Carbohydrate metabolism

3.1 Diabetes mellitus

Lothar Thomas

Diabetes mellitus is a chronic illness that requires continuing medical care, ongoing patient self management education and support to prevent acute complications and to reduce the risk of long-term complications. Long-term chronic hyperglycemia includes dysfunction and damage of organs such as retinopathy, nephropathy, peripheral neuropathy, atherosclerotic cardiovasculardisease, peripheral arterial, and cerebrovascular disease /1/.

3.1.1 Classification of diabetes mellitus

Diabetes can be classified into the following general categories /2/:

• Type 1 diabetes: β-cell destruction leads to absolute insulin deficiency
• Type 2 diabetes: progressive loss of adequate β-cell insulin secretion. The background is insulin resistance. Prediabetes screening is recommended
• Specific types of diabetes due to other causes e.g., neonatal diabetes, maturity onset diabetes of the young
• Gestational diabetes mellitus: diabetes diagnosed in the second or third timester of pregnancy.

Refer to Tab. 3.1-1 – Classification of diabetes mellitus.

3.1.2 Epidemiology of diabetes mellitus

According to estimates /3/, the world prevalence of diabetes among adults aged 20–79 is 6.4%. It varies between continents: North America 10.2%, Middle East (EMME) 9.3%, South-East Asia 7.6%, Europe 6.9%, South America 6.6%, and Africa 3.8%. More than 90% have type 2 diabetes. This type, formerly known as adult-onset diabetes, not only affects adults, but increasingly also children and adolescents. The most common risk factors for type 2 are overweight and lack of physical activity. Estimated incidence rates from the United States of type 1 diabetes increased by 1.4% annually and of type 2 diabetes among non Hispanic whites by 0,6% annually in the 2002–2012 period. The incidences increased particularly among youths of minority racial and ethnic groups /4/.

Prediabetes and type 2 diabetes have reached epidemic levels and are associated with major morbidity and mortality. The US Preventive Services Task Force (USPSTF) and the American Diabetes Association (ADA) recommended lowering the starting age for diabetes screening to 35 years to facilitate earlier detection and treatement of diabetes /4/.

Secondary diseases of diabetes include /2/:

• Cardiovascular disease; three thirds will die of it
• Stroke; occurs four times more frequently in diabetics than in the general population
• Dementia; the risk is 3-fold higher, or 11-fold higher in the presence of hypertension
• The incidence of depression and Parkinson’s disease is twice as high in patients with diabetes than in the general population.

3.1.3 Type 1 diabetes (T1D)

This type of diabetes is a continuum characterized by a long subclinical phase with islet autoimmunity and beta-cell loss, detectable months to years before the development of clinical symptoms and dysglycemia /86/. In this stage (stage I) multiple autoantibodies are recognized, e.g. glutamic acid decarboxylase (GAD), islet cell cytoplasmatic (ICA), insulinoma associated-2T tyrosine phosphate (IA-2), insulin (IAA), and zinc transporter-8 (ZnT8).

Type 1 has two subtypes (Tab. 3.1-1 – Classification of diabetes mellitus):

• Type 1A, the immune-mediated form
• Type 1B, the idiopathic form.

Type 1A

The autoimmune form of diabetes (type 1a) is characterized by immune-mediated destruction of β-cells initiated by as-yet-unconfirmed triggering events. Patients present with absolute insulin deficiency and 30–40% with ketoacidosis. There is a preponderance of genes regulating immune response, especially HLA DR3-DQ2 and DR4-DQ8 which account for approximately 50% of hereditary predisposition. However, only about 10% of patients with new-onset diabetes have a first-degree relative with type 1 diabetes mellitus /1/. The rate of β-cell destruction is variable, being rapid in some individuals and slow in others. In some patients, especially children and adolescents, the disease may first manifest as ketoacidosis. Others have modest fasting hyperglycemia which can rapidly progress to severe hyperglycemia with ketoacidosis in the presence of an infection or stress. Still others, in particular adults, may retain residual β-cell function sufficient to prevent ketoacidosis for many years and will not become dependent on insulin until later. At this latter stage of the disease, there is no more detectable insulin or C-peptide secretion. Although type 1A diabetes typically develops in children and adolescents, it can also occur later in life. The incidence is about the same for both sexes /5/.

Markers of the immune-mediated destruction of β-cells include the following islet cell antigen autoantibodies: anti-insulin, anti-glutamic acid decarboxylase (GAD65), anti-tyrosine phosphatase IA-2 and IA-2 β. At least one but usually several of these antibodies are present in 85–90% of patients when fasting hyperglycemia is initially detected.

Type 1A has a strong HLA association and shows familial aggregation. It is also associated with other autoimmune diseases such as Graves’ disease, Hashimoto’s thyroiditis, Addison’s disease, celiac disease, autoimmune hepatitis, myasthenia gravis, pernicious anemia or vitiligo in the patient him/herself or a close family member.

Type 1A is a multifactorial and polygenic disease. Polygenic means that multiple genetic mutations are required for the disease to manifest. Factors of the immune-mediated destruction of β-cells are /6/:

• Genetic predisposition for this disease
• Environmental factors that trigger the immunological process
• Expression and activation of target antigens to support the immune process.

With continuous glucose monitoring identifying individuals in the first stages of type 1A who are at risk of rapidly progressing to clinical diabetes, the potential health economic benefits of type 1A prevention and guiding the timing of initiation of insulin therapy the authors advocate for an increasing role of continuous glucose monitoring in presymptomatic type 1A /86/.

Type 1B

This type of diabetes has no known etiology. Multiple forms of β-cell dysfunction are associated with this type of diabetes. Only a small portion of type 1 diabetics fall into this category. Type 1B lacks evidence of an autoimmune process and has no HLA association, but it is strongly inherited. Most patients are of African or Asian ancestry. They suffer from episodic ketoacidosis and exhibit varying degrees of insulin deficiency between episodes. Type 1B belongs to the ketosis-prone diabetes group /5/.

3.1.3.1 Diagnostic criteria for type 1A diabetes /2/

Stage 1

• Multiple autoantibodies
• No impaired fasting glucose (IFG)
• No impaired glucose tolerance (IGT).

Stage 2

• Multiple autoantibodies
• Fasting plasma glucose (FPG) 100–125 mg/dL (5.5–6.9 mmol/L) and/or
• Two hour plasma glucose concentration 140–199 mg/dL (7.8–11.1 mmol/L) during an oral glucose tolerance test
• HbA_1C 5.7–6.4% (39–47 mmol/mol) or ≥ 10% increase

Stage 3

• Multiple autoantibodies or autoantibodies may become absent
• FPG ≥ 126 mg/dL (7.0 mmol/L)
• Two hour plasma glucose ≥ 200 mg/dL (11.1 mmol/L) during an oral glucose tolerance test
• Random glucose ≥ 200 mg/dL (11.1 mmol/L), with symptoms of polyuria and weight loss
• HbA_1C ≥ 6.5% (48 mmol/mol).

3.1.3.1.1 Recommended target levels for children with type 1 diabetes mellitus /7/

• Fasting, including bed time, nocturnal and preprandial concentrations: 70–126 mg/dL (3.9–7.0 mmol/L)
• Postprandial peak: 180 mg/dL (10.0 mmol/l)
• Time in Range, > 70% of a 24-hour day* 70–180 mg/dl (3.9–10.0 mmol/L) (* based on continuous glucose monitoring)
• HbA_1C < 7.0% (53 mmol/mol)

3.1.4 Categories with increased risk for type 2 diabetes (prediabetes)

Prediabetes is the term used for individuals whose glucose concentrations do not meet the criteria for diabetes yet have abnormal carbohydrate metabolism. Prediabetes is a condition of impaired glucose metabolism rather than an intermediate stage or risk factor for predicting /2, 8/:

• The development of type 2 diabetes (increased risk for diabetes)
• The increased risk of cardiovascular or microvascular disease.

Testing of prediabetes should be considered /2/:

• in adults with overweight or obesity (Body mass index ≥ 25 kg/m²)
• first degree relatives with diabetes
• high risk race/ethnicity
• history of cardiovascular disease
• hypertension (> 140/90 mmHg) or on therapy for hypertension
• HDL cholesterol concentration < 35 mg/dL (0.90 mmol/L) and/or a triglyceride concentration > 250 mg/dL (2.82 mmol/L)
• women with polycystic ovary syndrome
• physical inactivity
• other clinical conditions associated with insulin resistance
• patients with prediabetes (HbA_1c ≥ 5.7%; 39 mmol/mol)
• impaired fasting glucose or impaired oral glucose tolerance should be tested yearly.

3.1.5 Type 2 diabetes (T2D)

In patients with T2D the paths to β-cell demise and dysfunction are less well defined, but deficient β-cell insulin secretion, frequently in the setting of insulin resistance, appears to be the common denominator /2/. T2D is characterized by chronic hyperglycemia and often remains undiagnosed for years, because glycemia increases gradually and does not cause any symptoms in the early stages. These patients are nevertheless at increased risk of developing microvascular and macrovascular complications. T2D develops as follows (Fig. 3.1-4 – Development of type 2 diabetes in three stages):

• it starts with a genetic predisposition
• as insulin resistance develops, glucose tolerance becomes impaired, a condition called prediabetes
• subsequent to insulin resistance there is reduced β-cell function, resulting in the development of T2D
• Prediabetes and type 2 diabetes meet criteria for conditions in which early detection via screening is appropriate. Age is a major risk factor for type 2 diabetes.

3.1.5.1 Long-term complications in youth-onset T2D

In children 10 to 17 years with obesity in the period from 2002 to 2012 the incidence of type 2 diabetes increased by 4.8% each year. Type 2 diabetes was defined according to the American Diabetes Association 2002 criteria, a fasting C-peptide concentration of > 0.6 μg/L, and a negative test for pancreas antibodies. A prospective longitudinal study /10/ indicated increased risk of diabetes related complications. Microvascular complications increased steadily over time and affected most participants by the time of young adulthood. Complications were more common among participants of minority race and ethnic group and among those with hyperglycemia, hypertension, and dyslipidemia.

3.1.5.2 Screening for type 2 diabetes

Testing of diabetes type 2 should be considered according to the criteria of prediabetes /2/:

• Fasting plasma glucose (FPG): ≥ 126 mg/dL (7.0 mmol/L)
• 2h plasma glucose during 75 g oGTT: ≥ 200 mg/dL (11.1 mmol/L)
• HbA_1c: ≥ 6.5% (48 mmol/mol)
• Random plasma glucose: ≥ 200 mg/dL (11.1 mmol/L).

3.1.5.3 Testing for type 2 diabetes in asymptomatic children

Overweight (BMI > 85th percentile for age and sex, weight for height > 85th percentile or weight > 120% of ideal for height). Plus any two of the following risk factors /3/:

• Family history of type 2 diabetes in first-degree or second-degree relative
• Race and ethnicity (Native American, African American, Latino, Asian American, Pacific Islander)
• Signs of insulin resistance or conditions associated with insulin resistance (acanthosis nigricans, hypertension, dyslipidemia, polycystic ovary syndrome, or birth weight small for gestational age)
• Maternal history of diabetes or gestational diabetes during the child’s gestation
• Age of initiation: 10 years at onset of puberty, if puberty occurs at younger age.

Frequency of investigation: every 3 years.

3.1.6 Gestational diabetes mellitus (GDM)

GDM is initially diagnosed during pregnancy at 24–28 weeks of gestation and is clinically not overt diabetes mellitus. The condition is associated with complications for the mother, the fetus and the newborn. Refer also to:

• Section 38.1.5
• Tab. 38.1.7 – Effects of lower versus higher glycemic criteria in pregnancy.

The medical decision-making approach is as follows /9/:

• During the first prenatal visit, a screening test for diabetes mellitus is performed. Pregnant women with the criteria (Tab. 3.1-6 – Testing for diabetes in pregnant women) have overt diabetes and will receive treatment /11/.
• If the test results are not diagnostic of diabetes, a 75-g oGTT is performed at 24–28 weeks of gestation and evaluated according to the criteria in Tab. 3.1-6. A pathologic oGTT indicates GDM and the pregnant women will receive treatment.

Women with a history of GDM have a greatly increased subsequent risk for diabetes and should be followed up, to be reclassified into one of the following categories /9/:

• Normoglycemia
• Elevated fasting glucose
• Impaired glucose tolerance
• Diabetes mellitus.

According to studies in the U.S., diabetes complicates an average 7% of pregnancies, with prevalence varying between 1% and 14% depending on the population studied and the diabetes criteria selected. The risk is higher in pregnant women over 25 years and even higher in those with a positive family history. About 88% of pregnant women with hyperglycemia have GDM, the rest have overt diabetes, thereof 35% type 1 and 65% type 2 /12/. GDM patients with risk factors (positive family history, T2D, body mass index above 27 kg/m², over 35 years of age, GDM, preeclampsia, malformation, macrosomia or intrauterine fetal death in the previous pregnancy, hypertension or macrosomia in the current pregnancy) are at significantly higher risk of intrauterine fetal death, premature delivery, or elective cesarian section.

3.1.6.1 Diagnostic criteria for gestational diabetes (GDM)

GDM can be accomplished with either of two strategies:

• one step strategy using the 75 g OGTT (patient starts fasting in the morning); the 2 h plasma glucose should not exceed 153 mg/dl (8.5 mmol/L)
• two step strategy using the 100 g OGTT (the patient starts fasting). The diagnosis GDM is made when at least two of the following four plasma glucose concentrations are met or exceeded: fasting 95 mg/dL (5.3 mmol/L), 1 h 180 mg/dL (10.0 mmol/L), 2 h 155 mg/dL (8.6 mmol/L), 3 h 140 mg/dL (7.8 mmol/L).

3.1.7 Other specific types of diabetes

3.1.7.1 Maturity-onset diabetes of the young (MODY)

In addition to TD1 and T2D, MODY comprises a small group of non-insulin-dependent diabetics, in whom the disease manifests in childhood and young adulthood. Patients have highly penetrating autosomal dominant mutations in a single gene (monogenic diabetes) which lead to dysfunction of the β-cells /13, 14/. There are different phenotypes with different characteristics such as age of occurrence, severity of hyperglycemia, response to treatment, secondary diseases, and extra pancreatic diseases. The prevalence of MODY is estimated to be 0.6–2% of all diabetes cases, although in reality it is probably higher since MODY is often incorrectly classified as TD1 or TD2. Heterozygous mutations in the genes GCK and HNF1A/4A account for up to 80% of MODY cases. GCK encodes the intracellular enzyme glucokinase, which acts as a glucose sensor in the pancreatic β-cells. Mutations in GCK lead to mild, often asymptomatic hyperglycemia whereas mutations in the genes encoding the transcription factors hepatocyte nuclear factor-1α and -4α cause progressive insulin deficiency with hyperglycemia which can lead to vascular complications. The less common type of MODY, which results from mutations of the transcription factor gene HNF1B, is associated with extra pancreatic manifestations such as cystic kidney disease. The different types of MODY are listed in:

• Tab. 3.1-3 – Postabsorptive and posprandial glucose in non diabetics and diabetics
• Tab. 3.1-4 – Types of MODY and their clinical significance.

3.1.7.2 Differential diagnosis of T1D, T2D and MODY

Differentiation of MODY from T1D: MODY develops slowly, with mild hyperglycemia and an increase in insulin levels in response to glucose load, without progression to ketoacidosis and negative pancreatic autoantibodies. Type 1 manifests in children and adolescents of lean habitus and usually occurs acutely with ketoacidosis.

A differential diagnosis is shown in:

• Fig. 3.1-6 – Differential diagnosis of MODY, type 1 and type 2 diabetes, but it is not always straightforward
• Fig. 3.1-5 – Important organs and tissues involved in glucose metabolism.

Differentiation of MODY from T2D: T2D often manifests at a later age (over 40 years) than MODY and there are co-morbidities or overweight, which is less frequently the case in MODY. MODY is an autosomal dominant disease which is inherited through 3 generations. Patients with MODY are generally lean rather than overweight.

3.1.7.3 Latent autoimmune diabetes in adults (LADA)

The pathophysiology of LADA is far less understood than that of T1D and T2D. However, its clinical manifestation contains characteristics of both these types of diabetes. LADA thus presents as follows:

• Clinically: as T2D, with onset in adulthood and without insulin dependency at diagnosis
• Diagnostically: like T1D due to positive result for islet cell antibodies
• Genetically: through association with the gene locus TCF7L2 as well as MHC /15/.

8–10% of diagnosed T2D cases are likely to be LADA. The definition of LADA as a separate subgroup is based on the understanding that this type of diabetes, which presents in adulthood and with autoantibodies, is non-insulin-dependent at diagnosis and its progression to insulin dependency is slower than in T1D. The “adult age of onset” varies between 25 and 40 years.

The presence of glutamic acid decarboxylase antibodies (anti-GAD65) and islet cell antibodies (ICA) indicates a lack of insulin and/or relative need for insulin.

Studies show:

• in newly diagnosed patients with GAD65 antibodies, insulin dependency develops within 10 years in 50% of cases, whereas in patients without GAD65 antibodies it only develops in 3% of cases
• in the UK Prospective Diabetes Study, 52% of GADA-positive patients became insulin dependent within 6 years
• in a Finnish study, T2D patients with GAD65 antibodies had higher C-peptide levels and more symptoms of the metabolic syndrome than T1D patients with GAD65 antibodies.

3.18 Other types of diabetes with known cause

3.18.1 Disease of the exocrine pancreas

Any process that injures the pancreas and leads to a reduction in β-cell mass can cause diabetes. This applies to acquired processes such as pancreatitis, trauma, infection, pancreatectomy and pancreatic carcinoma. In pancreatic cancer (adenocarcinoma) other processes appear to play a role, because processes that involve only a small part of the organ are often already associated with diabetes /1/. The prevalence of diabetes in chronic pancreatitis is 30–70% /16/. It depends on the time of investigation, as shown in a study /17/ which reports an increase in incidence from 8% to 78% during a 10-year period of disease. The loss of exocrine and endocrine function does not occur at the same time. Patients with severe pancreatitis requiring enzyme substitution may have normal glucose tolerance.

3.1.8.2 Hereditary hemochromatosis

The prevalence of diabetes is 50–60%. The severity depends on the degree of injury caused to the liver and pancreas. The decreased extraction of insulin from portal blood with consecutively reduced insulin clearance leads to the development of chronic hyperinsulinism. This causes a reduction of the tissue receptor sensitivity, leading to insulin resistance. As iron deposition in the Langerhans islets increases, β-cells are destroyed, resulting in insulin deficiency /18/.

3.1.8.3 Endocrinopathy and diabetes

Hormones that antagonize insulin action can cause diabetes if they occur in elevated concentrations. This is the case, for example, with

• Acromegaly (excess growth hormone)
• Cushing’s syndrome (hypercortisolism)
• Pheochromocytoma (catecholamine excess).

Hypercortisolism-induced and growth hormone-induced hypokalemia can cause diabetes by inhibiting insulin secretion. Hyperglycemia generally resolves after successful treatment of the underlying disease.

3.1.8.4 Drug- or chemical-induced diabetes

Drugs and chemicals that have a toxic effect can cause direct injury to the β-cells and permanently destroy them. This is the case with the rat poison vacor or intravenous administration of pentamidine. More commonly, however, medications cause diabetes in individuals with existing insulin resistance by impairing insulin action. Examples are glucocorticoids, nicotinic acid, and interferon-α /2/.

3.1.8.5 Infection-induced diabetes

Generalized viral infections can sometimes cause sufficient inflammatory injury to the pancreas to lead to the development of diabetes. The infections can be caused by Coxsackievirus B, Adenovirus, Cytomegalovirus, and the Mumps virus. Congenital Rubella virus infection, in contrast, causes type 1A diabetes /2/.

3.1.8.6 Other genetic syndromes sometimes associated with diabetes

Many genetic syndromes can be associated with diabetes mellitus. Examples are listed in Tab. 3.1-1 – Classification of diabetes mellitus /2/.

Comments, criteria and laboratory findings for these types of diabetes are summarized in Tab. 3.1-5 – Laboratory findings in other types of diabetes.

3.1.9 Glycemic control of diabetes

The diagnostic symptoms of diabetes are persistent hyperglycemia and an elevated HbA_1c. Blood glucose must be lowered to near-normal levels to prevent the following complications /1/:

• impaired vision, polyuria, polydipsia, fatigue, weight loss with polyphagia, vaginitis, or balanitis
• risk of hypoglycemia, acute hyperglycemia in the form of diabetic ketoacidosis, hyperglycemic hyper osmolar non ketotic syndrome. Hyperglycemia is associated with increased morbidity and mortality.
• risk of cardiovascular disease, diabetic retinopathy, nephropathy, and neuropathy
• a lipid profile that is not associated with increased atherogenic risk.

The targets and concepts for the control of glycemia, lipidemia and blood pressure are shown in Tab. 3.1-9 – Glycemic goals in the treatment of diabetes.

3.1.9.1 Management of glycemic control

Self-monitoring of blood glucose and measurement of HbA_1c are important components in the glycemic control of diabetics.

Self-monitoring of glucose (SMBG): SMBG allows patients to monitor their individual response to treatment, to check whether glycemic goals are reached, and to prevent hypoglycemia under therapy. Recommendations for SMBG are as follows /1/:

• TD1, TD2 and GDM on multiple-dose insulin: 3–4 times daily prior to meals
• Patients who inject insulin only occasionally, take oral anti diabetics or control their diabetes through diet: occasionally for monitoring, mainly after meals.

Continuous glucose monitoring (CGM): an international panel of physicians, researchers, and individuals with diabetes recommended CGM as a robust research tool, and continuous glucose data should be recognized by governing bodies as a valuable and meaningful end point to be used in clinical trials of new drugs and devices for diabetes treatment /19/.

Determination of HbA_1c: the purpose of the measurement is to estimate the mean glucose level of the past 2–3 months. The HbA1c measurement at the beginning of treatment serves as the baseline value of glycemia. Testing at least twice per year is a criterion of whether metabolic control of diabetes has been achieved and whether the level is within the target range. The individual target of a diabetic should be as close as possible to the upper reference limit of 6% without provoking frequent hyperglycemic episodes. The relationship between plasma glucose levels averaged over a 2–3 month period and HbA_1c is shown in Tab. 3.1-10 – Correlation of plasma glucose with the HbA_1c level.

