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Indicators of Repeated Waking Cortisol

Measurements

DOI: 10.1097/PSY.0b013e31814c405c Source: PubMed

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Area Under the Curve and Other Summary Indicators of Repeated Waking

Cortisol Measurements

DESTA B. FEKEDULEGN, PHD, MICHAEL E. ANDREW, PHD, CECIL M. BURCHFIEL, PHD, JOHN M. VIOLANTI, PHD,

TARA A. HARTLEY, MPH, LUENDA E. CHARLES, PHD, AND DIANE B. MILLER, PHD

Objective: To derive the area under the curve and related summary measures of stress from saliva samples collected over time and

to provide insight into the interpretation of the derived parameters. In research designed to assess the health consequences of stress

these samples are often used as a physiologic indicator of the responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis. To

make these repeated measurements of salivary cortisol more useful in defining the relationships between stress and health there is

a need to derive two forms of area under the curve that summarize the measurements: area under the curve with respect to ground

(AUCG) and area under the curve with respect to increase (AUCI). The latter parameters, AUCI, however, is seldom used by

research scientists. Methods: In this study, interpretation and generic definition of the area under the curve was provided through

graphical analyses and examination of its association with other summary measures using data from the Buffalo Cardio-Metabolic

Occupational Police Stress (BCOPS) Pilot Study. In generic form, AUCI is derived as the area under the curve above the baseline

value minus the area above the curve below the baseline value. Results: The sign and magnitude of AUCI are related to the profile

and the rate of change of the measurements over time. The parameter showed significant associations with other summary indicators

that measure pattern or rate of change of the measurements over time. Conclusion: Principal components analyses revealed that

summary parameters derived from repeated cortisol measurements can be grouped into two meaningful general categories:

measures of the magnitude of response and measures of the pattern of response over time. Key words: repeated cortisol

measurements, area under the curve, principal component analysis, total hormonal secretion, time course of salivary cortisol.

AUCG area under the curve with respect to ground; AUCI area both pieces of information can be captured through the use of

under the curve with respect to increase; AUCB area under the AUC. The use of AUC simplifies the statistical analyses by

curve with respect to baseline; HPA hypothalamic-pituitary-adre- transforming the multivariate data into univariate space, espe-

nal; PCA principal component analyses. cially when the numbers of repeated measurements are high

and there is a need to summarize the information (7). This

INTRODUCTION

approach also reduces the number of statistical comparisons

M any studies utilizing the salivary cortisol measure as a

physiologic indicator of the responsiveness of the hy-

pothalamic-pituitary-adrenal (HPA) axis to determine the

between groups, which minimizes the need for adjustment of

the significance level. With AUC, the number of statistical

comparisons only depends on the number of groups to be

health consequences of stress obtain these cortisol values in

compared, as opposed to the original repeated data. In addi-

samples collected over time from the same subject. In research

tion, when the time interval between repeated measurements is

involving repeated measurements of a response variable, there

not identical, the use of AUC provides an alternative because

is a need to derive parameters that summarize the information

repeated measures analysis of variance, using the original data,

contained in the multivariate data. The area under the curve

has no proven method to adjust for these differences (10).

(AUC) computed with the trapezoidal formula is one of the

A recent study by Pruessner et al. (10) provides a simple

widely used parameters (19) but has had little application in

formula for the computation of two types of AUC that reveal

work involving repeated measurements of salivary cortisol.

different information embodied in the repeated multivariate

In repeated data, each measurement is comprised of two

measurements (Figure 1B): area under the curve with respect

types of information (Figure 1A); its distance from the ground

to ground (AUCG) and area under the curve with respect to

(i.e., the intensity or magnitude of the response) and its dis-

increase (AUCI). The formulas are basically simple additions

tance from its neighbor (i.e., changes over time). Unlike other

of areas consisting of triangles and rectangles. AUCG is the

summary measures (e.g., baseline value, average, maximum),

total area under the curve of all measurements. It takes into

account both sensitivity (the difference between the single

From the Biostatistics and Epidemiology Branch (D.B.F., M.E.A., C.M.B.,

T.A.H., L.E.C.), Toxicology and Molecular Biology Branch (D.B.M.), Health

measurements from each other) and intensity (the distance of

Effects Laboratory Division, National Institute for Occupational Safety and these measures from ground). AUCI is calculated with refer-

Health, Centers for Disease Control and Prevention, Morgantown, West ence to the baseline measurement and it ignores the distance

Virginia; Department of Social and Preventive Medicine (J.M.V.), School of

Public Health and Health Professions, State University of New York at

from zero for all measurements and emphasizes the changes

Buffalo, Buffalo, New York. over time. With endocrinological data, AUCG is assumed to

Address correspondence and reprint requests to Desta B. Fekedulegn, be a measure more related to total hormonal output, whereas

Biostatistics and Epidemiology Branch, National Institute for Occupational

Safety and Health, HELD/BEB, MS 4050, 1095 Willowdale Rd., Morgan- AUCI is a parameter that emphasizes the changes over time

town, WV 26505. E-mail: djf7@cdc.gov and is more related to sensitivity of the system.

