Environ Sci Pollut Res (2013) 20:4993–5002
DOI 10.1007/s11356-013-1472-9
RESEARCH ARTICLE
The function of digestive enzymes on Cu, Zn, and Pb release
from soil in in vitro digestion tests
Yi Li & Walelign Demisie & Ming-kui Zhang
Received: 4 September 2012 / Accepted: 7 January 2013 / Published online: 18 January 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract The bioaccessibility of soil heavy metals is the
solubility of soil heavy metals in synthetic human digestive
juice, which is usually determined using in vitro digestion
test. To reveal the effects of digestive enzymes on soil heavy
metals bioaccessibility, three representative in vitro diges-
tion tests, Simple Bioaccessibility Extraction Test (SBET),
Physiologically Based Extraction Test (PBET), and Simple
Gastrointestinal Extraction Test (SGET), were chosen. The
bioaccessibility of soil Cu, Zn, and Pb in each method were
respectively evaluated with and without digestive enzymes,
and the differences were compared. The results showed that
the effects of digestive enzymes varied with different meth-
ods and elements. Because of digestive enzymes addition,
the environmental change from acid gastric phase to neutral
intestinal phase of PBET did not result in apparently de-
crease of the bioaccessibility of soil Cu. However, the
solubility of soil Zn and Pb were pH-dependent. For
SGET, when digestive enzymes were added, its results
reflected more variations resulting from soil and element
types. The impacts of digestive enzymes on heavy metal
dissolution are mostly seen in the intestinal phase.
Therefore, digestive enzyme addition is indispensable to
the gastrointestinal digestion methods (PBET and SGET),
while the pepsin addition is not important for the methods
only comprised of gastric digestion (SBET).
Responsible editor: Zhihong Xu
Y. Li : W. Demisie : M.-k. Zhang (*)
Zhejiang Provincial Key Laboratory of Subtropical Soil
and Plant Nutrition, College of Environmental and Natural
Resource Sciences, Zhejiang University,
Hangzhou 310058, China
e-mail: mkzhang@zju.edu.cn
W. Demisie
Department of Dry Land Crop Science,
Jijiga University, Jijiga, Ethiopia
Keywords In vitro digestion tests . Digestive enzymes .
Soil . Heavy metals
Introduction
In vitro digestion test has been widely applied to the risk
assessment of As and metals contamination in soils, mine
wastes, and dusts in the past two decades (Williams et al.
1998; Kim et al. 2002; Kientz et al. 2003; Turner and Ip
2007; Bosso and Enzweiler 2008; Hu et al. 2011). Although
various in vitro digestion tests have been developed, a
complete in vitro digestion test is usually composed of three
steps: mouth, gastric phase, and intestinal phase. Mouth is
the first step of soil digestion. Considering that pH is the
major factor governing the solubility of heavy metals
(Chuan et al. 1996), the pH of saliva is nearly neutral and
the retention time in mouth is too short (Intawongse and
Dean 2006; Dean and Ma 2007), mouth digestion is omitted
in many in vitro digestion tests. Stomach, whose digestive
enzyme is pepsin, is the main site of digestion. The gastric
phase is the most important part of in vitro digestion tests
because of its acid environment (Ruby et al. 1996; Turner
and Ip 2007). The intestine is the main site where elements
are absorbed. The intestinal digestive enzymes are basically
composed of bile salts and pancreatin. Technically, bile salts
cannot be regarded as a kind of enzyme, but bile salts are
still called as digestive enzyme hereinafter for succinct
expression. Soil heavy metal mobilization is inhibited by
the neutral environment of the intestine, hence soluble met-
als in the stomach may be deposited after they enter the
intestine. Many in vitro digestion tests are made up of
gastric phase and intestinal phase. Based on the presence
or absence of pepsin, gastric juice falls into two categories:
acid solution without pepsin (the digestive juice of Simple
Bioaccessibility Extraction Test (SBET)) and acid solution
4994
with pepsin (the digestive juice of Physiologically Based
Extraction Test (PBET) and Simple Gastrointestinal
Extraction Test (SGET)). After gastric digestion and neu-
tralization, intestinal juice can be divided into two groups:
intestinal juice with bile salts and pancreatin, and intestinal
juice without bile salts and pancreatin.
It is understandable the in vitro digestion test with all
digestive enzymes (pepsin, bile salts and pancreatin) cer-
tainly reproduces a more similar situation to human diges-
tive tract; however, as a screening-level method, emphasis
of in vitro digestion test sometimes is measuring the bio-
accessibility easily, quickly, and reproducibly for the pur-
poses of precaution and protection. Therefore, there are
usually three kinds of opinions about whether digestive
