Electrolyte Methodology

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

Precautions in the Determination of Sodium

1. Na+ excretion decreases during exercise
2. anticoagulants lower Na+ levels like oxalates which tend to increase plasma volume
3. unpurified distilled water contains traces of Na+ ions which will erroneously elevate Na+
levels
4. mixing tubes using thumb as cover causes an elevation of Na + levels since NaCl is
present on the skin
5. glasswares may be contaminated with oxide of Na+

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: outdated because of large sample volume requirements and lack of precision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

: TCA (protein precipitant)

: zinc uranyl acetate
: PVA
: distilled water
: sodium uranyl acetate precipitate (yellow)

2. Magnesium-Uranyl acetate Method, Colour Test

 Principle. Na+ is precipitated with Mg-uranyl acetate; the uranyl ions remaining in
suspension form a yellow-brown complex with thioglycollic acid. The difference between
reagent blank (without precipitation of Na+) and analysis is proportional to the Na+
concentration.
Reagents
: magnesium uranyl acetate (precipitating solution)
: ammonium thioglycollate, ammonia (colour reagent)
: sodium standard (150mmol/L)

 Flame Emission Spectrophotometry (FES)

Principle. Ions are heated sufficiently causing the electrons to become excited to a
higher energy state within the atom. As these electrons drop back to ground state, they emit
light energy of specific wavelength. A detector measures the amount of light given off which
is proportional to the ions present in the solution.
1.1222 Na+ = 390nm
Purpose of Dilution (non-ionic diluting agent)
: adjust the concentration of a measured ion so that the light sensitivity lies within
the range of optimum sensitivity and instrument accuracy
: promotes uniform flow of sample and decreases atomizer plugging (in cases of
viscous samples)
: eliminates interference by sample constituents
 Ion Specific Electrode (ISE)
uses semipermeable membrane to develop a potential produced having different ion
concentrations on either side of the membrane
two electrodes are used; one has a constant potential making it the reference electrode
and the other is the measuring electrode
most analyzers use a glass ion-exchange membrane in its SE system for Na +
measurement

Types of ISE measurement based on the sample preparation

Direct
: provides an undiluted sample to interact with ISE membrane
Indirect
: diluted sample is used
*no significant difference in results except when samples are hyperlipidemic or
hyperproteinemic
Sources of Error
: protein build-up on the membrane through continuous use; protein coated
membrane cause poor selectivity resulting in poor reproducibility of results
= e.g., Vitros Analyzer (Ortho-Clinical Diagnostics), single-use direct ISE system
 Atomic Absorption Spectrophotometry (AAS)
 Gravimetric
 Titrimetric
 Sodium FS
Method: enzymatic photometric test
Principle: β-galactosidase catalyzes the conversion of o-nitrophenyl-β-D-
galactopyranoside (ONPG) to o-nitrophenol and galactose. The activity of β-galactosidase
depends on the Na+ concentration in the sample. The Abs increase at 405nm is proportional to
the Na+ concentration in the sample.
R1: buffer, chelator, β-galactosidase
R2: buffer, o-nitrophenyl-β-D-galactopyranoside

Procedure:
 blank sample/calibrator

Sample/calibrator ---- 40µL

Distilled Water 40µL ----

R1 900µL 900µL

Mix, incubate for 5 minutes at 37°C

R2 300µL 300µL

Mix, incubate for 5 minutes at 37°C, read Abs A1 after 1 minute

and start stopwatch. Read Abs A2 after 1 and 2 minutes at
37°C, and calculate ΔA/min. Read against reagent blank.

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

Precautions in Potassium determination

1. hemolyzed specimens are contaminated with K+ from RBCs
2. K+ levels in serum are slightly higher than those in plasma due to K+ released from
ruptured platelets during blood clotting 
3. proper collection and handling of samples for K+ analysis is extremely important because
there are many causes of artifactual hyperkalemia
4. coagulation process releases K+ from platelets so that serum K+ may be 0.1–0.7 mmol/L
higher than plasma K+ concentration. If patient platelet count is elevated
(thrombocytosis), serum K+ may be further elevated. may be avoided by using
 heparinized tube to prevent clotting of specimen; maintain at 25degrees Celsius;
separate plasma within minutes
5. If tourniquet is left on the arm too long during blood collection or if patients excessively
clench their fists or otherwise exercise their forearms before venipuncture, cells may
release K+ into the plasma
6. Storing of blood on ice promotes the release of K+ from cells, whole blood sample for K+
determinations should be stored at room temperature (never iced) and analyzed
promptly or centrifuged to remove the cells
7. if hemolysis occurs after the blood is drawn, K+ may be falsely elevated
8. excessive muscular activity prior to blood extraction increases K + levels

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

Test titration (mL)

Cl = Std titration (mL) x 100 = mEq/L

Precautions (for Schales and Schales)

1. effect of protein
= contribute to false increased value, precipitated with heavy metal
= remedy: use PFF or by ether extraction

2. interferences by other halogens

= bromine: most important culprit but seldom encountered
= remedy: run Cl¯ as it is, test for Br and subtract from earlier value to get the
corrected Cl¯ value

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)

2. Schoenfeld and Lewellen

Principle. Cl¯ ions in serum displaces thiocyanate ions from mercuric thiocyanate
forming mercuric chloride which is only very slightly ionized. The liberated thiocyanate ions
react with ferric ions to form the orange-yellow thiocyanate complex which is measured at
550nm against a water blank.

