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Lipids and Lipoproteins |
1
Pacific Biometrics, Inc., 220 West Harrison Street, Seattle, WA 98119.
2
Otsuka America Pharmaceutical, Inc., Rockville, MD
20850.
3
Cardiovascular Research Institute, University of
California, San Francisco, CA 94143.
a Author for correspondence. Fax 206-298-9838; e-mail ETL{at}PACBIO.COM.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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General application of these research findings has been limited by lack of facile methods for quantification of VLDL and chylomicron remnants. Although lipoprotein remnants are adequately defined in functional terms, no straightforward methods have been developed to separate them from other TG-rich lipoproteins (14). Preparative and analytical ultracentrifugation (UC) have been used to separate VLDL and intermediate-density lipoprotein (IDL), designating the small VLDL and IDL as lipoprotein remnants. Smaller TG-rich lipoproteins have also been separated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (15) or by electrophoresis in agarose gels, in which they have reduced mobility (16). These methods have limited applicability for routine analysis in clinical laboratories because they are labor-intensive, complex, and expensive.
Recently, a novel immunoseparation method for remnant lipoproteins was developed by Nakajima et al. (17). This method uses a monoclonal antibody to human apolipoprotein (apo) B-100 that recognizes an epitope near B-51 (18) to remove almost all LDL and most VLDL particles containing apo B-100 together with a monoclonal antibody to apo A-I to remove almost all HDL particles. Remaining unbound to these antibodies are principally all chylomicron remnants and a fraction of VLDL particles, both enriched in apo E, a characteristic of remnant lipoproteins (19). This unbound fraction is designated "remnant-like particles" (RLPs). The monoclonal antibodies are conjugated to Sepharose 4B to facilitate the separation of bound lipoproteins such as LDL and HDL from the RLP fraction. Cholesterol in the unbound fraction (RLP-C) is measured by an enzymatic cholesterol assay. With this immunoseparation method, researchers have found that RLP-C is increased in patients with CAD (20)(21), type III hyperlipoproteinemia (22), and diabetic nephropathy (23) and in patients undergoing hemodialysis (24)(25).
The objectives of this study were to evaluate the technical performance of this RLP-C assay and to assess the relationship between RLP-C and other atherogenic lipoproteins. A reference range of serum RLP-C for the US population was established, and the serum RLP-C concentrations were evaluated in patients with CAD confirmed by coronary angiography.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish US reference ranges of serum RLP-C concentrations, 364 men and 362 women older than 17 years were recruited in four different parts of the country (Austin, TX; Brighton, MA; Miami, FL; and Phoenix, AZ). These subjects were free of symptoms and signs of CAD and of endocrine or metabolic disorders that may affect lipid metabolism. However, lipid and lipoprotein concentrations per se were not part of the inclusion or exclusion criteria. No subject took any medication that may alter lipid or lipoprotein metabolism. Each subject was asked to participate in two visits: one after 12 h of fasting and one at random in relation to the last meal (either fasting or nonfasting). Serum concentrations of RLP-C, total cholesterol (TC), TGs, and HDL-C were measured at each visit.
To evaluate the clinical utility of RLP-C in predicting CAD, 203 adult patients with >20% stenosis of at least one coronary artery at coronary angiography were recruited from nine medical centers in the US (Boston, MA; Jacksonville, FL; Lombard, IL; Santa Rosa, CA; Houston, TX; Minneapolis MN; Winter Park, FL; Ocala, FL; and Clearwater, FL) and one medical center in Canada (Montreal, PQ). Control subjects (n = 477) with similar ages to the CAD patients were recruited from the same four US centers that participated in the reference range study. Subjects with serum TG blank values >500 mg/L (50 mg/dL; because of heparin used during angiography before specimen collection), who did not complete both visits, or who did not have a fasting specimen collected were excluded from the data analysis. The remaining population included 172 CAD patients and 469 controls.
