A simple and rapid method for the determination of 4-hydroxy-3-methoxymandelic acid (HMMA) in urine

Clinica Chimica Acta, 44 (1973) 179-182 Q Elsevier Scientific Publishing Company,

A

SIMPLE

AND

RAPID

METHOD

q-HYDROXY-3-METHOXYMANDELIC

J. E. BUTTERY

- Printed in The Netherlands

FOR

THE

DETERMINATION

ACID

(HMMA)

IN

179

OF

URINE

AND G. F. DE WITT

Department of Biochemistry. (Malaysia) (Received

Amsterdam

September

Institute for Medical Research, Pahang Road, Kuala Lumpuv

13, 1972)

SUMMARY

A simple and rapid method for the determination of urinary 4-hydroxy-3methoxymandelic acid (HMMA) using Dowex resin is outlined. The HMMA is measured as a colour complex with diazotised p-nitroaniline. The method also allows for the determination of high HMMA levels as well as repeat estimations from the eluate which can then be completed within 20 min. An average of six estimations can be done by one technician within 3 h. Repeatability is good and good correlation is found when compared with the method of Pisano. We have found the proposed method to be suitable for routine work.

The urinary level of 4-hydroxy-3-methoxymandelic acid (HMMA) is frequently measured by the method of Pisano I, but the procedure is fairly laborious. Wybenga and Pileggi2 introduced a method based on the adsorption of HMMA on to Dowex resin. The HMMA was eluted with 3 M sodium chloride, oxidised to vanillin and then reacted with indole phosphoric acid reagent in an ice bath. Good agreement with the Pisano method was claimed by these workers, but in our hands we have not been able to achieve this. The measurement of the colour complex at the low temperature was not possible under the prevailing high humidity of the tropics which caused misting on the surface of the cuvettes. At the temperature where misting did not occur, the colour complex showed progressive increase in absorbance. The method proposed in this paper employs the Dowex resin adsorption technique of Wybenga and Pileggi with modifications both in the chromatographic procedure and in colour development step. First, in the chromatographic procedure, standard disposable Pasteur pipettes were used. The resin column height was reduced from 6 cm to 3 cm and the urine volume reduced from 5 ml to 2 ml. This resulted in a substantial shortening of time taken to process the urine through the resin. The eluted HMMA was then made to react directly with diazotised p-nitroaniline according to the method of Woiwod and KnighV. The resultant complex was extracted into chloroform, re-extracted into alkali to produce the pink colour which was read colorimetric-

180

BUTTERY,DE

WITT

ally. Our modifications employed a single extraction step each with chloroform and alkali instead of using three and four extractions respectiveiy as was done by Woiwod and Knight. The overall working time of our method was shortened by half as when compared with the Wybenga and Pileggi procedure. The linearity between HMMA concentration and absorbance of the colour holds good up to a concentration equivalent to IOO mg HMMA/l. REAGENTS I. Dowex-AG xX4, IOO-zoo mesh (Bio-Rad Laboratories) for preparation to Wybenga and Pileggi. 2. Phosphate buffer, 0.1 M, pH 6.1. 3. Sodium chloride, 3 M solution. 4. Potassium carbonate, 10% solution. 5. Sodium hydroxide, 0.2 M solution. 6. Chloroform, Analar grade. 7. IIiVIRlA standard, 50 pg/ml. Dissolve 50 mg HMMA (Sigma) in water make up to I litre. Dispense into small tubes and store in deep freezer. Use one for a series of estimation. 8. ~-~itroaniline diazotised reagent. Prepared as according to ?Voiwod

