CLIN. CHEM. 36/1, 36-42 (1990)
Calculated Values for Low-Density LipoproteinCholesterol in the Assessment of Lipid
Abnormalities and Coronary Disease Risk
Judith R. McNamara,’ Jeffrey S. Cohn,’ Peter W. F. WIlson,2and ErnstJ. Schaefer”3
Low-density Ilpoprotein (LOL) cholesterol concentrations are
most commonly estimated by the formula LDL cholesterol
total cholesterol
[triglycerides (TG)/5 + high-density
lipoprotein cholesterol], although alternative factors such as
TG/6 have also been used. Using standardized, automated,
=
-
enzymatic lipid assays, we analyzed 4797 plasma samples
from normal and dyslipidemic adults, to compare LDL cholesterol concentrations obtained after ultracentrifugation with
those calculated by several such methods (i.e., TG/4-TG/8).
For TG concentrations O.50 g/L, TG/4 agreed best with the
direct assay; for TG of 0.51-2.00 g/L, TG/4.5 was best; and
for TG of 2.01-4.00 g/L, TG/5 was best. Differences in
estimated values were generally small, however. At TG
>4.00 g/L, none of the factors tested allowed a reliable
estimate of LDL cholesterol. When TG were 4.00 gIL, 86%
of estimated LDL cholesterol values were properly classified
according to National Cholesterol Education Program cutpoints when the factor TG/5 was used. We conclude that a
convenient direct method for measuring LDL cholesterol is
needed but, until one is available, use of the factor TGI5 will
assure that most individuals with TG 4.00 g/L, as measured
in a standardized laboratory, can be reasonably well classified for risk of coronary artery disease.
AdditIonal Keyphraees: estimation compared with direct assay
(post-u!tracentrifugation). screening
heart disease
risk
factors
Several studies have shown a significantly increased risk
of coronary artery disease (CAD) with increasing concentrations of cholesterol in serum or plasma.4 This increase in
risk is primarily due to increased low-density lipoprotein
(LDL) cholesterol (1-3). Moreover, prospective
studieshave
shown that when high LDL cholesterol concentrations are
decreased by use of diet and drugs, the subsequent incidence of CAD is diminished (4-8). Therefore, the National
Cholesterol Education Program’s (NCEP) Adult Treatment
Panel has recommended
the screening of adults for cholesterol abnormalities
to assess each individual’s CAD risk
(9). When the total cholesterol
concentration
exceeds 2.40
‘Lipid Metabolism Laboratory, USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington St.,
Boston, MA 02111.
2F’thngham
Heart Study, National Heart, Blood and Lung
Institute, Framingham, MA.
3Address correspondence to this author.
Presented in part at ClinChem-88, Springfield, MA, November
8, 1988.
4Nonstandard abbreviations: VLDL, LDL, and HDL, very-low,
low- and high-density lipoproteins; TG, triglycerides; CAD, coronary artery disease; HNRC, USDA Human Nutrition Research
Center on Aging at Tufts University; FHS, Framingham Heart
Study; NCEP, National Cholesterol Education Program; CDC,
Centers for Disease Control; and NHLBI, National Heart, Lung,
and Blood Institute, NIH.
Received June 28, 1989; accepted August 31, 1989.
36 CLINICAL CHEMISTRY, Vol.36, No. 1, 1990
g/L, or when it exceeds 2.00 g/L and two or more other CAD
risk factors are present, NCEP has recommended
further
lipoprotein testing to determine the concentration of LDL
cholesterol.
LDL cholesterol concentration then becomes
the basis for definitive dietary and drug treatment.
Currently,
LDL cholesterol concentration cannot be determined directly from serum or plasma without use of
cumbersome ultracentrifugation
procedures. The reference
procedure for measuring
LDL cholesterol is based on measurement of cholesterol in the density fraction >1.006 kgfL
after ultracentrifugation
(10). Because ultracentrifugation
is not available in most clinical laboratories, assessment of
LDL cholesterol usually is based on indirect
calculations
made from total cholesterol,
high-density
lipoprotein
(HDL) cholesterol, and triglycerides (TG), which are usually unblanked
for free glycerol (11, 12). The validity of
calculated LDL cholesterol values therefore does not depend on the accuracy of one direct assay, but rather on the
accuracy of three other assays, plus a mathematical calculation factor that estimates the amount of cholesterol in
very-low-density
lipoproteins (VLDL).
