Analysis of lipid oxidation during digestion by liquid chromatography–mass spectrometric and nuclear magnetic resonance spectroscopic techniques

Tarvainen Marko
Analysis of lipid oxidation during digestion by liquid chromatography–mass spectrometric and nuclear magnetic resonance spectroscopic techniques

Tarvainen Marko
Katso/Avaa
Lataukset: 

University of Turku
URI
http://urn.fi/URN:ISBN:978-951-29-5402-5
Näytä kaikki kuvailutiedot
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe2021042715409
Tiivistelmä
Lipid autoxidation is an unwanted process that affects the quality of food and has impact on

human health. Lipid oxidation has been studied extensively, but oxidation during digestion

has largely been ignored. Formation of oxidized lipids increases rapidly when protective

antioxidants are exhausted. On the other hand, the nature of antioxidants can lead to problems

when fortifying foods with too much antioxidants. Pro-oxidative effects of several

antioxidants have been observed when used in excessive amounts.

Methods for studying lipid oxidation are numerous. Among them are unspecific titrimetric

methods and highly specialized chromatographic and mass spectrometric methods. Nuclear

magnetic resonance (NMR) spectroscopy, especially the proton (1H) NMR, is a promising

technique for fast screening of lipid samples as it is non-destructive and because of the large

dynamic scale of the technique. Drawbacks of NMR are that relatively large amount of

sample is required for the analysis and that specific molecular structures may be difficult to

identify from complex spectrum. This thesis focuses on the study of in vitro lipid oxidation by

different chromatographic, mass spectrometric and nuclear magnetic resonance spectroscopic

methods.

The most significant findings of the studies in this thesis centre around oxidation, hydrolysis,

and behaviour of lipids in an artificial digestion model used in the studies. The model

simulates the digestion processes of human and can be used to study lipid oxidation in vitro.

Also of importance, are the lipid analysis techniques developed for the experiments, as the

techniques can be adopted to other fields of scientific studies as well for industrial uses.

Four major studies were conducted in this thesis: first an in vitro digestion model was adopted

to study the behaviour of differently oxidized rapeseed oils. Simultaneously, a novel HPLC–

evaporative light scattering detector–MS analysis technique was developed, which enabled

the analysis of native and oxidized free fatty acids, monoacylglycerols, diacylglycerols, and

triacylglycerols in the chyme produced by the digestion model. The main findings of the study

were that thermally oxidized rapeseed oil, chemically oxidized rapeseed oil and unoxidized

rapeseed oil were hydrolyzed in a similar manner. No hydroperoxides were detected in the

digested samples, even though they were present in the undigested oils. Also, the finding of

Abstract vii

large amounts of sn-1(3) monoacylglycerols was surprising, questioning the long believed

mechanism of triacylglycerol digestion and absorption.

In the second study, an ultra-high performance liquid chromatography (UHPLC) analysis

technique was developed to replace the previous HPLC method. Analysis time was reduced

by a factor of 5.5 without the loss of chromatographic resolution or detection sensitivity. Over

150 compounds were detected from digested and undigested oxidized rapeseed oils with the

method. Most significant finding was that toxic core aldehydes present in the undigested

oxidized oils were not detected in the extracted chyme. This implies that the aldehydic

functions were either lost during the hydrolysis of lipids or that the compounds formed

various complexes with other components of the chyme and were not detectable by the

analysis technique used.

In the third study, a series of antioxidants were assessed for the effects in the artificial

digestion model. An improved UHPLC–ESI–MS analysis method was developed, which used

lithium salt to greatly enhance the ionization and therefore the detection limits of the low level

analytes in electrospray ionization–mass spectrometry. The main findings were that native

(unoxidized) rapeseed oil can be oxidized during the digestion processes and that none of the

used antioxidants could completely prevent this oxidation. L-ascorbic acid, 6-palmitoyl-O-Lascorbic

acid, 3,5-di-tert-butyl-4-hydroxytoluene (BHT), DL-α-tocopherol, and DL-α-

tocopheryl acetate had different kinds of effects against this oxidation, as measured by the

concentration of oxidized lipids in the samples.

