Analyses of Clinical Features and Investigations on Potential Mechanisms in Patients with Alzheimer’s Disease and Olfactory Dysfunction

Abstract

Background:

OD is common in patients with Alzheimer’s disease (AD). However, the relationship between OD and clinical symptoms and the potential mechanisms of OD in AD patients are still unknown.

Objective:

To explore the relationship between OD and clinical symptoms and the potential mechanisms of OD in AD patients.

Methods:

We evaluated OD using the Hyposmia Rating Scale (HRS), classified patients into AD with OD (AD-OD) and AD with no OD (AD-NOD) groups, and detected the levels of free radicals and inflammatory factors, including hydroxyl radical (•OH), hydrogen peroxide (H₂O₂), nitric oxide, interleukin-1β, interleukin-6, tumor necrosis factor-α, and prostaglandin E₂ in serum from AD patients.

Results:

It was shown that the scores of the Mini-Mental State Examination, Animal Fluency Test, Boston Naming Test (BNT), and Auditory Verbal Learning Test-delayed recall were all significantly lower and the score of overall activity of daily living (ADL) and instrumental ADL were significantly higher in AD-OD group than those in AD-NOD group. Compared with AD-NOD group, •OH level in serum was prominently elevated, and H₂O₂ level was dramatically declined in AD-OD group. In the correlation analysis, HRS score was significantly and positively correlated with the score of BNT, and negatively correlated with •OH level in serum.

Conclusions:

AD-OD patients suffered from severe cognitive impairment in the domain of language. Oxidative stress might be correlated with AD-OD featured by the drastically increased •OH level in serum.

Keywords

Alzheimer’s disease language •OH olfactory dysfunction oxidative stress

INTRODUCTION

In Alzheimer’s disease (AD) patients, neuropathological changes occur in both peripheral and central systems that process olfactory information. Olfactory dysfunction (OD) was reported to be an early preclinical sign of AD. The first stage in the neurodegenerative progression of AD occurs in entorhinal cortex, a region initiating olfactory information processing. Prospective cohort studies established that OD inferred the risk of developing cognitive impairment [1, 2] and predicted conversion from mild cognitive impairment (MCI) to AD [3]. It was reported that 90% of AD patients had significant OD in an olfactory test; however, only 6% of AD patients complained of OD at early stage of disease [4]. Clinically, OD could be firstly assessed by the symptom rating scale and then evaluated by the tests, for example, the University of Pennsylvania Smell Identification Test (UPSIT) [5] and Sniffin’ Sticks Test (SST), etc. [6]. SST was regarded as the subjective method for OD detection. Nonetheless, this time-consuming test may not be readily available to most clinicians, or be practical for the use in large populations. Hence, a time-saving, widely used and applicable screening tool for OD is urgently needed. Thus, in this study, the Hyposmia Rating Scale (HRS), a validated self-administered scale [7], was used to evaluate olfactory function for AD patients.

Few studies have examined human olfactory pathology with respect to normal aging. Studies reported that OD with normal aging in humans appeared to localize to the olfactory epithelium and cortical areas [8]. Other studies suggested that OD in AD patients was correlated with the dysfunction in cortical areas, in addition to or despite pathologic change in the olfactory bulb and epithelium [8]. Therefore, it is a continuous process for OD processing. Although several studies focused on OD in AD patients [9 –11], the underlying mechanism is not fully understood yet. Thus, investigation of the potential mechanism underlying OD in patients with MCI and dementia due to AD might be very helpful for further enhancement of our understanding of the significance of OD as a preclinical marker of AD. In the future, we will confirm this finding in patients in preclinical stage. OD could be a predictor for earliest diagnosis of AD and conversion from MCI to dementia due to AD [3]. However, OD is frequently overlooked by both doctors and patients. Yet, there is no investigation exploring the clinical features of cognition in AD patients with OD.

