MEDICINE AND SURGERY "F"
Course of LABORATORY MEDICINE
Lipids and Lipoproteins

PLASMA LIPOPROTEINS

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      Lipids (essentially triglycerides and cholesterol) are required for tissue metabolism and membrane production; they are present in blood at relatively high concentrations (indicative reference values: <200 mg/dL triglycerides and <200 mg/dL cholesterol). Since lipids are insoluble in water, they are transported in the blood in the form of complexes with soluble proteins, called lipoproteins. Lipoproteins fall in different categories, based on their density (as measured with the ultracentrifuge), their electrophoretic mobility and their metabolic origin and destination. Excess concentration of lipids in the blood is associated with increased risk of atherosclerosis, myocardial infarction and other diseases; thus their measurement has prognostic value and may suggest that a therapy is indicated.

Audio: lipids in the blood plasma

      Chylomicrons are the lowest density lipoproteins. They are of alimentary origin, and are synthesized by the enterocytes with triglycerides and some cholesterol. The apolipoprotein subunits of chylomicrons (i.e. the proteic component of the particle) are called A-I, A-II, B-48, C-I, C-II, C-III and E: each is coded by a specific gene and presents its own aminoacid sequence. The density of chylomicrons, measured by ultracentrifugation is <0.95 g/mL.
      Chylomicrons are released both in the portal venous system and in the lymphatic vessels. Chylomicrons in the porta vein are removed by the liver which transfers their lipidic contento to other types of lipoproteins. Chylomicrons released in the intestinal lymph ("chyle") reach the thoracic duct that drains into the brachiocephalic vein and from this into the superior vena cava. They are thus distributed to all tissues that can extract triglycerides for their metabolic needs. The major consumers of triglycerides are the skeletal muscle and the heart. Adipocytes are able to extract and store the lipidic content of chylomicrons (and other lipoproteins) if in excess for the metabolic demands, Removal of triglycerides requires the enzyme lipoprotein lipase, which is activated by apolipoprotein C-II. As triglycerides are removed, the particles shrink and are enriched in cholesterol, forming the so-called chylomicron remnants, that are taken up by the liver.

      Very low density lipoproteins (VLDL) are composed of triglycerides, cholesterol and colesteryl-esters, bound to apolipoprotein components B-100, C-I, C-II, C-III and E. Their density is 0.95-1.006 g/mL. VLDLs are synthesized by the liver and their lipid component is degraded by lipoprotein lipase as it occurs for chylomicrons. As VLDLs shrink, they loose some apolipoprotein components and are successively converted to IDLs and LDLs.

      Intermediate density lipoproteins (IDL) are composed of triglycerides, cholesterol and colesteryl-esters, bound to apolipoprotein components B-100,C-III and E. Their density is 1.006-1.019 g/mL. They originate from the shrinking of VLDLs and have a similar fate.

      Low density lipoproteins (LDL) are composed of triglycerides, cholesterol and colesteryl-esters, bound to apolipoprotein component B-100. Their density is 1.019-1.063 g/mL. LDLs are rich in cholesterol and are absorbed by the cells via receptor mediated endocytosis, thanks to a specific LDL receptor residing on the cell membrane. The endocytic vesicle is degraded by fusion with lysosomes and the B-100 apolipoprotein component is digested by proteases. Cholesterol and triglycerides are used by the cell either for membrane turnover or for energy roduction (limited to the beta oxidation of fatty acids).

      High density lipoproteins (HDL) carry essentially cholesterol and its esters and are composed by the apolipoprotein components A-I, A-II, C-I, C-II, C-III, D and E. Their density is 1.063-1.210 g/mL. Their function is the transport of cholesterol from peripheral tissues to the liver: i.e. in the opposite direction of other lipoproteins. The liver requires substantial amounts of cholesterol for the biosynthesis of bile salts (cholate and deoxycholate). Bile salts are secreted in the intestine where they emulsify fat of alimentary origin and allow its digestion by lipases; they are reabsorbed by the intestine and re-utilized (on average 7-times), then are finally lost in the faeces. The main way of eliminating cholesterol from the organism is via the bile salts (i.e. the bile salts are the terminal metabolytes of cholesterol).

Audio: lipoproteins


Main traffic routes of lipids and lipoproteins.

