Do universal codon-usage patterns minimize the effects of mutation and translation error?

Background

Do species use codons that reduce the impact of errors in translation or replication? The genetic code is arranged in a way that minimizes errors, defined as the sum of the differences in amino-acid properties caused by single-base changes from each codon to each other codon. However, the extent to which organisms optimize the genetic messages written in this code has been far less studied. We tested whether codon and amino-acid usages from 457 bacteria, 264 eukaryotes, and 33 archaea minimize errors compared to random usages, and whether changes in genome G+C content influence these error values.

Results

We tested the hypotheses that organisms choose their codon usage to minimize errors, and that the large observed variation in G+C content in coding sequences, but the low variation in G+U or G+A content, is due to differences in the effects of variation along these axes on the error value. Surprisingly, the biological distribution of error values has far lower variance than randomized error values, but error values of actual codon and amino-acid usages are actually greater than would be expected by chance.

Conclusion

These unexpected findings suggest that selection against translation error has not produced codon or amino-acid usages that minimize the effects of errors, and that even messages with very different nucleotide compositions somehow maintain a relatively constant error value. They raise the question: why do all known organisms use highly error-minimizing genetic codes, but fail to minimize the errors in the mRNA messages they encode?

     

Pharmacometabolomics applied to zonisamide pharmacokinetic parameter prediction

Introduction

Zonisamide is a new-generation anticonvulsant antiepileptic drug metabolized primarily in the liver, with subsequent elimination via the renal route.

Objectives

Our objective was to evaluate the utility of pharmacometabolomics in the detection of zonisamide metabolites that could be related to its disposition and therefore, to its efficacy and toxicity.

Methods

This study was nested to a bioequivalence clinical trial with 28 healthy volunteers. Each participant received a single dose of zonisamide on two separate occasions (period 1 and period 2), with a washout period between them. Blood samples of zonisamide were obtained from all patients at baseline for each period, before volunteers were administered any medication, for metabolomics analysis.

Results

After a Lasso regression was applied, age, height, branched-chain amino acids, steroids, triacylglycerols, diacyl glycerophosphoethanolamine, glycerophospholipids susceptible to methylation, phosphatidylcholines with 20:4 FA (arachidonic acid) and cholesterol ester and lysophosphatidylcholine were obtained in both periods.

Conclusion

To our knowledge, this is the only research study to date that has attempted to link basal metabolomic status with pharmacokinetic parameters of zonisamide.

     

Profiling of faecal water and urine metabolites among Papua New Guinea highlanders believed to be adapted to low protein intake

Introduction

Adequate amount of proteins from foods are normally needed to maintain muscle mass of the human body. Although protein intakes of Papua New Guinea (PNG) highlanders are less than biologically adequate, protein deficiency related disorders have rarely been reported. It has been postulated that gut microbiota play a role in such low-protein-adaptation.

Objective

To explore underlying biological mechanisms of low-protein adaptation among PNG highlanders by investigating metabolomic profiles of faecal water and urine.

Methods

We performed metabolome analysis using faecal water extracted from faecal samples of PNG highlanders, PNG non-highlanders and Japanese subjects. We paid special attention to amino acids and other metabolites produced by gut microbiota, as well as to metabolites involved in nitrogen recycling in the human gut.

Results

Our results indicated that amino acid levels were higher in faecal water from PNG highlanders than PNG non-highlanders, but amino acid levels did not differ between PNG highlanders and Japanese subjects. Among PNG highlander samples, amino acid levels tended to be higher in those who consumed less protein.

Conclusion

We speculated that a greater proportion of urea was excreted to the intestine among the PNG highlanders than other groups, and that the urea was used for nitrogen salvage. Intestinal bacteria are essential for producing ammonia from urea and also for producing amino acids from ammonia, which is a key process in low-protein adaptation. Profiling the gut microbiota of PNG highlanders is an important avenue for further research into the mechanisms of low-protein adaptation.

     

Comprehensive metabolite profiling of onion bulbs (<Emphasis Type="Italic">Allium cepa</Emphasis>) using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry

Introduction

Onion (Allium cepa) represents one of the most important horticultural crops and is used as food, spice and medicinal plant almost worldwide. Onion bulbs accumulate a broad range of primary and secondary metabolites which impact nutritional, sensory and technological properties.

Objectives

To complement existing analytical methods targeting individual compound classes this work aimed at the development and validation of an analytical workflow for comprehensive metabolite profiling of onion bulbs.

