Catecholamines in urine; an evaluation of alumina-trihydroxyindole methods and a description of an improved method

137

Clinica Chimica Acta, 58 (1975) 137--144 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

CCA 6787 CATECHOLAMINES IN URINE; AN EVALUATION OF ALUMINATRIHYDROXYINDOLE METHODS AND A DESCRIPTION OF AN IMPROVED METHOD

EVELYN S.C. QUEK, J.E. BUTTERY and G.F. DE WITT Division of Biochemistry, Institute for Medical Research, Kuala Lumpur 02-14, (Malaysia)

(Received August, 13, 1974)

Summary A critical assessment of the accuracy and practicability of five alumina-trihydroxyindole methods for the estimation of urinary catecholamines, including our proposed method, was undertaken. The recovery of noradrenaline added to urine obtained by these methods was quantitated against different types of standards, and varied from 22.9% to 104.8%. The major analytical problems (fluorescence suppression and loss of catecholamines during column chromatography) were evaluated and related to the recovery of the respective procedures. The improved m e t h o d we developed is simple, rapid and reliable. The low fluorescence suppression and column losses of noradrenaline of this method, averaging 2.9% and 5.3%, respectively, resulted in a mean recovery Of 92.0% against an external standard. Both the chromatography step and fluorescence development are simple and short, and a batch of ten cases can be completed within 3 hours.

introduction The most sensitive estimations for urinary catecholamines are currently performed fluorimetrically, of which modifications of the t r i h y d r o x y i n d o l e (THI) methods of Ehrldn [1] and Lund [2] are extensively employed. Most of these methods are based on selective adsorption of the catecholamines on aluminium oxide at pH 8.0--8.5, elution with acid and subsequent oxidation of the amines to their highly fluorescent trihydroxyindole derivatives. In a preliminary survey of some of these methods, we f o u n d that despite their fundamental similarity in principle, the technical variables introduced resulted in considerable differences in the analyses. When comparing the recovery data by these methods on urine samples, a wide range of values was found, m a n y of which were poor or unsatisfactory. Thereupon, a critical study was undertaken to assess the reliability of these methods. This subsequently led to the develop-

138 men~ of an improved m e t h o d which we found to be simple, fast and accurate and very suitable for the clinical laboratory. The various methods studied in our investigation are: Method 1, Sobel and Henry [3]; M e t h o d 2 , Beckman Fluorimetric Procedures Manual [4]; Method 3, Oxford Kit m e t h o d [5] ; Method 4, yon Euler and Lishajko [6] and Method 5, our proposed method. Methods 1--4 were selected on the basis that they were manual methods employed routinely in hospital laboratories; they were methods using alumina columns, and had different approaches for quan. titation of the urinary catecholamines. From our investigation, the Oxford Kit procedure was the best of the four methods studied as judged from recovery studies. However, it was the most expensive m e t h o d with a lengthy chromatography procedure. We developed a m e t h o d modifying the chromatography step but developing the fluorescence according to the Oxford Kit. Our m e t h o d is rapid, accurate and economical. Fluorescence suppression and losses from the column are minimised, thus justifying the use of an external standard for quantitation. Details of our method will be presented in the latter part of this paper. Evaluation o f Methods

Experimental and Results Methods 1--4 were performed as described in the original papers. Throughout the investigation, all analyses were performed by one analyst after familiarization with each method. For chromatography, pyrex glass columns of suitable dimensions were used except in Method 3 where the Oxford Kit columns and funnels were used. Aluminium oxide (BDH, for chromatographic adsorption analysis, Brockman activity II) was preconditioned if necessary and used according to the respective procedures. Noradrenaline standards for fluorescence development were prepared in appropriate acetic acid strength and fluorescence reading was taken on the Beckman Model 772 Ratio Fluorometer. Recovery studies were done on four different pooled urines collected from laboratory, staff, preserved with acid and refrigerated. Each urine pool was used for a week to run five analyses, one for each of the five methods. For each m e t h o d evaluated, recoveries of added noradrenaline to urine were calculated taking into account three different types of standards: (a) internal standard, consisting of noradrenaline standard added to the column eluate prior to fluorescence development; (b) column standard, an aqueous standard passed through the column; (c) external standard, an aqueous standard introduced at the stage of fluorescence development. Losses due to fluorescence suppression and column chromatography, the major factors leading to poor recoveries, were also quantitated. The average time required to process each sample through the column was recorded. All these data have been tabulated in Table I. Experimental details of the evaluation were as follows: During the analysis of each method, three columns were set up, and the following specimens A, B and C were run through columns 1, 2 and 3, respectively. Specimen A (basal) consists of a pooled urine specimen; specimen B

139 TABLE I c o M P A R A T I V E D A T A O F R E C O V E R Y S T U D I E S ON F I V E A L U M I N A - - T H I M E T H O D S Added n o r a d r e n a l i n e w a s 2 0 0 - - 2 5 0 ~g/l. a n d s t u d i e s w e r e d o n e o n f o u r d i f f e r e n t p o o l e d urines.

