Cardiac diseases

2.1 Atherosclerosis

2.1.1 Development of atherosclerosis

Atherosclerosis is a leading cause of morbidity and mortality in industrialized countries. It is an illness that remains asymptomatic for decades. Clinical symptoms do not appear until an advanced stage, by which time it is too late for preventive measures. Atherosclerosis is a multifactorial disease whose development depends on hereditary and acquired risk factors. The main clinical manifestations of atherosclerosis are coronary heart disease, cerebrovascular disease, and peripheral arterial occlusive disease. The pathogenesis of atherosclerosis is most commonly explained using the “response to injury” model, which combines components of the once controversial lipid and inflammation theories. The Framingham Heart Study led to the identification of major risk factors for cardiovascular disease, including high cholesterol, hypertension, physical inactivity, and diabetes.

Atherosclerosis risk /1/

Important risk factors include genetic factors, age, gender, dyslipidemia, hypertension, smoking, chronic inflammation, diabetes mellitus, and chronic renal disease. These risk factors lead to vascular endothelial dysfunction. An individual’s risk of disease depends on the interplay between genetic factors, preexisting medical conditions, and lifestyle. This is one reason why the negative impact of an unhealthy lifestyle differs between individuals.

Biomarkers: cholesterol (LDL), triglycerides, Lp(a), HbA_1c or oral glucose tolerance test, homocysteine, C-reactive protein, and creatinine.

Mechanisms of atherosclerosis /1, 2/

Atherosclerotic plaques develop over the course of decades. The antecedents of atherosclerosis begin in utero and evolve throughout the lifespan, depending on genetic predisposition and, to a larger extent, behavioral factors and exposures. In contrast the thrombotic complications of atherosclerotic disease occur suddenly, often without warning. Atheroscerotic plaques destabilize without resulting in a clinical syndrome The occurrence of an acute coronary syndrome probably depends on the disruption of a balance between instability and healing of an atherosclerotic plaque.The two most frequent causes of thrombosis are plaque rupture and superficial erosion. Plaque rupture occurs when the fibrous cap covering the necrotic core fissures, exposing the highly thrombogenic core to the blood. Plaque erosion is caused by endothelial damage or denudation and overlying thrombosis in the absence of frank cap rupture.

Low-density lipoproteins (LDL) pass into the intima of arteries from where they are removed by the high-density lipoprotein (HDL) reverse transport process and macrophages if blood cholesterol levels are normal. According to the “response to retention” hypothesis, atherosclerosis develops as a result of LDL retention in the intima of arteries. The exudation of LDL into the intima is facilitated by vascular endothelial dysfunction caused by risk factors such as hypercholesterolemia, smoking, hypertension, and hyperglycemia. The cholesterol removal system is also overloaded.

LDL that has accumulated in the intima is oxidized (oxLDL) or enzymatically modified (eLDL). A multiple molecular networks activated by oxidized lipids, proinflammatory cytokines, and prothrombotic factors can induce important changes in the atherosclerotic plaque. The oxLDL or eLDL activates the innate immune system. A local inflammatory response occurs and an atherosclerotic lesion (atherosclerotic plaque) develops. As a result of the migration of inflammatory cells, the continuous accumulation of LDL, and the multiplication of local cells and connective tissue elements, the intima expands and the vessel wall thickens, first eccentrically and later concentrically. This latter process leads to local stenosis. Atherosclerotic plaques, characterized by marked intimal lipid accumulation and cell debris and a thin fibrous cap, are particularly prone to rupture.

Contact between the blood and the macrophages and activated endothelial cells releases tissue factors and contact with lipids and collagen of the exposed intima triggers platelet aggregation and activates clotting. The resulting acute partial vascular occlusion can cause acute cerebrovascular syndrome, unstable angina pectoris, myocardial infarction, sudden unexplained cardiac death, or stroke.

LDL starts to accumulate in the intima in the form of fatty streaks as early as the first years of life and continues to do so as shown by the FELIC study, faster in children of mothers with hypercholesterolemia than in children of mothers with normal cholesterol levels /3/. Atherosclerotic plaques do not develop until the age of 12–16 years.

2.1.2 Diseases caused by atherosclerosis

Acute coronary syndrome (ACS)

In the setting of a clinical presentation with ACS on the basis of the history, electrocardiogram and biochemical markers a case is classified as ST-segment elevation (STEMI), non-STEMI or non ischemic chest pain. The usual initiating mechanism for acute myocardial infarction (MI) is rupture or erosion of a vulnerable, lipid-laden atherosclerotic coronary plaque, resulting in exposure of circulating blood to highly thrombogenic core and matrix materials in the plaque. The inflammatory reaction in the plaque that leads to the rupture of its fibrous cap plays an important role. The inflammation activates the prothrombotic potential and inhibits the antifibrinolytic capacity of the hemostatic system and triggers thrombus formation on the plaque /4/.

Cerebrovascular atherosclerosis: ischemia is present in around 85% of strokes, mainly triggered by embolizing thrombi from the heart or carotid arteries.

2.1.2.1 Primary prevention of coronary heart disease

The goal of primary prevention is to prevent the occurrence of cardiovascular diseases. Independent, classic risk factors include age, male gender, smoking, family history of atherosclerotic vessel disease, diabetes mellitus, hypertension, hypercholesterolemia, high LDL cholesterol, and hypertriglyceridemia. A distinction is made between global risk (clinical significance of multiple risk factors in a risk profile) and the risk associated with a single, extremely elevated risk factor. Important isolated risk factors include the following:

• Cholesterol: individuals whose total cholesterol is ≥ 320 mg/dL (8.0 mmol/L) or LDL cholesterol is ≥ 240 mg/dL (6.22 mmol/L) are considered high-risk patients /5/. With respect to global risk assessment, an LDL cholesterol concentration of 100 mg/dL (2.6 mmol/L), 130 mg/dL (3.4 mmol/L), or 160 mg/dL (4.1 mmol/L), depending on the estimated risk or score value, requires treatment.
• Hypertension: patients whose blood pressure is higher than 180/110 mmHg are considered high-risk patients /5/. When global risk is assessed, a blood pressure ≥ 140/80 mmHg is considered pathological.

2.1.2.2 Risk stratification using algorithms and scores

With a view to preventing cardiovascular diseases, an individual’s global risk is determined and different risk factors are combined using algorithms or scores (Tab. 2.1-1 – Assessment of cardiovascular risk using algorithms and scores). The most widely recognized algorithms and scores have been derived from the German PROCAM study /6/ and the US Framingham study /7/. The ESC (European Society of Cardiology) score, the latest version of which was published in 2011, is widely used in Europe. The ESC score takes gender, age, smoking, blood cholesterol, blood pressure, and diabetes into account; the Framingham score considers gender, smoking, HDL cholesterol, blood pressure and family history; the PROCAM score also takes triglycerides and LDL cholesterol (rather than total cholesterol) into account.

Based on the severity of the risk factors, algorithms and scores are used to calculate a percentage 10-year risk of fatal or non-fatal myocardial infarction (PROCAM, Framingham) or death due to cardiovascular disease (European Society of Cardiology; ESC). The clinical significance of this risk is assessed as follows:

• Scores above 20% (PROCAM, Framingham) or 5% (ESC) identify high-risk patients
• Scores of 10–20% (PROCAM, Framingham) or 1–5% (ESC) indicate moderate risk
• Scores below 10% (PROCAM, Framingham) or 1% (ESC) indicate low risk.

High-risk patients also include individuals with diabetes mellitus and manifest atherosclerosis and those with a history of the following preexisting conditions:

• Stable or unstable angina pectoris
• Myocardial infarction
• Coronary artery bypass grafting or angioplasty
• Abdominal aortic aneurysm
• Ischemic stroke and transient ischemic attacks as well as carotid stenosis.

Strategies for prevention and treatment can be derived from the scores. Patients in the low-risk group should be encouraged to make lifestyle changes, patients at moderate risk should be strongly encouraged to make such changes and may also require medication, and intensive measures should be taken to reduce all risk factors in the high-risk group. The significance of the different scores varies. For example, the Framingham risk algorithm overestimates the risk of coronary heart disease by more than double for German men, whereas it is more reliable for the American population.

Overall, the algorithms and scores have a relatively good negative predictive value of over 90% for identifying low-risk patients. However, there is a significant false positive rate among individuals with moderate and high risk. A score for the assessment of the 10 years mortality risk is shown in Fig. 2.1-1 – European Society of Cardiology cardiovascular risk score.

2.1.2.3 New biomarkers

New markers for the primary prevention of coronary heart disease are being discussed (Tab. 2.1-2 – New risk markers for the primary prevention of cardiovascular disease (CVD)) /8/. These are known as “emerging markers.” Although some of these markers are statistically associated with cardiovascular risk (independently of the classic risk markers), they do not improve the prognostic efficiency of the algorithms derived from the classic risk factors, or they improve them marginally at most. In the current recommendations for primary prevention, therefore, their use is restricted to the sub stratification of medium-risk patients.

2.1.3 Pathophysiology of atherosclerosis

One popular hypothesis is that risk factors such as hypertension, cigarette smoking, insulin resistance, and type 2 diabetes lead to vascular endothelial damage and that LDL then accumulates in the vessel wall, where it is modified enzymatically or oxidatively. The modified LDL initiates and maintains an inflammatory response that leads to progression of the atherosclerosis and rupture of atherosclerotic plaques. There are three phases in the development of atherosclerosis /1, 15/:

• Initiation of atherosclerosis
• Progression to atherosclerotic plaque
• Plaque rupture and acute coronary syndrome.

Initiation of atherosclerosis

LDL accumulates in the arterial intima to form fatty streaks. These are asymptomatic and can disappear again or develop into atherosclerotic plaques. The LDL in the fatty streaks is sequestered from antioxidants in the blood, which promotes oxidation or chemical modification of the LDL. The modified LDL induces a local inflammatory response.

Leukocyte adhesion is another factor that contributes to the initiation of atherosclerosis. Leukocytes do not normally adhere to endothelial cells. However, pro inflammatory stimuli such as the risk factors for atherosclerosis or modified LDL trigger the expression of P-selectin and vascular cell adhesion molecules such as VCAM-1, which mediate reversible rolling or irreversible adhesion of lymphocytes and monocytes to the endothelium. Leukocyte binding to VCAM-1 and ICAM-1 activates signal transduction, which ultimately triggers the opening of intercellular junctions and diapedesis of the leukocytes through the endothelium.

The differentiation of intimal monocytes increases under the influence of inflammatory mediators and monocytes mature into macrophages under the influence of macrophage colony-stimulating factors. Oxidative enzymes (e.g., myeloperoxidase, NADPH peroxidase, inducible nitric oxide synthase, or lipoxygenases) as well as macrophage lipases and proteases intensify LDL modification. The modified LDL is taken up in an uncontrolled manner by scavenger receptors on macrophages. The cholesterol of the modified LDL is stored in cytosolic vacuoles in the form of cholesterol esters. The cholesterol-laden cells are known as “foam cells” due to their appearance on electron microscopy. These exacerbate inflammation by producing growth factors and inflammatory cytokines and chemokines, which in turn stimulate the further migration of monocytes and lymphocytes into the intima as well as the proliferation and migration of smooth muscle cells in the media. The migrating cells also include CD4⁺T-cells , which mediate a helper T-cell adaptive immune response.

The presence of foam cells and the development of atheromas mark the first phase of atherosclerosis (Fig. 2.1-2 – Initiation of atherosclerotic plaques).

Progression to a complex atherosclerotic lesion

While fatty streaks contain mainly foam cells, atheromatous plaques, which represent an advanced stage of atherosclerosis, are characterized by the presence of fibrous material (Fig. 2.1-3 – Progression to atherosclerotic plaque). The extracellular matrix of the plaque consists predominantly of smooth muscle cells. Activated by the platelet-derived growth factor (PDGF) produced by macrophages and endothelial cells and other mediators, the smooth muscle cells proliferate and migrate from the media to the intima, which results in the degradation of the extracellular matrix. The degradation is mediated by MMP-9 and other proteases. In the intima, the smooth muscle cells produce extracellular matrix proteins such as collagen. Atheromas produce increased amounts of interleukin-18, which in turn stimulates the production of interferon gamma by macrophages, T-cells, and smooth muscle cells, thereby maintaining the inflammatory response.

In the next step, the plaque, which is hypoxic due to its thickness, is vascularized by the vasa vasorum. Local bleeding occurs with the release of thrombin. This stimulates endothelial cells, smooth muscle cells, macrophage, and platelets to produce inflammatory markers such as CD40 ligand and RANTES (regulated on activation, normal T-cell expressed and secreted) and macrophage migration inhibitor factor (MIF). The effects of CD40 ligand, RANTES, and MIF promote plaque progression and possibly rupture of the fibrous cap with subsequent thrombus formation. Platelets are also involved in the synergistic interaction between inflammation and thrombosis by producing CD40 ligand and PDGF.

Plaque rupture and acute coronary syndrome /4/

The lipid-rich center of an atheromatous plaque (also known as the necrotic core) is covered by a fibrous cap. This prevents contact between the blood with its latently activated clotting potential and the lipid-rich center that contains some thrombogenic material. The fibrous cap is often only μm thick. Ruptured plaques usually have a large, lipid-rich core and contain many inflammatory cells and spotty or patchy calcification.

The fibrous cap, which protects the atheromatous plaque from rupture, consists of fibrillar collagen produced by smooth muscle cells in the artery wall. The stability of the fibrous cap depends on the balance between collagen synthesis and breakdown.

Inflammatory cells such as activated CD4⁺T-helper cells have a significant influence on the integrity of the fibrous cap:

• By synthesizing IFN-γ, CD4⁺T-helper cells inhibit the production of fibrillar collagen by smooth muscle cells
• By synthesizing CD40-ligand (CD154), which activates the corresponding CD40 receptor on macrophages, they stimulate the macrophages to increase the production of matrix metalloproteinases (MMP). The interstitial collagenases MMP-1, MMP-8, and MMP-13 in particular degrade fibrillary collagen and, by doing so, destroy the integrity of the fibrous cap.
• In summary, inflammation in the atheromatous plaque causes progressive thinning of the fibrous cap, which can lead to plaque rupture, thrombosis, and acute coronary syndrome.

2.1.3.1 Important new markers of atherosclerosis

New biomarkers have been recommended for the primary prevention of atherosclerosis and coronary heart disease. They are referred to as emerging markers because, although they are associated with increased risk, their causative, independent, and quantitative contributions are not as well documented as those of established criteria such as dyslipidemia, hypertension, and smoking. In addition, it has become evident that these markers and other mediators do not provide an advantage either for the early diagnosis of myocardial infarction (when compared to the necrosis marker troponin) or for risk management /15/. The pathophysiology of new biomarkers is described in Tab. 2.1-2 – New risk markers for the primary prevention of cardiovascular disease (CVD).

2.1.3.2 Clinical consequences of atherosclerosis

The sclerosis of coronary vessels is not a continuous process, but a disease that is characterized by alternating clinical phases of stability and instability. Acute coronary syndromes such as unstable angina pectoris and myocardial infarction are due to an acute or subacute reduction in myocardial oxygen supply caused by the rupture of an atherosclerotic plaque in combination with vasoconstriction, coronary thrombus formation, and distal coronary micro-embolization /10/.