Inflammation and oxidative stress in brain featured by the robust productions of highly toxic inflammatory factors and free radicals play vital roles on cognitive decline in population with AD [12]. Emerging evidence suggested that neuroinflammation early involved in the pathogenesis of AD and exerted a causal action in the pathogenesis of the disease [13]. Several investigations revealed that inflammatory biomarkers could be used to identify prodromal stages of AD [14, 15]. Moreover, elevated level of inflammatory factors, e.g., tumor necrosis factor (TNF)-α in cerebrospinal fluid, was reported to be related to the conversion from MCI to the dementia stage of AD [16].

Although the conclusion varied in different investigations, for instance, the levels of TNF-α and interleukin (IL)-6 were significantly elevated [17, 18], whereas their levels were dramatically declined [19] in serum from AD patients; the results implied the potential correlation between peripheral inflammation and AD.

As we all known, the olfactory epithelium is the first site contacting with external environment and thus may serve as the starting point of AD. Studies showed that local inflammatory processes in olfactory bulb could increase permeability of blood-brain barrier, facilitate the penetration of exogenous pathogens, trigger reactive microgliosis, and eventually cause global neuroinflammation [20]. One study reported an increased level of reactive oxygen species (ROS) in olfactory bulb and/or hippocampus in AD model [21]. However, there is no study investigating the relationship between OD and peripheral inflammatory in AD patients.

In this study, we evaluated OD by HRS, assessed clinical symptoms of AD by a variety of rating scales, detected the levels of oxidative and inflammatory factors, including hydroxyl radical (•OH), hydrogen peroxide (H₂O₂), nitric oxide (NO), IL-1β, IL-6, and TNF-α, in serum, and analyzed the correlations among the scores of HRS, clinical symptoms, and the levels of oxidative and inflammatory factors above in serum, with the aim to understand the clinical features and potential mechanisms of OD in AD patients.

MATERIALS AND METHODS

Subjects

Inclusion criteria: This study included MCI due to AD and dementia due to AD according to National Institute of Aging and Alzheimer’s Association (NIA-AA) criteria [22].

Exclusion criteria: 1) Acute respiratory infections within 3 weeks; 2) chronic nasitisand sinusitis, and chronic obstructive pulmonary disease; 3) long-term or significant exposure to volatile substances, such as pesticides, herbicides, metallic dusts, acid fumes, industrial solvents, cleaning products or sawdust; 4) severe head trauma, nasal surgery; 5) cigarette smoking and drug abuse; 6) other neuropsychiatric disorders affecting olfactory function, such as Parkinson’s disease, multiple sclerosis, and epilepsy, etc.; and 7) immunological and inflammatory diseases in the peripheral and central nervous systems.

A total of 86 AD patients were consecutively recruited from the Department of Neurology, Beijing Tiantan Hospital, Capital Medical University from November 2014 to February 2017. Four patients with severe nasal or sinus diseases and 3 patients with epilepsy were excluded. Finally, 79 AD patients were evaluated and analyzed in this study, among which, 48 cases were MCI due to AD and 31 cases were dementia due to AD.

Collection of demographic information

Demographic variables, including age, age of onset, sex, education, and disease duration of 79 AD patients were recorded.

Assessment of olfactory function

HRS was validated by the SST in Parkinson’s disease patients by Millar in 2012 [7] and used to evaluate OD of AD patients in this study. HRS is a simple and convenient tool for screening OD in AD patients. Total score of the HRS shows significant correlation with olfactory threshold, discrimination, identification, and total SST scores. HRS has 6 items with each one describing the level of OD from 0 to 4 point(s). Total HRS score was obtained by summing up the score of each item. The higher the HRS score, the better the function of olfaction. The best cut-off point for HRS was 22.5 with a sensitivity of 70% and a specificity of 85% [7]. Thus, AD patients with total HRS score ≥23 points and ≤22 points were defined as AD-OD and AD-NOD groups, respectively [7]. In this study, 29 and 50 cases were recruited into the AD-OD and AD-NOD groups, respectively.

