Analytical Errors due to Lipemia

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CE Update—Clinical Chemistry III
Analytical Errors due to Lipemia
Michael H. Creer, MD, and Jack Ladenson, PhD
he staff of the clinical laboratory
must understand and identify a
sample that may be subject to analytical error. They can then devise
mechanisms to obviate the error or
alert the clinician that it occurred.
Such errors commonly arise when lipemic samples are analyzed.
This article will review the nature
of hyperlipemia and consider the three
types of errors that can occur in hyperlipemic samples: electrolyte exclusion, partitioning, and light scattering
(Table I).
Nature of Hyperlipemia
Lipids are fatty, oily, or waxy substances generally possessing longchain hydrocarbon groups. 1 The major lipids in blood are triglycerides,
cholesterol esters, phospholipids, and
small amounts of free fatty acids. Triglycerides and cholesterol e s t e r s ,
which are hydrophobic lipids without
charged groups and therefore insoluble in water, are soluble in organic
solvents such as ether, chloroform, and
hexane. 1 - 2 Other lipids such as phospholipids and free fatty acids are ampiphilic, possessing ionic terminal head
groups. Such lipids have variable solubility in water but are generally soluble in one or more organic solvents. 3
The lipids in plasma exist as mac r o m o l e c u l a r complexes noncovalently bound to apolipoproteins. 4 These
proteins increase the solubility of their
a s s o c i a t e d lipids to p e r m i t t h e i r
transport in blood. The lipids in such
From Washington Univ and Barnes Hosp, Box 8118.
660 S Euclid, St. Louis, MO 63110.
complexes are generally arranged with
the long hydrocarbon chains self-associated to form a hydrophobic central core surrounded by the more polar
head groups. The polyionic apolipoproteins are then bound by electrostatic and hydrogen-bonding forces to
the neutral polar or charged head
groups, forming an outer mantle of
tightly packed polar moieties at the
aqueous solution interface. This surface coating of charged groups on the
apolipoprotein enables the complex to
remain in aqueous solution despite the
high content of nonpolar lipids. 3
Table I shows some properties of the
four major lipid classes in blood. The
large lipid particles—chylomicrons
and very low density lipoproteins
(VLDL)—are primarily composed of
triglycerides with small amounts of
protein, cholesterol, and phospholipid. Their low density makes separation
by
ultracentrifugation
relatively easy. The small lipoproteins — low density lipoprotein (LDL)
and high density lipoprotein (HDL) —
have a lower triglyceride content and
a higher content of protein, cholesterol, and phospholipid. Their density
slightly exceeds that of water which,
in combination with their small size,
makes separation by ultracentrifugation more difficult.
We consider a sample lipemic when,
due to increased concentration of one
or more of the lipid classes, it has a
turbidity that is detectable on visual
inspection. Hyperlipemia may be due
to a primary pathologic process but is
more frequently due to a secondary
process in association with other disease states. Some of the most common
conditions associated with hyperlipemia 4 are:
(1) Diabetes mellitus
(2) Ethanol abuse
(3) Chronic renal failure
(4) Hypothyroidism
(5) Pancreatitis
(6) Carcinoma of colon or liver
(7) Dysproteinemias (eg, multiple
myeloma)
(8) Primary biliary cirrhosis
(9) Systemic lupus erythematosis
(10) Total parenteral nutrition with
lipids such as Intralipid"
The properties of lipids are such that
three different types of errors may occur when analyzing a hyperlipemic
sample:
1. Electrolyte exclusion. Due to the
nonpolar nature of the internal
core region of circulating lipoproteins, a two-phase system is
created where the lipid phase
excludes virtually all ionic and
to a lesser degree most polar
substances, including water.
2. Partitioning. This occurs when
nonpolar substances with low
water solubility preferentially
distribute into the lipid phase.
P a r t i t i o n i n g v a r i e s with temperature, the lipid/H 2 0 distribution coefficient, the presence
and affinity of t r a n s p o r t proteins for the solute, and the analytical reagents used.
