3Acid-Base Biochemistry Definitions What is an acid?What is a base?
4Acid-Base Biochemistry Definitions Definitions of an acidTasteBoyleArrheniusBronsted-LowryLewis
5Acid-Base Biochemistry Definitions TasteAcere – tasting sourLemon juiceVinegarDefinition - Thousands of years old
6Acid-Base Biochemistry Definitions Robert Boyle 17th centuryAcids taste sour, are corrosive to metals, change litmus (a dye extracted from lichens) red, and become less acidic when mixed with bases (Alkali).Bases (Alkali) feel slippery, change litmus blue, and become less basic (alkaline) when mixed with acids.
7Acid-Base Biochemistry Definitions ArrheniusArrhenius suggested that acids are compounds that contain hydrogen and can dissolve in water to release hydrogen ions into solution. For example, hydrochloric acid (HCl) dissolves in water as follows:H2OHCl (g) → H+ (aq) + Cl-(aq)
8Acid-Base Biochemistry Definitions Arrhenius defined bases as substances that dissolve in water to release hydroxide ions (OH-) into solution. For example, a typical base according to the Arrhenius definition is sodium hydroxide (NaOH):H2ONaOH (s) → Na+ (aq) + OH-(aq)
9Acid-Base Biochemistry Definitions The Arrhenius definition of acids and bases explains a number of things. Arrhenius's theory explains why all acids have similar properties to each other (and, conversely, why all bases are similar): because all acids release H+ into solution (and all bases release OH-).
10Acid-Base Biochemistry Definitions The Arrhenius definition also explains Boyle's observation that acids and bases counteract each other. This idea, that a base can make an acid weaker, and vice versa, is called neutralization.
11Acid-Base Biochemistry Definitions Neutralization: As you can see from the equations, acids release H+ into solution and bases release OH-. If we were to mix an acid and base together, the H+ ion would combine with the OH- ion to make the molecule H2O, or plain water:H+ (aq) + OH-(aq) → H2O
12Acid-Base Biochemistry Definitions The neutralization reaction of an acid with a base will always produce water and a salt, as shown below:Acid Base Water SaltHCl + NaOH → H2O + NaClHBr + KOH → H2O + KBr
13Acid-Base Biochemistry Definitions Limitations of ArrheniusThe Arrhenius definition does not explain why some substances, such as common baking soda (NaHCO3), can act like a base even though they do not contain hydroxide ions.
14Acid-Base Biochemistry Definitions Brǿnsted-Lowry 1923An acid is any chemical species that donates a proton to another chemical species (proton donor)A base is any chemical species that accepts a proton from another chemical species (Proton acceptor)
15Acid-Base Biochemistry Definitions The Brønsted-Lowry definition of acids is very similar to the Arrhenius definition, any substance that can donate a hydrogen ion is an acid (under the Brønsted definition, acids are often referred to as proton donors because an H+ ion, hydrogen minus its electron, is simply a proton).
16Acid-Base Biochemistry Definitions The Brønsted definition of bases is, however, quite different from the Arrhenius definition. Arrhenius base releases hydroxyl ions whereas the Brønsted base is defined as any substance that can accept a hydrogen ion.
17Acid-Base Biochemistry Definitions The Brønsted-Lowry definition includes the Arrhenius bases soNaOH and KOH, as we saw above, would still be considered bases because they can accept an H+ from an acid to form water.But it extends the concept of a base and introduces the concept of conjugate acid-base pairs
18Acid-Base Biochemistry Definitions The removal of a proton (hydrogen ion) from an acid produces its conjugate base, which is the acid with a hydrogen ion removed, and the reception of a proton by a base produces its conjugate acid, which is the base with a hydrogen ion added
19Acid-Base Biochemistry Definitions The Brønsted-Lowry definition also explains why substances that do not contain OH- ions can act like bases. Baking soda (NaHCO3), for example, acts like a base by accepting a hydrogen ion from an acid as illustrated below:Acid Base SaltHCl + NaHCO3 → H2CO3 + NaCl
20Acid-Base Biochemistry Definitions Lewis definition 1923A substance that can accept an electron pair from a base; thus, AlCl3, BF3, and SO3 are acids.The Lewis theory defines an acid as a species that can accept an electron pair from another atom, and a base as a species that can donate an electron pair to complete the valence shell of another atom
21Acid-Base Biochemistry Definitions pH Under the Brønsted-Lowry definition, both acids and bases are related to the concentration of hydrogen ions present. Acids increase the concentration of hydrogen ions, while bases decrease the concentration of hydrogen ions (by accepting them). The acidity or basicity of something therefore can be measured by its hydrogen ion concentration.
