⑔ጟ⋵଻ஜⅣႺ⎇ⓥᚲᐕႎ╙ 31 ภ㧘61 㧙 65㧘2004ේ⪺⺰ᢥ↲⁁⣼ᧃࠍ฽᦭ߔࠆஜᐽ㘩ຠਛߩ࠻࡝࡛࡯࠼࠴ࡠ࠾ࡦ෸߮࠴ࡠࠠࠪࡦߩ
ಽᨆᴺ᫪↰㇌ᱜ㧘Ძ೑㓉⟤㧘ਛᎹ␞ሶㅒ⋧♽䈱㜞ㅦᶧ૕䉪䊨䊙䊃䉫䊤䊐䉕૶䈦䈩䋬↲⁁⣼ᧃࠍ฽᦭ߔࠆஜᐽ㘩ຠਛߩ 3,3',5-࠻࡝࡛࡯࠼࠴ࡠ࠾ࡦ෸߮࠴ࡠࠠࠪࡦߩಽᨆᴺࠍᬌ⸛ߒߚ㧚ஜᐽ㘩ຠߪࡔ࠲ࡁ࡯࡞෸߮᳓㧛ࡔ࠲ࡁ࡯࡞㧔1:1㧕ࠍ↪޿ߡᵞᵺᓟ㧘
Pronase ࠍ↪޿ߡട᳓ಽ⸃ߒߚ㧚3,3',5-࠻࡝࡛࡯࠼࠴ࡠ࠾ࡦ෸߮࠴ࡠࠠࠪࡦߪ 2 㧑ࠕࡦࡕ࠾ࠕ᳓ࡔ࠲ࡁ࡯࡞
ṁᶧߢ᛽಴ߒ㧘࿕⋧᛽಴ᴺ(Bond Elut C18 ࠞ࡯࠻࡝࠶ࠫ)ࠍ↪޿ߡࠢ࡝࡯ࡦࠕ࠶ࡊߒߚ㧚㜞ㅦᶧ૕䉪䊨䊙䊃䉫䊤䊐䈲䉦䊤䊛䈮 InertsilODS-3 (4.6x 150mm, 5µm)ࠍ㧘⒖േ⋧ߦ᳓㧛ࡔ࠲ࡁ࡯࡞㧛࡝ࡦ㉄㧔500:500:1, v/v㧕ࠍ↪޿㧘᷹ቯᵄ㐳 230nm ߢಽᨆߒߚ㧚ᧄᴺߦࠃࠆஜᐽ㘩ຠ߆ࠄߩ 3,3',5-࠻࡝࡛࡯࠼࠴ࡠ࠾ࡦ෸߮࠴ࡠࠠࠪࡦߩ࿁෼₸ߪ 70 㨪 80%㧘ቯ㊂ਅ㒢୯ߪ 3,3',5-࠻࡝࡛࡯࠼࠴ࡠ࠾ࡦ߇ 0.8µg/g㧘࠴ࡠࠠࠪࡦ߇ 1.4µg/g ߢ޽ߞߚ㧚㨇ࠠ࡯ࡢ࡯࠼ :↲⁁⣼㧘'-࠻࡝࡛࡯࠼࠴ࡠ࠾ࡦ㧘࠴ࡠࠠࠪࡦ㧘HPLC㧘࿕⋧᛽಴ᴺ㧘ஜᐽ㘩ຠ㨉㧝㧔USP: United States Pharmacopeia㧕ߦႎ๔ߐࠇߡ޿ࠆ 㧚ߪߓ߼ߦੇ῎↲⁁⣼ߪ㘩↪₞ߩ↲⁁⣼ࠍߣࠅੇ῎ߒߚ᷆㤛⦡㨪ߐࠄߦ㧘ⴊਛߩ T ࠍ࿕⋧᛽಴ߒ LC/MS ߢ᷹ቯߔࠆᣇᴺἯⶊ⦡ߩ☳ᧃߢ㧘ࠊߕ߆ߦ․⇣ߥ⡺⥇߇޽ࠆ㧚↲⁁⣼ౝ߿ஜᐽ㘩ຠਛߩ T෸߮ T ࠍ LC/MS ߢ᷹ቯߔࠆᣇᴺ ߇ߩ'-࠻࡝࡛࡯࠼࠴ࡠ࠾ࡦ T ෸߮࠴ࡠࠠࠪࡦ T ߪႎ๔ߐࠇߡ޿ࠆ㧚ߒ߆ߒ㧘↲⁁⣼ᧃ߇ᷙ౉ߐࠇߚஜᐽ㘩࠲ࡦࡄࠢ⾰ߣ⚿วߒߡ࠴ࡠࠣࡠࡉ࡝ࡦߩᒻߢࠈ⢩ౝߦ⾂ຠਛߦߪ㧘ฦ⒳ߩᅹኂᚑಽ߇ሽ࿷ߔࠆߚ߼㧘ߎࠇࠄߩᣇ⬿ߐࠇߡ޿ࠆ㧚࠴ࡠࠣࡠࡉ࡝ࡦߪ࠲ࡦࡄࠢಽ⸃㉂⚛ߩ૞ᴺߢ HPLC ࠍ ↪޿ߡ᷹ቯߔࠆߎߣߪ࿎㔍ߢ޽ࠆ㧚ߘߎ↪ߦࠃߞߡಽ⸃ߐࠇ㧘T ෸߮ T ߇ⴊਛߦㆆ㔌ߒ㧘ࡎ࡞ߢ㧘⒳㘃ߩஜᐽ㘩ຠࠍ↪޿ߡ㧘↲⁁⣼ᧃࠍ฽᦭ߔࠆஜࡕࡦ૞↪ࠍ๒ߔࠆ㧚ੇ῎↲⁁⣼ᧃߪ↢૕ߩၮ␆ઍ⻢ࠍ㜞ᐽ㘩ຠਛߩ T෸߮ Tߩ HPLC ಽᨆᴺࠍᬌ⸛ߒߚ㧚߼ࠆ૞↪߇޽ࠅ㧘☼ᶧ᳓⣲㧘ࠢ࡟࠴ࡦ∝㧘↲⁁⣼ᯏ⢻ૐਅ∝㧘↲⁁⣼⣲╬ߩᴦ≮⮎ߣߒߡㆡᔕߐࠇࠆ㧚ᴦ≮㐿ᆎ㧞ᤨ㧘ੇ῎↲⁁⣼ᧃߪ㨪mg/day ߆ ࠄᛩਈߒ㧘೽૞↪

