89
Ciinica Chimica Acta, 97 (1979) 89-91 0 Elsevier/North-Holland Biomedical Press
BRIEF TECHNICAL
NOTE
CCA 1093
A SENSITIVE AND ACCURATE PLASMA NORADRENALINE USING A MODIFIED FLUORIMETRIC METHOD
S. GHIONE
*, M. PELLEGRINI
and A. CLERIC0
C.N.R., Clinical Physiology Laboratory, (Received
January
15th,
ASSAY
Pisa (Italy)
1979)
Plasma noradrenaline (NA) can be currently assayed by fluorimetric and radioenzymatic methods. Miura et al. [l] have recently shown that Renzini’s fluorimetric assay [ 21 yields results as satisfactory as a radioenzymatic method. Mayor drawbacks of fluorimetric methods are the larger blood samples required and the difficult discrimination between adrenaline (A) and NA. In this report, we describe some improvements of Renzini’s two-step chromatographic trihydroxyindole fluorimetric assay [ 21, which permits NA determination in smaller plasma samples with negligible interference by A. When not otherwise stated the original procedure was adopted without changes [ 21. The first chromatographic step on Allumina (Merck, F.R.G.) (200 mg) was performed using glass columns with 4.5 mm internal diameter, the elution was obtained with 1.8 ml perchloric acid (0.5 mol/l) in a beaker containing 50 ~1 of 5% EDTA and 50 ~1 of 0.1% ascorbic acid. The second step on Amberlite CG 50 (BDH Chemicals Ltd., U.K.) was performed in capillary columns (internal diameter 1.5 mm, resine volume 50 pl), and elution was achieved with 200 ~1 boric acid (0.66 mol/l). After oxidation of 100 ~1 of the eluate, the sequence of the alkalinization and of the reduction/stabilization steps was inverted by adding 20 ~1 10 mol/l NaOH 3 min after the start of oxidation and 5 ~1 of a solution of 10% 2-mercaptoethanol in 19.5% formaldehyde, 15 set later. External standards of NA and A (1 ng), reagent, faded blanks and excitation fluorescence spectra were obtained according to the original method. Results and discussion Samples
faded
and reagent
* Correspondence should be addressed via Savi 6. 56100. Piss. Italy.
blanks to:
were constantly
Dr. Se&o
Ghione.
Laboratorio
lower than Fisiologia
10% with
Clinica.
C.N.R.,
90
mj of A or NA oxidized
01
0.2
05
0.7
10
fluorescence A/NA ration
100
7.2
3.6
72
5.1
mean
A/NA
ratio
6.62+2.43% ,..SD)
Catecholamine
assayed.ng
Fig. 1. Comparison of fluorescence of noradrenaline and adrenaline.
respect to the fluorescence of 1 ng of NA. With respect to NA, the fluorescence of A was less than 10% over a wide range of concentration (Fig. 1). The within and between assay variability was 3.5% (n = 5) and 10.5% (n = 14), respectively. A linear response has been demonstrated from 2 to 8 ml of plasma (r = 0.9898, n = 4). The recovery of NA added to catecholamine-free plasma (5 ml) was 74.2 + 5.3% (mean + SD., II = 5). NA concentrations in 23 normal subjects after one hour of recumbency and one further hour of upright posture were 205 + 67 rig/l and 375 ?r 128 rig/l,, respectively (values corrected for 74.2% recovery). By alkalinizing the reaction mixture just prior to the reduction/stabilization step with mercaptoethanol, advantage is taken of the fact that A fluorophor is much more poorly stabilized when in alkaline solution than the NA fluorophor [3]. The use of mercaptoethanol, instead of BAL, permits to obtain more stable and lower blanks, as already reported by other authors [ 1,4]. In the method of Renzini, A/NA discrimination is obtained on the basis of small differences of wavelength maxima of the excitation spectra of A and NA, but this differentiation required fluorescence readings at non-optimal wavelength, whereas, with our modification, NA fluorescence is read at its optimal wavelength. Obtaining a smaller volume of final eluate, the volume of plasma to be assayed can be considerably reduced down to 2 ml; routinely, however, we use 3-5 ml of plasma. With respect to the radioenzymatic assay, the fluorimetric assay offers the advantage of a lower cost; the number of determinations which can be performed in a working day are approximately the same (6-12 samples). Our results in normal subjects are quite comparable with those reported by other authors using fluorimetric or radioenzymatic methods.
