Journal of Alzheimer’s Disease xx (20xx) x–xx 1

DOI 10.3233/JAD-180441
IOS Press

1 The Distribution of Urinary

2 Alzheimer-Associated Neuronal Thread
Protein and Its Association with Common

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3

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4 Chronic Diseases in the General Population

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5 He Jina,c , Shaochen Guanb,c , Rong Wanga,c,d,∗ , Xianghua Fangb,c,∗ , Hongjun Liub,c , Yanchuan
6 Wua,c , Yanlei Zhangb,c and Chunxiao Liub,c
7
a Central Laboratory, Xuan Wu Hospital, Capital Medical University, Beijing, China
8
b Evidence-based Medical Center, Xuanwu Hospital, Capital Medical University, Beijing, China
c Beijing Geriatric Medical Research Center, Beijing, China

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9

10
d Beijing Institute for Brain Disorders, Beijing, China

Accepted 26 June 2018

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11 Abstract.
12 Background: Alzheimer-associated neuronal thread protein (AD7c-NTP) has been found to be a biomarker for Alzheimer’s
13 disease (AD).
14 Objective: The aim of the present study was to investigate the distribution of Alzheimer-associated neuronal thread protein
d

15 and its relationship to common chronic diseases in the general population.

16 Methods: Urine samples of 1,805 participants were collected from four districts (Xi Cheng, Fang Shan, Tong Zhou, and Yan
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17 Qing) in Beijing. The assessment in this study included a questionnaire that captured participants’ demographic informa-
18 tion, use of medication and histories of disease, neurological examinations, psychometric evaluations, physical and clinical
19 examinations, and laboratory tests.
20 Results: Urine AD7c-NTP level was increased among the population over 60 years old and females exhibited higher lev-
21 els than males. These results controlled for other demographic factors such as education levels, employment status, body
mass index and current residence. The urine AD7c-NTP levels exhibited no association with non-neurological diseases
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22

23 (0.3346 ± 0.4482 ng/ml), such as hypertension (0.3445 ± 0.4187), stroke (0.3652 ± 0.4010), diabetes (0.3319 ± 0.4371),
24 dyslipidemia (0.3440 ± 0.4314), renal insufficiency (0.3223 ± 0.3909), cancer (0.5055 ± 1.0006), chronic lung disease
25 (0.2911 ± 0.2852), chronic liver disease (0.5579 ± 0.6726), severe depression symptoms (0.5186 ± 0.7040), and mild depres-
26 sion symptoms (0.3669 ± 0.3811).
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27 Conclusions: Cut-off values for urine AD7c-NTP levels for different age groups and genders should be established.
28 AD7c-NTP levels proved relatively stable in the body and were not impacted by demographic factors or common chronic
29 diseases.
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30 Keywords: Alzheimer’s disease, Alzheimer-associated neuronal thread protein, biomarker, urine

INTRODUCTION 31

∗ Correspondence
to: Rong Wang and Xianghua Fang,
Alzheimer’s disease (AD) is a progressive and fatal 32
PhD, No.45, Changchun Street, Beijing 100053, P.R.
China. Tel.: +86 13611367292/+86 13661272893; E-mails: neurodegenerative disorder manifested by cognitive 33

rong wang72@aliyun.com and xhfang163@163.com. and memory deterioration, progressive impairment 34

ISSN 1387-2877/18/$35.00 © 2018 – IOS Press and the authors. All rights reserved
 2 H. Jin et al. / Urinary AD7c-NTP Distribution

35 of daily activities, and a variety of neuropsychiatric of a spectrum of the disease as well as of confounding 87

36 symptoms and behavioral disturbances [1]. Currently factors that could potentially impact AD7c-NTP lev- 88

37 there are more than 26.6 million AD patients world- els. The purpose of the survey was to investigate the 89

38 wide. As the world population ages, the number of level of urine AD7c-NTP in relation to demographic 90

39 AD patients is expected to reach more than 106.2 factors and common diseases among the Chinese 91

40 million by 2050; one of every 85 persons will live population. 92

41 with AD [2]. The onset of AD is often hidden and

42 the disease seems to be irreversible. To date, no

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43 effective treatments to prevent, halt, or reverse it are MATERIALS AND METHODS 93

