Clinical Biochemistry (1+1)

Clinical Biochemistry: Syllabus Outline

• Acid Base Balance and Associated Disorders
• Renal Function test
• Liver Function Tests
• Pancreatic Function Tests
• Cardiac Function Tests
• Clinical Enzymology
• Hormones and Associated Disorders
• Metabolic Disorders (Diabetes, Atherosclerosis, Jaundice,
Obesity, Fatty Liver formation)
• Metabolism of Xenobiotics
• Biochemical Techniques
• Heavy Metaal toxicity
• Immunity, Antigen, Antibody
 Books Recommended
• Clinical Biochemistry of Domestic Animals (J. Jerry
Kaneko, John W. Harvey, Michael L. Bruss)

• Harper’s Biochemistry

• Textbook of Medical Biochemistry (DM Vasudevan)

• Textbook of Medical Biochemistry (Dr U.

Satyanarayan)
Physiology of Acid Base Balance
and Associated Disorders

Naveen Shreevastva
Assistant Professor
Department of Vet Anatomy,
Physiology & Biochemistry
 Normal Arterial Plasma pH
Animals Plasma pH Plasma H+ ion
Concentration (nmol/L)
Dog 7.31-7.42 31 - 42
Cat 7.24 – 7.40 24 - 40
Horse 7.32 – 7.44 32 - 44
Ox 7.35 -7.50 35 - 50
Sheep 7.32 – 7.54 32 - 54
Pig 7.39 39
Human 7.35 – 7.45 35 - 45
• pH less than normal : Acidosis (Acidemia)

• pH more than normal : Alkalosis (Alkalemia)

 Mechanism of regulation of plasma pH

• Buffers of body fluids (First line defense mechanism)

• Respiratory system (Second line defense

mechanism)

• Renal system (Third line defense mechanism)

Buffers of Body Fluids
• Buffers are solutions that can resist change in
pH when acid or alkali is added.

Composition of Buffers:
• Mixtures of weak acids with their salt with a
strong base
• Mixtures of weak bases with their salt with a
strong acid
 Examples of buffers
• H2CO3/NaHCO3 (Bicarbonate buffer)

• CH3COOH/CH3COONa (Acetate buffer)

• Na2HPO4/NaH2PO4 (Phosphate buffer)

 Buffer System of The Body
EXTRACELLULAR FLUID INTRACELLULAR FLUID ERYTHROCYTE

NaHCO3 K2HPO4 K+Hb

-------------- ------------- ---------
H2CO3 KH2PO4 H+Hb
(BICARBONATE BUFFER) (PHOSPHATE BUFFER) (HEMOGLOBIN BUFFER)

Na2HPO4 K+PROTEIN K2HPO4

-------------- ----------------- -------------
NaH2PO4 H+PROTEIN KH2PO4
(PHOSPHATE BUFFER) (PROTEIN BUFFER) (PHOSPHATE BUFFER)

Na+ ALBUMIN KHCO3 KHCO3

-------------------- ----------- -----------
H+ ALBUMIN H2CO3 H2CO3
(PROTEIN BUFFER) (BICARBONATE BUFFER) (BICARBONATE BUFFER)
 Bicarbonate Buffer

• It is the major buffer of ECF

• It is found in the highest concentration in plasma and

has the highest effectiveness
HCO3 -
H2CO3

HCO3-
pCO2xS
S= solubility constant for CO2
S= 0.03

A/C to Henderson-Hasselbalch equation

pH = pKa + log HCO3-/pCO2x S

Mechanism of action of bicarbonate buffer
 Significance of bicarbonate buffer
• Highest concentration in ECF
• Represents alkali reserve
• Components are under physiological control
 Phosphate buffer

• Not important ECF buffer

• Major role in buffering renal tubular fluid and

ICF
 Protein Buffer
• Albumin shows greatest buffering action in
plasma (95% of total nonbicarbonate buffer)

• Effective group is histidine imidazole group

(pk=7.3)
 Hemoglobin Buffer
• Transport CO2 formed in tissues with minimum
change in PH
• Generate alkali reserve

