Method for the determination of the amino acids for myocardial infarction and diabetes type 2 with our Amino Acid Analyzer ARACUS
The aim of the study was to develop and validate a method for the determination of the amino acids homocysteine, valine, methionine, isoleucine, leucine, tyrosine and phenylalanine of the amino acids in human blood plasma. Cardiovascular diseases are the number one cause of mortality worldwide. Studies over the past 10 to 20 years have shown that elevated homocysteine is a marker of risk of cardiovascular disease. Homocysteine is an amino acid measured in plasma and the normal levels are in the range 5 – 15 nmol / ml blood plasma.
Elevated plasma homocysteine has been associated with endothelial dysfunction, oxidative stress, and increased thrombogenicity, contributing to the development of atherosclerosis (Refsum et al., 2006).
Sample Preparation and Analysis
500 µl blood plasma and 100 µl 4% dithiothreitol solution were mixed in a 1.5 mL tube and incubated by 40° for 30 min. Bound forms of homocysteine in the sample are reduced in form of free homocysteine by the use of 4% dithiothreitol solution. After the incubation 150 µL of precipitation solution were added and deposit in the refrigerator for 20 min for the protein precipitation. Then 500 µL of sample dilution buffer (including internal standard norleucin, 100 nmol/mL) were added and mixed. The supernatant was filtered with a membraSpin by centrifugation at 14000 rpm for five minutes. The particle free solution was used for the injection.
The samples were analyzed by the Amino Acid Analyzer ARACUS, manufactured and distributed by membraPure GmbH worldwide. ARACUS is using the classic routine analysis of amino acids by post-column derivatization with ninhydrin and the detection at 440 nm and 570 nm.
The use of post-column derivatization with ninhydrin is a well-established method to accurately quantify amino acids, including homocysteine, in clinical samples (Cohen & Michaud, 1993).
Figure 1: Amino Acid Analyzer ARACUS
Results & Discussion
Table 1 and Table 2 show the results from patients without and patients with myocardial infarction. The concentration of homocysteine increases by patients with myocardial infarction.
Elevated homocysteine in myocardial infarction patients has been shown to correlate with increased inflammatory markers and greater cardiovascular risk (Lentz, 2005).
Table 3 and Table 4 show the results from patients without and patients with diabetes type 2. The concentration of the AA Leucine and Tyrosine increases and the level of Phenylalanine decreases. Homocysteine increases, so the patients with diabetes type 2 have also a high risk of developing cardiovascular diseases.
Higher levels of branched-chain amino acids (BCAAs) like leucine and isoleucine, as well as aromatic amino acids such as tyrosine, have been linked to insulin resistance and the development of type 2 diabetes (Newgard et al., 2009).
Table 1: Amino acid concentration of valine, homocysteine, methionine, allo-isoleucine, isoleucine, leucine, tyrosine, and phenylalanine in nmol/mL plasma of patients without myocardial infarction.
Table 2: Amino acid concentration of valine, homocysteine, methionine, allo-isoleucine, isoleucine, leucine, tyrosine, and phenylalanine in nmol/mL plasma of patients with myocardial infarction.
Table 3: Amino acid concentration of valine, homocysteine, methionine, allo-isoleucine, isoleucine, leucine, tyrosine, and phenylalanine in nmol/mL plasma of patients without Diabetes Type 2.
Table 4: Amino acid concentration of valine, homocysteine, methionine, allo-isoleucine, isoleucine, leucine, tyrosine, and phenylalanine in nmol/mL plasma of patients with Diabetes Type 2.
References:
- Cohen SA, Michaud DP. “Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography.” Anal Biochem. 1993;211(2):279-287.
- Vinod Wali, et al. (2012) Serum Homocysteine Levels As a Novel Biomarker in Patients with Acute Myocardial Infarction. International Journal of Medical and Health Sciences. October 2012, Vol-1.
- Metabolite Profiles and the Risk of Developing Diabetes. Thomas J. Wang, et al. Nat Med. 2011 April; 17(4): 448–453.
- Refsum H, et al. “Homocysteine and cardiovascular disease.” Annu Rev Med. 2006;57: 625-654.
- Lentz SR. “Mechanisms of homocysteine-induced atherothrombosis.” J Thromb Haemost. 2005;3(8):1646-1654.
- Newgard CB, et al. “A branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance.” Cell Metab. 2009;9(4):311-326.