Abstract
We report the first high-precision characterization of molecular and intramolecular δ(15)N of nucleosides derived from mammalian DNA. The influence of dietary protein level on brain amino acids and deoxyribonucleosides was determined to investigate whether high protein turnover would alter amino acid (15)N or (13)C values. Pregnant guinea pig dams were fed control diets, or high or low levels of dietary protein throughout gestation, and all pups were fed control diets. The cerebellar DNA of offspring was extracted at 2 and 120 days of life, nucleosides isolated and δ(15)N and δ(13)C values characterized. Mean diet δ(15)N was 0.45 ± 0.33‰, compared with cerebellar whole tissue and DNA δ(15) N= +4.1 ± 0.7‰ and -4.5 ± 0.4‰, respectively. Cerebellar deoxythymidine (dT), deoxycytidine (dC), deoxyadenosine (dA), and deoxyguanosine (dG) δ(15)N were +1.4 ± 0.4, -2.1 ± 0.9, -7.2 ± 0.3, and -10.4 ± 0.5‰, respectively. There were no changes in amino acid or deoxyribonucleoside δ(15) N values due to dietary protein level. Using known metabolic relationships, we developed equations to calculate the intramolecular δ(15)N values originating from aspartate (asp) in purines (pur) or pyrimidines (pyr), glutamine (glu), and glycine (gly) to be δ(15)N(ASP-PUR), δ(15)N(ASP-PYR), δ(15) N(GLN), and δ(15) N(GLY) +11.9 ± 2.3‰, +7.0 ± 2.0‰, -9.1 ± 2.4‰, and -31.8 ± 8.9‰, respectively. A subset of twelve amino acids from food and brain had mean δ(15) N values of 4.3 ± 3.2‰ and 13.8 ± 3.1‰, respectively, and δ(15)N values for gly and asp were 12.6 ± 2.2‰ and 15.2 ± 0.8‰, respectively. A separate isotope tracer study detected no significant turnover of cerebellar DNA in the first six months of life. The large negative δ(15)N difference between gly and cerebellar purine N at the gly (7) position implies either that there is a major isotope effect during DNA synthesis, or that in utero gly has a different isotope ratio during rapid growth and metabolism from that in adult life. Our data show that cerebellar nucleoside intramolecular δ(15)N values vary over more than 40‰ and are not influenced by dietary protein level or age.