Perennial Baki™ Bean Safety for Human Consumption: Evidence from an Analysis of Heavy Metals, Folate, Canavanine, Mycotoxins, Microorganisms and Pesticides.

Global food production relies on annual grain crops. The reliability and productivity of these crops are threatened by adaptations to climate change and unsustainable rates of soil loss associated with their cultivation. Perennial grain crops, which do not require planting every year, have been proposed as a transformative solution to these challenges. Perennial grain crops typically rely on wild species as direct domesticates or as sources of perenniality in hybridization with annual grains. Onobrychis spp. (sainfoins) are a genus of perennial legumes domesticated as ancient forages. Baki™ bean is the tradename for pulses derived from sainfoins, with ongoing domestication underway to extend demonstrated benefits to sustainable agriculture. This study contributes to a growing body of evidence characterizing the nutritional quality of Baki™ bean. Through two studies, we investigated the safety of Baki™ bean for human consumption. We quantified heavy metals, folate, and canavanine for samples from commercial seed producers, and we quantified levels of mycotoxins, microorganisms, and pesticides in samples from a single year and seed producer, representing different varieties and production locations. The investigated analytes were not detectable or occurred at levels that do not pose a significant safety risk. Overall, this study supports the safety of Baki™ bean for human consumption as a novel pulse crop.

Succinic semialdehyde dehydrogenase deficiency in mice and in humans: An untargeted metabolomics perspective.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare neurometabolic disorder caused by disruption of the gamma-aminobutyric acid (GABA) pathway. A more detailed understanding of its pathophysiology, beyond the accumulation of GABA and gamma-hydroxybutyric acid (GHB), will increase our understanding of the disease and may support novel therapy development. To this end, we compared biochemical body fluid profiles from SSADHD patients with controls using next-generation metabolic screening (NGMS). Targeted analysis of NGMS data from cerebrospinal fluid (CSF) showed a moderate increase of aspartic acid, glutaric acid, glycolic acid, 4-guanidinobutanoic acid, and 2-hydroxyglutaric acid, and prominent elevations of GHB and 4,5-dihydroxyhexanoic acid (4,5-DHHA) in SSADHD samples. Remarkably, the intensities of 4,5-DHHA and GHB showed a significant positive correlation in control CSF, but not in patient CSF. In an established zebrafish epilepsy model, 4,5-DHHA showed increased mobility that may reflect limited epileptogenesis. Using untargeted metabolomics, we identified 12 features in CSF with high biomarker potential. These had comparable increased fold changes as GHB and 4,5-DHHA. For 10 of these features, a similar increase was found in plasma, urine and/or mouse brain tissue for SSADHD compared to controls. One of these was identified as the novel biomarker 4,5-dihydroxyheptanoic acid. The intensities of selected features in plasma and urine of SSADHD patients positively correlated with the clinical severity score of epilepsy and psychiatric symptoms of those patients, and also showed a high mutual correlation. Our findings provide new insights into the (neuro)metabolic disturbances in SSADHD and give leads for further research concerning SSADHD pathophysiology.

Distinguishing Oligosaccharide Isomers Using Far-Infrared Ion Spectroscopy: Identification of Biomarkers for Inborn Errors of Metabolism.

Distinguishing isomeric saccharides poses a major challenge for analytical workflows based on (liquid chromatography) mass spectrometry (LC-MS). In recent years, many studies have proposed infrared ion spectroscopy as a possible solution as the orthogonal, spectroscopic characterization of mass-selected ions can often distinguish isomeric species that remain unresolved using conventional MS. However, the high conformational flexibility and extensive hydrogen bonding in saccharides cause their room-temperature fingerprint infrared spectra to have broad features that often lack diagnostic value. Here, we show that room-temperature infrared spectra of ion-complexed saccharides recorded in the previously unexplored far-infrared wavelength range (300-1000 cm-1) provide well-resolved and highly diagnostic features. We show that this enables distinction of isomeric saccharides that differ either by their composition of monosaccharide units and/or the orientation of their glycosidic linkages. We demonstrate the utility of this approach from single monosaccharides up to isomeric tetrasaccharides differing only by the configuration of a single glycosidic linkage. Furthermore, through hyphenation with hydrophilic interaction liquid chromatography, we identify oligosaccharide biomarkers in patient body fluid samples, demonstrating a generalized and highly sensitive MS-based method for the identification of saccharides found in complex sample matrices.

Toward plant breeding for multicrop systems.