Whether myocardial necrosis develops in the area depends on the extent and duration of the reduced oxygen supply. Although the vessel is initially occluded as a result of platelet aggregation, fibrin must also be produced to stabilize the aggregation and form a thrombus. By secreting serotonin and thromboxane A₂, platelet-rich thrombi induce vasoconstriction at the site of the plaque rupture or more distally.

If embolization to distal arterioles and capillaries occurs as a result of an epicardial coronary artery thrombosis, focal micro necroses occur that can be demonstrated histologically and by means of a high sensitivity troponin assay. Small embolisms can cause micro necroses in the absence of epicardial coronary artery occlusion. As a result of blood stasis, fibrin-rich apposition thrombi can develop and occupy large areas of a vessel proximal and distal to a platelet-rich occlusive thrombus that was formed as the result of a plaque rupture.

2.1.3.3 Diabetes mellitus and atherosclerosis

Atherosclerotic vascular complications are the main causes of disability and mortality in patients with type 2 diabetes. The prevalence of coronary heart disease, stroke, and peripheral arterial occlusive disease is 2–4 times higher in these patients than in the general population. Traditional risk factors are:

• Hypertonia (test: blood pressure measurement)
• Hyperglycemia (tests: fasting glucose, HbA_1c, glucose tolerance)
• Dyslipidemia (cholesterin, LDL, triglycerides).

Pathophysiology of dysfunctions in patients with atherosclerosis:

• Insulin resistance (tests: HOMA, leptin, adiponectin)
• Destruction of islet cells (tests: insulin, proinsulin, glucagon)
• Inflammation of adipose cells (tests: CRP, TNF-alpha, etc.).

Vascular complications in type 2 diabetics can be quantified as follows:

• One in four patients shows signs of reduced cardiac perfusion, one in three has silent myocardial ischemia, and one in nine shows angiographic evidence of coronary stenosis /12/.
• One-third of strokes in women and one-sixth of strokes in men can be linked to diabetes.
• The prevalence of peripheral arterial occlusive disease is twice as high in diabetics as in the general population /16/.

References

1. Gibbons GH, Seidman CE, Topol EJ. Conquering atherosclerotic cardiovascular disease – 50 years of progress. N Engl J Med 2021; 384 (9): 785–8.

2. Vergalo R, Crea F. Atherosclerotic plaque healing. N Engl J Med 2020; 383: 846–57.

3. Napoli C, Glass CK, Witztum JL, Deutsch R, D’Armiento FP, Palinski W. Influence of maternal hypercholesterinemia during pregnancy on progression of early atherosclerotic lesions in childhood: Fate of Early Lesions in Children (FELIC) study. Lancet 1999; 354: 1234–41.

4. Libby P. Mechanisms of acute coronary syndromes and their implications for therapy. N Engl J Med 2013; 368: 2004–12.

5. Pearson TA, Bazzarre TL, Daniels SR, Fair JM, Fortmann SP, Franklin BA, et al. American Heart Association guide for improving cardiovascular health at the community level: a statement for practice health practitioners, healthcare providers, and health policy makers from the American Heart Association Expert Panel on Population and Prevention Science. Circulation 2003; 107: 645–51.

6. Assmann G, Cullen P, Schulte H. Simple scoring scheme for calculation of the risk of acute coronary events based on the 10-year follow-up of the prospective cardiovascular Münster (PROCAM) study. Circulation 2002; 105: 310–5.

7. Wilson PW, D’Agostino RB, Belanger AM, Silberschatz H, Kannel WB. Prediction of coronary heart disease using risk factor categories. Circulation 1998; 97: 1837–47.

8. Apple FS, Smith SW, Pearce LA, Murakami MM. Assessment of the multiple-biomarker approach for diagnosis of myocardial infarction in patients presenting with symptoms suggestive of acute coronary syndrome. Clin Chem 2009; 55: 93–100.

9. Reiner Z, et al. ESC/EAC guidelines. Eur Heart J 2011; 32: 1769–1818.

10. Myers GL, Christenson RHM, Cushman M, Ballantyne CM, Cooper GR, Pfeiffer CM, et al. National Academy of Clinical Biochemistry laboratory medicine practice guidelines: Emerging biomarkers for primary prevention of cardiovascular disease. Clin Chem 2009; 55: 378–84.

11. US Preventive Services Task Force. Using nontraditional risk factors in coronary heart disease risk assessment: US preventive services task force recommendation statement. Ann Intern Med 2009; 151: 474–82.

12. Murphy MJ, Hosking J, Metcalf BS, Voss LD, Jeffery AN, Sattar N, et al. Distribution of adinopectin, leptin, and metabolic correlates of insulin resistance: a longitudinal study in British children; 1. prepuberty (early bird 15). Clin Chem 2008; 54: 1298–1306.

13. Scirica BM, Sabatine MS, Jarolim P, Murphy SA, de Lemos JL, Braunwald E, Morrow DA. Assessment of multiple cardiac biomarkers in non-ST-segment elevation acute coronary syndromes: observations from the MERLIN-TIMI 36 trial. Eur Heart J 2011; 32: 697–705.

14. De Winther MPJ, Lutgens E. The link between hematopoiesis and atherosclerosis. N Engl J Med 2019; 380, 19: 1869–701.

15. Stampfer MJ, Hu FB, Manson JE, Rimm EB, Willett WC. Primary prevention of coronary heart disease in women through diet and lifestyle. N Engl J Med 2000; 343: 16–22.

16. Hirsch AT, Criqui MH, Treat-Jacobson D, Regensteiner JG, Creager MA, Olin JW, et al. Peripheral arterial disease detection, awareness and treatment in primary care. JAMA 2001; 286: 1317–24.

2.2 Metabolic syndrome

The metabolic syndrome (MetS) was first described as the coexistence of obesity, hyper-, and dyslipidemia, type 2 diabetes, gout and hypertension, associated with an increased incidence of atherosclerotic vascular diseases, fatty liver disease, and cholelithiasis due to a “common soil” of super nutrition and physical inactivity, and a genetic predisposition /1, 2/.

The pathophysiological features of the MetS are:

• Abdominal (central) obesity
• Atherogenic dyslipidemia
• Hypertension
• Impaired glucose tolerance or type 2 diabetes.

MetS is diagnosed if three or more of the following findings are present:

• Hyperglycemia
• Elevated triglycerides
• Low HDL cholesterol
• Arterial hypertension
• Visceral obesity.

Taken together, the features of the metabolic syndrome form an atherogenic network that increases the risk of non-alcoholic fatty liver disease, of diabetes mellitus and cardiovascular morbidity and mortality. In a study of 12,659 children with a mean age of 11.3 years, mortality rates up to the 55th year of life were 78% higher among children in the highest quartile of the cohort than those among children in the lowest quartile /3/. The assessment criteria were body mass index, hypertension, glucose intolerance, and serum cholesterol. In another study of overweight schoolchildren between the ages of 8 and 14 years, 6.5% had three or more features of the metabolic syndrome /4/.

The prevalence of the MetS is age-dependent and shows regional variation. In the USA /5/, it has a prevalence of 7%, 44%, and 42% in the age groups 20–29 years, 60–69 years, and over 70 years respectively, while in Hong Kong, the prevalence is only 6%. The prevalence of the metabolic syndrome in Germany is 22.7% among men and 18% among women /6/.

A MetS is not a single disease entity, but rather a constellation of interconnected clinical and laboratory findings that are associated with diabetes mellitus and coronary heart disease.

The definitions of MetS are listed in Tab. 2.2-1 – Definition of the metabolic vascular syndrome. In the population, however, the specific predictive models recommended for coronary heart disease and type 2 diabetes are better criteria to identify persons at increased risk of these diseases than the criteria for metabolic syndrome. This has been demonstrated by the San Antonio Heart Study and the Mexico City Diabetes Study /8/. In these studies, the Framingham Risk Score was better than the criteria for metabolic syndrome for predicting the risk of cardiovascular disease /9/ and the Diabetes Predicting Model /10/ was better for predicting the risk of type 2 diabetes. Furthermore, after adjustment for diabetes, the metabolic syndrome does not improve the prediction of mortality in preexisting cardiovascular disease /11/. In individuals who have the metabolic syndrome according the NECP-ATP III definition, most studies show an increased cardiovascular risk with a mean hazard ratio of 1.6 /12/. Depending on the definition of the metabolic syndrome, the risk of cardiovascular mortality is 2.09–1.51 for men and 1.53–1.09 for women /12/.

In addition to dyslipidemia, diabetes, and hypertension, chronic inflammation of adipose tissue also acts as a link between the MetS and cardiovascular disease /13/.

Refer to:

• Tab. 2.2-2 – Individual components of the metabolic syndrome
• Tab. 2.2-3 – Diseases associated with the metabolic vascular syndrome.

Cross sectional data of MetS with Lp(a) concentration of the participants of the two studies BASE-II and SHILP-O were sparse. There was an inverse association between MetS and Lp(a) /40/. Participants with MetS had 11.9 mmol/L lower Lp(a). Overall 27.6% of the participants in the two studies had MetS.

2.2.1 Adipokines

The term adipokines (adipose tissue cytokines) describes a group of polypeptides that are mainly, though not exclusively, secreted by adipose tissue /14, 15, 16/. While adipokines are secreted into the circulation specifically by adipocytes, cytokines are also secreted by other cells in the adipose tissue such as macrophages, fibroblasts, and infiltrated monocytes. The adipocytes in adipose tissue make up around one-third of the cells in the body and have the following important functions:

• Energy storage
• Hydrolysis of triglycerides and production of free fatty acids to supply energy to tissues
• Adipokine release. The main adipokines are leptin, resistin, and adiponectin.

Adipokines are involved in important physiological functions such as carbohydrate and fat metabolism, insulin sensitivity, appetite regulation, inflammatory processes, and cardiac function.

Ectopic adipose tissue, which in obese individuals is found mainly in the visceral fat of the omentum as well as the epicardial and mediastinal fat, plays an important role in adipokine production. Ectopic adipose tissue is responsible for the pathogenesis of many obesity-related diseases.

2.2.1.1 Adiponectin

Adiponectin is a peptide with a molecular weight of 30 kDa that circulates in the plasma in complexes of various sizes. Complexes with high molecular weight are the most active. Plasma adiponectin concentration is inversely related to obesity, insulin resistance, type 2 diabetes, and cardiovascular disease /17, 18/.

Pathophysiology: adiponectin exerts its effect via two receptors. The ADIPOR1 receptor is expressed by skeletal muscle and other tissues while the ADIPOR2 receptor is expressed by hepatocytes. Adiponectin inhibits hepatic glucose production and lipogenesis, stimulates insulin secretion, glucose uptake in the muscles, and fatty acid oxidation in the liver and muscles, modulates food intake and energy consumption, and inhibits the production of proinflammatory cytokines. Its secretion is stimulated by insulin sensitizers such as roglitazone. Adiponectin inhibits inflammation by stimulating the secretion of IL-10, blocks the activation of nuclear factor κB (see also Fig. 19.1-1), and inhibits the release of IL-6 and TNF-α. Conversely, adiponectin secretion is inhibited by inflammation, and the low-grade adipose inflammation in obese individuals in particular reduces its plasma concentration. In this way, adiponectin can be seen as a negative modulator of the systemic inflammation that characterizes the metabolic syndrome.

Indication: possible marker for assessing the risk of insulin resistance and coronary heart disease in obese individuals.

Specimen: serum: 1 mL

Reference interval: 7–12 mg/L, depending on the assay used /19/.

Clinical significance: adiponectin is an important marker for cardiovascular risk because it disrupts the dangerous cycle of inflammatory vascular damage that is associated with atherosclerosis and type 2 diabetes. Values below 4 mg/L are associated with a significant risk of atherosclerosis. Treating diabetics with insulin sensitizers increases adiponectin concentrations, reduces blood glucose and HbA_1C values, and reduces insulin resistance, thereby also reducing cardiovascular risk. Weight loss leads to increased adiponectin concentrations.

The adiponectin/leptin ratio can be used to distinguish between type 1 and type 2 diabetes in young people. Mean adiponectin values of 18 mg/L and an adiponectin/leptin ratio of 3.8 have been demonstrated in young people with type 1 diabetes while values of 9 mg/L and a ratio of 0.46 have been demonstrated in those with type 2 diabetes /20/.

Note: over the course of 30 days, adiponectin concentrations demonstrate intraindividual variation of /21/:

• 12.2% in overweight individuals with metabolic syndrome (equivalent to a change of 1.7 mg/L in the reference interval)
• 18.8% in normal individuals (equivalent to a change of 3.6 mg/L in the reference interval). In non-obese individuals, minor fluctuations only in adiponectin concentrations were observed within a period of 15 months (initial value 8.3 ± 2.9 mg/L compared to 8.2 ± 3.0 after 15 months). Diurnal and 3-hour postprandial fluctuations are small.

2.2.1.2 Leptin

Leptin is an adipocyte secretory product whose plasma concentration increases with the amount of adipose tissue in the body /15, 18/. It circulates as a peptide with 164 amino acids and has a molecular weight of 16 kDa.

Pathophysiology: leptin inhibits food intake, stimulates energy consumption, inhibits hepatic glucose production and fatty acid synthesis, activates fatty acid oxidation in liver and muscle, stimulates insulin secretion and glucose uptake by liver and muscle, stimulates the secretion of inflammatory cytokines, and inhibits the expression of resistin. Leptin has an overall anti-obesity effect that is mediated by hypothalamic pathways. Obese individuals have elevated leptin concentrations, which suggests that they are resistant to adipokine.

Indication: assessment of the amount of adipose tissue in relation to food intake.

Specimen: serum: 1 mL

Reference interval: 2–15 μg/L, depending on the assay used.

Clinical significance: the serum leptin concentration depends on the amount of body fat. Increases in leptin levels are governed by food intake, insulin concentration, and serum cortisol concentration. Eating causes leptin levels to rise.

The leptin concentration increases exponentially as the amount of body fat increases.

During fasting, leptin levels are low. In a study /22/, cardiometabolic biomarkers were measured in schoolchildren. Being overweight was associated with increased concentrations of leptin, CRP, and fibrinogen and reduced concentrations of ApoA1. The respective odds ratios were 59.8 (leptin), 6.3 (CRP), 2.8 (fibrinogen), and 2.6 (ApoA1).

The adiponectin/leptin ratio is being evaluated as a means of distinguishing between type 1 and type 2 diabetes in young people. Refer to Tab. 2.2-2 – Individual components of the metabolic syndrome (MetS).

Note: stable in serum at 4 °C for two months.

2.2.1.3 Resistin

Resistin received its name from the observation that it induced insulin resistance in mice. It is a 12.5 kDa peptide that belongs to the family of cysteine-rich proteins also known as resistin-like molecules (RELMs). Resistin circulates in plasma primarily in high molecular weight (hexamer) form /15, 18/.

Pathophysiology: resistin is produced by adipose tissue and placenta. It reduces tissue insulin sensitivity and increases glucose production in the liver. It also stimulates the synthesis of pro inflammatory cytokines such as IL-6 and TNF-α. In summary, resistin provides a link between obesity and insulin resistance. It is thought that resistin plays a role in the development of obesity and insulin resistance.

