Milk liposoluble components and their effects on human health

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Metabolism of lipids, vitamin A, and cholesterol in dairy ruminants

Milk fat consists predominantly of triacylglycerides (TAG) (> 95% of total milk lipids) containing about 500 individual fatty acids (FA), most of which are present in amounts of <1% of total lipids [26,27]. Only saturated FA (SFA) of chain lengths from 4 to 18 carbon atoms, cis-9-16:1, oleic acid (cis-9-18:1), trans-18:1, and linoleic acid (18:2 n-6) are present in amounts greater than 1% in milk fat [26]. Along with TAG, the lipid composition of milk comprises small amounts of diacylglycerides (DAG), monoacylglycerides (MAG), free fatty acids (FFA), phospholipids (PL), glycolipids and sterols (cholesterol esters, lanosterol, dihydrolanosterol, and 7-dehydrocholesterol). Minor lipids include waxes, carotenoids, liposoluble vitamins (A, D, E, K), and lipoproteins [16].
The lipids in milk occur in the form of globules, comprising a core of TAG and small amounts of cholesteryl esters, FFA and retinol esters, surrounded by a thin membrane [28]. The membrane called the milk fat globule membrane (MFGM) contains primarily phospho- and sphingolipids and membrane-specific proteins [29]. The diameter of the fat globules ranges from <1 to about 10 μm [26]. Some studies reported that the average fat globule size is smaller in goat milk (< 3.5 μm) than in cow milk (~4 μm) [30,31]. This characteristic supports the hypothesis that goat milk fat has a higher digestibility than cow milk fat [30].

Origins of milk fatty acids

Rumen lipid metabolism

Diets consumed by ruminants generally contain between 20 and 40 g lipid/kg dry matter (DM), with a high proportion of polyunsaturated FA (PUFA) [32]. The predominant PUFA in ruminant diets are linoleic acid (LA, 18:2 n-6 or cis-9,cis-12-18:2) and linolenic acid (ALA, 18:3 n-3 or cis-9,cis-12,cis-15-18:3), derived from forages, cereals, and oilseeds. Moreover, some oilseeds provide monounsaturated FA (MUFA) (mainly cis-9-18:1 from rapeseed oil), whereas marine products (fish oil, algae) provide long-chain PUFA (mainly 20:5 n-3 (eicosapentaenoic acid, EPA) and 22:6 n-3 (docosahexaenoic acid, DHA)) [18].
On entering the rumen, hydrolysis of the ester linkages found in TAG, PL, and glycolipids is the initial transformation dietary lipids undergo [33]. Following this lipolysis carried out by microbial and plant lipases, nonesterified FA (NEFA) are released into the rumen and adsorbed onto feed particles and hydrogenated or incorporated directly into bacterial lipids [21,28]. The second major step in dietary lipids metabolism is the ruminal biohydrogenation (RBH) of unsaturated NEFA [33]. For most diets, RBH averages 80% for LA and 92% for ALA [34]. The major pathways of RBH have been established as a result of numerous in vitro and in vivo studies. Metabolism of LA and ALA starts with the isomerisation of the cis-12 double bond and the formation of a conjugated 18:2 or 18:3 FA, respectively. Conjugated products are further hydrogenated into trans-11-18:1 and then into 18:0 as the final end product [35]. The final hydrogenation step is considered to be rate-limiting and therefore trans-18:1 intermediates can be accumulated and then flow out of the rumen [32]. RBH of dietary PUFA results in the formation of numerous FA intermediates that following formation in the rumen can be incorporated into milk fat [28]. The occurrence of a wide range of isomers of trans-18:1, 18:2, and 18:3 FA containing one or more trans double bonds suggests that the metabolic pathways of RBH are much complex than previously thought. More recent in vitro and in vivo studies have provided additional data regarding possible biochemical pathways accounting for the formation of specific intermediates during the metabolism of LA (Fig. 1) and ALA (Fig. 2) [32]. Fig.1. Putative pathways describing linoleic acid (LA, cis-9,cis-12-18:2) metabolism in the rumen. Arrows with solid lines highlight the major biohydrogenation pathway, whereas arrows with dashed lines describe the formation of minor fatty acid metabolites [32]
Regarding the RBH of oleic acid (cis-9-18:1), this FA is often shown to form directly stearic acid (18:0) [36]. However, more recent in vitro studies reported that cis-9-18:1 metabolism results in the formation of hydroxystearic (10-OH 18:0) and ketostearic (10-O 18:0) acids and multiple trans-18:1 intermediates with double bond positions from C6 to C16 [37,38]. Cis-9-18:1 RBH typically varies between 58% and 87% [32]. Moreover, RBH also occurs on 20- and 22-carbon FA having more than 3 double bonds, such as EPA and DHA in fish oil and marine algae. The RBH of these FA is extensive, but generally they do not become fully saturated [39]. Incubation of EPA (cis-5,cis-8,cis-11,cis-14,cis-17-20:5) and DHA (cis-4,cis-7,cis-10,cis-13,cis-16,cis-19-22:6) in cultures of mixed ruminal microorganisms caused the disappearance of these two FA as well as the accumulation of trans-18:1 [40]. If consistent with pathways for LA and ALA RBH, the initial isomerisation of EPA and DHA should produce isomers with five and six double bonds, including at least one trans double bond. Isomerisation should be followed by hydrogenation to isomers with four and five double bonds [32,36]. However, more research is required to elucidate the biochemical pathways of EPA and DHA rumen metabolism.

