lipid and water levels in five organs of albino rats bearing … · tracts of two organs from 29...

Lipid and Water Levels in Five Organs of Albino RatsBearing Walker Carcinosarcoma 256*

ELDONM. BOYD,E. M. KELLY,MONICAE. MURDOCH,ANDC. E. BOYD(Department of Pharmacology, Queen's University, Kingston, Ontario, Canada')

Based upon analyses of lipids and water inWalker carcinosarcoma 256 and in the testicle,skeletal muscle, and carcass of the host albino rat,Boyd, Boyd, Hill, and Ravinsky (2) have suggested "the production by the Walker tumor of a hy-

drolipotropic factor whose effect upon the tissuesof the host is to make their water and lipid concentrations become more like those in the tumor-component." The concept of a tumor's being ableto convert organs of the host into tumor-like tissue

Purina Fox Chow checkers and water ad libitum. The technicof tumor inoculation and of lipid and water analysis have beenpreviously noted (4, 5). The animals were studied in pairs oftwins, one twin inoculated in the groin and the other twin notinoculated with Walker carcinosarcoma 256. Data upon theage and weight of 89 pairs of rats and their tumors have beensummarized in Table 1. The rate of tumor growth was thesame (P > 0.05) in males as in females, body and host weightbeing greater (P < 0.001) in the males. The statistical methodsused were those of Croxton (6). The distribution of tumors maybe seen in Chart 1. Each pair of twins was sacrificed withchloroform, and alcohol-ether extracts of the organs were

TABLE 1

THEAGEANDWEIGHTOFALBINORATSBEARINGWALKERCARCINOSARCOMA256ANDOFNONTUMOR-BEARINGLITTERMATES

(Data are expressed as means Â±standard deviations)

i

26 Â±788.5 Â±64.2

175 Â±3450.8 Â±35.2

1.86Â± 1.06

MEASUREMENTMALES

(55 paira)

Age of tumor (days)Wt. of tumor (gm.)Wt. of host (gm.)Wt. of tumor, per cent host wt.Mean rate of increase in tumor wt.,

per cent host wt/dayFinal age of rat (days) 71 Â±11Initial body wt. (gm.) 143 Â±40Final body wt. (gm.) 264 Â±76

Initial body wt. (gm.) 138 Â±45Final body wt. (gm.) 225 Â±42

FEMALES MALES ANDFEMALES(34pairs)Tumor-bearing

albinorats24

Â±570.1+48.7183Â±2854.4Â±37.52.14Â±

1.1767

+10108+27203

Â±56Nontumor-bearinglittermatea108

Â±26156Â±23(89

paira)25

Â±680.7Â±58.9158

Â±3552.3+36.21.98+

1.1168

Â±11129+37238Â±75127

Â±35196Â±49

has been noted beforeâ€”-forexample, in Earle's

tissue culture studies on Walker carcinosarcoma(7) and in Greenstein's studies of enzymes in theliver of hepatoma-bearing rats (9). The objectiveof the work herein described was to determine theeffect, if any, of the presence of Walker carcinosarcoma 256 upon the water and lipid levels oftrachea-larynx, thymus gland, cervical lymphnodes, submaxillary salivary glands, and diaphragm of albino rats.

MATERIALS AND METHODSThe albino rats were originally of a Wistar strain which has

been inbred since 1937 in the animal quarters of the Department of Pharmacology at Queen's University. They were fed

Received for publication January 18, 1956.

prepared on the same day. Water and lipid levels were calculated as gm/100 gm nonlipid dry weight of tissue. The concentration of each compound in a control nontumor-bearingtwin was subtracted from the corresponding value in its tumor-bearing twin. The differences (D[T-Cj) were plotted againstthe mean rate of increase in tumor weight, and the significanceof mean differences was calculated for male twins, femaleÂ£wins,and for male and female twins combined. The mean rateof increase in tumor weight was calculated by dividing theweight of the tumor by the weight of the host, multiplying thequotient by 100, and dividing by the duration of tumor growthin days, which expressed the mean rate of increase in weight ofthe tumor as per cent of host weight per day.

* A paper upon this work was presented before the tumorsection of the Federation of American Societies for Experimental Biology, Atlantic City, April, 1956.

This work was supported by a grant from the NationalCancer Institute of Canada.

535

on April 12, 2020. © 1956 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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0â€¢¿�IOâ€¢¿�20â€¢¿�30

TRACHEA

<P Â°Â°â€¢¿�Â° o

WT.

