Acid In Whole Blood 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.

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.

Automating DUID methods using robotics for rapid high throughput sample preparation – Analysis of Delta-9-Tetrahydrocannabinol and its major metabolite Delta-9-tetrahydrocannabinol acid in whole blood

To fully automate, using liquid-handling robotics, a liquid chromatography tandem mass spectrometry method for the analysis of delta-9-tetrahydrocannabinol (THC) and delta-9-tetrahydrocannabinol acid (THC-A) in whole blood. Uptake in usage of ultra-high performance liquid chromatography (UHPLC) has led to development of methods with very fast (typically < 5 minutes) chromatographic run times used in the forensic toxicology laboratory. Nevertheless, use of whole blood for quantitative analysis continues to present challenges for delivering high throughput methods with rapid analysis times. Whole blood requires the use of solid phase extraction (SPE) for sample pre-treatment and preparation, which is time consuming and labour intensive. Recent developments in SPE technologies have produced 96-well format platforms that are used to extract greater number of samples in parallel and are ideally suitable for automated liquid handlers. With recent sensitivity increases in mass spectrometers, smaller volumes (< 250 μL per aliquot of blood) can be used for extraction in 96-well plates. This facilitates the use of robotic platforms together with rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) methods to give truly high throughput and fast analytical methods, increasing method efficiencies, reducing human intervention and errors. Here we present the development, validation and implementation of a fully automated robotic sample preparation method for whole blood for analysis of THC and THC-A in UK driving under the influence of drugs (DUID) cases. Aliquots (200 μL) of blank blood and casework samples previously dispensed into 96-well plates by a robotic system (BRAND) were prepared by the liquid handling system (Hamilton). Casework samples and blank blood were spiked with internal standard and blank blood samples were further spiked to produce calibrants and quality controls. The plate is then mixed with the addition of a cell lysis solution and the aliquots transferred to a protein/lipid depletion (PPT) plate (Waters). A positive pressure manifold was used to draw the solution through the PPT plate into a collection plate where the precipitate was dried. The precipitate was reconstituted and loaded by the Hamilton onto a C18 microelution SPE plate (Waters). Following extraction, the samples are reconstituted and the collection plate is placed in the autosampler for analysis. The LC-MS/MS method consists of a 2.1 mm × 50 mm, 1.7 μm, C18 column running a 4 minute gradient of water and acetonitrile with 0.1% formic acid. The method was successfully validated, with LLOQs for THC and THC-A of 1 μg/L and 5 μg/L, respectively. Precision (%CV) for THC was < 8% and < 6% for THC-A. Accuracy was within 15% of the spiked concentration for THC and THC-A across all validation batches ( n = 11). The robotic platform was able to undertake sample pre-treatment and extraction in 3.5 hours. A batch of 40 casework samples can be completed in 13 hours including the LC-MS/MS run time. The introduction of this method to our laboratory has resulted in a significant increase in the speed for a sample to be processed and results obtained. We can prepare and extract double the amount of samples in less than half the time taken by our manual method. We have reduced the amount of sample used and decreased our turnaround times all for our customer's benefit. Full automation of the sample preparation of whole blood samples using liquid handling robots can significantly improve laboratory throughput and capacity and efficiently utilise high speed chromatography providing rapid analysis from start to finish.

Enantioanalysis of Aspartic Acid Using 3D Stochastic Sensors

Enantioanalysis of aspartic acid is of high importance for the metabolomics involved in gastric cancer. Therefore, two 3D stochastic sensors based on sulfur-doped graphene modified with protoporphyrin IX are reported for the enantioanalysis of aspartic acid. The 3D stochastic sensors were enantioselective with high sensitivity (the highest 1011 and 108 s−1/mol L−1 for l‐ and d-aspartic acid) and limits of quantification of 1 fmol L−1 and 1 pmol L−1 for l‐ and d-aspartic acids. The whole blood screening method based on utilization of the 3D stochastic sensors was validated using blood collected from healthy people and from patients with confirmed gastric cancer. Recoveries higher than 98.00% were recorded for enantioanalysis of aspartic acid in whole blood.