Increasing cropping system diversity has great potential to address environmental problems associated with modern agriculture, such as erosion, soil carbon loss, nutrient runoff, water pollution, and loss of biodiversity. As with other agricultural sciences, plant breeding has primarily been conducted in the context of dominant monoculture cropping systems, with little focus on multicrop systems. Multicrop systems have increased temporal and/or spatial diversity and include a diverse set of crops and practices. In order to support a transition to multicrop systems, plant breeders must shift their breeding programs and objectives to better represent more diverse systems, including diverse rotations, alternate-season crops, ecosystem service crops, and intercropping systems. The degree to which breeding methods need to change will depend on the cropping system context in question. Plant breeding alone, however, cannot drive adoption of multicrop systems. Alongside shifts in breeding approaches, changes are needed within broader research, private sector, and policy contexts. These changes include policies and investments that support a transition to multicrop systems, increased collaboration across disciplines to support cropping system development, and leadership from both the public and private sectors to develop and promote adoption of new cultivars.

Novel cerebrospinal fluid biomarkers of glucose transporter type 1 deficiency syndrome: Implications beyond the brain's energy deficit.

We used next-generation metabolic screening to identify new biomarkers for improved diagnosis and pathophysiological understanding of glucose transporter type 1 deficiency syndrome (GLUT1DS), comparing metabolic cerebrospinal fluid (CSF) profiles from 12 patients to those of 116 controls. This confirmed decreased CSF glucose and lactate levels in patients with GLUT1DS and increased glutamine at group level. We identified three novel biomarkers significantly decreased in patients, namely gluconic + galactonic acid, xylose-α1-3-glucose, and xylose-α1-3-xylose-α1-3-glucose, of which the latter two have not previously been identified in body fluids. CSF concentrations of gluconic + galactonic acid may be reduced as these metabolites could serve as alternative substrates for the pentose phosphate pathway. Xylose-α1-3-glucose and xylose-α1-3-xylose-α1-3-glucose may originate from glycosylated proteins; their decreased levels are hypothetically the consequence of insufficient glucose, one of two substrates for O-glucosylation. Since many proteins are O-glucosylated, this deficiency may affect cellular processes and thus contribute to GLUT1DS pathophysiology. The novel CSF biomarkers have the potential to improve the biochemical diagnosis of GLUT1DS. Our findings imply that brain glucose deficiency in GLUT1DS may cause disruptions at the cellular level that go beyond energy metabolism, underlining the importance of developing treatment strategies that directly target cerebral glucose uptake.

Amino acid and fatty acid profiles of perennial Baki™ bean.

To realize the potential of sainfoins to contribute to sustainable agriculture and expand on demonstrated uses and benefits, de novo domestication is occurring to develop perennial Baki™ bean, the trade name used by The Land Institute for pulses (i.e., grain legumes) derived from sainfoins. The objective of this study was to characterize amino acid and fatty acid profiles of depodded seeds from commercial sainfoin (Onobrychis viciifolia) seed lots, and compare these results with data published in the Global Food Composition Database for Pulses. The fatty acid profile consisted primarily of polyunsaturated fatty acids (56.8%), compared to monounsaturated (29.0%) and saturated fatty acids (14.2%), and n-3 fatty acids (39.5%), compared to n-9 (28.4%) and n-6 (17.6%) fatty acids. The essential fatty acid linolenic acid (18,3 n-3) was the most abundant fatty acid (39.2%), followed by oleic acid (18,1 cis-9) (27.8%), and the essential fatty acid linoleic acid (18,2 n-6) (17.3%). The amino acid profile consisted primarily of the nonessential amino acids glutamic acid (18.3%), arginine (11.6%), and aspartic acid (10.8%), followed by the essential amino acids leucine (6.8%), and lysine (5.8%). Essential amino acid content met adult daily requirements for each amino acid. This indicates that sainfoin seeds may be a complete plant protein source. However, further research is necessary to better understand protein quality, defined by protein digestibility in addition to the amino acid profile. By demonstrating favorable fatty acid and amino acid profiles to human health, these results contribute to a growing body of evidence supporting the potential benefits of perennial Baki™ bean, a novel, perennial pulse derived from sainfoins.

Abnormal concentrations of acetylated amino acids in cerebrospinal fluid in acetyl-CoA transporter deficiency.

