Introduction:

Blood doping, which involves the use of erythropoietin (EPO) or blood transfusions, is a significant concern in the world of sports. The practice enhances an athlete’s endurance by increasing the oxygen-carrying capacity of their blood, providing an unfair advantage over competitors. In this blog post, we will delve into the advanced forensic techniques employed to detect recombinant EPO and other blood manipulation practices, such as isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Erythropoietin (EPO) and its role in blood doping:

Erythropoietin (EPO) is a glycoprotein hormone produced primarily in the kidneys, with a small amount produced in the liver. EPO stimulates the production of red blood cells (erythropoiesis) in the bone marrow, increasing the oxygen-carrying capacity of the blood. Athletes looking to gain an edge in endurance sports may resort to the administration of recombinant human EPO (rHuEPO) or blood transfusions to elevate their red blood cell count, leading to improved performance.

Detection of recombinant EPO:

Detecting rHuEPO use in athletes is challenging, as the recombinant form is nearly identical to the naturally occurring hormone. However, there are subtle differences in their glycosylation patterns, which advanced forensic techniques can exploit to distinguish between the two forms.

Glycosylation is a post-translational modification (PTM) of proteins, where carbohydrate moieties (oligosaccharides) are covalently attached to specific amino acid residues on the protein. Glycosylation can significantly impact the structure, stability, and function of a protein. In the case of erythropoietin (EPO), glycosylation plays a crucial role in determining its biological activity, half-life, and immunogenicity.

Erythropoietin produced naturally in the body (endogenous EPO) and recombinant human erythropoietin (rHuEPO) have very similar amino acid sequences. However, they exhibit subtle differences in their glycosylation patterns due to variations in the glycosylation machinery of the cells that produce them. Endogenous EPO is produced in the kidneys and liver, whereas rHuEPO is produced using recombinant DNA technology in non-human cell lines, such as Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells.

These different cell types possess unique glycosylation machinery, leading to variations in the types and structures of oligosaccharides attached to the protein, as well as differences in the extent of glycosylation (the number of glycosylation sites occupied). Consequently, the glycosylation patterns of endogenous EPO and rHuEPO can be exploited to distinguish between the two forms using advanced forensic techniques, such as isoelectric focusing (IEF), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and mass spectrometry (MS).

Glycosylation affects the isoelectric point (pI) of a protein, which is the pH at which the protein carries no net charge. Since endogenous EPO and rHuEPO have distinct glycosylation patterns, they display different pI values. Isoelectric focusing (IEF) can separate EPO isoforms based on their pI values, enabling the identification of rHuEPO in biological samples.

Mass spectrometry (MS) can provide detailed information about the molecular weight and structure of proteins and their attached oligosaccharides. By analyzing the mass spectra of endogenous EPO and rHuEPO, it is possible to identify specific glycopeptides and oligosaccharide structures that are characteristic of the recombinant form, thereby distinguishing it from the endogenous hormone.

In summary, the subtle differences in glycosylation patterns between endogenous EPO and rHuEPO can be exploited using advanced forensic techniques to identify the presence of recombinant erythropoietin in biological samples, ensuring fair competition in sports and preventing the abuse of performance-enhancing substances.

Isoelectric Focusing (IEF):

Isoelectric focusing (IEF) is an electrophoretic technique used to separate proteins based on their isoelectric points (pI) – the pH at which the protein carries no net charge. Since EPO isoforms exhibit different pI values due to variations in glycosylation, IEF can effectively separate and identify recombinant EPO forms.

In IEF, a sample containing EPO is applied to a gel with a pH gradient, and an electric field is applied across the gel. The proteins migrate through the gel until they reach the pH corresponding to their pI, where they become neutral and stop migrating. This results in a distinct pattern of bands, which can be visualized using specific staining methods. The presence of rHuEPO is indicated by the appearance of distinct bands that are not found in the normal EPO isoform distribution.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE):

SDS-PAGE is another electrophoretic technique used to separate proteins based on their molecular weights. While IEF separates proteins based on their charge, SDS-PAGE relies on differences in molecular size. In this technique, proteins are denatured and combined with sodium dodecyl sulfate (SDS), which imparts a uniform negative charge to the proteins. The SDS-treated proteins are then loaded onto a polyacrylamide gel, and an electric field is applied.

During electrophoresis, proteins migrate through the gel according to their size, with smaller proteins moving faster and further than larger ones. After electrophoresis, the gel is stained, and protein bands are visualized. SDS-PAGE can be employed as a complementary technique to IEF, providing additional evidence for the presence of rHuEPO based on its distinct molecular weight compared to endogenous EPO.

Conclusion:

Blood doping and the use of erythropoietin continue to pose challenges in maintaining fair competition in sports. Advanced forensic techniques like isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis are used to try to determine those you use EPO as a performance enhancing drug in sports.