Review

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A review of biomarkers of Alzheimer’s

disease in noninvasive samples
Samuel Hartmann1 & Tarso B Ledur Kist*,1
1
Laboratory of Methods, Department of Biophysics, Institute of Biosciences, Federal University of Rio Grande do Sul, 91.501–970,
Porto Alegre, RS, Brazil
*Author for correspondence: tarso@ufrgs.br

The discovery of biomarkers that confer high confidence of presymptomatic Alzheimer’s disease (AD)
diagnosis would be a valuable tool to study the etiology of the disease, to find risk factors, to discover
more treatments and medicines. The present work reviews the potential biomarkers of AD based on the
concentration changes of small molecules and chemical elements in noninvasive samples (urine, saliva,
hair and others). An updated table with 74 target compounds is produced and ranked. Until the present
date, there are a few biomarkers, present in urine, with the most promising potential: isoprostane 8,12-
iso-iPF2a-VI, total free amino acids, 8-hydroxy-2 -deoxyguanosine, glycine and enzymatic activity of NaCl-
stimulated PON1. All show increased levels in AD carriers, with the exception of NaCl-stimulated PON1.

3.0

HO
HO
2.5 COOH

AD
2.0 HO
Probability density

1.5
MCI

1.0

0.5

0.0
2 3 4 5 6
Concentration (ng/mg creatinine)

First draft submitted: 6 November 2017; Accepted for publication: 9 February 2018; Published online:
13 June 2018

Keywords: Alzheimer • biomarker • urine

As the life expectancy is increasing due to higher living standards and efficient prevention and treatment of other
diseases, the prevalence of AD is predicted to rise dramatically within the next years and place it among the major
public health concerns. The number of people worldwide with dementia was estimated to be 35.6 million in 2010

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C 2018 Future Medicine Ltd Biomark. Med. (Epub ahead of print) ISSN 1752-0363
Review Hartmann & Ledur Kist

by the WHO. This is projected to nearly double every 20 years, to 65.7 million in 2030 and 115.4 million in
2050 [1]. Dementia is characterized by the impairment of multiple brain functions, including memory, thought
processes, orientation, comprehension, learning capacity, language and judgment. Alzheimer’s disease (AD) is the
most common form of dementia among people aged 65 or more and probably contributes to around 60–70% of
cases. It should not be confused with normal age-related decline in cognitive functions. The pathophysiology of
AD is characterized by protein aggregation in the brain (amyloid β [Aβ] protein plaques and neurofibrillary tangles
comprising hyperphosphorylated tau proteins) [2,3], loss of neuronal tissue, usually initiating in the hippocampus
and progressively worsening over time until atrophy affects large regions of the brain [1,4]. Image analysis of the
brain shows progressive enlargement of the cerebral ventricles and the cerebral cortex, depending on the stage (early,
middle or late stage) of the disease [5]. The most common symptoms include loss of short-term memory, mood
swings and problems with language, behavior, motivation, self-care and disorientation. As the patient’s condition
declines, they withdraw from family and social interactions. Body functions are further gradually impaired, with
patients subjected to extreme apathy and exhaustion. The common causes of death are infections of pressure ulcers
and pneumonia, among other factors. In 2013, the average life expectancy following diagnosis was estimated at
between 3 and 9 years [6].
Familial AD accounts for only 0.5% of all cases, with the onset before the age of 65 and 50% of them
carry at least one mutation in the APP, PSEN1 or PSEN2 gene. Therefore, other important risk factors are still
under investigation, especially that one responsible for the so-called late-onset AD [7,8]. The current standard for
evaluating AD dementia in clinical practice is based on recommendations from the National Institute on Aging-
Alzheimer’s workgroups, first published in 1984, and last revised in 2011 by a workgroup of investigators of AD [9].
This standard, commonly referred to as NINCDS-ADRDA, comprises a general screening of patients, including
medical history, clinical examination, neuropsychological testing and laboratory assessments. Three categories were
proposed for AD patients: Probable AD dementia, Possible AD dementia, and Probable or Possible AD dementia
with evidence in pathophysiological processes. The first two categories include patients with dementia that present
a sudden onset of cognition impairment, affecting their language functions, visual abilities and their capacity to
acquire new information and handle complex tasks, ultimately leading to changes in personality and behavior. The
inclusion of biomarkers in the diagnosis of AD led to the definition of the third category, which resulted in increased
accuracy diagnoses, as well as the possibility to differentiate between the stages and progression of the disease. A
consensus report [10] of a working group on molecular and biochemical markers of AD listed the following desired
properties of an AD biomarker: It should detect a fundamental feature of neuropathology; It should be sensitive,
specific, reliable, reproducible and inexpensive; It should be obtained through simple noninvasive procedures.
While this report was published 20 years ago, there is still no known biomarker that meets all these criteria.
A biological marker, commonly referred to as a ‘biomarker’, is a substance, structure or process that can be
accurately and reproducibly measured in the body or its products. The occurrence of a specific biomarker at a certain
quantitative level serves as an indication of a normal or pathogenic biological process, a pharmacological response
to a therapeutic intervention, or a by-product of the metabolism of a medicine. Biomarkers are increasingly used
in the prognosis, diagnosis and monitoring of many diseases and treatments [11]. An ideal AD molecular biomarker
should indicate with 100% confidence if a patient does or does not have the AD in progression at the time of
the test. These biomarkers may be categorized as genetic, imaging, behavioral, molecular or chemical element. A
number of connections between cerebrospinal fluid (CSF) biomarkers and AD have been identified [12], for a review,
see [13]. The Aβ protein, total tau (t-tau) and phosphorylated tau (p-tau) proteins are currently the key biomarkers
used, where the decrease in Aβ protein concentration with increased levels of t-tau or p-tau differentiates AD from
other forms of dementia with accuracy of about 90%. Therefore, the search for new and additional biomarkers
continues, as there are still limitations due to the overlap in these biomarker levels with conditions other than AD.
Examples of other biomarkers that are being studied are: new Aβ isoforms [14], improved MRI techniques [15],
cerebral microbleeds [16], semantic memory decline [17], eye movement [18], retinal abnormalities [19] and alteration
in blood lymphocytes [20]. A review of genetic, imaging and molecular biomarkers is given in [21]. In the present
work, only molecular and chemical element biomarkers in noninvasive samples will be reviewed.
For noninvasive samples, this corresponds to: the presence, higher than normal levels, lower than normal levels
or complete absence of a chemical compound (protein, peptide, metabolite or chemical element). However, this is
hard to prove for most of the biomarkers. Real surveys are normally conduced with a very limited number (N) of
individuals (patients and controls) because chemical analysis of the samples remains slow, some of them are expensive
and have limited precision (with errors of up to 5% in the results of the analyses being accepted). In addition to this

