Biogenic Amine Analysis Service

What Are Biogenic Amines?

Biogenic amines refer to a broad category of nitrogen-containing small-molecule organic compounds. They can be classified into three main groups based on their structural characteristics: Aliphatic amines, including compounds like putrescine, cadaverine, spermine, and spermidine;Aromatic amines, such as tyramine and phenylethylamine; Heterocyclic amines, including histamine and serotonin.Biogenic Amine is the basic nitrogenous compounds, which is formed by decarboxylation of amino acids and amination and transamination of the aldehydes and ketones.Biogenic amines are naturally present in the tissues of various animals and plants. Trace amounts of these compounds serve as normal active constituents in organisms, including the human body, playing essential physiological roles.

Functions of Biogenic Amines

Biogenic amines serve as precursors for the synthesis of hormones, alkaloids, nucleic acids, and proteins within organisms. They exhibit significant physiological and toxicological effects. In small quantities, they benefit the body by promoting growth, metabolism, enhancing immune activity in the gastrointestinal system, and exerting various activities within the nervous system, including blood pressure regulation. However, excessive intake of biogenic amines can lead to adverse physiological reactions, and in severe cases, it can be life-threatening.

Biogenic amines, exemplified by spermine, spermidine, putrescine, and cadaverine, constitute indispensable cellular constituents within organisms. They undertake pivotal roles in the regulation of processes such as DNA, RNA, and protein synthesis, alongside the stabilization of biological membranes. Moreover, spermine assumes a regulatory function within the small intestine, while serotonin participates in the regulation of blood pressure. These biogenic amines are typically recognized for exerting their biological functions through mechanisms grounded in electrostatic reactions and interactions involving a spectrum of molecules, encompassing DNA, RNA, proteins, and components of cellular membranes. As such, the precise quantity of biogenic amines holds paramount significance in ensuring their effective functionality.

Biogenic Amine LC-MS/MS Analysis

Creative Proteomics has established an analytical approach primarily using ACQUITY UPLC/TripleQuad5500 (Waters/AB Sciex) to quantitatively and qualitatively analyze various biogenic amine substances. However, these polar amine metabolites exhibit poor retention on reverse-phase chromatography columns and unstable chromatographic behavior under normal-phase conditions, often resulting in poor sensitivity when analyzing complex biological matrices. To address these challenges in compound detection, we offer a comprehensive analysis solution that includes sample preparation, liquid chromatography, mass spectrometry methods, and data analysis.

Putrescine Analysis Service

Spermine Analysis Service

Tryptamine Analysis Service

Cadaverine Analysis Service

Spermidine Analysis Service

Analytical methods for the determination of biogenic amines in food products (Tırıs et al., 2022)

Advantages and Features of the Biogenic Amine Analysis Approach

Comprehensive Coverage: This approach facilitates the simultaneous detection of up to 90 amine metabolites, encompassing essential amino acids as well as their methylated and acetylated derivatives. This wide-ranging coverage offers a swift and thorough assessment.

Improved Precision and Sensitivity: The methodology enhances the precision of amine metabolite quantification and bolsters detection sensitivity, contributing to more reliable and precise results.

Versatility Across Biological Matrices: This method exhibits versatility across diverse biological sample types, such as blood, urine, and tissue, making it suitable for the swift profiling of amine metabolites and enabling differential analyses across various biological contexts.

Sample Types and Requirements

Serum, plasma, urine, bile, and bile acid samples: 10 microliters

Various tissues (e.g., blood, liver, brain) and feces: 10 milligrams

Samples from plants, yeast, microorganisms, etc.

Creative Proteomics's analytical scientists are highly experienced and knowledgeable in the application of HPLC analyses to a wide variety of industries ranging from Food & Beverage to Pharmaceuticals and much more. Experience, fast turnaround, clear concise written reports, and custom service are provided to help customers resolve your analytical and technical challenges.

References

1. Verma, Neelam, et al. "Enzymatic biosensors for the quantification of biogenic amines: A literature update." Critical reviews in biotechnology 40.1 (2020): 1-14.
2. Tırıs, Gizem, et al. "A review of the currently developed analytical methods for the determination of biogenic amines in food products." Food Chemistry (2022): 133919.

Characterization of Dnajc12 Knockout Mice, a Model of Hypodopaminergia

Journal: bioRxiv

Published: 2024

Background

Neurodegenerative diseases often feature protein inclusions that disrupt proteostasis. Heat shock proteins (HSPs), such as HSP90, HSP70, HSP60, HSP40, and small HSPs, are essential for protein folding, trafficking, and degradation. Variants in DNAJC12, an HSP40 family member, are linked to various neurological disorders including young-onset parkinsonism, dystonia, developmental delay, intellectual disability, and neuropsychiatric disorders. DNAJC12 also affects hyperphenylalaninemia, typically due to mutations in phenylalanine hydroxylase (PAH) and tetrahydrobiopterin (BH4). As a co-chaperone for aromatic amino acid hydroxylases (AAAHs) like PAH and tyrosine hydroxylase (TH), DNAJC12 is crucial for dopamine (DA) and serotonin (5-HT) synthesis. Mutations in DNAJC12 and other DNAJC family members are associated with movement disorders and vary in penetrance and presentation, necessitating further investigation into DA regulation.