Received for publication October 30, 2006; revision received May 10, Despite the simple computational formula for AUCI, the

2007.

This work was supported by Contract No. 200 to 2003 to 01580 from the parameter has had limited application in research compared

National Institute for Occupational Safety and Health. The findings and with AUCG. This could be attributed to the lack of a generic

conclusions in this report are those of the authors and do not necessarily formula and clear interpretation of the parameter and the

represent the views of the National Institute for Occupational Safety and

Health. limited literature on its association with other summary indi-

DOI: 10.1097/PSY.0b013e31814c405c cators. In addition, graphical representation of the region

0033-3174/07/6907-0651

Copyright 2007 by the American Psychosomatic Society

D. B. FEKEDULEGN et al.

where one or more of the measurements have values lower

than the baseline measurement. The main objectives of this

study, therefore, are to provide more insight into the interpre-

tation of AUCI by defining a generic formula for AUCI

through graphical analyses, to study its association with other

well defined cortisol parameters known to measure different

aspects of the multivariate data, and to select optimal param-

eters that capture meaningful information about the original

data using dimension reduction analytical techniques.

METHODS

Artificial Data

The study used two different data sets for analyses. First, a simulated data set

(Table 1) representing a variety of different waking cortisol measurement patterns

(at awakening, 15, 30, and 45 minutes after waking) was used to derive a generic

formula for AUCI and to demonstrate how the sign and magnitude of AUCI

changes with various cortisol profiles. The data were also used to illustrate the

parameters (summary measures) that can be computed to describe the information

contained in the waking measurements. The derived parameters include the

following: average of the four waking cortisol measurements (AVE); area under

the curve with respect to the baseline (AUCB); area under the curve with respect

to the ground (AUCG); area under the curve with respect to increase (AUCI); area

under the curve above the baseline (AUCAB); peak cortisol (PK); time in minutes

from baseline to peak (TBP); slope from baseline to peak (SBP); reactivity (RT);

slope of the line through the baseline and last measurement (SP1); intercept of the

regression line fitted through the raw cortisol data (INT); slope of the regression

line fitted through the raw cortisol data (SP2); and area under the curve of the

regression line fitted through the raw cortisol data (AUR).

Figure 1. Plot of repeated measurements indicating magnitude of response Average cortisol is the average of the four measurements. Peak cortisol is

or intensity (I1, I2, and I3) at each time point, changes in the response over the maximum value of the measurements regardless of when it occurred. Time

time or sensitivity (S1 and S2) (A) and the three forms of AUC (B). AUC from baseline, where baseline is the measurement at awakening, to peak is the

area under the curve.

TABLE 1. Simulated Data on Waking Cortisol Measurements at Awakening, 15, 30, and 45 Minutes After Waking and Derived Summary

Measures of the Data

Subjects (8 cases)

Characteristics

(a) (b) (c) (d) (e) (f) (g) (h)

Cortisol measurements

At awakening 9.60 9.60 9.60 9.60 14.1 14.1 14.1 14.1

15 min after waking 13.4 19.0 13.4 10.2 13.4 11.1 10.0 3.00

30 min after waking 16.5 11.1 11.1 9.10 11.1 9.10 6.10 6.10

45 min after waking 17.2 7.20 7.20 6.40 9.40 7.10 2.40 9.50

Derived cortisol parameters

AVE 14.18 11.73 10.33 8.83 12.00 10.35 8.15 8.18

AUCB 432 432 432 432 635 635 635 635

AUCG 650 578 494 410 544 462 365 314

AUCI 218 146 62 23 91 173 269 321

AUCAB 218 157 73 7 0 0 0 0

PK 17.20 19.00 13.40 10.20 14.10 14.10 14.10 14.10

TBP 45.00 15.00 15.00 15.00 0.00 0.00 0.00 0.00

SBP 0.17 0.63 0.25 0.04 0.00 0.00 0.00 0.00

RT 7.60 2.40 2.40 3.20 4.70 7.00 11.7 4.60

SP1 0.17 0.05 0.05 0.07 0.10 0.16 0.26 0.10

INT 10.29 13.99 11.75 10.43 14.46 13.80 14.00 9.78

SP2 0.17 0.10 0.06 0.07 0.11 0.15 0.26 0.07

AUR 638 528 465 397 540 466 367 368

AVE average of the four waking cortisol measurements; AUCB area under the curve with respect to the baseline; AUCG area under the curve with respect

