Title マウス骨格筋中のダプトマイシンの定量法開発に関する研究( 本文(Fulltext) ) Author(s) 阪井, 祐介 Report No.(Doctoral Degree) 博士(薬科学) 連創博甲第44号 Issue Date 2019-03-25 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/77970 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
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࣐࢘ࢫ㦵᱁➽୰ࡢࢲࣉࢺ࣐ࢩࣥࡢ
ᐃ㔞ἲ㛤Ⓨ㛵ࡍࡿ◊✲
Development of Quantification Method for Daptomycin in
Murine Skeletal Muscles
㐃ྜ⸆་⒪ሗ◊✲⛉ ⸆⛉Ꮫᑓᨷ
┠ ḟ ➨୍❶ ⥴ゝ ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭ 1 ➨❶ ỗ⏝ⓗᢳฟἲࢆ⏝࠸ࡓ࣐࢘ࢫ㦵᱁➽ࡽࡢࢲࣉࢺ࣐ࢩࣥࡢᅇ࣭࣭࣭࣭6 ➨୕❶ ࢺࣜࣉࢩࣥᾘࢆ⏝࠸ࡓ࣐࢘ࢫ㦵᱁➽ヨᩱࡢ๓ฎ⌮ἲ ➨୍⠇ ࢲࣉࢺ࣐ࢩࣥᅇ㐺ࡍࡿ㓝⣲ࡢ㑅ᐃ ࣭࣭࣭࣭࣭࣭࣭࣭9 ➨⠇ ࢺࣜࣉࢩࣥࡼࡿ㦵᱁➽ヨᩱ๓ฎ⌮᮲௳ࡢ᭱㐺࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭11 ➨୕⠇ ࣐࢘ࢫ㦵᱁➽ࢆ⏝࠸ࡓࢲࣉࢺ࣐ࢩࣥࡢῧຍᅇ ࣭࣭࣭࣭࣭࣭࣭࣭࣭13 ➨ᅄ❶ ⓶ୗᢞ࣐࢘ࢫ㦵᱁➽୰ࡢࢲࣉࢺ࣐ࢩࣥࡢᐃ㔞 ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭16 ➨❶ ⤖ㄽ ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭19 ᐇ㦂ࡢ㒊 ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭20 ᘬ⏝ᩥ⊩ ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭23 ㅰ㎡ ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭28
➨ ➨୍❶ ⥴ ゝ
ࢲࣉࢺ࣐ࢩࣥ㸦DAP㸧ࡣ Streptomyces roseosporus ࡼࡾ⏘⏕ࡉࢀࡿ࣏ࣜ࣌ࣉࢳ ࢻᢠ⏕≀㉁࡛ࠊ⌧ᅾࠊ၏୍⮫ᗋᛂ⏝ࡉࢀ࡚࠸ࡿࡶࡢ࡛࠶ࡿࠋDAP ࡣ࣓ࢳࢩࣜࣥ⪏ ᛶ㯤Ⰽࣈࢻ࢘⌫⳦㸦MRSA㸧ࢆྵࡴࢢ࣒ࣛ㝧ᛶ⌫⳦ᑐࡋࠊ㏿ࡸࡘ⃰ᗘ౫Ꮡ ⓗ࡞ẅ⳦స⏝ࢆ♧ࡋࠊ⓶࣭㌾㒊⤌⧊ឤᰁ1-3)ࠊMRSA ⳦⾑ࠊឤᰁᛶᚰෆ⭷⅖ ➼ 4-6)ࡢ㞴ᛶࡢឤᰁࡢ⒪⏝ࡉࢀ࡚࠸ࡿࠋDAP ࡣ㠀ࢱࣥࣃࢡᛶ࣑ࣀ㓟 ࢆྵࡴࢺࣜࢹ࢝࣌ࣉࢳࢻ࡛࠶ࡾࠊN ᮎ➃ࢹ࢝ࣀࣝ⬡⫫㓟ࢹ࢝࣌ࣉࢳࢻࣛࢡࢺ ࣥᵓ㐀ࢆ᭷ࡍࡿࠋ㢮ఝࡢ࣏ࣜ࣌ࣉࢳࢻᢠ⏕≀㉁ࡣࡳ࡞ྠᵝ N ᮎ➃ࢹ࢝ࣀࣝ⬡ ⫫㓟ࢹ࢝࣌ࣉࢳࢻࣛࢡࢺࣥࡲࡓࡣࣛࢡࢱ࣒ᵓ㐀ࢆᣢࡕࠊࡇࢀࡽࡀ࢝ࣝࢩ࣒࢘ ࡢ⤖ྜࡸᢠ⳦స⏝࠾࠸࡚㔜せ࡞ᙺࢆᯝࡓࡍࡉࢀ࡚࠸ࡿ 7)ࠋ⮫ᗋᛂ⏝ࡉࢀ࡚ ࠸࡞࠸ࡶࡢࡶྵࡵࠊ࣏ࣜ࣌ࣉࢳࢻᢠ⏕≀㉁ࡣ࣌ࣉࢳࢻ⎔ࡀ࣑ࢻ⤖ྜ࡛㛢ࡌ࡚ ࠸ࡿࢶࢩ࣐ࢩࣥ➼ࡢࢢ࣮ࣝࣉ࢚ࢫࢸࣝ⤖ྜ࡛㛢ࡌ࡚࠸ࡿ A21978C 㢮ࡢࢢࣝ ࣮ࣉࡢ2 ✀ࡢࢢ࣮ࣝࣉࡀ࠶ࡿࠋ๓⪅ࡣ࣌ࣉࢳࢻࢢࣜ࢝ࣥ๓㥑యࡢྜᡂࢆᢚไ ࡍࡿࡼࡾࠊ⣽⬊ቨྜᡂࢆ㜼ᐖࡍࡿࡢᑐࡋࠊDAP ࢆྵࡴᚋ⪅ࡣ࢝ࣝࢩ࣒࢘⃰ ᗘ౫Ꮡࡋࡓ⣽⳦ࡢ⣽⬊⭷ࡢ㏱㐣⬺ศᴟࢆࡋࡓࣘࢽ࣮ࢡ࡞ẅ⳦࣓࢝ࢽࢬ࣒ ࢆᣢࡘࡇࡀ᫂ࡽ࡞ࡗ࡚࠸ࡿ 5, 8)ࠋࡋࡋ࡞ࡀࡽࠊDAP ࡢ⓶ࡸ➽⫗࡞⏕ య⤌⧊୰ࡢᾐ㏱㛵㐃ࡋࡓάᛶࡢ࣓࢝ࢽࢬ࣒ࡣᚲࡎࡋࡶゎ᫂ࡉࢀ࡚࠸ࡿ ࡣ࠸࠼࡞࠸ࠋ୍᪉࡛ࠊDAP ࢆ⏝ࡋࡓ⒪࠾ࡅࡿࠊ≉␗ⓗ࡞స⏝ࡋ࡚ࠊ ➽㞀ᐖࡀᗈࡃ▱ࡽࢀ࡚࠾ࡾ6, 9-11) ࠊࡲࡓ➽㞀ᐖࡣ㦵᱁➽≉␗ⓗ㉳ࡇࡿࡇࡀ ሗ࿌ࡉࢀ࡚࠸ࡿ10)ࠋ
Fig.1. Structure of Daptomycin㸦DAP㸧
