The Science of the Total Environment, 50 (1986) 55--63 55 Elsevier Science Publishers B.V., Amste rdam -- Printed in The Nether lands

P L A S M A A N D T I S S U E L E V E L S O F S O M E L A N T H A N I D E E L E M E N T S

I N M A L I G N A N T A N D N O N - M A L I G N A N T H U M A N T I S S U E S *

M A U R O ESPOSITO** and P A O L A COLLECCHI

Istituto Nazionale per la Ricerca sul Cancro, Genova (Italy)

SILVIO B R E R A and ENZO M O R A

Clinica Otorinolaringoiatrica " B " dell'UniversitY, Genova (Italy)

A M B R O G I O M A Z Z U C O T E L L I

Istituto di Chimica Generale dell'UniversitY, Genova (Italy)

M A U R I Z I O CUTOLO

Centro Reumatologico I.S.M.L, UniversitY, Genova (Italy)

MASSIMO O D D O N E

Dipartimento di Chimica Generale delrUniversit~, Pavia (Italy)

(Received March 9th, 1985; accepted October 3rd, 1985)

A B S T R A C T

Lan thanum (La) levels in plasma, in e ry th rocy te hemolysa te and in tissue f rom heal thy subjects and pat ients with laryngeal carc inoma were de termined by neut ron activation analysis. Plasma lan thanum levels were significantly higher in laryngeal carcinomas than in ei ther heal thy controls or in subjects suffering f rom localized inf lammat ion (e.g. epicondyl i t is of the e lbow) (p ~ 0 . 0 0 1 ) . The mean La concent ra t ion in malignant tissue samples was 57.5 + 7.2 ngg-1 ; the corresponding level in normal adjacent tissue f rom the same organ was 94.6 + 12.0 ngg -1 . This 61% decrease in the concent ra t ion of La in malignant tissues was highly significant ( p ~ 0.001). In pat ients with laryngeal carcinoma we did not observe any detectable level of lanthanum in e ry th rocy te hemo- lysate; the mean La e ry th rocy te hemolysa te level in heal thy controls and in pat ients suffering f rom localized in f lammatory condi t ion was 14.3 and 33.2 ng ml - 1, respectively.

Fur the r studies are in progress to evaluate whether or not this e lement can serve as a marker for diagnosis or prognosis in cancer.

*Presented at the Sixth In ternat ional Sympos ium on the Prevent ion and Detec t ion of Cancer, 26--29 November 1984, Vienna, Austria+ **All cor respondence should be sent to Dr M. Esposito, Is t i tuto Nazionale per la Ricerca sul Cancro, Viale Benede t to XV, 10 - - 16132 Genova, Italy.

0048-9697/86/$03 . 50 © 1986 Elsevier Science Publishers B.V.

56

INTRODUCTION

In recent years there has been much interest concerning the presence and the role played by trace metals in human tissues and in body fluids, and the manner in which concentrations may be altered by malignant and other diseases. The ions of the lanthanide group, or rare-earth elements, have a high affinity for calcium binding sites [1, 2] ; in particular, the lanthanum ion has been used in a variety of studies to investigate the action of Ca 2+- dependent mechanisms [3].

Lanthanides occur in only trace amounts in organisms and play no known biochemical role [1]; recently, our preliminary studies have suggested that endogenous plasma levels of lanthanides are significantly affected by inflam- matory conditions, and that local procaine therapy could significantly influence the plasma lanthanum concentration [4].

The principal purpose of the present study was to evaluate the levels of La and other lanthanides in plasma, erythrocyte hemolysate and in non- malignant and malignant human tissues.