All clinical protocols were approved by the Institutional Review Board. Informed consent forms were signed by all participating subjects.
materials
The RLP-C Immunoseparation Reagent kits were provided by Japan
Immunoresearch Laboratories. The kit consists of immunoaffinity gel,
cholesterol reagent, a cholesterol calibrator, and a Tris-HCl buffer
solution (pH 7.4). The immunoaffinity gel consists of monoclonal
antibodies to human apo B-100 (JI-H) and apo A-I (H-12) conjugated to
Sepharose 4B beads as the solid phase (17). The cholesterol
reagent contains ascorbate oxidase, cholesterol esterase, and
cholesterol oxidase in R1, and horseradish peroxidase,
4-amino-antipyrine, and
N-ethyl-N-(3-methylphenyl)-N'-succinyl-ethylenediamine
in R2.
To monitor the imprecision of the RLP-C assay, fresh-frozen serum quality-control pools (RLP QC) were prepared and stored at -70 °C. They were supplemented with potassium EDTA (8 g/L) and sucrose (100 g/L) to preserve lipoprotein integrity during freeze-thaw cycles. Addition of sucrose has been shown to protect lipoproteins from damage during lyophilization (26). Diluted human serum pools were also prepared to monitor the imprecision of the cholesterol assay independent from the immunoseparation procedure (Low Cholesterol QC). Serum samples were diluted to cholesterol concentrations between 0.13 and 0.65 mmol/L (between 5 and 25 mg/dL), using normal saline with 50 g/L bovine serum albumin. EDTA and sucrose were also added into the diluted serum pools. The EDTA/sucrose-supplemented sera were then further diluted 61-fold with RLP buffer before freezing at -70 °C to simulate the dilution of samples during the RLP immunoseparation step. All frozen-serum QC pools were thawed and mixed on a shaker for 30 min at room temperature before use.
Hemoglobin and bilirubin (conjugated and unconjugated) were obtained from Kokusai Shiyaku Co., Ltd. (Interference Check-A Plus, cat. no. 5186). Glucose and ascorbic acid were obtained from Sigma Chemical Co.
rlp-c analysis
A 3-mm diameter stainless steel bead (Otsuka Electronics) was
added to each microsample cup (part no. 140-668-0001, Boehringer
Mannheim); 300 µL of the immunoaffinity gel and 5 µL of specimen
were then added to each cup. The mixture was incubated at room
temperature on a dedicated mixer with build-in magnetic bars effecting
mixing by driving the beads up and down in each cup (18).
After 2 h, the supernatant (unbound fraction) was transferred for
cholesterol analysis in a Cobas MIRA S chemistry analyzer (Roche
Analytical Instruments, Inc.). The cholesterol values were multiplied
61-fold to reflect dilution of the specimen at the immunoseparation
step (Table 1
).
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technical evaluation of the rlp-c assay
The detection limit of the RLP-C assay was defined as 3 SD above
the mean from 10 replicate analyses of the RLP buffer over 2 days. To
evaluate linearity, a serum with an increased RLP-C concentration was
mixed with a second serum with low RLP-C concentration or with normal
saline in various proportions and then analyzed in duplicate.
Within-run imprecision (CV) was evaluated by running the two RLP QC
pools in 20 replicates. The run-to-run imprecision of the RLP-C assay
was evaluated over a 3-month period on the same RLP QC pools by
multiple analysts. The CV of the cholesterol assay was evaluated using
the two Low Cholesterol QC pools without the immunoseparation step.
To compare the difference between serum and plasma in RLP-C measurement, specimens from 43 subjects were analyzed. Blood samples (fasting or nonfasting) were drawn into various Vacutainer Tubes (Becton Dickinson) in the following order: SST (for serum), EDTA, lithium heparin, EDTA, lithium heparin, SST. After the samples stood at room temperature for 30 min, serum or plasma were prepared by centrifugation to remove blood cells. The same specimen types were pooled and then aliquoted into cryogenic vials for storage at 4 °C or at -70 °C. TC, TGs (corrected for a glycerol blank), and HDL-C were measured in the fresh fasting sera. RLP-C was measured in all specimens within 4 h of collection and at specific intervals after storage.
