MEDICINE AND SURGERY "F"
Course of LABORATORY MEDICINE
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The analysis of exogenous substances, usually in the serum is routinely carried out in the clinical laboratory for several different reasons, among which the following three are paramount: (i) the concentration of
is measured in order to assess whether the therapeutic regime is appropriate, and to avoid overdosage; (ii)
or their metabolytes are measured because of forensic reasons; (iii) the concentration of
in body fluids is measured in cases of accidental, criminal, or professional intoxications.
present in the environment or contaiminating the patient (e.g. because of its job) are measured for preventive as well as diagnostic reasons. All these substances have very low or zero reference values, i.e. they should be almost absent in the body fluids of healthy people.
classification of toxic substances
is made difficult by they great variety. At least four classes of toxic compounds should be considered:
Aspecific toxic compounds.
Strong acids and bases and strong oxidizing agents chemically damage several components of the tissues, and especially the cell membranes. The toxic dose is usually elevated.
Specific toxic compounds.
Several toxic substances, either of biological or non-biological origin behave as enzyme inhibitors, agonists or antagonists of receptors, etc. They thus have one or more very specifical and identifiable biological target. Given that these substances act on macromolecules that are present in a limited number of copies in the organism, the toxic dose may be low. Specific poisons may be of inorganic, organic or biological nature: e.g. heavy metals, cyanide, carbon monoxide, mushroom poisons all belong to this group.
Mutagenic and carcinogenic compounds.
Compounds that selectively bind to DNA and interfere with its duplication may cause mutations and cancer. These compounds have an intermediate level of biological specificity with respect to categories (i) and (iii) in that they have a specific macromolecular target (DNA) to which they bind aspecifically (i.e. not to specific genes). As a consequence, mutagenic substances may cause virtually any mutation on any gene.
Bacterial, plant and animal toxins.
These are usually proteins having enzymatic activity against a selected substrate. Poisons belonging to this class have the lowest toxic dose, and it has been calculated that a single molecule of the plant toxin ricin could kill one cell (ricin is an hydrolytic enzyme that removes an adenine base from eukaryotic rRNA, irreversibly inactivating the ribosome). Diagnosis is usually difficult and relies strongly on the anamnesis. Toxins typically target only a few biological systems: e.g. the coagulation system (e.g. several snake poisons are coaugulases), synaptic transmission (e.g. tetanus toxin, an enzyme that digests the protein synaptobrevin), protein synthesis, membrane transporters.
ESSENTIAL CONCEPTS OF PHARMACODYNAMICS
Drugs are administered by several route (e.g. oral, intramuscular, intravenous, etc.) and are transported by the blood to their target organs or cells. The rate of absorption is usually limited by diffusion: the peak blood concentration may be immediate (in the case of intravenous administration) or may be delayed by some hours (in the case of oral administration).
As soon as the drugs appears in the blood the processes of its metabolic transformation and excretion also start. Urinary excretion is usually proportional to the drug concentration in the serum and obeys an exponential (i.e. first order) kinetic law. Enzymatic transformation (generally by the liver followed by excretion via the bile or the urine) is saturable and obeys a zero order kinetic law.
SPECIMEN COLLECTION AND ANALYTICAL METHODS
Each drug has a characteristic therapeutical range (i.e. a range of serum concentration in which it exerts its therapeutic action) and a toxic threshold (the serum concentration at which toxic or undesired effects appear). The therapeutic index is the ratio between the toxic and therapeutical concentrations and is widely different for different drugs: e.g. valium, when used as an ansiolytic is a very safe drug, with therapeutic index >100, whereas lithium ion, only has 2. The dosage, and therefore the therapeutic index of a drug, however, may vary depending on the clinical indications. For example, valium may be used as an antiepileptic drug, ad doses several tens of times higher than those used for its ansiolytic activity. In these cases its therapeutic index is substantially lower.
Accidental drug intoxications are common especially in the elderly and in children, and require prompt diagnosis and treatment. The anamnesis is crucial to identify the drugs that were available to the patient.
Determination of the serum (or other biological fluid) concentration of a drug is indicated if: (i) the therapeutc index is low; (ii) absorption or excretion exhibit large interindividual variability (especially in the presence of liver or kidney diseases); (iii) non-compliance or abuse by the patient are suspected; (iv) unexpected toxic effects appear.
The appropriate sample is usually the serum; in some cases the urine,bile or other body fluids. In some cases the drug concentration is effectively measured; in other cases a characteristic metabolyte is measured instead.
