Chemical and Biochemical Methods


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      Blood is probably the most informative biological specimen the physician can obtain. The procedure is moderately invasive as blood must be taken from venous, arterial or "capillary" vessels. Urine is very easy to collect using either invasive (vescical catheterism) and non invasive methods. Cerebro-spinal fluid collected by means of lumbar puncture may give information about infectious and other pathological conditions of the brain, the meninges and the spinal cord. Intestinal fluids may be collected using specially devised tubings: pancreatic fluid, bile, gastric fluid, intestinal fluid, etc. Faeces can be analyzed for the presence of intestinal parasites or their eggs and larvae. Bioptic samples may be collected during open or endoscopic surgery; the procedures are highly invasive, but these samples provide unequivocal microscopic information that is necessary to some diagnoses (e.g. cancer).
      The chosen artery (usual the radial artery at the wrist) is found by palpation and punctured with a gas-tight syringe. The main (only) indication for collection of an arterial blood sample is the analysis of respiratory gases (hemogas analysis), which also gives information on the blood pH and buffers.
      Venous blood is the standard sample for the majority of clinical analyses. Blood can be separated by centrifugation in a cellular fraction, containing the erythrocytes, leucocytes (granulocytes, lymphocytes and monocytes) and platelets, and a liquid fraction called plasma. If the blood is allowed to clot, the fibrinogen is removed from the plasma: de-fibrinated plasma is called serum.
      So called capillary blood, collected by digital puncture, is used for some analyses (e.g. blood smear, or hemoglobinometry, red cell count), when a venous puncture is unnecessary.
      Biological samples may be submitted to standard chemical analysis, either qualitative (to ascertain whether a substance or ion is present or absent) or quantitative (to determine the concentration of a substance or ion). If the substance of interest is a biological macromolecule, most often a protein, its qualitative or quantitative determination is often difficult, the reason being that biological macromolecules may be chemically very similar to each other and therefore difficult to differentiate, e.g. our genome codes for some 25,000 to 30,000 proteins, all with the same basic chemical structure of heteropolymers of aminoacids. In these analyses specifically designed biochemical methods are resorted to, as detailed below. In the following section the most common methods employed in Clinical Chemistry with be summarized.
      Chemical and physical methods are resorted to for the chemico-clinical analyses, whose objective is usually the measurement of the concentration of low molecular weight solutes: ions (electrolythemia); urea; glucose; creatinine; and many others.
      POTENTIOMETRIC METHODS. Potentiometric (or electrochemical) methods are safe, rapid and convenient. Since they are based on redox chemical reactions, they can only be applied when the substance one wants to measure undergoes a redox reaction suitable to be used in a galvanic cell. An example is the measurement of most blood electrolytes, e.g. sodium, potassium, calcium, magnesium and chloride. As an example we may redox couple Mg+2 / Mg that undergoes the semireaction:
Mg --> Mg+2 + 2 e-

This semireaction can be made to happen in a galvanic cell containing a solution of a magnesium salt (that dissociates into magnesium ion) and an electrode of elementary magnesium. The solution of magnesium salt, of course, may be any biological fluid (e.g. blood serum). The electric potential of this cell is determined by Nernst's equation:
E' = Eo + 0.06/n log [Mg+2]

E' may be measured using a potentiometer, i.e. an instrument that determines the electromotive force of a battery composed by the above cell, and a reference cell of known potential (E"):
f.e.m. = E" - E'

From the above equations one derives:
E' = E" - f.e.m.
log [Mg+2] = (E' - Eo) n/0.06

      Potentiometric methods are strongly selective: if an electrode of Mg is immersed in a sample of blood plasma, that contains dozens of different electrolytes, it will form a redox couple only with Mg+2, ignoring all other ions. They are also very fast: the instruments measures the f.e.m. and calculates [Mg+2] in a matter of seconds or less. Given that the working time of the technician is the most relevant parameter in determining the cost of the analysis, electrochemical methods are also cheap.
      There are two limits of electrochemical methods: (i) they cannot be applied to analytes that do not undergo convenient redox reactions or that undergo redox reactions unsuitable to be realized in galvanic cells; and (ii) they require a selective electrode that may not be cheap, easy to build, and durable. These limitations have often be overcame by means of electrodes capable of quantitatively exchanging the ion of interest with another, easier to measure. E.g. the instrument used to measure the pH (pHmeter) uses an electrode that exchanges H+ with K+, and measures the latter in place of the former, in order to avoid the necessity of the idrogen gas electrode.

