MEDICINE AND SURGERY "F" Course of LABORATORY MEDICINE Disturbances of water and electrolyte balance WATER AND ELECTROLYTES 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.       Water and electrolytes are subject to a rapid turnover due mainly to dietary intake and renal excretion. Other causes of water (and electrolytes) loss are faeces, respiration and sweating. Under pathological conditions water and electrolyte loss may occur because of several additional reasons that include: diarrhoea (e.g. cholera, bacterial enteritis or cholitis); vomiting, inability of the kidney to concentrate the urine (e.g. diabetes mellitus, diabetes insipidus), etc.       As a general rule, water is absorbed or excreted passively, thus it follows the main extracellular electrolytes, sodium and chloride, and water deficits usually occur together with sodium and chloride deficits. It is important to remark, however, that the intracellular water compartment is usually most affected and that symptoms may occur also under conditions in which the water and electrolyte deficits one may estimate from blood composition are relatively mild. The central nervous system is particularly sensitive to water and electrolyte unbalance and symptoms (e.g. irritability, convulsions, coma) may be severe.       Water accounts for approx. 65% of body weigth, and is divided in three compartments: blood plasma, extracellular fluids and intracellular fluids. The clinical laboratory can measure the volumes of blood; blood plus extracellular water; and total body water (TBW). We cannot measure the volume of intracellular water alone, but we can easily estimate it from the difference between TBW and blood plus extracellular fluid, if needed. The normal reference values (as percentages of body weigth) are as follows: blood 4%; extracellular water 16%; intracellular water 45%.       The principle of water measurement is the same for all compartments to be tested. To measure blood water (this means plasma volume, because intraerycthrocytic water belongs to the intracellular water) one proceeds as follows: a minimum amount of a suitable tracer (e.g. serum albumin labelled with radioactive cromium) is injected in a peripheral vein. After a few minutes a blood sample is taken from a different vein, in order to avoid contamination by the tracer that may be present on the skin, and its concentration is measured. The tracer, whose initial amount is known, is diluted in the plasma and the plasma volume can be calculated as follows (as you may remember from the course of Chemistry): volume = amount of tracer / concentration       Other compartments are measured exactly in the same way, but using tracers that evenly distribute in the compartment we are interested in. However no tracer distributes in the extracellular or intracellular compartment alone, and this explains the above point that one cannot measure the volume of intracellular or extracellular fluid alone. The characteristics of a good tracer are: (i) it evenly distributes in the selected compartment(s); and (ii) its concentration is easily measured. The timing is also important to guarantee the even distribution of the selected tracer (e.g. if waiting too long, Cr-albumin will partly extravasate and distribute in the extracellular fluid; but it is not a good tracer for this measurement because it attains different concentrations in the plasma and extracellular fluid). Audio: Water in our body       In practice the determination of TBW and other volumes is seldom carried out, because it has little diagnostic value; a much more important parameter to measure is serum osmolarity. If osmolarity is normal, excess water can be well tolerated, as it happens in oedematous or ascitic patients. The osmolarity of human body fluids is around 300 mOsMoles/L and is strictly regulated; changes of about 2% induce strong responses (thirst, and changes in the secretion of ADH). A change of serum osmolarity of about 10-15% may cause the hyperosmotic coma, a potentially lethal condition observed, for example in untreated type 2 diabetes mellitus, with glycemias >400 mg/dL. The principal osmolytes in body fluids are low molecular weigth ions: in the extracellular fluids the three most concentrated ions are sodium, chloride and bicarbonate, which account for 260 mOsMoles/L. In the intracellular fluids potassium largely replaces sodium. A practical approximate formula to calculate serum osmolarity is as follows: 2 x ([Na+] + [K+]) + [BUN]/2.8 + [Glucose]/18 where [Na+] and [K+] are expressed in mEq/L, whereas [BUN] (blood urea nitrogen) and [Glucose] are expressed in mg/dL. Audio: Water losses COMBINED DEFICIT OF SODIUM AND WATER: Loss from GI tract: vomiting, diarrhoea, medical practices (e.g.: colostomy) Loss form the skin: excess sweating Loss due to hemodialysis or peritoneal dialysis Acute or chronic renal failure: "salt wasting renal disease" Excess diuretic therapy Diabetes mellitus Diabetes insipidus (insufficient production of ADH) Adrenal disease: Addison disease (glucocorticoid deficiency); hypoaldosteronism       Measurement of electrolyte concentration in the blood is usually effected by spectroscopic or potentiometric methods, using the appropriate electrodes. Other methods are less commonly employed, e.g. flame atomic absorption.       Hyponatremia is a condition in which sodium losses exceed water losses; decrease of the electrolyte concentrations and hypo-osmolarity follow. This condition may occur in several types of renal failure, in heart failure because of the sodium retention in peripheral oedema, in several endocrine disease and in neoplastic diseases, in the course of diuretic therapy. An uncommon but important cause is the (usually paraneoplastic) syndrome of inappropriate secretion of antidiuretic hormone (ADH). Water intoxication occurs when the plasma osmolarity falls below 240 mOsM/L (note: some laboratories measure osmolalities instead of osmolarities; to a first approximation the values are the same, i.e. 240 mOsM/L more or less corresponds to 240 mOsm/Kg).       Hypernatremia (i.e. increased concentration of serum electrolytes, especially sodium) is typically due to water deprivation or increased and unreplaced water losses (e.g. profuse sweating). It requires prompt rehydration, in severe cases by intravenous administration of glucose isotonic solution (one cannot infuse distilled water because the local hypotonic shock would kill the blood and endothelial cells; infusion of an isotonic 5% glucose solution achieves the same effect of distilled water because the sugar is promptly removed by the liver, but avoids local damage).       Vasopressin (antidiuretic hormone, ADH) is a 9-aminacid residues polyeptide hormone produced in the hypothalamus and secreted by the posterior pituitary. The stimulus to ADH secretion is hyperosmolarity. ADH has two main effects: in the kidney it promotes reabsoprtion of water, sodium and urea, thus reducing the diuresis; at the level of the arterioles it causes constriction and increases the blood pressure. Increased ADH secretion (e.g. because of a benign tumor of the neurohypophisis) causes water and sodium retention; reduced secretion (e.g. because of a compressive lesion in the region of the sella turcica that destroys the gland) causes diabetes insipidus with polyuria and hypernatremia. Diabetes insipidus may also be of nephrogenic origin (the kidney fails to respond to ADH; usually due to chronic affections of the kidney e.g. polycystic kidney disease).       Potassium concentration is low in the serum (4-5 mEq/L) but high in the cell cytoplasm (140 mEq/L). This causes the body to have a huge reserve of this cation. All cells possess a sodium-potassium pump that exchanges these two cations (intracellular sodium is low, serum sodium is high, at 135 mEq/L). The regulation of serum potassium is very strict, because both hypokalemia and hyperkalemia cause severe cardiac arrhythmias and may result lethal. Potassium is abundant in the diet, but is efficiently excreted by the kidney, which is the principal regulator of its serum concentration. Potassium balance is regulated by hormones: 1) after a meal glucose and potassium concentrations in the serum increase. This causes the secretion of insulin that promotes the cellular uptake of both glucose and potassium (see the lecture on glycemia). Thus insulin lowers the potassiemia by causing this cation to be absorbed in the cell sytoplasm (most notably in the muscle cells). 2) Catecholamines regulate the cell absorption and secretion of K+: α-adrenergic receptors impair cell absorption of potassium, whereas β-adrenergic receptors stimulate this function. 3) Fine regulation of sodium and potassium in the serum is mainly operated by aldosterone, a steroid hormone produced by the adrenal cortex. The hormone promotes the exchange of Na+ with K+ in the distal tubule; this causes reabsorption of sodium from the urine and secretion of potassium in the urine. Ultimately aldosterone increases serum Na+ and lowers K+. The effects of aldosterone are antagonized by the atrial natriuretic peptide. For a review on serum potassium regulation you may check this link.       