AlKarkh University of Science

College of Science
Second year level
Clinical Biochemistry Lab
Supervisor: Dr. Tariq Salah
Assist: . Wesen Ibrahim Jaber
Lab -3-
Determination of Glucose Concentration in Serum
Introduction:-
• Glucose is the primary energy source for the human body.
• It is derived from the breakdown of carbohydrates in the diet and in the body stores
(glycogen), as well as by endogenous synthesis from protein or the glycerol moiety of
triglycerides.
• When energy intake exceeds expenditure, the excess is converted to fat and glycogen for
storage in adipose tissue and liver or muscle respectively.
• Insulin: a hormone produced by beta-cells of the islets of Langerhans in the pancreas that
allows your body to use sugar (glucose) from carbohydrates in the food that you eat for
energy or to store glucose for future use.
• Insulin helps keeps your blood sugar level from getting too high (hyperglycemia) or too
low (hypoglycemia.
• Another important action of insulin is to increase the permeability of cells to glucose. With
deficiency in effective insulin (diabetes), the fasting blood glucose levels tend to increase
(hyperglycemia) and the body shows less ability to metabolize carbohydrates. At the other
hand, an islet cell tumor can produce an excess of insulin, resulting in very low levels of
blood glucose (hypoglycemia).

Clinical Significance:
A- Hyperglycemia: may be due to
1. Diabetes mellitus (D.M.)
2. Intravenous administration of glucose-containing fluids.
3. Severe stress (Temporary effect) such as cerebrovascular accident (CVA).

B- Hypoglycemia:
1. Fasting hypoglycemia; symptoms occur at night or early morning, it may precipitated by
prolonged fast or vigorous exercise.
o In appropriate high (insulin)
 Pancreatic tumors → Insolinoma.
o Glucocorticoid deficiency
 Addison’s disease
o Severe liver disease
o Hyperlipidemia (↑ endogenous triglycerides and VLDL)
2. Non-fasting hypoglycemia; symptoms typically occurs within 5-6 hours after a meal and
may related to ingestion of particular type of food, or be associated with medication and
alcohol.
Normal Range:
[Link] (F)
<17 yr 17-60 60-90 >90 yr
Yr yr
60–100 74–100 82–115 75–121
mg/dl

[Link] (R)
<17 yr 17-60 60-90 >90 yr
Yr yr
mg/dl 105– 120– 128– 106–201
180 180 192

Principle:

Glucose is oxidized by glucose-oxidase to gluconate and hydrogen peroxide according to the

following equation:

Glucose + H2O + O2 GOD Gluconate + H2O2

The H2O2 formed, under catalysis of peroxidase, with phenol and 4- Amino-antipyrine (4AA)
2H2O2

4-AA (4 Amino-antipyrine) + phenol Peroxidas Quinonimine + H2O

Glassware and Equipment:

- Plain tube.
-Micropipette (50µl, 1000µl) and disposable tips.
-Timer.
-Incubate at 37ºC.
-Filter paper.
-Spectrophotometer.
Specimen Preparation & Stability:
• Serum (not hemolyzed). Plasma collected on heparin fluoride or heparin iodoacetate
(not hemolyzed) Spinal fluid.
• Fasting specimen: for adults the fasting time is usually 10-12 hrs.
• For children the fasting time is 6 hours unless longer timer is indicated.
• Post prandial specimen- blood collected 2 hours after a meal.
• Random specimen- blood sample collected at any time regard less of food intake.

Preparation and Stability:-

Dissolve each vial (R2) with buffer (R1), (in brown vial); Stability 8 weeks at 20-25°C, 8
month at 2-8°C.

Procedure:
Wave length: …………………………………505 nm (490-
550) Temperature: ………………………………… (20, 25,
37°C) Zero adjustment: blank reagent

Blank Standard Sample

Standard --- 10 µl ---
Sample --- --- 10 µl
Reagent 1 ml 1 ml 1 ml

Mix, incubate 10 min. at 37 °C or 30 min at room temperature (20-25°C).The colour is stable

30 min.

