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Sterol oxidation in infant milk formulas and milk cereals
Abstract
7-Ketocholesterol and 7-ketositosterol were chosen as reliable markers of the oxidation of cholesterol and phytosterols in infant milk formulas and infant milk cereals. A reversed-phase HPLC method was developed to measure them simultaneously in infant formulas. This method was then tested on a wide range of infant milk formulas and milk cereals on sale in Italy whose lipid composition is representative of the most common commercial formulas. The analytical results revealed no significant differences in the extent of oxidation of cholesterol and sitosterol. As the level of 7-ketocholesterol often followed the cholesterol level, a cholesterol content similar to that of human milk produced amounts of cholesterol oxides with possible negative effects on infant health. In contrast, the low cholesterol content of milk cereals never produced amounts of cholesterol oxides high enough to cause concern. The contents of phytosterols and hence their oxides were always low.
... Comparatively, we found total sterol concentrations of 6.46 ± 1.64 mg/100 mL of sample and 11.08 ± 2.11 mg/100 mL of IF liquid and powder, respectively. In contrast, cholesterol concentrations in IF may vary between 0.1 to 7.3 mg/g lipid (Scopesi et al., 2002;Zunin, Calcagno, & Evangelisti, 1998). Our results range from 0.3 to 2.14 mg of cholesterol per g of lipid (Table S3, Supplemental information). ...
... The copyright holder for this this version posted November 20, 2020. ; https://doi.org/10.1101/2020.11.18.20233528 doi: medRxiv preprint epoxycholesterol (5,6β-epoxy), cholestane-3β,5α,6β-triol (triol), and 25-hydroxycholesterol (25-OH) (Gorassini, Verardo, Fregolent, & Bortolomeazzi, 2017;Guardiola, Bou, Boatella, & Codony, 2004;Rodriguez-Estrada, Garcia-Llatas, & Lagarda, 2014) and 7-ketocholesterol are the most common COPs found in foods (Zunin et al., 1998). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. ...
... ;https://doi.org/10.1101https://doi.org/10. /2020 IF (Zunin et al., 1998). It is not possible to exclude that such discrepancies in COPs and other oxidative compounds between studies can be associated with the advancement in IF manufacturing process and formulation itself. ...
Approximately two-thirds of US infants receive infant formula (IF) as a primary or sole nutritional source during the first six months of life. IF is available in a variety of commercial presentations, although from a manufacturing standpoint, they can be categorized in powder-(PIF) or liquid-(LIF) based formulations. Herein, thirty commercial IFs were analyzed in their oxidative and non-oxidative lipidomics profiles. Results show that LIFs have a characteristic lipidomic fingerprint, enriched in an oxidated form of cholesterol, and a lower load of phytosterols. We identified 7-ketocholesterol-a major end-product of cholesterol oxidation-as a potential biomarker of IF manufacturing. Our data allowed re-classification of IF based on their metabolomic fingerprint, resulting in three groups assigned with low-to-high oxidative status. Finally, we modeled the dietary intake for cholesterol, sterols, and 7-ketocholesterol in the first year of life. The database provided in this study will be instrumental for scientists interested in infant nutrition, to establish bases for epidemiological studies aimed to find connections between nutrition and diet-associated diseases, such as sitosterolemia.
... In the few studies available on the profile of sterols in IF, the most abundant were found to be b-sitosterol (11-83 mg/L) and cholesterol (3-258 mg/L), followed by campesterol (7-32 mg/L), stigmasterol (3-12 mg/L), desmosterol (2.4-4.3 mg/L), brassicasterol (1-3 mg/L) and sitostanol (0.3-1.4 mg/L) (Huisman et al. 1996;Zunin et al. 1998;Scopesi et al. 2002;Maduko & Park 2007;Kamelska et al. 2011;Ramalho et al. 2011;Claumarchirant et al. 2015). ...
