doi: 10.1161/01.ATV.0000134391.01498.b8
© 2004 American Heart Association, Inc.
Letters to the Editor |
Associations Among Plasma Lipoprotein Subfractions as Characterized by Analytical Capillary Isotachophoresis, Apolipoprotein E Phenotype, Alzheimer Disease, and Mild Cognitive Impairment
Department of Cardiology, Fukuoka University School of Medicine, Fukuoka, Japan
Department of Neurology, Fukuoka University School of Medicine, Fukuoka, Japan
To the Editor:
Alzheimer disease (AD) is the most common form of dementia, and the central pathogenic event is the abnormal accumulation of amyloid ß–protein (Aß) in extracellular amyloid deposits and cerebral blood vessels.1 AD is a complex and genetically heterogeneous disease. Mild cognitive impairment (MCI), a cognitive disorder in the transition between normal cognition and AD, is a known risk factor for AD, with a conversion rate of 
10% per year.2
Apolipoprotein E (apoE), a lipid transporter, has been found to be contained in amyloid plaques. The apoE4 isoform or APOE 
4 allele is associated with the development of AD1 and an increased risk of MCI.3 Cholesterol has also been identified as a risk factor for AD1,4 and MCI.5 A direct role of cholesterol in the pathogenesis of AD has been suggested by studies in transgenic animal models of AD: cholesterol feeding increases Aß accumulation and accelerates AD-related pathology,6 whereas cholesterol lowering with statin reduces Aß pathology.7
Although CAD is a prevalent finding in AD,8 whether or not plasma lipoprotein subfractions are associated with MCI and AD has not yet been investigated. The separation and determination of lipoprotein subfractions are generally labor-intensive and time-consuming. Recently, however, the research group of Schmitz and coworkers developed a new automated technique to separate and quantify lipoprotein subfractions in minutes using capillary isotachophoresis (cITP).9,10 Therefore, in the present study, we investigated the associations among lipoprotein subfractions as determined by cITP, apoE phenotype, MCI, and AD.
Twenty-eight patients with MCI, 47 patients with AD, and 26 nondemented control subjects were evaluated at the Neurology Department of Fukuoka University Hospital between 2002 and 2003. Global cognitive impairment was assessed by the Mini-Mental State Examination (MMSE)11 and Clinical Dementia Rating (CDR).12 Patients with MCI (CDR=0.5) met the diagnostic criteria for MCI formulated by the Mayo Clinic Alzheimer Disease Research Center (MCADRC) in Rochester, MN.13 Patients with AD met the National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer Disease and Related Disorders Association (NINCDS-ADRDA) criteria for probable AD and had a CDR score >1. Two patients with AD and one patient with MCI received pravastatin. Nondemented control subjects were volunteers who underwent a standard medical check up and were judged to be in good health. They were required to have a CDR score of 0. This study was approved by the Ethics Committee of Fukuoka University, and samples were collected only after the participants had given their informed consent.
ApoE phenotypes were determined by using isoelectric focusing, as described previously,14 and were confirmed by apoE genotyping.15 ApoE phenotype–genotype nonconcordance was found in one control subject (E2/E3 and E3/E3), and the results of apoE genotyping were used. Capillary isotachophoresis of plasma lipoproteins was performed on a Beckman P/ACE MDQ system (Beckman-Coulter Inc.) according to the method of Bottcher et al9 with some modifications, as described previously.16 Plasma lipoprotein was stained with NBD C6-ceramide (Molecular Probes, Inc.), a lipophilic dye. The peak area for each cITP lipoprotein subfraction relative to that of the internal marker was presented as the level of the cITP lipoprotein. Agarose gel electrophoresis and differential staining were performed using a Rapid Electrophoresis system (REP, Helena Laboratories) according to the method of Kido et al.17 REP Lipo-30 plate and CHOL/TRIG COMBO (K.K. Helena Kenkyujo) were used as the agarose gel and staining reagents, respectively.
Patients with AD included significantly more females (87.2% versus 61.5%, P<0.05), were older (76.9±0.6 years versus 74.2±0.9 years), and had lower MMSE scores (18.8±0.8 versus 29.2±0.2) than nondemented control subjects. Patients with AD included a significantly higher percentage of subjects carrying the apoE4 isoform than controls and patients with MCI (Table). Serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and apoB in patients with both MCI and AD were significantly higher than those in controls as assessed by Scheffe’s multiple comparison test (Table). The differences among groups were significant after adjusting for age and sex by an analysis of covariance (Table).
