CLINICAL CHEMISTRY- LABORATORY

MIDTERM- ACID-BASE BALANCE

Mr. Rodmie Oliver E. Pumaras
 Oxygen saturation (O2 saturation) - 94 - 100 volume %
DEFINITION OF TERMS:
• Acid: a compound that could donate a H+ ion
• Base: a compound that could accept a H+ ion  Parameters used to assess adequate tissue oxygenation:
• Acid-Base Balance: a mechanism by which the pH of blood is maintained at 7.35- 1. Available atmospheric oxygen
7.45 for homeostasis 2. Adequate ventilation
• Buffer: a weak acid/base with its conjugate salt that resists changes in Ph 3. Gas exchange between lungs and arterial blood
Lungs: Respiratory control of CO2 excretion allows rapid and very sensitive adjustments in blood pH. 4. Loading of Oz ot Hb
Kidneys: excretion of acid, which is equivalent to generation of alkali or reabsorption of HCO3 from the 5. Adequate Hb
glomerular filtrate (proximal tubules of the kidneys) and add it to the blood. 6. Adequate transport (Cardiac output)
- Acid is excreted in the form of NH4+ and titrable acid. 7. Release of O2 to tissue
Blood pH. Blood gases and acid-base balance
Parameters definition  FORMS OF HEMOGLOBIN SPECIES

1. pH - a measure of the effective acidity and alkalinity of 1. O2Hb - O2 is reversible bounded to Hb

2. Total CO2 concentration (ctCO2)
a. Unionized fraction
b. Ionized fraction
3. CO2 combining power (CO2 Cp)
4. Partial Pressure of CO2 (PCO2)
5. Partial pressure of Oxygen (PO2)
 Oxygen content (O2 content)
a substance. - 2-3DPG is important in O2Hb dissociation
- negative Logarithm of Hydrogen ion 2. ННb deoxy-hemoglobin
concentration. 3. MetHb methemoglobin
Normal pH: 7.35- 7.45
4. СОНb carboxy hemoglobin
• pH 7.40 is the optimum level for arterial blood.
- refers to the total CO2 concentration in the
blood. ▪️pH
- carbonic acid (H≥CO3)/ cdCO2 - important in the body for proper metabolic functioning
- include un-dissociated carbonic acid and - normal blood pH 7.35 - 7.45
physiologically dissolved anhydrous CO2 - increase pH (alkalosis), decrease (acidosis)
- HCO3, the common form of CO2 dissolved in – minor alteration (0.1) alters the ff:
blood a. Oxygen transport
- Carbamino compounds (0.2 mmol/L) - CO3 b. Cellular transport of materials
(1/1000) c. Enzyme activity
- an index of the amount of CO2 that can be bound Sources of Hydrogen ions or protons (body)
as bicarbonate to serum, plasma or whole blood at -body produces 15-20 moles of hydrogen ion per day
partial CO2 pressure of 40 mmHg at 25.
- Alveolar CO2 - normal concentration in extracellular fluid is 36 - 44 nmol/L
- Arterial CO2 1. Diet
- pressure or tension exerted by CO2 gas dissolved
in the blood 2. Produce endogenously as a product of metabolism
- index of deficiency of gas in the lungs, not a
measure of CO2 conc. in the blood Metabolic processes:
Normal pCO2: 35-45 mmHg
• Increased CO2: use of elicit drugs like a. Glucose conversion to lactic acid 1 molecule glucose = 2 molecule LA
barbiturates and morphine, and alcoholism
- Alveolar and arterial Oxygen b. Fatty acid breakdown FA - - acetoacetic acid - - - acetate + H
- pressure or tension exerted by O2 gas dissolved in
arterial blood c. Amino acid metabolism AA---Urea +CO2 +H
Normal pO2: 81-100 mmHg (adequute
d. Mitochondrial release and uptake during oxidative
oxygenation)
phosphorylation
- reflects the availability of the gas in blood but
not its content
▪️Buffer System
- volume of O2 combined with Hemoglobin on
arterial blood - 15 - 23 volume % - normal pH in the body is maintained by different buffer systems

