Glycated hemoglobin

Glycated hemoglobin (hemoglobin A1c, HbA1c, A1C, or Hb1c; sometimes also HbA1c) is a form of hemoglobin that is measured primarily to identify the average plasma glucose concentration over prolonged periods of time. It is formed in a non-enzymatic glycation pathway by hemoglobin's exposure to plasma glucose. 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. This serves as a marker for average blood glucose levels over the previous months prior to the measurement.

In diabetes mellitus, higher amounts of glycated hemoglobin, indicating poorer control of blood glucose levels, have been associated with cardiovascular disease, nephropathy, and retinopathy. Monitoring HbA1c in type 1 diabetic patients may improve outcomes.

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.

Principle
Glycation of proteins is a frequent occurrence, but in the case of hemoglobin, a nonenzymatic reaction occurs between glucose and the N-end of the beta chain. This forms a Schiff base which is itself converted to 1-deoxyfructose. This rearrangement is known as 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.

Glucose levels are intermittently raised in portal vessels carrying absorbed glucose to the liver for regulation. Passing red cells will have increased glycation after sugary food intake.

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. The HbA1c level is proportional to average blood glucose concentration over the previous four weeks to three months. Some researchers state that the major proportion of its value is weighted toward the most recent 2 to 4 weeks. This is also supported by the data from actual practice showing that HbA1c level improved significantly already after 20 days since glucose-lowering treatment intensification.

Measuring A1C
A number of techniques are used to measure A1C.

Laboratories use:
 * High-performance liquid chromatography (HPLC): The HbA1c result is calculated as a ratio to total hemoglobin by using a chromatogram.
 * Immunoassay
 * Enzymatic
 * Capillary electrophoresis
 * Boronate affinity chromatography

Point of care (e.g., doctor's office) devices use:
 * Immunoassay
 * Boronate affinity chromatography

In the United States, A1C testing laboratories are certified by the National Glycohemoglobin Standardization Program (NGSP) to standardise them against the results of the 1993 Diabetes Control and Complications Trial (DCCT). An additional percentage scale, Mono S is in use by Sweden and KO500 is in Japan.

Switch to IFCC units
The American Diabetes Association (ADA), European Association for the Study of Diabetes (EASD) and International Diabetes Federation (IDF) have agreed that, in the future, HbA1c is to be reported in the International Federation of Clinical Chemistry (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:

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Interpretation of results
Laboratory results may differ depending on the analytical technique, the age of the subject, and biological variation among individuals. Two individuals with the same average blood sugar can have A1C values that differ by as much as 3 percentage points. Results can be unreliable in many circumstances, such as after blood loss, for example, 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. In general, the reference range (that found in healthy persons), is about 20–40 mmol/mol (4–5.9 DCCT %).

Higher levels of HbA1c 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 HbA1c values. A diabetic person with good glucose control has a HbA1c level that is close to or within the reference range. The International Diabetes Federation and American College of Endocrinology recommend HbA1c values below 48 mmol/mol (6.5 DCCT %), while American Diabetes Association recommends that the HbA1c be below 53 mmol/mol (7.0 DCCT %) for most patients. Recent results from large trials suggest that a target below 53 mmol/mol (7 DCCT %) may be excessive: Below 53 mmol/mol (7 DCCT %) 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 diabetes patients found that patients with an A1C 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 ACCORD, ADVANCE and VADT trials estimated that the risks of the main complications of diabetes (retinopathy, nephropathy, neuropathy and macrovascular disease) decreased with approximately 3% for every 1 mmol/mol decrease in HbA1c.

Practitioners must consider an individual patient's health, his/her risk of hypoglycemia, and his/her specific health risks when setting a target A1C level. Because patients are responsible for averting or responding to their own hypoglycemic episodes, the patient's input and the doctor's assessment of the patient's self-care skills are also important.

Persistent elevations in blood sugar (and, therefore, HbA1c) 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.

Lower-than-expected levels of HbA1c can be seen in people with shortened red blood cell lifespan, such as with glucose-6-phosphate dehydrogenase deficiency, sickle-cell disease, or any other condition causing premature red blood cell death. 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 HbA1c to underestimate the actual average levels. There may also be distortions resulting from blood donation which occurred as long as two months before 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 Vitamin B12 or folate deficiency.

The approximate mapping between HbA1c values given in DCCT percentage (%) and eAG (estimated average glucose) measurements is given by the following equation: eAG(mmol/l) = 1.59 × A1C − 2.59 Data in parentheses are 95% confidence intervals
 * eAG(mg/dl) = 28.7 × A1C − 46.7

The 2010 American Diabetes Association Standards of Medical Care in Diabetes added the A1c ≥ 48 mmol/mol (≥6.5 DCCT %) as another criterion for the diagnosis of diabetes.

Indications and use
Glycated hemoglobin testing is recommended for both (a) checking the blood sugar control in people who might be pre-diabetic and (b) monitoring blood sugar control in patients with more elevated levels, termed diabetes mellitus. There is a significant proportion of people who are unaware of their elevated HbA1c level before they have blood lab work. 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 two times a year in patients with diabetes that are meeting treatment goals (and that have stable glycemic control) and quarterly in patients with diabetes whose therapy has changed or that are not meeting glycemic goals.

