Supplementary Evaluations and Data for the article, A Critical Evaluation of the Creatinine Correction Approach: Can It Underestimate Intakes of Phthalates? A Case Study With Di-2-ethylhexyl phthalate, DEHP
1. Application of Creatinine Correction Approach to High Dose Individuals
Besides the extraction and evaluation of a subset of NHANES data, the capability of the creatinine approach can be demonstrated in another way. For the two individuals with known exposures, creatinine was also measured in the urine samples. So, the question arises, what would the creatinine correction approach say about the magnitude of the daily dose of DEHP if, for the sake of argument, all one had were these several urine measurements of metabolites and creatinine, but did not know what the intake dose was? To answer this question, the creatinine approach was applied to the data for these individuals. Daily intakes of DEHP derived from each urination event using the creatinine correction approach applied to the four metabolites and total concentration, are graphed in Figures S1 and S2. Several of the trends discussed in the article are seen here. Near the time of exposure, urine concentrations are the highest and imply daily total excretions that exceed the actual intake. This is an appropriate outcome, since urine concentrations of the metabolites are assumed to represent the daily average, according to the creatinine correction approach. However, after about 8 hours, the creatinine correction equation begins to underpredict the intake. This underprediction gets as high as an order of magnitude by about 20 hours following exposure, and up to two orders of magnitude into the second day after exposure for the self-dosed individual (Figure 4).
For more clarity on trends, the percent overpredictions and underpredictions in Figures S1 and S2 were quantified. The percent change was calculated as the difference between the “predicted” and “observed” intakes, divided by the “observed” intakes, and multiplied by 100 to convert to a percentage basis. Looking first at Figure S1, overpredictions occurred for the first 5 urine measurements until hour 5.5 after intake. The average overprediction for the 4 metabolites were (average, then range): MEHP: 175% (24 to 455%), 5OH-MEHP: 119% (-50 to 261%), 5oxo-MEHP: 111% (-60 to 254%), 5cx-MEPP: 133% (-60 to 237%), and sum of congeners: 128% (-48 to 258%). Keeping in mind that a 200% result suggests an overprediction by a factor of 2, these results suggest an average overprediction by less than a factor of 2, with some overpredictions up to 5 times. Underpredictions began at the 6th event, which occurred at about 8 hours after the dosing. Looking at the average and range of underprediction from this point until the end of 24 hours (which entailed the next 9 urination events), the results are: MEHP:
-75% (14 to -98%), 5OH-MEHP: -77% (-24 to -94%), 5oxo-MEHP: -79% (-33 to -95%), 5cx-MEPP: -69% (-18 to -89%), and sum of congeners: -75% (-21 to -92%). Keeping in mind that a result of -75% translates to an underprediction by a factor of 3, this suggests that from hours 8 to 24, the creatinine correction underpredicted by an average of a factor of 3 with several results showing more than a 10-fold (i.e., a number smaller than -90%) underprediction. Without presenting the results, all predictions had results between -90% and -99% for the 10 events after 24 hours for this individual. For the apheresis patient results shown in Figure S2, the results are broken up in the first 3 events up until about hour 7 showing an overprediction, followed by 3 events showing an underprediction up until hour 24. Without the details, the analysis showed that the average overprediction for each individual metabolite and the sum of all metabolites ranged from 42 to 166%, with some individual overpredictions above 200%. Average underpredictions for the metabolites ranged from -12 to -66%, with some as low as -75%. So, for this individual, overpredictions were mostly less than a factor of two, and underpredictions, less than a factor of three.
Figure S1. Predictions of DEHP intake for the self-dosed individual using the creatine correction approach, compared against the known intake of 650 µg/kg DEHP.
Figure S2. Predictions of DEHP intake for the individual undergoing the apheresis procedure for blood platelet donation, using the creatine correction approach, compared against the known intake of 31 µg/kg DEHP.
