MEDICINE AND SURGERY "F"
Course of LABORATORY MEDICINE

Standard blood tests; tests of organ function

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      This lecture presents some elements of clinical reasoning on "standard" blood tests. It is meant to illustrate two common conditions: preventive screening of an asymptomatic person, and the initial study of a patient whose clinical picture is relatively aspecific. A standard blood test is indicated in every patient; but of course if a patient presents strongly indicative symptoms and signs, e.g. obvious jaundice or the symptoms of congenital erythropoietic porphyria (do you remember them? if not go to the lecture on porphyrias!), you will also prescribe much more specific analyses.

      An important consideration is as follows: in the majority of cases, every organ is involved in more than a single function, and every function requires the activity of more than a single organ. For example, the liver plays a role in the excretion of bilirubin amd cholic acids, in the blood coagulation (because of the biosynthesis of coagulation factors), in the protidemia, in the regulation of glycemia, in the biosynthesis of urea and detoxification of ammonia, etc. At the same time the regulation of glycemia requires the cooperation of the liver, the endocrine secretion of the pancreas (insulin and glucagone), of the hypophysis (mainly because fo growth hormone) and of the adrenals (glycocorticoid hormones). As a consequence the same symptom or laboratory finding may point to lesions in several organs, and it is the association of many symptoms and laboratory signs that identifies the organ that is affected. Never try to interpret a single symptom, always refer to the complete clinical picture! However, within the general clinical picture two different diseases may coexist, especially in the elderly: in these cases try to assign each symptom or sign to its cause in order to establish the diagnoses. If necessary prescribe more tests.

      It is very common for the physician to prescribe a routine selection of "standard" blood tests, e.g. during general screening or as an instrument of preventive medicine aimed at the early diagnosis of yet asymptomatic conditions (secondary prevention).
      It is also common that a patient may present with aspecific symptoms: fever, fatigue, mild dispnoea, imprecisely localized pain, mental confusion or somnolence, etc. These symptoms offer a poor guide to diagnosis, and you may be uncertain on the analyses to prescribe. Some routine analyses may help you to better focus the clinical case.

      The aim of a standard blood test is to explore the function and the possible lesions of as many organs as possible; one should always reason in terms of the different organs and apparatuses, rather than taking the results as a sort of "generic" information about health. For example, a standard blood test will include the measurement of glycemia (many causes of hypo- and hyperglycemia exist, the most common being diabetes mellitus, and its therapy with insulin); bilirubinemia (measures liver function); blood urea nitrogen and creatininemia (estimate kidney function); electrolytes (usually regulated by hormones); red cells, white cells and platelets (bone marrow; anemias, leukemias); etc. It is important to distinguish functional information from information about anatomical lesions of the same organ. For example bilirubinemia tests the liver function; epatic enzymes (ALT, AST, etc.) if increased indicate a liver lesion that is killing epatocytes. Often, but not always, signs of impaired function and anatomical lesion are both present. Some organs and apparatuses are poorly represented in the standard blood tests; for example the gastro-intestinal tract. Unfortunately, for practical and economic reasons one cannot screen for every possible disease, thus the standard blood test will usually not provide the "final" diagnosis; rather it will suggest more specific investigations.

      The following table summarizes the representation of different organs in the standard blood tests; it will be noticed that some organs are poorly tested, if at all, and require more sepcific investigation.
 
    Organ           typical presenting symptom(s)             functionality tests      
 
Central and peripheral nervous system motor and sensory impairment; mental confusion serum enzymes (CPK, GOT)
Endocrine system variable requires specific tests; electrolytes, and glycemia may provide some clue
Lung and respiratory tree dispnoea; cyanosis; fatigue poorly represented in the standard blood test; requires hemogas analysis (reduced PO2; increased PCO2; respiratory acidosis); measurement of respiratory volumes; measurement of pulmonary blood flow
Heart perypheral oedema; dispnoea; altered blood pressure; altered pulse; thoracic pain; abnormal heart sounds determination of central venous pressure; determination of LDH, CPK and other typical heart enzymes; measurement of pulmonary blood flow; ECG; echography
Bone marrow anemia, pallor, fatigue, petechiae hemochromocytometric analysis; marrow biopsy
gastro-intestinal tract vomiting, diarrhoea, abdominal pain, etc. poorly represented in the standard blood test; requires specific tests
Liver jaundice; dark urine; itch serum bilirubin concentration; transaminases and other liver specific enzymes; virological tests (antibodies, antigens, viral RNA); liver biopsy.
In advanced stages: hyperammonemia, hypoalbuminemia, coagulation defects (reduced biosynthesis of coagulation factors)
Pancreas abdominal pain (exocrine pancreas); diabestes mellitus (endocrine pancreas) Serum amylase; glycemia
Kidney increased BUN; confusive state; hypertension BUN; glomerular filtration rate (creatinine clearance); anemia (due to reduced biosynthesis of erythropoietin)
Prostate variable, usually mild; blood in the semen Serum alkaline phosphatase; Prostatic antigen (PSA)
Ovary or testes infertility; cycle alterations poorly represented in the standard blood test; require specific tests
Uterus pain poorly represented in the standard blood test; requires specific tests

      Usually every finding may have several causes and be compatible with many diagnoses; thus the physician should imagine a list of the conditions which are compatible with the abnormal finding and explore all of them; this process is the differential diagnosis. For example hypoglycemia may be due to genetic defects of glycogen metabolism; to adrenal insufficiency (Addison's disease); to liver failure; to an insulinoma; to excess antidiabetic therapy; etc. Some signs may be compatible with common and uncommon diseases; don't be fooled into ignoring the latter! For example hyperglycemia is most usually due to diabetes mellitus type I or type II, thus these are the first hypotheses to consider; but it may also be due to primary hemocromatosis, to Cushing's disease, to lesions that destroy the pancreas (pancreatitis, cancer) ...

Audio: reasons to run a standard blood test

      A systematic approach to clinical reasonining is always advisable, and may provide an important guide. To approach an aspecifically symptomatic patient, or an asymptomatic person, you will first record the anamnesis, carry out the physical examination, take the blood pressure and body temperature, check for neurological symptoms and signs.
      Next, you can imagine your path as a sequence of questions. The first question you ask yourself is: is the patient condition acute or chronic? Is he/she an emergency or at risk to become an emergency? Medical emergencies usually give precise and important symptoms, thus they do not fall in the case we are presently discussing, and have their own specialistic approach. However there are conditions which, though not being actual emergencies, are at risk to become emergencies in a short time: e.g. a mild appendicitis may aggravate and cause a peritonitis in a few hours; a diabetic ketoacidosis may present itself as a mild confusional state and turn to ketoacidosis coma quite rapidly. Emergencies require the stabilization of the patient and the critcal decision on whether they require medical or surgical interventions, but we are not dealing with them here, as they are treated in dedicated courses.
      If the answer to your first question is negative (i.e. the patient's condition is not a medical or surgical urgency that requires prompt hospitalization and a dedicated procedure) your next step will probably be the prescription of a standard blood test. Standard blood tests may vary somewhat; however they usually include the blood cell count (the quantitation of red cells, of the various types of white cells and of platelets; hemoglobin concentration), total protein concentration, electrophoretic protidogram, glycemia, azotemia (BUN), creatininemia, transaminases (sGOT, sGPT), bilirubinemia, cholestrolemia, triglyceridemia, electrolythemia, and possibly other analyses. The general relevance of these routine tests is listed in the Table below.
 
