JPH04184256A - Method and device for measuring calcium in body fluid - Google Patents
Method and device for measuring calcium in body fluidInfo
- Publication number
- JPH04184256A JPH04184256A JP31375990A JP31375990A JPH04184256A JP H04184256 A JPH04184256 A JP H04184256A JP 31375990 A JP31375990 A JP 31375990A JP 31375990 A JP31375990 A JP 31375990A JP H04184256 A JPH04184256 A JP H04184256A
- Authority
- JP
- Japan
- Prior art keywords
- calcium
- calcification
- body fluid
- measuring
- diffusion
- 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.)
- Pending
Links
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 229910052791 calcium Inorganic materials 0.000 title claims abstract description 60
- 239000011575 calcium Substances 0.000 title claims abstract description 60
- 210000001124 body fluid Anatomy 0.000 title claims abstract description 40
- 239000010839 body fluid Substances 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims description 20
- 230000002308 calcification Effects 0.000 claims abstract description 40
- 238000009792 diffusion process Methods 0.000 claims abstract description 15
- 239000003112 inhibitor Substances 0.000 claims abstract description 10
- 239000011159 matrix material Substances 0.000 claims abstract description 8
- 239000000758 substrate Substances 0.000 claims description 23
- 239000000126 substance Substances 0.000 claims description 15
- 230000002401 inhibitory effect Effects 0.000 claims description 13
- 238000005259 measurement Methods 0.000 claims description 9
- 229920002401 polyacrylamide Polymers 0.000 claims description 9
- 229920000642 polymer Polymers 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 238000002834 transmittance Methods 0.000 claims description 4
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims 1
- 229910001424 calcium ion Inorganic materials 0.000 claims 1
- 239000012466 permeate Substances 0.000 claims 1
- 210000000988 bone and bone Anatomy 0.000 abstract description 8
- 238000001914 filtration Methods 0.000 abstract description 5
- 230000008021 deposition Effects 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 238000000151 deposition Methods 0.000 abstract 3
- 208000004434 Calcinosis Diseases 0.000 description 32
- 239000000499 gel Substances 0.000 description 19
- 210000002966 serum Anatomy 0.000 description 12
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical group NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 10
- 230000011164 ossification Effects 0.000 description 9
- 208000001132 Osteoporosis Diseases 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 2
- 235000011941 Tilia x europaea Nutrition 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
- 238000009534 blood test Methods 0.000 description 2
- 230000004097 bone metabolism Effects 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000004571 lime Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- LPNSCOVIJFIXTJ-UHFFFAOYSA-N 2-methylidenebutanamide Chemical compound CCC(=C)C(N)=O LPNSCOVIJFIXTJ-UHFFFAOYSA-N 0.000 description 1
- WHNPOQXWAMXPTA-UHFFFAOYSA-N 3-methylbut-2-enamide Chemical compound CC(C)=CC(N)=O WHNPOQXWAMXPTA-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000008468 bone growth Effects 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 208000037824 growth disorder Diseases 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004880 lymph fluid Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000006371 metabolic abnormality Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- AYGYHGXUJBFUJU-UHFFFAOYSA-N n-[2-(prop-2-enoylamino)ethyl]prop-2-enamide Chemical compound C=CC(=O)NCCNC(=O)C=C AYGYHGXUJBFUJU-UHFFFAOYSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000002188 osteogenic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 229960005382 phenolphthalein Drugs 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は１体液中のカルシウムの測定方法及びその測定
器具に関するものであり、より詳細には、骨粗髭症及び
骨軟化症などの骨代謝異常を診断するために、血清中の
血清自身のもつ石灰化沈着能を測定する方法及びその測
定器具に関する。Detailed Description of the Invention (Industrial Field of Application) The present invention relates to a method for measuring calcium in a body fluid and an instrument for measuring calcium, and more particularly, it relates to a method for measuring calcium in a body fluid and a device for measuring calcium. The present invention relates to a method for measuring the calcification capacity of serum itself in order to diagnose metabolic abnormalities, and a measuring device therefor.
