Pantothenic acid assay methods

For most assays, the incorporated pantothenic acid has to be Hberated en2ymatically. Usually, a combination of pantotheinase and alkaline phosphatase is used to hberate the bound pantothenic acid. The official method for pantothenic acid of the Association of Official Analytical Chemists (AOAC) is the microbiological assay that uses U. Plantarium (A.TCC 8014) as the test organism (71). Samples are extracted at 121°C at pH 5.6—5.7, proteins are precipitated at pH 4.5, and the resulting clear extracts are adjusted to pH 6.8 prior to assay. This procedure is only suitable to determine calcium pantothenate or other free forms of pantothenic acid. 

Microbiological assays are the most popular means of quantitating food pantothenate (188). Table 24 summarizes a recent HPLC method for determining pantothenic acid in infant formula. 

There are no convenient or reliable functional tests of pantothenic acid status, thus assessment is made by direct measurement of whole blood or urine pantothenic acid concentrations. Urine measurements are perhaps the easiest to conduct and interpret, and concentrations are closely related to dietary intake, Whole blood measurements are preferred to plasma, which contains only free pantothenic acid and is insensitive to changes in pantothenic acid intake. Concentrations of pantothenic acid in aU of the above fluids can be measured by microbiological assay, most commonly using Lactobacillus plantarum. Whole blood must first be treated with an enzyme preparation to release pantothenic acid fi om CoA. Other techniques that have been used to measure pantothenic acid in human samples include radioimmunoassay and gas chromatography, Other techniques that have been developed include gas chromatography-mass spectrometry and a stable isotope dilution assay. CoA and AGP can be measured by enzymatic methods. ... 

Several B vitamins, including folic acid, niacin, pyridoxine, and pantothenic acid, are routinely determined using microbiological assays, details of which can be found in the AOAC Official Methods of Analysis. Standard methods for thiamine determination using fluorimetric detection are also detailed in the AOAC methods in addition, LC techniques are now being used routinely for thiamine and other B vitamins, e.g., riboflavin. 

Coverage includes B vitamins and folate in the context of a historical background, disease, cardiovascular effects and the importance of vitamins in biochemistry as illustrated by a single vitamin. Thereafter there are chapters on the chemistry and biochemistry of thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, folate and cobalamin. Methodical aspects include characterization and assays of B vitamins and folate in foods of all kinds, dietary supplements, biological fluids and tissues. The techniques cover solid-phase extraction, spectrofluorimetry, mass spectrometry, HPLC, enzymatic assay, biosensor and chemiluminescence. In terms of fimction and effects or... 

Biological, chemical and physiological methods are available for the measurement of pantothenic acid (vitamin B5). However, the development of analytical methods for pantothenic acid lags far behind that for other vitamins. A classical microbiological assay remains the most practical method to measure pantothenic acid, mainly because there has been little interest in assessing pantothenic acid deficiency. 

Following the discovery of pantothenic acid in 1933, several biological methods were reported based on the growth response to pantothenic acid of chicks, yeast and bacteria such as Lactobacillus, Streptococcus and Proteus. Microbiological assays have been used more commonly than animal tests because they are cheaper, faster and simpler methods. Lactobacillus, in particular, responds well... 

Pantothenic acid and pantetheine may be assayed using several enzymatic tests with pantothenase (40). Immunoassays and, especially, radioimmunoassays have been used in the analysis of pantothenic acid in tissue fluids, because of their sensitivity, specificity, and high rates of sample throughput. The radioimmunoassay (RIA) methods have about the same sensitivity as microbiological tests (approximately 0.05 pg/mL). As an alternative to the radioimmunoassay, the enzyme-linked immunosorbent assay (ELISA) is particularly suitable for the routine analysis of foods (41-45). 

The determination of trimethylsilylated pantoyllactone by gas chromatography-mass fragmentography (GC-MF) with a packed column has been developed for the assay of pantothenic acid by Tarli et al. (23). Umeno et al. (24) used the same method to investigate hopantenic acid levels in serum and urine after administration of calcium hopantenate. 

Currently employed HPLC methods for pantothenic acid and/or pantothenates have been applied solely to pharmaceuticals and simple matrices such as fortified infant formulas, whereas assays of coenzyme A and its acyl analogs have also been successfully performed on animal tissues. In the last few years, chiral stationary phases have been developed for optical resolution of pantothenic acid and related compounds by HPLC, and also HPLC-MS has become a promising technique. However, the newly developed HPLC procedures still require increased sensitivity and selectivity to make them applicable for the analysis of the total vitamin content in complex matrices such as foods and feeds. 

Concentrations in body fluids are traditionally measured by microbiological assay using Lactobacillus plantarum. If CoA is present, enzymatic hydrolysis is needed to liberate free pantothenic acid for the microbiological assay. Other assay methods reported include gas chromatography (after conversion to a volatile derivative), radioimmunoassay (RIA), or enzyme-linked immunoabsorbent assay (ELISA). 

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