Extraction folates

Fazili, Z., PfeifTer, C.M., Zhang, M., and Jain, R., 2005. Erythrocyte folate extraction and quantitative determination by Kquid chromatography-tandem mass spectrometry Comparison of results with microbiologic assay. Clinical Chemistry. 51 2318-2325. 

Monch, S., and Rychlik, M., 2012. Improved Folate Extraction and Tracing Deconjugation Efficiency by Dual Label Isotope Dilution Assays in Foods. Journal of Agricultural and Food Chemistry. 60 1363-1372. 

The effects of pH in the oxidative destruction of folates has been reviewed by several authors (6,10,28). Each vitamer has its own unique pH stability, and it is therefore sometimes difficult to optimize extraction conditions for all folate forms in a single extraction. The pH of the extractant, presence of oxygen, the temperature used, and in addition, buffer type, can all affect the efficiency of folate extraction (8). Several folate derivatives can be altered during extraction. The presence of phosphate accelerates conversion of 10-HCO-H4-folate to (1) 5,10-CH+-H4-folate and (2) 5-HCO-H4-folate at pH values below pH 7 during extraction. Also, 5,10-CH2-H4 folates can be converted to H4 folate during extraction (29). As the use of ascorbic acid reduces 5-CH3-H2-folate to 5-CH3-H4-folate, the former can be quantified only in the 5-CH3-H4-folate pool (30). Thus, extraction conditions can affect the vitamer distribution and therefore need always to be carefully considered and fully reported. The chosen extraction conditions are dependent on the purpose of the analysis to be carried out. 

In historical terms, folates are among the most recently identified of the vitamins. Wills was the first to describe a form of anaemia associated with pregnancy and malnutrition which could be cured by yeast or liver extract (Wills, 1933 Wills et al, 1937). The active constituent of these dietary... 

Vitamins such as thiamin, biotin, and vitamin Bj2 are often added. Once again, the requirements of anaerobes are somewhat greater, and a more extensive range of vitamins that includes pantothenate, folate, and nicotinate is generally employed. In some cases, additions of low concentrations of peptones, yeast extract, casamino acids or rumen fluid may be used, though in higher concentrations, metabolic ambiguities may be introduced since these compounds may serve as additional carbon sources. 

The major sources of folate are green vegetables, citrus fruits, legumes, egg yolk, wheat germ, and yeast [417]. This vitamin can be added only in the form of pteroylmonoglutamic acid [402]. The multiplicity of forms, low stability, low concentration, and the complex extraction and detection techniques make the analysis of folate in food a difficult task. 

HPLC does not give a good response to long-chain derivatives of folate, therefore, conversion of the natural form of folate, polyglutamates, to mono- or di-glutamate is required. The enzyme used is y-glutamylcarboxypeptidase, extracted from chicken pancreas, hog kidney, or to a lesser extent from... 

Goyer, A., Navarre, D. A. (2007). Determination of folate concentrations in diverse potato germplasm using a trienzyme extraction and a microbiological assay. J. Agric. Food Chem., 55, 3523-3528. 

MTHF is separated from other folates by reverse-phase HPLC and quantified with EC detection [17]. Prior to HPLC, serum is extracted with Sep-Pak Cl 8 cartridges, while CSF is injected without pretreatment [18]. The two HPLC systems described are very similar, and both can be used for CSF or serum. 

Folacin extraction from foods generally begins with homogenization in neutral or mildly acidic solution (122-124,152,153). One study (152) demonstrated that an extraction at pH 7.85 provided the most efficient recovery of endogenous folate from plant and animal tissues a second extraction of the residual tissue significantly increased the completeness of the extraction procedure. The extractant should contain antioxidants, such as ascorbic acid, 2-mercaptoethanol, dithiothreitol, or a mixture thereof to protect the natural oxidation states of the folacin vitamers. 

