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The estimation of indoxyl sulphate in urine
CLINICA CHIMICA ACTA
THE
ESTIMATION
99
OF INDOXYL
SULPHATE
IN URINE
H. J. IlYLANCE
Rheumatic
Diseases
Unit,
Northern
General’ Hospital,
Ferry Road, Edinbovgh
4 (U.K.)
(Kevised manuscript received March 27, 1969)
SUMMARY
A method is presented for the determination of indoxyl sulphate in urine. The urine is passed down a Dowex 50-X8 column and the indoxyl sulphate, which is not retained, is estimated in the eluate with p-dimethylaminobenzaldehyde. The 24-h output from normal subjects was 77.8 & 21.7 mg.
INTRODUCTION
Urinary indoxyl sulphate (indican) appears as the eventual result of bacterial action on tryptophan in the gut. The excretion is elevated in conditions where increased bacterial metabolism is occurring ; an alteration in the normal gut flora may also modify the output. Curzon and Walsh1 state : “The determination of urinary indoxyl sulphate may provide a rough estimate of the bacterial contribution to the urinary excretion of aromatic substances in so far as it is influenced by nonspecific dietary factors, and the absorption and movement of intestinal contents.” Our own interest arises as a result of the study of tryptophan metabolism, in patients with rheumatoid arthritis, which has been investigated in this Unit over a number of years. Although indoxyl sulphate results from bacterial action it seemed desirable, for completeness, to investigate the output of this material. There are many methods available for the estimation of indoxyl sulphate in urine. Most involve either oxidation to indigo or various condensation reactions, with solvent extraction as part of the method. That of Curzon and Walshi, for example, used the reaction with fi-dimethylaminobenzaldehyde in acid solution followed by basification and petrol ether extraction, with a correction applied for urea interference. A rapid and specific method, preferably not requiring solvent extractions, would have obvious advantages. The only method along these lines was that of Bryan2, who after treatment of the urine with acid passed it through an ion-exchange column and estimated the indican by the Bratton and Marshall3 reaction. This method appeared accurate but rather tedious. It required 1% of the total 24-h urine specimen, pressure for passing the sample through the column,washing with 2 x 20 ml acid for elution, and finished with an estimation procedure which was read after 3 h. We suggest here a Clin. Ckim. Acta, 26 (IgSg) gg-103
more rapid, reliable method based on the use of a Dowex 5011: column, of indican in the eluate with ~-dimeth~laminc,benzaldeh?_de. MATERIALS
and estimation
AXD METHOD
Dowex 5o\%T-X8 (IOO-zoo mesh) resin was prepared as described by Siirbol and used as a column of dimensions 0.8 x 5 cm. P-Dimethylaminobenzaldehyde reagent : 0.8 g P-dimetl~ylaminobenzaldel~yde (Koch-Light Laboratories Ltd.) was dissolved in 30 ml ethanol and 30 ml concentrated llydr~)chloric acid added.
One ml of urine is added to the column and washed through with z-ml portions of distilled water until IO ml of eluate has been collected. Five ml $-dimethylaminobenzaldehyde reagent is added to j ml of the eluate, and after mixing, left for 20 min at room temperature. The extinction is then read at 480 met against a blank of 5 ml ~-dimethylan~inob~nzaldehyde reagent and j ml v&er. The indican concentration (as potassium salt) is obtained by comparison with a standard curve prepared from potassium indoxyl sulphate (Sigma). RESULTS AND DISCUSSION
The absorption curve for the indoxyl sulpllate-~-dinletl~ylat~~inobe~?zaldel~~d~ reaction product is shown in Fig. I in which are also shown the absorption curves obtained from a urine and blank submitted to the above procedure. There is no evidence of any other contributing structure. The rate of colour development was studied by measuring the extinction of a standard solution at IO, 15, 20, 30 and 40 min. The maxil~um extinction was obtained at zo min, with a slight decrease thereafter. The column elution conditions were checked by estimating the indoxylsulphate in fractions from the column after applying standard and urine samples. The indoxyl sulphate was completely eluted after 9 ml eluate had passed through. For convenience the eluate was collected until exactly IO ml was obtained. 1.8t
Wavelength(my) Fig. I. Absorption cwves of urine and standard indican-p-cii~nethylaminobenzaldrhydc products (determined on a Unicam SP Xoo Spectrophotometer).
