Acid-Base Balance

ACID, BASE, BUFFER

Information on a patient’s acid-base balance and blood gas homoeostasis is an important aspect in clinical
biochemistry. These data often are used to assess patients in life-threatening situations.
An acid is a substance that can donate protons in the form of hydronium ions (H+) while a base is a substance
that accepts protons and yields hydroxyl ions (OH-). Strong acids readily give H+ while strong bases readily
accept H+. A conjugate base of strong acid is a weak base and vice versa.
The pK of a solution is the negative logarithm of the ionization constant of a weak acid or base. It is the
relative strength of acids and bases, and their ability to dissociate in water. It is also the pH at which there is
equilibrium between an acid/base with its conjugate. Acids have pK values <7.0, whereas bases have pK values
>7.0.
The pH of a solution is defined as the negative logarithm of hydrogen ion concentration. The average pH of
blood (7.40) corresponds to a hydrogen concentration of 40 nmol/L. Potentiometric determination of blood
pH measure hydrogen activity; however, hydrogen activity is equal to hydrogen concentration.
pH = log 1 = -log cH+
cH+
The buffer, a combination of a weak acid or a weak base and its salt, is system that resists changes in pH. The
effectiveness of a buffer depends on the pK of the buffering system and the pH of the environment in which it
is placed. In plasma, the bicarbonate-carbonic acid system, having a pK of 6.1 is one of the principal buffers.
H2CO3 = HCO3 + H+
The normal concentration of H+ in the extracellular body fluids ranges from 36-44nmol/L (pH 7.34-7.44).
Through the exquisite mechanisms that involves the lungs and the kidneys; the body controls and excretes H+
in order to maintain pH homeostasis. Any H+ value outside this range will cause alterations in the rates of
chemical reactions within the cell and affect many metabolic processes of the body and can lead to alterations
in consciousness, neuromuscular irritability, tetany, coma and death.
An increase in H+ concentration decreases the pH. A blood pH below the reference range is called academia,
whereas above the reference range is alkalemia. Acidosis or alkalosis refers to the physiological state of the
body that results to academia or alkalemia.

BUFFER SYSTEMS: REGULATION OF H+

The body’s first line of defense against extreme changes in H+ concentration is the buffer systems present in
all body fluids. A great buffer has a pK that is near the desired pH; has an acid:base ratio of 10:1 to 1:10; has a
high concentration. All buffers consist of a weak acid, such as carbonic acid, and its salt or conjugate base,
bicarbonate (for the bicarbonate-carbonic acid system).

Bicarbonate/Carbonic Acid Buffer System

This system has a low buffering capacity, because of its low pK (6.1) and high base to acid ratio (20:1). What
makes this an effective buffer is its high concentration (>20mmol/L). When an acid is added to the
bicarbonate-carbonic acid system, the HCO3 will combine with H+ to form from the acid to form H2CO3. When
a base added, H2CO3 will combine with the OH group to form H2O and HCO3.

Phosphate Buffer
At a plasma pH of 7.4, the HPO4:H2PO4 is 4:1 (pK=6.8). The total concentration of this buffer in both
erythrocytes and the plasma is less than that of the other buffer systems, accounting for only 5% of the
noncarbonated buffer value of plasma. Organic phosphate, however, in the form of 2,3-diphosphoglycerate
(2,3-DPG) (present in erythrocytes in a concentration of about 4.5 mmol/L), accounts for about 16% of the
noncarbonated buffer value of the erythrocyte fluid.