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 A review of biomarkers of Alzheimer’s disease in noninvasive samples Review

bottleneck, there are many additional sources of errors which may under or overestimate the role of a biomarker.
As summarized by Nelson et al. [22], the environment where the study was performed (i.e., hospital-based, clinic or
random population) is one of the many aspects that may affect the results. In a statistically well-conducted survey,
the participating individuals should be randomly chosen from within the population, using a random number
generator (to avoid biased results). However, this is not possible due to practical and ethical issues. Although some
uncertainties and errors will still remain, even when all sorted individuals agree voluntarily to participate in a
planned survey. Some examples include the interference of other diseases that may share some common features,
errors during sample collection and labeling, variations between clinicians’ practices in sorting patients into the
control and AD group (particularly in the early stages), if AD is an intermittent neurodegenerative process or not,
to cite a few.
In the following sections, some molecular biomarkers of AD in noninvasive samples are reviewed and studied,
specifically samples from urine, saliva, hair and nails. No publications were found in the scope of the present review
(with at least a documented p-value, mean value and standard deviation) in relation to chemical biomarkers in
other noninvasive samples (nasal fluids, tears, breath, sweat or others). All those works published after 1984 and
included in the present review follow the NINCDS-ADRDA guidelines for evaluating AD (with the exception
of only three, shown in Table 1). There are many statistical studies that can be performed with the data if it is
presented as frequency histograms or scattering plots. Unfortunately, the authors presented only the p-values, mean
values and standard deviations. While a few also presented scatter diagrams, no one presented the data as frequency
histograms. This is unfortunate because a lot of information is lost when the dataset is reduced to averages and
standard deviations. It was also not possible to recover the frequency histograms from the few scatter plots found in
the reviewed literature, because more than 10% of the points are overlapped with other points. Therefore, only the
‘resolution’ and the overlap of the concentration of biomarkers between control and AD patients could be calculated
and tabulated. This was performed with the assumption of a normal probability density distribution. The overlap
of the distributions indicates the fraction (between 0 and 1) of the exams giving false-positive or false-negative
results when a particular biomarker is used. Resolution (R) is a parameter indicating how well the two distributions
(biomarker concentration in control and AD groups) are separated from each other. This is calculated by taking the
modulus of the difference between the mean values (μ) of the biomarker and dividing it by two-times the sum of
their standard deviations (σ), R = |μcontrol + μAD |/[2(σcontrol + σAD )]. Distributions with R = 1 are already resolved
from each other, but still with an important overlap, while R > 2 means well-resolved distributions with negligible
overlap (with negligible occurrence of false positives or false negatives in the clinical exams). The p-values found by
the authors of the reviewed papers are also cited within the text, but they are only an indication of whether the two
distributions (biomarker concentration in control group and AD) could be produced by pure chance. This is an
important parameter, but it should be taken simply as an indication of a sample’s compatibility with a hypothesis,
and not as a guarantee for the truth of that hypothesis [23].

Biomarkers in noninvasive samples

Studies of biomarkers of AD have traditionally focused on invasively obtained fluids, such as blood samples and
CSF. Exponent markers found in these fluids are used for the prediction of future cognitive impairment and the
progression of the disease, related to neurofibrillary and amyloid pathologies.
The use of noninvasive samples presents the potential to benefit millions of people worldwide, because these
samples do not require special storage conditions and are therefore easily collected and shipped to laboratories for
clinical analysis. Moreover, some of these noninvasive samples (like urine and saliva) are disposed of on a daily basis.
They could be readily used in small, affordable and disposable kits or sensing devices for AD testing at point-of-use
or point-of-discard. In the next sections, the literature reporting surveys of AD molecular biomarkers will be
reviewed, focusing on articles published until July 2017, and only taking into account those with documented
mean values, standard deviations and p-values.

Urine
The main functions of the urinary system are to eliminate waste from the body and to regulate the following
variables of the blood: its volume, pressure, pH and levels of both electrolytes and metabolites. Adult humans
produce an average of 1.4 L of urine per day, 91–96% of it consisting of water with the remainder made up of
electrolytes and organic compounds. Metabolites and hormones are among these organic compounds, and they
vary depending on physiological conditions and what is introduced into the body [37].

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Table 1. Alzheimer’s disease biomarkers in noninvasive samples ranked by resolution, R = |μcontrol -

