Nucleotide Metabolism


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      Nucleotides play important roles in the cell, both as the monomers of nucleic acids (DNA and RNA) and in their free form (e.g. ATP, cAMP, etc.). They are made up of a pentose sugar (ribose in RNA and ribonucleotides; deoxyribose in DNA and deoxyribonucleotides), an organic heterocyclic base similar to either pyrimidine or purine, and phosphoric acid.

      Gout is a relatively common metabolic syndrome caused by increased serum concentration of uric acid, the terminal metabolyte of the purine degradation (see below).
      There are different possible causes of hyperuricemia and gout, which therefore cannot be considered a precisely identified disease; rather it is a syndrome that may appear because of: (i) hydiopathic hyperuricemia (hydiopatic, meaning "of no known cause" in this case is probably to be explained as the effect of an unfavorable combination of allelic variants of otherwise functional enzymes in the purine degradation pathway); (ii) increased biosynthesis of purine bases (e.g. myeloproliferative diseases like leukemias and lymphomas; psoriasis); (iii) reduced renal clearance of uric acid (e.g. renal insufficiency); (iv) metabolic conditions such as reduced purine salvage (e.g. hereditary deficiency of hypoxantine-guanine phosphoribosyl transferase). Gout may be aggravated by a diet rich in nucleic acids (e.g. fish, meat) or alcohol and by obesity and diabetes.
      In most animals, plants and bacteria uric acid is not a terminal metabolyte, as this compound is converted to allantoin by the enzyme uricase; e.g. carnivora because of their diet produce large amounts of uric acid but do not suffer of gout because they further convert it to allantoin. Man and primates have a non functional gene for uricase; from an evolutionary point of view this is possibly explained by the advantage of retaining relatively high concentrations of uric acid and urates to take advantage of their antioxidant properties.

      Clinical laboratory determination of uricemia: uric acid is a reductant; thus it can react with several reagents whose reduction is associated to a color change, quantificed by absorbance spectrophotometry. An example of such reagents is sodium tungstate. Since the serum contains other reducing substances (e.g. ascorbic acid), this reaction has interferences and usually overestimates the concentration of uric acid. A much more selective and precise method is enzymatic, based on uricase. This enzyme converts uric acid to allantoin, and this causes a characteristic decrease of absorbance at 293 nm. Uricase is highly specific, thus this reaction has no interferences and leads to a precise estimate of uricemia.

      Considerations on hydiopathic hyperuricemia: it has been repeatedly asserted that "hydiopathic" is an elegant word to conceal our ignorance and that its meaning is "we do not know why". Nowadays, however we have quite a clear idea of what hydiopathic means, or could mean, at least in clinical contexts like hyperuricemia. The members of the human population differ because of the presence in their genomes of different allelic variants of the same genes. Even when these variants are fully functional and none can be considered pathological, there will be people having more functional combinations of alleles of different genes and people having less functional combinations. Thus the human population includes individuals who produce and excrete uric acid at a faster or a slower rate, and the urate concentration in the blood serum is distributed as a gaussian. This, coupled with the different dietary intake of purine bases, explains the hydiopathic hyperuricemia as the tail of the gaussian distribution. A quantitative example of such reasoning is presented elsewhere.
      Clinical features: gout usually manifests itself with a sudden and painful arthritis of one or more major joints (ankle, knee, elbow, wrist); involvement of the metatarsophalangeal joint of the first toe is common (podagra). Precipitating factors may be minor traumas, or administration of some drugs (penicillin, diuretics). The affected joint is actuely painful, red and swollen and on palpation characteristic hard calculi (gout tophi, consituted by urate salts) may be appreciated on palpation. Fever is common. Chronic cases present erosive joint deformities.
      Diagnosis: the clinical presentation is quite typical and suggest the correct diagnosis, which is confirmed by the finding of increased serum urate concentration (normal value in adult males < 7 mg/dL; slightly lower in women. This value is very close to the solubility of urate in the plasma), and of negatively birefringent needle-shaped urate crystals in the synovial fluid (birefringence is the property of a crystal whose refractive index varies with the direction of light polarization: observed in the microscope upon polarized light the crystal changes from bright to dark as the polarizing filter is rotated). Urate concentration is measured using an enzymatic assay (the enzyme uricase is used, that converts uric acid to allantoin; the reaction is monitored by absorbance spectroscopy at 293 nm; uricase is not produced by humans). Once a diagnosis of gout has been made, the possible underlying conditions should be looked for (e.g. leukemia, diabetes, etc.).
      DIFFERENTIAL DIAGNOSIS OF GOUT: gout is a non-febrile recurring acute arthritis, to be differentiated from septic arthritis, rheumatoid arthritis, rheumatic fever, autoimmune diseases. Increased urate in the serum and the urine and the presence of tophi are diagnostic. Moreover in gout autoantibodies characteristic of rheumatoid arthritis and other autoimmune diseases are absent. Rheumatic fever follows a streptococcus infection and the patient has elevated indexes of it (e.g. high Anti-Streptolysin O titer, ASO). Acute septic arthritis is a major infectious syndrome with fever and leukocytosis.
Differential diagnosis of gout arthritis
diseaseclinical findingslaboratory findings
Gout arthtritisacute onset; gout tophiincreased urate concentration in the serum; birefringent urate crystals in the synovial fluid
Septic arthtritisfever; general symptomspus in the synovial fluid
Rheumatic feveracute onset; major joints; heart involvement; follows streptococcal infectionelevated serum TAS
Rheumatoid arthtritis and other autoimmune arthrtitesslow onset; minor jointsautoantibodies and elevated inflammation markers in the serum

