Acid-Base Biochemistry Dr. Catherine Street Consultant Clinical Biochemist Colchester Hospital...

Acid-Base Acid-Base BiochemistryBiochemistry

Dr. Catherine StreetDr. Catherine StreetConsultant Clinical BiochemistConsultant Clinical Biochemist

Colchester Hospital university NHS Colchester Hospital university NHS Foundation Trust Foundation Trust

Acid-Base BiochemistryAcid-Base Biochemistry

►DefinitionsDefinitions►MethodsMethods►PhysiologyPhysiology►PathologyPathology

Acid-Base BiochemistryAcid-Base BiochemistryDefinitionsDefinitions

What is an acid?What is an acid?

What is a base?What is a base?

► Definitions of an acidDefinitions of an acid

1.1. TasteTaste

2.2. BoyleBoyle

3.3. ArrheniusArrhenius

4.4. Bronsted-LowryBronsted-Lowry

5.5. LewisLewis

►TasteTaste

Acere – tasting sour Acere – tasting sour

Lemon juice Lemon juice

VinegarVinegar

Definition - Thousands of years oldDefinition - Thousands of years old

►Robert Boyle 17Robert Boyle 17thth century century►AcidsAcids taste sour, are corrosive to taste sour, are corrosive to

metals, change litmus (a dye extracted metals, change litmus (a dye extracted from lichens) red, and become less from lichens) red, and become less acidic when mixed with acidic when mixed with bases (Alkali). (Alkali).

►BasesBases (Alkali) feel slippery, change (Alkali) feel slippery, change litmus blue, and become less basic litmus blue, and become less basic (alkaline) when mixed with (alkaline) when mixed with acids. .

ArrheniusArrhenius►Arrhenius suggested that Arrhenius suggested that acidsacids are are

compounds that contain hydrogen and compounds that contain hydrogen and can dissolve in water to release can dissolve in water to release hydrogen ions into hydrogen ions into solution. For . For example, hydrochloric example, hydrochloric acid (HCl) (HCl) dissolves in water as follows:dissolves in water as follows:

HH22OO

HCl HCl (g)(g) →→ H H+ + ((aqaq)) + Cl + Cl--((aqaq))

►Arrhenius defined Arrhenius defined bases as substances as substances that dissolve in water to release that dissolve in water to release hydroxide hydroxide ions (OH (OH--) into ) into solution. For . For example, a typical base according to example, a typical base according to the Arrhenius definition is sodium the Arrhenius definition is sodium hydroxide (NaOH): hydroxide (NaOH):

HH22OO

NaOH NaOH (s)(s) → → NaNa+ + ((aqaq)) + OH + OH--

((aqaq))

►The Arrhenius definition of The Arrhenius definition of acids and and bases explains a number of things. explains a number of things. Arrhenius's Arrhenius's theory explains why all explains why all acids have similar properties to each acids have similar properties to each other (and, conversely, why all bases other (and, conversely, why all bases are similar): because all acids release are similar): because all acids release HH++ into into solution (and all bases release (and all bases release OHOH--). ).

►The Arrhenius definition also explains The Arrhenius definition also explains Boyle's observation that acids and Boyle's observation that acids and bases counteract each other. This bases counteract each other. This idea, that a base can make an acid idea, that a base can make an acid weaker, and vice versa, is called weaker, and vice versa, is called neutralization..

►NeutralizationNeutralization: As you can see from : As you can see from the equations, the equations, acids release H release H++ into into solution and and bases release OH release OH--. If we . If we were to mix an acid and base were to mix an acid and base together, the Htogether, the H++ ion would combine would combine with the OHwith the OH-- ion to make the ion to make the molecule HH22O, or plain water:O, or plain water:

►HH+ + ((aqaq)) + OH + OH--

((aqaq) ) →→ H H22OO

►The The neutralization reaction of an reaction of an acid with a with a base will always produce water will always produce water and a and a salt, as shown below: , as shown below:

►Acid Base Water SaltAcid Base Water Salt►HCl + NaOH HCl + NaOH →→ H H22O + NaClO + NaCl

►HBr + KOH HBr + KOH →→ H H22O + KBrO + KBr

►Limitations of Arrhenius Limitations of Arrhenius ►The Arrhenius definition does not The Arrhenius definition does not

explain why some substances, such as explain why some substances, such as common baking soda (NaHCOcommon baking soda (NaHCO33), can ), can act like a act like a base even though they do even though they do not contain hydroxide not contain hydroxide ions. .

BrBrǿǿnsted-Lowry 1923nsted-Lowry 1923

An acid is any chemical species An acid is any chemical species that donates a proton to another that donates a proton to another chemical species (proton donor)chemical species (proton donor)

A base is any chemical species that A base is any chemical species that accepts a proton from another accepts a proton from another chemical species (Proton acceptor)chemical species (Proton acceptor)

►The Brønsted-Lowry definition of The Brønsted-Lowry definition of acids is very similar to the Arrhenius is very similar to the Arrhenius definition, any substance that can definition, any substance that can donate a hydrogen donate a hydrogen ion is an acid is an acid (under the Brønsted definition, acids (under the Brønsted definition, acids are often referred to as are often referred to as protonproton donors donors because an Hbecause an H++ ion, hydrogen minus its ion, hydrogen minus its electron, is simply a proton). , is simply a proton).

►The Brønsted definition of The Brønsted definition of basesbases is, is, however, quite different from the however, quite different from the Arrhenius definition. Arrhenius base Arrhenius definition. Arrhenius base releases hydroxyl ions whereas the releases hydroxyl ions whereas the Brønsted Brønsted base is defined as any is defined as any substance that can accept a hydrogen substance that can accept a hydrogen ion. .

