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The relationship of allopregnanolone immunoreactivity
and HPA-axis measures to experimental pain sensitivity:
Evidence for ethnic differences
q
Beth Mechlin
a,b
, A. Leslie Morrow
a
, William Maixner
c
, Susan S. Girdler
a,*
a
Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
b
Department of Psychology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
c
Department of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Received 13 June 2006; received in revised form 29 November 2006; accepted 27 December 2006
Abstract
In animal models, allopregnanolone (ALLO) negatively modulates the hypothalamic–pituitary–adrenal (HPA) axis and has
been shown to exert analgesic effects. The purpose of this study was to assess the relationship between plasma ALLO immu-
noreactivity (ALLO-ir), HPA-axis measures, and pain sensitivity in humans. Forty-five African Americans (21 men, 24 women)
and 39 non-Hispanic Whites (20 men, 19 women) were tested for pain sensitivity to tourniquet ischemia, thermal heat, and cold
pressor tests. Plasma ALLO-ir, cortisol, and b-endorphin concentrations were taken following an extended rest period. Lower
concentrations of ALLO-ir were associated with increased pain tolerance to all three pain tests and increased pain threshold to
the thermal heat pain task in the non-Hispanic Whites only (rs=.35 to .49, ps < .05). Also, only in the non-Hispanic Whites
was cortisol associated with thermal heat tolerance (r= +.39, p< .05) and threshold (r= +.50, p< .01) and cold pressor toler-
ance (r= +.32, p< .05), and were b-endorphin concentrations associated with cold pressor tolerance (r= +.33, p< .05). Media-
tional analyses revealed that higher cortisol levels mediated the relationship between lower ALLO-ir and increased thermal heat
pain threshold in the non-Hispanic Whites only. These results suggest that lower ALLO-ir concentrations are associated with
decreased pain sensitivity in humans, especially in non-Hispanic Whites, and that this relationship may be mediated by
HPA-axis function.
2007 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
Keywords: Allopregnanolone; Pain; African Americans; HPA-axis
1. Introduction
African Americans experience more clinical pain
(Edwards et al., 2001a; McCracken et al., 2001; Riley
et al., 2002) and report more pain associated with
chronic medical conditions (Edwards et al., 2001b). In
the laboratory, studies consistently indicate no ethnic
differences in pain onset (i.e., pain threshold) but that
African Americans have reduced pain tolerance relative
to Caucasians (Chapman and Jones, 1944; Woodrow
et al., 1972; Edwards and Fillingim, 1999; Sheffield
et al., 2000; Campbell et al., 2005; Mechlin et al.,
2005). Studies of biological mechanisms that may con-
tribute to these ethnic differences are scant (Mechlin
et al., 2005).
While there is a well-documented relationship
between higher blood pressure (BP) levels and reduced
0304-3959/$32.00 2007 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.pain.2006.12.027
q
This study was supported by NIH Grants NIDA R01 DA1375 and
GCRC RR00046. The authors are grateful to Dot Faulkner for her
assistance with manuscript preparation.
*
Corresponding author. Tel.: +1 919 966 2544; fax: +1 919 966
0708.
E-mail address: susan_girdler@med.unc.edu (S.S. Girdler).
www.elsevier.com/locate/pain
Pain 131 (2007) 142–152
pain sensitivity in Caucasians (Zamir and Shuber, 1980;
Maixner, 1991; Sheps et al., 1992; McCubbin and
Bruehl, 1994; Bragdon et al., 1997), thought to be medi-
ated by stimulation of mechanoreceptive afferents (i.e.,
baroreceptors; Randich and Maixner, 1986), a recent
report from our laboratory found no evidence for a rela-
tionship between BP and pain sensitivity in African
Americans, while the expected relationship held for a pri-
marily Caucasian sample (Mechlin et al., 2005). Greater
concentrations of stress-induced cortisol are also associ-
ated with decreased pain sensitivity in Caucasian samples
(al’Absi et al., 2002; Girdler et al., 2005; Mechlin et al.,
2005). However, our prior study found no evidence for
a relationship involving cortisol and pain sensitivity in
African Americans, which was documented in the
primarily Caucasian sample (Mechlin et al., 2005).
Taken together, the results of our previous study
suggest ethnic differences in the relationship of stress-
responsive biological measures, that may act to dimin-
ish pain as part of an integrated response during the
defense reaction (Maixner, 1991), to pain sensitivity.
Another stress-relevant neuroendocrine factor that
may modulate pain perception in humans, and which
constitutes the novel focus of this report, is allopreg-
nanalone (ALLO). ALLO is an A-ring-reduced metab-
olite of progesterone that is produced by ovary and
adrenals and also de novo in brain (Paul and Purdy,
1992). Owing to its lipophilicity, even peripherally
produced ALLO readily crosses into brain (Paul and
Purdy, 1992). ALLO is a potent modulator of GABA
A
receptors via dose-dependent enhancement of GABA-
induced Cl
ion channels (Morrow et al., 1987). ALLO
is stress sensitive in both animals (Purdy et al., 1991;
Barbaccia et al., 1996), and humans (Girlder et al.,
2001). Animal models indicate that stress-induced
increases in ALLO negatively modulate hypothalam-
ic–pituitary–adrenal (HPA)-axis activity, thereby facili-
tating the recovery of physiologic homeostasis in this
system following stress (Guo et al., 1995; Patchev
et al., 1996).
