Nagata et al. Biomarker Research (2018) 6:5 DOI 10.1186/s40364-018-0119-x RESEARCH Open Access Comparative analysis of cerebrospinal fluid metabolites in Alzheimer’s disease and idiopathic normal pressure hydrocephalus in a Japanese cohort Yuki Nagata1* , Akiyoshi Hirayama2, Satsuki Ikeda2, Aoi Shirahata2, Futaba Shoji2, Midori Maruyama2, Mitsunori Kayano3, Masahiko Bundo4, Kotaro Hattori5, Sumiko Yoshida5, Yu-ichi Goto5, Katsuya Urakami6, Tomoyoshi Soga2, Kouichi Ozaki1 and Shumpei Niida1 Abstract Background: Alzheimer’s disease (AD) is a most common dementia in elderly people. Since AD symptoms resemble those of other neurodegenerative diseases, including idiopathic normal pressure hydrocephalus (iNPH), it is difficult to distinguish AD from iNPH for a precise and early diagnosis. iNPH is caused by the accumulation of cerebrospinal fluid (CSF) and involves gait disturbance, urinary incontinence, and dementia. iNPH is treatable with shunt operation which removes accumulated CSF from the brain ventricles. Methods: We performed metabolomic analysis in the CSF of patients with AD and iNPH with capillary electrophoresis-mass spectrometry. We assessed metabolites to discriminate between AD and iNPH with Welch’s t-test, receiver operating characteristic (ROC) curve analysis, and multiple logistic regression analysis. Results: We found significant increased levels of glycerate and N-acetylneuraminate and significant decreased levels of serine and 2-hydroxybutyrate in the CSF of patients with AD compared to the CSF of patients with iNPH. The ROC curve analysis with these four metabolites showed that the area under the ROC curve was 0.90, indicating good discrimination between AD and iNPH. Conclusions: This study identified four metabolites that could possibly discriminate between AD and iNPH, whichpreviousresearchhasshownarecloselyrelatedto the risk factors, pathogenesis, and symptoms of AD. Analyzing pathway-specific metabolites in the CSF of patients with AD may further elucidate the mechanism and pathogenesis of AD. Keywords: Alzheimer’s disease, Idiopathic normal pressure hydrocephalus, Diagnostic marker, Cerebrospinal fluid, Serine, Glycerate, N-acetylneuraminate, 2-hydroxybutyrate Background according to the National Institute of Neurological Alzheimer’s disease (AD) is the most common type of de- and Communicative Disorders and Stroke and the mentia in the world, which concerns approximately 60–70% Alzheimer’s Disease and Related Disorders Association cases of dementia, and it is becoming a significant social criteria [1]. Currently, there is no curative or radical issue because of the growing aging population. treatment for AD, although some medicines have In general, AD is diagnosed based on the presence of been developed to reduce the symptoms [2]. However, cognitive impairment and by neuropsychological testing some symptoms of AD are similar to those of other neurodegenerative diseases, such as idiopathic normal * Correspondence: [email protected] pressure hydrocephalus (iNPH). iNPH is caused by the accu- 1Medical Genome Center, National Center for Geriatrics and Gerontology, mulation of cerebrospinal fluid (CSF) in the brain and causes 7-430 Morioka-cho, Obu, Aichi 474-8511, Japan Full list of author information is available at the end of the article gait disturbance, urinary incontinence, and dementia. In © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nagata et al. Biomarker Research (2018) 6:5 Page 2 of 11 contrast to other dementias, iNPH is treatable by shunt op- at different AD stages and found that choline, dimethylar- eration that removes the accumulated CSF [3]. Therefore, a ginine, arginine, valine, proline, serine, histidine, creatine, precise diagnosis that discriminates patients with AD from carnitine, and suberylglycine could be possible disease patients with iNPH is essential for proper treatment at the progression markers [16]. Furthermore, they conducted early stages of these diseases. Presently, increased phosphate ultra-high-performance liquid chromatography-time-of- tau (p-tau) and decreased amyloid-beta 1-42 (Aβ42) in the flight mass spectrometry (TOFMS) and found uracil and CSF are used as established AD diagnostic markers [4]. uridine were good candidate biomarkers for AD, as has However, p-tau accumulates after synaptic degeneration and been previously described [15, 17]. Aβ42 is difficult to accurately quantify. Therefore, we tried to However, different metabolomic platforms and methods find additional biomarkers, capable of precisely detecting AD detect different metabolites, thus utilizing multiple types before neurodegeneration progresses. of metabolomic platforms would provide a wider perspec- Recently, accumulated evidence has indicated that tive of metabolomics information under study. In addition, AD is a type of metabolic disease in