Microorganisms involved in rumen biohydrogenation

RBH involves only some species of rumen microorganisms, carrying out this process as a means of protection against the toxic effects of PUFA on microbial growth [41,42]. Several studies have shown that within the rumen microbial population, bacteria are mainly responsible for RBH when compared to protozoa and anaerobic fungi [36]. LA and ALA metabolism involves two groups of ruminal bacteria: Group A, which hydrogenates PUFA to trans-18:1 FA, and Group B, which hydrogenates trans-18:1 FA to 18:0 [35]. Nevertheless, more recent studies have reported that cellulolytic bacteria from Butyrivibrio group are of principal importance in RBH. Butyrivibrio fibrisolvens was identified to produce cis-9,trans-11-18:2 and trans-11-18:1 from LA, whilst is does not form 18:0 [36]. To the present, the rumen bacteria identified as having the capacity to produce 18:0 are Butyrivibrio hungatei and Clostridium proteoclasticum, reclassified as Butyrivibrio proteoclasticus [41,43].
The contribution of protozoa to RBH has been suggested to be due to the activity of ingested or associated bacteria [36]. However, recent data indicate that ruminal protozoal cells contain proportionally more cis-9,trans-11-18:2 and trans-11-18:1 than ruminal bacteria. The most likely explanation is that protozoa do not form these FA, but play an important role in the uptake/protection of the intermediates of bacterial RBH [44]. Moreover, an in vitro study demonstrated that rumen fungi have the ability to biohydrogenate LA, with Orpinomyces fungus being the most active. RBH is slower in fungi than in bacteria and has trans-11-18:1 as the end product [45].

Effect of diet on rumen biohydrogenation

Diet composition (nature of forage, forage:concentrate ratio), the level and type of lipids in diet, and interactions between these factors have an important influence on the predominant RBH pathways resulting in changes in the profile of FA available for absorption and incorporation into milk fat [28]. In this regard, the extent of RBH is mainly dependent on the percentage of concentrate in the diet. When concentrate exceeds 70%, RBH is strongly reduced [34]. This phenomenon is probably due to a decrease in rumen pH, which normally ranges between 6.0 and 6.7 [41]. At a pH < 6, rumen lipolysis has been reported to be low [46]. Low rumen pH has also been shown to have a negative effect on microbial growth, especially on the growth of cellulolytic bacteria [47]. It is well known that cellulolytic bacteria, the main ruminal biohydrogenating bacteria, are sensitive to acidic condition (pH < 6) in the rumen [48].
Besides diet composition, the nature of dietary FA is a major factor in the variation of RBH. The addition of plant oils or oilseeds rich in LA and ALA to ruminant diet is reported to lead to incomplete metabolism of dietary PUFA into 18:0, with the accumulation of trans-18:1 intermediates [18]. Supplementation of diet with fish oil rich in EPA and DHA is also reported to inhibit the complete RBH of 18 PUFA, causing an increase in the ruminal outflow of trans-18:1 at the duodenum [49]. This effect may involve alterations in total ruminal bacteria and Butyrivibrio populations, probably related to the toxicity of PUFA on rumen bacteria [50]. Fish oil has been shown to be a more potent inhibitor of the hydrogenation of trans-18:1 intermediates to 18:0 in the rumen than plant oils and oilseeds [28].
However, differences in milk FA composition responses when lipid supplement are fed also occur as a consequence of the composition of the basal diet [4]. In this respect, previous studies provided evidence that low-forage/high-concentrate diets supplemented with plant oils rich in PUFA are characterized by a shift in RBH towards trans-10-18:1 at the expense of trans-11-18:1 [51]. Likewise, on high-concentrate diets with marine lipids supply, trans-10-18:1 has often been reported to replace trans-11-18:1 as the major trans FA in milk fat [52].

