J. Phyeiol. (1979), 286, pp. 343-350 With 3 text-figures Printed in Great Britain
343
THE TRANSPORT OF THE LEAD CATION ACROSS THE INTESTINAL MEMBRANE
BY J. A. BLAIR, I. P. L. COLEMAN AND M. E. HILBURN From the Department of Chemistry, University of Aston in Birmingham, Gosta Green, Birmingham B4 7ET (Received 18 April 1978) SUMMARY
1. The transport of the lead cation has been investigated using the everted sac preparation of Wilson & Wiseman (1954). 2. Only a small percentage of lead was transported into the serosal compartment but there was a rapid and massive uptake onto the tissue. There was no significant difference in the amount of lead cations transported across different regions of the small intestine. 3. Both the rate of transport into the serosal compartment and the tissue uptake increased linearly with increasing concentration of the lead cation, from 10-7 M to 5 x 10-5 M. Little evidence for saturation of serosal transport or tissue uptake was found. 4. Lead transport into the serosal compartment appeared to be related to water movement, but was little affected by changes in glucose concentration and temperature. 5. It is concluded that lead is transported into the serosal space by a process of passive diffusion linked to water transport. 6. The interaction between lead ions and the intestinal tissue was extremely tenacious and displayed characteristics of covalent bonding. 7. It is suggested that the lead cation interacts with tissue phosphate ions thus removing lead ions from the transport pool. Chelation of lead to form a neutral species reduces this interaction and also promotes transport. INTRODUCTION
Adults normally absorb only 5-10% of ingested lead compounds (Rabinowitz, Wetherill & Kopple, 1975). However the absorption of dietary lead is the most important factor in determining the body burden of lead. Clinically evident lead poisoning usually results from the absorption of lead across the gastrointestinal tract rather than the respiratory tract (Hilburn, 1977). There is also evidence that age (Kostial, Simonovic & Pisonic, 1971) and dietary factors (Barltrop & Khoo, 1975; Garber & Wei, 1974) affect lead absorption. Nevertheless nothing is known about the mechanism involved in the transport of the lead cation, or how these factors could affect a transport mechanism. Nor is it known whether lead affects the transport of other substances across the intestine. The object of this study is
J. A. BLAIR, I. P. L. COLEMAN AND M. E. HILBURN 344 to establish the site and mechanism of absorption of the lead cation in the rat intestine using a concentration of lead comparable to dietary levels in normal man. METHODS
Intestinal preparation Male Wistar rats (190-220 g) were starved for 18 hr before anaesthesia by injection with (100 mg/kg). The duodenum was excised at the pyloric sphincter and the duodenaljejunal flexure. The remainder of the small intestine was removed by severing at the ileo-caecal junction. Both sections were transferred to Krebs-Henseleit bicarbonate buffer (Krebs & at 0 0C, gassed continuously with 5 % C02/95 % 02. Henseleit, 1932) containing 20 The duodenum was divided into two parts at the point of entry of the bile and the pancreatic ducts (DI proximal, D2 distal). The jejunum and ileum were flushed through with buffer and divided into twelve sections of approximately 8 cm in length (I proximal to XII distal); only three sections were taken from any one animal. The sections were everted and sacs prepared as described by Wilson & Wiseman (1954).
i.P.
Inactin
mM-glucose
Measurement of transmural potential difference and glucose transport Transmural p.d. was measured in Krebs-Henseleit bicarbonate buffer containing 20 mMglucose by the method of Barry, Dikstein, Matthews, Smyth & Wright (1964). Using a Pye Unicam of1
hr. The effect of digital voltmeter, readings were taken every 10 min over a period p.d. was investigated by adding lead acetate (10- to 5 x 10-5 M) to both sides of the intestinal preparation. The concentration of glucose in the mucosal and serosal solutions after 1 hr was measured by an automated Technicon Autoanalyser according to the method of Salway
lead on the
(1969).