We wanted to test the effect of using calculation factors
on the categorization
of subjects in a lipid clinic and in a
normal population, with respect to NCEP guidelines. Using
assays standardized
by the Centers for Disease ControlNational Heart, Lung, and Blood Institute (CDC-NHLBI)
(13), we compared LDL cholesterol values obtained after
ultracentrifugation
(10) with those obtained by estimation.
We tested several estimation
factors:
(a) the formula of
Friedewald et al. (11), (b) the method of DeLong et al. (12),
and (c) other similar factors to see which of these might
give more nearly accurate estimations of LDL cholesterol
at various concentrations of TG.
Materials and Methods
Study Populations
The two independent populations of adults, all older than
20 years,that were used for the present analysis had blood
sampled after a 12- to 14-h overnight fast.
The first population of 1469 individuals consisted of two
major subgroups: 731 dyslipidemic patients referred to the
Lipid Clinic at New England Medical Center, Boston, MA,
and 738 research study subjects from the USDA Human
Nutrition Research Center on Aging at Tufts University
(HNRC). Plasma lipids and lipoproteins for both subgroups
were determined by the HNRC Lipid Metabolism Laboratory.
The second population
consisted of 3328 adults who
participated
in Cycle 3 of the offspring follow-up of the
Framingham
Heart Study (FHS), Framinghain,
MA, for
whom lipid and lipoprotein analyses were performed in the
FHS laboratory.
The FHS population represented
an epidemiological
cross-section of a normally distributed
population (mean ±
SD: TG = 1.25 ± 1.21 g/L, LDL cholesterol = 1.34 ± 0.36
g/L). The HNRC population,
however, consisted of two
diverse subgroups: the Lipid Clinic patients had a high
predominance
of lipid abnormalities
(TG = 3.85 ± 6.61 g/L,
LDL cholesterol
=
1.52 ± 0.64 g/L), and the research
subjects generally had low-to-normal
lipid values (TG =
0.82 ± 0.51 g/L, LDL cholesterol = 1.03 ± 0.32 g/L). The
only samples excluded before this data set (n = 4797) was
compiled were samples drawn from non-fasting individuals, those for which ultracentrifugation
was not performed,
and those from individuals younger than 21 years.
Upoprotein Analysis
Blood was drawn into tubes containingEDTA (1.5 gIL
final concentration).
Plasma, separated after centrifugation (1000
g, 20 mm, 4#{176}C),
was stored at 4#{176}C
until
analysis. For both populations, lipid and lipoprotein analyses were performed by identical methodogy, but in separate laboratory facilities. An aliquot of each sample was
subjectedtoultracentrifugation
(18 h, 135 000 x g, 4 #{176}C)
at
a plasma density of 1.006 kgfL, with L8-80 ultracentrifuges
and 50.3Ti rotors (Beckman
Instruments,
Inc., Palo Alto,
CA). After ultracentrifugation,
the d <1.006 fraction was
separated
from the d >1.006 fraction by tube slicing
(Nuclear Supply & Service Co., Washington,
DC). Total
cholesterol,
TG (unblanked
for free glycerol), 1.006 kg/L
infranate cholesterol, and HDL cholesterol concentrations
were determined
with Abbott Diagnostics’
ABA-200
bichromatic analyzers and enzymatic reagents (Abbott’s“AGENT”), as previously described
(13), after precipitation
with dextran-Mg2
(14). Lipoprotein cholesterol was calculated by subtraction: total cholesterol
infranate cholesterol = VLDL cholesterol; infranate cholesterol
HDL
cholesterol
=
LDL cholesterol.
All HNRC lipid analyses
were standardized
through the Lipid Standardization
Program of the CDC-NHLBI.
In addition, LDL cholesterol concentrations
were estimated by seven different calculation formulas: (a) the
method of Friedewald et al. (11), where total cholesterol
(TG/5 + HDL cholesterol)
=
LDL cholesterol; (b) the
method of DeLong et al. (12) with the same formula, except
that TG/6 is used in place of TG/5; and (c-g) by use of the
same formula, but with TG/4, TGI4.5, TG/5.5, TG/7, or
X
-
-
-
TG/8.
