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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-. </p></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK593857_"><span class="title" itemprop="name">Quantitative determination of sphingomyelin</span></h1><div class="contrib half_rhythm"><span itemprop="author">Nozomu Okino</span>, Ph.D.<div class="affiliation small">Kyushu Univ<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-uhsuyk.rga@onikon" class="oemail">pj.ca.u-uhsuyk.rga@onikon</a></div></div><div class="small">Corresponding author.</div></div><div class="contrib half_rhythm"><span itemprop="author">Makoto Ito</span>, Ph.D.<div class="affiliation small">Kyushu Univ<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-uhsuyk.rga@iotokam" class="oemail">pj.ca.u-uhsuyk.rga@iotokam</a></div></div></div><p class="small">Created: <span itemprop="datePublished">September 28, 2021</span>; Last Revision: <span itemprop="dateModified">March 21, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g194-quantifysm.Introduction"><h2 id="_g194-quantifysm_Introduction_">Introduction</h2><p>Sphingomyelin is an important lipid component of plasma membrane that is synthesized from ceramide and phosphatidylcholine by sphingomyelin synthases and is hydrolyzed by sphingomyelinases into ceramide and phosphorylcholine. Ceramide is shared with glycosphingolipids (GSLs) as the precursor; therefore, sphingomyelin metabolism affects GSL metabolism and then GSL functions. Since sphingomyelin metabolism is crucial for cellular membrane turnover, the regulation of cell growth, and cellular differentiation, sensitive and reliable methods for the quantification of this lipid are of considerable importance.</p></div><div id="g194-quantifysm.Protocol"><h2 id="_g194-quantifysm_Protocol_">Protocol</h2><p>In this chapter, two kinds of protocols for determining sphingomyelin will be described: 1) using sphingomyelinase and <i>E. coli</i> diacylglycerol kinase (DGK) (<a class="bk_pop" href="#g194-quantifysm.REF.1">1</a>,<a class="bk_pop" href="#g194-quantifysm.REF.2">2</a>) and 2) using four types of enzymes (sphingomyelinase, alkaline phosphatase, choline oxidase, and horseradish peroxidase) (<a class="bk_pop" href="#g194-quantifysm.REF.3">3</a>,<a class="bk_pop" href="#g194-quantifysm.REF.4">4</a>) (<a class="figpopup" href="/books/NBK593857/figure/g194-quantifysm.F1/?report=objectonly" target="object" rid-figpopup="figg194quantifysmF1" rid-ob="figobg194quantifysmF1">Figure 1</a>). In the second protocol, first, bacterial sphingomyelinase is used to hydrolyze the sphingomyelin to obtain ceramide and phosphorylcholine. Second, alkaline phosphatase is used to generate choline from the phosphorylcholine. Third, choline oxidase is used to generate hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) from the choline. Finally, H<sub>2</sub>O<sub>2</sub>, in the presence of horseradish peroxidase, reacts with dihydroxyphenoxazine (Amplex Red) to generate a highly fluorescent product, resorufin, which is measured using a fluorescent microplate reader.</p><div id="g194-quantifysm.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Quantitative determination of sphingomyelin [I]</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin"><i>n</i>-Octylglucoside (#494459, Merck Millipore, Billerica, MA)</p></dd><dt>b.</dt><dd><p class="no_top_margin">Cardiolipin (C0563, Sigma-Aldrich, St. Louis, MO)</p></dd><dt>c.</dt><dd><p class="no_top_margin">Diethylenetriaminepentaacetic acid (DETAPAC) (D1133, Sigma-Aldrich)</p></dd><dt>d.</dt><dd><p class="no_top_margin">Sphingomyelin standard (C18-sphingomyelin, #1911, Matreya LLC, Pleasant Gap, PA): Make a 20-mM C18-sphingomyelin stock solution in ethanol and keep at −20°C.</p></dd><dt>e.</dt><dd><p class="no_top_margin"><i>Bacillus cereus</i> sphingomyelinase (S9396, Sigma-Aldrich)</p></dd><dt>f.</dt><dd><p class="no_top_margin"><i>E. coli</i> DGK (D3065, Sigma-Aldrich)</p></dd><dt>g.</dt><dd><p class="no_top_margin">Chloroform (CHCl<sub>3</sub>)</p></dd><dt>h.</dt><dd><p class="no_top_margin">Methanol (MeOH)</p></dd><dt>i.</dt><dd><p class="no_top_margin">CHCl<sub>3</sub>-MeOH mixture (C/M, volume/volume)</p></dd><dt>j.</dt><dd><p class="no_top_margin">0.1 M Imidazol-HCl, pH 6.6</p></dd><dt>k.</dt><dd><p class="no_top_margin">Distilled water (DW)</p></dd><dt>l.</dt><dd><p class="no_top_margin">Thin-layer chromatography (TLC) plates (Silicagel 60, 20 × 20 cm, Merck Millipore)</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Quantitative determination of sphingomyelin [II]</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Standard sphingomyelin (C18-sphingomyelin, #1911, Matreya LLC): Make a 20-mM C18-sphingomyelin stock solution in ethanol and keep at −20°C.