enzymes addition is necessary for in vitro digestive tests:
they are considered to be entirely, partially, or barely impor-
tant. For instance, Rodriguez and Basta (1999) preferred an
in vitro digestion test containing all digestive enzymes.
Turner and Simmonds (2006) chose an in vitro digestion
test only comprising pepsin, and Lamb et al. (2009) would
rather apply an in vitro digestion test without digestive
enzymes at all. Efforts were made to uncover the role of
digestive enzymes in in vitro digestion tests. Oomen et al.
(2003a) studied the variation of bioaccessibility with differ-
ent concentrations of bile, but a decreasing or increasing
trend was not observed. Oomen et al. (2004) compared the
effects of different animal origin bile on the results of in
vitro digestion test and considered the bioaccessibility dif-
ferences resulting from bile type to be irrelevant for risk
assessment purpose. Still, no definite conclusion has been
given on the function of digestive enzymes in in vitro
digestion test. According to the chemical character of diges-
tive enzymes, researchers are inclined to think they may
help heavy metals stay soluble in simulated digestive juice
as a result of forming complexes with metals (Ruby et al.
1993; Oomen et al. 2003b; Wragg et al. 2011). The concen-
trations and mobilizations of Cu, Zn, and Pb in soils are
relatively high, and excess accumulations of Cu, Zn, and Pb
in human body cause serious health damage. The bioacces-
sibility of soil Cu, Zn, and Pb were frequently evaluated and
discussed in previous researches (Turner and Ip 2007;
Poggio et al. 2009; Roussel et al. 2010; Sialelli et al.
2010; Pelfrene et al. 2011). Therefore, three in vitro diges-
tion tests, SBET, PBET, and SGET, were selected to study
the effects of digestive enzymes on the bioaccessibility of
soil Cu, Zn, and Pb (the bioaccessibility in this paper refers
to the amounts of soil heavy metals extracted by in vitro
digestion test). The designs of these three in vitro digestion
tests distinctly vary and just respectively fall into the three
opinions described above. Then the reaction between diges-
tive enzymes and heavy metals in in vitro digestion test was
discussed, some phenomena were explained, and the role of
digestive enzymes in in vitro digestion tests was revealed.
Environ Sci Pollut Res (2013) 20:4993–5002
This study differed from previous researches in laying stress
on the influences of environmental conditions, such as
method, element, and soil types.
Material and methods
Introductions of three in vitro digestion tests
SBET, a method without intestinal phase and digestive
enzymes, was adopted by British Geological Survey and
described by Oomen et al. (2002). PBET, a method includ-
ing gastric and intestinal phase with corresponding digestive
enzymes, was firstly described by Ruby et al. (1996), then
modified by Cave et al. (2003). Its details were depicted by
Intawongse and Dean (2008). The last method called as
SGET in this paper is composed of gastric phase with pepsin
and intestinal phase without bile salts and pancreatin. It was
from US Pharmacopoeia (USP XXII 1990) at the time it
merely consisted of gastric phase, then was adopted by
Hamel et al. (1998) and improved by Hamel et al. (1999)
who introduced saliva and intestinal juice, lastly adapted by
Bosso and Enzweiler (2008) who dispensed with saliva and
retained gastric and intestinal juice.
Soil samples
Three sampling locations were selected in Hangzhou and
Fuyang cities in Zhejiang province of China and would be
expressed as A, B, and C. They are contaminated by heavy
metals and their characteristics differ widely. Soil sample A
was collected on a road near a steel factory in Hangzhou
city. Soil sample B was collected from a copper industrial
zone in Fuyang city. Soil sample C was collected from a
lead-zinc mine in Fuyang city. Each of the samples consist-
ing of five subsamples was randomly taken in the surface
(0–5 cm depth) and in an area of 25 m2. Samples were air-
dried, homogenized and stored in polypropylene containers.
Some soil chemical and physical properties were given in
Table 1.
Characterizations of soil samples
Soil samples passing through 2-mm sieve were used to
analyze pH and cation exchange capacity (CEC). Soil sam-
ples passing through 0.15 mm sieve were used to measure
organic matter and total contents of heavy metals. Particle
size distribution was measured using hydrometer method.
Soil pH was determined by pH meter (Mettler Toledo,
FE20) in a 1:5 ratio of soil to distilled water. Soil organic
matter was characterized using the acid dichromate oxida-
tion method described by Yeomans and Bremner (1988).
CEC of soil was determined using the sodium acetate
Environ Sci Pollut Res (2013) 20:4993–5002 4995