Hypokalemia is due to excessive loss of potassium in the urine, faeces or sweat. Renal waste of potassium occcurs in Bartter syndrome (a disease of unknown origin) and in the presence of excess secretion of mineralocorticoid hormones such as Cushing disease or primary hyperaldosteronism (e.g. because of a benign tumour of the adrenal glands) and in primary disturbances of the kidney involving the proximal and distal tubuli. It may be iatrogenic, due to excess diuretic therapy (e.g. using thiazides or furosemide) and it is always advisable in prolonged diuretic therapy to associate a K-saving diuretic (e.g. spironolactone). Normal potassiemia is 5 mEq/L and in severe depletion (3 mEq/L or less) muscular weakness, and severe cardiac arrythmias, both due to the fact that excitable tissues function is compromised if the intra- extra-cellular potassium gradient is altered. Severe hypokalemia requires (careful !) intravenus administration of potassium.       Hyperkalemia may occur in type I diabetic patients or in the course of severe kidney disease with reduction of the GFR (e.g. glomerulonephritis, acute renal failure), given that the kidney excretes potassium very efficiently and, in the absence of severe disfunction, prevents or corrects this condition. Another possible important cause of hyperkalemia is adrenal insufficiency (Addison's disease). Pseudohyperkalemia is the transient increase of potassium concentration due to release of intracellular potassium by the red or white blood cells or by platelets. True hyperkalemia is a dangerous condition that requires prompt treatment, given that at potassiemia > 6.5 mEq/L severe arrythmias occur and ventricular fibrillation is possible. Hormones regulating sodium and potassium hormone target organ(s) effect on sodium effect on potassium aldosterone (adrenal cortex) kidney retention increased excretion; lowers serum K+ atrial natriuretic peptide kidney increased excretion retention; increases serum K+ antidiuretic hormone (ADH; neurohypophysis) kidney causes water retention; dilutes all electrolytes insulin most cell types   increases potassium import in cells; lowers serum K+ catecholamines / α-receptor muscle cells and other cell types   reduces cell uptake; increases serum K+ catecholamines / β-receptor muscle cells and other cell types   increases cell uptake; reduces serum K+       Disturbances of Calcium metabolism. The concentration of calcium in the blood serum in the healthy adult is approx. 2.5 mMoles/L (or 5 mEq/L or 10 mg/dL) and is strictly regulated by two hormones and one vitamin: the Parathyroid hormone (PTH) is a protein secreted by the parathyroid glands; it causes release of calcium from the skeletal deposits, and reabsorption from the urine and from the gut, thus causing the calcemia to increase. Thyrocalcitonin is a protein hormone produced by the parafollicular cells of the thyroid and causes calcium phophate deposition in the bone matrix, opposing the effect of PTH. Vitamin D promotes absorption of calcium from the diet. The serum concentration of calcium is carefully regulated because this ion is essential for the excitability of nervous and muscular tissues and changes in its concentration cause severe symptoms.       Calcium distribution in the body The human body contains approx. 1 kg (25 moles) of Calcium in the bone matrix, as calcium phosphate; the extracellular fluids contain approximately 22 mMoles of Calcium (9 mMoles in the blood). The intracellular fluids contain extremely low concentrations of the ion. The daily turnover is approximately 5 mMoles; loss is via the urine and the feces (via secretion in the bile), absorption occurs in the small intestine. Approximately 10 mMoles of Calcium are exchanged every day between the extracellular fluid and the bone matrix. The normal calcium concentration in the serum is 2.5 mM, of which approx. 70% is present as the free ion and the remaining 30% is bound to serum proteins.       Absorption of Calcium occurs in the small intestine and is stimulated by calcitriol, an hormone derived from vitamin D. The metabolic pathway is as follows: The final step of the conversion of vitamin D to calcitriol occurs in the kidney under the stimulus of parathyroid hormone (PTH), whose secretion is stimulated by hypocalcemia.       