Calculations:
Glucose concentration = Asample / Astd. × conc. Std.

(n) n = 100 mg/dl, 5.55 mmol/l and 1 g/l

AlKarkh University of Science
College of Science
Second year level
Clinical Biochemistry Lab
Supervisor: Dr. Tariq Salah
Assist: . Wesen Ibrahim Jaber
Lab -4-
Determination of Uric Acid Concentration in Serum
 Uric acid is the end product of purine metabolism (adenine, guanine) in the human.

 A normal diet some 5-6 mmol of urate is produced daily. Of this amount about 3-4 mmol
is produced from purines synthesized in the body, the remaining 1-2 mmol are contributed by
dietary purines.

 When humans lack uricase, the uric acid levels are high. It is because of the poor solubility
of urate that humans are prone to the clinical effects of hyperuricaemia, such as gout & renal
damage.

 The purines adenine & guanine are constituents of nucleic acid from DNA & RNA.

 Urinary excretion is slightly lower in males than in females, which may contribute to
the higher incidence of hyperuricaemia in men.

 75% of urate leave the body is in urine. The remaining 25% passes into the intestinal
lumen.

Clinical significance
A - Hyperuricaemia:
The factor that may contribute to hyperuricaemia are:
1- Increased synthesis of purines (gout).
2- Increased intake of purine.
3- Increased turnover of nucleic acid (Leukemia).
4- Increased rate of urate formation (Hypoxia, Acidosis).
5- Reduced rate of excretion.
B – Hypouricaemia
This is rare unless it is:
1- The result of the treatment of hyperuricaemia.
2- Associated with proximal renal tubular damage.
3- Can be seen with Fanconi's syndrome.
4- A finding in some patients receiving parenteral nutrition.
5- A finding pregnancy.
6 - Associated with type 1 diabetes mellitus.

Reference Range:

N.V. Child Adult

Male Female
mg/dl 2.0 – 5.0 3.5 – 7.2 2.6 – 6.0
mmol/l 0.12 – 0.32 0.21 –0.42 0.15 – 0.35

Urine 250-750mg/24h

Principle:
Uricase acts on uric acid to produce allantoin, carbon dioxide, and hydrogen peroxide.
Hydrogen peroxide in the presence of peroxidase reacts with chromogen (amino-antipyrine
and dichlorohydroxybenzen sulfonate) to yield quinoneimine (red complex).

Uric acid + O2 +2H2O Uricase Allantoin + H2O2 + CO2

AA + DCHS + Peroxidase H2O2 Quinoneimine

The intensity of the red color formed is proportional to the uric acid concentration in the
sample.
 Specimen:
1- Serum or plasma (heparin).
2- Uric acid in serum or plasma is stable up to days at 2-8ºC and for 6 months at -20ºC.

Reagents
Contents Initial Concentration of solutions

Vial R1: Enzymes

Potassium hexacyanoferrare (II) 42 ųmol/L
Uricase ≥ 120 U/L

Peroxidase ≥ 450 U/L

Amino-antipyrine 0.150mmol/l

Vial R2: Buffer

dichlorohydroxybenzen sulfonate 2 mmol/L

tris pH 8.0 at 25ºC 50 mmol/L
preservative

Vial R3: Standard

uric acid 10 mg/dl (595 ųmol/L)
Procedure:
Blank Sample Std.
Reagent 1.0 ml 1.0 ml 1.0 ml
Sample ― 25 µl ―
Std. ― ― 25µl

-Mix. Let stand 5 min at 25ºC.

-Read Abs (A) of the sample and std. at 520 nm against the blank.