Measurement Usual range Increases in Decreases in
Complete Blood Count (hemocytometer test)
Red blood cells 4.5-6 M/μL Primary or secondary polycytemia; increased erythropoietin stimulus because of kidney or lung disease; high O2 affinity hemoglobinopathies Anemias, acute and chronic hemorrages
Hemoglobin 13-16 g/dL see above (polycytemias) Anemias, acute and chronic hemorrages, iron deficiency, vitamin deficiency, thalassemias, etc.
Platelets 150-400 K/μL uncommon Damage of the bone marrow (e.g. leukemias, lymphomas, idiopathic medullary aplasia)
White cells (total) 4-10 K/μL Leukemias; infections (especially bacterial infections) Damage of the bone marrow (e.g. leukemias, lymphomas, idiopathic medullary aplasia)
White cells formula
Neutrophile granulocytes 2-8 K/μL (35-80%) acute bacterial infections; myeloid leukemias bone marrow aplasia; cancer; chemotherapy; autoimmune diseases
Eosinophile granulocytes 0-800/μL (0-7%) allergy; parasitic infection  
Basophile granulocytes 0-200/μL (0-2.5%) (myeloid leukemias)  
Lymphocytes 1-5 K/μL (10-50%) (Lymphatic leukemias) (bone marrow aplasia; cancer; some types of infection; autoimmune diseases)
Monocytes 160-1000/μL (0-12%) (chronic infections; autoimmune diseases) some types of infection; marrow aplasia; glucocorticoid therapy
 
Hematochemical tests
Glycemia 65-110 mg/dL Diabetes mellitus (type I; type II); Cushing syndrome insulinoma; excess insulin therapy in diabetic patients; Addison's disease; some glycogenoses
Azotemia (BUN) 5-20 mg/dL as urea nitrogen (10-50 mg/dL urea) any kidney disease causing renal insufficiency Late stages of liver failure; inherited defects of the urea cycle
Creatininemia 0.6-1.3 mg/dL any kidney disease causing renal insufficiency  
Bilirubinemia total 0.3-1 mg/dL (conjugated 0.1-0.3 mg/dL) jaundice; liver disease (increase of unconjugated bilirubin only: hemolytic crisis)  
sGOT < 37 U/L Hepatitis; biliary obstruction; diseases causing the death of liver cells  
sGPT < 55 U/L Hepatitis; biliary obstruction; diseases causing the death of liver cells  
cholesterol total <200 mg/dL Hypercholesterolemia of dietary or genetic origin Mevalonic aciduria or other defect of cholesterol biosynthesis
Protidemia total 6-8 g/dL Often due to the gamma globulin fraction (see electrophoresis) decreased biosynthesis because of malnutrition or liver insufficiency; accelerated loss because of severe burn or nephrotic syndrome
Protein fractionation by electrophoresis
Albumin 55-65% (decrease of other components?) malnutrition, liver insufficiency, nephrotic syndrome, severe burn
Alpha 1 globulins 3-5%    
Alpha 2 globulins 7-12%    
Beta 1 globulins 4.5-7%    
Beta 2 globulins 3-6%    
gamma globulins 11-19% Bacterial infections; multiple myeloma (monoclonal peak) Congenital and acquired immunodeficiencies
 
Inflammation markers
Erythrocyte sedimentation rate (ESR) 2 mm/hour inflammation, bacterial infection uncommon
C-reactive protein < 8 mg/L inflammation, bacterial infection uncommon
 
Electrolytes
Sodium 135 mEq/L hyperaldosteronism  
Potassium 4-5 mEq/L   sweating; hyperaldosteronism
Calcium 4-5 mEq/L (2-2.5 mM) hyperparathyroidism; hypervitaminosis D; milk-alkali syndrome Chronic renal insufficiency; hypoparathyroidism
Chloride 100 mEq/L loss of bicarbonate (catution: chloride may be normal and bicarbonate may be decreased if other acids are present, e.g. in ketoacidosis) - run hemogas analysis increase of bicarbonate (e.g. chronic respiratory acidosis) - run hemogas analysis


Audio: standard blood tests

 
      A very important complement to the study of organ pathology is provided by the determination of intracellular enzymes released in the blood plasma by the death of cells of the affected organ. This is because the enzymatic profile is organ-specific and identifies which organ is affected by disease. In the standard blood test only sGOT and sGPT are routinely measured, but testing some more is a good practice, because information on some organs would otherwise not be available. This subject has been considered in the lecture on plasma proteins, to which the student is referred for details. In a general screening usually six or seven enzymes are measured, as detailed in the Table below; these provide a gross indication of the organs that may be affected; in case of positive results, more enzymes can be tested (see the lecture on plasma proteins). The routine screening of 6-8 enzymes is important because some organs are poorly assessed in the standard blood test, and the alteration of their characteristic enzymes may be the only diagnostic clue to their disease: this applies in particular to the nervous system, the heart, and the gastrointestinal tract.

Principal enzymes that can be tested in routine screenings
  Heart Brain Prostate Placenta Intestine Bone Liver Kidney Pancreas Sarcoidosis Leukocytes
Creatine phosphokinase (CK, CPK) +++ +++                  
Aspartate transaminase (AST, GOT) ++ ++         +++        
Alanine transaminase (ALT, GPT)             +++        
Lactate dehydrogenase (LDH) ++                    
Alkaline phosphatase (ALP)       +++ +++ +++ +++ +++     +++
Acidic phosphatase (ACP)     +++                
γ Glutamyl transferase (γGT)             +++ +++      
Amilase                 +++    

Audio: blood enzymes and organ damage

EVALUATION OF THE STANDARD BLOOD TEST

      Your next step is to evaluate the blood test. Look first for correlations; then reason along two different lines: by function, and by organ. These lines of reasoning are aimed at suggesting not one but several diagnostic hypotheses, to be evaluated in subsequent analyses and tests. The general rule is as follows: several organs may participate to the same function, and each organ may participate to several functions; correlations between apparently unrelated signs of disease may point very precisely to the affectd organ. For esample anemia and hypertension may be associated in chronic kidney diseases (e.g. atherosclerotic kidney disease) because of reduced production of erythropoietin and impaired excretion of electrolytes and water; thus measurement of urea and creatinine concentration and GFR is strongly indicated.
      The possible diagnostic hypotheses suggested by the initial evaluation of the patient may or may not belong to the same nosographic category: don't be fooled by categories! For example hyerglycemias are usually due to endocrine disorders, e.g. diabetes, Cushing's disease, hyperthyroidism. Thus in the majority of cases hyperglycemias fit a single nosographic category. Hypoglycemias are less common and do not fit in a single nosographic category: two possible causes among many are Addison's disease (a endocrine disorder), and glycogenoses (inherited defects of metabolism).

