The determination of platinum in tissue of different human organs by means of neutron activation analysis 11sotope Division, Ris(21National Laboratory, Roskilde, and 2Department of Oncology, The Finsen Institute, University Hospital of Copenhagen, Copenhagen, Denmark
Key words neutron activation analysis - electrolysis - platinum distribution
Abstract. The aim of this study was to determine quantitatively the content of platinum of tissue samples from the autopsy ofpatients treated with cisplatin. Furthermore, it was important to determine the relationship between dosage, time and platinum concentrations in the tissues analyzed. It was demonstrated that radiochemical neutron activation analysis can be used for these studies because of its sensitivity and precision and a low detection limit for platinum (- 1 ng). Tissues of the following organs were analyzed for platinum: liver, kidney, testis, lung, pancreas and muscle. This study was perfomled at Riso National Laboratory, Isotope Division in cooperation with the University Hospital, Copenhagen.
cially when radiochemical separation is applied. When complete separation is achieved, the only significant interference comes from the second-order reaction of the naturally occurring gold in the sample matrix (double neutron capture): 197Au(n,y 198)Au(n,y) 199Au Correction for this interference is based on the counting ofa gold comparator for 24 h, so that the precision of the Pt determination for the tissue samples is not significantly affected.
Samples
Accepted for publication
July 16, 2001 Correspondence to Dr.B. Rietz Isotope Division,
Cisp latin-ci s-diamm inedichloroplatinum is one ofthe most used antineoplastic dmgs. It has been proven successful in the treatment of a number of malignant tumors, such as testicular carcinoma [Gregg et al. 1992, Jonson et al. 1988], head and neck tumors. Unf0l1unately, its use has also shown a number of chronic side effects, in particular nephro[Daugaard 1989], oto- [Laurell 1991] and neurotoxicity. Cisplatin is a heavy metal compound potentially accumulated in the different organs of the body [Cavanagh 1973]. In the actual study, quantitative measurements of plat in in different human tissue were carried out using radiochemical neutron activation analysis. The 199Audaughter ofl99Pt formed by the reaction: 19'1
Pt~
-
199
BUilding 202, P.O.B. 49,
19RPt(n,y)
DK-4000 Roskilde, Denmark bernd.
[email protected]
is a favorable indicator for the determination of platinum in biological material, espe-
All samples were post-mortal material. Legal mles according to guidelines approved by the regional Ethical Committee and The National Health Board were kept. Sampling of tissue specimens was carried out in connection with autopsy. In practice, the samples were cut with a sterile disposable scalpel (surgical steel blade II, single use, SwannMorten) with direct application in precleaned (demineralized water), pre-weighted and marked tight closing half-dram polyethylene vials (Lab Grade, Olympic Plastics Company, Inc., Los Angeles, Cf. 90016). The vials, containing the sample material, were weighted again, and the net "wet-weight" of the samples was calculated. All vials were frozen at once at -30°C.
Au The tissues were irradiated individually in the same weighted half-dram polyethylene
-+-u-~-
KlcNy
--..-
Testis
-D-Lung -X-P8llCRtllS
•• .••··MuscIe
100
200
300
400
OIly •• fter Tl'lIlItment Stop
Figure 1. Elimination of platinum from human tissues. All values normalized to 100 mg cisplatin doses/m2 body surface.
vials previously used for sample storage. Two half-dram polyvials, containing 0.02 mg of Au and 8 mg ofPt in 1 cm3 of2 molll HN03 (the Au and Pt standard, respectively) were placed in 2-dram polyethylene vials. All vials were heat-sealed, placed in separate aluminum irradiation containers and irradiated together for 6 h at 4.0 x 1017 n/m2s in the rotating activation facility of the DR 3 heavy-water moderated and cooled reactor operating at 10 MW. After decaying for about 45 h, the half-dram polyvials, containing the Au and Pt standards, were removed from the 2-dram polyethylene vials for counting. The samples were removed from the polyvials prior to radiochemical separation.
