Bound forms of vitamins

H. Mitsuda. Isolation from rice bran of OS088 a bound form of vitamin B-6 and its identification as 5 -0-(beta-D-gluco-pyranosyl) pyridoxine. Agr Biol Chem 1977 41 1061. 

The importance of vitamins to plants themselves is often overlooked, but they play the essential roles in a plant metabolism too (Smith et ah, 2007). Some algal species require different combinations of certain vitamins such as vitamins B12 and Bi. Because the concentration of these vitamins in the natural environment is quite low, their absorption is insufficient (Croft et ah, 2006). According to Yamada et ah (1996a), red algae Porphyra tenera can take up the free (not protein-bounded) form of vitamin B12 from the incubation medium by concentration- and temperature-dependent processes. The amoimt of uptake increases with the time of incubation. 

Owing to the desirability of converting the more labile forms of vitamin Bi2 to cyanocobalamin by cyanide treatment, it has been common to extract Bi2 from crude materials with KCN solutions. Skeggs, Driscoll, Chamey, and Wright (39) found the most effective treatment with cyanide to be extraction with 0.01% KCN (10 mg. of KCN for 10 y.g. of vitamin B12 activity) at 60 C. for 30 minutes. Higher temperatures are necessary to release bound forms of vitamin B12. Vitamin Bu has a maximum of stability at pH 4-5 an acetate buffer pH 4.6 is recommended when higher temperatures are used. 

In general, pyridoxamine and pyridoxin are more stable than pyridoxal. All vitamers are relatively heat-stable in acid media, but heat labile in alkaline media. All forms of vitamin B6 are destroyed by UV light in both neutral and alkaline solution. The majority of vitamin B6 in the human body is stored in the form of pyridoxal phosphate in the muscle, bound to glycogen phos-phorylase. 

Around 99% of calcium is contained in the bones, whereas the other 1% resides in the extracellular fluid. Of this extracellular calcium, approximately 40% is bound to albumin, and the remainder is in the ionized, physiologically active form. Normal calcium levels are maintained by three primary factors parathyroid hormone, 1,25-dihydroxyvitamin D, and calcitonin. Parathyroid hormone increases renal tubular calcium resorption and promotes bone resorption. The active form of vitamin D, 1,25-dihydroxyvitamin D, regulates absorption of calcium from the GI tract. Calcitonin serves as an inhibitory factor by suppressing osteoclast activity and stimulating calcium deposition into the bones. 

Knowledge of the coenzyme forms of vitamin Bi2 has increased steadily. The first coenzyme of Bi2 isolated from bacteria had similarities to pseudovitamin Bi2 it contained adenylic acid instead of 5,6-dimethyl-benzimidazole, but differed in lacking cyanide and having an extra molecule of adenine which was assumed to be bound to the cobalt atom by the coordination site, often occupied by cyanide (B24). This coenzyme, adenylcobamide, was completely inactive for Ochromonas malhamensis, but active for Escherichia coli 113-3. 

Liver tissue B12 is assayed by suspending 50 mg of lyophilized human liver in 100 ml of aconitate buffer to which 100 mg sodium metabisulfite is added. The suspension is autoclaved for 30 minutes at 16 psi to liberate the bound forms of the vitamin. Aliquots are diluted 1 10 after autoclaving to reduce metabisulfite toxicity on microorganism growth. Each milliliter now contains 50 pg of liver (w/v) 1.0, 1.5, and 2.0 ml of this hydrolyzate is added. After dilution to a volume of 5 ml, this represents an assay of 10, 15, and 20 ig of liver per milliliter of final solution. Normal liver contains between 2-14 mpg of vitamin B /mg liver powder. 

Coenzyme B12 is the cofactor form of vitamin B 2, which is unique among all the vitamins in that it contains not only a complex organic molecule but an essential trace element, cobalt. The complex corrin ring system of vitamin B12 (colored blue in Fig. 2), to which cobalt (as Co3+) is coordinated, is chemically related to the porphyrin ring system of heme and heme proteins (see Fig. 5-1). A fifth coordination position of cobalt is filled by dimethylbenzimidazole ribonucleotide (shaded yellow), bound covalently by its 3 -phosphate group to a side chain of the corrin ring, through aminoisopropanol. 

To replace losses, oxaloacetate can be synthesized from pyruvate and C02 in a reaction that uses ATP as an energy source. This is indicated by the heavy gray line leading downward to the right from pyruvate in Fig. 10-1 and at the top center of Fig. 10-6. This reaction depends upon yet another coenzyme, a bound form of the vitamin biotin. Pyruvate is formed from breakdown of carbohydrates such as glucose, and the need for oxaloacetate in the citric acid cycle makes the oxidation of fats in the human body dependent on the concurrent metabolism of carbohydrates. 