㧞࡮㧝ታ㛎ᣇᴺ⹜⮎ߩߥ޿ߎߣࠍ⏕⹺ߒ㧘㨪ㅳ㑆ߢ⛽ᜬ㊂㨪mg/dayੇ῎↲⁁⣼ᧃߪᏢ࿖⤳ེ⵾⮎㧔ᩣ㧕⵾ߩᣣᧄ⮎ዪᣇຠߦჇ㊂ߔࠆ㧚ㆊ㊂ߦߥࠆߣ↲⁁⣼ᯏ⢻੣ㅴᤨߦߺࠄࠇࠆࠍ↪޿ߚ㧚Tris hydroxymethyl aminomethane ߪ ๺శ⚐⮎ฦ⒳ߩ೽૞↪߇⃻ࠇࠆ㧚ࡃ࠮࠼࠙∛ࠃ߁ߩ∝⁁߿♖␹∛Ꮏᬺ㧔ᩣ㧕⵾㧘-Mercapto- -methylimidazole ߪ Aldrichࠃ߁∝⁁ࠍ઻߁㧚㗫⣂㧘ᔃᖪ੣ㅴ㧘᦬⚻ਇ㗅㧘ή᦬⚻㧘⵾㧘Pronase ߪ Calbiochem Novabiochem ⵾ No㧘ࡔ♖␹ਇ቟ቯ෸߮ਅ∯╬ߩ⻉∝⁁߇㗫ᐲ߇㜞޿㧚mg/day࠲ࡁ࡯࡞ߪ๺శ⚐⮎Ꮏᬺ ᩣ ⵾ߩ HPLC ಽᨆ↪ࠍ↪޿ߚ㧚એ਄ߩㅪ↪ߢ㧘ߥ߆ߦߪࡩ᦬ߊࠄ޿ߩ⍴ᦼ᦯↪ߢ߽⷗T ෸߮ T ᮡḰේᶧߪ ' -࠻࡝࡛࡯࠼࠴ࡠ࠾ࡦ࠽࠻ࠄࠇࠆ଀߇޽ࠆ 㧚ੇ῎↲⁁⣼ᧃߪᐕ㗃㧘∳り⋡⊛࡝࠙ࡓ㧔Sigma ⵾㧕ࠍmg㧘࠴ࡠࠠࠪࡦ࠽࠻࡝࠙ࡓ

ߢ‫ࠆ޽߇ߣߎߚࠇߐ↪ੂߡߒߣޠ⮎ߖ߿ޟ‬㧚ᐔᚑ㨪

᳓๺‛㧔Sigma ⵾㧕ࠍmg ⒊㊂ߒ㧘%ࠕࡦࡕ࠾ࠕ᳓-ᐕߦ࠳ࠗࠛ࠶࠻↪ஜᐽ㘩ຠߦᷙ౉ߐࠇ㧘ో࿖⊛ߦ↲⁁ࡔ࠲ࡁ࡯࡞ࠍട߃ߡml ߣߒ಄ᥧᚲߦ଻ሽߒߚ㧚↪ᤨ㧘⣼ᯏ⢻੣ㅴࠃ߁∝⁁ࠍ઻߁ஜᐽⵍኂ߇ᄙᢙ⊒↢ߒߚ㧚ߎߩᶧ㨪ml ࠍߣࠅ%ࠕࡦࡕ࠾ࠕ᳓-ࡔ࠲ࡁ࡯࡞ࠍട߃ੇ῎↲⁁⣼ᧃਛߩ T෸߮ T ࠍ᷹ቯߔࠆᣇᴺߣߒߡ㧘ߡml ߣߒߚ߽ߩࠍᮡḰṁᶧߣߒߚ㧚↲⁁⣼⚵❱ਛߩ࠴ࡠࠣࡠࡉ࡝ࡦࠍ࠲ࡦࡄࠢಽ⸃㉂⚛ࠍ↪

