Hyperlipidemia (hyperlipemia) - increase in concentration total lipids plasma like physiological phenomenon can be observed 1-4 hours after eating. Nutritional hyperlipemia is more pronounced, the lower the level of lipids in the patient’s blood on an empty stomach.
The concentration of lipids in the blood changes under a number of pathological conditions:
Nephrotic syndrome, lipoid nephrosis, acute and chronic nephritis;
Biliary cirrhosis of the liver, acute hepatitis;
Obesity - atherosclerosis;
Hypothyroidism;
Pancreatitis, etc.
The study of cholesterol (CH) levels reflects only the pathology of lipid metabolism in the body. Hypercholesterolemia is a documented risk factor coronary atherosclerosis. CS is an essential component of the membrane of all cells; the special physicochemical properties of CS crystals and the conformation of its molecules contribute to the orderliness and mobility of phospholipids in membranes when temperature changes, which allows the membrane to be in an intermediate phase state (“gel - liquid crystal”) and preserve physiological functions. CS is used as a precursor in the biosynthesis of steroid hormones (gluco- and mineralocorticoids, sex hormones), vitamin D 3, as well as bile acids. Conventionally, we can distinguish 3 pools of cholesterol:
A - quickly exchanging (30 g);
B – slowly exchanging (50 g);
B – very slowly exchanging (60 g).
Endogenous cholesterol is synthesized in significant quantities in the liver (80%). Exogenous cholesterol enters the body as part of animal products. Transport of cholesterol from the liver to extrahepatic tissues is carried out
LDL. The removal of cholesterol from the liver from extrahepatic tissues into the liver is produced by mature forms of HDL (50% - LDL, 25% HDL, 17% VLDL, 5% -CM).
Hyperlipoproteinemia and hypercholesterolemia (Fredrickson classification):
Type 1 – hyperchylomicronemia;
type 2 - a - hyper-β-lipoproteinemia, b - hyper-β and hyperpre-β-lipoproteinemia;
type 3 – dys-β-lipoproteinemia;
type 4 – hyper-pre-β-lipoproteinemia;
Type 5 – hyper-pre-β-lipoproteinemia and hyperchylomicronemia.
The most atherogenic are types 2 and 3.
Phospholipids are a group of lipids containing, in addition to phosphoric acid (an essential component), alcohol (usually glycerol), fatty acid residues and nitrogenous bases. In clinical and laboratory practice, there is a method for determining the level of total phospholipids, the level of which increases in patients with primary and secondary hyperlipoproteinemia IIa and IIb. A decrease occurs in a number of diseases:
Nutritional dystrophy;
Fatty liver degeneration,
Portal cirrhosis;
Progression of atherosclerosis;
Hyperthyroidism, etc.
Lipid peroxidation (LPO) is a free radical process, the initiation of which occurs with the formation of reactive oxygen species - superoxide ion O 2 . ; hydroxyl radical HO . ; hydroperoxide radical HO 2 . ; singlet oxygen O 2 ; hypochlorite ion ClO - . The main substrates of LPO are polyunsaturated fatty acids found in the structure of membrane phospholipids. The strongest catalyst is iron metal ions. SEX is a physiological process that has important for the body, as it regulates membrane permeability, affects cell division and growth, begins phagosynthesis, is a pathway for the biosynthesis of certain biological substances(prostaglandins, thromboxanes). The level of lipid peroxidation is controlled by the antioxidant system ( ascorbic acid, uric acid, β-carotene, etc.). Loss of balance between the two systems leads to the death of cells and cellular structures.
For diagnostic purposes, it is customary to determine the content of lipid peroxidation products (diene conjugates, malondialdehyde, Schiff bases) and the concentration of the main natural antioxidant - alpha-tocopherol in plasma and red blood cells with the calculation of the MDA/TF coefficient. An integral test for assessing LPO is determining the permeability of erythrocyte membranes.