Montalban, Kimberly N. 1
CLINICAL CHEMISTRY- LABORATORY
MIDTERM- ACID-BASE BALANCE
Mr. Rodmie Oliver E. Pumaras
- mixture of weak acid and its salts with the capability of combining with - refers to COz and O2 concentration
protons or releasing protons in response ot external shift in pH - during blood collection, temperature of patient must be noted. (required for the computation)
 Instruments
▪️Major buffer systems - most are automated machines and quantitated using special electrodes.
1.BI-CARBONATE BUFFER SYSTEM = H20 + CO2 <———> H2CO3<——> HCO3 +
H  pH determination
= increase in CO2, shift to the right producing more 1. Automated method
H and HCO3  - use of glass electrode sensitive to H - standard electrode
= the reaction will shift to the left: a. Ag and AgCI electrode
a. Increase in protons (^proton) b. Calomel electrode
b. Decrease in CO2 (Respiration) - electrodes are calibrated using PO4 buffer with known pH
2. HEMOGLOBIN/OXYHEMOGLOBIN - Related to oxygen transport 2. pH electrode (pH meter)
BUFFER SYSTEM - protons and O2 exchange in Hb - follow the principle of potentiometry
- protons are coming from cellular metabolism 3. Mathematical computation
(tissue) a. Seggard-Anderson alignment nomograms
-O2 is from respiration (lungs)
c. Henderson-Hasselbach equation
 Minor buffer system - pH =pka +log of HCO3/H2C03
- pH = рКа 6.1 НСО3 (salt) / H2C03 (acid) (PCO2 x 0.03)
1. Phosphate buffers (organic/in-organic - plays important role in plasma and erythrocytes
 Total CO2
2. Proteins - due ot amino and carbonyl group 1. Gasometric Method Natelson microgasometer
NH2 - captures H-- - NH3 - 10 % lactic acid (release CO2 from HCO3)
COOH - - - COO- + H - 12 % NaOH (total re-absorption of CO2)
- caprilic alcohol (prevents foaming)
Procedure:
• Acid-Base regulation - a. Introduction of sample and reagents to reaction chamber
- maintenance of acid-base balance si regulated by Kidneys and Lungs
- b. Agitation (release CO2 from the sample)
A. Respiratory Control (Lungs)
- c. Compression of liberated gas, measurement of
- pH is controlled through transport of Hand Hb and disposal of COz
o P1 (total pressure exerted by all gases)
B. Renal/Metabolic Control (Kidneys)
- d. Introduction of 12% NaOH into reaction chamber, then P2 is measured
- pH is controlled based on the excretion and secretion of electrolyte and H in PCT and DCT o P2 - pressure of non-CO2 gases
***** Paste page 19 and 20 here**** o PCO2 = P1 - P2
2. Automated machine (SMA)
 Arterial puncture Normal Value:
- appropriate method of blood collection Arterial whole blood - 19-24 mmol/L
Venous plasma/serum - 23 - 29 mmol/L
- done by experts (trained MT or Physicians) - sites: Venous whole blood - 22 - 29 mmol/L
a. Brachial artery
b. Radial artery  PCO2
c. Femoral artery 1. Henderson-Hasselbach equation
 Receptacle for collection 2. PCO2 electrode
1. Heparinized glass syringe (best) - severing hause PCOz electrode
2. Heparinized plastic syringe 3. Automated method
(gases leaks out of the plastic) - CO2 interact with NaHCO3 solution, change in pH is detected using specialized electrode
3. Oiled syringe (oil absorb COz) o Normal Value
4. Evacuation tube (Vacutainer)
 35 - 40 mmHg (SI expressed in KiloPascal)
(POz is affected due to Oz in the tube)
 increased :Hypercapnia
 decreased: Hypocapnia
 ABG (Arterial Blood Gas Analysis)
- commonly requested test
Montalban, Kimberly N. 2
CLINICAL CHEMISTRY- LABORATORY
MIDTERM- ACID-BASE BALANCE
Mr. Rodmie Oliver E. Pumaras
- modification of macro-electrode
 PO2 3. Optic sensors
1. Gasometric - based on reaction of CO2, O2 and H with fluorescent dye
2. Automated (Clarke PO2 electrode) - measurement is either increase in fluorescence or quenching of fluorescence
- polarographic principle
- O2 reacts with platinum cathode, generating a current flow which is proportional to the 0 2 content of
the sample
GENERAL TERMS
o N.V.: 95 - 100 mmHg
 (Hyperoxemia, hypoxemia) o Acidosis/Acidemia
 CO2 and HCO3 - blood pH of less than 7.35
1. Ion Selective Electrode (ISE)
2. Automated method o Alkalosis/ Alkalemia
- diffusion of CO2 into solution containing phenolphthalein indicator, reverse reaction occurs - Blood pH of more than 7.45
(regeneration of proton) result into change in pH and shift in color of indicator.
- Change in color is measured spectrophotometrically.
3. Enzymatic method (Dupont ACA) Respiratory Acidosis
- bicarbonate (HCO3) reacts with PEP producing Oxaloacetate and PO4
- Oxaloacetate + NADH producing malate and NAD (measure at 340 nm) o Accumulation of excess CO2 in the blood
o N.V.: o To decrease in ability of the lungs to exhale CO2
o Hypoventilation (decrease CO2 elimination)
- H2CO3 1.05 - 14.5 mmol/L
o Impairment in the respiration
- HCO3 21- 28 mmol/L
o Increase in CO2 causing shift to the right of the bicarbonate buffer

