)2(Calcium1

8/9/2019 )2(Calcium1

1/6

Calcium Dr. H Khouja

Calcium

Physiological Role and Function

98% of Ca is in the bone and teeth

1% is in the body fluids

1% in soft tissues

Most of the circulating Ca is in the plasma with little or no Ca in rbcs

Milk and meat are rich dietry sources of Ca

In the bone, Ca & Pi form the hydroxyapatite crystals which provide the strength of the bone

and serves as the major store for Ca

Blood coagulation requires Ca as factor IV

Ca is involved in the skeletal and heart muscle contractions

Ca is involved in the regulation of membrane ion transport and membrane permeability

Ca is also involved in milk production

Ca is involved in activation of cAMP secondary messenger mechanism of hormone action

It is involved in cellular secretion

o Circulating Ca also known as total Ca (tCa) is made up of;

~60% of tCa is Free or ionized Ca (Ca2+) which is the physiologically active

form

~35% of tCa is Bound Ca to proteins mainly albumin

~ 5% of tCa is complexed with citrate, oxalate, phosphate

Factors involved in Regulation of Circulating Levels of Ca

a) Non-hormonal factors;

1- pH-affects Ca2+ -increase pH -- decreased Ca2+ & increased bound Ca , tCa no change

-decrease pH -- increased Ca2+ & decreased bound Ca , tCa no change

for every 0.1 pH unit change there is ~0.1 mmol/L Ca2+ change

2- Total protein

-affects bound and tCa , does not affect Ca2+

- hyperproteinaemia-increased bound Ca, increased tCa no change in Ca2+

- hypoproteinaemia-decreased bound Ca, decreased tCa no change in Ca2+

3- Serum Pi levels

A reciprocal (inverse) relationship between Ca & Pi is present in serum or plasma

o Increased Pi results in decreased Ca and vice versa

o e.g. in uraemia, Pi is increased due to renal retention of Pi -- decreased Ca

o In susceptible persons increased Pi may result in kidney stone (Ca phosphate

precipitate),

o in others with alkaline urine may result in phosphate crystals and/or amorphous

phosphate

4- Oxalate and citrate levels

o Increased levels of citrate or oxalate result in decreased Ca because of binding and

formation of Ca citrate or Ca oxalateo In susceptible persons increased citrate or oxalate may result in kidney stone (Ca

citrate or oxalate precipitate)

1

8/9/2019 )2(Calcium1

2/6


o in others it may result in oxalate crystals and/or amorphous oxalate

a) Hormonal Control

1- Parathyroid Hormone (PTH)

Polypeptide hormone produced by the parathyroid glands

Released in response to decreased levels of circulating Ca2+

Net result is increased Ca by;

o Increased bone resorption (stimulation of formation and activation of osteoclasts)

o Increased Ca reabsorption by the renal tubules

o Decreased tubular reabasorption of Pi

o Stimulation of synthesis of 1,25-dihydroxycholecalciferol (1,25-(OH)2 VitD3)

2- 1,25-dihydroxycholecalciferol (1,25-(OH)2 VitD3)

Physiologically active form of Vit D3

Stimulated by the action of PTH

Net effect is increased Ca in circulationo Increased Ca2+ absorption from the intestine

o Increased bone resorption

o Increased Ca reabsorption by the renal tubules

o In chronic renal disease 1,25-(OH)2 VitD3 synthesis is decreased which results in

decreased absorption of Ca2+

3- Calcitonin

A polypeptide hormone produced by the parafolicular C cells in the

thyroid, parathyroid & thymus glands

Stimulated when the levels of Ca is increased in circulation Net effect is to reduce circulating levels of Ca by;

o Inhibition of bone resorption (inhibition of osteoclast formation and activity)

o Stimulation of bone formation (stimulation of osteoblast action)

o Mobilization of Ca & Pi from the ECF into bone & bone fluid

o Increase the secretion of Ca, Pi & Na through the kidneys into the urine

4- Thyroid hormones (T3 & T4)