The parathyroid hormone (PTH) is a small protein produced by the parathyroid glands as a response to low calcium concentration in the serum. The effects of PTH are meant to increase the concentration of calcium via: (i) increased absorption in the gut (mediated by calcitriol, see above); (ii) mobilization of the ion from the bone (demineralization); and (iii) increased reabsorption from the urine in the kidney.       Thyrocalcitonin is a small protein hormone produced by the thyroid as a response to high calcium concentration. It antagonizes the effects of PTH and promotes calcium deposition in the bone.       Hypocalcemia is an infrequent finding; it may depend on several causes, among which: (i) hypoparathyroidism (often associated to surgical removal of the parathyroid in the course of a thyroidectomy); (ii) vitamin D deficiency (e.g. rickets); (iii) renal tubular disease or renal failure; (iv) acute pancreatitis (calcium chelation by lipolytic products). Clinical symptoms include reduced cerebral function (pseudo-dementia), possibly with psychotic symptoms, and muscular tetany. Hypercalcemia is usually caused by hyperparathyroidism (often of neoplastic origin), and is associated to excessive bone matrix reabsorption (osteoporosis). Other neoplastic diseases, unrelated to the parathyroids, can cause osteolysis with hypercalcemia and osteoporosis, because of secretion of osteoclast activating factors (so called "humoral hypercalcemia of malignancy"). Hypervitaminosis D is another possible cause of hypercalcemia.       Osteoporosis is a condition caused by chronic loss of calcium phosphate in the bone matrix. It is frequent in the elederly and may lead to increased risk of bone fractures. Calcemia is usually normal, but calcium absorption in the gut is usually impaired, leading to a negative balance of the ion.       A list of important laboratory tests for calcium and the hormones that control its serum concentration is reported in the lecture on endocrine diseases.       Hypophosphatemia. Calcium phosphate is the main mineral component of the bone tissue, and mobilization of phosphate usually follows that of calcium: e.g. PTH causes reabsorption of both calcium and phosphate. However, in the intracellular milieu and in the diet calcium and phosphate have different distributions and thus changes in the serum concentrations of phosphate does not necessarily follow those of calcium. Hypophosphatemia is not uncommon but rarely severe or even symptomatic. The main cause is reduced renal reabsorption.       Disturbances of Magnesium metabolism. In the healthy adult the serum magnesium concentration is approx. 2 mEq/L and is regulated mainly at the level of urinary and fecal excretion. Hypomagnesemia may result from prolonged poor dietary intake or reduced intestinal absorption (diarrhoea, malnutrition, etc.) it may be aggravated by some physiological conditions of excess consumption (e.g. lactation). Hypermagnesemia may occur, together with other electrolyte disturbances, in chronic or acute renal failure.       The importance of chloride. Chloride is mainly present in the extracellular fluids, and attains low concentration in the cell cytoplasm. It has however a great importance for clinical reasoning and its concentration in the serum should be looked at with great attention. Our reasoning on chloride is based on three premises: (i) the osmolarity of body fluids is essentially constant; and (ii) the sum of positive charges equals that of negative charges (electroneutrality principle); (iii) the concentration of cations is quite strictly regulated, as is that of bicarbonate (see below, blood buffers). The kidney excretes or reabsorbs chloride in order to obey the above premises; thus chloride is the variable parameter that the kidney adjusts to compensate for changes in the concentration of other electrolytes. Changes in chloride concentration may be unimportant per se but tell us important information about electrolytes that were not directly measured. In particular: hypochloremia in the presence of normal sodium concentration suggests an increase of non-measured anions (e.g., bicarbonate, lactate, ketoacids); hyperchloremia suggests a decrease in bicarbonate. Alterations of chloride concentration are a strong indication to carry out a hemogas analysis. Caution: a variation of chloride concentration suggests an opposite varation of bicarbonate, hence a disturbance of blood pH, but a normal chloride