► The Brønsted-Lowry definition includes the The Brønsted-Lowry definition includes the Arrhenius bases soArrhenius bases so

►NaOH and KOH, as we saw above, would still NaOH and KOH, as we saw above, would still be considered bases because they can be considered bases because they can accept an Haccept an H++ from an acid to form water. from an acid to form water.

► But it extends the concept of a base and But it extends the concept of a base and introduces the concept of conjugate acid-introduces the concept of conjugate acid-base pairs base pairs

The removal of a proton (hydrogen ion) The removal of a proton (hydrogen ion) from an acid produces its from an acid produces its conjugate conjugate basebase, which is the acid with a , which is the acid with a hydrogen ion removed, and the hydrogen ion removed, and the reception of a proton by a base reception of a proton by a base produces its produces its conjugate acidconjugate acid, which is , which is the base with a hydrogen ion addedthe base with a hydrogen ion added

►The Brønsted-Lowry definition also The Brønsted-Lowry definition also explains why substances that do not explains why substances that do not contain OHcontain OH-- ions can act like bases. ions can act like bases.

►Baking soda (NaHCOBaking soda (NaHCO33), for example, acts ), for example, acts like a base by accepting a hydrogen ion like a base by accepting a hydrogen ion from an acid as illustrated below: from an acid as illustrated below:

►Acid Base SaltAcid Base Salt►HCl + NaHCOHCl + NaHCO33 →→ H H22COCO33 + NaCl + NaCl

► Lewis definition 1923Lewis definition 1923► A substance that can accept an electron pair A substance that can accept an electron pair

from a base; thus, AlCl3, BF3, and SO3 are from a base; thus, AlCl3, BF3, and SO3 are acids. acids.

► The Lewis theory defines an acid as a The Lewis theory defines an acid as a species that can accept an electron pair species that can accept an electron pair from another atom, and a base as a species from another atom, and a base as a species that can donate an electron pair to complete that can donate an electron pair to complete the valence shell of another atom the valence shell of another atom

pHpHUnder the Brønsted-Lowry definition, Under the Brønsted-Lowry definition, both both acids and and bases are related to the are related to the concentration of hydrogen concentration of hydrogen ions present. present. Acids increase the concentration of Acids increase the concentration of hydrogen ions, while bases decrease the hydrogen ions, while bases decrease the concentration of hydrogen ions (by concentration of hydrogen ions (by accepting them). The acidity or basicity accepting them). The acidity or basicity of something therefore can be measured of something therefore can be measured by its hydrogen ion concentration.by its hydrogen ion concentration.

► In 1909, the Danish biochemist Sören In 1909, the Danish biochemist Sören Sörensen invented the Sörensen invented the pH scale for scale for measuring acidity. The pH scale is measuring acidity. The pH scale is described by the formula:described by the formula:

► pH = -log [HpH = -log [H++]]►Note: concentration is commonly Note: concentration is commonly

abbreviated by using square brackets, thus abbreviated by using square brackets, thus [H[H++] = hydrogen ] = hydrogen ion concentration. When concentration. When measuring pH, [H+] is in units of moles of measuring pH, [H+] is in units of moles of H+ per litre of solution.H+ per litre of solution.

Acid-Base BiochemistryAcid-Base BiochemistryMethodsMethods

pH electrodepH electrode

Acid-Base BiochemistryAcid-Base BiochemistryMethods Methods

►pH electrodepH electrode

How the pH Electrode worksHow the pH Electrode works ►As the pH Glass comes into contact As the pH Glass comes into contact

with an aqueous substance to with an aqueous substance to measure, a gel layer forms on the measure, a gel layer forms on the membrane. This also happens on the membrane. This also happens on the inside of the glass layer. inside of the glass layer. ..

How the pH Electrode worksHow the pH Electrode works ►The pH value of the aqueous The pH value of the aqueous

solution will either force Hydrogen solution will either force Hydrogen Ions out of the gel layer or into Ions out of the gel layer or into this layer. The Internal buffer in this layer. The Internal buffer in the glass electrode has a constant the glass electrode has a constant pH value and this keeps the pH value and this keeps the potential at the inner surface of potential at the inner surface of the membrane constant.the membrane constant.

How the pH Electrode worksHow the pH Electrode works ►The membrane potential is therefore The membrane potential is therefore

the difference between the inner the difference between the inner and outer charge. If you then factor and outer charge. If you then factor in the stable potential of reference in the stable potential of reference electrode, you have a voltage electrode, you have a voltage proportional to the pH value of the proportional to the pH value of the solution being measured, this being solution being measured, this being approximately 58mV/pH unit @ 20ºC approximately 58mV/pH unit @ 20ºC

Other methods you need to know and Other methods you need to know and understandunderstand

►Carbon dioxide electrodeCarbon dioxide electrode►Oxygen electrodeOxygen electrode►Laboratory measurement of Laboratory measurement of

bicarbonatebicarbonate► Ion selective electrodes for KIon selective electrodes for K++ Na Na++ Cl Cl--

Acid-Base BiochemistryAcid-Base BiochemistryPhysiologyPhysiology

►What is Physiological pH range?What is Physiological pH range?

