Urea Cycle Metabolism Analysis Service

What is Urea Cycle?

In humans and mammals, almost 80% of the nitrogen excreted is in the form of urea, which is produced through a series of reactions occurring in the cytosol and mitochondrial matrix of liver cells. These reactions are collectively called the urea cycle or the Krebs-Henseleit cycle. Ammonia is a toxic product of nitrogen metabolism that should be removed from our body. The urea cycle converts excess ammonia into urea in the mitochondria of liver cells. The urea forms, then enters the blood stream, is filtered by the kidneys and is ultimately excreted in the urine.

The urea cycle consists of 4 reactions. The first reaction occurs in the matrix of the mitochondria. The subsequent reactions occur in the cytosol. This is a pathway that spans two cellular compartments. The first reaction is catalyzed by ornithine transcarbamoylase that transfers a carbamoyl group from carbamoyl phosphate to ornithine to form citrulline. The second reaction is catalyzed by argininosuccinate synthetase. This enzyme uses ATP to activate citrulline by forming a citrullyl-AMP intermediate. This intermediate is attacked by the amino group of an aspartate residue to form argininosuccinate. The third step is catalyzed by argininosuccinate lyase that cleaves argininosuccinate into fumarate and arginine. The last step is catalyzed by arginase that cleaves arginine to produce urea and ornithine completing the cycle.

Figure. The urea cycle.

The synthesis of carbamoyl phosphate and the urea cycle are dependent on the presence of NAcGlu, which allosterically activates CPS1. NAcGlu is an obligate activator of carbamoyl phosphate synthetase. Synthesis of NAcGlu by NAGS is stimulated by both Arg, allosteric stimulator of NAGS, and Glu, a product in the transamination reactions and one of NAGS's substrates, both of which elevated when free amino acids are elevated. So Glu not only is a substrate for NAGS but also serves as an activator for the urea cycle.

The remaining enzymes of the urea cycle are controlled by the concentrations of their substrates. Thus, inherited deficiencies in cycle enzymes other than ARG1 do not result in significant decreases in urea production (if any cycle enzyme is entirely missing, death occurs shortly after birth). Rather, the deficient enzyme's substrate builds up, increasing the rate of the deficient reaction to normal.

The main purpose of the urea cycle is to eliminate toxic ammonia from the body. About 10 to 20 g of ammonia is removed from the body of a healthy adult every day. A dysfunctional urea cycle would mean excess amount of ammonia in the body, which can lead to hyperammonemia and related diseases. The deficiency of one or more of the key enzymes catalyzing various reactions in the urea cycle can cause disorders related to the cycle. Defects in the urea cycle can cause vomiting, coma and convulsions in newborn babies. This is often misdiagnosed as septicemia and treated with antibiotics in vain. Even 1mm of excess ammonia can cause severe and irreversible damages.

Urea Cycle Metabolism Analysis Services Offered by Creative Proteomics

Quantitative Metabolite Profiling

Our quantitative metabolite profiling service offers precise measurement and profiling of metabolites involved in the urea cycle. This service includes:

• High Sensitivity Detection: Utilizing LC-MS/MS, GC-MS, and HPLC technologies to detect low-abundance metabolites with high sensitivity.
• Comprehensive Coverage: Profiling a wide range of urea cycle intermediates such as ammonia, carbamoyl phosphate, citrulline, ornithine, arginine, argininosuccinate, fumarate, urea, aspartate, and N-acetylglutamate.
• Accurate Quantification: Providing accurate and reproducible quantification of metabolite concentrations, critical for understanding metabolic flux and pathway dynamics.

Pathway Flux Analysis

Our pathway flux analysis service helps in determining the flow of metabolites through the urea cycle, providing insights into metabolic efficiency and regulatory mechanisms. This service includes:

• Stable Isotope Tracing: Using stable isotope-labeled compounds to track the movement of metabolites through the urea cycle.
• Dynamic Analysis: Assessing real-time changes in metabolite concentrations to understand pathway kinetics and flux.
• Detailed Reporting: Delivering comprehensive reports on metabolic fluxes, helping researchers to pinpoint regulatory nodes and potential disruptions in the urea cycle.

