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NMR protocol for determination of oxidation susceptibility of serum lipids and application of the protocol to a chocolate study.

Tynkkynen T, Mursu J, Nurmi T, Tuppurainen K, Laatikainen R, Soininen P - Metabolomics (2011)

Bottom Line: The oxidation susceptibility of serum lipids decreased in the HPC group, and there was a significant difference between the WC and HPC groups (P = 0.031).Furthermore, arachidonic, docosahexaenoic, docosapentaenoic and palmitic acids, gamma-glutamyl transferase, hemoglobin, HDL, phosphatidylcholine and choline containing phospholipids explained about 60% of the oxidation susceptibility values.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0323-2) contains supplementary material, which is available to authorized users.

View Article: PubMed Central - PubMed

ABSTRACT
A protocol for determination of oxidation susceptibility of serum lipids based on proton nuclear magnetic resonance ((1)H NMR) spectroscopy is presented and compared to the commonly used spectrophotometric method. Even though there are methodological differences between these two methods, the NMR-based oxidation susceptibility correlates well (r(2) = 0.73) with the lag time determined spectrophotometrically. In addition to the oxidizability of serum lipids, the NMR method provides also information about the lipid profile. The NMR oxidation assay was applied to the chocolate study including fasting serum samples (n = 45) from subjects who had consumed white (WC), dark (DC) or high-polyphenol chocolate (HPC) daily for 3 weeks. The oxidation susceptibility of serum lipids decreased in the HPC group, and there was a significant difference between the WC and HPC groups (P = 0.031). According to the random forest analysis, the consumption of the HPC chocolate induced changes to the amounts of HDL, phosphatidylcholine, sphingomyelin, and nervonic, docosahexaenoic and myristic acids. Furthermore, arachidonic, docosahexaenoic, docosapentaenoic and palmitic acids, gamma-glutamyl transferase, hemoglobin, HDL, phosphatidylcholine and choline containing phospholipids explained about 60% of the oxidation susceptibility values. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0323-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


A 1H NMR spectrum of extracted serum with signal assignments (a). Some essential parts of the spectra (b–e) before and after the oxidation are shown at the top of the figure. The signals arising from the double bond protons (b) and the bisallylic protons from PUFAs (c) decrease during oxidation. There are also changes in the amounts of different fatty acids (d). Cholesterol oxidizes slightly under the conditions used and these oxysterol C(18)H3 signals resonate at 0.61–0.69 ppm. The signal areas are referenced to total cholesterol C(18)H3 signals including also the oxidized forms (e). EC esterified cholesterol, FA fatty acid, FC free cholesterol, PC phosphatidylcholine, PG phosphoglyceride, sat saturated, TC total cholesterol, TG triglyceride
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Fig1: A 1H NMR spectrum of extracted serum with signal assignments (a). Some essential parts of the spectra (b–e) before and after the oxidation are shown at the top of the figure. The signals arising from the double bond protons (b) and the bisallylic protons from PUFAs (c) decrease during oxidation. There are also changes in the amounts of different fatty acids (d). Cholesterol oxidizes slightly under the conditions used and these oxysterol C(18)H3 signals resonate at 0.61–0.69 ppm. The signal areas are referenced to total cholesterol C(18)H3 signals including also the oxidized forms (e). EC esterified cholesterol, FA fatty acid, FC free cholesterol, PC phosphatidylcholine, PG phosphoglyceride, sat saturated, TC total cholesterol, TG triglyceride

Mentions: The areas of the lipid resonances were determined using lineshape fitting analysis (Tukiainen et al. 2008). The structures of some multiplets (for example, see Fig. 1), which were defined by the coupling constants, were used as constraints that enabled the quantitative analysis of severely overlapping peaks (Soininen et al. 2005). The signal areas of the bisallylic protons from PUFAs (APUFA) at 2.74–2.88 ppm were used to determine the NMR oxidation susceptibility. First, the difference between the amounts of PUFAs before (REF) and after (OX) the oxidation reaction was calculated. Then the difference was divided by the signal area of PUFAs from the spectrum of non-oxidized sample and converted to a percentage value ((APUFA(REF) − APUFA(OX))/APUFA(REF) × 100%). The obtained value describes the amount of oxidized PUFAs, and thus, the oxidation susceptibility of serum lipids. The signal areas were scaled so that the signal area of total cholesterol C(18)H3 protons was the same both in the spectra of non-oxidized and oxidized serum. It should be noted that many of the cholesterol oxidation products can be identified from 1H NMR spectrum since their C(18)H3 signals resonate at 0.61–0.69 ppm (Bradamante et al. 1992), next to the non-oxidized cholesterol C(18)H3 signals (0.676 and 0.678 ppm). Thus, it is important to take all these signal areas into account when calculating the total cholesterol amount that is used as a reference in the NMR protocol.Fig. 1


NMR protocol for determination of oxidation susceptibility of serum lipids and application of the protocol to a chocolate study.