MEANTUMORLEVEL

oÃ¤

5

i

+ 2â€¢¿�H

0

+4+ 2

0

3O

+ 600t300

0

01 2345MEAN RATE OF INCREASE IN TUMOR WEIGHT

% HOST WT. PER DAYCHART1.â€”Shifts in the wet weight and concentration of the value in its nontumor-bearing, control twin [D(T-C)].

neutral fat (NF), free cholesterol (FC), ester cholesterol (EC), The solid columns to the right indicate the mean level in Walk-phospholipid (P), and water (VV) in the trachea-larynx of er carcinosarcoma 256 minus the mean level in the trachea-albino rats bearing Walker carcinosarcoma 256. (The results larynx of nontumor-bearing albino rats.)are plotted as the value in each tumor-bearing twin minus



BOYO et al.â€”Lipid and Water Levels in Five Organs 537

RESULTSTrachea-larynx.â€”The trachea and larynx were

removed from 88 pairs of twin albino rats, of which33 pairs were females and 55 pairs were males. Thefresh wet weights of the organs are summarized inTable 2. The analytical error of lipid analyses wasrelatively high, because the minimal amounts ofthe various lipids necessary for a minimal analytical error (3) were not present in the extracts of onetrachea-larynx. To lessen the analytical error, extracts of two organs from 29 pairs of rats, each pairof the same litter, the same sex, and the sametumor status, were combined for lipid analysisafter determination of their individual dry weightsand water concentration.

The D(T-C) values were plotted against themean rate of increase in tumor weight, and exemplary scatterplots are shown in Chart 1. For calculation of D(T-C) values of lipids and water

hydrolipotropic shift consisted of a fall in the levelof neutral fat toward the low level in the tumorand an increase in the level of free cholesterol,ester cholesterol, phospholipid, and water towardthe high levels in the tumor. As Walker carcino-sarcoma 256 grew in these albino rats, the lipidand water levels in the trachea-larynx became likethose in the tumor.

A statistical analysis of the data as a group hasbeen given in Table 3. In order that the data upontrachea-larynx might be compared with those inthe tumor, the data upon Walker carcinosarcoma256, reported previously (4, 5) as gm/100 gm dryweight, were recalculated individually as gm/100gm nonlipid dry weight, and a statistical summary is given in Table 4. It may be seen that, asa group, lipid and water levels in the trachea-larynx of nontumor-bearing albino rats were different from those in Walker carcinosarcoma 256

TABLE 2

THE FRESH,WET WEIGHTIN GM.OFORGANSREMOVEDFROMTUMOR-ANDNONTUMOR-BEAHINGALBINORATS

DIFFERENCESm TUMOB-BEABISOBATS*

ORGANTrachea-larynxThymus glandCervical lymph

nodesSubmaxillary glandsDiaphragm

NONTDHOB-BEABINGBATS(MEANÂ±ST. DEV.)

0.160Â±O.OSO0.326 + 0.0790.224 + 0.069

0.356Â±0.0820.54310.151

MeanÂ±S.D.0.015

+0.0400.139+0.1170.048Â±0.0910.089

+0.0840.143Â±0.135P

(difference-0)0.001<0.001<0.001<0.001<0.001Percentchange-

9.4-42.6-21.4-25.0-26.3

* \Yeight in tumor-beuring twin minus weight in nontumor-bearing twin.

per unit nonlipid dry weight, there were availablesufficient data upon 24 pairs of female twins and 49pairs of male twins, insufficient data for these calculations being available upon the remaining fifteen pairs of twins. There was some evidence thatthe lipid and water levels of the trachea-larynxwere affected by tumors growing at rates of lessthan 1 per cent of host weight per day. The tumorsin this group of twelve pairs of rats consisted ofeleven which weighed less than 20 per cent of hostweight (average, 10 per cent) and one slow-growing tumor (the twelfth) which comprised 39 percent of host weight after 40 days of tumor growth.The weight of tumors growing at the rate of 1-1.99 per cent of host weight per day averaged 32per cent of host weight; at 2-2.99 they averaged60 per cent of host weight; and at 3 per cent andover they averaged 102 per cent of host weight, thelargest tumor being 153 per cent of host weight.

Loss of weight and shifts in the levels of lipidsand water became obvious as the rate of tumorgrowth increased to about 1 per cent of hostweight per day and after the weight of the tumorhad exceeded about 20 per cent of host weight. The

and shifted toward tumor levels in the tumor-bearing albino rats.