to the ground; AUCI area under the curve with respect to increase; AUCAB area under the curve above the baseline; PK peak cortisol; TBP time in

minutes from baseline to peak; SBP slope from baseline to peak; RT reactivity (the change in salivary cortisol during the observation period); SP1 slope

of the line through the baseline and last measurement; INT intercept of the regression line fitted through the raw cortisol data; SP2 slope of the regression

line fitted through the raw cortisol data; AUR area under the curve of the regression line fitted through the raw cortisol data.

ANALYSES OF AWAKENING CORTISOL MEASUREMENTS

number of minutes from the baseline to the peak value. Slope from baseline TABLE 2. Descriptive Summary Statistics of the Waking Cortisol

to peak is the slope of the line connecting the baseline to the peak value. The Response and Derived Cortisol Parameters, Buffalo Cardio-Metabolic

reactivity was defined as the change in salivary cortisol during the observation Occupational Police Stress (BCOPS) Pilot Study, 20012003

period and was calculated as the difference between the first and the last

sample (sample 4 sample 1). Slope of the line through the baseline and last Characteristics Minimum Maximum Mean SD

measurement is a scaled version of reactivity; it is reactivity divided by time

in minutes from baseline to last measurement. Cortisol measurements

Previous studies (3,11) used the slope from a fitted regression model and (n 68)

the time-weighted average to describe the pattern and the amount of cortisol At awakening 0.1 49.4 13.5 10.5

secreted, respectively. Hence, the last three parameters (INT, SP2, and AUR) 15 min after waking 0.1 74.5 16.6 13.7

are derived after fitting a simple linear regression model to the waking data. 30 min after waking 0.9 57.1 15.4 11.5

The area under the regression line (AUR) was computed by using the 45 min after waking 0.1 55.4 14.1 10.3

estimated equation and integrating the resulting function as follows: Derived parameters

(n 68)

AVE 0.9 47.0 14.9 9.9

AUR a bxdx a b

2

2 AUCB

AUCG

4.5

58.5

2223.0

2244.0

641.4

725.6

480.1

482.7

0

AUCI 670.5 834.8 84.2 278.1

AUCAB 0.0 834.8 167.5 195.7

where is the time interval in minutes from the baseline measurement to the PK 1.6 74.5 20.8 14.7

last measurement, x is time from baseline (predictor variable), a is the TBP 0.0 90.0 24.3 19.3

intercept, and b is the slope of the fitted regression line. SBP 0.0 1.7 0.3 0.4

RT 34.5 29.6 0.6 11.6

Data From BCOPS Study SP1 0.8 0.7 0.0 0.3

Endocrinological data from a recent study with 100 police officers were INT 1.4 63.7 14.9 12.2

chosen to study the association of AUCI with other cortisol parameters. SP2 1.0 0.7 0.0 0.3

Participants are 100 police officers from Buffalo, New York, who were AUR 57.6 2112.8 710.7 462.6

randomly selected in the Buffalo Cardio-Metabolic Occupational Police

Stress (BCOPS) Pilot Study and were examined between 2001 and 2003. The SD standard deviation; AVE average of the four waking cortisol

study design, methods, and participant characteristics are described by Vio- measurements; AUCB area under the curve with respect to the baseline;

lanti and colleagues (12). Cortisol was measured in saliva samples and was AUCG area under the curve with respect to the ground; AUCI area under

analyzed using a competitive chemiluminescence immunoassay technique. the curve with respect to increase; AUCAB area under the curve above the

The participants collected 13 saliva samples at various times during a 3-day baseline; PK peak cortisol; TBP time in minutes from baseline to peak;

period. On the second day, awakening cortisol measures were obtained from SBP slope from baseline to peak; RT reactivity (the change in salivary

four salivary samples taken at 15-minute intervals on first awakening (at cortisol during the observation period); SP1 slope of the line through the

awakening, 15, 30, and 45 minutes after waking). Morning cortisol response baseline and last measurement; INT intercept of the regression line fitted

is widely studied because it serves as a useful index of adrenocortical activity. through the raw cortisol data; SP2 slope of the regression line fitted through

Hence, the waking data were used for statistical analyses in this study. From the raw cortisol data; AUR area under the curve of the regression line fitted

the 100 participants, 68 subjects with complete (nonmissing) data on all four through the raw cortisol data.

waking cortisol samples and times of measurement were used in this study.

