Detecting Glycine in Biological Samples Using Enzymatic Assays

Glycine — a neutral, non-essential amino acid — is the only proteinogenic amino acid without a chiral center. Yet, it has been confirmed that glycine is a significant neurotransmitter found in the central nervous system in particular locations such as the brainstem and the spinal cord.

Glycine employs rapid postsynaptic inhibition that is required for pain transmission processes, and hearing processing. In addition, glycine is a potent glutamate coagonist on NMDA receptors. Abnormal concentrations are found in patients suffering from schizophrenia and hyperglycinemia. Conventional procedures for the quantitation of glycine involve MS, HPLC, NMR, and immunoassay methods. Conversely, these protocols are not perfect for compound screening or high-throughput screening of a large number of samples.

In this article, BioVision describes an easy, simple, and high-throughput enzymatic assay using an enhanced Glycine Oxidase (GO) Isozyme and a fluorophore in a 96-well format. The results show that glycine serum in control versus patient samples are analogous to those issued in literature. The assay provides researchers the ability to examine a large number of samples and offers an innovative tool in the area of neuroscience research.

Key Features

Detecting Glycine in Biological Samples Using Enzymatic Assays

  • Simple and Rapid: Sample Deproteinization → Add Detection Reagents → Read
  • Extremely Stable: Long shelf life
  • High-throughput adaptable
  • Reliable: Reproducible results with low intra- and inter-assay variability
  • Convenient: Non-radioactive, handling/disposal, no special instrumentation
  • Fluorometric Quantitation (Ex/Em = 535/587 nm)
  • Specific: Only glycine is detected
  • Ample reagents to execute 100 assays in 96-well plate format

Determination of Inter (A) and Inter Variability (B) Coefficient of Variations. Inter variability: Glycine Standard Curve was assayed in triplicate according to the protocols using the same components. Inter variability: Glycine Standard Curve was assayed in triplicate using three sets of reagents.

Figure 1. Determination of Inter (A) and Inter Variability (B) Coefficient of Variations. Inter variability: Glycine Standard Curve was assayed in triplicate according to the protocols using the same components. Inter variability: Glycine Standard Curve was assayed in triplicate using three sets of reagents.

A GO Isozyme is Used to Detect Glycine Over Sarcosine

(A) Determination of KM values using Glycine Oxidase Isozyme with Glycine and Sarcosine as Substrates. (B) Comparison of KM values using Wild-type and Isozyme Glycine Oxidase with Glycine and Sarcosine as substrates. The GO Isozyme is 17-fold more specific toward Glycine when compared KM-Glycine vs. KM-Sarcosine.

Figure 2. (A) Determination of KM values using Glycine Oxidase Isozyme with Glycine and Sarcosine as Substrates. (B) Comparison of KM values using Wild-type and Isozyme Glycine Oxidase with Glycine and Sarcosine as substrates. The GO Isozyme is 17-fold more specific toward Glycine when compared KM-Glycine vs. KM-Sarcosine.

Amino Acids and Other Metabolites do not Interfere with the Assay

(A) Effect of Amino acid interference on the estimation of Glycine. Final Amino Concentration (10 mM). (B) Effect of high abundant metabolites on the estimation of Glycine. Final Concentrations: [Creatinine]: 25 mM, [Ascorbic Acid]: 10 mM, [Glucose]: 10 mM, [Lactic Acid]: 10 mM, [Triglycerides]: 0.25 mM, [Bilirubin]: 5 mg/dl.

Figure 3. (A) Effect of Amino acid interference on the estimation of Glycine. Final Amino Concentration (10 mM). (B) Effect of high abundant metabolites on the estimation of Glycine. Final Concentrations: [Creatinine]: 25 mM, [Ascorbic Acid]: 10 mM, [Glucose]: 10 mM, [Lactic Acid]: 10 mM, [Triglycerides]: 0.25 mM, [Bilirubin]: 5 mg/dl.

Multiple Applications of In-Vitro Glycine Assay Kit

[Glycine]
(nmol/mg protein)
Hippocampus 8.1 ± 2.6
Cerebellum 9.9 ± 2.8
Brain Cortex 12.1 ± 3.1
Brain Stem 22.6 ± 6.2

Glycine localization in mouse brain using BioVision’s Glycine Assay Kit. Two C57BL/6J mice were sacrificed and their brain cortices, stems, hippocampi, and cerebella were collected. Tissue homogenate (100 mg tissue/ml were prepared for each sample. Homogenates deproteinized using 10 kDa spin columns. Deproteinized homogenates were serially diluted using Glycine Assay Buffer (Dilution Factor: 4-16). Ten microliters of each diluted sample were spiked with 0.3 nmol of Glycine Standard and assayed following the protocol. Our results demonstrate that brain Glycine is highly localized in Brain Stem, while Hippocampus showed the least amount of glycine per milligram of protein.