EXPERIMENTAL

Subjects

Three groups of subjects were considered. Group I -- laryngeal carcinoma: 15 male patients, without known exposure to toxic amounts of La, aged 43 to 76 years (mean + SD: 66.8 + 11.2). No patient had received any type of therapy at the time of the evaluation. Group I I - localized inflammatory condition: eight male patients, aged 45 to 58 years (mean + SD: 52.7 + 5.0), suffering from epicondylitis of the elbow; no t reatment with analgesic, steroidal or non-steroidal anti-inflammatory drugs had been given for at least 15 days. The longer half-life for non-steroidal anti-inflammatory drugs and analgesic is 36 h, and after 15 days no trace of the parent drug or its metabolites is detectable [5] . Group I I I - controls: 30 apparently healthy male individuals, aged 30 to 70 years (mean + SD: 54.6 + 18.0) were studied in order to obtain reference values for basal lanthanum levels in plasma and in erythrocyte hemolysate samples. All groups had a normal nutritional status and no or minimal history of alcohol intake. Smoking history was variable.

Nutritional and haematic status was evaluated by the following serum parameters: hematocrit , hemoglobin, ferritin, total iron, transferrin, cerulo- plasmin, total protein, albumin, a- and/3-globulin, and lipidic status. Cancer patients weighing 25% less than their usual body weight were excluded from this study.

Blood samples

Fasting blood samples (10ml) were collected, between 7 and 9 a.m. with plastic syringes, then transferred to plastic tubes containing 50pl

57

of K3-ethylenediaminetetracetic acid (K3-EDTA, Sclavo, Lot. No. 81800) as an anticoagulant. For all cancer patients the blood samples were collected on the morning of surgery. Plasma was separated by centrifugation at 2000g for 15 min; the cells were washed with isotonic saline solution, and cell lysis was carried out by adding an equal volume of cold distilled water.

Tissue samples

Histologically non-malignant and malignant human laryngeal tissues were obtained from the same individual at surgery and designated as "paired samples". Great care was taken to avoid contamination of samples during either collection or storage prior to analysis and, in order to minimize airborne contamination, all samples were kept inside a dust~free cabinet installed in a clean room [6--8]. TABLE 1 E X P E R I M E N T A L

Sampling-standard reference rnoteriols

{ Neutron octivotion I

Decay of irradiated samples

I Radlochemical seporotionl

1 week; thermol neutron f lux: 8x10 -12n cm - 2 s -1

2-3 doys

Mineralization (12 M HCI/14 T HN03)

JO.5 M HC I +0.01 M H2C204 I 2.0 M HNO 3

16.0 M HCI I

Sc,Co~Fe with 0.5 M HCI+O.02 hf H2C204

Interfering alcaline metals and As wi th 2,0 M HNO 3

I I

u

~0 ^ U

an E u

~ 7 " J~

0

+

dryness taken-up 0.5 M HCI (known amounts of lanthanides to be determined were added as carr iers)

Lanthonides with 6,0 M HCL $

I c~nt ing of r~:fic~ctivity (gamma-ray spectrometry)

58

Elements studied and method of assay

Lan thanum, Ce, Yb and Lu were de te rmined by radiochemical neu t ron act ivat ion analysis (KNAA). A nitric acid solut ion of all the rare-earth e lements s tudied was used as reference standard. Al iquots of this solut ion were irradiated toge ther with the samples and s tandard reference materials (NBS Orchard Leaves, NBS Bovine Liver) for 1 week in a thermal flux o f abou t 8 x 10 '2 n c m -2 s -1 Usually, 2 m l of p lasma or e r y t h r o c y t e hemo- lysate and be tween 100 and 3 0 0 m g of tissue sample were inserted into quar tz vials for irradiation. The choice of the cor rec t nuclear react ion was made according to Meloni et al. [ 10] . The details of the analyt ical p rocedure are given in Tables 1--3; induced radioact ivi ty was measured by gamma-ray spec t romet ry , using an 18% eff iciency Ge(Li) de t ec to r coupled to an analyzer- c o m p u t e r system. Af te r radiochemical separat ion and count ing, the samples, the s tandard materials and a lanthanide carrier solut ion were re-irradiated for 4 0 m i n at a thermal f lux of abou t 1 × 1012 n c m -2 s - ' to evaluate the chemical yield of the p rocedure (yield ob ta ined: 90 + 4%).