Summary statistics of the waking measurements and the derived parameters

for the BCOPS waking data are shown in Table 2. generic definition of AUCI suitable to all patterns. The first

We examined the association between the area measures (AUCI and AUCG) scenario (Figure 2) illustrates a pattern where all subsequent

and the other cortisol parameters by using correlation and cluster analyses. We measurements are greater than the baseline value. For such pat-

used cluster analysis (k-means clustering) to classify participants into two clus-

terns, AUCI is always the area under the curve above the baseline

ters, using AUCI as the clustering variable and Students t test to assess differ-

ences in other cortisol parameters between these two clusters. Principal compo- value and hence, AUCI AUCAB. In the second scenario

nent analysis (PCA) was employed to understand the correlation structure among (Figure 3), the last measurement is less than the baseline value.

the derived cortisol parameters and reduce the dimension of the summary mea- Generally, when one or more of the subsequent measurements

sures to a few new meaningful components. The Eigenvector loadings associated are less than the baseline value, AUCI is not the area under the

with the new components were examined graphically to assess what aspect of the

curve above the baseline value. For such patterns, AUCI is the

multivariate data the components measure. All statistical analyses were per-

formed using the SAS/STAT software, version 9.0 for Windows. difference of two areas: area under the curve above the baseline

value (AUCAB) minus area above the curve below the baseline

value. This is a generic definition for AUCI applicable to all

RESULTS patterns of measurements. The first scenario is a special case of

Generic Definition of AUCI this generic definition where the area above the curve below the

A generic formula for AUCI is illustrated using two scenarios. baseline value is zero. Note that for patterns where all subsequent

The purpose of this section is not to provide a formula for measurements are less than the baseline value, AUCAB is always

computation of AUCG and AUCI, as this is already presented by zero and consequently AUCI takes a negative value.

Pruessner and colleagues (10). Rather, we intend to show that the

existing definition of AUCI, area under the curve above the Scenario I

baseline value, is not applicable to all patterns of measurements. Four repeated measurements taken at 15-inute intervals (sam-

Here we treat the area under the curve above the baseline value ple 1 (baseline) measurement 9.6 at 0720 AM; sample 2

(AUCAB) as a separate parameter. We then provide a more 13.4 at 0735 AM; sample 3 16.5 at 0750 AM; and sample 4

D. B. FEKEDULEGN et al.

vals where the value of the last sample is less than the baseline measurement.

AUCI is the difference of two areas: area under the curve above the baseline

value (AT1 AT2 AR2 AT3) minus area above the curve below the

Figure 2. Pattern of four repeated measurements taken at 15-minute inter- baseline value (AT5).

vals where all subsequent measurements are larger than the baseline value.

AUCI is the area under the curve above the referent baseline measurement

(shaded region).

Scenario II

Four repeated measurements taken at 15-minute intervals

14.2 at 0805 AM). The pattern of the measurements is shown in (sample 1 (baseline) measurement 9.6 at 0720 AM; sample

Figure 2. The area under the curve with respect to the baseline 2 13.4 at 0735 AM; sample 3 11.1 at 0750 AM; and

(AUCB), area under the curve with respect to the ground (AUCG), sample 4 7.2 at 05 AM). The pattern of the measurements

area under the curve with respect to increase (AUCI), and area under is shown in Figure 3. As in scenario I, AUCB, AUCG and

the curve above the baseline (AUCAB) are computed as: AUCI are calculated using the computational formula as:

AUC B 9.6 15 15 15 432 AUC B 9.6 15 15 15 432

2 15 13.4 16.5

2 AUC G 9.6 13.4

2 15

15

16.5 14.2

2 15 627 13.4 11.1

2 15

AUC I AUC G AUC B 627 432 195

11.1 7.2

15 493.5

2

13.4 9.6

AUC AB 15 AUC I AUC G AUC B 493.5 432 61.5

2

2 15 However, to compute AUCAB and to illustrate the generic

definition of AUCI, the region between two sampling points is

partitioned into a series of triangles (T) and rectangles (R). The

2 15 area (A) of each triangle and rectangle was calculated and given as:

AT1 28.5, AT2 17.25, AT3 4.33, AT4 11.08, AT5 11.08,

AR1 144, AR2 22.5, AR3 144, AR4 13.85, and AR5 108.