Plasma Protein and Hemoglobin Buffer System

Proteins, especially albumin, account for the greatest portion (95%) of the noncarbonated buffer value in
plasma. The most important buffer groups of protein in the physiological pH range are the imidazole group of
histidines (pK = 7.3). Each albumin molecule contains 16 histidines.
Hemoglobin accounts for the major part of the noncarbonated buffer of the erythrocyte fluid, with the
remainder being contributed by 2,3-DPG. The imidazole groups of hemoglobin are quantitatively the most
important buffer groups.
REGULATION OF ACID-BASE BALANCE
Lungs
Inspired O2 diffuses form the alveoli into the blood and is bound to hemoglobin (oxyhemoglobin). The H+ that
was carried on the hemoglobin in the venous blood is released to recombine with HCO 3 to form H2CO3, which
dissociates into H2O and CO2. The CO2 diffuses into the alveoli and is eliminated through ventilation. The net
effect of these two buffering systems is a minimal change in H+ concentration between the venous and the
arterial circulation. When the lungs do not remove CO2 at the rate of its production (decreased in ventilation
or disease), it accumulates in the blood, causing an increase in H+ concentration. If, however, CO 2 removal is
faster than production (hyperventilation), the H+ concentration is decreased. Consequently, ventilation affects
the pH of blood. A change in the H+ concentration of blood that results from nonrespiratory disturbances
cause the respiratory center to respond by altering the ventilation in an effort to restore the blood pH to
normal. The lings, by responding within seconds, together with the buffer systems, provide the first line of
defense to changes in acid-base status.
Kidneys
The kidneys are also able to excrete variable amounts of acid or base, making them an important player in the
regulation of acid-base balance. The kidney’s main role in maintaining acid-base homoeostasis is to reclaim
HCO3 from the glomerular filtrate. Without this reclamation, the loss of HCO3 in the urine would result in the
excessive acid gain in the blood. The main of site for HCO3 reclamation is the proximal tubules. The glomerular
filtrate contains essentially same levels of HCO3 as the plasma. The process is not a direct transport of HCO3
across the tubular membrane into the blood. Instead, sodium in the glomerular filtrate is exchanged for H+ in
the tubular cell. The H+ combines with HCO3 in the filtrate to form H2CO3 which is converted into H2O and CO2
by carbonic anhydrase. The CO2 easily diffuses into the tubule and reacts with H2O to reform H2CO3 and then
HCO3 which is reabsorbed into the blood together with sodium. With alkalotic conditions, the kidneys excrete
H2CO3 to compensate for the elevated blood pH. The exchange between H+ and Na+ suggests, in part, why
clinicians order pH and blood gases together, along with electrolytes, to assess the patient.

PARAMETERS IN ASSESSING ACID-BASE BALANCE

Evaluate the pH
Normal blood pH: 7.35 – 7.45
<7.35 = Acidosis
>7.45 = Alkalosis
7.40 is optimum pH level of arterial blood.
To preserve the pH within the narrow physiologic range, short-term buffering capacity must neutralize acids as
they are generated, and long-term corrective measures must eliminate the acid permanently, but not on a
continuous basis.
pH is elevated:
Hyperventilation
Anxiety, Pain
Anemia
Shock
Myocardial Infarction
Hypokalemia
Gastric Suctioning or Vomiting
Antacid Administration
Aspirin Intoxication
pH is decreased:
Strenous Physical Excercise
Obesity
Starvation
Diarrhea
Ventilatory Failure
Pulmonary Edema
Cardiac Arrest
Renal Failure
Lactic Acidosis
Ketoacidosis in Diabetes
Evaluate the Ventilation
Normal pCO2: 35-45 mm/Hg
< 35 mm/Hg = Respiratory Alkalosis
> 45 mm/Hg = Respiratory Acidosis
pCO2 is an index of efficiency of gas exchange and not a measure of CO2 concentration in the blood.
H2CO3 is proportional to the partial pressure of CO2 (pCO2).
The lungs regulate the pH by retaining or eliminating CO2.
There are two factors that have a significant impact on pCO 2. The first is how rapidly the person is breathing
and the second is the lung capacity for freely exchanging CO2 across the cellular membrane.
Increased pCO2:
Pulmonary Edema
Obstructive Lung Disease
Decreased pCO2:
Hyperventilation
Hypoxia
Anxiety
Pregnancy
Pulmonary Embolism

Evaluate the Metabolic Process

Normal HCO3: 21-28 mmol/L
<21 mmol/L = Metabolic Acidosis
>28 mmol/L = Metabolic Alkalosis
The kidneys regulate the pH by excreting acid and reabsorbing HCO3 from the glomerular filtrate.
Increased HCO3:
Severe or Prolonged Vomiting
Lung Disease
Cushing syndrome
Decreased HCO3:
Chronic Diarrhea
Diabetic Ketoacidosis
Shock
Kidney Disease
Ethylene Glycol Poisoning
Aspirin Overdose