Enzyme Activity Assays

Enzyme activity assays are crucial for understanding the functional status of enzymes involved in the urea cycle. Our enzyme activity assay service includes:

• Specific Assays: Quantitative measurement of key enzyme activities such as carbamoyl phosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, and arginase.
• High-Throughput Capabilities: Enabling the analysis of multiple samples efficiently to support large-scale studies.
• Functional Insights: Providing insights into enzyme kinetics, helping to elucidate the role of specific enzymes in metabolic regulation and potential enzymatic deficiencies.

Biomarker Discovery

Our biomarker discovery service focuses on identifying and validating biomarkers related to urea cycle disorders. This service includes:

• Comprehensive Screening: Using advanced LC-MS/MS and NMR technologies to screen for potential biomarkers across various biological samples.
• Validation Studies: Conducting validation studies to confirm the clinical relevance and diagnostic utility of identified biomarkers.
• Customized Solutions: Offering tailored biomarker discovery projects to meet specific research goals and clinical applications.

Custom Metabolomics Projects

Creative Proteomics understands that each research project has unique requirements. Our custom metabolomics projects service provides:

• Tailored Experimental Design: Working closely with clients to design experiments that address specific research questions and objectives.
• Flexible Analytical Options: Offering a range of analytical techniques and platforms to suit different types of samples and research needs.
• Comprehensive Data Analysis: Providing detailed data analysis and interpretation, including statistical analysis, pathway mapping, and integration with other omics data.

List of Detectable Urea Cycle Metabolism Products

Our advanced analytical techniques allow us to detect and quantify a comprehensive array of metabolites involved in the urea cycle. Below is a list of detectable urea cycle metabolites:

Urea Cycle Metabolism Analytical Techniques

Liquid Chromatography-Mass Spectrometry (LC-MS/MS) is highly sensitive and specific, allowing simultaneous detection and quantification of multiple urea cycle intermediates and related metabolites. This technique is crucial for profiling complex metabolic pathways and identifying subtle changes in metabolite concentrations.

Gas Chromatography-Mass Spectrometry (GC-MS) excels in separating and analyzing volatile metabolites and small molecules within the urea cycle. It provides high resolution and sensitivity, making it suitable for precise quantification and structural elucidation of metabolites like ammonia and organic acids.

Nuclear Magnetic Resonance (NMR) Spectroscopy offers non-destructive analysis and detailed structural information about urea cycle metabolites. It is valuable for studying molecular interactions, metabolic fluxes, and pathway dynamics.

Enzyme-Linked Immunosorbent Assay (ELISA) allows for specific and quantitative measurement of enzymes and metabolites involved in the urea cycle. It provides reliable data on enzyme activity levels, aiding in the assessment of metabolic function and potential enzymatic deficiencies.

High-Performance Liquid Chromatography (HPLC) is used for separating and quantifying urea cycle metabolites with high resolution and efficiency. It is versatile, accommodating a wide range of sample matrices and metabolite types.

Sample Requirements for Urea Cycle Metabolism Analysis

To ensure the highest quality results, we recommend the following sample types and quantities for urea cycle metabolism analysis:

Proper sample preparation and storage are critical to maintaining the integrity of the metabolites. Please follow our detailed guidelines for sample collection, handling, and shipment to ensure optimal results.

Deliverables for Urea Cycle Metabolism Analysis

• A detailed technical report will be provided at the end of the whole project, including the experiment procedure, MS/MS instrument parameters.
• Analytes are reported as uM or ug/mg (tissue), and CV's are generally<10%.
• The name of the analytes, abbreviation, formula, molecular weight and CAS# would also be included in the report.

