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(Journal of Nutrition. 2001;131:1898-1902.)
© 2001 The American Society for Nutritional Sciences

Articles

Dietary Fats Affect Rat Plasma Lipoprotein Secondary Structure as Assessed by Fourier Transform Infrared Spectroscopy1

Gema López*, Rebeca Martínez*, Jesús Gallego*, María Jesús Tarancón{dagger}, Pedro Carmona** and María Victoria Fraile*2

* Departamento de Ciencias Básicas, Facultad de Ciencias Experimentales y Técnicas, Universidad S. Pablo-CEU, Madrid, Spain, {dagger} Hospital de la Fuenfría, Cercedilla, Madrid, Spain and ** Instituto de Estructura de la Materia (CSIC), 28006, Madrid, Spain

2To whom correspondence should be addressed. E-mail: vfradot{at}ceu.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
This investigation was undertaken to determine by Fourier transform infrared spectroscopy the effects of diets enriched with fish, sunflower or olive oils on the secondary structure of plasma HDL and LDL from rats, as well as the effects on lipid unsaturation and acyl chain lengths. Controls were fed a commercial diet. In HDL, random coil conformation was relatively high in rats fed the fish diet, probably due to the irregular geometry of polyunsaturated fatty acids interacting with apoproteins. Parallel structural behaviors were observed for rats fed control and olive oil diets. The lowest lipid unsaturation level was found in HDL of rats fed olive oil, and acyl chain lengths were slightly increased by the three fats. Rats fed olive oil had the lowest percentage of LDL ß-sheets and these were more abundant in rats fed the fish oil diet. The least lipid unsaturation in LDL was in rats fed the olive oil diet. No significant differences in acyl chain lengths were observed. Certain protein conformational changes and/or apoprotein composition differences due to dietary fat may affect the binding between lipoproteins and their receptors in cells.

KEY WORDS: • dietary fat • lipoproteins • rats • secondary structure • Fourier transform infrared spectroscopy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Plasma lipoproteins are particles that carry cholesterol to different tissues. Cardiovascular disease has been related to diet and lipoprotein composition. Numerous epidemiological studies have demonstrated that high levels of plasma cholesterol, especially that transported in LDL, and/or low concentrations of cholesterol transported in HDL are associated with an increase in the risk of artherosclerosis and cardiovascular disease. Fatty acids are the most important dietary component that determines the concentrations of plasma lipids. The association between diet, plasma lipid concentrations and atherosclerosis has been well documented and reviewed (1)Citation . Studies carried out by Parthasarathy et al. (2)Citation have shown the relationship between oleic and linoleic acid-rich diets and the lipoprotein composition associated with artherosclerotic risk, as well as the different composition of the dietary fat and the LDL oxidation susceptibility. This susceptibility is composition-dependent, depending mainly on the fatty acids of the phospholipids in the outer layer of the lipoprotein particles. Although lipoproteins in normal plasma present different LDL subclasses that vary in terms of size, lipid composition, apolipoprotein content and metabolic properties (3Citation 4Citation 5)Citation , we have studied the particles without previous separation into subclasses. In the present study, we used rats as an animal model for evaluating four dietary fats effects on apoprotein structure by infrared spectroscopy.

The objective of the present study was to establish the secondary structure of plasma apolipoproteins in relation to the dietary oil, as assessed by Fourier transform infrared, showing the possible structural changes that the fatty composition of the diet induces in the secondary structure of the apolipoproteins in LDL and HDL particles. Structural changes in LDL have been shown to alter their metabolism and atherogenic potential. We have used animal and not human models because it is very difficult to feed an appropriate number of persons particular oil diets and, moreover, human commercial food consists usually of heterogeneous fat components.

The infrared amide I band arises primarily from in-plane peptide C-O stretching vibrations, and partly from in-plane N-H bending vibrations (6)Citation . The exact location of the amide I band in the infrared spectrum depends on hydrogen bonding and the conformation of the protein backbone (6)Citation . In heteropolypeptides and in real-world proteins, there exist a variety of domains containing polypeptide fragments in different conformations. Thus, the observed amide I band contours of proteins are usually complex composites that consist of a number of overlapping component bands, representing helices, ß-sheets, turns and unordered structures. The currently available techniques of resolution enhancement, such as Fourier deconvolution and derivative spectroscopy, usually allow the identification of these otherwise hidden component bands. Moreover, band-fitting procedures allow for quantitative evaluation of the various components of protein secondary structure, such as {alpha}-helices, ß-structures and turns, on the basis of measuring the fractional areas (integrated intensities) of the fitted component bands. These areas are directly related to the relative populations of the conformational structures represented by these components.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Subjects.