      HYPERURICEMIA AND KIDNEY FAILURE. The relationship between gout and kidney failure is two-fold: hyperuricemia of whatever cause may be lead to chronic kidney failure because of the possible precipitation of urate crystals in the kidney tubules, leading to their death; kidney failure of whatever cause may lead to impaired elimination of uric acid and hyperuricemia. Thus these two conditions may aggravate each other. An indicative list of the principal causes of hyperuricemia is reported in the Table below. Notice that a purine rich diet is not a cause of hyperuricemia per se but may aggravate any of the conditions listed in the Table.
Some possible causes of hyperuricemia
hydiopathic gout
genetic defects of purine metabolism (e.g. Lesch Nyhan syndrome, see below)
myeloproliferattive disorders (e.g. leukemias, lymphomas)
chronic kidney failure
collateral effect of some drugs (e.g. ethambutol, cyclosporin)
may occur in patients suffering of hypothyroidism
may occur in patients suffering of Down syndrome
excess alcohol intake

      Therapy of the acute attack is very specific, as gout responds dramatically to colchicine (not more than 3-4 mg/die).

      All the nucleotide components can be synthesized ex-novo.
      Ribose 5'-phosphate is produced from glucose in the penthose phosphate pathway and is converted to 5'-phosphoribosyl 1'-pyrophosphate (PRPP) by the enzyme ribose phosphate pyrophosphokinase. Deoxyribose is not produced directly; rather ribonucleotides are synthesized and converted to deoxyribonucleotides by the enzyme ribonucleotide reductase that uses the small protein thioredoxin as the reductant. Oxidized thioredoxin is reduced by the NADPH-dependent enzyme thioredoxin reductase, whose inhibition (by drugs and poisons) slows down cell replication.
      Pyrimidine bases are produced in a specific biosynthetic pathway from carbamyl phosphate and aspartate; phosphoribosyl pyrophosphate is used as the donor of ribose (the enzymes or metabolytes most commonly affected by heritable diseases are underlined):

      Purine bases are produced in a specific biosynthetic pathway from glycine and aspartate; phosphoribosyl pyrophosphate is used as the donor of ribose:
Tetrahydrofolate is an important cofactor of nucleotide biosynthesis. An important observation on the biosythesis of nitrogenous bases is as follows: purine biosynthesis requires two formyl groups that are donated by N-formyl-tetrahydrofolate in the reactions catalyzed by GAR transformylase and AICAR transformylase. Moreover methyl-tetrahydrofolate is required for the methylationm of uracil to thymine. Tetrahydrofolate is derived from the vitamin B9 (formic acid). As a consequence avitaminosis B9 may disrupt the biosynthesis of purine nucleotides and produce symptoms similar to those of some genetic defects of nucleotide biosynthesis (megaloblastic anemia). The picture below illustrates the origin of carbon and nitrogen atoms in the aromatic rings of purines and pyrimidines:

