Stereospecific effects of (+)- and (−)-catechin on glycogen metabolism in isolated rat hepatocytes

50

Biochimtca et Blophvswa Acta, 763 (19~3} 50 57 Elsevier

BBA 11185

S T E R E O S P E C I F I C EFFECTS O F ( + ) - AND ( - ) - C A T E C H I N ON GLYCOGEN M E T A B O L I S M IN I S O L A T E D RAT H E P A T O C Y T E S F R I T Z NYFELER, U L R I C H K. MOSER and PAUL WALTER *

Department of Biochemistry, Vesalianum, University of Basel, Vesalgasse 1, CH- 4056 Basel (Switzerland) (Received March 12th, 1983)

Key words: Glycogen metabolism," Catechin; Glucagon; Glycogen phosphorylase," (Rat hepatocyte)

( + )- and ( - )-catechin showed opposite effects on glycogen metabolism in isolated rat hepatocytes. Addition of 0.5 mM catechin to hepatocytes from fasted rats resulted in the case of the (+)-isomer in a 90% stimulation and the case of the ( - )-isomer in a 90% inhibition of net glycogen production. When 0.5 mM of the two isomers were added to hepatocytes from fed rats, (+)-catechin inhibited glycogenolysis by 33%, whereas the ( - ) - i s o m e r stimulated the same process by 42%. At equal concentrations, the effects of ( - ) - c a t e c h i n were stronger than those of the (+)-isomer. (-)-Epicatechin acted in a manner similar to (+)-catechin; however, the effect was less pronounced. (+)-Catechin antagonized the inhibitory action of suboptimal doses of glucagon on glycogen production whereby no change in basal or glucagon-elevated cyclic A M P level was observed. The activities of glycogen synthase a and glycogen phosphorylase a were changed by ( + ) - and ( - ) - c a t e c h i n in a way corresponding to the changes in glycogen production and breakdown. ( - )-Catechin, however, stimulated the activities of both glycogen synthase a and glycogen phosphorylase a in hepatocytes from fed rats. A possible interaction of the flavonoids or of their metabolites with glycogen phosphorylase is discussed.

Introduction

Catechin belongs to the group of flavonoids which are known to be widely distributed in plants. In the past years, (+)-catechin has attracted special attention, since it has been shown to possess some interesting properties related to liver function, such as stabilization of the membranes of liver lysosomes in vivo [1], prevention of necrosis induced by paracetamol in liver slices [2] and inhibition of lipid peroxidation [3-5]. Furthermore, administration of (+)-catechin led to a faster recovery of decreased ATP levels and of N A D : N A D H ratios in rats With liver damage provoked by a low-protein/high-fat diet or by * To whom correspondence should be addressed. 0167-4889/83/$03.00 © 1983 Elsevier Science Publishers B.V.

administration of orotic acid or ethanol [6]. ( + ) Catechin is on the market today as a hepatoprotective drug under the name of (+)-cyanidanol-3 (for reviews on clinical aspects see Ref. 7). Information on the effects of catechin on carbohydrate metabolism is scarce. In one report, it was shown that administration of (+)-catechin in vivo increased the lowered glycogen content in rat liver during experimental steatosis [8]. We had previously observed some effects of catechin and other flavonoids on respiration and oxidative phosphorylation in rat liver mitochondria [9,10]. Further studies have now revealed a unique stereospecific effect of the enantiomers of catechin on glycogen metabolism in isolated rat hepatocytes. Some of the results have previously been reported in abstract form [11].