the brain [5, 6]. differences in ethnicity, culture, and education may influ- The brain is the most energy-consuming organ and ence decision making regarding the diagnosis, symptoms, glucose is an essential and dominant energy source and severity of AD [18]. Accordingly, accumulating infor- for the brain [7]. Progressive regional cerebral glu- mation related to AD pathology from differential ethnora- cose metabolism reduction, correlated with the cial cohorts and with differential methods is essential. symptom severity, has been found in the brain of pa- From these points of view, we performed metabolo- tients with AD [8, 9]. Mills et al. (2013) performed micanalysisoftheCSFofpatientswithADoriNPH RNA-Seq analysis in the parietal cortex of patients in Japanese cohorts with capillary electrophoresis- with AD and reported that two enzymes, ACOT1 TOFMS (CE-TOFMS). Our present findings would and ACOT2, which are involved in lipid metabolism, support the utility of metabolomics analysis for dis- were upregulated. They also found a downregulation criminate between AD and iNPH. of TERC, which is involved in the synthesis of very long chain fatty acids [10]. In addition, impairment Methods of the insulin response, as is seen in diabetes melli- Subjects tus and metabolic syndrome, was revealed as a risk The characteristics of the study patients with AD and factor for AD [11–13]. These reports suggested that iNPH are summarized in Table 1. AD and iNPH were the pathomechanism of AD is strongly related to a diagnosed according to previously published criteria [1, disturbance in brain energy metabolism and homeo- 19]. Bioresources were largely obtained from the biobank stasis. The disturbance could induce metabolite al- of the National Center for Geriatrics and Gerontology terations in the body fluids of patients with AD, (NCGG, Aichi, Japan) and partly donated by the such as in the plasma, serum, and CSF. Therefore, National Center of Neurology and Psychiatry (NCNP, metabolome analysis of body fluids in AD has been Tokyo, Japan) and the Department of Biological Regulation, actively performed to identify new diagnostic School of Medicine, Tottori University (Tottori, Japan). All markers. Especially, the CSF is thought to be a su- samples were obtained with the written informed consent of perior analyte than other body fluids because it is in the patients before sampling between 2011 and 2015. This direct contact with the extracellular space of the study was reviewed and approved by the ethics committees brain and thus directly reflects the biological of all participating institutes and biobanks. changes in the pathological brain processes in AD. So far, several pertinent reports have been pub- Table 1 Subject characteristics. lished and several diagnostic markers for AD have Metabolomics Validation been suggested. For example, D’ Aniello et al. (2005) AD iNPH AD iNPH conducted high performance liquid chromatography Subject No. 39 19 42 38 (HPLC) analysis and found L-glutamine was in- Mean age (SD) 73.5 (9.8) 77.8 (4.5) 74.1 (9.6) 77.9 (5.6) creased and L-asparate was decreased in the CSF of Age range 47-86 70-86 47-86 57-87 patients with AD than in the CSF of controls [14]. Czech et al. (2012) reported that increased cysteine P-value 0.03 0.03 with decreased uridine was the optimal combination Male/female 14/25 9/10 15/27 22/16 to identify mild AD, and increased cortisol levels P-value 0.24 0.09 were associated with the progression of AD in a Mean MMSE (SD) 21.4 (5.1) 23.5 (2.9) 21.2 (5.0) 22.1 (3.1) European cohort [15]. Ibanez et al. (2012) performed P-value 0.08 0.46 capillary electrophoresis-mass spectrometry (CE-MS) Missing values 6 0 6 0 to investigate metabolome changes in the CSF of patients Nagata et al. Biomarker Research (2018) 6:5 Page 3 of 11 Collection of CSF delivered as sheath liquid at 10 L/min. ESI-TOFMS was op- CSF was collected by lumbar puncture and centrifuged erated in the positive ion mode, and the capillary voltage to remove debris. The supernatant was aliquoted into was set at 4 kV. The flow rate of heated nitrogen gas low protein-binding tubes and immediately frozen in li- (heater temperature, 300 °C) was maintained at 10 psig. In quid nitrogen and stored at −80 °C until use. TOFMS, the fragmentor, skimmer, and Oct RF voltages were set at 75, 50, and 125 V, respectively. Automatic recal- Measurement of p-tau and Aβ42 ibration of each acquired spectrum was performed using To detect the concentration of p-tau and Aβ42 in the the masses of reference standards ([13C isotopic ion of pro- + CSF, the enzyme-linked immunosorbent assay systems of tonated methanol dimer (2CH3OH + H)] , m/z 66.06306) + INNOTEST β-AMYLOID(1-42) and INNOTEST PHOSPHO- and ([hexakis(2,2-difluoroethoxy)phosphazene + H] , m/z TAU(181P) were used according to the manufacturer’sinstruc- 622.02896).