In this chapter we are concerned, not primarily with vitamins per se, but with coenzymes. Many coenzymes are modified forms of vitamins. The modifications take place in the organism after ingestion of the vitamins. Coenzymes act in concert with enzymes to catalyze biochemical reactions. Tightly bound coenzymes are sometimes referred to as prosthetic groups. A coenzyme usually functions as a major component of the active site on the enzyme, which means that understanding the mechanism of coenzyme action usually requires a complete understanding of the catalytic process. 

Direct assays for vitamin B12, such as the competitive protein-binding immunoassays, detect all forms of vitamin B12 in the serum, including physiologically inactive analogues. The vitamin B12 bound to transcobalamin II (TCII) has been shown to be the physiologically... 

Q7 Calcium is present in both intracellular fluid (ICF) and ECF, but the concentration in the ECF is twice as high as that in the ICF. Calcium is found in both ionized and bound forms, and Ca2+ homeostasis is mainly controlled by parathyroid hormone, which increases absorption of calcium in the intestine and reabsorption in the nephron. Calcitonin also affects ECF calcium concentration by promoting renal excretion when there is an excess of calcium in the body. The normal kidney filters and reabsorbs most of the filtered calcium however, in renal disease this is reduced and blood calcium decreases. Calcium and phosphate imbalance can occur in patients with renal failure, leading to osteomalacia (defective mineralization of bone). Osteomalacia is mainly due to reduced production of 1,25-dihydroxycholecalciferol, an active form of vitamin D metabolized in the kidney. Deficiency of 1,25-dihydroxycholecalciferol reduces the absorption of calcium salts by the intestine. 

The predominant circulating form of vitamin Bg is pyridoxal phosphate. Absorbed pyridoxine is oxidized and phosphorylated in intestinal mucosal cells, liver, and erythrocytes. Pyridoxine enters hepatocytes and erythrocytes by passive diffusion and is mostly retained by phosphorylation. Pyridoxal phosphate is transported in the blood bound to albumin. The blood-brain barrier has limited permeability to pyridoxal. 

The possible existence of bound forms of ascorbic add in animal and plant tissues forms one of the most contradictory subjects in the biochemical literature. The very high concentrations of ascorbic acid within both plant and animal cells provide reasons to look for such combined forms. The realization that most of the water-soluble vitamins function... 

The final step in this pathway is the oxidation of PNP to PLP and is carried out by PdxH. Tbe recombinant enzyme from E. coli has been studied in vitro and is a 51 kDa homodimer that utilizes flavin mononucleotide (FMN) as a cofactor. PdxH can use either PNP or pyridoxamine 5 -phosphate (PMP) as a substrate with a of 2 and 105 pM and cat of 0.8 and 1.7 s for PNP and PMP, respectively. The structures of the enzyme from E. coli as well as homologues from Mycobacterium tuberculosis and humans have been solved. The E. coli enzyme with PLP and FMN bound is shown in Figure 6. PdxH is involved in both the biosynthetic and the salvage pathways and is further discussed in a section describing the transport, salvage, and interconversion of the various forms of vitamin Bg. 

Lipophilic hormones that use intracellular gene-specific transcription factors include the steroid hormones, thyroid hormone, retinoic acid (active form of vitamin A), and vitamin D (Fig. 11.8). Because these compounds are water-insoluble, they are transported in the blood bound to serum albumin, which has a hydrophobic binding pocket, or to a more specific transport protein, such as steroid hormonebinding globulin (SHBG) and thyroid hormone-binding globulin (TBG). The intracellular receptors for these hormones are structurally similar and are referred to as the steroid hormone/thyroid hormone superfamily of receptors. 

Fig. 45.5. A. Structures of vitamin K derivatives. Phylloquinone is found in green leaves, and intestinal bacteria synthesize menaquinone. Humans will convert menadione to a vitamin K active form. B. Vitamin K-dependent formation of y-carboxyglutamate residues. Thrombin, Factor VII, Factor IX, and Factor X are bound to their phospholipid activation sites on cell membranes by Ca ". The vitamin K-dependent carboxylase, which adds the extra carboxyl group, uses a reduced form of vitamin K (KH2) as the electron donor and converts vitamin K to an epxjx-ide. Vitamin K epoxide is reduced, in two steps, back to its active form by the enzymes vitamin K epoxide reductase and vitamin K reductase.

Bacteriorhodopsin is found in differentiated regions of the cytoplasmic membrane of several Halobacteria. These so-called purple membranes are composed of two major lipid species (25% by weight) and one protein component which contains retinal (the aldehyde form of vitamin A) as a covalently bound prosthetic group. The retinal is bound via a protonated Schiff-base linkage to a lysine residue of the protein moiety of bacteriorhodopsin and absorbs light with a maximum absorbance at 570 nm [18]. 

