JP2850128B2 - Aging measurement method - Google Patents
Aging measurement methodInfo
- Publication number
- JP2850128B2 JP2850128B2 JP33193388A JP33193388A JP2850128B2 JP 2850128 B2 JP2850128 B2 JP 2850128B2 JP 33193388 A JP33193388 A JP 33193388A JP 33193388 A JP33193388 A JP 33193388A JP 2850128 B2 JP2850128 B2 JP 2850128B2
- Authority
- JP
- Japan
- Prior art keywords
- aging
- urine
- 80hdg
- age
- degree
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 230000032683 aging Effects 0.000 title claims description 37
- 238000000691 measurement method Methods 0.000 title description 4
- 210000002700 urine Anatomy 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 17
- 230000036542 oxidative stress Effects 0.000 claims description 7
- HCAJQHYUCKICQH-VPENINKCSA-N 8-Oxo-7,8-dihydro-2'-deoxyguanosine Chemical compound C1=2NC(N)=NC(=O)C=2NC(=O)N1[C@H]1C[C@H](O)[C@@H](CO)O1 HCAJQHYUCKICQH-VPENINKCSA-N 0.000 claims description 6
- 238000012544 monitoring process Methods 0.000 claims description 2
- 230000009885 systemic effect Effects 0.000 claims description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 201000010099 disease Diseases 0.000 description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- -1 lipid peroxide Chemical class 0.000 description 7
- 229910052760 oxygen Inorganic materials 0.000 description 7
- 239000001301 oxygen Substances 0.000 description 7
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 4
- 206010012601 diabetes mellitus Diseases 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 208000024827 Alzheimer disease Diseases 0.000 description 3
- 208000023275 Autoimmune disease Diseases 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 208000023589 ischemic disease Diseases 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- RKEITGVZZHXKON-SKAWGCAZSA-N Thymidine glycol Chemical compound O=C1NC(=O)C(C)(O)C(O)N1[C@@H]1O[C@H](CO)[C@@H](O)C1 RKEITGVZZHXKON-SKAWGCAZSA-N 0.000 description 2
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 2
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 229940109239 creatinine Drugs 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- GUKSGXOLJNWRLZ-UHFFFAOYSA-N thymine glycol Chemical compound CC1(O)C(O)NC(=O)NC1=O GUKSGXOLJNWRLZ-UHFFFAOYSA-N 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- 229940116269 uric acid Drugs 0.000 description 2
- 230000002485 urinary effect Effects 0.000 description 2
- YKBGVTZYEHREMT-KVQBGUIXSA-N 2'-deoxyguanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 YKBGVTZYEHREMT-KVQBGUIXSA-N 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 201000010374 Down Syndrome Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101150096839 Fcmr gene Proteins 0.000 description 1
- 208000025500 Hutchinson-Gilford progeria syndrome Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 208000007932 Progeria Diseases 0.000 description 1
- 206010044688 Trisomy 21 Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000012223 aqueous fraction Substances 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000018823 dietary intake Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002032 methanolic fraction Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 201000008383 nephritis Diseases 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 230000003863 physical function Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000036561 sun exposure Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000005353 urine analysis Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measurement Of The Respiration, Hearing Ability, Form, And Blood Characteristics Of Living Organisms (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、尿中に排泄される核酸分解物（８ハイドロ
キシデオキシグアノシン）を簡易的に分析定量する測定
方法に関するものであり、特に、老化の進行度を予測す
るため、また、老化を遅らせる物質を探索するために利
用し、さらに糖尿病・循環器糸疾患・アルツハイマー病
・虚血性疾患・自己免疫疾患等の予測予防方法を探索す
るために利用するものである。Description: TECHNICAL FIELD The present invention relates to a measurement method for simply analyzing and quantifying a nucleic acid degradation product (8-hydroxydeoxyguanosine) excreted in urine, and particularly to aging. In order to predict the degree of progression of the disease, and to search for substances that delay aging, and to search for methods to predict and prevent diabetes, cardiovascular disease, Alzheimer's disease, ischemic disease, autoimmune disease, etc. To use.
