Glycated hemoglobin

Glycated hemoglobin, also called glycohemoglobin, is a form of hemoglobin that is chemically linked to a sugar. Most monosaccharides, including glucose, galactose, and fructose, spontaneously bond with hemoglobin when they are present in the bloodstream. However, glucose is only 21% as likely to do so as galactose and 13% as likely to do so as fructose, which may explain why glucose is used as the primary metabolic fuel in humans.
The formation of excess sugar-hemoglobin linkages indicates the presence of excessive sugar in the bloodstream and is an indicator of diabetes or other hormone diseases in high concentration. A1c is of particular interest because it is easy to detect. The process by which sugars attach to hemoglobin is called glycation and the reference system is based on HbA1c, defined as beta-N-1-deoxy fructosyl hemoglobin as a component.
There are several ways to measure glycated hemoglobin, of which HbA1c is a standard single test. HbA1c is measured primarily to determine the three-month average blood sugar level and is used as a standard diagnostic test for evaluating the risk of complications of diabetes and as an assessment of glycemic control. The test is considered a three-month average because the average lifespan of a red blood cell is three to four months. Normal levels of glucose produce a normal amount of glycated hemoglobin. As the average amount of plasma glucose increases, the fraction of glycated hemoglobin increases predictably. In diabetes, higher amounts of glycated hemoglobin, indicating higher blood glucose levels, have been associated with cardiovascular disease, nephropathy, neuropathy, and retinopathy.

Terminology

hemoglobin is preferred over glycosylated hemoglobin to reflect the correct process. Early literature often used glycosylated as it was unclear which process was involved until further research was performed. The terms are still sometimes used interchangeably in English-language literature.
The naming of HbA1c derives from hemoglobin type A being separated on cation exchange chromatography. The first fraction to separate, considered to be pure hemoglobin A, was designated HbA0, and the following fractions were designated HbA1a, HbA1b, and HbA1c, in their order of elution. Improved separation techniques have subsequently led to the isolation of more subfractions.

History

Hemoglobin A1c was first separated from other forms of hemoglobin by Huisman and Meyering in 1958 using a chromatographic column. It was first characterized as a glycoprotein by Bookchin and Gallop in 1968. Its increase in diabetes was first described in 1969 by Samuel Rahbar and coworkers. The reactions leading to its formation were characterized by Bunn and coworkers in 1975.
The use of hemoglobin A1c for monitoring the degree of control of glucose metabolism in diabetic patients was proposed in 1976 by Anthony Cerami, Ronald Koenig, and coworkers.

Damage mechanisms

Glycated hemoglobin causes an increase of highly reactive free radicals inside blood cells, altering the properties of their cell membranes. This leads to blood cell aggregation and increased blood viscosity, which results in impaired blood flow.
Another way glycated hemoglobin causes damage is via inflammation, which results in atherosclerotic plaque formation. Free-radical build-up promotes the excitation of Fe²⁺-hemoglobin through into abnormal ferryl hemoglobin. Fe⁴⁺ is unstable and reacts with specific amino acids in hemoglobin to regain its Fe³⁺ oxidation state. Hemoglobin molecules clump together via cross-linking reactions, and these hemoglobin clumps promote cell damage and the release of Fe⁴⁺-hemoglobin into the matrix of innermost layers of arteries and veins. This results in increased permeability of interior surface of blood vessels and production of pro-inflammatory monocyte adhesion proteins, which promote macrophage accumulation in blood vessel surfaces, ultimately leading to harmful plaques in these vessels.
Highly glycated Hb-AGEs go through vascular smooth muscle layer and inactivate acetylcholine-induced endothelium-dependent relaxation, possibly through binding to nitric oxide, preventing its normal function. NO is a potent vasodilator and also inhibits the formation of plaque-promoting LDLs oxidized form.
This overall degradation of blood cells also releases heme from them. Loose heme can cause oxidation of endothelial and LDL proteins, which results in plaques.

Principle in medical diagnostics

Glycation of proteins is a frequent occurrence, but in the case of hemoglobin, a nonenzymatic condensation reaction occurs between glucose and the N-end of the beta chain. This reaction produces a Schiff base, which is itself converted to 1-deoxyfructose. This second conversion is an example of an Amadori rearrangement.
When blood glucose levels are high, glucose molecules attach to the hemoglobin in red blood cells. The longer hyperglycemia occurs in blood, the more glucose binds to hemoglobin in the red blood cells and the higher the glycated hemoglobin.
Once a hemoglobin molecule is glycated, it remains that way. A buildup of glycated hemoglobin within the red cell, therefore, reflects the average level of glucose to which the cell has been exposed during its life-cycle. Measuring glycated hemoglobin assesses the effectiveness of therapy by monitoring long-term serum glucose regulation.
A1c is a weighted average of blood glucose levels during the life of the red blood cells. Therefore, glucose levels on days nearer to the test contribute substantially more to the level of A1c than the levels on days further from the test.
This is also supported by data from clinical practice showing that HbA1c levels improved significantly after 20 days from the start or intensification of glucose-lowering treatment.