Statistical Analysis
The variables utilized in this study were entered and
stored in a VAX 11/780 computer (Digital Equipment
Co.,
Maynard, MA) by using the scientific package RS/i (BBN
Research Systems).Mathematical
calculations
were made
with the RB/i software. Paired t-test analyses, performed
with the Statistical Analysis Systems (SAS) software (SAS
Institute, Cary, NC), were used to compare the significance
of the differences between the various calculated estimations of LDL cholesterol and the concentrations determined
after ultracentrifligation.
Results
After ultracentrifugation,
the LDL cholesterol
concen-
trations measured in the 1469 HNRC samples and the 3328
FHS samples were compared with values calculated by
each of the estimation methods described above. Because
the results for each population, based on unblanked
PG
concentration
and NCEP classification,
were very similar,
the data were combined. The values shown in Table 1
represent the percentage of samples in each unbianked PG
range in which the LDL cholesterol concentration by estimation differed by <10% from the measured concentration.
When TG concentrations were s0.50 gIL, use of the factor
TG/4 led to the highest percentage of individuals having
values for estimated LDL cholesterol that fell within 10%
of measured LDL cholesterol (87%, vs 86% for TG/4.5 and
84% for TG/5). The factor TG/4.5 gave the closest results,
89%, when PG concentrations were 0.51-2.00 g/L, vs 88%
for TG/4 and 86% for TG/5. However, the differences in the
estimated
LDL cholesterol values between these three
factors were generally small at PG concentrations
s2.#{174}
g/L. Use of the factor TG/5 (Friedewald) gave the closest
estimations when TG concentrations were 2.01-4.00 g/L:
73%, vs 56% for TGI4, 69% for TG/4.5, and 70% for TG/5.5.
Above 4.00 g/L, no single best estimation factor emerged,
but use of the factors TG/5 to PG/6 generally yieldedthe
highest percentages. In the TG range of 4.01-6.00 g/L, PG/6
had the highest percentage of values within 10% of the
measured values, but that encompassed
only 43% of the
samples with TG values in this range. Additionally, only
factors TG/4.5 and TG/5 had estimated values that were not
significantly different from measured values in the same
Table 1. Percent of Samples for Which Estimated LDL Cholesterol Was withIn 10% of Measured LDL Cholesterol
Factor used In estImation
TG, g/L
n
TG/4
TG/4.5
TG/5
TG/5.5
O.50
0.51-1.00
528
1908
87*b
86
84
83
88
89*
84
1.01-1.50
1.51-2.00
1016
540
84
87*b
86
86
79
60
40
86*b
84
77*
78
72
56
19
32b
41
6b
10b
2.01-3.00
3.01-4.00
4.01-6.00
6.01-8.00
8.01-10.00
10.01-15.00
>15.00
459
132
96
31
26
29
32
84
74
56
42
26*
8
0
10b
15’
l4*
iga
7
0
3
0
3
7b
TG/6
81
TGI7
TGI8
77
73
82
79
70
69
76
71
71
59
52
37
49
38
20
21
10
15
15
13
50
43*
23
3
3
3
8
0
0b
9*b
Within each TG range, a indicatestheestimation factor with the greatestnumber of samples with less than 10% difference between measured andestimated
LIX cholesterol concentration. b Estimation factors where the LDL cholesterol concentration was not significantly different(P >0.05) from the measured
concentration by paired ftest analysis.
a
CLINICAL CHEMISTRY, Vol. 36, No. 1, 1990 37
PG range by paired t-test analysis. At concentrations of
6.01-8.00 gIL, PG/5 and TG/5.5 gave the closest agreement
(26%); at concentrations of 8.01-10.00 g/L, TG/6 was closest
at 27%; the 10.01-15.00 g/L range had the best agreement
when estimation was calculated with the factor PG/5 (14%);
and forconcentrations
>15.00 g/L, PG/8 had the highest
percentage of agreement (9%). Similar results have previously been reported by Kuba and Frantz (15).
When the data were analyzed for percent error in the
estimated values, similar trends were noted (Table 2 and
Figure 1). PG concentrations
<0.50 g/L had the lowest
percent error when TG/4 was used. The percent errorwhen
PG were in the range of 0.51-1.00 g/L was evenly split
between TG/4 and TG/4.5. Concentrations of 1.01-2.00 g/L
also had the lowest percent error with TG/4.5. When PG
values were 2.01-4.00 g/L, LDL cholesterol concentrations
had the least error when TG/5 was used. When they were
>4.00 gIL, PG15 generally continued to give the smallest
mean percent error, but with increasingly large standard
deviations.