㉂ ⚛ ✭ ⴣ ᶧ ߪ M Ⴎ ൻ ࠽ ࠻ ࡝ ࠙ ࡓ 㧘 M Tris

޿ߡട᳓ಽ⸃ᓟ㧘HPLC ߢ ቯ㊂ߔࠆᣇᴺ߇☨࿖⮎ዪᣇhydroxymethyl aminomethane ෸ ߮ M2-Mercapto-⑔ጟ⋵଻ஜⅣႺ⎇ⓥᚲ㧔‫⑔ ޥ‬ጟ⋵ᄥቿᐭᏒᄢሼะ૒㊁㧕1-methylimidazole ࠍ 6M Ⴎ㉄ߢ㧘pH8.4±0.05 ߦ ⺞⵾ߒ಄㧞࡮㧟࡮㧟

6෸߮6ߩ*2.%᷹ቯ᧦ઙ

ᥧᚲߦ଻ሽߒߚ㧚Pronase ṁᶧߪ Pronase mg ࠍ⒊㊂ߒ㧘ࠞ࡜ࡓ

InertsilODS-

㉂⚛✭ⴣᶧࠍട߃ml ߣߒߚ߽ߩࠍ↪ᤨ⺞⵾ߒߚ㧚⒖േ⋧᳓㧛ࡔ࠲ࡁ࡯࡞㧛࡝ࡦ㉄㧔:: v/v㧕 mm

ᵹ㊂

 ml/minࠞ࡜ࡓ᷷ᐲ͠ᵈ౉㊂ Ǵ l฽߻ࡔ࠲ࡁ࡯࡞ṁᶧࠍ⺞⵾ߒߚ㧚᷹ቯᵄ㐳 nm㧞࡮㧞଻ᜬᤨ㑆T㧦ಽ㧘 T㧦ಽ࿕⋧᛽಴ߦߪ Varian ⵾ߩ Bond Elut C ࠞ࡯࠻࡝࠶ࠫ㧔 mg  ml㧕ࠍ↪޿ߚ㧚㧑ࠕࡦࡕ࠾ࠕ᳓ -ࡔ࠲ࡁ࡯࡞ṁᶧߪࠕࡦࡕ࠾ࠕࠍ㧑ⵝ⟎⚡ᄖๆశᐲᬌ಴ེ㧔 SPD- AV㧕෸߮࠺࡯࠲ಣℂⵝ⟎㧔C-R A㧕ઃ HPLC ߪፉᵤ⵾૞ᚲ㧔ᩣ㧕⵾ߩ LCA ࠍ 㧘㧟ࠞ࡜ࡓߪ GL ࠨࠗࠛࡦࠬ㧔ᩣ㧕⵾ߩ InertsilODS- 㧔㧟࡮㧝ߢ޽ࠆߎߣ߆ࠄ㧘㨪 mg ߩੇ῎↲⁁⣼ᧃߦ Pronaseታ㛎ᠲ૞㧞࡮㧟࡮㧝ੇ῎↲⁁⣼ᧃ߆ࠄ↢ᚑߐࠇࠆ6෸߮6㊂ᴦ≮⮎ߣߒߡߩੇ῎↲⁁⣼ᧃߩ↪㊂ߪ㨪 mg/day mm  Ǵ m㧕ࠍ૶↪ߒߚ㧚㧞࡮㧟⚿ᨐ෸߮⠨ኤੇ῎↲⁁⣼ᧃਛߩ T3 ෸߮ T4 ߩ ಽᨆᴺṁᶧml ࠍട߃㧘ታ㛎ᠲ૞ߦᓥߞߡੇ῎↲⁁⣼ᧃ߆ࠄ↢ੇ῎↲⁁⣼ᧃߩಽᨆᴺߪ USP ߩᣇᴺ  ߦḰߓߡ૞ᚑᚑߔࠆ T෸߮ T㊂ࠍ᷹ቯߒߚ㧔 n= 㧕㧚ߘߩ⚿ᨐ㧘ੇ῎ߒߚ߇㧘᛽಴ṁ೷ߪ %࡝ࡦ㉄ࠕ࠮࠻࠾࠻࡝࡞ṁᶧ߆ࠄ↲⁁⣼ᧃ㨪mg ߩ▸࿐ౝߢ߶߷৻ቯߩ T෸߮ Tߩ↢ࡔ࠲ࡁ࡯࡞ߦᄌᦝߒߚ㧚ട᳓ಽ⸃ᤨ㑆ߪዊဈࠄ ߩታ㛎ᚑ㊂߇ߺࠄࠇ㧘 g ߩੇ῎↲⁁⣼ᧃᒰߚࠅ T߇㨪⚿ᨐ߆ࠄ㨪ᤨ㑆ߣߒߚ㧚ੇ῎↲⁁⣼ᧃmg ࠍmlǴ g ෸߮ T߇㨪Ǵ g ↢ᚑߒߚ㧔࿑㧝㧕㧚ߩ⹜㛎▤ߦ⒊㊂ߒ㧘 Pronase ṁ ᶧml ࠍട߃㧘⓸⚛ࠟࠬ㪈㪋㪇㪇ࠍ⚂ಽ㑆ᵹߔ㧚⹜㛎▤ࠍㆤశߒ㧘͠ߢ㨪ᤨ㑆㧘ᤨᔃಽ㔌ᓟ㧘᳓ጀࠍណขߒᱷᷲߦࡔ࠲ࡁ࡯࡞ml ࠍട߃ᝄߣ߁᛽಴ߔࠆ㧚㆙ᔃಽ㔌ᓟ㧘ࡔ࠲ࡁ࡯࡞ጀࠍណขߒ᳓ጀߣวࠊߖ㧘㧑ࠕࡦࡕ࠾ࠕ᳓-ࡔ࠲ࡁ࡯࡞ࠍട߃ߡml ߣߔࠆ㧚㧞࡮㧟࡮㧞ੇ῎↲⁁⣼ᧃࠍ฽᦭ߔࠆஜᐽ㘩ຠਛߩ6෸