ࡑࡋ࡚ࠊࡑࡢస⏝ࡣ㐣㔞ᢞࡸ 10) ᢞ㛫㝸ࢆ▷ࡃࡋࡓⓎ⏕ࡋࡸࡍ࠸ࡇ ࡀ♧ࡉࢀ࡚࠸ࡿ11)ࠋ ࡲࡓࠊ㢖ᗘࡣᚲࡎࡋࡶ㧗ࡃ࡞࠸ࡀࠊDAP ⒪ࡢ㔜⠜࡞స⏝ࡋ࡚ᶓ⣠➽⼥ゎ ࡢⓎ㛵ࡍࡿࢣ࣮ࢫ࣏࣮ࣞࢺࡶ」ᩘሗ࿌ࡉࢀ࡚࠸ࡿ12-15)ࠋ ࡋࡋ࡞ࡀࡽࠊࡇࢀࡽࡢస⏝ࢆᘬࡁ㉳ࡇࡍせᅉࡢ≉ᐃࡣࠊᚋࡢ◊✲ㄢ㢟 ࡞ࡗ࡚࠸ࡿࠋKostrominova ࡽࡣࠊDAP ᢞࣛࢵࢺࡢ㦵᱁➽㞀ᐖ㛵ࡍࡿචᰁⰍ ἲࢆ⏝࠸ࡓ⌮⤌⧊Ꮫⓗᐇ㦂ࡽࠊDAP ⒪ࡢ᪩ᮇⓎ⏕ࡍࡿࣛࢵࢺ㦵᱁➽㞀ᐖ ࡣ➽⥺⥔㠧ࡢ⛛ᗎࡢ႙ኻ㉳ᅉࡍࡿࡇࢆሗ࿌ࡋ࡚࠸ࡿ16)ࠋࡲࡓࠊస⏝࡛࠶ࡿ 㦵᱁➽ࡢᙳ㡪ࢆ㑊ࡅࡿࡓࡵࡣࠊDAP ࡣ 1 ᪥ 1 ᅇࡢฎ᪉ࡀጇᙜ࡛࠶ࡿࡇࡶሗ ࿌ࡉࢀ࡚࠸ࡿ11)ࠋࡋࡋࠊ⾑₢୰DAP ࡢ᭱㧗⾑୰⃰ᗘ㸦Cmax㸧ࡸ⾑୰⃰ᗘ‐㛫 ᭤⥺ୗ㠃✚㸦AUC㸧ࡢୖ᪼ࡣࠊ⾑Ύࢡࣞࢳࢽࣥ࣍ࢫ࣍࢟ࢼ࣮ࢮάᛶࡢୖ᪼ࡸ➽ 㞀ᐖࡢⓎ⋡ࡣ༢⣧ࡣ㛵㐃ࡋ࡞࠸ࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ11)ࠋᚑࡗ࡚ࠊ➽⤌⧊ ࡢ⸆≀ࡢᾐ㏱ࡸศᕸࡢ≉ᛶࠊ࠶ࡿ࠸ࡣDAP ⒪࠾ࡅࡿ➽㞀ᐖࡢ࣓࢝ࢽࢬ࣒ゎ ᫂ࡢࡓࡵࡣື≀ࢆ⏝࠸ࡓヲ⣽࡞ᇶ♏ᐇ㦂ࡀᚲせ࡛࠶ࡾࠊࡑࡢࡓࡵࡣ㦵᱁➽୰ ࡢDAP ࡢṇ☜࡞ᐃ㔞ἲࡢ㛤ⓎࡀྍḞ࡞ࡿࠋ DAP ࡢᐃ㔞᪉ἲ㛵ࡍࡿ◊✲ࡣࠊ⾑₢୰ࡲࡓࡣ⾑Ύ୰ DAP ࢆᑐ㇟ࡋ࡚ࠊྛ ✀ࡢ᳨ฟἲࢆ⏝ࡋࡓ㧗㏿ᾮయࢡ࣐ࣟࢺࢢࣛࣇ࣮ἲ㸦HPLC ἲ㸧ࡸ㉸㧗㏿ᾮయ
ࢡ࣐ࣟࢺࢢࣛࣇ࣮ἲ㸦UHPLC ἲ㸧ࡀ㛤Ⓨࡉࢀ࡚࠸ࡿࠋ᳨ฟჾࡣࠊ⣸እ྾ග㸦UV㸧 17-20)ࠊ㉁㔞ศᯒ㸦MS㸧21)ࠊࢱࣥࢹ࣒㉁㔞ศᯒ㸦MS/MS㸧22, 23)ࡀ⏝࠸ࡽࢀ࡚࠸ࡿࠋ ㏆ᖺ࡛ࡣࠊᚤ⏕≀ࢆ⏝࠸ࡓẚ⃮ศᯒἲࡶ㛤Ⓨࡉࢀ࡚࠾ࡾࠊDAP ࡢᢠ⳦άᛶࡶేࡏ ࡚ホ౯ࡍࡿࡇࡀྍ⬟࡛࠶ࡿࡀࠊḞⅬࡋ࡚⏕యヨᩱ࡞ࡢࡼ࠺ዃ㞧≀ࢆከࡃ ྵࡴሙྜࡢᐃ㔞ࡣ㐺⏝ࡍࡿࡇࡀᅔ㞴࡛࠶ࡿ24)ࠋ⏕యヨᩱ㐺ᛂྍ⬟࡞ࢡ࣐ࣟ ࢺࢢࣛࣇἲࡢሗ࿌ࡢከࡃࡣࠊ⾑₢ࡸ⾑Ύ୰ࡢDAP ࢆ ᐃᑐ㇟ࡋ࡚࠾ࡾ๓ฎ⌮ ࡋ࡚࠸ࡎࢀࡢሗ࿌࠾࠸࡚ࡶ㢮ఝࡢᡭ㡰ࡀ⏝࠸ࡽࢀ࡚࠸ࡿࠋMartens-Lobenhoffer ࡽ ࡣ ࠊ ࣄ ࢺ ⾑ ₢ ࣓ ࢱ ࣀ ࣮ ࣝ ࡼ ࡿ 㝖 ࢱ ࣥ ࣃ ࢡ ࢆ ⾜ ࠸ ࠊ ୖ ࡳ ࢆ ࡑ ࡢ ࡲ ࡲ HPLC-UV ἲ࡚ศᯒࡍࡿࡢࡳ࡛ᴟࡵ࡚Ⰻዲ࡞ᅇ⋡ࡀᚓࡽࢀࡿࡇࢆሗ࿌ࡋ࡚ ࠸ࡿ17)ࠋ୍᪉ࠊE. Gikasetn ࡽࡣ࢘ࢧࢠ⾑Ύࡢ㝖ࢱࣥࣃࢡฎ⌮࠾࠸࡚ࠊ࣓ࢱࣀ ࣮ࣝࡢࡳ࡛ࡣዃ㞧≀ࡀቑ࠼ࠊࢭࢺࢽࢺࣜࣝࡢࡳ࡛ࡣᅇ⋡ࡀᝏࡋࡓࡇࡽ ࣓ࢱࣀ࣮ࣝࢭࢺࢽࢺࣜࣝ㸦1㸸2㸧ࡢΰྜᾮ࡚㝖ࢱࣥࣃࢡࢆ⾜ࡗࡓᚋࠊୖ ࡳࡢⓎᅛࢆ⾜࠸ࠊࡑࡢṧ´ࢆ⛣ື┦࡛ᕼ㔘ࡋ࡚ UHPLC-UV ὀධࡍࡿࡇ ࡼࡾࠊప⃰ᗘࠊ୰⃰ᗘࠊ㧗⃰ᗘ࡛ࡑࢀࡒࢀ 102.5 ± 4.3%, 100.6 ± 5.1%, 96.5 ± 3.6% ࡢ㧗࠸ᅇ⋡ࢆᚓ࡚࠸ࡿ18)ࠋBazoti ࡽࡶࠊࣄࢺ⾑₢ࢆ⏝࠸ࠊྠᵝࡢ๓ฎ⌮ࢆ⾜࠸ UHPLC-MS/MS ศᯒࡼࡾࠊప⃰ᗘࠊ୰⃰ᗘࠊ㧗⃰ᗘ࡛ࡑࢀࡒࢀ 96.1 ± 8.3%, 98.6 ± 7.5%, 93.5 ± 5.3%ࡢᅇ⋡ࢆᚓ࡚࠸ࡿ22)ࠋNaito ࡽࡣࣄࢺ⾑₢ࡢࡳ࡞ࡽࡎࣄࢺᒀ ୰ࡽࡢᐃ㔞ἲࢆሗ࿌ࡋ࡚࠾ࡾࠊ๓ฎ⌮ࡋ࡚⾑₢ࡣ㓑㓟ࢆῧຍࡋ࡚㐲ᚰศ㞳 ࢆ⾜࠸ࠊᒀࡣ࣓ࢱࣀ࣮ࣝࢆຍ࠼ࠊୖࡳࢆࣜࣥ㓟⦆⾪ᾮ࡛ᕼ㔘ࡍࡿࡇ࡛ࠊ UHPLC-CV ἲࡼࡾࡑࢀࡒࢀ 95.6%ࠊ105.4%Ⰻዲ࡞ᅇ⋡ࢆᚓ࡚࠸ࡿ20)ࠋBaietto ࡽࡣࠊHPLC-MS ࡼࡿࣄࢺ⾑₢୰ࡢ DAPࠊ࣑࢝ࢩࣥࠊࢤࣥࢱ࣐ࢩࣥࠊࣜࣇ ࣥࣆࢩࣥࡢከྠᐃ㔞ࢆヨࡳ࡚࠾ࡾࠊࢭࢺࢽࢺࣜࣝࡼࡾ㝖ࢱࣥࣃࢡࡋࡓ ୖࡳࢆỈࢺࣜࣇࣝ࢜ࣟ㓑㓟ΰᾮ㸦98㸸2㸧࡛ᕼ㔘ࡍࡿࡇࡼࡾࠊDAP ࡘ ࠸࡚90.5%ࡢᅇ⋡ࢆᚓ࡚࠸ࡿ21)ࠋࡲࡓࠊBaietto ࡽࡣࣄࢺ⾑₢ࢆᑐ㇟ࡋࢭࢺ ࢽࢺࣜࣝࠊ࣓ࢱࣀ࣮ࣝ࠾ࡼࡧỈࡢΰᾮ㸦10㸸1㸸1㸧ࢆຍ࠼ࠊ㐲ᚰศ㞳ࢆ⾜ࡗࡓୖ
ࡳࢆⓎᅛࡋࠊ⁐ゎࡢᚋࠊᗘࠊ㐲ᚰศ㞳ࡋ࡚ᚓࡓୖࡳࢆ UHPLC-UV ศᯒࡍࡿࡇ࡛94.48%ࡢᅇ⋡ࡀᚓࡽࢀࡓࡇࡶሗ࿌ࡋ࡚࠸ࡿ19)ࠋVerdier ࡽࡣࠊ ࣄࢺ⾑₢ࢆ⏝࠸ࠊࢭࢺࢽࢺࣜࣝࡼࡿ㝖ࢱࣥࣃࢡࡢᚋୖࡳࢆỈࢭࢺࢽ ࢺࣜࣝࡢΰᾮ㸦=1㸸1㸧࡛ᕼ㔘ࡋࠊప⃰ᗘࠊ୰⃰ᗘࠊ㧗⃰ᗘࡢ࠸ࡎࢀࡢ㡿ᇦ࡛ࡶ ࡑࢀࡒࢀ91.0 ± 1.4%, 93.5 ± 6.2%, 94.6 ± 7.7%ࡢ㧗࠸ᅇ⋡ࢆᚓ࡚࠸ࡿ23)ࠋࡇࢀࡽ ࡣ࠸ࡎࢀࡶࠊ⾑₢࡞ࡢ⾑ᾮヨᩱࡸᒀ୰ࡽDAP ࢆ㧗࠸ྜ࡛ᢳฟࡋ࡚࠾ࡾࠊ᳨ యࡢ✀㢮ࢆၥࢃࡎⰋዲ࡞ᅇ⋡ࡀᚓࡽࢀ࡚࠸ࡿࠋಶయࡽࡢᢳฟࡢሗ࿌ࡋ࡚ࡣࠊ ྾ỈᛶࡢࢹࣂࢫDAP ྵ᭷ࣄࢺ⾑₢ࢆῧຍࡋ⇱ᚋࠊ୍ᐃᮇ㛫ಖ⟶ࡋࡓࡶࡢ ࡽDAP ࢆᢳฟࡋࠊศᯒࡍࡿࡇࡀヨࡳࡽࢀ࡚࠸ࡿࠋࡇࢀࡣ⮫ᗋࡢ⌧ሙ࠾࠸࡚ࡣ DAP ࡢ TDM ࢆ⾜࠼ࡿタࡀ㊊ࡋ࡚࠸ࡿࡇࡸࠊヨᩱࢆ㛗ᮇ㛫Ᏻᐃࡋ࡚ಖ⟶ࡍ ࡿࡇࡀᅔ㞴࡛࠶ࡿࡇࡽࠊDAP ྵ᭷⾑₢ࢆᢅ࠸᫆࠸ᙧែࡋ࡚ಖᏑࡍࡿࡓࡵ ࡛࠶ࡿࠋࢹࣂࢫࡼࡾ㓑㓟࢚ࢳࣝࠊ㓑㓟ࠊࢭࢺࣥࠊỈ㸦50㸸20㸸20㸸10㸧ࡽ ࡞ࡿᾮ࡛DAP ࡢᢳฟࢆ⾜࠸ࠊୖࡳࢆⓎᅛࡋࠊ⁐ゎࡢᚋࠊᗘ㐲ᚰศ㞳ࡋ ࡚ᚓࡓୖࡳࢆUHPLC-UV ศᯒࡋࡓ⤖ᯝࠊࢹࣂࢫ୰ࡢ DAP ࡢಖᏑᛶࡣⰋዲ࡛ ࠶ࡗࡓࡀࠊᅇ⋡ࡣ 