Introduction

Q. What is clinical biochemistry? Give its importance.

Q. Mention the use of biochemical tests.
Q. State the role of biochemical laboratory investigations in medical practice. (RU-15Ja)
Ans.
Clinical biochemistry; Clinical biochemistry is that branch of laboratory medicine in which the chemical and
biochemical methods are applied to study the diseases.
Clinical biochemical tests comprise over l/J"1 of all hospital laboratory investigations. It is usually confined to
study on blood, urine and other body fluids such as C.S.F.
Other names: Clinical chemistry or chemical pathology.
Importance of clinical biochemistry; Biochemical tests may be of use to the clinician-
1. In diagnosis of diseases
2. To monitor the progress or response of treatment of disease
3. To assess the prognosis of disease
4. To screen for disease in seemingly healthy individual
5. In research for biochemical basis of disease
6. In clinical trial for new drugs.
(Ref: Lecture SSMC X- CMC)

Q. What are the modern techniques used in biochemistry? (SU-1 l/05Ju)

Ans.
Modern techniques used in biochemistry:
1. Chromatography
2. Electrophoresis
3. Photometry (Colorimeter & Spectrophotometer)
4. Fluorimetry
5. Flame photometry
6. Ultracentrifugation
7. Radioimmune assay assay (ELISA)
,CricA'

8. Enzyme-linked immunosorbant
9. Hybridoma technology. (Ref: SatyanarayanaZ3nl/719-730)

(DU-13Ja. SU-12Ja)
Q. Short note: Electrophoresis.
Ans. f chargcd particles (ions) in an electric field resulting in their micration
Electrophoresis: The move n js known as electrophoresis.
towards the oppositely charged eiectr

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480 | arifs representation on MEDICAL BIOCHEMISTRY
Molecules with a net positive charge (cations) move towards the negative cathode while those with net
negative charge (anions) migrate towards positive anode.
The rate of migration of ions in an electric field depends on several factors that includc-
Shape, size, net charge and solvation of the ions
Viscosity of the solution and
• Magnitude of the current employed.
Use: Electrophoresis is a widely used analytical technique for the separation of biological molecules such as
plasma proteins, lipoproteins and immunoglobulins.
Different ty pes of electrophoresis:
1. Zone electrophoresis: A simple and modified method of moving boundary electrophoresis is the zone
electrophoresis. An inert supporting material such as paper or gel arc used.
Paper electrophoresis
Gel electrophoresis
2. Isoelectric focusing: This technique is primarily based on the immobilization of the molecules at
isoelectric pH during electrophoresis.
3. Immunoelectrophoresis: This technique involves combination of the principles of electrophoresis and
immunological reactions. Immunoelectrophoresis is useful for the analysis of complex mixtures of
antigens and antibodies.
(Ref: Satyanarayana/3rd/724-725)

Q. Short note on: Hemoglobin electrophoresis. (RU-13Ju)

Ans.
Hemoglobin electrophoresis: Hemoglobin electrophoresis is a blood test that can detect different types of
hemoglobin. It uses the principles of gel electrophoresis to separate out the various types of hemoglobin and is
a type of native gel electrophoresis.
Usefulness:
The test can detect abnormal levels of HbS, the form associated with sickle-cell disease, as well as
other abnormal hemoglobin-related blood disorders, such as hemoglobin C.
It can also be used to determine whether there is a deficiency of any normal form of hemoglobin, as in
the group of diseases known as thalassemias.
The hemoglobin electrophoresis is also known to be thalessemia screening, this also can be helpful for
the patient who is frequently need of fresh blood transfusion.
Method: Different hemoglobins have different charges, and
according to those charges and the amount,
hemoglobins move at different speeds in the gel whether in
alkaline gel or acid gel. Electrophoresis is done by
the use of cellulose acetate. Thalassemia major HbF level and
HbA2 levels increase.
(Ref: http://en.wikipedia.org/wiki/Hcmoglobin_electrophoresis)

Q. Short note: Chromatography.

Ans.
Chromatography: It is an analytical technique dealing with
the separation of closely related compounds from
a mixture. These include proteins, peptides, amino acids, lipids, carbohydrates,
vitamins and drags.
It is one of the most useful and popular tools of Biochemistry.
Principals .nd cl.ssinc.tloq: Chromatography usually consists of a mobile phase and a stationary
phase. The mobile phase refers to the mixture of substances (to be separated),
dissolved
The stationary phase is a porous solid matrix through which the sample contained inin athe mobile
liquid or a gas.
phase
percolates. The interaction between the mobile and stationary phases results in the separation of the

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 CHAPTER 07: CLINICAL BIOCHEMISTRY | 481
Darthlon^/n001
partition,
m’Xturc‘ Thcsc interactions include the physicochemical principles such as adsorption,
lon-exchange, molecular sieving and affinity.

,| Partition | j i n —
[Adsorption], | lon-axchanpa] | filtration*] |
Gel Affinity | | HPLC |
+ 4-
Column TLC

Paper chromatography

•f 4-
Slngla dimensional Two dimensional

t Ascending
Descending

»• Thin layer chromatography

Gaa-llquld chromatography
Fig: Important types of chromatography.
(HPLE- High performance liquid chromatography; TLC- Thin layer chromatography)
(Ref: Satyanarayana/3rf/719-720)

Q. Short note: Autoanalyzer. (RU-12Ju)

Ans.
Autoanalyzer: The auto-analyzer is an automated analyzer using a special flow technique named continuous
flow analysis (CFA).
Application:
1. Clinical analysis: These instruments typically determine levels of (in blood serum or other bodily
samples) -
albumin
alkaline phosphatase
aspartate transaminase (AST)
blood urea nitrogen
bilirubin
calcium
cholesterol
creatinine
glucose
inorganic phosphorus
proteins
uric acid
2. Industrial analysis: To perform-
solvent extraction
distillation

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482 | ariFs representation on MEDICAL BIOCHEMISTRY
on-line filtration
UV digestion
soil, plant, tobacco, food, fertilizer and wine analysis.
Current uses: Autoanalyzers arc also commonly used in-
neonatal screening or Anti-D
soil testing laboratories
fertilizer analysis
process control
seawater analysis
air contaminants
tobacco leaf analysis.
Benefits: Autoanalyzers are used because they decrease costs, save time, conserve reagents and materials,
minimize errors, and improve productivity. An autoanalyzer can analyze hundreds of samples every day with
one operating technician.
(Ref: http://en.wikipedia.Org/wiki/AutoAnalyzer#Current_Uses)

Laboratory
Q. What are the common instruments used in a biochemistry laboratory?
Q. What are the common galsswares and equipments in laboratory?
Ans.
Common instruments used in biochemistry laboratory:
A. Common glass wares:
1. Pipette
2. Burette
3. Measuring cylinder
4. Volumetric flask
5. Beaker
6. Funnel
7. Reagent bottle, dropping bottle
8. Evaporating basin
9. Test-tube, centrifuge tube
10. Micropipette
11. Syringe
12. Stirrer
B. Others:
13. Plastic syringe
14. Test tube holder
C. Common equipments:
15. Centrifuge machine
16. Esbach’s albuminometer
17. Colorimeter / Spectophotometer I Flame photometer
1 8. Electrophoretic apparatus
19. Water bath with a thermostat
20. Autoclave
2 1 . pH meter
22. Blood gas analyzer
23. Hot air oven

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 Osmome^ HIAPTeR 07: CLINICAL BIOCII EM ISTR Y 483
26:
Analytical
Electronic
z/- Burner
28. Auto analyzer.

(Ref: Lecture SSMC &CMC)

Q. What are the
common
Q. What are the types of tests done in the biochemistry laboratory?
Ans. biochemical tests? Give examples of each.
Types of biochemical tests:
The biochemical tests arc
A. Core biochemical tests: grouped under three headings as follows-
Tests are carried out in every
1 . Estimation of blood glucose biochemical laboratory-
2. Blood urea & serum
creatinine
' Serum electrolytes (Na+,
3.
K\ Cl', HCOj )
4. Serum calcium & phosphate
5. Serum total protein, albumin,
A:G, prothrombin time (PT)
6. Serum bilirubin
7. Serum enzymes: ALT, AST,
ALP
8. Blood gas analysis
B. Special tests: Tests that are needed in the diagnosis
of rare disease and not every laboratory is equipped to
carry out all these tests, e.g. estimation of-
1 . Hormone (e.g. T3, T4, TSH, cortisol etc.)
2. Specific protein
3. Trace elements
4. Vitamins
5. DNA analysis
6. Lipid profile
7. Drugs
C. Emergency tests: Tests that the clinicians likely to take immediate action. All clinical biochemistry
laboratories provide facilities for urgent tests, e.g.
1. Blood urea & electrolytes
2. Serum creatinine
3. Blood glucose
4. Serum amylase
5. Blood gas analysis
6. Salicylates
7. Paracetamol
8 Serum calcium. (Ref: Lecture SSMC & DMC)

/ samples that can be analyzed in a clinical biochemistry

Q. Name the clinical specimen
oT^Yhe^m^used for biochemical
analysis. (DU-I8M)

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4S4
I ar*f*s representation on MEDICAL BIOCIII.AHSLBX—
)
VI
Clinical specimen that can he nnnhzcd In clinical bhicJicmjriry: (V.V.I. for
1. Blood: (venous, arterial, capillary)
a) Whole blood
b) Plasma
c) Scrum
2. Urine:
a) Random urine sample
b) Early morning sample
c) 24 hours sample
3 Stool
4 Sputum
5 Semen
6 Saliva
7 Gastric washings
8 Milk
0 Transcellular fluid
Spinal fluid (CSF)
Pleural fluid
Pericardial fluid
• Peritoneal fluid
Amniotic fluid
Synovial fluid
10 Calculi (stone) and tissues.
(Ref: Lecture SSMC & DMC)

Laboratory Hazards
Q. W hat do you mean by laboratory hazard or lab hazard? (V.V.I. for VIVA)
Q. Short note: Laboratory hazard. (DU-17Ja,16M/Ja,14Ja, SU-15M)
Ans.
Lab hazard / Laborators' hazard: Lab hazard is the biochemical hazard denotes infectious material or agents
and chemical that presents a risk or even a potential (directly or indirectly through the environment) risk to the
health of human or animal in the laboratory.
(Ref: Lecture SSMC)
Hazards arise from three main basic causes:
1 . From dangerous chemicals
2. From infected specimens sent for analysis, and
3. From faulty apparatus and instruments.
(Ref: Varley’s Clinical Biocheniistn76,k/1)

Q. Name the different types of laboratory hazards. (DU-18M,l5Ja,l2Ju, SU-15/08Ja)

Q. Mention the common hazards In clinical biochemistry’ laboratory. (DU-lOJa, SU-07Ju)
Ans.
Common laboratory hazards; (V.V.I. for VIVA)
Type of hazards Examples
Physical hazard Breaking of glassware producing cut injury
Bums

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 £» I A PIER (17; (LI NICW I. f HOCH EM LSTRY
c hcmicul ha7ar(l Inin non c g AIDS. ylrn[hgintitk (V.V.I. for MCQ)

Firc hn7nrd
* Damage to the lungs from inhaling vapours
* I kvtiic shock
_
Hnnnlhl effect by the toxic chimiiah and corrosive substances e.g.
IINOi, 1 1 >S( )| etc.

1 Injury from explosions

(Ref: Lecture SSMQ
Q. What are the hazardous
materials?
Ans.
Hazardous materials;
1. Explosive
2. Compressed gases
3. Flammable liquids
4. Flammable solids
5. Oxidized materials
6. Toxic materials
7. Radio-active materials
S. Corrosive materials
9. Miscellaneous materials.
(Ref: Lecture SSMC)

Q. Short note: Chemical hazards.

Ans.
Chemical hazards; Injury from chemicals results from:
1 . Direct contact:
with the skin, e.g. when pouring reagents or from breakage of containers;
with lips or mouth when pipetting (mouth pipetting);
with the esophagus and stomach if inadvertently swallowed.
2. Damage to the lungs from inhaling vapours or, less likely fine powders.
3. Toxic effects of substances absorbed from the lungs, alimentary tract or skin on other tissues such as
bone marrow, liver or kidney.
The main groups of harmful chemicals are:
1. Corrosive substances: strong acids or alkalies, e.g. nitric, sulphuric and hydrochloric acids etc.
2. Organic solvents: e.g. benzene, carbon tetrachloride and other halogenated hydrocarbons.
3. Explosive compounds: e.g. perchloric acid.
4. Poisons: e.g. cyanide and barbiturates.
5. Carcinogens: e.g. aromatic amines.

harmful chemicals
should always be taken when handling
Sl,°"'d bC C'“r'y '“MCd “me and a"y ha“rd
3
noted
4. Do not lick labels; clicmicals from the store
5.
6.
7. Never carry large bottlef
shelve
8. Keep bottles in use on

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_ | arifs representation on MEDICAL
BIOCHEMISTRY
C0n^cn^s ^'avc ^ccn used, empty and residue, rinse well with water and dispose of the
enntni
vomaincr.
(Ref: Varley’s Clinical Biochcmhtry/6"71-6)
Q*
^hfttis biological hazard? (DU-12Ju) What arc the biological hazards? How do you prevent
biological hazards? (RU-06Ja)
Q. What is biohazard? How biohazard can be prevented? (DU-15Ja) (V.V.I. for VIVA)
Q. Short note: Biological hazard. (DU-18Nv,l7M)
Q. Short note: Biohazard. (DU-19M)
Ans.
Biological hazard: Biological hazards, also known as biohazards, refer to biological substances that pose a
threat to the health of living organisms, primarily that of humans. This can include medical waste or samples of
a microorganism, virus or toxin (from a biological source) that can affect human health.
Biological hazards include HIV, Hepatitis B virus etc. may be transmitted by-
1 . Accidental inoculation
2. Contaminated needle
3. Accidental injection
4. Mouth pipetting
5. Accidental introduced by non-contact skin
6. Accidental inhalation of the air-bom droplet.
Prevention of biological hazards:
1. Never mouth the pipette
2. Treat infectious agent carefully to avoid spills and minimize aerosolization
3. Restrict use of needles, syringe to procedure where there is no alternative
4. Use protective coats, rubber gloves, face mask, eye shed etc.
5. Wash hand frequently
6. Decontaminate work surfaces before and after work
7 . Never eat, drink or smoke in the lab.
(Ref: Lecture SSMC)

Q. What are the safety measures in clinical laboratory? (SU-08Ja)

Q. What are the precautions should be taken to prevent laboratory hazards?
Q. What precautions to be taken during sample collection? (DU-19Nv)
Ans.
Precautions should be taken to prevent laboratory hazards:
1 . Every laboratory should be equipped with commercially available fire extinguisher
2. Should be kept sand bucket
3. Nearest flame should be 10 feet away from the flammable chemical
4. Flammable liquid should be heated in water bath
5. Mouth pipetting should be avoided
6. Protective eye glass and eye shell should be used
7. Corrosive chemicals should be added to w'ater to make diluted
8. Highly toxic chemical should be stored into locked copet
9. Hand should be properly washed after using highly toxic chemical & carcinogens
10. Protective plastic or rubber gloves, facemask, laboratory apron should be used
11. Switches & wires should be well insulated
12. Earthling of all essential electrical instrument is essential
13. Eating, drinking & smoking should be avoided in the laboratory.
(Ref: Lecture SSMC)

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 n un
8 nrC —th° CHAPTER
>i iai 07: CLINICAL BIOCHEMISTRY
Ans. cnuslnR Infection?
1 • Blood
2- Transccllular fluid-
Pleural fluid
Peritoneal fluid
Pericardial fluid
CSF.
(Ref: Lecture SSMC)
Q. What arc the infection hazards?
Q. What arc specimen-bora / blood-born infections?
Ans.
Infection,
hazard^. Specimen received by pathology laboratories arc all potentially infectious. The main
hazards in the clinical chemistry laboratory arc¬
fl) Viral hepatitis
(2) AIDS.
(Ref: Varley’s Clinical Biochemistry/6Tl710)
Sample Collection
Q- Write the methods of sample collection / blood collection.
Ans.
Methods of sample collection / blood collection: (V.V.I. for VIVA)
1. Identification of the patient as age, sex, address.
2. Ask the patient whether he/she is fasting or not (the best time for taking blood is after fasting
overnight).
3. Patient should be comfortably seated / supine for 20 minutes before specimen is drawn.
4. Note the date & time of collection.
5. Select the numbers of test tube needed.
6. Needle of 1 8 gauge is used.
7. Cleaning by ethanol or isopropanol.
8. Tourniquet is tied up.
9. Blood is drawn within 1 minute.
10. Blood is collected by inclining the syringe at 15° angle.

