Birgit Scholz

Gut metabolites and bacterial community networks during a pilot intervention study with flaxseeds in healthy adult men

SCOPE Flaxseeds contain the phytoestrogens lignans that must be activated to enterolignans by intestinal bacteria. We investigated the impact of flaxseeds on fecal bacterial communities and their associations with fecal and blood metabolites. METHODS AND RESULTS Nine healthy male adult subjects ingested 0.3 g/kg/day flaxseeds during 1 week. Gut bacteria as well as blood and fecal metabolites were analyzed. Ingestion of flaxseeds triggered a significant increase in the blood concentration of enterolignans, accompanied by fecal excretion of propionate and glycerol. Overall diversity and composition of dominant fecal bacteria remained individual specific throughout the study. Enterolactone production was linked to the abundance of two molecular species identified as Ruminococcus bromii and Ruminococcus lactaris. Most dominant species of the order Bacteroidales were positively associated with fecal concentrations of either acetic, isovaleric, or isobutyric acid, the latter being negatively correlated with blood levels of triglycerides. The relative sequence abundance of one Gemmiger species (Ruminococcaceae) and of Coprococcus comes (Lachnospiraceae) correlated positively with blood levels of LDL cholesterol and triglycerides, respectively. CONCLUSION Flaxseeds increase enterolignan production but do not markedly alter fecal metabolome and dominant bacterial communities. The data underline the possible role of members of the family Ruminococcaceae in the regulation of enterolignan production and blood lipids.

Simultaneous analysis of free phytosterols/phytostanols and intact phytosteryl/phytostanyl fatty acid and phenolic acid esters in cereals.

An approach based on solid-phase extraction for the effective separation of free phytosterols/phytostanols and phytosteryl/phytostanyl fatty acid and phenolic acid esters from cereal lipids was developed. The ester conjugates were analyzed in their intact form by means of capillary gas chromatography. Besides free sterols and stanols, up to 33 different fatty acid and phenolic acid esters were identified in four different cereal grains via gas chromatography-mass spectrometry. The majority (52-57%) of the sterols and stanols were present as fatty acid esters. The highest levels of all three sterol and stanol classes based on dry matter of ground kernels were determined in corn, whereas the oil extract of rye was 1.7 and 1.6 times richer in fatty acid esters and free sterols/stanols than the corn oil. The results showed that there are considerable differences in the sterols/stanols and their ester profiles and contents obtained from corn compared to rye, wheat, and spelt. The proposed method is useful for the quantification of a wide range of free phytosterols/phytostanols and intact phytosteryl/phytostanyl esters to characterize different types of grain.

Dietary fat and gut microbiota interactions determine diet-induced obesity in mice

Objective Gut microbiota may promote positive energy balance; however, germfree mice can be either resistant or susceptible to diet-induced obesity (DIO) depending on the type of dietary intervention. We here sought to identify the dietary constituents that determine the susceptibility to body fat accretion in germfree (GF) mice. Methods GF and specific pathogen free (SPF) male C57BL/6N mice were fed high-fat diets either based on lard or palm oil for 4 wks. Mice were metabolically characterized at the end of the feeding trial. FT-ICR-MS and UPLC-TOF-MS were used for cecal as well as hepatic metabolite profiling and cecal bile acids quantification, respectively. Hepatic gene expression was examined by qRT-PCR and cecal gut microbiota of SPF mice was analyzed by high-throughput 16S rRNA gene sequencing. Results GF mice, but not SPF mice, were completely DIO resistant when fed a cholesterol-rich lard-based high-fat diet, whereas on a cholesterol-free palm oil-based high-fat diet, DIO was independent of gut microbiota. In GF lard-fed mice, DIO resistance was conveyed by increased energy expenditure, preferential carbohydrate oxidation, and increased fecal fat and energy excretion. Cecal metabolite profiling revealed a shift in bile acid and steroid metabolites in these lean mice, with a significant rise in 17β-estradiol, which is known to stimulate energy expenditure and interfere with bile acid metabolism. Decreased cecal bile acid levels were associated with decreased hepatic expression of genes involved in bile acid synthesis. These metabolic adaptations were largely attenuated in GF mice fed the palm-oil based high-fat diet. We propose that an interaction of gut microbiota and cholesterol metabolism is essential for fat accretion in normal SPF mice fed cholesterol-rich lard as the main dietary fat source. This is supported by a positive correlation between bile acid levels and specific bacteria of the order Clostridiales (phylum Firmicutes) as a characteristic feature of normal SPF mice fed lard. Conclusions In conclusion, our study identified dietary cholesterol as a candidate ingredient affecting the crosstalk between gut microbiota and host metabolism.

