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: skhushu@yahoo.com

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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