3. Light scattering. The particle size
of circulating lipoproteins is on
the order of 100 to 1,000 nm,
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T
LABORATORY MEDICINE • VOL. 14, NO. 6, JUNE 1983 3 5 1
Table I: Characteristics of Human Plasma Lipoprote ns*
Surface
area
(A2 x 103)
Core
volume
3
(A x 10 s)
Density
range
(g/mL)
Lipoprotein
class
Diameter
(A)
Chylomicrons
7506000
25000
-4000
0.950.97
VLDL
250750
570
30
0.951.006
LDL
170260
145
3
HDL
70120
31
0.10
'Adapted from Weidman and Schonfeld1 and Eisent erg
Approximate Composition
(g/100g dry wt)f
Choi
PhL
Prot
TG
5
7
2
86
50
20
20
10
1.0061.063
8
40
30
22
1.0631.21
8
16
24
52
2
tTG = triglycerides, Choi = total cholesterol, PhL == phospholipids, Prot = protein
Electrolyte Exclusion Errors
For lipemic samples, a significant
fraction of the total solution volume
may be occupied by the central core
region of the lipid complexes. The hydrophobic nature of this lipid phase
prohibits entry of charged solutes due
to the large positive free energy req u i r e m e n t s needed to perform t h e
work of moving an ionic solute from
a polar H 2 0 environment with a high
dielectric constant to a nonpolar lipid
phase with a low dielectric constant.
Consequently, t h e c e n t r a l core regions of the lipoprotein complexes
present in the sample are devoid of
small ionic solutes. 2 - 3 From an analytical perspective, such a stable twophase system will not interfere with
direct m e a s u r e m e n t s of thermodynamic activity (eg, direct potentiometry using ion selective electrodes)
since ionic solute activity in the water
phase will be little affected by the inert liposomes in the sample. Methods
subject to analytical error are those
that require dilution of a sample aliquot of fixed volume prior to meas u r e m e n t , examples of which are
shown in Table II. This leads to a low
value for solute concentration that is
not reflective of the activity of the
species in the water phase of plasma.
This type of error is greatest for lipids with high particle volume such as
chylomicrons and VLDL (Table I).
Since triglycerides are the major lipid
component of chylomicrons and VLDL,
the magnitude of the error is related
to the triglyceride value. A perfect
correlation with triglyceride concentration would not be expected because
variation in fatty acyl chain length
and the degree of unsaturation both
affect the particle diameter by altering the packing of triglycerides in the
lipid core. 3
The error is always negative (ie, the
measured value is less than the true
value) and the magnitude of the error
expressed as a percentage should be
similar for all ionic solutes confined
to the water phase since the excluded
lipid volume affects them all equally.
In terms of absolute concentration
change however, it will be particularly noticeable when interpreting sodium values. The exclusion error will
generally be less than 5% until triglyceride concentration exceeds 2,500
mg/dL, 56 so normal postprandial triglyceride elevations will not cause
significant errors.
This type of error can be overcome
by removal of the chylomicra by ultracentrifugation at approximately 105
g,5 7 by electrolyte measurement us-
3 5 2 LABORATORY MEDICINE • VOL. 14, NO. 6, JUNE 1983
ing an ion selective electrode, 5 or by
correcting for the influence of the lipids. This can be accomplished by directly measuring the water content 8
or by measuring the triglyceride concentration and using the formula: %
error = .0021 x triglyceride concentration (mg/dL) - 0 . 6 . 6
Partitioning Errors
Partitioning errors may arise when
small molecules with low intrinsic
water solubility are redistributed from
the polar aqueous phase to the nonpolar interior of the solution liposomes. This partitioning lowers the
effective concentration of solute capable of reacting with analytical reagents confined to the water phase and
thus interferes with analysis. Examples of nonpolar substances subject to
partitioning errors include steroid
hormones and their synthetic analogues, and lipophilic drugs such as
d i l a n t i n , p h e n o b a r b i t a l , and digitoxin.
The methods most often used to
measure these nonpolar compounds
may be grouped into two major categories:
(1) Chromatographic techniques
(GPC, HPLC, etc)
(2) Immunoassay techniques (RIA,
EIA, CPB, etc)
Prior to analysis by either of these
Table II: Methods Subject to Electrolyte Exclusion Errors
Method
Flame photometry
Atomic Absorption
Analytes Measured
Na + , K+, Li +
Ca + + , Mg + +
& other divalent cations
Amperometry/Coulometry
(Chloridometer)
Indirect potentiometry
ciNa + , K + , C a + + , C I -
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which can lead to significant
scattering of light and interference in spectrophotometric analysis.
We will next consider each of these
three sources of error with regard to
the chemical properties of the analytes affected, a n a l y t i c a l methods
subject to error, magnitude of error,
relationship to the type and concentration of the lipid components, and
methods to eliminate or reduce error.
(1) Solute polarity
(2) Volume of the lipid phase
(3) Assay conditions that affect the
reversibility of solute exchange
between the aqueous and lipid
compartments.
