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 the blood plasma at relatively high concentrations (indicative reference values: <150 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.
Methods for measuring cholesterol and triglycerids. 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).

Methods for the quantification of lipoproteins. 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:

FUNCTIONS OF THE ADIPOSE TISSUE The adipose tissue has long been considered a relatively inert deposit of fat to be used for energy production. However, it is also an endocrine organ which produces several hormones that regulate the metabolism and hunger, the most important of which is the protein leptin; this is quite obvious in retrospective, since the amount of body energy reserves is relevant for several other body functions.
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.
A summary of the principal functions of the adipose tissue is as follows:
1) reservoir of triglicerides and cholesterol; principal energy storage organ.
2) Production of leptin the satiety hormone. Leptin suppresses the hunger sensation at the level of the hypotalamus, and inhibits the secretion of the hunger peptides Y. The more fat one has the less he/she should want to eat (unfortunately the hypotalamus adapts to persistenly high levels of leptin and obesity ensues).
3) Production of insulin desensitizing hormones (resistin, adiponectin). Excess fat stimulates the cells to use less glucose and more fat; obesity may be a cause of type II diabetes mellitus.
4) Production of hormones and mediators of inflammation.
5) Some of the above mentioned hormones (e.g. adiponectin) also act on the arterioles and may cause hypertension, which is often associated with obesity
6) Elaboration and storage of steroid hormones. Both obesity and excessive loss of adipose tissue may be associated with infertility in both sexes (e.g. amenorrhea in nervous anorexia)

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.

COMMON LABORATORY FINDINGS IN OBESITY Obesity is extremely common in developed countries, because of the easy availability of excess palatable food and reduced muscular work. We do not need laboratory data to diagnose obesity, which is conveniently identified by the body mass index (BMI): BMI = body weight (in kg) / squared height (in m)
e.g. a man who weights 70 kg and is 1,70 m tall has BMI = 70 / 1,7² = 24,2. A BMI>25 indicates overweight, a BMI>30 indicates obesity.

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. Insulin is involved in the leptin-peptide Y system and has a hunger-suppressione effect.

SPECIAL TYPES OF OBESITY may be related to endocrine or genetic reasons; these usually have specific laboratory markers.
The Babinsky-Froelich syndrome (adiposo-genital syndrome; Froelich syndrome) is usually associated to hypothalamic tumors occurring during adolescence; it causes disregulation of appetite, obesity, and hypogonadism with delayed puberty.

LABORATORY FINDINGS IN OBESITY are of two kinds: those possibly due to the (rare) specific diseases causing obesity: e.g. the Prader-Willi syndrome or the adiposo-genital syndrome (two inherited diseases affecting the endocrine system); and those that are consequences of the excess adipose tissue and its (endocrine) functions. We shall be mostly concerned with those laboratory findings which are consequences of obesity.
Inflammation markers are often increased in obese patients: C-reactive protein (hs-CRP), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). Thus obesity may simulate a chronic, low-grade inflammatory syndrome.
Endocrine alterations are frequent, especially those related to insulin resistance. Leptin and resistin are not usually tested in the clinical laboratory, but a useful parameter is the fasting glycemia to insulin ratio: G/I = glycemia (in mg/dL) / insuin (in μU/mL). Typical values are: healthy subjects: G/I ˜ 15; insulin resistance: G/I ˜ 3-5. In conclamated diabetes mellitus (i.e. with severely increased fasting glycemia) this index is not informative, because the insulin levels may be severy increased or severely decreased.

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.
Rosenbaum M., Leibel RL, and HirschJ. Obesity New England J. Med. 1997; 337:396-407

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).

is it possible to take gulonolactone oxidase to synthesize vitamin C
instead of vitamin C supplement?
Bellelli: no, this approach does not work. The main reason is that
the biosynthesis of vitamin C, as almost all other metabolic processes, occurs intracellularly.
If you administer the enzyme it will at most reach the extracellular fluid but will not be
transported inside the cells to any significant extent. Besides, there are other problems
in this type of therapy (e.g. the enzyme if administered orally, may be degraded by digestive
proteases; if administered parenterally, may cause the immune system to react against a
non-self protein). In theory one could think of a genetic modification of the inactive human
gene of gulonolactone oxidase, but the risk and cost of this intervention would not be
justified. In addition to these considerations, except for cases of shipwreckage or
other catastrophes, a proper diet or administration of tablets of vitamin C is effective,
risk-free and unexpensive, thus no alternative therapy is reasonable. However, I express my
congratulations for your search on the biosynthesis pathway of ascorbic acid.