►Extracellular fluid Extracellular fluid

pH 7.35 – 7.46 (35-45 nmol/L)pH 7.35 – 7.46 (35-45 nmol/L)

Does this apply to whole body Does this apply to whole body

?any different pH ranges elsewhere?any different pH ranges elsewhere

More extreme/variable pH rangeMore extreme/variable pH rangeDigestive tractDigestive tract

Gastric Juice 1.0-3.0Gastric Juice 1.0-3.0Pancreatic Juice 8.0-8.3Pancreatic Juice 8.0-8.3

Intercellular organellesIntercellular organellesLysosomal pH 4-5Lysosomal pH 4-5

Digestive and lysosomal enzymes Digestive and lysosomal enzymes function optimally at these pH function optimally at these pH

rangesranges

Traditionally use pH to measure acidityTraditionally use pH to measure acidity

Problem Problem

1. direction of pH change is1. direction of pH change is

opposite to increase/decrease of Hydrogenopposite to increase/decrease of Hydrogen

ion concentrationion concentration

2. Use of log scale ‘masks’ the extent of the 2. Use of log scale ‘masks’ the extent of the changechange

-change of 0.3 in pH represents doubling/halving -change of 0.3 in pH represents doubling/halving of hydrogen ion concentrationof hydrogen ion concentration

►More recently – use Hydrogen ion More recently – use Hydrogen ion concentration [Hconcentration [H++]]

►Traditionalists and older equipment Traditionalists and older equipment use pHuse pH

►For large pH changes may not register For large pH changes may not register change in units eg nmole/L to change in units eg nmole/L to moles/Lmoles/L

►Most practical - give bothMost practical - give both

►WHAT THE SOURCES OF ACID IN THE WHAT THE SOURCES OF ACID IN THE BODY?BODY?

►Sources of acidSources of acid Metabolism of foodMetabolism of food Metabolism of drugs Metabolism of drugs Inborn errors of metabolismInborn errors of metabolism

►Acid production from metabolism of Acid production from metabolism of foodfood Sulphuric acid from metabolism of Sulphuric acid from metabolism of

sulphur-containing amino acids of proteinssulphur-containing amino acids of proteins Lactic acid from sugarsLactic acid from sugars Ketoacids from fatsKetoacids from fats

►Acid production from metabolism of Acid production from metabolism of drugsdrugs Direct metabolism of drug to more acidic Direct metabolism of drug to more acidic

compound eg salicylates urates etccompound eg salicylates urates etc Induction of enzymes which metabolise Induction of enzymes which metabolise

other compounds (endogenous or other compounds (endogenous or exogenous) to acids exogenous) to acids

► Inborn errors of metabolism Inborn errors of metabolism Organic acid disordersOrganic acid disorders Lactic acidosisLactic acidosis

Greatest potential source of acid Greatest potential source of acid

Carbon dioxideCarbon dioxide

(1)(1)COCO22 + H + H22O O <=> H<=> H22COCO33

(2) H(2) H22COCO3 3 <=> H<=> H++ + HCO + HCO33--

Potentially 15,000 mmol/24 hoursPotentially 15,000 mmol/24 hours

►Hydrogen ion homeostasisHydrogen ion homeostasis►1. buffering1. buffering►2. excretion2. excretion

Buffering of hydrogen ionsBuffering of hydrogen ions

In health as hydrogen ions are In health as hydrogen ions are produced they are buffered – limiting produced they are buffered – limiting the rise in the rise in [H[H++]]

Buffer solutions consist of a weak acid Buffer solutions consist of a weak acid and its conjugate baseand its conjugate base

As hydrogen ions are added some will As hydrogen ions are added some will combine with the conjugate base and combine with the conjugate base and convert it to undissociated acidconvert it to undissociated acid

Bicarbonate – carbonic acid buffer systemBicarbonate – carbonic acid buffer system

HH++ + HCO + HCO33-- <=><=> H H22COCO33

►Addition of HAddition of H+ + drives reaction to the rightdrives reaction to the right

Conversely Conversely ►Fall in HFall in H+ + drives reaction to the left as drives reaction to the left as

carbonic acid dissociates producing carbonic acid dissociates producing more Hmore H+ +

►Buffering systems in bloodBuffering systems in blood Bicarbonate ions-most importantBicarbonate ions-most important Proteins including intracellular proteinsProteins including intracellular proteins Haemoglobin Haemoglobin

►Buffer solutions operate most Buffer solutions operate most efficiently at efficiently at [H[H++]] that result in that result in approximately equal concentration of approximately equal concentration of undissociated acid and conjugate base undissociated acid and conjugate base

►But at normal extracellular fluid pH But at normal extracellular fluid pH

[[HH22COCO33]] 1.2 mmol 1.2 mmol

whereas whereas [[HCOHCO33--] is twenty times ] is twenty times

greatergreater

Acid-Base BiochemistryAcid-Base Biochemistry Physiology Physiology

► The bicarbonate system is enhanced The bicarbonate system is enhanced by the fact that carbonic acid can be by the fact that carbonic acid can be formed from COformed from CO22 or disposed of by or disposed of by conversion to COconversion to CO22

COCO22 + H + H22O O <=> H<=> H22COCO33

►For every hydrogen ion buffered by For every hydrogen ion buffered by bicarbonate – a bicarbonate ion is bicarbonate – a bicarbonate ion is consumed.consumed.

►To maintain the capacity of the buffer To maintain the capacity of the buffer system, the bicarbonate must be system, the bicarbonate must be regenerated regenerated

►However, when bicarbonate is formed However, when bicarbonate is formed from carbonic acid (COfrom carbonic acid (CO22 and H and H22O) O) equimolar amounts of equimolar amounts of [H[H++]] are formed are formed

►Bicarbonate formation can only Bicarbonate formation can only continue if these hydrogen ions are continue if these hydrogen ions are removedremoved

►This process occurs in the cells of the This process occurs in the cells of the renal tubules where hydrogen ions are renal tubules where hydrogen ions are secreted into the urine and where secreted into the urine and where bicarbonate is generated and retained bicarbonate is generated and retained in the body in the body

►2 different processes2 different processes►Bicarbonate regeneration (incorrectly Bicarbonate regeneration (incorrectly

reabsorption)reabsorption)►Hydrogen ion excretionHydrogen ion excretion

Importance of Renal Bicarbonate Regeneration Importance of Renal Bicarbonate Regeneration ► Bicarbonate is freely filtered through the Bicarbonate is freely filtered through the

glomerulus so plasma and glomerular filtrate glomerulus so plasma and glomerular filtrate have the same bicarbonate concentration have the same bicarbonate concentration