195 The generic definition entails that AUCI is the difference of two

ANALYSES OF AWAKENING CORTISOL MEASUREMENTS

areas. The first component, area under the curve above the The x coordinate of the intersection point of the two lines

baseline value (AUCAB), is the sum of areas of T1, T2, T3 and is obtained by solving the simultaneous equations: y 9.6 and

R2 (28.5 17.25 4.33 22.5 72.58). The second y 18.9 0.26X. This yields a value of x 35.76923

component, area above the curve below the baseline, is the minutes from baseline.

area of T5 (11.08). AUCI is then 72.58 11.08 61.5; the

same result as in the computational formula.

Note that to compute the area of the triangles named T3, T4, Sign and Magnitude of AUCI

T5 and the rectangle named R4, the coordinates of the point Previous studies (1315) have shown that the response

labeled a (Figure 3) need to be known. The y coordinate that magnitude and time course of salivary cortisol levels after

represents the cortisol level is 9.6 but the x coordinate that awakening are significantly related to various psychological

represents time of measurement (in minutes) from baseline is and physical conditions (e.g., pain, stress). Therefore, the

unknown. The x coordinate was then computed by solving the change in the sign and magnitude of AUCI associated with

simultaneous equation of the two intersecting lines: the hori- various cortisol patterns is important in understanding the

zontal line through the baseline measurement (y 9.6) and definition of the parameter. In this section, the variation of

the line connecting the 3rd and 4th measurements. The equa- AUCI associated with various cortisol patterns is examined

tion of the line passing through the third and fourth measure- numerically and graphically. Artificial data on eight waking

ments is computed by solving the equation: cortisol patterns (Table 1) was used for illustration and the

corresponding graphical depiction of these patterns is shown

Y y1 y y2 y1 7.2 11.1 in Figure 4 (AH). Although simulated for analyses here, the

0.26

X x1 x x2 x1 45 30 patterns in Figure 4 can also be observed in real life. In each

where (x1, y1) and (x2, y2) are the coordinates of the 3rd and figure, the area represented by AUCI is shown. There are four

4th sample points, respectively. After algebraic manipulation, distinct patterns in Figure 4: a steadily increasing pattern

the equation of the line is (Figure A); a pattern where cortisol peaks 15 minutes after

waking and decreases thereafter (Figures BD); a steadily

Y 18.9 0.26X. decreasing pattern (Figures EG); and a pattern where

Figure 4. (AH). Various time courses of salivary cortisol and the sign and magnitude of AUCI associated with each profile. AUCI area under the curve

with respect to increase.

D. B. FEKEDULEGN et al.

cortisol drops 15 minutes after waking and increases there- TABLE 3. Correlation Coefficients of Waking AUCI and AUCG with

after (Figure H). the Raw Measurements and Other Derived Parameters, Buffalo

Cardio-Metabolic Occupational Police Stress (BCOPS) Pilot Study,

For the first example (A), AUCI is positive and clearly 20012003 (n 68)

defined; it is the area of the region under the curve above the

reference baseline measurement. The examples in (B), (C), AUCI AUCG

and (D) depict similar patterns but the AUCI values differ Cortisol

substantially. Note that, unlike the first example, AUCI is not Measurements Correlation Correlation

p p

Coefficient (r) Coefficient (r)

clearly defined in a single region but it is obtained through

subtraction of area from two regions: area above the baseline At awakening 0.296 .0143 0.810 .0001

and below the curve minus area below the baseline and above 15 min after waking 0.137 .2669 0.915 .0001

the curve. This illustrates that AUCI balances the amount of 30 min after waking 0.435 .0002 0.862 .0001

45 min after waking 0.619 .0001 0.741 .0001

increase versus decrease and can be considered as an

Derived parameters

index of change. AUCI therefore is the difference in area AVE 0.258 .0339 0.976 .0001

under and above the curve with reference to the baseline. In AUCB 0.280 .0206 0.833 .0001

(B) and (C), the amount of increase (area under the curve AUCG/AUCI 0.297 .0138 0.297 .0138

above the baseline) is larger than the amount of decrease AUCAB 0.88 .0001 0.58 .0001

PK 0.221 .0706 0.950 .0001

(area above the curve below the baseline). In (D), the amount

TBP 0.644 .0001 0.181 .1387

of increase is smaller than the amount of decrease; hence, SBP 0.493 .0001 0.655 .0001