Resorption and not reabsorption would lead to hypercalcemia ie bone matrix being broken down.
Bellelli: I am not sure to interpret your question correctly. Resorption indicates destruction of the bone matrix and release of calcium and
phosphate in the blood, thus it causes an increase of calcemia. Reabsorption usually means active transport of calcium from the renal tubuli to the blood, thus
it prevents calcium loss. It prevents hypocalcemia, and thus complement bone resorption. To avoid confusion it is better use the terms "bone resorption" and "
renal reabsorption of calcium". If you have a defect in renal reabsorption, parthyroid hormone will be released to maintain a normal calcium level by means of
bone resorption; the drawback is osteoporosis.

In Reed and Frost model: I haven't understood what is the relationship
between K and R reproductive index. Thank you Professor!
Bellelli: in the Reed and Frost model K is the theoretical upper limit of
R₀. R the reproductive index is the ratio (new cases)/(old cases) measured after
one serial generation time. R₀ is the value of R one measures at the beginning
of the epidemics, when in principle all the population is susceptible.

What is the link between nucleotide metabolism and immunodeficiencies and mental retardation?
Bellelli: the links may be quite complex, but the principal ones are as follows:
1) the immune response requires a replication burst of granulocytes and lymphocytes, which in turn requires
a sudden increase of nucleotide production, necessary for DNA replication. Defects of nucleotide metabolism
impair this phase of the immune defense. Notice that the mechanism is similar to the one responsible of
anemia which requires a sustained biosynthesis of nucleotides at a constant rate, rather than in a burst.
2) Mental retardation is mainly due to the accumulation of nulceotide precursors in the brain of the
newborn, due to the incompletely competent blood-brain barrier.

How can ornithine transaminase defects cause hyperammonemia? Is it due to the accumulation
of ornithine that blocks the urea cycle or for other reasons?
Bellelli: ornithine transaminase is required for the reversible interconversion of ornithine
and proline, and thus participates to both the biosynthesis and degradation of ornithine. The enzyme is
synthesized in the cytoplasm and imported in the mitochondrion. Depending on the metabolic conditions
the deficiency of this enzyme may cause both excess (when degradation would be necessary) or defect
(when biosynthesis would be necessary) of ornithine; in the latter case, the urea cycle slows down. Thus
there is the paradoxical condition in which alternation may occur between episodes of hyperammonemia
and of hyperornithinemia.

When we use the Berthelot's reaction to measure BUN do we also have to
measure the concentration of free ammonia before adding urease?
Bellelli: yes, in principle you should. Berthelot's reaction detects ammonia,
thus one should take two identical volumes of serum, use one to measure free ammonia,
the other to add urease and measure free ammonia plus ammonia released by urea. BUN is
obtained by difference. However, free ammonia in our blood is so much lower than urea that
you may omit the first sample, if you only want to measure BUN.

Why do we have abnormal electrolytes in hematological neoplasia e.g.
leukemia?
Bellelli: I do not have a good explanation for this effect, which may have
multiple causes. However, you should consider two factors: (i) acute leukemias cause a massive
proliferation of leukocytes (or lymphocytes depending on the cell type affected) with a very
shortened lifetime; thus you observe an excess death rate of the neoplastic cells. The dying
cells release in the bloodstream their content, which has an electrolyte composition different
from that of plasma: the cell cytoplasm is rich in K and poor in Na, thus causing hyperkalemia.
(ii) the kidney may be affected by the accumulation of neoplastic white cells or their lytic products.