      MANOMETRIC METHODS. Manometric methods rely on the measurement of gas volumes at constant (atmospheric) pressure or of gas pressures at constant volume. Obviously they are ideally suited for respiratory gases: O2>, CO2, N2. They are often coupled with potentiometric methods: e.g. the patient may breathe in a spirometer that measures the volumes of inspired and espired air, and the oxygen content may be determined using an oxygen electrode.
      Respiratory gases behave ideally, thus they obey the state law:
      P V = n R T
where P is the pressure in atm or mmHg, V the volume in litres, T the temperature in Kelvin, n the number of moles of particles contained in the gas sample and R the gas constant (0.082 L Atm / mol K). The number of moles of a given gas in a given sample results:
      n = P V / R T
E.g. if a patient inspires 5 L/min of air at 1 atm and 25 C (298 K) with an O2 content of 20% and expires an equal volume of air at the same pressure and temperature with O2 content of 15%, he has:
inspired nO2, insp = 1 x 5 x 0.2 / 0.082 x 298 = 0.041 moles O2 /min.
expired nO2, exp = 1 x 5 x 0.15 / 0.082 x 298 = 0.031 moles O2 /min.
the amount of oxygen retained (and used) by the organism in one minute is thus 0.041-0.031 = 0.010 moles.
Indirect calorimetry: the measurement of O2 consumption allows the physician to estimate the basal metabolism of the patient, given that the caloric equivalent of O2 in biological systems is essentially constant at 117 kcal / mole. E.g. the above measurement tells us that 10 mMoles of O2 /min. x 1440 min. /day x 117 kcal /mol = 1680 kcal /day.

      OSMOMETRY AND OTHER METHODS BASED ON COLLIGATIVE PROPERTIES. These methods are employed only in a limited range of analyses, e.g. the osmolarity of urine may be measured as a gross test of renal function. Measuring the true osmotic pressure of a solution by means of an osmotic balance usisng the appropriate semipermeable membrane is expensive, thus the density of the sample or its freezing temperature are measured instead.

      SPECTROPHOTOMETRIC METHODS. Spectrophotometric methods take advantage of the interaction with radiant energy to determine the qualitative and quantitative composition of matter. Given that the interaction between radiant energy and matter causes different effects depending on the wavelength, there are several different kinds of spectrophotometric methods.
      ABSORPTION SPECTROSCOPY IN THE UV/Vis WAVELENGTH RANGE. The most widely employed spectroscopy technique relies on the absorption of light in the visible and Ultra-Violet regions of the spectrum. This region of the spectrum corresponds to a wavelength range of 650-200 nm.
      A photon of this wavelength has energy enough to promote the bonding electrons of a molecule fom their ground state to an excited state: typically absorption of one photon excites one electron of a π bonding orbital to the corresponding π* antibonding orbital. Decay of the excited state to the ground state may occur radiatively (i.e. with emission of light, in phenomena called either fluorescence or phosphorescence) or non radiatively (i.e. with emission of heat). Other electronic transitions are possible in specific (less common) chromophores: e.g. in hemoproteins a d --> π* transition can be excited, promoting one electron residing in a d orbital of the iron to a π* orbital of the porphyrin. A substance which has absorption in the UV/Vis range is called a chromophore and appears colored to the eye, if it absorbs in the visible range of the spectrum.
      The instrument required for a measurement of absorption spectrophotometry can be represented as in this scheme:
The white light emitted by a lamp is passed through a monochromator, that splits it into its components (colors) and allows the operator to select the desired wavelength. The monochromatic light thus obtained passes through the sample (usually in solution) and the light which is transmitted by the sample is measured by a photomultiplier tube. If the sample is removed one measures the intensity of the incident light; when the sample is inserted one measures the intensity of the transmitted light. Transmittance (T) is defined as the fraction of incident light that corsses the sample:
      T = intensity of transmitted light / intensity of incident light
Since in this type of spectroscopy the sample can only absorb light, it is evident that 0 < T < 1 : i.e. the sample cannot transmit more light than it receives nor can it transmit less than zero.
      Absorbance (also named Optical Density) is defined as:
      Abs = -log10 T
      Absorption spectroscopy obeys the Lambert and Beer's law:
      Abs = ε C l
where ε represents the extinction coefficient of the chromophore at the wavelength used in the experiment, C its molar concentration and l the length of the optical path (usually 1 cm). A consequence of this law is that the concentration of a chromophore can be determined as:
      C = Abs / ε l
      By varying the wavelength of the incident light, one can collect the absorption spectrum of the sample, the Cartesian graph of Absorbance vs. wavelength, as in the absorbance spectra of oxy- and deoxy-hemoglobin, below:

