The metabolism of nickel carbonyl-14C

TOXICOLOGYANDAPPLIEDPHARMACOLOCY

The

15,295-303(1969)

Metabolism

of Nickel

KAZIMIERZ ~.KASPRZAK~AND

CarbonylJ4C1

F. WILLIAM SUNDERMA~T,JR.

Department of Laboratory Medicine, University of Connecticut School of Medicine, Hartford, Connecticut 06112 Received November 21, 1968

The Metabolism of Nickel Carbonyl-*4C. KASPRZAK, KAZIMIERZ S., and SUNDERMAN, F. WILLIAM, JR. (1969). Toxicol. Appl. Pharmacol. 15, 295-303.The metabolicfate of the carbonyl moiety of nickel carbonyl was investigatedin rats after intravenousinjection of 1 LD50 of Ni(t4C0)4 or 63Ni(C0)4.It wasfound that 36% of the administerednickel carbonyl was exhaledwithout metabolic alteration within 6 hours after injection. Most of the remaining Ni(CO)d underwent intracellular decomposition and oxidation to Ni2+ and CO. The carbon monoxide, which was gradually releasedfrom Ni(CO),, becameboundto hemoglobin,reachinga maximum saturation of 35% 2 hours after injection. Within 6 hours after injection, 49% of administeredt4C radioactivity wasexhaledas “CO. Only 1.1% of the administeredi4C radioacitvity wasexhaledast4COz.Lessthan 1% of the administeredi4C radioactivity wasexcretedin urine during 24 hours after injection of Ni(14C0)4. Investigations in our laboratory have partially elucidated the biochemical and pathologic mechanismsof nickel carbonyl toxicity (seereferencesin Sunderman et al., 1968). In a previous study (Sunderman and Selin, 1968), the distribution and excretion of 6sNi were measured in rats after administration of a 6sNi(C0)4 LD50, As a continuation of this study, we have undertaken the present investigation of the metabolic fate of the carbonyl moiety of nickel carbonyl. MATERIALS

AND METHODS

The experimental animals were 130male rats of the Sprague-Dawley strain weighing approximately 200 g. The rats were fasted for 24 hours before the administration of nickel carbonyl. The nickel carbonyl-14C 3 in LD50 (5 pl/lOO g) was injected into a tail vein by meansof a Hamilton microsyringe (Hackett and Sunderman, 1967, 1968). The specific activity of the Ni(14C0)4 was 0.01 mCi/ml. The metabolism chamber (Fig. 1, C) described by Sunderman and Selin (1968) was usedfor measurementsof i4C in excreta. The rat wasplaced in the metabolism chamber within 5-10 set after the injection of Ni(14C0)4. Urine was collected in a side-arm test 1Supported by U.S. Public Health ServiceResearchGrant (National Cancer Institute) CA-11250-01;by AmericanCancerSocietyGrant E374C;andby Atomic EnergyCommission Grant AT(3O-1)4051. z Presentaddress: Departmentof PathologicalAnatomy,MedicalAcademy,Prqbyszewskiego 49, Poznan,Poland. 3Synthesized by Mr. Karl Amlauer,IsotopeProductsLaboratories, LosAngeles,California. 295

296

KASPRZAK

AND

SUNDERMAN

tube (Fig. 1, B) and was prepared for liquid scintillation counting by the method of Hansen and Bush (1967). Exhaled 14C was collected by a trapping assembly (Fig. 1, D to I) based upon the techniques of McCullough and co-workers (1947) and Baggiolini and Bickel (1966). The expired air was bubbled through ethanolamine in the first 2 scintillation vials (Fig. 1, D and E) in order to trap 14C02. The expired air was then bubbled through concentrated sulfuric acid (Fig. 1, F), in order to trap any ethanolamine which evaporated from vials D and E, and to remove water vapor. The expired air was then passed through a hot column of red mercuric oxide (Fig. 1, G), in order to oxidize 14CO and Ni(14C0)4 to 14C02. The 14C02 thus generated was trapped in 2 scintillation vials (Fig. 1, H and I) which contained ethanolamine in methanol (20 % v/v). The