2. Pigment exchange a set of complex transformations of various colored substances in the human and animal body.
The most well-known blood pigment is hemoglobin (a chromoprotein that consists of the protein part of globin and a prosthetic group represented by 4 hemes, each heme consists of 4 pyrrole nuclei, which are interconnected by methine bridges, in the center there is an iron ion with an oxidation state of 2 +) . The average lifespan of an erythrocyte is 100-110 days. At the end of this period, destruction and destruction of hemoglobin occurs. The disintegration process begins already in vascular bed, ends in the cellular elements of the system of phagocytic mononuclear cells (Kupffer cells of the liver, histiocytes connective tissue, plasma cells bone marrow). Hemoglobin in the vascular bed binds to plasma haptoglobin and is retained in the vascular bed without passing through the renal filter. Due to the trypsin-like action of the beta chain of haptoglobin and the conformational changes caused by its influence in the porphyrin ring of the heme, conditions are created for easier destruction of hemoglobin in the cellular elements of the phagocytic mononuclear system. The resulting high-molecular green pigment verdoglobin(synonyms: verdohemoglobin, choleglobin, pseudohemoglobin) is a complex consisting of globin, a broken porphyrin ring system and ferric iron. Further transformations lead to the loss of iron and globin by verdoglobin, as a result of which the porphyrin ring unfolds into a chain and a low molecular weight green bile pigment is formed - biliverdin. Almost all of it is enzymatically restored into the most important red-yellow pigment of bile - bilirubin, which is a common component of blood plasma. On the surface plasma membrane hepatocyte undergoes dissociation. In this case, the released bilirubin forms a temporary associate with the lipids of the plasma membrane and moves through it due to the activity of certain enzyme systems. Further passage of free bilirubin into the cell occurs with the participation of two carrier proteins in this process: ligandin (it transports the main amount of bilirubin) and protein Z.
Ligandin and protein Z are also found in the kidneys and intestines, therefore, in case of insufficient liver function, they are free to compensate for the weakening of detoxification processes in this organ. Both are quite soluble in water, but lack the ability to move through the lipid layer of the membrane. By binding bilirubin to glucuronic acid, the inherent toxicity of free bilirubin is largely lost. Hydrophobic, lipophilic free bilirubin, easily dissolving in membrane lipids and consequently penetrating into mitochondria, uncouples respiration and oxidative phosphorylation in them, disrupts protein synthesis, the flow of potassium ions through the membrane of cells and organelles. This has a negative impact on the condition of the central nervous system, causing in patients a number of characteristic neurological symptoms.
Bilirubin glucuronides (or bound, conjugated bilirubin), unlike free bilirubin, immediately react with the diazo reagent (“direct” bilirubin). It should be borne in mind that in the blood plasma itself, bilirubin that is not conjugated with glucuronic acid can either be associated with albumin or not. The last fraction (bilirubin not associated with albumin, lipids, or other blood components) is the most toxic.
Bilirubin glucuronides, thanks to the enzyme systems of membranes, actively move through them (against the concentration gradient) into bile ducts, excreted along with bile into the intestinal lumen. In it, under the influence of enzymes produced by intestinal microflora, the glucuronide bond is broken. The released free bilirubin is reduced to form first mesobilirubin and then mesobilinogen (urobilinogen) in the small intestine. Normally, a certain part of mesobilinogen is absorbed in the small intestine and in the upper part of the colon, through the system portal vein enters the liver, where it is almost completely destroyed (by oxidation), turning into dipyrrolic compounds - propent-diopent and mesobileucane.
Mesobilinogen (urobilinogen) does not enter the general circulation. Part of it, together with the products of destruction, is again sent into the intestinal lumen as part of bile (enterohepotic circulation). However, even with the most minor changes in the liver, it barrier function is largely “removed” and mesobilinogen enters first into the general blood circulation and then into the urine. The bulk of it is directed from small intestine into the thick one, where under the influence of anaerobic microflora (Escherichia coli and other bacteria) it undergoes further reduction with the formation of stercobilinogen. The resulting stercobilinogen (daily amount 100-200 mg) is almost completely excreted in the feces. In air, it oxidizes and turns into stercobilin, which is one of the pigments of feces. A small part of stercobilinogen is absorbed through the mucous membrane of the large intestine into the inferior vena cava system, delivered in the blood to the kidneys and excreted in the urine.
So in the urine healthy person There is no mesobilinogen (urobilinogen), but it contains some stercobilin (which is often incorrectly called “urobilin”)
To determine the content of bilirubin in blood serum (plasma), chemical and physico-chemical methods studies, including colorimetric, spectrophotometric (manual and automated), chromatographic, fluorimetric and some others.
One of the important subjective signs of a disorder of pigment metabolism is the appearance of jaundice, which is usually noted when the level of bilirubin in the blood is 27-34 µmol/l or more. The causes of hyperbilirubinemia can be: 1) increased hemolysis of red blood cells (more than 80% total bilirubin represented by unconjugated pigment); 2) impaired liver cell function and 3) delayed bile outflow (hyperbilirubinemia is of hepatic origin if more than 80% of total bilirubin is conjugated bilirubin). In the first case, they talk about the so-called hemolytic jaundice, in the second – about parenchymal jaundice (can be caused by hereditary defects in the processes of transport of bilirubin and its glucuronidation), in the third – about mechanical (or obstructive, congestive) jaundice.