concentration does not exclude an alteration of blood pH: e.g. in acute respiratory acidosis chloride and bicarbonate may be within normal limits, yet the pH change may be severe! Reference electrolyte concentrations in human serum Sodium 135 mEq /L Potassium 5 mEq /L Calcium 5 mEq /L Magnesium 2 mEq /L Chloride 100 mEq /L Bicarbonate 26 mMoles /L Disturbances of electrolyte concentrations and of osmolarity ma be rapidly fatal! EVALUATION OF KIDNEY FUNCTION       The kidney plays a fundamental role in several physiological functions: function possible symptom(s) laboratory tests (i) production of urine; elimination of water-soluble waste products; resorption of useful solutes (e.g. glucose; aminoacids; electrolytes); this function actually requires two separate steps, each of which can be considered a function in itself: filtration (glomerular function) and secretion/resorption (tubular function) oedemas; palpebral oedema; mental confusion (due to increased BUN) measure urine production in 24 hours; GFR; loss of useful solutes in the urine (proteins, glucose, bicarbonate, aminoacids, etc.); the tubular function can be measured by the excretion of phenolsulfonphthalein a tracer that is not only fileterd by the glomerulus but also secreted by the tubuli (ii) homeostasis of total body water and osmolarity; control of the concentrations of sodium, potassium and the majority of other electrolytes symptoms of electrolyte imbalance: fatigue, weakness, muscle contraction, coma measure serum osmolarity and serum electrolytes (iii) Acid-base homeostasis (long term control of bicarbonate) possible renal tubular acidosis or other types of acid base imbalance (iv) Regulation of the arterial pressure (via control of sodium and water, and via the renin-angiotensin system) low arterial pressure; weakness; lipothymias measure blood pressure, serum concentration of angiotenisn II can be measured by radioimmunoassay, but this test is rarely carried out (v) Nitrogen balance; elimination of nitrogen (as urea, creatinine, ammonia and uric acid) increased BUN may cause mental confusion measure BUN, creatinine, ammonia, and uric acid; Reference values for low molecular weigth nitrogen-containing compounds in human plasma: urea < 20 mg/dL by nitrogen (< 7 mMol/L); creatinine < 1.2 mg/dL (< 110 μMol/L); ammonia < 50 μMol/L (in newborns and infants < 150-200 μMol/L); uric acid and urate ion < 7 mg/dL (v) Erythropoiesis and erythrocyte count via the biosynthesis of erythropoietin (EPO) anemia; weakness, palor, tachycardia measure red cell count; hemoglobin content; EPO by radioimmunoassay (vi) Elimination of water-soluble xenobiotics (e.g. drugs) possible drug toxicity measure GFR       Kidney diseases (e.g. glomerulonephritis, renal failure, etc.) may affect all these functions; usually blood urea nitrogen (BUN) is the first to be affected. Important pathological alterations occurring in later stages of chronic kidney diseases include anemia (because of reduced production of erythropoietin), or more rarely polycitemia; hypertension; and loss of bicarbonate with metabolic acidosis.       The fundamental parameter that measures the glomerular function is the glomerular filtration rate (GFR), i.e. the measurement of the volume of plasma filtered by the kidney in 1 minute. The median value for healthy adults is 120 mL/min, but this parameter decreases with age, and values of 60 mL/min in elderly patients should not be considered pathological.       To measure the GFR the physician should select a tracer that is entirely filtered by the glomerulus and neither reabsorbed nor secreted by the tubuli. For experimental studies the polysaccaride inulin is used, but in the clinical practice the physiological solute creatinine is routinely employed because it is physiologically produced at a constant rate by the accidental degradation of creatine in the striated muscle.       Creatine and creatinine. Creatine is synthesized by the liver starting from arginine, glycine and S-adenosilmethinine. The daily production of creatine is approximately 1 g/die. This compound is used mainly by the skeletal muscle as a reservoir of high energy phosphate bond, in the form of phosphocreatine (or creatine phosphate), that can rapidly and anaerobically replenish ATP (for burst energy consumption). Phosphocreatine may undergo spontaneous conversion to the terminal metabolyte creatinine, which cannot be utilized by the muscle and is secreted in the blood and eliminated in the urine. The formulas and reactions are as follows:       Creatinine is the second most important low-molecular weight nitrogen containing compound in the blood. In the healthy adult its serum concentration is 0.5-1.2 mg/dL (or 50-110 μMol/L).       