2. Data and Analysis on Individuals Who Fasted
In an unpublished study on fasting, three individuals fasted on a glass bottle water-only diet for two days. Their urine was sampled and measured for several phthalate metabolites, including metabolites for DEHP, DiNP, DnBP, DiBP, and BBzP. Data on four DEHP metabolites from all urination events for three individuals over about 48 hours were available for this effort. While the intakes of DEHP were, of course, not known for these individuals, all had similar patterns of DEHP metabolites in their urine which support the hypotheses in this study. That is, concentrations declined over time from their initial measurements, and 5cx-MEPP ultimately became the metabolite that had the highest concentration in urine. The consistent trend of decline in all individuals provided evidence that, at least for these individuals, food was likely to have been the dominant source explaining initial concentrations measured in their urine, and also that no other significant non-food exposures to DEHP occurred during the fast. The individuals were healthy individuals, with demographics (age/gender/body weight) as follows: 27/m/77 kg, 32/m/86 kg, and 29/f/60 kg. The detailed data from these individuals is:
Sex: M
Age at time of experiment: 27
Weight at time of experiment: 77
Hours after exposure / Urine Volume, mL / Creatinine, g/L / MEHP, µg/L / 5OH-MEHP, µg/L / 5cx-MEPP, µg/L / 5oxo-MEHP, µg/L / 2cx-MMHP, µg/L0.0 / 550 / 0.19 / 0.4 / 1.0 / 1.6 / 0.8 / 0.7
0.8 / 400 / 0.79 / 3.4 / 6.6 / 6.6 / 3.4 / 1.8
1.8 / 50 / 1.73 / 6.8 / 17.3 / 18.9 / 12.5 / 11.8
8.3 / 400 / 1.37 / 4.8 / 17.4 / 14.9 / 11.4 / 7.6
10.3 / 400 / 0.52 / 1.2 / 3.0 / 3.9 / 2.7 / 1.2
12.1 / 320 / 0.52 / 0.3 / 2.0 / 3.0 / 1.5 / 1.1
13.6 / 550 / 0.28 / 0.1 / 0.9 / 1.5 / 0.7 / 0.4
15.0 / 400 / 0.31 / 0.1 / 0.8 / 1.6 / 0.8 / 0.9
17.3 / 600 / 0.36 / 0.1 / 0.7 / 1.5 / 0.8 / 0.6
19.9 / 350 / 0.64 / 0.4 / 1.7 / 2.5 / 1.9 / 1.0
21.8 / 800 / 0.22 / 0.1 / 0.5 / 0.9 / 0.5 / 0.4
23.8 / 800 / 0.21 / 0.1 / 0.4 / 0.8 / 0.5 / 0.5
32.5 / 500 / 1.15 / 1.1 / 2.9 / 3.6 / 2.2 / 1.3
37.2 / 300 / 1.31 / 0.7 / 2.8 / 3.7 / 2.6 / 1.2
38.3 / 200 / 0.64 / 0.1 / 1.2 / 2.0 / 1.6 / 0.5
39.6 / 550 / 0.41 / 0.1 / 0.6 / 0.9 / 0.8 / 0.2
41.1 / 600 / 0.28 / 0.1 / 0.3 / 0.8 / 0.4 / 0.1
44.6 / 500 / 0.60 / 0.1 / 1.1 / 1.7 / 1.3 / 0.4
46.3 / 200 / 0.74 / 0.1 / 1.5 / 2.1 / 1.4 / 0.6
Sex: M
Age at time of experiment: 32
Weight at time of experiment: 86
Hours after exposure / Urine Volume, mL / Creatinine, g/L / MEHP, µg/L / 5OH-MEHP, µg/L / 5cx-MEPP, µg/L / 5oxo-MEHP, µg/L / 2cx-MMHP, µg/L0.0 / 100 / 1.72 / 18.3 / 75.0 / 73.1 / 35.1 / 34.3
2.4 / 130 / 2.39 / 31.3 / 135.8 / 92.8 / 48.0 / 44.1
3.9 / 120 / 1.68 / 30.6 / 103.3 / 79.5 / 52.2 / 30.3
9.8 / 220 / 1.26 / 17.0 / 59.1 / 55.7 / 29.2 / 15.7
12.2 / 250 / 1.45 / 19.5 / 60.3 / 64.1 / 29.0 / 17.1
14.4 / 150 / 1.92 / 18.9 / 46.7 / 57.5 / 27.7 / 22.9
16.2 / 230 / 0.78 / 4.8 / 10.0 / 14.6 / 6.7 / 3.9
17.2 / 240 / 0.39 / 1.8 / 3.8 / 6.9 / 2.7 / 2.5
17.8 / 250 / 0.32 / 1.5 / 2.1 / 4.9 / 1.6 / 1.9
19.2 / 150 / 0.81 / 3.9 / 5.3 / 10.8 / 4.5 / 3.6
20.5 / 240 / 0.44 / 1.4 / 2.7 / 6.1 / 2.0 / 2.3
22.1 / 170 / 0.86 / 3.3 / 3.8 / 11.0 / 4.2 / 3.8
