44 The formation of all these compounds has been completely blocked by aminoguanidine sulphate (10 -4 M), a potent inhibitor of DAO, which is consistent with the general accepted scheme of oxidative deamination running with an appropriate aldehyde formation. Thus it is evident that every further enzymatic transformation of 7-aminobutyraldehyde must lead to a drop of this portion of aminoaldehyde which is undergoing cyclization to Al-pyrroline and which in the Okuyama and Kobayashi method plays a role of an indicator of DAO activity. It has been suggested that the use of inhibitors of aldehyde metabolism may give a better approximation of the real values of DAO activity when tissue homogenates are used as a source of enzyme [4]. However, the compounds used in the present study, known as good inhibitors of monoamine-derived aldehyde-metabolizing enzymes, revealed that aldehyde dehydrogenase catalysis of y-aminobutyraldehyde oxidation seems to differ from AldDH catalysis of oxidation of monoamine-derived aldehydes. For example, CROW [6], using glyceraldehyde substrate for sheep liver AIdDH, obtained K i for chloral hydrate 5.7 • 10-6 and 7.3 • 10-6 M (cytoplasmic enzyme) and 4.5 x 10-6 and 4.7 • 10-6 M (mitochondrial enzyme). Such high concentration of inhibitors as has been used in this work suggests that their effect was rather unspecific. Phenobarbital used as an inhibitor of AldR expressed almost no activity, which favours the idea of the absence of AldR in guinea pig liver. However, the presence of AldR in liver of several mammalian species has been described. Therefore it may be

Histamine Metabolism

concluded that phenobarbital is not the most specific inhibitor of this AIdR which catalyses the reduction of aminoaldehyde. Received 20 June 1978.

References [1] T. OKUVAMA and Y. KOBAYASHI, Determination of

Diamine Oxidase by Liquid Scintillation Counting, Arch. Biochem. Biophys. 95, 242-250 (1961). [2] L.T.

KREMZNER, J.M.

HILLER and

E.J.

SIMON,

Metabolism of Polyamines in Mouse Neuroblastoma Cells in Culture. Formation of GABA and Putreanine, J. Neurochem. 25, 889-894 (1975). [3] N. SEILER and B. EICHENTOPF, 4-Aminobutyrate in Mammalian Putreseine Catabolism, Biochem. J. 152, 201-210 (1975). [4] W.A. FOGEL, T. BIEGA/qSKI, J. WO2;NIAK and Cz. MAgLI/qSKI, Interference of Aldehyde Metabolizing

Enzymes with Diamine Oxidase (Histaminase) Activity as Determined by ~4C Putreseine Method, Biochem. Pharmacol. 27, 1159-1162 (1978). [5] A.-CI-I. ANDERSSON, S. HENNINGSSON and E. ROSENGREN, The Influence of Some Drugs on the Determination of Diamine Oxidase Activity, 7th Histamine Club Meeting, Lbdi, 2-4 May, 1978. [6] K.E. CROW, T.M. KITSON, A.K.H. MACGIBBON and R.D. BATT, Intraeellular Localisation and Properties of Aldehyde Dehydrogenases from Sheep Liver, Biochim. Biophys. Acta 350, 121-128 (1974).

The Influence of Some Drugs on the Determination of Diamine Oxidase Activity by A.-CrI. ANDERSSON,S. HENNINGSSONand E. ROSENGREN Department of Physiolo.gy and Biophysics, University of Lurid, Lund, Sweden

The main route in the metabolism of putrescine seems to be a multi-step sequence with several enzymes involved where the first two steps of the route are catalysed by diamine oxidase (histaminase) and 7-aminobutyraldehyde dehydrogenase, respectively [1, 2]. On incubating putrescine and homogenate of tissues rich in diamine oxidase, the major metabolites formed are GABA and some unidentified compound(s); Al-pyrroline and CO 2 are also obtained [3]. Chloral hydrate and phenobarbital are reported to inhibit the metabolism of aldehydes. MA~LIIqSKI and coworkers [4] found a striking increase of Al-pyrroline on incubating homogenate of guinea-pig liver with ~4C-putrescine, using these inhibitors. In the present study homogenate of guinea-pig liver was incubated with ~4C-putrescine in order to examine the effect of phenobarbital and chloral hydrate on the various compounds derived from putrescine. In addition the effect of aminooxyacetic acid, an inhibitor of 7-aminobutyric acid transaminase, was studied. The results are presented in Figure 1. It appears that the effects of chloral hydrate and phenobarbital are essentially the same, i.e. an increased formation of A~-pyrroline and a decreased formation of GABA, the unidentified compound(s) and carbon dioxide, with increasing dose of the inhibitors. As to aminooxvacetic