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44 available. What therapies exist only alleviate or post-
45 pone clinical symptoms [3]. Diagnostic tests have To investigate the distribution of AD7c-NTP and 94

46 moved the field closer to the development of effec- its relationship to common chronic diseases in the 95

47 tive AD biomarkers to facilitate early diagnosis and general population, the urine AD7c-NTP levels of 96

48 to improve clinical care [4, 5]. Therefore, simple 1,805 participants were detected, and the assess- 97

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49 and non-invasive biomarker tests for early detection ment in this study included a questionnaire that 98

50 of AD would prove invaluable, especially those that captured participants’ demographic information, use 99

51 could be performed during a routine clinical screen- of medication and histories of disease, neurologi- 100

52 ing for the elderly. cal examinations, Mini Mental State Examinations 101

(MMSE), Montreal Cognitive Assessments (MoCA),

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53 Alzheimer-associated urine neuronal thread pro- 102

54 tein (AD7c-NTP) is a member of the neuronal Activities of Daily Living (ADL), psychometric 103

55 thread protein family with a molecular weight of evaluations, physical and clinical examinations, and 104

56 approximately 41 kD. As a potential biomarker laboratory tests. 105

57 of AD, it is closely related to the pathophysiol-

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58 ogy of the disease. Previous studies demonstrated
59 that AD7c-NTP immunoreactivity co-localized with Participants 106

60 tau-immunoreactive neurofibrillary tangles and dys-

61 trophic neurites, and abnormal AD7c-NTP gene Participants, aged 35–93 years (n = 1,805), were 107

62 expression precedes the formation of neurofibrillary recruited and evaluated from the general population 108

tangles [6, 7]. Elevated levels of AD7c-NTP are residing in four Beijing districts (Xi Cheng, Fang
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63 109

64 detectable in cortical neurons, brain tissue extracts, Shan, Tong Zhou, and Yan Qing) from August 2014 to 110
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65 cerebrospinal fluid (CSF), and urine in the early December 2015. The study encompassed 16 admin- 111

66 course of AD [8–10]. From 1996 when AD7c- istrative districts in Beijing: 14 urban and 2 rural. 112

67 NTP was first discovered and reported by de la The definitions of urban and rural areas were adopted 113

68 Monte et al. [6], to the present, a number of studies from government administrative data that categorized 114

69 have confirmed and further developed understand- settings according to their levels of urbanization and 115
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70 ings about the association between the protein and economic development. To ensure adequate represen- 116

71 AD; these studies have focused on the improvement tation of the population, the survey populations were 117

72 of detection methodologies [10–14], the validation selected using a 4-stage stratified random procedure, 118

73 of clinical multi-facility measurements [15–17], and details of the process were previously described [21, 119
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74 the combination of the protein with other biomarkers 22]. Exclusion criteria were participants with cogni- 120

75 [18–20]. tive impairment (mild cognitive impairment (MCI), 121

76 Due to a lack of large-scale epidemiological data AD, vascular dementia, or other types of dementia). 122

77 for urine AD7c-NTP and a dearth of studies on the A consensus expert panel of 2 neurologists (Fang 123
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78 physiological variations of urine, some important Li and Jing-Hong Ma) reviewed all of the data col- 124

79 questions remain. First, the distribution of urinary lected and determined diagnoses. All participants 125

80 AD7c-NTP in the general population is unclear. were informed of the purpose of the study at the 126

81 Second, whether or not the levels of urinary AD7c- time of recruitment and signed an informed con- 127

82 NTP are associated with non-neurological diseases sent form to participate in the survey. The study 128

83 is unknown. Thus, we measured the urinary concen- was conducted in accordance with relevant clinical 129

84 trations of AD7c-NTP in 1,805 human urine samples research regulations and was approved by the Ethics 130

85 obtained via a community-based survey. The focus on Committee of Xuanwu Hospital, Capital Medical 131

