- Email: [email protected]
Urinary vanilmandelic acid (VMA) excretion in children: Use of a simple semiquantitative test
844
The Journal of P E D I A T R I C S
Urinary vanilmandelic acid (VMA) excretion in children." Use of a simple semiquantitative test A simplified colorimetric method is described [or use in detecting excessive excretion of urinary VMA in children with suspected neurogenic tumors. Elevated levels are demonstrated in patients with neuroblastomas (13), ganglioneuroblastomas (3), ganglioneuromas (7), a pheochromocytoma (I), and a chemodectoma (1). Other patients studied included those with chronic diarrhea (15), hypertension (7), various tumors (10), cystic fibrosis of the pancreas (12), hypoxia (10), familial dysautonomia (2), and glycogen storage disease (1). In this miscellaneous group values were usually within the normal range with the exception o[ a Jew cases which are discussed in more detail.
R. B. Young, M.D., "x"D. D. Steiker, M.D., A. M. Bongiovanni, M.D., "~'x" C. E. Koop, M.D., and W. R. Eberlein, M.D. P IIILADELPYIIA~
PA.
I N 1 9 5 7 Armstrong t demonstrated the presence of vanilmandelict acid (VMA) in normal urine and showed this compound to be a major metabolite of norepinephrine. Subsequent investigators 2-G confirmed this finding and also showed that epinephrine is metabolized in essentially the same manner.
Supported by a training grant (2A-5197) National Institute of Arthritis and Metabolic Diseases, United States Public Health Service. From The Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania. ~United States Public Health Service Trainee, "Address, Department o[ Endocrinology, The Children's Hospital o] Philadelphia, 1740 Balnbridge Street, Philadelphia 46, Pa. t3-Methoxy-4-hydroxymandelle acid,
Isotope studies 4, ~ have shown that approximately 30 per cent of administered norepinephrine or epinephrine was excreted in the urine as VMA, whereas only 4 per cent was excreted as the free amine. This factor plus the meticulous care which must be exercised in the determination of free urinary catecholamines has encouraged the development of techniques for measuring urinary V M A in the routine chemistry laboratory. The clinical importance of this urinary metabolite is evident since it has been shown to be elevated in the urine of subjects with pheochromocytomas,1, ~-14 neuroblastomas,S, 12, la, 15-20 ganglioneuroblastomas,15, is ganglioneuromas, 2~ and in one carotid bodylike tumor. 24
Volume 62
Number 6
Various techniques have been developed for quantitation of urinary VMA. The original method of Armstrong, 25' 2s with twophase chromatography, has been used by several investigators 9' lo, 1.~,~, 21 employing various modifications. Single-phase chromatography has been usedl~; however, the details of this technique are in press 2~ at the time of this writing. The method of Sandler and Ruthven 8 employs initial separation with a cation exchange resin with the eventual oxidation of V M A to vanillin for quantitation. Others,1, 14, 2s have used similar techniques involving vanillin conversion. High-voltage eleetrophoresis has been employed by von Studnitz?2, ~9, 20 Isotope dilution techniques have been used by several investigators. 4' 6, 8o Gas chromatography has been suggested TM a2 although little has been reported regarding its use in the determination of urinary VMA. All of the foregoing tests are either very time consuming or require the use of equipment not usually available in a routine chemistry laboratory. In this laboratory we have employed a modification of the screening method of Gitiow.~..., 34 All values shown in this study and in prior communications 2~ 22, as were determined by the use of this method, which was employed as a semiquantitative screening technique. All specimens shown to have elevated values by the screening method were then subjected to two-phase chromatography for more discriminating evaluation. This communication describes a comparison of the simple screening procedure and chromatography as well as the application of these techniques to a group of children with and without neurogenic tumors. METHODS
The patient population consisted of children ranging from 1 week to 14 years of age at The Children's Hospital of Philadelphia. Indications for evaluation of V M A excretion, aside from the controls, incIuded thoracic and abdominal tumors, hypertension, chronic diarrhea and osteolytic lesions. No effort was made to restrict the physical activity of these children. Prior to and dur-
VMA
excretion in children
84 5
ing the collection of urine, foods containing caffeine, bananas, and vanilla were avoided. More recently citrus fruits have been excluded from the diet. Nonessential medications, particularly aspirin and sympathomimetic amines, were omitted, although several authors~0, sG have noted no significant interference with the latter compounds. Urinary collections were acidified to approximately p H 4 with 6N HC1 and stored at 8 to 10 ~ C. until tested. Twenty-four hour collections were preferred, although this was not possible in all cases. Urinary V M A determinations were performed by the following methods: Screening. An aliquot of urine (1 ml.) was adjusted to p H 1 (indicator paper) and extracted with ethyl acetate. The dried extract was dissolved in potassium carbonate solution and reacted with diazotized p-nitraniline to form a colored complex. The reaction mixture was subsequently extracted with n-amyl alcohol and the absorption of the final alcoholic extract determined after dehydration with granular anhydrous sodium sulfate. With a Beckman D U spectrophotometer the corrected reading for absorption at 545 m~ was determined as follows: Corrected Rd. 545 m/~ = (Rd. 500 m/~ + Rd. 590 m/~) Rd. 545 m / L 2 The details of the above method are essentially those described by Gitlow, 33' 34 with the omission of an initial extraction with ethyl acetate at p H 4 and the inclusion of V M A standards carried through the entire extraction procedure for quantitation. By the use of this method, absolute recoveries of 4 to 8 meg. V M A in water ranged from 80 to 100 per cent whereas recoveries of 12 mcg. were 76 to 78 per cent. Recoveries of 4 to 8 mcg. V M A added to urine ranged from 72 to 81 per cent. V M A standards maintained their potency in slightly acid ( p H 4 to 5) aqueous solution for 1 to 2 months at 8 to 10 ~ C. and would thereafter show a gradual decline. Standards maintained for 3 days at varying de-
846
June 1963
Y o u n g et al.
Table I. Control values in children with nonspecific minor conditions--24 hour collections (screening method)
Patient
JK BH RB BH TF BB MB BR GW VB RL DB DS JG G BH CC EF IL
Age 1 week 1 month 5 weeks 2} months 289 months 4 months 489 months 7 months 1 year 2 years 3 years 5 years 5 years 6 years 7 years 7 years 10 .years 12 years 14 years
24 hour ! volume mg./24 (ml.) hours 200 0.02 115 0.17 325 0.42 375 0.45 200 0.32 120 0.37 460 0.78 1.18 205 180 0.31 0.89 115 530 2.5 1.2 240 2.2 400 230 1.6 54O 2.4 1,070 2.8 1,080 3.3 4.5 855 900 4.8
mg./L.
