Examination of pain threshold and neuropeptides in patients with acute suicide risk

Abstract

Introduction

One of the main challenges in suicide prevention is the limited understanding of the biological mechanisms underlying suicide. Recent findings suggest impairments in pain processing in acutely suicidal patients. However, little is known about the biological factors that may drive these discrete physiological abnormalities. In this study, we examined plasma peptides involved in analgesic and inflammatory responses and physical pain threshold in acutely suicidal patients.

Methods

Thirty-seven depressed patients of both sexes hospitalized for severe suicidal ideation or a recent suicide attempt were characterized clinically including history of suicidal ideation and behavior. Psychological and physical pain, and pressure pain threshold was also measured. Plasma levels of β-endorphin, neurotensin, agouti-related protein (AgRP), C-reactive protein (CRP), adrenocorticotropic hormone (ACTH), and brain-derived neurotrophic factor (BDNF) were run in Milliplex multiplex assays.

Results

The number of lifetime suicide attempts was positively correlated with β-endorphin (r= 0.702; p=0.007), and neurotensin (r=.728, p=0.007) plasma levels. Higher pain threshold was measured in the suicide attempt group as compared to the suicidal ideation group. Pain threshold was strongly and negatively associated with CRP plasma levels (r=−0.548; p<0.001). In patients reporting chronic pain, lower AgRP levels and lower pain threshold were observed (t=4.472; p=0.001).

Conclusion

Our results suggest that abnormalities in the opioid and neurotensin systems may underlie the increase in pain threshold found in suicide attempters, and possibly risk for suicidal behavior. Targeting pain circuits and systems may provide therapeutic mechanisms for suicide prevention.

Introduction

A growing and persistent epidemic, suicide is the 2^nd leading cause of death among young adults. Almost 800,000 individuals die by suicide annually (2018). There are approximately 25 suicide attempts for every suicide (Drapeau and McIntosh, 2015, Maris, 2002). The complexities of suicidal behavior are highlighted by the multiple factors and potential mechanisms that underlie suicide risk.

Although risk for suicide is multifactorial, there is a growing literature regarding abnormalities in pain processing in suicidal individuals. A strong association is observed between psychological pain and suicidal behaviors (Ducasse et al., 2018). A recent meta-analysis showed a significant link between physical pain and suicidal thoughts and behaviors (Calati et al., 2015). Further, chronic pain is associated with increased suicidal ideation and attempts (Fishbain et al., 2014, Jimenez-Rodriguez et al., 2014). Previous studies have described higher suicide rates in patients with autoimmune disorders, which are characterized by persistent pain (Timonen et al., 2003). Additionally, higher pain tolerance and threshold, defined as the highest level an individual tolerates a given pain and the lowest level an individual feels pain, respectively, are found in individuals after recent suicidal behavior (Caceda et al., 2017, Orbach et al., 1997, Orbach et al., 1996a, Orbach et al., 1996b). This decrease in pain sensitivity seems to be transient (Caceda, Kordsmeier, 2017). We have previously shown that the elevated pressure pain threshold in recent suicide attempters returned within a week to levels comparable with those of non-suciidal patients and depressed patients with only suicidal ideation but no suicidal behavior (Caceda, Kordsmeier, 2017). Elucidating the biological mechanisms associated with pain processing abnormalities may provide insight into the underlying risk factors for suicide.

Reports indicate a pro-inflammatory state associated with suicidal behavior (Brundin et al., 2017). Treatment with interferon alpha has been shown to induce suicidal ideation in patients with hepatitis C (Dieperink et al., 2004). Moreover, increased C-reactive protein (CRP), a marker of systemic inflammation, and pro-inflammatory cytokines have been described in individuals following recent (Gibbs et al., 2016) and remote (Courtet et al., 2015) suicide attempts, (for review see (Black and Miller, 2015)). Recent suicide attempters show the most inflammation among suicidal patients (Gananca et al., 2016).

Peptides, in addition to modulating physiological processes (e.g. pain, immune function and endocrine regulation), are known to powerfully regulate an array of complex behaviors including social bonding, mating, parenting, aggression, and even self-harm (Macdonald and Macdonald, 2010, Panksepp et al., 2007, Stanley et al., 2010, van Anders et al., 2011). Alterations in pain modulating peptides such as β-endorphin are found in individuals endorsing chronic pain (Bruehl et al., 2012) and in patients diagnosed with psychiatric disorders (Colasanti et al., 2011, Merenlender-Wagner et al., 2009). It stands to reason that specific peptides may underlie changes in the pain processing and inflammatory pathways that influence the progression to suicidal behavior.