Analytical methods typically used in clinical toxicology include: chromatography (e.g. high pressure liquid chromatography; gas chromatography); potentiometry; spectrophotometry; mass spectrometry; radio-immunoassay and ther immunological methods.
SOME DRUGS THAT MOST COMMONLY REQUIRE TOXICOLOGICAL ANALYSES
Some drug categories require toxicological determinations, because of essentially two main reasons: (i) the therapeutic index is low, thus even small deviations from mean values may cause risk; or (ii) the pharmacodynamics exibits high inter-individual variability.
MEASUREMENT OF DRUG CONCENTRATIONS
10 ug/ml / >20 ug/ml
0.8 mEq/l / 1.5 mEq/l
0.5ng/ml / 5 ng/ml
10-20 ug/ml / >5 ul/ml
2 ug/ml / >30 ul/ml
Warfarin and related anticoagulants
Prothrombin time (or its normalized derivative INR)
Recreational drugs are taken by the patient in the absence of a physician's prescriptions and without therapeutical scope. Most of them are toxic to the central nervous system, and the patient may be brought to the emergency room, often in an unconscious state. As a consequence anamnestic information may not be available, and one has to guess whether a drug was used and which one. Detection of the drug used by the patient is essential for an appropriate therapy, and has forensic relevance. The following table lists some commonly used recreational drugs.
serum, expired air
gas chromatography, enzymatic
Chromatography (HPLC; gas chromatography)
Lysergic acid (LSD)
LS Diethylamide; 2-oxo 3-hydroxy LSD
delta 9 tetrahydrocannabinol
A related subject is the forensic analysis of recreational drugs seized or confiscated from illegal manufacturers. This analysis is carried out by specialized chemical laboratories, and is not a clinical analysis; the samples are usually powders or pastes. Information on this type of analyses can be obtained from the website of the
United Nations Office on Drugs and Crime
that reports the methods of choice for different substances in a
series of freely available manuals
POISONS AND ENVIRONMENTAL INTOXICANTS
Acute or chronic intoxications occur frequently because of several reasons: some commonly used detergents and bleaching agents are highly toxic (e.g. ipochlorite, common bleach), or may cause chemical burns (e.g. sodium hydroxide, hydrochloric acid, permanganate); some jobs entail risks of professional poisoning (e.g. miners, workers of chemical factories, etc.); food may be contaminated with pesticides and other compounds used in agriculture, or polutants; etc. In the following list only some of the most common poisons are included.
electrochemical; atomic absorption spectrometry
(serum) urine, tissues
electrochemical; atomic absorption spectrometry
HbCO/total Hb > 0.2
Blood, skin, nails
Atomic absorption spectrometry
detection of organophosphate metabolytes by chromatography; reduced esterase activity
Lead poisoning is a relatively common cronic (more rarely acute) professional disease. The disease affects several organs and tissues. Neurological symptoms are common, with insomnia, tremor and cognitive defects. Peripheral neuropathy may be responsible of painful crisis, usually referred to the abdomen (so called saturnine colic, often misdiagnosed as appendicitis). Anemia is frequent, as is kidney failure. In symptomatic cases lead is presnt in the blood at concentration > 30-40 ug/dL. The ion is measured in the blood, tissues and urine by means of potentiometry or atomic absorption spectroscopy. Chelation therapy is indicated.
Other toxic metals
Essentially every metal ion if absorbed in excess may cause toxicity. Some intoxications are uncommon: e.g. sodium, potassium, calcium, and magnesium are physiologically present at high concentration in our body and we have effective excretion routes, thus intoxication can only occur because of parenteral administration. Iron is physiologically present in our body and has no physiological excretion pathway: we loose iron because of hemorrages. However, the absorption of iron is strictly regulated, thus we do not risk iron intoxication except in two cases: (i) because of inherited defects in the regulation of absorption (primary hemochromatosis); or (ii) because of parenteral administation, usually in the form of blood transfusion (secondary hemocromatosis). Chelation therapy is indicated.
is a highly toxic metal, that may be absorbed from the environment where it is present as a free metal or in the form of its ogranometallic derivative (e.g. methylmercuric chloride). It reacts with Cys residues of enzymes and blocks their action. Acute mercury poisoning leads to kidney insufficiency and death. Early and specific chelation therapy is highly recommended (the chelator of choice is di-mercapto propanol). Mercury can be detected in the blood or in any tissue by means of atomic absorption spectroscopy.
and other transition metals may be responsible of human poisoning, usually in workers of specialized factories (e.g. varnish). Diagnosis is established by atomic absorption spectroscopy.