Method

Metabolite profiling was performed by liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC/ESI-QTOFMS). For annotation of metabolites accurate mass tandem mass spectrometry experiments were carried out.

Results

On the basis of LC/ESI-QTOFMS and two chromatographic methods an analytical workflow was developed which facilitates profiling of polar and semi-polar onion metabolites including fructooligosaccharides, proteinogenic amino acids, peptides, S-substituted cysteine conjugates, flavonoids and saponins. To minimize enzymatic conversion of S-alk(en)ylcysteine sulfoxides, a sample preparation and extraction protocol for fresh onions was developed comprising cryohomogenization and a low-temperature quenching step. A total of 123 metabolites were annotated and characterized by chromatographic and tandem mass spectral data. For validation, recovery rates and matrix effects were determined for 15 model compounds. Repeatability and linearity were assessed for more than 80 endogenous metabolites.

Conclusion

As exemplarily demonstrated by comparative metabolic analysis of six onion cultivars the established analytical workflow in combination with targeted and non-targeted data analysis strategies can be successfully applied for comprehensive metabolite profiling of onion bulbs.

     

The urinary metabolome in female mink (<Emphasis Type="Italic">Mustela neovison</Emphasis>) shows distinct changes in protein and lipid metabolism during the transition from diapause to implantation

Introduction

The mink exhibit an obligatory diapause. The metabolic changes during the transition from diapause to implantation and established pregnancy are currently unknown.

Objectives

The study aimed to characterize changes in the urinary metabolome in mink during the period from mating to early gestation and to identify the metabolites involved.

Methods

Urine samples were collected from 56 female mink on March 24, April 8, and April 15, covering the period from mating to early pregnancy. The urine samples were subjected to non-targeted LC-MS metabolomics. Processed data were evaluated by principal component analysis (PCA) and the peak area of identified metabolites were subjected to ANOVA.

Results

The samples showed clear clustering according to sampling date in a PCA scores plot, and 35 metabolites differing significantly between sampling days were identified. The excretion of dicarboxylic acids and acylcarnitines of dicarboxylic acids exhibited a decline on April 8, and the same trend was observed for four unidentified metabolites, two of which were putatively identified as acids of the furan fatty acid type. The decreased excretion of lipid components was suggested to be a result of increased oxidation of these compounds. In contrast, the excretion of amino acid-related metabolites showed an increase on April 8 which was attributed to increased metabolism of amino acids at this time point.

Conclusion

The urinary metabolic profile of mink showed distinct changes during the period studied. The major changes were observed at the time of implantation where increases in the lipid and protein metabolism were evident.

     

Quantitative metabolomics comparison of traditional blood draws and TAP capillary blood collection

Introduction

Mass spectrometry and computational biology have advanced significantly in the past ten years, bringing the field of metabolomics a step closer to personalized medicine applications. Despite these analytical advancements, collection of blood samples for routine clinical analysis is still performed through traditional blood draws.

Objective

TAP capillary blood collection has been recently introduced for the rapid, painless draw of small volumes of blood (~?100 μL), though little is known about the comparability of metabolic phenotypes of blood drawn via traditional venipuncture and TAP devices.

Methods

UHPLC-MS-targeted metabolomics analyses were performed on blood drawn traditionally or through TAP devices from 5 healthy volunteers. Absolute quantitation of 45 clinically-relevant metabolites was calculated against stable heavy isotope-labeled internal standards.

Results

Ranges for 39 out of 45 quantified metabolites overlapped between drawing methods. Pyruvate and succinate were over threefold higher in the TAP samples than in traditional blood draws. No significant changes were observed for other carboxylates, glucose or lactate. TAP samples were characterized by increases in reduced glutathione and decreases in urate and cystine, markers of oxidation of purines and cysteine—overall suggesting decreased oxidation during draws. The absolute levels of bile acids and acyl-carnitines, as well as almost all amino acids, perfectly correlated among groups (Spearman r?≥?0.95).

Conclusion

Though further more extensive studies will be mandatory, this pilot suggests that TAP-derived blood may be a logistically-friendly source of blood for large scale metabolomics studies—especially those addressing amino acids, glycemia and lactatemia as well as bile acids, acyl-carnitine levels.

     

Interactions of in vitro selected fluorogenic peptide aptamers with calmodulin

Objectives

We examined the importance of aptamer usage under the same condition as the selection process by employing the previously selected aptamers for calmodulin (CaM) which includes a non-natural fluorogenic amino acid, 7-nitro-2,1,3-benzoxadiazole.

