Formation of Lipoprotein and Cholesterol Levels
If cholesterol levels are too high for disposal into the bloodstream, these cells are filled with fatty deposits, which then hardens into a plaque, and eventually clogs blood vessels causing strokes, or heart attacks.
VLDL: very low density lipoproteins and are precursors of low density lipoproteins.
Formation of a lipoprotein
Both cholesterol and triglycerides are carried in blood as part of molecules called lipoproteins. These lipoproteins also consist of phospholipids, cholesterol, protein and apolipoprotein. According to the percentage share of different structural components, they are classified as chylomicrons (QM), low density lipoprotein (LDL), very low density lipoprotein (VLDL), high density lipoprotein (HDL) and intermediate density lipoprotein (IDL).
The lipid transport through the human body
Lipids can not move in body fluids due to its hydrophobic nature. Therefore, to allow transport in the body is combined with proteins called betaglobulins to form lipoproteins. Once the lipids have been absorbed through the gut, are combined in the blood plasma of polypeptide chains to produce a family of different lipoproteins, which are classified according to their density, determined by centrifugation. As lipids are less dense than proteins, there is an inverse relationship between lipid content and density for example, a high lipid content means low density particles.
The major classes of lipoproteins
Basically, lipoproteins are grouped into 3 main categories: Chylomicron (QM) and very low density protein ( "Very Low Density Lipropotein 'or VLDL). Are relatively low in protein, phospholipids and cholesterol but high triglycerides (55 to 95%). More broadly, these particles are called lipoproteins rich in triglycerides. ntermediate density lipoproteins ( "Intermediate Density Lipoproteins" or IDL) and LDL ( "Low Density Lipoproteins" or LDL). They are characterized by high levels of cholesterol, mainly in the form of cholesterol esters. The second way mentioned cholesterol (LDL) is highly insoluble. Given that up to 50% of the mass of LDL cholesterol is not surprising that LDL have a significant role in the development of atherosclerotic disease. High density lipoproteins ( "High Density Lipoproteins" or HDL). The highlights of these particles is their high protein content (50%) and relatively high content of phospholipids (30%). Generally, they are divided into two HDL subclasses HDL2 and HDL3. The HDL2 are larger and less dense; the HDL3 are smaller and denser.
Main functions of the lipoproteins
Chylomicrons and very low density lipoprotein (VLDL) transport triacylglycerol through the body from food and endogenous (produced by the body). Low density lipoprotein (LDL) and high density lipoproteins (HDL) carry cholesterol from food and endogenous. HDL and high density lipoprotein (VHDL) transport the ingested and endogenous phospholipids. Lipoproteins consist of a central hydrophobic lipids surrounded by a deck of polar lipids which, in turn, is surrounded by a protein shell. The proteins used in the transport of lipids are synthesized in the liver and are called "apolipoproteins" or "apo". Up to 8 apolipoproteins may be involved in shaping the structure of a lipoprotein. The proteins are called Apo A-1, Apo A-2, Apo B-48, Apo C-3, etc..
Taken together, retain lipoproteins in the blood lipid concentration of 500 mg of total lipids in 100 ml of blood. Of these 500, 120 mg are triacylglycerols (TAG), 220 mg cholesterol and 160 mg it is phospholipid. LDL contain, typically, 50-70% of total serum cholesterol and both are directly related to the risk of heart disease or coronary. HDL containing typically 20-30% of total cholesterol, HDL levels are inversely related to risk of heart disease or coronary. VLDLs contain 10-15% of total serum cholesterol and the majority of serum triglycerides in post-fasting VLDL are precursors of LDL, it is presumed that some forms of VLDL, particularly VLDL waste, are atherogenic.
Small amounts of cholesterol are transported also two minor lipoprotein classes: intermediate-density lipoproteins ( "Intermediate Density Lipoproteins" or IDL), density 1,006-1,019 kg / L and lipoprotein (a) of density 1.045 -- 1.080 kg / L. Chylomicrons (density <1.006 kg / L) appear in the blood transiently after a fat meal and usually disappear completely within 12 hours. They are rich in triglycerides and responsible for the increase postprandial (after eating) of triglycerides in plasma but does not normally have significant effect on total cholesterol concentration.
