Enzymic studies in small amounts of human tissue with the help of microanalytical methods

(‘T.ISIc‘.\

(‘HIllI(‘.\ .\l”r.\

The direct study of cnzymic processes in tissucl sampIes continues to make hendway in the field of experimental physiopathology. However, in the past its extension to human tissue has been limited to surgical samples or to skin’ and muscle tissue”. The possibility of removing parenchymal tissue by the newer techniques of needle biopsy and the sinlult~~ne~~ls (l~~v~l~~~rnei~tof ~~icr~~~nal~tical methods allowing work on tissue alicluots in the milligram range have paved the way for direct enzymic studies on human liver material. Little work has been done in this field. In 1954 ~VATI:I~LOW ASI) I'ATIIIC~I<~~ measured the activity of several liver enzymes in puncture material removed from -Jamaican chilclren suffering from kwashiorkor and gave values ohtained in 5 cases for succinic osidasc, malic dehydrogenase, lactic dehydrogenase, glutamic dehydrogenasc,, Slutamic-oxnlncctic transaminasc and DPN-q&chrome c rcductase. Glycolic acid oxidase, DPSH-dehydrogenasc, malic dehydrogenase, glutamic-oxalacctic transaminasc, xanthine oxidasc and r,-amino acid osidase have been studied more recentl!. by BLWCH et u/.~ in the same disease. Dipeptidase5, catalase, and &erase 8 have been measured in other ~~tli~)l~)~ic~~l conditions. Enzymic disturbances have heen dcscribed by SC.H~~IIL)T et nl.’ in a studv of 92 cases of hepatitis in various stages. ‘l‘hest~ authors ha\re recently compared the activities of aldolase, u-glycerophosphate dehydrogenase, alcohol dehydrogensso, Warburg’s “Zwischenferment”, enolase, pyruvatc kinasc, lactic dchydrogcnase, glutamic acid dehydrogcnase and glutamic-oxalacetic transaminase of normal human and normal rat liver tissue a. The present paper deals with enzymic ~listurbanc~s observed in livers of cirrhotic and precirrhotic patients. Previous clinical results”. lo corroborate the assumption that the disturbances of the carbohydrate metabolism observed in liver cirrhosis are connected with cnzymic disorders at the level of the glycolytic chain and the tricarboxylic cycle, thus entailing an impairment of the energetic metabolism of thr liver cell. Special attention was therefore dire&ccl to the determination of enzymes involved in these two metabolic procc*sst~sand to the study of the overall expression of cellular energetics like oxygen consumption, and oxidative resynthesis of A’I’P. The gltltamic--oxalacetic transaminasc and an enzyme involved directly in the respiratory chain, the I)Pi\;-cytochrome c reductasc,werc also considclcd.

VOL. 3

(1958)

ENZYMIC STUDIES

1X’ SMALt

AMOdNTS

OF TISSttE.

Ift.

487

METHODS The liver tissue was obtained from fasting patients under local anesthesia, by biopsy puncture using a Roholm needle. This technique usually supplies 15-40 mg of tissue, IO mg of which is witheld for histological examination. The remainder is kept at o” and weighed on a torsion balance. It is then homogenized by hand for 1 min chronometrically at 0’ in limbs’ saline solution using a “Misco” a&glass homogeniser. Aliquots of the homogenate are withdrawn in order to measure oxygen consumption and oxidative resynthesis of ATP by methods previously describedn. The remaining homogenate is diluted with IO times its volume of distilled water for the determination of enzymic activities and the protein content. Enzymic activities Seven enzymes have been considered : lactic dehydrogenase dehydrogenase, succinic dehydrogenase, fructose-r,6-diphosphate TABLE -_-.

METHODICAL __

Enzyme LDH’”

MDH’2

IDH’”

SDH’”

RED’J

GOT’”

ALD’r

DATA CONCERNING ..__._

THE ENZYMIC

Reaction medium (molar concentrations) lactate Na, pH 9.2 glycine buffer, pH DPN

malate Na, pH 10.2 glycine buffer, pH 10.2 DPN

* 10-l * 10-3 ’ 10-l 10-I

2.00 2.00.