Interference of hemoglobin, bilirubin, glucose, and ascorbic acid with the RLP-C analysis was evaluated by adding various concentrations of the respective interference substances or saline as control to two serum samples.
other lipid and lipoprotein analyses
TC and TGs were measured by CDC-standardized enzymatic methods.
HDL-C was measured after precipitation of non-HDL lipoproteins by
dextran sulfate (Mr 50 000)-Ca2+.
VLDL, IDL, and LDL were separated by UC at 250 000 g rpm
using a Beckman Ti 42.2 rotor for 4 h or a Beckman 40.3 rotor for
18 h (27). Serum was centrifuged, and then the
centrifuge tube was sliced with a Beckman tube slicer to yield a
d <1.006 kg/L fraction (containing chylomicrons and VLDL)
and a d 
1.006 kg/L fraction (containing IDL, LDL, and
HDL). The serum was also adjusted to a density of 1.019 kg/L with
potassium bromide solution and then centrifuged to yield a d
<1.019 kg/L fraction (containing chylomicrons, VLDL, and IDL) and a
d 1.019 kg/L fraction (containing LDL and HDL).
Cholesterol concentrations in each of the four fractions were measured
by an enzymatic cholesterol assay. VLDL-cholesterol (VLDL-C) was
calculated as [TC - cholesterol in d 1.006 kg/L
lipoproteins]. IDL-C was calculated as [cholesterol in d
1.006 kg/L lipoproteins - cholesterol in d 1.019
kg/L lipoproteins]. LDL-C was calculated as [cholesterol in
d 1.006 kg/L lipoproteins - HDL-C] or measured by
the Direct LDL-Cholesterol method (Genzyme Corp.).
Lipoproteins were also separated and measured by PAGE (LipoPhorTM by Quantimetrix) (28). Lipoprotein(a) was measured by an immunoturbidimetric assay from INCSTAR in a Cobas FARA automated chemistry analyzer (Roche Analytical Instruments, Inc.).
statistical analyses
Imprecision of the assays was expressed as CV. Comparison between
RLP-C and other lipoproteins was evaluated using the Pearson
correlation. The difference among different specimen types was
estimated using ANOVA. The Mann–Whitney U-test was used to
estimate the statistical difference between CAD patients and age- and
gender-matched controls in their lipid and lipoprotein values because
some of the values did not fit a gaussian distribution. A P
value <0.05 was considered statistically different.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We evaluated the effects of two different storage conditions (4 °C
and -70 °C) on RLP-C values. RLP-C concentrations in the nonfasting
samples tended to increase when stored at 4 °C (Fig. 1
). The increase of mean fasting RLP-C concentrations was <0.03
mmol/L (1.0 mg/dL) within 1 week of sample collection. Freezing at
-70 °C prevented the gradual increase seen in both fasting and
nonfasting samples with TG concentrations <4.52 mmol/L (400 mg/dL) for
up to 3 months (Fig. 1
). Fasting RLP-C concentrations rose after
freezing in three of the five samples with TG concentrations >4.52
mmol/L (400 mg/dL).
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technical performance of the rlp-c assay
The detection limit, defined as 3 SD above the mean value for the
RLP buffer, was 0.08 mmol/L (3.1 mg/dL). The assay was linear up to
3.37 mmol/L (130 mg/dL) when serum was used as the diluent or to 2.33
mmol/L (90 mg/dL) when normal saline was used as the diluent.
