Microbiological assays folates

Recently, radioassay methods have been refined to measure folates in biological samples. These techniques use radioactively labeled folates and competitive protein binding.80 Johnson et al.81 compare this method with traditional microbiological assay with L. casei. 

Some commonly used detectors are UV (at 280 nm), ELD, ED and microbiological assay of collected fractions. UV presents a low sensitivity, but all folate derivatives respond to this detection. ELD is used even if some compounds, like folic acid, do not fluoresce. Therefore, a postcolumn derivatization, involving hypochlorite to cleave folic acid, di- and tetra-hydrofolic acid oxidatively to fluorescence pterins [571], has been introduced. Eewer reports on the use of LC-MS for folate detection are available in the literature [578-580]. 

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. 

Kariluoto, M. S., Vahteristo, L. T, Piironen, V. 1. (2001). Applicability of microbiological assay and affinity chromatography purification followed by high-performance liquid chromatography (HPLC) in studying folate contents in rye. J. Sci. Food Agric., 81, 938-942. 

Folic Acid (folate). Chemically, folic acid is a pteryl-glutamic acid. The several forms that occur in nature depend on the numbers of glutamic acid units and methyl groups in the molecules. Because of its usual low concentration, folic acid is generally determined in food materials by the microbiological assay with lactobacillus casei and measured turbidimetrically or titrimetrically. Deficiency of this vitamin could result in... 

Methylcobalamin, 516 5-Melhyl-cytosine, deaminatitm, 894 Methylglyoxal, 836 Methylmalonic acid (MMA), 434, 522 MethylmalcHiy. CoA, 434, 517, 518 Mevalonic acid, 327, 328 Mg-ATP complex, 795-796 Micelles, 25,27-29 MLcroaulophagy, 444 Microbiological assays, 508 biotin determination, 541 folate status by, 509 thiamin status, 607 Microcytic anemia, 5H Microsomal ethanol-oxidizing system, 247 Microvilli, 58 Milk... 

Sulfonamides, in contrast to trimethoprim, do not interfere with the microbiological assay of folate (77). 

O Broin S, Kelleher B. Microbiological assay on microtitre plates of folate in serum and red cells. J Clin Pathol 1992 45 344-7. 

With the introduction in 1960 (B6) of an improved and sensitive microbiological assay for folate the measurement of formiminoglutamic acid was no longer used as a diagnostic test, although it continues to be used as a research tool. The microbiological assay has in turn now been replaced in many laboratories by isotope dilution techniques. 

As the clinical usefulness of the microbiological assay for folate became more widely recognized so the number of variations and modifications to the method increased. As a consequence there was a wide range in the reported values found in normal subjects and a considerable variation in the level below which a patient was considered folate deficient. Some of these are shown in Table 1. Some workers have even suggested that folate levels in serum be graded as low, intermediate, or normal. In general it appears that serum folate levels below 3.0 xg/liter reflect a deficient state. 

Waxman, Schreiber, and Herbert first described a radioisotopic assay for the measurement of serum folate in 1971 (W14) which they claimed gave almost identical results to those obtained by with the L. casei microbiological assay and could separate low, borderline, and normal folate levels. Serum was mixed with PHjmethyltetrahydrofolic acid in a phosphate buffer and a folate binder was added which, in this case, was Carnation brand instant powdered milk. Following incubation, free vitamin was separated from that which was bound using hemoglobin-coated charcoal. The radioactivity of the supernatant was determined, and from this the folate concentration in the serum sample could be calculated using appropriate standards and blanks. 

B7. Baril, L., and Carmel, R., Comparison of radioassay and microbiological assay for serum folate, with clinical assessment of discrepant results. Clin. Chem. (Winston-Salem, N.C.) 24, 2192-2196 (1978). 

Radiometric Microbiologic Assays for the Measurement of Vitamin Folate, and Niacin in Human Blood and Food... 