Forty male Wistar rats from an in-house colony were subdivided into four dietary groups (10 animals per group) weighing between 70 and 90 g for 21/2 mo until their killing. One group control and three experimental groups were fed synthetic diets with 5 g/100 g in fat prepared in our laboratory (7Citation ,8)Citation . The three diets differed primarily in their fatty acid composition: one with 42% (n-3) PUFA (fish oil) (9)Citation ; another with 60% (n-6) PUFA (sunflower oil) and the third with 75% monounsaturated fatty acid (olive oil) (9)Citation . RMN commercial rat diet (in pellet form) provided by Harlam Ibérica (Barcelona, Spain) was used as the commercial control with 2.2 g/100 g fat. The rats were allowed free access to food and water. The above animals were killed by decapitation, and the corresponding blood samples were immediately centrifuged to separate the plasma.

Lipoproteins.

IDL + LDL (d 1.006–1.063 kg/L) and HDL (d 1.063–1.21 kg/L) were isolated from 9 mL of plasma obtained at each time point by sequential ultracentifrugation in a Beckman TL100 ultracentrifuge using a NVTi rotor (Beckman Coulter, Fullerton, CA). These lipoproteins were then purified by dialysis against saline solution (pH 7.0) for 12 h.

Infrared spectroscopy.

Infrared spectra were recorded at room temperature with a Perkin-Elmer 2000 instrument (Norwalk, CT) using a high sensitivity deuterated triglycine sulfate detector and a spectral resolution of 2 cm-1. The spectra of LDL and HDL were recorded using the film procedure. This was followed by placing ~30 µL of lipoprotein aqueous suspension on an infrared window and evaporating under vacuum. The spectra were transferred to a personal computer, where small amounts of water vapor were substracted when needed. Quantitative information on protein structure was obtained through decomposition of the amide I band into its constituents. The spectral resolution enhancement was performed as described previously (10)Citation , and the amide I band decomposition was performed using the curvefit routine running under SpectraCalc (Galactic, Salem, NH). The amide I band components were fitted with weighted sums of Lorentz and Gaussian functions. The choice of the starting parameters was assisted by Fourier self-deconvolution and second derivative spectroscopy. The initial heights were set at ~90% of those in the original spectrum for the bands in the wings and for the most intense component, and at ~70% of the original intensity for the other bands. The mathematical solution of the decomposition may not be unique, but if restrictions are imposed such as the maintenance of the initial band positions in an interval of ± 1 cm-1, the preservation of the bandwidth within the expected limits, or the agreement with theoretical boundaries or predictions, the result becomes, in practice, unique.

Data analysis and statistics.

The area percentages of amide I band components are reported as the mean for each group. Student’s t tests were used to determine differences between experimental groups and controls.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Protein secondary structure and lipid/protein ratio

    HDL. The spectra of the HDL are shown in Figure 1Citation . These show three bands corresponding to the C-O stretching motions ({nu}C-O) of lipid carbonyl groups (band centered near 1735 cm-1), carbonyl groups of protein backbone (band centered near 1655 cm-1, which is the so-called amide I band) and N-H groups ({delta}N-H band centered at 1550 cm-1, so-called amide II). Although the height of the 1735 cm-1 band relative to that located at 1655 cm-1 was variable when controls were compared with those fed the experimental fats, the lipid carbonyl band area was smaller than that of the amide I band in each sample, as expected from the composition of the HDL. In fact, these usually contain ~50% apoprotein, 20% phospholipids, 10% cholesteryl esters and 20% unesterified cholesterol (4)Citation . We estimated average absorptivities for carbonyl and amide I bands of carbonyl lipid fraction and protein fraction, respectively, which showed that the absorptivity of the 1735 cm-1 lipid band relative to that of the amide I band was ~2. Because of this result and the above lipoprotein composition, it was expected that the area of the amide I band in each sample would be higher than that of the corresponding 1735 cm-1 lipid band.



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  		Figure 1. Infrared spectra of HDL of (from top to bottom) control fish oil, sunflower oil and olive oil diets.

