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1
„INTERNAL
ENVIRONMENT“
Claude Bernard
1813 - 1878
© Department of Biochemistry (V.P.), Faculty of Medicine, MU Brno 2009
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3
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)
4
Internal environment = ECF
(extracellular fluid)
external environment
ECF
cell
constancy
of internal
environment
5
Internal environment = ECF
(extracellular fluid)
external environment
acid-base regulation
= acid-base metabolism
(action)
ECF
cell
constancy
of internal
environment
acid-base equilibrium
(„ABE“) (state)
6
Carbonatedehydratase
= carboanhydr(at)ase)
= carbonate hydrolyase :
CO2 + H2O
H2CO3
erythrocyte
kidney
carbonate hydro-lyase EC 4.2.1.1
[laieis]
7
PUFRY
BUFFERS
8
Buffer
KA
(weak acid)
HA
H+
+
A-
(salt of weak acid)
BA
B+
+
A-
A weak acid = weak electrolyte  fractional dissociation only
reversible (two-way) reaction
A salt = strong electrolyte  dissotiation practically complete,
non-reversible (one-way) reaction
9
KA = [H+] . [A-] / [HA]
Keq = [H+] . [A-] / [HA] . [H2O]
10
Buffer – the reaction with an acid :
KA
(weak acid)
HA
H+
+
A-
(salt of weak acid)
BA
B+
+
AH+
Cl-
11
Buffer – the reaction with an acid :
KA
(weak acid)
HA
H+
+
A-
(salt of weak acid)
BA
B+
+
AH+
Cl-
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
12
Buffer – the reaction with a base :
KA
(weak acid)
HA
H+
+
A-
(salt of weak acid)
BA
B+
+
A-
Na+ OH13
Buffer – the reaction with a base :
KA
(weak acid)
HA
H+
+
A-
(salt of weak acid)
BA
B+
+
A-
H+
H2O
Na+ OH14
The titration
curve and
ability to
buffer
„pKA ± 1“
15
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
16
:
Remark:
[H+] is here given in nmol . l-1 (it is 10 –9 mol . l-1) ,
- do not mistake for mmol . l-1 , which
comprise million times higher concentration !!!
[H+] (nmol . l-1) = 10 (9 – pH)
pH = 9 – log [H+] (nmol . l-1)
17
Boundary values of pH (the whole blood)
pH = 7,40
[H+]  40 nmol . l-1
normal value
pH = 6,80
pH = 7,70
[H+]  160 nmol . l-1
[H+]  20 nmol . l-1
4multiple of normal [H+]
½ of normal [H+]18
:
The extreme values of pH
compatible with life
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 tolerance for acidemia (acidosis) is considerably higher,
therefore alkalemia (alkalosis) is of greater danger
19
CO2 in blood plasma
CO2 + H2O
H2CO3
800 mol
1 mol
H+ + HCO3-
0,03 mol
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 !
20
Carbonic acid in plasma:
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 )
21
Henderson – Hasselbalch equation
for HCO3- / H2CO3 in blood plasma :
pH = pK a + log
cs
ca
HCO3-
pH = pK H2CO3 + log
CO2 + H2CO3 
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HCO3-
pH = pK H2CO3 + log
CO2 + H2CO3 
HCO3-
pH = 6,10 + log
0,230 * pCO2
27
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) 28
The principle of ABE parameters determination :
calculated
HCO3-
pH = 6,10 + log
0,230 * pCO2
measured
29
pCO2 a pO2 cell
(„electrode“)
METHODS OF A „DIRECT
MEASUREMENT“
(not the Astrup´s method!)
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 O22- (formation of peroxide, the polarografic principle:
electric current – proportionate to pO2 - is measured
between Pt cathode and Ag / AgCl anode in phosphate
30
buffer).
PARAMETERS
of ABE
31
Basic parameters of ABE:
pH
= 7,40
 0,05
pCO2 = 5,33
 0,5 kPa
BE
 3 mmol . l-1
= 0
____________________________________________________
BE = base excess [beis ik´ses]
= „výchylka nárazníkových bazí“,
„výchylka pufrových bazí“ - původní význam „nadbytek
bazí“ zanikl spolu s pojmem
32
„base deficit“, BD
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
33
- some of them can be „actual“ and others „corrected“ !!
Parameters of ABE
act HCO3std HCO3std BE
=
=
=
actual / standard
24  3 mmol . l-1
24  3 mmol . l-1
0  3 mmol . l-1
(Under identical conditions [HCO3-] = 24 mmol . l-1
corresponds to the BE = 0 mmol . l-1 )
34
[HCO3-]
vs. BE :
[HCO3-] mmol/l … 21 22 23 24 25 26 27 …
BE
mmol/l … -3 -2 -1 0 +1 +2 +3 …
35
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 !!
36
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 °C
4/ sample of whole blood
(„anaerobic collection“)
37
Corrected parameters of ABE
are recalculated for the normal pCO2
38
Replenish data:
• pO2 = 9 – 15 kPa (age dependence)
• saturation of Hb with oxygen
= 0,95 – 0,98
• forms of Hb not transporting oxygen
39
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)
40
BBp = buffer base (plasma)  42 mmol . l-1
24 mmol . l-1 HCO3-
16 mmol . l-1 protein2 mmol . l-1
42
všechny ostatní pufrované báze
all other buffer bases
BBb = buffer base (blood)  48 mmol . l-1
41
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
42
of erythrocytes too).
Imidazole nucleus of His :
+
N
HN
N
H
+ H+
N
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).
43
Remark:
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-]
44
nepřímá měření
od 70./80. let
UŽ NE !!
indirect measurements
since 70th/80th years
NOT ANY
MORE !!
45
Sodium hydrogencarbonate
(„bicarbonate“) is alkalic :
NaHCO3 + H2O
H2CO3 + Na+ + OH-
(The carbonic acid in oval symbolizes a weak - practically undissociated
electrolyte. 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.)
46
Buffer capacity :
Buffer system
HCO3-/H2CO3 + CO2
IVF
whole blood erythrocytes
plasma
50 %
ISF
ICF
HCO3- HCO3-
17 %
33 %
Protein/HProtein
45 %
-
27 %
proteins
18 %
HPO42-/H2PO4-
5%
(inorg.)
1%
Concentration of buffer 48 ± 3
systems ( mmol . l-1 )
42 ± 3
3 % (org.)
1 % (inorg.)
~ 56
inorg.
phosphate
org.
phosphate
„interaction reaction“
among buffer systems
47
BBb
BBp
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
48
The buffers in different compartments:
buffer
Ery
plasma
ISF
ICF
bicarbonate