86 the general population permitted a deep examination University. 132

 H. Jin et al. / Urinary AD7c-NTP Distribution 3

133 Questionnaire An enzyme-linked immunosorbent assay (ELISA) 180

kit (Anqun Biological Technology Co. Ltd., Shen- 181

134 All of the enrolled participants were asked to zhen, China) was used to detect the level of urinary 182

135 complete a questionnaire conducted by well-trained AD7c-NTP. The method used for detection reflected 183

136 medical students using standardized methods. The standard procedures for ELISA kits [13]. Accord- 184

137 questionnaire covered a wide range of topics, ing to the introduction, 100 ␮l of standard or urine 185

138 including demographic characteristics (gender, age, specimen was added to the AD7c-NTP antibody 186

139 education level, employment status, and current resi- pre-coated plate and incubated at 37◦ C for 1 h. Fol- 187

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140 dence), and history of hypertension, stroke, diabetes, lowing five consecutive wash steps with PBS, 100 ␮l 188

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141 dyslipidemia, renal insufficiency, cancer, chronic of biotinylated rabbit anti-AD7c-NTP antibody was 189

142 lung disease, chronic liver disease, and the use of added and incubated at 37◦ C for another 1 h. Next, 190

143 medication. Depression symptoms were assessed 100 ␮l of horseradish peroxidase labeled avidin was 191

144 using the Geriatric Depression Scale (GDS) [23]. added after five times of washing with PBS and 192

incubated at 37◦ C for 30 min. Finally, 50 ␮l of chro- 193

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145 Physical examination and laboratory mogenic reagent A and B was added in turn after 194

146 measurements five times of washing with PBS, and then incubated 195

at 37◦ C for 15 min. The reaction was stopped by 196

147 Physical examination adding 50 ␮l of sulfuric acid as stop buffer. The OD 197
Before the examination, each participant was asked

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148 value of each sample was measured at 450 nm with 198
149 to rest for at least 20 min. Sitting blood pressure a microplate reader (Multiskan Spectrum, Thermo 199
150 (BP) was measured twice on the right arm at 2- to Fisher Scientific, Waltham, MA, USA). The AD7c- 200
151 5-min intervals, and the mean of the two measure- NTP concentration in a urine specimen was positively 201
152 ments was calculated for analysis. Blood pressure correlated with the absorbance value and calcu- 202
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153 was measured using a standard mercury sphygmo- lated according to the standard curve of recombinant 203
154 manometer. Height was measured without shoes human AD7c-NTP peptides with measured taken 204
155 using a standard right-angle device and a fixed mea- simultaneously. 205
156 surement tape (to the nearest 0.5 cm). Body weight
157 without heavy clothing was measured using a weight Definitions 206
measurement device (V-body HBF-371, Kyoto,
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158

159 Japan). Classification of population demographic 207

characteristics
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208

160 Blood samples laboratory measurement The 1,805 participants were divided into four 209

161 All blood samples were obtained from partici- groups according to age (Table 1); less than 60 years 210

162 pants in the morning following an overnight fast, of age, 60–69 years of age, 70–79 years of age, and 211

163 centrifuged to serum, stored in a refrigerator at 2◦ C to 80–89 years of age, respectively. Education levels 212

8◦ C, and transferred to a central laboratory (IPE Cen-

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164 were categorized as follows: less than a high school 213

165 ter for Clinical Laboratory, Beijing, China), which diploma (<high school), high school diploma, and 214

166 performed all analyses within 24 h. Total cholesterol college or higher (>college). Employment status were 215

167 (TC), triglyceride (TG), high-density lipoprotein categorized as employed, retired and unemployed. 216
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168 cholesterol (HDL-C), low-density lipoprotein choles- Current residence was classified as either urban or 217

169 terol (LDL-C), and fasting blood glucose (FBG) rural. Body mass index (BMI) was calculated as body 218

170 levels were determined by a Hitachi 7600 automatic weight in kilograms divided by the square of height 219

171 analyzer (Hitachi High-Technologies Corporation, in meters and divided into categories of underweight 220
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172 Tokyo, Japan). Serum creatinine level was measured (BMI < 18.5), normal weight (18.5 ≤ BMI < 23.9), 221

