Metabolic regulatory variations in rats due to acute cold stress & Tinospora Cordifolia...
Transcript of Metabolic regulatory variations in rats due to acute cold stress & Tinospora Cordifolia...
ORIGINAL ARTICLE
Metabolic regulatory variations in rats due to acute cold stress &Tinospora Cordifolia intervention: high resolution 1H NMRapproach
Sonia Gandhi • M. Memita Devi • Sunil Pal •
Rajendra P. Tripathi • Subash Khushu
Received: 15 March 2011 / Accepted: 7 June 2011 / Published online: 22 June 2011
� Springer Science+Business Media, LLC 2011
Abstract Acute cold stress may trigger systemic bio-
chemical and physiological changes in the living organ-
isms, which leads to rapid loss of homeostasis. These
changes may reverse due to self-regulatory mechanism of
the organism or by the intervention of suitable medication
in the form of herbs. The present study was undertaken to
assess the alterations in metabolites levels arising due to
acute cold stress and to monitor the restoration of these
changes by suitable herb intervention. Male Sprague-
Dawley rats were exposed to acute cold stress of -10�C for
3 h and urine samples were collected and analyzed by
NMR spectroscopy in conjugation with Principal Compo-
nent Analysis (PCA). The study revealed highly significant
biochemical changes in urinary metabolites and also
demonstrated the protective effects of Tinospora Cordifolia
(Tc) extract on the stressed rats. These changes suggest the
involvement of various metabolic pathways such as Tri-
carboxylic Acid (TCA) cycle, gut microbiota, renal func-
tion, catecholamines and muscle metabolism in the
metabolic alterations induced by cold stress and the com-
pensation required to restore homeostasis. The present
study forms the basis of future studies to establish potential
biomarkers for cold stress in humans and lay down the
optimum dosage of Tc to be administered for providing
immunity to the body as prophylactic and mitigating agent
against environmental insult such as cold stress.
Keywords Metabonomics � NMR spectroscopy � Urine �Cold stress � Tinospora Cordifolia � Principal component
analysis
Abbreviations
NMR Nuclear magnetic resonance
PCA Principal component analysis
SD Sprague-Dawley
TCA Tricarboxylic acid cycle
TSP 3-(Trimethylsilyl) propionic-2, 2, 3, 3-d4 acid
sodium salt
GIT Gastrointestinal tract
SNS Sympathetic nervous system
1 Introduction
Exposure to acute stress conditions is responsible for
affecting multiple biochemical regulatory systems and
triggering various disorders (Wang et al. 2009; Epel 2009).
Stress is a combination of events beginning with a stimu-
lus, which precipitates a reaction in the brain and subse-
quently results in the activation of certain physiologic
systems in the body i.e., stress response (Dhabhar et al.
1997). Stressors are responsible for perturbing the well-
balanced metabolism of living organism. Acute stress such
as extreme cold or heat, panic, toxins, tension may result in
development of neuro-psychiatric symptoms such as
depression and cognitive impairment (Pacak et al. 1995;
Joca et al. 2003). Evidence exist for the adverse effects of
S. Gandhi � M. M. Devi � R. P. Tripathi � S. Khushu (&)
Division of Radiological Imaging & Biomedical Engineering,
NMR Research Centre, Institute of Nuclear Medicine and Allied
Sciences (INMAS), Brig. S.K. Mazumdar Road,
Delhi 110054, India
e-mail: [email protected]
S. Pal
Division of Cyclotron & Radiopharmaceutical Sciences,
Institute of Nuclear Medicine and Allied Sciences (INMAS),
Brig. S.K. Mazumdar Road, Delhi 110054, India
123
Metabolomics (2012) 8:444–453
DOI 10.1007/s11306-011-0326-z
acute cold stress on human health including cardiovascular
and respiratory diseases like hypertension, asthma, diseases
related to immune system and diarrhea. These biochemical
changes occurring due to exposure to acute cold stress can
directly be reflected in biofluids like urine, blood or serum.