Phytosterol oxidation products in enriched foods: Occurrence, exposure, and biological effects

Hypercholesterolemia is an important risk factor for the development of cardiovascular diseases. Dietary intake of phytosterols/phytostanols and their fatty acid esters results in a reduction of the LDL and total plasma cholesterol levels. Therefore, these constituents are added to a broad spectrum of foods. As in the case of cholesterol, thermo-oxidative treatment of phytosterols may result in the formation of phytosterol oxidation products (POPs), i.e. keto-, hydroxy-, and epoxy-derivatives. This review summarizes and evaluates the current knowledge regarding POPs in the light of the potentially increasing dietary exposure to these constituents via the consumption of foods enriched with phytosterols/phytostanols and their esters. Data on the occurrence of POPs and approaches to assess the potential intake of POPs resulting from the consumption of enriched foods are described. The knowledge on the uptake of POPs and the presently available data on the impact of the consumption of enriched foods on the levels of POPs in humans are discussed. Biological effects of POPs, such as potential proatherogenic properties or the loss of the cholesterol-lowering effects compared to nonoxidized phytosterols, are discussed. Finally, knowledge gaps are outlined and recommendations for further research needed for a safety assessment of POPs are presented.

Online LC-GC analysis of free sterols/stanols and intact steryl/stanyl esters in cereals.

The suitability of online liquid chromatography-gas chromatography for the analysis of free sterols/stanols, steryl/stanyl fatty acid esters, and trans-steryl/stanyl ferulic acid esters in cereals is demonstrated. The silylated lipid extracts were fractionated via liquid chromatography on a normal phase, and the fractions containing the sterol classes were transferred online to the gas chromatograph for the analysis of their individual compositions. The study provides for the first time data on free sterols/stanols and intact steryl/stanyl esters in sweet corn, popcorn, and proso millet. Sweet corn revealed the highest contents of free sterols/stanols and steryl/stanyl fatty acid esters, and popcorn, in turn, the highest amounts of trans-steryl/stanyl ferulic acid esters. The distribution patterns of the proso millet samples revealed pronounced differences from those of sweet corn and popcorn as they particularly exhibited high proportions of free cholesterol and cholesteryl fatty acid esters. Furthermore, no trans-steryl/stanyl ferulic acid esters could be detected.

On-line liquid chromatography–gas chromatography: A novel approach for the analysis of phytosterol oxidation products in enriched foods

A novel methodology for the automated qualitative and quantitative determination of phytosterol oxidation products in enriched foods via on-line liquid chromatography-gas chromatography (LC-GC) was established. The approach is based on the LC pre-separation of acetylated phytosterols and their corresponding oxides using silica as stationary phase and a mixture of n-hexane/methyl tert-butyl ether/isopropanol as eluent. Two LC-fractions containing (i) 5,6-epoxy- and 7-hydroxyphytosterols, and (ii) 7-ketophytosterols are transferred on-line to the GC for the analysis of their individual compositions on a medium polar trifluoropropylmethyl polysiloxane capillary column. Thus, conventionally employed laborious off-line purification and enrichment steps can be avoided. Validation data, including recovery, repeatability, and reproducibility of the method, were elaborated using an enriched margarine as example. The margarine was subjected to a heating procedure in order to exemplarily monitor the formation of phytosterol oxidation products. Quantification was performed using on-line LC-GC-FID, identification of the analytes was based on on-line LC-GC-MS. The developed approach offers a new possibility for the reliable and fast analysis of phytosterol oxidation products in enriched foods.

Heating Two Types of Enriched Margarine: Complementary Analysis of Phytosteryl/Phytostanyl Fatty Acid Esters and Phytosterol/Phytostanol Oxidation Products

Two phytosteryl and/or phytostanyl fatty acid ester-enriched margarines were subjected to common heating procedures. UHPLC-APCI-MS analysis resulted for the first time in comprehensive quantitative data on the decreases of individual phytosteryl/-stanyl fatty acid esters upon heating of enriched foods. These data were complemented by determining the concurrently formed phytosterol/-stanol oxidation products (POPs) via online LC-GC. Microwave-heating led to the least decreases of esters of approximately 5% in both margarines. Oven-heating of the margarine in a casserole caused the greatest decreases, with 68 and 86% esters remaining, respectively; the impact on individual esters was more pronounced with increasing degree of unsaturation of the esterified fatty acids. In the phytosteryl/-stanyl ester-enriched margarine, approximately 20% of the ester losses could be explained by the formation of POPs; in the phytostanyl ester-enriched margarine, the POPs accounted for <1% of the observed ester decreases.

