The Value of Plasma Paraquat Concentration in
Predicting Therapeutic Effects of Haemoperfusion in
Patients with Acute Paraquat Poisoning
Yunying Shi1., Yangjuan Bai2., Yuangao Zou2, Bei Cai2, Fei Liu1, Ping Fu1, Lanlan Wang2*
1Department of Nephrology, West China Hospital of Sichuan University, Chengdu, People’s Republic of China, 2Department of Laboratory Medicine, West China Hospital
of Sichuan University, Chengdu, People’s Republic of China
Abstract
Objective: This study was aimed to analyze the scavenging effect of haemoperfusion on plasma paraquat (PQ) and to
evaluate the clinical significance of PQ examination in the treatment of patients with acute paraquat poisoning.
Methods: 85 patients with acute paraquat intoxication by oral ingestion were admitted in West China Hospital from Jun,
2010 to Mar, 2011. A standardized therapeutic regimen including emergency haemoperfusion was given on all subjects. A
total of 91 whole blood samples were taken before (0h), underway (1h after haemoperfusion beginning) and at the end (2h)
of the haemoperfusion therapy. The clearance rate was calculated and related factors were analyzed.
Results: As heamoperfusion was going on, the plasma paraquat concentration of the patients kept falling down. After
1 hour of haemoperfusion, the average clearance rate (R1) was 37.06621.81%. After 2 hours of haemoperfusion, the
average clearance rate (R2) was 45.99623.13%. The average of R1/R2 ratio was 76.61622.80%. In the high paraquat
concentration group (plasma paraquat concentration (C0) .300 ng/mL), both the averages of R1 and R2 were significantly
higher than those of the low paraquat concentration group (C0#200 ng/mL) (p,0.05), and there was no significant
difference of R1/R2 between these two groups (p.0.05).
Conclusions: The dynamic monitoring of plasma PQ concentration was not only critical in the clinical evaluation but also
helpful in guiding the treatment of patients with acute PQ intoxication. Haemoperfusion can effectively eliminate paraquat
from the plasma in patients with high initial plasma PQ concentration, while in patients with low initial plasma PQ
concentration (,200 ng/ml), the clearance effect of harmoperfusion was very limited. Increasing HP time might improve
the overall clearance rate of HP on plasma PQ yet decrease the elimination efficiency of HP, while repeated HP treatment
was helpful against the rebound phenomena.
Citation: Shi Y, Bai Y, Zou Y, Cai B, Liu F, et al. (2012) The Value of Plasma Paraquat Concentration in Predicting Therapeutic Effects of Haemoperfusion in Patients
with Acute Paraquat Poisoning. PLoS ONE 7(7): e40911. doi:10.1371/journal.pone.0040911
Editor: Aditya Bhushan Pant, Indian Institute of Toxicology Reserach, India
Received April 27, 2012; Accepted June 15, 2012; Published July 20, 2012
Copyright: � 2012 Shi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was sponsored by National Natural Science Foundation of China (Nos. 81072443, 81100536, 30772051, 30950010, 81001325) and
Foundation of Education Ministry of China (20090181120099, 20090181110026). The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: wangll87@126.com
. These authors contributed equally to this work.
Introduction
Paraquat (PQ, 1,19-dimethyl-4,49-bipyridinium chloride) was a
non-selective herbicide that has been widely used in countryside
since the 1960s. Although it has been proved safe in occupational
use, PQ poisoning has been observed in patients who ingest the
pesticide either accidentally or intentionally as a suicide attempt.
The mortality rate of PQ intoxication ranged from 50–90% and
there was no specific antidote [1]. The most characteristic feature
of PQ poisoning was lung damage, the mechanism of which lay in
the selective accumulation of PQ in alveolar cells, inducing the
production of large amount of toxic free radicals such as reactive
oxygen species (ROS) which lead to lipid peroxidation of cell
membrane, exhaustion of nicotinamide adenine dinucleotide
phosphate (NADPH) and hence to cell death [2,3,4].
The clinical manifestations and outcomes of acute PQ
intoxication are dependent on the exposure degree of PQ.
Ingestion of PQ .20 ml of a 20% preparation is likely to cause
death from multi-organ failure and cardiogenic shock within 1–
4 days, while smaller quantities (10–20 ml) may initiate an
irreversible lung fibrosis and renal failure that would result in
death within several weeks. Therefore, the ingestion volume and
plasma concentration of PQ were often used as important
indicators of patients’ prognosis [5,6,7,8,9].