Recovery b a s e d on

internal standard (%)

Recovery b a s e d o n

column standard (%)

Recovery b a s e d on

external standard (%)

Fluorescence suppression (%)

Column losses o f noradrenaline a d d e d to urine (%)

Column losses o f aqueous n o r a d r e n a l i n e standard (%)

Average t i m e for c o l u m n chromatography procedure Urine a l i q u o t p r o c e s s e d through c o l u m n

Method of Sobel a n d Henry

Method from Beckman Manual

Method from Oxford Kit

Method of yon Euler and Lishajko

Proposed method

69.4 62.1 63.8 73.0 ~-67.1 49.0 61.4 56.4 71.1 ~-59.5 26.0 28.1 22.9 27.8 ~-26.2 62.5 54.8 64.1 61.9 ~-60.8 30.6 37.9 36.2 26.9 ~-32.9 46.9 54.2 59.4 60.8 ~-55.3 7 0 rain

80.3 81.2 80.3 79.4 x- 8 0 . 3 92.7 93.1 84.6 83.3 ~- 8 8 . 4 60.0 65.8 61.8 55.6 ~" 6 0 . 8 25.3 18.9 23.0 30.0 ~-- 24.3 19.7 18.8 19.7 20.6 ~- 19.7 35.3 29.3 27.0 33.4 ~'- 3 1 . 3 105 rain

89.0 84.7 86.7 89.6 ~- 8 7 . 5 97.3 91.1 97.3 93.6 x- 9 4 . 8 83.9 81.8 80.0 79.3 ~- 8 1 . 3 5.7 3.4 7.8 11.4 ~7.1 11.0 15.3 13.2 10.4 x- 12.5 13.8 10.2 17.8 15.2 ~- 14.3 135 rain

65.2 61.2 62.5 61.8 ~'62.7 81.9 71.8 72.4 70.5 ~-74.1. 64.8 59.6 58.5 59.1 ~'60.5 0.5 2.7 6.4 4.3 x- 3.5 34.8 38.8 37.5 38.2 x'-37.3 20.9 17.0 19.1 16.1 x-18.3 60 rain

91.4 95.2 97.5 94.8 ~- 9 4 . 7 104.8 103.5 103.6 98.2 ~-102.5 87.4 95.2 92.1 93.2 ~- 9 2 . 0 4.3 0 5.6 1.7 ~" 2.9 8.6 4.8 2.5 5.2 ~" 5.3 12.5 8.1 11.1 5.1 x'- 9.2 35 rain

10 m l

10 m l m i x e d w i t h 5 m l 0.2 m o l / l sodium acetate

5 ml

25 m l

1 ml

(recovery) consists of the same pooled urine with a known a m o u n t of noradrenaline standard added; and specimen C (column standard) is an aqueous noradrenaline standard having a concentration as t h a t added in specimen B. Fluorescence development was carried out on the column eluates of A, B and C to obtain the recovery data tabulated in Table I. Blanks, whether faded or nonoxidized, were also set up according to the particular method. For each recovery experiment, ten tubes were set up as in Table II. Tubes 1, 4, 7 and 9 represent basal, recovery, column standard and external standard, respectively; tubes 3, 6, 8 and 10, their corresponding blanks. Tubes 2 and 5 are the so-called internal standards of basal and recovery, respectively. They contain pure standard added to the eluate prior to fluorescence development.

140

T A B L E II SCHEME FOR FLUORESCENCE T u b e No.

DEVELOPMENT

OF COLUMN ELUATES

C o n t a i n i n g X ml* eluate of c o l u m n

1 1 2 1, w i t h s t a n d a r d a d d e d to e l u a t e 3 1 4 2 5 2, w i t h s t a n d a r d a d d e d to e l u a t e 6 2 7 3 8 3 9, c o n t a i n i n g X m l w o r k i n g s t a n d a r d s o l u t i o n 10, c o n t a i n i n g X m l w o r k i n g s t a n d a r d s o l u t i o n

Representing basal internal standard basal blank recovery internal standard recovery blank column standard column standard external standard ex t e r n a l s t a n d a r d

o f basal

o f recovery

blank blank

* X ml is t h e c o l u m n e l u a t e a l i q u o t u s e d in a p a r t i c u l a r m e t h o d .