Gaussian curve: If it is bimodal is it more likely to be a "certain diagnosis" than if it is
unimodal or does it only show the distinguishment from health?
Bellelli an obviously bimodal Gaussian curve indicates that the disease is clearly
separated from health: usually it is a matter of how precise and clear-cut is the definition of the disease.
For example tuberculosis is the disease caused by M. tuberculosis, thus if the culture of the sputum is
positive for this bacterium you have a "certain" diagnosis (caution: the patient may suffer of two diseases,
e.g. tuberculosis and COPD diagnosis of the first does not exclude the second). However, in order to have
a "certain" diagnosis it is not enough that distribution of the parameter is bimodal, it is also required that the
patient's parameter is out of the range of the healthy condition: this is because a distribution can be
bimodal even though it is composed by two Gaussians that present a large overlap, and the patient's
parameter may fall in the overlapping region. Thus, in order to obtain a "certain" diagnosis you need to
consider not only the distribution of the parameter(s) but also the patient's values and the extent of the
overlapping region.

Prof can you please elaborate a bit more on the interhuman variability and its difference
with the interpopulation variability please?
Bellelli: every individual is a unique combination of different alleles of the same genes;
this is the source of interindividual variability. Every population is a group of individuals who intermarry and
share the same gene pool (better: allele pool). Every allele in a population has its own frequency. Two
population may differ because of the diffferent frequencies of the same alleles; in some cases one
population may completely lack some alleles. The number and frequencies of alleles of each gene
determine the variance. If you take two populations and calculate the cumulative interindividual variance
of the population the number you obtain is the sum of two contributions: the interindividual variance within each population, plus the interpopulation variance
between the means of the allele frequencies. For example, there are human population in which the frequency of blood group B is close to 0% and other populati
ons in which it is 30% or more.

Prof can you please explain again the graph you have showed us in class about thromboplastin?
(Y axis=abs X axis= time)
Bellelli: the graph that I crudely sketched in class represented the signal
of the instrument (an absorbance spectrophotometer) used to record the turbidity of the
sample (turbidimetry). The plasma is more or less transparent, before coagulation starts.
When calcium and the tissue factor (or collagen) are added. thrombin is activated and begins
digesting fibrinogen to fibrin; then fibrin aggregates. The macroscopic fibrin aggregates cause
the sample to become turbid, which means it scatters the incident light. The instrument reads
this as a decrease of transmitted light (i.re an increase of the apparent absorbance) and the
time profile of the signal presents an initial lag phase, which is called the protrombin or
thromboplastin time depending on the component which was added to start coagulation
(tissue factor or collagen).

Prof can you please explain the concept you have described in class about
the simultaneous hypercoagulation and hemorrhagic syndrome? How can this occur?
Bellelli: The condition you describe is observed only in the Disseminated
Intravascular Coagulation syndrome. Suppose that the patient experiences an episode of
acute pancreatitis: tripsin and chymotripsin are reabsorbed in the blood and proteolytically
activate coagulation causing an extensive consumption of fibrinogen and other coagulation
factors. Tripsin and chymotripsin also damage the vessel walls and may cause internal
hemorrages, but at that point the consumption of fibrinogen may have been so massive that
not enough is left to form the clot where the vessel has been damaged, causing an internal
hemorrage. Pancreatitis is a very severe, potentially lethal condition, and DIC is only one of
the reasons of its severity.

You said that certain drugs (ethanol, cocaine, cannabis, opiates...) cause a
necessity of higher and higher dosage, for two reasons: the enzyme in the liver is inducible and
the receptors in the brain are expressed less and less. So, first, I am not sure I got it right, and
second I did not understand how expressing less receptors leads to a necessity of higher
dosage.
Bellelli: You got it correctly, but the detailed mechanism of resistance may
vary among different substances, and not all drugs cause adaptation.
The reason why reducing the number of receptors may require an increased dosage of the drug
is as follows: suppose that a certain cell has 10,000 receptors for a drug. When bound to its
agonist/effector, each receptor produces an intracellular second messenger. Suppose that in
order for the cell to respond 1,000 receptors must be activated. The concentration of the
effector required is thus the concentration that produces 10% saturation. You can easily
calculate that this concentration is approximately 1/10 of the equilibrium dissociation constant
of the receptor-effector complex (its Kd), the law being
Fraction bound = [X] / ([X]+Kd)
where [X] is the concentration of the free drug.
After repeated administration, the subject becomes adapted to the drug, and his/her cells
express less receptors, say 5,000. The cell response will in any case require that 1,000
receptors are bound to the effector and activated, but this now represents 20% of the total
receptors, instead of 10%. The drug concentration required is now 1/4 of the Kd.
Continuing administration of the drug further reduces the cell receptors, but the absolute
number of activated receptors required to start the response is constant; thus the fewer
receptors on the cell membrane, the higher the fraction of activated receptors required.