173 by enzymatic assay, which was calibrated to the iso- overweight (24.0 ≤ BMI < 27.9), and obese (BMI 222

174 tope dilution mass spectrometry-traceable creatinine ≥ 28) as defined by the National Health and Fam- 223

175 assays. ily Planning Commission for Chinese adult weight 224

standards. 225

176 Urine AD7c-NTP laboratory measurement

177 The midstream urine samples were collected in Definitions of the diseases 226

178 an EP tube with boric acid (2 g/L) as a preserva- Hypertension was defined following the Joint 227

179 tive, refrigerated immediately, and maintained at 4◦ C. National Committee guideline VII36 to include 228
 4 H. Jin et al. / Urinary AD7c-NTP Distribution

Table 1
Baseline characteristics of study participants
Variables Subject group Age (y) Male:female no. (%) no. cases Percent (%)
All 65.7 ± 10.1 756:1042 1805 100.0
Age, y <60 50.8 ± 6.3 177:239 416 23.0
60–69 65.6 ± 2.6 290:428 718 39.8
70–79 73.7 ± 2.8 248:318 566 31.4
80–89 82.4 ± 2.7 48:57 105 5.8
Education level <High school 66.7 ± 9.4 493:745 1238 68.6

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High school 63.4 ± 10.5 163:213 376 20.8
63.8 ± 11.8

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>High school 98:75 173 9.6
Employment status employed 52.3 ± 10.0 95:69 164 9.1
retired 68.9 ± 7.0 452:587 1039 57.6
unemployed 63.8 ± 11.0 197:369 566 31.4
BMI (kg/m2 ) <18.5 71.0 ± 10.1 15:13 28 1.6
18.5–23.9 66.1 ± 10.1 204:246 450 24.9
24–27.9 65.6 ± 10.3 329:394 723 40.1

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≥28 65.0 ± 9.2 167:315 482 26.7
Current residence Urban 66.9 ± 9.4 391:579 970 53.7
Rural 64.2 ± 10.7 372:463 835 46.3
Hypertension + 67.6 ± 8.5 483:694 1177 65.2
– 62.2 ± 11.9 280:348 628 34.8
70.7 ± 6.9

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Stroke + 103:93 196 10.9
– 65.1 ± 10.2 652:941 1593 88.3
Diabetes + 67.5 ± 7.9 183:269 452 25.0
– 65.1 ± 10.7 580:773 1353 75.0
Dyslipidemia + 66.3 ± 9.4 419:673 1092 60.5
– 64.7 ± 11.1 344:369 713 39.5
71.7 ± 7.9
Renal insufficiency +
Au 113:246 359 19.9
– 64.2 ± 10.1 642:782 1424 78.9
Cancer# + 71.0 ± 5.8 14:23 37 3.2
– 71.2 ± 5.3 459:645 1104 96.8
Chronic lung disease# + 72.2 ± 5.8 35:23 58 5.1
– 71.2 ± 5.3 438:645 1083 94.9
Chronic liver disease# + 70.4 ± 4.6 9:17 26 2.3
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– 71.2 ± 5.4 464:651 1115 97.7

Depression symptoms# + 74.4 ± 6.9 2:9 11 1.0
–/+ 72.7 ± 5.6 13:34 47 4.1
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– 71.0 ± 5.2 453:621 1074 94.1

Notes: “+” represents the disease; for depression symptoms, “+” represents severe depression, “–/+” represents
mild depression; “#” represents that the health history with regard to these diseases were only obtained from
participants (n = 1,141) over 65 years old; age is expressed as a mean ± standard deviation.
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229 participants with systolic blood pressure ≥140 mm III guidelines [26]. Renal function was assessed 244

230 Hg or/and diastolic blood pressure ≥90 mm Hg or using the estimated glomerular filtration rate, which 245
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231 who had a history of hypertension or had taken was calculated by the Modification of Diet in 246

232 antihypertension medication [24]. The diagnosis Renal Disease formula as follows: 186 × (serum 247

