Acid In Body Fluids Research Articles

Common Organic Acidemias in a Tertiary Hospital in Egypt

Abstract Background Organic acidemias (OAs) are a heritable group of heterogeneous metabolic disorders characterized by the accumulation of organic acids in body fluids and tissues. They are rare disorders and infrequently reported worldwide. Methylmalonic acidemia (MMA), propionic acidemia (PA), isovaleric acidemia (IVA), 3-methylglutaconic aciduria (3-MGA), and glutaric acidemia type 1(GA1), are considered the more common OAs, their prevalence is higher in Arab populations due to the high consanguinity rate. Aim This study aimed to: • Evaluate the spectrum of common organic acidemias among pediatric patients who followed up at the metabolic clinic of a tertiary hospital in Egypt. • Present different clinical and biochemical characterizations and the time of onset of these disorders, so that helps in raising the high index of suspicion among pediatricians and health care practitioners to make an early diagnosis. Method We conducted a descriptive retrospective study by reviewing the medical records of pediatric aged group patients diagnosed with common forms of OAs, followed up in Metabolic Clinic, Ain Shams University Hospitals, Egypt, over a 4-year period, between January 2017 and January 2021. All patients were investigated by metabolic work up, including the acylcarnitine profile and urinary organic acid analysis. Results A total of 76 patients with common OAs were included in the study. There were (56.6%) males and (43.4%) females. Most patients presented after the first year of life (58.7%); whereas the median age at diagnosis was 15 months. GA-1 was the most frequent disorder (39.5%), followed by MMA (36.8%). There were relatively high rates of consanguinity (80%) and affected siblings (23%). The most frequent presenting symptoms were developmental delay (69.3%), poor feeding (65.7%), and vomiting (50.7%). The main observed complications among studied patients were delayed motor development (67.1%), delayed speech (22.4%) and faltering growth (15.8%). Regarding the disease course and patientś outcome; 55.3% of patients exhibited regressive course, whereas 84.2% of patients were alive and well. Conclusion GA-1 is the commonest organic acidemias in Egypt and is more evident due to high rate of consanguinity. This urges the need to enhance awareness among pediatricians, primary healthcare providers and devise a governmental newborn screening program for the early detection and prevention of such disorders.

A luminescent MOF-based nonenzymatic probe for colorimetric/photothermal/fluorescence triple-mode assay of uric acid in body fluids

Monitoring the levels of uric acid (UA) in body fluids is of great significance in the clinical diagnosis and therapy of related diseases. Herein, a novel nanocomposite R6G@Fe-MOF based nonenzymatic probe is presented to provide a ratiometric fluorescent, colorimetric, and photothermal triple read-out signal for the visual, sensitive, and convenient assay of UA. The framework structure of the in situ encapsulated R6G@Fe-MOF is found to decompose upon the addition of UA, resulting in the reduction of Fe3+ to Fe2+. This reduction will lead to a rapid increase in fluorescence emission (FL) at 430 nm. Simultaneously, the FL at 573 nm will decrease remarkably due to the inner filter effect (IFE) between UA and R6G@Fe-MOF. Furthermore, the reaction of the generated Fe2+ with potassium ferricyanide (K3 [Fe(CN)6]) can in situ generate Prussian blue (PBNPs) with outstanding color and photothermal properties, which allow for easy colorimetric and photothermal signal readout. The detection limits (LOD) for the colorimetric, fluorometric and photothermal detection are low at 1.68 μM, 0.236 μM, and 1.32 μM respectively. Ultimately, it is successfully employed to determine UA in urine, serum, and saliva, yielding satisfactory results. The constructed R6G@Fe-MOF sensor provides a simple, sensitive, and accurate determination of UA that can be tailored to meet the needs of various applications, and also provides new perspectives for the design and development of versatile sensors for diverse uses.

Mice, Rats and Guinea Pigs Exhibit Significant Variations in the Plasma, Urine and Tissue Levels of Taurine, Betaine, Sarcosine and Other Osmolyte-Active Amino Acids.