 
The relative intensities of {nu}(C-O) lipid and amide I bands were smaller in the rats fed the fish oil or sunflower oil diet than in controls or in the rats fed the olive oil diet. In contrast, the spectral profiles of the amide I and amide II bands differed in the experimental groups compared with controls, which means that these diets affected protein secondary structure and/or apoprotein composition.

With the aim of quantifying the changes observed in the spectral profiles, we have done the curve fitting of the amide I band generated by the protein backbone (Fig. 2Citation ). The assignment of the amide I component bands to conformational structures (Table 1Citation ) seems at first difficult because the number of bands is larger than the number of expected protein secondary structures, and because natural proteins do not always exhibit the same behavior as model molecules and homopolypeptides that are thought to reflect these secondary structures (10)Citation . Still, some of the bands could be unambiguously assigned (Table 1Citation ), whereas for others, reasonable approximations could be made in comparison with data from other techniques. Thus, the band ~1655 cm-1 corresponds to {alpha}-helix plus nonstructured peptide, but only to {alpha}-helix in N-deuterated polypeptide backbone. ß-Turns are located between 1660 and 1685 cm-1, whereas the extended structures (ß-strands) give rise to signals in the region 1610–1640 cm-1. Two different groups of bands are seen in this region, one in the 1625–1640 cm-1 range and the other group in the 1610–1625 cm-1 range. The first group of bands arise from intramolecular C = O vibrations of ß-sheets. The latter is found in denatured proteins (11Citation ,12)Citation but it is not so common in native proteins. In these, the bands falling in the 1610–1625 cm-1 range were first found in concanavalin A (13)Citation and were assigned to peptides in an extended configuration, with a hydrogen bonding pattern formed by peptide residues not taking part in intramolelcular ß-sheet but rather hydrogen-bonded to other molecular structures, e.g., forming intermolecular hydrogen bonding in monomer-monomer interaction. They were also found in triosephosphate isomerase (14)Citation , and even in human LDL (15)Citation . These low frequency bands were called "ß-edge" bands, typical of the outer strands of ß-sheets (13Citation ,14)Citation . This pattern also implies intermolecular hydrogen bonding, as postulated for the low-frequency bands in irreversibly aggregated proteins (10)Citation , or in monomer-monomer contacts, as in concanavalin A (13)Citation . Aromatic ring vibration of tyrosine residue can also generate a band usually appearing near 1615 cm-1 (16)Citation .



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  		Figure 2. Infrared spectra of the amide I band of HDL of control rats: (upper) curve-fitting of the absorption spectrum and (lower) second derivative spectrum.

 

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  		Table 1. Frequency positions of the amide I band components in the infrared spectra of HDL and LDL lipoproteins for different diets from rats fed control, fish oil, sunflower oil or olive oil diets1

 
Figure 3Citation shows, as a characteristic feature, similar area percentages for all the amide I band components in HDL proteins from olive oil and control diets. In contrast, the spectral behavior of HDL from rats fed fish oil in the 1645 cm-1 band component tended to differ from that of controls, where this secondary structure was less abundant. Only the fish oil diet generated a visible band at 1674 cm-1, and the most prominent bar of the 1650 amide I band component corresponded to HDL from rats fed this diet (Fig. 3)Citation . By contrast, its area percentage at 1681 cm-1 was the lowest one. The sunflower oil diet resulted in the most intense band components at 1622 and 1615 cm-1.



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  		Figure 3. Protein secondary structure percentages of HDL from rats fed control (), fish oil (), sunflower oil () or olive oil diets (). Values are means ± SE, n = 10.