refer

and tube and

Knight. METHODS

Standard disposable Pasteur pipette having an internal diameter of 5 mm and length cut to 13 cm is found suitable as a column for the Dowex resin. A little cotton wool is placed inside the column at the point where the constriction begins. A slurry of resin is poured into the column to a height of 3 cm by means of a Pasteur pipette and allowed to drain. The 24-h urine specimen collection contains IO ml glacial acetic acid. Pour about 30 ml of the urine into a beaker and adjust pH to 6.0-6.5 on pIi paper with 5 XI sodium hydroxide. Two nlillilitre urine specimen mixed with z ml phosphate buffer pH 6.1 is passed through the column. The eflluent is discarded. The column is washed with 1.0 ml of a mixture of I volume water and I volume phosphate buffer. The HMMA is now eluted with 8.0 ml of 3 M sodium chloride. Into a 3o-ml Quickfit tube containing TO ml water, is placed 2.0 ml of the eluate. The standard tube contains 2.0 ml 3 ICI sodium chloride, IO ml water and 0.1 ml standard EMMA. The colour development at this stage should be carried out in subdued light. One ml of the freshly prepared $-nitroaniline diazotised reagent is now added to each tube, mixed and allowed to stand for z min, after which 1.0 ml of 10% potassium carbonate is added, mixed and allowed to stand for a further 3 min. Ten ml of chloroform is now added to each tube and shaken for z min. Allow the two layers to separate out by twirling the tubes (if necessary) and aspirate off the top aqueous layer. Transfer 5.0 ml of the clear chloroform layer into a Is-ml Quickfit tube. Two and a half ml of 0.2 M sodium hydroxide is added and the tube shaken for 2 min. Read the absorbance of the alkali layer at 510 nm setting to zero with water.

181

HMMA IN URINE

Calculation

HMMA(m&v h) =

Test absorbance Std

absorbance

Test absorbance

Std. absorbance V is the volume of the 24 h urine specimen. RESULTS

5

x~ 1000 X-

X

5

V 100

AND DISCUSSION

The proposed method was compared

with the method of Pisano and the results “elevated” concentrations of I. Urine samples containing by mixing urines of high and low HMMA concentrations.

are outlined in Table HMMA were obtained

The correlation coefficient (Y) was 0.74 (9 < 0.001) for the test cases and for the “elevated” cases Y = 0.97 (9 < 0.001). The two methods gave good correlations which were significant at less than the o.IO/~ level.

Urine

Numbev cases

specimen

Test cases (q/z4 “Elevated” (q/l)

h)

20

‘4

of

Method of Pisano Mean * S.D.

Proposed Mean +

3.0 It I.3 27.9 f 7.’

4.0 + 24.4 *

method S.D. I.5 5.6

In one measurement of a high urinary HMMA level (> IOO mg/24 h), not included in Table I, repeat analyses by both methods were necessary. The proposed method permitted the repeat to be done within 20 min using a smaller eluate volume for the colour development. The repeat analysis by the method of Pisano using a smaller urine volume took half a working day to perform. When HMMA is added to an aliquot of the eluate, the recovery was generally poor. This led us to suspect that urinary inhibitors were suppressing the colour formation. With a sixfold dilution of the eluate (z ml eluate plus IO ml of water), the inhibition of the colour formation was kept minimal. Replicate analyses (n = 20) done on Hyland Urine Chemistry Control gave a mean value of 8.20, S.D. 5 0.16 and a coefficient of variation of 2.0%. The proposed method gave higher values when compared with the Pisano method for normal HMMA excretion levels (Table I). The difference of the mean by the two methods was +I.o, but for the “elevated” HMMA specimens, the proposed method gave a lower mean value, the difference being -3.5. These differences did not constitute any serious objection for the proposed method which showed good correlation with the Pisano method. Urinary HMMA levels below IO mg/24 h is taken as our normal excretory level. ACKNOWLEDGEMENTS

We wish to thank the Central Co-ordinating

Board of Trop-Med

(SEAMES)

for

BUTTERY,

182

financial assistance in support of this investigation. Weng Yoke for his technical assistance.

We also wish to thank

REFERENCES

I J. J. PISAKO, J. K. CROW ANU D. ABRAHAM, Cl&. Chim. Acta, 7 (1962) 285. 2 D. WYBEKGA AXD V. J. PILEGGI, C&z. Cl-km. Acta, 16 (1967) 147. 3 A. J, WOIWOD

ASD R. KNIGHT,

J. C&n. P&d,

1-1_(1961) 502.

DE WITT

Ah-. Chu