WATER BALANCE & ELECTROLYTES

Dehydration
 Pure water loss or deficit.
The daily obligatory losses are shown as follows:  Water and sodium loss
Skin 500 mL
Lungs 400 mL Overhydration
Gut 100 mL  excessive intake of water (polydipsia)
 excessive reabsorption of water such as in
Kidneys 500 mL cases of SIADH and ectopic ADH
Total 1500 mL secretion.

These obligatory losses are compensated by Function of Electrolytes

water taken from the following sources:  Maintenance of osmotic pressure and
Water from oxidative 400 mL hydration e.g., sodium
metabolism  Buffering functions e.g., HCO3
 Activators in enzyme reactions e.g., Mg
Minimum in diet 1100 mL
 Normal neuromuscular excitability e.g., Ca
 Redox reaction (electron transport) e.g.,
Total 1500 mL
Fe

ECF Osmolality Levels of electrolytes in the plasma

 The ECF osmolality is regulated by the (mmol/L)
level of sodium and associated anions
(e.g., HCO3), glucose, urea, and proteins.
 Of these solutes sodium is the major
determinant of plasma osmolality.

ECF Volume
The ECF volume is directly dependent on the
sodium content and is maintained by
 Regulation of renal excretion of sodium or
glomerular filtration rate.
 About 70% of filtered sodium is
reabsorbed by the earlier parts of the
renal tubules.
 Aldosterone via the RAA system. Anion Gap
 Aldosterone  The anion gap refers to the difference
- promotes sodium reabsorption between the sums of the concentrations of
- stimulating the release of potassium the principal cations (e.g., Na and K) and
and hydrogen. of the principal anions (e.g., CI and
HCO3).
  It represents the unmeasured net
negative charge on plasma proteins.
Methods of calculating Anion Gap. The anion
gap (AG) may be measured by any of the
following formula:
1. AG = Na+ - (Cl- + HCO3 -)
NV: 7-14 mmol/L
2. AG = (Na+ + K+)+ - (Cl- + HCO3-)
NV: 10-18 mmol/L
Sodium
 most abundant cation in the extracellular
fluid.
 accounts for about 92% of the osmotically
active solutes in the plasma. Its amount
also determines the ECF volume.
 70% of sodium is freely exchangeable
while 30% is complexed in the bone.
Regulators of Sodium Level
Diet
 Kidney
 renal threshold for sodium is 110-130
mmol/L.
 about 70-80% of filtered sodium is
reabsorbed at the proximal tubule
 RAA
 aldosterone promotes retention of sodium
in exchange of secretion of potassium or
hydrogen ions.
 Atrial natriuretic factor (ANF):
 this cardiac peptide promotes natriuresis
(urinary excretion of sodium) and
relaxation of the vascular smooth muscle
(vasodilatation)
sodium in the extracellular fluid is 135-145
mmol/L in the intracellular fluid, it is within 4-
10 mmol/L
Determination of Sodium
Serum sodium may be measured using
emission flame photometry (EFP), atomic
absorption spectrophotometry (AAS), or ion-
selective electrodes (ISE).
Colorimetric method called Albanese-Lein
 involves combining sodium with zinc
uranyl acetate to produce sodium uranyl
acetate precipitate. Addition of water to
the precipitate produces a yellow
solution.
Potassium
 Potassium is the major intracellular cation.
 It is about 20 times greater in
concentration inside the cells than outside.
 about 90% of potassium is free or
exchangeable
 only 10 % is bound in the red blood cells,
bone and brain tissues.
Normal values of potassium in serum samples are
in the range of 3.8-5.0 mmol/L.
 elevated levels of potassium (>7.5
mmol/L)can seriously inhibit the
irritability of muscles, including the heart
and may lead to paralysis or cessation of
heartbeat
 low serum potassium (<3.0 mmol/L)
may increase muscle irritability and cause
heartbeat during systole.
Measurement of Potassium
 Like sodium, potassium may be measured
by flame photometry, atomic absorption
spectrophotometry and ion-selective
electrode.
 Colorimetric procedure for potassium is
Lockhead and Purcell that uses sodium
cobaltinitrite.