Results

We added five amino acids at the N-terminus which was employed for the selection and then we tested the affinity and selectivity for CaM binding. Surface plasmon resonance and fluorescence measurements showed that the additional amino acids for one of the aptamers drastically improved binding affinity to CaM, indicating the importance of aptamer use under the same conditions as the selection process. Such drastic improvement in affinity was not observed for the sequence which had been reported previously. Nuclear magnetic resonance data identified that the primary binding site is located in a C-terminal of CaM and the additional residues enhance interactions with CaM.

Conclusions

We found that the addition of the common sequence, which was employed for ribosome display, makes the affinity of a selected peptide as strong as the previously reported peptide.

     

Habitual sleep and human plasma metabolomics

Introduction

Sleep plays an important role in cardiometabolic health. The sleep-wake cycle is partially driven by the endogenous circadian clock, which governs a range of metabolic pathways. The association between sleep and cardiometabolic health may be mediated by alterations of the human metabolome.

Objectives

To better understand the biological mechanism underlying the association between sleep and health, we examined human plasma metabolites in relation to sleep duration and sleep timing.

Methods

Using an untargeted approach, 329 fasting plasma metabolites were measured in 277 Chinese participants. We measured sleep timing (midpoint between bedtime and wake up time) using repeated time-use surveys (4 weeks during 1 year) and previous night sleep duration from questionnaires completed before sample donation.

Results

We found 64 metabolites that were associated with sleep timing with a false discovery rate of 0.2 or lower, after adjusting for potential confounders. Notably, we found that later sleep timing was associated with higher levels of multiple metabolites in amino acid metabolism, including branched chain amino acids and their gamma-glutamyl dipeptides. We also found widespread associations between sleep timing and numerous metabolites in lipid metabolism, including bile acids, carnitines and fatty acids. In contrast, previous night sleep duration was not associated with plasma metabolites in our study.

Conclusion

Sleep timing was associated with a large number of metabolites across a variety of biochemical pathways. Some metabolite associations are consistent with a relationship between late chronotype and adverse effects on cardiometabolic health.

     

A targeted metabolomic procedure for amino acid analysis in different biological specimens by ultra-high-performance liquid chromatography–tandem mass spectrometry

Introduction

Amino acid analysis in biological fluids is essential for the study of inborn errors of metabolism (IEM) and other diseases.

Objectives

Our aim was to develop a UPLC-MS/MS procedure for the analysis of 25 amino acids and identification of 17 related compounds.

Methods

Sample treatment conditions were optimized for plasma, urine, cerebrospinal fluid (CSF) and dried blood spots. Amino acids and related compounds were analyzed on a Waters ACQUITY UPLC H-class instrument with a reversed-phase C-18 column using water and acetonitrile with 0.1% formic acid as the mobile phases (run time?=?9 min). The detection was performed with a Waters Xevo TQD triple-quadrupole mass spectrometer using positive electrospray ionization in the multiple reaction monitoring mode.

Results

The method linearity, intra-assay and inter-assay precision, detection limit, quantification limit and trueness analysis displayed adequate results in both physiological and pathological conditions. Method comparison was performed between UPLC-MS/MS and ion exchange chromatography (IEC) with ninhydrin derivatization, and the methods showed good agreement, except for 4-hydroxyproline, aspartate and citrulline. Paediatrics age-related reference values in plasma, urine and CSF were established and patients with different IEM were easily identified.

Conclusion

We report a modified UPLC-MS/MS procedure for the analysis of 42 amino acids and related compounds in different specimens. The method is fast, sensitive and robust, and it has been validated to be an alternative to the traditional IEC procedure as the routine method used in metabolic laboratories. The method greatly decreases the run time of the analysis while displaying good metrological results.

     

An effective hybrid of hill climbing and genetic algorithm for 2D triangular protein structure prediction

Background

Proteins play fundamental and crucial roles in nearly all biological processes, such as, enzymatic catalysis, signaling transduction, DNA and RNA synthesis, and embryonic development. It has been a long-standing goal in molecular biology to predict the tertiary structure of a protein from its primary amino acid sequence. From visual comparison, it was found that a 2D triangular lattice model can give a better structure modeling and prediction for proteins with short primary amino acid sequences.

Methods

This paper proposes a hybrid of hill-climbing and genetic algorithm (HHGA) based on elite-based reproduction strategy for protein structure prediction on the 2D triangular lattice.

