Determination Of Amino Acids In Plasma Research Articles

An Oligomeric Diet Limits the Response to Injury in Traumatic Brain-Injured Rats

Adequate nutritional support is a major challenge in brain injury patients, because malnutrition cannot be reversed by standard enteral nutrition. We hypothesized that an oligomeric formula could improve nutritional status by restoring intestinal trophicity. Eighteen male Sprague-Dawley rats (300-330 g) underwent gastrostomy on day-7 (D-7) and traumatic brain injury (TBI) by hydraulic percussion (D0) and were then fed for 4 days with either a polymeric formula (Sondalis® HP, TBIP, n = 9), or an oligomeric formula (Peptamen® HN, TBIO, n = 9). In addition, a control group of healthy gastrostomized rats was fed the polymeric diet (control, n = 8). All rats were weighed daily. On D+4, the rats were euthanized. Blood was collected for plasma amino acid determination. Organs were removed and weighed. Intestinal morphometry was studied. Protein content was assessed on intestine and muscles. Enterobacterial translocation and dissemination were evaluated. Results were expressed as means ± SEM and compared using analysis of variance+Newman-Keuls test. TBI induced a significant decrease in whole body weight (TBIP vs. control, p < 0.05) that was totally blunted by the oligomeric diet (TBIP vs. TBIO, p < 0.01). Thymus weight significantly decreased after TBI (TBIP vs. control, p < 0.05) and was restored by the oligomeric formula (TBIO vs. TBIP, p < 0.05). Glutamine (GLN) concentration was improved by the oligomeric diet in both plasma (TBIO: 688 ± 19 vs. control: 591 ± 45 and TBIP: 615 ± 42 μmol/L, p < 0.05) and soleus muscle. These results show that the use of an oligomeric diet may limit response to injury after brain injury and could be a simple nutritional strategy in this setting.

Simple and rapid quantitative high-performance liquid chromatographic analysis of plasma amino acids

A simple and rapid high performance liquid chromatographic method for the determination of plasma amino acids was developed. The method uses minimal sample volume and automated online precolumn derivitization of amino acids with o-phthalaldehyde and fluorescent detection. Amino acids are separated by a simplified gradient without column heating. The assay is linear from 5 to 1000 μmol/L for all amino acids. Recovery of amino acids was between 91 and 108%, intra-assay coefficient of variation (CV) was 1–7%, and inter-assay CV was 2–12%. The simple sample preparation and minimal sample volume make the method useful for the quantitation of amino acids in both patient and experimental animal samples.

Are Plasma Citrulline and Glutamine Biomarkers of Intestinal Absorptive Function in Patients With Short Bowel Syndrome?

Sensitive biomarkers for intestinal absorptive function would be clinically useful in short bowel syndrome (SBS). Citrulline (Cit) is a product of the metabolism of glutamine (Gln) and derived amino acids by enterocytes. Cit is produced almost exclusively by the gut, which is also a major site of Gln metabolism. The goals of this study were to examine whether plasma Cit and Gln concentrations are biomarkers of residual small intestinal length and nutrient absorptive functions in adult SBS patients followed prospectively. We studied 24 stable adults with severe SBS receiving chronic parenteral nutrition (PN) in a double-blind, randomized trial of individualized dietary modification +/- recombinant human growth hormone (GH). During a baseline week, intestinal absorption studies (% absorption of fluid, kcal, nitrogen, fat, carbohydrate, sodium, phosphorus, and magnesium) were performed and concomitant plasma Cit and Gln concentrations determined. Individualized dietary modification and treatment with subcutaneous injection of placebo (n = 9) or GH (0.1 mg/kg daily x 21 days, then 3 times/week; n = 15) were then begun. PN weaning was initiated after week 4 and continued as tolerated for 24 weeks. Repeat plasma amino acid determination and nutrient absorption studies were performed at weeks 4 and 12. Residual small bowel length at baseline was positively correlated with baseline plasma Cit (r = 0.467; p = .028). However, no significant correlations between absolute Cit or Gln concentrations and the percent absorption of nutrient substrates at any time point were observed. Similarly, no correlation between the change in Cit or GLN concentration and the change in % nutrient absorption was observed (baseline vs weeks 4 and 12, respectively). By weeks 12 and 24, 7 and 13 subjects were weaned completely from PN, respectively. However, baseline plasma Cit or Gln did not predict PN weaning at these time points. We concluded that plasma Cit (but not Gln) concentrations appeared to be an indicator of small intestinal length in adult SBS. However, neither plasma Cit nor Gln was a biomarker for intestinal absorptive function in this cohort of patients with SBS.

