Pantothenic acid hydrolysis

Chemical methods are mainly based on analysis of pantothenic acid hydrolysis products by specttophotometry or fluorometry. Although relatively rapid to perform, these methods lack the specificity and sensitivity needed to determine vitamin in foods or differentiate between d and l forms. Several spectrophotomet-ric determinations of pantothenic acid and its salts have been developed. They are based on the reaction of pantolactone with hydroxylamine and 2,7-naphtha-lenediol. Most published methods use the determination of P-alanine. Ninhydrin, 1,2-naphthoquinone, o-phthalaldehyde, acetylacetone, and some other reagents have been introduced for the spectrophotometric determination of pantothenates via p-alanine (5,46,47). 

Pantothenate in blood and tissues is bound (R9) and released by autolysis or hydrolysis. More vitamin could be released by use of an alkaline phosphatase and an enzyme from avian liver (L6). This method liberates pantothenate from coenzyme A in a variety of foods and tissues (N3, N4). A comparison of hydrolytic methods in blood suggested autolysis to be the most advantageous method (N3) in our hands, treatment with Clarase gave more reliable results as compared with autolysis, acid hydrolysis, treatment with Mylase P, or combination of Clarase and papain, or liver enzyme and alkaline phosphatase. In urine, pantothenic acid is unbound our results show no increase with Clarase treatment. The vitamin has presumably a low threshold. Pantothenic acid shows the same concentration in blood and cerebrospinal fluid. 

Pantothenic acid occurs in foods both in the free form and bonded to coenzyme (CoA) or acyl carrier protein (ACP) therefore hydrolysis is needed to extract it totally. Since it is degraded by acid and alkaline hydrolysis, only an enzymatic digestion can be applied. Enzyme hydrolysis with papain, diastase, clarase, takadiastase, intestinal phosphatase, pigeon liver pantetheinase, or combination of them has been used. 

UV absorption occurs only below 220nm, thereby it is affected by the interference from mobile phase and from artifacts in complex foods. A multiwavelength UV detection has been experimented successfully for unambiguous evaluation of pantothenic acid [609]. However, UV detection presents a low sensitivity, compared to other techniques, like FLD or MS. FLD is applied by using a postcolumn derivatization. Pantothenic acid is converted to 3-alanine by hot alkaline hydrolysis and a reaction with OPA [610]. Also MS is successfully applied to increase the sensitivity of pantothenic acid analysis. 

Enzyme hydrolysis, with papain, diastase, clarase, takadiastase, intestinal phosphatase, or combinations thereof is most commonly used to release pantothenate from food proteins (186). A cold perchloric acid extraction was used to release pantothenic acid from tissue samples (187). Food spoilage prior to analysis may lead to inflated pantothenic acid levels (19). 

During the course of studies on the microbial production of chiral intermediates for D-pantothenic acid [2,134,135], Shimizu and co-workers found that several micro-organisms, such as Fusarium, Brevibacterium and so on, produce a novel enzyme that catalyzes the hydrolysis of aldonate lactones or aromatic lactones [136, 137],... 

About 85% of dietary pantothenic acid is as CoA or phosphopantetheine. In the intestinal lumen, these undergo hydrolysis to phosphopantetheine, then pantetheine (see Figure 12.2). Intestinal mucosal cells have a high panteth-einase activity and rapidly hydrolyze pantetheine to yield free pantothenic acid. 

Availability. Although commercially available via the degradation of pantothenic acid, (i )-pantolactone is also conveniently prepared by enantioselective reduction of its corresponding keto lactone employing homogeneous catalysis," " or by microbial methods. The (5)-enantiomer has been prepared by inversion of the natural product in 90% yield and 97% ee via triflate activation, acetate displacement, and Lithium Hydroxide hydrolysis. The enantiomers were also prepared by resolution of the race-mate with (R)- and (5)-phenethylamine. A gas chromatographic method exists for ee determination. ... 

The biologically active R- or 5 -pantothenic acid can be obtained upon hydrolysis of coenzyme A with a combiaation of two enzymes, alkaline phosphatase and pantotheiaase (13) (Fig. 1). The phosphatase catalyzes the selective cleavage of the phosphate bond ia coenzyme A to afford adenosin-3 5 -diphosphate (6) and 4-phosphopantetheiae (7). The latter substance is dephosphorylated enzymatically to yield pantetheiae (8), which is rapidly converted by pantotheiaase to pantothenic acid (1). Table 1 Hsts some physical properties of pantothenic acid and its derivatives. 

This definition should be corrected by eliminating the word one, for it is impossible to consider as protides a large number of compounds which liberate upon hydrolysis one molecule of amino acid, such as glycocholic acid which yields glycine and cholic acid pantothenic acid which liberates -alanine and phospholipides containing serine. At the most, hippuric acid can be considered as a natural, somewhat special peptide. 