91
References 1 2 3 4
Miura. Y.. Campese. V., De Quattro. V. and Mcijer, D. (19’77) J. Lab. Clin. Med. 89,421-427 Renzini, V., Brunori, C.A. and Valori, C. (1970) Clin. Chim. Acta 39.587-595 Laverty, R. and Taylor, K.M. (1968) Anal. Biochem. 22.269-279 Jiang, N.S., Stoffer, S.S., Pikler, G.M., Wadel. 0. and Sheps, S.G. (1973) Majo Clin. Proc. 48, 47-49
Ciinica Chimica Acta, 97 (1979) 89-91 0 Elsevier/North-Holland Biomedical Press
BRIEF TECHNICAL
NOTE
CCA 1093
A SENSITIVE AND ACCURATE PLASMA NORADRENALINE USING A MODIFIED FLUORIMETRIC METHOD
S. GHIONE
*, M. PELLEGRINI
and A. CLERIC0
C.N.R., Clinical Physiology Laboratory, (Received
January
15th,
ASSAY
Pisa (Italy)
1979)
Plasma noradrenaline (NA) can be currently assayed by fluorimetric and radioenzymatic methods. Miura et al. [l] have recently shown that Renzini’s fluorimetric assay [ 21 yields results as satisfactory as a radioenzymatic method. Mayor drawbacks of fluorimetric methods are the larger blood samples required and the difficult discrimination between adrenaline (A) and NA. In this report, we describe some improvements of Renzini’s two-step chromatographic trihydroxyindole fluorimetric assay [ 21, which permits NA determination in smaller plasma samples with negligible interference by A. When not otherwise stated the original procedure was adopted without changes [ 21. The first chromatographic step on Allumina (Merck, F.R.G.) (200 mg) was performed using glass columns with 4.5 mm internal diameter, the elution was obtained with 1.8 ml perchloric acid (0.5 mol/l) in a beaker containing 50 ~1 of 5% EDTA and 50 ~1 of 0.1% ascorbic acid. The second step on Amberlite CG 50 (BDH Chemicals Ltd., U.K.) was performed in capillary columns (internal diameter 1.5 mm, resine volume 50 pl), and elution was achieved with 200 ~1 boric acid (0.66 mol/l). After oxidation of 100 ~1 of the eluate, the sequence of the alkalinization and of the reduction/stabilization steps was inverted by adding 20 ~1 10 mol/l NaOH 3 min after the start of oxidation and 5 ~1 of a solution of 10% 2-mercaptoethanol in 19.5% formaldehyde, 15 set later. External standards of NA and A (1 ng), reagent, faded blanks and excitation fluorescence spectra were obtained according to the original method. Results and discussion Samples
faded
and reagent
* Correspondence should be addressed via Savi 6. 56100. Piss. Italy.
blanks to:
were constantly
Dr. Se&o
Ghione.
Laboratorio
lower than Fisiologia
10% with
Clinica.
C.N.R.,
90
mj of A or NA oxidized
01
0.2
05
0.7
10
fluorescence A/NA ration
100
7.2
3.6
72
5.1
mean
A/NA
ratio
6.62+2.43% ,..SD)
Catecholamine
assayed.ng
Fig. 1. Comparison of fluorescence of noradrenaline and adrenaline.
respect to the fluorescence of 1 ng of NA. With respect to NA, the fluorescence of A was less than 10% over a wide range of concentration (Fig. 1). The within and between assay variability was 3.5% (n = 5) and 10.5% (n = 14), respectively. A linear response has been demonstrated from 2 to 8 ml of plasma (r = 0.9898, n = 4). The recovery of NA added to catecholamine-free plasma (5 ml) was 74.2 + 5.3% (mean + SD., II = 5). NA concentrations in 23 normal subjects after one hour of recumbency and one further hour of upright posture were 205 + 67 rig/l and 375 ?r 128 rig/l,, respectively (values corrected for 74.2% recovery). By alkalinizing the reaction mixture just prior to the reduction/stabilization step with mercaptoethanol, advantage is taken of the fact that A fluorophor is much more poorly stabilized when in alkaline solution than the NA fluorophor [3]. The use of mercaptoethanol, instead of BAL, permits to obtain more stable and lower blanks, as already reported by other authors [ 1,4]. In the method of Renzini, A/NA discrimination is obtained on the basis of small differences of wavelength maxima of the excitation spectra of A and NA, but this differentiation required fluorescence readings at non-optimal wavelength, whereas, with our modification, NA fluorescence is read at its optimal wavelength. Obtaining a smaller volume of final eluate, the volume of plasma to be assayed can be considerably reduced down to 2 ml; routinely, however, we use 3-5 ml of plasma. With respect to the radioenzymatic assay, the fluorimetric assay offers the advantage of a lower cost; the number of determinations which can be performed in a working day are approximately the same (6-12 samples). Our results in normal subjects are quite comparable with those reported by other authors using fluorimetric or radioenzymatic methods.
91
References 1 2 3 4
Miura. Y.. Campese. V., De Quattro. V. and Mcijer, D. (19’77) J. Lab. Clin. Med. 89,421-427 Renzini, V., Brunori, C.A. and Valori, C. (1970) Clin. Chim. Acta 39.587-595 Laverty, R. and Taylor, K.M. (1968) Anal. Biochem. 22.269-279 Jiang, N.S., Stoffer, S.S., Pikler, G.M., Wadel. 0. and Sheps, S.G. (1973) Majo Clin. Proc. 48, 47-49