Acetyl-CoA transporter 1 (AT-1) is a transmembrane protein which regulates influx of acetyl-CoA from the cytosol to the lumen of the endoplasmic reticulum and is therefore important for the posttranslational modification of numerous proteins. Pathological variants in the SLC33A1 gene coding for AT-1 have been linked to a disorder called Huppke-Brendel syndrome, which is characterized by congenital cataracts, hearing loss, severe developmental delay and early death. It has been described in eight patients so far, who all had the abovementioned symptoms together with low serum copper and ceruloplasmin concentrations. The link between AT-1 and low ceruloplasmin concentrations is not clear, nor is the complex pathogenesis of the disease. Here we describe a further case of Huppke-Brendel syndrome with a novel and truncating homozygous gene variant and provide novel biochemical data on N-acetylated amino acids in cerebrospinal fluid (CSF) and plasma. Our results indicate that decreased levels of many N-acetylated amino acids in CSF are a typical metabolic fingerprint for AT-1 deficiency and are potential biomarkers for the defect. As acetyl-CoA is an important substrate for protein acetylation, we performed N-terminal proteomics, but found only minor effects on this particular protein modification. The acetyl-CoA content in patient's fibroblasts was insignificantly decreased. Our data may help to better understand the mechanisms underlying the metabolic disturbances, the pathophysiology and the clinical phenotype of the disease.

Untargeted metabolomics and infrared ion spectroscopy identify biomarkers for pyridoxine-dependent epilepsy.

BackgroundPyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine catabolism that presents with refractory epilepsy in newborns. Biallelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase/antiquitin, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important cofactor pyridoxal-5'-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but intellectual disability may still develop. Early diagnosis and treatment, preferably based on newborn screening, could optimize long-term clinical outcome. However, no suitable PDE-ALDH7A1 newborn screening biomarkers are currently available.MethodsWe combined the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy to discover and identify biomarkers in plasma that would allow for PDE-ALDH7A1 diagnosis in newborn screening.ResultsWe identified 2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) as a PDE-ALDH7A1 biomarker, and confirmed 6-oxopiperidine-2-carboxylic acid (6-oxoPIP) as a biomarker. The suitability of 2-OPP as a potential PDE-ALDH7A1 newborn screening biomarker in dried bloodspots was shown. Additionally, we found that 2-OPP accumulates in brain tissue of patients and Aldh7a1-knockout mice, and induced epilepsy-like behavior in a zebrafish model system.ConclusionThis study has opened the way to newborn screening for PDE-ALDH7A1. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients.FundingSociety for Inborn Errors of Metabolism for Netherlands and Belgium (ESN), United for Metabolic Diseases (UMD), Stofwisselkracht, Radboud University, Canadian Institutes of Health Research, Dutch Research Council (NWO), and the European Research Council (ERC).

Amadori rearrangement products as potential biomarkers for inborn errors of amino-acid metabolism.

The identification of disease biomarkers plays a crucial role in developing diagnostic strategies for inborn errors of metabolism and understanding their pathophysiology. A primary metabolite that accumulates in the inborn error phenylketonuria is phenylalanine, however its levels do not always directly correlate with clinical outcomes. Here we combine infrared ion spectroscopy and NMR spectroscopy to identify the Phe-glucose Amadori rearrangement product as a biomarker for phenylketonuria. Additionally, we find analogous amino acid-glucose metabolites formed in the body fluids of patients accumulating methionine, lysine, proline and citrulline. Amadori rearrangement products are well-known intermediates in the formation of advanced glycation end-products and have been associated with the pathophysiology of diabetes mellitus and ageing, but are now shown to also form under conditions of aminoacidemia. They represent a general class of metabolites for inborn errors of amino acid metabolism that show potential as biomarkers and may provide further insight in disease pathophysiology.

Novel Protein Biomarkers of Monoamine Metabolism Defects Correlate with Disease Severity.

