nutrients
Article
Hydration Status of Geriatric Patients Is Associated with
Changes in Plasma Proteome, Especially in Proteins Involved
in Coagulation
Laura Hoen 1 , Daniel Pfeffer 1,2 , Johannes R. Schmidt 3 , Johannes Kraft 2 , Janosch Hildebrand 1
Stefan Kalkhof 1,3, *
Citation: Hoen, L.; Pfeffer, D.;
Schmidt, J.R.; Kraft, J.; Hildebrand, J.;
Kalkhof, S. Hydration Status of
Geriatric Patients Is Associated with
Changes in Plasma Proteome,
Especially in Proteins Involved in
Coagulation. Nutrients 2023, 15, 3789.
https://doi.org/10.3390/
nu15173789
Academic Editor: Motoyuki Iemitsu
Received: 24 July 2023
Revised: 2 August 2023
Accepted: 4 August 2023
Published: 30 August 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Nutrients 2023, 15, 3789. https://doi.org/10.3390/nu15173789
and
1 Institute for Bioanalysis, Coburg University of Applied Sciences and Arts, Friedrich-Streib-Str. 2,
96450 Coburg, Germany
2 Division of Geriatrics, Klinikum Coburg GmbH, 96450 Coburg, Germany
3 Proteomics Unit, Department of Preclinical Development and Validation, Fraunhofer Institute for Cell
Therapy and Immunology—IZI, Perlickstr. 1, 04103 Leipzig, Germany
* Correspondence: stefan.kalkhof@izi.fraunhofer.de
Abstract: Due to multifactorial reasons, such as decreased thirst and decreased total body water,
elderly patients are vulnerable to dehydration. The study aims to investigate whether moderate dehy-
dration or hyperhydration affects the blood proteome. Blood samples, medication, and bioelectrical
impedance analysis (BIA) details were collected from 131 geriatric patients (77 women and 54 men
aged 81.1 ± 7.2 years). Based on an evaluation by Bioelectrical Impedance Vector Analyses (BIVAs)
of this cohort, for each hydration status (dehydrated, hyperhydrated, and control), five appropriate
blood plasma samples for both males and females were analyzed by liquid chromatography–mass
spectrometry (LC-MS). Overall, 262 proteins for female patients and 293 proteins for male patients
could be quantified. A total of 38 proteins had significantly different abundance, showing that
hydration status does indeed affect the plasma proteome. Protein enrichment analysis of the affected
proteins revealed “Wound Healing” and “Keratinization” as the two main biological processes being
dysregulated. Proteins involved in clot formation are especially affected by hydration status.
Keywords: proteomics; geriatric nutrition; chronic dehydration; coagulation
1. Introduction
Dehydration refers to a reduction in the total body water, which can be due to a
water deficit (water loss dehydration) or salt and water deficit (salt loss dehydration) [1].
There are two different forms of dehydration considering the blood sodium level: hyper-
natraemic (high blood sodium levels) or hyponatraemic (low blood sodium levels) [2].
Mostly, dehydration is the outcome of disease and/or the effects of medication [1], but the
dehydration risk increases with age [3]. This is because aging is accompanied by various
physical changes, including reduced thirst or decreased total body water and inadequate
water intake [3]. As the demographic change leads to an increasingly aging population
(age > 65) [4], the correct diagnosis of dehydration is becoming more important and, at the
same time, challenging since tools such as capillary refill time and abnormal skin turgor
or respiratory pattern cannot be applied to the elderly because of the aging process [3,5].
In addition to these external examinations, dehydration can be diagnosed with laboratory
testing of the blood; for example, the plasma urea–creatinine ratio is a good indicator of
whether the kidney is healthy [5]. However, older adults often have a raised ratio related
to renal failure, heart failure, or other age-associated diseases [5]. In addition, the sodium
level can be measured, but this is a less sensitive indicator than osmolality. Serum or plasma
osmolality is considered the gold standard for diagnosing dehydration, as it can indicate
dehydration from as low as 1% water loss [5]. Serum osmolality is calculated from lab
https://www.mdpi.com/journal/nutrients
Nutrients 2023, 15, 3789 2 of 13

values for blood urea nitrogen, serum bicarbonate, creatinine, glucose, sodium, calcium,
and potassium [1,5]. Nevertheless, osmolality has its limitations, as it was formerly used
for acute dehydration [6–8]. However, older adults have a more chronic dehydration [9,10],
which is associated with elevated markers of inflammation and coagulation [11].
The research aim of this study is as follows:
I. To find out whether and, if so, to what extent moderate dehydration or hyperhydration
affects the blood proteome;
II. To find which proteins are differentially abundant and could serve as potential
biomarkers;
III. To investigate which functions these dysregulations are related to.

