Patients Who Leave the Emergency Department Without Being Seen and Their Follow-Up Behavior: A Retrospective Descriptive Analysis

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Journal of Alzheimer’s Disease xx (20xx) x–xx DOI 10.3233/JAD-190401 IOS Press

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Urinary Alzheimer-Associated Neuronal Thread Protein is not Elevated in Patients with Subjective Cognitive Decline and Patients with Depressive State

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Yuxia Lia,b , Meimei Kanga , Hongxing Wangb , He Jina , Xiaozhen Wanga , Wenjing Gana , Mingyan Zhaob , Xing Zhaob , Rong Wanga,c,d,∗ and Ying Hanb,c,d,e,∗

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a Central

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b Department

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Laboratory, Xuanwu Hospital, Capital Medical University, Beijing, China of Neurology, Xuanwu Hospital of Capital Medical University, Beijing, China c Beijing Geriatric Medical Research Center, Beijing, China d Center of Alzheimer’s Disease, Beijing Institute for Brain Disorders, Beijing, China e National Clinical Research Center for Geriatric Disorders, Beijing, China

Accepted 31 July 2019

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Abstract. Subjective cognitive decline (SCD) is a risk factor for Alzheimer’s disease (AD). Urinary Alzheimer-associated neuronal thread protein (AD7c-NTP) has been identified as a biomarker for AD. It was hypothesized that if urinary AD7c-NTP were also elevated in SCD, as it is in prodromal AD (mild cognitive impairment stage), it could be a convenient and efficient clinical biomarker for the early diagnosis of SCD. SCD is often accompanied by a depressive state (DS), and the impact of DS on urinary AD7c-NTP levels remains unknown. A total of 297 right-handed Chinese Han subjects were recruited, including 98 subjects with SCD, 92 patients with DS, and 107 well-matched cognitively normal controls (NC). The levels of AD7c-NTP in urine samples were measured using an enzyme-linked immunosorbent assay AD7c-NTP kit. Our results demonstrated that urinary AD7c-NTP levels in the SCD group (0.7561 ± 0.5657 ng/mL) were not significantly higher than in either the DS (0.7527 ± 0.5607 ng/mL) or NC (0.7214 ± 0.5077 ng/mL) groups. Furthermore, urinary AD7c-NTP levels were not correlated with Hamilton Depression Rating Scale and Hamilton Anxiety Scale scores, but they were negatively associated with Mini-Mental State Examination scores (r = –0.222, p = 0.033) and Montreal Cognitive Assessment-Basic scores (r = –0.207, p = 0.048). Urinary AD7c-NTP level is not elevated in SCD and is unaffected by DS. Urinary AD7c-NTP may therefore not be a good potential biomarker for SCD and DS, although it may become elevated with more severe cognitive decline.

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Keywords: Alzheimer-associated neuronal thread protein, Alzheimer’s disease, biomarker, depressive state, subjective cognitive decline

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INTRODUCTION

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Subjective cognitive decline (SCD) is a risk factor for Alzheimer’s disease (AD) [1–3]. Many previ∗ Correspondence

to: Rong Wang and Ying Han, PhD, No.45, Changchun Street, Beijing 100053, P.R. China. Tel./Fax: +86 1063159572; E-mails: [email protected] (Rong Wang); [email protected] (Ying Han)

ous cross-sectional and longitudinal studies have confirmed that SCD can occur in the preclinical phase of AD [4, 5]. A meta-analysis reported that approximately 26.6% of SCD patients developed mild cognitive impairment (MCI), and 14.1% of SCD patients developed dementia within four years [4]. Patients with SCD have a two-times-higher risk of developing prodromal AD in one year compared

