Catecholamines Analysis Service

What are Catecholamines?

Catecholamines are biogenic amines synthesized primarily in the adrenal medulla and sympathetic nervous system from the amino acid tyrosine, with dopamine serving as a key intermediate. This group includes three major compounds: dopamine, epinephrine (adrenaline), and norepinephrine (noradrenaline). In response to physical and emotional stress, these catecholamines are released into the bloodstream, functioning as critical hormones and neurotransmitters that facilitate various physiological responses.

Physiological Roles of Catecholamines

Catecholamines exert their effects by binding to specific receptors on target cell membranes, influencing numerous bodily functions. They are vital for:

• Nerve Impulse Transmission: In the brain, catecholamines play a crucial role in transmitting nerve impulses.
• Physiological Responses: They promote the dilation of pupils and bronchioles, enhancing oxygen intake and visual acuity.
• Energy Release: Catecholamines stimulate the release of energy from glucose and fatty acids, providing a quick energy source during stress.
• Cardiovascular Regulation: Norepinephrine constricts blood vessels, leading to increased blood pressure and heart rate, thereby elevating metabolism and aiding the body's adaptation to acute and chronic stress.

Once they have fulfilled their roles, catecholamines are metabolized into inactive compounds: dopamine is converted to homovanillic acid (HVA), norepinephrine degrades into normetanephrine and vanillylmandelic acid (VMA), and epinephrine is transformed into metanephrine and VMA. These metabolites are subsequently excreted from the body through urine.

Under normal circumstances, catecholamines and their metabolites exist in low concentrations in the body, with levels spiking during stress. However, certain rare tumors, such as pheochromocytomas and paragangliomas, can lead to excessive catecholamine production, resulting in persistent or sudden surges in blood pressure. Symptoms of these tumors may include sweating, nausea, anxiety, severe headaches, and heart palpitations. Long-term exposure to elevated catecholamines can increase the risk of severe health issues like kidney damage, strokes, heart attacks, and heart failure. Therefore, accurate diagnosis and treatment of these tumors are crucial, as surgical removal can often resolve hypertension caused by them.

Catecholamines Analysis Services Offered by Creative Proteomics

Catecholamine Quantitative Analysis

We measure the concentrations of catecholamines in biological samples such as plasma, serum, urine, and tissue. This assessment is critical for determining baseline levels and identifying abnormalities. We employ advanced techniques to ensure high sensitivity and specificity for precise quantification.

Metabolite Profiling

Our metabolite profiling service analyzes the byproducts generated during catecholamine breakdown. This analysis provides insights into metabolic pathways and correlates catecholamine levels with specific health conditions and stress responses.

Catecholamine Structural Analysis

We characterize the chemical structures of catecholamines and their metabolites, assessing molecular compositions and identifying structural variants. This service is essential for understanding structure-activity relationships in drug development and pharmacological research.

Catecholamine Functional Analysis

We explore the biological effects of catecholamines on target cells and tissues, examining their interactions with specific receptors and downstream effects, such as changes in gene expression and enzyme activity.

Custom Analytical Solutions

We offer tailored analytical solutions to address specific research questions. Our experienced scientists can develop custom methodologies and assays to accommodate a wide range of study designs and experimental conditions.

List of Catecholamines We Can Detect

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Metabolomics Services

We provide unbiased non-targeted metabolomics and precise targeted metabolomics services to unravel the secrets of biological processes.

Our untargeted approach identifies and screens for differential metabolites, which are confirmed by standard methods. Follow-up targeted metabolomics studies validate important findings and support biomarker development.

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Characterization of Dnajc12 knockout mice, a model of hypodopaminergia

Journal: Scientific Reports

Published: 2021

Background

Pathogenic variants in the DNAJC12 gene, part of the HSP40 family, are associated with several neurological disorders including young-onset Parkinson's disease, infantile dystonia, developmental delay, intellectual disability, and neuropsychiatric disorders. DNAJC12 functions as a co-chaperone for aromatic amino acid hydroxylases, enzymes essential for the synthesis of biogenic amines such as dopamine (DA) and serotonin. Mutations in DNAJC12 can lead to reduced levels of these neurotransmitters, contributing to the clinical features of these disorders.

