Metabolomics (2017) 13:5

DOI 10.1007/s11306-016-1144-0

ORIGINAL ARTICLE

Metabolomics of the human aqueous humor

Olga A. Snytnikova1,2 · Anastasiya A. Khlichkina1,2 · Lyudmila V. Yanshole1,2 ·
Vadim V. Yanshole1,2 · Igor A. Iskakov3 · Elena V. Egorova3 · Denis A. Stepakov4 ·
Vladimir P. Novoselov4 · Yuri P. Tsentalovich1,2 

Received: 9 September 2016 / Accepted: 26 November 2016

© Springer Science+Business Media New York 2016

Abstract Results The concentrations of 71 most abundant metab-

Introduction The optical elements of the eye—cor-
nea, lens, and vitreous humor—are avascular tissues, and
their nutrition and waste removal are provided by aqueous
humor (AH). The AH production occurs through the active
secretion and the passive difusion/ultrailtration of blood
plasma. The comparison of the metabolomic proiles of AH
and plasma is important for understanding of the mecha-
nisms of biochemical processes and metabolite transport
taking place in vivo in ocular tissues.
Objectives The work is aimed at the determination of
concentrations of a wide range of most abundant metabo-
lites in the human AH, the comparison of the metabolomic
proiles of AH and serum, and the analysis of the post-mor-
tem metabolomic changes in these two biological luids.
Methods The quantitative metabolomic proiling was car-
ried out with the use of two independent methods—high-
frequency 1H NMR spectroscopy and HPLC with high-res-
olution ESI-MS detection.
olites in blood serum and AH from living patients and
human cadavers have been measured. It has been found that
the level of ascorbate in AH is by two orders of magnitude
higher than that in serum; the levels of other metabolites
are either similar to that in serum, or difer from that by a
factor of 2–5. The post-mortem metabolomic composition
of both serum and AH undergoes rapid and strong changes.
Conclusion The diferences between the metabolomic
proiles of AH and serum for majority of metabolites can
be attributed to the metabolic activity of the ocular tissues
leading to the lack or excess of some metabolites, while the
high concentration of ascorbate in AH demonstrates the
activity of ascorbate-speciic pumps at the blood-aqueous
border. The post-mortem metabolomic changes are caused
by the disruption of the major biochemical cycles and cell
lysis. These changes should be taken into account in the
analysis of disease-induced changes in post-mortem sam-
ples of the ocular tissues.

Keywords Human metabolomics · Serum · Aqueous

Electronic supplementary material The online version of this humor
article (doi:10.1007/s11306-016-1144-0) contains supplementary
material, which is available to authorized users.

* Yuri P. Tsentalovich 1 Introduction

yura@tomo.nsc.ru
1 Most of the human tissues are directly connected to the
International Tomography Center SB RAS, Institutskaya 3a,
Novosibirsk 630090, Russia vascular system, which provides the supply of tissues with
2 nutrients and other metabolites as well as the removal of
Novosibirsk State University, Pirogova 2,
Novosibirsk 630090, Russia the metabolic waste. The optical elements of the eye—
3 cornea, lens, and vitreous humor—are rare examples of
Multidisciplinary Science and Technology Complex “Eye
Microsurgery”, Kolchidskya 10, Novosibirsk 630096, Russia avascular tissues, and their nutrition and waste removal
4 are provided by aqueous humor (AH). AH is secreted by
Novosibirsk Regional Clinical Bureau of Forensic
Medical Examination, Nemirovicha-Danchenko 134, the ciliary processes into the posterior chamber, lows into
Novosibirsk 630087, Russia the anterior chamber, and drains out via the trabecular