14.18%ప࠸್࡛࠶ࡗࡓ 19)ࠋࡇࡢࡼ࠺⾑ᾮࠊᒀ୰࡞ࡢ DAP ࡢᐃ㔞ࡣάⓎ◊✲ࡀࡉࢀ࡚ࡁࡓࠋ୍᪉ࠊDAP ࡢ⤌⧊ࡢᾐ㏱㛵ࡍࡿࢹ࣮ ࢱࡣ㠀ᖖᑡ࡞ࡃࠊࡲࡓ⤌⧊୰ࡢDAP ⃰ᗘࡢ ᐃἲ㛤Ⓨ㛵ࡍࡿሗ࿌ࡶᴟࡵ࡚ ᑡ࡞࠸ࠋᏛ⾡ㄽᩥࡋ࡚ሗ࿌ࡉࢀ࡚࠸ࡿࡶࡢ࡛ࡣࠊࣛࢵࢺ⓶ࢆ⏝࠸ࡓ⸆≀ືែ ヨ㦂25)࠾ࡼࡧே㦵୰ࡢ DAP ࢆ ᐃࡋࡓ⮫ᗋヨ㦂㝈ࡽࢀ࡚࠸ࡿ 26)ࠋ๓⪅࠾ࡅ ࡿᢳฟἲࡣ⤌⧊ࢆpH 6.0 ࡢࣜࣥ㓟⦆⾪ᾮ 18 㛫ᾐₕࡋࡓᚋࠊ㐲ᚰศ㞳ࡍࡿ᪉ ἲ࡛࠶ࡾࠊṑ᰿⫗ⱆ⭘⤌⧊୰ࡢࣥࣆࢩࣜࣥᐃ㔞᪉ἲೌࡗࡓࡶࡢ࡛࠶ࡿ27)ࠋࡲ ࡓࠊᚋ⪅࠾࠸࡚ࡣࠊ୍ᐃ㔞ࡢ⢊○ࡋࡓ⭸㛵⠇㦵pH 6 ࡢࣜࣥ㓟⦆⾪ᾮࢆຍ࠼ࠊ ᨩᢾ㉸㡢Ἴฎ⌮ࢆࡋࡓࡶࡢࢆ2 㛫෭ⶶࡋࡓᚋࠊ㐲ᚰศ㞳ࢆ⾜࠸ࠊୖࡳࡢᅇ ࢆ⾜࠺୍㐃ࡢ᧯సࢆ3 ᅇ⧞ࡾ㏉ࡋ࡚࠸ࡿ26)ࠋࡇࢀࡽࡢሗ࿌࡛ࡣࡇࡢࡼ࠺ࡋ࡚ ᚓࡓୖࡳࢆHPLC-UV ἲ࡛ศᯒࡋ࡚࠸ࡿ25, 26) ࠋ ࡋࡋ࡞ࡀࡽࠊ⏝࠸ࡽࢀࡓᢳ
ฟἲࡼࡗ࡚ࠊ࣍ࣔࢪࢼࢬࡉࢀࡓ⓶⤌⧊ࡢᠱ⃮ᾮࡸ⢊○ࡉࢀࡓே㦵ࡽࡢ DAP ࡢᢳฟ⋡㛵ࡍࡿヲ⣽ࡣ࡛᫂࠶ࡿࠋᚑࡗ࡚ࠊሗ࿌࠶ࡿᢳฟ᪉ἲࡼࡗ࡚ ᐃࡉࢀࡓDAP ⃰ᗘࡢ┿ᗘࡣᐃ࡛ࡣ࡞࠸ࠋ Kato ࡽࡣឤᰁࣔࢹ࣐ࣝ࢘ࢫࢆ⏝࠸ࠊDAP ࡢึᮇ㈇Ⲵᢞࢆ⾜ࡗࡓணഛᐇ㦂 ࠾࠸࡚➽⤌⧊୰ࡢ DAP ࡢᐃ㔞ࢆヨࡳࡓ 28)ࠋࡋࡋ࡞ࡀࡽࠊ⏕య⤌⧊୰ࡢ⸆≀ศ ᯒࡢヨᩱㄪ〇᪉ἲࡋ࡚ᗈࡃ▱ࡽࢀ࡚࠸ࡿ࣍ࣔࢪࢼࢬ㉸㡢Ἴฎ⌮ࡼࡿᢳฟ ᪉ἲࢆࠊ➽⫗⤌⧊ᑐࡋ࡚㐺⏝ࡋࡓࡀࠊ ᐃࡋࡓࢡ࣐ࣟࢺࢢ࣒ࣛୖDAP ࡢࣆ࣮ ࢡࢆ᳨ฟࡍࡿࡇࡣ࡛ࡁࡎࠊ➽⫗୰ࡽDAP ࢆᢳฟࡍࡿࡇࡣ࡛ࡁ࡞ࡗࡓࠋᚑ ࡗ࡚ࠊ㦵᱁➽ࡽDAP ࢆຠᯝⓗᢳฟࡍࡿ๓ฎ⌮᪉ἲࢆ㛤Ⓨࡍࡿࡇࡣࠊ➽⤌⧊ ୰⛣⾜ࡋࡓDAP ࡢᐃ㔞ᚲせྍḞ࡛࠶ࡿ࡛࠶ࡿ⪃࠼ࡽࢀࡿࠋⴭ⪅ࡢ▱ࡿ㝈 ࡾࠊ㦵᱁➽୰ࡢDAP ⃰ᗘ ᐃἲࢆ☜❧ࡋࡼ࠺ࡋࡓヨࡳࡣሗ࿌ࡉࢀ࡚࠸࡞࠸ࠋ ᮏ◊✲࡛ࡣࠊDAP ࡢ➽⤌⧊୰ࡢ⸆≀⛣⾜ࡸ DAP ⒪ࡢ㦵᱁➽㞀ᐖ㸦స⏝㸧 ࡢ࣓࢝ࢽࢬ࣒ゎ᫂㈨ࡍࡿࡓࡵࠊ࣐࢘ࢫ㦵᱁➽୰ࡢṇ☜࡞ᐃ㔞ࡢࡓࡵࡢ๓ฎ⌮ἲ ࢆ㛤Ⓨࡋࡓࠋࡲࡓࠊ㛤Ⓨࡋࡓ᪉ἲࢆ⏝ࡋ࡚ࠊDAP ࢆ༢ᅇ⓶ୗᢞࡋࡓ࣐࢘ࢫ ࡽ᥇ྲྀࡋࡓ⭣➽ࢆヨᩱࡋ࡚ࠊᐇ㝿ࡢ⭣➽୰ࡢ ᐃࢆ⾜ࡗࡓࠋ
➨ ➨❶ ỗ⏝ⓗᢳฟἲࢆ⏝࠸ࡓ࣐࢘ࢫ㦵᱁➽ࡽࡢࢲࣉࢺ࣐ࢩࣥࡢᅇ ࣍ࣔࢪࢿ࣮ࢩࣙࣥ㉸㡢Ἴฎ⌮ࡼࡿᆺⓗ࡞ᾮ┦-ᅛ┦ᢳฟἲࡣࠊ⸆≀࡞ࡢ ᭷ᶵྜ≀ࢆ⏕యᅛయヨᩱ୰ࡽᢳฟࡍࡿᡭẁࡋ࡚⌧ᅾࡶỗ⏝ࡉࢀ࡚࠸ࡿ 25, 26)ࠋࡲࡓࢡ࣒࣓ࣟࣟ࣍ࣝࢱࣀ࣮ࣝ࠾ࡼࡧỈࢆ⏝࠸ࡓᢳฟἲࡣࠊ⬡⁐ᛶᡂศࢆྵ ࡴΰྜ≀ࡢศ㞳⏝࠸ࡽࢀ࡚࠾ࡾBlighDyer ἲࡤࢀ࡚࠸ࡿࠋᮏἲࡣ DAP ࡸ ࣂࣥࢥ࣐ࢩࣥ࡞ࡢỈ⁐ᛶ⸆≀ࢆỈᛶࡢୖࡳ⛣⾜ࡉࡏࡿࡇࡀ▱ࡽࢀ࡚ ࠸ࡿ 29)ࠋⴭ⪅ࡣᮏἲࢆࠊỗ⏝ⓗᢳฟἲࡋ࡚ᮏ◊✲ࡢᇶ♏ᢳฟἲࡋ࡚⏝࠸ࡓࠋ ỗ⏝ⓗᢳฟἲ࠾࠸࡚ࠊỈ⁐ᛶ࡛࠶ࡿDAP ࣂࣥࢥ࣐ࢩࣥሷ㓟ሷࡣ⭣➽࣍ࣔ ࢪࢿ࣮ࢺࡽᢳฟᾮࡢỈ┦ᢳฟࡉࢀࡿࡶࡢᮇᚅࡋࡓࠋⴭ⪅ࡣ➽⤌⧊୰ࡽࡢ DAP ᢳฟࡢࡓࡵࡢணഛヨ㦂ࡋ࡚ࣈࣛࣥࢡ⭣➽㸦0.2 g㸧ᑐࡋ 1.0 × 10-4 M ࡢ ࢫࢺࢵࢡ⁐ᾮ20 PL ࢆῧຍࡋࡓ࣐࢘ࢫ㦵᱁➽ࢆసᡂࡋ DAP ࠾ࡼࡧࣂࣥࢥ࣐ࢩࣥ ࡢῧຍᅇヨ㦂ࢆ⾜ࡗࡓࠋFig.2 ࡣࡑࡢᢳฟᡭ㡰ࢆ♧ࡋࡓࡶࡢ࡛࠶ࡿࠋHPLC ࠾ ࡅࡿ᳨㔞⥺ࡣࡑࢀࡒࢀࡢ⸆≀ࡢྛ⃰ᗘࡢᶆ‽ᾮෆᶆ‽ࣆ࣮ࢡ㧗ࡉࡢẚࡼࡾస ᡂࡋࡓࠋ᳨ฟ㝈⏺ࡣS/N ẚ 3 ௨ୖࡢ᭱ప⃰ᗘࡋࠊDAP ࠾ࡼࡧࣂࣥࢥ࣐ࢩࣥ ࠾࠸࡚ࡑࢀࡒࢀ0.3 PM ࠾ࡼࡧ 0.4 PM ࡛࠶ࡗࡓࠋ୧⸆࠾࠸࡚๓ண ࡉࢀ ࡓヨᩱ୰⃰ᗘᑐᛂࡋࡓᐃ㔞ୖ㝈࡛࠶ࡿ 0.75 PM ࡛ࡢ⢭ᗘࡣ 20%௨ෆ࡛࠶ࡗࡓࠋ ᳨㔞⥺ࡣ0.75 ࡽ 10 PM 㸦DAP㸸n = 6, r = 0.992, ࣂࣥࢥ࣐ࢩࣥ㸸n = 6, r = 0.999㸧 ࡢ⠊ᅖ࡛┤⥺ᛶࢆ♧ࡋࡓࠋ
⢭ᗘࡣ┦ᑐᶆ‽೫ᕪ㸦RSD㸧ࡼࡾ⟬ฟࡋࡓࠋ0.75 PMࠊ2.5 PMࠊ࠾ࡼࡧ 7.5 PM ࡢDAP ᶆ‽ᾮࢆ⏝࠸࡚ ᐃࡋࡓ RSD ್ࡣࡑࢀࡒࢀ 12.86%ࠊ6.76%ࠊ2.18%࡛࠶ࡗ ࡓࠋࡲࡓ0.75 PMࠊ2.5 PMࠊ࠾ࡼࡧ 7.5 PM ࡢࣂࣥࢥ࣐ࢩࣥᶆ‽ᾮࢆ⏝࠸࡚ ᐃ ࡋࡓ྾ගᗘࡢRSD ್ࡣࡑࢀࡒࢀ 15.01%ࠊ5.28%࠾ࡼࡧ 1.85%࡛࠶ࡗࡓࠋ Fig.3 ࡣ DAP ࠾ࡼࡧࣂࣥࢥ࣐ࢩࣥࡑࢀࡒࢀ 3 ᳨యࡘ࠸࡚ῧຍᚋ⤒㐣㛫 ᅇ⋡ࢆࣉࣟࢵࢺࡋࡓࡶࡢ࡛࠶ࡿࠋ ࡇࢀࡽࡢ⤖ᯝࡣࠊᅇ⋡ࡀ⸆ῧຍᚋࡢ⤒㐣㛫౫Ꮡࡍࡿࡇࢆ♧ࡋ࡚࠸ࡿࠋ Fig.3 ♧ࡍࡼ࠺ࠊࣂࣥࢥ࣐ࢩࣥࡢᖹᆒᅇ⋡ࡣ࠸ࡎࢀࡢ⤒㐣㛫࠾࠸࡚ࡶ 96%–104%࡛࠶ࡗࡓࠋ୍᪉ࠊDAP ࡢᅇ⋡ࡣ㛫ࡢ⤒㐣ඹᣦᩘ㛵ᩘⓗῶᑡ ࡋࠊ⣙25%᮰ࡋࡓࠋࡇࡢ⤖ᯝࡣ㦵᱁➽ 1 g ࠶ࡓࡾ 12 Pg ࡢ DAP ࡀఱࡽࡢ≉ ู࡞⤖ྜࡼࡾ➽⫗⤌⧊୰ྲྀࡾ㎸ࡲࢀ࡚࠾ࡾࠊࡑࢀࡽࡣศ㓄ᖹ⾮ࡼࡿ⁐ᾮᢳ ฟ࡛ࡣᅇᅔ㞴࡛࠶ࡿࡇࢆ♧၀ࡋ࡚࠸ࡿࠋ DAP ࡢ⾑₢ࢱࣥࣃࢡ⤖ྜ⋡ࡣ⣙ 92%㧗⋡࡛࠶ࡿࡇࡣᗈࡃ▱ࡽࢀ࡚࠸ࡿࡀ9)ࠊ ⾑₢୰ࡽࡣ࣓ࢱࣀ࣮ࣝࡸࢭࢺࢽࢺࣜࣝ➼ࡼࡿ㝖ࢱࣥࣃࢡ㐲ᚰศ㞳ࡼࡿ ㏻ᖖࡢ๓ฎ⌮࡛ࢱࣥࣃࢡ⤖ྜࡼࡾᖸ΅ࡉࢀࡿࡇ࡞ࡃࠊᐜ᫆⾑₢୰ࡽᅇ ྍ⬟࡛࠶ࡿ17-23)ࠋࡑࢀᨾࠊ⌧ẁ㝵࡛ࡣDAP ࡀ➽⤌⧊୰ྲྀࡾ㎸ࡲࢀࡿ≀⌮ Ꮫⓗᶵᗎࡘ࠸࡚ࡣ᫂ࡽ࡛ࡣ࡞࠸ࡀࠊࡇࡢᐇࡣ⮫ᗋศᯒᏛࡢほⅬࡽࡣ㠀 ᖖ⯆῝࠸ࠋ
