September 14, 2008
Urinary and Serum Metal Levels As Indicators of Embedded Tungsten Alloy Fragments
By Kalinich, John F Vergara, Vernieda B; Emond, Christy A
ABSTRACT Novel metal formulations are being used with increasing frequency on the modern battlefield. In many cases the health effects of these materials are not known, especially when they are embedded as fragments. Imaging techniques, although useful for determining location, provide no information regarding the composition of embedded fragments. In this report, we show that laboratory rats implanted with weapons-grade tungsten alloy (tungsten, nickel, and cobalt) pellets demonstrate significant increases in both urinary and serum levels of tungsten, nickel, and cobalt, which indicates that such measurements can provide information on the composition of embedded fragments. We also propose that, in addition to the requirements promulgated by the recent directive on analysis of metal fragments removed from Department of Defense personnel (Health Affairs policy 07-029), urine and blood/serum samples should be collected from personnel and analyzed for metal content. Such measurements could yield information on the composition of retained fragments and provide the basis for further treatment options. INTRODUCTIONTungsten has been used for a variety of functions throughout history.1 Combining tungsten with various other metals, particularly the transition metals nickel, cobalt, copper, and iron, produces tungsten alloys (WAs) with specific characteristics, some of which have military applications. Tungsten-containing materials have been used in some small-caliber ammunition (the "green bullet")2 and shot for waterfowl hunting,3 as well as in kinetic-energy penetrators.4 The composition of these materials varies widely with respect to the individual metals present and their concentrations. For example, shot for waterfowl hunting usually contains 20% to 70% tungsten, with the balance consisting of iron, nickel, tin, or bronze.3 In contrast, tungsten levels in kinetic-energy penetrators are well over 90%.5 Manufacturing methods for these applications also differ.
Widespread public concern regarding the potential health effects and environmental impact of the continued use of depleted uranium (DU) has led the militaries of many nations to replace DU in armor- penetrating munitions with WAs. Although only a limited number of studies have been reported, the prevailing theory is that elemental tungsten and insoluble tungsten compounds have only limited toxicity.6 However, an investigation reported that surgical implantation of pellets of weapons-grade WA (91.1% tungsten, 6.0% nickel, and 2.9% cobalt) into the leg muscles of laboratory rats resulted in the formation of highly aggressive, metastatic rhabdomyosarcomas around the implanted pellets.5 These results raise serious questions regarding the diagnosis and treatment of embedded WA-fragment wounds, because standard surgical procedures usually recommend leaving embedded fragments in place.
Clearly, a procedure to screen wounded personnel to determine the type of embedded metal fragment would be of great benefit to medical staff members. Examination of radiographs and magnetic resonance imaging scans can be used to detect fragments in tissues.7 Unfortunately, neither of these imaging procedures can be used to determine whether the embedded metal fragments consist of WAs. Experience with DU showed that, in a rodent model, embedded DU fragments rapidly degraded, and the resulting urinary DU levels were excellent indicators of fragment load.8 This was also proven to be the case with veterans with retained DU fragments from the First Persian Gulf War.9,10 Whether such a situation would hold for other metallic fragments, such as WAs, is not known. Therefore, the purpose of this study was to determine whether urinary and serum metal levels can be used as indicators of retained WA fragments. Further investigations can then assess whether these types of fragments need to be surgically removed and, if so, how rapidly.
All pellets were cylinders 1 mm in diameter and 2 mm in length. WA pellets were produced by Aerojet Ordnance Tennessee (Jonesborough, Tennessee) by using standard kineticenergy penetrator production processes. An average WA pellet weighed 27.5 mg and consisted of 91.1% tungsten, 6.0% nickel, and 2.9% cobalt. Tantalum pellets (99.95% tantalum; Alfa Aesar, Ward Hill, Massachusetts) were used as control pellets and weighed 27 mg, on average. Before implantation surgery, all pellets were cleaned and chemically sterilized.8
F344 rats (male, 6 weeks of age; Harlan, Frederick, Maryland) were maintained in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility, in accordance with the Guide for the Care and Use of Laboratory Animals.11 All procedures were approved by the Armed Forces Radiobiology Research Institute Animal Care and Use Committee. Upon arrival, animals were screened for common rodent pathogens. Rodents were pairedhoused in plastic microisolator cages, with hardwood chips for bedding, and were fed a certified NTP-2000 (Quality Laboratory Products, Elkridge, Maryland) diet.12 Acidified water was provided ad libitum. Animals were on a 12-hour light/dark cycle witii no twilight.