13
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5 Page 2 of 9
meshwork and Schlemm’s canal (Freddo 2013). Besides
the nutrition of the avascular ocular tissues, AH fulills an
important task to maintain the intraocular pressure via the
balance between the AH production and outlow. An imbal-
ance between the secretion and outlow may increase the
intraocular pressure causing glaucoma.
The AH production occurs in the ciliary epithelium
through both the active secretion (Na+, K+, and other ionic
pumps) and the passive difusion/ultrailtration of blood
plasma (Macknight et al. 2000; To et al. 2002; Gabelt et al.
2006; Goel et al. 2010) with the rate of 2–3 µL/min (Bru-
baker 1991). Thus, in general, the AH metabolomic com-
position should be similar to that of plasma. The diference
between the metabolomic proiles of AH and plasma can be
attributed to one of the following reasons: (1) the metabo-
lites are actively pumped into AH against the concentration
gradient; (2) the metabolites are produced by the avascu-
lar ocular tissues connected to AH; (3) the metabolites are
consumed by avascular ocular tissues faster than they enter
AH. Therefore, the comparison of the metabolomic pro-
iles of AH and plasma is important for understanding the
mechanisms of biochemical processes taking place in vivo
in ocular tissues. The metabolomic composition of serum
has been measured by diferent methods (Psychogios et al.
2011; Gowda and Raftery 2014; Gowda et  al. 2015); the
data are collected in online databases (“Human Metabo-
lome Database”, “Serum Metabolome Database”). Quali-
tative and semi-quantitative data on the AH metabolomic
composition and the concentrations of some metabolites in
AH can be found in literature (Brown et  al. 1986; Mack-
night et  al. 2000; Wakabayashi et  al. 2006; Reddy et  al.
1961; Gribbestad and Midelfart 1994; Hu et al. 2012; Tka-
dlecová et  al. 1999; Mayordomo-Febrer et  al. 2015; Song
et al. 2011; Keleş et al. 2011), but the comprehensive quan-
titative metabolomic proile of AH has not been reported.
After death, the metabolomic composition of plasma
undergoes deep and rapid changes (Donaldson and Lamont
2013, 2014, 2015; Zelentsova et  al. 2016), caused by the
disruption of oxygen-dependent enzymatic reactions (in
particular, the TCA cycle) and by cell lysis. Consequently,
the concentrations of some metabolites in plasma increase
ten-fold within several hours (Donaldson and Lamont 2013,
2014, 2015; Zelentsova et al. 2016). Since the AH secretion
is an ATP-dependent process, the post-mortem connection
between AH and plasma can be realized only through the
passive difusion/ultrailtration. The post-mortem changes
in the AH metabolomic composition can occur either
due to the decomposition of the ocular tissues, or due to
the metabolite difusion from the vascular system. Thus,
the analysis of the post-mortem metabolomic changes in
plasma and AH may help to estimate the eiciency of the
metabolite transport between plasma and AH via the pas-
sive difusion without the active secretion. The data on
13
O. A. Snytnikova et al.
the post-mortem changes in the AH metabolomic compo-
sition can be useful in the analysis of the disease-induced
metabolomic changes of the ocular tissues. Since healthy
samples of the ocular tissues are diicult to obtain for the
“case-control” study, the postoperational samples of dis-
eased tissues are often compared to the post-mortem sam-
ples from healthy donors (Streete et al. 2004; Kryczka et al.
2013; Tsentalovich et  al. 2015). Thus, it is important to
distinguish the metabolomic changes which are the results
of pathogenesis from the ones corresponding to the post-
mortem changes. The post-mortem metabolomic changes
in biological luids can also be used by forensic science
for the estimation of post-mortem interval (Donaldson and
Lamont 2013, 2015; Zelentsova et al. 2016).
In the present work, the quantitative metabolomic analy-
sis of human serum and AH was performed for the samples
taken from both living patients and human cadavers. The
quantitative metabolomic proiling was carried out with the
use of two independent methods—high-frequency 1H NMR
spectroscopy and HPLC with high-resolution ESI-MS
detection (LC-MS). The work is aimed at determining the
concentrations of a wide range of most abundant metabo-
lites in the human AH, the comparison of the metabolomic
proiles of AH and serum, and the analysis of the post-mor-
tem metabolomic changes in these two biological luids.
2 Experimental
2.1 Chemicals
All chemicals were purchased from Sigma–Aldrich (USA).
Acetonitrile HPLC grade was purchased from Panreac
(Spain). D2O 99.9% was purchased from Armar Chemicals
(Switzerland). H2O was deionized using Ultra pure water
system (SG water, Germany) to the quality of 18.2 MOhm.
2.2 Sample preparation
All procedures of this study were subjected to the Decla-
ration of Helsinki—ethical principles for medical research
involving human subjects and with the ethical approval
from International Tomography Center.
Blood and AH samples from living patients were col-
lected at the “Eye Microsurgery” clinic (Novosibirsk) from
6 non-diabetic (both genders) individuals with advanced
cataracts (age 55–84  years, average age 66). Aqueous
humor was collected during the extracapsular cataract
extraction surgery, blood samples were taken from antecu-
