Lipoprotein metabolism

4.1 Lipid disorders

4.1.1 Introduction

Lipids (fats) are transported in the plasma as water-soluble lipoprotein particles. Each particle includes one or a set of highly conserved apolipoproteins that provide structural integrity for the complex, allow for its assembly and secretion, and provide a mechanism for receptor binding. The lipoprotein particles moving cholesterol through the plasma carry hydrophobic cholesterol esters and triglycerides in a central core, enveloped with an external layer of hydrophilic phospholipids and free cholesterol.

Lipoprotein particles are classified according to their size and density, with

Chylomicrons
Chylomicron remnants
Very low density lipoproteins (VLDL), being relative large and light
Low density lipoproteins (LDL), particles which are smaller and heavier than VLDL. LDL particles are the main carrier of cholesterol to peripheral tissues where they are internalized through the LDL-receptor.
High density lipoproteins (HDL), particles which are smaller and heavier than LDL. HDL particles removes fat molecules and cholesterol from cells and transports them back to the liver for excretion or re-utilization.

Apolipoproteins and lipoprotein particles

Apo A is the major protein component of HDL particles in plasma and enables efflux of cholesterol by accepting cholesterol from cells of the tissues
Apo B-100 is the primary apolipoprotein of chylomicrons, VLDL, intermediate-density lipoprotein particles and LDL-particles, and as much is responsible for transport of fat molecules to all peripheral cells
Triglycerides play an important role as transporters of dietary fat and energy sources
Lp (a) is an LDL-like lipoprotein particle consisting of one apo A molecule covalently bound to apo B-100.

The lipoprotein transport system serves the following functions:

Transport of dietary fats from the intestine to the liver
The secondary transport of processed cholesterol particles to peripheral tissues for membrane synthesis and steroid hormone synthesis
The processing of free fatty acids which serve as a source of fuel.

4.1.2 Dyslipidemia

Dyslipidemia, a genetic or multifactorial disorder of lipoprotein metabolism, is defined by elevations in levels of total cholesterol, low density lipoprotein cholesterol (LDL-C), non-high-density lipoprotein cholesterol (non-HDL-C), triglycerides, or some combinations thereof, as well as lower levels of HDL cholesterol (HDL-C) /1/.

For lipid profiling the determination of total cholesterol, LDL-C, HDL-C, and triglycerides are recommended /2/.

According to the National Cholesterol Education Program (NCEP) of the U.S. /2/ none of the following markers is appropriate for routine screening: Lp(a), lipoprotein remnants, small LDL particles, HDL subspecies, apolipoprotein B, and apolipoprotein A-I.

4.1.3 Dyslipidemia and atherosclerotic vascular disease (ASCVD)

Coronary heart disease and stroke are the two leading causes of death worldwide and by the year 2030, they are projected to account for nearly 25% of deaths. The association of hypercholesterolemia with both diseases make them a global effector of the worldwide disease burden associated with increased mortality.

4.1.3.1 Indication

Children, adolescents or young adults with a family history of dyslipidemia or premature atherosclerosis
Lipid profiling in patients > 21 years of age at increased cardiovascular risk
Lipid profiling in order to asses total cardiovascular risk in men > 40 and women > 50 years of age with one of the following conditions: type 2 diabetes, established cardiovascular disease (CVD), hypertension, smoking, BMI ≥ 30 kg/m² or waist circumference > 94 cm for men, > 80 cm for women, family history of premature CVD, chronic inflammatory disease, chronic kidney disease, family history of familial hypercholesterolemia /3/.

4.1.3.2 Specimen

Serum, heparinized plasma, EDTA plasma: 1 mL

To improve patient compliance with lipid testing the routine use of non-fasting lipid profiles are recommended /4, 5/.

Fasting sampling may be considered:

When non-fasting triglycerides are > 440 mg/dL (5.0 mmol/l) /4/
In patients with non-fasting concentrations of non-HDL-C ≥ 220 mg/dL (5.7 mmol/l) or triglycerides ≥ 500 mg/dL (5.7 mmol/l) that a fasting sample was needed to inquire into an underlying genetic disorder /5/.

In situations for which fasting is preferred or may be required patients should fast for at least 8 h prior to blood collection.

Blood is collected from outpatients after 10–15 min. of rest in a seated position, since lipid levels are 12% higher in an upright position and 6% higher in a seated position than in a recumbent position. Approximately 2 min. of stasis causes an increase in lipid levels by up to 10%. Lipid levels are 5% lower in plasma than in serum /6/.

Acute inflammation changes the lipid pattern, however, in acute myocardial infarction, cholesterol and LDL-C can still be evaluated within the first 8 h from the onset of infarction before the acute phase, during which LDL-C levels decline by up to 30%. If no previous results are known and LDL-C was not measured in blood collected within the first 8 h, a valid evaluation of lipid metabolism will not be possible until 8–12 weeks after the myocardial infarction.

4.1.3.3 Reference interval

Lipid screening values /5/ are presented in Tab. 4.1-1 – Upper reference values and recommended ranges of lipids.

4.1.3.4 Clinical significance

Dyslipidemia is a genetic or multifactorial disorder of lipid metabolism. Elevations in levels of total cholesterol, LDL-C, and non-HDL-C are associated with the risk of atherosclerotic cardiovascular disease (ASCVD) in adults, as are lower levels of HDL-C and, to a lesser extent, elevated triglyceride levels /5/.

Large-scale intervention studies document that 80–90% of patients with clinically significant ASCVD have at least one of the following four classic risk factors:

Total cholesterol above 240 mg/dL (6.22 mmol/L)
Systolic blood pressure above 140 mmHg (hypertension)
Diabetes mellitus
Smoking.

4.1.3.4.1 Laboratory evaluation of atherosclerotic cardiovascular disease (ASCVD)

The goals of the American College of Cardiology (ACC), the American Heart Association (AHA) Guidelines, and the European Society of Cardiology (ESC) the European Atherosclerosis Society (EAS) Guidelines are to prevent ASCVD and to improve the management of people who have these diseases /3, 5/. Contributions arising from genetic and biochemical studies and observational epidemiological and ecological studies showed associated higher LDL-C levels with greater ASCVD risk. Besides life stile modification (i.e., adhering to a heart-healthy diet, regular exercise habits, avoidance of tobacco products, and maintenance of a healthy weight) statin therapy is an important tool to lower morbidity and total mortality when used as primary and secondary prevention.

The priorities of prevention are /7/:

1. Identification of patients with existing ASCVD.

2. Diagnosis of asymptomatic individuals with an increased risk for ASCVD. This includes:

Individuals with multiple risk factors. These are individuals who have a ≥ 5% risk of mortality from ASCVD within the next 10 years /3/ or a > 20% risk of suffering a fatal or non-fatal myocardial infarction /2/
Patients with diabetes type 2 and type 1 with micro albuminuria
Patients with a significantly elevated risk factor, in particular if it leads to end-organ damage.

3. Screening of near relatives of individuals with premature atherosclerosis for cardiovascular risk factors.’

4. Intensive statin therapy in certain groups of people.

4.1.4 Familial hypercholesterolemia

The main genetic (primary) dyslipidemias that lead to ASCVD include:

Familial hypercholesterolemia which occurs in up to 1 of 100 individuals in North America and Europe and includes highly elevated LDL-C levels, e.g., ≥ 190 mg/dL (4.92 mmol/l). Heterozygous familial hypercholesterolemia occurs in approximately 1 of every 200 to 500 individuals.
PCSK9 dyslipidemia. PCSK9 is a critical regulator of cholesterol metabolism through its interaction with the hepatic LDL receptor. PCSK9 prevents recycling of the LDL receptor to the cell surface, thereby attenuating LDL-C clearance. Higher circulating PCSK9 levels predict lower catabolism of apolipoprotein B, the main constituent of LDL.

Refer to Tab. 4.1-2 – Genetic dyslipidemias.

4.1.5 Multifactorial dyslipidemia

The multifactorial dyslipidemia is the main secondary dyslipidemia that leads to atherosclerotic cardio vascular disease (ASCVD). Multifactorial dyslipidemia is defined by levels of LDL-C ≥ 130 mg/dL (3.37 mmo/l), triglycerides ≥ 200 mg/dL (2.26 mmol/l) or both that are not attributable to familial hypercholesterolemia.

The risk for ASCVD is influenced by other factors not included among major, independent risk factors (cigarette smoking, hypertension, low HDL-C, family history of premature ASCVD). Diabetes is regarded as a ASCVD. Diabetes mellitus, metabolic syndrome and ASCVD are closely linked to dyslipidemia. Obesity is associated with slight elevations in LDL-C but more strongly related to elevated triglycerides and lower HDL-C.

The European Guidelines state /3, 8/:

There is a linear relationship between the glucose concentration, in particular the 2-h value in the oral glucose tolerance test, and the risk of ASCVD. This also applies to HbA_1c. The risk exists even with levels in the upper reference interval.
The relative risk of ASCVD is approximately 1.5 times higher with impaired glucose tolerance, 2 to 4 times higher with diabetes, and can be even higher in women.
The risk is related to diabetes and additional risk factors.
Ideally, the development of diabetes should be prevented.
Good metabolic control prevents microvascular complications.
Goals for lipids are total cholesterol below 175 mg/dL (4.5 mmol/L), preferably below 150 mg/dL (4.0 mmol/L), and LDL-C below 70 mg/dL (1.8 mmol/L).
Age above 45 years for men and above 55 years for women or premature menopause, are risk factors.
Smoking is a risk factor.
Hypertension above 140/90 mm Hg or antihypertensive treatment are risk factors.
A positive family history of ASCVD is a risk factor.

Refer to Tab. 4.1-3 – Multifactorial dyslipidemia.

4.1.6 Treatment of dyslipidemia

In general, the European Guidelines state /3/:

Total cholesterol and LDL-C are etiologically associated with ASCVD. The association is strong, independent, progressive and is the primary focus of treatment.
Reduction of total cholesterol and LDL-C reduces the incidence of ASCVD and stroke. Recommendations for treatment targets are shown in Tab. 4.1-4 – Thresholds for therapeutic lifestyle changes and drug therapy in different risk categories of ASCVD
In routine diagnosis, most dyslipidemias need not be differentiated, since there is only a limited number of therapeutic options. Therefore, in routine diagnostics only total cholesterol, triglycerides, LDL-C, and HDL-C need to be analyzed /9/.

4.1.6.1 Statin therapy in patients at increased atherosclerotic vascular disease (ASCVD) risk

There are four groups most likely to benefit from fixed doses of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) therapy /5, 7/:

Patients with any form of clinical ASCVD (including acute coronary syndrome, history of myocardial infarction, stable or unstable angina, coronary or other arterial vascularization, transient ischemic attack, or peripheral arterial disease presumed to be of atherosclerotic origin). High-intensity statin therapy.
Patients over 21 years of age at increased cardiovascular risk without heart failure (NYHA class II, III, or IV) or end-stage renal disease and LDL-C ≥ 190 mg/dL (4.92 mmol/l). High-intensity statin therapy.
Patients 40 to 75 years of age who have diabetes and LDL-C of 70 to 189 mg/dL (1.81 to 4.89 mmol/l). Calculate 10-year risk of ASCVD. If risk < 7.5% moderate-intensity statin therapy, if risk ≥ 7.5%, high intensity statin therapy.
Patients 40 to 75 years of age who have no diabetes and LDL-C of 70 to 189 mg/dL (1.81 to 4.89 mmol/l). Calculate 10-year risk of ASCVD. If risk ≥ 7.5% moderate to high-intensity statin therapy.

The first three groups are considered high risk, and treatment should focus on high-intensity statin therapy. The last group does not automatically receive statin therapy, but is engaged in a risk discussion with their physician.

The 10-year risk of ASCVD is calculated with the use of the risk calculators available at http://my.americanheart.org/cvriskcalculator or available at doi: 10.1093/eurheartj/ehr158). LDL particles are the main carrier of cholesterol to peripheral tissues where they are internalized through the LDL receptor, a crucial mediator of plasma LDL concentration. Genetic defects that result in loss of function within the LDL receptor are a major determinant of inherited dyslipidemis and ASCVD /3/.

Recommendations for treatment targets of dyslipidemia are shown in Tab. 4.1-5 – Intervention strategies as a function of ASCVD and LDL-C level.

Lipid assays are not evaluated on the basis of the concept of reference intervals, but using defined goals based on European and international consensus recommendations. Refer to Tab. 4.1-6 – Goals for primary and secondary prevention of cardiovascular disease.

4.1.7 Separation and classification of lipoproteins

Following their separation by ultracentrifugation, the lipoproteins are differentiated into the following classes in order of increasing density: chylomicrons < very low density lipoproteins (VLDL) < low density lipoproteins (LDL) < high density lipoproteins (HDL). The following differentiation is made based on the velocity of electrophoretic migration toward the anode in an alkaline buffer medium: alpha fraction (contains HDL) > pre-β fraction (contains VLDL) > β-fraction (contains LDL) and > chylomicrons (remain at origin).

Fredrickson classified hyperlipoproteinemias into 6 phenotypes which do not represent a defined disease description /10/. Each phenotype can have primary or secondary causes, and a single genetic defect can cause one or several phenotypes. Similarly, a single phenotype can be caused by different genetic defects. Therefore, the Fredrickson classification has been replaced by a differentiation between hypercholesterolemia, hypertriglyceridemia, and combined hyperlipoproteinemia, which is sufficient for therapeutic decision-making (Tab. 4.1-7 – Assays for the assessment of cholesterol-containing lipoproteins).

4.1.8 Clinical symptoms of dyslipidemias

There are few clinical symptoms. The following are indicative:

Xanthelasma in younger patients
Xanthomas (planar, tendinous, tuberous, eruptive, palmar)
Arcus lipoides, lipemia retinalis
Abdominal troubles (pancreatitis with hyperchylomicronemia)
Joint inflammation due to precipitation of cholesterol crystals in the synovial fluid.

4.1.9 Quantifying dyslipidemias

Diabetes, hypertension, smoking, and obesity are the strongest risk factors for premature coronary heart disease. Low density lipoprotein (LDL) cholesterol has been the main biomarker for determination of dyslipidemias and statins the main target of lipid-lowering therapy since the 1990s /36/.

According to statements and guidelines recommended by European states and the US guidelines laboratories should determine for quantifying and managing dyslipidemias the following biomarkers in plasma/serum /36, 37/:

total cholesterol
triglycerides
LDL cholesterol
HDL-cholesterol
Lp(a) which should be measured at least once in all individuals /38/
Non-HDL cholesterol
Remnant triglceride-rich lipoprotein (TRL) /36/.

References

1. U.S. Preventive Task Force. Screening for lipid disorders in children and adolescents. Am Family Physician 2016; 94: 1004A-1004E.

2. Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 2001; 285: 2486–97.

3. The task force for the management of dyslipidemias of the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS). ESC/EAS Guidelines for the management of dyslipidaemias. European Heart J 2011; 32: 1769–1818.

4. Nordestgaard BG, Langstedt A, Mora S, Kolovou G, Baum H, Bruckert E, et al. Fasting is not routinely required for determination of a lipid profile: Clinical and laboratory implications including flagging at desirable concentration cutpoints. A joint consensus statement from the European Atherosclerosis Society and European federation of clinical chemistry and laboratory medicine. Clin Chem 2016; 62: 930–46.

5. Stone NJ, Robinson JG, Lichtenstein AH, Bairy Merz CN, Blum CB, Eckel RH, et al. 2013 ACC/AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults: a report of the American College of Cardiology/American Heart Association Task Force on practice guidelines. Am J Coll Cardiol 2014; 63. 2889–934.

6. Nauck M, Wieland H. Die Differentialdiagnostik von Fettstoffwechselstörungen unter besonderer Berücksichtigung methodischer Aspekte. J Lab Med 2001; 25: 16–22.

7. McBride P, Sone NJ, Blum CB. Should family physicians follow the new ACC/AHA cholesterol treatment guidelines? Am Fam Physician 2014; 90: 212–6.

8. De Backer G. New European guidelines for cardiovascular disease prevention in clinical practice. Clin Chem Lab Med 2009; 47: 138–42.

9. März W, Von Eckardstein A. Laboratoriumsdiagnostik bei Fettstoffwechselstörungen. J Lab Med 2001; 25: 433–48.

10. Beaumont JL, Carlson LA, Cooper GR, Fejfar Z, Fredrickson DS, Strasser T. Classification of hyperlipidemias and hyperlipoproteinemias. Bull WHO 1970; 43: 891– 908.

11. Talmud PJ, Shah S, Whittall R, Futema M, Howard P, Cooper JA , et al. Use of low density lipoprotein cholesterol gene score to distinguish patients with polygenic and monogenic hypercholesterolemia. Lancet 2014/3; 381 (9874): 1293–1301.

12. Talmud PJ, Shah S, Whittall R, Futema M, Howard P, Cooper JA, , et al. The fine line between familial and polygenic hypercholesterolemia. Clin Lipidol 2013; 8: 303–6.

13. Youngblom E, Pariani M, Knowles JW. Familial hypercholesterolemia. Gene Reviews (Internet) 2016; University of Washington, Seattle.

14. deGoma EM, Ahmad ZS, O’Brien EC, Kindt I, Shrader P, Newman CB, et al. Treatment gaps in adults with heterozygous familial hypercholesterolemia in the United States data from the CASCADE-FH registry. Circ Cardiovasc Genet 2016; 9: 240–9.

15. Langsted A, Kamstrup PR, Benn M, Tybjaerg-Hansen A, Nordestgaard BG. High lipoprotein (a) as a possible cause of clinical familial hypercholesterolemia: a prospective cohort study. Lancet Diabetes Endocrinol 2016; 4: 577–87.

16. Fung M, Hill J, Cook D, Fröhlich J. Case series of type II hyperlipoproteinemia in children. BMJ Case Rep 2011; PMC free article: PMC3116222.

17. Hegele RA, Ban MR, Hsueh N, Kennedy BA, Cao H, Zou GY, et al. A polygenic basis for four classical Fredrickson hyperlipoproteinemia phenotypes that are characterized by hypertriglyceridemia. Hum Mol Genet 2009, 18. 189–94.

18. Ferns G, Keti V, Griffin B. Investigation and management of hypertriglyceridaemia. J Clin Pathol 2008; 61: 1174–83.

19. Stein O, Stein Y. Lipid transfer proteins (LTP) and atherosclerosis. Atherosclerosis 2005; 178: 217–30.

20. Horta BL, Victora CG, Lima RC, Post P. Weight gain in childhood and blood lipids in adolescence. Acta Paedriatica 2009; 98: 1024–8.

21. Franks PW, Hanson RL, Knowler WC, Sievers ML, Bennett PH, Looker HC. Childhood obesity, other cardiovascular risk factors, and premature death. N Engl J Med 2010; 362: 485–93.

22. Ingelsson E, Schaefer EJ, Contois JH; McNamara JR, Sullivan L, Keyes MJ, et al. Clinical utility of different lipid measures for prediction of coronary heart disease in men and women. JAMA 2007; 298: 776–85.

23. Sniderman AD, Scantlebury T, Cianflore K. Hypertriglyceridemic hyperapoB: the unappreciated atherogenic dyslipoproteinemia in type 2 diabetes mellitus. Ann Intern Med 2001; 135: 447–59.

24. Riley P, O’Donohue J, Crook M. A growing burden: the pathogenesis, investigation and management of non-alcoholic liver disease. J Clin Pathol 2007; 60: 1384–91.

25. Olbricht CJ. Pathophysiologie und Therapie von Lipidstoffwechselstörungen bei Nierenerkrankungen. Klin Wochenschr 1991; 69: 455–62.

26. Wheeler DC, Bernard DB. Lipid abnormalities in the nephrotic syndrome: causes, consequences and treatment. Am J Kidney Dis 1994; 23: 331–46.