Osmolytes are naturally occurring compounds that protect cells from osmotic stress in high-osmolarity tissues, such as the kidney medulla. Some amino acids, including taurine, betaine, glycine, alanine, and sarcosine, are known to act as osmolytes. This study aimed to establish the levels of these amino acids in body fluids and tissues of laboratory animals used as models for human diseases in biomedical research. Liquid chromatography coupled with mass spectrometry was used to quantify taurine, glycine, betaine, alanine, beta-alanine, and sarcosine in plasma, urine, and tissues of adult, male mice, rats and guinea pigs. Among the species analyzed, taurine was found to have the highest tissue concentrations across all compounds, with the heart containing the greatest amount. In guinea pigs, betaine levels were higher in the renal medulla than in the renal cortex (p < 0.01), while in rats and mice, there were no significant differences in betaine levels between the kidney cortex and medulla. The urine of guinea pigs had lower levels of sarcosine compared to rats (p < 0.001), while the plasma (p < 0.05; > 0.05), heart (p < 0.05; < 0.05), lungs (p < 0.01; < 0.01), liver (p < 0.001; < 0.05), and kidneys (p < 0.01; < 0.01) of rats exhibited notably higher concentrations of sarcosine compared to both mice and guinea pigs, respectively. There are pronounced differences in the concentrations of taurine, betaine, and other amino acids across the investigated species. It is important to acknowledge these differences when selecting animal models for preclinical studies and to account for variations in amino acid concentrations when selecting amino acids doses for interventional studies.

On-chip microfluidic dual detection of amino acid metabolism disorders using cell-free protein synthesis

Various metabolic diseases are associated with the accumulation of specific amino acids due to abnormal metabolic pathways, and thus can be diagnosed by measuring the level of amino acids in body fluids. However, present methods for amino acid analysis are not readily accessible because they require a complex experimental setup, expensive equipment, and a long processing time. Here, we present a dual sensing microfluidic device that enables fast, portable, and quantitative analysis of target amino acids, harnessing the biological mechanism of protein synthesis. In this device, the working principle of a finger-actuated pumping unit is applied, and the microchannels are designed to perform cell-free synthesis of a reporter protein in response to the target amino acids in the assay samples. Multiple steps required for the translational assay are controlled by the simple operation of two pushbuttons on the device. It is demonstrated that the developed microfluidic device provides precise quantification of two amino acids (methionine and phenylalanine) within 30 min at room temperature. We expect that the application of the presented device can be readily extended to the point-of-care testing of other metabolic compounds.

A versatile technique based on surface-enhanced Raman spectroscopy for label-free detection of amino acids and peptide formation in body fluids.

As an effective analytical method, surface-enhanced Raman spectroscopy (SERS) is widely used in the detection of nucleic acids, amino acids, and other biomolecules. However, obtaining the SERS signal of nonaromatic amino acids is still a challenge. In this work, excess sodium borohydride was used as a reducing agent to prevent the surface of silver nanoparticles from being coated with AgO to enable amino acid molecules to reach the surface of silver nano-substrates. Calcium ions were used as aggregators for silver nano-substrates to successfully achieve the label-free and accurate fingerprint determination of various nonaromatic amino acids. Different types of amino acids were distinguished based on the changes in their peak intensity that were obtainedusing colorless and transparent organic dichloromethane (DCM) as the internal standard. A Raman signal for low-concentration amino acids in body fluids was detected, and the detection limit for tyrosine was 5 ng/mL. Moreover, the physical and chemical properties of peptides and the formation of peptide chains were further analyzed. The proposed method can potentially be applied to protein sequencing.

Metabolite Identification Using Infrared Ion Spectroscopy─Novel Biomarkers for Pyridoxine-Dependent Epilepsy.

