Title: The Determination of Serum Calcium

Objective: To determine the serum calcium concentration by getting absorbance.

Introduction:
Calcium, Ca2+ perform different constituents in human physiology. In bone, it
combines with phosphorus to form hydroxyaptite crystals, giving strength to the bone
structure and providing a constant reservoir in the rest of all the body. It also play important
roles in blood coagulation, muscle contraction and membrane permeability. 99% of overall
calcium can be found in bone and teeth. Serum calcium exists in three different form, one is
free calcium ion, Ca2+, with percentage of 50 percent, 45% of albumin bounded calcium and
5% of complexed calcium. The ionized calcium is physiologically most significant but has
proven difficult to assay directly. It may be estimated from total calcium given a knowledge
of the protein content and pH of the blood which strongly affect the level of ionized calcium.
(Repository.uobabylon.edu.iq).
There are few method can be used to determine the calcium concentration. The method
that will be used in this experiment is the spectrophotometric determination of the serum
calcium. In this method, the absorbance of the Ca-oCPC complex will be read at 580 nm. The
resulting increase in absorbance of the reaction mixture is directly proportional to the calcium
concentration in the sample. A one-piece color reagent will be used. When a certain amount of
the serum add into the reagent, a steady color formation will take place.
This Calcium oCPC complex can be procedure by the reaction of calcium ions 𝐶𝑎2+ ,
reacting with o-cresolphthalein complexone in an alkaline solution to form an intense violet
colored complex which maximally absorbs at 578 nm. 2,3, 8-Hydroxyquinoline will be used
as chelator to remove interference by other divalent metal ions like magnesium and iron.
In the serum, it does not only contain calcium complexes, it also associated with the
hemoglobin complexes also with have similar absorbance reading which at 578 nm, thus, a
correction has to be made for this. This is when the EDTA play its role. The most common
anticoagulant to obtain plasma is ethylene diamine tetra acetic acid (EDTA).
In this experiment, the plasma sample provided is from a 58 year-old female patient
which has undergo a partial thyroidectomy as treatment for hyperthyroidism. She has now
being admitted to the hospital for cataract removal. She also showed signs of increased neuro-
muscular excitability. A determination of her serum calcium (and phosphate) is necessary to
confirm a tentative diagnosis of hypoparathyroidism (UTAR UDEE 2104 Metabolism 1 (Year
2 Semester 1). 2017).

Materials: Diethanolamine buffer (2 M, pH 11.7), chromogen, colour reagent, 0.1 M TCA,

4.0 mM calcium standard, 150 mM EDTA (10 mL), serum sample, spectrophotometer.
Procedures:
A B C D E F U1 U2
Colour 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
reagent
(mL)
Calcium 0 10 20 30 40 50 - -
standard
(μL)
H2O 50 40 30 20 10 0 - -
(μL)
Serum - - - - - - 50 50
sample
(μL)

Table 1: Table to set up the tubes by using 4 mM of standard calcium solution.

1. The tubes were mixed and stood for 30 minutes at room temperature.
2. The absorbance was read of all tubes against tube A at 578 nm (Absorbance 1).
3. Each solution was poured back into their respective tubes after reading.
4. 50 μL of 150 Mm EDTA was added to tube A, U1 and U2. Mixed well and read the
absorbance at 578 nm (Absorbance 2).
5. Calculate Abs 1- Abs 2 for the unknowns and tube A.
6. A standard curve was plotted and the unknown values were read off.

Results:
Tubes Absorbance 1 at 578nm Concentration (mM)
A (as a blank) 0.000
B 0.028 0.008
C 0.112 0.016
D 0.225 0.024
E 0.169 0.032
F 0.285 0.040
Unknown 1 (U1) 0.128 -
Unknown 2 (U2) 0.132 -
Table 2: Absorbance taken without EDTA added

Tubes Absorbance 2 at 578nm

A (as a blank)
Unknown 1 (U1) 0.015
Unknown 2 (U2) 0.017
Table 3: Absorbance taken after added EDTA
Calculations:
Concentration for tube B, Concentration for tube E,
M1V1 = M2V2 M1V1 = M2V2
(4.0mM)(10µl) = (M2)(5050µl)
(4.0mM)(40µl) = M2(5050µl)
M2 = 0.008 mM
M2 = 0.032 mM

Concentration for tube C,

Concentration for tube F,
M1V1 = M2V2
M1V1 = M2V2
(4.0mM)(20µl) =(M2)(5050µl)
(4.0mM)(50µl) = M2(5050µl)
M2 = 0.016 mM
M2 = 0.040 mM

Concentration for tube D,

M1V1 = M2V2
(4.0mM)(30µl) = (M2)(5050µl)
M2 = 0.024 mM

Graph of absorbance against concentration

0.3
y = 6.8818x
0.25 R² = 0.8865
Absorbance at 578nm

0.2

0.15

0.1

0.05

0
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045
Concentration of calcium standard (mM)

Graph 1: Concentration of calcium standard at different absorbance at 578nm

To find the absorbance of unknowns;
U1 = Absorbance1 - Absorbance2 U2 = Absorbance1 - Absorbance2
= 0.128-0.015 = 0.132-0.017
= 0.113 = 0.115

To calculate the concentration of unknowns, the equation y = 6.8818x is used.