Cholesterol levels in patients
The level of cholesterol in the blood is determined partly by inheritance and partly by acquired factors such as diet, calories and physical activity level. Factors that affect blood cholesterol include age, sex, weight, diet, alcohol and snuff, exercise, genetic factors, family history, medications, menopausal status, use of hormone replacement therapy and chronic disorders such as hypothyroidism, obstructive liver disease, pancreatic disease (including diabetes) and kidney disease. In many people, high blood cholesterol poses a high risk of developing a coronary artery disease. Blood levels of total cholesterol and various cholesterol fractions, particularly LDL cholesterol, HDL-cholesterol are useful in the evaluation and treatment monitoring of patients with cardiovascular and related diseases.
Blood levels of these components of cholesterol, including triglyceride have been separated in the 'desirable', 'limit' and 'high risk' by the National Institute of Heart, Lung, and Blood Institute of the United States In its report of 1993. These categories form a useful basis for evaluation and treatment of patients with hyperlipidemia (lipid levels above normal). Therapy to reduce these risk parameters includes diet, exercise and medication and reduction of body fat mass, which is particularly effective when combined with diet or exercise.
Lipoprotein concentrations in plasma
The level of lipids in plasma is the most commonly used clinical indicator to evaluate the potential risk of premature cardiovascular disease. The levels of triglycerides, cholesterol and HDL-cholesterol post-fasting can also be used to identify abnormalities. Typically, women with the lowest concentration of triglycerides (80 mg / Dl.) Regarding the men (120 mg / Dl.) Women also have higher HDL-cholesterol level (55mg/Dl. Versus 43 mg / Dl. for men). The newborn baby has levels of triglycerides and total cholesterol between half and one third of an adult. The HDL-cholesterol levels are relatively high in the newborn (35 mg / Dl.)
In which the ratio of total cholesterol and HDL-cholesterol is equal to 2, in adults the ratio is 3.5 for women and 4.6 for men. Lipid levels in infants are perhaps the 'ideal' birth, the plasma total cholesterol is low while HDL-cholesterol is relatively high. Except in the case of genetic abnormalities, the vascular walls of newborns are free of traces of fat. The accumulation of fat occurs during the first years of life, indicating that food intake and environmental factors probably influence the initiation and progression of atherosclerosis. At birth, no differences between male and female infants and that sex hormones apparently have a limited influence in this stage of development. The apolipoprotein (Apo) are structural components of plasma lipoproteins, which play an important role in regulating metabolism. Of the nine known apolipoproteins, all differ in their amino acid content and its molecular weight, their plasma concentration in healthy individuals is in the range from 0.03 to 0.15 g / l.
The apolipoproteins have a typical molecular conformation known as amphipathic alpha helix, in which hydrophobic portion integrates a high content of nonpolar amino acids and the hydrophilic portion integrates the polar residues of the amino acids are abundant. Each structure is essential for the integrity of the lipoprotein, to be able to interact with the lipids in the hydrophobic portion of the lipoprotein molecule and interact simultaneously with the aqueous environment.
Based on an alphabetical, apolipoproteins can be grouped into four families that include members of different structure, function and metabolic status.
Apolipoproteins A
Apolipoproteins A group of proteins are variably distributed on different lipoproteins, for example, Apo AI and Apo A-II Ia is found mainly in HDL, but also in chylomicrons. Apo A-IV in free form in plasma or lipoproteins.
Apolipoproteins
KDa molecular weight
Plasma concentration in g / l
Function
Apo AI
28.000
1.0 - 1.5
Activate LCAT enzyme
Apo AII
17.000
0.3 to 0.5
?
Apo AIV
46.000
0.15 to 0.20
Secretion of Chylomicrons and reverse cholesterol transport.
Apo B48
250.000
0.5
Secretion of chylomicrons
Apo B100
513.000
0.8 - 1.0
Interaction with LDL receptor
Apo CI
6.500
0.04 to 0.07
Activating LACT
Apo CII
8.500
0.03 to 0.08
Cofactor for LPL
Apo AIII
8.750
0.8 to 0.15
Inhibition of LPL and its receptor
Apo E
39.000
0.03 to 0.06
Interaction with LDL receptor and Apo E receptor
The Apo AI is the most abundant apolipoprotein in plasma, is present almost in full in HDL and is about 90% and 60-70% of the protein fraction in the subfractions HDL2 and HDL3, respectively. The plasma levels of Apo AI were generally higher in females and correlated positively with HDL-cholesterol concentration. This correlation is not valid in subjects with hypertriglyceridemia, where HDL is enriched with triglycerides and cholesterol almost absent. The Apo AI is synthesized initially in the liver and intestine as a precursor protein, which is degraded into its mature form in plasma, a single polypeptide chain containing 243 amino acids. As the most concentrated protein component of HDL, is actively involved in "reverse cholesterol transport," serving as an activator of the enzyme lecithin-cholesterol acetyltransferase (LCAT) and as a link to the HDL-receptor complex, located in the hepatocyte and various peripheral cells.