The first goal of the current study was to shed light into the biological underpinnings of suicidal behavior by examining plasma levels of peptides involved in regulation of pain and inflammatory responses. Specifically, we aimed to evaluate whether plasma peptides associated with inflammation and pain regulation were linked to suicidal ideation and behavior. The second goal of the study was to examine the relationship between these peptides with different components of the pain experience in suicidal patients, namely psychological and physical pain, and threshold for experimental pain. We chose markers related to analgesic and inflammatory responses for analyses: β-endorphin, neurotensin, agouti-related protein (AgRP), C-reactive protein (CRP), adrenocorticotropic hormone (ACTH), and brain-derived neurotrophic factor (BDNF). We hypothesized that a) recent suicide attempters (patients currently hospitalized for suicide attempt) as compared to recent suicidal ideators (patients currently hospitalized for suicidal ideation) have increased pain threshold and elevated plasma levels of pain regulatory and inflammatory markers; b) the number of lifetime suicide attempts, current/lifetime suicidal ideation severity, and lifetime suicide attempt lethality are correlated with elevated plasma levels of pain regulatory and inflammatory markers; and c) pain processing abnormalities (psychological and physical pain, and higher pain threshold) are correlated with elevated plasma levels of pain regulatory and inflammatory markers.

Methods

Participants

Depressed adult patients of both sexes, ages 18–62 years with current suicidal ideation or after a recent suicide attempt were recruited between September 2016 and June 2017 from the inpatient units of the Psychiatric Research Institute at the University of Arkansas for Medical Sciences (UAMS) within 24 hours of hospital admission.

Inclusion criteria were the following: a) presence of current suicidal ideation as defined by the suicidal ideation severity subscale of the Columbia Suicide Severity Rating Scale (C-SSRS) (Posner et al., 2011) or having had a recent suicide attempt (within the last three days) as defined by self-harm with the intent to die; and b) fulfill Diagnostic and Statistical Manual of Mental Disorders IV-TR (American Psychiatric Association, 2000) criteria for Major Depressive Episode, Major Depressive Disorder, or Bipolar Disorder. As our goal was to understand the biology of acutely suicidal behavior, we recruited patients within a few days of a suicide attempt. Exclusion criteria were the following: a) inability to speak, read and write English; b) inability to provide informed consent; c) history of dementia, neurovascular or neurodegenerative conditions; d) physical disabilities that prohibit task performance; e) history of non-suicidal self-harm; and f) and involuntary hospitalization. Patients were assigned to the recent suicide attempt group if they had attempted suicide within the last three days or assigned to the suicidal ideator group if they endorsed current suicidal ideation but had not attempted suicide for at least 6 months prior to study enrollment. The UAMS Institutional Review Board approved all study recruitment and procedures. The study abided by to the principles of the Declaration of Helsinki. Participants were compensated for their participation in the study.

Procedure

Consecutive patients were screened for hospital admission for suicidal ideation or suicide attempt to the inpatient units of the Psychiatric Research Institute at the UAMS within 24 hours of hospital admission. The research team contacted the treatment team for patient safety clearance and permission to approach the patients. The treatment team obtained approval from the patients to be approached by the research team. The research team then approached the patient and provided information about the study and written informed consent was obtained. The psychiatric diagnosis was recorded from the intake clinical interview performed by one of the three board-certified attending psychiatrists, as defined by the DSM-IV TR. Demographics, detailed psychiatric and medical history, cognitive measurements, and ratings for anxiety and hopelessness were also obtained. The C-SSRS and Beck Depression Inventory (BDI-2) (Beck et al., 1996) were used to characterize suicidal ideation and behavior and depression severity, respectively. Subjects also completed the Beck Anxiety Inventory (Beck and Steer, 1990) and Beck Hopelessness Scale (Beck, 1988). Physical and psychological pain were measured with the Physical and Psychological Pain Scale using numerical scales (Jollant et al., 2018, Olie et al., 2010).

The C-SSRS, a widely used interviewer-rated instrument with different subscales to quantify current and past history of self-harm thoughts and actions (Posner, Brown, 2011), was administered by RC or two research assistants who were trained and certified (http://cssrs.columbia.edu/training/training-research-setting/).