Reagents: hydrochloric acid (HCI), 37%, p.a.; nitric acid (HN03), 60%, p.a.; perchloric acid (HCI04), 60%, p.a. Au carrier: 0.200 g of gold wire (Advent, GB), 99.99%, dissolved in 10 cm3 of aqua regia. Platinum comparator: 0.080 g of platinum wire (Advent, GB), 99.99%, dissolved in 100 cm3 of aqua regia diluted 1 : 100 with 2 molll HN03· Gold comparator: 100 III of Au carrier, diluted to 100 cm3 with 2 molll HN03 in a volumetric glass flask. After shaking, 100 III of this solution was diluted to 100 cm3 with 2 mol/I HN03. All comparator solutions were stored in darkness and used within 1 week.
Digestion and separation After irradiation, complete decomposition of the irradiated material is achieved by digestion with a mixture of hydrochloric, nitric and perchlorie acids under strict temperature control in a modified Bethge digestion apparatus for 3 h. Before starting the digestion procedure, an inactive Au carrier solution with approximately 6 mg of Au is added to the sample. After digestion, gold is separated from the other radioactive species in the irradiated sample by electrolysis of the sample solution (I molll hydrochloric acid). Digestion and electrolysis procedure have been described before [Rietz et al. 1994].
All samples and standards were counted at a distance of 4.5 cm from a 60 cm3 y-X Ge detector, using 8,192 channels at a gain of 0.2 keY Ichannel. The detector has a resolution of 1.62 keY at 1,333 keY and a relative efficiency of 10%. The Au standard was counted for 24 h during the radiochemical separation of the sample and subsequently, the separated sample was counted for 24 h. Finally, the Pt standard was counted for I h.
Radiochemical yield The yield of the separation was determined by re-irradiation of the separated sample together with a reference containing the same amount of gold as was added to the sample. Irradiation for 10 s takes place in the pneumatic tube of the DR 3 reactor with a fluence rate of2 - 3 x 1017 n/m2s. Sample and reference were counted for 5 minutes at a distance of 17 cm from the detector.
The high sensitivity needed for the determination of platinum requires a long irradiation time at a high neutron flux density. However, the radiation decomposition ofbiological material limits the duration of the irradiation. Because the limit of detection for platinum is determined by the formation of
40
Rietz. Heydorn and Krarup-Hansen
Table 1.
Patient characteristic
Patient no.
Sex
1 2 3 4
Table 2.
Age
43 18 18 26
M M
F F
and cumulated doses of cisplatln chemotherapy.
Diagnosis
cisplatin mg/kg body weight
treatment (days)
197 364 457 53
5.1 11.9 15.2 4.4
3 93 137 28
testicular Cancer Sarcoma Liver cancer
Duration of cisplatin
Platinum content of tissue obtained from patients treated with the antineoplastic Cumulative cisplatin dose (mg/m2)
Treatment free period (days)
197
13
2
364
75
3
457
125
4
153
394
Patient no.
Cumul. dose of
Cumul. dose of cisplatin mg/m2 body surface
mg platinum/kg
197 2
364
13 75
3
457
125
4
153
394
13 75 125 394
compound post-mortal
56.00 14.00 11.50 99.25
cisplatin. tissue" (± SO)
Liver
Kidney
Testis
Lung
Pandcreas
Muscle
4.91 (0.16) 6.22 (0.21) 7.7 (0.25) 1.79 (0.06)
3.3 (0.11 ) 0.63 (0.02) 1.52 (0.05) 0.23 (0.01)
3.07 (0.10) 1.85 (0.06)
1.86 (0.22) 1.28 (0.04) 0.8 (0.03) 0.15 (0.006)
1.03 (0.04) 0.68' (0.02) 0.98 (0.03) 0.24 (0.008)
1.02 (0.04) 0.34 (0.02) 0.39 (0.01) 0.09 (0.004)
Table 3. "Platinum content of tissue obtained from patients treated with the antineoplastic of administered cisplatin. Patient CumUlative Treatment no. c1splatin dose free period (mg/m2) (days)
Cisplatin Start of freezing treatment free of tissues after period (days) death (hours)
compound cisplatin related to 100 mg doses
mg platlnumlkg postmortal tissue" x 100 mg cisplatin adm. (% of Pt content of all sample organstl) Liver
Kidney
Testis
Lung
Pancreas
Muscle
2.49 (40.0) 1.71 (68.1) 1.68 (67.0) 1.17 (68.8)
1.68 (27.3) 0.17 (6.8) 0.33 (13.3) 0.15 (5.9)
1.56
0.94 (15.3) 0.35 (13.9) 0.18 (7.2) 0.15 (5.9)
0.52 (8.5) 0.19 (7.6) 0.21 (8.4) 0.17 (10.0)
0.52 (8.5) 0.09 (3.6) 0.09 (3.6) 0.06 (3.5)
H 0.51
H
198Au, from which the I99Au indicator of Pt cannot be separated, an irradiation time of6 h at approximately 4 x 1017 n/m2s in the rotating facility ofthe DR 3 reactor was chosen as a compromise. Patient characteristics, doses of cisplatin per m2 body surface area and per kg body weight, treatment duration, the time from treatment stop to death and the time from death to the start Qf freezing the samples are shown in Table I. Surface area and body
Note
"fresh "wet"weight lIexctuding
testicular tissue
weight refer to the beginning of the cisplatin treatment. The concentrations of platinum per kg of fresh tissue in the different organs are shown in Table 2. The normalized value of platinum was calculated from the total content of platinum per 100 mg administered cumulative dose of cisplatin (mg platinum content of tissue/I 00 mg cisplatin/m2). The values are shown in Table 3. This Table shows also the distribution
Patient no.
