Annals of Clinical Biochemistry, 1979, 16, 38-43
Routine laboratory investigation of urinary catecholamine metabolites in sick children C. E. NIEHAUSl R. S. ERSSER, AND SHEILA M. ATHER DEN From the Hospital for Sick Children and Institute of Child Health, 30 Guilford Street, London SUMMARY Simple and rapid thin-layer chromatographic methods have been used to investigate the catecholamine metabolites present in the urine of sick children. A semi-quantitative method for 4-hydroxy-3-methoxy-mandelic acid (HMMA) has been devised and compared with the quantitative spectrophotometric procedure. The methods have been performed on both normal subjects and children with catecholamine-secreting tumours, without dietary restriction, and have led to a 50 ~;I reduction in the number of samples requiring laborious quantitative determination of HMMA excretion.
There are good clinical indications for determining the excretion of urinary catecholamine metabolites in both children and adults (Sunderman, 1964). In adults the catecholamine-secreting tumours are mainly phaeochromocytomas and rarely chemodectomas, whereas in children they form the neuroblastoma - ganglioneuroma - retinoblastoma group (Sandler, 1967). While several catecholamine metabolites appear in the urine of patients with such tumours (Wadman et al., 1976), 4-hydroxy-3-methoxy-mandelic acid (HMMA) excretion is commonly elevated. The quantitative and relatively specific method of Pisano et at. (1962) is satisfactory for the estimation of HMMA but it is time-consuming and unsuitable for total automation. In addition, a small number of these tumours in children secrete the dopamine metabolites homovanillic acid (HY A) and 3, 4-dihydroxy phenyl acetic acid (DOPAC) in the urine rather than HMMA (Wadman et al., 1976). Improvements in the therapy and long-term management of children with such tumours and the growing interest in hypertension during childhood has resulted in a large increase in requests for the estimation of urinary HMMA and other catecholamine metabolites. The routine service offered by our laboratory has therefore been re-assessed and more efficient methods of analysis have been sought. We describe the methods that have led to a 50 % reduction in the number of samples requiring quantitative determination of HMMA. This has lPresent address: Dr C. E. Niehaus, Clinical Laboratories, PO Box 4419, Pretoria, S. Africa.
enabled us to increase the number of patients who can be investigated without an increase in staff. Material and methods URINE SAMPLES
Twenty-four-hour urine collections, stored in bottles containing 5 ml of 6M hydrochloric acid, were obtained from 30 children with catecholaminesecreting tumours. Both newly diagnosed and treated patients were included. Urine samples from a group of 93 hospitalised children without tumours served as controls. METHODS
Urinary creatinine was estimated by the Technicon automated alkaline picrate method. Acid up to a final urine concentration of 2M did not interfere with the result. Quantitative estimation of HMMA was performed by the colorimetric method of Pisano et at. (1962). Chromatography was performed on aliquots of a concentrated ether extract of urine (Ersser et al., ] 970). Urine (2 ml) was acidified with concentrated hydrochloric acid (0'2 ml) saturated with sodium chloride, and the phenolic compounds were extracted by vigorous shaking with di-ethyl ether (8 ml). After centrifugation the ether layer was transferred to a wide-necked bottle and allowed to evaporate either overnight in a fume cupboard, or by gentle warming, or by use of a rotary evaporator. The residue was dissolved in 0'1 ml 50 % aqueous propan-Z-ol. Volumes of this solution, related to the creatinine content of the original urine, were used for chromatography (Table 1).
38
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Routine laboratory investigation of urinary catecholamine metabolites in sick children
Table I
Volume of extract applied to chromatograms
Creatinin« (mmollf)
Volume or extract (,./) J-dimensional (/()() mm)
(50 x 50 mm).
-HlO
18'0
6'0
1'01-1'~0
I~'O
o
2-dimensiona/
1'~1-2'OO
13'~
~'O 4'~
2'0l-2'~0
12-0
4-0
2·~1-2·7~
10'~
3'~
2'7~3'OO
9·0
3·0
7'~
2'~
J'~l-4'~O
6·0
2·0
4'~1-~'~0 ~'~1-7'OO
4'~
l-~
3·0
1·0
7'01-8'~0
2'2~
0-7~
8'~I+
1'~
O'~
lOl-3'~0
-Four times this volume is applied to layers] 00
'\Ie
100 mm in size.
A semi-quantitative estimate of HMMA concentration was obtained by chromatographing the extracts together with two standards of known concentration: one approximated to the upper limit of normal (2'5 JLI of a 0·007 mM solution of HMMA in water) and the other was twice this amount (5'0,....1). Aliquots of the standard solutions were stored frozen and discarded after use. Samples were applied, using a graduated microlitre syringe, as bands (15 mm long) to layers of silica gel (Kieselgel 60, F 254, E. Merck No. 5554, BDH, Poole, Dorset), 100 mm long, and dried with warm air from a fan. A single ascending development in amyl acetate: glacial acetic acid: water (60 : 20 : 6) was performed. A 75 mm solvent rise from the origin took approximately 40 minutes at 25°C. After drying with warm air the layer was viewed under short-wave ultraviolet light (254 nm). While only grossly elevated concentrations of HMMA were detected at this stage, the presence of possible interfering substances such as glycine conjugates of phenolic acids was revealed.
