DE1673015B1 - METHOD FOR RADIOCHEMICAL DETERMINATION OF VITAMIN B LOW 12 - Google Patents

Description

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covalent bonds are held together. Such preferably a vitamin B ₁₂ solution with known

Particles, even if they swell in water, are concentration as a standard solution,

are able to be completely insoluble in this, so that the radioactivity can be

the polymer material or substance to be voted by, e.g. B. by counting the scintillations. covalent forces bound to this material, 5 The amount of particles to which the bound

during the treatment stages, e.g. B. is bound during the vitamin B ₁₂ capable substance,

Washing, nothing is lost. Examples of such will depend, inter alia, on the for the

Polymer particles are polymer grains, the sensitivity required by trial selected,

wetting of substances with a large number of The amount of added labeled vitamin B ₁₂

Hydroxyl groups such as carbohydrates and sugars are selected, for example, so that about 40 to

alcohol, ζ. B. dextran, starch, dextrins and 60 ° / ₀ labeled vitamin B ₁₂ to the binding of

other polysaccharides as well as polyvinyl alcohol are bound with substance capable of _{vitamin B 12,}

a bifunctional compound can be obtained. if at the same time no unlabelled vitamin B ₁₂

Examples of bifunctional compounds are those that are present. Incubation can take place at different

with the general formula X - R - Z, in which 15 temperatures occur. It is preferable to lead them

X and Z e.g. B. halogen atoms or epoxy groups at temperatures between +4 and 25 ° C,

and R is the residue of a bifunctional compound. It is not necessary that the reaction between

means e.g. B. an aliphatic residue with 3 to the vitamin B ₁₂ and the substance that the vitamin B ₁₂

10 carbon atoms. able to bind until the end is carried out.

You can z. B. use grains of a product, 20 Incubation may e.g. B. after 2 hours under the Contains dextran, which is broken by glycerine-ether bridges. The interruption can be given networked is and by treatment of dextran with if also later, z. B. made after 24 hours Epichlorohydrin is obtained. Such products are. It is important that the response time and they swell in water, but they also represent temperature for the sample solutions and standard insoluble gel grains. They contain hydroxyl solutions.

groups that can easily be replaced by other groups, e.g. B. Since the method according to the invention can be replaced easily and quickly, amino groups or carboxyl groups, can be carried out in practice and can provide precise analyzes and is good for the formation of covalent results, it is also suitable for routine sub-bonds to substances which, in turn, are suitable for quantitative determinations . Ability to bind vitamin B _{12, e.g.} B. Intrinsic factor. 30 With the method according to the invention

Further examples are reactive derivatives, also examine very small sample amounts,

which one by treatment of a copolymer from With the method according to the invention is free

Dextran and epichlorohydrin with cyanide halides, vitamin B ₁₂ determined. In the serum z. B. is the vitamin

z. B. cyanogen bromide is obtained. These derivatives attach to B ₁₂ but are bound to a protein. For determination

easily deal with intrinsic factor. The vitamin B _{12 must} therefore be released, e.g. B.

The use of small particles is preferred, by heating with hydrochloric acid,

because this has a larger contact area available excess protein in the serum, which is necessary for binding

place. Capable of vitamin B ₁₂ , can with the help of the

The substance with the ability to bind by the method according to the invention can also be determined

Vitamin B ₁₂ is made by covalent bonds to the 40 den, in such a way that particles are attached to

Bound carrier particles, under conditions that enabled _{one to bind vitamin B 12}

which are so mild that their reactivity is not bound to the substance and appropriate amounts of

is significantly reduced. Due to the co-labeled vitamin B ₁₂ to the untreated serum

valent bonds, the substance can with the ability and, after the separation of the particles, the radio-

ability to bind vitamin B _{12 is} not lost 45 measures activity.

and not be washed out of the particles. As mentioned, the invention also relates to a

chemical binding of this substance to the polymer reagent for the determination of vitamin B ₁₂ according to

particles are used in the polymer contained in the process described, the particles of an in

contains reactive groups such as amino groups, hy- water-insoluble polymers to which

hydroxyl groups and carboxyl groups, whereby a 50 by covalent forces is used to bind

Bridge of covalent bonds between the _{substance capable of vitamin B 12} is bound, in

Binding of vitamin B ₁₂ capable substance (P) dried, z. B. lyophilized form,

and the polymer particle, e.g. B. disfigured as follows: The reagent can be in a closed ampoule

be included.