The within-run CV of the RLP-C assay was 6.1% at 0.13 mmol/L (4.9
mg/dL) and 3.5% at 0.51 mmol/L (19.8 mg/dL). The within-run CV (n
= 20) of the cholesterol assay, independent of immunoseparation, was
4.7% at 0.22 mmol/L (8.6 mg/dL) and 1.6% at 0.67 mmol/L (26.0 mg/dL;
Table 2
). The run-to-run CV over a representative 3-month period
(n = 36) was 10.2% at 0.15 mmol/L (5.9 mg/dL) and 5.6% at 0.51
mmol/L (19.7 mg/dL). The run-to-run CV attributable to the cholesterol
assay alone during the same period was 5.7% at 0.23 mmol/L (8.7 mg/dL)
and 3.3% at 0.62 mmol/L (23.9 mg/dL; Table 2
). The representative
run-to-run CV was ~10% at 85 mg/L (8.5 mg/dL) for VLDL-C plus IDL-C
by UC (n = 79) and 13% at 90 mg/L (9.0 mg/dL) for VLDL-C by
LipoPhor (n = 64).
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Hemoglobin up to 5000 mg/L (500 mg/dL), bilirubin (both conjugated and unconjugated) up to 342 mmol/L (20 mg/dL), glucose up to 66.6 mmol/L (1200 mg/dL), and ascorbic acid up to 170 mmol/L (3 mg/dL) did not interfere with the RLP-C assay. We showed previously that the binding capacity for HDL, expressed as its cholesterol concentration, was 2.59 mmol/L (100 mg/dL) (18). Above this concentration, a substantial amount of HDL appeared in the RLP fraction. By contrast, there was no clear cut point at which a relevant amount of LDL escaped antibody binding. Instead, the monoclonal antibody JI-H gradually lost its binding capacity with increased LDL (measured as LDL-C). Specifically, ~3% and 5% of the LDL were present in the unbound fraction in samples with LDL-C concentrations of 5.18 mmol/L (200 mg/dL) and 7.77 mmol/L (300 mg/dL), respectively (18).
relationships between rlp-c and other lipids and lipoproteins
To evaluate the relationship between RLP-C and other lipoproteins,
we compared RLP-C with other TG-rich lipoprotein fractions separated by
either UC or by PAGE (e.g., LipoPhor) in 157 CAD patients and 314 age-
and gender-matched controls. The correlation coefficients
(r) between RLP-C and VLDL-C were 0.88 and 0.77 for the VLDL
separated by UC and LipoPhor, respectively (Fig. 2
). RLP-C also moderately correlated with serum IDL-C by UC
(r = 0.46) or by LipoPhor (r = 0.59;
Fig. 2
). The correlation coefficients between RLP-C and total TG-rich
lipoproteins (VLDL-C + IDL-C) were 0.86 and 0.76 (measured by UC and
LipoPhor, respectively). RLP-C was also highly correlated with serum TG
(r = 0.87) but poorly correlated with TC
(r = 0.36) and LDL-C (r = 0.18). There
was no correlation between RLP-C and lipoprotein(a) (r
= -0.06).
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The lower correlation between RLP-C and IDL-C than with VLDL-C was
supported by the investigation of the distribution of RLP-C in
different lipoprotein fractions separated by UC. In 21 sera examined,
the concentration of RLP-C in the IDL fraction, calculated as (RLP-C in
the d <1.019 kg/L fraction - RLP-C in the
d <1.006 kg/L fraction), was negligible except in samples
with TG concentrations >5.65 mmol/L (500 mg/dL; Fig. 3
). RLP-C in the d <1.006 kg/L fraction was highly
correlated with serum TG concentrations (r = 0.95). At
higher serum TG concentrations, a larger percentage of RLP-C was in the
d <1.006 kg/L fraction.