      An important feature of absorption spectra is their nearly-Gaussian shape: i.e. the light required to promote the electronic transition(s) is not strictly monochromatic. This dependes on the energetic degeneracy of the ground and excited states of the chromophores. In the absence of degeneration (e.g. in atomic spectra) the absorption bands reduce to pure monochromatic lines.
      Absorbance is a very useful and inexpensive method for determinining the concentration of a substance that has significant values of ε at any wavelength in the 650-200 nm range. Unfortunately not all substances have significant extinction coefficients in this spectral regions; moreover biological samples usually contain mixtures of different substances and it may happen that the chromophore whose concentration we want to measure is mixed with other interfering chromophores which absorb in the same spectral range. The absorption of a mixture of different chromophores with overlapping extinction coefficients is:
      Abs = (ε1 C1 + ε2 C2 + ε3 C3 + ...) l
      Resolving the concentration of each chromophore (C1, C2, C3, ...) can be done by comparison of the absorbance values at different wavelengths, given that the extinction coefficients of each chromophore vary; but this is usually not an easy task. A very convenient method to overcome the problem of interference is to take advantage of a chemical reaction specific of the chromophore that we want to measure, that abolished its absorption: e.g. if our chromophore absorbs light because of a π --> π* transition and if a reaction exists that saturates the double bond and breaks the π orbital, we can measure:
      Absbefore reaction = (ε1 C1 + ε2 C2 + ε3 C3 + ...) l
      Absafter reaction = (ε2 C2 + ε3 C3 + ...) l
      Absbefore reaction - Absafter reaction = ε1 C1 l
      C1 = (Absbefore reaction - Absafter reaction) / ε1 l

      FLUORESCENCE SPECTROSCOPY IN THE UV/Vis WAVELENGTH RANGE. While most organic compounds absorb light (though often in regions of the spectrum that are poorly convenient in practice), only a few of them are fluorescent. Once an organic molecule has absorbed light and one of its electrons has been promoted from the ground to the excited state (most often in a π --> π* transition) it usually reverts to the ground state with emission of heat. Fluorescence is an uncommon consequence of absorption and consists in the emission of light during the process of de-excitation from the excited to the ground state: π* --> π + light. The wavelength of the light emitted by the excited sample is usually longer than that of the absorbed light (i.e. the emitted light has less energy per photon than the exciting light; law of Stokes). However, since both the absorption and fluorescence spectra have both a nearly Gaussian shape, and may partially overlap, there is a limited interval of wavelength in which the emitted light may have shorter wavelength than the exciting light (so called anti-Stokes region of the fluorescence spectrum).

      ATOMIC ABSORPTION (AND EMISSION) FLAME SPECTROSCOPY. If a sample (be it of biological origin or not) is heated on a hot flame, the chemical compounds it contains may be disrupted to isolated atoms (in gaseous phase). The absorption (or emission) spectrum of the gas thus obtained may be analyzed to reveal the lines characteristic of the quantum levels of isolated atoms. This method is occasionally used in medical toxicology (e.g. to reveal arsenic). In principle any monoatomic ion could be measured using this technique, but this is rarely done since electrochemical methods are equally selective and sensitive and much cheaper.

      CHROMATOGRAPHIC METHODS. Chromatography is a technique in which a liquid or gaseous solvent (the mobile phase) moves over a solid, gel or liquid phase (called the stationary phase). If a mixture of different solutes is applied to the stationary phase and then eluted with the mobile phase, each solute undergoes a partition equilibrium between the two phases according to its relative solubility. The more a solute is soluble in the mobile phase the earlier it is removed from the stationary phase and eluted. The mobile phase that leaves the stationary phase is continuously monitored for the presence and concentration of solute(s), usually by means of spectroscopic methods.

      Liquid chromatography is usually carried out using a highly solvated gel as the stationary phase, percolated with a liquid eluent (the mobile phase). The gel may have affinity for the substances to be separated because of their solubility (polarity chromatography), electrical charge (ion exchange chromatography), molecular mass (gel filtration chromatography) or specific binding (affinity chromatography). A short movie tutorial which presents the use of this technique to separate two substances (one brown, the other green-blue) can be downloaded from this link (29 MB - downloading can take time; codec Xvid required).

      Gas chromatography is rarely used in clinical analyses. The stationary phase is an organic solvent, the mobile phase is an inert gas; obviously the substances to be analyzed must have a high vapour pressure. Gas chromatography is important for toxicology.