233 of diabetes was based on the American Diabetes creatinine–1.154 ) × (age–0.203 ) × (0.742, if female), 248

234 Association criteria for people with high FBG with the serum creatinine concentration expressed 249
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235 (≥7.0 mmol/L [126 mg/dL]), a history of diabetes, in milligram per deciliter (mg/dL) [27]. Stroke, can- 250

236 or who had taken hypoglycemic medication [25]. cer, chronic lung disease and chronic liver disease 251

237 Participants who had high TC (serum level of TC were self-reported and the disease history was ver- 252

238 ≥6.21 mmol/L [240 mg/dL]), high TG (serum level ified with medical or hospital records. Depression 253

239 of TG ≥2.26 mmol/L [200 mg/dL]), high LDL-C symptoms were evaluated using the 30-item GDS 254

240 (serum level ≥4.14 mmol/L [160 mg/dL]), low HDL- Scale [23]. Scores ranged from 0 to 30. Scores 255

241 C (serum level <1.03 mmol/L [40 mg/dL]) or had of ten or lower were identified as normal, 11 to 256

242 taken lipid-lowering medication were diagnosed with 20 as mild depression, and 21 to 30 as severe 257

243 dyslipidemia according to the Adult Treatment Panel depression. 258

 H. Jin et al. / Urinary AD7c-NTP Distribution 5

259 Statistical analysis Additionally, the multivariate analysis of variance 303

between different age groups showed no differences 304

260 Analysis of the data was performed using the Sta- (p > 0.05). 305

261 tistical Package for the Social Sciences version 20.0

262 software (SPSS Inc., Chicago, IL, USA). Data are Urine AD7c-NTP levels by some other 306

263 expressed as mean ± standard deviation (SD). The demographic characteristics 307

264 urine level of AD7c-NTP between two groups were

265 compared by two independent sample t-test and three The urine AD7c-NTP levels among participants 308

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266 groups or more were analyzed with one-way ANOVA with different education levels, employment statuses, 309

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267 followed by a LSD’s post hoc test. Multivariate anal- BMI levels, current place of residence all showed no 310

268 ysis of variance was used for confounders analysis. significant differences (p > 0.05, Table 2). 311

269 A p value of <0.05 was considered statistically sig-

270 nificant. The association between urine AD7c-NTP level 312

and nine common diseases 313

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271 RESULTS
Urine AD7c-NTP levels did not show any dif- 314

272 Baseline characteristics of study participants ferences (p > 0.05, Table 2) in people with the 315

depression symptoms or any of the following dis- 316

eases: hypertension, stroke, diabetes, dyslipidemia,

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273 The 1,805 participants had an average age of 317

274 65.7 ± 10.1 years and 41.9% (n = 756) were male. renal insufficiency, cancer, chronic lung disease, and 318

275 The participants’ baseline characteristics and preva- chronic liver disease. 319

276 lence of disease are shown in Table 1. The history

277 of cancer, chronic lung disease, chronic liver disease, DISCUSSION 320
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278 and depression symptoms were obtained from par-
279 ticipants over 65 years of age. Those participants In this population-based cross-sectional study, we 321

280 (n = 1,141) completed an additional questionnaire measured the concentrations of AD7c-NTP in 1,805 322

281 relevant to the community management of chronic human urine samples and obtained demographic data 323

282 disease in the presence of geriatric syndrome. All and comorbidity information from participants. Our 324

questions and examinations yielded a response rate results demonstrated that urine AD7c-NTP had a
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283 325

284 of 93.3% to 100%. tendency to increase with age to some extent, and 326
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females exhibited higher levels than males. How- 327

285 Urine AD7c-NTP levels by gender ever, urine AD7c-NTP levels were not affected 328

by other demographic factors such as education 329

286 As shown in Table 2, compared to males, the urine levels, employment status, BMI, or current place 330

287 AD7c-NTP level in female subjects was significantly of residence. Moreover, there was no association 331
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288 higher (0.3790 ± 0.4873 and 0.2739 ± 0.3815 ng/ml, between AD7c-NTP levels and non-neurological 332