27. Simha V, Garg A. Lipodystrophy: lessons in lipid and energy metabolism. Curr Opin Lipidol 2006; 17: 162–9.

28. Penzak SR, Chuck SK. Hyperlipidemia associated with HIV protease inhibitor use: pathophysiology, prevalence, risk factors and treatment. Scand J Infect Dis 2000; 32: 111–23.

29. Series JJ, Biggart EM, O’Reilly DSST, Packard CJ, Shepherd J. Thyroid dysfunction and hypercholesterolemia in the general population of Glasgow, Scotland. Clin Chim Acta 1988; 172: 217–22.

30. Wallace RB, Hoover J, Barrett-Connor E, Rifkind B, Hunninghake DB, Machenthun A, Heiss G. Altered plasma lipid and lipoprotein levels associated with oral contraceptive and estrogen use. Lancet 1979; ii: 111–5.

31. Beaumont JL, Carlson LA, Cooper GR, Fejfar Z, Fredrickson DS, Strasser T. Classification of hyperlipidemias and hyperlipoproteinemias. Bull WHO 1970; 43: 891–908.

32. Gidding SS. Finding familial hypercholesterolemia. Clin Chem 2021; 67 (5): 710–2.

33. Paththinige S, Sirisena ND, Dissanayake V. Genetic determinants of inherited susceptibility to hypercholesterolemia – a comprehensive literature review. Lipids Health disease 2017; 16 (1): 103. doi: 10.1186/s12944-017-0488-4.

34. Benito-Vicente A, Uribe KB, Jebari S, Galicia-Garcia U, Ostolaza H, Martin C. Familial hypercholesterolemia: the most frequent cholesterol metabolism disorder caused disease. Int J Mol Sci 2018; 19: 3426. doi: 10.3390/ijms19113426.

35. Dullaart RPF. PCSK9 inhibition to reduce cardiovascular events. N Engl J Med 2017; 376: 1790–91.

36. Wulff AB, Nordestgaard BG. Remnants and triglyceride-rich lipoproteins in the prevention of premature coronary heart disease. Clin Chem 2022; 68 (2): 266–8.

37. Grundy SM; Stone NJ, Bailey AL, Beam C, Bircher KK, Blumenthal RS, et al. 2018 AHA / ACC / AACVPR / AAPA / ABC / ACPM / ADA / AGS / APHA / ASPC / NLA / PCNA guideline on the management of blood cholesterol: a report of the American college of cardiology/American heart association task force on clinical practice guidelines. Circulation 2019; 139: e1082–143.

38. Mach F, Baigent C, Catapano AL, Koskinas KC, Casula M, Badimon L, et al. ESC scientific document Group. 2019 ESC/EAS guidelines for the management of dyslipidemias to reduce cardiovascular risk. Eur Heart J 2020; 41: 111–88.

39. Langlois MR, Nordestgaard BG, Langsted A, Chapman MJ, Aakre KM, Baum H, et al. European Atherosclerosis Society (EAS) and the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) joint consensus initiative. Quantifying atherogenic lipoproteins for lipid lowering strategies: consensus based recommendations from EAS and EFLM. Clin Chem Lab Med 2020; 58: 496–517.

4.2 Total cholesterol (TC), LDL-C, HDL-C, Non-HDL-C, Triglyceride-rich lipoprotein (TRL)-C

Cholesterol

Due to its low water solubility, the movement of cholesterol through plasma is mediated by lipoprotein particles that carry hydrophobic cholesterol esters and triglycerides in a central core enveloped within an external layer of hydrophobic phospholipids and free cholesterol. Each lipid particle contains one or a set of apolipoproteins that provide structural integrity for the particle and a mechanism for receptor binding. The transport of cholesterol through plasma is mediated by apo B containing low-density lipoprotein particles (LDL) and apo A-containing high-density lipoprotein particles (HDL). Cholesterol is a moderate component of very-low-density lipoproteins (VLDL) and a minor component of chylomicrons /1/.

Cholesterol is mainly acquired from two sources: Dietary cholesterol or intracellular synthesized cholesterol. Cellular cholesterol is synthesized in the endoplasmic reticulum in a 19 step process. Dietary cholesterol is absorbed by the enterocytes and emulsified by bile acids. Intestinal lipases hydrolyze cholesterolesters to free cholesterol that is taken up by the enterocytes through Niemann-Pick C1-like (NPC1L1) protein which has a cholesterol binding site. Cholesterol is an essential component of cell membranes and a precursor for the synthesis of steroid hormones and bile acids. The sterol ring of cholesterol cannot be metabolized. Therefore, peripherally synthesized or intestinally reabsorbed cholesterol is transported to the liver and excreted directly or together with bile after being converted to bile acids.

LDL cholesterol (LDL-C)

Approximately two thirds of the cholesterol circulating in plasma is contained in LDL particles. Etiologically LDL-C is an important component in the initiation and progression of pathological changes in the vessel wall which lead to the formation of atherosclerotic plaques.

The atherogenic potential of LDL has been demonstrated in numerous epidemiological and clinical studies. Most studies show that, apart from age, LDL-C has the strongest association with the morbidity and mortality of atherosclerotic cardiovascular disease (ASCVD).

HDL cholesterol (HDL-C)

Approximately 25% of the cholesterol in serum is transported in HDL particles. HDL are responsible for the reverse transport of cholesterol i.e., the return of excess cholesterol from the macrophages back to the liver, from where it is excreted into the intestine via the biliary tract. In contrast to LDL, HDL is inversely associated with the occurrence of ASCVD. Low levels are associated with an increased risk of ASCVD.

Triglyceride-rich (TRL) cholesterol

TRL cholesterol is remnant cholesterol and plays a role in the clinical assessment of patients at cardiovascular risk.

4.2.1 Indication

In screening programs for athersclerotic cardio vasculardisease (ASCVD) risk the measurement of LDL-C and HDL-C is indicated instead of total cholesterol:

In healthy adults (males < 40 years, females > 50 years) without clinical or medical history related risk factors for ASCVD
In all patients with ASCVD risk factors e.g., diabetes prediabetes, metabolic syndrome, suspected stroke
In children with a family history of dyslipidemia or ASCVD
For monitoring lipid-lowering therapy.

4.2.2 Method of determination

4.2.2.1 Total cholesterol

Reference method: the primary reference method of the National Institute of Standards and Technology (NIST) in the USA is isotope dilution mass spectrometry (IDMS). The secondary reference method for cholesterol of the Centers for Disease Control (CDC) is a modification of the Abell-Kendall extraction method. The latter shows a positive bias of 1.6% compared to IDMS /2/.

Routine method: a fully enzymatic assay is used (Fig. 4.2-1 – Principle of enzymatic cholesterol assays).

Principle: cholesterol is determined enzymatically by use of cholesterol esterase and cholesterol oxidase, with spectrophotometric quantization of either Δ⁴-cholestenone or H₂O₂ formed in the reaction sequence. Measurement via H₂O₂, preferably the peroxidase catalyzed formation of a purple quinone imine dye from phenol and 4-amino antipyrine has been adopted (Trinder reaction) /3/.

4.2.2.2 HDL cholesterol

Reference method: the Working Group of the National Cholesterol Education Program (NCEP) in the USA recommends using the CDC-HDL reference method. This is a multi-step assay involving ultracentrifugation during which VLDL and chylomicrons are separated. The HDL are selectively removed from the sediment fraction (D ≥ 1.006 kg/L) by precipitating the non-HDL-lipoproteins [IDL, LDL, Lp(a)] with magnesium heparinate. HDL-C is measured in the clear supernatant using the Abell-Kendall method /4/.

Routine method: homogeneous (direct) method /3/.

Principle: the HDL-C assays use detergents, surfactants, or antibodies to modify the surface of chylomicrons, VLDL and LDL. The modified lipoproteins have reduced activity toward cholesterol oxidase and cholesterol esterase. HDL thus becomes a primary substrate for these enzymes, and its cholesterol content can be measured directly. If polyethylene glycol (PEG) and dextran sulfate are present in the reaction mixture, the enzymes cholesterol oxidase and cholesterol esterase are modified by PEG to exhibit selective activities toward the different lipoprotein fractions, with their reactivity in the fractions increasing in the following order: LDL < VLDL and chylomicrons < HDL. HDL-C is oxidized to Δ⁴ cholestenone and H₂O₂ by cholesterol oxidase.

4.2.2.3 LDL cholesterol

Reference method: the CDC reference method for HDL-C is also used for LDL-C. After HDL-C has been extracted from the sediment fraction, cholesterol is measured in the remaining β-fraction (sediment fraction cholesterol minus HDL-C = LDL-C) /2/.

Routine method: Homogeneous (direct) method

Principle: the LDL-C assays use detergents, surfactants, or antibodies to modify the surface of chylomicrons, VLDL and LDL. The modified lipoproteins have reduced activity toward cholesterol oxidase and cholesterol esterase. LDL thus becomes the primary substrate for these enzymes and its cholesterol content can be measured directly /5, 6/. If a detergent is involved in the enzymatic measurement of cholesterol, its reactivities in the lipoprotein fractions increase in the following order: HDL< chylomicrons < VLDL < LDL. In the presence of Mg⁺⁺, the enzymatic reaction of the cholesterol measurement (cholesterol esterase and cholesterol oxidase) is significantly reduced in the VLDL and chylomicrons. The combination of a sugar compound with detergent increases the selectivity of cholesterol oxidase for LDL-C.

4.2.2.4 LDL calculated using Friedewald formula /6/

LDL-C (mg/dL) = cholesterol (mg/dL) – [HDL-C (mg/dL) + triglycerides (mg/dL)/5]
LDL-C (mmol/L) = cholesterol (mmol/L) – [HDL-C (mmol/L) + triglycerides (mmol/L)/2.22]

Besides the direct determination of LDL-C, the Friedewald equation is commonly used to determine LDL-C. It has the following limitations:

If triglycerides exceed 400 mg/dL (4.6 mmol/L) or the patient is not in a fasting state at the time of blood collection, chylomicrons, chylomicron remnants, or VLDL remnants will be present. As a result, the triglyceride/cholesterol ratio in the VLDL will be higher than 5 : 1, leading to overestimation of VLDL cholesterol and underestimation of LDL-C.
A small number of patients have type III hyperlipidemia (remnant hyperlipidemia, dysbetalipidemia) in which cholesterol-rich VLDL are present. In these circumstances, VLDL cholesterol is underestimated, and consequently LDL-C is overestimated.

4.2.2.5 LDL-C calculated using Sampson equation /7/

Compared with β-quantification, the new equation is more accurate than other LDL-C equations (slope, 0.964; RMSE = 15.2 mg/dL; R2 = 0.9648; vs Friedewald equation: slope, 1.056; RMSE = 32 mg/dL; R2 = 0.8808; vs Martin equation: slope, 0.945; RMSE = 25.7 mg/dL; R2 = 0.9022), particularly for patients with hypertriglyceridemia (MAD = 24.9 mg/dL; vs Friedewald equation: MAD = 56.4 mg/dL; vs Martin equation: MAD = 44.8 mg/dL). The new equation calculates the LDL-C level in patients with TG levels up to 800 mg/dL as accurately as the Friedewald equation does for TG levels less than 400 mg/dL and was associated with 35% fewer misclassifications when patients with hypertriglyceridemia (TG levels, 400-800 mg/dL) were categorized into different LDL-C treatment groups.

4.2.2.6 Non-HDL cholesterol (non-HDL-C)

Non-HDL-C (mg/dL) = cholesterol (mg/dL) – HDL-C (mg/dL)

4.2.2.7 Ratio LDL-C/HDL-C

Ratio = LDL-C (mg/dL)/HDL-C (mg/dL)

4.2.2.8 Triglyceride-rich lipoprotein (TRL) cholesterol

Triglyceride-rich lipoprotein cholesterol is the cholesterol of VLDL remnants and chylomikron remnants. Therefore to calculate remnant cholesterol the concentration of LDL-cholesterol may be subtracted from the concentration of non-HDL cholesterol.

TRL-C (mg/dL) = Non-HDL-C (mg/dL) – LDL-C (mg/dL)

Triglycerides are major components of triglyceride lipoproteins (TRLs), including VLDL and chylomikrons, which are synthesized and secreted from the liver and intestinal enterocytes, respectively /31/. Lipoproteinlipase (LPL) promotes hydrolysis of triglycerides in VLDL and chylomikons, producing VLDl remnants and chylomikron remnants, both of which are enriched in cholesterol relative to triglycerides. Some of these particles may be taken up by the liver and cleared. Those that remain in the circulation are further modified by hepatic lipase and lipoprotein lipase (LPL), resulting in formation of cholesterol enriched LDL particles.

4.2.3 Specimen

Serum, heparinized plasma, EDTA plasma: 1 mL

4.2.4 Reference interval

Refer to Tab. 4.1-1 – Upper reference values and recommended ranges of lipids.

4.2.5 Clinical significance

The Adult Treatment Panel III of the Expert Panel of the National Cholesterol Education Program (NCEP) in the USA /8/ and the European Guidelines on Cardiovascular Disease Prevention in Clinical Practice /9/ have defined a group of risk factors associated with atherosclerotic cardiovascular disease (ASCVD). They include elevated LDL-C, cigarette smoking, hypertension, decreased HDL-C, family history of early ASCVD, and age.

4.2.5.1 Clinical significance of total cholesterol

Total cholesterol levels increase from birth, stabilize at approximately age 2 years, peak before puberty, and then decline slightly during adolescence /10/.

The total cholesterol concentration is a basic parameter which indicates whether further lipid metabolism assays need to be performed. Cholesterol levels ≥ 200 mg/dL (5.18 mmol/L) are associated with an increased risk of ASCVD. The risk increases with increasing cholesterol levels and in the presence of additional risk factors such as overweight, hypertension, smoking, insulin resistance, and diabetes mellitus. Hypercholesterolemia has a high prevalence in the population which increases with age. The majority of individuals with hypercholesterolemia are asymptomatic, but have additional risk factors of ASCVD. In screening programs, the total cholesterol level is recommended to be used to estimate total ASCVD risk by means of a score system. An individual with a 10-year ASCVD risk of 2% can have a cholesterol of 390 mg/dL (8.0 mmol/L) while another individual with a 10-year risk of 21% can have a cholesterol of only 193 mg/dL (5.0 mmol/L) /11/.

The total cholesterol may be misleading, especially in women who often have high HDL-C concentrations and in patients with diabetes and metabolic syndrome who often have a low HDL-C level. For adequate risk analysis, at least HDL-C and LDL-C should be analyzed /9/.

There is general consensus that ASCVD rarely occurs with cholesterol concentrations below 160 mg/dL (4.1 mmol/L), and that the risk of CVD increases moderately with concentrations equal to or above 190 mg/dL(4.9 mmol/L) and significantly with levels equal to or above 250 mg/dL (6.5 mmol/L) /12/. For treatment and progress monitoring, it is important to know that the mean intraindividual coefficient of variation for cholesterol is 8% /13/.

4.2.5.2 Clinical significance of LDL-cholesterol (LDLC)

LDLC has become a key criterion for the assessment of atherosclerotic cardiovascular disease (ASCVD). Elevated LDLC is strongly associated with an increased risk, development and progression of ASCVD. Guidelines from the American Heart Association, the American College of Cardiology and the European Society of Cardiology have recommended LDLC as the primary target for diagnosis and therapy of ASCVD. Cardiologists primarily prescribe statins to lower LDLC in patients with high risk.

LDL particles are the main carrier of cholesterol to peripheral tissues where they are internalized through the LDL receptor, a crucial mediator of plasma LDL concentration. Genetic defects that result in loss of function within the LDL receptor are a major determinant of familiar hypercholesterolemia and risk of ASCVD /14/.

The increase in LDL particles plays a central role in atherogenesis. The initial process is the subendothelial incorporation of apo B-containing particles within the intima of the vessels. In cases with elevated concentration of LDL particles in blood, an increasing number of LDL particles are incorporated within the intima where they bind to proteoglycan. Following oxidation and other modifications, the LDL particles are taken up by macrophages, causing these to transform into foam cells and start the atherosclerotic process. The higher a person’s LDL particle count, the higher their risk of ASCVD. The LDL-C concentration is a surrogate marker for LDL particle number.

For clinicians epidemiological evidence shows that elevated concentrations of LDL-C are associated with an increased risk of myocardial infarction and vascular death.

LDL-C cutoff values for adults

The cutoff levels for primary prevention of ASCVD are shown in Tab. 4.1-1 – Upper reference values and recommended ranges of lipids.

LDL-C cutoff values for children

If familial hypercholesterolemia is suspected based on the medical history, the National Cholesterol Education Program recommends the following limits /8/:

Borderline high 110–129 mg/dL (2.85–3.34 mmol/L)
High ≥ 130 mg/dL (3.37 mmol/L).

LDL-C treatment guidelines for the use of statin therapy

The European Atherosclerosis Society Consensus Panel recommends LDL-C < 100 mg/dL (2.5 mmol/l) or < 70 mg/dl (1.8 mmol/l) in adults with ASCVD. For children < 135 mg/dL (3.5 mmol/l) /15/. An LDL concentration of < 100 mg/dL (2.5 mmol/l) as the goal of therapy in secondary prevention is recommended in the Third Report of the National Cholesterol Education Program. The American College of Cardiology/American Heart Association Guidelines for the use of statin therapy in patients with increased ASCVD /9/ are shown in Fig. 4.2-2 – Guidelines for the use of statin therapy.

4.2.5.3 Clinical significance of non-HDL-C

The Third Report of the National Cholesterol Education Program recommends that non-HDL-C (cholesterol minus HDL-C) be measured if triglyceride levels are above 400 mg/dl (10.3 mmol/L), because in this case HDL-C cannot be determined correctly using the Friedewald equation /8/. Non-HDL-C comprises all apoprotein B-containing lipoproteins. According to the Lipoprotein Management in Patients with Cardiometabolic Risk consensus statement from the American Diabetes Association and the American College of Cardiology, non-HDL-C should be part of every lipid assay panel /8/. Data from prospective studies show that non-HDL-C is better suited for primary prevention of atherosclerotic cardio vascular disease (ASCVD) than LDL-C /16/. The National Cholesterol Education Program has defined the cutoff for non-HDL-C as 150 mg/dL (3.9 mmol/L). It has thereby, at its own discretion, set the target for non-HDL-C 30 mg/dL (0.8 mmol/L) higher than that for LDL-C under the argument that VLDL cholesterol is 30 mg/dL at the upper-threshold triglyceride level of 150 mg/dL (1.71 mmol/L).

4.2.5.4 Clinical significance of HDL-C

High density lipoproteins (HDLs) are a family of lipoprotein particles composed of lipid an protein components. HDL facilitates reverse cholesterol transport. In this process, free cholesterol from macrophages is transferred to HDL and transported to the liver where it is excreted into stool together with bile. HDL cholesterol (HDL-C) uptake into the liver is mediated either by the scavenger receptor or by the cholesterol ester transfer protein (CETP) mediated transfer of the cholesterol esters to apo B containing lipoproteins which are absorbed by the liver /17/. In plasma there are multiple subfractions of HDL that are differentiated by HDL size, density, and charge. The main subfractions are HDL-3 and HDL-2. The main apolipoproteins in terms of quantity are apo A-I and Apo A-II.

4.2.5.4.1 Atherosclerotic Cardiovascular Disease (ASCVD)

Epidemiological studies have found an inverse relationship between the concentration of HDL-C and the risk of ASCVD /18/. In the Framingham Heart Study, each 10 mg/dL (0.26 mmol/L) increase in HDL-C was associated with a 19% decrease in ASCVD mortality risk in men and a 28% decrease in women /18/. The prevalence of low HDL-C concentrations, defined by the National Cholesterol Education Program guidelines as ≤ 40 mg/dL (1.04 mmol/L) in men and ≤ 50 mg/dL (1.30 mmol/L) in women, was found to be as high as 66% in high-risk patients with ASCVD, irrespective of their absolute levels below the cutoff values /19/. The magnitude of HDL-C elevation above the threshold values is not an indicator of a higher protective effect with regard to CVD risk.

