Clinical Utility of Fructosamine and Glycated Albumin

Pathophysiology

HbA_1c is a product of nonenzymatic glycation of hemoglobin. RBCs have a lifespan of approximately 90 to 120 days; hence, HbA_1c indicates the mean blood glucose concentration over the lifespan of the RBC. HbA_1c is influenced by conditions affecting RBC survival. Conditions causing low RBC turnover, such as untreated iron, vitamin B12, or folic acid deficiency anemias, result in falsely high HbA_1c  values. On the other hand, conditions causing high RBC turnover, such as hemolytic anemia, can result in falsely low HbA_1c values. This phenomenon can occur in patients treated for iron, vitamin B12, or folate deficiencies, as well as in those treated with erythropoietin, such as individuals with chronic kidney disease.

Fructosamine (1-amino-1-deoxy fructose) is a stable ketoamine formed by the reaction between glucose and the amino group of protein, predominantly albumin, but also including globulins and lipoproteins. The attachment of the aldehyde group of the carbohydrate with the N-terminal amino acid of the protein forms the reversible Schiff base product, the aldimine intermediate. The Schiff base product may be converted back to glucose and protein, or undergo the Amadori rearrangement to form stable fructosamine. This process is known as nonenzymatic glycation and is also referred to as the Maillard reaction. The Maillard reaction causes the browning phenomenon that occurs in milk and other food products when heated. Glycated albumin refers to the formation of ketoamine, specifically involving the major circulating protein albumin (3.5-5 g/dL). Glycated albumin is an example of a fructosamine. Because albumin is the most abundant of the serum proteins, fructosamine is predominantly a measure of glycated albumin. The formation of fructosamine and glycated albumin represents post-translational protein modifications.

Non-immunoglobulin serum proteins have a much lower half-life, approximately 14 to 21 days.[6] The measurement of fructosamine or glycated albumin provides information on glucose control within the previous 2 to 3 weeks. Another important difference with HbA_1c is the rate of nonenzymatic glycation of albumin, which is approximately 9- to 10-fold higher than that of HbA_1c.[7][8]

Specimen Requirements and Procedure

Serum or plasma is the sample type used for the measurement of fructosamine and glycated albumin. Fasting specimens are not required.

Testing Procedures

Fructosamine

A colorimetric assay is the most commonly used method for measuring serum fructosamine, which uses the reduction of the dye nitroblue tetrazolium to formazan. The rate of formazan formation is directly proportional to the fructosamine concentration and is measured using the spectrophotometric technique.[9] These assays are widely available, can be automated, and are relatively inexpensive. The reference range for fructosamine in non-diabetic individuals is generally 200 to 285 µmol/L. However, unlike HbA_1c, there is a serious lack of standardization across the different fructosamine assays.

Glycated Albumin

Several assay methodologies are available for analyzing glycated albumin, including:

• Enzymatic assay
• High-performance liquid chromatography and affinity chromatography
• Immunoassay, including quantification by radioimmunoassay
• Enzyme-linked immunosorbent assay
• Enzyme-linked boronate immunoassay
• Colorimetry
• Electrochemical assay

The enzymatic assay (Lucica GA-L kit, Asahi Kasei Pharma, Tokyo, Japan) is easier to use, highly accurate, and automated.[10] First, there is the elimination of endogenous glycated amino acids and peroxide by a ketoamine oxidase, followed by a peroxidase reaction.[11] An albumin-specific proteinase hydrolyzes the glycated albumin. The products of this reaction are oxidized by ketoamine oxidase to hydrogen peroxide, which is then measured quantitatively using a colorimetric method. The albumin concentration is also measured concurrently. The final result is expressed as the ratio of glycated to total albumin.[12]

The normal value is around 14%, and it increases to greater than 17% in patients with diabetes mellitus. Values in diabetes mellitus can go as high as 2 to 5 times the upper limit of normal.

Interfering Factors

Fructosamine assays are influenced by changes in temperature and by the increased levels of reducing substances in serum, such as vitamin C and bilirubin. Fructosamine and glycated albumin lack standardized assays, and their measurements are influenced by any condition that alters serum albumin concentrations. However, this limitation is minimized for glycated albumin, as results are expressed as a percentage of total albumin. Fructosamine is unreliable when serum albumin is less than 3.0 g/dL. This limitation applies to conditions associated with reduced albumin synthesis, such as liver cirrhosis, as well as those involving albumin or protein loss, including nephrotic syndrome and protein-losing enteropathies. Fructosamine levels may also be influenced by conditions associated with elevated total protein levels, such as multiple myeloma (due to increased immunoglobulins) and polyclonal gammopathies.[13][14]

Results, Reporting, and Critical Findings

The reference range for fructosamine in non-diabetic individuals is typically 200 to 285 μmol/L. Although glycated albumin assays also suffer from standardization issues, the newer assay developed by Asahi Kasei has shown significant improvement. According to this assay, healthy individuals have values around 14% and those with diabetes mellitus have greater than 17%. In cases of diabetes mellitus, levels can reach as high as 2 to 5 times the upper limit of normal.

Clinical Significance

The clinical utility of fructosamine and glycated albumin includes monitoring of diabetes mellitus, diagnosis of pre-diabetes mellitus, and prediction of both the microvascular and macrovascular complications. An added advantage is that their measurement does not require a fasting sample.

