Continuous gradient temperature Raman spectroscopy and differential scanning calorimetry of N-3DPA and DHA from -100 to 10°C

Authors: Broadhurst, C; Schmidt, Walter; Nguyen, Julie; Qin, Jianwei - Tony Qin; Chao, Kuanglin - Kevin Chao; AUBUCHON, STEVEN - Waters Corporation; Kim, Moon

Submitted to: Chemistry and Physics of Lipids
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/8/2017
Publication Date: 3/28/2017
Citation: Broadhurst, C.L., Schmidt, W.F., Nguyen, J.K., Qin, J., Chao, K., Aubuchon, S.R., Kim, M.S. 2017. Continuous gradient temperature Raman spectroscopy and differential scanning calorimetry of N-3DPA and DHA from -100 to 10°C. Chemistry and Physics of Lipids. 204:94-104.

Interpretive Summary: Excluding water, the brains of all mammals are 60% fats. In particular, the cell membranes of the brain, central nervous system, eye and heart contain an abundance of highly polyunsaturated fatty acids from both the omega 3 and omega 6 series. One of the greatest unanswered questions in all biological science is why the omega polyunsaturated fatty acid docosahexaenoic acid (DHA; 22:6n-3) is absolutely necessary for constructing these fast signal processing tissues. Over 600 million years of evolution DHA has reigned supreme: everything from the simple eye spot of plankton to the human brain use DHA, yet it is abundant only in the cold water marine food chain. Two fatty acids of the same chain length with just one less double bond—the docosapentaenoic acids (DPA; 22:5n-3 and 22:5n-6)—are present in the marine and terrestrial food chains yet cannot substitute for DHA. When research animals are deprived of DHA but given DPA, they have permanent developmental disorders, brain and vision abnormalities, dementia, and memory loss. Thus far there is no acceptable chemical model as to why DHA differs so greatly from the DPAs. DHA, n-3DPA and n-6DPA are indistinguishable with conventional Raman or infrared spectroscopy techniques. Here at ARS Beltsville we have developed a new technique of continuous gradient temperature Raman spectroscopy (GTRS). We can analyze a sample’s Raman signal from liquid nitrogen temperatures to over 300°C. We found that DHA, n-3DPA and n-6DPA have different three dimensional structures when examined in solid state with GTRS. Only DHA has a fully helical structure which would facilitate its “enmeshing” into the helical structure of cell membrane proteins.

Technical Abstract: Docosahexaenoic acid (DHA, 22:6n-3) is exclusively utilized in fast signal processing tissues such as retinal, neural and cardiac. N-3 docosapentaenoic acid (n-3DPA, 22:5n-3), with just one less double bond, is also found in the marine food chain yet cannot substitute for DHA. Gradient Temperature Raman spectroscopy (GTRS) applies the temperature gradients utilized in differential scanning calorimetry (DSC) to Raman spectroscopy, providing a straightforward technique to identify molecular rearrangements that occur near and at phase transitions. Herein we apply GTRS and both conventional and modulated DSC to n-3DPA and DHA from -100 to 20°C. Three-dimensional data arrays with 0.2°C increments and first derivatives allowed complete assignment of solid, liquid and transition state vibrational modes. Melting temperatures n-3DPA (-45°C) and DHA (-46°C) are similar and show evidence for solid-state phase transitions not seen in n-6DPA (-27°C melt). The C6H2 site is an elastic marker for temperature perturbation of all three lipids, each of which has a distinct three dimensional structure. N-3 DPA shows the spectroscopic signature of saturated fatty acids from C1 to C6. DHA does not have three aliphatic carbons in sequence; n-6DPA does but they occur at the methyl end, and do not yield the characteristic signal. In n-3DPA strong bending and rocking is observed at both C6H2 and C9H2, and twisting occurs at C6H2, but n-6 DPA bends centered about C15H2. In contrast DHA appears to have a uniform twist from C6H2 to C12H2 to C18H2.