DE2737286C2 - Multi-layer analytical elements for the determination of triglycerides and glycerine in liquids - Google Patents

Description

in such a distribution that contains the liquid sample to be analyzed on the spreading layer applied triglyceride is hydrolyzed to glycerol, that the glycerol obtained in L-a-glycerophosphate is transferred and that the L-α-glycerophosphate with simultaneous reduction of the Eleklrancnakzeptors is oxidized to produce a measurable change.

2. Analytical element according to claim 1, characterized in that the Elcktronenakzcploraus 2,6-dichlorophenol indole phenol or 2- (p-indophenyl) -3- (p-nitrophenyl) -5-phenyI-2H-tetrazolium chloride.

3. Analytical element according to claim 1, characterized in that the electron acceptor is oxygen and that there is also an indicator system consisting of a substance with peroxidative activity and a Dye precursor compound, which in the presence of hydrogen peroxide and the substance with pcroxidaliver Activity brings about a measurable change.

4. Analytical element according to one of claims 1 to 3, characterized in that the σ-glyccrophosphate oxidase originates from Streptococcus faecalis.

5. Multi-layer analytical element for the determination of glycerine in liquids, with at least a spreading layer for spreading a sample of a liquid to be analyzed and a Reagent layer, characterized in that the element

(a) glycene kinase,

(b) adenosine triphosphate,

(c) ar-Glyrerophospf ^ t-Oxitiise and

(d) a hydrogen peroxide indicator

in such a distribution that the glycerol applied with the liquid sample to be analyzed is converted into L-fl-glycerophosphate and that the L-ar-glycerophosphate obtained in the presence of Oxygen is oxidized to peroxide, which in turn with the indicator causing a measurable Change reacts.

The invention relates to multilayer, analytical elements for the determination of triglycerides and glycerol in liquids with at least one spreading layer for spreading a sample of one to be analyzed Liquid and a reagent layer.

In the usual known methods of determining serum triglycerides, hydrolysis of the triglycerides takes place with the release of glycerine and reaction of the glycerine with certain Rcagcn / icn under F.r / cugung a compound which can be quantified by spectrophotometric means. Generally will a base is used to hydrolyze the triglycerides. On the US-PS 37 03 591 and 37 59 793 and the GB-PS However, in 14 41 642 it is also known to use enzymatic hydrolysis processes using a lipase alone (US-PS 37 59 793 and GB-PS 14 41 642) or in combination with a protease (US-PS 37 03 591) to be used. Further non-enzymatic hydrolysis processes are, for example, from DR-PS 22 29 849 and 23 609 known.

Three enzymatic methods can be used to determine glycerol, namely:

(a) Garland and Rändle method (P. B. Garland and P. J. Rändle, Nature, 196 [1962], pages 9X7-9XX).

Glycerol kinase
Glycerin + ATP ► La-glycerophosphate + ADP

;.; i pyruvate kinase

M ADP + phosphocnolpyruvate ► pyruvate + ATP

F "lactate

Pyruvate + NADH ► Lactate + NAD ⁺

y 65 dehydrogenase

f; ⁵ (b) method according to Weiland (ü. Weiland, Biochem. Z., [1957], 329, page 313).

§§ glycerol kinase

B (ilyccrin + ATP * L-tf-glyccrophosphate + ADP

ik: ir-glycerophosphate

y ^J L-ff-Glyccrophosphat + NAD ¹ * NADII + Dihydroxyacetonphosphat + H '

y dehydrogenase a

i ~ i (Ο Glycerin-D ^ hydrogcnase-method (JH Hagen and PB Hagen, Can. J. Biochem. and Physiology, 40 | l% 21, page 1129).

Glycenn

<i glycerine + NAD ⁴ * dihydroxyacetone + NADH + H "

7 * dehydrogenase '

Modifications of the method described under (a) are also known, for example, from DE-OS 26 65 556, GB-PS 13 22 462 and US-PS 37 59 793. In all cases the NADH formed or the disappearance of NADH is determined at 340 nm using a UV spectrophotometer. In method (a) three enzymes are used and in method (b) two. In the case of method (c), an enzyme is used. The last two methods mentioned are extremely sensitive to pH values and prone to failure, unless precise pH monitoring is carried out. In all three methods, especially in the case of method (a), not only the stability of the diagnostic enzyme but also the cofactor, NAD (II), is of great importance. The disadvantages of the enzymatic methods used are described in detail by HP Chen and SS El-Mequid in the journal Biochemical Medicine, 7, (1973), page 460.

From T. E. Barman, Enzyme Handbook, Vol. II, Springer Verlag, BerMn 1969, pp. 503-504; the US-PS 37 03 591

and Chemical Abstracts 75, 59705k (1971) it is also known that lipase is produced by surface-active compounds can be activated so that the enzyme only attacks emulsified trig | y_cerides and that the separation of the Lipids of lipid protein, for example by protease or by surface-active compounds, is possible.