      THEORETICAL BASES OF CHROMATOGRAPHIC SEPARATIONS. Chromatography is analogous to the partition of a solute between to non miscible solvents. Let us imagine that we have solution of two solutes named A and B in water. Prior to any attempt to separate A and B, let the concentration of A to be a1 and that of B to be b1. Suppose now that A and B are also soluble in chloroform, a solvent immiscible with water, and let the partition constants to be 4:1 for A and 1:4 for B. The definition of the partition constant is KA=[A]H2O / [A]CHCl3 = 4:1. Thus A is four times more soluble in water than in chloroform, while B is four times more soluble in chloroform than in water.
      If we thoroughly shake our water solution with an equal volume of chloroform, allow the two solvents to separate and measure the concentrations of A and B in each solvent we obtain:
a2H2O = 0.4 a1
a2CHCl3 = 0.1 a1
b2H2O = 0.1 b1
b2CHCl3 = 0.4 b1
Notice that the total concentrations of each solute are half the inital one (i.e. a2H2O + a2CHCl3 = 0.5 a1) because the total volume is twice as the initial one.
      The water phase presents an a2/b2 ratio of 4 (a remarkable increase with respect to the initial one a1/b1=1), whereas the opposite is true for the chloroform phase (a2/b2=0.25). If this enrichment of each solute in its preferred phase is not sufficient, the procedure can be repeated:
      The water sample is estracted with an equal volume of chloroform; one obtains:
a3H2O = 0.16 a1
a3CHCl3 = 0.04 a1
b3H2O = 0.04 b1
b3CHCl3 = 0.01 b1
a3H2O + a3CHCl3 = 0.5 a2H2O
a3H2O / b3H2O = 16
      The chloroform sample is extracted with an equal volume of water; one obtains:
a3H2O = 0.04 a1
a3CHCl3 = 0.01 a1
b3H2O = 0.04 b1
b3CHCl3 = 0.16 b1
b3CHCl3 + b3H2O = 0.5 b2CHCl3
a3CHCl3 / b3CHCl3 = 1:16
      The procedure can be repeated as many times as needed to reach the desired degree of enrichment of A over B or B over A.
      Since to reach partition equilibrium requires some time, and since in a chromatographic column the mobile phase moves over the stationary phase, equilibration occurs over a segment of the column, which is called the theoretical equivalent plate. A typical chromatographic column may contain some hundreds of plates and thus is equivalent to some hundreds of solvent extractions.
      TITRIMETRIC METHODS. A titration is a determination of the concentration of an analyte in a complex mixture by means of a specific chemical reaction; for example the determination of urea concentration may take advantage of its specific reaction with diacetyle:
Titrimetric methods must be associated to some of the above described methods for the detection of the reaction product: e.g. in the case of the determination of urea in the reaction above, diacetylurea is yellow and its concentration can be measured by means of absorption spectroscopy. The more specific the reaction, the lower the risk of interefrence by other compounds present in the biological sample, that might lead to an overestimation of the analyte concentration.
      Other classical reactions udsed in the clinical laboratory are: Jaffe's reaction for creatinine, Van Den Bergh reaction for bilirubin, etc.

      IMMUNOLOGICAL METHODS. Immunological methods are used to quantitate solutes with complex molecular structure, whose chemical analysis would be expensive and time consuming. These methods make use of antibodies specific for the substance of interest, and take advantage of their great specificity. To apply immunological methods, the analyte of interest must be an antigen, and the laboratory must have the antibody able to bind to it; thus immunological methods can be applied to proteins, hormones, viruses, bacteria, bacterial antigens, etc. but not to other common analytes such as glucose or electrolytes. A description of the immunological methods used in endocrinology can be found in the lecture on hormones. The antigen-antibody complex formed must be revealed by an artificially produced signal: e.g. the antibody used (or a second antibody) can be labelled with an enzyme that catalyzes a reaction which produces an absorbance or fluorescence signal or to a radioactive isotope. Alternatively one may add to the mixture a radioactively labelled antigen and run a competition test. Most often either the antigen or the antibody can be immobilized on a solid matrix, to allow an easy removal of the unbound excess.

      ENZYMATIC METHODS. The concentration of low molecular weight solutes that are substrates of enzymes can be selectively measured by adding the appropriate enzyme and monitoring the catalyzed reaction by absorbance or fluorescence spectroscopy (this implies that the reaction must produce a "signal"). An example of an enzymatic analysis is that of oxidation of glucose. The reactions are as follows:

glucose + ATP --> glucose-6-phosphate + ADP (enzyme: glucokinase or exokinase)
glucose-6-phosphate + NAPDP+ + H2O --> 6-phospho gluconic acid + NADPH + H+ (enzyme: glucose 6 phosphate dehydrogenase)

This assay is carried out by adding a small volume of the glucose containing sample to a spectrophotometric cuvette containing ATP, NADP+ and the two enzymes; the reaction is monitored by the absorbance change associated to reduction of NADP+.