0.1 1.5 1.3 1.2 1.6 3.1 1.7 5.8 1.7 5.9 4.7 5.0 5.4 6.9 4.4 2.4 3.1 5.3 5.3
grees of alkalinity (pH 7-9) showed 10 to 17 per cent loss. Excessive acidity in the reconstituted extract caused interference with the color development. Chromatography. One milliliter of urine was extracted repetitively with ethyl acetate as in the method of Gitlow. The dried residue was dissolved in ethanol, applied to a two-dimensional paper chromatogram by the method of Armstrong, 2~,2~ and developed at room temperature. After color development with potassium carbonate and diazotized p-nitroaniline, the areas corresponding to the position of pure V M A were removed, cut into small strips, and eluted in one of two ways: 1. n-Amyl alcohol with 1 per cent monoethanolamine (twice for 120 minutes with 4 ml. and once for 60 minutes with 2 ml.). 2. Two per cent aqueous sodium carbonate in ethyl alcohol (1:2) (with 2 ml. twice for 60 and 30 minutes, respectively, and once for 30 minutes with 1 ml.). Absolute recoveries of standards with each method of elution were 65 per cent and 50 per cent, respectively. Other areas representing various phenolic acids were also eluted and studied. Absorbance of the final
eluates were read against suitable blanks with a Beckman D U spectrophotometer. Suitable standards were chromatographed and eluted in the same way. This maneuver rendered the results comparable regardless of the method of elution. In specimens where only confirmation of significantly elevated levels was sought, visual comparison with standards stained on the paper by Armstrong's method was usually adequate. Compounds other than V M A eluted from the chromatograms were tentatively identified by their chromatographic mobility and color reaction with diazotized p-nitroaniline as described by ArmstrongY 5, 26 The specimens containing high levels of V M A usually showed relatively large amounts of homovanillic acid, although no precise quantitation of the latter was performed. Certain compounds were eluted and their maximal absorbancies determined. Where variance in absorbancies was noted in eluates from similar areas on different chromatograms it was assumed that this resulted from the admixture of two or more compounds occupying a common area. The following compounds were detected in some specimens: A. m-Hydroxyhippuric acid and p-hydroxymandelic acid occupying a common area (max. 500 mt~). B. m-Hydroxyphenylhydracrylic acid (max. 500 and 525 m/z). C. 3-Methoxy-4-hydroxyphenylhydracrylic acid (compound "C") (max. 545 m/z). D. p-Hydroxyphenylacetic acid (max. 475 and 545 m/z). E. Salicyluric acid (max. 475 m/z). F. 3-Methoxy-4-hydroxyphenylacetic acid (homovanillic acid) (max. 550 m/z). G. 3-Methoxy-4-hydroxybenzoic acid (vanillic acid) and 3-hydroxy-4-methoxybenzoic acid (isovanillic acid) occupying a common area (max. 545 m/z). Results. Comparison between values obtained with the screening method and chromatography with elution is shown in Fig. 1. The 6 specimens outlined by the oval show significant discrepancies between the two methods. Five of these specimens were de-
Volume 62 Number 6
liberately included since they represented either high normal or slightly elevated levels in patients with conditions not usually associated with high V M A excretion. All 6 specimens were shown to have normal amounts of V M A by chromatography. On the chromatograms 5 of the 6 showed significant amounts of compound " C " (above) which has been previously noted as a possible source of false elevations. In 4 specimens from patients with concentrations greater than 30 mg. per liter, there was poor correlation between the methods with chromatographic values which were 50 to 160 per cent of the Screening method values. In each of these specimens it was readily apparent by the screening method that they indeed contained a very high level of VMA. The remaining values showed satisfactory correlation. The clinical investigations are recorded in the various tables. Although neurogenic tumors in children comprised the major portion of the disorders, a variety of other conditions was studied. When possible the results are based on accurate 24 hour urine collections; however, in m a n y instances it has been necessary to express the findings as concentration per liter. The latter device proved useful in assessing disorders associated with elevated excretions of VMA, especially where preoperative circumstances were not suited to obtaining better collections of urine. In the evaluation of V M A excretion 24 hour collections probably give a more accurate reflection of the rate of production. In infants under 2 years of age, 24 hour specimens are believed to be essential for proper interpretation. Control values were obtained by the screening method from 65 children from 1 week to 14 years of age with nonspecific minor ailments. In the group under 2 years of age, values ranged from 0.1 to 8.6 mg. per liter (mean 2.3 + 2.4 rag. per liter). In those 2 to 14 years of age, the values ranged from 0 to 10.2 rag. per liter (mean 4.3 _+ 2.3 mg. per liter). The upper limits of normal were arbitrarily defined as 7.1 rag. and 8.9 mg. per liter, respectively. Table
VMA
excretion in children
84 7
I shows the values noted in 20 children from whom complete 24 hour collections were obtained. The results with the screening method in children with various neurogenic tumors are shown in Table II. In general, the patients with neuroblastomas which were large and inoperable or showed evidence of diffuse metastases presented with the highest V M A levels. Tumors which were incompletely removed continued to cause elevated urinary V M A levels although a marked decrease could be noted in comparison with preoperative levels. Recurrence or continued growth was reflected by rising V M A levels as illustrated in 2 cases (SC, M K ) shown in Fig. 2. Irradiation and chemotherapy caused a dramatic but temporary decrease in one of these cases ( M K ) . rThree patients with ganglioneuroblastomas were studied, including one in' whom only a normal postoperative specimen was available. T w o ( G T and DG) had high preoperative levels with a marked decrease following operative removal, as illustrated in Fig. 2. Because of intervertebral infiltration in one of these patients (DG), a small portion of the tumor was not removed. The V M A level 8 days after operation was 14 mg. per liter which while much diminished remained in the slightly elevated range. An25 t S" ~,s*'S
_.1
2O
E
9
E3
sS sS , s s S~ s,. SS
~E z z I,I n~" 0 (,n
s,,s S
ss 9
I0 9 ,.s s*' 9 9
5
":./(,
9 .
9
r 0 ~SS~
I 5
I I0
I 15
I 20
25
CHROMATOGRAPHY (mg/L)
Fig. 1. Comparison of VMA methods (patients and controls).
8 4 8 Young et al.
June
1963
Table II. VMA excretion (screening method)
I Age J J Turn~ Case (years) Sex origin L Neuroblastomas (13) 1 PB
46A2
F
Abd.
I ' =ses'l r,a men, lltervo'*l (reg.~L,) J M, B, RN S-I, irrad., VLB, M
- 3 days + 6 months
188 89 127 53 116
2 SA
8
F
Chest?
M, B, SC
Scalp biop., LC
+ +
3 EC
i~2
M
Abd.
B, M,
LC
- 9 days - 6 days + 2 weeks
4 EH
3~2
!V[
Abd.
SC
M, B
Mult. biop., C, irrad.
5 1 4 5
days day months months
140 90 -
Status
Died Tumor active
Died
- 1 month + 6 months + 9 months
23.2 26.4 -
Died
5 EL
16A2
iV[
Abd.
M, B, L Spinal
LC
+12 days
10.4
Died
6 AW
~2
M
Abd.
L, Liv. Testes
S-I, irrad., C
+ 2 months
19
Died
7 DW
56~2
M
Abd.
B, L
Cerv. nodes
Biop., C, irrad., + 6 months VLB +10 months +11 months
68.5 142 112
Died
B
S-I, LC
- 1 day + 3 days + 2 months
91.5 19.6 161
S-C
- 2 + 1 + 7 +14 +14
S-I, irrad.
- 1 day + 1 month + 2 months
8 SC
6A2
F
Abd.
9 FP
~2
M
Chest
10 CP
~/a2
F
Chest
11 M K
2~2
M
?
RN
Cerv. nodes, chest
S-I (RN)
GF
~2
M
Abd.
13
AM
~2
F
Abd.
RN
1.01w No apparent 0.38w recurrence 0.96w 1.61w 1.07w
days day months months months
8.1 w 2.2w
3.1w
Tumor active
+ 6 days + 9 days + 1 month +189 months + 2 months + 3 months + 5 months + 6 months
I3.2 9.9 5.3 8.4 26.7 1.3 66 -
Died
S-C
+ 7 days + 9 months +18 months
0.99w 1,2
Apparently "cured"
S-C
+ 6 days + 7 months + 1 year
4.7 5.5
Asymptomatic
S-I
- 5 - 3 - 2 + 8 + 3
s-i
(RN)
Irrad. + C., S-I (chest)
12
Tumor active
II. Ganglioneuroblastomas (3) 1 DG
2s~2
F
Chest
Intervertebral
days days days days months
150 164 120 14 8
Asymptomatic
Volume 62 Number 6
V M A excretion in children
84 9
Table II. Cont'd
Case
Age I Sex (years)
Tumor origin Metastases*
Interval~;
Treatmentf S-C
- 1 day + 6 days + 1 month
Chest
S-I (x2), irrad., M
7{ months after second operation
M
Chest
S-C
- 9 - 7 - 5 + 8 +11 +29 +389 +10 +16 +18
2 GT
1:)~2
M
Abd.
3 PW
2~2
F
RN
VMA I
(mg./L.)
Status
76 2.9
Asymptomatie
4.9
Asymptomatic
days days days days days days months months months months
20.0 15.3 15.3 5.1 6.3 7:4 4.5 1.5 4.9 5.9
Asymptomatic
1II. Ganglioneuromas (7) 1 DB
3~2
2 ML
10
F
Chest
S-C
- 3 + 3 + 5 +11
days days days months
19.8 7.2 4.5 3.2
Asymptomatic
3 KM
6
F
Chest
S-C
- 5 + 2 +14 +289 +689
days days days months months
7.2 2.1 4.7 0.25 7.5
Asymptomatie
4 DU
36~2
17
Chest
S-C
+ +
days days days months
7.7 6.2 3.3 5.3
Asymptomatic
5 KT
9
17
Chest
S-C
-13 days + 6 days + 2 months
10.8 1.7 4.5
6 DN
4
M
Chest
S-C
+3.I_, years
2.6
Asymptomatic
7 JB
10
M
Chest
S-I
- I + 5 +20 + 4
5.8 6.6 7.1
Asymptomatic
Intervertebral
3 2 7 7
day days days months
eM, marrow; B, bone; Rig, regional nodes; SC, subcutaneous tissues; L, lung; Liv., l i v e r . . ~'S-CI and S-I, operation with complete or incomplete removal of tumor; VLB, Velban (vmblastine sulfate, Lilly); M, mitomyein-C (Bristol Laboratories); LC, leurocristine sulfate (Lilly); C, Cytoxan (Mead Johnson). 1:Interval before or after initial treatment. w per 24 hours.
othe r specimen 3 months l a t e r showed a high n o r m a l level (8 rag. per liter). Seven patients with ganglioneuromas were studied an d of the 6 f r o m w h o m p r e o p e r a tive specimens were available only 3 showed elevated values. I t is noteworthy that none of the a b n o r m a l levels in these patients exceeded 20 mg. per liter a n d in one there was a level of 10.8 mg. per liter w h i c h is only slightly above the n o r m a l range. I n each
of the cases w i t h elevated levels t h er e was a notable p o st o p er at i v e drop to n o r m a l levels as is illustrated in Fig. 2 (DB, M L , K T ) . O t h e r r el at ed conditions i n c l u d e d a pheoc h r o m o c y t o m a a c c o m p a n i e d by a V M A level of 23.3 mg. p e r liter as well as e v a l u a t i o n of the free catecholamines. O n e subject with a c h e m o d e c t o m a showed V M A values which varied f r o m a high n o r m a l level of 7.2 mg. to significantly elevated values of 13.5 mg.