Variability
started to become noticeable when PG concentrations were >2.00 gIL, and when PG concentrations
were 3.01-4.00 g/L at least 41% of individuals had estimated LDL cholesterol concentrations that deviated from
the measured values by >10%. At TG concentrations >4.00
g/L this percentage increased to more than 57% of individuals (Table 1). As shown in Table 2 and Figure 1, the
potential
for significant
estimation
errors
increased
steadily with the PG concentration.
We then subdividedthe data (using NCEP criteria for
total cholesterol and PG) to separately evaluate normolipidemics (cholesterol
<2.40, TG 2.50
gIL, n = 3369),
hypercholesterolemics
(cholesterol
2.40,
TG 2.50
g/L,
n = 934), moderate
hypertriglyceridemics
(cholesterol
<2.40, PG 2.51-8.00 g/L, n = 178), and “combined” hyperlipidemics (cholesterol 2.40,
PG 2.51-8.00 g/L, n = 229).
In addition, a subset of the combined hyperlipidemics
with
VLDL cholesterol/PG ratios 0.30 (n = 28) were analyzed
separately
as possible Type III hyperlipoproteinemics.
Severe hypertriglyceridemics
(n = 87) were not analyzed in
these subgroups. The data are summarized in Table 3. The
accuracy of estimation for the normolipidemics
and the
hypercholesterolemics
was similar to that for the entire
group shown in Table 1. The best estimating factor for the
normolipidemics
was TG/4.5, with 86% having estimated
0
UI
I.2
w
0
-50
050
0514.00
101-1.50 1.51-2.00 2.01-3.00 3.01-400 4.01-6.00 6.01-8.00 8,01-10.00
TR00LYCERIDE
CONCENTRATION
(gel)
Fig. 1. Percent error for estimated LDL cholesterol associatedwith
seven estimationfactors(TG/4, TG/4.5, TG/5, TG/5.5, TGI6, TG/7,
TGI8) at variousTG ranges (gIL)
Each bar representsone standard deviationfrom the mean percent errorfor
each factor. The dotted linerepresents the 10% cutpoint used for evaluating
acceptable error.The #{149}
shows the bar(s) in each TG range withthe smallest
percent error
LDL cholesterol
Normolipidemics,
values within 10% of the measured values.
as defined here, represented
70% of our
entire population.
Hypercholesterolemics
had an estimation accuracy rate of 91% with the factor PG/4 and represented 19% of the population. Hypertriglyceridemics
with
PG 2.51-4.00 gIL were best estimated with factors TG/5.5
and TG/6, but only correctly estimated 58% of samples. The
hypertriglyceridemics
with PG >4.00 gIL were best estimated with the factor TG/6; however, only 37% were
correctly estimated within the 10% limits. Combined hyperlipidemics with PG values of 2.51-4.00 gIL were correctly estimated in 73% of cases using PG/4.5; for those
with PG 4.01-8.00 gIL, 43% were correctly estimated with
the factor TG/5. The subgroup of combined hyperlipidem-’
ics, who, in addition to above-normal cholesterol and PG
values, had VLDL cholesterolfFG ratios 0.30, had a much
lower rate of correct estimation, as would be expected. The
number of those with PG of 2.51-4.00 gIL was small (n =
16), but they were best estimated with TG/4, but at a rate
of only 31%, and markedly lower rates for all other factors.