reaction
INDOXYL
SULPHATE
A linear
IN
101
URIi%;E
response
of extinction
to change
in concentration
was obtained
as
shown in Fig. 2. Replication was exceedingly good-six replicates of one urine sample, carried through the complete method, gave identical results (72.0 mg indoxyl sulphate per 24-h urine). We have not found it necessary in the estimation of indoxyl sulphate to routinely
Fig.
L. Plot
dilute the urines.
of extinction
against
concentration
of indoxyl
sulphate
standards.
Examination of the eluate from 36 urines by ascending paper chromatography, using Fz-butanolLacetic acid-water (120 : 30 : 50) as solvent, showed no other material reacting with fi-dimethylaminobenzaldehyde within z h. No urea was detected, confirming SGrbo’s observation4 that the column retains this material. Several hours after spraying, a few chromatograms showed other Ehrlich-positive spots. This only occurred in 6 out of the 36; in 2 urines the additional spot was blue and in the other 4 yellow (but not urea, from RF value). A number of compounds, mainly indole derivatives, were tested to see if they interfered in the method; these were 3-indolyl acetic acid, 5-hydroxyindolyl acetic acid, kynurenic acid, xanthurenic acid, kynurenin, tryptophan, tryptamine, 3-hydroxy anthranilic acid, urea and hippuric acid. Fifty /lg samples were applied to columns and 5 ml of the IO ml eluate treated with fi-dimethylaminobenzaldehyde for 20 min at room temperature. None of these compounds interfered with the method, all either being retained by the column or not reacting with the reagent under the conditions of the estimation. In separate experiments a mixture of 50 /!g of each of the compounds plus 200 j/g of potassium indoxyl sulphate was estimated after passage through the column and also without the column treatment. No effect on the column performance was produced by these amounts of material and no quenching of the colour with p-dimethylaminobenzaldehyde observed. Column eluates from 6 urine specimens and the standard indoxyl sulphate were reacted with $-dimethylaminobenzaldehyde in the normal way, the solutions basified and extracted with ether. Thin-layer chromatography of the extracts on silica-gel GF254 of thickness 0.25 mm gave only spots corresponding to indoxyl sulphate and fi-dimethylaminobenzaldehyde in the urinary products. Two solvent systems were C/in.
China.
Acta,
26
(1969)
99-103
used (I) ether-petrol ether (100-120~) I:I and (2) 3”1,, ethanol in toluene. The h’,.. values of the product from the standard were in (I) 24 and in (2) 33; the urinary products gave in (I) 26 and in (2) 35. As a result of the alkali treatment before extraction, standard and urinary product appear as red spots on thin-layer chromatograpll~. The absorption
curves were also determined
(Fig. 3).
1.6 1.4 1.2 1.0 (l),
6 t; 0.8 c
(2), (4) from urine samples (3) standard indoxyl sulphare (5) blank
.$0.6 04 0.2
400
450 500 Wavelength (nm)
550
600
Fig. 3, Absorption curves for extracted material from reaction with P-dimethylaminol~enzaldeh~tlc. (Solution made alkaline, ether extracted and after evaporation at room temp. residue taken up in alcohol for spectrometry using a Unicam SF’ 800 Spectrophotometer.) Note: .4lkaline treatment results in A,,,. now being 516 m,u.
The chromatographic
evidence
confirms
the spectrophotometric
finding that
the coloured compounds derived both from standard indican and from urine eluates are identical, thus indicating the specificity of the method. Recovery experiments carried out gave good results (Table I). Examination of
0.060
-
Urine
concentriltion
Fig. 4. Plot of urine concentration C&L. Chim.
.4&z,
26
(1969)
gg-103
against extinction.