bital vein from the same patients. Written informed consent
was obtained from the patients. The samples of blood and
AH from cadavers (5–12  h post-mortem) were provided
by the Novosibirsk Clinical Bureau of Forensic Medical
Metabolomics of the human aqueous humor
Examination. A total of 6 donors were of both sexes, aged
18–81  years (average age 44  years). The collected blood
samples were immediately centrifuged (3000 g, 10 min) to
remove the blood cells. The obtained luid samples (plasma
and AH) were frozen and stored at −70 °C until analyzed.
The procedure used to purify the samples from proteins
and lipids is described in detail elsewhere (Tsentalovich
et al. 2015; Tamara et al. 2016; Zelentsova et al. 2016). The
proteins in AH and plasma samples were precipitated with
the addition of EtOH (4:1). The lipids were removed from
the protein-free plasma extracts by the layered chloroform/
ethanol/water mixture. The AH protein-free extracts were
analyzed without the delipidation procedure.
The extracts for NMR measurements were lyophilized,
re-dissolved in 500  μL of D2O containing 6 × 10−6  M
sodium 3-trimethylsilylpropane-1-sulfonate (DSS) as an
internal standard and 20  mM deuterated phosphate bufer
to maintain pH 7.4.
The extracts for LC-MS analysis were lyophilized, re-
dissolved in 50  μL of aqueous solution containing 10  μM
N-acetyl-tryptophan as an internal control (IC). The
extracts were then diluted with IC solution in the ratios: 1
(undiluted), 1/3, 1/10, and 1/50 to expand the quantiication
dynamic range for metabolite concentrations under study.
2.3 1H NMR and LC-MS measurements
The 1H NMR measurements were carried out with the use
of a NMR spectrometer AVANCE III HD 700 MHz (Bruker
BioSpin, Germany) equipped with a 16.44 T Ascend cryo-
magnet. The proton NMR spectra for each sample were
obtained with 200 accumulations. Temperature of the
sample during the data acquisition was kept at 25 °C, the
detection pulse was 90°. The repetition time between scans
was 30 s to allow for the complete relaxation of all spins.
Low power radiation at the water resonance frequency was
applied prior to acquisition to presaturate the water signal.
The signal assignment was conirmed by the addition of
standard samples into extract solutions. The concentrations
of metabolites in the samples were determined by the peak
area integration respectively to the internal standard DSS.
The detailed description of the LC-MS measurements
can be found in earlier papers (Tsentalovich et  al. 2015;
Tamara et  al. 2016; Zelentsova et  al. 2016). Briely, the
ion-pairing HPLC separation was performed on Agi-
lent Zorbax XDB-C18 column (4.6 × 250  mm, 80  Ǻ,
5  μm). Solvent A consisted of 0.1% formic acid and
5  mM of perluorooctanoic acid solution in H2O, sol-
vent B consisted of 0.1% formic acid solution in ace-
tonitrile. The gradient was (solvent B): 5% (0–10  min),
5–95% (10–55  min); the low rate was 200  µL/min, the
sample injection volume was 10  µL. The MS detection
was performed on an ESI-q-TOF high-resolution hybrid
Page 3 of 9 5
mass spectrometer maXis 4G (Bruker Daltonics, Ger-
many). The mass spectra were recorded in the positive
mode with 50–1000  m/z range. The MS setup, the cali-
bration procedure, and the data processing are described
in details in (Yanshole et  al. 2014). For the calibration
curve plotting, we prepared nine solutions containing an
equimolar mixture of metabolites with the concentrations
ranging from 0.068 to 34 μM. The present study includes
only the metabolites whose identity has been conirmed
with the commercially available chemical standards.
2.4 Statistical analysis
The data were analyzed using the statistical package Sta-
tistica 6.0 (StatSoft, USA). The comparison of groups
was performed with the standard independent t test.
3 Results
The metabolomic compositions of the serum and AH
samples taken from living patients and from cadavers
were measured with the use of two methods: high-fre-
quency NMR and LC-MS. NMR gives simple and reli-
able metabolite quantiication since the 1H NMR signal
from a metabolite is directly proportional to its concen-
tration. The advantages of the LC-MS method are the rel-
atively simple metabolite identiication procedure and its
high sensitivity, and, correspondingly, a much longer list
of identiied metabolites. However, the LC-MS signals
strongly depend on the metabolite ionization capability;
the quantiication requires the construction of calibra-
tion curves for each metabolite under study with the use
of commercially available chemical standards. Besides,
the intensities of MS signals might be inluenced by the
ion suppression efect (Annesley 2003). In this work, the
concentrations of 24 metabolites have been measured by
NMR only, 20—by LC-MS only, and 27—by both meth-
ods. For the majority of metabolites measured by both
methods, similar quantitative results have been obtained.
For several compounds (e.g. alanine, lactate, proline)
the diference was signiicant, it was attributed either to
the strong ion suppression efect (alanine, proline) or to
the poor ionization and fast signal saturation (lactate).
For that reason, we used the following approach to the
data selection: for metabolites demonstrating good non-
overlapping NMR signals, the data obtained by the NMR
method were taken. If the NMR signals were strongly