_; Another method for determining triglycerides is known from DE-PS 21 39 103. Be this

ί- 'Process takes place a H vdrolvse derTrielvceride, an oxidation of the glycerol obtained to formaldehyde and Conversion of formaldehyde with ammonia and a stable, water- and alcohol-soluble, colorless.-0 Metal complex of acetylacetone to produce a colored compound.

From the journal Journal of Bacteriology, 98: 3 (1969), pages 1063 to 1068 and the journal Archives of Biochemistry and Biophysics, 88, (1960), pages 250 to 255, the production of ar-glycerophosphate Oxidasc, ie an enzyme which is able to mediate an electron transfer from σ-glycerophosphate to O ₂ with the simultaneous generation of I1; O _: and dihydroxyacetone phosphate is known.

There are still /. B. from DE-OS 22 13 721 and DE-PS 22 64 438 detection systems with oxidases are known in which the reaction is indicated either via an electron acceptor or indirectly with the aid of an indicator reaction in which the hydrogen peroxide formed _{with the electron acceptor O :.} reacts with peroxilasc and chromogens.

After all, it is well known, e.g. B. from DE-OS 2J 32 760, BE-PS 8 01 742 and US-PS -10

M) 92 158, for the qualitative and quantitative analysis of liquids, e.g. B. blood serum and urine to use multilayer analytical elements which advantageously have a porous spreading layer which is in contact with a reagent layer which contains at least one compound capable of reacting with a component or a decomposition product of a component of the liquid. The patent specifications do not contain any suggestion for a triglyceride-glycerol determination.

The object of the invention is to provide multilayer analytical elements for the determination of triglycerides and glycerine in liquids that enable a quick and reliable determination of the triglyceride and / or Glycerinchallcs of liquids without the use of specially trained Personnel is required.

The invention is based on the knowledge that the task at hand can be achieved by means of multilayer analytics shear elements and with the help of a multi-stage process taking place in these, including the (lets ilycerophosphate oxidase decomposition dissolve.

The invention thus relates to multilayer analytical elements as described in the claims Marked are.

Contains an analytical element according to the invention for the determination of triglycerides in liquids ■ iomit three different enzymes, namely a lipase, glycerol kinase and α-glycerophosphate oxidase as well Adenosine triphosphate and an electron acceptor. The reagent system enables the determination of triglycerides through the following process steps:

1. the lipase splits triglycerides into glycerol and fatty acids; m)

2. The glycerol kinase also catalyzes the reaction of the glycerol produced in the first process stage Acnosine iriphosphate to L-α-glycerophosphate;

.V die (/ - (ilyccrophosphal-Oxidasc catalyzes the reaction of the generated in the second process stage l.-ii-Glyccrophosphhalcs with the electron acceptor to a determinable compound.

The active compounds which bring about triglyceride hydrolysis and which cause glycerol concentration are preferably present allow in the elements in the following ways:

(a) All compounds are contained in the Reugen layer or

(b) the enzyme hydrolyzing the triguceride is present in the slurry, and the compounds that are mil (lycerin and its oxidation products are able to react, are in one or more disks Reagent layers in front of or

(C) the compounds or substances required for hydrolysis of the triglycerides are in a discrete manner Layer between spreading layer and reagent layer (or reagent layers) in front of and all others active compounds are contained in a reagent layer.

The reagent layer preferably contains an indicator or an indicator system, the h / w. the mil iiem κι hydrogen peroxide reugiert, which is he / eugt in the enzymatically catalyzed oxidation of glycerol, if Oxygen is used as electron acceptor. whereby a color change is effected in the element, which differs from that of the Triglyceride and / or glycerine content of the liquid wedge sample applied to the element depends.

According to a particularly advantageous embodiment of the invention, these are the triglyceride hydrolysiereiuli · Enzyme and optionally other compounds, such as. B. a surface active compound and / or l'rotease in a spreading layer over a reagent layer, which in turn contains the active compounds, which are required for the determination of glyccrin.

In Fig. I a erfjndungsgcmäßes multilayer analytical element is a schematic cross section dargeslcHl 7 / ( "- igt FirhrtiirvRn fi.ir Tynki-ho ■ rfindiingsHemüße elements.

In their layer arrangement, the multilayer analytical elements according to the invention can z. B. from US-PS 39 92 158 known analytical elements correspond.

In the case of elements according to the invention, triglycerides and / or (ihcerin iiiianlilaliv determined by the following reactions:

Lipase
1. Triglyceride + HOH »glycerin + fatty acids

Glycerol kinase
2. Glycerin + ATP> L-cr-glycerophosphate 4 ADP

cr-GP oxidase

- ¹ U 3. Lu - Glycerophüsphat + electron acceptor "Dihydroxyacetonphc.sphat + determinable compound

If oxygen is used as the electron acceptor, then in the reaction equation (3) ΙΙΛ), is generated as a determinable compound, in which case the H ₂ O ₂ determination can advantageously be carried out according to the following reaction scheme:

Peroxidase
4. H _: O _: + H _: A (red.) - ► A (ox) + H_, O (determinable product)

• i- where mean:

HA (red.) = Dye precursor compound which is the reduced form of the dye;
A (ox) = dye produced by the oxidation of Ii ₂ A.

