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Dr.F.Iranmanesh

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Calcium,Physiologic chemistry Distribution: 5 th most common element Most prevalent cation in the body Healthy adult contain 1-1.3kg of calcim,99% in the form of hydroxyapatite,1% in ECF & Soft tissue Serum(Plasma) calcium exists in three forms: 1:Free(Ionized) #50% 2:Complex with anions #10% 3:Bound to plasma proteins#40%,Mostly Albumin,80%

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Calcium binds to negatively Charged sites of proteins,so dependent to PH & Protein cncentration. Alkalosis : binding so decreased free ca. Acidosis : Binding so Increased free ca. [Ca++][pr--]/[Capr]= Hostings &Mclean 1939 [Ca++]=[pr--]/[Capr]

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Calcium Function mineralization Blood coagulation Neural transmission Maintenance of normal tone and excitabilityof Skeletal and cardiac muscle. Glandular synthesis and regulation of exocrine & endocrine glands. Preservation of cell membrane integrity and permeability.

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Calcium intake Average dietary Intake : 600-800mg/Day Recommended 1200 mg during preg.& Lactation and 800-1200 mg during childhood. Ca absorption : Active transport in Duodenum and upper jejunum.(50%) Increased in pregnancy, lactation and rapid growth and decreased with advanced ages. Major stimulus of ca. absorption is vitamin D.

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Absorption enhanced by Growth hormone,acid medium,incresed protein intake. Decreased with:Ca/phos ratio >2 Phytic acid,Oxalate,Fatty acids,Cortisol, Excessive alkalinity of intestinal contents.

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Ca Excretion Sweath:15-100mg/day Major loss:Urine 100-200mg/day Wide variation in intake has little effect on U.Excretion Enhanced by: Acidosis,hypercalcemia,phosphate deprivation and glucocorticoids. Decresedby PTH,Diuretics,VitaminD

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Kidney Parathyroid Liver Thyroid c cells Intestine Hypocalemi a PTH 255255 Hyper ca Phosphorus Urine PT H Ca++ 25-OH-D3 1,25(OH)2D3 Ca++ 1,25(OH)2D3 Calcitonin Calcium Homeostasis

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Analytical techniques : Total Calcium Clark and collip method Today 3 methods: 1)Colorimetric analysis 2)Atomic absorption spectrometry(AAS) 3)Indirect Potentiometry

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Colorimetric Metallochromatic indicators: O-Cresolphthalein complexon(CPC) Red color in alkaline solution. Measured at 580nm. Addition of 8 -hydroxyquinolone: Mg. Arsenazo III,Ca-indicator complex: Measured at 650nm High specificity at slightly acidic PH Hemolysis,lipemia,icterus,paraproteins and Mg intrfere with colorimetric methods.

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Calcein forms fluorescent complex Stimulates at 490nm & emits at 590nm Titration of complex with EDTA AAS is the reference method Dilution with Lanthanum hydrochloride to reduce viscosity and interference from proteins and organic and inorganic ions. Ind.Potentiometry:An electrode selective for ca.measures a sample that is also measured against a Na selective electrode.

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Analytical techniques Ionized calcium Ion selective electrodes(ISE) Accurate,precise,automatic determination of ionized(Free)Ca. Consists of a membrane separating a reference solution (CaCl2,AgCl)and a reference electrode(Ag/AgCl or calomel) from the solution to be analyzed.

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Reference intervals Total calcium Total ca. in adults 8.8-10.3mg/dl(2.20-2.58mmol/L) Serum is the preferred Specimen Heparinized plasma is also acceptable. Citrate,Oxalate,EDTA interfere with commonly used methods. Hemolysis,icterus,lipemia,paraproteins and Mg interfere with colorimetric methods. Total ca.corrected for hypoalbuminemia=total ca(measured)+[(Normal Albumin-patient,sAlb.)x0.8] Normal albumin=4.4

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Reference interval Ionized calcium 4.6-5.3mg/dl(1.16-1.32 mmol/L) Whole blood,Heparinized plasma or serum are acceptable. Specimens should be collected anaerbically and transported on ice and stored at 4C to prevent loss of CO2 and glycolysis and stabilize PH.

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Reference interval Urinary calcium Varies with diet Average 300mg/day Urine collection with appropriate acidification to prevent calcium salt precipitation.(15 ml hydrochloric acid)

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Phosphorus Physiologic chemistry Adult body content :700mg 85% in Skeleton(Inorganic),15% in ECF & soft tissue(Organic) In blood,Plasma(Inorganic),cells (Organic) In serum ratio of H2PO4-:HPO4-- is pH dependent. 1:1 in acidosis,1:4 in pH 7.4,1:9 in alkalosis. Serum phosphorus 10% bound to proteins,35% complex with Na,calcium;Mg and 55% free. Only inorganic ph.is measured in routine.

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Function Skeleton Intra & extracellular role. Nucleic acid,phospholipid,phosphoproteins ATP and NADP.In various enzyme systems(Adenylate cyclase) Essential for normal muscle contractility,Neurologic function,Electrolyte transport and oxygen carrying by Hb.

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Phosphorus homeostasis Present in virtually all foods. Average dietary intake 800- 1400 mg/day. 60% -80% of intake is absorbed mainly by passive transport.Active transport stimulated by 1.25(OH)2D3 Freely filtered in glomerulus. >80% reabsorbed in proximal tubule and smaller in distal tubule. Proximal transport:(Na-P cotransport)mainly regulated by ph.intake and PTH. PTH inhibits Na-P Cotransport and causes phsphaturia.