（従来技術及び発明が解決しようとする問題点）従来、
骨代謝異常の血清検査による詮所には。(Prior art and problems to be solved by the invention) Conventionally,
For investigation by serum tests for abnormalities in bone metabolism.
血液中のカルシウム、リン、アルカリフォスファターゼ
、口ｔｌＮ、ＰＴＩＩ、１ 、２５ （ＯＨ）２０ｓの
直接測定が行われてきた。Direct measurements of calcium, phosphorus, alkaline phosphatase, oral tIN, PTII, 1,25(OH)20s in the blood have been performed.
しかしながら、これらの測定方法に基づく値は、かなら
ずしも骨代謝異常と相関しない。これは、これらの測定
値が骨形成能に関係する因子を正確に測定していないと
解せられるからである。また、血清中のカルシウム等は
骨形成に必要な成分であることは明かであり、骨形成能
に関与することは明かである。しかし、カルシウム成分
のみを直接血清中から測定しても、その値が骨形成能と
充分な相関、特に骨粗鬆症との相関をとらない場合があ
る。However, values based on these measurement methods do not necessarily correlate with abnormal bone metabolism. This is because these measured values do not accurately measure factors related to osteogenic ability. Furthermore, it is clear that calcium and the like in serum are necessary components for bone formation, and it is clear that they are involved in bone formation ability. However, even if only the calcium component is directly measured from serum, the value may not correlate sufficiently with bone forming ability, especially with osteoporosis.
したがって、本発明の目的は、体液中の骨形成能を正確
に測定することのできる体液中のカルシウムの測定方法
及びその測定器具を提供することにある。Therefore, an object of the present invention is to provide a method for measuring calcium in a body fluid and a measuring device therefor, which can accurately measure the bone forming ability in a body fluid.
本発明の目的はまた、骨粗鬆症等と相関が充分にとれた
体液中のカルシウムの測定方法及び測定器具を提供する
ことにある。Another object of the present invention is to provide a method and a measuring device for measuring calcium in body fluids that are sufficiently correlated with osteoporosis and the like.
（問題点を解決するための手段）
本発明によれば、体肢中に含まれる石灰化抑制物質を除
去或は拡散を阻止した後１体液中に含まれるカルシウム
成分を石灰化沈着して測定することを特徴とする体液中
のカルシウムの測定方法が提供される。(Means for Solving the Problems) According to the present invention, after removing or inhibiting the diffusion of calcification-inhibiting substances contained in body extremities, calcium components contained in body fluids are calcified and measured. A method for measuring calcium in a body fluid is provided.
本発明はまた。前記カルシウム成分の石灰化沈着におい
て、カルシウム成分が沈着し得る基質を用い、その基質
に生じる石灰化沈着物を光学的に測定することを特徴と
することができる。The present invention also includes: In the calcification of calcium components, a substrate on which the calcium components can be deposited may be used, and the calcification deposits formed on the substrate may be optically measured.
本発明によればまた。カルシウム成分が浸透可能で、且
つ石灰化沈着の抑制をするタンパク質系高分子の拡散が
前記カルシウム成分より小さい構造体基質であって、か
つ前記抑制物が減少した状態のときにカルシウムを石灰
化沈着するｌＩ構造体基質有することを特徴とした体液
中のカルシウム測定器具が提供される。Also according to the invention. Calcium is calcified when the calcium component is permeable to the structure matrix, and the diffusion of protein-based polymers that inhibit calcification is smaller than the calcium component, and the inhibitor is reduced. Provided is a device for measuring calcium in a body fluid, characterized by having an II structure substrate.
本発明は測定器具においてまた。前記構造体基質の光透
過率（厚さ１０ｍ１）を２０％以上の透明体とすること
を特徴とすることができる。The invention also relates to measuring instruments. The structure substrate may be a transparent body having a light transmittance (thickness of 10 m1) of 20% or more.
