Genes Nutr (2014) 9:417

DOI 10.1007/s12263-014-0417-3

RESEARCH PAPER

Blood cells transcriptomics as source of potential biomarkers

of articular health improvement: effects of oral intake of a rooster
combs extract rich in hyaluronic acid
Juana Sánchez • M. Luisa Bonet • Jaap Keijer • Evert M. van Schothorst •

Ingrid Mölller • Carles Chetrit • Daniel Martinez-Puig • Andreu Palou

Received: 26 February 2014 / Accepted: 2 July 2014 / Published online: 15 July 2014
 Springer-Verlag Berlin Heidelberg 2014

Abstract The aim of the study was to explore periph- and improved knee muscular strength indicators as
eral blood gene expression as a source of biomarkers of compared to placebo. About 157 coding genes were
joint health improvement related to glycosaminoglycan differentially expressed in blood cells between supple-
(GAG) intake in humans. Healthy individuals with joint mented and placebo groups post-intervention, but not
discomfort were enrolled in a randomized, double-blind, pre-intervention (p \ 0.05; fold change C1.2). Among
placebo-controlled intervention study in humans. Sub- them, a reduced gene expression of glucuronidase-beta
jects ate control yoghurt or yoghurt supplemented with a (GUSB), matrix metallopeptidase 23B (MMP23B), xy-
recently authorized novel food in Europe containing losyltransferase II (XYLT2), and heparan sulfate 6-O-
hyaluronic acid (65 %) from rooster comb (MobileeTM sulfotransferase 1 (HS6ST1) was found in the supple-
as commercial name) for 90 days. Effects on functional mented group. Correlation analysis indicated a direct
quality-of-life parameters related to joint health were relationship between blood cell gene expression of
assessed. Whole-genome microarray analysis of periph- MMP23B, involved in the breakdown of the extracellular
eral blood samples from a subset of 20 subjects (10 matrix, and pain intensity, and an inverse relationship
placebo and 10 supplemented) collected pre- and post- between blood cell gene expression of HS6ST1,
intervention was performed. MobileeTM supplementation responsible for 6-O-sulfation of heparan sulfate, and
reduced articular pain intensity and synovial effusion indicators of knee muscular strength. Expression levels
of specific genes in blood cells, in particular genes
related to GAG metabolism and extracellular matrix
Electronic supplementary material The online version of this dynamics, are potential biomarkers of beneficial effects
article (doi:10.1007/s12263-014-0417-3) contains supplementary on articular health.
material, which is available to authorized users.

J. Sánchez  M. L. Bonet  A. Palou (&) Keywords Blood cells  Transcriptomics  Joint

Laboratory of Molecular Biology, Nutrition and Biotechnology discomfort  Glycosaminoglycans  MobileeTM
(Nutrigenomics), University of the Balearic Islands and CIBER
Fisiopatologı́a de la Obesidad y Nutrición (CIBEROBN), Edifici
Mateu Orfila. Carretera de Valldemossa Km 7.5,
07122 Palma de Mallorca, Spain Introduction
e-mail: [email protected]
Degenerative joint disease (osteoarthritis, OA) is an
J. Keijer  E. M. van Schothorst
important, highly prevalent health problem (Goldring and
Department of Human and Animal Physiology, Wageningen
University, Wageningen, The Netherlands Goldring 2007; Das and Farooqi 2008; Bonet et al. 2011).
Osteoarthritis (OA) is a heterogeneous disease character-
I. Mölller ized by the degradation of articular cartilage, subchondral
Instituto Poal de Reumatologia, Barcelona, Spain
bone alterations, and inflammation of the synovial mem-
C. Chetrit  D. Martinez-Puig brane (Goldring and Goldring 2007; Das and Farooqi
Bioiberica S.A., Palafolls, Barcelona, Spain 2008). Osteoarthritis (OA) is a chronic and slowly