The study describes a conditional knockout mouse model for Dnajc12 (cDKO and DKO) that mimics the human mutation, disrupting DA synthesis and related behavior. This model offers insight into DA dysregulation, providing a foundation for exploring therapeutic strategies for DNAJC12-related dystonia and parkinsonism. The knockout mice exhibit impaired motor functions, altered neurotransmitter levels, and changes in biochemical markers, modeling the impact of Dnajc12 loss on DA metabolism.

Materials & Methods

Animals:

Dnajc12 knockout (DKO) mice were generated on a C57BL/6J background using CRISPR/Cas9 to insert loxP sites flanking exon 2. Cre recombination deleted exon 2. Mice were housed in enriched cages, and genotyped post-mortem.

Behavioral Tests:

• Open Field Test: Measured locomotion with infrared beams in a 30-minute session.
• Grip Strength Test: Assessed muscle strength by measuring the force required for mice to release a grip.
• Balance Beam Test: Evaluated balance and coordination on a narrow beam.

Antibodies:

Various monoclonal and polyclonal antibodies were used for immunoblotting key proteins like DNAJC12, tyrosine hydroxylase, and SNAP25.

Tissue Collection:

Mice were perfused, and midbrain and striatal tissues were extracted, homogenized, and centrifuged for protein analysis.

Cell Culture & Co-immunoprecipitation:

HEK293FT cells were transfected, and co-immunoprecipitation was performed to study protein interactions.

SDS-PAGE & Western Blotting:

Protein extracts were separated and analyzed using SDS-PAGE followed by immunoblotting.

Immunofluorescence & Confocal Microscopy:

Brain sections were stained with antibodies and visualized using confocal microscopy.

Electrophysiology:

Fast scan cyclic voltammetry measured dopamine release in striatal slices.

HPLC:

Dopamine and serotonin levels were quantified using HPLC.

Metabolomics:

Plasma metabolites were analyzed via UPLC-MRM/MS.

Statistical Analysis:

Data were analyzed using GraphPad Prism, presented as mean ± SEM.

Results

DNAJC12 Interactions:

DNAJC12 co-immunoprecipitated with tyrosine hydroxylase (TH) and HSC70 in both HEK293 cells and WT mouse brain tissue, confirming its interaction with dopaminergic synthesis machinery. Confocal microscopy revealed DNAJC12 localization within dopaminergic neurons.

Behavioral Impairments in DKO Mice:

DKO mice exhibited impaired locomotion and exploratory behavior, with reduced ambulatory distance, speed, and increased resting time. However, motor strength and coordination remained intact.

Altered TH Phosphorylation:

In DKO mice, TH phosphorylation at pSer40 increased in the striatum, while pSer31 decreased. In the midbrain, total TH and phosphorylation at both sites were elevated. Hsc70 and 14-3-3 protein levels were unchanged.

Dopamine and Serotonin Deficiency:

DKO mice showed reduced striatal dopamine, its metabolites (DOPAC, HVA), and serotonin, while phenylalanine levels in plasma increased. FSCV measurements showed diminished dopamine release, though reuptake kinetics remained unaffected.

Synaptic Protein Changes:

DKO mice had reduced SNAP25 and elevated endophilin 1A in the striatum, with no changes in other synaptic proteins like clathrin heavy chain, VAMP2, or DNAJC5.

Striatal serotonin (5-HT) levels and its metabolite are reduced in 3M DKO mice.

Dnajc12 knockout recapitulates hyperphenylalaninemia as exemplified in human patients with similar pathogenic variants, but doesn't alter plasma biogenic amines. Plasma levels of Phe (significant difference, p = 0.005), Tyr (no significant difference, p = 0.47), Trp (no significant difference, p = 0.41), and biogenic amines including DA (p = 0.31), NE (p = 0.18), Adrenaline (p > 0.10), and 5-HT (p = 0.84) in WT (n=9) and DKO (n=7) mice, 6-8 months old. Error bars indicate ±SEM.

Reference

1. Deng, Isaac Bul, et al. "Characterization of Dnajc12 knockout mice, a model of hypodopaminergia." bioRxiv (2024): 2024-07.