Lack of correlation between plasma DOPEG and urinary MOPEG levels in depressed patients

900

BloL PSYCHIATRY 19~6;2l:~~

Lack of Correlation between Plasma DOPEG and Urinary MOPEG Levels in Depressed Patients Henri Lao, Trevor Dennis, Jean-Marie Vanelle, Liliane Rouquier, Marie-France Poirier-Liter,

Maine

Garreau, Chawki Benkelfat,

Daniel Sechter, and Bernard Scatton -

T~e?~~~o~r-hoLfr

~rjna~

excretion

--

of 3-metho~,4-hydru~phenyLethyleneglycof

(MO-

PEG) and Erveis of free und conjugated plasma 3,4-dihydroxyphenylethyleneglycol

(DO-

PEG) were measured in 56 depressed patients to find a possible correlation between these two peripheral indices of cerebral noradrenergic uctivity. Plasma DOPEG was measured

at 9:00

MUP~G.

A/I depressed

to

DS~-IIf .No

and urinao depressed

AM

on the same day that urine was collected for the measurement

correlation

MOPEG

of

patients were diagnosed as having a~e~tive disorders according

was found between plasma free or conjugated DOPEG levels

output. This lack of correlation

putients (56),

in 45 patients diugnosed

was found

in the total sample of

as having major depressive

and in 24 depressed patients diagnosed as major depressive with melancholia. discuss the sign~~cance

of this lack qf’ correlation

between

two peripheral

episodes,

The authors indices

of

ventral ~~orudrenergi~ met~lboli.~m

Introduction The catecholaminergic theory of depression was formulated 20 years ago (Schildkraut 1965: Bunney and Davis 1976). Soon after, it was suggested that there were several bi~hemical types of depression with possible deficiencies in serotonin~rgic or noradrenergic transmission (Asberg et al. 1976; Goodwin et al. 1978). Monoaminergic dysfunction in depression is generally deduced by measurement of precursors and, especially, of catabolites of monoamines in the cerebrospinal fluid (CSF), blood, and urine of depressed patients. Two main methods for the investigation of central norepinephrine (NE) metabolism have been widely used in humans: the measurement of 3-methoxy,4-hy~oxypheny~e~yleneglycol (MOPEG) in 24-hr urine samples, and the measurement of plasma levels of 3,4-dihydroxyphenylethyleneglycol (DOPEG) in their free and conjugated forms. DOPEG results from the oxidative deamination of NE after its reuptake into the noradrenergic nerve endings (Li et al, 1981; Verbeuten and Vanhoutte 1982) by type A monoamine

From the Department of Mental Health and Therapeutics, Sainte-Anne Hospital, Paris-&chin Faculty (H.L., J.-M.1’. M-F.P.-I.. , M.G.. C.B., D.S .). Paris, and the Biochemical Pharmacology Gmup, Syntbklabo-LERS. (T.D., I-R., B.S. /. Bagneux, France. Address reprint requests to Dr. Hew L6o, Service de Sante Mentale et de Th&apeuttque. Centre Horpitnher Sainte-Anne. 1 meCabams, 75014 Paris, France. Received July IX, 1985. revised lkrrmher 10. 1985.

Correlationof DOPEG and MOPEG in Depression

BIOL PSYCHIATRY 1986:21:900-906

901

oxidase (MAO-A); this metabolite is then converted to MOPEG by catechol-O-methyltransferase (COMT). Animal studies have shown that brain DOPEG levels accurately reflect central noradrenergic activity in several mammalian species (Pardal et al. 1980; Scatton 1982). There have been only a few studies of plasma DOPEG in depression. Robinson et al. (1983) found a decrease of plasma DOPEG levels in depressed patients treated with amitriptyline or phenelzine. Loo et al. (1983) and Scatton et al. (1985) observed that mean free and sulfoconjugated plasma DOPEG levels were lower in depressed patients than in paired control subjects. Studies on urinary MOPEG raise questions as to the reliability of the biochemical determination of MOPEG, and the respective contribution of central and peripheral NE to MOPEG levels. It has been estimated that about 50% of urinary MOPEG originates from cerebral NE metabolism (Maas et al. 1979). Linnoila et al. (1983) reported relative stability of 24-hr urinary MOPEG levels in a given untreated individual from one day to another. In contrast, wide variations from day to day were observed in both healthy subjects (Linnoila et al. 1983) and in depressed patients (Hollister et al. 1978). These variations have been attributed in part to diet (Hollister et al. 1980), level of motor activity (Muscetolla et al. 1977), and age and sex (Ebert et al. 1972). The present study was undertaken to determine the correlation between urinary MOPEG excretion and plasma DOPEG levels in depressed patients, as both metabolites are believed to reflect the activity of central noradrenergic neurons.