The lipid and water levels available upon thetrachea-larynx of 25 female, nontumor-bearingalbino rats were not different (P difference equals0 was 0.2 to 0.6) from those in 50 male, nontumor-bearing albino rats. The D(T-C) values for lipidsand water in the trachea-larynx of 24 pairs of female twins likewise did not differ significantly (Pdifference equals 0 was 0.3 to 0.9) from those in the49 pairs of male twins.

Thymus gland.â€”Boyd, Fontaine, and Hill (3)have reported an increase in the mean level oftotal cholesterol (P = 0.01), free cholesterol (P <0.05), phospholipid (P = 0.05), and water (P <0.05) in the thymus gland of 27 pairs of twin albinorats, one twin bearing and one not bearing Walkercarcinosarcoma 256. The levels were calculated asgm/100 gm dry weight. The mean concentrationof ester cholesterol was also elevated, but therewere insufficient data to prove a significant increase except possibly in eight rats with tumors ofintermediate size. In these eight rats there werealso possible pyramidal peak increases in total



538 Cancer Research

cholesterol, free cholesterol, and phospholipidlevels.

The original data of Boyd, Fontaine, and Hill(3) were individually recalculated as gm/100 gmnonlipid dry weight, and data from 58 additionalpairs of twin albino rats were added. The totalseries included 85 pairs, of which 32 pairs werefemales and 53 were males. The age and weight ofthe animals and tumors were essentially the sameas the values given in Table 1, and the distributionof tumors similar to that shown in Chart 1. Thefresh, wet weights of the thymus glands are given

in Table 2. Twelve small thymus glands were combined for lipid analysis, two to an extract, in thesame manner as noted above for trachea-larynx.

Sufficient data were available upon 29 pairs offemale twins and 43 pairs of male twins for calculation of D(T-C) values, which are summarized inTable 5. There were found to be increases in thelevels of total cholesterol, ester cholesterol, freecholesterol, phospholipid, and water in the tumor-bearing animals. These increases were toward thelevels in Walker carcinosarcoma 256, as shown bythe data given in Table 5.

TABLE3LIFIDANDWATERLEVELSINTHETRACHEA-LARYNXOFALBINORATS

BEARINGWALKERCARCINOSARCOMA256NONTUMOH-BEAHINO RATB

Ml AM III Ml M

Total lipidNeutral fatTotal fatty acidsTotal cholesterolEster cholesterolFree cholesterolPhospholipidWater

MeanÂ±S.D.(gm/100 gm

nonlipid dry wt.)

17.77 Â±13.44 Â±15.04 Â±0.939Â±0.080Â±0.859Â±3.39 +

9.959.459.230.6550.6780.3251.54

364 Â±107

Mean difference fromWalker carcinosarcoma

ÃŽS6 (see Table 4)*

- 5(P = 0.4)

+ 196(P<0.001)+ 37(P = 0.001)- 64(P<0.001)- 92(P<0.001)- 48(P<0.001)- 63(P<0.001)- 45(P<0.001)

TUMOR-BEARING RATB

Mean of differencesfrom nontuznor-bearing twinsf

- 16(P<0.001)- 31 (P<0.001)- 21 (P<0.001)+ 45(P<0.001)+271 (P= 0.02)+ 23(P<0.001)+ 19(P<0.001)+ 33(P<0.001)

* Per cent of mean in AValkercarcinosarcoma 256 (P difference equals 0).t Per cent of mean in nontumor-bearing twinÂ«{P difference equals 0).

TABLE 4

LIPID AND WATERLEVELSIN THE NON-NECROTICZONE OF44 WALKERCAR

CINOSARCOMA256 TUMORS*

MeasurementTotal lipidNeutral fatTotal fatty acidsTotal cholesterolEster cholesterol5VeecholesterolPhospholipidWater

MeanÂ±S.D.(gm/100gm nonlipiddrywt}

16.89 Â±5.454 54 Â±4.35

11.02 Â±4.002.634 + 1.8280.997+ 1.8141.637+ 0.7229.13 Â±3.42

064 Â±99* Recalculated from the original data of Boyd and

McEwen (4) and of Boyd, McEwen, und Shanai (a).

When the D(T-C) values were plotted againstthe rate of increase in tumor weight, the increaseswere found to pyramid at rates between about 1and 3 per cent of host weight per day or in animalsbearing tumors which averaged about 40 to 50 percent of host weight. The D(T-C) values for estercholesterol have been given as an exemplary scat-terplot in Chart 2. The estimating equations Yeland Ye2, solved by the method of least squares,have been shown plus and minus their standarderrors (SY.X) as a solid black area which seemedbest to fit the scatter of values. The probabilitythat the 42 values in the pyramidal peak ("B"

TABLE5LIPIDANDWATERLEVELSINTHETHYMUSGLANDOFALBINORATS

BEARINGWALKERCARCINOSARCOMA256

MEASUREMENTTotal lipidNeutral fatTotal fatty acidsTotal cholesterolEster cholesterolFree cholesterolPhospholipidWater

NONTUMOR-BEARINGRATS

MeanÂ±S.D.(gm/100 gm

nonlipid dry wt.)