51

Materials and Methods

Hepatocytes from 24 h fasted rats were prepared as described earlier [12]. Those from normally fed rats were prepared identically, except that buffer solutions used during the isolation procedure contained 30 mM glucose to avoid endogenous glycogen breakdown. Male Wistar rats, weighing 180-260 g, were obtained from the Biological Department of the Swiss Vitamin Institute in Basel. Incubation procedure. (10-25)-106 cells were preincubated for 20 min in stoppered 25 ml Erlenmeyer flasks which were gassed with 95% Oz/5% CO 2 and shaken at 37°C. The medium for the preincubation (2.5 ml, pH 7.4) consisted of 11.34 mM NaC1, 6.48 mM KC1, 0.95 mM MgSO4, 0.95 mM NaH2PO 4, 30 mM N a H C O 3, 1.56 mM CaC12 and 4.8% albumin. After the preincubation, the incubations were started by the addition of varying amounts (see Results) of 0.3 M glucose and catechin (dissolved in 0.15 M NaC1 under gentle heating). Isotonic NaC1 solution was added to give a final incubation volume of 3 ml. The final incubation medium corresponded to a Krebs-Ringer bicarbonate buffer in which some of the Na ÷ and C1- was substituted by glucose. Unless otherwise stated, the incubation time was always 60 min. Analytical methods. For the determination of glycogen, 1 ml of the cell suspension was added to 1 ml of 54% KOH. After digestion, precipitation and hydrolysis according to standard procedures [13,14], glycogen was .measured as glucose [15]. ' N e t glycogen production' and 'glycogen breakdown' refer to the differences in glycogen content (expressed as glucose) after the incubation and the zero time (i.e., after preincubation). The activity of glycogen synthase a was determined by the incorporation of UDP[U-14C]glucose into glycogen in 0.2 ml aliquots of the cell suspensions which had been frozen in solid CO2/ acetone [16]. The activity of glycogen phosphorylase a was determined in identically frozen aliquots in the direction of glycogen synthesis by measuring the incorporation of [U-lac]glucose 1-phosphate into glycogen [17]. Radioactivity was counted on paper strips as described by Thomas et al. [18]. Cyclic AMP levels were measured by the competi-

tive protein binding assay [19]. Extracts for cyclic AMP determination were prepared as described by Avruch et al. [20]. Expression of results. All results are expressed per g liver wet weight, and a value of 125 • 10 6 cells per g liver wet weight was used for calculations [21,22]. Most results are given as mean + S.E. of a number of experiments with different cell preparations; in each of these experiments, at least triplicate incubations were carried out for each parameter tested. In some instances, the results of a representative experiment with a single cell preparation are given. Student's t-test on paired or unpaired data was used to determine statistical significance. Materials. All chemicals were of the highest grade commercially available. Bovine albumin (fraction V) was purchased from Miles (Cavenago, Brianza, Italy), purified according to Chen [23], dialyzed first against 0.15 M NaCI and then against water. ( + )-Catechin, ( - )-catechin and epicatechin were partly purchased from Roth GmbH, Karslruhe, F.R.G. and were partly a generous gift from Zyma SA, Nyon, Switzerland. The ultraviolet absorption spectra and molar extinctions of the two enantiomers of catechin were identical. Results

Stimulation of net glycogen production by (+)catechin Net glycogen production from glucose in isolated hepatocytes from 24-h-fasted rats was signifi-

TA B LE I E F F E C T OF ( + ) - C A T E C H I N ON N E T G L Y C O G E N PROD U C T I O N IN TH E P R E S E N C E OF VARIOUS G L U C O S E CONCENTRATIONS n = number of experiments with different cell preparations. Significance versus control (unpaired data): b p < 0.005, a p < 0.001. Glucose (mM)

10 20 30

Net glycogen production ( ~ m o l g l u c o s e . g - ' . h - i + S.E.) Control

+ 0.5 mM ( + )-catechin

0.96_+0.15 6.10_+0.52 9.54_+ 1.16

3.29+0.52 a 11.53_+0.71 a 16.74_+1.71 b

15 56 10

52 24

//

c

2C

•

. "I/Zt/ '°

u

E ~: 16

•//

'b~

o s

..¢':. L

0

o

• //

" *""O•

8

} .[;''. °o

'

~ o0 . ~ ~

'

'

'

'

Control r~te of net glycogen production (/Jmol glucose.g-l.h -1)

Fig. 1. Effect of ( + )-catechin on net glycogen production. The glucose concentration was 20 m M in all incubations. Each point represents a single experiment.

cantly enhanced in the presence of 0.5 mM (+)catechin (Table I). With 10, 20 or 30 mM glucose as substrate, mean stimulations of 240, 90 and 75%, respectively, were observed. As reported earlier [11], the control rates of net glycogen production in hepatocytes showed large variations between single experiments. In Fig. 1, the control rates with 20 mM glucose as substrate were therefore plotted against the stimulated rates in the T A B L E 1I S T I M U L A T I O N OF N E T G L Y C O G E N P R O D U C T I O N BY V A R I O U S C O N C E N T R A T I O N S OF ( + )-CATECHIN 20 m M glucose was present in all incubations. All values are m e a n s + S.E. of seven experiments with different cell preparations. Statistical analysis was performed by Student's t-test on paired data. ( + )Catechin (mM)

Net glycogen production (/xmolglucose'g-l'h -l)

Mean stimulation (%)

0

6.68 _+ 1.14 8.81 5:1.32 12.54 + 1.99 9.39 + 1.74

control 39+ I 1 98 + 24 60 5:23

0.1 0.5 1

P vs. control

< 0.005 < 0.005 < 0.025

~30

.