succinate Na phosphate buffer, pH 7.4 cytochrome c KCN

8.30. 10-Z 7.60. row5 3.90. x0-5

fructose-1,h-diphosphate collidine buffer, pH 7.4 DPNH mixture of glycerophosphate dehydrogenase and trioseisomerase (suspension)

volume

MAIN

REFERENCES

Amount qf I:IO diluted homogenate (~1)

so5

5

505

5

2.00 * 10-I

5.40. 10-d 9.00 . 10-Z

aspartate Na a-ketoglutarate Na phosphate buffer, pH 7.4 DPNH MDH (suspension)

reaction (Yl)

WITH

10-I

I.33 2.00

isocitrate Na collidine buffer, pH 7.4 TPN MnCl,

DPNH cytochrome c phosphate buffer, pH 7.4 KCN

I DETERMINATIONS,

Final

5.33.

9.2

*, malic dehydrogenase, isocitric aldolase, DPN-cytochrome c

I.jj

* TO-’

2.00

f x0-2

SIO

IO

785

50

605

5

570

5

500

5

* 10-2

2.22

5.80.

IO-~

6.60.

10-s

8.2 j * IO-’

4.13

1 10-Z

3.51

. 10-Z

7.00

* 10-Z

3.51 * IO-* 1.80.

10-d

5E”l

4.00 + 10-a 9.00. 10-a 2.56 + IO-* 10 pi

DPN. DPNH, MDH, fructose-r, &diphosphate, glycerophosphate dehydrogenase-trioseisomerase mixture were purchased from C. F. Bohringer and SBhne, Mannheim-Waldohf. Germany. Cytochrome 6, TPN and isocitrate were products of Sigma, St. Louis, MO. (U.S.A.) * The following abbreviations are used in this paper: ATP = adenosine ~iphosphate; DPN = dipho~h~yridine nucleotide; TPN = ~phosph~yridine nucleotide; LDH = lactic dehydrogenase; MDH = matic dehydrogenase: IDH = isocitric dehydrogenase; SDH = succinic dehydrogenase; ALD = Fructose-1,6_diphosphate aldolase; RED = DPN-cytochrome c reductase; GOT = Glutamic-oxalacetic transaminase. References Q. 49~

4ss

H. IIYSEK

\‘()I.. 3 (“)jN)

et ul.

reductase and glutamic-oxalacetic transaminase. All activities were measured by optical trsts. The composition of the different reaction media and the main references are given in Table I. l.nder the conditions indicated the activities are linear functions of time and enzyme concentration. The readings were made during 6 LO min on the l’ppendorf photometer at 360 m/r for lDH, i\lL)ll, IDH, GOT and ALD, and at 546 mp for RFD and SDH. The temperature was kept constant in thermoregulated micro-cells at 32” for all enzymes, except for SDH where a temperature of 25 was used. The activities of LDH and RED were measured within L h after homogenisation. For the other enzymes the I : IO diluted homogenate was frozen and thawed out in order to disrupt infrastructures and to liberate the structure-bound enzymes. The measurements were made within a period of 24 h for these enzymes. Expression

of the results

The oxygen consumption is expressed in ~1 O,/min, the enzyme activities in /~moles/min of converted coenzyme (DPN, DPNH, TPN or cytochrome c). These values are referred to I ml of undiluted homogenate, as well as to the protein content of I ml homogenate. The proteins are measured by the method of FOLIN AND CIOCALTELJ’*, as modified by LOWRY et al.i9 and adapted to our requirements. This very sensitive calorimetric method is based on the presence of tyrosine and tryptophane in parenchymal proteins. Ten ~1 of I : IO diluted homogenate are treated with joo ,nl of o. I N r\‘aOH at room temperature for L h. Then 500 ~1 of the carbonate--copper solution (reagent D of LOWRY) and 50 ~1 of the Folin reagent are added and the extinction is read at 750 m,n on the Beckman DU spectrophotometer in a IO mm-lightpath microcell. The results are expressed in mg of tyrosine per ml homogenate, this amino acid being taken as the standard. This method is easily adapted to microscale analysis and has been preferred because tyrosine and tryptophane content is very low in connective tissue, thus allowing a much more accurate estimation of the parenchymal protein in cirrhotic liver*. Duplicate determinations have been performed for the seven enzyme activities, oxygen con sumption and protein content. These tests together with single determinations of oxidative ATPTABLE

