Raman Spectroscopy of
Vitamin E


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Vitamin E is a fat soluble vitamin comprised of numerous closely related compounds known as tocopherols. Its primary role in the body is to mop up free radicals and prevent oxidation of sensitive molecules such as polyunsaturated fats, with a number of secondary roles including schematic diagram of lungcontrolling inflammation.

The lung is the organ responsible for absorption of oxygen in the body and is connected to the mouth and nose by the trachea (windpipe), as shown in Figure 1. The trachea branches to left and right and these branches become known as the bronchi. Each bronchus further divides into bronchioles that then finally terminate in small air filled sac called alveoli. The alveoli are the regions in the body where oxygen is absorbed from the air into the blood stream and consequently presents a very highly oxidative environment where free radicals can readily be generated. This makes redox control a critical aspect of homeostatis in this tissue.

The alveoli are supremely optimised for air absorption, having very thin walls one cell thick with a dense network of capillaries spreading over this layer, maintaining very close contact. In order for the oxygen to transfer to the blood it must first be dissolved into water from the air, so a special type of cell (the alveolar type II) secretes a surfactant solution. This surfactant minimises surface tension to allow rapid and ready transfer of gases between the gas and solution phase. Because of the highly oxidative environment this surfactant contains a very high proportion of saturated fatty acids and very few unsaturated ones. In addition the type II cell incorporates tocopherols into the solution for chemcial structure of tocopherolsadded protection.

Tocopherols belong to a homologous family (i.e. they share a common structural backbone), the basis of which is a chromanyl ring to which various side groups may be attached, and an extended branch 'phytyl' chain, as can be seen in Figure 2. In mammals, including humans, the predominant form of tocopherol that is used by the body is the alpha form as the transport proteins for the tocopherol have a much greater affinity for this form than any other. This leads to the interesting situation whereby ingesting large quantities of vitamin E rich vegetables is inefficient as vegetables generally have predominantly the gamma form. In order to fully understand the role of tocopherol in the lung it is essential to know exactly what form it is in, where it is located and what happens to it as it carries out its role. Typically this requires use of different extractions for different classes of compound or use of a number of different fluorescent labels. Raman spectroscopy has the advantage of being able to multiplex large amounts of information and be used for imaging. The important question is, what can it do in the world of tocopherols?

The Raman spectra of the tocopherolic compounds can readilyRaman spectra of tocopherol alpha gamma be related to their chemical structures. Figure 3 shows the Raman signals obtained from the two most important tocopherol homologues in the human body, alpha and gamma. The two molecules only differ in the methyl group at the top of the chromanyl ring in the alpha form, which is replaced by hydrogen for the gamma form. This slight change in the molecular structure does induce noticeable changes in the Raman signal, especially in the intensity of two peaks contributed to by the methyl group (highlighted by the red arrows), with one peak completely disappearing at 481 cm-1 and a significant reduction in intensity at 1335 cm-1. In addition the absence of the methyl affects the whole-ring vibrations that are observed at 1581 and 1612 cm-1 in the alpha form, shifting the position of these bands and causing their relative intensity to swap. In the alpha homologue the lower wavenumber band is more intense, in the gamma form the higher band is more intense. Both molecules share the extended branched phytyl chain and acyl chains give a characteristic doublet of bands at 2845 and 2865 cm-1.

Direct oxidation of tocopherol by oxygen results in the destruction oRaman spectrum tocopheol quinone vitamin E oxidationf the chromanyl ring, as shown in the chemical structures displayed in Figure 4. In this process the methyl group at the top of the chromanyl ring is retained, but the intensity that this accounted for in the alpha tocopherol spectrum is still lost in the alpha tocopherol quinone. This can be explained by the radical change induced by the conversion of the chromanyl ring to a quinone group. As previously demonstrated (3-5) the Raman spectrum is not a simple sum of its individual building blocks, rather they depend on the symmetry of the whole molecule, thus the bands arising from a particular group can be altered if the rest if the molecule undergoes significant alteration, even if that group is not itself affected directly. The quinone retains the phytyl chain, so the double at 2845 and 2865 is retained, although it these are less intense that the main CH stretching mode at 2920 cm-1. Not surprisingly the modes attributed to the aromatic chromanyl ring are eliminated and replaced by a much more intense doublet at 1633 and 1650 cm-1 that arises from the quinone.

Metabolism of tocopherol is readily distinguished from direct oxidRaman spectrum tocopherol metabolic product hydroxychromanation by the fact that metabolic processing of tocopherol is a single electron oxidation involving the removal of the phytyl chain to leave a hydroxychroman. Figure 5 shows the Raman spectra and chemical structures involved in this conversion. Again the alpha homologue of tocopherol retains its characteristic methyl group but again the intensity is disrupted, with a drop in intensity at 481cm-1 and an increase at 1335 cm-1, and this is despite the chemical changes being more remote from the methyl group than in the conversion to quinone. The chromanyl ring is retained and the doublet corresponding to this aromatic ring are retained, though their positions are shifted to slightly lower wavenumbers. The most significant change induced in the spectrum is the loss of the doublet at 2845 and 2865 cm-1, as the phytyl chain has now been eliminated from the molecule. In its place is an acid group that give the weak bands indicated by the white arrows.

