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HISTOCHEMICAL PROPERTIES OF AMYLOID IN THE SWISS ALBINO STRAIN

OF MOUSE

vivo and in vitro. This suggested that the heparin-like sub-stances would have completely prevented the periodic-acid/Schiff reaction.The staining properties peculiar to the acid mucopoly-

saccarides were distinctly weakened by neuraminidase, bothin vivo and in vitro in " second stage " and " late stageamyloid only.

In " late stage " amyloid, the fraction consisting ofchondroitin sulphate, types A and C, was masked by othersubstances, which were hydrolysed by pepsin. This fraction,on the whole, corresponded to the " indirect " or

" masked "

mucopolysaccharide fraction of human amyloid substance.The outstanding differences in the histochemical properties

of amyloid substance seem then to be due to the variations inits chemical composition, and they are strictly related to thedifferent evolutionary phases of the amyloid itself.2 2

In the recent Halle symposium amyloidosis was described asan anomalous process of fibrosis.

6

As in collagen tissue, in amyloid tissue, too, the compositionof the acid mucopolysaccharides varies, according to the

appearance at different stages of the process of different frac-tions. This is important for correct interpretation of histo-chemical and biochemical findings.

Indeed, the differing views of some workers on the nature ofthe acid mucopolysaccharides in amyloid arise probably fromobservations at different stages of the same process.The role of the mucopolysaccharides in amyloidosis as

well as in fibrosis remains obscure. Nevertheless, besidesheparin, some fractions of acid mucopolysaccharides,particularly the chondroitin sulphate, types A and C, area significant component of amyloid. They appear in theinitial stage of amyloidosis and make up the fractionseemingly more directly connected with the disease.

SALVATORE BATTAGLIALUIGI MATTURRI.

University Institute ofMorbid Anatomy,

Milan, Italy.6. Battaglia, S. in Fortschritte der Amyloidforschung. Aug. 23, 1965,

Halle. Acta Leopoldina (in the press).

RECOGNITION OF HETEROZYGOSITY IN

SPHINGOLIPOIDOSES

H.-R. WIEDEMANNH. GERKENE. GRAUCOBH.-G. HANSEN.

Pædiatric Department,Christian-Albrechts University,

Kiel, Western Germany.

SiR,—The possibility of a heterozygous state in clini-cally healthy relatives of a person with lipoidosis is of

great genetic interest. Recently we demonstrated typicalGaucher cells in the bone-marrow of the two clinicallynormal parents and a normal sister of two children withovert Gaucher’s disease, and of the two clinically normalparents and two normal sisters of a young man with

proved Gaucher’s disease. We regard our findings as anindication of heterozygosity.1-3

In another family sphingolipoidosis of the Niemann-Picktype was diagnosed. The clinically healthy parents are firstcousins. Several of their children died in early childhood inconsequence of Niemann-Pick disease. In the mother theserum-cholesterol level and the total lipid fraction were at theupper limit of normality; in the father these values were slightlyelevated (cholesterol 266-8 mg. per 100 ml. [normal 150-260],total lipids 1.18 g. per 100 ml. [normal 063-098]). The fatheralso had a raised serum-vitamin-A-ester level (44-6 I.u. per100 ml. [normal 0-40]).

In the bone-marrow of both parents, large storage-cells(15-45 fl.) with a coarse or fine vacuolised plasma, and most ofthem with a single nucleus, were seen here and there. Thesecells appeared to be more frequent in the bone-marrow of thefather than of the mother.

We interpret these findings as morphological evidenceof the heterozygous state in the parents.

CHORIONIC GONADOTROPINS IN PREGNANCY

SiRj—I wish to raise certain questions on the possibleuse of full-term amniotic fluid for the treatment of hydatid-mole, chorioadenoma destruens, and choriocarcinoma.The physiological role of chorionic gonadotropins during

pregnancy is said to be to prevent the atrophy of the corpusluteum in the ovary of the pregnant female.