Measurement

Several techniques are used to measure hemoglobin A1c. Laboratories may use high-performance liquid chromatography, immunoassay, enzymatic assay, capillary electrophoresis, or boronate affinity chromatography. Point of care devices use immunoassay boronate affinity chromatography.
In the United States, HbA_1c testing laboratories are certified by the National Glycohemoglobin Standardization Program to standardize them against the results of the 1993 Diabetes Control and Complications Trial. An additional percentage scale, Mono S has previously been in use by Sweden and KO500 is in use in Japan.

Switch to IFCC units

The American Diabetes Association, European Association for the Study of Diabetes, and International Diabetes Federation have agreed that, in the future, HbA_1c is to be reported in the International Federation of Clinical Chemistry and Laboratory Medicine units. IFCC reporting was introduced in Europe except for the UK in 2003; the UK carried out dual reporting from 1 June 2009 until 1 October 2011.
Conversion between DCCT and IFCC is by the following equation:

"IFCC" HbA_1c	"DCCT" HbA_1c	"Mono S" HbA_1c

10	3.1	2.0
20	4.0	2.9
30	4.9	3.9
40	5.8	4.8
45	6.3	5.3
50	6.7	5.8
55	7.2	6.3
60	7.6	6.8
65	8.1	7.2
70	8.6	7.7
80	9.5	8.7
90	10.4	9.6
100	11.3	10.6

Interpretation of results

Laboratory results may differ depending on the analytical technique, the age of the subject, and biological variation among individuals. Higher levels of HbA_1c are found in people with persistently elevated blood sugar, as in diabetes mellitus. While diabetic patient treatment goals vary, many include a target range of HbA_1c values. A diabetic person with good glucose control has an HbA_1c level that is close to or within the reference range.
The International Diabetes Federation and the American College of Endocrinology recommend HbA_1c values below 48 mmol/mol, while the American Diabetes Association recommends HbA_1c be below 53 mmol/mol for most patients. Results from large trials in suggested that a target below 53 mmol/mol for older adults with type 2 diabetes may be excessive: Below 53 mmol/mol, the health benefits of reduced A1c become smaller, and the intensive glycemic control required to reach this level leads to an increased rate of dangerous hypoglycemic episodes.
A retrospective study of 47,970 type 2 diabetes patients, aged 50 years and older, found that patients with an HbA_1c more than 48 mmol/mol had an increased mortality rate, but a later international study contradicted these findings.
A review of the UKPDS, Action to Control Cardiovascular Risk in Diabetes, Advance and Veterans Affairs Diabetes Trials estimated that the risks of the main complications of diabetes decreased by about 3% for every 1 mmol/mol decrease in HbA_1c.
However, a trial by ACCORD designed specifically to determine whether reducing HbA_1c below 42 mmol/mol using increased amounts of medication would reduce the rate of cardiovascular events found higher mortality with this intensive therapy, so much so that the trial was terminated 17 months early.
Practitioners must consider patients' health, their risk of hypoglycemia, and their specific health risks when setting a target HbA_1c level. Because patients are responsible for averting or responding to their own hypoglycemic episodes, their input and the doctors' assessments of the patients' self-care skills are also important.
Persistent elevations in blood sugar increase the risk of long-term vascular complications of diabetes, such as coronary disease, heart attack, stroke, heart failure, kidney failure, blindness, erectile dysfunction, neuropathy, gangrene, and gastroparesis. Poor blood glucose control also increases the risk of short-term complications of surgery, such as poor wound healing.
All-cause mortality is higher above 64 mmol/mol HbA1c as well as below 42 mmol/mol in diabetic patients, and above 42 mmol/mol as well as below 31 mmol/mol in non-diabetic persons, indicating the risks of hyperglycemia and hypoglycemia, respectively. Similar risk results are seen for cardiovascular disease.
The 2022 ADA guidelines reaffirmed the recommendation that HbA1c should be maintained below 7.0% for most patients. Higher target values are appropriate for children and adolescents, patients with extensive co-morbid illness and those with a history of severe hypoglycemia. More stringent targets are preferred for pregnant patients if this can be achieved without significant hypoglycemia.