Indication: research at present.

Specimen: serum: 1 mL

Reference interval: 5–15 μg/L, depending on the assay used.

Clinical significance: in theory, the resistin concentration should rise with increasing obesity and with the development of insulin resistance and type 2 diabetes. Treatment with the insulin sensitizer roglitazone down regulates resistin. In contrast to earlier studies, however, there is growing evidence that obesity and resistin concentrations are not related.

During pregnancy, insulin sensitivity falls continuously as the fetoplacental unit develops. In one study /23/, resistin concentrations in pregnant women with and without gestational diabetes (GDM) were compared. The women with normal glucose tolerance had resistin values of 9.3 ± 1.3 μg/L and those with GDM had values of 4.3 ± 1.6 μg/L. However, no relationship was found between insulin sensitivity and resistin concentration. Other investigators have found increased resistin levels in GDM.

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2.3 Cardiovascular disease

Cardiovascular disease (CVD) is the first cause of premature mortality in the world and represented 46% of the deaths attributed to non communicable diseases in 2012. Because CVD has a multifactorial origin and most cardiovascular events occur in asymptomatic individuals, health authorities recommend global approaches to evaluate a patient risk profile. Decisions about whether to take preventive measures are based on the risk of experiencing myocardial infarction (MI) or sudden unexplained cardiac death in the coming years. The risk profile involves estimating major risk factors such as age, sex, smoking, hypertension and predisposing risk factors such as familial background /1/.

Laboratory investigations play an important role in the CVD risk assessment and diagnosis of MI /22/:

• The diagnosis of myocardial necrosis by determining necrosis markers in the serum, in particular cardiac troponin
• Primary and secondary prevention of cardiovascular disease by determining blood markers, e.g., total cholesterol, low-density lipoprotein cholesterol, triglycerides, fasting plasma glucose, and HbA_1c.
• Assessment of cardiac function by determining the level of natriuretic peptide or NT-proBNP.

The causes of myocardial necrosis are listed in Tab. 2.3-1 – Myocardial necrosis due to myocardial damage.

2.3.1 Clinical aspects of acute coronary syndrome

Unstable angina, acute non-ST-elevation myocardial infarction (NSTEMI) and acute ST-elevation myocardial infarction (STEMI) are the three presentations of acute coronary syndrome (ACS). They are considered cardiac emergencies, requiring prompt interventions including revascularization with percutaneous coronary intervention (PCI), thrombolytic therapy or coronary artery bypass graft surgery.

Various risk calculators are available for estimating the overall risk, such as the Framingham Risk Score, PROCAM score or ESC score (Tab. 2.1-1 – Assessment of cardiovascular risk using algorithms and scores). Based on these scores, individuals without cardiovascular disease and without diabetes mellitus are classified into categories with a low (under 10%), intermediate (10–20%), or high (over 20%) probability of experiencing a cardiovascular event. The traditional criteria used are age, gender, blood pressure, cholesterol concentration, and smoking.

Refer to Section 2.1.2 – Diseases caused by atherosclerosis.

In the USA, 31% of asymptomatic men and 7% of asymptomatic women fall into the intermediate risk group. “Non-traditional” markers can be used to further clarify the correct risk group for these patients.

Refer to Tab. 2.3-2 – Cardiovascular disease, risk factors, and their frequency in the USA.

According to the U.S. Preventive Services Task Force /3/, CRP is the only useful marker. 11% of men in the intermediate group were reclassified into the high-risk group based on a CRP value of over 3 mg/L.

Refer to Tab. 2.1-2 – New risk markers for the primary prevention of cardiovascular disease.

High-sensitivity troponin and B-natriuretic peptide are of similar importance.

2.3.1.1 Genetic predisposition to cardiovascular disease

Various gene mutations are associated with cardiovascular disease. The following are recognized /2/:

• Genes associated with familial cardiomyopathies and arrhythmias
• Diseases (familial hypercholesterolemia, autosomal recessive hypercholesterolemia, familial defective apolipoprotein B-100, apolipoprotein A-1 deficiency, sitosterolemia, Tangier disease, homocystinuria) and their gene variants that are associated with an increased risk of cardiovascular disease
• Gene variants associated with an increased risk of cardiovascular disease.

2.3.1.2 Epidemiology of cardiovascular disease

In Germany, nearly one in two adults dies as a result of a cardiovascular disease (in decreasing order of frequency: CVD, stroke, heart failure, hypertension, peripheral arterial disease). The annual mortality rate from myocardial infarction is 107 per 100,000 of the population.

According to one study /4/, the average age for myocardial infarction was 76.1 years for women and 66.7 years for men. Frequent comorbid diseases were:

• Diabetes mellitus; women 38.8%, men 29.5%
• Hypertension; women 68.1%, men 66%
• Heart failure; women 45%, men 35.2%
• Renal insufficiency; women 22.1%, men 19.4%. Approximately 50% of patients with chronic kidney disease (CKD) of stages 4 and 5 develop cardiovascular disease (CVD).

The following mortality rates were observed following myocardial infarction:

• Hospital mortality 13.9%
• 30-day mortality 16.7%
• 90-day mortality 20.8%
• 1-year mortality 28.1%.

After adjustment for age, mortality rates are the same for men and women.

Globally, acute myocardial infarction has a mortality rate of 30–50% within the first month, with half of all fatalities occurring during the first two hours.

According to a systematic review /5/, the outcome of acute myocardial infarction in patients who are not adequately treated and followed up is as follows:

• 23% die before they reach hospital
• 13% die following admission to hospital
• After discharge from hospital, 10% die within the first year and a further 5% die during each subsequent year
• After 15 years, the cumulative mortality rate is 70%
• Following a second myocardial infarction, 33% die before reaching hospital, 20% die in hospital, and a further 20% die within one year of discharge. The annual mortality rate is 10% in each subsequent year.

2.3.2 Universal definition of myocardial infarction

The most recent guidelines recommend high-sensitivity cardiac troponin T (hs-cTnT) or cardiac troponin I (hs-cTnI) for the detection of acute myocardial infarction.

The Fourth universal definition of acute myocardial infarction defines myocardial injury as a distinct condition characterized by at least one hs-cTnI or hs-cTnT value above the 99th percentile of the biomarkers distribution values evaluated in a healthy adult population. The 99th upper reference limit (URL) value from 6-12 h less in a clinical setting is consistent with acute myocardial ischemia /23, 24/.

A combination of criteria is required to meet the diagnosis of myocardial infarction, namely the detection of an increase and/or decrease of (hs-cTnT) or (hs-cTnI), with at least one value above the 99th percentile of the upper reference limit and at least one of the following:

• Symptoms of ischemia
• New or presumed new significant ST-T wave changes or left bundle branch block on 12-lead ECG
• Development of pathological Q waves on ECG
• Imaging evidence of new or presumed new loss of viable myocardium or regional wall motion abnormality
• Intracoronary thrombus detected on angiography or autopsy.

The time to diagnosis of NON-ST-Elevation myocardial infarction (NSTEMI) from 6–12 h reduces with the use of hs-cTnTor hs-cTnI to less than 3 h in patients admitted to the emergency department. The 0–1 h algorithm allow to rule-in and rule-out NSTEMI in the shortest possible time /1/.

2.3.3 Types of myocardial ischemia /6/

• Unstable angina: defined as myocardial ischemia at rest or minimal exertion in the absence of cardiomyocyte necrosis. Among unselected patients presenting with suspected NSTEMI to the emergency department, the introduction of high sensitivity cardiac troponin measurements in place of standard troponin assays resulted in an increase in the detection of myocardial infaection (about 4% absolute and 20% relative increase) and a reciprocal decrease in the diagnosis of unstable angina. Compared with NSTEMI (non ST emergency myocardial infarction) patients, individuals with unstable angina do not experience myocardial necrosis, have a substantially lower risk of death and appear to derive less benefit from intensified antiplatelet therapy as well as early invasive strategy.
• Type 1 MI: characterized by atherosclerotic plaque rupture, ulceration, fissure, erosion or detection with resulting intraluminal thrombus in one or more coronary arteries leading to decreased myocardial blood flow and/or distal embolization and subsequent myocardial necrosis. The patient may have underlying severe coronary artery disease (CAD) but, on occasion (i.e. 5–20% of cases), there may be non-obstructive coronary atherosclerosis or no angiographic evidence of coronary artery disease (CAD), particularly in women.
• Type 2 MI is myocardial necrosis in which a condition other than coronary plaque instability contributes to an imbalance between myocardial oxygen supply and demand. Mechanisms include coronary artery spasms, coronary endothel dysfunction, tachyarrhythmias, bradyarrhythmias, anemia, respiratory failure, hypotension and severe hypertension. In addition in critically ill patients and in patients undergoing major non-cardiac surgery, myocardial necrosis may be related to injurious effects of pharmacological agents and toxins.

In clinical diagnostics the separation of NSTEMI from non-cardiac chest pain is a problem. A study /19/ showed that troponin algorithms using low baseline troponin concentrations and delta values showed improved clinical sensitivity for NSTEMI by impoved differentiation between patients with unstable angina pectoris and noncardiac chest pain.

2.3.3.1 Exclusion of myocardial infarction

An acute myocardial infarction can be ruled out as mentioned in Section 2.4.5.3.

2.3.3.2 Course of myocardial infarction

Progression over time

From a temporal perspective, myocardial infarction can be divided into the following phases:

• Acute phase (6 hours to 7 days)
• Recovery phase (7–28 days). ST elevation can persist during the recovery phase and biochemical markers of myocardial necrosis may still be abnormal.
• Healed (from day 29).

Classification of infarct size and location

Acute myocardial infarctions are classified by size as follows /7/:

• Microscopic (focal necrosis)
• Small (less than 10% of the left ventricle)
• Medium (10–30% of the left ventricle)
• Large (involving more than 30% of the left ventricle)
• Location: anterior, lateral, inferior, posterior, septal, or a combination of locations.

2.3.3.3 Risk assessment in patients with acute coronary syndrome

Scores are used to assess the future cardiac risk in patients with acute coronary syndrome.

TIMI risk score /8/

The Thrombolysis in Myocardial Infarction (TIMI) risk score assesses the risk of recurrent ischemia, infarction, or death of inpatients with acute coronary syndrome during the next 14 days using the following parameters: age over 65 years, three or more risk factors for cardiovascular disease (family history of cardiovascular disease, hypertension, diabetes, smoking), known cardiovascular disease, two or more episodes of chest pain in the previous 24 hours, use of aspirin in the 7 days before admission to hospital, ST segment deviation of more than 0.05 mV, a rise in a myocardial necrosis marker. One point is awarded for each criterion and the total number of points is calculated. The algorithm and its rating are available on the Internet.

GRACE risk model /9/

The Global Registry of Acute Coronary Events (GRACE) risk model assesses the risk of recurrent ischemia, MI or death of inpatients with acute coronary syndrome and the 6 months following the acute event using the following parameters: increased age, history of MI, cardiac failure, increased pulse rate, low systolic blood pressure, elevated serum creatinine, an elevated myocardial necrosis marker, ST segment depression on the ECG. The eight criteria are added to give an overall score, which is then compared with a reference nomogram.

2.3.3.4 Atherosclerotic cardiovascular disease (ASCVD) risk factor from AHA

The calculator determines the 10-year risk of heart disease or stroke. The prerequisites are age of 40–75 years and LDL-cholesterol below 190 mg/dL (4.9 mmol/L) Diabetes, sex, race, smoking, total cholesterol, HDL-cholesterol, and systolic and diastolic blood pressure are considered. At a risk threshold of 10% in 10 years, the ASCVD equation has a sensitivity to identify future CVD events of approximately 80%, with the highest specificity (69%) and positive predictive value (17%) among 9 calculations tested /10/.

2.3.3.4.1 Important prognostic factors

Along with age, the following factors are important prognostic indicators for the outcome of myocardial infarction:

• Medical history: first infarction, second infarction, diabetes mellitus, renal insufficiency
• Infarct size and location (anterior or posterior wall infarction)
• Low initial blood pressure
• Extent of ischemia; represented by biochemical markers and ST segment elevation
• Diabetes mellitus. Diabetics with acute coronary syndrome have a one-year mortality hazard ratio of 1.65 compared to non-diabetics with acute coronary syndrome /11/.

2.3.3.4.2 ECG and myocardial infarction

Acute ST segment elevation on the ECG in conjunction with continuing clinical symptoms has a high predictive value for MI and coronary reperfusion measures should be introduced immediately. This is because patients who present within 6 hours of the acute event may not yet show elevated biochemical necrosis markers unless sensitive or high-sensitivity cardiac troponin assays are used.

Persistent ST segment elevation indicates persistent occlusive thrombosis. It is a sensitive marker of myocardial ischemia and can be detected within minutes of the onset of clinical symptoms. It has a diagnostic sensitivity of 80–90% for type 1 MI. However, only 30–40% of patients with acute chest pain have ST segment elevation on admission. ST segment elevation in MI is more obvious and occurs more frequently in men than in women. In the presence of clinical symptoms, a normal ECG does not exclude MI and a rise or fall in biochemical cardiac markers is also sufficient to make a diagnosis /12/.

Assessment of infarct size

Comparative magnetic resonance imaging investigations in MI have shown that the extent and temporal course of increases in CK-MB mass and cTn correlate with the infarct size and reperfusion.

Diagnosis of early re-infarction /13/

The term “re-infarction” applies to an acute myocardial infarction that occurs within 28 days of an incident or recurrent myocardial infarction /13/. A re-infarction should be considered if ST segment elevation ≥ 0.1 mV or new pathological Q-waves recur in at least two leads. A cTn assay should be performed immediately and repeated 3–6 hours later. A rise in cTn of ≥ 20% in the second sample points to re-infarction /14/.

2.3.4 Biomarkers of myocardial necrosis

During myocyte necrosis, structural proteins, cytoplasmic proteins, and other proteins are released into the cardiac interstitium /15/. These proteins, which include cTn, CK-MB, CK, myoglobin, AST, and LD can be measured in the serum. cTn has proven to be the most reliable marker. Myoglobin and the enzymes are less specific than cTn. A typical course of the necrosis markers in MI is shown in Fig. 2.3-2 – Time profile of enzymes, cardiac troponin, and CK-MB mass in acute myocardial infarction.

2.3.5 Secondary prevention of coronary heart disease (CHD)

Patients with preexisting coronary heart disease present a significant problem. It is important to correctly stratify risk in individual patients and tailor treatment accordingly. Important predictive markers include BNP/NT-proBNP, CRP, cystatin C, and cTn. In one study /16/ 12 markers were determined to evaluate the risk of a coronary event occurring over 3.6 years in stable cardiovascular disease. The hazard ratios for each standard deviation increase were as follows:

• NT-proBNP 1.71; median without event 89 ng/L, with event 501 ng/L
• Cystatin C 1.43; median without event 0.81 mg/L, with event 0.86 mg/L
• CRP 1.33; median without event 2.09 mg/L, with event 3.86 mg/L.