O O H OH N H O NHCH3 H O H N H O H2N N H O H N O OH OH HO HN H HOOC O N H O H HO Cl O O O O OH NH2 OH OH OH Cl H HCl
(a) daptomycin (DAP) (b) vancomycin MW: 1620.6 MW: 1449.2
Fig. 3. Dependence of Recovery Ratios of DAP (a) and Vancomycin (b) on the Time to Start Conventional Extract after Spiking Murine Skeletal Muscles
The muscle samples were prepared by spiking analyte-free femoral muscle (0.2 g) with 20 PL of the 100 PM standard solution of the drug, and were treated according to procedure P1 shown in Fig. 1. In the case of vancomycin, 0.1 M HCl (100 PL) and 300 PL water were added instead of 400 PL water in the P1 procedure to dissociate vancomycin. Recovery calculated from the comparison with the peak height for standard solution prepared in the same concentration was evaluated by measuring three replicates of the samples. The error bars indicate the 95% confidence interval.
➨ ➨୕❶ ࢺࣜࣉࢩࣥᾘࢆ⏝࠸ࡓ࣐࢘ࢫ㦵᱁➽ヨᩱࡢ๓ฎ⌮ἲ ➨୍⠇ ࢲࣉࢺ࣐ࢩࣥᅇ㐺ࡍࡿ㓝⣲ࡢ㑅ᐃ ๓ ❶ࡢ ⤖ ᯝ ࡼࡾ ࠊ ⭣ ➽୰ ࡢ DAP ࡣࢭࢺࢽࢺࣜࣝࡼࡿ㝖ࢱࣥࣃࢡ BlighDyer ἲࡢࡳ࡛ࡣ༑ศ࡞ᢳฟࡀᅔ㞴࡛࠶ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ㢮ఝࡢ ࡋ࡚ࠊ∵⾑₢୰ࡽࢺࣜࢡࣟࣟ㓑㓟ࢆ⏝࠸ࠊ࣌ࣉࢳࢻࢆᢳฟࡋࡓሙྜࠊศᏊ㔞 3000 ௨ୗࡢศᏊࡣࡰᢳฟࡉࢀ࡞ࡗࡓ30)ࡸࠊ࣐࢘ࢫ⾑₢୰ࡽࢭࢺࢽࢺࣜ ࡛ࣝ㝖ࢱࣥࣃࢡࢆ⾜࠸ࠊ࣑ࣟࢻβ-ࣉࣟࢸࣥࣇࣛࢢ࣓ࣥࢺࡢᅇ⋡ࡀⴭࡋࡃ Ⰻ࡛࠶ࡗࡓ31)ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡑࡢᶵᗎࡋ࡚ࡣ᭷ᶵ⁐፹ῧຍࡼࡿ࣌ࣉ ࢳࢻࡢ⮬ᕫจ㞟ཪࡣ∝㞧ࢱࣥࣃࢡ㉁ࡀจ㞟ࡍࡿ㝿 ᐃᑐ㇟ࡢ࣌ࣉࢳࢻࡀඹỿࡍ ࡿࡓࡵ⪃࠼ࡽࢀ࡚࠸ࡿ 31)ࠋ๓❶ࡢ⤖ᯝࡽࡣศᏊ㔞ࡀྠ⛬ᗘࡢ DAP ࣂࣥࢥ ࣐ࢩ࡛ࣥ࠶ࡗ࡚ࡶDAP ࡢࡳ⤒ⓗ࡞ⴭࡋ࠸ᅇ⋡ࡢᝏࢆㄆࡵ࡚࠾ࡾࠊศᏊ㔞 ࡢࡳࡀࢱࣥࣃࢡ୰ࡽࡢ࣌ࣉࢳࢻࡢᅇࢆᅔ㞴ࡍࡿỴᐃᅉᏊ࡛ࡣ࡞࠸ࡇࡀ᥎ ᐹࡉࢀࡿࠋⴭ⪅ࡣ㦵᱁➽୰ྲྀࡾ㎸ࡲࢀࡓDAP ࢆᅇࡍࡿࡓࡵࠊࣜࣃ࣮ࢮࢆ⏝ ࠸⬡⁐ᛶ⸆≀ࢆ⬡⫫⤌⧊ࡽຠᯝⓗᢳฟࡋ࡚࠸ࡿ32)ࢆࣄࣥࢺࡋ࡚ࠊ㓝⣲ ᾘࢆ⏝࠸ࡓ๓ฎ⌮ἲ╔┠ࡋࡓࠋ 㓝⣲ᾘἲࢆ HPLC ศᯒࡢ๓ฎ⌮⏝࠸ࡓ࡛ࡣࠊ᭷ᶵ⁐፹33, 34)ࡼࡿ㝖ࢱࣥ ࣃࢡࡸ㉸㡢Ἴ34)ࡼࡿ๓ฎ⌮㐯Ⰽࡢ࡞࠸⤖ᯝࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋ ᮏ◊✲࠾࠸࡚ࠊ⏝ࡍࡿ㓝⣲ࡣ㦵᱁➽ᑐࡋᙉ࠸ᾘຊࢆᣢࡕࠊࡘ DAP ࢆศゎࡋ࡞࠸≉ᛶࡀồࡵࡽࢀࡿࠋࢺࣜࣉࢩࣥࡣ⮅㓝⣲࡛࠶ࡾࠊ➽⤌⧊ᑐࡋᙉ࠸ ᾘస⏝ࢆ♧ࡋࠊ⃰ᗘ౫Ꮡⓗ࡞ᾘ㏿ᗘࢆ♧ࡍ35, 36)ࠋศᏊస⏝ᶵᗎࡋ࡚ࡣࠊࣉ ࣟࣜࣥ௨እ㞄᥋ࡍࡿࠊࣜࢪࣥࡸࣝࢠࢽࣥࡢ࢝ࣝ࣎࢟ࢩࣝᇶഃࡢ࣌ࣉࢳࢻ㙐ࢆ ≉␗ⓗ㛤ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ 37)ࠋࡇࡢ≉ᛶࡣ DAP ࡢࢹ࢝࣌ࣉࢳࢻࣛࢡ