Pantothenic acid is taken in as dietary CoA compounds and dCphosphopantetheine and hydrolyzed by pyrophosphatase and phosphatase in the intestinal lumen to dephospho-CoA, phosphopantetheine, and pantetheine. This is further hydrolyzed to pantethenic acid. The vitamin is primarily absorbed as pantothenic acid by a saturable process at low concentrations and by simple diffusion at higher ones. The saturable process is facilitated by a sodium-dependent multivitamin transporter, for which biotin and lipoate compete. After absorption, pantothenic acid enters the circulation and is taken up by cells in a manner similar to its intestinal adsorption. The synthesis of CoA from pantothenate is regulated by pantothenate kinase, which itself is subject to negative feedback from the products CoA and acyi-CoA. The steps involved were outlined above. Pantothenic acid is excreted in the urine after hydrolysis of CoA compounds by enzymes that cleave phosphate and the cys-teamine moieties. Only a small fraction of pantothenate is secreted into milk and even less into colostrum. 

A pantothenic acid hydrolase (pantothenase) activity has been isolated from Pseudomonas fluorescens and other Pseudomonas strains. This enzyme hydrolyzes the amide bond of pantothenic acid 2 to form pantoic acid 5 (or pantoyl lactone) and /i-alanine 7 (EC 3.5.1.22) (Equation (10)). A detailed kinetic study of the reaction mechanism has shown that the reaction is partially reversible because of the formation of an acyl—enzyme (pantoyl-enzyme) intermediate during the course of catalysis, which may react with either water or / -alanine to form pantoic acid (the product hydrolysis) or pantothenic acid (the original substrate) Such a mechanism suggests that this enzyme could act as a pantothenate synthase, as reaction of the active site serine with pantoyl lactone would result in the formation of the pantoyl—enzyme intermediate. However, no biochemical or genetic evidence is currently available to support such a hypothesis. 

Minerals and vitamins are usually found in BSG [27]. The mineral elements include aluminum, barium, calcium, chromium, cobalt, copper, iron, magnesium, manganese, phosphorus, potassium, selenium, silicon, sodium, strontium, sulfur, and zinc, typically all in concentrations lower than 0.5%, except for silicon that is the major mineral present. The vitamins include biotin, choline, folic acid, niacin, pantothenic acid, riboflavin, thiamine, and pyridoxine. Although, many of the vitamins can be destroyed during the hydrolysis... 

Acrylonitrile can thus be converted to aminopropionitrile, which on hydrolysis with aqueous ammonia at 200°C yields /3-alanine, an important intermediate in the preparation of pantothenic acid. ... 

Pantolactone, an important intermediate for the synthesis of pantothenic acid (a constituent of Coenzyme A), is commercially available in only one optically active form. The (/ )-(— )-enantiomer currently sells for slightly less than 1.00 per gram. ( S)-( + )-Pantolactone (257) must be synthesized, and it is readily accessible from L-malic acid via the 3,3-dimethyl analog 231a [80]. Selective hydrolysis of the 1-ester furnishes the monoacid 256. Reduction of the 4-ester with L-Selectride followed by lactonization then gives 257 in 40% overall yield starting from dimethyl (5)-malate (2 231a 256 —> 257). 

Fluorescent compounds can be synthesized from pantothenic acid and the first HPLC-fluorimetric method was reported in 1995 (Blanco et al. 1995). In principle, pantothenic acid can be hydrolysed to (3-alanine and pantoic acid by hot alkaline hydrolysis and the product (3-alanine is reacted with u-phthal-dialdehyde and 3-mercaptopropionic acid (3-MPA) to form a fluorescent compound, l-alkylthio-2-alkylisoindole. This fluorescence is monitored at an excitation wavelength of 345 nm and emission wavelength of 455 nm (Figure 20.2). This method can determine the total pantothenic acid contents in foodstuffs and urinary pantothenic acid levels (Pakin et al. 2004 Takahashi et al. 2009). 

One of the first findings which gave a definite clue to the functioning of pantothenic acid in biological systems was the discovery of its presence as a fundamental constituent of coenzyme A. Initially preparations of this coenzyme failed to yield pantothenic acid, but in the author s laboratory a sample of coenzyme submitted by Dr. Lipmann was found to yield on acid hydrolysis a substantial amount of / -alanine , a fact strongly suggesting the presence of pantothenic acid, which was itself later released by enzymatic action . A further link in the chain was revealed in the discovery of pantothiene (the Lactobacillus bulgaricus factor) which has the formula indicated ... 

