Measuring B12: Why the Confusion?
- B12 Amounts Versus B12 Activity
- Methods for Measuring Amounts of B12
- Methods for Measuring B12 Activity of a Food
- Bacterial Contamination
Inactive B12 analogues in plant foods compromise the accuracy of traditional methods used to determine the vitamin B12 amounts and activity of a food. Bacterial contamination of a food can create the false impression that all such foods contain B12 analogues. The only reliable way to determine if a food is a source of active B12 is to test various batches of that food to see if it reduces methyl malonic acid (MMA) levels in humans.
It is important to distinguish between the amounts of B12 in a food, and the B12 activity of a food. There are a number of ways that have traditionally been used to measure B12 amounts:
- Microbiological Assay
- R-protein Assay
- Intrinsic Factor Assay
- Paper Chromatography
There are also a number of ways to measure B12 activity:
- Macrocytic Anemia Improvements
- Homocysteine Reduction
- Methylmalonic Acid (MMA) Reduction
Microbiological assays use a process in which B12 is measured by "feeding" a food to certain B12-dependent bacteria and measuring how well they grow. Various test organisms for measuring B12 have been used:
Table 1. Test Organisms for B12 Microbiological Assays Organism Organism Notes Escherichia coli mutant 113-31 bacteria Not recommended because it responds to so many inactive B12 analogues.1 Lactobacillus leichmannii 3261 bacteria May utilize some inactive B12 corrinoids.1 Lactobacillus delbrueckii ATCC 7830152 bacteria New name for Lactobacillus leichmannii2 Euglena gracilis Z-alga1 protozoa May utilize some inactive B12 corrinoids.1 Ochromonas malhamensis1 protozoa Most specific for cobalamins.1 Arthrobacter Lochhead 38 bacteria A 1959 study showed it to be similar to O. malhamensis.3
Some or all of these bacteria thrive on various inactive B12 analogues, making them unreliable for measuring the B12 content of plants, which often contain a variety of inactive B12 analogues. Despite this, many laboratories, especially those of private companies who want to market their product as containing B12, still use the less reliable of these methods when measuring the B12 content of plant foods and seaweeds.
The R-protein assay measures the B12 analogue content of a food by seeing how much will bind to R-protein, which is a B12 transport protein found in the mouth and other places. R-protein is known to bind to inactive B12 analogues.
The Instrinsic Factor (IF) assay measures the B12 analogue content of a food by seeing how much will bind to IF, which is a B12 transport protein secreted by the stomach. For a few years, it was thought that IF only binded to active B12 analogues. In fact, the difference in amounts between an R-protein and an IF assay was thought to tell how much inactive B12 analogue a food contained.
Dagnelie et al. (4)(1991, Netherlands) quickly changed the thought that intrinsic factor assays can predict the contribution of foods to B12 status. Macrobiotic toddlers (14-26 months old) with B12 deficient anemia (serum B12 < 184) were given foods that had been found to have some B12 analogue using IF assays. According to Dagnelie, as little as 0.1 µg/day of B12 can lead to a full therapeutic response in 1 month (4). 4-6 months after eating these foods their serum B12 levels had increased, but the anemia was worse in the vegan children:
Table 2. Results of Dagnelie et al.4 Subject µg/day of
B12 Analogue Given
Vegans 1 2.7 spirulina, nori worse 2 2.1 spirulina, nori worse 3 1.5 nori worse 4 0.3 nori worse 5 0.1 sourdough bread, kombu, barley malt syrup worse Non-Vegans 6 1.5 / 0.5 algae / fish & milk better 7 0.3 fish better 8 0.2 fish better 9 0.2 fish, milk better 10 0.15 fish, nori worse 11 0.2 supplement, fish, nori worse MCV - Mean Corpuscular Volume
A likely explanation for the poor response is that nori, spirulina, and kombu either contained no active B12 or they contained enough inactive B12 analogue that it overcame the active B12, producing an overall negative effect. Dagnelie et al. say, "It seems unjustified to advocate algae and other plant foods as a safe source of vitamin B12 because its bioavailability is questionable."
It should be noted that, based on a more recent study, intakes as high as .3 µg/day for infants 6-16 months old are probably not enough to prevent B12 deficiency (based on MMA levels) (5). Thus, some of the patients in Dagnelie et al.'s study above may have needed more B12 for a positive response.
Degnalie et al's study showed that it is important to measure a food's B12 activity in humans rather than to rely only on measurements of B12 amounts using assays. You simply do not know for sure until you test the food. To make things more complicated, batches of some foods may have B12 from bacterial contamination, while other batches of the same food do not. Tempeh is an example.
Baker et al. (6) compared Ochromonas malhamensis to an IF assay and found that IF gave a 44% higher reading than Ochromonas malhamensis. The authors suggest that O. malhamensis may be the most effective of the various assays at measuring metabolically active B12. Unfortunately, only one study on B12 in plant foods has used O. malhamensis . Still, it is not known whether O. malhamensis growth is an accurate measurement of B12 activity in humans.
For simplicity, paper chromatography is the term used here to refer to an array of methods that when combined can provide a fairly precise determination of the exact structure of a molecule. These methods are more reliable than those mentioned above, but are difficult to perform. Furthermore, they cannot tell the actual B12 activity of a given plant food as a whole; they can only look at the parts.
B12-deficiency can cause macrocytic anemia (large red blood cells). However, folate deficiency can do the same. If someone with macrocytic anemia is known to have adequate folate status, and is fed a food thought to have B12, and their anemia improves, it is a fairly safe bet that the food has some B12-activity for red blood cells. Unfortunately, it is not known for sure if B12 that is active for blood cells is always active for nerve cells. See the section Inactive Analogues: Worse than Useless in Vitamin B12 Analogues.
B12-deficiency can cause elevated homocysteine levels in the blood. However, folate and vitamin B6 deficiency can do the same. Reducing homocysteine levels might give a good idea as to the B12 activity of a food, but because folate and B6 can confound the results, it is not the safest test for determing B12 activity.
Because the biochemical pathway that reduces MMA levels in the blood uses only vitamin B12, lowering MMA levels is a test that is specific for B12 activity. Although it is not known for sure, it is likely that this biochemical pathway is an integral part of B12's nerve tissue function(s). Thus, if a food lowers MMA levels, it can be assumed to provide full B12 activity.
Because some bacteria produce B12 and might live in the growing medium or contaminate some foods (e.g., see section on tempeh) it is not enough simply to test a food one time to see if it reduces MMA levels. Numerous batches of the food from various regions, using various preparation methods, should be tested to make sure the B12 is consistently found in the food.
2. Watanabe F, Takenaka S, Kittaka-Katsura H, Ebara S, Miyamoto E. Characterization and bioavailability of vitamin B-12-compounds from edible algae. Journal Of Nutritional Science And Vitaminology. 2002(Oct);48(5): 325-331.3.
4. Dagnelie PC, van Staveren WA, van den Berg H. Vitamin B-12 from algae appears not to be bioavailable. Am J Clin Nutr. 1991;53:695-7. | Link
5. Food and Nutrition Board, Institute of Medicine. Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Washington, DC: National Academy Press; 2000.
6. Baker H, Frank O, Khalil F, DeAngelis B, Hutner SH. Determination of metabolically active B12 and inactive B12 analog titers in human blood using several microbial reagents and a radiodilution assay. J Am Coll Nutr. 1986;5(5):467-75.