Untargeted liquid chromatography–mass spectrometry (LC–MS)-based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing “grand challenge” in the utilization of this approach is metabolite identification—confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC–MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico-predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of α-aminoadipic semialdehyde (α-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While α-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics–IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids and that are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly provided novel insights into the pathophysiology of PDE-ALDH7A1.

Fabrication of Copper Oxide Modified Graphene Composite Sensor for Simultaneous Detection of Biometabolites from Urine

It is a common knowledge that the concentration of certain biomarkers in the body accounts for the presence of some diseases. For instance, the unusual levels of uric acid in body fluids may be indicative of gout and hyperuricemia. These biomarkers may be present within the body but may not be detected at very low concentrations and on time to enable proactive treatments, and the consequences may be dire. The current research report features a new approach developed towards fabricating a new copper oxide modified graphene composite sensor with unique nanostructures on a pencil electrode for sensing three biometabolites (xanthine, uric acid and urea) in urine at very low limit of detection (LOD). The surface structures of this composite sensor composed of several electroactive sites for adsorption of desired analytes. This sensor was appropriately characterized by means of Raman spectroscopy, scanning electron microscopy, cyclic voltammetry as well as localized and normal electrochemical impedance spectroscopy. This sensor was significantly sensitive towards uric acid ((LOD = 10 ppm) and urea (LOD = 20 ppm) sensing compared to xanthine (LOD = 50 ppm). Optimization of key sensor parameters towards efficient sensing (e.g. CuO content) were also examined. The efficiency of the composite sensor toward sensing these analytes in the presence of potential interference was investigated using real human urine. Biomolecular sensing of disease biomarkers at low LOD is important in the assessment of health conditions and monitoring treatment progress of patients. Our experimental results demonstrate the efficacy of simultaneously sensing multiple analytes in a complex biological sample. Keywords: Biosensing; Simultaneous detection; Biometabolites; Disease biomarker; Limit of detection

Sandwich-type detection of nucleic acids by bioorthogonal SERS probes

Nucleic acids in body fluids, such as circulating cell-free nucleic acids, viral DNA, and RNA have received much attention for their great potential as biomarkers in liquid biopsies of serious diseases. Although quantitative polymerase chain reaction (qPCR) has been traditionally used as a laboratory-based assay for measuring nucleic acids, there is a strong demand for techniques to qualitatively, rapidly, and simply measure the extremely low-abundance nucleic acids in order to realize the nucleic acid-based liquid biopsies. With this aim in mind, we developed a simple and highly sensitive sandwich-type assay for nucleic acids using a combination of surface-enhanced Raman scattering (SERS), which enhances Raman scattering by 108– to 1010–fold, and bioorthogonal Raman tags, which generate signals in the biologically silent region (1800–2800 cm−1). Using gold nanorods having approximately 240 strands of oligonucleotides and 4-cyano-N-(2-mercaptoethyl)benzamide (4CMB) as the bioorthogonal Raman tag, we successfully detected target nucleic acids in a sequence-selective manner.

Ultra-sensitive and rapid detection of nucleic acids and microorganisms in body fluids using single-molecule tethering

Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases. However, culture-based diagnostics that are time-consuming remain the gold standard approach in certain cases, such as sepsis. New culture-free methods are urgently needed. Here, we describe Single MOLecule Tethering or SMOLT, an amplification-free and purification-free molecular assay that can detect microorganisms in body fluids with high sensitivity without the need of culturing. The signal of SMOLT is generated by the displacement of micron-size beads tethered by DNA probes that are between 1 and 7 microns long. The molecular extension of thousands of DNA probes is determined with sub-micron precision using a robust and rapid optical approach. We demonstrate that SMOLT can detect nucleic acids directly in blood, urine and sputum at sub-femtomolar concentrations, and microorganisms in blood at 1 CFU mL−1 (colony forming unit per milliliter) threefold faster, with higher multiplexing capacity and with a more straight-forward protocol than amplified methodologies. SMOLT’s clinical utility is further demonstrated by developing a multiplex assay for simultaneous detection of sepsis-causing Candida species directly in whole blood.

