40
Br~ves communications - Kurze Mittcilungen EXPERIENTIA XVII/1Iproniazid verhindert die durch Reserpin verursachte Erniedrigung des F-Aminobutters~uregehaltes des Gehirns und der Krampfschwelle
Kontrollen Iproniazid 100 mg/kg (20 h) Reserpin 5 mg/kg (3 h) Reserpin 5 mg]kg (3 h) + Iproniazid 100 mg/kg (20 h) (siehe Methodik) Ver- suchs- zahl 60 7 20 12 ~-Aminobutter- s~iure txMol/g 3,49 -t- 0,14 3,92 :j= 0,19 2,45 -i- 0,22 3,48 ± 0,23 Elektroschock 100 Hz, 5 rose% 5 see 4,4 mA (4,2-4,6) 5,0 mA (4,8-5,2) 1,2 mA
(1,0-1,4)
5,9 mA (5,5-6,3)Methodik. "Weisse M~iuse (18-20 g). Reserpin (5 mg/kg) wurde subkutan, in den Versuchen, in denen schon 15rain n a c h der I n j e k t i o n G A B A - G e h a l t und KrampIschweUe b e s t i m m t wurden, intraperitoneal injiziert. Iproniazgd (100 mg/kg) intramuskul~ir 20 h v o r T 6 t u n g der Tiere (GABA-Bestimmnng) bzw. v o r Elektroschock.
G A B A - B e s t i m m u n g . ~thylalkoholische E x t r a k t e aus je 3 Gehirnen. N a c h E i n d a m p f u n g zur Trockne Aufnahme in 0,7 ml A q u a dest. + 0,2 ml HCtO4 (1,2 n). Hochspan- nungselektrophoretische A u f t r e n n u n g des in der Ultra- zentrifuge gewonnenen klaren lJberstandes. Q u a n t i t a t i v e 13estimmung m i t d e m Spinco-Analytrol (Beckman Instr.) nach Anf/irbung m i t N i n h y d r i n und Stabilisierung der Farbflecke m i t Cu-Reagens.
Elektroschock. Cornealelektroden. Reizung m i t 100 Hz, 5 msec., Reizdauer 5 sec. E r m i t t l u n g der StromstArke (mA), bei der 50% der Tiere krampften, an Gruppen yon mindestens je 15 Miiusen. Berechnung nach LITCHFIELD und WILCOXON 1,.
Summary. 1. I n mice, reserpine (5 mg/kg) causes a long- lasting depletion of the brain for 7-aminobutyric acid (GABA), which corresponds t e m p o r a l l y to t h e lowered seizure threshold (electroshock).
2. Iproniazid prohibits b o t h the depletion of G A B A and the lowering of seizure threshold.
3. Relations between amine- and G A B A - m e t a b o l i s m of the brain are pointed out.
H. BALZER, P . HOLTZ und D. PALM Pharmakologisches Institut der Universitiit Frank/urt a . M . (Deutschland), 1. August 1960.
O n t h e I n h i b i t i o n o f A l c o h o l i c F e r m e n t a t i o n in
Brettanomyces
Y e a s t s u n d e r A n a e r o b i cC o n d i t i o n s
I n W a r b u r g experiments (direct m e t h o d at 30°C) ana- erobiosis was obtained b y flushing for 30 rain with pure N,, freed from traces of O 3 b y means of a Pd-asbestos catalyst s a t u r a t e d with H v Assuming a respiratory quo- t i e n t (R. Q.) of 1 for the oxidation of carbohydrate, gas production under aerobic conditions essentially represents the aerobic fermentation. I f R. Q. would be lower t h a n I, e.g. in oxidation of ethanol, the actual aerobic fermen- tation would be even higher t h a n t h a t directly observed. Cells of Br. claussenii, h a r v e s t e d from aerobic shake cultures on various liquid media after different times of incubation a t 30°C, i n v a r i a b l y showed a n e g . P. E. (Fig. 1, curves A and t3) in f e r m e n t a t i o n of all sugars e x a m i n e d (galactose after adaptation), in distilled w a t e r as well as in phosphate solution a t p H 3.5 to 6.5 and in buffers a t p H 4.5, prepared w i t h a large n u m b e r of organic acids n. The rate of aerobic f e r m e n t a t i o n strongly depends on t h e concentrations of K + and Na+ present 6.
