Electrolyte Methodology Lecture Guide
Electrolyte Methodology Lecture Guide
Electrolyte Methodology Lecture Guide
1 Sodium (Na)
2 Potassium (K)
3 Chloride (Cl)
4 Bicarbonate (HCO3)
5 Magnesium (Mg)
6 Calcium (Ca)
7 Phosphate (PO3)
8 Lactate (C3H6O3)
SODIUM
Reference Ranges
serum, plasma = 136-145mmol/L (mEq/L)
24-hour urine = 40-220mmol/day (varies with diet)
CSF = 136-150mmol/L
Specimen
serum, plasma
: plasma must be obtained with lithium heparin, ammonium heparin and lithium oxalate
: hemolysis does not cause significant change in serum or plasma values as a result of
decreased levels of intracellular Na+
: whole blood may be used with some analyzers, check operations manual for
acceptability
24-hr collection is the preferred urine specimen
sweat is also suitable for analysis
Methods
Chemical or Colorimetric
: outdated because of large sample volume requirements and lack of precision
1. Albanese-Lein
Principle. Na+ is made to react with zinc uranyl acetate to produce sodium uranyl
acetate precipitate after the addition of polyvinyl alcohol. With the addition of water, a yellow
solution is formed which is then measured spectrophotometrically.
Reagents and Result
Procedure:
blank sample/calibrator
R1 900µL 900µL
R2 300µL 300µL
POTASSIUM
Reference Ranges
serum = 3.5–5.1 mmol/L (mEq/L)
plasma: Males = 3.5–4.5 mmol/L
Females = 3.4–4.4 mmol/L
urine (24 hour) = 25-125mmol/day
Specimen
serum, plasma
: hemolysis must be avoided because of high K+ content of RBCs
: heparin is anticoagulant of choice
: both give similar levels but the serum reference intervals tend to be slightly higher
: significantly elevated platelet counts may result in the release of K+ during clotting from
rupture of these cells causing spurious hyperkalemia
: whole blood samples may be used with some analyzers
urine
: 24-hour urine collection is used to eliminate the influence of diurnal
variation
Methods
ISE* current method of choice
: a valinomycin membrane is used to selectively bind K+, causing an impedance change
that can be correlated to K+ concentration
: KCl is the inner electrolyte solution
Colorimetric/Chemical Method
1. Lockhead and Purcell
Principle. K+ is reacted with sodium cobaltinitrite to produce sodium potassium
cobaltinitrite. With the addition of phenol, a blue color is produced and determined
spectrophotometrically.
Reagents and Result
: sodium cobaltinitrite
: sodium acetate
: glycine
: sodium carbonate
: phenol
: blue (end color)
Potassium FS
Method: enzymatic photometric test
Principle: PK is activated by K+ ions in the sample and subsequently catalyzes the
dephosphorylation of phosphoenolpyruvate to pyruvate. In a second step, pyruvate is
transformed to lactate under consumption of a NADH analogue. The signal decrease measured
at 340nm is proportional to the amount of K+ in the sample.
R1: buffer, NADH, PEP, ADP, LD
R2: buffer , PK
PK
ADP + PEP ATP + PYRUVATE
LD
+
PYRUVATE + NADH + H LACTATE + NAD
Procedure:
blank sample/calibrator
Sample/calibrator ---- 100µL
Distilled Water 100µL ----
R1 1000µL 1000µL
Mix, incubate for 5 minutes at 37°C
R2 250µL 250µL
Mix, incubate for 5 minutes at 37°C, read Abs A1 after 2 minutes and start
stopwatch. Read Abs A2 after 1, 2 and 3 minutes at 37°C, and calculate
ΔA/min.
CHLORIDE
Reference Ranges
plasma, serum: 98-107mmol/L
urine (24-hour): 110-250mmol/day (varies with diet)
Specimen
serum or plasma may be used
: lithium heparin as anticoagulant of choice
: hemolysis does not cause significant change in serum or plasma values as a result of
decreased levels of intracellular Cl¯
: marked hemolysis may cause levels to be decreased as a result of dilutional effect
: whole blood may be used with some analyzers
urine
: 24-hour collection is needed because of the large diurnal variation
sweat
Methods
ISE*
ion-exchange membrane is used to selectively bind Cl¯ ions
: solid-state electrode using membranes composed of AgCl**
: in the presence of Cl¯ anions, an oxidation-reduction reaction occurs, silver metal
forms Ag+ cation and electrons
: change in the potential is detected electronically and is taken as a reflection of
the ion concentration***
Amperometric-Coulometric Titration, aka Potentiometric Titration
uses coulometric generation of silver ions which combine with Cl¯ to quantitate chloride
ion concentration
: Ag ions are generated by an electrode system immersed in a solution containing
dilute HNO3 with the sample to be tested. Ag + ions will combine with all Cl¯ available until free
Ag+ ions (unreacted) appear in the solution. The presence of free Ag + ions results in a change in
potential. Such change cause a timer to turn off and automatically stop the titration. The length
of time the titrator generates Ag+ is directly proportional to serum Cl¯ concentration.