INDOXYL TABLE
SULPHATE 1
RECOVERY (.L\s
IN URINE
OF
potassium
ADDED
IPI‘DOXYL
Indoxyl Expected
Ierinc alone +lj/Ag
SULPHATE
indoxyl sulphate)
78
sulphate
(pg/ml)
Found 53 80
Expected
Found
114
89 II8
+jOpK 4~ 75 I%
I”3
‘07
139
139
12x
‘37
1%
I64
4
153
189
189
IOO$/‘h’
dilutions
163 ._
of a urine gave a linear relationship
(Fig. 4). The effect of aspirin metabolites
between concentration
on the estimation
and final reading
was investigated.
No inter-
ference was observed with salicylate (350 mg/Ioo ml), gentisate (5 mg/roo ml) or hydroxyhippurate (350 mg/Ioo ml) when added to urine. The values obtained from 13 normal volunteers for 24-h excretion of indoxyl sulphate were 77.8 & 21.7 mg. Twenty-three patients with rheumatoid arthritis gave values of 80.3 I 41 mg. (Most of the rheumatoid arthritis patients were receiving aspirin.) The value given for indoxyl sulphate output by Bryan2 was 83 f 36 mg/24 h (18 subjects). Our figures are comparable. ACKNOWLEDGEMENTS
I wish to thank Dr. D. B. Horn for helpful criticism D. Bertram for technical assistance.
of the manmcript
and Mrs.
During the period of this study, the Rheumatic Diseases Unit, Northern General Hospital, Edinburgh, was in receipt of grants from the Medical Research Council, the Arthritis and Rheumatism Council, the Nuffield Foundation, and Boots Pure Drug Company Limited, and the author was supported by the McRobert Fellowship awarded by the Arthritis and Rheumatism Council. REFERIXNCIX I G. Cu~zos AXD J. WALSH, Clin. Chim. Acta. 7 (1962) 2 G. T. RRYAS, Anal. B&hem., IO (1965) 120. 3 A. C. BRATTON AND E. Ii. MARSHALL, J. Biol. Chem., 4 TS. SBRRO, Clin. Chim. Acta, 6 (1961) 87.
657. 128
(1939)
537.
Clin. Chim.
Acta,
26 (1969)
99-103
THE
ESTIMATION
99
OF INDOXYL
SULPHATE
IN URINE
H. J. IlYLANCE
Rheumatic
Diseases
Unit,
Northern
General’ Hospital,
Ferry Road, Edinbovgh
4 (U.K.)
(Kevised manuscript received March 27, 1969)
SUMMARY
A method is presented for the determination of indoxyl sulphate in urine. The urine is passed down a Dowex 50-X8 column and the indoxyl sulphate, which is not retained, is estimated in the eluate with p-dimethylaminobenzaldehyde. The 24-h output from normal subjects was 77.8 & 21.7 mg.
INTRODUCTION
Urinary indoxyl sulphate (indican) appears as the eventual result of bacterial action on tryptophan in the gut. The excretion is elevated in conditions where increased bacterial metabolism is occurring ; an alteration in the normal gut flora may also modify the output. Curzon and Walsh1 state : “The determination of urinary indoxyl sulphate may provide a rough estimate of the bacterial contribution to the urinary excretion of aromatic substances in so far as it is influenced by nonspecific dietary factors, and the absorption and movement of intestinal contents.” Our own interest arises as a result of the study of tryptophan metabolism, in patients with rheumatoid arthritis, which has been investigated in this Unit over a number of years. Although indoxyl sulphate results from bacterial action it seemed desirable, for completeness, to investigate the output of this material. There are many methods available for the estimation of indoxyl sulphate in urine. Most involve either oxidation to indigo or various condensation reactions, with solvent extraction as part of the method. That of Curzon and Walshi, for example, used the reaction with fi-dimethylaminobenzaldehyde in acid solution followed by basification and petrol ether extraction, with a correction applied for urea interference. A rapid and specific method, preferably not requiring solvent extractions, would have obvious advantages. The only method along these lines was that of Bryan2, who after treatment of the urine with acid passed it through an ion-exchange column and estimated the indican by the Bratton and Marshall3 reaction. This method appeared accurate but rather tedious. It required 1% of the total 24-h urine specimen, pressure for passing the sample through the column,washing with 2 x 20 ml acid for elution, and finished with an estimation procedure which was read after 3 h. We suggest here a Clin. Ckim. Acta, 26 (IgSg) gg-103
more rapid, reliable method based on the use of a Dowex 5011: column, of indican in the eluate with ~-dimeth~laminc,benzaldeh?_de. MATERIALS
and estimation
AXD METHOD
Dowex 5o\%T-X8 (IOO-zoo mesh) resin was prepared as described by Siirbol and used as a column of dimensions 0.8 x 5 cm. P-Dimethylaminobenzaldehyde reagent : 0.8 g P-dimetl~ylaminobenzaldel~yde (Koch-Light Laboratories Ltd.) was dissolved in 30 ml ethanol and 30 ml concentrated llydr~)chloric acid added.