߮6ߩಽᨆᴺஜᐽ㘩ຠg ࠍml ߩ⹜㛎▤ߦ⒊㊂ߒ㧘ࡔ࠲ࡁ࡯࡞㪈㪉㪇㪇

T 3 ෸䈶 T4 Ớᐲ㩿㱘㪾㪆㪾㪀‫ޘ‬ᝄࠅᷙߗߥ߇ࠄട᳓ಽ⸃ߔࠆ㧚⹜ᢱߩ Pronase ṁᶧࠍ㆙㪈㪇㪇㪇㪏㪇㪇㪍㪇㪇㪋㪇㪇㪉㪇㪇㪇

ml ෸߮᳓㧛ࡔ࠲ࡁ࡯࡞㧔:v/v㧕ml ߢߘࠇߙࠇ࿁㪈㪇ᵞᵺߒ㧔ߘࠇߙࠇಽ㑆෸߮ಽ㑆ᝄߣ߁᛽಴ 㧕㧘㆙ᔃ㪉㪇㪊㪇㪋㪇㪌㪇ੇ῎↲⁁⣼㩷㩿㫄㪾㪀ಽ㔌ᓟ਄ጀߩᵞᵺᶧߪᝥߡࠆߎࠇߦ Pronase ṁᶧml ࠍ࿑䋱䋮ੇ῎↲⁁⣼ᧃ䈎䉌↢ᚑ䈘䉏䉎T3 ෸䈶T4 ㊂ട߃㧘⓸⚛ࠟࠬࠍ⚂ಽ㑆ᵹߔ㧚⹜㛎▤ࠍㆤశߒ㧘͠㪫㪊㪫㪋ߢ㨪ᤨ㑆㧘ᤨ‫ޘ‬ᝄࠅᷙߗߥ߇ࠄട᳓ಽ⸃ߔࠆ㧚⹜ᢱߩ Pronase ṁ ᶧࠍ㆙ᔃಽ㔌ᓟ㧘᳓ጀࠍណขߒᱷᷲߦ㧑ࠕࡦࡕ࠾ࠕ᳓-ࡔ࠲ࡁ࡯࡞ml ࠍട߃࿁ᝄߣ߁᛽಴ߔࠆ㧚㧟࡮㧞㧑ࠕࡦࡕ࠾ࠕ᳓ -ࡔ࠲ࡁ࡯࡞᛽಴ᶧࠍ⓸⚛ࠟࠬਛ㨪㧔㧕ᵞᵺ߇6෸߮6ߩ↢ᚑߦ෸߷ߔᓇ㗀ੇ῎↲⁁⣼ᧃߩࡔ࠲ࡁ࡯࡞෸߮᳓㧛ࡔ࠲ࡁ࡯࡞͠ߢml એਅߦỚ❗ᓟ㧘᳓ጀߣวࠊߖ㧘ࡔ࠲ࡁ࡯࡞ࠍஜᐽ㘩ຠߦߪฦ⒳ߩᬀ‛߿ߘߩ᛽಴ᚑಽ߇ᷙ౉ߐࠇ

ട߃ߡml ߣߔࠆ㧚Bond Elut C  ࠞ࡯࠻࡝࠶ࠫߪࡔ࠲HPLC ᷹ቯߩᅹኂߣߥࠅ߿ߔ޿㧚߹ߚ↲⁁⣼ᧃࠍ฽߻ஜࡁ࡯࡞ml㧘᳓ml ࠍ㗅ᰴᵹߒࠦࡦ࠺࡚ࠪ࠾ࡦࠣߔࠆ㧚mlᐽ㘩ຠߩ Pronase ಣℂߦ߅޿ߡ㧘࠴ࡠࠣࡠࡉ࡝ࡦߩട᳓

ߩ⹜ᢱ᛽಴ᶧ߆ࠄml ࠍណขߒ㧘᳓ࠍട߃ߡml ߦᏗ㉼ಽ⸃߇ᅹኂߐࠇࠆߎߣ߇ႎ๔ߐࠇߡ޿ࠆ  㧚ஜᐽ㘩ຠߦߔࠆ㧚ߎࠇࠍ Bond Elut C  ࠞ࡯࠻࡝࠶ࠫߦᵹߒ T  ෸฽߹ࠇࠆᄳ㔀ᚑಽࠍ޽ࠄ߆ߓ߼㒰෰ߔࠆ⋡⊛ߢ㧘ੇ῎↲

߮ T  ࠍ଻ᜬߐߖߚᓟ 㧘᳓ ml ෸߮᳓㧛ࡔ࠲ࡁ࡯࡞㧔  : ⁁⣼ᧃmg ࠍࡔ࠲ࡁ࡯࡞ml ߢ࿁㧔ߘࠇߙࠇಽ㑆෸v/v 㧕 ml ߢᵞᵺߔࠆ 㧚㧑ࠕࡦࡕ࠾ࠕ᳓ - ࡔ࠲ࡁ࡯࡞ ml߮ಽ㑆ᝄߣ߁᛽಴㧕ᵞᵺᓟ㧘ߐࠄߦ᳓㧛ࡔ࠲ࡁ࡯࡞