23.80 Â± 17.9117.70 Â± 16.6421.32 + 15.46

1.069Â± 0.4970.014Â±1.056Â±5.20 Â±

430

0.4670.4482.45

Â±143* Per cent of mean in Walker carcinosarcoma 256 (P difference equals 0).t Per cent of mean in nontumor-bearing twins (P difference equals 0).


856 (see Table 4)*+ 41 (P = 0.005)+ 290(P<0.001)+ 93(P<0.001)- 59(P<0.001)- 99(P<0.001)- 35 (P<0.001)- 43(P<0.001)- 35(P<0.001)

TUMOR-BEARING RATS

Mean of differencesfrom nontumor-bearing twinsf

0(P = 1.0)-18(P = 0.1)

-9(P = 0.4)+ 113 (P<0.001)

+3,780(P<0.001)+65(P<0.001)+35(P = 0.001)+34(P<0.001)



BOYOet al.â€”Lipid and Water Levels in Five Organs 539

values) were the same as the fifteen occurring before ("A" values) or fifteen after ("C" values) the

peak was 0.001. Similar results were obtained insimilar scatterplots of D(T-C) values for freecholesterol, phospholipid, and water levels.

The sex of the albino rats did not influence thelevels of water and lipids in the thymus glands ofthe nontumor-bearing animals (P differencesequals 0 was from 0.2 to 0.9), nor did it influence

the D(T-C) values (P differences equals 0 wasfrom 0.2 to 0.8). The results upon thymus glandconfirm both the certain and tentative conclusionsof Boyd, Fontaine, and Hill (3).

Cervical lymph nodes.â€”The cervical lymphnodes were dissected and extracted from 83 pairsof twin albino rats, of which 34 pairs were femalesand 49 pairs were males. The mean age and weightof the rats and the tumors were practically identi-

+ 4

ÃŒ

o

OO

KluO.

-2

THYMUS ESTER CHOLESTEROL

B â€¢¿� C

PÃ‚-B Â«0.001 PB-C * o.ooi

Yel- SY.X

,Ye2 + SY.X

Ye2

Ye2-SY.X

012345

MEAN RATE OF INCREASE IN TUMOR WEIGHT% HOST WT. PER DAY

CHAHT2.â€”Correlation of shifts in the level of ester cho- (The results are plotted as the value in each tumor-bearinglesterol in the thymus gland of albino rats bearing Walker car- twin minus the value in its nontumor-bearing, control twinriÃ±osaremua 256 with the rate of increase in tumor weight. [D(T-C)].)



540 Cancer Research

cal to the values listed in Table 1. The wet weightof the nodes was the same in male as in female non-tumor-bearing albino rats (Table 2), and as a result the nodes comprised 39 per cent more of bodyweight in females (0.143 + 0.048 per cent) than inmales (0.103 Â±0.033 per cent), with the probability of the difference being zero < 0.001. The meanloss of wet weight (D[T-C]) averaged 21 per centof the weight of the cervical lymph nodes in thenontumor-bearing twins (Table 2), the percentageof wet weight of nodes lost being greater in malesthan in females. The lymph nodes removed included the superficial submaxillary nodes of Job,the anterior submaxillary nodes and accessorynodes as found present (8). Several pairs of extracts of cervical lymph nodes were combined forlipid analysis in the same manner as were the extracts of trachea-larynx.

of increase in tumor weight exceeded 1 per cent ofhost weight per day, and thereafter remained ata low but level plateau. Cervical lymph nodes fromtumor-bearing rats had higher levels of free cholesterol and water even in animals bearing the smallest tumors studied. These shifts suggest a veryearly effect of the tumor upon the cervical lymphnodes of the host. The D(T-C) values for freecholesterol pyramided upward in the same manner as illustrated in Chart 2 and at about the samepoint in growth rate of Walker carcinosarcoma256.