Studies on t h e elimination of t h e neg. P. E. were per- formed in KH~PO~ buffer a t p H 4.5 with yeast, h a r v e s t e d from a s y n t h e t i c substrate containing glucose, minerals, biotin, and thiamine, a t a culture age (42-48 h) character- ized b y m a x i m u m f e r m e n t a t i o n a c t i v i t y per u n i t Of celt d r y weight.
T h e anaerobic f e r m e n t a t i o n is appreciably s t i m u l a t e d even b y m i n u t e a m o u n t s of O~ (e. g. 0.1% in t h e gas phase) or b y aerobic addition of glucose previous t o flushing w i t h N 3 (as in Saccharomyces ssp.6). This c a n n o t be due to storage of fermentable substrate within the cells u n d e r aerobic conditions, because on anaerobic addition of glu- cose a f t e r completed f e r m e n t a t i o n of this sugar, added aerobically, the anaerobic f e r m e n t a t i o n r a t e also ap- proaches the aerobic one. Iff addition, a s t i m u l a t i n g effect on anaerobic f e r m e n t a t i o n m a y be seen on anaerobic ad- dition of t h e clear supernatant, obtained b y shaking washed ceils of Br. claussenii or baker's yeast aerobically in glucose solution at 30°C for a few hours and centrifug- ing the suspension. W h e n suspensions of Br. claussenii and baker's yeast are mixed in t h e ~Varburg vessels, the rates of the aerobic fermentations appear to be additive, b u t the rate of the anaerobic f e r m e n t a t i o n of the m i x t u r e equals or even surpasses the sum of the anaerobic r a t e of baker's y e a s t and the aerobic rate of Br. claussenii (Fig. 1), These results suggest t h a t some substance(s), formed in f e r m e n t a t i o n of glucose, m a y replace O~ in eliminating the neg. P. E. This stimulation is n o t caused b y CO 2, e t h a n o l , or acetic acid, b u t can be produced b y acetaldehyde (10 -~ M) and b # p y r u v i c acid (5 × 10 -a M). I n order to s t u d y a possible effect of lipid components, acetone-ether extracts of Br. claussenii and baker's y e a s t were mixed with w a t e r and the solvents e v a p o r a t e d in vacuo, leaving emulsions of the e x t r a c t e d material, which strongly stimulated the anaerobic f e r m e n t a t i o n in Br. claussenii. However, in control experiments, acetone (10 J M) produced t h e same effect. E t h e r (5× 10 .3 M or e v e n t e s s ) a l s o stimulates to an appreciable extent. I n addition to acetaldehyde, acetone, and p y r u v i c acid, also otfier carbonyl compounds are active in eliminating the
As was shown b y COSTERS 1, t h e y e a s t Bmttanomyces clausseni$ ferments glucose a t a higher r a t e u n d e r aerobic t h a n u n d e r anaerobic conditions. CUSTERS introduced the t e r m ' n e g a t i v e Pasteur effect' (neg. P. E.) for this pheno- menon. As WIKI~N and :RICHARD 3"3 observed a similar effect in Saccharomyces strains u n d e r p a r t i c u l a r conditions, a closer s t u d y of the effect seemed to be of interest. As t h e neg. P. E. is characteristic of all Breltanomyces species 4, effortswere c o n c e n t r a t e d on Br. claussenii, strain C B S 76.
