BIOCHEMICAL
MEDICINE
18, 455-459
LETTER
(1977)
TO THE EDITOR
Quantitative Determination of Catecholamines by Gas-Liquid Chromatography Work in our laboratory is currently concerned with clinical studies on circulating plasma catecholamines, and we therefore would like to draw attention to what we consider to be three inconsistencies in the publication by Lovelady in this journal (I). First, the concentrations of epinephrine (E) and norepinephrine (NE) given by Lovelady as being present in human plasma are 18.00 ? 0.28 and 2.01 -+: 0.19 rig/ml, respectively, and these values are totally inconsistent with values reported in normal human subjects by workers using the more established and sensitive radioenzymatic assay for plasma E and NE. Those values are summarized in Table 1. Adding three standard deviations to the mean values given in Table I, it is apparent that the upper 99.7% confidence limit for plasma E concentration is less than OS rig/ml, and that for NE is less than 1. I rig/ml. The author makes no attempt to clarify this discrepancy between the values in his publication and those previously reported using radioenzymatic techniques. Furthermore, the method used by Lovelady (I) for plasma and red blood cell catecholamine determinations was the same as that which the author has previously published (2). It is therefore surprising to note significant discrepancies between the data for E and NE concentrations given in the two papers. For instance, the author stated that E and NE concentrations in human red blood cells (RBC) of the normal subjects examined were 4. IO + 0.19 and 1 I .32 + 0.72 rig/ml, while Lovelady and Foster (2) reported values in RBC (seemingly in a single normal subject) to be 0.364 -+ 0.16 and 0.466 + 0.35 rig/ml, respectively. Second, it is doubtful that a mixture of n-hexane and 4-methyl-2pentanone (methyl isobutyl ketone) is a suitable solvent for the extraction of catecholamines from an aqueous medium. For RBC, the author has reported a greater than 90% recovery of catecholamines into the organic phase following evaporation to near dryness of an aliquot of protein-free extract of RBC dissolved in perchloric acid solution and subsequent extraction of the residue with water:n-hexane:4-methyl-2-pentanone (I : I : 1). This is surprising since the organic solvent pair, n-hexane and 4.55 Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN OiM-2944
tG QI
Christensen. N. J.. C/in. SC.;. MO/. .2fd. (1973)
Siggers. D. C.. Salter. C.. and Toselnnd. P. A..
0.220 2 0.080 0.420 + 0.010 0.200 + 0.277 0.210 k 0.285 0.250 +_ 0.053
0.090 + 0.100 0.060 k 0.139 0.040 2 0.095
4
12 IO
7
16
Males Females
Males (8) and Females (8)
28, 356 (1976)
0.050 rt 0.040
Males (4) and Females (4)
Phannac..
Chim.
26, 53 (1970)
K..
and Portno).
( 1974) 45, 163
B.. C~iwrricrtiu,r
39, 107
Henry, D. P.. Starman. B. J.. Johnwn. D. G.. and Williams. R. H.. I$v Sk 16. 375 (1975)
Res.
Mt,rah.
Ac~tr 30. 373 (I970\
Ekfocrinol.
Engelman,
C/in.
C&n.
Cryer. P. E.. Santiago, H. V.. and Shah. S.. ./.
Pharm.
0.290 2 0.158 0.410 2 0.122 0.220 t- 0.057
0.170 e 0.063 0.130 ” 0.024 0.040 rf: 0.028
6 8
Males Females
-_---.Hortnagl. H.. Benedict, C‘. R.. Grahame-Smith. D. G.. and McGrath. B.. Br. J. C/i/l. Phrrw~rrt to appear Callingham, B. A.. and Barrand. M. .4.. .I
IO
0.444 + 0.129 0.550 + 0.087
0.124 t 0.076 0.130 -t 0.071
Reference
Males Females
NE
II 7
E
.__-
Sex
Plasma content (ngiml + SD)
TABLE I CONTENTSOF VENOUS PLASMA SAMPLES FROM NORMAL HUMAN SUBJECTS
No. of subjects
CATECHOLAMINE
P Y
Males
Males
Females
25
26
6
8
Males Females -
16 22 14
0.045 (0.0-O. 160) 0.058 (0.020-O. 190) 0.160 zt 0.057
0.058 _’ 0.030
-
0.258 (O.OSO-0.7SO) 0.238 (0.012-0.430) 0.160 zt 0.141
0.133 k 0.070
0.292 + 0.138
0.270 -t 0.164 0.307 -t 0.145
Moerman, E. J.. Bogaert, M. G., and De Schaepdryver. A. F., Clim. Chim. Arta 72, 89 ( 1976)
Ziegler. M. G.. Lake, C. R.. and Kopin. I. J.. Nofure 261, 333 (1976) Lake, C. R.. Ziegler, M. G.. and Kopin. I. J.. L(fe SC;. 18, 1315 (1976) Miura, Y., and DeQuattro. U., Jpn. Circulution J. 39, 583 (1975) Pedersen, E. B.. and Christensen, N. H., Acta Med. Stand. 198, 373 (1975)
458
LETTER
TO THE EDITOR
4-methyl-2-pentanone, would not be expected to have the required “polarity” to remove catecholamines from aqueous solution. The catecholamines would therefore remain in the water phase and not partition into the organic phase. To examine this possibility, we have prepared solutions of E (75 pg/ml) and NE (0. I pg/ml, containing 0.01 ~Cilml of tritiated NE) in 0.4 N perchloric acid. One-milliliter aliquots of each solution were evaporated to dryness and the residue was extracted with waterirz-hexane/4-methyl-2-pentanone as described by Lovelady (I). The phases were separated and evaporated to dryness. For E, the relative amount of E in each phase was determined using absorbance at 280 nm in 0. I N HCI (A,,, for E in acid solution) as shown in Table 2. Similarly, for NE the relative amounts in each phase was determined by liquid scintillation counting of tritium as shown in Table 2. It can be seen that for both E and NE there was negligible recovery (less than 2% for NE) into the organic phase since the bulk of the catecholamines remained in the aqueous solution. Dopamine would not be expected to behave differently, nor
TABLE NE
PARTITION
OF E AND
2 IN WATER:
d-METHYL-I-PENTANONE
Epinephrine (E) (Absorbance at 280 nm in 0.1 N HClb) Determination No. I 2 3
n-HEXANE:
(I: t : l)a
Norepinephrine (NE) (total dpmd)
Organic Phase
Aqueous Phase
Organic Phase
Aqueous Phase