Enzymes: Clinical Chemistry

CLINICAL
CHEMISTRY 1

3. Hydrolase – catalyze the hydrolysis of compound with introduction of water
ENZYMES into the structure
4. Lyases – removal of one group from the substrate without hydrolysis, leaving
Enzymes double bonds in the molecular structure of the products
-‐considered as biochemical catalysts (speed up chemical reaction) 5. Isomerase – catalyzes intramolecular rearrangement of the substrate
-‐protein in nature compound
Isoenzyme 6. Ligases – catalyze the joining together of two substrate molecules with a
Apoenzyme. simultaneous breakdown of pyrophosphate bond in ATP.
Holoenzyme-‐

Components: Measurement:
a. active site 1. Measurement of Enzyme Activity
________________________________________________________________________________. -‐ based on Michaelis –Menten Kinetics
b. allosteric site
____________________________________________________________________________. Saturation Point = up to the substrate concentration when enzyme is fully saturated
Characteristics: with substrate
1. Highly specific -‐ also called V max : the rate of reaction when all the things are
2. Measured by its activity rather than its concentration. Normally secreted in equal varies only with the enzyme concentration.
minute concentration.
3. Require substrate 3 indications of reaction:
4. Require activators or metallic ions that are loose bound to the enzymes and a. use of excess substrate to determine enzyme
help activate the enzyme. concentration.
5. Affected by pH and temperature b. Measurement of reaction products like NAD or NADH
6. Secreted by cells and tissues NADH is measured by its Use of a coupled enzyme
assay if the reaction do not utilize NAD /NADH.
Factors that affect enzyme reaction:
1. Substrate – molecules or substances that attached with enzymes (highly Example of COUPLED ENZYME ASSAY : (measurement of disappearance of excess
specific) NADH)
2. Temperature
3. pH Aspartate + a-‐ketoglutarate AST (addition – no utilization of NADH) -‐>
4. Activators/Cofactors oxaloacetate(OAA) + glutamate +NADH (MD + excess a-‐ketoglutarate + NADH) -‐-‐-‐à
5. Coenzyme malate + NAD
6. Inhibitors
Other examples of enzymes not utilizing NAD/NADH:
Enzyme nomenclature: a. ALP – use excess p-‐nitrophenyl phosphate at pH 10. (absorbance of p-‐
1. Name of the substance or group on which the enzyme acts plus suffix “ase” nitrophenol at 405nm).
2. Type of reaction involved b. ACP – use excess p-‐nitorphenyl phosphate at pH 5.0
3. Combination of the 1st and 2nd.
4. International Unit of Biochemistry (IUB) 2. Measurement of Enzyme Antigen Concentration
-‐ quantity of enzyme is proportional to enzyme activity
a. Usually by immunoassay
Classes of Enzyme: If quantity of enzyme do not agree with enzyme activity, it maybe
1. Oxidoreductase – catalyze oxidation-‐reduction reaction due to:
2. Transferase – catalyze the transfer of a chemical or functional group from one 1. presence of serum enzyme inhibitors
substance to another 2. deficiency of a necessary cofactor
K.B.R 1

2 CLINICAL CHEMISTRY

3. macroenzymes Reaction Catalyzed: transfer of an amino group in aspartic acid for an alpha-‐keto group
4. defective enzyme in alpha-‐ketoglutaric acid forming oxaloacetateand glutamate.
5. proteolytically inactivated enzymes
Tissue Sources:
a. organic -‐ present in cardiac muscle , liver , skeletal muscle , kidney, brain ,
b. inorganic or coenzymes lung and pancreas (in decreasing water)
Other enzymes subtypes: Laboratory Methods:
1. Macroenzyme 1.Karmen method – incorporates a coupled enzymatic reaction using MD as
2. Defective Enzyme: indicator enzyme and monitors the change in absorbance at 340 nm.
Type of Enzyme Inhibition a. NADH –strongly absorbs at 340 nm
1. Uninhibited enzyme: plotting of 1/V vs. 1 /[S] resulting into a straight line b. MD is used to reduce oxaloacetate to
which is called a Lineweaver-‐Burke Plot. Wherein y-‐intercept equals 1/V max malate
and x-‐intercept is equal to -‐1/Km. 2.Reaction with DNPH: Reitman –Frankel
-‐ketoacids formed after the catalyzed reaction are reacted with 2,4
2. Noncompetitive inhibition : due to an inhibitor that binds with the away DNPH to form ketoacid hydrazones which produce intense brown color in the
from the substrate binding site. presence of NaOH measured at 505nm.

3. Competitve inhibition : inhibitor and enzyme substrate compete for the 3.Coupling with Diazonnium Salts
same binding site. ketoacid + diazo compound -‐-‐-‐-‐-‐-‐-‐-‐-‐diazonium
derivative
4. Uncompetitive inhibition : substance that binds the enzyme-‐substrate(ES)
complex and stabilizes it.
II. ALT/ SGPT
FIRST Order Kinetics Reaction Catalyzed: transfer of an amino group of alanine for an alpha keto group in
__________________________________________________________________________________________. alpha ketoglutaric acid forming pyruvate and glutamate.
ZERO order kinetics Tissue Source: Liver
__________________________________________________________________________________________ Laboratory Methods:
a. Coupled Enzyme Reaction
Fixed Time vs Continuous : Substrate: ____________________.
__________________________________________________________________________________________. Indicator Reaction: LD as indicator enzyme catalyzes reduction of
pyruvate to lactate with simultaneous oxidation monitored at 340
nm.
b.Reaction with DNPH – keto-‐acid formation
c.Coupling with Diazonium Salts
Unit of Enzyme Measurement:
1. International Unit (IU) : amount of enzyme that catalyzes the conversion of 1 Clinical Significance:
micromole of substrate per minute Increased: hepatic parenchymal disease, viral hepatitis, obstructive
2. Katal (1 katal) : mount of enzyme that catalyzes the conversion of 1 mole of jaundice, Reye’s Syndrome, Heart failure or AMI, Infectious
substrate per second. Mononucleosis, Muscular Dystrophy
3. 1 IU = 16.7 nanokatals
Decreased: Not significant
NOTE:
Intra-‐individual variation is more significant in ALT than AST with marked diurnal
I.AST/SGOT variation (highest in the afternoon) and day to day variation of 30%.