289 respectively (p < 0.05)). diseases, such as hypertension, stroke, diabetes, 333

dyslipidemia, renal insufficiency, cancer, chronic 334

290 Urine AD7c-NTP levels by age lung disease, chronic liver disease, and depres- 335
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sion symptoms. This study represents the first one 336

291 According to one-way ANOVA, the greatest differ- to examine urine AD7c-NTP levels together with 337

292 ences (p = 0.002) of urine AD7c-NTP levels among possible confounding factors in a large sample of 338

293 the four age groups were between the groups <60 the elderly. The results provide a compelling case 339
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294 years of age and 80–89 years old. The urine AD7c- for the use of urinary AD7c-NTP as a biomarker 340

295 NTP levels in groups <60, 60–69, 70–79, and 80–89 for AD. 341

296 years old appear in Table 2. The urine AD7c- The key neuropathological hallmarks of the 342

297 NTP level in group <60 years old was significantly AD brain are diffuse and neuritic extracellular 343

298 lower (p = 0.046, p = 0.084, p = 0.000) and the group amyloid plaques—often surrounded by dystrophic 344

299 80–89 was significantly higher (p = 0.000, p = 0.005, neurites—and intracellular neurofibrillary tangles 345

300 p = 0.004) than other groups. There was no signifi- [28]. The expression of AD7c-NTP and related genes 346

301 cant differences of urine AD7c-NTP levels between are modulated with neuritic sprouting; an overexpres- 347

302 the group 60–69 and the group 70–79 (p = 0.839). sion of AD7c-NTP in neuronal cells causes neuritic 348
 6 H. Jin et al. / Urinary AD7c-NTP Distribution

Table 2
Urine Alzheimer-associated neuronal thread protein (AD7c-NTP) levels according to demographic characteristics and some non-neurological
diseases
Variables subject group AD7c-NTP (ng/ml) F/t(a) p(a) p(b)
Gender male 0.2739 ± 0.3815 –4.350 0.000 0.006
female 0.3790 ± 0.4873
Age (y) <60 0.2771 ± 0.2874 5.067 0.002 0.330
60–69 0.3424 ± 0.4992
70–79 0.3363 ± 0.3613

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80–89 0.4998 ± 0.8144
0.3346 ± 0.4722

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Education level <High school 2.352 0.095 0.236
High school 0.3026 ± 0.3892
>High school 0.4092 ± 0.4060
Employment status employed 0.2675 ± 0.2191 1.798 0.166 0.524
retired 0.3509 ± 0.4549
unemployed 0.3279 ± 0.4918
BMI (kg/m2 ) <18.5 0.4309 ± 0.6414 0.316 0.814 0.932

rP
18.5–23.9 0.3293 ± 0.4918
24–27.9 0.3396 ± 0.4421
≥28 0.3345 ± 0.4257
Current residence Urban 0.3468 ± 0.4397 1.050 0.294 0.918
Rural 0.3204 ± 0.4579
0.3445 ± 0.4187

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Hypertension + 1.358 0.175 0.993
– 0.3055 ± 0.4990
Stroke + 0.3652 ± 0.4010 0.841 0.400 0.426
– 0.3311 ± 0.4554
Diabetes + 0.3319 ± 0.4371 –0.121 0.903 0.690
– 0.3355 ± 0.4520
0.3440 ± 0.4314
Dyslipidemia +
Au 0.927 0.354 0.959
– 0.3202 ± 0.4730
Renal insufficiency + 0.3223 ± 0.3909 0.500 0.617 0.082
– 0.3381 ± 0.4639
Cancer + 0.5055 ± 1.0006 –0.889 0.380 0.103
– 0.3586 ± 0.4836
Chronic lung disease + 0.2911 ± 0.2852 1.051 0.294 0.462
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– 0.3672 ± 0.5176
Chronic liver disease + 0.5579 ± 0.6726 –1.498 0.146 0.142
0.3588 ± 0.5035
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–
Depression symptoms + 0.5186 ± 0.7040 0.594 0.552 0.582
–/+ 0.3669 ± 0.3811
– 0.3530 ± 0.4771
Notes: p(a) represents the value of one-way ANOVA; p(b) represents the value of multivariate analysis; Data are
expressed as mean ± SD.
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349 growth and cell death [6, 9]. In AD patients, the very little is known about confounding factors of 364

