Participants
Volunteers were recruited from the (blinded), Australia. The project was advertised by publicity and news media as “a study about mental health”. Inclusion criteria stated that participants were to be at least 18 years of age. Exclusion criteria were the presence of an acute medical illness or current medication for a mental illness.
Instruments
Background questionnaire.
Participants were asked to state their age (years), sex, and racial origin.
Depression.
Depression was assessed by the 20-item Zung Self-rating Depression Scale (SDS) 13. The SDS is based on data from factor analytic studies of MDD 14 and fits the most recent definitions of that disorder 15. Respondents indicate the frequency of each of those 20 items by answering: “None or a little of the time”, “Some of the time”, “Good part of the time”, or “Most or all of the time”, which produce numerical scores of 1 to 4, providing total raw scores from 20 to 80. SDS raw scores of 40 or above indicate the presence of “clinically significant depression” 16, p. 335. The SDS has demonstrated split-half reliability of .81 13, .79 17 and .94 18. Internal consistency (Cronbach alpha) has been reported as .88 for depressed patients and .93 for non-depressed patients 19 and as .84 for a previous Australian sample 20. The SDS has been shown to possess higher validity than the MMPI Depression Scale or the Beck Depression Inventory for assessing depression in male psychiatric inpatients, referenced to psychiatric diagnoses 19. SDS raw scores were used in this study.
Daily functioning
Two multi-item indices (mental health, physical health) of daily functioning were drawn from the SF-36, a well-validated indicator of health status that is used in studies of health economics to contribute to the Quality-Adjusted Life Years measure. The SF-36 has satisfactory validity in recognizing patients with good and poor health, and also reliability (Cronbach alpha) of .85 21, 22. The SF-36 has eight subscales, measuring various ‘domains’ of a person’s functioning in physical, social and emotional areas, plus mental health and pain. From these subscales, three items were developed to measure (i) global mental health on a five-point scale (excellent, very good, good, fair, poor), (ii) whether participant’s mental health had influenced their daily functioning by impeding the time they had spent on activities, their success on those activities, or the care they had taken with those activities, and (iii) whether their mental health had impeded their social activities (all ‘yes’ vs ‘no’ responses). The same questions were repeated for physical health to clarify the relative contributions of both aspects of health and functioning. The mental health functioning scale was called MHFS, and the physical health functioning scale was named PHMS.
Cortisol extraction
Cortisol in saliva was measured using a specific salivary cortisol ELISA kit from Abnova Corporation (KA1885, Taipei, Taiwan). This is a solid-phase ELISA using a polyclonal rabbit antibody directed against cortisol. The assay is based on the principle of competitive binding, and endogenous cortisol in the sample competes with a cortisol-horseradish peroxidase conjugate for binding to the antibody. This ELISA has an intra-assay variability of 8.27% and inter-assay variability of 8.33%, with a spiking recovery of 100% and calibration range of 0.1 to 30 ng/ml. Salivary cortisol was assayed according to the manufacturer’s instructions. Briefly, 50 µl of neat saliva from participants or cortisol standards (0.1–30 ng/ml) were transferred to the appropriate wells of the 96-well microtitre plate. Fifty µl enzyme-conjugate was dispensed into each well with thorough mixing. The plate was incubated at room temperature with gentle rocking for 60 minutes. The contents of each well were aspirated followed by four rinses with 300 µl wash solution. Substrate (200 µl) was added to each well and the plate was incubated at room temperature for 30 minutes. The reaction was stopped with the addition of 50 µl stop solution and absorbance was read at 450 nm immediately. All standards, controls and samples were assayed in duplicate and results were calculated using a 4-parameter logistics curve fit.
CRP assays
Blood samples were collected and centrifuged at 1000 g for 15 min. The sera were frozen at -80 °C until analysis of C-reactive protein. Serum concentrations of CRP were determined using a Siemens Dimension XPand Plus Autoanalyser (Siemens, Newark, USA), using the CRP extended range (RCRP) Flex reagent cartridge (Siemens Dimension, Newark, USA) according to the manufacturers’ instructions. This assay is based on the particle-enhanced turbidimetric immunoassay (PETIA) technique, where synthetic particles coated with anti-CRP antibodies aggregate in the presence of CRP, increasing turbidity in proportion to CRP concentration. Concentrations are reported in mg/L.
Procedure
From a list of 20,000 random names and addresses (balanced for equal numbers of males and females) supplied by the Australian Electoral Commission, sufficient participants were recruited to exceed the sample size required for Principal Components Analysis as used by 2. Recommendations for the ratio of participants to components range from 5:1 to 20:1 23, and the latter ratio was adopted here, so that there were at least 80 participants to test a four-factor solution. Additionally, the rule of thumb of 8M + 50 participants for regression analysis (where M = the number of variables entered into the regression equation) was followed so that, with four variables, the required sample size was 82 participants.
Participants received a link to an online portal or a copy of the questionnaire booklet containing an explanatory statement and consent form, plus the background questionnaire and the SDS, MHFS and PHFS, and a small container (Salivette) and written instructions for collection of morning cortisol saliva (about 45 minutes after waking in the morning and without eating or drinking before sampling), which represents the apex concentration in the diurnal rhythm (DR) of cortisol 24, and which may be used as an indicator of the overall cortisol-related stress level of the individual 25. Participants were asked to freeze their saliva samples until they came to the researchers’ lab a few days later to provide a blood sample for the CRP assay. Although some previous research has collected salivary cortisol data on several days, as well as placing an increased research burden upon participants and increasing the likelihood of participant drop-out in community samples, there are sufficient data from studies of repeated collections of salivary cortisol to argue that there is acceptable long-term agreement between concentrations of salivary cortisol over periods of several months 26, which is much longer than the few days that was the gap between saliva collection and serum collection in this study. CRP is also relatively stable over time and shows no DR 11. The project was approved by the University of New England Human Research Ethics Committee (approval no. HE14-010) in accordance with the Declaration of Helsinki (1964 and subsequent confirmations). All participants gave written consent to the study.
Statistical analyses
Data were tested for normality. Pearson correlation coefficients were calculated to test for any significant age effects on the DVs, and gender was also tested for its effect via MANOVA. The cortisol:CRP ratio was derived by dividing cortisol concentration by the CRP value as described by Suarez et al. 11, 27. Z-scores were calculated for the SDS, MHFS, PHFS, and the cortisol:CRP ratio to reduce the possibility of a confound due to the influence of one of the measures above the others. These were then entered into PCA, firstly without the cortisol:CRP ratio, and then with it included, to create two versions of the IBI-D based upon the component loadings from the PCA. Hierarchical regression was used to determine which of these two versions of the IBI-D contributed most strongly to the SDS raw score.