Alteration in plasma free amino acid levels and its association with gout
Mahbub et al. Environmental Health and Preventive Medicine
Alteration in plasma free amino acid levels and its association with gout
MH Mahbub 0
Natsu Yamaguchi 0
Hidekazu Takahashi 0
Ryosuke Hase 0
Yasutaka Ishimaru 0
Hiroshi Sunagawa 0
Tsuyoshi Tanabe 0
0 Department of Public Health and Preventive Medicine, Yamaguchi University Graduate School of Medicine , 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505 , Japan
Background: Studies on the association of plasma-free amino acids with gout are very limited and produced conflicting results. Therefore, we sought to explore and characterize the plasma-free amino acid (PFAA) profile in patients with gout and evaluate its association with the latter. Methods: Data from a total of 819 subjects (including 34 patients with gout) undergoing an annual health examination program in Shimane, Japan were considered for this study. Venous blood samples were collected from the subjects and concentrations of 19 plasma amino acids were determined by high-performance liquid chromatography-electrospray ionization-mass spectrometry. Student's t-test was applied for comparison of variables between patient and control groups. The relationships between the presence or absence of gout and individual amino acids were investigated by logistic regression analysis controlling for the effects of potential demographic confounders. Results: Among 19 amino acids, the levels of 10 amino acids (alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, tryptophan, valine) differed significantly (P < .001 to .05) between the patient and control groups. Univariate logistic regression analysis revealed that plasma levels of alanine, isoleucine, leucine, phenylalanine, tryptophan and valine had significant positive associations (P < .005 to .05) whereas glycine and serine had significant inverse association (P < .05) with gout. Conclusions: The observed significant changes in PFAA profiles may have important implications for improving our understanding of pathophysiology, diagnosis and prevention of gout. The findings of this study need further confirmation in future large-scale studies involving a larger number of patients with gout.
Amino acids; Plasma; Profile; Gout; Relationship
Gout, the most prevalent inflammatory joint disease is
usually characterized by recurrent attacks of intense
pain. It is predominant in men and also in older women
. Currently, the global prevalence of gout is estimated
to be at least 1–2% in the general population and 3–4%
in the adult population . The prevalence can be as
high as 7% among the male population over 75 years of
age . In Japan, the overall prevalence of gout was
found to be lower in a survey which was 0.51% overall
and 1.1% amongst men . The incidence and
prevalence of gout are found to be increasing in many
parts of the world [5, 6]. With the aging of world
population, the global burden of gout continues to
rise . Therefore, better understanding of various
factors and mechanisms underlying the
pathophysiology of gout is necessary for the proper diagnosis
and management of it.
The most important underlying mechanism in gout
involves elevated levels of uric acid in the blood. Such a
persistent increase in the levels of serum uric acid causes
crystallization of it and intra-articular formation and
deposition of monosodium urate (MSU) crystals which can
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trigger the painful attacks of gout . The serum uric
acid is the end product of an exogenous (from food)
pool of purines and endogenous (from liver, intestines,
muscles, kidneys and the vascular endothelium) purine
metabolism . Certain amino acids take part in the
biosynthesis of purine and subsequent formation of uric
acids. For example, amino acids like glutamine, glycine,
and serine are utilized in increased amounts for the
formation of uric acid in gout . Therefore, it is
reasonable to postulate that amino acids play important roles
in the pathogenesis of gout.
Plasma free amino acids (PFAAs) abundantly circulate as
a medium linking all organ systems in the human body; the
PFAA profiles have been shown to be influenced by
metabolic variations in specific organ systems induced by
specific diseases . PFAA profiles can be used as reliable
markers for monitoring the risks of various diseases in
various populations and also the improvements in physiological
states [12–14]. Therefore, amino acid profiles are
increasingly being used in the evaluation of various diseases.
Analysis of PFAA profiles with a high degree of validity and
reliability can be useful in understanding the underlying
pathophysiology and assessing the severity of a disorder.
