DE1767931C3 - Diagnostic means and method for the detection of urobilinogen bodies - Google Patents

Description

CHO

U)

in which R ₁ denotes the radical of a secondary, straight-chain or branched, open-chain or mono- or polycyclic alkyl or aralkyl group and R _{2 represents} a hydrogen atom or an alkyl group.

2. Diagnostic agent according to claim 1, characterized in that the absorbent carrier is impregnated with a solution which contains 0.001 to 1% of an aldehyde of the general formula I. and contains at least 3, preferably 15 to 30% of a strong, solid acid.

3. Method for the detection of urobilinogen bodies in body fluids on an absorbent Carrier, a reagent film or in solution in the presence of a strong, solid or even liquid acid with the help of a substance similar to Ehrlich's reagent with urobilinogen bodies provides a color change, characterized in that substances are used as color indicators of formula I.

CHO

(D

in which R _{1 is} the radical of a secondary, straight-chain or branched, open-chain or mono- or polycyclic alkyl or aralkyl group and R _{2 is} a hydrogen atom or an alkyl group, is used.

The invention relates to a diagnostic agent for the detection of urobilinogen bodies in body fluids, preferably in urine, consisting of an absorbent carrier or a film, a strong, solid acid and a substance which, in the manner of Ehrlich's test, with urobilinogen bodies provides a color change.

It is known that urobilinogen bodies (bilanes), indole, sulfonamides, porphobilinogen, urinary indican and 5-hydroxy-indole acetic acid with a solution of p-dimethylaminobenzaldehyde in hydrochloric acid can prove. This evidence is known in the literature as Ehrlich's reaction: he has especially in medical diagnostics as evidence of "increased urobilinogens" in the urine gained considerable importance. Although the sample is not very specific, it is considered the standard method for the diagnosis of liver and biliary diseases.

To simplify the test, it has already been suggested. Manufacture test tablets (British Patent specification 779 921), in which, in addition to p-dimethylaminobenzaldehyde, a solid inorganic or organic Acid is contained (cf. also USA.-Patent 2 320 282 - test tablets for the detection of Sulfonamides). If one dissolves such tablets in more or less diluted urine, one obtains Color reactions d roughly correspond to those of the original Ehrlich sample; however will For example, to evaluate the color reaction with sulfonamides, the use of a blue auxiliary color (e.g. methylene blue) recommended.

To further simplify Ehrlich's sample, the reaction is also already available on test papers transfer; see Fischl and P in to, Clinica Chim. Acta 2 (195η, pp. 527 to 533, as well as the French Patent documents 1 260 561 and 1 450 273. in which those of Fischl and P i η t ο used Phosphoric acid is replaced by oxalic acid or another strong organic acid.

Surprisingly, in the search for a further improvement in the test, it has now been found that certain derivatives of p-aminobenzaldehyde are much more sensitive and less prone to failure Provide evidence of urobilinogen as previously possible. As derivatives of p-aminobenzaldehyde Substances come into consideration which have a secondary alkyl or aralkyl group on the p-amino group carry.

The invention thus consists in a diagnostic agent of the type mentioned at the outset, which is carried out by a content of one or more substances of the formula (I)

45

CHO

in which R _{1 is} the remainder of a secondary, straight-chain or branched, open-chain or mono- or

polycyclic 'alkyl or aralkyl group and R _{2 represents} a hydrogen atom or an alkyl group, is indicated.

In a preferred embodiment of the new diagnostic agent, the absorbent carrier is with soaked in a solution containing 0.001 to 1% of an aldehyde of the general formula (I) and at least 3, preferably 15 to 30% of a strong, solid acid contains. As strong, solid acids z. B. in question: sulfosalicylic acid, p-toluenesulfonic acid, potassium bisulfate, Glutamic acid hydrochloride and especially oxalic acid.

The method according to the invention specified in claim 3 consists in general use of substances according to formula (I) for the detection of urobilinogen bodies. To carry out the procedure the new diagnostic means can be immersed in the investigated solution and the Read off the color change after a short time.

A particular advantage of the new substances (I) is that they do not display previously unknown metabolic products that occasionally occur in normal urine. It can also be described as surprising that the more basic derivatives of p-aminobenzaldehyde with a branched substituent R ₁ on the nitrogen atom do not react with urea, although, according to previous observations, more strongly basic aldehydes also react more strongly with urea.

For the detection of urobilinogen or urobilinogen bodies in solutions one can of course Replace p-dimethylaminobenzaldehyde with the substances of the formula (I) and thus the content photometrically determine exactly and without interference in the cuvette.

In the examples below are the method according to the invention and those according to the invention diagnostic means explained in more detail, with comparative experiments with previously known diagnostic Means the superiority over the state of the art is proven.

example 1

Filter paper is soaked with a solution of the following composition:

p-iCyclohexylaminoJ-benzaldehyde 0.05 g

Oxalic acid 20.0 g

Methanol, ad 100.0 ml

After drying, mar receives a white test paper, which with Uririen, the urobilinogen body contains, depending on the content, shows a meb- or less intense pink to purple color. Urobilinogen body arms Urine or a 3% aqueous urea solution do not change the color of the Test paper. Hamindikan gives no color reaction. The test papers can be used after 1 to 2 minutes can be read.