Mammary lipogenesis

Milk FA originate from two sources: the uptake from circulation of preformed FA (ca. 60%) and de novo synthesis within the mammary gland (ca. 40%) [39]. Precursors for de novo FA synthesis are acetate and butyrate, volatile FA (VFA) produced during microbial fermentation of cellulose and hemicellulose in the rumen [21,39]. Butyrate is converted to β-hydroxybutyrate in the rumen wall [26]. Acetate and β-hydroxybutyrate are used by mammary gland for the synthesis of 4:0 to 12:0 FA, most of myristic acid (14:0) (ca. 95%) and about a half of palmitic acid (16:0) in milk fat [53]. The remaining 16:0 and all of the long-chain FA (LCFA) derive from mammary uptake of circulating TAG-rich lipoproteins (very low-density lipoproteins (VLDL) and chylomicrons (CM)), and plasma albumin bound NEFA that arise from intestinal absorption of lipids and body fat mobilization [32,54]. Mammary lipoprotein lipase (LPL) allows TAG hydrolysis and NEFA uptake by the mammary gland [20].
Milk fat contains also odd- and branched-chain FA (OBCFA), which are largely synthesized de novo by rumen bacteria [11]. Odd-chain FA are synthesized through elongation of propionate or valerate. Branched-chain FA are formed from their precursors: the branched-chain amino acids (valine, leucine and isoleucine) and the branched-short-chain carboxylic acids (isobutyric, isovaleric and 2-methyl butyric acids) [55].
De novo synthesis of milk FA involves two key enzymes: acetyl-CoA carboxylase (ACC) and fatty acid synthetase (FAS) [20]. Acetate and β-hydroxybutyrate contribute equally to the initial four carbon unit. Acetate is converted to acetyl-CoA and used to extend the chain length of synthesized FA via the malonyl-CoA pathway, whereas β-hydroxybutyrate is incorporated directly following activation to butyryl-CoA [32]. ACC catalyses the formation of malonyl-CoA from acetate, and FAS catalyses condensation cycles of malonyl-CoA with either acetyl-CoA or butyryl-CoA [39]. LCFA containing 16 or more carbon atoms are known to lower mammary FA synthesis in bovine or caprine mammary epithelial cells in vitro due to direct inhibitory effects on ACC. The inhibitory effects have shown to be more potent when FA contain a longer carbon chain and/or have a higher degree of unsaturation [56]. This phenomenon explains the decrease in the concentrations of medium-chain FA (MCFA) in milk fat following an increase in the supply of LCFA to the mammary gland either from the diet, or from body fat mobilisation [39].