Furthermore, various indexes developed from PFAA
profiles have shown the potential for diagnosis of various
pathological conditions [11, 14, 15]. Understanding the
variations in PFAA profiles among various populations
including patients with gout seems to be important which may
guide to the diagnosis of gout. However, the number of
studies investigating the changes in PFAA profiles
associated with gout is very limited. Also, there is a lack of
published research works, especially in recent times, on the
relationship between the amino acid profiles and gout. A
few previous studies studies reported altered PFAA profiles
in patients with gout. Compared to control subjects, some
researchers reported a significant increase for some of the
amino acids and a significant decrease for the others in
patients with gout [10, 16], whereas other researchers
reported hyperaminoacidemia for all the investigated amino
acids in the patients or similar amino acid spectrums in
normal subjects and patients with gout [17, 18]. As
understandable, the findings are conflicting as the observed
changes in those studies are inconsistent for various amino
acids. Furthermore, those studies aimed at finding the
significant group differences between gout patients and
control subjects in the concentrations of various amino acids,
and did not investigate the relationship of amino acids with
gout. Therefore, the purpose of the present study was to
further explore and characterize the PFAA profiles in
patients with gout and evaluate its association with the latter.
A total of 831 subjects who underwent their annual
health check-up between June and July 2012 at different
health examination centers in Shimane Prefecture, Japan
and for whom workplace health examination was not
applicable, were considered for inclusion in this study. The
health examination included physical examination, clinical
and laboratory tests, and a self-administered questionnaire
containing personal and medical history. Based on the
questionnaire data, 12 subjects were excluded from the
study due to lack of information on gout. Finally, 34
subjects were identified as having an established diagnosis of
gout and were treated as the patient population, and the
rest 785 subjects were treated as the control population in
this study. Among the patients, 26 were currently taking
medications for gout and the rest 8, currently without any
medications for it. The subjects had no serious health
problems like cancer or renal failure.
An oral explanation of the study protocol was made in
detail to the study participants and written informed
consent was obtained from all of them. The current
study protocol was approved by the institutional review
board of Shimane University Hospital (No. H25-26-2).
Measurement of plasma amino acid concentrations
Five ml of blood samples were collected and analyzed for
plasma amino acid concentrations following the protocol
previously described elsewhere [11, 19–21]. Briefly, after
overnight fasting, venous blood samples were collected
from the cubital vein of the seated subjects in tubes which
contained ethylenediaminetetraacetic acid (EDTA; Termo,
Tokyo, Japan). The tubes were placed on ice immediately
and kept there for about 15 min. After centrifugation of
tubes under 4 °C at 3,000 rpm for 15 min, the plasma was
immediately separated into tubes and stored at −80 °C.
The tubes were kept there until (within 2 weeks to
2 months) the desired analysis for plasma amino acids.
The plasma samples were deproteinized using acetonitrile
at a final concentration of 80% before measurements. The
amino acid concentrations in the plasma were measured
by high-performance liquid chromatography–electrospray
ionization–mass spectrometry (HPLC–ESI–MS) followed
by precolumn derivatization which allows such
measurements with high accuracy. PFAA profiling included the
measurement of absolute concentrations (in μmol/L) of
the following 19 amino acids: alanine (Ala), arginine (Arg),
asparagine (Asn), Citrulline (Cit), glutamine (Gln), glycine
(Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine
(Lys), methionine (Met), ornithine (Orn), phenylalanine
(Phe), proline (Pro), serine (Ser), threonine (Thr),
tryptophan (Trp), tyrosine (Tyr), and valine (Val).
The continuous variables in this study showed a
nonnormal distribution by Kolmogorov-Smirnov and
ShapiroWilk tests. Hence, statistical analyses were performed with
those variables after logarithmic transformation of data.
The continuous variables were expressed as geometric
mean and its 95% confidence interval (95% CI). Smoking
status was categorized as current/past smokers and
nonsmokers, and alcohol consumption, as current drinkers and
non/rare-drinkers. For convenience of graphical
presentation of individual amino acids, they were divided into 3
groups according to their plasma geometric concentrations.
The differences between the patient and control groups
were assessed with Student’s t-test and Chi-square (χ2) test
for continuous and categorical variables, respectively. To
further explore the amino acids associated with the
outcome (gout), logistic regression analysis was carried out
with the individual amino acids that did differ between the
two groups at P < .05. The models were adjusted for the
potential confounding demographic factors and
corresponding odds ratios (OR), 95% confidence interval (CI) and
Pvalues were obtained. All statistical tests were considered as
two-tailed, and the significance level was set at the value of
P < .05. The software package SPSS version 22 for
Windows (SPSS Inc., Chicago, IL, USA) was used to
perform the statistical analyses.