Younget al.
850
June 1963
4
DG
120
80
/
8 ~- 3o
I
/
/
/
/
A GANGLIONEUROMA
IRRAD. +
/
(,~ Z
/
/
9 NEUROBLA.STOMA o r GANGLIO-' NEUROBLASTOMA
/
DB
2o QK
I0 doys ~'~'PRE-OP
20 doys
I months
6
II
16
18
POST-OP INTERVAL
Fig. 2. VMA excretion in 7 cases of neurogenic tumors. per liter (9.8 rag./15 hr.). Five patients with neurofibromatosis, including one with hypertension, all showed values (2.5 to 8.5 rag. per liter) within the normal range. Ten patients with miscellaneous tumors and pseudotumors showed a normal range of values (0.37 to 7.2 mg. per liter). This group included sarcoma of the mandible, subhepatic abscess, acute myelogenous leukemia, adrenal hemorrhage with hematoma, xanthogranuloma with extensive abdominal spread, third ventricle astrocytoma, intestinal obstruction, metastatic Wilm's tumor, ruptured appendix and uterine rhabdomyosarcoma. Seven children with idiopathic hypertension, ranging in age from 5 to 15 years, showed VMA values ranging from 1.3 to 8.1 mg. per liter. Two other children showed levels of 9.5 and 12.4 mg. per liter by the screening method, but chromatographic evaluation revealed values of 3 and 4 mg. per liter, respectively. Both specimens
showed relatively large amounts of what was vanillic and isovanillic acid according to chromatographic mobility and stains. These have been previously noted as possible sources of interference. Four patients with the celiac syndrome had values ranging from 2.8 to 8.4 mg. per liter. Eleven patients with diarrhea, including 2 with ulcerative colitis and one with ileocolitis, excreted amounts of VMA ranging from 0.86 to 8,1 mg. per liter. Two other specimens in the latter group showed slight elevations Of 11.6 and 14.7 mg. per liter by the screening method. Chromatography, however, revealed values within normal limits. Twelve patients with mucoviscidosis and moderate to severe pulmonary involvement showed values ranging from 1.2 to 14.4 nag. per liter (mean 6.6). Three of these specimens showed borderline values (8.0, 9.0 and 9.4 mg. per liter) while one specimen was in the slightly elevated range (14.4 mg. per liter).
Volume 62
Number 6
Of 10 patients with various conditions which would cause hypoxia (i.e., atelectasis, cyanotic congenital heart disease, lobar pneumonia, acute asthmatic attack) only 2 showed values (9.5 and 10 mg. per liter) which slightly exceeded normal values. T w o patients with familial dysautonomia showed normal values (4.4 and 6.7 mg. per liter). A i-year-old infant with glycogen storage disease showed an excretion of 1.65 mg. per day which represents a slight elevation for age. DISCUSSION
A simplified method has been used in evaluating 249 urine specimens from 147 patients as a relatively rapid means of detecting excessive excretion of V M A . I t has proved to be useful in the diagnostic evaluation of children with thoracic and abdominal tumors, hypertension, chronic diarrhea, and osteolytic lesions for the possible presence of neural crest tumors. Serial determinations following the removal of such tumors have assisted in the follow-up care of these children. I n one recent study, 1~ 25 patients with neuroblastomas were reported to have significantly elevated levels of urinary VMA, whereas 3 patients with ganglioneuromas showed normal levels. It was suggested that V M A excretion could be used as a means of distinguishing these two tumor types preoperatively. The results herein presented do not support such a sharp differentiation. Kogut and Kaplan 37 reviewed the cases of 29 patients with adrenergic symptoms and neurogenic tumors, of which 6 were ganglioneuromas. Of this latter group, only one 3s was studied and shown to have a significantly elevated leveI of urinary V M A . Voorhess 23 has demonstrated that a few patients with neuroblastomas do not exhibit elevated levels of V M A in the urine and a small number show no elevation of any of the known catecholamines or metabolites. In a more recent report, a9 she emphasized the potential value of other urinary metabolites in guiding clinical care. The determination of dopamine and norepinephrine would seem
VMA
excretion in children
85 1
helpful in detecting those few patients in whom V M A excretion is not elevated. Recently von Studnitz ~9 has called attention to the fact that homovanillic acid excretion is usually elevated in patients with neuroblastomas. Of 25 patients whom he studied, 24 had elevations of urinary V M A and 17 had elevations of homovanillic acid. Of the latter group, there was a normal V M A excretion in one. He and other investigators17, 19 have shown that homovanillic acid excretion is not elevated in patients with pheochromocytoma who have high V M A levels. I n the current chromatographic studies, specimens with high V M A levels generally showed large amounts of homovanillic acid, although precise quantitation of each was not performed. In the present study one patient with a chemodectoma showed an elevated level of V M A in one specimen and several specimens with high normal values. There was a large thoracic mass with intervertebral extension, erosion of adjacent ribs and vertebral bodies with the eventual development of paraplegia. Moderate hypertension was present. This type of tumor is thought to arise from the chemoreceptor system and usually is non-chromaffin staining. In this instance, the tumor demonstrated chromaffin staining material, as did a carotid body-like tumor with elevated V M A excretion which was recently reported. 2. The latter patient also had moderate hypertension. The screening of hypertensive patients should include one or more tests to rule out the possibility of a pheochromocytoma. Several investigators sla have recommended the use of V M A excretion as a practical screening test for this condition but free urinary catecholamines should be determined in doubtful cases. It is of interest that the V M A excretion m a y be elevated in the presence of virtually normal values for urinary catecholamines a3, 3, and the reverse situation m a y also be found in a small number of cases?* I n the current study one patient with a pheochromoeytoma showed a significantly elevated level of VMA, as well as of urinary catechotamines. Five pa-
852
Young et al.
tients with neurofibromas, including one with hypertension and extensive intra-abdominal neurofibromatosis, showed no significant elevation of VMA. It is of interest that, in the previously mentioned review ~7 of neurogenic tumors with adrenergic symptoms, 15 of the 99 showed hypertension. Two patients (DG, DB) with neurogenic tumors were originally admitted for evaluation of gastrointestinal dysfunction. One patient, previously reported upon, 22 was admitted for evaluation of hypercalcemia, celiac syndrome, and growth retardation. An intravenous pyelogram incidentally revealed the presence of a posterior mediastinal mass which was completely removed at operation and was shown to be a ganglioneuroma. There was improvement in growth rate and bowel symptoms thereafter. The second, a 3-year-old girl, was admitted for evaluation of chronic diarrhea of 6 months' duration. A posterior mediastinal mass was noted on a routine flat plate of the abdomen. This proved to be a ganglioneuroblastoma. The gastrointestinal improvement was striking following subtotal surgical removal. In both patients a mild-to-moderate degree of acidosis was noted. A more detailed discussion of these two cases will be published. In view of the findings in these last 2 patients and similar reports in the literature, 37 a number of children with chronic diarrhea in this hospital have had urinary V M A determinations. Four patients with the celiac syndrome and 11 with diarrhea, including 2 with ulcerative colitis and one with ileocolitis, have shown normal values. Of 12 patients with mucoviscidosis studied, questionable or slight elevations of urinary V M A (8.9 to 13.4 mg. per liter) were found in 4. Each of the latter group showed moderate to severe pulmonary involvement. Of 10 patients with various other conditions causing hypoxia, 2 were shown to have borderline elevations (9.5 and 10 mg. per liter). Since hypoxia is a stimulus to catecholamine production, this would seem a likely explanation for these elevated levels. A previous investigator s~ has noted that seriously ill patients with severe pulmonary
]une 1963