      The essential clinical information about lipoproteins can be summarized as follows: chylomicrons essentially carry dietary triglycerides, are synthesized by the enterocytes, and are the largest and less dense lipoproteins; their proteic component is relatively complex and involves many different subunits. All other lipoproteins are synthesized by the liver and modified by the adipocytes. LDL, IDL and VLDL strongly resemble each other; they carry both cholesterol and triglycerides, and their apoliprotein component always involves apoliproptein B-100. Their affinity for cholesterol is relatively low and they may release some of their cholesterol content to the endothelial wall (so called "bad" cholesterol, associated to increased risk of atherosclerosis). HDL carry essentially cholesterol, have a very specific apolipoprotein composition, different from that of VLDL, IDL, and LDL. They bind cholesterol with high affinity and may be able to remove it from the endothelial wall (so called "good" cholesterol, it reduces the risk of atherosclerosis).
      Approximately 60-75% of the total cholesterol in blood is bound to LDL and is thus "bad" cholesterol; 20-25% is bound to HDL and is thus "good" cholesterol; the remaining is carried by VLDL and IDL. It is important to remark that atherosclerosis is a complex process, initiated by cholesterol deposition in the arterial wall by LDL (and possibly removed by HDL). Cholesterol in the cell wall undergoes oxidative damage (partially prevented by vitamin E) and this, in turn, causes an inflammatory reaction. Ultimately the inflamed cholesterol plaque becomes organized because of collagen deposition by fibroblasts, and the vessel stricture becomes irreversible.

Audio: LDL-cholesterol and HDL-cholesterol

      Triglycerides are used mainly for energy production and their fatty acids are oxidized by the beta-oxidation and the Krebs cycle: they are thus expelled as CO2 and H2O. The skeletal muscle and the heart are among the organs that use mostly fatty acids for their catabolic metabolism. triglycerides are also be used for the turnover of cell membrane components, after conversion to phospholipids.
      Cholesterol has three main functions: it is used in the cell membrane, as the precursor of steroid hormones, and as the precursor of bile salts (that also constitute its main elimination pathway).

DISLIPIDEMIAS
      Dislipidemias of clinical relevance are always associated with increased serum concentrations of lipids and lipoproteins, as these conditions may favor atherosclerosis and other disturbances; on the contrary, lower than normal levels of blood lipids are not considered clinically relevant, unless they are associated to other conditions (e.g. hyponutrition, anorexia, malabsorption, etc.). The usual classification of hyperlipoproteinemias is due to Fredrickson, but has been greatly improved over time. It is summarized in the Table below.

FREDRICKSON'S CLASSIFICATION OF HYPERLIPIDEMIAS
type defect increased lipoprotein lipidic pattern possible complications
I decreased lipoprotein lipase activity (autosomal recessive) chylomicrons moderately increased cholesterol; greatly increased triglycerides Pancreatitis; eruptive xanthomas
II poorly functional LDL receptor (autosomal dominant) LDL (possibly also VLDL) greatly increased cholesterol; normal or slightly increased triglycerides Severe atherosclerosis
III decreased or absent apolipoprotein E2 (mode of inheritance unclear) IDL greatly increased cholesterol and triglycerides Severe atherosclerosis
IV (genetically heterogeneous) VLDL normal or moderately increased cholesterol; greatly increased triglycerides Glucose intolerance; hyperuricemia
V (genetically heterogeneous) VLDL and chylomicrons normal or moderately increased cholesterol; greatly increased triglycerides Pancreatitis; eruptive xanthomas; Glucose intolerance; hyperuricemia

      We observe that:
1) the main clinical manifestations of hyperlipidemias group in three families: atherosclerosis; hypergycemia, up to a type 2 diabetes mellitus (possibly associated with hyperuricemia); and pancreatitis.
2) Hypertriglyceridemia preferentially associates with increased risk of pancreatitis.
3) Hypercholesterolemia preferentially associates with atherosclerosis.
4) Increased VLDL concentration preferentially associates with hyperglycemia; this association is probably indicative of events occurring inthe adipose tissue, rather than to an effect of lipids per se (see below: metabolic syndrome).

Audio: hyperlipidemias

      A rare genetic form of hypercholesterolemia different from the ones in Fredrickson classification is due to lecithin cholesterolacyltransferase deficiency. This disease is inherited as an autosomal recessive trait and causes hypercholesterolemia and hypertriglyceridemia. It causes anemia and cataract; liver or kidney failure may occur.