Nice to Know:
Types of blood may be: (V.V.I. for VIVA)
1. Capillary blood
2. Venous blood
3. Arterial blood
Capillary or venous blood is used for most determinations made on blood.
Capillary blood:
When used / Indications:
1. When small amount of blood is needed.
of blood glucose by glucose.
3. Totes, BT, CT. blood grouping & es^adon

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488 I ariPs representation on MEDICAL BIOCHEMISHtY,
Sites:
1. Thumb (about 5 mm from the nail edge)
2. Finger tip (index <6 middle finger).
3. Ear lobule.
4. Sole of foot (in ease of infant and young children).
Procedure:
1. Site is cleaned with isopropyl alcohol and allowed to dry.
gentle press to the thumb.
2. Prick the skin with a disposable sterile blood lancet and apply collected.
3. First few drops of blood are wiped away and then the sample
is

is called venipuncture.
Venous blood: It is the commonest. Collection of blood from vein
Site:
1. Any prominent vein (e.g. anticubital vein)
2. Dorsum of the hand.
3. Ankle
4. Jugal vein, when the patient is in shock.
• Arterial blood: This is less frequently examined.
1. Used in blood gas analysis e.g. Pcoj, P02.
Site: Radial, brachial orfemoral artery. .
(Ref: Lecture SSMC; Varley’s Clinical Biochemistry/6 /312-313)

Q. What is prolonged venous stasis?

Ans.
Prolonged venous stasis; Keeping of tourniquet for more than 2 minutes causes prolonged venous stasis.
Effects:
1) It causes t cone, of protein & protein bound substances in blood.
2) TK*.
(Ref: Lecture SSMC)

Q. What are the biochemical tests of blood specimen?

Ans.
Biochemical tests of blood specimen:

1. Whole blood - Blood gas analysis

-pH
- NPN test
- NH3 test
- Hb estimation
2. Plasma - - Prothrombin time
Obtained by centrifugation - Glucose estimation
after addition of appropriate -cr
anticoagulants - HCO3~
3. Serum (commonest)- - Serum electrolytes ( Na\ K+, Ca2\ Mg2\ SO?’, Cl', HCO3)
Obtained by centrifugation -Serum glucose
after clot formation - Serum albumin
- Serum globulin
- Serum bilirubin
- Serum urea

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 489

Blood Collection
Q- Mention the fitters that can affect the correctness of test results on blood.

Fact ore ;hi: ci~i a feet the enrrvetrw of teM reunite on blood:
1. hxerreer p a-emre technique
2. E=v:we:RBC
• Dree ccOewico
• Durmg mansferrmg to cvciamer
• Durmg processing of sample
•. CcO-con of specimen in a wrong container
a Znsmhhty of some cheminaJ constituents in whole blood, serum or plasma.
Sc. to get a va-id and reliable test results care must be taken during the whole procedure of blood collection,
mxessfnz. meservabon & forwurdinc to the laboratory.
(Ref: Lecture SSMC)

Q. VX rite down the precautions to be taken during blood sample collection. (DU-19Nv)

Precautions to be taken during blood sample collection:

a I The Sjninze. needle and test tube must be dry. sterilized and clean.
b) The needle should have sufficient wide bore.
c) Tccrmouer should nec be applied so tightly and should not be tied prolonged time (more than 1 minute)
d) Area cf prink should be cried.
e) Blood should be drawn into die syringe with gentle pull within 1 minute.
fl Acer removing cf needle. blood should be poured on test tube (to avoid break down of RBC) with the
tip touching the side of the test tube gently.
si e arrdccegulam should be avoided.
h) of plasma by centrifuge moderately at 2000-3000 per minute.
i) it is better to separate serum within 30-45 minutes. Serum should be well protected.
j) Blood should not be freezing for prolonged time.
k) Cdl and serum should be separated before presen e.
I) Discard the used needle into a sharps
container.
(Ref: Lecture SSMC)

sample if it is kept for long time whi.

Q- What changes occur in bloodVIVA) " '“““t Processing faefor
Mahsis?(DL-lj/12Ja)(V.V.I. for
in blood sample in prolonged stand* ^^-IGNy)
Q- Mention the changes that occur in blood sample in delayed processing"*
Q- Mention the changes that
occur J* 14Ju) •

Ans. in collected blood when estimation delaid

^Schemical chaoses occur
k
may ChanS« tai- .
C P 3Ce~
CO: consent of plasma from rhe /
J- Loss of CO. /as the the cells to the plasma. This lass mates rhe bl a^Hne)
P to the
atmosphere arJ from
2. ter ions in
3. Conversion
the cel! ldi»e to shift of CT ions
of glucose and pyr^are
A ^Plasma inorganic
phosphate
—
into rhe cell to preserve el
into lactic acid Increase lactic

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90 I
5.

6.
arifs representation on MEDICAL BIOCHEMISTRY__^^—
Formation of ammonia from nitrogenous substances (of which urea is the chief)
concentration
—
Diffusion of K* ion from the cell * Increase K* ion
—
— Increase ammonia
7. Decrease of RBC, pH, protein factor, Na+, DPG
8. Inactivation of Na*-K+ pump
9. Decrease clotting factors , , . . ...
10. Many hormones, particularly peptides, arc affected by proteases in the thcm
(Ref: Varley’s Clinical Biochcmistry/6 *7314-315; Lecture SSMC)

Q. How hemolysis is prevented in clinical lab during blood collection and processing? (DU-15M)
Q. How do you avoid hemolysis in collected blood sample? (DU-12Ja)

Prevention of hemolysis in collected blood sample: Hemolysis is minimized by avoiding mechanical

breakdown of red cells and movement of water out of or into the cells.
1) The syringe and needle should be dry
2) When taking blood, constrict the arm minimally
3) Tourniquet is placed just above the vein of the elbow and should be just tight enough to stop the blood
flow
4) Draw the blood slowly and steadily into the syringe and expel it slowly and gently into the container
with the syringe tip touching the side so that the blood runs slowly down
5) Do not expel blood through the needle
6) Excessive amount of anticoagulant must be avoided
7) The anticoagulant should be mixed with blood by gentle rotation or inversion; not by vigorous shaking
8) Whole blood should not be freezed
9) If plasma is to be separated, centrifuge at moderate speeds (2000-3000 rpm)
10) It is better to separate the serum within 30-45 minutes.
(Ref: Lecture SSMC; Varley’s Clinical Biochemistry/6,h/312-313)
(Ref: Lecture SSMC, DMC, ShSMC)
Q. What are the differences between serum and plasma? (DU-19M)
Ans.
Differences between serum and plasma: (V.V.I. for VIVA)
Traits Plasma
1. Definition
Serum
It is the part of the blood that Blood serum is blood plasma without I
contains both the serum and clotting factors
clotting factors
2. Clotting factors Contains all the blood clotting It does not contain fibrinogen and some
factors other clotting factors like factor-II, V and
VIII etc.
3. Serotonin content Low High (due to breakdown of platelets during
clotting)
4. Clot Can clot because it has all the blood Does
not clot due to lack of some of the
clotting factors clotting factors like fibrinogen
5. Colour Yellowish Straw
6. Usage in medicine It is most often used for transfusion It
is used in numerous diagnostic tests, as
(e.g. fresh frozen plasma) well as blood typing
Q. For what test you will choose what type of blood sample?
Ans.

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 7? CHAPTER 07: CLINICAL BIOCHEMISTRY 491
Plasma' f L”1' 'T” CXCCpttime
2) iasma - for
coaKu,nlion study.
' prothrombin test.

An" *S th° mcd,an cubital vein used for blood

collection?
^lan^ibhnl
1•
vein Is used for blood coHecdon; Bccausc-
1 he vein issuperficially placed
2. Fixed to cubital fossa
3. Wall of the vein is thin
4. Pressure of blood is low
5. More volumetric.

Blood Gas Analysis

Q. Short note: Blood gas analysis.
(RU-19M,13Ja)
Q^Short note: Arterial blood gas analysis. (RU-15M)
Bl^^£2s_ana!isis. An arterial blood gas (ABG) analysis is a blood test that is performed using blood from
^at measures the arterial oxygen tension (PaO2), carbon dioxide tension (PaCO2), and acidity
(pH). In addition, arterial oxyhemoglobin saturation (SaO2) can be determined.
ABG testing is mainly used in pulmonology and critical care medicine to determine gas exchange which reflect
gas exchange across the alveolar-capillary membrane. ABG testing also has a variety of applications in other
areas of medicine.
Site of blood collection: For blood gas analysis, a sample of arterial blood is used collected from-
Radial artery in the forearm, (most commonly) or
(Less commonly) from the femoral artery in the leg.
Parameters: The biochemical profile measured are-
pH of the blood (H* ion concentration)
Partial pressure of carbon dioxide (PCO2)
Partial pressure of oxygen (PO2)
Serum bicarbonate level.
Many blood gas analyzers will also report concentrations of lactate, hemoglobin,
several electrolytes, oxyhemoglobin, carboxyhemoglobin and methemoglobin.
Importance: The measurement of blood gas is an important investigation in the laboratory service. In certain
acid-base disorders, blood gas (CO2 and O2) measurement
conditions associated with respiratory failure and/or
when caring for patients with critical
assumes significance Such information is vital severity of the condition, oxygen illness or respiratory
C on the results obtained
Rased and the treatment or artificial
IStion is carried out. As a
intensive care units (ICUs).
result, the ABG is one of the most common tests performed on patients in

gas analysis:
Reference ranges of arterial blood

[H’] 35-43 mmoi/L alkalemic (H* < 35)

The pH indicates if a patient is acidemic (pH < 7 35) or
Lph

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PCO: 4.5-6.0 kPa A hit’ll
"',lia
Pat t). indicates hyper-
’-ventilation _
or <>
is not oxygenating
PO; '16'5-13.5 kPa \ low PnO; indicates
that ---^
the p t

ptopcrly, and is problem is

Bicarbonate 24-30 mmol/l. The UCO. Ion A low
acidosis, » high HCOf

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Clotting Factors and Coagulation Disorders

Q. Name the blood clotting factors chronologically.
Ans.
Blood clotting factors:
Blood clotting factors Svnonvm 1
Fibrinogen Factor 1
Prothrombin Factor 11
Tissue factor Factor HI; tissue thromboplastin
Calcium Factor IV
Factor V Proaccelerin; labile factor
Factor NII Stable factor; proconvertin; Serum prothrombin conversion accelerator
(SPCA)
Factor VIII Antihemophilic factor (AHF); Antihemophilic globulin (AHG);
Antihemophilic factor A.
Factor IX Christmas factor; Antihemophilic factor B; Plasma thromboplastin
component (PTC)
Factor X Stuart factor; Stuart-Prower factor
Factor XI Antihemophilic factor C; Plasma thromboplastin antecedent (PTA)
Factor XII Hageman factor
Factor XIII Fibrin-stabilizing factor
Prekaliikrein Fletcher factor
High-molecular wt. kinlnogen Fitzgerald factor
platelets
(Ref: Guyton/12"7452)

Q. Name the primary / essential blood clotting factors.

Ans.
Primary / essential blood clotting factors:
I . Fibrinogen- factor /
2. Prothrombin- factor 1!
3. Tissue factor- factor III
4. Calcium- factor IV.
(Ref: S. Wright’s/13,h/23)

Q. Name the vitamin K dependant clotting factors. (V.V.I. for VIVA)

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 493
CHAPTER 07: CLINICAL BIOCHEMISTRY
Ans.
th
cToVing blood clotting factor: Vitamin K is required by the liver for the synthesis of
1 . Factor 11 (Prothrombin)
2. Factor Vil (Stable factor)
3. Factor IX (Christmas factor)
4. Factor X (Stuart-prowcr factor)
(Ref: Ganong/23n'/532; Guyton/12,b/457)

Q. Why is blood clotting abnormal in a patient with vitamin K deficiency?

Ans.
Blood clotting is abnormal in a pntlent with vitamin K deficiency: Vitamin-K is essential for y-
carboxylation (post-translational modification) of factors II, VII, IX and X. so, in partents with vitamin-K
deficiency, there is reduction in the active factors II, VII, IX and X, and therefore blood clotting becomes
abnormal.
(Ref: Guyton/12,b/457)

the laboratory tests done in hemorrhagic disorder. (RU-08Ju)

Laboratory tests done in hemorrhagic disorder:
1 . Routine blood test (Hb%, ESR, total count ofWBC, differential count ofWBC)
2. Screening tests-
a) Platelet count
b) Bleeding time (BT)
c) Clotting time (CT)
d) Prothrombin time (PT)
e) Activated partial thromboplastin time (APTT)
f) International normalization ratio (INR)
g) Thrombin time (TT)
h) Tourniquet test
3. Measurement of plasma levels of specific clotting factors
4. Determination of platelet function
4. Presence of circulating anticoagulants.