Above PG of 4.00 gIL, the number of individuals
was
equally small (n = 12), and none of the factors correctly
estimated any of the samples. The entire group of combined
Table 2. Mean (± SD) Percent Deviation of Estimated LDL Cholesterol from Measured LDL Cholesterola
Factor used In estImation
TO, g/L
0.50
0.51-1.00
1.01-1.50
1.51-2.00
n
528
1908
1016
510
TG/4
17*b
TG/4.5
TG/5.5
TG/6
TO/i
TG/8
27b
37b
47b
58b
6±8
6±8
4±8
5±8
6±8
6±9
7±8
9±8
7±9
9±8
11±8
TG/5
18*b
28b
4#{247}gb
0#{247}8*01
28b
08*b
28b
4±8
4±8
110*b
4±10
7±11
14±11
459
-7±11
11±11
0#{247}13*b
20±14
-6±15
5±14
9±13
15±14
3.01-4.00
132
13±16
5 ± 28*
13 ± 28
38 ± 32
-5 ± 29*b
20 ± 29
31 ± 31
96
-17 ± 31
4.01-6.00
27 ± 66
58 ± 69
5 ± 66*)
12 ± 66
39 ± 67
31
-28 ± 68
72 ± 72
6.01-8.00
4 ± 60
64 ± 69
43 ± 63
-26 ± 63
14 ± 59
-53 ± 72
26
-87 ± 86
8.01-1 0.00
97 ± 96
32 ± 90
47 ± 76
127 ± 100
-36 ± 90
7 ± 72*
10.01-1 5.00
29
-88 ± 80
-437 ± 628
-88 ± 431
72 ± 361
-600 ± 731
-301 ± 547
-799 ± 861
32
-1048 ± 1027
>15.00
a Within each TG range, * indicatesthe estimation factor withthe lowest percent error. Estimation factors where the mean estimated LDL cholesterol
and the
mean measuredLDL cholesterolconcentrationsvaried by 0.O5 g/L.
2.01-3.00
b
38 CLINICAL CHEMISTRY, Vol. 36, No. 1, 1990
Table 3. Percent of Samples for Which LDL Cholesterol Was within 10% of Measured LDL Cholesterol,
Lipldemic Type’
Factor used In estimatIon
10/4
10/4.5
TG/5
TG/5.5
82
86*
84
82
Normo.0’
s2.50
70
89
86
s2.50
19
91
90
Hyperchol.0
43
55
58
HyperTGt’
4.00
3
24
>4.00
1
2
20
25
30
s4.00
3
64
71
67
Combinedcd
73*
40
>4.00
2
25
29
43*
VLDLC/TG
4.00
0.3
31*
6
0
0
030c.d
>4.00
0.3
0
0
0
0
aW,.J.ln each lipid category, * IndIcates the estimationfactorwith the greatest number of samples with less than 10%
estimated LOLcholesterolconcentration. b Total cholesterolconcentrations<2.40 g/L. Total
cholesterol concentrations 2.40
2.50 and 8.00 g/L.
Classif.
TO, g/l.
% Pop.
C
TQ/6
78
82
58
37*
56
26
0
0
Grouped by
TO!?
10/8
71
73
44
25
38
13
0
0
64
67
34
20
9
0
0
difference between measured and
g/
d16
concentrations
between
classification. This is due in part to misclassification
errors
in these ranges being unidirectional
and in part to these
categories containing
more samples with concentrations
that were farther from the decision levels of 1.30, 1.60, and
1.90 g/L. Samples with measured
LDL cholesterol values in
the middle ranges of 1.30 to 1.90 gIL had the possibility of
being misclassified bidirectionally. Pable 4B indicates that
samples with measured LDL cholesterol concentrations
differing by >0.10 gIL from a decision level were correctly
classified
84-96% of the time, and that PG/4.5 and PG/5
were the two best estimation
factors (96%). When mea-
hyperlipidemics
with a VLDL cholesterollTG
ratio 0.30
(potentially Type Ill individuals) represented
only 0.6% of
our entire population,
all of them coming from the HNRC
population.
