Whole Blood Amino Acid Research Articles

Development and validation of a platform based on 3D stochastic sensors for fast screening of whole blood and brain tissue for brain cancer

A novel stochastic platform was assembled by integrating a sensor based on single-wall carbon nanotube paste and ZnO modified with (Z)-N-[2-(4-hydroxyphenyl) ethyl] octadec-9-enamide for the enantioanalysis of two amino acids in whole blood and brain tissue tumor samples. The stochastic platform was utilized to obtain wide concentration ranges for the L-tryptophan, D-tryptophan, L-glutamine, and D-glutamine. The reported sensitivities for both isomers of glutamine exhibited a comparable magnitude order of 107. In contrast, among the four enantiomers, D-Trp displayed the highest sensitivity, measuring at 4.43 × 108. The limits of quantification were in the order of 1.00 × 10−18 mol•L−1. The efficacy of the platform was demonstrated through successful testing on biological samples. The advantages of using this type of platform for such analysis are the possibility of identifying and quantifying in one run different types of biomarkers as well as other substances of biological importance usually requested by medical doctors to be quantified, which can take the test from a fast-screening method to a fast diagnostic method. The analysis is performed within minutes, which is very important for illnesses such as brain cancer that can develop quickly in the body. The early detection of brain tumors can save lives. Furthermore, the portability of the developed detection technology makes it suitable for on-site analysis.

Steady state microdialysis of microliter volumes of body fluids for monitoring of amino acids by capillary electrophoresis with contactless conductivity detection

BackgroundThe availability of dialysis membranes in the form of hollow fibres with diameters compatible with the fused silica capillaries used in capillary electrophoresis is very limited. However, haemodialysis bicarbonate cartridges commonly used in human medicine containing polysulfone hollow fibres are available on the market and are used for the fabrication of coaxial microdialysis probes. The miniature probe design ensures that steady-state conditions are achieved during microdialysis of minimal volumes of body fluids. ResultsA coaxial microdialysis probe with a length of 5 cm and an inner diameter of 200 μm is used for microdialysis of 10 μL of body fluid collected into a sampling fused silica capillary with an inner diameter 430 μm. Microdialysis is performed into 0.01 M HCl as a perfusate at stopped flow and 2 μL of the resulting microdialysate are subjected to analysis by capillary electrophoresis with contactless conductivity detection. Microdialysis pre-treatment is verified by analysis of 11 common amino acids at a 100 μM concentration level, resulting in recoveries of 98.3–102.5%. The electrophoretic separation of amino acids is performed in 8.5 M acetic acid at pH 1.37 as a background electrolyte with analysis time up to 4.5 min and LOD in the range of 0.12–0.28 μM. The reproducibility of the developed technique determined for the peak area ranges from 1.2 to 4.5%. Applicability is tested in the quantification of valine and leucine in plasma during fasting and subsequent reconvalescence. SignificanceThe fabrication of a coaxial microdialysis probe for the laboratory preparation of microliter volumes of various types of clinical samples is described, which is coupled off-line with capillary electrophoretic monitoring of amino acids in 2 μL volumes of microdialysate. The developed methodology is suitable for quantification of 20 amino acids in whole human blood, plasma, tears and has potential for analysis of dry blood spots captured on hollow fibre.

Amino acid profiling as a screening and prognostic biomarker in active tuberculosis patients

