IL34839A - Process for the determination of catecholamine and serotonin metabolites - Google Patents

Description

Process for the determination of catecholamine and serotonin metabolites This invention is concerned with a process for the determination of catecholamine and serotonin metabolites.

In clinical chemistry, "catecholamines" means noradrenalin (NA) , adrenalin (A) and dopamine. The most numerous metabolites, and the most important diagnostically at the present time, are metadrenalin, normetadrenalin and the phenolcarboxylic acids, -hydroxy-5-methoxymandelic acid (HMMA or VMA - vanillin-mandelio acid), vanillic acid (VA) and homovanillic acid (HVA). The excretion of these metabolites is increased in various diseases, for example phaeochromocytoma, neuroblastoma and ganglioneuroma.

The most important serotonin metabolite from the diagnostic point of view is 5-hydroxyindolacetic acid (HIAA).

The excretion of HIAA is greatly increased, for example in the case of cancer of the lung and carcinoid of the small intestine.

There are many methods for determining these metabolites. The fluorimetric and spectral-photometrio methods, which are most widely used at the present time, have certain disadvantages. They are either relatively simple, but non= specific and therefore not very informative, or else they are specific, but very complicated and involve a great deal of work.

A method is known by which metabolites still carrying an amino group, i.e. metadrenalin («» metanephrine) and normetadrenalin (° normetanephrine) , can be detected by the use of a cation exchanger, followed by alkaline treatment. But these metabolites are only excreted in increased quan° tities during the attack. HMMA, on the other hand, is between attacks. The simultaneous determination of HMMA is difficult, because many other phenols occur in the urine, which form colouring agents with the reagents involved, and because the basic ion exchangers do not bind the phenol-carboxylio acids particularly firmly in the presence of the urine salts.

In comparison with these methods using cation exchangers, purely chromatographic methods have the advantage that several metabolites present in the same specimen can be specifically determined simultaneously. Several methods for the determination of these metabolites using paper and thin layer chromatography are described in the literature. According to these known methods, extraction of the urine is generally followed by drying, filtering and concentration of the extract, followed by separation by paper or thin layer chromatography on silica gel or cellulose. The metabolites are detected in the usual way with a colour reagent, the colouration being compared with a standard. j It has also been proposed to omit the pretreatment of the urine before application to a cellulose layer. This is a substantial simplification,but only permits unsatisfactory separation, because other urine components, for example salts and urea, distort the separated zones., Moreover, only the HMMA can be determined and its identification is relatively uncertain. Quantitative estimation is also very uncertain and only excessive excretion of HMMA can be detected. The lower detection limit is about 10 mg /day, but the normal value is -4- mg %, so that values of about or less than 10 mg % a day should also be determined.

We have now sur risin l found that the use of a particular sorption agent, namely cellulose impregnated with polyethylene-imine, for the investigation of body fluids containing such metabolites, enables good chroma-tograms to be obtained with a high degree of zone sharpness and very good separation. We have further found surprisingly that urine can be applied to the sorption agent directly without any pretreatment. The detection limits of metabolites are also very low and the procedure provides a particularly · advantageous method for the simultaneous determination of catecholamine and serotonin meta-, bolites. The new method is extremely simple to carry out and can be used for all the metabolites that are of diagnostic .importance in this connection. · The chromatograph-ically separated substances can be identified and quantitatively estimated with great certainty.

According to the present invention, we provide a method for the determination of catecholamine and serotonin metabolites in' body fluids, which comprises subjecting a body fluid to thin layer chromatographic separation using cellulose impregnated with polyethylene-imine as the sorption agent and applying a colour reagent to the chromatogram obtained to colour the separated zones.

An acid mixture, preferably consisting of at least three components, is used to develop the chromatogram, at least in the first separation. The developer should preferably contain, besides at least 5% water, at least one other solvent from at least one of the following groups.

A) solvents that are only slightly miscible or immiscible with water, with organic solvents, 0) acids, preferably lower alkanecarboxylic acids.

The method according to the invention iB particularly applicable to the examination of urine, but other body fluids, for example cerebrospinal fluid, may also be investigated.

