Description
Principle of the Assay: Branched Chain Amino Acid Assay Kit measures BCCAs within food or biological samples. L-Leucine, L-valine, and L-isoleucine are converted by Leucine Dehydrogenase (in the presence of excess NAD+) into their corresponding alpha-keto products (alpha-ketoisocaproate, alpha-ketovalerate, or alpha-ketoisovalerate) plus ammonia and NADH. The converted NADH is then detected colorimetrically with WST-1 which is converted to the formazan form in the presence of an electron mediator. Samples are compared to a known concentration of L-Leucine standard within the 96-well microtiter plate format. Samples and standards are then read with a standard 96-well colorimetric plate reader (Figure 1).
Background/Introduction: Amino acids are organic compounds that contain amine (-NH2) and carboxyl (-COOH) functional groups, as well as a side-chain (R group) which confers uniqueness to each amino acid. The main elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, although other elements can be found in some amino acids. About 500 amino acids are known, but only 20 are coded in the human genome. A Branched Chain Amino Acid (BCAA) contains a branch structure side chain with one central carbon connected to at least three other carbon atoms. The only three BCAAs coded by the human genome are leucine, valine, and isoleucine. BCAAs play key roles in animal metabolism and physiology. BCAAs enhance protein synthesis, signaling pathways, and glucose metabolism. On a physiological level, BCAAs function within the immune system. BCAAs are degraded by dehydrogenase and decarboxylase enzymes, are found in immune cells, and are required for lymphocyte growth, proliferation, and activity. BCAAs are transported into the brain by the same enzyme used to transport aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Once in the brain BCAAs contribute to protein synthesis, neurotransmitter synthesis, and energy production. In humans, rats and mice models, BCAA levels in the blood are elevated in obese, insulin resistant models of diet-induced diabetes. This result suggests the possibility that BCAAs promote obesity and diabetes pathogenesis. In mice fed limited amounts of BCAAs, glucose tolerance is improved and weight loss is observed. Branched Chain Amino Acid Assay Kit is a simple colorimetric assay that measures the total amount of free BCAAs (Leucine, Isoleucine, and Valine) present in foods or biological samples in a 96-well microtiter plate format. BCAAs in polypeptide chains (peptides and proteins) are not detected. Each kit provides sufficient reagents to perform up to 192 assays*, including blanks, L-Leucine standards and unknown samples. Sample BCAA concentrations are determined by comparison with a known L-Leucine standard. The kit has a detection sensitivity limit of 15.6 uM BCAAs