Calculation

Uric acid conc.= A sample / A sta. × n (Conc. Std.)

n= 10 mg/dl
AlKarkh University of Science
College of Science
Second year level
Clinical Biochemistry Lab
Supervisor: Dr. Tariq Salah
Assist: . Wesen Ibrahim Jaber

Lab -5-
Determination of Urea Concentration in Serum
Introduction:-

1- Urea is the major metabolic product of nitrogen substances in the body. It is

formed in the liver via KrebsHenseleit (urea) cycle form ammonia. Urea is
freely filtered through the glomeruli, and 40 % -50% of urea are passively
reabsorbed in proximal tubules.
2- Urea or carbamide is an organic compound with the chemical formula
CO(NH2)2. The molecule has two —NH2 groups joined by a carbonyl (C=O)
functional group.
3- Urea is found in body fluid and tissues in approximately equal concentration. In
the serum of healthy individuals, the concentration of urea ranges between 3.3
and 7.8 mmol/l.
4- The concentration of urea in whole blood is lower, as erythrocytes contain less
urea. Urea is excreted in urine.
5- The blood level of urea is proportional to the dietary content of protein, i.e. to the
relationship of protein catabolism and liver function. In healthy subjects taking
an average diet, the level of urea is a highly sensitive indicator for the
assessment of renal function. The increase in the level of urea usually precedes
the detection of significant changes in the level of creatinine.

Range of expected Values

1. Serum or plasma
Infants: 6-18 mg/dl (1.0-3.0 mmol/l)
Children: 15-33 mg/dl (2.5-5.5 mmol/l)
Adults: 15-45 mg/dl (2.5-7.5 mmol/l)

2. Urine
• 250-500 mmol/24 h
• 15-30 g/24 h
Clinical Significance:
1. Physiologic changes in urea concentration:
The concentration of urea depends on diet. Urea is normally lower in children
and women because of their lower muscular mass than in men.
A. Increased levels:
• In healthy individuals, the blood urea concentration increases with dietary
protein.
• The concentration of urea increases with age.
• In late pregnancy because of increased protein intake.

B. Decreased levels:
• Blood urea concentration is lower in children during growth than in adults.
• In early normal pregnancy.
• In individuals on a low- protein high- carbohydrate diet.
2. Pathologic changes in urea concentration:

A. Increased urea levels in (Hyperuremia):

• Excessive urea production

 Intake of high-protein diet 12 hours before blood sampling.
 Enhanced catabolism of body protein.
• Incomplete urea clearance from the body.
 Prerenal failure: mild dehydration, high protein diet, reabsorption of
blood protein after a gastrointestinal hemorrhage, cardiac failure,
vomiting and shock.
 Renal failure: glomerulonephritis, acute or chronic nephritis.
 Postrenal failure: obstruction of urinary tract.

B. Decreased urea levels in (Hypouremia):

• Acute and subacute necrosis of the liver
• After hemodialysis
• Cystic fibrosis
• Eclampsia
• Hepatic failure
• Hepatolenticular degeneration
• Liver cirrhosis and liver disease
• Nephrotic syndrome
• Per-eclampsia
• Toxic hepatitis
 Risks Factor:

Increased values Decreased values

• Dehydration • Diurnal rhythm: morning
• Stress • Fasting
• Trauma • Pregnancy
• Shock • Smoking
• Diurnal rhythm: afternoon
• Physical exercise
• Season: summer
• Nephrotoxic drugs

Principle:

Urea is hydrolyzed by urease

Urea + H2O urease 2NH3 + CO2

Salicylate and hypochlorite in the react with ammonium ions to form 2,2-dicarboxy- indophenol.
The quantities of this green compound is portional to the urea concentration.

Nitroprusside
NH4 + Salycilate + NaClO Indophenol + NaCl
OHˉ

Specimen:
o Use serum or plasma (EDTA, Citrated or heparinized). Serum stable (1day
at room temperature, several day in refrigerator, 1month when frozen).

Contents Initial Concentration of solutions

R1: Standard Urea 8.325 mmol/l or 50 mg /dl

R2: Urease 3000µ / l
R3: Color Reagent

Phosphate PH 8 50 mmol/l
EDTA 1 mmol/l

Sodium salicylate 62 mmol/l

Sodium nitroprussiate 3.35 mmol/l

R4 :Alkaline reagent
Sodium hypochlorite NaClO 24.8 mmol/l
 Glassware and Equipment:

-Glass tubes or plain tube.