      Given that the biosynthesis of nucleotide bases is energy-expensive, all animals (man included) possess biochemical pathawys that allow the recovery of nucleotides derived from DNA degradation (either because of dietary apport or because of cell and tissue turnover). In man two enzymes are responsible for the salvage of purine bases: adenine phosphoribosyl transferase and hypoxantine-guanine phosphoribosyl transferase. The chemical reactions catalyzed by these anzymes are as follows:

      All the metabolic pathways involved in the biosynthesis of nucleotides can be subject to hereditary disturbances; however most defects in such important bisynthetic pathways are likely to be non viable and thus few diseases are described.. The most common inherited disturbance of the pentose phosphate pathway, responsible for the biosynthesis of ribose, is the genetic anomaly of glucose-6-phosphate dehydrogenase known as favism, which is described under the diseases of glucose metabolism.
      The most important disease of nucleotide biosynthesis/salvage is the hereditary, X-linked deficiency of hypoxantine-guanine phosphoribosyl transferase. The genetic defect varies and, depending on the amount of the enzyme that is produced and its activity, the severity of the resulting disease also varies from mild sex-linked uric aciduria (a variant of gout, see below), to the lethal Lesch Nyhan syndrome, associated with renal insufficiency, severe mental retardation, automutilation and death in the infancy. Diagnosis is based on clinical grounds and is confirmed by genetic analysis.
      Another important hereditary disease of nucleotide bisynthesis is Orotic aciduria, that causes megaloblastic anemia, mental retardation, and stunted growth. This condition is due to hereditary deficiency of the enzyme orotate phosphoribosyl transferase, a bifunctional enzyme that catalyzes two reactions in the pyrimidine biosynthesis pathway: conjugation of orotic acid to ribose 5'-phosphate and decarboxylation of orotidine 5-phosphate to uridine 5-phosphate. The disease may be suspected because of the finding of a megaloblastic anemia in the newborn, resistent to the administration of folate and vitamin B12, and is confirmed by the finding of high amounts of orotic acid in the urine. Gene sequencing is also possible and reveals the mutation.
      Increased activity of Phosphoribosyl Pirophosphate Synthetase is a rare hereditary, X-linked genetic defect causing a genetic form of gout. The enzyme is allosteric and downregulated by ADP and 2,3-DPG. The abnormal variant is constitutively active, irrespective of the concentration of its allosteric inhibitors. The disease may be apparent also in female newuborns, given that the effect of the mutation is dominant. Diagnosis is by gene sequencing.
      Adenylosuccinase Deficiency is a rare hereditary defect, associated with mental retardation. Diagnosis is established by gene sequencing.

      Differential diagnosis
      Disturbances of the biosynthesis and salvage pathways cause diseases in which the following symptoms predominate: mental retardation, gout, megaloblastic anemia. The symptoms appear very early, within the first months of life. Aside from the case of Lesch Nyhan syndrome, in which major neurological and psychiatric symptoms predominate, the other conditions are quite uncharacteristic. The main difficulty for diagnosis is that the physician rarely considers these hereditary defects as possible causes of stunted growth and mild mental retardation.
      In a newborn or infant presenting stunted growth, a blood test should be required as a routine analysis. Finding of megaloblastic anemia is an important indication, because this condition occurs in folate or B12 deficiency or in hereditary diseases causing slowed cell cycle (reduced rate of DNA biosynthesis). Indeed the molecular mechanism underlying megaloblastic anemia is the same for the two conditions, because folic acid and vitamin B12 are required cofactors for the biosyntehsis of nucleotides. Folate deficiency in the newborn occurs under conditions of severe social deprivation and usually is associated to malnutrition of the infant and his/her parents. Vitamin B12 deficiency in the newborn or infant may be due to malnutrition, or to ideological factors: e.g. vegan parents may undergo mild vitamin B12 deficiency, which, through lactation may affect their baby. However, the serum concentration of both folate and vitamin B12 can be determined: if these are low, the diagnosis is of a deficiency megaloblastic anemia, if they are normal, a hereditary condition should be suspected and investigated by gene sequencing.

