Hemoglobin (Hb) is a globular protein from the group of hemeproteins and represents most of the protein content of red blood cells, also called erythrocytes (Ery), corresponding to around 95% of the average volume of these cells (Yoshida, Prudent, & D’alessandro, 2019). Its structure consists of a tetramer, and each of the four subunits contains a heme group (Fig. 1) (Kettisen and Bülow, 2021, Longo et al., 2021). Hb, through the heme group, performs its primary function, the transportation of O2 from the lungs to the body’s tissues and organs, as well as assisting in the transport of CO2 from the tissues back to the lungs to remove this gas from the body (Ahmed, Ghatge, & Safo, 2020). Hb is also involved in several other functions, including the regulation of oxygen-dependent NO metabolism, maintenance of serum pH, and the ability to participate in body heat exchange during the oxygenation/deoxygenation cycle (Kosmachevskaya & Topunov, 2018).

There are different isoforms of Hb, which differ in subunit composition and expression, varying during human development. The most common type in healthy adults (95–98%) is hemoglobin A (HbA), composed of two α subunits and two β subunits. Hemoglobin A2 (HbA2) is found in a smaller proportion (2–3%), comprised of two α subunits and two δ subunits, and has a lower affinity for O2 than HbA. The main Hb isoform in fetuses and newborns is fetal hemoglobin (HbF), comprised of two α subunits and two γ subunits. HbF has a greater affinity for oxygen than HbA, facilitating the transfer of O2 from the mother’s blood to the fetus during pregnancy (Guo et al., 2019, Kaufman et al., 2023). Mutations in the β-globin gene can lead to the formation of HbA variants, resulting in different forms of hemoglobin. Among the most common variants are hemoglobin S (HbS), which is responsible for sickle cell anemia, and hemoglobin C (HbC), which can cause mild hemolytic anemia (Ding et al., 2018).

A fraction of HbA can undergo covalent modification through a spontaneous chemical reaction due to adding a free amine group from the protein to the carbonyl of reducing sugars. This process occurs through an irreversible non-enzymatic reaction called glycation, leading to the formation of the total fraction of glycated hemoglobin (HbA1) and various subtypes such as HbA1a, HbA1b and HbA1c (Heo et al., 2021), with HbA1c being defined as the most stable adduct of this reaction (Ding et al., 2018).

The first record of HbA1c was in 1955 when Kunkel and Wallenius observed a new Hb, in small quantities in adult human blood, but with electrophoretic properties different from HbA (Kunkel & Wallenius, 1955). In 1957, hemoglobin fractions that eluted faster than HbA were identified (Allen et al., 1958). About 10 years later, already termed HbA1c, glycated hemoglobin was defined as the fraction of HbA modified by the addition of a hexose to the N-terminal group of the β-chain and unique both as a glycoprotein and in the mode of sugar binding to the protein (Bookchin & Gallop, 1968). It also was observed that HbA1c was elevated in the erythrocytes of diabetic patients (Rahbar et al., 1969), and for the first time, the relationship between HbA1c, average glycemia, and chronic complications in diabetic patients was described (Trivelli et al., 1971), while Koenig et al. (1976) observed a correlation between HbA1c and average blood glucose levels. In 1993, the American Association for Clinical Chemistry (AACC) established a subcommittee to set an international standard for the measurement of HbA1c (Little et al., 2001). The American Diabetes Association (ADA) (2010) recommended HbA1c levels for diagnosing diabetes and pre-diabetes. Considering the impact of diabetes as a public health problem worldwide, in 2021, the World Health Organization (WHO) (2023) launched the Global Diabetes Compact to promote improvements in the prevention and control of the disease, especially in low- and middle-income countries. In addition, in 2022, the World Health Assembly endorsed five global targets for diabetes coverage and treatment by 2030 (WHO, 2023). Fig. 2 illustrates a timeline of the scientific evolution of the clinical use of HbA1c in diabetes from its discovery to the present day.

The process of Hb glycation leads to structural and, consequently, conformational changes in the protein’s native structure, compromising its function, such as oxygen transportation by the heme group. In diabetic patients, Hb glycation is even more evident due to hyperglycemia and is associated with various complications. In this context, it is important to evaluate the consequences of glycation on Hb by exploring different experimental strategies for monitoring these changes, which could provide valuable insights into understanding the pathophysiology of the disease and help develop more effective and targeted therapeutic strategies.

This chapter discusses the main concepts involved in the process of glycation and carbonyl stress related to the formation of HbA1c and its use as a marker for diabetes. This process is closely related to the conformational changes that occur in the protein, so topics such as in vitro protocols for Hb glycation, structural changes in glycated Hb, and the main biophysical assays for assessing changes in protein structure are covered.