The BDI-II is a 21 item self-report instrument to measure severity of depression (Beck, Steer, 1996).

The Beck Anxiety Inventory (BAI) is a 21 item self-report instrument to measure severity of anxiety (Beck and Steer, 1990).

The Beck Hopelessness Scale (BHS) is a 20 item self-report instrument to assess the level of positive and negative beliefs about the future (Beck, 1988).

The Physical and Psychological Pain Scale was used to measure self-reported states of physical and psychological pain currently and during the past 15 days (Olie, Guillaume, 2010).

Measure of pressure pain sensitivity

The utilization of experimental pain provides an objective measure of pain sensitivity. Pressure pain threshold was used to examine pain sensitivity differences between the two groups using standard procedures (Aweid et al., 2014). Briefly, a hand-held gauge with a 1-cm² rubber tip (Wagner Instruments, Greenwich, Conn., USA) was used for the pressure application. Patients were placed supine and alternate pressure (3 times per leg) was applied 5 inches below the patella in the medial border of both tibias. The patients were instructed to verbally report when the pressure sensation was painful. Six measurements (three per leg) were averaged to calculate the mean pressure pain threshold for each patient.

Biological samples processing

Blood was drawn for analysis of markers associated with inflammation and pain regulation including: β-endorphin, neurotensin, agouti-related protein (AgRP), C-reactive protein (CRP), adrenocorticotropic hormone (ACTH), and brain-derived neurotrophic factor (BDNF). Following collection, whole blood was placed immediately on ice and processed within 30 minutes. Plasma was separated from the cellular components by centrifugation at 1,200 rpm for 10 min at 4°C and stored at −80°C and assayed through commercially available ELISA kits (Human Neuropeptide Magnetic, Human Cardiovascular 3 MAG, Human Pituitary MAG and Huam Myokine Magnetic; Millipore, Billerica, MA). Absorbance was read at 450 nm on a BioRad Benchmark plus microplate spectrophotometer. The concentration for each sample was quantified based on optical density values calculated from the standard curve.

Data analysis

Descriptive statistics were calculated. Student T-tests were performed for the suicide attempt and suicidal ideation groups on peptide plasma levels and pain threshold. Independent-samples Mann-Whitney U tests were used for the suicide attempt and suicidal ideation groups on clinical ordinal measures (psychological and physical pain, suicidal ideation severity, and highest suicide attempt lethality). Student T-tests were performed for presence of chronic pain on plasma levels and pain threshold. Independent-samples Mann-Whitney U tests were used for presence of chronic pain on clinical ordinal measures listed above. Pearson correlation analyses were used for number of suicide attempts, pain threshold, and plasma levels. Spearman correlation analyses were used for continuous variables when violation of normality was noted. Specifically, suicidal ideation severity, psychological and physical pain, and lifetime suicide attempt highest lethality (ordinal variables) were correlated with plasma levels. Bonferroni correction was used to control for multiple comparisons. All analyses were conducted using SPSS 24 (SPSS Inc., Chicago, IL, USA).

Results

A total of 37 depressed patients with current suicidal ideation (n=26) or following a recent suicide attempt (n=11) were examined. Table 1 presents the demographic and clinical characteristics of the study sample. The mean age was 39 years old, with similar proportion of males and females. Student t-test revealed that higher pain threshold was measured in the suicide attempt group (p=.028; Hedges’ g=0.89) as compared to the suicidal ideation group. The suicidal ideation group had higher severity of current suicidal ideation (p=.012; Hedges’ g=.86) as compared to the suicide attempt group. There were no differences in peptide plasma levels and measures of current psychological or physical pain between the two groups (Table 1). Because of this lack of differences between the suicide attempt and suicidal ideation groups, and with most of the study population having had a lifetime suicide attempt (except 6 individuals), both groups were combined for the further analyses.

Table 1.

Demographic and clinical characteristics of study sample.