HbA_1c monitoring /1/:

• At least two times a year in patients who are meeting treatment goals (and who have stable glycemic control)
• Quarterly in patients whose therapy has changed or who are not meeting glycemic goals.

Gliflozins in the management of T2D

The reabsorption of glucose from the glomerular filtrate is an active process linked to Na⁺ and requires a carrier protein, referred to as sodium-glucose cotransporter (SGLT) /84/. The SGLT2 is localized in the epithelial cells of the proximal renal tubule, where it is responsible for 65% of the Na⁺ reabsorption and > 90% of glucose reabsorption.The SGLTs are coupled to accessory protein MAP17, which is required for glucose transport, and are encoded by genes in the SLC5A family. Orally absorbed SGLT2 inhibitors, also referred as gliflozins, reduce glycated hemoglobin levels by approximately 0.5 to 1.1% in T2D and do not cause hypoglycemia.

3.1.9.2 Benefits of good glycemic control

The Diabetes Control and Complication Trial (DCCT) demonstrated that a regimen of intensive therapy aimed at maintaining near-normal glycemic control reduces the risk of development or progression of retinopathy, nephropathy and neuropathy in T1D by 50–70% /1/. This regimen achieved a median HbA_1c of 7.2% compared with conventional therapy with a median HbA_1c of 9%. The DCCT reference interval for HbA_1c was 4–6%.

For T2D, the United Kingdom Prospective Diabetes Study (UKPDS) demonstrated that improved glycemic control reduces the risk of developing retinopathy, nephropathy and possibly neuropathy. With tighter glycemic control, the overall microvascular complication rate was decreased by 25% compared with conventional treatment. Epidemiological analysis showed a continuous relationship between hyperglycemia and the rate of microvascular complications, such that for every percentage point decrease in HbA1c (e.g., 9% to 8%), there was a 35% reduction in the risk of microvascular complications.

3.1.10 Acute complications of diabetes

Diabetes is associated with acute and chronic complications. In addition, metabolic stress responses can be expected, which occur with acute diseases.

The main acute complications of diabetes are hypoglycemia, metabolic stress response, diabetic ketoacidosis, and hyperglycemic hyper osmolar non ketotic syndrome.

3.1.10.1 Iatrogenic hypoglycemia

In healthy individuals, regulating mechanisms that increase glucose levels are activated when blood glucose falls below 70 mg/dL (3.9 mmol/L). They prevent hypoglycemia and insufficient supply of the brain with high-energy substrate. In people with diabetes therapeutic insulin excess caused by treatment with insulin, sulfonylureas and glinides can initiate hypoglycemic episodes /20/.

3.1.10.2 Defective glucose counter regulation

Typically hypoglycemia occurs during less marked or even relative therapeutic insulin excess in patients with diminished exogenous glucose supply, decreased endogenous glucose production, increased glucose consumption or increased insulin sensitivity. Such patients, those with T1D or advanced T2D have β-cell failure or absolute insulin deficiency and compromised defenses against hypoglycemia. As glucose levels fall, the compromised physiologic defenses include failure of insulin levels to fall, failure of glucagon secretion to increase and attenuated epinephrine secretion. This combination of compromised physiologic defenses cause the syndrome of defective glucose counter regulation with increased risk of recurrent hypoglycemia. An important finding is that prior hypoglycemia weakens the body’s defense against subsequent hypoglycemia in type 1 diabetics and non diabetics. This led to the concept of hypoglycemia associated autonomic failure (HAAF). According to this concept recent hypoglycemia causes both defective counter regulation and hypoglycemia unawareness.

3.1.10.3 Rate of hypoglycemia

The rate of hypoglycemia is about 10 times higher in T1D than in T2D. The rate of severe hypoglycemia (requiring the assistance of another person) is 62–170 episodes per 100 years in T1D and 3–73 in T2D. While in T1D glucose counter regulation is impaired at an early stage, in T2D it will become compromised only in absolute insulin deficiency. The risk of iatrogenic hypoglycemia is similar to that in T1D. Children under 6–7 years of age usually have hypoglycemia unawareness. The ADA recommends treatment with glucose or carbohydrate-containing foods if glucose levels are below 70 mg/dL (3.9 mmol/L).

Nocturnal hypoglycemia is a significant problem. In the Diabetes Control and Complication Trial, more than half of severe hypoglycemic events in T1D occurred during sleep. They were caused by the glucose-lowering effects of evening exercise, sleep-induced defects in counter regulatory hormone responses to hypoglycemia, and missed bedtime snacks. A 12-month study /21/ of children and adults with T1D showed the following findings:

• 7.4% of participants had hypoglycemic events during 8.5% of nights
• The duration of hypoglycemia was longer than 2 h in 23% of nights with hypoglycemia
• Hypoglycemia was defined as two consecutive glucose readings ≤ 60 mg/dL (3.3 mmol/L) in 20 minutes.

3.1.10.4 Metabolic stress response

Surgery or other stressful events can induce a metabolic stress response in critically ill patients, which is caused by the increase of insulin counter regulatory hormones such as noradrenaline, glucagon, cortisol and growth hormone. These hormones have a catabolic effect and increase gluconeogenesis and lipolysis, leading to elevated levels of glucose, free fatty acids and ketone bodies in blood. The increase in these substrates impairs the insulin secretory response of the islet cells. Combined with acidosis, which may develop due to the increased levels of lactate and ketone bodies, this will result in lower insulin sensitivity and increasing insulin resistance of the tissues, thereby worsening the metabolic situation.

To differentiate diabetes from stress-induced hyperglycemia, glucose and HbA_1c concentrations should be determined. An elevated HbA_1c level in the presence of hyperglycemia is indicative of pre-existing diabetes while a normal HbA_1c level generally precludes it.

More than 90% of critically ill patients have hyperglycemia (glucose > 126 mg/dL; 7.0 mmol/L) due to a stress response elicited by surgery or other events.

For adults, the NICE-Sugar Study /22/ demonstrated that tight insulin-based glycemic control with a target blood glucose range of 81–108 mg/dL (4.5–6.0 mmol/L) is associated with higher mortality than glycemic control with a target of ≤ 180 mg/dL (10.0 mmol/L).

For children aged 0–3 years who have undergone heart surgery, insulin-based glycemic control with a target blood glucose range of 81–108 mg/dL (4.5–6.0 mmol/L) showed no benefit compared to higher levels. The length of hospitalization, the rate of infections and mortality are the same, but the rate of hypoglycemic events was significantly higher.

3.1.10.5 Diabetic ketoacidosis (DKA)

DKA is defined as the occurrence of metabolic acidosis with ketonemia. The main symptoms are ketonemia/ketonuria, metabolic acidosis and dehydration. In 35–40% of children, DKA is detected at diagnosis of T1D. While blood glucose concentrations in individuals with DKA are usually in the range of 400–500 mg/dL (22.2–27.8 mmol/L), it has been shown that some individuals may have levels under 300 mg/dL (16.7 mmol/L), above 800 mg/dL (44.4 mmol/L), or even within the reference interval /23/. The latter may be the case when there is severe dehydration with a reduced glomerular filtration rate. Hyperglycemia causes dilutional hyponatremia. For every 100 mg/dL (5.6 mmol/L) increase in glucose, the sodium concentration decreases by 1.6 mmol/L /23/. Further information on DKA can be found in Section 5.5 – Ketone bodies.

3.1.10.6 Hyperglycemic hyperosmolar non ketotic syndrome (HHNS)

HHNS is an acute, life-threatening complication of diabetes. It is caused by a relative or absolute insulin deficiency and elevated levels of insulin counter regulatory hormones such as glucagon, catecholamines, growth hormone, and cortisol. Although HHNS may occur in both T1D and T2D, DKA is generally associated with T1D, and HHNS with T2D /24/. Patients are often unaware of having diabetes. HHNS mainly affects individuals over the age of 55. Blood glucose is usually above 600 mg/dL (33.3 mmol/L) and serum osmolality is elevated above 330 mmoL/kg. In recent years, HHNS has been increasingly diagnosed in children with T2D. In these cases, T2D initially manifests with symptoms and findings of HHNS. It mainly affects overweight children aged 10 years and older of African-American descent /25/. Further information on HHNS can be found in Section 5.5 – Ketone bodies.

3.1.11 Chronic complications of diabetes

Pathophysiological mechanisms associated with diabetes comprise endothelial vascular dysfunction, low-grade inflammation, and thrombocyte dysfunction.

There are three main categories of chronic diabetes complications:

• microvascular complications in type2 diabetes, in particular retinopathy and nephropathy.
• macrovascular complications in type 1 diabetes, in particular cardiovascular disease, cerebrovascular disease, peripheral vascular diseases, and neuropathies of both the peripheral and the autonomic nervous system.

The overwhelming body of evidence on glycemic control in end stage renal disease has been obtained using HbA_1c /26/.

3.1.11.1 Microvascular disease in TD2

Even though diabetics usually die from macrovascular complications, microvascular complications such as retinopathy and nephropathy play an important role because they significantly restrict the quality of life. Microvascular disease develop as a result of poor metabolic control.

Early changes include hyperglycemia-induced vascular dilatation with increased blood flow, as well as increased intravascular pressure in the capillaries of the retina and the renal glomeruli. As a result, there is increased leakage of proteins from the capillaries, measurable by the presence of micro albuminuria.

Long-term hyperglycemia-induced changes include altered structuring of the extracellular matrix, in particular the basal membrane of the vessels. The toxic effect of glucose is caused by various mechanisms /21/:

• The direct i.e., not enzyme-mediated, reaction of glucose with proteins. This reaction, also known as non-enzymatic glycosylation, leads to increased glycation, in particular of long-life proteins such as collagen.
• The glycation of proteins is the starting point for the gradual formation of advanced glycation end products (AGEs), which are responsible for the vascular damage that is caused when the AGEs bind to the AGE receptors in the vascular cells, activating the synthesis of inflammatory cytokines (also refer to Section 3.6 – Hemoglobin A_1c).

Glycemic control /1/: strict glycemic control plays an important role in retarding the development and progression of microvascular complications. The Epidemiology of Diabetes Interventions and Complications (EDIC) study showed that tight glycemic control significantly reduced the progression of retinopathy and nephropathy in patients with T1D. This was also the case in the UK Prospective Diabetes Study (UKPDS) of patients with T2D and in the Veterans Affairs Diabetes Trial (VADT), in which glycemic control with a mean HbA_1c of 6.9% resulted in a significant reduction in micro albuminuria. The Action in Diabetes and Vascular Disease (ADVANCE) study similarly showed a significant reduction in albuminuria in patients with T2D when the mean HbA_1c was reduced from the general goal of 7.0% to the stringent goal of 6.3%.

3.1.11.2 Diabetic retinopathy

Diabetic retinopathy is a specific microvascular complication of diabetes. In the industrialized countries it is the leading cause of blindness among adults aged 20–65 /27/. The risk of developing diabetic retinopathy after 20 years of diabetes is nearly 100% in T1D and over 60% in T2D. Diabetic retinopathy progresses /28/:

• From a mild non-proliferative form, characterized by increased vascular permeability
• To moderate and severe non-proliferative retinopathy, characterized by vascular closure
• To proliferative retinopathy, characterized by the growth of new blood vessels on the retina and posterior surface of the vitreous.

A significant factor contributing to the pathophysiology of the disease is the activation of angiotensin II in the retina where it mediates vascular growth and accelerates the development of proliferative retinopathy. The permeability of the retinal capillaries for proteins is increased, thereby promoting the development of macular edema.

The DCCT showed that stringent glycemic control can reduce or prevent the development of diabetic retinopathy by 27% and the progression of it in 34–76% of cases. This improvement was achieved with an average 10% reduction in HbA1c from 8% to 7.2% (upper reference limit 6.5%). The UKPDS showed that for every percentage point decrease in HbA1c there was a 35% reduction in the risk of microvascular complications /28/.

3.1.11.3 Diabetic nephropathy

About 20–30% of patients with T1D develop diabetic nephropathy about 20 years following diagnosis of the disease. Diabetic kidney disease occurs in about 40% of patients with T2D. Approximately 60% of patients with end stage renal disease (ESRD) belong to this type. African Americans and Hispanics with T2D progress to dialysis earlier than non-Hispanic Caucasians. The number of diabetics requiring dialysis is on the rise, since the prevalence of T2D is increasing and these patients increasingly live longer. However, at the stage of chronic renal failure, only 20% of patients have a life expectancy of more than 5 years /29/.

In addition to hyperglycemia, the increased production of angiotensin II also plays an important role in diabetic microangiopathy. It causes contraction of the efferent arterioles in the kidney, which increases the filtration pressure in the glomeruli, resulting in increased excretion of albumin. Angiotensin II also increases systemic blood pressure and induces endothelial dysfunction and glomerular injury.

The earliest evidence of nephropathy is albuminuria. Normoalbuminuria is defined as an excretion rate of < 30 mg/24 h.

Diabetic nephropathy develops in the following stages:

• persistent albuminuria with an excretion rate of 30–299 mg/24 h. This is the stage of incipient diabetic nephropathy. It is usually accompanied by glomerular hyperfiltration and early hypertension. Albuminuria is classified as persistent if it is detectable in at least two out of three urine samples within 6 months.
• persistent albuminuria with an excretion rate of ≥ 300 mg/24 h. This is the stage of early overt diabetic nephropathy. Albuminuria ≥ 300 mg/24 h is also referred to as clinical albuminuria. Hypertension is also present. In T1D this stage develops untreated over a period of 10–15 years following diagnosis of persistent albuminuria, with albuminuria increasing at a rate of 10–20% per year. Only 20–40% of T2D patients progress to this stage.
• advanced diabetic nephropathy. This stage is characterized by the progressive increase of proteinuria and hypertension. The glomerular filtration rate (GFR) gradually falls over a period of several years at a rate that shows yearly interindividual variability of 2–20 [mL × min^–1 × (1.73 m²)^–1]. The decreasing GFR can be detected early by determining the creatinine or cystatin C based estimated GFR.
• end stage renal disease (ESRD). Without treatment, 50% of type 1 diabetics with clinical nephropathy will progress to ESRD within 10 years, and over 75% within 20 years, whereas in T2D, only 20% of diabetics with clinical nephropathy will develop ESRD within 20 years.

It must be noted that transient albuminuria can also be caused by exercise, urinary tract infections, short-term hyperglycemia, marked hypertension, heart failure, and acute febrile illness.

Persistent albuminuria is not only the earliest indicator of diabetic nephropathy, but also a marker of elevated cardiovascular morbidity and mortality of diabetics.

Early therapeutic intervention in diabetics can delay the development of renal complications and reduce the progression of the disease. The UKPDS, ADVANCE, and STENO-2 studies have shown that stringent blood glucose and blood pressure control reduce the incidence and progression of diabetic nephropathy. In patients with T2D, inhibition of the renin-angiotensin-aldosterone system by angiotensin-converting enzyme (ACE) inhibitors or an ACE receptor blocker delayed the occurrence of albuminuria and retarded the progression to ESRD. The use of ACE inhibitors and ACE receptor blockers is therefore part of the standard treatment of patients with T2D. Also refer to Section 12 – Kidney and urinary tract.

End stage renal disease

The overwhelming body of evidence on glycemic control in end stage renal disease has been obtained using HbA_1c /26/.

3.1.12 Macrovascular and other diseases

Macrovascular diseases in patients with T1D and T2D include cardiovascular disease, cerebrovascular and peripheral vascular diseases. If diabetes is diagnosed, life expectancy is reduced by 30%, with cardiovascular disease being the leading cause of death. In Sweden from 1998 through 2014, mortality and the incidence of cardiovascular outcomes declined. Patients with T1D had roughly 40% greater reduction in cardiovascular outcomes than controls and patients with T2D had roughly 20% greater reductions than controls. Reductions in fatal outcomes were similar in patients with diabetes and controls /30/.

3.1.12.1 Cardiovascular disease (CVD)

The reduction in life expectancy in diabetics is mainly due to cardiovascular events. The risk for CVD is 2–3 times higher in male diabetics and even 3–5 times higher in female diabetics than in non diabetics of the same age. An increased risk for CVD exists even before T2D is diagnosed. 40% of patients with newly diagnosed T2D have CVD, and 80% of patients with CVD have T2D or prediabetes. More than 60% of patients with T2D will die of CVD /1, 31/. Interventions such as change of lifestyle, control of blood pressure, reduction of lipids, and treatment with anti-platelet agents can reduce the progression and complications of T2D. Results of studies investigating the effect of strict glycemic control have been unsatisfactory, but data from the UKPDS have shown that it has a protective effect /33, 34/. In the ADVANCE, VADT and Action to Control Cardiovascular Disease in Diabetes (ACCORD) studies, however, strict glycemic control was associated with increased mortality and an increased risk for CVD.

Myocardial infarction (MI)

Diabetics with AMI and a glucose levels above the range of 124–180 mg/dL (6.9–10.0 mmol/L) at admission are at nearly twice the risk for congestive heart failure, cardiogenic shock or in hospital death compared to non diabetics /34/. If a patient with AMI has a blood glucose level > 180 mg/dL (10.0 mmol/L) at admission, this is usually not due to stress-induced hyperglycemia, but undiagnosed diabetes /35/. It is important that hyperglycemia in patients admitted with AMI is normalized by appropriate treatment. The Diabetes Insulin Glucose in Acute Myocardial Infarction (DIGAMI) study has shown that immediate reduction of blood glucose levels to below 198 mg/dL (11.0 mmol/L) by insulin treatment reduces mortality by about 30% /35/.

Coronary bypass surgery

According to data from the National Cardiac Surgery Database (NCSD), diabetics undergoing bypass surgery have an approx. 50% higher mortality rate than non diabetics /22/.

3.1.12.2 Neuropathies and cerebrovascular disease

Neurological complications of diabetes include neuropathy, stroke and Alzheimer’s disease.

Neuropathy

The diabetic neuropathies are heterogeneous with diverse clinical manifestations. There are two main types of neuropathy /11, 36/:

• Distal symmetric polyneuropathy (DPN). There is progressive loss of myelinated nerve fibers, and segmental demyelination develops. Early detection is important since stringent diabetic control retards the progression. Moreover, DPN is asymptomatic in up to 50% of cases. There may be painless numbness starting from the toes and feet, and impaired perception of pain and temperature with the risk of painless ulcers developing. Data from the DCCT show that strict diabetic control can reduce the development and progression of clinical neuropathies by 64% in T1D and by 42% in T2D /36, 37/.
• Autonomic neuropathy. It can affect any organ and system in the body. Clinical symptoms include tachycardia at rest, exercise intolerance, orthostatic hypotension, constipation, gastroparesis, erectile dysfunction, and urination problems. Autonomic neuropathy is also a risk factor for CVD.

Stroke

According to epidemiological studies, the prevalence of thromboembolic, not hemorrhagic, stroke is 2–6 times higher in diabetics than in non diabetics. In the Framingham study, the incidence of stroke was 3.6 times higher in diabetic women and 2.5 times higher in diabetic men than in non diabetics of the same age group /38/.

The following relationships exist between blood glucose levels and stroke /38/:

• Hypoglycemia worsens the prognosis for stroke. In the presence of global ischemia, glucose concentrations below 65 mg/dL (3.6 mmol/L) lead to higher mortality and a worse functional outcome /37, 38/.
• Acute hyperglycemia increases mortality and worsens the prognosis for cerebral hemorrhages.

Alzheimer’s disease

Patients with T2D have a 2–2.5-fold higher risk of developing Alzheimer’s disease and vascular dementia than non- diabetics of the same age group. The cause is reported to be microvascular infarctions in subcortical structures /39/.

3.1.12.3 Arterial thrombosis

In diabetes there is an imbalance between procoagulants and the factors of fibrinolysis, leading to the deposition of fibrin clots along the vessel wall. This is thought to induce the formation of arterial thrombi even before the endothelium is compromised and structural damage is caused to the intima. The formation of fibrin clots activates the release of mitogens and mediators of inflammation which then cause alterations in the intima /40/. Therefore, as an example, the prognosis for stroke patients with prediabetes and T2D is worse in comparison to patients with normoglycemia, and T2D is reported to double the risk of stroke /41/. The risk of mortality also is reported to be twice as high if venous plasma glucose levels are > 144 mg/dL (8.0 mmol/L) at admission, irrespective of age, type and severity of the stroke /41/.

Stroke

According to epidemiological studies, the prevalence of thromboembolic, not hemorrhagic, stroke is 2–6 times higher in diabetics than in non diabetics. In the Framingham study, the incidence of stroke was 3.6 times higher in diabetic women and 2.5 times higher in diabetic men than in non diabetics of the same age group /43/.

The following relationships exist between blood glucose levels and stroke /35/:

• Hypoglycemia worsens the prognosis for stroke. In the presence of global ischemia, glucose concentrations below 65 mg/dL (3.6 mmol/L) lead to higher mortality and a worse functional outcome /40/.
• Acute hyperglycemia increases mortality and worsens the prognosis for cerebral hemorrhages.

Alzheimer’s disease

Patients with T2D have a 2–2.5-fold higher risk of developing Alzheimer’s disease and vascular dementia than non diabetics of the same age group. The cause is reported to be microvascular infarctions in subcortical structures /41/.

3.1.12.4 Polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a heterogeneous disorder which affects 5–10% of women of childbearing age /40/. It manifests clinically as chronic anovulation with oligo-/amenorrhea, infertility, and hyperandrogenism. 40–50% of women with PCOS are insulin resistant and may develop symptoms of metabolic syndrome along with cardiovascular disease, hypertension, vascular dysfunction and obstructive sleep apnea in addition to having an increased prevalence of endometrial carcinoma.

PCOS is a prediabetic state with a 31–35% prevalence of impaired glucose tolerance (IGT) and a 7.5–10% prevalence of T2D. The rate of conversion from IGT to T2D is reported to be 5–10 times higher in women with PCOS than in those without this syndrome /41/. Insulin resistance is assessed using the Homeostasis Model Assessment (HOMA) test (see Tab. 3.7-6 – Interpretation of baseline and glucagon-stimulated C-peptide levels in diabetics).

3.1.12.5 Autoimmune polyglandular syndromes and diabetes

The autoimmune polyglandular syndrome (APS) is characterized by the immune-mediated destruction of endocrine tissues. A distinction is made between type 1 (APS-1) and type 2 APS (APS-2).

