Journal Elsevier

ofAffective Disorders, 20 (1990) 115 - 120

115

JAD 00746

Seasonal variation

and the results of the dexamethasone suppression test

Brian Harris ‘, Sara Watkins ‘, Robert Newcombe 2, Nigel Cook 3, Graham Read 3 and Diana Riad-Fahmy 3 ’ Department of Psychological Medicine, ’ Department of Medical Computing and Statistics and -’ Supraregional Assay Laboratoty, Tenovur Institute,

University of Wales College of Medicine, Heath Park, Cardiff, UK. (Received 27 December 1989) (Revision received 21 May 1990) (Accepted 30 May 1990)

Summary There is controversy over the presence of a circannual rhythm in cortisol values in samples provided by depressed patients after a standard dexamethasone suppression test (DST). Post-DST cortisol values from patients admitted to an acute psychiatric ward over a 2-year period have therefore been analysed by appropriate statistical tests. No evidence was found for significant seasonal variation.

Key words: Cortisol;

Depression;

Seasonal

variation;

Introduction Interpretation of hormonal data must be made in the light of the phenomenon of chronobiological rhythmicity. The dexamethasone suppression test (DST), modified for psychiatric practice by Carroll et al. (1981), takes account of the normal circadian variation of adrenal activity and of cortisol secretion. Thus, the test is standardised by taking all samples at a fixed time of day, namely 16.00 h. There is evidence that some biological parameters oscillate over the course of a 12-month period.

Address for correspondence: Brian Harris, MRCPsych, Department of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff, U.K. 0165-0327/90/$03.50

0 1990 Elsevier Science Publishers

Dexamethasone

suppression

test

This circannual variation is seen in such widely divergent phenomena as food intake in children (Young et al., 1951) and melatonin secretion in man (Arendt et al., 1979; Arendt and Marks, 1982). The latter is clearly linked to the light/dark cycle, increased levels of melatonin being associated with winter dark phases. Wirz-Justice and Richter (1979) have drawn attention to greater variation within the circannual rhythm of some hormones than has previously been described between normal and abnormal subjects. Defining circannual rhythms presents difficulties, and studies have been basically of two kinds. Firstly, in longitudinal studies, small groups of individuals may be followed intensively over relatively long periods of time, account being taken of life stresses and changes in individual circumstances. For example, Reinberg et al. (1978)

B.V. (Biomedical

Division)

116

studied five normal healthy men living in Paris over a period of 14 months, urine samples being obtained on a 24-hourly basis once a month and blood monitored for 28-h periods (4-hourly samples) once every 2 months. Using this technique, they reported significant circannual variations in cortisol, testosterone, thyroxine, renin, FSH and LH. Secondly, in transverse studies results obtained from large numbers of subjects for various times of the year may be compared with one another. Reinberg et al. (1988) examined normal men presenting for vasectomy over a 2-year period where a single pre-vasectomy blood sample was taken. Again, a significant circannual variation was described in plasma testosterone. As far as cortisol is concerned, evidence supporting a clear circannual variation in normal subjects, living in non-polar latitudes, is conflicting (Agrimonti et al., 1982; Guagano et al., 1983; Haus et al., 1988). However, there is evidence that there are depressed individuals who show exacerbation of symptoms during the winter months (Wehr and Rosenthal, 1989). These patients with seasonal affective disorder (SAD) would be expected to show higher rates of DST non-suppression in the winter months when depression is most severe. Any unselected group of depressed patients would be expected to contain some individuals with SAD. These individuals would have higher post-DST cortisol values and would thus increase the mean cortisol concentration for the group in the winter months. It is thus surprising to find that Swade et al. (1987) report increased rates of suppression and lower cortisol levels in the winter months. Other workers, however, have failed to confirm this (Van Bemmel et al., 1988) and these conflicting findings, coupled with major reservations on the appropriateness of the statistical analysis adopted by Swade et al. (1987) prompted us to re-examine data from a previous study from this laboratory, involving 280 patients admitted to an acute psychiatric ward (Harris et al., 1990) for evidence of a circannual rhythm. Materials and methods Patients (n = 280) admitted over the course of a 2-year period to a typical acute psychiatric unit were classified according to DSM-III criteria