Apolipoprotein Apo A-II is the second largest protein component of HDL concentration, but is absent in the HDL2 subfraction, this is the third part as a protein component of HDL3. Apo A-II is found in lower concentrations in plasma for Apo AI, and plasma levels do not correlate with HDL-cholesterol levels. From a structural standpoint, the Apo A-II is different from other lipid binding proteins that is the only plasma apolipoproteins present as dimer. Apo A-II is composed of two polypeptide chains of 77 amino acids, linked by a disulfide bond of cysteine residues in position 6. The specific function of Apo-II is not clearly specified, but recent studies indicate a role in regulating the activity of hepatic lipase. However, a complete absence of Apo-A-II was observed in a Japanese family, non-associated with a metabolic disorder or significant medical condition. This confirms that the Apo A-Il has a low participation in the metabolism of lipids.
Apolipoprotein A-IV is found in minute concentrations in plasma and this is where it circulates freely, and also is bound to chylomicrons HDLA (about 50%). Apo A-IV is composed of a polypeptide chain composed of 376 amino acids, strongly shaped like an alpha-helix amphipathic nature, a condition that is needed to join chylomicrons in the intestinal cells and participate in the reverse transport of cholesterol or counterflow , encouraging interaction between HDL and cells.
Apolipoprotein B
Apolipoprotein B is a high molecular weight protein present in chylomicrons, VLDL and LDL lipoproteins. The plasma concentrations of Apo B are in the range of 0.8 - 1.0 g / l in normolipidemic individuals. Its concentration is directly correlative with the values of total cholesterol and HDL cholesterol.
Two molecular forms called Apo B100 and Apo B48, exist in plasma. The first is a single chain polypeptide of 4,536 amino acids is one of the largest proteins in plasma are synthesized in the liver and secreted in VLDL. This is quantitatively maintained during the conversion of VLDL to IDL to LDL, which is the only protein component. The Apo B 100 is essential for the coupling of the lipoprotein particles (VLDL). This plays an important role as a molecule, ligand for LDL and its receptor. It also participates in the regulation of cholesterol blood level.
Apo B48 is composed of a polypeptide chain of amino acids 2.152 (these amino acids are similar to those of Apo B 100, therefore, Apo B48 is 48% similar with respect to Apo B 100). The plasma levels of Apo B48 in a normal subject over a period of fasting, is 50 times smaller compared to the concentration of Apo B 100. This concentration has a notable increase during the postprandial period. Apo B48 is synthesized in the intestine and is a molecule essential for the formation of chylomicrons.
Apolipoprotein C
A family of low molecular weight proteins including Apo CI, C-Il and C-III. All three apolipoproteins differ in their molecular weight, amino acid composition and function. The C apolipoproteins are synthesized in greater proportion in the liver and to a lesser extent in intestine are present in lipoproteins that comprise mostly triglycerides, as in the case of chylomicrons, VLDL, HDL. Apo C in plasma has an important role in maintaining the dynamic balance between HDL and VLDL quiomicrones. The plasma concentration in normal subjects is very low, 0.03 g / l for Apo C-II and 0.15 g / l for Apo C-III. You can only see an increase in postprandial periods and in patients with hypertriglyceridemia. Apo apolipoprotein CI is smaller, is composed of 57 amino acids. In vitro processes can activate the enzyme lecithin-cholesterol acetyltransferase (LCAT). This situation indicates that performs the same function in vivo, but the concentration and affinity for the enzyme is higher than that of Apo AI.
Apo C-Il is a polypeptide of 79 amino acids, which are variably distributed according to the different classes of lipoproteins. This plays an important role in regulating metabolism of triglycerides, is in fact an essential cofactor for lipoprotein lipase activity, the enzyme responsible for hydrolysis of triglycerides in lipoproteins, and is critical for the catabolism as chylomicrons and VLDL.