2 K.B.R

CLINICAL CHEMISTRY 3

III. Lactate Dehydrogenase Skeletal Muscle Disease: muscular dystrophy
Reaction Catalyzed: oxidation of L-‐lactate to pyruvate with the mediation of NAD or Brain and CNS disease: CVA
hydrogen receptor (reversible reaction)
VI. Alkaline Phosphatase:
Tissue source: liver, heart, skeletal muscle , kidney, erythrocytes Reaction Catalyzed: hydrolysis of organic phosphate esters with formation of alcohol
Laboratory Methods: and phosphate ion at alkaline pH
a. Wacker Method :
b. Wroblewski LaDue Method. Tissue Sources: bone, liver, intestine kidney, placenta, carcinoplacental ALP (Regan –
Other Methods: Lung CA, Nagao – adenocarcinoma , Kasahara-‐ gastrointestinal cancer)
a. Heat Denaturation
Isoenzymes: Laboratory Methods:
LD1 – HHHH: cardiac muscle, RBC, kidney 1.bessey-‐Lowry-‐Brock: p-‐nitrophenylphosphate
LD2-‐ HHHM: cardiac muscle, kidney, RBC 2. Bowers-‐McComb: p-‐nitrophenylphosphate
LD3 –HHMM: spleen, kidneys, lungs , RBC 3. King-‐Armstrong : phenylphosphate
LD4 –HMMM: spleen, lungs, kidney 4. Sinowara-‐Jones-‐Reinhart: B-‐glycerophosphate
LD5 – MMMM: liver, skeletal muscle

Clinical Significance: Isoenzyme Differentiation:
Increased: a.Phenylalanine inhibits intestinal (75%) and placental (80%)
Cardiac Muscle: AMI, CHF, Myocarditis b. Electrophoresis: Liver ALP migrates fastest followed by bone, placental and
Skeletal Muscle Diseases: muscular dystrophy , muscle intestinal fractions
trauma c.Urea(2M) inactivates bone (90%) more than liver (60%) fraction
Hepatic Parenchymal Diseases: Viral Hepatitis, Cirrhosis, d. Heating to 56C for 15 mins = bone and liver ALP fraction loses activity,
Obstructive Jaundice , placental ALP most is the most stable can resist Heat Denaturation at 60C for
Infectious Mononucleosis 10 mins.
Others: megaloblastic and pernicious anemia
Decreased: not clinically significant
Clinical Significance:
NOTE: Elevated LD1 and LD5 : due to AMI complicated by liver congestion and chronic
alcoholism complicated by liver damage and megaloblastoc anemia Increased: obstructive jaundice, cholestasis, viral hepatitis, alcoholic cirrhosis, hepatic
malignancy , hyperparathyroidism , bone tumors , rickets , osteomalacia, osteitic
V. Creatine Kinase (2.7.3.2 Transferase) deformans (Paget’s Disease), pregnancy (3rd trimester)
Reaction Catalyzed: reversible phosphorylation of Creatine by ATP in the presence of
magnesium ions. Decreased: not significant
Tissue source: skeletal muscle, myocardium (cardiac muscle) , brain
VII. Acid Phosphatase
Laboratory Methods: Reaction Catalyzed: hydrolysis of organic phosphate esters with formation of alcohol
1.Tanzer Gilvarg Assay and phosphate ion at an acid pH.
Types:
2.Oliver-‐Rosalki Assay a.Red Cell Acid Phosphatase
-‐ distuinguished form other acid phospahatss
Isoenzymes: CK-‐BB –brain, CK-‐MM muscle, CK-‐MB-‐ Heart b. prostatic Acid Phosphatase
Increased: Tissue Source: prostate gland , non-‐prostatic source: RBC, platelets, liver, spleen ,
Cardiac Muscle: AMI kidney , bone marrow
K.B.R 3


Laboratory Methods: Laboratory Methods:
1. Bodansky – B-‐glycerophosphate 1.Turbidimetric Enzyme Reaction
2. Gutman, King Armstrong –phenylphosphate Substrate: olive oil or triolein
3. Babson and Reed – a-‐ naphthylphosphate Indicator Reaction: decreased turbidity as LPS hydrolyzes the turbid substrate
4. Roy-‐thymolphthalein monophosphate fat emulsion
2.Titiration : Cherry-‐Crandal Method (classic Method)
uses olive oil substrate and measure liberated fatty acid by titration
after 24 incubation.

Chemical Inhibition by tartrate: PAP fraction is inhibited by tartrate Increased: acute pancreatitis, pancreatic cyst or pseudocyst, obstructive jaundice,
Total ACP –ACP activity after tartrate inhibition = prostatic ACP peritonitis, intetsinal obstruction
RBC ACP Prostatic ACP Decreased: pancreatic insufficiency.