Anticoagulants
Q. Name the anticoagulants commonly used in clinical laboratory (RU-09Ju) with their uses and
mechanism of action.
Q. Short note on: Anticoagulant. (DU-17/15/13Ja,08Ju,07Ja) (V.V.I. for VIVA)
Ans.
Anticoagulant: Anticoagulants are the drugs which are
used to prevent unwanted coagulation. The mostly
used anticoagulants are-
(EDTA)
I . Ethylene-diamine-tetra-acetic acid

3.
’
Heller mixture/ Double oxalate (NH4-oxalate : K-oxalate = 3:1)
4. NaF (NaF with K+oxalate) K-citrate)
5. Salts of citrate (Na-citrate, K-oxlate)
6. Salts of oxalate (Na-oxlate,

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7. Acid citrate dextrose (ACD)
Use of anticoagulant:
1. Anticoagulants arc used in different hematological, biochemical and serological investigation
2. They arc used for the preparation of plasma
3. They are used for transfusion of blood.
Mechanism of action & use of some commonly used anticoagulants:
I) EDTA: Mostly used, cheapest, most stable. It acts by preventing calcium ionization by chelation. It is
used in total count of RBC, WBC, DC, ESR etc.
ii) Heparin: Best & most expensive, natural anticoagulant. Combining with antithrombin III it removes
thrombin and prevent fibrin production and removes several activated factors IX, X, XI, XII.
iil) Paul Hcllar’s mixture: Prevent coagulation by precipitating ionic Ca“ and forming insoluble salts.
Most commonly used in determination of cell volume (PCV, MCV, MCH, MCHC etc.)
iv) NaF with K* oxalate: Used in determination of blood glucose level, by acting inhibition of glycolysis
by inactivating the enzyme phosphofructokinasc.
v) Salts of citrate, oxalate: Prevents coagulation by removing calcium by chelation.
vi) Acid citrate dextrose: Used in blood collecting bags. Dextrose is supplied for nutrition of RBC.
vii) Sodium citrate: (1) Used in blood transfusion.
(2) Used in blood bank.
(3) Used in prothrombin time, partial prothrombin time determination.
(Ref: Lecture SSMC)

Q. Name the anticoagulants used in blood collecting bag.

Ans.
Anticoagulants used in blood collecting bag:
1. ACD (Acid Citrate & Dextrose): It is used in blood collecting bag. Dextrose is supplied for nutrition of
red blood cells. Citrate acts as an anticoagulant.
2. CPD (Citrate Phosphate & Dextrose)
3. CPD-A (Citrate Phosphate Dextrose & Adenine).

Q. In pregnancy which anticoagulant can be used?

Ans.
In pregnancy, heparin should be used, as it has no teratogenecity.

Nice to Know:
Injectable anticoagulants: Heparin, ancord.
Ora! anticoagulants: Warfarin, dicoumarol.

Urine Analysis
Q. What are the indications of urine examination?
Ans.
Indications of urine examination:
1 . Diseases of urinary tract
2. Nephrotic syndrome
3. Metabolic and systemic diseases, e.g.

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 „W|,~
pinlwcR Imipldui
‘
Jaundice
'•■ Pregnancy test.
(Rrf: lecture SSMC, DMC)
to Know-
Th'pcs of urine sample: (V.V.I.
for VIVA)
I) Early morning sample
2) 24 hours sample
3) Random sample
• Early morning sample;
Indication;
exam,nallon
'2 Diagnosis of pregnancy by detecting (protein <6 glucose mainly)
HCG in urine
24 hours urine sample:
Indication:
I. Determination of daily urine
output polyuria, olguria (less than 500 ml/day), anuria
2. Creatinine clearance test
3. End product of hormone
4. Urinary total protein
Procedure of collection:
1. At least 3-4 L bottle or container with wide mouth and screw cap is required
2. All urine should be collected
3. The container should be labeled
4. Before collection, the patient is advised to avoid some drugs
5. Preservation: To prevent bacterial contamination and urea decomposition
6. Bottle should be kept capped.
Random sample:
Indication:
1. Qualitative test
2. Detection of abnormal constituents of urine e.g. glucose, protein.
(Ref: Lecture SSMC)

Q. What are the tests of urine?

Q. Name the test to detect sugar, protein, ketone body and bile salt in urine. (DU-16Ju)
Ans
Laboratory testing for routine urine analysis: (V.V.I. for VIVA)
A. Physical examination:
ml/day)
a. Volume: 1 - 2.5 L/day (average 1 500
b. Colour: Straw (pale yellow)
c. Odour: Pungent
transparent
d. Appearance: Clear/
e. Sedimentation: Absent
L Specific gravity: 1.010-1.020
g. Osmolarity: 600-900
mosm/L

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Chemical examination:
a. pH - Litmus paper test
—— —Acidic (45-6.8)
Albumin Heat coagulation test Usually nil or in a trace amount (<30 mg/day)
-

— —
c. Sugar - Benedict test » Nil
<1- Excess phosphate Nil

—
c* Bile salts- Hay’s surface tension test
f. Bile pigment - Foucher’s test
—
Nil
g. Ketone bodies - Rothera’s test * Nil
—
h. Blood - Orthotolidin test » Nil
Nil

C- Microscopic examination:
Cclls-
a. RBC - Absent
b. Pus cells - 0-3/ HPF (in male), 0-5 (in female)
c. Epithelial cell — Occasional
Casts-
a. RBC casts - Absent
b. WBC casts - Absent
c. Epithelial casts - Absent
d. Granular casts - Absent
e. Hyaline casts - Absent
Crystals and others-
a. Urates - Absent
b. Uric acid - Absent
c. Calcium oxalate - Absent.

ForVIVA:
A- Physical examination:
I. Volume: May be polyuria. oligouria. anuria.
• Polyuria (> 3000 ml'day) found m:
a. Diabetes mellitus
b. Diabetes insipidus
Oliguria (< 500 ml /day) found in:
a. Renal failure and cardiac failure
b. Excessive loss of fluid in diarrhea, vomiting or bum
c. zXcute glomerulonephritis ( AGN)
d. Nephrotic syndrome
c. Urinary tract obstruction
• Anuria (< 50 ml day ) found in:
a. Shock
b. Bilateral obstruction of ureters.
2. Colour:
.Vrrmrco/ourrNwmalurincductourochronicandurobilinoecn
Ydhnr: In jaundice (due to bilirubin or excess urobilinogen)
Red: Due to lib and RBC
• Milky: Due to chy luria
• Pinkish: Duc to rifampicin fanti-TB druz)

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 < H AP I I H 07: CLINICAL niWIIEMISTRV I 422
3* Appcaraneei
1 cell#. ctccmivc pin cell
• htnftM In urinary ‘

115?®»
Albumin -Tien testISbS’*'1'0" “"<1P™1'1'*
Sugar - Benedict
acidosis.
tCSt P^lnurla) in
< DA»
Bile salts- th
5- Bile pigment (bilirubM^ t*'0*1
OUcl'cl s
'" ^otl"tl P03'1^ in obstructive jaundice)
(Tottnd positive in conjugated hyperbilirubinemia)
b- Ketone bodies - Roti
7' Blood - Orthotnli<i;»
>” ’
S ,CS*
,cst

Positive in ketosis and ketoacidosis)

051 (f°und positive in hematuria, hemoglobinuria).
।

Stool Examination
1. Fecal fat estimation (impairment
in fat digestion)
2. Detection of reducing substances (e.g.
Lactose)
3. Routine examination.

Organ Function Tests

Liver Function Tests (LFT)
(V.V.I. for VIVA)

Q. State liver function tests briefly. (DU-17Ja,16Ju,15Nv, RU-16Nv, SU-16Nv) What are the
indications of liver function tests? (CU-15Nv)
Q. Enumerate the liver function tests with their utility. (DU-16Nv,13Ju)
Q. Write the liver function tests (DU-18/08Ja, CU-17Nv, SU-14Ja,12Ju,ll/0SJa, RU-14Ja) with
their interpretation. (SU-18/17M, CU-17M)
Q. Name some clinically important liver function tests. (RU-19M, CU-16Nv)
Q. Name the liver function tests with their normal values & clinical importance. (SU-17M, CU-
15M,09Ja,07Ju)
Q. What are the commonly done liver function tests? (DU-lOJa, SU-08Ja)
Q. How liver function tests are assessed in the laboratory. (DU-16Ja)
(RU-14Ju)
Q. Discuss the tests for evaluating chronic liver disease.
the excretory and synthetic functions of liver. (RU-
Q. Describe the biochemical tests to assess
time and serum albumin level. (RU-17M)
0 Write the reference ranges of prothrombin
Q.Shortnote^Livcr function tests. (DU-19Nv,RU-15Ja,08Ja)

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g
Liver function tests: Liver function tests nre n group of biochemical investigation which is don
of liver disease.
Indications of liver function tests:
1. Differential diagnosis of jaundice
2. Diagnosis of liver disease
3. Assessment of the severity of liver disease
4. Assessment of the prognosis of liver disease
5. Treatment monitoring
6. Detection of complication.
Commonly done liver function tests with normal vales:
1. Scrum bilirubin concentration (normal = 0.6-1 mg/dl)
2. Serum total protein (normal = 6-8 g/dl)
3. Serum albumin level (normal = 3.5-5 g/dl)
4. Serum albumin-globulin ratio (normal = 1.5 : 1)
5. Prothrombin time (PT) (normal = 12-16 seconds)
6. Enzyme level-
- AST / SGOT - (10-45 IU/L)
ALT / SGPT - (70-50 IU/L)
ALP- (40-125 IU/L)
y-glutamyl transferase (y-GT) (5 - 55 IU/L)
7. Galactose tolerance test (for metabolic function of liver).
(Ref: Lecture SSMC)

|Or, The answer may be like the following pattenu]

The major liver function tests may be classified as follows-
1. Tests for synthetic functions:
a. Total plasma protein cone, (normal 6-8 gm/dl)
b. Serum albumin level (normal 3.5-5 gm/dl)
c. Albumin & globulin ratio (1.5: 1)
d. Prothrombin time (Normal 12-16 sec)
2. Tests for excretory function:
a. Serum bilirubin cone. (0.6 - 1 mg/dl)
b. Van den Bergh test
c. Urinary bilirubin and urobilinogen
d. Stercobilinogen (in stool)
3. Tests for enzymatic function:
a. Alanine aminotransferase (ALT) / Serum Glutamate Pyruvate Transaminase (SGPT)
b. Aspertate aminotransferase (AST) / Serum Glutamate Oxaloacctate Transaminase (SGOT)
c. Alkaline phosphatase (ALP)
d. y-glutamyl transferase (y-GT)
e. Serum 5-nucleotidase
4. Tests for metabolic function:
a. Glucose tolerance test
b. Galactose tolerance test
c. Serum ammonia

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e- Fecal fatcholBim,!S
S'W*™ 07: CLINICAL BIOCHEMISTRY I W
analysis

• nippunc netd test

b- NHj cone, in blood
61 Other Investigations:
a. Viral markers in hepatitis
A. B. C, D, and E.
(Ref: Sntynnarayana/3H/454; Lecture SSMC)
I. The liver function tests (LFT) n hi
FC n'^ncnuc®! investigations to assess the capacity of the liver to
- , . . j
lunrtinn it•/performs
carrv out any of the •wivuun JC

4.
To foK
Differential diagnosis and progress ofjanndiee.
(Ref: Satyanarayana/3rt/454)
f important ,iver function tests. (SU-09Ja)
O
O S
O Whot nr th
*
J
"Jes
fUnCd°n tests with interpretation. (SU-15Ja)
m’Jasurcd 'n liver function tests with their interpretation. (RU-19M)

each of them. (DU-10/09Ja)CaI *" hepatic disease? Write d°"n the interpretations of
Q. Draw a table showing the role of serum enzyme assay in differentiating various liver
disorders. (RU- 11 Ju)
Q. Mention the enzymes released from diseased liver tissue and state their clinical importance.
(RU-lOJu)
Ans.
Interpretation of some important liver function tests: (‘“V.V.I. for VIVA)
H Liver function tests (LFT) Normal value Interpretation
ALT/SGPT 10-50 IU/L Increase markedly in hepatic parenchymal liver
disease (e.g. hepatitis)
AST/SGOT 10-45 IU/L Increase markedly in hepatic parenchymal liver
disease. Also in non-hepatic diseases e.g. MI, muscle
disease
ALP 40-125 IU/L Increase markedly in hepatobiliary diseases (e.g.
biliary obstruction)
Increase in hepatobiliary disease, alcoholism
y-glutamyl transferase (y-GT) 5 - 55 IU/L (alcoholic liver disease)
3.5 - 5 g/dl Decrease in chronic liver disease
Serum albumin Altered in chronic liver diseases
Albumin-globulin ratio (A:G) 1.5: 1 mg/dl Increase in different types of jaundice
0.6 - 1
Serum bilirubin Hemolytic jaundice: Unconjugated
• Hepatocellular: Both conjugated and unconjugated
Obstructive: Conjugated
Differentiate types of jaundice;
Van den Bergh test Hemolytic jaundice: Indirect + ve
Hepatocellular jaundice: Biphasic
Obstructive jaundice: Direct + ve
(Ref: Lecture SSMC & DMC)

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Q. Mention some tests for cellular damage of liver. (SU-1 1 Ju)
Ans.
Tests for cellular damage of liver:
1 . Alanine aminotransferase (ALT)
2. Aspartate aminotransferase (AST)
3. Scrum albumin
4. Albumin-globulin ratio (A : G)
5. Scrum bilirubin.
(Ref: Lecture SSMC & DMC)
(CU-05Ju)
Q. Name the liver function tests required for a person with 12.5 mg/ dl of serum bilirubin.
Ans.
Liver function test (scrum bilirubin 12.5 mg/dl):
1 . Determination of scrum enzymes
a) ALT/SGPT
b) AST/SGOT
c) Alkaline phosphatase
d) y-glutamyl transferase (y-GT)
2. Serum total protein
3. Serum albumin
4. Albumin globulin ratio (A:G)
5. Prothrombin time (PT).
(Ref: Lecture SSMC & DMC)