Assessments of clinical symptoms for AD patients

Global cognitive level for AD participants was first of all rated by the Mini-Mental State Examination (MMSE) scale [23], which was followed by the assessment of individual cognitive domain by using a variety of rating scales below: 1) Auditory Verbal Learning Test for immediate and delayed verbal memory [24]; 2) Rey-Osterrieth Complex Figure Test (CFT)-Delayed Recall (CFT-DR) for visual delayed memory [25]; 3) Rey-Osterrieth Complex Figure Test (CFT)-Copy for visuospatial ability [25]; 4) Chinese modified version of the Trail Making Test (TMT)-A [25], Trail Making Test (TMT)-B [26], Stroop Color-Word Test-Chinese version (CWT)-Color time [26] for attention/executive function; 5) Animal Fluency Test (AFT) [27] and Boston Naming Test (BNT)-30 items for language [28]; 6) Symbol Digit Modality Test (SDMT) for visuomotor speed [29].

Overall psychological symptoms were assessed by the Neuropsychiatric Inventory (NPI), which was followed by the evaluation of individual psychological manifestation using a line of rating scales below: Hamilton Anxiety Scale (HAMA)-14 items for anxiety, Hamilton Depression Scale (HAMD)-24 items for depression [30], Apathy Scale (AS) for apathy, Pittsburgh Sleep Quality Index (PSQI) for overall sleep quality, Epworth Sleepiness Scale (ESS) for excessive daytime sleepiness, and Cohen-Mansfield Agitation Inventory (CMAI) for agitation.

Activities of daily living (ADL) includes basic ADL (BADL) and instrumental ADL (IADL), which were assessed by Katz BADL scale [31], and Lawton and Brody IADL scale [32], respectively.

Collections of serum samples

AD patients were requested to withhold the drugs of anti-cognitive impairment for at least 12–14 h if their condition allowed. Total 2 ml venous whole blood was collected in a polypropylene tube between 7 a.m. and 10 a.m. under fasting condition. Serum samples were centrifuged immediately at 3000 rpm at 4°C. Approximately 0.5 ml volume of supernatant of serum was aliquoted into separate Nunc cryotubes and kept frozen at –80°C until ready for assay. Each aliquot was dedicated to each measure to avoid freeze-thawing and potential degradation of protein.

Detections of the levels of free radicals and inflammatory factors in serum

The levels of free radicals, including •OH, H₂O₂, and NO in serum were measured by chemical colorimetric method. A018 kit for •OH, A064 kit for H₂O₂ and A012 kit for NO were from Nanjing Jiancheng Biological Engineering Research Institute (Nanjing, China).

The levels of inflammatory factors, including IL-1β, IL-6, TNF-α, and PGE₂ in serum were measured by enzyme-linked immunosorbent assay. 1R040 kit for IL-1β, 1R140 kit for IL-6, and 1R350 kit for TNF-α were from Langka Company (Shanghai, China). CSB-E07965 h kit for PGE₂ was from CUSABIO Company (Wuhan, China).

Data analyses

Statistical analyses were performed with SPSS Statistics 20.0 from IBM Corporation (New York, USA). p value was significant when it was <0.05.

Continuous variables, if they were normally distributed, were presented as means±standard deviations and compared by two-sample t test. Continuous variables, if they were not normally distributed, were presented as median (quartile) and compared by nonparametric test. Discrete variables were compared by Chi square test.

Demographics variables, the scores of olfactory function, cognitive symptoms, psychological manifestations and ADL by related rating scales and the levels of •OH, H₂O₂, NO, IL-1β, IL-6, TNF-α, and PGE₂ in serum were compared between AD-OD and AD-NOD groups.

Spearman correlation analyses were made between the scores of HRS and each clinical symptom of AD, and between HRS score and •OH level in serum in AD patients.

In multiple linear regression models, age, age of onset, sex, educational level, the level of •OH in serum and the scores of AFT and BNT were set as independent variables, and HRS score was set as a dependent variable.

RESULTS

Frequency of OD in AD patients

The frequency of OD in total AD patients was 36.71%. Further investigation revealed that the frequencies of OD in patients with MCI and dementia due to AD were 25.00% and 54.84%, respectively.

Evaluation of olfactory function in AD-OD and AD-NOD groups

The average score of HRS in AD-OD group and AD-NOD group were 20.00 (15.50∼21.00) and 24.00 (23.00∼24.00) points, respectively. The HRS score in AD-OD group was significantly lower than that in AD-NOD group (p = 0.00).