Apo C-III consists of 79 amino acids and is present in plasma in its glycosylated form. In relation to an isoelectric analysis, there are three isoforms III0 identifiable C-, C-III1 and C-III2, depending on the sialic acid molecules to which it is attached (which serves to facilitate its binding to its receptor or other molecules ). Apo C-II and C-III are involved in the regulation of lipoprotein lipase, generating an inhibitory effect on it.
Apolipoprotein E
The Apo E is a polypeptide of 299 amino acids, found in VLDL and LDL and HDL subfraction as a call HDL1. The plasma concentration in normal subjects is 0.03 to 0.07 g / l and reaches 2 to 3 fold increase in hyperlipoproteinemia and in a condition known as beta-wide disease, characterized by the presence of a thick band of lipoproteins that migrate to the region a pre-beta electrophoretic shift. The Apo E is found in humans three isoforms recognized by isoelectric analysis, called E2, E3 and E4. The three isoforms differ from each other by a single amino acid substitution (arginine to cysteine) in two specific positions in the sequence of Apo E. The presence of three isoforms, each encoded by a single allele, generating six different phenotypes, three homozygous (E2/E2, E3/E3 and e4/e4) and three heterozygous (E2/E3, E2/E4 and E3 / E4), variably distributed in the population. The E3/E3 phenotype is the most common (60% of the population) and the E2/E2 is the rarest and serves as an absolute test of hyperlipoproteinemia type III. The Apo E is known for its specific receptor (present in the liver responsible for the catabolism of waste.
The association between abnormalities of lipid metabolism and incidence of cardiovascular disease is well known all on the basis of numerous epidemiological studies that have been documented on paper has atherogenic LDL, as well as the protective or anti-atherogenic of HDL.
The direct relationship between increased lipid levels and atherosclerosis is confirmed by the studies carried out after the early 80's, which clearly showed that low levels of blood cholesterol are associated with reduced cardiovascular events and delayed atherosclerotic disorders. It is important that health authorities in each country in the world, through campaigns to inform people of the role of plasma lipids in the pathogenesis of atherosclerosis and establish criteria for identifying coronary risk.
Currently blood cholesterol concentrations related to different conditions are:
To clarify the cardiovascular risk in patients with cholesterol above 240 mg / dl (6.21 mmol / L) required as a next step to determining associated with atherogenic lipoprotein cholesterol as LDL cholesterol. To determine directly the analyte is necessary ultracentrifugation of the sample, since the use of alternative methods do not give real results, however, the required equipment is only available at specialized centers. LDL cholesterol concentrations can never be estimated with fair approximation by applying the well-known formula.
LDL Cholesterol = Total Cholesterol - HDL Cholesterol - Triglycerides / 5 results expressed in mg / dl
LDL Cholesterol = Total Cholesterol - HDL Cholesterol - Trigliceridos/2.2 results expressed in mmol / L
Note: the equation should never be used when triglycerides exceed 400 mg / dl (4.52 mmol / L)
LDL values determined are the key to making a clinical decision and start treatment to lower cholesterol levels. he consensus data from epidemiological studies suggest restricting cholesterol values as only data for a logical and realistic medical evacuation of the status of the individual, considering the values of LDL-cholesterol as an atherogenic high-value data. However, two other parameters such as lipid and lipoprotein triglycerides and HDL-cholesterol, play an important role in establishing cardiovascular risk in each individual. Today is known throughout the health personnel that raising levels of HDL-cholesterol is a protective factor for atherosclerosis. This also is set in an epidemiological nature (with application of retrospective and prospective studies). A mild form of hypo-alpha-lipo-proteinemia is a form of dyslipidemia diagnosed based on values less than 35 mg / dl, HDL cholesterol, a condition invariably associated with a high incidence of cardiovascular disease. However, this is not enough direct evidence that an increase in HDL-cholesterol is the cause of improvement in cardiovascular fitness.
Some studies have shown the predictive value of HDL-Cholestero figures in the identification of subjects with cardiovascular risk. However, some clinicians interested in this parameter have limited their use because they have observed individual changes in their assessments, depending on the method used.