39
The layer was sprayed with saturated aqueous sodium carbonate and allowed to dry. At this stage readily oxidisable di-hydroxy compounds such as DOPAC and homogentisic acid appeared as brown bands. The layer was then sprayed with a freshly made saturated solution (approximately 200 mg/lOO ml) of stabilised diazonium salt of p-nitroaniline (Koch-Light Laboratories, Colnbrook, Bucks) dissolved in 30% aqueous ethanol. In addition to the purple band of HMMA, a variety of colours were produced by other phenolic compounds present in the extract, as illustrated in Table 2. The colours started to fade within 15 minutes of location unless the layer was stored at -20°C. A two-dimensional chromatogram on microcrystalline cellulose (Schleicher and Schuell F.1440, Anderman and Co, E. Molesey, Surrey) was prepared using a second aliquot of the extract as described by Ersser et al. (1970). Layers of 50 mm square were used as the process could be completed in one hour. A solvent mixture of propan-Z-ol : butan-I-ol : 2-methyl-propan-2-o1 : 25 % ammonium hydroxide : water (40 : 20 : 20 : 10 : 20) was used in the first dimension (15 minutes for a 40 rom rise at 25°C) and benzene: acetic acid: water (70 : 29:1) was used in the second dimension (5 minutes). The layers were sequentially viewed under ultraviolet light (254 nm and 366 nm), dipped in a 1 % solution of acridine in acetone, dried and viewed under ultraviolet light (366 nm) to locate acidic compounds, sprayed with saturated aqueous sodium carbonate, dried, and finally sprayed with diazotised sulphanilic acid to locate phenolic compounds (Smith and Seakins, 1976). HMMA yielded an orange colour and HV A a purple colour. The reactions of other commonly occurring compounds are shown in Table 2. Chromatograms were assessed by visual
Table 2 Colour reactions of common phenolic acids with diazo reagents No. Acid
Colour with alkaline diazo reagents
Sulphanilic acid
p-Nitroaniline
1 Vanillic 2 HVA 3 js-Hydrcxyphenylacetic 4 s-Hydroxyhippuric ~ 3-Hydroxy+methoxy-phenylhydracrylic 6 m-Hydroxyhippuric 7 DOPAC 8 Homogentisic 9 HMMA 10 ~.Hydroxy-indolylacetic 11 Aceto-acetic 12 p-Hydroxypbenyllactic 13 e-Hydroxyphenylacetic 14 p-Hydroxybenzoic U p-Hydroxymandelic 16 m-Hydroxyphenylacetic
Orange
Purple Blue Mauve Red Red Red
Pink Red/brown Yellow Yellow Yellow
Brown Brown
Orange Red Red/brown Yellow Yellow Yellow Yellow
Downloaded from acb.sagepub.com by guest on June 4, 2016
Purple Purple Yellow Mauve Pink Red Orange Red
40
C. E. Niehaus, R. S. Ersser, and Sheila M. Atherden
comparison with normal samples run simultaneously. Two-dimensional chromatography for amino acids was also performed on the neat urine (Ersser and Smith, 1976) to investigate the excretion of cystathionine.
graphic system (Fig. 2). It is valuable for the investigation of disorders of phenylalanine and tyrosine metabolism and has been recommended for the detection of catecholamine metabolites (Ersser et al., 1970; Ebinger et al., 1975; Wadman et al., 1976). Its main disadvantages are: (a) that mild increases in HMMA excretion are not readily apparent on simple visual inspection; (b) semiquantitation of irregularly shaped spots is prone to unacceptably large errors; (c) the di-hydroxy compounds are destroyed by the alkaline solvent used in the first dimension; (d) the area of the chromatogram occupied by HMMA can be crowded with glycine conjugates of exogenous phenolic acids jf dietary restriction is impracticable (Seakins and Smith, 1976). The one-dimensional system (Fig. 1) overcomes these uncertainties of identification and semiquantitation of HMMA excretion with one minor exception. Iso-vanilloyl glycine, which was present in urine samples after the experimental ingestion of
Results and discussion Two complementary systems of TLC were required for the satisfactory investigation of catecholamine metabolites in the presence of exogenous and endogenous urinary phenolic compounds. Identification of individual components in complex urinary extracts is aided by the use of two thin-layer media with different separating characteristics and by the use of two diazo location reagents, each yielding a different pattern of colour reactions, in addition to the changes in migration produced by the three solvent mixtures. A good general separation of urinary phenolic acids is achieved by the two-dimensional chromate-
A
3
B
.:E=3
3 E::::31O
c
D
F
E
G
2:@ 13c=3 7' - -" '-. 5 5~
"e:::::::=;
c=:> 6~
6~6c:=:J
1
~9~9~~ c::::::>
D
lJ 0 D -lsI
~l
Fig. I Tracing ofone-dimensional chromatogram of urinary extracts separated on silica gel using amyl acetate/acetic acid solvent (see Table 2 for identity of bands): A = normal subject excreting salicyluric acid, B = alkaptonuria; C = HMMA standard-upper limit of normal concentration; D = neuroblastoma; E = phenylketonuria; F = HMMA standard - twice upper limit of normal concentration; G = neuroblastoma with increased H VA excretion but normal concentration of HMMA.