（従来の技術） 長年にわたり、これまで老化の程度を知るために、い
ろいろな方法が考案されている。老化は、老徴と言われ
る多くの外観的、機能的な特徴によを表現されており、
10年単位で各年齢を分類すれば、老徴は大別的に当ては
まる。しかし、同じ年代の高齢者間で見ると、それぞれ
寿命の差が大きいように、老徴も違っており、一概に老
化の程度はわからない。(Prior Art) Over the years, various methods have been devised to determine the degree of aging. Aging is described by many appearance and functional characteristics called aging signs,
If we classify each age in ten-year units, the old symptoms apply broadly. However, when looking at elderly people of the same age, the signs of aging are also different, as the differences in life expectancy are large, and the degree of aging is unclear.
大別的な老徴の分類は、例えば、表１のように報告さ
れている（亀山正邦 内科51,10221983）。Classification of senile genotypes is reported, for example, in Table 1 (Masakuni Kameyama Internal Medicine 51,10221983).
日野原は、身体の機能を表２のように16の項目に分類
し、各項目にスコアーを与え、合計点から老化度を算出
し、第６図の如き結果を得ている（日野原重明 日老医
誌16,2381979）。 Hinohara classifies the physical functions into 16 items as shown in Table 2, gives a score to each item, calculates the degree of aging from the total points, and obtains the results shown in Fig. 6 (Hinohara Shigeaki Hirohara Medical journal 16,2381979).
一方、老化を起こす因子として、近年、フリーラジカ
ルの存在とそのかかわり合いが論じられて来ている。 On the other hand, in recent years, the existence of free radicals and their involvement have been discussed as factors that cause aging.
生体内に、発生する活性酸素は、正常な細胞、組織に
損傷を与え（酸化的ストレス）、老化を起こす原因の一
つに考えられている（カトラー：第２回協和発酵バイオ
サイエンスシンポジウム、東京 1985,9、ソハール;Bas
ic Life Sci,35:75,1985）。Reactive oxygen generated in living organisms is considered to be one of the causes of damage to normal cells and tissues (oxidative stress) and aging (Cutler: 2nd Kyowa Hakko Bioscience Symposium, Tokyo, Japan) 1985, 9, Sohar; Bas
ic Life Sci, 35:75, 1985).
また、糖尿病、虚血性疾患、腎炎、動脈硬化症自己免
疫疾患、癌等、成人病と称される疾患は、その原因機序
に活性酸素が関わることが強く示唆されている（コーエ
ンら;J.Biol.Chem.,249:2447,1974,マエストロ;Acta.Ph
ysiol.Scand.492（suppl.）:153,1980,トーマスら;Adv.
in Free radi−cal＆Biol Med.,;2.347.1986.ステファ
ンら;J.Nenrological Sci.,67;319,1985.末松ら；医学
の歩み、142:729,1987,阿部ら；医学の歩み、142;742、
1987）。活性酸素は、細胞の核酸を修飾し、或いは細胞
死に至らしめることが推測され、核酸由来とみられる修
飾塩基；チミングリコール、チミジングリコールが尿中
に検出され、動物種の酸素消費量と相関が認められ、老
化とのかかわり合いを示唆している（エーデルマンら、
Proc,Natl.Acd.Sci.USA,85,2706,1988）。また８−ハイ
ドロキシデオキシグアノシン，チミングリコールそして
チミジングリコールは、核酸に放射線を照射した時に生
成されることも知られている（葛西ら；癌、75:1037.19
4.フレンケルら;Biochemistry,20:7566.1981）。血液中
の過酸化脂質と老化とのかかわり合いはよく知られてい
るが（ストレーラーら;J.Gerontol,14.430,1959）、こ
の過酸化脂質と酸化的ストレスとの間に相関性もあるこ
とが我々の研究により強く示唆されている。In addition, it has been strongly suggested that diseases called adult diseases, such as diabetes, ischemic disease, nephritis, autoimmune disease of arteriosclerosis, and cancer, involve active oxygen in the causative mechanism thereof (Cohen et al .; J. .Biol.Chem., 249: 2447,1974, Maestro; Acta.Ph
ysiol. Scand. 492 (suppl.): 153, 1980, Thomas et al .; Adv.