Example 2

Filter paper is soaked with a solution of the following composition:

p- (sec-butylamino) benzaldehyde .. 0.05 g

Potassium bisulfate 15.0 g

Polyethylene glycol 5.0 g

Water, ad 100.0 ml

After drying, a white test paper is obtained which has the same properties as the test paper produced according to Example 1.

Example 3

Filter paper is filled with solutions of different concentrations, each λ 'part of p- (isopropylamino) benzaldehyde (i-PABA), p- (n-Propylamino) -benzaldehyde (n-PABA) or p- (dimethylamino) -benzaldehyde (DABA) and 20 parts of oxalic acid in 100 parts of methanol, as described in Example 1, impregnated. the dry test papers are immersed in a 3% urine solution. The color changes observed are compiled in the following table:

X i-PABA n-PABA DABA 0.05 White yellowish yellow 0.075 White yellowish yellow ο, ι White yellow yellow 0.15 weak- yellow strong yellow yellowishL

As can be clearly seen from this, the test paper according to the invention with i-PABA is essential less sensitive to urea and therefore reacts more sensitively and specifically to urobilinogen bodies, because the pink color reaction with urobilinogen is not due to the more or less strong yellow Color reaction is covered with urea.

Example 4

Filter paper is impregnated with methanolic solutions containing 0.05 parts of the solution in 100 parts the following substance (I) and 20 parts of oxalic acid. White to yellowish test papers are obtained. The color reactions observed when these test papers are immersed become apparent after 1 to 2 minutes and are compiled in the following table:

Substance (I)

White*)

pale yellowish
pale yellowish
yellowish
yellow

pale yellowish yellowish almost white *) pale yellowish yellowish yellowish yellow
yellowish *) The strips dipped into the urine show a pale yellow color, which is due to the inherent color of the

p- (isopropylamino) benzaldehyde

p- (Isopropylamino) -m-methylbenzaldehyde ....

p- (sec-butylamino) benzaldehyde

p- (pentyl-3-amino) benzaldehyde

p- (2,4-Dimethylpentyl-3-amino) -benzaldehyde ..

p-f-CyclopentylaminoJ-benzaldehyde

p- (2-Methylcyclopentyl-amino) -benzaldehyde ..

p- (Cyclohexylamino) benzaldehyde

p- (Cyclohexylamino) -m-methylbenzaldehyde ...
p-iCyclohexylaminoJ-m-isopropylbenzaldehyde.
p- (2-Methylcyclohexyl-amino) -benzaldehyde ...

p-menthylamino-benzaldehyde

p- (l-Cyclohexylpropyl-2-amino) -benzaldehyde ..

Reaction with 3%

Urea solution
or usually poor urine

Usually containing urine

pink

pink

pink

pink

Red

pink violet

pink violet

pink violet

pink violet

pink violet

pink violet

orange

pink

Urine is related.

continuation

Substance (I) Reaction with 3%
Urea solution
or usually poor urine Usually containing urine p-iCyclododecylaminoJ-benzaldehyde
p- (Norbornyl-2-amino) benzaldehyde
p- (u-methylbenzylamino) benzaldehyde
p- (α-Isopropylbenzylamino) benzaldehyde
p- (1-Phenylpropyl-2-aniino) benzaldehyde
p-flndanyl - ^ - aminoJ-be ^ aldehyde pale yellowish
yellow
yellowish
yellowish
yellowish
yellowish pink
pink
pink
pink
pink
pink

Example 5

A mixture is used to produce a film-like reagent layer for the detection of urobilinogen prepared from the following components:

p- (Cyclohexylamino) benzaldehyde .. 0.05 g

Colloidal silica 1.00 g

Oxalic acid 10.00 g

Polyvinyl butyracetal 0.5 g

Polyethylene glycol 6000 0.5 g

Methanol 30.0 ml

The above mixture is applied about 1 mm thick to a white polyvinyl chloride film and then applied dried with warm air, creating a water-insoluble layer. Will this with a Drops of urobilinogen-containing urine are wetted and after 1 to 2 minutes a red color appears. With No staining is observed in urobilinogen-free urine.

Example 6

25 ml urine are acidified with 12 ml glacial acetic acid and extracted with 40 ml ether. The extract will Washed twice with 20 ml of water each time and made up to 40 ml with ether. Take 20 ml of this, mixed with 0.5 ml of a solution of 1 g of p- (cyclopentylamino) benzaldehyde in 5 ml of conc. hydrochloric acid and shakes vigorously. Then 2.5 ml of semi-saturated sodium acetate solution and 3.5 ml are added Add water and shake again, the red dye collecting in the aqueous phase. To After the separation, the ether is washed again with 2 ml of water. The combined aqueous extracts are made up to 20 ml, at 557 nm in the photometer measured and evaluated using a calibration curve drawn up beforehand.

Claims (1)

Patent claims: 1. Diagnostic agent for the detection of urobilinogen bodies in body fluids, preferably in urine, consisting of an absorbent carrier or film, a strong, solid acid and a substance which, in the manner of Ehrlich's test, with urobilinogen bodies provides a color change, characterized by the content of one or more Substances of formula I