	Suicide Attempt Group	Suicidal Ideation Group	p value	Hedges’ g
N	11	26
Age	34.55±10.36	41.96±12.89	p=.100a	0.61
Gender (f)	4 (36.7%)	13 (50.0%)
Education in years	12.45±2.02	12.15±3.04	p=.781b	0.11
Depression (BDI-2)	29.73±12.99	37.46±9.87	p=.056a	0.71
Anxiety (BAI)	24.18±13.47	32.19±14.33	p=.123a	0.57
Hopelessness (BHS)	10.00±1.90	10.03±2.49	p=.964a	0.13
Current suicidal ideation severity	1.27±1.95	2.81±1.72	p=.012b	0.86
Number of lifetime suicide attempts	9.82±14.95	8.38±22.01	p=.845a	0.07
Highest suicide attempt lethality	1.45±1.51	2.25±1.45	p=.145b	0.54
Current physical pain	3.64±3.17	4.73±3.18	p=.366b	0.34
Current psychological pain	5.27±3.82	6.62±2.97	p=.366b	0.42
Pain Threshold	18.05±4.62	13.96±4.59	p=.028a	0.89

^aStudent T-test

^bIndependent-Samples Mann-Whitney U Test

Pearson correlation revealed a positive correlation between number of suicide attempts and β-endorphin (r= .702; p_corrected <.001), and neurotensin (r=.728, p_corrected <.001). Both survived multiple comparisons correction (Bonferroni-corrected alpha level for significance was p=.008). There were no significant correlations between number of suicide attempts and AGRP, CRP, ACTH, and BDNF.

Pain threshold was negatively correlated with CRP levels (r=−.548, p_corrected =.002). There were no significant correlations between pain threshold and other plasma peptides. Spearman correlation revealed that current physical pain was associated positively with BDNF (r=.365; p=.026) and negatively with ACTH (r=−.425; p=.009) and AGRP (r=−.369; p=.024) plasma levels. None of these associations survived multiple comparison correction. There were no significant correlations between current physical pain and β-endorphin, neurotensin CRP, and AGRP levels. Current psychological pain was not significantly associated with any plasma peptide levels. Lifetime highest suicide attempt lethality was negatively associated with β-endorphin (r=−.398, p=.027) and neurotensin (r=−.397, p=.047) plasma levels. However, no associations with suicide attempt lethality survived multiple comparisons correction. Suicidal ideation severity was not significantly associated with any plasma peptide levels.

Patients reporting chronic pain (n=21) showed elevated levels of neurotensin (t=2.476, p=.020) and β-endorphin (t=2.137, p=.041) while there was a decrease in ACTH (t=2.230, p= .044) and AGRP (t=4.472, p<.001) levels. Only the AGRP level analysis survived multiple comparison correction. In patients reporting chronic pain, the independent-samples Mann-Whitney U test revealed higher current physical pain (p=.001; Hedges’ g=1.35) and student t-test revealed lower pain threshold (p=.009; Hedges’ g=1.01) (Table 3). There were no significant differences between patient reporting chronic pain and measures of psychological pain.

Table 3.

Presence of chronic pain on clinical and plasma measures.

	Presence of chronic pain	No chronic pain	p-value	Hedges’ g
N	21	13
Current physical pain	6.00±2.35	2.46±3.04	P<.001b	1.35
Current psychological pain	6.52±2.64	6.38±3.50	p=.834b	0.05
Pain Threshold	13.05±4.30	17.52±4.58	p=.009a	1.01
C-reactive protein (pg/ml)	0.22±0.24	0.11±0.16	p=.175a	0.52
BDNF (pg/ml)	1172.52±663.08	837.74±526.54	p=.133a	0.54
ACTH (pg/ml)	3.03±1.10	5.79±4.38	p=.044a	0.98
AGRP (pg/ml)	9.45±5.26	21.48±8.77	P<.001a	1.77
β-Endorphin (pg/ml)	207.13±158.82	124.46±62.01	p=.041a	0.63
Neurotensin (pg/ml)	144.19±83.75	94.80±28.76	p=.020a	0.72

^aStudent T-test

^bIndependent-Samples Mann-Whitney U Test

Discussion

The present study sought to define the association between suicidal thoughts and actions and plasma peptides involved in the regulation of pain and the inflammatory response. Additionally, we examined the relationships between pain experience and levels of plasma peptides. We report: a) higher pain threshold and lower suicidal ideation severity in the suicide attempt group as compared to the suicidal ideation group, b) a positive correlation of number of lifetime suicide attempts with β-endorphin and neurotensin levels, and c) a negative correlation between pain threshold and CRP levels. In patients reporting chronic pain, there was a decrease in AGRP levels as compared to patients not reporting chronic pain. Additionally, higher levels of current physical pain and lower pain threshold were reported by patients with chronic pain.