ࠍᵹߒ T  ෸߮ T  ࠍṁ಴ߔࠆ㧔ೋᶧ - Ṣߪᝥߡࠆ 㧕㧚ṁ㧔: v/v㧕ml ߢ࿁㧔ߘࠇߙࠇಽ㑆෸߮ಽ㑆ᝄߣ಴ᶧߪ⓸⚛ࠟࠬ᳇ᵹਅ㨪͠ߢ ml એਅ߹ߢỚ❗ᓟ 㧘߁᛽಴㧕ᵞ޿㧘ᵞᵺߦࠃࠆ T෸߮ Tߩ↢ᚑ߳ߩᓇ㗀ࠍ㧑ࠕࡦࡕ࠾ࠕ᳓ -ࡔ࠲ࡁ࡯࡞ࠍട߃ߡ ml ߦߒ⹜ᢱṁᬌ⸛ߒߚ㧚࿑ A ߦ T෸߮ TߩᮡḰṁᶧ㧘࿑ B ߦੇᶧߣߔࠆ㧚῎↲⁁⣼ᧃߩ⹜ᢱṁᶧߩ HPLC ࠢࡠࡑ࠻ࠣ࡜ࡓࠍ␜ߔ㧚⴫㧝ੇ῎↲⁁⣼ᧃߩࡔ࠲ࡁ࡯࡞෸߮᳓㧛ࡔ࠲ࡁ࡯࡞㧔㧕
ᵞᵺߦࠃࠆ6෸߮6↢ᚑ߳ߩᓇ㗀T3

T4µg/g ± ᮡḰ஍Ꮕੇ῎↲⁁⣼ᧃ㧔ᧂᵞᵺ㧕357.4±7.61218.7±31.3

ੇ῎↲⁁⣼ᧃ㧔ᵞᵺ㧕357.0±8.11222.6±14.2

ੇ῎↲⁁⣼ᧃ㊂ 20mg㧘ታ㛎࿁ᢙ㧔n=5㧕ᧂ ᵞ ᵺ ߩ ੇ ῎ ↲ ⁁ ⣼ ᧃ ߩ T ෸ ߮ T ㊂ ߪ ߘ ࠇ ߙ ࠇǴ g/g ෸߮Ǵg/g㧘ᄌേଥᢙ㧑એౝߢ޽ߞߚ㧔⴫ 㧕㧚৻ᣇ㧘ᵞ ᵺ ߒ ߚ ੇ ῎ ↲ ⁁ ⣼ ᧃ ߩ႐ ว㧘T෸߮ T㊂ߪߘࠇߙࠇǴ g/g ෸߮Ǵ g/g㧘ᄌേଥᢙ㧑એౝߢ޽ߞߚ㧚ᵞᵺߦࠃࠆੇ῎↲⁁⣼ᧃߩ៊ᄬ෸߮ട᳓ಽ⸃߳ߩᓇ㗀ߪ⹺߼ࠄࠇߥ߆ߞߚ㧚Ꮢ⽼ߐࠇߡ޿ࠆஜᐽ㘩ຠ A㧔⚊⼺⩶ࠍ฽᦭ߔࠆ࠳ࠗࠛ࠶࠻↪ஜᐽ㘩ຠ㧕g ߦੇ῎↲⁁⣼ᧃmg ࠍᷝടᓟ㧘㧞࡮㧟࡮㧞ߩታ㛎ᠲ૞ߦᓥߞߡ⹜ᢱṁᶧࠍ⺞ᢛߒߚ㧚࿑㧔C㧕ߦࡔ࠲ࡁ࡯࡞෸߮᳓㧛ࡔ࠲ࡁ࡯࡞ߢᵞᵺߒߚஜᐽ㘩ຠ A ߩ HPLC ࠢࡠࡑ࠻ࠣ࡜ࡓࠍ␜ߔ㧚࿑ B ߩੇ῎↲⁁⣼ᧃߩࠢࡠࡑ࠻ࠣ࡜ࡓߣᲧߴߡ T ߩ೨ߩࡇ࡯ࠢ߇ᵞᵺߩലᨐߦࠃࠅ㒰෰ߐࠇߡ޿ࠆ㧚࿑ E ߩࡔ࠲ࡁ࡯࡞෸߮᳓㧛ࡔ࠲ࡁ࡯࡞ᧂᵞᵺߩஜᐽ㘩ຠ A ߩࠢࡠࡑ࠻ࠣ࡜ࡓߢߪ T෸߮ Tߩࡇ࡯ࠢ߇ᄙߊߩᅹኂᚑಽߣ㊀ߥࠅቯ㊂ߔࠆߎߣߪߢ߈ߥ޿㧚ߎߩᵞᵺᣇᴺࠍ↪޿ߚ႐ว㧘Pronase ߦ ࠃࠆട᳓ಽ⸃ᤨߩੇ῎↲⁁⣼ᧃ߆ࠄ↢ߓࠆᚑಽ෸߮ஜᐽ㘩ຠߦሽ࿷ߔࠆ HPLC ᷹ቯߩᅹኂᚑಽࠍ߆ߥࠅ㒰ߌࠆ߽ߩߣ⠨߃ࠄࠇߚ㧚㧟࡮㧟࿑㧞㉂⚛ಣℂᓟߩ౒ሽ‛⾰ߩ㒰෰ੇ῎↲⁁⣼ᧃߩ↪㊂㧔⛽ᜬ㊂㧕ߪ⚂mg/day ߢ޽㧚ᮡḰṁᶧ㧔㧭㧘6෸߮6ฦ‫ ޘ‬PI㧕㧘ੇ῎↲⁁⣼ᧃ OI㧔㧮 㧕㧘ࡔ࠲ࡁ࡯࡞෸߮᳓㧛ࡔ࠲ࡁ࡯࡞ߢᵞᵺߒߚஜᐽ㘩ຠ㧔㧯 㧕㧘$QPF 'NWV % ࠞ࡯࠻࡝࠶ࠫಣℂߒߚஜᐽ㘩ຠ㧔㧰㧕෸߮ࡔ࠲ࡁ࡯࡞෸߮᳓㧛ࡔ࠲ࡁ࡯࡞ᧂᵞᵺߩஜᐽ㘩ຠ㧔㧱㧕ߩ*2.%ࠢࡠࡑ࠻ࠣ࡜ࡓ
㧝㧘6㧧 㧞㧘6ஜᐽ㘩ຠߦߪᣣᒰߚࠅߩ↪㊂߇㍤એ਄ߣᄙ޿߽ߩ߇޽ࠆ㧚↪㊂߇ᄙ޿ஜᐽ㘩ຠ㧔ੇ῎↲⁁⣼ᧃmg/g/⓸⚛ࠟࠬ᳇ᵹਅߢỚ❗ᓟ㧘㧑ࠕࡦࡕ࠾ࠕ᳓ -ࡔ࠲ࡁ࡯࡞㍤㧕ߩ႐ว㧘⹜ᢱ g ࠍ⒊㊂ߔࠆߣੇ῎↲⁁⣼ᧃߣߒࠍട߃ml ߦߒߚ㧚࿑ D ߦ Bond Elut C  ࠞ࡯࠻࡝࠶ߡmg㧘⴫㧝߆ࠄ T෸߮ T㊂ߣߒߡߘࠇߙࠇ⚂Ǵgࠫߢಣℂߒߚஜᐽ㘩ຠ A ߩ HPLC ࠢࡠࡑ࠻ࠣ࡜ࡓࠍ␜෸߮Ǵg ߣߥࠅ㧘HPLC ߢቯ㊂ߔࠆߚ߼ߦߪ⹜ᢱṁᶧߔ㧚࿑ C ߩࡔ࠲ࡁ࡯࡞෸߮᳓㧛ࡔ࠲ࡁ࡯࡞ߢᵞᵺᓟࠍỚ❗ߔࠆᔅⷐ߇޽ࠆ㧚ߎߩߎߣ߆ࠄ㧘Bond Elut C ߩࠢࡠࡑ࠻ࠣ࡜ࡓߣᲧߴߡ D ߩࠢࡠࡑ࠻ࠣ࡜ࡓߪ Tߩࠞ࡯࠻࡝࠶ࠫࠍ↪޿ߡࠢ࡝࡯ࡦࠕ࠶ࡊߒ㧘ṁ಴ᶧࠍỚ❗

