CLINICAL BIOCHEMISTRY CLO 3&4 PDF

CLO4 Ion-selective electrodes IOn selective electrodes (ise): Are membrane electrodes that converts activity of specific ion dissolved in solution into an electrical potential. These include probes that measure specific ions and gases in solution. The most commonly used ISE is the pH probe or vo...

CLO4 Ion-selective electrodes IOn selective electrodes (ise): Are membrane electrodes that converts activity of specific ion dissolved in solution into an electrical potential. These include probes that measure specific ions and gases in solution. The most commonly used ISE is the pH probe or voltmeter. The use of Ion Selective Electrodes in clinical analysis offer several advantages over other methods of analysis. Advantages of ‘ise’ Low Few matrix Wide range of Accuracy & Measure both Unaffected by Used in aqueous cost, simple, modifications concentration/ precision levels of + & - ions sample colour solutions over a easy, and fast needed applications/ or turbidity wide temperature temperature +-2 or 3% range e.g. ideal for blood gas analysis Crystal membranes: Basic ise setup 0°C - 80°C plastic membranes: 0°C - 50°C - Meter Probe Various consumables (Capable of (Selective for each (Used for pH or ionic reading millivolts) analyte for interest) strength adjustments) list of the main areas in ISE uses: Pollution Monitoring: CN, F, S, Cl, NO3 etc., in effluents, and natural waters. Agriculture: NO3, Cl, NH4, K, Ca, I, CN in soils, plant material, fertilisers and feedstuffs. Food Processing: NO3, NO2 in meat preservatives. Salt content of meat, fish, dairy products, fruit juices, brewing solutions. F in drinking water and other drinks. Ca in dairy products and beer. K in fruit juices and wine making. Corrosive effect of NO3 in canned foods. Detergent Manufacture: Ca, Ba, F for studying effects on water quality. Paper Manufacture: S and Cl in pulping and recovery-cycle liquors. Explosives: F, Cl, NO3 in explosive materials and combustion products. Electroplating: F and Cl in etching baths; S in anodising baths. Biomedical Laboratories: Ca, K, Cl in body fluids (blood, plasma, serum, sweat). Education and Research: Wide range of applications. F in skeletal and dental studies. They are particularly useful in applications where it is only necessary to know that a particular ion is below a certain concentration level. Useful in biological/medical applications; they measure activity of ion directly, rather than concentration. Invaluable for the continuous monitoring of changes in concentration. How to achieve accuracy & precision - Careful use Frequent calibration Awareness of limitations The sensing part of the electrode is usually made of an ion- specific membrane, along with a reference electrode.  Types of ion-selective membrane Glass membrane Saturated calomel Crystalline membranes Ion-exchange resin Enzyme electrodes Good selectivity, but only electrode (SCE) Are made from mono- or membrane Are not true ion-selective for several single- Is a reference electrode poly-crystallites of a Are based on special electrodes but usually are charged cations; mainly based on the reaction single substance. organic polymer considered within the ion- H+, Na+, and Ag+. between elemental mercury membranes which specific electrode topic. and mercury (I) chloride. They have good contain a specific ion- Chalcogenide glass selectivity; only ions exchange substance Such an electrode has a has selectivity for which can introduce (resin). "double reaction" mechanism The aqueous phase in double-charged metal contact with the mercury and themselves into the - an enzyme reacts with a ions, such as Pb2+, the mercury (I) chloride is a crystal structure can The most widespread specific substance, and the interfere with the electrode, specially with and Cd2+. saturated solution of anionic selectivity. product of this reaction electrode response. potassium chloride in water. (usually H+ or OH-) is The glass membrane Usage of specific detected by a true ion- Selectivity of crystalline resins allows has excellent chemical selective electrode, such as a membranes can be for preparation of selective durability and can work both cation and anion of electrodes for tens of pH-selective electrodes. in very aggressive different ions, both the membrane-forming media. single-atom or multi- substance. e.g. pH glass electrode. atom. e.g. fluoride selective Low chemical, physical electrode based on LaF3 durability, and “survival crystals. time". e.g. potassium selective electrode, based on valinomycin as an ion- exchange agent.  CLO5 The metabolism of calcium, inorganic phosphate and magnesium Calcium: Is the most abundant mineral in the body. In an average man of 70 Kgs. The approximate weight of calcium compared to other minerals would be…… major functions - Bone Blood Neuromuscular Muscle Cellular contraction/ formation clotting activity metabolism nerve impulse (muscles & heart) transmission Calcium in I - Bone calcium has a mechanical role. Hydroxyapatite Plasma Body makes the bone rigid and strong and it acts 45% in free-ionized form (the physiologically active form) 99% in skeleton as a reservoir of 45% bound to proteins (predominantly albumin) 0.9 % intracellular calcium for the plasma. 