A multilayer analytical element according to the invention has conventional, known methods essential advantages for the determination of triglycerides:

First of all, any electron acceptor can be used with α-glycerophosphate in the presence of Oxidase is able to react to produce a determinable change. As a result, the Measure the response at one of several wavelengths in the visible region of the spectrum, depending on the

5u tronen donor choice. Measurements in the visible range of the spectrum are subject to less interference,>. N than those performed at 340 nm.

Second, the stability of NAD * or NADH is no longer a problem, since O ₂ or another electron acceptor is the cofactor in the o-glycerophosphate oxidase reaction. Serum components which use NAD 'or NADH (e.g. lactate plus lactate hydrogenase), which can interfere with the known reaction sequences of the prior art, do not interfere with the reaction sequences taking place in the analytical elements according to the invention.

Finally, the enzymes used in the case of the elements according to the invention are within a wide range pH value range is active, which is why, in the case of the elements according to the invention, precise monitoring of the pI value is not required.

hO The lipase can be used alone or in combination with a hydrolysis stimulator, e.g. B. a protease or an effector from a surface-active compound can be used. Since lipase preparations, proteases, surface-active compounds and other reactive substances are readily available in lyophilized form, they can easily be incorporated into analytical elements of the type described here.
Particularly advantageous preparations for the hydrolysis of serum triglycerides in the elements according to the invention, in particular for the hydrolysis of protein-bound triglycerides, consist of a mixture of a lipase that is able to hydrolyze protein-associated triglycerides, such as those found in serum, for example, and a surface-active compound, and lipases which do not undergo hydrolysis in one can also be used when a surface-active compound is used at the same time

would cause an acceptably slow response rate.

Lipases of vegetable and animal origin can be used. Preferably willow for production inven- (limg.sgcmäUcr Elements used microbiological lipases, e.g. B. the lipase from Candida rugosa. Such a one Lipase has proven to be particularly advantageous when combined with a surface-active Connection is used. Advantageous lipases are also lipases from Chromobacterium viscosum, Vn'-anl paralipoytieum, raw or purified, also the lipase from Rhizopus arrhizus (Variant delemari. cleaned, as detailed, for example, by Fukumoto and coworkers in the magazine J. (Ion. Appli. Microbiol. 10, (1964), 257-265 and also lipase preparations with corresponding activities.

Further suitable lipases which can be used for the production of elements according to the invention, as well as Process / and their production are from US-PS 28 88 385, 31 68 448, 31 89 529, 32 62 863, 35 13 073, in 37 03 591 and 37 59 793 known. Commercially available lipases can also be used.

Since it has been shown that.! certain surface-active compounds reduce the hydrolysis activity of certain Ability to inhibit lipase preparations, it is necessary, before using a combination of one surface-active compound and a lipase to check whether the two components are compatible with each other are.

Surface-active compounds that are able to stimulate the hydrolysis activity of lipase preparations, are non-ionic and anionic surface-active compounds, for example natural surface-active compounds Compounds such as the bile salts including deoxycholate, chenodeoxycholate, choiates and raw bile salt mixtures as well as synthetic surface-active compounds, e.g. B. the sodium salts of Alkylarylpolyethersull'onates and also alkylphenoxypolyethoxyethanols. : n

Proteases can also advantageously be used as effective effectors for lipase preparations, typical suitable proteases are, for example, chymotrypsin, Streptomyces griseus protease (commercially available available under the name “Pronase”), proteases from Aspergillus oryzac and Bacillus subtilis. Elastase. Papain and bromelain. Mixtures of such enzymes can also be used.

The optimal concentration of lipase and effector in the lün / cll'alle depends on various factors for example the period of time available for carrying out the determination, the purity and the Activity of the enzyme preparation used and the nature of the triglycerides.

Furthermore, practically any glycerol kinase is suitable for the implementation of elements according to the invention. As special It has previously been shown to use the glycerol kinase from E. coü and Candida mycodermea. Other suitable glycerine kinasc enzymes are known, for example from the book by T. E. Barman. Enzymes hand- .H) book, I, Springer-Verlag, N.Y. (1969), pages 401-402.

As Elekironcnakzcptoren practically all electron acceptors in question, which an oxidation of the «/ - (ilycerophosphalcs in the presence of the oxidase enzyme with simultaneous induction of a measurable Enable change. Compounds have proven to be particularly advantageous electron acceptors which lead to the formation of a colored reaction product or a chemical intermediate compound which .15 I not colored itself, but due to a reaction or due to reactions that lead to color formation. ren, can be determined.

The usability of an electron acceptor can be determined by experiments with potentially suitable electrons determine.

A particularly advantageous electron acceptor is oxygen, which absorbs the L-α-glycerophosphate in the presence of the 4ü Oxidase oxidizes to dihydroxyacetone phosphate and hydrogen peroxide. Procedure for determining the presence and the concentration of hydrogen peroxide as a result of such reactions are known.