The percentage
of values classified correctly according to
NCEP guidelines for estimated vs measured LDL cholesterol concentrations, utilizing all of the factors, are shown
in Pables 4 and 5, and Figure 2. Table 44 provides the data
for samples with PG s4.0O g/L and indicates that samples
with the highest (1.90
g/L) and the lowest (<1.30 g/L)
LDL cholesterol concentrations had the best rates of correct
Table 4. Percent of Samples Properly Classified for LDL Cholesterol Content According to NCEP Guldellnes
Classified
by U/C
Classified by
estimatIon
g/L
g/L
Estimation factors (% classIfied)
n
10/4
TG/4.5
96*
94
10/5
10/5.5
10/6
1017
10/8
92
8
0
0
90
87
6
o
0
10
0
0
13
83
16
0
0
0
0
0
16
74
10
0
10
76
13
1
8
75
16
1
6
73
21
1
3
69
27
1
2
64
33
2
0
12
79
10
0
7
0
4
80*
77
14
19
0
2
71
27
0
1
65
34
0
0
2
0
0
0
98
100
79
75
60
89
79
56
85
75
line In each
A) For TG 4.00 g/L
<1.30
<1.30
1.30-1.59
1.60-1.89
2281
o
i .90
0
<1.30
1.30-1.59
1.30-1.59
1.60-1.89
24
1219
<1.30
1.30-1.59
686
1.60-i .89
i .90
<1.30
1.90
Overall
71
6
0
i .90
1.60-1.89
4
o
o
29
67
4
18
75
6
0
0
0
0
1.60-1.89
19
12
6
0
0
5
1 .90
81
88
94
96
84
86
86*
85
0
0
3
97
83
69*
96
86*
67
94
85
65
93
83
1.30-1.59
397
4583
B) For measuredLDL cholesterol<0.10 or 0.10 gIL froma decisionlevel
<0.10
1831
65
68
0.10
2752
95
96*
Overall
4583
84
86
0
o
a WIthIneach NCEP category, * indicatestheestimationfactor with the highestpercentof properlyclassifiedsamples. In Tables 4 and 5, the primary
groupIng Is in boldface, to simplifyunderstanding. U/C, ultracentrlfugation.
81
19
CLINICAL CHEMISTRY, Vol. 36, No. 1, 1990 39
Table 5. Percent of Samples Properly ClassIfiedfor LDL Cholesterol According to NCEP Guidelines,
for TG >4.00 g/La
Classified by
Estimation
Classified
by U/C
g/L
g/L
A) For TG,
Estimation Factors (% classified)
n
10/4
10/4.5
10/5
10/5.5
10/6
1017
88
5
4
4
84
6
6
4
81
8
6
5
75
13
6
6
65
20
5
10
51
30
9
10
20
12
56
16
20
32
36
16
12
52*
16
8
44
24
0
36
32
0
16
40
4
8
16
20
24
32
44
27
13
13
27
40
13
20
47
0
13
53*
0
13
27
20
20
20
33
60
0
13
20
67
0
13
7
80
0
29
0
14
0
0
0
0
0
0
43
29
29
29
29
0
0
0
0
0
0
29
57
71
71
71
63
66
69
72
63
100*
56
100*
42
98*
94
90
66
3
3
1
3
4
3
4
87
5
3
5
85
0
4
5
6
18
5
11
52
22
9
18
67
0
0
67
0
0
33
33
0
33
33
0
0
33
33
0
0
67
0
0
33
33
33
33
33
33
33
67
50
50
0
0
0
50
0
50
50
0
0
0
0
0
0
50
0
0
0
50
0
50
50
50
50
50
0
100
108
4.01-8.00 gIL
<1.30
1.30-1.59
<1.30
80
1.60-1.89
1.90
<1.30
1.30-1.59
1.60-1.89
1.90
1.30-1.59
1
64
25
<1.30
1.60-1.89
1.30-1.59
15
1.60-1.89
1.90
<1.30
1.30-1.59
1.90
7
1.60-1.89
i
.90
Overall
89*
6
4
127
40
B) For TG >8.00
<1.30
<1.30
1.30-1.59
1.60-1.89
i.90
79
0
<1.30
1.30-1.59
1.30-1.59
3
1.60-1.89
i
1.60-1.89
.90
<1.30
1.30-1.59
1.60-1.89
i.90
2
<1.30
1.90
33
33
33
33
0
0
0
0
0
0
33
0
1.60-1.89
0
0
0
0
0
33
i.90
67
67
67
67
67
67
89
85
83
82
63
87
91
each NCEPcategory, * Indicatesthe estimationfactorwith the highest percent of properlyclassifiedsamples. U/C, ultracentrifugation.
1.30-1.59
Overall
a Within
3
sured concentrations
were within 0.10 g/L of a decision
level, however, samples were correctly classified in only
56-69% of cases, with TG/5 having the highestrateat 69%.