23.0 / 240 / 0.36 / 1.3 / 1.9 / 3.9 / 1.5 / 1.9
25.8 / 230 / 1.04 / 2.7 / 5.8 / 10.7 / 4.0 / 3.8
26.8 / 240 / 0.52 / 1.4 / 2.5 / 4.8 / 1.8 / 2.0
27.9 / 150 / 0.68 / 1.8 / 3.3 / 6.0 / 2.3 / 2.5
31.8 / 280 / 0.84 / 2.4 / 4.6 / 6.2 / 2.9 / 2.3
36.3 / 280 / 1.20 / 3.3 / 5.0 / 7.6 / 3.6 / 2.8
39.6 / 140 / 2.12 / 4.6 / 7.9 / 12.5 / 6.5 / 5.5
40.6 / 200 / 0.55 / 1.5 / 1.5 / 3.2 / 1.4 / 1.0
41.3 / 240 / 0.27 / 0.8 / 0.8 / 1.9 / 0.7 / 0.5
42.4 / 140 / 0.78 / 1.8 / 2.3 / 4.1 / 2.3 / 1.0
44.6 / 200 / 1.22 / 1.6 / 2.5 / 5.2 / 2.7 / 1.4
46.8 / 70 / 2.44 / 3.9 / 12.1 / 11.0 / 6.4 / 5.9
48.1 / 90 / 1.44 / 2.7 / 5.7 / 7.1 / 4.2 / 2.4
Sex: F
Age at time of experiment: 29
Weight at time of experiment: 60
Hours after exposure / Urine Volume, mL / Creatinine, g/L / MEHP, µg/L / 5OH-MEHP, µg/L / 5cx-MEPP, µg/L / 5oxo-MEHP, µg/L / 2cx-MMHP, µg/L0.0 / 425 / 0.17 / 2.2 / 3.3 / 3.7 / 3.0 / 1.6
2.3 / 375 / 0.37 / 8.7 / 16.3 / 13.7 / 12.7 / 2.7
7.3 / 425 / 0.58 / 4.1 / 15.1 / 18.2 / 12.4 / 4.9
9.0 / 420 / 0.51 / 1.0 / 3.3 / 3.9 / 2.7 / 1.8
10.8 / 325 / 0.40 / 1.0 / 2.5 / 4.1 / 2.6 / 1.9
19.0 / 410 / 1.02 / 2.1 / 6.6 / 8.5 / 6.3 / 3.3
24.8 / 250 / 1.31 / 2.2 / 6.4 / 7.6 / 6.5 / 2.3
27.8 / 300 / 0.47 / 0.5 / 1.7 / 2.7 / 2.1 / 0.9
33.5 / 400 / 0.74 / 1.0 / 4.1 / 5.7 / 4.2 / 1.4
35.5 / 600 / 0.40 / 0.3 / 0.8 / 1.4 / 0.8 / 0.3
44.3 / 220 / 1.53 / 2.2 / 7.3 / 10.2 / 6.1 / 2.6
47.0 / 100 / 1.22 / 2.2 / 4.0 / 7.7 / 7.2 / 2.3
Figure S1 shows the ratio of the tertiary metabolite, 5cx-MEPP, to the secondary metabolite, 5OH-MEHP. The trends seen in this Figure might, in fact, reflect a “chronic” general population exposure to DEHP. The decline in concentrations when fasting does suggest food to be the exposure media for DEHP, but the trend in the ratio is not the same as the two individuals who were highly exposed. While generally starting out at 1 and below suggesting perhaps a recent exposure and then rising to 1.5 and more, there is a dip in the ratio for all three individuals after about a day (not below 1.0, however) and then a second rise until the end of the experiment. The second rise occurs from hours 32 to 44, when the ratio again approaches and exceeds 2.0. This dip during day 2 could reflect a trend with chronic exposures of delayed metabolism, release of stored phthalate, or some other phenomena that could also have occurred with the self-dosed individual (as discussed above). In any case, we see with these fasting individuals, the trend being highlighted for this study: initially higher concentrations of 5OH-MEHP than 5cx-MEPP, which then reverses at about 8 hours post exposure staying greater than 1.0 and mostly above 1.5 during a time when one might reasonably hypothesize that there was no exposure to DEHP.
Figure S3. Ratio of the secondary metabolites, 5cx-MEPP to 5OH-MEHP, for three fasting individuals (the circle, triangle, and square are the data points for each individual; see text for more detail).
3. Raw Urine Data from the Two High-Dose Individuals
The raw data from the two individuals used to develop the alternate model to estimate intakes, and also estimate the intakes using the creatinine correction data, are provided in this section.
