A precise catecholamine assay for small plasma samples

The time, temperature, and pH of the trihydroxyindole reaction were controlled to minimize interference between norepinephrine (NE) and epinephrine (E) so that, in plasma catecholamine assay within normal human physiologic range, NE contributes less than 1 per cent to the fluorescence produced from E, and E contributes only 0.5 to 10 per cent to NE fluorescence; assay and blank measurements show superior stability. Elution from alumina and instrumental factors, such as optical filters, were optimized so that a single 5 ml. plasma sample sufficed for triplicate analyses of both NE and E, with duplicate blanks for each. This triplicate fluorescence assay was combined with duplicate recovery measurements of radioactive catecholamines added in tracer quantities to the plasma so as to make possible reliable estimates of the precision of measurement of each individual sample. The mean levels found for men aged 40 to 60 years from antecubital vein blood drawn during the subject's working hours (but after a brief rest) were, for NE 0.38 ng. per milliliter, range 0.15 to 0.54 ng. per millilter, and for E 0.09 ng./ml, range 0.01 to 0.27 ng. per millilter. The high precision and small amounts of blood required by this method made possible the measurement of basal plasma levels of E and NE in rats under two-stage phenobarbital anesthesia. The mean levels for 13 male and 4 female Long-Evans-stain rats weighing 250 to 350 grams were, for NE 0.26 ng. per millilter, range 0.16 to 0.37 ng. per millilter, and for E 0.36 ng. per millilter, range 0.14 to ng. per millilter. These rat values are called basal because they are only 1/5 to 1/10 as large as values obtained on plasma secured by single-stage pentobarbital anesthesia or from unanesthetized rats. Eight to 11 ml. of whole blood obtained from each rat were sufficient for individual analyses, an improvement over most other reported methods which require blood from a number of animals to be pooled. The average precision of an individual determination of NE was plus or minus 10 per cent in the range of 0.20 to 0.40 ng. per millilter. The average recovery for NE was 65 per cent and for E it was 55 per cent. Major factors influencing recovery were quantitatively investigated and discussed.