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progressive disease. However, prior to the onset of OA, Materials and methods
there is a preclinical condition characterized by joint dis-
comfort in which no structural lesions are apparent that Subjects
may or may not progress to OA (Das and Farooqi 2008).
Intervention at this stage appears to be of great importance Inclusion criteria
as a target for functional health claims made on food.
The glycosaminoglycan (GAG) hyaluronic acid (HA) • Adults of both sexes aged between 20 and 70.
is used in the therapeutics of OA, mostly following intra- • Mild joint discomfort (visual analog scale (VAS)
articular injection (Dougados et al. 1993; Altman and between 3 and 5 cm).
Moskowitz 1998). Recently, however, beneficial effect of • Symptomatic joint discomfort for a minimum of
HA following oral administration has been also demon- 6 months.
strated (Tashiro et al. 2012; Martinez-Puig et al. 2013). In
fact, there is evidence that high-molecular-weight HA is
Exclusion criteria
absorbed and distributed to connective tissues after its
oral administration (Balogh et al. 2008). In addition, oral
• Subjects who have clinical OA or any other degenera-
supplementation with HA (as MobileeTM formulation)
tive joint disease.
reduced the degree of synovial effusion and increased the
• Subjects receiving treatment with anti-inflammatory or
concentration of HA in synovial fluid in horses diagnosed
chondroprotective drugs (chondroitin sulfate, glucosa-
with osteochondritis (Martı́nez-Puig et al. 2007). More-
mine, HA, diacerein) 2 weeks before the selection.
over, a placebo-controlled nutritional study confirmed in
• Subjects receiving intra-articular injections of the knee
humans that 3-month oral administration of HA (as Mo-
joint in the 3 months prior to the study.
bileeTM) provides improvement in knee muscle strength
• Subjects with cardiovascular, hepatic, renal, respira-
in individuals with mild joint discomfort (Martinez-Puig
tory, or hematologic illness, or other medical or
et al. 2013).
psychiatric condition that, in the opinion of the
Despite the increasing prevalence of OA, early and
investigator, would compromise participation or be
readily accessible biomarkers that can be used in disease or
likely to lead to hospitalization during the course of the
pre-disease states prognosis and monitoring and/or as tools
study.
to evaluate the responsiveness to treatments are still lack-
ing. This is an active area of research, and several potential
biomarkers are currently at various stages of qualification Study design
and validation, including circulating or urinary levels of
certain OA-related proteins or protein-degradation products The study was a double-blind, prospective nutritional
such as cartilage oligomeric matrix protein (COMP), intervention study carried out in accordance with the
C-terminal cross-linked type I or type II collagen (CTX-I principles of the Declaration of Helsinki. All the partici-
and CTX-II), type 2 Coll2-1, myeloperoxidase, or matrix pants gave their informed consent for the study, and the
metalloproteinase-3, among others (reviewed in (Kraus Fundació Gol i Gurina Ethical Review Committee
et al. 2011; Rousseau and Garnero 2012; Ramonda et al. approved the study protocol.
2013)). Between September 2009 and March 2010, subjects
Blood cells are increasingly being investigated as a met all inclusion criteria and none of the exclusion criteria
noninvasive source of biomarkers of health/disease, were randomly assigned to one of two experimental
nutritional exposure, and response to therapy. In fact, the groups. The supplemented group ate one yoghurt supple-
expression levels of specific genes in blood cells have mented with 80 mg of MobileeTM (Bioiberica S.A., Pala-
been proposed as markers of body metabolic status, folls, Spain) per day for a period of 90 days. The stability
potentially providing an early warning of future disorders of HA in the yoghurt matrix for a period of 4 weeks was
(Sanchez et al. 2012). However, its potential relevance in assessed before the beginning of the study. The control
the area of preclinical articular health or comfort has not group ate one yoghurt per day without any supplement.
been explored so far. Here, we aimed to explore the fea- Subjects were told not to take analgesics or nonsteroidal
sibility of using total human blood RNA as a source of anti-inflammatory drugs. Only paracetamol (500 mg) was
biomarkers of articular health improvement related to allowed as a pain reliever. Physical therapy was not carried
MobileeTM intake. MobileeTM is a rooster comb extract out during the study period. The subjects that fulfilled the
containing HA (65 %), polysaccharides, and collagen, inclusion criteria without presenting any exclusion criteria
recently authorized as a novel food in Europe (EFSA were entered into a randomization scheme for the admin-
2013). istration of supplemented or unsupplemented yoghurt.

