MEDICINE AND SURGERY "F" Course of LABORATORY MEDICINE Anemias and hemoglobinopathies ANEMIAS THE REGISTRATION OF ATTENDANCE TO THIS ON-LINE LECTURE IS ACTIVE!       To register your attendance please type in your surname and matricola number. Surname: Matricola: Notice that your attendance will be registered only if you completed the reading, questions, and audios, and that you cannot interrupt and resume the session (but you can repeat it as many times as you like). Remember to press the [send] button before leaving this page! A confirmation message will appear at the end of this page.       A comment section has been added at the end of this lecture. Adding a comment or question does not require registration with your matricola number, feel free to comment whenever you like.         Anemia is a pathological condition characterized by reduced hemoglobin content in the blood. Physiological values of hemoglobin concentration range between >13 g/dL for adult males to >11 g/dL for infants and pregnant women. Hemoglobin concentrations below approx. 9 g/dL are symptomatic, and cause fatigue, dispnea and tachycardia. The increased cardiac work associated to reduced oxigenation of the blood perfusing the myocardium may cause angina pectoris and, occasionally, myocardial infarction. Blood transfusion is indicated when hemoglobin is <7 g/dL.       Anemia is neither a disease, nor a diagnosis: it is a clinical condition that may be present in many different diseases, and may have a number of causes (summarized in the table below). An essential distinction is made between those conditions that impair the biosynthesis of red blood cells and those that reduce the red blood cell lifespan. TABLE 1: SOME DISEASES CAUSING ANEMIA   Cause of anemia Pathogenesis Test(s) for diagnosis Blood loss: Acute or chronic hemorrages, internal or external Demonstration of blood loss (e.g. fecal occult blood; hematuria; hematemesis)   Hemolysis, toxic or allergic Demonstration of hemoglobinuria and reduced erythrocyte lifespan   Deficitary hemopoiesis: Bone marrow aplasia Biopsy of the bone marrow   Leukemias (and lymphomas) Demonstration of neoplastic cells in the blood (or the bone marrow)   Talassemias Demonstration of lack of one of the subunits of hemoglobin in the erythrocytes (by electrophoresis); demonstration of abnormal hemoglobin genes   Chronic kidney diseases Low erythropoietin levels in the serum; renal failure   Folate or vitamin B12 deficiency Erythrociytes of increased size (megalocytes); measurement of serum levels of folate; deficit of the intrinsic factor in the gastric secretion   Iron deficiency Low iron level in the serum   Lead poisoning High lead concentration in the serum   Reduced erythrocyte lifespan: Hemoglobinopathies Abnormal electrophoretic mobility of hemoglobin; demonstration of abnormal hemoglobin genes   Talassemias (see above)   Spherocytosis and ellissocytosis Direct observation by optical microscopy   Genetic defects of red cell enzymes Demonstration of abnormal enzyme genes (e.g. Glucose 6-phosphate dehydrogenase in favism)       It is important to remark that some diseases cause anemias because of more than a single reason: e.g. thalassemias cause premature death of the red cells but also defective erythropoiesis (hence these diseases appear twice in the table).       The lifespan of erythrocytes does not exceed 120 days, on average: thus every month the bone marrow has to replace one fourth of the circulating red cells. In some diseases causing ineffective erythropoiesis (i.e. destruction of abnormal erythrocytes during their differentiation in the bone marrow) an abnormal expansion of the marrow is observed: e.g. this is the case with beta-talassemia and the so-called "brush" skull. Diseases in which the biosynthesis of DNA is impaired (e.g. deficiency of folate or of vitamin B12) cause a delay in the differentiation of red blood cells, that result fewer, larger and paler (because of lower hemoglobin content) than in the healthy condition.       Anemias caused by reduced erythrocyte lifespan cause a compensatory increase in the rate of red cells production in the marrow, and release of an increased fraction of reticulocytes (immature erythrocytes) in the blood; by contrast reticulocytes are scarce or absent in anemias due to bone marrow aplasia.       As stated above, anemia is not a diagnosis (or it is a very imprecise one), but an easily recognized pathological condition. The identification of the disease underlying a condition of anemia is a differential diagnosis among the conditions listed in table 1, and requires the demonstration of the essential features of the disease. Some relevant cases are individually discussed below.       