ࢺࣥ⎔ࢆ㛤ࡋ࡞࠸ࡶࡢ⪃࠼ࡽࢀࡓࠋࡲࡓDAP ࡣ㓝⣲ࡉࢀࡓ㝿ࠊ࢟ࣔࢺࣜ ࣉࢩࣥࡸ I ᆺࣉࣟࢸ࣮ࢮࡣຍỈศゎࡉࢀࡿࡶ㛵ࢃࡽࡎࠊࢺࣜࣉࢩࣥࡼࡿ ຍỈศゎࡢᙳ㡪ࡣཷࡅ࡞࠸ࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ38)ࠋࡉࡽࢺࣜࣉࢩࣥࡣ࢝ࣝࢩ ࣒࢘࢜ࣥࡸ࣐ࢢࢿࢩ࣒࢘࢜ࣥ㜼ᐖࡉࢀࡿࡇࡣᗈࡃ▱ࡽࢀ࡚࠾ࡾࠊࡑࡢࡓ ࡵࠊⴭ⪅ࡣ㦵᱁➽ᾘฎ⌮᭱ࡶ㐺ࡍࡿ⪃࠼EDTA ࢆྵࡴࢺࣜࣉࢩࣥ⁐ᾮࢆᑐ ㇟㓝⣲ࡋ࡚㑅ᐃࡋࡓࠋ
➨⠇ ࢺࣜࣉࢩࣥࡼࡿ㦵᱁➽ヨᩱ๓ฎ⌮᮲௳ࡢ᭱㐺 ࢺࣜࣉࢩࣥᾘ᮲௳ࡢ᭱㐺ࡣ➽⤌⧊ࡢ DAP ࡢྲྀࡾ㎸ࡳࡀ᮰ࡋ࡚࠸ࡿ ⪃࠼ࡽࢀࡿDAP ῧຍᚋ 3 㛫⤒㐣ࡋࡓ㦵᱁➽ࢆ⏝࠸࡚⾜ࡗࡓࠋࡇࢀࡽᑐࡋࠊࢺ ࣜࣉࢩࣥᶆ‽ᾮࢆῧຍᚋࠊ࣮ࣥ࢟ࣗ࣋ࢩࣙࣥࢆ⾜࠸ࠊᘬࡁ⥆ࡁỗ⏝ⓗᢳฟἲࢆ ⾜ࡗࡓࠋFigure 4a ࡣ 37 °C ࡛ 10 ศ㛫㓝⣲ฎ⌮ࡋࡓࡢ ࢺࣜࣉࢩࣥᶆ‽ᾮࡢῧຍ 㔞➽ヨᩱࡽࡢDAP ࡢᅇ⋡ࢆࣉࣟࢵࢺࡋࡓࡶࡢ࡛࠶ࡿࠋࡑࡢ⤖ᯝࠊ100 PL ௨ ୖࡢࢺࣜࣉࢩࣥᶆ‽ᾮࡢῧຍࡼࡾᣦᩘ㛵ᩘⓗDAP ࡢᅇ⋡ࡢቑຍࢆㄆࡵࡓࠋ ࡇࡢ⌧㇟ࡢヲ⣽࡞ᶵᗎࡣ࡛᫂࠶ࡿࡀࠊ㓝⣲ᛂ࠾࠸࡚ࠊᛂ㏿ᗘࡣᛂศ Ꮚ⃰ᗘ౫Ꮡࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ39)ࠋ (a) (b)
Fig. 4. Dependences of Recovery of DAP from the Muscle Spiked with DAP on the Amounts of Trypsin Standard Solution Used for Digestion (a) and on Incubation Time for Muscle Digestion (b)
The additional recovery samples were prepared by using analyte-free muscle (0.2 g) spiked with a 20 PL aliquot of 50 PM DAP standard solution, and treated with trypsin followed by the conventional extraction, as shown in Fig. 1. (a) Trypsin was used for muscle digestion involving incubation for 10 min at 37°C. (b) Incubation time was measured for muscle digestion with 150 PL trypsin standard solution at 37℃.
ࢺࣜࣉࢩࣥῧຍ㔞ࡢቑຍࡣ➽⤌⧊ࡢ㏿ࡸ࡞ᔂቯᾮ≧ࢆಁ㐍ࡋࠊᇶ㉁࡞ ࡿ DAP-➽⤌⧊⤖ྜ㒊⃰ᗘࡀ㏿ࡸୖ᪼ࡍࡿࡓࡵࠊ㓝⣲ᛂ㏿ᗘࡀୖ᪼ࡍࡿ ࡶࡢ⪃࠼ࡽࢀࡿࠋࢺࣜࣉࢩࣥᇶ㉁࡛࠶ࡿ DAP-➽⤌⧊⤖ྜ㒊ࡣ」ྜయࢆᙧ ᡂࡋࡓᚋࠊࡑࡢ≉␗ⓗ࡞࣌ࣉࢳࢻ⤖ྜࡢ㛤ࢆక࠺ࢱࣥࣃࢡᵓ㐀ࡢኚࢆ㉳ࡇࡋࠊ DAP ࡀ▐㛫ⓗᨺฟࡉࢀࡿࡶࡢ᥎ᐹࡉࢀࡿࠋ ࢱࣥࣃࢡ㉁ࡢᾘ㐣⛬ DAP ࡢ㐟㞳ᛂࡣ➇ྜ㜼ᐖ࡛࠶ࡿࡓࡵࠊヲ⣽࡞㏿ᗘㄽ ⓗゎᯒࡣᐜ࡛᫆ࡣ࡞࠸ࡀࠊࡇࢀࡽ୍㐃ࡢᛂࡢ⤖ᯝࠊ୍ᐃࡢᛂ㛫ୗ࠾࠸࡚ࠊ ࢺࣜࣉࢩࣥῧຍ㔞ᑐࡋᣦᩘ㛵ᩘⓗ࡞ᅇ⋡ࡢୖ᪼ࢆㄆࡵࡓࡶࡢ⪃࠼ࡽࢀࡿࠋ ୍᪉࡛ࠊFig.4b ࡣᾘฎ⌮ࡢ㛫 DAP ᅇ⋡ࡢᙳ㡪ࢆ♧ࡋ࡚࠸ࡿࠋࢺࣜࣉ ࢩࣥᶆ‽ᾮ150 PLࠊ37 °Cࠊ30 ศ㛫ࡢฎ⌮࡛ 0.2 g ࡢ⭣➽ࡢ࡞ᾘ⣙ 100% ࡢᅇ⋡ࡀほᐹࡉࢀࡓࠋ࡞࠾ࠊ␃ពࡍࡁ⤖ᯝࡋ࡚ࠊ1 㛫௨ୖࡢᾘฎ⌮࡛ ࡣDAP ࡢᅇ⋡ࡢపୗࢆㄆࡵ࡚࠸ࡿࠋࡇࢀࡽࡢ⤖ᯝࡼࡾࠊ0.2 g ࡢ࣐࢘ࢫ㦵᱁➽ ࡢᾘࡣ150 PL ࢺࣜࣉࢩࣥ ᶆ‽ᾮࢆ⏝࠸ࡓࡣ 37 °C ࡛ 30 ศࡢฎ⌮ࡀ᭱㐺 ᮲௳࡛࠶ࡿࡇࡀ᫂ࡽ࡞ࡾࠊ௨ᚋࡢ᳨ウࡣࡇࡢ᮲௳ࢆ⏝࠸ࡓࠋFig.5 ࡣࡑࡢ ฎ⌮ᡭ㡰ࢆ♧ࡋࡓࡶࡢ࡛࠶ࡿࠋ
Fig. 5. Procedure for pretreatment involving the conventional extraction (Bligh-Dyer method) after trypsin digestion before HPLC analysis of DAP in mice skeletal muscle.
