<!-- class: birds-title --> <!-- <div id="vantajs"></div> --> <!-- <div id="birds-writing"> --> <!-- <h1>Acid-base physiology</h1> --> <!-- <h3>UCL Physiology Course 2023</h3> --> <!-- <h3>Robert W Hunter</h3> --> <!-- </div> --> <!-- <script> --> <!-- VANTA.WAVES({ --> <!-- el: "#vantajs", --> <!-- mouseControls: true, --> <!-- touchControls: true, --> <!-- gyroControls: false, --> <!-- minHeight: 200.00, --> <!-- minWidth: 200.00, --> <!-- scale: 1.00, --> <!-- scaleMobile: 1.00, --> <!-- color: 0x252b2d --> <!-- }); --> <!-- </script> --> <!-- --- -->  # .black[Acid-base physiology] ### UCL Physiology Course 2023 | Robert W Hunter --- # Learning objectives - GCSE chemistry revision - physiological approach - sources of acids and alkalis - carbonic acid buffer system - respiratory control of acid-base balance - renal tubular control of acid-base balance - slides at: https://www.kidneyfish.net/talks/ .RWH_footer_pearl[clinical pearls] --- # What is an acid?  .RWH_footnote_right[.RWH_footer_style[Eknoyan (NDT 2022)]] ??? In antiquity, acidity [defined crudely](http://pubsapp.acs.org/subscribe/archive/tcaw/12/i03/pdf/303chronicles.pdf?) according to sour taste / ability to corrode metals. Acids were sour; alkali from water extract of wood ashes. C17th notion (as in wood etching) that acids caused diseases and alkalis cured them. --- # GCSE Chemistry `$$\tag{Sorensen, 1909} pH = -log_{10}[H^+]$$` `$$\color{#549ed6}{pH_i = 7.2 \equiv 80 \space nM \space [H^+]}$$` `$$\color{#549ed6}{pH_o = 7.4 \equiv 40 \space nM \space [H^+]}$$` -- <br> `$$\underset{\color{#549ed6}{acid}}{HA} \rightleftharpoons H^+ + \underset{\color{#549ed6}{base}}{A^-} \tag{Bronsted | Lowry, 1923}$$` -- <br> `$$\tag{Henderson, 1908} Ka = \frac{[H^+][A^-]}{[HA]}$$` `$$\tag{Hasselbalch, 1917} pH = pKa+log \frac{[A^-]}{[HA]}$$` ??? Sorensen proposed pH scale as a way of converting wide range of [H⁺] into convenient numerical scale. On this scale, pH_o = 7.4 (c.f. neutral pH 6.8 at 37deg). Dissociation constant and pK. # Notes [H⁺] in nM is ~1 million times less concentrated than other salts (Na, K, Cl, HCO3 etc. - all measured in mM). Probably so tightly controlled because protein structure (and therefore function) highly pH-dependent - particularly histidine residues. Bronsted and Lowry developed theory independently. An acid donates a proton to a conjugate base. HA is the acid, A^- is the conjugate base. Confusingly, sometimes written as `$$HB \rightleftharpoons H^+ + B^-$$`. The dissociation constant, Ka = measure of how strong an acid is. A high K (i.e. low pK) = strong acid = full dissociation; a low K (i.e. high pK) = weak acid = not fully dissociated. When pH = pH, 50% dissociated. Good buffering at pH = pK +/- 1 unit. When pH above pKa, the acid is mainly dissociated (e.g. carbonic acid, ketones in plasma). When pH is below pKa, acid is mainly undissociated (e.g. NH4⁺ in urine). Titration of an acid is the process of determining its concentration by neutralising it with a base of known concentration. An acid salt is the product of a reaction between a strong acid and a weak base (e.g. NH4Cl). --- # Physiological approach **Core tenets:** - HCO₃^- and PaCO₂ determine pH - loss of HCO₃^- from ECF = gain of H⁺ - HCO₃ reabsorption in the renal tubule = H⁺ secretion - kidneys determine H⁺ / HCO₃^- balance - don't forget chloride! -- <br> **Alternatives:** i) base excess (Siggard-Andersen) ii) physiochemical (Stewart) ??? Main reason for preferring physiological approach is that it fits well with our model of how the renal tubular controls balance of HCO₃^- and H⁺. In physiochemical approach, HCO₃^- is a dependent variable. The independent variables are: PaCO₂, A_tot (total weak acid concentration) and SID. --- class: center, middle, inverse # .white[Sources of acid and alkali] ---  ??? Complete metabolism of CHO and fat -> H₂O & CO₂. This produces 20 moles (i.e. 