Table 1 Chemical and physical properties of samples 1:1 HCl. After 1 h gastric incubation, intestinal so-
Sample A B C lution was obtained by adjusting the gastric solution
to pH7.0 with saturated NaHCO3 solution, then add-
pH 8.73 7.87 2.39 ing 52.5 mg bile salts (Fluka, 48305) and 15 mg
−1
Organic matter (gkg ) 111 31.5 11.1 pancreatin (Sigma, P3292). The incubation time of
CEC (cmol kg−1) 8.69 10.9 14.7 intestinal phase was 2 h.
Particle size distribution (%) PBET design 2 (P2): The simulated gastric solution
>2 mm 12.0 8.14 7.00 was prepared as the same as P1 (1 L mixed solution
0.2–2 mm 26.0 57.5 28.0 of 1.25 g pepsin, 0.5 g citrate, 0.5 g malate, 0.42 ml
0.02–0.2 mm 42.8 10.1 21.6 lactic acid, and 0.5 ml acetic acid, pH2.5). But after
0.002–0.02 mm 13.1 12.1 39.3 1 h gastric incubation, intestinal solution was obtained
<0.002 mm 6.07 12.2 4.13 just by adjusting the gastric solution to pH7.0 with
The total content of heavy metals, Mean (SE) saturated NaHCO3 solution. No bile salts and pancre-
Cu (mgkg−1) 132 (5.73) 375 (18.6) 105 (2.24) atin were added. The incubation time of intestinal
Zn (mgkg−1) 1034 (62.0) 722 (10.5) 1281 (51.4) phase was 2 h.
Pb (mgkg )−1
140 (13.6) 2711 (4.04) 259 (6.65) PBET design 3 (P3): The simulated gastric solution
was prepared by dissolving 0.5 g citrate, 0.5 g ma-
SE standard error late, 0.42 ml lactic acid, and 0.5 ml acetic acid in
1 L distilled water. The pH of gastric solution was
method (Nakos 1987). The total contents of heavy metals in adjusted to 2.5 with 1:1 HCl. After 1 h gastric
soil were measured after digestion procedure (HCl-HNO3- incubation, intestinal solution was obtained by adjust-
HF-HClO4) described in SEPA (1997). ing the gastric solution to pH 7.0 with saturated
NaHCO3 solution, then adding 52.5 mg bile salts
In vitro digestion tests and 15 mg pancreatin. The incubation time of intes-
tinal phase was 2 h.
For the better understanding of the influence of digestive PBET design 4 (P4): The simulated gastric solution was
enzymes, the methods composed of two phases (PBET the same as P3 (1 L mixed solution of 0.5 g citrate, 0.5 g
and SGET) were carried out in two batches. One of malate, 0.42 ml lactic acid and 0.5 ml acetic acid, pH2.5).
batches only performed the gastric digestion, and the After 1 h gastric incubation, intestinal solution was
other batch completed the whole gastrointestinal diges- obtained by adjusting the gastric solution to pH7.0 with
tion. Thereby, the bioaccessibility of heavy metals in the saturated NaHCO3 solution. The incubation time of in-
gastric phase and gastrointestinal phase were both testinal phase was 2 h.
measured. 2 SGET
In vitro digestion tests were performed with sacimple
size<0.25 mm, which would adhere to hands, then be avail- SGET design 1 (SG1): The gastric solution was made
able for digestion (Schroder et al. 2004). Sample and simu- by dissolving 3.2 g pepsin, 7.0 ml concentrated HCl
late digestive juice were mixed at a solid–liquid ratio of and 2.0 g NaCl in 1 L distilled water. Although the
1:100 (in gram per milliliter) and shaken in water bath pH of gastric solution was not strictly controlled, the
(100 rpm, 37 °C). After digestion, 20 ml digestive juice of theoretical pH was 1.08 according to the composition
gastric phase or gastrointestinal phase were collected, centri- (Morrison and Gulson 2007). The pH of gastric so-
fuged (4000 rpm, 10 min), then filtered through 0.45-μm lution prepared in our lab was measured, and it was
membrane filter for analysis. about 1.1. After 2 h gastric digestion, intestinal solu-
To understand the behaviors of digestive enzymes in in tion was obtained by neutralizing the gastric solution
vitro digestion tests, PBET and SGET were performed in to pH7.0 with saturated NaHCO3 solution, then add-
four designs, and SBET was carried out in three designs. ing 52.5 mg bile salts and 15 mg pancreatin. Bile
The specifics were described below: salts and pancreatin were absent in the original de-
sign of SGET (SG2). The amounts of bile salts and
1 PBET pancreatin added were from PBET. Incubation time
PBET design 1 (P1): The simulated gastric solution of intestinal solution was also 2 h.
was prepared by dissolving 1.25 g pepsin (Sigma, SGET design 2 (SG2): The simulated gastric solution
P7125), 0.5 g citrate, 0.5 g malate, 0.42 ml lactic was made in accordance with SG1 (1 L mixed solu-
acid, and 0.5 ml acetic acid in 1 L distilled water. tion of 3.2 g pepsin, 7.0 ml concentrated HCl, and
The pH of gastric solution was adjusted to 2.5 with 2.0 g NaCl, pH 1.1). After 2 h gastric digestion,
4996
intestinal solution was obtained just by neutralizing
the gastric solution to pH7.0 with saturated NaHCO3
solution. Incubation time of intestinal solution was
2 h.
SGET design 3 (SG3): 7.0 ml concentrated HCl and
2.0 g NaCl were dissolved in 1 L distilled water to
prepare the simulated gastric solution. The pH of
gastric solution was about 1.1. After 2 h gastric