Q. Which tests differentiate between chronic and acute liver disease? Explain. (V.V.I. for VIVA)
Ans.
Serum albumin & globulin ratio
Serum prothrombin time (PT).
Normal A:G ratio is 1.5:1; in chronic liver disease it becomes reverse, e.g. 0.8:1 or 0.7:1. If A:G ratio is not
reversed a diagnosis of chronic liver disease can never be made.
Liver synthesizes almost all of the coagulation factors. In chronic liver disease plasma concentration of
coagulation factors are reduced which is recognized by prolongation of prothombin time (PT).
(Ref: Lecture CMC)

Q. A patient with history of jaundice 6 months ago found with A: G ratio 0.6:1, what may be
the cause?
Ans.
Chronic liver disease.
(Ref: Lecture CMC)

Q. Which important enzymes rise in hepatic parenchymal diseases (e.g. hepatitis)?

Ans.
In hepatic parenchymal diseases serum level of following enzymes rise:
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Lactate dehydrogenase
Isocitrate dehydrogenase.
(Ref: Lecture CMC)

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 Q. Which onC ,S CHAPTER 07; CLINICAL BIOCHEMISTRY J 501
Ans. morc sPecinc liver function test- ALT
or AST? Explain. (V.V.I. for VIVA)
ALT is
addition to liver1 Tl^°r°f°rC’
failure and nnisrlr.
dama8c ^an AST. AST is also present in heart, brain, kidney and muscle in
Jcvc'to liverAST also can be seen in myocardial infarction, congestive cardiac
J ry, v
in addition damage. That's whyALT is morc specific that AST.
(Ref: Lecture CMC)
Q. Which enzyme Increase
slgnlllcanfly In biliary obsfruedon? (V.V.I. for VIVA)
caiMbcuii^nT^
synthesized in the hemtnr
increases significantly in biliary obstruction. It is present primarily in bile
lbranc of hepatocytes. In biliary tract obstruction at any level, new ALP
*
the main indicator of WUaiytacS^
j
is cscaPcd into thc b|o°d. A greatly increased plasma ALP is therefore
Destruction though it docs not provide information about thc site of obstruction.
(Ref: Lecture CMC)
h
Aris"indicates
ALT
biochemical test indicates that liver is inflammed? (V.V.I. for VIVA)
that the liver is inflammed.
(Ref: Lecture CMC)
*S acu^e v*rai hepatitis? What bio-chemical
test will you do in acute viral hepatitis?

^cute x iral hepatitis: Acute viral hepatitis

hepatitis A, B, C, D or E viruses.
is a disease in which acute inflammation of liver occurs due to

Tests for viral hepatitis: The following bio-chemical test should be

done for the diagnosis of acute viral
hepatitis: (V.V.I. for VIVA)

- ALT (markedly increase)

AST
ALP
Serum bilirubin level.
(Ref: Lecture CMC)

Q. Enumerate the importance of prothrombin time and serum albumin level in relation to
hepatic failure. (RU-17M)
Ans.
Prothrombin time in relation to hepatic failure: The clotting factors involved in the prothrombin time (PT)
are factor I, II, V, VII and X. All are produced by liver and for synthesis of factor II, VII, IX and X vitamin K
is needed. Prolonged PT indicates the deficiency of concerned clotting factors due to hepatic failure or vitamin
K deficiency. If PT remains increased even after administration of vitamin-K, then hepatic failure is confirm.
Serum albumin level in relation to hepatic failure: Synthesis of albumin exclusively happens in liver. So, in
hepatic failure serum concentration of total protein and albumin decreases markedly.

Renal Function Tests

O Name the kidney function test.
(DU-05Ja, RU-19Nv,13Ja, CU-19Nv)
n
Q. Briefly dLuss
p • nv discusi the biochemical tests done
to assess kidney function. (DU-09/08Ju)
assessed in biochemistry laboratory? (DU-11 Ju)
Q. How renal func on can be ?
Q. How renal functions are awe

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arirs representation on MEDICAL BIOCHEMISTRY ...
• Give their interpretation.
Q. Name the tests done in biochemistry lab to assess renal functions.
(DU-17M,14Ja)
Q. Short note: Renal function test. (DU-18M)
Ans.
Please see the chapter of “Renal Biochemistry".
Q. Name the renal plasma clearance test with their clinical importance. (DU
Ans.
Please sec the chapter of “Renal Biochemistry"
Q. Short note: Proteinuria. (SU-17Nv, RU-16/15Nv)
Ans.
Please see the chapter of “Renal Biochemistry".

Thyroid Function Tests

Q. Enumerate the thyroid function tests. (DU- 05 Ja) ,/n<-T
Q. List the thyroid function tests. Give the clinical importance of each test. (LU-Ub/injaj
Ans.
Please see the chapter of “Clinical Endocrinology”.

Colorimetry & Photometry

Q. What is colorimetry? What are the operations involved in its procedure.
Q. Short note: Colorimetry. (V.V.I. for VIVA)
Ans.
Colorimetry: Colorimetry is based upon the matching of a coloured solution representing an unknown
concentration of the substance undergoing analysis with a standard solution of specific colour representing the
substance of known concentration.
Colorimetric procedure involves 3 operations:
a) Preparation of the coloured substance to represent the unknown (test)
b) Obtaining the suitable colour (standard)
c) Colour matching by an instrument which is called the colorimeter.
(Ref: Lecture SSMC)
Q. What is colorimeter? (V.V.I. for VIVA)
Ans.
Colorimeter: Colorimeter is the instrument used to facilitate exact matching of two coloured solution and used
for quantitative estimation in colorimetric analysis.
The working of colorimeter is based on the principle of Beer-Lambert law (discussed below).

Q. What are the types of colorimeter? (V.V.I. for VIVA)

Ans.
Types of colorimeter:
(i) Visual colorimeter: Estimation of Hb by acid haematin method.
(ii) Photoelectric colorimeter: Blood sugar estimation by Folin
Wu method.
Q. What are the parts of colorimeter or photometer?

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 503
CHAPTER 07; CLINICAL BIOCHEMIS TRY
Ans.
Parts of colorimeter or photometer: (V.V.I. for VIVA)
a) A source of light (c.g. a
filament lamp Is usually serves a //«/»/ source)
b) A means of selecting monochromatic light of suitable wave length (filter) c niters allow
the
passage of a small range of wave length as incident light)
c) Cuvette
d) Sample holder
c) Detectors (photo cells)
f) Single beam or double beam optical path
g) Display (a suitable Galvanometer which can display the light absorbance).
The diagrammatic representation of a colorimeter is depicted in below:

Light Display
source * Filter
* Cuvette
* Detector

Fig: Diagrammatic representation of the components in a colorimeter.

(Ref: Lecture SSMC; Satyanarayana/3rd/727)

Q. Draw and label the basic parts of a photoelectric colorimeter. (DU-18Nv)

Ans.
Basic parts of a photoelectric colorimeter:
Polychromatic light Monochromatic light

Light source holder

Fig: Basic parts of a photoelectric colorimeter.

for VIVA)
Q. What is filter? (V.V.I.
Ans.
selecting near monochromatic light of suitable web length.
Filter: It is means of

density (OD)? (SU-13Ja)

Q. What is optical density (O.D). (V.V.I.
for VIVA)
Q. Short note: Optical
the intensity of the incident to the
Ans. density (O.D) is the logarithm of the ratio of
Optical density:Optical
light.
emergent (transmitted)
O.D = log y-

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— | ariPs representation on MEDICAL BIOCHEMISTRY
Here, Io” Intensity of incident light,
h ” Intensity of emergent light.
Optical density or absorbance or extinction is the amount of light which is absorbed by particles ot a coloured
solution while passing through it (within colorimeter).
It is inversely & logarithmically related with the concentration of the compound in a solution.

O.D or A= log —T1

When the light path is constant, the optical density or absorbance is directly proportional to the concentration
of the substance in solution.
, OD of the unknown subs tan ce x cone, of the standard.
r'
Concentration ot~ the unknown substance =
O.D of the known substance

Q. Observation in colorimeter of an unknown supplied sample is as follows: ODc = 1.10; ODs =

0.45. Given cone, of standard cholesterol is 200 mg/dl. (OD= optical density). Find out the cone,
of that sample and give your comment on result. (CU-14Ju)
_
Ans.
. ,
Concentration of the unknown substance =
OJD of theunknown substance cone,
x
,
ot the standard.
O.D of theknown substance
_ 0-45 x 2qq mg/j| = 81.82 mg/dl.
1.10

Q. Define photometry. (DU-19Nv,18/17M,15Ja, SU-13Ja) (V.V.I. for VIVA)

Q. Write a brief about photometry. (DU-07Ja)
Q. Short note: Photometry I Spectophotometry. (DU-16Ju,08/07Ja, CU-19Nv, SU-16Nv/M)
Ans.
Spectophotometry / photometry': It is an analytical procedure based upon the direct measurement of light
absorption at specific wave length (regions of specton) are known as photometric or spectophotometric
procedures and the instruments used are called photometer or spectrophotometer.
Principle of photometry’: Please see later.
It involves the folloyying operations:
1. Separation of the substance undergo analysis-
a) Precipitation
b) Absorption
c) Dialysis
d) Adsorption
2. Separated substances are coloured or made to be coloured
3. Measurement of light absorption by photometer
4. Calculation.
(Ref: Lecture SSMC)
Q. Write down the principles of colorimetry. (DU-19Nv,18/17M,15/05Ja) (V.V.I. for VIVA)
Q. Define Beer’s layv and Lambert’s layv. (SU-05Ju)
Ans.

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 1
Principles or
the absorption of ligln10^' ^CICnt'^c ^as's
CHAPTER 07; CLINICAL BIOCHEMISTRY
colorimeter is the laws of light. The two basic laws
Jgoverning
505

1) Beer’s law
*■) Lambert’s law
Peer’ law; The amount of absorption of light is
of solution. The directly proportional to the concentration of the substance
°fabsorP*’on of light increases exponentially with increasing concentration of the
substance in the soluf
2. j^ambert s law. The intensity of the
transmitted (emergent) light decreases exponentially with the
increasing length of light path (thickness of the medium).
(Ref: Lecture SSMC)
Q. State the basic principles of photometry. (DU-16M, SU-13Ja)
Q’?' °"n Photometric principle and the laws applied to the photometry.
Photometric principle: (V.V.I. for VIVA)
a. The substances in solution are colored or made to be colored.
of the color depends on the concentration of the substance.
c. ro ored substance absorbs light at a definite wave length. The degree of absorption depends on the
concentration of the substance.
Laws of photometry’:
I. Beer’s law: Please see above.
2. Lambert’s law: Please see above.

Q. Write down the differences between spectrophotometer and colorimeter.

Ans.
Differences between spectrophotometer and colorimeter: (V.V.I. for VIVA)
H Subjects Spectrophotometer Colorimeter
1. Monochromator 1 . Prism 1. Filter
2. Spectral purity 2. High degree 2. low (relatively)
3. Slit pores 3. Two partition of slit pores 3. No partition of slit pores
4. Straight light in the exit beem 4. Minimum 4. Not minimum
5. Accuracy 5. Greater 5. Lesser
6. Sample needed 6. Small amount 6. Relatively larger amount
7. Auto calibrated (we can programme) 7. Not auto calibrated
7. Calibration
8. Easier 8. Relatively not easier
8. To handle
9. Advantages 9. Absorbance can be measured with 9. It is not possible
different wave length at the same time
continuously
(Ref: Lecture SSMC)

Transmittance
Q. Define transmittance.
Ans. is inversely and logarithmically
related with the concentration of the
Transmittance; Transmittance
compound in a solution.

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Quality Control
(V.V.I. for VIVA)
Q« XMlat do you mean bv quality control of biochemical lab? Write about its components in short.
(SU-l5Ja,12Ju)
Q. Define accuracy, precision, sensitivity & specificity. (DU-18M,15/l3/06Ja, SU-12/06Ja)
Q. Short note: Quality control. (DU-13Ju,l lJa,09/08Ju, SU-16Nv,15M,13Ja,09Ju)
Ans.
Quality control: It is the system to reduce the variants (errors) in the laboratory results by improving
accuracy, precision, sensitivity and specificity, which involves number of systems.
Components: Quality control comprises four interrelated factors namely accuracy, precision, sensitivity and
specificity.
Accuracy: The degree of closeness of measured value to true value is known as accuracy, e.g. if true blood
urea level is 40 mg/dl. the laboratory reporting 35 mg/dl is more accurate than the one reporting 30 mg/dl.
Precision: The agreement between the replicate measurements on the same or identical sample by repeated
tests is known as precision. For instance, the person is good, if the blood glucose level is 80, 82 & 84 on
replicates.
Sensitivity: It is the analytical ability by which smallest amount of analyte can be measured in the sample by
an analytical method.
Specificity: The degree of accuracy by which a definite analyte can be measured without interference from the
co-existing substance in the solution of sample is known as specificity.
Methods of quality control:
1. Internal quality control: Internal quality control refers to the analysis of the same pooled sample on
different days in a laboratory; the results should vary within a narrow range.
The procedure of utilizing the results of only one laboratory minimize for quality control purpose
which analyze the specimen utilized e.g. If the specimen from DMCH is taken to the DMC clinical
pathology department.
2. External quality control: It deals with the analysis of a sample received from outside, usually from a
national or regional quality control centre. The results obtained are then compared.
The procedure of utilizing the results of different laboratories for quality control purpose which
analyze the specimen utilized e.g. if the specimen from Mitford Hospital is taken to the BIRDEM
Hospital for diagnosis.
(Ref: Lecture SSMC)

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 Reliability: Continuous maintenance
c ot the accuracy,
„
precision, sensitivity & specificity for a long period.
Variance: Disagrecment/differenree ,
among .1.
the results of the measure of an analyte in same sample.
(Ref: Lecture SSMC)

Normal Value
Q. What is normal value? (V.V.I. for
VIVA)
Q. Short note: Normal value.
Ans.
^ojmal •value. A particular value that is normal for a healthy single person without any signs and symptoms
of disease is called normal value.
Or,
The concentration of a substance in normal condition in respect of age and sex is called normal value.
Normal value = Mean value ± 2SD. (SD= Standard deviation)
Factors influencing the normal value:
1. Patient’s age, sex, race, social class, diet, physiological factors (pregnancy or environment), posture,
diurnal and other cyclic variations, fasting or postprandial state, drugs and level of exercise.
2. Laboratory analytical method employed.
3. Method of collection and preservation of the specimen.
4. Time of collection.
5. Handling of sample for analysis.
6. Lab accuracy.
7. Seasonal change.

Q. What is reference value? (RU-16Nv)

are the values of particular quantity obtained from the individuals in
Reference value: Reference values
different states of health.