I. Self-Dosed Individual
The study involving the self-dosing at 48.5 mg DEHP is described in Koch et al (2004; 2005b).
Sex: M
Age at time of experiment: 67
Weight at time of experiment: 87 kg
Administration of DEHP: on buttered toast.
Hours after exposure / Urine Volume, mL / Creatinine, g/L / MEHP, mg/L / 5OH-MEHP, mg/L / 5cx-MEPP, mg/L / 5oxo-MEHP, mg/L / 2cx-MMHP, mg/L0.0 / 140 / 0.82 / 0.001 / 0.001 / 0.001 / 0.001 / 0.001
1.2 / 90 / 1.23 / 2.639 / 4.392 / 2.821 / 2.264 / 0.117
1.6 / 150 / 0.38 / 3.627 / 6.630 / 6.874 / 4.350 / 0.270
3.0 / 210 / 0.34 / 2.370 / 8.774 / 6.512 / 5.543 / 0.474
4.1 / 140 / 0.51 / 1.460 / 10.036 / 8.161 / 6.227 / 0.805
5.3 / 180 / 0.58 / 1.236 / 6.782 / 6.225 / 3.936 / 0.825
8.1 / 240 / 1.03 / 2.017 / 5.540 / 4.821 / 3.164 / 0.927
10.2 / 190 / 1.19 / 0.976 / 3.299 / 3.395 / 1.993 / 0.918
12.1 / 250 / 0.71 / 0.277 / 1.447 / 1.386 / 0.838 / 0.455
13.2 / 180 / 0.66 / 0.164 / 0.781 / 0.898 / 0.439 / 0.409
14.1 / 60 / 0.96 / 0.243 / 1.255 / 1.499 / 0.687 / 0.641
18.1 / 460 / 0.45 / 0.048 / 0.346 / 0.510 / 0.209 / 0.241
20.2 / 230 / 0.78 / 0.052 / 0.615 / 0.685 / 0.309 / 0.421
21.3 / 140 / 0.78 / 0.026 / 0.316 / 0.510 / 0.164 / 0.407
23.3 / 130 / 1.09 / 0.031 / 0.474 / 0.983 / 0.229 / 1.199
25.3 / 250 / 0.61 / 0.008 / 0.150 / 0.367 / 0.091 / 0.393
27.5 / 240 / 0.62 / 0.004 / 0.167 / 0.312 / 0.099 / 0.314
30.1 / 180 / 1.04 / 0.006 / 0.222 / 0.366 / 0.121 / 0.579
31.1 / 180 / 0.61 / 0.006 / 0.072 / 0.184 / 0.040 / 0.241
31.4 / 150 / 0.45 / 0.002 / 0.035 / 0.113 / 0.026 / 0.171
34.5 / 120 / 2.25 / 0.007 / 0.345 / 0.749 / 0.156 / 0.805
36.2 / 230 / 0.81 / 0.003 / 0.046 / 0.110 / 0.039 / 0.171
39.3 / 260 / 0.97 / 0.001 / 0.066 / 0.149 / 0.036 / 0.246
43.2 / 190 / 1.28 / 0.002 / 0.090 / 0.136 / 0.050 / 0.214
44.2 / 110 / 1.29 / 0.002 / 0.081 / 0.113 / 0.054 / 0.168
II. Apheresis Patient
The study involving an apheresis procedure to donate platelets which introduced 2.6 mg DEHP is described in Koch et al (2005a,c).
Sex: M
Age at time of experiment: 35
Weight at time of experiment: 83 kg
Administration of DEHP: through plastic tubing directly into the bloodstream
Hours after exposure / Urine Volume, mL / Creatinine, g/L / MEHP, µg/L / 5OH-MEHP, µg/L / 5cx-MEPP, µg/L / 5oxo-MEHP, µg/L / 2cx-MMHP, µg/L0.0 / 160 / 0.41 / 25.4 / 77.7 / 46.1 / 67.1 / 4.5
3.8 / 240 / 1.32 / 388.0 / 822.0 / 577.0 / 729.0 / 125.0
7.7 / 260 / 2.08 / 158.0 / 314.0 / 460.0 / 275.0 / 201.0
11.8 / 210 / 1.40 / 76.0 / 119.0 / 178.0 / 105.0 / 90.9
18.5 / 280 / 0.98 / 59.6 / 90.5 / 144.0 / 74.6 / 74.4
23.9 / 320 / 0.66 / 19.1 / 32.3 / 60.4 / 26.6 / 32.4