Copper Other Enzymes:
Tartrate ion 1. Cholinesterase
2. Glucose-‐6-‐phosphate Dehydrogenase
3. Aldolase
4. Ornithine Carbamoyl Transferase
Clinical Significance: 5. Leucine Aminopeptidase
Increased: metastatic carcinoma of the prostate, bone disease : osteoporosis,
multiple myeloma, Paget’s disease, Gaucher’s Disease
Decreased: not clinically significant

VII. Amylase
Reaction Catalyzed: hydrolysis of polysaccharides such as amylase, amylopectin,
glycogen .
Tissue Sources: pancreas, salivary gland

Laboratory Methods: Calcium and Chloride are both activators
1. Amyloclastic – time required for the disappearance of colored starch iodine
complex upon hydrolysis of amylase (+) blue color disappears
2. Saccharogenic – measure the amount of reducing sugars formed after
hydrolysis .
3. Chromogenic –uses chromogen
4. Coupled enzyme reaction test

Increased: acute non-‐hemorrhagic pancreatitis, bacterial parotitis
Decreased: pancreatic insufficiency

VIII. Lipase
Reaction Catalyzed: hydrolysis of fats into fatty acids and monoglycerides
Tissue Sources: pancreas, stomach , small intestine

4 K.B.R


MAJOR DETERMINANT OF PLASMA OSMOLALITY?______
FLUIDS and ELECTROLYTES
Osmolaility of body fluids includes: _________________
Body Water
-osmolalilty is determined by particle number not the nature or weight of the
-bulk of the body mass is water
solute.
-can vary from 40%- 70% of the total body weight depending on the amount of fat
-measure specific gravity in urine to measure osmolality.
contained in the tissues.
Urine :Range in normal individuals:
Importance:
Plasma :
– it constitutes the medium by which solutes are dissolved
Ratio of urine to plasma:
– By which metabolic reactions take place
FORMULA:__________________________
Normal Reference values
NOTE: Increase pure water loss = increase ECF osmolality
` Venous -INCREASED in ECF osmolality triggers the ff physiological responses:
Sodium 135-148 mEq/L – Antidiuretic hormone/vasopressin release via hypothalamic
Potassium 3.5-5.3 mEq/L osmoreceptors
Chloride 98-106 mEq/L – Stimulation of the hypothalamic thirst center
Bicarbonate 19-25 mEq/L – Redistribution of water from the ICF compartment
Anion Gap 12-18 mEq/L
Serum Osmolality 285-310 mOsm/kg H2O
b.ECF volume
Obtained from: -directly dependent on the sodium content.
1.diet • Maintained by the ff:
-drink-1L – Regulation of renal excretion of sodium or glomerular filtration rate
-food -1-1.2 L – Aldosterone via the RAA system
-fat metabolism -100ml/100g DISORDERS OF WATER BALANCE
-protein metabolism -44ml/100g a. Dehydration
-carbohydrate metabolism-60ml/100g b. Overhydration
2. oxidation of food
ELECTROLYTES
Water Excretion:
1.skin
2.lungs Cations – ions w/ positive charge
3.GI tract Anions – ions w/negative charge
4. Kidneys -readily absorbed in the GIT into the circulation
-initially filtered at the glomerulus since they are very small and readily pass through
Distributed in 2 compartments: membrane pores.
1.) Intracellular
2.) Extracellular 2 processes:
a. Intravascular a.reabsorption
b. Interstitial b.excretion
2 properties of ECF
a. ECF osmolality FUNCTIONS:
• -Regulated by the levels of sodium and associated anions, ________________________________________________________
glucose, urea and proteins ____________________.
OSMOLALITY -___________________
K.B.R 5


Hormonal Regulation
Two Major hormone involved: Laboratory Determination.
1.Antidiuretic hormone (ADH) 1.Flame Emission Photometry
2.Ion-selective Electrode.
2.Renin-angiotensin aldosterone system 3. Colorimetric method:
– Sodium + zinc uranyl acetate = sodium uranyl acetate precipitate
ANION GAP – sodium uranyl acetate precipitate + water = yellow solution
• -Difference between the sums of the concentrations of the principal cations
and of the principal anions
• Represents the unmeasured net negative charge on plasma proteins POTASSIUM
Anion gap_________________________________ • Major intracellular cation
INCREASED in: Concentration:
• Uremia – CELL - Inside (20x greater) > Outside
• Ketoacidosis – 90% free or exchangeable; 10% bound to RBC, bone and brain
• Methanol & aspirin poisoning tissues
• Severe dehydration • Kidney - primary that controls extracellular K+
• Lactic acidosis • Proximal tubule
DECREASED in: • Distal tubule
• Multiple myeloma • Secreted in the gastric juice and reabsorbed by the small intestines
• Protein error • Influenced by pH
• Instrument error • ACIDOSIS
SODIUM • ALKALOSIS
• Most abundant cation in the ECF /chief base of the blood. • Normal value:
• Main function are water pull Function:
– Osmotic activity of ECF a. nueromuscular excitability, contraction of the heart. Intracellular fluid
– Blood volume regulation volume and hydrogen ion concentration.
– Neuromuscular excitability Clinical Implication:
Levels are regulated by: 1. Hyperkalemia/Hyperpotassemia –inc. K levels
– Diet -inadequate excretion (renal failure)
++
– Kidney: renal threshold for Na is 110 - 130 mmol/L -cell damages (burns, accidents, surgery, chemotherapy)
– RAA system -acidosis
– Atrial natriuretic factor 2.Hypokalemia/ Hypopotassemia-dec. K levels
Normal value: ________________L -GI loss associated w/ diarrhea , severe vomiting , starvation ,
malabsorption
Clinical Implication; -Increased secretion of adrenal steroids primarily aldosterone
1.)Hypernatremia -severe burns
a.severe dehydration -insulin treatment
b.Cushing’s Syndrome (hyperadrenalism) – Laboratory Examinations:
c.Diabetic coma after insulin treatment 1.Colorimetric Method- K is determined in a specifically prepared mixture of sodium
d.Diabetes insipidus(def. of ADH) tetraphenylboron producing a colloidal suspension whose turbidity is proportional to
2)Hyponatremia –dec. sodium levels the potassium concentration in the sample.
a.severe burns, severe diarrhea • Lockhead and Purcell
b.Addison’s disease 2. FES
c.Diabetic acidosis 3. ISE
d.Renal Tubular Disease a.glass that is permeable selectively to sodium
6 K.B.R