One ml of urine is added to the column and washed through with z-ml portions of distilled water until IO ml of eluate has been collected. Five ml $-dimethylaminobenzaldehyde reagent is added to j ml of the eluate, and after mixing, left for 20 min at room temperature. The extinction is then read at 480 met against a blank of 5 ml ~-dimethylan~inob~nzaldehyde reagent and j ml v&er. The indican concentration (as potassium salt) is obtained by comparison with a standard curve prepared from potassium indoxyl sulphate (Sigma). RESULTS AND DISCUSSION
The absorption curve for the indoxyl sulpllate-~-dinletl~ylat~~inobe~?zaldel~~d~ reaction product is shown in Fig. I in which are also shown the absorption curves obtained from a urine and blank submitted to the above procedure. There is no evidence of any other contributing structure. The rate of colour development was studied by measuring the extinction of a standard solution at IO, 15, 20, 30 and 40 min. The maxil~um extinction was obtained at zo min, with a slight decrease thereafter. The column elution conditions were checked by estimating the indoxylsulphate in fractions from the column after applying standard and urine samples. The indoxyl sulphate was completely eluted after 9 ml eluate had passed through. For convenience the eluate was collected until exactly IO ml was obtained. 1.8t
Wavelength(my) Fig. I. Absorption cwves of urine and standard indican-p-cii~nethylaminobenzaldrhydc products (determined on a Unicam SP Xoo Spectrophotometer).
reaction
INDOXYL
SULPHATE
A linear
IN
101
URIi%;E
response
of extinction
to change
in concentration
was obtained
as
shown in Fig. 2. Replication was exceedingly good-six replicates of one urine sample, carried through the complete method, gave identical results (72.0 mg indoxyl sulphate per 24-h urine). We have not found it necessary in the estimation of indoxyl sulphate to routinely
Fig.
L. Plot
dilute the urines.
of extinction
against
concentration
of indoxyl
sulphate
standards.
Examination of the eluate from 36 urines by ascending paper chromatography, using Fz-butanolLacetic acid-water (120 : 30 : 50) as solvent, showed no other material reacting with fi-dimethylaminobenzaldehyde within z h. No urea was detected, confirming SGrbo’s observation4 that the column retains this material. Several hours after spraying, a few chromatograms showed other Ehrlich-positive spots. This only occurred in 6 out of the 36; in 2 urines the additional spot was blue and in the other 4 yellow (but not urea, from RF value). A number of compounds, mainly indole derivatives, were tested to see if they interfered in the method; these were 3-indolyl acetic acid, 5-hydroxyindolyl acetic acid, kynurenic acid, xanthurenic acid, kynurenin, tryptophan, tryptamine, 3-hydroxy anthranilic acid, urea and hippuric acid. Fifty /lg samples were applied to columns and 5 ml of the IO ml eluate treated with fi-dimethylaminobenzaldehyde for 20 min at room temperature. None of these compounds interfered with the method, all either being retained by the column or not reacting with the reagent under the conditions of the estimation. In separate experiments a mixture of 50 /!g of each of the compounds plus 200 j/g of potassium indoxyl sulphate was estimated after passage through the column and also without the column treatment. No effect on the column performance was produced by these amounts of material and no quenching of the colour with p-dimethylaminobenzaldehyde observed. Column eluates from 6 urine specimens and the standard indoxyl sulphate were reacted with $-dimethylaminobenzaldehyde in the normal way, the solutions basified and extracted with ether. Thin-layer chromatography of the extracts on silica-gel GF254 of thickness 0.25 mm gave only spots corresponding to indoxyl sulphate and fi-dimethylaminobenzaldehyde in the urinary products. Two solvent systems were C/in.