Radiometric microbiological assays (RMA) for measuring the levels of vitamin B12, folate, and niacin in biological fluids and food were developed using Lactobacillus leichmannii, casei, and plantarum, respectively- In the presence of the appropriate Cdabeled substrate, these organisms readily produce directly proportional to the amount of the... 

After repeated failures to obtain comparable results with the CPBA and the TMA for vitamin B12 (23,24), we decided to approach the microbiological assay in a new way. Our studies have led to the development of RMA for the essential nutrients vitamin B12, folate, and niacin. 

CHEN AND MciNTYBE Vitamin Bft, Folate, 6- Niacin in Blood 481 Microbiological Assay (30)... 

Table VIII. Radiometric Microbiological Assay Reproducibility of Erythrocytic Folate Levels (26)...

As reviewed in detail by Rosenberg and Godwin (R18), folate absorption has been measured by three basically different methods (1) measurement of rises in blood folate after an oral dose, (2) measurement of folate compounds in urine after an oral dose, and (3) administration of isotopically labeled folate by mouth followed by measurement of isotope appearing in plasma and excreted in urine and feees. Folate in plasma and urine is assayed with bacteria, usually strains of Lactobacillus casei or Streptococcus faecalis, which require folate for growth. They differ somewhat in the forms of folate they can utilize, but in general these microbiological assays measure unconjugated folate in either their reduced or unreduced forms. 

There are various analytical platforms used to measure folate concentrations in food, each with its advantages and drawbacks. Microbiological assay makes use of bacteria (e.g. Lactobacillus easel) that are very dependent on folate availability for their growth. This assay is commonly used to determine the total folate contents in food, which is expressed in terms of folic acid equivalents. The most important limitation of microbiological assay is that the various vitamers are not distinguishable... 

Microbiological assay Based on the microbial growth obtained an amount of folate in the medium. 

A 96-well microtitre plate assay with microcomputer analysis has simplified the microbiological assay making it less laborious, less time-consuming and more reproducible. Recently, an automated 96-well plate isotope-dilution tandem mass spectrometry method and an ultra-performance liquid chromatography-tandem mass spectrometry method have been developed and validated for the quantification of synthetic folic add in folate-fortified breads (Fazili and Pfeiffer 2004 Chandra-Hioe et al. 2011). 

Folate can be measured in plasma or serum by microbiological assay using L. casei as the test organism, but this test can be confounded if the subject is on antibiotic treatment. Serum folate values reflect recent dietary intake and a vitamin deficiency is ascribed only where serum folate remains low over a period of time. Plasma folate levels are thought to reflect the day-to-day variations in dietary folate levels while red blood cell folate is a better indicator of long-term tissue storage levels. 

Radioassay kits are used extensively in clinical laboratories. Folate values obtained by using the dilferent commercially available kits fluctuate considerably and therefore no absolute values can be given to indicate folate deficiency. Each laboratory has to define its own lower limits based on a large number of sample assays from a representative population of normal subjects (Shane 2011). At present, the microbiological assay using L. casei is one of the best and most versatile methods for the determination of food folate. 

Tamura, T., and Hyun, T.H., 2005. Trienzyme extraction in combination with microbiologic assay in food folate analysis An updated review. Experimental... 

The most widely used method to quantitate folates is a microbiological assay (MBA), which provides a total figure for all folate vitamers. During this bioassay, the growth of Lactobacillus casei ssp. rhamnosus, for which folate is essential, is measured in a specifically deficient nutrient solution. The medium is then supplemented with a sample or a folate standard, and the resulting growth is measured at intervals by turbidity, which conveniently can be measured in an ultraviolet-visible (UV-vis) spectrophotometer. 

Method comparison between SIDA, radiobinding assay (A) or microbiological assay (B) for serum folates. The results are presented as Bland-Altman difference plots solid line represents the mean difference between the two methods. The SIDA method is used as the comparison method. Dashed lines represent the limits of agreement or 0.95 central intervals of the differences between the two methods (mean difference = 2 SD). A total of 46 serum samples were used in the analysis in A and 49 for the analysis in B. 

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