According to a particularly advantageous embodiment of the invention, a colored electron acceptor is used or non-colored material used that is subject to a color change or leads to a color generation, when it is reduced in the presence of the enzyme and the substrate. Suitable electron acceptors this Type are for example indole phenols, potassium ferricyanide and certain tetrazolium salts. As particularly beneficial For example, the use of 2,6-dichlorophenolindophenol alone or in combination has proven itself with phenyl in methosulfate and the use of 2- (p-indophenyl) -3- (p-nitrophenyl) -5-phenyl-2H-tetrazolium chloride, Proven either alone or in combination with phenazine methosulfate.

L-ff-glycerophosphate oxidase, a microbiological enzyme, can be of various origins. In so L-s-glycerophosphate oxidases from Streptococcaceae, Lactobacillaceae and Use Pcdiococcus. The enzyme from cultures of Streptococcus has proven to be a particularly advantageous enzyme Laccalis-proven cultures of this type are available, for example, from the American Type Culture Collection. Particularly advantageous enzymes have proven to be those derived from the strains ATCC 11 700, ATCC 19 634 and ATCC 12 755 are derived. As can be seen from the examples below, the enzyme is shown from ATCC 12 755 exhibited activity within a somewhat broader pH range than enzymes from the other two Strains, which is why the enzyme from ATCC 12,755 has proven particularly advantageous.

Suitable enzymes and suitable processes for their preparation and extractions are for example described in more detail and L. K. Koditschek and W. W. Umbreit, "a-glycerophosphate oxidase in Streptococcus faecium, F 24 ", in Journal of Bacteriology, Volume 98, No. 3, (1969), pages 1063-1068 and N. J. Jacobs and P. J. Van Denmark, "The Purification and Properties of the a-Glycerophosphate Oxidizing Enzyme of Streptococcus faecalis, 10 Cl ", in Archives of Biochemistry and Biophysics Volume 88 (1960), pages 250-255.

If an enzyme preparation of unknown origin is used, care should be taken to ensure that impurities, which could affect the method of determination are extracted. So, for example has shown that certain preparations of L-ff glycerophosphate oxidase occasionally produce such high levels of Kon-h5 may have concentrations of impurities that the crude preparations according to customary known fractionation methods and column separation procedures must be cleaned before they can be used to determine the Triglyccride content of blood serum can be used without causing disturbances.

For the production of analytical elements according to the invention suitable indicators for the determination of produced hydrogen peroxide are known. For example, such indicators can be used that can also be used in the enzymatic determination of glucose and uric acid and IV from the US-PS 30 92 465 and 29 81 606 are known.

The hydrogen peroxide indicators generally consist of a substance with a pcmxkiliven activity, preferably peroxidase, and a dye precursor component which is subject to color formation or color change in the presence of hydrogen peroxide and the substance with peroxidative activity There are several substances which, when oxidized in the presence of H ₂ O ₂ and peroxidase, do not cause any significant change in color

ίο which, however, in their oxidized state with a color-forming or color-changing substance, z. B. react a color coupler, which enables a qualitative and preferably quantitative analysis will. Such indicators or indicator systems are known, for example, from US Pat. No. 2,981,606. In the Color precursor components mentioned in this patent, which lead to color formation via a coupling reaction lead, are particularly advantageous for the production of crfindungsgemül.icr Hlemenlc.

In addition to peroxidases, other substances that are not themselves enzymes but have peroxidative activity are suitable have, are: iron sulfocyanate, iron tannate, ferroferrocyanide and chromium salts, e.g. H. Kaliunichromisullal, adsorbed on silica gel.

Dye precursor components that cause color formation in the presence of hydrogen peroxide and a Substances with peroxidative activity and possibly a coupler compound are:

1. Monoamines ,? .. B. aniline and aniline derivatives, ortho-toluidine and para-toluidine;

2. diamines, e.g. B. ortho-phenylenediamine; Ν, Ν'-dimethyl-paraphenylenediamine; N ₁ N'-diilhylphynylamine; Benzidine and dianisidine;

3. phenols, e.g. B. Phenol itself. Thymol, ortho-, meta- and para-cresols, ^ -naphthol and / ^ -naphthol;

4. Polyphenols, e.g. B. catechol, guaiacol, orcin, pyrogallol, p, p-dihydroxydiphenyl and phloroglucin;

5. aromatic acids, e.g. B. salicylic acid, 2,3-dihydroxybenzoic acid and bile acids;

6. Leuco dyes, e.g. B. Leueomalachite Green and Leucophenolphthalein;

7. dyes, e.g. B. 2,6-dichlorophenol indophenol;

8. various biological substances, e.g. B. epinphrine, flavones, tyrosine, dihydroxyphenylalanine and Tryptophan;

9. other substances such as gum guaiac, guaiaconic acid, potassium, sodium and other water-soluble Iodides as well as bilirubin and

10. special dyes such as 2,2'-azin-di (3-ethylbenzothiazoline- (6) -sulfonic acid) and 3,3'-diaminobenzidine.

