1 . 2 Claude Bernard 1813 - 1878 „INTERNAL ENVIRONMENT“ © Department of Biochemistry (V.P.), Faculty of Medicine, MU Brno 2009.

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Claude Bernard1813 - 1878

„INTERNAL ENVIRONMENT“

© Department of Biochemistry (V.P.), Faculty of Medicine, MU Brno 2009

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Internal environment :

Claude Bernard, 1877-78: ● „it is …. blood plasma and all interstitial fluids…“ ● „the constancy+) of the internal environment is the condition for a free and independent life.“

it is the existence of living substance, which is not affected by changes of external environment

+) the name „homeostasis“ was introduced more than 60 years later (→ Walter Cannon)

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Internal environment = ECF (extracellular fluid)

external environment ECF cell

constancy of internal environment

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Internal environment = ECF (extracellular fluid)

external environment ECF cell

constancy of internal environmentacid-base regulation

= acid-base metabolism (action) acid-base equilibrium

(„ABE“) (state)

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carbonate hydro-lyase EC 4.2.1.1

Carbonatedehydratase = carboanhydr(at)ase) = carbonate hydrolyase :

CO2 + H2O H2CO3

erythrocytekidney

[laieis]

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P U F R YB U F F E R S

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(weak acid) HA H+ + A-

(salt of weak acid) BA B+ + A-

Buffer

A weak acid = weak electrolyte fractional dissociation only reversible (two-way) reactionA salt = strong electrolyte dissotiation practically complete, non-reversible (one-way) reaction

KA

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KA = [H+] . [A-] / [HA] Keq = [H+] . [A-] / [HA] . [H2O]

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(weak acid) HA H+ + A-

(salt of weak acid) BA B+ + A-

H+ Cl-

Buffer – the reaction with an acid :

KA

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(weak acid) HA H+ + A-

(salt of weak acid) BA B+ + A-

H+ Cl-

Buffer – the reaction with an acid :

KA

the part of molecules of an acid, which is not dissociated, does not influence on pH ! „excess“ H+ iontes can be removed as an undissociated acid

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(weak acid) HA H+ + A-

(salt of weak acid) BA B+ + A-

Na+ OH-

Buffer – the reaction with a base :

KA

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(weak acid) HA H+ + A-

(salt of weak acid) BA B+ + A-

H+

H2ONa+ OH-

Buffer – the reaction with a base :

KA

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The titrationcurve andability tobuffer

„pKA ± 1“

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Boundary values of pH (the whole blood) :

pH = 7,40 [H+] 40 nmol . l-1

pH = 6,80[H+] 160 nmol . l-1

pH = 7,70[H+] 20 nmol . l-1

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Remark:

[H+] is here given in nmol . l-1 (it is 10 –9 mol . l-1) ,

- do not mistake for mmol . l-1 , whichcomprise million times higher concentration !!!

[H+] (nmol . l-1) = 10 (9 – pH)

pH = 9 – log [H+] (nmol . l-1)

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Boundary values of pH (the whole blood) :

pH = 7,40 [H+] 40 nmol . l-1

normal value

pH = 6,80 [H+] 160 nmol . l-1

4multiple of normal [H+]

pH = 7,70[H+] 20 nmol . l-1

½ of normal [H+]

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pH = 6,80 [H+] 160 nmol . l-1

4multiple of normal [H+]

pH = 7,70[H+] 20 nmol . l-1

½ of normal [H+]

The extreme values of pH compatible with life

the tolerance for acidemia (acidosis) is considerably higher, therefore alkalemia (alkalosis) is of greater danger

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CO2 + H2O H2CO3 H+ + HCO3-

800 mol 1 mol 0,03 mol

CO2 in blood plasma

This model idea would be valid in total closed system only (see next !). In the living organism it is the not attainable state.However it is used to underline being of „effective concentration“of carbonic acid (next picture). It is increased at every retention of CO2 , when the system stops to be total open( e.g. the need of increase of HCO3

- concentration in an ionic disorder).

At being (total) open system the ratio [CO2] / [HCO3-]

will not be 800 / 0,03 , however 1 / 20 (as correspond to pH = 7,40).Do not mistake for: normal ratio [HCO3

-] / [H2CO3 + CO2] = 24 / 1,2 = 20 . # log 20 = 1,3 - see next !