Correlations: is there more than a single abnormal value? If so, the two or more abnormal values may depend on one and the same disease or do they point to the coexistence of two diseases? Multiple diseases are rare in young patients, frequent in the elderly. Examples of typical associations due to single diseases are: increased BUN and anemia (kidney insufficiency with reduced production of erythorpoietin); increased bilirubin and hypoprotidemia (liver failure); reduced erythrocytes and platelets with increased white cells (leukemias); etc.

Reason next on the patient's condition, by function and ethiopathogenesis: are there anomalies in his/her blood test? Which type of anomaly did you notice?
      - Metabolic? Metabolic defects may be genetic, inherited (usually in children); toxic; endocrine. Are there alterations of glycemia (diabetes), lipidemia, electrolytes, urine or blood osmolarity?
      - Infectious? Is there leukocytosis, altered white cell's formula, increase of gamma globulins in the electrophoresis, increase of acute phase proteins, increase of Erythrocyte Sedimentation Rate (ESR)? Consider Tbc, subacute viral infections (e.g. cytomegalovirus). Has the patient visited a country where an infectious or parasitic disease is endemic (e.g. schistosomiasis, malaria)?
      - Due to malabsorption or malnutrition? Are there signs of avitaminoses?
      - Due to a chronic or acute inflammatory disease (possibly autoimmune)? Crohn disease? Lupus? Rheumatoid arthritis? Are there markers of acute or chronic inflammation? Prescribe an analysis of autoantibodies.
      - Neoplastic? Look for tumor markers; scintigraphy; imaging methods (e.g. total body NMR). Leukemias and lymphomas usually cause severe alterations in the blood cell count and usually affect all the corpuscles (pancytopenia); selective decrease of just one type of corpuscles (e.g. erythrocytes or platelets) usually suggest a different diagnosis, even though in the early stage of a leukemia or lymphoma one cell type may be affected more than the others.

Reason next on patient's condition by organ (this mode of reasoning is not alternative to the preceding one; carry out both, and cross your results):
      - Might the disturbance be neurological? Peripheral or central? Of ischemic, metabolic, toxic, infectious origin? Is liquor analysis indicated? The standard blood test carries little information about brain function, except for the possible presence of brain enzymes. If meningitis or brain hemorrage are suspected an exam of the cerebrospinal fluid is indicated; however this is an invasive procedure and it is probably advisable to carry out imaging investigations first.
      - Might the disturbance be of cardiac origin? Circulatory? Is hypertension present? Are there abnormalities in the electrolytes? Are the heart enzymes increased?
      - Lung? Lung is poorly tested in standard blood tests, and requires the hemogas analysis; however a saturimeter may rapidly measure O2 saturation. An indirect information on lung function in the standard blood test may be given by chloride. The clinical reasoning is as follows:
[Na+] + [K+] = [Cl-] + bicarbonate + anion gap
Thus:
bicarbonate + anion gap = [Na+] + [K+] - [Cl-]
Abnormal chloride, in the presence of normal or nearly normal sodium and potassium points to an abnormal bicarbonate or anion gap or both and strongly indicates that a hemogas analysis is necessary. Unfortunately, this indication is not exhaustive of lung function and acid-base equilibrium because several acidoses and alkaloses may be normochloremic (e.g. acute respiratory acidosis and alkalosis; metabolic acidoses in which an increased anion gap may compensate for a decreased bicarbonate).
      - Is the gastrointestinal system affected? Abdominal pain? Diarrhoea or stipsis? Malabsorption? Diarrhoea or vomiting may cause major alterations in the electrolytes.
      - Liver? Is bilirubin increased? Is the protidogram normal? Are liver enzymes increased?
      - Kidney? Are BUN, and creatinine elevated? Kidney insufficiency may be associated to either increased or decreased production of erythropoietin, and hence to increased or decreased red cell count, in the absence of abnormalities in the platelet and leukocyte count.
      - Pancreas? Pancreas enzymes, notably amylase, are indicative of chronic or acute pancreatitis; hyperglycemia may be due to tipe I diabetes.
      - Endocrine system? Often, an endocrine system dysfunction manifests itself via metabolic disturbances (see above), and/or electrolyte disturbances.
      - Bone marrow and erythropoiesis are well represented in the standard blood test: essentially any alteration in the cell count (erythrocytes, platelets, and leukocytes) reflects either a malfunctioning of the bone marrow or an increased cell loss. Leukemias, lymphomas, bone marrow aplasia usually cause a severe reduction of all blood corpuscles (pancytopenia), with the possible exception of the cell clone affected by the leukemia.

Audio: reasoning on organs and functions

      In a young subject usually think of a single disease, possibly acute; in an elderly subject think of multiple diseases, at least some of them chronic: e.g. atheroslerosis, with chronic ischemic damage of heart ad kidney, possibly coupled with COPD.

      Assessment of organ function
      If the standard blood test suggests that some alteration of organ function is present, you should pursue this line by specific tests. Indeed specific tests are essential to assess organ functions. Moreover, in many cases the physician may suspect a disease affecting a specific organ, but he may not have a clue on its cause, which is essential for diagnosis: e.g. jaundice suggests a defect in liver function, increased azotemia (Blood Urea Nitrogen, BUN) suggests a defect in kidney function, peripheral oedema suggests cardiac failure, etc. None of these considerations is a diagnosis: diagnosis is liver cirrhosis or acute viral hepatitis, not "liver dysfunction". Thus organ diagnosis is extremely important and helps focusing a more precise ethiological diagnosis. In order to evaluate laboratory results and to prescribe the appropriate tests, the physician should be familiar with the various functions of different organs, that may be simultaneously altered by one and the same disease: e.g. liver failure may cause hyperbilirubinemia and jaundice (because of reduced bilirubin excretion), but also hypoalbuminemia and coagulation disorders (because of reduced biosynthesis), and hyperammonemia (because of impaired biosynthesis of urea); or acute pancreatitis, caused by the release of proteolytic enzymes inside the pancreas may cause a form of type I diabetes mellitus (because of destruction of the Langerhans islets). Organ functions were listed in the pertinent lectures; a summary of the appropriate links is as follows:
Lungs
Kidneys
Liver
Pancreas
Adipose tissue