We replicated the finding of higher pain threshold in recent suicide attempters (Caceda, Kordsmeier, 2017, Orbach, Mikulincer, 1997, Orbach, Palgi, 1996a, Orbach, Stein, 1996b). Similarly, a recent meta-analysis found that that individuals who engaged in self-injurious behaviors reported less pain and had higher threshold and tolerance to experimental pain as compared to healthy controls (Koenig et al., 2016). The results indicate lower pain sensitivity associated with self-injurious and suicidal behavior. However, patients who reported chronic pain had lower pain thresholds to experimental pain as compared to those who did not report chronic pain. There is high comorbidity of pain with psychiatric disorders including depression (Stubbs et al., 2015a, Thompson et al., 2016) and schizophrenia (Stubbs et al., 2015b) and with chronic conditions such as fibromyalgia (Kindler et al., 2011). The lower pain thresholds associated with chronic pain may be attributed to hypersensitization (Kindler, Bennett, 2011). As chronic pain may increase pain sensitivity, those who engage in suicidal behavior may have show pain processing and regulation that increases risk for suicide.

The suicide attempt group had less severe suicidal ideation than the suicidal ideation group. Similar findings are reported from a previous study from our group in a different sample (Cáceda et al., 2014) and by recent suicide attempters as compared to distant attempters (Paashaus et al., 2019), Suicidal ideation tends to be transient and fluctuates (Kleiman and Nock, 2018, Kleiman et al., 2017). Moreover, for many patients suicide attempt is a cathartic event and suicidal ideation and intention decreased considerably shortly after a failed suicide attempt (Rosen, 1976). Our study recruited patients who were hospitalized for very high suicide risk: ongoing suicidal ideation and /or recent suicide attempts. However, there may be a qualitative difference in individuals who engage in suicide attempts that does not entail severity of suicidal ideation. For instance, the capability for suicide may serve as a strong risk factor for suicide (Ribeiro et al., 2015, Van Orden et al., 2008). Additionally, while previous self-injurious thoughts and behaviors are known risk factors for future suicidal ideation and behavior, the predictive effects have been shown to be weak (Ribeiro et al., 2016). The capability of suicide may play a transient role in increased suicide risk, and fearlessness of pain may also serve as a driving force for engaging in suicide attempts. Our findings of higher pain threshold in recent suicide attempters as compared to suicidal ideators suggest that higher risk for suicide may be associated with pain processing abnormalities, and not necessarily the severity of suicidal ideation.

In this study, we found higher levels of β-endorphin and neurotensin with increased number of suicide attempts. Both neuropeptides have been shown to have analgesic and antinociceptive properties (Bruehl, Burns, 2012, Kleczkowska and Lipkowski, 2013). β-endorphin produces a clinically useful analgesic effect when binding to μ-opioid receptors. The analgesic impact of opioids such as β-endorphin occur by suppressing GABAergic neurotransmission, and in turn disinhibiting the periaqueductal gray (PAG) and rostral ventromedial medulla (RVM) projections, which allows for analgesic signal output to the spinal cord (Lau and Vaughan, 2014). A recent clinical trial demonstrated that a four week administration of buprenorphine, a μ-opioid partial agonist and κ-antagonist, reduced suicidal ideation as well as mental pain (Yovell et al., 2016). On the other hand, neurotensin has been implicated in pain modulation and analgesia (Cáceda et al., 2006). The antinociceptive effects of neurotensin are through direct excitation of the PAG neurons innervating the RVM (Li et al., 2001), which projects to the dorsal horn of the spinal cord where pain signal transmission is inhibited (Fields and Heinricher, 1985). Even though we did not directly explore the dopamine system, it is worth noting that β-endorphin and neurotensin modulate neurotransmission in the ventral tegmental area and PAG regions, affecting not only reward, but also pain processing and regulation (Korotkova et al., 2003, Latimer et al., 1987, St-Gelais et al., 2006, Zahm et al., 2001). The positive association between plasma levels of β-endorphin and neurotensin and the number of suicide attempts may suggest dysregulation of pain processing circuits that increases risk for suicidal behavior.

