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Articles |
1
Central Clinical Laboratories, Shinshu University Hospital, 3-1-1 Asahi, Matsumoto 390-8621, Japan.
2
Department of Functional Polymer Science, Faculty of
Textile Science and Technology, Shinshu University, 3-15-1 Tokita, Ueda
386-0018, Japan.
3
Department of Laboratory Medicine, Shinshu University
School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan.
a Author for correspondence. Fax 81-263-34-5316; e-mail mtozuka{at}hsp.md.shinshu-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We adapted an assay for CSF apoE sensitivity using an ELISA. It allowed us to measure CSF apoE with sufficient reproducibility and precision.
Results: The within- and between-run CVs were <7%, and the detection limit was 0.025 mg/L. No cross-reaction was found for other apolipoproteins. No significant differences related to sex or apoE phenotype were observed in the CSF apoE concentration. The mean CSF apoE concentration was significantly higher in the 0–5 year group (n = 6; 18.47 ± 1.14 mg/L, mean ± SD) than in the >5 year group (n = 34; 8.82 ± 3.31 mg/L). The mean concentrations of total cholesterol (TC) and phospholipid (PL) in CSF were 2.68 ± 2.16 and 6.50 ± 2.84 mg/L (n = 52), respectively. Although no significant differences in TC or PL in the CSF were found with respect to sex or age, the concentrations in subjects with the apoE phenotype E4/E3 were significantly lower than in those with E3/E3 and E3/E2. The concentrations of apoE, TC, and PL in CSF did not correlate with those in plasma. The time-related fluctuations in CSF apoE were independent of those in total protein and IgG. CSF apoE was significantly correlated with TC and PL concentrations in the CSF, but not with the number of cells in the CSF.
Conclusions: These findings support the idea that apoE and lipids are unable to cross the blood-brain barrier and that their concentrations in CSF may reflect production in central nervous tissue.© 1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A subject's apoE phenotype is known to affect the plasma total cholesterol (TC) concentration as follows: E2/E2 < E3/E2 < E3/E3 < E4/E3 < E4/E4 (except in type III hyperlipidemia) (7). apoE reportedly exists in the cerebrospinal fluid (CSF) as a component of HDL subfraction 1 (8)(9) and transports cholesterol and phospholipid (PL) through the LDL receptors expressed in elements of the nervous system such as neurons and glia (9)(10)(11)(12). Over the past 10 or so years, several groups have measured CSF apoE to investigate its clinical relevance to neurological diseases such as multiple sclerosis (13)(14)(15) and Alzheimer disease (AD) (16)(17). apoE expressed in the brain is also thought to contribute to the growth and repair of the nervous system (18)(19)(20). However, information relating to CSF apoE, such as data on the influence of apoE phenotype on apoE and lipid concentrations in the CSF or on the relationship between CSF apoE and lipid concentrations, has not been reported in as much detail as that relating to plasma apoE.
The concentration of apoE in the CSF is approximately one-tenth of that in the plasma (15); therefore, the assay method for CSF apoE requires greater sensitivity. We devised an improved assay for apoE by adapting an ELISA method originally developed by Bury et al. (21), which enabled us to use CSF without high multiple predilution. In addition, the TC and PL concentrations in the CSF were also examined, and the composition of the lipoprotein content of the CSF was assessed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subjects
One hundred sixteen CSF samples with no contamination by
erythrocytes were obtained from subjects 0–82 years of age (mean
± SD, 45.1 ± 22.3 years). In 40 of these subjects, serum samples
were obtained in pairs with the CSF samples on the same day. These
samples were stored at -80 °C before use. All subjects gave their
informed consent before participation. One hundred ten of these CSF
samples were obtained from subjects >5 years of age; these were used
to compare apoE concentrations between groups with (n = 76) or
without (n = 34) neurological disease. TC and PL were measured in
the 52 of the 116 CSF samples that were of sufficient volume.
elisa procedure
Sample preparation.
All samples were diluted
with an equal volume of phosphate-buffered saline (PBS), pH 7.4,
containing 1.0 g/L Tween 20 (PBS-Tween 20).
Calibrators.
Calibrators were prepared by dilution of
reference serum (purchased from Daiichi Pure Chemicals Co. Ltd.) to 0,
0.05, 0.10, 0.50, 5.0, and 25.0 mg/L using PBS-Tween 20.
Assay procedure.