when all patient Cl¯ ions are bound to Ag ions, excess or free Ag ions are used to
indicate the endpoint as Ag ions accumulate, the coulometric generator and timer are turned
off the elapsed time is used to calculate the concentration of Cl¯ ions in the sample
: Cotlove Chloridometer/Titrator (Buchler Instruments) uses this principle in Cl¯ analysis
Mercurimetric Titration
1. Schales and Schales
Principle. Cl¯ in the sample combines with the added Hg ++ to form the soluble HgCl2
complex. Excess unreacted added Hg++ combines with an indicator such as
diphenylcarbazone to form a blue violet/purple end point of titration.
Reagents
: mercuric nitrate (Hg(NO3)2; titrant)
: 1% diphenylcarbazone (indicator)
: 1N HNO3 (acidifies medium and render HgCl2 more soluble)
: ethyl ether
: chloride standard (100mEq/L; 0.1N HCl, 100mEq/L NaCl)
Procedure
flasks (std, test)
into each flask, place: 2mL H2O, 0.2mL standard/ serum, 1 drop indicator, 2mL ether
titrate with Hg(NO3)2 to violet end point (end point should persist in the solution)
note the volume of titrant used in both flasks
Colorimetry
1. CHLORIDE liquicolor, Photometric colorimetric test, TPTZ method
Principle. Cl¯ ions react with a mercury (II)-2,4,6-tri-(2-pyridil)-s-triazine (TPTZ) complex
to form mercury (II)-chloride. The liberated TPTZ reacts with iron (II) ions yielding a blue colored
complex. The resulting Abs change at 590nm is directly proportional to the amount of Cl¯ ions
in the sample.
Reagents
colour reagent
: 2,4,6-tri-(2-pyridil)-s-triazine [TPTZ; partially as mercury(II) complex]
: iron (II) sulfate
chloride standard (100mmol/L or 355mg/dL)
Sweat Test
single most accepted common diagnostic tool for the clinical identification of CF
normally, the coiled lower part of the sweat gland secretes a “presweat” upon
cholinergic stimulation. As the presweat traverses the ductal part of the gland going through
the dermis, various constituents are resorbed.
In CF, the electrolytes, most notably Cl¯ and Na ions, are improperly resorbed owing to a
mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which
controls a cyclic AMP–regulated Cl¯ channel
a standard method has been suggested by the Cystic Fibrosis Foundation based on the
pilocarpine nitrate iontophoresis method of Gibson and Cooke
: process of using electricity to force the drug into the skin for the purpose of
inducing sweating
: pilocarpine is a cholinergic-like drug used to stimulate the sweat glands
sweat is absorbed on a gauze pad during the procedure
after collection by iontophoresis, Cl¯ analysis is performed
generally, sweat is leached into a known volume of distilled water and analyzed for Cl¯
(chloridometer)
in general, values greater than 60 mmol/L are considered positive
Chloride 21 FS
Method: photometric test using ferric perchlorate
Principle: Cl¯ forms with ferric ions a yellow complex whose Abs is measured at 340nm.
A discoloring agent in R2 displaces Cl¯ from the complex thereby removing color from the
solution. The difference in Abs between the colored and discoloured state of the solution is
proportional to the concentration of Cl¯ in the sample.
R1: methanesulfonic acid, ferric perchlorate
R2: inorganic salt
Procedure:
blank sample/calibrator
Sample/calibrator ---- 40µL
Distilled Water 40µL ----
R1 900µL 900µL
Mix, incubate for 5 minutes at 37°C, read Abs A1, then add
R2 225µL 225µL
Mix, incubate for 1 minute at 37°C, then read Abs A2. Read against reagent blank.