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Subjects were assigned to treatment by a sequential ran- vacutainer tubes (Qiagen, Izasa-Barcelona, Spain) via
domization schedule in blocks of 4 following the confir- antecubital fossa venipuncture. PAXgene tubes contain a
mation of eligibility, before study yoghurts were provided. proprietary reagent that immediately stabilizes RNA, thus
Randomization was performed and maintained by an reducing RNA degradation and minimizing changes in
independent statistician. The study yoghurts were provided gene expression following phlebotomy (Rainen et al.
in kits containing a randomization code. The sponsor, the 2002). After the blood was drawn, the tubes were inverted
investigator, and all study staff having a role in the day-to- 20 times, allowed to sit for 2 h at room temperature, and
day conduct of the study remained blinded to treatment. then stored at -70 C until processed. Total RNA was
isolated using the PAXgene blood RNA kit according to
Clinical assessment the manufacturer’s instructions (Qiagen). The RNA that is
isolated with this protocol comes from all cells in whole
Clinical assessment was documented by the same investi- blood. RNA quality and purity were analyzed by spectro-
gator for every subject during the whole study. Clinical photometry using the Nanodrop ND-1000 (NanoDrop
assessment included the following criteria: Techonologies, Inc., Wilmington, DE), and its integrity
was measured on an ExperionTM system using RNA Std-
• Primary efficacy assessment: isokinetic test. The evaluation
Sens chips (Bio-Rad, Madrid, Spain) just before microarray
was carried out with a Biodex system 3 Pro Isokinetic
processing.
dynamometer (Biodex Medical Systems, New York, USA)
using five repetitions at two angular velocities (180/s and
Microarray processing
240/s). The subject assumed a sitting position with the
hips flexed at 90. The degree of freedom of the knee was
Whole-genome microarray analysis of blood samples from
restricted to extension/flexion of 0–0–90. A break of
a subset of 20 subjects (11 placebo and 9 supplemented)
2 min was allowed between sets. Based on the data
collected pre- and post-intervention was conducted. Sam-
retrieved from all the sets, the maximum work load (J),
ples were randomly selected ensuring an equal distribution
maximum peak torque (Nm), and power (W) at 180/s and
between groups and that, for each subject, paired pre- and
240/s were determined. The maximum peak torque (Nm)
post-intervention mRNA samples of high quality were
was defined as the maximum force produced by the tested
available (amount, purity, and integrity). For microarray
musculature at the two different angular velocities.
hybridization, 1 lg of RNA from each sample was reverse-
• Secondary efficacy assessments: ultrasonographic eval-
transcribed to complementary DNA (cDNA) using the
uation of the knee. A rheumatologist (medical special-
Agilent Quick Amp Labeling kit (Agilent Technologies,
ist) performed ultrasonography of the study knee
Inc., CA, USA), according to the manufacturer’s protocol.
according to the prespecified ultrasonography parame-
Thereafter, cDNA samples were split into 2 equal amounts,
ters (details in (D’Agostino et al. 2005)). In addition,
to synthesize Cyanine 3-CTP (Cy3)- and Cyanine 5-CTP
pain was assessed using the VAS.
(Cy5)-labeled cRNA as described previously (van Scho-
• Other secondary efficacy parameters include overall
thorst et al. 2007). Transcription and labeling were carried
quality-of-life assessment with subscales using the SF-
out at 40 C for 2 h. Labeled cRNA was purified using
36 survey (McHorney et al. 1993).
Qiagen Rneasy MiniSpin columns (Qiagen, Venlo, the
The clinical data, pain assessment using the VAS, and Netherlands). The incorporation of dyes and cRNA
inclusion/exclusion criteria were obtained at the selection concentration was measured using the ‘‘microarray mea-
visit. The selected participants were randomly distributed surement mode’’ of the NanoDrop ND 1000 spectropho-
and given the isokinetic test, US evaluation, and SF-36 tometer. About 2,500 ng of every Cy3-labeled cRNA
survey at the inclusion visit, 1 day prior to the consumption sample (with a yield C825 ng and activity C6.0 pmol per
of the first yoghurt. The US evaluation, pain assessment, and lg cRNA) was pooled, and the pool was used as a common
SF-36 survey were repeated in weeks 4 and 8 after the start internal reference. One of the samples (from placebo pre-
of the nutritional intervention period. At the end of the study intervention group) did not reach the standard quality
(12 weeks), a complete clinical assessment was repeated, parameters at this stage and was excluded. Thus, hybrid-
including a global subject-satisfaction assessment. ization was conducted with a total number of 39 samples.
Individual samples containing 825 ng of Cy5-labeled
Blood sampling and processing for gene expression cRNA and 825 ng of the Cy3-labeled cRNA pool were
analysis hybridized on 4x44 K G4845A human whole-genome
Agilent microarrays (Agilent Technologies, Inc., Santa
For each participant, a total of 2.5 mL peripheral blood Clara, CA, USA) for 17 h at 65 C in hybridization
was collected under fasting conditions into PAXgene chambers in an oven rotating at 10 rpm (Agilent