Determination of free amino acids in pig plasma by precolumn derivatization with 6- N-aminoquinolyl- N-hydroxysuccinimidyl carbamate and high-performance liquid chromatography

A high-performance liquid chromatographic method for measuring amino acids in pig plasma has been developed by using 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate as a precolumn derivatization reagent. With the method presented here it was possible to separate 24 amino acids in pig plasma within one single run in 45 min, while only 18 amino acids were properly separated with the original AccQ.Tag conditions used for analysis of hydrolysate samples.The recovery was above 90% for most amino acids, and the mean coefficient of variation of the retention times below 0.30%. The within- and between-assay reproducibility for the determination of plasma amino acids showed C.V.s below 2.2% and 4.5%, respectively, for most amino acids analyzed. In the present work, most of the plasma amino acids were assayed with high sensitivity, accuracy and good reproducibility in a relatively short time and on very small amounts of sample.

Pharmacological characterization of phenytoin-resistant amygdala-kindled rats, a new model of drug-resistant partial epilepsy

By repeated treatment with phenytoin, subgroups of animals with different response to phenytoin were selected from a large group of amygdala-kindled rats. In one subgroup (‘phenytoin responders’) phenytoin induced reproducible increases in focal seizure threshold, while in another subgroup (‘phenytoin nonresponders’) animals were resistant to phenytoin. These phenytoin nonresponders, which comprised about 12% of the kindled animals tested, did not differ from phenytoin responders in drug levels, drug adverse effects or location of the stimulation electrode in the amygdala. Treatment of phenytoin responders and nonresponders with other primary antiepileptic drugs showed that valproate and phenobarbital induced much smaller increases in focal seizure threshold in phenytoin nonresponders than in responders, whereas carbamazepine induced about the same threshold increase in both groups. The novel antiepileptic drug vigabatrin, which acts by increasing GABA levels, exerted anticonvulsant effects in phenytoin responders but was inactive in nonresponders. Determination of plasma amino acids before and after vigabatrin treatment demonstrated marked differences in biochemical responses to vigabatrin although drug levels were about the same in both groups. The data demonstrate that amygdalakindled rats with phenytoin resistance offer a basic approach to the investigation of mechanisms of drug resistance in epilepsy. Furthermore, these animals may be used in the evaluation of new anticonvulsant drugs for treatment of partial seizures which do not respond to the currently available therapies.

Automatic Pre-Column Derivatization and Reversed-Phase High Performance Liquid Chromatography of Primary and Secondary Amino Acids in Plasma with Photo-Diode Array and Fluorescence Detection

Abstract An automatic pre-column double derivative procedure has been established for the determination of plasma amino acids by reversed-phase high performance liquid chromatography. O-phthaldialdehyde/3-mercaptopropionic acid and fluorenylmethyl-chloroformate were used as reagents for the derivatization. The optimal conditions for separation and detection and the stability of derivative were investigated. This method has the advantages of automatic pre-column derivatization and analysis; simple and economic mobile phase; shorter analysis time with optimal separation; high precision for retention time and peak area; higher sensitivity and reliable peak identification and simultaneous determination of both primary and secondary amino acids. Plasma amino acid levels in 160 healthy children were determined and the results showed a good agreement with reference values reported previously by using an ion-exchange chromatographic method. Patients suffering from several type of amino acid metabolic abnormality ...

Gas chromatography of amino acids: Use of a fused‐silica capillary column in the determination of plasma amino acids

AbstractA capillary chromatographic procedure using a fused silica column is described which can be used to quantitatively determine amino acids in plasma following the pre‐chromatographic “clean‐up” described in a recent paper [1]. In substituting this procedure for that involving a packed column, advantage has been taken of the greater resolving power to separate amino acids from background component peaks. In order to extend this advantage and provide a sound basis for quantitative analysis, the technique of cold on‐column injection was employed. As a result, good precision of standard analysis was obtained with relative standard deviation (RSD) values for all amino acids of less than 4%. Application of the entire procedure to plasma samples yields RSD values of better than 10% for all amino acids with recoveries ranging from 72% to 104%. Simultaneous determination of plasma amino acid levels by gas chromatography (GC) using capillary columns and by classical ion exchange (CIE) showed reasonable agreement. Statistical evaluation showed no significant difference between twelve amino acids. Values for the remaining two, namely, phenylalanine and histidine are significantly different (p &lt; 0.005). Comparison of the values obtained from GC capillary and packed columns reveals no significant difference between fourteen amino acids. Significant differences exist between results for phenylalanine and tyrosine (p &lt; 0.001). It is concluded that there is good agreement between data obtained by GC capillary and CIE techniques and that differences between results for phenylalanine and histidine are method related.