b. membrane impregnated w/ valinomycin ( has a charged activity just the Laboratory Determinations:
right size to admit K and exclude others. 1.)ISE method.
4.AAS 2.) Coulometric-amperometric method
3.) Colorimetric Method
ISE for Na and K • Zall color reaction
• Based on the activity of the ion Colorimetric method
– Its dissociated or completely ionized fraction per unit volume of Result:
water Schales and Schales
+
• Na : glass electrode – Mercuric nitrate titration method
+: - + +
• K liquid ion-exchange membrane electrode, incorporating the antibiotic – Cl + Hg (mercuric nitrate) = Mercuric chloride + Hg (mercuric
valinomycin nitrate) = colorless or faint pink to violet
2 methods: CALCIUM
1. DIRECT METHOD • Most abundant cation in the body
2. INDIRECT METHOD • 99% bound to the skeleton
++
• Bone: Ca + phosphates = hydroxyapatite crystals; to provide strength to
REMEMBER:_____________________________ the bone
-impt. activator of the coagulation system.
CHLORIDE --skeletal and cardiac muscle contractility
• Major extracellular anion ,exist as NaCl or HCl -neuromuscular conduction and activation.
• Functions:
– Maintenance of electrolyte balance Reference range (serum): 9-11 mg/dl or 4.5 -5.5 mEq/L
– Hydration 3 forms:
– Maintenance of osmotic pressure 1.) bound
– Only known anion to serve as an enzyme activator (stimulating the 2.) Ionized
starch hydrolysis reaction catalyzed by the enzyme amylase 3.) Complexed
– Has the reciprocal power of increasing or decreasing in
concentration whenever changes occur in the concantration of Factors Affecting Calcium Levels
other anions. 1.Parathyroid Hormone – mobilizes calcium from the bones in the circulation, acts on
– Ex. metabolic acidosis –inc. Cl :HCO3 dec. the kidney inhibiting the tubular reabsorption of phosphate while enhancing the
• Normal value reabsorption of calcium and magnesium and stimulating the formation of a vitamin D
Clinical Implication: derivative
1.)Hyperchloridemia
a.dehydration 2.Vit D3 /Calcitriol
b.Cushing’s syndrome 3.Calcitonin – peptide hormone produced by specific cells in the thyroid gland
c.Hyperventilation(respiratory alkalosis)
d.anemia Clinical Implications:
e.Cardiac decompensation 1.Hypercalcemia
2.)Hypochloridemia
a.severe vomiting, severe diarrhea , severe burns 2.Hypocalcemia
b.heat exhaustion
c.Diabetic Acidosis LABORATORY DETERMINATION:
d.acute infection(pneumonia) 1. Formation of colored complexes between Ca++ and a variety of dyes
e.Diuretics
* Followed by colorimetric method
K.B.R 7


-phosphorus in the form of inorganic phosphates is allowed to react with -one of the buffering system
molybdic acid producing a phosphomolybdate complex in the presence of a reducing -reported as mg/dl
agent.
a.Fiske –Subbarow Function?
b.Gomori MOdificatio
2.ISE QUESTION? Why is there a need to avoid opening tubes used for ionized calcium?
3.AAS .
4.FES IRON
5.Precipitation of Ca++ as an insoluble compound using • 70% in the RBC and 25% in the RES, incorporated with ferritin and
hemosiderin as stored iron
• After precipitation it is followed by titration or colorimetric • cofactors for enzymes
method • 2 forms in the body:
• c. Ferrous iron -
6. Removal of calcium from colored complex by titration with a chelating agent d. Ferric iron Ferritin = iron storing protein(soluble)
– EDTA Transferrin
– EGTA Haptoglobin
• Endpoint is reached by recovery of the original color of the dye or the Hemopexin
disappearance of the fluorescence of the calcium-dye complex Hemosidirin)
CLINICAL IMPLICATION
MAGNESIUM 1.)Iron Overload
• 4th most abundant cation in the body
• Essential activator of several enzymes
• Therapeutic agent and anti-convulsant, laxative and antacid effects 2,) Iron deficiency
• Involved in protein synthesis and DNA metabolism
REFERENCE RANGE:______________________ Laboratory assays:
• Serum Iron
CLINICAL IMPLICATION: • Total Iron Binding Capacity
1.)Hypermagnesia • Transferrin Saturation
2.)Hypomagnesia %Saturation=Serum Fe/TIBC x 100

• TIBC = 260 - 440 ug/dL
Laboratory Determination: • % saturation = 20 - 50%
1. AAS or ISE
2.Fluorometric analysis: Nephelometric Assay for Transferrin
3.Colorimetric methods: a.IRMA and enzyme liked systems
Examples of dyes
a.Calgamite -
b.Methylthymol blue
c.Titan yellow
d..Formazan Dye

PHOSPHORUS
-has an inverse relationship with Calcium
-measured in body fluids as a mixture of the phosphates HPO4 and H2PO4.
8 K.B.R


-‐Proteins are amphoteric , carrying both acidic and basic charge and in this way
are able to find or release excess hydrogen as required.
• Imidazole groups of Histidine are present in plasma proteins which
Acid –Base Disorders are negative in charge and capable of binding with H+.

