Quantification of Copper in Biological Materials by Use of Electron Spin Resonance

Abstract

Plasma copper is reportedly increased in pregnancy, infections, and inflammation and decreased in corticosteroid and corticotropin treatment (1). The usual and convenient methods for measurement of metal ions are colorimetry and atomic absorption spectrophotometry (AAS). These methods, however, are not suitable for copper. For example, the molar absorptivity at 480 nm of copper complex with bathocuproine disulfonic acid, the most suitable reagent, is not strong (e = 1.3 × 104) (2)(3)(4), and the atomic absorption at 324.7 nm specific to copper is marginal. As a consequence, both methods require 2 mL of serum, although the copper concentration in plasma is fairly high, e.g., 15.7 μmol/L (1 μg/mL). A more sensitive but selective and convenient method is needed for quantification of small amounts of copper in blood as well as in polluted water, food, and tissues. Electron spin resonance (ESR) is a very sensitive method that requires only 109 spins for detection. We examined the use of ESR for measurement of copper because Cu2+ is paramagnetic, and thus can be measured by ESR, although Cu+ is diamagnetic. We confirmed the results of a previous report that diethyldithiocarbamate (DDC) reacts with Cu2+ at pH 0–10 (5). The Cu2+-DDC complex could be extracted with most organic solvents at these pHs (5)(6). We found that the addition of Na2SO4 facilitated the extraction of the complex with 1-butanol. As shown in Fig. 1A⇓ (spectrum a), Cu2+-DDC in 1-butanol shows a characteristic four-line ESR spectrum with hyperfine splitting of 80 gauss and a splitting factor (g) of 2.047. The height of the peak indicated by the arrow at the highest magnetic field of 3350 gauss was used for the quantification of copper. To liberate copper from bound protein …