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CO2 = physically dissolved CO2

(chemically not affected) H2CO3 = CO2 reacted on an acid CO2 + H2CO3 = „effective concentration of carbonic acid“ ( „Effective“ means, that as carbonic acid will be used its molecules replenished from the excess of CO2 too )

Carbonic acid in plasma:

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Henderson – Hasselbalch equation for HCO3

- / H2CO3 in blood plasma :

pH = pK a + log

cs

ca

HCO3-

pH = pK + log CO2 + H2CO3

H2CO3

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HCO3-

pH = pK + log CO2 + H2CO3

H2CO3

HCO3-

pH = 6,10 + log 0,230 * pCO2

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HCO3-

pH = 6,10 + log 0,230 * pCO2

24 log = log 20 = 1,30 1,2

HCO3- is not given in mol . l-1

(as it is common in other pH calculations) , however in mmol . l-1 (its usual dimension)

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HCO3-

pH = 6,10 + log 0,230 * pCO2

calculated

measured

The principle of ABE parameters determination :

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pCO2 a pO2 cell („electrode“)

pCO2 silicone membrane the change of pH is measured (combined glass and Ag /AgCl electrode in bicarbonate solution) pO2 polypropylene membrane oxygen is reduced to O2

2- (formation of peroxide, the polarografic principle: electric current – proportionate to pO2 - is measured between Pt cathode and Ag / AgCl anode in phosphate buffer).

METHODS OF A „DIRECTMEASUREMENT“ (not the Astrup´s method!)

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PARAMETERSof ABE

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pH = 7,40 0,05

pCO2 = 5,33 0,5 kPa

BE = 0 3 mmol . l-1

Basic parameters of ABE:

____________________________________________________BE = base excess [beis ik´ses] = „výchylka nárazníkových bazí“, „výchylka pufrových bazí“ - původní význam „nadbytek bazí“ zanikl spolu s pojmem

„base deficit“, BD

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Parameters of ABE:1/ pH is a crucial parameter metabolism in cells is determined by enzymes, which have their pH optimum we must lead all our actions to the normalization of pH (~ 7,40)

2/ pCO2 and BE are the basic parameters inform of the way, how was the resulting pH obtained together with pH allow to asses the type of ABE disturbance

3/ all other parameters are helping ones- some of them can be „actual“ and others „corrected“ !!

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Parameters of ABE actual / standard

act HCO3- = 24 3 mmol . l-1

std HCO3- = 24 3 mmol . l-1

std BE = 0 3 mmol . l-1

(Under identical conditions [HCO3-] = 24 mmol . l-1

corresponds to the BE = 0 mmol . l-1 )

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[HCO3-] mmol/l … 21 22 23 24 25 26 27 …

BE mmol/l … -3 -2 -1 0 +1 +2 +3 …

[HCO3-] vs. BE :

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Actual parameters of ABE : „act“ = actual it is in the given state, which does not correspond to standard More simple: in praxis it is the value of some parameter of ABE at the pCO2 , which differs from its normal value (pCO2 5,33 kPa !!)The analyzer secures some standard conditions during the measurement (pO2 and the temperature of the whole blood sample). - The standard way of sample collection and handling the sample must be allways strictly kept !!

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Standard parameters of ABE :

„std“ = standard = corrected, expressed under standard conditions The standard conditions:1/ pCO2 = 5,33 kPa (normal)2/ pO2 (the blood saturated with oxygen)3/ t = 37,0 °C4/ sample of whole blood („anaerobic collection“)

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Corrected parameters of ABE

are recalculated for the normal pCO2

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Replenish data:

• pO2 = 9 – 15 kPa (age dependence)

• saturation of Hb with oxygen = 0,95 – 0,98

• forms of Hb not transporting oxygen

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Dřívější údaje / former data :

BBb = buffer base (blood) 48 mmol . l-1

´bafә beis blad souhrn konjugovaných pufrových bazí

(plné krve)

BBp = buffer base (plasma) 42 mmol . l-1

´bafә beis plaezmә souhrn konjugovaných pufrových bazí (plazmy)

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BBp = buffer base (plasma) 42 mmol . l-1

24 mmol . l-1 HCO3-

25 mmol . l-1 protein-

2 mmol . l-1 všechny ostatní pufrované báze

all other buffer bases

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BBb = buffer base (blood) 48 mmol . l-1

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Hb - concentration and buffer capacity MHb = 64.458 g . mol-1 ( 4 Fe)

[Hb] = 140 g . l-1

140 / 64.458 = 0,002 17 mol . l-1 = 2,2 mmol . l-1

Hb has behavior of polyprotic conjugated base: it has 38 His. At the pH of plasma the carboxyls and the aminogroups of side chains are completely ionized and they do not buffer. The buffer capacity of Hb is so produced by imidazole nuclei of His.(For computation of part of buffer capacity of Hb in blood is necessery to know a hematokrit and possibly a density of erythrocytes too).