APS-1 /42/

Autoimmune poly endocrinopathy-candidiasis-ectodermal-dystrophy syndrome (APECED) is characterized by mucocutaneous candidiasis, autoimmune destruction, in particular of the endocrine glands, and ectodermal dystrophy. APECED is a rare, autosomal recessive disease, which occurs with equal frequency in both sexes. It is caused by mutations in the autoimmune regulator (AIRE) gene on chromosome 21q22.3, which is involved in the induction and maintenance of immunotolerance. The most common clinical manifestations are hypoparathyroidism, candidiasis, Addison’s disease, alopecia, and hypogonadism. The incidence of T1D is 5–10%. Early symptoms occur in the first decade of life. Most patients have 3–5 disease components.

APS-2 /42/

This syndrome is characterized by the coexistence of Addison’s disease, autoimmune thyroid disease, and T1D. The prevalence of T1D is 50–60%. Other less common diseases associated with APS-2 include pernicious anemia, vitiligo, celiac disease, alopecia, gonadal insufficiency, hypophysitis, and myasthenia gravis. The prevalence of APS-2 is 15–45 per 1 million inhabitants. Women are affected 1.6–3 times more than men. The autoimmunopathies often manifest beginning from the second to the third decade of life. Biomarkers for the diagnosis of diabetes include screening for hyperglycemia and, if necessary, autoantibodies against islet cell antigens. Thyroid disease associated with the APS-2 syndrome can manifest as hypo- or hyperthyroidism. APS-2 is a genetic disease. It is thought to be caused by multifactorial genetic factors. There is a strong association with HLA B8, DRB1*0301 (DR3) and DQA1*0501-DQB1*0201 (DQ2).

3.1.13 Pathobiochemistry and pathophysiology

3.1.13.1 Postprandial and postabsorptive glucose

The behavior of plasma glucose and insulin in the post absorptive (fasting) state (fasting plasma glucose, FPG) and postprandial following glucose load in the oral glucose tolerance test (oGTT) are important criteria for assessing insulin secretion (fasting level) and insulin resistance (2-h level). Glucose ingestion leads to increased insulin secretion. Normally, plasma glucose is taken up by the peripheral insulin-sensitive tissues in the postprandial state. If there is insulin resistance, glucose clearance is delayed, resulting in hyperglycemic glucose levels. Combined FPG and oGTT testing provides information about insulin secretion via the FPG level and information about insulin resistance via the 2-h glucose level from the oGTT.

This can produce the following results, which are indicative of the presence of prediabetes:

• Elevated FPG with normal 2-h glucose level; a condition termed impaired fasting glucose (IFG).
• Normal FPG with elevated 2-h glucose level; a condition termed impaired glucose tolerance (IGT).
• Combination of IFG and IGT without reaching the diagnostic thresholds for type 2 diabetes; a condition termed IFG/IGT.

Tab. 3.1-3 – Post absorptive and postprandial glucose in non-diabetics and diabetics shows the post absorptive and postprandial behavior of glucose in healthy individuals and diabetics. Criteria and tests for prediabetes screening in asymptomatic individuals are shown in Tab. 3.1-2 – Diagnosis of prediabetes and diabetes mellitus based on ADA criteria.

3.1.13.2 Genetic predisposition

Type 1A is thought to be caused by an interaction between different genetic and environmental factors. For example, the concordance rate among monozygotic twins is less than 100%, and even though type 1A aggregates in the family, there is no clear mode of inheritance /6, 43/. There is a clear association between the genes on chromosome 6p21 and type 1A. These are class II genes which encode HLA-DR and HLA-DQ markers. Therefore, one of the key focuses in predicting the genetic risk of type 1A is the genotyping of the HLA-DR and HLA-DQ loci. For example, children who carry both high-risk haplotypes (DR3-DQ2 and DR4-DQ8) have a 1 : 20 risk of developing type 1A by the age of 15. If one monozygotic twin is diabetic and the other carries both haplotypes, the risk of developing diabetes is as high as 55%. About 35% of type 1A diabetics among the white population in the USA are DR3-DR4 heterozygous compared to 2.4% of the general population. It is thus possible to identify high-risk groups in combination with autoantibody testing.

A number of non-HLA-associated gene loci that are associated with type 1A were identified. The odds ratio of the loci for the association with type 1A is only 1.2–1.5, with the exception of that of the loci INS and PTPN22, which is 2–2.5. INS is the gene for insulin and the primary autoantigen in type 1A. CTLA4 plays a role in the T-cell development and autoantigen recognition, and PTPN22 encodes the tyrosine phosphatase of T-cells and is involved in T-cell receptor signaling.

It is thought that 1–5% of the Caucasian population carry genes associated with a high risk of type 1A.

The following proportions are reported for the risk of type 1A /2, 44/:

• total population 0.4%
• familial aggregation, defined as the risk in siblings as compared to the total population (6%/0.4%; risk is about 15 times higher)
• HLA DR3-DR4 heterozygous individuals 7%
• first-degree relatives (siblings or parents with type 1A diabetes) 6%, child born to a mother with type 1A diabetes 1.3–4%, child born to a father with type 1A diabetes 6–9%
• first-degree relatives (siblings or parents with type 1A) with HLA DR3–DR4 heterozygosity 20–30%
• monozygotic twin of a type 1A diabetic 30–70%.

However, 85% of cases of type 1A diabetes occur sporadically i.e., in the absence of a first-degree relative with immune-mediated diabetes. Therefore the determination of genetic markers is currently considered of little value in the diagnosis of type 1A.

3.1.13.3 Genetic factors

The significance of genetic factors is reflected by the fact that ethnic populations, such as the Pima Indians, have a diabetes prevalence of up to 21%. The strong genetic basis of type 2 diabetes has also been demonstrated by tests on twins which show that the concordance of T2D in monozygotic twins is 70% compared with only up to 30% in dizygotic twins. T2D is a polygenic disease which is caused by the concurrent occurrence of many DNA sequence variations in different genes /45/. Each of these variations alone has only a moderate effect on the relevant function or expression of the gene, but in combination they lead to higher sensitivity to adverse environmental factors. Currently there are over 60 gene loci with variants that influence the risk of developing type 2 diabetes. Usually these are single nucleotide substitutions of one base for another (single nucleotide polymorphisms, SNPs) which are of different relevance for the risk of diabetes for the different ethnic groups. Relevant SNPs are known in the following loci: PPARG, KCNJ11, TCF7L2, HHEX, CDKALI, CDKN2A/B, IGF2BP2, SCL30A8 and WFS1. However, the associations are weak, and each variant increases the risk for diabetes only by a factor of 1.05–1.4. Even though these gene variants contribute to the risk of diabetes, they do not allow a better prediction of the risk than the clinical risk factors.

3.1.13.4 Epigenetic factors

The main epigenetic factors are older age, obesity, and lifestyle factors. Obesity is caused by excess consumption of calories, in particular unsaturated fats, sugary or starchy foods, reduced consumption of dietary fiber, a sedentary lifestyle, and reduced physical activity /46/. Although obesity is an important factor, 10% of type 2 diabetics are of normal weight, and not every obese person will develop diabetes. Psychosocial factors such as sleep deprivation and depression are also important factors while smoking and infections play a minor role.

Symptoms: because T2D has few symptoms, its clinical diagnosis is delayed. Only marked hyperglycemia will lead to symptoms such as fatigue, weakness, polyuria and polydipsia. Ketoacidosis is rare and only occurs when there is an infection, another disease or a stress situation /47/. The results of the United Kingdom Prospective Diabetes Study allow the conclusion that patients with type 2 diabetes still have 50% of normal β-cell function at diagnosis, but only 25% 6 years later. Extrapolation back to 100% β-cell function allows the conclusion that the decline of insulin secretion began 10–12 years before clinical symptoms appeared /48/.

3.1.13.5 Insulin action

Hyperglycemia can attribute glucose toxicity in renal tubular cells, neurons, hepatocytes, and immune cells that take up glucose in a manner that is insulin-independent. The gradient is created by a difference between extracellular and intracellular concentration of glucose.

Rare genetic disorders that are accompanied by altered peripheral insulin action are usually associated with mutations of the insulin receptor /11/. Associated metabolic abnormalities can range from hyperinsulinemia and mild hyperglycemia to severe diabetes. Some individuals with these mutations may have hyperglycemia together with acanthosis nigricans, virilism or polycystic ovaries, or they may present with leprechaunism or Rabson-Mendenhall syndrome.

3.1.13.6 Type 1 diabetes

Evidence of environmental factors contributing to the development of type 1A is based on the observation of seasonal and geographical variations in the incidence of the disease. For example, the incidence is lower in infants who received breast milk compared to those who were fed formula.

Viral infections are considered to be an important trigger of type 1A. The virus either directly destroys the β-cells through its cytopathogenic effect, or it triggers a chronic inflammatory process during which the β-cells are damaged. Finally, the destruction of islet cells can also result from a similar antigen structure of viral and β-cells (molecular mimicry).

Viral infection: molecular mimicry is reported to be a main cause of virus-induced autoimmune-mediated diabetes. Examples of viral infections that can trigger type 1A /49/:

• Congenital Rubella virus infection. More than 90% of genetically predisposed children (HLA DR3 or DR4 positive) with a congenital rubella infection develop diabetes
• Severe Coxsackie B virus infection. There are cross-reacting antigens between Coxsackie B4 virus proteins and the β-cell auto antigen GAD65.

The immune system begins to produce antibodies to β-cell antigens long before clinical symptoms of type 1A diabetes appear. These autoantibodies, also known as islet cell antibodies (ICA), are directed against sequestrated antigens of the β-cells and sustain an immune response which ultimately leads to the destruction of the β-cells. Clinical symptoms of diabetes appear when about 80% of the β-cells are dysfunctional. Since ICA are stimulated by antigens, their titer decreases over time. There is as yet no clear evidence that the ICA play a causal role in type 1A. They are, however, disease markers that are produced in the destruction of β-cells as an immune-mediated response to the release of β-cell proteins /43/.

The inflammatory injury of the pancreas that occurs in type 1A is known as insulitis. The early stage of inflammation is characterized by mononuclear cell infiltration of the acini (peri-insulitis) which surround the islets of Langerhans. Once these cells penetrate the islets, their destruction begins. The preclinical and clinical phases associated with the loss of islet cell mass are shown in Fig. 3.1-1 – Development of type 1A diabetes.

Criteria of type 1A diabetes and relevant screening tests are shown in:

• Tab. 3.1-1 – Classification of diabetes
• Tab. 3.1-2 – Diagnosis of prediabetes and diabetes based on ADA criteria.

3.1.13.7Type 2 diabetes

T2D is characterized by insulin resistance, impaired insulin secretion and reduced β-cell function.

Insulin resistance: refer to prediabetes and Section 2.2 – Metabolic syndrome /50/.

β-cell mass and T2D /51/: the cause of the diminished β-cell function in T2D is unknown. One significant factor is the reduction in the number of β-cells: in obese individuals with elevated fasting glucose levels, the number of β-cells is reduced by 50% compared to healthy individuals, and the United Kingdom Prospective Diabetes study showed that insulin secretion in type 2 diabetics is also reduced to 50% at diagnosis. The diminished β-cell function leads to the following disorders in T2D:

• pulsatile insulin secretion is impaired, a behavior that can manifest in the early stages of type 2
• fasting insulin levels are normal, low or elevated, but basal insulin secretion is too low in relation to the degree of overweight and hyperglycemia
• the effect of glucose stimulation on insulin secretion is diminished. Type 2 diabetics with fasting hyperglycemia thus have no first-phase insulin response to glucose load (see also Tab. 3.7-6 – Interpretation of baseline and glucagon-stimulated C-peptide levels in diabetics). Even when comparing the dose-response relationship between glucose concentration and insulin secretion following maximum stimulation by non-glucose secretagogues such as arginine in normal individuals compared to T2D, it is apparent that the ability of glucose to increase the insulin response of arginine is considerably reduced in T2D (Fig. 3.1-3 – Relationship between maximum insulin response to arginine stimulation and the plasma glucose concentration in healthy controls and type 2 diabetics). Maximum insulin secretion is reduced compared to normal individuals, and studies show that the β-cell defect is inversely related to fasting glucose: the higher the fasting glucose concentration, the lower the insulin secretion /52/.

The apoptosis of β-cells in T2D is reported to be attributable to the intracellular oligomerization of islet amyloid polypeptide (IAPP). IAPP is co-expressed and co-secreted with insulin by the islet cells. IAPP inhibits insulin secretion by exerting a direct paracrine effect on these cells. One assumption is that, in T2D, IAPP is misfolded in the cells, causing it to form cytotoxic aggregates and induce the apoptosis of the β-cells /53/.

3.1.13.7.1 Risk of progression from prediabetes to type 2 diabetes

Studies on the prevention of prediabetes and T2D have shown that the progression from prediabetes to T2D can be prevented or delayed through lifestyle intervention (change of diet, weight loss, increased physical activity), pharmacotherapy (e.g., metformin) or, in the case of heavy obesity, through bariatric surgery. In addition to the components of the metabolic syndrome (visceral obesity, hypertension, dyslipidemia), biomarkers also are important indicators for evaluating the risk for diabetes (see also Section 2.2 – Metabolic syndrome). Risk scores are used in particular to identify high-risk patients in order to alleviate secondary complications.

Two of these risk scores for diabetes are:

• The diabetes risk test (DRT; webtool, www.dife.de), the German adaptation of the Finnish score (FINDRISK). It applies for individuals aged 35 and older and determines the overall risk of developing T2D within the next 5 years /54/. The overall risk considers age, anthropometric data (waist circumference, height), hypertension as well as nutrition and lifestyle related variables (frequency of consuming wholemeal bread, red meat, coffee, alcohol, smoking, activity profile). Based on a cutoff score of ≥ 49, the test identifies the T2D cases expected in the normal population over the next 5 years with a diagnostic sensitivity of 85% and a specificity of 68%. By additionally performing biomarkers such as plasma glucose, HbA_1c, HDL cholesterol, triglycerides, GGT and ALT, the diagnostic sensitivity and specificity are improved. A fasting plasma glucose ≥ 100 mg/dL (5.6 mmol/L) alone increases the diagnostic specificity to 85%.
• The scores derived from the US Atherosclerosis Risk in Communities (ARIC) study. The base score contains the parameters hip circumference, maternal diabetes, hypertension, paternal diabetes, microsomia, black race, age > 55, overweight, increased heart rate, and smoking. The incidence of type 2 diabetes in the highest quintile is 33% for the subsequent 10 years. Addition of the biomarkers glucose, triglycerides, HDL cholesterol and uric acid to the score increases the incidence to 46.1%, if plasma glucose is ≥ 106 mg/dL (5.88 mmol/L), triglycerides are ≥ 179 mg/dL (2.02 mmol/L), HDL cholesterol is < 40 mg/dL (1.02 mmol/L) and uric acid ≥ 7.8 mg/dL (464 μmol/L) in men /50/.

3.1.13.8 Gestational Diabetes

Pregnancy has a profound effect on carbohydrate metabolism. The developing fetus relies on maternal supply of glucose, amino acids and lipids, which is primarily regulated by insulin. In the first trimester, during which the levels of human chorionic gonadotropin (hCG), estrogens and progesterone are increased, insulin sensitivity of the tissues is normal or increased. During the course of the pregnancy the woman becomes more insulin resistant, resulting in increased insulin secretion /55/. Insulin resistance is highest in the third trimester and is due to the increase in progesterone, prolactin, cortisol and human placental lactogen (hPL) levels. The main cause of insulin resistance is likely to be hPL as its structure is nearly homologous to that of growth hormone, which acts as an antagonist to insulin /56/. To maintain glucose homeostasis, more insulin must be secreted in order to overcome insulin resistance. Insulin resistance is linked to an increase in free fatty acids which intensify insulin resistance. The main cause that triggers GDM in some pregnant women appears to be a deficient reserve of insulin. Pregnant women with GDM exhibit a diminished first-phase insulin response to glucose load /55/. The similarities between GDM and T2D suggest that GDM is a prodromal form of type 2 diabetes being unmasked by pregnancy.

3.1.13.8.1 Maternal risks and complications

Women with GDM are at increased risk of miscarriage due to hyperglycemia. After the pregnancy they are at increased risk of developing diabetes, in particular type 2. Overweight and other risk factors for insulin resistance increase the likelihood of developing T2D while the presence of islet cell antibodies increases the probability of developing T1D and LADA /57/. GDM patients should have their carbohydrate metabolism evaluated with the 75-g oGTT 6–12 weeks following delivery and then as described in Tab. 3.1-2 – Diagnosis of prediabetes and diabetes mellitus based on ADA criteria for diabetes screening.

Most women with GDM return to normoglycemia within a short period after childbirth. However, some will develop GDM again in a subsequent pregnancy.

Approximately 20% of patients with GDM still have impaired glucose tolerance during the early postpartum phase, and 17–63% will develop manifest diabetes within the following 5–16 years. In a Swedish study /9/, 30% of patients with GDM had developed diabetes and 51% had developed impaired glucose tolerance within 5 years post partum. Independent predictors in pregnancy were fasting blood glucose levels > 94 mg/dL (5.2 mmol/L) and HbA_1c levels ≥ 5.7%.

Risk factors for the subsequent development of impaired glucose tolerance in pregnant women include /55/:

• insulin requirement during pregnancy
• elevated fasting glucose levels during pregnancy and post partum
• diagnosis of GDM during early pregnancy
• maternal overweight.

3.1.13.8.2 Pregnancy with overt diabetes

Women with T1D or T2D who are planning to become pregnant have to consider the effect of the pregnancy on their diabetes and vice versa /56/.

Maternal risks and complications

Since about two-thirds of pregnancies in women with diabetes are unplanned, diabetics of childbearing age should be counseled at an early stage to plan for a desired pregnancy. To ensure a complication-free pregnancy and optimal development of the embryo and fetus, good glycemic control in the preconceptional phase and during pregnancy is of paramount importance.

During the 1st trimester of pregnancy in insulin-dependent diabetic women, the need for insulin usually decreases and there is a tendency to develop hyperglycemia. From the 2nd trimester, however, insulin demand increases steadily up to the preconceptional level.

3.1.13.8.3 Laboratory findings

To evaluate the metabolic situation and possible diabetes-related complications of the pregnant women, the following laboratory tests should be performed /9/:

• Blood glucose measurements. The target levels are listed in Tab. 3.1-6 – Testing for diabetes in pregnant women. T2D patients receiving oral antidiabetic treatment and dietary counseling should be changed to insulin therapy, if they do not reach these targets. Blood sugar should be tested by a doctor to validate the patient’s self-monitoring results. The frequency of hypo- or hyperglycemic episodes must be inquired from the patient and investigated. Severe, frequent or inexplicable hypoglycemia can be caused by deficient counter regulation, impaired awareness, insulin medication errors, and excessive alcohol consumption.
• Ketone bodies in urine and/or blood. The elevated concentration of ketone bodies in diabetic women with hyperglycemia is indicative of the onset or presence of ketoacidosis. Ketone bodies are indicative of absolute or relative insulin deficiency. Testing for ketone bodies is of particular importance in diabetic women who self-monitor their carbohydrate metabolism and in those with blood glucose levels > 200 mg/dL (11.1 mmol/L). Pregnant women with higher levels than these may have diabetic ketoacidosis, which is linked to a high fetal mortality rate.
• HbA_1c test. For preconceptional metabolic control, this test should be performed every four weeks, and then every 6–8 weeks once glycemic control has been achieved. Good control means levels below 6%; the target level is below 7%. During pregnancy, the HbA_1c level should be determined monthly and should be below 6% /1/.
• Serum creatinine to evaluate glomerular function. Due to the elevated filtration rate during pregnancy, the kidneys are more susceptible to dysfunction. Pregnancy is not advised in women who have diabetic nephropathy with a creatinine clearance < 50 [mL × min.^–1 × (1.73 m²)^–1] or serum creatinine > 3.0 mg/dL (51 mmol/L) prior to conception.
• Urinary albumin in relation to creatinine excretion to evaluate the effect of pregnancy on renal function. If renal function is normal prior to conception, there will be no renal dysfunction during pregnancy, with the exception of mild proteinuria, which will normalize post partum. If persistent albuminuria (30–300 mg/24 h; 20–200 μg/min.; 30–300 mg/g creatinine) is present prior to conception, about one-third of women with this condition will progress to proteinuria with excretions in the range of g/L by the end of the pregnancy. If persistent albuminuria (> 300 mg/24 h, > 200 μg/min., > 300 mg/g creatinine) is present prior to conception, the likelihood of preeclampsia developing close to the date of childbirth is 30%. Some women develop retinopathy and nephrotic proteinuria.
• Investigation for hypertension, which is a common concomitant complication in diabetic pregnancies. This applies in particular if persistent albuminuria is present prior to conception. Pregnant women with T1D usually develop hypertension in combination with diabetic nephropathy, which is accompanied by persistent albuminuria. Pregnant women with T2D are more likely to have hypertension than those with T1D.
• TSH in T1D; 5–10% of these women have hypo- or hyperthyroidism.

3.1.13.9 Fetal risks and neonatal complications in diabetic pregnancies

Diabetic embryopathy

During organogenesis, a diabetic metabolism increases the rate of miscarriage. The risk increases linearly with the hyperglycemia measurable based on the HbA1c level, and the number of damaged organs increases with increasing maternal glucose levels. Anomalies in diabetic embryopathy include defects of the neural tube, omphalocele, musculoskeletal anomalies, deformities of the kidneys and urinary system, and conotruncal heart defects. The increased rate of malformations contributes significantly to perinatal mortality [death between pregnancy week 20 (22) and 7th day of life].

Since organ development is not complete until about 6 weeks post conception, good diabetic control prior to conception is important. Ideally, the HbA_1c level prior to conception should be < 7%, or better < 6% /11/. Since gestational diabetes does not develop until the second half of pregnancy, impaired glucose tolerance in the earlier months is uncommon and therefore does not lead to congenital malformations. Where such malformations are reported, T2D is usually present. T2D is becoming more prevalent in younger pregnant women. Although glucose levels in these women are more stable than in those with T1D, hyperglycemia is nevertheless present and there is a risk of congenital malformations.

Diabetic fetopathy

Maternal hyperglycemia in the second half of pregnancy causes diabetic fetopathy /56/. In addition to macrosomia, respiratory distress syndrome and postnatal hypoglycemia, main findings also include hyperbilirubinemia, polyglobulia, hypocalcemia, and hypogmagnesemia (Tab. 3.1-7 – Tests for the risk assessment of infants born to diabetic mothers).

