Organic Acids In Plasma Research Articles

Galactosemia Diagnosis by Whole Exome Sequencing Later in Life

Galactosemia Diagnosis by Whole Exome Sequencing Later in Life

Quantitative Analysis of Lactate and Other Organic Acids in Plasma: Preanalytical Challenges

Abstract Background and Objectives We developed a gas chromatography–mass spectrometry (GC-MS) method for the analysis of organic acids (OAs) in plasma using liquid-liquid extraction with acidified methanol. This method allows for the simultaneous analysis of several metabolites associated with energy metabolism, including lactic and pyruvic acids. Here, we evaluated the effect of different preanalytical variables (anticoagulants, delayed plasma separation, fasting vs nonfasting status) on plasma organic acid quantitation. Methods Nine organic acids (lactic, pyruvic, 3-hydroxybutyric, acetoacetic, 2-ketoisovaleric, 2-keto-3-methylvaleric, 2-ketoisocaproic, succinic, and glutaric) were extracted by deproteinizing with acidified methanol, oximated to preserve ketoacids, converted to volatile trimethylsilyl derivatives, and detected by GC-MS. Compound identification was obtained by retention time and fragmentation spectra using Agilent MassHunter software. We performed several comparison studies using blood collected from self-reported healthy participants (24-60 years of age; 14 females, 13 males). The effect of fasting/nonfasting status was evaluated in 14 participants by comparing samples collected after overnight fasting and 4 hours after meal. Paired t test was used for the statistical analysis of results. Results When compared to the preferred sample type, sodium heparin (green top) plasma EDTA plasma (purple top, n = 9) and serum (red top or serum separator tube; n = 5) displayed an unacceptable variability for several analytes. The biggest differences were observed for lactic and pyruvic acids. Lactic acid was significantly lower in EDTA plasma (9%-35% decrease; P = .0001), but higher in serum (4%-63% increase; P = .014), while pyruvic acid was significantly higher in EDTA plasma (4%-145% increase; P = .0002). The measurements of lactic and pyruvic acids were also affected by delayed separation of plasma in samples kept at room temperature. We observed a marked increase in lactate and decrease in pyruvate levels (+42% and –43% mean change, respectively) in plasma separated from whole blood after 2 hours of collection compared to samples processed within 30 minutes. Plasma separated 1 hour postcollection was acceptable. Overnight fasting (12-14 hours) did not have a significant impact on ketones (3-hydroxybutyric and acetoacetic acids) or branched-chain ketoacids (2-keto-3-methylvaleric, 2-ketoisocaproic, 2-ketoisovaleric acids). In contrast, fasting samples displayed higher lactic acid concentrations compared to nonfasting samples (24% increase on average, P = .0072), although in most fasting samples (13 out of 14), lactate concentrations were still within established normal range (600-2,600 µmol/L). Conclusions We identified several preanalytical factors influencing plasma organic acid quantitation, particularly lactic and pyruvic acids. Since those are critical analytes, suggestive of primary or secondary metabolic acidosis, appropriate steps should be taken to ensure sample quality during collection and processing.

The role of previously unmeasured organic acids in the pathogenesis of severe malaria.

IntroductionSevere falciparum malaria is commonly complicated by metabolic acidosis. Together with lactic acid (LA), other previously unmeasured acids have been implicated in the pathogenesis of falciparum malaria.MethodsIn this prospective study, we characterised organic acids in adults with severe falciparum malaria in India and Bangladesh. Liquid chromatography-mass spectrometry was used to measure organic acids in plasma and urine. Patients were followed until recovery or death.ResultsPatients with severe malaria (n=138), uncomplicated malaria (n=102), sepsis (n=32) and febrile encephalopathy (n=35) were included. Strong ion gap (mean±SD) was elevated in severe malaria (8.2 mEq/L±4.5) and severe sepsis (8.6 mEq/L±7.7) compared with uncomplicated malaria (6.0 mEq/L±5.1) and encephalopathy (6.6 mEq/L±4.7). Compared with uncomplicated malaria, severe malaria was characterised by elevated plasma LA, hydroxyphenyllactic acid (HPLA), α-hydroxybutyric acid and β-hydroxybutyric acid (all P<0.05). In urine, concentrations of methylmalonic, ethylmalonic and α-ketoglutaric acids were also elevated. Multivariate logistic regression showed that plasma HPLA was a strong independent predictor of death (odds ratio [OR] 3.5, 95 % confidence interval [CI] 1.6–7.5, P=0.001), comparable to LA (OR 3.5, 95 % CI 1.5–7.8, P=0.003) (combined area under the receiver operating characteristic curve 0.81).ConclusionsNewly identified acids, in addition to LA, are elevated in patients with severe malaria and are highly predictive of fatal outcome. Further characterisation of their sources and metabolic pathways is now needed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-1023-5) contains supplementary material, which is available to authorized users.

Liquid chromatographic-mass spectrometric method for simultaneous determination of small organic acids potentially contributing to acidosis in severe malaria.

