Journal of Pediatric Ophthalmology and Strabismus

Catecholamine Metabolites in the Aqueous of Retinoblastoma

David H Abramson, MD; Philip A Piro, MS; Robert DeCresce, MD; Robert M Ellsworth, MD; David Kitchin, MD

Abstract

Catecholamines are a group of metabolically related compounds occurring naturally in a variety of tissues including the central nervous system, the peripheral nervous system, and the adrenal medulla.1 Many of the catabolic products of catecholamines are normally found in the serum and urine.2 The main catabolic products of norepinephrine and epinephrine are noremetanephrine (NM), and metanephrine (M), 3-methoxy, 4-hydroxy mandelic acid and vanilmandelic acid (VMA).

Since 1957 it has been recognized that a variety of tumors of neural crest origin secrete large amounts of catecholamines into the blood and urine.1-6 Among these tumors are pheochromocytoma, neuroblastoma, and ganglioneuroma. In cases of neuroblastoma, the determination of VMA may have prognostic implications and may aid in detecting metastasis and recurrences prior to the time they are clinically obvious.6 For example, urinary VMA levels may return to normal after successful surgical removal of a neuroblastoma.6 The first evidence of catecholamine containing tissue in the human retina was presented in 1966.7 Using silver staining techniques, catecholamine-containing cell bodies were found in the innermost cell row of the inner nuclear layer among the amacrine cells. Retinoblastoma is known to have many similarities to neuroblastoma, and while there is no direct evidence that retinoblastoma tissue either contains catecholamines or secretes catecholamines, there is indirect evidence which suggests that certain cases of retinoblastoma may be associated with elevated catecholamine levels.

In 1962, one case was presented with retinoblastoma and elevated urinary VMA levels.6

In 1966, 12 retinoblastoma patients were studied, and nine had total urine VMA and HVA levels above normal. R In one patient with elevated VMA and HVA levels prior to surgery, the urinary VMA and HVA levels returned to normal after enucleation of the eye containing the tumor. A statistical analysis, however, revealed these levels to be insignificant, perhaps due to the small sample number. There has been considerable interest in aqueous aspiration to aid in the diagnosis and management of retinoblastoma. Recently, it has been shown that lactic dehydrogenase (LDH) is elevated in the aqueous of 92 percent of eyes harboring retinoblastoma.' It was hoped that in this pilot study, detectable levels of catecholamine by-products would be found in the aqueous humor of retinoblastoma patients.

MATERIALS AND METHODS

The following catecholamine by-products were assayed in this study: normetanephrine, metanephrine, VMA and 3-methoxy, 4-hydroxy phenoglycol. The specimens were approximately 0.2 cc of aqueous humor obtained using a 30 gauge needle and a 1 cc tuberculin syringe at the time of enucleation. In all five cases, the clinical diagnosis of retinoblastoma was confirmed pathologically. Simultaneous analysis of lactic dehydrogenase was performed in each of these eyes. The level of catabolic products was determined using standard techniques.10 High voltage electrophoresis is used to separate normetanephrine, metanephrine, VMA, and 3methoxy, 4-hydroxy phenoglycol. When separated, the bands are stained with a diazotized P-nitro-analine spray revealing a purple color. The test is sensitive to 1 /xg/ml.

RESULTS

There were no detectable levels of normetanephrine, metanephrine, VMA,or3-methoxy, 4-hydroxy phenoglycol in any of the five aqueous samples.

DISCUSSION

Because of two prior studies6'8 demonstrating elevated urinary levels of VMA and HVA in patients with retinoblastoma, it has been postulated that retinoblastoma is a catecholamine secreting tumor. In this pilot study of catecholamine catabolic products, no detectable levels were seen in the aqueous of each of the four metabolic products. This technique does not detect the presence of HVA, and additional studies will be needed to determine whether HVA is present in the aqueous of eyes containing retinoblastoma. It has been shown that in patients with neuroblastoma, there may be a correlation between the appearance of rosettes and the levels of catecholamine…

Catecholamines are a group of metabolically related compounds occurring naturally in a variety of tissues including the central nervous system, the peripheral nervous system, and the adrenal medulla.1 Many of the catabolic products of catecholamines are normally found in the serum and urine.2 The main catabolic products of norepinephrine and epinephrine are noremetanephrine (NM), and metanephrine (M), 3-methoxy, 4-hydroxy mandelic acid and vanilmandelic acid (VMA).

Since 1957 it has been recognized that a variety of tumors of neural crest origin secrete large amounts of catecholamines into the blood and urine.1-6 Among these tumors are pheochromocytoma, neuroblastoma, and ganglioneuroma. In cases of neuroblastoma, the determination of VMA may have prognostic implications and may aid in detecting metastasis and recurrences prior to the time they are clinically obvious.6 For example, urinary VMA levels may return to normal after successful surgical removal of a neuroblastoma.6 The first evidence of catecholamine containing tissue in the human retina was presented in 1966.7 Using silver staining techniques, catecholamine-containing cell bodies were found in the innermost cell row of the inner nuclear layer among the amacrine cells. Retinoblastoma is known to have many similarities to neuroblastoma, and while there is no direct evidence that retinoblastoma tissue either contains catecholamines or secretes catecholamines, there is indirect evidence which suggests that certain cases of retinoblastoma may be associated with elevated catecholamine levels.