... The cholesterol concentrations found in our study were comparable to those reported by other authors (3-258 mg/L; 3.0-8.9 mg/100 g reconstituted IF) in bovine milk-based IFs (Huisman et al. 1996;Zunin et al. 1998;Scopesi et al. 2002;Kamelska et al. 2011;Ramalho et al. 2011;Ahn et al. 2012;Jeong et al. 2012;Claumarchirant et al. 2015) and lower than those found in caprine milk-based IFs (9.7-9.9 mg/100 g reconstituted IF) (Maduko & Park 2007) and in mature HM (46-283 mg/L) (Mellies et al. 1979;Clark et al. 1983;Kallio et al. 1989;Huisman et al. 1996;Scopesi et al. 2002;Laitinen et al. 2009;Ramalho et al. 2011;Kamelska et al. 2013;Alvarez-Sala et al. 2015). In this regard, breastfed infants have significant higher total cholesterol and LDL-cholesterol than infants fed with infant formulas (Kallio et al. 1992;Wong et al. 1993) or mixed fed (HM and IFs) (Harit et al. 2008). ...
... However, IF-2, IF-5, IF-6 and IF-7 presented cholesterol contents higher than those of b-sitosterol, and in IF-5 and IF-6 (with MFGM), the animal sterol content was higher than the total plant sterols. This tendency was found also in IFs analyzed by Zunin et al. (1998) which had higher cholesterol content than b-sitosterol. Furthermore, the sterol profile in IFs with MFGM (IF-5 and IF-6) may be closer to that of HM (animal sterols > plant sterols), though cholesterol contents in HM are generally higher than in IFs. ...
Aim:
To validate a gas chromatographic method with flame ionization detection for the determination of animal (cholesterol and desmosterol) and plant sterols (brassicasterol, campesterol, stigmasterol, β-sitosterol and sitostanol) found in IFs. All correlation coefficients obtained for the calibration curves of sterols studied were >0.99. Limits of detection (<1 μg/100 mL) and quantification (<4 μg/100 mL) are suitable for sterols determination in IFs. The within-assay precision ranged from 1.6% to 8.8%, while the between-assay precision was <10% for most of sterols. Accuracy was satisfactory and was calculated by recovery assays (ranging 93-108%). The analytical parameters obtained showed the suitability of the proposed method for the determination of sterols in IFs.
... Fatty acids in the lipid fractions were transesterified as described by Zunin et al. [23] and analyzed with a gas chromatograph (DANI, Milan, Italy) equipped with a ZB Vax column and a FID detector (Thermoscientific, Milan, Italy) [24]. The resulting chromatograms allowed identifying the different methyl ethers through the retention time and quantifying them through the peak areas. ...
The rapid development of urbanization and industrialization leads to the production of large amounts of wastewater all over the world, which could be exploited to grow microalgae, thereby reducing their cultivation costs and making biofuel production more feasible. In this research work, a co-culture of Chlorella vulgaris and Arthrospira platensis was grown in a continuous flow column membrane photobioreactor using 20% (v/v) of winery wastewater as a medium with the aim of reducing its polluting impact. Three different hydraulic retention times were investigated, namely 4.6, 2,0 and, 1.4 days. Chemical oxygen demand was reduced by more than 75%, and a biomass concentration higher than 4 g Dry Weight/L was obtained with a lipid content higher than 20 g Lipid/100 g Dry Weight. Fatty acids were mainly saturated. The results obtained suggest that C. vulgaris and A. platensis can grow in winery wastewaters, leading to a low-cost biomass production and to a reduction in the environmental impact of the industrial effluent used in this study.
... Conversely, our data is lower than that reported by others (1.2-11.2 µg/g of lipids), in a study performed on six IF (Zunin, Calcagno, & Evangelisti, 1998). It is not possible to exclude that such discrepancies in COPs and other oxidative compounds between studies can be associated with the advancement in IF manufacturing process and formulation itself. ...
Approximately two-thirds of US infants receive infant formula (IF) as a primary or sole nutritional source during the first six months of life. IF is available in a variety of commercial presentations; from a manufacturing standpoint, they can be categorized as powder- (PIF) or liquid- (LIF) based formulations. Thirty commercial IFs were analyzed in their oxidative and non-oxidative lipid profiles. We identified 7-ketocholesterol – a major end-product of cholesterol oxidation – as a potential biomarker of IF manufacturing. The statistical analysis allowed a re-classification of IF based on their metabolomic fingerprint, resulting in three groups assigned with low-to-high oxidative status. Finally, we modeled the dietary intake of cholesterol, sterols, and 7-ketocholesterol in the first year of life. The database provided in this study will be instrumental for scientists interested in infant nutrition, to establish bases for epidemiological studies aimed to find connections between nutrition and diet-associated diseases, such as sitosterolemia.