If we accept that chorionic gonadotropins have a trophiceffect on embrionic tissue per se, then may we not assume thatthe trophic action of chorionic gonadotropins is primarily onthe implanted ovum in the endometrium, close to the site ofproduction of these hormones in the cytotrophoblast, and onlysecondarily, via the maternal circulation, on the corpus luteumpreventing its atrophy ?The exceptionally high titres of chorionic gonadotropins

found with hydatid mole, in which there is no fcetal circulationin the placenta, and with some other conditions, confirms thatchorionic gonadotropins have a hypertrophic influence on thehuman embryo analogous to their anti-atrophic effect on thecorpus luteum of the pregnant mother. But we must assumethere also exists in the normal foetus, and possibly in theamniotic fluid, an anti-chorionic-gonadotropin principle, per-haps an enzyme which neutralises the trophic effects ofchorionic gonadotropins on the foetus, progressively from aboutthe 12th week of pregnancy to term. The atrophy of the corpusluteum at this stage, in normal pregnancy, would indicate the

activity of an antigonadotropic principle, since the placentalcells which produce chorionic gonadotropins are not removeduntil after birth with the expulsion of the placenta. The peaklevel of chorionic gonadotropins between the 50th and 80th daysof normal pregnancy further confirms that the antigonadotropicprinciple may begin to act normally at this stage of foetal

development, when the sex of the foetus is determined. Canthis hypothetical antigonadotropic principle be a precursor ofandrogens or oestrogens, depending on its biochemical reactionswith the chorionic gonadotropins ?1. Wiedemann, H.-R., Gerken, H. Lancet, 1964, ii, 866.2. Gerken, H., Graucob, E., Wiedemann, H.-R. Br. med. J. 1964, ii, 1594.3. Gerken, H., Wiedemann, H.-R. Ann. pœdiatr. 1964, 203, 328.

1284

If we interpret the pathological conditions referred toabove as manifestations of progressive intoxication withchorionic gonadotropins, in the absence of the

antigonadotropic principle-reaching a climax in chorio-carcinoma where the cells producing chorionic gonado-tropins react to their own product as a further stimulusfor production-would not local application of full-termamniotic fluid, provided it contains the antichorionicgonadotropin principle, break the vicious circle ?

ADAN GRAETZ.Cuernavaca, Mor,Mexico.

EFFECT OF QUINIDINE ON CORONARY FLOW

SIR,-There is scanty and controversial experimentalevidence of the effect of quinidine on coronary flow.1 2Rowe et al. have shown pronounced increases in coronaryflow in animals, while Szekeres and Lenard, 4 byperfusing fibrillating hearts at varying temperatures, haveshown a paradoxical effect on myocardial blood-flow-a prolonged decrease inflow at normal temperatures anda marked increase at a low temperature (26°C).We have measured coronary flows in seven canine heart/lung

preparations. The coronary sinus was cannulated by a straightMarowitz metal cannula (lateral diameter 4-5 mm.) via the rightatrium. Sinus blood was drained into the leucite chamber of acalibrated Andrews outflow recorder.5 Special care was takento adjust the level of the outflow tubing so as to avoid obstruct-ing the coronary-sinus outflow. Continuous flow-tracings wereobtained in these experiments during control and quinidine

CORONARY-SINUS FLOW BEFORE AND AFTER PERFUSION WITH QUINIDINE

infusion periods after allowance of ample time (20-30 minutes)for stabilisation of the preparation.

After a steady coronary flow was ascertained, treatment withquinidine sulphate or gluconate (25-30 mg. per litre of blood)was instituted either as a single injection into the aortic outflowtubing or as a continuous drip (0-25% in physiological salinesolution at 40 drops per minute) into the reservoir blood untilelectrocardiographic evidence of early quinidine effects wasnoticed.

In two control experiments in these denervated hearts

lasting from 40 to 140 minutes, coronary flow remained

essentially unaltered as long as the mean arterial pressure waskept constant and a glucose-saline-insulin infusion was

administered. 6 Treatment with quinidine invariably broughtabout an initial and almost immediate increase in coronaryflow lasting about 45 to 75 seconds, followed by a slower butpermanent significant decrease in flow of 32-49% of thecontrol values (see accompanying table). The changes in flowwere apparently independent of mean arterial pressure and

always preceded any observed changes in cardiac output. Thisis similar to the "

spontaneous " increase in coronary flow

1. Bodo, R. J. Physiol. 1927-28, 64, 365.2. Kountz, W. B. J. Pharmacol. 1932, 45, 65.3. Rowe, G. G., Emanuel, D. A., Maxwell, J. F., Castillo, C., Schuster, B.,

Murphy, Q. R., Crumpton, C. W. J. clin. Invest. 1957, 36, 844.4. Székères, L., Lenard, G. Experientia, 1958, 14, 338.5. Andrews, W. H. H. J. Physiol. 1952, 117, 45P.6. Bayliss, L. E., Muller, E. A., Starling, E. H. ibid. 1928, 65, 33.

found by Laurent et al. concomitant with a decrease in cardiacoxygen consumption when the limit of normal physiologicaladjustment of cardiac oxygen consumption to oxygenavailability is surpassed.The possibility of a specific yet reversible effect of

quinidine on coronary smooth muscle is at present underinvestigation.