With parenchymal form of jaundice destructive-dystrophic changes are noted in the parenchymal cells of the liver and infiltrative ones in the stroma, leading to an increase in pressure in the liver bile ducts. Stagnation of bilirubin in the liver is also facilitated by a sharp weakening of metabolic processes in affected hepatocytes, which lose the ability to normally perform various biochemical and physiological processes, in particular, transfer bound bilirubin from cells into bile against a concentration gradient. An increase in the concentration of conjugated bilirubin in the blood leads to its appearance in the urine.
The most “subtle” sign of liver damage in hepatitis is the appearance mesobilinogen(urobilinogen) in the urine.
With parenchymal jaundice, the concentration of bound (conjugated) bilirubin in the blood increases mainly. The content of free bilirubin increases, but to a lesser extent.
The pathogenesis of obstructive jaundice is based on the cessation of bile flow into the intestine, which leads to the disappearance of stercobilinogen from the urine. With congestive jaundice, the content of conjugated bilirubin in the blood increases mainly. Extrahepatic cholestatic jaundice is accompanied by a triad clinical signs: Discolored stool, dark urine and itchy skin. Intrahepatic cholestasis is clinically manifested by skin itching and jaundice. At laboratory research hyperbilirubinemia (due to associated), bilirubinuria, increased alkaline phosphatase with normal values transaminases in blood serum.
Hemolytic jaundice are caused by hemolysis of red blood cells and, as a consequence, increased formation of bilirubin. An increase in free bilirubin is one of the main signs of hemolytic jaundice.
IN clinical practice distinguish congenital and acquired functional hyperbilirubinemia, caused by a violation of the elimination of bilirubin from the body (the presence of defects in enzyme and other systems for the transfer of bilirubin through cell membranes and its glucuronidation in them). Gilbert's syndrome is a hereditary benign chronic disease that occurs with moderate non-hemolytic unconjugated hyperbilirubinemia. Post-hepatitis hyperbilirubinemia Kalka - acquired enzyme defect leading to an increase in the level of free bilirubin in the blood, congenital familial non-hemolytic jaundice of Crigler - Nayjar (absence of glucuronyltransferase in hepatocytes), jaundice with congenital hypothyroidism (thyroxine stimulates the enzyme glucuronyltransferase system), physiological jaundice of newborns, drug jaundice, etc. .
Disturbances in pigment metabolism can be caused by changes not only in the processes of heme decomposition, but also in the formation of its precursors - porphyrins (cyclic organic compounds based on a porphin ring consisting of 4 pyrroles connected by methine bridges). Porfiria – group hereditary diseases, accompanied by a genetic deficiency in the activity of enzymes involved in the biosynthesis of heme, in which an increase in the content of porphyrins or their precursors is detected in the body, which causes a number of clinical signs (excessive formation of metabolic products, causes the development of neurological symptoms and (or) increased photosensitivity of the skin).
The most widely used methods for the determination of bilirubin are based on its interaction with a diazoreagent (Ehrlich's reagent). The Jendrassik-Grof method has become widespread. In this method, a mixture of caffeine and sodium benzoate in acetate buffer is used as a “liberator” of bilirubin. The enzymatic determination of bilirubin is based on its oxidation by bilirubin oxidase. It is possible to determine unconjugated bilirubin by other methods of enzymatic oxidation.
Currently, the determination of bilirubin using “dry chemistry” methods is becoming increasingly widespread, especially in rapid diagnostics.
Vitamins.
Vitamins are essential low-molecular substances that enter the body with food from the outside and are involved in the regulation of biochemical processes at the enzyme level.
Similarities and differences between vitamins and hormones.
Similarities– regulate metabolism in the human body through enzymes:
· Vitamins are part of enzymes and are coenzymes or cofactors;
· Hormones or regulate the activity of existing enzymes in the cell, or are inducers or repressors in the biosynthesis of necessary enzymes.
Difference:
· Vitamins– low molecular weight organic compounds, exogenous factors regulating metabolism and come from food from the outside.
· Hormones– high molecular weight organic compounds, endogenous factors, synthesized in the endocrine glands of the body in response to changes in external or internal environment the human body, and also regulate metabolism.