HDL cholesterol can be considered as a parameter for lipid-lowering therapies, but the change may be minimal in a positive way. There is more controversy over whether the triglycerides have an important role in defining cardiovascular risk. While the American College of Pathologists triglycerides does not consider an independent risk factor, the European School (particularly Scandinavian) for many years has identified a factor in hypertriglyceridemia primitive and independent coronary risk. The mechanisms responsible for the atherogenicity resulting from increased blood triglycerides, you probably are in the structural and functional modifications of lipoproteins that become more atherogenic risk factor for patients with hypertriglyceridemia. For example in the LDL lipoprotein, when the content is high triglycerides and low in cholesterol esters, exhibits a reduced affinity for its specific receptors and is more susceptible to catabolism, causing accumulation of lipids in the walls of the veins.
In a more constant and evident is the reduction of HDL-cholesterol levels in patients with hypertriglyceridemia, where values are frequently below 35 mg / dl (0.91 mmol / L). The clinical significance of this phenomenon is not well established, since the reduction of HDL-cholesterol levels did not reflect a decreased number of circulating particles but a simple reduction in cholesterol content.
Hypertriglyceridemia is also associated with impaired carbohydrate metabolism and coagulation, a situation that may at any time aggravate the vascular condition of the individual. It is therefore very important to define the role or not of triglycerides in the development of atherosclerosis.
Recently, the list of lipid and lipoprotein parameters that give us a prognostic value of atherosclerosis has been extended to other components of lipoprotein, particularly toward the apolipoproteins and lipoproteins abnormal or malignant called lipoprotein a or Lp a that is a macromolecular complex formed by LDL, bound to a glycoprotein.
Lipoprotein a is structurally homologous to plasminogen and exists in plasma in several isoforms of different molecular weight, ranging from 200 to 700 KD. Blood levels are also variable ranges from 0 to 1.0 g / L. The physiological role of Lp (a) is not well defined, but a plasma concentration of 0.3 g / dl is associated with a high incidence of atherosclerosis in coronary and peripheral level. Lipoprotein (a) appears to be involved in atherosclerotic plaque formation by a dual mechanism: inhibition of fibrinolysis and lipid accumulation as part of the atherosclerotic plaque.
Clinical significance of analysis of apolipoproteins
The plasma concentration of Apo AI is reduced by 15% and the concentration of Apo B, increased 43% in patients with myocardial infarction, when compared the concentration of healthy control individuals. The relationship Apo AI / B (ratio of anti-atherogenic apolipoproteins and atherogenic) was reduced by 40% in patients with myocardial infarction, these levels and their relationship show discriminating value between normal patients and cardiac risk, with greater evidence and clearness that the classic lipid and lipoprotein parameters.
Several studies have confirmed the decline, sometimes moderate, Apo AI and significant and continuous increase in the levels of Apo B in patients with myocardial infarction and vascular complications generally manifest.
Consequently, the relationship Apo AI / B is considered by many authors as a powerful indicator of coronary risk.
Evaluation of apolipoproteins in patients subjected to coronary angiography has indicated a strong correlation of values Apo AI and Apo B as the marker that represents a deterioration in coronary arterial system. The relationship Apo AI / B should be used not only to identify subjects with coronary risk, but also as an important index of the severity and progression of atherosclerotic disease. Other assessments of apolipoproteins that have been added to the classical measurements of Apo AI and Apo B for years, are the analysis of Apo A-Il and Apo E but so far there is no correlation of results to determine cardiovascular risk.
Other reasons for assessing the apolipoproteins and give clinically useful information is that the apolipoprotein is the best index for estimating the number of particles in the bloodstream, particularly important in the case of patients with hypertriglyceridemia in which lipoproteins such as LDL, HDL, high triglycerides show, so they exhibit less of cholesterol, therefore, the result of HDL-cholesterol is wrongly interpreted in this sense, the determination of the concentration of Apo AI provides accurate information to estimate the HDL level in the patient.
The determination of Apo A-Il and the ratio AI / A-II is an alternative to the difficult assessment of Ultracentrifugation recommended methodology for estimating the fractions of HDL. The determination of apolipoproteins is a specific marker to distinguish abnormalities of lipid metabolism. In the case of a hiperapobetalipoproteinemia, a disorder associated with high coronary risk, the plasma concentration of Apo B is elevated in the presence of normal levels of total cholesterol and LDL cholesterol.
Practical problems in the analysis of apolipoproteins
The clinical utility of the determination of apolipoproteins is mainly the identification of cardiovascular risk and determine the condition of individual metabolism, but there are some technical aspects that are important to consider, depending on the characteristics of the methodology used for evaluation. The first point to consider is that some characteristics of lipoproteins may affect the determination of apolipoproteins.
