Home Email this page Print this page Bookmark this page Decrease font size Default font size Increase font size
Noise & Health  
 CURRENT ISSUE    PAST ISSUES    AHEAD OF PRINT    SEARCH   GET E-ALERTS    
 
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Email Alert *
Add to My List *
* Registration required (free)  
 


 
   Abstract
   Conclusions
   Acknowledgments
   References
   Article Figures
 

 Article Access Statistics
    Viewed13963    
    Printed488    
    Emailed18    
    PDF Downloaded436    
    Comments [Add]    

Recommend this journal

 


 
ARTICLES Table of Contents   
Year : 1999  |  Volume : 1  |  Issue : 4  |  Page : 57-65
Noise and stress - salivary cortisol as a non-invasive measure of allostatic load

Center for Psychobiological and Psychosomatic Research, University of Trier, Germany

Click here for correspondence address and email
 
  Abstract 

The psychobiology of stress has received increasing attention throughout the past two decades. Physiological pathways and subjective response patterns are described in more details aiming at a better understanding of the pathways leading to health or disease under prolonged periods of stress. Technical advances in the laboratory have significantly contributed to this development. One of these methodological advances is the measurement of cortisol in saliva which has promoted psychobiological stress research both in the laboratory and in the field. The present paper provides a brief methodological background and the use of salivary cortisol assessment as an indicator of stress in human studies from this research centre. It is suggested that research on health consequences of noise exposure should include salivary cortisol as a sensitive measure of allostatic load.

Keywords: Saliva, Stress, TSST, Cortisol, Allostasis

How to cite this article:
Kirschbaum C, Hellhammer DH. Noise and stress - salivary cortisol as a non-invasive measure of allostatic load. Noise Health 1999;1:57-65

How to cite this URL:
Kirschbaum C, Hellhammer DH. Noise and stress - salivary cortisol as a non-invasive measure of allostatic load. Noise Health [serial online] 1999 [cited 2018 May 21];1:57-65. Available from: http://www.noiseandhealth.org/text.asp?1999/1/4/57/31723
Cortisol - a Player of Major Importance in Allostasis

Different avenues of research suggest that prolonged exposure to stress may eventually lead to the development of disease in prone individuals. The most compelling theoretical model to explain the process of disease development as a result of stress available today is the so called 'allostasis' model (Schulkin et al. 1994). [Figure - 1] gives a schematic picture of the model. This model assumes that the organism attempts to maintain function through change. The organism responds to any stimulus or situation which may put the individual at risk of being damaged (both physically or psychologically) or even killed, with a pathway of changes. When the threatening situation is over, the stress responses have to be shut off, the organism calmed down. The wear and tear on the body systems which provide the physiological changes in response to stress is referred to as the allostatic load.

A larger number of physiological processes change in response to environmental stress, including the production of stress-related hormones. Among other effects, this allows the body to spend more energy in times of increased demand. While on a short-term basis this response appears highly adaptive, there is a price the organism has to pay in the long-run. Diseases which may result from this 'allostatic load' include osteoporosis, coronary heart disease, hypertension, atherosclerosis, diabetes and immune-related diseases (McEwen, 1998). Although other bodily systems are also involved, it appears that the activity of the hypothalamus­pituitary-adrenal (HPA) axis plays a central role in this process. While corticotropin-releasing hormone and adrenocorticotropin (ACTH) may also contribute to the development of these disorders, the end product of this endocrine axis, cortisol, appears to be closely associated with the majority of symptoms observed in stress­associated health problems (McEwen, 1998; Rosmond et al. 1998; Chrousos and Gold, 1998).

Environmental conditions which impose an allostatic load on the body that leads to chronically increased HPA activity can therefore contribute to disease onset and/or progression. Noise, as a major environmental stress, needs to be scrutinised as a potential source for allostatic load. Changes in HPA activity induced by exposure to constant or intermittent noise would be of prime importance to both, the basic researcher and the clinician. Since accumulating evidence suggests that the measurement of cortisol in saliva reliably reflects the biologically active fraction of the major HPA effector hormone, we suggest to include salivary cortisol measurements in studies of the biological effects of noise stress. In the following, we first give a brief characterization of this hormone including technical recommendations for use of saliva samples in laboratory and field studies.

