The Chemical Estimation of Epinephrine and Norepinephrine in Human and Canine Plasma. I. A Critique of the Ethylenediamine Condensation Method 1

Abstract

The recovery of epinephrine (E) and norepinephrine (N) from plasma on activated alumina is reproducible and this adsorption technique can be made reasonably specific for catechols. The development of chemical methods for the assay of E and N in peripheral plasma has been hampered, however, by: 1) the low concentrations of E and N normally present (1 ,jg. per L.-or less); 2) the lack of specificity of the methods for E and N in the presence of other catechols which are excreted in urine (1, 2) and thus presumably are present in blood; and 3) difficulty in obtaining "blanks" from plasma extracts or eluates, a condition usually met in methods designed to measure endogenous substances.In 1949 and 1950, Lund (3-5) described a method based on the conversion of E and N to their fluorescent trihydroxyindole derivatives. Only dihydroxyphenyl compounds possessing a side chain which contains both an hydroxyl and an amino group appear to be detected by this method, and "blanks" can be obtained by allowing oxidation of the trihydroxyindole derivatives of E and N to proceed to a non-fluorescent state. However, the sensitivity of the method appears insufficient to permit measurements of E and N in small quantities of normal peripheral plasma (5). Simultaneously, Natelson, Lugovoy, and Pincus (6) described the condensation of E with ethylenediamine (EDA) to form a highly fluorescent product. In 1952 and1953, Weil-Malherbe and Bone (7, 8) extended the use of this procedure to the simultaneous estimation of E and N in blood by measuring the intensity of fluorescence at two different wave lengths. Modifications (9, 10) have been reported, and the method appears to be in fre- quent use. It is less specific than the Lund (trihydroxyindole) method, and will detect the presence of dihydroxyphenyl compounds other than E and N (7).The levels of E and N found in normal human plasma by Weil-Malherbe and Bone (1.3 and 5.2 micrograms per liter, respectively) differ from those observed by others (10-12) using essentially the same method, and from the levels obtained by bioassay (13). In view of the fact that a reliable method for estimation of E and N can assume importance in the interpretation of physiological changes in man, it was considered necessary to re-evaluate the EDA method and to compare the results obtained with this technique with those obtained by the trihydroxyindole method. Our results with both dog and human plasma indicate that the EDA method is not specific for N, and may not be so for E. METHODSBlood was collected in heparinized syringes, the cells separated by centrifugation, and to a measured amount of the plasma one-half its volume of sodium fluoride (2 per cent)-sodium thiosulfate (3 per cent) solution was added.Removal of E and N from plasma was accomplished by adsorption of the catechol amines on activated alumina at pH 8.0 to 8.5, followed by elution from the alumina column with acetic acid as described by Weil-Malherbe and Bone (7). Woelm alumina, non-alkaline, Grade I (Alupharm Chemical Co., Elmont, L. I.) was ...

The catecholamine content of the peripheral plasma in human subjects with complete transverse lesions of the spinal cord

“…More recently Born & Hornykiewicz (1957) Table 1 suggests that where great care is taken to maintain the platelets and the integrity of the plasma generally (Valk & Price, 1956), the adrenaline and noradrenaline values are at their lowest. Where maximum disruption of platelets is likely to occur, as in the fluoride solution, maximum catecholamine values are found.…”

The Uptake of Adrenaline and Noradrenaline by Blood Platelets of the Pig

“…The question arises whether the catechol amines which are found in blood (Holzbauer and Vogt, 1954) are likewise concentrated in the platelets. Results obtained with human blood by WeilMalherbe and Bone (1954) and by Robinson and Stott (1958) suggested that this was so, although Valk and Price (1956) found no catechol amines in human platelets.…”

Arteriovenous Differences of the Norepineprine-Like Material From Normal Plasma and Infused Norepinephrine*

“…In this respect, the EDA method is inferior to the THI method (6,8,9), a fact which is emphasized by the false high readings of the former in uremia (10). However, the THI method may measure dopamine (3, 4), dihydroxyphenylalanine (2-4), isopropylnorepinephrine (2-4, 9), serotonin (2) or aureomycin (11) modification used.…”

“…In this respect, the EDA method is inferior to the THI method (6,8,9), a fact which is emphasized by the false high readings of the former in uremia (10). However, the THI method may measure dopamine (3,4), dihydroxyphenylalanine (2)(3)(4), isopropylnorepinephrine (2-4, 9), serotonin (2) or aureomycin (11), depending on the * This investigation was supported (in part) by a grant from the Life Insurance Medical Research Fund, and by a research grant (H-4807) and a training grant (HTS-5369) from the National Heart Institute, Bethesda, Md. modification used.…”