Vitamin B Vitamin Bg is the generic name for six naturally occurring vitamers which are derivatives of 2-methyl-3-hydroxy pyridone. All forms of vitamin Bg have been detected in human plasma with the exception of pyridoxine phosphate. Both free and protein bound forms can be extracted with trichloroacetic acid or perchloric acid, the excess of which should be removed prior to chromatography. Cation-exchange chromatography with linear gradients of hydrochloric acid and phosphate buffers produces a profile of the vitamin Bg compounds in 50 min. 

Vitamin E In man a-tocopherol is the major form of vitamin E activity found in plasma and cells with fl- and y-tocopherol being minor components all of which are bound to lipoproteins. With a plasma concentration of 5-15 mg 1 a-tocopherol has the highest plasma concentration of any of the fat soluble vitamins. For the separation of the tocopherols, reversed-phase HPLC can be used. Though the molar... 

To give but one example of the importance of imines in biological systems, the active form of vitamin A aldehyde (retinal) is bound to the protein opsin in the human retina in the form of an imine called rhodopsin or visual purple (see Chemical Connections 4B). The amino acid lysine (see Table 18.1) provides the primary amino group for this reaction ... 

Most structural (and spectroscopic) investigations have concerned the diamagnetic Co -corrins, in which a hexacoordinate (corrin-bound) Co center carries two axial ligands (12,16,17). In the thermodynamically stable forms of vitamin Bi2 (1), coenzyme B12 (2), and methylcobalamin (3), all representatives of the (nucleotide containing) complete cobalamins in their base-on form, the nucleotide base coordinates at the lower a-face of the cobalt center. The second... 

From a nutritional standpoint, it is significant that five of the B-complex vitamins (riboflavin, nicotinamide, thiamine, vitamin Be, and pantothenic acid) have been shown to be constituents of the coenzymes. The nutritional requirement of these vitamins is explained on the basis of their coenzyme function. In all cases the coenzyme form appears to be the sole bound form of the vitamin, and this then becomes the only metabolically active form for these particular vitamins. 

The vitamin, biotin, has been shown to infiuence a variety of enzymatic reactions. Although a bound form of biotin, biocytin, has been obtained in crystalline form, no conclusive proof of the existence of a coenzyme form... 

Bussi et al. (1953) and Cox (1953) divided pernicious anemia serum into protein and nonprotein fractions by ultrafiltration. The inhibitor was usually in the ultrafiltrate. Cox (1953) found that the protein fraction, rid of this inhibitor by ultrafiltration, had maturing properties. Microbiological assays showed that this fraction contained not only bound vitamin B12 but bound forms of the folic acid group of substances including citrovorum factor (Cox, Hornsby et al., unpublished). 

In the plasma, vitamin A is transported from its major depots in the liver to tissues in several forms, primarily as a 1 1 molar complex of all-frans-retinol with RBP. Low concentrations of dll-trans- and 13-ds-retinoic acid, probably bound to albumin, and retinyl and retinoyl p-glucuronides, are also present. Holo-RBP also interacts strongly with transthyretin in the plasma. All of these forms of vitamin A can be taken up by various tissue cells. Several tissues besides the parenchymal cells of the liver can also synthesize RBP, as evidenced by the presence of mRNA for RBP within such cells. Thus, the extensive recycling of retinol between the liver and peripheral tissue cells may well occur as complexes with RBP. Another possibility is that retinyl ester, which is synthesized in essentially all cells of the body, might be carried back to the liver in lipoproteins. 

The only biologically active form of vitamin Bs is D-(- -)-biotin, a unique steroisomer foimd in nature among the eight isomers theoretically possible [1,40]. In animal and plant tissues, most biotin is covalently bound to a lysine residue, free (D-biocytin) or belonging to biotin-dependent enzymes through an amide attachment. Biotin is also the form used for food enrichment [4]. 

Meat, meat products, offal and egg yolk are rich sources of vitamin Bg (Table 5.8). In foods of animal origin, the main compounds are pyridoxal and pyridoxamine, especially in the form of phosphate esters. For example, in meat the main form is present as pyridoxal 5 -phosphate (about two thirds of this vitamin are prosthetic groups of enzymes) bound to various proteins (such as imine), to a lesser extent as free pyridoxal 5 -phosphate, followed by pyridoxamine 5 -phosphate. In contrast, milk contains only about 10% of vitamin in bound forms. The vitamin content in milk and cheeses is relatively low. 

The main form of vitamin Bg in cereals is free pyridoxol and pyridoxol bound to glucose. The vitamin content of flour depends on the degree of milling. In dough and also in bread, the losses are small and do not exceed 15%. Vitamin retention in cooked pasta is 50-70%. 

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