FIG. 1. Apparatus for trapping 1% after injection of Ni(l%0)4 (see Sunderman and Selin, 1968). A: Detector probe of mass flowmeter (Matheson Co., East Rutherford, New Jersey), used to monotor the rate of air flow. Air enters the apparatus via the inlet tube on the left side of the detector probe (arrow). B: Side-arm test tube to collect urine. C: Metabolism chamber. D and E: Scintillation vials, each containing 2.5 ml of ethanolamine. F: Rinsing tube, containing 20 ml of concentrated sulfuric acid. 0: Pyrex glass pipe containing red HgO. The glass pipe (diameter = 1 cm, length = 14 cm, length of HgO column = 10 cm) is heated electrically by a Nichrome heating tape, and adjusted to 200” by regulation of applied voltage through a variable transformer. H and I: Scintillation vials, each containing 10 ml of 20% (v/v) ethanolamine in methanol. The outlet tube from the last vial is connected (arrow) to a vacuum pump, and the air flow is maintained at 250 ml/min.

expired air was sampled for 0.5, 1, 2, 3, 4, 5, and 6 hours after the injection. At the conclusion of each collection period, the scintillation vials were rapidly replaced by fresh vials. The volumes in the scintillation vials were adjusted to 10 ml by addition of methanol, and 10 ml of scintillation fluid [3.8 g of 2,2,Sdiphenyloxazole (PPO) and 0.2 g of dimethyldiphenyloxazolylbenzene (dimethyl-POPOP) per liter of toluene] was added to each vial. In another series of experiments, rats were exsanguinated by cardiac puncture at 0.25, 1, 2, 3, 4, 5, 6, 9, and 24 hours after injection of Ni(14C0)4. Carbon monoxide saturation of hemoglobin was measured by the Dubowski method (1964). Hematocrits were measured in heparinized capillary tubes with an International microhematocrit centrifuge. The WZ activity in whole blood or serum was measured by means of the apparatus diagrammed in Fig. 1, modified by replacing the metabolism chamber (C) by a 50-ml Erlenmeyer flask. The Erlenmeyer flask, which served as a reaction vessel,

METABOLISM

OF NICKEL

CARBONYL-14C

297

was fitted with inlet and outlet tubes and was placed upon a magnetic stirring apparatus. The outlet tube was connected to the train of scintillation vials. Whole blood or serum (1.5 ml) wasplaced in the reaction vessel. A potassium ferricyanide reagent was prepared just before use by mixing equal volumes of: (a) citrate buffer, 0.1 M, pH 5.0; and (b) a solution containing 48 g of KJFe (CN)(, and 1 g of saponin per 100 ml of water. The ferricyanide reagent (10 ml) was added to the reaction vessel, and the evolved gases were flushed out by a stream of air for 2 hours and were trapped as previously described. In order to standardize the technique for trapping i4C, known amounts of Ni(14C0)4, 14C0, and i4C02 were added to the reaction vessel or the metabolism chamber and collected. The recovery in vials H and I of 1% from Ni(14C0)4 averaged 95 % (SE f 5). Only 0.7 & 0.2 % of r4C from Ni(i4C0)4 was recovered in the first two ethanolamine traps (vials D and E). The recovery in vials H and I of 14C from r4C0 averaged 94 f 4 % and negligible r4C from r4CO was recovered in vials D and E. The recovery in vials D and E of i4C from i4C02 averaged 86 f lx, and 1.7 f 0.2 % of i4C from r4C02 was recovered in vials H and I. Experimentswith 63Ni(C0)4showed that nickel carbonyl passed through the ethanolamine and sulfuric acid traps (D, E, and F) without detectable decomposition. The 63Ni was deposited on the mercuric oxide just after entering the oxidizing pipe. The red mercuric oxide did not undergo any diminution of oxidation efficiency after 10 successive experiments. Nevertheless, the mercuric oxide was replaced after every 4 or 5 experiments. Water vapour from expired air did not have any detectable effect upon the efficiency or reproducibility of the trapping procedure. Previous measurements (Sunderman and Selin, 1968) of 63Ni exhalation following intravenous injection of 63Ni(C0)4 were repeated during the present investigation. The specific activity of the 63Ni(C0)4 was 0.16 mCi/ml. Exhaled 63Ni was collected as 63Ni12 by bubbling the expired air through a series of 3 scintillation vials, each containing 10 ml of a solution of iodine in methanol (lx, w/v). Excess iodine was decolorized by reduction with an aqueous solution of sodium thiosulfate [0.5 ml of 20 % (w/v) Na2S203.5H20 per vial]. The recovery of 63Ni following addition of known amounts of 63Ni(C0)4 to the rat chamber averaged 92 % (SE & 3). A Beckman model CPM-100 liquid scintillation spectrometer was employed for counting r4C or 63Ni within an error of (i2 SD) of 1%. The counting efficiency of each sample was determined by addition of 20 ~1 of an internal standard of tolueneJ4C (5.26 x 10s dpm/ml) or 63Ni(C0)4 (9.67 x 103 dpm/ml). After correction for counting efficiency and trapping efficiency, the amounts of 14C or 63Ni were expressed as a percentage of the original dose of Ni(C0)4. RESULTS Exhalation of I‘T and63Ni Measurements of i‘+C and 63Ni in expired air after intravenous injections of Ni(14C0)4 or 63Ni(C0)4 are summarized in Table 1. The exhalation of 1% was consistently greater than 63Ni. By the end of the sixth hour after injection, a mean of 86% of the administered 14C was recovered in expired air, compared with 36% of administered 63Ni. 4 During this 6-hour period, the exhalation of i4C as 14COz amounted to 1.1% of 4 The corresponding mean value for exhalation of 63Ni in previously reported experiments (Sunderman and Selin, 1968) was 38% (SE + 3).