-Micropipette (10µl, 1000µl) and disposable tips.
-Incubator at 37ºC.
-Timer.
-Spectrophotometer.

Preparation of working solution:

Dispense one vial of Reagent 2 into one vial of Reagent 3 and mix by turning
upside down several times.

Procedure:
Reagent Standard Sample
blank
Standard ----- 10 µl ---
Sample ---- --- 10 µl
Working solution 1ml 1ml 1ml
Mix, incubate 5 min. at 37◦C or 10 min. at 20-25 ◦C. Then add :
Reagent 4 200 µl 200 µl 200 µl
Mix, incubate 5 min. at 37◦C or 10 min. at 20-25 ◦C.
Measure the standard (Astd.) and sample (Asample ) against reagent blank (580 nm).
Coloration is stable 2 hours in dark.

Calculations:
Urea concentration = (Asample / Astd. ) × conc.
Std. (n) (mmol/l, mg/dl, g/l)

Conversion factor:
mmol/l x 0.06= g/l, g/l x 16.67= mmol/l, mmol/l x= mg/dl, mg/dl x 0.167 =
mmol/l
AlKarkh University of Science
College of Science
Second year level
Clinical Biochemistry Lab
Supervisor: Dr. Tariq Salah
Assist: . Wesen Ibrahim Jaber

Lab -6-
Determination of Creatinine Concentration in Serum
Introduction:-
 Serum creatinine (a blood measurement) is an important indicator of renal health because it
is an easily measured byproduct of muscle metabolism that is excreted unchanged by the
kidneys.
 Creatinine itself is produced via a biological system involving creatine, phosphocreatine
(also known as creatine phosphate).
 Creatine is synthesized primarily in the liver and then transported through blood to the
other organs, muscle, and brain, where, through phosphorylation, it becomes the high-
energy compound phosphocreatine.
 During the reaction, creatine and phosphocreatine are catalyzed by creatine kinase, and a
spontaneous conversion to creatinine may occur. Creatinine is removed from the blood
chiefly by the kidneys, primarily by glomerular filtration. If the filtration in the kidney is
deficient, creatinine blood levels rise.
 General formula of creatinine is C4H7N3O
Range of expected Values
1- Serum
mg/dl µmol/l
<5yr 0.04-0.40 4-40
6-9yr 0.20-0.52 18-46
10-17yr 0.22-0.59 19-52
Male 0.62-1.10 55-96
female 0.45-0.75 40-66

2- Urine
15-25 mg/kg/24 h

Clinical Significance:
Higher than normal level may be due to:

1. Blocked urinary tract.

2. Kidney problems, such as kidney damage or failure, infection, or reduced blood flow.
3. Loss of body fluid (dehydration)
4. Muscle problems, such as breakdown of muscle fibers.
5. Problems during pregancy, such as seizures (eclampsia), or high blood pressure caused by
pregnancy (preeclampsia).

Lower than normal level may be due to:

1. Conditions involving the muscles and the nerves that control them.
2. Muscle problems, such as late stage muscle loss.

There are many other conditions for which the test may be ordered, such as high blood pressure,
diabetes or medication overdose. Your health care provider will tell you more, if needed.
 Principle:
Creatinine in a basic picrate solution forms a colored complex. The ∆ extinction at predetermined
times during conversion is proportional to the concentration of creatinine in the sample.

Glassware and Equipment:

-Glass tubes or plain tube.
-Micropipette (10µl, 1000µl) and disposable tips.
-Incubator at 37ºC.
-Timer.
-Spectrophotometer.