T o m i n i m i s e d i f f e r e n c e s in v o l u m e in t h e v a r i o u s t u b e s , t h e standard a d d e d t o t u b e s 2 a n d 5 is e i t h e r c o m p e n s a t e d w i t h t h e s a m e v o l u m e of d e i o n i z e d w a t e r in t h e o t h e r t u b e s w h e n specified b y t h e m e t h o d , or its volume k e p t v e r y small ( n o t e x c e e d i n g 1 0 0 pl). T h e a b s o l u t e a m o u n t s o f a d d e d n o r a d r e n a l i n e c o n t a i n e d in the internal standard, c o l u m n standard a n d e x t e r n a l standard w e r e the s a m e . This facilitated direct comparison and quantitation. T h e d a t a o b t a i n e d were p r o c e s s e d as follows: (i) C o r r e c t e d f l u o r e s c e n c e = s a m p l e f l u o r e s c e n c e - - b l a n k f l u o r e s c e n c e e.g. c o r r e c t e d f l u o r e s c e n c e o f basal = {Tube 1 - - T u b e 3) (ii) R e c o v e r y o f n o r a d r e n a l i n e a d d e d to urine, R = C o r r e c t e d f l u o r e s c e n c e o f r e c o v e r y - - C o r r e c t e d f l u o r e s c e n c e o f basal = ( T u b e 4 - - T u b e 6) - - ( T u b e 1 -- T u b e 3) (iii) C o r r e c t e d f l u o r e s c e n c e o f internal s t a n d a r d , X = {Tube 2 - - T u b e 1) or X = ( T u b e 5 - - T u b e 4) T h e m e a n o f t h e two readings is used. (iv) C o r r e c t e d f l u o r e s c e n c e o f c o l u m n standard, Y = ( T u b e 7 - - T u b e 8) (v) C o r r e c t e d f l u o r e s c e n c e o f e x t e r n a l standard, Z = ( T u b e 9 - - T u b e 10) (vi) R e c o v e r y b a s e d o n internal standard (%) = 1 0 0 R / X (vii) R e c o v e r y b a s e d on c o l u m n standard (%) = 100 R / Y (viii) R e c o v e r y b a s e d on e x t e r n a l standard (%) = 1 0 0 R / Z (ix) F l u o r e s c e n c e s u p p r e s s i o n (%) = 100 ( Z - X ) / Z (x) C o l u m n losses o f n o r a d r e n a l i n e a d d e d to u r i n e (%) = 1 0 0 - - ( 1 0 0 R / X ) (xi) C o l u m n losses o f a q u e o u s n o r a d r e n a l i n e s t a n d a r d (%) = 100 (Z -

g)/z T h e t i m e r e q u i r e d t o process each s a m p l e t h r o u g h t h e c o l u m n was recorded a n d t h e average t i m e c a l c u l a t e d .

Discussion In o u r p r e s e n t s t u d y o f five a l u m i n a - - T H I m e t h o d s f o r u r i n a r y catecholamines, several variables have b e e n e v a l u a t e d on a c o m p a r a t i v e basis, t h e most e x t e n s i v e being the r e c o v e r y e x p e r i m e n t w i t h r e c o v e r i e s ranging f r o m 22.9 to

141 104.8% (Table I). The low recoveries are mainly due to fluorescence suppression by interfering urinary constituents in the eluate and losses of catecholamines from the column. These factors have led workers to adopt alternative approaches to quantitation by using an internal standard in the eluate or a standard passed through the column, instead of an external standard. These approaches are at best compromises for methods t h a t are n o t analytically accurate. The critical assessment of each m e t h o d follows.

Method 1, Sobel and Henry This m e t h o d showed a high degree of fluorescence suppression (mean 60%} with losses of catecholamines during the column run. Hence the low recovery obtained (mean 26.2%) when an external standard was used for quantitation. The recovery improved to 59.5% and 67.1% when quantitation was based on column standard or standard added to eluate, respectively. Thus the internal standard which Sobel and Henry proposed for each urine eluate seems appropriate for correcting fluorescence suppression but it does n o t account for losses through the column which is about 33%. The average time required to process one urine sample through the column is about 70 min. In the fluorescence development the extra tubes for the internal standards of the specimens limit the batch size t h a t can be handled. Centrifugation is necessary prior to fluorimetric reading and the delay may affect the result as the fluorescence is unstable after 10--20 min. Method 1 is therefore not reliable but it has been useful as a basis for several modifications, including Method 2.