Evaluate the Degree of Oxygenation

Normal pO2: 81-100 mmHg
Mild Hypoxemia = 61-80 mmHg
Moderate Hypoxemia = 41-60 mmHg
Severe Hypoxemia = <40mmHg
pO2 reflects the amount of oxygen gas dissolved in the blood.
It measures the effectiveness of the lungs in pulling oxygen into the blood stream from the atmosphere.
It also measures the degree of dissociation of O2 with hemoglobin.
Increased pO2:
Increased oxygen levels in the inhaled air
Polycythemia
Decreased pO2:
Decreased oxygen levels in the inhaled air
Anemia
Heart Decompensation
Chronic Obstructive Pulmonary Disease
Restrictive Pulmonary Disease
Hypoventilation
Henderson-Hasselbalch Equation
The Henderson-Hasselbalch equation expresses acid-base relationships in a mathematical formula. It relates
the pH of a solution to the dissociation properties of a weak acid. It indicates that pH depends on the ratio of
HCO3/pCO2. When the kidneys and the lungs are functioning properly, a 20:1 ratio of HCO 3 to H2CO3 will be
maintained, and it is expressed in the Henderson-Hasselbalch equation.

pH= pK + log conjugate base

weak acid
 In the plasma at 37°C pKa of the bicarbonate/carbonic acid buffer system is 6.1.
 Concentration of H2CO3 is proportional to the pressure exerted by pCO2.
 H2CO3 in mmol/L can be converted to pCO2 in mmHg by the conversion factor 0.0307 mmol/L/mmHg
 Total CO2 can be determined by the sum of H2CO3 and HCO3.

OXYGEN AND GAS EXCHANGE

• Evaluation of patient's oxygen status is done by measuring pCO2 in blood gas analysis (together with pCO2
and pH)
• For adequate tissue oxygenation, the following are necessary:
1. Available atmospheric oxygen
- Amount of O2 available in atmospheric air depends on barometric pressure (BP)
- At sea level, BP is 760 mm Hg
- Dalton's law states that the total atmospheric pressure is the sum of individual gas pressures
- One atmosphere exerts 760 mm Hg pressure and is made up of 78.1% nitrogen, 20.93% O2, 0.03%
CO2, 1% inert gases; percentage of each gas is the same at all altitudes
- Partial pressure of each gas is equal to the BP at a particular altitude times the appropriate
percentage for each gas
- Vapor pressure of water (47 mm Hg at 37*C) must be accounted for in calculating partial pressure
for each gas; in the body, these gases are always fully saturated with water
- Partial pressure of O2 in the body at sea level:
(760 mmHg - 47 mm Hg) x 20.93% = 149 mm Hg at 37*C
- Partial pressure of CO2 in the body at sea level:
(760 mm Hg - 47 mm Hg) x 0.03% = 2 mm Hg at 37*C
1. Adequate ventilation
- During inspiration, the thoracic cavity expands creating a negative pressure gradient causing air to
enter alveoli
- During inspiration, airways are still filled with air retained from the previous expired breath; this is
called dead space air; dead space air dilutes the air being inspired; air being inspired is also fully
saturated with water vapor causing inspired air to have only a pO2 of 110 mm Hg (instead of 149
mm Hg)
- Three factors influence pO2 in alveoli:
• percentage of O2 in inspired air
• amount of pCO2 in the expired air
• ratio of the volume of inspired air to the volume of dead space air
- Percentage of O2 in inspired air / fraction of inspired O2 / FiO2 can be increased by breathing gas
mixtures of up to 100% O2
- Conditions that influence pCO2 in expired air: increased metabolism, hyperthermia produce more
CO2 than can be eliminated
- Ratio of volume of inspired air to the volume of dead space air is usually constant; but people
with shallow breaths have less fresh air entering the lungs than those breathing deeply
2. Gas exchange between the lungs and arterial blood
- Influenced by:
• destruction of alveoli (emphysema)
 • pulmonary edema
• airway blockage (asthma, bronchitis)
• inadequate blood supply (pulmonary embolism, pulmonary hypertension, CHF)
• diffusion of CO2 and O2 (O2 diffuses 20 times slower than CO2)