_, .. _TTT ". _TTT _,.,, 55 Such an ampoule can be used in a test pack

P - NH · CS · NH - polymer particles ^ _{Determination of vitamin Bu ent} h. _{to be} old. These

P - NH - CO · NH - polymer particles packing consists of one or more closed

ρ _ _N _ _N _ polymer particles ampoules in which in dried, e.g. B. lyophilized

Form particles from a water-insoluble 60 polymer contained, attached by covalent

It is not necessary that the nature of the bond linkages one for binding of vitamin B ₁₂ is capable bound between the groups capable of binding of vitamin B ₁₂ substance, as well as of one or more substance, for. B. Intrinsic factor and the polymer ampoules with vitamin B ₁₂ , which is determined more precisely with a radioisotope. There are many variations - marked and also in dried, z. B. possibilities. It is only necessary that the form is lyophilized through the 65.

Binding the substance that _{bind the vitamin B 12} The following examples showing the determination

capable of not being washed out. of vitamin B ₁₂ in blood serum

The invention is used to carry out the analysis.

example 1

Determination of vitamin B ₁₂ in blood serum

A. Production of particles to which a substance capable of _{binding vitamin B 12 is bound by covalent forces}

Finely ground particles made of a dextran crosslinked with glycerine ether bridges, which is formed by p-nitrophenoxy-hydroxypropyl ether groups is substituted (degree of substitution 200 μΜοΙ nitro groups per gram Dry matter), serve as the starting material. 10 g of this product were mixed with 50 ml of water in a Given two-necked flask. The temperature of the mixture was then brought to 35 ° C. The mixture was stirred with 25 ml of a 5 normal aqueous sodium hydroxide solution and then 6 g of sodium dithionite were added so that the nitro groups were reduced to amino groups. To Another 5 grams of sodium dithionite was added about 30 minutes. The reduction was after about one Hour, after which it was neutralized with dilute hydrochloric acid. The solid substance was filtered off and washed with distilled water on a suction filter.

10 g of the p-aminophenoxy-hydroxypropyl-substituted product obtained in this way were placed in a flask with 100 ml of a 10% strength solution of thiophosgene in carbon tetrachloride. The flask was stopped with a stopper and the mixture was stirred or agitated for about 2 hours. The mixture was then cooled in an ice bath. The flask was then opened and the contents filtered. The residue on the filter was washed with a 0.1 M aqueous sodium bicarbonate solution, distilled water and acetone and then dried in a drying oven at 60 to 8O ⁰ C.

2 g of the product obtained, substituted with p-isothiocyanate-phenoxyhydroxypropyl groups, were allowed to swell in 6 ml of a 0.1 M aqueous sodium bicarbonate solution. After the stirrer was started, 4 ml of the same sodium bicarbonate solution containing 95 mg of intrinsic factor was added dropwise. The mixture was ^{stirred at 20 °} C. for 24 hours and then filtered. The residue on the filter was washed with a 0.5M sodium bicarbonate aqueous solution to remove unreacted matter. The product can then be carefully dried, e.g. B. by lyophilization.

B. Determination

The analyzes are best carried out in glass or plastic test tubes measuring 50 x 10 mm. As the diluent was a 0.05M Tris buffer pH 7.4 containing 0.9% sodium chloride, 0.1% porcine serum albumin and 0.01 ° / ₀ sodium azide was used. Prior to analysis, the vitamin B was ₁₂ separated in the serum from the serum protein by heating the serum with hydrochloric acid as follows: 0.5 ml serum + 0.5 ml of 0.9 ° / ₀ sodium chloride solution were mixed with 2 micrograms of sodium per milliliter and 1 ml of 0.1 N hydrochloric acid are added. This mixture was placed in a boiling water bath for 20 minutes and then cooled under cold running water.