Method 2, Beckman fluorimetric procedure This m e t h o d is more tedious and lengthier than Method 1. Columns are not allowed to drain completely during analysis, thus limiting the batch size that can be processed. Recoveries, however, are much better as there is less fluorescence suppression and losses via the column. The mean recovery based on external standard was only 60.8%, but with column standard and internal standard the recoveries increased to 88.4% and 80.3%, respectively. As losses of noradrenaline in aqueous standard passed through the column exceeded that in urine, quantitation with this column standard gave an apparently better recovery yield. There is a time-consuming step of adjusting all solutions to pH 6.5 on pH paper prior to fluorescence development. Besides, the centrifugation step of the previous m e t h o d is retained.

Method 3, Oxford Kit procedure The Kit procedure had the best recovery for the four selected methods, averaging 81.3% on the external standard. With internal standards, the recovery increased to 87.5% and with column standards, to 94.8%. Losses of noradreaaline from the column for aqueous standard averaged 14.3% and for urine 12.5%. Thus the use of column standards as suggested by the Kit was appropriate. The chromatography procedure was lengthy and t o o k a b o u t 135 min as it involved several voluminous washes. Fluorescence development was, however,

142 simple and rapid. Our proposed m e t h o d is an improvement of this Kit proce. dure. Method 4, yon Euler and Lishajko This m e t h o d gave unsatisfactory recoveries {mean 60.5%) when based 0n an external standard as recommended by the authors. This poor result was due to losses of urinary noradrenaline via the column which averaged 37.3%. The original workers did n o t correct for this loss in their method. We have found that quantitation using column standard did n o t improve much the recovery values as the noradrenaline losses from the column standard were much less than the corresponding losses from urine specimen. It might be significant to note that the fluorescence suppression by this m e t h o d was low (3.5%) but in achieving this, losses of urinary noradrenaline from the column were appre. ciable. The fluorimetry step of this m e t h o d was generally tedious and difficult, especially the pH adjustment of the eluate with ammonia. Method 5, our proposed method The details of our m e t h o d are described in the next section. Mean re. coveries based on internal, external or column standard were better than 90%. Fluorescence suppression was low {2.9%} and column losses of noradrenaline averaged 5.3%. As such, the use of an external standard is acceptable and this further simplifies the method. The column run is rapid and takes about 35 min. The fluorimetry procedure is simple and rapid, being essentially similar to that of the Kit procedure. Proposed m e t h o d Materials and Procedure Reagents 1. Aluminium oxide (BDH), washed several times with deionized water to remove fine particles and dried at 100--120 °. Store in a tightly stoppered brown bottle. 2. EDTA (disodium salt), 100 g/1. 3. Sodium hydroxide, 1 mol/l and 5 mol/l. 4. Sodium acetate, 0.2 mol/1. Store in refrigerator. 5. Acetic acid, 0.4 mol/1. 6. Disodium tetraborate (Na2B40~" 10H20), 30 g/1. 7. Potassium ferricyanide, 5 g/1. Prepare fresh every month. 8. Alkaline ascorbate mixture. Prepare just before use. Weigh 100 mg L-ascorbic acid and dissolve in 5 ml deionized water. Mix 1 part ascorbic acid solution with 9 parts 5 mol/l NaOH. 9. Noradrenaline stock standard (= 1 g noradrenaline/1). Weigh 188.7 mg L-arterenol bitartrate (Sigma Chemical Co.), dissolve and make up to 100 ml with 0.1 mol/l HCI. Store in refrigerator and renew after 6 months. Substock (- 2 mg/1). Dilute 0.1 ml stock solution to 50 ml with 0.1 mol/l HC1. Store in refrigerator and renew every m o n t h .

143

Working standard (= 20 pg/1). Prepare fresh by diluting 0.1 ml substock to 10 ml with 0.4 mol/1 acetic acid.

Apparatus Chromatography columns, Pyrex (obtainable from Griffin and George Ltd, Middlesex, England, or Fisher Scientific Co., Pittsburg, Pa.). Borosilicate glass, internal diameter 1.0--1.1 cm, height 30--40 cm, with sealed-in sinteredglass disc of No. 1 porosity at the lower end. The column height was cut to 20 crfl.

Beckman Model 772 Ratio Fluorometer, with mercury lamp and the phosphor sleeve at position Hg. Primary filters: for reference beam: Schott UG 11; for sample beam: Schott GG 13, Coming CS 5-58 and Coming CS 7-51; (-= 405 11121),

Secondary filters: Coming CS 3-71 and Coming CS 4-72; ( - 495 nm). Matched premium cuvettes {Beckman), 12 mm b y 75 mm.