Working reagent
Mix proportionally 1 : 1 ; the reagent R1 and R2 Stability : 1 month at 20-25°C

Procedure:
Wavelength: ………………… 492 nm (490-510)
Calculations:
Having calculated the ∆OD=OD2-OD1. The samples creatinine concentration will
be obtained using the following formula.
 Lab -7-
Determination of Calcium Concentration in
Serum
Introduction:-
Calcium is essential for all living organisms. 99% of calcium is found in bones and teeth
with t remaining 1% found in the blood and soft tissue. The amount of calcium in the
human body between 1 - 1.5 kg
Calcium in the body is classified into the following:
Blood ionized calcium (Ca+ +), which contributes as an important factor in the
mechanisms blood clotting and stop the bloodshed and very necessary for the conduct
the movement of t heart muscles…etc.
Blood non-ionized calcium or protein-bound blood like that associated with blood
albumin.
Bone calcium stored in bone cells in the form of organic compounds and compounds
phosphate and other compounds, very essential for the safety and the health and
durability bones and teeth and gums, calcium compounds necessary for healthy skin, nails
and hair as th enter in some compositions skin.

Men and women (ages 19 to 50 years): each requiring about: 1000 to 1200 mg per day.
Children: Birth to 6 months need about: (200 mg daily) from 7 to 12 months, they need about:
(270 - 300 mg daily), 1-3 years: (500 mg daily), 4-8 years: (800 mg daily), 9-18 years: (1200
mg daily

Clinical Significant:-
A- Hyper Calcemia:-
1. Hyper Para thyroidism.
2. Calcification in the heart and kidneys.
3. High concentration of cholesterol in the blood.

B- Hypo Calcemia:-
1. Hypo Para thyroidism.
2. Kidney diseases (nephritis).
3. Deficiency of vitamin D.
4. Pregnancy and lactation.

Normal value: (8.0-12.0 mg/dL)

Principle for calcium:
The method is based on the specific binding of Ortho-cresolphthalein
complexone (OC Calcium forms a purple-colored complex with OCC in an alkaline medium.

OCC + Calcium PH 10.7 OCC- Calcium Complex

Glassware and Equipment:

- Plain tube.
-Micropipette (50µl, 1000µl) and disposable tips.
-Timer.
-Incubate at 37ºC.
-Spectrophotometer.

Specimen Preparation & Stability:

• Serum or plasma (heparin)
• Fresh serum collected in the fasting state is the preferred specimen.
• Serum or plasma should be separated from blood cells as soon as
possible, becau prolonged contact with the clot may cause lower calcium value.
• Calcium in serum is stable for 7 days at room temperature, 10 days refrigerated at 4°C a
for one month when frozen.
Reagent Composition:-

Contents Initial Concentration of solutions

CAL: Standard 2.46mmol/l

R1: Buffer
2-amino-2-methyl-propan-1-ol 3.5mol/l, PH 10.7

Propan-1-ol, pH 10.7

R2: Chromogen
O-Cresolphthalein complexone 0.16mmol/l
8- Hydroxyquinoline 6.89mmol/l

Hydrochloric acid 60mmol/l

R3: EDTA 150mmol/l

Working Reagent (R): Mix 1 vol. R1+ 1 vol. R2 to give enough reagents for the number
samples bring run.

Procedure:

Blank Sample Std.

Reagent 1.0 ml 1.0 ml 1.0 ml
Sample ― 20 µl ―
Std. ― ― 20 µl
-Mix, stands 5 min at room temp.
-Read Abs of the sample and sta. at 570 nm against the blank.

Calculation:

Calculate the calcium concentration in serum by using the following formulae:

Serum Calcium Concentration = ASample /AStd × conc. of std (9.82 mg/dl).

2. Unit conversion: mg/dl x 0.25 = mmol/l

AlKarkh University of Science
College of Science
Second year level
Clinical Biochemistry Lab
Supervisor: Dr. Tariq Salah
Assist: . Wesen Ibrahim Jaber
Lab -8-

Determination of Phosphorus Concentration in Serum

Alternative Names
Phosphorus - serum; HPO4-2, PO4-3; Inorganic phosphate; Phosphorus blood test;
Serum phosphorus.