The findings of our second study were supported by the fourth study in where 1H NMR

spectroscopy was used along UHPLC–ESI–MS analyses to study the behaviour of core

aldehyde-rich oils in the artificial digestion model. Again, no compounds with aldehydic

functions were detected by UHPLC–ESI–MS analyses of the digested oils even when high

amounts of core aldehydes were present in the original oil. However, 1H NMR analyses of

several samples revealed that there were some remaining carbonyl functions in the digested

samples. The combined results of these analyses techniques strongly hinted that Schiff bases

and Michael addition products were formed in the digestion mixture. Overall, the scientific

studies conducted in this thesis have increased the knowledge of lipid oxidation and especially

provided more detailed information on possible oxidation during lipid digestion. The findings

merit for more research in the fieLipid autoxidation is an unwanted process that affects the quality of food and has impact on human health. Lipid oxidation has been studied extensively, but oxidation during digestion has largely been ignored. Formation of oxidized lipids increases rapidly when protective antioxidants are exhausted. On the other hand, the nature of antioxidants can lead to problems when fortifying foods with too much antioxidants. Pro-oxidative effects of several antioxidants have been observed when used in excessive amounts.

Methods for studying lipid oxidation are numerous. Among them are unspecific titrimetric methods and highly specialized chromatographic and mass spectrometric methods. Nuclear magnetic resonance (NMR) spectroscopy, especially the proton (1H) NMR, is a promising technique for fast screening of lipid samples as it is non-destructive and because of the large dynamic scale of the technique. Drawbacks of NMR are that relatively large amount of sample is required for the analysis and that specific molecular structures may be difficult to identify from complex spectrum. This thesis focuses on the study of in vitro lipid oxidation by different chromatographic, mass spectrometric and nuclear magnetic resonance spectroscopic methods.

The most significant findings of the studies in this thesis centre around oxidation, hydrolysis, and behaviour of lipids in an artificial digestion model used in the studies. The model simulates the digestion processes of human and can be used to study lipid oxidation in vitro. Also of importance, are the lipid analysis techniques developed for the experiments, as the techniques can be adopted to other fields of scientific studies as well for industrial uses.

Four major studies were conducted in this thesis: first an in vitro digestion model was adopted to study the behaviour of differently oxidized rapeseed oils. Simultaneously, a novel HPLC–evaporative light scattering detector–MS analysis technique was developed, which enabled the analysis of native and oxidized free fatty acids, monoacylglycerols, diacylglycerols, and triacylglycerols in the chyme produced by the digestion model. The main findings of the study were that thermally oxidized rapeseed oil, chemically oxidized rapeseed oil and unoxidized rapeseed oil were hydrolyzed in a similar manner. No hydroperoxides were detected in the digested samples, even though they were present in the undigested oils. Also, the finding of large amounts of sn-1(3) monoacylglycerols was surprising, questioning the long believed mechanism of triacylglycerol digestion and absorption.

In the second study, an ultra-high performance liquid chromatography (UHPLC) analysis technique was developed to replace the previous HPLC method. Analysis time was reduced by a factor of 5.5 without the loss of chromatographic resolution or detection sensitivity. Over 150 compounds were detected from digested and undigested oxidized rapeseed oils with the method. Most significant finding was that toxic core aldehydes present in the undigested oxidized oils were not detected in the extracted chyme. This implies that the aldehydic functions were either lost during the hydrolysis of lipids or that the compounds formed various complexes with other components of the chyme and were not detectable by the analysis technique used.

In the third study, a series of antioxidants were assessed for the effects in the artificial digestion model. An improved UHPLC–ESI–MS analysis method was developed, which used lithium salt to greatly enhance the ionization and therefore the detection limits of the low level analytes in electrospray ionization–mass spectrometry. The main findings were that native (unoxidized) rapeseed oil can be oxidized during the digestion processes and that none of the used antioxidants could completely prevent this oxidation. L-ascorbic acid, 6-palmitoyl-O-Lascorbic acid, 3,5-di-tert-butyl-4-hydroxytoluene (BHT), DL-α-tocopherol, and DL-α-tocopheryl acetate had different kinds of effects against this oxidation, as measured by the concentration of oxidized lipids in the samples.

The findings of our second study were supported by the fourth study in where 1H NMR spectroscopy was used along UHPLC–ESI–MS analyses to study the behaviour of core aldehyde-rich oils in the artificial digestion model. Again, no compounds with aldehydic functions were detected by UHPLC–ESI–MS analyses of the digested oils even when high amounts of core aldehydes were present in the original oil. However, 1H NMR analyses of several samples revealed that there were some remaining carbonyl functions in the digested samples. The combined results of these analyses techniques strongly hinted that Schiff bases and Michael addition products were formed in the digestion mixture. Overall, the scientific studies conducted in this thesis have increased the knowledge of lipid oxidation and especially provided more detailed information on possible oxidation during lipid digestion. The findings merit for more research in the field.

 

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