There are some congenital diseases in which HDL-C concentrations are reduced without there being an increased risk for ASCVD. These include lecithin-cholesterol acyl transferase deficiency, apo A-I Milano mutation, and Tangier disease (mutation of the ATP-binding cassette transporter A1).

Patients with ASCVD have smaller and denser HDL particles, while large particles are thought to be inversely associated with the risk of ASCVD. HDL particles can be differentiated into three size classes by nuclear magnetic resonance (NMR) spectroscopy. However, a study /20/ showed that individuals with very large HDL particles are at increased risk of ASCVD.

Although epidemiological studies indicated that higher HDL-C serum concentrations are associated with a reduced risk for cardiovascular events, a recent study documented that genetic mechanisms raising HDL-C plasma concentrations are not associated with a lower risk of ASCVD /21/. The results of a study /22/ provide new evidence that the remodeling of the HDL proteome in patients with coronary heart disease has important functional implications with respect to the effects of HDL on endothelial cell survival.

4.2.5.4.2 Chronic kidney disease (CKD)

In CKD lower concentrations of HDL-C are measured in serum. HDLs in CKD tend to be dysfunctional lacking anti-inflammatory and cholesterol efflux capabilities. In a study /34/ signicant associations between albuminuria, but not eGfR and the lipid composition of HDL in patients with CKD were found.

4.2.6 Hereditary diseases of cholesterol metabolism

Malfunctions of cholesterol metabolism result from inherited susceptibility to hypercholesterolemia e.g.,

Familial hypercholesterolemia (FH); FH is a common inherited autosomal co-dominant disorder characterized by high plasma levels of low-density lipoprotein cholesterol due to its reduced metabolism
Sitosterolemia type C; a rare autosomal recessively inherited disease
Niemann-Pick type C1 disease; disturbance of the intracellular transport of lipids causes storage of cholesterol and sphingolipids in the lysosomes.

Refer to:

Table 4.1-2 – Genetic predisposition of dyslipidemia
Table 4.1-3 – Multifactorial secondary dyslipidemias
Table 4.2-1 – Criteria of familial hypercholesterolemia.

4.2.6.1Clinical significance of TRL-Cholesterol

Remnant/TRL-cholesterol can cause atherosclerosis by combination of the two pathways /32/:

Triglycerides can be a risk factor due to increased inflammation in the vascular wall caused by the production of free fatty acids by lipoprotein lipase lipolysis
Remnant/TRL particles enter the inflammed arterial intima.
Inflammation and remnant/TRL cholesterol deposition in the arterial wall work together. For women with coronary heart disease at age < 55 years increased risk of coronary heart disease was 1.8-fold higher in comparison to LDL-cholesterol /33/.

4.2.7 Comments and problems

Blood sampling

Refer to Section 4.1.3.2.

Intraindividual variation

Total cholesterol 14.9%, HDL-C 19.7%, LDL-C 25.7% /23/.

Method of determination

LDL-C

Most LDL-C direct assays met the National Cholesterol Education Program total error goals for non diseased individuals. All assays failed to meet these goals for diseased individuals, however, because of lack of specificity towards abnormal lipoproteins /24/.

Direct measurement of LDL-C has the following advantages and disadvantages /24/:

HDL-C can be performed on samples with triglyceride levels up to 1.200 mg/dL (13.6 mmol/l)
Different specificity for LDL-C (recovery 87–105%)
Levels are reported to be 6 mg/dL (0.15 mmol/L) lower than those calculated with the Friedewald equation
VLDL cholesterol is partly included in the measurement
31–64% of IDL cholesterol is measured
Lp (a) is included in the measurement at different degrees.

Limitations of the Friedewald equation are listed under 4.2.2. The equation proposed by Sampson et al for calculation of LDL-C has two major strengths: first it alows accurate determination of LDL-C over the range of triglyceride concentrations up to almost 800 mg/dL (9.1 mmol/L) and second, it provides relable estimation of LDL-C at concentrations well below 50 mg/dL (0.57 mmol/L) /25/

HDL-C

Most HDL-C direct assays met the National Cholesterol Education Program total error goals for non diseased individuals. All assays failed to meet these goals for diseased individuals, however, because of lack of specificity towards abnormal lipoproteins /24/.

The direct measurement of HDL-C has the following advantages and disadvantages:

Elevated levels of immunoglobulins and monoclonal gammopathies (in particular IgM) , can lead to artificially increased HDL-C results
Levels are reported to be 6 mg/dL (0.15 mmol/L) lower than those calculated with the Friedewald equation.

Stability

Plasma and serum can be stored for up to 4 days at 4 °C.

References

1. Genest J. Lipoprotein disorders and cardiovascular risk. J Inherit Metab Dis 2003; 26: 267–87.

2. Warnick GR, Kimberly MM, Waymack PP, Leary ET, Myers GL. Standardization of measurements for cholesterol, triglycerides, and major lipoproteins. Labmedicine 2008; 39: 481–9.

3. Bergmeyer HU. Methods of enzymatic analysis, 3rd edition, vol. VIII. Verlag Chemie, Weinheim 1983.

4. Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol.Clin Chem 1974; 20: 470–5.

5. Okada M, Matsui H, Ito Y, Fujiwara A, Inano K. Low density lipoprotein cholesterol can be chemically measured. A new superior method. J Lab Clin Med 1998; 132: 195–201.

6. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma without use of the ultracentrifuge. Clin Chem 1972; 18: 499–502.

7. Sampson M, Ling C, Sun Q, Harb R, Ashmaig M, Warnik R, et al. A new equation for calculation of low-density lipoprotein cholesterol in patients with normolipidemia and/or hypertriglyceridemia. JAMA Cardiol 2020; 5. 540-8.

8. Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 2001; 285: 2486–97.

9. The task force for the management of dyslipidemias of the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS). ESC/EAS Guidelines for the management of dyslipidaemias. European Heart J 2011; 32: 1769–1818.

10. U.S. Preventive Task Force. Screening for lipid disorders in children and adolescents. Am Family Physician 2016; 94: 1004A-1004E.

11. De Backer G. New European guidelines for cardiovascular disease prevention in clinical practice. Clin Chem Lab Med 2009; 47: 138–42.

12. Assmann G, ed. Lipid metabolism disorders and coronary heart disease, 2nd edition. Stuttgart; MMV 1993.

13. See www.athero.org and www.chd-taskforce.com

14. Ridker PM. Lipids in cardiovascular disease 1. LDL cholesterol: controversies and future therapeutic directions. Lancet 2014; 384: 607–17.

15. Cuchel M, Bruckner E, Ginsberg HN, et al. European Atherosclerosis Society Consensus Panel on Familial Hypercholesterolemia. Homozygous familial hypercholesterolemia: new insights and guidance for clinicians to improve detection and clinical management. A position paper from the Consensus Panel on Familial Hypercholesterolemia of the European Atherosclerosis Society. Eur Heart J 2014; 35: 2146–57.

16. Contois JH, McConnell JP, Sethi AA, Csako G, Devaraj S, Hoefner DM, Warnick GR. Apolipoprotein B and cardiovascular disease risk: position statement from the AACC Lipoproteins and Vascular Disease Division Working Group on Best Practices. Clin Chem 2009; 55: 407–19.

17. Movva R, Rader Dj. Laboratory assessment of HDL heterogeneity and function. Clin Chem 2008; 54: 788–800.

18. Wilson PW, Abbott RD, Castelli WP. High density lipoprotein cholesterol and mortality. The Framingham Heart Study. Arteriosclerosis 1988; 8: 737–41.

19. Alsheikh-Ali AA, Lin JL, Abourjaily P, Ahearn D, Kuvin JT, Karas RH. Prevalence of high density lipoprotein cholesterol in patients with documented coronary heart disease or risk equivalent and controlled low-density lipoprotein cholesterol. Am J Cardiol 2007; 100: 1499–1501.

20. van der Steeg WA, Holme I, Boekholdt SM, Larsen ML, Lindahl C, Stroes ES, et al. High density lipoprotein cholesterol, high density lipoprotein particle size, and apolipoprotein A-I: significance for CV risk: the IDEAL and EPIC-Norfolk studies. J Am Coll Cardiol 2008; 51: 634–42.

21. Voight BF, Peloso GM, Ortho-Melander M, Frikke-Schmidt R, Barbalic M, Jensen MK, et al. Plasma HDL-cholesterol and risk of myocardial infarction: a mendelian randomization study. Lancet 2012; 380: 572–80.

22. Riwanto M, Rohrer L, Roschitzki B, Besler C, Mocharla P, Mueller M, et al. Altered activation of endothelial anti- and proapoptotic pathways by high-density lipoprotein from patients with coronary artey disease. Circulation 2013; 127: 891–904.

23. Technology and Training for Healthcare Laboratories. Westgard QC. www.westgard.com/biodatabase1.htm

24. Miller WG, Myers GL, Sakurabayashi I, Bachmann LM, Caudill SP, Dziekonski A, et al. Seven direct methods for measuring HDL and LDL cholesterol compared with ultracentrifugation reference measurement procedures. Clin Chem 2010; 56: 977–86.

25. Chapman MJ, Giral G, Therond P. LDL cholesterol: The times they are changin. Clin Cem 2020; 66: 1136–9.

26. Keaney JF, Curfman GD, Jarcho J. A pragmatic view on the new cholesterol treatment guidelines.N Engl J Med 2014; 370: 275–8.

27. Gidding SS.Finding familial hypercholesterolemia. Clin Chem 2021; 67 (5): 710–2.

28. Paththinige S, Sirisena ND, Dissanayake V. Genetic determinants of inherited susceptibility to hypercholesterolemia – a comprehensive literature review. Lipids Health disease 2017; 16 (1): 103. doi: 10.1186/s12944-017-0488-4.

29. Benito-Vicente A, Uribe KB, Jebari S, Galicia-Garcia U, Ostolaza H, Martin C. Familial hypercholesterolemia: the most frequent cholesterol metabolism disorder caused disease. Int J Mol Sci 2018; 19: 3426. doi: 10.3390/ijms19113426.

30. Alonso R, Diaz-Diaz JL, Arrieta F, Fuentes-Jiminez F, Illan F, Mata P. Current therapy for homozyous familial hypercholesterolaemia. EMJ 2021; 6 (4): 76–86.

31. Budoff M. Triglycerides and triglyceride-rich lipoproteins in the causal pathway of cardiovascular disease. Am J Cardiol 2016; 118: 138–45.

32. Wulff AB, Nordestgaard BG. Remnants and triglyceride-rich lipoproteins in the prevention of premature coronary heart disease. Clin Chem 2022; 68 (2): 266–8.

33. Dugani SB, Moorthy MV, Li C, Demler OV, Alsheikh-Ali AA, Ridker PM, et al. Association of lipid, inflammatory, and metabolic biomarkers with age at onset for incident coronary heart disease in women. JAMA Cardiol 2021; 6: 437–47.

34. Lindgard B, Hoofnagle AN, Zelnick LR, de Boer IH, Fretts AM, Kestenbaum BR, et al. High-density lipoprotein lipidomics in chronic kidney disease. Clin Chem 2023; 69 (3): 273–82.

4.3 Triglycerides

Triglycerides, also called triacylglycerol or neutral lipids, are esters derived from glycerol and three mono carboxylic acids (esters of fatty acids). They have a mean molecular weight of 875 D. The lipids occurring in nature consist of triglycerides and small amounts of mono- and diacylglycerides.

In human adipose tissue, fatty acid esters mainly consist of even-numbered, unbranched mono carboxylic acids with 18 or 16 carbon atoms with or without double bonds, in particular oleic acid and palmitic acid. The triglycerides are poorly soluble in water and are transported in plasma bound to apolipoproteins. Triglycerides are mostly carried in chylomicrons, very low density lipoproteins (VLDL), and chylomicron and VLDL remnants.

Triglycerides and postprandial state

One of the main sources of the triglycerides circulating in postprandial plasma is dietary fat. The triglycerides are hydrolyzed into free fatty acids and glycerol, absorbed by the microvilli in the small intestine and repackaged into chylomicron particles via which they pass into the venous blood through the thoracic duct, bypassing the liver. From the bloodstream, the triglycerides are then deposited in adipose or muscle tissue where endothelial lipoprotein lipase removes the triglyceride component. The residual particles i.e., the chylomicron and VLDL remnants, which are rich in cholesterol esters, are released into the circulation and taken up by the liver.

Triglycerides and fasting state

In the fasting state, triglycerides and cholesterol are supplied to the tissues via the endogenous pathway. The liver forms VLDL particles and releases them into the circulation where the triglyceride component is removed by lipoprotein lipase. The VLDL remnants are either directly absorbed by the liver or converted to intermediate-density lipoproteins (IDL) and then taken up by the liver.

During lipoprotein electrophoresis, VLDL migrate in the pre-β-fraction (pre-β-lipoproteins) and chylomicrons remain at the application point.

4.3.1 Indication

Diagnosis of primary and secondary dyslipidemias

Primary and secondary prevention of atherosclerotic cardiovascular disease (ASCVD)
Risk marker of the metabolic syndrome
Calculation of LDL-C using the Friedewald formula
Monitoring of lipid-lowering dietary and drug therapy.

4.3.2 Method of determination

Principle: microbial lipase hydrolyzes triglycerides quantitatively to glycerol and free fatty acids /1/. The indicator systems for the determination of glycerol are based on enzymatic methods (Fig. 4-3-1 – Indicator systems for determination of free glycerol).

4.3.3 Specimen

Serum, plasma: 1 mL

There has been the assumption that triglycerides should be measured in the fasting state because the levels of fasting triglycerides are lower and possibly less variable from measurement to measurement compared with triglycerides measured in the non fasting. However, recent large prospective studies reported that non-fasting triglycerides predict cardiovascular disease risk more strongly than the fasting triglycerides state /4/.

4.3.4 Reference interval

Triglycerides (fasting) ≤ 150 mg/dL (1.71 mmol/L)* /2/
Triglycerides (non-fasting) ≤ 175 mg/dL (1.98 mmol/L) /3/

* This is the recommended upper threshold value.

Upper threshold values for children. Refer to Tab. 4.3-1 – Triglyceride levels for males and females 5–19 years of age.

Conversion triglycerides: mg/dL × 0.01129 = mmol/l

4.3.5 Clinical significance

High triglycerides may promote atherosclerosis via the accumulation of triglyceride-rich remnant particles within the endothelium. Although smaller triglyceride-rich particles can cross the subintimal space and be found in atherosclerotic plaques, most plaque lipid is cholesterol ester and not triglycerides.

4.3.5.1 Hereditary (primary) and secondary hypertriglyceridemias

It is often difficult to differentiate between primary and secondary hypertriglyceridemias. Secondary hypertriglyceridemias occur with many types of organ damage (e.g., hepatopathy, nephropathy, hypothyroidism, pancreatitis). Primary hypertriglyceridemias are less common than the secondary type /13/.

Hypertriglyceridemia is due to an overproduction of chylomicrons and/or VLDL particles. Most cases result from increased concentrations of VLDL due to increased, often endogenous, synthesis. Elevated triglycerides are associated with a reduced concentration of HDL particles and smaller LDL and HDL particles.

High triglyceride concentrations are often associated with low HDL-cholesterol and high levels of dense LDL particles.

Triglyceride-rich lipoproteins are increasingly considered as a direct driver of atherosclerosis in diabetic patients. The atherogenic dyslipidemia is often comorbid with hyperglycemia and low HDL-cholesterol in patients with diabetes type 2, namely associated with obesity, insulin resistance, hyperinsulinism, and the metabolic syndrome /5/.

The prevalence of fasting hypertriglyceridemia > 150 mg/dL (1.71 mmol/L) is 30% in individuals over the age of 20 and increases to 43% in individuals over the age of 50 /6/.

Based on the National Cholesterol Education Program (adult treatment panel III) /2/ triglyceride levels are classified as follows in the fasting state:

Normal: ≤ 150 mg/dL (1.7 mmol/l)

Borderline: 151–193 mg/dL (1.7–2.2 mmol/L).
High: 194–480 mg/dL (2.3–5.5 mmol/L).
Very high: 481–960 mg/dL (5.6–11.0 mmol/L).
Severe: Above 960 mg/dL (11.0 mmol/L).

Following food intake, triglyceride level increases rapidly in the postprandial phase, peaks at 4 h and subsequently returns to baseline level within 8 h after food intake in women and within 9 h in men. From this point on, a metabolic state exists in which the triglycerides are only present in the form of VLDL particles and not as chylomicrons /7/.

Severe hypertriglyceridemias occur predominantly with genetic dyslipidemias. In these cases, chylomicronemia (Fredrickson type I and V) is present and there is an increased risk of acute pancreatitis. Refer to Tab. 4.3-2 – Classification of hyperlipoproteinemia according to Fredrickson.

Hypertriglyceridemia (type IV) is a disease of adulthood. type IV is often associated with the metabolic syndrome, diabetes mellitus and obesity. Triglyceride levels are generally 200–500 mg/dL (2.3–5.7 mmol/L).

Mild to moderate hypertriglyceridemia of 150–500 mg/dL (1.7–5.6 mmol/L) is causally related to increased risk of acute pancreatitis, most likely as a marker of future severe hypertriglyceridemia /15/.
Levels of triglycerides > 500 mg/dL (5.7 mmol/l) are found at 5% of the population and concentrations > 1,000 mg/dL (11.4 mmol/l) at approximately 0.05% of the population.

Metabolic syndrome as defined by the National Cholesterol Education Program (adult treatment panel III) /2/ is present if three of the following five criteria are met:

Abdominal obesity
Triglycerides ≥ 150 mg/dL (1.71 mmol/L)
HDL-C below 40 mg/dL (1.0 mmol/L) in men and below 50 mg/dL (1.3 mmol/L) in women
Hypertension ≥ 130/85 or antihypertensive therapy.
Fasting glucose ≥ 100 mg/dL (5.6 mmol/L).

4.3.5.2 Hypertriglyceridemia and atherosclerotic cardio vascular disease (ASCVD)

High triglycerides may promote atherosclerosis via the accumulation of triglyceride-rich remnant particles within the vascular endothelium /3/. An increase in the plasma concentration of triglycerides is an established risk factor for ASCVD /8/. This applies in particular if postprandial triglyceride levels are used as a criterion. The effect of triglycerides is independent of the concentration of LDL and HDL particles /9/. Patients with high triglyceride levels > 2.212 mg/dL (25 mmol/L) and those with familial chylomicronemia syndrome rarely develop atherosclerosis, probably because their lipoprotein particles are too large to accumulate into the vascular intima.

In contrast, patients with moderate hypertriglyceridemia i.e., metabolic syndrome, familial hypertriglyceridemia and familial combined hypertriglyceridemia, and patients with elevated chylomicron remnants and VLDL remnants develop atherosclerosis more frequently, because they have smaller lipoprotein particles that are able to pass into the vascular intima /9/.

Because these conditions are present in the post- prandial state, it is important that triglycerides are measured within about 4 h of food intake.

Tab. 4.3-3 – Postprandial triglycerides and hazard ratios for cardiovascular disease and death shows the hazard ratios of the Copenhagen Risk Study for cardiac ischemia, cardiac infarction and death as a function of postprandial hypertriglyceridemia. In Women’s Health study /10/ the postprandial triglyceride levels were divided into tertiles ≤ 104 mg/dL (1.19 mmol/L), 105–170 mg/dL (1.20–1.71 mmol/L) and ≥ 171 mg/dL (1.95 mmol/L). Compared to the first tertile, the hazard ratios for cardiovascular events were 1.44 (0.90–2.29) in the second tertile and 1.98 (1.21–3.25) in the third tertile.