Measurement of lead transport and water transport The everted sacs D2, V and XI were selected for studies of the transport of lead ions, as being with representative of the distal duodenum, mid-jejunum and distal ileum respectively, and 500 #1. Krebs-Henseleit bicarbonate buffer containing 20 Each sac was incubated, for various periods of time up to hr at either 37 or 27 'C in shaking at 60 oscillations per 10 ml. Krebs-Henseleit bicarbonate buffer and gassed continuously with 5 % C02/95 % 02. 1 of the radioisotope 203Pb was used as a tracer and added to the mucosal buffer solution Lead was estimated in the total ing between 10-7 and 5 x 10-5 lead acetate and 20 serosal fluid and the entire sac by counting the 279 keV gamma emission of in a Nuclear
min,
M
filled
mM-glucose.1
#ac
contain-
mM-glucose.
203Pb
Enterprises NE 8312 counter. Total counts were corrected at 30 min intervals to account for isotope decay. Water transport in the presence and absence of Pb2+ was assessed from appropriate weighings of the sacs on a Whites Instruments torsion balance. The effect of glucose on the transport of in the presence of zero, 10, 20, 30 and 40 mM-glucose. The effect of the Pb2+ was investigated chelating agent trisodium Mcalcium diethylenetriaminepentaacetate (DTPA) was measured in the presence of 10-6 and 10-5
DTPA.
The effect of washing the incubated sacs
The everted sacs D2, V and XI were incubated as described previously at 37 'C for 30 min in Krebs-Henseleit bicarbonate buffer containing 20 acetate and 1 10-6 as tracer. After incubation the sacs were transferred for a further 30 min incubation period at 37 'C in 10 ml. of either the same buffer or Krebs-Henseleit bicarbonate buffer containing but no radioactive 10-6 M-lead or 10-6 M-DTPA. The washing solutions contained 20 tracer. Aliquots were taken from the washing medium at 5 min intervals and the transport of
mM-glucose,
lead
M-lead mM-glucose
eac 203Pb
away from the tissue compartment was assessed as described previously.
Materials
was supplied by the M.R.C. Cyclotron Unit, Hammersmith Hospital, London, W. 12. 203PbCl2 grade lead acetate and buffer salts were supplied by BDH Chemicals; Inactin by Pro-
Analar
INTESTINAL ABSORPTION OF LEAD
345
monta; DTPA by Ciba-Geigy. Wistar rats were maintained on Heygates Diet 41B and water ad lib. in an animal house at 25 'C. All results presented are the mean of six observations and are expressed as mean + s.E. of mean. Student's t test was used to analyse the results and regression lines were achieved using the method of least squares. Statistical calculations were performed on an Olivetti Programma 101 computer.
RESULTS
The viability of everted sac preparations, taken from the whole of the small intestine was confirmed over a 1 hr experimental period by the presence of a steady transmural p.d., active glucose transport and a linear uptake of water. Over a range of lead concentrations (1I0 to 5 x 1O-5 M) these same parameters were largely unaffected indicating that the preparations were suitable for lead transport studies.
Lead transport The entry of lead into the serosal compartment and uptake by the intestinal tissue were assessed separately at different lead concentrations. Only a small percentage of lead was transported into the serosal compartment but there was a rapid and massive uptake on to the tissue (Table 1). Although generally the amount of lead transported across the duodenum was greater than across other regions, this difference was not statistically significant. The jejunum was observed to take up more lead onto the tissue than the other sacs but again the difference was not significant (Table 1). Table 1. Tissue uptake (n-mole Pb/g initial wet wt.) and serosal transport (p-mole Pb/g initial wet wt.) of the lead cation after 30 min incubation of the everted sac preparation at 37 0C in Krebs-Henseleit buffer containing 10-7-5 x 10- M-lead labelled with 203Pb. Each value is the mean of six experimental observations + s.E. of mean.