Produced by the thyroid glands in response to TSH stimulation

Increases mobilization of Ca2+ into the bone

5- Glucocorticoids

Produced by the adrenal cortex glands

Chief glucocorticoid is cortisol

Increases bone resorption

Decreases bone formation

Decreases serum concentration of Ca

Decreases levels of 1,25-(OH)2 VitD36- Sex hormones

Androgens (Testosterone) in males and estrogen (estradiol) in women stimulate Ca & Pi

mobilization into bone

They promote bone formation and maintainence

At menopause estorogen levels decrease which results in increased bone resorption

(osteoprosis)

2

8/9/2019 )2(Calcium1

3/6


At old age men testosterone levels decrease which results in increased bone resorption

Clinical Correlations

1-Hypercalcemia: Increased levels of Ca is associated with;

a) primary hyperparathyroidism

b) hypervitaminosis D

c) bone neoplasmd) milk-alkali syndrome (in treatment of peptic ulcers)

e) thyrotoxicosis

f) multiple myeloma

g) sarcoidosis

h) polycythaemia Vera

i) ectopic production of PTH by various tumors

Symptoms associated with hypercalcemia;

- nausea -vomiting -abdominal pain - polyuria

-CaPi stone formation -abnormal calcification of soft tissues

Lab findings in 1ry hyperparathyroidism

o increased Ca2+ -increased tCa -increased urinary Ca & Pi

o decreased serum Pi -increased PTH

Lab findings in Hypervitaminosis D & multiple myeloma

-increased tCa & Pi -PTH normal or slightly decreased - no change in Ca2+

2- Hypocalcemia; low levels of Ca is associated with;

o hypoparathyroidism -vit D deficiency -hypomagnesemia -steatorrhea

o

nepherosis (loss of protein in urine)o nepheritis (decreased reabsorption of Ca) -pancreatitis (formation of Ca soaps)

o Physiological (non-pathological) -pregnancy -lactation (give Ca supplements)

Symptoms:

-increased neuromuscular irritability -tetany -convulsions - cataract -depression

Lab findings in hypoparathyroidism

-decreased PTH, tCa, Ca2+, urinary Ca & Pi -increased Pi or normal

lab findings in the other conditions

-decreased bound & total Ca -increased PTH -Ca2+ normal

Determination of Calcium

Specimen Requirements.

tCa;

Fasting sample required.

- Serum OR -plasma use Li-heparin Urine 24hour acidify with 10 mL 6M HCl

-Never use oxalate, citrate (precipitate Ca)

-Never use EDTA (chelate Ca)

-Avoid prolonged tourniquet application (haemoconcentration --- increased tCa & bound Ca)

by ~ 0.1-0.15 mmol/L

-do not expose whole blood to open air; change of pH affects Ca2+

3

8/9/2019 )2(Calcium1

4/6


-Posture : recumbent 0.1-0.15 mmol/L LOWER THAN standing up.

Methods for tCa

1) Atomic Absorption Spectrophotometry (reference method)

2) Colourimetric procedures

3) Titration methods4) Precipitation methods

5) FES

1) Atomic Absorption Spectrophotometry (reference method) (AAS)

Principle: Ca-containing compounds, when introduced into a flame, dissociate to give ground

state Ca atoms. Ca atoms absorb light at a specific characteristic wave length (422.7 nm), which

is produced from a hallow cathode Ca lamp. The amount of absorbed light is directly

proportional to Ca concentration.

Major interferences (Sources of errors)a) Phosphate forms highly non-dissociable compounds with Ca.

To eliminate this interference add lanthanum chloride (LaCl3) as La binds preferentially to

Pi and forms tight complex with Pi.

b) Proteins in the sample result in clogging of the tube and produces false low tCa.

To eliminate this interference precipitate the proteins with trichoroacetic acid, this improves

the precision and accuracy of the procedure.