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Imidazole nucleus of His :

H+

H

N

N

N

NH

+ H+

The pKA of His is in environment of blood plasma approximately in the range 7 > pKA > 6 (in water: 6,1).Imidazole is an exclusive group of aminoacids able to buffer at physiological pH of blood ( 7,4).

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Contemporary methods of „direct measurement“ of ABE parameters do not allow to quantify BBb and BBp by analyzers.(However they can be calculated by replenish some other values - [Hb], ionts *) ).

The values BBb and BBp were used in the days of „equilibration method“ according to Astrup (approximately at the end of 70th. That time both values were enumerated among ABE parameters together with other results).

Up today both values give usefull information about buffer properties of blood and plasma respectively.

*) BBp = [Na+] + [K+] - [Cl-]

Remark:

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nepřímá měření od 70./80. let

UŽ NE !!

indirect measurements since 70th/80th years

NOT ANYMORE !!

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Sodium hydrogencarbonate („bicarbonate“) is alkalic :

NaHCO3 + H2O H2CO3 + Na+ + OH-

(The carbonic acid in oval symbolizes a weak - practically undissociatedelectrolyte. The sodium hydroxide is a strong base, it is almost total dissociated electrolyte. - In the aqueous solution the excess of OH- ions makes the solution basic.)

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Buffer system

IVF

whole blood erythrocytes

plasma

ISF ICF

HCO3-/H2CO3 + CO2 50 % 17 %

33 %

HCO3- HCO3

-

Protein/HProtein 45 % 27 %

18 %

- proteins

HPO42-/H2PO4

- 5 % (inorg.) 3 % (org.)

1 % 1 % (inorg.)

inorg.

phosphate

org.

phosphate

Concentration of buffer

systems ( mmol . l-1 )

48 ± 3 ~ 56

42 ± 3

Buffer capacity :

BBb BBp

„interaction reaction“among buffer systems

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Remark

At the evaluation of part of BBp (= 42 mmol . l-1)and the buffer capacity of erythrocytes ( 56 mmol . l-1)on BBb (it is on the total buffer capacity of blood) we should take in consideration the hematocrit ( 0,45) at least:

0,45 of volume are erythrocytes,their part in BBb is: 56 · 0,45 = 25,2 25 mmol . l-1

0,55 of volume is plasma,its part in BBb is: 42 · 0,55 = 23,1 23 mmol . l-1

the sum ( BBb ) 48 mmol . l-1

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buffer Ery plasma ISF ICF

bicarbonate Hb

phosphate protein

Ery = erythrocyteISF = interstitial fluidICF = intracellular fluid

The buffers in different compartments:

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acidémie

alkalémie

Pufry v bb.

Buffers in the cell

(směna H+ a K+)(the change H+ for K+)

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PORUCHY ABR

ACID-BASE DISORDERS

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Anaerobic sampling of blood (1) :

Keeping of samples :• at room temperature: measurement of pO2 within 5 min, the other acid-base parameters within 30 min• in ice-cold water: up to 4 h after sampling

from the heel in infants

„arterialized“ capillary bloodfron the ear lobe / finger

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Anaerobic sampling of blood (2) :

heparinizingof blood

closingof capillary

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Basal terms:deviations from the normal pH: acidemia (pH 7,36)

alkalemia (pH 7,44)

processes evoking these deviations: acidosis („Ac“) alkalosis („Alk“)

respiratory process („R“): the primary disorder is in the changing of pCO2

metabolic process („M“):the primary disorder is in the changing of [HCO3

-] or [H+]

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HCO3-

pH = pK + log CO2 + H2CO3

H2CO3

„acidosis“ (pH 7,36) metabolic disorder

„alkalosis“ (pH 7,44) respiratory disorder

The classification of ABE disorders (1):

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Sources of acids in metabolism :1/ volatile carbonic acid: ● the source is CO2 - from decarboxylations ● CO2 with water gives weak volatile carbonic acid ● the exchange of CO2 ( 15 – 25 mol . d-1 ) between the blood and the external environment secure the lungs

2/ nonvolatile acids: ● sulfuric acid - from sulfur-containing amino acids (Cys + Met) ● phosphoric acid - from phosphorus-containing compounds ● carboxylic acids (e.g. lactate, acetoacetate, 3-hydroxybutyrate), unless they are completely oxidized to CO2

and water ● nonvolatile acids cannot be removed through the lungs, they are excreted by the kidney into the urine ( 40 – 80 mmol . d-1 )

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15-25 (~ 20) mol . d-1

40-80 (~ 60) mmol . d-1

mmol vs. mol !!