disease inheritance biochemical defect symptoms laboratory findings
Lesch Nyhan s. X-linked (male only) hypoxantine-guanine phosphoribosyl transferase (purine salvage pathway) gout, mental retardation, psychotic autoaggressive symptoms increased urate serum
orotic aciduria autosomal recessive orotate phosphoribosyl transferase (pyrimidine biosynthesis pathway) megaloblastic anemia, mental retardation, stunted growth orotic acid in serum and urine
Increased activity of Phosphoribosyl Pirophosphate Synthetase X-linked, dominant (female affected) Phosphoribosyl Pirophosphate Synthetase (purine and pyrimidine biosynthesis pathway) familial gout increased urate serum
Adenylosuccinase Deficiency autosomal recessive Adenylosuccinate lyase (purine biosynthesis pathway) mental retardation, epilepsy succinylated purine derivatives in the urine
Severe Combined Immunodeficiency Disease (SCID) autosomal recessive Adenosine Deaminase (ADA; purine degradation pathway) repeated infections (may be lethal in the absence of bone marrow transplant) reduced white cell counts; greatly reduced immunoglobulin fraction in the serum electropherogram; reduced ADA activity in the hemolysate
Purine Nucleoside Phosphorylase Deficiency autosomal recessive Purine Nucleoside Phosphorylase (purine degradation pathway) immunodeficiency; repeated infections reduced T lyphocyte count
Pyrimidine 5' Nucleotidase Deficiency autosomal recessive Pyrimidine 5' Nucleotidase (pyrimidine degradation pathway) hemolytic anemia basophilic stippling in the erytrocytes due to accumulation of pyrimidine nucleotides
Dihydropyrimidine Dehydrogenase Deficiency autosomal recessive Dihydropyrimidine Dehydrogenase (pyrimidine degradation pathway) mental retardation elevated pirimidine bases in the serum

      Excess nucleotides introduced with the diet or produced by tissue turnover are degraded in specific biochemical pathways. The metabolytes of pyrimidine bases are derivatives of 3-amino propanoic acid which are converted to either malonyl-CoA (utilized for fatty acid biosynthesis) or methylmalonyl-CoA (converted to succinyl-CoA):

      Purines are converted to uric acid which is excreted by the kidney:

      Clinical manifestations of genetic defects of the purine and pyrimidine degradation pathways. Defects of the nucleotide degradation pathways are inherited as autosomal recessive traits and in the homozygous state become clinically manifest in the first months of life. The most natable syndromes are those of immunodeficiency, that may be masked while the baby is milk-fed by the mother, thanks to the maternal antibodys present in the milk. Other possible syndromes are those of anemia (in the case of Pyrimidine 5' Nucleotidase Deficiency) and mental retardation (Dihydropyrimidine Dehydrogenase Deficiency). A summary of the clinical manifestations of the diseases of nucleotide metabolism is presented in the following table:
symptompossible genetic defect(s)
mental retardationLesch Nyhan s.; orotic aciduria; Adenylosuccinase Deficiency; Dihydropyrimidine Dehydrogenase Deficiency
megaloblastic anemiaorotic aciduria
hemolytic anemiaPyrimidine 5' Nucleotidase Deficiency
goutLesch Nyan s.; increased activity of Phosphoribosyl Pirophosphate Synthetase
immunodeficiencyAdenosine Deaminase Deficiency; Purine Nucleoside Phosphorylase Deficiency