Introduction:-
Living cells use phosphate to transport cellular energy in the form of adenosine
triphosphate (ATP).
Foods rich in phosphorous include tuna, salmon, sardines, liver, turkey, chicken, eggs and
cheese.
An average adult human contains about 0.7 kg of phosphorus, about 85–90% of which is
present in bones and teeth in the form of apatite, and the remainder in soft tissues and
blood (~1%).
Why the Test is performed
This test is performed to see how much phosphorus in blood. Kidney, liver, and certain
bone diseases can cause abnormal phosphorus levels.
The kidney help control the amount of phosphate in the blood. Extra phosphate is filtered
by the kidneys and passes out of the body in the urine. A high level of phosphate in the
blood is usually caused by a kidney problem.
The amount of phosphate in the blood affects the level of Ca in the blood. Calcium and
phosphate in the body react in opposite ways: As blood calcium levels rise, phosphate
levels fall. A hormone called parathyroid hormone (PTH) regulates the levels of calcium
and phosphorus in your blood. When the phosphorus level is measured, sometimes a PTH
level is measured at the same time.
 The relation between calcium and phosphate may be disrupted by some diseases or
infections. For this reason, phosphate and calcium levels are usually measured at the same
time.
Children: 0-6 months need about: (300 mg daily) from 7 to 12 months, they need about:
(500 mg daily), 1-3 years: (800 mg daily), 4-10 years: (800 mg daily), 11-18 years: (1200
mg daily).

Men and women: each requiring about: 800 to 1000 mg per day.
Normal Values
Normal values range from 2.4 - 4.5 milligrams per deciliter (mg/dL).

Males
1-4 years: 4.3-5.4 mg/dL
5-13 years: 3.7-5.4 mg/dL
14-15 years: 3.5-5.3 mg/dL
16-17 years: 3.1-4.7 mg/dL
> or =18 years: 2.5-4.5 mg/dL

Females
1-7 years: 4.3-5.4 mg/dL
8-13 years: 4.0-5.2 mg/dL
14-15 years: 3.5-4.9 mg/dL
• years: 3.1-4.7 mg/dL
> or =18 years: 2.5-4.5 mg/dL

Phosphorus levels less than 1.0 mg/dL are potentially life-threatening and are considered a
critical value in the Mayo Health System.
What abnormal results mean?
Higher than normal levels (hyperphosphatemia) may indicate:
• Acute renal failure and chronic
• Taking vitamin "d" with a large amount
• During fracture healing
• Hypocalcemia
• Hypoparathyroidism
• Liver disease
Lower than normal levels (hypophosphatemia) may indicate:
• Hypercalcemia
• Hyperinsulinism
• Hyperparathyroidism
• Cases of rickets and osteomalacia
• Cases of poor digestion and absorption
• Dependence on intravenous nutrition for a long time

Principle
Inorganic Phosphorus forms a colored complex when treated with Ammonium molybdate which
is reduced by hydroxylamine to molybdenum blue.

Phosphate + Molybdate H+ Heteropolyacid complex

Glassware and Equipment:

- Plain tube.
-Micropipette (50µl, 1000µl) and disposable tips.
-Timer.
-Incubate at 37ºC.
-Spectrophotometer.
 Reagents

R1:

Ammonium 4.5 g/l

molybdate
Detergent and S.Q.
conservator

R2:

hydroxylamine 0.1 mol/l

R3:

Sodium hydroxyde 1.9 mol/l

R4:
Standard 5.0 mg/dl

Procedure:
Blank Standard Sample
Sample - - 0.1 ml
Satndard - 0.1 ml -
R1 Solution 1.0 ml 1.0 ml 1.0 ml

R2 Solution 1.0 ml 1.0 ml 1.0 ml

Mix and after 2 minutes incubation (do not worry about turbid)
Blank Standard Sample
R3 Solution 1.0 ml 1.0 ml 1.0 ml

Mix stand for 10 min at 25 ºC, read Abs. on 650 nm against blank. The color is stable for 30
minutes at room temperature.
Calculation:
[Link] Phosphat conc. = O.D Sample X Standard concentration
O.D Standard
OD (optical density) = Absorbance
mg/dl phosphorus X 0.323 = mmol/L phosphorus