in Free radi-cal & Biol Med.,; 2.347.1986. Stefan et al .; J. Nenrological Sci., 67; 319, 1985. Suematsu et al. 742,
1987). Active oxygen is presumed to modify cell nucleic acids or lead to cell death. Modified bases that are considered to be derived from nucleic acids; thymine glycol and thymidine glycol are detected in urine and correlated with the oxygen consumption of animal species. And suggest a connection with aging (Edelman et al.,
Proc, Natl. Accd. Sci. USA, 85, 2706, 1988). It is also known that 8-hydroxydeoxyguanosine, thymine glycol and thymidine glycol are produced when a nucleic acid is irradiated (Kasai et al., Cancer, 75: 1037.19).
4. Frenkel et al .; Biochemistry, 20: 7566.1981). Although the relationship between blood lipid peroxide and aging is well known (Strahler et al .; J. Gerontol, 14.430, 1959), there may be a correlation between this lipid peroxide and oxidative stress. It is strongly suggested by our research.
（発明が解決しようとする課題） 老化度測定法は、老化の基準を客観的に設定すること
で一致しているが、臓器機能の加齢変化は、個体差が著
しいので、ある臓器の機能から個体差を推定するのは不
可能である。又、スコアー方式についても多次元ベクト
ルとしての老化の尺度に一歩接近しているとは言えるも
のの、情報の重みが科学的に決定されていない欠点があ
る。以上のごとく、これまでも老化度の測定をいろいろ
試みられてはいたが、満足すべきものが得られていな
い。(Problems to be Solved by the Invention) The aging degree measurement method agrees with the objective setting of the aging criteria. It is impossible to estimate individual differences from. In addition, although the score method can be said to be one step closer to the scale of aging as a multidimensional vector, there is a disadvantage that the weight of information is not scientifically determined. As described above, various attempts have been made to measure the degree of aging, but no satisfactory results have been obtained.
老化防止策は、食物摂取の栄養的な観点、筋肉・骨組
織機能維持の運動整生学的な観点、住宅環境の改善、太
陽光線被爆防止などの物理学的な観点等、いろいろ方法
がある。しかし、その最適化をモニターする実用的な方
法はない。There are various methods for preventing aging, such as nutritional aspects of food intake, exercise rehabilitation aspects of maintaining muscle and bone tissue function, improvement of housing environment, and physical aspects such as prevention of sun exposure. . However, there is no practical way to monitor that optimization.
上記のように、個人々々について、体の遺伝的背景、
ライフスタイル、栄養の摂取状況が違っており、このよ
うな状況下で老化を防止する為の実用的なモニターの方
法は未だ提案されていない。As mentioned above, for each individual, the genetic background of the body,
Due to differences in lifestyle and nutritional intake, practical monitoring methods for preventing aging under such circumstances have not yet been proposed.
（課題を解決する為の手段） 本発明は、尿中の核酸分解物;8−ハイドロキシデオキ
シグアノシンについて、老化とのかかわり合いを種々検
討した結果、老化促進度の指標となることを見いだし、
上述した老化度の測定法の問題点を解決し、本発明を完
成した。(Means for Solving the Problems) The present invention, as a result of various studies on the relationship between nucleases in urine; 8-hydroxydeoxyguanosine and aging, has been found to be an index of the degree of aging,
The present invention has been completed by solving the above-mentioned problems of the method for measuring the degree of aging.
