Calcium, Phosphate and Alkaline phosphatase Learning objectives Explain the diagnostic importance of determining the level of each of calcium & phosphate in serum & urine and principles of these estimations. Describe the function of alkaline phosphatase in bone structure & the diagnostic importance of its estimation in blood as well as the principle of this estimation. 4/16/2017 KMF,09

Calcium Widely distributed in food substances : Milk, Milk products, Egg yolk, beans, nuts, figs, cabbage Calcium- Most abundant mineral in human body Average adult body contains 25,000 mmol, Total calcium content of ECF 22.5 mmol, (Plasma 9 mmol) About 99% of the body's calcium is stored in the bones, most of which exists as complex inorganic hydrated calcium salts (hydroxyapatite), Ca10(PO4)6(OH)2. Plasma Ca+2 concentration : 8.5 -10.5 mg% ( 2.1 - 2.6 mmol / l ) Calcium in the plasma: 50 % in ionized form (the physiologically active form) 45% bound to proteins (predominantly albumin) 5 % complexed with anions (citrate, sulfate, phosphate) 4/16/2017 KMF,10

Functions Ca+2 plays a role in /as Formation of bone and teeth Muscle contraction Normal functioning of many enzymes (cofactors) Blood clotting Normal heart rhythm Intracellular secondary messenger 4/16/2017 KMF,09

Calcium homeostasis The body precisely controls the amount of calcium in cells and blood. The body moves calcium out of bones into blood as needed to maintain a steady level of calcium in the blood. If we do not consume enough calcium, too much calcium is mobilized from the bones, weakening them. Osteoporosis can result. To maintain a normal level of calcium in blood without weakening the bones, we need to consume at least 1,000 to 1,500 milligrams of calcium a day. 4/16/2017 KMF,09

Calcium homeostasis is maintained by an interplay of : Intestinal secretion (6mmol/24hrs) and absorption (12 mmol/24 hrs) Renal excretion: Glomerular filtration of ionized Ca2+ (240 mmol/24hrs), reabsorption (236 mmol/24hrs), Excretion (4mmol/24 hrs) Bone remodeling (exchange(500 mmol/24 hrs), formation (7.5 mmol/24 hrs) and resorption (7.5 mmol/24 hrs) 4/16/2017 KMF,09

Regulation of Calcium Parathyroid hormone does the following: Parathyroid hormone is produced by parathyroid glands, located around the thyroid gland in the neck. Ca2+ parathyroid hormone Parathyroid hormone does the following: Stimulates bones to release calcium into blood Causes the kidneys to excrete less calcium in urine Stimulates the digestive tract to absorb more calcium Causes the kidneys to activate vitamin D, which enables the digestive tract to absorb more calcium Calcitonin is produced by cells of the thyroid gland. Its level increases when serum calcium is more than 2.25 mmol/l. It suppresses bone resorption and increases excretion . It lowers the calcium level in blood. 4/16/2017 KMF,09

The level of calcium in blood is regulated primarily by two hormones: Parathyroid hormone and calcitonin. 4/16/2017 KMF,09

Diagnostic importance Normal value: 8.5 – 10.5 mg/dl ( 2.1 – 2.6 mmol/L) Hypocalcemia (<7.5 mg/dl) Hypocalcemia can affect the brain and cause neurologic or psychologic symptoms, such as confusion, memory loss, delirium, depression, and hallucinations. These symptoms disappear if the calcium level is restored. An extremely low calcium level may cause tingling (often in the lips, tongue, fingers, and feet), muscle aches, spasms of the muscles in the throat (leading to difficulty in breathing), stiffening and spasms of muscles (tetany), seizures, and abnormal heart rhythms. Hypercalcemia (>11.5 mg/dl) The earliest symptoms are usually constipation, nausea, vomiting, abdominal pain, and loss of appetite. Very severe hypercalcemia often causes brain dysfunction with confusion, emotional disturbances, delirium, hallucinations, and coma. Muscle weakness may occur, and abnormal heart rhythms and death can follow. 4/16/2017 KMF,09

Causes Hypercalcemia: A high calcium level may result from a problem with the parathyroid glands (hyperparathyroidism), as well as from diet, cancer, or disorders affecting bone. Hypervitaminosis D, Excessive bone mineralization, muscle weakness Hypocalcemia: A low calcium level may result from a problem with the parathyroid glands (Hypoparathyroidism, Pseudohypoparathyroidism) , as well as from diet (Inadequate consumption of calcium, Vitamin D defficiency), kidney disorders, or certain drugs. 4/16/20174/16/2017 KMF,09

Differential Diagnosis: Estimation of Calcium Sample: Fasting serum or heparinized plasma (Only heparin used as anticoagulant) Photometric methods Principle: Release bound/complexed calcium by diluting with acid and buffer with an organic base. Ca2+ combines with O-cresolphthalein complexone in alkaline solution and forms red chromophore that absorbs maximally at around 580 nm. Mg2+ interference reduced by adding 8-hydroxy quinoline, Buffering near pH 12 Measuring at 580 nm. Differential Diagnosis: Blood tests are done to evaluate kidney function and to measure magnesium, phosphate, parathyroid hormone, and vitamin D levels. Other substances in blood may be measured to help determine the cause. 4/16/2017 KMF,09