本発明の前記構造体基質は複数の充填穴を備えたプレー
トに充填してなることを特徴とすることができる。The structure substrate of the present invention may be characterized in that it is formed by filling a plate with a plurality of filling holes.
本発明は前記構造体基質がポリアクリルアミドゲルであ
ることを特徴とすることができる。The present invention may be characterized in that the structure substrate is a polyacrylamide gel.
（作用）
本発明は１体液中、特に血清中のカルシウム成分を石灰
化沈着させてカルシウム成分の測定をしたことが重要で
あり、しかもこの測定に際して体液中に存在する石灰化
抑制物質を実質的に除いた状態としたことが重要である
。このような測定をすることによって、骨形成能に関す
る病状、特に骨粗鬆症との相関が一層高まり、これらの
測定方法及び器具が極めて血液検査に有意義になるとい
ものである。従来、血清中のカルシウム等を測定しても
、骨形成との相関はあまりみられず、充分な骨形成能測
定にはなり得なかった６本発明は骨形成に必要な体液中
の燐酸とカルシウムを石灰沈着能により、骨形成能を直
接測定する方法と実質同じと考えられる。以下、本発明
の作用について詳述する。(Function) It is important that the present invention measures the calcium component by calcifying the calcium component in one body fluid, especially the serum, and moreover, in this measurement, the calcification-inhibiting substances present in the body fluid are substantially removed. It is important to make sure that the condition is removed. By performing such measurements, the correlation with pathological conditions related to bone formation ability, especially osteoporosis, will be further enhanced, and these measurement methods and instruments will become extremely useful for blood tests. Conventionally, even when measuring serum calcium, etc., there was not much correlation with bone formation and it was not possible to measure bone forming ability sufficiently. This method is considered to be substantially the same as the method of directly measuring bone formation ability using calcium calcification ability. Hereinafter, the effects of the present invention will be explained in detail.
体液中には骨を構成しているカルシウムと＃Ｉｌ酸とが
存在していること、そして、本発明は体液が骨形成化を
抑制する物質も含んでいるという新たな事実に着目した
ことに特徴がある。また、ここで骨形成化とは曖昧な表
現ではあるが、骨にカルシウム成分が沈着する能力を意
味し１本発明はこの骨形成化を実質的に石灰化沈着能と
同質であるとしたことにも特徴がある。The present invention focuses on the new fact that calcium and #Il acid, which make up bones, exist in body fluids, and that body fluids also contain substances that inhibit bone formation. It has characteristics. In addition, although osteogenicity is an ambiguous term, it refers to the ability to deposit calcium components in bones, and the present invention considers this osteogenicity to be substantially the same as the ability to deposit minerals. There are also characteristics.
体液中において、この石灰化（骨形成化）抑制因子があ
るが由に通常は石灰化がまぬがれているが、この石灰化
抑制因子が物理的、化学的に除かれ、しかもそこに石灰
化の基質になり得る物質が存在すれば、そこに石灰沈着
が起こる。成体内における骨形成における石灰沈着もこ
の原理に基づいていると考えられる。In body fluids, calcification is usually avoided because of the presence of this calcification (bone formation) inhibitory factor, but when this calcification inhibitor is physically and chemically removed, there is also a calcification inhibitor. If a substance that can serve as a substrate is present, calcification will occur there. Calcification during bone formation in adults is also thought to be based on this principle.
体液中に存在する石灰化抑制物質は分子ｊ１１万以上と
考えられ１本発明に於いては、この物質を体液中から実
質的に除去あるいはその物質の拡散を阻止するものであ
る。除去方法としては、濾過等があり、拡散阻止として
は、ゲル構造体での浸透を抑制するものがある。また、
濾過等の場合は体液中のカルシウム成分がふるい分けら
れずに通過するものでなければならない。また、構造体
においては、少なくとも石灰化抑制物質の拡散に比して
構造体での拡散が大きくなければならない。The calcification-inhibiting substance present in body fluids is thought to have a molecular weight of 110,000 or more, and the present invention aims to substantially remove this substance from body fluids or prevent its diffusion. Examples of removal methods include filtration, and diffusion prevention methods include suppressing penetration through a gel structure. Also,
In the case of filtration, etc., calcium components in body fluids must be able to pass through without being sieved. Furthermore, in the structure, the diffusion in the structure must be at least greater than the diffusion of the calcification-inhibiting substance.