Q. Mention the reference serum levels of (in both SI and conventional uni.)- (V.V.I. for VIVA)
Triacylglycerol (RU-13/12Ja)
. Glucose / fasting glucose
Urea(RU-16Nv,13Ja,SU-05Ju)
£y-'6Nv) - Bilirubin (RU-16Nv,15M,13Ja,12Ju)
• Plasma osmolality
BUN(Rdu;R3S^ Serum total protein (RU-15M,13Ja)
Serum albumin (RU-13Ja,12Ju)
Serum creatinine (RU-16Nv,12Ju)
- Serum calcium
(RU-13Ja)

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—
MIK I

Ans.
| Name
I
„ .p
^^presentation on MEDICAL IIKK
SCr“m phosPhnte (RU-IMn)
III.M1STRV
. F.ulng blood glucose (RU-l5M,13Ja)

OjnventlonnHmlHmgA^
Blood urea 2. 5-6.6 mmol 1 1 5-40 mg/dl
" ‘ '
BUN 7-21 mg/dl
Scrum uric acid Men: 0.18-0.54 mmol/L Men: 3-7 mg/dl
W omen: 0.15-0.45 mmol/L Women: 2-5 mg/dl
Scrum ervaum ne 60-120 pmol 1 0,7- 1.4 mg/dl
[Total scrum cholesterol Z8-8.3 mmol/L 150-220 mg/dl
' Scrum tnglyecnde 0.0-2.0 mmol/L 60-150 mg/dl
1 ni bosmolality
i 1inibin 3.5-20.5 ptnol/L 0.6-1 mg/dl
Plasma 285-295 mosnVkg
| Scrum total protein 64-83 gm/L 6.4-8. 3 g/dl
i Scrum albumin 35-50 gm/L 3.5-5 g/dl
| Scrum elobulin 26-41 gm/L 2.6-4. 1 g/dl
Scrum fibrinogen 2-6 gnVL (average 3 gin/L) 0.2-0. 6 g/dl (average 0.3 gm/dl)
Scrum prothrombin 1 -2 gm/L 0.1 -0.2 g/dl
Scrum calcium 2.4 mmol/L 9- 1 1 mg/dl
Scrum phosphate 1 .0 mmol/L 3.5 mg/dl
* Fasting blood glucose 3.6-6. 1 mmol/L 65-1 10 mg/dl
(Ref: Harper’s/25,h/Appendix; Lecture SSMC)

Q. Mention the common serum electrolytes with their normal values in SI unit.
Q. Mention the normal values of serum electrolytes.
Q. Mention the reference serum levels of Na+ (RU-16Nv,12Ju), K+ (RU-16Nv), Cl' (RU-16Nv),
HCO3 . Ca:* (RU-16Nv,13Ja,12Ju), Phosphate (RU-13Ja)
Ans.
Common serum electrolytes with their normal values: (V.V.I. for VIVA)
Constituents SU unit (mmol/L) Non-SI unit (mEq/L) I
Na' 135-145 mmol/L 135-145 mEq/L
K* 3.5-5 mmol/L 3.5-5 mEq/L
Ca*' 2. 1-2.6 mmol/L 4.5-6 mEq/L (9-11 mg/dl)
Cl 95-106 mmol/L 95-106 mEq/L
HCOj 24-28 mmol/L 24-28 mEq/L
POZ 1-3 mmol/L 3-9 mEq/L

Plasma Proteins
Q. Give the values of plasma proteins. List their important functions.
Q. Name the plasma proteins and state the functions of albumin. (RU-18Nv)
Q. Short note: Plasma proteins. (RU-15Ja)
Ans.
Plasma proteins: Plasma proteins are a complex mixture (over 200) of proteins that are structurally and
functionally different from each other.
| Major plasma proteins
| Albumin
Other plasma proteins 1
| Angiotensinogen 1

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 TRY 509
CHAPTER 07: CLINICAL HIOCHEMIS
Globulin Thromboplastin
a, globulin, c.g. arantitrypsin Immunoglobulin
a? globulin, c.g. haptoglobin Transferrin
• P globulin, eg transferrin Ceruloplasmin
y globulin, c.g. immunoglobulins (antibodies) Haptoglobin
Fibrinogen Antithrombin-Ill
Prothrombin CRP (C-rcactivc protein)
Coagulation factor VII. IX, X
Thyroid binding globulin (TBG) etc.
Nnrm.nl values of plasma protein:

| Total plasma protein 64-83 gm/L

Non-SI unit (grn/dl)^^^
6.4-8.3 grn/ai
_
3.5-5 gm/dl
1 Scrurnalbumin (50% of plasma protein) 35-50 gm/L
26-4,1 gm/dl
1 Scrum globulin 26-41 gmzL
/ Scrum fibrinogen 2-6 gm'L (average 3 0.2-0.6 g/dl (axerage 0.3
gm/L)
[ Scrum prothrombin 1-2 gm/L 0,1 -0.2 gdl

Source of plasma proteins: All plasma proteins are produced by liver except the y-globulin which is produced
by plasma cells (B-cells).
Functions ofcach of the plasma proteins: (V.V.I. for VIVA)
1. Albumin a. Maintains 75-80% colloidal osmotic pressure in plasma
b. Maintains viscosity of blood
c. Acts as a buffer and maintains acid-base balance
d. Acts as a carrier of calcium, fatty acid, bilirubin, hormone and drugs
e. Acts as a protein reservoir
2. Globulin 1 . Maintains colloidal osmotic pressure (20%)
2. Maintains viscosity of blood
3. di-globulin includes antiprotease and transports lipid & steroids
4. dz-globulin transports Cu as ceruloplasmin
5. p-globulin transports Fe as transferring
6. y-globulin acts as antibody
3. Fibrinogen 1 . Essential for blood coagulation
2. Increases ESR and helps to maintain viscosity of blood
4. Prothrombin 1 . Essential for blood coagulation
(Ref: Samson Wright's/ 13^/21; Harper’s/291!>)

Q. Give the properties of plasma proteins.

Ans.
Properties of plasma proteins:
1. Precipitation by salt
2. Fractional precipitation
3. Sedimentation by ultracentrifuge
4. Iso-electric point
5. Buffer action: Plasma proteins act as buffers by virtue of their power of H* acceptance, but
account for less than one-sixth of the total buffering power of the blood.
6. Molecular weight and shape:

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510 arifs representation on MEDICAL BIOCHEMISTRY
Plasma proteins Molecular weight I
Albumin 69,000
1 Fibrinogen J 3,30,000

7. Osmotic effect: The plasma proteins normally have an osmotic effect of 25 mm Hg and thus influence
the exchange of fluid between the blood and tissue spaces.
8. X iscosity: The resistance to the flow of fluid (at constant velocity) through a capillary (at constant
bore) depends almost entirely on the viscosity of the fluid. The viscosity of the blood is thus a factor in
maintaining the peripheral resistance and thereby, the arterial blood pressure. The viscosity of protein
solution depends far more on the shape of the protein than on its size. The less symmetrical the
molecule, the greater is its viscosity.
9. Electrophoretic mobility
10. Transport action: Plasma proteins combine loosely with many chemical agents, including hormones,
e.g. thyroxin & cortisol; metals e.g. iron & copper; and numerous drugs.
(Ref: Samson Wrigh’s/13,b/20-22)

Q. Give the applicable clinical importance of plasma protein. (CU-15M)

Q. State the clinical importance of albumin. (RU-18Nv)
Ans.
Clinical importance of plasma protein (albumin):
A. Hvpoproteinaemia:
1. Increased loss:
GIT: Protein loosing enteropathy.
Kidney' (proteinuria): Microalbuminuria (e.g. early diabetic nephropathy, hypertension etc.),
nephritic syndrome, nephrotic syndrome.
Skin: Bum.
2. Increased catabolism: Infection / trauma / thyrotoxicosis.
3. Decreased synthesis:
Liver: Chronic liver disease (CLD).
GIT: Protein energy malnutrition (PEM).
B. Hyperproteinaemia: Increased immunoglobulin synthesis (hypergammaglobulinaemia) e.g. in
multiple myeloma.

Q. What are immunoglobulins? Name the immunoglobiulins. State their important functions.
Q. Short note: Immunoglobulin. (RU-03M)
Ans.
Immunoglobulin: The antibodies are gamma globulins called immunoglobulins.
They usually constitute 20%
of all the plasma proteins.
(Ref: Guvton/12rt/437)
Or,
These are globulin of animal origin endowed antibody activity or structurally similar proteins having no
antibody activity are also called Ig.
(Ref: Akram’s Immunology/2“d/29)
Classification: 1g are divided into 5 classes-
1. IgG contains y chain
2. IgA contains a chain
3. IgM contains p chain
4. IgE contains e chain

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 CHAPTER 07: CLINICAT BIOCHEMISTRY 511
5, IgD contains 6 chain

Functions of Immunoglobulin;
1. Neutralize toxin and viruses
2. Opsonize microbes so thev arc mnr.C
Cns y Phagocytoscd
3. Activate complement
4. Prevent the Lohmen, of microbra

l^arlont funedont
7^G(75%)
Crosses the^nccnt^
* Complement activifini! PrUVK
cnsicr to Phagocytosc .. .
Cs immunjty 10 the infants upto 6 months after birth

72(15%) B protects 1111irnnS mr.inh-nn.1 f T ; — 7

TgM (9%) of
boctena and vtrwtes
2 Neutralization of toxins
r^D(02°/o) l unction is
uncertain
IgE (0.004%)
-—rpT^Ct as anl^cn receptor on the surface of B cell
mnl'T
P™uOr!
"’1mcdiatc hypersensitivity (anaphylactic reaction) by causing release of
mast cclls & basophils upon exposure to antigen (allergen)
r
'l2Yld£^elense against parasitic infections
(Ref: Park/20"7100; Akram’s Immunology/2nd/31-36; Lecture SSMC)

Blood Lipids & Lipid Profile

Q. Name the blood lipids (plasma lipids) with their normal value.
Q. List the plasma lipids with reference values. (DU-13Ju/Ja, SU-14Ju, RU-09/08Ju)
Ans.
Blood lipids with their normal value: Total lipid: 450-1000 mg/dl.
1 Blood lipids SI unit (mmol/L) Conventional unit (mg/dl) ,

1. Triacylglycerol (45%) 0.9-2.0 mmol/L 60-150 mg/dl

2. Cholesterol (15%) 2.8-8.3 mmol/L 150-220 mg/dl
3. Phospholipid (35%) 1.8-5.8 mmol/L 145-200 mg/dl
4. Free fatty acid (5%) 0.2-0.6 mmol/L 6-16 mg/dl
(Ref: Harper’s/25"7269)

Q. What is lipid profile? Give their normal reference value. (DU-18Nv,17Nv/M/Ja,16M/Ja,15M/

Ja, CU-15Nv,06Ju) (V.V.I. for VIVA)
Q. List the components of lipid profile with reference value. (CU-19Nv, RU-19M) Mention the
indications of lipid profile measurement. (RU-19M)
Q. Give the values of lipid profile. Mention their clinical importance. (CU-12Ju)
Q. Briefly discuss the clinical significance of scrum lipid profile. (RU-15M)
Q. Short note: Lipid Profile. (DU-19Nv,18M,14Ja, RU-19/18/17Nv,16M,15Ju,12Ja, SU-
16M,15Ja,12/09Ju)
^d profile: The term -Lipid profile- means the pattern or status of the lipids in plasma.
The r profile means “group of laboratory tests done to assess lipid status of an individual and to
ihe term lipid Q
evaluate risk factors for atherosclerosis ana
coroi y artery disease (CAD)’’.
coronary

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_
.
Components of lipid profile v ith their normal values: Lipid
following parameters-
profile k commonly determined
oy me—
Parameters
SI unit (mmol/L)
1. Total cholesterol ( TC ) 3.9 - J-
150-220
2. Triacylglyccrol (TAG) 60- 150
0.6-1.7
3. LDL-cholesterol < 130 <3.38
4. HDL-cholestcrol >40 >1.0
(To convert mg/dl into mmol/L multiply by 0.026)
Indications / objectives of lipid profile:
1. Diagnosis of dyslipidacmia
2. Assessment of prognosis for a known dyslipidacmic patient
3. Assessment of risk for atherosclerosis and coronary artery disease.
Preparation of a patient for lipid profile testing:
1 . 10-12 hours fasting prior to test
2. Usual diet in previous 3 days (no dietary change allowed)
3. Usual activities in previous 3 days
4. Individual must be free from trauma, surgery, acute infection, pregnancy.
Procedure:
1 . Collect fasting morning blood sample
2. Blood is taken in a plain test tube & allowed to clot
3. Serum is separated and lipid profile components are measured.
Clinical importance:
1 . Screening for cardiovascular disease
2. Increased LDL directly related to cardiovascular
disease
3. Increased HDL inversely related to
cardiovascular disease.
(Ref: Davidson’s/21S‘/45I,1296; Harper’s/25th/869)

Dyslipidemia
Q. What is dyslipidemia?
hyperlipidemia? (CU-15Nv) (DU-16/15M) What are the common causes of secondary
Q. Mention the causes of hyperlipidaemia.
Q. Classify dyslipidemics. (CU-19Nv)
Q. State dyslipidemia. (RU-17M)
(RU-15Ju/Ja)
Q. Short note: Dyslipidemia.
Ans. (RU-14/13Ju)
Dyslipidemia (dvslipoprotcinaemia);
lipoprotein and lipid Group of disorders
concentrations. characterized by abnormal (high or low) plasma
Classification of dyslipidemia
A. Primary hyperlipidemia (dvslipoprotcinaemia):
(primary hyperlipoproteinaemia):
1. Type I: Familial Inherited
2.
hyperchylomicronemia
Type Ila: Familial hyper
3. Type lib: Familial cholesterolemia
4. combined hyperlipidemia
Type III: Familial
5. dysbetalipoprotcinemia (broad beta
Type IV: Familial hyper
triacylglycerolemia disease / remnant removal disease)

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 CHAPTER 07; CLINICAL BIOCHEMISTRY | 5.12
6. Type V: Familial combined hyperlipidemia