ISSN 1387-2877/19/$35.00 © 2019 – IOS Press and the authors. All rights reserved

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chological tests is normal [3]. This cognitive decline is persistent and unrelated to acute events. However, SCD is heterogeneously defined [15–17] and is often accompanied by a depressive state (DS) [18]. There is currently very little published research investigating whether DS correlates with urinary AD7c-NTP levels. DS is a common, debilitating psychiatric disorder that involves feeling unhappy, having a DS, a lack of interest in things, insomnia, fatigue, listlessness, a feeling of uselessness, inattention, indecision, and weight loss or weight gain without dieting. Concurrently, DS patients also have impaired memory and other kinds of cognitive decline [19]. No studies have compared differences in urinary AD7c-NTP levels between DS and SCD subjects. Thus, the primary aim of this study is 1) to explore urinary AD7c-NTP levels in SCD subjects, 2) to assess urinary AD7c-NTP levels in DS subjects, and 3) to analyze whether urinary AD7c-NTP levels are affected by DS or cognitive impairment.

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MATERIALS AND METHODS

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Participants

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This study was approved by the Research Ethics Review Board of Xuanwu Hospital (ClinicalTrials.gov identifier: NCT03370744) and all subjects gave their written informed consent. In the study, 297 right-handed Chinese Han subjects were recruited, including 98 SCD subjects, 92 DS subjects, and 107 well-matched cognitively normal controls (NC). Both the SCD and DS subjects were diagnosed by two neurologists from the Neurology Department, Xuanwu Hospital, Capital Medical University, Beijing, China. NC subjects were recruited from the local community via broadcast advertisements and web advertising. Diagnoses of SCD were made by experienced neurologists according to the SCD Plus diagnostic framework suggested by the Subjective Cognitive Decline Initiative [2, 20]. The inclusion criteria for SCD included: 1) older than 60, right-handedness, Han nationality; 2) presence of a self-perceived continuous cognitive decline compared with a previously normal status, and this decline was unrelated to an acute event; 3) memory loss was the primary symptom, rather than any other cognitive domain; 4) the subject had concerns or worries associated with the memory complaint; 5) the subject reported that their cognitive function was worse than others in the same age group; 6) memory loss was confirmed by an informed person; 7) the subject failed to meet the cri-

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with those without any subjective cognitive impairment complaints [4]. SCD Plus was considered more likely to convert to MCI or dementia. The conversion rate for SCD Plus (18.9%) to MCI was significantly higher than SCD (5.6%) and normal control (NC) (4.9%) [5]. The current treatments available for AD do not modify the course of the disease, and clinical trials of drugs to treat AD have been unsuccessful, which may be because it is too late to treat patients who are already in the irreversible phase [6]. Researchers are beginning to study the prodromal and preclinical stages of AD, and SCD is now receiving more research attention. In 2011, the National Institute on Aging-Alzheimer’s Association workgroups (NIA-AA) began to place a greater emphasis on the role of biomarkers for the early diagnosis of AD [7]. Since then, possible early AD biomarkers have attracted increasing attention, and biomarker evidence is expected to enhance our understanding of the pathophysiology of AD and aid in its diagnosis. Urinary Alzheimer-associated neuronal thread protein (AD7c-NTP) has been identified as a possible biomarker for early AD [8, 9]. Previous studies demonstrated that AD7c-NTP immunoreactivity colocalizes with neurofibrillary tangles and dystrophic neurites, and abnormal AD7c-NTP levels are associated with tau-immunoreactive neurofibrillary tangles [10, 11]. AD7c-NTP is found in cerebrospinal fluid (CSF) as well as in urine, and previous studies have shown that both CSF and urine levels of AD7CNTP have high sensitivity and specificity as AD biomarkers, and give similar results [12]. Because of its noninvasive, nonradioactive, economic, sensitive, and repeatable nature, the urinary AD7c-NTP test is a safe and promising biomarker for early AD [8]. It has been previously established that urinary AD7cNTP levels in AD (2.25 [0.43–8.62] ␮g/L) are higher than those in normal controls (0.82 [0.47–2.77] ␮g/L, p < 0.01). Besides, previous research has demonstrated that MCI patients have higher levels of urinary AD7c-NTP compared with normal controls [13]. It is thus suggested that urinary AD7c-NTP may be an important biomarker for the early diagnosis of both MCI and AD [13, 14]. However, although there are many reports in the literature on the outcome of increased AD7c-NTP levels, most are restricted to MCI and AD. It is still unclear whether AD7c-NTP is elevated in SCD subjects, and whether AD7c-NTP can be used as a biomarker for the early diagnosis of SCD. SCD refers to a self-reported persistent cognitive decline, although objective performance in neuropsy-