 A blue sodium potassium cobaltinitrite
is produced with addition of phenol
reagent.
Measurement of Potassium
 Turbidimetric assay
 Uses Na tetraphenylboron as reagent
 Production of a colloidal suspension of K
tetraphenylboron
Sodium and Potassium Assay Notes
- anticoagulants which tend to increase
plasma volume e.g., oxalates, lowers
sodium levels.
- blood samples taken after physical
exercise or muscular activity have lower
sodium.
- Water used in the assay must be free of
electrolyte traces and the thumb must not
be used when mixing the tubes since the
skin may contain sodium chloride.
- serum potassium levels are usually higher
than those obtained using plasma samples
due to platelets release potassium during
the clotting process
- among the causes of spuriously high
potassium are:
 Increased platelet count
  Prolonged application of tourniquet
due to juxtavenular cellular injury
production leakage of potassium
 Increased muscular activity e.g.,
repeated or excessive clenching of
fist prior to and during drawing of
blood
 Hemolyzed specimen (greatest
error)
 Contamination with potassium
EDTA
Chloride
 Chloride is the major extracellular anion.
 Maintenance of electrolytes balance
 Hydration
 Maintenance of osmotic pressure
Measurement of Chloride
Chloride may be measured by the ISE method
 membrane of the chloride ISE is a
composite of silver sulfide and silver
chloride
 only bromide can possibly cause
interference.
Coulometric-amperometric method with an
adaptation of this - the Cotlove titrator or
chloridometer.
Zall color reaction has been used in some
semi-automated chloride analyzers
- reagent contains mercuric thiocyanate
and ferric nitrate
- the chloride ions displace the thiocyanate
ions to form soluble but undissociated
mercuric chloride
- releasing in the process an equivalent
amount of thiocyanate
- this reacts with ferric ions derived from
the ferric nitrate to produce reddish
brown ferric thiocyanate
Mercuric nitrate titration method of Schales
and Schales
 the mercuric nitrate forms soluble but
virtually undissociated mercuric
chloride, which does not affect the
indicator diphenylcarbazone
 as soon as all the chloride ions are
thus combined, the next drop of
mercuric nitrate added will cause the
indicator to change to from colorless or
faint pink to violet
 the volume of mercuric nitrate
required to produce the endpoint is a
measure of the amount of chloride
present
CSF Chloride
 normally higher than that of serum
because the protein concentration in
cerebrospinal fluid is low, hence, there are
practically no proteinate anions.
 about 115-132 mmol/L
 falls to about serum levels in cases of
bacterial meningitis when protein
concentration in the CSF is elevated
Sweat Chloride
 screens for cystic fibrosis
 about 50 mg sweat is needed to test for
chloride
 sweat-inducing drug, pilocarpine, is
introduced into a limited area of the skin
by means of an electric current flowing
between two electrodes attached to a limb
(of a child) or the back (on an infant),
technique is called iontophoresis
 normal sweat chloride is about 5-40
mmol/L and most patients with cystic
fibrosis have levels above 60 mmol/L.
Calcium
 Calcium is the most abundant cation in
the body.
 It is 90% bound to the skeleton.
 In the bone, its combines with phosphates
to form the hydroxyapatite crystals which
provide strength to the bone.
 Functions of calcium (Marshall, 1995)
Parathyroid Hormone
 increases blood calcium levels
 secretion is inhibited by hypercalcemia
and calcitriol
Calcitriol or Activated Vitamin D3
 also known as 1.25-didydroxy-
cholecalciferol
 it is stimulated by increased PTH and
decreased phosphate
Calcitonin
 produced by the C cells of the thyroid
gland
 decreases blood calcium level
(hypocalcemic hormone)
 calcium-dye complex, and ethanol to
Actions of parathyroid decrease the absorbance of the blank