また、カルシウム成分の石灰化をもたらす基質は、カル
シウムが沈着し得るものであれば良いが、特に好ましく
は透明体である。基質が透明体であれば、その後のカル
シウムの沈着の程度を光学的に測定しやすくなるからで
ある。Further, the substrate that causes calcification of the calcium component may be any substrate on which calcium can be deposited, and is particularly preferably a transparent substrate. This is because if the substrate is transparent, it becomes easier to optically measure the degree of subsequent calcium deposition.
また、本発明においては、石灰化抑制物質の拡散を阻止
する構造体と、カルシウム成分が沈着し得る基質とが同
−戒は併用可能であることが望ましい、前記構造体と基
質とが併用された同一物であると、体液中の石灰化抑制
物質が構造体に拡散が阻止され、被測定液中でのその濃
度が低下すると同時に、カルシウム成分が構造体（基質
）中に沈着をはじめ、その沈着量にはあまり左右されな
いことから、Ｉり定上ｒＪＪｇが生じないばかりか、測
定器具として簡単な構造を提供し得る。またこの場合に
も、構造体が一定の透明性を有していれば光学的な測定
も容易となることが理解される。Further, in the present invention, it is preferable that the structure that prevents the diffusion of calcification-inhibiting substances and the substrate on which calcium components can be deposited can be used together. If they are the same substance, the calcification-inhibiting substance in the body fluid will be prevented from diffusing into the structure, and its concentration in the fluid to be measured will decrease, and at the same time calcium components will begin to deposit in the structure (matrix). Since it does not depend much on the amount of deposition, not only does the I-representation rJJg not occur, but also a simple structure can be provided as a measuring instrument. Also in this case, it is understood that optical measurement becomes easy if the structure has a certain degree of transparency.
（実ＩＭ、態様）
以下、本発明に係る体液中のカルシウム成分の測定方法
及びその器具の好ましい実施態様を説明する。(Actual IM, Aspects) Hereinafter, preferred embodiments of the method for measuring calcium components in body fluids and the device according to the present invention will be described.
本発明に適用される体液としては、血液、リンパ液１組
織液、及び尿等を含むものであるが１本発明においては
特に血清等に使用される。体液は測定においてそれ自体
希釈あるいは濃縮して使用することができるが、通常そ
のまま使用することが操作上望ましい。Body fluids applicable to the present invention include blood, lymph fluid, tissue fluid, urine, etc. In the present invention, serum etc. are particularly used. Although body fluids can be used after being diluted or concentrated in the measurement, it is usually desirable for operation to use them as they are.
と の ゛ か゛の たｊ
石灰化抑制物質とは、実質的に骨形成化抑制タンパク質
系高分子であり、ここでタンパク質系高分子とは、分子
量が約１万以上のものである。また、タンパク質系高分
子とは、その分子内にＷＷＲ及び脂質を一部に結合させ
ているものであってもよい。このような高分子タンパク
はカルシウムと何等かの結合様式を取り得ると考えられ
る。The calcification-inhibiting substance is essentially a protein-based polymer that inhibits bone formation, and the protein-based polymer herein is one having a molecular weight of about 10,000 or more. Furthermore, the protein-based polymer may be one in which WWR and lipid are partially bound within the molecule. It is thought that such high-molecular proteins can bind to calcium in some manner.