digestion, intestinal solution was obtained by neutral-
izing the gastric solution to pH 7.0 with saturated
NaHCO3 solution, then adding 52.5 mg bile salts
and 15 mg pancreatin. Incubation time of intestinal
phase was 2 h.
SGET design 4 (SG4): the gastric solution was the
same as SG3 (1 L mixed solution of 7.0 ml concen-
trated HCl and 2.0 g NaCl, pH1.1). After 2 h gastric
digestion, intestinal solution was obtained by neutral-
izing the gastric solution to pH 7.0 with saturated
NaHCO3 solution. Incubation time of intestinal solu-
tion was 2 h.
3 SBET
SBET design 1 (SB1): the simulated gastric juice was
0.4 M glycine solution. The pH of gastric solution was
adjusted to 1.5 with 1:1 HCl. The incubation time was
1 h.
SBET design 2 (SB2): the gastric solution was prepared
by dissolving 1.25 g pepsin in 1 L of 0.4 M glycine
solution. The pH of gastric solution was adjusted to 1.5
with 1:1 HCl. Incubation time was 1 h. Because the
original design of SBET (SB1) did not include pepsin,
the content of pepsin followed PBET.
SBET design 3 (SB3): the gastric solution was made by
dissolving 3.20 g pepsin in 1 L of 0.4 M glycine solution.
Its pH was adjusted to 1.5 with 1:1 HCl. Incubation time
was 1 h. The content of pepsin was from SGET.
In short, P1 and SG1 contained all digestive enzymes;
P2, P3, SG2, and SG3 included either gastric or intesti-
nal enzyme; P4 and SG4 had no digestive enzymes at
all; no digestive enzyme was added to SB1; different
amounts of pepsin were added to SB2 and SB3; P1,
SG2, and SB1 are the original designs of PBET, SGET,
and SBET, respectively.
Quality control
The determination of soil physiochemical properties
were conducted in duplicate, and in vitro digestion tests
were conducted in triplicate. Standard reference material
(GBW 07405) was used for quality assurance of the
contents of total soil heavy metals measurement. The
recovery of heavy metals in standard reference material
Environ Sci Pollut Res (2013) 20:4993–5002
was between 90 and 110 %. The contents of total and
extracted soil heavy metals were determined by analysis
using flame atomic absorption spectrophotometer (AAS,
novAA 300, Jena), and the quality control of the mea-
surement was done by measuring the standard solution
every ten samples.
Statistical analysis
The statistical analysis of all data was processed by
Microsoft Office Excel 2003 and SPSS 16.0 for windows.
The differences between data were analyzed by one-way
analysis of variance. The significance criteria were p<0.05
and p<0.01. p>0.05 indicates no significant differences.
Results and discussions
Variations of soil heavy metal bioaccessibility after
digestive enzymes addition
After digestive enzymes addition, no clear trend was ob-
served in changes of bioaccessibility of soil Cu, Zn, and Pb
(Tables 2, 3, and 4). Protein is the third most abundant solid
in bile (Yamazaki et al. 1988). Pepsin and pancreatin are
proteins. In biological systems, heavy metals are bound to
proteins to forming metalloproteins (Fraga 2005), which
help metal ions stay soluble. However, heavy metals bind-
ing to protein may lead to protein precipitation. The protein
precipitation caused by metals was applied to protein puri-
fication (Kumar et al. 1998). The reaction between metal
and protein was described as a two-step process by Iyer and
Przybycien (1996). At first, metal ions and protons compete
to coordinate heteroatoms on the protein surface in a rapid
equilibrium binding process. Second, metal ion bound sites
on a protein coordinate with unbound sites on other proteins
to form a cross-linked network. The network formation
continues until the formation of an infinite or macroscopic
cluster results in precipitation. Therefore, the dissolution
process of soil heavy metals in in vitro digestive test after
the addition of digestive enzymes was clear. At the begin-
ning, heavy metals were extracted from soil by the synthetic
digestive juice. Thereafter, some dissolved soil heavy metals
began to be combined with digestive enzymes forming
complexes, which reduced the concentration of metal ions
in the digestive juice. Consequently, more heavy metals
were mobilized from soil to the digestive juice and the
metal–protein complexes keep gathering until precipitation
occurred. Then, these heavy metals deposited with digestive
enzymes became insoluble. The whole process depicted
above was presented in Fig. 1. An equation was proposed
to describe the effects of digestive enzyme based on this
process. It is assumed that the pH and ionic strength of
Environ Sci Pollut Res (2013) 20:4993–5002
solution is constant, no coprecipitation occurs, no compet-
itions exist among ions in solution, and only one specific
heavy metal involves in this entire process. The heavy
metals extracted from soil by digestive juice at the begin-
ning are E. The increased heavy metals, namely those mo-
bilized from soil into solution as a result of metal–protein