.

.

60.

.

IncubQt~ time (rain)

.

90

Fig. 2. Time-course of stimulation of glycogen production by (+)-catechin. 20 m M glucose was present in all incubations. All values are_+S.E, of four experiments with different cell preparations. • • , 20 m M glucose; • A, 20 m M glucose and 0.5 m M (+)-catechin. Significance (paired data): l0 rain, n.s.; 20 min, P < 0.05; 30 min, P < 0.025; 60 min, P < 0.01; 90 rain, P < 0.005.

presence of 0.5 mM (+)-catechin. The control rates of net glycogen production varied between about 2 and 14/~mol • g- 1. h - l, and it is apparent that there is a good correlation (P < 0.001) between the magnitude of the control rate and the stimulation by (+)-catechin. The dose-response experiments summarized in Table II show a maximal stimulation of net glycogen production by a catechin concentration of 0.5 mM. This maximal effect was confirmed in "additional experiments with 0.25, 0.50 and 0.75 mM (+)-catechin. Furthermore, at 1 mM, the stimulation decreased and, at higher concentrations, an inhibition was observed (results not shown). The hepatocytes were not damaged by the addition of the flavonoid up to a final concentration of 5 mM as judged by Trypan blue exclusion and leakage of lactate dehydrogenase from the ceils. The time study in Fig. 2 shows that the effect of (+)-catechin is not immediate, but takes about 20 min to become significant. It is noteworthy that the stimulation remained constant over a period of 90 min. When, however, the cells had been preincubated with (+)-catechin for 20 min, then centrifuged off and reincubated with 20 mM glucose, most of the

53

TABLE III EFFECT OF PRETREATMENT OF HEPATOCYTES WITH ( + ) - OR ( - ) - C A T E C H I N ON NET G L Y C O G E N PRODUCTION Hepatocytes were preincubated for 20 min with or without catechin. Afterwards the cells were centrifuged off (5 min at 200 × g) and resuspended in prewarmed incubation buffer containing 20 mM glucose and, where indicated, ( + ) - or ( - ) catechin. The values represent means + S.E. of five incubations with the same cell preparation. Cells preincubated with

0.5 mM ( + ) catechin 0.5 mM ( - )catechin

Additions during incubation

Net glycogen production (~ mol glucose. g-~.h -l )

20 mM glucose 20 mM glucose + 0.5 mM (+)-catechin 20 mM glucose + 0.5 mM ( - )-catechin

4.26 _+0.21

20 mM glucose

4.62 + 0.04

20 mM glucose

3.14+0.24

9.72+0.20 0.23 + 0.08

effect of catechin had disappeared (Table III). It appears, therefore, that the stimulation is reversible and occurs only in the continuous presence of

TABLE IV E F F E C T OF ( + ) - C A T E C H I N ON NET G L Y C O G E N PROD U C T I O N A N D CYCLIC AMP C O N T E N T IN PRESENCE A N D ABSENCE OF G L U C A G O N The glucose concentration was 20 mM in all incubations. When indicated, 0.5 mM (+)-catechin a n d / o r 3.10 -9 M glucagon were added. Glycogen was measured after 60 min of incubation, whereas cyclic AMP content was determined in an aliquot after 6 min of incubation. All values are the mean-1-S.E, of six experiments with different cells preparations. Significance (paired data): a p < 0.002 vs. control; b p < 0.02 vs. control; P < 0.01 vs. glucagon; d n.s. vs. control; e p < 0.005 vs. control; f n.s. vs. glucagon

(+)-catechin. In further experiments it was investigated whether ( + )-catechin interferes with the action of glucagon. This hormone is a well-known inhibitor of hepatic glycogen synthesis, producing its effect via a transiently enhanced cyclic AMP level. In our system, the addition of 0.5 mM (+)-catechin partly antagonized the inhibitory action of glucagon (Table IV). Neither the basal nor the glucagon-elevated cyclic AMP level was changed by the addition of the flavonoid, indicating that the stimulatory effect of catechin and the inhibitory activity of glucagon are based on different mechanisms.

