Predicted values of DU excretion from historic inhalation exposures.
Inhaled fine particulates of DU oxide have the potential to lodge deep in the lung, decompose slowly into solution into the blood stream, and become stored in bone, kidney and other organs to be excreted over time with organ and bone re-working, all while undergoing slow radioactive decay. The rate of dissolution is a key parameter that underpins the notion that DU can be detected in urine many years after exposure.
To test whether DU plays any role in GWI, it is first necessary to predict the concentration of DU expected in a urine sample for a given DU aerosol inhalation exposure as a function of the dose and oxide type of the original DU exposure, the time since exposure, and likely dietary intakes of NU in the run up to urine collection. Without this prediction, it is impossible to know whether a negative test is due to an inconsequential exposure or an insensitive urine assay method—a valid criticism of past research on this problem.
The defining studies by the U.S. Department of Defense [6], the World Health Organisation [21] and the British Royal Society [8], classified battlefield situations of potential DU aerosol exposure into 3 standard exposure levels likely to occur in combat situations: level I-high, level II-medium, and level III-low. Exposure level I includes direct inhalation of an impact aerosol; level II, inhalation of resuspended impact aerosol or oral ingestion within a contaminated vehicle; and level III, inhalation of an aerosol plume at a distance from an impact or fire or resuspension from ground contamination. Doses in milligrams of DU taken into the body in each of these situations were estimated with evidence from other studies, including field studies involving measurements during destruction of vehicles with DU penetrators (Table 1) [6].
Table 1
Current ranges of urinary total [U] and U isotopic ratios that identify DU estimated from different levels of DU inhalation exposure during the 1991 Gulf War, time since exposure, and daily dietary intake of natural uranium running up to urine sample collection.
Parameters specified in the prediction model
|
Ranges of parameters expected 18 years after specified DU inhalation exposure
|
Can expected levels of DU be detected by criteria used in prior studies?
|
Standard DU exposure level
|
Estimated DU intake (mg) for the exposure level in 1991 Gulf Wara
|
Daily dietary U excretion (ng) in run-up to testing
|
Total [U]
(ng U/g creatinine)
|
238U/235U
|
236U/238U x10− 6
|
Method 1: total [U] > 50
ng U/g creatinineb
|
Method 2: 238U/235U >166 by SF-ICP-MSc
|
Method 3: 238U/235U >140 by MC-ICP-MSd
|
Level I
|
250
|
2
|
14.5–74.5
|
367–467
|
25.9–29.2
|
rarely
|
yes
|
yes
|
|
|
8
|
20.5–80.5
|
247–397
|
18.3–27.0
|
rarely
|
yes
|
yes
|
Level II
|
10
|
2
|
2.5–4.9
|
161–241
|
6.0-17.8
|
no
|
yes
|
yes
|
|
|
8
|
8.5–10.9
|
144–171
|
1.8-8.0
|
no
|
no
|
yes
|
Level II
|
5
|
2
|
2.3–3.5
|
150–198
|
3.3–12.6
|
no
|
no
|
yes
|
|
|
8
|
8.3–9.5
|
141–155
|
0.9–4.6
|
no
|
no
|
yes
|
Level III
|
2
|
2
|
2.1–2.6
|
143–165
|
1.4–6.7
|
no
|
no
|
yes
|
|
|
8
|
8.1–8.6
|
139–145
|
0.4-2.0
|
no
|
no
|
yes
|
a Numerical values are derived from modelling [8, 22, 27] as illustrated in Fig. 1, described in more detail in on-line methods.
|
b In the most recent study of DU in Gulf War veterans, Dorsey et al. [20] screened urine total [U] for DU by this criterion.
|
c Dorsey et al. [20] identified the presence of DU with the 238U/235U ratio measured by lower precision sector field–inductively coupled plasma-mass spectrometry (SF-ICP-MS)
|
d The present study identified the presence of DU with the 238U/235U ratio measured by the high precision multiple collector–inductively coupled plasma-mass spectrometry (MC-ICP-MS)
|
We used the modelling of [8, 21, 27] to estimate the urinary excretion of uranium on the basis of an inhalation dose. We considered intake doses representing each of the Royal Society’s exposure levels at 18 years after exposure and at 2 likely dietary intake levels of NU in the run up to urine collection to predict current excretion rates of DU oxides (UO2, U3O8, etc.) in a range of excretion levels determined by the level of uncertainty in the ICRP models (Fig. 1). The model output allowed calculation of the current expected ranges of the following 3 parameters most used for detecting past DU exposure from urinary assays: total [U], the 238U/235U isotopic ratio, and the 236U/238U ratio, given in Table 1.