Pellet Implantation Surgery
At 9 weeks of age, all rats underwent implantation of a total of 20 pellets, evenly split between the hind legs. Experimental groups included tantalum (control, 20 tantalum pellets), low-dose WA (4 WA pellets and 16 tantalum pellets), and high-dose WA (20 WA pellets) groups. Tantalum was used as a negative implantation control because it is considered inert and has been used in human prostheses.13,14 For pellet implantation, anesthesia was induced through continuous administration of isoflurane by using an open circuit system with a scavenger/recapture system. All surgery was performed by using aseptic techniques. After the surgical sites were clipped and cleansed witii betadine, an incision was made through the skin to expose the gastrocnemius muscle. Pellets were implanted in the muscle, spread ~1.5 mm apart on the lateral side of each leg. The incision was closed wim sutures and surgical adhesive. Rats were closely monitored after surgery until they were ambulatory. An analgesic (buprenorphine hydrochloride; Reckitt and Colman, Hull, United Kingdom) was administered preoperatively and then as needed postoperatively. At various times after implantation or when moribund, rats were euthanized with an isoflurane overdose. As part of the complete gross pathology examination, a urine sample was removed from the bladder by using a syringe with a 25-gauge needle. In addition, a serum sample was obtained from whole blood taken from the abdominal aorta into a serum-separator tube (BD Biosciences Labware, Franklin Lakes, New Jersey). After being inverted five times, the tube was left at room temperature for 30 minutes before centrifugation for 10 minutes at 1,000 x g to obtain serum. Serum and urine samples were stored at -80[degrees]C until assayed.
Sample Preparation for Metal Analysis
Urine samples did not require any preparation, except dilution, before analysis. Serum samples, after the addition of rhodium as a recovery standard, were wet-ashed with 5 mL of 70% nitric acid (Optima ultrapure grade; Fisher Scientific, Pittsburgh, Pennsylvania) and 200 [mu]L of 30% hydrogen peroxide (semiconductor grade; Sigma-Aldrich, St. Louis, Missouri) by heating to just below boiling (to avoid sample splashing) until complete evaporation. After wet-ashing, samples were dry-ashed at 600[degrees]C for 8 to 12 hours in a muffle furnace (Fisher Isotemp muffle furnace; Fisher Scientific) and then were wet-ashed again with nitric acid and hydrogen peroxide. After the second wet-ashing procedure, the white residue was dissolved in 2% nitric acid and analyzed.
The metal content of the serum and urine samples was determined by using an inductively coupled plasma mass spectrometer (PQ ExCell system; ThermoElemental, Franklin, Massachusetts) equipped witii a Cetac ASX500 autosampler (Cetac Corporation, Omaha, Nebraska). High- pressure liquid argon (99.997%) was used as the plasma gas. Instrument operating parameters are given in Table I. The instrument was calibrated witii external standards of the appropriate metal in 2% nitric acid. The sample probe was washed wim a constant flow of 2% nitric acid between measurements. Quantitative analysis was performed with reference to the slope of the calibration curve (counts per second per nanogram per liter), as well as an internal standard. Serum data were normalized to the volume of serum analyzed, and urinary data were normalized to creatinine levels. Creatinine content was determined by using a modified Jaffe reaction,15,16 with a commercially available colorimetric kit (Oxford Biomedical Research, Oxford, Michigan).
As part of our laboratory's studies on the health effects of embedded fragments, F344 rats were implanted with eithier a low dose (4 pellets) or high dose (20 pellets) of weaponsgrade WA. The WA pellets, manufactured by using the same procedures as used to produce WA-based munitions, consisted of 91.1% tungsten, 6.0% nickel, and 2.9% cobalt. Another set of rats were implanted with 20 pellets of tantalum, an inert metal, to serve as a control group. At various times after pellet implantation or when necessary as a result of tumor development, rats were euthanized and urine and serum samples were collected. Samples were processed and metal contents were analyzed by using inductively coupled plasma mass spectrometry, under the conditions specified in Table I. Small but statistically significant decreases in WA pellet mass occurred after surgical implantation (Table II). Despite these decreases, the pellets did not appear remarkably different as a result of implantation. The appearance of WA pellets ~6 months after implantation in rat muscle is shown in Figure 1. As a result, we expected little increase in serum and urinary levels of tungsten, nickel, and cobalt; however, that was not the case. As shown in Figure 2, WA pellet-implanted rats excreted significantly higher levels (p
WA component metals were also found at elevated levels in the serum from pellet-implanted rats (Fig. 3), although not at the concentrations found in urine. Serum levels of tungsten, nickel, and cobalt peaked 1 month after implantation and decreased over time, with the exception of serum tungsten levels in the high-dose WA group 3 months after implantation. Serum tungsten levels at that time were extremely elevated, reaching levels similar to those in the urine samples.
The use of novel materials in munitions on the modern battlefield opens the possibility of wounds with embedded fragments whose health effects and toxicity characteristics have been investigated incompletely, if at all. The lack of this critical information puts physicians responsible for planning and implementing long-term treatment strategies at a distinct disadvantage when dealing with wounds of this type. Obviously, not every compound used on the battlefield can be tested for potential health effects as embedded fragments. However, compounds used in munitions, for which wounds containing embedded fragments are highly likely, should be tested.