Extracorporeal elimination such as hemodialysis (HD) and
haemoperfusion (HP) were common measurements used clinically
in treating acute PQ intoxication. Many researchers believed that
HP was more efficient in the clearance of plasma paraquat
[10,11,12,13]. Kang et al. proved that the PQ elimination by HP
was as effective as or more effective than the renal elimination and
PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40911
inferred that early HP must be provided for life saving treatment
in patients with acute PQ intoxication [14].
?twb .2w?>Recently, many studies focused on the relationship
between HP and the overall prognosis of PQ poisoning.
Unfortunately, the results were discouraging and controversial
opinions aroused on the therapeutic effects of HP in the PQ
intoxication treatment [15,16]. However, due to the lack in
standardized treatment regimen, it’s hard to make an evaluation
objectively and factually. Meanwhile, limited studies was found
focusing on the direct evaluation of the scavenging effect of HP on
plasma PQ, most of which were in vitro simulation studies without
dynamic in vivo data. Hence, doctors can only rely on their own
clinical experiences and patients’ symptoms to perform HP, which
was not beneficial for the development of such therapy or the
improvement of the patients’ prognosis.
We have established an accurate and fast method for the
quantitative detection of human plasma PQ concentration [17].
Therefore, in order to reveal the scavenging effects of HP on
plasma PQ more directly and accurately, we plan to study the
dynamic change of plasma PQ concentration during the HP
process in patients with acute PQ poisoning, which might
contribute to the theoretical basis of clinical regimen of acute
PQ intoxication and the exploration of more efficient treatment.
Meanwhile, we also hope to reveal the predictive significance of
plasma PQ concentration monitoring in the treatment of patients
with acute PQ intoxication.
Materials and Methods
Objectives
This study was aimed to analyze the scavenging effect of
haemoperfusion on plasma paraquat (PQ) and to evaluate the
clinical significance of PQ examination in the treatment of patients
with acute paraquat poisoning.
Participants
85 patients with acute paraquat intoxication by intended oral
ingestion, who admitted to the Emergency Department of West
China Hospital from Jun, 2010 to Mar, 2011, were included in this
study (including 33male and 52 female patients, with average age of
31.24613.21 years). A detailed history taking, including demo-
graphic characteristics, past medical history, and specific questions
about the paraquat poisoning (volume ingested, time of gastric
lavage in local hospital and clinical symptoms) was completed. The
exposure to PQ poisoning was assessed by the ingestion volume and
a blood paraquat test. After checking vital signs, routine lab tests
including completed blood cell counts, liver and renal function,
coagulation function, arterial blood gas and routine urine analysis
were conducted when admission. A unified therapeutic regimen
including gastric lavage, fluid replacement, antioxidants (VitaminC,
Vitamin B and L-Glutathione) and immunosuppressant (corticoste-
roids) was given to all the patients. Then emergency haemoperfusion
was performed on all the patients except ones who were diagnosed
withmultiple organ failure with unstable vital sign on admission.HP
was conducted through a double lumen femoral venous catheter
(Gambro, Germany) for 2 hours at a blood flow rate of 200 ml/min,
using AK-200 haemoperfusion machine(Gambro, Germany)and a
resin-containing column coated with polycarbonate (HA-230, Zhu
Hai Jian Fan, China). We followed up these patients after they
discharged from the hospital.
Sample Collection
A set of three whole blood samples were withdrawn from
poisoned patients and collected in heparinized tubes at the
beginning (0h), underway (1h after haemoperfusion beginning)
and the end (2h) of HP therapy, respectively, which were used for
the examination of plasma PQ concentration and marked
accordingly as C0, C1 and C2. 84 patients received emergent
haemoperfusion, among whom 6 patients received repeated
haemoperfusions (5 patients received twice and one patients
received three times), thus there were totally 91 sets of samples.