The emerging metabolite profiling method using NMR or
GC/MS in conjugation with computer based data reduction
and pattern recognition methods such as Principal Com-
ponent Analysis (PCA) has successfully captured bio-
chemical changes in psychologically stressed rats (Teague
et al. 2007).
Tinospora Cordifolia (Tc) Miers referred to as Guduchi
(In Sanskrit means plant which protects from disease)
belongs to Menispermaceae family and is widely used in
Indian ayurvedic medicine as a tonic, vitalizer and as a
remedy for metabolic disorders. According to some herb-
alists, Tc has adaptogenic effects, a term that indicates it
helps the body to adapt to the stress (Rege et al. 1999).
Besides anti-allergy effects, evidence hints that Tc may
have anti-cancer (Panchabhai et al. 2008), immune-stimu-
lating (Nair et al. 2004), nerve cell protecting (Rawal et al.
2004), anti-diabetic (Stanely and Menon 2001; Stanely and
Menon 2003; Rathi et al. 2002), cholesterol-lowering
(Stanely and Menon 2003) and liver protective actions
(Bishayi et al. 2002). Tc has also shown some evidence
of decreasing the tissue damage caused by radiation
(Subramanian et al. 2002; Goel et al. 2004; Pahadiya and
Sharma 2003), the side effects of some form of chemo-
therapy (Mathew and Kuttan 1998) and speeding the pro-
cess of healing of diabetic foot ulcers (Purandare and Supe
2007). It has also been reported that Tc is beneficial in
boosting immune system (Mathew and Kuttan 1999) and
preventing or alleviating the impairment of cold stress. Due
to all these beneficial effects of Tc, it is essential to
understand the metabolic alterations caused by acute cold
stress and dietary intervention of Tc.
Conventional biochemical approaches such as series of
targeted clinical assays for assessing metabolic responses
to stimuli like acute stress, are typically time-consuming
and fragmentary. It is now well-established that NMR
based metabonomic studies in conjugation with data
reduction techniques, offers a powerful approach for gen-
erating and analyzing high information density metabolic
data on biofluids (Holmes et al. 1998; Brindle et al. 2002;
Nicholson et al. 1984). This approach can simultaneously
be used to detect a wide range of low molecular weight
metabolites, thus acting as a metabolic fingerprinting
technique, providing a comprehensive and quantitative list
of metabolites that can be mapped to specific pathways.
Hence it provides biomarkers and/or mechanistic infor-
mation about a process (Nicholson and Wilson 2003). To
reduce the interpretational challenge presented by large
data sets, a strategy of data reduction followed by
multivariate analysis (PCA) is typically employed (Holmes
et al. 1998). Principal Component Analysis (PCA) is an
exploratory technique of dimension reduction, with each
principal component (PC) being linear combination of the
original variables with appropriate weighting coefficients
(Nicholson et al. 1999). Hence, NMR spectroscopy in
combination with PCA is a powerful tool for discriminat-
ing and investigating the metabolic state between samples
obtained from control and cold stress rats.
Present studies were conducted to get a comprehensive
analysis of urinary metabolites from Sprague-Dawley (SD)
rats exposed to acute cold stress, using NMR spectroscopy
thereby giving an insight of cold stress induced biochem-
ical responses and metabolic consequences. Also, pre-
liminary studies were done to study the holistic and
protective effects of Tc during cold stress and restoring
homeostasis.