Cortisol in Saliva

After stimulation by adrenocorticotropin (ACTH), the adrenal cortex synthesizes (among other steroid hormones) the main glucocorticoid hormone of the human body, cortisol. Within seconds after release into the blood stream, up to 95% of cortisol is bound to corticosteroid binding globulin (CBG) and nonspecific blood­-borne carriers. Although it is believed that cortisol bound to carriers constitutes a reservoir of this steroid for use in times of increased demands, the physiological significance of the bound cortisol fraction has yet to be determined. Only 5-15% of cortisol circulates unbound in blood and can freely enter all cells in the body (Walker et al. 1978). Since the vast majority of all physiological functions of cortisol known today require binding to an intracellular receptor and subsequent interaction with DNA domains, it is the 'free' cortisol fraction which exerts biological activity (Robbins and Rall, 1957; Ekins, 1990).

The biochemical properties of cortisol (low molecular weight, high lipid solubility) not only allow the unbound steroid to freely enter nucleated cells, but to also enter other bodily fluids, including saliva. Since the transfer from blood to saliva appears to occur by passive diffusion (Vining & McGinley, 1984), the concentration of salivary cortisol is independent of saliva flow rate. This means that under conditions of reduced saliva flow (e.g., induced by anxiety or psychoactive drugs) as well as in times of maximal stimulation of saliva glands the concentration of cortisol found in saliva closely reflects the levels of unbound cortisol in blood (Hiramatsu, 1981; Vining & McGinley, 1984). While the determination of free cortisol in blood is time consuming and special technical expertise is required, the analysis of free cortisol in saliva is rather uncomplicated because no CBG is present here and commercial assay kits can be adapted for use with saliva instead of serum or plasma (e.g., Kirschbaum et al. 1989). Salivary cortisol is thus an inexpensive, yet valid and reliable measure of the bioactive cortisol in the body.

Of major importance for use in stress research is the noninvasiveness and ease of sampling and storage of saliva. Samples can be obtained by subjects of a wide age range (- 3-90+ years) with a hygienic sampling device (Salivette™) or by using a small open container. Parents or hospital staff can assist infants, small children and other individuals unable to collect saliva. This method thus provides an excellent opportunity to measure stress responses from subjects of all age groups. Sampling saliva for 20-60 seconds is usually sufficient, since sensitive assay systems often require only 200 µl of sample volume or less for duplicate analysis.

Cortisol is a rather stable molecule in saliva. Although it is recommended to readily store samples at -20EC to avoid molding, saliva can be kept at room temperature for at least four weeks without a significant drop in cortisol levels (Kirschbaum and Hellhammer, 1989). This allows the collection of samples in a wide spectrum of field studies spanning from 'mundane' settings, e.g. at home or work to rather 'exquisite' sites like during off-shore sailing regattas, sky-diving, or space missions (Kirschbaum and Hellhammer, 1994; Deinzer et al. 1997) and subsequent mailing of the samples to a laboratory (even overseas). Moreover, since saliva can be collected at almost unlimited amounts and short intervals, the time course of stress responses is easily assessed in the field.

This is only one of several advantages of salivary over urinary cortisol analyses.

Salivary Cortisol in Stress Research

Salivary cortisol was first introduced to psychobiological stress research almost two decades ago. Among the pioneers to use this method, Stahl and Dorner (Stahl and Dorner, 1982) investigated changes in cortisol levels in response to medical diagnostic procedures in several patient populations. They were able to show that cortisol levels can increase manifold within short periods after onset of stimulation. Similar observations were made in the following years with cortisol 'sensitive' stress stimuli including academic exams, unpleasant video sequences, public speaking, computer-aided tasks and sky-diving (Kirschbaum and Hellhammer, 1994; Deinzer et al. 1997). A major focus of this research was the demonstration of the multitude of situations capable of inducing an adrenocortical stress response in adults, which eventually may lead to a better understanding of the biological and psychological factors underlying cortisol secretion. As summarised already three decades ago (Mason, 1968), there are certain situation markers which can be regarded as crucial for induction of a cortisol stress response under such circumstances. Novelty, unpredictability, uncontrollability and (probably most important) anticipation of negative consequences in a given situation will lead to significant rises of salivary cortisol levels in most study subjects.

