# Glycated hemoglobin

Glycated hemoglobin, also called glycohemoglobin, is a form of hemoglobin (Hb) that is chemically linked to a sugar. Most monosaccharides, including glucose, galactose, and fructose, spontaneously (that is, non-enzymatically) 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 (HbA1c > 6.4%). 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 component.
There are several ways to measure glycated hemoglobin, of which HbA1c (or simply A1c) 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 in a predictable way. In diabetes, higher amounts of glycated hemoglobin, indicating higher of blood glucose levels, have been associated with cardiovascular disease, nephropathy, neuropathy, and retinopathy.

## Terminology

Glycated hemoglobin is preferred over glycosylated hemoglobin to reflect the correct (non-enzymatic) 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, probably 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 et al. The reactions leading to its formation were characterized by Bunn and his 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 (atheroma) formation. Free-radical build-up promotes the excitation of Fe$^{2+}$-hemoglobin through Fe$^{3+}$-Hb into abnormal ferryl hemoglobin (Fe$^{4+}$-Hb). Fe$^{4+}$ is unstable and reacts with specific amino acids in hemoglobin to regain its Fe$^{3+}$ oxidation state. Hemoglobin molecules clump together via cross-linking reactions, and these hemoglobin clumps (multimers) promote cell damage and the release of Fe$^{4+}$-hemoglobin into the matrix of innermost layers (subendothelium) of arteries and veins. This results in increased permeability of interior surface (endothelium) 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 (NO), preventing its normal function. NO is a potent vasodilator and also inhibits formation of plaque-promoting LDLs (sometimes called "bad cholesterol") 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 (R−N=CHR', R=beta chain, CHR'=glucose-derived), 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 (117 days for men and 106 days in women). Therefore, glucose levels on days nearer to the test contribute substantially more to the level of A1c than the levels in days further from the test.
This is also supported by data from clinical practice showing that HbA1c levels improved significantly after 20 days from 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 (e.g., doctor's office) 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 (DCCT). 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 (IFCC) 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:
    $\mathrm {IFCC\ HBA1c} \,{\Big{\frac {\text{mmol}}{\text{mol}}}{\Big )}=[\mathrm {DCCT\ HBA1c} \,(\%)-2.14]\times 10.929$
| "IFCC" HbA$_{1c}$ | "DCCT" HbA$_{1c}$ | "Mono S" HbA$_{1c}$ |
| (mmol/mol) | (%) | (%) |
| 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 (6.5 DCCT %), while the American Diabetes Association recommends HbA$_{1c}$ be below 53 mmol/mol (7.0 DCCT %) for most patients. Results from large trials in 2008–09 suggested that a target below 53 mmol/mol (7.0 DCCT %) 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 (6.5 DCCT %) 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 (ACCORD), Advance and Veterans Affairs Diabetes Trials (VADT) estimated that the risks of the main complications of diabetes (diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, and macrovascular disease) 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 (6.0 DCCT %) 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 (and, therefore, HbA$_{1c}$) 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 (loss of sensation, especially in the feet), gangrene, and gastroparesis (slowed emptying of the stomach). 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 (8.0 DCCT%) HbA1c as well as below 42 mmol/mol (6.0 DCCT %) in diabetic patients, and above 42 mmol/mol (6.0 DCCT %) as well as below 31 mmol/mol (5.0 DCCT %) 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 (<6.0%) are preferred for pregnant patients if this can be achieved without significant hypoglycemia.