complexes formation are I. The heavy metals deposited
along with digestive enzyme precipitation are D. The final
heavy metal concentration in synthetic digestive juice after
digestive enzymes addition is F. Then, the equation can be
expressed as below:
F ¼EþI D
Therefore, the role of digestive enzymes can be evaluated
by the difference between I and D. If I−D>0, then the
existence of digestive enzymes will increase the heavy metal
bioaccessibility; if I−D=0, then the heavy metal bioacces-
sibility will not change when digestive enzymes are present;
if I−D<0, then the heavy metal bioaccessibility will be
diminished after digestive enzyme are added. The value of
I is mainly connected with soil characteristics and synthetic
digestive juice extraction ability. If it is easier for heavy
metals to be extracted from soil, then the value of I is bigger.
The value of D is determined by the reaction between metal
ions and digestive enzyme. The chance of precipitation
increases with concentrations of metal–protein complexes.
It must be pointed out that the equation has little application
value. First, the equation is established on a simplified
condition. To know the exact values of D and I is nearly
impossible. Second, to add digestive enzymes is far easier
than to predict the effects of digestive enzymes. The signif-
icance of the equation is to provide a better understanding of
in vitro digestion test and explanations for the observation
of this study and some phenomena:
1 The role of digestive enzymes in the solubility of soil
heavy metals can be regarded as a kind of regulatory and
protection mechanism of human body. The dissolution
of metals in intestine, where the elements are absorbed,
is hindered due to its neutral environment. So, digestive
enzymes help keep metals soluble by forming metal–
protein complexes when the concentration of metals in
solution is low and inhibit the dissolution of metals by
precipitation when the content of metals is high.
Turnlund (1998) found that the absorption of Cu was
much more efficient when intake is low and a tenfold
increase in dietary copper resulted in only twice as much
Cu absorbed.
2 Assessing the amounts of heavy metals available for
absorption is always the chief interest of in vitro diges-
tion test. It is believed that absorption is the first step for
heavy metals to harm human health. Actually, heavy
metals already start to harm human health when the
4997
precipitation of digestive enzymes is caused. There were
many literatures on the inhibition of digestive enzyme
activities by heavy metals (Chen et al. 2002; Krejpcio
and Wojciak 2002). Kojima et al. (1985) observed
that proteins depressed the absorption of Cd, and the
digestion of these proteins were depressed by Cd at
the same time, so it was pointed out that the inhibi-
tory effect of Cd on the digestion of proteins was one
of the causes of the inhibitory effects of the proteins
on the absorption of Cd.
3 The pH of gastric phase of in vitro digestion test is
usually seen between 1 and 4, like PBET, SGET, and
SBET. That is because human gastric pH ranges from 1
to 4 (Ruby et al. 1993; Intawongse and Dean 2006; Dean
and Ma 2007). But from another angle, pepsin is nega-
tively charged and available to bind metal ions only
when the environmental pH is higher than protein iso-
electric point of pepsin, which is 1.0, so the gastric pH
cannot be less than 1 even though lower pH is more
helpful for dissolution of heavy metals.
4 The role of digestive enzymes to keep heavy metals
soluble by forming metal–protein complexes is more
obvious in neutral intestinal phase due to the precip-
itation of metal ions resulting from the pH rise and
the lack of competition with H+ for binding sites. For
example, the bioaccessibility of soil heavy metals in
the gastrointestinal phase of SGET were all signifi-
cantly promoted when pepsin was added (Table 2).
Therefore, digestive enzymes were more meaningful
for the gastrointestinal digestion. However, the bioac-
cessibility may decline when pepsin, bile salts, and
pancreatin were all added (Table 3). This was because
the concentrations of metal–protein complexes, which
linked together to form precipitation, increased with
the content of protein.
Role of digestive enzymes in in vitro digestion test
PBET and SGET
Three points were noticed from Figs. 2 and 3:
1 The bioaccessibility of soil Zn and Pb are pH-dependent.
The solubility of soil Zn and Pb obviously dropped when
entered the intestinal phase due to the rise of pH no
matter whether digestive enzymes were added or not,
especially in SGET.
2 The response of Cu was different as compared with Zn
and Pb. The digestive enzymes resulted in soil Cu re-
main soluble or even promoted its dissolution in some
soil in the intestinal phase. For soil A and C in P1, and
soil B in SG1 and SG2, there were no significant differ-
ences (p > 0.05) between the extractable Cu by the
4998 Environ Sci Pollut Res (2013) 20:4993–5002

