PROTECTIT-E COLLOIDS IS CAXCER L. A. LIUNRO T h e Hendry-Connell Research Foundation, K i n g s t o n , Ontario, Canada Received February 24, 1964
Differences in the protective action of the colloids of spinal fluid in health and disease have been recognized and used for a long time in the Lange gold number technique. In cancer the constituent; of the blood serum differ from normal. F. L. Pllunro (19), working in these laboratories, obtained the values given below in a study of the sera of one hundred and thirty-five cancer patients: Peruin albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . Serum globulin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .i/G ratio. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Total protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
3.63 2.52 1.52 6.41
i 0.37 grains & 0.32 grains f 0.41 grams i 0.39 grams
per per per per
cent cent cent cent
4.70 2.45 2.02 7.04
f 0.21 grams per cent
For a group of thirty normals the values were: .-\lbuniiri . . . . . . . . . . . . . . . . . . . . . . . . . . Globulin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . A/G r a t i o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Total protein.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
~
,
~
i 0.30 grams per cent i 0.20 granis per cent f 0.26 grams per cent
These normal values, although from a relatively small group, are in agreement vith the results of Streef and Streef-Spaan (26), x h o reported the following: 4.66 2.33 2 0 7.3
I1buniin Globulin .i/G iatio Total protein
granis grams gianis grams
per per per per
cent cent cent cent
Furey ( S i , from a study of seventy-eight normals, gave slightly higher values: viz.,4.9, 2.4. 2.OG, and 7.4 grams per cent, respectively. Gutman et al. (12) gave 5 . 2 , 2.0, 2.60, and 7.2 grams per cent as mean values. Several norkers haT-e also noted a decrease in the total protein in cancer serum (4, 5, 6. 7 , 10, 14, 15, 18). There is considerable evidence that this lowering is chiefly due t o a loirer albumin (3, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 23, 24, 25). The globulin cdues are practically unchanged, although some workers have reported an increase in globulin (15, 20). Obviously, if the albumin decreases and the globulin is unchanged or is higher, the &\/G ratio will invariably he lower. Jfarchal. Paturel, Guerin, and Guerin (18) reported that in hens inoculated 11 ith sarcoma- a decrease in both albumin and globulin of from 25 to 40 per cent is obtained. 187
188
L. A. MUNRO
Rabb (21) found that the S / G ratio for carcinomatous dogs was only 0.74, whereas for normals the value was 1.11. Wladasch (27) gave 1.51-2.40 as the normal range,-the average being 1.9 grams per cent. This marked difference between normals and cancerous individuals should be indicated in the protective action of the sera against the coagulation of a solution by an electrolyte. That such is the case will be seen from the following results. EXPERIMENTAL
The test used in the experiments reported below is a modification of that used by Asai (1)in an attempt to detect the onset of malaria by such changes in the blood serum. The adaptation of this test mas undertaken because the author was using Congo red in other work on the reticulo-endothelial system. Other solutions or dyes could doubtless be used. To a mixture of 5 cc. of 0.1 per cent Congo red and 0.5 cc. of serum, Asai added 5 cc. of 0.3 per cent quinine hydrochloride. After 1 to 2 hr. a t room temperature, the mixture was centrifuged and the supernatant liquid compared in a colorimeter with 0.025 per cent Congo red. The test was reported positive within 4 days of infection. Various electrolytes, including other alkaloid sulfates and chlorides, have been tried in this laboratory, but so far the quinine hydrochloride seems to give better differentiation between the two groups. Tests are run using three different quantities of sera: 0.075, 0.10, and 0.15 ml. The serum is transferred to conical centrifuge tubes in duplicate with a micropipet. Five-milliliter portions of standard Congo red are then added and the mixture stirred well. Five milliliters of the electrolyte are pipetted into separate tubes. At zero minus 2 min. the electrolyte is dumped into the tube containing the largest amount of serum and mixed by pouring back and forth four times. This has been found to be better than shaking the tubes, probably owing to changes in concentration of serum induced by the frothing which occurs when the mixture is shaken. The tube containing the smallest amount of serum is mixed at zero time and the three tubes placed in a thermostat a t 30°C. for 30 min. The three tubes are then centrifuged for 4 min., and the color in the supernatant is evaluated in a Klett photoelectric colorimeter from a standard curve for Congo red, using a green filter S o . 59. The results for seventy-eight cancer sera and for seventy normal sera are given in tables 1 and 2. In column two the values are given in gammas of Congo red per milliliter. In columns three and four theresults are grouped to sholv extent of coagulation: 6f indicates residual color from 0 to 25 y;5f from 25 to 50 7; 4+ from 50 to 100 y;3+ from 100 to 200 y ;2+ from 200 to 350 y ; 1+ from 350 to 450 y ;=!= from 450 to 500 y, the theoretical maximum. Since before flocculation there is an increase in absorption, although the mixture appears clear, such an increase in N e t t reading v-ithout viiible precipitate is denoted a > ”trace” or tr. If the value = 500, .honing no coagulation, the reading is indicated by a minus sign. Xll sera u-ere prepared from “fasting” blood..