The measurement of GFR using creatinine as a tracer is as follows: creatine concentration is determined in a venous blood sample and in the urine collected over a short period of time (by cateterism, if necessary). The amount of plasma filtered in the collection time is given by the formula: GFR = Volumeurine x [creatinine]urine / [creatinine]plasma x time The measurement of creatinine is easily achieved using the classical Jaffe' reaction (formation of a red-coloured complex with picric acid):       The tubular function is estimated by measuring the amoung of useful solutes lost in the urine collected over 24 hours (glucose, sodium, potassium, bicarbonate, phosphate, aminoacids). These amounts should be considered in relation to the plasma concentration of the same solutes; e.g glucose may appear in the urine because of excess filtration if the patient has hyperglycemia (diabetes mellitus) or because of impaired resorption is glycemia is normal (nephrogenic mellituria). Glomerular and tubular function are often both lost at the same time, but usually not to the same extent; thus in some diseases the reduction of GFR may be predominant and the tubular function may be maintained (e.g. glomerulonephritis); in other diseases the opposite may occur (e.g. in mercury and other metal poisoning). Clinical examples 1) 80 year old patient complainng of nausea, tiredness, weight loss, episodes of mental confusion. Blood test reveals: hemoglobin 7.8 g/dL * erythrocytes 3 x 106 /mmc * white cell count 6.3 x 103 /mmc platelets 250 x 103 /mmc sodium 136 mEq/L potassium 4.8 mEq/L calcium 4 mEq/L BUN 130 mg/dL (46 mMol/L) * creatinine 7.8 mg/dL * glucose 84 mg/dL Analysis of the case: the main findings are anemia, and strongly increased BUN and creatinine (asteriscs). This suggests chronic kidney failure, a diagnosis that is fully compatible with the symptoms. BUN and creatinine are increased because of diminished urinary excretion; anemia is likely due to reduced production of erythropoietin. Suggested next step is measuremente of GFR (expect significant decrease), and possibly echography or NMR of the abdomen (expect shrinked kidneys). Hemodialysis may be indicated. 2) In the last few months vomiting and weight loss. The serum electrolytes are as follows: sodium 130 mEq/L potassium 3.0 mEq/L* calcium 4.8 mEq/L chloride 80 mEq/L * bicarbonate 40 mEq/L * phosphate 1.2 mMol/L /mmc Analysis of the case: prolonged vomiting causes loss of hydrochloric acid and metabolic alkalosis (hence low serum chloride and high bicarbonate), and loss of cations (hence hypokalemia). Investigate the stomach to look for gastric cancer or gastric ulcer, causing a stricture of the pylorus. NMR or gastroscopy are indicated. Carry out a complete screen of serum enzymes. 3) Acute trauma; crushing syndrome. The serum electrolytes are as follows: sodium 135 mEq/L potassium 7.5 mEq/L* calcium 3.2 mEq/L* chloride 95 mEq/L bicarbonate 15 mEq/L * phosphate 1.8 mMol/L Analysis of the case: massive destruction of muscle tissue releases intracellular potassium. Phospholipids from the cell membrae become clogged in the renal tubuli causing acute kidney failure. Restore electrolytes; hemodialysis indicated. Caution: this is a life-threatening condition! Further readings 1) Gumz ML, Rabinowitz L, and Wingo CS An Integrated View of Potassium Homeostasis N Engl J Med. 2015; 373: 60–72. Questions and exercises: 1) The osmolarity of biological fluids is: 150 mOsM 300 mOsM 450 mOsM 2) Indicative concentration values for the three major ions in human blood are: sodium=135 mEq/L; chloride=100 mEq/L; bicarbonate=26 mM sodium=135 mEq/L; bicarbonate=100 mEq/L; chloride=26 mM chloride=135 mEq/L; sodium=100 mEq/L; bicarbonate=26 mM 3) Potassium and calcium are very important ions regulating the membrane potential of nerve and cardiac cells. Their normal serum concentration is: 5 mEq/L for both 2.5 mEq/L for both 5 mEq/L for K, 2.5 mEq/L for Ca 4) The serum concentrations of urea and creatinine in a healthy adult are < 40 mg/dL and < 2 mg/dL < 20 mg/dL and < 2 mg/dL < 40 mg/dL and < 1 mg/dL your score: 10 Attendance not registered because of insufficient score - Retry! 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 Slides for this lecture: