Scientists in Perm have discovered a new method for identifying neurodegenerative diseases.

Amino acids, the building blocks of proteins, can exist in two forms known as L and D. For a long time, it was believed that only L-amino acids occur naturally, while D-amino acids could only be synthesized in a laboratory. However, since the 1960s, the second type has been found in small quantities in bacteria, plants, animals, and humans. Doctors suggest that elevated levels of D-amino acids may indicate or even contribute to the development of diseases such as Alzheimer’s, Pick’s disease, and others. To better understand the accumulation of D-amino acids in nerve tissues, it is essential to determine their concentration. Researchers at Perm Polytechnic University have discovered a new, previously untested method that allows for rapid and highly sensitive detection of these amino acids.

2025-02-27 10:00:12https://naked-science.ru/article/column/sposob-vyyavleniya-nejrod

The article has been published in Journal of Chromatography A. The research was conducted with the support of the Russian Scientific Foundation.

Today, high-performance liquid chromatography with fluorescent or ultraviolet detectors is employed to determine the amino acid content in various liquids. The method involves introducing the sample mixture into a tube filled with a special substance. Within this tube, the components of the mixture are separated and exit sequentially to the detector, which records their appearance as peaks. This sequence of peaks is known as a chromatogram.

For the fluorescent detector to identify the substance being analyzed, it must emit bright light in response to irradiation; however, most natural amino acids (such as serine) do not possess these properties. Therefore, they are chemically labeled with a fluorophore using reagents—a compound capable of emitting light when irradiated.

Dansyl chloride has often been used as such a reagent. However, the products of its reaction with amino acids are unstable, significantly reducing measurement accuracy. This issue can be addressed using its analog, dabsyl chloride. It was previously thought that dabsyl chloride could not be used for detecting amino acids via fluorescent methods, as it "quenches" the fluorescence, resulting in the detector not "seeing" anything. Nevertheless, polytechnic researchers discovered that dabsyl chloride's light absorption is so strong that it even suppresses background radiation, to the extent that the detector signal drops below the baseline, creating what are known as negative peaks on the chromatogram. These negative peaks can also be used to determine the presence of the desired substance, similar to positive peaks.

Scientists from Perm Polytechnic University decided to investigate the feasibility of using this method to detect D-amino acids and compare it with the traditional ultraviolet method. They prepared several solutions containing mixtures of L- and D-serine and recorded chromatograms using both ultraviolet and fluorescent detectors.

“On the first detector, we observed standard peaks pointing upwards, while on the second, they pointed downwards. The depth of the peak is proportional to the amino acid content, allowing for quantitative analysis. We repeated the experiment multiple times for five concentrations of serine ranging from 0.01 to 0.08 mg/ml, which corresponds to actual amino acid quantities in biological samples. We found that the error in determining D-serine using the ultraviolet and fluorescent detectors is virtually identical, not exceeding 8% for the lowest concentration (0.01 mg/ml) and less than one percent for the highest concentration (0.08 mg/ml),” comments Leonid Asnin, Associate Professor of the Department of “Chemistry and Biotechnology” at PNIPU, PhD in Chemistry.

The results indicate that the fluorescent method is as effective for detecting D-amino acids as the ultraviolet method. This enables verification of test results through different methods, enhancing their accuracy.

The method has so far been developed using model solutions, but it will be adapted for real biological fluids, such as cerebrospinal fluid or blood plasma, as well as for animal nervous tissues. This could enable its use in medicine and neurophysiology to study neurodegenerative diseases and metabolic processes in the brain.

The findings from PNIPU researchers will aid in the development of objective diagnostic methods (based on the content of D-amino acids in biological samples) for socially significant diseases, such as Alzheimer's and Pick's disease or discirculatory encephalopathy.