೨ߩᚑಽ߇ലᨐ⊛ߦ㒰෰ߐࠇߡ޿ࠆ㧚ߒ߆ߒ㧘ᣣᒰࠅߔࠆᣇᴺࠍᬌ⸛ߒߚ㧚ੇ῎↲⁁⣼ᧃmg ࠍ↪޿ߡ㧘ੇߩ↪㊂߇㍤޽ࠆ޿ߪࠞࡊ࠮࡞એਅߩஜᐽ㘩ຠ߿౒ሽ‛῎↲⁁⣼ᧃߩታ㛎ᠲ૞ߦᓥߞߡ⺞⵾ߒߚ T෸߮ Tߩ⹜⾰߇ዋߥ޿ߣ⠨߃ࠄࠇࠆஜᐽ㘩ຠߩ႐วߪ㧘Bond Elut C㛎ṁᶧࠍ↪޿ߡ㧘㧑ࠕࡦࡕ࠾ࠕ᳓ -ࡔ࠲ࡁ࡯࡞ߩṁ಴㊂ߩᠲ૞ࠍ⋭޿ߡ T෸߮ Tࠍቯ㊂ߔࠆߎߣ߇චಽน⢻ࠍᬌ⸛ߒߚ㧚ߘߩ⚿ᨐ㧘T෸߮ Tߪ㨪ml ߦ㧑ṁ಴ߢ޽ࠆ㧚ߒߚߎߣ߆ࠄ㧘㧑ࠕࡦࡕ࠾ࠕ᳓ -ࡔ࠲ࡁ࡯࡞ṁᶧ㊂ߪ㧟࡮㧠ml ߣߒߚ㧚ታ⹜ᢱߩ᷹ቯᤨߦߪṁ಴ᶧࠍ࿁෼₸ߣౣ⃻ᕈஜᐽ㘩ຠ A ෸߮ B ⚊⼺⩶ࠍ฽᦭ߔࠆ࠳ࠗࠛ࠶࠻↪ஜᐽ㘩ຠ ࠍ↪޿ߡᷝട࿁෼ታ㛎ࠍⴕߞߚ㧚ஜᐽ㘩ຠg߇ๆ⌕ߒ㧘᛽಴߇㕖Ᏹߦ࿎㔍ߥߎߣߦࠃࠆ߽ߩߣ⠨߃ࠄߦੇ῎↲⁁⣼ᧃmg ࠍᷝടᓟ㧘ታ㛎ᠲ૞ߦᓥߞߡ࿁෼ࠇߚ㧚ታ㛎ᠲ૞㧞࡮㧟࡮㧞ߢ㧑ࠕࡦࡕ࠾ࠕ᳓ -ࡔ࠲ࡁ࡯₸ࠍ᳞߼ߚ㧚➅ࠅ㄰ߒታ㛎ߪ࿁ⴕ޿㧘ߘߩ⚿ᨐࠍ⴫ߦ࡞᛽಴ᶧߩઍࠊࠅߦੇ῎↲⁁⣼ᧃߩಽᨆᴺߦ␜ߒߚࡔ࠲ࡁ࡯࡞ࠍ↪޿ߚ႐ว㧘ஜᐽ㘩ຠ A ߩ T ෸߮ Tߩ࿁෼₸␜ߒߚ㧚߇㨪㧑ૐਅߒߚ㧚ߘ ࠇࠁ߃ታ⹜ᢱߩ᛽಴ᶧߦ⴫㧞 ஜᐽ㘩ຠ߆ࠄߩ6෸߮6ߩᷝട࿁෼⹜㛎⚿ᨐߪ㧑ࠕࡦࡕ࠾ࠕ᳓ -ࡔ࠲ࡁ࡯࡞ṁᶧࠍ↪޿ߚ㧚ᧄᴺߦࠃ
ࠆ T  ෸߮ T  ߩቯ㊂ਅ㒢୯ߪ T  ߇Ǵg/g ෸߮ T߇