Definition of Terms:

a.Acid – substance that can yield a hydrogen ion (H+) or Hydronium Ion when
dissolved in water. 3. Phosphate Buffer System
b. Base – substance that can yield a hydroxyl ions (OH-‐). -‐Excess H+ combines in renal tubules with Na2PO4
-‐Sodium is reabsorbed and H+ is passed into urine.
c. K value or ionization constant – describes the relative strength of an acid -‐ plays a role in plasma and RBC which is involved in the
and a base. It also describes their ability to dissociate(separate) in water. exchange of sodium ion in the urine H+ filtrate.
-‐
d. pKa – defined as the negative log of the ionization constant (level of 4. Hemoglobin –Oxyhemoglobin Buffer System
concentration for protonated and unprotonated are equal). -‐ Venous content has higher CO2 content and bicarbonate ion concentration than
Ex. strong acid – less than pKa 3.0 ( if the pH becomes more than the areterial blood. The pH is the same since oxyhemoglobin (acid) in RBC has taken over some
pKa it will cause the acid to dissociate and yield H+) anion function which was provided by excess bicarbonate venous plasma.
Ex. strong base – greater than pKa 9.0 (if the pH becomes less than
the pKa it will cause release of OH-‐).
ACID BASE BALANCE
e. Buffer -‐ combination of a weak acid and weak base and its salt. It prevents Maintenance of H+
changes in the pH. -‐Reference Range : 34 – 44 nmol/L ( pH 7.34 – 7.44)
-‐ Principal Buffer system : Bicarbonate-‐Carbonic Acid System (pKa of 6.1) -‐ major role in maintaining pH thru the lungs and kidneys.
H2CO3 ß-‐-‐-‐-‐à HCO3-‐ + H+ Formula to Compute pH when H+ is given:
Reference value : pH of 7.40 or H+ of 40 nmol/L(arterial blood) pH = log 1 / H+ = -‐log H+ (pH is the negative log of H+)

Major Buffer Systems: TAKE NOTE!
Since pH if the negative log of H+:
1.Bicarbonate –Carbonic Acid Buffer System -‐ Increase in H+ concentration ____________the pH (< pH 7.34 –
It is the principal buffer system of the body. When a strong acid such as HCl enters acidosis)
the body, H+ combines with HCO3 ions of NaHCO3 yielding carbonic acid and -‐ Decrease in H+ concentration ___________ the pH (> pH 7.44 –
neutral salt. alkalosis)

HCL + NAHCO3 H2CO3 + NACl
The Bicarbonate –Carbonic Acid system (HCO3-‐ and H2CO3)
Weak acid like H2CO3 does not release H+ as readily as strong acid like HCl , thus -‐ main role is to regulate H+
ionizes effectively . When a strong base such as NaOH is added to the system, it is -‐ H2CO3 – is a weak acid , do not dissociate easily into H+ and
neutralized by carbonic acid yielding water and HCO3 from a salt. HCO3-‐

Functions:
In cases of imbalance, before Renal and Pulmonary compensatory mechanisms
1. H2CO3 dissociates into CO2 an H2O so CO2 can be eliminated by
the lungs alter the ratio of HCO3 to denominator H2CO3 by blowing off CO2.
the Lungs and H+ as water.
2. Respiration or Ventilation Rate is affected by changes in CO2
2. Protein Buffer System 3. HCO3 can be altered in the kidneys
K.B.R 9


4. Buffering system immediately counters the effects of fixed non-‐
volatile acids (H+ A-‐) by binding the dissociated hydrogen ion
(H+A-‐ + HCO3-‐ = H2CO3 + A-‐)
5. The resultant H2CO3 dissociates and H+ neutralized by the
buffering capacity of hemoglobin.

2 components:
a. HCO3 : HCO3 + H-‐ -‐> H2CO3
b. H2CO3: H2CO3 + OH -‐> H2O + HCO3

1. CO2 (by product of aerobic metabolism) diffuses in tissue
2. CO2 combines with H20 forming H2CO3 then dissociates to HCO3 + H by
carbonic anhydrase found in RBC.
3. HCO3 diffuses in plasma
4. H+ is increased and Cl-‐ is diffuses in the cell (Chloride shift , this is to maintain
electroneutrality or maintaining same number of + and – charged ions)
5. Plasma Proteins and plasma buffers also binds with free H+ to maintain stable
pH.

