modified by palmitoylation of a cysteine residue(s) within the first 10 amino acids of the protein (in addition to amino-terminal myristoylation that is a common modification of the Src family of PTKs). This is now extended to three other members of this family by showing incorporation of [3H]palmitate into p59fyn, p55fgr, and p56hck, but not into p60src. The [3H]palmitate was released by treatment with neutral hydroxylamine, indicating a thioester linkage to the protein. Individual replacement of the two cysteine residues within the first 10 amino acids of p59fyn and p56lck with serine indicated that Cys3 was the major determinant of palmitoylation, as well as association of the PTK with glycosyl-phosphatidylinositol-anchored proteins. Introduction of Cys3 into p60src led to its palmitoylation. p59fyn but not p60src partitioned into Triton-insoluble complexes that contain caveolae, microinvaginations of the plasma membrane. Mapping of the requirement for partitioning into caveolae demonstrated that the amino-terminal sequence Met-Gly-Cys is both necessary and sufficient within the context of a Src family PTK to confer localization into caveolae. Palmitoylation of this motif in p59fyn also modestly increased its overall avidity for membranes. These results highlight the role of the amino-terminal motif Met-Gly-Cys in determining the structure and properties of members of the Src family of PTKs.

Caveolae are subdomains of the plasma membrane which concentrate cholesterol, glycosphingolipids, and glycosylphosphatidylinositol-linked proteins. It has recently been demonstrated that specific members of the Src family of protein tyrosine kinases require palmitoylation of NH2-terminal cysteine residues to localize in caveolae. Here we report that caveolin, an integral membrane protein which forms part of the coat of caveolae, also incorporates palmitate through linkage to cysteine residues. Caveolin contains only three cysteine residues which are all located on the COOH-terminal side of the hydrophobic transmembrane region. Immunofluorescent staining of cells transfected with caveolin indicated that, like the NH2 terminus, this COOH-terminal region is located on the cytoplasmic side of the plasma membrane. Studies of cysteine substitution mutants showed that all three cysteines are capable of incorporating palmitate and that the juxtamembrane Cys133 residue is the predominant site of palmitoylation. Simultaneous mutation of all three cysteine residues in caveolin resulted in the loss of ability to incorporate palmitate; however, this did not affect localization of the protein. Thus, palmitoylation of cysteine residues in nonmembrane spanning Src family protein tyrosine kinases has different consequences than in the transmembrane protein caveolin. Caveolae are subdomains of the plasma membrane which concentrate cholesterol, glycosphingolipids, and glycosylphosphatidylinositol-linked proteins. It has recently been demonstrated that specific members of the Src family of protein tyrosine kinases require palmitoylation of NH2-terminal cysteine residues to localize in caveolae. Here we report that caveolin, an integral membrane protein which forms part of the coat of caveolae, also incorporates palmitate through linkage to cysteine residues. Caveolin contains only three cysteine residues which are all located on the COOH-terminal side of the hydrophobic transmembrane region. Immunofluorescent staining of cells transfected with caveolin indicated that, like the NH2 terminus, this COOH-terminal region is located on the cytoplasmic side of the plasma membrane. Studies of cysteine substitution mutants showed that all three cysteines are capable of incorporating palmitate and that the juxtamembrane Cys133 residue is the predominant site of palmitoylation. Simultaneous mutation of all three cysteine residues in caveolin resulted in the loss of ability to incorporate palmitate; however, this did not affect localization of the protein. Thus, palmitoylation of cysteine residues in nonmembrane spanning Src family protein tyrosine kinases has different consequences than in the transmembrane protein caveolin.

Niemann-Pick type C1 (NPC1) disease is a rare progressive neurodegenerative disorder characterized by accumulation of cholesterol in the endolysosomes. Previous studies implicating oxidative stress in NPC1 disease pathogenesis raised the possibility that nonenzymatic formation of cholesterol oxidation products could serve as disease biomarkers. We measured these metabolites in the plasma and tissues of the Npc1(-/-) mouse model and found several cholesterol oxidation products that were elevated in Npc1(-/-) mice, were detectable before the onset of symptoms, and were associated with disease progression. Nonenzymatically formed cholesterol oxidation products were similarly increased in the plasma of all human NPC1 subjects studied and delineated an oxysterol profile specific for NPC1 disease. This oxysterol profile also correlated with the age of disease onset and disease severity. We further show that the plasma oxysterol markers decreased in response to an established therapeutic intervention in the NPC1 feline model. These cholesterol oxidation products are robust blood-based biochemical markers for NPC1 disease that may prove transformative for diagnosis and treatment of this disorder, and as outcome measures to monitor response to therapy.