Normal serum total calcium (Cat) falls within

the range of 8.5 to 10.4 mg/dL (2.13 to 2.60
mmol/L).
Multiply mg/dL by 0.25 to convert to mmol/L
Ionized calcium (Ca2+) in plasma, serum or
whole blood is within the normal range of 4.68-
5.32 mg/dL (1.17 to 1.33 mmol/L)

Phosphate
 in the body exists only as inorganic
phosphate esters.
 about 80% of the phosphates are
Measurement of Calcium
incorporated into the bone together with
Aside from ISE (reference method), atomic
calcium.
absorption spectrophotometry and flame
 most organic phosphates are present
photometry.
inside the cells as components of
Three approaches
molecules e.g., the DNA, phospholipids,
 Precipitation of calcium as an insoluble
ATP, etc.
compound followed by titration or
 in contrast, most inorganic phosphates
colorimetric methods
are mostly confined in the extracellular
 ammonium oxalate (Clark-Collip)
fluid where they act as buffers.
 chloranilic acid (Ferro-Ham
 excreted principally via the urine.
method)
 phosphate homeostasis is closely linked
 picrolonic acid
with calcium regulation since the same
 Formation of colored complexes calcium
hormones regulate the levels of the two
and a variety of dyes followed by
minerals.
colorimetric determination of the complex
 PTH, for example, stimulates the kidney
 Alizarin
to excrete phosphate while conserving
 O-cresolphthalein complexone
calcium.
 Calcein (Diehl-Ellingboe method)
 Usually, the relationship between calcium
 Ammonium purpurate (Murexide)
and phosphorus is inverse.
 Nuclear fast red
 Removal of calcium from a colored
Measurement of Phosphates
complex by titration with a chelating agent
 Fiske-Subbarow method
 EDTA (ethylene diamine tetraacetic
 protein-free filtrate is prepared using
acid)
trichloroacetic acid
 EGTA (ethylene glyco-bis(2-
 conversion of the inorganic phosphate in
aminoethyl ether)- tetraacetic
the sample to the heteromolybdenum
acid)
blue by a reaction with ammonium
 Dyes
molybdate and the reducing agent,
The endpoint is reached by recovery of the
aminonaphthol sulfonic acid (pictol)
original color of the dye or the disappearance of
 the absorbance of the complex is
the fluorescence of the calcium-dye complex.
measured at 700 nm