In 1962, one case was presented with retinoblastoma and elevated urinary VMA levels.6

In 1966, 12 retinoblastoma patients were studied, and nine had total urine VMA and HVA levels above normal. R In one patient with elevated VMA and HVA levels prior to surgery, the urinary VMA and HVA levels returned to normal after enucleation of the eye containing the tumor. A statistical analysis, however, revealed these levels to be insignificant, perhaps due to the small sample number. There has been considerable interest in aqueous aspiration to aid in the diagnosis and management of retinoblastoma. Recently, it has been shown that lactic dehydrogenase (LDH) is elevated in the aqueous of 92 percent of eyes harboring retinoblastoma.' It was hoped that in this pilot study, detectable levels of catecholamine by-products would be found in the aqueous humor of retinoblastoma patients.

MATERIALS AND METHODS

The following catecholamine by-products were assayed in this study: normetanephrine, metanephrine, VMA and 3-methoxy, 4-hydroxy phenoglycol. The specimens were approximately 0.2 cc of aqueous humor obtained using a 30 gauge needle and a 1 cc tuberculin syringe at the time of enucleation. In all five cases, the clinical diagnosis of retinoblastoma was confirmed pathologically. Simultaneous analysis of lactic dehydrogenase was performed in each of these eyes. The level of catabolic products was determined using standard techniques.10 High voltage electrophoresis is used to separate normetanephrine, metanephrine, VMA, and 3methoxy, 4-hydroxy phenoglycol. When separated, the bands are stained with a diazotized P-nitro-analine spray revealing a purple color. The test is sensitive to 1 /xg/ml.

RESULTS

There were no detectable levels of normetanephrine, metanephrine, VMA,or3-methoxy, 4-hydroxy phenoglycol in any of the five aqueous samples.

DISCUSSION

Because of two prior studies6'8 demonstrating elevated urinary levels of VMA and HVA in patients with retinoblastoma, it has been postulated that retinoblastoma is a catecholamine secreting tumor. In this pilot study of catecholamine catabolic products, no detectable levels were seen in the aqueous of each of the four metabolic products. This technique does not detect the presence of HVA, and additional studies will be needed to determine whether HVA is present in the aqueous of eyes containing retinoblastoma. It has been shown that in patients with neuroblastoma, there may be a correlation between the appearance of rosettes and the levels of catecholamine in the urine. Retinoblastoma is a tumor that is well known to form rosettes, although at times no rosettes can be identified in a globe completely filled with tumor. It is possiblethat with a larger sample, elevated levels of catabolic products will be found and possible correlations with tumor pathology will be made. It should be mentioned that when assaying for the metabolic products of these substances, the samples should be collected more than 24 hours after the ingestion of aspirin, bananas, or vanilla.

SUMMARY

The aqueous humor of five patients with histologically proven retinoblastoma were analyzed for the main catabolic products of norephinephrine and epinephrine. In each case, there were no detectable levels of normethanephrine, metanephrine, vanilmandelic acid (VMA), and 3-methoxy, 4hydroxy phenoglycol. The techniques are sensitive to 1 Mg/ml but will not detect the presence of homovanillic acid (HVA).

REFERENCES

1. Bloom FE, GiarmanNJ: Physiologic and pharmacologic considerations of biogenic amines in the nervous system. Ann Rev Pharmocol 8:229, 1968.

2. Armstrong MD, McMillan A, Shaw KN: 3Methoxy, 4-Hydroxy mandelic acid, urinary metabolite of norepinephrine. Biochem Biophy Acta 25:422-423, 1975.

3. Mason GA, et al: Adrenaline-secreting neuroblastoma in infant. Lancet 2:322-325, 1957.

4. Greenberg RE, Gardner LI: New diagnostic test for neural tumors of infancy; increased urinary excretion of 3-Methoxy, 4-Hydroxymandelic acid and norepinephrine in ganglioneuroma with chronic diarrhea. Pediatr 24:683-684, 1959.

5. Voorhees ML, Gardner LI: Urinary excretion of norepinephrine, epinephrine and 3-methoxy, 4hydroxymandelic acid by children with neuroblastoma. J Clin Endocrinol 21:321-335, 1961.

6. Kontras SB: Urinary excretion of 3-Methoxy, 4Hydroxymandelic acid in children with retinoblastoma. Cancer 15:978-986, 1962.

7. Ehinger B: Adrenergic nerves to the eye and to related structures in man and in the cynomolgus monkey. Invest Ophthalmol 5:42, 1966.

8. Brown DH: The urinary excretion of vanilmandelic acid (VMA) and homovanillic acid (HVA) in children with retinoblastoma. Am J Ophthalmol 62:239-243.

9. Abramson DH et al: LDH levels and isoenzyme patterns in retinoblastoma. (In press.)

10. Gitlow SE et al: Diagnosis of neuroblastoma by qualitative and quantitative determinations of catecholamine metabolites in urine. Cancer 25:1377-1383, 1970.

10.3928/0191-3913-19780301-05

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