The demographic and clinical characteristics of the
current study subjects have been presented in table 1.
Most study participants in both patient and control
groups were elderly for whom smoking and drinking
were common. Statistically significant differences
between the patient and control groups were observed for
gender, BMI, and smoking and drinking status (P < .001
to .01). Furthermore, significant differences in terms of
alanine aminotransferase/ALT, gamma-glutamyl
transpeptidase/γ-GTP, high-density lipoprotein cholesterol/
HDLC, and triglycerides/TG (P < .001 to .05) were also
observed between the two groups. Among the
participants of this study, the commonly used medications
were for hypertension (patients, 20/34 or 58.8%;
controls, 329/785 or 41.9%), diabetes mellitus (patients, 5/34
Table 1 Demographic and clinical characteristics of study subjects. Values are expressed as geometric mean and 95%CI for
continuous variables, and number (n) and percent (%) for categorical variables
Glucose a (mg/dl)
*Bold values indicate statistically significant difference
ALT, alanine aminotransferase, AST, aspartate aminotransferase, BMI body mass index, DBP diastolic blood pressure, γ-GTP gamma-glutamyl transpeptidase; Glucose
fasting blood glucose, HbA1C haemoglobin A1C, HDLC high-density lipoprotein cholesterol, LDLC low-density lipoprotein cholesterol, SBP systolic blood pressure,
a Glucose: 4 missing values for control group
b HbA1C: 1 missing value for patient group and 17, for control group
or 14.7%; controls, 75/785 or 9.6%), and dyslipidemia
(patients, 10/34 or 29.4%; controls, 272/785 or 34.6%).
With respect to the use of medications, the patients and
controls did not differ significantly for diabetes mellitus
(χ2 = .981; P = .322), dyslipidemia (χ2 = .396; P = .585), or
for hypertension (χ2 = 3.812; P = .075).
Data on plasma uric acid were available only for a
limited number of subjects: 19 (19/34 or 55.9%) patients and
392 (392/785 or 49.9%) control subjects. Overall, the
concentration of uric acid was higher in the patients
compared to the controls which also significantly differed
between the two groups (geometric mean and 95%CI for
patients and control groups were 6.2 mg/dl and 5.7–6.7
mg/dl, and 4.9 and 4.7–5.0 mg/dl, respectively; P < .001 by
Figure 1 shows the concentrations of the individual
amino acids for the two groups. Significant differences
between patients and controls were found for the plasma
levels of Ala, Gly Ile, Leu, Met, Phe, Pro, Ser, Trp, and
Val (P < .001 to .05). Compared to the corresponding
values in the control subjects, the concentrations of Ala,
Ile, Leu, Met, Phe, Pro, Trp and Val were significantly
higher in the plasma samples of the patients with gout;
in contrast, the concentrations of Gly and Ser were
significantly lower in the patients.
To identify the association between the selected amino
acid levels and gout, we performed logistic regression
analysis without and with adjustment for the demographic
factors that significantly differed between the patient and
control groups (as in table 1). Before adjustment, all the
amino acids that significantly differed between the two
groups showed their association with gout. When the
logistic regression model was adjusted for 3 demographic
factors (BMI, smoking status, and alcohol consumption),
all the amino acids retained their association with gout
except for Met and Pro. Among them, plasma levels of Ala,
Ile, Leu, Phe, Trp and Val had significant positive
associations [OR between 1.50 and 1.78; 95% CI between 1.02
and 1.25 (lower) and 2.20 to 2.53 (upper); P < .005 to .05)],
whereas Gly (OR 0.60; 95% CI 0.38 to 0.94; P < .05) and
Ser (OR 0.67; 95% CI 0.48 to 0.95; P < .05) had significant
inverse association with gout (Table 2). However, when
this model was further adjusted for gender, the association
was just significant for plasma levels of Ile (P = .049) and
Ser (P = .048) and remarkable for Gly (P = .061) and Phe
(P = .050) (Table 2).