China.
Acta,
26
(1969)
99-103
used (I) ether-petrol ether (100-120~) I:I and (2) 3”1,, ethanol in toluene. The h’,.. values of the product from the standard were in (I) 24 and in (2) 33; the urinary products gave in (I) 26 and in (2) 35. As a result of the alkali treatment before extraction, standard and urinary product appear as red spots on thin-layer chromatograpll~. The absorption
curves were also determined
(Fig. 3).
1.6 1.4 1.2 1.0 (l),
6 t; 0.8 c
(2), (4) from urine samples (3) standard indoxyl sulphare (5) blank
.$0.6 04 0.2
400
450 500 Wavelength (nm)
550
600
Fig. 3, Absorption curves for extracted material from reaction with P-dimethylaminol~enzaldeh~tlc. (Solution made alkaline, ether extracted and after evaporation at room temp. residue taken up in alcohol for spectrometry using a Unicam SF’ 800 Spectrophotometer.) Note: .4lkaline treatment results in A,,,. now being 516 m,u.
The chromatographic
evidence
confirms
the spectrophotometric
finding that
the coloured compounds derived both from standard indican and from urine eluates are identical, thus indicating the specificity of the method. Recovery experiments carried out gave good results (Table I). Examination of
0.060
-
Urine
concentriltion
Fig. 4. Plot of urine concentration C&L. Chim.
.4&z,
26
(1969)
gg-103
against extinction.
INDOXYL TABLE
SULPHATE 1
RECOVERY (.L\s
IN URINE
OF
potassium
ADDED
IPI‘DOXYL
Indoxyl Expected
Ierinc alone +lj/Ag
SULPHATE
indoxyl sulphate)
78
sulphate
(pg/ml)
Found 53 80
Expected
Found
114
89 II8
+jOpK 4~ 75 I%
I”3
‘07
139
139
12x
‘37
1%
I64
4
153
189
189
IOO$/‘h’
dilutions
163 ._
of a urine gave a linear relationship
(Fig. 4). The effect of aspirin metabolites
between concentration
on the estimation
and final reading
was investigated.
No inter-
ference was observed with salicylate (350 mg/Ioo ml), gentisate (5 mg/roo ml) or hydroxyhippurate (350 mg/Ioo ml) when added to urine. The values obtained from 13 normal volunteers for 24-h excretion of indoxyl sulphate were 77.8 & 21.7 mg. Twenty-three patients with rheumatoid arthritis gave values of 80.3 I 41 mg. (Most of the rheumatoid arthritis patients were receiving aspirin.) The value given for indoxyl sulphate output by Bryan2 was 83 f 36 mg/24 h (18 subjects). Our figures are comparable. ACKNOWLEDGEMENTS
I wish to thank Dr. D. B. Horn for helpful criticism D. Bertram for technical assistance.
of the manmcript
and Mrs.
During the period of this study, the Rheumatic Diseases Unit, Northern General Hospital, Edinburgh, was in receipt of grants from the Medical Research Council, the Arthritis and Rheumatism Council, the Nuffield Foundation, and Boots Pure Drug Company Limited, and the author was supported by the McRobert Fellowship awarded by the Arthritis and Rheumatism Council. REFERIXNCIX I G. Cu~zos AXD J. WALSH, Clin. Chim. Acta. 7 (1962) 2 G. T. RRYAS, Anal. B&hem., IO (1965) 120. 3 A. C. BRATTON AND E. Ii. MARSHALL, J. Biol. Chem., 4 TS. SBRRO, Clin. Chim. Acta, 6 (1961) 87.
657. 128
(1939)
537.
Clin. Chim.
Acta,
26 (1969)
99-103