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease. Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease. Niemann-Pick type C (NPC) disease is a rare, primarily pediatric disorder characterized by accumulation of cholesterol and other lipids in the viscera and central nervous system. Approximately 95% of NPC cases are caused by mutations of the NPC1 gene, whereas the remaining 5% are caused by mutations in the NPC2 gene (1Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. et al.Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.Science. 1997; 277: 228-231Crossref PubMed Scopus (1216) Google Scholar, 2Naureckiene S. Sleat D.E. Lackland H. Fensom A. Vanier M.T. Wattiaux R. Jadot M. Lobel P. Identification of HE1 as the second gene of Niemann-Pick C disease.Science. 2000; 290: 2298-2301Crossref PubMed Scopus (701) Google Scholar). Affected individuals typically present in early childhood with ataxia and progressive impairment of motor and intellectual function and usually die in adolescence. There are currently no FDA-approved therapies for this progressively fatal neurodegenerative disorder. However, a recent controlled study and a series of case reports suggests efficacy for miglustat (3Patterson M.C. Vecchio D. Prady H. Abel L. Wraith J.E. Miglustat for treatment of Niemann-Pick C disease: a randomised controlled study.Lancet Neurol. 2007; 6: 765-772Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar), an inhibitor of glycosphingolipid biosynthesis that is now licensed for use as a disease modifying therapy in multiple countries, including the European Union, Russia, Brazil, Australia, Canada, and Taiwan. A major barrier to developing new therapies for NPC1 disease has been the lack of an inexpensive and noninvasive diagnostic test. The current diagnostic standard for NPC1 is an invasive skin biopsy and filipin staining, which is only available in one of several specialized clinical laboratories world-wide (4Wraith J.E. Baumgartner M.R. Bembi B. Covanis A. Levade T. Mengel E. Pineda M. Sedel F. Topcu M. Vanier M.T. et al.Recommendations on the diagnosis and management of Niemann-Pick disease type C.Mol. Genet. Metab. 2009; 98: 152-165Crossref PubMed Scopus (197) Google Scholar). This approach is confounded by ”variant” NPC cases that have normal filipin staining and, therefore, require another diagnostic method for their detection. Together, the complex and time-consuming processing of the biopsy and lack of sensitivity of the filipin test have contributed to significant diagnostic delays in pediatric cohorts (>4 years) and in adults (>6 years) with NPC1 disease (5Sevin M. Lesca G. Baumann N. Millat G. Lyon-Caen O. Vanier M.T. Sedel F. The adult form of Niemann-Pick disease type C.Brain. 2007; 130: 120-133Crossref PubMed Scopus (319) Google Scholar, 6Yanjanin N.M. Velez J.I. Gropman A. King K. Bianconi S.E. Conley S.K. Brewer C.C. Solomon B. Pavan W.J. Arcos-Burgos M. et al.Linear clinical progression, independent of age of onset, in Niemann-Pick disease, type C.Am. J. Med. Genet. B. Neuropsychiatr. Genet. 2010; 153B: 132-140Crossref PubMed Scopus (138) Google Scholar). The delay in diagnosis is unfortunate as early intervention is likely to yield the most benefit in this disease. Loss of NPC1 function results in endolysosomal accumulation of unesterified cholesterol (7Ory D.S. Niemann-Pick type C disease: a disorder of cellular cholesterol trafficking.Biochim. Biophys. Acta. 2000; 1529: 331-339Crossref PubMed Scopus (122) Google Scholar, 8Sturley S.L. Patterson M.C. Pentchev P. Unraveling the sterol-trafficking defect in Niemann-Pick C disease.Proc. Natl. Acad. Sci. USA. 2009; 106: 2093-2094Crossref PubMed Scopus (19) Google Scholar), which is the hallmark of NPC disease. In NPC1-deficient cells, cholesterol accumulation is associated with cellular oxidative stress, as evidenced by increased production of reactive oxygen species (ROS), oxidative damage, and a gene expression profile indicative of oxidative stress (9Reddy J.V. Ganley I.G. Pfeffer S.R. Clues to neuro-degeneration in Niemann-Pick Type C disease from global gene expression profiling.PLoS ONE. 2006; 1: e19Crossref PubMed Scopus (90) Google Scholar, 10Zampieri S. Mellon S.H. Butters T.D. Nevyjel M. Covey D.F. Bembi B. Dardis A. Oxidative stress in NPC1 deficient cells: protective effect of allopregnanolone.J. Cell. Mol. Med. 2009; 13: 