TABLE 2 NUCLEAR DATA OF LANTHANIDES NEUTRON ACTIVATION ANALYSIS

DETERMINED BY RADIOCHEMICAL

Element Abundance Activation Produced Half-life of stable cross-section radionuclide (days) nuclide (%) (barn)

Measured gamma-ray energy (keV) and relative intensities

La 99.9 8.9 140-La 1.68 1595 (100), 487 (48) Ce 88.48 0.6 141-Ce 32.5 145 (100) Yb 0.14 11000 169-Yb 32.6 64(100), 198 (85) Lu 2.6 2100 177-Lu 6.75 208 (100)

TABLE 3 SENSITIVITIES FOR LANTHANIDES NEUTRON ACTIVATION ANALYSIS

DETERMINED BY RADIOCHEMICAL

Element Radionuclide Measured gamma ray Sensitivitya(ng) used (keV)

La 140-La 1595 0.200 Ce 141-Ce 145 0.100 Yb 169-Yb 198 0.020 Lu 177-Lu 208 0.004

aEvaluated under the experimental conditions described in this paper.

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Statistical methods

T h e t w o s a m p l e " t " t e s t ( c o m p a r i s o n of t w o means , u n p a i r e d case) was u s e d to t e s t t h e s ign i f i cance o f d i f f e r ences in m e a n p l a s m a a n d e r y t h r o c y t e h e m o l y s a t e c o n c e n t r a t i o n s o f all s u b j e c t g roups . T i ssue d a t a o b t a i n e d f r o m p a t i e n t s w i t h l a r y n g e a l c a r c i n o m a were s t a t i s t i ca l ly a n a l y z e d w i t h t he p a i r e d " t " tes t .

RESULTS

T h e l a n t h a n i d e c o n t e n t of t he s t a n d a r d r e f e r e n c e ma te r i a l , NBS O r c h a r d L e a v e s - - N B S B o v i n e Liver, c o m p a r e d w i t h l i t e r a t u r e va lues [9] is r e p o r t e d in

TABLE 4 LANTHANIDE CONTENT (pg g- 1 dry wt.) OF STANDARD REFERENCE MATERIALS

Element Orchard leaves NBS-SRM 1571 Bovine Liver NBS-SRM 1577

This work Gladney [9 ] This work Gladney [9 ] (pgg-~ +SD) (#gg-~ +SD)

La 1.4100 + 0.0200 1.200 0.0310 + 0.0040 0.028 Ce 1.1800 + 0.0300 1.000 0.0510 +- 0.0050 0.046 Yb 0.0290 + 0.0050 0.027 0.0028 + 0.0006 0.005--0.830 Lu 0.0086 + 0.0003 0.0061--0.010 0.0064 + 0.0007 ~ 0.001

TABLE 5 PLASMA CONCENTRATIONS (ngml - l ) OF LANTHANUM, CERIUM, LUTETIUM AND YTTERBIUM IN PATIENTS WITH LARYNGEAL CARCINOMA

Patient La Ce Yb Lu

1 71.0 56.0 6.7 0.3 2 69.0 62.0 6.9 0.4 3 .73.0 61.0 6.6 0.4 4 67.0 57.0 6.7 0.4 5 60.0 51.0 6.1 0.3 6 81.0 71.0 6.7 0.3 7 69.0 58.0 6.9 0.3 8 42.1 47.3 3.1 0.5 9 75.0 60.0 5.7 0.4

10 63.5 57.2 6.8 0.4 11 70.0 54.0 6.6 0.3 12 65.0 68.0 6.2 0.5 13 68.2 63.9 7.0 0.4 14 79.0 60.0 6.7 0.3 15 47.0 45.0 5.5 0.3

Mean 66.6 58.1 6.3 0.4 SD 10.5 7.0 1.0 0.1

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Table 4. Table 5 shows plasma lanthanides' concentrations in patients with laryngeal carcinoma. The results of plasma and erythrocyte hemolysate La levels for all patient groups are shown in Table 6.