► At normal GFR approx 4300 mmol of bicarbonate At normal GFR approx 4300 mmol of bicarbonate would be filtered in 24 hr would be filtered in 24 hr

► Without re-generation of bicarbonate the Without re-generation of bicarbonate the buffering capacity of the body would be depleted buffering capacity of the body would be depleted causing acidotic statecausing acidotic state

► In health virtually all the filtered bicarbonate is In health virtually all the filtered bicarbonate is recoveredrecovered

► Renal Bicarbonate Regeneration involves the Renal Bicarbonate Regeneration involves the enzyme carbonate dehydratase (carbonic enzyme carbonate dehydratase (carbonic anhydrase) anhydrase)

► Luminal side of the renal tubular cells Luminal side of the renal tubular cells impermeable to bicarbonate ionsimpermeable to bicarbonate ions

► Carbonate dehydratase catalyses the Carbonate dehydratase catalyses the formation of COformation of CO22 and H and H22O from carbonic acid O from carbonic acid (H(H22COCO33) in the renal tubular lumen ) in the renal tubular lumen

► COCO2 2 diffuses across the luminal membrane diffuses across the luminal membrane into the tubular cellsinto the tubular cells

► within the renal tubular cells carbonate dehydratase within the renal tubular cells carbonate dehydratase catalyses the formation of carbonic acid (Hcatalyses the formation of carbonic acid (H22COCO33) ) from COfrom CO22 and H and H22OO

► Carbonic acid then dissociates into HCarbonic acid then dissociates into H++ and HCO3 and HCO3--

► The bicarbonate ions pass into the extracellular fluid The bicarbonate ions pass into the extracellular fluid and the hydrogen ions are secreted back into the and the hydrogen ions are secreted back into the lumen in exchange for sodium ions which pass into lumen in exchange for sodium ions which pass into the extracellular fluidthe extracellular fluid

► Exchange of sodium and hydrogen ions an active Exchange of sodium and hydrogen ions an active process involving Naprocess involving Na++/K/K++/H/H++ ATP pump ATP pump

► KK++ important in electrolyte disturbances of acid-base important in electrolyte disturbances of acid-base

► Regeneration of bicarbonate does not involve net Regeneration of bicarbonate does not involve net excretion of hydrogen ionsexcretion of hydrogen ions

► Hydrogen ion excretion requires the same reactions Hydrogen ion excretion requires the same reactions occurring in the renal tubular cells but also requires occurring in the renal tubular cells but also requires a suitable buffer in urinea suitable buffer in urine

► Principal buffer system in urine is phosphatePrincipal buffer system in urine is phosphate► 80% of phosphate in glomerular filtrate is in the 80% of phosphate in glomerular filtrate is in the

form of the divalent anion HPOform of the divalent anion HPO442-2-

► This combines with hydrogen ionsThis combines with hydrogen ions► HPOHPO44

2- 2- + H+ H++ ↔ H ↔ H22POPO44--

►Hydrogen ion excretion capacityHydrogen ion excretion capacity►The minimum urine pH that Can be The minimum urine pH that Can be

generated is 4.6 ( 25µmol/L)generated is 4.6 ( 25µmol/L)►Normal urine output is 1.5LNormal urine output is 1.5L►Without the phosphate buffer system Without the phosphate buffer system

the free excretion of Hydrogen ions is the free excretion of Hydrogen ions is less than 1/1000 of the acid produced less than 1/1000 of the acid produced by normal metabolismby normal metabolism

►The phosphate buffer system The phosphate buffer system increases hydrogen ion excretion increases hydrogen ion excretion capacity to 30-40 mmol/24 hourscapacity to 30-40 mmol/24 hours

► In times of chronic overproduction of In times of chronic overproduction of acid another urine buffer systemacid another urine buffer system

►Ammonia Ammonia

►Ammonia produced by deamination of Ammonia produced by deamination of glutamine in renal tubular cells glutamine in renal tubular cells

►Catalysed by glutaminase which is Catalysed by glutaminase which is induced by chronic acidosisinduced by chronic acidosis

►Allows increased ammonia production Allows increased ammonia production and hence increased hydrogen ion and hence increased hydrogen ion excretion via ammonium ionsexcretion via ammonium ions

►NHNH33 + H + H++ ↔ NH ↔ NH44++

► At normal intracellular pH most ammonia is present At normal intracellular pH most ammonia is present as ammonium ions which can’t diffuse out of the as ammonium ions which can’t diffuse out of the cellcell

► Diffusion of ammonia out of the cell disturbs the Diffusion of ammonia out of the cell disturbs the equilibrium between ammonia and ammonium ions equilibrium between ammonia and ammonium ions causing more ammonia to be formedcausing more ammonia to be formed

► Hydrogen ions formed at the same time!Hydrogen ions formed at the same time!► These are used up by the deamination of glutamine These are used up by the deamination of glutamine

to glutamate during gluconeogenesisto glutamate during gluconeogenesis

► Carbon dioxide transportCarbon dioxide transport► Carbon dioxide produced by aerobic respiration Carbon dioxide produced by aerobic respiration

diffuses out of cells and into the ECFdiffuses out of cells and into the ECF► A small amount combines with water to form A small amount combines with water to form

carbonic acid decreasing the pH of ECFcarbonic acid decreasing the pH of ECF► In red blood cells metabolism is anaerobic and In red blood cells metabolism is anaerobic and

very little COvery little CO22 is produced hence it diffuses into is produced hence it diffuses into red cells down a concentration gradient to form red cells down a concentration gradient to form carbonic acid (carbonate dehydratase) buffered carbonic acid (carbonate dehydratase) buffered by haemoglobin . by haemoglobin .