AUCI takes a negative value. RT 0.818 .0001 0.072 .5581

The examples in (E), (F), and (G) also present similar SP1 0.809 .0001 0.091 .4590

patterns but the AUCI values show large differences in mag- INT 0.180 .1418 0.851 .0001

SP2 0.767 .0001 0.137 .2650

nitude. Note that, for these cases, the amount of increase (area

AUR 0.287 .0176 0.996 .0001

above the baseline AUCAB) is zero; hence, AUCI is the

amount of decrease and takes a negative value. The slope of AVE average of the four waking cortisol measurements; AUCB area

the regression line fitted to the patterns in (E), (F), and (G) are under the curve with respect to the baseline; AUCG area under the curve

0.11, 0.15, and 0.26, respectively (Table 1). The rate of with respect to the ground; AUCI area under the curve with respect to

increase; AUCAB area under the curve above the baseline; PK peak

decrease is highest for (G) because the cortisol value de- cortisol; TBP time in minutes from baseline to peak; SBP slope from

creases by 0.26 nmol/l for 1- minute increase in time. AUCI baseline to peak; RT reactivity (the change in salivary cortisol during the

has the largest magnitude for the curve that has the steepest observation period); SP1 slope of the line through the baseline and last

slope. This illustrates that AUCI also measures or indicates the measurement; INT intercept of the regression line fitted through the raw

rate of decrease relative to the baseline. The large differences cortisol data; SP2 slope of the regression line fitted through the raw cortisol

data; AUR area under the curve of the regression line fitted through the raw

in magnitude of AUCI for these three examples are related to cortisol data.

the differences in the rate of decrease. The pattern depicted in

(H) is a good example illustrating that large and negative

AUCI does not necessarily imply that the pattern of the mea- ground; rather, it only considers distance of the measurements

surements is strictly decreasing. In this example, cortisol de- from the reference baseline. A similar result was reported by

creased 15 minutes after waking and then picked up. There is Pruessner et al. (10). Note that area under the curve above the

a stronger decrease than increase and, hence, a negative AUCI. baseline (AUCAB) shows stronger correlation to AUCI than

In summary, AUCI is an index of change that takes negative AUCG because it is the first component in the generic formula

or positive values and translating the magnitude of AUCI into for AUCI.

patterns over time in reference to the baseline requires caution. Five parameters largely measure the pattern of the mea-

surements over time: area under the curve above the baseline

Association of AUCI with Other Cortisol Parameters (AUCAB); time from baseline to peak; reactivity; slope from

A correlation analysis of AUCI and AUCG with each of the baseline to last measurement; and slope of the regression line

original measurements as well as with other derived parame- fitted to the data. The slope of the regression line fitted to the

ters was performed to gain further insight in interpreting the data can be thought of as a more informative measure of

parameter (Table 3). The correlations with the raw measure- pattern than the other four because it utilizes all the informa-

ments indicate that AUCI is negatively and significantly cor- tion in the multivariate data. Large positive values for all four

related to the baseline measurement. This can be seen by parameters generally indicate an increasing trend in the mea-

examining the computational formula for AUCI (AUCI surements over time and, hence, a positive or large AUCI.

AUCG AUCB). AUCB is an area solely determined by the Two observations can be ascertained based on the correla-

baseline value and larger baseline measurements yield larger tion of the two area measures with the other derived param-

AUCB and, hence, smaller AUCI. It also shows that the eters (1). AUCI showed strong positive correlations with the

association between AUCI and the raw measurements is four parameters (reactivity, slope from baseline to last mea-

weaker compared with AUCG. This is because AUCI does not surement, slope of the regression line fitted to the data, and

take into account the distance of the measurements from the time from baseline to peak) although the corresponding cor-

ANALYSES OF AWAKENING CORTISOL MEASUREMENTS

relations with AUCG were weak or negligible (2). AUCG total hormonal secretion and parameters indicating the time

showed significant positive correlations with the intercept of course of the measurements. A formal analytical tool, princi-

the regression line fitted to the data (r .85) and the corre- pal component analysis (PCA), was used to uncover if there

sponding area under the regression line (AUR) (r .99) are two underlying sets of parameters. The Eigenvalues and

although the associations of AUCI with these two parameters the Eigenvectors of the correlation matrix of the 13 derived

were weaker. The intercept from the regression line is a model cortisol parameters were computed. Only the first two princi-

estimate of the baseline value; hence, the two parameters (INT pal components had Eigenvalues 1.0 and the majority of the

and baseline cortisol) are strongly correlated (r .97, p variability (nearly 90%) among the 13 parameters was ac-