JF Gregory, R Engelhardt, SD Bhandari, DB Sartain, SK Gustafson. Adequacy of extraction techniques for determination of folate in foods and other biological materials. J Food Comp Anal 3 134— 144, 1990. 

CM Pfeiffer, LM Rogers, JF Gregory. Determination of folate in cereal-grain food products using trienzyme extraction and combined affinity and reversed-phase liquid chromatography. J Agric Food Chem 45 407-413, 1997. 

Intracellular protozoa of the phylum Apicomplexa such as plasmodium, toxoplasma, and eimeria have long been known to respond to sulfonamides and sulfones. This has led to the assumption that Apicomplexa must synthesize their own folate in order to survive. The reaction of 2-amino-4-hydroxy-6-hydroxymethyl-dihydropteridine diphosphate with />aminobenzoate to form 7,8-dihydropteroate has been demonstrated in cell-free extracts of the human malaria parasite Plasmodium falciparum. 2-Amino-4-hydroxy-6-hydroxymethyl-dihydropteridine pyrophosphokinase and 7,8-dihydropteroate synthase have also been identified. Sulfathiazole, sulfaguanidine, and sulfanilamide act as competitive inhibitors of p-aminobenzoate. It has not been possible to demonstrate dihydrofolate synthase activity in the parasites, which raises the possibility that 7,8-dihydropteroate may have substituted for dihydrofolate in malaria parasites. Similar lack of recognition of folate as substrate was also observed in the dihydrofolate reductase of Eimeria tenella, a parasite of chickens. 

The possible relationship of the methane fermentation with the more conventional examples of one-carbon metabolism as catalyzed by folate and vitamin B12 cofactors has been long apparent. 5-Methyl tetrahydro-folate, 5,10-methylene tetrahydrofolate, and methyl vitamin B12 are converted to methane by cell-free extracts of M. barkeri 32) and M. omeli-anskii (33). The involvement of vitamin B12 is further implicated by its high cellular level in methane bacteria and by the isolation of B12-containing proteins in extracts of M. barkeri 30) which stimulate methane evolution from methyl vitamin B12. The components and pathways that can be demonstrated in cell-free M. barkeri extracts 32) are listed below. 

The involvement of folate in the methane fermentation is less clear. 5-Methyltetrahydrofolate is used only moderately as a methane source by extracts of M. barkeri 14). No folate-containing factor has been demon-... 

Vitamin M Vitamin M is also called pteroylglutaminic add or folic acid. It was isolated from yeast extract by Wills in 1930. Its structure was described by Anger in 1946. Folic add is made up of pteridine + p-aminobenzoic add + glutamic add. There are several known derivatives, called folates, which are capable of mutual restructuring. The coenzyme tetrahydrofolic acid, which plays a role in many biochemical reactions, is formed with the help of Bi2. Around 50% of total body folate are stored in the liver. A folate-binding protein (FBP) is available for transport. Folate undergoes enterohepatic circulation. The release of folate from the liver cells is stimulated by alcohol, which increases urine excretion. Folate deficiency (e.g. in the case of alcohol abuse) is accompanied by the development of macrocytosis. 

Some itamirLS are water soluble, while others are fat soluble. This classification is valuable as it indicates whether the vitamin is likely to be absorbed similarly to lipids or like other water-soluble nutrients. The fat-soluble vitamins are A, D, E, and K. The water-soluble vitamins arc ascorbic acid, biotin, folate, niacin, pantothenic acid, riboflavin, thiamin, vitamin B i, and vitamin B 2. The classification is also valuable, as it helps chemists decide on the best way to extract and analyze a particular vitamin in foods and biological tissues. Aside from having some bearing on the path ways of absorption and distribution throughout the body, the question of whether a particular vitamin is fat soluble or water soluble has little or no relevance to its function in the body. 

It has been observed several years ago81 that cell-free extracts of bacteria were able to incorporate sulfonamides into folate-like compounds. That this did not constitute a case of metabolic activation (see Section 2.3.), was... 

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