Analysing postprandial amino acid responses in crossover studies with the aaresponse package for R.

Nowadays, a healthier and more sustainable lifestyle is the subject of much research. One example is the use of crossover trials to investigate the uptake of proteins, usually from alternatives to animal-based sources, by healthy volunteers. The data analysis is complex and requires many decisions on the part of the scientists involved. Such a process can be streamlined and made more objective and reproducible through bespoke software. This paper describes such a software package, aaresponse , for the R environment, available as open source. It features ample visualization functions, supports consistent curation strategies, and compares amino acid uptake of different protein meals (interventions) through the use of mixed models analysing parameters of interest like the area under the curve (AUC). The defining feature is the use of parametric curves to fit the amino acid levels over time, increasing the robustness of the approach and allowing for more strict quality control strategies.

Is the Brain Undernourished in Alzheimer's Disease?

Cerebrospinal fluid (CSF) amino acid (AA) levels and CSF/plasma AA ratios in Alzheimer Disease (AD) in relation to nutritional state are not known. Methods: In 30 fasting patients with AD (46% males, 74.4 ± 8.2 years; 3.4 ± 3.2 years from diagnosis) and nine control (CTRL) matched subjects, CSF and venous blood samples were drawn for AA measurements. Patients were stratified according to nutritional state (Mini Nutritional Assessment, MNA, scores). Results: Total CSF/plasma AA ratios were lower in the AD subpopulations than in NON-AD (p < 0.003 to 0.017. In combined malnourished (16.7%; MNA < 17) and at risk for malnutrition (36.6%, MNA 17−24) groups (CG), compared to CTRL, all essential amino acids (EAAs) and 30% of non-EAAs were lower (p < 0.018 to 0.0001), whereas in normo-nourished ADs (46.7%, MNA > 24) the CSF levels of 10% of EAAs and 25% of NON-EAAs were decreased (p < 0.05 to 0.00021). CG compared to normo-nourished ADs, had lower CSF aspartic acid, glutamic acid and Branched-Chain AA levels (all, p < 0.05 to 0.003). CSF/plasma AA ratios were <1 in NON-AD but even lower in the AD population. Conclusions: Compared to CTRL, ADs had decreased CSF AA Levels and CSF/plasma AA ratios, the degree of which depended on nutritional state.

Gestational Leucylation Suppresses Embryonic T-Box Transcription Factor 5 Signal and Causes Congenital Heart Disease.

Dysregulated maternal nutrition, such as vitamin deficiencies and excessive levels of glucose and fatty acids, increases the risk for congenital heart disease (CHD) in the offspring. However, the association between maternal amino-acid levels and CHD is unclear. Here, it is shown that increased leucine levels in maternal plasma during the first trimester are associated with elevated CHD risk in the offspring. High levels of maternal leucine increase embryonic lysine-leucylation (K-Leu), which is catalyzed by leucyl-tRNA synthetase (LARS). LARS preferentially binds to and catalyzes K-Leu modification of lysine 339 within T-box transcription factor TBX5, whereas SIRT3 removes K-Leu from TBX5. Reversible leucylation retains TBX5 in the cytoplasm and inhibits its transcriptional activity. Increasing embryonic K-Leu levels in high-leucine-diet fed or Sirt3 knockout mice causes CHD in the offspring. Targeting K-Leu using the leucine analogue leucinol can inhibit LARS activity, reverse TBX5 K-Leu modification, and decrease the occurrence of CHD in high-leucine-diet fed mice. This study reveals that increased maternal leucine levels increases CHD risk in the offspring through inhibition of embryonic TBX5 signaling, indicating that leucylation exerts teratogenic effects during heart development and may be an intervening target of CHD.

Increased immunoreactivity of glutamate receptors, neuronal nuclear protein and glial fibrillary acidic protein in the hippocampus of epileptic rats with fast ripple activity.