The optimal threshold for assessing increased risk of cardiovascular disease is non fasting hypertriglyceridemia of 175 mg/dL (1.98 mmol/L) /3/.

4.3.5.3 Acute promyelocytic leukemia

Patients with acute promyelocytic leukemia are often obese or overweight and have metabolic disorders, such as high triglyceride concentrations.and a positive correlation between triglyceride levels and leukocyte counts. Hypertriglyceridemia serves as a risk factor for early death in acute promyelocytic leukemia patients. Peroxisomeproliferator-activated receptor-a (PPRa) is a regulator of cell metabolism and transcription factor involved in cancer development. PPRa knockdown should affect the expression of target genes responsible for acute promyelocytic leukemia proliferation. The findings emphasize the influence of obesity and dyslipidemia on acute promyelocytic leukemia proliferation and expand the knowledge of the mechanisms responsible for the obesity-cancer association /14/.

4.3.6 Comments and problems

Blood sampling

Blood should be sampled in the fasting state. For cardiovascular risk assessment, blood withdrawal is best carried after 4 h of food intake.

Intraindividual variation

The intraindividual variation for triglycerides is 19.7%.

Specimen

Fresh serum is required.

Method of determination

In serum, free glycerol is present in a concentration of approximately 10 mg/dL (1.1 mmol/L). Free glycerol must be subtracted from total glycerol to obtain the component from the triglycerides. In healthy individuals, this correction may be within the limits of tolerable error. Patients with diabetes and hepatopathies can have hyperglycerolemia where falsely very high values of triglycerides are obtained. Therefore a blank measurement should be carried out without hydrolysis.

Free glycerol produced by spontaneous hydrolysis (e.g., when the sample is stored at room temperature for extended periods of time) should not be subtracted from the triglyceride value, since it comes from the triglycerides.

Interfering factors /1/

Elevated alkaline phosphatase levels in the patient sample can lead to falsely increased triglycerides. Therefore, special attention must be paid to pediatric samples.

Bilirubin and ascorbic acid interfere chemically and spectrophotometrically with assays using colorimetric indicator reactions, leading to a decrease in the triglyceride concentration.

Stability

Concentration remains unchanged if the sample is stored at 4 °C for 4 days.

4.3.7 Pathophysiology

Triglycerides are hydrophobic fatty acids organized and transportet by lipoproteins /15/.

The synthesis of triglycerides is mainly regulated by two enzymes:

monoacylglycerol acyltransferase that facilitates addition of a fatty acid to monoacylglycerol
diacyl glycerol transferase that facilitates the addition of a fatty acid to diacylglycerol, resulting in the formation of triglycerides
triglcerides are packed into lipoproteins and transported as lipoproteins in plasma /15/.

Triglycerides enter the plasma in large lipoprotein particles, the chylomicrons, containing the apolipoprotein B48. After fat absorption from food chylomicrons are assembled in the intestine, bypass the liver and enter the circulation via the lymph through the thoracic duct. The tissues are able of utilizing free fatty acids from lipoprotein lipase facilitated triglyceride hydrolysis. The free fatty acids are either metabolized as energy in muscles or stored in fat tissue.The remaining triglycerides of the chylomicron remnants are later resecreted into the circulation by the liver in apoB100 containing very low density lipoprotein (VLDL) particles Triglycerides in these particles are again hydrolyzed by lipoprotein lipase with liberation of free fatty acids thereby producing smaller very low-density lipoprotein remnants and finally low-density lipoproteins (LDL) a particle that is removed from the circulation. Refer to Fig. 4.9-2 – Metabolism of triglyceride-rich chylomicrons and VLDL by heparin-sensitive (endothelial) lipoprotein lipase.

References

1. Klotzsch SG, McNamara JR. Triglyceride measurements: a review of methods and interferences. Clin Chem 1990; 36: 1605–13.

3. White KD, Moorthy MV, Akinkuolie AO, Demler O, Ridker PM, Cook NR, Mora S. Identifying an optimal cutpoint for the diagnosis of hypertriglyceridemia in the nonfasting state. Clin Chem 2015; 61: 1156–63.

4. Langsted A, Nordestgaard BG. Nonfasting lipid profiles: the way of the future. Clin Chem 2015; 61: 1123–5.

5. Hermans MP, Valensi P. Elevated triglycerides and low high-density lipoprotein cholesterol level as marker of very high risk in type 2 diabetes. Curr Opin Endocrinol Diabetes Obes 2018; Apr; 25 (2): 118–29.

6. Ford ES, Giles WH, Dietz WH. Prevalence of the metabolic syndrome among US adults – Findings from the Third National Health and Nutrition Examination Survey. JAMA 2002; 287: 356–9.

7. Sundvall J, Laatikainen T, Hakala S, Leiviskä J, Alfthan G. Systematic error of serum triglyceride measurements during three decades and the effect of fasting on serum triglycerides in population studies. Clin Chim Acta 2008; 397: 55–9.

8. Nordestgaard BG, Varbo A. Triglycerides and cardiovascular disease. Lancet 2014; 384: 626–35.

9. Nordestgaard BG, Benn M, Schnohr P, Tybjaerg-Hansen A. Nonfasting triglycerides and risk of myocardial infarction, ischemic heart disease and death in men and women. JAMA 2007; 298: 299–308.

10. Bensal S, Buring JE, Rifai N, Mora S, Sacks FM, Ridker PM. Fasting compared with nonfasting triglycerides and risk of cardiovascular events. JAMA 2007; 298: 309–16.

11. Beaumont JL, Carlson LA, Cooper GR, Fejfar Z, Fredrickson DS, Strasser T. Classification of hyperlipidemias and hyperlipoproteinemias. Bull WHO 1970; 43: 891–908.

12. Tamir I, Heiss G, Glueck CJ, Christensen B, Kwiterovich P, Rifkind B. Lipid and lipoprotein distributions in white children ages 6–19 years: the Lipid Research Clinics Program Prevalence study. J Chron Dis 1981; 34: 27–39.

13. Parhofer KG, Laufs U. The diagnosis and treatment of hypertriglyceridemia. Dtsch Arztebl Int 2019; 116: 825–32.

14. Wu S, Li S, Peng J, Yi Z, Li C, Jin W, Li J, Wang K. Interplay between hypertriglyceridemia and acute promyelocytic leukemia mediated by cooperation of peroxisome proliferator-activated receptor-a with the PML/RAR a fusion protein on super-enhancers. Haematologica 2022; 107: 2589–2600.

15. Hansen SEJ, Varbo A, Nordestgaard BG,Langsted A. Hypertriglyceridemia-associated pancreatitis: New concepts and potential mechanisms. Clin Chem 2023; 69 (10): 1132-44.

4.4 Lipoprotein electrophoresis

The electrophoretic separation of lipoproteins is based on the classification of hyperlipoproteinemias according to Fredrickson.

4.4.1 Indication

Differential diagnosis of dyslipoproteinemia, in particular type III hyperlipoproteinemia.

4.4.2 Method of determination

Principle: the lipoproteins are separated on agarose gel by migration toward the anode at alkaline pH /1/. The lipoprotein fractions are then precipitated by poly anions in the gel and densitometrically quantified. The lipoproteins of the α-fraction correspond with HDL, those of the pre-β-fraction with VLDL and those of the β-fraction with LDL. Chylomicrons in serum remain at the application point of the serum.

4.4.3 Specimen

Serum: 1 mL

4.4.4 Reference interval

No dyslipidemia. Refer to Tab. 4.4-1 – Primary hyperlipoproteinemia: classification according to Fredrickson.

4.4.5 Clinical significance

Quantitative lipoprotein electrophoresis allows the classification of hyperlipoproteinemia into the Fredrickson phenotypes (Tab. 4.4-1) /2/.

4.4.6 Comments and problems

Fresh serum is required. Heparin alters the mobility of the lipoproteins.

References

1. Aufenanger J, Haux P, Kattermann R. Improved method for enzymatic determination of cholesterol in lipoproteins separated on thin layer agarose gels. J Clin Chem Clin Biochem 1989; 27: 807–13.

2. Riesen WF. Fettstoffwechsel. In Thomas L, ed. Labor und Diagnose. Frankfurt 2008; TH-Books, 225–48.

4.5 Lipoprotein (a) (Lp(a))

4.5.1 Indication

Atherosclerotic cardiovascular disease (ASCVD)
Cardiovascular outcomes
Aortic stenosis
Testing at least once in adults.

4.5.2 Method of determination

Lp(a) quantitative: immunoassay (ELISA, electroimmunoassay, immunonephelometric and immunoturbidimetric assays).

Number of K-IV repeats: isoelectro focusing followed by immunoblotting /1/.

4.5.3 Specimen

Serum: 1 mL (should be measured at least once in all individuals according to the Canadian and European guidelines) /2/.

4.5.4 Reference interval

Lp(a) has significant size variability, a conversion of mmol/L to mg/dL is therefore not possible. The concentration of Lp(a) is specified either in nmol/L or mg/dL. The recommended classification is:

not elevated: < 75 nmol/L (30 mg/dL)
borderline: 75–125 nmol/L (30–50 mg/dL)
elevated: > 125 nmol/L (> 50 mg/dL).

4.5.5 Clinical significance

Lp (a) plays an important role in dyslipidemias, especially in atherosclerotic cardiovascular disease an aortic stenosis.

Refer to Tab. 4.5-2 – Biochemistry of Lp(a).

4.5.5.1 Lp(a) and atherosclerotic cardiovascular disease (ASCVD)

Prospective epidemiological and genetic studies strongly support an continuous association between the concentration of Lp(a) and ASCVD in different ethnicities. Elevated Lp(a) is a risk factor even at very low concentrations of low-density lipoprotein cholesterol. High Lp(a) is associated both with microcalcification and macrocalcification of the aortic valve. According to the 2022 guidelines of the Euopean Society of Cardiology endorsed by the European Society of Anesthesiology and Intensive are (ESAIC) increased serum levels of Lp(a) are associated with increased risk of ASCVD /3/.

In Caucasians, the threshold for increased risk of ASCVD is reported to be as low as ≥ 75 nmol/L (30 mg/dL). The thresholds used for Blacks are higher and those for Asians are lower than the thresholds for Caucasians. An even higher risk is reported when elevated Lp(a) levels are accompanied by additional risk factors such as elevated LDL-C, decreased HDL-C, and hyperfibrinogenemia /4/.

A great number of reports were published examining the role of Lp(a) as a risk factor for atherosclerosis. A comprehensive meta-analysis of prospective studies found incidence rates of ASCVD in the top and bottom tertiles of baseline Lp(a) of 4.4 and 5.6 per 1,000 patient years. The authors concluded that a continuous independent and modest association of the Lp(a) concentration with the risk of ASCVD and stroke exists /5/.

Initiation of statin therapy reduces LDL-cholesterol without a significant change in Lp(a). Associations of baseline and on-statin treatment Lp(a) with cardiovascular risk are approximately linear, with increased risk at Lp(a) values of 50 mg/dL. A study /6/ has shown the following results: initiation of statin therapy reduced LDL cholesterol by 39%, and the hazard ratios of cardiovascular events adjusted for age and sex were 1.04 for 15 to < 30 mg/dL, 1.11 for 30 to < 50 mg/dL, and 1.31 for 50 mg/dL or higher. A possible interpretation of the study indicated that in statin treated patients Lp(a) has an independent approximately linear relation with cardiovascular disease risk.

Recommendations from guidelines show no unanimous agreement when to measure Lp(a) and how to deal with increased Lp(a) values (Tab. 4.5-1 – Recommendations from guidelines when to measure Lp(a) and how to deal with increased values) /7/.

Lp(a) variants play an important role in the development of ASCVD. For example, the chromosomal regions 6q26–27 and 1p13 are strongly associated with the risk of ASCVD. The LPA locus of 6q26–27, which encodes Lp(a), shows the strongest association.

Variants rs10455872 and rs3798220 of the LPA locus have an odds ratio for ASCVD of 1.70 (1.49–1.95) and 1.92 (1.48–2.49). Both variants are strongly associated with elevated Lp(a) levels, small Lp(a) particle size and a reduced number of LPA copies (these determine the number of type 2 kringle IV repeats) /8/.

Lipid apheresis (LA) is performed in patients with Lp(a) levels above 60 mg/dL whose ASCVD is progressive despite receiving maximum drug therapy. LA removes Lp(a) and LDL simultaneously /9/.

Particular attention should be paid to patients with renal disease and hemodialysis patients. They have a 2 to 3-fold increase in Lp(a). The most important next step is to treat traditional modifiable risk factors such as LDL-C, hypertension, smoking, diabetes, and obesity. Niacin and LDL apheresis can be used to lower Lp(a) in selected patients /7/.

The comment of some authors for screening for Lp(a) levels in individuals at low risk for ASCVD is as follows: There are no approved drugs for lowering elevated Lp(a) and no randomized controlled study demonstrating that lowering Lp(a) levels has any clinical benefit in either primary or secondary prevention of cardiovascular disease /10/.

Lp(a) is a potential factor in the pathogenesis of aortic valve disease.

4.5.5.2 Secondary changes in Lp(a) concentration

The influence of diseases, hormones and drugs on Lp(a) is shown in Tab. 4.5-3 – Factors influencing Lp(a).

4.5.6 Comments and problems

Comparison of LP(a)

In a study /11/, Lp(a) serum concentrations using six different assays, providing Lp(a) in mg/dl or in nmol/L were measured. All assays relied of five-point calibrations using calibrators provided by the manufacturers. While the imprecision of all assays was in an acceptable range, the actually obtained concentration due to calibration differed most likely due to biases. The assays applied produced remarkably different values obtained for the PRM-1 reference standard, which has assigned concentrations of 43.3 mg/dL or 96.6 nmol/L, respectively.

Standard

Based on the above reference method, a secondary reference material, SRM 2B, was developed. This material is the first WHO/IFCC reference reagent for Lp(a) for the calibration of immunoassays and allows the calibration on a molar basis. SRM 2B consists of Lp(a) isoforms of the three main apo A polymorphisms (containing 16, 17 and 18 KIV kringles, respectively) and three less common polymorphisms (containing 14, 20 and 34 KIV kringles, respectively) /13/.

Reference range

There is no consensus on the threshold for defining elevated Lp(a). Scientific guidelines differ on normal levels (< 30 mg/dL or < 50 mg/dL). It is recommended that Lp(a) no longer be expressed as mass in mg/dL but as concentration in nmol/L (1 mg/L corresponds to about 3.17 nmol/L) /5/. The reason for this is the size heterogeneity of apo(a) and the resulting multitude of isoforms of Lp(a). Most Lp(a) immunoassay manufacturers use an antibody that detects the kringle IV type 2 epitope. As a result, the Lp(a) isotypes of individuals are detected differently which can lead to reduced or increased levels of Lp(a). The broad heterogeneity of Lp(a) results from the genetic coding of kringles IV and V. Apo(a), synthesized in different individuals, can have between 1 to 10 kringle IV repeats, and consequently the Lp(a) particle can vary in size. Therefore it is important to employ assays that are independent of the apo(a) polymorphism and use standards that are calibrated on a molar basis /15/.

Stability

After deep-frozen storage, Lp(a) mass decreases by 23% at concentrations in the range of 41 to 345 mg/L /12/.

4.5.7 Structure and function of Lp(a)

Lp(a) comprises an LDL particle covalently linked by disulfide bridges to the highly glycosylated apolipoprotein (a) called Apo(a), which is under tight genetic regulation. Lp(a) has pro inflammatory, pro oxidative and atherothrombogenic properties. The plasma concentration of Lp(a) is determined by the rate of hepatic secretion of Apo(a) that in turn is inversely related to the size of Apo(a) and hence the copy number of genetic variants that encode the number of K-IV type 2 repeats of the Apo(a) protein /13/. This copy number variation in the K-IV type 2 protein domain, encoding Apo(a) isoforms of varying size, results in a varying number of up to about 40 copies of identical K-IV type 2 repeats. The plasma concentration of Lp(a) is determined by the rate of hepatic secretion of Apo(a) and inversely related to the copy number of genetic variants that encode the number of K-IV type 2 repeats of the Apo(a) protein /14/.

Apo(a), a highly polymorphic glycoprotein component of Lp(a), consists of repetitive protein segments, so called kringles that are highly homologous to kringle IV of plasminogen. One kringle contains 110 amino acids forming a secondary structure. In humans more than 40 genetically determined isoforms of Apo(a) exist, giving rise to substantial size heterogeneity. There is an inverse correlation between the molecular weight of a given Lp(a) molecule and the number of type IV kringles. Individuals with a high Apo(a) molecular weight have a low plasma concentration of Lp(a) while those with a low Apo(a) molecular weight have a high plasma concentration of Lp(a). Apo(a) is produced almost exclusively in the liver and follows classical steps of glycoprotein biosynthesis /5/.

Lp(a) consists of an LDL-like core lipoprotein and glycoprotein Apo(a) covalently linked by a disulfide bridge /7/. The lipid core of Lp(a) is similar in structure and composition to LDL. The composition of Lp(a) is as follows: 30% protein, 35.5% cholesterol esters, 8.5% free cholesterol, 19.5% phospholipids, 2% triglycerides.

An essential aspect of the Lp(a) molecule is the tail of the Apo(a) moiety containing kringle proteins IV and V. Kringle IV consists of 10 subtypes or segments (numbered 1–10), of which subtype 2 has an individually variable number of copies (3–40). Levels of Lp(a) mass may vary up to a 1.000-fold between individuals /9/. The length of kringle IV type 2 repeats is genetically determined and not influenced by life stile.

Lp(a) is involved in various processes related to atherosclerosis and vascular disease, with an overall pro atherogenic effect that is similar to LDL-C, as well as having a prothrombotic effect. Studies demonstrate strong support for Lp(a) as a causal risk factor for atherosclerotic cardiovascular disease (ASCVD). For ASCVD prevention it is important to determine whether it is high Lp(a)concentration or the number of K-IV type 2 repeats that account for the increased risk of ASCVD associated with increased Lp(a) levels /16, 17/.

Blood levels of Lp(a) are highly heritable and are chiefly determined by copy number variation at the LPA locus at chromosome 6.

Lipid oxidation is the hallmark of for atherosclerotic diseases. Lp(a) promotes the development of atherosclerosis because significant amounts of oxidized phospholipids that exert pro-inflammatory actions are bound to Lp(a). In the vessel wall Lp(a), via its apo(a) component, binds to glycoproteins such as laminin, induces the activation of inflammatory cells and smooth muscle cells and causes increased synthesis of alkaline phosphatase, which catalyzes a calcification process. The accumulation of Lp(a) in the vessel wall generally leads to increased proliferation and migration of inflammatory cells and smooth muscle cells and promotes the development of atherosclerotic plaques /15, 16/. In addition, Lp(a) is an LDL-like particle that is capable of transporting significant amounts of cholesterol. As a rule of thumb, the amount of cholesterol Lp(a) transports corresponds to a third of its, measured as protein concentration (mg/L); at a concentration of 30 mg/dL, Lp(a) contributes 10 mg/dL of LDL cholesterol. From a pharmacological standpoint it is important to know that this amount cannot be corrected by statin therapy. Very high concentrations of Lp(a) can therefore be the reason for statin resistance.

Competing with plasminogen, Lp(a) can inhibit the binding of plasminogen to fibrinogen and fibrin as well as the activation of plasminogen to plasmin by the tissue plasminogen activator. This may be the reason why elevated Lp(a) is associated with an increased risk for venous thrombosis and stroke, at least in children and adolescents.