P Primary hypolipidemia (primary hypolipoprotcinaemia): Inherited

1. Familial hypobctallpoprotclncmln: Harmless
2. Abetallpoprotelncmla: Associated with decreased absorption of fat and fat soluble vitamins.
3. Familial alpha-lipoprotein deficiency (Tangier disease): Increased risk of atherosclerosis
diseases, c.g.
f. Secondary dyslipidemia: Secondary acquired lipoprotein disorders arc due to some other
1. Diabetes mcllitus
2. Nephrotic syndrome
3. Atherosclerosis
4. Hypothyroidism etc.
(Ref: llnrpcr’s/30"7275; Satayanarayana/3,,’/321)
Clinical importance of dyslipidemia: It contributes to-
1. /Xthcrosclcrotic disorders, c.g.
• Coronary artery disease (CAD)
Cerebrovascular disease (c.g. stroke)
• Peripheral vascular disease (PVD)
2. Fatty liver disease
3. Acute pancreatitis.
(RU-08Ju)
Q. Name the important “Hyperlipoproteinaemias” with their causes and effects.
(SU-13Ja)
Q. Name the main types of hyperlipoproteinaemias and what are the causes?
Q. Short note: Hyperlipoproteinaemia. (RU-12Ja)
Ans.
Hyper lipoproteinaemias: Elevation in one or more of the lipoprotein fractions constitutes lipoproteinaemias.
These disorders may be either primary or secondary.
Name Causes Effects
I Familial hyperchylomicronemia Deficiency Increase in plasma chylomicron and 1
of
lipoprotein triacylglycerol level and no increased risk,
lipase
of coronary disease
Ila Familial hyper cholesterolemia/ Deficiency of Elevated LDL & cholesterol level causing
familial hyper beta LDL receptors atherosclerosis and coronary disease
lipoproteinemia
Hb Familial combined Overproduction Elevation of both LDL and VLDL along
hyperlipidemia of apo-B with plasma cholesterol and TAG causing 1
atherosclerosis and coronary disease
III Familial Abnormality in Elevation of IDL along with cholesterol and |
dysbetalipoproteinemia (broad apo-E TAG causing very high risk of
beta disease / remnant removal atherosclerosis (Mostly in peripheral
disease) vessels)
IV Familial hyper Overproduction of Elevation of TG with a concomitant
triacyl glycerolemia rise in
endogenous TG VLDL and is associated with obesity, DM.
V Familial combined Overproduction of Both chylomicrons and VLDL
are elevated
hyperlipidemia
r and associated with obesity, DM, coronary
1 heart disease etc
(Ret. Harper s/30 "/275;
Satayanarayana/3rt/321)

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causes of secondary hyper,
Q- What do you mean by hypercholMterclenda? Give the
cholesterolemia. (RU-I7M)
Q. What arc the causes of hypercholesterolemia? (DU-18Nv)
Q. State hypercholesterolemia. (RU-15Ja)
Ans. level (150 220 mg/dl or 3.9-
Hypercholesterolemia: Presence of cholesterol in the blood above its normal
5.72 mmol/L) is known ns hypercholesterolemia.
.Causes: It is observed in many disorders-
I . Primary' cause - idiopathic (familial hyper cholesterolemia)
2. Secondary' cause -
Hypothyroidism
Nephrotic syndrome
Chronic renal failure
Diabetes mellitus
Obstructive jaundice
Obesity.
Clinical importance: It contributes to atherosclerotic disorders, e.g.
Coronary artery disease (CAD)
Cerebrovascular disease (e.g. stroke)
Peripheral vascular disease (PVD).
Control of hypercholesterolemia:
I . Consumption of PUFA (polyunsaturated fatty acids)
2. Avoidance of cholesterol rich foods (<300 mg/day)
3. High intake of dietary fiber & plant sterols
4. Avoidance of high carbohydrate diet (e.g. sucrose)
etc.)
5. Lifestyle changing (smoking cessation, regular exercise, blood pressure control
6. Moderate alcohol consumption
7. Use of lipid lowering drugs.
(Ref: Satayanarayana/4,l7316)

Q. State the factors promoting elevation of blood lipids. (CU-17Nv)

Ans.
Factors promoting elevation of blood lipids: cholesterol)
1. Nutritional factors (e.g. excess calories in diet, excess consumption of saturated fat or
2. Hepatic diseases (e.g. hepatitis, obstructive jaundice etc.)
3. Obesity, alcohol abuse
4. Diabetes mellitus
5. Renal diseases (e.g. chronic renal failure, nephrotic syndrome)
6. Endocrine disorders (e.g. hypothyroidism, Cushing syndrome etc.)
7. Drug (e.g. steroid, oral contraceptives etc.)

Q. Enumerate lipid lowering drugs. S

Q. Name two important lipid lowering drugs. (CU-15Nv)
;
Ans.
•
Lipid lowering drugs:
1. HMG-CoA reductase inhibitors (“statins”): \
»
Atorvastatin (widely used in our country)
Lovastatin

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 515
* CHAFFER 07: CLINICAL BIOCIIEMISTKV
Simvastatin
Pravastatin
Fluvastatin
Rosuvastatin
2. Fibrlc ncld derivatives (Flbrafes)
• Fenofibmte
Bczafibratc
• Ciprofibratc
• Gemfibrozil
3. Niacin (nicntlnic acid)
4. Bile acid sequestrants (resins)
• Cholestyramine
Colestipol
Colcscvclam
5. Cholesterol absorption inhibitors
Ezetimibe
6. Highly poly-unsaturated long chain co-3 fatty acids
Eicosapentaenoic acid
Docosahexaenoic acid.
(Ref: Lippincott’s/5"7265,268-273; Laurence/10,l'/474-476; Satayanarayana/4rb/316)

Blood Urea & Serum Creatinine

Q. Write down the normal levels of blood urea and creatinine.
Ans.
Normal levels of blood urea and creatinine: (V.V.I. for VIVA)
Blood urea: 15 - 40 mg /dl or, 2.5-6.6 pmol/L
Serum creatinine: 0.7 - 1.4 mg /dl or, 60-120 pmol/L.

Nice to know:
Normal value of BUN is 7-21 mg/dl.
(Ref: Lecture SSMC)

Q. Give a short description about the clinical interpretation of blood urea assay. (RU-13Ja)
Q. Write the cause of high blood urea.
Ans.
also be increased by:
Causes of high blood urea: Besides renal failure, the level of urea in the blood can
1 . Increased production of urea in the liver, due to:
high protein diet
bum, cancer)
increased protein breakdown (surgery, infection, trauma,
gastrointestinal bleeding
drugs (e.g. tetracyclines and corticosteroids)
2. Decreased elimination of urea, due to: (e.g. hypotension, cardiac failure)
decreased blood flow through kidney
urinary outflow obstruction
bladder rupture
3. Dehydration

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 | anTs representation on MEDICAL BIOCHEMISTRY
4. Chronic infection of the kidney such ns chronic pyelonephritis.

Q. What is uraemia? Write down its causes.

Q. Short note: Uremia. (RU-14Ju)
Ans.
Uraemia:Presence of excess urea in blood above normal is called uraemia.
Or,
syndrome
When the breakdown products of protein metabolism accumulate in blood and develop clinical
called uremia.
Causes of uraemia: (V.V.!. for VIVA)
a. Pre-renal:
i) High protein diet
ii) Severe vomiting
iii) Prolonged diarrhoea
iv) Shock
v) Severe bum
vi) Haemorrhage
b. Renal:
i) Acute renal failure
ii) Nephrotic syndrome
iii) Glomerulonephritis
iv) Pylonephritis
c. Post-renal:
i) Prostatic enlargement
ii) Urinary calculi
iii) Urethral stricture
iv) Tumour.
Symptoms of uremia:
1. Lethargy
2. Anorexia
3. Nausea and vomiting
4. Mental deterioration and confusion.
5. Muscle twitching
6. Convulsion
7. Coma.

Q. What do you mean by BUN and how it is calculated? (V.V.I. for VIVA)
Ans.
BUN: BUN (Blood Urea Nitrogen) means the amount of nitrogen present as urea in blood.
Calculation:
o Amount of N in urea x cone, of urea
t>UN
Molecular wt. of urea
No.of N atom x Atomic wt.of N x cone, of urea
=
Molecular wt. of urea
2x14 x cone, of urea.
=
60

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 517
CHAPTER 07: CLINICAL BIOCHEMISTRY
0.4666 x cone, of urea.
Q. Write the clinical importance of scrum creatinine.
Q. Short note: Scrum creatinine.
Ans.
Creatinine: Creatinine is a break-down product of creatine phosphate in muscle, and is usually produced at a
fairly constant rate by the body (depending on muscle mass).
Normal level: 0.7 - 1.4 mg/dl (60-120 pmol/l). (V.V.I. for VIVA)
Reduced excretion In:
1) Renal failure
2) Competition for tubular secretion (c.g. trimethoprim, cimetidine)
High production in:
1) Rhabdomyolysis
2) Large muscle mass.
High production in:
a. Small muscle mass.
(Ref: Davidson’s/20"7461; Lecture SSMC)

Q. Why serum creatinine level is more specific than serum urea level as a renal function test?
(For VIVA)
Ans.
Serum creatinine level is more specific than serum urea level as a renal function test: Blood urea is a poor
guide to renal excretory function as it is varies with protein intake, liver metabolic capacity and renal
perfusion.
Serum creatinine is more specific as it is produced from muscle at a constant rate and almost completely filtered
at the glomerulus. If muscle mass remains constant, changes in creatinine concentration reflect changes in GFR.
(Ref: Davidson’s/20"7461)

Q. Why serum creatinine level is estimated? (For VIVA)

Ans.
To detect impaired renal function.

Renal Failure
Q. What is renal failure? What are the types of renal failure?
Q. Discuss the laboratory diagnosis of chronic renal failure. (RU-15M,14Ja)
Q. How will you diagnose a case of renal failure? (CU-19Nv,15Ja)
Q. Discuss interpretation of chronic kidney disease. (RU-14Ju)
Q. Short note: Renal failure.
Ans.
Renal failure (RF): Failure of the excretory and other functions of kidney leading to retention of nitrogenous
fluid, electrolyte and acid base balance.
waste products of metabolism with derangements of
Types of RF:
1 . Acute renal failure (ARF)
2. Chrome renal failure (CRF)

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 nriris ^presentation on
MEDICAL BIOCHEMISTRY
/ Acute kidney Injun (AKI): Sudden, rapid and usually reversible (non-
ne ol GFR and loss of renal function w hich develops over a period of days or weeks.
Common causes of .-fRF;
E Prc-rcnal causes:
Hypoxolcmia due to renal or extra-renal salt and water loss
Cardiac failure
Systemic vasodilatation due to sepsis, anaphylaxis etc.
* Hypcr-viscosity syndrome, c.g. polycythemia, multiple myeloma
2. Renal causes, c.g. renal diseases.
3. Post-renal causes, c.g. urinary tract obstruction.
Laboratory' findings in ARF:
Scrum creatinine: High
- Blood urca BUN: High
Renal clearance of creatinine/ urea: Low
• Hyponatremia, hyperkalemia, hyperphosphatemia and hypocalcemia
Features of metabolic acidosis:
a. Blood pH: Low
b. Serum bicarbonate: Low
c. PCO< Low
d. Plasma anion gap: High
Chronic renal failure (CRF) / Chronic kidney disease (CKD): It is an irreversible deterioration in renal
function which classically develops over a period of years.
Common causes of CRF:
a. Chronic glomerulonephritis
b. Chronic pyelonephritis
c. Chronic interstitial nephritis
d. Diabetes mellitus
e. Hypertension
f. Multisystem diseases, e.g. SLE (systemic lupus erythematosus)
g Metabolic conditions, e.g. nephrocalcinosis, multiple myeloma etc.
Laboratory findings of CRF:
1. Serum creatinine: High
2. Blood urea/BUN: High
3. Renal clearance of creatinine/urea: Low
4. Hyponatremia, hyperkalemia, hyperphosphatemia and hypocalcemia
5. Features of metabolic acidosis:
Blood pH: Low
Serum bicarbonate: Low
PCO2: Low
Plasma anion gap: High
6. Serum uric acid: High
7. Proteinuria
of sediments, e.g. cells, casts.
8. Urinary excretion

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 — CHAPTER 07: CLINICAL BIOCHEMISTRY
Blood Urea Nitrogen (BUN) Test
519

urca nitrogen (BUN) test reveals important information about how well your
eys an iver arc working.
It measures the level of urca nitrogen in a sample of the patient’s blood.
Urea is a substance that is formed in the liver when the body breaks down protein. Urca then circulates in the
blood in the form of urca nitrogen. In healthy people, most urca nitrogen is filtered out by the kidneys and
leaves the body in the urine. If the patient's kidneys are not functioning properly or if the body is using large
amounts of protein, the BUN level will rise. If the patient has severe liver disease, the BUN will drop.

BUN is an indication of renal health. If Glomerular Filtration Rate (GFR)and blood volume decrease
(hypovolemia) then BUN will increase. Other factors
responsible for its increment are fever,
increased catabolism, high-protein diet and gastrointestinal bleeding.
Purpose:
.
A BUN test can reveal whether your urea nitrogen levels are higher than normal, suggesting that your kidneys
or liver may not be working properly.
The BUN level may be checked in order to assess or monitor:
the presence or progression of kidney or liver disease.
blockage of urine flow.
mental confusion. Patients with kidney failure are sometimes disoriented andconfused.
abnormal loss of water from the body (dehydration).
recovery from severe burns. The body uses larger than normal amounts of protein following serious
bums.
Abnormally low BUN: Low' levels of BUN may indicate overhydration, malnutrition, celiac disease , liver
damage or disease, or use of corticosteroids. Low BUN may also occur in early pregnancy.

Porphyrins & Porphyrias

Q. Define porphyrin. Name the porphyrins.

Ans.
Porphyrins: Porphyrins are cyclic molecules fonned by the linkage of four pyrrole rings through methenyl
bridges
6 (=HC-)
v and readily bind metal ions- usually Fe2* or Fe\
(Ref: Lippincott s/s /277)
magnesium-containing porphyrin
Examples are the iron porphyrins such as heme of hemoglobin and the
chlorophyll, the photosynthetic pigment of plants. (Ref: Harper’s/30“7323)

Q. Define and classify porphyria. (SU-12Ju)

Q. Short note: Porphyrias. (SU-14Ju)
. _ . group vi disease caused by abnormalities in the pathway of biosynthesis of
Porphyrias: The porphyrias are a group of
,

various porphyrins. (Ref: Harper’s/30,h/329)

Or,
produced due to the deficiency of enzymes involved in biosynthesis of
It is a heterogenous group of disorders
„mu|atiOn and excretion of the intermediate compounds (porphyrins) prior
heme leading to overproduction,
to the enzyme block.