Sac D2 V XI
Compartment Serosal (p-mole) Tissue (n-mole) Serosal (p-mole) Tissue (n-mole) Serosal (p-mole)
10-7 m-Pb 1-92 + 0-28
5 x 10-7 M-Pb
0-23+0-03 1-54+0-24 0-33 ± 0-03 1-68 + 0-34
1-99±0-49 7-69 ± 0-67 2-98 ± 0-26 6-73 ± 0-67
6-30±2-36 20-0 + 2-8 5-14 + 0-63 15-4 ± 1-0
Tissue
0-34 ± 0-05
2-75 + 0-55
4-28 + 0-87
5 x 106 M-Pb 96-6± 25 23-8 ± 4-8
10-5 M-Pb 136 ± 27-0 26-7 + 2-8 150±49-0 39-0 + 6-4 110± 18-0 38-8+5-8
5 x 10-5 M-Pb 462 ± 115 102-4 ± 13-5 410± 63 166-3 ± 12-5 423±96
(n-mole)
Sac
Compartment
D2
Serosal (p-mole) Tissue (n-mole) Serosal (p-mole) Tissue (n-mole) Serosal (p-mole) Tissue (n-mole)
V XI
68-8+ 9-0
26-5 + 13-5 78-3+ 14-0
23-4+9-1
9-13±1-35
10-6 M-Pb 23-6 ± 3-0
121-6+26-0
Time based studies indicated that there was a linear transport of Pb2+ into the serosal compartment (Fig. 1). Extrapolation of the regression line to the Y axis produces an intercept some distance from the origin. The probable interpretation of the data is that the maximum rate of lead transport to the serosal compartment occurs during the first 10 min of incubation. Little evidence of saturation was
346 J. A. BLAIR, I. P. L. COLEMAN AND M. E. HILBURN observed after 60 min incubation over a lead concentration range of 10-7-10-5 M. A good correlation between lead transport and water movement into the serosal compartment over the incubation period of 1 hr, for all regions of the small intestine, was also observed (r > 0.92) (as illustrated for the mid-jejunum in Fig. 2). The maximum rate of transport to the serosal compartment coincided with a very rapid uptake of lead onto the intestinal tissue, equilibration of tissue and mucosal lead occurring after approximately 20 min incubation (Fig. 3). The same pattern of lead uptake by the intestinal tissue occurred at all lead concentrations studied. 55r 50
r>096
-
45 Fa)
40
3
[-
n
CA
35 1-
._
30
-
4-
.0
E1 25 F0 4,
20
-
15
-
10 5 BE
I
I
I
I
10
20
30 Time (min)
45
60
Fig. 1. Transport of 10-1 M-lead labelled with 2'Pb into the mid-jejunal serosal compartment of the everted sac after incubation for periods up to 60 min at 37 IC. Each value is the mean of six experimental observations ± S.E. of mean represented by vertical bars.
Efflux experiments Everted sacs incubated for 30 min in buffer containing 106 M-lead labelled with mPb were transferred to non-radioactive buffer and the amount of activity appearing in the bathing medium was measured during a further period of 30 min. Efflux of lead from the tissue was not affected by either the presence or absence of lead in the bathing medium or the presence of DTPA (106 M). Approximately 25 % of the lead was removed from each region of the intestine after 30 min incubation. Half
347 INTESTINAL ABSORPTION OF LEAD of this amount was removed during the first 5 min, after which the appearance of lead in the bathing medium followed a linear trend. A greater percentage of lead was removed from the duodenum than from the jejunum or ileum but the difference was not statistically significant. 60 rm
r>092
5050
0.-
* 40 ¢ 30 C cm
20
0
E 6. 10
0
100
500
1000
Water movement (mg HO/g initial wet wt. of tissue)
Fig. 2. Correlation between the transport of water and lead across the mid-jejunum after incubation periods of either 10, 20, 30, 45 or 60 min at 37 00 in 104 M-lead labelled with 20SPb. Each value is the mean of six experimental observations + s.E. of mean appropriately represented by horizontal and vertical bars. The incubation time associated with each experimental point is indicated in parentheses. an
r-
03
149
'0
8
C 0.
5 4
+.
_6 5 0,6 6.