Examination of Plasma PQ Concentration
We used an improved approach for extraction and analysis of
paraquat in human plasma [17]. A high performance liquid
chromatographic (HPLC) system (Shimadzu, Japan) was used,
using diethyl paraquat as an internal standard. The sample
separation was achieved on a XtimateTM C18 column
(25064.6 mm, 5 um particle size)(Shimadzu, Japan). PQ and the
internal standard (IS) were eluted under 35uC at a flow-rate of
1.0 ml/min and monitored by UV absorption at 256 nm. The
mobile phase containing 150 mg sodium dodecyl sulfate (SDS)
and 0.1 M orthophosphoric acid in 850 ml of deionized water was
made, of which the pH was adjusted to 3.0 with the addition of
triethylamine, and then acetonitrile was added to yield a 15% (v/v)
proportion. Stock standard solutions (200 ug/ml) of PQ and IS
were prepared in deionized water and stored at 4uC till use. The
plasma samples (0.5 ml/sample) were spiked with 10 ul of known
amounts of IS to yield a final concentration of 10 ug/ml. Samples
were vortex-mixed after adding 1 ml acetonitrile and then
centrifuged for 5 min at 12000 rpm in an Eppendorf 5810R
Centrifuge (Eppendorfchina Ltd., Shanghai, China), Supernatants
were transferred into a round bottom glass tube containing 3 ml
methylene chloride, vortex-mixed for 5 min and centrifuged for
5 min at 2500 rpm. Finally, 100 ul upper aqueous phase was
injected to HPLC analysis.
The detection limit (LOD) of this method was 10 ng/ml and the
calibration curve obtained from extraction of plasma containing
known amounts of PQ was linear over the quantities ranged from
20 ng/ml to 80000 ng/ml (R= 0.9999). The intra-day precision
was less than 4.26%. The inter-day precision at all concentrations
examined was less than 4.17%. The recovery rate of this method
ranged from 88% to 115%.
The Clearance Rate of Haemoperfusion on Plasma PQ (R)
We calculated the clearance rate according to the following
formula: Rx = 1006(C02Cx)/C0 (%). Rx could be R1 or R2,
representing the clearance rate of 1 hour HP or 2 hour HP on
plasma PQ. C0 stood for the plasma PQ concentration at the
beginning of HP treatment, while Cx stood for C1 or C2,
representing the plasma PQ concentration at corresponding time
point.
Ethics Statement
The ethics committee of West China Hospital of Sichuan
University specifically approved the study of ‘‘The value of plasma
paraquat concentration in predicting therapeutic effects of
haemoperfusion in patients with acute plasma paraquat poison-
ing’’. All the 85 patients collected from Jun, 2010 to Mar, 2011 in
this study were fully aware of the purpose and content of the study,
and the clinical investigation was conducted according to the
principles expressed in the Declaration of Helsinki. All the invasive
therapeutic procedures were conducted with their voluntary
permission and written informed consent. Because patients were
all admitted from the emergency department in an urgent
situation, and the test of plasma paraquat concentration was
necessary to the treatment and prognosis, as well as no extra costs
or procedures would be generated besides necessary treatment in
Dynamic Monitoring on Plasma Paraquat Level
PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40911
the study, so the permission to the use of patients’ paraquat
concentration data in this study were obtained by verbal informed
consent instead of written form from all the patients, and all the
data were analyzed anonymously. The ethics committee under-
stood the situation and specifically approved such procedure.
Statistical Analysis
The results were presented as mean 6 standard error (X6SD).
The differences among 3 groups were tested by one-way ANOVA
while the comparison between 2 groups was achieved by Dunnett
T3 or Tamhane’s T2. The data was analyzed using the Statistical
Package for the Social Sciences version 14.0 (SPSS Inc, Chicago,
IL, USA) statistical software. A p-value ,0.05 was considered to
be statistically significant.
Results
Till May 2nd, 2011, 44 out of 85 patients died. The overall
fatality rate was 48.24% and their medial survival time was 81.00
(2.00–201.50) days. The medial amount of PQ ingestion was 20
(10–50) ml. The average time from poisoning to gastric lavage was
0.5 (0.5–2.0) hour, while to the starting of HP was 6.75 (5.00–
9.88) hours.
The Change of Plasma PQ Concentration as
Haemoperfusion Going on
In our study, the medial plasma PQ concentration of the
poisoned patients before HP treatment (C0) was 921.06 (201.57–
5446.51) ng/ml. The average PQ concentration of all the 85
patients declined about 37.06621.81% after 1 hour of HP
treatment. Then the decrease of PQ concentration slowed down
dramatically in the second hour. As shown by Figure 1, there was
an obvious inflection point after the first hour. The average PQ
clearance rate after 2 hours of HP (R2) was 45.99623.13% and
the average R1/R2 was 76.61622.80%.
The Relationship Between Plasma PQ Clearance Rate and
C0
By analyzing the plasma PQ clearance rate of patients with
different C0 (Figure 2), we found that the PQ clearance rate (R2) of
patients with C0 lower than 200 ng/mL were all below 40%, while
for patients with C0 higher than 300 ng/mL, their R2 were all
above 40%. Thus, according to C0, we classified the patients into 3
groups: group A with C0#200 ng/mL, group C with
C0.300 ng/mL and group B in between.