The Lp(a) level is a causal risk factor for ASCVD. It is important whether it is high Lp(a) levels or the number of kringle IV (K-IV) repeats [apo(a) isoform size] that account for the increased risk of ASCVD associated with increased Lp(a) levels. The results of a study /14/ suggest that absolute levels of Lp(a), rather than apo(a) isoform size, are the main determinant of ASCVD risk.

References

1. Langer C, Tambyrayah B, Nowak-Gottl U. Testing for apolipoprotein(a) phenotype using isoelectric focussing and immunoblotting technique. Methods Mol Biol 2013, 992: 407–12.

2. Mach F, Baigent C, Catapano AL, Koskinas KC, Casula M, Badimon L, et al.ESC Scientific Document Group 2019 ESC/EA guidelines for the management of dyslipidemias to reduce cardiovascular risk. Eur Heart J 2020; 41: 111–88.

3. Crea F. Lipoprotein (a) in atheroscleotic cardiovascular disease and in aortic stenosis: an emerging therapeutic target. Eur Heart J 2022; 43 (9): 3815–18.

4. Clarke R, Peden JF, Hopewell JC, Kyriakou T, Goel A, Heath SC, Parish S, et al. Genetic variants associated with Lp(a) lipoprotein level and coronary disease. N Engl J Med 2009; 361: 2518–28.

5. Emerging Risk Factors Collaboration. Erqou S, Kaptoge S, Perry PL, Di Angelantonio E, Thompson A, White IR, et al. Lipoprotein (a) concentration and the risk of coronary heart disease, stroke and nonvascular mortality. JAMA 2009; 302: 412–23.

6. Willeit P, Ridker PM, Nestel PJ, Simes J, Tonkin AM, Pedersen TR, et al. Baseline and on-statin treatment lipoprotein (a) levels for prediction of cardiovascular events: individual patient data meta-analysis of statin outcome trials. Lancet 2018; 13; 392 (10155) 1311–20.

7. Kostner KM, März W, Kostner GM. When should we measure lipoprotein (a)? Eur Heart J 2013; 34: 3268–76.

8. Clarke R, Peden JF, Hopewell JC, Kyriakou T, Goel A, Heath SC, Parish S, et al. Genetic variants associated with Lp(a) lipoprotein level and coronary disease. N Engl J Med 2009; 361: 2518–28.

9. Stefanutti C, Julius U, Watts GF, Harada-Shiba M, Schettler VJ, Soran H, et al. Toward an international consensus – Integrating lipoprotein apheresis and new lipid-lowering drugs. J Clin Lipidol 2017; 11: 858–71.

10. Furlan L, Azcarate P, Redberg F. Sceening for lipoprotein (a) levels in persons at low risk for cardiovascular disease.JAMA 2021; 181 (4): 539.

11. Scharnagl H, Stojakovic T, Dieplinger B, Dieplinger H, Erhart G, Kostner G, et al. Comparison of lipoprotein (a) serum concentrations measured by six commercially available immunoassays. Atherosclerosis 2019; 289: 206–13.

12. Siekmeier R, Scharnagl H, Kostner GM, Grammer T, Stojakovic T, März W. Lipoprotein (a) – Struktur, Epidemiologie und Funktion. J Lab Med 2007; 31: 109–24.

13. Marcovina SM, Albers JJ, Gabel B, Koschinsky ML, Gaur VP. Effect of the number of apolipoprotein (a) Kringle 4 domains on immunochemical measurements of lipoprotein (a). Clin Chem 1995; 41: 246–55.

14. Hopewell JC, Seedorf U, Farrall M, Parish S, Kyriakou T, Goel A, et al. Impact of lipoprotein (a) levels and apolipoprotein (a) isoform size on risk of coronary heart disease. J Intern Med 2014; 276: 260–8.

15. Marcovina SM, Koschinsky ML, Albers JJ, Skarlatos S. Report of the National Heart, Lung, and Blood Institute Workshop on Lipoprotein (a) and Cardiovascular Disease: recent advances and future directions. Clin Chem 2003; 49: 1785–96.

16. Greenland P, Alpert JS, Beller GA, Benjamin EJ, Budoff MJ, Fayad ZA, et al. 2010 ACCF/AHA guideline for assessment of cardiovascular risk in asymptomatic adults: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation 2010; 122e: e584–e636.

17. AACE Lipid and Atherosclerosis Guidelines. Endocr Pract 2012; 18, suppl 11: 1–78.

18. Nordestgaard BG, Chapman MJ, Ray K, Boren J, Andreotti F, Watts GT, et al. European Athersclerosis Society consensus P. Lipoprotein (a) as a cardiovascular risk factor: current status. Eur Heart J 2010; 31: 2844–53.

4.6 Apolipoproteins

The movement of lipids through plasma is mediated by lipoprotein particles. Each particle typically contains a set of apolipoproteins that provide structural integrity for the complex. Apolipoproteins are synthesized in the liver and besides albumin and immunoglobulins, apolipoproteins are the third largest protein fraction in plasma.

Functions of the apolipoproteins include:

Ligand for the interaction of lipoprotein particles with receptors of the cell membrane
Activation or inhibition of enzymes of lipid metabolism.

Since apolipoproteins have an important function in lipid metabolism, their measurement should be important markers in risk prediction of atherosclerotic cardiovascular disease (ASCVD). However apolipoprotein determination provides no benefit beyond traditional lipid markers e.g., LDL-C and Lp(a), in prevention of ASCVD /1/.

The biochemical properties and functions of apolipoproteins and their association with diseases are shown in Tab. 4.6-1 – Biochemical properties and functions of apolipoproteins.

4.6.1 Apolipoprotein B (apo B)

There are two isoforms of apo B in plasma: apo B-100 and apo B-48. The first isoform is synthesized in the liver and is the structural protein of VLDL and LDL. The latter is produced in the mucosa of the small intestine and is the structural protein of the chylomicrons. The plasma concentration of apo B-48 barely exceeds 5% of total apo B.

In fasting plasma, 90–95% of apo B is present in LDL and 5–10% in VLDL. Consequently, the apo B concentration is a good indicator of the LDL concentration. Since each LDL particle contains only one apo B molecule, the apo B concentration is also an indicator of the number of LDL particles.

4.6.1.1 Indication

ApoB should be considered as an alternative risk marker, especially in combined hyperlipidemia, e.g., diabetes, metabolic syndrome, ASCVD /1/.

4.6.1.2 Method of determination

Immunonephelometric or immunoturbidimetric immunoassays /2/.

4.6.1.3 Specimen

Serum: 1 mL

4.6.1.4 Reference interval

Apolipoprotein		Interval	50. Percentile
Apo B* /2/	Male	0.66–1.44	1.03
Apo B* /2/	Female	0.60–1.41	0.93

Data expressed in g/L; interval values are 5th and 95th percentiles.

* Related to the IFCC reference preparation SP3-07 (WHO International Reference Reagent) for apolipoprotein B.

4.6.1.5 Clinical significance

The dyslipidemia management in patients with atherosclerotic cardiovascular disease is based on the lipid profile total cholesterol, HDL-cholesterol, LDL-cholesterol, and total triglycerides. In elderly populations with several comorbidities i.e.,obesity, diabetes, chronic kidney disease the traditional lipid profile is a weak predictor of atherosclerotic vascular disease (ASCVD). The IFCC working group on apolipoproteins by mass spectrometry has established a scientific and technical basis for SI-standardization of 7 relevant apolipoproteins to facilitate the future adoption of clinical apolipoprotein profiling for precision medicine, and development of strategies for accurate testing /8/.

Apo B is the major apolipoprotein of the atherogenic lipoprotein families LDL, IDL, and VLDL. The level of apo B is a good representative of the number of circulating LDL particles, since apo B accounts for 95% of the total protein content of LDL and each LDL particle contains only one apo B molecule /3/. Prospective studies have shown that the number of LDL particles is an important factor in atherogenesis and the concentration of apo B is a more representative biomarker of the particle number than the concentration of LDL-C. Apo B has been shown in several prospective studies to be equal to LDL-C in risk prediction of ASCVD.

The major disadvantages of apo B are:

that it is not include in algorithms for calculation of global risk of ASCVD
it has not been a pre-defined treatment target in controlled trials
it has not been evaluated as a primary target in statin trials.

4.6.1.6 Comments and problems

Routine assays measure either total apo B (apo B-100 plus Apo B-48) or apo B-100 alone.

Reference material

International reference material SP3-07, a human serum preparation developed by the IFCC and endorsed by WHO. Contains apo B 1.22 g/L (3.95 mmol/L) /2/.

Stability

Serum can be stored for at least 3 days at 4 °C. In the presence of antibiotics and antioxidants, apo B will remain stable for at least 6 months at –20 °C. Frozen storage at –80 °C is preferable.

4.6.2 Apolipoprotein A-1 (Apo A-1)

Apo A-1 is the main structural protein of HDL. It is synthesized in the intestinal mucosa and in the liver and induces the efflux of cholesterol from cells. Apo A-1 activates the enzyme lecithin-cholesterol acyl transferase.

4.6.2.1 Indication

Detection of atherosclerosis risk, in particular as part of the apoB/apoA-I ratio.
Characterization of rare HDL deficiency syndromes.

4.6.2.2 Method of determination

Immunonephelometric or immunoturbidimetric immunoassays /4/.

4.6.2.3 Specimen

Serum: 1 mL

4.6.2.4 Reference interval

Apolipoprotein		Interval	50. Percentile
Apo A-I* /4/	Male	1.02–1.75	1.32
Apo A-I* /4/	Female	1.15–2.07	1.51

Data expressed in g/l; interval values are 5th and 95th percentiles.

* Related to the IFCC First International Reference Material for apo A-I.

4.6.2.5 Clinical significance

The concentration of apo A-I correlates well with the level of HDL particles and the HDL-C concentration /5/. If HDL-C cannot be measured in serum that is turbid due to hypertriglyceridemia, apo A-I can be determined as an alternative.

The ratio of apo B/apo A-1 combines the risk information of apo B and apo A-1 and may be recommended as an alternative analysis for atherosclerotic cardio vascular disease (ASCVD) screening /1/.

If HDL cholesterol is very low or not detectable, the concentration of apo A-I can provide etiological clues, although without allowing a diagnosis. With apo A-I deficiency, apo A-I is not detectable. In patients with Tangier disease apo A-I is often below 10 mg/L, and with LCAT deficiency it is generally in the range of 40–50 mg/L.

4.6.2.6 Comments and problems

Stability

4.6.3 Apolipoprotein E (Apo E)

Apo E mediates the uptake of chylomicron remnants and VLDL remnants in the liver and is involved in the transformation into LDL particles. Apo E is a ligand for the LDL receptor. Apo E shows a genetic polymorphism and occurs in three alleles (epsilon 2, epsilon 3, and epsilon 4) which encode six phenotypes: Apo E_2/2, E_2/3, E_2/4, E_3/3, E_3/4, E_4/4.

The binding of apo E to the LDL receptor is an important mechanism for clearing remnants from the circulation.

4.6.3.1 Indication

Diagnosis of hyperlipoproteinemia type III, in particular apo E2 homozygosity.

4.6.3.2 Method of determination

Quantification of apo E

Immunonephelometry, immunoturbidimetry.

Phenotyping of apo E

Immunoblotting following isoelectric focusing.

Genotyping of apo E

DNA hybridization of allele-specific PCR.

4.6.3.3 Specimen

Serum: 1 mL

4.6.3.4 Reference interval

Apolipoprotein	Median	Percentiles 2.5 and 97.5
Apo E	74	10–389 /6/
Apo E/Apo B	0.050	0.007–0.178 /6/

Data expressed in mg/L

4.6.3.5 Clinical significance

The determination of apo E and the apo E/apo B ratio are criteria for the diagnosis of hyperlipoproteinemia type III. This type of hyperlipoproteinemia is suspected if cholesterol and triglyceride levels are between 250 mg/dl and 800 mg/dL and lipoprotein electrophoresis detects a broad β-band. The band is sensitive, but not very specific. Clinically, 50% of cases present with cutaneous xanthomas.

Hyperlipoproteinemia type III is caused by a dysfunctional isoform of apo E, a glycoprotein with a molecular weight of 34 kDa. Over 90% of type III patients are homozygous for the E2 isoform. Lipoprotein particles with this isoform do not bind to the apolipoprotein B, E receptors, resulting in delayed clearance of chylomicron and VLDL remnants from the plasma. A study /6/ showed an apo E/apo B ratio of 0.056 ± 0.037 in controls and of 0.197 ± 0.073 in type III patients.

However, the apo E_2/2 phenotype is far more common than hyperlipoproteinemia type III. Therefore, other genetic, metabolic or environmental factors (e.g., diabetes mellitus, hypothyroidism or LDL receptor mutation) play a significant role in the severity of hyperlipoproteinemia type III.

References

1. The task force for the management of dyslipidemias of the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS). ESC/EAS Guidelines for the management of dyslipidaemias. European Heart J 2011; 32: 1769–1818.

2. Contois JH, McNamara JR, Lammi-Keefe CJ, Wilson PWF, Massov T, Schaefer EJ. Reference intervals for plasma apolipoprotein B determined with a standard commercial immunoturbidimetric assay: results from the Framingham Offspring study. Clin Chem 1996; 42: 515–23.

3. Contois JH, McConnell JP, Sethi AA, Csako G, Devaraj S, Hoefner DM, Warnick GR. Apolipoprotein B and cardiovascular disease risk: position statement from the AACC Lipoproteins and Vascular Disease Division Working Group on Best Practices. Clin Chem 2009; 55: 407–19.

4. Contois JH, McNamara JR, Lammi-Keefe CJ, Wilson PWF, Massov T, Schaefer EJ. Reference intervals for plasma apolipoprotein A-I determined with a standard commercial immunoturbidimetric assay: results from the Framingham Offspring Study. Clin Chem 1996; 42: 507–14.

5. März W, Von Eckardstein A. Laboratoriumsdiagnostik bei Fettstoffwechselstörungen. J Lab Med 2001; 25: 433–48.

6. März W, Feussner G, Siekmeier R, Donnerhak B, Schaaf L, Ruzicka V, Groß W. Apolipoprotein E to B ratio: a marker of Type III hyperlipoproteinaemia. Eur J Clin Chem Clin Biochem 1993; 31: 743–7.

7. Riesen WF. Fettstoffwechsel. In Thomas L, ed. Labor und Diagnose. Frankfurt 2008; TH-Books, 225–48.

8. Cobbaert CM, Althaus H, Begcevic-Brkovic I, Ceglarek U, Coassin S, Delatour V, et al. Towards an SI-traceable reference measurement system for seven apolipoproteins using bottom-up quantitative proteomics: conceptual approach enabled by cross-disciplinary/cross-sector collaboration. Clin Chem 2021; 67 (3): 478–89.

4.7 Enzymes of lipoprotein metabolism

Intravenous administration of heparin causes the endothelium to release the triglyceride lipases lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL). Both enzymes act as clearing factors and ensure that the tissues are supplied with free fatty acids via the hydrolysis of triglyceride-rich lipoprotein particles and HDL.

4.7.1 Lipoprotein lipase (LPL)

LPL (EC 3.1.1.34), an enzyme with glycerol ester hydrolase activity, is located on the endothelium surfaces of extrahepatic capillaries, in particular in adipose tissue and skeletal and heart muscles. The enzyme catalyzes the hydrolysis of lipoprotein associated triglycerides, yielding free fatty acids (FFS) for supply of the tissues.

LPL is not actually produced by the endothelia, but by adipocytes and muscle cells, and is transported to the endothelia of the vessels via transcytosis on the luminal side. LPL is activated by apolipoprotein C-II and regulated mainly by insulin. In the postprandial phase the LPL activity of adipose tissue is high in order to store the FFS produced after food ingestion in the tissues. In the post absorptive phase it is high in muscle in order to provide sufficient FFS to supply the myocytes with energy under load /1/.

4.7.1.1 Indication

Clinical symptoms such as recurrent upper abdominal pain (pancreatitis), eruptive xanthomas, hepatosplenomegaly, lipemia retinalis, in particular in children.

4.7.1.2 Method of determination

The lipolytic activity of LPL is triggered by injection of 60–100 U of heparin/kg of body weight. Blood is collected 10 min (15 min) after injection. LPL activity is measured in the amount of radio labeled fatty acids released from glycerol-tri-[1-14C] oleate substrate per time unit following inhibition of HTGL with sodium dodecyl sulfate (SDS) /2/.

4.7.1.3 Specimen

EDTA plasma, cool and centrifuge, deep-freeze and transport to laboratory: 2 mL

4.7.1.4 Clinical significance

LPL activities of less than 25% of normal values indicate LPL deficiency which, however, needs to be confirmed by molecular genetic analysis. In heterozygous mutation carriers, LPL activity is reduced to half of normal values and triglycerides are only slightly to moderately elevated.

4.7.1.5 Comments and problems

Stability

At least 3 months at –70 °C.

4.7.2 Hepatic triglyceride lipase

HTGL (EC 3.1.1.3) is located on the capillary endothelia of liver cells and, like LPL, is released by heparin. Its function is the hydrolysis of triglyceride-rich lipoproteins and the degradation of HDL particles, in particular HDL₂. It is measured analogously to LPL, but LPL is inactivated by NaCl 1.0 mol/L. Genetic HTGL deficiency is very rare.

4.7.3 Lecithin-cholesterol acyl transferase (LCAT)

Two thirds of cholesterol in plasma is esterified with free fatty acids. This is mediated by LCAT (EC 2.3.1.43), which is synthesized in the liver. The cofactor is apo A-I. LCAT plays a key role in the metabolism of lipoproteins, in particular HDL.

4.7.3.1 Indication

Suspected LCAT deficiency or fish-eye disease, if the following symptoms are present: low HDL, cloudy cornea, renal dysfunction, hemolytic anemia, xanthomas.

4.7.3.2 Method of determination

The transfer of fatty acids (e.g., oleic acids) to radioactive cholesterol from phosphatidylcholine is measured. This was either equilibrated with endogenous lipoproteins (cholesterol esterification rate) or obtained in the form of apo A-I, phosphatidylcholine and cholesterol-containing artificial HDL, to which exogenous plasma was added (LCAT activity in a narrower sense).

4.7.3.3 Specimen

Plasma, cool and centrifuge, deep-freeze and transport to laboratory: 2 mL

4.7.3.4 Clinical significance

In classic LCAT deficiency, LCAT can neither esterify cholesterol equilibrated with endogenous lipoproteins nor unesterified radioactive cholesterol added in the form of exogenous HDL.

In fish-eye disease (partial LCAT deficiency), this endogenous cholesterol esterification rate is normal, but the LCAT activity proper measured with exogenous proteoliposomes is reduced.

4.7.3.5 Comments and problems

Stability

1 week at 4 °C, long-term storage requires a temperature of –20 °C.

References

1. Pilz S, März W. Free fatty acids as a cardiovascular risk factor. Clin Chem Lab Med 2008; 46: 429–34.

2. Assmann G, Jabs HU. Lipoproteinlipase (Postheparinlipase). In: Bergmeyer HU, ed. Methods of enzymatic analysis Vol IV. Weinheim 1984; Verlag Chemie: 42–51.

4.8 Low-density lipoprotein receptor

The low-density lipoprotein receptor (LDL receptor) is a glycoprotein of the cell membrane consisting of 839 amino acids. The receptor binds apo B-100 and Apo E. The receptor is synthesized as a precursor protein in the endoplasmic reticulum and transported to the cell membrane within 45 min. After the binding of LDL, an endocytotic vesicle is formed within 3 to 5 min. which transports LDL to the inside of the cell where it is then released.

The synthesis of the LDL receptor is encoded by the LDLR gene located on chromosome 19. More than 1,000 mutations of the LDLR gene have been identified in patients with familial hypercholesterolemia. The resulting functional defects of the receptor are differentiated into 5 classes /1/:

Defect in the expression of protein synthesis
Impaired transport of the receptor from the endoplasmic reticulum to the cell membrane
Defective receptor binding of apo B and apo E
Defective internalization of the receptor
Defective recycling of the receptor.