Polystyrene microtiter plates (Nunc TM) were
coated with anti-apoE polyclonal antibody (rabbit; Dako) in 0.1 mol/L
Na2CO3, pH 9.6 (1.2 mg
protein/L) and incubated at 4 °C overnight. Plates were washed five
times with PBS-Tween 20 after each of the subsequent incubation steps.
Unoccupied sites were blocked with 10 g/L skim milk in PBS-Tween 20 for
2 h at room temperature. The calibrators and samples were then
added at 100 µL/well and incubated for 2 h at 25 °C.
Anti-apoE monoclonal antibody in PBS (0.25 mg protein/L) was added at
100 µL/well and incubated for 1 h at 37 °C.
Peroxidase-conjugated anti-mouse IgG (goat; MBL), diluted 3000-fold
with PBS, was then added at 100 µL/well and incubated for 1 h at
37 °C. After the final washing, the color reaction was developed
with 100 µL/well of 5 g/L t-methylbenzidine
dihydrochloride and hydrogen peroxide, followed by 100 µL/well of 0.4
mol/L sulfuric acid to stop the reaction. The absorbance at 450 nm was
measured by a Behring ELISA processor II (Behringwerke). A calibration
curve was generated using a semilogarithmic scale, and the apoE
concentration in the CSF was calculated from the curve. Each assay was
carried out in triplicate.
isoelectric focusing
Sample preparation.
CSF (15 µL) was incubated with 5 µL of
neuraminidase (20 kU/L; Nakarai Chemical) in 0.1 mol/L citric acid
buffer containing 10 g/L Tween 20 for 12 h at 37 °C, and then
treated with 5 µL of 75 mmol/L dithiothreitol (Wako Pure Chemicals)
in 10 g/L Tween 20 for 1 h at room temperature.
Electrophoresis and immunoblotting.
Immunoblotting was carried out as described previously
(22). Briefly, 20 µL of the prepared sample
was electrophoresed on a 4.8% polyacrylamide gel containing 8 mol/L
urea and 20 mL/L ampholine (pH 4~6) using 3.3 mmol/L phosphoric acid
as the anode buffer and 20 mmol/L NaOH as the cathode buffer.
Electrophoresis was performed overnight at 4 °C under constant
voltage (200 V). The separated proteins were electrophoretically
transferred onto nitrocellulose membranes, which were then incubated
with 50 mmol/L Tris-HCl, pH 8.0, containing 20 g/L skim milk (blocking
buffer) for 30 min at room temperature. The membranes were then
incubated with anti-apoE polyclonal antibody in blocking buffer for
1 h at room temperature. After washing, the membranes were
incubated with peroxidase conjugated anti-rabbit IgG for 1 h at
room temperature. The bands representing the various apoE isoforms were
developed using 3,3'-diaminobenzidine tetrahydrochloride (Dojin
Chemical Co.) and hydrogen peroxide (Wako).
other assays
Serum apoE.
The serum apoE concentration was determined by
turbidimetric immunoassay using a commercially available kit (ApoE Auto
N DAIICHI; Daiichi) and a Hitachi 7170 automated analyzer.
Total protein in CSF.
The total protein concentration in CSF
was determined by the Pyrogallol red method (Wako) using a Hitachi 7170
automated analyzer.
TC and PL in CSF.
The TC and PL concentrations in CSF were
determined, respectively, by the cholesterol-oxidase method (Kyowa
Medex Co. Ltd.) and the choline-oxidase method (Wako) using a Hitachi
7170 automated analyzer. The assay conditions were modified by
increasing the ratio between sample volume and reagent volume to
15-fold higher than the ratio used for serum.
IgG in CSF.
IgG in CSF was determined by laser nephelometric
immunoassay using a Behring Nephelometer-Analyzer (Behringwerke).
statistical methods
The CSF apoE value for each phenotype was expressed as mean and
SD, as calculated using Microsoft Excel Ver.
5.0® (Microsoft). apoE values obtained by this
method in diluted patient sera and by turbidimetric immunoassay were
compared by linear regression analysis. The relationships among CSF
apoE, serum apoE, CSF lipid, and CSF total protein were determined
from the correlation coefficient obtained by linear regression
analysis. The statistical difference between any two groups was
assessed by the Student or Welche t test, P
<0.05 being considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The absorbance of the zero calibrator (PBS-Tween 20), as
measured on five different plates as nonspecific binding, was 9.4
± 1.4 (x 10-3, mean ± SD). The minimum
detection limit, calculated from the mean absorbance of the zero
calibrator plus 2 SD (based on 10 replicate measurements of PBS-Tween
20), was 0.025 mg/L. The upper limit of the assay, as determined
by a series of dilutions, was 25 mg/L.