</p></dd><dt>b.</dt><dd><p class="no_top_margin"><i>Bacillus cereus</i> sphingomyelinase (S9396, Sigma-Aldrich)</p></dd><dt>c.</dt><dd><p class="no_top_margin">Calf intestinal alkaline phosphatase (2250A, Takara Bio Inc., Otsu, Japan)</p></dd><dt>d.</dt><dd><p class="no_top_margin">Choline oxidase (#26986, Sigma-Aldrich)</p></dd><dt>e.</dt><dd><p class="no_top_margin">Horseradish peroxidase (HRP, #77334, Sigma-Aldrich)</p></dd><dt>f.</dt><dd><p class="no_top_margin">Amplex Red Reagent (<a href="/nuccore/492583" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">A12222</a>, Invitrogen/Life Technologies, Carlsbad, CA): Make a 20-mM stock solution in dimethyl sulfoxide (DMSO) and keep at −20°C (<b>Note 1</b>)</p></dd><dt>g.</dt><dd><p class="no_top_margin">Enzyme cocktail: 12.5 mU of <i>B. cereus</i> sphingomyelinase, 400 mU of alkaline phosphatase, 120 mU of choline oxidase, 200 mU of horseradish peroxidase, and 20 nmol of Amplex Red Reagent in 100 μL of the reaction buffer</p></dd><dt>h.</dt><dd><p class="no_top_margin">Reaction buffer: 50 mM of Tris-HCl, pH 7.4, which contains 5 mM of MgCl<sub>2</sub></p></dd><dt>i.</dt><dd><p class="no_top_margin">Standard dilution buffer: 50 mM of Tris-HCl, pH 7.4, which contains 2% Triton X-100 and 5 mM of MgCl<sub>2</sub></p></dd><dt>j.</dt><dd><p class="no_top_margin">96-Well microplate (Black plate)</p></dd></dl></dd></dl></div><div id="g194-quantifysm.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Imaging analyzer (FLA 5000, Fujifilm, Tokyo, Japan)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Sonic bath</p></dd><dt>3.</dt><dd><p class="no_top_margin">Speed Vac concentrator (Thermo Fisher Scientific Inc., Waltham, MA)</p></dd><dt>4.</dt><dd><p class="no_top_margin">ARVO fluorescence microplate reader (PerkinElmer, Waltham, MA)</p></dd></dl></div><div id="g194-quantifysm.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Quantitative determination of sphingomyelin [I]</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Dissolve lipids whose sphingomyelin content is measured in 10 μL of 7.5% n-octyl-β-D-glucopyranoside, 5 mM of cardiolipin, and 1 mM of DETAPAC.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Mix with 30 μL of 0.1 M imidazole–HCl, pH 6.6, containing 0.1 M of NaCl, 25 mM of MgCl<sub>2</sub>, 4 mM of DTT, and 2 mM of EGTA.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Add 5 μL of enzyme (5 mU of <i>E. coli</i> DGK and 100 mU of <i>B. cereus</i> sphingomyelinase) in 10 mM of imidazole–HCl, pH 6.6, containing 1 mM of DETAPAC, 1 mM of DTT, and 10% glycerol.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Initiate the reaction by adding 5 μL of 5 mM “cold” adenosine triphosphate (ATP) and 0.1 μCi of [γ-<sup>32</sup>P]ATP in the buffer described in Step c (the final 50-μL reaction mixture contains 0.5 mM of ATP and 0.1 μCi of [γ-<sup>32</sup>P]ATP).</p></dd><dt>e.</dt><dd><p class="no_top_margin">Incubate at 25°C for 1 h.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Add 0.6 mL of CHCl<sub>3</sub>/MeOH (1/1, v/v).</p></dd><dt>g.</dt><dd><p class="no_top_margin">Vortex for a few seconds, and add 250 μL of 1 M KCl in 20 mM of Mops, pH 7.0.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Centrifuge at 10,000 ×<i>g</i> for 5 min.</p></dd><dt>i.</dt><dd><p class="no_top_margin">Transfer the organic phase to an Eppendorf tube and dry under a stream of N<sub>2</sub> gas.</p></dd><dt>j.</dt><dd><p class="no_top_margin">Dissolve the sample in 20 μL of CHCl<sub>3</sub>/MeOH (1/1, v/v), and apply an aliquot of the solution on a TLC plate.</p></dd><dt>k.</dt><dd><p class="no_top_margin">Develop the TLC plate with CHCl<sub>3</sub>/acetone/ MeOH /acetic acid/water (10/4/3/2/1, v/v/v/v/v).</p></dd><dt>l.</dt><dd><p class="no_top_margin">Dry the TLC plate, and cover it with a wrap.</p></dd><dt>m.</dt><dd><p class="no_top_margin">Expose the TLC plate to an imaging plate.</p></dd><dt>n.</dt><dd><p class="no_top_margin">Determine the radioactivity of the bands corresponding to [<sup>32</sup>P]Cer-1-P with an imaging analyzer, such as the FLA5000 (<b>Note 2</b>).</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Quantitative determination of sphingomyelin [II]</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Homogenize a sample whose sphingomyelin content is measured in a 20-fold volume of 0.2% Triton X-100.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Centrifuge at 10,000 ×g for 5 min.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Withdraw the supernatant.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Add 100 μL of enzyme cocktail to each well of a 96-well microtiter plate.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Add 5 μL of the sample homogenate to each well.