μAD |/[2(␴control + ␴AD )].
Ranking Sample Biomarker Unit Control group AD patients R % % % Exact
False False results
posit. neg.
n μ ␴ n μ ␴
1 u 8,12-iso-iPF2␣-VI† [24] ng/mg 40 1.5 0.1 50 4.6 0.2 5.17 0.0 0.0 100.0
creat.
2 u Total free amino acids‡ [25] nmol/dL 8 28.94 6.44 8 54,262.9 5757.35 4.70 0.0 0.0 100.0
3 u 8-Hydroxy-2 - nmol/L 20 24.8 8.3 21 129 30 1.36 0.2 0.4 99.4
deoxyguanosine [26]
4 u Glycine [25] nmol/dL 8 8.5 1.3 8 15.6 1.7 1.18 0.7 1.1 98.2
5 u NaCl-stimulated PON1 [26] U/L 20 444 11.4 21 388 15.6 1.04 2.2 2.0 95.8
6 u 8-Hydroxy-2 - μ mol/mol 20 9.28 2.23 21 115.7 50 1.02 1.3 0.1 98.6
deoxyguanosine [26] creat.
7 u PON1 [26] U/L 20 104.1 2.9 21 94.1 3.1 0.83 5.0 4.7 90.4
8 u Protein concentration [25] mg/mL 8 0.088 0.012 8 0.135 0.026 0.62 4.8 15.0 80.3
9 h Lead§ [27] μ g/g dry 60 0.74 0.47 62 0.14 0.13 0.50 17.4 3.8 78.8
tissue
10 h Spermidine/Spermine ratio [28] – 18 1.86 0.95 34 0.66 0.33 0.47 22.4 5.3 72.2
11 u Pseudouridine [29] nmol/μ mol 34 6.168 5.275 36 20.433 13.184 0.39 7.6 27.4 65.0
creat.
12 u Triglycerides [26] nmol/L 20 1.73 0.15 21 1.53 0.12 0.37 42.4 9.0 48.6
13 u N2,N2-dimethylguanosine§ [29] nmol/μ mol 34 0.349 0.299 36 1.934 1.886 0.36 2.7 17.3 80.0
creat.
14 u Non-HDL-cholesterol [26] nmol/L 20 3.73 0.23 21 3.48 0.22 0.28 44.9 16.3 38.9
15 u AD7c-NTP [30] μ g/mL 22 18.1 6.7 49 26.8 9.4 0.27 19.0 38.2 42.9
16 n Copper [26] μ g/g dry 60 40.04 32.74 62 17.49 11.87 0.25
tissue
17 h Selenium [31] μ g/g 31 0.6 0.1 37 0.5 0.1 0.25
18 u 2-Deoxyguanosine [29] nmol/μ mol 34 3.087 2.812 36 7.218 6.004 0.23
creat.
19 u Total cholesterol [26] nmol/L 20 4.93 0.25 21 4.73 0.21 0.22
20 n Manganese [27] μ g/g dry 60 6.76 9.57 62 2.02 1.61 0.21
tissue
21 n Iron [27] μ g/g dry 60 384.62 332.12 62 171.76 177.73 0.21
tissue
22 h Manganese [31] μ g/g 33 0.4 0.2 41 0.6 0.3 0.20
23 s Morning cortisol [32] μ g/dL 34 10.31 4.14 19 16.55 12.38 0.19
24 h Sodium [27] μ g/g dry 60 42.79 48.6 62 89.45 76.24 0.19
tissue
25 u 3-Methyluridine [29] nmol/μ mol 34 0.226 0.174 36 0.375 0.242 0.18
creat.
26 h Aluminum [33] μ g/g 35 6.2 3.6 71 3.9 2.96 0.18
27 h Copper [31] μ g/g 31 10.3 2.4 41 12.8 4.8 0.17
28 s Evening cortisol [32] μ g/dL 34 2.59 2.55 19 4.73 3.73 0.17
29 u 8-Hydroxy-2 - nmol/μ mol 34 0.139 0.126 36 0.239 0.199 0.15
deoxyguanosine [29] creat.
30 u 1-Methyladenosine [29] nmol/μ mol 34 0.623 0.354 36 0.967 0.786 0.15
creat.
The references are given in the brackets after each biomarker’s name. The sample sizes (number of individuals, N), concentration mean values (μ ) and standard deviations (␴) are
shown for control and AD groups. The percentage of false positives and false negatives for AD that would be observed in each survey if the respective biomarker would be used are
also shown (a normal density probability distribution is assumed).
†
In this study, the biomarker levels were also measured in 33 mild cognitive impairment carriers; the average level found was located between the control group and the AD carriers
(shown in Figure 1).
‡ Total free amino acids including citrulline and ornithine.
§ Poisson density probability distribution would be a best fit compared with normal distribution (Gaussian).
¶ Did not follow the NINCDS-ADRDA guidelines.
#
Measuring units for albumin is missing.
††
Data not available.
AD: Alzheimer’s disease; Creat.: Creatinine; h: Hair; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; n: Nail; R: Resolution; s: Saliva; TSH: Thyroid-stimulating hormone;
u: Urine.

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 A review of biomarkers of Alzheimer’s disease in noninvasive samples Review

Table 1. Alzheimer’s disease biomarkers in noninvasive samples ranked by resolution, R = |μcontrol -