Phosphate In the body, almost all phosphorus is combined with oxygen, forming phosphate. Bone contains about 85% of the body's phosphate. The rest is located primarily inside cells, where it is involved in energy production. Phosphate is necessary for the formation of bone and teeth. Phosphate is also used as a building block for several important substances, including those used by the cell for energy(ATP), cell membranes, DNA & RNA, secondary messengers, modulation of enzyme activity, biological buffers etc. The body obtains phosphate from foods and excretes it in urine and stool. 4/16/2017 KMF,09

Overview of Phosphate Balance 4/16/2017 KMF,09

Sample: Fasting serum or heparinized plasma Assay of Phosphate Sample: Fasting serum or heparinized plasma Colorimetric method Principle: Phosphate + Ammonium molybdate under acidic conditions forms phosphomolybdate that reacts with ferrous ammonium sulphate , a reducing agent, to form a blue colored Molybdenum blue complex. The blue colored complex absorbs maximally at 670 nm. H+ Phosphate + Ammonium molybdate Phosphomolybdate Phosphomolybdate + Ferrous ammonium sulphate Molybdenum blue ( λmax= 670 nm) 4/16/2017 KMF,09

Diagnostic importance Normal value: 0.9-1.3 mmol /L Hyperphosphatemia Increased GI Intake Decreased Urinary Excretion Renal Failure Low PTH (hypoparathyroidism) Cell Lysis Rhabdomyolysis Tumor lysis syndrome 4/16/2017 KMF,09

Hypophosphatemia Decreased GI Absorption Decreased dietary intake Diarrhea / Malabsorption Phosphate binders (calcium acetate, Al & Mg containing antacids) Decreased Bone Resorption / Increased Bone Mineralization Vitamin D deficiency / low calcitriol Hungry bones syndrome Osteoblastic metastases Increased Urinary Excretion Elevated PTH (as in primary hyperparathyroidism) Fanconi syndrome 4/16/2017 KMF,09

When hypophosphatemia (low phosphorus) is present, muscle weakness, rhabdomyolysis (skeletal muscle death), bone pain and rickets (failure to form and maintain bone) may present. These can be accompanied by lethargy, confusion and impaired sphincter control. In severe cases, a generalized impairment of energy metabolism may occur. Limiting Pi means limited ATP formation and reduced oxidative phosphorylation. In the blood, this may have serious consequences. In white cells, a reduction in ATP results in an impairment of bactericidal activity and predisposition to infection. In red cells, 2,3-diphosphoglycerate (DPG) is reduced, which shifts the oxygen dissociation curve, making it more difficult to release bound oxygen. Hypophosphatemia occurs during alkalosis, when cells take up circulating Pi and the kidneys do not resorb enough to account for the shortfall. . Hyperphosphatemia Hyperphosphatemia induces metabolic acidosis. Symptoms include muscle cramps, tetany, and the suppression of Vit D formation. One specific danger involves the abnormal deposition of CaP complexes in non-osseous tissue. This is particularly troublesome in blood vessels. 4/16/2017 KMF,10

Alkaline phosphatase Phosphatases are enzymes which catalyse the splitting of a phosphate from mono-phosphoric esters. Alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in liver, bile duct, kidney, bone, and the placenta. Alkaline phosphatase (ALP), a mixture of isoenzymes from liver, bone, intestine and placenta, has maximum enzyme activity at about pH 10.5. ALP is important in mineralization of bones Serum ALP measurements are of particular interest in the investigation of hepatobillary and bone diseases. 4/16/2017 KMF,09

Estimation of ALP Sample; Serum or heparinized plasma can be used. Stable for 7 days at 2- 80C. The activity increases if samples are left at room temperature (25-350C) for several hours. Colorimetric method Principle Paranitrophenyl phosphate, which is colorless, is hydrolyzed by alkaline phosphatase at pH 10.5 & 370C to form free paranitrophenol, which is yellow colored . The addition of NaOH produces p-nitrophenoxide ions. and the final color shows maximum absorbance at 410 nm. pH (10.5), 370C Paranitrophenyl phosphate paranitrophenol + PO4 (Colorless) ALP ( Yellow, λmax= 410nm) 4/16/2017 KMF,09

Diagnostic significance Serum alkaline phosphatase (adults) - 40-125 U/L. (Levels up to 3 times this may be normal in children) Liver, bone and placenta contain very high concentrations of ALP. Therefore, increase in ALP activity is usually related to hepatobiliary and bone disorders. Increased ALP levels are observed in liver diseases, osteomalacia, rickets and bone disorders. Moderate elevations are sometimes noted in congestive heart failure, intestinal disease and intra-abdominal bacterial infections. 4/16/2017 KMF,09

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