RBC
Relative capacity of buffer systems
• 52% buffer capacity in tissue cells
• 6% in RBC
• 42% in ECF

• Buffers can respond immediately but do not maintain PH

completely

• For final maintenance of PH, respiratory & renal systems are

essential
 Respiratory Regulation of PH

• Elimination or retention of CO2

• Acidosis : Hyperventilation

• Alkalosis : Hypoventilation
 Alkalemia
Acidemia

Stimulation of central & Inhibition of central &

peripheral chemoreceptors
peripheral chemoreceptors

Stimulation of respiratory Inhibition of respiratory

center
center

hypoventilation
hyperventilation

HCO3- = Normal ratio

pCO2xS
 Renal Regulation of pH

A. Excretion of hydrogen ion in the form of

i. Titratable acids (NaH2PO4) and

ii. Ammonium ions (NH4+)

B. Reclamation (Reabsorption)of bicarbonate

Excretion of hydrogen ion
• The secretion of hydrogen ions in PCT, thick
ascending limb of loop of Henle and early part
of distal tubules takes place by the help of
NHE (Sodium hydrogen exchanger)
• NHE are present on the luminal surface of
tubular cell, which helps to secrete one
hydrogen from tubular cell to tubular lumen
and one sodium from tubular lumen to
tubular cell.
• In the tubular cell, water and carbon dioxide react to
form carbonic acid , which further breaks down to
form hydrogen ion and bicarbonate ion.
• The bicarbonate ion is reabsorbed into plasma of
peritubular capillary by sodium bicarbonate
cotransporter.
• The majority of secreted hydrogen ions in tubular
lumen binds with bicarbonate, few hydrogen ions
bind to NaHPO4- to form NaH2PO4 and few bind to
NH3 to form NH4+.
• In acidosis, upregulation of NHE takes place, which
causes more secretion of hydrogen into tubular
lumen. The opposite occurs in alkalosis
Fig. Excretion of Hydrogen ions in late DCT and collecting tubules by
intercalated cell
• The intercalated cells in late DCT and
collecting duct secrete hydrogen ions in
tubular lumen by hydrogen ATPase pump.

• The generated bicarbonate in the tubular cell

enters into plasma by chloride-bicarbonate
countertransporter.
Reclamation of bicarbonate
• The glomerular filtrate conatains NaHCO3.
• The HCO3- binds to H+ ions in the presence of
carbonic anhydrase to form H2CO3.
• H2CO3 breaks to form H2O and CO2.
• CO2 diffuses into tubular cells to form H2CO3 ,
followed by the formation of H+ and HCO3-.
• Now HCO3- is transported into plasma.
• In acidosis, the reclamation of bicarbonate in
more and the opposite occurs in alkalosis.
Fig. Reabsorption of bicarbonate in the different segments of nephrons
Excretion of Titratable acids(NaH2PO4)
• The glomerular filtrate contains Na2HPO4,
which dissociates to form Na+ and NaHPO4-.
• The released NaHPO4- binds to H+ ion to form
NaH2PO4 (titratable acid)
• In acidosis, there is increase in urinary
titratable acid and the opposite occurs in
alkalosis.
Excretion of ammonium ions
• Glutamine is present in tubular cell, which is acted
upon by glutaminase to release NH3.
• Being volatile in nature, NH3 diffuses into tubular
lumen, where it binds hydrogen ions to from NH4+
ions.
• NH4+ ions bind anions such chloride to get excreted
through urine.
• In acidosis, glutaminase is stimulated to release more
NH3 and the opposite occurs in alkalosis.
DISORDERS OF ACID BASE BALANCE
ACIDOSIS: (plasma pH <7.35)
• Metabolic acidosis (↓HCO3-)
• Respiratory acidosis (↑H2CO3)

ALKALOSIS : (plasma pH >7.45)

• Metabolic alkalosis (↑HCO3-)
• Respiratory alkalosis (↓H2CO3)
 METABOLIC ACIDOSIS
(Non-respiratory acidosis)
Metabolic acidosis is characterized by decrease
in plasma pH and Bicarbonate.