The determination was then carried out as follows:

1. In each of two test tubes, 0.25 ml of the serum solution treated as above was added filled.

2. In each of two test tubes, 0.25 ml of standard solutions with different concentrations of vitamin B ₁₂ , e.g. B. 1000, 400, 100, 40, 10 and 0 pg / ml, which were diluted with the buffer solution described above, filled with the simultaneous addition of 2 micrograms of sodium chloride per milliliter.

3. 0.1 ml of a solution which per milliliter contained INanograms of vitamin B ₁₂ , marked with 58 Co and had been diluted with the buffer solution with the addition of 2 micrograms per milliliter, was added to all test tubes.

4. 1 ml of a homogenized suspension of the polymer particles (1 mg per milliliter) to the Intrinsic factor bound by covalent forces was added to all test tubes.

5. The test tubes were incubated for 3 hours at room temperature or 4 ° C and in the process in slow rotation.

6. The particles were centrifuged at 3000 rpm for 5 minutes.

7. The particles were washed twice with a 0.9% strength sodium chloride solution. After the last suction of the supernatant liquid, the test tubes were placed in counting tubes in order to determine _{the radiation of the bound, labeled vitamin B 12.}

8. The number of counts in the unit of time that were determined with the standard test tubes was plotted in a "count-dose diagram" with a lin-log scale, from which the amount of vitamin B ₁₂ in the unknown samples will later be calculated can.

It is also possible, following the centrifugation mentioned under 6, to transfer 1 ml of the supernatant liquid into the counter tubes so that the radiation of the free labeled vitamin B ₁₂ can be determined. "Counts" from the standard tubes can be transferred in the same way to a diagram with a lin-log scale, so that the amount of vitamin B ₁₂ in the unknown test samples can be determined graphically in the manner mentioned.

Example 2
Determination of vitamin B ₁₂ in an aqueous sample

A. Preparation of the particles to which a substance capable of _{binding - 5} of vitamin B _{12 is bound}

g of a copolymer obtained by reacting dextran with epichlorohydrin were allowed to swell for 3 minutes with stirring in 200 ml of a cyanobromide solution which contained 10 g of this compound in 100 ml of water each time. An aqueous 5M sodium hydroxide solution was then added with stirring until the pH was 10.7. This value was held constant for minutes. The temperature was kept at 20 ^° C. during the entire process.

The mixture was then placed on a glass filter and carefully washed neutral with water. The separated particles were shrunk by washing with acetone. Then the

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Particles carefully dried; they could e.g. B. in the case of filtration residue, an aqueous 0.5m

-20 ° C. Sodium bicarbonate solution, so as not to

Remove 2 g of the cyanogen bromide activated set substance obtained in this way. Subsequently was

Particles were allowed to dry carefully in 6ml of an aqueous 0.1m- the product, e.g. by lyophilization

Swell sodium bicarbonate solution. Philize after 5.

When the stirrer was set, 4 ml of the g determination were added dropwise
same sodium bicarbonate solution in which

100 mg intrinsic factor were included. The mixture The determination was carried out in the example 1, B,

was stirred for 24 hours and then filtered. Performed the specified way.

Claims (5)