298

KASPRZAK

AND

SUNDERMAN

the administered dose. Nickel in expired air has previously been shown by gas chromatography to be present only in the form of Ni(C0)4 (Sunderman et al., 1968). Therefore, the proportion of the administered 1% activity found in expired air was distributed as follows: 1% as W02; 36 % as Ni(14C0)4, and 49 % as 14C0. The rates of exhalation of total 14C, 63Ni(C0)4, and ‘4CO (by difference) are plotted on a semilogarithmic scale in Fig. 2. TABLE 1 EXHALATION INJECTION

OF 1% AND OF RADIOACTIVE

63Ni AFTER Ni(C0)4

Cumulative exhalation (% of dose”vb) Hours after injection 0.5 1 2 3 4 5 6

1%

63Ni

30* 11 46 zt 10” 67 dc7 80 i 8* 80 rt 5 83 1-4 86 * 4’

16zt4 25 i 5 33 *2 35i I 36 xt 1 36zk 1 36 -c 1

rl Intravenous injection of Ni (14CO)4or 63Ni(C0)4 in dosageof 5 $/lo0 g. b Mean & SE; N = 4. c 14C excretedas 14C02= 0.46 * 0.05°/0of dose. * 14C excretedas 14CO2= 0.89 f 0.03% of dose. e 14C excretedas 14CO2= 1 .lO i O.OS’A of dose. Urinary Excretion

qf 1% and 63Ni

During the 24 hours after injection of Ni(14C0)4, less than 1% of administered 1% was excreted in urine. In contrast, during the same period after injection of 63Ni(C0)4, 27 % (SE f 2) of administered 63Ni was excreted in urine (Sunderman and Selin, 1968). Measurements

of 14C and 63Ni in Blood

As indicated in Table 2, an average of 10.4 % of administered 14C was present in the blood volume 1 hour after injection of Ni(14C0)4. Thereafter, the 1% within the vascular space diminished rapidly, reaching 0.02% of the administered dose 24 hours after injection. During the entire period from 1 to 24 hours, the proportion of blood 1% which was found within the erythrocytes remained essentially constant, averaging 95.4 % (SE f 1.6, N = 20). In comparison, an average of 6.5. % of administered 63Ni was present in the blood volume 1 hour after injection of 63Ni(CO)4. Thereafter, the 63Ni in the blood diminished very slowly, reaching 1.9 % of the administered dose at 24 hours. During this period there was a translocation of 63Ni from erythrocytes to serum. The proportion of blood 63Ni in the erythrocytes was 48 % (SE k 4.5) at 1 hour, and 8 $: 0.3 ‘A at 6 hours (Sunderman and Selin, 1968). The translocation of 63Ni from

METABOLISM

OF NICKEL

Hours

299

CARBONYL-14C

after

injection

of

Ni(C0)4

FIG. 2. Rates of exhalation of total l4C (x--x), 63Ni(C0)4, (O-O), and i4C04 (- - -) after injection of radioactive nickel carbonyl. Each point for total 14C and 6sNi(C0)4 represents the mean + SE (N = 4). The curve for t4C0 exhalation is computed as the difference between the rates of exhalation of total 14C and 6sNi(C0)4.