22Acid-Base Biochemistry Definitions In 1909, the Danish biochemist Sören Sörensen invented the pH scale for measuring acidity. The pH scale is described by the formula:pH = -log [H+]Note: concentration is commonly abbreviated by using square brackets, thus [H+] = hydrogen ion concentration. When measuring pH, [H+] is in units of moles of H+ per litre of solution.
25Acid-Base Biochemistry Methods How the pH Electrode worksAs the pH Glass comes into contact with an aqueous substance to measure, a gel layer forms on the membrane. This also happens on the inside of the glass layer. .
26Acid-Base Biochemistry Methods How the pH Electrode worksThe pH value of the aqueous solution will either force Hydrogen Ions out of the gel layer or into this layer. The Internal buffer in the glass electrode has a constant pH value and this keeps the potential at the inner surface of the membrane constant.
27Acid-Base Biochemistry Methods How the pH Electrode worksThe membrane potential is therefore the difference between the inner and outer charge. If you then factor in the stable potential of reference electrode, you have a voltage proportional to the pH value of the solution being measured, this being approximately 58mV/pH 20ºC
28Acid-Base Biochemistry Methods Other methods you need to know and understandCarbon dioxide electrodeOxygen electrodeLaboratory measurement of bicarbonateIon selective electrodes for K+ Na+ Cl-
29Acid-Base Biochemistry Physiology What is Physiological pH range?
30Acid-Base Biochemistry Physiology Extracellular fluidpH 7.35 – 7.46 (35-45 nmol/L)Does this apply to whole body?any different pH ranges elsewhere
31Acid-Base Biochemistry Physiology More extreme/variable pH rangeDigestive tractGastric JuicePancreatic JuiceIntercellular organellesLysosomal pH 4-5Digestive and lysosomal enzymes function optimally at these pH ranges
32Acid-Base Biochemistry Physiology Traditionally use pH to measure acidityProblem1. direction of pH change isopposite to increase/decrease of Hydrogenion concentration2. Use of log scale ‘masks’ the extent of the change-change of 0.3 in pH represents doubling/halving of hydrogen ion concentration
33Acid-Base Biochemistry Physiology More recently – use Hydrogen ion concentration [H+]Traditionalists and older equipment use pHFor large pH changes may not register change in units eg nmole/L to moles/LMost practical - give both
34Acid-Base Biochemistry Physiology WHAT THE SOURCES OF ACID IN THE BODY?