►Haemoglobin has greatest buffering Haemoglobin has greatest buffering capacity when it is dexoygenated hence the capacity when it is dexoygenated hence the buffering capacity increases as oxygen is buffering capacity increases as oxygen is lost to the tissues lost to the tissues

►Net effect is that carbon dioxide is Net effect is that carbon dioxide is converted to bicarbonate in red cellsconverted to bicarbonate in red cells

► Bicarbonate diffuses out of red cells down Bicarbonate diffuses out of red cells down concentration gradient and chloride ions concentration gradient and chloride ions diffuse in to maintain electrochemical diffuse in to maintain electrochemical neutrality (chloride shift)neutrality (chloride shift)

•Acid-Base BiochemistryAcid-Base BiochemistryPhysiologyPhysiology

► In the lungs this process is reversedIn the lungs this process is reversed►Haemoglobin is oxygenated reducing its Haemoglobin is oxygenated reducing its

buffering capacity and generating buffering capacity and generating hydrogen ionshydrogen ions

►These combine with bicarbonate to form These combine with bicarbonate to form CO2 which diffuses into the alveoliCO2 which diffuses into the alveoli

►Bicarbonate diffuses into the cells from Bicarbonate diffuses into the cells from the plasma the plasma

►Most of the carbon dioxide in the blood is Most of the carbon dioxide in the blood is present as bicarbonate present as bicarbonate

► Carbon dioxide, carbonic acid and Carbon dioxide, carbonic acid and carbamino compounds less than 1/10 th of carbamino compounds less than 1/10 th of the totalthe total

► Bicarbonate /total COBicarbonate /total CO22 used interchangeably used interchangeably though not strictly the same though not strictly the same

►Most analytical methods actually measure Most analytical methods actually measure total COtotal CO2 2 as bicarbonate difficult to measureas bicarbonate difficult to measure

The hydrogen ion concentration of plasma The hydrogen ion concentration of plasma is directly proportional to the PCOis directly proportional to the PCO22 and and inversely proportional to bicarbonateinversely proportional to bicarbonate

[H[H++] = k pCO] = k pCO22/[HCO/[HCO33--]]

[H[H++] in nmoles/L, [HCO] in nmoles/L, [HCO33--] in mmoles/L] in mmoles/L

pCOpCO2 2 in kPa k = 180in kPa k = 180

pCOpCO2 2 in mm Hg k= 24in mm Hg k= 24

►Derived bicarbonateDerived bicarbonate►Possible to use the equation to Possible to use the equation to

calculate the bicarbonate calculate the bicarbonate concentration from the pCOconcentration from the pCO2 2 and pH and pH (blood gas analysers)(blood gas analysers)

►?how valid in non-ideal solutions?how valid in non-ideal solutions►Auto analysers – measured Auto analysers – measured

bicarbonatebicarbonate

►The relationship between [HThe relationship between [H++], pCO], pCO22 and bicarbonate fundamental to and bicarbonate fundamental to understanding pathophysiology of understanding pathophysiology of hydrogen ion homeostasishydrogen ion homeostasis

Acid-Base BiochemistryAcid-Base BiochemistryPathologyPathology

►4 Components to acid-base disorders4 Components to acid-base disorders GenerationGeneration BufferingBuffering CompensationCompensation CorrectionCorrection

Occurring concurrentlyOccurring concurrently

►Classification of acid-base disordersClassification of acid-base disorders►Acidosis Acidosis ►[H+] above normal, pH below normal[H+] above normal, pH below normal►AlkalosisAlkalosis►[H+] below normal, pH above normal [H+] below normal, pH above normal

►Further classified asFurther classified as RespiratoryRespiratory Non-respiratory (metabolic)Non-respiratory (metabolic) Mixed – difficult to distinguish between Mixed – difficult to distinguish between

primary mixed condition and primary mixed condition and compensated disordercompensated disorder

►Respiratory disorders involve a change Respiratory disorders involve a change in pCOin pCO22

►Metabolic disorders involve change in Metabolic disorders involve change in production or excretion of hydrogen production or excretion of hydrogen ions or bothions or both

►Non-respiratory acidosisNon-respiratory acidosis► Increased production/reduced Increased production/reduced

excretion of acidexcretion of acid►?causes?causes

►Non-respiratory acidosisNon-respiratory acidosis►Overproduction of acidOverproduction of acid

Keto acidosis (diabetes, starvation, alcohol)Keto acidosis (diabetes, starvation, alcohol) Lactic acidosis (inherited metabolic defect Lactic acidosis (inherited metabolic defect

or drugs)or drugs) Inherited organic acidosesInherited organic acidoses Poisoning (salicylate, ethylene glycol, Poisoning (salicylate, ethylene glycol,

alcohol)alcohol) Excessive parenteral amino acids Excessive parenteral amino acids

Acid-Base BiochemistryAcid-Base Biochemistry Pathology Pathology

►Non-respiratory acidosisNon-respiratory acidosis►Reduced excretion of acidReduced excretion of acid

Generalised renal failure Generalised renal failure Renal tubular acidosesRenal tubular acidoses Carbonate dehydratase inhibitorsCarbonate dehydratase inhibitors

►Non-respiratory acidosisNon-respiratory acidosis►Loss of BicarbonateLoss of Bicarbonate

DiarrhoeaDiarrhoea Pancreatic, intestinal, biliary fistula or Pancreatic, intestinal, biliary fistula or

drainagedrainage

►Compensation of non-respiratory Compensation of non-respiratory acidosisacidosis

Excess hydrogen ions are buffered by Excess hydrogen ions are buffered by bicarbonate forming carbonic acid which bicarbonate forming carbonic acid which dissociates to carbon dioxide to be lost dissociates to carbon dioxide to be lost in expired airin expired air