➨୕⠇ ࣐࢘ࢫ㦵᱁➽ࢆ⏝࠸ࡓࢲࣉࢺ࣐ࢩࣥࡢῧຍᅇ ⴭ⪅ࡣ㦵᱁➽ᙉᅛྲྀࡾ㎸ࡲࢀࡓ DAP ࢆᢳฟࡍࡿࡓࡵࠊࢺࣜࣉࢩࣥฎ⌮ࡀ 㠀ᖖຠᯝⓗ࡛࠶ࡿࡇࢆぢฟࡋࡓࠋࡇࡢ▱ぢࡼࡾࠊⴭ⪅ࡣDAP ῧຍ⭣➽ࡽ ࡢᅇᐇ㦂ࢆ⾜ࡗࡓࠋヨᩱࡣࣈࣛࣥࢡ⭣➽㸦0.2 g㸧 50 PM DAP ᶆ‽ᾮ 20 PL ࢆῧຍࡋࠊ3 㛫෭ⶶࡋㄪ〇ࡋࡓࠋFigure 6 ࡣ๓ฎ⌮ࡼࡿᅇ⋡ࢆẚ㍑᳨ウࡍࡿ ࡓࡵࠊ➽⫗ヨᩱ⾜ࡗࡓ๓ฎ⌮ᡭ㡰ࢆ♧ࡋࡓࡶࡢ࡛࠶ࡿࠋFig. 6A ࠾ࡼࡧ Fig. 6C ࡣࡑࢀࡒࢀࠊỗ⏝ⓗᢳฟἲ࠾ࡼࡧࢺࣜࣉࢩࣥฎ⌮ࡢᚋỗ⏝ⓗᢳฟἲࢆ㐺⏝ࡋ࡚ ㄪ〇ࡋࡓヨᩱ㸦4 ࢧࣥࣉࣝ㸧ࢆ HPLC ศᯒࡋࡓ⤖ᯝ࡛࠶ࡿࠋ
Fig. 6. Experimental Scheme for Recovery Assays from Skeletal Muscle Samples
The muscle samples were analyte-free femoral muscles spiked with 1.62 Pg DAP. The measured mean values (n = 4) are shown in red.
ࡉࡽࠊFig. 6B ࡣỗ⏝ⓗᢳฟἲࡼࡿฎ⌮ᚋࠊࢧࣥࣉࣝࢳ࣮ࣗࣈෆṧࡗࡓ ➽⤌⧊ᑐࡋ࡚ࢺࣜ ࣉࢩࣥฎ⌮ỗ⏝ⓗ ᢳฟἲࢆ ⾜ࡗ࡚ㄪ〇 ࡋࡓヨᩱ⁐ᾮࢆ HPLC ศᯒࡋࡓࡶࡢ࡛࠶ࡿࠋ࡞࠾ࠊFig. 6 ᖹᆒࡢᅇ⋡ࡶ♧ࡋ࡚࠸ࡿࠋ⯆῝࠸ ࡇࠊỗ⏝ⓗᢳฟἲࡢࡳࢆ⾜ࡗࡓA ࡑࡢṧ´ࢺࣜࣉࢩࣥฎ⌮࠾ࡼࡧỗ⏝ⓗ ᢳฟἲࢆ⧞ࡾ㏉ࡋࡓB ࡢྜィࡣࠊࢺࣜࣉࢩࣥฎ⌮࠾ࡼࡧỗ⏝ⓗᢳฟἲࢆ⾜ࡗࡓ C ࡢᅇ⋡ྠ⛬ᗘ࡛࠶ࡗࡓࠋࡇࡢ⤖ᯝࡣ2/3 ௨ୖࡢ DAP ࡀ➽⫗ࢱࣥࣃࢡ୰ᙉᅛ ⤖ྜࡋ࡚Ꮡᅾࡋ࡚࠾ࡾࠊࡑࢀࡽࡣࢺࣜࣉࢩࣥࡼࡿᾘࡀ࡞ࡃ࡚ࡣᢳฟࡀᅔ㞴 ࡛࠶ࡿࡇࢆ♧၀ࡋ࡚࠸ࡿࠋ Table 1 ࡣ 3 ẁ㝵ࡢ␗࡞ࡿ⃰ᗘ࡛ DAP ࢆῧຍࡋࡓ⭣➽ヨᩱࡽࡢᅇᐇ㦂ࡢ ⤖ᯝ࡛࠶ࡿࠋ
Table 1. Recovery Data and the RSD of DAP from Murine Skeletal Muscles Spiked with 0.064, 0.16, and 1.62 Pg DAP. Addition amounts (Pg) Recovery (%) a) SD RSD (%) 1 2 3 4 Mean 0.064b) 75.2 42.3 87.3 52.8 64.4 20.52 31.87 0.16c) 86.3 84.4 103.1 94.3 92.0 8.54 9.28 1.6d) 97.5 99.5 101.1 100.3 99.6 1.54 1.55
a) Data obtained from HPLC analysis after sample pretreatment shown in Figs. 2 and 5. b) Prepared by spiking a 20 PL aliquot of a 2.0 PM DAP standard solution. The concentration of this muscle sample is calculated as 0.32 Pg/g (muscle).
c) Prepared by spiking a 20 PL aliquot of a 5.0 PM DAP standard solution. The concentration of this muscle sample is calculated as 0.80 Pg/g (muscle).
d) Prepared by spiking a 20 PL aliquot of a 50 PM DAP standard solution. The concentration of this muscle sample is calculated as 8.0 Pg/g (muscle). The mean value shown in Fig. 4C was obtained from these experiments.
0.064 Pg ῧຍ㸦᭱ࡶప⃰ᗘࡢሙྜ㸧௨እࡣ⏕≀Ꮫศᯒࡢᇶ‽ࢆ‶ࡓࡋ࡚࠸ࡓࠋ ࡇࡢ⤖ᯝࡽDAP ῧຍ➽⤌⧊ࡢࢺࣜࣉࢩࣥฎ⌮ࡣ 0.88 Pg/g㸦➽⫗㸧ࡢ⠊ᅖ ࠾࠸࡚ࠊ㧗ࡃᏳᐃࡋࡓᅇ⋡ࡀᚓࡽࢀࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ୍᪉࡛ࠊTable 1 ♧ࡋࡓ㏻ࡾࠊ0.08 Pg ࡢῧຍࡢ RSD ࡣᇶ‽ࢆ‶ࡓࡉ࡞ࡗࡓࠋᮏᐇ㦂⣔࠾ ࡅࡿHPLC ὀධࡢ᭱⤊⃰ᗘࡣ 0.8 PM ࢆୗᅇࡗ࡚࠾ࡾࠊࡇࢀࡣ⌧ᅾࡢ HPLC-UV ἲࡢᐃ㔞ୗ㝈㸦0.75 PM㸧㏆࠸ࡶࡢ࡛࠶ࡗࡓࠋ ࡇࢀࡽࡢ⤖ᯝࡽࠊᐇ㝿ࡢ⸆≀ᢞ࠾࠸࡚ࡶ➽⫗┤᥋ῧຍࡍࡿྠᵝ DAP ࡀ➽⤌⧊⛣⾜ࡍࡿ࠸࠺௬ᐃࡢඖ࡛ࠊࢺࣜࣉࢩࣥᾘࢆྵࡴ๓ฎ⌮᪉ἲ ࡣࠊ࣐࢘ࢫ㦵᱁➽୰ࡢDAP ࢆṇ☜ᐃ㔞ྍ⬟ࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ
➨ ➨ᅄ❶ ⓶ୗᢞ࣐࢘ࢫ㦵᱁➽୰ࡢࢲࣉࢺ࣐ࢩࣥࡢᐃ㔞 ⴭ⪅ࡣ᭱㐺ࡉࢀࡓ᮲௳ୗ࠾࠸࡚ࠊDAP ⓶ୗᢞ࣐࢘ࢫ㦵᱁➽୰ࡢᐃ㔞ࢆヨ ࡳࡓࠋDAP ࢆ 100 mg/kg 㔞࡞ࡿࡼ࠺༢ᅇ⓶ୗᢞࡋࠊᢞ 2 㛫ᚋ࣐࢘ࢫ ⭣➽ࢆ᥇ྲྀࡋࠊFig. 5 ♧ࡋࡓࢺࣜࣉࢩࣥฎ⌮ỗ⏝ⓗᢳฟἲࡼࡿ୍㐃ࡢᢳ ฟࢆ⾜࠸ࠊDAP ࡢᐃ㔞ࢆ⾜ࡗࡓࠋFigure 7 ࡣ➽⫗ヨᩱࡢ๓ฎ⌮ࡢࢫ࣮࣒࢟ HPLC ศᯒࡽᚓࡽࢀࡓ⤖ᯝ࡛࠶ࡿࠋ
Fig. 7. Experimental Scheme for the HPLC Analyses of the Determination of DAP in Murine Skeletal Muscles Collected from Mice (n = 5) Administered a Single Subcutaneous Dose of 100 mg/kg DAP
ῧຍᐇ㦂ྠᵝࠊA ࡣỗ⏝ⓗᢳฟἲࡢࡳࢆ⾜ࡗࡓࡶࡢࠊB ࡣࡑࡢṧ´ࢺࣜ ࣉࢩࣥฎ⌮࠾ࡼࡧỗ⏝ⓗᢳฟἲࢆ⧞ࡾ㏉ࡋࡓࡶࡢࠊC ࡣࢺࣜࣉࢩࣥฎ⌮࠾ࡼࡧỗ
⏝ⓗᢳฟἲࢆ⾜ࡗࡓࡶࡢ࡛࠶ࡿࠋTable 2 ࡣᚓࡽࢀࡓ ᐃ್ࢆ♧ࡋࡓࠋ
in vivo ࡢ➽⫗⤌⧊ࡢศᯒ⤖ᯝ࠾࠸࡚ࡣࠊ➽⤌⧊ෆࡢከᵝᛶࡸ㠀᳨య≀㉁ࡢᒁ ᅾ➼ࡼࡿㄗᕪࡀ㉳ࡇࡾᚓࡿࡓࡵࠊ࠶ࡿ⛬ᗘࡢ⤖ᯝࡢࡤࡽࡘࡁࡣண ࡉࢀࡓࠋ
ᚓࡽࢀࡓ DAP ⃰ᗘࡣỗ⏝ⓗᢳฟἲࡼࡿ➽⫗๓ฎ⌮㸦Fig. 7A㸧ẚࠊࢺࣜࣉ
ࢩࣥᾘἲ(Fig. 7C㸧ࡢ᪉ࡀ㧗ࡗࡓࠋ ࡲࡓࠊῧຍᐇ㦂ྠᵝࠊ࡚ࡢ᳨య࠾࠸࡚ࠊࡑࢀࡒࢀ A B ࡢྜィࡣ C ㏆ఝࡍࡿ࠸࠺㛵ಀᛶࡀぢࡽࢀࡓࠋ ࡇࡢ⤖ᯝࡣࠊᐇ㝿ື≀ DAP ࢆᢞࡋࡓ㝿ࡶࠊఱࡽࡢ➽ࢱࣥࣃࢡࡢ┦ స⏝ࡼࡾࠊ➽⤌⧊୰DAP ࡀྲྀࡾ㎸ࡲࢀ࡚࠾ࡾࠊࡑࢀࡽࡢṇ☜࡞⃰ᗘࢆᐃ㔞 ࡍࡿୖ࡛ࢺࣜࣉࢩࣥฎ⌮ࡀྍḞ࡛࠶ࡿࡇࢆ♧၀ࡋ࡚࠸ࡿࠋ୍᪉࡛ࠊDAP ࡢ⾑ ₢࠾ࡼࡧ⾑Ύ୰⃰ᗘࡣᩘ㛫࡛᫂☜పୗࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࡇࡼࡾ 23, 28)ࠊᢞᚋ1 㛫 2 㛫ࡢ➽⫗୰⃰ᗘࡣࡁࡃኚືࡍࡿࡶࡢண ࡋࡓࠋTable 2 ࡣᢞᚋ 1 㛫ࡢ࣐࢘ࢫࡼࡾ᥇ྲྀࡋࡓ➽⤌⧊ࡢ⤖ᯝࡶ୍㒊♧ࡋ࡚࠸ࡿࠋ
Table 2. DAP Concentrations in Murine Femoral Muscles Collected 2 h after the Single Subcutaneous Administration of DAP (100 mg/kg)
Procedurea) Measured values of DAP (Pg/g)b) SD RSD(%)
1 2 3 4 5 Mean
A 1.1 1.3 1.4 1.5 1.1 1.3 0.18 14.0
B 1.5 1.9 2.2 1.8 1.4 1.8 0.32 18.2
C 2.5 3.0 3.5 3.3 2.4 2.9 0.48 16.4
C (1 h)c) 3.0 2.8 3.8 - - 3.2 0.53 16.5
a) Sample preparation procedures shown in Figs. 2 and 5.