BackgroundTuberculosis (TB) is one of the world's most deadly chronic infectious diseases; early diagnosis contributes to reducing disease transmission among populations. However, discovering novel diagnostic and prognostic biomarkers is still an important topic in the field of TB. Amino acid is the basic unit of protein composition, and its structure and physicochemical characteristics are more stable. Therefore, it is a potential target for TB diagnosis and the prediction of TB development. MethodsIn this study, the blood of healthy people (HC), TB patients (TB), cured TB (RxTB), and other non-TB pneumonia patients (PN) were collected to detect the levels of amino acids in whole blood and plasma using ultra-high performance liquid chromatography coupled with mass spectrometry. ResultsWe detected that the amino acid levels correlated with participants status (TB, HC, RxTB, or PN) and the degree of lung damage. The results showed that phenylalanine had a good effect on the screening of TB (AUC = 0.924). We then built a TB prediction model. The model, which was based on the ratio of plasma amino acid content to whole blood amino acid content, showed good performance for the screening of TB, with 84% (95% CI = 60–97) sensitivity and 97% (95% CI = 83–100) specificity. The result of correlation between the HRCT score and amino acid level indicated that the glutamine content of plasma was significantly inversely associated with disease severity. Additionally, ornithine levels in the plasma of RxTB group reduced and four amino acids of which the ratio in plasma to whole blood showed significantly changed. ConclusionsTaken together, amino acid profiling can be used for TB screening, and a multiparameter profiling model is better. The profiling can also reflect the severity of lung damage. Moreover, the amino acid profile is useful for reflecting the efficacy of TB treatment.

Simple and rapid analytical method for detection of amino acids in blood using blood spot on filter paper, fast-GC/MS and isotope dilution technique

A simple and rapid method for quantitative analysis of amino acids, including valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met) and phenylalanine (Phe), in whole blood has been developed using GC/MS. In this method, whole blood was collected using a filter paper technique, and a 1/8 in. blood spot punch was used for sample preparation. Amino acids were extracted from the sample, and the extracts were purified using cation-exchange resins. The isotope dilution method using 2H 8-Val, 2H 3-Leu, 2H 3-Met and 2H 5-Phe as internal standards was applied. Following propyl chloroformate derivatization, the derivatives were analyzed using fast-GC/MS. The extraction recoveries using these techniques ranged from 69.8% to 87.9%, and analysis time for each sample was approximately 26 min. Calibration curves at concentrations from 0.0 to 1666.7 μmol/l for Val, Leu, Ile and Phe and from 0.0 to 333.3 μmol/l for Met showed good linearity with regression coefficients = 1. The method detection limits for Val, Leu, Ile, Met and Phe were 24.2, 16.7, 8.7, 1.5 and 12.9 μmol/l, respectively. This method was applied to blood spot samples obtained from patients with phenylketonuria (PKU), maple syrup urine disease (MSUD), hypermethionine and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and the analysis results showed that the concentrations of amino acids that characterize these diseases were increased. These results indicate that this method provides a simple and rapid procedure for precise determination of amino acids in whole blood.

Gender related differences in the effect of aging on blood amino acid compartmentation☆

This work has been focused on the study of the variations in blood amino acid compartmentation (plasma and blood cells) with aging, both in men and women. Aging is a situation which, under the influence of gender, involves a decline in body weight functions and variations in energy metabolism with a deterioration of muscular metabolism leading to changes in amino acid handling. We determined the blood levels of individual amino acids in whole blood, plasma compartment and blood cell compartment of 51 men and 51 women. Subjects were classified in three age groups—AG1 (18 to 35 y), AG2 (35–50 y) and AG3 (more than 50 y). Aging was accompanied by significant changes in blood levels of amino acids showing gender-linked differences which were distinct for both blood compartments (plasma and blood cells). In men, aging was accompanied by a drop in blood levels of several amino acids, due mainly to the plasma compartment, whereas in women aging brought about a rise in blood levels of various amino acids mainly in blood cell compartment. This paper contributes to enhancing the physiological importance of the blood cell pool in the handling of amino acids.

Blood amino acid compartmentation in men and women with different degrees of obesity

The role of blood cells in the interorgan transport of amino acids and the important size of such a pool has lead us to investigate the possibility that obesity causes alterations in amino acid blood compartmentation in humans and that such alterations show gender-linked differences given the special features of the obese state for each gender. We determined the blood levels of amino acids in whole blood and in the blood cell compartment of 42 men and 46 women. Subjects were classified according to their body mass index (BMI) into one of three groups: lean, overweight, or obese. Obesity was found to increase the levels of blood amino acids in both men and women, although not in the same way: In men, moderate overweight was accompanied by a significant increase of the amino acid blood levels, which were practically unchanged by a greater BMI; however, in women this same increase was observed only in the obese group. This effect is more marked in the cell compartment, where these alterations are actually reflected because plasma contribution values do not change as a whole. In conclusion, total blood and blood cell amino acids were altered in obesity, but in a different way, in both men and women depending on the degree of obesity. This profile of changes is seen to be particularly marked in the cellular pool, which contributes to enhancing the importance of this pool in the handling of amino acids.