20,000 mmoles) per day! ---  ??? Incomplete metabolism of CHO and fats (i.e. in hypoxia or insulin deficiency) -> pyruvic acid, lactic acid, keto-acids. Protein metabolism -> HCl, H₂SO₄. (Sulphuric acid from sulphur in methionine and cysteine.) In rarer circumstances, some other endogenous acids (e.g. pyroglutamic acid). These non-volatile acids = 70 mmol per day (i.e. 1 mmol per kg) on an omniverous Western diet. In ketosis, up to 1000 mmol per day. Kidney has to excrete these acids but cannot do so simply as unbuffered H⁺ (as for 70 mmol would require 2000L urine at pH 4.0). ## Notes ### Lactic and keto-acidosis Understanding the physiology of lactic and ketoacidosis is tricky. Two key concepts are important. #### Lactate and ketone bodies do not cause acidosis Convincing arguments that for both [lactic acidosis](https://pubmed.ncbi.nlm.nih.gov/15308499/) and [ketoacidosis](https://pubmed.ncbi.nlm.nih.gov/30744927/), the lactate or ketones are *associated* with acidosis but are NOT the cause of the acidosis. Rather, lactate / acetoacetate / `\(\beta\)`-hydroxybutyrate are produced as anions (i.e. conjugate bases) and would tend to *correct* rather than cause any acidosis. The excess protons in lactic acidosis arise when ATP hydrolysis (which releases protons) is not balanced by mitochrondrial oxidative phosphorylation (in which protons are used to maintain the proton gradient). In ketoacidosis, the excess protons come from lipolysis, `\(\beta\)`-oxidation and CoA synthesis. The pKa of lactate, acetoacetate and `\(\beta\)`-hydroxybutyrate are 3.9, 3.6 and 4.7 respectively; so these are found almost entirely in their dissociated (de-protonated) forms *in vivo*. (NB Acetone is a neutral molecule and therefore cannot function as an acid.) Therefore, were they produced as acids, they would generate H⁺ ions (which would then be buffered by HCO₃^- and converted to volatile acid, CO₂). However, they are instead produced as salts (e.g. sodium salts). ### Lactate and ketone bodies represent potential sources of bicarbonate Lactate and ketones can be metabolised back into bicarbonate during the corrective phase of an acidosis. Lactate is converted back to pyruvate in the liver ([Cori cycle](https://pubmed.ncbi.nlm.nih.gov/29021365/)) and then used as a substrate for gluconeogenesis. Ketones can be metabolised to acetyl-CoA during [ketolysis](https://pubmed.ncbi.nlm.nih.gov/34350876/). Either way, these substrates can feed into the Kreb's cycle, leading to the generation of (equimolar) bicarbonate. Therefore lactate and ketones can be thought of as ["potential bicarbonate"](https://pubmed.ncbi.nlm.nih.gov/18322160/). In DKA, urinary ketone losses result in an HCO₃^- deficiency, and hence in a hyperchloraemic acidosis in the later phases. ---  ??? Non-volatile acids consume HCO₃^-. The kidney must then regenerate HCO₃^- through one of two mechanisms (excretion of TA to reabsorb filtered HCO₃ or *de novo* synthesis through ammoniagenesis). And then the conjugate base must be excreted or metabolised. Therefore, non-volatile acids are disposed of as follows: `$$HA + NaCO_3 \rightarrow NaA + H_2O + CO_2 \rightarrow NH_4A + HA \rightarrow NaHCO_3$$` ---  -- .RWH_footer_pearl[anion gap <br> diet <br> NaHCO₃] ??? Anion gap can be used to infer presence of organic anion. In CKD, when kidneys struggling, we need to supplement HCO₃. 1g tds NaHCO₃ = 36 mmoles. In a plant-based diet, organic ions (e.g. citrate) are converted to HCO₃^- in the liver. Disposed of as KOA in urine. Claude Bernarde's Rabbits! --- class: center, middle, inverse # .white[Buffers] --- # Buffers `$$\color{#549ed6}{\underset{\color{grey}{acid}}{HA} \rightleftharpoons H^+ + \underset{\color{grey}{base}}{A^-}}$$` -- <br> `$$\tag{carbonic acid} H_2CO_3 \underset{\color{red}{pKa \sim 6}}{\rightleftharpoons} H^+ + HCO_3$$` `$$\tag{phosphoric acid} H_2PO_4^- \underset{\color{red}{pKa \sim 7}}{\rightleftharpoons} H^+ + HPO_4^{2-}$$` `$$\tag{haemoglobin} HHb + O_2 \underset{\color{red}{pKa \sim 7}}{\rightleftharpoons} H^+ + O_2Hb^{-}$$` -- <br> `$$\tag{\(\beta\)-OH butyric acid} C_4H_8O_3 \underset{\color{red}{pKa \sim 4.7}}{\rightleftharpoons} H^+ + C_4H_7O_3^-$$` <!