2. Material and Methods

2.1. Study Design
With the focus on an investigation of hydration-associated diseases and changes,
a geriatric study (ethical approval of the ethics committee of the University of Applied
Sciences Coburg to the project “Storage and analysis of blood samples from dehydrated
and adequately hydrated patients” from 3 December 2019, Code: HC_Kalkhof_03122019)
was conducted in 2020. This included 131 female patients and male patients (all non-
smokers aged 65 to 98 years). The study center was the Department of Geriatrics of
Prof. Dr. Kraft (Regiomed Klinikum Coburg, Coburg, Germany). Of these patients, body
composition, including hydration status, was recorded with BIA. In addition, data on
medication, diagnoses, and blood values were recorded. In addition, blood samples were
taken for subsequent proteomic analyses.
The patients were not conditioned in the context of diets, sports, etc., and none of
them were initially hospitalized because of their hydration status. The examinations and
sampling took place within the framework of regular ambulatory examinations. If patients
had several examinations in 2020, several samples were also included, which ultimately
led to a total number of 272 collected samples (Table S1). A total of 71 of the 131 patients
were measured at least twice.

2.2. Bioelectrical Impedance Vector Analyses (BIVAs)

Bioelectrical Impedance Vector Analyses and classification of patients according to
their hydration status were performed according to Hoen et al. [12].
The InBody S10 (InBody 770, Cerritos, CA, USA), a multifrequency (1, 5, 50, 250, 500,
and 1000 kHz) four-point bioelectrical impedance device, was used for all BIA approaches
as several patients were not able to walk. Analyses were performed a maximum of 1 h after
blood sampling. BIVA values were calculated as described by Piccoli et al. [13]. The whole-
body resistance was calculated as previously described with the values for the right arm,
right leg, and trunk at 50 kHz measured with an electric current intensity of 80 µA [12,14].
The formulas provided by Piccoli were used for the BIVA [15]. As the reference population,
the Coburg cohort containing 130 male and 115 female patients with a BMI between 25 and
28 kg/m2 and an age between 65 and 98 was applied as previously described [12].

2.3. Selection and Characteristics of Analysed Samples

From the total 272 blood plasma samples, 5 samples of each hydration status (hy-
perhydrated, dehydrated, and control) in both genders were selected for further analysis.
Only the first sample of each patient was considered because prolonged hospitalization
would be expected to normalize hydration status. Alongside the hydration status, the
medical condition was crucial. Exclusion criteria were tumor diagnosis, missing limbs,
and incomplete patient information (for example, missing medication). The controls were
matched as well as possible by age to the other hydration states.
The average age and number of drugs prescribed are shown in Table 1. The main
diagnosis of all the patients analyzed included mobility impairment (e.g., due to fractures);
only one of the hyperhydrated men was admitted for cerebral infarction.
Nutrients 2023, 15, 3789 3 of 13

Table 1. Average age and number of drugs prescribed in the different groups with standard devia-
tion (SD).

Group Age ± SD Drugs ± SD

Normal hydrated men (control) 89.0 ± 3.5 8.4 ± 4.6
Hyperhydrated men 78.2 ± 5.8 8.8 ± 5.6
Dehydrated men 80.6 ± 4.7 13.4 ± 2.0
Normal hydrated female (control) 84.0 ± 3.6 10.4 ± 4.4
Hyperhydrated female 80.6 ± 5.1 5.8 ± 7.4
Dehydrated female 86.2 ± 5.9 10.2 ± 3.3

2.4. Preparation of Blood Samples for Proteomic Analyses

Samples were collected during standard blood collection in the Department of Geri-
atrics. They were kept cold (4 ◦ C) until they were centrifuged at 2500× g for 30 min at 4 ◦ C
on the same day within 3 h after collection. Afterward, the supernatant (blood plasma) was
transferred to 1.5 mL Tubes (Greiner, Kremsmünster, Austria) and frozen at −80 ◦ C.
The top 14 abundant proteins of each sample were depleted according to the man-
ufacturer’s protocol with the High SelectTM Top14 Abundant Protein Depletion Mini
Spin Columns (Thermo Fisher Scientific, Waltham, MA, USA). Total protein quantitation
was determined using the Pierce 660 nm Protein Assay (Thermo Scientific Fisher, Bre-
men, Germany). After depletion, a modified filter-aided sample preparation (FASP) was
conducted [16]. An ultrafiltration using a Vivacon 500 filter unit (30 kDa MWCO) was
performed. A corresponding volume of 30 µg of depleted plasma proteins of each sample
was added to the filter, and the buffer was changed to 8 M urea in 0.5 M TCEP ([16] methods
3.2, steps 1–3). This was followed by a thiol-alkylation with 50 mM iodoacetamide (IAA) in
the adjusted buffer ([16] methods 3.2, step 4), which was then incubated for 30 min without
mixing in the dark. To remove the excess IAA, the filter was rinsed twice with 8 M urea
in 0.5 M TCEP with pH 8.0. Then, the filter units were transferred to a new collection
tube, and 10 µL of urea buffer and 80 µL of trypsin (with an enzyme-to-protein ratio of
1:50) in 50 mM TEAB buffer was added. The tubes were sealed with parafilm to prevent
evaporation and incubated overnight at 37 ◦ C, mixing at 600 rpm. After centrifugation, the
collected peptide samples were stored at −80 ◦ C.
Subsequently, the samples were labeled with the TMT11plex Label Reagents (Thermo
Scientific Fisher, Germany) in accordance with the manufacturer’s protocol (section C
peptide labeling) with the following changes: 27 µL of the appropriate 10-plex solution was
mixed to the samples, and 5 µL of the quenching solution was used.
A Common Reference was generated from aliquots of all samples (except for 2, as
too little material remained from these, which concerns one dehydrated female and one
hyperhydrated male). Therefore, 10 µL were taken from each sample and pooled. A total
of 200 µL of this reference was labeled with 80 µL of TMT10 -131 and quenched with 6 µL
quenching solution.
After labeling, the samples were analyzed by liquid chromatography–mass spectrome-
try (LC-MS) as previously described [17]. Briefly, 1 µg of labeled peptides were injected into
an Easy-nLC 1200 coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific,
Germany). The peptides were separated on a 20 cm analytical HPLC column packed
in-house using ReproSil-Pur C18-AQ 1.9-µm silica beads (Dr. Maisch GmbH, Ammerbuch,
Germany). Separation was achieved by applying a 120 min multistep gradient from 10%
solvent A to 90% solvent B at a constant flow rate of 200 nL/min. As mobile phases, 0.1%
formic acid in water (solvent A) and 80% ACN, supplemented with 0.1% formic acid in wa-
ter (solvent B), were applied. The eluting peptides were ionized by electrospray ionization
at 3.2 kV. Data were acquired in data-dependent mode and controlled by XCalibur software
(version 2.9). The survey scans were acquired in a scan range of 300–1650 m/z, a mass
resolution of 60,000, an AGC target of 3 × 106 , and a maximum injection time of 25 ms. The
Nutrients 2023, 15, 3789 4 of 13