Method 24-h urine specimens are collected in brown bottles containing 15 ml 50% concentrated HCI as preservative and kept refrigerated. Place a 25-ml urine aliquot in a beaker, add 5 drops of EDTA solution, and adjust urine to pH 8.2--8.5 with reagent 3 using pH paper. Rapidly centrifuge a b o u t 10 ml of the urine and use within an hour. Pour 1.5 g aluminium oxide into the glass column arranged vertically on a rack. Prime the alumina with 5 ml deionized water followed by 5 ml 0.2 mol/1 sodium acetate. Introduce 1.0 ml of the pretreated urine into the column and allow to percolate. Wash the column with 10 ml of 0.2 mol/1 sodium acetate and 10 ml deionized water, respectively, w i t h o u t disturbing the alumina. Allow the column to drain completely between additions. Discard all column effluents up to this stage. The adsorbed catecholamines are subsequently eluted with 10 ml 0.4 mol/l acetic acid and collected. Fluorescence development m a y be delayed as the catecholamines in the acid eluate are stable for 24 h at 4 °. Fluorimetry is according to the Oxford Kit procedure as outlined in Table III.

TABLE IIl

Standard (S)

Standard blank (SB)

Urine test (T)

Urine blank (TB)

Column eluate --1.0 ml 1.0 ml Working standard 1.0 ml 1.0 m! --Disodium tetraborate 2.5 ml 2.5 ml 2.5 ml 2.5 ml Potassium ferricyanide 0.1 m l -0.1 m l -Seal w i t h p a r a f l l m , m i x b y inversion and l e t s t a n d f o r 3 - - 5 r a i n . Alkaline a s c o r b a t e 1.0 ml 1.0 ml 1.0 ml 1.0 m l Mix a g a i n b y inversion a n d r e a d f l u o r e s c e n c e w i t h i n 2 0 r a i n , e a c h test f o l l o w e d by its c o r r e s p o n d i n g blank.

144

Calculation T - - TB Urinary catecholamines (pg/24 h) - S -- S-----B×

20 x

24-h urine volume (ml) 0.1

_ T T--_____B_B x 24-h urine volume (ml) S - - SB Results and Discussion

Accuracy The accuracy of the proposed m e t h o d was determined by assaying a freeze-dried control urine (Bio-Reagents and Diagnostics Inc., California) in duplicates in three different runs. The value obtained was 570 +- 30 pg/1 as compared with the reference value of 600 +- 100 pg/1. Recovery studies were also done on aliquots of a pooled urine specimen containing different amounts of added noradrenaline. The mean percentage recoveries based on triplicate assays were 92.6, 88.7, 92.0, 89.8 and 85.7% for added noradrenaline equivalent to 50, 100, 200, 250 and 500 pg/l, respectively. Precision Duplicate assays were done on 23 ward cases (range 4.2---600 pg/24 h) and the coefficient of variation (CV) and the "95% limits" calculated according to Henry and Dryer [7] were 5.39% and 149.3 ± 11.15%, respectively. The between-batch precision was also assessed on a urine control (mean 627 pg/24 h) during six different estimations. The CV was 4.15% and the "95% limits" was 627 ± 10.67%. Our improved m e t h o d is thus simple, rapid and reliable. The normal urinary excretion of catecholamines is below 100 pg/24 h. References 1 2 3 4

I. Ehrl~n, F a r m . R e v y , 4 7 ( 1 9 4 8 ) 2 4 2 A. L u n d , A c t a P h a r m a c o l . ( K b h . ) , 5 ( 1 9 4 9 ) 2 3 1 C. S o b e l a n d R . J . H e n r y , A m . J. Clin. P a t h o l . 27 ( 1 9 5 7 ) 2 4 0 B i o - S c i e n c e L a b o r a t o r i e s , C a l i f o r n i a ( E d s ) , Clinical A n a l y s i s f o r C a t e c h o l a m i n e s in Urine, in B e c k m a n Fluorimetric Procedures Manual 1447 5 O x f o r d L a b o r a t o r i e s , F o s t e r C i t y , C a l i f o r n i a , U . S . A . , Kit for d e t e r m i n a t i o n o f free c a t e c h o l a m i n e s in urine 6 U.S. v o n E u l e r a n d F. L i s h a j k o , A c t a P h y s i o l . S c a n d . , 51 ( 1 9 6 1 ) 3 4 8 7 R . J . H e n r y a n d R . L . D r y e r , in D. S e l i g s o n ( E d . ) , S t a n d a r d M e t h o d s o f Clinical C h e m i s t r y , A c a d e m i c Press, N e w Y o r k , Vol. 4, 1 9 6 3 , p p . 2 1 6 a n d 2 1 7