Vitamins are classified into:
1. Fat soluble: A, D, E, K, A.
2. Water-soluble: group B, PP, H, C, THFA (tetrahydrofolic acid), pantothenic acid(B 3), P (rutin).
Vitamin A (retinol, antixerophthalmic) – the chemical structure is represented by a β-ionone ring and 2 isoprene residues; The body's need is 2.5-30 mg per day.
The earliest and specific sign hypovitaminosis A - hemeralopia (night blindness) - impaired twilight vision. Occurs due to a lack visual pigment- rhodopsin. Rhodopsin contains retinal (vitamin A aldehyde) as an active group - located in the retinal rods. These cells (rods) perceive low-intensity light signals.
Rhodopsin = opsin (protein) + cis-retinal.
When rhodopsin is excited by light, cis-retinal, as a result of enzymatic rearrangements inside the molecule, transforms into all-trans-retinal (in the light). This leads to a conformational rearrangement of the entire rhodopsin molecule. Rhodopsin dissociates into opsin and trans-retinal, which is a trigger that excites in the endings optic nerve an impulse that is then transmitted to the brain.
In the dark, as a result of enzymatic reactions, trans-retinal is converted back into cis-retinal and, combining with opsin, forms rhodopsin.
Vitamin A also affects growth and development processes cover epithelium. Therefore, with vitamin deficiency, damage to the skin, mucous membranes and eyes is observed, which manifests itself in pathological keratinization of the skin and mucous membranes. Patients develop xerophthalmia - dryness of the cornea of the eye, as the lacrimal canal becomes blocked as a result of keratinization of the epithelium. Since the eye ceases to be washed with tears, which have a bactericidal effect, conjunctivitis, ulceration and softening of the cornea - keratomalacia - develop. With vitamin A deficiency there may also be damage to the gastrointestinal mucosa, respiratory and genitourinary tract. The resistance of all tissues to infections is impaired. With the development of vitamin deficiency in childhood, growth retardation occurs.
Currently, the participation of vitamin A in protecting cell membranes from oxidants has been shown - that is, vitamin A has an antioxidant function.
Lipids are called fats that enter the body with food and are formed in the liver. Blood (plasma or serum) contains 3 main classes of lipids: triglycerides (TG), cholesterol (CS) and its esters, phospholipids (PL).
Lipids are able to attract water, but most of them do not dissolve in the blood. They are transported in a protein-bound state (in the form of lipoproteins or, in other words, lipoproteins). Lipoproteins differ not only in composition, but also in size and density, but their structure is almost the same. central part(core) is represented by cholesterol and its esters, fatty acids, triglycerides. The shell of the molecule consists of proteins (apoproteins) and water-soluble lipids (phospholipids and non-esterified cholesterol). The outer part of apoproteins is capable of forming hydrogen bonds with water molecules. Thus, lipoproteins can be partially dissolved in fats and partially in water.
Chylomicrons, after entering the blood, break down into glycerol and fatty acids, resulting in the formation of lipoproteins. Cholesterol-containing chylomicron residues are processed in the liver.
Cholesterol and triglycerides are formed in the liver into very low-density lipoproteins (VLDL), which release some of the triglycerides to peripheral tissues, while the remainder goes back to the liver and is converted into low-density lipoproteins (LDL).
L PN II are transporters of cholesterol for peripheral tissues, which is used to build cell membranes and metabolic reactions. In this case, non-esterified cholesterol enters the blood plasma and binds to high-density lipoproteins (HDL). Esterified cholesterol (bound to esters) is converted into VLDL. Then the cycle repeats.
The blood also contains intermediate density lipoproteins (IDL), which are remnants of chylomicrons and VLDL and contain large amounts of cholesterol. DILI in liver cells with the participation of lipase are converted into LDL.
Blood plasma contains 3.5-8 g/l of lipids. An increase in blood lipid levels is called hyperlipidemia, and a decrease is called hypolipidemia. The indicator of total blood lipids does not provide a detailed picture of the state of fat metabolism in the body.
Quantitative determination of specific lipids is of diagnostic importance. The lipid composition of blood plasma is presented in the table.