1 M. Ta. J. CUSTERS, Onderzoekingen over bet gistgeslacht Brettano- myces, Thesis Delft (1940).
3 T, WIKI~H and O. RICHARD, Exper. 9, 417 (1953).
T. WIK~N alld O. RICHARD, Antonie van Leeuwenhoek 20, 385 (1954); Schweiz. Z. atlg. Path. Bakt. 17, 475 (1954).
T. WtK~N, W. A. SCH~F~RS, and A. J. M. VERHAAR, to be published before long.
S T. WIK~N, A. J. M. VERHAAR, and W. A. SCHE~FERS, to be published before long.
15. I. 1961 Brevi comunicazioni - Brief Reports
41
neg. P. E., e. g. f o r m a l d e h y d e (10-z M ) , a c e t o i n (10 -s M ) , d i h y d r o x y a c e t o n e (10-~ M ) , m e t h y l e t h y l k e t o n e (10-= M ) , a n d ~ - k e t o g l u t a r i c a c i d (5 × 10 -~ M ) . O n t h e o t h e r h a n d , e t h a n o l , iso-propanol, l a c t i c acids, m e t h a n o l , 2, 3 - b u t a n - ediol, a n d glycerol do n o t s t i m u l a t e . O x i d i z i h g a g e n t s s u c h as f e r r i c y a n i d e ( I 0 - ~ - i 0 -z M ) a n d q u i n o n e ( 2 × I 0 -~ t o 10 -z M ) h a v e no s t i m u l a t i n g effect, w h e r e a s m e t h y l e n e b l u e ( 5 × i 0 - ¢ - 5 x 10 -z ~r) h a s o n l y a m o d e r a t e o n e ; a t t h e h i g h e r c o n c e n t r a t i o n s m e n t i o n e d , t h e s e a g e n t s a l r e a d y c o n s i d e r a b l y r e d u c e t h e r a t e of a e r o b i c CO~ p r o d u c t i o n . T h e e f f e c t on a n a e r o b i c f e r m e n t a t i o n b y p y r u v i c a c i d (5 × 10 -s M ) o r a c e t o i n (I0 -s M ) is a b o l i s h e d b y previous, b u t n o t b y s u b s e q u e n t a d d i t i o n of K F (10 -~ M ) . T h e results s u g g e s t a n a c t i o n 0f t h e c a r b o n y l c o m p o u n d s as H - a c c e p t e r s in e n z y m a t i c d e h y d r o g e n a t i o n , c o m p a r a b l e w i t h t h e So-called p h y t o c h e m i c a l r e d u c t i o n ~. O x i d i z e d c o e n z y m e I ( D P N ) e n h a n c e s a n a e r o b i c f e r m e n t a t i o n to a n e x t e n t d e p e n d i n g o n i t s c o n c e n t r a t i o n (Fig. 2). R e - d u c e d c o e n z y m e I ( D P N H ) h a s o n l y a s m a l l effect, cor- r e s p o n d i n g t o t h e k n o w n a m o u n t of D P N p r e s e n t as a n i m p u r i t y . I n c o n n e c t i o n w i t h t h e s e results, i t m a y b e m e n t i o n e d t h a t i t was r e c e n t l y d e m o n s t r a t e d ~ t h a t ceU-ffee e x t r a c t s of S. cemvisiae as well as c r y s t a l l i n e y e a s t a l c o h o l d e h y d r o - g e n u s • o x i d i z e D P N H o n a d d i t i o n of l o w e r a l i p h a t i c a l d e h y d e s . As s u p p o s e d b y s e v e r a l a u t h o r s ~,z,~, t h e i n h i b i t i o n of f e r m e n t a t i o n f o u n d u n d e r a n a e r o b i c c o n d i t i o n s i n dif- f e r e n t y e a s t s m i g h t b e d u e t o a t o o m u c h r e d u c e d s t a t e of o n e o r m o r e c a t a l y t i c c o m p o n e n t s in t h e e n z y m e s y s t e m concerned. I n v i e w of t h e a b o v e findings, i t is t e n t a t i v e l y s u g g e s t e d t