The buffering limits the rise in [H+] at the The buffering limits the rise in [H+] at the expense of reduction in bicarbonate expense of reduction in bicarbonate

► Compensation of non-respiratory acidosisCompensation of non-respiratory acidosis►Hyperventilation increases removal of COHyperventilation increases removal of CO22

lowering pCOlowering pCO22

► PCOPCO22 / [HCO / [HCO33--] ratio falls reducing [H] ratio falls reducing [H++]]

►Hyperventilation is the direct result of Hyperventilation is the direct result of increased [Hincreased [H++] stimulating the respiratory ] stimulating the respiratory centre (Kussmaul respiration)centre (Kussmaul respiration)

Respiratory compensation of non-respiratory Respiratory compensation of non-respiratory acidosisacidosis

► Compensation of non-respiratory acidosisCompensation of non-respiratory acidosis► LimitationsLimitations► Respiratory compensation cannot Respiratory compensation cannot

completely normalise the [Hcompletely normalise the [H++] because the ] because the hyperventilation is stimulated by the hyperventilation is stimulated by the increase in [Hincrease in [H++] and as this falls the drive on ] and as this falls the drive on the respiratory centre is reducedthe respiratory centre is reduced

► Increased work of respiratory muscles Increased work of respiratory muscles during hyperventilation produces COduring hyperventilation produces CO22 limiting the degree to which PCOlimiting the degree to which PCO22 can be can be loweredlowered

►The degree of compensation may be The degree of compensation may be limited further if respiratory function is limited further if respiratory function is compromisedcompromised

► If it is not possible to correct the cause If it is not possible to correct the cause of the acidosis may get a new steady of the acidosis may get a new steady state of chronic acidosisstate of chronic acidosis

[H[H++] ] [HCO[HCO33--] and ↓PCO2] and ↓PCO2

► In the absence of acidosis - hyperventilation In the absence of acidosis - hyperventilation would normally generate a respiratory would normally generate a respiratory alkalosis alkalosis

► Compensatory mechanisms usually involve Compensatory mechanisms usually involve generation of a second opposing generation of a second opposing disturbance disturbance

► In non-respiratory acidosis the In non-respiratory acidosis the hyperventilation limits the severity of the hyperventilation limits the severity of the acidosis but is not great enough to cause acidosis but is not great enough to cause alkalosis in the patient alkalosis in the patient

►Non-respiratory compensation of non-Non-respiratory compensation of non-respiratory acidosisrespiratory acidosis

► If renal function is normal excess [HIf renal function is normal excess [H++] ] can be excreted by the kidneyscan be excreted by the kidneys

►But renal function is often impaired But renal function is often impaired even if not the primary cause of the even if not the primary cause of the acidosisacidosis

►Correction of acidosis Correction of acidosis ►Complete correction requires reversal Complete correction requires reversal

or removal of the underlying causeor removal of the underlying cause►Ethylene glycol poisoning – slow the Ethylene glycol poisoning – slow the

rate of metabolism with ethanol rate of metabolism with ethanol ►Diabetes – rehydration and insulinDiabetes – rehydration and insulin

►Summary of non-respiratory acidosisSummary of non-respiratory acidosis►pH pH ►[H[H++] ] ►PCOPCO22 ► [HCO[HCO33

►Management of non-respiratory Management of non-respiratory acidosisacidosis

►1. Removal of cause1. Removal of cause►2. Administration of Bicarbonate – only 2. Administration of Bicarbonate – only

in severe cases pH <7.0 and where 1 in severe cases pH <7.0 and where 1 is not possibleis not possible

►Must be given in small quantities with Must be given in small quantities with constant monitoring of pHconstant monitoring of pH

►Respiratory acidosisRespiratory acidosis►Primarily an increase in PCOPrimarily an increase in PCO22

►Number of different causesNumber of different causes

►Retention of CORetention of CO22

►Production of carbonic acidProduction of carbonic acid►For every hydrogen ion produced a For every hydrogen ion produced a

bicarbonate ion is generatedbicarbonate ion is generated►Most of the [HMost of the [H++] is buffered by ] is buffered by

intracellular buffers (haemoglobin)intracellular buffers (haemoglobin)►Development of renal compensation if Development of renal compensation if

renal function is normalrenal function is normal

►Acute respiratory acidosis Acute respiratory acidosis

For every KPa increase in PCOFor every KPa increase in PCO22

increase in bicarbonate < 1 mmoleincrease in bicarbonate < 1 mmole Increase in [H+] 5.5 nmol/LIncrease in [H+] 5.5 nmol/L

►ChronicChronic

For every KPa increase in PCOFor every KPa increase in PCO22

increase in bicarbonate 2-3 mmoleincrease in bicarbonate 2-3 mmole Increase in [H+] 2.5 nmol/LIncrease in [H+] 2.5 nmol/L

►Compensation of respiratory acidosisCompensation of respiratory acidosis► Increased renal excretion of hydrogen Increased renal excretion of hydrogen

ionsions

►Management of respiratory acidosisManagement of respiratory acidosis►With reduced ventilation it is usually With reduced ventilation it is usually

the hypoxaemia that is life threatening the hypoxaemia that is life threatening 4 mins if ventilation ceases 4 mins if ventilation ceases

► Improve alveolar ventilation Improve alveolar ventilation bronchodilators and antibioticsbronchodilators and antibiotics

►Artificial ventilation close monitoring Artificial ventilation close monitoring required to avoid over correction esp required to avoid over correction esp in chronic acidosisin chronic acidosis

Summary of respiratory acidosisSummary of respiratory acidosis

AcuteAcute ChronicChronic

pHpH Slight Slight or low or low normalnormal

[H[H++]] Slight Slight or or high normalhigh normal

PCOPCO22

[HCO[HCO33--]] Slight Slight

►Non respiratory alkalosisNon respiratory alkalosis►Loss of un-buffered hydrogen ionsLoss of un-buffered hydrogen ions