-- `$$\tag{ammonium chloride} NH_4Cl \underset{\color{red}{pKa \sim 9.3}}{\rightleftharpoons} NH_3 + HCl$$` --> ??? A buffer is a solution of a weak acid and its conjugate base (or a weak base and its conjugate acid). Main plasma buffers all have pKa ~ 7. (Other buffers important in other circumstances - e.g. ketones in acid urine.) Isohydric principle: all buffers are in equilibrium. Therefore can determine status of whole system by looking at any one buffer (i.e. carbonic acid). --- # Carbonic acid `$$H^+\color{white}{_1} + HCO_3 \rightleftharpoons H_2CO_3 \stackrel{\color{red}{CA}}{\rightleftharpoons} H_2O + CO_2 \color{white}{\Longrightarrow \text{open}}$$` -- <br> `$$\underset{\color{green}{40 \space nM \space \space \space}}{H^+\color{white}{_1}} + \underset{\color{green}{24 \space mM}}{HCO_3} \color{grey}{\rightleftharpoons H_2CO_3} \stackrel{\color{red}{CA}}{\rightleftharpoons} H_2O + \underset{\color{green}{1.2 \space mM}}{CO_2} \color{#549ed6}{\Longrightarrow \text{open}}$$` -- <br> `$$pH = pKa+log \frac{[A^-]}{[HA]}$$` $$pH = 6.1 + log (\frac{24}{1.2}) = 6.1 +log(20) \approx 6.1+1.3 = 7.4 $$ `$$\color{red}{pH = 6.1 + log \frac{[HCO_3]}{\alpha .PaCO_2}}$$` ??? $$\alpha = 0.23 \space mM \space kPa ^{-1} \approx 1.2 \space mM \text{ (for 5.2 kPa)} $$ Carbonic anhydrase in RBCs. At pH 7.4, reaction shifted predominantly to the left, so that HCO₃ forms the vast majority of TCO<sub>2</sub. Dissolved CO₂ is around 1.2 mM. Hydrogen ions present at 1 million times less concentrated than bicarbonate (or most other extracellular solutes - measures in mM). Total body H⁺ around 3 micromol (c.f. 750 mmol for HCO<sub>3</sub). How can hydrogen ion concentration be less than that of HCO3? Because i) H+ will be buffered by other systems; ii) HCO3 can be delivered from acid salts (e.g. NaHCO₃)? Most HCO₃^- exists in dissociated form. Undissociated NaHCO₃ around 1.2 mM (Wimberley, 1985). --- `$$pH = 6.1 + log \frac{[HCO_3]}{\alpha .PaCO_2}$$`  ??? Can solve and equation with three variables in 3D space. ---  .RWH_footnote_right[.RWH_footer_style[Davenport (c. 1958)]] ??? This plot (pH vs. HCO₃ with PaCO₂ isopleths) as per Horace Davenport after Van Slyke (c. 1958). An alternative configuration (pH vs. PaCO₂ with HCO₃ or BE nomogram lines) preferred by Siggard-Andersen (1971). Main principle is that to get a complete understanding of any acid-base disturbance, must look at the respiratory and metabolic components. <br> ### Notes The prompt to Astrup and Siggard-Andersen to develop a better method for analysing acid-base status was the 1951 Copenhagen Polio epidemic. During this epidemic, patients initially erroneously identified as having an alkalosis because PaCO₂ not measured. They introduced concept of base excess (or THID = titratable H⁺ ion difference). The slope of the "buffer line", `\(-\beta\)`, is determined by the sum of all the non-bicarbonate buffers, principally Hb. Van Slyke defined buffer capacity, as `\(\frac{\Delta H^+}{\Delta pH}\)`, in 1922. Buffer capacity is therefore [measured in 'slykes'](https://pubmed.ncbi.nlm.nih.gov/15308499/). Siggard-Anderson proposed an equation that he called the "Van Slyke" equation, to define the relationship between pH, HCO₃, BE and Hb. This is used by ABG machines to compute BE. On the Davenport plot, `\(-\beta = -25 \space mM \space pH \space unit ^{-1}\)` and is approximately linear over physiological pH ranges. The gradient is steeper with increasing [Hb]. For comparison, the line is flat in a closed HCO₃-only system: `\(\approx - 5.4 \space mM \space pH \space unit ^{-1}\)`. ---  ---  ---  -- .RWH_footer_pearl[VBG *vs.