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(SCD-D) and SCD without DS (SCD-ND), based on HAMD scores [25] and depressive symptoms. The HAMD scores of subjects in the SCD-D group were ≥7, and in the SCD-ND group, they were <7. DS subjects were divided into two groups: DS with cognitive impairment (DS-CI) and DS without cognitive impairment (DS-NCI), based on total Montreal Cognitive Assessment-Basic (MoCA-B) scores [26] and their symptoms of cognitive impairment. MoCA-B overall scores for subjects with a college education were <24 in the DS-CI group and ≥24 in the DS-NCI group. For subjects with middle school education, MoCA-B total scores were <22 in the DS-CI group and ≥22 in the DS-NCI group. When subjects had primary education or were illiterate, MoCA-B total scores were <19 in the DS-CI group and ≥19 in the DS-NCI group [26]. Measures

All subjects underwent a standardized clinical and neuropsychological evaluation, including the Mini-Mental State Examination (MMSE), MoCAB, AVLT-H, FAQ, Shape trails test-A, Shape trails test-B, 17-item HAMD, Hamilton Anxiety Scale (HAMA), Animal Verbal Fluency Test, and Boston Naming Test (30-items). Next, to exclude other diseases that cause memory loss, blood samples were obtained from all participants in the morning. Laboratory tests included blood biochemistry, blood routine, thyroid series, antibody tests for syphilis and human immunodeficiency virus, homocysteine, serum folate assay, and serum vitamin B12 determination.

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teria for MCI [21]. The diagnostic criteria for MCI included: 1) having impaired scores (defined as >1 standard deviation below the age-corrected normative mean) on both measures within at least one cognitive domain (i.e., memory, language, or executive function) using the Auditory Verbal Learning Test-HuaShan (AVLT-H) to test memory, the Animal Verbal Fluency Test and Boston Naming Test (30-items) to check language, and the Shape trails tests-A and -B to test executive function; 2) having impaired scores in each of the three cognitive domains sampled (memory, language, and executive function); and 3) having a rating ≥9 in the Functional Activities Questionnaire (FAQ) [22]. NC was screened in a structured interview to confirm the lifelong absence of psychiatric and neurological illness, as previously described in the Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV) Non-patient Edition. NC inclusion criteria included: 1) older than 60, right-handedness, Han nationality; 2) no cognitive decline complaints, with neither worry nor concern about their cognition; 3) scores within the normal range for standardized neuropsychological tests, scale-adjusted for age, sex, and education; 4) negative for general and nervous system physical examinations; 5) a review of the medical history and family history was negative and without history of stroke, severe psychiatric disease, or other neurological disorder; and 6) accessory examination revealed no diseases that could cause cognitive decline. Inclusion criteria for DS subjects included: 1) a current episode of the major depressive disorder according to the DSM-IV standards based on a clinical interview [23] and (2) a score ≥18 on the 17-item Hamilton Depression Rating Scale (HAMD) [24]. DS could be either self-reported or observed by others, and the depressive symptoms included feeling unhappy, a DS, a lack of interest in things, insomnia, fatigue, listlessness, feelings of uselessness, inattention, indecision, and weight loss or weight gain without dieting. The exclusion criteria that were applied to all subjects were: history of stroke, serious psychiatric disease, another neurological disorder (such as Parkinson’s disease, multiple sclerosis, or a brain tumor), alcohol or drug abuse, and systemic disease (such as severe anemia or thyroid dysfunction), syphilis, or acquired immune deficiency syndrome. Furthermore, to clarify the relationship between cognitive impairment and depression, we further divided the two groups into subgroups. SCD subjects were divided into two groups: SCD with DS

