Metabolomic profiles in Jamaican children with and without autism spectrum disorder.

Background: Autism spectrum disorder (ASD) is a complex neurodevelopmental condition with a wide range of behavioral and cognitive impairments. While genetic and environmental factors are known to contribute to its etiology, the underlying metabolic perturbations associated with ASD which can potentially connect genetic and environmental factors, remain poorly understood. Therefore, we conducted a metabolomic case-control study and performed a comprehensive analysis to identify significant alterations in metabolite profiles between children with ASD and typically developing (TD) controls. Objective: To elucidate potential metabolomic signatures associated with ASD in children and identify specific metabolites that may serve as biomarkers for the disorder. Methods: We conducted metabolomic profiling on plasma samples from participants in the second phase of Epidemiological Research on Autism in Jamaica (ERAJ-2), which was a 1:1 age (±6 months)-and sex-matched cohort of 200 children with ASD and 200 TD controls (2-8 years old). Using high-throughput liquid chromatography-mass spectrometry techniques, we performed a targeted metabolite analysis, encompassing amino acids, lipids, carbohydrates, and other key metabolic compounds. After quality control and imputation of missing values, we performed univariable and multivariable analysis using normalized metabolites while adjusting for covariates, age, sex, socioeconomic status, and child's parish of birth. Results: Our findings revealed unique metabolic patterns in children with ASD for four metabolites compared to TD controls. Notably, three of these metabolites were fatty acids, including myristoleic acid, eicosatetraenoic acid, and octadecenoic acid. Additionally, the amino acid sarcosine exhibited a significant association with ASD. Conclusions: These findings highlight the role of metabolites in the etiology of ASD and suggest opportunities for the development of targeted interventions.

Dysregulation of the kallikrein-kinin system in bronchoalveolar lavage fluid of patients with severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the angiotensin-converting enzyme 2 (ACE2) receptor, a critical component of the kallikrein-kinin system. Its dysregulation may lead to increased vascular permeability and release of inflammatory chemokines. Interactions between the kallikrein-kinin and the coagulation system might further contribute to thromboembolic complications in COVID-19. METHODS: In this observational study, we measured plasma and tissue kallikrein hydrolytic activity, levels of kinin peptides, and myeloperoxidase (MPO)-DNA complexes as a biomarker for neutrophil extracellular traps (NETs), in bronchoalveolar lavage (BAL) fluid from patients with and without COVID-19. FINDINGS: In BAL fluid from patients with severe COVID-19 (n = 21, of which 19 were mechanically ventilated), we observed higher tissue kallikrein activity (18·2 pM [1·2-1535·0], median [range], n = 9 vs 3·8 [0·0-22·0], n = 11; p = 0·030), higher levels of the kinin peptide bradykinin-(1-5) (89·6 [0·0-2425·0], n = 21 vs 0·0 [0·0-374·0], n = 19, p = 0·001), and higher levels of MPO-DNA complexes (699·0 ng/mL [66·0-142621·0], n = 21 vs 70·5 [9·9-960·0], n = 19, p < 0·001) compared to patients without COVID-19. INTERPRETATION: Our observations support the hypothesis that dysregulation of the kallikrein-kinin system might occur in mechanically ventilated patients with severe pulmonary disease, which might help to explain the clinical presentation of patients with severe COVID-19 developing pulmonary oedema and thromboembolic complications. Therefore, targeting the kallikrein-kinin system should be further explored as a potential treatment option for patients with severe COVID-19. FUNDING: Research Foundation-Flanders (G0G4720N, 1843418N), KU Leuven COVID research fund.

Picomolar Sensitivity Analysis of Multiple Bradykinin-Related Peptides in the Blood Plasma of Patients With Hereditary Angioedema in Remission: A Pilot Study.

Background: Hereditary angioedema (HAE) is a rare autosomal dominant disease; the most well understood forms concern the haplodeficiency of C1 esterase inhibitor (C1INH) and a gain of function mutation of factor XII (FXII). The acute forms of these conditions are mediated by an excessive bradykinin (BK) formation by plasma kallikrein. Methods: A validated LC-MS/MS platform of picomolar sensitivity developed for the analysis of eleven bradykinin-related peptides was applied to the plasma of HAE-C1INH and HAE-FXII sampled during remission. Results: In HAE-C1INH plasma, the concentrations of the relatively stable BK1-5 fragment (mean ± S.E.M.: 12.0 ± 4.2 pmol/L), of BK2-9 (0.7 ± 0.2 pmol/L) and of the sums of BK and its tested fragments (18.0 ± 6.4 pmol/L) are significantly greater than those recorded in the plasma of healthy volunteers (1.9 ± 0.6, 0.03 ± 0.03 and 4.3 ± 0.8 pmol/L, respectively), consistent with the previous evidence of permanent plasma kallikrein activity in this disease. Kinin levels in the plasma of HAE-FXII patients did not differ from controls, suggesting that triggering factors for contact system activation are not active during remission. Conclusion: BK1-5, BK2-9 and the sum of BK and its fragments determined by the sensitive LC-MS/MS technique are proposed as potential biomarkers of HAE-C1INH in remission while this was not applicable to HAE-FXII patients.

