Phase I: Initial cross-sectional cohort
Baseline characteristics
BLL was screened in a total of 720 participants. A total of 180 participants had a BLL > 40 μg/dl. This level indicated that chronic lead toxicity was present and that chelation therapy was required (Figure 1). Among the 180 participants, 86 participants met the inclusion and exclusion criteria and were enrolled in the study. Table 1 shows the baseline characteristics of the 86 participants. Most of the participants were males (n=71, 85%), and the average age was 37.6 ± 7.3 years. The mean BLL was 81.4 ± 9.8 μg/dl. Twenty-six (30.2%) participants had hepatitis; they had SGOT levels above the ULN (>35 mg/dl, n=14) and/or SGPT above the ULN (>40 mg/dl, n=17).
The mean LS value of the 86 participants was 5.4 ± 0.9 kPa. Notably, 23 participants (26.7%) from this cohort had significant fibrosis (i.e., the LS value was > 6.1 kPa). The mean CAP was 225.1 ± 49.3 dB/m. Forty-four (51.2%) of the participants had CAP > 213 dB/m, indicating that there was significant liver steatosis. Among those with significant fibrosis, 42 participants (48.8%) had no steatosis (S0), while 30 (34.8%) and 15 (17.4%) participants had mild-moderate steatosis (S1-2) and severe steatosis (S3), respectively. The numbers of participants with LS and CAP are shown in Figure 2.
Table 1 Baseline pretreatment laboratory profiles of 86 participants in the initial cross-sectional cohort
Parameters
|
Mean ± SD
|
Age (years)
|
37.6 ± 7.3
|
Waist (cm)
|
80.2 ± 12.0
|
BMI (kg/m2)
|
24.2 ± 4.9
|
Blood lead level (µg/dL)
|
81.4 ± 9.8
|
Hemoglobin (g/dL)
|
13.4 ± 1.6
|
White blood cell count (103/μL)
|
8.0 ± 1.8
|
Platelets (1012/L)
|
285.6 ± 58.4
|
Creatinine (mg/dL)
|
0.9 ± 0.3
|
Total bilirubin (mg/dL)
|
0.8 ± 0.4
|
Direct bilirubin (mg/dL)
|
0.3 ± 0.1
|
Alkaline phosphatase (U/L)
|
68.8 ± 19.5
|
SGOT (U/L)
|
30.7 ± 28.9
|
SGPT (U/L)
|
33.3 ± 33.2
|
Liver stiffness (kPa)
|
5.4 ± 0.9
|
CAP (dB/m)
|
225.1 ± 49.3
|
SGOT, serum glutamate oxaloacetate transaminase; SGPT, serum glutamate pyruvate aminotransferase; CAP, controlled attenuation parameters
Factors associated with liver fibrosis and steatosis
In the univariate analysis, the duration of lead exposure, but not BLL, was significantly associated with the degree of hepatic fibrosis (Pearson’s r=0.249, p=0.021). Other factors associated with liver fibrosis included age, BMI, liver steatosis, SGPT, and ALP (Table 2). In the multivariate analysis, only the duration of lead exposure and the SGPT level remained independently associated with liver fibrosis, with Pearson’s r values of 0.229 and 0.317 (p= 0.026 and 0.002), respectively.
The following variables were found to be significantly associated with the presence of liver steatosis in the univariate model: sex, WC, BMI, liver fibrosis, SGOT, and SGPT. WC and SGPT were independently associated with liver steatosis, with Pearson’s r values of 0.524 and 0.397 (p<0.001 and 0.018), respectively. We did not detect any association between lead-related parameters and liver steatosis (Table 2).
Table 2 Potential factors that may be associated with liver fibrosis and steatosis in the initial cross-sectional cohort
Parameters
|
Factors associated with liver fibrosis
|
Factors associated with liver steatosis
|
Univariate analysis
|
Multivariate analysis
|
Univariate analysis
|
Multivariate analysis
|
Pearson Correlation
|
P-value
|
Pearson Correlation
|
P-value
|
Pearson Correlation
|
P-value
|
Pearson Correlation
|
P-value
|
Age
|
0.215
|
0.047
|
0.195
|
0.060
|
-0.042
|
0.703
|
-
|
-
|
Sex
|
-0.155
|
0.153
|
-
|
-
|
-0.237
|
0.028
|
-0.161
|
0.062
|
BLL
|
-0.086
|
0.430
|
-
|
-
|
-0.075
|
0.493
|
-
|
-
|
Duration of lead exposure
|
0.249
|
0.021
|
0.229
|
0.026
|
0.020
|
0.854
|
-
|
-
|
Waist circumference
|
0.220
|
0.076
|
-
|
-
|
0.702
|
<0.001
|
0.524
|
<0.001
|
BMI
|
0.226
|
0.037
|
0.107
|
0.350
|
0.660
|
<0.001
|
2.064
|
0.360
|
Liver steatosis
|
0.242
|
0.025
|
0.080
|
0.525
|
1
|
-
|
-
|
-
|
Liver fibrosis
|
1
|
-
|
-
|
-
|
0.242
|
0.025
|
2.155
|
0.656
|
SGOT
|
0.161
|
0.142
|
-
|
-
|
0.329
|
0.002
|
-0.531
|
0.273
|
SGPT
|
0.332
|
0.002
|
0.317
|
0.002
|
0.566
|
<0.001
|
0.397
|
0.018
|
ALP
|
0.222
|
0.041
|
0.107
|
0.347
|
0.154
|
0.161
|
-
|
-
|
BLL, blood lead level; BMI, body mass index; SGOT, serum glutamate oxaloacetate transaminase; SGPT, serum glutamate pyruvate aminotransferase; ALP, alkaline phosphatase
Phase II. Interventional prospective cohort
Effects of chelation therapy on liver fibrosis and steatosis
An association between liver fibrosis and steatosis was detected in our initial cross-sectional cohort (Pearson’s r =0.242, p=0.025). After excluding participants with severe fatty liver and those with high-risk features for metabolic syndrome as described previously, a total of 70 participants were enrolled in this study. Correlation analyses were repeated to confirm the independent effects between the degree of liver fibrosis and steatosis. We found that there was no significant association between the degree of fibrosis and steatosis. The Pearson’s r between the level of LS and CAP at the prechelation phase was -0.039 (p = 0.75), and at the postchelation phase, it was 0.151 (p = 0.21). Pearson’s correlation analysis between the degree of postchelation LS reduction and the degree of postchelation CAP reduction was 0.160 (p = 0.19) (Supplementary table 1).