2 Experimental
2.1 Animal handling and sample collection
Eight-week-old male Sprague-Dawley rats (for stable
metabolic status) (200 ± 20 g) were obtained from
experimental animal facility of the institute and were
housed individually in the cages, and fed with a certified
standard rat chow and tap water ab libitum. All the animal
studies and handling were conducted in accordance with
institutional animal ethical committee guidelines. Room
temperature and humidity were regulated at 25 ± 2�C and
40 ± 15%, respectively. A light cycle of 12 h light and
12 h dark was set with lights on at 8.30 a.m. After 1 week
of acclimatization in metabolic cages, rats were randomly
divided into two groups viz control (C, n = 6) and
Tinospora group (Tc, n = 6). Aqueous extract of Tc was
prepared by dissolving 5 g of Tc powder (standardized dried
powder supplied by Nidco pharmaceuticals, Dehardun,
India) in 100 ml of distilled water. The suspension was
stirred at room temperature overnight and then boiled for
15 min. The procedure was repeated twice with the residue
and all the three filtrates were pooled together. The
supernatant was filtered and clear supernatant was evapo-
rated to dryness. The dried filtrate was reconstituted in dis-
tilled water (20 mg/ml). Orally a daily dose of 100 mg/Kg
of body weight was administered from day 1 till day 15 to
Tc group using a gavage. C group received the same vol-
ume of vehicle daily. C and TC groups were treated exactly
the same throughout the entire investigation, from time of
arrival through dosing (i.e., vehicle vs. treatment) and
sample collection. On day 16, all the rats were exposed to
-10�C for 3 h, and immediately returned to metabolic
cages at room temperature. 0–12 h urine sample from each
Cold stress induced changes in urine & restorative effect of T. Cordifolia 445
123
animal in both the groups were collected pre and post-cold
exposure in vials containing 1 ml of 0.1% sodium azide.
Particle contaminants were removed by centrifuging the
urine samples at 8,000 rpm for 10 min and the resultant
supernatants were stored at -80�C for NMR analysis.
Similar experiment was repeated on different group of rats
to check the reproducibility of the results.
2.2 Sample preparation and 1H NMR spectral
acquisition
300 ll of urine sample was mixed with 300 ll of buffered
deuterium oxide (0.2 M Na2HPO4/0.2 M NaH2PO4, pH 7.0,
D2O, Aldrich, 99.9%) and transferred to 5 mm NMR tubes
containing 1 mM TSP as an external standard in a co-axial
capillary tube for spectral acquisition. 1H NMR spectra were
acquired on each sample at 400.13 MHz on a Bruker Avance
400 spectrometer at a probe temperature of 298 K. Water
suppression was achieved using 1D NOESYPR pulse
sequence. Standard one-dimensional water peak pre-satu-
ration pulse sequence (90�–t1–90�–tm–90�–ACQ) was
applied. Interpulse delay t1 was 3 ls, and the mixing time tmwas 100 ms. Weak irradiation field was applied at the water
resonance frequency during both the mixing time and the
recycle delay. For each sample, 64 transients were collected
into 32 K data points with relaxation delay of 2 s and flip
angle of 90�. Relative concentration of each metabolite was
calculated by identifying the peaks, integrating with respect
to TSP and using these integral values in the following
equation:
C½ �X¼ C½ �TSPNTSP:IX= NX:ITSP½ �
where [C]X is the concentration of metabolite X, IX and
ITSP are the NMR signal intensities of X and TSP,
respectively. NX is the number of protons per molecule
giving rise to the integrated signal and NTSP = 9 (Sharma
et al. 2001).
2.3 Spectral processing, data reduction and pattern
recognition
For all 1D 1H NMR spectra, free induction decays were
multiplied by an exponential function corresponding to
0.3 Hz line broadening prior to Fourier Transformation,
were phased and baseline-corrected using TOPSPIN (Bru-
ker, Germany). The spectra were referenced to the TSP
resonance at 0 ppm. Normalization of the spectra to a con-
stant sum was carried out on these data and the spectra were
reduced to 250 integrated regions of equal width (0.04 ppm)
corresponding to the region d0–10. The region d4.6–5.5 was
removed to avoid residual spectral effects of imperfect water
suppression. Normalization of the spectra to a constant sum
and Principal Component Analysis (PCA) was done using
MATLAB 7.1 (Mathworks, USA). The data obtained was
visualized in the form of the Principal Component (PC)
scores plots and loadings plots (Holmes et al. 1998,
Robertson et al. 2005). Scores plots of the PC were con-
structed to visualize any inherent clustering of the samples
between Control and Tc group pre and post-cold exposure.