すなわち、尿中に排泄される修飾された核酸塩基;8−
ハイドロキシデオキシグアノシンを高速液クロにより電
気的・紫外部検出器を用いて測定し、これを全身的な酸
化的ストレスの指標とし、個人、体の能力に応じて、老
化度を測定する方法を提供するものである。活性酸素に
よる酸化的ストレスは、従来の技術の所で記載したよう
に、成人病はじめ数多くの疾患の原因機序であり、著者
らは、老化すなわち細胞核酸の損傷をおこす活性酸素と
その防禦について次の概念を推測するに至っている。That is, modified nucleobases excreted in urine; 8-
Provides a method for measuring hydroxydeoxyguanosine by high-performance liquid chromatography using an electric / ultraviolet detector and using this as an index of systemic oxidative stress, and measuring the degree of aging according to individual and body abilities. Is what you do. Oxidative stress caused by reactive oxygen, as described in the prior art, is a causative mechanism of many diseases including adult diseases.The authors discuss active oxygen and its protection that cause aging, or damage to cellular nucleic acids. The following concept has been speculated.
酸化的ストレスは以下のように定義される。 Oxidative stress is defined as follows.
乃至はOS＝Ｋ′（Σ［活性酸素］−Σ［抗酸化性物
質］）。Ｋ及びＫ′は常数である。 Or OS = K '(Σ [active oxygen] -Σ [antioxidant]). K and K 'are constants.
以下、この経緯を詳細に述べる。Hereinafter, this process will be described in detail.
o8−ハイドロキシデオキシグアノシン（80HdG）の調製 標準物質として用いた80HdGは、池原らの方法（Chem.
Pharm.Bull.13.1140−1142,1965）に従い合成した。Preparation of o8-hydroxydeoxyguanosine (80HdG) 80HdG used as a standard substance was prepared by the method of Ikehara et al. (Chem.
Pharm. Bull. 13.1140-1142, 1965).
すなわち、1gのデオキシグアノシンを原料とし、780m
lの0.13M燐酸緩衝液（pH6.8）に溶解後、140mlの0.1Mア
スコルビン酸、0.1MFeSO4と65mlの0.1M EDTAをそれぞれ
加え37℃下で酸素を吹き込みながら３時間反応を行っ
た。終了後、遮光で1N塩酸でpH3.7とし、10gの活性炭を
加え、十分撹拌後、それをガラスカラムに通し濾過し
た。カラムの残留物は、蒸溜水で数回洗浄後、500mlの
水・アセトン（1:1、v/v）で、溶出させた。溶出液は、
エバポレーターを用いて濃縮乾固し、さらに高速液体ク
ロマトグラフィーを用いて、精製を行った（colum:supe
luco社、ODS4.6×100mm、solvenl:15％MeOH）。その結
果、80HdGは、約90mgの白色結晶として得た。That is, 1 g of deoxyguanosine as a raw material, 780 m
After dissolving in 1 l of 0.13 M phosphate buffer (pH 6.8), 140 ml of 0.1 M ascorbic acid, 0.1 M FeSO 4 and 65 ml of 0.1 M EDTA were added, and the reaction was carried out at 37 ° C. for 3 hours while blowing oxygen. After the completion, the pH was adjusted to 3.7 with 1N hydrochloric acid in the absence of light, 10 g of activated carbon was added, and after sufficient stirring, the mixture was filtered through a glass column. The column residue was washed several times with distilled water and then eluted with 500 ml of water / acetone (1: 1, v / v). The eluate is
The solution was concentrated to dryness using an evaporator, and further purified using high performance liquid chromatography (colum: supe
luco, ODS 4.6 x 100 mm, solvent: 15% MeOH). As a result, 80HdG was obtained as about 90 mg of white crystals.
本物質のNMRスペクトル、UVスペクトル、HPLCクロマ
トグラムを第１図、第２図、第３図に示す。The NMR spectrum, UV spectrum and HPLC chromatogram of this substance are shown in FIG. 1, FIG. 2, and FIG.
ｏ尿中の８−0HdGの定量法 次に、人の尿に微量存在する80HdGの分析方法につい
て記載する。o Quantitative method of 8-0HdG in urine Next, a method of analyzing 80HdG present in a trace amount in human urine will be described.