In ruminants, FA in milk fat that are taken up from circulation are derived mostly from the digestive absorption of dietary and microbial FA [57]. When reaching the intestine, these FA are usually in the unesterified form. They are absorbed in the duodenum, esterified in the enterocyte, and used in conjunction with PL and cholesterol esters in the assembly of VLDL and CM that pass into the peripheral blood [58]. Before esterification, 18:0 can be desaturated to cis-9-18:1 within the enterocyte, but only to a limited extent [59]. The remainder of the circulating FA originates from the mobilisation of body fat reserves, which typically accounts for less than 10% of milk FA [52]. Nevertheless, the contribution from mobilized FA increases when cows are in early lactation and/or in negative energy balance [54]. As 18:0 and cis-9-18:1 are the main FA stored in ruminant adipose tissue, body fat mobilisation induces a sharp increase in these FA concentrations in milk [60].
LCFA entering the mammary secretory cells can be desaturated, whereas preformed FA cannot undergo elongation (e.g. 16:0 to 18:0) within the mammary gland [20]. Mammary secretory cells contain the stearoyl-CoA desaturase (SCD) complex, also known as Δ-9 desaturase, an enzyme that acts by adding a cis double bond between carbon atoms 9 and 10 of the FA chain [28]. Δ-9 desaturase activity in the ruminant mammary gland is assumed to occur as a mechanism to ensure the liquidity of milk for efficient utilization by the offspring [61]. In this respect, the mammary gland converts 18:0 into cis-9-18:1 and contributes to 60% to 80% of all the oleic acid secreted in milk [53,62]. Likewise, the activity of Δ-9 desaturase is estimated to contribute to 90% of cis-9-14:1 and 50% of cis-9-16:1 in milk fat [63]. Other FA shorter than 18 carbon chain length, such as 10:0, 12:0, 14:0, 15:0, and 17:0, can also be used as substrates for Δ-9 desaturase [32]. Moreover, it is estimated that 25% of the vaccenic acid (trans-11-18:1) formed in the rumen is desaturated in the mammary gland to rumenic acid (cis-9,trans-11-18:2), the main isomer of conjugated linoleic acid (CLA) in milk [64]. Endogenous synthesis in the mammary gland from trans-11-18:1 is responsible for 70% to 95% of the milk cis-9,trans-11-18:2 [53].
The above mentioned metabolic pathways (de novo synthesis, uptake from circulation and desaturation) allow the formation of a pool of FA further used to form TAG through glycerol esterification [20]. The central carbon atom of TAG (sn-2) shows chirality, resulting in an asymmetrical TAG molecule, if two different FA are in the primary positions (sn-1 and sn-3) of the molecule [31]. The distribution of FA within TAG synthesis is not random. 8:0, 10:0, 12:0, and 14:0 FA are preferentially esterified at sn-2 position, 18:0 is preferentially esterified at sn-1 position, whereas the distribution of 16:0 between sn-1 and sn-2 positions is equal. Short-chain FA (SCFA) (4:0 and 6:0) and cis-9-18:1 are more abundant in the sn-3 position of TAG [26]. FA asymmetrical distribution on the glycerol molecule, as for example the preferential esterification of SCFA and oleic acid on the sn-3 position, influences the physical properties of milk fat. It decreases milk fat melting point at or below the body temperature of the cow (39°C), thus ensuring its fluidity [61].