350 elevated levels of AD7c-NTP in the CSF and urine AD7c-NTP, as very few studies with large samples 365
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351 may reflect the neurodegenerative processes. The cor- have been conducted. 366

352 relation between AD7c-NTP levels and the severity The findings indicated that AD7c-NTP levels did 367

353 of the dementia makes it a potential biomarker for AD not increase with age among participants between the 368

354 diagnosis [8, 15–18]. The urine AD7c-NTP ELISA ages of 60 and 79. However, in a previous study, we 369
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355 test is recognized as sensitive, economical, and non- have reported that AD7c-NTP levels did increase with 370

356 invasive. Moreover, urine does not form part of the age in a healthy Chinese population [31]. The discrep- 371

357 body’s homeostatic mechanisms, yet reflects changes ancy could be explained by the fact that the number 372

358 such as pregnancy, daily rhythms, and kidney dis- of samples in the previous study was small (n = 294) 373

359 eases [29]. Because the brain and urine seem so and treated all subjects >60 years old together in 374

360 anatomically distant and distinct, a direct relation- one group. For the population as a whole, the inci- 375

361 ship between the two has proven difficult to establish dence of AD/MCI increases exponentially with age, 376

362 [30]. Additionally, physiological variations in urine with the most pronounced increase occurring dur- 377

363 have not been studied extensively [29]. Moreover, ing the seventh and eighth decades of life [28, 32]. 378
 H. Jin et al. / Urinary AD7c-NTP Distribution 7

379 This may be consistent with the age-relatedness of Nevertheless, it remains unclear whether or not the 431

380 urine AD7c-NTP levels. However, multivariate anal- level of AD7c-NTP in urine could be affected by 432

381 ysis of variance suggested that urinary AD7c-NTP other diseases. AD7c-NTP shows up in urine from 433

382 levels are not related to age, so further study needs to blood by filtration and the kidneys/urinary tracts by 434

383 be done. Female urine AD7c-NTP levels were higher secretion. Even though the brain and urine seem to be 435

384 than those of males, consistent with findings from completely separate, we cannot rule out the possibil- 436

385 the earlier study [31]. The higher AD7c-NTP lev- ity that changes present in the brain could somehow 437

386 els in females also prove consistent with the higher be reflected in urine [30]. Therefore, it would be 438

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387 incidence of AD among females [33]. This result sug- necessary to co-analyze urine AD7c-NTP with other 439

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388 gests the need to establish different AD7c-NTP cutoff diseases, especially the risk factors of AD with large- 440

389 values for males and females. scale population data. Previous studies have shown 441

390 Some epidemiological studies have suggested that that depression symptoms and all of the diseases 442

391 low education is a risk factor for AD. A Chi- included in this study were associated with the inci- 443

392 nese population-based cross-sectional survey also dence of AD [28, 40–45]. However, whether those 444

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393 reported that the higher incidence of dementia and diseases in other parts of the body could influence 445

394 AD in rural areas compared to urban ones might the vitro excretion or detection of urinary AD7c-NTP 446

395 be due to differences in educational levels [34]. remained unclear. 447

396 However, other studies have demonstrated that more Our results suggested that the urinary AD7c-NTP 448

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397 highly educated people may have a greater cognitive levels were no different between subjects who have 449

398 reserve that possibly delays the clinical manifesta- or do not have symptoms of depression or non- 450

399 tion of dementia [35]. Our results demonstrated that neurological diseases, such as hypertension, stroke, 451

400 urinary AD7c-NTP concentrations were not related diabetes, dyslipidemia, renal insufficiency, cancer, 452

401 to educational attainment, place of residence, envi- chronic lung disease, and chronic liver disease. These 453
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402 ronment, or employment status. The comparison of results demonstrated that AD7c-NTP was relatively 454