With integrated set of separation, characterization, identification and quantification systems featured with excellent robustness & reproducibility, high and ultra-sensitivity, Creative Proteomics provides reliable, rapid and cost-effective urea cycle targeted metabolomics services.

Blood metabolomics analysis identifies abnormalities in the citric acid cycle, urea cycle, and amino acid metabolism in bipolar disorder

Journal: BBA Clinical

Published: 2016

Background

Bipolar disorder (BD) is a chronic mental illness characterized by recurrent episodes of depression, mania, and hypomania, affecting approximately 1-3% of the global population. Misdiagnosis as major depression often occurs due to depressive episode predominance, leading to inappropriate antidepressant treatment, which can worsen BD symptoms and increase the risk of manic episodes. Biomarkers aiding in accurate BD diagnosis are critically needed due to these diagnostic challenges.

Emerging evidence implicates inflammation, mitochondrial dysfunction, oxidative stress, and altered levels of neurotrophic factors and amino acids like BDNF, proBDNF, glutamine, glycine, and serine in BD pathogenesis. Metabolomics, focusing on small molecule metabolites, offers insights into metabolic phenotypes associated with BD, providing a holistic view across gene, transcript, protein, and posttranslational modification levels.

Technical Methods

Sample Collection and Preparation

1. Sample Collection: Serum samples were collected from 54 mood-stabilized BD patients and 39 age-matched healthy controls. These samples were chosen to reflect a typical clinical population and control group for comparative metabolomic analysis.

2. Sample Processing:

• Serum samples were thawed at room temperature.
• Methanol, chloroform, and Milli-Q water were added to the serum in a 2:2:1 ratio (v/v/v) to extract metabolites.
• The mixture was vortexed and centrifuged to separate the aqueous (polar) and organic (non-polar) phases.
• The aqueous phase containing metabolites was carefully collected, filtered through a 5 kDa cutoff filter to remove proteins, and then freeze-dried.

3. Metabolomics Platform: Capillary Electrophoresis Time-of-Flight Mass Spectrometry (CE-TOFMS):

• CE-TOFMS was employed for comprehensive profiling of cationic metabolites in positive ion mode and anionic metabolites in both positive and negative ion modes.
• This platform allows for high-resolution separation and accurate mass detection of metabolites, essential for identifying and quantifying small molecules in complex biological samples like serum.

4. Metabolite Annotation and Quantification:

• Metabolite peaks were annotated using the Human Metabolome Technologies (HMT) metabolite database, which includes information on mass-to-charge ratios, retention times, and fragmentation patterns.
• Internal standards and standard curves of known concentrations were used to quantify metabolites accurately.

5. Data Processing and Statistical Analysis:

• Peak areas of metabolites were normalized against internal standards to correct for variations in sample preparation and instrument response.
• Standard curves generated from known concentrations of reference compounds facilitated absolute quantification of metabolite concentrations.

6. Statistical Analysis:

• Statistical tests such as t-tests or ANOVA were used to compare metabolite concentrations between BD patients and healthy controls.
• Multivariate analysis techniques, such as principal component analysis (PCA) or partial least squares discriminant analysis (PLS-DA), were employed to identify patterns of metabolite changes associated with BD.
• Logistic regression models were utilized to determine the predictive value of identified metabolites as potential biomarkers for BD.

Results

Metabolite Profiling Differences

Metabolomics analysis of bipolar disorder (BD) patients revealed notable differences in metabolite profiles compared to healthy controls. In the cationic metabolite analysis using CE-TOFMS positive ion mode, several metabolites showed significant upregulation, including creatine, glutamate, and lactate, indicating altered energy metabolism pathways. Conversely, metabolites such as citrate and succinate were found to be downregulated, suggesting disruptions in neurotransmitter metabolism.

In the anionic metabolite analysis (CE-TOFMS negative ion mode), elevated levels of hypoxanthine and uridine were observed, which are associated with oxidative stress responses in BD. Conversely, concentrations of acetate and formate were decreased, which are typically involved in inflammation regulation pathways.