4.8.1 Indication

Type II hyperlipoproteinemia with tendon xanthomas and suspected familial hypercholesterolemia.

4.8.2 Method of determination

Determination of the binding of radio labeled LDL particles to cultured fibroblasts in the patient.

An alternative method is the flow cytometric measurement of LDL receptors on the surface of monocytes as well as sequencing of the LDL receptor gene.

4.8.3 Specimen

Skin biopsy sample from which fibroblasts are derived.

EDTA blood: 5 mL

4.8.4 Clinical significance

Homozygous patients with familial hypercholesterolemia exhibit only little binding activity compared to normal controls. Heterozygous mutation carriers, in contrast, exhibit 50–60% binding activity.

References

1. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science 1986; 232: 34–47.

4.9 Pathophysiology of lipoprotein metabolism

Lipids include free fatty acids, neutral glycerides, free cholesterol, cholesterol esters, glycerophospholipids, sphingolipids, and glycolipids. None of these lipids have significant aqueous solubility and so their carriage from blood into and through tissues acquires an association with proteins. A lipoprotein transport system and lipid transfer system are differentiated /1/.

4.9.1 Lipid transport system

Proteins with which the lipids associate are plasma lipoproteins, such as chylomicrons, chylomicron remnants, VLDL, and LDL. Although these lipoproteins are remodeled in the plasma by transfer proteins, their uptake into cells and tissues is mediated by specific receptors e.g., LDL receptor. The receptors primarily recognize protein of the lipoprotein particle as ligand /1/. Refer also to Tab. 4.9-1 – Pathophysiology of lipoprotein particles /2/.

4.9.2 Lipid transfer system

The lipid transfer proteins primarily recognize lipid, while the receptors recognize protein as the primary ligand. Transfer proteins may be categorized into two groups /1/:

Proteins that mediate the assembly and metabolisms of lipoproteins
Proteins which are engaged in the distribution of lipids within the cell and that may modify the lipid composition of individual cellular membranes

4.9.3 Transfer of lipid from diet to tissue

Lipids from diet, mostly triglycerides and cholesterol esters in the intestinal lumen are hydrolyzed yielding free fatty acids and free cholesterol. These products enter the enterocyte and are re-sythesized into triglycerides and cholesterol esters. The triglycerides are either packaged into lipoprotein particles containing the apolipoprotein Apo B48, or are stored as droplets that may later be mobilized for the assembly of chylomicrons /1/. The coupling of triglycerides with apo B48 is mediated by the microsomal triglyceride transfer protein (MTTP). The surface of the chylomicron is coated by a monolayer of phospholipid that is mediated by the phospholipid transfer protein (PLTP) /1/:

Free cholesterol in the intestinal lumen is released by the hydrolysis of dietary cholesterol ester and taken up into the enterocyte mediated by the Niemann-Pick C1 Like-1 (NPC1L1) transfer protein. The enterocyte is also a source of HDL particles. About 30% of the HDL particles in plasma are generated via the action of the ABCA1 transporter providing glycerphospholipid and cholesterol to the nascent HDL particle /1/.

Circulating lipoprotein particles are complexes including a hydrophobic core of triglyceride and/or cholesterol ester. The surface contains amphipathic phospholipids, free cholesterol, apo B or a set of exchangeable apoproteins. Lipoproteins transport dietary fats from the intestine to the liver and the processed particles to the peripheral tissues. Before entering the cells plasma lipoprotein particles are remodeled by the exchange of their surface components. The core components are remodeled mediated by the cholesterol ester transfer protein (CETP). The CETP mediates the exchange of cholesterol ester for triglycerides between HDL and VLDL and LDL. Individuals lacking CETP have elevated levels of HDL-C and low levels of LDL-C /3/.

The metabolism of lipoprotein particles in blood is divided into an exogenous and endogenous pathway and reverse lipid transport.

4.9.3.1 Exogenous pathway

This transport takes place in the postprandial phase, starting in the small intestine (Fig. 4.9-1 – Exogenous and endogenous pathway of lipoprotein metabolism). After dietary lipids, mainly triglycerides, have been hydrolyzed, they are absorbed by the mucosa of the small intestine and resynthesized by mucosa cells and packaged into chylomicrons together with a set of apolipoproteins (A-I, A-IV, B, C, E).

Bypassing the liver, the chylomicrons pass into blood via the thoracic duct. In blood they are present only during the post- prandial phase i.e., for up to 4 h following food ingestion. The presence of apo C-III in the chylomicrons prevents premature interaction of these triglyceride-rich particles with the liver.

The triglycerides of the chylomicrons are consecutively hydrolyzed to chylomicron remnants in the tissues, in particular adipose tissue and muscle, by lipoprotein lipase (LPL) at the surface of the capillary endothelium. The activation of LPL is mediated by apo C-II of the chylomicrons. The hydrolyzed fatty acids are available as energy sources for muscle and adipose tissue (Fig. 4.9-2 – Metabolism of triglyceride-rich chylomicrons and VLDL by heparin-sensitive lipoprotein lipase in blood).

The resulting chylomicron remnants are cleared by the liver through LDL receptor mediated endocytosis. The binding to the receptor is mediated by apo E.

4.9.3.2 Endogenous pathway

In the fasting state (at least 8–9 h after food ingestion), the endogenous pathway supplies the tissues with triglycerides and cholesterol (Fig. 4.9-1 – Exogenous and endogenous pathway of lipoprotein metabolism). The liver releases triglyceride-rich VLDL particles containing apo B-100 and apo E into the blood. Like the chylomicrons, the triglycerides of VLDL are also hydrolyzed by the enzyme LPL, leaving VLDL remnants (intermediate-density lipoproteins, IDL). Due to the apolipoprotein E, one part of the IDL bind to the LDL receptor, mainly of the liver, and enter the hepatocytes. The other part is remodeled into LDL-particles and taken up by the liver via the LDL receptor. The ligand for the LDL receptor is apo B-100. About two thirds of the cholesterol in the blood is transported by LDL particles, and three quarters are converted to bile acids following absorption by the liver via the LDL receptor, or directly excreted into the gall bladder. The remainder of dietary cholesterol or synthesized daily serves for steroid hormone production and membrane synthesis.

4.9.4 Reverse cholesterol transport

The reverse transport of excess cholesterol from the peripheral tissues, in particular macrophages, to the liver starts with the synthesis of pre-β HDL particles, in particular in the liver and the enterocytes of the small intestine. The pre-β HDL particles consist of apolipoprotein A-I and phospholipids. They are loaded with phosphatidylcholine and free cholesterol from cell membranes by the ATP-binding cassette transporter A1 (ABCA1) (Fig. 4.9-3 – Reverse transport of cholesterol by HDL particles). Cholesterol is then esterified in the resulting discoidal HDL, catalyzed by lecithin-cholesterol acyltransferase (LCAT). This results in small dense particles (HDL₃) which grow in size as they are loaded with lipids from cells and lipoproteins (HDL₂). Part of the HDL are taken up by the liver via the scavenger receptor BI, while another part is transferred to triglyceride-rich lipoprotein particles mediated by cholesterol ester transfer protein (CETP), and the remnants or LDL are absorbed by the liver.

References

1. Getz GS. Thematic review series on lipid transfer proteins. J Lipid Res 2018; doi: 10.1194%2Fjlr.R084020.

2. Kostner GM, März W. Zusammensetzung und Stoffwechsel der Lipoproteine. In: Schwandt P, Richter WO, Parhofer K (eds). Handbuch der Fettstoffwechselstörungen. Stuttgart 2001; Schattauer: 3–57.

3. Mabuchi H, Nohara A, Inazu A. Cholesterylester transfer protein (CETP). Deficiency and CETP inhibitors. Mol Cells 2014; 37: 777–84.

4. Rashid S, Uffelman KD, Lewis GF. The mechanism of HDL-lowering in hypertriglyceridemic, insulin-resistant states. J Diabetes Complict 2002; 16: 24–8.

5. Contois JH, McConnell JP, Sethi AA, Csako G, Devaraj S, Hoefner DM, Warnick GR. Apolipoprotein B and cardiovascular disease risk: position statement from the AACC Lipoproteins and Vascular Disease Division Working Group on Best Practices. Clin Chem 2009; 55: 407–19.

6. Charlton-Menys, Betteridge DJ, Colhoun H, Fuller J, France M, Hitman GA, et al. Targets of statin therapy. LDL cholesterol, non-HDL-cholesterol, and apolipoprotein B in type 2 diabetes in the Collaborative Atorvastatin Diabetes Study (CARDS). Clin Chem 2009; 55: 473–80.

7. Ferns G, Keti V. HDL-cholesterol modulation and its impact on the management of cardiovascular risk. Ann Clin Biochem 2008; 45: 122–8.

8. Keary JF, Curfman GD, Jarcho JA. A pragmatic view of the new cholesterol treatment guidelines. N Engl J Med 2014; 370: 275–8.

9. Braun JEA, Severson DL. Regulation of the synthesis, processing and translocation of lipoprotein lipase. Biochem J 1992; 287: 337–47.

Table 4.1-1 Upper reference values and recommended ranges of lipids /2/

LDL cholesterol	Assessment
< 100 (2.59)	Optimal
100–129 (2.59–3.34)	Near or above optimal
130–159 (3.37–4.12)	Borderline High
160–189 (4.12–4.90)	High
≥ 190 (4.92)	Very high
HDL cholesterol
< 40 (1.04)	Low
≥ 60 (1.55)	High
Total cholesterol
< 200 (5.18)	Desirable
200–239 (5.18–6.19)	Borderline high
≥ 240 (6.22)	High
Triglycerides
< 150 (1.69)	Normal
150–199 (1.69–2.25)	Borderline
200–399 (2.26–4.51)	High
≥ 400 (4.52)	Very high

The upper reference values for children are according to the National Cholesterol Education Program 1992: LDL-C < 130 mg/dL (3.36 mmol/L), triglycerides < 200 mg/dL (2.26 mmol/L).

Table 4.1-2 Genetic dyslipidemias

Clinical and laboratory findings

Polygenic hypercholesterolemia

Polygenic hypercholesterolemia is the most common cause of elevated total cholesterol. LDL-C elevations are 140–300 mg/dL (3.63–7.77 mmol/l). The condition is caused by 12 LDL-C raising altered genes (polygenic) that might be involved /11/. If a patient has inherited several of these altered genes, each may have a small additive effect raising the serum cholesterol level. The 12 single nucleotide polymorphism LDL-C gene score might be a good tool for distinguishing between polygenic and monogenic hypercholesterolemia /12/. Polygenic hypercholesterolemia is the most common cause of primary (hereditary) hypercholesterolemia, even with familial presentation and familial hypercholesterolemia clinical diagnosis, when the genetic screening for pathogenic variants of LDLR, APOB and PCSK9 are negative, and the number of genes involved in these phenotypes seems low enough to study them in certain circumstances /12/.

Heterozygous Familial hypercholesterolemia (HeFH) /13/

HeFH also referred to as heterozygous FH, refers to familial hypercholesterolemia resulting from the inheritance of mutations in one allel of genes regulating LDLR, APOB and PCSK9. HeFH is a relatively common disorder (prevalence 1 : 200 to 1 : 250).

LDLR and APOB code for apoB protein that acts as a ligand for the LDL receptor, PCSK9 codes the proprotein convertase subtilisin/kexin 9 that regulates LDL receptor cycling.

The major determinants in that system are:

APOB 1–5%. Penetrance for HeFH can be incomplete in individuals with a heterozygous APOB pathogenic variant
LDLR 80–85%. LDLR (LDL receptor gene) with more than 3,000 variants is one of the key genes responsible of FH development. LDLR mutations are associated with increased concentration of LDL cholesterol (LDL-C), as absorption of LDL-C from the blood is reduced.
PCSK9 0.2%. The gene encoding proprotein convertase subtilisin-kexin type 9 (PCSK9) is a critical regulator of cholesterol metabolism through its interaction with the hepatic LDL receptor (LDLR). Circulating PCSK9, a serin protease, binds to the extracellular EGF-like domain of LDLR, followed by endocytosis and lysosomal degradation of the PCSK9-LDLR complex. Consequently, PCSK9 prevents recycling of the LDLR to the cell surface, thereby attenuating LDL clearance. PSCK9 is present in human plasma and higher circulating levels predict lower catabolism of apoB, the main constituent of LDL. Statins and PCSK9 inhibitors both accelerate apoB catabolism, but only PCSK9 inhibition attenuates apoB production. PCSK9 inhibitors decrease incidence of cardiovascular disease. Mutations of the gene PCSK9 cause autosomal dominant hypercholesterolemia. Penetrance is approximately 90% in individuals heterozygous for the c.381 T>A (p.Ser 127 Arg) pathogenic variant, lower in individuals heterozygous for the p.Asp 374Tyr pathogenic variant, and unknown for other PCSK9 variants, respectively /35/.
Unknown 20–40%

A large proportion of individuals with the clinical diagnosis of HeFH do not have a causative mutation in LDLR, APOB and PCSK9. Typically, HeFH individuals with a positive mutation have higher LDL-C levels and a higher presence of tendon xanthomas than individuals with clinical diagnosis but without mutations in these genes.

Clinical findings

HeFH is characterized by severely elevated LDL-C concentrations that lead to atherosclerotic plaque deposition in the coronary artery and proximal aorta at an early age.

Physical findings: xanthomas (patches of yellowish cholesterol buildup) around the eyelids, tendons of the elbows, hands, knees and feet. Corneal arcus (white, gray, or blue opaque ring in the corneal margin)
History of premature coronary artery disease: angina pectoris, myocardial infarction, peripheral vascular disease
Family history: familial hypercholesterolemia, high concentration of LDL-C, early onset i.e., coronary artery disease, especially myocardial infarction at age below 50 years. median age of 47 years and in 29% of women with a median age of onset of 55 years /14/. More than 61% of adults with FH have at least one modifiable ASCVD risk factor. Die ASCVD is diagnosed in 47% of men with FH, with a median age of 47 years and in 29% of women with a median age of onset of 55 years /14/.

Establishing diagnosis

Three formal diagnostic criteria for HeFH which rely on the diagnostic criteria (e.g., extreme hypercholesterolemia, history of premature coronary artery disease, findings on physical examination) are used:

US MEDPED program
UK Simon Broome Familial Hypercholetrolaemia Registry
Dutch Lipid Clinic Network

Laboratory findings

FH should be suspected in individuals with the following findings:

Adults (untreated): total cholesterol > 310 mg/dl (8.03 mmol/l), LDL-C > 190 mg/dL (4.92 mmol/l)
Children/adolescents (untreated): total cholesterol > 230 mg/dl (5.96 mmol/l), LDL-C > 130 mg/dL (3.37 mmol/l)
Elevation of two consecutive LDL-C concentrations is often recommended to confirm the diagnosis
Identification of a pathogenic variant in a gene known to be associated with FH is the gold standard for diagnosis. Molecular testing approaches are serial single-gene testing or the use of a multi-gene panel that includes LDLR, APOB and PCSK9.

Homozygous Familial hypercholesterolemia (HoFH) /30/

HoFH is a rare and aggressive disorder, resulting from the inheritance of mutations in the two alleles of genes regulating the low-density lipoprpotein receptor (LDLR) function. HoFH is caused by variants in the LDLR gene in more than 90% of cases and less frequently in APOB, PCSK9 and LDLRAP1 genes. HoFH is a rare disorder (prevalence 1 : 300,000). LDL-cholesterol concentrations are high (> 500 mg/dL, 12.9 mmol/L). Cutaneous and and tendon xanthomas are detected before the age of about 10 years. Both parents have heterozygous familial hypercholesterolemia and high cholesterol concentrations. HoFH patients are characterized by high frequency and early onset (children of about 10 years with cardiovascular disease and aortic and supraaortic valve disease). The major determinant of survival is on-treatment of total cholesterol. The survival is 6.2 times lower in cases with total cholesterol > 585 mg/dL (15.1 mmol/L) than those with total cholesterol < 314 mg/dL (8.1 mmol/l). Therapeutic LDL cholesterol goals are: < 70 mg/dL (1.8 mmol/l) if clinical cardiovascular disease or type 2 diabetes, < 100 mg/dL (2.5 mmol/L) in adults, < 135 mg/dL (3.5 mmol/L) in children.

Conditions with clinical findings similar to those of FH /13/

27-hydroxylase deficiency

The cerebrotendinous xanthomatosis is characterized by xanthomas.

Clinical findings: dementia ataxia and cataracts

Laboratory finding: normal LDL-C.

Familial hyperlipoproteinemia type III

Refer to column familial hyperliporoteinemia type III (familial dyslipoproteinemia)

Sitosterolemia

Biallelic pathogenic variants in either ABCG5 or ABCG8 are causative. Autosomal recessive inheritance

Laboratory findings: normal or mildly elevated LDL-C.

Polygenic hypercholesterolemia

Refer to column polygenic hypercholesterolemia

Extremely elevated lipoprotein a Lp(a) /15/

The disorder is caused by variants in the number of kringle IV type 2 repeats in LPL. Autosomal dominant inheritance.

Clinical findings: family history or personal history of cardiovascular disease.

Laboratory findings: very elevated LDL-C concentration. High Lp(a) levels are appreciated to synergistically increase risk in individuals with FH.

Conditions with laboratory findings similar to those of FH /13/

Hypercholesterolemia secondary to obesity, diabetes mellitus, hypothyroidism, kidney disease or drugs (refer to Tab. 4.1-3 – Multifactorial dyslipidemia)

Autosomal recessive hypercholesterolemia

Hypercholesterolemia is caused by biallelic pathogenic variants LDLRAP1.

Laboratory findings: LDL-C > 400 mg/dL (10.36 mmol/l). Heterozygotes have normal LDL-C concentration

Familial combined hyperlipidemia

Refer to column Familial combined hyperlipidemia

Familial hyperliporoteinemia type III (familial dyslipoproteinemia)

Apo E occurs in various isoforms. Apo E3 is the wild-type form (normal type), apo E4 is a common variant, and apo E2 is the least common form. Apo E are encoded by the alleles E4, E3 and E2, and therefore the following occur in plasma /16/:

The homozygous phenotypes E2/2, E3/3 and E4/4.
The heterozygous phenotypes E2/3, E2/4 and E3/4.

The allelic forms apo E4 and apo E2 differ from the wild type in the amino acids 112 and 158: the wild type contains cysteine at position 112 and arginine at position 158. Apo E2 contains cysteine at both positions, and apoE4 has arginine at both positions. Apo E4 is associated with elevated LDL-C and an increased risk of Alzheimer’s disease. Apo E2 is associated with type III hyperlipoproteinemia (HLP). The incidence of the apo E2/2 phenotype is about 1 : 100. Since only about 2% of these individuals develop type III hyperlipoproteinemia, the prevalence is only 1 : 5000.

Apo E is a protein of chylomicrons and VLDL as well as of the remnants and of HDL. It is produced by the liver, the macrophages and the astrocytes of the central nervous system. Its main function is that of ligand for the hepatic lipoprotein receptors for the uptake of chylomicrons and IDL remnants. Apo E2 and apo E4 carriers can have reduced binding capacity to the receptors. As a result, remnants of triglyceride-rich lipoproteins accumulate in plasma of individuals with type III HLP.

Clinical findings: symptoms manifest from age 20 onwards. Characteristic manifestations include palmar xanthomas (xanthoma striata palmaris), tuberous and tubero-eruptive xanthomas. These patients are at high risk for atherosclerosis and many have cardiovascular disease or peripheral arterial vascular occlusions at diagnosis. The manifestation of type III HLP requires several other conditions in addition to apoE2/2 homozygosity (e.g., diabetes mellitus, hypothyroidism, LDL receptor mutations, and hemochromatosis).