G
V r 2nd
13
@J~odj•. . . . - . - t st
0';·
8G l~a65
Downloaded from acb.sagepub.com by guest on June 4, 2016
r
2nd
Fig.2 Tracing of two-dimensional chromatograms ofurinary extracts of two patients with neuroblastomas. Separation on microcrystalline cellulose. Solvent 1 - mixed alcohols: ammonia; Solvent 2 - benzene: acetic acid (see Table 2 for identity of spots). D = elevated excretion of HMMA and HVA; G = elevated excretion of HVA but normal concentration of HMMA.
Routine laboratory investigation of urinary catecholamine metabolites in sick children
iso-vanillic acid, has the same Rr value as HMMA in this system and yields a similar purple colour when reacted with alkaline diazotised p-nitroaniline. The small amount of this compound, derived from dietary sources, which may normally be present in the urine is insufficient to cause a false-positive result. It is resolved from HMMA in the twodimensional system and produces a distinctly different shade of orange when reacted with alkaline diazotised sulphanilic acid. Gutteridge and Wright (1970) originally described the one-dimensional system (Fig. I) for the separation of methyl-malonic acid. It also resolves other compounds with active hydrogen atoms such as acetoacetate, which react with acid diazo-reagents (Seakins and Ersser, 1976). It gives a good separation of phenolic acids which have low Rr values in both the solvents used in the two-dimensional technique but does not resolve HVA and 5HIAA from common urinary phenolic acids. Both DOPAC and homogentisic acid can be detected by this technique (Fig. I). Mild increases in HMMA excretion are easily detected when samples are compared with the two concentrations of standard recommended. In agreement with Addanki et al. (1977) and Ong and Dupont (1975), the p-nitroaniline 'spot test' for the detection of HMMA in neat urine spotted on to filter paper was rejected as a screening method. False-positive results were occasionally obtained due to the presence of other phenolic compounds in the
41
urine. Dilute urine samples may give false-negative results (Addanki et al., 1977). The quantitative urinary excretion of HMMA (Pisano et al., 1962) by hospitalised children without tumours and on an unrestricted diet is summarised in Table 3. The excretion of catecholamine metabolites by children with confirmed tumours is summarised in Table 4. Complete agreement between assessment by TLC and the quantitative spectrophotometric determination of HMMA excretion was obtained when both values were related to creatinine content. Serious discrepancies occurred, however, when the quantitative values were expressed as 24-hour output. This was due mainly to extremes in urine volume (Sunderman, 1964). High urine volumes resulted from excessive fluid intake or diuretic therapy, while low volumes in the seriously ill child were due to copious perspiration, dehydration, or difficulties in obtaining complete 24-hour urine collection. Results from a 10-month-old girl with a neuroblastoma illustrate this problem (Table 5). The most reliable information was obtained when 24-hour urine samples were collected and results for HMMA were expressed as ,umol HMMAjmmol creatinine. This ratio was more variable in random samples (Sunderman, 1964) but could still be helpful in cases of clinical urgency. Semi-quantitative assessment of urinary cystathionine excretion provided additional diagnostic information. Cystathioninuria occurs in up to 50%
Table 3 Quantitative urinary excretion of HMMA (Pisano et al., 1962) by 93 hospitalised children without tumours on an unrestricted diet Urinary HMMA excretion (umol)
Age (years)
per 24 hours
n
Mean
(}.!
t- 6 7-11 I2-adult
SD
5·9 8'7 16·4 24·8
14 48 23 8
per mmoJ creatinine
5·8
5-8 7·9 10·0
Range
Mean
SD
Range
0'3-21·0 1·5-21·0 6·9-30·7 16'1-48·4
6'8 5'6 4·4 3'1
4·1 2·9 1·6 1·5
1·8-16'3 1,9-13,7 3·3- 7·6 1·2- 6·7
Table 4 Urinary metabolites detected in 30 children with catecholamine-secreting tumours
Status
No.