### Factors other than glucose that affect A1c

Lower-than-expected levels of HbA$_{1c}$ can be seen in people with shortened red blood cell lifespans, such as with glucose-6-phosphate dehydrogenase deficiency, sickle-cell disease, or any other condition causing premature red blood cell death. For these patients, alternate assessment with fructosamine or glycated albumin is recommended; these methods reflect glycemic control over the preceding 2-3 weeks. Blood donation will result in rapid replacement of lost RBCs with newly formed red blood cells. Since these new RBCs will have only existed for a short period of time, their presence will lead HbA$_{1c}$ to underestimate the actual average levels. There may also be distortions resulting from blood donation during the preceding two months, due to an abnormal synchronization of the age of the RBCs, resulting in an older than normal average blood cell life (resulting in an overestimate of actual average blood glucose levels). Conversely, higher-than-expected levels can be seen in people with a longer red blood cell lifespan, such as with iron deficiency.
Results can be unreliable in many circumstances, for example after blood loss, after surgery, blood transfusions, anemia, or high erythrocyte turnover; in the presence of chronic renal or liver disease; after administration of high-dose vitamin C; or erythropoetin treatment. Hypothyroidism can artificially raise the A1c. In general, the reference range (that found in healthy young persons), is about 30–33 mmol/mol (4.9–5.2 DCCT %). The mean HbA$_{1c}$ for diabetics type 1 in Sweden in 2014 was 63 mmol/mol (7.9 DCCT%) and for type 2, 61 mmol/mol (7.7 DCCT%). HbA1c levels show a small, but statistically significant, progressive uptick with age; the clinical importance of this increase is unclear.

### Mapping from A1c to estimated average glucose

The approximate mapping between HbA$_{1c}$ values given in DCCT percentage (%) and eAG (estimated average glucose) measurements is given by the following equation:
    eAG(mg/dL) = 28.7 × A1C − 46.7 eAG(mmol/L) = 1.59 × A1C − 2.59 (Data in parentheses are 95% confidence intervals>)
| HbA$_{1c}$ | HbA$_{1c}$ | eAG | eAG |
| % | mmol/mol | mmol/L | mg/dL |
| 5 | 31 | 5.4 (4.2–6.7) | 97 (76–120) |
| 6 | 42 | 7.0 (5.5–8.5) | 126 (100–152) |
| 7 | 53 | 8.6 (6.8–10.3) | 154 (123–185) |
| 8 | 64 | 10.2 (8.1–12.1) | 183 (147–217) |
| 9 | 75 | 11.8 (9.4–13.9) | 212 (170–249) |
| 10 | 86 | 13.4 (10.7–15.7) | 240 (193–282) |
| 11 | 97 | 14.9 (12.0–17.5) | 269 (217–314) |
| 12 | 108 | 16.5 (13.3–19.3) | 298 (240–347) |
| 13 | 119 | 18.1 (15–21) | 326 (260–380) |
| 14 | 130 | 19.7 (16–23) | 355 (290–410) |
| 15 | 140 | 21.3 (17–25) | 384 (310–440) |
| 16 | 151 | 22.9 (19–26) | 413 (330–480) |
| 17 | 162 | 24.5 (20–28) | 441 (460–510) |
| 18 | 173 | 26.1 (21–30) | 470 (380–540) |
| 19 | 184 | 27.7 (23–32) | 499 (410–570) |

### Normal, prediabetic, and diabetic ranges

The 2010 American Diabetes Association Standards of Medical Care in Diabetes added the HbA$_{1c}$ ≥ 48 mmol/mol (≥6.5 DCCT %) as another criterion for the diagnosis of diabetes.
Diagnostic standard for HbA1c in diabetes :
| Diagnosis | "IFCC" HbA$_{1c}$ | "DCCT" HbA$_{1c}$ | "Mono S" HbA$_{1c}$ |
| Normal | < 40 mmol/mol | < 5.7% | < 4.7% |
| Prediabetes | 40–47 mmol/mol | 5.7–6.4% | 4.7–5.4% |
| Diabetes | ≥ 48 mmol/mol | ≥ 6.5% | > 5.5% |