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Reference intervals Adults:2.8-4.5 mg/dl(0.89-1.44 mmol/L) Higher in growing children(4.0-7.0) Serum phosphate has DIURNAL VARIATION. Higer levels in afternoon and evenings. Best measured in FASTING MORNING. Levels are influenced by dietary intake,meals,and exercise.

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Analytical techniques Reaction of inorganic phosphate with ammonium molibdate to form phosphomolibdate complex measured at 340 nm in autoanalyzers. Complex can be reduced to form molibdenum blue measured at 600 to 700 nm. Enzymatic methods. Serum is preferred. Most anticoagulants(Except heparin) interfere Prolonged storage with cells at room temperature causes Ph. Hemolyzed specimens are Unacceptable (RBC organic esters hydrolize to inorganic phosphate during storage.)

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Disorders of mineral metabolism Hypercalcemia Serum ca is associated with: Anorexia,Nausea,vomiting,Constipation,hypotonia,depression,high voltage T waves on ECG,lethargy,coma Persistent hyperca. Causes ectopic deposition of ca(vessels,connective tissue ad joints,gastric mucosa,kidney) Most common causes:Primary hyperpara,Malignancy Others :Renal Failure,Diuretics,Endocrine disorderes,Vitamin A and D intoxication,Lithium therapy,Milk alkali synd.,immobilization,Hyperthyroidism,familial hypercalciuric hypercalcemia.

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Primary Hyperparathyroidism(PHPT) PTH in the absence of an appropriate physiologic stimulus causing generalized disorder of Ca,Ph,Bone metabolism. 100,000 case/Year in USA F/M : 2/1 Majority caused by solitary parathyroid adenoma. Others:Multiple adenoma,Hyperplasia,Rarely carcinoma. Ca,Phosphate,Mild acidosis(Renal Bicarbonate reabsorption) Ca due to :1)Direct action PTH on Bone,increased resorption.2)PTH activated renal reabsorption 3)PTH stimulated increased renal biosynthesis of 1,25(OH)2D3 which increases intestinal calcium absorption or more are asymptomatic.

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Hyperparathyroidism PHPT:Sporadic MEN1 (Pituitary &pancreas tumors,Zollinger Ellison synd.)MEN2A(Pheo. &Medullary CA of thyroid.) Secondary Hyperparathyroidism: Resistance to PTH: RF,VIT D deficincy, Low to normal Ca,High phosphate. Renal osteodistrophy

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Malignancy :the most frequent cause of Hpercalcemia in the hospital inpatient population. Malignancy associated hypercalcemia: With and without bony metastasis. With B.M:Hemathologic(Multiple Myeloma,Lymphoma,lukemia)breast,Lung,others Osteoclast activating factor,tumor necrosis factor,IL1 Without B.M:Humoral hypercalcemia of malignancy; Renal,hepatic,epidermoid of head,neck,lung and ilet cell of pancreasPTH-rP Urinary CAMP excretion + or normal PTH

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Vitamin D intoxication Granulomatous disorders(Sarcoidosis) Milk alkali syndrome(Serumca,U.ca,Azotemia,Alkalosis) Lab tests in diff DX of hypercalcemia: Serum total & Ionized ca.,Urine ca. Serum &urine phosphorus Alkaline phospatase,Albumin,PTH,PTH-rP,Urine CAMP VitaminD,cortisol,GH,

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Magnesium 4th most abundant cation in the body(after Na,K,Ca) 2 nd most prevalent intracellular cation. Normal body content:1000mmol (22.66mg) 50-60% in Bone,40-50% in soft tissue. 1/3 skeletal Mg is exchangeable.Reservoir for extracellular Mg(1% of total body Mg) Serum:55% Ionized(Mg2+),15%complex with phosphate,citrate,,30% protein bound(Albumin) 45% of TB Mg, is intracellular.(ATP,Nucleus,mith0chondria;RE)

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Function,Mg Essential for >300 cellular Enzymes. (Transfer of phosphate groups,DNA replication,transcription,RNA translation,ATP) Cellular energy metabolism,Membrane,nerve conduction,Cardiac muscle(K pump) Mg after cardiac Surgury,causes refractory plasma electrolyte abnormalities(K)and arrythmia

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Mg GI absorption,Renal Excretion MG:diatery intake:300-350 mg/day Sturable transport system and passive diffusion Renal excretion:120-140 mg/24hour Thick ascending loop of henle(60-70%) Distal tubule(10%),Major regulation site. Mg2+ the most important regulator.(PTH,Calcitonin,glucagon,)

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Analytical techniques Serum is preferred over plasma. Anticoagulants interfere. Methods: AAS,Reference method(remove of ph. With lanthanum) Photometric methods,Routine,Metallochromatic indicators(Calmagite:collor in Alk.sol.520nm) Ionized(Free)Mg:ISE(Neutral ionophores selective for Mg2+)Interference with ca.

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Reference interval,Mg Total Mg:1.7-2.2mg/dl(0.75-0.95 mmol/L) No age or sex difference in total Mg concentration. CSF Mg:2.0-2.7mg/dl Ionized Mg:0.44-0.60 mmol/L

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Thank you for your attention