Canine HbA1c

Canine HbA1c




HbA1c is a type of hemoglobin on which several monosaccharides such as glucose, galactose and fructose tend to bind with and exist in the bloodstream. The chemical linking process between sugar and hemoglobin is named glycation. HbA1c is actually an indicator of the beta-N-1-deoxy fructosyl on hemoglobin, which has been used as diagnostic measurement of long-term glycemic control for patients with diabetes mellitus. The increasing number of HbA1c in bloodstream represent the elevated plasma glucose level that usually indicating a poor diabetic management may lead to severer conditions. Due to the lifecycle of red blood cells is average four months, the HbA1c test could effectively present the real blood sugar degree in latest three months. In 1980, Wood and Smith first demonstrated the applicable method of examining canine diabetes with monitoring glycosylated haemoglobin. Later, Smith et al 1982, Mahaffey and Cornelius 1982, Dennis 1989, Jensen 1995 all confirmed this method is workable on diagnosing diabetic dogs.


Molecular structure of HbA1c

Figure 1 showed an aldimine linkage between glucose and hemoglobin (Figure1).

Figure 1. Glycation process between Hemoglobin and glucose

As we previously discussed that different monosaccharide would form different chemical structure with blood cells. Thus, while using cation exchange chromatography to separate Hemoglobin type A, different fractions would be separate out like HbA0, HbA1a, HbA1a2, HbA1b, and HbA1c. Fractions were named after eluting order (Figure 2).


Figure 2. Different separated fractions of Hemoglobin type A with cation exchange chromatography


Glycated hemoglobin forming harmful factors in human body

Highly reactive free radicals promote the formation of abnormal ferryl Hb (Fe4+-Hb), which enhance macrophage accumulation in blood vessel. Both macrophage accumulation and accumulation of glycated hemoglobin would elevate blood viscosity and slow down normal blood flow. Thus, atherosclerotic plaque would gradually occur in blood vessel.


History of standardized HbA1c as diagnostic tool for diabetes

Diabetes has become a serious health issue in entire world, due to over 220 million patients are suffering of it. Among all cases of diabetes, type 2 diabetes take majority part (90% to 95%). Besides, worsen type 2 diabetes could also cause extra complications, such as cardiovascular diseases, peripheral neuropathy, nephropathy, optic neuropathy, diabetic foot and even unto death. Several risks factor related to diabetes including high fat diet, obesity, smoking, elevated cholesterol levels, high blood pressure and lack of regular exercise.


For preventing and relieving conditions of diabetes, taking healthy diet and regular exercise are important. In addition, diabetic patients’ blood sugar condition should be monitored regularly all the time. There are many clinical methods to evaluate glycemia like urine glucose, random or fasting plasma glucose etc. However, HbA1c is considered to be one of the most accurate and efficient method for measuring long-term blood sugar level (3 to 4 months). From 1894 to 1993, Diabetes Control and Complications Trial (DCCT) established the big data of diabetic patients’ HbA1c values to mean blood glucose resulted in making HbA1c a reliable index of mean blood glucose. However, DCCT haven’t standardized the HbA1c assay methods for labs and clinics. Later, the American Association for Clinical Chemistry (AACC) Standards Committee established a HbA1c Standardization Subcommittee to develop a plan for standardizing HbA1c assay that clinical laboratories could take advantage of it to perform precise glycemic evaluation and control. Now, the National Glycohemoglobin Standardization Program (NGSP) continue to develop reliable assays of HbA1c.


Diagnosing canine diabetes with HbA1c

2018 American Animal Hospital Association suggested guidelines of diagnosis and assessment for animal diabetes. Clinical evaluation of animal diabetes (cats and dogs) include hyperglycemia, physical exam, complete blood count [CBC], Elevated blood glucose (BG), glucosuria, chemistry with electrolytes, urine analysis with culture, urine protein creatinine ratio (UPC), triglycerides, blood pressure (BP), and thyroxine (T4). While the level of BG concentration elevated to 200 mg/dL in dogs and 250–300 mg/dL in cats, glucosuria will typically occur. Pets with persistent glucosuria, persistent hyperglycemia, and presence clinical signs would be judged as diabetes mellitus (DM).


Furthermore, HbA1c now have become a crucial glycemic indicator for diabetic dogs. Neslihan Tascene et al. revealed that blood HbA1c levels of diabetic were found to be 3.11±0.4 %, whereas the normal dog was 1.07±0.08 % respectively. Besides, the blood serum glucose level of diabetic dogs was around 526.71±22 mg/dl, whereas blood sugar in control dogs were around 97.80±2.93 mg/dl. Hasegawa S. demonstrated 6.41% HbA1c in diabetic dogs, whereas normal dogs with 2.6% (mean HbA1c of total Hb, %). And mean HbA1 values of normal dogs and diabetic dogs were 3.58 and 7.41%.