Results

The simulation results show that the proposed HHGA can successfully deal with the protein structure prediction problems. Specifically, HHGA significantly outperforms conventional genetic algorithms and is comparable to the state-of-the-art method in terms of free energy.

Conclusions

Thanks to the enhancement of local search on the global search, the proposed HHGA achieves promising results on the 2D triangular protein structure prediction problem. The satisfactory simulation results demonstrate the effectiveness of the proposed HHGA and the utility of the 2D triangular lattice model for protein structure prediction.

     

Nucleotide binding domain 1 pharmacophore modeling for visualization and analysis of P-glycoprotein–flavonoid molecular interactions

Background

P-glycoprotein (P-gp) is a 170-kDa membrane protein. It provides a barrier function and help to excrete toxins from the body as a transporter. Some bioflavonoids have been shown to block P-gp activity.

Objective

To evaluate the important amino acid residues within nucleotide binding domain 1 (NBD1) of P-gp that play a key role in molecular interactions with flavonoids using structure-based pharmacophore model.

Methods

In the molecular docking with NBD1 models, a putative binding site of flavonoids was proposed and compared with the site for ATP. The binding modes for ligands were achieved using LigandScout to generate the P-gp–flavonoid pharmacophore models.

Results

The binding pocket for flavonoids was investigated and found these inhibitors compete with the ATP for binding site in NBD1 including the NBD1 amino acid residues identified by the in silico techniques to be involved in the hydrogen bonding and van der Waals (hydrophobic) interactions with flavonoids.

Conclusion

These flavonoids occupy with the same binding site of ATP in NBD1 proffering that they may act as an ATP competitive inhibitor.

     

Granulocyte colony-stimulating factor alters the systemic metabolomic profile in healthy donors

Introduction

Peripheral blood stem cells mobilized by granulocyte colony-stimulating factor (G-CSF) from healthy donors are commonly used for allogeneic stem cell transplantation. The effect of G-CSF administration on global serum metabolite profiles has not been investigated before.

Objectives

This study aims to examine the systemic metabolomic profiles prior to and following administration of G-CSF in healthy adults.

Methods

Blood samples were collected from 15 healthy stem cell donors prior to and after administration of G-CSF 10 µg/kg/day for 4 days. Using a non-targeted metabolomics approach, metabolite levels in serum were determined using ultrahigh performance liquid chromatography-tandem mass spectrometry and gas chromatography/mass spectrometry.

Results

Comparison of the metabolite profiles of donors before and after G-CSF treatment revealed 239 metabolites that were significantly altered. The major changes of the metabolite profiles following G-CSF administration included alteration of several fatty acids, including increased levels of several medium and long-chain fatty acids, as well as polyunsaturated fatty acids; while there were lower levels of other lipid metabolites such as phospholipids, lysolipids, sphingolipids. Furthermore, there were significantly lower levels of several amino acids and/or their metabolites, including several amino acids with known immunoregulatory functions (methionine, tryptophan, valine). Lastly, the levels of several nucleotides and nucleotide metabolites (guanosine, adenosine, inosine) were also decreased after G-CSF administration, while methylated products were increased. Some of these altered products/metabolites may potentially have angioregulatory effects whereas others may suggest altered intracellular epigenetic regulation.

Conclusion

Our results show that G-CSF treatment alters biochemical serum profiles, in particular amino acid, lipid and nucleotide metabolism. Additional studies are needed to further evaluate the relevance of these changes in healthy donors.

     

Non-invasive urinary metabolomic profiling discriminates prostate cancer from benign prostatic hyperplasia

Introduction

Prostate cancer (PCa) is one of the most common malignancies in men worldwide. Serum prostate specific antigen (PSA) level has been extensively used as a biomarker to detect PCa. However, PSA is not cancer-specific and various non-malignant conditions, including benign prostatic hyperplasia (BPH), can cause a rise in PSA blood levels, thus leading to many false positive results.

Objectives

In this study, we evaluated the potential of urinary metabolomic profiling for discriminating PCa from BPH.

Methods

Urine samples from 64 PCa patients and 51 individuals diagnosed with BPH were analysed using ¹H nuclear magnetic resonance (¹H-NMR). Comparative analysis of urinary metabolomic profiles was carried out using multivariate and univariate statistical approaches.

Results

The urine metabolomic profile of PCa patients is characterised by increased concentrations of branched-chain amino acids (BCAA), glutamate and pseudouridine, and decreased concentrations of glycine, dimethylglycine, fumarate and 4-imidazole-acetate compared with individuals diagnosed with BPH.