Fig. 1 Comparison between gout patients (n = 34) and control subjects (n = 784; 1 missing value) for individual amino acids: a, amino acids with
mean concentration <90 μmol/L; b, amino acids with mean concentration between 90 and 150 μmol/L; c, amino acids with mean concentration
>150 μmol/L. Values are shown as geometric mean and 95% CI. Significantly different from the corresponding control value: *P < .05, **P < .005
and ***P < .001
Val 1.92 1.36 2.72 <.001 1.50 1.02
*Bold P-values indicate statistically significant level of association with CI not including 1
a Model I: without adjustment
bModel II: adjusted for BMI, smoking status, and alcohol consumption
cModel III: adjusted for gender, BMI, smoking status, and alcohol consumption
dRounded to 1
Plasma free amino acids play important physiological
roles in the biosynthesis and catabolism of various
metabolites and regulators of many metabolic pathways
. A disease state causes specific metabolic changes
and subsequent alterations in PFAA profiles in the
human body. In this study, we measured the plasma
concentrations of 19 amino acids by using the advanced
HPLC-ESI-MS technique and investigated the possible
association between PFAAs and gout that has not been
As revealed in our study, there were significant
elevations in the levels of ALT, γ-GTP and TG levels, and a
significantly lower HDLC level in patients, compared
with the control subjects. These findings are in line with
the existing literature as gout is associated with
increased levels of uric acid in the blood, and
hyperuricemic men and women have shown the coexistence of
hypertriglyceridemia, hypercholesterolemia, and
hypoHDLC ; and an increased level γ-GTP might be
associated with the latter . In a recent study, Chen
et al. found a significant association between
hyperuricemia and ALT elevation . The authors postulated that
an increased oxidative stress in hyperuricemia might be
the underlying cause for such an elevation in ALT levels;
moreover, it may also stimulate the synthesis of γ-GTP
in hyperuricemic subjects [23, 24].
In this study, we observed significantly different
patterns in the levels of a number of amino acid between
the patients with gout and control subjects. Our results
correspond to the findings from the previous research
works, in which a number of amino acids in serum or
plasma of gout patients and control subjects have been
measured and compared which indicated varying
patterns of amino acid concentrations among the
participants. Our findings are consistent with those of Kaplan
et al. , who found significantly elevated levels of
serum Ala, Ile and Leu, Val, Tyr, Phe and Lys in patients
with gout. In a study, Yü et al.  compared PFAA
concentrations in male patients with primary gout with
those of control males, and found significant (P < .05 to
.01) increases in Ala and Ile in the patients as also
observed in our study. Similarly, in another study including
a small number of subjects (7 patients with primary gout
and 6 control males), Yü et al.  observed a
significantly (P = 0.01) elevated level of only Ile and a
remarkable (P = 0.05) increase in Leu in the patients.
In this study, compared to the control subjects, we
observed lower levels of Gly and Ser among the
patients with gout, which is also supported by the study
of Yü et al. , who found significant (P < .05 to
.01) decreases in the mean concentrations of Gly and
Ser among such patients. However, in the other study
by Yü et al. , the level of depression in Gly was
just significant (P = 0.05). On the other hand, Kaplan
et al.  observed a significant increase in serum
Gly level among such patients. Similar to the
observation in our study, the concentration of plasma Gln
was found to be normal among the patients in a
study conducted by Pagliara and Goodman . In
contrast to the findings of all the above-mentioned
studies, Derrick and Hanley  did not reveal any
significant differences in amino acid profiles between
gout patients and normal control subjects.
The overall findings for the trend of differences in
various amino acid levels between gout patients and
control subjects were more or less similar across studies
except the study of Kaplan et al.  for the level of
serum Gly. However, considering the relevant changes in
amino acid concentrations in different studies including
the current one, we postulate that the amino acid profile
is altered in gout causing an elevation in a number of
amino acid concentrations and a depression in others.
Gly and Ser play important roles in the biosynthesis of
purine; they are the precursors of uric acid. Lower
concentrations of these amino acids in patients with gout is
probably due to the fact that they donate either amide
nitrogen or carbon or both to the purine ring which are
utilized in increasing amounts for the formation of uric
acid in gout . Furthermore, there is a possibility that
the alterations in plasma amino acid balance caused by
deficiency of particular amino acids in the plasma due to
their involvement in purine biosynthesis affect the
plasma amino acid profile and lead to the increased level
of some other amino acids as observed in our study.