β - THALASSEMIA (Cooley disease). Thalassemias (or talassemias) are diseases caused by an imbalance of the synthesis of the alpha and beta subunits of hemoglobin. Since hemoglobin is a tetrameric protein, composed by two α and two β subunits, it is necessary that during the maturation of the erythrocyte, the two types of subunit are produced in equal amounts. Thalassemias are genetic diseases due to mutations of the genes of the α or the β subunit of hemoglobin, that may occur in the promoter region of the gene (lowering its transcription efficiency) or in the coding sequence (usually because of the mutation of a coding codon to a stop codon). The aploid human genome contains two copies of the α subunits gene and only one copy of the β subunit gene, on different chromosomes.       Thalassemia has autosomal recessive Mendelian transmission, and α thalassemia (in which the alpha subunits are lacking) is uncommon due to the redundancy of the alpha genes; thus β thalassemia is the most common of thalassemias. β talassemia in the heterozygous state, causes mild anemia and reduced volume erythrocytes (microcytosis). This form is asymptomatic or nearly so and is called talassemia minor. In the homozygous state the erythrocytes of the patient contain mostly unpaired alpha subunits, that precipitate inside the cell, forming Heinz bodies. This significantly shortens the lifespan of the affected erythrocytes and causes a very severe anemia. If untreated, β thalassemia major (Cooley disease) is fatal before puberty. In some cases the severity of a beta thalassemia major may be reduced by the hereditary persistence of foetal hemoglobin (made up by two α and two γ subunits, thus being independent of the biosynthesis of beta subunits). Diagnosis is made by finding the microcytosis in a blood smear and is confirmed by sequencing of the β globin gene. A frequently associated finding is the expansion of the bone marrow, with increased thickness of the bones where it is contained (e.g. the skull) and its protrusion in the periostium (e.g. in the vertebrae). In some regions or countries along the coast of the Mediterranean sea beta thalassemia is (or was) quite common, due to the fact that the minor, heterozygous form offers protection against malaria, which was formerly endemic in the region. The reason of protection probably lies in the reduced resistence of the affected erythrocytes which, when infected by the Plasmodium may undergo hemolysis before the parasite has completed its intraerythrocyte development, leading to an abortive infection.       An important indication may be obtained by electrophoresis; this is because the α and β subunits of hemoglobin have different electrophoretic mobility, and in the blood of a thalassemic patient one may observe normal human hemoglobin, and an excess of either subunit, revealed as an additional band with lower (α subunits excess in β-thalassemia) or higher mobility (β subunits excess in α-thalassemia).       FOLATE DEFICIENCY. Folate is a vitamin required for the biosynthesis of nucleotides, hence of DNA, and deficiency of folate slows down the proliferation of stem cells of various tissues. The effect is particularly evident for red blood cells because of their rapid turnover, hence the anemia. Due to the reduced rate of replication the erythrocytes are few, large and pale and immature forms (reticulocytes) in peripheral blood are increased. Folate deficiency may occur in pregnant women, due to the high requirement of the vitamin by the foetus, and oral supplementation is indicated.       VITAMIN B12 DEFICIENCY (pernicious anemia, Brill disease)       The biological function of vitamin B12 is somewhat similar to that of folate, since it is a methyl group donor required for the biosynthesis of nitrogen bases of DNA. The pathogenetic mechanism of the anemia and the microscopic appearance of the red cells is also similar, i.e. this is a magalocytic, megaloblastic anemia. At variance with folate deficiency, pernicious anemia also causes neurological symptoms: paraesthesias, irritability, depression, and dementia. The disease is fatal if untreated.       Vitamin B12 deficiency is rarely due to insufficient dietary apport, although this can occur in strict vegan diets. More often vitamin B12 deficiency is due to atrophic gastritis, that impairs the production of the intrinsic factor (a protein required for the absorption of the vitamin) by the gastric mucosa. Because of this peculiarity, which is not shared by any other vitamin, oral administration of vitamin B12 may prove ineffective and intramuscular administration is required.       