In addition, Na-Yon Kim et al. showed significantly higher HbA1c concentrations of diabetic dogs (>6.2%) than non-diabetic dogs (p < 0.001) with commercial HbA1c testing system. Furthermore, Chao-Nan Lin et al. present the stability of canine glycosylated hemoglobin sample at room (25°C) and refrigerator (4°C) temperatures over 14 days. Besides, different purified methods would cause slight variation in measuring HbA1c value.


Monitoring of pets’ diabetes

Monitoring options include performance of blood glucose curves (BGCs), monitoring urine glucose (UG), measuring fructosamine, and assessment of clinical signs and weight.


a. Blood glucose (BG) levels:

Blood glucose levels fluctuate and could be used for indicating short periods of hyperglycemia. Normal BG were 63~110mg/dL in dogs; 47~151mg/dL in cats. As we mentioned, when the BG concentration goes over approximately 200 mg/dL in dogs and 250–300 mg/dL in cats, Glucosuria will occur. Blood Glucose Curves should be established during insulin treatment.


b. Threshold of urine glucose (UG):

UG concentration reflects only the average BG. Thus, it’s not recommended to solely rely on UG measurements is not recommended. Regardless, UG concentration can assist in assessment of DM together with other evaluation parameters. The threshold of urine glucose (UG) would fall in 180mg/dL in dogs; 252mg/dL in cats.


c. Fructosamine:

Fructosamine is a glycosylated protein formed by nonenzymatic, irreversible binding of glucose to serum albumin. It able to discern normal glycemic level from diabetes with chronic hyperglycemia and won’t be affected by transient hyperglycemia. The rate of formation related to the average BG level and the half-life of albumin. Thus, it reflects blood sugar control around previous 1–2 weeks. Reference normal range of fructosamine is 225-365 mumol/L in dogs (Table 1) and 340 mumol/L in cats. Usually, animals with fructosamine over 500 mumol/L represent poor metabolic control.


Table 1. Referring fructosamine level in dogs

Dogs Fructosamine values (μmol/L)
Normal dog without diabetes 225–365
Newly diagnosed dog 320–850
Treated dogs with diabetes:
Excellent control 350–400
Good control 400–450
Fair control 450–500
Poor control >500


However, fructosamine can’t reveal transient abnormalities in the blood glucose concentration and also not able to discern dysregulation and Somogyi effect. This would require serial measurement of blood glucose concentrations. Besides, canine with hypoalbuminemia would also show decreased fructosamine level (false negative).


d. HbA1c:

HbA1c test won’t be affected by fluctuation of blood glucose concentrations short after eating or drinking. Thus, taking advantage in demonstrating long-term blood sugar level within 2 to 4 months. Also, it won’t be fluctuating due to the conditions of stress-related or postprandial hyperglycemia. The diagnostic standard for HbA1c in human diabetes, HbA1c under <5.7 belongs to normal condition;whereas, prediabetes patients would demonstrate HbA1c with 5.7-6.4% and over 6.5% would be judged as diabetes. Besides, veterinarians suggested normal HbA1c in dogs would fall 1.0 % to 3.8 %, 4.0 % to 6.0% would considered as pre-diabetes and over 6.0% should be diagnosed as diabetes. In controlled diabetic dogs, level of HbA1c should be 3.9 % to 5.8%. However, limitation of measuring HbA1c still exist, HbA1c would be affected by hemoglobin concentrations, e.g. polycythemia or anemia.


Figure 3. HbA1c range for canine and feline


Measurement of HbA1c

Currently scientists have classified 3 main available HbA1c testing methods to clinical laboratories:


a. Chromatography based assay

HbA1c can be distinct from other hemoglobin A by different charge and size with chromatographic method, such as high pressure (or performance) liquid chromatography (HPLC) and boronate affinity chromatograph. Using ion exchange or affinity column with an HPLC instrument could separate and measure the ratio of HbA1c peak area to the total hemoglobin. Boronate affinity chromatography separates two fractions, glycated and non-glycated proteins, and comparing the glycated fraction to the total are % HbA1c (HbA1c detection range is 5.3% to 17%).


b. Antibody based immunoassay

HbA1c specific antibodies could be coated on latex membrane for binding and detecting molecules (HbA1c). HbA1c (%) could be measured by calculating the immuno-fluorescent level of HbA1c (Figure 2). Sensitive commercial ELISA kit has been widely used on detecting HbA1c. Furthermore, Jo et al. utilized Luminescent Resonance Energy Transfer (LRET) -based biosensor method to detect HbA1c.


Figure 4. HbA1c antibody based immunoassay


c. Enzyme based HbA1c assay:

The fructosyl valine oxidase (FVO) would catalyze glycated valines of blood samples exposed by proteolytic digestion and produce hydrogen peroxide. Then, hydrogen peroxide would react to horseradish peroxidase and demonstrate biomarking dye with chromogen (Figure 3).