T3

T4

Ǵg/g ߢ޽ߞߚ㧚࿁෼₸ ± ᮡḰ஍Ꮕஜᐽⵍኂߩ⊒↢ߔࠆ߅ߘࠇ߇ุቯߢ߈ߥ޿ਛ࿖⵾࠳ࠗ
ࠛ࠶࠻↪ஜᐽ㘩ຠߣߒߡෘ↢ഭ௛⋭ߩࡎ࡯ࡓࡍ࡯ࠫ਄ߦ

ஜᐽ㘩ຠ A౏⴫ߐࠇߡ޿ࠆஜᐽ㘩ຠຠ⋡ࠍ౉ᚻߒ᷹ቯߒߚ㧚ߘߩ

Bond Elut C18 ಣ ℂ೨70.7±2.569.8±0.6⚿ᨐ㧘ߘߩ฽᦭㊂ߪ T߇Ǵg/g㧔㨪Ǵg/g㧕㧘T

Bond Elut C18 ಣ ℂᓟ71.1±0.9

68.0±1.0߇Ǵ g/g㧔㨪Ǵ g/g㧕ߢ޽ߞߚ㧚ੇ῎↲⁁⣼

Bond Elut C18 ಣ ℂ೨77.2±1.779.8±1.9⋧ᒰߔࠆ㧚ߎߩຠ⋡ߩ᷹ቯ୯ߪ⹜ᢱࠍࡔ࠲ࡁ࡯࡞෸߮

Bond Elut C18 ಣ ℂᓟ81.5±2.778.2±2.8᳓㧛ࡔ࠲ࡁ࡯࡞ߢᵞᵺߒߚ߽ߩ޽ࠆ޿ߪᧂᵞᵺߩ߽ߩߢ

ஜᐽ㘩ຠ Bᧃ㊂ߦ឵▚ߔࠆߣ㧘⚂mg/g㧔 㨪mg/g㧕 ߦ޽ࠅ㧘޿ߕࠇ߽ Bond Elut C ᧂಣℂߩᬌ૕ߢ޽ࠆ㧚ߥஜᐽ㘩ຠ#෸߮$ߦ߅޿ߡ6 ෸߮6 ߪᧂᬌ಴ߢ޽ߞߚ㧚ஜᐽ㘩ຠIߦੇ῎↲⁁⣼ᧃOIࠍᷝടߒߚ㧚ታ㛎࿁ᢙ㧔P㧕㧚߅㧘ᧄᴺࠍᔕ↪ߔࠆ㓙ߩᵈᗧὐߣߒߡ㧘T ෸߮ T ߪᵞ೷ߢ⪭ߜߦߊ޿ߚ߼㧘⹜ 㛎 ߦ ૶ ↪ ߒ ߚ ࠟ ࡜ ࠬ ེ ౕ ߪ㨪 N ߩ⎫㉄╬ߢߔߔ߉ᵞ޿ߔࠆᔅⷐ߇޽ࠆ