      Several pathological conditions may cause quantitative and/or qualitative changes in the protein content of body fluids, most notably blood plasma. Diagnosis of these conditions requires identification of the abnormal protein and measurement of its concentration. Qualitative changes include the presence of a protein in a fluid where it is normally absent, or presence of a pathological genetic variant of a specific protein (e.g. hemoglobinopathies). Quantitative changes include absence or great diminution of the concentration of a protein that should be normally present in the fluid (e.g. thalassemias), or increase in the concentration of a protein (e.g. transaminases in the blood serum).
      Increases in the concentration of a protein in a body fluid above their normal range may be due to overproduction (e.g. multiple myeloma) or to the death of cells that contain the protein, and consequent release in the blood (e.g. transaminases during acute hepatitis). Decreases in the concentration of a protein may be due to diminished biosynthesis, as in thalassemias or hemophylias, or accelerated destruction or loss (e.g. hypoalbuminemia in the course of nephrotic syndrome). Since multiple mechanisms may lead to similar conditions, differential diagnosis is never simple or based on a single finding and requires multiple laboratory and clinical investigations.

      TOTAL PROTEIN CONTENT. Total protein content (concentration) is measured in the plasma, urine or cerebrospinal fluid. The normal values of total protein concentration in these fluids, together with examples of pathological conditions that cause significant changes in this parameter are reported in the table below:
Total protein content in important body fluids
Blood plasma Urine Cerebro Spinal Fluid
Physiological protein concentration 7 g / dL    
may be higher because of multiple myeloma nephrotic syndrome bacterial meningitis
may be lower because of nephrotic syndrome
chronic liver disease
extensive burns

      Electrophoresis is a method to separate charged macromolecules (e.g. proteins, DNA) by means of their rate of migration in a stationary electric field. Nucleic acids are negatively charged because of their phosphate groups; proteins can be positively or negatively charged, depending of the pH of the medium, because of their acidic (aspartate and glutamate) or basic aminoacid (lysine, arginine) residues. A photographic tutorial which describes the technique can be looked at
this link.

      In a typical experiment the protein mixture to be subjected to the analysis, dissolved in a buffer at the desired pH is applied to a gel matrix (in the figure above a gel of polyacrylamide) equilibrated in the same buffer. Current is applied via the electrodes at the top (negative electrode in the figure above) and at the bottom of the gel (positive electrode in the figure above) and the proteins migrate with a velocity that is proportional to their net negative charge and inversely proportional to their molecular mass. A tracking dye (bromophenol blue, evident as a series of spots in the picture above) is added, that migrates faster than any protein in the mixture because of its very low molecular mass, in order to indicate the migration front, and the run is stopped when the dye has reached the bottom of the gel. The proteins trapped in the gel are then stained with a suitable dye and their concentration is quantitated by means of absorbance or reflectance spectroscopy (see the figure below).



      Molecular biology is mainly concerned with the study of genetic material. The key method for the study of gene variants is their selective amplification using the DNA Polymerase Chain Reaction (PCR) followed by sequencing. The main clinical indication for this type of analysis is the diagnosis of inherited genetic defects, e.g. hemoglobinopathies, thalassemias, cistic fibrosis, favism, phenylketonuria and the like. The analysis requires a bloof sample to prepare DNA (from the leukocytes), and can be carried out on the patient and on his/her parents; in prenatal genetic conseling on the parents only.
      The polymerase chain reaction requires that RNA primers specific for the gene of interest be added to the patient's DNA, together with a heat-resistant DNA polymerase (e.g. from the thermophylic bacterium Thermococcus thermophylus). The sample is heated to separate the DNA double strand, then cooled to allow the primers to couple with the desired genetic region, and the polymerase to synthesize new DNA; then the process can be repeated as many times as necessary. At the end of the procedure the DNA fragment identified by the artificial primers provided by the researcher will be amplified by hundreds- or thousands-folds.

Questions and exercises:
1) Electrolytes can be measured by:
potentiometry, spectroscopic methods
electrophoresis, chromatography
manometric techniques

2) Electrophoresis can be used for:
glucose and other sugars
biological macromolecules

3) The principal advantage of enzymatic methods for the study of low molecular weigth compounds is:
low cost

4) Immunological methods may be used to quantitate:
proteins, hormones
nucleic acids
glucose and its metabolytes

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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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