Naturwissenschaften 79, 379- 381 (1992) © Springer-Verlag 1992

Psychosocial Stress Affects Urinary Pteridines in Tree Shrews E. Fuchs and O. J0hren Deutsches Primatenzentrum, W-3400 G0ttingen, FRG M. Goldberg Institut for Tierphysiologie der Universit~tt, W-8000 Mtinchen, FRG

Pteridines were first described as pigments of butterflies. They belong to a group of compounds containing a pyrazino[2,3-d]pyrimidine ring. Unlike folates, which also contain a pterin ring, in vertebrates most pteridines are synthesized in vivo from guanosine triphosphate; the only exceptions are pterin and 6-hydroxymethylpterin which also may derive to some extent from endogenous folates [1]. In recent years, there has been increasing interest in quantifying pteridines in body fluids from mammals, including man. The main attention has been focused on neopterin, which may serve as a marker for the activation of the T-lymphocytemacrophage system. Although neopterin does not appear to play a role in the immune process, its measurement in urine and blood has proved useful for monitoring diseases affecting the immune system [2]. Another member of the pteridine family is biopterin. In its fully reduced form, tetrahydrobiopterin, it is a cofactor for the pteridinedependent monooxygenases which play important roles in the biosynthesis of biogenic monoamines such as catecholamines and indoleamines [1]. Stimulation of the hypothalamo-pituitary-adrenal (HPA) axis by physical stressors or by injection of ACTH resulted in increased urinary neopterin and biopterin concentrations [3, 4]. In the present study we investigated whether a more "natural" stressor such as chronic psychosocial conflict may also affect urinary pteridines. We therefore analyzed the excretion pattern of biopterin, 6-hydroxymethylpterin, isoxanthopterin, neopterin, and pterin before and during chronic social conflict. As indicator for the intensity of stress urinary cortisol, norepinephrine, and epinephrine were quantified simultaneously. For our experi-

merits we used male tree shrews (Tupaia belangeri, n = 12). Their value as a model for studying the consequences of psychosocial stress has been demonstrated previously [5]. The animals were kept individually in cages, without visual contact to their neighbors, in airconditioned rooms under artificial illumination (for details, see [6]). Body weight was recorded daily between 7:45 and 8:00 am, before the lights were switched on; morning urine was collected after a light massage of the hypogastrium. The experiments regarding chronic social confrontation were carried out as follows. During the control situation, animals were kept individually, urine was collected daily, and they were weighed as described above. After 10 days, the wire mesh and the partition between the cages of two males, which were unknown to each other, were removed. This enlargement of the territories resulted in low-intensity attacks and within a few hours, a stable dominance structure was established. In this situation the subordinate animal had a reduced sphere of action and withdrew from situations which could evoke attacks from the dominant animal. This phase of social confrontation lasted 10 days, and urine was collected and body weight was recorded daily. For determination of pteridines, a 500-/zl aliquot of the morning urine was immediately snap-frozen and stored in the dark until analysis. The five pteridines biopterin, 6-hydroxymethylpterin, isoxanthopterin, neopterin, and pterin were separated by high-performance liquid chromatography (HPLC) and quantified with fluorescence detection as described [7]. For determination of epinephrine and norepinephrine, a 190-/xl aliquot of the morning urine was added to 10/zl 6N