Mobile healthcare system based on the combination of a lateral flow pad and smartphone for rapid detection of uric acid in whole blood

Excessive production of uric acid (UA) in blood may lead to gout, hyperuricaemia and kidney disorder; thus, a fast, simple and reliable biosensor is needed to routinely determine the UA concentration in blood without pretreatment. The purpose of this study was to develop a mobile healthcare (mHealth) system using a drop of blood, which comprised a lateral flow pad (LFP), mesoporous Prussian blue nanoparticles (MPBs) as artificial nanozymes and auto-calculation software for on-site determination of UA in blood and data management. A standard curve was found to be linear in the range of 1.5–8.5 mg/dL UA, and convenience, cloud computing and personal information management were simultaneously achieved for the proposed mHealth system. Our mHealth system appropriately met the requirements of application in patients’ homes, with the potential of real-time monitoring by their primary care physicians (PCPs).

Fatty acid profile and estimated desaturase activities in whole blood are associated with metabolic health

BackgroundThe aim was to investigate if fatty acid profile and estimated desaturase activities; stearoyl CoA-desaturase (SCD), delta-5-desaturase and delta-6-desaturase (D5D; D6D), differ between individuals with metabolically healthy (MH) and unhealthy (MU) phenotypes. We also explored these associations according to BMI categories.MethodsMen and women at moderately elevated risk of cardiovascular disease were included in this cross-sectional study (n = 321). If subjects met ≥4 out of 5 criteria (elevated triglycerides, total and LDL-cholesterol, HbA1c and low HDL-cholesterol), they were classified as MU (n = 52). If levels were within reference ranges for ≥3 of the same criteria, subjects were classified as MH (n = 150). Utilizing the entire population, a score ranging from 0 to 5 denoting the number of MU criteria met was computed. Estimated desaturase activities were calculated as product-to-precursor ratio of fatty acids in whole blood (SCD16 [16:1n7/16:0], SCD18 [18:1n9/18:0], D5D [18:3n6/18:2n6], D6D [20:4n6/20:3n6]).ResultsIndividuals with MH had lower estimated SCD16 and SCD18 activities, whereas estimated D6D activity was higher compared to MU. Similar, SCD16 and SCD18 increased, whereas D6D decreased with increasing criteria of MU. Trends were similar across BMI categories.ConclusionsThis study supports the notion of estimated desaturase activities as possible novel biomarkers of metabolic health irrespectively of BMI.

Non-enzymatic electrochemical sensor for the simultaneous determination of adenosine, adenine and uric acid in whole blood and urine

Herein we report for the first time, a simultaneous voltammetric sensor for adenosine, adenine and uric acid using a pencil graphite (PG) substrate electropolymerised with 2-amino-5-mercapto-1,3,4-thiadiazole in phosphate buffer with pH 7. The developed sensor was characterized morphologically, structurally and electrochemically. Under the optimum conditions, a linear concentration range of 0.2–25.6 µM, 0.05–12.8 µM and 5–1200 µM was obtained with a detection limit of 0.19 µM, 0.039 µM and 2.74 µM for adenosine, adenine and uric acid respectively. Viability of the sensor was also tested in blood and urine samples, and it was found that the sensor is an excellent tool for the simultaneous determination of the above mentioned targets.

Nanopore Determination of Nucleic Acids in Whole Blood Based on a Displacement Reaction Strategy

Nanopore stochastic sensing has attracted substantial interest as a label-free versatile tool to explore a wide variety of applications. However, utilizing nanopore sensors to analyze clinical samples remains a significant challenge due to the severe matrix effect. Here, we take advantage of displacement chemical reaction to selectively extract the target nucleic acid from whole blood, which can then be accurately detected by nanopore sensor. Our method is simple and sensitive: does not involve tedious centrifugation separation and elution procedures or require sample dilution. Given the importance of nucleic acids as valuable biomarkers for various diseases, our developed displacement chemistry-based nanopore sensing strategy offers the potential for fieldable / point-of-care diagnostic application.