top 10 most abundant precursor ions were selected for isolation (window of 0.7 amu) and
fragmentation by higher-energy collisional dissociation (normalized collision energy: 34).
For MS2 scans in orbitrap mass analyzer, AGC target and maximum injection time were
set to 1 × 105 and 110 ms, respectively. A dynamic exclusion for MS2 scans was set to 30 s.
The proteomics raw data were analyzed using MaxQuant software (version 1.6.2.3), and
the integrated protein identification algorithm Andromeda was utilized. The experimental
spectra were matched against reference proteome of H. sapiens. Trypsin was defined as a
position-specific protease in fixed mode with a tolerance of up to two missed cleavages.
Carbamidomethylation (cysteine) was set as fixed modification, whereas oxidation (me-
thionine) and acetylation (protein N-terminus) were set as optional modifications. A mass
tolerance of 20 ppm (first search and fragment ions) and 4.5 ppm (main search) was allowed.
Two peptides, including at least one unique peptide, were required for identification, con-
trolling the false discovery rate (FDR) to 0.05 for peptide spectrum matches, and protein
identification. The identified proteins were filtered to exclude potential contaminants and
decoy entries.

2.5. Data Analysis

The proteomics MS data were deposited in the ProteomeXchange Consortium via the
PRIDE [18] partner repository with the dataset identifier PXD043728.
Samples with less than 200 protein quantity values greater than zero (valid values)
and significant sample outliers according to the Grubbs test were not included in further
analysis. Each criterion excluded one of the hyperhydrated samples (both female and male).
A minimum of three remaining samples in each group (hyperhydrated, dehydrated, and
control) was ensured.
A common reference scaling was performed first, followed by a median adjustment
using R (version 3.5.1). The means of each group (hyperhydrated vs. control, dehydrated
vs. control, and dehydrated vs. hyperhydrated) were then tested for significance (p < 0.05)
using a two-tailed t-test.
The significant differentially abundant proteins (DAPs) were then further analyzed
using STRING to identify affected biological processes. Enrichment analysis was used to
identify the biological processes involving most of the proteins in the apparent clusters.

3. Results
3.1. Protein Abundance Is Linked with Hydration Status
A total of 272 blood samples were collected from 131 patients. Further, bioelectrical
impedance analyses were performed for every patient within one hour after blood collection.
The average age of the patients was 81.1 ± 7.2 years. A total of 54 patients were male (41.2%),
whereas 77 were female (58.8%). Based on BIVAs considering our previously published
reference cohort [12], the samples were assigned to a hydration status (hyperhydrated
patients in the lower left and dehydrated patients in the upper right—Figure 1). The groups
range from highly dehydrated (14 samples, 8♀and 6♂), moderately dehydrated (22 samples,
12♀and 10♂), to slightly dehydrated (9 samples, 4♀and 5♂). A total of 77 samples (45♀and
32♂) are normally hydrated. A total of 33 samples were highly hyperhydrated (18♀and
15♂), 22 samples (15♀and 7♂) were moderately hyperhydrated, and 16 samples (9♀and 7♂)
were slightly hyperhydrated. Because hydration status changes during hospitalization, only
the first examination of each patient was considered for the proteomic approach because it
is expected that hydration status should return to normal during hospitalization. Due to
this exclusion criteria, only five out of seven male dehydrated patients were suitable for
further investigations. Therefore, each group (dehydrated, hyperhydrated, and control) in
each gender was delimited to five samples to keep the groups the same size for the statistics.
Nine female patients were dehydrated on their first examination. None of these patients
were initially hospitalized because of dehydration, which underlines the aspect of chronic
dehydration in patients. A total of 9 male and 12 female patients were hyperhydrated, and
26 female and 17 male patients were normally hydrated on the first day of examination.