Temporal changes in concentrations of amino acids in plasma and whole blood of healthy neonatal foals from birth to two days of age

Summary Temporal changes, as well as differences in distribution, in concentrations of 24 amino acids in plasma and whole blood of neonatal foals were determined from birth to 2 days of age. In addition, differences in concentrations of amino acids in plasma between mare and foal pairs were determined at birth. Significant (P < 0.05) hypoaminoacidemia existed for 15 amino acids in plasma of foals at birth, compared with mares (paired t-test). Concentrations of 7 amino acids (aspartate, glutamate, glutamine, glycine, hydroxyproline, phenylalanine, proline) in plasma of foals were higher (P < 0.05) at birth than in mares, and concentrations of 2 (taurine, tryptophan) were not different (P > 0.05). Significant (P < 0.05) temporal changes for concentrations of 19 of 24 amino acids in plasma were observed during the 48-hour period. Concentrations of 13 of the 19 amino acids in plasma that had significant changes were higher (P < 0.05) at 48 hours. Significant (P > 0.05) effect of time on concentration of 5 amino acids (alanine, methionine, phenylalanine, taurine, threonine) in plasma was not found after birth. Temporal changes in concentrations of 7 amino acids (alanine, asparagine, glutamine, histidine, hydroxyproline, methionine, and threonine) in whole blood were not significantly (P > 0.05) different from those in plasma. Temporal changes for concentrations of the remaining 17 amino acids in whole blood were significantly (P < 0.05) different, compared with plasma. Distribution of the concentrations of 18 amino acids between whole blood and plasma was significantly (P < 0.05) different. Concentrations of 5 amino acids (citrulline, cystine, glutamine, methionine, tryptophan) were significantly (P < 0.05) lower in whole blood than in plasma, whereas concentrations of 13 amino acids were significantly (P < 0.05) higher in whole blood vs plasma. Concentrations of 6 amino acids (asparagine, isoleucine, leucine, proline, serine, valine) in whole blood were not significantly different from concentrations in plasma. Significant differences in temporal patterns of concentrations of amino adds in plasma and whole blood may be attributable to nutritional or physiologic changes associated with parturition. Significant differences between concentrations of amino acids in whole blood and plasma may be attributable to ontogeny or specificity of transport systems across cell membranes.

Concentrations of amino acids in plasma and whole blood in response to food deprivation and refeeding in healthy two-day-old foals

Summary Concentrations of amino acids in plasma and whole blood in response to 10 hours of food deprivation were determined in healthy 2-day-old foals (n = 8) and were compared with control values in foals of the same age (n = 8) allowed free access to suckle. In addition, response of concentrations of amino acids in plasma to 15 minutes of free-access suckling was determined at the end of the 10-hour period in both groups. Response of 13 amino acids in plasma of food-deprived foals was significantly (P < 0.05) different, compared with that in control foals. Concentrations of 3 amino acids (alanine, glycine, and phenylalanine) in plasma increased significantly (P < 0.05), whereas concentrations of 7 amino acids (asparagine, citrulline, histidine, ornithine, proline, tryptophan, and tyrosine) in plasma decreased significantly (P < 0.05) during food deprivation. Response of concentrations of 2 amino acids (glycine and histidine) in whole blood was significantly (P < 0.05) different from that in plasma of food-deprived vs control foals. Refeeding of food-deprived foals resulted in significantly (P < 0.05) different responses for concentrations of all but 2 amino acids (cystine and taurine) in plasma, compared with responses in controls. Changes in concentrations of amino acids in plasma and whole blood of foals in response to food deprivation are similar to those in foals with septicemia and in children with grade 1 or 2 kwashiorkor. The significantly different response of food-deprived foals to refeeding may be attributable to increased protein intake or altered physiologic state.