LUNGS (net effect is minimal change in H+ concentration in venous and
Regulation of Acid-‐Base Balance by Lungs and Kidneys arterial blood).
-‐ depicted by the Henderson –Hasselbach Equation 1. Inspired O2 binds with Hgb forming O2Hgb.
o pH = pKa + log A/ HA or HCO3/ pCO2 (H2CO3) 2. H+ from reduced Hgb in venous blood combines with HCO3-‐ forming
-‐ LUNGS: regulate pH by retention or elimination of CO2 by H2CO3 which dissociates again to H2O and CO2 . CO2 diffuses in alveoli
changing the rate and volume of ventilation and eliminated by ventilation/expiration.
o Represented by pCO2 (H2CO3) 2 conditions:
§ Denotes Lung Function
-‐ KIDNEYS: regulate pH by excreting acid , primarily in Ammonium a. Increase H+ / decreased pH
ion and reclaiming HCO3 from the glomerular filtrate. § lungs do not remove CO2 or decreased
o Represented by HCO3 ventilation
§ Denotes Kidney Function § Hypoventilation
b. Decrease H+/ increased pH
§ CO2 removal is increased or
hyperventilation.
§ Hyperventilation

10 K.B.R


NOTE: if the change in H+ is non-‐respiratory origin, lungs compensate by altering -‐ The remaining H+ combines with HPO4(2-‐) or monohydrogen
ventilation and regulating the pH. (first line of defense in acid-‐base status phosphate and (NH3) ammonia.
-‐ HPO4 (2-‐) becomes H2PO4(-‐) and NH3 becomes NH4 which is
*LUNGS MAINTAIN BALANCE BY ALTERING VENTILATION excreted. Remember daily excretion of H+ is mainly influenced
by the amount of NH4+ formed this is due to capability of renal
KIDNEYS tubular cells to generate NH3 from glutamine and other amino
-‐ main role is to reclaim HCO3-‐ from the glomerular filtrate. acid.
-‐ Main site for HCO3-‐ reabsorption is Proximal Tubules * Excretion of H+ in urine depends on the amount of NH4+
-‐ Transport of HCO3 in tubules is indirect by exchanging one formed.
molecule of H+ for Na+. * Concentration of NH3 will increase in response to a decreased
-‐ The H+ combines with HCO3-‐ to form H2CO3 which dissociates blood pH.
to H2O and CO2 ( by carbonic anhydrase) . The CO2 diffuses into Decrease pH = Increase NH3
the tubule and reacts with H2O to reform H2CO3 then
dissociates to HCO3-‐ and H+. HCO3-‐ formed is reabsorbed into -‐ LUNGS – regulate CO2 (hyperventilation or hypoventilation)
the blood together with sodium. -‐ KIDNEYS –regulate HCO3-‐ (reabsorption or
2 conditions: excretion/secretion)
a. Increased HCO3-‐ , Increased pH
b. Decreased HCO3 , Decreased pH Assessment of Acid -‐Base Homeostasis

other factors affecting HCO3-‐ reabsorption: Use: Henderson-‐Hasselbalch Equation
1. when HCO3-‐ is higher than 26-‐ 30 mmol/L.
2. Exception to this compensatory mechanism
(retention of HCO3-‐ even when it is increased
is when the condition is chronic lung Reference Range:
disease.) Parameter Normal Range at 37C
Increased levels of HCO3: pH 7.35-‐7.45
1. Increased HCO3 maybe due to IV infusion of lactate , acetate and HCO3-‐. pCO2 (mmHg) 35-‐45
2.Increased HCO3 may be due to excess loss of Chloride ( sweating , vomiting, HCO3-‐ (mmol/L) 22-‐ 26
prolonged nasogastric suction) . HCO3 is retained in the tubules to preserve Total CO2 content mmol/L 23-‐27
electroneutrality. pO2 (mmol/L) 80-‐110
Decreased levels of HCO3: SO2 (%) >95
1. use of diuretics O2Hb >95
2. excessive loss of cations

3. kidney dysfunction like chronic nephritis or infections (HCO3-‐
*lungs – control blood pH by Hyper or Hypoventilation
reabsorption is impaired).
* kidneys – control HCO3 concentration.

Important terms: (determines the pH)

a. Reabsorption and Reclamation – process of re-‐entering the blood.
ACID –BASE Disorders : Acidosis and Alkalosis
b. Secretion or Excretion – happens in the tubules by removing substances

from the filtrate.
2 conditions:

a. Acidemia -‐ <pH , excess acid concentration or H+ concentration
Notes: § Primary Respiratory Acidosis – change in pCO2
-‐ Normal Next excess of H+ : 50-‐100 mmol/L (this must be
excreted because minimum pH is 4.5)
K.B.R 11


o Increased PCO2 (decrease alveolar elimination or § Secondary compensation occurs when the original organ
hypoventilation leading to decreased elimination of with problem begins to correct the ratio by retaining
CO2 by the lungs) bicarbonate.
o Decreased pH
o Less than 20:1 ratio b. Alkalemia -‐ >pH , excess base concentration .
§ Primary Respiratory Alkalosis – change in pCO2
o Due to excessive elimination of CO2
Cause: o Decrease pCO2
-‐ COPD – desctruction of airways and alveolar wall increase the o Increase pH
size of alveolar air spaces resulting to the reduction of lung o Increase 20:1 ratio
surface area available for gas exchange. This leads to retention
of CO2 causing hypercarbia (elevated pCO2). Cause:
-‐ Bronchopneumonia – impeded gas exchange because of ü -‐hypoxia
secretions such as WBC , bacteria and fibrin in the alveoli wall ü -‐chemical stimulation by salicyates
-‐ Use of Barbiturates , morphine ,alcohol – promotes ü -‐increase environmental temperature
hypoventilation increasing blood pCO2 ü -‐fever
-‐ Mechanical Obstruction or Asphyxiation (strangulation or ü -‐hysteria (hyperventilation)
aspiration) ü -‐pulmonary emboli and fibrosis
-‐ Congestive Heart Failure – less blood being presented to the
lungs for gas exchange increasing pCO2. Compensatory Organ: Kidneys ( excretion of HCO33-‐) in the urine and
reclaiming H+ in the blood.
Compensatory Organ: Kidneys
-‐ Increase excretion of H+ and increase reabsorption og Treatment: In hysterical hyperventialtion –breathing into a paper
HCO3-‐ bag

