What is a Biomarker?

Biomarkers are the foundation of the biological sciences, from basic microbiology experiments to clinical research. A biomarker is a molecular, cellular or biochemical change that can be measured accurately and reproducibly and can be used to identify and monitor physiological and pathogenic processes or responses to pharmacological interventions. The key message here is that biomarkers not only aid in the diagnosis of disease (diagnostic biomarkers), but also in identifying potential treatments as well as tracking disease progression, regression, and outcome after intervention. Biomarkers include carbohydrates, proteins, lipids, genes, DNA, RNA, platelets, enzymes, hormones, and other biomolecules. Any substance that contributes to the identification of a disease can serve as a biomarker, whether it is a metabolite, a change in a biological structure or process, or a characteristic. Biomarkers can be categorized in a number of ways. Biomarker tests can be performed using a variety of methods, from blood tests to magnetic resonance imaging (MRI), all of which affect their usability.

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Biomarker Testing Methods

Starting from the first discovery of protein cancer marker-urine periplasmic protein in 1847, after more than 150 years of development, the completion of the sequencing of the human genome has opened the journey of discovery of genetic biomarkers.

Non-protein-based Biomarker Testing

Non-protein biomarkers contain endogenous substances such as lipids, polysaccharides, amino acids, fatty acids, and vitamins. Metabolomics, as a bridge connecting genotype and phenotype, is the science of studying the types and quantities of metabolites (non-protein endogenous metabolites) and their patterns of change when an organism is perturbed (e.g., by genetic changes or environmental changes). Currently, there are three major detection platforms for metabolomics: NMR, LC-MS and GC-MS. LC-MS is the main detection platform for metabolomics because of its high sensitivity and wide detection range.

Protein-based Biomarker Testing

For free-state protein-based biomarker detection platforms are ELISA, MSD, SIMOA, Luminex, Ella, etc. The principles and characteristics of these detection platforms are shown in Table 2. For non-free state biomarkers, the detection platforms include flow cytometry, fluorescence microscopy and laser confocal microscopy.

Uses of Stable Isotopes in Biomarker Analysis

In a variety of applications, stable isotopes have entered the field of biomarker analysis relatively late, but their accuracy, reliability, and depth are constantly improving. Their applications range from clinical diagnosis to metabolomics and drug development. One of the most important is the use of stable isotopes in biomarker analysis.

Metabolic Pathway Analysis

Stable isotopes are integral to understanding metabolic pathways. By using isotopically labeled substrates, the researchers were able to trace the flow of metabolites in biochemical pathways. For example, isotope labeling of carbon-13 (13C) glucose can track metabolic flux and thus help understand nutritional abnormalities such as diabetes. This application provides insights on how metabolic processes change in disease at the cellular level and could also lead to the identification of new biomarkers.

Protein Labeling and Tracking

Stable isotopes are also used in the field of proteomics, primarily for protein labeling and tracking. Isotopically labeled amino acids can be used to distinguish proteins in complex mixtures, for example, cell culture amino acid Stable isotope labeling (SILAC). It is of great significance for the study of protein synthesis, turnover and interaction. Nitrogen-15 (15N) labeling is commonly used as an internal standard for measuring protein dynamics and post-translational modifications to infer disease mechanism or function.

Quantitative Mass Spectrometry

Quantitative mass spectrometry is often complemented by the use of stable isotopes. Isotope dilution mass spectrometry (IDMS) uses isotopically labeled standards for precise quantification of biomolecules. This application is critical for accurate and repeatable quantification of biomarkers in clinical samples. This includes increasing the analytical sensitivity of mass spectrometers through isotopic labeling, such as deuterium (2H), to detect low-abundance biomarkers needed for early disease detection and surveillance.

Structural and Functional Analysis

In terms of structure and function of biomolecules, stable isotopes play an important role in the analysis of structure and function of biomolecules. 1H NMR spectroscopy is often used to study molecular flexibility and conformational changes, where 2H is the nuclear magnetic resonance active spin, allowing tracking of isotope-labeled sites in dynamic exchange. Such applications could provide insights into protein-ligand interactions and enzyme mechanisms, which could aid biological understanding and drug development efforts.

Drug Development and Metabolism

Using stable isotopes to study drug metabolism and pharmacokinetics, sequencing digestive products will help determine the epitopes that are expected to be released during actual digestion of isotopically labeled drugs, and thus determine the ADME profile of isotopically labeled drugs, including absorption distribution, metabolic excretion, etc. For example, oxygen-18 (18O) labeling of drug molecules is a formulation-optimized way to monitor metabolism. This can help speed up the development of new therapies and improve the safety and effectiveness of drugs.

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Types of Stable Isotopes in Biomarker Analysis

Carbon-13

Carbon-13 is a stable isotope of carbon with a natural abundance of approximately 1.1%. It is extensively used in metabolic studies and protein labeling. In biomarker analysis, 13C-labeling helps track metabolic pathways and quantify biomolecular interactions with high precision. For example, 13C-labeled glucose can trace metabolic fluxes in cells, providing insights into metabolic disorders.

Deuterium

Deuterium, or heavy hydrogen, is utilized in various analytical techniques due to its distinctive NMR signal. It is particularly useful in studying protein dynamics and structural conformations. In biomarker analysis, deuterium-labeled compounds enhance the sensitivity and specificity of NMR and mass spectrometry, aiding in the detailed characterization of biomolecules.

Nitrogen-15

Nitrogen-15 is a stable isotope commonly used in protein studies. It is incorporated into proteins through metabolic labeling, allowing researchers to analyze protein synthesis, degradation, and turnover. 15N-labeling is also instrumental in elucidating protein-protein interactions and post-translational modifications, making it invaluable in biomarker research.

Oxygen-18

Oxygen-18 is utilized in studying enzymatic reactions and metabolic processes. Its incorporation into biomolecules provides insights into biochemical pathways and reaction mechanisms. In biomarker analysis, 18O-labeling aids in tracing metabolic products and understanding molecular interactions with high resolution.

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Benefits of Stable Isotopes in Biomarker Analysis

Enhanced Sensitivity and Specificity

Stable isotopes improve the sensitivity and specificity of biomarker analysis by providing clear, unambiguous signals. The use of isotopically labeled standards reduces interference and enhances the ability to detect low-abundance biomarkers.

Accurate Quantification

Stable isotope techniques offer accurate quantification of biomarkers by allowing for precise comparison between labeled and unlabeled molecules. This accuracy is crucial for reliable biomarker measurement and subsequent data interpretation.

Multiplexing Capabilities

Stable isotopes enable multiplexing, the simultaneous analysis of multiple biomarkers in a single experiment. This capability increases throughput and efficiency, making it possible to analyze complex biomarker panels and obtain comprehensive data from a single sample.

Improved Dynamic Range

Stable isotopes extend the dynamic range of biomarker assays, allowing for the detection and quantification of biomarkers over a wide concentration range. This expanded range is beneficial for studying biomarkers present at both high and low levels.

Validation and Reproducibility

The use of stable isotopes as internal standards enhances the validation and reproducibility of biomarker assays. By providing a consistent reference point, stable isotopes ensure that measurements are accurate and reliable across different experiments and conditions.

Biomarkers have now become an important tool for drug research and development, and with the wide application of biomarkers, more and more signaling molecules and pathways have been confirmed as therapeutic targets for drugs. Moreover, as a probe that can indicate the biopharmacological activity of a drug, biomarkers can be used to precisely study drug activity and are highly predictive for clinical trials. The addition of stable isotope labeling has made it easier to detect and monitor biomarkers, which will greatly facilitate the process of new drug development.