尿検体の調整；尿の採取は、午後０時から翌日の午後
０時までの24時間の全量を同一容器に採取し、その少量
を、フィルター（0.22μｍ）で濾過した。尿は、予めメ
タノールと蒸溜水で洗浄したカラム（C18:sep−pack,ミ
リポア社）に通し、蒸溜水と35％メタノールで溶出さ
せ、それぞれの濾液を、水分画、35％メタノール分画と
した。HPLCの設定；ヒューレット・パッカード社のHPLC
1090で、カラムは、supeluco社のODS系のプレカラムとC
18S 250×4.6mmを用いた。ソルベントは、50mM燐酸緩衝
液（pH5.5）と10％メタノール同緩衝液あるいは、12.5m
Mクエン酸緩衝液（pH5.1）と10％メタノール同緩衝液
で、０−60分間のグラディエントをかけ、紫外部ダイオ
ードアレイ（205,231,293nm）とEC検出器（東ソ社;EC−
8000）で測定した。標準物質として、80HdGの他に、尿
酸そしてクレアチニンを用いた。Preparation of urine sample; urine was collected in the same container over the entire 24-hour period from midnight to midnight the following day, and a small amount thereof was filtered through a filter (0.22 μm). Urine was passed through a column (C18: sep-pack, Millipore) previously washed with methanol and distilled water, eluted with distilled water and 35% methanol, and the respective filtrates were used as a water fraction and a 35% methanol fraction. . HPLC setup; Hewlett-Packard HPLC
In 1090, the column is a pre-column of supeluco ODS system and C
18S 250 × 4.6 mm was used. Solvent: 50mM phosphate buffer (pH 5.5) and 10% methanol same buffer or 12.5mM
A gradient of 0 to 60 minutes was applied with an M citrate buffer (pH 5.1) and the same buffer of 10% methanol, and an ultraviolet diode array (205, 231, 293 nm) and an EC detector (Toso; EC-
8000). Uric acid and creatinine were used as standard substances in addition to 80HdG.
本発明は、測定手段として、高速液体クロマトグラフ
によりODS系カラムLC18S等、20cm〜30cm×4.6mm（１〜
２本）を用い、リン酸等の酸性側緩衝液（pH4.5〜6.0）
に10％メタノール等のグラディエントを（０〜60分間）
かけ、電気化学的（印加電圧550mV〜700mV）および紫外
部吸収検出器（231,243,293nm）により、修飾塩基、尿
酸、クレアチニンの同時測定を可能とする条件を用いる
ことを特徴とする老化度測定法である。The present invention uses a high-performance liquid chromatograph, such as an ODS column LC18S or the like, as a measuring means, 20 cm to 30 cm × 4.6 mm (1 to
2) using acidic buffer such as phosphoric acid (pH 4.5-6.0)
10% methanol gradient (0-60 minutes)
Aging measurement method characterized by using conditions that allow simultaneous measurement of modified base, uric acid, and creatinine by electrochemical (applied voltage 550 mV to 700 mV) and ultraviolet absorption detector (231,243,293 nm) is there.
また本発明の測定器の構成は、上記のごとく詳しく述
べたが、A.尿の処理工程、B.高速液クロのカラムによる
分離、C.検出器として電気化学的もしくは紫外部吸収に
よるシステムである。糖尿病・循環器系疾患・アルツハ
イマー病・虚血性疾患・自己免疫疾患等、加令に伴う諸
疾患の予知が出来るようにした老化度測定器である。Although the configuration of the measuring instrument of the present invention has been described in detail as described above, A. urine processing step, B. separation by high-performance liquid chromatography column, and C. system using electrochemical or ultraviolet absorption as a detector. is there. This is an aging degree measuring instrument that can predict various diseases associated with aging, such as diabetes, circulatory disease, Alzheimer's disease, ischemic disease, and autoimmune disease.