In another study /17/, the prognostic value of a high-sensitive cTn assay was evaluated. Patients had acute coronary syndrome without ST segment elevation. Patients whose baseline cTnI value was ≥ 0.04 μg/L were at significantly higher risk of myocardial infarction/cardiac death at day 30 than patients with a lower value. After adjusting for the TIMI risk score, a baseline cTnI value ≥ 0.04 μg/L was associated with a 3-fold (2.2–4) increase in the risk of myocardial infarction/cardiac death at 30 days. These preliminary findings demonstrate the high predictive value of sensitive cTn assays.

Inflammation is one of the central pathomechanisms of atherosclerosis. Elevation of high sensitivity CRP and interleukin-1β are associated with increased risk of cardiovascular events. Statin therapy relates to both a reduction in cholesterol level and inflammation inhibition. Reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Reducing vascular inflammation in the absence of concomitant lipid lowering reduces the rates of cardiovascular events. In a study /18/ patients with a history of myocardial infarction and a high sensitivity CRP concentration of more than 2 mg/L were treated with canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β. Treatment with canakinumab led to a significantly lower rate of recurrent cardiovascular events than placebo.

2.3.6 Primary prevention of coronary heart disease (CHD)

Individual risk assessment is currently based on scoring systems such as the ESC, Framingham, or PROCAM scores. However, biomarkers can also demonstrate an association with the occurrence of cardiovascular events in the longer term in population groups.

This was investigated in the MONICA, risk, genetics, archiving, and monograph (MORGAM) biomarker project /19/. Thirty biomarkers were evaluated and integrated into one score and the risk of cardiovascular events was followed up for 10 years. The score was based on the markers with the highest prognostic significance: NT-proBNP (hazard ratio 1.23), CRP (hazard ratio 1.23), and sensitive troponin (hazard ratio 1.18).

2.3.7 Myocarditis

Myocarditis can result from infectious and non-infectious diseases /20/ e.g.

• viruses: adenoviruses, enteroviruses (e.g., Coxsackie virus), vasculotropic viruses (e.g. Parvovirus B19), lymphotropic viruses (e.g., Cytomegalo virus, Epstein Barr virus, Herpes virus 6) cardiotoxic viruses (e.g., Hepatitis C virus, HIV, and Influenza viruses) and possibly angiotensin converting enzyme 2 tropic cardiotoxic viruses (e.g., Coronaviruses)
• bacteria (e.g., Corynebacterium diphtheriae, Borrelia burgdorferi
• parasites (e.g., Trypanosoma cruzi)
• poststreptococcal autoimmune rheumatic carditis
• exposure to drugs and toxins
• amyloidosis
• thyrotoxicosis
• immune stimulation (vaccines and cancer therapies)
• autoimmunity (sarcoidosis).

Clinical findings: The incidence of myocarditis among patients varies from 0.3 to 4.0% according to the age and country. Among patients with chest pain, who are seen in the emergency department, 3% have acute myocarditis and pericarditis. Main clinical manifestations are chest pain with an otherwise uncomplicated clinical picture.

Laboratory findings /20/: Markers of inflammation (erythrocyte sedimentation rate, C-reactive protein) are not specific and not increased in myocarditis, high-sensitivity troponin is a valuable test to diagnose myocyte injury. A study /21/ found that human homologue (hsa-MiR-Chr8:96) synthesized by type 17 helper T cells could be used to distinguish patients with myocarditis from those with myocardial infarction and healthy controls.

References

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2. 2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation. Eur Heart J 2015; doi: 10.1093/eurheartj/ehv320.

3. US Preventive Services Task Force. Using nontraditional risk factors in coronary heart disease risk assessment: US preventive services task force recommendation statement. Ann Intern Med 2009; 151: 474–82.

4. Heller G, Babitsch B, Günster C, Möckel M. Sterblichkeitsrisiko von Frauen und Männern nach Myokardinfarkt. Dtsch Ärztebl 2008; 105: 279–91.

5. Law, MR, Watt HC, Wald NJ. The underlying risk of death after myocardial infarction in the absence of treatment. Arch Intern Med 2002; 162: 2405–10.

6. Roffi M, et al. 2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation. Eur Heart J 2016; 37: 267–315.

7. Chapman AR, Anand A, Boeddinghaus J, Ferry AV, Sandeman D, Adamson PD, et al. Comparison of the efficacy and safety of early rule-out pathways for acute myocardial infarction. Circulation 2017; 135: 1586–96.

8. Antman E, Cohen M, Bernink PJLM, McCabe CH, Horacek T, Papuchis G, et al. The TIMI risk score for unstable angina/non-ST elevation MI: a method for prognostication and therapeutic decision making. JAMA 2000; 284: 835–42.

9. Eagle KA, Lim MJ, Dabbous OH, Pieper KS, Goldberg RJ, van de Werf F, et al. A validated prediction model for all forms of acute coronary syndrome. JAMA 2004; 291: 2727–33.

10. Grammer TB, Dressel A, Gergei I, Kleber ME, Laufs U, Scharnagl H, et al. Cardiovascular risk algorithms in primary care: results from the DETECT study. Nature Scientific Reports 2019; doi: 10.1038/s41598-018-37092-7.

11. Battler A. European Heart Survey of acute coronary syndromes. Eur Heart J 2002; 23: 1109–201.

12. Anderson JL, Morrow DA. Acute myocardial infarction. N Engl J Med 2017; 376: 2053–64.

13. Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, WHite HD;, the writing group on behalf of the Joint ESC/ACCF/AHA/WHF Task Force for the Universal Definition of Myocardial Infarction. Third universal definition of myocardial infarction. Eur Heart J 2012; 33: 2551–67.

14. Morrow DA, Cannon CP, Jesse RL, Newby LK, Ravkilde J, et al. National Academy of Clinical Biochemistry Laboratory Medicine Practice Guideline: Clinical characteristics and utilization of biochemical markers in acute coronary syndromes. Clin Chem 2007; 53: 552–74.

15. Jaffe AS. Third universal definition of myocardial infarction. Clin Biochem 2013; 46: 1–4.

16. Bonaca M, Scirica B, Sabatine M, Dalby A, Spinar J, Murphy SA, et al. Prospective evaluation of the prognostic implications of improved assay performance with a sensitive assay for cardiac troponin I. J Am Coll Cardiol 2010; 55: 2118–24.

17. Ridker PM, Everett BM, Thuren T, MacFadyen JG, Chang WH, Ballantyne C, et al. Antiinflammatory therapy with canakinumab for atherosclerotic disease. N Engl J Med 2017; 377: 1119–30.

18. Blankenberg S, Zeller T, Saarela O, Havulinna AS, Knee F, Tunstall-Pedoe H, et al. Contribution of 30 biomarkers to 10-year cardiovascular risk estimation in 2 population cohorts. The MONICA, risk, genetics, archiving, and monograph (MORGAM) biomarker project. Circulation 2010; 121: 2388–97.

19. Tjora HL, Steiro OT, Langorgen J, Bjorneklett RO, Skadberg O, Bonarjee VVS, et al. Diagnostic performance of novel troponin algorithms for the rule-out of non-ST-elevation acute coronary syndrome. Clin Chem 2022; 68 (2): 291–302.

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23. Thygesen K, Alpert JS, Jaffe AS, Chaitman BR, Bax JJ, Morrow DA, et al. Fourth universal definition of mycardial infarction (2018). Eur Heart J 2019; 237–69.

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2.4 Cardiac troponin (cTn)

The preferred biomarker for myocardial injury and myocardial infarction is cardiac troponin (cTn), which has both high myocardial tissue specificity and clinical sensitivity, although not decisive for a disease /1/. Two isoforms of cTn are differentiated: cardiac troponin T (cTnT) and cardiac troponin I (cTnI). Both troponins are muscle proteins that form part of the complex that regulates muscular contractility. Troponin and tropomyosin make up the thin filament of the contractile structure of striated muscle (Fig. 2.4-1 – Structure of a thin filament of troponin complex).

In the difference to conventional immunoassays cTn can be detected im blood with high-sensitivity immunoassays (hs-cTnI and hs-cTnT) in concentrations < 5 ng/L. The use of hs-cTnI and hs-cTnT has allowed to progressively reduce the time to diagnosis of Non-ST-Myocardial Infarction (NSTEMI) /2/.

For patent-related reasons, only cTnT assays of one manufacturer are commercially available, whereas cTnI assays are produced by a number of diagnostics manufacturers.

2.4.1 Indication

Investigation of acute myocardial necrosis:

• Diagnosis and course of myocardial infarction (MI)
• Detection of focal necrosis following invasive cardiologic interventions
• Detection of subclinical myocardial necrosis
• Detection of stress-induced myocardial ischemia
• Detection of toxic myocardial necrosis
• Suspected myocarditis
• Evaluation of the success of thrombolytic therapy
• Primary and secondary prevention of cardiovascular disease (CVD).

2.4.2 Method of determination

On basis of the lower detection limit hs-cTnI and hs-cTnT are differetiated from conventional sensitive cTn immunoassays.

2.4.2.1 Sensitive conventional immunoassays

Sensitive conventional immunoassays for the diagnosis of MI have to meet two basic criteria /3/.

• The total imprecision at the 99th percentile value for healthy individuals should be ≤ 10%
• Measurable concentrations below the 99th percentile should be attainable with an assay at a concentration value above the assay’s limit of detection for at least 50% (and ideally > 95%) of healthy individuals to attain the highest level of scorecard designation.

2.4.2.2 High-sensitivity immunoassays

High sensitivity cardiac troponin (hs-cTn) reflects the characteristics of the respective assay and is not related to the methodology with which the concentration is measured. The concentration of hs-cTn is measured in ng/L.

Important features of hs-cTnI and hs-cTnT assays are /3/:

• The reduction of the time to diagnosis of NSTEMI /2/
• The significant higher precision at the upper reference limit (URL) for healthy individuals (99th percentile of the upper reference limit; URL) /3/
• The higher predictive value for NSTEMI and the 4% absolute and 20% relative increase in the detection of MI type 1
• A corresponding decrease in the diagnosis of unstable angina
• The 2-fold increase in the detection rate of NSTEMI.

Refer to:

• conventional sensitive immunoassays (Tab. 2.4-1 – Analytical characteristics of sensitive cTn assays)
• high-sensitivity cTn immunoassays (Tab. 2.4-2 – Analytical characteristics of high-sensitivity cTn assays).

2.4.2.3 Point-of-care immunoassays

Point of care immunoassays are analyzed in the hospital, in the doctor’s practice, and in the emergency medicine. They have a detection sensitivity comparable to that of laboratory tests.

2.4.2.3.1 Point of care cTnT tests in whole blood

Principle: whole blood applied to the test pad releases cTnT antibodies a biotinylated labeled and a gold-labeled monoclonal antibody. Cellular blood components are retained by a filter. In the presence of cTnT, immune complexes are formed when the biotin-labeled cTnT antibody complexes bind to immobilized streptavidin. The gold-labeled indicator antibody binds to the immobilized cTnT antibody complex and makes the reaction visible in the results window. Another test pad is used as a positive control on which the gold-labeled TnT antibody binds directly to immobilized cTnT /4/. Accurate results are measured when the hematocrit is 14–55%.

2.4.2.3.2 Sensitive cTnT assay in serum/plasma /5/

Principle: the quantitative cTnT assay is performed as a one-step sandwich assay using streptavidin technology. The antibodies recognize two adjacent epitopes in the central part of the cTnT molecule. Therefore, the assay is not susceptible to proteolytic degradation of cTnT. The assay detects free and complex-bound cTnT. The point of care test and automated assay are compatible with each other.

2.4.2.3.3 High-sensitivity cTnI assay in serum/plasma

Principle: the cTnI assay is performed as a one or two-step immunoassay using two monoclonal or polyclonal antibodies against different epitopes of the cTnI molecule. Because the cTnI molecule is degraded in the blood, antibodies that recognize the central part of the molecule should be used. Because cTnI is present in the blood as part of a binary cTnI-TnC complex, the antibodies used should also recognize this complex.

2.4.2.3.4 High-sensitivity cTnT assays /6/

The hs-cTnT assay uses the Fab fragments of two monoclonal mouse antibodies. They are directed against epitopes in the central region of the TnT molecule. The capture antibody is biotinylated and directed against epitopes of amino acids 125–131 and the detection antibody is directed against an epitope of amino acids 136–147.

2.4.2.3.5 High-sensitivity cTnI assays

An evaluated hs-cTnI assay uses three different monoclonal antibodies to detect cTn epitopes: one against amino acids (AA) 30–35, one against AA 41–56, and one against AA 171–190. The lower detection limit is 0.8 ng/L and the upper reference interval value is 48 ng/L. Detectable hs-cTnI concentrations are present in 93% of healthy individuals /5/.

2.4.3 Specimen

Laboratory tests: serum, plasma (heparin, citrate): 1 mL

Point of care test (POCT): whole blood: 0.05–1 mL

2.4.4 Concepts of troponin testing

The definition of myocardial infarction (MI) requires a cardiac troponin value that exceeds the 99th percentile and undergoes significant rise.

2.4.4.1 Rapid rule-in and rule-out algorithms of acute myocardial infarction

Strategies for implementation of high sensitivity cardiac troponin (hs-cTn) for rapid diagnosis of MI are recommended /3/.

2.4.4.2 Rule-in strategy using conventional sensitive immunoassays for cTn measurement

The admission measurement, often referred to as “one and done” was originally proposed for single-sample rule-out /4/. The use of commercially available imunoassays for the determination hs-cTnI and hs-cTnT has progessively reduced the time to diagnose of NSTEMI /3/.

2.4.4.3 Rule-out strategy using conventional sensitive immunoassays for cTn measurement

At 3 hours from admission, combined with calculation of the change (delta) between values. The option is a composite approach using admission measurement followed by serial measurement at 3 hours but using the 99th percentile /7/. Rule-out MI is either on admission measurement alone or both values remain below the 99th percentile with a low delta value. A high delta with a rise to a value exceeding the 99th percentile rules in MI /3/.

2.4.4.4 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation /8/

It is recommended to use the 0h/3h algorithm as an alternative. 0h/1h assessments are recommended when high-sensitivity cTn assays with a validated algorithm are available. The positive predictive value for MI in those patients meeting the rule-in criteria was 75–80%. Most of the rule in patients with diagnosis other than MI did have conditions that usually require inpatient coronary angiography for accurate diagnosis.

2.4.5 Clinical assessment

2.4.5.1 Troponin in the diagnosis of myocardial infarction (MI)

Myocardial injury with necrosis may occur either in the presence of overt ischemia, or in the absence of overt ischemia from MI accompanying other conditions /9/. Working groups of the WHO established a primarily electrocardiographic (ECG) based definition of MI /10/. If an acute coronary symptom is suspected MI is classified on the basis of the presence or absence of ST-segment elevation.

• MI Type 1 is due to coronary atherothrombosis and is based on ST-segment elevation /11/.
• MI Type 2 is a condition without ST-segment elevation and occurs in the clinical setting without overt myocardial ischemia where a condition other than an acute atherothrombotic event is the major contributor to a significant imbalance between myocardial oxygen supply and/or demand /9/.

2.4.5.2 Determination of hs-cTnI or hs-cTnT on admission and 1 h to 3 h after admission

The high-sensitivity immunoassays for hs-cTnI and hs-cTnT are actually recommended by the most guidelines as gold standa laboratory methods for the detection of myocardial injury and the diagnosid myocardial infarction (MI) /2/.