HavRaman spectroscopy PLS regression tocopherol A549 palmitic acid methyl estering determined the relationship between chemical structure and Raman signal for these important compounds we now need to address how well we can detect and identify these within biological tissues. To do this we supplemented an immortalised lung cell line (A549) with a range of concentrations of alpha tocopherol and measured the cells by Raman and by HPLC. The HPLC confirmed that the cells took up the alpha tocopherol in a linear relationship with the level of supplementation (0 – 50 mM) and also provided reference values for calibrating the Raman signal. The Raman signal gave a strong correlation with tocopherol concentration (R2validation = 0.95, see Figure 6) in the A549 cells. In order to test the correlation in a lipid matrix alpha tocopherol was mixed with palmitic acid methyl ester (palmitic acid accounts for 80% of lung surfactant), which gave an R2validation of 0.99.

In order to assess traman spectroscopy map tocopherol lung alveolar type II cellhe biochemistry of tocopherols in the lung the alveolar region was mapped in low resolution in order to identify where the type II cells were located. Since these cells only account for <5 % of the total alveolar cells it was important to carry out this initial rapid survey of the tissue. Figure 7 shows the h+e stained tissue with the red box indicating the area scanned by the Raman mapping. In the image green represents nuclei, red represents porphyrins (haemoglobin and cytochromes) and blue represents the tocopherol. There are a number of deposits of blue within the scanned region. Those in the upper part of the map were found associated with intense orange indicating a high level of porphyrin, which suggests this is a capillary wall and not an alveolus. In the lower right corner the deposit is surrounded by green, so this region was chosen for a higher resolution mapping.

Figure 8 shows this alveolus mapped in high resolution, in this figure alpha and gamma are shortened to a and g, tocopherol abbreviated to T and quinone to Q. The alpha and gamma homologues are co-localised (aT,gT), with the gamma form present in 1/10th of the concentration (hence much of its distribution is below the detection limit). In contrast the distribution of the tocopherols and their oxidation/metabolic products rarely overlaps, with only a few points showing orange in the combined aT,aTQ map. The metabolic product was identified by the absence of the doublet at  2845 and 2865 cm-1, but the remainder of the spectrum made it clear it was not present in chromanol form, but further oxidised as the quinone equivalent. This metabolic product (aT,aCEHCQ) was closely associated with the tocopherol quinone, hence distinct from the aT. The overlay of the porphyrin map and the quinone map revealed that this redox enzyme showed a very strong association with the quinones. The quinones only appeared where there was a porphyrin adjacent and the tocopherols were located further from these enzymes. This suggests that the porphyrins play a critical role in controlling redox reactions in the alveolus and that tocopherols are an important part of this role. The maps discussed so far have beenRaman spectroscopy tocopherol alpha gamma quinone hydroxychroman lung alveolus based on principal component analysis of the unnormalized data, so the intensities reflect the absolute amount of each constituent present. However, it is often informative to understand the concentrations relative to other constituents and so the final two maps employ the regressions described above to predict the concentration of alpha tocopherol relative to surfactant (aT/PAME, where PAME models the surfactant closely) and relative to cellular proteins (aT/Protein, where cellular protein is modelled by the A549 cells). It is clear that the impression of tocopherol concentration given is very different. While the largest absolute amount of tocopherol is present within the type II cell, the secreted surfactant that lines the alveolus contains a higher concentration relative to the fatty acids. This is important as the highest oxygen tension would be expected at the very interface at which oxygen absorption occurs. The tocopherol concentration relative to the proteins is even more elevated in the lining as the protein concentration is higher inside the cells and little is secreted in the surfactant. The map also indicates that the protein concentration is low inside the deposits of lipids within the type II cells.

This work is continuing with exploration of other tocopherolic compounds and components of surfactants.

1.            Beattie, J. R., and Schock, B. C. (2009) Identifying the spatial distribution of Vitamin E, pulmonary surfactant and membrane lipids by Confocal Raman Microscopy In Lipidomics (Armstrong, D., ed) p. 402, Humana Press

2.            Beattie, J. R., Maguire, C., Gilchrist, S., Barrett, L. J., Cross, C. E., Possmayer, F., Ennis, M., Elborn, J. S., Curry, W. J., McGarvey, J. J., and Schock, B. C. (2007) The use of Raman microscopy to determine and localize vitamin E in biological samples. Faseb J. 21, 766-776

3.            Beattie, J. R., Bell, S. J., and Moss, B. W. (2004) A Critical Evaluation of Raman Spectroscopy for the Analysis of Lipids: Fatty Acid Methyl Esters. Lipids 39, 407-419

4.            Oakes, R. E., Beattie, J. R., Moss, B. W., and Bell, S. E. J. (2003) DFT Studies Of Long-Chain Fames: Theoretical Justification For Determining Chain Length And Unsaturation From Experimental Raman Spectra. Theochem-J. Mol. Struct. 626, 27-45

5.            Oakes, R. E., Beattie, J. R., Moss, B., and Bell, S. E. J. (2002) Conformations, Vibrational Frequencies And Raman Intensities Of Short Chain Fatty Acid Methyl Esters Using DFT With 6-31 G(d) And Sadlej pVTZ Basis Sets. Journal of Molecular Structure 586, 91-110

 


   

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