Methods Fifty-six inpatients (32 women and 24 men, aged 25-77 years; mean ? SD 48 + 12 years) were included in the trial, which took place in the period January 1982-January 1984. All cases were diagnosed as major affective disorders according to DSM-III criteria. The distribution into diagnostic subcategories was as follows: 45 major depressive episode; 15 bipolar disorder, depressed; 21 major depression, recurrent; 9 major depression, single episode; and 11 nonmajor depressive episode. Severity of depression was assessed with the Hamilton Rating Scale (NIMH version 1967-3) of 25 items and was arrived at by consensus between two scorers. All subjects had a total score above 40 (absence of symptoms is scored as 24). None of the patients had any somatic disease. All patients gave their informed consent to participate in this study. Plasma DOPEG and urinary MOPEG levels were measured after a therapeutic washout period of at least 4 days. The only treatment allowed during this period consisted of benzodiazepines (oxazepam 50-150 mg/per day). Certain patients also received laudanum of Sydenham (opium extract, 50-150 drops/day; 40 drops is equivalent to 10 mg morphine) because of severe anxiety and suicidal risk. An earlier study (Scatton et al. 1986) had failed to reveal any difference in DOPEG levels between depressed patients receiving benzodiazepines and laudanum and depressed patients receiving benzodiazepines alone. The blood sample for measurement of DOPEG was drawn at 9:00 AM, with the patient lying in a horizontal position after resting for 15 min. Ten milliliters of blood was drawn by venipuncture into a heparinized tube. Plasma was frozen at - 25°C until assayed. Free and sulfoconjugated DOPEG levels were measured by the radioenzymatic method of Dennis and Scatton (1982). Results were expressed in picograms per milliliter. Twentyfour-hour urine samples were collected between 8:00 AM on the first day and 8:OOAM on the second day, and were kept at -25°C until analyzed. Creatinine levels were measured in each urine sample with the aid of a Creatinine-Kit (Bio Merieux).

902

BIOL PSYCHIATRI 198621:‘M&906

H. L&I et ai

The urinary (deconjugated) monoamine metabolites were assayed as blind duplicates by high-performance liquid chromatography with electrochemical detection. Briefly, the experimental conditions were as follows. Enzymatic hydrolysis of the urine samples was achieved by overnight incubation of urine with P-glucuronidase/arylsulfatase from H. porn&a. After acidification, a number of samples were spiked with known concentrations of standards in order to correct for the efficiency of extraction. Samples were extracted with water-saturated ethyl acetate, following which the organic phase was evaporated. The dry residue was reconstituted with the mobile phase, and samples were injected directly into the chromatographic system. The optimal chromatographic separation of the chemical species was achieved by using a citrate-phosphate (0.2 M, pH 4.2) eluent containing 6% methanol. The flow rate of the mobile phase was 1 ml/min, and the column (1.5 cm x 4.6 mm, packed with Cl8 Spherisorb, 5 pm) was maintained at 30°C. Under these experimental conditions, we were able to separate vanillylmandelic acid, MOPEG. 5hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) (respective retention times: 2.25, 4.4, 10, and 12.2 min) with efficiencies of extraction of 92% +- 3%‘. 85% rt 2%, 68% -+ 3% and 92% -+ 2%, respectively. The results were expressed in micrograms of metabolite per milligram creatinine. Statistical analysis of the data was performed by using the Student’s t-test for large samples, following a normal law and a non-parametric permutation test (Fisher test) for smaller samples. Correlations were caiculated by using a least-squares regression analysis.

Results Earlier studies have shown an influence of sex and age on the formation of MOPEG (Ebert et al. 1972). Plasma DOPEG does not appear to be influenced by sex in depressed patients, but a significant correlation has previously been found between age and plasma free DOPEG (MO et al. 1983). Furthermore, the washout period was not of uniform duration in the patients studied. Therefore, it was necessary to rule out any possible influence of these factors on the biochemical parameters measured before attempting to correlate them.

lnjluence of Age There was no significant correlation between plasma free or conjugated DOPEG levels and age (r = 0.17), but urinary total MOPEG excretion was positively correlated with age (r = 0.38; p < 0.01). Using Hotelling’s test, we failed to observe a significant difference between these two correlation coefficients.

lnjluence of Sex Values of plasma DOPEG levels and urinary MOPEG excretion in male and female patients are given in Table 1. No significant sex-related differences were observed.