2.4.5.2.1 Two-hour algorithm

In a multicentre study using high-sensitivity cTnI a two-hour algorithm for triage toward rule-out and rule-in of MI was investigated /13/. Baseline values as well as absolute changes after 2 hours were incorporated. The 2-h algorithm classified 56% of patients as rule-out, 17% as rule-in and 27% as observation. Resulting diagnostic sensitivity and negative predictive value were 99.2% and 99.8% for rule- out; specificity and positive predictive value were 95.2% and 75.8% for rule-in. Refer to Fig. 2.4-2 – The use of the 0h/3h rule.

2.4.5.2.2 One-hour algorithm

In a multicentre study using high-sensitivity cTnT a 1-h- algorithm for triage toward rule-out and rule-in of MI was investigated /14/. Baseline values as well as absolute changes after 1 h were incorporated. The 1-h algorithm classified 59.5% of patients as rule-out, 16.4% as rule-in and 24,1% as observation. Resulting diagnostic sensitivity and negative predictive value were 99.6% and 99.9% for rule-out; specificity and positive predictive value were 95.7% and 78.2% for rule-in.

Refer to Fig. 2.4-3 – The use of the 0h/1h assessment.

2.4.5.3 Rule-in measurement and determination after 1 to 3 h

A high-sensitivity immunoassay for cardiac troponin (hs-cTn) is recommended by all the most recent guidelines as gold standard laboratory method for the detection of myocardial injury and the diagnosis of MI using high-sensitivity cTn assays /46/. A single low cTn value excludes up to 50% of chestpain patients without MI /2/.

A review of high-sensitivity cTnI at presentation in patients with suspected acute coronary syndrome showed the following results /15/: in patients without MI at presentation hs-cTnI concentrations were less than 5 ng/L in 61% with a negative predictive value of 99.6% for the primary outcome. At 1 year, these patients had a lower risk of MI and cardiac death than did those with a cTnI concentration of 5 ng/L and more.

In a second study a pooled analysis of five international prospective, observational cohort studies with blinded outcome assessment and 30-day follow-up was reported /16/. Eligible patients had non-ischemic ECG determined and high-sensitivity cTnI measured at presentation. The lower limit of detection of cTnI was 1.2 ng/L. MI developed in 9.2% of patients. The 1.2 ng/L limit of detection gave a sensitivity of 99.0% and a negative predictive value of 99.5%. This cutoff level would allow for early discharge of 18.8% of patients. All higher cutoff values had a negative predictive value less than 98.0%.

Testing with one single criterion has decreased diagnostic specificity for MI, since high-sensitivity assays detect the presence of cTn in most normal persons. In a number of disorders, other than MI high-sensitivity cTn concentrations are measured. Refer to Tab. 2.4-3 – cTn in acute coronary syndrome and other types of myocardial injury.

2.4.5.4 Extracardiac elevation of cTn

Patients with MI-Type 2 can have elevated cTn concentrations without cardiovascular disease /9/, e.g., infection, sepsis, congestive heart failure, chronic kidney disease (Tab. 2.4-4 – Conditions other than acute myocardial infarction associated with cTn elevation).

2.4.5.5 hs-cTn as a prognostic marker

Adults without cardiovascular disease

High sensitivity cTn assays are positive in many adults without recognized cardiovascular disease. In the Dallas Heart Study /12/, 27% of individuals aged 30–65 years were positive for hs-cTnT and 3.4% of those had values above the 99th percentile URL. In the Atherosclerosis Risk in Communities (ARIC) study /13/ and the Cardiovascular Health Study /14/, 66% of middle-aged and older individuals had detectable hs-cTnT levels. In the ARIC study, 7.4% had values > 99th percentile URL and in the Cardiovascular Health Study, 16.6% had hs-cTnT values of over 12.9 ng/L. These studies and others have shown that advanced age, male gender, previous ischemic or non-ischemic cardiovascular disease, chronic kidney disease, and cardiovascular risk factors are associated with detectable or elevated cTn values.

Patients with stable cardiovascular disease

High sensitivity cTn assays can also be used to identify patients with cardiovascular disease (CVD) who are at increased risk of MI in the future. For example, patients with hs-cTnT concentrations of 8–14 ng/L (normal range) have a hazard ratio of 1.47 for future myocardial infarction /15/. In the Prevention of Events with Angiotensin Converting Enzyme Inhibition (PEACE) trial /16/, hs-cTnT values above 6.3 ng/L were associated with an increased cumulative incidence of cardiovascular death in individuals with stable cardiovascular disease. In the ARIC study, hs-cTnT values above 3 ng/L were associated with increased mortality and hospitalization and values above 6 ng/L with the risk of ischemic heart disease. For risk stratification of CVD. Refer to Tab. 2.4-5 – Risk stratification of patients/individuals with/without ACS.

Patients with suspected acute coronary syndrome (ACS)

Early risk stratification in patients with suspected ACS is carried out by means of a cTn assay and evaluation of the results with respect to the 99th percentile URL and the clinical symptoms /17/. cTn assays in patients whose clinical history was consistent with ACS have shown that patients with values in the lower range have a higher risk of recurrent cardiac events than those with undetectable cTn levels. In the Treat Angina with Aggrastat and Determine Cost of Therapy with an Invasive or Conservative Strategy (TACTICS-TIMI 18) study, patients whose cTnI was just above the 99th percentile URL (0.1 μg/L, CV 20%) had a > 3-fold increased risk of recurrent cardiac events or cardiac mortality compared to patients with lower cTn levels /18/.

In a further study /19/, patients with ACS associated with Type 2-MI and hs-cTnI values ≥ 40 ng/L on admission had a 3-fold increased risk of further MI or cardiac death within 30 days.

2.4.5.6 Cardiac troponins and the treatment of acute coronary syndrome

The choice of cTn value as of which treatment takes place is a decisive factor in therapeutic success. For example, in the TACTICS-TIMI 18 study, 25% more patients had a positive outcome when percutaneous revascularization was carried out at a cTnT value of just 0.01 μg/L instead of 0.1 μg/L /18/. Treatment guidelines have been defined by the American Heart Association /20/.

2.4.5.7 Cardiac surgery

The three primary criteria for diagnosis of MI are chest symptoms, ECG changes and abnormal biomarkers (cardiac troponin, CK-MB). Perioperative myocardial infarction is an important complication after cardiac surgery and a challenging diagnosis because preoperative ischemia, insufficient interoperative myocardial protection, sternotomy pain and sedation may mask ischemic symptoms /44/. Recommended thresholds for diagnosis of perioperative myocardial infarction have been similar for troponin and CK-MB and are > 10 times the upper reference limit. Alternative recommendation from the Academic Research Consortium-2 /45/ have addressed this by suggesting troponin ratios of 35 or higher for perioperative myocardial infarction (with evidence of ischemia) or 70 or higher for significant myocardial injury (without evidence of of ischemia). However few data are available that evaluate these higher thresholds.

Distinct from perioperative myocardial infarction, the nebulous term “distinct perioperative myocardial injury” is used to describe markedly abnormal levels of cardiac troponin and CK-MB that occur without supportive evidence of ischemia but that also are associated with an excess risk of perioperative death and higher long-term mortality /44/.

2.4.6 Comments and problems

Specimen

Since heparin can interfere with cTn assays, in particular due to the formation of micro fibrin clots, serum samples are recommended for some commercial assays. Because EDTA splits the troponin complex, EDTA blood samples are not recommended for cTnI assays.

Method of determination /19/

cTnI: many commercial assays are available for automated analyzers and point-of-care testing with antibodies against various cTnI epitopes. However, because the amino and carboxy terminal parts of cTnI are susceptible to proteolysis, which, in turn, depends on the degree of ischemia, the assays behave differently during MI. Because they are not standardized, cTnI assays cannot be compared.

The hs-cTn assays do not necessarily translate into higher clinical sensitivities at presentation compared with contemporary assays in emergency department populations of patients presenting with diverse pathophysiology for myocardial injury and more type 2 myocardial infarctions /21/.

cTnT: because cTnT assays are produced by one manufacturer only, the quantitative assays performed automated analyzers and point-of-care testing show the same results since they are calibrated using the same reference material. Since the assays use two monoclonal antibodies that are directed against central cTn antigens, there is little cross-reactivity with skeletal muscle TnT. The assay is robust in the face of molecular changes and TnT degradation products.

Point of care test: POCT should be implemented in a hospital if the in-house laboratory cannot determine cTn within 30 minutes.

Stability

Depending on the assay, storing the sample at room temperature for several hours can lead to an increase or decrease in the concentration due to cTn degradation.

Hemolysis

Hemolysis can interfere with cTnI and cTnT assays. Follow the manufacturer’s instructions.

Standardization of cTnI

The National Institute of Standards and Technology (NIST) together with the American Association of Clinical Chemistry (AACC) and International Federation of Clinical Chemistry (IFCC) has created certified reference material for cTnI (SRM 2921) for standardization purposes /22/. The cTnI concentration is 31.2 mg/L.

False positive cTn results /23/

The prevalence of false positive cTn results is 0.2–3%. False positives result from analytical interferences due to fibrin clots and micro particles in the sample, heterophile antibodies, human anti-animal (mouse) antibodies, rheumatoid factors, hyperbilirubinemia, hemolysis, lipemia, alkaline phosphatase, and the formation of macro immune complexes. These interferences are manufacturer-specific and do not apply to all cTn assays.

Autoantibodies against cTn

Autoantibodies against cTnI and cTnT are present in the serum of approximately 10% of individuals, mostly directed against cTnI or cTnT, but also to both cTn in around 1%. They can interfere with cTn assays and cause falsely low or even false negative cTn measurements in patients with acute coronary syndrome /24/. Persistent autoantibodies can still be detected in patients with acute coronary syndrome 3–12 months after admission.

Criterion for the diagnosis of acute myocardial infarction

To support the diagnosis of MI the 99th percentile URL remains the best-established approach given the absence of cTn assays standardization /21/.

2.4.7 Pathophysiology

The troponin complex consists of three different structural proteins that are located in the thin filament of the contractile apparatus (Fig. 2.4-1 – Structure of a thin filament of troponin complex). Both in cardiac muscle and skeletal muscle, each of the proteins is coded by a specific gene. The three proteins are cTnT, cTnI, and cTnC, with molecular weights of 39 kDa, 26 kDa, and 19 kDa respectively. Specific isoforms of TnT and TnI are present in cardiac and skeletal muscle. TnC is identical in cardiac and skeletal muscle and is therefore not suitable for diagnosing myocardial injury. A significant proportion of cTnT and cTnI is structure-bound in the thin muscle filament.

Following myocardial damage, cTn is rapidly broken down and released into the circulation. Only 6–8% of cTnT and 3–4% of cTnI is present in a soluble form in the cytoplasm. The different compartmentalization of cTnT and cTnI is one reason for the biphasic release of cTnT and the monophasic release of cTnI. The majority of the cTn is not released from the myofibrils until around 12 hours later, as binary cTnI/C or ternary cTnI/C/T complexes following proteolytic degradation. The ternary complex clearly predominates (Fig. 2.4-4 – Compartmentalization and release of cTnT and cTnI following myocardial injury). Binary and ternary cTn are responsible for the protracted cTn elevation in acute myocardial infarction. Some of the cTnI released from the cytosol combines with soluble cTnC (sTnC) to form the binary complex cTnI/sTnC in the circulation, which is detected to different degrees by the commercially available immunoassays /21/.

cTn is released from cardiac muscle in various ways. In acute ischemia with myocyte necrosis, it is released following irreversible damage to the cell membrane. Transient ischemia can cause cTn release due to temporary cell membrane leakage. This mechanism is possibly also present in multi-organ failure e.g., in sepsis. hs-cTn values < 99th percentile URL are a sign of ongoing myocardial damage due to reduced blood flow in atherosclerosis.

Autoantibodies against cTnI are directed against the central fragment of cTnI, in particular the C-terminal part.

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2.5 CK-MB mass

CK-MB is an isoenzyme of CK that is present mainly in cardiac muscle. CK-MB is released during myocardial necrosis (for CK and CK-MB, see also Tab. 1.8-4 – CK and CK-MB activities in acute myocardial injury).

The CK-MB mass is analyzed by determining the protein concentration of the CK-MB isoenzyme. Compared with the measurement of CK-MB enzyme activity, this offers improved sensitivity and specificity for diagnosing acute myocardial infarction (MI) /1, 2/.

2.5.1 Indication

If cardiac troponin determination is not possible:

• Early diagnosis of acute myocardial infarction
• Monitoring of acute myocardial infarction for early identification of re infarction
• Monitoring of thrombolytic therapy
• Infarct diagnosis following coronary artery bypass surgery
• Prognosis in unstable angina pectoris.

2.5.2 Method of determination

Point of care test in whole blood

Principle: whole blood applied to the test pad releases both a monoclonal, immobilized, CK-MB-specific antibody and an indicator antibody labeled with gold, for example. Cellular blood components are retained by a filter. If CK-MB is present, it binds to the immobilized antibody to form immune complexes. The second, gold-labeled antibody binds to the immobilized CK-MB-antibody complex and makes the reaction visible in the results window.

Quantitative assay in serum/plasma

Principle: in a one-step sandwich assay, CK-MB in the specimen reacts with both a biotinylated monoclonal CK-MB-specific antibody and a CK-MB antibody labeled with a ruthenium complex to form a sandwich complex. The immune complex is bound to streptavidin coated paramagnetic micro particles using biotin. The electrochemiluminescence of the sandwich complexes is measured. However, other procedures also exist.

CK-MB isoforms

To improve the diagnostic sensitivity of CK-MB in the early stage of myocardial infarction, it is separated into its two isoforms, CK-MB 2 and CK-MB 1, using high voltage electrophoresis. These isoforms result from post synthetic modification of the primary CK-MB structure. CK-MB is transformed into the two isoforms by the enzymatic action of the enzyme carboxypeptidase. The electrophoresis gel is scanned with ultraviolet light to establish the CK-MB 2/CK-MB 1 ratio. This procedure is used in the differential diagnosis of increased CK activity that cannot be explained clinically.

2.5.3 Specimen

• Serum, plasma: 1 mL
• Whole blood (EDTA or heparin): 0.1 mL

2.5.4 Reference interval

CK-MB mass: Below 5–8 μg/L /3, 4/ (depending on the manufacturer) CK-MB-2/CK-MB-1 ratio: Below 1.7

2.5.5 Clinical significance

According to the guidelines of the European Society of Cardiology and the American Heart Association/American College of Cardiology (ESC/ACC), CK-MB mass assay has an important role in acute coronary syndrome in cases where cTn assay is not available /5/. This applies to diagnosis, risk stratification, and the choice of treatment methods for acute coronary syndrome with or without ST segment elevation on the ECG /6/. Although the CK-MB mass is less specific for the myocardium than cTn, it has higher diagnostic specificity for ruling out acute myocardial infarction /5/. As with cTn, an increased CK-MB mass concentration is defined as a value above the 99th percentile of upper reference limit (URL) for a healthy control group. In the acute coronary syndrome, the CK-MB mass should be determined on admission, after 2–4 h, after 6–9 h, and after 12 h. The behavior of the CK-MB mass in acute coronary syndrome is shown in Tab. 2.5-1 – CK-MB mass in acute coronary syndrome and other muscular damage.