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Precision.
The within-run reproducibility was determined by
making 20 replicate measurements of three diluted serum samples on the
same plate (CV, 2.4–7.0%). The between-run reproducibility was
determined by making triplicate measurements of two diluted serum
samples on each of 10 consecutive days (CV, 5.7–9.0%; Table 1
).
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Accuracy.
The accuracy of this assay was confirmed by
analytical recovery studies using purified human apoE. The recovery
rates from samples to which 0.50 and 1.45 mg/L of apoE had been added
were 84.0% and 89.0%, respectively.
Lot-to-lot variability.
To assess the lot-to-lot variability,
we analyzed 32 CSF samples twice using two different plates coated with
anti-apoE polyclonal antibody on different occasions. The mean apoE
values were 10.9 ± 5.6 and 10.3 ± 5.4 mg/L (mean ±
SD), respectively. The linear regression equation was:
y = 0.960x - 0.133; r
= 0.998; Sy|x = 0.3285.
Specificity.
Assay specificity was estimated by measuring the
cross-reactivity with other apos. The absorbance values measured for
apoAI, apoAII, and apoB at a maximum concentration of 500 mg/L, and for
apoCII and apoCIII at a maximum concentration of 100 mg/L in place of
apoE were roughly similar to that obtained for the zero calibrator
(data not shown).
Intermethod comparison.
We examined the correlation between
the apoE values obtained using the present assay (y) and
those obtained using a turbidimetric immunoassay (x) using
diluted and undiluted serum samples, respectively. The mean values for
apoE obtained using the present assay and the turbidimetric immunoassay
were 51.6 ± 20.2 and 53.4 ± 23.7 mg/L (mean ± SD),
respectively. The linear regression equation was: y =
0.768x + 10.521; r = 0.903.
clinical observations
apoE concentration.
The age-related distribution of CSF
apoE for the 41 of 116 subjects who had no neurological disease is
shown in Fig. 2
. The concentration in the 0–5 year age group (n = 6; age,
3.1 ± 2.0 years) was significantly higher (mean concentration,
18.47 ± 1.14 mg/L) than that in the group >5 years of age
(n = 34; 8.82 ± 3.31 mg/L). No significant difference in CSF
apoE concentration was observed between males and females or among the
three major apoE phenotypes; however, a significant difference was
observed between the controls and those with a neurological disease
(Table 2
) (23).
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Lipid concentration in CSF.
The within- and between-run CVs
for the TC and PL assays using diluted serum (mean TC, 1.56 mg/L; mean
PL, 1.99 mg/L) were <6.3% and 9.7%, respectively. The minimum
detection limits for TC and PL, calculated from the mean values for the
zero calibrator plus 2 SD (based on 10 replicate measurements of PBS),
were 0.10 and 0.08 mg/L, respectively. The mean concentrations (with
SD) of TC and PL are shown in Table 3
. Significantly higher concentrations of both lipids were found
in phenotype E3/E2 (or E3/E3) than in E4/E3. However, age, sex, and the
presence of neurological disease had no effect on these concentrations
(data not shown).
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Relationship between CSF apoE and other components.
As shown
in Fig. 3
, the apoE, TC, and PL concentrations in the CSF showed no
correlation with those in the serum (n = 40; apoE,
r = 0.100; TC, r = 0.221; PL,
r = 0.161). However, the lipid concentrations in the
CSF correlated significantly with the CSF apoE (n = 40; vs TC,
r = 0.604, P <0.001; vs PL,
r = 0.702, P <0.001; Fig. 4
). The relationships between apoE and other major components
(total protein, IgG, and number of cells in the CSF) were investigated
by linear regression analysis. CSF apoE showed extremely weak
correlations with total protein (n = 112; r =
0.288; P <0.01) and IgG (n = 95; r =
0.273; P <0.01), but no correlation with the number of
cells (n = 95; r = 0.028). The time-related
fluctuations in apoE, total protein, and IgG concentrations in the CSF
of three different patients with neurological disease are shown in Fig. 5
. The changes in these values seemed to be quite independent of
each other in all three cases. In each case, apoE varied more than
total protein.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The reference interval calculated from the CSF apoE concentrations of the subjects without neurological disease was similar to previous data presented by Carlsson et al. (15). Although the CSF apoE concentrations were not sex-related, they were significantly higher for children <5 years of age than for subjects >5 years of age. Neuronal growth, such as the extension of neurons, is remarkably active at an early age. Previous studies have also demonstrated that apoE synthesized within the CNS plays an important role in the growth, repair, and maintenance of neuronal tissues (9)(10)(11)(12). This could account for the relatively high concentration of CSF apoE seen at an early age. Indeed, in newborn infants CSF apoE concentrations would be expected to be even higher than the concentrations reported here.
