Retinyl stearate

This method is also used to measure ex vivo low-density lipoprotein (LDL) oxidation. LDL is isolated fresh from blood samples, oxidation is initiated by Cu(II) or AAPH, and peroxidation of the lipid components is followed at 234 nm for conjugated dienes (Prior and others 2005). In this specific case the procedure can be used to assess the interaction of certain antioxidant compounds, such as vitamin E, carotenoids, and retinyl stearate, exerting a protective effect on LDL (Esterbauer and others 1989). Hence, Viana and others (1996) studied the in vitro antioxidative effects of an extract rich in flavonoids. Similarly, Pearson and others (1999) assessed the ability of compounds in apple juices and extracts from fresh apple to protect LDL. Wang and Goodman (1999) examined the antioxidant properties of 26 common dietary phenolic agents in an ex vivo LDL oxidation model. Salleh and others (2002) screened 12 edible plant extracts rich in polyphenols for their potential to inhibit oxidation of LDL in vitro. Gongalves and others (2004) observed that phenolic extracts from cherry inhibited LDL oxidation in vitro in a dose-dependent manner. Yildirin and others (2007) demonstrated that grapes inhibited oxidation of human LDL at a level comparable to wine. Coinu and others (2007) studied the antioxidant properties of extracts obtained from artichoke leaves and outer bracts measured on human oxidized LDL. Milde and others (2007) showed that many phenolics, as well as carotenoids, enhance resistance to LDL oxidation. 

Fio. 35. HPLC record of a standard mixture of retinol and retinyl esters. Conditions column, 10 un octadecyl silica flowrate, I ml/min . mobile phase, (A) CHtOH/58.9 x I0- Af [Ag ], (B) CHsOH/23.5 x lO" W [Ag ], (C) CH,OH detection 330 hm. Peak identity (I) retinol (2) retinyl propionate (3) retinyl linoleate (4) retinyl lauratc (S) retinyl oleate (6) retinyl myristate (7) retinyl palmitate and (8) retinyl stearate. Reprinted with permission from DeRuyter and DeLeenheer (242), Anal. Chem. Copyright 1979 by the American Chemical Society. 

Fig. 2.15. Relative importance of plasma antioxidants. Time-course of the consumption of antioxidants during oxidation of plasma low-density lipoproteins (a) tocopherols, (b) lycopene, (c) retinyl stearate, (d) /5-carotene. (Adapted from Esterbauer et al., 1989.)...

Reversed-phase packing materials (Cig and Cg) are utilized especially for the separation of the different retinyl esters and for combined vitamin A and carotenoid mixtures such as those in food extracts and clinical samples. Most semiaqueous mobile phases, consisting of mixtures of methanol or acetonitrile and water, result in chromatographic runs of more than 1 h to elute retinyl stearate. Gradient elution shortens this elution time but results in a loss of separation of two predominant esters, retinyl palmitate and retinyl stearate. More recently, isocratic non-aqueous reversed-phase (NARP) conditions with an acetonitrile-dichloromethane mixture resulted in a successful separation of the different esters within 15 min. The big advantage of NARP conditions over semiaqueous systems is the increased solubility of lipids. To compensate for this increased affinity of the compounds of low polarity for the eluent, highly retentive stationary phases are recommended. 

Waters ODS column (1.2 x 10 cm) at 2 mL/mm with a linear gradient of 15% water m methanol to methanol over 20 mm, followed by 30 mm of methanol. This water/methanol-based system resolves 5,6-epoxyretmol, all-fran -retinol, all-tran5-retmal, anhydroretmol, and the esters retmyl docosahexanoate, retinyl palmitoleate, retinyl linoleate, and retinyl stearate, whereas retinyl palmitate and retinyl oleate elute as a single peak (Fig. 2). 

Fig. 2. HPLC analysis of neutral retinoids in tissues and plasma Samples were eluted from a reverse-phase Waters ODS column (1.2 x 10 cm) at 2 mL/min with a linear gradient of 15% water in methanol to methanol over 20 min, followed by 30 min of methanol. 1, 5,6-epoxyretinol 2, aU-trans-retinol, 3, all-/ra -retinal, 4, anhydroretinol, 5, retinyl docosahexanoate 6, retinyl palmitoleate 7, retinyl linoleate 8, retinyl palmitate and retinyl oleate, 9, retinyl stearate These specific examples illustrate the analyses of neutral-tissue retinoids equilibrated with orally fed [ HJretinol and extracted from (A) rat small-intestine mucosa (dashed line) or kidney (solid line) (B) plasma (dashed line) or liver (solid line) The concentrations of retinol and most retinyl esters in tissues and blood, however, are sufficiently large for detection by UV at 325 nm.