TABLE

2

~~CAND QNi INBLOODA~RINJECTIONOFRADIOACTIVE

Ni(C0)4

Percent of dosein blood volumes-c Hours after injection 1 3 6 9 24

14C 10.4 8.0 4.4 1.2 0.02

i 0.Y h 0.8 z??0.5’ rto.3 * 0.02f

63Ni 6.5 6.1 5.5 5.0 1.9

i 1.0 * 0.9 i 0.8 zk 0.8 37 0.8

a Intravenous injection of Ni(i4C0)4 or 63Ni(C0)4 in dosage of 5 /11/100 g. * Mean f SE ; N = 4 for 14C and 5 for 63Ni. c Assuming blood volume = 6.4 ml/l00 g (Wang, 1959). d Percent of blood i4C present as l4CO2 = 0.2 f 0.07. ’ Percent of blood 14C present as 14COa = 2.0 i 0.8. ’ Percent of blood 14C present as t4C02 = 13.0 & 6.

300

KASPRZAK

AND

SUNDERMAN

erythrocytes to serum is shown graphically in Fig. 3, which furnishes new data for the nickel partition 2, 3, 5, and 7 hours after injection of Ni(C0)4, together with the data at 1 and 6 hours that were previously reported (Sunderman and Selin, 1968).

9T % 2l ,u

‘O86-

-x-

_e--

4-

EL

-

:

P’ -0 B P

.r," 2

total I

-*--

__--

blood 63Ni ”

___---

-x

\

serwn63Ni

< \

2-

l0.80.6-

Od-

Hours

FIG. 3. Contents of 63Ni in volume (- - -) after injection 5 determinations. Each point for serum 63Ni is computed erythrocyte volumes.

after

injection

of “3Ni(CO).,

whole blood volume (x---x), erythrocyte volume (O--O), and serum of 63Ni(CO)+ Each point for whole blood 63Ni represents the mean of for erythrocyte 63Ni represents the mean of 3 determinations. Thecurve as the difference between the contents of 63Ni in the whole blood and

Measurementsof Carbon Monoxide Hemoglobin

Measurements of carbon monoxide saturation of hemoglobin are illustrated in Fig. 4. The CO saturation reached a peak of approximately 35 % during the second hour after injection of-Ni(C0)4. Thereafter, the CO saturation decreased exponentially with a T,/, of 90 min. As shown in Fig. 4, the curve of CO saturation parallels the dotted-line curve, which represents the exhalation rate of 14CO previously derived (Fig. 1). DISCUSSION Nickel tetracarbonyl, Ni(C0)4, is a colorless, volatile and flammable liquid (melting point: -19.2”, boiling point: 43.2”), which is miscible in all proportions with most organic solvents (Matheson Gas Data Book, 1966). Nickel carbonyl is slightly soluble in water (18 mg/lOO g of water at 9.8’); its solubility in blood and serum is approximately 2.5 times that in water (Armit, 1907; Sazegar, 1961). Owing to the nature of its

301

METABOLISM OF NICKEL CARBONYL-14C .E -a4

E b P

I 1

I 2

I 3

Hours

I 4

after

injection

I 5

c 6

of N&O),

FIG. 4. Measurements of carbon monoxide saturation of hemoglobin (x-x) after injection of Ni(W0)4. Each point for carbon monoxide saturation represents the mean + SE (N = 6). In order to facilitate comparison, the rate of exhalation of 1% (- - -) is replotted from Fig. 2.