Materials & Methods

Animals

All procedures adhered to the Institutional Animal Care and Use Committee guidelines at the University of Florida.

Dnajc12 knockout (DKO) mice were created on a C57BL/6J background using CRISPR/Cas9 to insert loxP sites flanking exon 2, followed by Cre recombination to ablate the sequence.

The Dnajc12 gene is located on chromosome 10, encoding 198 amino acids in the main isoform (NM_013888.3) and 116 amino acids in a shorter isoform (NM_001253685.1). The homozygous mutation DNAJC12 c.79-2A>G (p.V27Wfs14) causes exon 2 skipping, leading to a stop mutation (p.V27Wfs44) in mice.

Behavior

Open Field Test: Assesses locomotion and motor activity using an Activity Monitor System with infrared photo beams. Data is analyzed based on zones within the arena.

Grip Strength Test: Measures neuromuscular function and strength by recording the peak force required for the mouse to release a grip.

Balance Beam: Evaluates balance and coordination by having mice traverse an elevated narrow beam, with performance recorded and analyzed for foot slips.

Antibodies

Various antibodies were used including those against 14-3-3, clathrin heavy chain, DNAJC5, DNAJC12, Endophilin 1, GFP, HSC70, myelin basic protein, olig2, Synaptobrevin 2, tyrosine hydroxylase, and β-tubulin.

Tissue Collection and Homogenization

Mice were anesthetized, perfused with PBS, and microdissected for midbrain and striatum. Tissues were homogenized in Cell Signaling Lysis Buffer, cleared by centrifugation, and protein concentrations measured using a BCA assay.

Cell Culture

HEK293FT cells were maintained in high glucose DMEM with supplements and transfected using Lipofectamine 2000 or RNAiMAX. Co-immunoprecipitation was performed to recover GFP-tagged proteins using GFP-Trap beads.

SDS-PAGE and Western Blotting

Proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with primary antibodies followed by fluorescent secondary antibodies. Blots were scanned using a ChemiDoc MP imaging platform.

Immunofluorescence with Confocal Microscopy

Mouse brains were fixed, cryoprotected, sectioned, and stained with anti-DNAJC12 and anti-TH antibodies. Images were acquired using confocal microscopy and analyzed with ImageJ software.

Electrophysiology

Fast scan cyclic voltammetry (FSCV) was used to assess dopamine release and reuptake in striatal slices from DKO mice. Slices were perfused with aCSF, and data were recorded and analyzed for dopamine levels.

High-Performance Liquid Chromatography (HPLC)

Striatal tissue samples were processed and analyzed for dopamine, serotonin, and metabolites using HPLC with electrochemical detection at the Emory HPLC Bioanalytical Core.

Metabolomics

Plasma samples were analyzed for free amino acids and related metabolites using UPLC-MRM/MS. Samples were prepared with dansyl chloride and analyzed using a Waters Acquity UPLC system coupled to a Sciex QTRAP 6500 Plus mass spectrometer.

Statistical Analysis

Data were analyzed using GraphPad Prism 10. Automated tests, grip strength measurements, and FSCV traces were analyzed with specialized software, and results were presented as mean ± SEM.

Results

Identification of DNAJC12 Interacting Partners In Vitro

Mass spectrometry of HEK293 cells overexpressing DNAJC12 identified its interaction with tyrosine hydroxylase (TH). Validation via immunoprecipitation with GFP-DNAJC12 and FLAG-TH confirmed the interaction with TH and endogenous HSC70. Immunoprecipitation from wild-type mouse brain lysates and Western blotting corroborated these findings. Confocal microscopy of midbrain slices from wild-type mice showed DNAJC12 localized within TH-positive dopaminergic neurons.

Behavioral Analysis of DKO Mice

CRISPR/Cas9-generated DKO mice exhibited impaired locomotion and exploratory behavior at 3 months of age. Behavioral tests showed reduced ambulatory distance, time, number of rears, and average speed, with increased resting time, while grip strength and motor coordination were unaffected.

Alterations in TH Protein Levels and Phosphorylation

In DKO mice, TH levels in the striatum remained unchanged, but phosphorylation at Ser40 was increased and Ser31 reduced. Midbrain TH levels and phosphorylation at Ser31 and Ser40 were elevated. HSC70 and 14-3-3 levels were similar between DKO and WT mice.

Striatal Dopamine and Metabolite Levels

HPLC analysis revealed reduced striatal dopamine (DA) (43.70 vs. 58.86 ng/mg), DOPAC (3.20 vs. 5.62 ng/mg), and homovanillic acid (HVA) (4.052 vs. 6.823 ng/mg) in DKO mice compared to WT. Reduced striatal serotonin (5-HT) and increased plasma phenylalanine (Phe) levels were observed. DA release in ex-vivo striatal slices was lower in DKO mice (0.140 vs. 0.30 µM), with unaltered reuptake kinetics.

Dnajc12 knockout reduces the levels of striatal DA, and its metabolites, and the peak amplitude of its release in 3M DKO mice.

Synaptic Protein Expression in the Striatum

No changes were observed in clathrin heavy chain (Chc), vesicular associated membrane protein 2 (Vamp2), and cysteine string protein-a (Dnajc5). However, Snap25 was reduced, and endophilin 1A was increased in DKO mice compared to WT.

Dnajc12 knockout on the expression of synaptic proteins in the striatum (STR).

Reference

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