Macrosomia: this abnormal condition is defined as a birth weight > 90th percentile of the gestational age. Since insulin stimulates tissue growth, the increased fetal insulin production associated with the mother’s diabetes leads to increased growth, in particular of the trunk. The main determinants of macrosomia are maternal weight and maternal weight gain, gestational age, and maternal glucose concentrations, the latter being the main treatable determinant of macrosomia. Approximately 20% of untreated diabetic women give birth to a macrosomic child. For fetuses of pregnant women with T1D the risk of being macrosomic is 25% and that of having hyperglycemia is 8% /56/.

Neonatal hypoglycemia: this state results from hyperplasia of the islet organs due to increased glucose supply by the diabetic mother. Even though newborns can have glucose levels < 45 mg/dL (2.5 mmol/L), 5–24% of infants born to diabetic mothers still have lower levels during the neonatal period (see also Tab. 3.2-2 – Hypoglycemia syndromes in childhood and infancy). Neonatal hypoglycemia can cause subsequent disorders of the central nervous system.

Respiratory distress syndrome: the syndrome is due to delayed pulmonary maturity and has an incidence of 1.6%, which is comparable to that of nondiabetic pregnancies.

3.1.13.10 Neonatal diabetes mellitus (NDM)

Uncontrolled hyperglycemia within the first 6 months of life occurs in all races and ethnic groups /60/. The majority of newborns present with intrauterine growth retardation, failure to thrive, lack of subcutaneous fat, and low or undetectable C-peptide levels. In most cases of NDM, the disease is monogenic and there is no evidence of islet cell autoantibodies. There are mutations in genes that are responsible for the development of the pancreas, β-cell apoptosis and the regulation of insulin production. The incidence is about 1 per every 300,000 to 500,000 live births.

A differentiation must be made between the above described permanent neonatal diabetes mellitus (PNDM) and the transient form (TNDM). In a large proportion of infants with TNDM, blood glucose returns to normal within the first few months, but they go on to develop T2D years after this initial hyperglycemia. About 57% of NDM cases belong to the TNDM group. They initially require treatment with insulin, but this can usually be discontinued within less than 18 months.

3.1.13.11 Diabetes management in chronic kidney disease

Refer to Table 3.1-12 – Diabetes management in chronic kidney disease.

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3.2 Hypoglycemia syndromes

Lothar Thomas

The loss of consciousness includes syncope and nonsyncopal causes.

Syncope is caused by a period of inadequate cerebral perfusion /34/.

• Intoxikation syncopal causes are orthostatic syncope, reflex syncope, cardiac arrhythmia, and structural cardiopulmonary disease
• Nonsyncopal causes are divided in seizure, intoxication (alcohol, sepsis, medication) psychiatric conditions, and metabolic conditions e.g., hypoglycemia.

See also Section 3.7.

3.2.1 Definition of hypoglycemia

The term hypoglycemia refers to a low blood glucose concentration associated with clinical symptoms. Hypoglycemia is the result of an imbalance between the inflow of glucose into the bloodstream due to decreased endogenous glucose production or deficient glucose uptake, and the consumption of glucose by the tissues. Hypoglycemia is prevented by a complex regulatory system /1/.

The post absorptive glucose concentration range is 70–100 mg/dL (4.0–5.6 mmol/L). The glucose threshold for a decrease in the blood insulin concentration is approx. 81 mg/dL (4.5 mmol/L). When the glucose level falls to about 65 mg/dL (3.5 mmol/L), there is increased secretion of the counter regulatory hormones glucagon, catecholamines, cortisol, and growth hormone. Glucagon and catecholamines raise the blood glucose level within minutes by stimulating hepatic glycogenolysis and gluconeogenesis as well as renal gluconeogenesis. The substrates of gluconeogenesis are glycerol, free fatty acids, and amino acids. Cortisol and growth hormone reduce the glucose consumption of insulin-sensitive tissues and lead to an increase in blood glucose within hours.

The main source of energy for the brain is glucose, and there are protective mechanisms to maintain glucose homeostasis. When glucose falls to ≤ 57 mg/dL (3.2 mmol/L) in capillary blood /2/ and to ≤ 54 mg/dL (3.0 mmol/L) in venous whole blood /3/, the autonomic (i.e. the sympathoadrenal) nervous system is activated, leading to hypoglycemic symptoms such as anxiety, sweating, tremor, fast heartbeat, and hunger. These end-organ responses, also called autonomic symptoms, can progress to neuroglycopenic symptoms including behavioral changes, cognitive dysfunction, seizures, and coma. However, the threshold for cognitive dysfunction depends on various clinical aspects and psychometric tests.

3.2.1.1 Threshold glycemic levels for symptoms

A study /4/ of 30 healthy subjects who had their capillary blood glucose levels measured 17–18 times per day, showed a daily mean with a standard deviation of 75 ± 14 mg/dL (4.2 ± 0.8 mmol/L). The physiological nadir was reached at 5 PM the level was 70 ± 11 mg/dL (3.9 ± 0.6 mmol/L) and the peak level at 2 PM was 88 ± 18 mg/dL (4.9 ± 1.0 mmol/L). Overall, 5% of glucose levels were below 54 mg/dL (3.0 mmol/L), and 2.8% were below 50 mg/dL (2.8 mmol/L). 33% of participants had levels below 54 mg/dL (3.0 mmol/L), while 17% were below 50 mg/dL (2.8 mmol/L). Since in 95% of cases, blood glucose levels were above 54 mg/dL (3.0 mmol/L), it would make sense to define this concentration as the diagnostic threshold for hypoglycemia /5/. However, consensus statements have defined thresholds of 40 mg/dL (2.2 mmol/L) for venous and capillary whole blood and 50 mg/dL (2.8 mmol/L) for venous plasma in non diabetics /6/. The clinical symptoms associated with a decrease in glucose concentrations are shown in Fig. 3.2-1 – Activation of glucose counter regulatory hormones.

3.2.2 Differentiation of hypoglycemia

Clinical hypoglycemia is present if:

• There are autonomic and glycopenic symptoms
• Glucose concentrations are ≤ 40 mg/dL (2.2 mmol/L) in capillary and venous whole blood, and ≤ 50 mg/dL (2.8 mmol/L) in venous plasma.
• Symptoms resolve after glucose ingestion/administration.

The aforementioned glucose levels are a highly specific criterion for hypoglycemia. According to studies /5/, depending on the specimen (capillary blood, venous blood), even concentrations in the range of 54–63 mg/dL (3.0–3.5 mmol/L) require further investigation if there are clinical symptoms suggestive of hypoglycemia. If levels are below the thresholds suggested by Whipple, further clinical investigations are necessary, even in the absence of hypoglycemia symptoms.

Hypoglycemia is not a diagnosis but a pathological state, the cause of which must be determined. The causes of hypoglycemia are manifold and can be differentiated into /34/:

• Spontaneous symptomatic hypoglycemia: elevated insulin and C-peptide concentration at the time of hypoglycemia
• Insulin autoimmune syndrome (Hirata disease): autoantibodies are present against endogeneous insulin
• Type B insulin resistance syndrome
• Exogenous insulin
• Beta cell hypertrophy
• Insulin secretagogues: salicylates, pentamidine, β-receptor blockers
• Other factors: physical exercise, alcohol, medications, liver cirrhosis, glucocorticoid deficiency, large tumors, malnutrition, parenteral nutrition, sepsis, shock, pseudo-hypoglycemia, adrenergic polyprandial syndrome, renal glucosuria, insulinoma, hyperinsulinemic hypoglycemia following gastric bypass surgery.

3.2.2.1 Hypoglycemia syndrome in adults

The most common diagnoses at admission in patients presenting with hypoglycemia are diabetes mellitus, alcoholism, sepsis, and reactive hypoglycemia. Insulinomas are very rare, with a prevalence of 4 cases per 1 million population per year. For evaluation refer to Section 3.7 – Insulin, C-peptide, proinsulin. Iatrogenic hypoglycemia in diabetics is evaluated based on medical history.

To differentiate the hypoglycemia syndrome, patients should be assigned to one of the following categories based on their medical history and clinical presentation /8/:

• The healthy appearing patient. If the patient has no pre-existing illness, then drug-associated hypoglycemia, alcoholism, reactive hypoglycemia, renal glucosuria, pseudo hypoglycemia, adrenergic postprandial syndrome and insulinoma must be considered primarily. Apparently clinically healthy patients need to undergo intensive laboratory testing for the confirmation and differential diagnosis of hypoglycemia.
• The ill patient. In this group of patients, hypoglycemia can be associated with the medication of the existing illness (hypertension, diabetes, malaria), or it can be due to a paraneoplastic syndrome (non-islet cell tumor hypoglycemia, NICTH), a congenital disorder of carbohydrate metabolism, or an endocrine disorder. Once the diagnosis is known, no further diagnostic evaluation of the hypoglycemia is required.
• The hospitalized patients who often have serious, multi­systemic illnesses. The main causes of the hypoglycemia, apart from diabetes mellitus, are sepsis, shock, liver disease, and renal failure. In these cases, constant blood glucose monitoring is necessary to detect the risk of hypoglycemia.

Hypoglycemia syndromes which are due to an insulinoma predominantly occur in the fasting state, rarely in the fasting plus postprandial state, and very rarely only in the postprandial state.

Postprandial symptoms, which occur 2–4 h after meals are classified as food-stimulated and those which occur more than 5 h after meals are classified as food-deprived. Autonomous symptoms without hypoglycemia, also known as pseudo-hypoglycemia, which occur after meals usually cannot confirmed as arising from hypoglycemia. If postprandial hypoglycemia occurs with blood glucose levels below 45 mg/dL (2.5 mmol/L), then the hypoglycemia is stimulated by food intake (e.g., in the case of hereditary fructose intolerance, crop poisoning, or in patients who have had Billroth II surgery) /8, 9/.

The flow chart in Fig. 3.2-2 – Flow chart for the differentiation of hypoglycemia in adults recommends a diagnostic workflow, Tab. 3.2-1 – Hypoglycemia syndromes in adults shows hypoglycemia syndromes in adults.

3.2.2.2 Biomarkers and functional tests for the evaluation of hypoglycemia

Blood glucose: detection of hypoglycemia. If the classic symptoms of hypoglycemia are present, the hypoglycemia etiology is confirmed if at least one of several values is below 45 mg/dL (2.2 mmol/L) in capillary or venous whole blood, and below 50 mg/dL (2.8 mmol/L) in venous plasma, and the remaining values are in the range of 45–54 mg/dL (2.5–3.0 mmol/L). If all values are above 45 mg/dL (2.2 mmol/L) in capillary whole blood, or above 50 mg/dL (2.8 mmol/L) in venous plasma, then hypoglycemia is not confirmed. In this case, the 72-h fast or another functional test should be performed.

In the case of suspected postprandial reactive hypoglycemia, blood glucose self-monitoring is reliable /10/.

The cutoff for the diagnosis of diabetic hypoglycemia is 70 mg/dL (3.9 mmol/L). Findings on hypoglycemia in adults and drug-associated hypoglycemia and their diagnostic significance are listed in Tab. 3.2-1 – Hypoglycemia syndromes in adults.

72-h fast: the test is the mainstay for the evaluation of food-deprived hypoglycemia. Distinction between normal individuals and those with hypoglycemic disorders is based on normal individuals tolerating 3 days food withdrawal without the development of symptoms, whereas patients with hypoglycemic disorder manifest Whipple’s triad usually well short of 72 h /8/. Detection and differentiation of hypoglycemia by determination of insulin, C-peptide and β-hydroxy butyrate (Tab. 3.7-2 – Insulin, C-peptide and proinsulin reference intervals).

C-peptide suppression test, intravenous tolbutamide test, glucagon test: these tests are performed if the 72-h fast is not conclusive.

3.2.2.3 Hypoglycemia in childhood and infancy

Neonates

The practical threshold for neonates and children is considered to be a blood glucose concentration of 45 mg/dL (2.5 mmol/L). Changes in the cerebral blood flow occur with levels < 30 mg/dL (1.7 mmol/L). All newborns with suspicious clinical symptoms should have their blood glucose maintained at levels > 45 mg/dL (2.5 mmol/L). During the neonatal period, infants of diabetic mothers should have a glucose concentration > 63 mg/dL (3.5 mmol/L), since they have low levels of substrates such as glucose, lactate, alanine and ketone bodies. Every child with a glucose level < 36 mg/dL (2.0 mmol/L) needs to be monitored, even in the absence of clinical symptoms /11/.

Due to the physiological decline in the blood glucose concentration with a nadir 1–2 h after birth, glucose measurements in newborns of nondiabetic mothers should not be performed until 3–4 h after birth, when the physiological hypoglycemia has been overcome /12/. Following enteral feeding, blood glucose levels cycle, with a peak occurring about 1 h after food intake. If hypoglycemia is suspected, a blood sample should be taken just before the second food intake. Low glucose levels in the first 24–48 h are not uncommon in normally developing newborns who are breast-fed.

Infants and adolescents /32/

Every year, approx. 30 in 100,000 children are admitted to hospital with a reduced level of consciousness or non-traumatic coma.

The main etiologies are infections, drug-induced intoxications, seizures, and metabolic disorders. If the condition is not diagnosed timely and correctly, the mortality rate is up to 40%.

3.2.2.4 Tests at hospital admission in children with a reduced consciousness

• Blood glucose and blood gas analysis.
• Sodium, potassium, creatinine
• Urea, ammonia, lactate, ketone bodies
• AST, LD, GGT, bilirubin
• Blood count and differential
• Urinalysis
• Blood culture.

Freezing of 10 mL urine and 2 mL serum/plasma for possible additional tests.

The possibility of a disease-associated hypoglycemia should be considered if the child has the following history /13/:

• Short, changing hypoglycemic episodes (2–4 h) not associated with food intake; they occur in hyperinsulinism and type I glycogenosis.
• Fasting phases of 6–8 h or even 14–16 h are tolerated, but that is not the case in enzyme defect, glycogenolysis or impaired gluconeogenesis
• Morning fasting hypoglycemia or hypoglycemia during intercurrent illnesses; can be suggestive of impaired gluconeogenesis
• Postprandial, reactive hypoglycemia; can be suggestive of hereditary fructose intolerance or leucine-sensitive hypoglycemia.

For information on childhood hypoglycemia syndromes refer to:

• Tab. 3.2-2 – Hypoglycemia syndromes in childhood and infancy
• Tab. 3.2-3 – Laboratory findings and suggestion of disorder in childhood hypoglycemia.

For the molecular basis of glucose homeostasis and incidence of congenital hypoglycemia see Ref. /33/.

References

1. Rosen SG, Clutter WE, Berk MA, Shah SD, Cryer PE. Epinephrine supports the post absorptive plasma glucose concentration and prevents hypoglycemia when glucagon secretion is deficient in man. J Clin Invest 1984; 72: 405–11.

2. Mitrakou A, Ryan C, Veneman T, et al. Hierarchy of glycemic thresholds for counter regulatory hormone secretion, symptoms, and cerebral dysfunction. Am J Physiol 1991; 260: E 67–74.

3. Brun JF, Baccara MT, Blacon C, Orsetti A. Niveaux de glycemie veineuse physioligiquement associes aux signes fonctionelles d’hypoglycemie. Comparaison avec des hypoglycemies reactionelles (Abstract). Diabetes Metab 1995; 21 A.

4. Marks V. Glycemic stability in healthy subjects: fluctuations in blood glucose during day. In: Andreani D, Marks V, Lefebvre PJ, eds. Hypoglycemia. New York; Raven Press 1987: 19–24.

5. Brun JF, Fedou C, Mercier J. Postprandial reactive hypoglycemia. Diabetes and Metabolism (Paris) 2000; 26: 337–51.

6. Whipple AO. The surgical therapy of hyperinsulinism. J Internat Chirol 1938; 3: 237.

7. Heller SR. Diabetic hypoglycemia. Bailliaire’s Clinical Endocrinology and Metabolism. 1999; 13: 295–308.

8. Service FJ. Hypoglycemic disorders. N Engl J Med 1995; 332: 1144–52.

9. Comi RJ. Approach to acute hypoglycemia. Endocrinol Metab Clin North Am 1993; 22: 247–62.

10. Palardy J, Havrankova J, Lepage R, Matte R, Belanger R, d’Amour P, Ste Marie LG. Blood glucose measurements during symptomatic episodes in patients with suspected postprandial hypoglycemia. N Engl J Med 1989; 321: 1421–5.

11. Deshpande S, Platt MW. The investigation and management of neonatal hypoglycemia. Semin Fetal & Neonatal Medicine 2005; 10: 351–6.

12. Wendel U. Diagnostisches Vorgehen bei kindlichen Hypoglykämien. Monatsschr Kinderheilkd 1988; 136: 592–6.

13. Cryer PE. Mechanisms of hypoglycemia-associated autonomic failure and its component syndromes in diabetes. Diabetes 2005; 54: 3592–3601.

14. American Diabetes Association Workgroup on Hypoglycemia. Defining and reporting hypoglycemia in diabetes: a report from the American Diabetes Association Workgroup on Hypoglycemia. Diabetes Care 2005; 28: 1245–9.

15. Weitzman ER, Kelemen S, Quinn M, Eggleston EM, Mandl KD. Participatory surveillance of hypoglycemia and harms in an online social network. JAMA Intern Med 2013; 173: 345–51.

16. Service GJ, Thompson GB, Service FJ, Andrews JC, Collazo-Clavell ML, Lloyd RV. Hyperinsulinemic hypoglycemia with nesidioblastosis after gastric-bypass surgery. N Engl J Med 2005; 353: 249–54.

17. Toft-Nielsen M, Madsbad S, Holst JJ. Exaggerated secretion of glucagon-like peptide-1 (GLP-1) could cause reactive hypoglycaemia. Diabetologica 1998; 41: 1180–6.

18. Bergman RN. Toward physiological understanding of glucose tolerance. Minimal model approach. Diabetes 1986; 38: 1512–27.

19. Ahmadpour S, Kabadi UM. Pancreatic alpha-cell function in idiopathic reactive hypoglycemia. Metabolism 1997; 46: 639–43.

20. Sasaki M, Moki T, Wada Y, Hirosawa I, Koizumi A. An endemic condition of biochemical hypoglycemia among male volunteers. Ind Health 1996; 34: 323–33.

21. Marimee TJ, Tyson JE. Hypoglycemia in men. Pathologic and physiologic variants. Diabetes 1977; 26: 161–5.

22. Escalande Polido JM, Alpizar Salazar M. Changes in insulin sensitivity, secretion and glucose effective ness during menstrual cycle. Arch Med Res 1999; 30: 19–22.

23. Zapf J, Futo E, Peter M, Froesch ER. Can big endothelin growth factor II in serum of tumor patients account for the development of extrapancreatic tumor hypoglycemia? J Clin Invest 1992; 90: 2574–84.

24. White Jr JR, Campbell RK. Dangerous and common drug interactions in patients with diabetes mellitus. Endocrinol Metab Clin North Am 2000; 29: 789–802.

25. Bonham JR. The investigation of hypoglycemia during childhood. Ann Clin Biochem 1993; 30: 238–47.

26. Gesellschaft für Neonatologie, pädiatrische Intensivmedizin, et al. Betreuung von Neugeborenen diabetischer Mütter. AWMF-Leitlinie 2010.

27. Roe TF, NG WG, Smit PGA. Disorders of carbohydrate and glycogen metabolism. In: Blau N, Duran M, Blaskovics ME, Gibson KM, eds. Physician’s guide to the laboratory diagnosis of metabolic diseases. Berlin; Springer 2002; 335–55.

28. Duran M. Disorders of mitochondrial fatty acid oxidation and ketone body handling. In: Blau N, Duran M, Blaskovics ME, Gibson KM, eds. Physician’s guide to the laboratory diagnosis of metabolic diseases. Berlin, Springer 2002; 309–34.

29. Birkebaek NH, Simonsen H, Gregersen N. Hypoglycaemia and elevated urine ethylmalonic acid in a child homozygous for the short-chain acyl-CoA dehydrogenase 625G>A gene variation. Acta Paediatr 2002; 91: 480–6.

30. Ryan C, Gurtunca S, Becker D. Hypoglycemia: a complication of diabetes therapy in children. Pediatr Clin N Am 2005; 1705–33.

31. Diabetes Control and and Complications Trial Research Group. Hypoglycemia in the Diabetes Control and Complications Trial. Diabetes 1997; 46: 271–86.

32. Bowker R, Green A, Bonham JR. Guidelines for the investigation and management of reduced level of consciousness in children: implications for clinical biochemistry laboratories. Ann Clin Biochem 2007; 44: 506–11.

33. Lang TF. Update on investigating hypoglycemia in childhood. Ann Clin Biochem 2011; 48: 200–11.

34. Haverkamp GLG, Ijzerman RG, Kooter J, Krul-Poel YHM. The after-dinner dip. N Engl J Med 2022; 386 (22): 2130–6.

3.3 Blood glucose

Lothar Thomas

Conventional blood glucose should be determined in capillary whole blood or venous plasma /1/. With other types of samples, different glucose concentrations are measured at the same sampling time in the same individual /2/. This must be taken into account during the clinical evaluation.

A blood glucose test is a measure of glucose concentration present in an the blood of an individual at a given point of time. Diagnostic laboratory tests for diabetes are (Tab. 3.3-1 – Blood glucose specimen for evaluation of the metabolic state):

• Fasting glucose (FG), an excellent test for "in moment glucose"
• 2-hour glucose during a 75-g oral glucose tolerance test (oGGT) for diagnosis of impaired glucose tolerance
• Random glucose (measurement of glucose at not given point)
• Continuous glucose monitoring using a chip implanted under the skin.

3.3.1 Indication

Suspected hyperglycemia

• Screening for diabetes mellitus in ambulant patient care and hospitals
• Monitoring of diabetes therapy
• Evaluation of carbohydrate metabolism (e.g., pregnant women, patients with obesity, hyperlipidemia, cardiovascular disease, stroke, renal failure, patients with reduced consciousness or in a coma, patients with liver disease, acute hepatitis, acute pancreatitis, chronic pancreatopathy, autoimmune polyglandular syndrome, acromegaly, Addison’s disease, panhypopituitarism, therapy with corticosteroids and drugs that induce hyperglycemia, stress response).

