40 research outputs found
Metabolomics reveals the depletion of intracellular metabolites in HepG2 cells after treatment with gold nanoparticles
No full textStudies on the safety of gold nanoparticles (GNPs) are plentiful due to their successful application in drug delivery and treatment of diseases in trials. Cytotoxicity caused by GNPs has been studied on the physiological and biochemical level; yet, the effect of GNPs (particularly gold nano-spheres) on the metabolome of living organisms remains understudied. In this investigation, metabolomics was used to comprehensively study the metabolic alterations in HepG2 cells caused by GNPs; and to investigate the role of representative GNP coatings. GNPs were synthesized, coated and characterized before use on HepG2 cell cultures. Cells were treated for 3 h with citrate-, poly-(sodiumsterene sulfunate)-, and poly-vinylpyrrolidone (PVP)-capped GNPs, respectively. The internalization of the different GNPs and their effect on mitochondrial respiration and the metabolome were studied. Results indicated that the PVP-capped GNPs internalized more and also caused a more observable effect on the metabolome. Conversely, it was the citrate- and poly-(sodiumsterene sulfunate) coated particles that influenced ATP production in addition to the metabolomic changes. A holistic depletion of intracellular metabolites was observed regardless of GNP coating, which hints to the binding of certain metabolites to the particle
Systemic and organ specific metabolic variation in metallothionein knockout mice challenged with swimming exercise
No full textMetallothioneins (MTs) are ubiquitous, multifunctional
proteins with key roles in metal homeostasis and
redox regulation. Their involvement in cellular energy
metabolism is evident from the observation that metallothionein-
knockout (MTKO) mice become moderately obese.
Transcriptomic studies have also indicated that several genes
associated with energy metabolism are differentially expressed
in these mice. Although single varying metabolites have
been reported, the roles of MTs on a global metabolic level
have not been investigated before. In this study, an untargeted,
hypothesis-generating metabolomics approach was
used to identify and report all metabolites that differ in relative
concentration between MT1?2KO, MT3KO and wildtype
(WT) mice before and after an exercise (1 h swim)
perturbation. PCA and univariate results confirmed that the
metabolism of the MTKO mice differs from the WT during
unchallenged conditions and hypothetically pointed to
increased anabolic activity which could contribute to their
previously reported tendency to become obese. Furthermore,
the metabolic differences observed in the liver after the 1 h
swim indicated that catabolic activity might be impaired in
these mice, which could be a consequence of a dysfunction of
a common catabolic signal. The MT3KO mice did not show
the same metabolic pattern as the MT1?2KO mice as most
metabolite concentrations in the brain of these mice were
lower after the 1 h swim. The reported metabolic variation
contributes to better understand the diverse functionality of
these ubiquitous proteins on a global phenotypic level
Abalone growth and associated aspects: now from a metabolic perspective
No full textWorldwide, there are approximately 100 Haliotis species, more commonly known as abalone or ‘Paua’ in New Zealand, ‘Venus's‐ears’ in Greece, ‘Awabi’ in Japan, ‘Perlemoen’ in South Africa and ‘Ormers’ in Europe. Regardless of what they are called in any part of the world, a high monetary value is coupled to this animal, because it is largely considered a seafood delicacy. Subsequently, a great deal of research primarily focused on improving the health and growth rates of abalone were carried out to maximise productivity of the commercial farming efforts in various countries. In this review, we comprehensively describe the most recent available scientific literature on abalone biology, and those aspects related to the growth of this organism; more specifically, those factors related to the uptake and breakdown of metabolic products which ensures long‐term growth. We subsequently discuss this in terms of basic animal design, farming outcomes, feeding, cellular growth mechanisms and the unique metabolic processes that exist in these species. Using this information and the knowledge of the metabolic processes in other organisms, we additionally make a number of new hypotheses regarding how these metabolic processes may function in terms of abalone growth. Based on the information presented in this review, we also identify major research opportunities and gaps in the existing knowledge of abalone metabolism, which when elucidated may not only serve the purpose of better understanding these organisms growth but also could potentially lead to increased productivity of the abalone commercial farming secto
DNA methylation associated with mitochondrial dysfunction in a south african autism spectrum disorder cohort
