Observable Metabolites and Metabolomic Sampling Protocols for Managed African Savanna Elephant (Loxodonta africana) Whole Blood Using H-NMR Spectroscopy

Abstract

We used nuclear magnetic spectroscopy (NMR) to evaluate the metabolomics of heparinized whole blood drawn from six African savanna elephants (Loxodonta africana) maintained on a well characterized diet. Whole blood samples obtained under behavioral restraint, then quickly frozen in liquid nitrogen, were stored at -80 °C until analysis. Frozen samples were thawed under controlled conditions and extracted with methanol and chloroform to separate the polar and non-polar metabolites. We identified 18 polar metabolites and 14 non-polar lipids using one-dimensional (1D) and two-dimensional (2D) NMR spectra. Despite unexpected rouleaux formation in the thawed frozen samples, spectra were consistent among animals and did not vary dramatically with age or the sex of the animal.

Keywords: African elephant; metabolomics; proton-NMR.

Figure 1

Representative 1D 600 MHz proton NMR spectra of the polar extract of whole blood from an 18-year-old female African savanna elephant (Loxodonta africana). The labeled peaks are as follows: (1) isoleucine, (2) valine, (3) alanine, (4) leucine, (5) lysine, (6) glycine, (7) tyrosine, (8a) α-glucose, (8b) β-glucose, (9) creatine, (10) creatinine ^§, (11) adenosine triphosphate, (12) acetate, (13) citrate, (14) urea, (15) benzoate, (16) formate, (17) lactate, (18) succinate, (19) methanol. ^§ Singlet peaks at 3.03 and 4.06, visible on expanded spectrum, not flagged due to label congestion in the region.

Figure 2

2D TOCSY proton NMR spectra of the polar extract of whole blood from an 18-year-old female African savanna elephant (Loxodonta africana). Representative labeled peaks are valine, lactate, leucine, and adenosine triphosphate (ATP).

Figure 3

1D 600 MHz proton NMR spectrum of non-polar extract of whole blood from a 47-year-old male African savanna elephant (Loxodonta africana). The labeled peaks are as follows: (1) cholesterol methyl of C18, (2) methyls of saturated and monosaturated omega-9 and or omega-7 chains and unsaturated omega-6 acyl groups, (3) C19 methyl of free cholesterol, (4) CH₂ in fatty acyl groups, (5) CO-CH₂-CH₂ in fatty acyl chain, in a complex peak that overlaps with water, (6) double bonds in fatty acyl chains except for DHA acyl groups, (7) ethylene methyl in fatty acyl chain except for DHA acyl group, (8) double bonds in fatty acyl chain of 18:2, 20:4, and 22:6, (9) phosphatidylcholine, (10) carbon 1 and 2 in glycerol backbone of triglycerides and phospholipids, (11) carbon 1 and carbon 3 in glycerol backbone of triglycerides, (12) esterified cholesterol, (13) glycerol groups in the backbone of triglycerides and phospholipids, (14) double bonds in fatty acyl chains.