Optimised liquid chromatography tandem mass spectrometry method for the simultaneous quantification of serum vitamin D analogues while also accounting for epimers and isobars.

The accurate quantification of multiple vitamin D analogues simultaneously is challenging. This study set out to use liquid chromatography-tandem mass spectrometry (LC-MS/MS) to develop a method capable of measuring a comprehensive vitamin D profile, encompassing twelve vitamin D analogues (vitamin D2, D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi-25(OH)D3, 7αC4 and1α(OH)D3) in a single run. Serum samples were prepared using double liquid-liquid extraction and analysed on an Agilent 6460 QQQ LC-MS/MS equipped with a Pursuit 3 Pentafluorophenyl (4.6 x 100 mm, 3 µm) column. Recovery rates for all analytes were above 95 % with a coefficient of variation (CV) below 10 %. The method exhibited good linearity (r > 0.995) and had a range of detection limits between 0.01 and 0.35 ng/mL and quantification limits between 0.15 and 0.96 ng/mL. Repeatability and within-lab precision were acceptable, with CV values below 10 % and 15 %, respectively. Method accuracy was excellent, with a systematic error below 6.60 %. additionally, all analytes-maintained stability for 48 h following sample preparation, and no interferences were observed among co-eluting analytes. Lastly, this method achieved "world-class" status according to the Sigma metric scale specifications, requiring minimal quality control to ensure data quality. This successfully validated method has the potential not only for improving vitamin D profiling procedures but also for aiding in the diagnosis of other genetic disorders where measuring beyond 25(OH)D is crucial.

Metabolite Changes of Perna canaliculus Following a Laboratory Marine Heatwave Exposure: Insights from Metabolomic Analyses.

Temperature is considered to be a major abiotic factor influencing aquatic life. Marine heatwaves are emerging as threats to sustainable shellfish aquaculture, affecting the farming of New Zealand's green-lipped mussel [Perna canaliculus (Gmelin, 1791)]. In this study, P. canaliculus were gradually exposed to high-temperature stress, mimicking a five-day marine heatwave event, to better understand the effects of heat stress on the metabolome of mussels. Following liquid chromatography-tandem mass spectrometry analyses of haemolymph samples, key sugar-based metabolites supported energy production via the glycolysis pathway and TCA cycle by 24 h and 48 h of heat stress. Anaerobic metabolism also fulfilled the role of energy production. Antioxidant molecules acted within thermally stressed mussels to mitigate oxidative stress. Purine metabolism supported tissue protection and energy replenishment. Pyrimidine metabolism supported the protection of nucleic acids and protein synthesis. Amino acids ensured balanced intracellular osmolality at 24 h and ammonia detoxification at 48 h. Altogether, this work provides evidence that P. canaliculus has the potential to adapt to heat stress up to 24 °C by regulating its energy metabolism, balancing nucleotide production, and implementing oxidative stress mechanisms over time. The data reported herein can also be used to evaluate the risks of heatwaves and improve mitigation strategies for aquaculture.

The cross-tissue metabolic response of abalone (Haliotis midae) to functional hypoxia.

Functional hypoxia is a stress condition caused by the abalone itself as a result of increased muscle activity, which generally necessitates the employment of anaerobic metabolism if the activity is sustained for prolonged periods. With that being said, abalone are highly reliant on anaerobic metabolism to provide partial compensation for energy production during oxygen-deprived episodes. However, current knowledge on the holistic metabolic response for energy metabolism during functional hypoxia, and the contribution of different metabolic pathways and various abalone tissues towards the overall accumulation of anaerobic end-products in abalone are scarce. Metabolomics analysis of adductor muscle, foot muscle, left gill, right gill, haemolymph and epipodial tissue samples indicated that South African abalone (Haliotis midae) subjected to functional hypoxia utilises predominantly anaerobic metabolism, and depends on all of the main metabolite classes (proteins, carbohydrates and lipids) for energy supply. Functional hypoxia caused increased levels of anaerobic end-products: lactate, alanopine, tauropine, succinate and alanine. Also, elevation in arginine levels was detected, confirming that abalone use phosphoarginine to generate energy during functional hypoxia. Different tissues showed varied metabolic responses to hypoxia, with functional hypoxia showing excessive changes in the adductor muscle and gills. From this metabolomics investigation, it becomes evident that abalone are metabolically able to produce sufficient amounts of energy when functional hypoxia is experienced. Also, tissue interplay enables the adjustment of H. midae energy requirements as their metabolism shifts from aerobic to anaerobic respiration during functional hypoxia.This article has an associated First Person interview with the first author of the paper.

