MEDICINE AND SURGERY "F"
Course of LABORATORY MEDICINE
Nucleotide Metabolism

METABOLISM OF NUCLEOTIDES AND RELATED DISEASES

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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
      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.

      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

      Therapy of the acute attack is very specific, as gout responds dramatically to colchicine (not more than 3-4 mg/die).
 
 
INHERITED DEFECTS OF PURINE AND PIRIMIDINE BASES METABOLISM

      BIOSYNTHESIS OF NUCLEOTIDES
      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:

 
 
      SALVAGE PATHWAYS
      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:

 
 
      DISTURBANCES OF THE BIOSYNTHESIS AND SALVAGE OF NUCLEOTIDES
      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 Nyan 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.

GENETIC DISEASES OF NUCLEOTIDE METABOLISM: A SUMMARY
disease inheritance biochemical defect symptoms laboratory findings
Lesch Nyan 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

 
 
      NUCLEOTIDE CATABOLISM
      Excess nucleotides introduced with the diet or produced by tissue turnover are degraded in specific biochemical pathways. The terminal metabolytes of pyrimidine bases are derivatives of 3-amino propanoic acid:

 
      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 Nyan 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.
 
      DISTURBANCES OF THE DEGRADATION OF NUCLEOTIDES
      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.

      DIFFERENTIAL DIAGNOSIS OF GENETIC DISTURBANCES OF PURINE AND PIRIMIDINE METABOLISM
      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.
 

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).



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