insufficiency m a y show elevated levels of V M A excretion. In a prior study from this laboratory, *~ medullary hyperplasia and elevated content of catecholamines in the adrenal gland were shown to be present in 5 o f 6 patients with cystic fibrosis who were fortuitously included in an evaluation of adrenal medulla hyperplasia seen at autopsy. The data herein presented are not sufficient for further conjecture, but it appears that catecholamine hypersecretion is not a prim a r y factor in this disease. The possibility of an altered metabolic disposition cannot be completely discounted. Pertinent to this question is the recent observation of Selye, 41 that prolonged administration of isoproterenol caused the secretion of viscous saliva and excessive stimulation of saliva~3r gland growth in rats. Other conditions which have been noted by previous investigators to cause small elevations of V M A are the carcinoid syndrome,14, 2, paroxysmal tachycardia, 14 and burns24 SUMMARY
Urinary V M A excretion has been determined by a semiquantitative screening method in 249 urine specimens from 147 children, some of whom were evaluated for the presence of neurogenic tumors. A discussion of the method and its limitations as well as chromatogenic comparisons is presented. False elevations in a small number of samples were shown to be caused by urinary phenolic acids probably derived from dietary sources. Thirteen patients with neuroblastomas were studied extensively. All pretreatment levels of V M A excretion were elevated. T w o of 3 patients with ganglioneuroblastoma were evaluated preoperatively and both showed augmented excretion. Six of 7 children with ganglioneuroma were evaluated preoperatively. Only 3 of the latter group showed elevated levels of V M A excretion. One child with a pheochromocytoma and another with a chemodectoma also showed elevated levels. Results in children with various other conditions are discussed. These conditions
Volume 62
Number 6
include miscellaneous tumors, hypertension, diarrhea, cystic fibrosis of the pancreas, hypoxia, familial dysautonomia, a n d glycogen storage disease. We wish to acknowledge the assistance of Dr. William Yakovac who performed the histologic examinations of the tumors presented. REFERENCES
1. Armstrong, M. 13., McMillan, A., and Shaw, K. N. F.: 3-Methoxy-4-hydroxy-D-mandelic acid, a urinary metabolite of nor-epinephrine, Biochlm. et biophys, acta 25: 422, 1957. 2. Axelrod, ]'.: O-Methylation of epinephrine and other catechols in vitro and in vlvo, Science 126: 400, 1957. 3. Axelrod, J., Inscoe, J. K., Senoh, S., and Witkop, B.: O-Methylation, the principal pathway for the metabolism of epinephrine and norepinephrine in the rat, Biochim. et biophys, acta 27: 210, 1958. 4. Kirschner, N., Goodall, McC., and Rosen, L.: Metabolism of d, 1-adenaline-2-C TM in the human, Proc. Soc. Exper. Biol. & Med. 98: 627, 1958. 5. LaBrosse, E. H., Axelrod, J,, and Kety, S. S.: O-Methylation, the principal route of metabolism of epinephrine in man, Science 128: 593, 1958. 6. Goodall, M., Kirshner, N., and Rosen, L.: Metabolism of noradrenaline in the human, J. Clin. Invest. 38: 707, 1959. 7. LaBrosse, E. H., Axelrod, J., and Sjoerdsma, A.: Urinary excretion of normetanephrine by man, Fed. Proc. 17: 386, 1958. 8. Sandler, M., and Ruthven, C. R. J.: Quantitative colorimetrie method for estimation of 3-methoxy-4-hydroxymandelic acid in urine. Value in diagnosis of pheochromocytoma, Lancet 2: 114, 1959. 9. Robinson, R., Ratcliffe, J., and Smith, P.: A screening test for pheochromocytoma, J. Clin. Path. 12: 541, 1959. 10. Gitlow, S. E., Mendlowitz, M., Khassis, S., Cohen, G., and Sha, J.: The diagnosis of pheochromocytoma by determination of urinary 3-methoxy-4-hydroxymandelic acid, J. Clin. Invest. 39: 221, 1960. 11. Sunderman, F. W., Jr., Cleveland, P. D., Law, N. C., and Sunderman, F. W.: A method for the determination of 3-methoxy4-hydroxymandelic acid (vanilmandelic acid) for the diagnosis of pheochromocytoma, Am. J. Clin. Path. 34: 293, 1960. 12. von Studnitz, W.: Methodische and klinische Untersuchungen fiber die Ausscheidung der 3-methoxy-4-hydroxymandels~iure im Urin, Scandinav. J. Clin. & Lab. Invest. 12: 1, 1960 (supp. 48). 13. Jacobs, S. L., Sobel, C., and Henry, R. J.: Excretion of 3-methoxy-4-hydroxymandelic acid and catecholamines in patients with
V M A excretion in children
14.
15.
16. 17.
18. 19. 20.
21.
22.
23.
24. 25. 26.
27.
28.
29.
853
pheochromocytoma, J. Clin. EndocrinoL 21: 315, 1961. Crout, J. R., Pisano, J. J., and Sjoerdsma, A.: Urinary" excretion of catecholamines and their metabolites in pheochromocytoma, Am. Heart J. 61: 375, 1961. Voorhess, M, L., and Gardner, L. I.: Urinary excretion of norepinephrine, epinephrine and 3-methoxy-4~hydroxymandelic acid by children with neuroblastoma, J. Clin. Endocrinol. 21: 321, 1961. Bell, M., and Steward, J. K.: Neuroblastoma and catecholamine excretion, Lancet 1: 1061, t961. Bettex, M., and Kaser, H.: Diagnostic and prognostic value of the determination of urinary output of vanillylmandelic acid in tumors of sympathetic nervous system, Arch. Dis. Childhood 37: 138, 1962. Kaser, H., and von Studnitz, W.: Urine of children with sympathetic tumors, A. M. A. J. Dis. Child. 102" 199, 1961. von Studnitz, W.: Neuroblastoma and catecholamine excretion, Lancet 2: 215, 1961. Steiker, D. D., and Eberlein, W. R.: Urinary VMA excretion in children with neural tumors. Program of the 43rd Meeting of the Endocrine Society, New York, N. Y., June, 1961, p. 102. Greenberg, R. E., and Gardner, L. I.: New diagnostic test for neural tumors of infancy: Increased urinary excretion of 3-methoxy-4hydroxymandelic acid and norepinephrine in ganglioneuroma with chronic diarrhea, Pediatrics 24: 683, 1959. Leboeuf, G., Eberlein, W. R., Steiker, D. D., and Bongiovanni, A. M.: Functioning ganglloneuroma, celiac syndrome and renal acidosis, presented at the American Pediatric Society, 71st Annual Meeting, Atlantic City, May, 1961. Voorhess, M. L., and Gardner, L. I.: Studies of catechotamine excretion by children with neural tumors, J. Clin. Endoerinol. 22: 126, 1962. Glenner, G. G.: A functional carotid-bodylike tumor, A. M. A. Arch. Path. 73: 230, 1960. Armstrong, M. D., Shaw, K. N. F., and Wall, P.E.: The phenolic acids of human urine, J. Biol. Chem. 218" 293, 1956. Armstrong, M. D.: Estimation of 3-methoxy4-hydroxymandelie acid (VMA) for the detection of phenochromacytoma. Pamphlet privately circulated by the author, Fels Research Institute, Yellow Springs, Ohio (8 pp.). Kaser, H.: Eine eindimensionale papierehromatographische Method zur Bestimmung der 3-Methoxy-4-hydroxymandelic Sfiure, Experientia (in press). O'Brien, D., and Ibbott, F. A.: Laboratory manual of pediatric micro and ultramicrobiochemical techniques, ed. 3, New York, 1962, Harper and Row, p. 74. von Studnitz, W., and Hanson, A.: Deter-
8 54
30.
31.
32.
33.
34.
35.
Young et al.
mination of 3-methoxy-4-hydroxymandelic acid in urine by high voltage paper electrophoresis, Scandinav. J. Clin. & Lab. Invest. 11: 101, 1959. Smith, A. A., Schweitzer, J. W., and Wortis, S. B.: The determination of norepinephrine metabolites by isotope dilution, Fed. Proc. 18: 145, t959. Williams, C. M., and Sweeley, C. C.: A new method for the determination of urinary aromatic acids by gas chromatography, J. Clin. Endocrinol. 21. 1500, 1961. Greet, M., and WiIliarns, C. M.: Catecholamine metabolism in neuroblastoma. Program of the 32nd Annual Meeting of the Society for Pediatric Research, Atlantic City, May, 1962, p. 115. Gitlow, S. E., Ornstein, L., Mendlowitz, M., Khassis, S., and Kruk, E.: A simple colorimetric urine test for pheochromoeytoma, Am. J. Med. 28: 921, 1960. Gitlow, S. E., Mendlowitz, M., Kruk, E., and Khassis, S.: Diagnosis of pheochromocytoma by assay of catecholamine metabotites, Circulation Res. 9: 746, 1961. Young, R. B., Steiker, D. D., Koop, C. E., and Bongiovanni, A. M.: Evaluation of a simple colorimetric determination of urinary
June 1963
36.
37. 38. 39.
40.
41.
3-methoxy-4-hydroxymandelic acid. Program of the 32nd Annual Meeting of the Society for Pediatric Research, Atlantic City, May, 1962, p. 71. Sandler, M., and Ruthven, C. R. J.: 3-Methoxy-4-hydroxymandelic acid excretion in pheochromoeytoma. Adrenergic mechanisms, Ciba Foundation Symposium, Boston, 1960, Little, Brown & Company, p. 40. Kogut, M. D., and Kaplan, S. A.: Systemic manifestations of neurogenic tumors, J. PEDIAT. 60: 694, 1962. Greenberg, R. E., and Gardner, L. I.: Catecholamine metabolism in a functional neural tumor, J. Clin. Invest. 39: 1729, 1960. Voorhess, M. L., and Gardner, L. I.: The value of serial catecholamine determinations in children with neuroblastoma, Pediatrics 30" 241, 1962. Bongiovanni, A. M., Yakovac, W. C., and Steiker, D. D.: Study of adrenal glands in childhood: Hormonal content correlated with morphologic characteristics, Lab. Invest. 10: 956, 1961. Selye, H., Veilleux, R,, and Cantin, M.: Excessive stimulation of salivary gland growth by isoproterenol, Science 133: 44, 1961.