 ctCO2
- Bicarbonate + H2CO3 Specific disorders under Respiratory acidosis

 CO2 combining power 1. Pneumonia

a. Sample is placed in separatory funnel (increase the surface area of the sample) 2. Emphysema
3. Asthma
b. Equilibrated with CO2 by 5.2% CO2 (blowing)
4. Chronic obstructive lung disease
c. Determination of ctCO2 (any method)
5. Myasthenia gravis
- Secondary effect to the impairment of the transmission of nerve impulses
 An-ion gap 6. Drugs (narcotic, morphine) depressed RC
- mathematical approximation of difference between the an-ions and cat-ions routinely measured in
serum
 Cat-ions: Na and K, An-ions: Cl and HCO3 B. Respiratory Alkalosis
- difference between an-ion and cat-ion should be less than 17 mmol/L (N.V.) - hyperventilation which causes increase exhalation of CO2
- average of unmeasured (+) ions (at least 7 mmol/L) - decrease CO2, causes shift to the left of the bicarbonate buffer
- unmeasured (-) ions (at least 24 mmol/L)

 Significance of an-ion gap 6.Hypoxia

- increase in an-ion gap occurs: acute: pneumonia, asthma
a. Uremia
b. Lactic acidosis chronic: pulmonary fibrosis, high altitudes
c. Ingestion of toxins
d. Increase plasma protein (dehydration) 7. Aspirin intoxication (overdose)
- Quality control for results in Na and K Cl and CO2
- respiratory center stimulation
 New Technology in Blood Gas Measurement
1. Macro-electrode sensors C. Metabolic Acidosis
- designed to minimize the required sample volume
2. Thick and Thin film technology - excessive metabolic hydrogen (H) production

Montalban, Kimberly N. 3
CLINICAL CHEMISTRY- LABORATORY
MIDTERM- ACID-BASE BALANCE
Mr. Rodmie Oliver E. Pumaras
- loss of bicarbonate ion
- Hydrogen ion retention may be due to retention of normally excreted materials
which may carry Hydrogen ion

• Specific disorders
1. Renal failure and Renal tubular dysfunction
2. Excessive loss of Na which
retains H instead
3. Ketoacidosis (D.M., alcohol, starvation)
- increase amount of B-hydroxy butyrate and
hydrogen ion which causes the loss of Na, K
4. Metabolism of toxic materials
a. Methanol (formic acid)
b. Ethanol (Acetic Acid)
c. Aspirin (Salicylic Acid)
5. Lactic Acidosis
- common cause of metabolic acidosis
- not converted to pyruvate (hypoxia)
D. Metabolic Alkalosis
- excess Hydrogen ion or excess alkali intake
- increase bicarbonate ion

Specific disorders
1. Patient with peptic ulcer treated with antacids which is rich in bicarbonate
2. Diuretic therapy
- due to excessive excretion of acid by kidneys leaving bicarbonate
3. Cortisol metabolism disorder
- increase in aldosterone which increases K and Cl excretion. To balance ionic equilibrium, HCO3 is
produced causing shift to the left of bicarbonate buffer

Types of Pipettes Graduated Pipettes Serologic Pipettes

Picture
Type Measuring Delivery/ Blow-out

Montalban, Kimberly N. 4
CLINICAL CHEMISTRY- LABORATORY
MIDTERM- ACID-BASE BALANCE
Mr. Rodmie Oliver E. Pumaras

Calibration “To deliver” – no “To contain” – AUTOMATIC

blow-out needed; needs blow-out for
accuracy not accurate
required delivery
Measuring:
1. Standards
2. Control
3. Unknown
specimens
or filtrates
Additional info  Has larger
orifice/
opening

 Has frosted
ring at non
calibrated
end

 Usually
empties
through
gravity

 Depending
on
calibration,
last drop
may need to
be expelled
to deliver
volume
-
PIPETTING TECHNIQUES

MANUAL

Montalban, Kimberly N. 5