MEDICINE AND SURGERY "F"
Course of LABORATORY MEDICINE
Toxicology

TOXICOLOGY

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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 drugs is measured in order to assess whether the therapeutic regime is appropriate, and to avoid overdosage; (ii) recreational drugs or their metabolytes are measured because of forensic reasons; (iii) the concentration of poisons in body fluids is measured in cases of accidental, criminal, or professional intoxications. Toxic substances 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.

      A satisfactory classification of toxic substances is made difficult by they great variety. At least four classes of toxic compounds should be considered:
(i) 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.
(ii) 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.
(iii) 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.
(iv) 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
     
Drug Dose Ther./Toxic Metabolyte specimen method
Methotrexate Methotrexate serum
Tacrolimus Tacrolimus whole blood Chromatography (HPLC)
Cyclosporine Cyclosporine Chromatography (HPLC)
Phenytoin 10 ug/ml / >20 ug/ml
Barbiturates 20 ug/ml Barbiturates serum spectrophotometric
Carbamazepine
Lithium ion 0.8 mEq/l / 1.5 mEq/l Lithium ion serum electrochemical
Digoxin 0.5ng/ml / 5 ng/ml Digoxin serum immunological
Quinidine 10-20 ug/ml / >5 ul/ml Quinidine serum immunological
Theophylline 2 ug/ml / >30 ul/ml Theophylline serum immunological

 
      RECREATIONAL DRUGS
      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.
Drug Toxic level Metabolyte specimen method
Ethanol 80 mg/dl Ethanol, acetaldehyde serum, expired air gas chromatography, enzymatic
Amphetamines Amphetamines serum immunological
Cocaine Benzoylecgonine serum Chromatography (HPLC)
Opiates Morphine, Heroine urine, serum Chromatography (HPLC; gas chromatography)
Lysergic acid (LSD) LS Diethylamide; 2-oxo 3-hydroxy LSD urine, serum Chromatography (HPLC)
Cannabis delta 9 tetrahydrocannabinol urine Chromatography (HPLC)

 
      DOSAGE OF 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.

Substance Toxic level Metabolyte specimen method
Lead > 30ug/dl Lead ion serum electrochemical; atomic absorption spectrometry
Mercury > 0.6ug/dl Mercury (serum) urine, tissues electrochemical; atomic absorption spectrometry
Carbon monoxide HbCO/total Hb > 0.2 Carboxy-hemoglobin blood spectrophotometric
Arsenicals Arsenic Blood, skin, nails Atomic absorption spectrometry
Organophosphates Blood, urine detection of organophosphate metabolytes by chromatography; reduced esterase activity

      Lead poisoning
      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.
      Mercury 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.
      Cadmium, chromium 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.
      Arsenic is a special case because it is a metalloid and presents metallic and non-metallic behaviour in different compounds. 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 (AsO3), is an inibitor of pyruvate dehydrogenase and succynate dehydrogenase; thus it blocks the Krebs cycle and causes cell death. The arsenate ion (AsO3) binds to enzymes at the same sites as phosphate and is thus an inhibitor of kinases. Arsenic is detected in blood and other tissues and fluids by meand of atomic absorption spectroscopy or X-ray fluorescence.

      Organophosphate and organochlorine pesticides
      Organophosphoric compounds 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.
      Organochlorine pesticides (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.


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:
0.5 mEq/L
1 mEq/L
2 mEq/L

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
amphetamines, cannabis

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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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