Laboratory findings: serum is turbid, cholesterol is elevated in the range of 300–600 mg/dL (7.8–15.5 mmol/L), triglycerides are also in a range of 300–600 mg/dL (3.4–6.9 mmol/L), LDL-C is low. Diagnosis of type III HLP by lipoprotein electrophoresis, which shows a broad pre-β fraction. Apo E genotyping.

Familial apolipoprotein B-100 defect (FDB)

Apo B-100 is part of the VLDL, IDL and LDL and is produced in the liver. The percentage of apo B-100 in the protein content of VLDL is 40–50%, increasing to 85–94% during the metabolization of VLDL to LDL via IDL. Apo B-100 mediates the uptake of LDL by the liver and the peripheral tissues by interacting with the LDL receptor. FDB is an autosomal dominant disease in which the affinity of apo B-100 for the LDL receptor is reduced. A common cause of this is a substitution in position 3500 of apo B-100 of arginine by glutamine. The prevalence in the total population is 1: 500; in hypercholesterolemia it is 1 : 100. Clinically, FDB is less severe than FH.

Laboratory findings: usually, type II hyperlipoproteinemia is present, with cholesterol concentrations in the range of 250–300 mg/dL (6.5–7.8 mmol/L) in carriers with heterozygous mutation. To differentiate FDB from FH, molecular genetic tests are required.

Familial combined hyperlipidemia (FCHLP) /17/

The cause of this disease is not well understood. It is believed to be due to increased synthesis of apo B, the main protein component of VLDL and LDL. The increased formation of VLDL consequently leads to an increase in its metabolic product, the LDL. If, in addition to this, the metabolism of VLDL is impaired, the reduced conversion to LDL also results in elevated VLDL. Therefore, different phenotypes can occur within a family (IIa, IIb, IV).

Clinical findings: approximately 10% of patients with myocardial infarction have FCHLP. Patients with FCHLP frequently have a metabolic syndrome (obesity, hypertension, impaired glucose tolerance) and hyperuricemia.

Laboratory findings: LDL-C and/or triglycerides are moderately elevated compared to the age- and gender-related upper reference limits.

Familial hypertriglyceridemia (FHT) /18/

FHT is an autosomal dominantly inherited disease whose etiology is most likely oligogenic. Its clinical manifestation is age dependent and is triggered by environmental factors such as a diet high in carbohydrates, alcohol consumption, and obesity. There is an overproduction of VLDL, possibly combined with reduced breakdown of VLDL. The VLDL particles contain more triglycerides, and the triglyceride/apoB ratio, which is normally 10 mg/mg, is 26 mg/mg. Secondary hypertriglyceridemia must be excluded prior to diagnosing FHT.

Laboratory findings: triglycerides elevated, LDL-C below 120 mg/dL (3.0 mmol/L), HDL-C < 40 mg/dL (1.0 mmol/L).

Chylomicronemia /18/

Chylomicronemia is a rare form of hyperlipidemia corresponding to Fredrickson type I or type V. Patients with type I in particular, but also many patients with type V hyperlipoproteinemia, have a deficiency in lipoprotein lipase (LPL), in the activating cofactors apo CII or apo A-V, or GPI-HBP. There is an increased risk of acute pancreatitis and dermatologic stigmata such as eruptive xanthomas. Decreased activity of LPL leads to reduced lipolysis of chylomicrons and VLDL (in type V). In addition there is reduced release of apo A-I and apo A-II from the remnants for the formation of HDL.

Laboratory findings: triglycerides around 1,000 mg/dL (11.4 mmol/L), keep serum refrigerated, chylomicrons form a creamy top layer, the sub natant is clear in type I or turbid in type V, LDL-C reduced, HDL-C reduced.

HDL dyslipoproteinemia

HDL dyslipoproteinemias with reduced HDL-C levels. Reduced HDL-C is a risk factor for cardiovascular disease.

In lipoprotein electrophoresis, the HDL migrate in the alpha-fraction i.e., fastest towards the anode. HDL are the smallest and most dense lipoproteins. The apo A-I and apo A-II of HDL account for 80–90% of the protein content. Minor HDL protein components include apoproteins such as apo A-IV and V, apo C, apo D, and apo E. HDL mediate a process called reverse cholesterol transport (RCT). Non-esterified cholesterol of the peripheral tissues, in particular the macrophages, is taken up by the HDL and transported to the liver, from where it is excreted in stool via the gallbladder. This process involves lipid transport proteins and the ATP-binding cassette transporter A1. The following proteins are involved /19/:

Lecithin-cholesterol-acyl transferase (LCAT). In the RCT pathway, non-esterified cholesterol released by the peripheral tissue cells is esterified by HDL-bound LCAT before it is transported to the liver. The hydrophobic esterified cholesterol migrates to the core of the HDL particle, thus enlarging the particle. The enzyme LCAT is activated by apo A-I.
Cholesterol ester transfer protein (CETP). This protein mediates the transfer of esterified cholesterol from HDL particles to VLDL particles in exchange for triglycerides. Because VLDL, in the form of apo B-containing particles, are catabolized by the liver, this is one pathway for the return of cholesterol from HDL to the liver.
Phospholipid transfer protein (PLTP). This protein maintains the concentration of HDL by transferring phospholipids from the remnants to HDL. It plays an important role in the reconfiguration and formation of lipid-poor apo A1-containing particles.
ATP-binding cassette transporter A1 (ABCA1). This is the preferred donor of cellular cholesterol, which it mediates to a small sub fraction of lipid-poor apo A-I.

Causes leading to reduced HDL levels in blood

Mutations in the gene for apo A-I, the principal structural protein of HDL. Individuals with heterozygosity for functionally relevant mutations in apo A-I have half-normal levels of HDL-C. Biallelic defects lead to complete HDL deficiency. Some mutations are associated with an increased risk of cardiovascular disease, others such as apo A-I Milano or apo A-I Paris are not. Some mutations are associated with familial amyloidosis. Individuals with Milano mutation of the genes APO A-I and APO A-I Paris have HDL-C levels below 20 mg/dL (0.52 mmol/L).
Homozygous defects in the gene LCAT. These patients have significantly reduced or undetectable HDL-C levels and corneal cloudiness. Mutations that contain residual LCAT activity on HDL do not lead to further clinical symptoms. The associated syndrome is called fish-eye disease. Mutations that lead to complete loss of LCAT activity cause the development of proteinuria, renal insufficiency, and hemolytic anemia. Heterozygous mutation carriers are not at increased risk of developing cardiovascular disease despite half-normal concentrations of HDL-C.
Functional defects in the gene encoding ABCA1 prevent the efflux of cholesterol from macrophages, causing the latter to store increasing amounts of cholesterol. Clinically, biallelic mutations manifest as Tangier disease (α-lipoprotein deficiency). HDL is undetectable in serum, cholesterol levels are below 100 mg/dL (2.6 mmol/L), triglycerides are normal to slightly elevated (above 200 mg/dL; 2.3 mmol/L). Patients present with yellow/orange-colored tonsils, peripheral neuropathy, splenomegaly or premature cardiovascular disease. Heterozygous mutation carriers are not at increased risk of developing cardiovascular disease despite half-normal levels of HDL-C.
Functionally relevant mutations in the gene CETP are relatively common in Japan, but rare in Europe. They lead to moderately elevated HDL-C in heterozygous carriers and significantly elevated HDL-C levels in homozygous carriers. Polymorphisms in CETP are associated with slightly changed HDL-C and LDL-C and a correspondingly changed risk for cardiovascular disease.

Table 4.1-3 Multifactorial dyslipidemias

Clinical and laboratory findings

Obesity

Adults with a body mass index (BMI) above 25 kg/m² are classified as overweight and those with a BMI above 30 kg/m² as obese. In children, overweight is defined as having a BMI above the sex- and age-specific 85th percentile. Children with excessive weight gain between 20–42 months of age have lower HDL-C, higher VLDL-C and a reduced HDL/LDL ratio from age 18 compared to normal-weight children of the same age /20/. In adults, obesity, glucose intolerance and hypertension are associated with increased mortality /21/.

Metabolic syndrome /22/

The individual components of the metabolic syndrome are overweight or obesity, hypertriglyceridemia, HDL dyslipidemia, hypertension, and impaired glucose tolerance or type 2 diabetes. In the setting of insulin resistance, hypertriglyceridemia results from hyperinsulinism-induced increased hepatic VLDL production, increased intestinal synthesis of triglyceride-rich lipoproteins and decreased lipoprotein lipase (LPL) activity. In addition, there is an increased flow of free fatty acids from adipose tissue to the liver due to increased lipolysis in the adipose tissue.

Laboratory findings: metabolic components of dyslipidemia according to WHO criteria include: triglycerides ≥ 150 mg/dL (1.7 mmol/L) and HDL cholesterol < 35 mg/dL (0.9 mmol/L) in men and < 40 mg/dL (1.0 mmol/L) in women.

Diabetes mellitus

Hyperlipidemia in diabetes is a combined disorder resulting from increased VLDL synthesis in the liver and reduced clearance. In patients with type 1 diabetes, the reduced activity of lipoprotein lipase is the most relevant factor. In type 2, hyperinsulinism and elevated concentration of free fatty acids lead to increased production of VLDL /23/. In the setting of insulin resistance, the free fatty acids are released by adipose tissue and the muscles.

Laboratory findings: patients with type 2 diabetes have elevated triglycerides, low HDL-C and a prevalence of small dense LDL particles. In type 2 diabetes, the prevalence of hypertriglyceridemia is at least twice as high as in type 1.

Liver disease /24/

Non-alcoholic steatohepatitis (NASH): this disease is linked to primary or secondary hypertriglyceridemia. The causes are increased hepatic VLDL synthesis or increased triglyceride synthesis.

Laboratory findings: triglycerides elevated, HDL-C decreased, slight increase in ALT.

Cholestatic liver disease: HDL-C is reduced and total cholesterol and LDL-C are elevated, especially in primary biliary cirrhosis. The cholesterol concentration is inversely correlated with albumin.

Acute hepatitis: total cholesterol and LDL-C normal or reduced, HDL-C reduced, triglycerides elevated. Total cholesterol correlates positively with the albumin concentration. There is a defect in the breakdown of triglyceride-rich lipoproteins (IDL), caused by hepatic lipase deficiency.

Cirrhosis of the liver: total cholesterol, HDL-C and triglycerides are normal or reduced.

Alcoholism and alcoholic liver disease: excessive alcohol consumption is one of the most common causes of hypertriglyceridemia, often associated with alcoholic fatty liver. Both Fredrickson type IV and type V hyperlipidemias occur, sometimes accompanied by pancreatitis and eruptive xanthomas. In rare cases, the hyperlipidemia is associated with jaundice and hemolytic anemia (Zieve’s syndrome). Steady, moderate consumption of alcohol also leads to elevated triglycerides, with the hypertriglyceridemic effect being especially marked in the setting of existing type IV and becoming even more pronounced after consumption of dietary lipids. The metabolism of ethanol inhibits the oxidation of fatty acids in the liver and leads to increased amounts of fatty acids being available for triglyceride synthesis. Cessation of alcohol consumption results in a rapid decrease in triglyceride levels. Elevated HDL-C levels are often associated with regular consumption of alcohol.

Renal disease /25/

Chronic renal disease: these patients develop a form of dyslipidemia that is characterized by high triglycerides (high VLDL and IDL levels) and low HDL-C. The hypertriglyceridemia is multifactorial and is due to increased production of VLDL and apo B-100 and reduced clearance of triglyceride-rich lipoproteins by hepatic triglyceride lipase and LPL. A significant number of patients with a GFR below 60 [ml × min^–1 × (1.73 m²)^–1] have elevated triglycerides. There is little change in the pattern and severity of dyslipidemia under hemodialysis, while under peritoneal dialysis the cholesterol level rises. About half of all patients who have undergone a kidney transplant have persistently elevated total cholesterol, LDL-C and triglyceride levels as a result of weight gain and administration of corticosteroids and cyclosporine. Cyclosporine inhibits the conversion of cholesterol to bile acids. As a result of this, intracellular cholesterol is elevated, which leads to decreased uptake of LDL by the liver and to an increase in LDL-C in plasma. Corticosteroids inhibit the lipoprotein lipase activity and increase the synthesis of VLDL, thus causing triglyceride levels to rise.

Nephrotic syndrome /26/: the main finding is elevated total cholesterol. An increase in triglycerides occurs especially with more severe proteinuria. The extent of the hyperlipidemia depends on the severity of the nephrotic syndrome. LDL-C is always elevated, often accompanied by increased VLDL levels. The cause is multifactorial and includes both increased synthesis and reduced breakdown of LDL particles.

Lipodystrophy /27/

Lipodystrophies are acquired or congenital disorders of adipocyte metabolism which are characterized by generalized or local loss of adipose tissue. Often, the condition is accompanied by metabolic syndrome, diabetes mellitus, hepatic steatosis, and dyslipidemia. The dyslipidemia is partly due to insulin resistance of these patients.

Laboratory findings: approximately 70% of patients have significantly elevated triglycerides and reduced HDL-C levels.

Chronic infection

Chronic infection is associated with a dyslipidemia that is characterized by a decrease in HDL-C and LDL-C and a slight, usually delayed, increase in triglycerides. This is also the case in acute systemic infections. Therefore, no lipid diagnostics should be performed in patients with systemic infection (CRP elevated) because, like albumin and transferrin, apolipoproteins are negative acute-phase proteins, and lipid concentrations may be falsely low.

HIV infection /28/

During intensive HIV therapy, marked hypertriglyceridemia occurs, and 40% of patients develop lipodystrophy. Insulin resistance also develops. The dyslipidemia associated with this condition often is of mixed nature, with low HDL-C, or hypercholesterolemia. The cause is thought to be deregulation of fatty acid metabolism with translocation into the liver and muscles. This leads to an accumulation of lipids in the myocytes, to insulin resistance, hepatic steatosis, and increased secretion of VLDL by the liver.

Hypothyroidism /29/

Due to a reduced receptor-induced breakdown of LDL, total cholesterol and LDL-C are elevated, and in some cases triglycerides are also slightly elevated. In terms of Fredrickson phenotypes, type IIa or IIb is present. However, type III or IV are also possible. Individuals with hypothyroidism can have total cholesterol levels above 300 mg/dL (7.8 mmol/L).

Hyperthyroidism

Due to increased LDL receptor activity, patients with hyperthyreosis tend to have low LDL-C levels.

Pregnancy

Due to higher estrogen levels, there is an increase in VLDL, LDL and HDL, resulting in a moderate rise in total cholesterol and triglycerides. These return to normal post partum.

Contraceptives /30/

Oral contraceptives increase LDL-C and VLDL as well as HDL-C levels. Progesterone has the opposite effect. In postmenopausal women under hormone replacement therapy, estrogens reduce LDL-C concentrations, but can lead to hypertriglyceridemia in women predisposed to elevated triglycerides.

Corticosteroids

If administered over a prolonged period of time, corticosteroids cause hypertriglyceridemia and a decrease in HDL-C levels due to insulin resistance and steroid-induced synthesis of VLDL. Similar changes occur in Cushing’s disease.

Various medications

Phenytoin, barbiturates, cimetidine (not ranitidine) cause an increase in HDL-C levels. Cimetidine can also cause chylomicronemia. Retinoids cause a significant increase in triglycerides, especially in the setting of existing type IV hyperlipidemia.

Table 4.1-4 Thresholds for therapeutic lifestyle changes (TLC) and drug therapy in different risk categories of cardiovascular disease (CVD) according to NCEP guidelines /2/

Risk category	LDL-C mg/dL (mmol/L) at which to initiate TLC	LDL-C mg/dL (mmol/L) at which to consider therapy	Triglycerides mg/dL (mmol/L)
0–1 Risk factor	≥ 160 (4.14) Goal: < 160 (4.14)	≥ 190 (4.91) [160–189; 4.14–4.89: drug optional]; 10-year risk < 10%	150 (1.71)
≥ 2 Risk factors and (10-year risk ≤ 20%)	≥ 130 (3.36) Goal: < 130 (3.36)	10-year risk 10–20%: ≥ 130 (3.36); 10-year risk < 10%: ≥ 160 (4.14)	150 (1.71)
CVD or CVD risk equivalents (10-year risk > 20%)	≥ 100 (2.59) Goal: < 100 (2.59)	≥ 130 (3.36): drug optional 100–129 (2.59–3.34)	150 (1.71)

NCEP, National Cholesterol Education Program of the USA; risk assessment in% according to Framingham or PROCAM score

Table 4.1-5 Intervention strategies as a function of ASCVD risk and LDL-C level /2/

Risk	ASCVD disease risk group (Score card according to Fig. 1 and Fig. 2 in Ref /3/)	Score card 10 years risk	Score card 10 years risk	LDL-C goal mg/dL (mmol/l)
Very high	Documented ASCVD, previous myocardial infarction, coronary revascularization, coronary artery bypass graft and other arterial re-vascularization procedures, ischemic stroke Type 2 diabetes Type 1 diabetes with organ damage (microalbuminuria) Patients with GFR < 60 mL/min/1.73 m² A calculated 10 year risk score ≥ 10	> 30%	≥ 10%	70 (1.81)
High	Markedly elevated single risk factors such as familial dyslipidemia and severe hypertension A calculated score ≥ 5% and < 10% for 10 year risk of fatal ASCVD	10–30%	5–10%	100 (2.59)
Moderate	Score ≥ 1% and < 5% at 10 years. Many middle-aged individuals belong to this risk category. The risk is further modulated by a family history of premature coronary artery disease, abdominal obesity, physical activity pattern, the levels of HDL-C, triglycerides, CRP, Lp(a), fibrinogen, homocysteine, apoB, and the social class of the person.	3–15%	1–5%	115 (2.98)
Low	Score < 1%.	< 3%	< 1%	115 (2.98)

Score charts of 10 year risk of fatal cardiovascular disease (CVD) in populations ad high CVD risk and at low CVD risk are presented in Reference /3/. ASCVD, atherosclerotic cardiovascular disease.

Table 4.1-6 Goals for primary and secondary prevention of cardiovascular disease /3/

Criterion	Primary prevention	Secondary prevention
Smoking	Avoid	Avoid
Dietary change	High-fiber diet	High-fiber diet
Physical activity	At least 30 min. per day	At least 30 min. per day
Body mass index	Below 25 kg/m²	Below 25 kg/m²
Blood pressure	Below 140/90 mmHg	Below 130/80 mmHg
Total cholesterol	Below 190 mg/dL (5.0 mmol/L)	Below 175 mg/dL (4.5 mmol/L)
LDL-C	Depending on risk	Below 70 mg/dL (1.8 mmol/L)
Fasting glucose	Below 110 mg/dL (6.0 mmol/L)	Below 110 mg/dL (6.0 mmol/L)
Hb A_1c		Below 6.0%

Table 4.1-7 Assays for the assessment of cholesterol-containing lipoprotein particles

Assay	Clinical and laboratory significance
Total cholesterol	Detects all cholesterol-containing lipoprotein particles, including non-atherogenic particles. The cholesterol concentration can vary significantly among patients with the same particle count.
LDL-C Friedewald formula Homogeneous assay	Homogeneous assay: standard for cardiovascular risk assessment. Advantage: automated assay. The Friedewald equation cannot be used for type III or if triglyceride levels are > 400 mg/dL (4.6 mmol/L).
Non-HDL-C	This assay evaluates C and HDL-C and the difference of total cholesterol minus HDL-C. Recommended by NCEP ATPIII as a secondary target in individuals with triglycerides > 400 mg/dL (4.6 mmol/L). Detects all atherogenic lipid particles.
Apolipoprotein B	Is a criterion for the number of atherogenic LDL particles, since each particle contains only one apo B molecule. Is supposed to be a better predictor of CHD risk than LDL-C.
LDL particle count	Determines CVD risk more directly than LDL-C. Different methods are used for this. Only few laboratories use the nuclear magnetic resonance (NMR) technique.