(1980). All the usual psychiatric diagnoses were represented. The DST protocol was strictly comparable to that of Swade et al. (Metcalf, personal communication) since (i) the patients remained on medication at the time of the test, (ii) the test was carried out 5-7 days after admission and (iii) dexamethasone (1 mg) was administered between 22.00 and 23.00 h and, on the following day, a blood sample was taken at 16.00 h. Plasma from such samples was stored at - 20 o C until analysed for cortisol. Plasma cortisol was determined by a simple, robust in-house radioimmunoassay (RIA) (RiadFahmy et al., 1979). This assay used an antiserum raised against a cortisol-3-( O-carboxymethyl) oxime/ bovine serum albumin conjugate, coupled to microcellulose, together with the homologous ‘251-radioligand. The assay did not involve solvent extraction; plasma samples and standards were incubated with the antiserum and 125I-radioligand in a low-pH buffer, which denatured cortisol-binding globulins. The assay not only satisfied accepted validation criteria but also provided results in good agreement with those of a reference gaschromatographic mass-spectrometric (GCMS) technique (Y = 0.968; cortisol RIA = cortisol GCMS + 2 nmol/l). Quality control samples having high (1096 nmol/l), medium (419 nmol/l) and low (281 nmol/l) plasma cortisol concentrations had acceptable coefficients of variation both within (4.7%; 6.2%; 4.9%) and between (10.5%; 6.8%; 12.0%) assays. Assay sensitivity, defined as the least amount distinguishable from zero at the 95% confidence level, was 28 nmol/l. External assessment of assay performance in the laboratory may be judged by data derived from the U.K. National External Quality Assurance (NEQAS) programme. At monthly intervlas, participants (n = 210) in NEQAS each receive samples (n = 6) for measurement of cortisol. Information returned to the laboratory by this programme indicated that, during the year 1985 (when many of the samples included in this study were processed), the laboratory had zero ‘blunders’ and a small consistent negative bias (mean < 5%) from ‘all laboratories trimmed mean’ values. Statistical methods Patients were

classified

according

to

the

117 TABLE

calender month in which the DST was performed. A non-parametric Kruskal-Wallis analysis of variance, yielding a &i-square statistic with 11 df, was first used to compare the 12 months, disregarding their cyclical order; a 2-df component was then extracted, representing the fit of a sinusoidal model with optimal amplitude and phase to the mean ranks obtained for the 12 months.

1

COMPARISON OF POST-DST CORTISOL SWADE ET AL., 1987 (a) vs. PRESENT STUDY Time of test

Study

6 Mar-Apr ; May-June

The incidence of non-suppression (defined by plasma cortisol > 137 nmol/l = 50 ng/ml) in all 280 subjects was 25%. Non-suppression occurred in all diagnostic groups, but was significantly more frequent in the group ‘Major depression with melancholia’ (n = 43, 21 non-suppressors) than in the remainder (n = 237; 59 non-suppressors); x2 = 18.55, df = 1, P < 0.001). The other main diagnostic categories were: major depression unspecified (n = 60, 9 non-suppressors), major depression without melancholia (n = 19, 3 non-suppressors), bipolar disorder, manic (n = 19, 4 nonsuppressors), paranoid schizophrenia (n = 35, 5 non-suppressors), schizophreniform psychosis (n = 17, 5 non-suppressors) and substance abuse (n = 29, 9-non-suppressors). In the combined group (n = 103), major depression with melancholia together with major depression unspecified, the overall rate of non-suppression was 29%. This combined group, classified according to DSM-III criteria, was considered the most appropriate for TABLE

Jul-Aug

Post-DST cortisol

15 19 32 13 26 13 38 16 21 20 24 20

Jan- Feb

Results

n

; a b

Sep-Ott ; Nov-Dee ;