Figure 5. Direct enzymatic HbA1c method


d. Capillary electrophoresis

According to charge-to-mass ratio, HbA1c could also be separated by capillary electrophoresis (CE) method. It separates hemoglobin A1C from HbA0, HbD, HbA2 and other hemoglobins by charge difference. This analytic method takes advantages on clearly detecting the HbA1c peak without being influenced by protein fractions or other hemoglobin derivatives (Figure 6).


Figure 6. Separating and analyzing HbA1c by capillary electrophoresis method


e. Other detecting methods – electrochemical biosensor for HbA1c

Shaivya Gupta et al. summarized several studies with electrochemical biosensing tools for detecting HbA1c molecule (Table 2).


Table 2. Various methods for detection of glycated hemoglobin.

Year Title Patent No Original Assignee
1981 Reagent and test kit for determining glycosylated hemoglobin US 4255385 A Abbott Laboratories
1993 Determination of glycated hemoglobin by fluorescence quenching WO 1993018407 A1 Abbott Lab
1994 Rapid determination of glycated hemoglobin EP 0590047 A1 Abbott Laboratories
1994 Combined glycated hemoglobin and immunoturbidometric glycated albumin assay from whole blood lysate US 5284777 A Isolab, Inc.
1995 Determination of glycated hemoglobin by fluorescence quenching US5478754 A Abbot Laboratories
1996 Method for preparing a glycated hemoglobin solution US 5589393 A Abbott Laboratories
1997 Methods and reagents for the rapid determination of glycated hemoglobin US 5686316 A Abbott Laboratories
1999 Measurement of glycated hemoglobin WO 1999022242 A2 Abbott Lab
2000 Determination of % glycated hemoglobin US6162645 A Abbott Laboratories
2001 Measurement of glycated hemoglobin US6174734 B1 Abbott Laboratories
2003 Method for quantitative determination of glycated hemoglobin US 6562581 B2 Portascience
2004 Method for measurement of glycated hemoglobin by a rapid strip test procedure US 6677158 B2 Exocell Inc.
2006 Method for the determination of glycated hemoglobin US 7005273 B2 Therasense, Inc.
2008 Cellular controls for glycated hemoglobin Hb A1c US 7361513 B2 Streck, Inc.
2008 Determination of glycated hemoglobin by fluorescence quenching CA 2102417 C BiBTeX, EndNote, RefMan
2009 Anti-glycated hemoglobin pan-specific monoclonal antibody EP 1414860 B1 Dako Denmark A/S
2009 Methods for the detection of glycated hemoglobin WO 2009067421 A1 Siemens Heathcare Diagnostics, Eddy Chapoteau, Richard Edwards, Chester Swirski, Wolodymyr Zazulak
2011 Method of measuring glycated hemoglobin concentration US 8021887 B2 Arkray, Inc.
2012 Methods for assaying percentage of glycated hemoglobin US 8318501 B2 General Atomics
2012 Device for the determination of glycated hemoglobin US 8206563 B2 Abbott Diabetes Care Inc.
2012 Method of measuring glycated hemoglobin concentration and concentration measuring apparatus US8268625 B2 Arkray, Inc.
2013 Cis di-ahl modified controls for glycated hemoglobin S-A1c derived from healthy blood cells US 8551784 B2 Streck, Inc.
2013 Method for measuring glycated hemoglobin US 8557590 B2 Infopia Co., Ltd.
2013 Methods for assaying percentage of glycated hemoglobin EP 2044444 B1 General Atomics
2013 Method of preparing controls for glycated hemoglobin S-A1c derived from healthy blood cells US 8546144 B2 Streck, Inc.
2014 Low cost electrochemical disposable sensor for measuring glycated hemoglobin US 8702931 B2 Indian Institute Of Science
2014 Methods for the detection of glycated hemoglobin US 8715942 B2 Siemens Healthcare Diagnostics Inc.
2014 Systems and methods for determining the percentage of glycated hemoglobin US 20140186862 A1 Relia Diagnostic Systems, Inc.
2014 Reaction cassette for measuring the concentration of glycated hemoglobin and measuring method thereof US 8846380 B2 Infopia Co., Ltd.


Suggested animal diabetes treatment


a. Diet:

Veterinarian usually recommend the diabetic diet that may able to decrease absorption rate of glucose include good-quality protein, fiber and complex carbohydrate and low fat content. Soluble and fermentable fibers are also considered to be prebiotics that feed the beneficial bacteria, probiotics, assist digestive function, body’s immune defenses and produce beneficial short-chain fatty acids. Supplementary foods are also recommended such as L-Carnitine, Chromium supplements, Zinc, cranberry extract, omega-3 fatty acids EPA and DHA


b. Exercise:

A moderate and consistent exercise may help maintain stable glycemic range and avoid sudden fluctuation of glucose levels of diabetic dogs.


c. Injections:

Insulin injection is a daily routine administration for diabetic dogs that should be executed by vet’s instruction.



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