      Laboratory findings in some common pathological conditions
      Infectious diseases
      Infectious diseases are probably the most common ailment of infants and young adults. they may be due to bacteria (protozoans), viruses, parasites or fungi. They usually present fever and laboratory signs of inflammation (increased C reactive protein, increased erythrosyte sedimentation rate. Additional findings depend on the causative agent.
      Bacterial diseases usually present localized pain in the infected organ, fever, possible rashes, and leukocytosis, with marked increase of neutrophil granulocytes (typical WBC count in excess of 10,000 cells / μL); antibodies against the pathogen may be found, and in acute infections IgM appear before IgG; poistivity for igG but not for IgM usually indicates a remote infection, which may have healed and be unrelated to the present clinical case. Since every bacterium has its own organ preference, bacterial infections are usually localized, and cause localized pain; imaging (X-rays, NMR) may provide precious information. Special attention should be paid to atypical cases, e.g. bacterial infections without leukocytosis. These may occur because the bacterium itself doed not cause leukocytosis (e.g. Brucella melitensis) or because of an impaired response of the patient (e.g. in the elderly; in the course of immunosuppressive therapy or lymphoma; in the course of sepsis).
      Viral diseases present with diffuse pain (to the back, to all muscles or joints, etc.), inconstant local symptoms (e.g. because of instestitial pneumonia), fever; possibly cutaneous rashes. Laboratory findings reveal increased ESR, and C reactive protein, and a moderate leukocytosis, with predominace of lymphocytes and monocytes. Viral antigens and sntiviral antibodies are present and the viral genome may be revealed in the peripheral blood by PCR and sequencing.
      Parasitic diseases are common in tropical and sub-tropical countries but relatively rare in temperate or cold climates; they may be acquired during a trip to a tropical country and brought back in a cold one. Fever, malaise, anemia and jaoundice may occur in several parasitoses (e.g. malaria, schistosomiasis). The laboratory diagnosis requires the identification of the parasite, or its eggs in the affected tissues or excretions (blood, urine, feces, etc.).
      Diseases due to fungi usually affect immunocompromised hosts or are limited to cutaneous infections.

      Clinical example: an atypical bacterial infection. 55 years old male, presents high fever (38oC in the morning, 40oC) resistant to antipiretics. No localized signs, no pain. Moderate cough. Clinical examination is poor: sparse bilateral areas of obtuse sound at toracic percussion; negative for neurological signs. Abdomen treatable, peristalsis present.
Laboratory findings:
hemoglobin 12 g/dL
RBC count 3.88x10o / μL;
mcv 88 fL
WBC 5,900 / μL;
WBC formula: Neu 81%; Eos 0.9%; Bas 0.6%; Lym 13.1%; Mono 4.1%
Platelets 180,000/ μL;
Erythrocyte Sedimentation Rate (ESR) 38 mm/h
C reactive protein 221 mg/L
Procalcitonin 0.24 ng/mL (normal range < 0.1)
Total bilirubin 1.44 mg/dL
sGOT 33 U/L
sGPT 35 U/L
δGT (gamma glutamyl transferase) 40 U/L
anti cytomegalovirus: IgG 12.8 AU/mL (++); IgM 0.03 AU/mL (-)
anti Epstein Barr virus EBVCA: IgG 59.9 AU/mL (++); IgM 0.29 AU/mL (-)
      Comment: the clinical picture strongly suggest a viral infection, with a possible localization at the level of lungs and bronchi. The hypothesis is also consistent with the low WBC count. The laboratory confirms the active inflammatory status (increased ESR and C reactive protein). Antibodies against cytomegalovirus and Epstein Barr virus are indicative of pregress, probably not presently active infection (positive IgG but negative IgM). Other plausible diagnostic hypotheses are: lymphoma (consistent with fever, inflammatory markers, pancytopenia; prescribe bone marrow biopsy); autoimmune disease (e.g. lupus erythematosus; look for autoanntibodies);
A chest X ray and an abdominal ecography were prescribed. The abdomen was negative, whereas multiple patches of pulmonary addensation were observed. Bronchial aspirate turned out positive for infection by the bacterium Legionella pneumophyla.
This case is interesting because of its atypical features: the bacterial infection seemed at the beginning an unlikely hypothesis because of the low WBC count, but one should always take into account that: (i) unlikely does not mean impossible; and (ii) some bacteria may not cause major leuocytosis.

      Arterial hypertension
      In 80% of the cases arterial hypertension has no other objective finding than the increase of pressure itself, and laboratory findings are negative (idiopathic hypertension); however, given the risks associated to chronic hypertension, an effort to ascertain possible causes is justified. The laboratory investigation of arterial hypertension points in two directions: endocrine disturbances, and kidney disfunctions acting via the renin-angiotensin system. Hormones that affect the arterial pressure include adrenaline and noradrenaline (e.g. in pheochromocytoma), and glycocorticoids (e.g. in Cshing's syndrome). All these hormones can be measured, but their blood leves vary significantly in the course of the day and in response to stimuli. The increase of noradrenaline in pheochromocytoma occurs in irregular bursts, associated to hypertensive crises, and followed by increased elimination of noradrenaline metabolytes (e.g. vanillylmandelic acid) in the urine.
      Renin is an proteolytic enzyme produced and released in the blood by the glomerulus as a response to low arterial pressure. In some kidney diseases (e.g. aterosclerotic kidney disease, stenosis of the renal artery) either or both kidneys may produce renin in excess. Renin digest the liver-produced serum protein angiotensinogen, releasing and endecapeptide fragment (angiotensin I), further converted in the lung to angiotensin II. The angiotensins are powerful vasoconstrictors and cause an increase of the arterial pressure (and a depression of the microcirculation). Renin inhibitors, converting enzyme inhibitors, and angiotensin antagonists are all available and may be used as a therapy of idiopatic and/or kidney-derived hypertension. The clinical laboratory can measure the level of angiotensins in the blood and urine.

      Chronic kidney failure
      Is common in the elderly, usually because of aterosclerotic kidney disease. The typical laboratory findings increased urea and creatinine. GFR is decreased. Anemia (due to reduced production of erythropoietin) and hypertension (due to excess production of renin) may be present. For a summary ofkidney functions and their clinical evaluation see the lecture on electrolytes. Moreover, a test of serum enzymes may reveal the presence of kidney enzymes (e.g. alkaline phosphatase); see the lecture on plasma proteins.

      Chronic liver failure, cirrhosis
      Presents increased bilirubin with jaundice, elevated liver enzymes, and, in advanced stages hypoalbuminemia, and hyperammonemia (due to reduced production of urea). For a summary of liver functions and their evaluation see the lecture on bilirubin and jaundice. The enzymes of the liver are highly indicative and are usually altered in chronic liver disease, thus confirming the diagnosis; see the lecture on plasma proteins.