§ Primary Non-‐Respiratory Acidosis /Metabolic / Renal § Primary Non-‐Respiratory Alkalosis/Metabolic/Renal
Function – change in HCO3-‐ Function – change in HCO3-‐
o Decrease HCO3 (< 24 mmol/L)
o Decreased pH o Increase HCO3-‐
o Less than 20:1 ratio o Increase pH
Cause: o Increase 20:1 ratio
• Administration of acid-‐producing substance like ammonium
chloride and calcium chloride Cause:
• Excessive formation of organic acids common in diabetic -‐ excess administration of sodium bicarbonate
ketoacidosis and starvation. -‐ ingestion of bicarbonate producing salts such as sodium lactate,
• Reduced excretion of acids like in renal tubular acidosis citrate and acetate
• Excessive loss of Bicarbonate due to diarrhea , drainage from -‐ Loss of acid through vomiting , nasogastric suctioning or
biliary , pancreatic or intestinal fistula. prolonged use of diuretics

Compensatory Organ: Lungs , Hypervantilation Compensatory Organ: Lungs – hypoventilation or retention of CO2
§ increase rate or depth of breathing
§ Blowing off CO2

12 K.B.R


5. Adequate hemoglobin
*compensation – body accomplishes by altering the factor NOT primarily affected by 6. Adequate transport (cardiac output)
the pathologic process to restore acid-‐base homeostasis. 7. Release of O2 into the tissue

REMEMBER: Notes:
If the imbalance is nonrespiratory origin the body compensates by altering -‐ *any alteration will cause poor tissue oxygenation
ventilation. For disturbances of the respiratory component , the kidneys compensate by • Amount of Oxygen available in atmospheric air depends on the Barometric
selectively excreting or reabsorbing anions and cations. The lungs can compensate pressure
immediately but the response is short term and incomplete. The kidneys are slower to • Dalton’s Law states that total atmospheric pressure is the sum of individual
respond (2-‐4 days) but the response is long term and potentially complete. gas pressures.
-‐ One Atmosphere (760 mmHg pressure) contains:
o Fully Compensated – pH has returned to normal range -‐ O2 – 20.93%
(20:1 range) -‐ CO2 – 0.03%
o Partially Compensated – pH has not yet returned to normal -‐ Nitrogen – 78.1%
-‐ Inert gases – approximately 1%
Summary of Acid-‐Base Disorders: • Partial Pressure = Barometric
Pressure at a particular altitude X
4 Major categories of acid-base disorders appropriate percentage for each
1. Respiratory acidosis gas.
• Vapor Pressure of water (47 mmHg
2. Respiratory alkalosis at 37C) is used to calculate partial
pressure.
3.Metabolic Acidosis Ex. Partial pressure of Oxygen at Sea Level (in the body )
= (760 mmHg – 47 mm Hg ) x 20 .93%
= 149 mm Hg (at 37C)
4. Metabolic Alkalosis

Partial Pressure of CO2 at sea level (in the body)
Oxygen and Gas Exchange
= (760 mm Hg -‐47 mm Hg) x0. 03%
Role of Oxygen: inside the mitochondria after oxidation of NADH and FADH2 , electron
= 2 mm Hg (at 37 C)
pairs are transferred to molecular oxygen causing release of the energy used to

synthesize ATP from phosphorylation of ADP.
Factors that influence amount of Oxygen moving in the alveoli into the blood and

into the tissue.
What is measured?
1. Destruction of alveoli – decrease surface area leading to inadequate amount of
pO2, pCO2 and pH
Oxygen moving into the blood

2. Pulmonary Edema – there is a barrier in the diffusion of gas between the

alveoli and capillary wall

3. Airway Blockage – prevention of air from reaching the alveoli like in Asthma

and Bronchitis

4. Inadequate Blood Supply – not enough blood to carry oxygen in the tissues

like in pulmonary embolism , pulmonary hypertension and heart failure
Seven Conditions Necessary for Adequate Tissue Oxygenation:
5. Diffusion of CO2 and O2-‐ decrease O2 diffusion
1. available atmospheric oxygen

2. adequate ventilation
Oxygen Transport
3. gas exchange between lungs and arterial blood
-‐ Oxygen is primarily transported in the tissue by Hemoglobin
4. loading of O2 in Hgb
K.B.R 13