189'
PROTECTIVE COLLOIDS IS CANCER
TABLE 1 Cancer patients
-
(2)
(1)
RESIDUAL DYE (0.075 YL.
PATIENT N O .
SERUX)
(3)
(4)
DYE REMOVED (0.10 YL. SERUM)
DYE REMOVED (0.15 ML. SERUM)
6+* 2+ 5+
tr tr
y per millilifer
1. . . . . . . . . . . . . . . . . . . . . . . I c 2 .. c 3. . . . . . . . . . . . . . . . . . . . . .
c
c 4. . . . . . . . . . . . . . . . . . . . . . c 5. . . . . . . . . . . . . . . . . . . . . . C 6. . . . . . . . . . . . . . . . . . . . . . c 7 C 8 c 9. . . . . . . . . . . . . . . . . . . . . . c 1 0 . .. . . . . . . . . . . . . . . . . . . c 11... . . . . . . . . . . . . . . . . . . C 13... . . . . . . . . . . . . . . . . . . C 14... . . . . . . . . . . . . . . . . . . C 1 5 . .. . . . . . . . . . . . . . . . . . . C 1 6 . .. . . . . . . . . . . . . . . . . . . C 17... . . . . c 18.. . . . . . . . . . . . . . . . . . . .
c 19... . . . . . . . . . . . . . . . . . .
c 21.. . . . . . . . . . . . . . . . . . . . C 23.. . . . . . . . . . . . . . . . . . . . C 24.. . . . . . . . . C 25.. . . . . . . . . C 26.. . . . . . . . . . . . . . . . . . . .
C C C C: C
c c
28... . . . . . . . . . . . . . . . . . . 2 9 . .. . . . . . . . . . . . . . . . . . . 30.. . . . . . . . . . . . . . . . . . . . 31.,. . . . . . . . . . . . . . . . . . . 32.. . . . . . . . . . . . . . . . . . . . 33.. . . . . . . . . . . . . . . . . . . . 34... . . . . . . . . . . . . . . . . . .
C 36 . . . . . . . . . . . . . . . . . . . . . c 37... . . . . . . . . . . . . . . . . . . c 3 6 . .. . . . . . . . . . . . . . . . . . . (' 3 9 . . . . . . . . . . . . . . . . . . . . c 40.. . . . . . . . . . . . . . . .
i
3.4 26. 13.3 5.6 9.0 4.3 8.1 6.3 8.1 8.5 2.6 3.1 6.0 6.1 3.5 8.4 7.2 6.3 4.3 4.3 6.0 7.2 6.7 3.6 6.1 15. 5.0 7.8 7.5 5.4 4.0 9.5 6.7 10.2 7.3 6.0 7.1 11.4 8.2 10.3 6.4 4 .7 4.6
5+ 6+ 6+ 2+ 5+ 6+ 4+ 6+ 6+
5+ 3+ 6+
5+ 2+ 4+ 6+ 6+ 6+ 5+
6+ 6+ 6+ 2+ 6+ B+ 2+
4+ 6+ 6+ 5+
*
5+ 5+ 6+ 3+ 5+ 3+ 6+
6+ 6+
3.6
G+
4.6 9.3 7.3
6+ 3+ 5+
++ +++ tr + tr tr tr
+++ tr tr tr tr tr tr tr tr
++++ +++++ tr tr
+++++ ++ ++ +++++ +++ ++++ tr tr tr tr tr tr tr tr tr
++ tr t,r tr tr tr tr
+++++
190
L. A. JlUlURO
TABLE I-Concluded (2)
(1)
PESIDU.4L DYE (0.075 ML. SERUX)
PATIENT KO.
