RESULTS AND ANALYSIS OF TUMOUR LEVELS OF ASCOR81C ACfD M, J. Moriarty, S. Mulgrew, J. R. M~lone and M. K. O'Connor

Saint Luke's Hospital, Dublin 6; Department of Physics, College of Technology, Kevin St., Dublin 8, and The Meath Hospital, Dublin 8. Stmlrnary UMOUR ascorbic acid estimations T w e r e carried out in 36 patients with malignant epithelial tumours (carcinomas). These levels were compared to surrounding normal tissue levels. The tumour levers were in all but one case markedly greater than those of the adjacent normal tissue (ratio of 2.4: 1). Tumour levels were noted to be higher in younger patients and in these who had no previous irradiation to that area. A reasonable correlation of plasma ascorbic acid levels to tumour levels was also obtained. The significance of these findJngs in relation to radiosensitivgy and to the administration of ascorbic acid was then discussed. Int~ducilon It has been known for some time that plasma and leucocyte levels of ascorbie acid are decreased in malignant disease (Wilson, 1974). In a previous study we have demonstrated that these blood levels are affected by a course of radiation therapy in such a way as to suggest marked release from the tumour when the latter has been damaged by treatment (KakeretaL, I97~). ~r~experimental animals the ascorbic acid content of turnout tissue is high (Musalin et el., 1936) and Kakar and Wilson (1974) have reported an accumulation of ascorbic acid in cancer tissue in human beings. In view of this we decided to investigate more fully tumour levels of ascorbic acid in a significant number of patients and attempt to correlate these results with various factors. This paper presents these results and analysis.

Patients arid Methods All the patients had their tumou rs re moved under general anaesthesia. Within 30 minutes of removal the macroscopic tumour was divided with the one piece going for histology and the other reserved for ascorbic acid estimation. The surrounding normat tissue was treated slmilarty, Le. one specJrnen to confirm normal histology and the other for ascorbic acid estimEttion. The tissues, then sent for aseorbic acid estimation, were cleared of surplus blood, weighed and cut into minute pieces. Two mls. of 5 per cent trichlroacetic acid was then added and with the aid of a glass rod the tissue was then emulsified as fully as possible It was then left at 4~ overnight. Twenty four hours later it was centrifuged at 3,060 r.p m for 10 rains., 1.5 m/s. of the supernatant was taken off and the ascorbic acid content then estimated by the dinitrophenylhydrazine procedure of Denson and Bowers (19,61). It must be stated that this method estimates both ascorbJc acid and dehydro-ascorbfc acid as the former is oxidized to the latter in this estimation. By tradition the term ascorbie acid only is used in this estimation and this will be adhered to in this paper. The result is expressed per 100 m9. weight tissue and therefore includes both interstitial and intra cellular levels. '

Results Tabte I indicates the overall resutts in the patients studied. As can be seen the bulk of turnouts studied were skin turnours with, however, a small range of other turnouts Histological examination showed that all the turnouts were car-

T U M O U B LEVELS OF A S B O R B I C ACID TABLE I Histology Patient

Age

Site

Tumor Suroundins Tissue Tissue Ascorbic Ascorbic Acid L e v i Acid Level /zgs/100 rag. pg8/100 mg

1

71

Skin - I~p

S q care.

5.0

2.6

2 3 4

58 75 22

Breast Skin - leg Skin~- nose

Adeno c a r e Sq. care. Sq, care.

11.6 4 167

2.2 1.5 12.6

5 6 7

62 47 52

Uterus - c e ~ i x Skin - lip Skin - c h e e k

Sq. care. Sq. care. Basal cell c a r e

7.6 3.3 17.9

8.1 33 7.9

8 9 10 11

64 57 62 75

Skin - ear Uterus - cervix Rectum Axilfa ry Gland

Sq. c a r e Sq. care. Adeno care, Sq. c a r e

208 8.7 2.4 3.2

5.5 24 1.3 1.1

/2

8g

Skin - l e g

Sq. c a r e

101

13 14 15 16

80 89 69 50

Skin - nose Skin - tempJe Skin - t h u m b Oropharynx

Sq. Sq. Sq Sq

care. care care care

4.7 5.7 13.1 42

53 25 2.8 6.2 24

17 18 19 20

74 41 85 80

Skin - leg Skin - ear Skin - leg B u c c a l cavity

Sq. Sq Sq Sq

carc care, care care

384 53.6 -23

63 8.7 6 10.9

21 22 2~

67 70 62

Skin - e a t S k i n - ear Skin 9 ear

Sq. c a r e S q care. S q carc.

14.9 18.2 10

4.3 9.8 3.5

24 25 26 27

63 81 7O 70

Skin Skin Skin Skin

Sq. Sq Sq. Sq.

care. carc care. care.