Data for samples with PG of 4.01-8.00 g/L (Table 5A), and
for samples with TG >8.00 gIL (Table SB) show similar
distribution patterns. In all cases, regardless of PG concentrations, samples with the lowest measured LDL cholesterols
were best classified with a low estimating factor (i.e., TG/4)
and, conversely, those with the highest measured values were
best classified with a high factor (i.e., TG/8). Of course, using
TG/4 causes one to estimate a lower LDL cholesterol and,
conversely, using TG/8 causes one to estimate a higher LDL
40
CLINICAL CHEMISTRY, Vol.36, No. 1, 1990
0
0
0
100
51
cholesterol value for any given set of variables. Therefore,
these latter findings are not surprising. Overall, PG/5.5 was
the best factor for PG values in the range 4.01-8.00 g/L, and
PG/4 was the best classification factor when PG values were
>8.00 g/L. The number of samples in both of these groups was
quite small, however, and the data were skewed, particularly
in the lattergroup.Proper classification
into NCEP categories should not supercede the methods for most nearlyaccurate estimation, and our data indicate that at PG values
>4.00 g/L none of these methods allows for accurate estimates. At PG <4.00 g/L, the factor PG/5 provides a reasonable assessment of LDL cholesterol.
100
LU
II-
Cl)
LU
80
>.
-J
I-
0
LU
60
0
0
(1)
LU
40
-j
0.
4
C/)
20
LL
0
I-
z
LU
0
0
LU
0
NCEP LDL CHOLESTEROL
CUTPOINTS
Fig. 2. Effect of estimatedLDL cholesterolconcentrationson correct
NCEP classificationfor each estimationfactor(TGI4, TGI4.5, TGI5,
TGI5.5, TGI6, TG/7, TG/8)
Open bars representsamples with TG c4.00 gIL; filled bars represent
samples with TG 4.01-8.00 gIL Eachgroupof barsrepresentsone category
of riskas definedby NCEP
Discussion
LDL cholesterol concentration has been recommended by
the NCEP Adult Preatment
Panel as the determining
factor in initiating dietary and drug treatment (9), so it is
important
to understand
the validity and limitations of
reported values for LDL cholesterol. There is no method
currentlyavailable for measuring LDL cholesterol directly
from plasma or serum; thus this analyte must be measured
after its separation
by ultracentrifugation.
Ultracentrifugation methodology was used by the Lipid Research Clinics
in the Prevalence and Primary Prevention studies (4,5,16)
and is generally regarded
as the standard
reference
method. The ultracentrifugation
technique
depends on the
accuracy
of centrifugation,
HDL precipitation,
and the
cholesterol assay. Owing to the expense and lack of availability of ultracentrifugation,
however, methods were developed to estimate LDL cholesterol concentration.
These
estimation
techniques
must rely on the accuracy
of the
cholesterol and TG assays, the HDL precipitation,
and also
on an additional
mathematical
factor that is used in an
attempt to estimate the VLDL cholesterol concentration.
There has been some controversy as to the most nearly
accurate estimation factor to use, and attempts
have been
made to evaluate and refine the original estimation calculations proposed by Friedewald et al. to decrease potential
estimation errors (12, 15, 17-19). However, the most frequently used estimation method in clinical laboratories
continues to be that of Friedewald et al.
The present
study was designed to re-evaluate
the
Friedewald
and other estimation factors with regard to
their impact on NCEP guidelines, and to examine the level
of potential error associated with these mathematical
estimation
factors. We have previously confirmed
that the
Friedewald
formula of TG/5 is an appropriate factor for
estimating VLDL and LDL cholesterol in individuals with
PG 4.00
g/L (20). Additionally, PGI5 also gave approximations of LDL cholesterol for individuals with elevated
PG, but with a large margin of error (21). In the present
study, we have re-examined those issues, using both normolipidemic and dyslipidemic adults (21-81 y), in conjunction with the examination of other calculation factors and
NCEP adult classification guidelines. In addition, we have
used automated enzymatic lipid assays of the type used in
most clinical chemistry laboratories, including unblanked
TG measurements,
which differ from chemical methods
used in our previous studies and in the Lipid Research
Clinic studies. Because the identical values for total cholesterol, TG, and HDL cholesterol were used for both
measuring
and estimating each sample, and because these
assays were CDC standardized,
errors due to inaccuracy,
imprecision,
and biological variability were not assessed in
this study. In clinical practice, however, variability
must
be minimized
as much as possible with regard
to assay
reliability, duration of fasting before sampling (22,23), and
interlaboratory
standardization
(24).
When comparing the estimated values with the measured values, we examined the data from two perspectives.
The first analysis assessed how closely each estimated
LDL cholesterol values approximated the measured value.