Diagnosis is suspected on clinical grounds (anemia associated to atrophic gastritis and neurological symptoms) and on the microscopic examination of a blood smear. It is confirmed by the measurement of vitamin B12 concentration in the serum (normal range 20-90 ng/dL). The preferred method for measurement is by radioimmunoassay. Some drugs may interfere with this test: e.g. Colchicine, Neomycin, Para-aminosalicylic acid, and Phenytoin.       IRON DEFICIENCY (sideropenic anemia)       Mammals have no physiological way to eliminate iron, and the iron content in the organism is regulated by its uptake in the gut. Iron is lost with hemorrages and iron deficiency may occur in the presence of insufficient uptake or increased loss by chronic or acute hemorrages. Fertle women have increased iron requirements. The anemia is microcytic (small erythrocytes) and diagnosis is confirmed by low values of sideremia (normal range in male adults: 65-175 ug/dL; female adults 50-170 ug/dL), and low iron saturation of circulating transferrin (normal concentration 200-360 mg/dL; normal saturation > 60%).       Combined post-hemorragic sideropenic anemia. A chronic unrecognized bleeding may lead to severe anemia because of the combination of two reasons: chronic loss of erythrocytes and loss of iron. The anemia is sideropenic, and possibly microcytic.       REDUCED HEME BIOSYNTHESIS       Insufficient production of the heme is observed in erythropoietic porphyrias and chronic lead poisoning. From a clinical view point these anemias are hypochromic and lack specific chracteristics; however the clinical picture entails several other symptoms (e.g. photosensitivity of the skin in eryhtropoietic porphyrias) that may suggest the diagnosis. Specific laboratory tests are available: measurement of heme precursors in the blood and urine for poprhyrias, measurement of lead in the blood, urine and tissues for lead poisoning.       INSUFFICIENT PRODUCTION OF RED BLOOD CELLS       Insufficient production of red blood cells is caused by diseases that destroy the bone marrow: idiopathic bone marrow aplasia and hematological tumors (e.g. leukemias, lymphomas, etc.). Often these anemias are accompanied by deficiency of other cell types in the blood (reduced platelet count, reduced count of white cells); in leukemias the blood contains tumor-specific cells. The presenting symptom may often be a thrombocytopenic purpura. Diagnosis is suspected because of the results of hemochromocytometric analysis, and is confirmed by bone marrow biopsy.       DIFFERENTIAL DIAGNOSIS OF ANEMIAS       Anemias provide a very useful example to illustrate clinical reasoning. Let's suppose that we have a patient suffering of faticability and dispnoea. We prescribe a standard blood test in which Hb concentration and erythrocyte count are performed (see the lecture on the standard blood tests); usually together with erythrocye count the laboratory provides also an estimate of the mean erythrocyte volume, which tells us if the patient's condition is microcytic, normocytic or macrocytic. From this point onwards the clinical reasoning follows a linear path: 1) If Hb is low (< 13 g/dL) the patient has anemia. 2) In anemias the erythrocyte count is usually low, thus this datum adds little to your knowledge. 3) If the other blood cells are also low we have pancytopenia, which usually points to a disease of the bone marrow. 4) If the mean red cell volume is significantly increased, the patient's anemia is macrocytic. The most important causes of macrocytic anemia are selective nutritional deficiencies, causing decreased production of erythrocytes: (i) folate deficiency; (ii) vitamin B12 deficiency; (iii) scurvy (deficiency of vitamin C); (iv) copper deficiency. The two former conditions are the most frequently observed. The next possible step in the diagnosis pathway is the measurement of vitamins and copper concentrations in the serum and the urine (see the lecture on vitamins). However, given that the administration of vitamins and minerals has virtually no contraindications, it is reasonable to prescribe vitamin and mineral supplements and see if this has the desired effect of curing the anemia (diagnosis ex juvantibus, literally "from the intervention that cures the disease"). It is also important to reevaluate the anamnesis, and possibly to ask the patient some more specific questions. Folate deficiency is more common during pregnancy, vitamin B12 is observed in vegans, etc. A very important consideration is that vitamin B12 is absorbed after binding to the intrinsic factor, a protein produced by the gastric mucosa; atrophic gastritis, gastric cancer, gastric surgery, etc. may be causes of vitamin B12 deficiency resistent to oral administration of the vitamin (intramuscular administration may be required). 