The average score of HRS in patients with MCI and dementia due to AD were 21.00 (19.50∼22.00) and 18.00 (14.50∼20.00) points, respectively. The HRS score in dementia due to AD group was significantly lower than that in MCI group (p = 0.018).

Demographic variables of AD-OD and AD-NOD groups

Among 79 AD patients, 59 cases (74.68%) were female and 20 cases (25.32%) were male. The disease duration varied from 6 month to 12 years, with a median of 3.0 years [interquartile range (IQR): 3.625 years].

Demographic variables, including age, age of onset, sex, educational level and disease duration of AD-OD and AD-NOD groups were compared (Table 1). The age and age of onset in AD-OD group were older than those in AD-NOD group. No significant differences in sex, educational level, and disease duration were found between AD-OD and AD-NOD groups.

Table 1

Demographic variables of AD-OD and AD-NOD groups

	AD-OD group (29 cases)	AD-NOD group (50 cases)	p
Age (y, mean±SD	69.77±9.78	65.54±10.02	0.08
Age of onset (y, mean±SD)	66.35±10.04	60.73±13.64	0.07
Male/Total [cases/total (%)]	12/29 (41.38%)	15/50 (30.00%)	0.33
Educational level (N, %)
Primary school and below	9/29 (31.03%)	8/50 (16.00%)	0.24
Middle and high school	14/29 (48.28%)	26/50 (52.00%)
Bachelor’s degree and above	6/29 (20.69%)	16/50 (32.00%)
Disease duration	3.93±4.93	3.56±2.90	0.73

Clinical symptoms of AD-OD and AD-NOD groups

Global cognitive function and each cognitive domain of AD-OD and AD-NOD groups were evaluated by a body of rating scales (Table 2). It was showed that AD-OD group had significantly lower scores of MMSE, AVLT-delayed recall, AFT, and BNT than AD-NOD group. There were no significant differences in the scores of AVLT-immediate recall, CFT-delayed recall, CFT, the time of TMT-A, TMT-B, CWT-C, and SDMT.

Table 2

Scores of clinical symptoms in AD-OD and AD-NOD groups

	AD-OD group (29 cases)	AD-NOD group (50 cases)	p
1. Cognitive function
Overall cognitive level
MMSE (scores, mean±SD)	18.31±8.85	23.94±5.44	0.001 ^*
Cognitive domain
Memory
Verbal memory
AVLT-immediate recall [scores, median (quartile)]	10.00 (7.00∼12.00)	11.00 (9.00∼16.00)	0.22
AVLT-delayed recall (scores, mean±SD)	1.36±2.72	3.00±3.24	0.045 ^*
Visual delayed recall
CFT-delayed recall (scores, mean±SD)	5.31±7.26	6.69±8.48	0.54
Visuospatial ability
CFT (scores, mean±SD)	22.93±12.89	20.32±14.02	0.53
Executive function/attention
TMT-A time (s, mean±SD)	124.81±71.98	111.20±72.44	0.47
TMT-B time [scores, median (quartile)]	240.00 (190.00∼240.00)	230.00 (176.50∼240.00)	0.52
CWT-C time (s, mean±SD)	138.00±82.19	109.98±58.22	0.14
Language
AFT (scores, mean±SD)	11.46±5.25	15.2 2±5.26	0.005 ^*
BNT (scores, mean±SD)	19.44±7.01	23.38±4.79	0.006 ^*
Visuomotor speed
SDMT (scores, mean±SD)	20.00±16.49	25.05±13.35	0.24
2. Psychological symptoms
NPI (scores, mean±SD)	5.08±8.02	4.74±6.40	0.85
HAMA (scores, mean±SD)	6.58±7.03	6.56±6.86	0.99
HAMD (scores, mean±SD)	8.27±8.67	7.16±6.47	0.57
AS (scores, mean±SD)	16.52±10.35	13.90±12.67	0.38
PSQI (scores, mean±SD)	9.76±3.98	10.42±4.03	0.5
ESS (scores, mean±SD)	4.92±5.64	3.44±3.90	0.24
CMAI (scores, mean±SD)	33.88±9.57	30.83±6.33	0.15
3. ADL
Overall ADL (scores, mean±SD)	31.63±15.97	23.39±8.72	0.025 ^*
BADL (scores, mean±SD)	7.65±3.77	6.32±1.45	0.09
IADL (scores, mean±SD)	14.88±8.03	10.02±4.83	0.008 ^**