obscured by other compounds, or the metabolite con-
centrations were too low for the reliable NMR measure-
ments, the LC-MS data were taken.
13
5 Page 4 of 9
3.1 Metabolomic composition of serum and AH
of living patients
The concentrations of the most abundant metabolites found
in the human serum and AH are collected in Table  1;
the complete list of quantiied metabolites includes 71
compounds, it is presented in Supporting Information,
Table  1ES. The data in Tables are mean ± standard devia-
tion. The symbols “a”, “b” and “c” in the tables indicate
whether the data have been taken from NMR and con-
irmed by LC-MS, from NMR only or from LC-MS only.
The obtained data on the serum metabolomic composition
are in a good agreement with previous reports (Gowda and
Raftery 2014; Gowda et al. 2015). The highest concentra-
tions in serum were found for glucose, lactate and urate,
followed by the most abundant amino acids. Glucose is
the major energy source for metabolomic processes. Dur-
ing the glycolysis, its oxidation in the presence of oxygen
leads to the formation of pyruvate, while under anaerobic
conditions the formation of lactate takes place. Urate is the
end product of purine catabolism in the human body. One
should note that the data scattering for diferent metabo-
lites in the human serum difer signiicantly: the standard
deviation for the majority of amino acids does not exceed
20–30%, while the concentrations of some compounds (e.g.
glucose, ascorbate, 3-hydroxy-butyrate) vary by a factor of
3–5. High data scattering for these metabolites should be
attributed either to the strong dependence of their levels on
the dietary intake, or to the diferent rates of their produc-
tion and removal from the vascular system.
Figure  1 shows typical NMR spectra of samples con-
taining serum and AH extracts. As expected, the metabo-
lomic compositions of serum and AH are rather similar.
For the quantitative estimation of the similarity between
the serum and AH metabolomes, we calculated the ratios
of the metabolite concentrations in AH to that in serum of
the same patient (AH/serum), and then the obtained values
were averaged. For the majority of compounds, the ratio
AH/serum was close to unit. The AH/serum ratios statis-
tically diferent from unit are presented in Fig.  2 (empty
bars). The most dramatic diference is found for ascorbate,
which level in AH is two orders of magnitude higher than
that in serum. The elevated levels in AH was also observed
for glutamate, lactate, pyruvate and some other compounds,
while the lowest values of AH/serum ratio were found for
hypoxanthine and glycine (Fig. 2).
3.2 Metabolomic composition of serum and AH
in post-mortem samples
The data on the concentrations of metabolites found in
serum and AH post mortem are given in Tables 1 and 1ES.
The following features should be noticed:
13
O. A. Snytnikova et al.
1. The concentrations of many metabolites in both serum
and AH in the post-mortem samples are much higher
than that in the samples from the living patients. Thir-
teen compounds in serum and four compounds in AH
demonstrate more than tenfold increase. The most
dramatic increase in serum was found for succinate,
hypoxanthine, phosphocholine, glutamate and creatine,
and in AH—for hypoxanthine, choline, glycerol and
phosphocholine.
2. The concentration of lactate in the post-mortem sam-
ples is by far greater than the concentration of any
other compound.
3. For few compounds (arginine, glucose, guanine, ino-
sine, ketoleucine) the concentrations in the post-mor-
tem samples are noticeably lower than that in the sam-
ples from the living patients.
4. The value of the AH/serum ratio for ascorbate in the
post-mortem samples is approximately tenfold lower
than that for the living patients.
5. The standard deviations for the post-mortem samples
are signiicantly greater.
3.3 Statistical analysis
The data given in Table 1, 1ES and 2ES were subjected to
statistical analysis with the use of the standard independ-
ent t test. The samples were divided into four groups: (1)
serum from living patients; (2) AH from living patients; (3)
post-mortem serum; (4) post-mortem AH. The signs *, †,
‡, § in Tables  <link rid="tb1">1</link and 1ES indicate
the statistically signiicant diference between two groups.
One sign corresponds to p value <0.05, two signs—to p
value <0.01, three signs—to p value <0.005. The ratios
AH/serum for living patients and post-mortem in Table 2S
were compared in a similar way.
4 Discussion
The AH formation occurs by both passive and active mech-
anisms. The passive mechanisms include the solute difu-
sion along a concentration gradient and the ultrailtration
of substances across fenestrated ciliary capillary endothelia
(Macknight et al. 2000; Civan and Macknight 2004). It has
been estimated that the contribution of the passive mecha-
nisms to the AH formation does not exceed 10–20% (Mark
2010). The major role in the AH formation is played by the
active secretion, mediated by transport proteins located in
the cellular membranes. The mechanism of the secretion
includes the active transfer of NaCl from ciliary stroma into
posterior chamber with water following the created osmotic
gradient. The energy required for the transportation is gen-
erated by the ATP hydrolysis.
Metabolomics of the human aqueous humor Page 5 of 9 5