The Journal of P E D I A T R I C S
Urinary vanilmandelic acid (VMA) excretion in children." Use of a simple semiquantitative test A simplified colorimetric method is described [or use in detecting excessive excretion of urinary VMA in children with suspected neurogenic tumors. Elevated levels are demonstrated in patients with neuroblastomas (13), ganglioneuroblastomas (3), ganglioneuromas (7), a pheochromocytoma (I), and a chemodectoma (1). Other patients studied included those with chronic diarrhea (15), hypertension (7), various tumors (10), cystic fibrosis of the pancreas (12), hypoxia (10), familial dysautonomia (2), and glycogen storage disease (1). In this miscellaneous group values were usually within the normal range with the exception o[ a Jew cases which are discussed in more detail.
R. B. Young, M.D., "x"D. D. Steiker, M.D., A. M. Bongiovanni, M.D., "~'x" C. E. Koop, M.D., and W. R. Eberlein, M.D. P IIILADELPYIIA~
PA.
I N 1 9 5 7 Armstrong t demonstrated the presence of vanilmandelict acid (VMA) in normal urine and showed this compound to be a major metabolite of norepinephrine. Subsequent investigators 2-G confirmed this finding and also showed that epinephrine is metabolized in essentially the same manner.
Supported by a training grant (2A-5197) National Institute of Arthritis and Metabolic Diseases, United States Public Health Service. From The Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania. ~United States Public Health Service Trainee, "Address, Department o[ Endocrinology, The Children's Hospital o] Philadelphia, 1740 Balnbridge Street, Philadelphia 46, Pa. t3-Methoxy-4-hydroxymandelle acid,
Isotope studies 4, ~ have shown that approximately 30 per cent of administered norepinephrine or epinephrine was excreted in the urine as VMA, whereas only 4 per cent was excreted as the free amine. This factor plus the meticulous care which must be exercised in the determination of free urinary catecholamines has encouraged the development of techniques for measuring urinary V M A in the routine chemistry laboratory. The clinical importance of this urinary metabolite is evident since it has been shown to be elevated in the urine of subjects with pheochromocytomas,1, ~-14 neuroblastomas,S, 12, la, 15-20 ganglioneuroblastomas,15, is ganglioneuromas, 2~ and in one carotid bodylike tumor. 24
Volume 62
Number 6
Various techniques have been developed for quantitation of urinary VMA. The original method of Armstrong, 25' 2s with twophase chromatography, has been used by several investigators 9' lo, 1.~,~, 21 employing various modifications. Single-phase chromatography has been usedl~; however, the details of this technique are in press 2~ at the time of this writing. The method of Sandler and Ruthven 8 employs initial separation with a cation exchange resin with the eventual oxidation of V M A to vanillin for quantitation. Others,1, 14, 2s have used similar techniques involving vanillin conversion. High-voltage eleetrophoresis has been employed by von Studnitz?2, ~9, 20 Isotope dilution techniques have been used by several investigators. 4' 6, 8o Gas chromatography has been suggested TM a2 although little has been reported regarding its use in the determination of urinary VMA. All of the foregoing tests are either very time consuming or require the use of equipment not usually available in a routine chemistry laboratory. In this laboratory we have employed a modification of the screening method of Gitiow.~..., 34 All values shown in this study and in prior communications 2~ 22, as were determined by the use of this method, which was employed as a semiquantitative screening technique. All specimens shown to have elevated values by the screening method were then subjected to two-phase chromatography for more discriminating evaluation. This communication describes a comparison of the simple screening procedure and chromatography as well as the application of these techniques to a group of children with and without neurogenic tumors. METHODS
The patient population consisted of children ranging from 1 week to 14 years of age at The Children's Hospital of Philadelphia. Indications for evaluation of V M A excretion, aside from the controls, incIuded thoracic and abdominal tumors, hypertension, chronic diarrhea and osteolytic lesions. No effort was made to restrict the physical activity of these children. Prior to and dur-
VMA
excretion in children
84 5
ing the collection of urine, foods containing caffeine, bananas, and vanilla were avoided. More recently citrus fruits have been excluded from the diet. Nonessential medications, particularly aspirin and sympathomimetic amines, were omitted, although several authors~0, sG have noted no significant interference with the latter compounds. Urinary collections were acidified to approximately p H 4 with 6N HC1 and stored at 8 to 10 ~ C. until tested. Twenty-four hour collections were preferred, although this was not possible in all cases. Urinary V M A determinations were performed by the following methods: Screening. An aliquot of urine (1 ml.) was adjusted to p H 1 (indicator paper) and extracted with ethyl acetate. The dried extract was dissolved in potassium carbonate solution and reacted with diazotized p-nitraniline to form a colored complex. The reaction mixture was subsequently extracted with n-amyl alcohol and the absorption of the final alcoholic extract determined after dehydration with granular anhydrous sodium sulfate. With a Beckman D U spectrophotometer the corrected reading for absorption at 545 m~ was determined as follows: Corrected Rd. 545 m/~ = (Rd. 500 m/~ + Rd. 590 m/~) Rd. 545 m / L 2 The details of the above method are essentially those described by Gitlow, 33' 34 with the omission of an initial extraction with ethyl acetate at p H 4 and the inclusion of V M A standards carried through the entire extraction procedure for quantitation. By the use of this method, absolute recoveries of 4 to 8 meg. V M A in water ranged from 80 to 100 per cent whereas recoveries of 12 mcg. were 76 to 78 per cent. Recoveries of 4 to 8 mcg. V M A added to urine ranged from 72 to 81 per cent. V M A standards maintained their potency in slightly acid ( p H 4 to 5) aqueous solution for 1 to 2 months at 8 to 10 ~ C. and would thereafter show a gradual decline. Standards maintained for 3 days at varying de-
846
June 1963
Y o u n g et al.
Table I. Control values in children with nonspecific minor conditions--24 hour collections (screening method)
Patient
JK BH RB BH TF BB MB BR GW VB RL DB DS JG G BH CC EF IL
Age 1 week 1 month 5 weeks 2} months 289 months 4 months 489 months 7 months 1 year 2 years 3 years 5 years 5 years 6 years 7 years 7 years 10 .years 12 years 14 years
24 hour ! volume mg./24 (ml.) hours 200 0.02 115 0.17 325 0.42 375 0.45 200 0.32 120 0.37 460 0.78 1.18 205 180 0.31 0.89 115 530 2.5 1.2 240 2.2 400 230 1.6 54O 2.4 1,070 2.8 1,080 3.3 4.5 855 900 4.8
mg./L.