Suspected hypoglycemia

• Diabetes therapy and occurrence of hypoglycemia symptoms
• Exclusion of hypoglycemia syndrome in clinically apparently healthy individuals (exclusion of insulinoma)
• Hypoglycemia symptoms in the critically ill
• Diagnosis of neonatal hypoglycemia
• Suspected congenital metabolic disorder
• Treatment with drugs that induce hypoglycemia.

3.3.2 Method of determination

There are different methods for the determination of glucose in blood and body fluids /1/.

Glucose oxidase method

Principle: the enzyme glucose oxidase catalyzes the oxidation of glucose to gluconic acid and H₂O₂. In the subsequent peroxidase-mediated indicator reaction, H₂O₂ oxidizes a reduced chromogen to produce a colored compound, which is measured using a photometer. The color intensity of the oxidized chromogen is proportional to the glucose concentration /3/.

Hexokinase method /4/

Principle: hexokinase in the presence of ATP phosphorylates glucose to form glucose-6-phosphate. The latter reacts with NADP to form 6-phosphogluconate and NADPH₂. This reaction is catalyzed by glucose-6-phosphate dehydrogenase (G-6-PD). The measurand is NADPH₂, the increase in NADPH₂ is measured at the endpoint of the reaction. The increase in absorbance determined is proportional to the glucose concentration in the test sample.

Glucose dehydrogenase (Gluc-DH) method /5/

Principle: glucose is oxidized to gluconolactone by Gluc-DH. The hydrogen released in the reaction is transferred to NAD, producing NADH₂. The increase in NADH₂ is measured using the principle of continuous absorbance registration. The increase in absorbance is proportional to the glucose concentration in the test sample. In contrast to the end point method addition of mutarotase to the reagents is not necessary.

Gluc-DH only reduces β-D-glucose. In aqueous solution, glucose is present in the α- and β-form. As the β-D-glucose is consumed, an equilibrium between the two forms is established again as a function of time. To prevent this reaction from becoming the determining factor for the speed of the Gluc-DH reaction, the reagent contains mutarotase. This enzyme accelerates the rate at which equilibrium is reached.

Measurement with a glucose sensor /6/

Biosensors are analytical devices that incorporate a biological material (e.g., the enzyme glucose oxidase) and are connected to an optical or electrochemical detection system.

Principle of the glucose sensor: in the first step, glucose reacts with the oxidized form of the enzyme glucose oxidase (GOD) to form gluconic acid. In this process, two electrons and two protons are released, and GOD is reduced. In the second step, O₂ which is present in the surrounding fluid reacts with GOD accepting the aforementioned electrons and protons leading to form H₂O₂ and regenerating oxidized GOD, which is ready to react once more with glucose. The glucose concentration in the test sample determines the amount of H₂O₂. This is detected following oxidation at the surface of a platinum electrode which causes a change in the electrochemical potential.

Measurement with glucose meters

Analyzers in which glucose is determined using readable strip and reflectance photometer are used for:

• Point-of-care testing in intensive care units in hospitals or outpatient clinics, in facilities for chronic disease management, and in doctor’s office
• Self-monitoring of blood glucose (SMBG) at home, at work, or at school. In the USA, national standards were developed for SMBG /8/.

Glucose tests with glucose meters are based on the photometric measurement of the color development of a chromogen or on the principle of the glucose electrode /9/. With the photometric measurement, glucose is enzymatically oxidized to gluconolactone by the enzymes glucose peroxidase or glucose dehydrogenase.

In the subsequent indicator reactions,

• the H₂O₂ produced, catalyzed by peroxidase, oxidizes the 3,3’, 5,5’ tetra methyl benzidine to a blue dye whose intensity is measured with a reflectometer,

or

• the NADH produced, catalyzed by diaphorase, reduces the dye 3-(4’,4’-dimethylthiazole-2-yl)-2,4-diphenyl tetrazolium bromide to a formazan dye.

The optimal sample is capillary blood. Modern glucose meters for the self-monitoring of blood glucose allow the storage and processing of the measured values and the calculation of mean blood glucose (MBG) and mean amplitude of glucose excursions (MAGE).

Continuous glucose monitoring (CGM) /10/

Continuous glucose monitoring (CGM) systems have been recognized as the ideal monitoring systems for glycemic control of diabetic patients. The CGM system measures blood glucose levels in subcutaneous tissue by attaching a CGM sensor to the skin, allowing the patient to make appropriate modifications to their medical interventions according to experience or empirically derived algorithms. The principles of glucose sensing employed in the commercially available CGM systems are mainly electrochemical and employ the enzyme glucoseoxidase as the glucose sensing molecule with the combination of hydrogen peroxide monitoring or with the combination of redox mediator harboring hydrogel.

3.3.3 Specimen

Depending on the method of determination, the following are used:

• Capillary blood, depending on the sampling procedure: 0.01–0.02 mL
• Venous (rarely capillary) plasma: 0.01–0.05 mL

3.3.4 Reference interval

Refer to Tab. 3.3-2 – Blood glucose reference intervals.

3.3.5 Clinical significance

The blood level of glucose depends on the metabolic state of an individual. The following states are possible (Tab. 3.3-1 – Blood glucose specimen as a function of the metabolic state):

• Post absorptive state; period of 6–12 h after the start of food intake. During this period the transition from the postprandial to the fasting state occurs.
• Postprandial state; this comprises a period of 2–3 h after the start of food intake. During this period, the blood glucose level in plasma rises up to 200 mg/dL (11.1 mmol/L) beginning 10 min. after the start of a meal. Even though glucose levels return to preprandial levels within 2–3 h after food intake, it takes about 6 h for a meal to be completely assimilated and for the post absorptive state to be restored. Compared to the fasting state, there is hyperglycemia. This depends on the type of carbohydrates consumed, the amount of fat and proteins, the size of the meal, and the time of day. Postprandial glucose accounts for approx. 30–40% of the total daytime hyperglycemia.
• Fasting state; comprises a period of 8–10 h after the last food intake. The glucose concentration of non diabetics is below 100 mg/dL (5.6 mmol/L).

The behavior of blood glucose in different conditions is shown in:

• Tab. 3.3-3 – Blood glucose in diabetes mellitus and other categories of glucose regulation,

and the diagnostic significance of glucose in the fasting and postprandial state in:

• Tab. 3.3-4 – Clinical significance of fasting and postprandial blood glucose.

Glucose levels within the reference interval do not rule out diabetes, and concentrations above the reference interval do not confirm it /13/. This is due to intraindividual variations of blood glucose levels which are greater than those of other blood parameters as they are influenced by physical activity and the length of time since the last food intake. For example, in healthy individuals the intraindividual (biological) and inter individual variation of fasting capillary glucose [mean glucose of 88 mg/dL (4.9 mmol/L)] is 4.8–6.1% and 7.5–7.8% respectively /14, 15/. The biological variability of plasma glucose is thus higher than the analytical imprecision. Moreover, the fasting plasma glucose concentration increases continuously with age from the third to the sixth decade of life. Dysregulations such as insulin resistance, hyperinsulinism and diabetes as well as pregnancy further increase the variations. In newly diagnosed type 2 diabetics, the intraindividual variation of fasting glucose is 13.7%, and the inter individual variation 14.8% /16/. The interpretation of blood glucose levels also depends on the type of sample examined.

Glycemic index (GI) and glycemic load (GL)

The GI is a measure of how much 50 g of carbohydrate from a specific food raises the blood glucose level. The lower the GI, the less the concentration of blood glucose increases. After consuming a certain food it is usually measured how high the increase of glucose is. A diet with a high GI is associated with an increased risk of cardiovascular disease and death.

The glycemic load (GL) is the product of GI and the consumed carbohydrates and a measure of insulin needs. The GL is calculated by multiplying the mean net carbohydrate intake (as measured in grams per day) by the GI and then dividing by 100.

Test procedure: Blood glucose increase is measured several times after ingestion of the specific food within 2 hours and compared with blood glucose increase after ingestion of 50 g glucose. The GI is calculated from the quotient 100 times the area of specific food/area of glucose. The assessment of GI is as follows:

• GI > 70; high glycemic load, associated with higher risk of cardiovascular disease compared to normal
• GI > 50–70 medium glyemic load
• GI < 50 low glycemic load

In a study /37/ diets with a high GI were associated with a higher risk of cardiovascular disease and death than diets with a lower GI.

3.3.6 Comments and problems

Sample materials

The concentration of glucose in blood depends on the type of sample examined. Due to the higher water content compared to the red blood cells, glucose concentration measured in venous plasma is generally 10–18% higher than in venous whole blood. Arterial whole blood has a higher glucose concentration than venous blood; the glucose concentration of capillary whole blood sampled from the finger tip is in between the two. Measurements in capillary whole blood and venous plasma result in similar glucose levels within the reference interval.

The following types of specimen are used in the different countries for determining blood glucose in routine diagnosis: Capillary whole blood, venous whole blood, and plasma from venous whole blood.

Capillary whole blood: samples should be collected by skin puncture from the finger or from the heel (infants only). In the fasting state there is no arteriovenous difference between arterial and venous blood. Therefore, the concentrations measured in venous and capillary whole blood are nearly identical. In the postprandial state, however, there may be a 20–70% difference /26/. The arteriovenous difference is greatest in lean nondiabetic individuals, smallest in diabetics, and larger with blood sampled from deep veins compared to blood from superficial veins /1/.

Compared to glucose measured in plasma, the glucose concentration in whole blood is influenced by the hematocrit (Hct), by proteins, lipoproteins and other dissolved and corpuscular components. The corresponding values for a glucose concentration in water of 180 mg/dL (10.0 mmol/L) are as follows /1/:

• In plasma: 168 mg/dL (9.3 mmol/L).
• In whole blood with 0.30 Hct: 155 mg/dL (8.6 mmol/L).
• In whole blood with 0.45 Hct: 150 mg/dL (8.3 mmol/L).
• In whole blood with 0.60 Hct: 144 mg/dL (8.0 mmol/L).

Venous plasma: the molality of glucose in whole blood and plasma is identical. However, the volume of water is about 11% higher in plasma than in whole blood. Therefore, glucose levels are also about 11% higher in plasma than in whole blood at a hematocrit of 0.43.

Serum: glucose levels are 5% lower in serum than in heparinized plasma /27/.

Sensor variability in continuous glucose monitoring (CGM): substantial variation is observed within sensors over time and across 2 different sensors worn simultaneously on the same individuals. In a study /41/ at baseline mean glucose for both sensors compared mean glucose was apparently 150 mg/dL (8.3 mmol/L) and time in range 70–180 mg/dL (3.9–10.0 mmol/L) was just below 80%. When comparing the same sensor at two different time points (two 2-week periods, 3 months apart), the within-person coefficient of variation (CVw) in mean glucose was 17.4% in the sensor of company one and 14.2% in the sensor for company two. CVw for percent time in range was 20.1% for sensor of company one and 18.6% for company two.

3.3.6.1 Glucose levels in different types of sample

A study /28/ has established the following conversion factors for the sample types capillary whole blood, venous whole blood, and venous plasma:

• Venous plasma/venous whole blood in diabetics and non diabetics: 1.148.
• Venous plasma/capillary whole blood in non diabetics: 0.997.
• Venous plasma/capillary whole blood in diabetics: 1.089 and overall mean 1.048.
• Capillary whole blood/venous whole blood in non diabetics: 1.173.
• Capillary whole blood/venous whole blood in diabetics: 1.055 and overall mean 1.155.

Recommendations

The International Federation of Clinical Chemistry (IFCC) recommends reporting the concentration of glucose in plasma, irrespective of the sample type and assay /29/. A constant factor of 1.11 is used to convert concentration in whole blood to the equivalent concentration in plasma. This applies to a Hct of 0.43. In the case of higher Hct values, as are typical in neonates, this factor must be increased by multiplication by the following correction factor (cf):

cf = 0.84/(0.93–0.22 Hct)

With a Hct of 0.70 and multiplication of 1.11 with cf, the new factor to convert concentration in whole blood to the equivalent concentration in plasma is 1.19.

According to the IFCC, it is possible to convert whole blood glucose and biosensor glucose to plasma glucose, but not whole blood glucose to biosensor glucose (Fig. 3.3-1 – Conversion factors for glucose). According to recommendations of the WHO, the cutoffs for fasting glucose and for the oral glucose tolerance test are identical for capillary whole blood and venous plasma.

Blood sampling

Fasting glucose: sampling 7 a.m. to 8 a.m. after at least 8 h of fasting.

Postprandial glucose: 1–2 h after a meal.

Type of sample

Capillary whole blood: only draw blood if blood circulation is good; finger must be warm. 0.01–0.02 mL of blood is added to hemolyzing solution.

Venous blood: is analyzed in the form of whole blood, plasma, and serum. In plasma and serum following blood collection is recommended in separator tubes. The collection tubes for determining glucose in whole blood contain NaF to prevent glycolysis, and potassium oxalate or Na₂EDTA to inhibit clotting. NaF acts by inhibiting glycolytic enzymes, in particular enolase, although the effect is minor in the first 2 h after blood collection. A better effect than with NaF alone is achieved by cooling the sample, by acidifying it, or by using citrate tubes for blood collection. If both glucose and lactate levels are to be determined, collection tubes containing NaF and citrate are suited best /30/.

Method of determination

The reference method is the hexokinase method, or in some countries the glucose oxidase method. Possible methodological errors are shown in Tab. 3.3-5 – Methodological errors in glucose measurement.

Drugs

In the glucose oxidase method, Novaminsulfon (metamizole) and ascorbic acid in concentrations > 0.4 g/l and α-methyldopa > 0.2 g/l can cause a decrease in the glucose level of up to 50% /31/.

Icodextrin /32/: the glucose polymer icodextrin is often added to the dialysis fluid during peritoneal dialysis so that an osmotic gradient can be maintained along the dialysis membrane and the ultrafiltration time can be prolonged. However, small amounts of icodextrin can get into the bloodstream via the lymphatic system. In the bloodstream it is hydrolyzed to glucose oligomers such as maltose and maltotriose. These oligomers cause falsely high glucose readings in some point-of-care glucometers.

Stability

Due to glycolysis, the glucose concentration in whole blood decreases by 5–7% (approx. 10 mg/dL; 0.6 mmol/L) per hour after blood sampling. At 4 °C there is only a slight decrease during the first 2 h and approx. 20% after 24 h /33/. The decrease depends on the glucose concentration, the ambient temperature, and the leukocyte count /13/. The decrease in glucose in whole blood within the first 2 h after blood sampling is approximately the same with and without NaF /34/.

In EDTA-coated collection tubes there is no significant decrease within 24 h in the presence of maleinimide.

Glucose concentrations measured in the serum/plasma of blood collected in tubes containing separator gel were comparable to those measured in plasma collected in tubes containing NaF and potassium oxalate /35/.

At 4 °C blood deproteinized by perchloric acid gives stable values in the supernatant, obtained by centrifugation, for at least 5 days.

Capillary blood, stabilized in the mentioned hemolyzation solutions shows stable glucose values for 48 h /2/.

In newborns, measurement of blood glucose should be performed as soon as possible after blood collection, since the rate of glycolysis of erythrocytes in newborns is considerably higher than in adults so that the glycolysis inhibitors cannot be as effective. Some cases of neonatal hypoglycemia are reported to be falsely low, particularly in those newborns with high Hct values /36/.

Sampling time

The biological variation in plasma glucose levels results from complex interaction of genetically anchored metabolic processes that are subject to strict hormone-controlled regulation. Diurnal variations expressed as amplitude/ Midline Estimating Statistic of Rhythm (MESOR) ratio, averaged 7.7% (range 5.9–9.3%). The amplitude of glucose levels decreased with increasing concentrations. Between 6.00 and 10.00 h the average decrease of 4% has to be considered. Nocturnal glucose samples accounted for only 5% of the total amount but contributed to 19.5% of all findings over 200 mg/dL (11.1 mmol/L) /40/.

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29. D’Orazio P, Burnett RW, Fogh-Andersen N, Jacobs E, Kuwa K, Külpmann WR, et al. Approved IFCC recommendation on reporting results for blood glucose Clin Chem Lab Med 2006; 44: 1486–90.

30. Bruns DE. Are fluoride-containing blood tubes still needed for glucose testing? Clin Biochem 20131; 46: 289–90.

31. Szasz G, Huth K, Busch EW, Koller PU, Stähler F, Vollmar J. In vivo drug interference with various glucose determinations compared with in vivo results. Z Klin Chem Klin Biochem 1974; 12: 256–61.

32. Apperloo JJ, Vader HL. A quantitative appraisal of interference by icodextrin metabolites in point-of-care glucose analysis. Clin Chem Lab Med 2005; 43: 314–8.

33. Miller SA, Wallace RJ, Musker DM, Septimus EJ, Kohl S, Baughn R. Hypoglycemia as a manifestation of sepsis. Am J Med 1980; 68: 649–54.

34. Mikesh LM, Bruns DE. Stabilization of glucose in blood specimens: mechanisms of delay in fluoride inhibition of glycolysis. Clin Chem 2008; 54: 930–2.

35. Chan AYW, Ho CS, Cockram CS, Swaminathan R. Handling of blood specimens for blood glucose analysis. J Clin Chem Clin Biochem 1990; 28: 185–6.

36. Fernandez L, Jee P, Klein MJ, Fischer P, Brooks SPJ. A comparison of glucose concentration in paired specimens collected in serum separator and fluoride/potassium oxalate blood containing tubes under survey field conditions. Clin Biochem 2013; 46: 285–8.

37. Tate PF, Clemens CA, Walters JE. Accuracy of home blood glucose monitoring. Diabetes Care; 1992; 15: 536–8.

38. Kane MJO, Pickup J. Self-monitoring of blood glucose in diabetes: is it worth it? Ann Clin Biochem 2009; 46: 2730–82.

39. Jenkins DJA, Deghan M, Mente A, Bangdiwala SI, Rangarajan S, Srichaikul K, et al. Glycemic index, glycemic load, and cardiovascular disease and mortality. N Engl J Med 2021. doi: 10.1056/NEJMoa2007123.

40. Özcürümez M, Arzideh F, Torge A, Figge A, Haeckel R, Streichert T. The influence of sampling time on indirect reference limits, and the estimation of biological variation of random plasma glucose concentrations. J Lab Med 2021; 45 (2): 111–9.

41. Selvin E, Wang D, Rooney MR, Fang M, Echouffo-Tcheugui JB, Zeger S, et al. Within person and between-sensor variability in continuous glucose monitoring metrics. Clin Chem 2023; 69 (2): 180–8.

3.4 Glucose in urine and extravascular fluids

Lothar Thomas

3.4.1 Indication

Urinary glucose

• Detection of glucosuria of unknown etiology
• Monitoring of diabetes mellitus therapy in patients who are unable to self-monitor their blood glucose levels.

Glucose in cerebrospinal fluid: suspected bacterial meningitis.

Glucose in extravascular fluids: suspected bacterial infection.

3.4.2 Method of determination

Qualitative, semi quantitative determination in urine

Test strip methods /1/: these methods are based on a glucose oxidase/peroxidase reaction with tetra methyl benzidine as redox indicator. The color of the reagent pad changes from yellow to green with increasing glucose concentration in the sample.

Other test strips use a potassium iodide chromogen instead of tetra methyl benzidine. In this case, the peroxidase-catalyzed oxidation causes the chromogen to turn from green to brown with increasing glucose concentration.

The semiquantitative tests work by the same principle of reaction. The intensity of the color that develops on the test pad indicates the glucose concentration in g/l.

Quantitative determination in urine

See blood glucose in Section 3.3 – Blood glucose.

3.4.3 Specimen

First- or second-void urine or urine of defined sampling periods, indication of collection volume, supplementation with 1 g of sodium azide per 24 h sampling period.

Other body fluids such as ascites: 0.1–1 mL

3.4.4 Reference interval

Refer to Tab. 3.4-1 – Glucose reference intervals in urine and body fluids.

3.4.5 Clinical significance

3.4.5.1 Urinary glucose and diabetes mellitus

The extent of glucosuria is the result of the glomerular filtration and tubular reabsorption of glucose. Up to a blood glucose concentration of 160–180 mg/dL (8.9–10.0 mmol/L), also called the renal threshold, all filtered glucose is reabsorbed by the renal tubules. When the blood glucose level exceeds the renal threshold, glucosuria occurs, which is an indirect indicator of hyperglycemia (Tab. 3.4-2 – Diseases and conditions associated with glucosuria).

Therefore, the finding of glucosuria is suggestive of the presence of diabetes and always requires further investigation. The urine test strip is unsuitable as a method of screening for diabetes. According to a study /3/, the diagnostic sensitivity is 55% with a specificity of 99%, the positive predictive value is 29% and the negative predictive value is 95%. This is due to the fact that in diabetics, especially elderly people, the renal threshold is raised.

Urinary glucose is no longer of relevance in the monitoring of glucose control in diabetics, because it is only a rough indicator of the glycemic state/4/. On the one hand, its usefulness is limited by the renal threshold of about 180 mg/dL (10.0 mmol/L), on the other hand a certain correlation between blood glucose concentration and urinary glucose excretion exists only with blood glucose levels up to about 140 mg/dL (7.8 mmol/L) (Fig. 3.4-1 – Urinary glucose excretion as a function of blood glucose concentration) /5/.

Pre-term infants are given glucose infusions for nutritional purposes. The amount of glucose administered should be limited such that the glucose concentration in urine does not exceed 20 g/l (see also Tab. 3.3-3 – Blood glucose in diabetes and various conditions).

3.4.5.2 Renal glucosuria

Renal glucosuria associated with normoglycemia /5/:

• Glucose-phosphate diabetes (glucosuria and phosphaturia)
• Fanconi syndrome (glucosuria, phosphaturia, aminoaciduria)
• Acquired tubular injury (pyelonephritis, glomerulonephritis, intoxication)
• Pregnancy
• Renal diabetes.

Renal diabetes is the most common of the various forms of glucosuria with normoglycemia.

Renal diabetes

Renal diabetes is a dominantly inherited form of glucosuria which mainly occurs in men. It is due to diminished reabsorption of glucose in the proximal tubules. In healthy individuals glucose is nearly completely reabsorbed in the proximal tubules and with a daily mean blood glucose level of 100 mg/dL (5.6 mmol/L) and a glomerular filtrate of 125 [mL × min.^–1 × (1,73 m²)^–1] 180 g of glucose is reabsorbed daily.