* ABG] ??? ### What is on the blood gas report? pH, PaCO₂, PaO₂ all measured directly. The [other parameters](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5873626/) are calculated: - HCO₃act = what the bicarb is in the sample (but will be higher as PaCO₂ higher) - HCO₃std = what the bicarb would have been if PaCO₂ was normal (i.e. under standard conditions: T37, fully oxygenated, PaCO₂ 5.3 kPa) - BE = the amount of strong acid that must be added to each liter of fully oxygenated blood to return the pH to 7.40 under standard conditions - BEact = the actual BE in the sample - BEstd = assuming Hb 5 g/dL (rationale being that Hb buffers all ECF, not just plasma) HCO₃std and BEstd give the best metrics of the metabolic component of any disturbance. c.f. TCO₂ - measured as the amount of CO₂ (detected with indicator dye) liberated from plasma by the addition of a very strong acid (to shift pH well below pKa). ### VBG vs. ABG Generally [good agreement](https://www.uptodate.com/contents/venous-blood-gases-and-other-alternatives-to-arterial-blood-gases#H1942481) between VBG and ABG. Should expect pH to be ~0.04 pH units lower and PaCO2 to be ~0.6 kPa higher (and HCO3 to be the same). However, can be large discrepancies in patients with shock or extremes of acid-base disturbance. --- class: center, middle, inverse # .white[Respiratory control of acid-base] --- # Respiratory acid excretion  .RWH_footnote_right[.RWH_footer_style[Fencl (AJP 1966)]] ??? Seminal experiments in goats showing that CSF pH sets alveolvar ventilation rate. Now have quite a sophisticated understanding of the regulatory neural networks. --- class: center, middle, inverse # .white[Renal control of acid-base] --- # Renal acid excretion **Core principles:** - main job is to regenerate HCO₃ for the ECF - keep urine pH ~ 6 <br> **How the kidneys do this:** `$$NAE = V \times (U_{NH_{4}^+} + U_{TA\color{white}{_{1}^+}} - U_{HCO_{3}^-})$$` --- # H⁺ out = HCO₃^- in  --- # H⁺ out = HCO₃^- in  ??? ### Reabsorption of HCO₃^- \~90% HCO₃^- re-absorption is in the PCT. The main mechanism is as follows: H⁺ excretion through NHE3; H⁺ combines with HCO₃^- through CAIV tethered to the brush border; CO₂ diffuses into PCT cell and is converted back into HCO₃^- through cytoplasmic CAII; exits through the basolateral membrane through NBCe1-A. (H⁺ therefore re-cycling across the apical membrane.) --- # Titratable H⁺  --- # Titratable H⁺  .RWH_footnote_right[.RWH_footer_style[Hamm (Am J Physiol 1987)]] ??? NH₄⁺ not included in TA as pK 9.2. Ketoacids constitute a large portion of TA during ketosis. --- # NH₄⁺ out = HCO₃^- in  ??? ### Ammoniagenesis Glutamine deaminated to NH₄⁺ and `\(\alpha\)`-ketoglutarate. `\(\alpha\)`-KG reacts with H⁺, to leave HCO₃^- to enter the plasma; NH₄⁺ is secreted into the tubular lumen. If NH₄⁺ then excreted in the urine, then there is therefore a net gain of HCO₃^-. (If NH₄⁺ is reabsorbed then will combine with HCO₃^- in the liver to produce urea, resulting in no net HCO₃^- gain.) May have [evolved](https://doi.org/10.1002/bies.201900161) as acid-stress resistance mechanism from ammonatelic fish. ---  .RWH_footnote_right[.RWH_footer_style[Gottshalk (Am J Physiol 1960)]] ??? Acid urine generated in distal renal tubule. However, effective acid secretion along the length of the tubule... --- # H⁺ and HCO₃^- secretion in CD  -- .RWH_footer_pearl[vomiting] ??? In vomiting, get alkalosis and chloride depletion. Cannot excrete the bicarbonate until chloride depletion corrected. ### Intercalated cells ICs are rich in mitochondria and CA. `\(\alpha\)`-ICs secrete acid through apical H⁺-ATPase and H⁺/K⁺-ATPase (and basolateral Cl^-/HCO₃^- exchanger). `\(\beta\)`-ICs secrete HCO₃^- through the Cl^-/HCO₃^- exchanger pendrin (and basolateral H⁺-ATPase). --- # H⁺ and HCO₃^- secretion in CD  -- .RWH_footer_pearl[F+F test for dRTA] ??? [Furosemide + fludrocortisone