10% complexed with anions (citrate, sulfate, phosphate) 0.1% extracellular Dietary sources - Milk Milk products They combine with calcium E.g. cheese, butter, yogurt, and ice-cream in the lumen of the intestine and form insoluble calcium- phosphate complexes. Digestion and absorption - Impaired; if ingested About 1 g of This happens in the Vitamin D is needed with foods containing calcium is ingested small intestine. for absorption. phosphates (PO4). per day but only e.g. Phytate found in 0.5 g is absorbed. cereals Osteoclasts: Cells seek out old bone or damaged bone tissue and destroy it, leaving small spaces. Osteoblasts: Cells use minerals like calcium, phosphorus, and vitamin D to fill in the spaces with new bone. Calcium & phosphorus The body continually replaces bone Hormones, PTH, Vit D, through a complex interaction among Calcitonin, etc minerals in the blood, particularly… Specialized bone cells called osteoclasts and osteoblasts Rickets: The mineral matter in bones is deficient due to a deficiency of Vitamin D. Therefore, he two flexible leg bones bend under the weight of the body. Osteoporosis: In older persons, the rate of bone break down exceeds that of bone formation. Symptoms include a reduction of height and a tendency to fracture. Vitamin D deficiency, or rickets, occurs only rarely in tropical climates where Comparison of sunlight is abundant, but it Microarchitectur was once common among e in Normal and children of northern cities Osteoporotic Bone: before the use of vitamin D- fortified milk. Acute radiation syndrome: When excessive amounts of radiation may be stored in bone tissue and initiate a cancer. Many radioactive elements have an affinity for bone. bone marrow is particularly susceptible to radiation. A single exposure of 25 roentgens produces a detectable drop of circulating lymphocytes (white blood cells). # of WBCs will be reduces. Then, RBCs. Both symptoms are directly related to damage done to the blood- forming tissue of bone marrow. The area around a break swells and discolors, but some fractures can be detected only by X ray. The weakened bones of the elderly are especially susceptible to fractures. 3 forms of calcium in plasma - Ionized calcium Albumin bound calcium Organic ion bound calcium (50%) (40%) (10%) Only ionised calcium directly Acts as a reservoir to replace E.g. ions such as citrate, effects neuromuscular activity. the ionised calcium if it drops lactate and bicarb (HCO3). too low in plasma. Also they controls PTH secretion. It is the physiologically active form it is important not to have of plasma calcium. prolonged venostasis when taking a blood sample for calcium analysis.; will increase plasma albumin levels that will increase plasma calcium levels. Parathyroid hormone (pth): A peptide hormone from the parathyroid glands that carefully maintain calcium levels in plasma along with vitamin D. PTH secretion: Stimulated directly by low plasma calcium; by acting on kidney and bone. Inhibited by high plasma calcium. Regulation of calcium (homeostasis): There is constant interchange of calcium ions between bone and that bound to albumin - not fixed permanently. Reference range: 2.20 - 2.55 mmol/L The free-ionized calcium stimulates the release of PTH. Kidney is an endocrine gland; this explains why we see drop in the plasma calcium in renal disease. 2 effects of pth on proximal tubes in kidney (low ca) - Reduces the clearance of Decreases the reabsorption of calcium via the tubules. phosphate at the proximal tubules. 1. It is first synthesized as pre-pro PTH. 2. It looses 25 amino acids to form pro-PTH. 3. It looses 6 more amino acids to form PTH.  Disturbances of calcium metabolism can be related to the concentration of free-ionized calcium, PTH and renal function. pth involved in calcium metabolism by - Releasing calcium Decreasing renal Decreasing reabsorption Promoting Vitamin D from bone clearance of calcium of phosphate in kidney Vitamin d (dhcc-dihydroxycholecalciferol): Is a group of fat-soluble seco-steroids responsible for increasing intestinal absorption of calcium, magnesium, and phosphate, and for many other biological effects. Is a pro-hormone that can be photochemically produced in skin (under sunlight) as well as ingested through diet (fats such Cheese, Milk, Fish.). Also, manufactured in the body when sterols migrate to the skin and become irradiated. Functions = Normal bone formation Retention of calcium & Protects the teeth & bones phosphorus in body against effects of low calcium in take 2 forms by making more effective use of calcium and phosphorus. The majority of vitamin D (up to 90%) is acquired Vitamin D2 Vitamin D3 via the action of sunlight (Ergocalciferol) (Cholecalciferol) on the skin, converting to vitamin D3. Naturally present in foods such as oily fish, milk, eggs and wild mushrooms. Vitamin D3, from all sources, then under-goes two hydroxylation processes, in the liver & kidneys, to become the active form vitamin D.  Vitamin D is derived from cholesterol. → Is transported to the liver in the blood. → An -OH group is added. Vitamin D is made in three different sites: The Skin, Liver and Kidney. → Another hydroxyl group os added. Inappropriately high PTH in plasma: 1. Primary hyperparathyroidism. 