Ordinary cellulose for chromatographic purposes impregnated with polyethylene-imine (PEI cellulose) is used as the sorption agent. Any cellulose suitable for chromatographic purposes, preferably microcrystalline cellulose, may be impregnated with polyethylene-imine as described in the literature (see, for example, Biochimica Biophysica Acta. 61 , pp 852-85 (1962)). Impregnation is most simply effected with a commercial, approximately 50%, aqueous solution of polyethylene-imine. This solution is mixed thoroughly with cellulose, if required after dilution with salt-free water.

The resulting suspension may be used directly for coating the chromatographic plates. The proportion of polyethylener imine with respeot to the sorption material is suitably from 0.15 to 0%, and preferably from 0.5 to 3.5%, by weight.

It is important not to use a PEI cellulose neutralized with hydrochloric acid as is customary in chromatography.

Acoording to the present invention it is essential to use a PEI cellulose that does not contain a too firmly bound ion of the opposite charge. The PEI cellulose is therefore advantageously in the free form or in the OH form or only contain anions that have a lower selectivity coefficient than the chloride anion. The selectivity coefficient, of course, provides a measure of the relative affinity of the ions in relation to the exchanger. Suitable anions in this butyrate.

The composition of the developer used influences the separation of the zones on the chromatogram. It has proved particularly advantageous to use, at least for the first separation of urine, an acid mixture consisting of several components, preferably at least three. The addition of at least 5%» scad preferably from 5 and- 20%, by volume of water has proved particularly advantageous. The desired separations can be obtained very well if developer is used that contains, besides water, at least one solvent that is only slightly or not at all miscible with water, at least one solvent that is miscible with both water and organic solvents, and at least one acid, preferably a lower alkanecarboxylic acid. The proportion of the acid in relation to the total mixture is as a rule from 1 and 40%, preferably from 5 to.20%, by volume.

Suitable solvents that are only slightly or not miscible with water (group A) include, for example, chlorinated hydrocarbons, such as chloroform and methylene chloride) aliphatic alcohols containing more than 3 carbon atoms, for example butanols or amyl alcohols; esters, such as ethyl acetate or other esters of lower aliphatic carboxylic acids, particularly acetic acid, e.g. acetio acid butyl or isoamyl esters; ethers; benzene and derivatives thereof, such as toluene or xylene.

Suitable solvents that are miscible with water and with organic solvents (group B), include, for example, for the present purpose» lower aliphatic alcohols, particularly methanol, ethanol, n-propanol and isopropanol; ketones, s tetra ydrofura } . acetonitrile , pyridine ; dimethyl formamide j and dimethyl sulphoxide. Suitable acids include, for example ι formic acid, acetic acid and propionic acid and substituted . carboxylic acids , such as methoxy-acetic acid. The separations are , as a rule , less satisfactory with mineral acids.

If the simultaneous separation of the less polar metabolites e .g. homovanillic acid, is dispensed with, one of the above-mentioned groups of solvents (A or B) may also be omitted from the developer mixture.

According to a particularly preferred embodiment , - a mixture of chloroform/n-butanol/ethanol/glacial acetic acid/ water is used in the ratio by volume 10/55/5/15/15. Other suitable mixtures are, for example, n-bu anol/ieopropanol/ glacial acetic acid/water in the ratio by volume 60/10/15/15 or 0/30/15/15 J n-butanol/ethyl acetate/glacial acetic acid/ water in the proportion by volume 60/10/1 / t n-butanol/ c glacial acetic acid/water in the ratio by volume 70/15/15» and n-butanol/ethanol/glacial acetic acid/water in the ratio by volume 60/5/1 O/25.

The viscosity of the developer mixture should be between 0.2 and 5 centipoise at 20°Cj the boiling range should be between 30 and 150°0.

Separation by the developer is preferably carried out twice and a basic developer mixture may therefore be used for the second separation. It is, in any case , important for urine to be applied to the PEI cellulose with an acid developer mixture , since it is applied without pretreatment. Further treatment of the chromatogram, particularly in the case of two-dimensional chromatography, on the other hand, or "basic developers. Particularly good separations can be obtained, for example, if the basic developer mixtures used are those that contain the components mentioned, above for the acid developers, the acid portions being, however, replaced by a base, preferably ammonia. Thus, the basic developer advantageously contains, besides the base, from to 20% of water, at least one solvent that is only slightly or not miscible with water (group A) and at, least one solvent that is miscible with both water and organic solvents (group B). A mixture of ethyl acetate/n-butanol/ isopropanol/25% aqueous ammonium hydroxide in the ratio by volume 30/20/25/25 has, for example, proved satisfactory.