35Acid-Base Biochemistry Physiology Sources of acidMetabolism of foodMetabolism of drugsInborn errors of metabolism
36Acid-Base Biochemistry Physiology Acid production from metabolism of foodSulphuric acid from metabolism of sulphur-containing amino acids of proteinsLactic acid from sugarsKetoacids from fats
37Acid-Base Biochemistry Physiology Acid production from metabolism of drugsDirect metabolism of drug to more acidic compound eg salicylates urates etcInduction of enzymes which metabolise other compounds (endogenous or exogenous) to acids
38Acid-Base Biochemistry Physiology Inborn errors of metabolismOrganic acid disordersLactic acidosis
40Acid-Base Biochemistry Physiology Hydrogen ion homeostasis1. buffering2. excretion
41Acid-Base Biochemistry Physiology Buffering of hydrogen ionsIn health as hydrogen ions are produced they are buffered – limiting the rise in [H+]
42Acid-Base Biochemistry Physiology Buffer solutions consist of a weak acid and its conjugate baseAs hydrogen ions are added some will combine with the conjugate base and convert it to undissociated acid
43Acid-Base Biochemistry Physiology Bicarbonate – carbonic acid buffer systemH+ + HCO3- <=> H2CO3Addition of H+ drives reaction to the rightConverselyFall in H+ drives reaction to the left as carbonic acid dissociates producing more H+
44Acid-Base Biochemistry Physiology Buffering systems in bloodBicarbonate ions-most importantProteins including intracellular proteinsHaemoglobin
45Acid-Base Biochemistry Physiology Buffer solutions operate most efficiently at [H+] that result in approximately equal concentration of undissociated acid and conjugate baseBut at normal extracellular fluid pH[H2CO3] 1.2 mmolwhereas [HCO3-] is twenty times greater
46Acid-Base Biochemistry Physiology The bicarbonate system is enhanced by the fact that carbonic acid can be formed from CO2 or disposed of by conversion to CO2CO2 + H2O <=> H2CO3
47Acid-Base Biochemistry Physiology For every hydrogen ion buffered by bicarbonate – a bicarbonate ion is consumed.To maintain the capacity of the buffer system, the bicarbonate must be regeneratedHowever, when bicarbonate is formed from carbonic acid (CO2 and H2O) equimolar amounts of [H+] are formed
48Acid-Base Biochemistry Physiology Bicarbonate formation can only continue if these hydrogen ions are removedThis process occurs in the cells of the renal tubules where hydrogen ions are secreted into the urine and where bicarbonate is generated and retained in the body
49Acid-Base Biochemistry Physiology 2 different processesBicarbonate regeneration (incorrectly reabsorption)Hydrogen ion excretion
50Acid-Base Biochemistry Physiology Importance of Renal Bicarbonate RegenerationBicarbonate is freely filtered through the glomerulus so plasma and glomerular filtrate have the same bicarbonate concentrationAt normal GFR approx 4300 mmol of bicarbonate would be filtered in 24 hrWithout re-generation of bicarbonate the buffering capacity of the body would be depleted causing acidotic stateIn health virtually all the filtered bicarbonate is recovered
52Acid-Base Biochemistry Physiology Renal Bicarbonate Regeneration involves the enzyme carbonate dehydratase (carbonic anhydrase)Luminal side of the renal tubular cells impermeable to bicarbonate ionsCarbonate dehydratase catalyses the formation of CO2 and H2O from carbonic acid (H2CO3) in the renal tubular lumenCO2 diffuses across the luminal membrane into the tubular cells
53Acid-Base Biochemistry Physiology within the renal tubular cells carbonate dehydratase catalyses the formation of carbonic acid (H2CO3) from CO2 and H2OCarbonic acid then dissociates into H+ and HCO3-The bicarbonate ions pass into the extracellular fluid and the hydrogen ions are secreted back into the lumen in exchange for sodium ions which pass into the extracellular fluidExchange of sodium and hydrogen ions an active process involving Na+/K+/H+ ATP pumpK+ important in electrolyte disturbances of acid-base
54Acid-Base Biochemistry Physiology Regeneration of bicarbonate does not involve net excretion of hydrogen ionsHydrogen ion excretion requires the same reactions occurring in the renal tubular cells but also requires a suitable buffer in urinePrincipal buffer system in urine is phosphate80% of phosphate in glomerular filtrate is in the form of the divalent anion HPO42-This combines with hydrogen ionsHPO42- + H+ ↔ H2PO4-
56Acid-Base Biochemistry Physiology Hydrogen ion excretion capacityThe minimum urine pH that Can be generated is 4.6 ( 25µmol/L)Normal urine output is 1.5LWithout the phosphate buffer system the free excretion of Hydrogen ions is less than 1/1000 of the acid produced by normal metabolism
57Acid-Base Biochemistry Physiology The phosphate buffer system increases hydrogen ion excretion capacity to mmol/24 hoursIn times of chronic overproduction of acid another urine buffer systemAmmonia
58Acid-Base Biochemistry Physiology Ammonia produced by deamination of glutamine in renal tubular cellsCatalysed by glutaminase which is induced by chronic acidosisAllows increased ammonia production and hence increased hydrogen ion excretion via ammonium ionsNH3 + H+ ↔ NH4+
59Acid-Base Biochemistry Physiology At normal intracellular pH most ammonia is present as ammonium ions which can’t diffuse out of the cellDiffusion of ammonia out of the cell disturbs the equilibrium between ammonia and ammonium ions causing more ammonia to be formedHydrogen ions formed at the same time!These are used up by the deamination of glutamine to glutamate during gluconeogenesis
61Acid-Base Biochemistry Physiology Carbon dioxide transportCarbon dioxide produced by aerobic respiration diffuses out of cells and into the ECFA small amount combines with water to form carbonic acid decreasing the pH of ECFIn red blood cells metabolism is anaerobic and very little CO2 is produced hence it diffuses into red cells down a concentration gradient to form carbonic acid (carbonate dehydratase) buffered by haemoglobin .