MMSE, Mini-Mental State Examination; AVLT, Auditory Verbal Learning Test; CFT, Rey-Osterrieth Complex Figure Test; TMT-A, Chinese modified version of the Trail Making Test (TMT)-A; TMT-B, Chinese modified version of Trail Making Test (TMT)-B; CWT-C, Stroop Color-Word Test-Chinese version (CWT)-Color; AFT, Animal Fluency Test; BNT, Boston Naming Test; SDMT, Symbol Digit Modalities Test; NPI, Neuropsychiatric Inventory; HAMA, Hamilton Anxiety Scale; HAMD, Hamilton Depression Scale; AS, Apathy Scale; PSQI, Pittsburgh Sleep Quality Index; ESS, Epworth Sleepiness Scale; CMAI, Cohen-Mansfield Agitation Inventory; ADL, Activities of Daily Living; BADL, basic ADL; IADL, instrumental ADL. ^*p < 0.05, ^**p < 0.01.

Overall psychological symptoms and each individual psychological manifestation were assessed by an array of rating scales (Table 2). There were no significant differences in the scores of NPI, HAMA, HAMD, AS, PSQI, ESS, and CMAI between AD-OD and AD-NOD groups.

ADL was assessed by the corresponding rating scales (Table 2). It was observed that AD-OD group had significantly higher scores of overall ADL and IADL than AD-NOD group. However, there was no significant difference in BADL score between the two groups.

Levels of free radicals and inflammatory factors in serum from AD-OD and AD-NOD groups

The levels of free radicals and inflammatory factors, including OH, H₂O₂, NO, IL-1β, IL-6, TNF-α, and PGE₂ in serum were compared between AD-OD and AD-NOD groups (Table 3). It was revealed that AD-OD group had significantly declined H₂O₂ level in serum and elevated •OH level than those in AD-NOD group (p <0.05). The levels of NO, IL–1β, IL-6, TNF-α, and PGE₂ in serum did not differ between AD-OD and AD-NOD groups.

Table 3

Levels of oxidative and inflammatory factors in serum from AD-OD and AD-NOD groups

	AD-OD group (29 cases)	AD-NOD group (50 cases)	p
•OH (U / mL, mean±SD)	713.85±307.59	564.54±310.88	0.042 ^*
H₂O₂ [mmol /l, median (quartile)]	24.51±9.78	33.85±18.12	0.003 ^**
NO (mmol/L, mean±SD)	60.05±17.57	57.03±17.63	0.46
IL-1β [pg/ml, median (quartile)]	9.44(7.86∼14.92)	9.17(7.18∼13.79)	0.88
IL-6 [pg/ml, median (quartile)]	2.56(1.59∼6.07)	2.53(1.30∼5.10)	0.88
TNF-α (pg/ml, mean±SD)	38.00±59.00	34.69±55.80	0.81
PGE₂ [pg/ml, median (quartile)]	7.39(2.68∼12.36)	5.53(2.79∼24.48)	0.63

•OH, hydroxyl radical; H₂O₂, hydrogen peroxide; NO, nitric oxide; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; prostaglandinE₂, PGE₂. ^*p < 0.05, ^**p < 0.01.

Relationship between OD and clinical symptoms

The relationships between HRS score and scores of cognitive symptoms were investigated. In the multiple linear regression analysis, it indicated that HRS score was significantly and positively correlated with the scores of MMSE (r = 0.361, p = 0.003), AFT (r = 0.355, p = 0.004), and BNT (r = 0.407, p = 0.001) after adjusting for age, age of onset, sex, and education level.

The relationships between HRS score and scores of psychological symptoms were explored. Analyses displayed no correlation between HRS score and the scores of NPI, HAMA, HAMD, AS, PSQI, ESS, and CMAI.