μAD |/[2(␴control + ␴AD )] (cont.).
Ranking Sample Biomarker Unit Control group AD patients R % % % Exact
False False results
posit. neg.
n μ ␴ n μ ␴
31 h Zinc [31] μ g/g 33 98 54 41 75 29 0.14
32 n Zinc [27] μ g/g dry 60 91.45 41.59 62 74.47 21.68 0.13
tissue
33 n Magnesium [27] μ g/g dry 60 1352.1 1705 62 778.57 497.77 0.13
tissue
34 n Cadmium [27] μ g/g dry 60 0.73 0.82 62 0.45 0.35 0.12
tissue
35 u 5-Deoxyadenosine [29] nmol/μ mol 34 0.020 0.019 36 0.028 0.017 0.11
creat.
36 u LDL-cholesterol [26] nmol/L 20 2.88 0.21 21 2.79 0.22 0.10
37 h Manganese [27] μ g/g dry 60 1.15 4.16 62 0.22 0.45 0.10
tissue
38 h Dry tissue copper [27] μ g/g 60 22.83 22.39 62 16.24 13.72 0.09
39 h Potassium [27] μ g/g dry 60 621.16 972.71 62 1010.7 1176.9 0.09
tissue
40 n TGF [34] mg/dL 89 161.64 102.59 43 133.14 62.12 0.09
41 n Sodium [27] μ g/g dry 60 392.08 396.85 62 594.51 813.62 0.08
tissue
42 n LDL-cholesterol [34] mg/dL 89 133.14 62.12 43 116.28 40.57 0.08
43 h Aluminum [27] μ g/g dry 60 251.15 474.28 62 134.26 255.52 0.08
tissue
44 s A␤42 [35] pg/mL 38 2.89 4.96 21 6.81 20.04 0.08
45 n HDL-cholesterol [34] mg/dL 89 56.42 13.92 43 61.1 16.31 0.08
46 n TSH [34] μ IU/mL 89 2.39 4.06 43 1.61 1.11 0.08
47 s A␤40 [35] pg/mL 38 20.82 5.55 21 22.3 4.88 0.07
48 u HDL-cholesterol [26] nmol/L 20 1.26 0.073 21 1.24 0.078 0.07
49 h Magnesium [27] μ g/g dry 60 572.72 699 62 839.4 1540.3 0.06
tissue
50 n Ferritin [34] ng/mL 89 80.76 80.17 43 95.51 61.04 0.05
51 n Mercury [27] μ g/g dry 60 0.28 0.32 62 0.23 0.17 0.05
tissue
52 h Mercury [27] μ g/g dry 60 0.19 0.29 62 0.14 0.26 0.05
tissue
53 n Aluminum [27] μ g/g dry 60 939.92 1112.1 62 1246.2 2575.4 0.04
tissue
54 n Calcium [27] μ g/g dry 60 1376.4 1453.6 62 1169.4 1054.7 0.04
tissue
55 h Calcium [31] μ g/g dry 60 622.06 876.59 62 495.41 880.85 0.04
tissue
56 h Cobalt [27] μ g/g dry 60 0.41 0.42 62 0.34 0.7 0.03
tissue
57 n Zinc [33] ppm 89 123.86 77.98 43 117.99 73.44 0.02
58 u PON1/HDL [26] nmol/L 20 87.6 5.4 21 87.2 5.17 0.02
The references are given in the brackets after each biomarker’s name. The sample sizes (number of individuals, N), concentration mean values (μ ) and standard deviations (␴) are
shown for control and AD groups. The percentage of false positives and false negatives for AD that would be observed in each survey if the respective biomarker would be used are
also shown (a normal density probability distribution is assumed).
†
In this study, the biomarker levels were also measured in 33 mild cognitive impairment carriers; the average level found was located between the control group and the AD carriers
(shown in Figure 1).
‡ Total free amino acids including citrulline and ornithine.
§ Poisson density probability distribution would be a best fit compared with normal distribution (Gaussian).
¶ Did not follow the NINCDS-ADRDA guidelines.
#
Measuring units for albumin is missing.
††
Data not available.
AD: Alzheimer’s disease; Creat.: Creatinine; h: Hair; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; n: Nail; R: Resolution; s: Saliva; TSH: Thyroid-stimulating hormone;
u: Urine.

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Review Hartmann & Ledur Kist

Table 1. Alzheimer’s disease biomarkers in noninvasive samples ranked by resolution, R = |μcontrol -

μAD |/[2(␴control + ␴AD )] (cont.).
Ranking Sample Biomarker Unit Control group AD patients R % % % Exact
False False results
posit. neg.
n μ ␴ n μ ␴
59 n Cobalt [27] μ g/g dry 60 0.62 0.62 62 0.67 0.81 0.02
tissue
60 n Homocysteine [33] μ mol/L 89 15.11 5.23 43 15.41 3.58 0.02
61 n Albumin¶ ,# [33] 89 4.13 0.42 43 4.11 0.47 0.01
62 h Cadmium [27] μ g/g dry 60 0.31 0.32 62 0.29 0.59 0.01
tissue
63 u 5-Methylcytidine [29] nmol/μ mol 34 0.225 0.166 36 0.231 0.137 0.01
creat.
64 h Zinc [27] μ g/g dry 60 100.38 64.4 62 98.49 49.4 0.01
tissue
65 h Iron [27] μ g/g dry 60 332.98 352.73 62 339.15 673.1 0.00
tissue
66 n Potassium [27] μ g/g dry 60 519.47 361.78 62 521.42 315.28 0.00
tissue
67 n Lead [27] μ g/g dry 60 3.32 2.01 62 3.32 1.84 0.00
tissue
68 u 5-Methyl-2 -deoxycytidine [29] nmol/μ mol 34 0.262 0.156 36 0.262 0.241 0.00
creat.
†† ††
69 u F2-isoprostanes [36] ng/mg 34 32 1.3 0.2 –
creat.
††
70 u 3-Methyl-histidine [25] nmol/dL 8 8 36,759.7 2580.93 –
24,945.1
††
71 h Putrescine¶ [28] ng/g 18 48.03 34 173.01 e –
††
72 u Histidine [25] nmol/dL 8 8 46,379.5 6615.54 –
35,035.1
††
73 u 1-Methyl-histidine [25] nmol/dL 8 8 23,879.4 6141.94 –
33,001.3
††
74 u Carnosine [25] nmol/dL 8 8 28,603.3 15,669.3 –
13,443.5
The references are given in the brackets after each biomarker’s name. The sample sizes (number of individuals, N), concentration mean values (μ ) and standard deviations (␴) are
shown for control and AD groups. The percentage of false positives and false negatives for AD that would be observed in each survey if the respective biomarker would be used are
also shown (a normal density probability distribution is assumed).
†
In this study, the biomarker levels were also measured in 33 mild cognitive impairment carriers; the average level found was located between the control group and the AD carriers
(shown in Figure 1).
‡ Total free amino acids including citrulline and ornithine.
§ Poisson density probability distribution would be a best fit compared with normal distribution (Gaussian).
¶ Did not follow the NINCDS-ADRDA guidelines.
#
Measuring units for albumin is missing.
††
Data not available.
AD: Alzheimer’s disease; Creat.: Creatinine; h: Hair; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; n: Nail; R: Resolution; s: Saliva; TSH: Thyroid-stimulating hormone;
u: Urine.