I1

ATP-resynthesis Oxygen consumption Lactic dehydrogenase Jlalic dehydrogenase Isocitric dehydrogenase Succin dehydrogenase DPN-cytochrome c reductase Glutamic-oxalacetic transaminase Aldolase of fructose- I ,h-diphosphate Protein-tyrosine

L5-_jO

Total amount

about

* For duplicate

3.4 334 IO0 IO0 IO0 IO0 50 50 IO0

mg mg* y* y* y* y* Y* Y* y* y*

10 mg

determinations.

resynthesis required about IO mg of fresh tissue (see Table II). It was not always possible to express ATP-resynthesis in absolute values using Krebs’ equation”. The yield of oxidative phosphorylation was therefore given throughout in relative values referred to a standard concentration of homogenate. Contvol

subjects

In this clinic, puncture biopsies were performed mainly to secure a histological diagnosis in undetermined liver diseases, or to detect granulomatous liver changes in cases where a non-typical tuberculosis or sarcoidosis were suspected. The latter group supplied some cases with normal liver histology and normal liver function tests, that could be considered as control subjects. Normal liver tissue was obtained also from surgical biopsies, performed during gastric resections in patients suffering from gastroduodenal ulcers, showing neither history nor clinical signs of liver disfunction. * Recent data of our laboratory (A. BAER) suggest that the enzymic the tissue is not always negligible. The variations in the blood content been measured our experiments.

References p. 4g.j

activity of the blood in of liver samples have not

VOL.

3 (1958)

ENZYMIC STUDIES IN SMALL AMOUNTS OF TISSUE. III.

489

RESULTS

The oxidative ATP-resynthesis has been measured in the total liver homogenate in a group of II cirrhotic and precirrhotic patients and compared with 12 control subjects. The histological picture showed 2 cases of typical Laennec cirrhosis, I case of pigment cirrhosis, 6 cases of precirrhotic fatty liver changes due to chronic alcoholism and 2 cases of early stages of liver fibrosis. The average phosphorylation of these cases lies far below the norm. In Fig. I the relative incorporation rates of inorganic labeled phosphate into ATP are plotted against the incubation time. The differences are highly

to

Fig. I. Oxidative

a

ATP-resynthesis

40

30

50

in liver homogenates (see text).

*

Control

0

Cirrhotic and prccirrhotic states (II eases I

60

subjects

(12 cases)

min

of cirrhotic

and precirrhotic

patients

significant. The vertical indices between the 2 curves express the minimal mean differences which are statistically significant in 99% of the cases in the t-test. As these relative values are not based on protein content it remained to be proved that the significant difference indicated by Fig. I is not due only to histological liver involvement. Accordingly 2 other groups of patients showing disturbed hepatic function tests but no gross histological liver changes were considered. The first group includes preTABLE III OXYGEN

CONSUMPTION AND OXIDATIVE ATP-RESYNTHESIS MEASURED WITHANDWITHOUTGROSSHISTOLOGICALCHANGES

Examined subjects (number of cases)

I. Control subjects II. Cirrhotic subjects (~6 in t-test)

(6-7) (8-18)

III. Alcoholic subjects without histological changes (5) (.sd in t-test) IV.

Protein-tyrosine (mgiml homogenate)

OS-uptake (plimg tyrosine) 1min)

r .94

163

0.463

1.26 (0.48, P 0.01)

44 (64, P 0.001)

0.294 (0.‘59. P 0.05)

-

Non-cirrhotic diseases without histological changes (7-r I) I .46 (cd in t-test) (not significant)

References p. 493

A TP-resynthesis (relative values)

IN LIVER SAMPLES

63 (67, P 0.001)

(55, I%oor)

-

0.312 (0.139,P 0.01)

LDH MDH IDH SDH RED (X)T ALL)

3.80 3.1.5 2.81

,.a] 2.j t

‘.i I .‘>j

o.‘pY 0.7’) 0.t)’ 0. to O.JH

(O.OOl) jo.or) (o.oot) (0.w) [O.Ol)

‘.Ji 0. 10 0.88

t.;.i t .s,1 t. to 0. t j 0.72

0.30 1.44

t.3i 0. tr> 0.1, t

X..H

4,L.j

3.34

io.ot)

4.2;

