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BIOCHEMISTRY OF RESPIRATION & pH REGULATION

SADIAH ACHMAD

DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE

UNISBA

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RESPIRATION

Respiration is the interchange of 2 gases : O2 & CO2, between the body & the atmosphere. The respiratory system consists of :

• A gases interchange system : organized by the lungs• A gases transport system : organized by the blood

O2 : atmosphere → alveoli → alveolar capillary → blood circulation → cells CO2 : cells → blood circulation → alveolar capillary → alveoli → atmosphere

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The gases interchange system Function of the lungs :

• Interchange gases (O2 & CO2) between the alveolar capillary blood & the atmosphere• Keep the blood pH at its normal level

Gas exchanges between alveoli & alveolar capillaries by : simple diffusion

Alveolar capillary

pO2 : 40 mmHg pO2 : 100 mmHgpCO2 : 45 mmHg pCO2 : 40 mmHg

Alveoli : pO2 : 100 mmHg pCO2 : 40 mmHg.

Atmosphere : pO2 : 160 mmHgpCO2 : 0,2 mmHg

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The gases transport system Transport of O2

O2 is not soluble enough in plasma (only about 2,3 ml O2 can be dissolved in 1 L of plasma at 38◦C ) → O2 need a carrier to meet the body’s needs : hemoglobine (Hb)

• O2 dissolved in plasma : 1-1.5 %

• O2 bound to Hb : 98.5 %

Hb performs 2 major transport functions : 1. transport of O2 from resp. organ to periph.tissues 2. transport of CO2 & H+ from periph. tissues to resp. organ

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Hb is a tetramer of polypeptides

Hb adult : Hb A, Hb A2, Hb F

Hb A consists of 2 subunits & 2 subunits . Hb A2 consists of 2 subunits α & 2 subunits δ .

Hb F consists of 2 subunits α & 2 subunits γ . Each subunit contains 1 heme group with a central Fe2+ in the heme pocket .

1 subunit heme binds 1 O2 mol 1 mol Hb binds 4O2 molecules.

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100

80

60

40

20

0 20 40 60 80 100

Partial pressure of oxygen (mm Hg)

Reduced bloodreturning from tissues

Oxygenated bloodleaving the lungs

Perc

ent s

atur

atio

nOxygen binding curve of hemoglobin A

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pO2 periph. cap P50pO2 alv. cappO2 ven.bl.

50

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O2 binding curve of Hb in normal blood : a sigmoid curve

- 98% saturated in the lungs

- 33% saturated in working muscle

50% of total Hb is saturated with O2 at pO2 : 26 mmHg

P50 HbA = 26 mmHg expressing O2 affinity of Hb

P50 Hb F = 20 mm Hg this difference permits Hb F in the

fetal blood to extract O2 from Hb A of placental blood.

Post partum : Hb F is unsuitable because of its high O2

affinity, makes HbF delivers less O2 to the tissues

deliver 65% of O2

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The cooperative effect of Hb The 4 subunits of Hb generates a “cooperative effect” in

binding O2 (cooperative binding kinetics of Hb)

- The binding of the first O2 to each Hb mol. (sub units)

enhances the binding of subsequent O2 molecules

- Hb binds O2 weakly at low O2 tension & tightly at high

O2 tension

- Hb binds maximal quantity of O2 at the respiratory organ

& delivers maximal quantity of O2 at peripheral tissues

The cooperativity effect happens because Hb exist in 2 forms :

T form and R form → Quaternary structure

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Quaternary structure of Hb : Hb exists in 2 different forms

deoxy Hb : T (tense) form oxy Hb : R (relax) form

Binding of O2 is accompanied by conformational changes of Hb

C- terminal residues of subunits are held together by salt bonds

1.O2 binding rupture of salt bonds between C–terminal residues of all 4 subunits - binding of O2 to 1 subunit of T - form local conformation changes weakens the association between subunits rupture of bonds - increasing PO2 more molecules are converted to R-form

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2. Upon O2 binding Fe2+ of deoxy Hb moves into the plane of the heme - ring