A CK-MB 2/CK-MB 1 ratio above 1.7 is an early indicator of myocardial necrosis.

2.5.6 Comments and problems

Point of care test in whole blood

CK-MB concentrations above 5 μg/L produce a positive test result. Assays are available that can determine the CK-MB mass, myoglobin, and cTnI simultaneously in a serum sample.

Quantitative assay in serum/plasma

Because of the high analytical specificity of enzyme immunoassays, CKMM, CKBB, macro CK type 1 and type 2, and adenylate kinase only interfere at very high concentrations.

Interference factors

Hemoglobin ≤ 10 g/L (0.63 mmol/L), bilirubin ≤ 500 mg/L (850 μmol/L), triglycerides ≤ 1,350 mg/dL (15.4 mmol/L), rheumatoid factors ≤ 500 U/mL, and commonly used pharmaceuticals do not interfere with the assays. Samples that contain precipitates must be centrifuged before the assay. Fibrin clots interfere with the assay.

Stability

At room temperature for at least 12 hours, at 2–8 °C for at least 3 days. For longer-term storage, must be frozen (at –20 °C is stable for at least 12 months, freeze and thaw once only).

2.5.7 Pathophysiology

The CK-MB mass constitutes 3–5% of skeletal muscle and 5% of cardiac muscle. However, in pathologically altered cardiac muscle, the CK-MB mass proportion is 20–30% /3/. This explains why individuals without a history of heart disease can show only slight increases in CK-MB following myocardial infarction. Causes of an increased CK-MB proportion include chronic cardiac stress due to ventricular hypertrophy and coronary heart disease /3/.

Chronic stress also induces an increase in the CK-MB proportion of skeletal muscle. This is the case, for example, in marathon runners and other endurance athletes as well as in individuals who carry out a lot of physical work in everyday life. The CK-MB proportion can also be as high as 20–30% in myopathies such as Duchenne muscular dystrophy and polymyositis /4, 7/. These examples explain why increased CK-MB mass concentrations can be detected not only in myocardial necrosis but also in skeletal muscle damage.

References

1. Mair J, Artner-Dworzak E, Dienstl A, et al. Early detection of acute myocardial infarction by measurement of mass concentration of creatine kinase-MB. Am J Cardiol 1991; 68: 1545–50.

2. Ravkilde J, Nissen H, Horder M, Thygesen K. Independent prognostic value of serum creatine kinase isoenzyme MB mass, cardiac troponin T and myosin light chain levels in suspected myocardial infarction. J Am Coll Cardiol 1995; 25: 574–81.

3. Ingwall JS, Kramer MF, Fifer MA, et al. The creatine kinase system in normal and diseased human myocardium. N Engl J Med 1985; 313: 1050–4.

4. Somer H, Duboeitz V, Donner M. Creatine kinase isoenzymes in neuromuscular diseases. J Neurol Sci 1976; 29: 129–36.

5. Bertrand ME, Simoons ML, Fox KAA, Wallentin LC, Hamm CW, McFadden E, et al. Management of acute coronary syndromes in patients presenting without persistent ST-segment elevation. Task Force Report. European Heart J 2002; 23: 1809–40.

6. Myocardial Infarction redefined: a consensus document of the Joint European Society of Cardiology/American College of Cardiology Committee for the redefinition of myocardial infarction. J Am Coll Cardiol 2000; 36: 959–69.

7. Neumeier D, Jockers-Wretou E. Tissue specific and subcellular distribution of creatine kinase isoenzymes. In: Lang H, ed. Creatine kinase isoenzymes – pathophysiology and clinical application. Berlin; Springer 1981: 85–129.

8. Gibler WB, Young GP, Hedges JR, et al. Acute myocardial infarction in chest pain patients with non-diagnostic ECGs: Serial CK-MB sampling in the emergency department. Ann Emerg Med 1992; 21: 504–12.

9. Mair J, Morandell D, Genser N, Lechleitner P, Dienstl F, Puschendorf B. Equivalent early sensitivities of myoglobin, creatine kinase MB mass, creatine kinase isoform ratios, cardiac troponin I and T for acute myocardial infarction. Clin Chem 1995; 41: 1266–72.

10. Stewart J, French JK, Theroux P, et al. Early noninvasive identification of failed reperfusion after intravenous thrombolytic therapy in acute myocardial infarction. J Am Coll Cardiol 1998; 31: 1499–1505.

11. Brener SJ, Lytle BW, Schneider JP, Ellis GE, Topol EJ. Association between CK-MB elevation after percutaneous or surgical revascularisation and three-year mortality. J Am Coll Cardiol 2002; 40: 1961–7.

12. Thygesen K, Alpert JS, White HD, Joint ESC/ACCF/AHA/WHF Task Force for the Redifinition of Myocardial Infarction. Third universal definition of myocardial infarction. Circulation 2012; 126: 2020–35.

2.6 Myoglobin

Myoglobin is a protein found in cardiac and skeletal muscle that functions as a reservoir for oxygen.

2.6.1 Indication

Myoglobin has an important role in addition to the cardiac troponin in multi marker diagnostics for:

• Early diagnosis or exclusion of myocardial necrosis in acute coronary syndrome (ACS)
• Detection of re-infarction
• Monitoring thrombolytic therapy for myocardial infarction
• Risk stratification in acute coronary syndrome in combination with cardiac troponin and/or CK-MB mass.

2.6.2 Method of determination

Qualitative and quantitative point of care test in whole blood

Principle: see cardiac troponin. Myoglobin assay is also available in combination with cardiac troponin (cTn) and CK-MB mass on one test pad.

Immunoturbidimetric assay

Principle: polystyrene particles coated with antibodies to myoglobin form an immune complex with myoglobin in the serum by forming agglutinates. The increased turbidity caused by the agglutinates is measured photometrically and evaluated using a calibration curve.

Immunonephelometric assay

Principle: agglutination reaction between myoglobin and myoglobin antibodies that are bound covalently to plastic particles. Following incubation, light scattering measurement is used to calculate the extent of the agglutination using a logit-log function.

Enzyme immunoassay

Principle: myoglobin is determined using homogeneous or heterogeneous assays. One-step sandwich assays are commonly used.

2.6.3 Specimen

• Serum or plasma: 1 mL
• Whole blood: 0.1 mL
• Urine if myoglobinuria due to skeletal muscle damage is suspected: 10 mL

2.6.4 Reference interval

Serum, plasma < 70–110 μg/L /1/

2.6.5 Clinical significance

Serial myoglobin measurements have no advantage over cTn in patients with acute coronary syndrome.

Behavior of myoglobin in myocardial infarction:

• Myocardial necrosis within 6 hours can be reliably excluded if myoglobin assays performed on admission and at 2, 4, and 6 hours after the acute onset of pain are all normal
• Unstable angina pectoris is suggested if no significant elevation is evident after 2–4 hours.

The behavior of myoglobin in acute coronary syndrome and other conditions is shown in Tab. 2.6-1 – Myoglobin in acute coronary syndrome and other muscular damage.

The disadvantages of myoglobin determination in patients with acute coronary syndrome are as follows:

• The release kinetics of myoglobin are fast and its release into the blood is characterized by a “staccato phenomenon” with peaks that are often short-lived, lasting only 1–2 hours. Consequently, blood must be collected at short time intervals of 2 hours at most.
• An increase in myoglobin that occurs for the first time 6–10 hours after the onset of chest pain is of limited significance since it could also be due to skeletal muscle damage.
• A normal myoglobin concentration is of no value if it is measured for the first time 10 hours after the acute onset of pain.
• It can be difficult to diagnose a re-infarction since the potential increase in myoglobin can show a high degree of variability.
• Myoglobin has low diagnostic specificity since it is also increased following skeletal muscle trauma.

2.6.6 Comments and problems

Interference factors: hemoglobin > 3 g/L (0.18 mmol/L), bilirubin > 32.2 mg/dL (550 μmol/L), and high rheumatoid factor concentrations interfere with immunonephelometric and immunoturbidimetric assays and enzyme immunoassays. Lipemic serum must be centrifuged (for 10 min at 15,000 g).

Stability: in serum and plasma at room temperature, at least 2 days; at 4 °C, at least 1 month; and at –20 °C, for longer.

2.6.7 Pathophysiology

Myoglobin is an oxygen-binding heme protein found in striated muscle (skeletal and cardiac muscle). It has a molecular weight of 17.8 kDa and accounts for 2% of the total muscle protein /2/. Myoglobin is located in the cytoplasm of muscle cells and rapidly permeates the extracellular space in the event of damage to the cell membrane. Therefore, myoglobinemia occurs relatively quickly following injury to striated muscle.

Myoglobin reaches pathological values before the muscle enzymes and the cardiac proteins such as troponin that are bound to cell structures are activated. Myoglobin levels also return to the reference interval sooner than those of the enzymes and structural proteins mentioned since it is filtered rapidly by the kidneys due to its low molecular weight. Therefore, myoglobin levels can increase significantly in end-stage renal disease.

The physiological importance of myoglobin is based on its ability to bind molecular oxygen reversibly with greater affinity than hemoglobin. Myoglobin therefore plays an important role in transporting and storing oxygen in striated muscle.

In coronary artery surgery, myoglobin assays can diagnose perioperative myocardial infarction earlier and determine the time of infarction more effectively than assays of other biochemical cardiac markers /3/.

Myoglobin’s short biological half-life of 10–20 minutes compared to that of the CK-MB mass (approx. 12 hours) has diagnostic advantages. During percutaneous coronary intervention, changes in micro perfusion can be detected with minimal delay in the serum by means of a rapid succession of myoglobin peaks (staccato phenomenon) caused by variable perfusion of the infarct vessel. Therefore, the course of myocardial necrosis and its response to treatment over time can be monitored more effectively than with the cardiac troponin and CK-MB mass /4/.

References

1. Laperche T, Steg PG, Dehoux M, et al. A study of biochemical markers of reperfusion early after thrombolysis for acute myocardial infarction. Circulation 1995; 92: 2079–86.

2. Sylven C, Jansson E, Book K. Myoglobin content in human skeletal muscle and myocardium: relation to fibre and size and oxidative capacity. Cardiovasc Res 1984; 18: 443–6.

3. Mair P, Mair J, Seibt I, Balogh D, Puschendorf B. Early and rapid diagnosis of perioperative myocardial infarction in aortocoronary bypass surgery by immunoturbidimetric myoglobin measurements. Chest 1993; 103: 1508–11.

4. Drexel H, Dworzak E, Kirchmair M, et al. Myoglobinemia in the very early phase of acute myocardial infarction. Am Heart J 1983; 105: 642–51.

5. Ng, SM, Krishnaswamy P, Morissey R, et al. Ninety-minute accelerated critical pathway for chest pain evaluation. Am J Cardiol 2001; 88: 611–7.

6. McCord J, Nowak R, McCullough P, et al. Ninety-minute exclusion of acute myocardial infarction by use of quantitative point-of-care testing of myoglobin and troponin I. Circulation 2001; 104: 1483–8.

7. Zabel M, Hohnloser SH, Röster W, Prinz M, Kasper W, Just H. Analysis of creatine kinase, CK-MB, myoglobin, and troponin T time-activity curves for early assessment of coronary artery reperfusion after intravenous thrombolysis. Circulation 1993; 87: 1542–50.

2.7 Heart failure (HF)

2.7.1 Definition of heart failure

Heart failure is a complex clinical syndrome with symptoms and signs that result from any structural or functional impairment of ventricular filling or ejection of blood. The American College of Cardiology (ACC) and the American Heart Association (AHA) stages of heart failure emphazise the development and progress of disease, and advanced stages and progression are associated with reduced survival /1/. The ACC/AHA stages of heart failure emphazise the develpment and progression of disease. Refer to Tab. 2.7-7 – Stages of heart failure. Therapeutic interventions in each stage aim to modify risk factors /1/:

• Stage A: treat risk and structural heart disease to prevent heart failure
• Stage B: pre heart failure, reduce symptoms and morbidity of structural heart disease
• Stages C and D: reduce morbidity and mortality of structural heart disease.

2.7.2 Chronic heart failure

The typical symptoms of chronic heart failure are dyspnea or fatigue, either at rest or on exertion, and signs of fluid retention such as leg edema /2/. However, because the diagnostic specificity of these symptoms is low, the clinical findings can only suggest, but not confirm, heart failure. Although dyspnea on exertion and orthopnea are useful indicators of left ventricular dysfunction, more than 30 causes exist for dyspnea and its prevalence in the population is 3–25%. However, once chronic heart failure has been diagnosed, the clinical symptoms can be used to classify the severity and monitor the response to therapy /3/.

2.7.2.1 Epidemiology

In the Framingham Study, the annual incidence rate of chronic heart failure was 2.3/1,000 in men and 1.8/1,000 in women /4/. In general, the expected annual incidence rate in Europe and North America is 1–4 new cases per 1,000 of the population. The prevalence depends on age. In Germany, the prevalence was 0.127% in individuals aged 45–65 years and 1.55% in those over 65 years of age, based on hospitalizations in 2006 /5/. Heart failure is the reason for about 20% of all hospital admissions among persons older than 65 in the United States. According to the Glasgow study /6/, the overall prevalence of left ventricular systolic dysfunction determined by echo cardiography was 2.9%. The left ventricular systolic dysfunction was symptomatic in 1.5% of patients and asymptomatic in 1.4%.

2.7.2.2 Etiology

The etiologies of chronic heart failure are listed in Tab. 2.7-1 – Etiology of chronic cardiac failure. For most patients, more than one of the etiological factors are relevant. The main cause is coronary heart disease. Not all patients have left ventricular contractile dysfunction and a reduced ejection fraction. Many also have cardiac valvular disease, aortic stenosis or mitral regurgitation resulting in diastolic heart failure. Around 30% of heart failure patients have a conduction defect that leads to delayed left or right ventricular systole.

Some 20–50% of patients have preserved left ventricular function or a normal left ventricular ejection fraction. Although their hearts contract normally, cardiac relaxation (diastole) is abnormal. The American College of Cardiology/American Heart Association Guidelines for the Diagnosis and Management of Heart Failure recommend using the term “heart failure with preserved ejection fraction” instead of “diastolic heart failure” for this form of chronic heart failure /7/.

Many patients with chronic heart failure are over 60 years of age and 75% have hypertension. In the Glasgow study /5/, 50% of cases had a history of myocardial infarction and 62% had a history of angina pectoris. Of the 280,000 individuals who suffer a myocardial infarction each year in Germany, 56,000 of those who survive are left with significantly reduced left ventricular function and an ejection fraction below 40%; 5–7% have a severely reduced ejection fraction of below 30%.

2.7.2.3 Classification and prognosis

Chronic heart failure in adults is classified using the New York Heart Association (NYHA) classification /8/ or the American College of Cardiology/American Heart Association classification /9/.

The NYHA classification differentiates symptoms of chronic heart failure into four stages of severity that correspond to the degree of exercise intolerance (Tab. 2.7-2 – New York Heart Association (NYHA) classification of heart failure). This in turn correlates with the patient’s prognosis. The 1-year mortality for severe heart failure is 50% and the 4-year mortality is 40–50% for moderate heart failure and 20–30% for mild heart failure.