• HCO3-↓: H2CO3 = less than 20 :1

Causes :
• Production of organic acids that exceeds the rate of
elimination

• Reduced excretion of acids

• Excessive loss of bicarbonate

 ANION GAP

{(Na+ + K+) - (Cl- + HCO3-)}

• Normal value: 10-20 mmol/L

• The unmeasured anions constitute the anion
gap.

• The unmeasured anions are protein anions ,

sulfate, phosphate and organic acids etc.
• On the basis of values of anion gap,
Metabolic acidosis is classified into two groups :

A. High anion gap metabolic acidosis

B. Normal anion gap metabolic acidosis
 HIGH ANION GAP ACIDOSIS (Organic acidoses)
Conditions : (MUDPILES)

Methanol poisoning
 Uremia of renal failure
Diabetes or ketoacidosis
Paraldehyde toxicity
Isoniazid, iron or ischemia
Lactic acidosis
Ethylene glycol
Salicylate intoxication (aspirin intoxication)
Conditions Retained acids

Methanol toxicity Formate

Uraemia of renal failure Sulfuric acid, phosphoric acid, organic

acid

Diabetes mellitus and ketoacidosis Acetoacetate and Beta hydroxybutyrate

Paraldehyde toxicity Beta hydroxybutyrate

Isoniazide or iron toxicity , Ischaemia Organic acid, mainly lactate

Lactic acidosis Lactate

Ethylene glycol toxicity Hippurate, glycolate, oxalate

Salicylate toxicity Salicylate, organic acid

 NORMAL ANION GAP ACIDOSIS

Conditions :
• Diarrhoea
• Renal tubular acidosis
• Carbonic Anhydrase inhibitors (Acetazolamide)
• A profound metabolic acidosis without
dehydration leading to depression,
recumbency and death has been described in
goat kids and calves.

• The cause was undetermined, but the acidosis

was usually associated with high anion gap.
 Compensatory mechanism in metabolis acidosis

• Buffer systems of blood :

Buffers of plasma (mainly Bicarbonate buffer) minimise
change in pH.

• Respiratory compensatory mechanism :

The respiratory system causes hyperventilation that leads to
reduction in pCO2 within minutes. This helps correct the ratio
in bicarbonate buffer to maintain normal pH. The protective
effects of respiratory system is short lived, lasting only a few
days.
Long term correction requires the role of renal system.
• Renal compensatory mechanism :- kidneys
respond to restore the nomal pH by :
Increased excretion of acid
Preservation of bicarbonate
Increased ammonia formation and increased urinary
ammonia
Increased titratable acidity of urine
Systemic effects of metabolic acidosis
Effects on CVS :
• Causes arteriolar vasodilatation, constriction of
peripheral veins

Oxygen delivery to the tissues:

• Acidosis causes a right shift of ODC. (immediate
effect)
• Acidosis also decreases the synthesis of 2,3-BPG
(late effect)
Effect on nervous system:
• Impaired consciousness of varying degree

Potassium homeostasis :
Acidosis causes Hyperakalemia

Effect on Bones :
• Bone decalcification, bone disease.

Other effects : leukocytosis, insulin resistance,

negative nitrogen balance
 Biochemical findings

Urinary findings:
In uncompensated state,
• H+ : high, pH : low , pCO2 : N/ • Urinary ammonia : high
low • Urinary titratable
acidity : high
• In fully Compensated state :
 Ratio : normal (20:1) , pH :
Normal ,
PCO2 : low, HCO3- : low

• Serum ionised Ca increases

• Hyperkalemia
 Treatment
• Correction of underlying cause
• Administration of bicarbonate (NaHCO3)
 METABOLIC ALKALOSIS
(Non-respiratory alkalosis)

• HCO3- ↑:H2CO3 = more than normal

• Metabolic alkalosis is characterized by increase in

plasma pH and bicarbonate.
 CAUSES OF METABOLIC ALKALOSIS

Excess base is added to the system

Base elimination is decreased
Acid rich fluids are lost
 Conditions of metabolic alkalosis

