Vitamin polar metabolites

In intestinal cells, carotenoids can be incorporated into CMs as intact molecules or metabolized into mainly retinol (or vitamin A), but also in retinoic acid and apoc-arotenals (see below for carotenoid cleavage reactions). These polar metabolites are directly secreted into the blood stream via the portal vein (Figure 3.2.2). Within intestinal cells, retinol can be also esterified into retinyl esters. 

A second major vitamin D metabolite is 24R,25-dihydroxyvitamin D3, a compound that circulates in the blood at a concentration 10 times higher than that of the 1,25-isomer.a b However, no biological function has been discovered, and like a series of other polar metabolites (>30) it is probably on a pathway of inactivation and degradation of vitamin D. la,25-Dihydroxyvitamin D is also hydroxylated... 

Vitamin D, along with parathyroid hormone and calcitonin, plays a primary role in calcium and phosphorus homeostasis in the body. Intensive research efforts over the past several years have elucidated a role for vitamin D in many other physiological processes as well. The biological actions of this seco-steroid are mediated primarily through the action of its polar metabolite, 1,25-dihydroxy vitamin D3 (l,25(OH)2D3). There is emerging evidence that l,25(OH)2D3 has many more target tissues than those involved in its classical role in the control of mineral metabolism. In addition, some of the actions of l,25(OH)2D3 may be mediated by mechanisms other than the classical steroid-receptor interaction. In this chapter we will provide a brief overview of the multiple actions of vitamin D3 and the pleiotropic mechanisms by which these actions are accomplished. 

The first indication that vitamin D3 might require further metabolic alteration before it could be active came from experiments showing a lag between the time of vitamin D3 administration and the first observed biological response [19]. The lag time could be shortened by intravenous administration (9 h) compared with oral administration (18 h) of vitamin D3, but not completely eliminated [20]. A major polar metabolite fraction... 

Most vitamin D is excreted in the bile less than 5% is excreted as water-soluble metabolites in urine. Some 2% to 3% of the vitamin D in bUe is cholecalciferol, calcidiol, and calcitriol, but most is a variety of polar metabolites and their glucuronide conjugates. In most tissues, the major pathway for inactivation of calcitriol is by way of 24-hydroxylation to calcitetrol, then onward oxidation byway of the 24-oxo-derivative, 23-hydroxylation, and oxidation to calcitroic acid (see Figure 3.3). In addition, a variety of hydroxylated and other polar metabolites have been identified in bile, and many of these onward oxidation products also undergo glucuronide conjugation in the liver (Reddy and Tserng, 1989). 

The interaction between alcohol and vitamin A is complex. They have overlapping metabolic pathways a similar 2-step process is involved in the metabolism of both alcohol and vitamin A, with alcohol dehydrogenases and acetaldehyde dehydrogenases being implicated in the conversion of vitamin A to retinoic acid. Alcohol appears to act as a competitive inhibitor of vitamin A oxidation. In addition, chronic alcohol intake can induce cytochrome P450 isoenzymes that appear to increase the breakdown of vitamin A (retinol and retinoic acid) into more polar metabolites in the liver, which can cause hepatocyte death. So chronic alcohol consumption may enhance the intrinsic hepatotoxicity of high-dose vitamin A. Alcohol has also been shown to alter retinoid homoeostasis by increasing vitamin A mobilisation from the liver to extrahepatic tissues, which could result in depletion of hepatic stores of vitamin A. ... 

Liquid chromatography On silica or polar bonded phases (nitro, cyano), the separation of vitamin D metabolites occurs according to the number and position of hydroxyl groups in the molecule. Binary mobile phases are usually based on hexane-2-propanol mixtures. Improved resolution of normally co-eluting D2 and D3 hydroxylated metabolites is afforded by ternary solvent systems containing dichloromethane as a third component. However, the D2 and D3 parent compounds remain unresolved in any normal-phase system. 

If a sorbent has the potential to interact with analytes through more than one retention mechanism, it should be possible to adjust the chromatographic conditions in order to strengthen just one of the interactions and thereby obtain different selectivity between the analytes. This is illustrated here for the case of the elution of vitamin A metabolites 1-5 from a stationary phase with embedded polar groups at different mobile phase compositions (see Fig. 2). 

Clearly, though solvents in the second class are desirable to minimize contamination of vitamin D extract, they pose problems in that they tend to provide efficient extraction for one particular metabolite or group of metabolites (e.g., the dihydroxylated metabolites) and poorly extract those of a different polarity. When simultaneous analysis of several vitamin D metabolites with a wide range of polarities is required, a total lipid extraction (83) may be necessary. The high efficiency of the Bligh and Dyer technique (83) for total lipid extraction (i.e., methanol chloroform 2 1, v/v) is probably due to the formation of a monophasic dispersion of sample in extracting solvent, followed by return to the classic two-phase system by addition of extra chloroform and saturated KCl. All hydroxylated nonacidic vitamin D metabolites can be extracted quantitatively by this technique. 