The stability of HIV-1 nucleic acid in whole blood and improved detection of HIV-1 in alternative specimen types when compared to Dried Blood Spot (DBS) specimens

BackgroundCommercially-available kits for HIV-1 detection include instructions for detecting HIV-1 in plasma and DBS, but don’t support other specimen types. ObjectivesShow quantitative stability of HIV-1 total nucleic acid (TNA) in blood and improved HIV-1 detection in alternative specimen types. Study designWhole blood and DBS specimens, tested as part of an external quality assurance program for qualitative HIV-1 detection, were used to evaluated error rates (false negative [FN], false positive [FP] and indeterminant [IND] results) across assays (internally developed [IH], Roche Amplicor [RA], and Roche TaqMan Qual [TQ]) and specimen types (frozen whole blood [BLD], DBS and cell pellets [PEL]). A modified Roche TaqMan HIV-1 assay was used to quantify HIV-1 TNA. ResultsSignificantly higher error rates were noted in DBS across all of the assays (4% vs. 0% for DBS and PEL, IH, p = 0.005; 4% vs. 0.1% for DBS and PEL, RA, p < 0.001; 10% vs. 1% for DBS and PEL or BLD, TQ, p < 0.001). HIV TNA concentration is stable in BLD (day 1 vs. day 10, p = 0.39) and higher than DBS (p < 0.001). ConclusionsTransporting refrigerated whole blood for centralized processing into alternative specimen types will improve the sensitivitiy of HIV-1 detection in samples with low virus loads.

A multiplex paper-based nanobiocatalytic system for simultaneous determination of glucose and uric acid in whole blood.

In this work, a versatile point-of-care assay platform based on a microfluidic paper-based analytic device (μPAD) was developed for the simultaneous detection of multiple targets. The μPAD with a central zone and six test zones is fabricated by a simple and inexpensive wax printing method. A flower-like hybrid nanocomplex synthesized with specific dual enzymes and Cu3(PO4)2 inorganic nanocrystals is spotted in the test zones on the μPAD, followed by the introduction of assay targets. Using dual-enzyme inorganic hybrid nanoflowers in the μPAD as nanobiocatalysts, which preserve the activity and enhance the stability of the enzymes, based on the H2O2-mediated catalytic oxidizing chromogenic reaction produced by glucose/uric acid, the developed multiplex paper-based nanobiocatalytic system is demonstrated to enable simultaneous and sensitive detection of glucose and uric acid with a detection limit of 60 and 25 μM, respectively. More importantly, it has been successfully used for detecting glucose and uric acid levels in human whole blood samples. The developed multiplex paper-based nanobiocatalytic system features very easy fabrication and operation, low cost, and high sensitivity and has promising prospects for a clinical multianalyte point-of-care test.

Molecular recognition of pyruvic acid and folic acid in whole blood

Cyclodextrins immobilized in TiO2Pt–graphene nanocomposite paste simultaneously detect folic and pyruvic acids in whole blood samples.

Essential fatty acid composition and correlates in children with severe acute malnutrition

Severe acute malnutrition (SAM) is a common condition in children living in low-income countries and may be associated with reduced polyunsaturated fatty acids (PUFA) blood levels. The purpose of this study was to describe whole blood fatty acid composition and correlates of PUFA in children admitted with SAM. We conducted a cross-sectional study among children admitted with SAM at Mulago National Referral Hospital and healthy controls. Whole blood fatty acid composition was measured and correlated with clinical data such as oedema, levels of haemoglobin, C-reactive protein and HIV-infection status. Multiple linear regression analyses were used to identify correlates of PUFA. The relative contribution of saturated fatty acid to the fatty acids in whole blood (FA%) were lower in 108 children with SAM compared to 24 well-nourished controls whereas most monounsaturated fatty acids were higher in children with SAM. Total and all n-6 PUFA including linoleic (18:2n-6, LA) and arachidonic acid (20:4n-6, AA), as well as total n-3 PUFA and docosahexaenoic acid (22:6n-3, DHA) were lower in children with SAM. The n-6:n-3 PUFA ratio was also lower in the children with SAM. Haemoglobin was a positive correlate of AA, n-3 docosapentaenoic acid (22:5n-3, n-3 DPA), DHA, total n-6 long chain (LC) PUFA and total n-3 LCPUFA. HIV infected children had 0.87 (0.47; 1.58) %-points less n-6 LCPUFA and 0.61 (0.03; 1.19) %-points less AA than the un-infected children. Children with SAM presented with lower FA% of LCPUFA. HIV infection and low haemoglobin were also associated with lower FA% of LCPUFA, which may be related to lower numbers of blood cells. Nutrition rehabilitation interventions need to pay more attention to the intake of PUFA.