BACKGROUND: Genetic defects of monoamine neurotransmitters are rare neurological diseases amenable to treatment with variable response. They are major causes of early parkinsonism and other spectrum of movement disorders including dopa-responsive dystonia. OBJECTIVES: The objective of this study was to conduct proteomic studies in cerebrospinal fluid (CSF) samples of patients with monoamine defects to detect biomarkers involved in pathophysiology, clinical phenotypes, and treatment response. METHODS: A total of 90 patients from diverse centers of the International Working Group on Neurotransmitter Related Disorders were included in the study (37 untreated before CSF collection, 48 treated and 5 unknown at the collection time). Clinical and molecular metadata were related to the protein abundances in the CSF. RESULTS: Concentrations of 4 proteins were significantly altered, detected by mass spectrometry, and confirmed by immunoassays. First, decreased levels of apolipoprotein D were found in severe cases of aromatic L-amino acid decarboxylase deficiency. Second, low levels of apolipoprotein H were observed in patients with the severe phenotype of tyrosine hydroxylase deficiency, whereas increased concentrations of oligodendrocyte myelin glycoprotein were found in the same subset of patients with tyrosine hydroxylase deficiency. Third, decreased levels of collagen6A3 were observed in treated patients with tetrahydrobiopterin deficiency. CONCLUSION: This study with the largest cohort of patients with monoamine defects studied so far reports the proteomic characterization of CSF and identifies 4 novel biomarkers that bring new insights into the consequences of early dopaminergic deprivation in the developing brain. They open new possibilities to understand their role in the pathophysiology of these disorders, and they may serve as potential predictors of disease severity and therapies. © 2020 International Parkinson and Movement Disorder Society.

Confirmation of neurometabolic diagnoses using age-dependent cerebrospinal fluid metabolomic profiles.

Timely diagnosis is essential for patients with neurometabolic disorders to enable targeted treatment. Next-Generation Metabolic Screening (NGMS) allows for simultaneous screening of multiple diseases and yields a holistic view of disturbed metabolic pathways. We applied this technique to define a cerebrospinal fluid (CSF) reference metabolome and validated our approach with patients with known neurometabolic disorders. Samples were measured using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry followed by (un)targeted analysis. For the reference metabolome, CSF samples from patients with normal general chemistry results and no neurometabolic diagnosis were selected and grouped based on sex and age (0-2/2-5/5-10/10-15 years). We checked the levels of known biomarkers in CSF from seven patients with five different neurometabolic disorders to confirm the suitability of our method for diagnosis. Untargeted analysis of 87 control CSF samples yielded 8036 features for semiquantitative analysis. No sex differences were found, but 1782 features (22%) were different between age groups (q < 0.05). We identified 206 diagnostic metabolites in targeted analysis. In a subset of 20 high-intensity metabolites and 10 biomarkers, 17 (57%) were age-dependent. For each neurometabolic patient, ≥1 specific biomarker(s) could be identified in CSF, thus confirming the diagnosis. In two cases, age-matching was essential for correct interpretation of the metabolomic profile. In conclusion, NGMS in CSF is a powerful tool in defining a diagnosis for neurometabolic disorders. Using our database with many (age-dependent) features in CSF, our untargeted approach will facilitate biomarker discovery and further understanding of mechanisms of neurometabolic disorders.

Thrombin dynamics in children with liver disease or extrahepatic portal vein obstruction or shunt.

INTRODUCTION: Changes in the coagulation profile in children with liver disease and extrahepatic portal vein obstruction or shunt result in both bleeding and thrombosis. Routine coagulation tests do not accurately predict bleeding risk, as they are not sensitive to changes in anticoagulant factors. The thrombin generation assay could be suitable for describing the overall balance of coagulation in children with liver disease. This study aims to characterise the mechanism of thrombin generation in this population, focusing on prothrombin conversion and thrombin inhibition. METHODS: Patients were categorised as: severe (paediatric end stage liver disease score > 15) and mild disease, or portal vein obstruction or shunt. Age and gender matched healthy controls were used. The thrombin generation assay was performed in plasma samples from patients and controls with and without exogenous thrombomodulin and the results were further analysed with the computational thrombin dynamics method. RESULTS: A total of 42 patients (severe, n = 5; mild, n = 29, obstruction/shunt, n = 8) and 20 controls were included in this study. The total prothrombin conversion, thrombin-antithrombin formation and the thrombin decay capacity, in the presence and absence of thrombomodulin were reduced in children with severe liver disease. The rate of prothrombin conversion was increased and thrombin decay capacity was decreased in patients with portal vein obstruction or shunt compared to controls. CONCLUSION: This study demonstrates changes in the mechanism in thrombin generation seen in severe chronic liver disease. The changes vary in parenchymal versus non parenchymal liver disease and further study assessing the clinical significance of these variations in mechanism is required.

Prothrombin conversion is accelerated in the antiphospholipid syndrome and insensitive to thrombomodulin.