His. F8 & its associated residues are pulled along with Fe2+

Fe

N

HCCH

N

F helix

Steric repulsion

Porphyrinplane

His F8

C N

HC CHN

F helix

O O

+O2

Fe

C

T form of Hb R form of Hb

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Effect of BPG (2,3 biphosphoglycerate)

RBC contain high levels of BPG, nearly equimolar with Hb

In peripheral tissues, O2 shortage ↑ accumulation of BPG

1 mol of BPG is bound per Hb tetramer in the central cavity of

Hb formed by all 4 subunits

The cavity is sufficient for BPG, only when the space between

chains is wide : Hb in T - form (deoxy Hb)

The cavity is lined with (+) charged groups (i.e. N – terminal

amino groups Val NA 1, Lys EF 6, His H 21 of chains) that

could firmly bind 1 mol of (-) charged BPG by salt bridges

BPG stabilizes deoxy Hb by cross linking its chains

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BPG binds more weakly to Hb F : H21 residue of chains is

Ser. rather than His. (serine is a neutral amino acid)

- can not form salt bridges with BPG

- less effect on stabilization of deoxy Hb

- Hb F has a higher affinity for O2 than HbA

Concentration of BPG in RBC in tissue hypoxia (anemia,

cardiopulmonary insuf, high altitude )

- enhance formation of deoxy Hb at low PO2

- Hb deliver more O2 to the tissues

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Physical factors affecting O2 binding1. temperature

- high temperature weakens O2 affinity of Hb enhance

unloading of O2

- below normal temperature : the binding is tighter

In fever or excercised muscle (elevated temp.) : additional

O2 is needed to support high metabolic rate.

In a hypothermic conditions : Hb’s diminished ability to

release O2 is compensated by :

- O2 utilization within the body

- solubility of O2 in plasma

- solubility of CO2 acidifies the blood

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2. pH

H+ concentration influences the O2 binding of Hb

- low pH weakens O2 affinity of Hb enhancing O2

delivery

- high pH O2 delivery

Decreased in pH is often associated with O2 demand.

Increased metabolic rate production of CO2 & lactic

acid pH ↓ → O2 is released to support the

metabolism

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Transport of CO2

CO2 transport is closely tied to Hb

acid – base balance

CO2 in blood is present in 3 major forms :

- dissolved CO2 : 5.5 %

- bicarbonate ion (HCO3-) : 89 %

- carbamino Hb : 5.5 %

Each is present both in arterial blood & venous blood

Net transport to the lung for excretion : concentration

difference between arterial & venous blood

Venous blood contains only 10% more than total CO2

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• Bicarbonate formation CO2, a metabolic product, enters the bloodstream & diffuses into RBC generates H+, most come from formation of HCO3

-

. This reaction is catalyzed by carbonic anhydrase.

CO2 + H2O H2C03 H+ + HC03-

Carbonic anhydrase : - found abundantly inside RBC - a very active enzyme : reaction

reaches equilibrium within 1 sec

- contains Zn

Most of H+ generated during HCO3- production is handled by

buffer action and / or other processes

carbonic anhydrase Spontaneous

rapid

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Carbamino - Hb formation, also generates H+

CO2 forms carbamino substances with amino groups of any

proteins within RBC, mostly Hb

Deoxy - Hb forms carbamino - Hb more readily than oxy Hb

Oxygenation causes release of CO2 in carbamino Hb

Carb. Hb formation occurs only with uncharged aliphatic

amino – groups, not with charged form R - NH3+

H HR – N + CO2 R – N + H+

H C – O ll carbamino

O substancecov. bond

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- N–terminal amino groups of globin chains are the

principle sites of carbamination

If block chemically by cyanate carbamino formation

does not occur

- N–terminal amino group of -globin is also the binding site

for BPG CO2 competes with BPG in binding Hb mol.