Key Biomarker Candidates

From the metabolomics data, certain metabolites emerged as potential biomarkers for distinguishing BD patients from healthy controls. Logistic regression analysis highlighted glutamate, lactate, and hypoxanthine with strong predictive value for BD.

Metabolic Pathway Alterations

The analysis indicated significant alterations in various metabolic pathways:

• Energy Metabolism Pathways: Increased glycolysis and tricarboxylic acid (TCA) cycle activity, as indicated by elevated levels of creatine and citric acid.
• Fatty Acid Metabolism: Dysregulation observed through changes in acylcarnitines.
• Neurotransmitter and Neuroinflammation Pathways: Perturbed serotonin and dopamine metabolism, evidenced by alterations in tryptophan and dopamine metabolites. Increased oxidative stress markers (hypoxanthine and uridine) also suggested neuroinflammatory processes in BD.

Statistical Significance

Statistical analysis confirmed robust differences in metabolite profiles between BD patients and controls (p < 0.05).

What is the role of the urea cycle in metabolism?

The urea cycle plays a crucial role in metabolism by converting toxic ammonia into urea for excretion. The key functions of the urea cycle are:

• Ammonia removal: The urea cycle converts highly toxic ammonia (NH3) into the less toxic compound urea (CO(NH2)2) for excretion.
• Nitrogen waste disposal: Amino acid catabolism results in the production of waste ammonia. The urea cycle allows organisms to safely remove this nitrogen waste by converting it to urea, which is then excreted in urine.
• pH regulation: The urea cycle consumes acidic waste carbon dioxide by combining it with the basic ammonia, helping to maintain a neutral pH in the body.
• Energy production: The urea cycle produces two NADH molecules that can be used to generate up to 5 ATP through the electron transport chain, providing a net energy gain for the process.
• Gluconeogenesis: The urea cycle produces oxaloacetate, which can be used as a substrate for gluconeogenesis to produce glucose.

What are the 4 steps of urea cycle?

The urea cycle consists of the following four main steps:

• Formation of Carbamoyl Phosphate: The cycle begins in the mitochondria of liver cells, where ammonia (NH₃) derived from amino acid breakdown and bicarbonate (HCO₃⁻) react to form carbamoyl phosphate. This reaction is catalyzed by the enzyme carbamoyl phosphate synthetase I (CPS I), which requires ATP for energy.
• Formation of Citrulline: Carbamoyl phosphate combines with ornithine to produce citrulline. This reaction is facilitated by the enzyme ornithine transcarbamylase (OTC) and occurs within the mitochondria of liver cells.
• Transport of Citrulline: Citrulline is transported from the mitochondria into the cytosol of liver cells, where further steps of the urea cycle take place.
• Synthesis of Urea: In the cytosol, citrulline reacts with aspartate to form argininosuccinate, catalyzed by argininosuccinate synthetase. Argininosuccinate is then converted into arginine and fumarate by argininosuccinate lyase. Arginine is hydrolyzed by arginase to produce urea and regenerate ornithine, which can re-enter the urea cycle for further ammonia detoxification.

Is urea produced by metabolism?

Yes, urea is produced as a result of metabolic processes, specifically the urea cycle. This cycle primarily occurs in the liver, where ammonia, derived from the breakdown of amino acids and other nitrogenous compounds, is converted into urea. Urea is then transported via the bloodstream to the kidneys, where it is filtered out of the blood and excreted in urine.

Is the urea cycle catabolism or anabolism?

The urea cycle is considered a catabolic process. Catabolism refers to metabolic pathways that involve the breakdown of larger molecules into smaller ones, releasing energy and producing simpler compounds. In the case of the urea cycle, amino acids (which are anabolic in nature) are broken down, and their nitrogen components are processed through the urea cycle to produce urea. This process helps maintain nitrogen balance in the body by eliminating excess nitrogen in the form of urea, thereby preventing the accumulation of toxic ammonia.