Lipid composition of blood plasma
| Lipid fraction | Normal indicator | |
| General lipids | 4.6-10.4 mmol/l | |
| Phospholipids | 1.95-4.9 mmol/l | |
| Lipid phosphorus | 1.97-4.68 mmol/l | |
| Neutral fats | 0-200 mg% | |
| Triglycerides | 0.565-1.695 mmol/l (serum) | |
| Non-esterified fatty acids | 400-800 mmol/l | |
| Free fatty acids | 0.3-0.8 µmol/l | |
| Total cholesterol (there are age-specific norms) | 3.9-6.5 mmol/l (unified method) | |
| Free cholesterol | 1.04-2.33 mmol/l | |
| Cholesterol esters | 2.33-3.49 mmol/l | |
| HDL | M | 1.25-4.25 g/l |
| AND | 2.5-6.5 g/l | |
| LDL | 3-4.5 g/l | |
Dyslipidemias are divided into primary, associated with inborn errors of metabolism, and secondary. The causes of secondary dyslipidemia are physical inactivity and excess nutrition, alcoholism, diabetes mellitus, hyperthyroidism, liver cirrhosis, and chronic renal failure. In addition, they can develop during treatment with glucocorticosteroids, B-blockers, progestins and estrogens. The classification of dyslipidemias is presented in the table.
Classification of dyslipidemias
| Type | Increased blood levels | |
| Lipoproteins | Lipids | |
| I | Chylomicrons | Cholesterol, triglycerides |
| On | LDL | Cholesterol (not always) |
| Type | Increased blood levels | |
| Lipoproteins | Lipids | |
| Nb | LDL, VLDL | Cholesterol, triglycerides |
| III | VLDL, LPPP | Cholesterol, triglycerides |
| IV | VLDL | Cholesterol (not always), triglycerides |
| V | Chylomicrons, VLDL | Cholesterol, triglycerides |
– a group of heterogeneous chemical structure and physical and chemical properties of substances. In blood serum they are represented mainly by fatty acids, triglycerides, cholesterol and phospholipids.
Triglycerides are the main form of lipid storage in adipose tissue and lipid transport in the blood. A study of triglyceride levels is necessary to determine the type of hyperlipoproteinemia and assess the risk of developing cardiovascular diseases.
Cholesterol performs essential functions: included in cell membranes, is a precursor of bile acids, steroid hormones and vitamin D, and acts as an antioxidant. About 10% of the Russian population have increased level cholesterol in the blood. This condition is asymptomatic and can lead to serious illnesses(atherosclerotic vascular lesions, coronary heart disease).
Lipids are insoluble in water, so they are transported by blood serum in combination with proteins. Lipid+protein complexes are called lipoproteins. And proteins that are involved in lipid transport are called apoproteins.
Several classes are present in blood serum lipoproteins: chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL).
Each lipoprotein fraction has its own function. synthesized in the liver and transport mainly triglycerides. playing important role in atherogenesis. Low-density lipoproteins (LDL) rich in cholesterol, deliver cholesterol to peripheral tissues. Levels of VLDL and LDL promote the deposition of cholesterol in the vascular wall and are considered atherogenic factors. High density lipoproteins (HDL) participate in the reverse transport of cholesterol from tissues, taking it away from overloaded tissue cells and transferring it to the liver, which “utilizes” it and removes it from the body. High HDL level is considered as an antiatherogenic factor (protects the body from atherosclerosis).
The role of cholesterol and the risk of developing atherosclerosis depends on which lipoprotein fractions it is included in. To assess the ratio of atherogenic and antiatherogenic lipoproteins, it is used atherogenic index.
Apolipoproteins- These are proteins that are located on the surface of lipoproteins.
Apolipoprotein A (ApoA protein) is the main protein component of lipoproteins (HDL), which transports cholesterol from peripheral tissue cells to the liver.
Apolipoprotein B (ApoB protein) is part of lipoproteins that transport lipids to peripheral tissues.
Measuring the concentration of apolipoprotein A and apolipoprotein B in blood serum provides the most accurate and unambiguous determination of the ratio of atherogenic and antiatherogenic properties of lipoproteins, which is assessed as the risk of developing atherosclerotic vascular lesions and coronary heart disease over the next five years.
To the study lipid profile includes the following indicators: cholesterol, triglycerides, VLDL, LDL, HDL, atherogenicity coefficient, cholesterol/triglycerides ratio, glucose. This profile gives full information about lipid metabolism, allows you to determine the risks of developing atherosclerotic vascular lesions, coronary heart disease, identify the presence of dyslipoproteinemia and type it, and also, if necessary, select the right lipid-lowering therapy.
Indications
Increased concentrationcholesterol It has diagnostic value with primary familial hyperlipidemia (hereditary forms of the disease); pregnancy, hypothyroidism, nephrotic syndrome, obstructive liver diseases, pancreatic diseases ( chronic pancreatitis, malignant neoplasms), diabetes mellitus.