It has long been known that vitamin A is stored mainly in liver as retinyl esters, and that the liver is capable of storing large quantities of retinol in ester form. A study of the composition of retinyl esters isolated from the livers of members of a number of vertebrate species (calf, sheep, rabbit, rat, human, frog, trout, and cat) was reported by Futterman and Andrews (1964). In all species examined, except the cat, livers contained substantial stores of retinol, of which 95% or more was present in esterified form. Cat liver contained very little vitamin A, with approximately equal amounts present as bee retinol and as retinyl ester. The palmitate ester was the predominant retinyl ester in all species, comprising 66% of the retinyl esters in the human and 69% in the rat. Retinyl stearate and oleate were the next most common esters together with retinyl palmitate these three esters accounted for approximately 90% of all retinyl esters in the species examined. 

Typical chromatograms obtained by high-performance liquid chromatography (HPLC) of the retinyl esters from human RPE are illustrated in Fig. 5. When conditions are optimized for isomer separation (normal phase, uj r half of Fig. 5), there are two major peaks (2, 4), each with a shoulder on the leading edge. Peak 2 is 11-cw-retinyl palmitate the shoulder corresponds to 11-cij-retinyl stearate. Peak 4 is all-trans-retinyl palmitate the shoulder corresponds to all-trans retinyl stearate. Peak 5 is all-rra/u-retinyl stearate, and peak 3 (which is sometimes composite) is unidentified at present. Peaks 2 and 4 were collected separately and injected onto a reverse-phase column. Under these conditions the two component esters were completely resolved, as shown in the lower half of Fig. 5 (peaks 2.1, 2.2 and 4.1, 4.2). 

Fig. S. Retinyl esters stored in human RPE. Analysis by high-performance liquid chromatography (HPLC). (A) Normal-phase column peak 2, ll-c -retinyl palmitate and stearate (shoulder) peak 3, unidentified peak 4, all-rraru-retinyl plamitate and stearate (shoulder) peak 5, ail-trans-retinyl oleate. (B) Peaks 2 and 4 from the normal-phase HPLC eluent were collected and analyzed spearately in a reverse-phase system. The standards consisted of 11-cis-retinyl palmitate (P) and stearate (S). The 11 -cis and all-trans isomers have the same retention time in this system. Peak 2.1, 11-cis-retinyl palmitate peak 2.2, 1 l-cis-retinyl stearate peak 4.1, all-rrons-retinyl palmitate peak 4.2, all-traru retinyl stearate. (From Bridges et al., 1982.)...

As mentioned above, the eye loses its retinoids only very slowly under conditions of hypovitaminosis A. In rats, where the dark-adapted stores of retinyl esters are very small, the loss is virtually absent in darkness but is accelerated in the light (Noell et al., 1971). In light, most of the retinoid is transferred fixim the rhodopsin in the ROS to the RPE (Dowling, 1960), where it resides as retinyl stearate and palmitate (Alvarez etal., 1981). It returns to the ROS when rfiodop-... 

Structurally, vitamin A and many synthetic retinoids consist of a (3-ionone ring, a polyunsaturated polyene chain, and a polar end group. The polar end group can exist in several oxidation states, as retinol, retinal, or retinoic acid. Retinol and retinyl esters are the most abundant vitamin A forms found in the body (Blaner and Olson, 1994). Retinol can be esterified with long-chain fatty acids (mainly palmitate, oleate, and stearate) to form retinyl esters, which are the body s storage form of vitamin A. Retinol also can undergo oxidation to retinal, which can be oxidized further to retinoic acid. The active... 

Within the enterocyte, retinol is bound to cellular retinol binding protein (CRBP 11) and is esterified by lecithin retinol acyltransferase (LRAT), which uses phosphatidylcholine as the fatty acid donor, mainly yielding retinyl palmitate, although small amounts of stearate and oleate are also formed. At unphysiologically high levels of retinol, when CRBP 11 is saturated, acyl coenzyme A (CoA) retinol acyltransferase (ARAT) esterifies the free retinol that accumulates in intracellular membranes. Then the retinyl esters enter the lymphatic circulation and then the bloodstream (in chylomicrons), together with dietary lipid and carotenoids (Norum et al., 1986 Olson, 1986 Blomhoff et al., 1991 Green et al., 1993 Harrison and Hussain, 2001). 

Studies in vitamin A replete animals suggest that most of the retinol is transferred from hepatocytes to the perisinusoidal stellate cells of the liver. Here, it is again esterified by LRAT to form mainly retinyl palmitate (76% to 80%), with smaller amounts of stearate (9% to 12%), oleate (5% to 7%), and linoleate (3% to 4%). The stellate cells contain 90% to 95% of hepatic vitamin A, as cytoplasmic lipid droplets that consist of between 12% to 65% retinyl esters (Batres and Olson, 1987). Studies with [ C]retinyl palmitate show that much of the recently ingested retinol appears more or less immediately in the... 

Most dietary retinoids are stored in the liver as esters of long-chain fatty acids (e.g. palmitate, stearate, oleate). Fish also store retinyl esters in their kidneys71. Measurement of hepatic retinyl ester concentrations provides an indication of total body reserves. However, with both retinoid and didehydroretinoid forms mediating physiological functions17,44, all varieties require consideration when assessing the retinoid status of fish. A study with brook trout (Salvelinus fontinalis) characterized... 