Stress-induced increases in ALLO may be adaptive
since ALLO has analgesic properties when administered
in animals (Kavaliers and Wiebe, 1987). Furthermore,
animal models clearly indicate the involvement of cen-
tral GABA
A
receptors in modulating pain sensitivity
(Yokoro et al., 2001). To date, no studies have examined
ALLO-associated analgesia in humans. The primary
aim of this report is to examine the relationship of
ALLO to experimental pain sensitivity in humans and
determine whether ethnic differences are present. A sec-
ondary aim of this report involves examining the inter-
relationships of plasma cortisol and b-endorphin, in
the relationship of ALLO to pain sensitivity. To our
knowledge, the relationship of b-endorphin concentra-
tions to pain sensitivity in different ethnic groups has
not been studied.
2. Methods
2.1. Subjects
Subjects that comprise the present report represent a large
sub-sample of the participants included in our recent report
on ethnicity, pain sensitivity, and endogenous pain regulatory
mechanisms described above (Mechlin et al., 2005). While that
prior study compared African Americans with all ‘Other’ eth-
nic groups, given the evidence that Hispanic populations differ
in clinical pain systems (Hernandez and Sachs-Ericsson, 2006),
the evidence for ethnic differences between Asian Americans
and European Americans in stress-responsive measures
(Stoney et al., 2002; Shen et al., 2004), and since the numbers
of Hispanics (n= 2) and Asians (n= 6) in our study did not
allow for valid analyses, the present report compares African
Americans with non-Hispanic Whites only. Of the 107 subjects
included in that earlier report, we were able to analyze ALLO
immunoreactivity (ALLO-ir) in plasma from 85 subjects who
self-reported as African American or non-Hispanic White.
Thus, the present report represents new measurements (ALLO
and b-endorphin) in a large subset of those subjects on which
we previously reported.
The participants were recruited through newspaper adver-
tisements seeking male and female non-smokers for a study
on pain perception. Non-smokers were recruited based on
self-report. Smokers were excluded since cigarette smoking
has been shown to have analgesic properties (Girdler et al.,
2005). The sub-sample of subjects providing ALLO-ir samples
was composed of 41 men and 44 women, aged 18–47 years.
Approximately half (n= 45) of the subjects was African Amer-
ican (21 men, 24 women) while the other half (n= 39; 20 men,
19 women) was non-Hispanic White.
There were no gender or ethnic group differences in
age (range 18–47) or diastolic blood pressure (DBP; range
50–87 mmHg), or heart rate (range 50–92 bpm). There were
ethnic differences in body mass index (BMI), since African
Americans had higher BMIs than non-Hispanic Whites (28.9
vs. 25.1; F(3,82) = 7.92, p< .01). Also, men had higher resting
systolic blood pressure (SBP) than women (120 mmHg vs.
112 mmHg; F(3,82) = 10.85, p< .01). No ethnic ·gender
interactions were observed for any of the above biological
measures.
All subjects were medically healthy, with no more than
mildly elevated BP (<160/90 mmHg) as determined during
an initial screening session. Only 4 subjects (2 non-Hispanic
Whites and 2 African Americans) had elevated BP, defined
as SBP > 135 mmHg and/or DBP > 85 mmHg. Additionally,
subjects were not taking any prescription medication, includ-
ing oral contraceptives or psychotropics, and none took any
over-the-counter medication more than four times per month.
Consistent with other studies on experimental pain (France
et al., 2002; al’Absi et al., 2004; Fillingim et al., 2005)we
instructed subjects to refrain from using any analgesic or other
over-the-counter medications for 24 h prior to pain testing.
This was verified based on self-report. All women reported reg-
ular menstrual cycles. Excluded from participating were sub-
jects with chronic pain conditions (e.g., temporomandibular
joint disorder, fibromyalgia, arthritis) and those exhibiting
signs of depression or anxiety based on Hamilton rating scales
for depression (score > 7) and anxiety (>9).
B. Mechlin et al. / Pain 131 (2007) 142–152 143
The protocol was approved by the institution’s Institutional
Review Board and all subjects provided informed, written con-
sent prior to participating. Subjects received $150 compensation.
2.2. Procedures
Women were tested three times, once during the early follic-
ular, once during the late follicular, and once during the luteal
phase of their menstrual cycles. Cycle phases were subsequent-
ly confirmed using serum estradiol and progesterone concen-
trations. Men were also tested three times, matched for
number of days between test sessions. Since ALLO-ir levels
are non-detectable in a substantial proportion of women in
their follicular phase (Girlder et al., 2001), only luteal phase
data are included in the present report. There were no signifi-
cant differences in the proportion of African American versus
non-Hispanic White women whose luteal phase session was the
first test session (9 vs. 6, respectively), second test session (5 vs.
9), or last test session (10 vs. 4; v2
ð2Þ¼3:78, p= .15).
One goal of the larger parent study (Mechlin et al., 2005)
was to examine stress-induced analgesia (SIA). Thus, pain test-
ing occurred twice; once following a modified Trier Social
Stress Test (TSST; Kirschbaum et al., 1993a,b, 1995a,b) and
once following a time equivalent rest control period. Half of
the subjects received the rest condition first, while the other
half received the stress condition first. The order of stress first
versus rest first was fully counter-balanced within gender and
ethnic groups. Analyses were limited to the pain sensitivity
measures obtained following the TSST (see Fig. 1) since
ALLO-ir and other neuroendocrine factors were assessed dur-
ing the extended baseline rest period that immediately preced-
ed the TSST. Since no blood samples were taken during the
baseline period that preceded the rest control period, measures
of pain sensitivity following the TSST were more temporally
contiguous with assessment of neuroendocrine factors.