      Primary hypolipoproteinemias are rare familial disorders, and should be distinguished from the more common secondary forms due to malnutrition or malabsorption:
Hypobetalipoproteinemia is an uncommon, mild genetic condition inherited as a dominant autosomal character, and characterized by reduced levels of LDL. Plasma cholesterol ranges between 70 and 120 mg/dL. This condition is asymptomatica and offers protection against atherosclerosis. No treatment is required.
Abetalipoproteinemia is a severe autosomal recessive genetic disease different from the preceding one, and characterized by the complete absence of chylomicrons and lipoproteins of the VLDL/IDL/LDL family. It causes retinitis pigmentosa, ataxia and mental retardation; moreover the erythrocytes have an abnormal morphology (acanthocytosis).
Tangier disease is a familial apha lipoprotein (HDL) deficiency. It causes recurrent peripheral polyneuropathies and hepatosplenomegaly.

ANALYTICAL METHODS FOR LIPIDS AND LIPOPTOTEINS
      It is customary to measure lipid concentrations in our blood, rather that lipoprotein concentrations; when fractionation is required we speak of cholesterol bound to HDL (HDL-cholesterol), or to LDL (LDL-cholesterol), etc. Cholesterol, triglycerides and phosphoglycerids can be extracted from the human serum using organic solvents (usually mixtures of hexane and 2-propanol), and their concentration can be measured by chemical or enzymatic methods (after dilution in the appropriate buffers). Examples of the methods used to quantitate Cholesterol and Triglycerids are as follows. Cholesterol can be measured using the enzyme cholesterol oxidase that uses molecular oxygen to convert cholesterol to cholestenone; the reaction produces one mol of hydrogen peroxide per mol of cholesterol, which is easily measured by with standard assays (e.g. using horseradish peroxidase and the fluorescent probe o-dianisidine). Triglycerides should be hydrolysed after extraction, to yield glycerol and fatty acids (this can be obtained either chemically or enzymatically, with lipiprotein lipases); the concentration of glycerol is then measured using the microbial enzymes glycerol kinase (that uses ATP to produce Glycerol-1-phosphate) and Glycerol-1P oxidase (that uses O2 and yields dihydroxyacetone phosphate and hydrogen peroxide, measured using standard methods).

      As their names imply, lipoproteins were originally classified according to their density, determined by ultracentrifugation in sucrose gradients. However, they can be separated equally well by electrophoresis, a method that is less expensive and faster, and thus is preferred.

Comparison of ultracentrifuge distribution and electrophoresis migration of lipoproteins.

Audio: lipoproteins

      The lipoproteins can be submitted to electrophoresis and separated in their components; however their quantitation requires specific procedures because they co-migrate with other protein fractions and stain poorly with the usual protein-specific dyes, due the relatively modest amount of protein (by weight). To quantitate the lipoproteins, the electrophoretic preparation must be stained using lipid-specific dyes (e.g. Sudan black). These do not react with the protein fractions and only stain the lipid content of lipoproteins.


Lipoproteins migration in the electrophoresis of serum proteins is as in the picture: HDL migrate with α1 globulins; VLDL migrate in the pre-β band; LDL migrate with the β globulins; and chylomicrons migrate in a poorly populated region between the β and γ bands.

      The electrophoretic patterns characteristic of the different hyperlipidemias are as follows:



THE METABOLIC SYNDROME (from the NIH website)
      The metabolic syndrome is a complex clinical condition that causes increased risk of myocardial infarction and other caridovascular diseases. It has the following markers
(i) A large waistline. This also is called abdominal obesity or "having an apple shape." Excess fat in the stomach area is a greater risk factor for heart disease than excess fat in other parts of the body, such as on the hips.
(ii) A high triglyceride level (or you're on medicine to treat high triglycerides).
(iii) A low HDL or "good" cholesterol level (or you're on medicine to treat low HDL cholesterol). HDL-cholesterol is cholesterol that has been removed from peripheral tissues and is carried to the liver for disposal after conversion to bile salts. A low HDL-cholesterol level raises your risk for heart disease.
(iv) High blood pressure (or you're on medicine to treat high blood pressure). This may favor heart failure and the build-up of atheromatous plaques in the arteries.
(v) High fasting blood sugar (or you're on medicine to treat high blood sugar). Mildly high blood sugar may be an early sign of type 2 diabetes.
      The metabolic syndrome depends on a genetic predisposition, and environmental factors.