3.4.5.3 Fractional glucose extraction

Renal glucosuria is characterized by determining its fractional glucose extraction (FE_G). This is the ratio of the glucose excreted in urine and the glomerular filtered glucose. The FE_G is determined in first-void morning urine and calculated using the following equation:

Cr, creatinine; G, glucose

The reference interval of FE_G (%) =(1.5–7.5) × 10^–4. In renal glucosuria, the FE_G is generally decreased. In gestational glucosuria it is on average 22.9 × 10^–4 /6/.

Diagnostic information about glucose in cerebrospinal fluid and other extravascular body fluids are shown in Tab. 3.4-3 – Diagnostic significance of glucose measurements in CSF and extravascular fluids.

3.4.6 Comments and problems

Method of determination

The lower detection limit of the glucose test strip methods is 30–50 mg/dL (1.7–2.8 mmol/L). Concentrations over 250 mg/dL (13.9 mmol/L) are measured with increased imprecision.

The hexokinase and glucose dehydrogenase methods have no interference from substances physiologically occurring in urine, and negligible interference from drugs. Interferences with the use of reagent strips are shown in Tab. 3.4-4 – Interferences in urine glucose determination using the glucose oxidase method.

Both methods can be used for quantitative determination of glucose in urine. The hexokinase method is used to detect elevated levels in the rare condition of fructosuria. Fructose excretion is also increased if high doses of fructose (e.g., diabetic sweets) are ingested orally.

Stability in urine

The measurement should be performed within 2 h after urine collection. Unless the urine contains stabilizing additives, approx. 40% of glucose is lost within 24 h /11/. In the presence of bacteriuria, leukocyturia or hematuria there is an even greater decrease in glucose levels. To stabilize the urine sample, it is recommended to add sodium azide so that its final concentration in urine is approximately 1%.

References

1. Smalley DL, Bradley ME. New test for urinary glucose (BM 33071) evaluated. Clin Chem 1985; 31: 90–2.

2. Heimsoth VH, Graffe-Achelis Ch, Banauch D, Vollmar J. Referenzwerte für die Glucosekonzentration im Urin von Erwachsenen. Med Labor 1978; 31: 236–40.

3. Mehnert H, Sewering H, Reichstein W, Vogt H. Früherfassung von Diabetikern in München. Dtsch Med Wschr 1968; 93: 2044–7.

4. American Diabetes Association. Tests of glycemia in diabetes. Diabetes Care 1999; 22: S77–S79.

5. Küntzel W, Mitzkat HJ. Zur Diagnostik der renalen Glucosurie. Dtsch Med Wschr 1971; 96: 1130–2.

6. Renschler H. Der Einfluss der Nierenfunktion auf die Glucoseausscheidung Gesunder. Habilitationsschrift; Heidelberg 1964.

7. Kornelisse RF, de Groot R, Neijens HJ. Bacterial meningitis: mechanisms of disease and therapy. Eur J Pediatr 1995; 154: 85–96.

8. Runyon BA, Hoefs JC, Morgan TR. Ascites fluid analysis in malignancy related ascites. Hepatology 1988; 8: 1104–9.

9. Boggs DS, Kinasewitz GT. Review: pathophysiology of the pleural space. Am J Med Sci 1995; 309: 53–9.

10. Berg B. Ascorbate interference in the estimation of urinary glucose by test strips. J Clin Chem Clin Biochem 1986; 24: 89–96.

11. Lodd JA, Turner K. Evaluation of Trinder’s glucose oxidase method for measuring glucose in serum and urine. Clin Chem 1975; 21: 1754–6.

3.5 Oral glucose tolerance test (oGTT)

Lothar Thomas

The oGTT is the standard test for the diagnosis of impaired glucose tolerance (IGT) and describes the postprandial glucose state. In prediabetes and diabetes mellitus glucose tolerance is impaired. The American Diabetes Association (ADA) recommends fasting plasma glucose (FPG) as an acceptable screening test for prediabetes and diabetes and classifies an elevated FPG level as impaired fasting glucose (IFG) /1/. The oGTT is recommended for confirming IGT in patients with IFG. In Europe the oGTT is the preferred screening test for prediabetes and diabetes as it allows the detection of both IFG and IGT in a single test /2/.

3.5.1 Indication

The ADA recommends the performance of an oGTT in /3, 4, 5/:

• Individuals with IFG (100–125 mg/dL; 5.6–6.9 mmol/L)
• Individuals ≥ 45 years
• All individuals, regardless of age, with a body mass index (BMI) ≥ 25 kg/m² and at least one additional risk factor. Risk factors include: first-degree relatives with type 2 diabetes (T2D), arterial hypertension, dyslipidemia, cardiovascular disease, history of gestational diabetes, member of an ethnic group with a high prevalence of diabetes (see also Tab. 3.1-2 – Diagnosis of prediabetes and diabetes mellitus based on ADA criteria).
• In pregnant women at 24–28 weeks of gestation
• Individuals with glucosuria and normal FPG.

3.5.2 Test procedure

Preparation of the patient

To oGTT will provide valuable results if the following requirements have been met by the patient prior to the test:

• No caloric intake for at least 10–16 h.
• Maintenance of usual eating habits for at least 3 days (≥ 150 g carbohydrates per day)
• Dis continuation of interfering medications for at least 3 days prior to the test if this is possible without risk to the patient
• Test should be performed with the patient seated or lying down (no muscular effort); no smoking prior to or during the test
• A time interval to menstruation of at least 3 days.

Performance of the oGTT

After the collection of capillary or venous blood samples to determine fasting glucose, the patient drinks the following solutions, dissolved in 250–300 mL of water, over 5 minutes:

• 75 g of water-free glucose, or
• 82.5 g of glucose monohydrate, or
• an isocaloric amount of hydrolyzed starch.

Children are given 1.75 g per kg of body weight, up to a maximum of 75 g.

The test begins in the morning, between 8 a.m. and 9 a.m. After a fasting blood sample has been collected and the individual has started to drink the glucose solution, another blood sample is taken after 120 min. During the test, the patient should be at rest without stress.

Perfomance of oGTT for gestational diabetes

in Europe the 75-g oGTT is performed. Blood samples are collected at fasting as well as 60 and 120 min. after the start of the glucose drink.

In North America diagnosis of gestational diabetes can be accomplished with either of two strategies:

• One-step 75 g oGTT or
• Two-step approach with a 50-g (non-fasting) screen followed by a 100-g oGTT for those who screen positive.

3.5.3 Specimen

Capillary or venous blood for determining blood glucose, per sample: 0.01–0.02 mL

3.5.4 Clinical significance

3.5.4.1 Diagnosis of prediabetes and diabetes

The oGTT combines the measurements of fasting plasma glucose (FPG) and postprandial glucose (2-h level). The FPG value provides information about insulin secretion while the 2-h value is an indicator of insulin resistance. The following results are possible (Tab. 3.5-1 – Diagnosis of diabetes mellitus and impaired glucose tolerance using 75 g oGTT):

• Elevated FPG with normal 2-h value, the condition of impaired fasting glucose (IFG).
• Normal FPG with elevated 2-h value, the condition of impaired glucose tolerance (IGT).
• Combination of IFG and IGT without reaching diabetic glucose levels; a condition termed IFG/IGT.

In contrast to the ADA, the European Association for the Study of Diabetes prefers to recommend the oGTT for diabetes screening. The ADA recommends performing the oGTT after the FPG or the HbA_1c in cases where the risk of diabetes needs to be better differentiated /3/.

Since the oGTT measures both IFG and IGT, it identifies more individuals at risk of developing T2D than the IFG test.

To diagnose gestational diabetes in pregnant women, the ADA alternatively recommends /3/:

• The two-step approach, in which the 50-g oGTT is performed initially, followed by the 100-g oGTT for women who screen positive on the initial test
• The one-step approach, in which the 75-g oGTT is performed primarily. Blood samples are collected at fasting as well as 60 and 120 min. after the start of the glucose drink.

When interpreting the glucose results of the oGTT, the influencing factors listed in Tab. 3.5-2 – Screening for and diagnosis of gestational diabetes using 75 g oGTT must be taken into consideration.

3.5.4.2 Fasting plasma glucose (FPG)

The FPG value provides information on whether insulin secretion is diminished or there is increased hepatic glucose production /8/. If insulin secretion is diminished, hepatic glucose production in the post absorptive state will be increased, resulting in hyperglycemia. Individuals with normal FPG (below 100 mg/dL; 5.6 mmol/L) only have a 5.5% risk of developing T2D, while the risk is much higher in individuals with concentrations ≥ 126 mg/dL (7.0 mmol/L). The result of the oGTT is of importance in individuals with FPG levels in the range of 100–109 mg/dL (5.6–6.0 mmol/L) and 110–125 mg/dL (6.1–6.9 mmol/L), since there is a 9% respectively 26% chance that the oGTT result is diagnostic of diabetes /9/.

3.5.4.3 2-h postprandial glucose value

The 2-h glucose value from the oGTT is an indicator of impaired glucose tolerance (IGT) and results from the insulin resistance of the tissues, in particular muscle, liver and fat tissues /8/. The glucose load stimulates insulin secretion. This does, however, not lead to glucose absorption by the tissues, since the tissue insulin receptors do not respond adequately. As a result, there is prolonged hyperglycemia due to reduced glucose clearance. In the postprandial period, the rate of glucose clearance depends on the insulin-sensitive tissues. In diabetes, the postprandial state is characterized by a significant and prolonged increase in blood glucose concentration. The postprandial peak blood glucose levels are closely related to atherosclerosis and cardiovascular complications.

A differential diagnosis is made based on the 2-h glucose concentration:

• Impaired glucose tolerance (IGT), which is an indicator of prediabetes and insulin resistance.
• Diabetic glucose tolerance, which is usually indicative of the presence of T2D.

The prevalence of IGT is higher in women than in men; with IFG the reverse is the case. The higher prevalence of IFG in men is reported to be due to the fact that men have a lower hepatic insulin sensitivity than women. The higher prevalence of IGT in women is reported to be due to their shorter body height and different fat distribution with the same glucose load /10/.

3.5.4.4 Diagnostic value of the oGTT

Glucose intolerance proceeds continuously within the wide range of normal glucose tolerance up to pathological glucose levels. In this process, the two pathophysiologically important disorders, insulin resistance and β-cell dysfunction progressively worsen. Compared to individuals with normal glucose tolerance, those with IGT in the top quartile of glucose levels only have 20% of their β-cell function left /11/.

Only 28% of 25,000 patients who underwent an oGTT met both IGT and IFG criteria /12/. Lean elderly individuals were more likely to have IFG while obese middle-aged individuals were more likely to have IGT. The prevalence of newly detected T2D cases according to different criteria is shown in Fig. 3.5-1 – Prevalence of newly detected type 2 diabetes, impaired glucose tolerance and impaired fasting glucose and individuals with normal oGTT.

3.5.4.5 Cardiovascular disease and oGTT

One of the main goals in the diagnosis of prediabetes and T2D is the prevention of long-term complications. Epidemiological studies show that IGT is an earlier predictor of future diabetic complications than IFG. This applies to the assessment of the risk of cardiovascular disease (CVD) /13/, increased mortality /14/, and macrosomia /15/. For example, women with isolated postprandial hyperglycemia have a 3.2-fold higher risk for CVD /16/. A high percentage of patients with acute coronary syndrome (ACS) have a pathological oGTT at admission to hospital. A study /17/ showed that 32% of patients with ACS had T2D, 37% had IGT, and 8% had isolated IFG.

3.5.4.6 Hepatic steatosis and oGTT

In obese adolescents with hepatic steatosis, the 2-h oGTT glucose level increases with increasing steatosis. Approximately 73% of those with extended fatty liver met the criteria of metabolic syndrome /18/. A high prevalence of hepatic steatosis in association with T2D was found in adults /19/.

3.5.4.7 Gestational diabetes mellitus (GDM)

One-step strategy: a 75-g oGTT is performed /7/:

Glucose measurement when patient is fasting and at 1 and 2 hours. The diagnosis of GDM is made when any of the following plasma glucose values are met or exceeded:

• Fasting: 92 mg/dL (5.1 mmol/L)
• 1 h: 180 mg/dL (10.0 mmol/L)
• 2 h: 153 mg/dL (8.5 mmol/L) in capillary plasma

and no factors influence the glucose tolerance test (Tab. 3.5-2 – Factors influencing glucose tolerance). The criteria of the oGTT depend on the specimen collection (Tab. 3.5-3 – Criteria of 75 g oGTT depending on specimen selection).

Two-step strategy

A 50-g oGTT with plasma glucose measurement at 1 h, at 24–28 weeks of gestation in women not previously diagnosed with overt diabetes is performed. A 100-g oGTT is performed if plasma glucose level measured 1 h after load are met or exceeded 130 mg/dL, 135 mg/dL, or 140 mg/dL (7.2 mmol/l, 7.5 mmol/L, or 7.8 mmol/L).

Women who screen positive should then undergo the 100-g oGTT on a subsequent day to confirm the diagnosis of GDM. The ADA recommends performing the 100-g oGTT as a confirmatory test for women who screen positive on the 50-g oGTT. GDM is present when any of the following thresholds are exceeded /1/:

• Fasting ≥ 95 mg/dL (5.3 mmol/L)
• 1 h ≥ 180 mg/dL (10.0 mmol/L)
• 2 h ≥ 155 mg/dL (8.6 mmol/L)
• 3 h ≥ 140 mg/dL (7.8 mmol/L).

There is only weak diagnostic agreement between the results obtained with the 75-g oGTT and the 100-g oGTT. While the 100-g oGTT was diagnostic for GDM in 60 out of 484 pregnant women, the 75-g oGTT only identified 26 women as having GDM.

3.5.5 Comments and problems

Test procedure /20/

The oGTT provides only acceptable reproducibility. The main reason is the large inter individual variability of the glucose concentration, the influence of gastric emptying following ingestion of the hyper osmolar glucose solution, the ambient temperature, and imprecision of the glucose measurement.

The oGTT shows a wide range of variation in the 2-h glucose value in repeat tests, which is usually due to the failure to strictly adhere to the test conditions. Thus, the reproducibility was 49% in pre diabetics, 73% in diabetics, and 93% in normal individuals /21/. Differences in the speed of drinking and incorrect timing of blood collection after 2 h can lead to variations in the blood glucose concentration of up to 20% /22/. This leads to incorrect classification especially in the threshold range. As a result, a pathological result may not always be confirmed by the repeat test.

Factors influencing the oGTT

Refer to:

• Tab. 3.5-4 – Influencing factors that can cause a false pathological result in the oGTT
• Tab. 3.5-5 – Influencing factors that can lead to a false normal oGTT result.

References

1. American Diabetes Association. Classification and diagnosis of diabetes: standards of medical care in diabetes –2018. Diabetes Care 2018; 41, Suppl 1: S13-S27.

2. World Health Organisation. Definition, diagnosis and classification of diabetes mellitus and its complications: Report of a WHO consultation. Part 1: Diagnosis and classification of diabetes mellitus. Geneva; World Health Organisation: 1999.

3. Standards of medical care in diabetes – 2012. Diabetes Care 2012; 35, Suppl 1: S11–S63.

4. Executive Summary: Standards of medical care in diabetes: Diabetes Care 2012; 35, Suppl 1: S3–S10.

5. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2012; 33, Suppl 1: S64–S71.

6. American Diabetes Association. Standards of medical care in diabetes. Diabetes Care 2006; 29, Suppl 1: S4–42.

7. Deutsche Diabetes-Gesellschaft. Praxis-Leitlinien. Diabetes and Stoffwechsel 2002; 11 Suppl 2: 1–37.

8. Bartoli E, Castello L, Sainaghi PP, Schianca GPC. Progression from hidden to overt type 2 diabetes mellitus: significance of screening and importance of the laboratory. Clin Lab 2005; 51: 613–24.

9. Ryan J, Siriwardhana D, Vasikaran SD. An audit on oral glucose tolerance test at a large teaching hospital: indications, outcomes, and confounding factors. Ann Clin Biochem 2009; 46: 390–3.

10. Faerch K, Borch-Johnson K, Vaag A, Jorgensen T, Witte DR. Sex differences in glucose levels: a consequence of physiology or methodological convenience? The Inter99 Study. Diabetologia 2010; 53: 858–65.

11. DeFronzo RA, Banerji MA, Bray GA, Buchanan TA, Clement Ss, Henry RR, et al. Determinants of glucose tolerance in impaired glucose tolerance at baseline in the Actos Now for Prevention of Diabetes (ACT NOW) study. Diabetologia 2010; 53: 435–45.

12. DECODE Study Group on behalf of the European Diabetes Epidemiology Study Group. Will new diagnostic criteria for diabetes mellitus change phenotype of patients with diabetes? Reanalysis of European epidemiological data. BMJ 1998; 317: 371–5.

13. Barzilay JI, Spiekerman CF, Wahl PW, Kuller LH, Cushman M, Furberg CD, et al. Cardiovascular disease in older persons with glucose disorders: Comparison of American Diabetes Association criteria for diabetes mellitus with WHO criteria. Lancet 1999; 354: 622–5.

14. DECODE Study Group. Glucose tolerance and mortality: comparison of WHO and American Diabetes Association diagnostic criteria. The DECODE Study Group. European Diabetes Epidemiology Study Group. Diabetes epidemiology: Collaborative analysis of diagnostic criteria in Europe. Lancet 1999; 353: 617–21.

15. Metzger BE. International Association of Diabetes and Pregnancy Study Groups recommendations on the diagnosis and classification of hyperglycemia in pregnancy. Diabetes Care 2010; 33: 676–82.

16. Barrett-Connor E, Ferrara A. Isolated postchallenge hyperglycemia and the risk of fatal cardiovascular disease in older women and men. The Rancho Bernardo Study. Diabetes Care 1998; 21: 1236–9.

17. Ilany J, Marai I, Cohen O, Matetzky, Gorfine M, Erez I, et al. Glucose homeostasis abnormalities in cardiac intensive care unit patients. Acta Diabetol 2009; 46: 209–16.

18. Cali AMG, De Oliveira AM, Kim H, Chen S, Reyes-Mugica M, Escalera S, et al. Glucose dysregulation and hepatic steatosis in obese adolescents: is there a link? Hepatology 2009; 49: 1896–1903.

19. Kotronen A, Juurinen L, Hakkarainen A, Westerbacka J, Corner A, Bergholm R, et al. Liver fat is increased in type 2 diabetic patients and underestimated by serum alanine aminotransferase compared with equally obese nondiabetic subjects. Diabetes Care 2008; 21: 165–9.

20. Koehler C, Temelkova-Kirktschiev T, Schaper F, Fücker K, Hanefeld M. Prävalenz von neu entdecktem Typ 2 Diabetes, gestörter Glucosetoleranz and gestörter Nüchternglucose in einer Risikopopulation. Dtsch Med Wschr 1999; 124: 1057–1061.

21. Balion CM, Raina PS, Gerstein HC, Santaguida PL, Morrison KM, Booker L, et al. Reproducibility of impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) classification: a systematic review. Clin Chem Lab Med 2007; 45: 1180–5.

22. Harris PE, Kitange HM, Fulcher G, Alberti KGMM. The oral glucose tolerance test: effects of different glucose loads, reproducibility and the timing of blood glucose measurements. Diab Nutr Metab 1991; 4: 293–6.

3.6 Hemoglobin A_1c

Lothar Thomas

Hemoglobin A_1c (HbA_1c) is glycated hemoglobin in which glucose is attached to the N-terminal valine residue of each β-chain of hemoglobin A /1/. Measurement of HbA_1c is integral to the diagnosis and management of diabetes because it reflects the average glucose over the preceding 120 days. The HbA_1c is now recommended as a standard of care for testing and monitoring diabetes, specifically the type 2 diabetes /2/. Further the mean HbA_1c over time provides a reliable measure of chronic glycemia and correlates well with the risk of long term diabetes complications, so that it is currently considered the test of choice for monitoring and chronic management of diabetes /3/.

3.6.1 Indication

• Diagnosis of diabetes risk and manifest diabetes /4/ (Fig. 3.6-1 – Diagnosis of diabetes based on HbA_1c, fasting plasma glucose, and the oral glucose tolerance test).
• Monitoring of long-term glycemic status
• Determine whether the glycemic control regimen for an individual diabetic is adequate
• Determine whether a diabetic meets the treatment goals and has stable glycemic control
• Assessment of the risk of diabetic complications, in particular cardiovascular disease.

The HbA_1c is recommended to be performed at least twice a year in diabetes patients with stable blood glucose levels.

3.6.2 Method of determination

HbA_1c can be measured by several analytical methods including immunoassays, ion-exchange chromatography, boronate affinity chromatography, enzymatic methods, and capillary electrophoresis.

Cation exchange chromatography

Principle: glycation results in loss of positive charges on the surface of the Hb molecule. On weak cation exchangers and with increasing ion concentrations and/or decreasing pH, glycohemoglobins are eluted before non glycated hemoglobins. Following separation total Hb and HbA_1c are measured separately with a spectrophotometer. The proportion of HbA_1c is then calculated as the ratio of HbA_1c to total Hb. A methodology commonly used is reversed-phase chromatography, in which mixtures of aqueous buffers and organic solvents are used as the mobile phase.

Immunoassay

Principle: hemoglobin glycated at the β-N-terminal valine provides a well-defined epitope for antibodies. The determination can be performed by an enzyme immunoassays using monoclonal or polyclonal antibodies which specifically recognize epitopes consisting of the last 4 to 8 amino acids of the glycated N-terminal end of the β-chain of HbA_1c.

In a first step, the N-terminal of the glycosylated β-chain is cleaved off by a peptidase. The epitope is bound by specific antibodies, and determined in a latex enhanced immunoassay or solid-phase immunoassay.