h a t t h e i n h i b i t i o n o f t h e s t a r t of ~ e r m e n t a t i o n in B r . claussenii u n d e r a n a e r o b i c c o n d i t i o n s is, a t l e a s t in p a r t , d u e t o a s h o r t a g e in D P N . T h i s i n h i b i t i o n is a b o l - ished on a d d i t i o n of 0¢ or of o t h e r s u b s t a n c e s a b l e t o o x i d i z e D P N H e n z y m a t i c a l l y . A r e s p i r a t i o n - d e f i c i e n t m u t a n t of Br. claussenii w a s s t u d i e d in o r d e r to d e t e r m i n e if r e s p i r a t i o n is i n v o l v e d in t h e a b o v e o x i d a t i o n b y Oz, as m i g h t b e e x p e c t e d . I n t h i s m u t a n t t h e r a t e s of a e r o b i c a n d a n a e r o b i c f e r m e n t a t i o n a r e n e a r l y e q u a l , a n d b o t h processes a r e s t i m u l a t e d b y a c e t o i n or D P N (Fig. 3), w h e r e a s D P N H h a s p r a c t i c a l l y no influence. T h e p r e s e n t i n v e s t i g a t i o n is b e i n g c o n t i n u e d . I000 I~LCOz 800 fi 500 / ~ / " 200 ~ ' . . . - ' " " 0 " ~ - ' " - ; ; ; ' 15 30 G5 60 rain Fig. 1. Brei~anom~ces clanssenii and baker's yeast, potassium phos-phate buffer, 0.18 M, pH 4.5, glucose 0.1 M.
• - - - e aerobic fermentation. • • anaerobic fermentation. A) Br. claussen~, ~0 mg wet weight/vessel. B) idem. C) baker's yeast, 5 rag wet weight/vessel. D) Br. chzuss~nii, ~O mg wet weight/vessel + baker's yeast, 5 mg Wet weight/vessel. E) calculated: A + C .
1000 ptC02 800 , / . ~ C t s =1
.:.a~;;"
~
C
~oo/.
¢~'-;/
200 ""-" 15 30 ~5 60 75 90 minFig. ~. Br. clauss~nii, 20 mg wet weight/vessel, potassium phosphate buffer, 0.05 M, pH 4.5.
o - - - e aerobic fermentation. • • anaerobic fermentation.
A) glucose, 0.05 M. B) idem + DPN, 0.02 M. C) glucose, 0.05 M + DPNH, 0.02 M. D) glueose, 0.05 M. E) idem + DPN, 0.02 M. F) glucose, 0.05 M + DPN, 0.001 M. G) glucoses 0.05 M + DPNH, 0.02 M. I000 p{COz 80O F D 600 .'" .~ C /+OO # " .~ "" B 200 . ~ . - .-" .. I ; ; ~ " 15 30 45 60 75 90 rain
Fig. 3. Br.
claussenli,
respiration-deficient mutant, 20rag wet weight/vessel, potassium phosphate buffer, 0,05/~a', pH 4.5. • - - - * aerobic fermentation. • . • anaerobi~ fermentation. A) glucose, 0.05 M. B) idem. C) idem + DPN, 0.02 M. D) idem + idem. E) glucose, 0.05 M + acetylmethylearbinol, 0.05 M. F) idem + idem. G) acetylmethylcarbinol, 0.05 M. H) idem.
gC. NEUBSRG, Adv. Carbohydr. Chem. 4, 75 (1949). S. SENTHE SIZANMUOANATHAN, Biochem. J. 74, 568 (1960). SA. J. KLUYVER and M. TH. J. CUSTERS, Antonie van Leeuwenhoek
6, 121 (1940).
10The author expresses his thanks to Prof. T. WZK~N for stimulating interest, to Mr.'iC. J. E. A. B~LDE~for kindly supplying the respi- ration-deficient strain of Br. ctaussenil, and to Miss H. BRAKMAN for valuable technical assistance. The author is indebted to the Koninkli}ke Nederlandsche Gist- en Spiritusfabriek N. V., Delft, for a gift of pure DPN and DPNH. Part of the work was supported by the Delfts Hogesehoolfonds and by the Netherlands Organi- zation for Pure Research (Z. W. 0.).