Organophosphate and organochlorine pesticides
are selective covalent inhibitors of Ser- and Tyr- esterases, most typically of acetyl cholinesterase. These compounds are widely employed in agriculture as pesticides and herbicides, thus professional or accidental poisoning is common. Some compounds of this class have been used as tosic gases for chemical warfare or terroristic attacks. The symptoms of acute organophosphate poisoning is the cholinergic crisis, due to excess activity of the cholinergic (parasympatic) system: convulsions, ataxia, depression of respiration and circulation, tremor, general weakness, possibly coma and death. Diagnosis relies on two tests: (i) detection of the metabolytes of organophosphates in the blood and urine, by means of liquid chromatography; and (ii) measurement of reduced pseudocholinesterase activity in the serum.
(e.g. DDT) are banned in Europe and USA, but have been widely employed in the past and have long persistence in the environment. These compounds target the peripheral nervous system, acting as agonists of sodium channels or as antagonists of chloride channels. They have significant toxicity for mammals, and may cause liver insufficiency and reduced fertility.
is a very toxic non-metallic element, and a common environmental pollutant. It may be released in the environment in the form of inorganic and organic compounds, the latter less oxic than the former. Examples of inorganic (and highly toxic) inorganic compounds containing arsenic are arsenates (e.g. sodium arsenate, Na
), arsenites (e.g. sodium arsenite, Na
), arsenous acid (As(OH)
), and arsine (AsH
). Arsenic is a member of the fifth group of the periodic table, like phosphorus and nitrogen, and may replace phosphorus in many compounds (compare the formulas of arsenates and phosphates). Many enzymes that require phosphorylation or utilize phosphate as a substrate are inhibited by arsenates.
Toxicity depends on the arsenic compound that has been absorbed. The atom itself can form complexes with sulfur and may inhibit enzymes whose activity requires a Cys residue. Arsenic, and in particular the arsenite ion (AsO
), is an inibitor of pyruvate dehydrogenase and succynate dehydrogenase; thus it blocks the Krebs cycle and causes cell death. The arsenate ion (AsO
) binds to enzymes at the same sites as phosphate and is thus an inhibitor of kinases.
Arsenic is a common natural pollutant
, present in many geological formations, that may be dissolved by rainwater; the effect is that some water sources may be contaminated by this element, even in the absence of human intervention; contaminated water sources are common especially in South East Asia and in some countries of South America. The case of the bay of Bengal (South-East India and Bangladesh) is of special interest. Himalayan rocks are rich in arsenic minerals. Arsenic salts are extracted and carried downstream by Himalayan rivers, tributaries of the Gange-Bramaputra river systems in whose estuaries they deposit and pollute water sources. It is estimated that
50 milllion people use contaminated water in Bangladesh alone
may be acute or chronic. Chronic poisoning may result in skin lesions, intestinal lesions (manifested as vomiting and diarrhoea), neuropathies, and cancer; it usually results from ingestion of contaminated water or from consumption of animals (fishes, clams) living in contaminated waters. Arsenic is also used for several industrial applications (e.g. glass, pigments, textiles, etc.) and both chronic and acute intoxications may occur as professional diseases.
relies on the demonstration of arsenic in the tissues. Acute assumption of arsenic is best tested by measuring As in the urine (collect the urine of the 24 hours following suspected As ingestion). Arsenic is detected in blood, urine, and other tissues and fluids by means of atomic absorption spectroscopy or X-ray fluorescence.
British Association for Clinical Biochemistry and Laboratory Medicine
has a good webpage on
National Center for Forensic Science
, University of Central Florida, FL, USA.
Bureau of Criminal Apprehension
, the Minnesota Department of Public Safety.
Questions and exercises:
1) A patient presents severe crises of abdominal pain and anemia. You suspect that this clinical picture may result from chronic poisoning and prescribe the measurement of
Lead concentration in the serum
Phenytoin concentration in the serum
Mercury concentration in the serum
2) Lithium is used in the treatment of major depression; however, due to its toxicity lithemia is periodically controlled in order not to exceed:
3) Carbon monoxide poisoning is detected by:
measurement of CO concentration in the serum
measurement of CO concentration in the urine
measurement of the fraction of carboxy-hemoglobin
4) A patient is in a state of coma, and you suspect abuse of recreational drugs; you prescribe the measurement of serum concentration of:
cocaine, benzoylecgonine, LSD
opiates, barbiturates, ethanol
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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
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
. R the reproductive index is the ratio (new cases)/(old cases) measured after
one serial generation time. R
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
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 (i.re 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
) + 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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