Pisano umol per mmol creatinine
Untreated
I 4 12 2
102-1I5 53-177 13·2-57 10·1-16'3 4·8 1·2-8,6
Treated
Other metabolites by TLC
HMMA excretion
Patients
I
10
ByTLC
HVA
t t t t t t t
t t t t t
N N
N
t N
f excretion greater than twice normal
N = normal excretion
Downloaded from acb.sagepub.com by guest on June 4, 2016
DOPAC
t t
N N N N
CYSTA
t t
N N N N N
42
C. E. Niehaus, R. S. Ersser, and Sheila M. Atherden
Table 5 Urinary HMMA excretion by different ways
1I
ltl-month-old in/ant with a neuroblastoma; results expressed in three HMMA (I'motl
Urine volume (ml per Z4 hl Pre-operative a b Post-operative a b Reference range
85 32 280 200 200-400
Metabolites by TLC
24 h
mmol crf"atininf'
15·5 19'5
53-4 108'3
5·5 2·1 1'5-21
8·6 3'7 1'9-9·2
of patients with neuroblastoma or ganglioneuroma and, in common with HV A, its excretion by some tumours may be elevated while HMMA excretion is within normal limits (Geiser and Efron, 1968). Its continued presence or reappearance after treatment indicates persistent active disease (Geiser and Efron, 1968). Conclusions
Routine investigation of children for disturbances in catecholamine metabolism is efficiently achieved by the progressive scheme described in this paper. The simple and quick systems using TLC are satisfactory for the majority of clinical requests and can be applied to large numbers of samples collected without initial dietary restriction. The same 24-hour urine collection can be used for subsequent quantitation of HMMA, which is performed on samples from all known tumour cases in order to monitor progress, in addition to those giving abnormal results by TLC. More detailed studies are reserved for particularly interesting or clinically complex patients and are performed, after dietary restriction, on suitable samples collected under specified conditions (Wadman et 01., 1976). The success of this system is in part due to the fact that large quantities of catecholamine metabolites are excreted by the majority of children with neuroblastomas. Prior restriction of dietary phenolic compounds would be prudent when screening adult populations as milder elevations in HMMA excretion are encountered in patients with phaeochromocytomas (Sandler, 1967). This would result in a narrower, but lower, reference range. Measurement of the urinary metadrenalines is also more valuable in adults (Sandler, 1967). We thank Professor Barbara E. Clayton and Dr J. W. T. Seakins for their advice and interest in this project. Mrs E. Hogg, Mr M. Cranfield, and Mr E. Layward contributed able technical assistance.
HMMA
HVA
t t
.
N N
N N
;
DOPAC
t t t N N
References Addanki, S., Gombos, R. L., Hinnenkamp, E. R .. and Sotos, J. F. (1977). Screening tests for vanillylrnandelic acid. Journal of Pediatrics, 90, 955-957 Ebinger, G., Veheyden, R., and Maurus, R. (1975). Thin-layer chromatography for diagnosis of secreting neuroblastoma. European Journal of Paediatrics, I:Zl, 63-67. Ersser, R. S.• Oakley, S. E., and Seakins, J. W. T. (1970). Urinary phenolic acids by thin-layer chromatography. Clinica Chimica Acta, 3D, 243-249. Ersser, R. S., and Smith, l. (1976). Aminoacids and related compounds. In Chromatographic and Electrophoretic Techniques, 4th edition, edited by I. Smith and J. W. T. Seakins, Volume I, pp, 75-109. William Heinemann Medical Books, London. Geiser, C. F. and Efron, M. L. (1968). Cystathioninuria in patients with neuroblastoma or ganglioneuroblastoma: its correlation to vanilrnandelic acid excretion and its value in diagnosis and therapy. Cancer, 22, 856-860. Guttedrige, J. M., and Wright, E. B. (1970). A simple and rapid thin-layer chromatographic technique for the detection of methylmalonic acid in urine. Clinica Chimica Acta, 27, 289-291. Ong, M., and Dupont, C. L. (1975). The La Brosse VMA spot test revisited. Journal of Pediatrics, 86, 238-240. Pisano, J. J., Crout, J. R., and Abraham, D. (1962). Determination of 3-methoxy-4-hydroxymandelic acid in urine. Clinlca Chimica Acta, 7, 285-291. Sandler, M. (1967). Catecholamine-secreting tumours. Proceedings of the Royal Society of Medicine, 60, 795-796. Seakins, J. W. T., and Ersser, R. S. (1976). Organic acids. In Chromatographic and Electrophoretic Techniques, 4th edition, edited by I. Smith and J. W. T. Seakins, Volume I, pp, 253-273. William Heinemann Medical Books, London. Seakins, J. W. T., and Smith, I. (1976). Phenolic acids. In Chromatographic and Electrophoretic Techniques, 4th edition, edited by I. Smith and J. W. T. Seakins, Volume I, pp, 218-244. William Heinemann Medical Books, London. Sunderman, F. W. Jr. (1964). Measurement ofvanilmandelic acid for the diagnosis of pheochromocytoma and neurohlastoma. American Journal of Clinical Pathology, 42, 481-497. Wadman, S. K., Ketting, D., and Voute, P. A. (1976). Gas chromatographic determination of urinary vanilglycolic acid, vanilglycol, vanilacetic acid and vanillactic acidchemical parameters for the diagnosis of neurogenic tumours and the evaluation of their treatment. Clinica Chimica Acta, 72, 49-68. Accepted for publication 5 October 1978
Downloaded from acb.sagepub.com by guest on June 4, 2016
Routine lahoratory investigation of urinary catecholamine metabolites in sick children
43
Reference
Addendum Since the completion of this work, benzene has been replaced by toluene, which is Jess toxic. Rr values in the toluene/acetic acid solvent are slightly lower but spots are more compact (Ersser, Seakins, and Manraj, 1978).
Ersser, R. S., Seakins, J. W. T., and Manraj, B. (19781. Thinlayer chromatography of phenolic acids with use of toluene instead of benzene. Clinical Chemistry, 24. 836.