For all subjects, lab testing began between 12pm and 2pm.
An intravenous line (i.v.) was then established in an arm vein
and once in place, a curtain was drawn to hide the i.v. and arm,
and to minimize awareness of blood sampling.
The sequence of laboratory events was as follows (see
Fig. 1): (1) Instrumentation and acclimation to the testing
chamber; (2) i.v. setup and recovery from venipuncture
(10 min); (3) Baseline (10 min quiet rest); (4) TSST (20 min);
(5) Recovery (10 min); (6) Pain Testing; These events are
described fully below.
2.2.1. Baseline
Immediately following the i.v. setup, a 10 min recovery
from venipuncture ensued, followed by a 10-min baseline rest
period. Blood was sampled at minute 10 of this rest period for
baseline ALLO-ir, cortisol and b-endorphin concentrations.
2.2.2. The Trier Social Stress Test (TSST)
The TSST is a stress test that reliably induces large and con-
sistent HPA and cardiovascular responses (Kirschbaum et al.,
1993a,b, 1995a,b). The TSST involved the following compo-
nents (see Mechlin et al., 2005 for detailed description of stress-
or events): (1) Pre-Task Instructions (5 min); (2) Speech
Preparation Period (5 min); (3) Job Speech (5 min); (4) Paced
Auditory Serial Addition Test (PASAT; Gronwall, 1977)
(8.5 min).
2.2.3. Stress recovery (10 min)
Subjects rested quietly alone. Blood was also sampled for
stress-induced increases in cortisol and ALLO-ir at the end
of this recovery period.
1
2.2.4. Pain testing procedures
Immediately after the recovery periods that followed the
TSST, subjects were exposed to the three different pain tests.
One of three task orders (i.e., 1– tourniquet, thermal, cold; 2
– thermal, cold, tourniquet; or 3 – cold, tourniquet, thermal)
was randomly assigned to each subject, ensuring that equal
numbers of men and women and African Americans and
non-Hispanic Whites received each of the three orders. There
was a 5-min recovery period following each pain test.
Instrument
/ i.v. set-up
Venipuncture
recovery
Baseline Trier Social
Stress Test (a)
Recovery
Pain
Testing
0 ' 10 ' 20 ' 45 ' 55 ' 100 '
Prep Speech Math
Rest
Control (b)
Recovery
Pain
Testing
120 ' 130 ' 175 '25 '
Instructions
(5 min)
Plasma ALLO-ir
cortisol, and
β-endorphin
Fig. 1. Schematic diagram representing the sequence of events during laboratory testing. Half of each ethnic and gender group had the order of stress
test and then rest control period as indicated above (a followed by b), while the other half had rest control first (b followed by a).
1
Relationships involving ALLO-ir, cortisol, and pain sensitivity as a
function of ethnicity did not differ in any appreciable way when
analyses were conducted using post-stress samples (post-stress samples
of b-endorphin were not obtained). However, preliminary data from
our laboratory indicated that the post-stress sample obtained for
ALLO-ir was delayed and did not capture the peak ALLO response to
stress (Girdler et al., unpublished data). Thus, in order to reduce
further Type I error rates, to increase the generalizability of our
findings to the existing literature on neuroendocrine factors and pain
perception, and because the ALLO response was not sampled when at
peak (Girdler et al. unpublished data), the present report focuses
exclusively on the relationship of resting baseline samples to pain
sensitivity.
144 B. Mechlin et al. / Pain 131 (2007) 142–152
2.2.4.1. The submaximal effort tourniquet procedure. In this
procedure, as described previously (Maixner et al., 1990), a
tourniquet cuff was positioned on the subject’s arm and the
arm placed to the side. Prior to inflating the tourniquet cuff
to 200 mmHg (Hokanson E20 Rapid Cuff Inflator), the sub-
ject’s arm was raised for 30 s to promote venous drainage,
and then the cuff was inflated, the experimenter’s stopwatch
started, and the arm returned to the side. To promote forearm
ischemia, subjects engaged in 20 handgrip exercises at 30% of
their maximum force with an inter-squeeze interval of 2 s. Sub-
jects were instructed to indicate when the sensations in their
arm first became painful (pain threshold) and when they were
no longer willing or able to tolerate the pain (pain tolerance).
A maximum time limit of 20 min was enforced, though
subjects were not informed of this limit.
2.2.4.2. Hand cold pressor. The apparatus for the cold pressor
consisted of a container filled with ice and water that was
maintained at 4 C as recorded immediately prior to initiating
the test. The use of a water circulator prevented the water from
warming near the subject’s hand. At the onset of the test, sub-
jects were instructed to submerge their hand to the marked line
on their wrist and to remain still. Subjects were instructed to
indicate to the experimenter when the sensations in their hand
first became painful (pain threshold) and to also indicate when
they were no longer willing or able to tolerate the pain by say-
ing ‘stop’ (pain tolerance). A maximum time limit of 5 min was
imposed, though subjects were not informed of this limit.