 
    LDL. Figure 4Citation includes the infrared spectra of LDL in the 1800–1500 cm-1 region. Apart from the fact that the lowest {nu}C-O intensity relative to that of amide I band was present in control samples, there seemed to be some differences within the amide I spectral region. This was particularly true for LDL from rats fed olive oil diet, which produced the lowest proportion of ß-structure compared with the rest of the samples. Thus, this diet generated 8.91% of the 1623 cm-1 band component (ß-structure), whereas the corresponding percentages in the LDL of rats fed the sunflower oil, fish oil and control diets were 15%, 14% and 11%, respectively (Fig. 5Citation ). A similar situation could be described for the other ß-structure band component at 1615 cm-1. The fish oil diet generated weaker intensities at 1651 cm-1 relative to controls. This band component can be attributable to {alpha}-helices and random coil polypeptide backbone. In contrast, it is obvious that random coil formation involves the presence of turns, which usually appear in the 1660–1675 cm-1 range. Because the band components at 1661 and 1671 cm-1 of LDL from rats fed the fish oil diet did not significantly differ from controls, we can tentatively assign the 1651 cm-1 intensity decrease in LDL of rats fed the fish oil diet to a concomitant decrease of {alpha}-helices. This can be easily explained by considering the lipid composition of this diet. In fact, fish fats are rich in PUFA, which contain cis C-C conformation (turns) in their hydrocarbon chains (16)Citation . In general, fatty acid composition in serum and plasma lipoproteins reflects, to a large extent, the fatty acid composition of the diet (17)Citation . Consequently, the lipids surrounding the apolipoproteins of rats fed fish and sunflower oil diets may adapt preferentially to random or ß-polypeptide conformation. In fact, we have found (unpublished results) that PUFA induce turns and ß-sheet structures in proteins.



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  		Figure 4. Infrared spectra of LDL of rats fed (from top to bottom) control, fish oil, sunflower oil or olive oil diets.

 


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  		Figure 5. Protein secondary structure percentages of LDL from rats fed control (), fish oil (), sunflower oil () or olive oil diets (). Values are means ± SE, n = 10.

 
The above diet-dependent protein conformational changes in LDL and HDL are not parallel. This can be explained by considering that apoprotein and lipid compositions in HDL and LDL particles are different, thus leading to particular lipid-protein interactions and subsequent protein structures adopted by each type of lipoprotein particles.

Unsaturation and acyl chains

With the aim of knowing the relationship between type of dietary fat and its effect on the amount of lipid unsaturation in lipoprotein particles, we have reported the intensity of the 3015 cm-1 band relative to that of 1745 cm-1 band (Fig. 6Citation ), which are generated by the stretching motions of C-H groups attached to C-C bonds and by the lipid {nu}C = O vibration, respectively. The most important differences in LDL were in those of rats fed the olive oil diet, which produced the least amount of unsaturation. This result is consistent with the lipid composition of the olive oil diet, which is less unsaturated than the others. By contrast, no differences were observed in HDL.



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  		Figure 6. I3015/I1745 intensity ratios for HDL and LDL from rats fed control, fish oil, sunflower oil or olive oil diets. Values are means ± SE, n = 10.

 
In contrast, Figure 7Citation shows the relative intensities of the 2954 and 2926 cm-1 bands, which depend on the acyl chain length and/or chain branching. The former is produced by the asymmetrical stretching vibration, {nu}asCH3, and the second is generated by the {nu}asCH2 mode with contribution from {nu}sCH3 (18)Citation . Consequently, the 2954/2926 cm-1 band ratio can be considered an indirect measurement of the amount of CH3 groups relative to CH2 groups; that is, an indirect measurement of the lipid acyl chain length or chain branching. In LDL there were no marked differences among diet groups. In HDL, controls contained hydrocarbon chains shorter than those of rats fed the experimental fats.



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  		Figure 7. I2954/I2926 intensity ratios for HDL and LDL from rats fed control, fish oil, sunflower oil or olive oil diets. Values are means ± SE, n = 10.

 
In conclusion, the effects of dietary fats (fish, sunflower and olive oils) on the secondary structures of HDL and LDL as well as on some structural properties of their lipids were studied by infrared spectroscopy. Concerning HDL structure, the most distinct spectroscopic behaviors were associated with the fish oil diet, which produced relatively high contents of random coil, probably due to the presence of the irregular geometries of PUFA. Other spectral features revealed similar structural behaviors in apoproteins from controls and rats fed the olive oil diet. Interestingly, this olive oil diet resulted in the lowest unsaturation level and acyl chains of HDL in rats fed the experimental diets seemed to be slightly longer than controls.

Regarding the protein secondary structure of LDL particles, olive oil produced the lowest proportion of ß-structure with this being more abundant in LDL from rats fed the fish oil diet. In contrast, no differences were observed in acyl chain lengths or branching, and as in HDL, the lowest level of unsaturation was is rats fed the olive oil.

These data suggest that certain protein conformational changes and/or apoprotein composition differences produced by some diets may be the primary factors that determine the magnitude of binding between these lipoproteins and their corresponding receptors in cells.