( 20.000 / 60 = 333, …)

( 99,7 % vs. 0,3 % )

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The classification of ABE disorders (2):

- according to time manifestation: acute (uncompensated) stabilized (compensated)

- completely pure metabolic disorders or completely pure respiratory disorders (it is the isolated acute disorders) practically do not exist, because the compensation processes begin nearly immediately, however the stabilization can also take some days (in dependence on the type of disorder)

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[acid]

[salt]

pH = 7,40

normal values*

[H2CO3 + CO2]

pCO2

BE

BE = 0

pCO2 = 5,33 kPa

Henderson – Hasselbalch equation

~

[HCO3-] ≈

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normalvalues

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overall evaluation (pH, pCO2 a pH)*) - by Astrup + Siggaard-Andersen [sigurd]

Evaluation of a disturbance and its compensation

*) from this only pCO2 is an independent variable, influencing the state of ABE

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M E T A B O L I C D I S O R D E R S

Primary metabolic disorder :

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R E S P I R A T O R Y

Primary respiratory disorder :

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Akutní poruchy ABR (modře) Acute disorders of ABE (in blue)

- - - - -

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Kompenzované poruchy ABR (červeně) Compensated disorders of ABE (in red)

- - - - -

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Nomenclature

disorder

compensation

correction

- - - - -

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Aktuální a standardní BEActual and standard BE

Pozor na metabolickou alkalózu !! Mind the metabolic alkalosis !!

- - - - -

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Časové hledisko úpravy poruch ABR :

„setrvačnost !!“

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The liver and ABE :

1/ acidemia: NH3 glutamin (Gln) (transport to kidneys, releasing NH4

+ by glutaminase ...)

2/ alkalemia: NH3 urea

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The liver and ABE – acidemia :

two ways of NH3 elimination in the liver

liver kidney urine

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NH

NH

4+

4+

NH

NH

2

2

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C O

OH

H +

+

+

HCO3-

The liver and ABE - alkalemia (1)

( In the contrary, in acidemia organism saves (basic) bicarbonates: → in acidemia the synthesis of urea will be reduced.)

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NH

NH

4+

4+

NH

NH

2

2

22

C O

OH

H +

+

+

HCO3-

The liver and ABE - alkalemia (2)

alkalic part

acidicpart

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NH

NH

4+

4+

NH

NH

2

2

22

C O

OH

H +

+

+

HCO3-

The liver and ABE - alkalemia (3)

MAlkcorrection of

MAlk

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The kidney and ABE :

antiportsymport

tubular cell tubular lumen

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MAc MAlk0

Kombinovaná porucha ABRMixed disorder of ABE

zdánlivě normální stavseemingly normal state

hlad / starvation

ketoacidóza / ketoacidosis

zvracení / vomiting

hypochlor(id)emic/ká MAlk

↑ [RA] ≈ ↓ [Cl-]

těhotenství / pregnancy

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Parameters of ABE and ionts

Usually at all time we complete the determinationof ABE parameters by the determinations of ions:

[Na+] (~ 140 mmol . l-1 ) [K+] (~ 4,4 mmol . l-1 ) [Cl-] (~ 100 mmol . l-1 )

The deviation of chlorides from the norm has a basic importance for detection of mixed disorders of ABE. Improved system is an extended evaluation according to Stewart and Fencl. (A simple procedure without computer is introduced.)

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IMPROVED EVALUATION

OF COMBINED DISORDERS OF ABE

(according to Stewart and Fencl)

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The principle of evaluation of parameters of ABE according to Stewart and Fencl : *)

1/ the calculated acide-base parameters [HCO3-] , BE and pH are dependent

on the values of independent valuables (it is on pCO2 , strong ions difference - SID and concentration of weak nonvolatile acids - Atot )

2/ the evaluation of this parameters, replenished with additional calculations of anions (above all with corrected chlorides - [Cl-]correc and unmeasured anions - [UA-]correc ), allows to find mixed disorders of ABE 3/ the changes of values of [Na+] and [Cl-] influence the acid-base state • the changes can result in simple or mixed disorders • their influence on acid-base state can become stronger or abolish • to the good diagnose contributes either calculation of corrected chlorides or (more simple) their reading from the diagram • the distinguishing simple and mixed disorders of ABE has not only the diagnostic, however the therapeutic importance too

*) výslovnost: P. A. STEWART byl Kanaďan ~ steward [,stjuəd] , ale na konci s –t # prof. MUDr. Vladimír FENCL byl Čech

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uses two systems:1/ the increase/decrease of hydrogencarbonate concentration = bicarbonate = [HCO3

-]2/ the values of base excess (BE)

Both systems are significantly limited:1/ first restriction: [HCO3

-], BE and pH event. [H+] are so called dependent variables and they are determined with several (independent) variables in plasma, which can change independently of one another. At the same time can exist influences of acidity or akality. The mixed abnormalities can escape our notice, if their influence on [HCO3

-], BE and pH is mutually canceled.