5) If the mean red cell volume is significantly decreased, the patient's anemia is microcytic. The most important causes of microcytic anemia are: (i) iron deficiency; (ii) thalassemias (consider in particular heterozygous β-thalassemia, β-thalassemia minor); (iii) inherited defects of the erythrocyte (e.g. spherocytosis);(iv) chronic lead (or other heavy metal) poisoning. Your next diagnostic step is to measure iron concentration in the blood (sideremia, serum transferrin), and to search for the relevant genetic diseases by gene sequencing. If the anamnesis suggests a possible professional intoxication, this should be tested by laboratory analyses. Caution: iron deficiency anemia may be the only sign of a chronic internal hemorrage, e.g. due to a bleeding colon cancer; a search of blood in the feces is indicated. 6) If the mean red cell volume is normal the patient anemia is normocytic. This is the largest group, and includes several possible diseases. Consider three possible cases: (i) chronic internal hemorrage (not associated to iron deficiency); (ii) increased destruction of red cells (reduced lifespan), possibly due to allergies, hemoglobinopathies, or other inherited conditions (e.g. defects of glucose-6-phosphate dehydrogenase); (iii) decreased production of red cells by the bone marrow because or aplasia, leukemias, lymphomas, etc. Clearly an important normocytic anemia requires a broad-scope investigation of several possible conditions. Normocytic anemia associated to reduced platelet count or pancytopenia strongly suggests a disease of the bone marrow. 7) If the mean red cell volume is increased the patient anemia is macrocytic. The group of macrocytic, megaloblastic anemias is relatively small: deficiency of vitamin B12, folate deficiency, chronic liver disease (probably becausethe liver stores vitamin B12). 8) If the mean red cell volume is marginally increased or decreased, and you do not feel confident in defining the patient's anemia macrocytic, normocytic, or microcyti, you apply the procedures of two cases, i.e. you carry out the analyses suggested for both macrocytic and normocytic anemias ( or microcytic and normocytic anemias).       Diagnosis is an iterative process: an alteration in the patient's parameters suggests a list of diagnostic possibilities, each of them requiring specific tests and investigations. It is important that our list is as complete as possible, because omissions may lead to incomplete searches and missed diagnoses. Anemias; an example of clinical reasoning The patient complains of fatigue, dispnoea, tachycardia, reduced resistance to muscular effort. The physical examination confirms dispnoea and tachycardia, and reveals pallor of the mucosae. A general blood test reveals reduced Hb concentration and reduced red cells count; thus the patient has anemia.   Check the mean erythrocyte volume (this is usually measured together with the erythrocyte count). Is it decrased, normal or increased? decreased mean red cell volume: microcytic anemia normal mean red cell volume: normocytic anemia increased mean red cell volume: macrocytic anemia - Iron deficiency? Measure sideremia - Copper deficiency? - Thalassemia minor? Sequence Hb genes - Chronic lead poisoning? ... Reduced erythropoiesis: - Reduced erythrocyte biosynthesis? Pancytopenia? Leukemia? Bone marrow aplasia? Perform bone marrow biopsy - Chronic kidney disease? Measure erythropoietin; GFR - Porphyrias? Sequence genes of heme biosynthesis Reduced erythrocyte lifespan: - Hemoglobinopathies? Sequence Hb genes; check for Heinz bodies - Genetic enzymatic defects? G6PDH deficiency? Increased erythrocyte consumption: - Chronic internal hemorrage? - Hemolysis? Possibly allergic or toxic? Drug induced? ... - Folate or vitamin B12 deficiency? - Chronic liver disease? Cirrhosis? ...       Searching for clinical or laboratory signs tha may be associated with altered erythropoiesis       If the patient's anemia is due to a defective erythropoiesis, the next step of clinical reasoning is by organ. Indeed many functions in our body depend on the cooperation of different organs, and the same organ may participate to more than a single function. Thus a lesion of an organ that participates to erythropoiesis may be associated to abnormalities in other functions played by the same organ. Clinical and laboratory findings and associations may very precisely indicate which organ is affected. We ask two crucial questions: i) Which organs participate to eruthropoiesis? ii) Which other functions are played by these organs? Answering these questions indicates the other signs that we have to search and the way to the diagnostic procedures