Stanley E. Gitlow

The biochemical techniques for detecting and establishing the presence of a pheochromocytoma. A review of ten years' experience.

Abstract Pheochromocytoma, fatal if undetected, is a rare cause of hypertension, paroxysmal symptoms and a hypermetabolic state. The clinical signs and symptoms associated with this tumor rarely result in its detection. Advances in knowledge of catecholamine metabolism have markedly improved the diagnostic reliability for tumors of the neural crest. Large numbers of patients must be screened and the diagnosis ultimately proved by biochemical assays for the catecholamines and their catabolites, vanillylmandelic acid, total metanephrines, 3-methoxy-4-hydroxyphenylethyleneglycol and homovanillic acid. Many assay procedures are nonspecific, and therefore do not reliably differentiate the rare patient with pheochromocytoma from patients with more common maladies. We reviewed laboratory screening procedures in 92 subjects with proved pheochromocytoma and 9,500 control subjects. Excretion of the total metanephrines, although uniformly increased in the presence of a pheochromocytoma, was occasionally increased in its absence. Vanillylmandelic acid excretion, rarely increased in the absence of a neural crest tumor, was within normal limits in about 3 percent of patients with pheochromocytomas. Nonspecific techniques for colorimetric assay of vanillylmandelic acid, now widely used, had little diagnostic value in over 30 percent of cases of pheochromocytoma. Familiarity with the various biochemical procedures for assay of the catecholamines and their catabolites is essential in establishing a diagnosis of sufficient reliability to warrant surgical exploration.

Diagnosis of neuroblastoma by qualitative and quantitative determination of catecholamine metabolites in urine

Neuroblastoma, one of the most common solid malignant tumors of childhood, has been frequently confused with other malignancies as well as nonneoplastic diseases. The observation that neural crest tumors resulted in excretion of elevated quantities of the catecholamines and their by‐products opened the way for development of biochemical techniques for their detection. Vanillylmandelic acid, metanephrines, homovanillic acid, and 3‐methoxy‐4‐hydroxy‐phenylethyleneglycol excretions of 180 normal children were compared with those of 62 subjects suffering from illnesses commonly confused with neuroblastoma as well as 41 patients with neural crest lesions. A simple, bedside chemical technique for detecting a neuroblastoma resulted in no false positive and 91% reliability among those patients with this tumor. Although quantitative assay for 3‐methoxy‐4‐hydroxyphenylethyleneglycol appeared to offer the greatest reliability for biochemical diagnosis of neuroblastoma, broad application of the screening test might significantly improve the ease of detection and differential diagnosis of neuroblastoma.

A gas-liquid chromatographic method for the separation and quantitation of normetanephrine and metanephrine in human urine

Abstract A gas-liquid Chromatographic method for the separation and quantitation of urinary normetanephrine and metanephrine as their trifluoroacetyl derivatives is presented. After a preliminary column separation of basic from neutral and acidic compounds in hydrolyzed urine, the trifluoroacetylated amines (normetanephrine, metanephrine, octopamine, tyramine, 3-methoxytyramine, tryptamine and serotonin) were separated on a 1:1 w/w (3% QF-1: 2% XF-1105) column. An electron capture detection system was employed for maximal sensitivity. Normal human urinary values for normetanephrine ranged from 0.086 to 0.177 μg/mg creatinine (mean = 0.116 ± 0.029) and for metanephrine ranged from 0.059 to 0.145 μg/mg creatinine (mean = 0.093 ± 0.027). Patients with pheochromocytoma and neuroblastoma had elevated urinary excretions of normetanephrine and/or metanephrine.

Endogenous levels of free and conjugated urinary 3-methoxy-4-hydroxyphenylethyleneglycol in control subjects and patients with pheochromocytoma determined by reversed-phase liquid chromatography with electrochemical detection.

