title: An enzyme combination assay for serum sphingomyelin: Improved specificity through avoiding the interference with lysophosphatidylcholine.
abstract: Serum sphingomyelin (SM) has predictive value in the development of atherosclerosis. Furthermore, SM plays important roles in cell membrane structure, signal transduction pathways, and lipid raft formation. A convenient enzymatic method for SM is available for routine laboratory practice, but the enzyme specificity is not sufficient because of nonspecific reactions with lysophosphatidylcholine (LPC). Based on the differential specificity of selected enzymes toward choline-containing phospholipids, a two-step assay for measuring SM was constructed and its performance was evaluated using sera from healthy individuals on a Hitachi 7170 autoanalyzer. Results from this assay were highly correlated with theoretical serum SM concentrations estimated by subtracting phosphatidylcholine (PC) and LPC concentrations from that of total phospholipids determined using previously established methods. There was a good correlation between the results of SM assayed by the proposed method and the existing enzymatic method in sera from healthy individuals. Moreover, the proposed method was superior to the existing method in preventing nonspecific reactions with LPC present in sera. The proposed method does not require any pretreatment, uses 2.5 μl of serum samples, and requires only 10 min on an autoanalyzer. This high-throughput method can measure serum SM with sufficient specificity for clinical purposes and is applicable in routine laboratory practice.
authors.affiliation: Research & Development Department, Kyowa Medex, Tokyo 104-6004, Japan. Electronic address: takehide.kimura@kyowa-kirin.co.jp.
authors.forename: Takehide
authors.initials: T
authors.lastname: Kimura
authors.affiliation: Research Laboratories, Kyowa Medex, Shizuoka 411-0932, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Kuwata
authors.affiliation: Research Laboratories, Kyowa Medex, Shizuoka 411-0932, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Miyauchi
authors.affiliation: Research Laboratories, Kyowa Medex, Shizuoka 411-0932, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Katayama
authors.affiliation: Research & Development Department, Kyowa Medex, Tokyo 104-6004, Japan.
authors.forename: Norihiko
authors.initials: N
authors.lastname: Kayahara
authors.affiliation: Kumamoto Health Science University, Kumamoto 861-5598, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sugiuchi
authors.affiliation: Kumamoto Health Science University, Kumamoto 861-5598, Japan.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Matsushima
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kondo
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Ishitsuka
authors.affiliation: College of Pharmaceutical Sciences, Daiichi University, Fukuoka 815-8511, Japan.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Irikura
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan; Center for Clinical Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan.
authors.forename: Tetsumi
authors.initials: T
authors.lastname: Irie
mesh_terms: D000328:Adult; D016477:Artifacts; D001322:Autoanalysis; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D057075:Enzyme Assays; D005260:Female; D006801:Humans; D008244:Lysophosphatidylcholines / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013109:Sphingomyelins / Q000097:blood* / Q000378:metabolism; D055815:Young Adult
chemical_list: D008244:Lysophosphatidylcholines; D013109:Sphingomyelins; D013108:Sphingomyelin Phosphodiesterase
keywords: High-throughput enzymatic assay; Improved specificity; Phospholipase D; Prevention of nonspecific reaction with lysophosphatidylcholine; Sphingomyelin