2.2.4.3. Thermal heat pain testing. Thermal heat pain threshold
and tolerance were determined by an ascending method of lim-
its using a 1-cm diameter contact thermode with the capability
for a rise time of 10 C/s (Medoc TSA-II Neurosensory Ana-
lyzer). The thermode was controlled by a personal computer,
and thermal probe applied to the left volar forearm. During
the pain testing, an adapting temperature of 38 C was main-
tained for 10 s. Then, the temperature increased directly to
41.5 C and from that point on increased 0.5 C every 5 s until
it reached 53 C or until the subject reached his/her tolerance.
To determine thermal pain onset (threshold), subjects were
instructed to press a mouse button (which terminated the stim-
ulus) when the thermal percept first became painful. This was
repeated three times and averaged to calculate thermal pain
thresholds. Then, three series to determine average thermal
pain tolerance were conducted by instructing the subject to
press a mouse button when they were no long able to tolerate
the pain.
2.2.5. Hormone and neuroendocrine assays
2.2.5.1. Plasma ALLO-ir. Plasma ALLO-ir (3a,5a-THP) was
assessed by radioimmunoassay (RIA) following extraction
and purification by column chromatography as previously
described (Janis et al., 1998; Girlder et al., 2001). The 3a,
5a-THP antiserum has previously been shown to produce min-
imal cross reactivity with other circulating steroids (Janis et al.,
1998). Cross-reactivity with progesterone (<3%), as well as the
stereochemical isomers of 3a,5a-THP, is minimal (3a,5b-THP
6.6%; 3b,5a-THP 2.8%; 3b,5b-THP 0.5%). In contrast, the
steroid 3a-hydroxy-4-pregnen-20-one binds to the antibody
to a greater degree than 3a,5a-THP (169% of 3a,5a-THP). It
is unknown, however, whether 3a-hydroxy-4-pregnen-20-one
exists in human serum. If the steroid does exist in human
serum, then it may contribute to the measurement of ALLO;
however, because both ALLO-ir and the pregnen-4 com-
pound are equally efficacious agonists of GABA
A
receptor
mediated Cl
-uptake (Morrow et al., 1990), they would be
expected to produce similar effects. The intra- and inter-assay
coefficients of variation from the assay are 6.2% and 5.9%,
respectively.
2.2.5.2. Plasma cortisol. Plasma cortisol was determined using
RIA techniques (MP Biomedicals, Inc.). The intra- and inter-
assay coefficients of variation from the assay are 4.7% and
7.6%, respectively. The sensitivity of the assay is 0.07 lg/dL,
and the specificity high, showing 0.05–2.2% cross-reactivity
with most similar compounds.
2.2.5.3. Plasma b-endorphin. Plasma b-endorphin was deter-
mined following extraction by RIA using a commercial kit
(INCSTAR Corp.). The intra- and inter-assay coefficients
of variation from the assay are approximately 10% and
15%, respectively, and the assay sensitivity is 3 pmol/L. There
is less than 0.01% cross-reactivity with most other peptides.
Since b-endorphin is released in a pulsatile manner, and
therefore can vary greatly over time, b-endorphin concentra-
tions were averaged across all three test sessions, and this
average b-endorphin concentration was used in subsequent
analyses.
2.3. Data reduction and analyses
Our first analytical strategy involved comparing groups for
differences that existed in demographic and baseline measures.
For each dependent measure, a 2(gender) ·2(ethnicity) analy-
sis of variance (ANOVA) was employed. Chi-square analyses
were also employed where indicated to examine ethnic differ-
ences in the proportion of subjects falling into various cells.
In order to investigate the relationship between ALLO and
pain tolerance, as well as the relationships involving b-endor-
phin, cortisol, and pain tolerance, a series of Pearson product
moment correlational analyses were employed. Since our prior
report (Mechlin et al., 2005) documented that ethnic differences
in pain sensitivity were evident in both genders, and since ethnic
differences existed only for measures of pain tolerance and not
pain threshold, to reduce Type I error rates our primary anal-
yses focused exclusively on differences between African Ameri-
cans and non-Hispanic Whites, collapsing across gender
though summary statistics by gender are provided in Table 1.
Results revealed a significant order effect for sensitivity to
the cold pressor task (F(1, 83) = 4.15, p< .05), since cold pres-
sor tolerance was lowest for subjects who had the order ther-
mal, cold pressor, tourniquet (mean cold pressor
tolerance = 19 s; standard error of the mean (SEM) = 2), com-
pared with those who had the order cold pressor, tourniquet,
and thermal (mean cold pressor tolerance = 39 s; SEM = 11),
and those who had the order tourniquet, thermal then cold
pressor (mean cold pressor tolerance = 79 s; SEM = 20).
Therefore, order of pain testing was partialed out of all corre-
lational analyses involving pain tolerance for all three pain
tasks, and was used as a covariate for all ANOVAs involving
pain sensitivity.
B. Mechlin et al. / Pain 131 (2007) 142–152 145
Where significant inter-correlations involving ALLO-ir,
pain tolerance and either cortisol or b-endorphins emerged,
we used the approach recommended by Baron and Kenny
(1986), including the Sobel test, which takes the standard error
of regression coefficients into account, and conducted media-
tional analyses to test whether the relationship between
ALLO-ir and pain tolerance was influenced by either cortisol
or b-endorphin concentrations. This analysis generates a Stu-
dent’s tvalue, an unstandardized regression coefficient (b)
which is the value of predicted change in the dependent vari-
able given a one unit change in the independent variable of
interest when all other independent variables are held constant,
and a standardized regression coefficient (b) is similar to an
unstandardized regression coefficient, however, since it is stan-
dardized it allows for the comparison between independent
variables of their relative contribution to the dependent vari-
able, for each variable in the model. Additionally, the percent-
age of variance of the dependent variable accounted for by the
independent variable and mediator (R
2
) is obtained, as well as
az-statistic from the Sobel test, which when significant indi-
cates that the mediator does in fact carry an influence from
the independent variable to the dependent variable.