Changes in metabolites under cold stress condition were
identified from the value of the PC loadings and NMR spectral
regions. Each co-ordinate on the scores plot represents an
individual sample and each co-ordinate on the loading plots
represents one NMR spectral region. Thus the loadings plots
provide information on spectral regions responsible for the
position of co-ordinates or clusters of samples in the corre-
sponding scores plots. In addition to this, classical one way
analysis of variance (ANOVA) was also utilized to judge
whether the results were statistically significant. In this study,
the threshold value of P for significance was set to 0.05.
3 Results and discussion
3.1 Results
3.1.1 1H NMR analysis of rat urine following acute cold
stress
Perturbations on 1H NMR spectra were seen on exposure to
acute cold stress indicating alterations in low molecular
weight metabolite profiles in response to stress. Figure 1
shows the representative 1D 400 MHz 1H NMR spectra of
control (C) group pre and post-cold exposure. Various
metabolites in rat urine were assigned using previous lit-
erature (Nicholson et al. 1991; Zuppi et al. 1997; Williams
et al. 2003; Mahdi et al. 2008; Liu et al. 2010; Holmes et al.
1997). Comparative spectra clearly indicate the changes in
the intensity of resonance peaks for the metabolites of
samples from C group pre and post-cold stress. This indi-
cates substantial alterations in urine metabolite profile,
consistent with a perturbation of homeostasis. The changes
in the relative concentration of various metabolites of urine
samples obtained from C group rats pre and post-cold
exposure are tabulated in Table 1 in the form of
mean ± standard deviation. Significant difference between
the metabolites pre and post-cold exposure was calculated
using one way ANOVA, where P B 0.05 was defined as
significant difference. Following acute cold stress, visual
comparison of NMR spectra for the urine samples of C
group pre and post-cold exposure showed a significant
decrease in resonance peak intensity of pyruvate, citrate,
2-oxoglutarate, succinate, fumarate, N-methylnicotinamide,
creatinine, hippurate, phenylalanine, b-hydroxybutyrate,
TMAO and acetoacetate. Changes in the relative concen-
tration of other low molecular weight metabolites were not
446 S. Gandhi et al.
123
significant at 0.05 levels. Since visual analysis of the 1H
NMR spectra is a subjective process and inter-animal
variation can easily distort interpretation of these data,
multivariate data analysis of NMR spectra was performed
in order to form a general overview of metabolite patterns
of the effects of cold stress.
3.1.2 Principal component analysis (PCA) of 1H NMR
spectra
Three dimensional PCA plots were generated for 1H NMR
urine spectra to systemically address the metabolic
responses to acute cold stress. Clear separation was
observed in the first principal component (PC), implying
that exposure to cold stress may lead to a systemic varia-
tion of living systems (Fig. 2). Metabolites responsible for
the separation in PC scores plot were identified by plotting
PC loading versus Chemical shifts for C group pre and
post-cold stress to compare the regions of the spectra
(Fig. 3). Loading plot suggested that the urine samples
obtained from cold stressed rats contained altered con-
centration of pyruvate (d2.38), citrate (d2.67), 2-oxoglu-
tarate (d3.01), succinate (d2.43), fumarate (d6.53),
N-methylnicotinamide (d8.19, d8.90), creatinine (d4.06),
hippurate (d7.55, d7.64, d7.84), phenylalanine (d7.34),
b-hydroxybutyrate (d1.22), TMAO (d3.27) and acetoace-
tate (d3.45).