The mean lipid and water levels in the cervicallymph nodes of the nontumor-bearing female albino rats were all lower than those in the males,but the differences were not sufficiently great to bedifferent from zero by the "t" test (P differencesequal 0 ranged from 0.1 to 0.4). There was the pos-

TABLE6LIPIDANDWATERLEVELSINTHECERVICALLYMPHNODESOFALBINORATS

BEARINGWALKERCARCINOSARCOMA256


* Per cent of mean in

t Per cent of mean in

NONTCMOR-BEMean

Â±S.D.(gm/100gmnonlipid

drywt.)84.71Â±18.2217.02+13.6620.60+15.821.228+

0.7650.084+0.7671.142+0.5016.56

+6.00445Â±162ARINO

RATSMean

differencefromWalkercarcinoaarcomaÂ«56

(see Table4)*+

46(P =0.001)+275(P<0.001)+

87(P<0.001)-53(P<0.001)-92(P<0.001)-SO(P<0.001)-

28(P =0.005)-33(P<0.001)

TDUOR-BEARINORATS

Mean of differencesfrom nontumor-bearing twins'

- 22(P = 0.005)- 38(P<0.001)- 28(P = 0.001)+ 20(P<0.05)-124(P = 0.6)+ 30(P = 0.005)-I- 6(P = 0.6)+ 20(P = 0.001)

'Wulker carcinosarcoma 356 (P difference equals 0).

nontumor-bearing twins (P difference equals 0).

For the calculation of D(T-C) values per unitnonlipid dry weight, sufficient data were availableupon 31 pairs of female twins and 44 pairs of maletwins. The distribution of mean rate of increase intumor weights was similar to that shown in Chart1. A summary of these data for all rats as tumor-and nontumor-bearing groups is given in Table 6.All the lipid and water levels in the cervical lymphnodes of the nontumor-bearing albino rats weredifferent from the levels in Walker carcinosarcoma256. The levels of neutral fat decreased toward thelow levels in Walker carcinosarcoma 256, and thelevels of free cholesterol and water increased toward the higher levels in Walker carcinosarcoma256. There were no significant shifts in the levels ofester cholesterol and phospholipid.

Scatterplots of the D(T-C) values for neutralfat, free cholesterol, and water against mean rateof increase in tumor weight revealed correlationssimilar to those illustrated in both Charts 1 and 2.The D(T-C) values for the level of neutral fat fellto negative in relation to controls when the rate

sibility that increasing the value of N might ormight not reveal a difference due to sex in theselymph nodes. The cervical lymph nodes of themale tumor-bearing albino rats showed a greaterincrease (P = 0.02) in the level of free cholesterolthan did those of the female rats. Otherwise, theD(T-C) values per unit nonlipid dry weight werethe same (P differences equals 0 was 0.2 to 0.9) inmales as in females.

Submaxillary salivary glands.â€”The submaxillary salivary glands were removed from 54 pairsof twin albino rats, of which twenty pairs werefemales and 34 pairs were males. The ages andweights of the animals and tumors were essentially the same as those given in Table 1 and the distribution of tumors as shown in Chart 1. Dataupon the wet weights of the submaxillary salivaryglands have been summarized in Table 2. Theaverage loss of wet weight in the tumor-bearingtwins was 25 per cent of the wet weight of thesalivary glands in the nontumor-bearing twins.None of the extracts of submaxillary salivary




glands nor of diaphragm was combined for lipidanalysis.

Data upon 48 pairs of twins, nineteen femaleand 29 male, were available for calculations perunit nonlipid dry weight, which are summarized inTable 7. The lipid levels of submaxillary salivaryglands in nontumor-bearing albino rats were similar to those in Walker carcinosarcoma 256 exceptthat the submaxillary salivary glands had higherlevels of neutral fat and lower levels of estercholesterol. In the tumor-bearing albino rats themean level of free cholesterol increased by 29 percent, which kept the level of free cholesterol withinthe range of its level in Walker carcinosarcoma256. There were no other proved changes in lipidlevels in the submaxillary gland of the tumor-bearing albino rats, but the water level increased 16per cent toward the higher level in the tumor. Thesubmaxillary salivary gland, therefore, participated to a lesser extent in the hydrolipotropic shiftsof host tissues.

The sex of the albino rat had no effect (P difference equals 0 was from 0.2 to 0.8) upon the lipidand water levels of submaxillary salivary glands inthe nontumor-bearing animals. In the tumor-bearing rats, the level of phospholipid was augmented

more in the female than in the male animals (P difference equals 0 was 0.02). The mean level ofphospholipid had been lower in the female than inthe male nontumor-bearing rats, and, while theprobability of this difference being 0 was 0.2, itmay have accounted for the sex difference in thetumor-bearing rats.