Decreased concentrationcholesterol has diagnostic value for liver diseases (cirrhosis, hepatitis), starvation, sepsis, hyperthyroidism, megaloblastic anemia.
Increased concentrationtriglycerides has diagnostic value for primary hyperlipidemia (hereditary forms of the disease); obesity, excessive consumption carbohydrates, alcoholism, diabetes mellitus, hypothyroidism, nephrotic syndrome, chronic renal failure, gout, acute and chronic pancreatitis.
Decreased concentrationtriglycerides has diagnostic value for hypolipoproteinemia, hyperthyroidism, malabsorption syndrome.
Very low density lipoproteins (VLDL) used to diagnose dyslipidemia (types IIb, III, IV and V). High concentrations of VLDL in the blood serum indirectly reflect the atherogenic properties of the serum.
Increased concentrationlow density lipoprotein (LDL) has diagnostic value for primary hypercholesterolemia, dislipoproteinemia (types IIa and IIb); for obesity, obstructive jaundice, nephrotic syndrome, diabetes mellitus, hypothyroidism. Determination of LDL levels is necessary for prescribing long-term treatment, the goal of which is to reduce lipid concentrations.
Increased concentration has diagnostic value for liver cirrhosis and alcoholism.
Decreased concentrationhigh density lipoprotein (HDL) has diagnostic value for hypertriglyceridemia, atherosclerosis, nephrotic syndrome, diabetes mellitus, acute infections, obesity, smoking.
Level determination apolipoprotein A indicated for early assessment of the risk of coronary heart disease; identifying patients with a hereditary predisposition to atherosclerosis in a relatively at a young age; monitoring treatment with lipid-lowering drugs.
Increased concentrationapolipoprotein A has diagnostic value for liver diseases and pregnancy.
Decreased concentrationapolipoprotein A has diagnostic value for nephrotic syndrome, chronic renal failure, triglyceridemia, cholestasis, sepsis.
Diagnostic valueapolipoprotein B- the most accurate indicator of the risk of developing cardiovascular diseases, is also the most adequate indicator of the effectiveness of statin therapy.
Increased concentrationapolipoprotein B has diagnostic value for dyslipoproteinemia (IIa, IIb, IV and V types), coronary heart disease, diabetes mellitus, hypothyroidism, nephrotic syndrome, liver diseases, Itsenko-Cushing syndrome, porphyria.
Decreased concentrationapolipoprotein B has diagnostic value for hyperthyroidism, malabsorption syndrome, chronic anemia, inflammatory diseases joints, multiple myeloma.
Methodology
The determination is carried out on the “Architect 8000” biochemical analyzer.
Preparation
to study the lipid profile (cholesterol, triglycerides, HDL-C, LDL-C, Apo-proteins of lipoproteins (Apo A1 and Apo-B)
It is necessary to refrain from physical activity, drinking alcohol, smoking and medicines, dietary changes for at least two weeks before blood collection.
Blood is taken only on an empty stomach, 12-14 hours after the last meal.
Preferably morning reception medicines carry out after drawing blood (if possible).
The following procedures should not be performed before donating blood: injections, punctures, general massage body, endoscopy, biopsy, ECG, x-ray examination, especially with the introduction of a contrast agent, dialysis.
If it was still insignificant exercise stress– You need to rest for at least 15 minutes before donating blood.
Lipid testing is not performed when infectious diseases, since there is a decrease in the level of total cholesterol and HDL-C, regardless of the type of infectious agent or the clinical condition of the patient. The lipid profile should only be checked after full recovery patient.
It is very important that these recommendations are strictly followed, since only in this case will reliable blood test results be obtained.
For the quantitative determination of total lipids in blood serum, the colorimetric method with a phosphovanillin reagent is most often used. Common lipids react after hydrolysis with sulfuric acid with a phosphovanillin reagent to form a red color. The color intensity is proportional to the content of total lipids in the blood serum.
1. Add reagents into three test tubes according to following diagram:
2. Mix the contents of the test tubes and leave in the dark for 40-60 minutes. (the color of the solution changes from yellow to pink).
3. Mix again and measure the optical density at 500-560 nm (green filter) against a blind sample in a cuvette with a layer thickness of 5 mm.
4. Calculate the amount of total lipids using the formula:
where D 1 is the extinction of the experimental sample in the cuvette;
D 2 – extinction of the calibration solution of lipids in the cuvette;
X is the concentration of total lipids in the standard solution.