（実施例１） ここで健康人男子25名（20〜35才:7名、36〜60才代:1
8名）について、尿中の80HdGを測定し、80HdGと年齢と
の相関関係を調べ、統計学的に検討を行った。その結
果、35歳以降においては、加齢に伴う24時間尿中の80Hd
G/kg体重の消長変化に有意の正相関が見られた（α＝0.
65,n＝18,p＜0.01,y＝0.052χ−１）（第４図）。すな
わち、若々しさが失われてきて、老徴が顕著に出現しだ
す約35歳以降は、８−0HdGは加齢とともに増加が見られ
ることがわかった。成長過程（20歳から30歳前後）にお
いては、代謝が活発に行われ、細胞の交代が烈しいため
80HdGの排泄はむしろ、増加し、80HdGは高く維持されて
いる（みかけの老化）。30歳ごろに生物学的に成熟が終
わり、35歳ごろまで80HdGの変動はみられるが、相関は
ない。35歳以降では、80HdGのレベルは必ずしも高くは
ないが、上昇を続け、50歳以降では、顕著になる。これ
は、明らかに加齢に伴う変化とすることが出来る。(Example 1) Here, 25 healthy males (20-35 years old: 7 people, 36-60 years old: 1
For 8 subjects), 80HdG in urine was measured, the correlation between 80HdG and age was examined, and statistically examined. As a result, after 35 years, 80Hd in urine for 24 hours with aging
A significant positive correlation was observed with the change in the change in G / kg body weight (α = 0.
65, n = 18, p <0.01, y = 0.052χ-1) (FIG. 4). That is, it was found that after about 35 years of age when the youth was lost and the senile signs began to appear remarkably, 8-0HdG increased with age. During the growth process (around the age of 20 to 30), metabolism is active and cell replacement is intense.
Rather, 80HdG excretion is increased and 80HdG is maintained high (apparent aging). Biological maturity ends around the age of 30, and 80HdG fluctuates until around the age of 35, but there is no correlation. After the age of 35, the level of 80HdG is not necessarily high, but continues to rise, becoming more pronounced after the age of 50. This can clearly be a change with age.
（実施例２） 上記、健康人について、血清中の過酸化脂質を八木法
（過酸化脂質実験法、金田ら、1984年、医歯薬出版）に
より測定し各々の80HdG値と対比を行った。その結果を
第５図に示す。血清中の過酸化脂質の上昇と尿中の80Hd
Gは、相関を有することがわかった。(Example 2) For the above healthy persons, lipid peroxide in serum was measured by the Yagi method (Lipid peroxide experiment method, Kaneda et al., 1984, Medical and Dental Medicine Publishing Co., Ltd.) and compared with each 80HdG value. . The results are shown in FIG. Elevation of serum lipid peroxide and urinary 80Hd
G was found to have a correlation.
老化の指標として、過酸化脂質値が使われることがあ
るが、採血を経なければならず、その点尿中の修飾塩基
の測定法は非侵襲的であるのできわめて便利に利用でき
る。Lipid peroxide levels may be used as an indicator of aging, but blood sampling must be performed, and the method for measuring modified bases in urinary spot urine is non-invasive and can be used very conveniently.
（実施例３） 早老症として知られる疾患；ダウン症患者の尿中の８
−0HdGを経時的に測定したところ表３のように年齢を問
わず健康人に対し排泄量が多く、老化が促進されている
のが明らかに認められた。Example 3 Disease known as progeria; 8 in urine of Down's syndrome patients
When −0HdG was measured over time, as shown in Table 3, excretion was large for healthy people regardless of age, and it was clearly observed that aging was promoted.
（実施例４） 放射線による照射治療を受けた患者の尿中の８−0HdG
を治療前後に亘って測定した。照射にしたがって、８−
0HdGの増加傾向が見られた（表４）。 (Example 4) 8-0HdG in urine of a patient who received irradiation treatment with radiation
Was measured before and after treatment. 8--
An increasing trend of 0HdG was observed (Table 4).