to be followed. Principal organs involved in erythropoiesis Other functions played by these organs to be investigated bone marrow production of all blood cells and platelets (is anemia associated to pancytopenia? bone marrow biopsy is indicated) kidney (because of erythropoietin) excretion of urea and creatinine (measure serum concentration of these substances; measure GFR); regulation of blood pH and electrolytes; regulation of blood pressure (because of renin and because of the control of electrolytes) GI tract and liver (absorption and storage of vitamins, notably B12, folate, ascorbic acid) The first sign of liver failure is usually hyperbilirubinemia (jaundice); other possible signs are hypoproteinemia, hyperammoniemia (because of reduced biosynthesis of urea); etc.       If anemia is due to reduced erythrocyte lifespan or increased loss, a similar reasoning is applied; the organs responsible for erythrocyte removal are the reticuloendothelial cells and the spleen; hemorrages may occur everywhere in the organism, but occult hemorrages most commonly occur in the GI tract. A search of occult blood in the feces is mandatory. HEMOGLOBINOPATHIES       Hemoglobinopathies are hereditary defects of hemoglobin subunits (the alpha and beta polypeptide chains), usually due to single site mutations. They are inherited as Mendelian recessive traits. Hereditary defects of hemoglobin, due to the high solubility and rich functional behaviour of the protein, are somewhat exceptional with respect to those of other proteins and enzymes. Indeed the most usual effect of random point mutation in proteins is a drastic decrease of stability and solubility and the mutant protein is absent or present at very low concentration in the cell. In hemoglobin this is by no means the only possible effect of mutation and hemoglobinopathies can be classed into at least five groups:       1) Mutations which cause diminished stability. The intracellular Hb precipitates and form Heinz bodies adhering to the red cell membrane. The resulting damage of the membrane reduces the lifespan of the erythrocyte, causing an hereditary anemia.       2) Mutations which cause increased oxygen affinity. The mutant Hb uploads oxygen in the lungs but delivers few oxygen to the tissues, causing chronic ischemia. The ischemic kidney produces more erythropoietin to stimulate the bone marrow and these conditions are often associated to polycytemia (increased blood cell count), increased hematocrit, and increased Hb content.             3) Mutations which cause reduced oxygen affinity. The mutant Hb is not fully saturated with oxygen in the lung capillaries and the blood oxygen content is decreased in spite of a normal arterial oxygen pressure. The patient has cyanosis, but the amount of oxygen released to the tissues is normal and no ischemia is present (in general); the red cell count and hemoglobin content are usually normal or nearly so. The patient has fatigue because oxygen transport under strenuous muscular effort is reduced (a consequence of the reduced arterial oxygen content).       4) Mutations which cause iron oxidation (methemoglobinemias). Mutation of the proximal or distal Histidine of either subunit to Tyrosine causes the heme iron to oxidize to the ferric state. Other mutations that increase the polarity of the heme pocket or allow water to flow in have the same effect. Methemoglobin (Hb with the heme iron in the ferric state) has low oxygen affinity and carries oxygen only on the hemes of the normal subunits, the ferric heme iron being unreactive. Thus the oxygen carrying capacity is halved. The patient presents a characteristic brown cyanosis of the mucosae (black mouth), due to the brownish colour of metHb; other symptoms are as in the case of reduced affinity Hbs, and polycytemia may be present to compensate for the reduced oxygen carrying capacity.       5) Mutations that cause other effects. This is a residual category that includes many different conditions. An important one is sickle cell Hb, the mutation of the sixth residue of the beta chain from Glu to Val. This mutation causes deoxyHb to polymerize and the resulting Hb polymer distorts the red cells, which assume a characteristic sickle shape. Sickled cells may stack in the venulae blocking the circulation: painful microinfarctions follow.       