Nutrients 2023, 15, 3789
out of seven male dehydrated patients were suitable for further investigations. Therefore,
each group (dehydrated, hyperhydrated, and control) in each gender was delimited to
five samples to keep the groups the same size for the statistics. Nine female patients were
dehydrated on their first examination. None of these patients were initially hospitalized
because of dehydration, which underlines the aspect of chronic dehydration in patients.
5 of 13
A total of 9 male and 12 female patients were hyperhydrated, and 26 female and 17 male
patients were normally hydrated on the first day of examination.

Figure 1.
Figure 1. Distribution
Distributionofofthe
thepatients
patientsononeach
each day
day of of
thethe sample
sample collection
collection in Bioelectrical
in Bioelectrical Imped-
Impedance
ance Vector Analyses (male (A), female (B)) plotted against the tolerance ellipses of Coburg’s
Vector Analyses (male (A), female (B)) plotted against the tolerance ellipses of Coburg’s cohort [12]; co-
hort [12]; Proteomic samples are highlighted in red, where the patients in the inner circle are the
Proteomic samples are highlighted in red, where the patients in the inner circle are the controls, the
controls, the upper right ones are the dehydrated, and the lower left ones are the hyperhydrated
upper right ones are the dehydrated, and the lower left ones are the hyperhydrated patient samples.
patient samples.
After common reference scaling and median adjustment, 262 proteins in the female
After common reference scaling and median adjustment, 262 proteins in the female
patients and 293 proteins in the male patients could be quantified, of which 20 are sig-
nificantlyand
patients 293 proteins
differentially in the male
abundant (p < patients
0.05) in could
femalebepatients,
quantified,
and of
23which 20 are sig-
are significantly
nificantly differentially
differentially abundant (p abundant
< 0.05) in(pmale
< 0.05) in female
patients. Fivepatients,
of these and 23 are significantly
differentially abundant
differentially abundant (p < 0.05) in male patients. Five of these differentially
proteins (DAPs) are significant in both genders. Overall, 38 proteins were significantly abundant
proteins (DAPs) are significant in both genders. Overall, 38 proteins were
altered in their abundance according to their p-value (Table 2). For each of these DAPs, thesignificantly
altered
effect in their
size, abundance
confidence according
intervals, to theirdeviation
and standard p-value (Table 2). For each
were calculated of these
(Table DAPs,
S2). As the
the effect size, confidence intervals, and standard deviation were calculated
significance of each DAP between the different hydration statuses (dehydrated vs. control, (Table S2).
As the significance
hyperhydrated of each and
vs. control, DAPdehydrated
between thevs. different hydrationwas
hyperhydrated) statuses
tested,(dehydrated
the amountvs. of
control, hyperhydrated
significant DAP differs invs. control,
these groupsand dehydrated
(Table 2). Somevs. hyperhydrated)
DAPs are significantwasfor tested, the
more than
amount
one of theoftested
significant DAP differs in these groups (Table 2). Some DAPs are significant
contrasts.
for more than one of the tested contrasts.
Table 2. Mean normalized protein intensity of significant proteins in female (=F) and male (=M) for
dehydrated (=D), hyperhydrated (=H), and control (=C) patients. Differential abundant proteins
(DAP) were identified by p-values < 0.05 of t-tests between dehydrated vs. control, hyperhydrated vs.
control, and dehydrated vs. hyperhydrated; p < 0.05 is highlighted in grey and black highlights are
proteins found significant in both genders.

Female Male
Mean p-Value Mean p-Value
Gene Gene DM HM DM
Name DF vs. HF vs. DF vs. Name
DF HF CF DM HM CM vs. vs. vs.
CF CF HF
CM CM HM
F2 20.27 20.24 20.92 0.010 0.201 0.947 F2 20.94 21.97 21.50 0.171 0.153 0.026
SERPINC1 21.04 21.33 21.89 0.028 0.111 0.168 SERPINC1 21.58 22.54 21.74 0.717 0.069 0.046
PZP 18.18 19.59 18.33 0.818 0.093 0.039 PZP 20.30 18.55 19.71 0.231 0.019 0.006
GC 20.73 20.91 21.40 0.003 0.080 0.412 GC 21.15 22.18 21.38 0.605 0.116 0.014
HSPA5 15.71 15.52 16.21 0.047 0.019 0.019 HSPA5 16.49 17.04 16.53 0.869 0.092 0.042
QSOX1 15.40 14.52 15.80 0.407 0.028 0.018 SERPINA1 22.43 21.27 22.14 0.514 0.024 0.026
Nutrients 2023, 15, 3789 6 of 13

Table 2. Cont.