|
The Figure shows typical electropherograms of plasma lipoprotein subfractions as characterized by cITP (left) and serum lipid profiles analyzed by agarose gel electrophoresis (right) for a young normal healthy subject (A), a patient with MCI (B), and a patient with AD (C). As shown, plasma lipoproteins were separated into 7 fractions by capillary isotachophoresis: three high-density lipoprotein (HDL) fractions (peaks 1 to 3: fast [f]-, intermediate [i]-, and slow [s]-migrating HDL), a chylomicron/remnants fraction (peak 4), a very low density lipoprotein (VLDL)/intermediate density lipoprotein (IDL) fraction (peak 5), and two LDL fractions (peaks 6 to 7: fast- and slow-migrating LDL). The three cITP HDL subfractions (fHDL, iHDL, and sHDL) corresponded to 
lipoprotein in agarose gel electrophoresis (Figure). The fLDL and sLDL corresponded to ß lipoprotein in agarose gel electrophoresis (Figure). The peak area relative to internal marker (RPA) for each lipoprotein fraction in patients with MCI and AD and control subjects is shown in the Table. Patients with AD had a significantly higher RPA for fHDL than control subjects, and patients with both AD and MCI had a significantly higher RPA for sLDL than control subjects. A multiple logistic regression analysis showed that the associations between cITP sLDL and MCI (odds ratio [95% CI]: 10 [2.4 to 40]) and between cITP sLDL and AD (9.6 [2.5 to 48]) were independent of apoE phenotype (P<0.01) after adjusting for age and sex.
|
This is the first examination of the association between charged-based LDL subfractions and MCI or AD. Because cITP separates plasma lipoproteins based on electric mobility, the fLDL fraction carries a more-negative electric charge than the sLDL fraction. The electronegative fLDL could be an atherogenic LDL because we found that this LDL fraction inversely correlates with the size of LDL and increases with the oxidation of LDL (unpublished data). We did not detect a significant association between the cITP fLDL subfraction and either MCI or AD, possibly because of the relatively small number of control subjects (Table). However, we found that the major LDL (sLDL) fraction was associated with both MCI and AD. Our findings support the notion that the increased synthesis of cholesterol is related to the progression of cognitive impairment. Our finding that the association between the cITP sLDL subfraction and AD was independent of the apoE4 isoform is consistent with other reports that the association between the serum TC level and AD is independent of the apoE genotype.18
Our finding that the cITP sLDL subfraction was increased in both MCI and AD patients suggests that early lipid-lowering therapy in MCI patients could be beneficial, considering that MCI is a known risk factor for AD. In fact, simvastatin has been shown to significantly decrease Aß40 in the cerebrospinal fluid of patients with mild Alzheimer disease, but not in severely affected patients.19
In conclusion, the charge-based major LDL subfraction as characterized by cITP was associated with both MCI and AD, independent of apoE isoforms, suggesting that the combination of apoE4 and cITP sLDL may be a better indicator of AD than the apoE4 isoform alone. Further large-scale studies are needed to confirm these findings.
Acknowledgments
Acknowledgments
This work was supported by grants-in-aid from the Ministry of Education, Science, and Culture of Japan (11670724 and 15790403), by a research grant from the Clinical Research (2003), by research grants from the Ministry of Health and Welfare, by a grant from Uehara Memorial Foundation (2002), and by research grants (996006 and 026001) from the Central Research Institute of Fukuoka University.
References
1. Puglielli L, Tanzi RE, Kovacs DM. Alzheimer’s disease: the cholesterol connection. Nat Neurosci. 2003; 6: 345–351.[CrossRef][Medline] [Order article via Infotrieve]
2. Turner RS. Biomarkers of Alzheimer’s disease and mild cognitive impairment: are we there yet? Exp Neurol. 2003; 183: 7–10.[CrossRef][Medline] [Order article via Infotrieve]
3. Tervo S, Kivipelto M, Hanninen T, Vanhanen M, Hallikainen M, Mannermaa A, Soininen H. Incidence and risk factors for mild cognitive impairment: a population-based three-year follow-up study of cognitively healthy elderly subjects. Dement Geriatr Cogn Disord. 2004; 17: 196–203.[CrossRef][Medline] [Order article via Infotrieve]
4. Koudinov AR, Berezov TT, Koudinova NV. Cholesterol and Alzheimer’s disease: is there a link? Neurology. 2002; 58: 1135.
5. Kivipelto M, Helkala EL, Hanninen T, Laakso MP, Hallikainen M, Alhainen K, Soininen H, Tuomilehto J, Nissinen A. Midlife vascular risk factors and late-life mild cognitive impairment: a population-based study. Neurology. 2001; 56: 1683–1689.
6. Refolo LM, Malester B, LaFrancois J, Bryant-Thomas T, Wang R, Tint GS, Sambamurti K, Duff K, Pappolla MA. Hypercholesterolemia accelerates the Alzheimer’s amyloid pathology in a transgenic mouse model. Neurobiol Dis. 2000; 7: 321–331.[CrossRef][Medline] [Order article via Infotrieve]
7. Refolo LM, Pappolla MA, LaFrancois J, Malester B, Schmidt SD, Thomas-Bryant T, Tint GS, Wang R, Mercken M, Petanceska SS, Duff KE. A cholesterol-lowering drug reduces ß-amyloid pathology in a transgenic mouse model of Alzheimer’s disease. Neurobiol Dis. 2001; 8: 890–899.[CrossRef][Medline] [Order article via Infotrieve]
8. Sparks DL, Martin TA, Gross DR, Hunsaker JC 3rd. Link between heart disease, cholesterol, and Alzheimer’s disease: a review. Microsc Res Tech. 2000; 50: 287–290.[CrossRef][Medline] [Order article via Infotrieve]
9. Bottcher A, Schlosser J, Kronenberg F, Dieplinger H, Knipping G, Lackner KJ, Schmitz G. Preparative free-solution isotachophoresis for separation of human plasma lipoproteins: apolipoprotein and lipid composition of HDL subfractions. J Lipid Res. 2000; 41: 905–915.