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520 | arirs representation on MEDICAL BIOCHEMISTRY
Classification of porphi rias;
Hepatic porphyrias Enihrnpoktic
1. Acute intermittent porphyria (A1P) 1. Congenital erythropoietic porphyria (CEP)
4. Porphyria cutanea tarda (PCT) 2. Erythropoietic protoporphyria
3. Hereditary coproporphyria (HCP)
2. Variegate porphyria (VP)
(Ref: Harpcr'sTO VllQ)

Porphyrias can also be classified as acute or cutaneous on the basis of their clinical features.
Major signs and ss mptoms;
1 . Photosensitivity
2. Neuropsychiatric disorders including peripheral neuropathy, confusion or psychosis
3. Abdominal pain
4. /Viaemia.
(Ref: Harper’s/27'‘/283. 285, 286)

Q. Name the heme containing proteins/ hemoproteins.

Ans.
Heme containing proteins/ hemoproteins:
Hemoprotcins Functions 1
1. Hemoglobin Transport of oxygen in blood
2. Myoglobin Storage of oxygen in muscle
3. Cytochrome c Involvement in electron transport chain
4. Cytochrome P450 Hydroxylation of xenobiotics
5. Catalase Degradation of hydrogen peroxide
6. Tryptophan pyrrolase Oxidation of tryptophan
(Ref: Harper’s^O^/SZS)

Haemoglobin
Normal range of haemoglobin in different age group:
Neonate: 23 gm/dl
• Children (1 year): 12 gm/dl
Adult male: 14 - 18 gm/dl
Adult female: 12 - 16 gm/dl.
(Ref: S. Wright’s/13,h/36; de Gruchy/^/138; Lecture SSMC)
Q. Give the steps of haemoglobin synthesis. (SU-16M)
Q. How haemoglobin is synthesized? (SU-17M)
Q. How heme is synthesized? (SU-12Ju)
Ans.
Hemoglobin
synthesi^. Hb synthesis begins in the pro-erythroblast stage and is completed in the
orthochromatic erythroblast stage.
Heme synthesis:
1. Raw materials: Succinyl CoA, glycine and iron.
2. Site of synthesis: In mitochondria.
3. Steps:
Acetic acid is converted to succinyl Co-A in the Krebs cycle.

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 CHATOR
.
,
.
2 Succinyl Co-A + 2 glycine
4 pyrrole compound -♦ -
Pyrrole
07;
compound.
Protoporphyrin IX
protoporphyrin IX + Fe24 1
leinc.
CLINICAL BIOCHEMISTRY
”
521

. .
Raw materials: Amino
A
acids.
2 Site of synthesis: Ribosome of ER
j. Steps:
. -
DNA transcription > mRNA "translation
. > p0|ypcplidc (Globin)
*—
Heme + Polypeptide Hemoglobin
chain
—
2a chains + 2p chains * Hemoglobin
A.
(a or P)

(Ref: Guyton/J3"7449)

Bilirubin Metabolism
Q. How bilirubin is synthesized? (SU-17M)
Q. How bilirubin is metabolized in our body? (SU-14Ja)
Q. Discuss in short about the metabolism of bilirubin.
Q. Illustrate and discuss the catabolism of heme. (RU-14Ju)
Q. Discuss the fate of hemoglobin after breakdown of RBC.
Ans.
Bilirubin Metabolism: (V.V.I. for VIVA)
Normal level of serum bilirubin: 0.6- 1.1 mg/dl.
Daily production: 250-350 mg/day.

Source of bilirubin:
1 . Mainly from hemoglobin (normal breakdown of RBC), a5so from
2. Ineffective erythropoiesis.
3. Catabolism of other heme containing proteins such as myoglobin, cytochrome P450, catalaze etc.
Steps of bilirubin metabolism:
1 . Formation of bilirubin in reticuloendothelial cells
2. Transport of bilirubin with albumin
3. Hepatic uptake and conjugation
4. Biliary excretion and events in intestine.
Fragile RBC

j- Reticuloendothelial system
Hemoglobin
Globin -> amino acid pool

Heme
I Heme oxidase
Biliverdin
I Biliverdin reductase
Bilirubin

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 j22 [ ariPs representation on MEDICAL BIOCHEMISTRY
Bilirubin

Blood Bilirubin-albumin complex

Albumin goes to circulation

Liver Bilirubin
+
Glucuronic acid
|Bilirubin glucuronyl transferase
Bilirubin di-glucuronide
(Conjugated bilirubin)

Stercobilinogen Urobilinogen
Oxidized by intestinal bacteria
Oxidation
Stercobilin
(Intestinal content) Urobilin

To feces To urine

(Ref: Harper’s/30“7330; Satyanarayana/3rd/215,216; Guyton/12,h/840,841)

Nice to Know:
/. / gm of hemoglobin yields 35 mg of bilirubin.
2. Normal production of bilirubin = 250-350 mg/day.
3. Maximum capacity of liver to conjugate and excrete dilirubin is over 3000 mg/ day.
4. Urobilinogen and stercobilinogen is colourless.
5. Darkening of feces upon standing in air is due to the oxidation of residual urobilinogens to
urobilins.
(Ref: Harper,s/27'l'/287; Lippincott's/5/h/282)

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 CHAPTER 07: o nnr vL BIOCHEMISTRY
Vet* t<* Know:
Determination of bilirubin concentration: Bilirubin is aunt tvnmnb dctcrunncd br theoofroan
reaction. in which diazotized sulfanilic acid (Ehrlich's diazn rrugmt reacts with hdindwn
coloured azobilirubin that can be measured ndorimctrnallv
1' Direct-acting bilirubin: In aqueous wltition. conjugated bilirubin being outer- sohdde. |
directly and rapidly with the diazonogent t within one uunutef io fam the cohn’ rvnpmoi
said to be “direct-acting."
2. Indirect acting bilirubin: The unconjugatcd bilirubin, which is meh less sohdde in,
solution. reacts with the reagent slowly. This reacts only after the addition of mthmd
bt)th iinconjugpted bilirubin and the diazo reagent are soluble in ethmoL As it m^cds ad^iuon
methanol to react with the reagent. umonjugated bilirubin is said Io be “indintf mtiw^ .
(Ref: Lippincoa's/^ f2R5: Harper's

Jaundice
(V.V.l. for VW A)

Q. Define (SU-15Ju, CU-17Nv, RU-19/18/17Nv,16M.15 08Ja) & classify jaundice. (RU-19 IS

17Nv,16M,15Nv,08Ja, SU-13Ja, CU-17Nv.08/06Ja)
Q. State some major causes of jaundice. (RU-14Ju)
Q. Short note: Jaundice. (RU-04S)
Ans.
Jaundice: Jaundice is a clinical condition characterized by yellow diseelcmdcn of skm. sclera and mezens
membrane of body due to elevated bilirubin level in the blood (>3 mz dl).
(ReE Harper s30*332)
Or.
Jaundice (also called icterus) refers to the yellow color of the skin, rail beds and sclera caused by depcsiri" of
bilirubin, secondary to increased bilirubin level in the blood. Jaundice is not a disease but usuaZv a of
underlying disorders.
Normal serum bilirubin: Less than 1 mg'dl (0.6 - 1 mg'cl) or. 17.1 trrvdl
(ReE Lippincott's. S’.IS-l)
Mechanism of jaundice:
1 . Increased production of bilirubin
2. Impaired excretion of bilirubin
(Ref: Guytooir^/SS-l)

Classification of jaundice:
1 . Hemolytic / pre hepatic jaundice
2. Hepatocellular jaundice
3. Obstructive/ cholestatic/ post hepatic jaundice.
Hemolytic jaundice: This is caused by increased destruction of red blood cells
with ra">;d
into the blood. * ««se oi bilirubin
Causes:
1 . Incompatible blood transfusion.
2. Sickle cell anemia.
3. Malaria.

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 4. Pyruvate kinase deficiency.
5. Glucose-6-phosphate dehydrogenase deficiency.
6. Autoimmune hemolytic disease.
7. Certain drugs. to increase in the
unconjugatcd bilirubin level
Hepatocellular jaundice: Damage to the liver cells can cause
blood as a result of decreased conjugation.
Causes:
1. Viral hepatitis
2. Cirrhosis of liver
3. Paracetamol poisoning
4. Cardiac failure
5. Chloroform. CCU phosphorus poisoning.
of the bile duct that prevents the passage of
Obstructive / Cholestatic jaundice: This is due to an obstruction
bile into the intestine.
Causes:
Intrahcpatic cholestasis:
1. Viral hepatitis.
2. Drug induced (e.g. methyltestosterone, estrogen, oral contraceptives).
3. Alcohol.
4. Primary biliary cirrhosis
5. Secondary biliary cirrhosis.
6. Pregnancy.
7. Post operative.
Extrahepatic cholestasis:
Gall stone.
Stone is common bile duct.
Carcinoma in common bile duct, ampulla of vater and head of pancreas.
Inflammatory strictures of bile ducts.
Cysts in bile ducts.
Parasite infection.
Traumatic biliary stricture.
(Ref: Harper’s/30"7333; Lippincott’s/5th/284; Satyanarayana/3n’/216,217; Guyton/U^l)

Q. What do you mean by clinical jaundice and latent jaundice?

Q. What is latent jaundice? (CU-15Nv)
Ans.
Clinical jaundice: Clinically jaundice is detected when serum bilirubin is 3 mg/dl, so it is called
> clinical
jaundice.
Latent jaundice: Serum bilirubin concentration is 1-3 mg/dl is regarded as latent jaundice.

Xice to Know:
Choluric jaundice: Jaundice with presence of bilirubin in urine is called choluric jaundice. So. hepatitis
and obstruction of the common bile duct (obstructive jaundice) causes choluric jaundice.
A choluricj aundice:Jaundice without presence of bilirubin in urine is called acholuric jaundice.
Hemolytic jaundice is acholuric type.

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 CHAPTER 07: CLINICAL BIOCHEMISTRY
Q. Show the biochemical findings of different types of jaundice in tabulated form. (D
16Nv,14/12Ju, RU-l9/18/17Nv, SU-13Ja) .
Q. Differentiate different types of jaundice biochemically. (DU- 13 Ju, RU-15Nv, SU-1 v,
Q. Give the difference between three types of jaundice. (SU-15Ju)
Q. How will you investigate a case of jaundice? (CU-08Ja)
Q. List the necessary investigations for a case of jaundice. (CU-18M)
Q. Give the laboratory findings of obstructive jaundice. (RU-16M,15Ju/Ja)
(
Q. Differentiate the types of jaundice from each other on the basis of biochemical findings.
I Ua. RU-08Ja)
Q. Differentiate between hemolytic and obstructive jaundice on the basis of laboratory findings.
(CU-15Nv)
Q. Draw a table showing the role of serum enzyme assay in differentiating various liver
disorder. (RU-1 Uu)
Q. Draw a table showing biochemical tests in differentiation various hepatic disorders. (RU-
12Ju)
Ans.
Important tests to differentiate different types of jaundice: (V.V.I. for VIVA)
A. Blood biochemistry;
I Test Hemolytic jaundice Hepatocellular jaundice Obstructive jaundice
Serum bilirubin Raised, mostly Raised, both conjugated Raised, conjugated
unconjugated and unconjugated
ALT(SGPT) Normal Marked raised Slightly ?ed
AST (SGOT) Normal Marked raised Slightly Ted
Alkaline phosphatase Normal High Very high
Prothrombin time Normal Prolonged Prolonged
Serum albumin Normal Reduced Normal or reduced
Plasma y-globulin Normal Increased Normal
B. Urine:
I Test Hemolytic jaundice Hepatocellular jaundice Obstructive jaundice
Urobilinogen Increased Normal or increased Decreased
Bile salts Absent Present Present
Bilirubin Absent Present Present
Van den Berg Indirect positive Direct positive Direct positive
reaction
C. Stool:
Test II emolytic jaundice Hepatocellular jaundice Obstructive jaundice
Stercobilinogen Increased Reduced Reduced or absent
Naked eye Dark brown colour Normal color or pale Pale or normal colour,
bulky, frothy due to high
fat content

(Ref: Samson Wright’s/13/46; Lecture SSMC)

Q- Short note: Hemolytic jaundice. (RU-17M)

Ans.

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 arirs representation on MEDICAL BIOCHEMISTRY
Hemolytic laundicc: Please write from
above.
Q. Short note: Hyperbilirubinemia.
Ans.
Hyperbilirubinemia: When bilirubin in the blood exceeds I mg/dl (17.1 pmol/L) hypcr-bilirubincmia exists.
Causes:
1 . Production of more bilirubin than the normal liver can excrete.
2. Failure of a damaged liver to excrete bilirubin produced in normal amounts.
3. Obstruction of the excretory ducts of the liver- leads to prevention of the excretion of bilirubin.
(Ref: Harpcr’s/30"7332)

Q. Short note: Neonatal jaundice.

Ans.
Neonatal jaundice/ Jaundice in newborns: Newborn infants, particularly premature babies, often accumulate
bilirubin due to an accelerated hemolysis around the time of birth and an immature hepatic system for the
uptake, conjugation, and secretion of bilirubin. The enzyme billirubin glucuronyl transferase is low at birth.
It reaches adult levels in about 4 weeks of age.
Consequence: Since the increased amount of bilirubin is unconjugated, it is capable of penetrating the blood¬
brain barrier when its concentration in plasma exceeds that which can be tightly bound by albumin (20-25
mg'dl). This can result in a hyperbilirubinemic toxic encephalopathy (Kernicterus), which can cause mental
retardation.
Treatment: Phenobarbital has been administered to jaundiced neonatesand is effective in this disorder.
In addition, exposure to blue light (phototherapy) promotes the hepatic excretion of unconjugated bilirubin by
converting some of the bilirubin to other derivatives such as maleimide fragments and geometric isomers that
are excreted in the bile.
(Ref: Harper/30"7332; Lippincott’s/5,h/285)
(N.B: Crigler-Najjar syndrome is caused by a genetic deficiency of hepatic bilirubin glucuronyl transferase).
(Ref: Harper/30,h/333)

Clinical Enzymology
Q. List the enzymes having diagnostic importance. (RU-15Nv)
Q. Name the clinically important enzymes with their normal tissue location. (CU-15Nv)
Ans.
Enzymes having diagnostic importance: (V.V.I. for VIVA)
a. Cardiac enzymes:
Creatine kinase (CK) / creatine phosphokinase (CPK.)
Aspertate aminotransferase (AST)
Lactate dehydrogenase (LDH)
b. Hepatic enzymes:
i) Serum transaminases-
• AST/ SGOT
• ALT/ SGPT
ii) Serum ALP
iii) Serum LDH
iv) y-Glutamyl transferase (GGT)

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 CHAKI ER 07; CLIMCAL BIOCHEMISTRY
c. Muscle enn me?:
• CPK
/AST
• LDH
• Aldolase
d. Bone enzymes:
• Scrum acid phosphatase (ACP)
• Scrum ALP
c. Pancreatic enn m«:
• Scrum amylase
• Lipase.
Q. Enumerate the clinically important enzy mes with their disease association. (RU-19M)
Q. Aame the serum enzymes of diagnostic importance. State the normal values and the
conditions in which the values are raised.
Q. State the clinical significance of alanine transaminase. LDH, alkaline phosphatase, creatine
kinase (CK) (SU-12Ju). acid phosphatase & serum amy lase.
Q. Gise the clinical importance of AST, ALT (SU-12Ju) & serum amylase.
Q. Give the importance of acid phosphatase & alkaline phosphatase.
Q. Write four causes of high LDH.