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Technologies). After hybridization, the arrays were washed RT-qPCR in blood cells. Quantitative RT-PCR was per-
with ‘‘GE wash buffer 2’’ for 1 min at 37 C, followed by formed as previously described (Sanchez et al. 2009). The
acetonitrile for 10 s at room temperature, and finally with a threshold cycle (Ct) was calculated using the instrument’s
solution for stabilization and drying for 30 s at room software (StepOne Software version 2.2), and the relative
temperature, according to the manufacturer’s protocol expression ratio of a target gene was calculated based on
(Agilent Technologies). the corresponding real-time PCR efficiency and Ct devia-
tion of an unknown sample versus mean Ct of all samples
Microarray data analysis and expressed in comparison with a reference gene (Pfaffl
2001). Ribosomal protein, large, P0 (RPLPO) was chosen
The arrays were scanned with an Agilent Microarray as reference gene because it has been validated for human
Scanner (Agilent Technologies). Scanned images were studies (Dheda et al. 2004; Sanchez et al. 2012) and
examined for visible defects and proper grid alignment. The appeared to be stable expressed by microarray analysis. All
intensities of the signals from each spot were quantified, and primers were obtained from Sigma Genosys (Sigma-
the raw data were extracted using Feature Extraction Soft- Aldrich Quimica SA, Madrid, Spain). Sequences of the
ware version 10.5.1.1 (Agilent Technologies). Quality primers used were as follows: GUSB, forward: 50 -CCAA
control was performed for each of the arrays using Lim- GAGCCAGTTCCTCATC-30 , reverse: 50 -GGTAGTGGCT
maGUI package in R from Bioconductor Software version GGTACGGAAA-30 ; MMP23B, forward: 50 -GCCTGATG
2.1. All the arrays passed quality control based on MA plot CACTCACAACAC-30 , reverse: 50 -GAAGGGGAATTCG
and signal intensity distribution (Allison et al. 2006). Thus, TAGCAGA-30 ; XYLT2, forward: 50 -CAGCCTCCTGAGG
in total, dataset from 39 arrays passed to the next step of ATGTACC-30 , reverse: 50 -TTGTCCCGGTTCTTGGAT
analysis. Data were exported into GeneMaths XT 2.12 AG-30 ; HS6ST1, forward: 50 -CAGCAGCGCTACCAGTA
(Applied Maths, Sint-Martens-Latem, Belgium) for back- CAA-30 , reverse: 50 -GGACCTGTCGTCTGTCCTGT-30 ;
ground correction and normalization. Locally weighted RPLPO, forward: 50 -ACAACCCAGCTCTGGAGAAA-30 ,
linear regression (lowess) analysis was chosen as a nor- reverse: 50 -TGCCCCTGGAGATTTTAGTG-30 .
malization method, which enables intensity-dependent
effects in the log2 (ratio) values to be removed (Yang et al. Statistical analysis
2002). Then, the values were converted to log2 values, and
the target samples (Cy5) intensities were normalized against Descriptive results were expressed as mean ± standard
the intensities of reference samples (Cy3), as described deviation (SD) or percentages, according to the variable
previously (Pellis et al. 2003). To search for biomarkers in being measured. To compare the effects of the two products
articular health, single t test comparisons were performed. (test and placebo) on the efficacy of the principal variable,
We focused only on those genes differentially expressed in as well as on the main secondary efficacy variables, an
peripheral blood between supplemented and placebo groups analysis of covariance was performed (ANCOVA) with the
post-intervention, without pre-intervention differences. The baseline value as covariate. For the rest of efficacy vari-
threshold of significance for this statistical test was set at ables, hypotheses were tested using Fisher’s exact test for
p B 0.05. Fold change (FC) calculation between both categorical variables, the Student’s t test for continuous
groups (supplemented vs. placebo subjects) was performed variables, and Mann–Whitney U test for ordinal variables.
by exponential conversion of the signal log ratio; FC equals Pearson’s correlation coefficient was used to determine the
the gene expression ratio between supplemented and pla- association between the expression pattern of selected
cebo in the case of increase and -1/this ratio, in the case of genes in peripheral blood and (1) the degree of synovial
decrease. Subsequently, a computer-generated list of genes effusion; (2) pain; and (3) indicators of muscular strength of
(p \ 0.05 and absolute fold change C1.2) was manually the affected knee, namely maximum work load, maximum
analyzed for their biological information, obtained with the peak torque, and power. The significance level was fixed at
use of available databases (NCBI-Gene and Medline-, p = 0.05. The SAS 6.10 statistics program (SAS Institute,
Genecards and UniProt) based on key biological domains, Inc., Cary, NC, USA) was used for data analysis. The sta-
such as molecular function and biological process. tistical analysis of microarray data has been described in
detail in the section referred to microarray data analysis.
Real-time quantitative RT-PCR analysis