Here also, however, one of the group A or B solvents may be omitted, because sufficient separation' is effected even with such omission in many cases. The following may be mentioned, by way of example, as other suitable basic developer mixtures t chloroform/n-butanol/ethanoi/25% aqueous ammonium hydroxide/ water in the ratio by volume 20/30/30/15/10; ethyl acetate/ n-butanol/isopropanol/25% ammonium hydroxide in the ratio by volume 30/20/25/25 ; n-butanol/acetone/25% ammonium hydroxide/ water in the ratio by volume 70/10/10/10; n-butanol/methylethyl ketone/25% ammonium hydroxide/water in the ratio by volume /4-0/15/15 ; acetic acid isoamyl, ester/isopropanol/25% ammonium hydroxide in the ratio by volume 30/45/25; isopropanol 25% ammonium hydroxide in the ratio by volume 80/20.

The thin layer chromatography plates used in carrying out the process according to the invention are prepared in the usual manner. The sorption agent suspension is applied by any suitable coating procedure to a support of glass, plastics v of the sorption agent layer is generally from 50 to 200^.

The plate is then dried for several hours at room temperature or for a shorter time at an elevated temperature, for example for several minutes at 80-120°C. As the layer is photosensitive, it is advantageous to keep the plate protected from light.

The chromatographic separation is carried out in the usual manner. Urine is applied to the sorption agent layer without pretreatment . In the case of quantitative analysis, the urine specimen may he diluted with water, which is preferably salt-free, to have comparable volumes and facilitate conversion. Advantageously, for example, the 24-hour excretion is made up to a whole litre.

About 1 to 5 of each urine specimen are applied; it is advisable to dry the plate at intervals if more than 2μ, are applied to a point. It is advisable to apply a standard solution besides two to three urine specimens. For development the plates (one or several) are placed in ordinary chambers containing the developer mixture. . Advantageously, the chamber is closed by a cover plate during development.

The developer front has generally reached the upper edge of the plate (8.5 cm height of ascent with a plate size of x 10 or 20 x 10 cm) after about 1 to 2 hours. The plate is then dried and, advantageously, drying is followed by a second development in the same manner, if desired using a different, for example, a basic, developer.

A colour reagent is then sprayed on to the plate in the usual manner to develop the colour of the separated zones of the tested liquid and the standard. These colour .;' %, usually in stabilized form, Diazotized p-nitroanilines are most commonly used, for example, p-nitrophenyldiazonium fluoborate, 4—diazobenzenesulphonic acid, 4~diazo-n-monoethyl orthotoluidine fluoborate, -diazo-N,N-diethylaniline fluo- 5 borate, and ~diazo-N-ethyl-N-(P-hydroxyethyl)-aniline; This will show with certainty whether the increase was simulated by masking by another component of the body fluid investigated.

It is also advantageous to carry out two-dimensional chromatography twice in each dimension. Here also, a basic eluting agent may be used in the second dimension and/or the second development..

Advantageously, in two-dimensional chromatography the thin layer plate is divided into two parts and two spatially separate urine specimens and. a standard solution, are first developed in known manner. Another standard is then applied and chromatography in the second dimension carried out in the usual manner. The spots of the urine specimens obtained in thie way can now easily be identified by the two corresponding spots of the standard solutions, i.e. by two co-ordinates. The separated substances can also be quantitatively estimated with great certainty in this way.

The present invention thus makes it possible for the first time to recognize and quantitatively estimate both the catecholamine and the serotonin metabolites present in the same specimen, without having to treat the urine beforehand, thus eliminating a further source of error. The new method is suitable, because of its simplicity and the rapidity with which it can be carried out, both for series tests and also for individual determinations in the case of pathological findings. The low detection limits make it possible to use the new method for a wide variety of problems and differential diagnoses.' tration only. The plates used , in these examples were prepared as followst Preparation of thin layer plates 3 1 . of salt-free water was added to 150 g of a 0% aqueous solution of polyethylene-imine . The solution was transferred to a larger vessel and there made up to a total of 17·5 k w th salt-free water. 4.8 kg of micro- crystalline cellulose were added to this polyethylene-imine solution. The mixture was agitated for 20 seconds in a high speed mixer. Glass plates measuring 20 x 20 cm were • r coated with the resulting suspension. The coating apparatus was adjusted to give a layer thickness of 2 0^. The plates were dried at about 90°C in a drying cabinet or overnight at room temperature . , The dried layer thickness was 80-120 /.