Mean plasma La levels were significantly higher in the group with laryngeal carcinoma ( 6 6 . 6 + 1 0 . 7 n g m l - ' ) than in either the inflammatory group ( 1 3 . 8 + 4 . 5 n g m 1 - 1 ) or the control group ( 5 . 5 + 1 . 2 n g m 1 - 1 ) ( p ~ 0 . 0 0 1 laryngeal carcinoma group versus either of the other two groups).

Lanthanum levels in erythrocyte hemolysate were not detected (levels below detect ion limit by this method) in patients with laryngeal carcinoma, while erythrocyte hemolysate La levels in the inflammatory group revealed a significant increase when compared with normal subjects.

Lanthanide concentrations in malignant and in non-malignant laryngeal tissues are reported in Table 7. When a comparison was made between histologically non-malignant laryngeal and malignant laryngeal tissues from the same patient, the La levels were found to be significantly lower in the tumor sample (p ~ 0.001); whereas a wide range of noticeable variability in concentration between non-malignant and malignant tissues existed for the other lanthanides (Table 8).

DISCUSSION

Mean La concentrations in plasma and erythrocyte hemolysate of patients with laryngeal carcinoma and in patients suffering from localized inflam- matory condition were significantly different from those detected in normal subjects.

In the literature a wide range of trace element concentrations in human tissue are reported [11--13] . In this study, to overcome the problem due to the differences in age, sex, diet, and other genetic and environmental factors,

T A B L E 6 MEAN PLASMA AND E R Y T H R O C Y T E H E M O L Y S A T E LEVELS OF L A N T H A N U M IN N O R M A L SUBJECTS AND IN PATIENTS WITH I N F L A M M A T O R Y CONDITION (EPICONDYLITIS) OR L A R Y N G E A L C A R C I N O M A

Lanthanum (ng m1-1)

Plasma Ery th rocy te hemolysa te

State a N I b M b, c N I b M Number of subjects 30 8 15 9 8 15 Mean 5.5 13.8 66.6 14.3 33.2 ND d Standard deviat ion 1.2 4.5 10.66 5.0 4.1

aN = normal subjects; I = in f lammatory condi t ion; M = laryngeal carcinoma. bp ~ 0.001 compared wi th normal subjects (Student ' s " t " test). Cp ~ 0.001 compared with in f lammatory condi t ion (S tudent ' s " t " test). dND = no t detected.

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TABLE 7

LANTHANUM, CERIUM, YTTERBIUM AND LUTETIUM CONCENTRATIONS (ng g - l dry weight) IN MALIGNANT AND NON-MALIGNANT HUMAN L A R Y N G E A L TISSUE

Pat ient Tissue a La Ce Yb Lu

1 N 75 34 7.3 0.6 M 49 81 4.1 0.4

2 N ND b ND ND ND M 51 91 3.9 0.3

3 N 91 41 8.1 0.5 M 56 39 6.3 0.6

4 N 101 91 9.2 0.6 M 57 49 10.1 0.9

5 N 103 94 11.6 0.4 M 59 61 11.2 0.6

6 N 103 89 13.8 0.9 M 61 71 12.3 0.9

7 N ND ND ND ND M 71 88 13.1 0.7

8 N 92 71 9.3 0.7 M 59 52 6.2 0.6

9 N 95 89 8.0 0.6 M 55 50 4.0 0.5

10 N 98 56 8.3 0.5 M 53 66 12.7 0.7

11 N 105 90 12.5 0.8 M 70 80 7.9 0.9

12 N 108 92 11.2 0.9 M 55 42 11.6 0.3

13 N 95 61 9.0 0.6 M 68 63 6.5 0.8

14 N 69 45 6.5 0.6 M 50 70 10.0 0.4

15 N ND ND ND ND M 49 57 8.0 0.8

N mean 94.6 71.1 9.6 0.6 SD 11.8 22.6 2.2 0.2 M mean 57.5 64.0 8.5 0.6 SD 7.3 16.2 3.3 0.2

aN = non-mal ignant tissue; M -- mal ignant tissue. bND -- no t de te rmined .