Downloaded from acb.sagepub.com by guest on June 4, 2016
Routine laboratory investigation of urinary catecholamine metabolites in sick children C. E. NIEHAUSl R. S. ERSSER, AND SHEILA M. ATHER DEN From the Hospital for Sick Children and Institute of Child Health, 30 Guilford Street, London SUMMARY Simple and rapid thin-layer chromatographic methods have been used to investigate the catecholamine metabolites present in the urine of sick children. A semi-quantitative method for 4-hydroxy-3-methoxy-mandelic acid (HMMA) has been devised and compared with the quantitative spectrophotometric procedure. The methods have been performed on both normal subjects and children with catecholamine-secreting tumours, without dietary restriction, and have led to a 50 ~;I reduction in the number of samples requiring laborious quantitative determination of HMMA excretion.
There are good clinical indications for determining the excretion of urinary catecholamine metabolites in both children and adults (Sunderman, 1964). In adults the catecholamine-secreting tumours are mainly phaeochromocytomas and rarely chemodectomas, whereas in children they form the neuroblastoma - ganglioneuroma - retinoblastoma group (Sandler, 1967). While several catecholamine metabolites appear in the urine of patients with such tumours (Wadman et al., 1976), 4-hydroxy-3-methoxy-mandelic acid (HMMA) excretion is commonly elevated. The quantitative and relatively specific method of Pisano et at. (1962) is satisfactory for the estimation of HMMA but it is time-consuming and unsuitable for total automation. In addition, a small number of these tumours in children secrete the dopamine metabolites homovanillic acid (HY A) and 3, 4-dihydroxy phenyl acetic acid (DOPAC) in the urine rather than HMMA (Wadman et al., 1976). Improvements in the therapy and long-term management of children with such tumours and the growing interest in hypertension during childhood has resulted in a large increase in requests for the estimation of urinary HMMA and other catecholamine metabolites. The routine service offered by our laboratory has therefore been re-assessed and more efficient methods of analysis have been sought. We describe the methods that have led to a 50 % reduction in the number of samples requiring quantitative determination of HMMA. This has lPresent address: Dr C. E. Niehaus, Clinical Laboratories, PO Box 4419, Pretoria, S. Africa.
enabled us to increase the number of patients who can be investigated without an increase in staff. Material and methods URINE SAMPLES
Twenty-four-hour urine collections, stored in bottles containing 5 ml of 6M hydrochloric acid, were obtained from 30 children with catecholaminesecreting tumours. Both newly diagnosed and treated patients were included. Urine samples from a group of 93 hospitalised children without tumours served as controls. METHODS
Urinary creatinine was estimated by the Technicon automated alkaline picrate method. Acid up to a final urine concentration of 2M did not interfere with the result. Quantitative estimation of HMMA was performed by the colorimetric method of Pisano et at. (1962). Chromatography was performed on aliquots of a concentrated ether extract of urine (Ersser et al., ] 970). Urine (2 ml) was acidified with concentrated hydrochloric acid (0'2 ml) saturated with sodium chloride, and the phenolic compounds were extracted by vigorous shaking with di-ethyl ether (8 ml). After centrifugation the ether layer was transferred to a wide-necked bottle and allowed to evaporate either overnight in a fume cupboard, or by gentle warming, or by use of a rotary evaporator. The residue was dissolved in 0'1 ml 50 % aqueous propan-Z-ol. Volumes of this solution, related to the creatinine content of the original urine, were used for chromatography (Table 1).
38
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Routine laboratory investigation of urinary catecholamine metabolites in sick children
Table I
Volume of extract applied to chromatograms
Creatinin« (mmollf)
Volume or extract (,./) J-dimensional (/()() mm)
(50 x 50 mm).
-HlO
18'0
6'0
1'01-1'~0
I~'O
o
2-dimensiona/
1'~1-2'OO
13'~
~'O 4'~
2'0l-2'~0
12-0
4-0
2·~1-2·7~
10'~
3'~
2'7~3'OO
9·0
3·0
7'~
2'~
J'~l-4'~O
6·0
2·0
4'~1-~'~0 ~'~1-7'OO
4'~
l-~
3·0
1·0
7'01-8'~0
2'2~
0-7~
8'~I+
1'~
O'~
lOl-3'~0
-Four times this volume is applied to layers] 00
'\Ie
100 mm in size.
A semi-quantitative estimate of HMMA concentration was obtained by chromatographing the extracts together with two standards of known concentration: one approximated to the upper limit of normal (2'5 JLI of a 0·007 mM solution of HMMA in water) and the other was twice this amount (5'0,....1). Aliquots of the standard solutions were stored frozen and discarded after use. Samples were applied, using a graduated microlitre syringe, as bands (15 mm long) to layers of silica gel (Kieselgel 60, F 254, E. Merck No. 5554, BDH, Poole, Dorset), 100 mm long, and dried with warm air from a fan. A single ascending development in amyl acetate: glacial acetic acid: water (60 : 20 : 6) was performed. A 75 mm solvent rise from the origin took approximately 40 minutes at 25°C. After drying with warm air the layer was viewed under short-wave ultraviolet light (254 nm). While only grossly elevated concentrations of HMMA were detected at this stage, the presence of possible interfering substances such as glycine conjugates of phenolic acids was revealed.