BICARBONATE
Reference Ranges
serum, plasma:CO2,venous 23 to 29 mmol/L
Specimen
venous serum or plasma determinations
: serum or lithium heparin plasma is suitable for analysis
: specimens should be anaerobic for the highest accuracy, many current analyzers
(excluding blood gas analyzers) do not permit anaerobic sample handling
Methods: CO2 measurements may be obtained in several ways; however, the actual
portion of the total CO2 being measured may vary with the method used
ISE
measures total CO2 using an acid reagent to convert all the forms of CO2 to CO2 gas and
is measured by a pCO2 electrode
enzymatic
-method alkalinizes the sample to convert all forms of CO2 to HCO3¯
-HCO3¯ is used to carboxylate phosphoenolpyruvate (PEP) in the presence of PEP
carboxylase, which catalyzes the formation of oxaloacetate
PEP carboxylase
Phosphoenolpyruvate + HCO3¯ Oxaloacetate + H2PO4¯
-coupled to this reaction, in which NADH is consumed as a result of the action of malate
dehydrogenase (MDH) MDH
+
Oxaloacetate + NADH + H Malate + NAD+
-of change in absorbance of NADH is proportional to the concentration of HCO 3¯
MAGNESIUM
Reference Ranges
serum, colorimetric: 0.63-1.0mmol/L (1.26–2.10 mEq/L)
Specimen
nonhemolyzed serum or lithium heparin plasma
: Mg++ concentration in RBC is 10x > than that in ECF, therefore, hemolysis should be
avoided and serum should be separated from clot as soon as possible
: serum is preferred over plasma because anticoagulants interfere with most procedures*
urine
: 24-hour is preferred because of a diurnal variation in excretion
: urine must be acidified with HCl to avoid precipitation
Tests:
1. total Mg++
: usually measured by photometry
: reference method for total Mg++ is AAS
: most labs use a photometric method on an automated analyzer
2. ionized (free) Mg++
: can be measured with Mg++ ISEs that have been incorporated into several
commercial clinical analyzers
: employ a neutral carrier ionophore that is selective for Mg++
: these ISEs also measure Ca++ thus requiring a chemometric correction to
calculate the true free Mg++ levels in the sample
Three most common dyes for colorimetric measurement of total serum Mg ++
: these methods use metallochromic indicators or dyes that change color upon
++
binding Mg from sample
: some of the chromophores used include calmagite, methylthymol blue,
formazan dye and magon
calmagite
Mg++ binds calmagite in alkaline solution to form a reddish-violet complex that be read
at 520nm
formazan dye
: Mg++ binds with the dye to form a colored complex that may be read at 660nm
methylthymol blue
: Mg++ binds with the chromogen to form a colored complex
Methods
Dye-Lake Method (Titan Yellow)
Principle. A TCA filtrate is treated with a titan yellow dye (methylbenzothiazide diazo
amino bensol disulfonic acid) in an alkaline solution. The red lake formed is adsorbed at the
surface of the Mg++ particles which are kept in solution with theaddition of polyvinyl alcohol.
Titan yellow/ Clayton/ thiazole yellow – dye used
Fluorometric and Complexometric Method
Principle. Mg++ ions and 8-hydroxyquinolone sulfonic acid reacts to form a fluorescence.
Ca , which will interfere in the determination of Mg++, is eliminated by complexing with
++
: most methods use a Ca++ shelter to prohibit interference from this divalent cation
: limitations
= because approximately 25% of Mg++ is protein bound, total Mg++ may not reflect the
physiologically active free ionized Mg
= because Mg++ is primarily an intracellular ion, serum concentrations will not necessarily
reflect the status of the intracellular Mg++ even when tissue and cellular Mg++ is depleted by as
much as 20%, serum Mg++ concentrations may remain normal
CALCIUM
Reference Ranges
total Ca++ (serum, plasma)
child: 2.2-2.7mmol/L (8.9-10.8mg/dL)
adult: 2.15-2.5mmol/L (8.5-10mg/dL)
++
ionized Ca (serum)
child: 1.20–1.38 mmol/L (4.8–5.5 mg/dL)
adult: 1.16–1.32 mmol/L (4.6–5.3 mg/dL)
++
ionized Ca (plasma)
adult: 1.03–1.23 mmol/L (4.1–4.9 mg/dL)
++
ionized Ca (whole blood)
adult: 1.15–1.27 mmol/L (4.6–5.1 mg/dL)
urine (24hr): 2.5-7.5mmol/day (100-300mg/day; varies with diet)
Specimen
preferred specimen for total Ca++ is either serum or lithium heparin plasma collected
without venous stasis
Methods
Precipitation and Redox Titration (Clark Collip Precipitation Method)
Principle. Ammonium oxalate is added to the diluted serum sample wherein Ca ++ is
precipitated as calcium oxalate. The precipitate is then washed with diluted ammonium
hydroxide to remove excess oxalates. This prevents interference of Mg++ which precipitates with
excess oxalates to form magnesium oxalate.