Other indicators that can be oxidized and detected by peroxides in the presence of peroxidase Reactions result, for example, consist of compounds that, when oxidized in the presence of peroxidase be able to couple with themselves or with their reduced form with the formation of a color stick. Such self-coupling compounds include, for example, hydroxylated compounds,

z. B. ortho-aminophenols, 4-alkoxynaphthols, 4-amino-5-pyrazolones, cresols, pyrogallol, guaiacol, orcin, Pyrocatechol, phloroglucinol, ρ, ρ-dihydroxydiphenyl, bile acid, 2,3-dihydroxybenzoic acid and salicylic acid. Such compounds are described in greater detail, for example, in the book by Mces and James "The Theory of the Photographic Process", (1966), especially in Chapter 17. For the production of elements according to the invention, so-called oxichromogenic compounds are also suitable Markable leuco dyes, as are known, for example, from US Pat. No. 3,880,658. Such connections can be made diffusible by certain substituents.

According to an advantageous embodiment of the invention, the measurable change is brought about by an indicator or an indicator system comprising a compound which is oxidizable in the presence of peroxidase and is subject to oxidative condensation with a coupler compound, for example with coupler compounds with phenolic groups or activated methylene groups. Examples of such oxidizable compounds are compounds such as benzidine and its homologues, p-phenylenediamines, p-aminophanols and 4-aminoantipyrines. Such couplers, including self-coupling compounds, are exemplified in the already mentioned book by Mees and James and in the book by Kosar "Light-Sensitive Systems ", 1965, pages 215-249.

Preferred dye precursor components are 4-methoxy-1-naphthol; 2- (3,5-Dimethoxy-4-hydroxyphynyl) -4,5-bis (4-dimethylaminophenyl) imidazole, also a combination of 1,7-dihydroxynaphthalene and 4-aminotipyrine (HCl) and also 4-isopropoxy-l-naphthol.

The more optimal in individual cases. Concentrations of the components of the various indicator systems in the Systems according to the invention depend on the glycerol concentration in the sample to be analyzed, the type the determination device and the dye to be produced.

The optimal concentrations of the other components required for the preparation of the elements according to the invention can also depend on the solution to be analyzed, e.g. Ii. of whether the solution to be analyzed is blood serum in the undiluted or diluted state or other complex aqueous solutions vr _; n glycerine and / or triglycerides.

The following table I gives an overview of generally applicable and preferably applicable concentration ranges of the various components in elements of the invention.

Iiibellc I

Lipasc (ilyeerinkiniisc (■ lycerophosphhal-Oxidasc

Protease ta nnn_ ir »<; nnn τ> ηηη-, οηηηη

Pcroxidasc

Surface active compound

In the above table i, an international unit υ of an EiUy Γι ι cn is defined as the amount of enzyme which converts one micromole of substrate in one minute at 37 ° C and pH 7.

It is well known that every enzyme has a pH-activity profile; H. the changes in the The activity of an enzyme as a function of the pH value can be shown graphically.

The pII activity profile of L-α-glycerophosphate oxidase peaks between a pH value of 5 and 8.5. The result is the optimum pH range in which an enzyme which can be used according to the invention is active from the following table II.

Table II

Suitable area Preferred area Rev / m ² Rev / m ² 9,000-27,000 13,000-26,000 100-1 500 250-800 800-5,500 1,500-3,300 36,000-105,000 72,000-90,000 3,000-11,000 6,000-7,500 g / m ² g / m ² 1.5-10 4-6

PH value

Lipase 5-9

Glycerin Kinase 7-9

L-tf glycerophosphate oxidase 6.3-8.0

Pcroxidase 6-8

The table above shows that it is generally desirable to use the layer or layers of the invention Hlemente with the appropriate reagents to pH values from 6.0 to 8.0 and in particular 7.0 to 8.0 buffering. Suitable methods of buffering are known. They are based on a solution or dispersing suitable concentrations of buffer substances in the masses used for the preparation of the

, Layers of an element according to the invention are used.

The spreading layer of an analytical element according to the invention is a spreading layer in the usual sense, d. H. a spreading layer that is porous, for example isotropically porous, and the excite Can absorb liquid sample, regardless of whether it is applied directly to the spreading layer or is fed through a layer or layers with which the spreading layer is in liquid contact, where within the layer the solvent or dispersion medium and at least one dissolved or dispersed component is distributed so that a uniform concentration of the component on the surface the spreading layer facing the reagent layer or layers of the element, is produced. Such spreading layers are z. B. from DE-OS 23 32 760 known.

The reagent layer or layers of an element according to the invention generally exist from a matrix in which the enzymes and / or other reagents that react with triglycerides, glycerine or decay products of the glycerol are able to react, distributed, d. H. are dissolved or dispersed. Layers that only serve as an aulhahmc layer for the absorption of measurable compounds or substances, will be used here referred to as registration layers.

The distribution of reactive compounds (i.e. enzymes and other reaction components) can be achieved by dissolving or dispersing in the matrix material. Although a uniform distribution of the reactive components is often preferred, such a uniform distribution is not absolutely necessary if the reactive substance, for example an enzyme, such as. B. Lipase, Glyce- ; V is rinkinasc or c-glycerophosphate oxidase, which is not consumed in the course of a reaction, but rather Γ rather only serves as a catalyst.