3. Whitehorn Titration Method

Principle. Cl¯ in the sample is made to react with mercuric thiocyanate. Mercuric
chloride and free thiocyanate are produced in the reaction. Thiocyanate ions combine with the
ferric ions to form ferric thiocyanate (reddish brown complex) which has strong absorption at
480nm which is directly proportional to the Cl¯ concentration.


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

: HCO3¯ is the second most abundant anion in the ECF

: total CO2 comprises the bicarbonate ion (HCO3¯), carbonic acid (H2CO3), and
dissolved CO2 , with HCO3¯ accounting for more than 90% of the total CO2 at
physiologic pH
: because HCO3¯ composes the largest fraction of total CO2, total CO2 measurement
-
is indicative of HCO3¯ measurement

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
++

ethylene bis-(oxyethylenenitrilo) tetraacetic acid (EGTA). 

 Atomic Absorption Spectrophotometry
Principle. This is the method of choice (reference method) for the determination of Mg +
+
. After deproteinization with either TCA or HCl and removal of phosphate ions with a
lanthanum salt, the diluted filtrate is analysed using the 285.2nm line of a Mg ++ hollow cathode
lamp.

: 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

Precautions in Calcium Determination

1. prolonged venous occlusion causes hemoconcentration which leads to increase in Ca ++
levels
2. EDTA or oxalate bind Ca++ tightly and interfere with measurement
***proper collection of samples for ionized Ca++ measurements requires greater care
: because loss of CO2 will increase pH, samples must be collected anaerobically
: although heparinized whole blood is the preferred sample, serum from sealed
evacuated blood collection tubes may be used if clotting and centrifugation are done quickly
(<30 minutes) and at room temperature 
= dry heparin products are available titrated with small amounts of Ca++ or Zn++
ions or with small amounts of heparin dispersed in an inert puff that essentially eliminates the
interference by heparin
 3. serum should be separated immediately from the clot to remove the possibility of Ca ++
influx into the cells as their permeability changes
4. bilirubin interferes with all fluorometric end point Ca ++ assay methods
5. for Ca++ in urine, an accurately timed urine collection is preferred
: urine should be acidified with 6mol/L HCl, 1mL/100mL urine

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

: hemolysis should be avoided because of high concentration in RC circulating PO4¯

levels are subject to circadian rhythm with highest levels late in the morning and lowest in the
evening*
: inorganic PO4¯ levels increase in serum due to action of phosphatases, hence
serum must be separated from the coagulum
 urine
: 24-hour collection because of the significant diurnal variation

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)

Arterial 0.5–1.6 mmol/L <1.3 mmol/L

(4.5–14.4 mg/dL) (<11.3 mg/dL)
 CSF 1.0–2.9 mmol/L (9–26 mg/dL)

: lactate is a by-product of an emergency mechanism that produces a small amount of

ATP when oxygen delivery is severely diminished
: pyruvate is the normal end product of glucose metabolism (glycolysis)
: conversion of pyruvate to lactate is activated when a deficiency of oxygen leads to an
accumulation of excess NADH
= sufficient oxygen maintains a favorably high ratio of NAD to NADH
= under these conditions, pyruvate is converted to acetyl-coenzyme A (CoA), which
enters the citric acid cycle and produces 38 moles of ATP for each mole of glucose oxidized
= under hypoxic conditions, acetyl-CoA formation does not occur and NADH
accumulates, favoring the conversion of pyruvate to lactate through anaerobic metabolism
: as a result, only 2 Moles of ATP Are produced for each mole of glucose metabolized to
lactate, with the excess lactate released into the blood
: release of lactate into blood has clinical importance because the accumulation of excess
lactate in blood is an early, sensitive, and quantitative indicator of the severity of oxygen
deprivation
: because lactate is a by-product of anaerobic metabolism, it is not specifically regulated,
as with K or Ca, for example
: as oxygen delivery decreases below a critical level, blood lactate concentrations rise
rapidly and indicate tissue hypoxia earlier than pH
: the liver is the major organ for removing lactate by converting lactate back to glucose
by a process called gluconeogenesis

measurements of blood lactate are useful for

1. metabolic monitoring in critically ill patients
2. indicating the severity of the illness
3. objectively determining patient prognosis

Types of lactic acidosis

 Type A is associated with hypoxic conditions, such as shock, myocardial infarction,
severe congestive heart failure, pulmonary edema, or severe blood loss
 Type B is of metabolic origin, such as with diabetes mellitus, severe infection, leukemia,
liver or renal disease, and toxins (ethanol, methanol, or salicylate poisoning)

Specimen

Precautions in Lactate Determination:

: special care should be practiced when collecting and handling specimens for lactate
analysis
1. ideally, a tourniquet should be not be used because venous stasis will increase lactate
levels. If a tourniquet is used, blood should be collected immediately and the should not
exercise the hand before or during collection
2. after sample collection, glucose is converted to lactose by way of anaerobic glycolysis
and should be prevented
3. heparinized blood may be used but must be delivered on ice and the plasma must be
quickly separated
4. iodoacetate or fluoride, which inhibit glycolysis without affecting coagulation, are
usually satisfactory additives, but the specific method directions must be consulted

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