Statistical Optimization of Urinary Organic Acids Analysis by a Multi-Factorial Design of Experiment

The analysis of urinary organic acids is useful for patients suspected to have inborn errors of metabolism known as organic acidurias. These diseases cause an accumulation of organic acids in body fluids and their abnormal excretion in urines. By means of chemometrics tools, such as principal component analysis and multiple linear regression, it was concluded that the conditions used in our laboratory are really the most suitable to achieve high yields of analytes.

Identification of a novel homozygous nonsense variant in a Chinese patient with ethylmalonic encephalopathy and a genotype-phenotype spectrum review

Ethylmalonic encephalopathy (EE) is a rare and devastating neurodegenerative disease caused by mutations in the ETHE1 gene. It is characterized by early-onset encephalopathy, chronic diarrhea, petechiae, orthostatic acrocyanosis, and high levels of methylsuccinic, lactic, and ethylmalonic acids in body fluids. In this study, we report a patient with EE, who was identified through newborn screening, and the diagnosis was confirmed by targeted next-generation sequencing (NGS). The patient displayed recurrent petechiae, intermittent jaundice, protracted diarrhea, and extensive developmental regression. Genetic testing identified a homozygous nonsense variant, c.295C > T (p. Q99*), in the ETHE1 gene. A review of all known ETHE1 variants observed in other EE patients was conducted. This revealed the current difficulties in EE diagnosis. Besides, it also showed that patients with truncated variants of ETHE1 might exhibit pathological symptoms earlier and present more severe manifestations. Finally, a novel nonsense variant was identified, which supported and expanded our current knowledge of the variant spectrum for ETHE1. This novel variant also deepened our understanding of the genotype-phenotype associations that occur in EE patients.

Highly specific enrichment of rare nucleic acid fractions using Thermus thermophilus argonaute with applications in cancer diagnostics.

Detection of disease-associated, cell-free nucleic acids in body fluids enables early diagnostics, genotyping and personalized therapy, but is challenged by the low concentrations of clinically significant nucleic acids and their sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for increasing the fractions of Nucleic Acids of clinical interest Via DNA-Guided Argonaute from Thermus thermophilus (TtAgo). TtAgo cleaves specifically guide-complementary DNA and RNA with single nucleotide precision, greatly increasing the fractions of rare alleles and, enhancing the sensitivity of downstream detection methods such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of the cancer biomarker KRAS G12D and ∼100-fold increased sensitivity of Peptide Nucleic Acid (PNA) and Xenonucleic Acid (XNA) clamp PCR, enabling detection of low-frequency (<0.01%) mutant alleles (∼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-based assays (e.g. DASH, Depletion of Abundant Sequences by Hybridization), identifying more mutation-positive samples when combined with XNA-PCR. Moreover, TtAgo does not require targets to contain any specific protospacer-adjacent motifs (PAM); is a multi-turnover enzyme; cleaves ssDNA, dsDNA and RNA targets in a single assay; and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.

Determination of cholic acid in body fluids by β‑cyclodextrin-modified N-doped carbon dot fluorescent probes.

An easy, dependable, and sensitive cholic acid activity experiment was designed based on β‑cyclodextrin-modified carbon dot (β‑CD-CD) nanoprobes with specific host-guest recognizing ability and photoelectron transfer capability. The β‑CD-CD nanoprobes were characterized by infrared, ultraviolet-visible, and fluorescence spectroscopy and transmission electron microscopy. The fluorescence of the probes under optimized conditions linearly responded to cholic acid concentration from 0 to 650 μmol·L-1 with a detection limit of 25 nmol·L-1. The probes also performed well in detecting cholic acid in serum and urine samples with an average recovery rate of 97.1%-103.4%. Thus, this study provides a reliable, rapid, and easy method of cholic acid detection in body fluids that can be potentially applied in medical studies.