2) Colorimetric procedures [o-cresolphthalein complexone method] (most commonly used in

routine clin labs)

Principle:

Ca in serum forms a coloured complex with o-cresolphthalein complexone at an alkaline pH

(10-12) in the presence of diethylamine buffer. Absorbance is measured at 570nm. Abs is

directly proportional to Ca concentration.

Interference: Mg also reacts with o-cresolphthalein complexone. To eliminate this interference,

8-hydroxyquinoline is added to bind Mg and prevent it from reacting with the dye.

3) Titration methods: EGTA titration method.(used in single element determination or

research)

Principle:In alkaline conditions, the dye calcein is fluorescent. When a sample containing Ca is added,

calcein binds Ca and the fluorescence is quenched. EGTA (ethylene glycol, 2-amino ethylether,

tetra acetate) has higher affinity for Ca than calcein. EGTA is added to the reaction cuvette until

all Ca present bind to EGTA and the initial fluorescence is restored to the original value.

The amount of EGTA added to reach the end-point is proportional to the Ca concentration.

4) Precipitation methods

a. Oxalate precipitation followed by redox titration

b. Precipitation with chloranilic acid

5) Flame emission spectrophotometry (FES)

4

8/9/2019 )2(Calcium1

5/6


Same principle as Na & K but lacks sensitivity (not commonly used in clinical or biological

applications)

Ionized Ca (Ca2+) Determination.

1) Determination by using the McLean-Hastings Formula;

The McLean-Hastings Formula can be used to determine Ca2+ when;

a) the tCa (mg/dL) & tProtein (g/dL) are known.

b) pH of the blood 7.35-7.45

c) measurements of the tCa & tProtein are carried out at 25oC

d) protein concentration is within the normal range

e) no protein-disorders (normal constituents)

f) lipid concentration within normal range (Cephalin binds Ca similar to protein)

-convert Ca concentration from mmol/L --

mg/dL (multiply by 4)-convert protein concentration from g/L - g/dL (divide by 10)

Ca2+ (mg/dL) =[(6 X tCa) (1/3 X tProtein)]

/(tProtein + 6)

-convert the Ca2+ from mg/dL -- mmol/L (divide by 4)

EXAMPLE

Calculate the Ca2+ for a patient blood which has a tCa = 2.36 mmol/L and total protein = 76 g/L.

All other conditions are satisfied.

2) Determination of Ca2+ by ISE

-Fasting blood specimen is collected anaerobically in a plain tube to prevent changes in the pH.

-Sample is allowed to clot at room temp.

-immediately after formation of clot, the serum is separated into 1mL tuberculin syringe.

-analyze immediately using the ISE

-the serum can be kept capped in the fridge for ~4hours, or at -20oC for 4days

-the activity of Ca2+ is measured so to convert to concentration compare to the activity of a

standard solution

-the standard solution is isotonic which also contains Na+ & Mg2+ because they also interfere with

electrode.

Correction for Albumin.

Because albumin is the major protein that binds Ca, its level affects bound and tCa, but not Ca2+. In

cases of abnormal albumin concentrations, it may be desirable to know the normal Ca

concentration OR corrected Ca. The following formulae are used to correct for albumin;

Corrected Ca (mmol/L) = [Ca]+ {0.02X(40-[Alb])} when Albumin < 40 g/L

Corrected Ca (mmol/L) = [Ca]- {0.02X([Alb]-45)} when Albumin > 45 g/L

Normal range (adults):

tCa = 2.10 2.55 mmol/L

Ca2+= 1.16-1.32 mmol/L

Higher values in children and the elderly

Lower values in pregnant and lactating mothers.

5

8/9/2019 )2(Calcium1

6/6


To obtain good diagnostic picture of Ca;

-determine tCa, Ca2+ ,acid-base status, tProtein, Albumin

-check if patient is taking certain drugs that affect Ca concentration, such as phenobarbital &phenotoyn which inhibit the synthesis of 1,25-DHCC & thus decrease the absorption of Ca and lead

to hypocalcaemia

-also check if the patient is under hormonal therapy that affects the Ca concentration.

6

)2(Calcium1

Documents

Transcript of )2(Calcium1