Antiphospholipid syndrome (APS) is a condition in which the presence of antibodies against phospholipid-binding proteins is associated with thrombophilia and/or pregnancy morbidity. Although antiphospholipid antibodies have anticoagulant characteristics in vitro, they are associated with thromboembolic complications. Thrombin generation (TG) is a sensitive global test of coagulation, and elevated TG is associated with thrombosis. Increased TG can be caused by increased prothrombin conversion, decreased thrombin inactivation, or a combination of both. In this study, we measured TG in APS patients and healthy controls with and without vitamin K antagonist (VKA) treatment at 1 and 5 pM tissue factor and with thrombomodulin. Prothrombin conversion and thrombin inactivation were determined by thrombin dynamics analysis. The TG peak was increased in nontreated APS patients at 1 pM TF compared with nontreated controls. Prothrombin conversion was significantly increased in nontreated APS patients. In contrast, prothrombin conversion did not differ in controls and patients that were on VKA therapy. Thrombin inactivation was comparable between controls and APS patients in the presence and absence of VKAs. Both TG (peak and ETP) and prothrombin conversion were significantly higher in APS patients with prior thrombosis compared with patients without a history of thrombosis. In this study, we demonstrate that in APS, the hemostatic balance shifts toward a more prothrombotic phenotype due to elevated prothrombin conversion but unchanged thrombin inactivation rates. Within the group of APS patients, increased TG and prothrombin conversion are associated with a history of thrombosis.

A Stepwise Procedure to Define a Data Collection Framework for a Clinical Biobank.

INTRODUCTION: Current guidelines for clinical biobanking have a strong focus on obtaining, handling, and storage of biospecimens. However, to allow for research tying biomarker analysis to clinical decision making, there should be more focus on collection of data on donor characteristics. Therefore, our aim was to develop a stepwise procedure to define a framework as a tool to help start the data collection process in clinical biobanking. MATERIALS AND METHODS: The Radboud Biobank (RB) is a central clinical biobanking facility designed in accordance with the standards set by the Parelsnoer Institute, a Dutch national biobank originally initiated with eight different disease cohorts. To organize the information of these cohorts, we used our experience and knowledge in the field of biobanking and translational research to identify research domains and information categories to classify data. We extended this classification system to a stepwise procedure for defining a data collection framework and examined its utility for existing RB biobanks. RESULTS: Our approach resulted in the definition of a three-step procedure: (1) Identification of research domains and relevant questions within the field that may benefit from biobank samples. (2) Identification of information categories and accompanying subcategories that are relevant for answering questions in identified research domains. (3) Reduction to an efficient framework based on essentiality and quality criteria. We showed the utility of the procedure for three existing RB biobanks. DISCUSSION: We developed guidelines for the definition of a framework that supports the standardization of the biobank data collection process. Connecting the biobank database to pertinent information collected from the electronic health record will improve data quality and efficiency for both care and research. This is crucial when using the corresponding biospecimens for scientific research. Further, it also facilitates the combination of different clinical biobanks for a specific disease.

Iron refractory iron deficiency anemia: a heterogeneous disease that is not always iron refractory.

TMPRSS6 variants that affect protein function result in impaired matriptase-2 function and consequently uninhibited hepcidin production, leading to iron refractory iron deficiency anemia (IRIDA). This disease is characterized by microcytic, hypochromic anemia and serum hepcidin values that are inappropriately high for body iron levels. Much is still unknown about its pathophysiology, genotype-phenotype correlation, and optimal clinical management. We describe 14 different TMPRSS6 variants, of which 9 are novel, in 21 phenotypically affected IRIDA patients from 20 families living in the Netherlands; 16 out of 21 patients were female. In 7 out of 21 cases DNA sequencing and multiplex ligation dependent probe amplification demonstrated only heterozygous TMPRSS6 variants. The age at presentation, disease severity, and response to iron supplementation were highly variable, even for patients and relatives with similar TMPRSS6 genotypes. Mono-allelic IRIDA patients had a milder phenotype with respect to hemoglobin and MCV and presented significantly later in life with anemia than bi-allelic patients. Transferrin saturation (TSAT)/hepcidin ratios were lower in IRIDA probands than in healthy relatives. Most patients required parenteral iron. Genotype alone was not predictive for the response to oral iron. We conclude that IRIDA is a genotypically and phenotypically heterogeneous disease. The high proportion of female patients and the discrepancy between phenotypes of probands and relatives with the same genotype, suggest a complex interplay between genetic and acquired factors in the pathogenesis of IRIDA. In the absence of inflammation, the TSAT/hepcidin ratio is a promising diagnostic tool, even after iron supplementation has been given. Am. J. Hematol. 91:E482-E490, 2016. © 2016 Wiley Periodicals, Inc.