PURA N. SUÁREZ ROLDÁN

ERIC OGDEN.

Department of Physiology,School of Medicine,

University of Puerto Rico,San Juan, Puerto Rico, 00905.Environmental Biology,National Aeronautics andSpace Administration,Ames Research Center,Moffett Field, California.

MECHANISM OF ACTION OF PHALLOIDIN

SIR,-While investigating the pathogenetic mechanismof some hepatotoxic substances I became interested inthe action of phalloidin, a toxin of Amanita phalloides(a poisonous mushroom, " Death Cap " fungus).

This phytotoxin is a cyclopeptide whose formula has beenidentified by Wieland 8 9; when injected in a lethal dose (2 jjt.g.per g. body-weight) either intraperitoneally or intravenously,it kills the rat within 2-4 hours. Microscopic examinationreveals widespread necrosis of the liver, whereas all other

organs-except the kidney, which is hyperxmic-are undam-aged. In-vitro addition of phalloidin to heart fibroblasts I orto bovine kidney cells," even at comparatively enormous doses,produces no effect.To explain why phalloidin should damage the liver alone,

I suggest that this polypeptide is not toxic in itself, but is insome way changed into a toxic compound by enzymes presentin the liver-i.e., drug-metabolising enzymes-and not in otherorgans. This toxic compound would be exclusively formed inthe liver, and would therefore damage that organ alone. Thismode of action has been demonstrated for dimethyl-nitrosam-ine,l1 a toxic substance which causes liver necrosis without

damaging other organs.In order to check the validity of my suggestion I injected

phalloidin in newborn rats, whose system of drug-metabolisingenzymes is very rudimentary.12 Fifteen Wistar albino rats

(eleven females and four males), aged 7 days and weighingfrom 9 to 11 g., were intraperitoneally injected with 100 Ilg.phalloidin (a generous gift from Professor T. Wieland) in 0.1ml. of saline solution. (I took care to use very thin needlesand to hold tightly for a few minutes between thumb andfinger a small fold of the abdominal wall at the site of injection,to prevent the fluid leaking out.) None of the newborn ratsdied, although each had received phalloidin in an amountabout five times the lethal dose (2 Ilg. per g.) for a young oradult rat.

This result points strongly to the assumption that

phalloidin is not toxic in itself, but is changed into a toxiccompound by drug-metabolising enzymes of the liver.This hypothesis is supported by the observation that

the alloxan-treated rat survives a lethal dose of eitherAmanita phalloides extract 13 or phalloidin,14 It has in factbeen observed that, in the rat, administration of alloxanreduces the activity of drug-metabolising enzymes.15

L. FIUME.Institute of General Pathology,

University of Bologna,Bologna, Italy.

7. Laurent, D., Bolene-Williams, C., William, F. L., Katz, L. N. Am.J. Physiol. 1956, 185, 355.

8. Wieland, T., Wieland, O. Pharmacol. Rev. 1959, 11, 87.9. Wieland, T. Pure appl. Chem. 1963, 6, 339.

10. Fiume, L. Unpublished.11. Magee, P. N. in Cancer, Progress Volume (edited by R. W. Raven);

p. 56. London, 1963.12. Kato, R., Vassanelli, P., Frontino, G., Chiesara, E. Biochem. Pharmacol.

1964, 13, 1037.13. Verne, J., Ceccaldi, P. F., Hebert, S. C.R. Soc. Biol., Paris, 1950, 144,

645.14. Wieland, O., Matschinsky, F. Unpublished. Reported in Wieland, T.,

Wieland, O. Pharmacol. Rev. 1959, 11, 87.15. Dixon, R. L., Hart, L. G., Fouts, J. R. J. Pharmacol. exp. Therap.

1961, 133, 7.