.001). counted by these two principal components. The magnitude

The two clusters of participants created using AUCI as a and sign of the Eigenvector loadings were examined to assign

clustering variable were compared with respect to the mea- meaningful interpretation to the two components. Several obser-

surements and the derived parameters (Table 4). The first vations can be made based on the results of the PCAs. The first

cluster contained 38 participants and the average AUCI for the observation is that the first principal component has large load-

subjects in the first cluster was 109.5 167. The second ings for the average of the waking measurements, AUCB, AUCG,

cluster had 30 subjects and an average AUCI of 329.6 178. peak cortisol, intercept of the regression line, and AUR. Thus,

The largest differences between the two clusters of partici- the first principal component, which accounts for 49.3% of the

pants, as shown by the test statistic, were observed for the variability, seems to dominantly measure total hormonal se-

following parameters: area under the curve above the baseline; cretion. Second, the second principal component has large

time from baseline to peak; reactivity; slope between the 1st loadings for AUCI, reactivity, SP1, SP2, and AUCAB. The

and 4th sample; and slope of the regression line. The result is second component that accounts for 39.5% of the variabil-

in agreement with the data in Table 3 that these parameters are ity in the original data seems to dominantly measure time

strongly correlated to AUCI and they all measure a common course of salivary cortisol. Finally, the results from PCAs

aspect of the repeated measurements: time course of salivary demonstrate that the large number of parameters can be

cortisol (profile). The baseline measurement, the area under reduced to two components: the first principal component

the curve with respect to the baseline (AUCB), and the inter- measures intensity or total hormonal secretion and a second

cept from the fitted regression model (INT) showed the least component measures sensitivity or profile of cortisol over

differences between the two clusters (Table 4). time (Table 5).

Based on the data in Tables 3 and 4, there seem to be two This study focused on interpretation of AUCI, the associ-

broad categories of derived parameters: parameters related to ation of area measures with other cortisol parameters, and

TABLE 4. Characteristics (Mean Standard Deviation, Test Statistic, and p Value) of Cortisol Measurements and Derived Parameters by the Two

Clusters of Participants Created Using AUCI as the Clustering Variable

15 min after waking 15.1 15.8 18.4 10.3 1.02 .3139

30 min after waking 11.6 10.2 20.3 11.4 3.31 .0015

45 min after waking 8.6 6.3 21.1 10.3 5.81 .0001

Derived parameters

AVE 12.6 8.5 17.7 6.7 2.18 .0332

AUCB 626.9 852.9 477.5 368.6 0.95 .3469

AUCG 605.5 508.6 877.7 406.7 2.39 .0198

AUCAB 35.1 63.3 335.3 176.7 8.87 .0001

PK 17.7 15.9 24.9 12.2 2.05 .0446

TBP 14.5 15.4 36.7 16.5 5.73 .0001

SBP 0.14 0.3 0.45 0.4 3.73 .0004

RT 6.6 8.8 9.7 7.6 8.09 .0001

SP1 0.14 0.19 0.20 0.16 7.87 .0001

INT 16.2 14.7 13.1 8.1 1.09 .2803

SP2 0.15 0.22 0.20 0.17 7.35 .0001

AUR 594.3 480.6 858.2 399.4 2.42 .0183

AVE average of the four waking cortisol measurements; AUCB area under the curve with respect to the baseline; AUCG area under the curve with respect

to the ground; AUCI area under the curve with respect to increase; AUCAB area under the curve above the baseline; PK peak cortisol; TBP time in

minutes from baseline to peak; SBP slope from baseline to peak; RT reactivity (the change in salivary cortisol during the observation period); SP1 slope

of the line through the baseline and last measurement; INT intercept of the regression line fitted through the raw cortisol data; SP2 slope of the regression

line fitted through the raw cortisol data; AUR area under the curve of the regression line fitted through the raw cortisol data.

D. B. FEKEDULEGN et al.

TABLE 5. Wakening Cortisol Summary Parameter Ranks Based on substantial differences in the magnitude of their AUCI values.

Their Relative Importance as Measures of Total Hormonal Secretion This is because AUCI takes into account the vertical distances

and Pattern of Response

of each measurement from the reference baseline.