Diaphragm.â€”The diaphragm, including itscrura, was analyzed in 55 pairs of twin albino rats,of which twenty pairs were female and 35 weremale. The ages and weights of the animals and tumors were similar to those summarized in Table 1and the distribution of tumors to those shown inChart 1. Values for the fresh, wet weights of thediaphragms have been summarized in Table 2. Theaverage loss of weight of this muscle in the tumor-bearing rats was 26 per cent.

Data upon the lipid and water levels of the diaphragm in nineteen pairs of female and 30 pairs ofmale twins have been given in Table 8. The diaphragm of nontumor-bearing albino rats had higher levels of neutral fat and lower levels of thecholesterol fractions, phospholipid, and water thanwere found in Walker carcinosarcoma 256. In thetumor-bearing twins it was possible to prove thatthere occurred a mean decrease of 40 per cent in

TABLE 7

LIPIDANDWATERLEVELSINTHESUBMAXILLARYSALIVARYGLANDSOFALBINORATSBEARINGWALKERCARCINOSARCOMA256

MEASUREMENTTotal lipidNeutral fatTotal fatty acidsTotal cholesterolEster cholesterolFree cholesterolFhospholipidWater

* Per cent of mean

t Per cent of mean

NoNTOMOR-BEARmO RATS TuMOR-BEARING RATS

Mean+S.D. Mean difference from Mean of differencesWalker carcinosarcoma from nontumor-

256 (see Table 4)* bearing twins t

+ 19(P = 0.05) - 10(P = O.S)+124(P<0.001) -28(P=0.2)+ 38(P=0.005) - 15(P = 0.3)â€”¿�43(P<0.001) +20(P = 0.005)-91(P<0.001) -131(P = 0.1)- 14(P = 0.1) + 29(P<0.001)- 7(P=O.S) + 8(P = 0.3)-41(P<0.001) + 16(P = 0.005)

(gm/100 gmnonlipiddry wt.)

20.15 Â±10.2610.18 Â± 9.0915.32 Â±

1.496Â±0.086Â±1.410 +8.53 +

395

9.180.4420.6000.5293.35

Â±100in Walker carcinosarcoma 256 (P difference equals 0).in nontumor-bearing twins (P difference equals 0).

' TABLE 8

LIPIDANDWATERLEVELSINTHEDIAPHRAGMOFALBINORATSBEARINGWALKERCARCINOSARCOMA256


* Per cent of mean int Per cent of mean in

NONTCMOR-]MeanÂ±S.D.

(gm/100 gmnonlipid dry wt.)

14.92 Â±7.609.74 + 6.51

12.37 Â±6.960.536+ 0.2670.004+ 0.2960.532+ 0.2534.56 + 1.90

343 + 62

Â¡EARING RATS


256 (see Table 4)*- 12(P = 0.2)+115(P<0.001)+ 12(P=O.S)- 80(P<0.001)-100(P<0.001)- 68(P<0.001)- 50(P<0.001)- 48(P<0.001)

Walker carcinosarcoma 256 (P difference equals 0).nontumor-bearing twins (P difference equals 0).

TUMOR-BEARINGRATSMeanof differences

fromnontumor-bearingtwinsf-20(P=0.01)-40(P<0.001)-26(P=0.001)+47 (P = 0.001)

+1,150 (P=0.3)+38(P=0.005)+17(P=0.2)+41 (P< 0.001)



542 Cancer Research

neutral fat levels, a mean increase of 47 per centin total cholesterol levels, a mean increase of 38per cent in free cholesterol levels, and a mean increase of 41 per cent in water levels. When theD(T-C) values were plotted against mean rate ofincrease in tumor weight, scatterplots similar tothat shown in Chart 1 were obtained.

The sex of the albino rat had no effect upon thelipid and water levels in the diaphragm of non-tumor-bearing albino rats (P difference equals 0wasfrom 0.2 to 0.9) nor of tumor-bearing animals(corresponding P values ranged from 0.5 to 1.0).

DISCUSSIONThe results described indicate that there was a

significant loss of fresh, wet weight in all the tissues studied, from 9 per cent in trachea-larynx to43 per cent in thymus gland, in the tumor-bearinganimals. The water levels in all the tissues of thenontumor-bearing albino rats were lower than thewater levels in Walker carcinosarcoma 256. Thewater levels increased in all the tissues studied inthe tumor-bearing rats, from 16 per cent in submaxillary salivary glands to 41 per cent in diaphragm. The total cholesterol levels were lowerthan those of Walker carcinosarcoma 256 in all thetissues studied in the nontumor-bearing albinorats, from 43 per cent in submaxillary glands to 80per cent in diaphragm. The total cholesterol levelswere increased in all five tissues of the tumor-bearing albino rats, from 20 per cent in cervicallymph nodes and submaxillary glands to 113 percent in thymus gland. The level of free cholesterolwas lower than that of Walker carcinosarcoma 256in four of the five tissues studied in the nontumor-bearing albino rats, and in the fifth tissue, submaxillary salivary glands, it was in the low rangeof tumor values. The level of free cholesterol wasincreased in all five tissues of the tumor-bearingalbino rats.