The American College of Cardiology/American Heart Association classification divides heart failure into four stages and identifies factors that can be used to identify high-risk patients. According to this classification, patients with chronic heart failure can progress from stage A to stage D but cannot return to a previous stage. According to the NYHA classification, however, patients with class 4 symptoms can improve quickly in response to diuretic therapy alone and return to class 3.

Refer also to Tab. 2.7-3 – Classification of chronic heart failure according to the criteria of the European Society of Cardiology.

Patients who receive treatment when they are asymptomatic or when their ventricular function is only moderately reduced have a better prognosis and quality of life. This is why it is so important to detect chronic heart failure in the early stages of ventricular dysfunction.

Right ventricular failure

The main determinants of right ventricular function include – like those of left ventricular function – preload, afterload, contractility, and active relaxation. Processes that initiate or promote right ventricular failure are hypertrophy, fibrosis, ischemia, neurohormonal activation, inflammation and shifts in metabolic substrates /16/.

Symptoms include dyspnea, early satiety, abdominal fullness, lower-extremity edema, right upper-quadrant tenderness, fatique, exertional intolerance.

Laboratory findings: elevated serum levels of Brain natriuretic Peptide (BNP) and NT-proBNP.

2.7.3 Pathophysiology

Various models are combined to explain the pathophysiology of chronic heart failure /9/:

• The hemodynamic model, which is based on an altered volume load on the failing ventricle and the remodeling that results from this
• The neurohormonal model, in which activation of the renin-angiotensin-aldosterone system and the sympathetic nervous system plays an important etiological role
• Finally, the failure of autocrine and paracrine vasoactive substances that are synthesized by the myocardium in response to the stretching of cardiac muscle plays a role. Among these substances is B-type natriuretic peptide.

Cardiac muscle remodeling

When the heart muscle is injured by myocardial necrosis or as a result of pressure and volume overload, blood supply to perfuse organs adequately is not possible. This condition induces left ventricular remodeling, a process in which the size, structure, and function of the ventricle are altered by mechanical, neurohormonal, and possibly genetic factors. The aim of this process is to preserve the cardiac output. This type of process continues for months after myocardial infarction and the associated ventricular alterations can adversely affect the heart’s ability to pump blood /10/. Mitral valve insufficiency or conduction defects, for example, can occur as a result of remodeling.

During remodeling, myocytes respond with eccentric rather than physiological hypertrophy at cellular level. Physiological hypertrophy is characterized by a proportional increase in the length and width of cardiac myocytes. The assembly of contractile-protein units in series characterizes the eccentric hypertrophy. The length of myocytes is increased more than the width. When the ventricle is subject to pressure overload, new contractile proteins are added to myocytes in parallel so that there is a relative increase in the width of myocytes resulting in concentric hypertrophy of the ventricle /11/.

In hypertrophic cardiomyopathy, mutated contractile proteins are produced. This disturbs the arrangement of myofibrils, which leads to secondary hypertrophy of myocytes. This type of cardiac hypertrophy features increased expression of embryonic genes in the myocytes (e.g., for natriuretic peptide or fetal contractile proteins). The induction of genes for producing natriuretic peptides and thus the production of these peptides is a prognostic indicator of the clinical severity of dilated cardiomyopathy /11/.

Hemodynamic and hormonal mechanisms in chronic heart failure

Diminished cardiac output in low-output heart failure or reduced peripheral vascular resistance in high-output heart failure is associated with atrial under filling /12/. Baroreceptor-mediated neurohumeral events are initiated, particularly the activation of the sympathetic nervous system, the activation of the renin-angiotensin-aldosterone system, and the non osmotic release of vasopressin. All the systems maintain arterial perfusion of vital organs. These neurohumeral reflexes may have deleterious affects. In the acute phase arterial blood pressure is increased and ensures that vital organs are adequately perfused. However, chronic stimulation of these systems leads to persistent inadequate peripheral vasoconstriction, volume retention, and inefficient inotropic stimulation. The consequences include pulmonary edema, hyponatremia, increased cardiac after load, and cardiac remodeling. This altered hemodynamic, functional, and metabolic status gives rise to the cardinal symptoms of chronic heart failure such as exercise intolerance, easy fatigability, and dyspnea /13/.

Blood volume in chronic heart failure

Congestive heart failure is characterized by increased total body-fluid in combination with ventricular insufficiency. In patients with chronic heart failure, Na⁺ and water are paradoxically retained despite increased intravascular volume /12/. This is because the integrity of the arterial circulation, in which the left ventricular ejection fraction and peripheral arterial resistance play a crucial role, is the main determinant of renal water and Na⁺ excretion (Fig. 2.7-1 – Causes and consequences of reduced arterial blood volume in chronic heart failure).

Renin-angiotensin-aldosterone system (RAAS) and chronic heart failure

Patients with mild heart failure may have little or no increase in plasma renin and aldosterone /12/. Unlike primary hyperaldosteronism, the hyperaldosteronism associated with chronic heart failure and Na⁺ and water retention is persistent (see also Chapter 31 – Mineralocorticoid excess).

In primary hyperaldosteronism, Na⁺ retention initially leads to an increase of 1.5–2 liters in the extracellular fluid volume. However, Na⁺ retention than ceases Na⁺ balance is reestablished and there is no edema (escape phenomenon).

In chronic heart failure, this “escape” from the action of aldosterone does not occur and therefore patients continue to retain Na⁺ in response to aldosterone /12/. This failure of the escape phenomenon in chronic heart failure is caused by increased Na⁺ reabsorption in the proximal tubule, which means that less Na⁺ reaches the collecting ducts.

The increased reabsorption of Na⁺ in the proximal tubule is due to increased α-adrenergic and angiotensin II stimulation in chronic heart failure (Fig. 2.7-2 – Mechanisms by which arterial hypovolemia leads to reduced delivery of sodium and water at the distal tubule of the kidney). In patients with chronic heart failure, angiotensin II causes constriction of the afferent and efferent arterioles. It stimulates contraction of glomerular mesangial cells, which leads to a reduced glomerular filtration surface.

Arginine vasopressin release in chronic heart failure

Non-osmotic release of arginine vasopressin in chronic heart failure may lead to increased water retention and hyponatremia. Hyponatremia may be caused by increased water intake in response to increased thirst /12/. In normal individuals, arginine vasopressin secretion should be inhibited in the case of plasma hypo osmolality. This is not the case in chronic heart failure. On the contrary, persistently elevated concentrations of arginine vasopressin are recorded.

Natriuretic peptides and chronic heart failure

Type A and type B natriuretic peptides (e.g., ANP, BNP) are released by cardiomyocyte stretching in the atria and ventricles and their concentration in the blood increases when intraatrial pressure is increased. The natriuretic peptides exert an effect on the kidneys /12/. They dilate the afferent arterioles and constrict the efferent arterioles in the glomeruli, which increases the glomerular filtration rate. ANP and BNP lead to reduced reabsorption of Na⁺ in the collecting ducts and, thereby, increasing Na⁺ excretion. Because even in the early phase of chronic heart failure, concentrations of natriuretic peptides are increased in the blood, ANP and BNP are sensitive markers of chronic heart failure. See also Tab. 2.8-2 – Behavior of BNP and NT-proBNP in disease states.

Endothelial hormones and chronic heart failure

Prostaglandin E and prostacyclin are produced from arachidonic acid in many cells. Both hormones have a vasodilatory effect and therefore counteract the neurohumoral renal vasoconstriction effects that are present in chronic heart failure /12/. Nitric oxide (NO) synthesized by endothelial cells is also a vasodilator. It works together with the natriuretic peptides to antagonize the neurohormonal compensation mechanisms in chronic heart failure.

Endothelin, on the other hand, is a potent vasoconstrictor that is found in high concentrations in the blood of patients in NYHA classes III and IV. High endothelin levels are associated with a poor prognosis.

Effect of regulatory and counter-regulatory mechanisms in chronic heart failure

As long as the neurohormonal systems that cause vasoconstriction and fluid retention are completely antagonized by the natriuretic peptides, chronic heart failure remains asymptomatic and cardiac remodeling is slowed down.

If the natriuretic peptides can no longer completely counteract these neurohormonal systems, patients develop the typical heart failure symptoms described in NYHA II. In advanced heart failure, concentrations of catecholamines, renin and aldosterone, and endothelin are elevated.

As heart failure progresses (from NYHA III on), renal perfusion is compromised. This results in even greater stimulation of the renin-angiotensin-aldosterone and sympathetic nervous systems as well as increased peripheral vascular resistance.

Even though the natriuretic peptides are produced in large amounts, they begin to lose their effect on the kidneys, which leads to increased retention of sodium and water. This increases the intravascular volume and promotes cardiac dilatation, which leads to a further deterioration in cardiac function.

Severity of chronic heart failure

With respect to the severity of chronic heart failure, there is a clear relationship between clinical symptoms, the extent of the dysfunction, and the patient’s prognosis. The best predictor of the severity and prognosis of chronic heart failure is left ventricular function, which is assessed by determining the ejection fraction (EF).

Biomarkers in chronic heart failure

Biomarkers can be used to:

• Estimate the extent of the disturbances in water and Na⁺ balance caused by chronic heart failure
• Ascertain the supply of O₂ to cardiac muscle
• Assess the severity of chronic heart failure
• Monitor therapy.

The assessment of specific biomarkers in chronic heart failure is the shown in:

• Tab. 2.7-4 – Common non-specific findings in chronic heart failure
• Tab. 2.7-5 – Biomarkers and their clinical significance in chronic heart failure.

Co-morbidities in chronic heart failure

Heart failure patients often suffer from multiple co-existent diseases and/or conditions that may complicate management and outcome (Tab. 2.7-6 – Co-morbidities in chronic heart failure).

Right ventricular failure

Pathophysiological processes that initiate or promote right ventricular failure, like those of left ventricular failure include myocyte hypertrophy, fibrosis, ischemia, neurohormonal activation, inflammation, and shifts in metabolic substrates /16/.

Clinical findings: dyspnoe, early satiety, abdominal fullness, lower extremity edema, right-upper-quadrant tenderness, exercise intolerance, fatique.

Laboratory findings: serum elevated brain natriuretic peptide (BMP) concentration or NT-proBNP in serum.

References

1. Heidenreich PA, Bozkurt B. 2022 AHA/ACC/HFSA guideline for the management of heart failure. J Am College Cardiology 2022; 79: e263–e421.

2. Von Scheidt W. Diagnostik der Herzinsuffizienz. Internist 2000; 41: 115–26.

3. Khunti K, Baker R, Grimshaw G. Diagnosis of patients with chronic heart failure in primary care: usefulness of history, examination, and investigations. Br J General Practice 2000; 50: 50–4.

4. McKee PA, Kastelli WP, Mc Namara PM, Kannel WB. The natural history of congestive heart failure: the Framingham Study. N Engl J Med 1971; 285: 1441–6.

5. Neumann T, Biermann J, Neumann A, Wasem J, Ertl G, Dietz R, Erbel R. Herzinsuffizienz: Häufigster Grund für Krankenhausaufenthalte. Dtsch Ärztebl 2009; 106: 269–75.

6. McDonagh TA, Morrison CE, Lawrence A, et al. Symptomatic and asymptomatic left-ventricular systolic dysfunction in an urban population. Lancet 1997; 350: 829–33.

7. Hunt SA, Abraham WT, Chin MH, Feldman AM, Francis GS, Ganists TG, et al. ACC/AHA guideline update for the diagnosis and management of chronic heart failure in the adult: a report of the American College of Cardiology and the American Heart Association Task Force on Practise Guidelines (Writing Committee to update the 2001 Guidelines for the Evaluation and Management of Heart Failure). Circulation 2005; 112, 12: e154-e235.

8. Pinokowski P, Voors AA, Anker SD, Bueno H, Cleland JGF, Coats AJS, et al. 2016 ESC guidelines for the diagnosis and treatment of acute and chronic heart failure: the task force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC) developed with the special contribution of the Heart Failure Association (HFA) of the ESC. European Heart Journal 2016; 37 (27): 2129–2200.

9. Hunt SA, Baker DW, Chin MH, et al. ACC/AHA guidelines for the evaluation and management of chronic heart failure in the adult: executive summary: a report of the American College of Cardiology and the American Heart Association Task Force on Practise Guidelines (Committee to revise the 1995 Guidelines for the Evaluation and Management of Heart Failure). J Am Coll Cardiol 2001; 38: 2101–13.

10. Jessup M, Brozena S. Heart failure. N Engl J Med 2003; 348: 2007–18.

11. Hunter JJ, Chien KR. Signalling pathways for cardiac hypertrophy and failure. N Engl J Med 1999; 341: 1276–83.

12. Schrier RW, Abraham WT. Hormones and hemodynamics in heart failure. N Engl J Med 1999; 341: 577–85.

13. Mair J, Puschendorf B. Kardiale Diagnostik. In: Thomas L, ed. Labor und Diagnose, S 121–58. Frankfurt 2005; TH-Books.

14. Triposkiadis F, Giamouzis G, Parissis J, Starling RC, Boudoulas H, Skoularigis J, Butler J, Filippatos G. Reframing the association and significance of co-morbidities in heart failure. European J of Heart Failure 2016; 18: 744-58.

15. Chan CHH, Pieper IL, Robinson CR, Friedmann Y, Kanamarlapudi V, Thornton C, et al. Shear stress-induced total blood trauma in multiple species. Artificial Organs 2017; 41 (10) 934–47.

16. Houston BA, Brittain EL, Tedford RJ. Right ventricular failure. N Engl J Med 2023: 388 (12): 1111–25.

2.8 B-type natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP)

BNP is a 32 amino acid cardiac peptide is produced in the ventricular myocardium and is stored in modest quantities. BNP reduces blood pressure and increases Na⁺ excretion. In response to hemodynamic load in the ventricles, the pre-proBNP is converted into proBNP, which is rapidly cleaved to form BNP and NT-proBNP. BNP is hormonally active, whereas NT-proBNP is inactive. BNP functions physiologically as a natriuretic peptide (NP). The natriuretic peptides are natural antagonists of the renin-angiotensin-aldosterone system and sympathetic nervous system. They act in a coordinated manner centrally and peripherally to control fluid and electrolyte balance.

BNP and NT-proBNP are released from cardiac myocytes in response to ventricular wall stress. Wall stress is directly related to the ventricular diameter and transmural pressure and inversely correlated with ventricular thickness. Increased left ventricular diameter and pressure increase the production of BNP and NT-proBNP, which leads to higher concentrations in the blood.

BNP and NT-proBNP levels are increased in heart failure (HF) and their concentration in the blood rises with the extent and duration of ventricular dysfunction.

2.8.1 Indication

Conditions of the heart accompanied by volume and pressure overload /1, 2, 3, 4/:

• Aid in the diagnosis of individuals suspected of having heart failure (e.g., in acute onset of dyspnea)
• Detection of mild forms of cardiac dysfunction
• Aid in the assessment of heart failure severity
• Risk stratification of patients with acute coronary syndrome and congestive heart failure
• Monitoring of treatment in patients with left ventricular dysfunction.