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Detection of urinary AD7c-NTP Clean midstream urine specimens were collected from all subjects in the morning in Eppendorf tubes containing boric acid (2 g/L) as a preservative. Samples were immediately centrifuged before being stored at 4◦ C. Levels of AD7c-NTP in urine samples were measured using an enzymelinked immunosorbent assay (ELISA) AD7c-NTP kit (Anqun Biological Technology Co. Ltd., Shenzhen, China) according to the manufacturer’s instructions. Samples were added to the kit and then incubated at 37◦ C for 1 h before washing thoroughly with phosphate-buffered saline. Samples were then incubated with biotinylated rabbit anti-AD7c-NTP antibody at 37◦ C for 1 h. Following thorough washing with PBS, horseradish-peroxidase-labeled avidin was added to samples and incubated at 37◦ C for 30 min.

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Statistical analysis

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The data were analyzed using the Statistical Package for Social Sciences (SPSS), Version 25.0. Data were expressed as the mean ± standard deviation (SD) in continuous variables, and if the data not distributed normally, we would represent them by the median and interquartile range, which was analyzed using a non-parametric Mann-Whitney U test. The two independent sample t-test was used to compare the levels of urinary AD7c-NTP between two groups, such as SCD-D and SCD-ND groups, DS-CI and DSNCI groups, and for analyses between three groups we used an analysis of variance (ANOVA) followed by a least significant difference post hoc test, such as SCD, DS, and NC groups. Statistical significance was analyzed using Chi-square tests to compare counting data as appropriate. Pearson’s correlation analysis was used for correlation analyses between urinary AD7c-NTP levels and HAMD, HAMA, MMSE, and MoCA-B total scores. A p value of <0.05 was considered statistically significant. RESULTS Participant clinical and demographic data

Comparison of urinary AD7c-NTP levels among NC, SCD, and DS groups After controlling for the potentially confounding effects of age, education level, and sex, urinary AD7c-NTP levels in the SCD group (0.7561 ± 0.5657 ng/mL) were not significantly higher than in the DS (0.7527 ± 0.5607 ng/mL) or NC (0.7214 ± 0.5077 ng/mL) groups (Table 2; oneway ANOVA). Further, pairwise comparison of three groups by post hoc test showed no significant difference using the least significant difference (Table 2). Comparison of urinary AD7c-NTP and neuropsychological assessment scales between the SCD-D and SCD-ND groups According to their HAMD score, SCD subjects were divided into SCD-D and SCD-ND groups. As can be observed in Table 3, there were no significant differences in age, sex, and years of education, and there were no significant differences in urinary AD7c-NTP levels between the SCD-D (0.6736 ± 0.41 ng/mL) and SCD-ND (0.7836 ± 0.61 ng/mL) groups. Two independent sample t-tests were used to compare neuropsychological assessment scale scores between the SCD-D and SCD-ND groups. The HAMD and HAMA scores in SCD-D group were significantly higher than those in SCD-ND group (p < 0.001). In contrast, there were no significant differences in MMSE or MoCA-B scores between the SCD-D and SCD-ND groups (p > 0.05).

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In this study, 297 subjects were recruited and divided into three groups: SCD (n = 98), DS (n = 92), and NC (n = 107). Participants had an average age of 64.66 ± 6.08 years, and 49.2% (n = 146) were male. The comparisons of basic demographic and clinical characteristics between groups are shown in Table 1. There were no significant differences in age, sex, and years of education, which were comparable among these groups. However, there were significant differences in MMSE, total MoCA-B, HAMD, and HAMA scores among groups (p values <0.05). Laboratory tests including blood biochemistry, blood routine, thyroid series, antibody tests for syphilis and human immunodeficiency virus, homocysteine, serum folate assay, and serum vitamin B12 determination were all in the normal range, which was used in order to exclude other causes of cognitive dysfunction.