! It has proven to be advantageous if the reagent layers for the spread or spread out

■: Components are uniformly permeable. One speaks of a uniform permeability of a layer

then when a homogeneous liquid or medium uniformly the surface of the layer t? is fed. Measurement of the concentration of the liquid or medium within the layer, dur-SS performs one with the same device and under identical conditions, but in different areas J «surface of the layer, lead to the same or practically the same results. In the case of a uniform ; f | Permeability are undesirable concentration gradients within, for example, a reagent layer,

lg avoided.

The selection of the matrix material for the reagent or recording layers depends on the type of use

of the element to be produced, as well as the reactive components used in the individual case, which in the reagent layer are to be incorporated. Advantageous matrix materials are, for example, hydrophilic Substances, both natural and synthetic, such as gelatine, üelatinederivale, hydrophilic cellulose derivatives, polysaccharides, for example dextran, gum arabic and agarose on the one hand as well as water-soluble polyvinyl compounds, e.g. B. poly (vinyl alcohol). Polyvinylpyrrolidone) and acrylamide polymers on the other hand. Organophilic strips can also be used in an advantageous manner, for example Cellulose ester.

The elements according to the invention can be self-supporting elements or the spreading layer, reagent layer and, if appropriate, further layers can be applied to a layer support. as

ίο Supports, the usual known support materials can be used, for example paper, papers coated with polyolefins, as well as a wide variety of polymeric supports, e.g. B. cellulose acetate, poly (ethylene terephthalate), polycarbonates and polyvinyl compounds, such as polystyrene.
The support can be opaque or opaque or transparent to light and other energy, depending on the method of determination to be used when the element is used. It is advantageous to use transparent layer supports which are permeable to electromagnetic radiation of wavelengths in a range from approximately 200 nm to approximately 900 nm. Of course, the transparent layer supports do not need to be permeable for the entire range from 200 to 900 nm, but only for the wavelength range of the radiation, which is not used in the corresponding determination.

If an element according to the invention has a layer support, the reagent / layer is located in the element between the support and the Ausfareitschicht. Particularly cool Durchüissig & eiishtireichc! Ür Elements according to the invention result from the indicator systems. Support a thickness of about 0.0254 mm to about 0.254 mm have proven to be particularly advantageous.

According to an advantageous embodiment of the invention, the analytical elements have one

: 5 structure so that they can be used in the context of analytical procedures in which spectropholometric Analytical methods can be applied. Accordingly, the elements according to the invention have a reflective layer which provides a suitable background for spectrophotometric measurements Meiert. If an element according to the invention has a layer support, the measurements are generally carried out through the substrate. Using a reflective layer enables the reflective

• Layer the passage of triglycerides, glycerine and / or decomposition products of the glycerine / ur reagent / or Indicator layer (i.e. a layer under a reagent layer containing enzymes that prevent the breakdown of triglycerides or glycerine and only the components for the determination of hydrogen peroxide: » contains). A white background has proven to be particularly beneficial for this purpose. With regard this function as a background for an indicator in the reagent or indicator layer is a rcilektie-

. "5 layer normally placed between the spreading and reagent or recording layers however, such a layer can advantageously also be placed between a reagent layer and an indicator layer be arranged. The reflection can also be brought about by a layer that has the function has a spreading layer. Pigments such as titanium dioxide and Use barium sulfate as a reflective pigment to create a reflective layer. Also fcön-

In some cases, blush polymers may be suitable substances for building up a reflective layer.

Optionally, an element according to the invention can also have one or more filter layers. Of the The structure and composition of such layers are known. Such layers have the task of to remove constituents from the sample to be analyzed which interfere with the reactions taking place in the element or could otherwise interfere with the determination process. For example, in

15 a multilayer analytical element according to the invention for the determination of the triglyceride level A separate filter layer is provided for the blood, which has the purpose of removing red blood cells / u or separate while the serum passes into the underlying layer. In the case of the analysis of Uluiserum or other liquids, a filter layer can serve to remove unwanted components / u, which interfere or could interfere with the primary display response.

5 (i The addition of a protease to a reagent layer whose matrix, for example, is primarily; ius gelatin or consists of other natural or synthetic substances that are attacked by protease Normal proleolytic reactions occur when the reagent layer is moistened, for example through Applying a sample to be examined to the element. Although the use of an element that which contains protease in a gelatin or corresponding reagent matrix is possible, it has been found to be necessary proved when the hydrolyzing component is accommodated in a layer which is resistant against the action of the protease and if as a further measure to protect the gelatin (or similar) Matrix of the reagent or another layer in front of the protease, a separating layer permeable to the analyte that selectively prevents protease from interacting with the gelatin or other proteinases Matrix substances of the element come into contact. In the case of such an element, it can also be

It is imperative to never place the glycerol-degrading enes in one layer with the hydrolysing components, so that it is only necessary for the method of determination. that the comparatively small hydrogen peroxide molecules penetrate the separating layer and / or reach an underlying layer, while the larger protease-ηΛ mmolckiilc are prevented from migrating into the protease-sensitive layer / u. If necessary, the reagents used for glycerol determination can be stored in a reagent

ι.- layer are accommodated, while the hydrolysis reagents are contained in the Ausbreilschichl.