Summary Measures Magnitude Rank Pattern Rank The effect of both measurement error and biological vari-

ability are important to consider. As in any other scientific

Average of the four waking 3 12 measurements, each awakening cortisol observation is com-

cortisol measurements (AVE)

Area under the curve with respect 6 8

posed of the true value plus some random error value (X

to the baseline (AUCB) T e). Random error is caused by any factor that affects the

Area under the curve with respect 2 11 measurement of the variable across the sample (subjects) and

to the ground (AUCG) is always part of any measurement. The accuracy of a cortisol

Area under the curve with respect 12 1

measurement may be as important as the measurement itself.

to increase (AUCI)

Area under the curve above the 8 5 In addition, the inherent (biological) variability associated

baseline (AUCAB) with each measurement is another important source of varia-

Peak cortisol (PK) 1 13 tion. For example, the baseline waking cortisol measurement

Time in minutes from baseline to 13 6 may have the largest variability of all cortisol measurements

peak (TBP)

Slope from baseline to peak (SBP) 7 7

within a day as it is obtained during the time of rapid rise from

Reactivity (RT) 11 2 the late night nadir to the morning peak. Multiple-day awak-

Slope of the line through the 10 3 ening cortisol sampling would be ideal to estimate and under-

baseline and last measurement stand the biological variation associated with each measurement.

(SP1)

Such extensive observations involving multiple day sampling

Intercept of the regression line 4 9

fitted through the data (INT) of cortisol may not be economically feasible and contribute to

Slope of the regression line fitted 9 4 participant burden. The analyses in this study were based on 1

through the data (SP2) day of awakening cortisol sampling (no retest reliability);

Area under the fitted regression 5 10 hence, we cannot provide an estimate of the variability asso-

line (AUR)

ciated with each of the waking measurements. However, we

The summary measures were ranked using the first and second principal believe that the variability in the baseline waking measure-

components. Rank 1 is the most important and rank 13 is the least important. ment has the largest influence in the magnitude of AUCI. This

is because AUCI is the difference between AUCG and AUCB

and the baseline measurement solely determines AUCB. For

application of PCAs to reduce the large number of summary

example, if we assume a random error of 1.5 nmol/l for the

indicators of repeated cortisol measurements to a smaller

baseline value of the data shown in Figure 3, the value of

meaningful set. Generically, AUCI was defined as the area

under the curve above the baseline minus the area above the AUCI could change by 56.3 area units.

curve below the baseline. The formula implies that AUCI In analyses of multivariate data, PCA can serve a dual role:

takes into account both sensitivity and intensity (the distance 1) it is often used to reduce the dimension of a correlated set

of the measurements from baseline value). It is a measure of of variables; and 2) PCA is also used to partition experimental

the pattern or rate of change of the repeated measurements units into subgroups so that units that are similar are in the

over time. The statistical analyses using the BCOPS data same group. In the latter case, the principal components can be

demonstrated that AUCI is highly and significantly correlated used as input into clustering programs (16). In this study, the

to those summary indicators that are known to measure the technique significantly reduced the number of summary mea-

pattern or rate of change of the measurements over time (e.g., sures and the new components explained the majority of the

reactivity and slope of the fitted regression line). Based on the variability in the original measures and had meaningful bio-

results of this study, the following conclusions can be made logical interpretation. According to PCA, the most important

regarding AUCI as a cortisol parameter. First, AUCI is always measures of total hormonal secretion are area under the curve

negative for patterns that are steadily decreasing over time. with respect to the ground (AUCG), peak cortisol, average of

The variability in magnitude of AUCI among these cases is the measurements, and intercept of the fitted regression line

directly related to variation in slope; the highest magnitude (INT), whereas the corresponding measures of time course of

being associated with a pattern having the steepest slope. salivary cortisol are area under the curve with respect to

Second, AUCI is always positive for patterns that are steadily increase (AUCI), reactivity (RT), SP1 and SP2. This result is

increasing over time. The magnitude of AUCI increases with consistent with previous studies (3,11). Note that the impor-

the rate of increase (slope) of the pattern. Third, for patterns tant measures of total hormonal secretion (AUCG, PK, AVE,

that exhibit a series of increases and decreases over time, and INT) are strongly correlated with the baseline measure-

AUCI can be positive or negative depending on the amount of ment (r .81 for AUCG; r .83 for PK; r .84 for AVE;

increase relative to decrease. Fourth, subjects with similar and r .97 for INT; p .0001 for all cases). This suggests

patterns do not necessarily have the same AUCI values. Two that the baseline measurement is a natural measure of total

patterns can have a strong positive correlation and still show waking hormonal concentration. PCA was rerun including the

ANALYSES OF AWAKENING CORTISOL MEASUREMENTS

baseline measurement in the list of cortisol parameters and the awakening in relation to different challenge tests and a 12-hour cortisol

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