acid, this compound, as expected, increased the content of GABA and consequently the release of CO 2 was inhibited. An increase in the formation of Al-pyrroline was observed. It should be noted that considerable amounts of GABA, the unidentified compound(s) and CO 2 are formed in spite of the rather high concentrations of chloral hydrate and phenobarbital used. These findings support the view that determination of the amount of Al-pyrroline is not well suited as a measure of the diamine oxidase activity in tissues.

Received 15 June 1978.

References [1] N. SEILER and B. EICHENTOPF, Biochem. J. 152, 201 (1975). [2] S. HENNINGSSON and E. ROSENGREN, Br. J. Pharmac. 58, 401 (1976). [3] A.-CH. ANDERSSON,S. HENNINGSSON,L. PERSSONand E. ROSENGREN,Acta physiol, scand. 102, 159 (1978). [4] W.A. FOGEL, T. BIEGANSKI, J. WOZNIAK and Cz. MA~H~SKI, Agents and Actions (in press).

45

Histamine Metabolism

100 000"

50 O00-

|0

A

1O0000t

~io-4 M 80 000

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[~ 10-2 M

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GABA

CO2

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U.C.

GABA

CO2

PYR.

U.C,

GABA

L

CO2

PYR.

Figure 1 Rate of in vitro formation of different metabolites from 14C-putrescine in guinea-pig fiver after addition of phenobarbital (A), chloral hydrate (B) and aminooxyacetic acid (C). The metabolites were GABA, CO2, ALpyrroline (PYR). and some unidentified compound(s) (U.C.). Each column is the mean + S.E.M. of three observations.

Diamine Oxidase Activity During Wound Healing in Guinea Pig Skin by JANINA WOZNIAK,T. BIEGAI~SKI,CZ. MASLI/~ISKI Department of Biogenic Amines, Polish Academy of Sciences, 60 Narutowicza, 90-136 L6d$, Poland

Introduction Putrescine (1,4-diaminobutane), a precursor of the polyamines spermidine and spermine, plays an important role in cell physiology. Its formation in mammalian tissues has been extensively studied; its catabolism, however, is less well known. Nevertheless it has been suggested that the diamine oxidase (DAO) may control the intracellular level of putrescine [1] which in turn affects the metabolism of polyamines [2]. The occurrence of diamines and polyamines is implicated in various kinds of normal and neoplastic growth. In the present work attempts were made to relate changes of both diamine oxidase activity and polyamine levels during a wound-heaiing process. Methods Experiments were made on guinea pigs, weighing 250-350 g. Wound model In the dorsal side region, under fight ether anaesthesia, eight circular wounds were made. The skin from the wound was taken on the 1st, 3rd, 7th, 14th and 21st day of heafing and divided into two portions for the determination of DAO activity and polyamines.

Diamine oxidase assay DAO activity was measured by the modified isotope assay of OKUYAMAand KOBAYASHI [3] using 14C-putrescine and unlabelled putrescine as substrates. One of the reaction products, dl-pyrroline, together with its polymers, was extracted into toluene and measured by liquid scintillation counting. Determination of polyamines Polyamines (spermidine, spermine) were determined after butanol extraction by the method of RAINA [4], based on separation by paper electrophoresis followed by staining with ninhydrin. After elution the intensity of the colour was measured at 505 nm. Results I. DAO activity and polyamine concentration durlng wound healing DAO activity undergoes biophasic changes during wound healing (Table 1). On the 1st day DAO activity is lower than in control, but from the 3rd day of healing its activity rises significantly up to 177% of control and these and ever higher values remain until the 21st day of healing, the last day of observation. The levels of spermidine and