3. Loading of O2 onto Hb
- Each Hb molecule can combine reversibly with four O2 molecules
- Actual amount of O2 loaded onto Hb dependent on:
• availability of O2
• concentration and type of Hb
• presence of interfering substances (CO)
• pH
• temperature of blood
• levels of pCO2 and 2,3-diphosphoglycerate
- Normally more than 95% of functional Hb (Hb capable of reversible binding O2) will bind O2
- Increasing FiO2 further saturates Hb; but once Hb is 100% saturated, the increased O2 in alveoli
only increases concentration of dO2 in the arterial blood
- Prolonged administration of high concentrations of O2 causes oxygen toxicity or decreased
ventilation that leads to hypercarbia
- Hb in blood exists either as:
• oxyhemoglobin (O2Hb) - with bound O2
• Deoxyhemoglobin (HHb; reduced Hb) - Hb not bound to O2 but capable of binding O2 when
available
• Carboxyhemoglobin (COHb) - with bound CO
• Methemoglobin (metHb) - unable to bind O2 because iron is oxidized
4. Adequate Hb
5. Adequate transport (cardiac output)
6. Release of O2 to the tissues
• Any disturbance in these conditions result to poor tissue oxygenation

ASSESSING OXYGEN STATUS

• Parameters commonly used to asses a patient's oxygen status:

1. Oxygen saturation (SO2; O2sat)
- Represents the ratio of O2 that is bound to Hb compared to the total amount of Hb capable of
binding O2
- Blood gas instruments uses algorithms to calculate SO2 from pO2, pH and temperature of sample;
but does not account for presence of other Hb types that do not reversibly bind O2
2. Measured fractional / percent oxyhemoglobin (FO2HB)
- Ratio of the concentration of oxyhemoglobin to the concentration of total Hb
3. Trends in oxygen saturation measured by transcutaneous pulse oximetry (SpO2)
- Pulse oximeters pass light of two or more wavelengths through the tissues in the capillary bed of the
toe, finger or ear
- Measures O2Hb and HHb; newer machines can now measure COHb and metHb
- Accuracy compromised by poor perfusion and severe anemia
4. Amount of O2 dissolved in plasma (pO2)
- Healthy adult breathing room air will have a pO2 of 90-95 mm Hg
- With a blood volume of 5 L, only 13.5 ml of O2 will be available from pO2 in plasma; 1,000 ml of O2
is carried as O2Hb
• Hemoglobin oxygen binding capacity: the maximum amount of O2 that can be carried by Hb in a given
quantity of blood; 1 gram of Hb carries 1.39 ml of O2
- Total Hb is 15 g/dL: when Hb is 100% saturated with O2, the O2 capacity is 20.8 ml O2 / 100 ml of
blood
• Oxygen content: is the total O2 in blood; determined from the sum of O2 bound to Hb (O2Hb) and the
amount of O2 dissolved in plasma (pO2)
- For every mm Hg of pO2, 0.00314 ml of O2 will be dissolved in 100 ml of plasma at 37*C; if pO2 is 100
mm Hg, 0.3 ml of O2 is dissolved in every 100 ml of plasma
- Amount of dissolved O2 is usually not clinically significant; but in instances when total Hb is low or in
hyperbaric conditions, dissolved O2 becomes an important source of oxygen to tissues
- When 97-99% of available Hb is saturated with O2 and total Hb is 15 g/dL, the oxygen content per 100
ml of plasma is 20.5 ml