GastrointestinalGastrointestinal

- vomiting with pyloric stenosis- vomiting with pyloric stenosis

- diarrhoeadiarrhoea

- nasogastric aspiration- nasogastric aspiration

Causes of non respiratory alkalosisCauses of non respiratory alkalosisRenal Renal Mineralo-corticoid excessMineralo-corticoid excess

Conn’s syndromeConn’s syndromeCushings syndromeCushings syndrome

Drugs with mineralocorticoid activityDrugs with mineralocorticoid activityDiuretic therapy (not KDiuretic therapy (not K++ sparing) sparing)

Causes of non respiratory alkalosisCauses of non respiratory alkalosis

Administration of alkaliAdministration of alkali Over-treatment of acidosisOver-treatment of acidosis

Chronic alkali ingestion (antacids)Chronic alkali ingestion (antacids)

►Non respiratory alkalosisNon respiratory alkalosis►Characterised by primary increase in Characterised by primary increase in

ECF bicarbonateECF bicarbonate►Consequent reduction in [HConsequent reduction in [H++]]►Normally increase in bicarbonate Normally increase in bicarbonate

causes reduction in renal bicarbonate causes reduction in renal bicarbonate regeneration and increased urinary regeneration and increased urinary excretion of bicarbonateexcretion of bicarbonate

►non respiratory alkalosisnon respiratory alkalosis►Maintenance requires inappropriate Maintenance requires inappropriate

renal bicarbonate renal bicarbonate reabsorption/regenerationreabsorption/regeneration

- decrease in ECF volume - decrease in ECF volume (hypovolaemia)(hypovolaemia)

- mineralocorticoid excessmineralocorticoid excess

- potassium depletionpotassium depletion

►non respiratory alkalosisnon respiratory alkalosis►Hypovolaemia Hypovolaemia

Increased stimulus to sodium reabsorption Increased stimulus to sodium reabsorption Dependant on adequate anionsDependant on adequate anions If chloride deficient (GI losses) If chloride deficient (GI losses)

electrochemical neutrality during Naelectrochemical neutrality during Na++ absorption maintained by increased absorption maintained by increased bicarbonate absorption and by Hbicarbonate absorption and by H++ and K and K++ excretionexcretion

►non respiratory alkalosisnon respiratory alkalosis►Mineralocorticoid excessMineralocorticoid excess

Alkalosis perpetuated by increased Alkalosis perpetuated by increased hydrogen ion excretion secondary to hydrogen ion excretion secondary to increased sodium reabsorptionincreased sodium reabsorption

Potassium depletionPotassium depletion

Potassium and hydrogen ion excretion Potassium and hydrogen ion excretion compete for exchange with sodium so compete for exchange with sodium so depletion of potassium causes increased Hdepletion of potassium causes increased H++ excretionexcretion

► non respiratory alkalosisnon respiratory alkalosis► CompensationCompensation► Low HLow H++ inhibits the respiratory centre causing inhibits the respiratory centre causing

hypoventilation and increase in PCOhypoventilation and increase in PCO22

► Self- limiting as increase in PCOSelf- limiting as increase in PCO2 2 increases drive on increases drive on respiratory centrerespiratory centre

► In chronic state development of reduced sensitivity In chronic state development of reduced sensitivity to PCOto PCO22 – more significant compensation BUT – more significant compensation BUT

► Hypoventilation causing hypoxaemia will provide Hypoventilation causing hypoxaemia will provide stimulation of RC and prevent further compensationstimulation of RC and prevent further compensation

►non respiratory alkalosisnon respiratory alkalosis►ManagementManagement►Dependent on severity and causeDependent on severity and cause►- severe hypovolaemia - severe hypovolaemia

/hypochloraemia correct with saline /hypochloraemia correct with saline infusioninfusion

►- potassium supplements/removal of - potassium supplements/removal of diureticsdiuretics

►Summary of non respiratory alkalosisSummary of non respiratory alkalosis►[H[H++] ] ►pHpH ►PCOPCO22

►[HCO[HCO33--] ]

►Respiratory alkalosisRespiratory alkalosis►Causes Causes ►HypoxiaHypoxia

High altitudeHigh altitude Severe anaemiaSevere anaemia Pulmonary diseasePulmonary disease

►Respiratory alkalosisRespiratory alkalosis►CausesCauses► Increased respiratory driveIncreased respiratory drive

Stimulants eg salicylatesStimulants eg salicylates Cerebral – trauma, infection, tumoursCerebral – trauma, infection, tumours Hepatic failureHepatic failure

►Respiratory alkalosis Respiratory alkalosis ►CausesCauses

Pulmonary diseasePulmonary disease

- Pulmonary oedema- Pulmonary oedema

- Pulmonary embolism- Pulmonary embolism

Mechanical over-ventilationMechanical over-ventilation

►Respiratory alkalosisRespiratory alkalosis►Characterised by reduction in PCOCharacterised by reduction in PCO22

►Reduces the PCOReduces the PCO22/ [HCO/ [HCO33--] ratio] ratio

For every KPa decrease in PCOFor every KPa decrease in PCO22

decrease in [H+] 5.5 nmol/Ldecrease in [H+] 5.5 nmol/L Small decrease in bicarbonateSmall decrease in bicarbonate

►Respiratory alkalosisRespiratory alkalosis►CompensationCompensation

-reduction in renal hydrogen ion -reduction in renal hydrogen ion excretion excretion