A second avenue of research was dedicated to the investigation of factors which can help to explain the large heterogeneity and complexity of cortisol stress responses. While some individuals show a large increase in free cortisol levels after, e.g., a laboratory stressor, there are other subjects who will show a moderate, blunted or even absent cortisol response (with similar cardiovascular responses in those subjects). Most interestingly, these endocrine response differences can not be attributed to differences in subjective stress responses as assessed by questionnaires. As shown in [Figure - 2], this response heterogeneity is not typical for psychosocial stress responses, but can be regarded a characteristic of the HPA axis. Similar inter-individual differences are observed in response to pharmacological or physical challenges.

Several studies from this laboratory have been able to identify some of the factors responsible for the heterogeneity in salivary cortisol response patterns. For this research program, the development of a laboratory stress protocol which induces significant responses of HPA hormones was indispensable. The "Trier Social Stress Test" (TSST) was finally found to provide large enough response magnitudes and between­subject variation for studies on stress modulators (Kirschbaum et al. 1993). The TSST is a laboratory stress protocol which mainly consists of a public speaking and a mental arithmetic task. Of prime importance to the study of HPA responses is that subjects have to perform this task in front of an "evaluation panel" of 2-3 persons with mock video and audio taping of the subject's performance. This 13-minute stress protocol induces sizable increases in ACTH, total and free cortisol, prolactin, growth hormone, catecholamines and other hormones as well as heart rate (Kirschbaum et al. 1994; Kudielka et al. 1998).

In numerous studies the TSST was used for an in-depth look at the factors which are associated with or responsible for a hypo- and hyper­reactive HPA axis. As is known from animal studies showing significant differences in HPA activity in different strains of mice or rats, also in humans genetic factors may influence the HPA axis. A study of mono and dizygotic twins revealed a rather high genetic determination for salivary cortisol baseline levels but only moderate to low resemblance of TSST responses in identical twins (Kirschbaum et al. 1992). In contrast, female sex steroids appear to exert a more profound influence on the cortisol stress response. Women using estrogen-containing oral contraceptives show blunted or absent salivary cortisol responses to the TSST as well as to physical exercise (Kirschbaum et al. 1995; Kirschbaum et al. 1996). This response decrement is not due to smaller ACTH and/or cortisol secretion, but rather a consequence of the medication-induced rise in CBG levels. Thus, women on oral contraceptives show the same endocrine response cascade to psychosocial stress, however, with an apparently important difference at the target tissue level: Increased CBG levels bind more cortisol released from the adrenal cortex in response to stress, thus there is a weaker cortisol signal to the target tissue in women on oral contraceptives, because cortisol bound to CBG can not exert its effects on the cell. Further experimental evidence for a strong impact of female sex steroids on cortisol responses came from a study in which young men received an estradiol or placebo patch 24-48 hours before being exposed to the TSST. Estradiol-treated men showed a clear-cut increase in ACTH and salivary cortisol responses to the TSST, again without a significant difference in subjective stress responses between the two groups (Kirschbaum et al. 1996).

Also a number of psychological variables were found to be closely associated with the individual salivary cortisol stress response. For example, acute social support can buffer against the consequences of stress exposure in men while the opposite may be true for women (Kirschbaum et al. 1995). On the other hand, social support may also be associated with cortisol reactivity on a longer-term basis as shown in a laboratory study with fire-fighters (Roy et al. 1998). Moreover, personality traits appear to have a bearing on the salivary cortisol response as suggested by a number of studies. Although the cortisol stress response is rarely found to consistently correlate with certain personality traits when the subjects are exposed to stress once (Kirschbaum et al. 1992), the impact of personality on cortisol responses are more readily observed if the subjects are repeatedly stressed. In a recent study this laboratory reported increasing correlations between personality traits like self-esteem or extraversion and the integrated salivary cortisol response to five repeated TSST sessions (Pruessner et al. 1997). These data suggest that the novelty component of a laboratory stress session can mask the contribution of other psychological factors on the cortisol response, e.g., personality. It is therefore necessary to exclude novelty as a potential source of response variation if the influence of personality traits on salivary cortisol responses are to be investigated. Currently, repeated stress exposure may be the best research strategy available to circumvent the novelty-associated 'contamination' of the endocrine response pattern. summarizes some of the many factors now known to modulate the cortisol response to acute psychosocial stress in humans.