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Milk fat depression

Feeding cow diets containing rations rich in readily digestible carbohydrates and poor in fibrous components and supplemented with plant oils, or marine oils can result in a decrease in milk fat content and yield but also in changes in milk FA composition. This phenomenon is commonly referred to as milk fat depression (MFD) [57]. Decreases in milk fat secretion during diet-induced MFD often occur within a few days, and in more severe cases milk fat yield can be lowered by up to 50% [52].
In contrast to cow, MFD is not frequently reported in goat, even when diets providing large amounts of starch and supplemented with plant oils or marine oils are fed [60]. Moreover, diets causing MFD in cow usually increase milk fat secretion in goat [18]. The differences between bovine and caprine regarding the impact of diet composition on milk fat synthesis appear to be attributed to differences between these two ruminant species in rumen lipid metabolism and mammary gland sensitivity towards components with anti-lipogenic activity [32,65].
Several theories have been proposed to explain the causes of diet-induced MFD. Owing to the importance as a substrate for de novo FA synthesis in the mammary gland, one theory attributes diet-induced MFD to a decrease in acetate and butyrate supply [57]. In this respect, previous studies reported that continuous ruminal infusions of acetate and butyrate increase milk fat concentration and yield [66]. However, these data indicate only that increases in acetate and butyrate supply stimulate mammary lipogenesis, which does not necessarily prove that MFD is caused by a deficit of precursors for the mammary FA synthesis. Diets causing MFD, such as high-concentrate diets, frequently induce variations in molar proportions of VFA in the rumen, but they also induce alterations in ruminal outflow of RBH intermediates and end products [67,68]. Since trans FA as RBH intermediates are shown to exert anti-lipogenic effects, discrimination between the contribution of decreases in acetate and butyrate supply and increases in ruminal trans FA outflow in the occurrence of diet-induced MFD is difficult [52].
The second theory proposed to explain MFD is the glucogenic-insulin theory, which supports that increased rumen production of propionate and enhanced circulating glucose levels cause an increase in circulating insulin concentrations. Elevated insulin secretion induces further a deficit of precursors for mammary synthesis of milk fat, as it stimulates the partitioning of FA towards adipose tissue rather than mammary gland [54]. One approach to examine the glucogenic-insulin theory has involved providing exogenous propionate and glucose through continuous intraruminal infusions. This experiment reported a decrease in milk fat concentration and yield, which was attributed to an increase in insulin secretion [66]. Nevertheless, results from studies using hyperinsulinemic-euglycemic clamps do not support the glucogenic-insulin theory of diet-induced MFD and indicate that decreases in milk fat content previously observed with propionate and glucose infusions are most likely due to the capacity of insulin to inhibit lipolysis, therefore limiting the availability of preformed FA mobilised from body fat stores [69].
The most recent theory trying to elucidate the mechanisms underlying diet-induced MFD is the biohydrogenation theory, which appears to offer a more plausible explanation for MFD over a wide range of diets. The biohydrogenation theory states that mammary synthesis of milk fat is inhibited directly by specific trans FA formed during RBH of dietary PUFA [54]. This theory is supported by studies showing that trans-10,cis-12-CLA, an intermediate in LA rumen metabolism, is a potent inhibitor of milk fat synthesis in lactating cows [70]. Further research has established that abomasal infusion of trans-10,cis-12-CLA decreases milk fat synthesis in the lactating cow in a dose-dependent curvilinear manner [71].
Moreover, several studies reported that diet-induced MFD can also occur in the absence of or after relatively small increases in milk fat trans-10,cis-12 CLA content, suggesting that other intermediates of RBH may also inhibit milk fat synthesis [52]. In this regard, diet-induced MFD is consistently associated with increases in milk fat trans-10-18:1 concentration [51,72]. However, abomasal infusion of trans-10-18:1 in lactating cows was found to increase the concentration of this trans FA in milk, whereas it had no effect on milk fat secretion, offering therefore little support for the anti-lipogenic effect of trans-10-18:1 [73]. Furthermore, post-ruminal infusion experiments provided evidence that cis-10,trans-12-CLA and trans-9,cis-11-CLA inhibit milk fat synthesis, but the collective ruminal outflow of these intermediates in LA rumen metabolism does not fully explain the milk fat decreases observed during MFD [74,75].
Nevertheless, to the present, the biohydrogenation theory is considered to be the most robust of all the theories trying to explain diet-induced MFD [32]. Yet, further studies are required to identify and characterise the anti-lipogenic effect of different RBH intermediates, as well as mechanisms other than direct inhibition that could be involved in diet-induced MFD [52]. An example of such a mechanism would be the decrease in the availability of 18:0 for mammary cis-9-18:1 synthesis reported to occur when PUFA-rich lipid supplements, particularly fish oil, inhibit the hydrogenation of trans-18:1 isomer. Reduced 18:0 and associated increases in trans-18:1 have been suggested to inhibit milk fat secretion due to incapacity of the mammary gland to maintain an adequate milk fat fluidity [76].

Origins of milk vitamin A

Vitamin A in milk has multiple origins. It may be derived from ruminant diet, more precisely from forages, concentrates (cereals, oilseeds) and/or mineral-vitamin supplements [21]. The distribution of a mineral-vitamin supplement is recommended as it has been demonstrated that ruminants requirements for vitamin A are in general not entirely covered by diet, except for pasture diet [77]. Ruminants are also able to synthesize vitamin A from precursors in diet. Thus, vitamin A is formed in the enterocytes by enzymatic hydrolysis of various isomers of β-carotene. The isomer all-trans is the major form, because it has the highest concentration in food and it is the best enzyme substrate [21]. Moreover, studies on lactating dairy cows indicated that β-carotene conversion into vitamin A may also occur in the mammary gland [78].