chemical bonds, nickel carbonyl has the properties of both nickel and carbon monoxide. Although the C-O bond in Ni(C0)4 is similar to that in carbon monoxide, the CO groups in Ni(C0)4 are more reactive than free carbon monoxide. Thus, atmospheric oxygen, especially in the presence of alkali, can oxidize nickel carbonyl to COZ and NiO (Thorne, 1924; Remy, 1956; Kirby, 1961). The CO groups in nickel carbonyl also react with Grignard reagents, whereas free carbon monoxide is nonreactive (Gilliland and Blanchard, 1926). Since the Ni-C bond is relatively weak, nickel carbonyl slowly decomposes to nickel and carbon monoxide at temperatures above 36” (Mellor, 1924). An excess of CO is needed to prevent this decomposition. Oxidizing agents, such as concentrated nitric acid or halogens, rapidly decompose Ni(CO)4 to Niz+ salts and CO (Sazegar, 1961). The CO groups in Ni(C0)4 may also be partially replaced by ammonia, hydrazine, hydroxylamine, amines, or alcohols (Mellor, 1924; Hieber et al., 1952; Remy, 1956). In the previous investigation, (Sunderman and Selin, 1968), precise data were obtained regarding the distribution and routes of excretion of 63Ni after administration of 63Ni(C0)4 to rats. These data did not, however, settle the long-existing controversy whether Ni(C0)4 is decomposed in uivo to yield carbon monoxide or carbon dioxide (Langlois, 1891); Henriot and Richet, 1891; McKendrick and Snodgrass, 1891; Vahlen, 1902; Mittasch, 1903; Armit, 1907; Garland, 1933). The results described in the present paper have shown that metabolism of Ni(CO)4 results in a gradual release of carbon monoxide, with formation of only traces of carbon dioxide. Therefore, the above-mentioned controversy has finally been resolved.

302

KASPRZAK AND SUNDERMAN

Based upon the present study as well as the results of previous investigations @underman, 1963; 1964; Sunderman and Selin, 1968; Sunderman et al., 1968), the following summarization is proposed regarding the metabolic fate of nickel carbonyl. After intravenous injection of Ni(C0)4 in LD50 dosage, approximately 36 % of the dose is exhaled without metabolic alteration. The remainder of the Ni(C0)4 slowly undergoes intracellular decomposition [Ni(CO), --f Ni” + 4 CO]. In the erythrocytes, the dissociation of Ni(C0)4 is accelerated by the presence of hemoglobin, which acts as a receptor for the released carbon monoxide. The hepatic CO-binding pigment (cytochrome P-450) has not been found to be a significant factor in binding the CO released from Ni(C0)4 (Sunderman, 1968). After dissociation of Ni(CO),, the released Ni” is oxidized to Ni2+ by intracellular oxidation systems which are, as yet, unidentified. It may be noted that the electrode potential for the oxidation reaction, Ni” --f Nizf + 2 e, is $0.25 V (Handbook of Chemistry and Physics, 196465), which is higher than the electrode potentials of typical intracellular electron acceptors : DPN+ (-0.32 V), glutathione (-0.23 V), dihydroascorbic acid (+O.OS V), and cytochrome b, (+0.12 V) (White et al., 1968). Some of the Ni2+ which is derived from Ni(C0)4 combines with nucleic acids and proteins, but most of the Niz+ is transported into the plasma, where it becomes bound to albumin (Sunderman and Selin, 1968). Ultimately, the Ni ‘2+ is excreted in the urine and feces. The carbon monoxide, which is released from Ni(C0)4 becomes bound to hemoglobin, and ultimately is exhaled. A very minor metabolic pathway of the carbonyl moiety of Ni(i4C0)4 results in formation of 14C02, which is also exhaled. This probably represents secondary oxidation of the carbon monoxide (Tzagoloff and Wharton, 1965; Luomanmaki, 1966; Coburn, 1967). The traces of 14C that are excreted in urine are presumed (but not proved) to represent Hi%O-s. Experiments are currently in progress in an attempt to identify the mechanisms whereby Ni” from Ni(C0)4 undergoes intracellular oxidation to Ni2+. REFERENCES ARMIT, H. W. (1907). The toxicology of nickel carbonyl. J. Hyg. 7,525-551. BAGGIOLINI, M., and BICKEL, M. H. (1966). A new type of incubation apparatus for the determination of metabolically produced i4C02. Anal: Biochem.14,290-295. COBURN, F. F. (1967). Endogenous carbon monoxide production and body CO stores. Acra

Med. Stand., Suppl. 472, pp. 269-282. DUBOWSKI, K. M. (1964). Measurements of hemoglobin derivatives. PrecursorsandMetabolites (F. W. Sunderman and F. W. Sunderman,

In: Hemoglobin,Its

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carbonyl,

carbon monoxide

and

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scin-

METABOLISM HENRIOT,

M.,

and

RICHET,

OF NICKEL

CARBONYL-14C

C. (1891). Deseffectsphysiologiqueset

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