Epilepsy is a neurological disorder in which an imbalance between excitatory and inhibitory transmission is observed. Glutamate is the principal excitatory neurotransmitter that acts through ionic and metabotropic receptors; both types of receptors are involved in temporal lobe epilepsy (TLE). High frequency oscillations called fast ripples (FR, 250-600 Hz) have been observed, particularly in the hippocampus, and they are involved in epileptogenesis. The present study analyzed the immunoreactivity of the principal glutamate receptors associated with epilepsy in epileptic animals with FR activity. Male Swiss-Wistar rats (210-250 gr) were injected with pilocarpine (2.4 mg/2 µl) and were video monitored (24/7) until the appearance of spontaneous and recurrent seizures. Then, a deep microelectrode implantation surgery was performed in the DG, CA3 and CA1 regions, and FR activity was observed 1-, 2-, 3-, 7-, and 14-day postsurgery. The animals were sacrificed on day 15, and fluorescence immunohistochemistry was carried out in the hippocampus for the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and mGlu-R5 glutamate receptors as well as Neuronal Nuclear Protein (NeuN) and Glial Fibrillary Acidic Protein (GFAP). An increase in the immunoreactivity for the three receptors was found. However, the AMPA receptor showed an increase in the three regions analyzed (i.e., DG, CA1 and CA3). The findings showed a decrease of NeuN in the DG and an increase of GFAP. These results suggest an important role of glutamate receptors in the hippocampus of epileptic rats with FR activity.

Effects of dietary amino acid levels and ambient temperature on mixed muscle protein turnover in Pectoralis major during finisher feeding period in two broiler lines.

Two broiler lines A and B were fed experimental diets from 21 to 42 days with an objective to determine Pectoralis major protein turnover (PT) as affected by the dietary amino acid (AA) levels and ambient temperature. Experimental diets (n = 9 replicate pens per diet) were formulated to 3,150 kcal/kg with five levels of digestible lysine (dLys) -80, 90, 100, 110 and 120% of recommended AA level giving g dlys/Mcal values of 2.53, 2.85, 3.17, 3.48 and 3.80 respectively. All other AA was formulated to a fixed ratio to dLys. Fractional synthesis or degradation rates (FSR or FDR) of P. major were measured on day 36 and day 42 for all dietary treatment levels for both broiler lines using stable isotope of AA (15 N-phenylalanine) as metabolic tracer. Experimental feeding studies were conducted once in hot season (24-hr mean ~ 85.3°F; 80.9% RH) and repeated in cool season (24-hr mean ~ 71.6°F; 61.7% RH) of the year. The FSR values increased (p < .05) as digestible AA in diet increased for both broiler lines in hot season until break point FSR occurring at 106.2% AA level. The average FSR values measured were higher for Line B at day 36 (20.98%/D for Line B vs. 20.69%/D for Line A) and at day 42 (16.07%/D for Line B vs. 12.47% D for Line A). FDR values observed at day 36 and day 42 were not different between lines (p > .05). Similar trends but elevated values of FSR and FDR in cool season than in hot season were recorded for both the lines. Line B showed the higher mixed muscle protein accretion (%/D) than Line A by actually increasing the FSR which was correlated by higher lean mass deposition and higher feed intake (p < .05). The overall findings indicated that PT response in P. major due to effects of digestible AA levels and ambient temperature was different and line-specific.

d-Amino Acid Levels in Perfused Mouse Brain Tissue and Blood: A Comparative Study.

The l-enantiomer is the predominant type of amino acid in all living systems. However, d-amino acids, once thought to be "unnatural", have been found to be indigenous even in mammalian systems and increasingly appear to be functioning in essential biological and neurological roles. Both d- and l-amino acid levels in the hippocampus, cortex, and blood samples from NIH Swiss mice are reported. Perfused brain tissues were analyzed for the first time, thereby eliminating artifacts due to endogenous blood, and decreased the mouse-to-mouse variability in amino acid levels. Total amino acid levels (l- plus d-enantiomers) in brain tissue are up to 10 times higher than in blood. However, all measured d-amino acid levels in brain tissue are typically ∼10 to 2000 times higher than blood levels. There was a 13% reduction in almost all measured d-amino acid levels in the cortex compared to those in the hippocampus. There is an approximate inverse relationship between the prevalence of an amino acid and the percentage of its d-enantiomeric form. Interestingly, glutamic acid, unlike all other amino acids, had no quantifiable level of its d-antipode. The bioneurological reason for the unique and conspicuous absence/removal of this d-amino acid is yet unknown. However, results suggest that d-glutamate metabolism is likely a unidirectional process and not a cycle, as per the l-glutamate/glutamine cycle. The results suggest that there might be unreported d-amino acid racemases in mammalian brains. The regulation and function of specific other d-amino acids are discussed.