（発明の効果） 本発明によれば、尿中に排泄される核酸分解物を簡易
に測定するものであるから、老化の進行度を予測するこ
ととができると共に、老化を遅らせる物質を探索する為
に使用し、糖尿病、循環器系疾患、アルツハイマー病、
その他の老人病に有効に対処し、これを予防し得る効果
がある。 (Effects of the Invention) According to the present invention, since a nucleic acid degradation product excreted in urine is simply measured, the progress of aging can be predicted, and a substance that delays aging is searched for. Used for diabetes, cardiovascular disease, Alzheimer's disease,
It is effective in effectively treating and preventing other geriatric diseases.
【図面の簡単な説明】 第１図は80HdG標品のNMRスペクトル、第２図は80HdG標
品のUVスペクトル、第３図は80HdG標品ののHPLCスペク
トル、第４図は健康人24時間当りの尿中の80HdG値、測
定による年齢とのクラスター分析、第５図は健康人24時
間当りの80HdG値と血液中の過酸化脂質値との相関関係
を示す図、第６図は機能上の老化度測定の判定点と年齢
とのグラフである。BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the NMR spectrum of the 80HdG standard, FIG. 2 shows the UV spectrum of the 80HdG standard, FIG. 3 shows the HPLC spectrum of the 80HdG standard, and FIG. 80HdG value in urine and cluster analysis with age by measurement, FIG. 5 is a diagram showing the correlation between 80HdG value per 24 hours in healthy subjects and lipid peroxide value in blood, and FIG. It is a graph of the determination point of aging degree measurement and age.
Claims (2)
シデオキシグアノシンを指標として全身的な酸化的スト
レスの程度を測定することを特徴とする老化度測定方
法。1. A method for measuring the degree of aging, comprising measuring the degree of systemic oxidative stress using 8-hydroxydeoxyguanosine as a modified nucleobase in urine as an index.
飾核酸塩基である８−ハイドロキシデオキシグアノシン
を測定し、老化進行の予防・防止方法をモニターするこ
とを特徴とする老化度測定方法。2. A method for measuring the degree of aging, comprising measuring 8-hydroxydeoxyguanosine, which is a modified nucleobase derived from oxidative stress excreted in urine, and monitoring a method for preventing or preventing the progress of aging.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33193388A JP2850128B2 (en) | 1988-12-28 | 1988-12-28 | Aging measurement method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33193388A JP2850128B2 (en) | 1988-12-28 | 1988-12-28 | Aging measurement method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02177950A JPH02177950A (en) | 1990-07-11 |
| JP2850128B2 true JP2850128B2 (en) | 1999-01-27 |
Family
ID=18249265
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP33193388A Expired - Fee Related JP2850128B2 (en) | 1988-12-28 | 1988-12-28 | Aging measurement method |
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| Country | Link |
|---|---|
| JP (1) | JP2850128B2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4529104B2 (en) * | 2000-03-16 | 2010-08-25 | 株式会社エイコム | Simple and sensitive measurement system for DNA oxidative damage index in biological samples |
| AU2003220877A1 (en) * | 2002-03-14 | 2003-09-22 | Hiroshi Kasai | Method of purifying oxidatively injured guanine nucleoside, method of measuring the same and analyzer for the embodiment thereof |
| DE102005009616A1 (en) * | 2005-03-03 | 2006-09-07 | Wittner, Robert, Dr. | Program minimizing biological ageing and maximizing human wellbeing involves control of nutritional, urine pH, fitness and stress management factors |
| JP2007271287A (en) * | 2006-03-30 | 2007-10-18 | Kitakyushu Foundation For The Advancement Of Industry Science & Technology | Oxidative stress substance detection sensor |
| EP2642293A1 (en) | 2012-03-22 | 2013-09-25 | Nestec S.A. | 9-oxo-octadecadienoic acid (9-oxo-HODE)as as biomarker for healthy ageing |
-
1988
- 1988-12-28 JP JP33193388A patent/JP2850128B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02177950A (en) | 1990-07-11 |
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