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Samples were washed with PBS before being incubated at 37◦ C for 15 min in 50 mL of chromogenic reagents A and B, in turn. Finally, the reaction was stopped by adding 50 mL of sulfuric acid as the stop buffer. A microplate reader at 450 nm wavelength was used to measure the absorbance (A value) and AD7c-NTP concentration.

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Comparison of urinary AD7c-NTP and neuropsychological assessment scales between the DS-CI and DS-NCI groups According to their MoCA-B total score and their depressive symptoms, DS subjects were divided into DS-CI and DS-NCI groups. There were no significant differences in age, sex, years of education, and HAMD or HAMA scores between the two groups. However, there were significant differences in urinary AD7c-NTP levels between the DS-CI (1.017 ± 0.7081 ng/mL) and DS-NCI (0.4574 ± 0.1571 ng/mL) groups, and the MMSE and total MoCA-B scores in DS-CI group were significantly lower than these in DS-NCI group (p < 0.001; Table 4).

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Table 1 Demographics and clinical features of the NC, SCD, and DS groups SCD (n = 98)

DS (n = 92)

NC (n = 107)

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47 (48.0%) 64.80 ± 6.03 13.46 ± 3.09

40 (43.5%) 63.73 ± 6.56 12.83 ± 3.03

59 (55.1%) 65.3 ± 5.64 12.79 ± 2.83

0.250 0.174 0.206

29.00 (2.00) 26.00 (3.00) 3.50 (5.25) 4.00 (4.00)

27.00 (4.00) 23.00 (3.00) 19.00 (5.00) 17.00 (15.00)

29.00 (2.00) 26.00 (3.00) 2.00 (3.00) 1.00 (3.00)

<0.001 <0.001 <0.001 <0.001

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Male sex, n (%) Age (y), mean ± SD Education (y), mean ± SD Testing Results MMSE, median (IQR) MoCA-B, median (IQR) HAMD, median (IQR) HAMA, median (IQR)

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SCD, subjective cognitive decline; DS, patients with the depressive state; NC, normal control; MMSE, Mini-Mental State Examination; MoCA-B, Montreal Cognitive Assessment-Basic; HAMD, Hamilton Depression Rating Scale; HAMA, Hamilton Anxiety Rating Scale; SD, standard deviation; IQR, interquartile range. Sex distribution was analyzed using the chi-square test. Age and education were analyzed using ANOVA followed by post hoc test for pairwise comparison. MMSE, MoCA-B, HAMD, and HAMA were analyzed using the Mann-Whitney U test. Table 2 Comparisons of urinary AD7c-NTP levels among the NC, SCD, and DS groups AD7c-NTP (ng/ml), mean ± SD

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NC (n = 107)

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0.7561 ± 0.5657

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0.7214 ± 0.5077

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NC versus SCD SCD versus DS NC versus DS

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AD7c-NTP, Alzheimer-associated neuronal thread protein; SD, standard deviation; SCD, subjective cognitive decline; DS, patients with the depressive state; NC, normal control. The urinary AD7c-NTP were analyzed using ANOVA followed by post hoc test for pairwise comparison. Table 3 Demographics and clinical features of the SCD-D and SCD-ND groups SCD-ND (n = 73)

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35 (47.9%) 64.78 ± 6.16 13.43 ± 3.18

0.996 0.818 0.600

0.6736 ± 0.4102 29.00 (2.00) 26.00 (4.50) 9.00 (2.50) 9.00 (5.50)

0.7836 ± 0.6099 29.00 (2.00) 26.00 (3.00) 2.00 (3.00) 4.00 (3.00)

0.526 0.832 0.628 <0.001 <0.001

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SCD-D (n = 25) Male sex, n (%) Age (y), mean ± SD Education (y), mean ± SD Testing Results AD7c-NTP (ng/ml) MMSE, median (IQR) MoCA-B, median (IQR) HAMD, median (IQR) HAMA, median (IQR)