Define the concept of “total lipids”. Compare the value you obtained with the normal values. What biochemical processes can be judged by this indicator?
Experiment 4. Determination of the content of b- and pre-b-lipoproteins in blood serum.
2. Set of pipettes.
3. Glass rod.
5. Cuvettes, 0.5 cm.
Reagents. 1. Blood serum.
2. Calcium chloride, 0.025 M solution.
3. Heparin, 1% solution.
4. Distilled water.
1. Pour 2 ml of 0.025 M calcium chloride into a test tube and add 0.2 ml of blood serum.
2. Mix and measure the optical density of the sample (D 1) on FEC-e at a wavelength of 630-690 nm (red filter) in a cuvette with a layer thickness of 0.5 cm against distilled water. Record the optical density D 1 value.
3. Then add 0.04 ml of a 1% heparin solution (1000 units in 1 ml) to the cuvette and measure the optical density D2 again exactly after 4 minutes.
The difference in values (D 2 – D 1) corresponds to the optical density due to the sediment of b-lipoproteins.
Calculate the content of b- and pre-b-lipoproteins using the formula:
where 12 is the coefficient for conversion to g/l.
Indicate the place of biosynthesis of b-lipoproteins. What function do they perform in the human and animal body? Compare the value you obtained with the normal values. In what cases are deviations from normal values observed?
Lesson No. 16. “Lipid metabolism (part 2)”
Purpose of the lesson: study the processes of catabolism and anabolism of fatty acids.
QUESTIONS FOR THE TEST:
1. Biochemical mechanism of fatty acid oxidation.
2. Metabolism of ketone bodies: formation, biochemical purpose. What factors predispose to the development of ketosis in animals?
3. Biochemical mechanism of fatty acid synthesis.
4. Biosynthesis of triacylglycerols. Biochemical role of this process.
5. Biosynthesis of phospholipids. Biochemical role of this process.
Date of completion ________ Point ____ Teacher's signature ____________
Experimental work.
Experiment 1. Express method for determining ketone bodies in urine, milk, blood serum (Lestrade test).
Devices. 1. Rack with test tubes.
2. Set of pipettes.
3. Glass rod.
4. Filter paper.
Reagents. 1. Reagent powder.
3. Blood serum.
4. Milk.
1. Place a small amount (0.1-0.2 g) of reagent powder on the filter paper at the tip of the scalpel.
2. Transfer a few drops of blood serum to the reagent powder.
The minimum level of ketone bodies in the blood that gives positive reaction, equal to 10 mg/100 ml (10 mg%). The rate of color development and its intensity are proportional to the concentration of ketone bodies in the test sample: if the violet color appears immediately - the content is 50-80 mg% or more; if it appears after 1 minute, the sample contains 30-50 mg%; the development of a faint color after 3 minutes indicates the presence of 10-30 mg% ketone bodies.
It should be remembered that the test is more than 3 times more sensitive when determining aceto acetic acid than acetone. Of all the ketone bodies in human serum, acetoacetic acid is predominant, but in the blood of healthy cows, 70-90% of ketone bodies are b-hydroxybutyric acid, and in milk it accounts for 87-92%.
Draw a conclusion based on the results of your research. Explain why excessive formation of ketone bodies is dangerous in the human and animal body?
They have different densities and are indicators of lipid metabolism. There are various methods for the quantitative determination of total lipids: colorimetric, nephelometric.
Principle of the method. The hydrolysis products of unsaturated lipids form a red compound with the phosphovanillin reagent, the color intensity of which is directly proportional to the content of total lipids.
Most lipids are not found in the blood in a free state, but as part of protein-lipid complexes: chylomicrons, α-lipoproteins, β-lipoproteins. Lipoproteins can be separated by various methods: centrifugation in saline solutions various densities, electrophoresis, thin layer chromatography. During ultracentrifugation, chylomicrons and lipoproteins of different densities are isolated: high (HDL - α-lipoproteins), low (LDL - β-lipoproteins), very low (VLDL - pre-β-lipoproteins), etc.
Lipoprotein fractions differ in the amount of protein, the relative molecular weight of the lipoproteins, and the percentage of individual lipid components. Thus, α-lipoproteins, containing a large amount of protein (50-60%), have a higher relative density (1.063-1.21), while β-lipoproteins and pre-β-lipoproteins contain less protein and a significant amount of lipids - up to 95% of the total relative molecular weight and low relative density (1.01-1.063).
Principle of the method. When serum LDL interacts with the heparin reagent, turbidity appears, the intensity of which is determined photometrically. Heparin reagent is a mixture of heparin and calcium chloride.