42
Br~ves communications - Kurze Mitteilungen EXPERIENTIA XVII/IZusammen[assung.
Der <~negative Pasteureffekt,~ inBret-
tanomyces-Hefen
kann durch O v G~rprodukte der Hefen, Diitthyliither, Aldehyde, Ketone und andere Carbonyl- verbindungen, sowie d u t c h D P N beseitigt werden.W , A. SCHEFFERS 10
Laboratory of Microbiology,
Technological University,
Delft (Netherlands), August 10, 1960.
D i e W i r k u n g v o n H y b r i d e n w e i n a u f die L e b e r U n t e r H y b r i d e n w e i n v e r s t e h t m a n das G~rungsprodukt des Traubensaftes y o n Kreuzungen aus.europi~ischen, so- genannten Edelreben und resistenten amerikanischen Reben. Mehrere A u t o r e n (PAGES, L~OBARDY, BREIDER et al.) behaupten, dass nach Genuss solcher Weine bei ge- sunden Organismen Leberschiidigungen auftreten wiirden. Diese Frage ist aber aus manchen Griinden noch keines- wegs abgekl~rt. 1Dber die W i r k u n g y o n A t h a n o l auI eine vorgeschAdigte L e b e r liegen bereits zahlreiche Untersu- chungen v o r (l:)bersicht KLIEWE4). Da aber noch unbe- k a n n t ist, ob und w a r u m Lebersch~deu nach Trinken v o n H y b r i d e n w e i n auftreten kOnnen, wurde u n t e r anderem zun~chst untersucht, ob eine d u t c h Allylalkohol vorge- sch~digte L e b e r i m Vergleich zu A t h a n o l und Edelwein d u r c h H y b r i d e n w e i n unterschiedlich ungfinstig beein-
flusst wird.
VCeisse m~innliche R a t t e n in den engen Gewichtsgrenzen v o n 145-155 g erhielten 3 :rage lang das hochwertige S t a n d a r d f u t t e r , A l t r o m i n , , a m T a g v o r Versuchsbeginn Gerste. U n m i t t e l b a r v o r dem Versuch wurden sie n a c h d e m Gewicht aussortiert. Alle LSsungen v e r a b r e i c h t e n wir m i t der Schlundsonde. Jedes T i e r erhielt 2 m l der zu t e s t e n d e n L6sung (Kontrolle: Wasser). I h danach wurden 0,6 ml eines frisch hergestellten, 3, 5%igen Allylalkohols (,Merck,) appliziert. Danach blieben die Tiere 10 h lang otme F u t t e r und Fliissigkeit; nach weiteren 38 h wurden sie nach Kopfschlag und E n t b l u t e n - j e w e i l s ein Kollektiv einzeitig - aufgearbeitet.
I n geringer Abweichung v o n den Angaben von EGER 5 wurde die Oberfl~iche der gesamten L e b e r (Ober- und Unterseite) sowie d i e der nekrotischen Stellen n a c h Ii~- chenhafter P r o j e k t i o n ohne Beriicksichtigung der L a p p e n - wSlbungen planimetrisch erfasst u n d der prozentuMe An- tell der Nekrosefi~chen an der Gesamtoberfliiehe zur sta- tistischen Auswertung herangezogen.
Ats Testl6sungen dienten ein weisser H y b r i d e n w e i n y o n einer FS4-Hybride (Athanol nach der A D H - M e t h o d e = 12,79 VoL%, I n v e r t z u c k e r = 2,6 g/l, freie SO2 = 49 rag/l), ein Edehvein (Siebeldinger M6nchspfad, A t h a n o l nach der A D H - M e t h o d e = 11,65 Vol~%, I n v e r t z u c k e r = 1,7 g/l und Ireie SO 2 = 50 mgfl. Zucker- und Atkohotgehalt wurden an den H y b r i d e n w e i n angeglichen. Zur Kontrolle diente unvergiilltes Athanol (12,79 Vol.%) und Wasser (Ergeb- Ilisse siehe Tab. I u n d II).