Instead of micro crys al line cellulose , othe celluloses suitable for thin layer chromatography may be used.

, The proportion of polyethylene-imine in relation to the total sorption agent is about .1 «5% by weight .

Implementation of method Example 1 , Use as a clinical test to ascertain increased renal excretion of catecholamine and serotonin metabolites .

The collected 24-hour, urine of a patient was made up to 1 , 2 or 3 whole litres with distilled water. 2jjX of urine per litre of this mixture were applied in spots to the longitudinal side of a 10 x 20 cm PEI cellulose glass plate (prepared as described above) at . 1.5 C1& from the bottom edge. A distance of 1.5 cm was kept between the applied dots. A applied beside every other urine specimen; every urine specimen was thus adjacent to a standard solution,, and 8 urine specimens could be applied to one plate. The plate was then developed twice with the eluting agent mixture, chloroform butanol/ethanol/glacial acetic acid/water (5/55/10/15/15 by volume) up to the top edge of the plate. , The plate was then dried and sprayed with an 0.05 molar solution of p-nitrobenzenediazonium tetrafluoborate in 0.3 M aqueous agCOj solution. After warming for a short time, the individual metabolites appeared separately as spots of different colours. The intensity of the coloration and size of the spots were used as a guide in determining whether one of the* urine specimens contained a metabolite in a higher concentration than the standard.

The standard was prepared by dissolving the metabolites in question in water. . The standard corresponded to the upper normal range, with the exception of metadrenalin and normet drenalin, which were added in excess quantities for better determination. The composition of the standard and the heights of ascent of the standard substances in the developer used are shown in the following table.

Table 1 In the present case all the urine ■ specimens had values within the standard range.

Example 2 Semiquantitative estimate of renal excretion of catecholamine and serotonin metabolites.

Standard solutions of the following composition and concentration were first prepared.

Table 2 Concentration of standard solution (ST) in mp i.

STT ST2 ST5 saw- ST5 Vanillic acid 10 5 20 30 50 Homovanillic acid 15 22.5 30 5 75 Metadrenalin 5. 7.5 10 ' 15 25 Normetadrenalin 5 . 7.5 10 15 25 -hydroxyindolacetic acid 10 15 20 30 50 4-hydroxy-3-methoxymandelic acid 10 15 20 30 50 2†yl of urine per litre of daily excretion and one of the different standard solutions were applied alternately ■ ' in increasing concentrations i the manner described in Example 1. The plate was developed and colour development was effected as described in Example 1 . The excretion of the different metabolites could be estimated without difficulty and with great certainty by means of the standards of different concentrations.

Four of the urine specimens used had values within the normal range for the six metabolites indicated, while .one • - —specimen, to which homovanillic acid had been added, had an increased concentration, which was classified between standard 2 and standard 3 (light blue spot). This corresponded to the addition of 25 mg/Ι· of homovanillic acid to this urine specimen.

-', Example 3 ., · Two-dimensional chromatography A 10 x 20 cm glass plate coated with PEI cellulose as described above was divided by a pencil line into two halves of 10 x 10 cm each. At least of untreated urine were applied to the left side of the plate at a distance of ■',· · 1 .5 cm from the left edge and from the bottom edge. The right half of the plate was treated similarly, but 2.jtJL of , a standard solution (ST 3 in Example 2) were applied here at a distance of 15 mm from the point of application of the urine. The plate was developed twice in the usual manner as far as the upper edge of the plate with the longitudinal side downwards with the aluting agent, chloroform/butanol/ ethanol/glacial acetic acid/water (5/55/10/15/1 by volume) . solution 3 were then applied to the places where the urine had been applied on the left side of the plate. The left half of the plate was then developed at right angles to the first development direction as far as the upper end, i.e. as far as the pencil line, this being done twice (with intermediate drying) with the developer mixture, ethyl acetate/n-butanol/isopropanol/25% aqueous ammonium hydroxide (30 20/25 25 by volume).