For this we used a 10% deviation from the measured
concentration
as a cutpoint. This 10% range represented an
allowable LDL cholesterol fluctuation of approximately
0.10 to 0.20 g/L (mean = 0.13 ± 0.05 gIL; range = 0.03 to
0.39 g/L). Not surprisingly, the ability to correctly estimate
LDL cholesterol concentration
varied inversely with the
PG concentration in the plasma. As seen in Table 1 and
Figure 1, the proportion of samples falling within the fairly
broad 10% range decreased continuously with increased
PG concentration,
irrespective of the calculation factor
used. The standard deviation of the variation increased
from ±7% for PG 0.50
gIL, to ±10-15% at TG concentrations of 3.01-4.00 g/L (Table 2). Above 4.00 g/L, the standard deviation increased to ±28% to more than ± 1000%
error.
When we subdivided
individuals
according
to their type
of dyslipidemia
(Table 3), we found, as expected, that those
with low PG concentrations
(i.e., normolipidemics
and
hypercholesterolemics)
usually were correctly estimated.
Those with increased TG concentrations (i.e., hypertriglyceridemics
and combined hyperlipidemics),
however, were
much less often correctly estimated,
and the subgroup of
combined hyperlipidemics
with an abnormally high VLDL
cholesterolfFG ratio even more so. Fortunately,
individuals
in the latter subgroup are encountered
infrequently
in the
general population-0.6%
in our study-and
their treatment would not differ substantially from that used for other
combined hyperlipidemics.
Unfortunately,
these individuals are impossible
to distinguish
from other combined
hyperlipidemics
without ultracentrifugation
and apolipoprotein E phenotyping.
Because the NCEP Adult Treatment
Panel guidelines
are now being used as a basis for identifying and determining treatment of dyslipidemic individuals, our second assessment involved an examination of the accuracy of classification of LDL cholesterol values, derived through estimation, into various NCEP categories. Because the data
used by NCEP for LDL cholesterol cutpoints were derived
primarily from samples analyzed after ultracentrifugation,
we believed it important to examine how individuals would
be classified if ultracentrifugation
were not available. Interestingly, as shown in Tables 4 and 5 and Figure 2, most
individuals were properly classified, even though the estiCLINICAL CHEMISTRY, Vol.36, No. 1, 1990 41
mated LDL cholesterol concentrations were less accurate
than one would wish. Those with values farthest from a
decision level (i.e., very low or very high values) were most
accurately classified. Individuals
with values close to the
decision points, where accuracy is most critical, were naturally at the highest risk of misclassification
(Table 4B).
Because of this, it is particularly
important
that these
individuals have their values rechecked to minimize inappropriate counseling. Additionally,
it is extremely important that patients adhere to 12- to 14-h fasts, because those
with higher PG concentrations
are in double jeopardy of
being misclassified:
they (a) are subject to a higher rate of
inaccurate LDL cholesterol estimation
due to the elevation
in PG and (b) are generally more sensitive to changes in
plasma PG from dietary fat intake, often requiring a longer
time to return to baseline after a meal (22,23).
Our data clearly indicate the need for a convenient direct
assay for measuring
LDL cholesterol.
However, at the
present time, utilization
of the original formula of Friedewald et al., with the factor TG/5, appears to provide a
reasonable
alternative
for individuals with PG <4.00 g/L.
For those with PG >4.00 g/L, TG/5 is still the factor that
gives results most closely matching
the measured LDL
cholesterol, but the potential error is so great for any given
individual that we do not recommend its use. It should be
noted that the prevalence of LDL cholesterol values 1.60
g/L in subjects with TO >4.00 gIL is reasonably low (13%,
Table 5). We recommend that these individuals (4.5% of our
entire study; 1.8% of the normal Framingham
population)
and those suspected of having Type ifi hyperlipoproteinemia be referred to specialized laboratories
with facffities
available for ultracentrifugal
analysis if the type of lipsprotein abnormality
is to be properly assessed (9).
We thank Dr. Mary M. Schaefer (HNRC) for carrying out the
statistical analysis, and Albina Mariano and Babe Bravo for
performing all of the lipid and lipoprotein determinations
for the
FHS samples.
This work was supported by Grant HL 35243 and Subcontract
RFP NHLBI HV 83-03 from the National Heart, Lung and Blood
Institute, and Contract 53-3K06-5-1O from the U.S. Dept. of
Agriculture Research Service.
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