Salivary Cortisol as a Measure of Allostatic Load

While the cortisol response to acute stress may be an important piece of information to include for a more precise prediction of disease susceptibility of an individual, also data on basal HPA functioning should be considered. Of potential importance are several measures which could reflect the degree of allostatic load: (a) the cortisol acrophase (highest levels in the circadian rhythm), (b) the cortisol nadir (lowest levels), (c) the morning-evening difference as an indicator of the steepness of the cortisol rhythm. While also the cortisol nadir and morning-evening differences may bear important information of HPA functioning, the following section will only discuss a novel method of measuring the cortisol acrophase using saliva samples.

With regard to the acrophase, standard measures of cortisol baseline levels were traditionally more or less restricted to a single-point assessment between 7 and 9 a.m. in the past. This sampling protocol is often dictated to the investigator by routine blood sampling regimen employed in hospitals for convenience reasons (e.g., clinical laboratory working hours). It has been recently shown, however, that cortisol levels show large variation during the first hour after awakening due to brief ACTH secretory episodes (Spath-Schwalbe et al. 1992). Within 30-45 minutes after awakening, salivary cortisol increases by 50-75% or more (Pruessner et al. 1997). [Figure - 4] depicts a typical cortisol response to awakening. Thus, morning cortisol levels may not be representative of the individual's acrophase when sampled at fixed times independent of the subject's sleep-wake rhythm. In order to be able to measure 'real' acrophase levels with increased consistency over repeated assessments, samples must be obtained with strict reference to the time of awakening. Sampling saliva repeatedly during the first hour (e.g., at 1, 15, 30, 45 and 60 minutes after awakening) may provide the researcher with important information of HPA activity related to allostatic load. In a recent study, adults reporting a high level of chronic stress due to work overload had a significantly greater cortisol increase after awakening compared to a low stress group (Schulz et al. 1998). Combining the feedback sensitivity measure by using the synthetic glucocorticoid dexamethasone (DEX) and the morning cortisol assessment, we were further able to show that continued long-term stress can also lead to a super-sensitive adrenocortex. Teachers scoring high on burnout scales tend to have lower cortisol levels after awakening and a higher feedback sensitivity to DEX than colleagues not suffering from burnout (Pruessner et al. in press). These data strongly suggest that the measurement of basal HPA (re)activity as reflected in the early morning wakening response, can add important information to adaptive changes in the body which may reflect the individual level of allostatic load.

Noise, stress research and salivary cortisol

Accumulating evidence suggests that noise can affect HPA function and hence cortisol levels acutely as well as chronically. For example, salivary cortisol levels were enhanced in response to mental work performed under 90 dB(A) white noise but not under quiet conditions (Miki et al. 1998). Also in the field the positive association between high noise exposure and enhance cortisol output has been documented. Industrial workers who were chronically exposed to an ambient noise level > 85 dB(A) failed to show the expected circadian decline in cortisol level in the afternoon. Following an intervention which caused reduction of noise to 30-33 dB(A) by the use of earmuffs, afternoon cortisol levels dropped significantly in these workers (Melamed and Bruhis, 1996). Moreover, noise not only has activating but also organizing effects on the HPA axis: Offspring of rhesus monkey mothers, who were stressed by unpredictable noise during mid to late gestation, showed enhanced HPA responses to stressors later in life (Clarke et al. 1994).


  Conclusions Top


This brief overview underscores the need for further research into the effects of noise exposure on the HPA axis. Since allostatic load of chronic noise exposure very likely has a major correlate in free cortisol levels with multiple effects on several target tissues, we suggest to include repeated cortisol assessment in future studies. For ease of sampling and biochemical analysis of the free hormone fraction, salivary cortisol should prove the perfect choice as a read-out system.