Uncovering the metabolic response of abalone (Haliotis midae) to environmental hypoxia through metabolomics.

INTRODUCTION: Oxygen is essential for metabolic processes and in the absence thereof alternative metabolic pathways are required for energy production, as seen in marine invertebrates like abalone. Even though hypoxia has been responsible for significant losses to the aquaculture industry, the overall metabolic adaptations of abalone in response to environmental hypoxia are as yet, not fully elucidated. OBJECTIVE: To use a multiplatform metabolomics approach to characterize the metabolic changes associated with energy production in abalone (Haliotis midae) when exposed to environmental hypoxia. METHODS: Metabolomics analysis of abalone adductor and foot muscle, left and right gill, hemolymph, and epipodial tissue samples were conducted using a multiplatform approach, which included untargeted NMR spectroscopy, untargeted and targeted LC-MS spectrometry, and untargeted and semi-targeted GC-MS spectrometric analyses. RESULTS: Increased levels of anaerobic end-products specific to marine animals were found which include alanopine, strombine, tauropine and octopine. These were accompanied by elevated lactate, succinate and arginine, of which the latter is a product of phosphoarginine breakdown in abalone. Primarily amino acid metabolism was affected, with carbohydrate and lipid metabolism assisting with anaerobic energy production to a lesser extent. Different tissues showed varied metabolic responses to hypoxia, with the largest metabolic changes in the adductor muscle. CONCLUSIONS: From this investigation, it becomes evident that abalone have well-developed (yet understudied) metabolic mechanisms for surviving hypoxic periods. Furthermore, metabolomics serves as a powerful tool for investigating the altered metabolic processes in abalone.

From untargeted LC-QTOF analysis to characterisation of opines in abalone adductor muscle: Theory meets practice.

Abalone have a unique ability to use pyruvate, various amino acids and dehydrogenases, to produce opines as means to prevent the accumulation of NADH during anaerobic conditions. In this study, the theoretical masses, formulae and fragment patterns of butylated opines were used to predict which of these compounds could be found in the abalone adductor muscle using untargeted liquid chromatography quadrupole time-of flight-mass spectrometry. These findings were validated using synthesised opine standards. In essence alanopine, lysopine, strombine and tauropine produced in abalone adductor muscle could be characterised using the highest identification confidence levels.

Foraging at wastewater treatment works affects brown adipose tissue fatty acid profiles in banana bats.

In this study we tested the hypothesis that the decrease in habitat quality at wastewater treatment works (WWTW), such as limited prey diversity and exposure to the toxic cocktail of pollutants, affect fatty acid profiles of interscapular brown adipose tissue (iBrAT) in bats. Further, the antioxidant capacity of oxidative tissues such as pectoral and cardiac muscle may not be adequate to protect those tissues against reactive molecules resulting from polyunsaturated fatty acid auto-oxidation in the WWTW bats. Bats were sampled at two urban WWTW, and two unpolluted reference sites in KwaZulu-Natal, South Africa. Brown adipose tissue (BrAT) mass was lower in WWTW bats than in reference site bats. We found lower levels of saturated phospholipid fatty acids and higher levels of mono- and polyunsaturated fatty acids in WWTW bats than in reference site bats, while C18 desaturation and n-6 to n-3 ratios were higher in the WWTW bats. This was not associated with high lipid peroxidation levels in pectoral and cardiac muscle. Combined, these results indicate that WWTW bats rely on iBrAT as an energy source, and opportunistic foraging on abundant, pollutant-tolerant prey may change fatty acid profiles in their tissue, with possible effects on mitochondrial functioning, torpor and energy usage.

The simultaneous detection and quantification of p-aminobenzoic acid and its phase 2 biotransformation metabolites in human urine using LC-MS/MS.