The TC and PL concentrations in serum generally are known to increase with age and to be distinctly different between males and females (24). However, we did not observe these tendencies in the CSF. Our data indicate that the lipid concentrations in the CSF are not affected by the physiological status of the individual and that they are regulated at a relatively constant concentration in each individual. Interestingly, significant differences were recognized in both TC and PL concentrations among the three major apoE phenotypes. Serum TC concentration, except in the case of type III hyperlipidemia, is known to vary in the rank order E2/E2 < E3/E2 < E3/E3 < E4/E3 < E4/E4 (7). In contrast, the CSF TC concentration was higher in E3/E2 (or E3/E3) than in E4/E3.
apoE4 is known to be a risk factor for AD, because the frequency with which the apoE-4 allele occurs in AD patients is significantly higher than in control subjects (25)(26)(27)(28). Recently, the isoform-specific binding of apoE to ß-amyloid peptides, which form a major component of senile plaque and neurofibrillary tangles (24)(29), has been studied (30)(31)(32) to elucidate the mechanism underlying the participation of apoE in AD and the differences in the effects of the various apoE isoforms on the development of AD. However, the conclusions reached in these studies are not entirely consistent with each other. It is believed that ß-amyloid peptides bind to the domain near the carboxy terminus of apoE (33), which is also the region with the highest potential for lipid binding (1). This indicates that competition may exist between ß-amyloid peptides and lipids for apoE. Our finding of a significant difference in CSF lipid concentrations between the apoE phenotypes E4/E3 and E3/E2 (or E3/E3), could be the key to the mechanism underlying the participation of apoE in the development of AD.
The mean (TC + PL)/apoE ratio in the CSF was 1.17. This supports the idea that the main lipoprotein in the CSF may consist of lipids and apoE in a ratio of ~1:1. This result agrees with previous reports that apoE exists in CSF in the form of HDL1 (8)(9). Recently, apoJ and traces of apoAI and AII have been identified in CSF, in addition to apoE (9)(34)(35). LaDu et al. (34) suggested that the CSF lipoprotein-containing apoE exists in large particles, that apoAI and apoAII are localized in smaller particles, and that apoJ is evenly distributed among all particle sizes. In addition, Guyton et al. (36) identified a novel large apoE-containing lipoprotein in CSF with a density of 1.006–1.060 kg/L in addition to HDL1, which has a density of 1.063–1.21 kg/L. In the present study, a significant correlation was observed between lipids and apoE, but the scatter around the regression lines was quite large. Taken together, these findings indicate that CSF lipoproteins may be microheterogeneous in size and composition and that the various particle sizes may be in a different ratio in each individual. A child <5 years of age and a subject with apoE phenotype E4/E3 would be expected to be extreme cases and would help to verify these ideas.
The apoE, TC, and PL concentrations in CSF did not correlate with those in serum. As indicated previously (15), such findings suggest that the apoE and lipids in the plasma are unable to cross the blood-brain barrier. Thus, the concentrations would reflect the local production within the CNS. The lack of strong correlations between apoE and total protein, IgG, and the number of cells in the CSF also indicates an independent regulation of CSF apoE.
Rifai et al. (13) suggested that the "apoE Index" (calculated from the concentration of apoE and albumin in CSF and serum) is useful in discriminating between remission and exacerbation in multiple sclerosis patients. Blennow et al. (16) and Lehtimäki et al. (17) showed a significant reduction in CSF apoE concentrations in AD patients compared with controls. These results indicate why we need a simple assay for CSF apoE of the kind described here, i.e., because of the importance of CSF apoE as an independent parameter in CNS disease.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, reference serum; 
, purified apoE; 
, patient CSF.
, apoE; 
, IgG.