Inhibition of net glycogen production by ( - ) catechin The effect of the ( - ) i s o m e r of catechin was opposite to that of the (+)-isomer, as it strongly inhibited net glycogen production from glucose in hepatocytes from 24-h-fasted rats. The results of the experiments summarized in Table V show that with glucose concentrations up to 30 mM, net glycogen production was practically abolished by the addition of 0.5 mM (-)-catechin. At higher glucose concentrations, this inhibition was somewhat less pronounced, but even with 60 mM glucose, 0.5 mM ( - )-catechin still decreased glycogen production by more than 70%. Further studies on the correlation between the inhibitory action of (-)-catechin and glucose show (Fig. 3) that ( - ) catechin shifted the dose-response curve of net glycogen production towards the right. This effect

TABLE V EFFECT OF ( - ) - C A T E C H I N ON NET G L Y C O G E N PROD U C T I O N IN THE PRESENCE OF VARIOUS GLUCOSE CONCENTRATIONS n = number of experiments with different cell preparations. Significance versus control (unpaired data) for all values P < 0.001.

Addition

Net glycogen production (/~ mol glucose. g-l.h-J )

Cyclic AMP content (nmol- g - i )

Glucose (mM)

Control (+)-Catechin Glucagon ( + )-Catechin + glucagon

4.42 _ 1.23 8.83+0.91 a 2.64 + 0.84 b 4.54+0.72 c

0.31 + 0.10 0.27 +0.10 d 3.48 + 0.57 ¢ 3.12+0.64 r

20 30 40 60

Net glycogen production (t-tmol glucose.g- l - h - 1 + S.E.) Control

+ 0.5 mM ( - ) - C a t e c h i n

6.03+0.79 11.09 + 1.74 15.90+2.87 24.13+3.51

0.57+0.13 1.04 + 0.34 2.08+0.49 6.94+ 1.03

21 9 7 6

54

.25[

TABLE VI E F F E C T OF (-)-EPICATECHIN, ( + ) - A N D I - I CATECHIN ON NET G L Y C O G E N PRODUCTION A N D G L Y C O G E N BREAKDOWN Two experiments are combined. Cells from fasted rats incubated in 20 mM glucose were used for the measurements of net glycogen production and cells from fed animals for the glycogen breakdown determination. In each experiment, values are means-+ S.E. of four incubations with the same cell preparation. Significance unpaired data: a p < 0.05: b p < 0.02: P < 0.01; d p < 0.001: e not significant.

-//~b

Net glycogen production (~mol glucoseg 1.h I)

Change in glycogen (/lmol glucoseg I.h-I )

Control 0.01 ( - )-epicatechin 0.1 (-)-epicatechin 0.5 (-)-epicatechin 1.0 (-)-epicatechin 2.0 ( -- )-epicatechin 0.5 ( + )-catechin 0.5 ( -)-catechin 1.0 ( _+)-catechin

3.08 _+0.18 3.62+0.16" 3.86_+0.17 b 2.74_+0.10 e 2.27-+0.21 d 0.44_+0.10 d 6.77 _+0.32 o 0.18_+0.14 J 0.30 -+ 0.07 d

59.61 + 1.25

2b 3~ 4%- 6~o

Glucose concentrotk~ OmM)

Fig. 3. Inhibition of glycogen production by various concentrations of (-)-catechin. The experiment was performed with a single cell preparation and each point represents the mean of three incubations. ( - ) - C a t e c h i n concentrations (mM): • • , none (control); • • , 0.1; • I , 0.5;

•

Flavonoid (mM)

• , 1.0.

was already apparent with 0.1 mM (-)-catechin. At high glucose concentrations, of 40-60 raM, the slope of the curve with 0.5 mM ( - ) - c a t e c h i n was comparable to that of the control curve. This inhibitory pattern can tentatively be explained by a competition of ( - ) - c a t e c h i n and glucose for a common site of action. The results in Table III furthermore shown that the inhibition was reversible, i.e., when cells preincubated with the inhibitor were centrifuged off and resuspended in incubation buffer, most of the inhibitory properties were lost. In this respect, the two enantiomers of catechin behaved identically. Effect of ( - )-epicatechin and of simultaneously added ( + )- and ( - )-catechin on glycogen production Catechin has two asymmetric centers, in posiOH