Tynkkynen T, Mursu J, Nurmi T, Tuppurainen K, Laatikainen R, Soininen P - Metabolomics (2011)

A 1H NMR spectrum of extracted serum with signal assignments (a). Some essential parts of the spectra (b–e) before and after the oxidation are shown at the top of the figure. The signals arising from the double bond protons (b) and the bisallylic protons from PUFAs (c) decrease during oxidation. There are also changes in the amounts of different fatty acids (d). Cholesterol oxidizes slightly under the conditions used and these oxysterol C(18)H3 signals resonate at 0.61–0.69 ppm. The signal areas are referenced to total cholesterol C(18)H3 signals including also the oxidized forms (e). EC esterified cholesterol, FA fatty acid, FC free cholesterol, PC phosphatidylcholine, PG phosphoglyceride, sat saturated, TC total cholesterol, TG triglyceride
© Copyright Policy
Related In: Results  -  Collection

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Fig1: A 1H NMR spectrum of extracted serum with signal assignments (a). Some essential parts of the spectra (b–e) before and after the oxidation are shown at the top of the figure. The signals arising from the double bond protons (b) and the bisallylic protons from PUFAs (c) decrease during oxidation. There are also changes in the amounts of different fatty acids (d). Cholesterol oxidizes slightly under the conditions used and these oxysterol C(18)H3 signals resonate at 0.61–0.69 ppm. The signal areas are referenced to total cholesterol C(18)H3 signals including also the oxidized forms (e). EC esterified cholesterol, FA fatty acid, FC free cholesterol, PC phosphatidylcholine, PG phosphoglyceride, sat saturated, TC total cholesterol, TG triglyceride
Mentions: The areas of the lipid resonances were determined using lineshape fitting analysis (Tukiainen et al. 2008). The structures of some multiplets (for example, see Fig. 1), which were defined by the coupling constants, were used as constraints that enabled the quantitative analysis of severely overlapping peaks (Soininen et al. 2005). The signal areas of the bisallylic protons from PUFAs (APUFA) at 2.74–2.88 ppm were used to determine the NMR oxidation susceptibility. First, the difference between the amounts of PUFAs before (REF) and after (OX) the oxidation reaction was calculated. Then the difference was divided by the signal area of PUFAs from the spectrum of non-oxidized sample and converted to a percentage value ((APUFA(REF) − APUFA(OX))/APUFA(REF) × 100%). The obtained value describes the amount of oxidized PUFAs, and thus, the oxidation susceptibility of serum lipids. The signal areas were scaled so that the signal area of total cholesterol C(18)H3 protons was the same both in the spectra of non-oxidized and oxidized serum. It should be noted that many of the cholesterol oxidation products can be identified from 1H NMR spectrum since their C(18)H3 signals resonate at 0.61–0.69 ppm (Bradamante et al. 1992), next to the non-oxidized cholesterol C(18)H3 signals (0.676 and 0.678 ppm). Thus, it is important to take all these signal areas into account when calculating the total cholesterol amount that is used as a reference in the NMR protocol.Fig. 1

Bottom Line: The oxidation susceptibility of serum lipids decreased in the HPC group, and there was a significant difference between the WC and HPC groups (P = 0.031).Furthermore, arachidonic, docosahexaenoic, docosapentaenoic and palmitic acids, gamma-glutamyl transferase, hemoglobin, HDL, phosphatidylcholine and choline containing phospholipids explained about 60% of the oxidation susceptibility values.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0323-2) contains supplementary material, which is available to authorized users.

View Article: PubMed Central - PubMed

ABSTRACT
A protocol for determination of oxidation susceptibility of serum lipids based on proton nuclear magnetic resonance ((1)H NMR) spectroscopy is presented and compared to the commonly used spectrophotometric method. Even though there are methodological differences between these two methods, the NMR-based oxidation susceptibility correlates well (r(2) = 0.73) with the lag time determined spectrophotometrically. In addition to the oxidizability of serum lipids, the NMR method provides also information about the lipid profile. The NMR oxidation assay was applied to the chocolate study including fasting serum samples (n = 45) from subjects who had consumed white (WC), dark (DC) or high-polyphenol chocolate (HPC) daily for 3 weeks. The oxidation susceptibility of serum lipids decreased in the HPC group, and there was a significant difference between the WC and HPC groups (P = 0.031). According to the random forest analysis, the consumption of the HPC chocolate induced changes to the amounts of HDL, phosphatidylcholine, sphingomyelin, and nervonic, docosahexaenoic and myristic acids. Furthermore, arachidonic, docosahexaenoic, docosapentaenoic and palmitic acids, gamma-glutamyl transferase, hemoglobin, HDL, phosphatidylcholine and choline containing phospholipids explained about 60% of the oxidation susceptibility values. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0323-2) contains supplementary material, which is available to authorized users.

No MeSH data available.