-‐ One Adult Hemoglobin can be filled up by Four Molecules of 1.Blood gas analysis routinely made @ 37C
Oxygen. (reversible ) 2. for each grade of fever in the patinet, PO2 will fall by 7% and CO2 will rise 3%.
o Binding is affected by:
§ Availability of oxygen
§ Concentration and type of Hemoglobin Measure by blood gas instrument:
present a. pH
§ Presence of interfering substances such as b. pCO2
CO, pH, Temperature , pCO2 and 2,3-‐DPG. c. pO2
§ Increase levels of Oxygen when hemoglobin
is fully saturated is toxic.
§ 4 forms of hemoglobin: NOTE: HCO3 and CO2 content can be obtained by nomogram after pH + pCO2
• Oxyhemoglobin – O2 bound to HgB -‐ measurement or as a direct readout from a calculation programmed in the instrument.
reversible
• Deoxyhemoglobin – Reduced Total CO2 Content:
Hemoglobin (not bound) -‐reversible
• Carboxyhemoglobin – CO2 bound to A. Automated Enzymatic Method
Hgb –reversible All forms of CO2 present in serum are converted
• Methemoglobin – Do not bind with HCOs by the addition of a base. The HCO3 is then converted to oxaloacetic acid by
O2 because Fe3+ / oxidized rather phosphoenolpyruvate carboxylase. The extent of oxaloacetic acid formation can be
than reduced binds with Hgb. monitored spectrophotomterically by measuring its conversion to malate by malate
Methemoglobin reductase can dehydrogenase with a corresponding consumption of NADH, a UV light chromogen.
reduce Fe3+ which is found in RBC.
B. Automated Colorimetric Method
Methods of Measurement of Acid-‐Base Balance The CO2 appearing in serum as HCO3 , H2CO3 or carbaminbound CO2 is released by
a.requires arterial blood sample (pO2 testing) addition of acid. The gaseous CO2 is dialyzed through a silicone-‐rubber gas dialysis
b. venous blood maybe taken for pH and CO2 provided it is drawn without stasis (no membrane into a buffer solution of cresol res at pH 9.2 . The CO2 diffuses through the
torniquet) and without patient clenching the fist. membrane and lowers the pH of buffered cresol red solution. The decrease in color
intensity is proportional to the CO2 content and is measured in a spectrophotometer
b. Arterialized venous blood maybe obtained by heating the hand or forearm in water at at 430 nm. Other continuous flow methods have used phenolphthalein as the
indicator.
45C for 5 minutes then drawing blood from dilated veins on the back of the hand.

c. Capillary blood is arterialized by warming the ear, finger or heel at 45C before taking the

sample.

d. Dead space in the needle should be filled with a needle with sterile anticoagulant to
prevent formation of air bubbles. ( maintain anaerobic environment)

e. Samples are placed in a tray if ice for analysis in the laboratory.

Specimen & Patient Consideration:
§ most desirable specimen is arterial blood ( it reflects the status of gas
§ ________________________________________.
§ ________________________________________.
For Patient:
14 K.B.R


MERCURY
TRACE and TOXIC ELEMENTS
Manganese
§ Levels varies based on: Absorption, Transport ,
Distribution, Metabolism and Elimination. Molybdenum
§ Deficiency maybe due to:
o Decreased Intake SELENIUM
o Impaired Absorption
o Increased Excretion Zinc
o Genetic Abnormality
2 types: Questions:
1. Essential 1. Discuss the disorders associated with the excess and deficiency of each trace
a. Associated with cofactors such as Metalloenzyme element/metal.
(enzyme) and Metalloprotein (protein) as cofactors. 2. Determine the specimen collection and preparation in the measurement of
2. Non-‐Essential each trace element and metal.
a. Usually toxic 3. Enumerate the different laboratory tests used in the measurement of each
Laboratory Determination: trace element/metal.
Specimen: Serum , Plasma and Urine 4. Discuss the absorption, transport and excretion/elimination of each trace
_____________________________________________________________________________________________________ element/metal.
____________________
INSTRUMENTATION
Methods of Instrumentation
1. AAS
2. AES (Atomic Emission Spectroscopy)
3. FAAS (Flame Atomic Absorption Spectroscopy)
4. GFAAS ( graphite furnace atomic absorption spectroscopy)
5. ICP-‐AES (Inductively Coupled Plasma Atomic Emission Spectroscopy)
6. ICP-‐ MS (Inductively Coupled Plasma Mass Spectroscopy)

Trace and Toxic Elements

ALUMINUM

ARSENIC

CADMIUM

Chromium

COPPER

IRON

LEAD
K.B.R 15


5. Epidermal growth factor receptor (EGFR)
TUMOR MARKERS • also known as HER1

• a receptor found on cells that helps them grow
AFP (a-‐fetoprotein)
• used to guide treatment and predict outcomes of non-‐small
cell lung, head and neck, colon, pancreas, or breast cancers
Cancer Antigen 125
6. S-‐100
Carcinoembryonic Antigen • protein found in most melanoma cells
• elevated in most patients with metastatic melanoma
Human Chorionic Gonadotropin

Prostate-‐Specific Antigen

CA 125

CA 19-‐9

OTHER TUMOR MARKERS
1. Bcr-‐abl -‐ abnormal gene , increased in chronic myeloid leukemia
§ Philadelphia chromosome or Philadelphia translocation –
defect in chromosome 22 (reciprocal translocation between
chromosome 9 and chromosome 22. – t (9;22) (q34;q11)-‐
common in CML
§ Breakpoitn cluster region-‐Abl 1 gene or a fusion gene
2.Beta-‐2-‐microglobulin (B2M)
• Elevated in multiple myeloma and chronic lymphocytic
leukemia
• Increased in kidney disease and hepatitis
1. Bladder tumor antigen (BTA)
• found in the urine of many patients with bladder cancer
2. CA 27.29
• used to follow patients with breast cancer during or after
treatment
3. CA 72-‐4
• ovarian and pancreatic cancer
• cancers starting in the digestive tract, especially stomach
cancer
4. CALCITONIN
• Produced by parafollicular cells
• In medullary thyroid carcinoma (MTC)

16 K.B.R

Enzymes: Clinical Chemistry

Uploaded by

Copyright:

Available Formats

Enzymes: Clinical Chemistry

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Enzymes: Clinical Chemistry

Uploaded by

Copyright:

Available Formats

CLINICAL

2.)Hypomagnesia %Saturation=Serum Fe/TIBC x 100

You might also like