3.1.3 Effects of Tc intervention on metabolic alterations
of cold stress
In the Tc group, comparison of the 1H NMR spectra of
urine samples shows little variation between resonance
peaks of metabolites for pre and post-exposure in response
Fig. 1 Comparison of expanded 1H NMR spectra of control
(C) group urine samples showing decreased/increased intensity of
metabolite resonances in control group pre and post cold exposure.
a complete 1H NMR spectra of C group pre and post cold exposure,
b expanded region of 1H NMR spectra from d0.5 to 5.0 ppm,
c expanded region of 1H NMR spectra from d6.0 to 10.0 ppm
Cold stress induced changes in urine & restorative effect of T. Cordifolia 447
123
to cold stress. The NMR spectra showed no interference
from exogenous chemical compounds of Tc with the
endogenous metabolites. 3D PCA scores plot (Fig. 4)
showed no separation between pre-exposure and post-
exposure for Tc group in comparison to pre and post-
exposure Control (C) group, indicating reduced variation in
systemic metabolic profiles on cold stress after Tc inter-
vention. Univariate statistical methods, classical one-way
ANOVA was used to confirm non-significance between the
metabolite relative concentrations (Table 1). Results sug-
gest that the relative concentration of metabolites showing
variation in the Control group were comparable between
pre and post exposure to the cold stress for Tc group.
Also, PCA plots were generated between Control versus
Tc group (Fig. 5) and all groups (Fig. 6). The 3D PCA plot
between Control versus Tc group (Pre cold stress) (Fig. 5)
shows clear separation between both the groups suggesting
systemic metabolic change by Tc intervention that possibly
causes adaptogenic and immune simulating response
thereby providing protection against cold stress before the
exposure.
3D PCA plot between all the groups i.e., Control before
stress, Control after stress, Tc before stress and Tc after
stress (Fig. 6) showed a clear separation between Control
before and after stress indicating that exposure to cold
stress may lead to a systemic variation of living systems
whereas Tc before and after stress showed no separation
between pre-exposure and post-exposure for Tc group
indicating reduced variation in systemic metabolic profiles
on cold stress after Tc intervention.
These findings were reconfirmed by generating 3D PCA
plots between the relative concentrations of all the four
groups i.e., Control before stress, Control after stress, Tc
before stress and Tc after stress (Fig. 7). Similar findings
were reported showing clear separation between Control
Table 1 The concentration of various metabolites (mmol/ll) for urine samples expressed as mean ± standard deviation with significant
difference (P) at 0.05 level obtained from control (C) and Tinospora Cordifolia (Tc) group, pre and post cold exposure of -10�C for 3 h
Metabolites Control group (n = 6) (3 h at -10�C) Tinospora Cordifolia (Tc) group (3 h at -10�C)
Pre-cold
exposure
Post-cold
exposure
P (one-way
ANOVA)
Pre-cold
exposure
Post-cold
exposure
P (one-way
ANOVA)
Pyruvate 1.77 ± 0.79 0.75 ± 0.52* 0.0451 2.31 ± 0.63 2.15 ± 0.23 NS
Citrate 40.81 ± 1.42 18.62 ± 6.46* 0.0013 28.42 ± 0.91 25.12 ± 1.43 NS
2-oxoglutarate 8.76 ± 4.25 3.27 ± 0.99* 0.0292 9.58 ± 2.87 8.71 ± 1.21 NS
Succinate 9.13 ± 4.67 4.04 ± 1.08* 0.0386 11.39 ± 1.26 9.12 ± 3.21 NS
Fumarate 1.39 ± 0.34 0.15 ± 0.09* 0.0487 3.91 ± 0.17 3.08 ± 1.82 NS
N-methylnicotinamide 3.46 ± 1.05 1.04 ± 0.12* 0.0323 5.85 ± 0.17 3.95 ± 2.10 NS
Creatinine 12.11 ± 2.98 2.99 ± 1.06* 0.0051 10.23 ± 1.19 9.58 ± 0.58 NS
Hippurate 6.77 ± 1.64 2.14 ± 0.85* 0.0132 9.49 ± 3.78 7.45 ± 1.29 NS
Tyrosine 9.88 ± 2.89 8.76 ± 0.32 NS 2.90 ± 0.96 1.91 ± 0.91 NS
Phenylalanine 7.60 ± 1.63 2.59 ± 1.62* 0.0226 10.74 ± 3.21 8.41 ± 0.22 NS
b-hydroxy butyrate 6.75 ± 2.85 1.86 ± 1.30* 0.0125 12.10 ± 2.19 11.23 ± 0.12 NS
TMAO 4.10 ± 1.11 1.19 ± 0.51* 0.0487 5.23 ± 1.98 5.01 ± 3.21 NS
N-isovaleryl glycine 6.18 ± 2.00 1.93 ± 0.92* 0.0025 10.21 ± 2.83 4.53 ± 1.82* 0.031