Conclusion

PCa patients have a specific urinary metabolomic profile. The results of our study underscore the clinical potential of metabolomic profiling to uncover metabolic changes that could be useful to discriminate PCa from BPH in a clinical context.

     

The altered human serum metabolome induced by a marathon

Introduction

Endurance races have been associated with a substantial amount of adverse effects which could lead to chronic disease and long-term performance impairment. However, little is known about the holistic metabolic changes occurring within the serum metabolome of athletes after the completion of a marathon.

Objectives

Considering this, the aim of this study was to better characterize the acute metabolic changes induced by a marathon.

Methods

Using an untargeted two dimensional gas chromatography time-of-flight mass spectrometry metabolomics approach, pre- and post-marathon serum samples of 31 athletes were analyzed and compared to identify those metabolites varying the most after the marathon perturbation.

Results

Principle component analysis of the comparative groups indicated natural differentiation due to variation in the total metabolite profiles. Elevated concentrations of carbohydrates, fatty acids, tricarboxylic acid cycle intermediates, ketones and reduced concentrations of amino acids indicated a metabolic shift between various fuel substrate systems. Additionally, elevated odd-chain fatty acids and α-hydroxy acids indicated the utilization of α-oxidation and autophagy as alternative energy-producing mechanisms. Adaptations in gut microbe-associated markers were also observed and correlated with the metabolic flexibility of the athlete.

Conclusion

From these results it is evident that a marathon places immense strain on the energy-producing pathways of the athlete, leading to extensive protein degradation, oxidative stress, mammalian target of rapamycin complex 1 inhibition and autophagy. A better understanding of this metabolic shift could provide new insights for optimizing athletic performance, developing more efficient nutrition regimens and identify strategies to improve recovery.

     

Italian cohort of patients affected by inflammatory bowel disease is characterised by variation in glycerophospholipid,free fatty acids and amino acid levels

Background

Inflammatory bowel disease is a group of pathologies characterised by chronic inflammation of the intestine and an unclear aetiology. Its main manifestations are Crohn’s disease and ulcerative colitis. Currently, biopsies are the most used diagnostic tests for these diseases and metabolomics could represent a less invasive approach to identify biomarkers of disease presence and progression.

Objectives

The lipid and the polar metabolite profile of plasma samples of patients affected by inflammatory bowel disease have been compared with healthy individuals with the aim to find their metabolomic differences. Also, a selected sub-set of samples was analysed following solid phase extraction to further characterise differences between pathological samples.

Methods

A total of 200 plasma samples were analysed using drift tube ion mobility coupled with time of flight mass spectrometry and liquid chromatography for the lipid metabolite profile analysis, while liquid chromatography coupled with triple quadrupole mass spectrometry was used for the polar metabolite profile analysis.

Results

Variations in the lipid profile between inflammatory bowel disease and healthy individuals were highlighted. Phosphatidylcholines, lyso-phosphatidylcholines and fatty acids were significantly changed among pathological samples suggesting changes in phospholipase A₂ and arachidonic acid metabolic pathways. Variations in the levels of cholesteryl esters and glycerophospholipids were also found. Furthermore, a decrease in amino acids levels suggests mucosal damage in inflammatory bowel disease.

Conclusions

Given good statistical results and predictive power of the model produced in our study, metabolomics can be considered as a valid tool to investigate inflammatory bowel disease.

     

Analysis of the free fatty acid metabolome in the plasma of patients with systemic lupus erythematosus and fever

Introduction

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease with heterogeneous clinical manifestations mediated by immune dysregulation.

Objectives

We aimed to analyze the metabolomic differences in free fatty acids (FFAs) between patients with SLE and healthy controls (HCs).

Methods

In this study, the levels of 24 FFAs, as their tert-butyldimethylsilyl derivatives, in the plasma of 41 patients with SLE and 41 HCs, were investigated using gas chromatography with mass spectrometry in selected-ion monitoring mode.

Results

The results showed that patients with SLE and HCs had significantly different levels of 13 of the 24 FFAs. The levels of myristic, palmitoleic, oleic, and eicosenoic acids were significantly higher, whereas the levels of caproic, caprylic, linoleic, stearic, arachidonic, eicosanoic, behenic, lignoceric, and hexacosanoic acids were significantly lower in patients with SLE, than in the HCs. In the partial-correlation analysis of the FFA profiles and markers of disease activity of SLE, several metabolic markers correlated with SLE disease activity.