Female Male
Mean p-Value Mean p-Value
Gene Gene DM HM DM
Name DF vs. HF vs. DF vs. Name
DF HF CF DM HM CM vs. vs. vs.
CF CF HF
CM CM HM
C3 23.28 23.91 23.83 0.015 0.811 0.134 KRT14 19.57 19.79 19.16 0.069 0.047 0.399
C4b 16.16 15.38 16.23 0.732 0.012 0.017 APOA1 22.71 22.42 23.25 0.049 0.435 0.769
KNG1 19.08 19.47 19.93 0.032 0.239 0.212 APOE 21.58 21.27 21.98 0.281 0.009 0.378
IGHG2 20.35 20.22 17.69 0.049 0.146 0.920 APOA2 20.34 21.08 20.59 0.428 0.148 0.048
VTN 19.60 19.92 20.14 0.001 0.411 0.287 FGG 22.31 21.68 22.17 0.641 0.139 0.011
CLU 20.03 20.11 20.81 0.005 0.027 0.727 ORM1 19.89 18.43 19.23 0.249 0.053 0.025
F5 19.22 19.34 20.30 0.038 0.159 0.825 SERPINA7 18.61 19.49 18.92 0.416 0.137 0.029
F9 17.32 16.83 18.38 0.047 0.245 0.673 KRT5 19.81 19.92 19.33 0.045 0.002 0.561
LCN1 16.62 16.28 16.58 0.855 0.273 0.040 LCP1 15.15 16.32 15.90 0.038 0.701 0.335
CDH5 17.10 16.66 17.55 0.207 0.036 0.144 JUP 16.85 16.64 16.23 0.022 0.061 0.247
SELENOP 17.93 17.89 18.60 0.007 0.015 0.861 AZGP1 19.57 20.38 19.72 0.731 0.187 0.046
Krt86 16.68 16.49 16.63 0.829 0.483 0.023 DAG1 14.61 14.19 14.58 0.909 0.174 0.043
MMRN2 15.81 15.42 16.39 0.104 0.019 0.056 SPARCL1 14.32 15.27 14.81 0.152 0.248 0.035
DSP 17.30 17.09 17.23 0.816 0.670 0.016 dnaK 15.83 16.80 16.25 0.385 0.236 0.019
HRNR 18.01 18.24 17.61 0.054 0.047 0.353
KIAA2013 15.69 15.81 15.35 0.139 0.044 0.419
FBXO30 16.57 14.87 15.55 0.011 0.657 0.365

3.2. DAPs Are Involved in Wound Repair and Blood Coagulation Are Associated with Hydration
Protein–protein interaction analyses of DAPs using STRING [19] resulted in a highly
significant clustering (PPI enrichment value of p < 1.0 × 10−16 ) consisting of 34 nodes and
126 edges (Figure 2). Overall, 63 biological processes from Gene Ontology with a false
discovery rate (FDR) < 10−3 , at least five involved genes, and p < 0.005 were covered by the
network of DAPs. In total, 16 main processes could be identified (subtermini, processes
included by these main processes, were excluded) (Table 3). Two subtermini of these
biological processes (response to wounding and keratinization) cover most of the proteins
in the two clusters, which were built according to the STRING analysis (Figure 2). Beyond
the wound healing process and keratinization, blood coagulation, negative regulation of
blood coagulation, fibrin clot formation, regulation of body fluid levels, or blood coag-
ulation intrinsic pathway, which would cover some more of the DAPs, could be found
significant (p < 0.005) in the network (not shown because of redundancy in the main pro-
teins involved). Prothrombin and Antithrombin-III were significantly lower in dehydrated
men vs. hyperhydrated men and dehydrated women vs. control. The fibrinogen gamma
chain was significantly higher in dehydrated men vs. hyperhydrated men. In addition,
coagulation factors V and IX were found to be significantly lower in dehydrated women vs.
control (Figure 3). Furthermore, keratins, which are the key structural material for the outer
layer of the skin, were found to be significantly higher in hyperhydrated men: Keratin
Type I cytoskeletal 14 and Keratin Type II cytoskeletal 5. In addition, Hornerin (HRNR, a
component of the epidermal cornified cell envelope) was significantly more abundant in
hyperhydrated men vs. control.
 two dehydrated men, two female controls, three male controls, no hyperhydrated female,
and two hyperhydrated men (one of whom was not considered in the analyses because of
the criteria (2.4)) who were taking anticoagulant drugs. Acetylsalicylic acid (ASA), in a
dose of a maximum of 100 mg per day, can be used as a platelet aggregation inhibitor as
Nutrients 2023, 15, 3789 well [20]. A total of 74 samples were receiving ASA as a platelet aggregation inhibitor, from 7 of 13
which 1 was dehydrated, 11 were hyperhydrated, and 26 were controls.

Figure 2. Protein–protein interaction clustering and functional annotation of all differentially abun-
Figure 2. Protein–protein
dant proteins found interaction
in males andclustering
females ofand
eachfunctional
comparisonannotation of vs.
(dehydrated allcontrol,
differentially
dehydrated vs.
abundant proteins found in males and females of each comparison (dehydrated vs. control, dehy- −8
hyperhydrated, and hyperhydrated vs. control); blue = response to wounding (p = 1.74 × 10 ) and
drated vs. hyperhydrated, and hyperhydrated vs. control); blue = response to wounding (p = 1.74 ×
green = keratinization (p = 4.58 × 10−5 ). Created with STRING [19] version 11.5. Red circles mark
10−8) and green = keratinization (p = 4.58 × 10−5). Created with STRING [19] version 11.5. Red circles
proteins
mark proteins being
being differentially
differentially abundant
abundant inin males
males and
and females.
females.