Comparison of the ability of three methods to distinguish DU from NU in urine
Applying the criterion of finding > 50 ng U/g creatinine of total [U] in urine will detect only veterans excreting the very highest total [U] in those with Level I inhalation exposures in the Gulf War, but would be unable to distinguish any with Level II or III exposures from NU excretion (Table 1).
The relatively low precision SF-ICP-MS method would be able to differentiate DU from NU in all veterans with Level I exposures in the Gulf War and most in the upper half with Level II exposures but only if they are consuming a diet low in NU (average 2 ng NU per day) so that DU is ≥ 25% of the total U excreted. It would thus be unable to differentiate most with Level II and all with Level III exposures from NU excretion (Table 1).
The high precision MC-ICP-MS method combined with chemical separation and purification of U from urine, allows the detection of excreted DU at a rate of > 0.068 ng/day, our methodological DU detection limit. Our prediction model indicates that it would detect an initial inhalation exposure of as little as 0.40 mg of DU from the 1991 Gulf War. Thus, it would be able to identify DU in all veterans with Gulf War exposures far less than the lowest Level III exposure (2 mg DU inhaled during the Gulf War) even if consuming a diet relatively high in NU before urine collection (Table 1). It follows then that only mass spectrometry with high precision MC-ICP-MS is capable of detecting DU from inhalation exposure in the Gulf War or confirming its absence in most Gulf War veterans.
Cohort of GWI cases and controls and battlefield deployment
We measured the uranium isotope composition in urine samples from a nested case-control sample of Gulf War veterans selected in a 3-stage stratified random sample of the 1991 U.S. military population studied in the U.S. Military Health Survey (USMHS). The methods of sample selection at the 3 stages have been published [3, 23]. The first stage involved a computer-assisted computer interview (CATI) survey (N = 8,020) which included questions covering battlefield situations likely to involve inhalation of various levels of DU, from which we assigned the standard DU exposure levels. The second stage involved blood collection from all veterans whose symptoms met the 3 widely used case definitions of GWI (cases) and an approximately 10% random sample of those not meeting it (controls), including both deployed and not deployed to the KTO. The third stage constituted a smaller representative sample selected from the larger stage 2 sample, and included 106 who met the 3 standard case definitions of GWI [2, 24–25] in which cases comprised of 31 with syndrome variant 1 (“impaired cognition”); 42 with syndrome variant 2 (“confusion-ataxia”); 33 with syndrome variant 3 (“central pain”) [2]; and 47 control veterans comprised of 26 deployed to the war theatre and 21 non-deployed not meeting the case definitions. Between November 2008 and June 2010, the 154 veterans in the stage 3 sample were studied extensively in a 7-day clinical research protocol in which each travelled to Dallas, Texas (USA) to be hospitalized in the UT Southwestern Medical Center’s Clinical and Translational Research Center. In addition to diverse neuropsychological, autonomic and neuroimaging studies, a 24-hour urine sample was collected in urine containers prewashed with nitric acid to remove any trace uranium; creatinine was measured on an aliquot shortly after collection.
Effectiveness of total [U] of urine to rule out past DU exposure.
The first method we used to detect evidence of past DU exposure was to screen the 154 GWI cases and control subjects’ urine for an increase in total [U] excretion (Table 2). The distribution of total [U] was similar to that of the U.S. population [19], and no values exceeded its 95th percentile (Fig. 2a). The geometric mean of total [U] of the cases meeting the 3 case definitions of GWI was 1.78 ng U/g creatinine, statistically indistinguishable from the combined deployed and non-deployed controls (geometric mean 1.57 ng U/g creatinine, t-test P = 0.18). These values of total [U] are consistent with lack of DU contamination but do not exclude the possibility of the small amounts of DU expected from our prediction model with Level II and III inhalation exposures (Table 1). Although continuing dissolution of DU shrapnel in metallic form retained in the body usually increases total [U] beyond the population’s 95% percentile, as concluded by [20], our findings further confirm that screening of total [U] is not useful for detecting the far smaller intake doses and the time-limited exposure situations involved in inhalation exposures to DU aerosols (Table 1).