本発明において、石灰化抑制物質を除去するには、約１
万の分子量の大きさの通過を阻止し得る渡過膜、透析膜
あるいは、経時的に高分子の流れを遅延し得るゲル濾過
等によって分離することができる。また、本発明に使用
される拡散阻止ａ運休としてはゲル等があり、例えば、
ポリアクリルアミドゲル、寒天ゲル等の合成ゲル或は天
然ゲルが挙げられる。特に１本発明においては、ポリア
クリルアミドゲルが望ましい。In the present invention, in order to remove the calcification inhibitor, approximately 1
The separation can be carried out by a transfer membrane, a dialysis membrane, which can prevent the passage of molecules with a molecular weight of 10,000,000, or by gel filtration, which can retard the flow of macromolecules over time. In addition, the diffusion prevention material used in the present invention includes gels, etc., for example,
Examples include synthetic gels such as polyacrylamide gel and agar gel, and natural gels. Particularly in the present invention, polyacrylamide gel is preferred.
ポリアクリルアミドゲルは１通常のアクリルアミドモノ
マーに所定量の架橋性アクリルアミドモノマーを添加す
ることによって、即ち公知の方法によってゲル中のボア
の大きさを調整でき、本発明に必要とされる分子量約１
万程度の拡散阻止を簡単に達成することができる１本発
明に使用されるアクリルアミドモノマーとしては、アク
リルアミド、ジメチルアクリルアミド、エチルアクリル
アミド等があり、架橋性モノマーとしてはＮ、Ｎ’ −
メチレンビスアクリルアミド、Ｎ、Ｎ’ −エチレンビ
スアクリルアミド等が挙げられ１本発明においては特に
アクリルアミド及びＮ、Ｎ’ −メチレンビスアクリル
アミドが使用上望ましい。The size of the pores in the polyacrylamide gel can be adjusted by adding a predetermined amount of crosslinkable acrylamide monomer to a normal acrylamide monomer, that is, by a known method, and the molecular weight required for the present invention is about 1.
Acrylamide monomers used in the present invention include acrylamide, dimethylacrylamide, ethylacrylamide, etc., and crosslinking monomers include N, N'-
Examples include methylenebisacrylamide, N,N'-ethylenebisacrylamide, etc.Acrylamide and N,N'-methylenebisacrylamide are particularly preferred in the present invention.
ポリアクリルアミドゲルの製造においては、それ自体公
知の方法によって作成されるが、本発明においては、ア
クリルアミドの元溶液に対する濃度は２０乃至６０％の
範囲が望ましく、また、架橋性アクリルアミドモノマー
は０．５乃至１．７％の範囲で含有させるのが望ましい
。Polyacrylamide gel is produced by a method known per se, but in the present invention, the concentration of acrylamide relative to the original solution is preferably in the range of 20 to 60%, and the crosslinkable acrylamide monomer is 0.5%. The content is preferably in the range of 1.7% to 1.7%.
゛ カルシ ム の
本発明に用いられる石灰化沈着用基質としては、その基
質内にカルシウム成分が存在すると、そのカルシウム成
分を白濁、即ち沈着させ得るものである。このような機
能を有するものとしては、前述のポリアクリルアミドゲ
ル構造体が代表として挙げられるが、通常このような基
質と成り得る構造体には、少なくとも高分子ゲル内にカ
ルボキシル基を有していることが望ましく、特にアミド
基の存在が望ましい。The matrix for calcification of calcium used in the present invention is one that, if a calcium component is present in the matrix, can cause the calcium component to become cloudy, that is, to be deposited. The above-mentioned polyacrylamide gel structure is a representative example of a structure having such a function, but structures that can be used as a substrate usually have at least carboxyl groups in the polymer gel. The presence of an amide group is particularly desirable.
また５本発明においては、基質（構造体）上でカルシウ
ム成分の白濁が確認できる程度の透明感を有している基
質であれば問題なく肉眼で測定使用できる。しかし、本
発明において光学的にその評価をすることが望ましい。Furthermore, in the present invention, the substrate (structure) can be used for measurement with the naked eye without any problem as long as the substrate is transparent to the extent that the cloudiness of the calcium component can be confirmed on the substrate. However, in the present invention, it is desirable to perform the evaluation optically.