Aging process and neurological diseases, such as AD, are related to an increase of free-radical reactions and the
enhancement of protein and DNA oxidative damages, confirmed by postmortem analysis of brain tissues. This
knowledge has led to a search for markers of the oxidative processes that occur during the development of the
disease, considering different hypotheses for the pathology.
A well-known biomarker for DNA oxidative damage is the 8-hydroxy-2 -deoxyguanosine (8-OHdG), making it a
promising biomarker for AD. In a study published in 2007, Lee et al. [29] enrolled 36 patients with AD and 34 healthy
controls. The results showed an increased level of 8-OHdG in patients with AD when compared with controls
(0.239 ± 0.199 nmol/μmol creatinine and 0.139 ± 0.126 nmol/μmol creatinine, respectively, with p < 0.05).
Similar results were found by Zengi et al. in 2011 [26], by comparing the mean urinary levels of 8-OHdG in a group of
21 patients with AD to 20 control patients. The AD group showed a mean urinary level of 115.7 ± 50 nmol/μmol
creatinine for 8-OHdG, while the control group had a mean level of 9.28 ± 2.23 nmol/μmol creatinine, with
p < 0.001.

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 A review of biomarkers of Alzheimer’s disease in noninvasive samples Review

Another suitable hypothesis in the context of free-radical reactions and oxidative damage is the lipid peroxidation
process. According to this proposition, a free-radical-induced injury to the phospholipid membrane could induce the
fragmentation of lipids, and some fragments could undergo an internal cyclization process producing isoprostanes
and neuroprostanes. In 1998, two independent studies indicated an increased level of F2-isoprostanes in CSF
samples of patients with AD (Praticò et al. [38] and Montine et al. [39]).
In 2002, these same authors published new results including levels of isoprostanes in the urine of patients with
probable AD. The methodology implemented by Montine et al. [36] was the comparison of F2-isoprostanes and
F4-neuroprostanes in plasma and urine levels using gas chromatography, negative ion chemical ionization mass
spectrometry and selective ion monitoring. The test was performed on 56 subjects with probable AD, and 34
healthy controls, and no difference was found between probable AD patients and healthy controls. In addition,
they induced an elevation of these substances in the brains of rats, aiming to test the function of the blood–brain
barrier in isolating prostanes in the brain from peripheral fluids. The results from the rats did not indicate an
increase in peripheral levels of prostanes, suggesting that peripheral levels do not reflect the CNS concentrations.
Another article published in 2002 by Praticò et al. [24] showed conflicting results with Montine et al.’s findings.
Praticò et al. collected urine and blood samples from 50 patients with AD, 33 with mild cognitive impairment
(MCI) and 40 healthy controls. MCI is characterized by an abnormal memory loss with no clinical evidence for a
diagnosis of AD, although it is believed that subjects in this stage have a higher probability of developing AD over
a short time period. In addition to the isoprostanes analysis, they also collected peripheral leukocytes aiming to
classify patients according to their ApoE-4 genotype. Using GC–MS analysis, it was found that levels of isoprostanes
in urine significantly increased from controls to MCI and AD patients. The mean level of isoprostanes in controls,
MCI subjects and AD patients was 1.5 ± 0.1 ng/mg of creatinine (p < 0.001), 3.6 ± 0.3 ng/mg of creatinine
(p < 0.01) and 4.6 ± 0.2 ng/mg of creatinine (p < 0.001), respectively. These findings suggest that isoprostanes
could be reliable markers of in vivo peroxidation processes, and their increased levels could be detectable even in
the early stages of the disease.
Since the discovery and isolation of a new membrane-associated protein by De La Monte et al. in 1997 [40],
the approximately 41-kDa protein called AD7c-NTP received a lot of attention from researchers and companies
around the world. This protein is involved in cell proliferation, differentiation and brain development, and its
associated Alu sequence is found to be overexpressed in brains of AD and CSF patients with the disease. A pioneer
study published in 2007 by Goodman et al. [41] measured AD7c-NTP levels in the urine of patients with AD. One
hundred and sixty-eight patients diagnosed with probable AD, possible AD or MCI were enrolled, in addition to
122 clinically healthy controls. The results showed a significantly higher level of AD7c-NTP in the urine of subjects
with probable AD, possible AD and MCI when compared with definite non-AD patients (33.35 μg/mLfor probable
AD, 22.15 μg/mL for possible AD, 24.84 μg/mL for MCI and 18.30 μg/mL for definite non-AD, p < 0.0001).
As the role of the neuronal thread protein AD7c-NTP in the development of dementias in still not clear,
Youn et al. [30] conducted a study with 49 patients with AD, 22 healthy controls and 20 patients diagnosed with
Parkinson’s disease (PD). The results showed that levels of the protein in AD subjects are significantly higher than
in PD and healthy controls (26.8 ± 9.4 μg/mL for AD, 18.1 ± 6.7 μg/mL for PD and 21.0 ± 8.5 μg/mLfor
healthy controls, p < 0.001 and < 0.05, respectively).
In another study published in 2014 by Ma et al. [42], the researchers traced the levels of AD7c-NTP according
to the stage of the disease. 45 AD subjects, 60 MCI and 65 controls were enrolled in their study. The mean level
of the protein in the urine of AD and MCI subjects was significantly higher than in healthy controls (2.14 ng/mL
for AD, 1.57 ng/mL for MCI and 0.53 ng/mL for controls, p < 0.05).
In a study published in 2007 by Fonteh et al. [25], the changes in free amino acids (FAAs) and dipeptides in
CSF, plasma and urine of probable AD patients were investigated. It is believed that FAAs can play a role in the
early detection of AD, as they are involved in neurotransmission and neurotoxicity mechanisms. Concentrations of
FAAs and dipeptides were measured with LC- and ESI–MS/MS in eight patients with probable AD and in eight
controls. They found a significantly increased level of glycine (p < 0.0054) in the urine of patients with probable
AD (8.5 ± 1.3 nmol/dL) compared with healthy controls (15.6 ± 1.7 nmol/dL). Concentrations of urinary
histidine, 3-methyl-histidine and carnosine were also elevated in AD patients (p < 0.05), while the concentration
of 1-methyl-histidine was decreased (p < 0.05).