To validate microarray data, mRNA expression levels of Results

glucuronidase-beta (GUSB), matrix metallopeptidase 23B
(MMP23B), xylosyltransferase II (XYLT2), and heparan Seventy-seven participants were included in the study.
sulfate 6-O-sulfotransferase 1 (HS6ST1) were measured by Nine were excluded from the intended-to-treat population

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Screened
n=77

Randomized
n=77

Placebo Mobilee
n=38 n=39

Safety: 38 Safety: 39 Total: 77

Excluded (n=4) Excluded (n=5)

Not last main efficacy Not last main efficacy
measurement measurement

ITT: 34 ITT: 34 Total: 68

Excluded (n=1) Excluded (n=1)

Not allowed concomitant Not allowed concomitant
medication medication

PP: 33 PP: 33 Total: 66

Fig. 1 Disposition of patients and reasons for exclusion

due to the lack of compliance, and two were excluded from 32.5 ± 4.96 cm; p = 0.005) and specially at the third month
protocol population due to protocol violation (Fig. 1). The (31.9 ± 15.81 vs. 21.1 ± 12.36 cm; p = 0.0005) (Fig. 2).
average age was documented as 54.8 ± 8.56 years. There The ultrasonographic assessment revealed a significant
were no significant changes in body weight or clinical reduction in the degree of synovial effusion associated with
parameters (pulse rate, blood pressure) after eating non- the eating of yoghurts supplemented with MobileeTM as
supplemented or supplemented yoghurt (Table 1). Only compared to placebo. The total number of participants with
one participant per group took paracetamol during the a non-physiologic degree of synovial effusion ([4 mm) was
whole study period, which is both low and nonsignificantly reduced in both groups after 3 months of supplementation.
different. However, the average reduction was of 44 % for the Mo-
The daily eating of yoghurt supplemented with 80 mg of bileeTM group, and 22 % for the placebo group, resulting in
MobileeTM significantly reduced pain intensity as com- significant between-group differences (p \ 0.05).
pared to the eating of yoghurts supplemented with placebo As shown in Fig. 3, the mean reduction in the synovial
from the second month of treatment (34.0 ± 3.85 vs. effusion after 3 months of supplementation was double in

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Table 1 Basal characteristics of intended-to-treat population (ITT) 0

population (mean ± SD)
Placebo MobileeTM p value -0.5
(n = 34) (n = 34)

Gender (male/female) 8/26 13/21 0.195 -1

synovial effusion (mm)

Age 53.6 ± 8.94 56.1 ± 8.08 0.458
Muscular strength of the 64.1 ± 24.83 65.4 ± 27.75 0.838 -1.5
affected knee (Nm) (peak
torque, extension at 240/
s) -2

Degree of synovial effusion 3.7 ± 2.15 4.2 ± 2.3 0.285

(mm) -2.5
Pain intensity (cm) 39.6 ± 4.86 42.3 ± 5.67 0.414
Quality-of-life SF-36 Placebo
-3
Mobilee
Physic component (PCS- 42.6 ± 10.26 41.2 ± 9.34 0.798
36)
Mental component 44.9 ± 12.79 50.1 ± 8.12 0.194
-3.5 *
(MCS-36)
Fig. 3 Degree of synovial effusion after 3 months compared to basal
values (mean ± SD). Statistics: *p \ 0.05
0
0 1 2 3

MobileeTM had an increased level of synovial fluid

-5 ([4 mm in the suprapatellar recess). The excess of syno-
vial fluid could produce pain and therefore interfere with
isokinetic assessment of the muscular strength. Sub-ana-
pain VAS (cm)