Table 2
Urinary excretion rate of total U by Gulf War veterans’ clinical group
|
|
Total [U]
|
Total [U] adjusted for creatinine
|
Clinical group
|
Sample size
|
Mean
|
95% CI
|
5th and 95th percentile
|
range
|
Median
|
Mean
|
Non-deployed controls
|
21
|
2.37
|
-1.04 / +1.86
|
0.24–11.2
|
0.2–30.2
|
1.25
|
1.24
|
Deployed controls
|
27
|
3.80
|
-1.60 / +2.78
|
0.42–50.2
|
0.32–96.4
|
2.16
|
1.87
|
GWI syndrome 1
|
31
|
2.53
|
-0.78 / +1.12
|
0.58–10.7
|
0.22–13.3
|
1.38
|
1.41
|
GWI syndrome 2
|
42
|
4.00
|
-1.25 / +1.81
|
0.77–21.7
|
0.70–28.8
|
2.05
|
2.15
|
GWI syndrome 3
|
33
|
3.33
|
-1.05 / +1.54
|
0.65-12.0
|
0.44–15.1
|
1.95
|
1.74
|
All controls
|
49
|
2.98
|
-0.98 / +1.45
|
0.36–25.9
|
0.17–96.4
|
1.86
|
1.57
|
All GWI
|
106
|
3.30
|
-0.57 / +0.69
|
0.69–16.2
|
0.13–23.9
|
1.84
|
1.78
|
All samples
|
154
|
3.24
|
-0.56/ +0.68
|
0.57–17.6
|
0.17–96.4
|
1.84
|
1.71
|
Use of high precision 238U/235U to determine significant inhalation exposure.
The second method we used to detect past DU exposure was analysis of the 238U/235U isotopic ratio measured by the high precision MC-ICP-MS method (Table 3). The uncertainty in the 238U/235U ratio, measured by its 95% confidence interval, increases (precision decreases) as the urinary total [U] decreases (Fig. 2b), but it was less than ± 1% for 95% of the samples (Fig. 2b). For urine samples with total [U] above 1 ng/g creatinine and a ± 1% uncertainty, values of the 238U/235U ratio above 139–140 including their lower uncertainty bound are considered likely to represent the presence of DU (Fig. 2b). This threshold of 139–140 for confirmation of DU is more robust and > 10 times more sensitive than the threshold of 166 applied by Dorsey et al. using the lower precision SF-ICP-MS (Table 1) [20].
Table 3
Urinary uranium isotope ratios by Gulf War veterans’ clinical group
|
|
238U / 235U
|
236U / 238U
|
Clinical group
|
Sample size
|
Median
|
Meana
|
95% CIa
|
Meana,c
|
95% CIa
|
Non-deployed controls
|
21
|
137.79
|
137.62
|
0.26
|
<LOD
|
n/a
|
Deployed controls
|
27
|
137.84
|
137.75
|
0.25
|
<LOD
|
n/a
|
GWI syndrome 1
|
31
|
137.50
|
137.51
|
0.16
|
<LOD
|
n/a
|
GWI syndrome 2
|
42
|
137.62
|
137.68
|
0.16
|
<LOD
|
n/a
|
GWI syndrome 3
|
33
|
137.62
|
137.71
|
0.19
|
<LOD
|
n/a
|
All controls
|
49
|
137.79
|
137.70
|
0.18
|
<LOD
|
n/a
|
All GWI
|
106
|
137.55
|
137.64
|
0.10
|
<LOD
|
n/a
|
All samples
|
154
|
137.63
|
137.66
|
0.09
|
<LOD
|
n/a
|
In house urine no IRMM184 d
|
4
|
137.70
|
137.68
|
0.27
|
<LOD
|
n/a
|
2 ppb IRMM184 + 233Ue
|
125
|
137.70
|
137.71
|
0.05
|
1.11E-07
|
1.3E-08
|
IRMM184 certified f
|
|
|
137.70
|
0.04
|
1.25E-07
|
5.3E-10
|
Natural uranium
|
|
|
137.82
|
~0.06
|
<10− 8
|
n/a
|
DU in munitions
|
|
|
~500
|
|
~ 0.000030
|
|
DU, depleted uranium; IRMM, European Commission's Institute for Reference Materials and Measurements; LOD, limit of detection; n/a, not applicable; U, uranium
|
aGeometric means with 95% confidence interval
|
bSince 238U/ 235U uncertainties are not significantly asymmetrical and are reported as a single value.
|
cThe 236U/ 238U ratios were overwhelmingly below the 0.0000015 limit of detection after all corrections and uncertainty propagations were made.
|
dThe 4 analyses of in-house urine that had no additional IRMM184 added.
|
eIRMM184 + 233 U values reported are corrected for very minor contributions of other isotopes in the 233U that was added.
|
fIRMM certified value from https://crm.jrc.ec.europa.eu/p/40454/40475/By-application-field/Nuclear/IRMM-184-URANIUM-238-NATURAL-ISOTOPIC-NITRATE-SOLUTION/IRMM-184
|
When predictions of DU excretion are applied to groups of veterans with different standard levels of DU inhalation exposures, the 238U/235U ratios are below 139 for veterans with all 3 inhalation exposure levels (Fig. 2c; Kruskal-Wallis test P = 0.74). The 3 veterans with outlying values nearest the threshold between 139 and 140 all had very low values of total [U] and thus very wide uncertainty intervals that generously overlap the 238U/235U of NU (~ 137.8). When the battlefield exposure situations from which the standard exposure levels were generated were broken out, the distributions of the 238U/235U ratios showed no pattern suggesting any departure from NU outside of methodological uncertainty (Fig. 2d).