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reference ranges of rlp-c in a us population
In 364 healthy men and 362 healthy women recruited from four US
centers, median fasting TC, TG, LDL-C, and HDL-C concentrations were
5.21 mmol/L (201 mg/dL), 1.10 mmol/L (97 mg/dL), 3.34 mmol/L (129
mg/dL), and 1.22 mmol/L (47 mg/dL), respectively. The 25th through 75th
percentiles were 4.48–5.96 mmol/L (173–230 mg/dL) for TC, 0.80–1.63
mmol/L (71–144 mg/dL) for TGs, 2.77–3.99 mmol/L (107–154 mg/dL) for
LDL-C, and 1.01–1.45 mmol/L (39–56 mg/dL) for HDL-C. The median RLP-C
concentrations were 0.13 mmol/L (5.1 mg/dL) in fasting samples and 0.16
mmol/L (6.2 mg/dL) in random samples. The 25th-75th-95th percentile
concentrations were 0.10-0.17-0.32 mmol/L (3.9-6.6-12.3 mg/dL) in
fasting samples and 0.13-0.23-0.46 mmol/L (4.9-8.8-17.6 mg/dL) in
random samples.
There was a significant difference in RLP-C concentrations between men
and women and between different age groups (Table 3
). In fasting samples, there was no difference in RLP-C
concentrations among the three age groups tested for the men (18–34,
35–54, and 55 years). RLP-C concentrations increased with age in
women. The median RLP-C concentration in women 55 and older was no
different from that in men of the same age group. In contrast, random
RLP-C concentrations increased with age in both men and women. In
addition to the effects of gender and age, fasting and random RLP-C
concentrations were higher in Hispanics (n = 173) and lower in
African Americans (n = 67) than in Caucasians (n = 475). The
median fasting RLP-C concentrations in the three populations were 0.14
mmol/L (5.4 mg/dL), 0.10 mmol/L (4.0 mg/dL), and 0.13 mmol/L (5.1
mg/dL), respectively.
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rlp-c in cad patients and controls
Of the 172 eligible CAD patients, 161 (94%) were Caucasians, 7
(4%) were African Americans, 2 (1%) were Hispanics, and 2 (1%) were
other races. In comparison, Caucasians accounted for only 58% of the
control population of the clinical utility study. To provide vigorous
comparison between the CAD and control groups, we matched each
Caucasian CAD patient with two Caucasian controls, either from the
reference range study or the clinical utility study, by their gender
and age (±1 year). The average age was 63.4 ± 8.6 years for both
groups; 77% were men and 23% were women.
Median fasting and random RLP-C concentrations were significantly
higher in CAD patients than in controls (P <0.05 and
P <0.001, respectively; Table 4
). Serum TG concentrations, fasting or random, were also
significantly higher in CAD patients. HDL-C concentrations were
significantly lower in CAD patients. LDL-C concentrations were slightly
lower in CAD patients. There were no differences between the two groups
in serum TC concentrations.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lipoprotein remnants are a group of highly heterogeneous particles varying in size, hydrated density, electrophoretic mobility, chemical composition, and recognition by receptors (14). Each of the available separation methods measures lipoprotein remnants on the basis of different physical and chemical properties. UC separates lipoproteins by their hydrated density. Researchers typically have used IDL [hydrated density between 1.006 and 1.019 kg/L, or Svedberg flotation rates (Sv) of 12–20 units] as a surrogate to reflect remnant status because sequential hydrolysis of VLDL leads to a group of smaller, more dense, and cholesteryl ester-rich lipoprotein remnants. Chylomicron remnants, with a size and density generally larger than those of VLDL, do not appear in appreciable amounts as lipoproteins in the 1.006–1.019 kg/L density range. Some investigators have suggested that lipoproteins of Sv between 12 and 60 units reflect remnant status better than lipoproteins of Sv between 12 and 20 units (29). This range of lipoproteins includes both IDL (1.006 < d < 1.019 kg/L) and smaller VLDL particles found in the d <1.006 kg/L fraction.
In contrast to UC, PAGE separates lipoproteins by their molecular sizes. With this method, lipoprotein remnants are often measured as "IDL", which includes any lipoprotein particles with the a size between VLDL and LDL (28). In the population studied, IDL measured by LipoPhor correlates only moderately with IDL isolated by UC, with a Pearson correlation coefficient of 0.57 (data not shown). In samples from the type III hyperlipoproteinemic patients, IDL-C measured by the two methods are highly correlated, with a Pearson correlation coefficient of 0.93 (data not shown).