1 2 sample liquid are separated and the radio claims: activity of the particles is determined. The phrase "one for binding vitamin B12 1. Method for radiochemical determination capable substance «here means a substance of vitamin B ₁₂ in an aqueous sample according to the 5 contains the proteins or polypeptides or preferably known isotope dilution method by carbohydrates, which the specific ability to mix the sample with a certain to bind Have vitamin B ₁₂ . A known amount of an example of such a substance labeled with a radioisotope is intrinsic factor, vitamin B ₁₂ , isolation of a mixture of a mucoprotein from the gastric mucosa, and also labeled and unlabelled vitamin B ₁₂ from a protein fraction from blood plasma which corresponds to that of the sample and determining the radioactivity ability has to bind vitamin B _12, characterized in that with the binding of the VitaminsB ₁₂ to these substances the labeled vitamin B ₁₂ mixed sub encounters, regardless of whether the vitamin B ₁₂ suchungsprobe with particles of a insoluble in water is marked with a radioisotope or not. The loan polymers to which one is bound to the binding 15 binding of labeled or unlabelled substance capable of vitamin B ₁₂ through co vitamin B ₁₂ is carried out depending on the valence forces is reacted by the labeled concentration, and the unlabeled the implementation of the particles from the sample liquid vitamin B ₁₂ . ability to be separated and the radioactivity The main advantage of the invention the particle is determined. 20 isolation method consists in that the 2. The method according to claim 1, _{characterized in that the substance capable of vitamin B 12} is not indicated in that the radioactivity of the carrier material is present in place of or in addition to the radio solution, but rather the radioactivity of the carrier material is very firmly bound to an insoluble activity of the particles. Hence the marked liquid can be determined. Vitamin B ₁₂ , which is used to bind vitamin B ₁₂ 3. The method according to claim 1, characterized marked 35 capable substance was bound, easily distinguished from the fact that for the determination of a vitamin B ₁₂ unbound labeled vitamin B _{12 is used} separated, which are radioactive with a, namely z. B. is marked cobalt isotope simply by centrifugation. or filtering. The determination according to the invention 4. The method as claimed in claim 1, characterized in that the method is so easy to carry out because it is shown that the _{amounts of particles determined for binding vitamin B 12} 30, to which the substance capable of binding to the polymer particles binding of vitamin B ₁₂ capable substance is bound by covalent forces, is intrinsic bound, e.g. B. put in test tubes and factor is. this can store without affecting the ability to bind. 5. reagent for the determination of vitamin B _{12 of} the introduced substance is lost, whereupon the entire method according to the method according to claims 1 35, including the separation of the up to 4, characterized in that it is free of particles labeled vitamin B ₁₂ and the bound one Contains water-insoluble polymers, to labeled vitamin B ₁₂ in one and the same Rew which can be carried out by covalent forces for binding a tube, without a _{substance capable of vitamin B 12} is bound, additional precipitants and the like are necessary. in dried, e.g. B. lyophilized form. 40 The inventive method can be used both for qualitative as well as quantitative determination can be used. According to one embodiment of the invention can instead of or in addition to the radioactivity of the particles 45, the radioactivity of the liquid is also determined The invention relates to a method for being radio. chemical determination of vitamin B ₁₂ in a The invention also relates to a reagent for aqueous sample after the isotope dilution implementation of the method described, method by mixing the sample with a The labeling of vitamin B ₁₂ can be in the usual _{certain amount of a vitamin B 12} labeled with a radioisotope 50 way made with a suitable isotope, isolation of a mixture, in particular with a radioactive isotope of labeled and unlabelled vitamin B ₁₂ of cobalt. With a radioactive cobalt isotope from the sample and determining the radioactivity. labeled vitamin B ₁₂ is commercially available, and Such methods are already known and z. B. most hospital laboratories now have them by F. Hecht and M. K. Z a ehe rl, Handbuch 55 on the necessary equipment for measurement der Mikrochemischen Methode, Volume 2, Vienna, 1955, this isotope. S. 142, 143, 155, and in "The Analyst", Vol. 85, 1960, As a carrier for the binding of vitamin B ₁₂ Pp. 393 and 394. In the case of the known capable substances, particles are made from water processes the mixture is isolated after insoluble polymers are used. The polymer will usual methods. These are cumbersome and with 60 selected so that there are suitable reactive ones Difficulties associated. Groups, e.g. B. amino groups, hydroxyl groups The invention provides a simplified insulation. or contains carboxyl groups or ver-It is characterized in that the can be seen with the, so that the substance capable of _{binding the labeled vitamin B 12} mixed investigation vitamin B _{12, z.} B. Intrinsic factor, sample with particles of a water-insoluble polymer, 65 bound by covalent forces to the polymer to which one can be able to bind vitamin B ₁₂ . Substance is bound by covalent forces in order to- It is particularly favorable if the polymer particles is set, after the implementation of the particles from which have a three-dimensional network structure, which by