If the separating layer serves to inhibit protease migration, the layer made of ilen can be different Most studs that are compatible with the various components of the element are advantageous! In this way, hydrophilic polymeric studs are suitable for building up such layers, which allow migration.

Allow hydrogen peroxide or glycerine while preventing the passage of the protease enzyme. Agaro.se and Polyacrylamide} has been shown to be poly (isopropyl acrylamide).

The analytical elements according to the invention are presented by known methods. An advantageous one Method consists in first applying a layer to a surface from which it is possible to or to apply to a layer support, whereupon the other layers are applied successively.

If the spreading layer has the function of a filter layer and a spreading layer, this layer is advantageously produced in that two layers or plies of a binder, e.g. B. cellulose acetate, dissolved in a mixture of organic solvents, can be applied to achieve "blush" polymer layers. Such a method simplifies the manufacturing operation, in which the multiple coating of a carrier is replaced by a coating operation, while producing a highly effective spreading and filtering layer. Optionally, one or both of the discrete layers can contain a reflective pigment, e.g. B. TiO ₂ , contained dispersed.

The thickness of the spreading layer can vary widely and depends at least in part on the sample volume that the spreading layer is able to absorb for reasons of convenience and cleanliness should be, as well as the pore volume of the layer, which also affects the amount of sample, that of 4 Layer can be absorbed. Spreading layers with a thickness of 50; xm to about 300 ^ m have been found to be proved particularly suitable.

The thickness of the reagent layer and its degree of permeability can also be very different. It has proven to be advantageous if the layer thickness , measured in the dry state, is about 10 ^ m to about ίΟΟ -jm.

The compounds required for hydrolysis can be accommodated in the reagent layer. According to In a particularly advantageous embodiment of the invention, however, they are accommodated in the spreading layer, for example in that the enzymes in a lyophilized state in the coating medium which is used for Hr / cugung the spreading layer, are dispersed, whereupon the coating mass is applied to the reagent layer.

If such an element is used, the elements applied to the element are spread out Sample and the hydrolysis of the triglycerides practically simultaneously and the glycerin of the sample, regardless of whether if it was present in the sample as a fatty acid ester or free glycerine, it is transferred to the reagent layer. A such Klcnicni takes advantage of the time required to spread the sample in order for it to react Prepare with the glycine determination reagents in the layer.

According to a further advantageous embodiment of the invention, there is a special layer that contains the hydrolysis component, between the spreading layer and which contains the glycerol-determining reagents containing layer. In this case, the hydrolysis takes place before the sample passes the glycerol determination reagent / ien achieved.

Regardless of where the hydrolysis component is located, optimal results are then obtained when the component is buffered to a pH between 6 and 8.0 and preferably between 7.0 and 8.0 will. The hydrolysis component is thus accommodated in the reagent layer or in another layer with the glycine determination reagents, a pH of about 7.0 gives optimal results. Appropriate pH values have proven to be advantageous if the hydrolyzing component in a second Kcagcn / schichl or is housed in the spreading layer.

The con / cniralion at Lipasc in the layer can be very different. In general, lipase concentrations of about 9,000 to about 27,000 U / m ^{2 have} proven to be advantageous. At lower concentrations, the hydrolysis cannot proceed to completion.

In a particularly advantageous embodiment of the invention, lipase concentrations of about 13,000 to about 26,000 U / nv are used. If protease is used as a hydrolysis stimulator in combination with lipase, it has proven advantageous to use the protease in concentrations of about 36,000 to about 105,000 I Viiv. According to a particularly advantageous embodiment of the invention-is P ^r otease in Kon / entralmnen of about 72 000 to about 90 000 r / m ^{2 was} used.

Since the layers described are preferably produced on the basis of solutions or dispersions, for example, by methods such as those known from US Pat. No. 39 92 158, h: U it is often expedient Proven to add coating auxiliaries to solutions or dispersions in order to provide layers with as much as possible to obtain uniform characteristics.

Surface-active compounds, which can be used as hydrolysis stimulants, can at the same time serve as coating aids that improve the coating characteristics of the materials to be produced. Hei use as coating aids have concentrations of the surface-active compounds in the order of about 0.1 g / m ² and about l, 0g / ^m: proven advantageous. Particularly advantageous concentrations of surface-active compounds which perform the function of coating auxiliaries are from about 0.3 g / m ³ to about 0.6 g / m 3.