Table 3. Main biological processes from Gene Ontology with a false discovery rate (FDR) < 10−3 , at
least 5 involved genes, and p < 0.005 found in the network of significant proteins; relevant subtermini
included are marked with an arrow.

# Genes Description FDR Value p-Value

27 Localization 3.10 × 10−6 1.21 × 10−9
27 Multicellular organismal process 7.80 × 10−5 2.04 × 10−7
27 Response to stimulus 0.0011 6.15 × 10−6
10 → Response to Wounding 1.12 × 10−5 1.74 × 10−8
7 → Blood Coagulation 2.80 × 10−4 8.63 × 10−7
22 Regulation of biological quality 1.91 × 10−5 3.49 × 10−8
22 Negative regulation of biological process 0.0012 6.96 × 10−6
21 Anatomical structure development 0.0037 3.32 × 10−5
5 → Keratinization 0.0048 4.58 × 10−5
Nutrients 2023, 15, 3789 8 of 13

Table 3. Cont.

# Genes Description FDR Value p-Value

19 Protein metabolic process 0.0023 1.53 × 10−5
14 Cellular component assembly 0.0027 1.83 × 10−5
14 Regulation of catalytic activity 0.0029 2.08 × 10−5
14
Nutrients 2023, 15, x FOR PEER REVIEW Immune system process 0.0037 8 of 13
3.24 × 10−5
11 Cell activation 3.30 × 10−4 1.26 × 10−6
Positiveprocesses
Table 3. Main biological regulation of multicellular
from Gene Ontology with a false discovery rate (FDR) < 10−3, at
11
least 5 involved genes, and p < 0.005 found
0.0113 1.30 × 10−4
in the network of significant proteins; relevant sub-
organismal process
termini included are marked with an arrow.
10 Regulation of body fluid levels 8.22 × 10−6 1.15 × 10−8
# Genes Description FDR Value p-Value
Positive regulation of cellular component
27 Localization 3.10 × 10 0.0135 × 10
1.21 1.70 × 10−4
−6 −9
9
27 organization
Multicellular organismal process 7.80 × 10−5 2.04 × 10−7
27 8 Response to stimulusCell adhesion 0.0011 0.01266.15 × 10−6 1.50 × 10−4
10  Response to Wounding 1.12 × 10−5 1.74 × 10−8
7
5  Blood Coagulation
Regeneration 2.80 × 10−4
0.00148.63 × 10−7 8.31 × 10−6
22 Regulation of biological quality 1.91 × 10−5 3.49 × 10−8
22 Negative regulation of could
As coagulation biological
be process 0.0012
influenced by anticoagulants, we had 6.96
a ×closer
10−6 look at them.
21 Anatomical structure development 0.0037 3.32 × 10−5
In total, 47 patients were receiving anticoagulant drugs (Apixaban, Edoxaban, Rivaroxaban,
5  Keratinization 0.0048 4.58 × 10−5
19
Heparin,Protein
and Phenprocoumon),
metabolic process
corresponding to 112
0.0023
of 272 samples taken,
1.53 × 10 −5
of which 8
14 were classified as dehydrated,
Cellular component assembly 38 were classified as hyperhydrated,
0.0027 and 21
1.83 × 10 −5 were classified
14 as control. Out of the five proteomic
Regulation of catalytic activity samples chosen, there
0.0029 was one dehydrated
2.08 × 10 −5 female,
14 two dehydrated men, two
Immune system female controls, three male
process controls, no3.24
0.0037 hyperhydrated
× 10−5 female,
11 and two hyperhydrated
Cell activation men (one of whom was not 3.30 considered
× 10−4 in the
1.26 × 10analyses
−6
because
11 Positive regulation
of the of (2.4))
criteria multicellular organismal
who were process
taking anticoagulant 0.0113 1.30 × 10−4 acid (ASA), in
drugs. Acetylsalicylic
10 a doseRegulation of body fluid
of a maximum levels
of 100 mg per day, can be used8.22 ×as10 a platelet
−6 1.15 × 10−8
aggregation inhibitor
9 Positive regulation of cellular component organization 0.0135
as well [20]. A total of 74 samples were receiving ASA as a platelet1.70 × 10−4
aggregation inhibitor,
8 Cell adhesion 0.0126 1.50 × 10−4
from which 1 was dehydrated, 11 were hyperhydrated, and 26 were controls.
5 Regeneration 0.0014 8.31 × 10−6