 Contraction alkalosis
o Prolonged vomiting (in small animals)
o Sequestration of chloride rich fluids in abomasum
of ruminants.
Mineralocorticoid excess:
o Primary hyperaldosteronism
o Secondary hyperaldosteronism
Exogenous base :
o Bicarbonate containing IV therapy

Hypokalemia
 Compensatory mechanism in
metabolic alkalosis
• Buffers of blood :
minimise the blood pH
• Respiratory compensatory mechanism :
Increase in pH depresses the respiratory
center
Leads to retention of CO2 and increase in
H2CO3
 Renal compensatory mechanism
• Kidneys respond by :
Decreased secretion of H+ ions
Decreased formation of ammonia
Decreased reclamation of bicarbonate
Decreased titratable acidity of urine
 Systemic effects in metabolic alkalosis:

Hypokalemia
Features of hypocalcemia (Low ionized
calcium, normal total calcium)
Paresthesia, muscle cramps, tetany
 Biochemical characteristics
In uncompensated state,
• H+ = decreases
• pCO2 = slightly high
• HCO3- = very high
• pH = high
In fully compensated state,
• pCO2 = high
• HCO3- = high
• Ratio of the components of bicarbonate
buffer : normal (20:1)
• pH = normal
Treatment :
Treatment of underlying cause
Expansion of ECF volume with isotonic saline
(in contraction alkalosis)
Maintenance of plasma potassium
 RESPIRATORY ACIDOSIS

HCO3 - = < 20
H2CO3↑ 1 (plasma pH decreases)

• Respiratory acidosis is charaterised by a

decrease in pH and increase in pCO2.
• It develops because of defective alveolar
ventilation.
 CAUSES:

FACTORS THAT DEPRESS RESPIRATORY CENTER:

• CNS trauma, degenerative disorders

FACTORS THAT AFFECT RESPIRATORY APPARATUS:

• Pulmonary fibrosis
• Diseases of upper airways
• Chronic obstructive pulmonary disease
• An additional cause of special importance in
veterinary importance is general anesthesia
with volatile agents using a closed system.
 COMPENSATORY MECHANISM
BUFFER SYSTEM :
 Excess carbonic acid is buffered by buffers of blood

RENAL MECHANISM :
Kidneys Respond By :
 Increased ammonium ion excretion
 Increased titratable acidity of urine
 Increased reclamation of bicarbonate
• Respiratory mechanism :
 The increase in pCO2 stimulates the respiratory center and
results in increased pulmonary rate and depth of respiration
provided that the primary defect is not in the respiratory
center

 The elimination of CO2 through the lungs results in a decrease

in H2CO3
Laboratory findings in respiratory acidosis

• pCO2 : increased

 PH : low
 HCO3- is high normal or slightly high
In fully Compensated state :
• HCO3-/H2CO3 = normal
• pH : normal
• pCO2 : high
• Hyperkalemia

Urinary findings :
• Urine pH : low
• Urine ammonia : high
 Treatment
• Treatment of underlying disease

• Administration of sodium bicarbonate

• In cases of bronchospasm or COPD, use of

bronchodilators

• Mechanical ventilation may be needed

 RESPIRATORY ALKALOSIS
HCO3 - = > 20
H2CO3↓ 1 (plasma pH high)

Respiratory alkalosis is associated with increase

in pH and decrease in pCO2.
 Causes for respiratory alkalosis
• Basic cause is abnormally rapid or deep
respiration (Hyperventilation) leading to
excess elimination of CO2
• Hyperventilation may be seen in dogs and other
nonsweating animals because they employ respiratory
evaporative process for heat loss to prevent overheating.