Analysis of phytostanyl fatty acid esters in enriched foods via UHPLC-APCI-MS.

A method for the analysis of phytostanyl fatty acid esters, the functional ingredients of cholesterol-lowering enriched foods, was developed. The procedure is based on (i) separation of the intact esters via reversed-phase ultrahigh-performance liquid chromatography; (ii) detection by atmospheric pressure chemical ionization-mass spectrometry; and (iii) quantitation using selected ion monitoring (SIM) mode. Employing a C8 column, phytostanyl fatty acid esters sharing the same stanol nucleus could be separated according to the esterified fatty acids while esters with different stanol moieties could be distinguished via SIM based on the formation of an intense fragment ion [M - fatty acid + H](+). The suitability of the approach was demonstrated using different types of enriched foods reflecting the diversity in potential matrices (skimmed milk drinking yogurt, margarine, and soft-cheese-style spread). The developed methodology extends the analytical basis for authenticity and quality assessments of functional foods enriched with phytostanyl fatty acid esters.

An approach based on ultrahigh performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry allowing the quantification of both individual phytosteryl and phytostanyl fatty acid esters in complex mixtures.

A method for the analysis of both individual phytosteryl and phytostanyl fatty acid esters in complex mixtures was established. The approach was based on a previously not described combination of three elements: (i) the formation of [M-FA+H](+) fragment ions via APCI (atmospheric pressure chemical ionization), (ii) a highly efficient UHPLC-based separation on a 1.7 μ C8 column, previously established for phytostanyl fatty acid esters, allowing the distinction of individual fatty acid esters sharing the same sterol/stanol nucleus and of isotope peaks of phytosteryl fatty acid esters and corresponding phytostanyl fatty acid esters based on these [M-FA+H](+) fragment ions, and (iii) the adjustment of the APCI conditions allowing the differential APCI-MS-SIM (single ion monitoring) detection of phytostanyl esters of linoleic and linolenic acid based on their distinct formation of a [M+H](+) ion. The usefulness of the methodology was demonstrated by the analysis of a commercially available enriched margarine. Two runs per sample allowed the quantification of 35 target analytes; the total amounts of esters were between 124.7 and 125.3g/kg, being in good agreement with the labelled 125 g/kg. Validation data were elaborated for 35 individual fatty acid esters of sitosterol, campesterol, brassicasterol, stigmasterol, sitostanol and campestanol. Recovery rates ranged from 95 to 106%; the coefficients of variation were consistently <5%, except for stigmasteryl-18:1. The approach describes for the first time a quantification of both individual phytosteryl and phytostanyl fatty acid esters and thus closes an analytical gap related to this class of health-relevant food constituents.

Quantitation of Acyl Chain Oxidation Products Formed upon Thermo-oxidation of Phytosteryl/-stanyl Oleates and Linoleates

A GC-based approach involving preseparation via solid-phase extraction was established for the quantitation of acyl chain oxidation products (ACOPs) formed upon thermo-oxidation (180 °C, 40 min) of oleates and linoleates of phytostanols and phytosterols. The concentrations of ACOPs resulting from initially formed 9-hydroperoxides (octanoates, 8-hydroxyoctanoates, 9-oxononanoates) were higher than those from 8-hydroperoxides (heptanoates, 7-hydroxy- and 7-oxoheptanoates, 8-oxooctanoates) in both oleates and linoleates. Significantly higher amounts of ACOPs were found in heat-treated linoleates compared to oleates. However, despite lower thermally induced losses of stanyl oleates and linoleates compared to the respective steryl esters, higher concentrations of ACOPs (approximately 9 and 10% of the ester losses, respectively) were observed in the heat-treated stanyl esters. In contrast, in the heated steryl oleates and linoleates the contribution of the ACOPs to the ester losses was lower (approximately 3 and 5%, respectively), and there was a more pronounced formation of oxidation products of the sterol moieties (approximately 26 and 18% of the ester losses, respectively).