L-Tyrosine DL-a-Valine L-Valine Wheat (Triticum vulqare) qerm oil Zinc acetate Zinc citrate Zinc gluconate Zinc methionine sulfate Zinc oxide Zinc stearate Zinc sulfate Zinc sulfate heptahydrate dietary supplement, food special dietary use D-Pantothenamide dietary supplement, gelatin capsules Retinyl palmitate... 

Ferric phosphate L-Lysine Nicotinic acid Retinyl acetate DL- rine nutrient, fermentation Methyl eicosanoate Methyl stearate ... 

Metasap Aluminum Stearate 537 Synpro Aluminum Monostearate 505 USP Synpro Aluminum Monostearate NF Synpro Aluminum Monostearate NF Gellant Synpro Aluminum Stearate 303 Synpro Aluminum Stearate LO GEL II Synpro Aluminum Stearate USP 230-338-6 Basic blue 6 230-363-2 Retinyl propionate 230-364-8 Arbanol ... 

Fig. 4. Chromatogram of a mixture of retinyl ester standards. Column, Supelcosil LC-8 (5 jLm) mobile phase, acetonitrile water (88 12) from origin to arrow and acetonitrile water (98 2) for rest of chromatography flow rate, 3 ml/min. The esters of retinol are 1, acetate, 2 0 2, laurate, 12 0 3, 7-linolenate, 18 3 4, myristate, 14 0, 5, palmitoleate, 16 1 6, linoleate, 18 2 7, palmitate, 16 0 8, oleate, 18 1 9, stearate, 18 0 10, arachidonate, 20 4. (Reprinted with permission from Ross, 1981.)...

In both man (Goodman et al., 1966b) and the rat (Huang and Goodman, 1965), the composition of lymph retinyl esters was remarkably constant, regardless of the fatty acid composition of the test meal, and regardless of whether the retinyl esters were derived from retinol or from p-carotene. Retinyl palmitate predominated, and saturated retinyl esters (retinyl palmitate and stearate, in a ratio of about 2 1) comprised approximately three-fourths of the labeled esters. Small amounts of retinyl oleate and linoleate were also found. 

The retinoids in dark-adapted eyes occur predominantly in the photoreceptor cell outer segments, which contain visual pigment, and in the adjacent retinal pigment epithelium (RPE) cells, which contain variable amounts of retinyl esters. These esters consist mainly of palmitate (A4) and stearate and are sometimes sequestered in lipid droplets (Bridges, 1975 Young and Bok, 1979 Alvarez etal., 1981 Bridges etal., 1982). The only major isomers present aieall-trans and 11-cis. The 11-cis isomer is restricted to ocular tissues. 

Fig. 16. Selective loss of I l-cis-retinyl ester (peak 2) in the RPE storage depots of eyes from a donor with dominantly inherited bilateral chorioretinal degeneration similar to retinitis pigmentosa (RPI37). (A) HPLC of retinyl esters from the affected tissue (B) HPLC of letinyl esters from normal tissue. Approximate amounts injected were 0.8 nmol for trace (A) and 19.5 nmol for trace (B). Peak I, 13-cis-retinyl palmitate and stearate peak 2, Il-cis-retinyl palmitate and stearate peak 3, not identified peak 4, all-rrons-retinyl palmitate and stearate peak S, all-frons-retinyl oleate. (From Bridges and Alvarez, 1982a.)...

The retinoid stored in the RPE from these eyes was predominantly retinyl ester. No accumulation of free retinol was observed. As in normal eyes (Bridges et al., 1982), the esters consisted mainly of palmitate with a smaller proportion of stearate. The rate of esteriHcation of aIl-rran5-[ H]retinol was measured in RPE homogenates and was not considered to be significantly lower than normals. 

Biesalski and Weiser separated a -trans from l3-cis, W-cis, and 9-cis isomers of retinyl esters (retinyl palmitate, stearate, oleate, palmitoleate, and linole-ate) by isocratic adsorption HPLC (126). Bridges et al. used step gradients to separate geometric isomers of retinyl esters, retinal, retinal oximes, and retinal (both vitamin Ai and vitamin A2 forms), also by adsorption ( normal-phase ) HPLC (139,140). 

The effects of common excipients and other foreign ions were checked under the optimized conditions containing a standard solution of 0.2 Xg/mL of vitamin E. No interference was observed at 250-fold for glycerol, Na+, K+, Ca, Mg +, Zn +, NHJ, S04", PO ", NO3, HCO3, Cl, methylcellulose, cholesterol, stearic acid, sucrose, glucose, starch, gum acacia, magnesium stearate at 100-fold for vitamin D, sorbitol, mannitol, oleic acid, triolein, EPA, DHA, polyvinyl alcohol, albumin, retinyl palmitate at 10-fold... 

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