MOLECULAR MECHANISMS OF HEMOGLOBINOPATHIES       Hemoglobin is among the most deeply understood proteins, and hemoglobinopathies have significantly contributed to the study of structure-function relationships in hemoglobin. The macromolecule is a heterotetramer made up of two α and two β subunits arranged in a α2β2 tetramer. The α and β subunits are globular proteins and are made up of 8 (or 7) α-helices. The subunits are arranged in the tetramer in a distorted tetrahedral geometry, and two types of α-β contacts occur. The α1-β1 contact involves helices B, G and H from both participating subunits; the α1-β2 contact involves helix C and the interhelical FG corner from both participating subunits. The regulation of oxygen affinity and cooperativity largely depends on (i) the residues lining the heme pocket of each subunit, (ii) the residues at the α1-β2 interface, which is responsible for the quaternary structural change associated to cooperative oxygen binding, and (iii) the two C-terminal residues of each subunit, that in deoxy Hb pack in proximity of the α1-β2 interface. The above listed regions of the macromolecule are thus those where single aminoacid mutations tend to exhert the most important functional effects. Examples of high oxygen affinity mutants are: Hb McKees Rocks, that bears the deletion of the last two C-terminal residues of the β, and Hb Chesapeake in which α Arg 92(FG3) is replaced by Leu; both mutations destabilize the low affinity T quaternary conformation and favor the transition to the high affinity R conformation. Examples of low oxygen affinity mutants are Hb Presbyterian that bears the mutation β 108 Asn to Lys, in proximity of the FG corner, and Hb Kansas that bears the mutation β 102 Asn to Thr; in the former the affinity is reduced because of stabilization of the T quaternary structure, in the latter presumably because G4 Asn is a heme contacting residue.             Several hemoglobinopathies, together with other hereditary diseases of the red cell (e.g. glucose 6 phosphate dehydrogenase deficiency, thalassemias) cause the red cell to become fragile and reduce its lifespan. While this effect usually results in an anemia, it may protect the heterozygous patient from malaria, because the damaged red cell does not support the full erythrocytic cycle of the parasite leading to an abortive effect. Because of this reason, in regions where malaria is or was endemic, an abnormally high frequency of any of these conditions may be observed (so called "polymorphism due to heterosis", i.e. higher fitness of the heterozygous carrier state that either the healthy or affected homozygous individual).             DIAGNOSIS OF HEMOGLOBINOPATHIES       The diagnosis is suspected on the basis of the clinical information, and confirmed by the finding of a hemoglobin with abnormal electrophoretic mobility. Protein sequencing and finger-printing are nowadays obsolete, and the diagnosis of the specific mutation depends on the sequencing of the genes coding for the α and β subunits. Clinical examples 1) 5 year old boy, asymptomatic, comes for routine screening. Blood test reveals: hemoglobin 11 g/dL * erythrocyte count 3.5 x 106 /mmc * mean corpuscular volume 75 fL (normal value 80-100 fL)* white cell count 6 x 103 /mmc platelets 190 x 103 /mmc Analysis of the case: the boy is well but presents an asymptomatic, mild microcytic anemia. Possible causes of microcytic anemia include: a) thalassemia minor - inquire familial anamnesis; sequence hemoglobin genes b) iron deficiency - inquire about diet; measure sideremia, transferrin saturation c) vitamin C deficiency - inquire about diet; measure vitamin C in blood. In this case β-thalassemia minor may appear the most likely starting hypothesis, but you should never disregard less likely hypotheses; thus all the analyses indicated above should be carried out. The genese of β-thalassemia reach high prevalence in areas where malaria was formerly or still is frequent; thus you should inquire for the genetic origin of the patient; the diagnosis is confirmed by sequencing the genes of hemoglobin subunits. If positive, extend the same analysis to the boy's parents and brothers / sisters. Iron deficiency is a common cause of microcytic anemia in the elderly; less so in children. To confirm or exclude this possibility, measure serum iron and transferrin saturation; inquire about diet. Common causes of iron deficiency are atrophic gastritis (an acidic gastric juice is required to solubilize iron) and hemorragies; chronic internal hemorragies, of which the patient may not be aware, are to be particularly considered. These occur mainly in the gut, because of diverticultis, ulcers, cancers, etc.; thus a search for occult blood in the feces is indicated. 