Table 2 Changes of soil heavy metal bioaccessibility in PBET and SGET when pepsin was added (P1, SG2) compared to the time when pepsin was
absent (P3, SG4)

Element Sample P3 P1 Sig. Trend SG4 SG2 Sig. Trend

GP Cu A 7.45 (0.44) 7.34 (0.32) 28.0 (1.63) 26.7 (1.52)

B 57.6 (0.50) 55.8 (4.96) 123 (4.16) 113 (9.21)
C 25.5 (1.34) 23.6 (1.08) 26.2 (0.26) 20.0 (0.34) ** ↘
Zn A 77.3 (5.15) 145 (4.85) ** ↗ 315 (20.0) 367 (12.3) * ↗
B 87.3 (2.61) 150 (7.07) ** ↗ 227 (11.5) 290 (24.1) * ↗
C 493 (3.87) 465 (14.0) * ↘ 506 (3.19) 480 (11.0) * ↘
Pb A 36.1 (3.18) 37.6 (0.60) 44.7 (2.59) 51.2 (5.06)
B 20.6 (1.92) 22.5 (1.99) 103 (9.97) 120 (7.63)
C 15.8 (1.03) 15.3 (0.82) 22.6 (1.46) 21.3 (1.71)
GiP Cu A 2.22 (0.19) 7.21 (0.51) ** ↗ 2.49 (0.26) 6.24 (0.37) ** ↗
B 72.1 (4.97) 78.9 (4.87) 13.4 (0.47) 106 (4.44) ** ↗
C 24.3 (0.63) 22.6 (1.18) 1.59 (0.13) 11.1 (0.84) ** ↗
Zn A 30.0 (2.63) 71.0 (3.63) ** ↗ 1.99 (0.15) 8.51 (0.77) ** ↗
B 70.3 (3.99) 127 (9.91) ** ↗ 8.69 (0.45) 17.1 (0.71) ** ↗
C 266 (6.43) 272 (21.4) 22.2 (0.93) 29.5 (2.93) * ↗
Pb A 10.5 (0.61) 10.5 (0.69) 2.14 (0.11) 3.15 (0.04) ** ↗
B 4.80 (0.43) 5.35 (0.24) 5.42 (0.39) 8.31 (0.45) ** ↗
C 7.94 (0.54) 7.29 (0.60) 1.76 (0.16) 3.39 (0.07) ** ↗

GP Gastric Phase, GiP Gastrointestinal Phase, Sig. Significance, ↗: increased, ↘: decreased

*p<0.05; **p<0.01

gastrointestinal phase and by the corresponding gastric
phase. The bioaccessibility of Cu in soil B in the gastro-
intestinal phase of P1, P2, P3, and P4 respectively, in-
creased by 41.36, 20.44, 25.19, and 14.42 % compared
to those in the corresponding gastric phase. The extent of
rise in P1, where all digestive enzymes were added, was
the greatest. The Cu extracted from soil B by SG3 and SG4
fell by 56.26 and 89.14 %, respectively, when entered the
intestinal phase. The extent of decline in SG4, where no
digestive enzymes were added at all, was far greater.
The different behavior of Cu were also observed in
previous studies (Turner and Ip 2007; Poggio et al. 2009;
Roussel et al. 2010; Sialelli et al. 2010; Pelfrene et al.
2011). The distinct impacts of digestive enzymes on Cu
may have two main causes. First, the ability of metal ions
bound to protein was different. The Cu2+ has unpaired
electrons that allow it to form more stable complex, while
Zn2+ and Pb2+ have no unpaired electrons. It was found
that binding sites of protein combined with Zn2+ were
three or more orders of magnitude more weakly than with

Table 3 Changes of soil heavy metal bioaccessibility in PBET and SGET when bile salts and pancreatin was added (P1, SG1) compared to the time
when they were absent (P2, SG2)