  Acknowledgments Top


This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ki 537/6-1).[33]

 
  References Top

1.Chrousos, G.P. and Gold, P.W. (1998) A healthy body in a healthy mind - and vice versa - the damaging power of "uncontrollable" stress. J.Clin.Endocrinol.Metab. 86, 1842-1845.  Back to cited text no. 1    
2.Clarke, A.S., Wittwer, D.J., Abbott, D.H. and Schneider, M.L. (1994) Long-term effects of prenatal stress on HPA axis activity in juvenile rhesus monkeys. Dev.Psychobiol. 27, 257-269.  Back to cited text no. 2    
3.Deinzer, R., Kirschbaum, C., Gresele, C. and Hellhammer, D.H. (1997) Adrenocortical responses to repeated parachute jumping and subsequent h-CRH challenge in inexperienced healthy subjects. Physiology and Behavior 61, 507-511.  Back to cited text no. 3    
4.Ekins, R. (1990) Measurement of free hormones in blood. Endocr.Rev. 11, 5-46.  Back to cited text no. 4    
5.Hiramatsu, R. (1981) Direct assay of cortisol in human saliva by solid phase radioimmunoassay and its clinical applications. Clin.Chim.Acta 117, 239-249.  Back to cited text no. 5    
6.Kirschbaum, C., Bartussek, D. and Strasburger, C.J. (1992) Cortisol responses to psychological stress and correlations with personality traits. Person.individ.Diff. 13, 1353-1357.  Back to cited text no. 6    
7.Kirschbaum, C. and Hellhammer, D.H. (1989) Salivary cortisol in psychobiological research: an overview. Neuropsychobiol. 22, 150-169.  Back to cited text no. 7    
8.Kirschbaum, C. and Hellhammer, D.H. (1994) Salivary cortisol in psychoneuroendocrine research: recent developments and applications. Psychoneuroendocrinol. 19, 313-333.  Back to cited text no. 8    
9.Kirschbaum, C., Klauer, T., Filip, S.-H. and Hellhammer, D.H. (1995) Sex-specific effects of social support on cortisol and subjective responses to acute psychological stress. Psychosom.Med. 57, 23-31.  Back to cited text no. 9    
10.Kirschbaum, C., Pirke, K.-M. and Hellhammer, D.H. (1993) The 'Trier Social Stress Test'- a tool for investigating psychobiological stress responses in a laboratory setting. Neuropsychobiol. 28, 76-81.  Back to cited text no. 10    
11.Kirschbaum, C., Pirke, K.-M. and Hellhammer, D.H. (1995) Preliminary evidence for reduced cortisol responsivity to psychological stress in women using oral contraceptive medication. Psychoneuroendocrinol. 20, 509-514.  Back to cited text no. 11    
12.Kirschbaum, C., Platte, P., Pirke, K.-M. and Hellhammer, D.H. (1996) Adrenocortical activation following stressful exercise: further evidence for attenuated free cortisol responses in women using oral contraceptives. Stress Med. 12, 137-143.  Back to cited text no. 12    
13.Kirschbaum, C., Scherer, G. and Strasburger, C.J. (1994) Pituitary and adrenal hormone responses to pharmacological, physical, and psychological stimulation in habitual smokers and nonsmokers. Clin. Investigator 72, 804-810.  Back to cited text no. 13    
14.Kirschbaum, C., Schommer, N., Federenko, I., Gaab, J., Neumann, O., Oellers, M., Rohleder, N., Untied, A., Hanker, J., Pirke, K.-M. and Hellhammer, D.H. (1996) Short term estradiol treatment enhances pituitary-adrenal axis and sympathetic responses to psychosocial stress in healthy young men. J.Clin.Endocrinol.Metab. 81, 3639-­3643.  Back to cited text no. 14    
15.Kirschbaum, C., Strasburger, C.J., Jammers, W. and Hellhammer, D.H. (1989) Cortisol and behavior: 1. Adaptation of a radioimmunoassay kit for reliable and inexpensive salivary cortisol determination. Pharmacol.Biochem.Behav. 34, 747-751.  Back to cited text no. 15    
16.Kirschbaum, C., Wust, S., Faig, H.-G. and Hellhammer, D.H. (1992) Heritability of cortisol responses to h-CRH, ergometry, and psychological stress in humans. J.Clin.Endocrinol.Metab. 75, 1526-1530.  Back to cited text no. 16    
17.Kudielka, B.M., Hellhammer, J., Hellhammer, D.H., Wolf, O.T., Pirke, K.-M., Varadi, E., Pilz, J. and Kirschbaum, C. (1998) Sex differences in endocrine and psychological responses to psychosocial stress in healthy elderly subjects and the impact of a two week DHEA treatment. J.Clin.Endocrinol.Metab. 83, 1756-1761.  Back to cited text no. 17    