y is the absorbance at 578nm while x is the concentration calcium standard.
Concentration of U1, Concentration of U2,
y = 6.8818x y = 6.8818x
0.113 = 6.8818x 0.115 = 6.8818x
x = 0.016mM x = 0.017 mM

Discussion:
Calcium is essential to all cells to carry out their activities. Calcium helps to build strong
bones and teeth. It is vital for heart function, and helps in muscle contraction, nerve signalling
and blood clotting. There are various methods developed for the determination of calcium
include colorimetric, fluorescent, gravimetric, ion selective, titrimetric and atomic absorption.
However, the atomic absorption method has been recommended as the reference method but it
requires complicated and expensive equipment. Rapid, convenient, simple and inexpensive
method is the based on interaction of calcium with a suitable chromogenic agent.
In this experiment, we are required to determine the concentration of calcium of the
unknown sample. The unknown sample is the plasma sample that took from the 58 years old
female. A determination of her serum calcium (and phosphate) 9s necessary to confirm a
tentative diagnosis of hypoparathyroidism. Hypoparathyroidism is decreased function of
the parathyroid glands with underproduction of parathyroid hormone. This is due to low levels
of calcium in the blood, often causing cramping and twitching of muscles
or tetany (involuntary muscle contraction) and several other symptoms (En.wikipedia.org,
2017).
In order to determine the concentration of calcium in unknown serum sample, a
standard calcium solution was prepared and determine their absorbance at 578nm. Calcium
solution were prepared by adding colour reagent, calcium standard and water (H20). The colour
reagent is mixing of equal amount of diethanolamine buffer and chromogen. In
diethanoalamine buffer, it contains urea, distilled water and diethanolamine. We used
diethanoalamine as a buffer to maintain the pH and also to enhance the colour intensity.Mixture
of urea and diethanoalanime could form more stable and strong hydrogen bonds with starch
molecules. Glacial acetic acid were added to the mixture when the pH is adjusted to 11.7
because to make it more stable due to their acid/base properties. Diethanolamine is a base so it
reacts with acetic acid (acid). Chromogen was prepared by dissoloving O-cresolphthalein
complexone and hydroxyquinone in ethanol. O-Cresolphthalein Complexone was used as a
complex reagent and hydroxyquinone was used to remove magnesium interference. Urea was
used in the colour reagent to diminish turbidity produced by the lipaemic specimens and to
improve the complex formation. Ethanol is used to inhibit the colour development in the blank.
Calcium + ο-Cresolphthalein Complexone Calcium-Cresolphthalein Complexone
Complex
Calcium reacts with ο-Cresolphthalein Complexone in an alkaline medium to form a
purple-colour solution that absorbs at 570nm. The intensity of the colour is proportional to the
calcium concentration (Pointe Scientific,INC., 2017). Besides, calcium standard was prepared
by dissolving anhydrous AR grade calcium carbonate in 0.1M TCA. At the end, EDTA
(Potassium ethylenediaminetetraacetic acid) was added to the serum sample (U1 andU2).
EDTA is a sample tube anticoagulant used for many laboratory analyses. Gross potassium
EDTA contamination of blood samples is easily recognised by marked hyperkalaemia and
hypocalcaemia. However, subtle contamination is a relatively common, often unrecognised
erroneous cause of spurious hyperkalaemia(Sharratt CL, 2017). EDTA was added to serum
sample to dissociate the calcium-phthalein purple complex. We measured again the absorbance
due to presence of haemoglobin complexes only. Concentration of calcium in serum sample
was measured by the difference between the initial absorbance reading and the final absorbance
reading.
As the result, we obtained 0.016mM and0.017mM for the serum sample (U1 and U2).
According to the reference range, a normol calcium level in body should be from 2.1- 2.6 mM.
It seems like the calcium concentration that we calculated was lesser than the normal range, it
is confirmed that she is suffering from hypoparathyroidism. Hypoparathyroidism is a condition
of parathyroid hormone (PTH) deficiency. Primary hypoparathyroidism is a state of inadequate
PTH activity. In the absence of adequate PTH activity, the ionized calcium concentration in
the extracellular fluid falls below the reference range (2.1-2.6mM). Primary
hypoparathyroidism is a syndrome resulting from iatrogenic causes or one of many rare
diseases. Secondary hypoparathyroidism is a physiologic state in which PTH levels are low in
response to a primary process that causes hypercalcemia. Treatment of patients with
hypoparathyroidism involves correcting the hypocalcemia by administering calcium and
vitamin D. Recombinant human PTH is commercially available in the United States and is
indicated as an adjunct to calcium and vitamin D to control hypocalcemia in patients with
hypoparathyroidism (Gonzalez-Campoy, 1994).

Precaution steps should be taken to minimise error. Colour reagent may be irritation to
skin. Avoid contact. Every time use blank as first reading before measure the absorbance
reading for the solution to avoid error occur in spectrophotometer. Handling the
spectrophotometer with care and carefully as it is sensitive to light. Ensure the correct micro-
pipetting technique when adding the solution into test tube. Prevent using the same tip when
pipetting different solution.
Conclusion:
By having the correction reading of the absorbance for the serum calcium, the unknown
concentration was determined to be 0.016mM for U1 and 0.017mM for U2. Since the
concentration value obtained is way lesser than the normal range value, it can be concluded
that she is suffering from hypoparathyroidism.
References:
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