Influence of anoxia on free amino acid levels in blood, liver and skeletal muscles of the goldfish, Carassius aurantus L.

1. 1. Fiften free amino acids were quantified in blood, liver, white muscle and red muscle of fasting goldfish subjected to 12 hr of anoxia. Normoxic goldfish were used as controls. 2. 2. The anoxia treatment significantly raised the total pool of free amino acids in whole blood and liver. 3. 3. A general trend is observed in all the anoxic tissues: the free amino acids, for the most part, increase in absolute amounts. A strong accumulation is observed for some specific (groups of) free amino acids: alanine, the branched-chain amino acids and the aromatic free amino acids. 4. 4. Only the amide glutamine decreases substantially in the white muscle. 5. 5. These measurements indicate proteolysis of tissue proteins in anoxic goldfish and are interpreted as evidence for a coupling of glycolysis and amino acid catabolism.

Free amino acids in whole blood and plasma of two cyprinids, Cyprinus carpio and Carassius auratus (Cyprinidae: Teleostei)

1. 1. Free amino acids were measured using a reversed-phase HPLC system in whole blood, plasma and erythrocytes of two fresh water cyprinids ( Cyprinus carpio, the common carp and Carassius auratus, the goldfish). 2. 2. Free amino acid recoveries were determined for whole blood, plasma and erythrocytes. Recoveries were high (80–90%) and were used for correction of free amino acid amounts in the biological samples. 3. 3. Taurine (2-aminoethanesulfonic acid) is the most common free amino acid in common carp and goldfish blood (respectively 9.2 and 5.5 mmol/1); the total amount of “protein-derived” free amino acids in whole blood of the common carp is significantly higher than that of the goldfish (5.3 ± 0.6 mmol/1 versus 3.9 ± 0.2 mmol/1; P < 0.01). The same holds for plasma free amino acids (common carp: 5.0 ± 1.0 mmol/1; goldfish: 3.6 ± 0.3 mmol/1). 4. 4. In these cyprinids, the essential free amino acids are more concentrated in the plasma, while the acidic free amino acids, as well as taurine and glycine, are restricted to the red blood cells. 5. 5. Some data on the free amino acid pools in the blood compartments of the crucian carp ( Carassius carassius) and the tilapia ( Sarotherodon mossambicus) show that the amino acid pattern of Carassius auratus is close to the other cyprinids but that tilapia has a far different pattern.

Abnormal regulation of venous alanine after glucose ingestion in myotonic dystrophy.

The concentration of amino acids in whole blood was measured before and during standard 5 h oral glucose tolerance testing in six male patients with myotonic dystrophy and five normal males. The plasma levels of insulin and glucose were also determined. From 90 to 240 min after glucose ingestion there was a striking decline in venous alanine concentration in the patients with myotonic dystrophy in contrast to a slight rise in alanine in the normal group. The patients displayed normal glucose tolerance, and there was a sustained fall in the venous concentration of the insulin-sensitive amino acids comparable with that seen in the normal controls. However, the patients showed a threefold increase of plasma insulin after glucose. These data indicate an abnormal regulation of alanine in myotonic dystrophy which may be the result of an alteration in muscle synthesis of this amino acid. This defect in alanine synthesis may be due to a decreased availability of intracellular pyruvate caused by the insulin resistance that exists in these patients.

Fetal and Maternal Amino Acid Concentrations During Fasting in the Ewe

The present investigation was undertaken to quantitate the whole-blood amino acid concentrations of the mother and fetus over an extended period of fasting using a chronic sheep preparation. Fetal amino acid levels generally increased or remained relatively high during the fast (e.g., alanine, glycine, valine, leucine). In contrast to the situation in the fetus, most amino acids (alanine, glycine, glutamine, serine, and threonine) decreased in concentration in the ewe with fasting. Alanine levels changed most dramatically in the ewe, decreasing to less than 50% of the fed state values within 72 h of fasting. Therefore, while the supply of gluconeogenic amino acids becomes limited within the maternal circulation during fasting, levels of these amino acids remain high or increase in the fetal arterial blood.