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SCD-D, subjective cognitive decline with depressive state; SCD-ND, subjective cognitive decline without depressive state; AD7c-NTP, Alzheimer-associated neuronal thread protein; MMSE, Mini-Mental State Examination; MoCA-B, Montreal Cognitive Assessment-Basic; HAMD, Hamilton Depression Rating Scale; HAMA, Hamilton Anxiety Rating Scale; SD, standard deviation; IQR, interquartile range. Sex distribution was analyzed using the chi-square test. Age, education, and urinary AD7c-NTP were analyzed using ANOVA followed by post hoc test for pairwise comparison. MMSE, MoCA-B, HAMD, and HAMA were analyzed using the MannWhitney U test.

Correlation between urinary AD7c-NTP levels and MMSE, MoCA-B, HAMD, and HAMA scores Urinary AD7c-NTP levels were negatively associated with MMSE (r = –0.222, p = 0.033) and MoCA-B (r = –0.207, p = 0.048) scores in the DS group. However, there were no significant correlations between urinary AD7c-NTP levels and

HAMD (r = 0.012, p = 0.903) or HAMA (r = –0.018, p = 0.862) scores in the SCD group (Table 5). There were no significant differences in MMSE or total MoCA-B scores between the SCD and NC groups (Table 1), and there were no significant correlations between urinary AD7c-NTP levels and MMSE (r = –0.025, p = 0.805) or total MoCA-B (r = –0.090, p = 0.379) in the SCD group (Table 5).

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Y. Li et al. / Urinary AD7c-NTP is not Elevated in SCD and DS Table 4 Demographics and clinical features of the DS-CI and DS-NCI groups DS-CI (n = 49)

DS-NCI (n = 43)

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21(42.9%) 64.22 ± 7.42 12.47 ± 2.78

19 (44.2%) 63.16 ± 5.45 13.23 ± 3.29

0.898 0.442 0.231

1.017 ± 0.7081 26.00 (3.00) 22.00 (1.25) 19.00 (7.00) 18.00 (15.50)

0.4574 ± 0.1571 28.00 (2.25) 26.00 (1.00) 19.00 (3.00) 16.50 (15.50)

<0.001 <0.001 <0.001 0.812 0.395

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Table 5 Correlation of AD7c-NTP and MMSE, MoCA-B, HAMD, and HAMA in the SCD, DS, and NC group SCD MMSE MoCA-B HAMD HAMA

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r

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–0.025 –0.090 0.012 –0.018

0.805 0.379 0.903 0.862

–0.222 –0.207 0.155 0.193

0.033 0.048 0.141 0.065

–0.090 0.033 –0.00 –0.025

0.359 0.739 0.997 0.797

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DISCUSSION

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Urinary AD7c-NTP levels in the SCD group

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The present study was designed to investigate whether urinary AD7c-NTP levels were elevated in SCD and DS, and to elucidate whether DS influences urinary AD7c-NTP levels. We demonstrated that 1) urinary AD7c-NTP levels in the SCD and DS groups were not significantly higher than those in the NC group; 2) there was no significant correlation between urinary AD7c-NTP and HAMD or HAMA scores; 3) there was a significant negative correlation between urinary AD7c-NTP and MMSE and total MoCA-B scores.