Shifts in the levels of ester cholesterol, neutralfat, and phospholipid were not quite so consistentas those of weight, water, total cholesterol, andfree cholesterol. The level of ester cholesterol wasbelow that of the tumor in all five tissues of thenontumor-bearing albino rats. In the tumor-bearing twins, ester cholesterol levels were significantlyincreased only in trachea-larynx and in thymusgland. The neutral fat levels were consistentlyabove those of Walker carcinosarcoma 256 in allfive tissues of nontumor-bearing albino rats. In thetumor-bearing twins, neutral fat levels were lowered in three of the five tissues. In the remainingtwo tissues, thymus gland and submaxillary salivary glands, the mean levels of neutral fat fell inthe tumor-bearing albino rats but not to the extent

of being statistically significant. Inability to demonstrate significant shifts in the levels of estercholesterol and neutral fat may, or may not, bedue to the high analytical error involved. The levelof each of these lipids is calculated from, respectively, two and four direct measurements uponextracts of lipids and, hence, the calculated values inherit the analytical errors in two or moreanalyses.

The levels of phospholipid were lower thanthose of Walker carcinosarcoma 256 in four of thefive tissues of nontumor-bearing albino rats, thelevel in submaxillary salivary glands being of thesame order as that in the tumor. In two of the fourtissues, the level of phospholipid increased in thetumor-bearing animals. In the remaining two tissues, cervical lymph nodes and diaphragm, therewere no significant changes in the tumor-bearinganimals, although the mean differences were ineach instance on the positive side. Of all the valuesdetermined, the levels of phospholipid were leastdifferent in nontumor-bearing albino rats fromthose in Walker carcinosarcoma 256, the averagebeing only one-third lower. If the conclusion thatthe effect of the tumor is to make phospholipidlevels of tissues become like those of the tumormay be assumed, for argument's sake, then a sig

nificant increase in phospholipid levels in these fivetissues would be the most difficult to prove. Thismay or may not be the reason that shifts in phospholipid levels were not proved present in cervicallymph nodes and submaxillary glands.

Values for total lipid and total fatty acids havebeen tabulated for reference purposes but not discussed separately, because they are dependentupon the values of the lipids discussed.

A summation of the levels of neutral fat, totalcholesterol, free cholesterol, phospholipid, andwater which were different from those of Walkercarcinosarcoma 256 in the nontumor-bearing animals indicates that hydrolipotropic shifts werepossible in 23 instances. In nineteen of these 23instances, shifts toward tumor levels occurred inthe tumor-bearing animals. In no instance wasthere a shift away from the tumor level in thetumor-bearing rats. These data add further evidence in support of the suggestions of Boyd, Boyd,Hill, and Ravinsky (2) noted in the first paragraphof this paper.

The significance of these hydrolipotropic shiftsremains to be proved, and possible factors will notbe discussed in detail. Bloor and Haven (1) havesuggested two other alternatives, namely, an increased activity of host tissues and, secondly, agreater production of certain lipids, such as phospholipid, for the use of the tumor. Nakahara and




Fukuoka (e.g. [10]) and others (9) have suggestedthat changes in the host are due to production of atumor toxohormone. It is possible that the shiftsin host tissues may be due to shifts in histologicallydemonstrable and countable cell types. Loss of atleast part of the neutral fat may be, and in manytissues certainly is, due to loss of storage fat inadipose tissue.

The general conclusion which emerges fromthese and previous studies upon hydrolipotropicshifts in the Department of Pharmacology atQueen's University is that, insofar as lipid and

water levels are concerned, the tissues of the tumor-bearing host gradually acquire that samenessof composition which characterizes the tumor. Inmost tissues and to some varying extent, this new,uniform composition is like that of the tumor. Ineffect, Walker carcinosarcoma 256 makes the hosttissues become more and more like Walker carcinosarcoma 256.

SUMMARYThe trachea-larynx, thymus gland, cervical

lymph nodes, submaxillary salivary glands, anddiaphragm of 54-88 pairs of twin albino rats, onetwin inoculated and the other twin not inoculatedwith Walker carcinosarcoma 256, were weighedand analyzed for lipid and water levels expressedas gm/100 gm nonlipid dry weight. The tumorsvaried from one to 153 (average 52) per cent ofhost weight.