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Saliva
Saliva is an aqueous biofluid made up of roughly 99% water and 1% of organic and inorganic compounds. Due to its
noninvasiveness and very straightforward collection and storage, it has many advantages as a sample for biomarker
research and clinical applications when compared with other fluids such as CSF and blood. Saliva collection can
be done at home by the patients themselves, or with the support of caregivers. This is done simply by spitting
in a plastic tube and storing in a refrigerator, if there are no conservative additives available. Although the fluids
presented in saliva are generated in tissues separated by many barriers from the neuronal tissue compartment, they
remain very sensitive to metabolic changes. Therefore, many previous studies have tried to describe the connection
between saliva’s components and AD.
In a study published in 2001 by Giubilei et al. [32], cortisol levels of saliva were measured in the morning and
evening from 18 patients with probable AD and 18 controls. It was found that the cortisol level increased both in
the morning and evening in AD patients (p < 0.05) when compared with the controls. The study also showed that
evening cortisol levels were significantly lower than morning levels for both AD patients and controls (p < 0.0001).
A significant correlation was found between morning salivary cortisol levels in AD patients and both Mini-Mental
State Examination and Global Deterioration Score, with r = -0.539 and 0.623, respectively.
In a more recent study on salivary cortisol levels and AD, published in 2008 by Wahbeh et al. [43], the Cortisol
Awakening Response was investigated in 19 mild AD patients, their 19 caregivers and 15 controls. The Cortisol
Awakening Response is characterized by a rapid increase in the cortisol levels during the first 30 min after awakening,
which may be related to alterations in the hypothalamic-pituitary-adrenal axis caused by a disorder or stress. The
study found that the salivary level of cortisol was higher both in the AD group (p < 0.01) and in their caregivers
(p < 0.03) during the 30 min after awakening, while no significant change was found in the control group. This
increase of caregivers’ cortisol level suggests that this could be a response to the psychological stress to which they
are subjected. Moreover, recent studies with urine samples of AD patients found higher levels of cortisol in these
samples of these subjects [44]. On the other hand, additional studies should be done in order to clarify if the AD
pathology causes an increase of cortisol level in patients with dementia, or whether the cortisol levels contribute to
the dementia progress itself.
Bermejo-Pareja et al. [45] published an article in 2010 comparing the levels of Aβ40 and Aβ42 in the saliva of 70
AD patients, 51 PD patients and 56 healthy controls. The concentrations were measured with an enzyme-linked
immunoassay kit, and showed that the concentration of Aβ42 in saliva significantly increased (p < 0.05) in the
early stages of AD compared with PD patients and controls. They used this result to reinforce that changes in Aβ42
levels are specific to AD, and no other neurodegenerative disorders.
In a study published in 2011 by Shi et al. [35], salivary levels of the forms of tau proteins in AD patients were
investigated using MS. The results of 21 patients with AD and 38 healthy controls showed that the difference in
t-tau and p-tau between the two groups was not statistically significant, but the ratio between the phosphorylated
form of the proteins and its total amount in saliva was higher in AD patients (p < 0.05).
Liang et al. [46] published the results of their study on saliva metabolomics and AD in 2015. Their approach was
based on fast ultra-HPLC coupled with TOF-MS, and compared the results from 256 patients with AD to 218
healthy controls. Among more than 3000 peaks found in the saliva of the subjects, six metabolites were found to
show a significant difference between AD patients and the control group. Three of them showed an increased level
in AD patients: sphinganine-1-phosphate (p = 0.0001), ornithine (p = 0.003) and phenyllactic acid (p = 0.0045),
and three of them showed lower levels in AD patients: inosine (p < 0.0001), 3-dehydrocarnitine (p = 0.003) and
hypoxanthine (p = 0.0064). The researchers reinforced the idea of using fast ultra-HPLC coupled with TOF-MS
as a high-throughput manner of identifying saliva markers of the disease.

Hair
Hair consists of a protein called keratin surrounded by an amorphous matrix. This structure has certain diagnostic
advantages when compared with other body tissues and fluids like blood, saliva and urine. Concentration of trace
elements in these latter materials vary greatly according to the environmental exposure of the subject, and in some
cases, these variations can preclude their use as a diagnostic tool. On the other hand, hair is an inert tissue and trace
elements are fixed into its structure. Their concentrations do not fluctuate over a short time scale, and thus they
can be used on a long-term diagnostic scale. Additionally, hair can be easily collected and stored, able to be widely
and cheaply used in clinical diagnostic centers around the world [47].

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 A review of biomarkers of Alzheimer’s disease in noninvasive samples Review

Hair has been investigated as a diagnostic tool for many diseases, with most studies relying on trace element
concentrations. Trace elements are those with very low concentrations within organisms, and even small variations
in these concentrations could be harmful or a sign of a disease.
The relationship between trace elements and neurological diseases has been studied since the 1970s. Burnet [48]
alleged that dementia could be a cascade effect resulting from the loss of zinc in the metalloenzymes in charge of
DNA replication, repair and transcription. After many studies comparing both serum, plasma and CSF levels of
trace elements and AD, Shore et al. [49] published the first work relating trace elements in hair and Alzheimer’s-type
dementia in 1984. They did not find any differences between serum and hair concentration of copper, zinc, calcium
and magnesium, alleging that it did not appear to be of use in Alzheimer’s diagnosis. On the other hand, they
found positive correlations between manganese and age in both AD patients and controls.
Four years later in 1988, Vance et al. [50] compared the results of hair from 63 patients with AD and 117
controls, finding that the levels of cobalt and calcium significantly decreased in AD patients (p < 0.01 and < 0.05,
respectively), while levels of bromine and zinc increased (p < 0.05). No significant difference was found in the
concentrations of sodium, iron, selenium, silver, arsenic, gold, chromium, antimony and scandium.
Koç et al. [31], in a study with 45 patients with AD and 33 controls, found that levels of copper, iron and
manganese were significantly lower in AD patients (p = 0.007 for Cu, p = 0.009 for Fe and p = 0.016 for Mn)
than in controls. Levels of boron in hair were significantly higher (p = 0.0001) in AD patients, while no significant
difference was found in the levels of zinc, aluminum and magnesium in hair.
In a recent study conducted by Koseoglu et al. [27], using 62 AD subjects and 60 healthy controls, hair levels of
sodium and potassium were significantly higher (p < 0.001 and < 0.01, respectively) in AD patients, while levels
of aluminum, lead, cobalt, iron, manganese, mercury, copper and cadmium were significantly lower (p < 0.01 for
Al, Pb and Co, p = 0.001 for Fe and Mn and p < 0.01 for Hg, Cu and Cd).