The RLP-C assay separates lipoprotein remnants on the basis of their
surface apo components. apo A-I-containing lipoproteins (namely HDL and
chylomicrons) are removed by anti-apo A-I; most apo B-100-containing
lipoproteins (namely LDL and most VLDL) are removed by anti-apo B-100
(JI-H) (17). This assay measures mainly chylomicron remnants
and larger VLDL remnants (19). These particles are rich in
apo E as compared with the bound VLDL (19)(30).
However, in samples from patients with diabetic (18) and
type III hyperlipoproteinemia (22), particles of IDL size
are found in the RLP fraction. In our nondiabetic subjects who did not
have type III hyperlipoproteinemia, the IDL fraction (1.006 kg/L
< d < 1.019 kg/L) contained little RLP-C (Fig. 3
).
RLP-C is thus better correlated with total TG-rich lipoproteins (VLDL +
IDL) than with IDL-C alone (r = 0.86 vs 0.46 by UC or
r = 0.76 vs 0.59 by LipoPhor (Fig. 2
).
Although IDL concentrations have been shown to predict the presence (10) and progression (31) of CAD in some studies, evidence that these particles are more atherogenic than other TG-rich lipoproteins is limited. Lipoprotein remnants with d <1.006 kg/L, on the other hand, have been found to possess proatherogenic characteristics: they increase permeability of the endothelial barrier (32) and are taken up by macrophages by receptor-mediated mechanisms (33)(34). Furthermore, lipoproteins isolated from human atherosclerotic plaque structurally resemble TG-rich lipoprotein remnants (35)(36). Clinical studies have also suggested that the concentration of remnants in the VLDL plus IDL range predict coronary atherosclerosis lesion progression (11)(13) or clinical coronary heart disease events (11) better than LDL.
RLPs isolated by the immunoaffinity mixed gel have been demonstrated to have biological properties of atherogenic lipoprotein remnants, including inhibition of endothelium-dependent vasorelaxation (37)(38)(39), enhancement of platelet aggregation in whole blood (40)(41), and uptake by macrophages without modification (42)(43). All of these properties of RLPs are potentially proatherogenic. In addition to in vitro evidence, clinical studies have also demonstrated that RLP-C concentrations are higher in CAD patients (17)(20)(21). Serum RLP-C concentrations have also been found to be substantially higher in patients with non-insulin-dependent diabetes mellitus and microalbuminuria (23), undergoing chronic hemodialysis (24), or with intermittent claudication (44).
To help us evaluate the clinical utility of the RLP-C assay in North
American populations, we conducted a multicenter program to establish
reference ranges and to compare RLP-C concentrations in patients with
CAD and in controls with similar ages. Consistent with other studies
(17)(20)(21), RLP-C concentrations,
particularly in random samples, were higher in Caucasian CAD patients
than in age- and gender-matched Caucasian controls (Table 4
).
Because of the large size of chylomicron-derived particles in nonfasting samples, VLDL-C cannot be calculated with reasonable accuracy in the postprandial state. To permit estimation of LDL-C, plasma lipid analyses are ordinarily performed on fasting samples in the clinical laboratory. A potential disadvantage of this practice is that it may reduce the opportunity to detect defects in lipoprotein remnant removal. It has been hypothesized that atherosclerotic disease can result from postprandial accumulation of remnant particles (45)(46). Previous studies have shown that postprandial RLP-C concentrations are substantially increased above fasting concentrations in CAD patients but not in healthy controls, where the fasting concentrations were considered within reference values in both groups (47)(48). Random sampling provides an alternative to cumbersome fat-loading studies in measuring postprandial RLP-C concentrations.