 Daly-Ertingshausen method
O-cresolphthalein complexone method
 the inorganic phosphates is converted into
 the dye binds calcium tightly in alkaline
phosphomolybdate polyacid by a reaction
solution to form a highly colored complex
with ammonium molybdate in sulfuric acid
with an absorbance at 578 nm
 precipitation of proteins is prevented using
 the reaction mixture contains 8-
a wetting agent called Tween 80
hydroxyquinoline to bind magnesium
 OD of the phosphomolybdic acid is
and prevent its interference, urea to
measured at 340 nm
decrease the turbidity of a lipemic serum
and increase color intensity of the
 Magnesium
 the 4th most abundant cation in the body
 majority of this mineral is stored in the
bones in complex with calcium and
phosphate
 about 70% of magnesium is free and only
30% is bound to protein
 most of the magnesium in the body is
located within the cell.
 an essential activator of several enzymes
e.g., phosphateses, kinases,
phosphorylases and enolases.
 also necessary in the oxidative
phosphorylation occurring in the
mitochondria.
 therapeutic agent and has an anti-
convulsant laxative and antacid effects
Measurement of Magnesium
Magnesium may be measured by ISE, AAS,
colorimetric or fluorometric analysis.
 Calmagite or 3-hydroxy-4 [(6-
hydroxy-m-toly)azo]-1-naphthalene-
O-sulfonic acid
 in the presence of
polyvinylpyrrolidone (used to
minimize the effects of seram
proteins), a violet complex forms
which absorbs light at 520 nm
 the reagent used contains
amphoteric betaine detergent
Empigen BB to shift the
wavelength of the blank,
strontium chelate to mask the
effect of calcium, and
triethanolamine to mask the
effects of iron.
Methylthymol blue
 complex formed is measured at 510 nm
 used in the DuPont aca analyzer.
Titan yellow
 method is called Dye-Lake method
 magnesium reacts with an alkaline
solution of titan yellow in the presence of
polyvinylpyrrolidone to form a red lake
colloidal precipitate
Fluorometric analysis include the use of the
either hydroxyquinoline or calcein.Normal
values magnesium fall within the range of 1.3 to
2.1 mEq/L or 0.65 to 1.05 mmol/L
Copper
 In the blood, copper is seen in red blood
cells or is bound to transport proteins e.g.,
albumin, and ceruloplasmin.
 Ceruloplasmin is necessary for the
absorption of iron to the ferric state, a
prerequisite for binding by transferrin. It
has a peroxidase activity.
 Copper is important in erythropoiesis
(hemoglobin synthesis) and catalytic
activity of several enzymes e.g.,
cytochrome oxidase and uricase.
 Serum copper may be measured by AAS
 To convert to SI, multiply ug/dL by 0.157
to get values in umol/L
Iron
 Total body iron in humans is
approximately 3-5 g with
 about 70% incorporated in the red blood
cells, and about 25% is found in the
reticuloendothelial system,
incorporated with ferritin and hemosiderin
as store iron
 Two forms of iron in the body are:
 Ferrous iron: found in oxyhemoglobin
and reduced hemoglobin
 Ferric iron: found in ferritin,
hemosiderin, transferrin and met-
hemoglobin
 The two other proteins that are involved in
the transport of iron are:
 Haptoglobin: This binds hemoglobin and
services to facilitate disposal of the iron
from this molecule
 Hemopexin: This binds heme to avoid to
aid its removal from the circulation
Measurement of Iron
 Ferrozine method
 where serum proteins are precipitated in
an acid solution containing thioglycolic
acid that reduces ferric to ferrous ion,
thereby dissociating the iron from its
binding to transferrin
 chromogen ferrozine is then added to
the supernate to form a highly colored
ferrous complex which is measured at 562
nm.
Normal serum iron concentration falls within
65 to 165 ug/dL (11.6-29.5 umol/L) for men
and 45 to 160 ug/fL (8.1-28.6 umol/L) for
women.
Higher values are obtained in the morning due
to diurnal variations.
 Total Iron Binding Capacity (TIBC) and
Transferrin Saturation
 A known amount of ferric ions, more than
sufficient to fully saturate the serum
transferrin with iron, is added to a serum
sample.
 The excess ferric ions, not bound to
transferrin, is removed by addition of a
small amount of buffered ion-exchange
resin.
 The sample is diluted and centrifuged, and
an aliquot of the supernate is analyzed for
iron content of the fully saturated
transferrin - this value is the TIBC.
 The % saturation of transferrin is
measured as follows:
Transferrin Saturation = Serum Fe x 10/TIBC
(% saturation)
 TIBC varies from 260 to 440 ug/dL
(46.5-78.8 umol/L).
 Transferrin saturation ranges from 20-
50%.
;where the brackets represent the molar
concentration. The equation tells that the higher
the hydrogen ion concentration, the higher is the
 Sulfonated bathophenanthroline 2,4,6-
acidity constant.
tripyridyl-s-traiazine (TPTZ)
 Terosite
Sensitive tests for iron
Zinc Protopophyrin/Heme Ratio (ZPP/H)
 excellent screening test for detecting iron
deficiency anemia and for monitoring the
course of therapy.
 hemoglobin in a drop of blood is converted
to cyanmethemoglobin by treatment with
cyanide containing reagent
 portion of the mixture is placed on a cover
slip and introduced into a ProtoFlour
hematofluorometer which measure
simultaneously the light absorbed by the
film of cyanmethemoglobin at 424 nm and
the fluorescent light emitted at 595 nm by
zinc protoporphyrin.
 the results are displayed as a ratio of umol
ZPP/mol heme. The normal ratio is 30 to
80 umol ZPP/mol heme.
 is normal in thalassemia but abnormal in
iron deficiency anemia.
 the ratio is elevated in all types of iron
deficiency syndromes and chronic
exposure to lead.
ACID-BASE BALANCE
 Blood gas analysis routinely involves
analysis of blood gases oxygen and carbon
dioxide, and blood pH.
 The preferred sample if arterial blood
collected in heparinized tubes.
 The normal arterial pH falls within the
range of 7.35 – 7.45 (average of pH 7.4).
This is equivalent to a molar hydrogen ion
concentration of 4.5 x 10-8M to 3.5 x 10-
8M buffer.
 The bicarbonate buffer system is
illustrated as follows:
 H2CO3 and HCO3 act as the conjugate
acid-base pair with the latter acting as the
base. The Ka (acidity constant) for the
equation can be written as follows:
 If the hydrogen ion concentration is to be
solved then the equation becomes
 Since pH = –log [H+], then;
 pH = - log Ka – log [H2CO3]/[HCO3- ]
Equation 4
 pH = [HCO3- ]/pKa + log [H2CO3]
Equation 5
 pH = pKa + log [HCO3- ]/[H2CO3]
 This is called the Henderson-
Hasselbalch equation.
 The pKa is a constant and it depends on
the buffer involved.
 The pKa is the pH at which the molar
concentration of the acid is equal to the
molar concentration of its conjugate base.
 It is at this pH where the system exerts its
maximum buffering activity. Usually the
range of pH at which a buffer is effective
is within pKa + 1 pH unit.
 The bicarbonate buffer has a pKa of 6.1
which means that it is effective in
maintaining the pH of a solution within the
range of pH 5.1 to pH 7.1.