39
The layer was sprayed with saturated aqueous sodium carbonate and allowed to dry. At this stage readily oxidisable di-hydroxy compounds such as DOPAC and homogentisic acid appeared as brown bands. The layer was then sprayed with a freshly made saturated solution (approximately 200 mg/lOO ml) of stabilised diazonium salt of p-nitroaniline (Koch-Light Laboratories, Colnbrook, Bucks) dissolved in 30% aqueous ethanol. In addition to the purple band of HMMA, a variety of colours were produced by other phenolic compounds present in the extract, as illustrated in Table 2. The colours started to fade within 15 minutes of location unless the layer was stored at -20°C. A two-dimensional chromatogram on microcrystalline cellulose (Schleicher and Schuell F.1440, Anderman and Co, E. Molesey, Surrey) was prepared using a second aliquot of the extract as described by Ersser et al. (1970). Layers of 50 mm square were used as the process could be completed in one hour. A solvent mixture of propan-Z-ol : butan-I-ol : 2-methyl-propan-2-o1 : 25 % ammonium hydroxide : water (40 : 20 : 20 : 10 : 20) was used in the first dimension (15 minutes for a 40 rom rise at 25°C) and benzene: acetic acid: water (70 : 29:1) was used in the second dimension (5 minutes). The layers were sequentially viewed under ultraviolet light (254 nm and 366 nm), dipped in a 1 % solution of acridine in acetone, dried and viewed under ultraviolet light (366 nm) to locate acidic compounds, sprayed with saturated aqueous sodium carbonate, dried, and finally sprayed with diazotised sulphanilic acid to locate phenolic compounds (Smith and Seakins, 1976). HMMA yielded an orange colour and HV A a purple colour. The reactions of other commonly occurring compounds are shown in Table 2. Chromatograms were assessed by visual
Table 2 Colour reactions of common phenolic acids with diazo reagents No. Acid
Colour with alkaline diazo reagents
Sulphanilic acid
p-Nitroaniline
1 Vanillic 2 HVA 3 js-Hydrcxyphenylacetic 4 s-Hydroxyhippuric ~ 3-Hydroxy+methoxy-phenylhydracrylic 6 m-Hydroxyhippuric 7 DOPAC 8 Homogentisic 9 HMMA 10 ~.Hydroxy-indolylacetic 11 Aceto-acetic 12 p-Hydroxypbenyllactic 13 e-Hydroxyphenylacetic 14 p-Hydroxybenzoic U p-Hydroxymandelic 16 m-Hydroxyphenylacetic
Orange
Purple Blue Mauve Red Red Red
Pink Red/brown Yellow Yellow Yellow
Brown Brown
Orange Red Red/brown Yellow Yellow Yellow Yellow
Downloaded from acb.sagepub.com by guest on June 4, 2016
Purple Purple Yellow Mauve Pink Red Orange Red
40
C. E. Niehaus, R. S. Ersser, and Sheila M. Atherden
comparison with normal samples run simultaneously. Two-dimensional chromatography for amino acids was also performed on the neat urine (Ersser and Smith, 1976) to investigate the excretion of cystathionine.
graphic system (Fig. 2). It is valuable for the investigation of disorders of phenylalanine and tyrosine metabolism and has been recommended for the detection of catecholamine metabolites (Ersser et al., 1970; Ebinger et al., 1975; Wadman et al., 1976). Its main disadvantages are: (a) that mild increases in HMMA excretion are not readily apparent on simple visual inspection; (b) semiquantitation of irregularly shaped spots is prone to unacceptably large errors; (c) the di-hydroxy compounds are destroyed by the alkaline solvent used in the first dimension; (d) the area of the chromatogram occupied by HMMA can be crowded with glycine conjugates of exogenous phenolic acids jf dietary restriction is impracticable (Seakins and Smith, 1976). The one-dimensional system (Fig. 1) overcomes these uncertainties of identification and semiquantitation of HMMA excretion with one minor exception. Iso-vanilloyl glycine, which was present in urine samples after the experimental ingestion of
Results and discussion Two complementary systems of TLC were required for the satisfactory investigation of catecholamine metabolites in the presence of exogenous and endogenous urinary phenolic compounds. Identification of individual components in complex urinary extracts is aided by the use of two thin-layer media with different separating characteristics and by the use of two diazo location reagents, each yielding a different pattern of colour reactions, in addition to the changes in migration produced by the three solvent mixtures. A good general separation of urinary phenolic acids is achieved by the two-dimensional chromate-
A
3
B
.:E=3
3 E::::31O
c
D
F
E
G
2:@ 13c=3 7' - -" '-. 5 5~
"e:::::::=;
c=:> 6~
6~6c:=:J
1
~9~9~~ c::::::>
D
lJ 0 D -lsI
~l
Fig. I Tracing ofone-dimensional chromatogram of urinary extracts separated on silica gel using amyl acetate/acetic acid solvent (see Table 2 for identity of bands): A = normal subject excreting salicyluric acid, B = alkaptonuria; C = HMMA standard-upper limit of normal concentration; D = neuroblastoma; E = phenylketonuria; F = HMMA standard - twice upper limit of normal concentration; G = neuroblastoma with increased H VA excretion but normal concentration of HMMA.
