Ir. J. Med. Sc. June, 1%3, pp. 251-255 Illustrated Printed in the Republic of Ireland

RIBONUCLEIC ACID LEVELS IN BLOOD OF CANCER PATIENTS

By JAi~IES O 'DONNELL, M.Sc.

Uni~ of Cell Metabolism, Dept. of Biochemistry, University College, Dublin

Introduction

M UCH effort has been directed to the problem of finding a generalised biochemical test for the diagnosis of cancer. Greenstein (1954) reviews the literature and more recently

Bodansky (in Greenberg and Harper, 1960) has commented on the results achieved in the field of blood serum enzyme levels. Serum lactic dehydrogen,~se (Hill, 1957) and adenosine deaminase (Straub et al., 1957) have been claimed to be elevated in cancer, but not without exception. In general two reservations must apply to increases in enzyme levels. They may not be specific to malignant disease, or it may not be possible to indicate in the laboratory the source of the increase. The same considerations apply to changes in non-enzymic components in the blood. Amino-acid patterns in a large number of cancer cases were investigated by Filteau (]961), but the differences from normal were not specific to cancer.

Mandel and Metals (1948) determined the levels of plasma Ribonu- cleic acid in 6 normal subjects and in 4 cases of cancer (among other pathological states). Because of the small numbers of subjects in their study and because of the availability of spectrophotometric methods for the determination of ribonucleic acids, it was decided to repeat the assay in a number of normal subjects and cases of malignant disease. Serial determinations of plasma ribonucleic acid (RNA) were also made on 2 normal and 2 cancer subjects to find the variation in indivi- dual levels over a period of time.

Materials and Methods

Normal subjects, of both sexes, ages ranging 30-70 years, were chosen at random. Cancer blood samples were obtained from the Research Laboratory of St. Luke's Hospital, Dublin. The hepar~nised samples were spun in a clinical centrifuge one hour approximately after being drawn and 2 ml. (or other convenient volume) withdrawn. Plasma was analysed for RNA 2-3 hours after the drawing of the blood sample.

Extraction followed the general method of Schmidt and Tannhauser as given in The Nucleic Acids (Chargaff and Davidson, 1960). The extracted residue was made up to 2 ml. with distilled water, and then 16 ml. of IN KOH added and hydrolysis allowed to proceed for 18 hours at 37~ An absorbing standard from plasma was prepared according to the method of Tsanev and Markov (1960), and the RNA content of the hydrolysed extract measured by the two-wavelength method of these

251

252 I R I S H J O U R N A L O F M E D I C A L S C I E N C E

authors. A commercial RNA (B.D.H.) was purified (Kirby, ]960) and used as a standard.

In this method absorption is measured, in neutral solution, of the hydrolysed RNA at 260 m~. and at a second wavelength found by experiment for a particular preparation, in this case 283 m~. The difference in magnitude of absorption is a function of RNA concentra- tion. Absorption was measured in the Beckman spectrophotometer, and RNA values calculated as mg. RNA per 100 ml. of plasma.

T A B L E I

Plasma R N A in Cancer Subjects

M a l i g n a n c y

M y e l o m a . . M y e l o m a . . M y e l o m a . . M y e l o m a . . M y e l o m a . . M y e l o m a . . M y e l o m a . . M y e l o m a . . M y e l o m a . . M y e l o m a . .

M e l a n o m a . . . . M e l a n o m a . . . . M e l a n o m a . . . . M e l a n o m a . . . .

L e u k a e m i a . . . . L e u k a e m i a L e u k a e m i a ( l y m p h a t i c ) * " L e u k a e m i a ( l y m p h a t i c )

C a n c e r o v a r y . . . . C a n c e r o v a r y . . . . C a n c e r o v a r y . . . . C a n c e r o v a r y . .

C a n c e r b r e a s t . . . . C a n c e r b r e a s t . . . . C a n c e r b r e a s t . . . .

H o d g k i n ' s d i s e a s e . . H o d g k i n ' s d i s e a s e . . H o d g k i n ' s d i s e a s e . .