... 7-Ketositosterol, followed by 7βhydroxysitosterol, 7α-hydroxysitosterol and 5,6-epoxysitosterol were the main sitosterol oxides found in the SFO during frying. This oxidative behaviour agrees with what has already been observed in previous studies of COP (Zunin et al. 1998), where the 7-keto derivative was pointed out as a tracer of the oxidation process. In addition, 7-keto derivative was the major POP in emulsified spreads (Conchillo et al. 2005). ...
Fried foods, both deep-fried and pan-fried, are enjoyed by people worldwide. Frying is one of the main factors leading to formation of phytosterols (PS) oxidation products (POP) in vegetable oils. The aim of this study was to measure the oxidation of β-sitosterol (24α-ethyl-5-cholesten-3β-ol) and campesterol (24α-methyl-5-cholesten-3β-ol) in commercial sunflower oil (SFO) during deep- and pan-frying of French fries for different periods (30, 60, 120 and 240 min). The total amount of PS in SFO was 4732 μg/g, wherein the major PS were β-sitosterol and campesterol. The results of POP were confirmed by the GC-MS analysis that monitored the formation of oxides during frying. Upon frying, total PS content decreased whereas the highest decrease was measured after 240 min of frying. The oxidative stability (OS) of different sitosterol and campesterol during both frying methods was evaluated. In general, pan frying resulted in more PS oxidation than deep frying. β-Sitosterol oxides predominated while campesterol oxides were formed to a lesser extent. 7-Ketositosterol, followed by 7β-hydroxysitosterol, 5,6-epoxy derivatives and 7α-hydroxysitosterol were the main POP induced during frying. The proportion of 7-keto derivatives decreased during frying while the proportion of 7β-hydroxy derivatives increased. The formation of POP might be a limiting factor for frying in SFO for long periods.
... These trends are similar to the absorption of non-oxidized phytosterols, implying that increasing the side chain length of either the PS or POPs, decreased their absorption and that the type of oxidation relates to the degree of absorption(Ryan et al., 2009). POPs may be related to the inflammation processes, dyslipidemia, atherosclerosis, apoptosis, and cell toxicity(Alemany et al., 2014;Zunin, Calcagno, & Evangelisti, 1998).Adcox, Boyd, Oehrl, Allen, and Fenner (2001) demonstrated that POPs affect protein synthesis and damage the cell membrane, while measuring total protein content and LDL leakage(Adcox et al., 2001). Recently, it has been hypothesized that due to the structural similarity between POPs and cholesterol oxidation products (COPs), POPs could potentially contribute to the onset of metabolic and neurologic diseases by an irreversible accumulation in the central nervous system. ...
Infant formulations are enriched with vegetable oils that confer not only calories, but also peculiar chemical attributes. Vegetable oils are particularly rich in phytosterols, a class of triterpene molecules analogous to cholesterol, but with different and largely unknown biological effects. The preparation and sterilization of infant formula provide opportune physicochemical conditions for oxidative reactions to occur. Oxidation of phytosterols during the preparation of infant formula can led to oxidized derivatives, known as phytosterol oxidation products [POPs], which harmful effects can be exacerbated given the wide variety of infant formulas characterized by their exclusivity of milk surrogates, required to fulfill specific needs in the nutritional development of a baby. In this review, the state‐of‐the‐art regarding phytosterols and their presence in infant formulation is revised, stressing the need of further investigation in the field of food processing. Reconsidering infant formula manufacturing in the context of phytosterols oxidation will lead to several opportunities for food engineers and technologists in the food safety.
Practical applications
Phytosterols are plant‐based bioactive lipids with health benefits. Therefore, they have been implemented as a supplement in infant formula through the addition of vegetable oils. Processing, packaging, and storage contribute to the oxidative process of these compounds. A surveillance of the entire food chain is needed to reduce the oxidative load in the final product.