1 2 3 4
Treatment free period in days
liver
Kidney
13 75 125 394
2.49 1.71 1.68 1.17
1.68 0.17 0.33 0.15
of platinum in percentage of all sample organs excluding the testicular tissue. The data of the cisplatin content of 4 patients are shown in Figure I in relation to the time from last treatment day to death. All organs showed high concentrations of platinum just after treatment stop. The content of platinum was very high especially in the liver tissue of all patients. Short time after treatment stop (day 13), a high content of platinum was found in the kidney of patient TN (1.68 mg platinum/kg kidney tissue x 100 mg administered cisplatin = 27.3% of all organs investigated). At day 75 and later, the content of platinum was reduced to 0.17 - 0.33 mglkg (6.8 - 13.3%) in the renal tissue quite parallel to the platinum content of other tissues such as lung and pancreas. Despite the initially high platinum concentrations found in kidney, a rapid decrease of these concentrations with time can be observed, and the rate of decline of the platinum content was the steepest decrement of all tissues. The renal blood flow combined with a larger urine production may explain the rapid elimination of platinum from the renal tissue. At any time, the overall platinum content was the highest in the liver and the concentration of platinum seems to increase relatively to the platinum content of the other tissues, e.g. lung, pancreas and muscle, tending to' be a chronic accumulation of platinum. The explanation of this phenomenon is based on the hepatic function, filtering the redistributed platinum from the blood, the second-pass effect. The platinum content of the testicular tissues was higher than that of the tissues of the lung, the pancreas and the muscle. This suggests that the testicular organ has an input of platinum, which is higher than the
mg platinumlk Testis
1.56 0.51
tissue Lung
0.94 0.35 0.18 0.15
Pancreas
Muscle
0.52 0.19 0.21 0.17
0.52 0.09 0.09 0.06
output rate. This accumulation of platinum may explain the decreased fertility years after treatment stop with cisplatin.
The radiochemical neutron activation analysis method described permits the simul-, taneous determination of Au and Pt in biological material with high accuracy and reasonable sensitivity. The analytical technique used in this investigation has provided quantitative information about the distribution of platinum among the different kinds of human tissues.
Cavanagh JB 1973 Peripheral neuropathy caused by chemical agents. Crit Rev Toxicol 365-417 Daugaard G 1989 Cisplatin nephrotoxicity: experimental and clinical studies. L'geforcningens Forlag, Copenhagcn, pp 1-15 Gregg RI¥. Molepo JM. Monpetit VJA. Mikael NZ. Redmond D. Gadia M 1992 Cisplatin neurotoxicity: the relationship between dosage, time and platinum concentration in neurologic tissues, and morphologic evidence of toxicity. J Clin Oncol 10: 795-803 Jonson R. Mattson S. UlIsgaard B 1988 A method for in vivo analysis of platinum after chemotherapy with cisplatin. Phys Med Bioi 33: 847-857 Laurell G 1991 Ototoxicity of the anticancer drug cisplatin. Clinical and experimental aspects. Almqvist and Wiksell Periodical Company, Stockholm Rietz B. Heydorn K. Krarup-Hansen A 1994 Determination of platinum by neutron activation analysis in nerve tissue from rats treated with cisplatin. Biological trace element research. Humana Press, Totowa, New Jersey, pp 343-350