Given a pCO2 of 44 mmHg and total CO2 of 29
mmol/L. Solve for the pH.
 first, convert pCO2 in mmHg to dissolved
In the peripheral tissues
 the bicarbonate diffuses out of the red
blood cell, to maintain electrical neutrality,
this diffusion of bicarbonate is
accompanied by as shift of chloride into
the red blood cells (chloride shift) which is
mediated by a transport protein located in
membrane of the red blood cell.
In addition to the respiratory component of the
acid-base balance, the levels of bicarbonates in
the blood is also closely regulated by the kidney.
 bicarbonates are readily filtered in the
glomerulus but it is absorbed in the
proximal tubule especially when a lot of
the base needed e.g., in cases of acidosis.
 this mechanism forms the metabolic
component of the acid-base balance.
In summary, acid-base balance is controlled by
chemical buffers primarily bicarbonate, the lungs
and the kidney. It can be represented as follows:
[HCO3-] which is a function of the kidney
(metabolic component)
pH =
[H2CO3] which is a function of the lungs
(respiratory component)
Normally, the levels of bicarbonate and carbonic
acid are maintained at a ratio of 20:1.
 Total CO2 consists of the HCO3 ,
undissociated H2CO3, dissolved CO2, and
carbamino-bound CO2.
 The bicarbonate is by far the largest
(~95% of the total) and accounts for all
but approximately 2 mmol/L of the CO2
content.
 The carbamino fraction is negligible in
serum, but is appreciable in whole blood
because of the presence of hemoglobin.
 Generally, the CO2 content is measured
by automated methods or using a CO2
electrode.
 Routinely, the total CO2 content is
assumed to be equal to the sum of the
dissolved CO2 and bicarbonate.
 This can be expressed in mmol/L. The
normal value of total CO2 is 23 – 27
mmol/L.
pCO2 or dissolved CO2
 constitutes about 5 – 10% of the total
CO2 content usually expressed in mmHg
 the normal value of 35 – 45 mmHg.
CO2 by multiplying the solubility
coefficient of CO2 gas which is a constant
(0.03 mmol/L/mmHg) i.e.,
 then determine the HCO3 concentration
by finding the difference between total
CO2 and dissolved CO2 concentration
29 mmol/l – 1.32 mmol/L = 27.68 mmol/L
 Calculate the pH as follows:
Physiologic Buffers
 Bicarbonate Buffer.
 Hemoglobin
- this is a major buffer localized inside the
red blood cells.
- in the peripheral tissues, carbon dioxide
accumulates as a waste product of
metabolism.
- as the pressure of carbon dioxide
increases in the plasma, the gas diffuses
into the red blood cells where it reacts
with water to form carbonic acid as
catalyzed by carbonic anhydrase (also
known as carbonic dehydratase).
- the carbonic acid readily splits into
hydrogen ions and bicarbonate.
- the hydrogen ion combines with
hemoglobin which then releases the
oxygen for the tissues. Hemoglobin
therefore can also act as buffer.
 Phosphate Buffer
 This buffer system has a minor role in the
blood. Instead, along with plasma
proteins containing especially the amino
acid glutamine, it is important for the
excretion of hydrogen ions in the kidney.
The conjugate acid-base pair of this buffer
is shown as follows:
 The oxygen-hemoglobin dissociation curve