Clinical importance of enzvmes: (V.V.I. for VIVA)

w Enzymes Normal saluc (IL7L) Increased in 1

1. Aspartate 10-45 a. Myocardial infarction (markedly raised)1
transaminase (Ref: Davidson’s/ZO^/nZO) b. Non-viral hepatitis
(AST/SGOT) c. Alcoholic liver disease
d. Cirrhosis of liver
e. Neoplastic disease of liver
(Ref: Davidson's/ZO^a)
2. Alanine 10-50 a. Hepatocellular damage
transaminase (Ref: Davidson's/ 20^/1320) Acute viral hepatitis
(ALT/SGPT) Chronic hepatitis C
Chronic hepatitis B
(Ref: Davidson’s/ZO^ 940,943)
a. Obstructive jaundice
3. Alkaline 40- 125 b. Primary biliary cirrhosis (PBC)
phosphatase (Ref: Davidson’s/ZO^/BZO) b. Metastatic carcinoma of liver
(ALP) c. Non-alcoholic fatty liver disease
(NAFLD)
d. Primary sclerosing cholangitis
e. Paget’s disease
f. Metastatic carcinoma of bone
g. Osteomalacia and rickets
~
(Ref: Davidson’s/20“7943,972,981.985)
4. Acid 0.5-4 a. Carcinoma of prostate
phosphatase

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 4ISTRY _
528 1 ariPs representationonjUEDICAI^BIOCH^jy'^T^cutTrnyocardiaTi^
5. Lactate 208 - 460 b. Acute infective hepatitis
dehydrogenase (Ref: Davidson’s/20* /1320) c. Muscle disease
(LDH) d. Acute leukaemia
c. Malignancy
f. Megaloblastic anaemia
g. Pernicious anaemia
h. Haemolytic anaemia
i. Pulmonary infection
j. Renal necrosis
(Ref: Davidson’s/20 /594;
Satyanarayana/3nl/107J08; Lecture SSMQ_
Male: 55-170 a. Myocardial infarction
6. Creatine
kinase (CK) Female: 30-135 b. Muscular dystrophy
(Ref: Davidson’s/20"71320) c. Inflammatory myositis and myopathy
d. Motor neuron disease
e. Alcohol, drugs
f. Trauma, strenuous exercise
g. Hypothyroidism
(Ref: Davidson’s/20tll/1075,1076) j
7. y-glutamyl 5-55 a. Liver disease /
transferase (Ref: Davidson’s/20,h/1320) b. Last stage of MI
(GGT) (Ref: Davidson’s/20th/940)
8. Serum amylase <100 a. Acute pancreatitis
(Ref: Davidson’s/20,h/1320) b. Intestinal ischaemia
c. Perforated peptic ulcer
d. Ruptured ovarian cyst
(Ref:Davidson’s/20th/905)
Q. What are the enzymes used to assay myocardial infarction?
Q. How plasma enzymes help in the diagnosis of myocardial infarction? (CU-15Nv)
Q. List the biochemical markers of acute myocardial infarction with their normal values. (CU-
16Nv)
Q. Name the cardiac enzymes I cardiac markers. (SU-18M) State their clinical importance. (RU-
09/07Ju) Evaluate their role in management of MI. (SU-18M)
Q. Enumerate the enzymatic function in MI. (SU-1
1 Ja)
Q. Show the pattern of cardiac markers
following myocardial infarction (MI). (DU-19M)
Q. A 58 year old man presented to the
emergency department with sudden onset of chest pain,
left arm pain, dyspnoea and
with their interpretation.
weakness. Name the cardiac markers used to diagnose the patient
Q. Write in short about
(RU-14Ju)
cardiac markers with their interpretation. (RU-19M,17Nv,16M,15Nv)
Q. Short note: Cardiac
enzymes. (DU-15Nv)
Q. Short note: Cardiac
marker. (RU-18Nv) (V.V.I. for VIVA)
Ans.
£ardiac markers: These are intracellular enzymes
due t0 ischemia (e.g. MI) or other or proteins released to blood following cardiac muscle
. assaults and shows serial change in plasma concentration with an
initial rise followed by a fall.

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 CHAPTER 07: CLINICAL BIOCHEMISTRY
Type* of cardiac markers:
1. Enzymes:
• AST (aspartate transaminase)
CK-MB (cardiac-specific isoform of CPK)
LDH (lactate dehydrogenase)
2. Other proteins:
• Myoglobin
• Troponin-I & Troponin-T.
Kinetic behavior / pattern of cardiac markers following myocardial infarction (MJ1-
t-U Return to normal
Myoglobin 2-4 hours of Ml 8-12hours 1 By 24 hours I
Troponin-1 4-6 hours of MI 12-24 hours 1 5-10 days I
CK-MB
AST
4-6 hours of Ml
6-8 hours of Ml
12 hours
24-48 hours
|
By 4 day
—1
LDH 8-12 hours of Ml 48-72 hours \ By 7th day onward |
1 Troponin-T 4-6 hours of Ml By 48 hours 10-15 days j

Nice to Know:
Characteristics of ideal cardiac markers:
1. Cardio specific with high concentration in myocardium.
2. Highly sensitive (capable to detect even mild myocardial damage).
3. Normally absent or present in trace amount in blood.
4. Rate of release is proportional to the extent of damage.
5. Stable and persist for reasonable period in blood.
6. Easy to measure and inexpensive.

Q. Which cardiac marker rises at first in acute MI? (For VIVA)

Ans.
Myoglobin (within 2-4 hours of MI).

Q. A 55 year man attended to emergency with the complaint of chest pain for 1 hour. How you
will proceed to investigate the case? (RU-15Ju)
Ans.
Investigation of the case:
1. History: Criteria of chest pain (site, radiation, character, aggravating or reliving factors). •
2. ECG
3. Cardiac markers (please write from above).

Q. Mention the clinical importance of ALT. (SU-15M,12Ju) (For VIVA)

Q. Short note: SGPT & SGOT. (CU-09Ja)
Ans.
SGPT & SGOT: Aspartate transaminase (AST / SGOT) & alanine transaminase (ALT / SGPT)
function
normally to transfer the amino group from an amino acid, alanine in the case of ALT and aspartate in the
of AST, to a ketoacid, producing pyruvate and oxaloacetate respectively. case

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530
_
mirsre^resentation^or^MEDICA^ —————— enzymes are
SGPT are located in the cytoplasm of the hepatocyte. Aldiough both transaminasethere orc this
Both SGOT & and
widely distributed in other tissues of the body, the activities
of ALT outside the liver are low.
enzyme is considered more specific for hepatocellular damage. (Ref:Davidson’s/20”7940)
Normal values and conditions " here SGPT & SGOT are increased:
Please "rite from above.

Q. Mention the clinical importance of CPK / CK. (RU-15Nv, SU-15M,12Ju)

Q. Short note: CPK / CK. (RU-08Ju)
Ans.
Creatine kinase (CK): Creatine kinase is an enzyme that is highly concentrated in brain, myocardium,
and
skeletal muscle and is composed of two dimers, designated “M” and “B”.
(Ref: Robbin’s/7,h/583)
Types / Isoforms:
(CK-MM): Found in skeletal muscle.
(CK-MB): Found in cardiac muscle.
(CK-BB): Found in brain.
(Ref: Davidson’s/20,h/I075; Robin’s/7,h/583)
Normal value:
Male: 55-170
Female: 30-135
(Ref: Davidson’s/20”1/ 1320)
Clinical importance: It is raised in- (For VIVA)
a. Myocardial infarction (MI)
b. Muscular dystrophy
c. Inflammatory myositis and myopathy
d. Motor neuron disease
e. Alcohol, drugs
f. Trauma, strenuous exercise
g. Hypothyroidism.

(Ref: Davidson’s/20"71 075, 1 076)

Q. What is the cut off value I
Q. Short note: Troponin.
reference range of Troponin-I? (CU-15Nv)
Ans.
Troponin: Troponins are proteins
that regulate calcium-mediated
Types: It is of 3 typcs- contraction of cardiac and skeletal muscle.
Troponin 1 (Tnl): It is totally
Troponin T (TnT): It is not cardio-specific.
Troponin C (TnC): It is not totally cardio-specific.
used as cardiac marker.
Clinical importance:
The new & preferred
(TnT), because these biomarkers for myocardial damage are Troponin I (Tnl) and Troponin T
markers have nearly complete cardiac
Troponin I (Tnl) and Troponin tissue specificity and high sensitivity.
levels of both cardiac troponins T are not normally detectable in the circulation, but after acute MI,
rise.
Initial rise
Troponin-I Time to peak Return to normal
By 48 hours

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 CHAPTER 07; CLINICAL BIOCHEMISTRY |
531
The troponins arc also released, to a minor degree, in unstable angina with minimal myocardial
damage.
(Ref: Lecture SSMC; Robbln’s/7"V5«3; DnvIdson’s/ZO' /593)

Q. What arc the investigations that will be performed In ease of a diabetic man of 50 years
having chest pain for 5 days?
Ans.
Investigations in case of a diabetic man of 50 years having chest pain for 5 days:
I . Estimation of scrum LDH level (normal value 60-250 IU/L).
2. Estimation of scrum y-GTT (normal value: male = 10-17 IU/L; female 7-30 IU/L).
3. Scrum cholesterol level.
4. OGTT.

Q. Give the lab findings in a fair, fatty, fertile female of 40 years suffering from jaundice.
Ans.
Lab findings:
1. Large increase of scrum alkaline phoshatase.
2. Increased level of scrum conjugated bilirubin.
3. Bilirubin present in urine.
4. Bile salt present in urine.
Diagnosis: Obstructive type of jaundice.

Fatty Liver Disease

Q. What is fatty liver? (SU-08Ju)
Q. Short note: Fatty’ liver.
Ans.
Fattv liver: Normal liver contains about 4-5% of total lipid. Of which 3/4* is phospholipid and 1/4* is neutral
lipid. However in certain conditions, lipids especially the TG accumulate excessively in the liver resulting in
fatty liver.
Factors that tend to increase liver fat:
1. Influx of dietary lipid.
2. Synthesis of fatty acid from CHO & protein.
3. Mobilization ot fatty acid from depots to liver.
Factors that tend to decrease liver fat:
1 . Mobilization of liver fat to the depot area
2. Degradation of fatty acid in the liver.
Types of fatty liver:
Type I: Overfeeding of fat.
Type II: Over-synthesis of fat from CHO.
Type III: Over-metabolism from depots to liver.
Type IV: Under metabolism from liver to depot area.
Type V: Under utilization in the liver.
Causes of fatty liver:
1 . Alcohol abuse.
2. Malnutrition-as protein energy malnutrition, deficiency of essential fatty acid and vitamins.
3. Diabetes mellitus.

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 4. Obesity.
5. Hepatotoxins and drugs.
Consequence: When accumulation of lipid in the liver becomes chronic, fibrotic changes occur in the cells
that progress to cirrhosis and impaired liver function.
Treatment: By lipotropic factor/ agent.
(Ref: Harpcr’s/30,h/260; Lecture SSMC)

Q. Short note: Lipotropic factor or lipotropic agent.

Lipotropic agent / factor: The substances which prevent accumulation of fat in liver can said to be lipotropic
agents.
Example: Choline. Methionine. Inositol.
Functions: These arc the substances which have the apparent effect of facilitating the removal of fat from liver
and thus prevent accumulation of fat in liver.
(Ref: Harper/30 h/261; Lecture SSMC)

Tumour Marker
Q. Short note: Tumour markers. (SU-12Ju)
Ans.
Tumour marker: The biochemical indicators are employed to detect the presence of cancers are collectively
referred to as tumour markers. These are the abnormally produced molecules of tumour cells such as surface
antigens, cytoplasmic proteins, enzymes and hormones.
(Ref: Satyanarayana/3rd/691)
Or,
Many cancers are associated with the abnormal production of enzymes, proteins, and hormones that can be
measured in plasma or serum. These molecules are known as tumour markers.
(Ref: Harper/30,h/739)
Clinical use of tumour markers:
1 . Screening for tumour
2. Diagnosis of tumour
3. Staging of malignant tumour
4. Monitoring the response to therapy
5. Early detection of recurrence.
(Ref: Saty,anarayana/3rd/691)
Some clinically useful tumour markers:
Tumour markers Associated cancer
Alpha-fetoprotein (AFP) Hepatocellular carcinoma
Germ cell tumour
Calcitonin (CT) Medullary carcinoma of thyroid
Carcinoembryonic antigen (CEA) Carcinoma colon
Carcinoma lung
Carcinoma breast
Carcinoma pancreas
Carcinoma ovary
Cancer antigen- 125 (CA-125)
Ovarian cancer

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 Clinical criteria of an ideal tumour marker:
1 . Disease sensitivity (positive in malignancy)
2. Disease specificity (negative in absence of malignancy)
3. Tumour specific and organ specific.

Reducing Substances in Urine

Q. What arc the reducing agents that can be found in urine? (Very Important for MCQ)
Q. Short note: Reducing substance in urine. (V.V.I. for VIVA)
Ans.
Reducing substances: Reducing substances arc the substances that can reduce Benedict solution.
The most important urinary reducing substances arc:
1. Sugar substances: Glucose, lactose, pentose, fructose & galactose.
2. Non-sugar substances:
Homogentisic acid (present in alkaptonuria)
Nalidixic acid
Drugs - Cephalosporin, aspirin therapy
Vitamin C (ascorbic acid).
(N.B: Normally reducing substances are absent in urine, but in certain conditions they are present in urine.
Glycosuria is most common. Lactosuria occurs in pregnancy and during lactation. Pentosuria, fructosuria,
and galactosuria are rare inborn errors of metabolism.)
(Ref: Lecture SSMC; Prof. Khaleque/202)

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