Course of Laboratory Medicine
Medical School course F - Faculty of Pharmacy and Medicine
Prof. Andrea Bellelli

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     In this purely theoretical exercise, we shall investigate one among the possible causes of inter-individual variability.

The reaction scheme
      Let us suppose that the terminal part of a metabolic pathway converts irreversibly metabolyte A into metabolyte B and then into terminal product C that is excreted. The reaction scheme is as follows:
A → B → C

      Reaction 1 irreversibly converts A to B and is catalyzed by enzyme E1; reaction irreversibly 2 converts B to C and is catalyzed by enzyme E2.
      We further suppose that the concentration of metabolyte A is regulated by a negative feedback mechanism and is maintained constant. Moreover since product C is excreted and the reactions leading to its production are irreversible, we can neglect its concentration. We want to investigate the steady-state concentration of metabolyte B.
      Let us assume that both E1 and E2 operate under steady-state conditions and obey a simple Michaelis and Menten equation. The rate of change of the concentration of metabolyte B is described by the following differential kinetic equation:
          (eqn. 1)
where the first term describes the rate of formation of B from A (whose concentration is assumed to be constant) and the second term describes the rate of degradation of B. Vmax,1 and KM,1 are the Michaelis and Menten parameters for E1 and Vmax,2 and KM,2 those for E2.

      Under steady-state conditions, the concentration of B is constant, i.e. the differential equation describing its change equals zero. Thus, we can solve the above equation for [B] to obtain:
          (eqn. 2)

      The above formula allows us to calculate the concentration of metabolyte B for any set of conditions. For example, if we assume the following set of parameters:
steady-state concentration of A       KM,1       Vmax,1       KM,2       Vmax,2      
1 mM 1 mM 10 s-1 1 mM 10 s-1
we can easily calculate from eqn. 2 that the steady-state concentration of B is 1 mM.

Presence of allelic variants of the enzyme(s) and their distribution in the population
      Let us assume that in the population under study there are two genetic variants of E1 and two of E2, that we call E1, e1, E2 and e2 respectively. All variants are functional, but they differ because of slight changes in Vmax and KM, e.g.:
E1: Vmax,1=11 s-1; KM,1=0.9 mM                   e1: Vmax,1=9 s-1; KM,1=1.1 mM
E2: Vmax,2=11 s-1; KM,2=0.9 mM                   e2: Vmax,2=9 s-1; KM,2=1.1 mM

      Let us further assume that the concentration of each enzyme is constant and that function of enzyme 1 in the heterozygous individual having an equimolar mixture of E1 and e1 can be approximated by a Michaelis and Menten equation with averaged parameters (i.e. Vmax,1=10 s-1; KM,1=1 mM). The same applies to the function of enzyme 2. These approximations are very rough, but sufficient for our present purpose:

      To simulate the distribution of the concentration of B in the population we need one further information, namely the gene frequencies for E1 and e1, E2 and e2. Let us assume that the gene frequencies are 50% for each gene; the Hardy-Weinberg law allows us to calculate the phenotype frequencies as follows:
E1E1: 0.52=0.25       E1e1: 0.5 x 0.5 x 2 = 0.5       e1e1: 0.52=0.25
E2E2: 0.52=0.25       E2e2: 0.5 x 0.5 x 2 = 0.5       e2e2: 0.52=0.25

      The frequencies of the different two-gene phenotypes are then calculated by multiplying those of the pertinent single-gene phenotypes; e.g.:
E1e1 / E2e2 = 0.5 x 0.5 = 0.25

      The distribution of metabolyte B concentration in the population can be calculated as follows:

1) A very simple model of a metabolic pathway including only three chemical intermediates and two enzymes, under the assumption of two very similar allelic variants of each enzyme, can generate a surprisingly wide variability of the intermediate metabolyte.
2) The distribution of the intermediate metabolyte concentration is described by a skewed Gaussian curve.
3) As predictable, the concentration of the intermediate metabolyte is highest in subjects whose genetic constitution is such that they are homozygous for the more effective variant of the producing enzyme (E1E1), and again homozygous for the less effective variant of the degrading enzyme (e2e2).
4) If the concentration of the intermediate metabolyte is in any way related to susceptibility to a specific disease the model would partially explain the incidence of the disease.
5) The frequencies of the highest and lowest values of the intermediate metabolyte concentrations do not correlate with the frequency of any single gene variant (because they correlate with the products of the gene frequencies).