G
V r 2nd
13
@J~odj•. . . . - . - t st
0';·
8G l~a65
Downloaded from acb.sagepub.com by guest on June 4, 2016
r
2nd
Fig.2 Tracing of two-dimensional chromatograms ofurinary extracts of two patients with neuroblastomas. Separation on microcrystalline cellulose. Solvent 1 - mixed alcohols: ammonia; Solvent 2 - benzene: acetic acid (see Table 2 for identity of spots). D = elevated excretion of HMMA and HVA; G = elevated excretion of HVA but normal concentration of HMMA.
Routine laboratory investigation of urinary catecholamine metabolites in sick children
iso-vanillic acid, has the same Rr value as HMMA in this system and yields a similar purple colour when reacted with alkaline diazotised p-nitroaniline. The small amount of this compound, derived from dietary sources, which may normally be present in the urine is insufficient to cause a false-positive result. It is resolved from HMMA in the twodimensional system and produces a distinctly different shade of orange when reacted with alkaline diazotised sulphanilic acid. Gutteridge and Wright (1970) originally described the one-dimensional system (Fig. I) for the separation of methyl-malonic acid. It also resolves other compounds with active hydrogen atoms such as acetoacetate, which react with acid diazo-reagents (Seakins and Ersser, 1976). It gives a good separation of phenolic acids which have low Rr values in both the solvents used in the two-dimensional technique but does not resolve HVA and 5HIAA from common urinary phenolic acids. Both DOPAC and homogentisic acid can be detected by this technique (Fig. I). Mild increases in HMMA excretion are easily detected when samples are compared with the two concentrations of standard recommended. In agreement with Addanki et al. (1977) and Ong and Dupont (1975), the p-nitroaniline 'spot test' for the detection of HMMA in neat urine spotted on to filter paper was rejected as a screening method. False-positive results were occasionally obtained due to the presence of other phenolic compounds in the
41
urine. Dilute urine samples may give false-negative results (Addanki et al., 1977). The quantitative urinary excretion of HMMA (Pisano et al., 1962) by hospitalised children without tumours and on an unrestricted diet is summarised in Table 3. The excretion of catecholamine metabolites by children with confirmed tumours is summarised in Table 4. Complete agreement between assessment by TLC and the quantitative spectrophotometric determination of HMMA excretion was obtained when both values were related to creatinine content. Serious discrepancies occurred, however, when the quantitative values were expressed as 24-hour output. This was due mainly to extremes in urine volume (Sunderman, 1964). High urine volumes resulted from excessive fluid intake or diuretic therapy, while low volumes in the seriously ill child were due to copious perspiration, dehydration, or difficulties in obtaining complete 24-hour urine collection. Results from a 10-month-old girl with a neuroblastoma illustrate this problem (Table 5). The most reliable information was obtained when 24-hour urine samples were collected and results for HMMA were expressed as ,umol HMMAjmmol creatinine. This ratio was more variable in random samples (Sunderman, 1964) but could still be helpful in cases of clinical urgency. Semi-quantitative assessment of urinary cystathionine excretion provided additional diagnostic information. Cystathioninuria occurs in up to 50%
Table 3 Quantitative urinary excretion of HMMA (Pisano et al., 1962) by 93 hospitalised children without tumours on an unrestricted diet Urinary HMMA excretion (umol)
Age (years)
per 24 hours
n
Mean
(}.!
t- 6 7-11 I2-adult
SD
5·9 8'7 16·4 24·8
14 48 23 8
per mmoJ creatinine
5·8
5-8 7·9 10·0
Range
Mean
SD
Range
0'3-21·0 1·5-21·0 6·9-30·7 16'1-48·4
6'8 5'6 4·4 3'1
4·1 2·9 1·6 1·5
1·8-16'3 1,9-13,7 3·3- 7·6 1·2- 6·7
Table 4 Urinary metabolites detected in 30 children with catecholamine-secreting tumours
Status
No.