Naturwissenschaften 79 (1992) © Springer-Verlag 1992

HC104 centrifuged immediately (10000 g; 4 °C) and the supernatant was stored at - 2 0 ° C . Urinary catecholamines were extracted by cation-exchange chromatography with Bio-Rex 70 resin (Bio-Rad, Munich, FRG), separated by reversed-phase HPLC (250 x 4 mm column packet with 5/zm Lichrospher 60 RP-Select B; Merck, Darmstadt, FRG) and quantified with electrochemical detection according to [8] with a coulometric detector (ESA 5100A, high-sensitivity cell 5001, Bischof, Leonberg, FRG) in the redox-mode (electrode 1 - 0.2 V, electrode 2 + 0.25 V). For the determination of cortisol, a 500-/xl aliquot of the urine sample was transferred to a glass tube and stored at - 2 0 ° C . Analysis of urinary cortisol was carried out by a two-step HPLC as recommended [9]. The first step was a cleanup of cortisol on a normal-phase column (125 x 40 mm, 5 /zm Lichrospher 100 DIOL, Merck) according to [10] after organic extraction of the urine with dichloromethane. In the second step, cortisol was separated by reversed-phase HPLC (250 x 40 mm, 5 #m Lichrospher 100 RP-18, Merck) and quantified with UV detection [11]. Urinary creatinine was determined with a Beckman Creatinine Analyzer 2. The concentrations of pteridines, catecholamines, and cortisol were related to the creatinine concentration so that the various urine samples were comparable regardless of physiological dilutions of the urine. In the urine of the tree shrews biopterin was the most abundant pteridine followed by isoxanthopterin; neopterin was only detectable in minor amounts (Table 1). The neopterin/biopterin ratio of 0.006 is at least ten times lower than in primates, including man, and is comparable to results reported for rodents [12]. In primates the ratio is higher because guanosine triphosphate cyclohydrolase is not the rate-limiting enzyme as it is in other mammals [13]. As expected [5, 6] during social confrontation, subordinate animals showed a clear loss in body weight of up to 10 %, whereas the body weight of the dominant animals remained more or less constant (Fig. 1). In subordinate animals, elevated urinary cortisol and norepinephrine concentrations are indicative of an activation of the H P A axis and the peripheral sympathetic nervous system, respectively. Urinary 379

200

110

RELATIVE BODY WEIGHT

EPINEPHRINE

BIOPTERIN

(% of Control)

(pmol/pmol Creatinine)

(nmol//J,mol Creotinine)

100 100

90

TI

co 5

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10

15

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20

CORTISOL (ng//~mol Creatinine)

5

400

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I

10

15

20

fl

co 5

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s,

IT

10

15

20

0.8 NOREPINEPHRINE

ISOXANTHOPTERIN

(pmol//~mol Creotinine)

(nrnol/p,mol Creotinine)

0.8

0.6 200

0.4

0.0

0.4

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co 5

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sl

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(Days) Fig. 1. Relative body weight and concentrations of cortisol, epinephrine, norepinephrine, biopterin, and isoxanthopterin in the morning urine of male tree shrews (mean +_ SD) during control (CO) and social interaction (SO. Each period lasted 10 days. Animals were classified as dominant ( V ; n = 6) and subordinate ( • ; n = 6) according to their body weight during social interaction

Table 1. Pteridine concentration (/zmol/mmol/creatinine) in the morning urine of tree shrews during control situation (n = 38; mean _+ SD) Pteridine concentration

Pteridine content

[%1 Biopterin 6-Hydroxymethylpterin Isoxanthopterin Neopterin Pterin

4.016 0.082 0.313 0.023 0.095

+ 0.89 +_ 0.03 _+ 0.08 +_ 0.01 + 0.04

epinephrine, representative for the activity of the adrenomedullary system, was elevated immediately after the onset of social conflict in both dominant and subordinate tree shrews. In subdominants, however, epinephrine was elevated for a longer period o f time than in dominants. A m o n g urinary pteridines, neopterin, pterin, and 6-hydroxymethylpterin remained unchanged during the 10 days o f chronic conflict in both groups o f animals (data not shown). This kind o f stress, however, affected biopterin and isoxanthopterin (the end product o f the pteridine metabolism) excretion only in subordinate animals. Biopterin was 380