Inter-population variability
      Human populations usually present different frequencies of the same alleles of the same genes. It is quite uncommon that an allele is present in a population and completely absent in another (if this happens, this points to a very ancient separation in the evolutionary history of Homo sapiens).
      We can easily simulate this condition using the same data we used for simulating inter-individual variability, but changing the gene frequencies. An important consequence of the above consideration is that it is very difficult to assign a single individual to any given population. What one can do is to estimate the probability that a single individual is a member of a given population. Obviously, the more allele variants and the more genes are considered, the more reliable this estimate is.

Populations are not races
      The concept of race applied to human populations has a releatively short (and infamous) history: it was initially used by De Gobineau in France and Knox in England (both circa 1850). A race is an artificial group, obtained in zootechnology or agriculture by rigorous control over reproduction. As a consequence it cannot be applied to human populations and we can state that human races do not exist. The difference between a race and a population or ethnic group is evident to genetists but quite subtle for non-specialists. We may explain this difference by comparing two examples, one for two populations, the other for two races.
      The distribution of blood groups in Italy is fairly homogeneous, except for Sardinia, which is different, as shown in the table below:
blood groups:0ABAB
Italy except Sardinia46%42%9%3%

      Clearly, the Sardinian population stands apart from the rest of Italy, but you cannot find this property in any single individual. A population differs from another because of the allelic frequencies of the same genes. Allele frequencies are properties of groups, not of individuals. Obviously, two populations differ because of the allele frequencies of a large number of genes, not only one.
      By contrast let's consider the case of two races, e.g. basset-hounds and terrier. All and every basset-hounds have a mutation of the gene FGFR3, and carry the same genetic defect of human achondroplasia, which causes a reduced growth of long bones. By contrast no terrier has this mutation. Thus race is a genetic property of the population and of each of its members. Clearly, a race can be maintained only if mating is strictly controlled, either by humans or by physical barriers. Again, two races of the same species differ because of allele variants of many genes, not only one.
      As a consequence of the above distinction, we can confidently state that an individual animal is a member of a specific race (and eventually how "pure" it is), whereas in the case of individual humans we can determine which alleles he/she posesses for relevant genes and infer the probability that he/she is a member of a population. Moreover, in order to define the human populations we also need non-genetic criteria (e.g. language or geographical considerations).
      Races, but not populations, have an impoverished gene pool. Applying strict control over mating one can further reduce the gene pool of a group of animals and obtain the strain. This is only done for laboratory animals; all individuals belonging to the same strain are genetically identical or almost so and they accept transplants from individuals of the same strain without rejection (which is not the case with animal of the same race).
      The genetic of populations is extremely important in medicine because different alleles may be differently related to susceptibility to diseases; thus disease prevalences may vary among different populations, because of their characteristic allele frequencies. This in turn affects the reliability of our diagnostic hypotheses, because the prevalence appears as the pre-test probability in Bayes' formula.

Questions and exercises:
1) Inter-human variability is a consequence of:
the distribution of allelic variants of the same genes
the presence of non-functional genes in the population
environmental factors

2) Inter-population variability is a consequence of:
different frequencies of (the same) allelic variants of genes
environmental factors
the presence of different genes in the different populations

3) The Hardy Weinberg law
describes the genotype frequencies
correlates the phenotype frequencies to the gene frequencies
describes allelic frequencies

4) The variability of analytes' concentrations among different individuals
is determined by stochastic phenomena
is genetically determined because of allelic variants of the analytes
is genetically determined because of allelic variants of the enzymes responsible of the metabolism of analytes

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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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