Walnuts are grown in various countries, and as product origin information is becoming more important to consumers, new techniques to differentiate walnut geographical authenticity are needed. We conducted bulk stable isotope analysis (BSIA) and compound-specific stable isotope analysis (CSIA) on walnuts grown in seven countries. The BSIA consisted of δ13Cbulk, δ15Nbulk, and δ34Sbulk, and CSIA covered δ2Hfatty acid, δ13Cfatty acid, δ13Camino acid, δ15Namino acid, and δ2Hamino acid. Analysis of variance (ANOVA) and linear discriminant analysis (LDA) were used for statistical analysis to compare samples from the USA and China. Parameters that yielded significant variations are δ2HC18:1n-9, δ13CC18:2n-6, δ13CC18:3n-3, δ13CGly, δ13CLeu, δ13CVal, δ2HGlu, δ2HIle, δ2HLeu, and δ2HThr. Our findings suggested that CSIA of fatty acids and amino acids can be useful to differentiate the geographical provenance of walnuts.

Stable (15)N isotopes have been used to examine movement of nitrogen (N) through various pools of the global N cycle. A central reaction in the cycle involves the reduction of nitrate (NO(-) 3) to nitrite (NO(-) 2) catalyzed by nitrate reductase (NR). Discrimination against (15)N by NR is a major determinant of isotopic differences among N pools. Here, we measured in vitro (15)N discrimination by several NRs purified from plants, fungi, and a bacterium to determine the intrinsic (15)N discrimination by the enzyme and to evaluate the validity of measurements made using (15)N-enriched NO(-) 3. Observed NR isotope discrimination ranged from 22 to 32‰ (kinetic isotope effects of 1.022-1.032) among the different isozymes at natural abundance (15)N (0.37%). As the fractional (15)N content of substrate NO(-) 3 increased from natural abundance, the product (15)N fraction deviated significantly from that expected based on substrate enrichment and (15)N discrimination measured at natural abundance. Additionally, isotopic discrimination by denitrifying bacteria used to reduce NO(-) 3 and NO(-) 2 in some protocols became a greater source of error as (15)N enrichment increased. We briefly discuss potential causes of the experimental artifacts with enriched (15)N and recommend against the use of highly enriched (15)N tracers to study N discrimination in plants or soils.