In metabolic diagnostics, there is an emerging need for a comprehensive test to acquire a complete view of metabolite status. Here, we describe a non-quantitative direct-infusion high-resolution mass spectrometry (DI-HRMS) based metabolomics method and evaluate the method for both dried blood spots (DBS) and plasma. 110 DBS of 42 patients harboring 23 different inborn errors of metabolism (IEM) and 86 plasma samples of 38 patients harboring 21 different IEM were analyzed using DI-HRMS. A peak calling pipeline developed in R programming language provided Z-scores for ~1875 mass peaks corresponding to ~3835 metabolite annotations (including isomers) per sample. Based on metabolite Z-scores, patients were assigned a ‘most probable diagnosis’ by an investigator blinded for the known diagnoses of the patients. Based on DBS sample analysis, 37/42 of the patients, corresponding to 22/23 IEM, could be correctly assigned a ‘most probable diagnosis’. Plasma sample analysis, resulted in a correct ‘most probable diagnosis’ in 32/38 of the patients, corresponding to 19/21 IEM. The added clinical value of the method was illustrated by a case wherein DI-HRMS metabolomics aided interpretation of a variant of unknown significance (VUS) identified by whole-exome sequencing. In summary, non-quantitative DI-HRMS metabolomics in DBS and plasma is a very consistent, high-throughput and nonselective method for investigating the metabolome in genetic disease.

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Background: Pathophysiology of life-threatening acute metabolic decompensations (AMD) in propionic acidemia (PA) and isolated methylmalonic acidemia (MMA) is insufficiently understood. Here, we study the metabolomes of PA and MMA patients over time, to improve insight in which biochemical processes are at play during AMD. Methods: Longitudinal data from clinical chemistry analyses and metabolic assays over the life-course of 11 PA and 13 MMA patients were studied retrospectively. Direct-infusion high-resolution mass spectrometry was performed on 234 and 154 remnant dried blood spot and plasma samples of PA and MMA patients, respectively. In addition, a systematic literature search was performed on reported biomarkers. All results were integrated in an assessment of biochemical processes at play during AMD. Results: We confirmed many of the metabolite alterations reported in literature, including increases of plasma valine and isoleucine during AMD in PA patients. We revealed that plasma leucine and phenylalanine, and urinary pyruvic acid were increased during AMD in PA patients. 3-hydroxyisovaleric acid correlated positively with plasma ammonia. We found that known diagnostic biomarkers were not significantly further increased, while intermediates of the branched-chain amino acid (BCAA) degradation pathway were significantly increased during AMD. Conclusions: We revealed that during AMD in PA and MMA, BCAA and BCAA intermediates accumulate, while known diagnostic biomarkers remain essentially unaltered. This implies that these acidic BCAA intermediates are responsible for metabolic acidosis. Based on this, we suggest to measure plasma 3-hydroxyisovaleric acid and urinary ketones or 3-hydroxybutyric acid for the biochemical follow-up of a patient's metabolic stability.

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Plant sterols are added as their FA esters to vegetable oil table spreads at levels of approximately 8% as a means to reduce blood cholesterol levels. A new chromatographic method was designed to quantify quickly the level of plant sterol FA esters in incoming (raw) materials and to monitor their processing and final product quality with respect to total sterol level. The method shows a significant improvement in elapsed time and thus labor cost over the classical methods for sterols published in normative references. This improvement was obtained together with high performance characteristics, as shown by the internal method validation for recovery and repeatability. Its validity and robustness were further tested and confirmed in an international collaborative test. The method allows monitoring of sterol content of raw materials, fat-blends, and consumer products at the target level, with a range of 10% or less around this target. The calculated within-and betweenlaboratory reproducibility were 0.680 and 1.194 w/w%, respectively, for sterol-containing spreads. The results afforded by this method can be used for setting tight product specifications or to monitor trade between companies. We propose to add this new and fast method for total 4-desmethyl sterol(s) to analytical method collections as an adjunct to methods already listed for more detailed sterol analysis.Plant sterols (PS) and plant sterol FA esters (PSE) are recognized for their therapeutic efficacy in lowering blood cholesterol levels. Recent reviews on the pharmacological properties of PS and their fully saturated isomers (stanols) focus on this cholesterol-lowering effect (1,2). The blood cholesterol-lowering (BCL) effect of sterols, in particular the 4-desmethyl sterol(s) such as sitosterol, has been known since the 1950s. With the use of the FA esters instead of the free PS, a wider range of formulation options exists. The new interest and formulation options have resulted in a range of consumer products, such as vegetable-oil table spreads, designed to lower total blood cholesterol and LDL-cholesterol (3). The normal daily intake of about 20 g of a vegetable fat spread containing 8% PS, equivalent to 13-14% PSE, reduces the LDL-cholesterol by about 10% (4). With the use of PS and PSE at these elevated levels, these natural edible oil constituents have become functional food ingredients. Sterols used for the BCL effect are mixtures of mainly 4-desmethyl sterol(s) (sitosterol, campesterol, stigmasterol, and brassicasterol).To support quality control of raw materials, processing, and final consumer product control at these levels, a new and fast total sterol analysis for the content of mainly 4-desmethyl sterol(s) has been developed, validated, and collaboratively tested. The method is designed for a total content of these 4-desmethyl sterol(s) in a range of approximately 15-20% for vegetable oil blends, 8% in consumer products such as spreads, and approximately 60% in PSE concentrates. A new method was necessary because current analytical methods...

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