Material under study: blood serum.
Reagents: 0.27% CaCl 2 solution, 1% heparin solution.
Equipment: micropipette, FEC, cuvette with an optical path length of 5 mm, test tubes.
PROGRESS. Add 2 ml of a 0.27% CaCl 2 solution and 0.2 ml of blood serum into a test tube and mix. Determine the optical density of the solution (E 1) against a 0.27% CaCl 2 solution in cuvettes using a red filter (630 nm). The solution from the cuvette is poured into a test tube, 0.04 ml of a 1% heparin solution is added with a micropipette, mixed, and exactly 4 minutes later, the optical density of the solution (E 2) is determined again under the same conditions.
The difference in optical density is calculated and multiplied by 1000 - an empirical coefficient proposed by Ledvina, since constructing a calibration curve is associated with a number of difficulties. The answer is expressed in g/l.
x(g/l) = (E 2 - E 1) 1000.
. The content of LDL (b-lipoproteins) in the blood varies depending on age, gender and is normally 3.0-4.5 g/l. An increase in LDL concentration is observed in atherosclerosis, obstructive jaundice, acute hepatitis, chronic diseases liver, diabetes, glycogenosis, xanthomatosis and obesity, decreased in b-plasmocytoma. The average LDL cholesterol content is about 47%.
Determination of total cholesterol in blood serum based on the Liebermann-Burkhard reaction (Ilk method)
Exogenous cholesterol in the amount of 0.3-0.5 g comes from food products, and endogenous is synthesized in the body in an amount of 0.8-2 g per day. Especially a lot of cholesterol is synthesized in the liver, kidneys, adrenal glands, and arterial wall. Cholesterol is synthesized from 18 molecules of acetyl-CoA, 14 molecules of NADPH, 18 molecules of ATP.
When acetic anhydride and concentrated sulfuric acid are added to blood serum, the liquid turns successively red, blue and finally green color. The reaction is caused by the formation of green sulfonic acid cholesterylene.
Reagents: Liebermann-Burkhard reagent (a mixture of glacial acetic acid, acetic anhydride and concentrated sulfuric acid in a ratio of 1:5:1), standard (1.8 g/l) cholesterol solution.
Equipment: dry test tubes, dry pipettes, FEC, cuvettes with an optical path length of 5 mm, thermostat.
PROGRESS. All test tubes, pipettes, cuvettes must be dry. You need to be very careful when working with the Liebermann-Burkhard reagent. 2.1 ml of Liebermann-Burkhard reagent is placed in a dry test tube, 0.1 ml of non-hemolyzed blood serum is added very slowly along the wall of the test tube, the test tube is shaken vigorously, and then thermostated for 20 minutes at 37ºC. An emerald green color develops, which is colorimeterized on FEC with a red filter (630-690 nm) against the Liebermann-Burkhard reagent. The optical density obtained on the FEC is used to determine the cholesterol concentration according to the calibration graph. The found cholesterol concentration is multiplied by 1000, since 0.1 ml of serum is taken into the experiment. The conversion factor to SI units (mmol/l) is 0.0258. Normal content total cholesterol (free and esterified) in blood serum 2.97-8.79 mmol/l (115-340 mg%).
Building a calibration graph. From a standard cholesterol solution, where 1 ml contains 1.8 mg of cholesterol, take 0.05; 0.1; 0.15; 0.2; 0.25 ml and adjusted to a volume of 2.2 ml with the Liebermann-Burkhard reagent (2.15; 2.1; 2.05; 2.0; 1.95 ml, respectively). The amount of cholesterol in the sample is 0.09; 0.18; 0.27; 0.36; 0.45 mg. The resulting standard cholesterol solutions, as well as the test tubes, are shaken vigorously and placed in a thermostat for 20 minutes, after which they are photometered. The calibration graph is constructed based on the extinction values obtained as a result of photometry of standard solutions.
Clinical and diagnostic value. If lipid metabolism is disrupted, cholesterol can accumulate in the blood. An increase in cholesterol in the blood (hypercholesterolemia) is observed in atherosclerosis, diabetes mellitus, obstructive jaundice, nephritis, nephrosis (especially lipoid nephrosis), hypothyroidism. A decrease in cholesterol in the blood (hypocholesterolemia) is observed with anemia, fasting, tuberculosis, hyperthyroidism, cancer cachexia, parenchymal jaundice, damage to the central nervous system, febrile states, when administered