The Comparison Among Three Groups with Different C0
No significant difference was found in gender, age, time from
intoxication to gastric lavage, the time from intoxication to the
starting of HP among three groups. As for ingestion volume, C1
and survival time, there were significant differences among 3
groups (Table 1). Patients in group A ingested much less PQ
compared to patients in group B and C (p,0.05). C1 of patients in
three groups followed a tendency as below: group C.group
B.group A (p,0.05). The survival time of patients in group C
were much shorter than that in group A and B (p,0.05).
Table 2 showed the average plasma PQ clearance rate in each
group. Both R1 and R2 were the highest in group C and the
lowest in group A, while the levels in group B were in between. R1
in group C was obviously higher than that in group A and B
(p,0.05). Though R1 in group B was higher than that in group A,
there was no significant difference (p.0.05). There was a statistical
significance in R2 among 3 groups (p,0.05). No significant
difference was found in R1/R2 among 3 groups (p.0.05). The
relevance between R1, R2 and C0 was poor (r: 0.172,0.332), so
was the relevance between C0 and the clearance rate in each
group (r: 0.076,0.381).
The Plasma PQ Concentrations of Patients Receiving
Repeated HP
There were 6 patients in our study who received repeated HP. 5
of them received the second session of HP the next day, and one
patient received HP once a day for continuous three days. The
plasma PQ concentrations prior to the next session of HP (C0–2) of
most patients (71.42%) were higher than their concentrations after
the first session of HP (C2–1), while the rebound rates varied from
27.56% to 69.80% (Table 3).
Discussion
Since the outcome of the patients with acute PQ intoxication is
closely related to the plasma PQ concentration, the therapeutic
effect of HP is closely associated with the elimination efficiency of
HP on plasma PQ. The elimination efficiency of HP on plasma
PQ was affected by many factors, while the haemoperfusion time
was an important one among them. An in vitro study had reported
that during the 2-hour HP treatment, the plasma PQ decreased
dramatically as time went by (0.5, 1.0, 1.5h), but after 2 hours
(2.5h, 3.0h), there was no obvious change in plasma PQ
concentration anymore [7]. Considering the adverse effect and
the limited advantage of long time HP, the HP time of the patients
with acute PQ poisoning was set for 2 hours in our dialysis center.
During the 2-hour process of haemoperfusion, the plasma PQ
concentration of all the poisoned patients in our study declined
continuously as HP going on,which inferred that PQ could be
eliminated persistently from patients’ plasma during HP. After
2 hours of HP, the average clearance rate of plasma PQ (R2) was
45.99623.13%. But the result of an in vitro study showed that the
clearance rate of PQ after 2 hours of HP could reach
97.69,99.52%, which was obviously higher than our data [18].
It inferred that the elimination ability of HP on plasma PQ
differed from in vivo to in vitro. Thus, the direct observation on
the elimination effect of HP on plasma PQ of poisoned patients is
more significant and necessary in clinical guidance. Sae-Yong
Honga et al. reported that the PQ clearance rate of patients in
survival group after 4-hour HP was 80.3619.9% compared to
67.2619.2% in non-survival group, which was slightly higher than
our result [10]. The prolonging of the perfusion time could
increase the effective contact time between plasma toxin and resin
particles, so that more toxins could be adsorbed on resin and then
eliminated from plasma. Therefore, we inferred that different
haemoperfusion time might be the reason to the difference in the
overall PQ clearance rate between two studies. The advantage of
long time HP was limited, because the complications such as
imbalance of coagulative function and low blood pressure would
increase and the elimination efficiency would decrease as the time
went on. A recent in vitro study had reported that the elimination
of HP on plasma could decrease from 215 ml/min at 30 min of
HP to 22 ml/min at the 6th hour [18]. Similar result was also
found in our study. In our study, we also found that the clearance
rate in the first hour of HP took up about three quarters of the
overall PQ clearance rate and the decrease of PQ concentration
slowed down dramatically in the second hour (Figure 1), which
might infer that the elimination efficiency of HP was higher in the
early phase and would decrease as time went on. At the beginning
of HP, the resin particles were blank and their binding capacity
was strong. The toxin can be adsorbed quickly and firmly by the
Dynamic Monitoring on Plasma Paraquat Level
PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40911
Figure 1. The change of average plasma PQ concentration as HP going on. The X axis stands for the treatment time of haemoperfusion
(hour), while the Y axis stands for average plasma PQ concentration (ng/ml).
doi:10.1371/journal.pone.0040911.g001
Figure 2.