この場合、構造体の光透過率（厚み１０賦）は少なくと
も２０％以上、より好ましく５０％以上であることが望
ましい。In this case, it is desirable that the light transmittance (thickness: 10 mm) of the structure is at least 20% or more, more preferably 50% or more.
このような透過率を有するものとしては前述したポリア
クリルアミドゲル等があげられる。Examples of materials having such a transmittance include the aforementioned polyacrylamide gel.
本発明においてはまた、前記石灰化抑制物質の拡散防止
用構造体と石灰化沈着させる基質とを同一のもの、即ち
両方の機能を備えた構造体とすることが望ましく重要で
ある。このような構造体として代表されるものとしてポ
リアクリルアミドゲルなどが挙げられる。In the present invention, it is also desirable and important that the structure for preventing diffusion of the calcification-inhibiting substance and the substrate for calcification are the same, that is, a structure that has both functions. A typical example of such a structure is polyacrylamide gel.
前記のように構成した本発明においては、前述した構造
体は、ガラスプレート上、試験管、或は９６穴培養用プ
レート等に充填され、その上に被測定液である体液１例
えば血清等が所定量加えられる。この体液を加えた状態
で一定時間置くと、石灰化抑制物質はその構造体での拡
散が防止される一方、体液中のカルシウム成分は構造体
内に移行すると同時に、石灰化抑制物質の虹皆を受けな
くなる。このため構造体内にはカルシウム成分の石灰化
沈着による白濁が生じる。したがって、この白濁状態を
肉眼的に或は光学的に測定することによって、体液中の
カルシウム成分量を求めることができ、しかも骨形成能
を知ることができる。In the present invention configured as described above, the above-described structure is filled in a glass plate, a test tube, a 96-well culture plate, etc., and a body fluid 1, such as serum, as a liquid to be measured is placed on it. A predetermined amount is added. When this body fluid is added and left for a certain period of time, the calcification-inhibiting substance is prevented from diffusing in the structure, while the calcium component in the body fluid is transferred into the structure, and at the same time, the calcification-inhibiting substance is released. I won't receive it. For this reason, cloudiness occurs within the structure due to calcification of calcium components. Therefore, by visually or optically measuring this cloudy state, it is possible to determine the amount of calcium components in the body fluid, and also to know the bone forming ability.
このため、骨粗鬆症の診断も容易となる。Therefore, the diagnosis of osteoporosis becomes easy.
なお、体液を加えた後の反応条件は、温度が２０℃乃至
４０℃、特に好ましくは３０’Ｃ乃至３９℃の範囲にあ
ることが望ましく、更に、１乃至１０％の００２、特に
１乃至８％の存在下で反応を行うことも重要である。炭
酸ガスが存在すると１反応初期において容易に炭酸カル
シウムとして沈澱を生じるからであると解せられる。Note that the reaction conditions after adding the body fluid are such that the temperature is in the range of 20°C to 40°C, particularly preferably 30'C to 39°C, and furthermore, 1 to 10% of 002, especially 1 to 8%. It is also important to carry out the reaction in the presence of %. This is believed to be because the presence of carbon dioxide gas easily precipitates calcium carbonate at the initial stage of one reaction.
以下、具体的な実施例を示す。Specific examples will be shown below.