0.1 1.5 1.3 1.2 1.6 3.1 1.7 5.8 1.7 5.9 4.7 5.0 5.4 6.9 4.4 2.4 3.1 5.3 5.3
grees of alkalinity (pH 7-9) showed 10 to 17 per cent loss. Excessive acidity in the reconstituted extract caused interference with the color development. Chromatography. One milliliter of urine was extracted repetitively with ethyl acetate as in the method of Gitlow. The dried residue was dissolved in ethanol, applied to a two-dimensional paper chromatogram by the method of Armstrong, 2~,2~ and developed at room temperature. After color development with potassium carbonate and diazotized p-nitroaniline, the areas corresponding to the position of pure V M A were removed, cut into small strips, and eluted in one of two ways: 1. n-Amyl alcohol with 1 per cent monoethanolamine (twice for 120 minutes with 4 ml. and once for 60 minutes with 2 ml.). 2. Two per cent aqueous sodium carbonate in ethyl alcohol (1:2) (with 2 ml. twice for 60 and 30 minutes, respectively, and once for 30 minutes with 1 ml.). Absolute recoveries of standards with each method of elution were 65 per cent and 50 per cent, respectively. Other areas representing various phenolic acids were also eluted and studied. Absorbance of the final
eluates were read against suitable blanks with a Beckman D U spectrophotometer. Suitable standards were chromatographed and eluted in the same way. This maneuver rendered the results comparable regardless of the method of elution. In specimens where only confirmation of significantly elevated levels was sought, visual comparison with standards stained on the paper by Armstrong's method was usually adequate. Compounds other than V M A eluted from the chromatograms were tentatively identified by their chromatographic mobility and color reaction with diazotized p-nitroaniline as described by ArmstrongY 5, 26 The specimens containing high levels of V M A usually showed relatively large amounts of homovanillic acid, although no precise quantitation of the latter was performed. Certain compounds were eluted and their maximal absorbancies determined. Where variance in absorbancies was noted in eluates from similar areas on different chromatograms it was assumed that this resulted from the admixture of two or more compounds occupying a common area. The following compounds were detected in some specimens: A. m-Hydroxyhippuric acid and p-hydroxymandelic acid occupying a common area (max. 500 mt~). B. m-Hydroxyphenylhydracrylic acid (max. 500 and 525 m/z). C. 3-Methoxy-4-hydroxyphenylhydracrylic acid (compound "C") (max. 545 m/z). D. p-Hydroxyphenylacetic acid (max. 475 and 545 m/z). E. Salicyluric acid (max. 475 m/z). F. 3-Methoxy-4-hydroxyphenylacetic acid (homovanillic acid) (max. 550 m/z). G. 3-Methoxy-4-hydroxybenzoic acid (vanillic acid) and 3-hydroxy-4-methoxybenzoic acid (isovanillic acid) occupying a common area (max. 545 m/z). Results. Comparison between values obtained with the screening method and chromatography with elution is shown in Fig. 1. The 6 specimens outlined by the oval show significant discrepancies between the two methods. Five of these specimens were de-
Volume 62 Number 6
liberately included since they represented either high normal or slightly elevated levels in patients with conditions not usually associated with high V M A excretion. All 6 specimens were shown to have normal amounts of V M A by chromatography. On the chromatograms 5 of the 6 showed significant amounts of compound " C " (above) which has been previously noted as a possible source of false elevations. In 4 specimens from patients with concentrations greater than 30 mg. per liter, there was poor correlation between the methods with chromatographic values which were 50 to 160 per cent of the Screening method values. In each of these specimens it was readily apparent by the screening method that they indeed contained a very high level of VMA. The remaining values showed satisfactory correlation. The clinical investigations are recorded in the various tables. Although neurogenic tumors in children comprised the major portion of the disorders, a variety of other conditions was studied. When possible the results are based on accurate 24 hour urine collections; however, in m a n y instances it has been necessary to express the findings as concentration per liter. The latter device proved useful in assessing disorders associated with elevated excretions of VMA, especially where preoperative circumstances were not suited to obtaining better collections of urine. In the evaluation of V M A excretion 24 hour collections probably give a more accurate reflection of the rate of production. In infants under 2 years of age, 24 hour specimens are believed to be essential for proper interpretation. Control values were obtained by the screening method from 65 children from 1 week to 14 years of age with nonspecific minor ailments. In the group under 2 years of age, values ranged from 0.1 to 8.6 mg. per liter (mean 2.3 + 2.4 rag. per liter). In those 2 to 14 years of age, the values ranged from 0 to 10.2 rag. per liter (mean 4.3 _+ 2.3 mg. per liter). The upper limits of normal were arbitrarily defined as 7.1 rag. and 8.9 mg. per liter, respectively. Table
VMA
excretion in children
84 7
I shows the values noted in 20 children from whom complete 24 hour collections were obtained. The results with the screening method in children with various neurogenic tumors are shown in Table II. In general, the patients with neuroblastomas which were large and inoperable or showed evidence of diffuse metastases presented with the highest V M A levels. Tumors which were incompletely removed continued to cause elevated urinary V M A levels although a marked decrease could be noted in comparison with preoperative levels. Recurrence or continued growth was reflected by rising V M A levels as illustrated in 2 cases (SC, M K ) shown in Fig. 2. Irradiation and chemotherapy caused a dramatic but temporary decrease in one of these cases ( M K ) . rThree patients with ganglioneuroblastomas were studied, including one in' whom only a normal postoperative specimen was available. T w o ( G T and DG) had high preoperative levels with a marked decrease following operative removal, as illustrated in Fig. 2. Because of intervertebral infiltration in one of these patients (DG), a small portion of the tumor was not removed. The V M A level 8 days after operation was 14 mg. per liter which while much diminished remained in the slightly elevated range. An25 t S" ~,s*'S
_.1
2O
E
9
E3
sS sS , s s S~ s,. SS
~E z z I,I n~" 0 (,n
s,,s S
ss 9
I0 9 ,.s s*' 9 9
5
":./(,
9 .
9
r 0 ~SS~
I 5
I I0
I 15
I 20
25
CHROMATOGRAPHY (mg/L)
Fig. 1. Comparison of VMA methods (patients and controls).
8 4 8 Young et al.
June
1963
Table II. VMA excretion (screening method)
I Age J J Turn~ Case (years) Sex origin L Neuroblastomas (13) 1 PB
46A2
F
Abd.
I ' =ses'l r,a men, lltervo'*l (reg.~L,) J M, B, RN S-I, irrad., VLB, M
- 3 days + 6 months
188 89 127 53 116
2 SA
8
F
Chest?
M, B, SC
Scalp biop., LC
+ +
3 EC
i~2
M
Abd.
B, M,
LC
- 9 days - 6 days + 2 weeks
4 EH
3~2
!V[
Abd.
SC
M, B
Mult. biop., C, irrad.
5 1 4 5
days day months months
140 90 -
Status
Died Tumor active
Died
- 1 month + 6 months + 9 months
23.2 26.4 -
Died
5 EL
16A2
iV[
Abd.
M, B, L Spinal
LC
+12 days
10.4
Died
6 AW
~2
M
Abd.
L, Liv. Testes
S-I, irrad., C
+ 2 months
19
Died
7 DW
56~2
M
Abd.
B, L
Cerv. nodes
Biop., C, irrad., + 6 months VLB +10 months +11 months
68.5 142 112
Died
B
S-I, LC
- 1 day + 3 days + 2 months
91.5 19.6 161
S-C
- 2 + 1 + 7 +14 +14
S-I, irrad.
- 1 day + 1 month + 2 months
8 SC
6A2
F
Abd.
9 FP
~2
M
Chest
10 CP
~/a2
F
Chest
11 M K
2~2
M
?
RN
Cerv. nodes, chest
S-I (RN)
GF
~2
M
Abd.
13
AM
~2
F
Abd.
RN
1.01w No apparent 0.38w recurrence 0.96w 1.61w 1.07w
days day months months months
8.1 w 2.2w
3.1w
Tumor active
+ 6 days + 9 days + 1 month +189 months + 2 months + 3 months + 5 months + 6 months
I3.2 9.9 5.3 8.4 26.7 1.3 66 -
Died
S-C
+ 7 days + 9 months +18 months
0.99w 1,2
Apparently "cured"
S-C
+ 6 days + 7 months + 1 year
4.7 5.5
Asymptomatic
S-I
- 5 - 3 - 2 + 8 + 3
s-i
(RN)
Irrad. + C., S-I (chest)
12
Tumor active
II. Ganglioneuroblastomas (3) 1 DG
2s~2
F
Chest
Intervertebral
days days days days months
150 164 120 14 8
Asymptomatic
Volume 62 Number 6
V M A excretion in children
84 9
Table II. Cont'd
Case
Age I Sex (years)
Tumor origin Metastases*
Interval~;
Treatmentf S-C
- 1 day + 6 days + 1 month
Chest
S-I (x2), irrad., M
7{ months after second operation
M
Chest
S-C
- 9 - 7 - 5 + 8 +11 +29 +389 +10 +16 +18
2 GT
1:)~2
M
Abd.
3 PW
2~2
F
RN
VMA I
(mg./L.)
Status
76 2.9
Asymptomatie
4.9
Asymptomatic
days days days days days days months months months months
20.0 15.3 15.3 5.1 6.3 7:4 4.5 1.5 4.9 5.9
Asymptomatic
1II. Ganglioneuromas (7) 1 DB
3~2
2 ML
10
F
Chest
S-C
- 3 + 3 + 5 +11
days days days months
19.8 7.2 4.5 3.2
Asymptomatic
3 KM
6
F
Chest
S-C
- 5 + 2 +14 +289 +689
days days days months months
7.2 2.1 4.7 0.25 7.5
Asymptomatie
4 DU
36~2
17
Chest
S-C
+ +
days days days months
7.7 6.2 3.3 5.3
Asymptomatic
5 KT
9
17
Chest
S-C
-13 days + 6 days + 2 months
10.8 1.7 4.5
6 DN
4
M
Chest
S-C
+3.I_, years
2.6
Asymptomatic
7 JB
10
M
Chest
S-I
- I + 5 +20 + 4
5.8 6.6 7.1
Asymptomatic
Intervertebral
3 2 7 7
day days days months
eM, marrow; B, bone; Rig, regional nodes; SC, subcutaneous tissues; L, lung; Liv., l i v e r . . ~'S-CI and S-I, operation with complete or incomplete removal of tumor; VLB, Velban (vmblastine sulfate, Lilly); M, mitomyein-C (Bristol Laboratories); LC, leurocristine sulfate (Lilly); C, Cytoxan (Mead Johnson). 1:Interval before or after initial treatment. w per 24 hours.
othe r specimen 3 months l a t e r showed a high n o r m a l level (8 rag. per liter). Seven patients with ganglioneuromas were studied an d of the 6 f r o m w h o m p r e o p e r a tive specimens were available only 3 showed elevated values. I t is noteworthy that none of the a b n o r m a l levels in these patients exceeded 20 mg. per liter a n d in one there was a level of 10.8 mg. per liter w h i c h is only slightly above the n o r m a l range. I n each
of the cases w i t h elevated levels t h er e was a notable p o st o p er at i v e drop to n o r m a l levels as is illustrated in Fig. 2 (DB, M L , K T ) . O t h e r r el at ed conditions i n c l u d e d a pheoc h r o m o c y t o m a a c c o m p a n i e d by a V M A level of 23.3 mg. p e r liter as well as e v a l u a t i o n of the free catecholamines. O n e subject with a c h e m o d e c t o m a showed V M A values which varied f r o m a high n o r m a l level of 7.2 mg. to significantly elevated values of 13.5 mg.