The polarity of the solute will be
the principal factor that determines
the ratio of solute concentration in the
lipid and aqueous compartments, ie,
the liposome/H 2 0 distribution coefficient (KD). As the nonpolar nature of
the solute increases, the KD and thus
the fraction of total solute in the lipid
phase will increase. One would then
expect an inverse relationship to exist
between the magnitude of the partitioning error and the polarity of the
solute. This has been confirmed by
Rash and co-workers 10 who found t h a t
the magnitude of the partitioning interference that arises in the RIA measurement of C-21 steroids decreased
as the number of hydroxyl substituents increased.
The volume of the lipid phase available for solute partitioning is determined by the size and shape of the
solution liposomes. These factors are
difficult to accurately predict due to
the heterogeneous composition of the
extracted lipids and variability in assay conditions.
The assay temperature is the major
factor governing the reversibility of
mass transfer of solute between the
two phases. 11 At low temperatures
n o n e q u i l i b r i u m p a r t i t i o n i n g dominates. As the temperature increases
from 4 °C to 37 °C, solute exchange
becomes more reversible, probably as
a result of the increased mobility of
the hydrocarbon chains that maintain the fluid-like state of the lipid
core necessary for it to function as a
nonpolar solvent. 3 When solute partitioning is a reversible equilibrium
process, immunoassay methods will
have potentially "correctable" partitioning errors because both labeled and
unlabeled solute will be distributed
between the lipid and aqueous phases
in the same fashion, thereby allowing
correction for the nonspecific binding
of labeled solute by the solution liposomes. 1 1 When n o n e q u i l i b r i u m
trapping of solute occurs, labeled solute will not partition into preformed
liposomes and conversely unlabeled
solute already incorporated in the liposome interior will not exchange with
the aqueous solute pool. A binding
correction is thus not possible. For this
type of partitioning process the order
in which reagents are added becomes
a crucial factor in determining the
magnitude of error. 11
The relationship between the magnitude of error and sample lipid concentration is complex and differs from
assay to assay. 10 It has been reported,
however, that testosterone and progesterone measurement by RIA can
be significantly affected by only a
twofold increase (above normal) in
sample lipid content. 10 Postprandial
lipid elevations can result in significant errors, as illustrated by the finding that the circadian rhythm in serum
estriol concentration d u r i n g preg-
nancy initially observed was artefactual and could be accounted for by the
partitioning errors that arise in samples drawn within a few hours after
eating. 11
Successful reduction of partitioning
errors depends on the solute being
measured, as well as the particular
assay method used. The original lite r a t u r e should be t h o r o u g h l y reviewed before using approaches such
as disrupting liposomes with dextrancoated charcoal, 12 or selectively extracting steroids based on differential
solubility properties. 13
Light Scattering Errors
The s c a t t e r i n g of l i g h t by suspended lipid particles is the most freq u e n t l y e n c o u n t e r e d s o u r c e of
analytical error in lipemic samples.
Unfortunately, although abstracts reporting such errors are abundant, 1 4 1 9
there are few, if any, published studies describing the manner in which
analytical errors arise despite the use
of conventional means of blank correction.
Light scattering generates errors in
spectrophotometric methods by decreasing the amount of light transmitted in a straight line from source
to detector. Since light scattering and
absorption both reduce the amount of
light reaching the detector, the light
scattering interference will manifest
as an apparent increase in sample absorbance. The final error that results
will be positive for methods in which
increases in sample analyte result in
increased absorption and the error will
be negative for the alternative case.
The fraction of incident light scattered at a given angle from a particle
source can be described by the following equation. 20
i(8)
=
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general methods, the analyte is frequently extracted from the aqueous
sample using an immiscible organic
solvent such as d i c h l o r o m e t h a n e ,
ether, or chloroform, in order to separate the solute of interest from associated p r o t e i n s and other polar
substances that might interfere with
the analysis. The nonpolar nature of
the sample lipids, however, results in
their coextraction following dissociation from their lipoprotein counterparts. In chromatographic methods the
coextracted lipids do not generate
partitioning errors as long as a nonaqueous mobile phase is employed for
the separation since liposomes will not
form in such a nonpolar environment.