-10
lysis of the muscular strength evolution excluding those
participants with a non-physiologic degree of synovial
Placebo **
-15 fluid at baseline indicated differences between groups
Mobilee
(p \ 0.05). In the placebo group, muscular strength
was reduced after 3 months of supplementation (-2.3 ±
-20
2.71 Nm), while in the MobileeTM group, it was increased
(2.9 ± 1.67 Nm) (Table 3).
-25
months ***
Microarray results
Fig. 2 Time evolution of the pain intensity (VAS). Results are
expressed as difference from baseline (mean ± SEM). Statistics: Of the 43,118 probes tested on the array, 24,640 had an
**p \ 0.01; ***p \ 0.001 expression value twice above the background and were
further taken into account. Of these, 640 probes were
the MobileeTM group than in the placebo group (-2.7 ± differentially expressed between supplemented and pla-
0.42 vs. -1.1 ± 0.54 mm; p = 0.041). cebo groups post-intervention, but not pre-intervention
No differences among SF-36 subscale scores were (p \ 0.05; Student’s t test). We further restricted the ana-
detected during the study or between treatment groups lysis to the differentially expressed probes (p \ 0.05) with
(Table 2). The primary analysis of isokinetic evaluation of an absolute fold change C1.2, resulting in a total of 264
muscular strength of the affected joint in extended position, probes representing 206 unique genes. Of these, 49 corre-
applying an angle speed of 2408 per second (absolute sponded to predicted genes with unknown function (43) or
change from baseline), is detailed in Table 2. No statistical to noncoding RNA (6) and 157 corresponded to known
difference was observed after the intervention. The average protein coding genes. Among the latter, 57 genes exhibited
value increased 1.435 Nm for the MobileeTM group and up-regulation and 100 down-regulation in the supple-
1.109 for the placebo (p = 0.9064). However, the analysis mented group as compared to the placebo group (Supple-
of subpopulations revealed that evolution of muscular mentary table 1). The fold change in these genes ranged up
strength could be interfered by the basal degree of synovial to 1.96, an expected small effect since nutritional studies
effusion on the affected knee. At baseline, 53 % of the within the physiological range in general result in small
participants in the placebo group and 47 % in the differences in transcript levels (Keijer et al. 2010). The 157

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Table 2 Results of the efficacy Parameter Basal 1 month 2 month 3 month

parameters registered on the
intended-to-treat (ITT) Muscular strength (Nm)
population (mean ± SD,
Peak torque, extension at 240/s
n = 34 in each group)
Placebo 64.1 ± 24.83 65.3 ± 23.73
MobileeTM 65.4 ± 27.75 66.7 ± 27.12
p value 0.906
Synovial effusion (mm)
Placebo 3.8 ± 2.15 2.7 ± 2.80
MobileeTM 4.2 ± 2.3 1.6 ± 1.51
p value 0.029
Pain VAS (cm)
Placebo 39.6 ± 4.86 36.1 ± 3.96 34.0 ± 3.85 31.9 ± 15.81
MobileeTM 42.3 ± 5.67 36.7 ± 5.99 32.5 ± 4.96 21.1 ± 12.36
p value 0.278 0.005 <0.001
Quality-of-life SF-36
Physical component (PCS-36)
Placebo 42.6 ± 10.26 44.5 ± 10.95 44.8 ± 10.56 44.0 ± 10.62
TM
Mobilee 41.2 ± 9.34 42.9 ± 8.67 43.4 ± 9.13 45.5 ± 9.05
p value 0.727 0.824 0.093
Mental component (MCS-36)
Placebo 44.9 ± 12.79 47.5 ± 11.82 47.5 ± 12.69 49.5 ± 10.79
MobileeTM 50.1 ± 8.12 50.4 ± 9.59 49.5 ± 9.97 50.2 ± 10.68
p values \ 0.05 are highlighted p value 0.881 0.876 0.295
in bold

Table 3 Absolute values of muscular strength (peak torque) in blood gene expression of MMP23B and XYLT2 following
extension at an angular velocity of 240/s of the participants without MobileeTM intervention compared to placebo (Fig. 5); a
synovial effusion at baseline (mean ± SD, n = 34 in each group) similar trend was evidenced for GUSB gene expression.
Muscular strength (peak torque, Basal 3 months
extension at 240/s)
Association studies of gene expression levels in blood
Placebo 57.13 ± 4.51 54.83 ± 3.25
TM
cells with articular health-related parameters
Mobilee 63.53 ± 7.82 66.46 ± 7.46
p value 0.0324
Pearson’s correlations were calculated to determine the
p values \ 0.05 are highlighted in bold relationship between the expression levels in blood cells of
identified genes related to GAG metabolism and extracel-
protein-encoding genes were manually annotated to bio- lular matrix dynamics possibly affected by MobileeTM
logical processes according to their function. As shown in intervention (GUSB, HS6ST1, MMP23B, XYLT2) and artic-
Fig. 4, the processes with the highest number of differen- ular health-related parameters (Table 5). The expression
tially expressed genes were related to cell signaling, cell levels in blood cells of HS6ST1 and MMP23B showed a
turnover, immune system, transcription and translation, and strong positive correlation with pain sensation, so that the
transport. Strikingly, among the differentially expressed higher the expression of these two genes, the higher the pain,
genes, there were 4 genes for proteins well known to be and vice versa (r = 0.356, p \ 0.05 for HS6ST1; and
related to GAG metabolism and extracellular matrix r = 0.522, p \ 0.001 for MMP23B). Additionally, HS6ST1
dynamics (Table 4). In particular, a lower peripheral blood expression was negatively correlated with all the muscular
gene expression of matrix metallopeptidase 23B strength indicators studied in the affected knee, namely peak
(MMP23B), glucuronidase-beta (GUSB), xylosyltransfer- torque, total work, and mean power at 240/s (r = - 0.368,
ase II (XYLT2), and heparan sulfate 6-O-sulfotransferase 1 -0.351, and -0.388, respectively, p \ 0.05), so that the
(HS6ST1) was found in the supplemented group post- lower the HS6ST1 expression, the higher these muscular
intervention compared to the placebo group. Additionally, strength indicators. No significant correlation between
RT-qPCR analysis on paired samples pre- and post-inter- peripheral blood expression of prioritised genes and synovial
vention indicated a significant reduction in peripheral effusion values was detected (data not shown). Pearson’s