Hb
phosphate
protein
Ery = erythrocyte
ISF = interstitial fluid
ICF = intracellular fluid
49
acidémie
Pufry v bb.
Buffers in
the cell
alkalémie
(směna H+ a K+)
(the change H+ for K+)
50
51
PORUCHY ABR
ACID-BASE DISORDERS
52
Anaerobic sampling of blood (1) :
from the heel
in infants
„arterialized“ capillary blood
fron the ear lobe / finger
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
53
Anaerobic sampling of blood (2) :
closing
of capillary
heparinizing
of blood
54
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+]
55
The classification of ABE disorders (1):
„acidosis“ (pH  7,36)
metabolic disorder
HCO3-
pH = pK H2CO3 + log
„alkalosis“ (pH  7,44)
CO2 + H2CO3 
respiratory disorder
56
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 )
57
40-80 (~ 60)
mmol . d-1
mmol
vs. mol !!
( 20.000 / 60 = 333, …)
( 99,7 % vs. 0,3 % )
15-25 (~ 20)
mol . d-1
58
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)
59
[acid]
Henderson – Hasselbalch equation
pH = 7,40
[H2CO3 + CO2]
~ pCO2
pCO2 = 5,33 kPa
*
normal values
BE = 0
[salt]
[HCO3-] ≈ BE
60
normal
values
61
Evaluation of a disturbance and its compensation
 overall evaluation (pH, pCO2 a pH)*)
- by Astrup + Siggaard-Andersen [sigurd]
62
*)
from this only pCO2 is an independent variable, influencing the state of ABE
Primary metabolic disorder :
M ETAB O LI C
DISORDERS
63
Primary respiratory disorder :
R
E
S
P
I
R
A
T
O
R
Y
64
Akutní poruchy ABR (modře)
Acute disorders of ABE (in blue)
-
-
-
-
-
65
Kompenzované poruchy ABR (červeně)
Compensated disorders of ABE (in red)
-
-
-
-
66
Nomenclature
disorder
compensation
correction
-
-
-
-
-
67
Aktuální a standardní BE
Actual and standard BE
-
-
-
-
-
68
Pozor na metabolickou alkalózu !! Mind the metabolic alkalosis
!!
Časové hledisko úpravy poruch ABR :
„setrvačnost !!“
69
The liver and ABE :
1/ acidemia:
NH3