   FOOTNOTES
 
1 Supported by Project 8/99 from the Universidad S. Pablo-CEU and Química Farmacéutica Bayer, S.A. Back

Manuscript received November 27, 2000. Initial review completed January 29, 2001. Revision accepted April 16, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

1. Steimberg D., Witztum J. L. Lipoprotein and atherogenesis: current concepts. J. Am. Med. Assoc. 1990;264:3047-3052[Abstract/Free Full Text]

2. Parthasarathy S., Khoo J. C., Miller E., Barnett J., Witztum J. L., Steimberg D. Low density lipoprotein rich in oleic acid is protected against oxidative modification: implications for dietary prevention of atherosclerosis. Proc. Natl. Acad. Sci. USA 1990;87:3894-3898[Abstract/Free Full Text]

3. Morrisett J. D., Jackson R. L., Gotto A. M., JR Lipoproteins: structure and function. Ann. Rev. Biochem. 1975;47:183-207

4. Smith L. C., Pownall H. J., Gotto A. M., JR The plasma lipoproteins: structure and metabolism. Ann. Rev. Biochem. 1978;47:751-777[Medline]

5. Nowicka G., Bruening T., Böttcher A., Kahl G., Schmitz G. Macrophage interaction of HDL subclasses separated by free flow isotachophoresis. J. Lipid Res. 1990;31:1947-1963[Abstract]

6. Krimm S., Bandekar J. Vibrational spectroscopy and conformation of peptides, polypeptides and proteins. Adv. Protein Chem. 1986;38:181-364[Medline]

7. Scanu A., Spector A. Biochemistry and Biology of Plasma Lipoproteins 1986 Dekker New York, NY.

8. Amusquivar E., Rupérez F. J., Barbas C., Herrera E. Low arachidonic acid rather than tocopherol is responsible for the delayed postnatal development in offspring of rats fed fish oil instead of olive oil during pregnancy and lactation. J. Nutr. 2000;130:2855-2865[Abstract/Free Full Text]

9. Vognild E., Elvevoll E. O., Brox J., Olsen R., Barstad H., Aursand M., Østerud B. Effects of dietary marine oils and olive oil on fatty acid composition, platelet membrane fluidity, platelet responses, and serum lipids in healthy humans. Lipids 1998;33:427-436[Medline]

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11. Muga A., Arrondo J.L.R., Bellon T., Sancho J., Bernabeu C. Structural and functional studies on the interaction of sodium dodecyl sulfate with ß-galactosidase. Arch. Biochem. Biophys. 1993;300:451-457[Medline]

12. Naumann D., Schultz C., Görne-Tschelnokov U., Hucho F. Secondary structure and temperature behaviour of the acetylcholine receptor by Fourier transform infrared spectroscopy. Biochemistry 1993;32:3162-3168[Medline]

13. Arrondo J.L.R., Young N. M., Mantsch H. H. The solution structure of concanavalin A probed by FTIR spectroscopy. Biochim. Biophys. Acta 1988;952:261-268[Medline]

14. Castresana J., Muga A., Arrondo J.L.R. The structure of proteins in aqueous solution: an assessment of triose phosphate isomerase structure by fourier-transform infrared spectroscopy. Biochem. Biophys. Res. Commun. 1988;152:69-75[Medline]

15. Goormaghtigh E., De Meutter J., Vanloo B., Brasseur R., Rosseneu M., Ruysschaert J. M. Evaluation of the secondary structure of Spo B-100 in low-density lipoprotein (LDL) by infrared spectroscopy. Biochim. Biophys. Acta 1989;1006:147-150[Medline]

16. Horvat S., Jakas A., Vass E., Samu J., Hollósi M. CD and FTIR spectroscopic studies of amadori compounds related to the opioid peptides. J. Chem. Soc. Perkin Trans. 1997;2:1523-1528

17. Seiquer I., Mañas M., Martínez-Victoria E., Ballesta M. C., Mataix F. J. Long-term influence of dietary fat (sunflower oil, olive oil, lard and fish oil) in the serum fatty acid composition and in the different lipidic fractions, in miniature swine. Int. J. Vit. Nutr. Res. 1996;66:171-179

18. Fraile M. V., Patrón-Gallardo B., López-Rodríguez G., Carmona P. FT-IR study of multilamellar lipid dispersions containing cholesteryl linoleate and dipalmitoylphosphatidylcholine. Chem. Phys. Lipids 1999;97:119-128





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