InJluence of the Washout Period The washout period ranged from 4 to 60 days: 16 patients had a washout of 4-6 days (5 +- 1 days), 25 had a washout of 7 days, and 15 had a washout of 8-60 days (14 ? 13 days). As shown in Table 2, the patients with a washout period of 7 days were significantly older (p < 0.02) than those with a shorter washout period. Urinary concentrations of total MOPEG were significantly higher in patients with a washout of 7 days as compared

BIOL PSYCHlATRY 198~21:~~

Correlation of DOPEG and MOPEG in Depression

903

Table 1. Lack of Correlation between Plasma Levels of Free or Sulfoconjugated DOPEG and Urinarv Concentrations of Total MOPEG in 56 Depressed Patients Plasma free DOPEG (pglml)

5% + 252 556 r 204 573 + 225”

Men (n = 24) Women (n = 32) All patients (n = 56) Plasma conjugated DOPEG (pglml)

Men (n = 24) Women (n = 32) Ail patients (n = 56)

966 r 791 707 rt 422 821 + 6156

Total urinary MOPEG (pglmg creatinine)

Men (n = 24) Women (n = 32) All patients (n = 56)

1.08 h 0.65 1.07 k 0.55 1.08 rt 0.59

Resahs repwent mean k so. a “ws”Sc: r = +0.03.5. b ver~w c: r =

+0.013.

with subjects with less than 7 days of washout (p < O.Ol), but values were similar in

patients with 4-6 days and 8-60 days of washout. The well~s~blished positive correlation between MHPG and age could account for the higher urinary MOPEG levels observed at 7 days of washout. The plasma levels of free DOPEG and conjugated DOPEG showed no significant difference in relation to the duration of therapeutic washout. Seven of the patients had previously received antipsychotics, 18 tricyclic antidepressants, 8 other ~tidep~ss~ts, 5 ~tipsych~i~s with ~tidep~ss~~, and 18 no antipsychoties or antidepressants. Analysis of the respective DOPEG and MOPEG values (Table

Table 2. influence of the Duration of Washout on Plasma DOPEG and Urinary MOPE% Levels and Their Correlation Washout period 4-6 Days

7 Days (n = 25)

S-60 Days (n = 15)

52 rt 11’ 576 rt 223d

48 -c 10 525 k 1788

858 2 671b

729 + 46Y

937 t 776h

0.86 2 o/w’

1.27 + 0.62,’

1.00 2 0.62’

(n = 16)

Age Plasma free DGPEG (P&m0 Plasma conjugated DOPEG (Pg/mI) Urinary total MOPEG (pg/mg creatinine) Correlations

jFisber’sexact probability:p = ‘Fisher’sexact probability:p = NS, not significant. Results are means 2 SD.

0.02. 0.01.

a vs c: r = 0.18

d vsf: r = 0.23

NS b vs c: r = 0.41 NS

NS e vsf: r = 0.01 NS

gvsi:r

= -0.49 NS h vs i: r = -0.13 NS

904

H. Looctal

BIOL PSYCHIATRY 1986;2 1900-906

Table 3. Influence of Previous Drug Treatment on Plasma DOPEG and Urinary MOPEG Levels (Mean ? SD) Antipsychotics associated with antidepressants

Antipsychotica

Urinary

total

MHPG

Other antidepressants

No treatment

7

5

IX

x

18

676 rt 307

577 -t 278

518 ? 192

614 ?z 243

568 -+ 206

1288 -r- 271

654 2 532

go5 Y?71s

722 rt 32X

736 ? 475

0.45

1.43 + 0.78

I.0 +- 0.62

1.16 f o.s7

0.98 t 0.56

n

Plasma free DOPEG (pgiml) Plasma conjugated DOPEG (pg/ml)

Tricyclic antidepressants

1.18

?