After 3 months of chelation therapy, the mean BLL decreased from 81.8 + 9.9 to 56.6 + 16.8 µg/dL (30.8%). After treatment, the degree of LS decreased significantly from 5.33 + 0.9 to 4.8 + 1.4 kPa (p=0.001). We did not find significant improvement in liver steatosis after chelation therapy (mean pre- and postchelation CAP levels were 208.6 + 31.7 and 207.0 + 45.0 dB/m, p=0.738, respectively) (Table 3).
Table 3 Comparison of lead-related parameters between pre- and postchelation therapy
Parameters
|
Prechelation
(mean + SD)
|
Postchelation (mean + SD)
|
Mean difference between post- and prechelation (95% confidence interval)
|
p-value
|
Blood lead level (µg/dL)
|
81.8 ± 9.9
|
56.6 ± 16.8
|
-25.2 + 13.8 (21.9 – 28.5)
|
<0.001
|
Liver stiffness (kPa)
|
5.3 ± 0.9
|
4.8 ± 1.4
|
- 0.5 + 1.2 (0.2-0.8)
|
0.001
|
Steatosis (dB/m)
|
208.6 ± 31.7
|
207.0 ± 45.0
|
-1.6 + 41.0 (-8.1-11.4)
|
0.738
|
TNF-α (pg/mL)
|
371.6 ± 211.3
|
215.8 ± 142.7
|
-155.8 + 137.4 (120.9-190.7)
|
<0.001
|
Interleukin-1β (pg/mL)
|
29.8 ± 1.7
|
25.9 ± 4.3
|
-3.8 + 3.7 (2.9-4.8)
|
<0.001
|
Interleukin-6 (pg/mL)
|
46.8 ± 10.2
|
35.0 ± 11.9
|
-11.8 + 10.6 (9.1-14.5)
|
<0.001
|
Glutathione (µg/mL)
|
3.3 ± 3.3
|
13.1 ± 3.7
|
9.8 + 3.7 (10.8 - 8.9)
|
<0.001
|
TNF, tumor necrosis factor
Effects of chelation therapy on oxidative stress and inflammatory markers
The mean levels of the inflammatory biomarkers TNF-α, IL-1β and IL-6 were significantly reduced after chelation therapy by 41.93% (371.6 + 211.3 to 215.8 + 142.7 pg/mL), 13.09% (29.8 + 1.7 to 25.9 + 4.3 pg/mL), and 25.21% (46.8 + 10.2 to 35.0 + 11.9 pg/mL), respectively. On the other hand, the mean GSH level significantly increased after chelation therapy from 3.3 + 3.3 to 13.1 + 3.7 µg/mL (297.0%) (Table 3). However, the correlation between the degree of change in BLL and the reductions in TNF-α and IL-6 levels was not significant. The increase in the level of GSH was also not significant (Table 4).
Table 4 Degree of correlation between reduced BLL, LS, and CAP for each inflammatory marker studied
Inflammatory markers affected by chelation treatment
|
Reduced BLL
|
Reduced LS
|
Reduced CAP
|
Pearson’s Correlation
|
p-value
|
Pearson’s Correlation
|
p-value
|
Pearson’s Correlation
|
p-value
|
TNF-α
|
0.212
|
0.919
|
-0.014
|
0.909
|
-0.237
|
0.048
|
Interleukin-1β
|
0.034
|
0.778
|
0.045
|
0.714
|
0.008
|
0.945
|
Interleukin-6
|
0.118
|
0.332
|
0.020
|
0.872
|
0.055
|
0.652
|
Glutathione
|
-0.100
|
0.410
|
-0.030
|
0.802
|
-0.079
|
0.517
|
BLL, blood lead level; LS, liver stiffness; CAP, controlled attenuation parameters; TNF, tumor necrosis factor