VALUES: (b)

Abnormal response

(ng/ml)

(5%)

86.0*13 36.0+ 9 123.0+17 43.0*16 103.0+ 14 3O.Ok 8 103.0*11 53.0+16 108.0 i 14 34.0* 9 57.0f 10 31.0*11

67 26 75 31 69 27 71 31 71 35 58 15

Study of (a) Swade et al. (1987) included 156 patients classified as major depressive disorder (RDC); (b) Cardiff group included 103 patients classified as major depression with melancholia, major depression unspecified (DSM-III).

comparison with the patients of Swade et al. (1987) who used RDC criteria. To facilitate comparison, data from the present study have been presented in a manner strictly comparable to that of Swade et al. (1987); cortisol concentrations are presented in ng/ml and data summarised as mean values + SEM (Table 1). Despite grave reservations regarding the validity of the t-test in this context, application of this test indicated that in the present

2

ANALYSIS

OF POST-DST

PLASMA

All subjects (n = 280) All subjects with depressive diagnoses d (n =134) Subjects with major depression (definite and probable melancholic) (n = 103)

CORTISOL

VALUES

BASED

ON KRUSKAL-WALLIS

TEST

x2 (11 d/) comparing 12 months

x2 (2 df) a cyclical trend

Angular displacement b of peak from mid-January

Time of fitted peak ’

11.56

2.09

+ 51

Early March

8.79

1.84

+91

Mid April

9.66

0.77

+55

Early March

a x2 (2 df) cyclical trend represents the fit of a sinusoidal model with optimal amplitude and phase to the mean ranks of post-DST cortisol values obtained for the 12 months. b Angular displacement represents the displacement (in degrees) of the peak of the sine curve from an orbitary origin (mid-January). ’ Time at which the sine curve filled through the plot of cortisol values vs. time of the year achieves a maximum. d There were 31 subjects with other depressive diagnoses, viz., major depression without melancholia (n = 19), adjustment disorders (n = 10) and dysthymic disorder (n = 2).

118

study, in contrast to that of Swade et al. (1987) cortisol levels in November-December were not significantly different from those in any other 2-month period (P > 0.2). The results for the 4 months from November to February were also not significantly different from those in the rest of the year (P > 0.2). In a more appropriate statistical analysis for significance of a circannual rhythm, a non-parametric method was used, namely Kruskal-Wallis ANOVA. This analysis, which is based on ranks, unlike the t-test, does not rely on the assumption of normality of data and has the added advantage of being able to cope with results which fall below assay sensitivity. Shown in Table 2 is the 11-df chi-square statistic comparing all 12 months disregarding their cyclical order. Also shown is the 2-df component representing a sinusoidal cyclical trend: this did not reach significance. (NB. This was also true for the whole group of 280 patients.) Discussion If there were a pronounced circannual rhythm in post-DST cortisol levels, this would have a profound influence on data interpretation. Not only would an optimum ‘cut-off value’ have to be calculated for each month, but diagnostic criteria, including sensitivity, specificity and positive predictive value, would also have to be assessed for each month. Furthermore, these parameters could fluctuate markedly, even when the appropriate ‘cut-off values’ were used, if circannual rhythms in post-DST cortisol values differed in depressed patients and their controls. It is therefore a matter of some importance to confirm or refute the report of Swade et al. (1987) which purports to demonstrate such a rhythm. Comparison of data (Table 1) from our study and those of Swade et al. (1987) reveals gross discrepancies in mean cortisol values and rates of non-suppression. A possible explanation for the discrepancies in Table 1 is that there are differences in the selection criteria for patients. Research Diagnostic Criteria (RDC) (Spitzer et al., 1978) were used by Swade et al. (1987) but they give no information concerning the subtypes of depression, e.g., agitated, situational, endogenous.