</p></dd><dt>f.</dt><dd><p class="no_top_margin">For controls, add 5 μL of the sample homogenate to the enzyme cocktail without sphingomyelinase.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Incubate at 37°C for 20 min.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Measure the fluorescence in the wells of microtiter plate with a microplate reader (set excitation and emission wavelengths at 544 and 590 nm, respectively) (<b>Notes 3–5</b>).</p></dd></dl></dd></dl></div><div id="g194-quantifysm.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">All stock solutions are prepared in the reaction buffer except Amplex Red, which is in DMSO.</p></dd><dt>2.</dt><dd><p class="no_top_margin">To determine the sphingomyelin content of the samples, the amount of endogenous ceramide, determined in a control experiment wherein the reaction is conducted without <i>Bacillus</i> sphingomyelinase, should be subtracted. The quantification of sphingomyelin can be determined from the standard curve using a known amount of standard sphingomyelin (C18-sphingomyelin). With this procedure, 10–1,000 pmol of sphingomyelin can be determined.</p></dd><dt>3.</dt><dd><p class="no_top_margin">Sphingomyelin content is calculated from the test value after subtracting the control value.</p></dd><dt>4.</dt><dd><p class="no_top_margin">Quantification of sphingomyelin can be determined from the standard curve using a known amount of standard sphingomyelin (C18-sphingomyelin): 2, 4, 10, 20, 40, 100, 200, and 400 pmol/μL (10, 20, 50, 100, 200, 500, 1,000, and 2,000 pmol/5 μL/well).</p></dd><dt>5.</dt><dd><p class="no_top_margin">Using this procedure, 20–1,000 pmol/well of sphingomyelin can be determined.</p></dd></dl></div></div><div id="g194-quantifysm.References"><h2 id="_g194-quantifysm_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g194-quantifysm.REF.1">He X, Chen F, Gatt S, Schuchman EH. An Enzymatic Assay for Quantifying Sphingomyelin in Tissues and Plasma from Humans and Mice with Niemann–Pick Disease. <span><span class="ref-journal">Anal Biochem. </span>2001;<span class="ref-vol">293</span>(2):204–11.</span> doi: https://doi.orgPMID. [<a href="https://pubmed.ncbi.nlm.nih.gov/11399033" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11399033</span></a>] [<a href="http://dx.crossref.org/10.1006/abio.2001.5108" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g194-quantifysm.REF.2">Okino N, Ito M. Quantitative determination of sphingomyelin I. GlycoPOD, 2014. <a href="https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t28" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">https://jcggdb<wbr style="display:inline-block"></wbr>.jp/GlycoPOD/protocolShow<wbr style="display:inline-block"></wbr>.action?nodeId=t28</a>.</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g194-quantifysm.REF.3">He X, Chen F, McGovern MM, Schuchman EH. A fluorescence-based, high-throughput sphingomyelin assay for the analysis of Niemann-Pick disease and other disorders of sphingomyelin metabolism. <span><span class="ref-journal">Anal Biochem. </span>2002;<span class="ref-vol">306</span>(1):115–23.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12069422" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12069422</span></a>] [<a href="http://dx.crossref.org/10.1006/abio.2002.5686" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g194-quantifysm.REF.4">Okino N, Ito M. Quantitative determination of sphingomyelin II. GlycoPOD, 2014. <a href="https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t29" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">https://jcggdb<wbr style="display:inline-block"></wbr>.jp/GlycoPOD/protocolShow<wbr style="display:inline-block"></wbr>.action?nodeId=t29</a>.</div></dd></dl></div><h2 id="NBK593857_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g194-quantifysm.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div class="bk_prnt_sctn"><h2>Figures</h2><div class="whole_rhythm bk_prnt_obj bk_first_prnt_obj"><div id="g194-quantifysm.F1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201%3A%20.%20Scheme%20for%20sphingomyelin%20determination.&p=BOOKS&id=593857_g194-quantifysm-Image001.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK593857/bin/g194-quantifysm-Image001.jpg" alt="Figure 1: . Scheme for sphingomyelin determination." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Scheme for sphingomyelin determination. (A) Quantification of sphingomyelin using sphingomyelinase and diacylglycerol kinase. (B) Quantification of sphingomyelin through four enzymatic steps.</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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