both malignant and non-malignant laryngeal tissues from the same individual were compared [12,14]. From our findings, there were significant differ- ences only for La (mean difference, non-mal ignant- -mal ignant tissue: + 36.9 ngg -1 dry wt, p ~ 0.001).

Studies by Amellal and Landry [15] suggest that lanthanides at a con- centration of 10 -s to 2 × 10 -4 M are useful not only for inhibiting cellular calcium influx, as currently claimed, but also as probes of intracellular

62

T A B L E 8

L A N T H A N I D E C O N T E N T ( n g g - ' d ry weight ) OF THE N O N - M A L I G N A N T LARYN- G E A L T I S S U E C O M P A R E D WITH M A L I G N A N T L A R Y N G E A L TISSUE O F THE SAME PATIENT, EACH P A T I E N T A C T I N G AS HIS OWN C O N T R O L

La Ce Yb Lu

Mean n o n - m a l i g n a n t laryngeal t issue 94. 6 71.1 9.6 0. 6

To ta l range n o n - m a l i g n a n t laryngeal t issue 69 - -108 34- -94 6 .5- -13 .8 0 .4- -0 .9

Mean ma l ignan t laryngeal t issue 57.5 64.0 8.5 0.6

To ta l range ma l ignan t laryngeal t issue 4 9 - 7 1 3 9 - 9 1 3 . 9 - 1 2 . 7 0. 3--0. 9

Mean d i f fe rence non- m a l i g n a n t - - ma l ignan t t issue + 36.9 % 35.1 + 1.0 + 0.01

Signif icance of paired " t " test, p < 0 .001 0.20 0 .20 0 .30

NS a NS NS

aNS ---- no t significant.

calcium binding protein. Other studies by E1-Fakahany et al. [16] show that pharmacological intervention could affect La binding to the membranes of murine neuroblastoma cells; indeed, the La binding observed after cells had been incubated with 10 -3M LaC13 was significantly reduced when the cells were pretreated, for 30min at 37°C, with 10 -7 M atropine, 10 -3M carbachol, 2 x 10 -7 M phenoxybenzamine or 5 × 10 -6 M dibenamine.

On the basis of these findings, one would predict La to have biological activity, notwithstanding its normal inability to penetrate the cellular membrane of living cells [1].

In addition, our results suggest that endogenous levels of La are signifi- cantly affected either by inflammatory or by cancerous (Laryngeal Carcin- oma) conditions.

Medical studies on the lanthanides are incomplete [17] ; in view of these data, however, we believe that investigations on the basic mechanism of the observed La alterations in laryngeal carcinoma and in inflammatory con- ditions might be clinically useful.

A C K N O W L E D G E M E N T S

We thank Dr Mattia Rocco for the revision of the manuscript and Ms Susanna Caprile for preparing the typescript.

The main part of the analytical work was performed by means of an RNAA technique at the University Research Reactor, Pavia, Italy.

63

Th i s w o r k was s u p p o r t e d , in pa r t , b y t he N a t i o n a l Resea rch C o u n c i l (CNR, R o m e , I t a ly ) w i t h i n t h e " P r o g e t t o F i n a l i z z a t o : P r e v e n t i v e a n d R e h a b i l i t a t i v e Medic ine . S o t t o p r o g e t t o SP8: C o n t r o l o f P a i n " , C o n t r a c t No. 8 4 . 0 2 3 7 1 . 5 6 .

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