Folate, measurement microbiological assay

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. 

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... 

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. 

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. 

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... 

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... 

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. 

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. 

The most common analytical method for folates is a microbiological assay, which measures the growth of Lactobacillus casei ssp. rhamnosus in a specifically deficient nutrient solution. 

JI Rader, CM Weaver, G Angyal. Use of microbiological assay with tri-enzyme extraction for measurement of pre-fortification levels of folates in enriched cereal-grain products. Food Chem 62 451 65, 1998. 

Measurement of the serum or red blood cell concentration of folate is the method of choice, and a number of simple and reliable radioligand-binding assays have been developed. There are a number of problems involved in radioligand-binding assays for folate, and in some centres microbiological determination of plasma or whole-blood folates is the preferred technique. 

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