      HUNGER AND BODY WEIGHT
      The sensation of hunger is regulated by hormones, the most important being the Y polypeptides produced by the hypothalamus. Increased hypothalamic production of Y polypeptides and hormones causes hunger.
      The production of hunger hormones by the hypothalamus is regulated by the adipose tissue in a negative feedback loop. The adipose tissue produces a protein hormone called LEPTIN that inhibits the production and secretion of Y polypeptides. Genetic defects of leptin are known that cause hereditary obesity (the gene coding for leptin is called Ob because it was identified in strains of genetically obese rats).
      Excess (or a satisfactory amount of) fat tissue produces excess leptin and inhibits production of Y peptides, causing the sensation of satiation. Unfortunately, the hypothalamus may become adapted to the higher level of leptin, and may stop responding, causing obesity.

      ENDOCRINE FUNCTIONS OF ADIPOSE TISSUE
      Following the discovery of leptin, several endocrine functions have been attributed to the adipose tissue. The protein factors secreted by the adipose tissue include cytokines, adiponectin, proteins of the renin-angiotensin system, and resistin; these contribute to the regulation of metabolism and of the use of energy reserves. Moreover, the adipose tissue participates to the metabolism of steroid hormones and is involved in fertility (especially in women).
      The endocrine functions of the adipose tissue help explaining the relationships between obesity and hypertension or diabetes. Indeed, obesity shares some endocrine responses of chronic inflammatory diseases (because of the production of cytokines). Moreover, some of the cytokines produced by the adipose tissue (e.g. adiponectin and resistin) induce insulin resistance in peripheral tissues. Obesity increases the production of cytokines and this is the most important factor relating obesity to type 2 diabetes.

     
SOME CLINICALLY IMPORTANT CORRELATIONS: A SUMMARY
      In this lecture some important correlations have been described, which we may summarize as follows:
1) an increase of LDL-cholesterol (that usually account for 60-75% of total blood cholesterol) is associated to atherosclerosis: this is the so-called "bad" cholesterol; an increase of HDL-cholesterol (usually 20-25% of total cholesterol) is protective against atherosclerosis.
2) An increase in triglycerides is associated to increased risk of pancreatitis, a very serious and potentially lethal disease.
3) Obesity is weakly associated to hyperlipidemias, but has important associations with arterial hypertension, type 2 diabetes, and possibly hyperuricemia. These associations are mediated by the endocrine functions of adipose tissue which play a role in regulating the metabolism and usage of fat stores. Moreover leptin secreted by adipose tissue regulates hunger and food intake.

Audio: important correlations: a summary

Further readings
Hajer G.R, van Haeften T.W., and Visseren F.L.J. Adipose tissue disfunction in obesity, diabetes and vascular diseases.

Questions and exercises:
1) The role of VLDL is:
transport of lipids derived from food from the gut to the liver
transport of lipids from the liver to the adipose and other tissues
transport of lipids from the tissues to the liver

2) Indicative concentration values for triglycerides and cholesterol in the blood of healthy humans are:
75 and 100 mg/dL, respectively
150 and 200 mg/dL, respectively
300 and 400 mg/dL, respectively

3) Hyperlipidemias most frequently associated with atherosclerosis in Fredrickson's classification are:
types IV and V
types III and IV
types II and III
types I and II

4) Obesity and type 2 diabetes are associated because:
the adipose tissue produces hormones that induce insulin resistance
the adipose tissue controls hunger via the leptin - Y peptide system
type 2 diabetes stimulates the tissues to consume more glucose and less triglycerides

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Thank you Professor (lecture on bilirubin and jaundice).

The fourth recorded part, the one on hyper and hypoglycemias is not working.
Bellelli: I checked and in my computer it seems to work. Can you better specify
the problem you observe?

This Presentation (electrolytes and blood pH) feels longer than previous lectures
Bellelli: it is indeed. Some subjects require more information than others. I was
thinking of splitting it in two nest year.