Plasma amino acids are highly correlated with each
other. Armstrong and Stave  in their study
observed high interrelations among a large group of
amino acids in healthy children and adults. Also, in
this study, we observed significant correlations
between the levels of selective amino acids in both
patient and control groups (results not shown).
Therefore, instead of multivariate analysis, we
performed univariate logistic regression analysis to
explore the association between selective individual
amino acids with gout. The logistic regression model
with adjustment for BMI, smoking status and alcohol
consumption revealed both positive and negative
association between specific amino acids and gout. As
those amino acids lost their significance or some
remained marginally significant for the association
with gout after further adjustment of the model for
the variable gender, this might have been caused by
the fact that a small number of female patients with
gout could be included in this study. Nonetheless, as
we believe, the role of those amino acids in gout
should not be underestimated as they showed highly
significant differences between the patient and control
groups in this study and also in a number of studies
as discussed earlier. However, direct comparison of
the current findings for the association between
amino acids and gout with those of other research
works is not possible currently as such an association
was not investigated in the previous studies. In our
study, among the group of amino acids showing
positive relations with gout, Ile, Leu and Val are branched
chain amino acids, and Phe and Trp are aromatic
amino acids. PFAA, especially branched chain amino
acids and aromatic amino acids are thought to be
associated with lifestyle-related diseases . Therefore,
the association between lifestyle-related diseases and
gout needs to be explored in future research works
including a lager sample of patients with gouts.
Limitations to the study findings
The present findings should be interpreted in the light
of several possible study limitations. Firstly, both
patients and controls included in this study were mainly of
older age groups, and thus the generalizability of the
current study findings is uncertain among younger
populations. Secondly, a good percentage of the included
subjects in both groups were under medications for
diseases like hypertension, diabetes mellitus and
dyslipidemia. There is a possibility that the results were
confounded by those factors. However, we believe, this
had little impact on the study results as the groups did
not differ largely with respect to those characteristics.
Thirdly, the small number of included subjects with gout
is also a limitation of the present investigation. However,
this was the natural outcome of the annual health
examination data used in this study. Fourthly, we could not
investigate the relationship of plasma uric acid level with
amino acid levels in the study populations due to a
limited number of available data on it. Lastly, the present
study did not examine the mechanisms involved in the
responses in amino acid profile caused by gout.
Therefore, the exact reasons for the observed differences
between the studies or the underlying mechanisms cannot
be determined from the study findings.
Taking all the findings of this study together, we
conclude that significant alterations in plasma amino acid
profile occurred in gout. Plasma levels of Ala, Ile, Leu,
Phe, Trp, and Val had significant positive associations
whereas Gly and Ser had significant inverse association
with gout. The observed changes in PFAA profiles may
have important implications for improving our
understanding of pathophysiology and prevention of gout.
Also, the specific variations in PFAA profiles and their
association with gout as observed in our study might be
useful in the diagnosis of this disease. The findings of
this study need further confirmation in future large-scale
studies involving a larger number of patients with gout.
The authors would like to express their appreciation to the participants of
this study and the staffs of all organizations cooperating in this project.
TT designed the study after discussion with MHM, NY, HT and RH. TT, HK and YF
performed initial selection of subjects. TT, NY, HA, MMK, HK and YF participated in
coordination and collection of blood samples and other data. HY, MY, SK, AI, NK
and MN provided essential materials and supported in the analysis of blood
samples. The plan for statistical analysis was designed by MHM, NY, HT, YI
and HS. MHM also conducted the final statistical analyses of data and
wrote the preliminary draft of the manuscript. All authors contributed in
the interpretation of the findings and critical revision of the manuscript.
Also, all authors read and approved the final manuscript.
HY, MY, SK, AI, NK, MN are employees of Ajinomoto Co., Inc. TT, HA, and YF
received research grants from Ajinomoto Co., Inc. This does not alter the
authors’ adherences to all of the journal policies. The authors declare that
they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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