Figure 4. Glycine localization in mouse brain using BioVision’s Glycine Assay Kit. Two C57BL/6J mice were sacrificed and their brain cortices, stems, hippocampi, and cerebella were collected. Tissue homogenate (100 mg tissue/ml were prepared for each sample. Homogenates deproteinized using 10 kDa spin columns. Deproteinized homogenates were serially diluted using Glycine Assay Buffer (Dilution Factor: 4-16). Ten microliters of each diluted sample were spiked with 0.3 nmol of Glycine Standard and assayed following the protocol. Our results demonstrate that brain Glycine is highly localized in Brain Stem, while Hippocampus showed the least amount of glycine per milligram of protein.

Glycine Concentration in Serum Samples of Patients Suffering from Neurological Conditions

Glycine concentration was evaluated using BioVision’s Glycine Assay Kit in human serum samples from patients known to have Neurological conditions. Serum Samples were serially diluted using Glycine Assay Buffer (Dilution Factor Range: 4-128). Twenty-five microliters of each diluted sample were spiked with 0.3 nmol of Glycine Standard and assayed following the protocol. Our assay demonstrates a significant decrease of Glycine in patients suffering from Depression and Schizophrenia, and higher concentration of glycine was found in the sample from a patient suffering from Alzheimer’s Disease. Similar trends have been shown in recent literature

Figure 5. Glycine concentration was evaluated using BioVision’s Glycine Assay Kit in human serum samples from patients known to have Neurological conditions. Serum Samples were serially diluted using Glycine Assay Buffer (Dilution Factor Range: 4-128). Twenty-five microliters of each diluted sample were spiked with 0.3 nmol of Glycine Standard and assayed following the protocol. Our assay demonstrates a significant decrease of Glycine in patients suffering from Depression and Schizophrenia, and higher concentration of glycine was found in the sample from a patient suffering from Alzheimer’s Disease. Similar trends have been shown in recent literature

Demographics characteristics of seven Caucasian male patients and Healthy-Volunteer Control.

Patient ID Diagnosis [Glycine]
(μM)
Age Medications
Control --- 197 54 None
A Multiple Sclerosis 233 58 Copaxone
B Parkinson’s Disease 238 60 Carbidopa;Selegiline
C Major Depression 129 56 Prozac
D Schizophrenia 155 54 Prozac, Geodon, Perphenazine, Thioridazine
E Amyotrophic lateral sclerosis 213 57 None
F Alzheimer’s D. 344 53 Donepezil

Glycine Concentration in Biological Fluids

Estimation of Glycine concentration in human serum, saliva, and urine. Samples were deproteinized using 10 kDa spin columns and diluted using Glycine Assay Buffer (Serum: 64-fold; Saliva: 32-fold, Urine 128-fold. Twenty-five microliters of each diluted sample were spiked with 0.3 nmol of Glycine Standard and assayed following the protocol. Glycine concentrations are: 224 ± 21 μM, Saliva: 149 ± 21 μM, Urine: 54 ±4 μM/mM Creatinine..

Figure 6. Estimation of Glycine concentration in human serum, saliva, and urine. Samples were deproteinized using 10 kDa spin columns and diluted using Glycine Assay Buffer (Serum: 64-fold; Saliva: 32-fold, Urine 128-fold. Twenty-five microliters of each diluted sample were spiked with 0.3 nmol of Glycine Standard and assayed following the protocol. Glycine concentrations are: 224 ± 21 μM, Saliva: 149 ± 21 μM, Urine: 54 ±4 μM/mM Creatinine.

Conclusion

  • The Glycine Assay Kit offered by BioVision is a high-throughput screening assay designed for measuring the concentration of glycine in various biological samples using an enzyme that particularly detects this amino acid and a fluorophore.
  • Various factors that affect glycine metabolism can be studied with the help of this kit.
  • Glycine Assay Kit offers a safe, easy, reproducible and sensitive method to measure glycine.
  • This kit can be employed for screening patient serum and small molecules inhibitors/activators, and for studying treatment progression.
  • BioVision’s Glycine Assay Kit addresses the significance of glycine as a potential biomarker and potent neurotransmitter in diseases like depression and schizophrenia.
  • This kit may be employed for studying factors and signaling pathways that up or down regulate to alter the upregulation or downregulation of glycine.
  • The entire assay kit is commercially available at www.biovision.com catalog # K589-100.

BioVision Incorporated

BioVision, Inc., is a privately held Life Science company headquartered in the beautiful San Francisco Bay Area.

BioVision develops and offers a wide variety of products including assay kits, antibodies, recombinant proteins & enzymes, and other innovative research tools for studying Apoptosis, Metabolism, Cell Proliferation, Cellular Stress, Cell Damage and Repair, Diabetes, Obesity and Metabolic Syndrome, Stem Cell Biology, Gene Regulation, Signal Transduction, etc. BioVision's products are currently being sold in more than 60 countries worldwide.


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Last updated: Mar 23, 2020 at 9:05 AM

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