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Technical Briefs |
1
Department of Laboratory Medicine, Niigata University School of Medicine, Asahimachi 1-757, Niigata City, Niigata 951-8510, Japan;
2
Central Clinical Laboratory and
3
Division of Pharmacy, Niigata University Medical Hospital, Niigata 951-8520, Japan;
4
Department of Pediatrics, Niigata Cancer Center Hospital, Niigata 951-8566, Japan;
5
Department of Internal Medicine, Kashiwazaki Central Hospital, Kashiwazaki 945-0055, Japan;
6
Third Internal Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan;
a author for correspondence: fax 81-223-0996, e-mail miida{at}med.niigata-u.ac.jp
The blood-brain barrier keeps the protein concentrations in cerebrospinal fluid (CSF) much lower than in serum (1)(2)(3)(4). However, the CSF apolipoprotein E (apoE) concentration is approximately one-tenth to one-twentieth of the serum apoE concentration (5)(6)(7)(8)(9)(10)(11)(12)(13). Mainly glia cells secrete apoE in the central nervous system (14)(15)(16). CSF apoE is carried exclusively on HDL, which is the major lipoprotein in the CSF (17)(18). The CSF apoE concentration varies in neurological disorders such as central nervous system inflammatory diseases (5)(19) and Alzheimer disease (7)(8)(9)(10)(11)(12)(13). However, the clinical significance of the CSF apoE concentration is still unclear.
Recent studies have suggested that the apoE content of CSF HDL is more important than the CSF apoE concentration (20). HDL enriched with apoE promotes nerve growth factor-induced neurite outgrowth (20). Because the number of synapses increases markedly in childhood (21), the apoE content of CSF HDL might be higher in children than in adults. To address this question, we measured the apoE and phospholipid (PL) concentrations in CSF simultaneously.
Samples were obtained from 59 neurologically normal subjects (42 males and 17 females, ages 2–86 years). Of the 59 subjects, 30 were patients with acute leukemia in complete remission, the others were patients who underwent surgery under lumbar anesthesia. The serum albumin and C-reactive protein concentrations were within the reference intervals in all of the subjects. Informed consent was obtained from all of the subjects or their parents. CSF was collected by lumbar puncture and centrifuged at 1500g for 5 min. We excluded samples that had macroscopic contamination with red blood cells. In all samples, the laboratory values (including cell counts and glucose, protein, and electrolyte concentrations) were within the reference intervals for CSF (data not shown). Simultaneous blood samples were obtained from all the subjects.
The apoE concentration was determined by a turbidimetric immunoassay (ApoAuto Daiichi; Daiichi Pure Chemicals), using an automated analyzer (Hitachi 7170; Hitachi). The calibration curve was linear in the range 1.0–15.0 mg/L. The PL concentration was measured enzymatically using a commercial kit (Phospholipid C-test Wako; Wako Pure Chemical). To measure extremely low PL concentrations, we modified the manufacturer's protocols in the following way. Reagents containing enzymes were dissolved at 10-fold concentrations with the buffers. Each sample was mixed with the enzyme solution in a 2:1 ratio (333 mL/L), whereas the ratio in the original protocol was 1: 150 (6.6 mL/L). The mixture was incubated at 37 °C for 5 min. The absorbance was measured at 600 nm. The calibration curve was linear in the range 1.0–15.0 mg/L (2.6–38.8 µmol/L). The albumin concentration was measured on a Hitachi 7170 with a turbidimetric immunoassay.
The lipoproteins were separated by agarose gel electrophoresis and electrically blotted onto nitrocellulose paper (pore size; 0.45 µm; Sartorius) (22). The apolipoprotein distributions were detected by the immunoblot. The particle size distribution was analyzed by high-speed chemical derivatization chromatography (CCPD; Tosoh) using a gel permeation column (TSKgel Lipopropak, 300 mm x 7.5 mm; Tosoh). Undiluted CSF was applied to the HPLC system. The postcolumn effluent was mixed with the enzyme solution (Determiner TC 555) at 45 °C to detect cholesterol. The absorbance at 550 nm was measured.
With agarose gel electrophoresis, the apoE band was detected together
with the apoAI and AII bands at the slow 
position. In HPLC
analysis, HDL was the major lipoprotein in CSF. CSF HDL was eluted a
little more slowly than serum HDL (Fig. 1
).
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Although the concentrations of the CSF components were very low, we could measure them with sufficient accuracy. The intra- and interassay coefficient of variations (CVs) were <3% and <7% for apoE, <2% and <5% for PL, and <2% and <2% for albumin, respectively. The CSF apoE concentration was not correlated with the serum apoE concentration (r = 0.151). Moreover, the CSF apoE concentration was not correlated with the CSF albumin concentration (r = 0.093), although the CSF albumin concentration was positively correlated with the serum albumin concentration (r = 0.309; P <0.05).
Age was the important factor in determining CSF HDL concentrations and
composition. The CSF apoE concentration was higher in children than in
adults 20–59 years of age (Table 1
). On the other hand, CSF PL was highest in the group 
60 years
of age. The apoE/PL ratio was higher in children than in adults. The
CSF/serum albumin ratio, a marker for the impaired blood-brain barrier,
increased with age.
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This study is unique in that we measured the concentrations of both the
protein (apoE) and lipid (PL) components in CSF. Some previous
investigators measured only the CSF apoE concentration
(5)(6)(7)(8)(9)(10)(11)(12)(13), whereas others measured only the CSF cholesterol or
PL concentration (23)(24). The reported values
were quite similar to ours (5)(6)(7)(8)(9)(10)(11)(12)(13)(24). Because
we measured both concentrations, we were able to calculate the CSF
apoE/PL ratio. The majority of the CSF lipoprotein is apoE-containing
HDL (Fig. 1
). In addition, PL is the major lipid component of HDL
(25). Thus, the CSF apoE/PL ratio is likely to reflect the
apoE content of CSF HDL.
Earlier studies suggested that the apoE content of CSF HDL is more
important than the CSF apoE concentration (20). In cultured
neuronal cells, nerve growth factor stimulates neurite outgrowth. When
either CSF HDL or apoE alone was added to neuronal cells, nerve growth
factor-dependent neurite outgrowth did not change at all. When CSF HDL
and apoE were added to the cells simultaneously, the outgrowth was
enhanced (20). Using the CSF apoE/PL ratio, the apoE content
of CSF HDL was ~70% higher in children than in adults 20–59 years
of age (Table 1
). Therefore, it is possible that CSF HDL in children
promotes neurite outgrowth without exogenous apoE.
The high apoE/PL ratio in children is not caused by an immature
blood-brain barrier. An impaired blood-brain barrier can be diagnosed
using the CSF/serum albumin ratio (26). This ratio increases
slightly after birth but falls to adult values by age 6 months and
gradually increases again after age 40 (26). In our study,
the CSF/serum albumin ratio was the lowest in children (Table 1
).
Therefore, the high apoE/PL ratio in children was not the result of
increased permeability of the blood-brain barrier. In addition, the CSF
apoE concentration was not correlated with serum apoE or CSF albumin.
These results also support our hypothesis. We conclude that the apoE
content of CSF HDL is higher in children than in adults. We speculate
that apoE-enriched CSF HDL might favor central nervous system
development in children.
Acknowledgments
This research was supported by a grant from the Kurozumi Medical Foundation (1998).
References
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