Itaconic acid indicates cellular but not systemic immune system activation

Itaconic acid is produced by mammalian leukocytes upon pro-inflammatory activation. It appears to inhibit bacterial growth and to rewire the metabolism of the host cell by inhibiting succinate dehydrogenase. Yet, it is unknown whether itaconic acid acts only intracellularly, locally in a paracrine fashion, or whether it is even secreted from the inflammatory cells at meaningful levels in peripheral blood of patients with severe inflammation or sepsis.The aim of this study was to determine the release rate of itaconic acid from pro-inflammatory activated macrophages in vitro and to test for the abundance of itaconic acid in bodyfluids of patients suffering from acute inflammation.We demonstrate that excretion of itaconic acid happens at a low rate and that it cannot be detected in significant amounts in plasma or urine of septic patients or in liquid from bronchial lavage of patients with pulmonary inflammation.We conclude that itaconic acid may serve as a pro-inflammatory marker in immune cells but that it does not qualify as a biomarker in the tested body fluids.

Fatal zolpidem poisoning due to its intravenous self-injection: Postmortem distribution/redistribution of zolpidem and its predominant metabolite zolpidem phenyl-4-carboxylic acid in body fluids and solid tissues in an autopsy case

We experienced a curious fatal case, in which a male in his 20s self-administered zolpidem intravenously. The victim was found dead lying on floor of his apartment room, with a tourniquet band and new injection marks on his right forearm. Nearby the body, a medical disposal syringe containing small-volume solution dissolving crushed zolpidem tablets was found. The postmortem interval was estimated at about two days.The direct cause of his death was judged as asphyxia due to the aspiration of stomach contents into the trachea and bronchi. The specimens dealt with were body fluids and solid tissues including femoral vein blood, right and left heart blood, pericardial fluid, urine, bile, stomach contents, the brain, lung, heart muscle, liver, spleen, kidney, pancreas and skeletal muscle. For the extractions of zolpidem, zolpidem phenyl-4-carboxylic acid, deuterated internal standards zolpidem-d7 and zolpidem phenyl-4-carboxylic acid-d4, a modified QuEChERS method was used, followed by the analysis by liquid chromatography–tandem mass spectrometry. Because this study included various kinds of human matrices with quite different properties, the standard addition method was most preferable to overcome the matrix effects and recovery rates, and also did not need to use blank human matrices for validation experiments. The concentration of zolpidem and its phenyl-4-carboxylic acid metabolite in various specimens tested were generally extreme higher than those of reported fatal cases, supporting that the victim had died of intravenous zolpidem injection. The concentrations of zolpidem in femoral vein blood and right and left heart blood specimens in the present case were 9.55, 28.5 and 46.9μg/mL, respectively, which far exceeded estimated fatal levels. The present study also showed the postmortem distribution/redistribution of zolpidem and its phenyl-4-carboxylic acid metabolite in 15 body fluid and solid tissue specimens including stomach contents. Although a number of published literatures dealt with zolpidem poisoning cases due to oral ingestion of the drug, this is the first report on fatal intravenous zolpidem injection case and postmortem distribution of zolpidem and its predominant metabolite.

Response to Comment on “Direct Monitoring of Exogenous γ-Hydroxybutyric Acid in Body Fluids by NMR Spectroscopy”: Several Issues to Consider When Quantifying γ-Hydroxybutyric Acid in Biological Matrixes

In response to the letter from Pichini and Busardo, we would like to stress that we do not suggest NMR as an alternative technique to GC-MS or LC-MS, but complementary to them (which could provide additional valuable information), as was stated in the last sentence of the conclusions of the published article.

Comment on "Direct Monitoring of Exogenous γ-Hydroxybutyric Acid in Body Fluids by NMR Spectroscopy": Several Issues to Consider When Quantifying γ-Hydroxybutyric Acid in Biological Matrixes.

Comment on "Direct Monitoring of Exogenous γ-Hydroxybutyric Acid in Body Fluids by NMR Spectroscopy": Several Issues to Consider When Quantifying γ-Hydroxybutyric Acid in Biological Matrixes.