Nonetheless, major depressive disorder according to RDC is essentially the same as major depressive disorder according to DSM-III criteria, except that one extra feature is required to make a diagnosis. It is unlikely therefore that the minor difference in diagnostic categories accounts for the higher rates of non-suppression in the Swade study (70% compared with 29%). It is also unlikely that the severity of depression accounts for the difference since mean Hamilton scores indicated mild to moderate depression. The latter information was not available for patients in our study, but they all required admission to an in-patient unit, indicating at least the same severity of illness. An alternative explanation for discrepancies in the data (Table 1) is that they may be due to major differences in the bias of the immunoassay procedure used. The assay for cortisol used in our study has been shown to give results in close agreement with a reference GCMS technique. Furthermore, the assay has been monitored by an external quality assurance scheme and, at the time at which samples in this study were assayed, results were within accepted limits of bias and variability of bias (VAR). Since Swade et al. (1987) have provided no details of their assay procedure, it could be that their assay has a positive bias; this would explain the high rates of non-suppression and the cortisol values at 4 p.m., which are high even for subjects not receiving dexamethasone. If the assay of Swade et al. (1987) has a positive bias then the conventional ‘cut-off value’ (50 ng/ml) would not be optimal, and would give high rates of escape in both melancholies and controls. This parameter cannot he checked because no reference is made to diagnostic specificity by these authors. By contrast, the ‘cut-off value’ used in our studies has been shown to have an optimal combination of sensitivity and specificity using receiver operator characteristic curves (Harris et al., 1990). Regardless of the merits of the immunoassays used and of the ‘cut-off values’ adopted, validity of the conclusions made by Swade et al. (1987) is critically dependent on the appropriateness of the statistical tests performed. The statistical analysis employed by this group is open to criticism on several counts, namely: (1) Failure to transform plasma cortisol values to

I19

improve the fit to a Gaussian distribution or to use non-parametric methods. (2) Loss of information in aggregating the data into 2-month intervals. (3) Use of multiple t-test, considering time intervals in pairs, instead of methods designed to examine the circannual data as a whole. Detailed analysis of the Cardiff data shows that the normal model is a very poor fit to untransformed and even log-transformed cortisol values, largely because of the high proportion of values at or below the limit of detection. The use of parametric methods is therefore inappropriate. Since Swade et al. (1987) have higher rates of non-suppression, their data may approximate more nearly to a normal distribution, thus justifying the use of the t-tests. However, normality should not have been assumed but should have been demonstrated. There are more appropriate ways to test seasonal trend than to perform several r-tests. The technique of comparing against the period with the lowest mean is likely to produce a nominally significant difference between whichever 2-month periods turn out to yield the lowest and highest mean values. This technique also takes no account of the natural ordering of the periods under study. In our study we have used the non-parametric Kruskal-Wallis ANOVA technique and demonstrated no significant differences between 2monthly periods. The 2-df component representing cyclical trends also failed to reach significance. Thus, using appropriate statistical tests, we have found no evidence for a significant circannual rhythm in post-DST cortisol values. This conclusion is in accord with the negative findings of Van Bemmel et al. (1988). In any study of circannual rhythms, apparent shifts in mean monthly values - even when statistically significant - do not offer conclusive proof of a biological rhythm. To exclude the possibility that any apparent shifts in monthly means are the results of changes in assay performance, it is mandatory that appropriate internal quality control data be collected and presented. A minimal requirement would be the collection of long-term quality control data derived by processing an appropriately targeted plasma pool with each batch of samples assayed. Demonstration that the target value had not shifted would reduce the possibility