Develops slowly maximal in 36-72 hoursDevelops slowly maximal in 36-72 hours

►Respiratory alkalosis managementRespiratory alkalosis management►Mainly removal of underlying causeMainly removal of underlying cause► Increasing inspired PCOIncreasing inspired PCO2 2 by by

rebreathing of expired air for rebreathing of expired air for temporary measure temporary measure

- Prolonged – risk of hypoxia- Prolonged – risk of hypoxia

►Summary of respiratory alkalosisSummary of respiratory alkalosis

► AcuteAcute ChronicChronic►pHpH Slight Slight or low or low

normalnormal►[H[H++]] Slight Slight or high normal or high normal►PCOPCO22 ► [HCO[HCO33

--]] Slight Slight

►Mixed acid base disordersMixed acid base disorders

respiratory alkalosis with metabolic acidosisrespiratory alkalosis with metabolic acidosis

e.g. salicylate poisoning causes respiratory e.g. salicylate poisoning causes respiratory alkalosis by directly stimulating the alkalosis by directly stimulating the hypothalamic respiratory centre causing hypothalamic respiratory centre causing over-breathing and increased excretion of over-breathing and increased excretion of COCO22

Salicylate metabolised to acidsSalicylate metabolised to acids

Interpretation of resultsInterpretation of results

►Reference rangesReference ranges►pH 7.35 – 7.46pH 7.35 – 7.46►[H[H++] 35-45 nmol/L] 35-45 nmol/L►pCO2 4.5-6.0 kPa (35-46 mm Hg)pCO2 4.5-6.0 kPa (35-46 mm Hg)►pO2 11-15 kPa (85-105 mm Hg) pO2 11-15 kPa (85-105 mm Hg) ►Total Bicarbonate (COTotal Bicarbonate (CO22) 22-30 mmol/L) 22-30 mmol/L

Further informationFurther information

A further algorithm for interpretation A further algorithm for interpretation of acid-base data and a number of of acid-base data and a number of clinical cases were provided as hard-clinical cases were provided as hard-copy. copy.

These can be found in Marshall (see These can be found in Marshall (see recommended reading) recommended reading)

► The polarographic (Clark) oxygen electrode The polarographic (Clark) oxygen electrode measures the measures the oxygen partial pressure in a blood or gas sample. A platinum oxygen partial pressure in a blood or gas sample. A platinum cathode and a silver/silver chloride anode are placed in a cathode and a silver/silver chloride anode are placed in a sodium chloride electrolyte solution, and a voltage of 700 mv is sodium chloride electrolyte solution, and a voltage of 700 mv is applied (Figure 1). The following reactions occur.applied (Figure 1). The following reactions occur.

► At the cathode: OAt the cathode: O22 + 2H + 2H22O + 4eO + 4e–– = 4OH = 4OH––. . ► In the electrolyte: NaCl + OH– = NaOH + ClIn the electrolyte: NaCl + OH– = NaOH + Cl––. . ► At the anode: Ag + ClAt the anode: Ag + Cl–– = AgCl + e = AgCl + e––..► Electrons are taken up at the cathode and the current Electrons are taken up at the cathode and the current

generated is proportional to oxygen tension. A membrane generated is proportional to oxygen tension. A membrane separates the electrode from blood, preventing deposition of separates the electrode from blood, preventing deposition of protein but allowing the oxygen tension in the blood to protein but allowing the oxygen tension in the blood to equilibrate with the electrolyte solution. The electrode is kept at equilibrate with the electrolyte solution. The electrode is kept at a constant temperature of 37°C and regular checks of the a constant temperature of 37°C and regular checks of the membrane are required to ensure it is not perforated or coated membrane are required to ensure it is not perforated or coated in proteins. Sampling two gas mixtures of known oxygen tension in proteins. Sampling two gas mixtures of known oxygen tension allows calibration.allows calibration.

► The Severinghaus or carbon dioxide electrode The Severinghaus or carbon dioxide electrode is a is a modified pH electrode in contact with sodium bicarbonate modified pH electrode in contact with sodium bicarbonate solution and separated from the blood specimen by a rubber solution and separated from the blood specimen by a rubber or Teflon semipermeable membrane. Carbon dioxide, but not or Teflon semipermeable membrane. Carbon dioxide, but not hydrogen ions, diffuses from the blood sample across the hydrogen ions, diffuses from the blood sample across the membrane into the sodium bicarbonate solution, producing membrane into the sodium bicarbonate solution, producing hydrogen ions and a change in pH.hydrogen ions and a change in pH.

Hydrogen ions are produced in proportion to the pCO2 and Hydrogen ions are produced in proportion to the pCO2 and are measured by the pH-sensitive glass electrode. As with the are measured by the pH-sensitive glass electrode. As with the pH electrode, the Severinghaus electrode must be maintained pH electrode, the Severinghaus electrode must be maintained at 37°C, be calibrated with gases of known pCO2 and the at 37°C, be calibrated with gases of known pCO2 and the integrity of the membrane is essential. Because diffusion of integrity of the membrane is essential. Because diffusion of the CO2 into the electrolyte solution is required the response the CO2 into the electrolyte solution is required the response time is slow at 2–3 minutes.time is slow at 2–3 minutes.

ION SELECTIVE ELECTRODEION SELECTIVE ELECTRODE

Acid-Base BiochemistryAcid-Base BiochemistryRECOMMENDED READINGRECOMMENDED READING

►Analytical/methods Analytical/methods Tietz Textbook of Clinical Chemistry Tietz Textbook of Clinical Chemistry

by Carl A. Burtis (Author), Edward R. by Carl A. Burtis (Author), Edward R. Ashwood (Author) Ashwood (Author)

►ClinicalClinical Clinical Biochemistry by William J. Clinical Biochemistry by William J.

Marshall and Stephen BangertMarshall and Stephen Bangert

Acid-Base Biochemistry Dr. Catherine Street Consultant Clinical Biochemist Colchester Hospital...

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