Why does hyperosmolarity happen in type 2 diabetes and not in type 1?
Bellelli: Hyperosmolarity can occur also in type 1 diabetes, albeit
infrequently. The approximate formula for plasma osmolarity is reported in the lecture on
electrolytes:
osmolarity = 2 x (Na⁺ + K⁺) + BUN/2.8 + glucose/18
this is expressed in the usual clinical laboratory units (mEq/L for electrolytes, g/dL for non-
electrolytes). The normal values are:
osmolarity = 2 x (135 + 5) + 15/2.8 + 100/18 = 280 + 5.4 + 5.6 = 291 mOsmol/L
Let's imagine a diabetic patient having normal values for electrolytes and BUN, and glycemia=400 mg/dL:
osmolarity = 280 + 5.4 + 22.4 = 307.8 mOsmol/L
The hyperosmolarity in diabetes is mainly due to hyperglycemia, even though other factors
may contribute (e.g. diabetic nefropathy); however the contribution of glucose to osmolarity is
relatively small. As a consequence in order to observe hyperosmolarity the hyperglycemia
should be extremely high; this is more often observed in type 2 than in type 1 diabetes, for
several reasons, the most relevant of which is that in type 1 diabetes all cells are starved of
glucose, and the global reserve of glycogen in the body is impoverished: there is too much
glucose in the blood and too few everywhere else, thus reducing, but not abolishing, the risk of
extreme hyperglycemia. Usually in type 2 diabetes the glycogen reserve in the organism is not
impoverished, thus the risk of extreme hyperglycemia is higher.

Hemostasis and Thrombosis lecture: I don't understand why is sodium citrate
added to the serum solution to measure the prothrombin time.
Bellelli: in order to measure PT or PTT you want to be able to start the
coagulation process at an arbitrary time zero, and measure the increase in turbidity of the
serum sample. To do so you need (i) to prevent spontaneous coagulation with an anticoagulant;
and (ii) to be able to overcome the anticoagulant at your will. Citrate (or oxaloacetate; or EDTA)
has the required characteristics: it chelates calcium, and in this way it prevents coagulation;
but you can revert its effect at your will by adding CaCl₂ in excess to the amount
of citrate. You cannot obtain the same effect with other anticoagulants (e.g. heparin) whose
action cannot be easily overcome.

Dear professor I cannot do the self evaluation test because it says the the
time has expired It is not possible because I havent even started them
Bellelli: this is due to the fact that the program registers your name and
matricola number from previous attempts. I shall fix this bug. Meanwhile try to use a fake
matricola number.

How is nephrotic syndrome associated hypoalbuminemia as you described
in methods of analysis of protein because seems counterintuitive
Bellelli: nephrotic syndrome is an autoimmune disease in which the
glomerulus is damaged and the filtration barrier is disrupted; diuresis is normal but there is
loss of proteins (mostly albumin) in the urine.
I m sorry i confused polyurea with hypoalbuminemia but my question still
stands during glomerulonephritis you mentioned something of polyurea as compensation
i could not follow how this compensation mechanism works and collapse after some time in
glomerulonephritis
Bellelli: the condition you describe is NOT characteristic of acute
glomerulonephritis. In glomerulonephritis there is damage of the glomerulus and severely
impaired GFR. Thus the diuresis is severely reduced, and due to impaired filtration proteins
appear in the urine.
The condition you describe corresponds to the initial stage of chronic kidney failure,
usually due to atherosclerosis, diabetes, hypertension or other type of damage of the kidney
tissue. In this case GFR is impaired, albeit to a lesser extent than in glomerulonephritis, and the
excretion of urea is reduced. This leads to increased BUN. However the increased concentration
of urea reduces the ability of the tubuli to reabsorb water, because of osmotic reasons, yielding
compensatory polyuria. The patient has reduced GFR but normal or increased diuresis (urine
volume in 24 hours). To some extent this effect is beneficial, as it favors the elimination of
urea; however it cannot completely solve the problem and in any case the progression of the
disease leads to kidney insufficiency. In its essence the point is that a moderately reduced GFR
can be partially compensated by reduced tubular reabsorption; a severely reduced GFR cannot.

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