Eicosapentaenoic acid and docosahexaenoic acid in whole blood are differentially and sex-specifically associated with cardiometabolic risk markers in 8-11-year-old danish children.

n-3 long-chain polyunsaturated fatty acids improve cardiovascular risk markers in adults. These effects may differ between eicosapentaenoic acid (EPA, 20∶5n-3) and docosahexaenoic acid (DHA, 22∶6n-3), but we lack evidence in children. Using baseline data from the OPUS School Meal Study we 1) investigated associations between EPA and DHA in whole blood and early cardiometabolic risk markers in 713 children aged 8–11 years and 2) explored potential mediation through waist circumference and physical activity and potential dietary confounding. We collected data on parental education, pubertal stage, 7-day dietary records, physical activity by accelerometry and measured anthropometry, blood pressure, and heart rate. Blood samples were analyzed for whole blood fatty acid composition, cholesterols, triacylglycerol, insulin resistance by the homeostatic model of assessment (HOMA-IR), and inflammatory markers. Whole blood EPA was associated with a 2.7 mmHg (95% CI 0.4; 5.1) higher diastolic blood pressure per weight% EPA, but only in boys. Heart rate was negatively associated with both EPA and DHA status (P = 0.02 and P = 0.002, respectively). Whole blood EPA was negatively associated with triacylglycerol (P = 0.003) and positively with total cholesterol, low density and high density lipoprotein (HDL) cholesterol and HDL:triacylglycerol (all P<0.01) whereas DHA was negatively associated with insulin and HOMA-IR (P = 0.003) and tended to be negatively associated with a metabolic syndrome-score (P = 0.05). Adjustment for waist circumference and physical activity did not change the associations. The association between DHA and HOMA-IR was attenuated but remained after adjustment for fiber intake and none of the other associations were confounded by dietary fat, protein, fiber or energy intake. This study showed that EPA status was negatively associated with triacylglycerol and positively with cholesterols whereas DHA was negatively associated with insulin resistance, and both were inversely associated with heart rate in children. The sex-specific associations with blood pressure confirm our previous findings and warrant further investigation.

Acute organ injury is associated with alterations in the cell-free plasma transcriptome.

BackgroundDespite a genomic revolution in biological sciences, clinical medicine has yet to integrate diagnostics based upon gene expression into practice. While commonly used plasma protein assays rely on organ-specific origins, nearly all nucleic acid in whole blood is derived from white blood cells limiting their utility to diagnose non-immune disorders. The aim of the study was to use cell-free plasma to define circulating messenger RNA sequences diagnostic of acute organ injury, including myocardial infarction (MI) and acute kidney injury (AKI).MethodsIn healthy human subjects (N = 4) and patients with acute MI (N = 4), we characterized the concentration and nature of circulating plasma RNA through spectrophotometry and chromatography. Through reverse transcriptase polymerase chain reaction (RT-PCR) of amplicons up to 939 base pairs, we determined whether this mRNA was intact but of insufficient quantity to sequence. In mice, we induced an acute anterior myocardial infarction through 1 h of ischemia followed by reperfusion of the left anterior descending (LAD) artery. We compared the cell-free plasma transcriptome using cDNA microarray in sham-operated mice compared to ischemia upon reperfusion and at 1 and 4 h. To determine organ specificity, we compared this profile to acute ischemia-reperfusion of the kidney.ResultsIn humans, there is more plasma RNA in those with acute MI than in healthy controls. In mice, ischemia-reperfusion of the LAD artery resulted in a time-dependent regulation of 589 circulating mRNA transcripts with less than a 5% overlap in sequences from acute ischemia-reperfusion injury of the kidney.ConclusionsThe mRNA derived from cell-free plasma defines organ injury in a time and injury-specific pattern.Electronic supplementary materialThe online version of this article (doi:10.1186/2197-425X-2-7) contains supplementary material, which is available to authorized users.