The resulting spots were made visible by spraying with an 0.05 molar solution of p-nitrophenyldiazonium fluoborate. Each spot could be characterized distinctly by the two-dimensionally accompanying standard solutions by two co-ordinates.

In the present case it was clear that the increased excretion of hydroxymethoxymandelic acid (blue spot) recorded in a preceding, single-dimensional chromatogram, was simulated by another metabolite with the same height of ascent. The spot of hydroxymethoxymandelic acid in the two-dimensional chromatogram was within the normal range as shown by its colour and size. .

Exampleι 4- ·· , 10024-hour urine specimens were tested in the manner described in Example 1 , except ,that the standard solution used in Example 1 was replaced by a standard of the following composition! ΗΜΜΛ, HIAA, HVA and VA in a concentration of 5 mg/1. in each case.

The following results were obtained after development and coloration of the chromatograms as in Example 1 i the 100 urine s ecimens 22 contained about m l. of HIAA, 2 about 7 m » of HIAA, and the remainder leas than 5 mg/1. of HIAA. All the specimens that contained · 5 mg/1. of HIAA or more were then chromatographed two-dimenBionally in the manner described in Example 3· It was found that an increase in HIAA above 5 mg/Ι· had been simulated in only one case in the uni-dimensional chroma-togram. 21 Of the specimens contained 5 mg/Ι· or more HMMA after evaluation of the uni-dimensional chromatogram. A check by two-dimensional chromatography showed that an interfering substance, which was separated by two-dimensional chromatography, was present in five cases in the uni-dimensional chromato rams. The two-dimensional chromatograms thus showed that in these five cases, excretion was within the normal range. Thus only 16 of the 100 specimens actually showed slightly increased excretion of HMMA; this was confirmed by other tests.

, HVA was found in only 10 of the 100 specimens by the size and intensity of the light-blue spots. A red substance was found in the case of many of the specimens with uni-dimensional chromatography a little below the HVA spots. To be quite sure that this red substance did not mask a spot due to HVA, 5» 10, 15 and 20 mg of HVA l. were added to the urine specimens in question. After the development of the chromatogram it was found that the detection of HVA was not impaired, in the method of the invention, by this red substance, because the concentration differences were readily visible. In one case a red substance was present at exactly the height of the HVA, but a two-dimensional contain substantial quantities of HVA.

In none of the 100 specimens, therefore, did the method of the invention Indicate pathological excretion. This proves that false positive results are not to ,be expected. This is all the more remarkable because the urine came from patients whose diets or drug-taking had not been deliberately modified prior to the test. False negative results are also impossible, because of the high detection sensitivity of the method of the invention.

Example 5 Semiquantitative estimation of renal excretion of catech-olamine and serotonin metabolites Standard solutions of the composition and concen-tration indicated in Example 2 were prepared. 2 ^Λ. of urine per litre of daily excretion of a urine marked by an increased content of homovanillio acid, and one of the different standard solutions were applied alternately in the manner described in Example 1 in an increasing concentration. The plate was developed and the colour reaction was carried out as described in Example 1. The quantity of the different metabolites excreted could be estimated with great certainty and without difficulty by means of the standards of different concentrations.

The urine was classified between standard 2 and standard 5 according to the intensity of the homovanillic acid. spot (light blue spot). This corresponded .to a content of 25 mg/1. of homovanillic acid in this specimen.

Claims (1)

1. 6. A method according to any of claims 3 to » n which the acidic developer mixture consists of chloroform n-butanol/ethanol/glacial acetic acid/water in the' ratio, by volume, of 10/55/5/15/5·. 7. A method according to any of claims 1 to 6, in which development of the chromatogram is oarried out twice in each case. 8. A method according to claim 7, in which the first development is carried out with an acidic developer mixture and the second development is carried out with a basio developer mixture. 9. A method according to claim 6, in which the basio developer mixture comprises, in addition to at least 5% of water, at least one other solvent from at least one of the following groupsi (a) solvents that are slightly or not miscible with water, (b) . solvents that are miscible with water and with organic solvents, and (c) bases. 10» A method according to claim 9» in which the base is ammonia. 11. A method according to any of claims 8 to 10, in which the basio developer mixture consists of ethyl acetate/ n-butanol/isopropanol/25?0 aqueous ammonium hydroxide in the