OH

53.37-+ 1.13 " 40.64-+2.91 d 31.95--+ 1.15 d 30.77 -+ 0.78 d 84.19-+0.44 d 68.54 -+ 0.95 ,I

tions 2 and 3 (see formula). The compounds have been named catechins or epicatechins depending on whether the hydroxyl group and the B-ring are in trans or cis positions. As shown in Table VI, 0.1 m M (-)-epicatechin had a stimulatory effect on glycogen production which was less pronounced than that of (+)-catechin. However, with only 0.5 mM (-)-epicatechin, the stimulation turned into an inhibition. When 0.5 mM each of ( + ) - and ( - ) - c a t e c h i n were added together, the effect was about the same as with ( - ) - c a t e c h i n alone (Table VI). However, when they were added sequentially, a different picture arose (Fig. 4). When hepatocytes were incubated for 20 min with glucose and thereafter for 40 min with (-)-catechin, glycogen production stopped completely and the glycogen levels actually decreased. However, when, during the first 20 min, glucose and (+)-catechin were added together, subsequent addition of the ( - ) - i s o m e r was much less effective and glycogen production continued at a low rate. In other experiments not shown, the simultaneous addition of suboptimal amounts of ( - ) - c a t e c h i n and increasing amounts

55

4o0 r

v ~6 (3_

z

0

20

40 60 Incubation t i m e (mirO

Fig. 4. Sequential addition of ( + ) - and (-)-catechin. The incubations contained either 30 mM glucose (O O) or 30 mM glucose and 0.5 mM (+)-catechin ( • •). After 20 min of incubation, 0.5 mM (-)-catechin was added where indicated (arrows). Control values of net glycogen production without added (-)-catechin at the end of 60 min incubation: with 30 mM glucose, 14.6 + 0.2/xmol glucose, g - l ; with 30 mM glucose and 0.5 mM ( + )-catechin, 24.2 + 0.8/~mol glucose, g - I. All values are mean + S.E. of four incubations of a single cell preparation.

10 0

a~ 40

s3ok

F"

~ 20 / 0

of (+)-catechin showed a partial but always small release of the inhibition.

Effect of (+)- and (-)-catechin on glycogen breakdown (+)- and (-)-catechin also affected the breakdown of glycogen in hepatocytes isolated from fed rats. As shown in Table VII, there was again a stere•specific effect. While 0.5 mM (+)-catechin inhibited glycogenolysis by 33%, the same concentration of the (-)-isomer stimulated the rate of glycogen breakdown by 42%. In further experi-

TABLE VII EFFECT OF ( + ) - A N D ( - ) - C A T E C H I N ON G L Y C O G E N BREAKDOWN n = number of experiments with different cell preparations. Significance (unpaired data) for both values P < 0.001. Additions

Change in glycogen % change n (~mol glucose. (mean:t: S.E.) g - l . h - I + S.E.)

None 0.5 mM (+)-catechin

- 91.24 _+5.04 -62.34+4.08

-33+3

16 16

None 0.5 m M ( - ) - c a t e c h i n

- 89.62 + 5.79 -122.64+6.33

+42+5

12 12

/Z

~ i i i 30 60 90 Incubation time (min)

Fig. 5. Effect of ( + )- and ( - )-cat~lfin on glycogen breakdown and on the activities of glycogen phosphorylase a and glycogen synthase a. Hepatocytes from fed rats were incubated for the times indicated; • • , no addition (control); • •, with 0.5 mM (+)-catechin; • • , with 0.5 mM ( - ) catechin. All values are the mean of three, incubations with a single cell preparation.

ments not shown in detail, higher doses of ( + ) catechin resulted in a somewhat stronger inhibition of glycogen breakdown and the effect reached a plateau at concentrations between 1-5 mM. ( - )-Catechin was most effective at 0.5 m M and its stimulatory action decreased at higher concentrations; when 5 m M ( - ) - c a t e c h i n was added, glycogen breakdown occurred at the same rate as without catechin. As shown in Table VI, addition of (+)-catechin resulted in a somewhat weaker stimulation than with the ( - ) - i s o m e r alone. Furthermore, 1 m M ( - ) - e p i c a t e c h i n inhibited glycogenolysis to about the same extent as 0.5 m M ( + )-catechin. Fig. 5 illustrates the time-course of the catechin effect on glycogen breakdown and on the activity of glycogen phosphorylase a and glycogen synthase a. On one hand, addition of (+)-catechin