Table 1 Levels of the most Compound Living patients Post-mortem

abundant metabolites (in mM)
found in the human serum and Serum AH Serum AH
AH
b
2-Hydroxy-butyrate 0.06 ± 0.04 0.06 ± 0.04 0.06 ± 0.03 0.14 ± 0.12
3-Hydroxy-butyrateb 0.05 ± 0.03 0.03 ± 0.01 0.07 ± 0.02 0.07 ± 0.06
Acetateb 0.08 ± 0.02 0.15 ± 0.04*** 0.9 ± 0.4††† 0.6 ± 0.8
Alanineb 0.38 ± 0.15 0.36 ± 0.04 1.5 ± 0.5††† 0.6 ± 0.3‡‡‡
Argininec 0.28 ± 0.10 0.36 ± 0.14 0.14 ± 0.10 0.10 ± 0.03§§
Ascorbatea 0.003 ± 0.004 0.3 ± 0.2** 0.07 ± 0.03††† 0.5 ± 0.3‡‡‡
Asparaginea 0.025 ± 0.004 0.018 ± 0.007* 0.17 ± 0.05††† 0.06 ± 0.03‡,§§
Aspartatec 0.017 ± 0.003 0.03 ± 0.03 0.6 ± 0.4† 0.034 ± 0.015‡
Betainea 0.04 ± 0.01 0.011 ± 0.002*** 0.09 ± 0.03††† 0.04 ± 0.03‡,§
Carnitinea 0.06 ± 0.02 0.022 ± 0.003*** 0.16 ± 0.07†† 0.05 ± 0.03‡‡
Cholineb 0.07 ± 0.05 0.008 ± 0.002* 0.3 ± 0.1††† 0.1 ± 0.1§
Creatinea 0.03 ± 0.01 0.11 ± 0.02*** 1.4 ± 1.2† 0.2 ± 0.2
Creatinineb 0.06 ± 0.01 0.044 ± 0.008** 0.20 ± 0.08††† 0.12 ± 0.10
Cysteinec 0.079 ± 0.005 0.033 ± 0.008* 0.04 ± 0.03 0.034 ± 0.007
Formateb 0.04 ± 0.02 0.010 ± 0.004* 0.05 ± 0.02 0.03 ± 0.01§§§
Glucoseb 6±4 5±4 3±3 1±1
Glutamatec 0.02 ± 0.01 0.08 ± 0.07 1.5 ± 1.2† 0.3 ± 0.3§
Glutaminea 0.42 ± 0.08 0.55 ± 0.14 1.0 ± 0.5† 0.6 ± 0.2
Glycerolb 0.06 ± 0.04 0.05 ± 0.03 1.6 ± 0.8††† 0.8 ± 0.6§
Glycineb 0.21 ± 0.09 0.03 ± 0.02*** 0.7 ± 0.4† 0.21 ± 0.09‡,§§§
Histidinea 0.053 ± 0.008 0.06 ± 0.01 0.15 ± 0.06††† 0.07 ± 0.02‡
Inosinec 0.06 ± 0.02 0.013 ± 0.005*** 0.03 ± 0.02† 0.04 ± 0.01§§§
Isoleucinea 0.06 ± 0.02 0.06 ± 0.01 0.15 ± 0.06†† 0.10 ± 0.06
Lactateb 1.6 ± 0.5 7 ± 2*** 27 ± 7††† 24 ± 8§§§
Leucinea 0.12 ± 0.02 0.16 ± 0.03* 0.4 ± 0.1††† 0.21 ± 0.12‡
Lysinea 0.19 ± 0.04 0.20 ± 0.07 0.34 ± 0.16 0.10 ± 0.04‡,§
myo-Inositola 0.07 ± 0.03 0.15 ± 0.03*** 0.9 ± 0.9 0.5 ± 0.3§
Ornithineb 0.03 ± 0.01 0.018 ± 0.007 0.19 ± 0.06††† 0.03 ± 0.02‡‡‡
Phenylalaninea 0.07 ± 0.02 0.14 ± 0.02*** 0.18 ± 0.06††† 0.14 ± 0.06
Prolineb 0.14 ± 0.04 NQ 0.37 ± 0.13††† 0.12 ± 0.05‡
Pyruvateb 0.03 ± 0.01 0.14 ± 0.03*** 0.05 ± 0.01 0.03 ± 0.02§§§
Taurinec 0.095 ± 0.014 0.10 ± 0.04 0. 7 ± 0.5† 0.7 ± 0.9
Threonineb 0.10 ± 0.04 0.16 ± 0.04* 0.3 ± 0.2† 0.14 ± 0.11‡
Tryptophana 0.05 ± 0.01 0.033 ± 0.007* 0.04 ± 0.01 0.037 ± 0.015
Tyrosinea 0.06 ± 0.02 0.11 ± 0.02*** 0.17 ± 0.07†† 0.09 ± 0.03‡
Uratec 0.43 ± 0.07 0.22 ± 0.08*** 0.18 ± 0.15† 0.22 ± 0.17
Valinea 0.21 ± 0.04 0.31 ± 0.08* 0.4 ± 0.2† 0.24 ± 0.10