Validated method for the determination of misoprostol acid in whole blood by ultra performance liquid chromatography–tandem mass spectrometry

Misoprostol is a pharmaceutical synthetic compound, analog of prostaglandin E1, frequently used as an abortifacient in not medically supervised or self-induced abortions, particularly in countries with restrictive abortion laws representing a serious public health problem. The aim of this study was to develop and validate a sensitive analytical method for the determination of misoprostol acid in whole blood samples. The samples were prepared by SPE and the chromatographic separation was performed by UPLC–MS/MS using ESI- and MRM mode with an Acquity UPLC® BEH C18 (50mm×2.1mm i.d., 1.7μm) column using a methanol–ammonium 0.1% solution gradient in a total run time of 7.0min. The method showed to be selective and linear in range 25–2000ng/L. The LOD and LOQ were 10ng/L and 25ng/L, respectively. The recovery ranged from 89 to 97%. No carryover and significant matrix effect were observed. The intra- and inter-assay precisions and the inter-assay accuracy results were 4.0% and 5.4%, 5.5% and 4.1%, and −1.4% and −2.8%, for the concentrations 50 and 500ng/L, respectively. The method developed allows the analysis of misoprostol acid in whole blood samples with adequate sensitivity to the concentration range obtained from therapeutic doses. The method was successfully used in a controlled misoprostol administration study and has been applied in our laboratory in the forensic toxicology field.

Enantioselective Determination of Methylphenidate and Ritalinic Acid in Whole Blood from Forensic Cases Using Automated Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry

A chiral liquid chromatography tandem mass spectrometry (LC-MS-MS) method was developed and validated for quantifying methylphenidate and its major metabolite ritalinic acid in blood from forensic cases. Blood samples were prepared in a fully automated system by protein precipitation followed by solid-phase extraction. The LC-MS-MS method was linear in the range of 0.5 to 500 ng/g for the enantiomers of both analytes. For concentrations above the limit of quantification, coefficients of variation were 15% or less, and the accuracy was 89 to 94%. For 12 postmortem samples in which methylphenidate was not determined to be related to the cause of death, the femoral blood concentration of d-methylphenidate ranged from 5 to 58 ng/g, and from undetected to 48 ng/g for l-methylphenidate (median d/l-ratio 5.9). Ritalinic acid was present at concentrations 10-20 times higher with roughly equal amounts of the d- and l-forms. In blood from 10 living subjects that were not suspected of being intoxicated by methylphenidate, the concentration ranges and patterns were similar to those of the postmortem cases. Thus, methylphenidate does not appear to undergo significant postmortem redistribution.

Assay of whole blood (6S)-5-CH3-H4folate using ultra performance liquid chromatography tandem mass spectrometry

