Porphyrias: diseases related to heme biosynthesis

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      Congenital or acquired disturbances of heme biosynthesis are called porphyrias. The reason why these disturbances may be congenital or acquired is that the same enzyme may be mutated or, in some cases, may be inhibited by exernal poisons, usually as a result of professional intoxications. The heme (iron protoporphyrin IX) is the prostethic group of hemoglobin and myoglobin; heme variants are present in many respiratory enzymes. The heme is so important that absence of its biosynthesis is incompatible with life. As a consequence in porphyrias the biosynthesis is deranged but not blocked (as blockage would cause intrauterine death at an early stage of embrionic development rather than a congenital disease), and symptoms are due to accumulation of wrong products or precursors in the tissues. Heme precursors or wrong metabolytes accumulate mainly in the skin, causing cutaneous porphyrias and/or in the peripheral nerves, causing neurological porphyrias.
      Porphyrins are synthesized by all cells because of the requirement of heme by the mitochondrion; however, major sites of biosynthesis are the red cell precursors and the liver, and this allows one to distinguish erythropoietic porphyrias (congenital erythropoietic porphyria and erythropoietic protoporphyria) from hepatic porphyrias (all other forms). You may notice that, with the exception of porphyria cutanea tarda, which is atypical in many respects (e.g. it may be acquired) all neurological porphyrias are of the hepatic variety. Cutaneous porphyrias may be erytropoietic (most often) or hepatic (especially porphyria cutanea tarda, which is atypical in many respects, e.g. because it may be acquired).

Audio: definition of porphyrias

      The main clinical symptom of cutaneous porphyrias is photosensytivity; the main symptom of neurological porphyrias is pain. The following table collects the different types of porphyrias, ordering them according to their main symptoms: neurological, mixed, and cutaneous.
DiseaseEnzymatic defectInheritance Symptoms
Acute Intermittent P.Uroporphyrinogen synthase Dominant Neu
Hereditary CoproporphyriaCoproporphyrinogen oxidase Dominant (Skin)/Neu
Variegate P.Protoporphyrinogen oxidase Dominant (Skin)/Neu
Congenital Erythropoietic P.Uroporphyrinogen III cosynthase Recessive Skin
P. cutanea tardaUroporphyrinogen decarboxylase Dominant or acquired Skin
Erythropoietic ProtoporphyriaFerrochelatase Dominant Skin
Symptoms: skin=major cutaneous lesions due to photosensitivity; Neu=neurological symptoms (mainly pain); (Skin)/Neu=minor cutaneous symptoms associated to major neurological symptoms
Pay attention not to confuse the similarly named congenital erythropoietic porphyria and erythropoietic PROTOporphyria!

Audio: classification of porphyrias

      In order to properly locate each disease along the heme biosynthesis pathway, and thus to identify the precursor(s) that accumulate, or the wrong metabolyte(s) that may be produced you may refer to the following picture that summarizes the complex sequence of reactions that, starting from glycine and succinyl-CoA, leads to heme biosynthesis. The process involves 8 main enzymes, and occurs partly in the mitochondrion and partly in the cytoplasm, thus membrane transporters are also necessary.
      The enzymes of heme biosynthesis have been named and renamed several times and some confusion exists in the literature. The first step of heme biosynthesis is the production of δ-aminolevulinic acid (ALA) from glycine and succinyl-CoA (from the Krebs cycle); the reaction is catalyzed by ALA synthaseand occurs in the mitochondrion. Two molecules of ALA are combined by the enzyme porphobilinogen synthase (also called ALA dehydratase) to yield porphobilinogen (PBG), a pyrrole derivative. Four molecules of PBG are deaminated and joined together to form hydroxymethyl bilane (HMB), a linear tetrapyrrole. The enzyme is HMB synthase (formerly called uroporphyrinogen synthase). HMB would spontaneously close to the tetrapyrrole ring of uroporphyrinogen I, but is instead processed by uroporphyrinogen III cosynthase that inverts the orientation of ring D and closes the tetrapyrrole ring as uroporphyrinogen III (notice that the two propionate residues of pyrroles C and D are adjacent to each other in uroporphyrinogen III while thay are not in HMB). A series of decarboxylation and oxidation reaction convert uroporphyrinogen III to protoporphyrin IX to which iron is finally added by ferrochelatase.