ND not detected
NQ not quantiied
*The levels in serum and AH from living patients are statistically diferent with p value < 0.05; **with p
value < 0.01; *** with p-value < 0.005
†
The levels in serum from living patients and post-mortem are statistically diferent with p value < 0.05;
††
with p value < 0.01; ††† with p value < 0.005
‡ ‡‡
The levels in serum and AH post-mortem are statistically diferent with p value < 0.05; with p
value < 0.01; ‡‡‡ with p value < 0.005
§
The levels in AH from living patients and post-mortem are statistically diferent with p value < 0.05;
§§
with p value < 0.01; §§§ with p value < 0.005
a
Data are taken from NMR measurements and validated by LC-MS measurements
b
Data are taken from NMR measurements
c
Data are taken from LC-MS measurements

13
5 Page 6 of 9 O. A. Snytnikova et al.

Lactate
AH
AH

Asc
Val
EtOH
Ala

Glucose

Lactate
Pyruvate

Glucose
Gln

Met

3-OH-But

Leu

2-OH-But

2-OH-3-Met-But

Mannose
Succinate

Me-Var
I-But

Kle
Ppg

Ile

Pyr
5,3 5,2 5,1 5,0 4,9 4,8 4,7 4,6 4,5 4,4 4,3 4,2 4,1
2,5 2,4 2,3 2,2 1,5 1,4 1,3 1,2 1,1 1,0 0,9 0,8 Chemical shift (ppm)
Chemical shift (ppm)

Serum

Serum
Met

Kle

Pro
Glu

Thr
2,5 2,4 2,3 2,2 1,5 1,4 1,3 1,2 1,1 1,0 0,9 0,8 5,3 5,2 5,1 5,0 4,9 4,8 4,7 4,6 4,5 4,4 4,3 4,2 4,1
Chemical shift (ppm) Chemical shift (ppm)

Tyr
Phe
AH

Tyr
AH
Glucose
EtOH

His
His
For
Glycerol
M- In

Cre

Uridine

Trp
Sc- In

Trp

Trp
Cre

Lys
Car

Crn
Ser

4,0 3,9 3,8 3,7 3,6 3,5 3,4 3,3 3,2 3,1 3,0 8,5 8,4 8,3 8,2 8,1 8,0 7,9 7,8 7,7 7,6 7,5 7,4 7,3 7,2 7,1 7,0 6,9
Chemical shift (ppm) Chemical shift (ppm)
CHCl3

Serum Serum
Gly

Betaine

Ac-Car

H-Xan
Cho

Orn

4,0 3,9 3,8 3,7 3,6 3,5 3,4 3,3 3,2 3,1 3,0 8,5 8,4 8,3 8,2 8,1 8,0 7,9 7,8 7,7 7,6 7,5 7,4 7,3 7,2 7,1 7,0 6,9
Chemical shift (ppm) Chemical shift (ppm)