7 per
C 48
c 49
C 50 C 51
c 52
c 53 c 54 c 55 c 50
I
I
c' 57 c 58 (? 59
, ~
c 60 c
61
c; 62 c 63
1
I
C 64 C 65 CJ 66
(4)
(3)
DYE REMOVED (0.15 ML. SERUM)
DYE REXOVED (0.10 1IL. SERUY)
ntilliliter
o+
4.7 6.3 9.0 10.7 23.0 9.3 17.5
tr tr tr tr tr tr tr tr
5+ 3+ 5+ 3+ 5+
tr
ll.G
G+
16.5 14.7 11.6 14.0 10.0 12.0
6+ 6+ 5+ 4+ G+ 5 iG+ i 5+ 6+
4.6
34. 11.2 7.4 24.0 14.2 22.2 8.3 11.5 6.0 9.0 12.2
++ +++ tr tr i tr
++++ tr +++ +++ tr tr tr
4c
C; 67 3+ c' 68 3+ C 69 6+ -I+ c 70 C 71 GS c; 72 I 5+ o+ C' 7 3 I * c' 74 I .3 G+ 10.5 c: 75 6+ 7.3 c' 76 ti+ 10.7 c 77 I 6+ 11.9 c 78 G+ ____ - __ * T h e significance of tlir notations used is given below: ~
,
tr tr
+++ tr ++++ ++++t ++++ +++ ++ +++ 7 PER YILLILITEH
-
~
6+
5+ 4+ 3+
~
0-25 25-50 50-100 I 00-200
~
_..
___
.
~
.
-
350-450
I
f tr
I I I
.\pparent 500 Original
DISCUSSIOS
The results show that tlic protective colloids in riorninl serum are much inorc effective than in the serum of cnnceroiis individuals. Thiq will be seen from the
PIiOTCCTIVI;
191
COLLOIDS 1s CABCEIl
:\\wages and from the distribution curves for the first and second tubes (table 4; tigures 1 and 2). For the 30-min. incubation, the 0.15 ml. of serum dfords protcctioii in all but some of the cancer sera. T.IBLE 2 -Yornial patzsnts
I SfRUXIO.
s
1 . .. . . . . . . .
s2
....... S8. . . . . . . s4 . . . . . . . . S 5 .........! s R ........ si. ....... s s. . . . . . . . . . 0. . . . . . . s 10 . . . . . . . . s 1 1 . .. . . . . . . . ~
.I
s s
1 2 . , . . . . . . . .1 S 1 3 . .. . . . . . . 1 s 1 4 . ,. . . . . . . S 15.. . . . . . .. ! s 16 . . . . . . . . . . K 1 7 . .. . . . . . . , , S 1s... . . . . . . .I s 1 9 . .. . . . . . . s 20. . . . . . . . . ' K21. . . . . . 1 ?; 2 2 . . . . . . . . . S 23 . . . . . . ' S 24 . . . . . . . . s 2 5 . .. . . .. . . I S 26... . . . . . . S 27. . . . . . . . s 28. . . . . . . i s 2 9 . .. . . . . . S 3 0 . .. . . . . . . . S31. . . . . . . . s 32. . . . . . . . s 33. . . . . . .
1
~
i
~
~
S 35.. . . . . .
227 27 15 28 48 79 39
474+ tr
4si
22 16
tr 6+ tr
40
26 37 18 10 80 41 15 24 33 20 26
i
tr 5+
I
tr
I
tr
2+
I
30 10
;:
~
,
~
I
S 46 s 47 s 48 s 49 s 50
1
:; 26 53 17 18
s 51
56
1-53 s 54
68
,-
tr tr ir tr tr
-\
-r
JJ
1
tr tr tr
3+ ........
S 56
s 57 ~
* ir
~
tr
+
18 23 10 34
s 52
tr
tr
0.075 ml.
~
s 44 s 45
I
1 1
X 36 1-37 s 3s s 39 S 40 s 41 1-42 h- 43
~
tr
1s 26 17 41 15 17 4s 25 15 82 142 33 26
'
tr
+
SERUM AYOUNTS
___
I
'' 1'
S 58 1-59 S 60
~
S 61 S 62 S 63 S 64
tr
1 65
tr tr tr tr ir
S 66
S S S S
67 68 60 70
..
7n I'J
L..
20 85 43 22 59
3+ tr
b l
3+ tr
E 38 50
tr tr 2+ 4+
40
4+
,
120
1
tr tr
I ~
i, tr tr tr tr tr
-
& h i csamination of the data for tht. 0.07'5 nil. of serum in table 3 shows that the diffrrcnces between the amounts of 1.WidlIi11 dye are significant. The difference hetv een nieans for the cancer patienti and group h of the normals is over swen t inies the standard error, and the differcncc in the case of group B of the normals is over ten times the qtandard eiror.
192
L. A. MUNRO
FIG. 1 . Distribution curves for residual dye after 30 miri. incubat,ion with electrolyte at 30°C. in the presence of 0.075 ml. of serum.
FIG.2. Distribution curves showing percentage of cases in the different coagulation groups in the presence of 0.10 in]. of serum.