3

0.80

“..li

0..32

(o.oot)

o._+z

( 1 .! ;-

3.1

not sigtlilicant not si~lii~~~~~llt “..li (0.O.j) twt significant not significant

not signilicant 0. I .<

(0.01)

referred to a wet-weight hasis. Vk%en referred to l’rc’tcin-tyr:,sin~~, onlv 2 out of 7 enzyme activities remain significantly reduced, namely .%LI) and II)H, Tahic IV (1)). An activity loss of more than zo”;, WE noticed also for (XJT and LI>If. In some cases, especially in patients with obvious clinical signs of chronic alcoholism but no microscopic liver changes, the activities of MI)H and SI)H were incrcasc-4 by amounts greater than z sta.ndard deviations of control subjects. An attempt was made to correlate the re4ts of these fn ;~i~ro In~~~surern~nts with the data of the usual clinical tests. For t?k purpose all the casts were divided into 3 groups according to the clinical evaluation of their liver function. The first group included patients with normal or slightly impaired liver function ; the second group, cases with moderately impaired liver function, whereas the third group consisted of patients in whom liver function w~as considered to lx greatly disturbed. This classili~fmnm p.@,j

VOL.

3 (1958)

fication

ENZYMIC

was achieved

determinations.

STUDIES

by clinicians

IN SMALL AhIOC’KTS OF TISSUE.

III.

49’

who were not aware of the results of the in vitro

The average values of oxidative

ATP-resynthesis,

oxygen

consump-

tion, and ALD activity, for these 3 groups are shown in Table V. The differences between groups I and II and even more between I and I II are highly significant for both overall functions and for ALD-activity. The latter value as well as the oxygen conTABLE CORRELATION

BETWEEN

THE

APPRECIATION

Liver function tests (number of cases)

I. Normal or little impaired (6-16) II. Moderately impaired (18-15) (ed in t-test) III.

BIOCHEMICAL OF

THE

Protein-tyrosine (mg/ml homegenate)

V

RESULTS

CASES

ON

OBTAINED

THE

BASIS

A TP-resynthesis (relative oalues)

I .94

OF

i?Z

(0,4j,

71

1.13 (0.49, P 0.001)

18 (98, P 0.001)

P

0.01)

THE

CLINICAL

TESTS

Oxygen uptake (plimg tyrosine j&k)

Aldolase actimty (pnolpng tyrosinelmin)

0.46

0.426

I2j

I.33 (0.44, P 0.001)

Uitf’oAND

FUNCTION

0.30

0.260

(0.17, P 0.001)

(O.12, P 0.01)

Severely impaired (3-6) (cd in t-test)

sumption

are based upon protein-tyrosine

0.21

P 0.01)

(0.20,

levels in order to correct

variations

due

to gross parenchymal loss. This excellent correlation between clinical and in vitro results supports the view that enzymic studies on human tissue could be developed as valuable

clinical tests. This point will be discussed

later.

DISCUSSION

As shown by the histological examination and further indicated by the fall in the protein-tyrosine content of liver aliquots, the active parenchyma in liver cirrhosis is partly replaced by enzymically neutral connective or fat tissue. A reduction of enzyme activities was therefore expected when referred to tissue wet weight. A drop was in fact observed for all enzymes considered, thus showing that the remaining parenchyma does not compensate the tissue loss by adaptive increase of metabolic activity.

If a partial compensatory

increase occurred,

the enzyme activities

would be

increased when referred to protein-tyrosine. In our cases, however, most of the activities remained lowered, suggesting a metabolic impairment of the remaining tissue. The significant

drop in IDH and ALD provides

evidence

for a lowered cellular con-

centration of these enzymes. Furthermore, since the coenzyme of IDH is added in excess for this determination, the optical test is likely to measure the cellular concentration of the apoenzyme. The same is probably

true for ALD. The view that a fall in cellular

enzyme concentration could possibly result from a disturbed protein synthesis in the damaged liver cell has recently been substantiated by experimental data obtained from rats fed a low-protein diet 20, 21.‘These data may also be related to the impaired utilisation of amino acids observed in cirrhotic patientsZ2. Clinical and experimental studies on carbohydrate metabolism in cirrhotic patients suggest disturbances at the level of the glycolytic chain and the tricarboxylic cycle8, lo. The finding of a significant decrease of ALD and IDH activity may be considered as further evidence for this view. It is generally assumed that a disturbed reaction sequence in the tricarboxylic References p. 49.j

cycle may affect the energetic

metabolism

as well. in our experiments

the cirrhotic

liver samples showed in fact an impaired ability to resynthesize ATP. Whether this defect is directly correlated with a fall in IDH and ALD activity remains to be shown. No data are available

at the present

time for oxidative

phosphorylation

and

oxygen consumption of human liver tissue. The oxidative phosphorylation has been studied with the hexokinase method in total liver homogenates of rats showing expcrimental

liver steatosis=.