~

. ~

~

~ 1 7 6

~

~

tNA

0 . 9 4 0 . 5 2 1.40 5 . 3 2 7 .30 2 . 5 0 4 . 2 5 6 -00 3 -20 3 . 6 4

2 . 5 0 2 . 8 0 7 . 5 0 5 . 5 0

4 -08 5 -72 5 . 3 4 2 . 8 6

2 . 0 8 5 . 0 0 2.83 2 . 3 2

5 -50 2 . 1 0 5 4 0

1 .05 3 -08 2 . 5 0

M a l i g n a n c y

C a n c e r b l a d d e r . . C a n c e r b l a d d e r . . C a n c e r b l a d d e r . .

C a n c e r l u n g . . . . C a n c e r l u n g . . . . C a n c e r l u n g . . . .

C a n c e r u t e r u s . . . . C a n c e r u t e r u s . . . .

R e t . C e l l s a r c o m a . . R e t . C e l l s a r c o m a . .

C a n c e r s k i n . . . . C a n c e r s k i n . . C a n c e r p r o s t a t e ' - . . C a n c e r l i v e r __ . . C a n c e r p a n c r e a s " . . C a n c e r t h r o a t . . . . C a n c e r f e m u r . . . . C a n c e r l a r y n x . . . . S a r c o m a b o n e . . S a r c o m a f e m u r . . S a r c o m a t e s t e s . . M a l i g n a n t l e s i o n s . . L e g l e s i o n s . . . . M o u t h g l a n d s . . . . ~ e e k g l a n d s . . . . H y s t e r e c t o m y . . . . H y s t e r e c t o m y . . . . O o p h o r e c t o m y . . M a e r o g l o b u l i n a e m i a . .

R N A

6-66 5 . 1 2 1-45

4 .40 4 .26 3.34

1.45 1.60

2 . 5 0 2.36

7 ' 3 6 2 . 5 4 2"03 3 ' 0 2 1 '36 3 '39 2'83 4"20 3"50 2.85 3"14 1"13 2"37 3"63 5-10 6.98 2 . 1 8 1-45 1.74

Non.Malignant Subjects. P e r i - a r t h r i t i s . . . . . . 3 -75 R h e u m a t o i d a r t h r i t i s . . . . 3 -45

Results

Plasma RNA was measured in 43 nm~nal subjects, and the frequency distribution of levels is plotted in Fig. 1. The median value is 3.43 mg./100 ml. and the mean 3.49 (-4-1.03) in a range 1.05-5.81 mg./100 ml. Table I lists the values obtained in 57 cases of malignancy, accord- ing to type, and in 2 cases of non-malignant disease. In Fig. 2 the

RIBONUCLEIC ACID LEVELS IN BLOOD OF CANCER PATIENTS 253

o

FtCUKE

- + + +

- + + + + +

- + + + + + +

- + + + + +

+ -I- + + + + - + + + + + + -I- 4- -I-

+ + + + + + + + + +

13.o I,.o Is.o I. FREQUENGY DISTRIBUTION OF PLASMA R N A (me/IOOmD

IN 43 BORMAL SUBJECTS.

O MYELOMA

+ OTHER CASES

0 + B

+ - +

+ - 0 +

0 +

0 + +

+ + + + 0 + + 0 -I- + + + 0 0 + + + + + + + + 0

+ + + + + + + + + + + + + -I" + + + + 0 4- -I-

Lo Lj o *,.o l,.o FDURs 2 . FIL~..QUENCY DISTRIBUTION OF PLASMA R N A(millOOml.) IN

CANCER CASES.

f requency distribution of levels in cancer is plotted. No difference in sexes was found.

To find the variat ion in levels with time, 2 normal and 2 melanoma sllbjccts were chosen and plasma RNA determinations made at intervals over a period of 30 days. The results are given in Fig. 3.

Discussion

There is a considerable degree of scatter in the normal distribution, but the mean value of 3.49 mg. RNA/100 ml. plasma is of the same order as that found by Mandel and ~r when their figures are converted f rom phosphate to nucleic acid concentrations. The levels in cancer cases are scat tered over a wider range than the normals, i.e. f rom 0.52-7.50 mg. mean 3.49 ~ .88 with one case of leukacmia giving a level of 14.08 mg. Whereas the normal values are concentrated around the mean figure, cancer levels occur more f requent ly above and below the normal mean.