Determination of plasma amino acids by gas chromatography

A complete methodology, incorporating a novel clean-up technique, for quantitative determination of amino acids in plasma by gas chromatography is described. Glucose, a component causing major analytical interference, is removed by an enzymic reaction included in the pre-chromatographic clean-up. The procedure for derivatisation of amino acid standards is shown to be reproducible down to a level of 2.5 μg for each amino acid, relative standard deviations for all amino acids except arginine and histidine being 3% or lower. For the entire procedure applied to plasma, relative standard deviations for most amino acids are below 5% with recoveries ranging from 90 to 120%. Normal values, obtained using eighteen plasma samples, are in reasonable agreement with published data. Plasma amino acid values were determined simultaneously by gas chromatographic and ion-exchange chromatographic techniques. Statistical evaluation shows there to be no significant difference between corresponding values for eleven amino acids. Values for tyrosine, histidine and particularly phenylalanine show significant differences ( p < 0.001).

Evaluation of conditions for the HPLC determination of plasma amino acids using precolumn derivatization

AbstractFor the determination of free amino acids in plasma, the conditions for precolumn derivatization of the amino acids and the chromatographic separation were examined. The isoindole products, formed by the reaction of the primary amino acids with orthophthalaldehyde (OPA), were readily separated by RPLC and detected spectrofluorometrically using an excitation wave‐length of 300 nm and an emission cut‐off filter of 440 nm. Since the sensitivity of this method permits determination of amino acids in the femtomole range, the analysis can be performed on samples as small as 10 μl of filtered plasma or serum. The separation is achieved in approximately 35 minutes with good precision for the majority of the amino acids.

702 POSTPRANDIAL PLASMA AMINO ACID PATTERNS IN INFANTS FED CASEIN- AND WHEY-BASED FORMULAS

The postprandial plasma amino acid patterns in infants fed a casein-or whey-predominant formula were assessed to determine the order of the limiting amino acids. Healthy fullterm infants were studied at birth, 1, 2, and 3 months of age. All infants were fed solely a casein or whey formula. At the first feed of the day, fasting, 1-, and 2-hr postprandial venous blood samples were obtained for the determination of plasma amino acids by standard automated techniques. The volume of milk consumed was determined by pre- and post-feeding weights of the infants using an electronic balance. Results of whey-fed infants expressed as change over baseline (Mean ± SD) are shown: Postprandial plasma tryptophan and cystine levels showed marked depressions at 3 months of age; plasma glycine levels fell persistently at 1 and 3 months of age. Thus, nonessential nitrogen and glycine may be limiting nutrients in infants fed a cow's milk whey-predominant formula.

Plasma amino acid pattern in burn subjects: Influence of septicemia

A comparison of plasma amino acid levels was made between two groups of burn subjects, septic and non-septic. On day 4 after injury, results do not show any differences between patients who later went on to develop sepsis on day 7 and those who remained non-septic. Similarly, no differences between these two groups were discernable on day 7. Thus metabolic variations induced by septicemia do not seem to modify the usual burn-induced amino acid variations; consequently, plasma amino acid determination in burn patients do not appear to predict septicemia.

Plasma amino acid pattern in burn subjects: Influence of septicemia

<h2>Abstract</h2> A comparison of plasma amino acid levels was made between two groups of burn subjects, septic and non-septic. On day 4 after injury, results do not show any differences between patients who later went on to develop sepsis on day 7 and those who remained non-septic. Similarly, no differences between these two groups were discernable on day 7. Thus metabolic variations induced by septicemia do not seem to modify the usual burn-induced amino acid variations; consequently, plasma amino acid determination in burn patients do not appear to predict septicemia.

Plasma amino acid levels in the first few days after burn injury and their predictive value.

Plasma amino acid concentrations were studied in 27 burn subjects on the day of injury and on day 4, as a function of the burn surface area and the outcome. On the day of injury, compared to normal subjects, plasma amino acid levels were generally higher in the most severely burned patients (significantly for ALA, GLY, HYP and PHE) and in nonsurvivors (significantly for ALA, PHE, GLY, PRO and ORN). In burn patients all initial values were higher in nonsurvivors than in survivors; for ALA, GLY, and LEU, the difference was significant. Between the day of injury and day 4, different variations were noticed depending on the amino acid in question and the outcome. On day 4, plasma amino acid levels were lower in nonsurvivors, significantly for GLU + GLN, THR, SER, PRO, ASP + ASN, LEU, ILEU and VAL. Our data suggest that plasma amino acid determination could be used as an accurate element of prognosis after burn injury.

A rapid and sensitive procedure for the quantitative determination of plasma amino acids

A rapid and sensitive procedure for the quantitative determination of plasma amino acids

Determination of plasma amino acids in small samples with the use of Dansyl-chloride

A radiochemical method based on dansylation of plasma samples with Dansyl-chloride and thin alyer chromatography on polyamide sheets is presented for the quantitative individual determination of plasma amino acids in very small blood samples. The results are first corrected with a norvaline internal standard and secondly with specific recovery factors for each amino acid. The results agree slosely with other methods and with already published plasma normal amino acids values in healthy adult rats.