Direct Monitoring of Exogenous γ-Hydroxybutyric Acid in Body Fluids by NMR Spectroscopy.

γ-Hydroxybutyric acid (GHB) is a popular drug increasingly associated with cases of drug-facilitated sexual assault (DFSA). Currently, expanding procedures of analysis and having forensic evidence of GHB intake in a long term are mandatory. Up to now, most studies have been performed using GC/MS and LC-MS as analytical platforms, which involve significant manipulation of the sample and, often, indirect measurements. In this work, procedures used in NMR-based metabolomics were applied to a GHB clinical trial on urine and serum. Detection, identification, and quantification of the drug by NMR methods were surveyed, as well as the use of NMR-based metabolomics for the search of potential surrogate biomarkers of GHB consumption. Results demonstrated the suitability of NMR spectroscopy, as a robust nondestructive technique, to fast and directly monitor (detect, identify, and quantify) exogenous GHB in almost intact body fluids and its high potential in the search for metabolites associated with GHB intake.

Experimental evidence that overexpression of NR2B glutamate receptor subunit is associated with brain vacuolation in adult glutaryl-CoA dehydrogenase deficient mice: A potential role for glutamatergic-induced excitotoxicity in GA I neuropathology

Glutaric aciduria type I (GA I) is biochemically characterized by accumulation of glutaric and 3-hydroxyglutaric acids in body fluids and tissues, particularly in the brain. Affected patients show progressive cortical leukoencephalopathy and chronic degeneration of the basal ganglia whose pathogenesis is still unclear. In the present work we investigated parameters of bioenergetics and redox homeostasis in various cerebral structures (cerebral cortex, striatum and hippocampus) and heart of adult wild type (Gcdh+/+) and glutaryl-CoA dehydrogenase deficient knockout (Gcdh−/−) mice fed a baseline chow. Oxidative stress parameters were also measured after acute lysine overload. Finally, mRNA expression of NMDA subunits and GLT1 transporter was determined in cerebral cortex and striatum of these animals fed a baseline or high lysine (4.7%) chow. No significant alterations of bioenergetics or redox status were observed in these mice. In contrast, mRNA expression of the NR2B glutamate receptor subunit and of the GLT1 glutamate transporter was higher in cerebral cortex of Gcdh−/− mice. Furthermore, NR2B expression was markedly elevated in striatum of Gcdh−/− animals receiving chronic Lys overload. These data indicate higher susceptibility of Gcdh−/− mice to excitotoxic damage, implying that this pathomechanism may contribute to the cortical and striatum alterations observed in GA I patients.

Preparation of poly(3,4-ethylenedioxythiophene) nanofibers modified pencil graphite electrode and investigation of over-oxidation conditions for the selective and sensitive determination of uric acid in body fluids

In this study, we have performed the preparation of over-oxidized poly(3,4-ethylenedioxythiophene) nanofibers modified pencil graphite electrode (Ox-PEDOT-nf/PGE) to develop a selective and sensitive voltammetric uric acid (UA) sensor. It was noted that the over-oxidation potential and time had a prominent effect on the UA response of the Ox-PEDOT-nf/PGE. Characterizations of PEDOT-nf/PGE and Ox-PEDOT-nf/PGE have been performed by cyclic voltammetry, electrochemical impedance spectroscopy, scanning electron microscopy, Fourier transform infrared spectroscopy and Raman spectroscopy. The highest voltammetric response of UA was obtained at pH 2.0. A linear relationship between the concentration of UA and oxidation peak currents was observed in the concentration range of 0.01–20.0 μM. The detection limit (1.3 nM according to S/N = 3) and reproducibility (RSD: 4.6 % for N:10) have also been determined. The effects of different substances on the determination of UA have been investigated. A very high peak separation value of 423 mV was obtained between UA and ascorbic acid which is the major interfering substance for UA. The use of Ox-PEDOT-nf/PGE has been successfully tested in the determination of UA in human blood serum and urine samples for the first time in the literature.