10. Schmitz G, Mollers C, Richter V. Analytical capillary isotachophoresis of human serum lipoproteins. Electrophoresis. 1997; 18: 1807–1813.[CrossRef][Medline] [Order article via Infotrieve]
11. Folstein MF, Folstein SE, McHugh PR. "Mini-mental state." A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res. 1975; 12: 189–198.[CrossRef][Medline] [Order article via Infotrieve]
12. Fernandes MA, Proenca MT, Nogueira AJ, Oliveira LM, Santiago B, Santana I, Oliveira CR. Effects of apolipoprotein E genotype on blood lipid composition and membrane platelet fluidity in Alzheimer’s disease. Biochim Biophys Acta. 1999; 1454: 89–96.[Medline] [Order article via Infotrieve]
13. Hanninen T, Hallikainen M, Tuomainen S, Vanhanen M, Soininen H. Prevalence of mild cognitive impairment: a population-based study in elderly subjects. Acta Neurol Scand. 2002; 106: 148–154.[CrossRef][Medline] [Order article via Infotrieve]
14. Matsunaga A, Sasaki J, Moriyama K, Arakawa F, Takada Y, Nishi K, Hidaka K, Arakawa K. Population frequency of apolipoprotein E5 (Glu3–>Lys) and E7 (Glu244–>Lys, Glu245–>Lys) variants in western Japan. Clin Genet. 1995; 48: 93–99.[Medline] [Order article via Infotrieve]
15. Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI. J Lipid Res. 1990; 31: 545–548.[Abstract]
16. Zhang B, Noda K, Saku K. Effect of atorvastatin on total lipid profiles assessed by analytical capillary isotachophoresis. Cardiology. 2003; 99: 211–213.[CrossRef][Medline] [Order article via Infotrieve]
17. Kido T, Kurata H, Matsumoto A, Tobiyama R, Musha T, Hayashi K, Tamai S, Utsunomiya K, Tajima N, Fidge N, Itakura H, Kondo K. Lipoprotein analysis using agarose gel electrophoresis and differential staining of lipids. J Atheroscler Thromb. 2001; 8: 7–13.[Medline] [Order article via Infotrieve]
18. Kivipelto M, Helkala EL, Laakso MP, Hanninen T, Hallikainen M, Alhainen K, Iivonen S, Mannermaa A, Tuomilehto J, Nissinen A, Soininen H. Apolipoprotein E 4 allele, elevated midlife total cholesterol level, and high midlife systolic blood pressure are independent risk factors for late-life Alzheimer disease. Ann Intern Med. 2002; 137: 149–155.
19. Simons M, Schwarzler F, Lutjohann D, von Bergmann K, Beyreuther K, Dichgans J, Wormstall H, Hartmann T, Schulz JB. Treatment with simvastatin in normocholesterolemic patients with Alzheimer’s disease: a 26-week randomized, placebo-controlled, double-blind trial. Ann Neurol. 2002; 52: 346–350.[CrossRef][Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  B. Zhang, A. Matsunaga, D. L. Rainwater, S.-i. Miura, K. Noda, H. Nishikawa, Y. Uehara, K. Shirai, M. Ogawa, and K. Saku Effects of rosuvastatin on electronegative LDL as characterized by capillary isotachophoresis: the ROSARY Study J. Lipid Res., September 1, 2009; 50(9): 1832 - 1841. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Imaizumi, S.-i. Miura, K. Nakamura, Y. Kiya, Y. Uehara, B. Zhang, Y. Matsuo, H. Urata, M. Ideishi, K.-A. Rye, et al. Antiarrhythmogenic effect of reconstituted high-density lipoprotein against ischemia/reperfusion in rats. J. Am. Coll. Cardiol., April 22, 2008; 51(16): 1604 - 1612. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Zhang, Y. Uehara, S. Hida, S.-i. Miura, D. L. Rainwater, M. Segawa, K. Kumagai, K.-A. Rye, and K. Saku Effects of reconstituted HDL on charge-based LDL subfractions as characterized by capillary isotachophoresis J. Lipid Res., May 1, 2007; 48(5): 1175 - 1189. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Zhang, T. Kaneshi, T. Ohta, and K. Saku Relation between insulin resistance and fast-migrating LDL subfraction as characterized by capillary isotachophoresis J. Lipid Res., October 1, 2005; 46(10): 2265 - 2277. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||