338 Bratn Reseat(h, 240 (1982) 3 ~;8 ~41 FNe~lel Biomedical Pres~ Stable-isotope dilution measurement of zinc and lead in rat hippocampus and spinal cord C J FREDERICKSON, W I MANTON, M H FREDERICKSON, G A HOWELL and M A MALLORY Laboratories for Geochronology and Neurobtology, Umverstty of Teras at Dallas, Rwhard~on, TX 75080 ( U S A ) (Accepted May 4th, 1982) Key words zinc -- lead -- hlppocampus -- stable-isotopes -- rat Zinc and lead concentrations m hippocarnpus and spinal cord of rats were measured using the highly-accurate method of stable isotope dflunon mass-spectrometry In hlppocampus, average zinc concentratmn was 72 7 ppm (dry weight), average lead, 0 053 ppm, m spinal cord, zme averaged 26 1 ppm, lead, 0 018 ppm Possible explanations for apparent overesttmanons of rat CNS metal content m prewously pubhshed work were dmcussed The presence of a quahtattvely distinct pool of zinc in the hlppocampus has been amply demon- strated by hlstoanalytlc 1°,11 and radlograpblc methods 2,6 Further, there are convergmg data from developmental-anatomic studies2,11, 25, light and electron microscopic studies 14, and electrophyslolo- gtcal workS, t5 which indicate that the distractive zinc in the h~ppocampus is closely assocmted with the mossy-fiber axons, possibly concentrated directly within the mossy fiber terminal bags ~3,as The pre- sent work was undertaken as a first step towards quantitative mvestlgatmns (of uptake, turnover, etc ) of the mossy-fiber related zinc, and the princi- pal goal of the work was to determine the zinc con- tent of the hlppocampus of the normal, adult rat For comparison wlth the putatively zinc-rich tuppo- campus, spinal cord tissue was also assayed for zinc Also, because zinc-dependent processes may be a site of actxon of heavy metal toxins 2z, the amounts of the heavy metal, lead, were measured m both tissues for comparison w~th the zinc To measure the metals, we used the highly-accu- rate method of geochemlsts, stable ~sotope ddutmn mass-spectrometry, as previously modified for use with bmlogleal tissues 21 The principle of the meth- od is straightforward a known amount (or 'spike') of a stable isotope of the to-be-measured element is added to the t~ssue sample, the tissue is decomposed (allowing the atoms of the spike to mix freely with the atoms of the endogenous isotopes), and then the ratio of the atoms of the spike to the atoms of an endogenous isotope 1S measured on an isotope-ratio mass-spectrometer The procedure was as follows Tissue (whole hlppocampus trimmed of psalte- rium, fimbna, and sublculum, whole cord trimmed of nerve roots) was dissected fresh, tyophdtzed and weighed, and then a precisely-measured spike of purified stable isotope (ZN-64 and/or Pb-206, Oak R~dge) was added to the tissue vessel Tissue and spike together were then Immersed m HNO3 (2-3 ml) and gently boiled in a refluxmg vessel until the mixture was colorless Next, the HNO3 was evapo- rated, the residue was treated m hot HC104 (0 5 ml) to decompose residual organics, and the HCIO 4 was evaporated, leaving a white, crystalline, mineral resi- due Finally, the zinc and the lead were separated from the other minerals by ion exchange, and each metal was loaded onto the filament of a mass- spectrometer for measurement of Isotope ratios. Contamination was minimized by performing all tissue preparation in a clean room in which pressu- rized room air was filtered, personnel wore clean over-garments, and all dissolutions and evapora- tmns were carried out under filtered air All surfaces