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Fig. 4 Classification into number of genes

biological processes of genes 0 5 10 15 20 25 30 35
that significantly change in
MobileeTM supplemented Cell signaling
versus control group post- Cell turnover
intervention but not pre-
Immune system
intervention
Transciption and Translation
Other, various
Transport
Lipid and CH metabolism
Membrane
Nervous System
Cytoskeleton
Up Regulated
Protein Metabolism Down regulated
Redox Homeostasis
Gycosamingycan metabolism and ECM
RNA Metabolism

Table 4 Genes related to GAG metabolism and extracellular matrix dynamics differentially expressed between MobileeTM and placebo groups
post-intervention (p \ 0.05), but not pre-intervention
Gene Name Systematic Function Fold change MobileeTM vs. p value
name placebo

GUSB Glucuronidase-beta NM_000181 Hydrolase that degradates GAG -1.22 0.027

HS6ST1 Heparan sulfate 6-O- NM_004807 Enzyme responsible for 6-O-sulfation -1.22 0.009
sulfotransferase 1 of heparan sulfate
MMP23B Matrix metallopeptidase 23B NM_006983 Protease involved in the breakdown of -1.30 0.028
the extracellular matrix
XYLT2 Xylosyltransferase II NM_022167 Enzyme that initiates the biosynthesis -1.32 0.004
of GAGs including chondroitin
sulfate, heparan sulfate, heparin, and
dermantan sulfate in proteoglycans

correlations gave similar results using either microarray or biomarkers of articular health improvement related to
RT-qPCR gene expression data, further validating them. MobileeTM intake. For this purpose, whole-genome
microarray analysis was performed on blood samples from
a subset of 20 women (OA frequently affects middle-age
Discussion women (Das and Farooqi 2008)), 10 non-supplemented,
and 10 supplemented with MobileeTM. On the basis of their
Healthy individuals with joint discomfort were enrolled in differential expression in MobileeTM and placebo groups
a prospective nutritional randomized, double-blind, pla- post-intervention and of correlations detected with articular
cebo-controlled study. The supplemented group ate one pain and affected muscle strength indicators, we propose
yoghurt containing MobileeTM—a rooster comb extract, gene expression of 4 proteins in GAG metabolism
containing HA (65 %), polysaccharides, and collagen—per and extracellular matrix remodeling—GUSB, HS6ST1,
day for 90 days. Similar to the outcome of a previous study MMP23B, and XYLT2—as potential blood cell transcript-
with another set of volunteers (Martinez-Puig et al. 2013), based biomarkers of beneficial joint responsiveness to the
the daily supplementation with MobileeTM significantly tested oral intervention.
reduced pain intensity (one of the primary symptoms of Blood cells travel through the body and are able to
joint discomfort) and the degree of synovial effusion (a respond to internal and external signals. Studies indicate
clinical finding linked to inflammation) and improved the that blood cells can reflect gene expression signa-
muscular strength parameters compared to placebo. Here, tures characteristic of different nutritional conditions,
we were particularly interested in novel and accessible physiological disorders, or diseases (Caimari et al. 2010;

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Genes Nutr (2014) 9:417 Page 9 of 12 417