thesis computed

In our experiments

for 9 patients

the mean value of oxidative

with histological

signs of steatosis

.%TP-resyn-

was lowered to 3201

of the normal value. This result compares favorably with the data obtained for rat liver homogenate in similar conditions as far as substrate and temperature conditions areconcernedZa. It is worth noting that several substances known as uncouplers oxidative phosphorylation (e.g. dinitrophenol, thyroxine, etc.) are also thought

of to

produce experimental liver steatosis 24. In those particular instances, an impairment of oxidative phosphorylation could be considered as primary damage in the development of steatosis. uncoupling of oxidative phosphorylation has been demonstrated in several pathological conditions like experimental hyperthyroidism2j, in vitamin E deficienciesz6, 27, late stages of thiamine28 and riboflavine deficiencieszs, radiation damages0 and various

intoxicationsXJ.

Similarly

in our experiments

a decrease

of ATP-resyn-

thesis has been observed in several diseases other than liver cirrhosis and steatosis, (see Table III,group III). Significantly lowered values were found in the livers of cancerous patients with no histological signs of liver involvement31,Values beyond 2 standard deviations from the norm have been found in several cases of hypo- and dysproteinemia of different origins, in pernicious anemia, in several cases of liver reticulosis, and in the late stages of biliary obstruction 31. The fundamental importance of oxidative

phosphorylation

pairment reactions

of this process would be specific for any pathological condition. Overall of this kind are more likely to give an index of the functional ability of a

in cellular

energetics

makes it very unlikely

that an im-

tissue or cell. They could therefore be of interest if developed as clinical tests. The obvious correlation between our in vitro results and the clinical data, as illustrated by Table V, support oxidative reactions

this view and may be accounted

for in the following

manner:

The

ATP-resynthesis produces the energy required for most of the metabolic studied by the usual function tests: the energy needed in the synthesis of

cholesterol, prothrombine, urea, as well as the energy available for the utilisation galactose, the transformation of benzoate into hippurate, etc. It is reasonable

of to

assume that an impairment of the energy-supplying processes will influence the dependent metabolic pathways. The measurement of this main process should thus provide a better test of cellular function than the study of related reactions. ilcI(~OWLEI)GE~~SPEh'~S

We wish to express our gratitude to Dr. P. MACNENAT for performing the needle biopsies; to Prof. J. L. NICOD and Dr. A. REYNIND for interpreting the histology of the liver samples; to Prof. P. DECKER, Prof. I;. SAEGESSER, Dr. A. JOST, Dr. R. MOSIMANN and Dr. P. RYNCKI, for their cooperation in supplying us with surgical material. Our thanks are also due to Miss M. S~HWEIZER for her skilful technical assistance. This work was made possible by a grant of the Ponds National Suisse de la Recherche Scientifiqne. Kl?frrrrtrC.s p. 49.3

VOL.

3

ENZYMIC

(1958)

STUDIES

IN SMALL AMOUNTS

OF TISSUE.

III.

493

SUMMARY Using microanalytical methods that allowed work on tissue aliquots of IO mg, the oxidative ATP-resynthesis, oxygen consumption, and the activity of seven liver enzymes have been measured in total homogenates of human liver samples removed by needle biopsy. A significant decrease of oxidative ATP-resynthesis and oxygen consumption has been found 2. in cirrhotic and precirrhotic patients showing gross histological liver changes, as well as in precirrhotic states with no evident microscopical involvement. This result provides new evidence of an impaired energetic liver metabolism in cirrhotic diseases. 3. The activities of lactic dehydrogenase, malic dehydrogenase, isocitric dehydrogenase, succinic dehydrogenase, aldolase, DPN-cytochrome c reductase, and glutamic-oxalacetic transaminase were found to be significantly reduced with reference to a tissue wet weight basis. When calculated in terms of parenchymal protein content this reduction remained significant for aldolase and isocitric dehydrogenase. Thus 2 steps of the glycolytic chain and tricarboxylic cycle appear to be impaired in the remaining parenchyma of cirrhotic and precirrhotic livers. oxygen uptake and aldolase activity does not 4, The reduction of oxidative ATP-resynthesis, seem to be specific for cirrhotic and precirrhotic states. In a group of 40 patients including noncirrhotic diseases, a significant correlation was found between the results of these in vitro determinations and the clinical evaluation of liver function. 5. It is assumed that the direct in vitro measurement of an overall cellular process like oxidative ATP-resynthesis in human liver samples could provide a more accurate and more representative evaluation of the liver function than most of the usual clinical tests. I.