193
PROTECTIVE COLLOIDS I N CANCER
Figure 1 gives the distribution of data in the first coluniri of each half of table 4 arranged in groups differing in concentration of residual color by 5 y per milliliter. It will be noted that 89.6 per cent of thc cancer patients have values less TABLE 3 Statistical significance
0.f
data
___~ .-__ NORMALS
____
C.3NCER PATIFSTS
A
Suniher of c a s e s . ,. . . . . . . . . . . . . . . . . . . . . . I AIean, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Standarc1 deviation . . . . . . . . . . . . . . . . . . Standard error of m e a n . . . . . . . . . . . . . . . . .
78 9.26 5.54
~
0.63
1
Standard error of t h c difference bctween inenns (1) cILiiccr and ( 2 ) cnncer and Differencc between iiieaiis : (3) c:incer and (4) rnncer and
* Omitting
-I 1
70 40.52 33.19 3.96
~
I
~
~
norinals norninls noiinals normals
B'
66 33.86 18.35 2.26
.I
4.01 2.35 31.26 24.00
I3 .I 13
the four high v d u e s
TAB1,I: 4 Distributzoii o j cases in the cliffereti1 coagitlulioii groups CANCER PATIENTS
1
0.075 ml. of serum
NORMALS
0.10 ml. of serum
~1
0.U;: ml. of hcrum
0.10 mi. of serum ~
Residual dye y per milliliter
'er cen if
total
)ye removed
!urnbe (70)
'er cent Nf total
__
~~
e(Miss R. O’C.)where canccr was suspected, a ‘(I?. C.” bloocl tc*t \va\ made, giving values of 9 y for thr firyt tube and 4+ for the second tube .In opeisation was ruhsequently performed and the biopsy showed malignancy. In another cabc ( A h . U.) where cancei’ was suspected, a value ~vellup in tlie noriiial range \va> obtained. Biopsy proof shoiyed a benign cyst. While the average total protein :tnd alhumin are lower for cancer patienti; than for normals, the A/G ratio is nioic. consistent. :In examination of this ratio on some sixty of the above cancer patients >lio\vs that there is only a very rough agreement between the A/G ratio and the protective colloid index. The protective action indicate5 more than changes in amounts of the above serum constituents. Induced qualitative variations in the state of aggregation or even in molecular species may poqsibly occur, owing to differences in protein metabolism. Further experiments are in piogress to adapt thi\ test to micro amounts of serum (0.02 cc.) and to substitute another coagulant for the quinine hydrochloride. SUJlhIARE’
The protective colloids of sevrnty-eight canccr \era have been compared with normal sera from seventy individuals. The addition of 0.075 ml. of serum is made to 5 ml. of Congo red and after addition of the coagulating electrolyte and incubation a t 30°C. for 30 niin., the residiial color is determined in a photoelectric. colorimeter. Average values of 9.26 and 40.52 y per milliliter, respectively, were obtained, 89.6 per cent of the canccr patients and 4.3 per cent of normals giving values below 15 y per milliliter. The -I/G ratio s h o w a rather poor parallel to the protective colloid (P.C.) index. It is suggested that other factors such tls molecular dispersity and PI-en molecular specics are mort iinportant. The author nishes to ucknon.ledge his debt t o Dr. W C. Iiruger of the Toronto Western Hospital and member< of hi\ staff for the major number of the clancerous bloods; to Agnes Medley, 13 1 , who carried out the protein analy to Gnendolyn Dan., B.A., for technical ai4stance. The n.ork was made possible by n grant from the Pro\-incc ot Ontario through tlic Department of Health. l~~:Fl!,l~I,h-cEs (1)
‘\>\I,
tJ A‘Ietl
RI
(2) .kI B H I ,
l s s o c Folnlost 40, 4.554; Clierl~ it).itIJcts 36, 5541 (1342)
R I B ~ R FCOlllpt lelld bO( hl0l 123, 327-5 (1976) W i m rned K‘ochic.11i 81, 1355 (1931)
‘rilRODET, AYD
(3) B ~ C TK , I1
CLL-STEII FOIIII.\TIOh7 IS THE .IDSORBED STATE
1%
CLUSTER FORlI.ITIOS ASI) PHASE TRANSITIONS IS THE ADSORBED STATE 1I.ISS 11. CASSEI, 11.26 East 46th Street, Chicago 15, Illinois
Receiced Jfarch 8, 19& I . I N S U F F I C I E N C Y O F CURRENT M C L T I L A Y E R T H E O R I E S
I t is well known that in Langmuir’s derivation of an isotherm for molecules adsorbed from the gas phase on a plane crystalline surface the interaction of neighboring adatoms is neglected. Consequently, the equation of state of the monolayer corresponds to that of a compressed gas above its critical point. As this holds no matter what the magnitude of the adsorption energy, the question naturally arises: How can extensions of the simple Langmuh mechanism t o the treatment of multilayers in the adsorption oi vapors account for the final liquefuction of the adsorbates?