Acidosis is an important cause of mortality in severe falciparum malaria. Lactic acid is a major contributor to metabolic acidosis, but accounts for only one-quarter of the strong anion gap. Other unidentified organic acids have an independent strong prognostic significance for a fatal outcome. In this study, a simultaneous bio-analytical method for qualitative and quantitative assessment in plasma and urine of eight small organic acids potentially contributing to acidosis in severe malaria was developed and validated. High-throughput strong anion exchange solid-phase extraction in a 96-well plate format was used for sample preparation. Hydrophilic interaction liquid chromatography (HILIC) coupled to negative mass spectroscopy was utilized for separation and detection. Eight possible small organic acids; l-lactic acid (LA), α-hydroxybutyric acid (aHBA), β-hydroxybutyric acid (bHBA), p-hydroxyphenyllactic acid (pHPLA), malonic acid (MA), methylmalonic acid (MMA), ethylmalonic acid (EMA) and α-ketoglutaric acid (aKGA) were analyzed simultaneously using a ZIC-HILIC column with an isocratic elution containing acetonitrile and ammonium acetate buffer. This method was validated according to U.S. Food and Drug Administration guidelines with additional validation procedures for endogenous substances. Accuracy for all eight acids ranged from 93.1% to 104.0%, and the within-day and between-day precisions (i.e. relative standard deviations) were lower than 5.5% at all tested concentrations. The calibration ranges were: 2.5-2500μg/mL for LA, 0.125-125μg/mL for aHBA, 7.5-375μg/mL for bHBA, 0.1-100μg/mL for pHPLA, 1-1000μg/mL for MA, 0.25-250μg/mL for MMA, 0.25-100μg/mL for EMA, and 30-1500μg/mL for aKGA. Clinical applicability was demonstrated by analyzing plasma and urine samples from five patients with severe falciparum malaria; five acids had increased concentrations in plasma (range LA=177-1169μg/mL, aHBA=4.70-38.4μg/mL, bHBA=7.70-38.0μg/mL, pHPLA=0.900-4.30μg/mL and aKGA=30.2-32.0) and seven in urine samples (range LA=11.2-513μg/mL, aHBA=1.50-69.5μg/mL, bHBA=8.10-111μg/mL, pHPLA=4.30-27.7μg/mL, MMA=0.300-13.3μg/mL, EMA=0.300-48.1μg/mL and aKGA=30.4-107μg/mL). In conclusion, a novel bioanalytical method was developed and validated which allows for simultaneous quantification of eight small organic acids in plasma and urine. This new method may be a useful tool for the assessment of acidosis in patients with severe malaria, and other conditions complicated by acidosis.

Metabolic analysis of body fluids by capillary electrophoresis using noncovalently coated capillaries

The potential of capillaries noncovalently coated with charged polymers for the metabolic analysis of body fluids by CE is illustrated. Firstly, the usefulness of a coating consisting of a triple layer of polybrene–dextran sulfate–polybrene for the fast analysis of organic acids is described. The CE system allowed direct injections of CSF, plasma and urine samples, yielding good separation efficiencies. RSDs for migration times and peak areas of organic acids in plasma were <3% and <5%, respectively. The usefulness of the system is illustrated by the profiling of organic acids in plasma and urine samples. Secondly, a CE system comprising a bilayer coating of polybrene–poly(vinylsulfonate), which provides a considerable EOF at low pH is described. This system was combined with TOF-MS and used for the fast analysis of amino acids in cerebrospinal fluid (CSF) and urine with minimal sample pretreatment. RSDs for migration times and peak areas of amino acids in CSF and urine were <2% and <10%, respectively. The applicability of the system is demonstrated by the profiling of endogenous low-molecular weight metabolites in CSF from a healthy individual and a patient with complex regional pain syndrome.

Cis-4-decenoic acid in plasma: a characteristic metabolite in medium-chain acyl-CoA dehydrogenase deficiency.

The profile of organic acids in plasma of patients with a deficiency of medium-chain acyl-CoA dehydrogenase (EC 1.3.99.3) was determined by gas-liquid chromatography of trimethylsilylated derivatives of the acids isolated by ethyl acetate extraction. All 13 patients had increased concentrations of free octanoate, cis-4-decenoate, and decanoate in their plasma. Cis-4-decenoate, an intermediary metabolite of linoleic acid, is pathognomonic of medium-chain acyl-CoA dehydrogenase deficiency. This metabolite does not accumulate in plasma after oral loading with medium-chain triglycerides, in contrast to octanoate and decanoate. Two postmortem plasma samples from victims of infant sudden-death syndrome had detectable octanoate and decanoate, but cis-4-decenoate could not be detected. The identification of cis-4-decenoate in plasma may be an aid in the diagnosis of an inherited defect in oxidation of medium-chain fatty acids.

Liquid-chromatographic determination of organic acids in plasma.

Journal Article Liquid-chromatographic determination of organic acids in plasma. Get access H J Halls, H J Halls Search for other works by this author on: Oxford Academic Google Scholar J K Teubner J K Teubner Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 32, Issue 10, 1 October 1986, Page 1979, https://doi.org/10.1093/clinchem/32.10.1979a Published: 01 October 1986

Gas chromatographic and gas chromatographic—mass spectrometric analysis of organic acids in plasma of patients with chronic renal failure

Apart from increased concentrations of aliphatic dicarboxylic acids and phenolic aromatic acids in plasma from patients with chronic renal failure, there is large elevation of a furanoid acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid, and of hippuric acid. The levels of 3-hydroxy- and 4-hydroxyhippuric acid are also raised. The quantitative results are as follows: furanoid acid in hemodialysis patients, 1.4 ± 0.6 mg/dl; in healthy individuals, 0.2 ± 0.1 mg/dl; hippuric acid in hemodialysis patients, 9.8 ± 6.5 mg/dl; in healthy individuals, 0.4 ± 0.5 mg/dl. Both compounds are dialysable, but less effectively than creatinine and urea. The mean elimination rate of the furanoid acid is only 21%.