CO2 ↑ : effect of BPG

BPG ↑: the ability of Hb to form carbamino-Hb

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Two processes regulate [ H+ ] derived from CO2 transport

1. Buffering Hb carrying O2

carrying CO2 as carb. Hb handling H+ produced by CO2 transport :

- buffering- isohydric mechanism

Hb buffer capacity is provided by its ionizable groups : - 4 N - terminal amino groups

- imidazole side chains of His. res (38 His. per Hb tetramer)

The buffer system handles 60% of H+ produced by CO2 transport : buffered by Hb : 50%

other buffers : 10% - HCO3

- / CO2

- organic - P in RBC - plasma proteins

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2. Isohydric mechanism

The remainder of H+ (40%) arising from CO2 is taken

up by Hb : isohydric carriage of CO2

This system takes up H+ ions with no change in blood

pH, through the operation of Bohr Effect

Hb binds 2 protons for every 4 O2 molecules released.

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2H2O + 2CO2 CO2

2H2CO3

2HCO3–

+ 2H+

Hb.4O2

Hb.2H+ + 4O2 O2

Hb.2H+ + 4O2 O2

Hb.4O2

2HCO3- + 2H+

2H2CO3

2H2O + 2CO2 CO2

HCO3–

Cl– Cl–

Cl– Cl–

HCO3–

exhaled toatmosphere

to peripheraltissues

generated bythe Krebs cycle

ArteryVeinVenous retum

Chloride shift

pulmonarycapillaries

extra pulmonarycapillaries

From atmosphere

The Bohr effect

Carb. anh.

carb. anh.

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pH REGULATIONMaintenance of relatively constant blood pH value is essential

for health, since changes in blood pH will affect intracell pH

alter : - protein conformation

- enzyme activity, metabolism

- equilibria of reactions that consume / generate H+

(oxidation - reduction reaction)

Maintenance of a constant blood pH is, in part, achieved by :

- The buffer system in the blood control short - term

changes in blood pH.

- for long term changes : balancing proton (H+) loss &

gain by the lung & the kidney

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pH value of blood may be affected by :

- malfunctioning of the blood buffer system or

- disturbance of acid-base balance due to diseases

e.g. - kidney disease or

- altered breathing frequency

( hypo / hyperventilation)

Normal arterial plasma pH : 7.40 0.05

- pH < 7.35 acidosis

- pH > 7.45 alkalosis

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BUFFER SYSTEM

3 major body water components : - plasma : within circulating system - interstitial fluid : fluid that bathes cells - intracellular fluidComposition : - plasma : - major cation : Na+

- small amounts : K+, Ca2+, Mg2+

- dominant anions : HCO3-, Cl-

- small amount anion : protein, HPO42-, SO4

2-

- mixture organic anions - interstitial fluid : similar with plasma, but contain less protein plasma & interst. fluid extracell. fluid - intracell fluid : - major cation : K+

- major anions : - organic phosphates - protein

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The buffer system in the blood• Blood plasma is a mixed buffer system :

H2CO3 / HCO3- , H2PO4

- / HPO42- , H protein / protein-

* Major buffer of plasma: H2CO3 / HCO3- system

- an open system: pCO2 is adjusted to meet the body,s

need by increasing / decreasing respiration.

- effective in controlling pH changes caused by other

than pCO2 changes

* Major buffer inside RBC : Hb buffer system

(HHb/Hb) → also responsible for buffering pCO2 changes

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A buffer system consists of : - a weak acid : H A

- its conjugate base : A-

e.g. Acetic acid / acetate-, NH4+

/ NH3, H2PO4- / HPO4

2-

The weak acid might be neutral, (+) charged, (-) charged

The conjugate base : 1 less (+) charge or 1 more (-) charge

than its weak acid

The Henderson - Hesselbalch equation :

pH = pKa + log

direct relationship between pH & ratio [conj. base] / [acid]

Ratio ↑ → pH ↑ or Ratio ↓ → pH ↓

[conj. base ] [acid]

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[HCO3-] / [CO2] buffer system :

Blood pH : 7,4

p.K.H2CO3 : 6,1 (1.3 = log 20)

In order to keep the blood pH at its normal level (7.4) →

[HCO3-] / [CO2] ratio must be 20/1

Every changes in [HCO3-] or [CO2] changes the ratio

changes pH body compensation is needed to

normalize blood pH.