This paper deal with the main aspects of the analysis of oxysterols. This analytical problem is quite new and many questions involved are still controversial. Consequently, many methodologies exist that still need improvement and standarization. Therefore, it is interesting to discuss the analytical methodologies proposed by different authors for the extraction, purification and determination (gas chromatography and high performance liquid chromatography) of oxysterols.
Sitosterylstearat wurde 1 Stunde bei 150°C im Sauerstoff-Strom oxidiert. Nach dünnschicht-chromatographischer Trennung der Oxidationsprodukte erfolgte deren Identifizierung. Im Gegensatz zur Oxidation des freien Sitosterins unter gleichen Bedingungen fehlen hier alle Oxidationsprodukte, bei denen die 3β-Hydroxigruppe zur Ketogruppe oxidiert und die Δ5 Doppelbindung nach Δ4 verlagert ist. Auch die Bildung des 6-Hydroperoxids, das bei der Oxidation des freien Sterins eines der wichtigsten Primärprodukte ist, wurde nicht beobachtet. Dafür treten in großer Menge die 5,6-Epoxide auf, auch mit zusätzlicher Oxidation in 7-Stellung.Autoxidation of Sitosterol III: SitosterylstearateSitosterylstearate was oxidized in a stream of oxygen for one hour at 150°C. The oxidation products were separated by thin-layer chromatography and identified. In contrast to oxidation of free sitosterol under identical conditions which yielded oxidation products, in which the 3β-hydroxy group was oxidized to a keto group and the Δ5 double bond was displaced to Δ4, none of these products were formed by the oxidation of β-sitosteryl ester. Moreover, the formation of 6-hydroperoxide, an important primary product of oxidation of the free sterol, was not observed. However, large amounts of the 5,6-epoxide are formed, also with additional oxidation in the 7-position.
Cholesterol is an important constituent of animal food products and has often been implicated in the etiology of atherosclerosis and coronary heart diseases. Recent reports have, however, shown the possible role of cholesterol oxidation products (COP), rather than cholesterol, in the initiation of atherosclerotic plaque formation. Cholestan-3β,5α,6β-triol and 25-hydroxycholesterol have been reported as the most potent atherogenic agents. Inhibition of HMG-CoA reductase (EC 1.1.1.34) enzyme activity and cholesterol biosynthesis by cholesterol oxidation products has also been thoroughly investigated in cultured cells. Various animal food products, viz meat products, egg products and dairy products (especially butter, butter oil, ghee, cheese etc) have been reported to contain various COP developed during certain processing treatments. The literature on the presence of COP in food products and their cytotoxic and atherogenic effects is reviewed.
High performance liquid chromatography of oxysterols of current biochemical and biomedical interest is reviewed.
The quantity of blocked lysine in spray-dried infant formulas in Italy was calculated by the furosine method. This quantity was then correlated with the composition of the carbohydrate fraction. In formulas containing only lactose as carbohydrate its presence in large quantities (6.7–7.5 g/100 mL of formula as fed), with a lysine-rich protein fraction, may lead to amino acid blocking of >20%. In formulas in which lactose was substituted with low-DE maltodextrin a reduction of lysine blocking was possible. Addition of glucose may result in high levels of blocked lysine.
The degree of cholesterol oxidation in commercial sweet baked foods (biscuits and snacks) and in laboratory baked biscuits, all containing fresh or powdered eggs, was determined. 7-Ketocholesterol was used as index of cholesterol oxidation and detected by two analytical methods. The analysis of the biscuits showed higher levels of 7-ketocholesterol and a more marked oxidative instability of cholesterol when prepared with powdered eggs. The significant amounts of 7-ketocholesterol found in some samples of commercial biscuits were attributed to the use of powdered eggs. These data are of importance to industries using eggs in sweet baked products which are mainly consumed by children.
As a class of compounds, oxysterols have demonstrated a wide variety of biological properties. Due to the general interest
in these compounds, new methods of chemical synthesis have been developed to provide them for biological investigation. The
specific inhibition by oxysterols of cholesterol biosynthesis in mammalian cells has been shown to result primarily from a
decrease in cellular levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. Recent evidence suggests
these cellular responses may be mediated by an oxysterol binding protein found in the cytosol of many lines of cultured cells.