Mass spectrometric study of variation in kinin peptide profiles in nasal fluids and plasma of adult healthy individuals.

BACKGROUND: The kallikrein-kinin system is assumed to have a multifunctional role in health and disease, but its in vivo role in humans currently remains unclear owing to the divergence of plasma kinin level data published ranging from the low picomolar to high nanomolar range, even in healthy volunteers. Moreover, existing data are often restricted on reporting levels of single kinins, thus neglecting the distinct effects of active kinins on bradykinin (BK) receptors considering diverse metabolic pathways. A well-characterized and comprehensively evaluated healthy cohort is imperative for a better understanding of the biological variability of kinin profiles to enable reliable differentiation concerning disease-specific kinin profiles. METHODS: To study biological levels and variability of kinin profiles comprehensively, 28 healthy adult volunteers were enrolled. Nasal lavage fluid and plasma were sampled in customized protease inhibitor prespiked tubes using standardized protocols, proven to limit inter-day and interindividual variability significantly. Nine kinins were quantitatively assessed using validated LC-MS/MS platforms: kallidin (KD), Hyp4-KD, KD1-9, BK, Hyp3-BK, BK1-8, BK1-7, BK1-5, and BK2-9. Kinin concentrations in nasal epithelial lining fluid were estimated by correlation using urea. RESULTS: Circulating plasma kinin levels were confirmed in the very low picomolar range with levels below 4.2 pM for BK and even lower levels for the other kinins. Endogenous kinin levels in nasal epithelial lining fluids were substantially higher, including median levels of 80.0 pM for KD and 139.1 pM for BK. Hydroxylated BK levels were higher than mean BK concentrations (Hyp3-BK/BK = 1.6), but hydroxylated KD levels were substantially lower than KD (Hyp4-KD/KD = 0.37). No gender-specific differences on endogenous kinin levels were found. CONCLUSIONS: This well-characterized healthy cohort enables investigation of the potential of kinins as biomarkers and would provide a valid control group to study alterations of kinin profiles in diseases, such as angioedema, sepsis, stroke, Alzheimer's disease, and COVID-19.

Reliable measurement of plasma kinin peptides: Importance of preanalytical variables.

BACKGROUND: The kallikrein-kinin system is involved in many (patho)physiological processes and kinin peptides are considered potential clinical biomarkers. Variance in blood specimen collection and processing, artificial ex vivo bradykinin formation, and rapid degradation of kinins have contributed to divergence in published plasma levels, therefore limiting their significance. Thus, reliable preanalytical settings are highly required. OBJECTIVES: This study aimed to develop and evaluate a standardized preanalytical procedure for reliable kinin quantification. The procedure was based on identification of the most impactful variables on ex vivo plasma level alterations. METHODS: Suitable protease inhibitors and blood specimen collection and handling conditions were systematically investigated. Their influence on plasma levels of seven kinins was monitored using an established in-house liquid chromatography-tandem mass spectrometry platform. RESULTS: In nonstandardized settings, ex vivo rise of bradykinin was found to already occur 30 seconds after blood sampling with high interindividual variation. The screening of 17 protease inhibitors resulted in a customized seven-component protease inhibitor, which efficiently stabilized ex vivo kinin levels. The reliability of kinin levels was substantially jeopardized by prolonged rest time until centrifugation, phlebotomy methodology (eg, straight needles, catheters), vacuum sampling technique, or any time delays during venipuncture. The subsequently developed standardized procedure was applied to healthy volunteers and proved it significantly limited interday and interindividual kinin level variability. CONCLUSION: The developed procedure for blood specimen collection and handling is feasible in clinical settings and allows for determination of reliable kinin levels. It may contribute to further elucidating the role of the kallikrein-kinin system in diseases like angioedema, sepsis, or coronavirus disease 2019.

Targeted LC-MS/MS platform for the comprehensive determination of peptides in the kallikrein-kinin system.