      Anemias
      Reduced hemoglobin and erythrocyte count. Possibly associated with the reduction of other blood cells (platelets, leukocytes). Anemia is a generic term, not a diagnosis, and the finding of anemia at a routine blood test indicates further analyses. The student is referred to the lecture on anemias in this course.

      Thrombocytopenic purpura
      Reduced platelet count is associated to purpura and may be due to several causes: bone marrow fibrosis, leukemias and lymphomas, autoimmune or drug-induced platelet destruction, etc.

THE URINE TEST (URINALYSIS)
      The urine is an important biological sample, which is easy to collect and complements the information provided by blood tests; it is customary practice to prescribe a urine test together with a blood test.
      The urine test provides information on:
(i) kidney function and kidney disease; it is essential to determine the glomerular filtration rate (usually using creatinine as the tracer); it may provide information on urinary tract infections, and glomerular filtration defects (e.g. glomerulonephritis, nephrotic syndrome). Moreover comparison of the urine analysis with blood analysis, may reveal defects of resoprtion of useful solutes (tubular function insufficiency; e.gg. Hartnup's disease).
(ii) Metabolic diseases, inherited or acquired. Many metabolytes are easily detectable in the urine, and provide information on possible metabolic diseases (e.g. disturbances of aminoacid metabolism; disturbances of nucleotide metabolism; gout; diabetes; etc.).
(iii) Pregnancy, endocrine diseases. Many hormones are more easily and more meaningfully measured in the urine than in blood, because they attain higher concentration and because urine collection over 24 hours averages the hormone concentration, which may experience wide fluctuations in blood.
(iv) Intoxications, acute or chronic; drug abuse; excess dosage of therapeutical drugs.
(v) Hemolytic diseases (hemoglobinuria).

Reference values of analytes commonly measured in the urine
analytenormal valueincreased indecreased in
glucoseabsentdiabetes type I and II; nephrogenic diabetes  -  
protein< 150 mg/dayglomerulonephritis; nephrotic syndrome; pyelonephritis  -  
creatinine14-26 mg/kg/day  -  chronic kdney failure
Vanillylmandelic acid1-9 mg/daypheochromocytoma  -  
chorionic gonadotropinabsentpregnancy  -  

      Practical considerations
      (i) Urine collection: collect either the first urine emission in the morning or the whole urine production over 24 hours. In the latter case the volume provides important information (normal daily urine production 1,000-2,000 mL; decreased volume suggests dehydration or impaired GFR, e.g. glomerulonephritis, atherosclerotic kidney disease; increased volume may suggest excess drinking, diabetes insipidus, or osmotic diuresis, e.g. because of diabetes mellitus or because of increased BUN and chronic compensated kidney insufficiency). Collection over 24 hours is indicated to average the concentration of hormones whose secretion experiences large oscillations (e.g. catecholamine metabolytes in pheocromocytoma). Sterile collection is required only if a culture of microorganisms is to be carried out.
      (ii) Visual inspection reveals color and cloudiness. Color is determined by the presence of pigments (e.g. bilirubin, hemoglobin, etc.)
      (iii) Chemical analysis includes the measurement of pH (normal range 5-8; varies in conditions of acidosis and alkalosis; extreme values indicate an hemogasanalysis), and glucose concentration; other substances as required by the physician.
      (iv) Microscopic examination of the sediment (after centrifugation) reveals the presence of red (hematuria) or white cells (possibly indicative of urinary tract infections). Pyuria (presence of pus in the urine) is observed in bacterial infections of the urinary tract (pyelonephritis, cystitis, uretritis; consider also sexually transmitted infections, e.g. gonorrhea).

      Some common findings:
      Proteinuria: protein in the urine should be less than 100 mg/m2.day. Excess protein in the urine may occur in two conditions: (i) increased filtration of protein because of a damage of the glomerular membrane (e.g. glomerulonephritis, nephrotic syndrome, etc.); in this case the main protein presnet in the urine is albumin (albuminuria). (ii) Excess presence in the blood of low molecular weight protein that escape the healthy glomerular membrane; in this case the main protein present in the urine should be identified because it is diagnostic of an extra-renal condition (e.g. light chain disease, myoglobinuria, hemoglobinuria, etc.).

      Glycosuria and other melliturias: glycosuria is the presence of glucose in the urine. Glucose is normally filtered in the glomerulus and reabsorbed in the tubuli. Glycosuria may be due to hyperglycemia (filtration exceeds the ability of the tubules to reabsorb the solute; occurs in all types of diabetes mellitus), or to tubular insufficiency in the presence of normal glycemia (nephrogenic diabetes mellitus). Mellituria refer to the presence in the urine of other monosaccharides (fructose, galactose, etc.) it is usually a consequence of an inherited genetic defect of sugar metabolism.

      Loss of bicarbonate and aminoacids: is typically an acquired or inherited tubular function insufficiency (e.g. Hartnup's disease). May cause renal tubular acidosis a type of NAGMA (see the lecture on acidosis and alkalosis).





      Clinical examples
1) Compare the two following patients, who present apparently similar laboratory results:
Laboratory findingPatient #1Patient #2
hemoglobin8.2 g/dL * 8.3 g/dL *
erythrocyte count2.5 x 106 /mmc *2.7 x 106 /mmc *
mean corpuscular volume85 fL (normal value 80-100 fL)88 fL (normal value 80-100 fL)
white cell count2 x 103 /mmc *52 x 103 /mmc *
platelets20 x 103 /mmc *30 x 103 /mmc *

Analysis of the two cases: both patients present severe anemia and thrombocytopenia. Patient #1 presents also a severe reduction of the white cells count; thus in this case we have a pancytopenia (all blood cells are reduced). Possible causes of pancytopenia are: bone marrow aplasia, lymphomas, erythroid leukemia. Patient #2 presents an important increase of white cells coupled to anemia and thromobocytopenia; the most likely cause is a leukemia which invades the bone marrow and causes the destruction of the erythorid and magakaryiocytic cell lines, repaced by leukemic cells. In both cases a bone marrow biopsy is strongly indicated; in Patient #2 cytologic examination of circulating white cells is also indicated.

2) Strong abdominal pain, 38.5oC fever. Blood test reveals:
erythrocytes  4x106 / μL
hemoglobin  13.4 g/dL
total leukocyte count  27x103 / μL *
leuckocyte formula: 
      neutrophile granulocytes  80%
      eosinophile granulocytes  2%
      basophile granulocytes  1%
      lymphocytes  12%
      monoocytes  5%
platelets100x103 / μL *
erythrocyte sedimentation rate40 mm /h

      Comment: this patient has fever, abdominal pain, a very significantly increased total leukocyte count and an increased ESR. The increase in leukocytes is almost entirely accounted for by neutrophils. Other values are unremarkable. The most likely diagnosis is an acute bacterial infection of an abdominal organ. Bacterial infections should be suspected in the presence of fever and localized pain (which may point to the site of the infection), and usually cause marked neutrophilia. Consider: acute appendicitis, diverticulitis, empyema of the gallbladder, liver abscess, peritonitis or peritoneal abscess, etc. Less likely diagnoses include myelocytic leukemia, intestinal infarction, volvulus, intussusception, posterior myocardial infarction, pyelonefritis, gynecological conditions in women.