The 2 cases of non-malignant disease gave values close to the normal mean. The wide range of normal values, and the fact that cancer

254 IRISH JOURNAL OF MEDICAL SCIENCE

RNA mg/1OOml

m / m ~ . m i . - --------- i

: / _ i\mm.____-m

0. v v

3 _ i/m-----m~m

alp B

m

I

FIGURE 3.

- n - - i - ! , . i

i I I I I I I t I I i 15

TIME (DAYS)

VARIATION IN PLASMA" R N A OVER A PERIOD OF 30 DAYS.

�9 NOR MAL �9 MELANOMA

I

levels are in the same range, indicates no significant difference, in general, f r om the normal levels.

I f type of mal ignancy is considered, i t is found tha t the plasma RN~A level m a y be in th~ uppe r or the lower pa r t of the range e.g. in 10 cases of advanced myel~ma one was as low as 0.52, while another gave a value of 7.30 rag. I n one case of leukaemia the level was exceptionally high at 14.08 mg., but 3 other cases of ]eukaemia were wi thin the normal range. Since all cases studied were advanced malignancies, the levels could not be correlated with the extent of mal ignancy. The serial determinat ions pe r fo rmed on two normal and two melanoma subjects showed tha t there was l i t t le var ia t ion in the normal individuals. I t is interest ing to note tha t in melanoma I - - s e e Fig. 3 - - the re was a notice- able increase in the level at a t ime when there was a breakdown of turnout. A t tha t t ime the p lasma adenosine deaminase (A.D.) was also being determined in this laboratory, for the same two melanoma cases (Smyth, 1963). I n melanoma case I there was also a marked rise of p lasma A.D. at this time. Melanoma case 2, in which there was a growth in t umour dur ing the per iod of serial determinations, showed no marked increase in plasma RNA, al though there was rise in p lasma A.D. at tha t time.

RIBONUCLEIC ACID LEVELS IN BLOOD OF CANCER PATIENTS 255

I t might be fu r the r noted tha t cancer plasmas which gave com- para t ive ly high values for RNA, e.g. 14.08 mg. in a case of ]eukaemia, did not show elevated adenosine deaminase.

Summary Levels of p lasma R N A were determined in 43 normal subjects and

in 57 cases of advanced mal ignan t disease. Normal levels were found to be within the range 1.0-5.8 mg. /100 ml. plasma. Cancer levels were found to be above and below the normal mean (3.49• in a sl ightly wider range than the normal. I n cancer eases the level could not be correlated with the na ture or the extent of malignancy, but in a case of melanoma there was a marked rise in RNA dur ing a period of t umour breakdown. This finding was in agreement with an increase in plasma adenosine deaminase observed at the same t ime by Dr. Smyth in this laboratory. A second case of melanoma, in which there was rise in p lasma adenosine deaminase dur ing tumour growth, showed no signifi- cant change in plasma RNA.

Acknowledgements

Thanks ate due to Professor E. J . Conway, F.1%.S., for discussion and direction. Blood samples were obtained through the courtesy of Professor 1%. A. Q. O'Meara

and Dr. 1~. Thornes. The author also acknowledges the tenure of a whole-time fellowship of the Medical

Research Council of Ireland.

Reference8

Greenstein, J . P. (1954). Biochem~t~J of Cancer, 2nd Edtn. N.Y. Ac~demic Press. Bodansky, O., in D. Greenberg and H. Harper (Eds.) (1960). Enzymes in Health and

Disease, Springfield III , C. C. Thomas. Hill, J. 1%. (1957}. J. Nat. Can. Inst., 18, 306. Straub, F., Stephanoek, O. and Acs, G. (1957). Biokimiya, 22 II. Filteau, G. (1961}. Laval Medical, 31, 648. Mandel, 1 ). and Metals, P. (1948). Compt. Rend. soc. biol., 14,2, 241. Tsanev, 1%. and Markov, G. {1960). Biochim. Biophys. Aeta. 42, 442. Chargaff, E. and Davidson, J . N. (Eds.} (1960). The Nucleic Acids, 2rid Edition. l~Yew

York, Academic Press, Vol. I II . Kirby, I-I. {1960}. Biochim. Biophysica. Aeta., 40, 193. Smyth, I-I. (1963}. Irish J . of Med. Science. 6th Series, 446, 59.