Catecholamine Metabolism in Neuroblastoma: Kinetics of Conversion of 3H-3-methoxy-4-hydroxyphenylglycol to 3 H-3-methoxy-4-hydroxymandelic Acid ELWOOD H. L A B R O S S E Center for the Study of Trauma, University of Maryland School of Medicine, Baltimore, Maryland 21201
the iv injection of 3H-VMA itself, and following iv 3H-MHPG. In addition, this patient3 was normotensive and his tumor converted H-NE to 3H-MHPG in tissue culture. These findings provide quantitative information on the in vivo conversion of MHPG to VMA in a patient with neuroblastoma and suggest that assay of urinary MHPG, after hydrolysis of the conjugates, may be of considerable value in the diagnosis of patients with neuroblastoma. (J Clin Endocr 30: 580, 1970)
ABSTRACT. Previous studies of the metabolic in tissue culture activity of neuroblastoma tumor 3 H-norepinephrine have revealed that added (3H-NE) is partially converted to 3H-3-methoxy4-hydroxyphenylglycol (3H-MHPG), but not to 3-methoxy-4-hydroxymandelic3 acid. In addition, the in vivo conversion of H-MHPG to 3HVMA has been described. This report presents quantitative information on the in vivo conversion of 3H-MHPG to 3H-VMA3 (27%) based on the results of assay of urinary H-VM A following
T
HE ELEVATED urinary excretion of 3-methoxy-4-hydroxymandelic acid (VMA)1 in patients with neuroblastoma has been reported by numerous authors (1-9), and it has usually been assumed that norepinephrine (NE1), or norepinephrine plus epinephrine, in the tumors was the source of this metabolite. The metabolic pathways from NE to VMA are presented in Fig. 1. In contrast to thefindingsin patients with pheochromocytoma, only a very small percentage of patients with neuroblastoma have hypertension even though a majority of the cases have considerable elevation of urinary VMA. One explanation for the paradoxical finding of a marked increase in the urinary excretion of a metabolite of NE in the absence of physiological symptoms of NE excess, such as hypertension, pallor or hyperpnea, is that the NE is metabolized within the tumor before release into the blood stream. This explanation is supported by the finding that neuroReceived October 3, 1969. 1 Abbreviations in consecutive order: VMA = 3methoxy-4-hydroxymandelic acid, NE = norepinephrine, 3H-MHPG =D,L-7-3H-3-methoxy-4-hydroxyphenylglycol, MHPG = 3-methoxy-4-hydroxyphenylglycol,
and
3
H-VMA=D,L-7- 3 H-3-
methoxy-4-hydroxymandelic acid.
blastoma tumor in tissue culture has been found to metabolize added 7-3H-NE, and the major metabolic product was found to be 3H-MHPG (10).1 In addition, it has been found that the tumor content of MHPG1 was about nine times that of VMA and the blood content of MHPG was two times that of VMA (11). This evidence has indicated that NE is synthesized within the neuroblastoma tumor and metabolized to MHPG; the latter is released into the blood and subsequently metabolized to VMA. The turnover of 3H-VMA,1 and kinetics of conversion of 3H-MHPG to 3HVMA following the intravenous administration of 3H-VMA and of 3H-MHPG to a patient with neuroblastoma are reported here. Materials and Methods 3
The 7- H-VMA and 7-3H-MHPG were synthesized from vanilloylformic acid and from 3 - methoxy - 4 - hydroxyphenylformylmethanol gas and a palladium on charcoal using tritium catalyst.2 The 3H-labeled compounds were purified by paper chromatography, and each showed only one peak of radioactivity which corresponded to the authentic compounds in at 2
Synthesized on contract by New England Nuclear Corp., Boston, Mass. 580
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May 1970
CATECHOLAMINE METABOLISM IN NEUROBLASTOMA
least 3 different solvent systems. The systems used were isopropanol-ammonia-water (8:1:1), butanol-ethanol-water (4:1:1), and butanolacetic acid-water (4:1:1). The patient was a 4-month-old white male, who, on routine examination, was found to have an enlarged liver, palpable at 5 cm below the right costal margin. The patient was the child of a gravid I, Para 1 female and had a normal birth weight of 7 pounds 3 ounces. The patient gained normally and appeared to be a perfectly healthy, well-nourished child of the stated age. Liver function tests, including glucagon stimulation, were within normal limits. The blood count was in the normal range: hemoglobin of 13.2 g/100 ml, WBC 12,500/mm3, with a normal3differential count, and platelets of 145,000 mm . An intravenous pyelogram revealed normal kidney size and function, with the left kidney being slightly lower than normal but this was felt at the time to be a congenital variation, within normal limits. A spot test on the urine for elevated catecholamine metabolites was positive (12). An ethyl acetate extract of acidified urine was prepared, and a 2-dimensional paper chromatogram of this extract revealed 2 strongly reactive spots; these corresponded to VMA and 3-methoxy-4hydroxyphenylacetic acid (HVA).3 These results strongly indicated the presence of neuroblastoma with metastatic involvement of the liver. Following the finding of the elevated levels of VMA and HVA in this patient's urine, he was admitted to the Clinical Study Center at the University of Maryland Hospital for further study. A repeat intravenous pyelogram with laminograms revealed normal kidney size and function, and, in addition, a shadow at the upper pole of the left kidney which appeared to be approximately 3X2 cm in size. Roentgenograms of the abdomen confirmed hepatomegaly, but no areas of calcification were found. To study the turnover of VMA in this patient, 102.6 /xCi (2.98 Mg) of 7-3H-VMA was administered intravenously, and serial urine specimens were collected for 45 hr. Because earlier studies on other patients had revealed that MHPG was partly converted to VMA, the turnover and metabolism of 7-3H-MHPG was also studied in this patient by an intravenous administration of 27.4 /xCi (0.58 ng) of 3HMHPG, and serial urine specimens were collected for a period of 46 hr. Aliquots of the urine specimens were acidified with HC1 to pH 2 and extracted with ethyl acetate. The ethyl acetate extracts were evaporated to dryness; the resi-
OH
OH
CH-CHJ-NHJ
tH-CHO
581 OH OH
JH-CH,
CONJUGATES
tOH
y^
CH-CHJ.NHJ
NOREPINEPHRINE
S
. O H NORMETANEPHRINE
3-METHOXY.4-HYDROXY. MANDELALDEHYDE
OH CH-CHO
\^T)H
OH OH / CH-CHj /
\^0H
OH N-METHYLATION, ETC.