Origins of milk cholesterol

Despite the extensive knowledge in humans, cholesterol metabolism in ruminants is nowadays still poorly documented. Milk cholesterol may be derived from mammary de novo synthesis from acetate, but the amount of milk cholesterol synthesised in the mammary gland has been estimated to represent only 20% of the total [79,80]. Nevertheless, studies conducted to investigate the origin of milk cholesterol in ruminants suggested that this liposoluble component in milk is derived principally from the uptake of serum cholesterol. Furthermore, serum cholesterol originates mainly from synthesis within the liver [8,79].

Table of contents :

INTRODUCTION 
REVIEW OF THE LITERATURE
1. Metabolism of lipids, vitamin A, and cholesterol in dairy ruminants
1.1. Origins of milk fatty acids
1.1.1. Rumen lipid metabolism
1.1.1.1. Microorganisms involved in rumen biohydrogenation
1.1.1.2. Effect of diet on rumen biohydrogenation
1.1.2. Mammary lipogenesis
1.1.3. Milk fat depression
1.2. Origins of milk vitamin A
1.3. Origins of milk cholesterol
2. Factors affecting milk fatty acid, vitamin A, and cholesterol composition
2.1. Breed
2.2. Species
2.3. Stage of lactation
2.4. Diet
2.4.1. Influence of nature of forage
2.4.2. Influence of diet supplementation with lipids
3. Milk liposoluble components and their effects on human health
3.1. Milk fat digestion
3.2. Milk fatty acids and their health effects
3.2.1. Butyric acid
3.2.2. Caproic, caprylic, and capric acids
3.2.3. Lauric, miristic, palmitic, and stearic acids
3.2.4. Odd- and branched-chain fatty acids
3.2.5. Oleic acid
3.2.6. Trans fatty acids
3.2.7. Conjugated linoleic acid (CLA)
3.2.8. Conjugated alpha-linolenic acid (CLnA)
3.2.9. n-3 and n-6 fatty acids
3.2.10. Antimicrobial fatty acids
3.3. Milk vitamin A and its health effects
3.4. Milk cholesterol and its health effects
PERSONAL CONTRIBUTIONS
1. Aims
2. Study 1. Influence of calf presence during milking on fatty acid profile and lipolytic system of milk in Prim’Holstein and Salers cow breeds
2.1. Introduction
2.2. Aims
2.3. Materials and methods
2.3.1. Cows and diets
2.3.2. Sampling, measurement and analyses
2.3.3. Statistical analyses
2.4. Results
2.4.1. Milk yield and composition
2.4.2. Milk fatty acid profile
2.4.3. Milk lipolysis
2.5. Discussion
2.5.1. Milk yield and composition
2.5.2. Milk fatty acid profile
2.5.3. Milk lipolysis
2.6. Conclusions
3. Study 2. The effects of calf presence during milking, cow parity and season on milk fatty acid composition and the lipolytic system in Salers cows
3.1. Introduction
3.2. Aims
3.3. Materials and methods
3.3.1. Cows and diets
3.3.2. Sampling, measurement and analyses
3.3.3. Statistical analyses
3.4. Results
3.4.1. Characteristics of animals, milk yield and composition
3.4.2. Milk fatty acid composition
3.4.3. Milk lipolytic system
3.5. Discussion
3.5.1. Milk yield and composition
3.5.2. Milk fatty acid composition
3.5.3. Milk lipolytic system
3.6. Conclusions
4. Study 3. Fatty acid, vitamin A, and cholesterol concentrations and oxidative stability in milk fat from Carpathian goats fed alfalfa hay-based diets supplemented with hemp seed oil
4.1. Introduction
4.2. Aims
4.3. Materials and methods
4.3.1. Goats and diets
4.3.2. Sampling, measurement and analyses
4.3.3. Statistical analyses
4.4. Results
4.4.1. Animal performance
4.4.2. Milk fatty acid concentrations
4.4.3. Milk vitamin A and cholesterol concentrations
4.4.4. Milk MDA concentration
4.5. Discussion
4.5.1. Animal performance
4.5.2. Milk fatty acid concentrations
4.5.3. Milk vitamin A and cholesterol concentrations
4.5.4. Milk MDA concentration
4.6. Conclusions
5. General conclusions
6. Originality and innovative contributions of the thesis
REFERENCES

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