Monitoring Glucose Control in Diabetes Mellitus

Fructosamine and glycated albumin can be used as short-term markers of glucose control. Both correlate significantly with HbA_1c levels. Although HbA_1c reflects glucose control over the preceding 8 to 12 weeks, fructosamine reflects the average glycemia over the preceding 2 to 3 weeks. This difference is a result of the inherent shorter half-life of albumin compared to hemoglobin in the erythrocyte.

Fructosamine has largely been used as an alternative to HbA_1c in certain conditions that limit the reliability of HbA_1c, such as hemoglobin variants and alterations in erythrocyte lifespan. Unlike HbA_1c, fructosamine and glycated albumin are unaffected by hemoglobin levels or RBC characteristics, making them reliable markers in conditions such as hemoglobinopathies, sickle cell anemia, and anemias due to iron, vitamin B12, or folate deficiency. Additionally, fructosamine has clinical utility in conditions where information regarding short-term glucose control is important in the management of the patient, such as in pregnancy or recent medication adjustment. Fructosamine and glycated albumin can also be useful in monitoring individuals with diabetes mellitus with fluctuating or poorly controlled diabetes.

Diagnosis of Diabetes Mellitus

Recent studies have investigated the use of the alternate glycaemic markers, such as fructosamine and glycated albumin, for diagnosing diabetes mellitus. During the diagnosis of diabetes, serum glycated albumin measurements have been reported to aid in determining the need for an oral glucose tolerance test. Glycated albumin shows a negative correlation with body mass index, which may lead to an underestimation of glycemic status in individuals with obesity. Currently, no guidelines support the use of glycated albumin or fructosamine for diagnosing diabetes mellitus or pre-diabetes mellitus.[12]

Diabetes Mellitus–Related Outcomes

Previously, limited evidence was available regarding the relationship of fructosamine and glycated albumin with diabetes mellitus complications and long-term outcomes. Recent studies, for example, the Atherosclerosis Risk in Communities Study (ARIC), have demonstrated that fructosamine and glycated albumin were strongly associated with retinopathy as well as significantly associated with the risk of incident chronic kidney disease and incident diabetes mellitus. Besides, both fructosamine and glycated albumin, even after adjustment for HbA_1c, are significant prognosticators of cardiovascular outcomes and mortality.[15]

Quality Control and Lab Safety

Quality assurance is crucial for ensuring the accuracy and reliability of fructosamine testing, which primarily involves 2 key components—internal quality control (IQC) and proficiency testing. IQC is conducted using control materials provided either by the assay manufacturer or third-party suppliers. These materials are used to verify system performance after the analyzer has been calibrated or standardized for fructosamine measurement.[16]

Controls are run at specified intervals, with the frequency determined through a risk assessment of the testing process. The results are recorded on Levey–Jennings charts and assessed using statistical criteria such as the Westgard rules. Any rule violations trigger a root cause analysis to identify whether the error is random or systematic. Appropriate corrective measures are then implemented, and the controls are repeated until the results fall within acceptable limits. Only after this can patient testing resume, following the supervising pathologist's approval.[17]

Patient samples must not be tested if control results are out of range, as this could produce inaccurate results. In compliance with ISO 15189 standards, all patient samples processed after the last acceptable IQC run must be reviewed and retested once the problem is resolved. Laboratories must select IQC materials that are stable, closely mimic patient sample matrices and behavior, present clinically relevant challenges near decision thresholds, and preferably span the assay's measurement range.[18][19]

Equally important is participation in proficiency testing or external quality assessment programs. These programs allow laboratories to benchmark their performance against peers, detect possible biases, and enhance assay reliability. Laboratories are required to have well-defined policies for enrollment, participation, and management of unsatisfactory results.[20][21]

Additionally, laboratory safety is vital in clinical testing. Staff must consistently use proper personal protective equipment, maintain good hand hygiene, and be trained in safety procedures, including the use of eyewash stations, biological and chemical spill kits, and precautions against bloodborne pathogens. A biosafety manual should be accessible to all personnel, with regular training and competency evaluations to ensure compliance with safe laboratory practices.[22][23]

Enhancing Healthcare Team Outcomes

Healthcare professionals, including nurse practitioners, should be familiar with the diagnosis of diabetes mellitus. Fructosamine and glycated albumin can be used as alternative markers in patients where the HbA_1c assay is unreliable. Additionally, they can identify poor glucose control more rapidly than HbA_1c, ie, short-term hyperglycemia. A major promise of the tests is their ability to predict those pre-diabetic patients who progress to clinical diabetes, as this could lead to major lifestyle and pharmacological interventions to prevent the onset of diabetes and its complications. Finally, they may also have a role in the management of diabetes mellitus during pregnancy, as pregnant patients need frequent glucose monitoring. Unlike HbA_1c, which reflects glycemia over 8 to 12 weeks, fructosamine and glycated albumin provide insight into glucose levels over a shorter period of 2 to 3 weeks.[24]

Glycated albumin has been reported to be a better marker than HbA_1c for the assessment of glucose control in people with diabetes mellitus who have chronic kidney disease and those on hemodialysis and peritoneal dialysis.[25][26]