The relationships between the scores of HRS and ADL were studied. Correlation analysis reflected that HRS score was significantly and negatively correlated with IADL score (r = –0.146, p = 0.019). There was no correlation between the scores of HRS with overall ADL and BADL.

The relationships between HRS score and the levels of free radicals and inflammatory factor in serum were finally analyzed. Correlation analysis presented that HRS score was significantly and negatively correlated with •OH level in serum (r = –0.256, p = 0.018). In multiple linear regression models, age, age of onset, sex, educational level, the level of •OH in serum and the scores of AFT and BNT were set as independent variables, and HRS score was set as a dependent variable. We still found HRS score was significantly and negatively correlated with •OH level in serum (r = –0.256, p = 0.023), and positively correlated with the score of BNT (r = 0.305, p = 0.031) after adjusting for confounders.

DISCUSSION

The frequency of OD in AD patients

The average prevalence of OD in the population of normally aging individuals was 24.5% [33], and OD increased the risk of MCI by 50%. A study showed only 6% AD patients reported OD, however, another study showed that 90% of AD patients had different degrees of OD by examining the first cranial nerve [34]. It indicated that AD patients were not aware of the debut of their OD, nor of the severity of their impairment. It needed more attention and screening of OD in these patients. Epidemiological surveys of OD in patients with MCI due to AD have been rarely reported.

In this study, the frequency of OD in patients of total AD patients was 36.71%, patients with dementia due to AD was 54.84%, while patients with MCI due to AD was 25.00%. Previous study showed that the prevalence of OD in AD ranged from 48–85% [35], in MCI was 24% [9], which was in line with our results. It indicated that AD patients have a high prevalence of OD, patients with dementia due to AD had a higher prevalence of OD than patients with MCI due to AD. However, the OD was often ignored by both clinicians and patients. The difference among studies might be accounted for the different methods for testing olfactory function and different demographic and sociological data.

The symptoms of OD in AD patients

In this study, HRS score in AD-OD group was significantly higher than that in AD-NOD group, and HRS score in patients with dementia due to AD was prominently higher than that in patients with MCI due to AD, illustrating that OD worsened with disease progression [36, 37].

Demographics of AD-OD and AD-NOD groups

In this study, demographic variables, including age, age of onset, and sex were not different between AD-OD and AD-NOD group. However, other study showed that the prevalence of OD increased with aging in elderly patients [37]. There is rare study investigating the relationship between age and OD in AD patients. The average age in this study was 66.99 ±10.8 years old, which was younger than that in a previous study, therefore, OD in this study might be affected little by aging.

In human population, OD was more severe in male than in female individuals because of the differences in the number of olfactory bulb cells [38]. However, this study failed to see correlation between OD and sex, which might be because that female individuals were more likely to suffer from AD and related OD during lifetime, leading to no relationship between sex and OD in AD patients [39].

Additionally, AD-OD group and AD-NOD groups presented no difference in disease duration, indicating that disease duration did not determine the occurrence of OD in AD patients.

Clinical symptoms of AD-OD and AD-NOD groups

In this study, MMSE score in AD-OD group was significantly lower than that in AD-NOD group, indicating that overall cognitive function in AD-OD group was significantly compromised than that in AD-NOD group. It has been known that normal olfactory function required intact cognitive function, including working memory, judgment and decision making, etc. Thus, dysfunction of olfaction might reflect the generalized impairment of cognitive function [40].

We then investigated the association between OD and each cognitive domain. AD-OD group showed prominently reduced AVLT-delayed recall score than that in AD-NOD group, implying that verbal delayed memory was seriously damaged in AD-OD group. However, HRS score was not decreased with the score of AVLT-delayed recall after adjusting for some factors, e.g., age, age of onset, sex, and education level. Delayed memory decline is a clinical hallmark of AD, but was not closely related with OD.