194 2.9 81 4.3

6 1.2 1.5 27

28 29 30

70 71 71

Skin - ear Skin - ear Skin - l e g

Sq. care. Sq. care. Sq care.

4.6 61 21

2.5 2.9 5.7

-ear - wrist - ear - hand

Mean 12.1 Mean 4 9 5 Ratio 2.4 : 1

cinomas. No sarcomas or lymphomas were available for examination. The ascorbic acid content of tumour tissue and surrounding normal tissue is presented a n d in a l l b u t o n e c a s e t h e t u r n o u r t i s s u e had a higher concentration of ascerbic

acid than the surrounding normal tissue w i t t a m e a n r a t i o n o f 2 . 4 : 1. S t a t i s t i c a l l y this is significant at the P
76

IRISH JOURNAL OF MEDICALSCIENCE turnouts would be necessary to answer fully this question. In a number of cases (11) it was possible to examine the relationship between )lasma ascorblc acid levels with

9

_~3o

I

<20

~

10

40 .

:!

:I

"~ ;

!~

30

so-so 61~7o 71-eo AGE - YEARS Fig.

"

,~20 this figure, there is a definite trend suggesting that the younger the patient the higher the ascorbic acid content of carcinomatous tissue, with a gradual fall in tumour levels as age increases. Patients in this series who developed a histoJogicelly proven recurrence in a previously irradiated area had their turnout asGorbic acid levels compared with turnouts which were not recurrent and in whom therefore no previous irradiation had been given to that area. These results are presented in Fig. 2 and it is noted that there is a significar~f difference between the two groups (P<0.0O5) indicating that prior irradiation )owers these recurrent tumeur tissue levels of ascorbJc acid. This, though not presented here, also app)ies to surrounding normal tissue ascerbic acid levels so that we can positively state that any tissue that has been irradiated is iow }n ascorbic acid. Histological appearances and site of turnours were also examined in relation to tumour ascorbic acid levels but no significant difference in tumour levels was found betweer~ the various types of histological features (e.g. differentiation) or between different sites (e.g. ear lesions compared with cheek lesions), It must be stated however that the bulk of lesions examined in this series were squamous carcinomas of the skin and a greater

?-i10 =

0

previous R T

no p r e y , c u e R/T

F~g.2 rartge of histoIogicaI Iypes and sites of turnout tissue and surrounding normal tissue eseorbie acid levels and this presented in F i g 3. No significant correlation of plasma ascerbic acid levels with normal tissue levels was found but a clear correlation of tumour ascorbic acid levels with pFasma levels does occur with co-efficient 0.87 omitting one point and 0.57 including all points. Lastly, in a number of patients the turnours were sufficiently large to enable us to take multiple sections from different areas of the tumour for ascorbic acid measurement and thereby assess the uniformity of ascorbic acid in these turnours, The results are shown in Tables II and Ill which also give the results of rnuh tipfe sections taken from surrounding normal tissue. The figures indicate a satisfactory degree of uniformity,

TUMOUR LEVELS OF ASCORBIC ACID r

TABLE Ill

~= m =

20

Adjacenl Noraml Tissues

~,~

"~

Sample*

:

-.-

20

E

10

mg %

Discussion

The data presented strong]y indicates that m a l i g n a n t epithelial t u m o u r s have a high concentration of ascorbic acid relative to s u r o u n d i n g normal t i s s u e This high t u m o u r s u r r o u n d i n g tissue ratio TABLE II Tumours

Sample*

1 g 3

A.

B

C.

D.

15t 1.7"

5.8 49

27 2.~

15 1,0

TAll figures are #0/100 mg. el tissue. A, B, C, D, are 4 tissues adjacent to tumours from different patients. '1, 2, are g sampJes taken from d0terent parts eom each tissue respeebvely

" 9

Plasma AA Fig. g

1 2

A

B

C

15.0t" 149 147

11,4 100 11.6

4.6 4.5 5.0

tAll ligures are /z0/100 mg of tissue. A, B, C, are 3 different tumours '1, 2, 3, sre 3 samples taken from different parts from eacb turnout respectively,

( 2 4 : 1) Could be due to genuinely high turnout revels of aseorbic acid Or a combination of both factors. Normal body tissue levels of ascorbic acid are reprod u c e d in Table IV (Geigy Documenta, 1970) a n d these figures c o m p a r e closely with the figures we have presented for s u r r o u n d i n g normal tissue. This therefore suggests that the normal tissue surrounda turnout is not necessarily low in ascotb i c acid. Our t u m o u r asoorbic acid levels are similar to leucocyte ascorbic acid levels and t h o u g h lower than organs noted for high ascorbic acid storage (e.g. adrenal g l a n d } these turneur leve[s are h i g h e r than expected from their tissue of origin, The relationship of t u m o u r ascorbic acid levels with plasma ascorbic acid levels and the close comparative figures of leucocyte ascorbio acid a n d turnout ascorbic acid levels m i g h t suggest that the raised turnout levels f o u n d in this series is due simply to increased vascularity with more blood in the t u m o u r compared to the s u r r o u n d i n g