Analytical errors may arise if the
sample lipids coelute in the chromatographic procedure with the analyte,
as has been reported for the measurement of dilantin and phenobarbital in
lipemic sera by HPLC following a single-step extraction with acetonitrile. 9
In immunoassay techniques requiring an aqueous medium, the organic
solvent must first be removed from the
initial sample extract by evaporation
or other suitable means. When the
dried protein-free extract is reconstituted in an aqueous buffer system,
partitioning errors may arise due to
the spontaneous self-association of the
lipids. Since antibody or other binding proteins react only with solute in
the aqueous phase, low values could
be obtained if the solute partitions into
the lipid phase. The magnitude of the
resulting error is directly related to
the fraction of total solute present in
the lipid phase, which is primarily determined by the following three factors:
9-rr(Np)r» ( M 2 - l )
( 1 + cos8)
i7 ^o^(^T2)
where i(0)/I o = fraction of incident
light scattered at a given angle (9), r
= particle radius, N p = particle concentration, X = light wavelength, d =
distance from source to detector, and
M = refractive index of particle/refractive index of solution.
In the classic equation of Rayleigh for
particles of uniform size and shape at
very low concentration, a = 6 and b
= 4. Unfortunately for lipemic sera
t h e exponents a and b are r a r e l y
LABORATORY MEDICINE • VOL. 14, NO. 6, JUNE 1983 3 5 3
s c a t t e r on t h e particle r a d i u s and
wavelength is t h a t the value of exponents a and b will be a unique feature of the individual sample. Several
workers have been able to demonstrate a linear relationship between
the log of the sample turbidity and
the log of the wavelength in individual patient samples, but the slope of
the regression line varies from 1.8 to
5.2 in different samples. 2629 Thus, for
effective blank correction, this emphasizes the importance of using the
patient's own serum to prepare the
sample blank, since the use of other
lipemic sera with different particle size
distributions would not be appropriate.
The spatial relationship of the light
source, sample, and measuring device
(the "detector geometry") also influences the light scattering error. In addition to the reduction in intensity of
scattered light with the square of the
source to detector distance (as expected for a radiative process), the
fraction of scattered light detected is
also governed by the shape of the
scattering envelope. The solid angle
subtended by the scattering source at
the exit slit determines which scattered rays will reach the detector. The
remainder of the scattered light will
not be measured, thus leading to an
artefactual increase in absorption. Insofar as instruments differ in regard
to dimensions and the spatial relationship of detector components with
the sample and light source, one would
expect differences to arise in the magnitude of the resulting error. Hubsch
and colleagues 30 show that this is indeed the case.
In summary, the magnitude of light
scattering interferences is as follows:
Sample dependent due to differences in particle size distribution and
concentration of liposome particles as
determined by dietary factors, age, sex,
activity level, and type of hyperlipidemia.
Method dependent due to differences in scattered light intensity at
the different wavelengths used and,
in all likelihood, to the effects of pH,
ionic strength, temperature, and added
reagents on the particle size distribution.
Instrument dependent due to differences in detector geometry.
In most cases, the relative contribution of the various factors to the
3 5 4 LABORATORY MEDICINE • VOL. 14, NO. 6, JUNE 1983
magnitude of the light scattering error has not been systematically examined. One would expect however,
t h a t the magnitude of the error will
decrease when
(1) Samples are dialyzed using a
d i a l y s i s m e m b r a n e of sufficiently small pore size to exclude the liposomes present in
the sample.
(2) Samples are diluted prior to
analysis. In addition to ensuring t h a t blank and sample absorbance fall within the linear
range of the detector where instrument error is at a minimum, 3 1 s a m p l e d i l u t i o n will
reduce the intensity of light
s c a t t e r e d by a given sample
since particle concentration will
be decreased.
(3) Single wavelengths are used for
measurement or small differences exist between wavelengths used in a polychromatic
method.
(4) Longer wavelengths are used.
(5) Double beam instruments are
employed for absorbance measurements. These instruments
have constant absorbance errors that are of smaller magnitude t h a n the transmittance
errors found in single beam instruments. 3 1
Two other general empirical approaches may reduce or eliminate light
scattering errors. The first involves
correcting for blank absorbance and
is based on the assumption that the
measured sample absorbance is the
algebraic sum of the absorbance resulting from two separate, nonintera c t i n g processes, n a m e l y , l i g h t
absorption by the colorimetric indicator t h a t conforms to Beer's law and
turbidity arising from light scattering. When these assumptions hold, one
can determine the indicator absorbance as the difference between measured total sample absorbance and a
turbidity (ie, blank) measurement.
This approach assumes that the liposomes do not directly affect light
absorption by the indicator (eg, the
indicator must not partition in the
lipid phase), and t h a t the indicator
substance has no effect on lipid particle size distribution. For virtually all
methods, the validity of these two ass u m p t i o n s r e m a i n s to be demonstrated.