7,4 = 6,1 + log 20/1

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ACID – BASE BALANCE & ITS MAINTENANCE

• Acidosis : excess acid or def. of alkali in the body

• Alkalosis : excess alkali or def. of acid in the body

There exist mechanisms where the body normally rids itself of

excess acid or alkali

• Metabolism in individuals produces large amounts of acids

• Major acid: H2CO3 (CO2) volatile: excreted by the lungs

Disorder of the lungs respiratory acidosis or alkalosis

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Respiratory acidosis : hypoventilation CO2 accumulates

within the blood → shift direction of the reaction :

H2O + CO2 ↔ H2CO3 ↔ H+ + HCO3- → to the right

Hypoventilation occurs when depth or rate of respiration ↓

- airway obstruction

- neuromuscular disorders

- diseases of CNS

- chronic obstr lung disease → chronic resp acidosis

- inhalation of gas with high pCO2 → resp. acidosis

Acute resp. acidosis

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Respiratory alkalosis : excess of breathing → CO2 will be

exhaled abundantly. Direction of reaction will shift to the left

H2O + CO2 ↔ H2CO3 ↔ H+ + HCO3-

Hyperventilation : - anxiety : most common cause

- CNS injury involving resp. center

- salicylate poisoning

- fever

- artificial ventilation

High altitude alv. pCO2 chronic resp. alkalosis

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Metabolic acidosis

The body metabolism produces many nonvolatile (fixed) acids

- protein metabolism → H+ + SO42-

- hydrolysis of phosphate-esters phosphoric acid

- metabolism - lactic acid

- acetoacetic acid

- -hydr. butyric acid

- administration of : NH4Cl / Arg-HCl / Lys-HCl → urea + HCl

produced in excess accumulation acidosis

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- ingestion of salicylate, methyl alcohol, ethylene glycol

production of strong organic acid

accumulation of nonvolatile acids metabolic acidosis

- abnormal loss of base (HCO3-)

- renal tubular acidosis : abnormal amount of HCO3-

escape from blood into urine

- severe diarrhea fecal excretion of HCO3-

→ HCO3 -

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Metabolic alkalosis

- intake excess of alkali / salt of organic acid :

- NaHCO3

- Na – lactate

- abnormal loss of acids : vomiting, gastric lavage

- rapid loss of body water : diuresis temporary [HCO3-]

in blood

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Causes of acid – base imbalance

Acidosis :

Respiratory : alveolar hypoventilation

Metabolic : - H+ overproduction

- HCO3- overexcretion

Alkalosis :

Respiratory : alveolar hyperventilation

Metabolic : - alkali ingestion

- H+ overexcretion

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Normal

Acidosis- Respiratory- Metabolic

Alkalosis- Respiratory- Metabolic

Blood pH

7,4

Urine pH

6 –7

[HCO3- ]/

[H2CO3]

20/1

20 / > 1< 20 / 1

20 / < 1>20 / 1

Cause

HypoventilationH+ production orHCO3

- excretion

HyperventilationAlkali ingestion or H+ excretion

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(1)

(10)

(<1)

(20)

(>1)

(20)

(1)

(20)

(1)

(>20)

(+) (-) (+) (-) (+) (-) (+) (-) (+) (-)Normal Resp.

acidResp.alkal

Met.acid

Metalkal

= H+

= HCO3-

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Role of kidney in the acid–base balance system

Excess of nonvolatile (fixed) acids & bicarbonates are excreted by the kidney pH urine varies between 4.4 - 8.0 Daily urine volume : 1,2 L

• Formation of urine Fundamental functional unit of kidney : the nephron :

- filter the blood - modify the glomerular filtrate to become urine

• Glomerulus : capillaries enclosed by glomerular capsule Water & low molecular weight solutes (inorganic ions, urea, sugars, amino acids pass the capillaries membrane into the capsular space ultra filtrate

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• Proximal convoluted tubule :

- most of water & solutes are reabsorbed tubular

fluid continues through the loop of Henle

• Distal convoluted tubule :

- further reabsorption of solutes

- secretion

• Collecting tubule :

- additional concentration urine :