Amino acid metabolism in the ovine fetus.

Quantitative measurements of the umbilical uptake of a variety of substrates were made in the ovine fetus in both the fed state and after 5 days of maternal fasting. Blood flow was measured in both study periods using the antipyrine method, with the simultaneous measurement of umbilical vein and arterial concentrations of glucose, oxygen, and 25 amino acids in whole blood. Umbilical blood flow and fetal oxygen consumption remained unchanged between the fed and fasted states. Umbilical glucose uptake decreased from a mean of 6.85 to 2.79 mg X kg-1 X min-1 with a concomitant fall in the glucose:oxygen quotient from a mean of 0.70 to 0.31. Whereas the majority of gluconeogenic amino acids decreased in concentration in the maternal circulation during fasting, the branched-chain amino acids, as well as 3-methylhistidine, increased. In contrast the majority of amino acids in the fetal arterial circulation either increased significantly or remained unchanged in concentration. Although the fetal excretion of urea increased by 70% during the period of maternal fasting, the total fetal uptake of nitrogen in the form of free amino acids via the umbilical circulation did not change (1,337 vs. 1,027 mg X kg-1 X day-1). These data suggest that the supply of amino acids from the mother to the fetus does not change dramatically during maternal fasting. Therefore, the fetus must either catabolize amino acids previously destined for fetal tissue synthesis and/or begin catabolizing its own protein stores.

Free amino-nitrogen exchange across the hindquarters of fed and fasted sheep and pigs

SUMMARYFree amino·N estimations were used to monitor the movement of free amino acids in blood passing through the hindquarters of sheep and growing pigs. The net uptakeor release of free amino·N was calculated from arterio-venous differences in the concentration of free amino·N measured in whole blood and plasma at short intervals of time.In both species the net change was an apparent release of free amino·N into venous blood. The release increased on fasting. Net uptake of free amino·N from blood was occasionally found at single sampling times due to loss from the cell compartment in sheep and from the plasma compartment in pigs. The uptake measured in whole blood was always less than in a single compartment of blood. The greatest fluctuation in free amino·N concentration occurred in the cells of aorta blood.It is concluded that both blood cells and plasma have independent roles in the net flux of free amino·N requiring determinations of amino acids in both whole blood and plasma to describe the exchange of blood free amino acids with tissue. A negative arteriovenous difference of free amino·N in blood across skeletal muscle is normal for healthy well-fed animals. Fasting increases the negativity.

Postprandial distribution of free amino acids between plasma and erythrocytes of common carp ( Cyprinus Carpio L.)

1. 1. Free amino acid concentration in whole blood, plasma and erythrocytes washed with saline were determined in common carp of an individual weight of 500 g. 2. 2. Glutamate and aspartate have shown the highest concentrations within the erythrocytes. 3. 3. Among these amino acids which have shown the concentration ratio (erythrocyte-plasma) below unity were leucine, valine, histidine and lysine. 4. 4. Post-prandial level has shown the maximum concentration of free amino acids more or less 3 hr after feeding. 5. 5. Several amino acids have shown significant correlations between whole blood and plasma, and whole blood and erythrocyte concentrations. 6. 6. Levels of essential free amino acids in whole blood and plasma were positively correlated with their levels in the diet.

Free amino acids in the blood of fetal and maternal Weddell seals.