Element Sample P2 P1 Sig. Trend SG2 SG1 Sig. Trend

Cu A 7.05 (0.53) 7.21 (0.51) 6.24 (0.37) 1.02 (0.10) ** ↘

B 67.2 (0.51) 78.9 (4.87) * ↗ 106 (4.44) 103 (3.37)
C 18.5 (1.35) 22.6 (1.18) * ↗ 11.1 (0.84) 18.6 (0.53) ** ↗
Zn A 29.9 (2.78) 71.0 (3.63) ** ↗ 8.51 (0.77) 5.35 (0.38) ** ↘
B 106 (1.94) 127 (9.91) * ↗ 17.1 (0.71) 19.1 (1.72)
C 325 (11.3) 272(21.4) * ↘ 29.5 (5.85) 43.1 (2.78) ** ↗
Pb A 6.90 (0.55) 10.5 (0.69) ** ↗ 3.15 (0.04) 21.4 (0.57) ** ↗
B 5.17 (0.51) 5.35 (0.24) 8.31 (0.45) 9.43 (0.52) * ↗
C 7.13 (0.53) 7.29 (0.60) 3.39 (0.07) 16.3 (0.62) ** ↗

GP Gastric Phase, GiP Gastrointestinal Phase, Sig. Significance, ↗: increased, ↘: decreased

*p<0.05; **p<0.01
Environ Sci Pollut Res (2013) 20:4993–5002 4999

Table 4 Changes of soil heavy metal bioaccessibility in SBET when 1.25 g (SB2) or 3.0 g (SB3) pepsin was added compared to the time when
pepsin was absent (SB1)

Element Sample SB1 SB2 SB3 Sig.

* **

Cu A 19.8 (1.77) b 29.8 (1.50) a 26.5 (1.59) a SB1 SB2, SB1 SB3
B 94.8 (0.80) b 84.3 (7.45) c 105 (1.25) a SB1 SB2, SB1 SB3 SB2 SB3
C 16.5 (0.47) c 27.0 (1.34) a 22.2 (0.68) b SB1 SB2, SB1 SB3, SB2 SB3
Zn A 356 (10.9) a 306 (24.2) b 287 (12.2) b SB1 SB2 SB1 SB3
B 236 (3.69) a 198 (14.9) b 224 (9.73) a SB2 SB3 SB1 SB2
C 469(19.4) a 504 (25.7) a 499 (5.26) a
Pb A 31.8 (1.58) a 34.7 (0.88) a 35.4 (3.45) a
B 56.6 (2.05) b 30.5 (0.93) c 64.5 (6.07) a SB1 SB3 SB1 SB2, SB2 SB3
C 5.91 (0.40) a 0.63 (0.03) c 2.04 (0.03) b SB1 SB2, SB1 SB3, SB2 SB3

Different lower-case letters mean there were significant differences between these two data, and it means no significant differences (p>0.05) existed
between them when the lower-case letters were the same
Sig. Significance
*p<0.05; **p<0.01

Cu2+ (Jackson et al. 2001). Steinhart et al. (1975) com-
pared the affinity of Cu with pepsin and soja-protein and
found Cu formed complexes with pepsin and zinc and was
loosely bound to both proteins. Second, Soil pH may also
have an important impact on the behavior of Cu besides
the biochemical characters of Cu. The influences of diges-
tive enzymes on Cu in soil B were more remarkable than
in soils A and C. The pH of soils A, B, and C were 8.73,
7.87, and 2.39, respectively. The acid soil could provide
H+ to compete with Cu2+ for the binding sites of protein,
and the sorption for Cu to alkali soil is strong, so the Cu in
neutral soil is more susceptible to digestive enzymes. The
pH of samples tested by Poggio et al. (2009) was 6.7±0.12
where the bioaccessibility of Cu was found higher in the
gastrointestinal phase than in the gastric phase.
3 The results of SGET containing all corresponding diges-
tive enzymes reflected more soil heavy metal bioacces-
sibility variations resulting from soil and element types.
After entering the intestinal phase, the heavy metals
mobilized from soils by SG4 averagely fell by 91.39±
2.41 % for Cu, 97.05±2.03 % for Zn, and 94.06±1.61 %
for Pb. The extents of decline for the three elements were
great and close. Moreover, the standard errors were all
small. The characteristics of soils tested were dissimilar,
although there were only three soil samples tested.
Therefore, it implies that the decline in SG4 was caused
by the rise of pH. The soil or element type scarcely
affected the results when digestive enzymes were absent.
However, compared to the gastric phase, the soil heavy
metals extracted by the gastrointestinal phase of SG1

DE
DE DE DE HM
DE HM
HM DE HM HM
HM HM HM HM
HM DE HM
Digestive HM
HM
Juice HM HM DE—HM
HM DE—HM
HM E HM

HM HM HM HM I
Soil P
HM HM HM HM HM HM HM HM HM HM HM HM
D
HM HM HM HM HM HM HM HM HM HM HM HM HM HM HM HM

1 2 3 4

Fig. 1 The process of reaction between soil heavy metals and digestive enzymes in in vitro digestion test. HM, Heavy Metal; DE, Digestive
Enzyme; P, Precipitation; E, I and D: the parameters in equation
5000 Environ Sci Pollut Res (2013) 20:4993–5002