2.8.2 Method of determination

BNP assays use different antibodies and materials for peptide detection /5/. There is a paucity of standardization and differences up to 50% across various BNP assays are reported.

Laboratory BNP assay

Principle: BNP in the sample reacts with a BNP-specific antibody bound to a solid phase and a second, labeled indicator antibody to form a sandwich complex. The most frequent methods use antibodies specific for two distantly located epitopes of the peptide. One of these antibodies is specific for the intact cysteine ring, while the other antibody recognizes the C-terminus of BNP /5/. The indicator antibody is labeled using an enzyme, a luminescence label, or radioactivity /6/.

Point of care BNP assay /8/

Principle: sandwich immunoassay with two monoclonal antibodies that are directed against different epitopes of the BNP ring structure. The indicator antibody is labeled with a fluorescence label. The fluorescence emitted is measured using a detector. Whole blood is used as the sample material.

Laboratory NT-proBNP assay /9/

Principle: NT-proBNP in the sample reacts with a biotinylated polyclonal NT-proBNP-specific antibody and a ruthenium-labeled antibody to form a sandwich complex. Streptavidin-coated micro particles are then added and the sandwich complex binds to the micro particles by means of an interaction between streptavidin and biotin. The micro particles attach magnetically to the surface of an electrode in the measuring cell of the analyzer. Once the unbound components have been removed, chemiluminescence is induced by applying a voltage and the chemiluminescence emission is measured using a photomultiplier. The antibodies are of monoclonal origin and recognize the amino acids 22–28 and 42–46 of the central region of NT-proBNP.

Point of care NT-proBNP assay /10/

The assay is performed as an immunoassay with reagents on multi layer film slides. A gold-labeled monoclonal antibody recognizes amino acid sequence 27–31 on NT-proBNP and the biotinylated polyclonal antibody recognizes the sequence 42–46.

2.8.3 Specimen

Point of care test (POCT)

• BNP: EDTA blood: 0.1–1 mL
• NT-proBNP: EDTA or heparinized blood: 0.1–1 mL

Automated analyzer assays

• BNP: EDTA plasma: 1 mL
• NT-proBNP: EDTA or heparinized blood: 1 mL

2.8.4 Reference interval

The reference intervals depend on the assay used; refer to the instructions provided by the assay manufacturer (Tab. 2.8-1 – Age and manufacturer-specific upper reference limits for BNP and NT-proBNP).

2.8.5 Clinical significance

The results of the BNP and NT-proBNP assays are diagnostically and prognostically important in cardiology.

2.8.5.1 Diagnostic significance of BNP and NT-proBNP

Physiological and pathological conditions associated with volume expansion and/or stretching of the left ventricular myocardium lead to the release of BNP and NT-proBNP. Age and gender are the main factors that determine the concentration of BNP and NT-proBNP in healthy individuals.

Since the assays for these cardiac markers are not standardized, the manufacturer’s specifications regarding the upper reference limits must be observed when evaluating the results. The concentrations of BNP and NT-proBNP increase with increasing age and are higher in women than in men. Because of these factors and differences between assays, standardized upper reference limits for BNP and NT-proBNP for diagnosing heart failure do not exist. However, diagnostic thresholds are available. Refer to Tab. 2.8-1 – Age and manufacturer-specific upper reference limits for BNP and NT-proBNP.

According to the European Society of Cardiology (ESC) guidelines a single NT-proBNP threshold value of 125 ng/L in patients < 75 years and 450 ng/L in patients ≥ 75 years are elevated /59/. When considering a threshold value of 125 ng/L in patients of < 75 years and of 450 ng/L in those ≥ 75 years 65.6% of patients had elevated NT-proBNP concentrations /16/.

Measured BNP or NT-proBNP concentrations must always be assessed in conjunction with the history, clinical picture, and other investigations (e.g., echo-cardiography and ECG.)

A flow diagram for diagnosing untreated patients with suspected heart failure and the differential diagnostic significance of BNP and NT-proBNP are shown in Fig. 2.8-1 – Interpretation of BNP and NT-proBNP concentrations in patients with acute dyspnea /4/. In patients who present with dyspnea and reduced exercise tolerance, NT-proBNP has a diagnostic sensitivity of 88%, a specificity of 92%, a positive predictive value of 96.7%, and a negative predictive value of 80.6% /13/. The negative predictive value of a NT-proBNP concentration below 300 ng/L is 98%. If values are greatly increased, systolic heart failure is often present.

Many patients with heart failure have normal systolic function but abnormal diastolic function. Diastolic heart failure has a similar mortality to systolic heart failure. Echo cardiography reveals a ventricular filling defect. BNP concentrations above 100 ng/L and NT-proBNP concentrations above 220 ng/L point to this type of heart failure, but echo cardiography is required to confirm it.

• BNP and NT-proBNP values correlate with the New York Heart Association (NYHA) heart failure classification:
• Fig. 2.8-2 – Median concentrations, highest values, and lowest values of BNP
• Fig. 2.8-3 – Median concentrations, highest values, and lowest values of NT-proBNP.

Patients with acute dyspnea and congestive heart failure generally have higher BNP and NT-proBNP concentrations than those with non-acute heart failure. When BNP and NT-proBNP assays with age and gender-specific cutoff values are used as a screening test for heart failure in the population (aged ≥ 45 years), they have a diagnostic sensitivity and specificity of 75–100% for detecting individuals with a left ventricular ejection fraction of ≤ 40%. Both parameters have approximately the same significance /14/.

When patients with acute dyspnea are admitted to hospital, a BNP or NT-proBNP determination can be used to distinguish between those with and without cardiac dysfunction (Fig. 2.8-1 – Interpretation of BNP and NT-proBNP concentrations in patients with acute dyspnea).

An important factor to consider when evaluating the BNP and NT-proBNP concentration is whether renal insufficiency is present. The concentrations of both biomarkers rise as the glomerular filtration rate decreases. In a study /16/ dependent of the glomerular filtration rate (GFR) in patients older than 20 years the concentration of NT-proBNP was:

• 4489 (1994–11,688) ng/L in the (GFR) subgroup below 30 [ml × min^–1 × (1,73 m²)^–1]
• 2455 (951–5671) ng/L in the GFR subgroup 30–50 [ml × min^–1 × (1,73 m²)^–1]
• 1018 (336–2578) ng/L in the GFR subgroup higher than 50 [ml × min^–1 × (1,73 m²)^–1]

Obese patients have lower BNP and NT-proBNP values /17/. A possible reason for this is that BNP is cleared more quickly from adipose tissue.

The behavior of BNP and NT-proBNP in cardiac dysfunction is shown in Tab. 2.8-2 – Behavior of BNP and NT-proBNP in disease states. The behavior of BNP and NT-proBNP in dyspnea is shown in Tab. 2.8-3 – NT-proBNP in patients with acute dyspnea with and without heart failure (HF).

The in-hospital mortality of acute coronary syndrome as a function of the BNP value is shown in:

• Tab. 2.8-4 – In-hospital mortality of patients with acute coronary syndrome as a function of the BNP value
• Fig. 2.8-4 – Kaplan-Meier curves showing survival rate in patients with acute heart failure.

Both BNP and NT-proBNP perform well to rule-out, but less to rule-in, in the diagnosis of heart failure among persons presenting to emergency departments or urgent care centers. For BNP, 100 ng/L appears to be a consensus point. No clear consensus has emerged for NT-proBNP, but the age-adjusted cutoffs of 450 ng/L for < 50 years, 900 ng/L for 50–75 years, and 1,800 ng/L for > 75 years appear promising /2/. In patients with septic shock and gold standard therapy NT-proBNP levels > 1,000 ng/L at 72 hours were associated with adverse outcome (mortality at 28 days) /7/.

2.8.5.2 Prognostic significance of BNP and NT-proBNP

BNP and NT-proBNP are better prognostic indicators than the NYHA classification in patients with chronic heart failure. In patients with stable angina pectoris, BNP and NT-proBNP concentrations provide information about cardiovascular events in the longer term and about mortality. In patients with a recent history of myocardial infarction, both markers provide information about left ventricular function, infarct size, and survival. BNP and NT-proBNP are also useful prognostic markers in volume overload associated with acute coronary syndrome, atrial fibrillation, and pulmonary embolism.

2.8.6 Comments and problems

Specimen

For BNP assays, EDTA plasma is the recommended specimen and for NT-proBNP the recommended specimen is serum.

Method of determination

The BNP and NT-proBNP values depend on the type of assay used, the antibody specificity, and the calibration sample. To use these markers correctly in everyday clinical practice, it is important to observe the manufacturer’s specifications regarding reference intervals /12, 46/. Overall, according to a systematic review /47/, BNP and NT-proBNP demonstrate a high degree of diagnostic accuracy without significant difference in the odds ratio (OR) for diagnosing chronic heart failure (OR_BNP 8.4; OR_NT-proBNP 23.4) and acute heart failure (OR_BNP 16.5; OR_NT-proBNP 18,6).

BNP or NT-proBNP results are not transferable among the current existing immunoassays owing to their differences in cross-reactivity and ability to detect various glycosylated forms of proBNP-derived fragments /48/. NT-proBNP assays are generally considered harmonized because all utilize one manufacturers antibodies and calibrators which are then configured to other manufacturers’immunoassay platforms.

BNP sampling

If an immunoassay with a combination of antibodies against epitopes of the amino acids 90–97 and 103–107 is used, the blood should be drawn in a plastic tube that contains EDTA /49/.

NT-proBNP sampling: serum tube or tube that contains lithium or NH₄⁺ heparin. Separator gel does not interfere with the assay. Values can be up to 10% lower in EDTA plasma than in serum.

Stability

Stability of BNP /49/: measurements should be performed within 4 hours if blood is stored at room temperature. If this is not possible, the plasma should be separated from the corpuscular components and can be stored in a tube containing a kallikrein or serine protease inhibitor at 4 °C for up to 72 hours. Storage at –80 °C up to 1 year.

NT-proBNP stability /50/: stable in whole blood and plasma at room temperature or 4 °C for up to 72 hours –80 °C.

Influence factors

Blood sampling: should not take place following stressful investigations such as ergometry or stress echo cardiography. Under such conditions, BNP or NT-proBNP levels can also be increased in healthy individuals /51/.

Medications: diuretics, ACE inhibitors, and beta-blockers can reduce the plasma BNP concentration. A baseline value should be determined before treatment is started. The administration of synthetic BNP (natrecor, nesiritide) can lead to elevated BNP values. Administration of neutral endopeptidase inhibitors (e.g., omapatrilat) can influence the plasma concentration of BNP by inhibiting its breakdown. Treatment with these medications does not affect NT-proBNP values.

Dietary sodium loading: increased dietary sodium intake from 10 g (171 mmol)/day to 30 g (513 mmol)/day over a period of 5 days leads to a 53% increase in the BNP concentration. Although NT-proBNP is released in equimolar amounts, it has a longer half-life than BNP, so its concentration increases more steeply than that of BNP in response to chronic dietary sodium loading /52/.

Age and gender /53/: every 10 years from the age of 45, BNP increases by up to 50% and NT-pro-BNP increases by up to 74% on average. Women have higher BNP and NT-proBNP values than men of the same age.

Physical exertion: a rise in BNP concentration of 143% compared to baseline can be measured in subjects 1 minute after completing a standard treadmill ergometry protocol /54/. BNP values measured 15 minutes after a marathon are up to 211% higher than baseline values /51/. The heart rate also influences the concentration of BNP and NT-proBNP. For every increase in heart rate of 10 beats per minute, the concentration of BNP falls by 9% and the concentration of NT-proBNP falls by 15% /53/.

Body mass index: obesity is associated with glomerular hyper filtration and lower age-related NT-proBNP values. The estimated glomerular filtration rate (eGFR) should therefore be taken into account when evaluating NT-proBNP values. However, the Cockcroft-Gault formula should be used since it takes age and weight into account. A fall in the eGFR_C-G of 10% is associated with a 9% increase in the NT-proBNP concentration /55/.

Intraindividual variation: in healthy individuals and patients with stable heart failure, the coefficient of variation (%) from week to week was 40% for BNP /56/ and 35% for NT-proBNP /5/.

Hospitalization days and costs of patients with elevated NT-proBNP: patients with heart failure (HF) are reportedly at high risk for all-cause re-hospitalization. Compared to HF-negative patients, the HF-positive patients had longer total hospitalization days (median 18 versus median 30). Medical costs for hospitalization were 0.76 million yen versus 2.38 million yen /57/.

2.8.7 Pathophysiology

The natriuretic peptides ANP, BNP, and C-CNP are characterized by a 17 amino acid ring structure with a disulfide bond between 2 cysteine residues (Fig. 2.8-5 – Cleavage of B-type natriuretic pro hormone (proBNP 1–108)). The ring structure is crucial for receptor binding and biological functions. The pro hormones are coded by separate genes. ANP and BNP are synthesized by ventricular myocytes during periods of increased vascular stretch and wall tension. In left ventricular dysfunction, ventricular hypertrophy, and other cardiac dysfunctions with chronically increased hemodynamic pressure or volume overload, ventricular myocytes undergo modification and start to re express fetal genes that code for increased synthesis of ANP and BNP /2/.

ANP is stored in secretory granules in the cardiac myocytes and is released rapidly in response to volume overload in the extracellular space. BNP, however, is not secreted from storage granules, but is released in bursts that are regulated by gene expression. Left ventricular myocardial stretching acts as a stimulus for this.

The natriuretic peptides exert their effects via three cell membrane receptors that transmit their signals to the inside of the cell by means of the guanylate cyclase pathway. Natriuretic peptide receptor A is activated preferentially by BNP and ANP and receptor B is activated with higher affinity by CNP and lower affinity by ANP and BNP. Natriuretic peptide receptor C is located in the liver, lungs, renal tubules, and vascular endothelium and removes natriuretic peptides from the circulation.

ANP and BNP act as antagonists of the renin-angiotensin-aldosterone system and sympathetic nervous system. Guanylate cyclase type A receptors are distributed throughout the body but the highest receptor density is found in the zona glomerulosa of the adrenal cortex and in the collecting ducts of the inner renal medulla. Atrial natriuretic peptides exerts its effects at the levels /58/:

• Of the glomerulus, it causes efferent arteriolar constriction and afferent arteriolar dilation, thereby causing a temporary increase of the glomerular filtration. The excretion of is Na⁺ increased.
• In the collecting duct, it causes a long-term decrease of Na⁺ reabsorption, thereby increasing Na⁺ excretion.

The physiological effects of ANP and BNP are listed in Tab. 2.8-5 – Effects of ANP and BNP.

The BNP gene encodes a 134 amino acid pre-proBNP precursor, which is converted to a 108-amino acid proBNP by the cleavage of a 26-amino acid signal peptide. The proBNP is cleaved by the serine protease corin in the cardiomyocytes to form BNP (amino acids 77–108) and the biologically inactive NT-proBNP (amino acids 1–76). BNP has a molecular weight of 3.5 kDa and NT-proBNP has a molecular weight of 8.5 kDa. Both peptides are released in equimolar amounts into the circulation. The half-life of BNP is around 20 minutes and the half-life of NT-proBNP is 1–2 hours /2/.

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