Nail
Nails are made by keratin and contain traces of many minerals and some metabolites. They grow at an average rate
of 3 mm (0.12 in) a month. As nails are very suitable for storing trace elements over a long period, they are of great
importance in the study of diseases that involve those elements. Zinc is the most abundant trace element in the
brain, and has a vital function in enzymatic processes involving the degradation of Aβ peptide, and the processing
of the amyloid precursor protein.
In the same study including hair samples, Koseoglu et al. [27] investigated the concentration of seven trace
elements in the nails of 62 AD patients and compared them with 60 healthy controls. Among them, only sodium
showed an increased concentration in AD patients (p < 0.01). The other five trace elements showed lower levels
in AD patients: manganese, iron, copper, cadmium, mercury (p < 0.001) and zinc (p < 0.01).

Performance of the biomarkers

All biomarkers reviewed in the previous sections are listed in Table 1. A total of 74 average values, standard
deviations and associated p-values were found across dozens of surveys. They are ranked by R, which is an objective
parameter to compare them among themselves. Resolution is defined as R = |μcontrol - μAD |/[2(σcontrol + σAD )],
where μ is the average value and σ is the standard deviation of a biomarker from the frequency histograms (or
probability density distributions) observed for the control and AD groups. R is easily calculated and has a direct
relationship with the minimum rate of failure of an exam, based on each biomarker. Figure 1 shows the size of
each group (n), the averages (μ) and standard deviation (σ) of a survey taken from [36]. The normalized density
probability distributions in the hypothesis of a normal distribution (or Gaussian distribution). In this case, the
R between the concentration of isoprostane 8,12-iso-iPF2a-VI (ng/mg creatinine) in urine of the control and
MCI groups is 2.63, between the control and AD groups is 5.17 and between the MCI and AD groups is exactly
1.00. In the population of this survey, the rate of failure of an exam to distinguish the control from MCI or AD
groups is negligible. However, there is a partial overlap between the MCI and AD groups (orange and red curves in
Figure 1), and an intercept with a significant value in the ordinate (probability density) can be observed. If 4.2 ng
of isoprostrane 8,12-iso-iPF2a-IV per mg creatinine is used as the threshold, then a fraction of 2.7% of the MCI
carriers would receive a false positive for AD (area under the orange distribution lying at right of the threshold).
Conversely, 1.8% of the AD carriers would receive a false result of MDI patients (area under the red distribution
lying at left of the threshold).

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4.5

4.0
Control
n = 40, μ = 1.5, σ = 0.1
3.5

3.0
Probability density

2.5
AD
n = 50, μ = 4.6, σ = 0.2
2.0

MCI
1.5 n = 33, μ = 3.6, σ = 0.3

Threshold
1.0

0.5

0.0
0 1 2 3 4 4.17 5 6
Isoprostrane 8,12-iso-iPF2a-VI
(ng/mg creatinine)

Figure 1. Normalized probability density distributions of isoprostane 8,12-iso-iPF2a-VI (ng per mg of creatinine) in
urine of a control, mild cognitive impairment and Alzheimer’s disease groups as an example. The sample sizes (n),
average values (μ) and standard deviations (σ) are shown. The resolution between the control and MCI groups is 2.63,
between the control and AD groups is 5.17 and between the MCI and AD groups is exactly 1.00. This corresponds to
the entry 1 in Table 1.
AD: Alzheimer’s disease; MCI: Mild cognitive impairment.

Therefore, the percentage of the false positives and false negatives shown in Table 1 was calculated as illustrated
in Figure 1: the intercept of the probability density distributions of the control and AD groups was taken as the
threshold point. This was used to express the result as either positive or negative for AD. The area under the tail of
the control curve that penetrates the AD group and limited by the threshold line gives the fraction of false positives,
while the area under the tail of the AD curve that penetrates the control group and limited by the same threshold
line gives the fraction of false negatives.
All the surveys found with a published p-value smaller than 0.05 are shown in Table 1. The samples used in the
surveys are hair (h), nail (n), saliva (s) and urine (u). The data are assumed to follow a normal density probability
distribution and the biomarkers are ranked by the resolution between the two distributions of control and AD
groups. The minimum expected percentage of false positives and false negatives was calculated for all biomarkers
exhibiting a resolution higher than 0.25. This estimation is based on the normal density probability distributions
obtained from averages (μ) and standard deviations (σ) documented in the reviewed articles. The results would
be more in accordance with real data if the histograms of frequencies would have been accessible. It is common
to observe differences between the results if the real data are taken (histogram of frequencies) instead the normal
probability distributions calculated from (μ and σ).
Based on the results shown in Table 1, increased levels of 8,12-iso-iPF2a-VI15, total FAAs, 8-OHdG and glycine
are connected to AD. The first two should give a confident result of the diagnosis for the individuals of this survey.
However, due to the small sample sizes (40 and 8, respectively), much more work must be done to produce more
precise frequency histograms or density probability distributions. Therefore, the combined analyses of two or more
of these biomarkers would improve the accuracy of the clinical exams. Moreover, the chemical nature of many of
the biomarkers shown on the top of Table 1 are oxidized nucleosides and polyunsaturated fatty acids, suggesting

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 A review of biomarkers of Alzheimer’s disease in noninvasive samples Review

that AD is characterized by an oxidative imbalance in neuronal tissue. This information, and information gained
from this type of survey, in general, can contribute to studies devoted to understanding the underlining biochemical
processes of AD, to help determine if there are independent processes at work or if they are all a consequence of
the neurodegenerative process per se.