2) 5 year old boy of african descent with very painful swelling of some fingers. Blood test reveals: hemoglobin 8 g/dL * erythrocyte count 2.5 x 106 /mmc * mean corpuscular volume 85 fL (normal value 80-100 fL) white cell count 7 x 103 /mmc platelets 200 x 103 /mmc Analysis of the case: the boy has an important normocytic anemia, associated to a painful crisis of the extremities. Although the two findings may be uncorrelated, the association, and the ethnic origin of the patient, suggest sickle cell anemia: carry out electrophoresis of hemoglobin; sequence hemoglobin genes. Extend genetic investigation to the boy's parents and brothers/sisters. Measure serum iron and ferritin. Questions and exercises: 1) Megalocytic megaloblastic anemia is due to: iron deficiency or thalassemias reduced biosynthesis of DNA and lowered cell replication rate, possibly caused by folate or vitamin B12 deficiency inherited hemoglobinopathies 2) Microcytic anemia may be due to iron deficiency or thalassemias reduced biosynthesis of DNA and lowered cell replication rate, possibly caused by folate or vitamin B12 deficiency inherited hemoglobinopathies 3) The normal concentration of hemoglobin in the blood of an healthy adult is: > 8 g/dL > 12 g/dL > 16 g/dL 4) Pancytopenia, i.e. reduced count of all cell types in blood may be due to: inherited immunodeficiencies inherited hemoglobinopathies lesions of the bone marrow (e.g. leukemias, lymphomas, aplasia) your score: 0 Attendance not registered because matricola was not entered. You can type in a comment or question below (max. length=160 chars.): All comments posted on the different subjects have been edited and moved to this web page (for optimal reading try to have at least 80 characters per line)! 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. leukemia? 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 (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 dosage. 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 electrolytes: 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. Hemostasis and Thrombosis lecture: I don't understand why is sodium citrate added to the serum solution to measure the prothrombin time. Bellelli: in order to measure PT or PTT you want to be able to start the coagulation process at an arbitrary time zero, and measure the increase in turbidity of the serum sample. To do so you need (i) to prevent spontaneous coagulation with an anticoagulant; and (ii) to be able to overcome the anticoagulant at your will. Citrate (or oxaloacetate; or EDTA) has the required characteristics: it chelates calcium, and in this way it prevents coagulation; but you can revert its effect at your will by adding CaCl2 in excess to the amount of citrate. You cannot obtain the same effect with other anticoagulants (e.g. heparin) whose action cannot be easily overcome. Dear professor I cannot do the self evaluation test because it says the the time has expired It is not possible because I havent even started them Bellelli: this is due to the fact that the program registers your name and matricola number from previous attempts. I shall fix this bug. Meanwhile try to use a fake matricola number. How is nephrotic syndrome associated hypoalbuminemia as you described in methods of analysis of protein because seems counterintuitive Bellelli: nephrotic syndrome is an autoimmune disease in which the glomerulus is damaged and the filtration barrier is disrupted; diuresis is normal but there is loss of proteins (mostly albumin) in the urine. I m sorry i confused polyurea with hypoalbuminemia but my question still stands during glomerulonephritis you mentioned something of polyurea as compensation i could not follow how this compensation mechanism works and collapse after some time in glomerulonephritis Bellelli: the condition you describe is NOT characteristic of acute glomerulonephritis. In glomerulonephritis there is damage of the glomerulus and severely impaired GFR. Thus the diuresis is severely reduced, and due to impaired filtration proteins appear in the urine. The condition you describe corresponds to the initial stage of chronic kidney failure, usually due to atherosclerosis, diabetes, hypertension or other type of damage of the kidney tissue. In this case GFR is impaired, albeit to a lesser extent than in glomerulonephritis, and the excretion of urea is reduced. This leads to increased BUN. However the increased concentration of urea reduces the ability of the tubuli to reabsorb water, because of osmotic reasons, yielding compensatory polyuria. The patient has reduced GFR but normal or increased diuresis (urine volume in 24 hours). To some extent this effect is beneficial, as it favors the elimination of urea; however it cannot completely solve the problem and in any case the progression of the disease leads to kidney insufficiency. In its essence the point is that a moderately reduced GFR can be partially compensated by reduced tubular reabsorption; a severely reduced GFR cannot.       Home of this course