The advantage of immunochemical assays is that there is no interference by abnormal hemoglobins or post translationally modified hemoglobins if a specific antibody is selected. The antibody detects only the first four amino acids of the β-chain, the keto amine bond and the glucose. The glycation at the -N-terminal valine can be accurately determined in sickle cell disease, because (β 6Glu-Val) and HbC (β 6Glu-Lys) only occur from position 6 /5/ (Fig. 3.6-2 – Reference material: Endoproteinase Glu-C cleaves the remaining 140 amino acids from HbA₀ and HbA_1c at the C-terminal side of glutamine). To prevent interference in the determination of HbA_1c by HbS and HbC variants, which occur in over 10% of African-Americans and in people of Arabic and Indian descent, some manufacturers use a special technology. The immunoassays, in which the antibodies are directed against a longer peptide, a second peptidase hydrolyzes the HbA_1c of the sample to a glycated pentapeptide which competes with the agglutinator (HbA_1c-loaded particle) for the anti-HbA_1c antibody, thereby reducing the rate of agglutinator. The immunochemical assays also measure the amount of HbA_2c, although the concentration is low and usually insignificant.

Capillary electrophoresis (CE)

The high resolution capability of CE allows the quantification of HbA_1c even in the presence of labile HbA_1c, acetylated and carbamylated hemoglobins and major hemoglobin variants /30/. These variants are commonly recognized as causes of interference in HbA_1c. By using an alkaline pH buffer, normal and abnormal or variant hemoglobins are detected in the following order from cathode to anode: A2/C, E, S/D, F, A0 other Hb and then HbA_1c /31/.

3.6.2.1 Standardization of HbA_1c

The HbA_1c test should be performed using a method that is certified by the U.S. National Glycohemoglobin Standardization Program (NGSP) and standardized or traceable to the Diabetes Control and Complications Trial reference assay /6/. A working group of the International Federation of Clinical Chemistry (IFCC) has developed a reference material for the standardization of HbA_1c which meets the requirements of the European Union Directive on in-vitro diagnostic medical devices (IVD) and follows the concept of metrological traceability.

The analyte measured is a hemoglobin molecule having a stable adduct of glucose to the N-terminal valine of the hemoglobin β-chain (βN-1-deoxyfructosyl-hemoglobin) /5/ (Fig. 3.6-2 – The endoproteinase Glu-C cleaves the N-terminal 6 amino acids from HbA₀ and HbA_1c). Pure HbA_1c and pure HbA₀ are isolated from human blood and mixed in well defined proportions to produce the certified primary reference material (PRMS) used for the reference measurement procedure. The PRMS values are assigned to secondary reference materials (SRMs) and the SRMs are used by the manufacturers to calibrate their instruments.

Result reporting

The results of the HbA_1c determination are reported in SI units (mmol/mol) according to IFCC, and related to NGSP units (% and one decimal) using the IFCC-NGSP master equation:

3.6.2.2 Reference to NGSP

Previously, all tests were related to the NGSP3.6.2.1 whose standard is an HbA_1c value determined with chromatographic assays in the Diabetes Control and Complication Trial (DCCT). The IFCC Working Group on HbA_1c Standardization prepared primary reference materials of pure HbA_1c and HbA₀ and developed a reference method for HbA_1c. Since the NGSP standard contains impurities, the values measured with the IFCC standardization program are 1.5–2% lower. It was agreed in a consensus statement that the values obtained with the IFCC standard program should be traced back to the NGSP, and HbA_1c should be reported both in % and in mmol HbA_1c per mol Hb.

Reference method /7/

Principle: the determination is performed in three steps: First, the N-terminal end of the hemoglobin β-chain, which is obtained from washed and lysed erythrocytes of the sample, is cleaved off by endoproteinase Gluc-C. High-pressure liquid chromatography, followed by quantification by electrospray ionization mass spectrometry or capillary electrophoresis. Hb A1c is measured as the ratio of glycated to non glycated N-terminal peptide and is reported as a percentage.

In the last step, the glycated (HbA_1c peptide) and non glycated (HbA₀ peptide) hexapeptides are quantified using mass spectrometry or capillary electrophoresis and UV detection. The percentage of HbA_1c is determined based on the ratio of glycated to non glycated N-terminal hexapeptides of the hemoglobin β-chain.

3.6.3 Specimen

EDTA blood: 1 mL (non-fasting patient)

3.6.4 Reference interval

Refer to Ref. /8/ and Tab. 3.6-1 – Standard interpretation of HbA_1c.

3.6.5 Clinical significance

The HbA_1c value reflects the average blood glucose level of the past 2–3 months. A multinational study /9/ investigated the relationship between HbA_1c and the average blood glucose (AG) over 3 months. The best relationship provided the following linear regression between HbA_1c and AG:

Although the relationship between HbA_1c and average glucose over the preceding 120 days is linear, wide variation was observed between individuals. An HbA_1c value of 6.5%, the threshold for diagnosis of diabetes, was associated with an average glucose from 125–175 mg/dL (6.9–9.7 mmol/l) while HbA_1c concentrations ranged between 5.5% and 8.0% when average glucose was 150 mg/dL /9/. See Tab. 3.1-10 – Correlation of plasma glucose level with the HbA_1c concentration.

Three-month testing intervals can determine if glycemic targets are met and maintained although frequency of testing is based on clinical assessment and the specific testing plan.

3.6.5.1 Assessment of the HbA_1c level

Mathematical models and practical experience show that a glycemic change on day 1 of the 120-day life span of the erythrocytes is not fully reflected in a change in HbA_1c levels. A significant increase or decrease in mean blood glucose leads to a relatively rapid and significant change in HbA_1c. Regardless of the initial HbA_1c, it takes 30–35 days to obtain a mean between the initial value and the new final value. Consequently, it only takes 1–2 weeks, not 3–4 months, before a significant change in blood glucose is reflected in a marked change in the HbA_1c value. Although the HbA_1c level in principle reflects the average blood glucose levels of the past 120 days, hyperglycemic events in days 1–30 contribute approximately 50% to the final result, while events in days 90–120 only contribute about 10% /10/. The level of HbA_1c correlates more strongly with fasting glucose than with postprandial glucose.

Physiological and pathophysiological conditions can change HbA_1c concentration independently of glucose (e.g., race, advanced age, hemoglobin variants, iron deficiency with and without anemia, red blood cell turnover, chronic kidney disease, and cardiovascular events).

• A reduced life span, as is the case in hemolytic anemias (autoimmune hemolytic anemia, hereditary spherocytosis, sickle-cell anemia, thalassemia), iron deficiency anemia, and blood loss, shortens the time during which Hb is in contact with glucose in the bloodstream, leading to falsely low HbA_1c levels.
• An increase in the life span of the red blood cells (iron deficiency, vitamin B₁₂ deficiency and folic acid deficiency) prolongs the contact between Hb and the glucose in the bloodstream, leading to falsely elevated HbA_1c values.

In conditions associated with increased red blood cell turnover, such as sickle cell disease, pregnancy (second and third trimesters) , hemodialysis, recent blood loss or transfusion, or erythropoietin therapy, only plasma glucose criteria should be used to diagnose diabetes /2/.

Due to the intraindividual changes of the Hb levels and the imprecision of the assays, variations between measurements must be at least 0.5% HbA_1c before they can be considered to be of clinical relevance. Therefore there should be an interval of 4 to 6 weeks between two HbA_1c measurements.

Since the results of the HbA_1c measurements are calculated as a ratio of HbA_1c to total hemoglobin, they are not influenced by body position or by sampling, whether venous or capillary blood is collected.

3.6.5.2 HbA_1c level and diagnosis of diabetes

The ADA has recommended HbA_1c with a cutoff ≥ 6.5% for diagnosing diabetes as an alternative to fasting plasma glucose (FPG) ≥ 126 mg/dL (7.0 mmol/L). FPG and oral glucose tolerance tests are recommended for the diagnosis of diabetes only if HbA_1c testing is not possible due to patient factors that preclude its interpretation and during pregnancy /2, 3/. The HbA_1c cutoff of ≥ 6.5% was associated with 3.8% false negative predictions, while the majority of false negative patients had borderline FPG (7.0–8.0 mmol/L) and HBA_1c (6.0%–6.5%), and therefore belonged to at-risk category on the basis of HbA_1c alone criteria /11/. See Tab. 3.6-2 – HbA_1c and hyperglycemia.

3.6.5.3 Metabolic control of type 2 diabetes

The HbA_1c test should be performed for metabolic control at least twice per year. Apart from providing an assessment of the glycemic status of the past 2–3 months and of the risk of future diabetic complications, the HbA_1c per se also contributes to improving glycemic control. Thus, it has been shown that patients who know their HbA_1c value and know how to interpret it have better glycemic control than patients who don’t /12/. A position paper by the ADA and the European Association for the Study of Diabetes recommends the following glycemic targets /13/:

• HbA_1c of 6.0–6.5% in selected patients (e.g., those with short diabetes history, long life expectancy, no significant cardiovascular disease)
• HbA_1c < 7.0% in most patients to reduce the incidence of microvascular complications
• HbA_1c of 7.5–8.0% in patients with a history of severe hypoglycemia, extensive comorbid conditions, advanced secondary complications, and limited life expectancy.

3.6.6 Comments and problems

Point-of-care HbA_1c

The American Diabetes Association (ADA) guidelines include use of Clinical Laboratory Improvement Amendments (CLIA)-waived point-of-care HbA_1c testing for diabetes monitoring but not diagnosis /2/.

Stability

During storage of the sample over 3–4 days, the decline of erythrocyte metabolism leads to the formation of glutathione adducts of Hb (HbA_1d/HbA₃), which can interfere with HbA_1c especially if chromatographic assays are used. Hemolysates are more unstable as whole blood.

Hemoglobinopathy

Over 1,200 hemoglobin variants have been identified; the gene β is involved in about 70% of these. While the vast majority are uncommon or rare, certain Hb variants, particularly HbAS, HbAC, HbAD, and HbAE, occur at relatively high frequencies in some populations. One cannot measure HbA_1c in individuals who are homozygous for these common variants or who have HbSC disease because they have no HbA /14/.

Disorders of Hb synthesis can be due to:

• Reduced or absent α- or β-chain production (α- or β-thalassemia), due to homozygous or heterozygous inheritance
• Changes in the structure of Hb. Hemoglobin anomalies, also named Hb variants, such as HbS, HbC (Africans), HbE (South East Asians), HbD, or Hb defects such as Hb New York are produced. Homozygous patients for these variants cannot be tested for HbA_1c. Patients who are heterozygous for these variants can be tested for HbA_1c with suitable methods. These include immunological assays, affinity chromatography, and some reversed-phase HPLC assays. However, immunological assays have also been reported to be susceptible to interference from Hb variants such as Hb Okayama (ASβ2; His/Gln); Hb Graz (ASβ2; His/Leu).
• In newborns, fetal hemoglobin (HbF) accounts for approximately 80% of total Hb, with the percentage falling to below 1% within the first months of life.

HbF interference in chromatographic assays is likely:

• In infants up to 9 month of age
• In adults with a rare HbF persistence
• In compensatory overproduction of HbF in thalassemia.

Drugs, stimulants, chemicals

Stimulants or environmental chemicals can form adducts with Hb which can then interfere with chromatographic assays in particular. This is the case with acetylsalicylic acid (acetylated hemoglobin) and alcohol (acetaldehyde adducts of Hb). Interference should be taken into consideration in the case of individuals who chronically ingest large amounts of these substances /15/.

Renal failure

Urea partly spontaneously decomposes to cyanate and ammonium ions. Cyanate, in the form of isocyanate, forms stable bonds with numerous proteins by carbamylation. In individuals with renal failure and elevated urea levels, the cyanate concentration increases, leading to the presence of carbamylated hemoglobins, which can interfere with HbA_1c if chromatographic assays are used /16/. Patients with renal failure, in particular uremic patients, often have impaired erythrocyte kinetics and a reduced erythrocyte life span. This can complicate interpretation of HbA_1c results.

Iron deficiency

The presence of iron deficiency with or without anemia leads to an increase in HbA_1c values compared to controls, with no concomitant rise in glucose indices /27/. Iron deficiency is associated with shifts in HbA_1c distribution from < 5.5 to ≥ 5.5% /28/.

Biological and analytical variation

HbA_1c levels increase by 0.1% per decade after 30 years of age /2/. African Americans with diabetes have significantly higher HbA_1c concentrations than white patients. The mean between-group difference was estimated to be approximately 0.65% HbA_1c. Black patients heterozygous for the common Hb Variant HbS may have lower HbA_1c by about 0.35 than those without this the trait /14/.

Biological variability: intraindividual variability 1.7%, inter individual variability 4% /17/.

Analytical goal: see Tab. 3.6-3 – Analytical goals for an HbA_1c method with reference to the IFCC reference method.

Misinterpretations

Falsely low HbA_1c values are the result of /18/:

• Rapidly developing diabetes
• Reduced erythrocyte life span or hyper regenerative erythropoiesis (hemolytic anemia, recent blood loss, blood transfusion, erythropoietin therapy, malaria, folic acid or vitamin B₁₂ deficiency).
• Increase in reticulocyte count higher than 3.2% reduce HbA_1c values.

Falsely high HbA_1c concentrations are the result of:

• Iron deficiency and iron deficient anemia, but decline in HbA_1c under therapy
• Renal failure
• Splenectomy
• Anti-retroviral therapy in HIV patients.

Glycated albumin

Studies have recommended glycated albumin (GA) as a useful alternative to HbA_1c. GA has a good diagnostic accuracy. A GA of 17.1% may be considered optimal for diagnosing diabetes in previously undiagnosed individuals /29/.

Myeloproliferative neoplasm

Myeloproliferative neoplasms such as chronic myeloid leukemia can cause an abnormal HbA_1c-result. In a study /31/ electropherogram from the HbA_1c-analysis by capillary electrophoresis showed unusual shifts in hemoglobin peak migration times. The right shift of the profile could not determine a HbA1c-value because peak assignments within standard zones for HbA_1c. The authors hypothesized that patients with hyperleukocytosis may have an accelerated migration speed of their sample, or that hyperviscosity was an other potential interference /31/.

3.6.7 Pathophysiology

The International Union of Pure and Applied Chemistry has recommended to use the term glycohemoglobin for the spontaneously i.e., non-enzymatically occurring glycation of hemoglobin (Hb). All hemoglobins glycated both at the N-terminal end of the β-chain as well as at other free amino groups are referred to as total glycohemoglobin /19/. The total glycohemoglobin is subdivided into sub fractions depending each on the glycation sites and reaction partners. The native (nonglycated) Hb is A₀. The sub fractions (HbA_{1a1 ,} HbA_{1a2 ,} HbA_1ab and HbA_1ac ) are produced by glycation of the amino group of the N-terminal amino acid valine of the Hb β-chain with different carbohydrates. The sum of these sub fractions are called HbA_1.

Proteins are frequently linked to sugar molecules during various enzymatic and nonenzymatic reactions that alter protein function. Glycosylation refers to an enzyme-mediated modification. Glycation refers to a monosaccharide (usually glucose) attaching nonenzymatically. In the case of Hb the glycation occurs by the reaction between the glucose and the N-terminal end of the β-chain, which forms a Schiff base. During the rearrangement, the Schiff base is converted into Amadori products.

Two steps are important (Fig. 3.6-3 – Reaction scheme of the glycation of N-terminal valine of Hb with glucose and subsequent Amadori rearrangement):

• In the first step glucose in the open chain format binds to the N-terminal to form an aldimine in a reversible reaction
• In the second step aldimine is gradually converted into the stable keto amine form (Amadori product), namely glycohemoglobin.

The major sites of Hb glycation are β-Val-1, β-Lys-66 and α-Lys 61. The glycation occurs continuously in vivo. However, as the average plasma glucose increases, so does the amount of glycated Hb in the red cells. Glucose passes from plasma through the red cell membrane and binds to Hb, forming an unstable product called aldimine, which then undergoes an Amadori rearrangement to produce a stable keto amine, namely glycohemoglobin). The life of Hb is defined by the erythrocyte survival time which is relatively constant at 10 to 120 days. The degree of glycation , apart from the life time of erythrocytes, depends essentially on the degree as well as the duration of the blood glucose elevation.

The glycation is irreversible and enzyme reactions for the degradation of hemoglobins are not known. The formation of HbA_1c within the red cell, therefore, reflects an estimate of the average level of glucose to which the red cell has been exposed. The rate of formation of HbA_1c is directly proportional to the glucose level in the blood and represents integrated values for glucose over the preceding 8–12 weeks before blood sampling. The concentration of HbA_1c is relatively constant compared to that of glucose and, unlike glucose, is not influenced by food intake or physical activity. The clinical value of HbA_1c was evaluated in the Diabetes Control and Complications Trial, which demonstrated a direct relationship between the blood glucose concentration, measured as HbA_1c, and microvascular complications of type 1 diabetes (T1D) /20/. Subsequent studies found that there is also a correlation between HbA_1c and microvascular complications of type 2 diabetes (T2D) /21/.

References

1. Sacks DB, Bebu I, Lachin JM. Refining measurement of hemoglobin A1c. Clin Chem 2017; 63: 1433–5.

2. American Diabetes Association. 2. Classification and diagnosis of diabetes: standards of medical care in diabetes – 2018. Diabetes Care 2018; 41, Suppl 1: S13-S27.

3. Sherwani SI, Khan HA, Ekhzaimy A, Masood A, Sakharkar MK. Significance of HbA_1c test in diagnosis and prognosis of diabetes patients. Biomarkers Insights 2016; 11: 95- 104.

4. Kerner W, Brückel J. Definition, Klassifikation und Diagnostik des Diabetes mellitus. Diabetologie 2011; 6: S 107–S110.

5. Schleicher E. Neuer Parameter für die Stoffwechseleinstellung: Durchschnittsglucose statt HbA_1c? Klinische Chemie Mitteilungen 2009; 40 (3): 63–7.

6. Hanas R, John G. 2010 consensus statement on the worldwide standardization of hemoglobin A_1c measurement. Ann Clin Biochem 2010; 47: 290–1.

7. Hoelzel W, Weykamp C, Jeppsson JO, Miedema K, Barr JR, Goodall I, et al. IFCC reference system for measurement of hemoglobin A_1c in human blood and the national standard schemes in the United States, Japan, and Sweden: A method-comparison study. Clin Chem 2004; 50: 166–74.

8. The International Expert Committee. International Expert Committee Report on the role of A1c assay in the diagnosis of diabetes. Diabetes Care 2009; 32: 1327–34.

9. Nathan DM, Kuenen J, Borg R, Zheng H, Schoenfeld D, Heine RJ. Translating the hemoglobin A1c assay into estimated average glucose values. Diabetes Care 2008; 31: 1473–8.

10. Goldstein DE, Little RR, Lorenz RA, Malone JI, Nathan D, Peterson CM, Sacks DB. Tests of glycemia in diabetes. Diabetes Care 2004; 27: 1761–73.

11. Khan HA, Ola MS, Alhomida AS Sobki SH, Khan SA. Evaluation of HbA_1c criteria for diagnosis of diabetes mellitus: a retrospective study of 12785 type 2 Saudi male patients. Endocr Res 2014; 39: 62–6.

12. Inzucchi SE, Bergenstal RM, Buse JB, Diamant M, Ferrannini E, Nauck M, et al. Management of hyperglycemia in type 2 diabetes: a patient-centered approach. Diabetes Care 2012; 35: 1364–79.

13. Costorino K, Jovanovic L. Pregnancy and diabetes management: advances and controversies. Clin Chem 2011; 57: 221–30.

14. Welsh KJ, Kirkman MS, Sacks DB.Role of glycated proteins in the diagnosis and management of diabetes: research gaps and future directions. Diabetes Care 2016; 39: 1299–1306.

15. Niederau Cm, Coe A, Katayama Y. Interferences of non-glucose adducts on the determination of glycated hemoglobind. Klin Lab 1993; 39: 1015–23.

16. Flückiger R, Harmon W, Meier W, Loo S, Gabbay KH. Hemoglobin carbamylation in uremia. N Engl J Med 1981; 304: 823–7.

17. Braga F, Dolci A, Montagnana M, Pagani F, Paleari R, Guidi GC, et al. Revaluation of biological variation of glycated hemoglobin (HbA_1c) using an accurately designed protocol and an assay traceable to the IFCC reference system. Clin Chim Acta 2011; 412: 1412–6.

18. Lapolla A, Mosca A, Fedele D. The general use of glycated haemoglobin for the diagnosis of diabetes and other categories of glucose intolerance: Still a long way to go. Nutrition, Metabolism & Cardiovascular diseases 2011; 21: 467–75.

19. Sacks DB. Translating hemoglobin A1c into average blood glucose: implications for clinical chemistry. Clin Chem 2008; 54: 1756–8.

20. The Diabetes Control and Complications Trial (DCCT). The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993; 329: 977–86.

21. UK Prospective Diabetes Study Group. UKPDS 33: Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes. Lancet 1998; 352: 837–52.

22. American Diabetes Association. Standards of medical care in diabetes – 2014. Diabetes Care 2014; 37, Suppl 1: S14–S80.

23. Selvin E, Steffens MW, Zhu H, Matsushita K, Wagenknecht L, Pankow J, et al. Glycated hemoglobin, diabetes, and cardiovascular risk in nondiabetic adults. N Engl J Med 2010; 362: 800–11.

24. Selvin E, Ning Y, Steffens MW, Bash LD, Klein R, Womg TY, et al. Glycated hemoglobin and the risk of kidney disease and retinopathy in adults with and without diabetes. Diabetes 2011; 60: 298–305.

25. Riddle MC, Ambrosius WT, Brillon DJ, Buse JB, Byington RB, Cohen RM, et al. Epidemiologic relationships between A1c and all-cause mortality during a median 3.4-year follow-up of glycemic treatment in the ACCORD trial. Diabetes Care 2010; 33: 983–90.

26. Zhang X, Gregg EW, Williamson DF, Barker LE, Thomas W, Bullard KM, et al. A1c level and future risk of diabetes: a systematic review. Diabetes Care 2010; 33: 1665–73.

27. English E, Idris I, Smith G, Dhatariya K, Kilpatrick ES, John WG. The effect of anemia and abnormalities of erythrocyte indices on HbA_1c analysis: a systematic review. Diabetologia 2015; 58: 1409–28.

28. Kim C, McKeever Bullard K, Herman WH, Beckles GL. Association between iron deficiency and A1c levels among adults without diabetes in the National Health and Nutrition Examination Survey, 1999–2006. Diabetes Care 2010; 33: 780–5.

29. Chume FC, Freitas PAC, Schiavenin LG, Pimentel AL,Camargo JL. Glycated albumin in diabetes mellitus: a metaanalysis of diagnostic test accuracy. Clin Chem Lab Med 2022; 60 (7): 961–74.