The kallikrein-kinin system (KKS) is involved in many physiological and pathophysiological processes and is assumed to be connected to the development of clinical symptoms of angioedema or COVID-19, among other diseases. However, despite its diverse role in the regulation of physiological and pathophysiological functions, knowledge about the KKS in vivo remains limited. The short half-lives of kinins, their low abundance and structural similarities and the artificial generation of the kinin bradykinin greatly hinder reliable and accurate determination of kinin levels in plasma. To address these issues, a sensitive LC-MS/MS platform for the comprehensive and simultaneous determination of the four active kinins bradykinin, kallidin, des-Arg(9)-bradykinin and des-Arg(10)-kallidin and their major metabolites bradykinin 2-9, bradykinin 1-7 and bradykinin 1-5 was developed. This platform was validated according to the bioanalytical guideline of the US Food and Drug Administration regarding linearity, accuracy, precision, sensitivity, carry-over, recovery, parallelism, matrix effects and stability in plasma of healthy volunteers. The validated platform encompassed a broad calibration curve range from 2.0-15.3 pg/mL (depending on the kinin) up to 1000 pg/mL, covering the expected concentrations in disease states. No source-dependent matrix effects were identified, and suitable stability of the analytes in plasma was observed. The applicability of the developed platform was proven by the determination of endogenous levels in healthy volunteers, whose plasma kinin levels were successfully detected in the low pg/mL range. The established platform facilitates the investigation of kinin-mediated diseases (e.g. angioedema, COVID-19) and enables the assessment of the impact of altered enzyme activities on the formation or degradation of kinins.

Sensitive mass spectrometric determination of kinin-kallikrein system peptides in light of COVID-19.

The outbreak of COVID-19 has raised interest in the kinin-kallikrein system. Viral blockade of the angiotensin-converting enzyme 2 impedes degradation of the active kinin des-Arg(9)-bradykinin, which thus increasingly activates bradykinin receptors known to promote inflammation, cough, and edema-symptoms that are commonly observed in COVID-19. However, lean and reliable investigation of the postulated alterations is currently hindered by non-specific peptide adsorption, lacking sensitivity, and cross-reactivity of applicable assays. Here, an LC-MS/MS method was established to determine the following kinins in respiratory lavage fluids: kallidin, bradykinin, des-Arg(10)-kallidin, des-Arg(9)-bradykinin, bradykinin 1-7, bradykinin 2-9 and bradykinin 1-5. This method was fully validated according to regulatory bioanalytical guidelines of the European Medicine Agency and the US Food and Drug Administration and has a broad calibration curve range (up to a factor of 103), encompassing low quantification limits of 4.4-22.8 pg/mL (depending on the individual kinin). The application of the developed LC-MS/MS method to nasal lavage fluid allowed for the rapid (~ 2 h), comprehensive and low-volume (100 µL) determination of kinins. Hence, this novel assay may support current efforts to investigate the pathophysiology of COVID-19, but can also be extended to other diseases.

Low-volume LC-MS/MS method for the pharmacokinetic investigation of carvedilol, enalapril and their metabolites in whole blood and plasma: Application to a paediatric clinical trial.

Evidence-based pharmacotherapy with carvedilol and enalapril in children suffering from heart failure is insufficient owing to limited pharmacokinetic data. Although a few data sets regarding enalapril, its metabolite enalaprilat and carvedilol in children have been published, pharmacokinetic data on carvedilol metabolites are missing. However, for both drug substances, their active metabolites contribute substantially to drug efficacy. As data can hardly be derived from adults owing to the unknown impacts of enzymatic maturation and ontogeny during childhood, customised assays are important to facilitate paediatric evidence-based pharmacotherapy. Considering ethical paediatric constraints, a low-volume liquid chromatography coupled to mass spectrometry (LC-MS/MS) assay was developed using whole blood or plasma for the quantification of enalapril, enalaprilat, carvedilol, O-desmethyl carvedilol, 4- and 5-hydroxyphenyl carvedilol as well as 3- and 8-hydroxy carvedilol. To facilitate broader applications in adults, the elderly and children, a wide calibration range-between 0.024/0.049 and 50.000 ng/ml-was achieved with good linearity (r ≥ 0.995 for all analytes). In compliance with international bioanalytical guidelines, accuracy, precision, sensitivity and internal standard normalised matrix effects were further successfully validated with the exception of those for 3-hydroxy carvedilol, which was therefore assessed semi-quantitatively. Distinct haematocrits did not impact matrix effects or recoveries when analysing whole blood. Blood-to-plasma ratios were determined for all analytes to form the basis for pharmacokinetic modelling. Finally, incurred sample reanalysis of paediatric samples confirmed the reproducibility of the developed low-volume LC-MS/MS method during study sample analysis. The assay facilitates the reliable generation of important data and contributes towards a safe drug therapy in children.

Improving sensitivity for the targeted LC-MS/MS analysis of the peptide bradykinin using a design of experiments approach.