Folates act as essential coenzymes in many biological pathways, including the synthesis and methylation of DNA. Low folate concentration in serum and whole blood (WB) is associated with several disease conditions. We describe a stable-isotope-dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantification of (6S)-5-CH(3)-H(4)folate (where H(4)folate is tetrahydrofolate) and non-CH(3)-H(4)folate [sum of HCO-H(4)folate, (6R)-5,10-CH(+)-H(4)folate, (6R)-5,10-CH(2)-H(4)folate, (6S)-H(4)folate, dihydrofolate, and folic acid] in WB. The assay includes a solid-phase extraction procedure after the hemolysis and deconjugation. The method was linear over the concentration range from 0.2 to 200 nmol/L. The limits of detection were 0.40 nmol/L or lower for the folate forms. The interassay coefficients of variation were 7.4% for (6S)-5-CH(3)-H(4)folate and 15.4% for non-CH(3)-H(4)folate. For the folate forms, the recoveries were between 97.1% and 102.7%. Sample preparation caused the generation of artificial folic acid in WB and serum in a dose-dependent manner, which can lead to misinterpretation of the results. The use of antioxidants could not prevent the formation of folic acid. The median fasting WB folate concentrations from 42 nonsupplemented and nonfortified adults were 576 nmol/L (6S)-5-CH(3)-H(4)folate and 73.6 nmol/L non-CH(3)-H(4)folate, and 1,206 nmol/L (6S)-5-CH(3)-H(4)folate and 155 nmol/L non-CH(3)-H(4)folate for 35 adults who had taken 500 μg of folic acid, 50 mg of vitamin B(6), and 500 μg of vitamin B(12) per day orally for 6 months. In conclusion, the UPLC-MS/MS method is fast and has a good sensitivity and selectivity for WB folates. We observed a dose-dependent oxidation of (6S)-H(4)folate, which resulted in the formation of artificial folic acid in serum and WB. To minimize this effect, we recommend a fast sample preparation.

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.

The antimicrobial peptide LL37 and its truncated derivatives potentiates proinflammatory cytokine induction by lipoteichoic acid in whole blood

Interactions of bacterial and host products in activating the innate immune system is an important area to address. The role of lipoteichoic acid (LTA) in these interactions is particularly important because it is understudied in comparison to other factors. This study evaluated the effect of cationic peptides (CPs) on LTA-induced proinflammatory cytokine production in human whole blood and on purified leukocytes. Four different CPs of truncated derivatives from the known peptides LL37, BPI, and CP207 were used. Two of the CPs (IG33 and LL33), derivatives from LL37, potentiated S. aureus LTA induced TNFα, IL-6 and IL-1β production in whole blood. The release of TNFα was increased 30-fold after 16 hours incubation. Intact LL37 also increased LTA-induced TNFα and IL-1β in a time dependent manner. LTA in combination with either LL33 or IG23 demonstrated a synergistic enhanced TNFα and IL-1β secretion on isolated leukocytes but not on purified monocytes. When complexed with IG23 and LL33, the electrophoretic mobility of LTA was altered in a non-denaturating gel electrophoresis. LTA was disaggregated and migrated more rapidly, suggesting an amphiphilic effect of CPs on LTA. In conclusion, LTA synergizes with LL37 and its truncated derivatives and this may lead to proinflammatory cytokine production and cause problems in sepsis therapy.

Effects of one-day reperfusion after transient forebrain ischemia on circulatory system in the rat

Although ischemia/reperfusion injury remains incompletely understood, it appears that reactive oxygen species produced mainly during postischemic recirculation play a critical role. The present study examined the impact of forebrain ischemia and subsequent one-day reperfusion on several blood parameters. We determined glutamate concentration in whole blood, measured Cu/Zn- and Mn-SOD (superoxide dismutase) activity in blood cells as well as plasma, and investigated the prevalence of single and double strand breaks of lymphocyte DNA. The results of our experiment showed that the concentration of glutamic acid in whole blood was increased by about 25%. Antioxidant activity of total SOD and Cu/Zn-SOD was reduced in blood cells and plasma. Mn-SOD activity in blood cells was not affected by ischemic insult and one-day reperfusion, but we detected its significantly lower activity in samples of plasma. We observed a weakly reduced level of double and a significantly elevated level of single strand breaks of lymphocyte DNA. In conclusion, one day of recovery after the ischemic attack failed to return peripheral circulatory system to physiological conditions. Reduced antioxidant capacity in the blood and an elevated level of excitotoxic amino acid glutamate may cause lymphocyte DNA damage, and probably contribute to insufficient postischemic recovery of brain tissue.