Audio: heme biosynthesis

      There are important differences in the heme biosynthesis in the liver and other cells and in the erythropoietic stem cells. Notably ALA synthase exists in two isoforms: ALAS1 in the liver and ALAS2 in the erythropoietic stem cell; these are differently regulated: ALAS1 in inhibited by the heme, the final product of the pathway, whereas ALAS2 is positively regulated by iron; thu the heme biosynthesis is differently regulated in the liver (and other tissues) and in erythropoietic stem cells. This allows erythropoietc cells to accumulate heme for hemoglobin biosynthesis (which continues after the mitochondria have been eliminated) In the liver, ALA synthase activity is stimulated, rather than inhibited, by insufficient heme production. Thus ALA and PBG levels are increased in hepatic (i.e. neurological) porphyrias.

      It is important to recall that porphyrins and their precursors are poorly soluble in water, thus urinary excretion is usually insufficient, and accumulation in the tissues occurs. Indeed elimination of heme catabolytes: (i) requires degradation to biliverdin, and then to bilirubin, followed by conjugation to glucuronic acid to increase bilirubin's solubility; and (ii) is mainly via the faeces, where solubility is a minor concern. Our organism has scarce capability of disposing of heme precursors, as these are poor substrates for heme oxygenase, the enzyme that converts heme to biliverdin.

      Porphyrias are either genetic or acquired diseases. Genetic porphyrias are due to the patient inheriting a poorly functioning or unstable variant of one of the enzymes involved in the biosynthesis of the heme. Acquired porphyrias are usually due to intoxication with substances capable of inactivating the same enzymes (e.g. chronic lead intoxication; chronic alcholism). Depending on the enzyme affected, and the intermediate which is accumulated, one may distinguish between cutaneous and hepatic/neurological porphyrias.
      Two types of porphyrias are particularly noteworthy. Congenital erythropoietic porphyria, is due to a defect of uroporphyrinogen III cosynthase. This enzyme guarantees the appropriate orientation of the pyrroles in the biosynthesis of uroporphyrinogen III. In the absence of the enzyme, uroporphyrinogen synthase produces mostly uroporphyrinogen I, an isomer differing in the position of acetate and propionate side chains, that cannot be used in the heme biosynthesis and accumulates in the tissues. This is the most severe form of cutaneous porphyria. Congenital protoporphyria is interesting because it leads to accumulation of iron-free protoporphyrin IX that should be recognized as a substrate by heme oxygenase; however the substance accumulates not only in the erythrocytes, but also in tissues where it is not metabolyzed to biliverdin, e.g. in the liver, and this may lead to progressive liver failure.

      Most porphyrias are hereditary and dominant; thus, multiple cases occur among the patient's relatives, and the familial anamnesis provides important diagnostic clues.
      Cutaneous porphyrias are easily suspected on a purely clinical basis. Porphyrin precursors accumulate in the skin and, given their florescence properties, they harvest sun light and transfer the radiant energy to the surrounding cells causing damage of the DNA (e.g. dimerization of timine) and necrosis. The resulting dermatitis is severe, with extensive ulcerations. The patient becomes aware of his/her condition in the early infancy and avoids direct sunlight: he/she leaves home after sunset and uses extensive clothing.

      A cardinal sign of erythropoietic congenital porphyria is erythrodonthia, a reddish coloration of the teeth, due to accumulation of porphyrinogens in the teeth. Illumination with blue light reveals the reddish fluorescence of the teeth and is of diagnostic value (no other disease causes this phenomenon).