62Acid-Base Biochemistry Physiology Haemoglobin has greatest buffering capacity when it is dexoygenated hence the buffering capacity increases as oxygen is lost to the tissuesNet effect is that carbon dioxide is converted to bicarbonate in red cellsBicarbonate diffuses out of red cells down concentration gradient and chloride ions diffuse in to maintain electrochemical neutrality (chloride shift)
64Acid-Base Biochemistry Physiology In the lungs this process is reversedHaemoglobin is oxygenated reducing its buffering capacity and generating hydrogen ionsThese combine with bicarbonate to form CO2 which diffuses into the alveoliBicarbonate diffuses into the cells from the plasma
65Acid-Base Biochemistry Physiology Most of the carbon dioxide in the blood is present as bicarbonateCarbon dioxide, carbonic acid and carbamino compounds less than 1/10 th of the totalBicarbonate /total CO2 used interchangeably though not strictly the sameMost analytical methods actually measure total CO2 as bicarbonate difficult to measure
66Acid-Base Biochemistry Physiology The hydrogen ion concentration of plasma is directly proportional to the PCO2 and inversely proportional to bicarbonate [H+] = k pCO2/[HCO3-] [H+] in nmoles/L, [HCO3-] in mmoles/L pCO2 in kPa k = 180 pCO2 in mm Hg k= 24
67Acid-Base Biochemistry Physiology Derived bicarbonatePossible to use the equation to calculate the bicarbonate concentration from the pCO2 and pH (blood gas analysers)?how valid in non-ideal solutionsAuto analysers – measured bicarbonate
68Acid-Base Biochemistry Physiology The relationship between [H+], pCO2 and bicarbonate fundamental to understanding pathophysiology of hydrogen ion homeostasis
69Acid-Base Biochemistry Pathology 4 Components to acid-base disordersGenerationBufferingCompensationCorrectionOccurring concurrently
70Acid-Base Biochemistry Pathology Classification of acid-base disordersAcidosis[H+] above normal, pH below normalAlkalosis[H+] below normal, pH above normal
71Acid-Base Biochemistry Pathology Further classified asRespiratoryNon-respiratory (metabolic)Mixed – difficult to distinguish between primary mixed condition and compensated disorder
72Acid-Base Biochemistry Pathology Respiratory disorders involve a change in pCO2Metabolic disorders involve change in production or excretion of hydrogen ions or both
73Acid-Base Biochemistry Pathology Non-respiratory acidosisIncreased production/reduced excretion of acid?causes
76Acid-Base Biochemistry Pathology Non-respiratory acidosisLoss of BicarbonateDiarrhoeaPancreatic, intestinal, biliary fistula or drainage
77Acid-Base Biochemistry Pathology Compensation of non-respiratory acidosisExcess hydrogen ions are buffered by bicarbonate forming carbonic acid which dissociates to carbon dioxide to be lost in expired airThe buffering limits the rise in [H+] at the expense of reduction in bicarbonate
78Acid-Base Biochemistry Pathology Compensation of non-respiratory acidosisHyperventilation increases removal of CO2lowering pCO2PCO2 / [HCO3-] ratio falls reducing [H+]Hyperventilation is the direct result of increased [H+] stimulating the respiratory centre (Kussmaul respiration)Respiratory compensation of non-respiratory acidosis
79Acid-Base Biochemistry Pathology Compensation of non-respiratory acidosisLimitationsRespiratory compensation cannot completely normalise the [H+] because the hyperventilation is stimulated by the increase in [H+] and as this falls the drive on the respiratory centre is reducedIncreased work of respiratory muscles during hyperventilation produces CO2 limiting the degree to which PCO2 can be lowered
80Acid-Base Biochemistry Pathology The degree of compensation may be limited further if respiratory function is compromisedIf it is not possible to correct the cause of the acidosis may get a new steady state of chronic acidosis [H+] [HCO3-] and ↓PCO2