Calcium absorption was studied in 28 adult male epileptics on chronic anticonvulsant therapy. In 16 patients on phenytoin alone calcium absorption was abnormal in 9. In 12 patients on both phenytoin and phenobarbitone calcium absorption was abnormal in 3. Hypocalcaemia (<8.5 mg/ 100 ml) occurred in only 2 patients, while serum alkaline phosphatase was elevated in 7 patients. The findings support the suggestion that rickets and osteomalacia reported in patients on chronic anticonvulsant therapy results from reduced calcium absorpion. The effect of these drugs appear to be the increased metabolism of vitamin D via enzyme induction, and an increase in the excretion of polar metabolites 25-hydroxycholecalciferol and 1,25-dihy-droxycholecalciferol which are necessary for normal calcium absorption (40 ). 

The principal function of vitamin D is in the control of calcium metabolism. This is accomplished through the mediation of polar, hydroxylat-ed metabolites, the most important of which is... 

Solid-phase extraction effectively separates vitamin D from its more polar 25-hydroxy metabolite. In the analysis of human milk (64), the dried lipid fraction of milk was dissolved in 35% dichloromethane in hexane and then applied to a preconditioned silica cartridge. The sample was fractionated using the following elution sequence 9 ml hexane (discard), 3 ml 7% ethyl acetate in hexane (discard), 15 ml 7% ethyl acetate in hexane (vitamins D2 and D3), 25 ml 15% ethyl acetate in hexane (discard), and 9.5 ml 3% 2-propanol in hexane (25-hydroxyvitamin D2 and 25-hydroxy vitamin D3). 

Normal-phase HPLC, using either silica or polar-bonded stationary phases, separates, isocrati-cally, vitamin D2 or D3 from their respective previtamins and inactive isomers (207). Vitamin D (D2 + D3), 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 can be separated from one another and from other hydroxylated metabolites (215), but vitamins D2 and D3 cannot be resolved from one another. The inability to resolve vitamins D2 and D3 means that one vitamer cannot be used as an internal standard for the other. 

There are many examples in the literature for applications of LC-NMR in the pharmaceutical industry. In the area of natural products, LC-NMR has been applied to screen plant constituents from crude extracts [54,57,67,68] and to analyze plant and marine alkaloids [69-72], flavonoids [73], sesquiterpene lactones [74,75], saponins [58,76], vitamin E homologues [77], and antifungal and bacterial constituents [56,78,79] as examples. In the field of drug metabolism, LC-NMR has been extensively applied for the identihcation of metabolites [42, 80-88] and even polar [89] or unstable metabolites [43]. And hnally, LC-NMR has been used for areas such degradation products [90-93], drug impurities [94-102], drug discovery [103,104], and food analysis [105-107]. 

It has been suggested that vitamin D3 (378) is metabolized into a more polar substance before stimulating calcium transport to the intestine. The principal metabolite from the blood, produced by the liver, has been found to be 25-hydroxycholecalciferol (379), whereas the trihydroxy-derivative (380) is the principal metabolite from the intestine. Autoxidation of cholesterol via hydroperoxide intermediates afforded a variety of hydroxylated cholesterol derivatives and products of side-chain degradation. ... 

The differences in their polarities, because of the number of hydroxyl groups, have been used to separate vitamin D and its metabolites. With three hydroxyl groups, l,25(OH)2D is more polar than 25(OH)D, with two hydroxyls, which is more polar than vitamin D, with one hydroxyl group. 

Beyond its physiologic role in the intestinal digestion of lipids and lipid-soluble vitamins, the bUe also plays an important role in the excretion of xenobio-tics, including drugs and their metabolites. This includes a diverse array of compounds, both polar and lipophilic, including, anions, cations, and neutral molecules. In humans, the molecular threshold is approximately 500 to 600, with renal excretion being the primary route of excretion for smaller molecules. 

Solvents commonly used in normal phase chromatography are aliphatic hydrocarbons, such as hexane and heptane, halogenated hydrocarbons (e.g., chloroform and dichloromethane), and oxygenated solvents such as diethyl ether, ethyl acetate, and butyl acetate. More polar mobile phase additives such as isopropanol, acetone, and methanol are frequently used see Table 2). The technique is particularly suited to analytes that are very hydrophobic, e.g., fat-soluble vitamins such as tocopherols (6J and other hydrocarbon-rich metabolites that exhibit poor solubility in the water-miscible solvents employed in other separation modes. In addition, since the geometry of the polar adsorbent surface is fixed, the technique is useful for the separation of positional isomers the proximity of functional groups to the adsorbent surface, and hence the strength of interaction, may well differ between isomers. 

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