A sensitive and specific reversed-phase liquid-chromatography (HPLC) method for the determination of urinary free and conjugated 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) was developed. Sample preparation is minimal and the method is ideally suited for routine clinical assays of this catecholamine metabolite. Reported in this paper are excretion profiles of controls subjects and patients with pheochromocytoma. Quantitative results were expressed as microgram MHPG per mg creatinine, in order to eliminate variations associated with the 24-h urine collection. The levels of free and conjugated MHPG, determined in 20 control subjects ranged between 0.04--0.11 microgram/mg of creatinine and 0.48--1.25 microgram/mg of creatinine, respectively. The corresponding levels of free and conjugated MHPG in 8 patients with pheochromocytoma were 0.29--1.21 microgram/mg of creatinine and 2.47--15.98 microgram/mg of creatinine, respectively. The described liquid-chromatographic analysis, coupled with the highly sensitive electrochemical detection, provides a simple and reliable method for metabolic profiling of catecholamine by-products at their endogenous levels. This offers an attractive possibility for the detection of neural crest lesions which may cause problems in diagnosis because of their small size, infrequent occurrence and symptoms similar to those of essential hypertension.

Analysis of urinary metanephrines by reversed-phase high-performance liquid chromatography and electrochemical detection

A sensitive and specific direct analysis of urinary normetanephrine (NMN) and metanephrine (MN) was achieved utilizing reversed-phase high performance liquid chromatography and electrochemical detection. Individual specimens from control subjects and those with pheochromocytoma were hydrolyzed and the metanephrines separated from other urinary constituents by elution with ammonia from a Dowex CG-50 resin. Chromatographic peaks were identified by retention behavior, co-chromatography with reference compounds, ratio of responses at various oxidation potentials and stopped-flow UV spectra of the collected fractions. The NMN and MN content for the control subjects was between 0.086 and 0.21 (mean - 0.14) microgram/mg creatinine and 0.012 and 0.092 (mean = 0.039) microgram/mg creatinine, respectively. The values for subjects with pheochromocytoma varied from 1.5 to 27.5 (mean = 9.9) microgram/mg creatinine for NMN and 0.10 to 1.60 (mean = 0.86) microgram/mg creatine for MN. The patient with ganglioneuroma had an NMN of 4.1 and an MN of 0.80 microgram/mg creatinine. While this method permits discrimination between those patients with pheochromocytoma and the overwhelming majority of hypertensive patients, it may ultimately be further extended to separate normal subjects from those with more subtle derangements in catecholamine metabolism.

Persistence of abnormal REM sleep response to ethanol as a result of previous ethanol ingestion

Ethanol was administered to Sprague-Dawley rats for 4–6 weeks following which these animals thrived in a manner comparable to a naive control group. Over 6 months after an ethanol-free interval, the alcohol-exposed animals responded to a test dose of ethanol with a greater disturbance in REM sleep than noted in the naive group.

Quantitative determination of 3-methoxy-4-hydroxyphenylethyleneglycol and its sulfate conjugate in human lumbar cerebrospinal fluid using liquid chromatography with amperometric detection

A sensitive and direct reversed-phase liquid chromatographic method with amperometric detection was developed for the determination of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG). The concentrations of the free and sulfate conjugate of MHPG were measured in human lumbar cerebrospinal fluid. All samples were preconcentrated by extraction with ethyl acetate. Deconjugation of the sulfate form of MHPG was achieved by enzymatic hydrolysis with sulfatase. Peaks were identified on the basis of chromatographic behavior, ratio of responses at several oxidation potentials and the stopped-flow UV spectra of the collected fractions. The free MHPG content of 20 cerebrospinal fluid samples ranged between 0.720 and 19.51 ng/ml with the mean of 5.126 +/- 4.652 (S.D.) ng/ml. The sulfate conjugate of MHPG in 12 samples of cerebrospinal fluid ranged between 0.08 and 0.850 ng/ml with the mean value of 0.2365 +/- 0.2269 (S.D.) ng/ml. Although our results correlate well with the literature values, no attempt was made to interpret the quantitative data since samples were obtained from routine, diagnostic testing of patients admitted to the medical or neurologic services at the Mount Sinai Hospital.