81Acid-Base Biochemistry Pathology In the absence of acidosis - hyperventilation would normally generate a respiratory alkalosisCompensatory mechanisms usually involve generation of a second opposing disturbanceIn non-respiratory acidosis the hyperventilation limits the severity of the acidosis but is not great enough to cause alkalosis in the patient
82Acid-Base Biochemistry Pathology Non-respiratory compensation of non-respiratory acidosisIf renal function is normal excess [H+] can be excreted by the kidneysBut renal function is often impaired even if not the primary cause of the acidosis
83Acid-Base Biochemistry Pathology Correction of acidosisComplete correction requires reversal or removal of the underlying causeEthylene glycol poisoning – slow the rate of metabolism with ethanolDiabetes – rehydration and insulin
85Acid-Base Biochemistry Pathology Management of non-respiratory acidosis1. Removal of cause2. Administration of Bicarbonate – only in severe cases pH <7.0 and where 1 is not possibleMust be given in small quantities with constant monitoring of pH
86Acid-Base Biochemistry Pathology Respiratory acidosisPrimarily an increase in PCO2Number of different causes
88Acid-Base Biochemistry Pathology Retention of CO2Production of carbonic acidFor every hydrogen ion produced a bicarbonate ion is generatedMost of the [H+] is buffered by intracellular buffers (haemoglobin)Development of renal compensation if renal function is normal
89Acid-Base Biochemistry Pathology Acute respiratory acidosisFor every KPa increase in PCO2increase in bicarbonate < 1 mmoleIncrease in [H+] 5.5 nmol/LChronicincrease in bicarbonate 2-3 mmoleIncrease in [H+] 2.5 nmol/L
90Acid-Base Biochemistry Pathology Compensation of respiratory acidosisIncreased renal excretion of hydrogen ions
91Acid-Base Biochemistry Pathology Management of respiratory acidosisWith reduced ventilation it is usually the hypoxaemia that is life threatening 4 mins if ventilation ceasesImprove alveolar ventilation bronchodilators and antibioticsArtificial ventilation close monitoring required to avoid over correction esp in chronic acidosis
92Acid-Base Biochemistry Pathology Summary of respiratory acidosisAcuteChronicpHSlight or low normal[H+]Slight or high normalPCO2[HCO3-]Slight
93Acid-Base Biochemistry Pathology Non respiratory alkalosisLoss of un-buffered hydrogen ionsGastrointestinal- vomiting with pyloric stenosis- diarrhoea- nasogastric aspiration
94Acid-Base Biochemistry Pathology Causes of non respiratory alkalosisRenalMineralo-corticoid excessConn’s syndromeCushings syndromeDrugs with mineralocorticoid activityDiuretic therapy (not K+ sparing)
95Acid-Base Biochemistry Pathology Causes of non respiratory alkalosisAdministration of alkaliOver-treatment of acidosisChronic alkali ingestion (antacids)
96Acid-Base Biochemistry Pathology Non respiratory alkalosisCharacterised by primary increase in ECF bicarbonateConsequent reduction in [H+]Normally increase in bicarbonate causes reduction in renal bicarbonate regeneration and increased urinary excretion of bicarbonate
97Acid-Base Biochemistry Pathology non respiratory alkalosisMaintenance requires inappropriate renal bicarbonate reabsorption/regeneration- decrease in ECF volume (hypovolaemia)- mineralocorticoid excess- potassium depletion
98Acid-Base Biochemistry Pathology non respiratory alkalosisHypovolaemiaIncreased stimulus to sodium reabsorptionDependant on adequate anionsIf chloride deficient (GI losses) electrochemical neutrality during Na+ absorption maintained by increased bicarbonate absorption and by H+ and K+ excretion
99Acid-Base Biochemistry Pathology non respiratory alkalosisMineralocorticoid excessAlkalosis perpetuated by increased hydrogen ion excretion secondary to increased sodium reabsorptionPotassium depletionPotassium and hydrogen ion excretion compete for exchange with sodium so depletion of potassium causes increased H+ excretion