3.4-OIHYDROXY. MANDELALDEHYDE
OH
N.
1
OH CH-COOH
\^-VHJ OH
3,4-OIHYOROXYPHENYLGLVCOL
K
J.METHOXY-4-HYDROXY PHENYLCLYCOL
J-METH0XY-4-HYDR0XYMANDELIC ACID (VMA)
T
W-CO'
CL FIG. 1. Metabolic pathways of norepinephrine (modified from Kopin, I. J., Science 131: 1372, 1960).
dues were dissolved in a small volume of ethanol, and aliquots of the ethanol extract were transferred to 8 in. X8 in. sheets of Whatman #3 MM filter paper and developed bidimensionally in the isopropanol-ammonia- water (8:1:1) and benzene-propionic acid-water (1000:700:41) systems (13). The chromatograms were dried and examined under ultraviolet light after chilling in liquid nitrogen. The area corresponding to authentic VMA, and which showed its characteristic phosphorescence, was cut out and eluted with 70% ethanol. Aliquots of the ethanol-water eluate were assayed colorimetrically for VMA and for tritium content by adding an aliquot to a fluor solution consisting of toluene with 0.4% 3,5-diphenyloxazole and 0.01% 3',5'-diphenyloxazolyl benzene 4and using a liquid scintillation spectrometer. The specific activities of the 3H-VMA in each of the urine specimens were calculated from the data on VMA and tritium contents. An exploratory laparotomy was performed and a tumor mass was found above the left kidney; this mass was removed completely and was found to measure 3.5 X2.5 X2.5 cm. A portion of this tumor was explanted to tissue culture. Examination of the liver revealed extensive involvement with nodular masses, and a small nodule in the liver was biopsied. The tumor mass was found to contain a narrow rim of normal adrenal tissue, but the remainder of the mass was neuroblastoma. The metastatic area in the liver was neuroblastoma and this lesion contained pseudorosettes. 4
Tri-Carb Scintillation Spectrometer, Model 3002, Packard Instrument Company, LaGrange, 3 HVA = 3-methoxy-4-hydroxyphenylacetic acid. 111.
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LABROSSE
582
Volume 30
TABLE 1. Urinary excretion of tritium after iv administration of 3H-VMA (102..6 nCi) to neuroblastoma patient SS Spec. no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Avg
time (hr) 0.150 0.634 1.067 1.292 1.626 2.168 2.876 3.501 3.918 4.292 4.626 7.126 9.626 11.501 14.251 16.026 17.560 19.910 22.460 24.460 25.876 27.501 29.501 33.251 37.501 39.876 41.842 43.759 Totals % of inj. dose
Total 3H in spec. (nCi) 34,706.1 32,627.3 6,437.2 7,787.8
4,763.8
4,172.4 6,747.5 485.8 273.8 400.4 464.1 1,153.2 106.1 279.3 188.7 75.8 39.4 120.0 82.6 68.6 31.8 74.8 48.0 94.2 78.2
50.3 49.9 39.4
Total 3H excr. rt. (nCi/min) 1928
3
H as 3H2O in spec. (nCi) 150.0 239.9 580.6 834.8 589.6 158.8 95.89 20.37 21.60 40.61 119.15 63.61 13.42 70.11 41.65 32.23 10.25 27.60 34.81 24.29 25.97 40.04 21.36 35.10 45.92 29.41 41.92 22.63 3,432.
816 536 519 190 104 150
16.2 13.7 16.0 30.9 4.05 7.07 1.33 1.57 0.815 0.433 0.628 0.718 0.549 0.707 0.499 0.533 0.262 0.521 0.373 0.494 0.305
101,466. 98.9
To study the metabolism of the tumor in tissue culture, 3H-NE, 14C-NE and 14C-tyrosine were added to tubes containing media and tumor and to control tubes which contained media only. Twenty-nine hr following the addition of the labeled compounds, portions of the media were removed and were subjected to paper chromatography in the n-butanol-ethanol- water (4:1:1) and n-butanol-ethanolwater-acetic acid (80:20:20:1)3 systems. The tritium-labeled water ( H23O) content of the urine, collected after both H-VMA and 3 H-MHPG, was assayed as follows: Aliquots of the urine (1-2 ml) were transferred to small bottles and the bottles were fitted with stoppers through which test tubes were inserted, so that the bottom of the test tubes came within 1.5 cm of the liquid within the bottles. The test tubes were made to function as "cold finger condensers" by filling them with crushed dry ice and this simple apparatus permitted a lowtemperature distillation (