Alanine 2.07 ± 0.88 0.69 ± 0.52 NS 1.67 ± 0.32 0.55 ± 0.18 NS
Acetoacetate 4.13 ± 1.35 0.63 ± 0.38* 0.0491 8.65 ± 1.86 2.31 ± 1.98 0.0173
Alantoin 25.02 ± 7.84 10.48 ± 3.74* 0.0078 30.12 ± 3.21 27.86 ± 1.87 NS
Formate 0.67 ± 0.27 0.40 ± 0.22 NS 1.82 ± 0.11 0.32 ± 0.02 0.054
* Significant to 0.05 level, NS non-significant
Fig. 2 3D-PCA scores plot for control group before and after-cold
exposure showing clear separation which indicates variation in
metabolites due to cold stress
448 S. Gandhi et al.
123
group pre and post-exposure whereas no distinct separation
was observed between Tc group pre and post-exposure
thereby reconfirming that Tc intervention provides pro-
tection against cold stress.
3.2 Discussion
Metabonomic studies on profiling of drug toxicity (Robosky
et al. 2002), several disorders such as those from inborn
errors of metabolism (Vangala and Tonelli 2007) and
evaluating biomarkers of clinical relevance have been of
main interest till date. A growing awareness about physi-
ological and psychological stress caused due to fast-paced
lifestyles, has led to an increasing number of experimental
studies on various stress-induced diseases (Teague et al.
2007). Studying the effects of acute cold stress can give an
insight to metabolic alterations, which may lead to devel-
opmental and depressive disorders (Kanayama et al. 1999).
The primary aim of this study was to firstly investigate the
metabolic alterations caused due to acute cold exposure
and secondly to verify the hypothesis that Tc intervention
can boost immune system and protect individuals against
adverse effects of cold exposure.
3.2.1 Metabolic response to acute cold stress
From previous literature, evidence exists for the adverse
effects of acute cold stress on human health including
cardiovascular and respiratory diseases (Cheng and Su
2010), rapid heart rate (Huang et al. 2011), tensed muscles
Fig. 3 Loading plots from analysis of 1H NMR spectra of urine
samples from control group pre and post-cold exposure representing
chemical shift positions corresponding to metabolites altered due to
cold stress. a Chemical shift region from d0.0 to d5.0 ppm.
b Chemical shift region from d6.0 to d9.5 ppm
Fig. 4 3D-PCA scores plot for Tc group before and after-cold
exposure showing no separation between the groups which indicates
no variation between the metabolites due to cold stress in Tc group
Fig. 5 3D-PCA scores plot for control and Tc group (pre-cold
exposure) showing clear separation which indicates systemic meta-
bolic change by Tc intervention providing protection against cold
stress
Cold stress induced changes in urine & restorative effect of T. Cordifolia 449
123
and increased alertness. Result from the present study
demonstrates the impact of cold stress at -10�C for 3 h on
the metabolic pathways. This is indicated by up or down-
regulated levels of low molecular weight metabolites in C
group pre and post-cold exposure. Cold stress showed
significant effects on the metabolites involved in several
pathways such as Tricarboxylic Acid (TCA) cycle (citrate,
2-oxoglutarate, succinate, fumarate, pyruvate) (Michaud
et al. 2008; Gibala et al. 1997), gut microflora (hippurate),
muscle metabolism (creatinine), phenylalanine, b-
hydroxybutyrate and trimethylamine-N-oxide (TMAO)
levels which is in agreement with previous literature on
alterations induced by various physiological stress (Bollard
et al. 2005; Janus et al. 2005; Sterling and Eyer 1988).