Conclusions

Our results provide a comprehensive understanding of the relationship between FFAs and markers of SLE disease activity. Thus, this approach has promising potential for the discovery of metabolic biomarkers of SLE.

     

Amino acid composition in endothermic vertebrates is biased in the same direction as in thermophilic prokaryotes

Background

Among bacteria and archaea, amino acid usage is correlated with habitat temperatures. In particular, protein surfaces in species thriving at higher temperatures appear to be enriched in amino acids that stabilize protein structure and depleted in amino acids that decrease thermostability. Does this observation reflect a causal relationship, or could the apparent trend be caused by phylogenetic relatedness among sampled organisms living at different temperatures? And do proteins from endothermic and exothermic vertebrates show similar differences?

Results

We find that the observed correlations between the frequencies of individual amino acids and prokaryotic habitat temperature are strongly influenced by evolutionary relatedness between the species analysed; however, a proteome-wide bias towards increased thermostability remains after controlling for phylogeny. Do eukaryotes show similar effects of thermal adaptation? A small shift of amino acid usage in the expected direction is observed in endothermic ('warm-blooded') mammals and chicken compared to ectothermic ('cold-blooded') vertebrates with lower body temperatures; this shift is not simply explained by nucleotide usage biases.

Conclusion

Protein homologs operating at different temperatures have different amino acid composition, both in prokaryotes and in vertebrates. Thus, during the transition from ectothermic to endothermic life styles, the ancestors of mammals and of birds may have experienced weak genome-wide positive selection to increase the thermostability of their proteins.

     

A lack of peptide binding and decreased thermostability suggests that the CASKIN2 scaffolding protein SH3 domain may be vestigial

Background

CASKIN2 is a neuronal signaling scaffolding protein comprised of multiple ankyrin repeats, two SAM domains, and one SH3 domain. The CASKIN2 SH3 domain for an NMR structural determination because its peptide-binding cleft appeared to deviate from the repertoire of aromatic enriched amino acids that typically bind polyproline-rich sequences.

Results

The structure demonstrated that two non-canonical basic amino acids (K290/R319) in the binding cleft were accommodated well in the SH3 fold. An K290Y/R319W double mutant restoring the typical aromatic amino acids found in the binding cleft resulted in a 20 °C relative increase in the thermal stability. Considering the reduced stability, we speculated that the CASKIN2 SH3 could be a nonfunctional remnant in this scaffolding protein.

Conclusions

While the NMR structure demonstrates that the CASKIN2 SH3 domain is folded, its cleft has suffered two substitutions that prevent it from binding typical polyproline ligands. This observation led us to additionally survey and describe other SH3 domains in the Protein Data Bank that may have similarly lost their ability to promote protein-protein interactions.

     

Transient decrease of hepatic NAD<Superscript>+</Superscript> and amino acid alterations during treatment with valproate: new insights on drug-induced effects in vivo using targeted MS-based metabolomics

Background

Therapeutic administration of the drug valproate (VPA) results in metabolic changes at the hepatic level that have not been fully characterized. Interference of this branched-chain fatty acid with the oxidative metabolism of amino acids may have consequences for the downstream biosynthesis of essential cofactors.

Objectives

We aimed to evaluate the effect of VPA on amino acid and NAD⁺ metabolism using targeted MS-based metabolite profiling.

Methods

Plasma samples from patients under chronic treatment with VPA were analyzed. VPA was administered to Wistar rats mimicking prolonged and acute treatment, the latter with two different doses. Plasma and liver samples were collected for targeted metabolomics studies using UPLC-MS/MS and GC-FID.

Results

Analysis of amino acids in rat plasma and liver and in human plasma demonstrated that drug intake is associated with a particularly significant drop in the levels of tryptophan, and increased levels of glycine and lysine. The lowered plasma tryptophan levels prompted us to study the intracellular content of tryptophan and various nicotinamide adenine dinucleotides. A significant decrease of NAD⁺ and NADP⁺ was observed in the liver of rats after the single administration of VPA at two different doses, but not after repeated administration.

Conclusion

The observed accumulation of kynurenine intermediates in rat liver tissue suggests a drug-induced interference with the de novo pathway of NAD⁺ biosynthesis. These findings provide novel insights into the mechanisms of VPA associated hepatocellular dysfunction and/or toxicity, but with possible major relevance to the anticancer effects of the drug.