The next cognitive domain we examined was language. It was reported that language dysfunction might occur early in AD patients and was with predominant deficits in semantic fluency and naming [41]. A previous study showed that OD could predict language decline [42]. Importantly, we found direct evidence showing that the scores of AFT and BNT in the AD-OD group were significantly decreased than those in the AD-NOD group, and HRS score was evidently reduced as the score of BNT decreased after further correlation analysis. Thus, impaired language function, mainly involving semantic fluency and naming, was closely associated with OD in AD patients. It is known that dysfunction of semantic fluency is attributed to the destruction of key nodes in language networks within left temporal and parietal lobes, and coincidentally, entorhinal cortex included in temporal lobe is part of olfactory pathway [43], implying a common anatomical base for language impairment and OD in an individual with AD.

Results from another investigator showed that OD in MCI patients was associated with visuomotor speed and executive function [43]. Unfortunately, the linkage between OD and cognitive domains above were not observed in the current study. This difference might be explained because patients collected were in a different disease stage and the severity of cognitive impairment was different.

Neuronal pathways responsible for visuospatial ability originating from occipital lobe to parietal lobe do not have crosstalk with that area in charge of olfactory function, such as medial temporal lobe and hippocampus, etc. Thus, anatomical discrepancy might explain one of the results from this study indicating no correlation between OD and visuospatial ability.

The associations between OD and psychological symptoms, including depression, anxiety, sleep quality, excessive daytime sleepiness, irritation, and apathy, were also explored. Generally speaking, we failed to determine the correlation between OD and overall psychological condition reflected by NPI score.

There was no significant difference in depression and anxiety between the AD-OD and AD-NOD groups, which was insistent with a previous study [44].

Sleep disorders are a common psychological manifestation in AD patients. There are very few studies investigating the relationship between sleep disorder and OD in AD patients. Here, sleep quality and excessive daytime sleepiness was not related to OD. It is known that raphe nuclei in brainstem contributes to an ascending arousal system that promotes wakefulness and prevents excessive daytime sleepiness [45]; however, brain areas related to olfactory function, e.g., medial temporal lobe and hippocampus, are disconnected with that related to sleep. Accordingly, there was no structural basis establishing the relevance between OD and sleep disorders.

Irritation is a frequent psychological symptom in AD patients. Irritation in patients with MCI and dementia due to AD is associated with frontal lobe, insular lobe, cingulate gyrus, and hippocampus [46], which are also related to olfactory function. However, irritation scores showed no difference between AD-OD and AD-NOD groups in this study. This might be explained that patients included here were relatively mild (the average MMSE score was 22.01±7.26 points) and only 26.58% cases had irritation. Hence, it was difficult to find correlation between olfactory function and irritation. In the future, AD patients in different stages of the disease will be needed to investigate the actual relationship between olfactory function and irritation.

Apathy is a highly common neuropsychiatric manifestation of AD. A previous study showed that reduced odor identification tested by Sniffin’ Sticks was associated with increased apathy severity in AD patients [46]. However, this study did not find this relationship, which might be because different methods were used to test olfactory function. HRS is a relatively comprehensive scale to evaluate olfaction. These data demonstrated that apathy was highly correlated with odor identification, but not other aspect of OD in AD patients.

After analyzing the correlation of OD with cognitive symptoms and psychological manifestations, we evaluated the impact of OD on the ADL of AD patients. Overall ADL was severely compromised in the AD-OD group compared to the AD-NOD group. Further investigation implied that IADL, but not BADL, was correlated with OD since HRS score was drastically decreased when IADL score was greatly increased. The average score of MMSE in AD patients was 22.01±7.26 points, indicating that AD patients in this study were at relatively early stage, hence, the IADL but not BADL, was significantly impaired.

Levels of free radicals and inflammatory factors in serum from AD-OD and AD-NOD groups