TABLE fV Present Series Adjoining tissue Ttlmour tissue

---

Normal Levels 5.0 127'

Overall body CONG.of aseorbic acid beur ascorblc acid Adrenal ascorbio acid

*All units are expressed in #0/100 rag. of tissue

----

50* 12 4O

78

IRISH JOURNAL OF MEDICAL SCIENCE

normal tissue. This might be a factor in increasing t u r n o u t ascorbic acid levels but it probBhly is not the main or only reason as in o u r t e c h n i q u e for estimating ascorbic acid, all surplus b/ood is removed. Many of the t u m o u r s in this series (e.g. basal cell carcinomata} are certainly not vascular ones and the turno u r ascorbJc acid levels are stil/ raised in them. L~sgy, we have recenUy examined two b e n i g n tBmours (papi)loma, f)broma) and there was n o increase in ascorbic acid levels in these t u m o u r s relative to surrounding normal tigsue. It is not clear h o w turnouts come to have high ascorbic acid levels and what the exact role of ascorbic acid is in these cells. In ao vitro situation we (O'Connor et at., 1976) demonstrated a protect(re effect against radiation of increased ascorbic acid levels added to the media. We are presently p u r s u i n g this point in an in vivo situation and if this confirms the in vitro findings, than the raised turno u r tissue/surrounding tissue ratio of ascorbic acid m i g h t be an important factor d e t e r m i n i n g non-responsiveness to irradiation a n d attempts to c h a n g e this ratio might be considered (Miller and Sokoloff, 1955}. This whole area is of great interest a n d has possible important applications in this area of t u m o u r responsiveness to irradiation, ft is especially interesting in this regard that, as shown, tissues following irradiation are l o w in ascarbic acid a n d if this decrease o c c u r s d u r i n g ~ course of treatment it m i g h t partially e x p l a i n the effectiveness of fractionated radiotherapy rather than a single treatment. Our results as presented here are consis,eFt with t u m o u r tissue taking up ascorbic acid to an increased extent a n d this would appear to be related to plasma aseorbJe acid levels. In recent years C a m e r o n and P a u l i n g (1974a; 1974b) have advocated treating patients with ad-

v a n c e d m a l i g n a n c y with high doses of ascorbic acid. It is quite possible from this study that this increased intake m i g h t lead to increased plasma and turnout ascorbic acid levels. If ascorbic acid levels are raised by high intake, this m a y damage the t u m o u r tissue by saturation but of course it is also quite possible that it m a y stimulate t u m e u r growth. We are at present attempting to answer the question of what h a p p e n s to t u r n o u t and plasma ascorbic acid leve)s when o n e gives large doses of 8scorbic acid orally and these results when available m i g h t be a guide to the use or otherwise of ascorbic acid in m a l i g n a n t diseases. It is e pleasure to acknowledge the assistance given by Professor Wilson and Dr Kakar at the start of this work. I wish to thank Saint Luke's Cancer Research Fund for helping to make this work possible. References

Cameron, E. and Pauling, L. 1974a. The erthomolecular treatment of cancer. I. The role of ascorb}c acid in host resistance. Chem. ~iol. ~~e rao~o~s. 9 r ~T3. Cameron, E. and Campbell, A 19745 The or~homolecular treatment of cancer. Ik Clinical trial of high dosage ascorbic acid supplements in advanced human cancer. Chem. Biol. Interactions. 9, 985. DensJn, K W. and B0wers~ E F 1961. The determination of ascorbic acid in white blood cells. Cfin. Sci. 21, 157. Geigy Documenta. 1970. Ascorbic acid R- 490. Kakar, S. C. and Wilson, C. W. M. 1974. A~corbic acid metabolism in human cancer. Froc. Nu~r. Society. 33, ~19 A (19741, Kakah S. C., Wilson, C W. M. and Moriarty, M. J. 1975. Unpublished data. Miller, T. R. and Sokotoff, 8. 1955. A vitamin O free diet [n radiatlon therapy of malignant diseases. Am. d. Roentgen. 73, 472 MusalJn, R. R., Silver Slat5 a. King, C. E. and Wordward, G. E 1936 The uglisation and bioIogicaJ as~y o1 vit~mio C in tumour tissue. Am, J. Cancer. 27, 707. O'Connor, M. K., Malone, J. F., Moriarty, M. J. and Mulgrew, S, (1976). A radioprotective effect of vitamin C observed in Chinese hams,or ovary cells tn Press. Wilson, C W. M. 1974. Tissue saturation, metabelism and desaturation. Practigoner. 212, 481.