No full textAutism spectrum disorder (ASD) is characterized by phenotypic heterogeneity and a complex genetic architecture which includes distinctive epigenetic patterns. We report differential DNA methylation patterns associated with ASD in South African children. An exploratory whole‐epigenome methylation screen using the Illumina 450 K MethylationArray identified differentially methylated CpG sites between ASD and controls that mapped to 898 genes (P ≤ 0.05) which were enriched for nine canonical pathways converging on mitochondrial metabolism and protein ubiquitination. Targeted Next Generation Bisulfite Sequencing of 27 genes confirmed differential methylation between ASD and control in our cohort. DNA pyrosequencing of two of these genes, the mitochondrial enzyme Propionyl‐CoA Carboxylase subunit Beta (PCCB ) and Protocadherin Alpha 12 (PCDHA12 ), revealed a wide range of methylation levels (9–49% and 0–54%, respectively) in both ASD and controls. Three CpG loci were differentially methylated in PCCB (P ≤ 0.05), while PCDHA12 , previously linked to ASD, had two significantly different CpG sites (P ≤ 0.001) between ASD and control. Differentially methylated CpGs were hypomethylated in ASD. Metabolomic analysis of urinary organic acids revealed that three metabolites, 3‐hydroxy‐3‐methylglutaric acid (P = 0.008), 3‐methyglutaconic acid (P = 0.018), and ethylmalonic acid (P = 0.043) were significantly elevated in individuals with ASD. These metabolites are directly linked to mitochondrial respiratory chain disorders, with a putative link to PCCB , consistent with impaired mitochondrial function. Our data support an association between DNA methylation and mitochondrial dysfunction in the etiology of AS
Untargeted metabolite profiling of abalone using gas chromatography mass spectrometry
No full textAbalone meat is a delicacy worldwide, fetching high prices and a valuable source of income for the many countries farming and exporting this commodity. The quality of abalone is based on its unique sensory properties and an analytical metabolomics method for determining the compounds related to this would serve as a valuable tool for ensuring quality and consumer satisfaction. Metabolomics is a promising “omics” tool which can be applied towards this goal; however, widely applicable parameters for the evaluation of an untargeted gas chromatography mass spectrometry (GC-MS) metabolomic approach is still lacking. GC-MS is a popular and suitable metabolomics method due to its high separation power, reproducible retention times, and selective mass detection. The aim of this study was to establish a reliable untargeted GC-MS method for analyzing firstly a standard compound mixture consisting of 10 compounds representing various compound classes and secondly applying the method in an untargeted manner to abalone muscle samples. Using a standard compound mixture with a concentration range of 1 to 100 μg/mL, the limit of detection (LOD) ranged between 0.01 and 3.30 μg/mL, the limit of quantification (LOQ) resulted in values between 0.02 and 9.49 μg/mL, the accuracy determined was <1.5 μg/mL, and the precision displayed a coefficient of variance (CV) <25 %. When evaluating the method in terms of biological samples harvested, the repeatability and intermediate precision showed CV values <50 % for most compounds measured, allowing application of this method for metabolite profiling of abalone to answer important biological question
From untargeted LC-QTOF analysis to characterisation of opines in abalone adductor muscle: Theory meets practice
No full textAbalone 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 level
Investigating the effect of anaesthesia on the metabolism of zebrafish (D. rerio)
No full textMSc (Biochemistry), North-West University, Potchefstroom CampusIn recent years, zebrafish (D. rerio) have emerged as valuable research models, being widely
used as models for aquaculture research, but also to model human metabolic diseases. The
ethical guidelines stipulate that anaesthetics need to be applied before sampling of zebrafish. The
effect of the anaesthetics on the metabolic profiles of zebrafish and the implications that this may
have for zebrafish as research models, have mostly been overlooked. The primary hypothesis of
this study was that anaesthetics will have an effect on zebrafish metabolism. The possibility for
new hypotheses to be generated were not eliminated, since untargeted metabolomics are more
hypothesis-generating than hypothesis-testing techniques. In this study, multi-platform
metabolomics were used to investigate the metabolic alterations in zebrafish caused by three
commonly used anaesthetics namely tricaine methanesulfonate (MS-222), eugenol and 2-
phenoxyethanol (2-PE). The metabolomic analyses indicated that anaesthesia caused minimal
metabolic alterations, with the concentration of only a small number of metabolites altered. It is
hypothesized that these concentration changes are caused by ATP depletion and a stress
response, which both lead to the upregulation of protein and lipid catabolism. Glycolysis and
tricarboxylic acid cycle (TCA) intermediates remained relatively unaffected, but there are some
indications that gluconeogenesis may be slightly upregulated. These effects are, however, limited
by the short induction times of the anaesthetics. It was also determined that eugenol exhibited
significantly less metabolic perturbations than the other anaesthetics with only three significantly
altered metabolites, making it the preferred anaesthetic for metabolomic studies in zebrafish.