      An important caveat is that each of the above clinical symptoms may also have causes other than inherited defects of nucleotide metabolism; e.g. megaloblastic anemias may also be due to vitamin deficiencies (B12 and/or folate); congenital immunudeficiencies may also be due to other inherited enzyme defects (e.g. Bruton's agammaglobulinemia is due to the inherited X-linked deficit of tyrosine kinase), etc.
      Several hereditary diseases due to defects of nucleotide degradation are known.
      Severe Combined Immunodeficiency Disease (SCID) is the common name of several hereditary deficits. The most common form is a X-linked defect of the of IL2RG gene that codes for the so-called common gamma chain, a component of several receptors of the lymphocyte membrane. This form of the disease is not related to nucleotide metabolism. An autosomal recessive form of SCID is due to the genetic defect of adenosine deaminase, the enzyme that converts AMP to IMP and starts the degradation of adenosine. Pathogenesis is due to the accumulation in the cells of deoxyAdenosine triphosphate (dATP), that inhibits the enzyme ribonucleotide reductase and slows down the biosyntehsis of DNA. All rapidly regenerating tissues are affected and the precursors of granulocytes and lymphocytes are affected most. The disease is invariably fatal because of infections; therapy usually requires bone marrow transplant. Diagnosis is suspected on clinical grounds (severely reduced lymphocyte and granulocyte count) and confirmed by genetic analysis. Notice that since several forms of SCID exist, which cannot be distinguished on clinical grounds, the differential diagnosis relies on the type of inheritance, the demonstration of the biochemical alteration, and gene sequencing.
      Purine Nucleoside Phosphorylase Deficiency. Purine Nucleoside Phosphorylase is responsible for releasing inosine or guanosine from the corresponding ribonucleotides or deoxyribonucleotides at the very beginning of the purine degradation pathway. The disease causes accumulation of deoxyribonucleotides, especially dGTP and affects mostly the replication of T-lymphocytes. The clinical picture is that of an immune deficiency, less severe than SCID.
      Pyrimidine 5' Nucleotidase Deficiency is an hereditary disease whose main symptom is an hemolytic anemia of variable severity.
      Dihydropyrimidine Dehydrogenase Deficiency causes neurological symptoms and mental retardation. Diagnosis is based on the finding of elevated concentration of free pyrimidine bases in the serum.

      Genetic disturbances of the biosynthesis and degradation of nucleotide bases may cause at least three widely different clinical syndromes:
(i) mental retardation, possibly associated to gout and/or stunted growth. The Lesch Nyan syndrome is the severest form of this group.
(ii) Congenital immunodeficiency syndromes. SCID due to ADA deficiency is the severest and paradigmatic example.
(iii) Anemias, either megaloblastic or hemolytic.
      The differential diagnosis of mental retardation is difficult because this condition may be due to a host of different causes, not all of them known.
      A large number of congenital immunodeficiencies is described. Some of these affect selectively the B or T lymphocytes, others affect both. The crucial step is differentiating from acquired immunodeficiencies (e.g. congenital HIV/AIDS). The immunodeficiency usually becomes clinically evident with weaning, because the mother's milk contains immunoglobulins that the breast-feed newborn absorbs. It is uncommon for acquired immunodeficiencies to appear as early as during weaning, thus an hereditary cause should be considered for every early-onset immunodeficiency, making genetic testing mandatory.

Clinical examples
1) 65 year old patients presenting sudden, painful arthritis of the right elbow. Body temperature 36.7oC. Blood test reveals:
hemoglobin12 g/dL
erythrocyte count4 x 106 /mmc
mean corpuscular volume85 fL (normal value 80-100 fL)
white cell count13 x 103 /mmc
platelets190 x 103 /mmc
erythrocyte sedimentation rate (ESR)48 mm/h (normal value <10 mm/h)*
platelets190 x 103 /mmc
BUN20 mg/dL
creatinine1.1 mg/dL
glucose110 mg/dL
Analysis of the case: acute monoarthritis of a large joint, with clear laboratory evidence for inflammation (increased ESR), but no indication of bacterial infection (normal white cell count, normal body temperature). The most likely hypothesis is gout; less likely hypotheses to be considered: septic arthritis, trauma, with possible joint hemorrage, autoimmune arthritis. Measure urate in the serum and urine; test for autoantibodies e.g. for dsDNA; if possible take a sample of sinovial fluid (may contain pus, bacteria, blood, etc.).

Questions and exercises:
1) The concentration of urate in the serum of a healthy adult is:
< 7 mg/dL
< 10 mg/dL
< 15 mg/dL

2) Congenital immunodeficiencies are due to:
hereditary deficiency of adenosine deaminase
a large number of possible causes, one of which is hereditary deficiency of adenosine deaminase
infection by HIV during the fetal life

3) The mechanism of inheritance of the Lesch-Nyhan syndrome is:
autosomal recessive
sex-linked dominant
sex-linked recessive

4) The blood analysis of a young child reveals megaloblastic anemia. Possible causes to take into consideration for differential diagnosis are:
Orotic aciduria, and vitamin B12 or folate deficiency
Orotic aciduria, β-thalassemia, and iron deficiency
Orotic aciduria, folate deficiency, and sickle cell anemia

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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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