When using the elements, a sample of the order of about 5 to about 50 -Δ is advantageously applied to the spreading layer or outermost layer of the element, as can be seen from the examples below. The sample is normally applied in the form of a contact spot or in the form of a free drop. As it passes through the spreading layer, the applied sample is spread out and fed in spread form to the reagent layer underneath. During the passage through the spreading layer or the reagent / layer, depending on the design of the element used, the triglycerides contained in the sample are hydrolyzed to glycerine, whereupon the released glycerine and possibly other glycerine contained in the sample come into contact with the glycerine kinase, whereby l.-fr-Glyi'crophosphal is generated, which in turn comes into contact with a-glyccrophosphate oxidase and in

Presence of oxygen generated _{H 2} O _2. The change which is produced by the action of the indicator or the indicator system which _{reacts with the H 2} O ₂ can then be determined quantitatively using known methods, from which the concentration of the triglycerides and the glycerol in the analyzed sample can be determined .
The following examples are intended to illustrate the invention in more detail.

example 1

An analytical element for determining the total glycerol content of blood serum, including the glycerol present in the form of triglycerides, was produced by coating a 0.18 mm thick poly (ethylene terephthalate) film support with a gelatin adhesive layer with: a Rcagenzschicht with 21.5 g / m ² deionized gelatin, 0.54 g / m ² 4-isopropoxy-1-naphthol, 0.1 g / m ² 5,5-dimethyl-1,3-cyclohexanedione, 0.4 g / m ² alkylphenoxypolyethoxyethanol, 6994 U / m ² peroxidase, 1.3 g / m ² adenosine 5'-triphosphate, disodium salt, 1506 U / m 'ff-glycerophosphate oxidase and 645 U / m ² glycerol kinase (a glycerol kinase from Worthington Biochemical Company, which is used proved to be a satisfactory commercial supplier of the enzyme) in a 0.1 molar potassium phosphate buffer of pH value7.

A layer of 0.3 g / m ^{2 of} poly-n-isopropyl acrylamide was applied to and on the reagent layer

a spreading reflective layer made of 45.2 g of TiO ₂ , 6.5 g of cellulose acetate, 900 units of lipase and 5.4 g of Oclylphenoxypolyäthoxyäthanol each per m ² .

When the reaction components were used in the stated concentrations, the reactions were with the one applied to the spreading layer! Serum at 37 ° C essentially within 5 to 7 minutes completed and reactions with glycerin or L-a-glycerophosphate were at 37 ° C in less than 5 minutes completed.

Serum samples (10 al parts) were applied to the produced elements, whereupon reflection densities D _R at 660 nm were measured after 8 minutes at 37 ° C.

In addition, an Eicnkurve (Fig. 2) was made from H ₂ O ₂ , aqueous glycerine and L-tr-Glycerophosphhal-Slandards. The total cholesterol concentrations in the serum were determined from the curve.

The D _Ä values, the ₂ sample were obtained on the elements by applying a 10 al H ₂ O were subtracted in each case. A comparison of the results of a quantitative serum triglyceride determination obtained using the elements according to the invention with the results obtained using the semi-authoritative chemical method of Kessler and Lederer (see "Fluorimetric Measurement of Triglycerides", Automation in Analytical Chemistry, Techican Symposia, editor: LT Skcggs Jr, Mediad, Inc.i> JYNY [1966], o '. 341) is shown in Table III. As the results obtained show, there is a good relationship between the two methods, from which it follows that mil ile elements according to the invention or the serum triglyceride content must be determined quantitatively.

Table III

Comparison of the results of a quantitative serum triglyceride determination when using rincs according to the invention Element with the results of a semi-automatic chemical method

Serum sample triglyceride concentration mg / dl

Method (/ -Glyccrophosphai-Syslern

according to Kessler and Lederer according to i-rounding

1 115 105

2 260 275

3,490,450
50

Example 2

A further element was tested as described in Example 1, again using the method described in Example I. described procedure was worked.

Two serum triglyceride concentrations were used, one normal concentration of 90 mg / dl and a higher concentration of 560 mg / dl. The results of the tests carried out are in the following Table IV compiled.

W) Table IV

Triglyceride con / cntralion

90 mg / dl 560 mg / dl

Sample I 0.519 1.146

Sample 2 0.519 1.169

27 37 286 560 mg / dl continuation O* Triglyceride concentration 1,123 90 mg / dl 1.130 Dr 1.142 Sample 3 0.532 1.47 Sample 4 0.572 Average 0.5355 Deviation coefficient% 4.65

Dg = reflection density at 540 nm.

The results illustrate a quantitative response of the analytical methods of the present invention
Hlcmcnto when determining the triglyceride content of liquids.

Although in the above the use of the elements according to the invention for determining the glycerol
and triglyceride content of liquids has been described, it should be noted that elements can of course also be prepared which can be used for the quantitative determination of L-sr-glycerophosphate, ATP and other compounds that intervene in the reactions indicated on page 7, provided that the concentrations the other compounds are known, e.g. B. glycerophospholipids, phosphoenolpyruvates,
Lipasc and glycerol kinase.

1 sheet of drawings

Claims (1)

Patent claims: 1. Multi-layer analytical element for the determination of triglycerides in liquids with at least a spreading layer for spreading a sample of a liquid to be analyzed and a Reagent layer, characterized in that the element (a) a lipase that can cleave triglycerides, (b) glycerol kinase, (c) adenosine triphosphate, (d) ff-glycerophosphate oxidase and
(e) an electronics acceptor