General pathways impacted during anaesthesia include but are not limited to ẞ-oxidation,
gluconeogenesis, urea cycle and amino acid transamination. In conclusion, anaesthetics used
during sampling are not expected to have a significant effect on the suitability of zebrafish as
research models. Further investigations are needed to confirm the hypotheses generated in this
study.Master
Effect of proline‐enriched abalone feed on selected metabolite levels of slow‐growing adult Haliotis midae
No full textAbalone is currently considered South Africa's most successfully produced aquacul‐
ture export product, with a 76% share of the total value generated by the aquaculture
sector. A major risk factor for this sector is slow growth rates experienced during
farming. Abalone feeds are often supplemented with amino acids in an attempt to
enhance abalone growth. This is a first investigation of the effect of added proline to
standard abalone feed, on the metabolite profile of slow‐growing abalone. A targeted
liquid chromatography tandem mass spectrometry metabolomics research approach
was followed to recognise the metabolic response of abalone showing slower growth
performance. The addition of proline to the standard abalone diet was found to serve
as a substrate for amino acid catabolism in slower growing abalone, by means of pro‐
line breakdown to assist with energy production via the tricarboxylic acid cycle.
Other amino acids and urea cycle intermediates, that is, arginine, asparagine, ornith‐
ine and creatine further support energy production via the action of protein catabo‐
lism in slow‐growing abalone. Additionally, the importance of understanding how
abalone respond metabolically to modified feed highlights the use of metabolomics
to answer abalone aquaculture farming question
Mammalian cell cultures as models for metabolomic studies
No full textThesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2012.The use of cultured cells in metabolomic studies is receiving more and more
attention. There are many advantages when using cultured cells in metabolomic
studies, for example cultured cells can easily be manipulated for the purpose of the
experiment. This creates many opportunities for metabolomics studies, for example
cell cultures can offer an alternative manner of drug testing. Even though the use of
cultured cells in metabolomic studies is very promising and they create many
opportunities for metabolomic research, there are still challenges that create
obstacles in this research. One of the challenges is that present analytical
technologies do not always fully meet the requirements for metabolomics. There is,
however, much effort going into optimising the methods concerning cultured cells
and metabolomics, but there is a lack of attention when it comes to the sample
preparation which is initiated by quenching. The aim of this study was to investigate
cultured cells as models for metabolomics investigations and to standardise a proper
quenching method for a metabolomics analysis of mammalian cultured cells.
A quenching method adapted from the literature was evaluated for the cell line used
in this study, namely HeLa. Metabolites of the central carbon metabolism were
targeted, using a published list. This method was tested for its effectiveness by
introducing the samples to waiting periods (0, 3, 6 and 24 hours) before extraction
after immediate quenching. Results indicated that the entire metabolism under study
was not effectively quenched. The optimum composition and temperature for this
quenching method were also investigated by comparing three different quenching
methods derived from the literature. The results were contradicting. Cell cultures
were exposed to two perturbations (environmental and genetic) to investigate if
these perturbations can be captured and measured by using metabolomics as an
instrument. There was a significant difference between control groups and the
groups exposed to the different perturbations. The results gained from this study
indicate that it is definitely possible to use cultured cells in metabolomics studies.Master
Characterising the metabolic differences related to growth variation in farmed Haliotis midae
No full textThe South African abalone farming industry is entirely based on Haliotis midae, which has been commercially cultured with great success the last 20 years. Even though abalone are cultivated under identical farming conditions, originating from homogenous genetic stocks, variation is experienced in individual abalone growth rates, justifying further research into the mechanisms related to these growth differences. Insights into the biochemical processes of abalone would help to identify the various metabolic factors related to varied abalone growth rates. Metabolomics, aims to investigate the metabolism holistically, and is considered a powerful tool for better elucidation of observed phenotypical changes. A metabolomics approach including the use of untargeted gas chromatography-time of flight spectrometry, semi-targeted liquid chromatography-quadrupole time of flight mass spectrometry and targeted liquid chromatography-tandem mass spectrometry were used to indicate and better describe the metabolic variation associated with slow and fast growing abalone. The results obtained by metabolomics analysis of H. midae adductor muscle samples showed that faster growing individuals utilise energy pathways and reserves (via elevated insulin production) in such a way that they promote protein synthesis. In contrast the metabolic profile of slow growing individuals supports protein catabolism, where energy allocation for breakdown of metabolic products has priority over mechanisms utilised for abalone growth. Overall these results sets the stage for future work highlighting metabolic pathways which should be investigated in a qualitative manner ensuring reference values which can furthermore be monitored to assist with abalone growth prediction