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urinary AD7c-NTP levels are higher in AD than in healthy controls [14]. Another important previous finding was that urinary AD7c-NTP levels in MCI subjects (median 1.57 [range 0.4–4.15] ng/mL) are significantly higher than in healthy subjects, which suggests that urinary AD7c-NTP may be an essential biomarker for the early diagnosis of MCI. Urinary AD7c-NTP is considered to be promising because of its advantages: it is non-invasive, free from radiation, has high repeatability, is a simple operation, has low cost, and is safe and readily accepted by patients. It was hypothesized that if urinary AD7cNTP were also elevated in SCD, as it is in MCI, it could also be a convenient and efficient clinical biomarker for the early diagnosis of SCD. To the best of our knowledge, no previous studies have reported urinary AD7c-NTP levels in SCD subjects. However, contrary to our expectations, urinary AD7c-NTP levels in the SCD group were not elevated compared with controls. Possible reasons for this lack of an increase in urinary AD7c-NTP levels in SCD are as follows. First, according to the guidelines issued by the NIA-AA in 2011, AD can be divided into three phases [28], and SCD is considered a preclinical AD that occurs before objective cognitive impairment appears [3]. Previous studies have reported that urinary AD7cNTP levels are positively correlated with the degree of cognitive impairment and negatively correlated with MMSE scores [29, 30]. Neuropsychological test scores were normal in SCD patients, so it may, therefore, be understood that urinary AD7c-NTP is not elevated. Second, a recent study reported that there are no significant changes in cortical thick-

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SCD, subjective cognitive decline; DS, patients with the depressive state; NC, normal control; AD7c-NTP, Alzheimer-associated neuronal thread protein; MMSE, Mini-Mental State Examination; MoCA-B, Montreal Cognitive Assessment-Basic; HAMD, Hamilton Depression Rating Scale; HAMA, Hamilton Anxiety Rating Scale.

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DS-CI, depressive state patients with cognitive impairment; DS-NCI, depressive state patients without cognitive impairment; AD7c-NTP, Alzheimer-associated neuronal thread protein; MMSE, Mini-Mental State Examination; MoCA-B, Montreal Cognitive Assessment-Basic; HAMD, Hamilton Depression Rating Scale; HAMA, Hamilton Anxiety Rating Scale; SD, standard deviation; IQR, interquartile range. Sex distribution was analyzed using the chi-square test. Age, education, and urinary AD7c-NTP were analyzed using ANOVA followed by post hoc test for pairwise comparison. MMSE, MoCA-B, HAMD, and HAMA were analyzed using the Mann-Whitney U test.

Several reports have demonstrated that urinary AD7c-NTP is a sensitive and specific diagnostic biomarker for the early identification of probable AD and MCI [13, 14, 27]. An initial study reported that

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Urinary AD7c-NTP levels and DS Cognitive complaints are commonly reported in DS, and DS often accompanies SCD. There is a relationship between DS and cognitive impairment, so we investigated whether DS was related to urinary AD7c-NTP levels. An important finding in our study was that urinary AD7c-NTP levels in the DS group were not higher than those in the NC group and that there were no significant differences in urinary AD7c-NTP levels between the SCD-D and SCD-ND groups. Furthermore, multiple linear regression analysis among groups revealed no significant correlation between urinary AD7c-NTP levels and HAMD or HAMA scores. This finding is consistent with an earlier report by Jin, who also found no difference in urinary AD7c-NTP levels between subjects who did or did not have DS [9]. In a word, these results suggest that DS is not associated with increased urinary AD7c-NTP levels. Previous studies have shown that urinary AD7cNTP levels are associated with AD-related pathology. Overexpression of AD7c-NTP is associated with increased levels of phospho-tau protein, and abnormal AD7c-NTP expression is associated with AD neurodegeneration [10]. Results from the present study, as shown in Table 5, indicated that urinary

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Urinary AD7c-NTP levels and cognitive impairment