Fig. 1 Representative 1H NMR spectra of the protein-free lipid-free Cho choline, Cre creatine, Crn creatinine, For formate, H-Xan hypox-
extracts from the human AH and serum with the metabolite assign- anthine, I-But isobutyrate, Kle ketoleucine, M-In myo-inositol, Orn
ment: 2-OH-3-Met-But 2-hydroxy-3-methyl-butyrate, 2-OH-But ornithine, Ppg propylene glucol, Pyr pyroglutamate, Sc-In scyllo-ino-
2-hydroxy-butyrate, 3-OH-But 3-hydroxy-butyrate, Me-Var 3-methyl- sitol. For amino acids, standard 3-letter symbols are used
2-oxovalerate, Ac-Car acetyl-carnitine, Asc ascorbate, Car carnitine,

13
Metabolomics of the human aqueous humor Page 7 of 9 5

(a) 10 AH/Serum, living

oxidative stress mostly rely on endogenous and exogenous
480 AH/Serum, post-mortem antioxidants. The advantage of ascorbate over other anti-
360 8 oxidants is that besides being a good electron donor, ascor-
Ratio of concentrations

240
bate can quench reactive triplet states formed under UV
6
40 irradiation, and therefore protect proteins from UV-induced
30 4 damages (Snytnikova et al. 2007; Sherin et al. 2016). That
explains the high concentration of ascorbate in the ocular
20
2 tissues of diurnal animals in general and of humans in par-
10
ticular (Reiss et al. 1986; Varma 1987; Tsentalovich et al.
0
0 2015).
Asc

Val
Glu
Lac
Pyv
Ade

P-Cho
Asp

M-In

Ac
Phe

Leu
Pyr
Cre
Citrate
Uridine

Ser

Tyr
Thr
I-But

Met
Some other metabolites also demonstrate the elevated
levels in AH as compared to serum (Fig. 2a), but for these
(b) 6 compounds the diference between AH and serum is much
AH/Serum, living
AH/Serum, post-mortem more moderate. Most likely, the observed diference should
5
be attributed to the enhanced production of these com-
Ratio of concentrations

4 pounds in the ocular tissues rather than to the active pump-

ing from blood to AH. For example, the elevated level of
3
lactate in AH can be attributed to the enhanced role of
2 anaerobic glycolysis for the metabolic energy production in
1 the ocular tissues due to the low oxygen content in these
tissues (Fitch et al. 2000; McNulty et al. 2004). Glutamate
0
plays an important role in the nitrogen metabolism; it can
Asn
KN

Kle
Orn
Trp
Crn

Urate
Cys

Betaine
Ino
Cho
Me-Var

Car

For
H-Xan
Gly
Cyst

be formed in the reactions of glutamine deamination or

α-ketoglutarate transamination, or produced from amino
Fig. 2 Ratios of the metabolite concentrations in the samples: open acids arginine, proline, histidine.
bars AH/serum from living patients, hatched bars AH/serum post- The concentrations of compounds shown in Fig.  2b in
mortem. Me-Var 3-methyl-2-oxovalerate, Ac-Car acetyl-carnitine, AH are lower than in serum. For some metabolites (e.g.
Ade adenosine, Ac acetate, Asc ascorbate, Car carnitine, Cho choline,
glycine, choline, inosine, betaine), their relatively low lev-
Cre creatine, Crn creatinine, Cyst cystathionine, Cyd cytidine, For
formate, Fum fumarate, H-Tau hypotaurine, H-Xan hypoxanthine, Ino els in AH can be explained in terms of their fast consump-
inosine, I-But isobutyrate, Kle ketoleucine, KN kynurenine, Lac lac- tion by the ocular tissues for biosynthesis. The diference in
tate, M-In myo-inositol, Orn ornithine, Pyr pyroglutamate, Pyv pyru- the hypoxanthine concentrations in serum and AH probably
vate. For amino acids, standard 3-letter symbols are used
corresponds to metabolic processes taking place in blood
already in  vitro during the short time period between the
For the majority of metabolites, their concentrations in blood sampling and plasma separation: the lack of oxy-
serum and AH are similar. That demonstrates a good con- gen disrupts the purine catabolism and leads to very fast
nection between these biological luids, providing AH increase of hypoxanthine level (Donaldson and Lamont
with the suicient nutrient supply and with the efective 2013; Zelentsova et al. 2016).
removal of metabolic waste produced by the avascular ocu- The post-mortem increase of the metabolite levels in
lar tissues. plasma originates primarily from the disruption of many
Besides the Na+-K+ water pumps, other types of active metabolic reactions and processes under anaerobic condi-
pumps for selected substances can exist at the blood/ tions. In particular, anaerobic glycolysis causes signiicant
aqueous border. The results of the present work conirm increase of lactate. The lack of energy disables intracel-
the activity of ascorbate-speciic pumps (DiMattio 1989; lular Na+-K+ pumps, which results in the Na+ accumula-
Delamare 1996): the concentration of exogenous antioxi- tion inside the cells, cell swelling due to the osmotic pres-
dant ascorbate in AH is two orders of magnitude higher sure, membrane damages, and leakage of the intracellular
than in serum. High concentration of ascorbate inside the metabolites into plasma (Cotran et al. 1994). For example,
eye relects a high demand of the ocular tissues in antioxi- a dramatic increase of levels of the membrane components
dants. From one side, these tissues are permanently sub- choline and glycerol in the post-mortem samples should
jected to the solar irradiation, which includes UV-A and be attributed to the deterioration of the cell membranes.
UV-B spectral components. From the other side, the avas- Finally, the microbial activity can also contribute into post-
cular ocular tissues (vitreous, lens and cornea) are meta- mortem metabolomic changes in plasma.
bolically inert (with the exception of thin epithelial and The data present in Table 1 and 1ES and in Fig. 2 show
endothelial layers), and their protection against UV-induced that the post-mortem metabolomic changes in AH are