Using integral values for 1H NMR spectra, relative
concentration of each metabolite was calculated. One
way ANOVA (P \ 0.05) was used to find significant
changes between metabolites pre and post-exposure to
stress. It was seen that important metabolites of TCA
cycle were altered in rat urine post cold stress. The
urinary excretion of citrate, 2-oxoglutarate, succinate,
fumarate and pyruvate were decreased. From previous
literature, irrespective of any kind of physiological stress,
there is increased energy consumption to provide pro-
tection against internal and external stress (Michaud et al.
2008; Sterling and Eyer 1988). Also, increased gluco-
corticoid secretion and enhanced Sympathetic Nervous
System (SNS) activity are two major upstream metabolic
regulatory pathways activated due to cold stress exposure
(Miller and O’Callaghan 2002). Hence, decrease in
metabolites observed due to cold exposure may be
attributed to the increased energy consumption indicative
of up-regulated TCA cycle activity which may be due to
enhanced adrenergic nerve activity. Adrenergic activity is
reported to activate key TCA cycle enzymes such as
succinate dehydrogenase (Kulinskii et al. 1986). Trans-
ferring rats back to metabolic cages at room temperature
results in reduced responsiveness, suggesting slower
energy consumption period for metabolic regulatory
network. Short-term exposure to acute cold stress fol-
lowed by long room temperature recovery process leads
to overall lower level of TCA cycle metabolites in 12 h
urine sample. This shows that alteration of TCA cycle is
an important part of metabolic regulatory and compen-
satory mechanism in response to cold stress exposure. A
decrease in N-methylnicotinamide levels in rat urine was
also observed due to cold exposure. This may be attrib-
uted to increased need for nicotinamide, an important
precursor of the coenzyme NADH and NADPH which
are indispensible electron transporters involved in the
TCA cycle (Fedyk et al. 1996).
The results of the present study showed decrease in
phenylalanine levels (an essential amino acid) on exposure
to acute cold stress. Phenylalanine is first converted to
tyrosine by phenylalanine hydroxylase and then to cate-
cholamine (hormone released by adrenal glands in
response to stress) (Blomstrand and Newsholme 1992).
Decreased phenylalanine levels can be attributed to its
increased conversion to catecholamines in response to cold
stress. These alterations indicate enhanced SNS activity,
leading to an up-regulated catecholoamine metabolic
pathway (Wang et al. 2009). This may lead to instant
physiological effects such as rapid heart rate, tensed mus-
cles, increased alertness and constricted peripheral
vasculature.
Fig. 6 3D-PCA scores plot for control before and after stress, Tc
before and after stress groups showing clear separation between
Control before and after stress whereas no separation between Tc
group before and after stress
Fig. 7 3D-PCA scores plot for relative concentrations of control
before and after stress, Tc before and after stress groups showing clear
separation between control before and after stress whereas no
separation between Tc group before and after stress
450 S. Gandhi et al.
123
The results showed decrease in creatinine levels due to
cold stress exposure. Creatinine is a break-down product of
creatine phosphate in muscle, and is usually produced at a
fairly constant rate by the body (depending on muscle
mass). Creatinine levels in urine may be used to calculate
the creatinine clearance (CrCl), which reflects the glo-
merular filtration rate (GFR). The GFR is clinically
important because it is a measurement of renal function.
Reduced creatinine may be indicative of reduced glomer-
ular filtration rate and/or modifications of transport mech-
anism at tubular level, which may be related to altered
cellular function or low glucose in tubular lumen. Reduced
creatinine levels might also indicate reduced ability of
kidney to eliminate acids and may be considered as an
early marker for impaired renal function.
Also, our results showed decrease in hippurate levels.
Hippurate is believed to be metabolized by gut microbial
community indicating significant involvement and up-reg-
ulation of gut microbiota activity in response to cold stress
(Dumas et al. 2006). These observed effects on gut mic-
robiota are interlinked with stress-induced variation of
catecholamines and noradrenaline as they coexist with gut
microflora in gastrointestinal tract (GIT) (Lyte and Bailey
1997; Hawrelak and Myers 2004).