Review criteria
Searches were conducted in Scopus and PubMed databases for articles written in English with documented surveys
on biomarkers for AD in noninvasive samples. The following keywords were used: biomarker, noninvasive sample,
Alzheimer, urine, hair, nail, nasal fluids and saliva. Only the surveys providing at least a calculated p-value, mean
value and standard deviation were considered in the present study.

Conclusion
The biomarker candidates to diagnose AD in noninvasive samples were reviewed until July 2017 and include
samples of urine, saliva, hair and nails. No references with a calculated p-value were found for chemical biomarkers
in other noninvasive samples (nasal fluids, tears, breath, sweat and others). In most of the publications only the mean
values and standard deviations of the concentration of the biomarkers were given, which is unfortunate. In addition
to these data, at least the histograms of frequencies are expected, as much more information can be extracted from
them. Moreover, the majority of the surveys conducted up to date were based on a small number of AD carriers and
controls. Worse still, in the great majority of the surveys only one single sample was collected from each individual.
The levels of a biomarker may change over time after the onset of AD, namely during the early, throughout the
middle and up to the advanced stages. Finally, the progression of some diseases has an intermittent nature, and only
multiple samples taken along the time would clarify this issue. One important bottleneck to accomplish this is the
chemical analysis step. The number of analytical instruments with the ability to process a large number of samples
within a short time interval is very limited, especially for the isoprostanes class of compounds, deoxyribonucleoside
derivatives and polypeptides.
The results found in the literature were tabulated and ranked according to the ‘resolution’ between the probability
distributions of the control and AD carrier groups. This was calculated from published mean values and standard
deviations, with the assumption that the data followed a normal distribution. According to the resulting panoramic
view, urine performed better as a noninvasive sample, followed by hair, nails and saliva. Five biomarkers were found
with resolution higher than unity, all of them in urine: 8,12-iso-iPF2a-VI (R = 5.17, 40 control and 50 AD),
total free amino acids (R = 4.70, 8 control and 8 AD), 8-OHdG in nmol/L(R = 1.36, 20 control and 21 AD),
glycine (R = 1.18, 8 control and 8 AD), NaCl-stimulated PON1 (R = 1.04, 20 control and 21 AD) and 8-OHdG
in nmol/mol creatinine (R = 1.02, 20 control and 21 AD). All showed increased levels in AD carriers, with the
exception of the enzymatic activity of NaCl-stimulated PON1, which was found to show decreased levels in AD.
From the data available, it was calculated and shown that false positives or false negatives for AD are negligible ‘in
the group studied’ if the biomarkers 8,12-iso-iPF2a-VI and total free amino acids are used in clinical tests. The
percentage of failure (percentage of false positives plus false negatives) is also small for 8-OHdG when expressed
in nmol/L (<1%) or μmol/mol creatinine (<2.5%). However, much more and much larger surveys must be
conducted to exclude all other possible clinical conditions that may interfere with the results. Moreover, there are
some conflicting results within the literature. All of these show the interesting potential to find reliable biomarkers in
noninvasive samples for AD and MCI. However, many more studies must be conducted, with the data documented
in a proper manner (as frequency histograms), to allow the extraction of much more accurate information that
may eventually lead to a panel of compounds for producing high-confidence AD diagnoses, especially in the early
stages.

Future perspective
Noninvasive samples such as urine, saliva, nasal fluids, hair and nail clippings are readily available, and often
disposed of on a daily basis. The volunteer collection, transportation and storage of such samples are much easier
if compared with those required for blood or CSF. Moreover, current developments of diagnostic devices based on
small sensors and ion selective electrodes, to be used at the point-of-care or at point of discharge of noninvasive
samples, have the potential to disseminate the diagnostic tools to a large fraction of the population in the near
future.

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Review Hartmann & Ledur Kist

Remotely connected sensors installed in public toilets and sanitary sewer systems could provide valuable infor-
mation for public health agencies and policy makers. Sensitive and long-lasting sensors together with wearable
electronics incorporated into advanced textiles have an unimaginable potential. The life standards will experience
another significant forward push if such resources become accessible to all.

Executive summary
The studies of biomarkers in noninvasive samples
• Only the references with a calculated p-value smaller than 0.05 (p < 0.05) were considered. The condition p <
0.05 is an indication that the results found are unlikely to be produced just by chance.
• In ‘all’ articles of the literature (with a calculated p-value), the dispersions are presented merely as mean values
and standard deviations (with only two exceptions, were scatter plots are used – however, the data are useless
because there are overlapped peaks in the form in which the data are presented). This is unfortunate because a
lot of information is lost when the dataset is reduced to averages and standard deviations.
• The recommendation is to present the data as scattering plots or histogram of frequencies, as much more
information can be extracted out from this format.
The noninvasive samples
• Studies of Alzheimer’s disease (AD)’s chemical biomarkers with p < 0.05 were found for the following
noninvasive samples: urine, saliva, hair and nails. No such references were found for chemical biomarkers in nasal
fluids, tears, breath, sweat or any other noninvasive sample.
The ranking
• The resolution between two distributions for a given biomarker is defined as the ratio of the mean value
differences divided by two-times the sum of the standard deviations. This number shows how far apart the two
distributions are from each other (AD carriers from control group) and how good this biomarker will be to
accurately diagnose AD carriers in the respective total group (AD carries plus control individuals).
• A table with 74 biomarker candidates was produced and ranked by resolution.
The most promising biomarkers
• Five biomarkers found in urine until the present date exhibited some potential: isoprostane 8,12-iso-iPF2α-VI,
total free amino acids, 8-hydroxy-2 -deoxyguanosine (nmol/l), glycine, NaCl-stimulated PON1 and
8-hydroxy-2 -deoxyguanosine (μmol/mol creatinine).
The weakness of the studies so far
• Most of the biomarkers listed are from single studies.
• The majority of the studies are made using small sample sizes: only approximately 20 to approximately 60 of AD
carriers and similar numbers for the control group. Much larger surveys must be conducted in order to increase
the confidence of these biomarkers, as many other clinical conditions may interfere in the levels of such
biomarkers.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or finan-
cial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria,
stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.

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10.2217/bmm-2017-0388 Biomark. Med. (Epub ahead of print) future science group