3786-3796Crossref PubMed Scopus (88) Google Scholar). In the NPC1 mouse model, the oxidative stress is accompanied by elevated ROS and nonenzymatic oxidation of cholesterol in multiple tissues (11Tint G. Pentchev P. Xu G. Batta A. Shiefer S. Salen G. Honda A. Cholesterol and oxygenated cholesterol concentrations are markedly elevated in peripheral tissue but not in brain from mice with Niemann-Pick type C phenotype.J. Inherit. Metab. Dis. 1998; 21: 853-863Crossref PubMed Scopus (57) Google Scholar, 12Zhang J.R. Coleman T. Langmade S.J. Scherrer D.E. Lane L. Lanier M.H. Feng C. Sands M.S. Schaffer J.E. Semenkovich C.F. et al.Niemann-Pick C1 protects against atherosclerosis in mice via regulation of macrophage intracellular cholesterol trafficking.J. Clin. Invest. 2008; 118: 2281-2290PubMed Google Scholar, 13Porter F.D. Scherrer D.E. Lanier M.H. Langmade S.J. Molugu V. Gale S.E. Olzeski D. Sidhu R. Dietzen D.J. Fu R. Wassif C.A. Yanjanin N.M. Marso S.P. House J. Vite C. Schaffer J.E. Ory D.S. Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease.Sci. Transl. Med. 2010; 2: 56ra81Crossref PubMed Scopus (265) Google Scholar). These cholesterol oxi­dation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), similarly were markedly increased in the plasma of all human NPC1 subjects studied (13Porter F.D. Scherrer D.E. Lanier M.H. Langmade S.J. Molugu V. Gale S.E. Olzeski D. Sidhu R. Dietzen D.J. Fu R. Wassif C.A. Yanjanin N.M. Marso S.P. House J. Vite C. Schaffer J.E. Ory D.S. Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease.Sci. Transl. Med. 2010; 2: 56ra81Crossref PubMed Scopus (265) Google Scholar). The 3β,5α,6β-triol and 7-KC markers were not increased in the plasma of other neurode­generative or lysosomal storage diseases, correlated with severity and age of onset of disease, and, in a NPC1 disease model, were reduced in response to therapy (13Porter F.D. Scherrer D.E. Lanier M.H. Langmade S.J. Molugu V. Gale S.E. Olzeski D. Sidhu R. Dietzen D.J. Fu R. Wassif C.A. Yanjanin N.M. Marso S.P. House J. Vite C. Schaffer J.E. Ory D.S. Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease.Sci. Transl. Med. 2010; 2: 56ra81Crossref PubMed Scopus (265) Google Scholar). These findings indicate that 3β,5α,6β-triol and 7-KC are NPC1 disease-specific biochemical markers and suggest a role for these markers in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. To date, quantification of oxysterols in biological samples has been accomplished principally using GC-MS methods (14Bjorkhem I. Breuer O. Angelin B. Wikstrom S.A. Assay of unesterified cholesterol-5,6-epoxide in human serum by isotope dilution mass spectrometry. Levels in the healthy state and in hyperlipoproteinemia.J. Lipid Res. 1988; 29: 1031-1038Abstract Full Text PDF PubMed Google Scholar, 15Breuer O. Bjorkhem I. Simultaneous quantification of several cholesterol autoxidation and monohydroxylation products by isotope-dilution mass spectrometry.Steroids. 1990; 55: 185-192Crossref PubMed Scopus Google Scholar, S. Breuer O. E. of cholesterol oxidation products in human plasma by isotope PubMed Scopus Google Scholar, of and in of human by PubMed Scopus Google Scholar, M. C. I. D. of a method for the of oxidation products in Sci. 2008; PubMed Scopus Google Scholar). this sensitive detection of oxysterols in complex methods are by and and by for plasma we describe the development of a sensitive and specific LC-MS/MS method of quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. 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P. K. M. K. D. fragmentation 2009; PubMed Scopus Google Scholar, B. specificity in using mass PubMed Scopus Google Scholar, M. C. associated with the use of mass in the clinical 2010; PubMed Scopus Google Scholar). The of plasma samples were with the of the in the 3β,5α,6β-triol and 7-KC The sensitivity of the as by the was for 3β,5α,6β-triol and 7-KC using samples the in the standard were and with a and the the was for 3β,5α,6β-triol and and the was for 3β,5α,6β-triol and 7-KC A the is in and K. 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Scherrer D.E. Lanier M.H. Langmade S.J. Molugu V. Gale S.E. Olzeski D. Sidhu R. Dietzen D.J. Fu R. Wassif C.A. Yanjanin N.M. Marso S.P. House J. Vite C. Schaffer J.E. Ory D.S. Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease.Sci. Transl. Med. 2010; 2: 56ra81Crossref PubMed Scopus (265) Google Scholar). The plasma 3β,5α,6β-triol and 7-KC were highly correlated that production of these oxysterols in likely a (13Porter F.D. Scherrer D.E. Lanier M.H. Langmade S.J. Molugu V. Gale S.E. Olzeski D. Sidhu R. Dietzen D.J. Fu R. Wassif C.A. Yanjanin N.M. Marso S.P. House J. Vite C. Schaffer J.E. Ory D.S. Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease.Sci. Transl. Med. 2010; 2: 56ra81Crossref PubMed Scopus (265) Google Scholar). To the of the 3β,5α,6β-triol and 7-KC to discriminate NPC1 subjects from oxysterol concentrations in these were using and and was demonstrated that the the for 3β,5α,6β-triol was and for 7-KC was a of a sensitivity of and specificity of to discriminate between NPC1 subjects and for a of a sensitivity of and specificity of to discriminate between NPC1 subjects and These oxysterol disease markers were elevated in NPC1 7-KC as with of the NPC1 demonstrated plasma 3β,5α,6β-triol concentrations the and of the NPC1 demonstrated plasma 7-KC concentrations the and specificity of plasma 3β,5α,6β-triol for control and NPC1 the for 3β,5α,6β-triol of sensitivity is and specificity is and specificity of plasma 7-KC for control and NPC1 the for 7-KC of sensitivity is and specificity is of plasma 3β,5α,6β-triol and 7-KC concentrations in NPC1 for plasma 3β,5α,6β-triol concentrations for control and NPC1 for plasma 7-KC concentrations for control and NPC1 The cholesterol oxidation products, 3β,5α,6β-triol and have been to diagnostic markers for NPC1 disease (13Porter F.D. Scherrer D.E. Lanier M.H. Langmade S.J. Molugu V. Gale S.E. Olzeski D. Sidhu R. Dietzen D.J. Fu R. Wassif C.A. Yanjanin N.M. Marso S.P. House J. Vite C. Schaffer J.E. Ory D.S. Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease.Sci. Transl. Med. 2010; 2: 56ra81Crossref PubMed Scopus (265) Google Scholar). The of this study was to a method for detection of these in human plasma that in a clinical We show that derivatization with dimethylglycine successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma Ory D.S. of by ionization mass after derivatization with 2007; 21: PubMed Scopus Google Scholar). The LC-MS/MS method successfully resolved the oxysterol dimethylglycinates with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. of plasma from control and NPC1 subjects of elevated plasma oxysterols in NPC1 disease and demonstrated that this method discriminate between control and NPC1 subjects with high sensitivity and This method a for clinical of the oxysterol markers and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease. The current diagnostic standard for NPC1 disease is an invasive skin biopsy by filipin staining of the for cholesterol (4Wraith J.E. Baumgartner M.R. Bembi B. Covanis A. Levade T. Mengel E. Pineda M. Sedel F. Topcu M. Vanier M.T. et al.Recommendations on the diagnosis and management of Niemann-Pick disease type C.Mol. Genet. Metab. 2009; 98: 152-165Crossref PubMed Scopus (197) Google Scholar). The filipin has several The assay is in only a specialized world-wide and typically several from time of biopsy to the filipin test has is in NPC1 subjects with disease, and confounded by NPC1 test To of cholesterol accumulation in cells, has been as a diagnostic but this approach similarly is to discriminate and NPC1 cases from normal C. D. S. Mengel E. H. Niemann-Pick Type C disease: in with lysosomal cholesterol Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). which is to filipin test is but in and to mutations on in of NPC1 cases Patterson M.C. K. Identification of mutations in Niemann-Pick disease type with biochemical and of in PubMed Scopus Google Scholar). there is no in NPC1 disease and there are mutations in the NPC1 to between and the LC-MS/MS assay for the 3β,5α,6β-triol and 7-KC species a of the NPC1 cellular (13Porter F.D. Scherrer D.E. Lanier M.H. Langmade S.J. Molugu V. Gale S.E. Olzeski D. Sidhu R. Dietzen D.J. Fu R. Wassif C.A. Yanjanin N.M. Marso S.P. House J. Vite C. Schaffer J.E. Ory D.S. Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease.Sci. Transl. Med. 2010; 2: 56ra81Crossref PubMed Scopus (265) Google with sensitivity and specificity sensitivity and is by the filipin The oxysterol not require specialized is are and in samples with of plasma and require all that of the LC-MS/MS assay in clinical The of a noninvasive, and test has for diagnosis and treatment of NPC1 disease. The oxysterol assay in for NPC1 disease, as with or and adults with disease and in which the of NPC1 disease approach B. M.R. D. disease type C in a PubMed Scopus Google Scholar, P. T. P. S. R. K. M. A. Niemann-Pick type C is a diagnosis in and of the of Scholar). the clinical of NPC1 disease is to a of other lysosomal storage of plasma oxysterols a new diagnostic to to the diagnosis of NPC1 disease in with or or with a storage disorder. The oxysterol assay for response to therapy by biomarkers that as in clinical (13Porter F.D. Scherrer D.E. Lanier M.H. Langmade S.J. Molugu V. Gale S.E. Olzeski D. Sidhu R. Dietzen D.J. Fu R. Wassif C.A. Yanjanin N.M. Marso S.P. House J. Vite C. Schaffer J.E. Ory D.S. Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease.Sci. Transl. Med. 2010; 2: 56ra81Crossref PubMed Scopus (265) Google Scholar). the assay a for The of for elevated oxysterol is by the demonstrated of the oxysterol the sensitivity for detection in and the detection of NPC1 disease by an oxysterol test treatment in NPC1 subjects, individuals most likely to benefit from intervention N.M. Velez J.I. Gropman A. King K. Bianconi S.E. Conley S.K. Brewer C.C. Solomon B. Pavan W.J. Arcos-Burgos M. et al.Linear clinical progression, independent of age of onset, in Niemann-Pick disease, type C.Am. J. Med. Genet. B. Neuropsychiatr. Genet. 2010; 153B: 132-140Crossref PubMed Scopus (138) Google Scholar). of the for the first the of NPC1 disease, which has likely been to the in a oxysterol concentrations were elevated not only in NPC1 subjects but in NPC1 in oxysterol between and we that 3β,5α,6β-triol and 7-KC plasma concentrations in NPC1 were increased and as with control subjects, with findings (13Porter F.D. Scherrer D.E. Lanier M.H. Langmade S.J. Molugu V. Gale S.E. Olzeski D. Sidhu R. Dietzen D.J. Fu R. Wassif C.A. Yanjanin N.M. Marso S.P. House J. Vite C. Schaffer J.E. Ory D.S. Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease.Sci. Transl. Med. 2010; 2: 56ra81Crossref PubMed Scopus (265) Google Scholar). a significant of the demonstrated plasma 3β,5α,6β-triol and 7-KC concentrations that the of the normal that the oxysterol assay is able to discriminate a of NPC1 from the cholesterol oxidation products, as 3β,5α,6β-triol and from tissue oxidative stress, elevated oxysterol concentrations increased for disease atherosclerosis or and in on a NPC1 of we that the of NPC1 with elevated oxysterol to of the and and assay In with increased of NPC1 disease with the to this is and the of the oxysterol test is the other using a for 3β,5α,6β-triol of as the is This by the to which the sensitivity to but the to The NPC1 cases in the using a as another NPC1 disease-specific or of the NPC1 In we describe the development of a sensitive and specific LC-MS/MS method of quantifying 3β,5α,6β-triol and 7-KC human plasma. this assay is to the and of these oxysterol species in and NPC1 subjects, and findings of increased plasma 3β,5α,6β-triol and 7-KC concentrations in NPC1 of plasma oxysterols offers the first noninvasive, and highly sensitive method for detection of NPC1 disease and, we the filipin test as the diagnostic standard for NPC1 disease. The LC-MS/MS assay a to for NPC1 disease and has the to current to diagnosis and treatment of the disorder. The Covey for and for and Gale for in of with 7-ketocholesterol ionization of dimethylglycine high control of quantification control control multiple Type C control reactive oxygen species of quantification

Determination of the reticulocyte hemoglobin content (CHr) provides an early measure of functional iron deficiency because reticulocytes are the earliest erythrocytes released into blood and circulate for only 1 to 2 days. The CHr in 78 patients undergoing bone marrow examination was measured to assess its clinical utility for the diagnosis of iron deficiency. Twenty-eight patients were iron deficient, based on the lack of stainable iron in the aspirate. The diagnostic power of CHr is limited in patients with high mean cellular volume (MCV) or red cell disorders such as thalassemia. However, when patients with MCV more than 100 fL are excluded, receiver operator curve analysis of CHr, ferritin, transferrin saturation, and MCV demonstrates that CHr has the highest overall sensitivity and specificity of these peripheral blood tests for predicting the absence of bone marrow iron stores.