3. Results
3.1. Baseline characteristics
As summarized in Table 1, women in the luteal phase
of their menstrual cycle had significantly higher levels of
ALLO-ir than men (F(1, 82) = 119.12, p< .0001). Men
had higher concentrations of b-endorphin than women
(F(1, 83) = 23.23, p< .0001). As previously reported in
the larger sample (Mechlin et al., 2005), overall the sub-
set of men also had greater cortisol concentrations than
women (F(1, 82) = 25.81, p< .0001), greater pain thresh-
old (Fs(1,82) = 2.51–7.63, ps < .05) and tolerance to all
three pain tests (Fs(1, 82) = 4.94–12.13, ps < .01) col-
lapsing across ethnic groups (i.e., main effect of gender)
even after controlling for order of pain tests. Finally, as
previously observed in the larger sample, African Amer-
icans had lower tolerance values to all three pain tests
relative to non-Hispanic Whites, and this was true for
both genders and after controlling for order (main effect
of ethnicity: Fs(3, 92) = 3.32–10.53, ps < .05). There was
no main effect of ethnicity on pain threshold for the
thermal or tourniquet tasks (Fs(2, 81) = 1.04 and 0.82,
respectively), however there was a difference for cold
pressor pain threshold (F(2, 81) = 4.17, p< .01), with
African Americans exhibiting a lower pain threshold
than non-Hispanic Whites.
For the cold pressor task, significantly more non-
Hispanic White subjects than African American subjects
reached the 5-min time limit (7 vs. 0; v2
ð1Þ¼11:34,
p< .01). There were no ethnic differences in the propor-
tion of subjects that reached the 20-min time limit for
the tourniquet task (3 vs. 6; v2
ð1Þ¼1:75) or the 53 C
limit for the thermal heat pain task (1 vs. 0;
v2
ð1Þ¼1:19). When comparing subjects who had rest first
versus stress first, there were no significant differences in
thermal pain tolerance (48.4 C vs. 48.3 C, respective-
ly), cold pressor tolerance (62 s vs. 35 s), tourniquet pain
tolerance (524 s vs. 426 s), baseline ALLO-ir (0.91
ng/mL vs. 0.85 ng/mL), baseline cortisol (7.63 pg/mL
vs. 8.76 pg/mL), or baseline b-endorphin (7.96 pg/mL
vs. 7.49 pg/mL). Therefore, subsequent analyses did
not control for the order of stress versus rest.
3.2. Relationships involving neuroendocrine factors and
pain sensitivity
As summarized in Table 2, the only significant partial
correlations (partialing out order of pain tests) to
emerge involving plasma ALLO-ir, b-endorphin or
cortisol concentrations and pain tolerance were seen in
the non-Hispanic White subjects. ALLO-ir levels were
negatively correlated with thermal heat pain tolerance
(r=.47, p< .01), cold pressor pain tolerance
(r=.35, p< .05), and ischemic pain tolerance (r=
.43, p< .01) in the non-Hispanic Whites. We observed
a positive partial correlation between plasma b-endor-
phin concentrations and cold pressor tolerance levels
(r= +.38, p< .05), but only in the non-Hispanic Whites,
Table 1
Mean (±SEM) Biological and Pain Sensitivity Measures as a Function of Ethnicity and Gender
African American
Females (n= 24)
Non-Hispanic White
Females (n= 19)
African American
Males (n= 21)
Non-Hispanic White
Males (n= 20)
Baseline ALLO-ir
a
(ng/mL) 1.25 (0.08) 1.50 (0.10) 0.36 (0.09) 0.35 (0.09)
Baseline cortisol
b
(pg/mL) 6.85 (0.67) 5.92 (0.75) 9.27 (0.72) 11.01 (0.73)
Baseline b-endorphin
b
(pg/mL) 6.47 (0.60) 5.84 (0.69) 9.61 (0.65) 9.00 (0.67)
Thermal heat threshold
b
(C) 42.70 (0.59) 42.45 (0.69) 44.39 (0.64) 45.53 (0.65)
Thermal heat tolerance
b,c
(C) 46.88 (0.37) 48.02 (0.44) 48.47 (0.41) 50.26 (0.42)
Ischemic threshold
b
(s) 222 (51) 239 (62) 332 (56) 333 (59)
Ischemic tolerance
b,c
(s) 336 (67) 431 (78) 478 (73) 739 (75)
Cold pressor threshold
b,c
(s) 6 (4) 10 (4) 12 (4) 28 (4)
Cold pressor tolerance
b,c
(s) 12 (14) 42 (16) 25 (15) 121 (15)
a
Females > males, p< .0001.
b
Males > females, p< .05.
c
Non-Hispanic Whites > African Americans, p< .01.
146 B. Mechlin et al. / Pain 131 (2007) 142–152
who also showed the expected positive correlation
between plasma cortisol and thermal heat pain tolerance
(r= +.39, p< .05) and cold pressor pain tolerance
(r= +.32, p< .05). The partial correlation coefficients
relating ALLO-ir, b-endorphin, or cortisol concentra-
tions to pain tolerance were uniformly non-significant
in the African Americans.
