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.
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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
45% bound to proteins
5 % complexed with anions
(the physiologically active form)
(predominantly albumin)
(citrate, sulfate, phosphate)
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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
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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.
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Calcium homeostasis is maintained by an
interplay of :
Intestinal
secretion (6mmol/24hrs) and
absorption (12 mmol/24 hrs)
Renal
excretion:
Glomerular filtration of ionized Ca 2+
reabsorption (236 mmol/24hrs),
Excretion
(4mmol/24 hrs)
Bone
remodeling
(exchange(500 mmol/24 hrs),
formation (7.5 mmol/24 hrs)
resorption (7.5 mmol/24 hrs)
and
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(240 mmol/24hrs),
Regulation
of
Calcium
Parathyroid hormone is produced by parathyroid glands, located
around the thyroid gland in the neck.
Ca2+
parathyroid hormone
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.
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The level of calcium in blood is regulated primarily by two hormones:
Parathyroid hormone and calcitonin.
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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.
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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.
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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. Ca 2+ 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.
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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.
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Overview of Phosphate Balance
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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
blue
Molybdenum
( max= 670 nm)
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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
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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)
Vitamin D deficiency / low calcitriol
Fanconi syndrome
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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, 2,3diphosphoglycerate (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.
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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.
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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 & 37 0C 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
+ PO4
(Colorless)
paranitrophenol
ALP
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( Yellow, max= 410nm)
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.
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