100Acid-Base Biochemistry Pathology non respiratory alkalosisCompensationLow H+ inhibits the respiratory centre causing hypoventilation and increase in PCO2Self- limiting as increase in PCO2 increases drive on respiratory centreIn chronic state development of reduced sensitivity to PCO2 – more significant compensation BUTHypoventilation causing hypoxaemia will provide stimulation of RC and prevent further compensation
101Acid-Base Biochemistry Pathology non respiratory alkalosisManagementDependent on severity and cause- severe hypovolaemia /hypochloraemia correct with saline infusion- potassium supplements/removal of diuretics
102Acid-Base Biochemistry Pathology Summary of non respiratory alkalosis[H+] pH PCO2 [HCO3-]
106Acid-Base Biochemistry Pathology Respiratory alkalosisCharacterised by reduction in PCO2Reduces the PCO2/ [HCO3-] ratioFor every KPa decrease in PCO2decrease in [H+] 5.5 nmol/LSmall decrease in bicarbonate
107Acid-Base Biochemistry Pathology Respiratory alkalosisCompensation-reduction in renal hydrogen ion excretionDevelops slowly maximal in hours
108Acid-Base Biochemistry Pathology Respiratory alkalosis managementMainly removal of underlying causeIncreasing inspired PCO2 by rebreathing of expired air for temporary measure- Prolonged – risk of hypoxia
109Acid-Base Biochemistry Pathology Summary of respiratory alkalosisAcute ChronicpH Slight or low normal[H+] Slight or high normalPCO2 [HCO3-] Slight
110Acid-Base Biochemistry Pathology Mixed acid base disordersrespiratory alkalosis with metabolic acidosise.g. salicylate poisoning causes respiratory alkalosis by directly stimulating the hypothalamic respiratory centre causing over-breathing and increased excretion of CO2Salicylate metabolised to acids
115Acid-Base Biochemistry Methods The polarographic (Clark) oxygen electrode measures the oxygen partial pressure in a blood or gas sample. A platinum cathode and a silver/silver chloride anode are placed in a sodium chloride electrolyte solution, and a voltage of 700 mv is applied (Figure 1). The following reactions occur.At the cathode: O2 + 2H2O + 4e– = 4OH–.In the electrolyte: NaCl + OH– = NaOH + Cl–.At the anode: Ag + Cl– = AgCl + e–.Electrons are taken up at the cathode and the current generated is proportional to oxygen tension. A membrane separates the electrode from blood, preventing deposition of protein but allowing the oxygen tension in the blood to equilibrate with the electrolyte solution. The electrode is kept at a constant temperature of 37°C and regular checks of the membrane are required to ensure it is not perforated or coated in proteins. Sampling two gas mixtures of known oxygen tension allows calibration.
116Acid-Base Biochemistry Methods The Severinghaus or carbon dioxide electrode is a modified pH electrode in contact with sodium bicarbonate solution and separated from the blood specimen by a rubber or Teflon semipermeable membrane. Carbon dioxide, but not hydrogen ions, diffuses from the blood sample across the membrane into the sodium bicarbonate solution, producing hydrogen ions and a change in pH.Hydrogen ions are produced in proportion to the pCO2 and are measured by the pH-sensitive glass electrode. As with the pH electrode, the Severinghaus electrode must be maintained at 37°C, be calibrated with gases of known pCO2 and the integrity of the membrane is essential. Because diffusion of the CO2 into the electrolyte solution is required the response time is slow at 2–3 minutes.
117Acid-Base Biochemistry Methods ION SELECTIVE ELECTRODE
118Acid-Base Biochemistry RECOMMENDED READING Analytical/methodsTietz Textbook of Clinical Chemistry by Carl A. Burtis (Author), Edward R. Ashwood (Author)ClinicalClinical Biochemistry by William J. Marshall and Stephen Bangert