Audio: cutaneous porphyrias

      By contrast, neurological porphyrias are difficult to diagnose on clinical grounds alone. The chief manifestation is pain, due to peripheral neuropathy. Pain occurs suddenly in acute crises and is frequently misdiagnosed as an acute abdominal condition. Given the intensity of the syndrome, these patients often undergo repeated surgeries, because of suspected appendicitis, volvulus, biliary or urinary calculi, etc. Needless to say none of these conditions is the culprit, even though all of them may occasionally co-exist. The most dramatic cases are those caused by acute intermittent porphyria. Neurological porphyrias must be differentiated from: (i) acute abdominal conditions requiring surgery; and (ii) other non surgical conditions such as tabes dorsalis or the colica saturnina (in the course chronic lead poisoning). At variance with cutaneous porphyrias, whose onset is in the infancy (except for the late onset porphyria cutanea tarda), neurological porphyrias usually manifest themselves in the adolescence.

      Diagnosis of porphyrias is based on the demonstration of the increased concentration of one of the heme precursors in the serum or in the urine. These compounds are strongly fluorescent, and in acidic medium (HCl), they react with benzaldehyde derivatives (Ehrlich reaction) to yield a characteristic purplish pigment. If the biological sample is positive to these simple test, identification of the specific porphyrin can be achieved by chromatography. An alternative method is to oxidize colorless prophyriinogen derivatives to the corresponding reddish porphyrin derivatives using the Lugol's reagent (a mixture of iodine and potassium iodide).
Laboratory diagnostic features of porphyrias
Porphyriaurine ALA and PBGurine porphyrinsfecal porphyrinsred blood cell porphyrins
Acute intermittent porphyria increased increased uroporphyrinogen normal normal
Hereditary coproporphyria increased increased coproporphyrinogen increased coproporphyrinogen normal
Variegate porphyria increased increased coproporphyrinogen increased coproporphyrinogen and protoporphyrin normal
Congenital erythropoietic porphyria normal increased uroporphyrinogen and coproporphyrinogen increased coproporphyrinogen increased uroporphyrinogen and coproporphyrinogen
Porphyria cutanea tarda normal increased uroporphyrinogen increased isocoproporphyrin normal
Erythropoietic Protoporphyria normal normal increased coproporphyrinogen increased uroporphyrinogena and coproporphyrinogen

      We remark that:
(i) in neurological porphyrias the urinary excretion of porphobilinogen (PBG) is increased (not so in cutaneous porphyrias). The normal value of PBG in the urine of healthy humans is <2.5 mg/die or <2 mg/L (in a random urine sample). During acute attacks of neurological porphyrias the patient may excrete >50 mg/die of PBG in the urine.
(ii) in cutaneous porphyrias, with the exception of porphyria cutanea tarda, the red blood cells contain porphyrinogens, which are absent in neurological porphyrias and in healthy subjects.
(iii) Gene sequencing may be carried out to confirm the diagnosis, but the biochemical laboratory tests should be first carried out, in order to reduce the number of genes to be sequenced.
      The differential diagnosis of cutaneous porphyrias is relatively simple: few diseases cause such severe damage of the skin exposed to sunlight. Dermatological diseases may be aggravated by exposure to sunlight, but most often are not limited only to the light-exposed areas, whereas skin lesions due to cutaneous porphyrias only affect exposed areas. Autoimmune diseases such as lupus erythematosus disseminatus or pemphigus may cause skin lesions in the light-exposed areas, but these are less severe than those observed in porphyrias, and are associated to other symptoms (e.g. renal malfunctiong) which are absent in porphyrias. Pellagra, caused by dietary deficiency of the vitamin niacin (B3, nicotinamide), or by genetic defects of triptophan metabolism, e.g by Hartnup's disease (nicotinamide can be produced during the metabolismo of triptophan; see the lecture on genetic defects of aminoacid metabolism), may cause severe skin lesions, aggravated by exposure to sunlight and coupled to neurological symptoms. Since both pellagra and porphyrias may have genetic causes and be present at birth or shortly thereafter, the differential diagnosis is important. The laboratory demonstration of heme precursors in the blood and urine confirms the diagnosis.