Bellelli in response to a question raised by email: when we compare the blood pH
with the standard pH we do not mean to compare the "normal" blood pH (7.4)
with the standard pH. Rather we compare the actual blood pH of the patient, with
the pH of the same blood sample equilibrated under standard conditions.
Thus, if we say that standard pH is lower than pH we mean that equilibriation with
40 mmHg CO2 has caused absorption of CO2 and has lowered the pH with respect
to its value before equilibration.

(Lipoproteins) Is the production of leptin an indirect cause of type 2 diabetes since
it works as a stimulus to have more adipose tissue that produces hormones?
Bellelli: in a sense yes, sustained increase of leptin causes the hypothalamus to adapt
and to stop responding. Obesity ensues and this in turn may cause an increase in the
production of resistin and other insulin-suppressing protein hormones produced by the
adipose tissue. However, this is quite an indirect link, and most probably other factors
contribute as well.

(Urea cycle) what is the meaning of "dissimilatory pathway"?
Bellelli: a dissimilatory pathway is a catabolic pathway whose function is not to produce
energy, but to produce some terminal metabolyte that must be excreted. Dissimilatory
pathways are necessary for those metabolytes that cannot be excreted as such by the
kidney or the liver because they are toxic or poorly soluble. Examples of metabolytes
that require transformation before being eliminated are heme-bilirubin, ammonia,
sulfur and nitrogen oxides, etc.

Talking about IDDM linked neuropathy can be the C peptide absence considered a cause of it??
Bellelli: The C peptide released during the maturation of insulin, besides being an indicator
of the severity of diabetes, plays some incompletely understood physiological roles. For
example it has been hypothesized that it may play a role in the reparation of the
atherosclerotic damage of the small arteries. Thus said, I am not aware that it plays a direct
role in preventing diabetic polyneuropathy. Diabetic neuropathy has at least two causes: the
microvascular damage of the arteries of the nerve (the vasa nervorum), and a direct
effect of hyperglycemia and decreased and irregular insulin supply on the nerve metabolism.
Diabetic neuropathy is observed in both IDDM and NIDDM, and requires several years to
develop. Since the levels of the C peptide differ in IDDM and NIDDM, this would suggest
that the role of the C peptide in diabetic neuropathy is not a major one. If you do have
better information please share it on this site!

In acute intermitted porphyria and congenital erythropoietic porphyria why do the end product
of the affected enzymes accumulate instead of their substrate??
Bellelli: First of all, congratulations! This is an excellent question.
Remember that a condition is which the heme is not produced is lethal in the foetus; thus
the affected enzyme(s) must maintain some functionality for the patient
to be born and to come to medical attention. All known genetic defects of heme
biosynthesis derange but do not block this metabolic pathway.
Congenital Erythropoietc Porphyria (CEP) is a genetic defect of uroporphyrinogen
III cosynthase. This protein associates to uroporphyrinogen synthase (which is present
and functional in CEP) and guarantees that the appropriate uroporphyrinogen isomer is produced
(i.e. uroporphyrinogen III). In the absence of a functional uroporphyrinogen III
cosynthase other possible isomers of uroporphyrinogen are produced together with
uroporpyrinogen III, mostly uroporphyrinogen I. The isomers of uroporphyrinogen
that are produced differ because of the positions of propionate and acetate side chains,
and this in turn is due to the pseudo symmetric structure of porphobilinogen. Only
isomer III can be further used to produce protoporphyrin IX. Thus in the
case of CEP we observe accumulation of abnormal uroporphyrinogen derivatives, which, as
you correctly observed are the products of the enzymatic synthesis operated by
uroporphyrinogen synthase.
The case of Acute Intermittent Porphyria (AIP) is similar, although there may be variants
of this disease. What happens is that either the affected enzyme is a variant that does not
properly associate with uroporphyrinogen III cosynthase or presents active site mutations
that impair the proper alignement of the phoprphobilinogen substrates. In either case
abnormal isomers of uroporphyrinogen are produced, as in CEP.
Also remark that in both AIP and CEP we observe accumulation of the porphobilinogen
precursor: this is because the overall efficiency of the biosynthesis of uroporphyrinogens is
reduced. Thus: (i) less uroporphyrinogen is produced, and (ii) only a fraction of the
uroporphyrinogen that is produced is the correct isomer (uroporphyrinogen III).



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