The calcium oxalate is then dissolved in sulfuric acid forming oxalic acid and is titrated
with a standardized potassium permanganate (KMnO4). The appearance of a purple color is the
end point.
Colorimetric
: methods employed in automated analyzers which are based on color complex formation
between Ca++ and dyes such as o-cresolphthalein complexone, alizarin, arsenzo III dye at pH 5.5,
calcein, murexide (ammonium purpurate), and nuclear fast red
1. o-cresolphthalein complexone dye (Baginski, Marie, Clark and Zak)
Principle. Serum is mixed with 0.3M HCl to dissociate Ca++ from proteins then dialyzed
into a reagent stream containing o-cresolphthalein complexone and hydroxyquinolone in
diluted HCl. Hydroxyquinolone is added to bind magnesium which otherwise cause
interference. A colored complex between Ca ++ and the dye is formed and maintained after the
addition of diethylamine buffer.
2. EDTA Titration Method (Bachra, Dauer and Sobel)
Principle. A diluted serum sample is titrated with EDTA in the presence of an indicator
(calcein red) at an alkaline pH (this prevents Mg interference). The initial yellow green
fluorescence caused by the Ca++-calcein complex changes to a nonfluorescent salmon pink color
(free calcein) when all the Ca++ present has been chelated with EDTA.*
Reagents and Result
: calcein red (indicator)
: Versene (EDTA; chelating agent)
: KOH (for alkalinity)
: Ca-EDTA complex (end product)
Precautions (EDTA titration Method)
: method is susceptible to interferences by copper, zinc, iron and drugs such as
sulfadiazine, heparin and acetylsalicylic acid
AAS
: in terms of accuracy, precision and speed, this determination is the method of
choice for routine analysis and reference procedure
Flame Emission Photometry
Ca ISE
: system may use membranes impregnated with special molecules that selectively
but reversibly bind Ca ++ ions
: as Ca ++ ions bind to these membranes, an electric potential develops across the
membrane that is proportional to the ionized Ca ++ concentration
PHOSPHATE
Reference Ranges
serum, plasma
neonate: 1.45–2.91 mmol/L (4.5–9.0 mg/dL)
child: 1.07–1.74 mmol/L (3.3–5.4 mg/dL)
adult: 0.78–1.42 mmol/L (2.4–4.4 mg/dL)
urine (24-hour): 13-42mmol/day (0.4-1.3g/day)
Specimen
serum or lithium heparin plasma
: patient should be in fasting state since ingestion of PO4¯ -rich food increases
serum P concentration while a high carbohydrate meal can cause a decrease
: oxalate, citrate or EDTA anticoagulants interfere with the analytical method
Methods
Formation of Ammonium Molybdophosphate Complex
-this colorless complex can be measured by UV absorption at 340nm
-can be reduced to form molybdenum blue, a stable blue chromophore which is read
between 600-700nm
Fiske and Subbarow method
Principle. A TCA filtrate of serum or urine is treated with molybdate reagent which
reacts PO4¯ s to form ammonium phosphomolybdate. A reducing agent is added to form a blue
color of heteropolymolybdenum blue.
Reagents and Result
: TCA (protein precipitant and for acidity of medium)
: Pictol (amino naphthol sulfonic acid; reducing agent)*
= other reducing agents
*stannous chloride*
*Elon (p-methylamine phenol)
*p-semidine (n-phenyl-p-phenylenediaminehydrochloride)
*ascorbic acid
*ferrous sulphate
: ammonium molybdate (color reagent)
: heteropolymolybdenum blue (end product and color)
LACTATE
Reference Ranges
Enzymatic Method,
Colorimetric, Whole Blood
Plasma
Venous
0.5–2.2 mmol/L 0.9–1.7 mmol/L
(4.5–19.8 mg/dL) (8.1–15.3 mg/dL)
Specimen
Methods
: although lactate is a sensitive indicator of inadequate tissue oxygenation, the use of
blood lactate measurements has been hindered because older methods were slow and
laborious
: other means of following perfusion or oxygenation have been used, such as indwelling
catheters that measure blood flow, pulse oximeters, base excess determinations, and
measurements of oxygen consumption (VO2)
: current enzymatic methods make lactate determination readily available
most commonly used enzymatic method uses lactate oxidase to produce pyruvate and H 2O2
Lactate oxidase
Lactate + O2 pyruvate + H2O2
: one of two couple reactions may then be used. Peroxidase may be used to produce a
colored chromogen from H2O2
peroxidase
H2O2 + H donor + chromogen colored dye +2H2O