（実施例）
１．１ＸＮ、Ｎ’−メチレンビスアクリルアミドを含む
４０πアクリルアミド水溶液を調整し、次にこの溶液１
０１１取り、２％Ｎ、Ｎ、Ｎ−テトラメチレンジアミン
を０゜ｌｌ１１．ｉび１０％過硫酸アンモニウムを０．
１ｍｌ加えて攪はんし、すぐにこの溶液を９６ｗｅｌｌ
培養用ｐｌａｔｅの１ｗｅｌｌ当り３０ｕ１ずつ加えた
。前記アクリルアミド溶液を加えたプレートなＮ２ガス
中で２ｈｒ放置し、該溶液をゲル化させた。このゲル上
に、異なった年齢（１１オ一８０才）の骨粗鬆症及び他
の疾病患者及び正常人、計３５人より得た血清及び牛胎
児血清を３００ｕｌずつ加え、５％ＣＯ２のインキュベ
ーター内で７日間３７℃に加温し、アクリルアミドゲル
内に起こる石灰沈着量の違いを調べた。 石灰沈着量
は肉眼的にもゲル内の白濁の程度を見ることで判断でき
るが、さらに正確な値を調べるために、ゲル面を蒸留水
洗浄を行ったのち、０．０ＩＮ ＠ｌ！１１００ｕｌを
加えカルシウムを抽出したのち、このカルシウム量を、
Ｂ、Ｃ，Ｒａｙｓａｒｋａｒ （ライサカー）とＵ、Ｐ
、Ｓ。(Example) 1. A 40π acrylamide aqueous solution containing 1XN,N'-methylenebisacrylamide was prepared, and then this solution 1
011, and added 2% N, N, N-tetramethylene diamine to 0°ll11. i and 10% ammonium persulfate.
Add 1ml, stir, and immediately add this solution to 96 wells.
30ul was added per well of the culture plate. The acrylamide solution was added to the plate and left for 2 hours in N2 gas to gel the solution. On this gel, 300 ul of serum obtained from a total of 35 patients with osteoporosis and other diseases and normal people of different ages (11 to 80 years old) and fetal bovine serum were added, and the mixture was incubated in an incubator with 5% CO2 for 7 days. The gel was heated to 37°C for several days, and the difference in the amount of lime deposits occurring within the acrylamide gel was examined. The amount of lime deposit can be determined visually by looking at the degree of cloudiness within the gel, but in order to find out a more accurate value, the gel surface was washed with distilled water and then 0.0 IN @l! After adding 1100ul and extracting calcium, the amount of calcium was
B, C, Raysarkar and U, P
,S.
Ｃｈａｕｈａｎ （＾ｎａｌｙｔｉｃａｌ Ｂｉｏｃｈ
ｅｍｉｓｔｒｙ、２０，１５５−１６６．１９６７）の
へん法によりカルシウムを測定した。以下にその方法を
記す。Chauhan (^analytical Bioch)
Calcium was measured by the metric method of Emistry, 20, 155-166, 1967). The method is described below.
上記間ｐｌａｔｅのｗｅｌｌ当り１００ｕｌのアンモニ
アｖＩ街液ｐ１１１０．５を加え、さらに、 ２５ｕ
１の Ｑ−ｃｒｅｅｓｏｌ −ｐｈｔｈａｌｅｉｎを加
え、プレートリーダーを用い吸光度を測定した。コント
ロールとして同 ｐｌａｔｅに０．０．１２５．０．２
５．０．５．１．２５．２．５．５，１ｏ、２０ｕｌの
カルシウムを含む蒸留水３０ｕｌを加え同様の操作を行
った。そのコントロールの吸光度から各サンプルのカル
シウム量を算出した。測定はすべて各サンプル当り Ｚ
ｗｅｌｌずつ用いて行った。その結果を表１に示した。During the above period, add 100 ul of ammonia vI street solution p1110.5 per well of the plate, and further add 25 u
1 of Q-creesol-phthalin was added, and the absorbance was measured using a plate reader. 0.0.125.0.2 on the same plate as a control.
5.0.5.1.25.2.5.5, 1o, 30 ul of distilled water containing 20 ul of calcium was added and the same operation was performed. The amount of calcium in each sample was calculated from the absorbance of the control. All measurements are for each sample Z
Each well was used. The results are shown in Table 1.
表１
表１に示すように、全般的にみて血清の石灰沈着能は血
液提供者の年齢に強く依存し１年齢の低い人はど血液の
石灰沈着能が高く、加齢に伴ってその能力が減ってくる
ことがわかった。また、骨ｆｌＩ髭症の患者の血清の石
灰沈着能は同年齢の正常人血清を比べて有意に低値を示
しており（提供名番号２３．３２．３３）骨粗髭症を含
む骨代謝異常の血液検査によるお断に１本発明が有用で
あることが示された。Table 1 As shown in Table 1, overall, the calcification ability of serum strongly depends on the age of the blood donor, with younger people having a higher calcification ability, and increasing age. was found to be decreasing. In addition, the calcification ability of the serum of patients with osteoporosis is significantly lower than that of normal human serum of the same age (provided name number 23.32.33). It has been shown that the present invention is useful for identifying abnormalities through blood tests.