      Cases from the medical literature
1) High fever (body temperature 38.5o C), malaise, mild jaundice in a previously healthy woman (from Medscape).
Laboratory findings:
Hemocytometric analysis: red cells 2x106/μL;
hemoglobin 5.8 g/dL;
white blood cell count: 2500/μL (reference range, 3500-10,500/ μL), with 65% neutrophils (reference range, 30%-60% neutrophils) and 18% bands (immature granulocytes);
platelet count: 88,000/μL (reference range, 150,000-400,000/μL)
Hematochemistry: serum creatinine level: 2.9 mg/dL (reference range, 0.6-1.2 mg/dL);
BUN: 54 mg/dL (reference range, 7-26 mg/dL);
ALT: 124 U/L (reference range, 0-55 U/L);
AST: 673 U/L (reference range, 5-34 U/L);
bilirubin: 2.8 mg/dL (reference range, 0.2-1.2 mg/dL);
serum lipase: 167 U/L (reference range, 8-178 U/L);
lactate: 4.6 mmol/L (reference range, 0.5-2.2 mmol/L);
ferritin: >21,000 ng/mL (reference range, 13-24 ng/mL);
triglycerides: 385 mg/dL (reference range, <150 mg/dL).

Comment: many serum and whole blood parameters are altered, thus we observe a multi-organ failure. Notice in particular the altered values of liver markers (bilirubin, AST, ALT) and kidney markers (BUN, creatinine). In this case we should look for the likely primary cause, taking into account that this condition is of acute origin and the patient was well before the onset of disease. Fever and pancytopenia are the most likely primary symptoms and signs; they are compatible with a lymphoma, and aleukemic leukemia, or with a bacterial sepsis. Banded granulocytes are immature cells thus a sign of an hyperproliferating bone marrow, again fully compatible with all the above hypotheses. Thus, we may suppose that multi-organ failure is caused by bacterial or neoplastic invasion of the affected organs.
Granulocyte count in bacterial sepsis is poorly informative, because both production and consumption of these cells are strongly increased; a slight prevalence of either may cause the granulocyte count to be increased or decreased. Increased lactate and anemia are both compatible with sepsis because of the bacterial fermentation of glucose, and the ability of bacteria to cause hemolysis.
What next? You should prescribe a bone marrow biopsy (to confirm or exclude leukemia and lymphoma) and a hemoculture (to detect the presence of bacteria or other microorganism in the blood). Pay attention: both diseases may be present and in this case lymphoma is primary and the depletion of white cells is the favoring cause of sepsis, which is secondary.
In this case the final diagnosis was bacterial sepsis by a bacterium of genus Erlichia; no abnormalities were found in the bone marrow, except overstimulation.

2) Severe osteoporosis in a 52 year old patient
A52 old woman affected by severe osteoporosis, discovered at a routine check-up, undergoes an examination of kidney function. Blood and urine samples are taken.
The standard blood test reveals: calcium, 8.9 mg/dL (= 4.5 mEq/L); phosphorus, 1.9 mg/dL; uric acid, 0.8 mg/dL; magnesium, 2.8 mg/dL; cholesterol, 202 mg/dL; protein, 6.9 g/dL; albumin, 4.2 g/dL; BUN, 8.4 mg/dL; creatinine, 1.19 mg/dL; glucose, 104 mg/dL; and HbA1c, 4.7%. Her estimated glomerular filtration rate (eGFR) was 53 mL/min.
The urine analysis gives the following results: specific gravity = 1.024 g/mL, pH 7.0, albumin +, glucose ++, RBC 20-29/High Power Field, and WBC 0-2/HPF.
The urine seems to be the most informative sample, thus urine was collected for 24 hours and a complete laboratory analysis was carried out. The results are as follows: protein, 2935 mg; albumin, 153 mg; sodium, 142 mmol; potassium, 59 mmol; chloride, 132 mmol; calcium, 428 mg; magnesium, 83.5 mg; uric acid, 455 mg; glucose, 20797 mg; and creatinine, 1.0 g. Moreover elevated levels of the following aminoacids were detected: taurine, aspartic acid, threonine, serine, asparagine, glutamine, glycine, alanine, citrulline, α-aminobutyric acid, valine, cysteine, isoleucine, leucine, tyrosine, phenylalanine, ornithine, lysine, l-methylhistidine, histidine, and arginine.

Comment: in this case we have a grossly altered urine analysis in the presence of moderately altered blood parameters; thus we suspect a chronic kidney failure that affects both the glomerular (reduced GFR) and tubular functions (impaired resorption of solutes). The tubular defect appears prominent, and affects many aminoacids. In a child or newborn we would suspect an inherited tubular resorption defect (e.g. Hartnup disease; cystinuria; cystinosis; inherited Fanconi's syndrome); but this is an adult subject, thus we should consider an acquired Fanconi's syndrome. Acquired Fanconi's syndrome may be due to several causes that include chronic metal or organic solvents poisoning (usually a professional disease; mercury, cadmium, lead or, among solvents, toluene are often involved); side effects of some drugs; monoclonal gammapathy (e.g. in multiple myeloma or in Waldenstrom disease). For a reference on the laboratory values in this uncommon condition see this reference.

3) A 37 years old male presents pertension (150/90 mmHg), weakness, polyuria. The laboratory tests show:
Serum electrolytes: Na 147 mmol/L, K 1.91 mmol/L, Cl 108 mmol/L;
Calcium (8.7 mg/dL), magnesium (2.2 mg/dL), and phosphorus (3.8 mg/dL) were within normal levels.
total CO2 27.8 mmol/L;
BUN 11 mg/dL;
creatinine 0.7 mg/dL.
      Hemogas analysis:
pH 7.435,
PCO2 46.5 mmHg,
PO2 87.7 mmHg,
HCO3- 30.5 mmol/L,
O2 saturation 97.1%
      Urine chemistry:
high potassium level of 18.56 mmol/L,
osmolality 199 mOsm/kg (normal range 50-1,200 mOsm/kg).
      Urine collection of 24 h:
creatinine 317 mmol/day (normal range 500-2,000 mg/day),
urea nitrogen 2.8 g/day (normal range 12-20 g/day),
sodium 322 mEq/day (normal range 40-220 mEq /day),
potassium 88 mEq/d (normal range 25-125 mEq/day),
calcium 379 mg/day (normal range 5-15 mEq/day, or 100-300 mg/day).