13
5 Page 8 of 9
similar to that in serum. In particular, a dramatic increase
of levels of choline, glycerol, and hypoxanthine has been
observed, while the level of glucose decreases. At the same
time, for some compounds (e.g. alanine, creatine, ornithine)
the diference between AH and serum in post-mortem sam-
ples increases (Table 2ES). The decrease of the metabolite
concentrations in AH should be attributed to the metabolite
consumption in post-mortem processes such as anaerobic
glycolysis. The post-mortem increase of the metabolite
levels in AH is determined by two major factors: the lysis
of the ocular tissue cells and the metabolite difusion from
blood through the blood/aqueous border. The metabolite
exchange between blood and AH can be facilitated by the
destruction of the blood/aqueous barrier (Freddo 2013).
From the results obtained in the present work, it is dii-
cult to estimate the contributions of these factors into post-
mortem AH metabolomic composition. It has been recently
shown that for experimental animals both factors play an
important role (Zelentsova et  al. 2016); most likely, this
statement is correct for humans as well.
The development of the post-mortem changes in blood
and in AH is a complex dynamic process, and at diferent
post-mortem intervals the metabolomic composition of the
same tissue may change signiicantly. Since the sampling
was performed at diferent post-mortem intervals, that
explains the high values of the standard deviations for post-
mortem samples.
The obtained results demonstrate that already several
hours post mortem, the AH metabolomic composition
undergoes signiicant changes. Apparently, these changes
should inluence the metabolomic proiles of other ocular
tissues, including cornea, lens, and vitreous. The study of
the metabolomic compositions of these tissues and their
post-mortem changes will be published in our forthcoming
papers.
5 Conclusions
This work is the irst comprehensive report on the quan-
titative metabolomic composition of the human aqueous
humor. The concentrations of 71 most abundant metabo-
lites in blood serum and AH from living patients and
human cadavers have been measured. It has been shown
that in the living patients, the levels of majority of metabo-
lites in serum and AH are similar. This inding relects a
good connection between AH and blood, providing a suf-
icient nutrient supply and an efective waste removal from
the avascular ocular tissues. The highest diference between
AH and serum is found for ascorbate, which level in AH is
two orders of magnitude higher than in serum. This difer-
ence is attributed to the high demand of the ocular tissues
in this antioxidant, and to the activity of ascorbate-speciic
13
O. A. Snytnikova et al.
pumps transferring ascorbate from blood into AH against
the concentration gradient. The diference in the levels of
other metabolites between serum and AH does not exceed
the factor of ive, it can be explained in terms of metabolic
activity of the ocular tissues with either production or rapid
consumption of metabolites.
The post-mortem metabolomic changes are signiicant
for both serum and AH: already several hours post-mortem,
the concentrations of some metabolites increase tenfold.
These changes should be taken into account in the analysis
of disease-induced changes in post-mortem samples of the
ocular tissues.
Acknowledgements The work was supported by the Russian Sci-
entiic Foundation (project No. 14-14-00056) in NMR measurements,
by the President of RF (project MK-5367.2015.3) in LC-MS meas-
urements, and by FASO Russia (project 0333-2014-0001) in sample
preparation.
Compliance with ethical standards
Conlict of interest Authors declare that there is no conlict of inter-
est.
Informed consent All participants signed an informed consent form.
Research involving human participants and/or animals The study
was conducted in accordance with the Declaration of Helsinki (2013)
of the World Medical Association, and with the ethical approval from
International Tomography Center.
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