test](https://pubmed.ncbi.nlm.nih.gov/17410104/): 40 mg + 1 mg then collect urine for 6 hrs. Healthy controls drop urine pH to < 5.3 (from 6 -- 7). --- class: center, middle, inverse # .white[Response to alkalosis / acidosis] --- # Response to alkalosis `$$NAE = V \times (U_{NH_{4}^+} + U_{TA\color{white}{_{1}^+}} - \color{red}{U_{HCO_{3}^-}})$$`  -- .RWH_footer_pearl[U_K after vomiting] ??? There is an apparent Tm for HCO₃^-, set close to 25 mM but variable and influenced by various factors (GFR, luminal pH, hormonal factors etc.) For example, during volume depletion, stimulation of Na reabsorption (NHE activity) will increase HCO₃^- re-absorption, so leading to an increase in the apparent Tm for HCO₃^-. Under normal circumstances, FE_HCO3 is \< 0.1% (Lote). In alkalosis, bicarbonaturia can drive cation loss (e.g. hypokalaemia). --- # Response to acidosis  ??? rpTECS mount an intrinsic (i.e. cell-autonomous) response to acidosis, generating more ammonium. One key mechanism is that the mRNA for key ammoniagenic enzymes contain AU-repeat pH-sensitive elements so that these [mRNAs are stablised](https://doi.org/10.2215/CJN.10391012) in intracellular acidosis. This also explains, in part, the mechanism of type IV RTA. Hyperkalaemia (somehow) induces intra-cellular alkalinisation, thus [inhibiting ammoniagenesis](https://doi.org/10.1681/ASN.2008020166). This mechanism was fleshed out by [Welling & Weiner](https://doi.org/10.1681/ASN.2017111163) using DCT-SPAK knockout mice to show that the hyperkalaemia *per se* was capable of inducing a metabolic acidosis. --- # Response to acidosis `$$NAE = V \times (\color{red}{U_{NH_{4}^+}} + U_{TA\color{white}{_{1}^+}} - U_{HCO_{3}^-})$$`  .RWH_footnote_right[.RWH_footer_style[Elkinton (Am J Med 1960)]] -- .RWH_footer_pearl[U_AG = U_Na + U_K - U_Cl] ??? Why is NH₄⁺ upregulated to excrete an acid load and not TA? To keep urinary pH around 6.0. When urine pH very high, risk of CaHPO₄ precipitation; when urine pH very low, risk of uric acid precipitation. ---  ---  --- class: center, middle, inverse # .white[When this goes wrong...] --- # RTA - type I = failure of distal H⁺ secretion - type II = failure of proximal HCO₃^- reabsorption - type III = loss of CA (mixed type I & II) - type IV = hypoaldosteronism & impaired ammoniagenesis -- <br> - **basal state** = UAG slighly +ve (UNa + UK > UCl) - **acidosis plus UAG -ve** = appropriate urinary acidification (UNa + UK < UCl) - **acidosis plus UAG +ve** = possible RTA ??? [UAG](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967442/) normally slightly positive in health (due to unmeasured sulphates, phosphates etc.) If extra-renal cause for acidosis (e.g. diarrhoea) then will be a compensatory rise in NH₄⁺ excretion, manifesting as a negative UAG. In the context of an acidosis, a positive UAG suggests failure to increase NH₄⁺ excretion - i.e. an RTA. (UAG may still be negative in some proximal and partial distal RTAs - so does not rule it out.) --- # Take-home points - lungs excrete 20 moles of acid per day - kidneys excrete c. 70 mmoles non-volatile acid per day - NAE determined by urinary NH₄⁺ + TA - HCO₃^- - preferentially generate new HCO₃^- in response to acidosis (to keep U_pH ~6) - ...resulting in urinary NH₄⁺ excretion - UAG as a proxy for urinary NH₄⁺ - chloride depletion limits HCO₃^- secretion (pendrin) .RWH_footnote_right[.RWH_footer_style[slides at: https://www.kidneyfish.net/talks/; see presenter notes for suggestions for futher reading]] ??? # Further reading [Hamm (CJASN, 2015)](https::/doi.org/10.2215/CJN.07400715) [Koeppen (Adv Phys Educ, 2009)](https::/doi.org/10.1152/advan.00054.2009) [Principles of Renal Physiology (Lote)](https://www.amazon.co.uk/Principles-Renal-Physiology-Christopher-Lote/dp/1461437849) [Kamel & Halperin book](https://www.sciencedirect.com/book/9780323355155/fluid-electrolyte-and-acid-base-physiology)