Considerable evidence implicated the role of oxidative stress and inflammation on the pathophysiology of AD. Studies of brain tissues from AD patients consistently showed the evidence of inflammation, as indicated by the presence of activated microglia and robust production of inflammatory factors [14]. Particularly, there was a great deal of evidence suggesting an important role of peripheral inflammation on the pathogenesis of AD. The action of peripheral inflammation, and indeed the inflammation in general, was still largely considered to be a contributor to the process of AD [47]. Yet, there was no study investigating the relationship between olfactory function and peripheral inflammation in AD patients. This study showed for the first time that the AD-OD group had significantly higher •OH levels in serum, and HRS score was prominently decreased with •OH levels in serum was robustly increased in AD subjects (r = –0.243, p = 0.036). Superoxide is synthesized into H₂O₂, which forms the fairly reactive and toxic •OH in the presence of a high concentration of free iron, causing damage to neurons [48]. Here, H₂O₂ levels in serum in the AD-OD group were significantly reduced than that in the AD-NOD group, implying that more H₂O₂ was converted to •OH during the oxidative event. However, there was no correlation between HRS score and H₂O₂ levels in serum, suggesting a pivotal effect of oxidative stress in AD-OD and •OH was a more sensitive indicator than H₂O₂ in the peripheral system for OD in AD patients. There are multiple mechanisms underlying the increased free radicals/inflammatory factors. Aβ deposition in the entorhinal cortex and the hippocampus could cause inflammation [42, 49], which might be further damage neurons, leading to OD as well as cognitive impairment. Additionally, iron-induced oxidative stress might modulate olfactory function [50, 51]. In this investigation, we found elevated levels of free radicals in AD-OD patients, which might be resulted from excessive depositions of Aβ or iron in the brain regions related to OD.

In the future, we will explore the relationship between the levels of free radicals/inflammatory factors and Aβ or iron in serum or cerebrospinal fluid, aiming to establish the link between them in AD-OD patients. No matter what the sources of free radicals/inflammatory factors, they may play pivotal roles on the degeneration and death of neurons in the olfaction and cognition-related brain regions by causing damage to membranes, and mitochondria, etc.

Limitations

This study had some limitations. Firstly, this study had a small sample size. This association needs to be verified in future studies with a larger sample size. Secondly, this is a cross-sectional design, prohibiting determination of causality or direction of the associations. Thirdly, the utility of the HRS compared to the UPSIT or SST had some subjectivity; larger studies that use SST are needed to confirm the results of the present study. Finally, this article is an exploratory study, aimed at finding more potential inflammatory factors participating in inflammation in patients with AD-OD. Therefore, statistical analysis is simple. In the future, we will enlarge the sample size and verify the conclusions.

Conclusions

In summary, the frequency of OD was high in patients with MCI and dementia due to AD. AD patients with OD had significant impairments in the cognitive domain of language. OD had a dramatic impact on IADL of AD patients. Oxidative stress played an important role on OD in AD patients and elevated •OH level in serum might be a potential predictor of OD in AD patients.

Fig.1

Correlations between the Hyposmia Rating Scale (HRS) score and the •OH level in serum in AD patients.

Footnotes

ACKNOWLEDGMENTS

This work was supported by The National Key Research and Development Program of China (2016YFC1306300, 2016YFC1306000), The National Natural Science Foundation of China (81571229, 81071015, 30770745), The Key Project of National Natural Science Foundation of China (81030062), The Key Project of Natural Science Foundation of Beijing, China (B) (kz201610025030), The Key Project of Natural Science Foundation of Beijing, China (4161004, kz200910025001), The Natural Science Foundation of Beijing, China (7082032), Project of Scientific and Technological Development of Traditional Chinese Medicine in Beijing (JJ2018-48), National Key Basic Research Program of China (2011CB504100), Important National Science & Technology Specific Projects (2011ZX09102-003-01), National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2013BAI09B03), Project of Beijing Institute for Brain Disorders (BIBD-PXM2013_014226_07_000084, High Level Technical Personnel Training Project of Beijing Health System, China (2009-3-26), Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges Under Beijing Municipality (IDHT20140514), Capital Clinical Characteristic Application Research (Z12110700100000, Z121107001012161), Beijing Healthcare Research Project, China (JING-15-2,JING-15-3), Excellent Personnel Training Project of Beijing, China (20071D0300400076), Basic-Clinical Research Cooperation Funding of Capital Medical University, China (2015-JL-PT-X04, 10JL49, 14JL15), Youth Research Funding, Beijing Tiantan Hospital, Capital Medical University, China (2014-YQN-YS-18, 2015-YQN-15, 2015-YQN-05, 2015-YQN-14, 2015-YQN-17).

Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/18-0425r2).

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