88.7 1.8 6.9 0.5 2.1

slightly and isoxanthopterin was drastically increased in the urine o f subordinates. As a result o f stress, creatinine concentration and urine volume were not reduced [6]. Therefore, the increased urinary concentrations of cortisol, catecholamines, and two pteridines in subordinate animals could not be explained as a result of an altered creatinine excretion. As demonstrated by this study, psychosocial stress affected the urinary excretion of biopterin and the catabolite isoxanthopterin without influencing urinary neopterin. The same result was reported recently from an investigation of pigs that were subjected to

physical exercise [14]. F r o m studies in rodents [4] it was concluded that stress per se resulted in elevated concentrations of neopterin and biopterin without affecting other members of the pteridine family. With regard to the present data, this idea, however, has to be revised and species-specific excretion patterns have to be taken into consideration. Depression [15] and various stressors modify the release and the catabolism of pteridines. Therefore, quantification of urinary pteridines could be a helpful means to assess alterations in "psychological well-being" and may offer an additional, noninvasive tool to supplement current methods of interpreting the duration and intensity of stress.

Received March 18, 1992 1. Hyland, K., Howells, D. W.: J. Chromatogr. Biomed. Appl. 429, 95 (1988) 2. MOiler, M. M., Curtius, H.-C., Herold, M., Huber, C. H.: Clin. Chim. Acta 201, 1 (1991)

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3. Rokos, H., Rokos, K., Kunze, R., in: Unconjugated pterins and related biogenic amines, p. 187 (H.-C. Curtius, N. Blau, R. A. Levine, eds.). Berlin: de Gruyter 1987 4. Koller, M. : Dissertation Univ. Mtinchen 1989 5. Holst, D. v., Fuchs, E., St6hr, W., in: Biobehavioral bases of coronary heart disease, p. 382 (T. M. Dembroski, T. H. Schmidt, G. Bliimchen, eds.). Basel: Karger 1983 6. Fuchs, E., Schumacher, M.: Physiol. Behav. 47, 713 (1990)

7. Goldberg, M., Gassner, F., Merkenschlager, M.: Pteridines 1, 29 (1989) 8. Schleicher, E. D., Kess, F. K., Wieland, O. H.: Clin. Chim. Acta 129, 295 (t983) 9. Nakamura, J., Yakata, M.: Clin. Chem. 28, 1497(1982) 10. Sch6neshofer, M., Weber, B.: J. Steroid Biochem. 18, 65 (1983) 11. Dingeon, B., Quenrad, M. T., Tome, H. :Ann. Biol. Clin. 46, 263 (1988) 12. Goldberg, M., Koller, M., Merkenschlager, M., in: Chemistry and biology of pteridines, p. 571 (H.-C. Curtius,

S. Ghisla, N. Blau, eds.). Berlin: de Gruyter 1990 13. Hasler, T., Niederwieser, A., in: Chemistry and biology of pteridines, p. 319 (B. H. Cooper, V. M. Whitehead, eds.). Berlin: de Gruyter 1986 14. Goldbert, M., Eichinger, H., Otten, W., Merkenschlager, M.: Tier~irztl. Prax. 19, 493 (1991) 15. Coppen, A., Swade, C., Armstrong, R. A., Blair, J. A., Leemin, R. J.: J. Affect. Disord. 16, 103 (1989)

Kohlendioxidhaushalts. Die Abh~ingigkeit der Lebewesen von den physikalischen Vorg~ingen im Ozean wird in einem Artikel fber Ringe von Wasserwirbeln am Rande des Golfstroms deutlich, mit denen Meeresorganismen in eine fur sie ungewOhnliche, oft t0dliche Umwelt verdriftet werden. Abschnitt 2 umfa6t sechs Aufsatze zum Thema Lebensraume und Lebensgemeinschaften. Sie zeigen, wie sich sessile Organismen an das Leben in der Brandungszone angepaBt haben, welche Faktoren Korallenriffe zu hochproduktiven Lebensr~iumen machen und welche Lebensformen sich durch die Symbiose zwischen wirbellosen Tieren und Schwefelbakterien an hydrothermalen Schloten der Tiefsee ausgebildet haben. Die Okologische Erforschung des Wattenmeeres l~iBt erkennen, wie menschliche Eingriffe, z.B. Deichbauten, Meeresverschmutzung und Einschleppen yon Organismen, den nattirlichen Wandel ver~ndert und beschleunigt haben, dem das Watt seit je unterliegt. Neue Ergebnisse polar-0kologischer Forschungen werden in einem Beitrag tiber das Meereis als Lebensraum pr~isentiert. Mit der Biologie von Fischen und Walen beschaftigen sich die Artikel des 3. Abschnittes. Anhand der Formen und Bewegungen des FischkOrpers werden verschiedene Antriebsweisen zur Fortbewegung im Wasser illustriert. Weitere Themen sind die Anpassungen der Fische der Gezeitenzone und der Antarktisfische an ihre speziellen Lebensbedingungen sowie der Nahrungser-