30. Sebia. CAPI 3 HbA1c-2019/12 Product insert. Sebia 2019.

31. Mathewson N, Pearson L, Doyle K. Abnormal HbA1c: Result from a diabetic patient leads to unexpected diagnosis. Clin Chem 2023; 69 (7): 684–9.

3.7 Insulin, C-peptide, proinsulin

Lothar Thomas

Pancreatic beta cells are specialized endocrine cells that continuously sense the concentration of blood glucose and other fuels. In response the beta cells secrete insulin to maintain normal fuel homeostasis. The insulin mRNA is translated as the single chain precursor pre proinsulin, and removal of its signal peptide during insertion into the endoplasmic reticulum generates proinsulin. The conversion of proinsulin leads to equimolar production of insulin and C-peptide.

Proinsulin has a direct biologic effect which is one-tenth as much as that of insulin. An increased serum proinsulin-to-insulin ratio is associated with beta-cell dysfunction.

C-peptide is a proinsulin cleavage product and released from the pancreas in amounts equimolar to insulin. C-peptide has no biologic activity on homologous or heterologous tissue and no ability to modify the action of insulin and/or proinsulin.

Insulin is secreted primarily in response to elevated concentrations of blood glucose and increased concentrations of other fuel molecules (e.g., fatty acids and amino acids). Insulin is in charge of facilitating glucose entry into cells.

3.7.1 Indication

Insulin, C-peptide and proinsulin measurements are indicated:

• Occasionally to estimate the insulin reserve in diabetics
• More frequently as part of functional tests for the evaluation the cause hypoglycemia
• In individuals with suspected prediabetes or diabetic metabolism.

Insulin

• Diagnosis and differentiation of hypoglycemia
• In the homeostasis model assessment (HOMA) for quantifying insulin resistance and β-cell function
• Determination of insulin sensitivity with the hyperinsulinemic clamp.

C-peptide

• Differentiation of hypoglycemia
• Estimation of the insulin reserve in diabetics.

Proinsulin

• Diagnosis of insulin resistance
• Differentiation of hypoglycemia.
• Suspicion of insulin-producing islet cell tumor (insulinoma)

3.7.2 Functional tests

Functional tests in which insulin, C-peptide and proinsulin levels are measured are listed in Tab. 3.7-1 – Functional tests for the diagnosis and differentiation of hypoglycemia syndrome.

3.7.3 Method of determination

Insulin

Immunoassay: Most assays use mouse monoclonal anti-insulin antibodies labeled e.g., with acridinium ester, alkaline phosphatase, or ruthenium. An anti-mouse antibody is coupled to a solid phase and binds the insulin-anti insulin antibody complex. After excess mouse monoclonal anti-insulin antibodies have been washed out, the signal emitted by the solid-phase bound insulin-anti-insulin antibody complex is measured. Many commercial assays are specific for insulin and have little cross-reactivity with proinsulin and proinsulin cleavage products /1/.

In the diagnosis of insulinoma, meaningful information can be obtained with assays that have cross-reactivity with des-31,32-split proinsulin and des-64,65 proinsulin, since insulinomas release fragments of insulin and proinsulin.

C-peptide

Immunoassay: these assays use monoclonal anti-C-peptide antibodies labeled e.g., with acridinium ester, alkaline phosphatase, or ruthenium. These antibodies compete with the C-peptide of the sample for binding to a solid-phase bound antibody. After excess anti-C-peptide antibodies have been washed out, the signal emitted by the bound C-peptide antibody complex is measured. The results generated by different commercial assays do not always agree, especially at higher concentrations of C-peptide /2/.

Proinsulin

Immunoassay: two-site immunoassays are described, in which one antibody is bound to the solid phase of the micro titer plate and the other is a soluble anti-proinsulin antibody labeled with acridinium ester. The intact proinsulin assay is specific for intact proinsulin while the total proinsulin assay measures all circulating forms of proinsulin /3/.

3.7.4 Specimen

Insulin

Serum (separate serum from whole blood within 2 h and store at 4 °C for same-day analysis), EDTA plasma is preferable due to higher stability: 1 mL

C-peptide

Serum (immediately separate serum from whole blood and freeze if analysis is not performed on the same day): 1 mL

Proinsulin

Serum (separate serum from whole blood within 2 h and store at 4 °C for same-day analysis): 1 mL

EDTA plasma (store at 4 °C if analysis is not performed on the same day): 1 mL

3.7.5 Reference interval

Refer to Tab. 3.7-2 – Insulin, C-peptide and proinsulin reference intervals.

3.7.6 Clinical significance

The secretion of insulin by the β-cells undergoes considerable physiological variations throughout the day which are reflected in fluctuations in insulin levels between 6 and 100 mU/L (42–700 pmol/L).

A pathological β-cell function can be associated with the following conditions and symptoms:

• Hyperinsulinemia associated with hypoglycemia
• Hyperinsulinemia associated with normoglycemia (insulin resistance)
• Hyperinsulinemia with impaired glucose tolerance (prediabetes)
• Hypoinsulinemia associated with hyperglycemia (overt diabetes).

The assessment of the functional tests is shown in

• Tab. 3.7-3 – Clinical significance of functional tests for hyperinsulinism-induced hypoglycemia, Tab. 3.7-4 – Interpretation of the 72-h fast
• Fig. 3.7-1 – Glucose, insulin, C-peptide, proinsulin and β-hydroxybutyrate at the end of the 72-h fast.

3.7.6.1 Hyperinsulinemia associated with hypoglycemia

Hypoglycemia can only develop if glucose consumption exceeds glucose production. In individuals with normal carbohydrate metabolism, a decline in plasma glucose levels to below 72 mg/dL (4.0 mmol/L) will cause insulin secretion to decrease and finally stop. However, due to the counter regulatory glucagon response, hypoglycemia only occurs after extreme stress, as a result of fasting in combination with alcohol consumption, and occasionally in pregnancy. In children, hypoglycemia can develop after only 12 h of fasting. Young women are sometimes diagnosed with asymptomatic hypoglycemia after fasting.

Insulinoma was once thought to be the only cause of the conditions of Whipple’s triad in apparently healthy individuals. This is no longer true, since there are other disorders of metabolism and of the endocrine glands that can lead to Whipple’s triad with inadequate insulin secretion /7/ (see Section 3.2 – Hypoglycemia syndromes). The Whipple’s triad is associated with characteristic clinical symptoms, provoked by fasting hypoglycemia with glucose levels of approximately below 50 mg/dL (2.8 mmol/L), which resolve after glucose is administered. Refer to

• Tab. 3.7-4 – Interpretation of the 72-h fast
• Tab. 3.7-5 – Diseases and conditions associated with hyperinsulinism and hypoglycemia.

The main problem with the diagnosis of hypoglycemia lies in its actual definition. It can be defined as glucose levels /8/:

• In the range of those of fasting healthy individuals
• In the range of those of patients with insulinoma
• In a range in which a physiological hormonal response occurs
• In a range in which clinical autonomic or neuroglycopenic symptoms first develop.

Since the threshold concentration of glucose is the cutoff for the decision as to when there is inadequate insulin response in hypoglycemia, it is important to know the thresholds for adequate hormone secretion.

According to two studies, these are as follows:

• At a glucose concentration ≥ 40 mg/dL (2.2 mmol/L) in venous plasma after a 72-h fast; below 36 pmol/L for insulin (measured using an unspecific radioimmunoassay), below 200 pmol/L for C-peptide, and below 5 pmol/L for proinsulin /7, 9/
• At a glucose concentration ≥ 45 mg/dL (2.5 mmol/L) after an 18-h fast and subsequent stationary cycling; below 30 pmol/L for insulin (specific assay), below 100 pmol/L for C-peptide, and below 20 pmol/L for proinsulin /7/.

Functional tests

To associate a hypoglycemia syndrome with hyperinsulinism and to carry out a diagnostic workup, functional tests, such as the fasting test (up to 72 h, or 18 h of fasting combined with cycling), C-peptide suppression test, tolbutamide test, and glucagon test, are performed depending on the patient’s situation. During these tests, it may be necessary to determine insulin, C-peptide, proinsulin, β-hydroxy butyrate and sulfonylurea concentrations. The additional determination of C-peptide levels is performed in order to confirm hyperinsulinism and to establish whether it is due to endogenous or exogenous causes.

3.7.6.2 Hypoinsulinemia associated with hyperglycemia

Based on the presence of autoantibodies (A⁺) or absence of them (A^–) and the presence of β-cell functional reserve (β⁺) or absence of β-cell functional reserve (β^–), there are four subgroups of diabetes mellitus /16/:

• A⁺ and β^– patients with autoantibodies but no β-cell reserve (insulin reserve)
• A⁺ and β⁺ patients with autoantibodies and preserved β-cell reserve (insulin reserve)
• A^– and β^– patients without autoantibodies and without β-cell reserve (insulin reserve).
• A^– and β⁺ patients without antibodies with β-cell reserve (insulin reserve).

Patients A⁺β^– and A^–β^– are genetically and immunologically different, but have the same clinical picture of type 1 diabetes. Patients A⁺β⁺ and A^–β⁺ are also immunologically and genetically different, but have the same clinical picture of type 2 diabetes with preserved insulin reserve.

The autoantibody diagnosis and determination of β-cell reserve are important classification and prognostic criteria for diagnosing the different phenotypes of diabetes mellitus. At initial manifestation, the presentation is as follows:

• LADA and type 1.5 diabetes (see Tab. 3.1-1 – Classification of diabetes) as well as the slowly progressing type 1 with autoantibodies but preserved β-cell reserve. LADA excludes patients who become dependent on insulin within the first six months after diagnosis.
• Phenotype A⁺β^– is always insulin dependent, and type A⁺β⁺ is insulin dependent in 90% of cases, if ketoacidosis is present as initial manifestation.

3.7.6.3 Determination of β-cell functional reserve

This determination allows clinicians to predict the short-term course of the disease. In the case of initial manifestation of diabetes mellitus and the possible presence of diabetic ketoacidosis (DKA), β-cell functional reserve is determined within 2–10 weeks following the correction of the DKA. For this purpose, baseline C-peptide levels are determined in fasting blood, and the time course of C-peptide levels in the glucagon test.

3.7.6.4 Glucagon test

In this test, blood is collected in the fasting state to determine baseline insulin levels, then 1 mg of glucagon is administered intravenously and blood is collected again at 5 and 10 min. to determine peak levels. The interpretation is shown in Tab. 3.7-6 – Interpretation of baseline and glucagon-stimulated C-peptide levels in diabetics. If insulin reserve (β-cell reserve) is positive, it can be expected to be preserved for 6 months to 1 year, depending on the phenotype.

3.7.6.5 Insulin resistance and β-cell dysfunction

Insulin resistance

Insulin resistance is a condition in which the body produces insulin, but the tissues, in particular muscle, liver and fat tissues, are insensitive to insulin, so that extra insulin is required to transport glucose into the cells. To compensate for the insulin resistance, the islet cells produce extra insulin over many years. Insulin resistance is a criterion of metabolic syndrome that can lead to type 2 diabetes by causing β-cell dysfunction. The following tests are important for early diagnosis of insulin resistance and β-cell dysfunction:

• Fasting proinsulin levels /17/. Levels above 11 pmol/L are indicative of diminished β-cell function caused by hyperglycemia-induced over stimulation. In this case, the cleavage capacity of carboxypeptidase H and other enzymes is exhausted, causing increasing amounts of unprocessed proinsulin to be released into the circulation /19/.
• The homeostasis model assessment (HOMA) for estimating insulin resistance (HOMA-IR) and β-cell function (HOMA-β). See Tab. 3.7-7 – Tests for the assessment of β-cell function and insulin resistance.

3.7.7 Comments and problems

Method of determination

Insulin: immunometric assays use monoclonal antibodies and have less cross-reactivity with proinsulin and proinsulin split products than the previously used radioimmunoassay (38–100%). Most assays have less than 2% cross-reactivity with proinsulin, 3% with des-31,32-split proinsulin, but over 40% with des-64,65 proinsulin. This is important in type 2 diabetes as well as impaired glucose tolerance, renal failure and liver cirrhosis, where the concentrations of proinsulin and proinsulin split products are several times higher than in healthy individuals.

However, the disadvantage of using specific assays for the diagnosis of insulinoma is that they do not detect the possibly increased amounts of proinsulin and its split products, making it more difficult to diagnose inadequate insulin secretion /1/. They also may not, or not fully, detect synthetic insulin such as lispro and can miss factitious hypoglycemia /8/.

Standardization

Insulin: the National Institute of Biological Standards and Controls (NIBSC) offers two standards which are used by the diagnostics manufacturers in the production of kit calibrators:

• First National Reference Preparation for Insulin 66/304, details of unit amounts are not yet available.
• First International Standard for Human Insulin 83/500. 1.0 g contains 26,500 units of insulin.
• Based on amino acid analyses, a conversion factor of 1 mU = 6.0 pmol is recommended. This conversion factor must, however, not be used for the preparation 66/304. In Great Britain, factors in the range of 6.0–7.5 are used, irrespective of the reference preparation /8/.

C-peptide: the first reference preparation has the code 84/510. The new standard preparation 76/561 is a synthetic 64-formyl lysin C-peptide and includes four extra basic amino acids, two at each end. The presence of formyl lysine at position 64 is reported to improve stability and solubility in aqueous solution /8/.

Proinsulin: the NIBSC offers a synthetic proinsulin, code 84/611, which is used for the production of most of the calibrators used in commercial assays /8/. A traceability scheme is proposed /35/.

Stability

Insulin: Up to 24 h in EDTA blood at 20 °C, up to 1 week at 4–8 °C, at least 3 months at –20 °C /27/.

C-peptide: C-peptide fragments are produced in vitro which react differently with the antibodies of the various immunoassays. Separation of the serum and storage at –20 °C if the analysis is not performed on the same day is recommended. Even at –20 to –25 °C, levels decrease by 2–26% or 28% within 4 weeks, depending on the assay /8/. Therefore, the sample must always be placed on ice immediately after collection for transport to the laboratory /8/.

Proinsulin: centrifuge within 4 h and store at 4 °C, better –20 °C /28/.

Hemolysis

Insulin: hemolysis results in the degradation of insulin due to the release of acid protease (EC 3.4.22.11) /29/.

C-peptide: hemolysis has no influence /29/.

Proinsulin: no effect has been observed. The acid protease has only little influence on proinsulin.

Biological variation

The European Biological Variation Study (EuBIVAS) /37/ determined for a mean concentration of insulin of 7.65 mU/L a within-subject variation (CVi) of 25.3% and a between-subject variation (CVG) of 33.4%.

Miscellaneous

For the assessment of insulin levels, the C-peptide concentration is important. Due to the longer half-life of C-peptide in plasma, insulin fluctuations caused by intermittent secretion can be better evaluated. In addition, the endogenous origin of increased insulin secretion can be confirmed based on the C-peptide concentration /8/.

The US Centers for Medicare and Medicaid Services require a C-peptide test for insulin pump approval. Due to the lack of comparability between the different assays, coverage for insulin pumps is provided only for patients whose C-peptide concentration is below the lower limit of the reference interval of the relevant assay, plus 10% for imprecision of the assay /28/.

The half-lifes and molecular weights are as follows:

• Insulin 3–4 min., MW 5808 Da
• Proinsulin approximately 17 min., MW 9390 Da
• C-peptide 30–40 min., MW 3018 Da.

3.7.8 Pathophysiology 

The secretion of insulin depends on the concentration of blood glucose, gastrointestinal hormones, islet cell hormones, and influence by the autonomic nervous system. Insulin is synthesized by the pancreatic islet cells which consist of the centrally located β- and peripheral α- and δ-cells. The α-cells synthesize glucagon, the β-cells insulin, and the δ-cells insulin growth factor.

In the pancreatic β-cell, the ATP-sensitive K⁺ channel plays an essential role in coupling membrane excitability with glucose-stimulated insulin secretion. An increase in glucose metabolism leads to elevated intracellular ATP/ADP ratio, closure of K⁺ ATP channels, and membrane depolarization. Consequent activation of voltage-dependent Ca²⁺ channels causes a rise in Ca²⁺ concentration, which stimulates insulin release. Conversely, a decrease in the metabolic signal is predicted to open K⁺ ATP channels and suppress the electric trigger of insulin secretion. Alterations in the metabolic signal, in the sensitivity of K⁺ ATP channels, could each disrupt electrical signalling in the β-cell and alter insulin release. Reduced or absent K⁺ ATP channel activity in the β-cell is causual in congenital hyperinsulism, a rare mostly recessive disorder /30/.

Glucose, hormones and certain drugs that reach the β-cells via arterial blood may cause release of insulin. This in turn is coupled to glucose metabolism. The higher the concentration of glucose, the more it is metabolized by glycolysis in the β-cell. This process produces signals that cause insulin secretion from the secretory granules and synthesis of new insulin /31/.

Like all neuroendocrine peptides, insulin is produced in the endoplasmic reticulum primarily as a single-chain precursor, and than is transported after folding and disulfide bonding to the secretory granules of the Golgi apparatus (Fig. 3.7-5 – Biosynthetic pathway of insulin). The precursor encoded in the Insulin gene is proinsulin, a molecule of 110 amino acids. It consists of a signal peptide that serves to direct the nascent polypeptide chain into the endoplasmic reticulum. There the signal peptide is cleaved off within 1 min., producing proinsulin which folds after approx. 20 min. before it is transported into the Golgi apparatus for storage. In the Golgi apparatus it is packed into the secretory granules together with two proprotein convertases (PC) and carboxypeptidase E (CPE). After approx. 2 h, the secretory granule has its final form and waits for secretion for hours or days /32/.

The granule is secreted in a complex reaction following stimulation of the β-cell by glucose, amino acids, fatty acids, and medications, such as sulfonylureas, which activate voltage-dependent Ca²⁺ channels by depolarization of the cell membrane potential. In the granules, the PC and CPE are autocatalytically activated. The combined action of the convertases produces des-64,65-split proinsulin, des-31,32-split proinsulin, insulin, and C-peptide (Fig. 3.7-6 – Processing of proinsulin to insulin and C-peptide). Insulin and C-peptide are released in equimolar amounts.

The processing of proinsulin shown in Fig. 3.7-6 does not take place completely. In healthy individuals, about 3% of the proinsulin is not converted to insulin and is released into the bloodstream together with the insulin. The same applies to the proinsulin split products produced in the processing. Elevated concentrations of proinsulin and proinsulin split products are measured in patients with insulinoma, type 2 diabetes, MODY (maturity-onset diabetes of the young), and pancreatic tumors associated with multiple endocrine type 1 neoplasia. Due to the longer half-life of proinsulin (17 min.) compared with insulin (2–3 min.), proinsulin accounts for approximately 10% of immunologically active insulin in plasma of healthy individuals. The long half-life of proinsulin and its low activity (3%) compared to insulin are due to low receptor affinity which is attributable to the connective peptide. The low clearance of proinsulin and its split products compared with insulin leads to their disproportionate increase in relation to secretion. Elevated des-31,32-split proinsulin levels have been found to be of diagnostic importance in the detection of β-cell stress due to over stimulation, such as occurs in type 2 diabetes (e.g., due to elevated concentrations of glucose and fatty acids).

C-peptide is biologically inactive. In contrast to insulin, it is not significantly metabolized by the liver; its main area of distribution is plasma. Although the plasma C-peptide level reflects β-cell function, it is only a limited marker of insulin secretion. The disadvantage is that, due to the (10 times) longer half-life of C-peptide compared to insulin, rapid changes in insulin secretion are not detected in the C-peptide test.

Insulin and C-peptide are secreted into the portal vein, and 40–60% of the secreted insulin is immediately absorbed by the liver. Regular oscillations of insulin secretion occur at 5 to 10 min. intervals. In non diabetics, these are in the range of approximately 3–7 mU/L /33/.

A distinction is made between the following effects of insulin action:

• Short-term effects occur within minutes at the plasma membrane (e.g., transport of glucose, amino acids, ions) and intracellular catalyzed enzymes. They serve to maintain glucose homeostasis, act directly at the cell membrane, increase the transport of glucose, amino acids and K⁺ and cause the activation of cytoplasmic enzymes such as of pyruvate dehydrogenase, glycogen synthase, acetyl-CoA-carboxylase, and phosphorylases.
• Insulin-mediated long-term effects. They require hours to days and include DNA and protein synthesis, the regulation of specific gene expression, and cell growth.

The effects of insulin on skeletal muscle cells, hepatocytes and adipocytes are mediated by receptors of heterogenous structure and function /34/.

The insulin receptor of the cell membrane has a heterotetramer structure consisting of two α-subunits of 135 kDa. These are connected with two transmembrane β-subunits with a MW of 95 kDa via disulfide bridges. If insulin binds to the α-subunit, conformational changes occur which are transferred on to the β-subunit. This leads to activation of the cytoplasmic enzyme tyrosine kinase, which passes on the signal via several postulated mechanisms.

Glucose-stimulated insulin secretion occurs in two phases. During the early phase lasting from seconds to about 10 min., preformed insulin is released from the secretory granules. Then, after a delay lasting from minutes up to 2 h, newly synthesized insulin is secreted, with plasma levels up to > 100 mU/L (700 pmol/L). The reduction in early insulin secretion can be an indicator of the functional disorder of the β-cells and an early symptom of diabetes which may develop in months or years later.

Type 1 diabetes is due to the increasing immune-mediated destruction of β-cells causing a decrease in insulin secretion to less than 1% of normal levels.

Type 2 diabetes is a complex and heterogenous disorder with the following mutually reinforcing or causative and probably consecutive manifestations:

• Loss of regular oscillations in insulin secretion, which leads to down regulation of the insulin receptors and impaired glucose tolerance and explains why pulsatile secretion of insulin has a greater hypoglycemic effect than continuous secretion /34/
• Diminished insulin secretion in relation to hyperglycemia after carbohydrate intake
• Insulin resistance (see Section 3.1 – Diabetes and prediabetes).
• Increased hepatic glucose synthesis despite the presence of hyperglycemia. This is caused by the stimulation of gluconeogenesis and glycogenolysis due to increased concentrations of free fatty acids.

The WHO hypothesizes that the clustering of type 2 diabetes, hypertension, dyslipidemia, and cardiovascular disease results from the common antecedent – insulin resistance. Insulin resistance across several organs (e.g., adipose tissue, muscle, liver, intestine) results in the metabolic syndrome phenotype /36/.

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