In certain instances, oxysterols have been shown to be produced in biological systems. These results support the supposition
that oxysterols may regulate sterol biosynthesis at the cellular level. Included herein are the inhibitory effects of 9α,
11α-epoxycholest-7-en-3β-ol cholest-8-en-3β-ol-7-one and cholest-8-en-3β-ol-11-one on HMG-CoA reductase activity and their
relative affinities for a cytosolic binding protein.
Soybean oil and wheat flour were analyzed for the content of sitosterol oxides. The method involved chromatography on a Lipidex-5000
column and enrichment on a disposable NH2-column, yielding a sterol fraction and a sterol oxide fraction. Each fraction was separated as trimethylsilyl-ethers on a
methyl silicone capillary column. Analysis of crude and freshly refined soybean oil showed no detectable levels of the isomeric
5,6-epoxysitosterols, the epimeric 7-hydroxysitosterols and 5,6-dihydroxysitosterol at the detection limit of 0.2 ppm. Storage
of a refined soybean oil for one year at 4°C caused no significant increase in the level of free sitosterol oxides when compared
to the freshly refined soybean oil. Analysis of three wheat flours (at 2, 8 and 36 months) revealed that the samples contained
variable levels of 5α,6α-epoxysitosterol (5.4–55 ppm in the lipids), 5β,6β-epoxysitosterol (0.2–29 ppm), 7α-hydroxysitosterol
(9.3–118 ppm) and 7β-hydroxysitosterol (9.7–126 ppm).
Two methods for routine analysis of free 7-ketocholesterol (7-k) in foods by high-performance liquid chromatography (HPLC)
were compared. The analyzed samples were egg noodles, biscuits prepared with eggs, sweet snacks, grated Parmesan cheeses,
and some ingredients utilized in the food industry (whole-milk and whole-egg powder). The enrichment of cholesterol oxides
was carried out by solid-phase separation of the total lipids with florisil and silica cartridges for methods A and B, respectively.
The 7-k analyses were run in normal-phase HPLC for method A, and a reverse-phase process was used in method B. Identification
of the 7-k peak was confirmed by gas chromatography/mass spectrometry analysis and peak purity checkvia spectral analysis with a diode array detector. The quantitation of 7-k was carried out with an internal standard for method
A and with a calibration curve for method B. The limit of quantitation (LQ) was 3 × 10−9 g/injection; the limit of detection was ten times lower than the LQ for both methods. The two methods showed good recovery
(99%) and good repeatability (coefficient of variation of 3.9 and 3.7% for methods A and B, respectively). These methods allow
a fast, sensitive, and reliable determination of one cholesterol oxidation product, which is present at a high concentration
level in the first stages of the oxidation process.
To clarify conflicting information regarding 7-ketocholesterol (7-KC) recovery from saponification, we evaluated the stability
of 7-KC in methanolic alkaline medium. We added 1 N alcoholic KOH to 7-KC or lard spiked with 7-KC and held the mixtures at
45, 55, 65, and 75°C for different times to simulate various saponification conditions. Gas-chromatographic determination
of residual 7-KC with 5α-cholestane as the internal standard (IS) showed that the higher the saponification temperature, the
greater the 7-KC degradation. Hot saponification at 75°C for 30 min caused extensive destruction and left only 31% 7-KC. 7-KC
loss during saponification could be described by pseudo first-order kinetics, and the dependence of degradation rate on temperature
(T, K) byk (h−1)=(2.6×1017) exp (−1.36×104/T). As predicted by the kinetic equation, 7-KC loss during room-temperature saponification (21°C) was practically negligible;
following the exposure of 7-KC or lard spiked with 7-KC to 1 N alcoholic KOH for 18 h, about 97% 7-KC was recovered. 6-Ketocholestanol,
when used as an IS, should be looked at carefully because of potential tautomerization, leading to the formation of two enol
isomers when in extended contact with trimethylsilyl derivatization reagents.