In non-Hispanic Whites, thermal heat pain threshold
was significantly correlated with both ALLO-ir
(r=.49, p< .01) and cortisol (r= +.50, p< .01), and
ischemic pain threshold was correlated with b-endor-
phin (r= +.37, p< .05). There were no significant par-
tial correlations involving pain threshold and
biological measures in African Americans (rs=.29
to +.24, ps > .05).
As depicted in Table 3, the expected relationship
between ALLO-ir concentrations and cortisol concen-
trations (i.e., negative) was only significant in non-His-
panic Whites (r=.50, p< .01; see Fig. 2), though the
direction of the relationship was similar in the African
Americans (r=.24; Fig. 2), but it was not significant.
Both groups showed negative correlations involving
ALLO-ir and b-endorphin concentrations (rs=.44
and .55, ps < .01; see Fig. 3), while neither group
showed a statistically significant relationship involving
plasma cortisol and b-endorphin.
3.3. Mediational analyses
Since only the non-Hispanic Whites showed signifi-
cant correlations among ALLO-ir, cortisol, b-endor-
phin, and pain tolerance, mediational analyses were
conducted in that sample only (Baron and Kenny,
1986). Because baseline cortisol and b-endorphin were
not correlated with each other, they were not considered
together in multiple mediation models but instead each
was examined as a simple mediator of the relationship
between ALLO-ir and pain tolerance. Despite the
numerous intercorrelations between ALLO-ir, cortisol,
b-endorphin, and pain tolerance in non-Hispanic
Whites, the only measure to meet strict criteria as a
mediator in the relationship between ALLO-ir and pain
sensitivity was cortisol.
For non-Hispanic Whites, since baseline ALLO-ir
was negatively correlated with baseline cortisol
(r=.50, p< .01), and since both baseline ALLO-ir
and baseline cortisol were correlated with thermal heat
pain threshold (rs=.49 and +.50, respectively,
ps < .01), a series of linear regression analyses were per-
formed (Baron and Kenny, 1986) to test whether corti-
sol served as a statistical mediator of the relationship
between ALLO-ir and thermal heat pain threshold.
These analyses indicated that the significant regression
Table 2
Partial correlations relating pain tolerance to neuroendocrine measures by ethnicity (controlling for order of pain tests)
Thermal
threshold
Thermal
tolerance
Cold pressor
threshold
Cold pressor
tolerance
Ischemic
threshold
Ischemic
tolerance
African Americans (n = 45)
Baseline ALLO-ir r=.24 r=.22 r=.29 r=.17 r=.18 r=.19
p=ns p=ns p=ns p=ns p=ns p=ns
Baseline cortisol r= +.18 r= +.21 r=.05 r= +.04 r= +.12 r= +.15
p=ns p=ns p=ns p=ns p=ns p=ns
Baseline b-endorphin r= +.18 r= +.14 r= +.24 r= +.18 r= +.14 r= +.02
p=ns p=ns p=ns p=ns p=ns p=ns
Non-Hispanic Whites (n = 39)
Baseline ALLO-ir r=.49
**
r=.47
**
r=.24 r=.35
*
r=.24 r=.43
**
p< .01 p< .01 p=ns p< .05 p=ns p< .01
Baseline cortisol r= +.50
**
r= +.39
*
r= +.24 r= +.32
*
r= +.15 r= +.19
p< .01 p< .05 p=ns p< .05 p=ns p=ns
Baseline b-endorphin r= +.07 r= +.06 r= +.24 r= +.38
*
r= +.37
*
r= +.22
p=ns p=ns p=ns p< .05 p< .05 p=ns
*
p< .05.
**
p< .01.
Table 3
Relationship of neuroendocrine factors as a function of ethnicity
African Americans (n= 45) Non-Hispanic Whites (n= 39)
Baseline ALLO-ir Baseline cortisol Baseline ALLO-ir Baseline cortisol
Baseline ALLO-ir – r=.24 – r=.50
**
–p=ns – p< .01
Baseline b-endorphin r=.44
**
r=.01 r=.55
**
r= +.30
p< .01 p=ns p< .01 p=ns
**
p< .01.
B. Mechlin et al. / Pain 131 (2007) 142–152 147
coefficient relating baseline ALLO-ir to thermal heat
pain threshold (t=2.85, p< .01; b=1.72;
b=0.43; R
2
= 0.16, p< .01) is reduced (t=1.34,
p> .05; b=0.85; b=0.21) and in fact no longer
significant, when baseline cortisol (t= 2.80, p< .01;
b= 0.34; b= 0.44) is added to the model. Thus, higher
baseline concentrations of cortisol met criteria for
mediating the relationship between lower baseline
ALLO-ir and higher thermal heat pain threshold
(z=2.18, p< .05). Baseline cortisol and baseline
ALLO-ir concentrations together accounted for 30%
of the variance in thermal heat pain tolerance
(F(2,35) = 8.74, p< .001).
When examining the relationship between ALLO-ir,
cortisol, and thermal heat pain tolerance; ALLO-ir,
cortisol, and cold pressor pain tolerance; and ALLO-
ir, b-endorphin, and cold pressor pain tolerance, analy-
ses indicated that cortisol did not meet statistical criteria
as a mediator in these relationships involving cortisol
and pain sensitivity.