Glycated hemoglobin measurement is not appropriate where there has been a change in diet or treatment within 6 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 that 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 hemoglobin A1C values at 64 mmol/mol or less should be provided additional testing to determine whether the HbA1c values are due to averaging out high blood glucose (hyperglycemia) with low blood glucose (hypoglycemia) or the HbA1c 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 but not by Medicare in the United States. The supplies tend to be expensive, since the sensors must be changed at least weekly. Another test that is useful in determining if HbA1c 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 renal failure, when measured by ion-exchange chromatography. The thiobarbituric acid method (a chemical method specific for the detection of glycation) shows that patients with renal 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 hemoglobin A1 (HbA1) is undetectable. Administration of prednisolone (PSL) 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 like International Expert Committee Report, drawn from the International Diabetes Federation (IDF), the European Association for the Study of diabetes (EASD), and the American Diabetes Association (ADA), suggests the A1C level of 48 mmol/mol (6.5 DCCT %) as a diagnostic level. The Committee Report further states that, when A1C testing cannot be done, the fasting and glucose tolerance tests be done.

Diagnosis of diabetes during pregnancy continues to require fasting and glucose tolerance measurements for gestational diabetes, and not the glycated hemoglobin.

Planning treatment response
A high A1c correlates with health problems. Because of this, when a patient has a high A1c, a physician may recommend treatment which would lead to the patient having improved health and therefore a lowered A1c.

For most adults age 65 or older, the use of medications to achieve an A1c lower than 7.5% is discouraged., which cites
 * For healthy adults in this age category and who have a long life expectancy, 7.0 – 7.5% is a reasonable glycemic target. For persons with a life expectancy of more than 10 years and some comorbidity, 7.5-8.0% is a good range, and 8.0-9.0% is reasonable for persons with less life expectancy and multiple morbidities. For older populations, evidence does not show that lowering A1c more than moderately will improve health but does show that it causes hypoglycemia.
 * For healthy adults in this age category and who have a long life expectancy, 7.0 – 7.5% is a reasonable glycemic target. For persons with a life expectancy of more than 10 years and some comorbidity, 7.5-8.0% is a good range, and 8.0-9.0% is reasonable for persons with less life expectancy and multiple morbidities. For older populations, evidence does not show that lowering A1c more than moderately will improve health but does show that it causes hypoglycemia.
 * For healthy adults in this age category and who have a long life expectancy, 7.0 – 7.5% is a reasonable glycemic target. For persons with a life expectancy of more than 10 years and some comorbidity, 7.5-8.0% is a good range, and 8.0-9.0% is reasonable for persons with less life expectancy and multiple morbidities. For older populations, evidence does not show that lowering A1c more than moderately will improve health but does show that it causes hypoglycemia.
 * For healthy adults in this age category and who have a long life expectancy, 7.0 – 7.5% is a reasonable glycemic target. For persons with a life expectancy of more than 10 years and some comorbidity, 7.5-8.0% is a good range, and 8.0-9.0% is reasonable for persons with less life expectancy and multiple morbidities. For older populations, evidence does not show that lowering A1c more than moderately will improve health but does show that it causes hypoglycemia.
 * For healthy adults in this age category and who have a long life expectancy, 7.0 – 7.5% is a reasonable glycemic target. For persons with a life expectancy of more than 10 years and some comorbidity, 7.5-8.0% is a good range, and 8.0-9.0% is reasonable for persons with less life expectancy and multiple morbidities. For older populations, evidence does not show that lowering A1c more than moderately will improve health but does show that it causes hypoglycemia.
 * For healthy adults in this age category and who have a long life expectancy, 7.0 – 7.5% is a reasonable glycemic target. For persons with a life expectancy of more than 10 years and some comorbidity, 7.5-8.0% is a good range, and 8.0-9.0% is reasonable for persons with less life expectancy and multiple morbidities. For older populations, evidence does not show that lowering A1c more than moderately will improve health but does show that it causes hypoglycemia.
 * For healthy adults in this age category and who have a long life expectancy, 7.0 – 7.5% is a reasonable glycemic target. For persons with a life expectancy of more than 10 years and some comorbidity, 7.5-8.0% is a good range, and 8.0-9.0% is reasonable for persons with less life expectancy and multiple morbidities. For older populations, evidence does not show that lowering A1c more than moderately will improve health but does show that it causes hypoglycemia.
 * For healthy adults in this age category and who have a long life expectancy, 7.0 – 7.5% is a reasonable glycemic target. For persons with a life expectancy of more than 10 years and some comorbidity, 7.5-8.0% is a good range, and 8.0-9.0% is reasonable for persons with less life expectancy and multiple morbidities. For older populations, evidence does not show that lowering A1c more than moderately will improve health but does show that it causes hypoglycemia.

For younger adults, evidence shows that harms including increased mortality can result from using medication to seek A1c below 7.0%.

Modification by exercise training
A meta-analysis of research done to identify the effect of two different kinds of training programs (combined aerobic and eccentric resistance exercise program and aerobic exercise only) on the glycated hemoglobin levels of individuals with T2DM found that the effect of combining resistance exercise with aerobic exercise improved the glucose control more than just the aerobics alone. The average effect of the training programs included reductions of glycated hemoglobin of 9 mmol/mol (0.8 percentage points), which was a result similar to that of long-term diet and drug or insulin therapy (which result in a reduction of 6.5–9.0 mmol/mol (i.e. 0.6–0.8 points).

Standardization and traceability
HbA1c is now standardized & traceable to IFCC methods HPLC-CE & HPLC-MS. A new unit (mmol/mol) is used as part of this standardization.