403 urinary AD7c-NTP levels between different BMI stable in the body and not affected by confounding 455

404 groups also failed to show any differences. This factors. 456

405 finding counters an understanding that an associa- An overview of the previous research conducted 457

406 tion exists between BMI and dementia/AD. Whereas on AD7c-NTP is shown in Fig. 1. The sample type 458

some epidemiological studies have shown that people selection, detection technology, and clinical verifica-
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407 459

408 with high BMI in midlife demonstrated an increased tion have been confirmed by several laboratories. This 460
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409 risk of dementia/AD in old age, no clear relationship study improved upon existing work on AD7c-NTP 461

410 between obesity in old age and AD has been proven through the information obtained from a large-scale 462

411 [36–38]. population since the metabolic process of AD7c- 463

412 It is noteworthy that urinary AD7c-NTP was asso- NTP in humans has been unclear. The search for 464

413 ciated with age and gender, but not with education brain disease biomarkers in urine poses challenges 465
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414 levels, place of residence, and BMI. These findings that derive from the possibility that a variety of fac- 466

415 support an understanding that urinary AD7c-NTP tors could temporarily affect urine. Additionally, the 467

416 represents a reliable and feasible biomarker for AD. pathological changes of human AD cannot be com- 468

417 Additionally, AD7c-NTP in human urine also could pletely replicated in experimental animals, which 469
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418 be affected by other factors, such as some diseases limits the possibilities for research on the mecha- 470

419 common among the elderly. For biomarker studies, nism of AD7C-NTP and AD development. Therefore, 471

420 the most important purpose is to identify a specific large-scale population data research represents the 472

421 and stable biomarker for a particular disease. The most compelling approach to be able to achieve an 473
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422 expression of neuronal thread protein in the brains objective understanding of AD7c-NTP. Our labora- 474

423 of people with AD has proven to be distinct from tory has been devoted to the development of the 475

424 those with other neurodegenerative diseases (Down’s urinary AD7c-NTP diagnostic kit in recent years 476

425 syndrome, Parkinson’s disease, Parkinson’s disease and it has been developed for the diagnosis of AD. 477

426 dementia, Huntington’s disease, multi-infract demen- The detection linear range was 0–10 ng/ml and the 478

427 tia, and schizophrenia) [8]. The concentration of urine normal reference value was ≤1.5 ng/ml. The sensi- 479

428 AD7c-NTP also has been proven to be higher than an tivity was 89.3% and specificity was 84.7% [13]. In 480

429 MCI group, a non-AD dementia control group, and a this study, we demonstrated that AD7c-NTP levels 481

430 non-dementia group [15–17, 39]. were higher for the population over the age of 60 482
 8 H. Jin et al. / Urinary AD7c-NTP Distribution

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Fig. 1. Research related to AD7c-NTP. CSF, cerebrospinal fluid; M-IRMA, immunoradiometric assay; ISHH, in situ hybridization his-
tochemistry; IH, immunohistochemical; RT-PCR, reverse transcriptase; NB, Northern blot; WB, Western blot; ELISA, enzyme-linked
immunosorbent assay; DN, Down’s syndrome; PD, Parkinson’s disease; PDD, PD dementia; HD, Huntington’s disease; MID, Multi-infarct
dementia.

483 and for females. Therefore, greater attention could Fuwai Hospital, National Center for Cardiovascular 510
Au
484 be devoted to the identification of different diagnos- Disease, Peking Union Medical College and Chinese 511

485 tic thresholds based on age and gender. The urine Academy of Medical Science: Drs. Zengwu Wang; 512

486 AD7c-NTP levels exhibited no association with non- Ming Guo; Ying Li. 513

487 neurological diseases. Therefore AD7c-NTP proved Authors’ disclosures available online (https:// 514

488 to represent a biomarker with good specificity; other www.j-alz.com/manuscript-disclosures/18-0441r2). 515

d

489 diseases did not interfere with the suspected pres-

490 ence of AD. These findings suggest the merits of a
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491 test like the AD7c-NTP ELISA kit for use in clinical REFERENCES 516

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