Methylphenidate Decreases ATP Levels and Impairs Glutamate Uptake and Na+,K+-ATPase Activity in Juvenile Rat Hippocampus.

The study of the long-term neurological consequences of early exposure with methylphenidate (MPH) is very important since this psychostimulant has been widely misused by children and adolescents who do not meet full diagnostic criteria for ADHD. The aim of this study was to examine the effect of early chronic exposure with MPH on amino acids profile, glutamatergic and Na+,K+-ATPase homeostasis, as well as redox and energy status in the hippocampus of juvenile rats. Wistar male rats received intraperitoneal injections of MPH (2.0 mg/kg) or saline solution (controls), once a day, from the 15th to the 45th day of age. Results showed that MPH altered amino acid profile in the hippocampus, decreasing glutamine levels. Glutamate uptake and Na+,K+-ATPase activity were decreased after chronic MPH exposure in the hippocampus of rats. No changes were observed in the immunocontents of glutamate transporters (GLAST and GLT-1), and catalytic subunits of Na+,K+-ATPase (α1, α2, and α3), as well as redox status. Moreover, MPH provoked a decrease in ATP levels in the hippocampus of chronically exposed rats, while citrate synthase, succinate dehydrogenase, respiratory chain complexes activities (II, II-III, and IV), as well as mitochondrial mass and mitochondrial membrane potential were not altered. Taken together, our results suggest that chronic MPH exposure at early age impairs glutamate uptake and Na+,K+-ATPase activity probably by decreasing in ATP levels observed in rat hippocampus.

Application of Pre-Column Labeling Liquid Chromatography for Canine Plasma-Free Amino Acid Analysis.

Plasma-free amino acid (PFAA) levels are a useful metric for diagnosing cancer and providing a prognosis. However, the use of analysis of PFAA levels has been limited in the veterinary medicine field. We addressed the application of liquid chromatography (LC) using a pre-column labeling technique for analysis of canine PFAA levels. This method significantly shortened the analysis time relative to conventional methods. No diurnal fluctuations were detected at 9:00 AM in most PFAA levels, and food intake increased the levels of some PFAAs, including valine, leucine, tyrosine, phenylalanine, and proline. These results indicate that LC with pre-column labeling is useful for measuring canine PFAA levels, for which time of day and interval after food intake must be taken into consideration.

Amniotic fluid amino acid levels in non-immune hydrops fetalis: a case-control study

In a prospective case-control study, we compared the amniotic fluid amino acid levels in non-immune hydrops fetalis (NIHF) and normal fetuses. Eighty fetuses underwent amniocentesis for different reasons at the prenatal diagnosis unit of the Department of Obstetrics and Gynecology, Faculty of Medicine, Dicle University. Forty of these fetuses were diagnosed with NIHF. The study included 40 women each in the NIHF (mean age: 27.69 ± 4.56 years) and control (27.52 ± 5.49 years) groups, who had abnormal double- or triple-screening test values with normal fetuses with gestational ages of 23.26 ± 1.98 and 23.68 ± 1.49 weeks at the time of sample collection, respectively. Amniotic fluid amino acid concentrations (intra-assay variation: 2.26-7.85 percent; interassay variation: 3.45-8.22 percent) were measured using EZ:faast kits (EZ:faast GC/FID free (physiological) amino acid kit; Phenomenex, USA) by gas chromatography. The standard for quantitation was a mixture of free amino acids from Phenomenex. The levels of 21 amino acids were measured. The mean phosphoserine and serine levels were significantly lower in the NIHF group, while the taurine, α-aminoadipic acid (aaa), glycine, cysteine, NH4, and arginine (Arg) levels were significantly higher compared to control. Significant risk variables for the NIHF group and odds coefficients were obtained using a binary logistic regression method. The respective odds ratios and 95 percent confidence intervals for the risk variables phosphoserine, taurine, aaa, Arg, and NH4 were 3.31 (1.84-5.97), 2.45 (1.56-3.86), 1.78 (1.18-2.68), 2.18 (1.56-3.04), and 2.41 (1.66-3.49), respectively. The significant difference between NIHF and control fetuses suggests that the amniotic fluid levels of some amino acids may be useful for the diagnosis of NIHF.