REFERENCES I J. M. PACHOUD AND B. SCHMIDLI, Dermatologica, IIO (1955) 323. 2 J, C. DREYFUSS, G. SCHAPIRA, F. SCHAPIRA AND J. DEMOS, Clin. Chim. Acta, I (1956) 434. 3 J. C. WATERLOW AND S. J. PATRICK, Ann. N.Y. Acad. Sk., 57 (x954) 750. 4 H. B. BURCH, G. ARROYAVE, R. SCHWARTZ, A. M. PADILLA, M. BELAR, F. VITERI AND U. S. SCRIMSHAW, Federation Proc.. 16 (1957) 160. 5 M. SPOSITO AND S. RUSSO-CAIA, Fegato, 2 (1956) 274. 6 G. B. DALE, Am. J. Med. Sci., 226 (1953) 42. 7 E. SCHMIDT, F. W. SCHMIDT AND W. WILDHIRT, Klin. Wochschr., 35 (1957) 842. 8 E. SCHMIDT, F. W. SCHMIDT AND W. WILDHIRT, Klin. Wochschr., 36 (1958) 172. g CL. REYMOND, CL. WILD, D. DJELALI, J. FREI AND TH. BBRAUD, Schweiz. med. Wochschr.,

IO II 12 13 14

15 16 17 18

rg 20 21 22 23 24

25 26

27 28

2g 30

85 (‘955)

325.

J. NORDMANN, Presse me’d., 61 (1953) 948. J. FREI AND H. RYSER, Clin. Chim. Acta, 3 (1958) 288, 294. J. L. STROMINGER AND 0. H. LOWRY, /. Biol. Chem., 213 (1955) 635. S. OCHOA, J. Biol. Chem., 174 (1948) 133. S. J. COOPERSTEIN, A. LAZAROW AND N. J. KURFESS, J. Biol. Chem., 186 (1950) 129. T. M. BRODY, R. J. H. WANG AND J. A. BAIN, J. Biol. Chem., r98 (1952) 821. A. KARMEN, J. C&z. Invest., 34 (1955) 131. G. BEISENHERZ, H. J. BOLTZE, TH. B~CHER, R. CZOK, K. H. GARBADE, E. MEYER-AREND AND G. PFLEIDERER, Z. Naturforsch., 8b (1953) 555. 0. FOLIN AND V. CIOCALTEU, J, Biol. Chem., 73 (1927) 627. 0. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. CL. BOREL, Thesis, Lausanne, 1958. W. E. KNOX, V. M. AUERBACH AND E. C. C. LINN, Physiol. Revs., 36 (1956) 164. CL. WILD, CL. REYMOND AND A. VANNOTTI, Schweiz. med. Wochschr., 85 (1955) 145. M. U. DIANZANI, Biochim. Biophys. Acta, I4 (1954) 514. TH. BRODY, Pharmacol. Revs., 7 (1955) 335. C. MARTIUS AND B. HESS, Arch. Biochem. Biophys., 33 (1951) 486; G. F. MALEY AND H. A. LARDY. J. Biol. Chem., 204 (1953) 453. C. MARTIUS. Proc. Intern. Congr. Biochem., 3rd Gong?‘., Brussels, 1955. J. FREI, Helv. Physiol. et Pharmacol. Acta, 15 (1957) C 18. J. FREI, Helv. Physiol. et Pharmacol. Acta, I4 (1956) 59. J. FREI AND M. SCHWEIZER. Helv. Physiol. et Pharmacol. Acta, 14 (1956) C 18. H. RYSER, B. SCHMIDLI, A. ZUPPINGER AND H. AEBI, Helv. Physiol. et Pharmacol. Acta, 13

(‘955)

270.

31 H. RYSER AND J. FREI, unpublished

data.

Received

April ISth, 1958