Likewise, veterans who continue to have potentially disabling symptoms of GWI had distributions of the 238U/235U ratio that did not differ from the deployed and non-deployed control veterans (Fig. 2e), and none had values that differed from NU. Moreover, the distribution of the 238U/235U ratios in the 3 GWI syndrome variant groups combined was similar to those of the deployed and non-deployed control groups (Kruskal-Wallis test P = 0.16).
Bivariate analysis of the 236U/238U and 235U/238U as a further test of DU exposure.
As a further test for the possibility of DU in these veterans, we studied their location on the bivariate distribution of the veterans’ ratios of both 236U/238U and 235U/238U isotopic ratios. This is an important procedure because, since 236U is found only in EU and DU but not in NU, it provides a further direct method of distinguishing DU from NU and from ascertaining whether EU may also have been involved. Since 236U constitutes only ~ 0.003% of DU used in munitions, it can only be measured by high precision MC-ICP-MS and thus has not previously been measured in research on GWI.
We applied this approach to our representative sample of Gulf War veterans in the context of a prior studies [7, 15, 31] of groups of U.S. civilians exposed to DU (and EU) aerosol pollution in New York State from a uranium fabrication plant in the 1960s and 1970s [32] and in part studied with MC-ICP-MS of urine samples using the same methods as in our study [7]. These studies demonstrated that significant doses of DU aerosols inhaled by factory workers and residents living nearby can be detected in urine ≥ 25 years after the plant was closed [7, 31], broadly consistent with predictions of the HRTM model.
In the bivariate plot in Fig. 2f, an individual excreting pure NU would be located at the point where 235U/238U = 0.00726 and 236U/238U = 0, reflecting the usual amount of 235U and the absence of 236U in natural U. A theoretical individual excreting pure DU would be located at a point defined by 235U/238U ≈ 0.002 and 236U/238U ≈ 0.00003, but most DU-exposed workers excrete a combination of DU and NU, which displaces them downward and to the right; whereas, EU added to the mix would displace them upward and to the right. Using published analyses of workers from the plant, mixtures of NU and DU had lower 235U and higher 236U, locating them along the solid line. Mixtures of NU and EU would have 235U/238U above that of NU (235U/238U > 0.00726), indicating substantially increased 235U, and variably increased 236U. Mixtures of NU, DU and EU would appear along the dashed line in the diagram.
Published studies of the DU plant workers and local town residents with proven non-military DU aerosol exposure [7, 31] are shown to illustrate where subjects with proven inhalation exposure to DU typically fall. All but 6 of the Gulf War veterans in our study are located in a narrow zone located exactly at 235U/238U = 0.00726, the value of natural U, and with 236U/238U ratios within the uncertainty zone around 236U/238U = 0 (Fig. 2f). The 6 exceptions have slightly elevated 236U/238U ratios and values of the 235U/238U incompatible with both DU and EU, indicating an artefact of measurement from organic molecule interference in these samples (see explanation in Additional materials, mass spectrometry).
Veteran with a Level I battlefield exposure and DU shrapnel wound.
One Gulf War veteran in our study was standing on an Abrams tank when it was hit in a “friendly fire” accident by a DU round which destroyed the tank, threw the individual several meters, peppered him with a mix of sand, pebbles and shrapnel, tattooing his skin and embedding 2 pea-sized pieces of DU shrapnel under his skin. He breathed the hot gases from the explosion for several minutes. Upon return to his base in the U.S. 4 months later, the shrapnel was removed. During his wartime deployment to the war theatre, however, he was also exposed to low-level sarin nerve agent, took pyridostigmine tablets, and has moderately low PON1 type Q isoenzyme level, which are typical risk factors for GWI [33–35]. The veteran had symptoms satisfying the case definitions of GWI, subclassified as variant syndrome 1 (“cognitive impairment”), but his urine showed a total [U] of 1.35 ng/g creatinine, a 238U/235U ratio of 137.8, and a 236U/238U ratio below detection limit (< 0.000001)—all typical of natural U with no DU. If residual DU were present it was being excreted at a rate of < 0.068 ng/day, our limit of detection, and our prediction model indicates that he could have absorbed no more than 0.40 mg of DU from the Gulf War from both inhalation and DU shrapnel.