Figure 3. Simplified Illustration of the coagulation cascade after Bauer et al. [21]. Significant regu-
3. Simplified
Figurelated Illustration of the coagulation cascade after Bauer et al. [21]. Significant regulated
proteins in the dehydration group summarized for men and women are shown with green
proteins in the
arrows dehydration
(=upregulated group summarized
in dehydration for men
state) and red arrows and womeninare
(=downregulated shown with
dehydration state). green arrows
Dashed arrows show the influencing proteins to the activation, and continuous
(=upregulated in dehydration state) and red arrows (=downregulated in dehydration arrows show acti-state). Dashed
vation pathways; inhibition is marked with a dead-end line, and coagulation factors are abbrevi-
arrowsated
showwith the influencing
Roman numerals. proteins to the activation, and continuous arrows show activation
pathways; inhibition is marked with a dead-end line, and coagulation factors are abbreviated with
Roman numerals.
Nutrients 2023, 15, 3789 9 of 13

3.3. Alzheimer Prediction Marker

Pregnancy zone protein (PZP), as a suggested marker in Alzheimer’s disease [22,23],
was also found to be differentially abundant. It was enriched in dehydrated men compared
to hyperhydrated men (p = 0.006) and in hyperhydrated women compared to dehydrated
women (p = 0.039). If the same criteria for dementia are applied as before [12], i.e., receiving
memantine, donepezil, Ginkgo biloba extract, rivastigmine or striking Mini-Mental State
Examination (MMSE) (Score ≤ 23), and/or Clock-Drawing Test (Score ≥ 3), then two of
the five dehydrated men had dementia, as did two of the three hyperhydrated men. No
hyperhydrated or dehydrated women in the samples analyzed had dementia.

4. Discussion
4.1. Plasma Proteome Is Affected by Moderate Dehydration and Hyperhydration
This study of 30 patients revealed 38 differential abundant proteins according to the
hydration status (dehydrated, hyperhydrated, and normally hydrated) based on proteomic
results. Furthermore, to the best of our knowledge, this is the first plasma proteome dataset
in moderately dehydrated and hyperhydrated geriatric patients. The selection strategy
of hydration status by BIVAs presented a representative subset for proteomics, although
geriatric patients are affected by different diseases (average drug prescription of 10.5). This
variance between the groups is also reflected by high standard deviations of the protein
quantities within the groups. Beyond this, it should be considered that previous studies
showed a lack of agreement between different BIA devices [24,25]. Although the reference
population used is measured with a standing BIA (InBody 770) [12] and the patients in this
study are measured with a supine BIA (InBody S10), both analyzers are multifrequency
devices, whereas the reported differences between bioimpedance analyzers are mainly
due to different measurement methods such as multifrequency, single frequency, and
bioimpedance spectroscopy rather than standing or supine. Nevertheless, the different
placement of the electrodes can have an impact, which should be kept in mind and has to
be investigated further. However, even considering the patient and device-related variance,
the proteome analysis of the blood plasma samples identified 38 DAPs, which indicates
that the protein expression is not random and the hydration status has an influence on the
abundance of blood plasma proteins. In turn, the DAPs identified interact in a highly inter-
connected network with significant pathways of hydration status. As several of the DAPs
for dehydration, as well as hyperhydration, are affected in the same direction, e.g., F2 is for
the females lower in both groups than in the control, the DAPs act as markers for general
hydration status changes rather than the consequence of specific hydration direction.

4.2. Wound Repair/Coagulation Is Affected by Hydration Status

As already known, hydration status is an important factor in wound healing [26–28].
This study revealed a coherence between the hydration status and the wound healing
process, especially the coagulation pathway, which is one of the main processes in wound
healing [29]. Two coagulation factors (V, IX), as well as prothrombin, were found to be
downregulated in dehydrated patients, which leads to an upregulation of Fibrinogen as
it cannot be converted into fibrin (Figure 3). The increase in Fibrinogen in dehydrated
humans has also been shown before [11]. Certainly, Antithrombin-III (ATIII) is also less
abundant in dehydrated patients. Nevertheless, antithrombin deficiency can occur due to
other conditions such as congenital antithrombin deficiency, liver damage, sepsis, renal
diseases, consumption coagulopathy, ingestion of ovulation inhibitors, heparin therapy,
and vitamin K deficiency [30]. Likewise, anticoagulation drugs inhibit the measurement
of ATIII [30]. The patients analyzed did not have sepsis, a known vitamin K deficiency, or
a known congenital antithrombin deficiency and did not take ovulation inhibitors. Only
one of the dehydrated female patients has a liver disease, which should not have had a
significant impact. The patient data showed that there was no difference in renal diseases
(according to the criteria by Hoen et al. [12]) in the female patients (three out of five controls
and dehydrated patients, as well as two out of the three analyzed hyperhydrated female
Nutrients 2023, 15, 3789 10 of 13