GP GiP GI GiP

90

Extracted Cu/mg kg -1
140
Extracted Cu/mg kg-1

80 120
70
60 100
50 80
40 60
30 40
20
10 20
0 0

SG1**
SG2**
SG3**
SG4**

SG3**
SG4**

SG4**
SG1
SG2

SG1*
SG2**
SG3**
P3**
P1

P2

P4**
P1**

P2*

P3**

P4**
P1

P2**

P3

P4*
A B C
A B C
600
Extracted Zn/mg kg-1

500 600

Extracted Zn/mg kg -1
400 500
400
300
300
200
200
100
100
0
0
P1**
P2**
P3**
P4**

P2**

P3**

P1**
P2**
P3**
P4**
P1*

P4*

SG1**
SG2**
SG3**
SG4**
SG1**
SG2**

SG3**
SG4**
SG1**
SG2**
SG3**
SG4**
A B C
A B C
45
40
-1
Extracted Pb/mg kg

35 140
Extracted Pb/mg kg-1

30 120
25 100
20 80
15
60
10
5 40
0 20
P1**

P2**

P3**

P4**
P1**

P2**

P3**

P4**
P1**

P2**

P3**

P4**

0
SG1**
SG2**
SG3**
SG4**
SG1**
SG2**

SG3**
SG4**
SG1**
SG2**
SG3**
SG4**
A B C

Fig. 2 Comparison of extracted Cu, Zn, and Pb from soils by the A B C

gastric and gastrointestinal phase of P1, P2, P3, and P4. GP, Gastric
Phase; GiP, Gastrointestinal Phase; *: p<0.05; **: p<0.01 Fig. 3 Comparison of extracted Cu, Zn, and Pb from soils by the
gastric and gastrointestinal phase of SG1, SG2, SG3 and SG4. GP,
Gastric Phase; GiP, Gastrointestinal Phase; *: p<0.05; **: p<0.01
averagely dropped by 37.39±50.93 % for Cu, 94.33±
3.84 % for Zn, and 57.95±34.26 % for Pb. The extent of
by SBET did not necessarily rise or decline when pepsin
decline for Cu, Zn, and Pb in SG1 were not the least or
was present and did not tend to increase or decrease when
greatest among these four designs, but they were much
the added pepsin increased from 1.25 to 3.20 g (Table 4).
dissimilar to one another, and the coefficients of varia-
But there are two reasons why digestive enzymes were not
tion were the highest. Similarly, the study of Li and
necessary for SBET. Firstly, the gastric digestion was usu-
Zhang (2012) showed the intestinal digestion results also
ally chosen to assess soil heavy metal pollution for the
reflected more variations resulting from soil and element
purposes of precaution and protection (Kim, et al. 2002;
types than gastric digestion results did.
Turner and Simmonds 2006; Lamb, et al. 2009), rather than
to mimic the real physiological situation and calculate the
SBET most likely absorbed amounts of heavy metals. Davis et al.
(1994) chose PBET with modifications to study the effects
The effects of pepsin added to SBET on the mobilization of of constituents added to the gastric juice on soil Pb bio-
heavy metals were not clear. The soil heavy metals extracted accessibility and noticed that the absence of either organic
Environ Sci Pollut Res (2013) 20:4993–5002
acids or enzyme, or both components together, resulted
in only a slight decrease in maximum Pb dissolution in
the stomach, which substantiated the theory that HCl
concentration is the primary factor controlling Pb disso-
lution in the stomach. Mercier et al. (2002) developed a
gastric digestion method called Gastric Juice Simulation
Test (GJST). Although the use of pepsin in the test
could ameliorate the GJST, pepsin addition was omitted,
because it would increase the complexity of the method
and the variability of results. More importantly, it is
inferred from the analysis of PBET and SGET that the
impacts of digestive enzymes on dissolution of heavy
metals are mostly seen in the intestinal phase.
Conclusions
The role of digestive enzymes varied with methods and ele-
ments. The solubility of Soil Cu in the intestinal phase of PBET
remained constant or was even promoted as a result of digestive
enzymes addition. But the solubility of soil Zn and Pb are pH-
dependent. The SGET containing all digestive enzymes can
reflect more variations of soil heavy metal bioaccessibility
resulting from soil and element types. In addition, it is inferred
from the analysis of PBET and SGET that the impacts of
digestive enzymes on dissolution of heavy metals are mostly
seen in the intestinal phase. Therefore, the addition of digestive
enzymes is indispensable to the gastrointestinal digestion tests,
such as PBET and SGET, while the addition of pepsin is not
important for gastric digestion test, such as SBET.
Acknowledgments This research is financially supported by the
National Natural Science Foundation of China (Grant NO. 21177108).
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