The nonapeptide bradykinin is endogenously present only in low picomolar plasma concentrations, subsequently making reliable detection using liquid chromatography coupled to mass spectrometry (LC-MS/MS) challenging. Furthermore, non-specific adsorption during sample preparation and storage can lead to unpredictable peptide losses. To overcome these issues, a design of experiments (DoE) approach was applied, which consisted of a screening to identify impacting factors, optimisation and confirmation runs. On the one hand, different injection solvent compositions and sample collection materials were investigated in order to decrease non-specific adsorption. On the other hand, the addition of modifiers, which are known to enhance the signal intensity in LC-MS/MS, to the chromatographic mobile phase was examined. Polypropylene was the most suitable material among those investigated and resulted in a factor increase of 12.0 compared to LC-MS glass. The advantages of protein low-binding polypropylene versus standard polypropylene were fully compensated by the optimisation of the injection solvent. The latter substantially contributed to a decrease of non-specific adsorption of bradykinin. In this regard, bradykinin further benefitted from an organic fraction and a high amount of formic acid. Based on the DoE results, the final optimised injection solvent-consisting of 8.7% formic acid in 49.4/5.3/36.6 water/methanol/dimethyl sulfoxide (v/v/v)-was established. Furthermore, optimisation of the mobile phase composition yielded a signal intensity increase by a factor of 7.7. The transferability of the optimisation results conducted in neat solutions were successfully confirmed in human plasma. The applicability of this approach was further supported by the successful determination of low-abundance endogenous bradykinin levels in human plasma using LC-MS/MS.

A quality control system for ligand-binding assay of plasma renin activity: Proof-of-concept within a pharmacodynamic study.

While the role of plasma renin activity (PRA) in heart failure has been widely studied in adults, comprehensive data on pediatric heart failure remain lacking. This drawback is increasingly being addressed by academic research. Nevertheless, such pediatric investigations are commonly conducted only once due to ethical constraints. Therefore, the quality of bioanalytical data must be ensured to acquire meaningful insights into maturing humoral parameters. However, appropriate post-validation assessment of bioanalytical runs is currently underrepresented by regulatory guidance. Thus, for applications in an academic environment, an easy-to-handle six-step bioanalytical quality control system was designed based on regulatory guidelines (e.g. U.S. Food and Drug Administration) combined with international recommendations (e.g. Clinical and Laboratory Standards Institute) and current scientific discussion. Its applicability to an enzyme-linked immunosorbent assay for determination of PRA was investigated within three pediatric trials of the EU-funded "Labeling of Enalapril in Neonates up to Adolescents" project. This quality control system identified 15 % bioanalytical runs as non-compliant to the predefined specifications and ensured the reliable quantification of 940 pharmacodynamic samples. The inter-run assessment of quality controls was able to demonstrate the comparability of the study results. Furthermore, 86 % of incurred sample reanalysis pairs complied with regulatory requirements (>67 %), thus underlining the long-term reproducibility of the utilized ligand-binding assay. Successful participation in interlaboratory testing confirmed the accuracy of the applied method throughout the entire study period. Further investigations showed no notable differences between the five applied lots of the PRA assay. The applicability of this quality control system was proven in an academic environment and ensured reliable results for PRA over the entire 24-month study period.

Potential and Limitations of Atrial Natriuretic Peptide as Biomarker in Pediatric Heart Failure-A Comparative Review.

Although B-type Natriuretic Peptide (BNP), N-terminal-proBNP (NT-proBNP), and mid-regional-proANP (MR-proANP) are included in current guidelines on heart failure in adults, no guideline considering these biomarkers in pediatric heart failure is available. A new drug class of neprilysin inhibitors as fixed-dose combination (Sacubitril/valsartan) has been introduced and is currently being investigated in children suffering from heart failure. Atrial Natriuretic Peptide (ANP) is discussed as a more useful alternative to BNP because it may grants better insights into the effects of this treatment. Thus, this review aimed to provide an overview of the current knowledge concerning ANP in pediatric heart failure and compares its suitability regarding diagnosis and prognosis of heart failure. A literature search using PubMed resulted in 147 publications of which 22 studies were classified as relevant. The review presents available ANP, NT-proANP, and MR-proANP level data in children (0-18 years). Summarizing, ANP shows only minor differences as marker for diagnosing and monitoring pediatric heart failure if compared to BNP. Due to its fast release, ANP offers the advantage of displaying rapid changes during therapy or operation. ANP is -like the other natriuretic peptides- influenced by age, presenting with the highest levels in very young infants. ANP also correlates with atrial pressure and volume overload in children. In addition, ANP determination in saliva appears to be a promising alternative to blood sampling. Similarly to NT-proBNP, NT-proANP, and MR-proANP offer better stability but only few data has been published in children and thus their potential is only presumable so far.