werb der Ftihlerfische, die ihre Beute angeln. Geschildert wird auch, wie sich Wale und Delphine an das Leben im Wasser angepa6t haben, welche Verhaltensweisen die groBen Bartenwale bei Nahrungssuche und Fortpflanzung zeigen und welche Funktion der WalrathOhle im Kopf des Pottwals vermutlich zukommt. Im 4. Abschnitt fiber Ernten aus dem Meer werden Probleme der Nutzung mariner Okosysteme am Beispiel der Antarktis dargestellt. Es folgen Beitr~ige fiber die MOglichkeiten der Aquakultur und tiber Algen als neue Nutz- und Kulturpflanzen. Den Aufs~itzen ist eine ausftihrliche Einffhrung von G. Hempel, dem Direktor des Alfred-Wegener-Instituts ffr Polar- und Meeresforschung in Bremerhaven, vorangestellt. In dieser Einffhrung werden die Inhalte der Einzelbeitr~ige in den Gesamtrahmen der modernen Meeresbiologie gestellt und etwas erweitert, zum Beispiel bezfglich der biologischen Folgen der Meeresverschmutzung. Das Buch ist mit zahlreichen, grOBtenteils farbigen Abbildungen reich illustriert. Bei dieser Ausstattung ist sein Preis nicht zu hoch. Als Manko empfindet der Rezensent, dab das Literaturverzeichnis nicht umfangreicher ist. Dem Nicht-Fachmann h~itte eine ausftihrlichere Liste den weiteren Einstieg in die Materie erleichtern kOnnen. Fazit: Ein empfehlenswertes Buch nicht nur ffr Biologen und Studenten, sondern ffr alle Interessierten. D. Sahrhage (Hamburg)

INlOtur

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Biologie der Meere. Mit einer Einffhrung von G. Hempel. Heidelberg: Spektrum der Wissenschaft Verlagsgesellschaft 1991. 224 S., 171 Abb., DM 44, Aus der Neuauflage von 18 Aufs~itzen, die - mit Ausnahme yon drei neuen Beitr~igen - in den vergangenen 12 Jahren in der Zeitschrift ,,Scientific American" bzw. ihrer deutschen Ausgabe ,,Spektrum der Wissenschaft" erschienen waren, entstand ein vielf~tltiges, interessantes, auch ffr den Laien gut verst~ndliches Lesebuch zu Fragen der Biologischen Meereskunde und marinen Biologie. Die beteiligten 31 Autoren, deren wissenschaftliche Werdeg~inge skizziert werden, kommen aus den USA, Grofibritannien und Deutschland. Das Werk umfaBt ein weites Spektrum yon Themen: Im ersten Abschnitt fiber den Ozean und seine Produktion werden der Stoffkreislauf des Ozeans mit den for die marinen Organismen wichtigsten chemischen Komponenten dargestellt und die dabei wirksamen Mechanismen beschrieben. Im Hinblick auf die gegenw~irtigen Diskussionen tiber den Treibhauseffekt durch vermehrte Kohlendioxidproduktion sind die Ausffhrungen fber die Ver~nderungen in der Chemie der Ozeane seit dem Ende der Eiszeit und in frfheren Erdzeitaltern besonders aktuell. Der n~chste Beitrag fiber die Prim~irproduktion der marinen Plankton-Algen besch~iftigt sich mit deren gro6er Bedeutung ffr den globalen Kreislauf des Kohlenstoffs und die Regulierung des

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