4. Discussion
The results of our study, which is the first to examine
the relationship of plasma ALLO-ir concentrations to
pain sensitivity in humans, suggest that there may be
ethnic differences in the degree to which ALLO interacts
with HPA-axis variables, cortisol and b-endorphin, to
influence pain perception.
4.1. Higher ALLO concentrations are associated with
lower pain tolerance
Contrary to expectations, and inconsistent with ani-
mal studies showing that increased concentrations of
GABAergic neurosteroids, including ALLO-ir, are asso-
ciated with a decrease in pain sensitivity (Kavaliers and
Wiebe, 1987), we found that higher ALLO-ir concentra-
tions were associated with increased pain sensitivity
(lower pain tolerance), at least in non-Hispanic Whites,
and this was true for all three pain tests. One possibility
for this discrepancy is that in the animal models, animals
are tested for pain sensitivity following the administra-
tion of exogenous ALLO (Kavaliers and Wiebe, 1987;
Wiebe and Kavaliers, 1988; Frye and Duncan, 1994),
while our study examined the relationship of endoge-
nous levels of ALLO-ir and pain perception. Thus, the
animal studies may have elicited supraphysiologic con-
centrations of ALLO. Since there is evidence for a
bimodal effect of ALLO concentration on GABA
A
-reg-
ulated mood in humans (Miczek et al., 1993, 1997, 2003;
Andre
´en et al., 2005), we speculate that lower endoge-
nous concentrations of ALLO, such as those seen during
the luteal phase, may be associated with enhanced sensi-
tivity to pain, while higher concentrations of ALLO,
such as seen during pregnancy, may be analgesic.
0121620
Baseline Cortisol (pg/mL)
0
1
2
3
Baseline ALLO-ir (ng/mL)
r = -.50
p < .01
Baseline Cortisol (p
g
/mL)
0
1
2
3
Baseline ALLO-ir (ng/mL)
r = -.24
p > .10
48
0121620
48
Fig. 2. A scatterplot of the relationship between baseline levels of
ALLO-ir and Cortisol separated by ethnicity with non-Hispanic
Whites displayed in the top panel, and African Americans displayed in
the bottom panel.
01215
Baseline Beta-Endorphin (pg/mL)
0.00
0.80
1.60
2.40
3.20
4.00
Baseline ALLO-ir (ng/mL)
r = -.55
p < .01
Baseline Beta-Endorphin (p
g
/mL)
0.00
0.80
1.60
2.40
3.20
4.00
Baseline ALLO-ir (ng/mL)
r = -.44
p < .01
369
01215
369
Fig. 3. A scatterplot of the relationship between baseline levels of
ALLO-ir and b-endorphin with non-Hispanic Whites displayed in the
top panel, and African Americans displayed in the bottom panel.
148 B. Mechlin et al. / Pain 131 (2007) 142–152
Dose–response studies in humans examining different
ALLO-ir concentration profiles and pain sensitivity will
be needed to test this hypothesis.
4.2. The relationship between ALLO and pain sensitivity
may be mediated by HPA-axis factors
Another, though not mutually exclusive, possibility
for finding an inverse relationship between ALLO-ir
concentrations and pain tolerance is indicated by our
mediational analyses conducted in the non-Hispanic
Whites. A number of prior studies have shown that mea-
sures reflecting increased activation of the HPA-axis,
specifically higher plasma cortisol and higher plasma
b-endorphin concentrations, are related to reduced sen-
sitivity to experimental pain procedures, including cold
pressor, ischemic, and thermal heat pain (al’Absi et al.,
2002; Straneva et al., 2002; Girdler et al., 2005; Mechlin
et al., 2005). We also observed significant positive corre-
lations involving greater cortisol and b-endorphin con-
centrations and greater tolerance to thermal heat and
cold pressor pain in the non-Hispanic Whites.
2
The
results of our mediational analyses extend the literature
on HPA-axis activation and pain perception by provid-
ing the first evidence in humans for an interrelationship
between ALLO and cortisol to influence pain sensitivity.
Though we found that lower ALLO-ir concentrations
were related to higher pain tolerance to all three pain
tests in non-Hispanic Whites, mediational analyses
indicated that at least for the thermal pain test, the
inverse relationship was mediated by greater plasma
cortisol.
While the exact mechanisms by which cortisol medi-
ates the relationship between ALLO and pain sensitivity
are unknown, one possibility involves the negative mod-
ulation of the HPA-axis by ALLO. Animal models dem-
onstrate that increases in ALLO facilitate the return of
HPA-axis activation to homeostasis following stress
(Guo et al., 1995; Patchev et al., 1996). Activation of
the HPA-axis with the associated pituitary release of
b-endorphin and/or increased corticotrophin-releasing
hormone (CRH) activity may be part of an integrated
adaptive mechanism since both are associated with
reduced pain sensitivity in humans (Hargreaves et al.,
1987; Rosa et al., 1988; Sheps et al., 1992; Lariviere
and Melzack, 2000; Straneva et al., 2002). It is now well
established that CRH acts on a large number of brain
structures involved in pain processing, including the
locus coeruleus, and that it can act both centrally and
peripherally to produce analgesia (see Lariviere and
Melzack, 2000 for review). Thus, since ALLO and corti-
sol are negatively related (Girlder et al., 2001; Girdler
et al., 2006), and ALLO modulates the HPA-axis at
multiple levels (Owens et al., 1992; Patchev et al.,
1994; Patchev et al., 1996; Calogero et al., 1998), higher
circulating ALLO would be expected to be associated
with increased pain sensitivity as we documented in
the non-Hispanic Whites. Regardless of mechanism,
ALLO-associated hyperalgesia in women could contrib-
ute to greater experimental pain sensitivity when they
are tested in their luteal versus follicular phase of the
menstrual cycle (Fillingim et al., 1997; Pfleeger et al.,
1997; Riley et al., 1999).