that the low values observed by Swade et al. (1979) in November-December were due to an assay artefact. We conclude that the case for a significant circannual rhythm in post-DST cortisol values remains unproven. References Agrimonti, F., Angeli, A., Frairia, R., Fazzari, A., Tamagnone, C., Fornaro, D. and Ceresa, F. (1982) Circannual rhythmicities of cortisol levels in the peripheral plasma of healthy subjects. Chronobiologia 9, 107-114. American Psychiatric Association (1980) Diagnostic and Statistical Manual of Mental Disorders, 3rd edn. APA, Washington, DC. Arendt, J. and Marks, V. (1982) Physiological changes underlying jet lag. Br. M. J. 284, 144146. Arendt, J., Wirz-Justice, A., Bradtke, J. and Kimemark, M. (1979) Long-term studies on immunoreactive human melatonin. Ann. Clin. B&hem. 16, 307-315. Carroll, B.J., Feinberg, M., Greden, J.F., Tarika, J., Albala, A.A., Haskett, R.F., James, N.McI., Kronfol, Z., Lohr, N., Steiner, M., DeVigne, J.P. and Young, E. (1981) A specific laboratory test for the diagnosis of melancholia. Arch. Gen. Psychiatry 38, 15-22. Guagano, M.T., Del Ponte, A., Mendani, P., Nuzzo, A., Palummeri, E., Angelucci, E., Vitacolonna, E. and Sensa, S. (1983) La Struttura temporale della secrezione endocrina: II. Variazioni circannuali delle frazioni libere della tri-tetraiodo tironina, de1 cortisolo, dell’human growth hormone e dell’insulina plasmatica in soggeti sani. Boll. Sot. Ital. Biol. Sper. 59, 1243-1247. Harris, B., Watkins, S., Cook, N.J., Walker, R.F., Read, G.F. and Riad-Fahmy, D. (1990) Comparisons of plasma and salivary cortisol determinations for the diagnostic efficacy of the DST. Biol. Psychiatry 27, 897-904. Haus, E., Nicolau, G.Y., Lakatua, D. and Sackett-Lundeen, L. (1988) Reference values for chronopharmacology. In: A. Reinberg, M. Smolensky and G. Labreque (Eds.), Annual Review of Chronopharmacology, Vol. 4. Pergamon Press, Oxford, pp. 391-424. Reinberg, A., Lagoguey, M., Cesselin, F., Touitou, Y., LeGrand, J.C., Delasalle, A., Andreassian, J. and Lagoguey, A. (1978) Circadian and circannual rhythms in plasma hormones and other variables of five healthy young males. Acta Endocrinol. 88, 417-427. Reinberg, A., Smolensky, M.H., Hallek, M., Smith. K.D. and Steinberger, E. (1988) Annual variation in semen characteristics and plasma hormone levels in men undergoing vasectomy. Fertil. Steril. 2, 309-315. Riad-Fahmy, D., Read, G.F., Gaskell, S.J., Dyas, J. and Hindawi, R. (1979) A simple direct radioimmunoassay for plasma cortisol, featuring an ‘251-radioligand and a solidphase separation technique. Clin. Chem. 25, 665-668. Spitzer, R.L., Endicott, J. and Robins, E. (1978) Research Diagnostic Criteria: rationale and reliability. Arch. Gen. Psychiatry 35, 773-782.

120 Swade, C., Metcalf, M., Coppen, A., Mendlewicz, J. and Linkowski, P. (1987) Seasonal variations in the dexamethasone suppression test. J. Affect. Disord. 13, 9-11. Van Bemmel, A.L., Van Diest, R., Smeets, E.H.J., Van Dongen, P.H.M. and Hilgersom, A.J.C. (1988) Seasonal variation of cortisol plasma levels in depressives. J. Affect. Disord. 15, 191-193. Wehr, T.A. and Rosenthal, N.E. (1989) Seasonality and affective illness. Am. J. Psychiatry 146, 829-839.

Wirz-Justice, A. and Richter, R. (1979) Seasonality in biochemical determinations: a source of variance and a clue to the temporal incidence of affective illness. Psychiatr. Res. 1, 53-60. Young, C.M., Smudski, V.L. and Steele, B.F. (1951) Fall and spring diets of school children in New York State. J. Am. Diet. Ass. 27, 289-292.

Seasonal variation and the results of the dexamethasone suppression test.

There is controversy over the presence of a circannual rhythm in cortisol values in samples provided by depressed patients after a standard dexamethas...
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