b) Measured values are indicated by the amount (Pg) of DAP contained in the 1g of muscle.
ࡋࡋ࡞ࡀࡽࠊ1 㛫ࡢ᳨య 2 㛫ࡢ᳨యࡢ DAP ⃰ᗘࡣྠ⛬ᗘ࡛࠶ࡗࡓࠋࡇࢀ ࡽࡣᮍࡔࣃࣟࢵࢺヨ㦂ࡢ⤖ᯝ࡛࠶ࡾࠊᮏ๓ฎ⌮᪉ἲࢆ⏝࠸ࠊ⸆ᢞᚋࡢ⾑₢ ࠾ࡼࡧ➽⫗୰ࡢ⸆≀ືែヨ㦂ࢆ⾜࠺ᚲせࡀ࠶ࡿࠋᅇࡢᐇ㦂⣔࠾࠸࡚ࡣࠊ㏻ᖖ
ࡢື≀ᐇ㦂ࡼࡾⱝᖸ㧗⏝㔞࡛ࡣ࠶ࡗࡓࡀࠊᮏ๓ฎ⌮᪉ἲࡢ㦵᱁➽୰ࡢDAP ࡢᐃ㔞
➨ ➨❶ ⤖ ㄽ ࣐࢘ࢫ㦵᱁➽୰ࡢ DAP ⏕యヨᩱศᯒࡢࡓࡵࡢ๓ฎ⌮ࡋ࡚ࡢ㓝⣲ฎ⌮ࡢ㐺ᛶ ࡘ࠸࡚ホ౯ࢆ⾜ࡗࡓࠋⴭ⪅ࡽࡢ⤖ᯝࡣࢺࣜࣉࢩࣥฎ⌮ࡀఱࡽࡢࢱࣥࣃࢡ㉁ ࡢ┦స⏝ࡼࡾ㦵᱁➽ᙉࡃྲྀࡾ㎸ࡲࢀࡓ DAP ࢆศᯒࡍࡿࡓࡵࡢ๓ฎ⌮᪉ἲ ࡋ࡚㠀ᖖ᭷ຠ࡛࠶ࡿࡇࢆ᫂ࡽࡋࡓࠋࢺࣜࣉࢩࣥᾘࡼࡿᮏ๓ฎ⌮ἲ ࡣᚋ DAP ࡢ㧗ឤᗘศᯒἲ⤌ࡳྜࢃࡏࡿࡇࡼࡾࠊDAP ࡀ㛵㐃ࡍࡿ➽㞀ᐖ ࢆ㉳ࡇࡋࡸࡍ࠸ᝈ⪅ࡢᅉᏊࡢ≉ᐃ⏝࡛ࡁࡿྍ⬟ᛶࡀ࠶ࡿࠋ 㓝⣲ᾘἲࡣ⡆౽ࡘᏳ౯࡞๓ฎ⌮ἲࡋ࡚ࠊ⣽⬊ᇵ㣴➼⏝࠸ࡽࢀ࡚࠸ࡿࡀ ⤌⧊ヨᩱ୰ࡽࡢ⸆≀ศᏊࡢ๓ฎ⌮ࡋ࡚⏝࠸ࡿヨࡳࡣᑡ࡞࠸32-34)ࠋYu ࡽࡣ⸆≀ ࡢ⏕యヨᩱศᯒࡢࡓࡵࡢ௦᭰ⓗ⤌⧊๓ฎ⌮᪉ἲࡋ࡚㓝⣲ᾘࡀ࣍ࣔࢪࢿ࣮ࢩࣙ ࣥྠ➼ࡢᢳฟຠ⋡ࢆ♧ࡍࡇࢆሗ࿌ࡋ࡚࠸ࡿ33)ࠋࡋࡋ࡞ࡀࡽࠊⴭ⪅ࡢ▱ࡿ㝈 ࡾࠊ⸆≀➽ࢱࣥࣃࢡࡢ┦స⏝ࡽゎ㞳ࡍࡿ᪉ἲࡋ࡚㓝⣲ᾘἲࢆホ౯ࡋࡓ ሗ࿌ࡣ࡞࠸ࠋᮏሗ࿌ࡣࢺࣜࣉࢩࣥᾘࢆ⏝࠸ࡓ➽ヨᩱࡢ๓ฎ⌮㛵ࡍࡿึࡢሗ࿌ ࡛࠶ࡿࠋ⌧ᅾࠊࡇࡢ➽ฎ⌮ἲࢆ⏝࠸ࡓࠊ⾑₢DAP 㦵᱁➽୰ࡢ DAP ⃰ᗘ㛵ࡍ ࡿ᭦࡞ࡿ⸆≀ືែᐇ㦂ࠊDAP ᙉᅛ┦స⏝ࢆ♧ࡍࢱࣥࣃࢡ㉁ࡢ≉ᐃࡀ㐍⾜ ࡋ࡚࠸ࡿࠋࡇࢀࡽຍ࠼ࠊDAP ࡢ㦵᱁➽⛣⾜㔞ࡣ UV ᳨ฟჾ᳨࡛▱ࡍࡿࡣᴟᚤ 㔞࡛࠶ࡿࡓࡵࠊࡇࡢ๓ฎ⌮᪉ἲే⏝ࡍࡿ LC-MS/MS ➼ࡢ㧗ឤᗘศᯒἲࡢ㛤Ⓨࡀ ᚲせ࡛࠶ࡿࠋ
ᐇ ᐇ㦂ࡢ㒊 㸯㸬ヨᩱ DAP ࡣࣇࢼࢥࢩ♫(ᮾி, ᪥ᮏ)ࡼࡾ㉎ධࡋࠊ᭦࡞ࡿ⢭〇࡞ࡣ⾜ࢃࡎ⏝ࡋࡓࠋ DAP ࡣ 100 PM ࡢỈ⁐ᾮࡋ࡚ 200 PL ࡢࢧࣥࣉࣝࢳ࣮ࣗࣈศὀࡋࡓࡶࡢࢆ෭ ಖᏑࡋࠊྛᐇ㦂๓㐺ษ࡞⃰ᗘㄪ〇ࡋࡓࠋStreptomyces orientalis ⏤᮶ࣂࣥ ࢥ࣐ࢩࣥሷ㓟ሷࡣSigma-Aldrich ࡼࡾධᡭࡋࠊ100 PM ࣂࣥࢥ࣐ࢩࣥሷ㓟ሷࢫ ࢺࢵࢡࢯ࣮ࣜࣗࢩࣙࣥࢆㄪ〇ࡋࠊ෭ಖᏑࡋࡓࠋHPLC ศᯒ⏝ෆᶆ‽ࡣ࢟ࢩࢲ Ꮫཬࡧࢼ࢝ࣛࢸࢫࢡ♫㸦ᮾி, ᪥ᮏ㸧ࡢศᯒ⏝ 4-ࢽࢺࣟࣇ࢙ࣀ࣮ࣝ 4-ࢽࢺ ࣟᏳᜥ㤶㓟ࢆࡑࢀࡒࢀ᭦࡞ࡿ⢭〇࡞ࡣ⾜ࢃࡎ⏝ࡋࡓࠋHPLC ⛣ື┦ࡣ㛵ᮾ Ꮫ♫(ᮾி, ᪥ᮏ)ࡢ HPLC-ࢢ࣮ࣞࢻࡢࢭࢺࢽࢺࣜࣝࢆ᭦࡞ࡿ⢭〇࡞ࡣ⾜ࢃ
ࡎ⏝ࡋࡓࠋᐇ㦂ࡣMilli-Q Academic A10 apparatus (Merck Millipore)࡚⢭〇ࡋ ࡓ㉸⣧Ỉࢆ⏝ࡋࡓࠋ⾑Ύࡣࢻࣛࢵࢢࣇ࣮࣐ࣜ࢘ࢫ⾑Ύ (Cosmo Bio Co., Ltd, ᮾ ி, ᪥ᮏ) ࢆ㉎ධࡋ⏝ࡋࡓࠋ
࣐࢘ࢫ㦵᱁➽࠾ࡼࡧ⾑Ύࡢ᥇ྲྀ
Charles River Laboratories Japan, Inc. (ᶓ, ᪥ᮏ) ࡼࡾ㉎ධࡋࡓ⣙ 22 g ࡢཎయ ࣇ࣮ࣜࢫࢫ࣐࢘ࢫࢆ⏝ࡋࡓࠋᐇ㦂ື≀ࡣ⡿◊✲ホ㆟᥎ዡࡢỈ‽࡛㣫⫱ࡋࠊ ⮬⏤⤥㣵ࠊ㣧Ỉࢆࡉࡏࡓࠋᮏᐇ㦂ࡣឡ▱་⛉Ꮫື≀ᐇ㦂ጤဨࡢᑂᰝᢎㄆ ࢆᚓ࡚ᐇࡋࡓࠋ10 ༉ࡢ࣐࢘ࢫࢆ⸆≀ࡣ⏝ࡏࡎ㓟Ⅳ⣲࡚ᏳᴦṚࡉࡏࡓᚋࠊ ⭣➽ࢆ㏿ࡸษ㝖ࡋࠊ2 mL ࡢ⁛⳦෭⏕⌮㣗ሷᾮ(0.9 w/v%)ඹ࣍ࣔࢪࢼ ࢬࡋࠊࡇࢀࡽࢆᅇᐇ㦂⏝ࡢศᯒᑐ㇟≀ࣇ࣮ࣜ⭣➽ࡋࡓࠋ࣍ࣔࢪࢼࢬࡉ ࢀࡓศᯒᑐ㇟≀ࣇ࣮ࣜ⭣➽ࡣ0.2 g ∦ศࡅࠊ⏝ࡲ࡛80 ºC ࡛ಖᏑࡋࡓࠋ ࡢ8 ༉ࡢ࣐࢘ࢫࡣ⏕⌮㣗ሷᾮ 200PL ⁐ゎࡋࡓ DAP 100 mg/kg ࢆ⓶ୗᢞࡋࠊ