ஜᐽ㘩ຠ A ߩ႐ว㧘Bond Elut Cಣℂ೨ߩ࿁෼₸ߪ T߇㧑 㨪㧑 ෸߮ T ߇㧑 㨪㧠߹ߣ߼㧑 ߢ޽ࠅ㧘Bond Elut C ಣℂᓟߩ࿁෼₸ߪ T߇㧑↲⁁⣼ᧃࠍ฽᦭ߔࠆஜᐽ㘩ຠਛߩ T෸߮ Tߩ HPLC㨪㧑 ෸߮ T ߇㧑 㨪㧑 ߢ޽ߞಽᨆᴺࠍᬌ⸛ߒߚ㧚ߘߩ⚿ᨐ㧘⹜ᢱࠍ Pronase ࠍ↪޿ߡߚ㧚ஜᐽ㘩ຠ B ߩ႐ว㧘Bond Elut C ಣℂ೨ߩ࿁෼₸ട᳓ಽ⸃ߔࠆ೨ߦࡔ࠲ࡁ࡯࡞෸߮᳓㧛ࡔ࠲ࡁ࡯࡞ࠍ↪޿ߪ T ߇㧑 㨪㧑 ෸߮ T ߇㧑 㨪ߡᵞᵺߔࠆߎߣߦࠃࠅ౒ሽ‛⾰ࠍലᨐ⊛ߦ㒰෰ߔࠆߎߣ㧑 ߢ޽ࠅ㧘Bond Elut Cಣℂᓟߩ࿁෼₸ߪ T ߇߇ߢ߈ߚ㧚߹ߚ㧘Pronase ߢട᳓ಽ⸃ᓟߩ᛽಴ᶧࠍ Bond㧑 㨪㧑 ෸߮ T ߇㧑 㨪㧑

Elut C ࠞ࡯࠻࡝࠶ࠫಣℂߔࠆߎߣߦࠃࠅ㧘ߐࠄߦ౒ߢ޽ߞߚ㧚ᄌേଥᢙߪ޿ߕࠇ߽㧑એౝߢ޽ࠅ߶߷ḩሽ‛⾰ࠍ㒰෰ߔࠆߎߣ߇ߢ߈ߚ㧚߹ߚ㧘࿁෼₸㧘ౣ⃻ᕈ⿷ߔࠆ⚿ᨐ߇߃ࠄࠇߚ㧚ߒ߆ߒ㧘࿁෼₸ߪ⧯ᐓૐ޿௑ะߣ߽ߦ߶߷ḩ⿷ߔࠆ⚿ᨐ߇ᓧࠄࠇߚ㧚ߦ޽ߞߚ㧚ዊဈࠄߪ T෸߮ Tߪๆ⌕ᕈ߇㜞ߊ㧘㈶㉄ࠛᢥ₂࠴࡞᛽಴ᶧࠍࠈ⚕ߢࠈㆊߔࠆߣࠈ⚕ߦๆ⌕ߒᭂ┵ߦ࿁෼₸߇ૐਅߔࠆߣႎ๔ߒߡ޿ࠆ 㧚ߘߎߢ㧘ஜᐽ㘩ຠࠍ㉂⚛ಣℂߒ㧑ࠕࡦࡕ࠾ࠕ᳓ -ࡔ࠲ᣣᧄ⮎ዪᣇ⸃⺑ᦠ✬㓸ᆔຬળ㧦╙ᡷᱜᣣᧄ⮎ዪᣇ⸃⺑ᦠ㧘㧔ppD - 㧕㧧᧲੩㧦ᑝᎹᦠᐫ㧘㧚ࡁ࡯࡞ml ߢ࿁᛽಴ߒߚᓟ㧘ߘߩᱷᷲߦ㧑ࠕࡦࡕ࠾ࠕThe United States Pharmacopeia  th㧦United States
Pharmacopeial Convention Inc  - ᳓-ࡔ࠲ࡁ࡯࡞ml ࠍട߃࿁᛽಴ߒߚ⚿ᨐ㧘T෸߮ T߇Tai S et al 㧦Clin Chem  - ஜᐽ㘩ຠ A ߩ႐ว⚂㧑㧘ஜᐽ㘩ຠ B ߩ႐ว⚂㧑࿁෼ዊဈᅱሶ࡮ᵿ↰ᵗᒾ㧦㘩ⴡ⹹㧘  - ߐࠇ㧘ዋ㊂߇᛽಴ᱷᷲߦᱷ⇐ߒߡ޿ࠆߎߣ߇ಽ߆ߞߚ㧚࿁෼₸ߩૐਅߩේ࿃ߪട᳓ಽ⸃ಣℂᓟߩ⹜ᢱߦ T෸߮ T

Fukuoka Institute of Health and Environmental Sciences,
Mukaizano 39, Dazaifu, Fukuoka 818-0135, JapanA reverse-phase HPLC method for determining 3,3',5-triiodo-L-thyronine (T3) and L-thyroxine (T4) in health foods isdescribed. A sample was washed with methanol and methanol/water (1:1, v/v), before hydrolyzing using a Pronase at 37 ͠ for
18 h. T3 and T4 was extracted with 2% ammonium hydroxide in methanol. The sample solution for HPLC was applied to solid
phase extractions (Bond Elut C18) with 2% ammonium hydroxide in methanol as mobile phase. The HPLC analysis was carriedout on a column of InertsilODS-3 (4.6 x 150 mm, 5 µm) with H2O/CH3OH/H3PO4 (650:350:5, v/v) as mobile phase and theeluate was monitored by UV detection at 230 nm. The average recovery of T3 and T4 from the health food sample was 70-80%.
The quantitation limits of this method of T3 and T4 were 0.8 and 1.4 µg/g, respectively.
[Key words; Thyroid 3,3',5-triiodo-L-thyronine, L-thyroxine, HPLC㧘Solid phase extractions, Health Food]