Younget al.
850
June 1963
4
DG
120
80
/
8 ~- 3o
I
/
/
/
/
A GANGLIONEUROMA
IRRAD. +
/
(,~ Z
/
/
9 NEUROBLA.STOMA o r GANGLIO-' NEUROBLASTOMA
/
DB
2o QK
I0 doys ~'~'PRE-OP
20 doys
I months
6
II
16
18
POST-OP INTERVAL
Fig. 2. VMA excretion in 7 cases of neurogenic tumors. per liter (9.8 rag./15 hr.). Five patients with neurofibromatosis, including one with hypertension, all showed values (2.5 to 8.5 rag. per liter) within the normal range. Ten patients with miscellaneous tumors and pseudotumors showed a normal range of values (0.37 to 7.2 mg. per liter). This group included sarcoma of the mandible, subhepatic abscess, acute myelogenous leukemia, adrenal hemorrhage with hematoma, xanthogranuloma with extensive abdominal spread, third ventricle astrocytoma, intestinal obstruction, metastatic Wilm's tumor, ruptured appendix and uterine rhabdomyosarcoma. Seven children with idiopathic hypertension, ranging in age from 5 to 15 years, showed VMA values ranging from 1.3 to 8.1 mg. per liter. Two other children showed levels of 9.5 and 12.4 mg. per liter by the screening method, but chromatographic evaluation revealed values of 3 and 4 mg. per liter, respectively. Both specimens
showed relatively large amounts of what was vanillic and isovanillic acid according to chromatographic mobility and stains. These have been previously noted as possible sources of interference. Four patients with the celiac syndrome had values ranging from 2.8 to 8.4 mg. per liter. Eleven patients with diarrhea, including 2 with ulcerative colitis and one with ileocolitis, excreted amounts of VMA ranging from 0.86 to 8,1 mg. per liter. Two other specimens in the latter group showed slight elevations Of 11.6 and 14.7 mg. per liter by the screening method. Chromatography, however, revealed values within normal limits. Twelve patients with mucoviscidosis and moderate to severe pulmonary involvement showed values ranging from 1.2 to 14.4 nag. per liter (mean 6.6). Three of these specimens showed borderline values (8.0, 9.0 and 9.4 mg. per liter) while one specimen was in the slightly elevated range (14.4 mg. per liter).
Volume 62
Number 6
Of 10 patients with various conditions which would cause hypoxia (i.e., atelectasis, cyanotic congenital heart disease, lobar pneumonia, acute asthmatic attack) only 2 showed values (9.5 and 10 mg. per liter) which slightly exceeded normal values. T w o patients with familial dysautonomia showed normal values (4.4 and 6.7 mg. per liter). A i-year-old infant with glycogen storage disease showed an excretion of 1.65 mg. per day which represents a slight elevation for age. DISCUSSION
A simplified method has been used in evaluating 249 urine specimens from 147 patients as a relatively rapid means of detecting excessive excretion of V M A . I t has proved to be useful in the diagnostic evaluation of children with thoracic and abdominal tumors, hypertension, chronic diarrhea, and osteolytic lesions for the possible presence of neural crest tumors. Serial determinations following the removal of such tumors have assisted in the follow-up care of these children. I n one recent study, 1~ 25 patients with neuroblastomas were reported to have significantly elevated levels of urinary VMA, whereas 3 patients with ganglioneuromas showed normal levels. It was suggested that V M A excretion could be used as a means of distinguishing these two tumor types preoperatively. The results herein presented do not support such a sharp differentiation. Kogut and Kaplan 37 reviewed the cases of 29 patients with adrenergic symptoms and neurogenic tumors, of which 6 were ganglioneuromas. Of this latter group, only one 3s was studied and shown to have a significantly elevated leveI of urinary V M A . Voorhess 23 has demonstrated that a few patients with neuroblastomas do not exhibit elevated levels of V M A in the urine and a small number show no elevation of any of the known catecholamines or metabolites. In a more recent report, a9 she emphasized the potential value of other urinary metabolites in guiding clinical care. The determination of dopamine and norepinephrine would seem
VMA
excretion in children
85 1
helpful in detecting those few patients in whom V M A excretion is not elevated. Recently von Studnitz ~9 has called attention to the fact that homovanillic acid excretion is usually elevated in patients with neuroblastomas. Of 25 patients whom he studied, 24 had elevations of urinary V M A and 17 had elevations of homovanillic acid. Of the latter group, there was a normal V M A excretion in one. He and other investigators17, 19 have shown that homovanillic acid excretion is not elevated in patients with pheochromocytoma who have high V M A levels. I n the current chromatographic studies, specimens with high V M A levels generally showed large amounts of homovanillic acid, although precise quantitation of each was not performed. In the present study one patient with a chemodectoma showed an elevated level of V M A in one specimen and several specimens with high normal values. There was a large thoracic mass with intervertebral extension, erosion of adjacent ribs and vertebral bodies with the eventual development of paraplegia. Moderate hypertension was present. This type of tumor is thought to arise from the chemoreceptor system and usually is non-chromaffin staining. In this instance, the tumor demonstrated chromaffin staining material, as did a carotid body-like tumor with elevated V M A excretion which was recently reported. 2. The latter patient also had moderate hypertension. The screening of hypertensive patients should include one or more tests to rule out the possibility of a pheochromocytoma. Several investigators sla have recommended the use of V M A excretion as a practical screening test for this condition but free urinary catecholamines should be determined in doubtful cases. It is of interest that the V M A excretion m a y be elevated in the presence of virtually normal values for urinary catecholamines a3, 3, and the reverse situation m a y also be found in a small number of cases?* I n the current study one patient with a pheochromoeytoma showed a significantly elevated level of VMA, as well as of urinary catechotamines. Five pa-
852
Young et al.
tients with neurofibromas, including one with hypertension and extensive intra-abdominal neurofibromatosis, showed no significant elevation of VMA. It is of interest that, in the previously mentioned review ~7 of neurogenic tumors with adrenergic symptoms, 15 of the 99 showed hypertension. Two patients (DG, DB) with neurogenic tumors were originally admitted for evaluation of gastrointestinal dysfunction. One patient, previously reported upon, 22 was admitted for evaluation of hypercalcemia, celiac syndrome, and growth retardation. An intravenous pyelogram incidentally revealed the presence of a posterior mediastinal mass which was completely removed at operation and was shown to be a ganglioneuroma. There was improvement in growth rate and bowel symptoms thereafter. The second, a 3-year-old girl, was admitted for evaluation of chronic diarrhea of 6 months' duration. A posterior mediastinal mass was noted on a routine flat plate of the abdomen. This proved to be a ganglioneuroblastoma. The gastrointestinal improvement was striking following subtotal surgical removal. In both patients a mild-to-moderate degree of acidosis was noted. A more detailed discussion of these two cases will be published. In view of the findings in these last 2 patients and similar reports in the literature, 37 a number of children with chronic diarrhea in this hospital have had urinary V M A determinations. Four patients with the celiac syndrome and 11 with diarrhea, including 2 with ulcerative colitis and one with ileocolitis, have shown normal values. Of 12 patients with mucoviscidosis studied, questionable or slight elevations of urinary V M A (8.9 to 13.4 mg. per liter) were found in 4. Each of the latter group showed moderate to severe pulmonary involvement. Of 10 patients with various other conditions causing hypoxia, 2 were shown to have borderline elevations (9.5 and 10 mg. per liter). Since hypoxia is a stimulus to catecholamine production, this would seem a likely explanation for these elevated levels. A previous investigator s~ has noted that seriously ill patients with severe pulmonary
]une 1963