Daraus ist zu ersehen, dass der Allylalkoholschaden der L e b e r durch Athanol, Edelwein und H y b r i d e n w e i n bei gleichem Volumen und gleichem Alkoliolgehalt in dieser Reihenfolge zunehmend verstiirkt wird. Dieser Befund ist m i t einer hohen Wahrscheinlichkeit statistisch gesichert. D e r Versuch m i t ,~thanol allein bestAtigt gleichzeitig die Ergebnisse y o n EGER 5.
Summary.
E x p e r i m e n t s on rats revealed t h a t liver d a m - age caused b y allyl alcohol (measured b y t h e rate of ne- crosis) is enhanced b y alcoholic beverages such as ethanol and E u r o p e a n or hybride wine. I n this respect, wine seems to cause more damage t h a n mere alcohol.H. KLIEWE, G. GILLISSEN und K. ~vVIENER
Hygiene-Institut der Ugiversitiit Mainz,
19.Oktober
1960.1 p. PAGES, C~ngr. int. pour l'4tude sei. du vin et du raisin (1957). J. DE LI~OBARDX', Bull. Office int. Vin
269,
3 (1953).3 H. BRXZDER, G. REUTHER uad E. WOLF, Der Ziichter
29,
317 (1959). 4 H. KLIEWE, ~rztl. PraMsI9,
671 (1959).6 W. EGER, Medicina exp. 1, 84 (1959); Virchow's Archly
328,
536 (1956) ; Arzneimittelforsch.10.
601 (1957).Tab. I. Der Einfluss alkoholischer Getrgnke auf den Allylalkohol- schaden der Leber
Testl6sung Allylalkohol atlein Athanol Edetwein Hybridenwein Mittlere Nekroserate in % 27,5 38,3 50,9 78,1 s.d. -~- 5,6 -4- 3,4
±
7,5
± 13,7Tab. II. Statistlsche Vergleiche
Vergleieh der t P Versuchsgruppen Allylalkohol: Athanol Allylalkohol: Edelwein Allylalkohol: Hybridenwein ~.thanol: Edelwein ~thanol: Hybridenwein Edelwein: Hybridenwein 5,070 7,228 10,257 4,628 8,487 4,666 < 0,001 < 0,001 < 0,001 < 0,01 < 0,001 < 0,01
(> 0,001)
(> 0,001)
T h e B l o o d F l o w of t h e C e r e b r a l C o r t e x D e t e r m i n e d b y R a d i o a c t i v e K r y p t o n s5 A q u a n t i t a t i v e m e t h o d for t h e d e t e r m i n a t i o n of regional blood flow in t h e brain has been developed b y I{ETY e t aLL T h e m e t h o d was based u p o n m e a s u r e m e n t of t h e incor- p o r a t i o n r a t e of an i n e r t r a d i o a c t i v e gas, a n d t h e t e c h - n i q u e required decapitation of the e x p e r i m e n t a l animM. Hence only one m e a s u r e m e n t per tissue could be m a d e in each animal. The m e t h o d described below is a modifi- cation of t h e m e t h o d of K~TY e t al. 1 in which tissue sampling has been replaced b y e x t e r n a l counting o v e r t h e exposed surlace of t h e brain, enabling multiple measure- merits of t h e blood flow in t h e same a r e a of t h e cerebral cortex t o be carried Out.T h e radioactive i n e r t gas used was k r y p t o # 5 (Kr*~) which emits practically only low energy fl-radiafion. Saline equilibrated w i t h K r 8~ Was injected into one com- m o n carotid a r t e r y in a cat under light n e m b u t a l anesthe- sia. A gyrus of t h e parietal cortex of t h e same side was exposed b y a small c r a n i o t o m y and resection of t h e dura.
* S. S. KETY, W.M. LANDAU, W. H. FREYGANG, L.P. ROWLAND, and L. SOKOLO~F, Fed. Proc. 14, 85 (1955).