## Indications and uses

Glycated hemoglobin testing is recommended for both checking the blood sugar control in people who might be prediabetic and monitoring blood sugar control in patients with more elevated levels, termed diabetes mellitus. For a single blood sample, it provides far more revealing information on glycemic behavior than a fasting blood sugar value. However, fasting blood sugar tests are crucial in making treatment decisions. The American Diabetes Association guidelines are similar to others in advising that the glycated hemoglobin test be performed at least twice a year in patients with diabetes who are meeting treatment goals (and who have stable glycemic control) and quarterly in patients with diabetes whose therapy has changed or who are not meeting glycemic goals.
Glycated hemoglobin measurement is not appropriate where a change in diet or treatment has been made within six weeks. Likewise, the test assumes a normal red blood cell aging process and mix of hemoglobin subtypes (predominantly HbA in normal adults). Hence, people with recent blood loss, hemolytic anemia, or genetic differences in the hemoglobin molecule (hemoglobinopathy) such as sickle-cell disease and other conditions, as well as those who have donated blood recently, are not suitable for this test.
Due to glycated hemoglobin's variability (as shown in the table above), additional measures should be checked in patients at or near recommended goals. People with HbA$_{1c}$ values at 64 mmol/mol or less should be provided additional testing to determine whether the HbA$_{1c}$ values are due to averaging out high blood glucose (hyperglycemia) with low blood glucose (hypoglycemia) or the HbA$_{1c}$ is more reflective of an elevated blood glucose that does not vary much throughout the day. Devices such as continuous blood glucose monitoring allow people with diabetes to determine their blood glucose levels on a continuous basis, testing every few minutes. Continuous use of blood glucose monitors is becoming more common, and the devices are covered by many health insurance plans, including Medicare in the United States. The supplies tend to be expensive, since the sensors must be changed at least every 2 weeks. Another useful test in determining if HbA$_{1c}$ values are due to wide variations of blood glucose throughout the day is 1,5-anhydroglucitol, also known as GlycoMark. GlycoMark reflects only the times that the person experiences hyperglycemia above 180 mg/dL over a two-week period.
Concentrations of hemoglobin A1 (HbA1) are increased, both in diabetic patients and in patients with kidney failure, when measured by ion-exchange chromatography. The thiobarbituric acid method (a chemical method specific for the detection of glycation) shows that patients with kidney failure have values for glycated hemoglobin similar to those observed in normal subjects, suggesting that the high values in these patients are a result of binding of something other than glucose to hemoglobin.
In autoimmune hemolytic anemia, concentrations of HbA1 is undetectable. Administration of prednisolone will allow the HbA1 to be detected. The alternative fructosamine test may be used in these circumstances and it also reflects an average of blood glucose levels over the preceding 2 to 3 weeks.
All the major institutions such as the International Expert Committee Report, drawn from the International Diabetes Federation, the European Association for the Study of Diabetes, and the American Diabetes Association, suggest the HbA$_{1c}$ level of 48 mmol/mol (6.5 DCCT %) as a diagnostic level. The Committee Report further states that, when HbA$_{1c}$ testing cannot be done, the fasting and glucose-tolerance tests be done. Screening for diabetes during pregnancy continues to require fasting and glucose-tolerance measurements for gestational diabetes at 24 to 28 weeks gestation, although glycated hemoglobin may be used for screening at the first prenatal visit.

## Modification by diet

Meta-analysis has shown probiotics to cause a statistically significant reduction in glycated hemoglobin in type-2 diabetics. Trials with multiple strains of probiotics had statistically significant reductions in glycated hemoglobin, whereas trials with single strains did not.

## Standardization and traceability

Most clinical studies recommend the use of HbA1c assays that are traceable to the DCCT assay. The National Glycohemoglobin Standardization Program (NGSP) and IFCC have improved assay standardization. For initial diagnosis of diabetes, only HbA1c methods that are NGSP-certified should be used, not point-of-care testing devices. Analytical performance has been a problem with earlier point-of-care devices for HbA1c testing, specifically large standard deviations and negative bias.

## Veterinary medicine

HbA1c testing has not been found useful in the monitoring during the treatment of cats and dogs with diabetes, and is not generally used; monitoring of fructosamine levels is favoured instead.