Blood collection and immunoassay procedures
Two tubes of blood were collected from all the participants at 7 to 12 weeks of gestation. Blood specimens placed into tubes with anticoagulant were centrifuged at 3000 g at 4℃, for 10 minutes, and, the supernatants were transferred to new tubes and frozen in liquid nitrogen and stored at -80℃ until analyzed.
Carnitine and Amino Acid Analyses
Levels of carnitine and FAA in the plasma specimens obtained were measured using LC-MS/MS (Shimadzu 8045, Japan) in line with La Marca et al. and Azzari et al. protocol [14,15].
Briefly, in a new tube, 50 µl from each specimen, 50 μl internal standard solution, and 700 μl Reagent-1 were mixed by vortex for 10 seconds, and the acquired solution was centrifuged at 4,000 rpm for 5 minutes. The data obtained were calculated based on internal standards on Shimadzu Neonatal software. Twenty-seven carnitine and 14 amino acid quantities were analyzed in a single injection.
The following 14 amino acids were analyzed: Methyl Glutaryl, aspartic acid (Asp), glutamic acid (Glu), ornitine (Orn), glycine (Gly), arginine (Arg), argininosuccinic acid (Asa), alanine (Ala), citrulline (Cit), tyrosine (Tyr), valine (Val), methionine (Met), leucine∕isoleucine (Leu∕Ile), and phenylalanine (Phe). The absolute concentration of each amino acid was expressed in µmol/L.