There was a loss of fresh, wet weight in all fivetissues of tumor-bearing animals, averaging from9 per cent (P = 0.001) in the trachea-larynx to 43per cent in the thymus gland.

In all five tissues of the nontumor-bearingtwins, the mean water, total cholesterol, estercholesterol, free cholesterol, and phospholipidlevels were significantly (P = 0.01 or less) lower,and the neutral fat levels higher, than those ofWalker carcinosarcoma 256, with the exceptionsof the submaxillary salivary glands, in which freecholesterol and phospholipid levels were in the lowrange of those in the tumor. Phospholipid levelswere on the average the least different and neutralfat levels the most different from those of thetumor.

In all five tissues of tumor-bearing twins therewere significant increases in the mean levels ofwater, total cholesterol, and free cholesterol andin two tissues of ester cholesterol and phospholipid. In three tissues there were significant declines in the mean level of neutral fat. In over 80per cent of the instances where shifts were possible, the water and lipid levels of all five tissuesin the tumor-bearing animals became more uni

formly like the composition of Walker carcinosarcoma 256.

The sex of the albino rat was practically without effect upon these shifts in water and lipidlevels.

The shifts in the tumor-bearing animals appeared most commonly in animals with tumorswhich had grown at the average rate of 1 per centor more of host weight per day or which weighed20 per cent or more of host weight. In thymusgland, and occasionally in cervical lymph nodes,the shifts pyramided in animals bearing tumorswhich weighed about 40 to 50 per cent of hostweight, the shifts in animals bearing larger tumorsbeing less marked.

These results indicate further that tissues ofalbino rats develop a sameness of lipid and waterlevels, like those of the tumor, as Walker carcinosarcoma 256 grows upon the host rat.

ACKNOWLEDGMENTSThe authors wish to acknowledge the cooperation of Eliza

beth A. Bentley, V. Fontaine, J. G. Hill, H. D. McEwen, P.Mott and A. O. Tikkala.

REFERENCES1. BLOOR,W. R., and HAVEN,F. L. The Weight and Lipid

Content of the Intestines in Rats with Walker Carcinoma256. Cancer Research, 16:173-76, 1965.

2. BOYD,E. M.; BOTD,C. E.; HILL,J. G.; and RAVINSKT,E.The Lipid and Water Content of Carcass, Skeletal Muscle,and Testicle in the Host Component of the Albino Rat-Walker Carcinoma 256 Dual Organism at ProgressiveStages of Tumor Growth. Can. J. Biochem. Physiol.,32:359-71, 1954.

3. BOTD,E. M.; FONTAINE,V.; and HILL, J. G. SignificantChanges in the Lipid and Water Content of the ThymusGland in Albino Rats Bearing Walker Carcinoma 256.Can. J. Biochem. Physiol., 33:69-82, 1955.

4. BOTD,E. M., and McEwEN, H. D. The Concentration andAccumulation of Lipids in the Tumor Component of aTumor-Host Organism, Walker Carcinoma 256 in AlbinoRats. Can. J. Med. Sc., 30:163-72, 1952.

5. BOTD, E. M.; McEwEN, H. D.; and SHANAS,M. N. TheLipid Composition and Water Content of Brain, Heart,Lung, Liver, Gut, and Skin in the Host Component of theAlbino Rat-Walker Carcinoma 256 Dual Organism. Can. J.Med. Sc., 31:493-504, 1953.

6. CROXTON,F. E. Elementary Statistics with Applicationsin Medicine. New York: Prentice-Hall, Inc., 1953.

7. EARL,W. R. A Further Study of the Walker Rat Mammary Carcinoma Nr. 256 in Vitro. Archiv, exper. Zell-forsch. GewebezUcht., 20:140-55, 1937.

8. GREENE,E. C. Anatomy of the Rat. New York: HafnerPublishing Co., 1955.

9. GREENSTEIN,J. P. Biochemistry of Cancer. 2d Ã©d.,pp.351-53. New York: Academic Press, Inc., 1954.

10. NAKAHARA,W., and FUKUOKA,F. Biosynthesis of Toxohormone. A Seventh Study on Toxohormone, the Characteristic Toxic Substance Produced by Cancer Tissue.Gann, 45:77-85, 1954.



1956;16:535-543. Cancer Res Eldon M. Boyd, E. M. Kelly, Monica E. Murdoch, et al. Walker Carcinosarcoma 256Lipid and Water Levels in Five Organs of Albino Rats Bearing

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