4.3. Ethnic differences in the relationship of ALLO to pain
sensitivity
Also regardless of mechanism, and in contrast to our
findings in the non-Hispanic Whites, our findings that
significant interrelationships involving ALLO, cortisol,
and pain sensitivity were not observed in African Amer-
icans are consistent with our other finding from this
same cohort indicating ethnic differences in the relation-
ship between pain sensitivity and stress-responsive bio-
logical factors. We had previously suggested (Mechlin
et al., 2005) that the ethnic differences in the relationship
involving plasma cortisol, NE, and BP and pain sensitiv-
ity may contribute to the greater clinical pain severity
(Mechlin et al., 2005) experienced by African Ameri-
cans. We have now extended those findings to include
ethnic differences in the relationships between both
ALLO and b-endorphin and pain sensitivity. At the
same time, it must be acknowledged that many of the
observed correlation coefficients in African Americans
involving pain tolerance and ALLO as well as the
HPA-axis variables were similar in direction to those
observed in the non-Hispanic Whites, though they did
not reach conventional levels of significance (Table 2).
While it is probable that these correlation coefficients
would have reached statistical significance if larger num-
bers of African Americans were included, low statistical
power to detect significant relationships cannot be the
sole or even primary explanation for the ethnic differenc-
es in the pattern of effects since our African American
sample was larger than the non-Hispanic White sample
and there was no evidence for ethnic differences in the
variability of the neuroendocrine or pain sensitivity
measures. Still, these findings should be replicated in
larger samples of African American and non-Hispanic
Whites. If confirmed, these results may have implica-
tions for the treatment of clinical pain in African
Americans.
2
It may be important to note that the relationship between baseline
cortisol and increased thermal heat and cold pressor pain tolerance in
non-Hispanic Whites in the present report was not found in our prior
report (Mechlin et al., 2005) from the larger sample. One distinct
possibility for this discrepancy concerns the influence of the menstrual
cycle on the HPA-axis (Kirschbaum et al., 1999; Kowalczyk et al.,
2006) since all samples included in the present report came from
women in the luteal phase of their menstrual cycle, while the prior
report, which focused on the first test session only, included women in
various stages of their cycles.
B. Mechlin et al. / Pain 131 (2007) 142–152 149
4.4. Limitations
There are also several limitations of the study, which
should be addressed. The primary limitation to this
report stems from the fact that the subjects represent a
large subset of those previously on which we previously
reported, where we observed the absence of relation-
ships involving plasma cortisol, NE, and BP and pain
sensitivity in African Americans. Thus, while the current
report extends our observations to include the absence
of relationships involving plasma ALLO-ir and b-endor-
phin with pain sensitivity in African Americans, the pos-
sibility exists that these findings are unique to this
particular cohort of African Americans for whatever
reason. Replication of these general findings in other
cohorts of African Americans will be needed before
any interpretations regarding ethnic differences in
endogenous pain regulatory mechanisms can or should
be made. Second, we relied exclusively on baseline and
not post-stress samples and this may account for the
unexpected inverse relationship we observed between
ALLO and pain tolerance in humans. Third, although
the intercorrelations involving ALLO-ir, cortisol, and
b-endorphin were significant in the non-Hispanic
Whites, the large range of values, particularly for corti-
sol and b-endorphin, may limit the predictability of
these measures. Fourth, another limitation to our study
relates to the use of laboratory testing procedures to
make inferences regarding clinical pain. For example,
although both male and female experimenters were ran-
domly used, only non-White Hispanic experimenters
administered the pain tests. Although the effect of the
experimenter’s ethnicity on pain tolerance is equivocal
(Zatzick and Dimsdale, 1990; Weisse et al., 2005), the
possibility does exist that the ethnicity of the experi-
menter or other non-specific effects of a laboratory envi-
ronment could have influenced our findings. Finally,
while one strength of our study was the use of multiple
pain tests, this strength was balanced by our finding of
an order effect involving sensitivity to the cold pressor
test. Though we did statistically control for the order
effects in all analyses, the reason for these effects remains
unexplained. Regardless of the reason for this finding, it
suggests the possibility for carryover effects from one
procedure to another in human clinical research that
should be carefully conducted and controlled for in sub-
sequent studies.
5. Conclusion
Despite the limitations to our study, the novel focus
of the relationship of ALLO to pain sensitivity in
humans, and the interactions involving the HPA-axis
are worth underscoring and may serve as important ear-
ly observations involving unfamiliar pain regulatory
mechanisms in humans. Our results suggest that lower
physiologic concentrations of plasma ALLO are
associated with increased pain threshold and tolerance
in non-Hispanic Whites, and that for thermal heat pain
threshold the relationship is mediated by higher cortisol
concentrations. The fact that we did not observe any
significant relationships involving ALLO-ir, cortisol or
b-endorphins and pain sensitivity in African Americans
adds to our prior report documenting ethnic differences
in the relationship of plasma NE, cortisol, and BP to
pain sensitivity (Mechlin et al., 2005), though, these
findings need to be replicated in separate cohorts of
African Americans and non-Hispanic Whites.
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