INFLUENCE OF ETHANOL ON HUMAN CATECHOLAMINE METABOLISM

Because ethanol is the most broadly used addicting sedative agent, i t is not surprising that its relationship to a central neurotransmitter, such as norepinephrine (KE) , has attracted the attention of a growing number of investigators during the past I5 years.?-x Unfortunately, these studies have offered diverse and occasionally contradictory results.y-14 Studies of the effects of alcohol on catecholamine metabolism in the intact human subject have demonstrated elevated N E excretion ? . J . I 5 and normal N E excretion,lh low vanillylmandelic acid ( V M A ) excretion,I7 and increased V M A excretion, in addition to other variations that were apparently determined by the subjects state of health, the quantity and duration of ethanol ingested, and the reliability of the assay procedures..?Jg Despite critical interest in the effect of ethanol on central neurotransmission and the awareness that ethanol penetrated the central nervous system (CSS) ,19 the status of catecholamine metabolism within the C N S of the human subject has not been investigated. The development of biochemical methods that use tritium-labeled N E ([JHINE). whereby catecholamine metabolism of the intact human subject could be more precisely evaluated.?o led to the in-depth study of two volunteer subjects during and after ingestion of more than 22 ounces of whiskey per day. Under normal circumstances. the administration of a tracer substance, [ H I S E , that failed to pass the blood-brain barrier resulted in the excretion of N E metabolites with specific activities lowered in proportion to the contribution of the unlabeled pool ( C N S ) . Prior studies of human volunteers to whom [ HINE had been administered intravenously suggested the C N S as the source of unlabeled 3-methoxy-4-hydroxyphenylethyleneglycol (G)?o that resulted in a lower specific activity of this substance relative to that of V M A . As little as I ounce of ethanol elicited a shift in the catabolic pathways of N E that favored the formation and excretion of G as opposed to V M A ( F I G I J K E l ) . 1 7 ~ 2 1 J ~ This shift in the biochemical degradation of both endogenous N E and the unevenly distributed exogenous tracer material would be expected to affect the relative specific activities of V M A and G in proportion to the modification of the metabolism of that N E within the C N S .

Benign pheochromocytoma associated with elevated excretion of homovanillic acid

The excretion of catecholamine metabolites was determined in nine children prior to and following resection of one or more well-encapsulated, benign pheochromocytomas. One third of these subjects presented with increased excretion of homovanillic acid as well as markedly increased excretion of vanillylmandelic acid and total metanephrines: biochemical evidence opposed to the presence of a benign pheochromocytoma. No significant difference in age, sex, clinical presentation, course, or postoperative biochemical evaluation could be detected between this group and the six remaining subjects. Elevated homovanillic acid excretion, indicative of the presence of a malignant pheochromocytoma in the adult, does not carry this serious prognostic import in childhood. In childhood, elevated homovanillic acid and markedly increased excration of vanillylmandelic acid and total metanephrines, usually associated with neuroblastoma, do not rule out the diagnostic possibility of a benign pheochromocytoma.

Simultaneous determination of 4-hydroxy-3-methoxy-phenylacetic (homovanillic) acid and other monoamine metabolites in human lumbar cerebrospinal fluid : An improved high-performance liquid chromatographic study with electrochemical detection

Abstract We describe a direct analysis for the simultaneous quantitative determination of 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid and other monoamine metabolites in human lumbar cerebrospinal fluid, utilizing reversed-phase high-performance liquid chromatography with amperometric detection. In addition, a rapid isocratic separation was developed for homovanillic acid in the presence of other endogenous compounds. Twenty-five unselected diagnostic specimens of human lumbar cerebrospinal fluid were extracted with ethyl acetate and subsequently analyzed using the described method. Chromatographic peaks were identified on the basis of retention behavior and ratio of responses at several oxidation potentials. Although our quantitative results correlate well with the literature values, the data were not interpreted clinically since samples were obtained from routine, diagnostic testing of patients admitted to the medical or neurologic services at the Mount Sinai Hospital.