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known, since they depend on the sample particle size distribution and other
variables t h a t cannot be readily determined.
Although it is an approximation, the
Rayleigh equation shows that the factors governing the magnitude of analytical error due to light scattering
are (in order of decreasing importance):
(1) particle size distribution
(2) wavelength of the measured
light energy
(3) distance from scattering source
to detector
(4) spatial distribution of scattered
light intensity (1 + cos2 9 term,
or the scattering envelope)
(5) particle concentration
The particle size distribution is extremely important since the amount
of light scattered depends exponentially on the particle radius with a
value for exponent a of between 2 and
g 20-24 T ^ p a r t i c i e s j z e distribution in
a lipemic sample reflects the proportions of chylomicrons, VLDL, LDL, and
HDL lipoproteins, and can differ
markedly from one sample to the next.
A relationship between the magnitude of analytical error and the type
of lipid particle has been experimentally confirmed by Nicholls. 25 He found
t h a t t h e r e l a t i o n s h i p between the
magnitude of observed error in hemoglobin concentration and triglyceride
concentration was different in a patient with type V hyperlipemia compared to patients receiving Intralipid.
Other factors affecting the particle size
distribution, such as fatty acyl chain
length and degree of unsaturation, and
such solution conditions as pH, temperature, and ionic strength, are important additional considerations that
have not been well characterized.
The intensity of scattered light also
varies with the inverse of the wavelength of light raised to the b power,
with b generally between 2 and 5. 2124
Thus, the light scattering interference is greatest for methods requiring
absorbance measurements in the ultraviolet (UV) or near-UV spectral
regions (eg, at 340 nm). In addition,
polychromatic methods require special consideration with regard to the
use of sample blank correction, since
the blank absorbance depends on the
wavelength of light measured.
Another important consequence of
the exponential dependence of light
Conclusion
In conclusion, analytical errors in
lipemic samples arise as a result of
the nonpolar nature of the long chain
acyl hydrocarbon moieties present in
most lipid molecules. This leads to low
water solubility and a tendency to aggregate to form macromolecular complexes which exclude electrolytes,
sequester other nonpolar solutes, and
scatter visible light. The magnitude
of the resulting error depends on the
individual sample being measured, the
method used, and the instrument employed for the analysis. Several effective strategies exist to reduce the
magnitude of lipid-related analytical
artefacts. Selection of an appropriate
error correction scheme must include
consideration of the mechanism responsible for generating the error, the
availability of alternative methods of
solute measurement that are not subject to lipemic interference, and the
accessibility of equipment needed to
separate the lipid and aqueous phases.
Table III: Reported Methods Subject to Light Scattering
Errors*
Test
Reference
Albumin
Alkaline phosphatase
Bilirubin, total & conjugated
Calcium, total
Chloride
Creatinine
Glucose
LDH
Phosphorus, inorganic
Protein, total
SGOT
SGPT
Urea nitrogen
Uric acid
15
15
14, 15, 32
15
15,33
33
15-17, 33
14,15
15,17
15
13,15
13-15
15,17
15
"reported for different instruments inc luc ing: Dacos, ACA
SMAC, and unidentified.
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18. Shukla R, Nario A, Bauer B, et al: Elimination
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The second approach to reduce or
eliminate error involves removal of the
lipid by ultracentrifugation prior to
analysis (several table-top, air-operated centrifuges are available for this
task). Ultracentrifugation would be
especially effective in removing the
large VLDL and chylomicrons, which
also have the lowest density. For
measurements at shorter wavelengths (eg, 340 nm), lipid separation
by ultracentrifugation may be less effective due to incomplete removal of
smaller lipid particles with densities
approaching that of water (Table I),
as these smaller particles also contribute significantly to the light scattering error at shorter wavelengths.
Since lipid separation and blank
correction represent two independent
approaches to error elimination, those
methods which do not use a proper
sample blank correction are most
likely to show a striking reduction in
analytic error when some means of
lipid separation is provided prior to
measurement.
Some of the reported interferences
due to light scatter are illustrated in
Table III. Most of these reports are
available only as abstracts. The laboratory should assume that light
scatter can interfere in any spectrophotometric method until it is established otherwise.
Supplier
a. Cutter Laboratories, Berkely, CA.
LABORATORY MEDICINE • VOL. 14, NO. 6, JUNE 1983 3 5 5
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