Urine contains 1% or less of water & solutes of

glomerular filtrate

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• The Kidney regulates acid - base balance by :- controlling bicarbonate (HCO3

-) reabsorption

- secreting acid (H+)

Both processes depend on the formation of H+ & HCO3- from

CO2 & H2O within the tubular cells

- H+ formed is actively secreted into tubular fluid in

exchange with Na+

- Na+ uptake transport by the tubule :

- partly passive

- partly active via antiport systems which reabsorb Na+

in exchange with H+,K+ or NH4+

- Na+ reabsorbed in exchange with H+ regeneration of

NaHCO3 within the tubular cell out of the cell plasma

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Fates of excreted H+

1. React with HCO3- H2CO3 ↔ H2O + CO2

CO2 will be absorbed from tubular fluid & reassociated

with H2O to form H2CO3. The dissociated HCO3- is

transported back to the plasma: reabsorption of NaHCO3.

Tubular fluid becomes depleted of HCO3- pH drops

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2. Buffered by HPO42- / H2PO4

- buffer system

H2PO4- is not reabsorbed passes out in the urine

represents net excretion of H+

In diabetic ketoacidosis : plasma acetoacetate & -hydroxy

butyrate & pass the glomerulus into tubular fluid → could

serve as buffers Effect of buffering : - excrete acid (H+)

- regenerate bicarbonate The amount of acid excreted could be measured → titratable acidity of urine : 1/3-1/2 of normal daily acid excretion

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3. Neutralization by NH3 H++ NH3 → NH4

+ elimination of NH4+ contributes

to net acid excretion

Tubular cells produce NH3 from amino acids, particularly glutamine ( tubular cells contain a lot of glutaminase)

NH4+ is the major urinary acid : ½ - 2/3 of daily acid excretion

This mechanism is more important in acidosis : - acid can be excreted without lowering pH of urine (formation of titratable acidity causes decrease in pH urine) - ammonia can be excreted in an enormous amount - spares body stores of Na+ & K+

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Compensatory mechanisms 4 primary types of acid – base imbalance

- resp. acidosis : caused by plasma pCO2

- resp. alkalosis : caused by plasma pCO2

- metab. acidosis : - addition of strong organic /

inorganic acid

- loss of HCO3-

plasma [HCO3-]

- metab. alkalosis : - loss of acid

- ingestion of alkali

plasma HCO3-

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Principles of acid-base imbalance compensation

Abnormal condition directly altered 1 term of [HCO3-] / [CO2]

If one of the term of [HCO3-] / [CO2] ratio is altered plasma pH can be readjusted back toward normal by compensatory alteration of the other term.

e.g ketoacidosis plasma [HCO3-]

Compensation : decreasing plasma [CO2] so that the ratio &

therefore pH is readjusted back toward normal.

Compensation does not have to return the HCO3- & CO2 blood

levels toward normal

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• If [HCO3-] changes to restore the [HCO3

-] / [CO2] ratio

is to change pCO2 in the same direction

• If primary change is pCO2 to restore the original ratio is

to alter [HCO3-] in the same direction

Specific compensatory processes• Acute resp. acidosis (eg breathing gas mixture containing

high level of CO2) pCO2 - plasma pH

- [HCO3-]

compensation : - renal excretion of H + & bicarbonate

reabsorption [HCO3-] , although its

level is already above normal

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• Acute resp. alkalosis pCO2 rapidly - pH

- [HCO3-]

Compensation : - renal excretion of [HCO3-] →

- plasma [HCO3-] →

- plasma pH toward normal• Metab. acidosis : 2 compensatory mechanisms in dealing with the excess acid

1. Renal H+ excretion : slow, inadequate to return [HCO3-]

& pH to normal

2. Respiratory compensation : hyperventilation pCO2

Compensation of metab. acidosis involves :

- pCO2

- small in [HCO3-]

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• Metab. alkalosis

- primary defect : plasma [HCO3-]

- immediate physiologic response : hypoventilation,

followed by renal excretion of HCO3-

Hypoventilation - pCO2

- small in [HCO3-]

Respiratory response to metabolic acid-base imbalance

is a rapid response

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