2/ second restriction: is in the inability to identify the different primary causes of MAc a MAlk. Either BE or [HCO3

-] do not give the directly information about individual primary cause of metabolic disorders.

The diagnostics of MAc / MAlk :

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Independent variablesdetermined the state of ABE :

Dependent variablesdetermined the state of ABE:

pCO2

SID weak nonvolatile acids = [Alb-] + [Pi

-]

pH , [H+] [HCO3

-] , BE

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None from next acid-base variables (it is pH, [HCO3

-], BE ) can be changed primarilly.

They are dependent values („dependent variables“) , which are changed only in dependence on the change of independent variables.

Dependent variablesdetermined the state of ABE:

improved procedure of evaluation of parametersof ABE by: P. A. Stewart (Canada) V. Fencl (Czech Rep.)

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are changed independently of one another

the system ofABE

1/

influence upon the systemfrom outside

X2/

X3/ are independent on the changes inside the system

4/ determinate the dependet variables (Only the changes in the independent variables can change dependent variables!)

dependent variables

[H+], pH[HCO3

-], BE

independent variables:

pCO2 , SID ,[Alb-], [Pi

-]

„weak nonvolatile acids“( It is fully abstract model constructed for the system of acidbase equilibrium! It gives only the relationships among variables. )

Independent variables :

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The procedure of evaluation of ABE parameters (1) :

1/ pH, pCO2, pH – according to Astrup + Siggaard-Andersen [sigurd]

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mmol . l-1

[Na+] 140

[Cl-]correc 100

[UA-]correc 8

[Pi-] 2

[Alb-] 12

The reference values :

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mmol . l-1 acidosis alkalosis

[Na+] 140 +

[Cl-]correc 100 +

[UA-]correc 8 +

[Pi-] 2 +

[Alb-] 12 +

The evaluation in patient (1) :the deviations of patient values from the reference values arefiled to the columns „acidosis“ / „alkalosis“ (according to their signs: „+“ for increase, „“ for decrease)

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mmol . l-1 patient acidose alkalose

[Na+] 140 129 11 +

[Cl-]correc 100 111 + 11

[UA-]correc 8 9 + 1

[Pi-] 2 1,7 + 0,3

[Alb-] 12 1,9 + 10,1

The evaluation in patient (2) :

„hypoalbuminemic MAlk + hyponatremic Ac“

pH = 7,367pCO2 = 5,25 kPaBE = - 2,5 mmol.l-1

= combined metabolic disorder with normal ABE parameters

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„hypoalbuminemic MAlk + hyponatremic Ac“

Why ?

hypoalbuminemia = decrease of one from „weak nonvolatile acids“ decrease is usually compensated by increase of concentration of (alkalic !) hydrogencarbonate MAlk

hyponatremia = consequence of dilution buffer systems are diluted decrease of buffer capacity related to volume at continual metabolic production of acids MAc (hyponatremic Ac = dilutional Ac)

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mmol . l-1 acidosis alkalosis

[Na+] 140 (1) +

[Cl-]correc 100 + (2)

[UA-]correc 8 + (3)

[Pi-] 2 + (4)

[Alb-] 12 + (5)

(1) hyponatremic Ac, „dilutional“ Ac(2) ~ hyperchlor(id)emic Ac ?(3) ~ normochlor(id)emic Ac ?(4 + 5) Ac from „weak nonvolatile acids“

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SID = [HCO3-] + [Alb-] + [Pi-]

„weak nonvolatile acids“

AG = [UA-] + [Alb-] + [Pi-]„weak nonvolatile acids“

[UA-] anions (mainly) of org. acids, completely dissociated

What is the content of constituent data derived from iontogram of plasma ?

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[UA-] anions (mainly) of org. acids, completely dissociated

hypoxia lactate -

ketoacidosis acetoacetate -

β-hydroxybutyrate -

renal insufficiency sulfate2 -

intoxication formiate -

salicylate -

…..

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