The correlation between urinary AD7c-NTP levels and cognitive impairment is still controversial. Most studies indicate that AD7c-NTP levels are positively correlated with AD severity [29, 30] and that urinary AD7c-NTP levels in advanced AD are significantly higher than in early-stage AD [34]. However, other studies have reported no correlation between urinary AD7c-NTP levels and cognitive impairment severity in AD [27, 29]. The results from the present study indicated a significant difference in urinary AD7c-NTP levels between the DS-CI (1.017 ± 0.7081 ng/mL) and DS-NCI (0.4574 ± 0.1571 ng/mL) groups, which is consistent with a previous study [35]. However, although urinary AD7c-NTP levels were significantly higher in the DS-CI group than in the DS-NCI group, they were still within the normal range. At present, the optimal cutoff value for urinary AD7c-NTP for the early diagnosis of AD is set at 1.5 ␮g/L (1.5 ng/mL), with 90.6% sensitivity and 91.8% specificity [34]. The mean urinary AD7c-NTP level in the DS-CI group in our study was 1.017 ± 0.7081 ng/mL, which is below the cutoff value above. de la Monte indicated that overexpression of AD7c-NTP might have a direct role in mediating some of the critical cell death cascades that are associated with AD neurodegeneration, and further established a link between AD7c-NTP overexpression and the accumulation of phosphotau in pro-apoptotic central nervous system neuronal cells [38]. Therefore, urinary AD7c-NTP levels are associated with disease progression and cognitive function impairment. Another important result in the current study was that there was a significant negative correlation between urinary AD7c-NTP levels and MMSE scores, which supports this view. Many previous studies also support a correlation between urinary AD7c-NTP and cognitive impairment [8, 36–38]. In the future, experiments with larger sample sizes are needed to confirm the correlation between the two groups.

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AD7c-NTP levels were not correlated with depressive and anxiety symptoms, but were associated with cognitive decline. This result suggests that DS is not a confounding factor that influences urinary AD7cNTP levels, and together, these findings support the idea that urinary AD7c-NTP is a reliable biomarker for AD-related pathology.

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ness in SCD [31], indicating that SCD is a very early stage of AD before AD-related pathological changes such as the accumulation of amyloid-␤ protein and hyperphosphorylated tau protein have not appeared. Thus, our finding that urinary AD7c-NTP levels were not increased in patients with SCD may be because SCD is still in the very early stage of the disease. It is, however, worth noting that SCD development is likely to accelerate memory impairment [32, 33]. A meta-analysis showed that SCD patients have twice the risk of developing dementia compared with those without subjective memory decline [4]; SCD has an annual conversion rate of 6.6% to MCI and 2.3% to dementia, compared with 1% in those without subjective memory decline [4]. Another study reported that the conversion rate to MCI for subjects with SCD Plus diagnosed by the International Working Group of SCD [2] was 18.9% during 13.1 months (range 10.7–22.4 months), while the conversion rate for NC was just 4.9% [5]. It will thus be important to follow up on these subjects in our study and observe the dynamic progress of the disease and changes in urinary AD7c-NTP levels over time.

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There are several limitations to our study. First, there was a relatively small sample size, and it is thus necessary to continue the research and expand the sample size. Second, as a cross-sectional study, any observation of the dynamic relationship between urinary AD7c-NTP levels and SCD progression was limited. Third, the SCD patients enrolled in this study were subjects diagnosed as SCD Plus, according to the SCD diagnostic framework, without the results of amyloid PET. Future studies should expand the sample size and carry out longitudinal studies to further confirm the relationship between urinary AD7c-NTP and cognitive impairment and DS.

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ACKNOWLEDGMENTS

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This article was supported by the National Key Research and Development Program of China (2016YFC1306300, 2016YFC0103000, 2018YFA0108503), National Natural Science Foundation of China (Grant 61633018, 81801052, 81522021, 81430037, 81471731), Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX201706), Beijing Nature Science Foundation (7161009), Beijing Municipal Commission of Health and Family Planning (PXM2019 026283 000002), China Postdoctoral Science Foundation (2018M641414) and Beijing Postdoctoral Research Foundation (ZZ2019-12). Authors’ disclosures available online (https:// www.j-alz.com/manuscript-disclosures/19-0401r2).

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In conclusion, this study demonstrated that urinary AD7c-NTP levels were not elevated in SCD and DS subjects. Urinary AD7c-NTP levels were not correlated with HAMD and HAMA scores but were associated with MMSE scores. Urinary AD7c-NTP may not be a potential biomarker for SCD and DS, but it may elevate with more severe cognitive decline.

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