Lipoproteins are a heterogeneous system in which the apolipoproteins are distributed in a different variable in particle size and structure. Because of this, it is important that the methods used for determining the levels of apolipoproteins are able to recognize and quantify apolipoproteins contained in the various lipoprotein particles.
Lipoproteins tend to aggregate in vitro, a condition that may manifest as a disadvantage for the storage of the sample or the proper preparation of a standard.
Finally, the real problem is to establish normal values and abnormal for the various apolipoproteins. The current analysis methods should be standardized and established international reference levels as requirements to achieve this clinical goal.
Overview of immunochemical methods
Today immunochemical methods have been expanded and appreciated in the clinical laboratory for sensitivity, specificity and quality. The main techniques to assess and quantify apolipoproteins Apo AI and Apo B include:
• Radioimmunoassay (RIA)
• immunoassay (ELISA)
• radial immunodiffusion (RID)
• Electroinmunodifusión (EID)
• nephelometry (INA)
• Immunoturbidimetry (ITA)
These methods are not free from criticism. The main problem is that the apolipoproteins are not present in the serum in isolation, but as large particles chemically heterogeneous. This causes variable response, sometimes significantly, depending on the characteristics of the samples (normolipemia or hyperlipidemia) antibody used, as well as calibration material. By employing an antibody in the identification of a specific protein allowed a more accurate measurement, despite being in a medium containing other proteins without the need for preliminary separation or purification. The specificity of the antibody directed towards the immunogen depends notably apolipoprotein or apolipoprotein used purified or not. The rapid development of various methods of imnunoensayo has generated a large number of facts and observations, although some discordant for nature and reactivity of the calibrators used. Unfortunately, at present clinical laboratories lack a simple method, no immunochemical, in order to properly ascertain the stability of plasma apolipoproteins, with which they can be often referred to the results obtained. Regarding the analytical precision of the immunoassay, is inferior to other techniques, the coefficient of variation (% CV) is generally above 2% for the results obtained in one run and increases of 7-8% in those who are processed in different runs. The inaccuracy is due not only to reagents (polyclonal antibodies), which show different avidity, specificity and affinity toward the antigen, but also wing predilection of the sample and the analytical procedure itself. Also consider the errors of the analytical phase.
Collection, storage and sample preservation
The variables tested for determination of Apo AI by radioimmunoassay (RIA) were:
a) The time elapsed between obtaining a blood specimen and the separation of plasma (which may be important in epidemiological studies where the sample is obtained away from the laboratory);
b) The presence of protease inhibitors (necessary to maintain the structural integrity of the Apo B);
c) The preservation at -70 º C for six weeks.
d) The addition of various preservatives or additives to the plasma such as antibiotics, bacteriostatic and antifungal (which can serve to protect the integrity of Apo AI during freezing and thawing).
The results of this study showed no significant effects for different variables, mentioned above, the authors note that this conclusion applies only to the method of RIA. The same study was also performed by Albers et. al. (1980) where they discussed the preservation of samples for the determination of Apo AI and Apo-II by radial immunodiffusion method (RID), The findings in this case indicated that the sample can be kept at 4 ° C for one month and half, and in the case to be free of bacterial contamination is stable for 2-3 years at 20 º C.
Studies by different authors on a recent "report of standardization for the determination of apolipoprotein AI and B" of this information is derived, that in the methods of nephelometry (INA) and radial immunodiffusion (RID) by using an antibody mono or polyclonal unaffected by the presence of anticoagulant (heparin or EDTA), however, the serum sample is recommended because the plasma provides a decrease of 3 to 4% in the results for sample dilution (plasma) as a result of the exit of intracellular fluid, the amount of anticoagulant used and the effect of freezing and thawing where active fibrin formation.
The concentrations of both apolipoproteins in the samples is not significantly changed after being kept 18 days at 4 º C. By contrast, when the samples stored for 6 months at -20 ° C or -70 ° C and analyzed by nephelometry show significant changes in concentration, while the results are constant radial immunodiffusion be preserved even after a year in the same conditions.
Recent research has shown that by employing the method of Immunoturbidimetry (ITA), preservation of samples for two months at -20 ° C or -70 ° C does not affect the concentration of Apo AI and Apo-B.