Comment: the laboratory data reveal a complex pattern that includes: mild metabolic alkalosis; early stage kidney insufficiency; hypokalemia and hypernatremia; excess urinary excretion of potassium and sodium retention. Several diagnostic hypotheses can be formulated: tubular kidney insufficiency, endocrine disorders (Cushing's disease, Conn's disease, thyroid disfunction). Thus a second round of tests was carried out which revealed:
Thyroid hormone level within normal limits;
plasma aldosterone (PA) level was 224.7 pg/mL (normal range up to 300 pg/mL)
plasma renin activity (PRA) 0.11 ng/mL (normal value 0.7-3.3),
high PA/PRA ratio.
For a more detailed discussion of aldosterone, renin and their ratio see this link; in short, aldosterone and renin are directly related and a high aldosterone/renin ratio is indicative of hyperaldosteronism.
These findings are compatible with primary hyperaldosteronism (Conn's disease) of moderate severity; thus abdominal computed tomography was prescribed, which revealed a left adrenal mass measuring 2 cm, confirming the diagnosis. Moreover impaired tubular function with production of a diluted, low osmolarity urine suggests nephrogenic diabetes insipidus. See this link for further details.

4) Weakness, flaccid paralysis.
A standard blood test reveals:
Electrolytes: sodium=140 mEq/L; potassium=2.6 mEq/L; chloride=115 mEq/L;
Nitrogenous compounds: BUN 22 mg/dL; creatinine 1.4 mg/dL.
Urine test: pH=6; sodium=35 mEq/L; potassium=40 mEq/L. Sodium/potassium ratio < 1 is very low; average value is 1.5-2 (caution: this parameter has high variability).
Comment: severe hypokalemia explains the symptoms; chloride is increased and this raises suspicion about possible bicarbonate alterations; indeed sodium+potassium-chloride=bicarbonate+AG=30 mEq/L. A hemogas analysis is indicated.

The hemogas analysis reveals:
PO2=90 mmHg;
PCO2=30 mmHg;
pH=7.32;
bicarbonate=17 mEq/L;
anion gap=13 mEq/L.
Comment: normal anion gap metabolic acidosis (NAGMA); see the possible causes of NAGMA in the lecture on hemogas analysis. In this case a renal tubular acidosis is likely, in view of the association with hypokalemia: the distal tubuli are responsible for the sodium/potassium exchange and for the chloride/bicarbonate exchange, and both are affected. Tubular renal disease may be a consequence of several conditions, possibly atherosclerotic kidney disease. Glomerular filtration rate may be measured but the modest increases of BUN and creatinine suggest that this function may not be severely compromised (case from Access Medicine).

Exercise
Try this 5 questions test (from Medscape).

Further readings
MedLine Plus: CBC blood test
Medscape: urinalysis

Questions and exercises:
1) Relevant abnormalities in the standard blood test of a patient were: leukocytosis, with marked increase of neutrophyles; increased C-reactive protein and ESR. These results suggest:
bacterial infection
a metabolic disease, possibly inherited
liver dysfunction

2) A patient presents hyperglycemia and reduced bicarbonate concentration. You prescribe further analysis to diagnose (or exclude):
kidney dysfunction
type 1 diabetes mellitus with ketoacidosis
type 2 diabets mellitus possibly associated to a metabolic syndrome

3) Reduced red blood cells and platelets, increased inflammation markers, abnormal electrolytes warrant further investigation for:
hematological neoplasia (e.g. leukemia)
kidney failure
heart failure

4) BUN and creatinine are markers of:
liver function
heart function
kidney function




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Thank you Professor (lecture on bilirubin and jaundice).

The fourth recorded part, the one on hyper and hypoglycemias is not working.
Bellelli: I checked and in my computer it seems to work. Can you better specify
the problem you observe?

This Presentation (electrolytes and blood pH) feels longer than previous lectures
Bellelli: it is indeed. Some subjects require more information than others. I was
thinking of splitting it in two nest year.

Bellelli in response to a question raised by email: when we compare the blood pH
with the standard pH we do not mean to compare the "normal" blood pH (7.4)
with the standard pH. Rather we compare the actual blood pH of the patient, with
the pH of the same blood sample equilibrated under standard conditions.
Thus, if we say that standard pH is lower than pH we mean that equilibriation with
40 mmHg CO2 has caused absorption of CO2 and has lowered the pH with respect
to its value before equilibration.

(Lipoproteins) Is the production of leptin an indirect cause of type 2 diabetes since
it works as a stimulus to have more adipose tissue that produces hormones?
Bellelli: in a sense yes, sustained increase of leptin causes the hypothalamus to adapt
and to stop responding. Obesity ensues and this in turn may cause an increase in the
production of resistin and other insulin-suppressing protein hormones produced by the
adipose tissue. However, this is quite an indirect link, and most probably other factors
contribute as well.

(Urea cycle) what is the meaning of "dissimilatory pathway"?
Bellelli: a dissimilatory pathway is a catabolic pathway whose function is not to produce
energy, but to produce some terminal metabolyte that must be excreted. Dissimilatory
pathways are necessary for those metabolytes that cannot be excreted as such by the
kidney or the liver because they are toxic or poorly soluble. Examples of metabolytes
that require transformation before being eliminated are heme-bilirubin, ammonia,
sulfur and nitrogen oxides, etc.

Talking about IDDM linked neuropathy can be the C peptide absence considered a cause of it??
Bellelli: The C peptide released during the maturation of insulin, besides being an indicator
of the severity of diabetes, plays some incompletely understood physiological roles. For
example it has been hypothesized that it may play a role in the reparation of the
atherosclerotic damage of the small arteries. Thus said, I am not aware that it plays a direct
role in preventing diabetic polyneuropathy. Diabetic neuropathy has at least two causes: the
microvascular damage of the arteries of the nerve (the vasa nervorum), and a direct
effect of hyperglycemia and decreased and irregular insulin supply on the nerve metabolism.
Diabetic neuropathy is observed in both IDDM and NIDDM, and requires several years to
develop. Since the levels of the C peptide differ in IDDM and NIDDM, this would suggest
that the role of the C peptide in diabetic neuropathy is not a major one. If you do have
better information please share it on this site!