Fig. 5 Percentage of change in percentage of change percentage of change

the expression of matrix MMP23B XYLT2
metallopeptidase 23B 10 40
(MMP23B), xylosyltransferase
0
II (XYLT2), glucuronidase-beta 20
-10
(GUSB), and heparan sulfate
-20 Placebo Placebo
6-O-sulfotransferase 1 0
-30 Mobilee Mobilee
(HS6ST1), on paired samples
pre- and post-intervention, -40 -20
analyzed by qRT-PCR, -50
-60
compared to basal values.
Statistics: *p \ 0.05, Student’s
* -40
*
t test percentage of change percentage of change
GUS HS6ST1
30 40

20 30

10 Placebo 20 Placebo
0 Mobilee 10 Mobilee

-10 0

-20 -10

Table 5 Pearson’s correlations between expression levels in blood cells of identified genes related to GAG metabolism and extracellular matrix
dynamics with pain and muscular strength indicators of the affected knee
Gene Name Pain Peak torque at 240/s Total work at 240/s Mean power at 240/s

GUSB Glucuronidase-beta n.s n.s n.s n.s

HS6ST1 Heparan sulfate 6-O-sulfotransferase 1 0.356* -0.368* -0.351* -0.388*
MMP23B Matrix metallopeptidase 23B 0.522*** n.s n.s n.s
XYLT2 Xylosyltransferase II n.s n.s n.s n.s
n.s not significant. * p \ 0.05; *** p \ 0.001

Oliver et al. 2013) and references therein). In particular, strategy toward biomarker discovery (Kraus et al. 2011).
because OA can be regarded as a systemic disorder in So far, transcriptome modification of white blood cells
which circulating blood cells (such as macrophages and T after dietary administration of curcumin and nonsteroidal
cells) accumulate in synovial tissues, subsequently result- anti-inflammatory drug has been assessed in OA affected
ing in cartilage degeneration and alterations of subchondral dogs (Colitti et al. 2012). The present work is, to our
bone (Sakkas and Platsoucas 2007), it is conceivably that, knowledge, first to apply this strategy to OA research or
even in preclinical stages, gene expression in blood cells articular discomfort research in healthy humans.
can both affect and reflect changes occurring in joint tis- At the end of the intervention period, but not prior
sues. In fact, changes in microRNA expression in periph- intervention, subjects in the MobileeTM group presented
eral mononuclear cells according to the progression of OA decreased expression levels of GUSB, HS6ST1, MMP23B,
have been reported (Okuhara et al. 2012), as well as dif- and XYLT2 in blood cells compared to subjects in the
ferences in peripheral blood gene expression signature control, placebo group. Interestingly, we found a strong
between OA cases and controls concerning genes related to correlation between the expression of MMP23B and
apoptotic pathways (Ramos et al. 2013), pro-inflammatory HS6ST1 with pain and indicators of muscular strength. In
cytokine expression (IL-1b) (Attur et al. 2011), and the particular, correlation analysis indicated that the lower the
expression of key bone-regulatory factors (bone morpho- expression of MMP23B and HS6ST1, the lower the pain
genetic proteins and Runx2) (Grcevic et al. 2010). Fur- intensity, and the lower the expression of HS6ST1, the
thermore, changes in peripheral white blood cells gene higher the indicators of knee muscular strength. Thus, it is
expression signature during the course of experimentally suggested that decreased transcript levels of these genes
induced OA have been documented in the horse (Kamm may be indicative of an improvement or an amelioration of
et al. 2013). Investigating biomarkers in the context of joint disease progression associated with MobileeTM
specific OA therapeutic treatments is considered a good consumption.

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Interestingly, the four prioritized genes encode proteins Conflict of interest JS, MLB, JK, EMvS, IM, and AP have no
previously related to joint diseases and specifically to OA conflict of interest, financial or scientific. DMP and CC are employed
by Bioiberica S.A., Palafolls, Barcelona, Spain, the supplier of Mo-
in a manner that suggests that down-regulation of its bileeTM. Bioiberica S.A., Palafolls, Barcelona, Spain, had no role in
expression may be beneficial. GUSB is a lysosomal the scientific interpretation of the results and did not influence the way
enzyme involved in the degradation of GAGs that plays these results were reported.
an essential role in the remodeling of the extracellular
Ethical standard All procedures followed were in accordance with
matrix components in both physiological and inflamma- the principles of the Declaration of Helsinki. All the participants gave
tory states (Naz et al. 2013). Increased GUSB activity their informed consent for the study, and the Fundació Gol i Gurina
(along with that of other glycosidases) is present in the Ethical Review Committee approved the study protocol.
synovial fluid of patients with rheumatoid arthritis (Or-
tutay et al. 2003) and OA (Kamada et al. 1993). HS6ST is
an enzyme involved in heparan sulfate biosynthesis whose
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