Pisano umol per mmol creatinine
Untreated
I 4 12 2
102-1I5 53-177 13·2-57 10·1-16'3 4·8 1·2-8,6
Treated
Other metabolites by TLC
HMMA excretion
Patients
I
10
ByTLC
HVA
t t t t t t t
t t t t t
N N
N
t N
f excretion greater than twice normal
N = normal excretion
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DOPAC
t t
N N N N
CYSTA
t t
N N N N N
42
C. E. Niehaus, R. S. Ersser, and Sheila M. Atherden
Table 5 Urinary HMMA excretion by different ways
1I
ltl-month-old in/ant with a neuroblastoma; results expressed in three HMMA (I'motl
Urine volume (ml per Z4 hl Pre-operative a b Post-operative a b Reference range
85 32 280 200 200-400
Metabolites by TLC
24 h
mmol crf"atininf'
15·5 19'5
53-4 108'3
5·5 2·1 1'5-21
8·6 3'7 1'9-9·2
of patients with neuroblastoma or ganglioneuroma and, in common with HV A, its excretion by some tumours may be elevated while HMMA excretion is within normal limits (Geiser and Efron, 1968). Its continued presence or reappearance after treatment indicates persistent active disease (Geiser and Efron, 1968). Conclusions
Routine investigation of children for disturbances in catecholamine metabolism is efficiently achieved by the progressive scheme described in this paper. The simple and quick systems using TLC are satisfactory for the majority of clinical requests and can be applied to large numbers of samples collected without initial dietary restriction. The same 24-hour urine collection can be used for subsequent quantitation of HMMA, which is performed on samples from all known tumour cases in order to monitor progress, in addition to those giving abnormal results by TLC. More detailed studies are reserved for particularly interesting or clinically complex patients and are performed, after dietary restriction, on suitable samples collected under specified conditions (Wadman et 01., 1976). The success of this system is in part due to the fact that large quantities of catecholamine metabolites are excreted by the majority of children with neuroblastomas. Prior restriction of dietary phenolic compounds would be prudent when screening adult populations as milder elevations in HMMA excretion are encountered in patients with phaeochromocytomas (Sandler, 1967). This would result in a narrower, but lower, reference range. Measurement of the urinary metadrenalines is also more valuable in adults (Sandler, 1967). We thank Professor Barbara E. Clayton and Dr J. W. T. Seakins for their advice and interest in this project. Mrs E. Hogg, Mr M. Cranfield, and Mr E. Layward contributed able technical assistance.
HMMA
HVA
t t
.
N N
N N
;
DOPAC
t t t N N
References Addanki, S., Gombos, R. L., Hinnenkamp, E. R .. and Sotos, J. F. (1977). Screening tests for vanillylrnandelic acid. Journal of Pediatrics, 90, 955-957 Ebinger, G., Veheyden, R., and Maurus, R. (1975). Thin-layer chromatography for diagnosis of secreting neuroblastoma. European Journal of Paediatrics, I:Zl, 63-67. Ersser, R. S.• Oakley, S. E., and Seakins, J. W. T. (1970). Urinary phenolic acids by thin-layer chromatography. Clinica Chimica Acta, 3D, 243-249. Ersser, R. S., and Smith, l. (1976). Aminoacids and related compounds. In Chromatographic and Electrophoretic Techniques, 4th edition, edited by I. Smith and J. W. T. Seakins, Volume I, pp, 75-109. William Heinemann Medical Books, London. Geiser, C. F. and Efron, M. L. (1968). Cystathioninuria in patients with neuroblastoma or ganglioneuroblastoma: its correlation to vanilrnandelic acid excretion and its value in diagnosis and therapy. Cancer, 22, 856-860. Guttedrige, J. M., and Wright, E. B. (1970). A simple and rapid thin-layer chromatographic technique for the detection of methylmalonic acid in urine. Clinica Chimica Acta, 27, 289-291. Ong, M., and Dupont, C. L. (1975). The La Brosse VMA spot test revisited. Journal of Pediatrics, 86, 238-240. Pisano, J. J., Crout, J. R., and Abraham, D. (1962). Determination of 3-methoxy-4-hydroxymandelic acid in urine. Clinlca Chimica Acta, 7, 285-291. Sandler, M. (1967). Catecholamine-secreting tumours. Proceedings of the Royal Society of Medicine, 60, 795-796. Seakins, J. W. T., and Ersser, R. S. (1976). Organic acids. In Chromatographic and Electrophoretic Techniques, 4th edition, edited by I. Smith and J. W. T. Seakins, Volume I, pp, 253-273. William Heinemann Medical Books, London. Seakins, J. W. T., and Smith, I. (1976). Phenolic acids. In Chromatographic and Electrophoretic Techniques, 4th edition, edited by I. Smith and J. W. T. Seakins, Volume I, pp, 218-244. William Heinemann Medical Books, London. Sunderman, F. W. Jr. (1964). Measurement ofvanilmandelic acid for the diagnosis of pheochromocytoma and neurohlastoma. American Journal of Clinical Pathology, 42, 481-497. Wadman, S. K., Ketting, D., and Voute, P. A. (1976). Gas chromatographic determination of urinary vanilglycolic acid, vanilglycol, vanilacetic acid and vanillactic acidchemical parameters for the diagnosis of neurogenic tumours and the evaluation of their treatment. Clinica Chimica Acta, 72, 49-68. Accepted for publication 5 October 1978
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Routine lahoratory investigation of urinary catecholamine metabolites in sick children
43
Reference
Addendum Since the completion of this work, benzene has been replaced by toluene, which is Jess toxic. Rr values in the toluene/acetic acid solvent are slightly lower but spots are more compact (Ersser, Seakins, and Manraj, 1978).
Ersser, R. S., Seakins, J. W. T., and Manraj, B. (19781. Thinlayer chromatography of phenolic acids with use of toluene instead of benzene. Clinical Chemistry, 24. 836.
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