glutamin (Gln)
(transport to kidneys,
releasing NH4+ by glutaminase ...)
2/ alkalemia: NH3
 urea
70
The liver and ABE – acidemia :
two ways of NH3 elimination in the liver
liver
kidney
urine
71
The liver and ABE - alkalemia (1)
+
NH 4
HCO 3
NH 4+
NH 2
+ 2 H 2O
C
O
+ H+
NH 2
( In the contrary, in acidemia organism saves (basic) bicarbonates:
→ in acidemia the synthesis of urea will be reduced.)
72
The liver and ABE - alkalemia (2)
+
NH 4
HCO 3
NH 4+
alkalic
part
NH 2
+ 2 H 2O
C
O
+ H+
NH 2
acidic
part
73
The liver and ABE - alkalemia (3)
+
NH 4
HCO 3
NH 4+
NH 2
+ 2 H 2O
C
O
+ H+
NH 2
MAlk
correction of
MAlk
74
The kidney and ABE :
tubular cell
tubular lumen
antiport
symport
75
Kombinovaná porucha ABR
Mixed disorder of ABE
0
MAc
těhotenství / pregnancy
MAlk
zdánlivě normální stav
seemingly normal state
hlad / starvation
zvracení / vomiting
ketoacidóza / ketoacidosis
↑ [RA]
hypochlor(id)emic/ká MAlk
≈ ↓ [Cl-]
76
Parameters of ABE and ionts
Usually at all time we complete the determination
of ABE parameters by the determinations of ions:
[Na+]
[K+]
[Cl-]
(~ 140 mmol . l-1 )
(~ 4,4 mmol . l-1 )
(~ 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
77
procedure without computer is introduced.)
IMPROVED EVALUATION
OF COMBINED DISORDERS OF ABE
(according to Stewart and Fencl)
78
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
79
*)
výslovnost: P. A. STEWART byl Kanaďan ~ steward [,stjuəd] , ale na konci s –t # prof. MUDr. Vladimír FENCL byl Čech
The diagnostics of MAc / MAlk :
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.
80
Independent variables
determined the state of ABE :
 pCO2
 SID
 weak nonvolatile acids = [Alb-] + [Pi-]
Dependent variables
determined the state of ABE:
 pH , [H+]
 [HCO3-] , BE
81
Dependent variables
determined the state of ABE:
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.
improved procedure of evaluation of parameters
of ABE by:
P. A. Stewart (Canada)
V. Fencl (Czech Rep.)
82
Independent variables :
2/
are changed
independently of
one another
X
1/
influence
upon the system
from outside
X
dependent variables
[H+], pH
[HCO3-], BE
independent variables:
pCO2 , SID ,
[Alb-], [Pi-]

„weak nonvolatile acids“
the system of
ABE
3/ are independent on
the changes
inside the system
4/
determinate the
dependet variables
(Only the changes in the independent
variables can change dependent variables!)
( It is fully abstract model constructed for the system of acidbase equilibrium!
It gives only the relationships among variables. )
83
The procedure of evaluation of ABE parameters (1) :
1/ pH, pCO2, pH – according to Astrup + Siggaard-Andersen
[sigurd]
85
The reference values :
mmol . l-1
[Na+]
[Cl-]correc
140
100
[UA-]correc
8
[Pi-]
[Alb-]
2
12
88
The evaluation in patient (1) :
the deviations of patient values from the reference values are
filed to the columns „acidosis“ / „alkalosis“
(according to their signs: „+“ for increase, „“ for decrease)
mmol . l-1 acidosis alkalosis
[Na+]
[Cl-]correc
140
100

+
+

[UA-]correc
8
+

[Pi-]
[Alb-]
2
12
+
+


89
The evaluation in patient (2) :
pH
= 7,367
pCO2 = 5,25 kPa
BE
= - 2,5 mmol.l-1
= combined metabolic disorder with normal ABE parameters
mmol . l-1 patient acidose alkalose
 11
+ 11
+

9
+

1,7
1,9
+
+
[Na+]
[Cl-]correc
140
100
129
111
[UA-]correc
8
[Pi-]
[Alb-]
2
12
1
„hypoalbuminemic MAlk
+ hyponatremic Ac“
 0,3
 10,1
91
Why
„hypoalbuminemic MAlk
+ hyponatremic Ac“
?
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)
92
mmol . l-1 acidosis alkalosis
[Na+]
[Cl-]correc
140
100

(1)
+
(2)
+

[UA-]correc
8
+
(3)

[Pi-]
[Alb-]
2
12
+
+
(4)
(5)


(1) hyponatremic Ac, „dilutional“ Ac
(2) ~ hyperchlor(id)emic Ac ?
(3) ~ normochlor(id)emic Ac ?
(4 + 5) Ac from „weak nonvolatile acids“
93
What is the content of constituent data
derived from iontogram of plasma ?
SID =
[HCO3-] + [Alb-] + [Pi-]

„weak nonvolatile acids“
AG = [UA-] + [Alb-] + [Pi-]

„weak nonvolatile acids“
[UA-] 
anions (mainly) of org. acids,
completely dissociated
94
[UA-]  anions (mainly) of org. acids,
completely dissociated
 hypoxia  lactate  ketoacidosis  acetoacetate β-hydroxybutyrate  renal insufficiency  sulfate2  intoxication  formiate salicylate  …..
95
100