Table 4.2-1 Findings in familial hypercholesterolemia (FH) /27, 28, 29/

Findings	Assessment
Definition	FH is a common inherited autosomal co-dominant disorder characterized by high plasma levels of low-density lipoprotein cholesterol (LDL-C) due to reduced catabolism. The prevalence has been estimated 1 : 300,000 in its homozygous and between 1 : 200 and 1,300 in its heterozygous form. If untreated, FH causes hypercholesterolemia and is associated with premature atherosclerotic cardiovascular disease (ASCVD).
Prevalence	The prevalence has been estimated 1 : 300,000 in its homozygous and between 1 : 200 and 1 : 300 in its heterozygous form. The prevalence of FH in patients with premature ASCVD is about 1 : 18.
Laboratory diagnostics	LDL-C thresholds: For children 190 mg/dL (4,92 mmol/L); for adults 220 mg/dL (5,70 mmol/L) or 190 mg/dL (4,92 mmol/L). Secondary causes of this level of increase (190 mg/dL; 4,92 mmol/L) are the presence of uncontrolled hypothyroidism, obstructive liver disease, or significant proteinuria. Genetic testing is required to identify FH patients with LDL-C levels below those thresholds. Approximately one-third of those with genetically confirmed FH have LDL-C concentrations below traditional thresholds. LDL-C concentrations are typically higher than 400 mg/dL (10,36 mmol/L) in patients with homozygous FH /27/.
Genetics of FH	The major determinants in FH are LDLR (80–85%), apoB100 (5–10%), and PCSK9 (2%).
Therapy	Treatment of heterozygous FH should begin at about 10 years of age, typically with statins. Treatment of homozyous FH begins at the time of diagnosis because of the severity of the condition when LDL-C levels are typically > 400 mg/dL (10.36 mmol/L).

Table 4.3-1 Triglyceride levels for males and females 5–19 years of age /12/

Males 5–9 years	Males 10–14 years	Males 15–19 years
85 (0.97)	111 (1.26)	143 (1.63)
Females 5–9 years	Females 10–14 years	Females 15–19 years
120 (1.37)	120 (1.37)	126 (1.44)

95th Percentile. Data expressed in mg/dl (mmol/l)

Table 4.3-2 Classification of hyperlipoproteinemia according to Fredrickson /11/

Phenotype	Type I	Type IIa	Type IIb	Type III	Type IV	Type V
Elevated LP fraction	Chylomicrons	LDL	LDL and VLDL	IDL	VLDL	VLDL, Chylo-microns
Cholesterol	N, 1+	2 to 4+	2 to 4+	3+	N, 1+	N, 3+, 4+
Triglycerides	4+	N	2+	3+	2+	4+
Incidence of HLP	< 1%	10%	40%	< 1%	45%	5%

Explanation of symbols: LP, lipoprotein; HLP, hyperlipoproteinemia, N, normal; 1+ slightly elevated; 2+ moderately elevated; 3+ markedly elevated; 4+, severely elevated

Following lipid measurement, keep serum refrigerated overnight. Serum is clear if only LDL are elevated (type IIa), and homogenously turbid if VLDL or remnants (IDL) are elevated (types IIb, III or IV). If chylomicrons are present, the serum has a milky white, creamy top layer (type I). Differentiation of type III requires lipoprotein electrophoresis.

Table 4.3-3 Postprandial triglycerides (Tgl) and hazard ratios for cardiovascular disease and death /9/

Triglycerides mg/dL (mmol/L)	Hazard ratio (women)			Hazard ratio (men)
Triglycerides mg/dL (mmol/L)	Ischemia	Infarction	Death	Ischemia	Infarction	Death
< 88.5 (< 1.0)	1.0	1.0	1.0	1.0	1.0	1.0
88.5–176.1 (1–1.99)	1.7	2.2	1.3	1.3	1.6	1.3
177–264.6 (2–2.99)	2.8	4.4	1.7	1.7	2.3	1.4
265.5–353 (3–3.99)	3.0	3.9	2.2	2.1	3.6	1.7
354–441.6 (4–4.99)	2.1	5.1	2.2	2.0	3.3	1.8
≥ 442.5 (≥ 5.04)	5.9	16.8	4.3	2.9	4.6	2.0

Table 4.4-1 Primary hyperlipoproteinemias: classification according to Fredrickson /2/

Type pattern	I		IIa	IIb	III	IV	V
Synonyms	Fat-induced hypertriglycerid- emia		Hyper- cholesterol- emia	Mixed hyperlipid_ emia	Broad-β disease	Endogenous hypertriglycerid- emia	Combined endogenous- exogenous hypertriglyceridemia
Appearance of fasting serum	Creamy top layer over clear serum		Clear	Slightly turbid	Turbid	Turbid	Turbid (creamy top layer)
Cholesterol	Normal		↑	↑	↑	Normal or ↑	Normal or ↑
Triglycerides	↑		Normal	↑	↑	↑	↑
LDL cholesterol	↓		↑	↑	Normal to ↑	Normal	Normal to ↑
HDL cholesterol	↓		Often ↓	Often ↓	Often ↓	Often ↓	Often ↓
Lipoprotein electrophoresis	+
	α	≡	≡	≡	≡	≡	≡
	pre-β	≡	≡	■	■	■	■
	β	≡	■	■		≡	≡
	chylo	■					■
	–
Xanthomas	Eruptive		Tendinous, tuberous	Tendinous, tuberous	Planar, tubero-eruptive	Tubero-eruptive	Tuberoeruptive
Atherogenicity	–		+++	+++	+++	++	+

Table 4.5-1 Recommendations when to measure Lp(a) and how to deal with increased values

Guideline	Recommendation
ATP-III NECP 2002 /18/	High Lp(a) counts as an additional risk factor that justifies a lower goal for LDL-C. The determination of Lp(a) is an option for selected patients, particularly if they have a strong family history of premature coronary heart disease (CHD) or suffer from familial hypercholesterolemia.
ACCF/AHA 2010 /16/	36 long-term prospective morbidity and mortality studies were considered. The study revealed modest associations of Lp(a) levels with increased risk for CHD and stroke.
ESC/EAS2011 /12/	The guidelines state that Lp(a) might not be a target for risk screening in the general population, yet it should be considered in individuals with elevated cardiovascular risk or a family history of premature vascular disease.
AACE 2012 /17/	Over the past 10 years a considerable body of evidence confirming the status of Lp(a) as a major risk factor independently of triglycerides, LDL-C and HDL-C has appeared. Testing of Lp(a) is not generally recommended, although it may provide useful information to ascribe risk in white patients with coronary artery disease (CAD) or in those with an unexplained family history of early CAD.

Table 4.5-2 Biochemistry of Lipoprotein(a) /2, 5/

Analysis	Assessment
Structure	Lp(a) comprises a single low-density lipoprotein cholesterol (LDL-C) particle linked by a disulfide bridge to a highly polymorphic apo(a) protein. The disulfide bridge links Cys4326 in apoB-100 with the only free Cys4057 in apo(a), located in kringle four (K-IV) type 9. Apo(a) is produced almost exclusively in the liver and follows classical steps of glycoprotein biosynthesis. Lp(a) levels highly correlate with apo(a) production.
Prevalence	Approximately 80% of the European population have a concentration of Lp(a) below 500 mg/L.
Impact	Increased Lp(a) concentrations stimulate pathogenetic mechanisms, that are associated with increased risk of coronary heart disease (CHD). The effects are: Proatherogenic, since there is a similar structure to the lDL-C particle Prothrombotic, since apo(a) has homology to plasminogen Proinflammatory, because the proportion of oxidized lipids are high in Lp(a). Oxidized lipids are the main constituents of atherosclerotic plaques. Elevated concentrations of Lp(a) are risk factors of mycordial infarction and stroke. The risk is as high than with the familial hypercholesterolemia.
Laboratory diagnostics	Lp(a) concentrations > 300 mg/L increase the risk of myocardial infarction regardless of other risk factors. The risk of CHD increases linear with the level of Lp(a) and concentrations higher than 500 mg/L increase the risk of myocardial infarction and aortic valve stenosis. Lp(a) concentrations are constant throughout life, which is why a single determination is sufficient for diagnosis. According to the recommendations of the European Association for Cardiology (ESC) and the European Atherosclerosis Society (EAS) the Lp(a) concentration should be determined for every individual once in a lifetime.
Therapy	Statins decrease Lp(a) levels by 10–20%. Lipoprotein apheresis of Lp(a) is a therapy option for severe elevation.

Table 4.5-3 Factors influencing Lp(a) /18/

Influencing factor	Behavior of Lp(a)
Hypothyreosis	Causes increase in Lp(a). Substitution with T4 leads to decrease.
Hyperthyreosis	Causes decrease in Lp(a), thyreostatics cause increase
Androgens, estrogens	Cause decrease in Lp(a), antiandrogens cause increase
FSH stimulation	Increase in Lp(a) in the luteal phase
Estrogen receptor-modulating substances	Tamoxifen, but not aromatase inhibitors (letrozole), cause increase in Lp(a)
Cholestatic liver disease	Caused by a high concentration of lipoprotein X, decrease in Lp(a)
Diabetic nephropathy, nephrotic syndrome	Increase in serum Lp(a) concentration
Alcoholism	Decrease in Lp(a), increase upon withdrawal
Nicotinic acid	Marked decrease in Lp(a)
Oral antidiabetics	Pioglitazone, rosiglitazone and troglitazone not only reduce postprandial glucose levels in type 2 diabetes, but also cause a decrease in Lp(a)
Statins	Statins alone do not reduce Lp(a)

Table 4.6-1 Biochemical properties and functions of apolipoproteins /7/

Apolipoprotein Occurrence	MW (kDa)	Site of synthesis	Serum concen- tration*	Function Association with disease
A-I HDL (chylo)	28.3	Intestine, liver	100–150	Structural protein of HDL, stimulation of cholesterol, efflux via ABCA1, LCAT activation, anti-inflammatory Apo A-I deficiency, amyloidosis
A-II HDL	17	Intestine, liver	30–50	Inhibition of hepatic lipase
A-IV Chylo, HDL	~ 46	Intestine	15	Regulation of appetite, anti-inflammatory?
B-100 LDL, VLDL	~ 549	Liver	60–140	Structural protein of LDL and VLDL and thus of cholesterol transport to the tissues, binding to apo B receptor and apo E receptor A-, hypo-β lipoproteinemia, familial apo B deficiency, β-lipoproteinemia, familial apo B defect
B-48 Chylo	~ 265	Intestine, liver	3–5	Major apoprotein of chylomicrons A-β-lipoproteinemia
C-I Chylo, VLDL, (HDL)	6.5	Liver	4–8	Activates LCAT* and lipoprotein lipase (LPL) A-β-lipoproteinemia
C-II Chylo, VLDL, (HDL)	8.8	Liver	3–8	The activator protein of LPL Type I hyperlipoproteinemia (HLP
C-III 0, 1, 2 Chylo, VLDL, (HDL)	8.9	Liver	8–15	Inhibition of remnant uptake and LPL Hypertriglyceridemia
D	20	?	10	Lipocalin
E (isoforms E₂, E₃, E₄) Chylo, VLDL, (HDL)	~ 34	Liver	3–5	Mediates the uptake of chylomicrons and VLDL remnants by interaction with the hepatic remnant receptor Type III HLP, Alzheimer’s disease

LCAT, lecithin-cholesterol acyltransferase; *serum concentration expressed in mg/dL;

MW, molecular weight

Table 4.9-1 Pathophysiology of lipoprotein particles

Lipoprotein particles

Chylomicrons

The chylomicrons are the largest lipoproteins (100–1,000 nm). They are formed in the enterocytes as food is digested and act as transport vehicles for the triglycerides consumed (approximately 100–150 g per day). In normal individuals, they are only present in postprandial plasma. Chylomicrons are poor in cholesterol. Important apolipoproteins include A-I and B48. The depletion of triglycerides from the chylomicrons by lipoprotein lipases results in the formation of chylomicron remnants which, like the VLDL remnants, have an atherogenic effect.

Very-low-density-lipoproteins (VLDL)

VLDL particles have a diameter of 30–90 nm and, like the chylomicrons, are rich in triglycerides. In contrast to the chylomicrons they are synthesized in the liver. VLDL float in the ultracentrifuge at a density of less than 1.006 g/mL. An increase in the VLDL fraction can lead to turbidity of the serum although, unlike with chylomicrons, no creamy layer forms over the turbid serum. VLDL transport the endogenous triglycerides formed in the liver to the peripheral tissues. As a result of the breakdown of the VLDL triglycerides, enrichment with cholesterol, and exchange of apolipoproteins and lipids with HDL and LDL, intermediate density lipoproteins (IDL) are formed. IDL can be taken up by the liver or converted to LDL. The protein content of VLDL consists of apoproteins B-100, CI, CII, CIII, and E.

VLDL have an atherogenic effect, since their metabolism produces small dense HDL particles and LDL particles as a result of an exchange of triglycerides. Cholesterol ester transfer protein (CETP) mediates the transfer of triglycerides from VLDL to HDL and LDL. The triglyceride-rich LDL and HDL are depleted of triglycerides by hepatic lipase, thus becoming atherogenic, small dense particles /4/.

Low-density lipoproteins (LDL) /5/

LDL particles have a diameter of 20–30 nm and float when centrifuged at a density of 1.006–1.063 g/mL. They are produced by hydrolysis of VLDL, predominantly in the post absorptive phase (fasting state). LDL contain a single molecule of apo B-100. This protein is the ligand for the LDL receptor. LDL particles transport cholesterol mainly to the liver, but also to peripheral body cells. The LDL particle number and particle size are important criteria in the primary and secondary prevention of atherosclerotic cardiovascular disease (ASCVD). The number of particles correlates with the apo B concentration.

Individuals with dyslipidemia who have an increased number of LDL particles, an elevated concentration of apo B and elevated LDL-C are predisposed for premature ASCVD. An increased number of LDL particles of normal size and density is found in patients with familial hypercholesterolemia. The cause are mutations in the LDL receptor gene (LDLR). Receptor-negative patients represent a more severe phenotype than receptor-defective mutations. The LDL receptor defect slows down the clearance of LDL particles. Another familial cause that is phenotypically similar to the receptor defect is a defective apo B-100 where the binding to the LDL receptor is reduced. The most common cause of elevated LDL particle number is familial combined hyperlipoproteinemia. In this disease there is increased secretion of apo B. Patients have mildly to moderately elevated cholesterol, sometimes also elevated triglycerides, and increased production of small dense LDL particles.

The native LDL particles are modified oxidatively, proteolytically and lipolytically in the intima of the arterial wall to become ligands of scavenger receptors and macrophages. Their uptake contributes to the formation of foam cells and ultimately fatty streaks and atheromatous plaques. Although, at present, diagnosis of the risk of atherosclerosis is focused mainly on the cholesterol content of LDL particles, apo B is a more important parameter.

Statins have a positive effect in the primary and secondary prevention of ASCVD. By competitively inhibiting hydroxy methyl glutaryl-CoA reductase, they reduce hepatic synthesis of cholesterol. As a result, the LDL receptor as well as the LDL-receptor-mediated catabolization of LDL particles are upregulated. Apo B has proven to be a better marker for monitoring statin therapy than LDL-C and non-HDL-C /6/.

High-density lipoproteins (HDL) /7, 8/

HDL particles are the smallest (7–10 nm) and most protein-rich lipoproteins. They float in the ultracentrifuge at a density of 1.063 to 1.21 g/mL. They consist of cholesterol and phospholipids. The main apoproteins are apo A-I and apo A-II. Minor components are apo C and apo E. The HDL₂ and HDL₃ classes are differentiated by density gradient ultracentrifugation. There are no convincing studies demonstrating diagnostic benefits of this.

The most important apoprotein of HDL particles is apo A-I, which is produced in the liver and small intestine. Secreted as a lipid-poor protein, it takes up phospholipids and free cholesterol mainly from the liver but also from the macrophages via the ABCA1 transporter, thus forming a discoidal particle (Fig. 4.9-3 – Reverse transport of cholesterol by HDL particles). This particle takes up further free cholesterol from the peripheral tissues, which is then esterified by lecithin-cholesterol acyl transferase (LCAT) which binds to the HDL particle. The discoidal particle thus becomes spherical. In the next step, cholesterol ester transfer protein (CETP) transfers esterified cholesterol to LDL and VLDL particles in exchange for triglycerides. These apo B-containing particles are taken up by the LDL receptor of the liver which then removes the cholesterol. The HDL particles are transformed to small dense particles as hepatic and endothelial lipoprotein lipase deplete the spherical LDL particles of triglycerides and phospholipids.

The HDL particles mediate the reverse transport of tissue cholesterol, which is mainly localized in macrophages, back to the liver. They thereby functionally antagonize the LDL. For this reason, HDL-C is thought to provide a protective effect against atherogenesis. While HDL-C concentrations above the threshold values provide atherogenic protection, significantly elevated levels do not translate into better atheroprotection. Low HDL-C concentrations are linked to increased risk of ASCVD. But there are exceptions. For example, LCAT deficiency is associated with a significant reduction in HDL-C, but not with increased risk of ASCVD. Elevated concentration of CETP, in contrast, are linked to increased prospective cardiac risk. Reduced concentrations of HDL-C can have a genetic origin (familial hypo-alpha lipoproteinemia, Tangier disease, LCAT deficiency) or, more likely, secondary causes (obesity, inactivity, smoking, insulin resistance, diabetes mellitus, renal disease).

Figure 4.2-1 Principle of enzymatic cholesterol assays.

Figure 4.2-2 American College of Cadiology/American Heart Association guidelines for the use of statin therapy in patients with increased atheroscleotic vascular risk (ASCVD). Modification according to Ref. /26/

Figure 4.3-1 Indicator systems for determination of free glycerol.

Figure 4.9-1 Exogenous and endogenous pathway of lipoprotein metabolism. Dietary lipids absorbed in the intestine are packaged into chylomicrons in the mucosa of the small intestine. In the blood, lipoprotein lipase (LPL) cleaves off free fatty acids (FFS), which are metabolized by the peripheral tissues. The remaining chylomicron remnants are taken up by the liver. In the endogenous pathway, VLDL are released by the liver, and free fatty acids are cleaved from them by lipoprotein lipase (LPL), resulting in VLDL remnants (intermediate density lipoproteins, IDL), which are either taken up by the liver via the remnant receptor or are converted to low density lipoproteins (LDL) by hepatic lipoprotein lipase (HL).

Figure 4.9-2 Metabolism of triglyceride-rich chylomicrons and VLDL by heparin-sensitive (endothelial) lipoprotein lipase (LPL) in blood /9/. FFS, free fatty acids; 2-MG, 2-monoacylglycerol, HDL, high density lipoprotein, VLDL, very low density lipoprotein.

Figure 4.9-3 Reverse transport of cholesterol by HDL particles. Lipid-poor apo A-I takes up free (non-esterified) cholesterol (FC), (e.g., from macrophages) via the ABCA1 transporter, forming a discoidal particle. This particle takes up further FC from the peripheral tissues, which is converted to cholesteryl ester (CE) by lecithin-cholesteryl acyltransferase (LCAT). The discoidal HDL particle becomes spherical. The HDL particle is either taken up by the hepatic SR-BI receptor, or the cholesteryl ester transfer protein (CETP) transfers CE from HDL to LDL and VLDL particles in exchange for triglycerides (TG), resulting in the HDL particles becoming small and dense. The apo B containing particles are taken up by the LDL receptor (LDL-R) of the liver which then eliminates the cholesterol.

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