HEMOGLOBIN-OXYGEN DISSOCIATION

• Hemoglobin must release O2 to the tissues

- increased H+ concentrations and pCO2 levels in tissues change the molecular configuration of
oxyhemoglobin facilitating oxygen release
• Oxygen dissociates from Hb in a characteristic fashion; graphed with pO2 on the x-axis and percent
saturated O2 on the y-axis; sigmoid curve
• Hb holds on to O2 until oxygen tension in tissues is about 60 mm Hg; below 60 mm Hg, O2 is released
rapidly
• The position of the curve reflects the affinity that Hb has for O2 and affects rate of dissociation
• Factors affecting position and shape of oxygen dissociation curve:
- H+ ion activity
- pCO2 and CO levels
- body temperature
- 2,3-DPG
• Shift to the right
- In actively metabolizing tissues, there is increased temperature, increase pCO2, increased 2,3DPG and
increased H+ (low pH)
- Hb has decreased affinity for O2 allowing release of O2 to tissues
• Shift to the left:
- In the lungs, temperature, pCO2, H+ and 2,3DPG decrease relative to tissue levels enhancing O2
binding to Hb and O2 uptake
• Hb in tissues (high CO2 and H+) release O2; release of O2 from Hb accelerates uptake of CO2 and H+ by Hb
• Hb in the lungs release CO2 and uptake O2
• Elevation of CO (smoking or CO exposure) causes a shift to the left with loss of the sigmoid curve making
release of O2 bound to Hb more difficult
• HbF causes a shift to the left

MEASUREMENT

SPECTROPHOTOMETRIC DETERMINATION OF OXYGEN SATURATION

• Actual percent oxyhemoglobin (O2Hb) can be determined spectrophotometrically using a CO-oximeter

designed to directly measure various Hb species
- Each Hb species has a characteristic absorbable curve
 - Number of Hb species measured will depend on number and specific wavelengths incorporated into
the instrument; ex. a two wavelength system can measure only O2Hb and HHb expressed as a fraction
or percentage of total Hb
- Instruments should have a minimum of four wavelengths; instruments with more than four
wavelengths can recognize dyes, pigments, turbidity, other Hb species and abnormal proteins
(interferences)
• Primary purpose of determining O2Hb is to asses oxygen transport from the lungs
• Measured O2Hb values reflect the patient's true status because calculated SO2 and O2Hb values will differ
in the presence of dyshemoglobins
• Samples are collected under anaerobic conditions and mixed immediately with heparin; samples must be
analyzed immediately to avoid consumption of oxygen by metabolizing cells
• Potential errors:
- Faulty instrument calibration
- Spectral interfering substances: substances that absorb light at the wavelengths used

BLOOD GAS ANALYZERS

• Uses electrodes (macroelectrochemical or microelectrochemical sensors) as sensing devices to measure

pO2, pCO2 and pH
• pO2 measurement is amperometric: meaning that the amount of current flow is an indication of the oxygen
present
• pCO2 and pH are potentiometric: a change in voltage indicates the activity of the analyte
• Calculate the following parameters: bicarbonate, total CO2, base excess, SO2
• Analyzers may include Hb, Hct, electrolytes, metabolites (glucose, lactate, creatinine, BUN) and CO-
oximetry
• Cathode: the negative electrode; a site to which cations tend to travel, site at which reduction occurs
• Reduction: is the gain of electrons by a particle (atom, molecule or ion)
• Anode: is the positive electrode; the site to which anions migrate; site at which oxidation occurs
• Oxidation: is the loss of electrons by a particle
• Electrochemical cell: is formed when two opposite electrodes are immersed in a liquid that conduct the
current

Measurement of pO2
• pO2 electrodes, called Clark electrodes, measure the amount of current flow in a circuit that is related to the
amount of O2 being reduced at the cathode
• A gas permeable membrane covering the tip of the electrode selectively allows O2 to diffuse into an
electrolyte and contact the cathode; electrons are drawn from the anode surface to the cathode surface to
reduce O2; a small constant polarizing potential is applied between the anode and cathode; a
microammeter placed between the anode and cathode measures the movement of electrons (current)
• Four electrons are drawn for every mole of O2 reduced making it possible to determine pO2
• Sources of error:
- Build-up of protein material on the surface of the membrane: retards diffusion and slows electrode
response
- Bacterial contamination within measuring chamber: consume O2
- Incorrect calibration
- Sample collection & handling: contamination of sample with room air, delay in analysis
• Pulse oximeters
 - Uses transcutaneous electrodes placed directly on the skin for continuous measurement of pO2
- Measurement depends on oxygen diffusing from capillary bed through the tissues to the electrodes
- Affected by: skin thickness and tissue perfusion with arterial blood; heated electrodes placed on skin
may enhance diffusion of O2 to electrodes
- pO2 measured by this method only reflects arterial pO2; these two values are not equivalent; arterial
pO2 affected by O2 consumption at electrode site, heating effects of electrodes, possible
hypoperfusion from cardiovascular instability