insufficiency m a y show elevated levels of V M A excretion. In a prior study from this laboratory, *~ medullary hyperplasia and elevated content of catecholamines in the adrenal gland were shown to be present in 5 o f 6 patients with cystic fibrosis who were fortuitously included in an evaluation of adrenal medulla hyperplasia seen at autopsy. The data herein presented are not sufficient for further conjecture, but it appears that catecholamine hypersecretion is not a prim a r y factor in this disease. The possibility of an altered metabolic disposition cannot be completely discounted. Pertinent to this question is the recent observation of Selye, 41 that prolonged administration of isoproterenol caused the secretion of viscous saliva and excessive stimulation of saliva~3r gland growth in rats. Other conditions which have been noted by previous investigators to cause small elevations of V M A are the carcinoid syndrome,14, 2, paroxysmal tachycardia, 14 and burns24 SUMMARY
Urinary V M A excretion has been determined by a semiquantitative screening method in 249 urine specimens from 147 children, some of whom were evaluated for the presence of neurogenic tumors. A discussion of the method and its limitations as well as chromatogenic comparisons is presented. False elevations in a small number of samples were shown to be caused by urinary phenolic acids probably derived from dietary sources. Thirteen patients with neuroblastomas were studied extensively. All pretreatment levels of V M A excretion were elevated. T w o of 3 patients with ganglioneuroblastoma were evaluated preoperatively and both showed augmented excretion. Six of 7 children with ganglioneuroma were evaluated preoperatively. Only 3 of the latter group showed elevated levels of V M A excretion. One child with a pheochromocytoma and another with a chemodectoma also showed elevated levels. Results in children with various other conditions are discussed. These conditions
Volume 62
Number 6
include miscellaneous tumors, hypertension, diarrhea, cystic fibrosis of the pancreas, hypoxia, familial dysautonomia, a n d glycogen storage disease. We wish to acknowledge the assistance of Dr. William Yakovac who performed the histologic examinations of the tumors presented. REFERENCES
1. Armstrong, M. 13., McMillan, A., and Shaw, K. N. F.: 3-Methoxy-4-hydroxy-D-mandelic acid, a urinary metabolite of nor-epinephrine, Biochlm. et biophys, acta 25: 422, 1957. 2. Axelrod, ]'.: O-Methylation of epinephrine and other catechols in vitro and in vlvo, Science 126: 400, 1957. 3. Axelrod, J., Inscoe, J. K., Senoh, S., and Witkop, B.: O-Methylation, the principal pathway for the metabolism of epinephrine and norepinephrine in the rat, Biochim. et biophys, acta 27: 210, 1958. 4. Kirschner, N., Goodall, McC., and Rosen, L.: Metabolism of d, 1-adenaline-2-C TM in the human, Proc. Soc. Exper. Biol. & Med. 98: 627, 1958. 5. LaBrosse, E. H., Axelrod, J,, and Kety, S. S.: O-Methylation, the principal route of metabolism of epinephrine in man, Science 128: 593, 1958. 6. Goodall, M., Kirshner, N., and Rosen, L.: Metabolism of noradrenaline in the human, J. Clin. Invest. 38: 707, 1959. 7. LaBrosse, E. H., Axelrod, J., and Sjoerdsma, A.: Urinary excretion of normetanephrine by man, Fed. Proc. 17: 386, 1958. 8. Sandler, M., and Ruthven, C. R. J.: Quantitative colorimetrie method for estimation of 3-methoxy-4-hydroxymandelic acid in urine. Value in diagnosis of pheochromocytoma, Lancet 2: 114, 1959. 9. Robinson, R., Ratcliffe, J., and Smith, P.: A screening test for pheochromocytoma, J. Clin. Path. 12: 541, 1959. 10. Gitlow, S. E., Mendlowitz, M., Khassis, S., Cohen, G., and Sha, J.: The diagnosis of pheochromocytoma by determination of urinary 3-methoxy-4-hydroxymandelic acid, J. Clin. Invest. 39: 221, 1960. 11. Sunderman, F. W., Jr., Cleveland, P. D., Law, N. C., and Sunderman, F. W.: A method for the determination of 3-methoxy4-hydroxymandelic acid (vanilmandelic acid) for the diagnosis of pheochromocytoma, Am. J. Clin. Path. 34: 293, 1960. 12. von Studnitz, W.: Methodische and klinische Untersuchungen fiber die Ausscheidung der 3-methoxy-4-hydroxymandels~iure im Urin, Scandinav. J. Clin. & Lab. Invest. 12: 1, 1960 (supp. 48). 13. Jacobs, S. L., Sobel, C., and Henry, R. J.: Excretion of 3-methoxy-4-hydroxymandelic acid and catecholamines in patients with
V M A excretion in children
14.
15.
16. 17.
18. 19. 20.
21.
22.
23.
24. 25. 26.
27.
28.
29.
853
pheochromocytoma, J. Clin. EndocrinoL 21: 315, 1961. Crout, J. R., Pisano, J. J., and Sjoerdsma, A.: Urinary" excretion of catecholamines and their metabolites in pheochromocytoma, Am. Heart J. 61: 375, 1961. Voorhess, M, L., and Gardner, L. I.: Urinary excretion of norepinephrine, epinephrine and 3-methoxy-4~hydroxymandelic acid by children with neuroblastoma, J. Clin. Endocrinol. 21: 321, 1961. Bell, M., and Steward, J. K.: Neuroblastoma and catecholamine excretion, Lancet 1: 1061, t961. Bettex, M., and Kaser, H.: Diagnostic and prognostic value of the determination of urinary output of vanillylmandelic acid in tumors of sympathetic nervous system, Arch. Dis. Childhood 37: 138, 1962. Kaser, H., and von Studnitz, W.: Urine of children with sympathetic tumors, A. M. A. J. Dis. Child. 102" 199, 1961. von Studnitz, W.: Neuroblastoma and catecholamine excretion, Lancet 2: 215, 1961. Steiker, D. D., and Eberlein, W. R.: Urinary VMA excretion in children with neural tumors. Program of the 43rd Meeting of the Endocrine Society, New York, N. Y., June, 1961, p. 102. Greenberg, R. E., and Gardner, L. I.: New diagnostic test for neural tumors of infancy: Increased urinary excretion of 3-methoxy-4hydroxymandelic acid and norepinephrine in ganglioneuroma with chronic diarrhea, Pediatrics 24: 683, 1959. Leboeuf, G., Eberlein, W. R., Steiker, D. D., and Bongiovanni, A. M.: Functioning ganglloneuroma, celiac syndrome and renal acidosis, presented at the American Pediatric Society, 71st Annual Meeting, Atlantic City, May, 1961. Voorhess, M. L., and Gardner, L. I.: Studies of catechotamine excretion by children with neural tumors, J. Clin. Endoerinol. 22: 126, 1962. Glenner, G. G.: A functional carotid-bodylike tumor, A. M. A. Arch. Path. 73: 230, 1960. Armstrong, M. D., Shaw, K. N. F., and Wall, P.E.: The phenolic acids of human urine, J. Biol. Chem. 218" 293, 1956. Armstrong, M. D.: Estimation of 3-methoxy4-hydroxymandelie acid (VMA) for the detection of phenochromacytoma. Pamphlet privately circulated by the author, Fels Research Institute, Yellow Springs, Ohio (8 pp.). Kaser, H.: Eine eindimensionale papierehromatographische Method zur Bestimmung der 3-Methoxy-4-hydroxymandelic Sfiure, Experientia (in press). O'Brien, D., and Ibbott, F. A.: Laboratory manual of pediatric micro and ultramicrobiochemical techniques, ed. 3, New York, 1962, Harper and Row, p. 74. von Studnitz, W., and Hanson, A.: Deter-
8 54
30.
31.
32.
33.
34.
35.
Young et al.
mination of 3-methoxy-4-hydroxymandelic acid in urine by high voltage paper electrophoresis, Scandinav. J. Clin. & Lab. Invest. 11: 101, 1959. Smith, A. A., Schweitzer, J. W., and Wortis, S. B.: The determination of norepinephrine metabolites by isotope dilution, Fed. Proc. 18: 145, t959. Williams, C. M., and Sweeley, C. C.: A new method for the determination of urinary aromatic acids by gas chromatography, J. Clin. Endocrinol. 21. 1500, 1961. Greet, M., and WiIliarns, C. M.: Catecholamine metabolism in neuroblastoma. Program of the 32nd Annual Meeting of the Society for Pediatric Research, Atlantic City, May, 1962, p. 115. Gitlow, S. E., Ornstein, L., Mendlowitz, M., Khassis, S., and Kruk, E.: A simple colorimetric urine test for pheochromoeytoma, Am. J. Med. 28: 921, 1960. Gitlow, S. E., Mendlowitz, M., Kruk, E., and Khassis, S.: Diagnosis of pheochromocytoma by assay of catecholamine metabotites, Circulation Res. 9: 746, 1961. Young, R. B., Steiker, D. D., Koop, C. E., and Bongiovanni, A. M.: Evaluation of a simple colorimetric determination of urinary
June 1963
36.
37. 38. 39.
40.
41.
3-methoxy-4-hydroxymandelic acid. Program of the 32nd Annual Meeting of the Society for Pediatric Research, Atlantic City, May, 1962, p. 71. Sandler, M., and Ruthven, C. R. J.: 3-Methoxy-4-hydroxymandelic acid excretion in pheochromoeytoma. Adrenergic mechanisms, Ciba Foundation Symposium, Boston, 1960, Little, Brown & Company, p. 40. Kogut, M. D., and Kaplan, S. A.: Systemic manifestations of neurogenic tumors, J. PEDIAT. 60: 694, 1962. Greenberg, R. E., and Gardner, L. I.: Catecholamine metabolism in a functional neural tumor, J. Clin. Invest. 39: 1729, 1960. Voorhess, M. L., and Gardner, L. I.: The value of serial catecholamine determinations in children with neuroblastoma, Pediatrics 30" 241, 1962. Bongiovanni, A. M., Yakovac, W. C., and Steiker, D. D.: Study of adrenal glands in childhood: Hormonal content correlated with morphologic characteristics, Lab. Invest. 10: 956, 1961. Selye, H., Veilleux, R,, and Cantin, M.: Excessive stimulation of salivary gland growth by isoproterenol, Science 133: 44, 1961.