Mammalian DNA δ¹⁵N exhibits 40‰ intramolecular variation and is unresponsive to dietary protein level

Abstract

We report the first high precision characterization of molecular and intramolecular δ15N 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 15N or 13C. 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. Cerebellar DNA of offspring was extracted at 2 and 120 days of life, nucleosides isolated and δ15N and δ13C characterized. Mean diet δ15N = 0.45±0.33‰, compared to cerebellar whole tissue and DNA δ15N = +4.1±0.7‰ and −4.5±0.4‰, respectively. Cerebellar deoxythymidine (dT), deoxycytidine (dC), deoxyadenosine (dA), and deoxyguanosine (dG) δ15N 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 δ15N due to dietary protein level. Using known metabolic relationships, we developed equations to calculate the intramolecular δ15N originating from aspartate (asp) in purines (pur) or pyrimidines (pyr), glutamine (glu), and glycine (gly) to be δ15NASP-PUR, δ15NASP-PYR, δ15NGLN, and δ15NGLY +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 δ15N of 4.3±3.2‰ and 13.8±3.1‰, respectively, and δ15N 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 δ15N 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 than in adult life. Our data show that cerebellar nucleoside intramolecular δ15N vary over more than 40‰ and are not influenced by dietary protein level or age.

The dietary protein paradox and threonine 15N‐depletion: Pyridoxal‐5'‐phosphate enzyme activity as a mechanism for the δ15N trophic level effect

“…[79] Thus, it stands to reason that pyridoxal-5'phosphate enzymes play pivotal roles in the unique δ 15 N fractionation patterns of these biomolecules; e.g., chitin [4] or the purines and pyrimidines of DNA. [80] To illustrate this point, recent research found that the total lipid extracts in a variety of marine organisms were highly 15 N-depleted compared with the bulk biomass. [81] These lipids contain a single atom of nitrogen that is primarily derived from Ser, and the δ 15 N Ser measurements were also found to be lower than those of the bulk biomass.…”

“…Pyridoxal‐5'‐phosphate enzymes are also involved in the biosynthesis and degradation pathways of a vast variety of different compounds including, but not limited to, amines, amino sugars, lipids, vitamins, antibiotics, heme, and chlorophyll . Thus, it stands to reason that pyridoxal‐5'‐phosphate enzymes play pivotal roles in the unique δ 15 N fractionation patterns of these biomolecules; e.g., chitin or the purines and pyrimidines of DNA . To illustrate this point, recent research found that the total lipid extracts in a variety of marine organisms were highly 15 N‐depleted compared with the bulk biomass .…”

Biochemical and physiological bases for the use of carbon and nitrogen isotopes in environmental and ecological studies

“…The analysis of a number of plants has shown that the mean value of β is −3.4 for the aquatic plants with a 1σ error of 0.9 (Figure 10a; Chikaraishi et al 2009). In terrestrial plants, the value of β was estimated to be 8.4 ± 1.6 (Figure 10b; Chikaraishi et al 2011;Steffan et al 2013). The difference in the β values of aquatic and terrestrial plants may be attributable to the differences in their metabolism of phenylalanine.…”

“…The isotopic signatures of cerebellar DNA acquired in utero are retained for at least the first 6 months of life (Strable et al 2011). It has also been demonstrated that the neuronal DNA in the human cerebellum turns over little after maturity (Slatkin et al 1985).…”

Possible Mechanisms of Biological Effects Observed in Living Systems during 2H/1H Isotope Fractionation and Deuterium Interactions with Other Biogenic Isotopes

“…The difference in the resonance effects shown by Zubarev [84], which is characterized by a 3-fold effect that is greater for 13 C compared to 15 N, can be explained (under the condition of equally pronounced neutron BADz phenomenon) by a much higher ability of the organism to absorb 13 C from the enriched 13 C diet (43.2–60.8% of the 80% enriched 13 C diet [110], and also in [111]), compared to its ability to absorb 15 N from the enriched 15 N-diet (by 250% from 95% enriched 15 N-diet [99,112]). At the same time, the co-enrichment of the medium with 13 C and 15 N, on the contrary, reduces the growth rate of the living organisms (for example, saprotrophic zygomycete fungus Absidia cylindrospora [113] or Poecilia reticulata guppies [86]), which is explained by the absence of a non-compensated neutron effect in these biological systems.…”