Measurement of pCO2 and pH

• An increased concentration or activity of the ions leads to an increase in the force exerted by these ions
• Potentiometric measurements measure how much force / energy / potential a given ion possesses
• Requires: reference electrode, an electrode responsive to the ion being measured, voltmeter (measures the
potential difference between the two electrodes)

Optical sensors
• Certain fluorescent dyes will react predictably with specific chemical such as O2, CO2 and H+
• Dye is separated from the sample by a membrane, analyte diffuses into the dye causing either and increase
or a quenching of fluorescence proportional to the amount of dye
• Optical technology is applied in in-dwelling blood gas systems (fiber optic catheters with sensors placed
within patient's arterial system)

Calculated parameters
• Instruments include algorithms to perform calculations of other parameters from pCO2 and pH
1. Bicarbonate (HCO3-): based on Henderson-Hasselbalch equation
2. Carbonic acid concentration: calculated using the solubility coefficient of CO2 in plasma at 37*C;
solubility constant to convert pCO2 to H2CO3 is 0.0307; temperature and an increase in lipids change
the solubility constant
3. Total carbon dioxide content (ctCO2): is bicarbonate plus dCO2 (carbonic acid) plus CO2 associated
with proteins (carbamates)
4. Base excess: used to asses the non-respiratory component; calculated from pH, pCO2 and Hb; a
positive value / base excess indicates an excess of bicarbonate or a relative deficit of noncarbonic acid
suggestive of non-respiratory alkalosis; a negative value / base deficit indicates a deficit of bicarbonate
or relative deficit of noncarbonic acids suggestive of non-respiratory acidosis

Correction for temperature

• Values for pH, pO2, pCO2 are temperature dependent; all measurements done at 37*C
• Most analyzer software perform correction
• Must use reference ranges for the patient's temperature for proper result interpretation; but reference
range is controversial and difficult to find

QUALITY ASSURANCE

• Proper patient identification

• Correct labeling
• Other information needed for interpretation: FiO2, ventilation (on room air or supplemental O2), body
temperature
• Collecting personnel
• Specimen for acid-base balance is arterial blood.
• Choice of site: radial, brachial, femoral or temporal arteries
• Venous blood gas samples: can be used if pulmonary function or O2 transport is not being assessed
• Capillary blood: to assess pH and pCO2; capillary pO2 does not correlate well with arterial pO2
• Central venous (pulmonary artery) blood samples: used to asses O2 consumption (calculated from O2
content of arterial and pulmonary artery blood and cardiac output)
• Collection and handling: collection device, form and concentration of heparin used, speed of syringe filling,
maintenance of anaerobic environment, mixing of sample, transport and storage time before analysis
- 1-3 ml self-filling plastic disposable syringe containing heparin; dry /lyophilized heparin better since
liquid heparin can dilute sample; evacuated collection tubes not appropriate
- Mix thoroughly with anticoagulant; mix immediately before analysis to resuspend cells
- Slow filling caused by mismatch of syringe an needle size; small needles cause bubbles to form
(hemolyze RBCs, affect pO2 and pCO2)
- Air trapped in syringe must be expelled immediately after blood draw
- transport time should be minimal to reduce cell metabolism (O2 and glucose used up, CO2 and lactate
produced)
- Place syringe in an ice water slurry to minimize metabolism; but may increase pO2 due to oxygen from
water entering pores of the plastic syringe
- Low temperatures cause increased oxygen solubility in blood and a shift to the left in the
oxyhemoglobin curve (more oxygen combining with Hb):causes falsely elevated pO2
- Air bubbles in syringe increases pO2 levels and decreases pCO2 causing increase in pH.
- Sample sitting more than 30 minutes and not on ice causes decrease in pO2 and increase in pCO2
resulting to decreased ph.
- Ice can inhibit glycolysis thus slowing the metabolism of cells.