A simple coloured-paper thin-layer chromotography test as a backup procedure for vanillylmandelic acid and homovanillic acid evaluation in a neuroblastoma screening program

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Clinica Chimica Acta 220 (1993) 229-232

Letter to the Editor

A simple coloured-paper thin-layer chromatography test as a backup procedure for vanillylmandelic acid and homovanillic acid evaluation in a neuroblastoma screening program Pierre Mathieu*“, Thierry Philipb, Jacques Greffea, Agnb Monteguea, Chantal Lacroixa “Sewire

de Biojogie G&kale

et Neurobiologie,

Centre hos~itai~er du Vinatier,

69677 f&-on-Lyon, France hCentre RPgional de lutte contre le Cancer, Centre Leon BPrard, 69008 Lyon, France

(Received 10 October 1992; revision received 2 June 1993; accepted 13 July 1993)

Ke_vwords: VanillylmandeIic acid; Homovaniliic acid; Neuroblastoma screening; Cancer

Dear Editor, Neuroblastoma is a malignant tumour derived from the primitive neural crest. In the majority of cases, there is an excessive urinary excretion of catecholamine metabolites [l-4], Abnormal urinary excretion of vanillylmandelic acid (VMA) and/or homovanillic acid (HVA), often occuring prior to clinical onset, are considered to be central for the diagnosis of neuroblastoma in infancy and have been proposed as the key to setting up screening programs [l-3]. In the Rhcine district of France, a pilot study for neuroblastoma screening was initiated in 1990, using HPLC analysis of urine with electrochemical detection (ECD). The analysis of VMA and HVA was performed on dilute urine at 1 part in 50 [5,6]. Since screening was started, 48,245 urine samples have been analysed. A small num* Corresponding author. 0009~8981/93/SO&OO SSDI

0009-898

0

1993

1(93)05639-V

Elsevier Science Publishers B.V. All rights reserved.

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ber of them showed one markedly increased peak with the same retention time (RT) as either VMA or HVA. These samples were collected from healthy babies. Therefore, a control for the HPLC procedure was needed. With the intention of making a simple backup procedure available, we have developed an inexpensive and rapid coloured-paper thin layer chromatography test, named the NB-test, which is described here. Urine was collected on a piece of filter paper (Whatman 17 CHR, 56 x 30 mm) and completely dried. The diazotized nitro-l-aniline (DZNA) step. The reagents were: Rl, a solution of 0.01 mol/l nitro-4-aniline in 0.2 mol/l HCl; R2, a solution of 0.2 mol/l sodium nitrite in HzO; R3, a solution of 0.3 mol/l sodium carbonate in H20; R4, a formaldehyde solution at about 37% by weight in Hz0 with lo- 15% methanol. RI and R2 should be stored in a refrigerator. Procedure: mix 1 ml each of Rl and R2, add 6.5 ml HzO, 10 ml R3 and 1.5 ml R4. Immediately and quickly dip the paper in the mixture and allow it to dry for about 30-60 min. A purple colour might indicate a VMA positive test, but the grey-blue colour of HVA, even if present in a large amount, is usually not seen. The thin-layer chromatography step must be done in all cases. One disk of 5 mm diameter was punched out from the coloured and dried filter paper. It was placed with 75 ~1 of methanol in a small stoppered glass tube; 15 min later, the methanolic solution of the azo derivatives was transferred to a 0.1 mm thick cellulose-coated TLC plastic sheet (Merck, art. 5565) and developed in the solvent system ethanol/lbutanol/ammonia/water, 6:2:1:2 by vol. After 1 h the azo derivatives of VMA (purple, Rs= 0.75) and of HVA (grey-blue, R, = 0.35) were separated from other compounds present. Control urines, containing known amounts of VMA and HVA, were treated in the same way, thus allowing semi-quantitative estimation. A lasting conservation of the coloured plates is obtained after a light spray using reagent R3. The low sensitivity and poor reliability of the spot-test described by La Brosse [7], which is still in use in some neuroblastoma screening centres [8], is due to the occurrence in urine of phenolic compounds originating from the diet or medical treatments, giving false positive tests. Otherwise urea lowers the activity of the DZNA reagent and hence lowers the sensitivity of the test. Urea destroys nitrites and nitrous acid [9,10) which is a major component of the DZNA reagent. In the NBtest, formaldehyde has been added to combine with urea and to block its activity, thereby increasing the formation of azo derivatives of the urinary phenolic acids with DZNA. Dipping the paper, instead of spraying it, achieves a faster and better colour reaction. In the second step, the azoic derivatives of the urinary phenolic compounds, freely soluble in a small volume of methanol, are extracted and transferred onto a TLC plate. The chromatographic separation is fast and simple because the separated substances are themselves coloured and a reagent spray is therefore not required. Sensitivity was approx. 0.7 pg for VMA and 1.5 pg for HVA. The NB-test clearly shows elevated spots from tested urine when excretion of VMA (> 10 mg/l) or HVA (> 20 mg/l) exceeds that from standard urine. False positive results have not been observed. None of the phenolic compounds listed in Ref. 11 with, in addition, isovanillylmandelic acid and iso-homovanillic acid which are the 4-O-methyl counter-

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parts of VMA and HVA, have the colours and/or the same R/values as those seen with VMA and HVA. Since the study was started, nine cases observed with an increased urinary excretion of VMA (> 10 mg/l) and/or HVA (> 20 mg/l, as indicated by the calculations) were found to show an obviously positive NB-test. After that, the occurence of a neuroblastoma was clinically confirmed in each case. Therefore, in these pathological cases, results from HPLC and the NB-test were both positive and in agreement with one another. In this series of 48,245 tests, there were 1I cases with calculated VMA peak values ranging from 46 to 142 mg/l and 6 cases with calculated HVA peak values ranging from 24 to 47 mg/l. These 17 samples, i.e. 1 in 2,838 urine analyses, were considered as false positives because (I) the NB-test has always been negative and, moreover, (2) a second or a third test on a new urine sample appeared as normal and (3) none of the babies whose urine showed these peaks was, or became, affected by a clinically diagnosed neuroblastoma. We conclude that these urine samples contained artifacts appearing on the HPLC tracings, but not being found in the NB-test. For ethical reasons. it is not permitted by our Control Committee to question families about the possible origin of these false VMA and HVA peaks. In conclusion, because cost/benefit reasons did not permit the purchase of GCMS equipment in a laboratory already fitted out with HPLC-ECD equipment for urinary VMA and HVA measurements as markers in a population-based study of neuroblastoma. we developed a test which, although making use of former practices, proved to be useful as a simple, rapid and cheap semi-quantitative method. This test may serve as a backup or control to the more precise but more costly procedures involved in the case of neuroblastoma screening in which small urine volumes are collected and mailed to the laboratory on dried filter paper pieces. References Sawada

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