 The pKa of this reaction is 6.8.

 Plasma Proteins. The amino acids
present in the proteins are amphoteric
and they can act as buffer.

Transport of Blood Gases

 The transport of oxygen and carbon
dioxide back and forth the lungs and the
peripheral tissues is a function of
hemoglobin in red blood cells.
Hemoglobin exists in two forms:
 “T” or taut structure (deoxyhemoglobin)
which has a low affinity for oxygen.
 “R” or relaxed structure
(oxyhemoglobin) which has high affinity
for oxygen.
 the curve is sigmoid in shape
 exhibits cooperative effects, in that, at
lower oxygen tension, the affinity of
hemoglobin with oxygen is very small,
however, once the hemoglobin molecule
has started binding with oxygen, its
affinity for the succeeding oxygen
molecules increases

Comparison of arterial and venous blood

Assay Notes
 Arterial blood is required for pO2
 Renal hydrogen ion excretion by the measurement.
phosphate and protein buffers.  Venous blood may be taken for pH and
pCO2 if it is drawn without stasis (no
In the peripheral tissues where the oxygen tourniquet) and without the patient
tension is very low because oxygen is utilized clenching the fist.
during metabolism, the hemoglobin molecule  “Arterialized” venous blood may be
exists in the taut form in order to prevent uptake obtained by heating the hand and forearm
of the delivered oxygen. in water at 45oc for 5 minutes and then
In contrast, the relaxed form is favored in the drawing blood from the dilated veins on
lungs where oxygen tension is very high. This the back of the hand.
allows uptake of oxygen by the hemoglobin  When the blood sample is left in open air,
molecule. carbon dioxide diffuses from the blood to
The affinity of hemoglobin with oxygen depends the surrounding air, reducing the pCO2 in
on many factors. This can be shown with the the blood thereby increasing the pH.
oxygen-hemoglobin dissociation curve.  In contrast, oxygen diffuses from the air
into the blood since pO2 in air is greater
than that in whole blood.
Measurements of blood pH, pCO2 and pO2 may
be done simultaneously in a blood gas
instrument. The pH is measured by a micro glass
electrode.
pCO2 is measured by a Severinghaus electrode
while pO2 is measured by the Clark electrode.
Natelson-Van Slyke method is an example of
a gasometric method for carbon dioxide.

Disorders of Hydrogen Ion Homeostasis

 When the bicarbonate level is primarily
defective, the condition is referred to as
metabolic in nature.
 If the level of carbonic acid is primarily
defective, the condition is classified as
respiratory in nature.

Metabolic Acidosis
 bicarbonate is very low resulting in a low
pH
 can be compensated for by the lungs by
hyperventilation lowers the carbonic acid
level restoring the pH.

Metabolic Alkalosis
 bicarbonate is very high resulting in high
pH
 can be compensated for by the lungs
hypoventilation which increases carbon
dioxide in the blood.

Respiratory Acidosis
 seen when the carbonic acid levels are
very high.
 can be compensated for by the kidneys by
reabsorb a lot of bicarbonates to restore
the pH of the blood.

Respiratory Alkalosis
 occurs when the level of carbonic acid is
very low.
 compensated for by the kidneys by
allowing more excretion of bicarbonates in
the kidney