56

showed the expected results: inhibition of glycogen breakdown and glycogen phosphorylase a activity as well as an activation of glycogen synthase a. On the other hand, addition of ( - ) - c a t e c h i n increased glycogen breakdown and the activity of glycogen phosphorylase a as expected; however, it also stimulated the activity of glycogen synthase a. It should be added that in hepatocytes from fasted rats, which contain a larger amount of synthase a than hepatocytes from fed rats [16,24], the activity of glycogen synthase a was increased by ( + ) catechin and decreased by (-)-catechin. A simultaneous stimulation of both phosphorylase a and synthase a activity has repeatedly been reported, e.g., after the addition of fructose [25,26] and glutamine [26,27]. The mechanism of this effect is not understood. A possible relation to ionic changes has been proposed by Hers [28]. Discussion

The results clearly show that ( + ) - and ( - ) catechin have opposite effects on glycogen synthesis as well as on glycogenolysis. The action of the stereoisomer (-)-epicatechin was more similar to that of (+)-catechin than that of (-)-catechin. The only difference between (+)-catechin and (-)-epicatechin is the configuration of the hydroxyl group in position 3. The results therefore indicate that this configuration is more important for the action pattern of the catechins than that of the B-ring in position 2. Unfortunately, (+)-epicatechin could not be tested because it is not commercially available. In earlier work, we had demonstrated the stimulatory effects of lithium [29] and of insulin [12] on net glycogen production in rat hepatocytes. As in the case of insulin and lithium, the stimulation of glycogen synthesis expressed as percentage was also with (+)-catechin most pronounced at low glucose concentrations and decreased with increasing substrate levels. When the effects of the three stimulating agents on glycogen synthesis are compared, it is obvious that (+)-catechin is the most potent effector of the three. The mechanism of the (+)-catechin stimulation appears to be different from that of insulin, since catechin did not decrease basal or glucagon-elevated cyclic AMP levels, as was found with insulin [12]. Lithium, too,

did not change cyclic AMP levels but, unlike ( + )catechin, it had no effect on glycogen breakdown in hepatocytes from fed rats (unpublished observation). It appears, therefore, that the mechanism of action of catechin is also different from that of lithium. At equal concentrations, (-)-catechin was clearly more effective than the (+)-isomer. The results in Fig. 4 indicate that the enantiomeric forms of catechin are to a certain extent competitive. It was furthermore found that ( - ) - c a t e c h i n also showed competitive characteristics with glucose (Fig. 3). A possible candidate for a common site of action is glycogen phosphorylase a. Stalmans et al. [30] have shown that glucose can bind to phosphorylase a, thus making it a better substrate for phosphorytase-phosphatase. This inactivation of phosphorylase a was claimed to be a prerequisite for an activation of glycogen synthase [t6]. It could therefore be speculated that glucose~ (+)-catechin and ( - ) - c a t e c h i n compete for a common binding site on phosphorylase a. Another possibility would be that ( + ) - and ( - ) - c a t e c h i n attach to a common binding site, thereby influencing the binding of glucose to its specific binding site in opposite ways. Our measurements of phosphorylase a activity in hepatocytes treated with the flavonoids (Fig. 5) are also in agreement with such hypotheses; however, as already discussed in the results section, the simultaneous stimulation of both phosphorylase a and synthase a activities in (-)-catechin-treated cells cannot be explained by such a mechanism. It should also be mentioned that various aromatic compounds have been shown to interfere with the action of phosphorylases a and b [31,32]. According to Mead et al. [32], it is also widely accepted that the active site of phosphorylase is located in a crevice between the two main domains in a hydrophobic pocket. Because part of the catechin molecule is also aromatic, it could very well have a similar but much more specific effect than the aromatic compounds described in the literature. It has been demonstrated by autoradiography of isolated hepatocytes incubated with [14C]catechin that this compound is rapidly adsorbed on the cell membrane and then transferred into the cytoplasmic area of the cell [33]. Moreover, in pharmacokinetic studies it has been shown that

57

several metabolites of catechin appear in the bile 0.5-1.5 h after intravenous administration of the flavonoid [34]. However, only one metabolite has so far been identified [35]. In preliminary experiments we could not observe any changes in the activities of phosphorylase a when either ( + ) - or ( - ) - c a t e c h i n was added directly to the assay systems for the enzyme measurements. The possibility must therefore be considered that a metabolite of catechin rather than catechin itself is the active compound.

Acknowledgements This work was supported by the Swiss National Science Foundation and by Zyma SA (Nyon, Switzerland). The authors wish to thank Miss E. Kux for her excellent technical assistance.

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