Audio: differential diagnosis of cutaneous porphyrias

      The differential diagnosis of neurological porphyrias is difficult, and many more common diseases may cause acute crises of abdominal pain. The first point the physician should address is whether the patient's symptoms indicate that peritoneal involvement is present (this usually suggests a surgical condition) or not (usually indicating a medical condition). An indicative diagnostic flow-chart is reported in the figure below.

Clinical examples
1) 45 year old patient arrives to the emergency department with very intense abdominal pain; no laboratory data available. Physical examination fails to reveal signs of peritoneal involvement (peristalsis present, Blumberg sign negative).
Analysis of the case: possible causes of intense abdominal pain that do not cause peritoneal involvement include: acute pancreatitis, neurological porphyrias (since this is an adult patient with no previous history this diagnosis is not particularly likely), urinary stones, posterior myocardial infarction, cancer, tabes dorsalis, lead (or other heavy metal) intoxication, etc. Prescribe a complete screening of serum enzymes (amylase is the marker of pancreatitis; include heart enzymes and markers), serum and urine porphyrinogens and porphobilinogen, analysis of serum heavy metals, echography, RX and NMR of the abdomen, electrocardiogram and echocardiography, Treponemal antigens and antibodies. Hope for the best. Reevaluate patient every six-twelve hours for the possible appearance of peritoneal signs.

2) 42 year-old woman with a history of episodes of severe weakness and abdominal pain. The patient is currently suffering of an intense abdominal crisis. The anamnesis reveals that her mother (presently deceased for unreported reasons) suffered of similar symptoms; this suggests a hereditary dominant condition. Porphyria was suspected and urinary porphobilinogen and δALA were measured:
urinary phorphobilinogen 188 μmol/L (normal level < 9 μmol/L)
urinary δALA 94 μmol/L (normal level < 50 μmol/L).
Chronic lead poisoning is a possible alternative explanation, even though it is usually a professional intoxication , not justified by the patient's history. The serum lead concentration was measured nevertheless, and resulted negative:
serum lead concentration 1.8 μg/dL (normal level < 5 μg/dL; chronic lead poisoning causes symptoms at concentrations of 40 μg/dL or higher).
Analysis of the case: the results of urinary heme precursors were compatible with a neurological porphyria, thus genetic testing was indicated. A genetic mutation of HMBS (hydroxymethyl bilane synthase) was found, thus confirming the diagnosis of autosomal dominant Acute Intermittent Porphyria. Important diagnostic clues in this case were: (i) the positive familial anamnesis; (ii) the fact that multiple similar episodes resulted in the remote pathological anamnesis; (iii) the fact that the urine analysis could be carried out during or immediately after the painful episode.

Further readings
Phillips JD. Heme biosynthesis and the porphyrias. Mol Genet Metab. 2019; 128: 164-177. doi: 10.1016/j.ymgme.2019.04.008.
D.A. Bryant, C.N. Hunter and M.J. Warren Biosynthesis of the modified tetrapyrroles—the pigments of life J. Biol. Chem. 2020, 295, 6888-6925.
T Wiederholt, P Poblete-Gutiérrez, K Gardlo, G Goerz, K Bolsen, H.F. Merk, J. Frank Identification of Mutations in the Uroporphyrinogen III Cosynthase Gene in German Patients With Congenital Erythropoietic Porphyria Physiol Res. 2006; 55 Suppl 2: S85-92.

Questions and exercises:
1) Abnormally elevated concentration of porphobilinogen (PBG) and δ-aminolevulinic acid (ALA) in the urine suggests:
a neurological porphyria
a cutaneous porphyria
Congenital erythropoietic porphyria

2) Erythrodontia is characteristic of:
Acute intermittent porphyria
Variegate prophyria
Congenital erythropoietic porphyria

3) Most porphyrias are inherited; the exception is porphyria cutanea tarda, which may be inherited or acquired. Acquired cases may be due to:
Viral infections
Chronic metal poisoning

4) Neurological porphyrias may simulate:
Acute abdominal crises, possibly requiring surgery
The cutaneous photosensistivity of lupus erythematosus disseminatus
Palsies due to stroke

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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.
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 ( 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
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
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.

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