Finally, reaction of the butyrolactone with the ethyl ester of P-alanine FI2NCFI2CFI2CO2CH2CFI3), a preparation of which is shown in Scheme 12.109, led to the preparation of the ethyl ester of pantothenic acid. The add itself formed on the basic hydrolysis of the ester. 

Structure of Coemyme A. The elucidation of the structure of CoA depended heavily on d radation by specific enzymes. The phosphate on carbon 3 of the adenosine was shown to be a monoester phosphate by hydrolysis with prostate phosphomonoesterase. The localization of the monoester at the 3 position was established by its sensitivity to a b nucleotidase that attacks only nucleoside 3 -pbosphates, not 2 - or 5 -phosphates. The original CoA molecule or the phosphatase product, depbospho CoA, can be split by nucleotide pyrophosphatases from potato or snake venom. These reactions permitted the identification of the adenosine phosphate portion of the molecule. The position of the phosphate on pantothenic acid cannot be determined enzymatically, but was established by studies on the synthesis of CoA from synthetic phos-phorylated pantetheines. Pantetheine is split to thiolethanolamine and pantothenic acid by an enzyme found in liver and kidney. This enzyme also attacks larger molecules, including CoA. 

An alcohol related to pantothenic acid and referred to as D-panthenol or D-pantothenyl alcohol also possesses vitamin activity (Fig. 5). It is commonly used in liquid pharmaceutical and cosmetic preparations. Panthenol is a hygroscopic viscous oil that can be crystallized. Panthenol is very slightly soluble in water but very soluble in alcohol. The products of hydrolysis of panthenol are pantoic acid and 3-amino-1-propanol (P-alanol). 

Pantothenic acid and pantothenates may also be analyzed following derivatization to extend the chromophore and hence allow UV detection at higher wavelengths or fluorometric detection. Hudson et al. (63) have attempted to analyze the vitamin as a P-alanine-fluorescamine complex. The derivatization procedure was lengthy and required extensive sample cleanup before the hydrolysis step due to the interference of riboflavin, niacinamide, and some minerals such as zinc, copper, manganese, and molybdenum. Although these interferences were eliminated, the method did not yield reproducible results. 

The first method is based on the conversion of racemic pantothenic acid and its derivatives (panthenol and pantolactone) to DL-pantoic acid. The hydrolysis is carried out in 0.5 M NaOH at 70°C for either 30 min (pantolactone) or 60 min (pantothenic acid and panthenol). Pantoic acid is directly resolved on a ligand-exchange chiral stationary phase MCI gel CRS lOW column (Fig. 12 Table 3). 

Two approaches are currently used for the preparation of volatile derivatives of the vitamin. The first approach is mostly based on the conversion of pantothenic acid and/or panthenol to acetyl (72) or trimethylsilyl derivatives (73). A simpler and more convenient approach for most applications seems to be the procedure based on the hydrolysis of the vitamin in acidic medium and analysis of the hydrolysis products. Pantothenic acid, its salts, and coenzyme A as well as its analogs undergo acid hydrolysis with formation of P-alanine and pantolactone. Panthenol breaks down to 3-amino-l-propanol (P-alanol) and pantolactone. For instance, P-alanine can be analyzed as the corresponding jv-trifluoroacetyl methyl ester or AMrifluoroacetyl butyl ester. Pantolactone is sufficiently volatile to be amenable to direct GLC (23,76,77), but it can also be analyzed as the corresponding trimethylsilyl ether, trifluoroacetyl, or isopropylurethane derivative (5,78). 

Determination of pantothenic acid in foods was described in detail by Davi-dek and Velisek (38). This method is also based upon the acid hydrolysis of the vitamin and the separation of pantolactone. The hydrolysis is performed in 25% (w/w) HCl for 4 to 5 h at 95 to 100°C. Pantolactone can be extracted either directly from the hydrolysate or from the neutralized hydrolysate (at pH 5) into dichloromethane. The extract may be further purified by chromatography on a silica-gel column (75,76) or directly analyzed by GLC (77) using polar stationary phases such as polyethylene glycols (Fig. 25). Methyl myristate and ethyl laurate can be used as internal standards. 

Pantothenic acid, also known as vitamin B5, is widely distributed in food, since it is a component in the coenzyme A structure. Therefore, it is essential to all organisms and its deficiency is imcommon. In addition, being part of this coenzyme, for the total vitamin B5 determination, an enzyme hydrolysis is necessary prior to analysis. Foods richest in pantothenic acid are organ meats, egg yolk, and whole grains. RP separations are employed to analyze pantothenic acid, which does not possess any specific UV—Vis absorption. To overcome this problem, either fluorescence detection or MS detection is employed. 

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