As a whole, our previous studies and the results of the current study suggest an association between a pro-inflammatory state and abnormalities in pain processing. CRP was correlated negatively with pain threshold. In other words, lower CRP levels were associated with higher pain threshold, indicating decreased pain sensitivity. We previously showed that CRP levels, a general inflammation marker, were positively correlated with current physical pain and number of lifetime suicide attempts (Caceda et al., 2018). Specifically, current physical pain and number of lifetime suicide attempts were associated with higher CRP levels. Abnormalities in the inflammatory response may modulate pain response, resulting in increased pain threshold and decrease in pain sensitivity, favoring progression to suicidal behavior. In patients reporting chronic pain, lower AgRP levels were observed. There are limited reports of AgRP in chronic pain in human studies. However, in rodents, a pro-inflammatory state decreases secretion of AgRP (Scarlett et al., 2008), suggesting the significant association between AgRP and inflammation. The findings from this study support further follow up interrogation of the dynamic roles of inflammatory processes and pain regulation in individuals with chronic pain and risk for suicidal behavior.

In this study, we did not find significant associations between BDNF or ACTH with any pain or suicidal ideation or behavior measures. Previous studies have shown mixed results regarding BDNF blood levels in suicidal behavior. Some studies have shown decreases in plasma BDNF in patients with MDD who had recently attempted suicide (Kim et al., 2007). However, recent meta-analyses indicate that while there was a decrease in plasma BDNF in patients who attempted suicide there were no differences in serum BDNF measures (Eisen et al., 2015, Salas-Magaña et al., 2017).

To our knowledge this is the first study evaluating plasma peptides in acutely suicidal individuals (e.g. within three days of a suicide attempt and/or with current suicidal ideation). Our findings build on previous work (Calati, Laglaoui Bakhiyi, 2015, Fishbain, Lewis, 2014, Ratcliffe et al., 2008), supporting the link between dysregulation of pain processes and suicidal behavior in acutely suicidal patients. Assessment of pain status and available coping mechanisms seem to be a valuable elements of suicide risk assessment and safety planning. The potential impact of targeting pain modulatory systems is exemplified by the reduction of suicidal ideation with a very low dose buprenorphine trial of patients at high risk for suicide (Ahmadi et al., 2018).

The limitations of our study include its cross-sectional design and limited sample size. An inherent limitation to all studies seeking to understand the biology of suicide is that individuals who survive a suicide attempt may differ from those who died by suicide. Three days post-attempt may be too long a delay to capture the state of mind and the neurobiology of an individual during the attempt itself. Correlation analyses limit causality inferences (Maurage et al., 2013). However, the associations observed between peptide levels and suicidal behavioral measures indicate that underlying neurobiological alterations in the pain system may play a pivotol role in priming an individual for suicide risk. Our study did not include subjects whose suicide attempts or subsequent treatment prevented them from being able to consent or be examined. Even though we controlled for use of antidepressant medications in this difficult to recruit population, this strategy is less optimal than studying medication-free individuals. Higher rates of suicidal behavior are observed in borderline personality disorder (an Axis II disorder) than in the general population (Pompili et al., 2005). Additionally, higher pain thresholds are observed in patients with borderline personality disorder (Ludäscher et al., 2007, Russ et al., 1992). This Axis II pathology was not systematically measured in this study, however we excluded subjects with nonsuicidal self harm.

To summarize, plasma levels of β-endorphin, and neurotensin, which are neuropeptides associated with pain modulation, were increased in recent suicide attempters and number of lifetime suicide attempts. Additionally, both recent suicide attempters and those reporting chronic pain showed altered physical pain threshold. Our results suggest that abnormalities in the opioid and neurotensin systems may underlie the decreased pain sensitivity found in recent suicide attempters, and possibly risk for suicidal behavior. Thus, targeting pain circuits and systems may provide therapeutic mechanisms for suicide prevention.

Table 2.

Plasma measures by group

	Suicide Attempt Group	Suicidal Ideation Group	p-value	Hedges’ g
N	11	26
C-reactive protein (pg/ml)	0.10±0.15	0.20±0.23	p=.211	0.46
BDNF (pg/ml)	850.62±404.03	1070.76±682.94	p=.327	0.36
ACTH (pg/ml)	9.05±15.97	3.91±2.75	p=.115	0.58
AGRP (pg/ml)	18.70±8.55	12.34±8.99	p=.054	0.71
β-Endorphin (pg/ml)	238.99±266.15	203.45±235.20	p=.674	0.16
Neurotensin (pg/ml)	150.49±111.70	154.91±179.36	p=.940	0.03