In summary, we have evaluated a novel immunoseparation method for the quantitative estimation of a lipoprotein remnant fraction. The RLP-C immunoseparation method is faster and easier to use than laboratory methods based on UC or electrophoresis. Additional clinical studies are needed to establish the usefulness of this novel assay.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  X.-Y. Zheng and L. Liu Remnant-like lipoprotein particles impair endothelial function: direct and indirect effects on nitric oxide synthase J. Lipid Res., August 1, 2007; 48(8): 1673 - 1680. [Abstract] [Full Text] [PDF]
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 B. C.H. Kwan, F. Kronenberg, S. Beddhu, and A. K. Cheung Lipoprotein Metabolism and Lipid Management in Chronic Kidney Disease J. Am. Soc. Nephrol., April 1, 2007; 18(4): 1246 - 1261. [Full Text] [PDF]
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 Y. Iwasaki, H. Matsuyama, and N. Nakashima Improved Specificity of a New Homogeneous Assay for LDL-Cholesterol in Serum with Abnormal Lipoproteins Clin. Chem., May 1, 2006; 52(5): 886 - 888. [Abstract] [Full Text] [PDF]
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 E. A. Brinton, M. N. Nanjee, and P. N. Hopkins Triglyceride-rich lipoprotein remnant levels and metabolism: Time to adopt these orphan risk factors? J. Am. Coll. Cardiol., June 16, 2004; 43(12): 2233 - 2235. [Full Text] [PDF]
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 T.B. Twickler, G.M. Dallinga-Thie, J.S. Cohn, and M.J. Chapman Elevated Remnant-Like Particle Cholesterol Concentration: A Characteristic Feature of the Atherogenic Lipoprotein Phenotype Circulation, April 27, 2004; 109(16): 1918 - 1925. [Full Text] [PDF]
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Y. Hirowatari, H. Yoshida, H. Kurosawa, K.-i. Doumitu, and N. Tada Measurement of cholesterol of major serum lipoprotein classes by anion-exchange HPLC with perchlorate ion-containing eluent J. Lipid Res., July 1, 2003; 44(7): 1404 - 1412. [Abstract] [Full Text] [PDF]
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P. R. W. de Sauvage Nolting, M. B. Twickler, G. M. Dallinga-Thie, R. J.A. Buirma, B. A. Hutten, J. J.P. Kastelein, and for the Examination of Probands and Relatives in S Elevated Remnant-Like Particles in Heterozygous Familial Hypercholesterolemia and Response to Statin Therapy Circulation, August 13, 2002; 106(7): 788 - 792. [Abstract] [Full Text] [PDF]
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D. C. Chan, G. F. Watts, P. H. R. Barrett, I. J. Martins, A. P. James, J. C. L. Mamo, T. A. Mori, and T. G. Redgrave Effect of atorvastatin on chylomicron remnant metabolism in visceral obesity: a study employing a new stable isotope breath test J. Lipid Res., May 1, 2002; 43(5): 706 - 712. [Abstract] [Full Text] [PDF]
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D. C. Chan, G. F. Watts, P. H. Barrett, J. C.L. Mamo, and T. G. Redgrave Markers of Triglyceride-rich Lipoprotein Remnant Metabolism in Visceral Obesity Clin. Chem., February 1, 2002; 48(2): 278 - 283. [Abstract] [Full Text] [PDF]
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T. G. Redgrave, G. F. Watts, I. J. Martins, P. H. R. Barrett, J. C. L. Mamo, S. B. Dimmitt, and A. D. Marais Chylomicron remnant metabolism in familial dyslipidemias studied with a remnant-like emulsion breath test J. Lipid Res., May 1, 2001; 42(5): 710 - 715. [Abstract] [Full Text]
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) and nonfasting (
) RLP-C
concentrations in serum, EDTA plasma, and heparin plasma from 38
subjects after storage at 4 °C up to 4 weeks (left) and
at -70 °C up to 6 months (right).
), and d
>1.019 kg/L (
) fractions. RLP-C was measured in each of the four
fractions. RLP-C concentrations in the 1.006–1.019 kg/L fraction were
calculated by subtracting the RLP-C in the d <1.006 kg/L
fraction from the RLP-C in the d <1.019 kg/L fraction.