3.2.2 Compensatory effect of Tinospora Cordifolia (Tc)
intervention
Tc is believed to be adaptogenic herb which helps in
boosting immune system. Second objective of these studies
was to understand the protective action of Tc against cold
stress. PCA analysis showed no distinction between Tc
group pre and post-exposure, suggesting no metabolic
variation on cold exposure for the Tc group of rats (Fig. 4),
whereas clear separation was observed in the Control group
of rats pre and post cold exposure (Fig. 2).
The chemical component of Tc aqueous extract, syrin-
gin (TC-4) and cardiol (TC-7) reduced immunohaemolysis
and gave rise to significant increase in IgG antibodies
(Kapil and Sharma 1997). Clerodane furano diterpene gly-
cosides (TC-1), cordioside (TC-2), cordifolioside (TC-5)
and cordifolioside B (TC-6) are found to have immune-
stimulating activities (Wazir et al. 1995). Tc prevented
gastric mucosal damage induced by cold stress. It is said to
decrease plasma cortisol level and activate the immune
cells directly without intervention of other mediators (Rege
et al. 1999). It can be inferred that protective effects of Tc
are partially due to the regulation of glucocorticoids and
the SNS system. Tc is also believed to down regulate the
plasma-corticosterone in stressed rats.
Our results showed decrease in creatinine levels due to
cold exposure. Creatinine is a marker for renal function and
altered catabolism of muscle proteins. Tc intervention
showed regulation of creatinine levels. This can be attrib-
uted to the fact that Tc can modify renal tissue architecture,
may protect the protein catabolism in muscle or it ame-
liorates the renal function in rats (Nagaraja et al. 2007).
The changes in hippurate levels showed a significant
involvement of gut microbiota in response to cold exposure
as hippurate is believed to be metabolized by gut micro-
flora (Dumas et al. 2006). These observed effects on gut
microbiota are interlinked with stress-induced variation of
catecholamines and noradrenaline as they coexist with gut
microflora in gastrointestinal tract (GIT) (Lyte and Bailey
1997). Hence GIT motility and secretions in response to
stress maybe the probable reason for these metabolite
alterations (Nagaraja et al. 2007). Most of the herbs like
Tc, when administered orally are absorbed via degradation
by gut microflora. It is observed that Tc treated rats did not
show significant changes in metabolites involved in gut
microflora pre and post-cold exposure showing protective
action of Tc against cold stress by mobilizing the symbiotic
microbial community.
4 Conclusion
Non-invasive monitoring of various biochemical pathways
can be done by studying the urinary metabolite profile
using NMR spectroscopy in conjugation with multivariate
statistical techniques. It reveals a subtle interplay of func-
tional metabolites and pathways leading to an under-
standing of systemic response to external stimuli, such as
cold stress. The results of this work show significant
alterations in metabolite patterns arising as a result of
stress-induced metabolite responses. Alterations in TCA
cycle metabolites, gut microflora, muscle metabolism and
renal function were observed in response to cold stress,
which can act as early biomarkers for cold stress induced
changes. Exposure to cold stress showed an up regulation
of TCA cycle, Catecholamine pathway and Gut Microflora
activity. Also, there is a reduction in glomerular filtration
rate which may be considered as an early marker for
impaired renal function. Further, our studies showed the
prophylactic action of Tc against cold stress, which is due
to its immunoregulatory response, ability to modify renal
tissue and interrelationship with symbiotic bacteria of gut
microflora. These studies will further be extended by cor-
relating the results with clinical parameters, to detect early
biomarkers for cold stress in humans and to develop the
dosage of Tc to be administered for providing immunity to
the body against environmental insult, thereby reducing the
response to cold stress.
Acknowledgments This work was performed as a part of DRDO
sponsored R & D project INM-308. The authors are thankful for the
Cold stress induced changes in urine & restorative effect of T. Cordifolia 451
123
financial support provided by Defence Research & Development
Organization (DRDO), Ministry of Defence, India.
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