(pg/mg creatinine)

3) with the Dunn’s Multiple Comparisons Test failed to reveal significant these biochemical parameters (p > 0.50) between these subgroups.

variation

of

Lack of Correlation between Plasma DOPEG Levels and Urinary MOPEG Excretion In our population, plasma free or conjugated DOPEG was not correlated with urinary MOPEG (Table 1). As urinary MOPEG excretion was correlated with age (see above). the variation in MOPEG excretion that was apparently related to washout duration might in fact be due to age. Nonetheless, the existence of a difference in urinary MOPEG excretion in relation to the duration of washout made it necessary to determine the DOPEG-MOPEG correlation independently for the three washout periods. Regardless 01 the washout duration, there was no correlation between plasma free or conjugated DOPEG levels and the urinary excretion of total MOPEG (Table 2). There was no correlation between plasma free DOPEG and urinary MOPEG, nor between plasma conjugated DOPEG and urinary MOPEG, in the 45 patients diagnosed as suffering from a major depression (21 men and 24 women, aged 49 + 11 years; total Hamilton Rating Scale score

Table 4. Lack of Correlation Major Depressed Patients

between Plasma DOPEG and Urinary MOPEG in

Plasma free DOPEG

fpgiml)

573 ? 234” 585 i 220’

MAD (n = 45) With melancholia tn = 24) Plasma conjugated

DOPEG

(pglml)

MAD (n = 45) With melancholia (n = 24) Urinary

MOPEG

(@mg

807 2 564h 780 5 508’

creafinine)

MAD (n = 45) With melancholia (n = 24) Results represent mean + w. NS. not significant. MAD, major affective disorder. u vs c: r = 0.06. NS h YS c: i- = 0.13, NS. d vsf: r = -0.26. NS < vrfi r = -0.13. NS

1.17 f 0.6tY 1.27 -c 0.54’

..

Correlationof DOPEG and MOPEG in Depression

BIOL PSYCHIATRY 1986;21:9C%906

905

50 IL 7) or in the 24 patients diagnosed as suffering from a major depression with melancholia (14 men and 10 women; total Hamilton Rating Scale score 53 + 7) (Table 4). Discussion In the present study, no correlation was found between the plasma levels of DOPEG and the urinary excretion of MOPEG, two metabolites believed to reflect the central metabolism of NE. This lack of correlation was found regardless of the subgroup of depressed patients studied. There was a wide interindividual variation in the urinary excretion of MOPEG and plasma levels of DOPEG, but values for these two NE catabolites were not correlated in the same depressed subjects. Urinary MOPEG concentrations were positively correlated with age in the different subgroups of depressed patients, but showed no difference between men and women. DOPEG levels were dependent upon neither age nor sex. The exact contribution of central NE metabolism to urinary MOPEG levels remains poorly defined. Different factors may cause variations in MOPEG and DOPEG levels, such as diet, motor activity, and anxiety. These factors can also modify the respective contributions of the central and peripheral nervous system to plasma DOPEG and urinary MOPEG. MOPEG is formed from DOPEG by COMT. The activity of this enzyme can be influenced by various factors; its activity might even be altered in major depression (Shulman et al. 1978). One study (Gershon 1978) reported that COMT activity in the red cells was higher in agitated depressed patients and lower in inhibited depressed patients. This could at least partly account for the lack of correlation between the levels of these two NE metabolities. In the present study, urinary MOPEG excretion was measured from a 24-hr collection, whereas plasma DOPEG was determined from a single venipuncture at a given time point of the day. The lack of correlation between these two biochemical parameters could thus be explained by the fact that the morning determination might bear a poor relationship with mean plasma DOPEG levels throughout the day. There may be variations over the course of the day and night in plasma DOPEG levels, as is the case for plasma NE (Shulman et al. 1978) and plasma MOPEG (Halaris 1984). Disturbances of some hormonal circadian rhythms in depressed patients would also appear to reflect cerebral monoaminergic circadian dysfunction (Checkley 1980; Halaris 1984). However, the existence of a plasma DOPEG cycle in the healthy individual and its possible disturbance in depressed patients are presently unknown. Although further studies are clearly needed in order to define the respective roles of plasma DOPEG and urinary MOPEG as peripheral indices of cerebral NE activity, one can conclude that the present study shows no correlation whatsoever between these two biochemical parameters in depressed patients. The authors wish to thank Dr. C. De Montigny for scientific advice and for stylistic correction of the manuscript; Mrs. Tort-ratfor the statistical analysis; the nursing staff of the department for their participation in this study; and Miss Bleasdale for typing this manuscript.

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in depressive

reactions.

A review.

A

Arch Gen

906

H. Loo et al.

BIOL PSYCHIATRY 1986:21:900-906

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levels are dependent

on central nor-

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activity in patients with

Verbeuten TJ. Vanhoutte PM ( 1982): Deamination of released jH-noradrenaline saphenous vein. Naunyn Schmiedebergs Arch Pharmacol3 18:148-I 57

in the canine