3 ༉ࢆ 1 㛫ᚋࠊ5 ༉ࢆ 2 㛫ᚋࡑࢀࡒࢀᒕẅࡋࡓࠋ➽⫗ࡣศᯒᑐ㇟≀ࣇ࣮ࣜ ⭣➽ྠࡌᡭ㡰ࡼࡾ᥇ྲྀࡋศᯒࡲ࡛80 °C ࡛෭ಖᏑࡋࡓࠋ ᢳฟἲ 3.1 ỗ⏝ⓗᢳฟἲ ‵㔜㔞⣙0.2 g ࡢ➽⤌⧊ࢆ๓ィ㔞ࡋࡓ 2 mL ࡢࢧࣥࣉࣝࢳ࣮ࣗࣈ㸦Watson㸧 ධࢀࠊṇ☜࡞㔜㔞ࢆ ᐃࡋࡓࠋ࣓ࢱࣀ࣮ࣝ㸦600 PL㸧 ࢡ࣒ࣟࣟ࣍ࣝ 㸦300 PL㸧 ࢆຍ࠼ࡓᚋࢆ㛢ࡵࠊ 10 ศ㛫 vortex ࡛᧠ᢾࡋࡓࠋ⥆࠸࡚ࠊෆᐜ≀ 300 PL ࡢ ࢡ࣒ࣟࣟ࣍ࣝ400 PL ࡢ㉸⣧Ỉࢆຍ࠼࡚ࠊ30 ⛊ vortex ࡛᧠ᢾࡋࠊࡑࡢᚋ 3,000 g ࡛5 ศ㐲ᚰศ㞳ࢆ⾜࠸ࠊ⸆≀ࢆỈ┦ᢳฟࡋࡓࠋࣂࣥࢥ࣐ࢩࣥࡢฎ⌮ࡣ ࣂࣥࢥ࣐ࢩࣥࢆゎ㞳ࡉࡏࡿࡓࡵࠊ400 PL ࡢ㉸⣧Ỉࡢ௦᭰ࡋ࡚ 0.1 M HCl 㸦100 PL㸧 300 PL ࡢ㉸⣧Ỉࢆῧຍࡋࡓࠋ ୖࡳ 㸦1.0 mL㸧 ࢆࣆ࣌ࢵࢺ࡛྾ ࠸ୖࡆࠊ࢚ࣂ࣏࣮ࣞࢱ࣮ࡼࡾⓎᅛࡋࡓࠋࢧࣥࣉࣝࢳ࣮ࣗࣈෆࡢṧ´ࢆෆᶆ ‽ࢆྵࡴ㉸⣧Ỉ50PL ࡛⁐ゎࡋࠊUltrafree-MC ࣇࣝࢱ࣮㸦Merck Millipore, 0.22 μm pore size㸧ࢆ⏝࠸ࠊ6,000 gࠊ4 °C ࡛ 10 ศ㐲ᚰศ㞳ࡋࡓࠋᚓࡽࢀࡓℐᾮ 20 PL ࢆ HPLC ࣒࢝ࣛὀධࡋࡓࠋ㸦Fig. 2㸧 3.2 ࢺࣜࣉࢩࣥᾘࢆే⏝ࡋࡓᢳฟἲ ࣇ࢙ࣀ࣮ࣝࣞࢵࢻࣇ࣮ࣜࡢ0.5 w/v% ࢺࣜࣉࢩࣥ࠾ࡼࡧ 5.3 mM EDTA·4Na ࢆྵ ᭷ࡍࡿࢺࣜࣉࢩࣥ-EDTA ⁐ᾮࡣᐩኈࣇ࣒ࣝග⣧⸆ 㸦㜰, ᪥ᮏ㸧ࡼࡾ㉎ධࡋࠊ ࢺࣜࣉࢩࣥᶆ‽ᾮࡋ࡚᭦࡞ࡿฎ⌮ࢆຍ࠼ࡿࡇ࡞ࡃ⏝ࡋࡓࠋṇ☜ィ㔞ࡋࡓ ➽࣍ࣔࢪࢿ࣮ࢺࢆྵࡴࢳ࣮ࣗࣈ㸦2 mL㸧ࠊࢆࡋࠊ๓ 37 Υ࡛ 30 ศຍ ࡋ ࡓࡶࡢࠊࢺࣜࣉࢩࣥᶆ‽ᾮ 㸦150 PL㸧 Tris-HCl ⦆⾪ᾮ 㸦pH 8; 0.1 M㸧 㸦200 PL㸧 ࢆῧຍࡋࡓࠋ⥆࠸࡚ࠊᆒ୍ࡢᠱ⃮ᾮࢆᚓࡿࡓࡵࠊvortex ᧠ᢾࢆ⾜ࡗࡓᚋ ๓㏙ࡋࡓỗ⏝ⓗᢳฟἲࢆ⾜ࡗࡓࠋ㸦Fig. 5㸧
ᅇ⋡ࡣศᯒᑐ㇟≀ࣇ࣮ࣜ⭣➽0.064 Pg, 0.16 Pg, ࠾ࡼࡧ 1.6 Pg ࡢ 3 ẁ㝵ࡢ㔞 ࡛ࡢDAP ࡢῧຍᅇヨ㦂ࢆ⾜࠸⟬ฟࡋࡓࠋῧຍᅇヨ㦂ࡢヨᩱࡣศᯒᑐ㇟≀ࣇࣜ ࣮⭣➽ 㸦0.2 g㸧 0.064 Pgࠊ0.16 Pgࠊ࠾ࡼࡧ 1.6 Pg ࡢࣞ࣋ࣝᑐᛂࡋࠊDAP ᶆ ‽ᾮࡽㄪ〇ࡋࡓ2.0 PMࠊ5 PM ࠾ࡼࡧ 50 PM ࡢᕼ㔘ᾮ 20 PL ࢆࡑࢀࡒࢀῧຍࡋ సᡂࡋࡓࠋྠᵝㄪ〇ࡋࡓヨᩱ4 Ⅼࢆ ᐃࡋ⟬ฟࡋࡓࠋ +3&/ศᯒ 10AD VP HPLC ࢩࢫࢸ࣒ 㸦ᓥὠ, ி㒔, ᪥ᮏ㸧 DGU-12A ࢹ࢞ࢵࢧ 㸦ᓥὠ㸧ࠊ CLASS M10A (SPD-MXA) ྾ගᗘ᳨ฟჾ 㸦ᓥὠ㸧ࠊ7125 ᆺ ࢩࣜࣥࢪ-࣮ࣟࢹࣥ ࢢࢧࣥࣉࣝࣥࢪ࢙ࢡࢱ࣮ 㸦Rheodyne, Cotati, CA, USA㸧ࢆ᥋⥆ࡋ⏝ࡋࡓࠋศ 㞳ࡣInertsil® ODS-3 㸦150 × 4.6 mm i.d., 5 Pm particle size㸧 ࣒࢝ࣛ 㸦GL Sciences Inc., ᮾி, ᪥ᮏ㸧ࢆ⏝ࡋࠊ⛣ື┦ࡣ DAP ࡣࠊࢭࢺࢽࢺࣜࣝ 36%ྵ᭷ࣜ ࣥ㓟⦆⾪ᾮ⦆⾪ᾮ 㸦pH 2.1; 0.01 M) ࢆࠊࣂࣥࢥ࣐ࢩࣥࡣࢭࢺࢽࢺࣜࣝ 10% ྵ᭷ࣜࣥ㓟⦆⾪ᾮ⦆⾪ᾮ 㸦pH 7.2; 0.01 M㸧ࢆࡑࢀࡒࢀ⏝࠸ࡓࠋ⛣ື┦ࡢὶ㏿ࡣ 1.0 mL/minࠊ࣒࢝ࣛ ᗘࡣ 40Υࡋ᳨ฟἼ㛗ࡣ DAP ࠾ࡼࡧࣂࣥࢥ࣐ࢩ࡛ࣥࡑࢀ ࡒࢀ221 nm ࠾ࡼࡧ 280 nm ࡋ Class M10A ࢩࢫࢸ࣒ 㸦ᓥὠ㸧 ࡚ࣆ࣮ࢡศᯒ ࢆ⾜ࡗࡓࠋ
ᘬ
ᘬ⏝ᩥ⊩
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