samples). The renal diseases differ in four out of five dehydrated male patients, but only
one of three hyperhydrated males was affected, and two of five controls had renal disease.
Furthermore, some of the patients analyzed were given oral anticoagulant drugs, which
also included heparin (3.2). As is already known, heparin activates antithrombin [31].
Heparin was taken by one hyperhydrated male, one control male, two control females,
and one dehydrated female patient. Nonetheless, the difference of one patient should
not influence the significance. Overall, more hyperhydrated samples (38 in total) than
dehydrated samples (8 in total) were receiving anticoagulant drugs, which is in line with
our previous study, where Apixaban, one of the anticoagulant drugs, was taken significantly
more often in hyperhydrated patients [12].
In total, four of five dehydrated females, two of three hyperhydrated females, and three
of five control females had at least one state that could influence ATIII levels, whereas four
of five dehydrated men, two of three hyperhydrated men, and three of five control men had
the same. In conclusion, the interaction of different influences of ATIII may play a role in the
measurement of ATIII. Moreover, an increased consumption of ATIII could appear, which
also leads to ATIII deficiency [32]. In addition, all of the patients analyzed were operated
on prior to the blood sampling. As Büller et al. [33] showed, surgery induces a decrease
in ATIII plasma levels to the lowest point at around the third postoperative day; this can
also affect the measurements. Although all groups should be affected by this, dehydrated
patients have lower levels than the other groups. In addition, Ruttmann et al. [34] reported
that hemodilution results in decreased activity of ATIII in vitro. Taken together, multiple
factors may interfere with the antithrombin abundance in patient’s plasma, especially in
elderly cohorts suffering from comorbidities and increased drug administration. However,
different studies have shown an effect of hydration status on the formation of venous
thromboembolism [35,36] and deep vein thrombosis (DVT) [34,37,38], which underlines
the relation of hydration status with the coagulation cascade.

4.3. Dehydration and Alzheimer

Ijsselstijn et al. [23] previously pointed out that serum levels of PZP are elevated in
persons who were developing Alzheimer’s disease (AD) in comparison to persons who
remained dementia-free. Dehydration decreases cognitive performance [39], which can
lead to a further deterioration in the cognitive state. In addition, patients with AD have
a major risk for current dehydration [40]. Dehydrated men showed increased levels of
PZP, although the proportion of patients already diagnosed with dementia is higher in
hyperhydrated men (66%) than in dehydrated men (40%). This is in line with our previous
study, where dehydration is linked with Resveratrol, a medication used to improve mild
cognitive impairment [12], and shows the unfavorable aspect of dehydration and cognitive
decline. This was also observed by Allen et al., where a 48% decrease in dementia was
predicted if the hydration was kept within normal levels [11]. Apart from that, PZP levels
were increased in hyperhydrated women. The mutual deterioration is not given in this
case, and it shows that PZP levels are not specifically linked with one hydration state.

4.4. Conclusions
In our study, we were able to show that the plasma proteome is indeed altered
in patients who are found to be moderately dehydrated as well as hyperhydrated. In
particular, the abundance of proteins functionally associated with wound healing and
coagulation was found to be altered. In fact, dehydration leads to decreased coagulation,
and hyperhydration is associated with increased coagulation, revealing proteins involved in
the coagulation pathway as potential biomarkers. In addition, proteins such as prothrombin
were differentially abundant in a gender-independent manner. Dehydration may also
exacerbate cognitive decline, as the Alzheimer’s predictive marker (PZP) was found in
dehydrated men. As a result, the study suggests that closer monitoring of hydration status
is needed to prevent adverse aspects, which is in line with the findings of Allen et al. [11].
Nutrients 2023, 15, 3789 11 of 13

In this regard, plasma protein analysis may serve as a more sensitive and effect-related
parameter compared to a general hydration analysis such as BIA.

Supplementary Materials: The following supporting information can be downloaded at https:

//www.mdpi.com/article/10.3390/nu15173789/s1. Table S1: Compilation of personal, clinical, and
bioimpedance data of the 272 samples included in the geriatric study. Table S2: Mean normalized
protein intensity, effect size, standard deviation, p-value, and confidence interval of differently
abundant proteins according to the p-value (<0.05).
Author Contributions: Conceptualization: S.K.; validation: L.H.; formal analysis: L.H.; investigation:
L.H., D.P. and J.R.S.; resources: J.K. and J.H.; data curation: D.P.; writing—original draft: L.H.;
writing—review and editing, D.P., J.R.S., J.H. and S.K.; supervision: J.K. and S.K.; project administra-
tion: J.K., J.H. and S.K.; funding acquisition: J.H. and S.K. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by the Bavarian State Ministry of Education and Culture, Science
and Art (BMUKK)—grant No. VII.2.F1116.CO/28/3 of the Collaborative Research Project “Measuring
health—methods for evidence-based health promotion” and by the Federal Ministry of Education
and Research, Germany (Bundesministerium für Bildung und Forschung, BMBF) (grant number
01ZX1910).
Institutional Review Board Statement: This study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of the
University of Applied Sciences Coburg for the project “Storage and analysis of blood samples from
dehydrated and adequately hydrated patients” from 3 December 2019.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The proteomics MS data were deposited to the ProteomeXchange
Consortium via the PRIDE [18] partner repository with the dataset identifier PXD043728.
Acknowledgments: The authors greatly appreciate the technical support of Bettina Völk, Melanie
Grosch (both Regiomed Clinics, Coburg), Carsten Geist (Fraunhofer Institute for Cell Therapy and
Immunology, Leipzig), and Richard Fry (Coburg University of Applied Sciences and Arts, Coburg).
Conflicts of Interest: The authors state no conflict of interest.

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