In acute intermitted porphyria and congenital erythropoietic porphyria why do the end product
of the affected enzymes accumulate instead of their substrate??
Bellelli: First of all, congratulations! This is an excellent question.
Remember that a condition is which the heme is not produced is lethal in the foetus; thus
the affected enzyme(s) must maintain some functionality for the patient
to be born and to come to medical attention. All known genetic defects of heme
biosynthesis derange but do not block this metabolic pathway.
Congenital Erythropoietc Porphyria (CEP) is a genetic defect of uroporphyrinogen
III cosynthase. This protein associates to uroporphyrinogen synthase (which is present
and functional in CEP) and guarantees that the appropriate uroporphyrinogen isomer is produced
(i.e. uroporphyrinogen III). In the absence of a functional uroporphyrinogen III
cosynthase other possible isomers of uroporphyrinogen are produced together with
uroporpyrinogen III, mostly uroporphyrinogen I. The isomers of uroporphyrinogen
that are produced differ because of the positions of propionate and acetate side chains,
and this in turn is due to the pseudo symmetric structure of porphobilinogen. Only
isomer III can be further used to produce protoporphyrin IX. Thus in the
case of CEP we observe accumulation of abnormal uroporphyrinogen derivatives, which, as
you correctly observed are the products of the enzymatic synthesis operated by
uroporphyrinogen synthase.
The case of Acute Intermittent Porphyria (AIP) is similar, although there may be variants
of this disease. What happens is that either the affected enzyme is a variant that does not
properly associate with uroporphyrinogen III cosynthase or presents active site mutations
that impair the proper alignement of the phoprphobilinogen substrates. In either case
abnormal isomers of uroporphyrinogen are produced, as in CEP.
Also remark that in both AIP and CEP we observe accumulation of the porphobilinogen
precursor: this is because the overall efficiency of the biosynthesis of uroporphyrinogens is
reduced. Thus: (i) less uroporphyrinogen is produced, and (ii) only a fraction of the
uroporphyrinogen that is produced is the correct isomer (uroporphyrinogen III).


is it possible to take gulonolactone oxidase to synthesize vitamin C
instead of vitamin C supplement?
Bellelli: no, this approach does not work. The main reason is that
the biosynthesis of vitamin C, as almost all other metabolic processes, occurs intracellularly.
If you administer the enzyme it will at most reach the extracellular fluid but will not be
transported inside the cells to any significant extent. Besides, there are other problems
in this type of therapy (e.g. the enzyme if administered orally, may be degraded by digestive
proteases; if administered parenterally, may cause the immune system to react against a
non-self protein). In theory one could think of a genetic modification of the inactive human
gene of gulonolactone oxidase, but the risk and cost of this intervention would not be
justified. In addition to these considerations, except for cases of shipwreckage or
other catastrophes, a proper diet or administration of tablets of vitamin C is effective,
risk-free and unexpensive, thus no alternative therapy is reasonable. However, I express my
congratulations for your search on the biosynthesis pathway of ascorbic acid.


Resorption and not reabsorption would lead to hypercalcemia ie bone matrix being broken down.
Bellelli: I am not sure to interpret your question correctly. Resorption indicates destruction of the bone matrix and release of calcium and
phosphate in the blood, thus it causes an increase of calcemia. Reabsorption usually means active transport of calcium from the renal tubuli to the blood, thus
it prevents calcium loss. It prevents hypocalcemia, and thus complement bone resorption. To avoid confusion it is better use the terms "bone resorption" and "
renal reabsorption of calcium". If you have a defect in renal reabsorption, parthyroid hormone will be released to maintain a normal calcium level by means of
bone resorption; the drawback is osteoporosis.

In Reed and Frost model: I haven't understood what is the relationship
between K and R reproductive index. Thank you Professor!
Bellelli: in the Reed and Frost model K is the theoretical upper limit of
R0. R the reproductive index is the ratio (new cases)/(old cases) measured after
one serial generation time. R0 is the value of R one measures at the beginning
of the epidemics, when in principle all the population is susceptible.

What is the link between nucleotide metabolism and immunodeficiencies and mental retardation?
Bellelli: the links may be quite complex, but the principal ones are as follows:
1) the immune response requires a replication burst of granulocytes and lymphocytes, which in turn requires
a sudden increase of nucleotide production, necessary for DNA replication. Defects of nucleotide metabolism
impair this phase of the immune defense. Notice that the mechanism is similar to the one responsible of
anemia which requires a sustained biosynthesis of nucleotides at a constant rate, rather than in a burst.
2) Mental retardation is mainly due to the accumulation of nulceotide precursors in the brain of the
newborn, due to the incompletely competent blood-brain barrier.

How can ornithine transaminase defects cause hyperammonemia? Is it due to the accumulation
of ornithine that blocks the urea cycle or for other reasons?
Bellelli: ornithine transaminase is required for the reversible interconversion of ornithine
and proline, and thus participates to both the biosynthesis and degradation of ornithine. The enzyme is
synthesized in the cytoplasm and imported in the mitochondrion. Depending on the metabolic conditions
the deficiency of this enzyme may cause both excess (when degradation would be necessary) or defect
(when biosynthesis would be necessary) of ornithine; in the latter case, the urea cycle slows down. Thus
there is the paradoxical condition in which alternation may occur between episodes of hyperammonemia
and of hyperornithinemia.

When we use the Berthelot's reaction to measure BUN do we also have to
measure the concentration of free ammonia before adding urease?
Bellelli: yes, in principle you should. Berthelot's reaction detects ammonia,
thus one should take two identical volumes of serum, use one to measure free ammonia,
the other to add urease and measure free ammonia plus ammonia released by urea. BUN is
obtained by difference. However, free ammonia in our blood is so much lower than urea that
you may omit the first sample, if you only want to measure BUN.

Why do we have abnormal electrolytes in hematological neoplasia e.g.
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.

Lecture on Hemogas analysis interpretation of complex cases standard pH
Why if PCO2 is less than 40 mmHg it is absorbed during equilibration? Thank you in advance
Bellelli: if PCO2 of the patient's blood sample is less than 40 mmHg, when
the machine equilibrates with 40 mmHg CO2 the gas is absorbed: i.e. the new PCO2 becomes
40 mmHg and the total CO2 of the sample increases; as CO2 is the acid of the buffer, the
standard pH (in this case) decreases, whereas standard bicarbonate will slightly increase.

Professor I don't understand how we arrive to this formula: Accuracy =
sensitivity x prevalence specificity x (1-prevalence)
Bellelli: ok, this relationship is poorly explained in your text, I shall improve its explanation.
We use the following definitions:
prevalence = sick individuals / total population;
accuracy = (true+ + true-) / total population;
sensitivity = true+ / sick individuals = true+ / total population x prevalence;
specificity = true- / healthy individuals = true- / total population x (1-prevalence);
thus we can rewrite:
sensitivity x prevalence = true+ / total population;
specificity x (1-prevalence) = true- / total population;
accuracy = sensitivity x prevalence + specificity x (1-prevalence)

Why do we use RNA primer in PCR and not DNA primers? I thought the
beginning of the sequence of the gene segment that is going to be formed is made of DNA
Bellelli: DNA polimerases require the RNA primers that are synthesized by
the enzyme primase. DNA primers do not exist in vivo and would not be recognized by DNA
polimerases.
You mentioned that 34;Insulin causes the liver to release glucose from glycogen34; Does insulin not cause the liver to convert glucose to glyc
ogen instead? ,



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