18.Mason, J.W. (1968) A review of psychoendocrine research on the pituitary-adrenal cortical system. Psychosom.Med. 30, 576-607.  Back to cited text no. 18    
19.McEwen, B.S. (1998) Protective and damaging effects of stress mediators. N.Engl.J.Med. 338, 171-179.  Back to cited text no. 19    
20.Melamed, S. and Bruhis, S. (1996) The effects of chronic industrial noise exposure on urinary cortisol, fatigue and irritability: a controlled field experiment. J.Occup.Environ.Med. 38, 252-256.  Back to cited text no. 20    
21.Miki, K., Kawamorita, K., Araga, Y., Musha, T. and Sudo, A. (1998) Urinary and salivary stress hormone levels while performing arithmetic calculation in a noisy environment. Ind.Health 36, 66-69.  Back to cited text no. 21    
22.Pruessner, J., Wolf, O.T., von Auer, K., Jobst, S., Kaspers, F., Buske-Kirschbaum, A., Hellhammer, D.H. and Kirschbaum, C. (1997) The cortisol response to awakening: consistency, reliability, and individual differences. Life Sci. 61, 2539-2549.  Back to cited text no. 22    
23.Pruessner, J.C., Gaab, J., Lintz, D., Schommer, N., Hellhammer, D.H. and Kirschbaum, C. (1997) Increasing correlations between personality traits and cortisol stress responses obtained by data aggregation. Psychoneuroendocrinol. 22, 615-625.  Back to cited text no. 23    
24.Pruessner, J.C., Hellhammer, D.H. and Kirschbaum, C. (in press) Burnout, perceived stress and cortisol responses to awakening. Psychosom.Med.  Back to cited text no. 24    
25. Robbins, J. and Rall, J.E. (1957) The interaction of the thyroid hormones and protein in biological fluids. Rec.Prog.Horm.Res. 13, 161-208.  Back to cited text no. 25    
26.Rosmond, R., Dallman, M.F. and Bjorntorp, P. (1998) Stress-related cortisol secretion in men: relationships with abdominal obesity and endocrine, metabolic and hemodynamic abnormalities. J.Clin.Endocrinol.Metab. 83, 1853-1859.  Back to cited text no. 26    
27.Roy, M.P., Steptoe, A. and Kirschbaum, C. (1998) Life events and social support as moderators of individual differences in cardiovascular and cortisol reactivity. J.Pers.Soc.Psychol. 75, 1273-1281.  Back to cited text no. 27    
28.Schulkin, J., McEwen, B.S. and Gold, P.W. (1994) Allostasis, amygdala, and anticipatory angst. Neurosci.Biobehav.Rev. 18, 385-396.  Back to cited text no. 28    
29.Schulz, P., Kirschbaum, C., Pruessner, J. and Hellhammer, D.H. (1998) Increased free cortisol secretion after awakening in chronically stressed individuals due to work overload. Stress Med.  Back to cited text no. 29    
30. Spath-Schwalbe, E., Scholler, T., Kern, W., Fehm, H.L. and Born, J. (1992) Nocturnal adrenocorticotropin and cortisol secretion depends on sleep duration and decreases in association with spontaneous awakening in the morning. J.Clin.Endocrinol.Metab. 75, 1431-1435.  Back to cited text no. 30    
31.Stahl, F. and Dorner, G. (1982) Responses of salivary cortisol levels to stress-situations. Endokrinologie (Leibzig) 80, 158-162.  Back to cited text no. 31    
32.Vining, R.F. and McGinley, R.A. (1984). Transport of steroids from blood to saliva. In Immunoassays of Steroids in Saliva. Read,G.F.; Riad-Fahmy,D.; Walker,R.F.; and Griffiths, K., eds. Alpha Omega, Cardiff, pp56-63.  Back to cited text no. 32    
33.Walker, R.F., Riad-Fahmy, D. and Read, G.F. (1978) Adrenal status assessed by direct radioimmunoassay of cortisol in whole saliva or parotid saliva. Clin.Chem. 24, 1460-1463.  Back to cited text no. 33    

Top
Correspondence Address:
Clemens Kirschbaum
Center for Psychobiological and Psychosomatic Research, University of Trier
Germany
Login to access the Email id

Source of Support: None, Conflict of Interest: None


PMID: 12689490

Rights and PermissionsRights and Permissions


    Figures

  [Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4]



 

Top