The content of free amino acids in whole blood was measured in near-term gravid female Weddell seals (Leptonychotes weddelli) and compared with fetal amino acid profiles during rest and during experimental diving. With the exception of taurine and glutathione, Ninhydrin-reactive components of acid extracts of blood occurred in higher concentrations on the fetal side of the placenta than on the maternal side. Compared with humans the Weddell seal displayed higher ratios of fetal arterial to maternal arterial levels for aspartate, glycine, alanine, valine, tyrosine, phenylalanine, and total branched-chain amino acids. In the resting state the total free amino acid concentration in maternal blood was only about 70% as large as the total amino acid concentration in fetal blood, compared with a value of over 80% for humans. Only modest changes in the concentrations of specific amino acids occurred during simulated awake diving, but the overall maternal pools of glycine, glutamate, and glutamine were augmented, creating favorable conditions for uptake by the fetus.

Free amino acid profiles in blood during diving and recovery in the Antarctic Weddell seal

The blood content of free amino acids was measured in the Antarctic Weddell seal (Leptonychotes weddelli) to assess their possible role in energy metabolism during simulated diving and recovery sequences. The total free amino acid pool size was similar to that in the common harbour seal and in man, but there were a few notable differences in amino acid contributions to the total pool. For example, in the Weddell seal alanine concentrations were unusually low. During diving, the content of free amino acids in whole blood changed hardly at all, unless glutathione levels also changed. In 5 of 10 dive sequences examined, glutathione was markedly depleted during diving, concomitant with a sharp rise in glutamate and glycine released on hydrolysis of this tripeptide. During recovery, glutathione levels were recharged quite rapidly. About half the glutamate and glycine expected from glutathione hydrolysis was expressed by elevated levels of these two amino acids; similarly, only a part of the glutathione reformed during recovery was represented by decreasing glutamate and glycine levels. The only other significant changes in amino acid levels during recovery were shown by alanine and glutamine, both of which increased in whole blood for periods of about 30–60 min. It was concluded that under most simulated diving conditions, free amino acid pools and profiles are not strongly perturbed. Metabolic conditions may arise, however, leading to large drops in glutathione levels, which in turn may perturb the free amino acid pool.

Transport of amino acids in whole blood and plasma of sheep.

Comparisons of amino acid transport in both whole blood and plasma were made across the portal-drained viscera, liver, kidneys, and hindquarters in sheep. Lyophilized samples could be stored at -20 degrees C for up to 17 wk with little or no changes observed on reanalysis. Only glutamine, taurine, and serine showed any loss (18, 23, and 16%) over a 3.5-yr period. However, there was no concomitant rise in glutamate over the same period, suggesting that glutamate does not simply deamidate. There were no differences in arterial concentrations of alanine, citrulline, isoleucine, and valine between whole blood and plasma. However, glutamate, asparagine, glycine, serine, threonine, taurine, ornithine, leucine, lysine, phenylalanine, and tyrosine were higher in whole blood compared to plasma, and glutamine and arginine were lower. Whole blood and plasma fluxes for all amino acids were positively correlated, and there were no differences in their regression lines among tissues. Plasma, however, underestimated amino acid transport glutamine, glutamate, and taurine at greater rates than plasma. It is concluded that blood cellular transport, per unit volume, was always concomitant to and usually at the same rate as plasma transport over all tissues measured. Plasma therefore reflects amino acid transport but underestimates total transport to about the extent of the packed cell volume and in cases of glutamine, glutamate, and taurine, perhaps even more.

Whole-blood and plasma amino acid analysis: gas-liquid and cation-exchange chromatography compared.

Whole-blood and plasma amino acid analysis: gas-liquid and cation-exchange chromatography compared.

Influence of dietary amino acid patterns on the free amino acid contents of blood and muscle of rainbow trout ( Salmo gairdnerii R.)

1. 1. Groups of rainbow trout were given one or other of two diets that differed in amino acid profile for two months; concentrations of free amino acids in whole blood and latero-dorsal muscle were then measured. The effects of temperature (12 and 18°C in freshwater) and salinity on the concentrations of free amino acids in these tissues were also observed. 2. 2. Although both diets apparently met the essential amino acid requirements of the trout and were isoenergetic, they nevertheless had different nutritional values for trout. 3. 3. Patterns of free amino acids in tissues of trout given the two diets were different. Blood and muscle amino acid fractions were affected differently by changes in dietary amino acids.