（発明の効果）
以上、説明したように本発明によれば、体液中のカルシ
ウム成分を、体液中の石灰化抑制物質の影響がない状態
で、その石灰化沈着によりｉｌｌ、１定したので、体液
中の骨形成能を正確に測定することのできる。また、本
発明の体液中のカルシウムの測定方法及びその測定器具
は、骨ｍｍ症等と充分に相関をとることができ、骨の発
育障害の診断に適用することができる。(Effects of the Invention) As explained above, according to the present invention, the calcium component in the body fluid is fixed by its calcification without the influence of the calcification inhibitor in the body fluid. The bone forming ability in body fluids can be accurately measured. Furthermore, the method for measuring calcium in body fluids and the measuring device thereof of the present invention can be sufficiently correlated with osteoporosis, etc., and can be applied to the diagnosis of bone growth disorders.
Claims (7)
を阻止した後、体液中に含まれるカルシウム成分を石灰
化沈着して測定することを特徴とする体液中のカルシウ
ムの測定方法。(1) A method for measuring calcium in a body fluid, which comprises removing or preventing diffusion of a calcification-inhibiting substance contained in the body fluid, and then calcifying and measuring the calcium component contained in the body fluid.
シウム成分が沈着し得る基質を用い、その基質に生じる
石灰化沈着物を光学的に測定することを特徴とする請求
項第１項記載のカルシウム測定方法。(2) Calcium measurement according to claim 1, characterized in that, in the calcification of the calcium component, a substrate on which the calcium component can be deposited is used, and the calcified deposit formed on the substrate is optically measured. Method.
８％の範囲で反応させて測定することを特徴とする請求
項第１項記載のカルシウム測定方法。(3) The method for measuring calcium according to claim 1, wherein the measurement is performed by reacting the calcified deposit at a CO_2 concentration in the range of 1 to 8%.
沈着の抑制をするタンパク質系高分子の拡散が前記カル
シウム成分より小さい構造体基質であって、かつ前記抑
制物が減少した状態のときにカルシウム成分を石灰化沈
着する構造体基質を有することを特徴とした体液中のカ
ルシウム測定器具。(4) A structure substrate through which calcium ion components can permeate and the diffusion of a protein-based polymer that suppresses calcification is smaller than the calcium component, and when the inhibitor is reduced, calcium A device for measuring calcium in body fluids, characterized by having a structured matrix that calcifies components.
０％以上の透明体であることを特徴とする請求項第４項
記載の測定器具。(5) The structure substrate has a light transmittance (thickness of 10 mm) of 2
5. The measuring instrument according to claim 4, wherein the measuring instrument is 0% or more transparent.
に充填してなることを特徴とする請求項第５項記載の測
定器具。(6) The measuring instrument according to claim 5, wherein the structure substrate is formed by filling a plate with a plurality of filling holes.
ことを特徴とする請求項第４項乃至６項記載の測定器具
。(7) The measuring instrument according to any one of claims 4 to 6, wherein the structure substrate is a polyacrylamide gel.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP31375990A JPH04184256A (en) | 1990-11-19 | 1990-11-19 | Method and device for measuring calcium in body fluid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP31375990A JPH04184256A (en) | 1990-11-19 | 1990-11-19 | Method and device for measuring calcium in body fluid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04184256A true JPH04184256A (en) | 1992-07-01 |
Family
ID=18045193
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP31375990A Pending JPH04184256A (en) | 1990-11-19 | 1990-11-19 | Method and device for measuring calcium in body fluid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04184256A (en) |
-
1990
- 1990-11-19 JP JP31375990A patent/JPH04184256A/en active Pending
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