One such example is WAs. The health effects of these unique materials, proposed as a surrogate for DU in armorpiercing munitions, have not been thoroughly tested. Work in our laboratory showed that, when embedded as fragments in laboratory rats, a WA composed of 91.1% tungsten, 6.0% nickel, and 2.9% cobalt induced a highly aggressive metastatic rhabdomyosarcoma.5 These results raise concerns regarding potential health effects if humans are wounded with fragments of this composition. Clearly, the first step in treating such injuries is to determine the composition of the embedded fragments.
Examination of radiographs and a variety of imaging techniques can be used to detect metallic fragments embedded in tissues,7,17 but such techniques provide no information regarding the composition of the embedded fragments. Because most embedded fragments are left in place, a procedure to identify fragment composition without surgical removal would be of great utility to those tasked with implementing treatment regimens. The results reported here indicate that urinary and serum metal measurements are useful indicators of embedded WA fragment composition. Although both urinary and serum measurements would provide a good indication of embedded fragment composition, urinary metal measurements would be preferable in the case of this particular type of WA, because of the ease of sample collection and preparation for analysis.
These results are not unique to WAs. Other metallic compounds, when embedded as fragments, also can be found in urine and blood. DU appears rapidly in the urine of laboratory rats with embedded DU fragments,8 and urinary uranium levels have been shown to be an excellent indicator of embedded DU in wounded veterans.9,10 Increased metal concentrations in blood are also found as a result of retained lead bullets18,19 and wear debris from orthopedic devices.20
In this study, the high concentrations of tungsten, nickel, and cobalt found in urine and serum might be surprising, in light of the relatively minor changes observed in WA pellet mass and appearance. However, this can be explained when the manufacturing processes and resulting microstructure of WA are considered. As noted earlier, the tungsten concentration in WA for military munitions is generally >90%, with the remainder being various metals, including nickel, cobalt, copper, and iron. Weapons-grade WA is made through a process known as liquid sintering.21 In this method, powders of tungsten and the appropriate alloying elements are mixed, compressed under high pressure, and heated under an inert atmosphere to ~1,500[degrees]C. This temperature is sufficient to melt the alloying elements. Tungsten does not melt under these conditions. However, some tungsten dissolves in the melted alloying metals. What results is a two-phase composite consisting of tungsten particles (tungsten phase) surrounded by a matrix of alloying elements and dissolved tungsten (binder phase). Studies conducted by Ogundipe et al.21 showed that the binder phase corrodes more rapidly than does the tungsten phase, releasing the alloying elements into solution. Based on the results reported here, we propose that a similar process occurs in vivo with embedded WA fragments. This would explain the high levels of nickel and cobalt in the urine and serum of pellet- embedded rats. We are continuing with studies examining the in vivo degradation of embedded WA.
A recent policy letter from the Assistant Secretary of Defense for Health Affairs mandates that all fragments removed from Department of Defense personnel be analyzed for metal content.22 We also suggest that urine and blood/ serum samples from personnel with retained fragments be analyzed for metal content, similar to recommendations for individuals with suspected DU fragments. Such data would provide metal exposure information that could be valuable in future treatment decisions. Finally, many of the metal composites found on the battlefield today are militarily unique; therefore, few if any toxicological data are available. We tested only one WA formulation. There are many more being used that have not been tested. In addition, if toxicity data are available, then they may not deal with likely battlefield exposure scenarios (e.g., embedded fragments or acute inhalation). Therefore, we think it is critical that new, militarily unique materials, especially those used in munitions, be screened for potential toxicity early in the development process. At a minimum, the dissolution characteristics and tissue distribution patterns of the metals constituting these materials should be investigated by using likely battlefield exposure scenarios. This information would be crucial in developing the framework within which future treatment decisions on retained fragments (surgical removal, pharmacological intervention, or status quo) could be made, so that wounded personnel receive appropriate care.
This work was supported by U.S. Army Medical Research and Materiel Command Grant DAMD17-01-1-0821.
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22. Department of Defense, Assistant Secretary of Defense for Health Affairs: Policy on analysis of metal fragments removed from Department of Defense personnel (Health Affairs policy 07-029), December 18, 2007. Available at http://mhs.osd.mil/Content/docs/ pdfs/policies/2007/07-029.pdf; accessed January 28, 2008.
John F. Kalinich, PhD; Vernieda B. Vergara, BS; Christy A. Emond, BS
Armed Forces Radiobiology Research Institute, Uniformed Services University, Bethesda, MD 20889-5603.
This work was presented at the Force Health Protection Embedded Fragment Working Group meeting, July 24, 2007, Falls Church, VA, and the Toxic Embedded Fragment Center Expert Panel meeting, January 9, 2008, Baltimore, MD.
This manuscript was received for review in February 2008. The revised manuscript was accepted for publication in April 2008.
Reprint & Copyright (c) by Association of Military Surgeons of U.S., 2008.
Copyright Association of Military Surgeons of the United States Aug 2008
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