BACKGROUND: p-Aminobenzoic acid (PABA) can be used as a probe substance to investigate glycine conjugation, a reaction of phase 2 biotransformation. METHODOLOGY/RESULTS: An LC-MS/MS method for simultaneous quantification of PABA and its metabolites from human urine was developed and validated. The metabolites can be quantified with acceptable precision and accuracy directly from human urine samples after ingestion of 550 mg PABA. CONCLUSION: The developed LC-MS/MS assay is to our knowledge the first method available for the simultaneous quantification of PABA and its glycine conjugation metabolites in human urine and provides important quantitative data for studies of this phase 2 biotransformation pathway.

Obesity and metabolomics: metallothioneins protect against high-fat diet-induced consequences in metallothionein knockout mice.

Obesity continues to rise as an alarming global epidemic. System level mechanisms, diagnostics, and therapeutics are sorely needed so as to identify at risk individuals and design appropriate population scale interventions. The present study evaluated the protective role of metallothioneins (MTs) against obesity and high-fat diet-induced effects such as insulin resistance in both male and female MT-1+2 knockout and MT-3 knockout mice. As the metabolome is closest to the functional phenotype, changes in metabolite levels were also evaluated, and the direct or indirect involvement of MTs in metabolism examined. MT-1+2-, MT-3 knockout, and wild-type mice were given a high-fat diet for 2 months. Variation in body weight gain, tissue weight, and response to oral glucose tolerance test and insulin tolerance test were determined and compared to mice that received the control diet. Effect of the high-fat diet on the knockout mice were investigated on the metabolome level in specific tissues using metabolomics. Both knockout mice strains were more susceptible to high-fat diet-induced effects, such as weight gain and moderate insulin resistance, with the MT-3 knockout mice most susceptible. Brain tissue of the knockout mice showed most metabolic variation and pointed to possible impairment of mitochondrial function. The protective effect of MTs against high-fat diet and obesity-induced effects such as insulin resistance was evident from our observations. The putative role MTs play in mitochondrial function is possibly the main contributor to the lack of these effects in wild-type mice. Considering the expression profiles of the MT isoforms and similarity in brain metabolic variation in the knockout strains, it appears that they promote mitochondrial function in the hypothalamus, thereby limiting weight gain and insulin resistance. Furthermore, metabolomics research in preclinical models of obesity and in the clinic is warranted in the near future.

Increased excretion of c4-carnitine species after a therapeutic acetylsalicylic Acid dose: evidence for an inhibitory effect on short-chain Fatty Acid metabolism.

Acetylsalicylic acid and/or its metabolites are implicated to have various effects on metabolism and, especially, on mitochondrial function. These effects include both inhibitory and stimulatory effects. We investigated the effect of both combined and separate oral acetylsalicylic acid and acetaminophen administration at therapeutic doses on the urinary metabolite profile of human subjects. In this paper, we provided in vivo evidence, in human subjects, of a statistically significant increase in isobutyrylcarnitine after the administration of a therapeutic dose of acetylsalicylic acid. We, therefore, propose an inhibitory effect of acetylsalicylic acid on the short-chain fatty acid metabolism, possibly at the level of isobutyryl-CoA dehydrogenase.

Quantitative determination of octadecenedioic acid in human skin and transdermal perfusates by gas chromatography-mass spectrometry.

A gas chromatographic (GC) method with mass spectrometric (MS) detection is developed and validated for the accurate and precise determination of octadecenedioic acid (C18:1 DIOIC) in human skin samples and transdermal perfusates. C18:1 DIOIC is extracted using methanol. The saturated analogue 1,18-octadecanedioic acid (C18:0 DIOIC) is added as internal standard. Prior to analysis, both compounds are converted to their trimethylsilylated derivatives using N,O-bis(trimethylsilyl)trifluoroacetamide with 15% trimethylchlorosilane. Quantitation is performed in selected ion monitoring mode with a limit of quantitation of 250 ng/mL. Linearity with a correlation coefficient of 0.998 is obtained over a concentration range of 250-2000 ng/mL. Values for within-day accuracy range from 94.5% to 102.4%, and from 97.5% to 105.8% for between-day accuracy. Within- and between-day precision values are better than 5% and 7%, respectively. The recovery values from the various matrices vary from 92.6% to 104.0%. The GC-MS method is employed for the determination of C18:1 DIOIC after application of an emulsion containing the active ingredient onto human skin in vitro. The results demonstrate that the method is suitable for the determination of C18:1 DIOIC in human skin samples and transdermal perfusates.