• Hypoxaemia seen in eg asthma, pneumonia, high altitude,

Cogestive heart failure, pulmonary disease, severe anemia
• Increased cerebral respiratory center drive eg, salicylate
intoxication, gram negative sepsis etc
 Compensatory mechanism
• Buffers of blood :
RBC & tissue buffers provide H+ ions to
consume small amount of HCO3-
 Renal mechanism

• Kidneys respond by :
 Decreased formation of ammonia & decreased excretion of
ammonium ions

 Decreased reclamation of bicarbonate

 Decreased titratable acidity of urine

• Respiratory mechanism :
Inhibition of respiratory rate causes
hypoventilation. This leads to accumulation of
CO2 and helps maintain plasma pH towards
normal.
 Laboratory in findings in respiratory
alkalosis
In Uncompensated or partially compensated
state :
• pCO2 : low
• HCO 3- : low normal or low
• pH : high
In Fully Compensated state :
• pCO2 : low
• HCO3- : low
• HCO3-/H2CO3 = normal
• pH : normal
Urinary findings :
• Urinary ammonium ion : decreases
• Urinary titratable acidity : decreases
 Mixed Acid base Disorders
1. Salicylate poisoning (Aspirin intoxication):
Metabolic acidosis and respiratory alkalosis

2. Alcoholism :
• An alcoholic who has been vomiting may develop metabolic
alkalosis.
• If such a patient were then to develop a superimposed
alcoholic ketoacidosis with high beta-hydroxybutyrate
concentration.
• He would develop metabolic acidosis.
ARTERIAL BLOOD GAS ANALYSIS
 ABG analysis :Indications
• In acid base disorders

• To differentiate metabolic acidosis from respiratory alkalosis

• To differentiate metabolic alkalosis from respiratory acidosis

• To monitor the treatment

• To guide artificial respiration

 ABG analysis :blood collection

• Arterial blood is collected for gas analysis

• Sites for blood collection :

A. cattle:
• Caudal auricular artery
• Carotid artery
• Brachial artery
• Common palmar digital artery
• Saphenous artery
B. Dog: Dorsal pedal artery
• Heparin & blood specimen in the syringe must be mixed

• Expel any air bubble & nozzle of the syringe must be

stoppered

• Perform the assay as soon as possible

• Keep the syringe in ice bath until analysis to lower the

metabolic activities of blood cells.
• Inject the blood in Arterial Blood Gas (ABG) analyser to get
the parameters of ABG.
 ABG analysis :parameters
• pH
• paCO2
• paO2
• cHCO3-
• Base excess
• Oxygen saturation (SaO2)

(It is done by ABG analyser)

Case Studies
 case 1
A case came to the emergency department with the
symptoms of persistent diarrhoea over the past 3 days
and rapid respiration.
Her ABG reports were
pH: 7.21 (7.35- 7.45)
pCO2 : 19 mmHg (35-45)
HCO3- : 7 mmol/L (22-26)
pO2 : 96 mmHg (70-100)

a) What is the patient’s acid base status?

 Case 2
A patient came to emergency because of increasing breathlessness. On
examination, central cyanosis and coughing copious of green phlegm
were found. The ABG results were

pH : 7.31 (7.35-7.45)
paCO2 : 9.3 kPa (4.6-6)
PaO2 : 6.9 kPa (9.3-13.3)
Bicarbonate : 37 mmol/L (24-32)

a. Name the acid-base disorder.

b. Comment on bicarbonate value.
 Case 3
A small animal admitted to hospital with a week long history of severe
vomitting. There was clinically severely dehydration and shallow
respiration.
ABG analysis showed

H+ : 28 nmol/L (35-45)
pCO2 : 7.2 kPa (4.6-6)
Bicarbonate : 43 mmol/L (21-28)
pO2 : 15 kPa (10.5-13.5)

What is the acid-base disorder?

Explain the rise in bicarbonate and pCO2 .
 Case 4
A patient was admitted unconscious to a casualty department. On
examination, hyperventilation was found . There was history of ethylene
glycol antifreeze ingestion. Blood results were:

plasma sodium : 136 mmol/L (135-145)

plasma potassium : 6 mmol/L (3.5-5)
Plasma bicarbonate : 10 mmol/L (24-32)
Plasma chloride : 93 mmol/L (95-105)
pH : 7.2 (7.35-7.45)
PaCO2 : 3.18 kPa (4.6-6)
PaO2 : 13.1 kPa (9.3-13.3)

Name the condition and calculate the anion gap.