2. Tertiary hyperparathyroidism. Low plasma PTH levels are found in Causes of calcium disorders 3. PTH from ectopic sites. Appropriately high PTH in plasma 1. Hypoparathyroidism - when plasma free-ionized calcium 2. Hypercalcaemia levels fall below normal. Hypercalcemia Hypocalcemia Total plasma calcium level should be interpreted by referring to the plasma albumin level. Pagets disease: A chronic disorder that typically results in enlarged and deformed bones. Thiazide diuretics: Are mainly used to treat high blood pressure, these drugs lower calcium excretion by the kidney. Excessive vitamin d consumption causes - Vitamin poisoning Kidney damage Lethargy Loss of appetite Magnesium: Is the second most abundant intracellular cation. Over 50% of the body magnesium is in the bone. About 1% is found in extracellular fluid (20-30% bound to protein; the rest free). The rest is in soft tissues, particularly muscle. The kidney controls the homeostasis of magnesium. The Plasma Reference Range: 0.74 - 1.00 mmol/L Dietary sources of magnesium: Vegetables & plants. Digestion and absorption - The daily intake of Magnesium is absorbed Excretion occurs magnesium is about in the small intestine mostly in urine. 250 mg/day. and large intestine. Only small amounts are excreted in the faeces. distribution magnesium in plasma - Concentration Plasma PTH may effect If plasma level of Magnesium moves of magnesium in magnesium is it’s absorption magnesium falls in and out of cells > plasma kept within very and excretion. too low, seizures cells = with potassium narrow limits may occur. bone = with calcium (0.70 - 1.00 mmol/L).  Hypomagnesemia (etiology) - Renal causes Extrarenal causes 1. Lack of magnesium in the diet. 2. Increased losses via the GIT due to malabsorption, chronic diarrhea or laxative abuse. 3. Increased cell uptake of magnesium is associated with alcohol withdrawal syndrome. 4. Increased storage in bone is seen after parathyroidectomy. Symptoms of Magnesium deficiency Rapid heart Weakness Muscle spasm Disorientation Nausea, Increase risk Develops beats vomiting and of slowly; we seizures osteoporosis store Mg people at risk - People using Those in very hot People with long Alcoholics. Diabetics. diuretics; they climate who spells of diarrhea increase Mg in sweat a lot. or vomiting. urine. Causes of - Magnesium deficiency Magnesium excess Small bowel resection: Surgical removal of a part of small intestine. Inorganic phosphorous: Phosphorus ion is mainly an intracellular ion. whereas Ca ion is mainly an extracellular ion. Bones contain > 85% of total body phosphorus [>99% of total body calcium both are present in hydroxyapatite (calcium phosphates)]. Organic phosphorus is present in - Cell membranes Nucleic acids Energy storage Glucose-6-phosphate (Phospholipids) (Adenosine Triphosphate - ATP) What influences phosphorus levels - Diet PTH Renal function phosphorous regulation: Parathyroid Hormone (PTH) affects phosphorus levels by influencing renal excretion of phosphorus and increasing calcium reabsorption in response to decreasing calcium levels in the extracellular fluid. Mobilisation of phosphorus from the bone is influenced by PTH levels. Calcium and phosphorus have an inverse relationship. phosphorous and kidney: Excretion by the kidney is controlled primarily by an overflow mechanism: The renal tubules have a normal transport maximum for reabsorbing phosphate of about 0.1 mM/min. When less than 0.1 mM/min is present all filtered phosphorus is reabsorbed. When more 0.1 mM/min is present the excess is excreted. Both calcium and phosphorus are absorbed in the upper small intestine. Vitamin D stimulates the intestinal absorption of Pi. PTH decreases the proximal tubule reabsorption of Pi. The calcium and phosphate levels in the blood are kept in careful balance by the body’s hormones and chemicals. This balance is like a see-saw (as one rises, the other often falls ).  Variations - Clinical variation Analytical variation within an individual and no test is perfect. All tests have some between individuals degree of variations for repeated measurements of the same sample. Final test results affected by factors - Pre-analytically At the time of test After test is completed Why analytical results vary Inter-individual Intra-individual Pre-analytical Analytical Post-analytical variation variation variation variation variation Age Diet Transport Random errors Transcriptions errors Sex Exercise Exposure to UV light Systematic errors Results reported to Race Drugs wrong patient Standing time before Genetics Sleep pattern separation of cells Long term health Posture Centrifugation time status Time of venipucture Storage conditions Length of time tourniquet is applied  Pre-analytical errors - Collection Identification – Was the right tube used? – Was the blood collected from the correct patient? – Was venipuncture performed correctly? – Was the blood correctly labeled? – Was the specimen properly stored? Patient name, ID, date, time of collection, phlebotomist The lab is required to have a clear and rational policy for identifying specimens, and handling misidentified specimens. Prolonged venous stasis: Blocking the flow of blood with the tourniquet will eventually lead to a sieving effect. Small molecules, water and ions are forced out blood vessels and larger molecules are concentrated. Increases Total Protein, proteins, iron (Fe), cholesterol, bilirubin. Decreases potassium. Supine vs sitting or standing: Going from lying (supine) to upright reduces total blood volume by about 700 ml. The following may decrease by 5-15% in the supine patient: –Total protein –Albumin –Lipids –Iron –Calcium –Enzymes Hemolysis: Rupture of red blood cell. Can be due to improper collection. End result is dumping cellular contents into blood. Mild dilution effect in some analytes. Significant increase in potassium, magnesium, phosphorous.