32
-0 0o
i CL
I
I
I
I
I
10
20
30 Time (min)
45
60
Fig. 3. Uptake of 10-6 M-lead labelled with 203Pb by the mid-jejunal tissue after incubation for time periods up to 60 min at 37 'C. Each value is the mean of six experimental observations ± s.E. of mean represented by vertical bars.
Factors affecting lead transport The transport of the lead cation onto the tissue and into the serosal compartment was measured over a range of glucose concentrations from zero to 40 mm. No significant effect was observed on lead transport into the serosal compartment during
348 J. A. BLAIR, I. P. L. COLEMAN AND M. E. HILBURN a 30 min incubation period. Both lead transport and water movement into the duodenal and jejunal serosal compartments were depressed however after 1 hr when glucose was absent from the bathing medium (Table 2). The passage of lead into the serosal and tissue compartments was not significantly affected by a depression of the incubation temperature. Transport of lead into the serosal compartment over a lead concentration range 106-10-5 M yielded Q10 Table 2. Effect of zero glucose and 20 mM-glucose on transport of lead and water across the duodenum (D2), jejunum (V) and ileum (XI) after 60 min incubation at 37 0C in Krebs-Henseleit buffer containing 10-6 M-Pb labelled with 203Pb. Lead transport expressed as p-mole Pb/g initial wet wt. Water movement expressed as mg H20/g initial wet wt. Each value is the mean of six experimental observations ± s.E. of mean. Transfer of lead to serosal compartment Glucose concentration (mM) Sac D2 V XI
0
25-9 ± 2-79 21-2 ± 3-31
25-9 + 6-11
20 347 +2-40 393 +4-81 278 +4-42
P value 0-05 0.02
0-8
Transfer of water to serosal compartment Glucose concentration (mM)
Sac D2 V XI
0 -104+ 59 120 +87 528 +174
20
59± 79 581+ 152 741 + 151
P value 0-05 0-02 0-3
Table 3. Effect of 10-i M-DTPA on transport of lead across the duodenum (D2), jejunum (V) and ileum (XI) after 30 min incubation at 37 'C in Krebs-Henseleit buffer containing 10-6 M-lead labelled with 203Pb. Lead transport expressed as p-mole Pb/g initial wet wt. Each value is the mean of six experimental observations + S.E. of mean. Sac D2 V XI
Transfer of lead into serosal compartment 10-5 M-DTPA Control 29-8 ± 6-25 49-0 6-73 13-2 + 1-83 44-8 +7-69 14-7 ± 3-61 38-9 5-77
Sac D2
Uptake of lead into tissue 10-5 M-DTPA Control 2-28+0-42 1-83+0-29
V XI
4-10 0-57 3-04 + 0-61
2-47± 035 1-64± 0-25
P
0-05 0-001 0-01 P 0-4 0-02 0-02
values between 1-43 (distal duodenum) and 0-68 (distal ileum). The uptake of lead by the intestinal tissue was partially reduced over the same concentration range, giving Q10 values between 1-8 (distal duodenum) and 1-2 (distal ileum). When the everted sac preparation was incubated for 30 min in the presence of an equimolar quantity of lead and DTPA (106 M) there was an increase in the transport
INTESTINAL ABSORPTION OF LEAD
349 of lead to the serosal compartment across all regions of the intestine. In the presence of a tenfold excess of DTPA the increase in the transport of Pb2+ became significant. At the same time there was a significant reduction in the amount of lead taken up by the jejunal and ileal tissue (Table 3). DISCUSSION
The transport of Pb2+ into the serosal compartment across selected sites of the small intestine is slow and dependent on the initial concentration of lead in the mucosal bathing medium. Lead transport appears to proceed in a linear manner over the 1 hr experimental period, although it is probable that there is a more rapid passage of Pb2+ into the serosal compartment during the first 10 min of incubation. There is no significant difference in the amount of Pb2+ transported across different regions of the small intestine. On the other hand there is a rapid massive uptake of lead by the intestinal tissue which again is dependent on the initial mucosal concentration of lead. More than half the total lead content is taken up by the tissue during the first 10 min of incubation. The data are comparable to those of Sahagian, Harding-Barlow & Perry (1967) who demonstrated by means of an in vitro perfusion of the intestine that there is a rapid uptake in zinc, cadmium and mercury by the intestinal tissue but only a slow rate of transport to the serosal compartment. It would appear that the passage of certain trace metals across the intestinal wall may be dependent upon two separate rate-limiting steps. The first is the uptake and binding of the metal by cellular surfaces, and the second, the transport of the metal across the intestinal membrane and subsequent accumulation in the serosal compartment. Therefore the transport of lead may largely depend upon (a) the affinity of lead for binding sites on the tissue, (b) the number and availability of particular binding sites and (c) the nature of the interaction between the lead cation and the intestinal tissue. The rapid massive binding of lead to the intestinal tissue suggests a surface adsorption process. If the interaction between the lead ions and the tissue is ionic a rapid exchange between the lead in the tissue and that in the mucosal medium would be expected. However the exchange is slow and incomplete. Neither is a large proportion of the tissue lead chelatable. Both facts are suggestive of a predominantly covalent bonding of lead to the tissue. The external surface of the mucosal epithelial cell is rich in phosphate ions due to the hydrolysis of ATP (Koenig & Vial, 1970). The lead ion in the tissue could therefore be sequestered in a covalent form as lead phosphate. This interaction is analogous to that which occurs between erythrocytes and Pb2+ (Clarkson & Kench, 1958). A consequence of lead being sequestered as lead phosphate would be that the Pb2+ would be less available for transport. Although it is often difficult to extrapolate from in vitro experimentation to the normal physiological condition (Forth & Rummel, 1975), the intestinal membrane may act as a protective mechanism against the passage of a large proportion of the lead present in the normal diet. Sequestered lead phosphate along the entire length of the small intestine may also reduce membrane permeability. The passage of the lead ion into the serosal compartment does not display saturation kinetics, nor does incubation at different temperatures appear to affect any
J. A. BLAIR, I. P. L. COLEMAN AND M. E. HILBURN transport processes. There is also a good correlation between lead transport and water movement across the intestinal wall. Over a range of glucose concentrations the movement of lead is unaffected during a 30 min incubation period; after 60 min incubation in the absence of glucose the transfer of lead across the duodenum and the jejunum is reduced, due to reduced water movement. The experimental data strongly suggest therefore, that the movement of lead across the intestinal membrane occurs by a passive diffusion process linked to some degree to the concomitant movement of water. Further indication of a non-active transfer system has been provided by Gruden & Stantic (1975) who reported serosal-mucosal ratio values of unity after incubating everted sacs for 60 min with equal concentrations of lead on both sides of the membrane. The presence of DTPA in the bathing medium increases the amount of lead transported to the serosal spaces but reduces tissue uptake of lead. Other investigators (Goodman & Gilman, 1965) have shown that DTPA is poorly absorbed by the intestine of man and experimental animals. It may be expected therefore that when the lead cation is complexed to form a neutral species, subsequent interaction with tissue phosphate ions will be reduced, whereas transport of the completed organo-lead moiety to the serosal compartment will be enhanced. 350
The work is supported by the European Economic Community (grant no. 167-77-1-NEV-UK), the Science Research Council and the City of Birmingham Environmental Department.
REFERENCES BARLTROP, D. & KHOO, H. E. (1975). The influence of nutritional factors on lead absorption. Post-grad. med. J. 51, 795-800. BARRY, R. J. C., DIKSTEIN, S., MATTHEWS, J., SMYTH, D. H. & WRIGHT, E. H. (1964). Electrical potentials associated with intestinal sugar transfer. J. Physiol. 171, 316-338. CLARKSON, T. W. & KENCH, J. E. (1958). Uptake of lead by human erythrocytes in vitro. Biochem. J. 69, 432-439. FORTH, W. & RUMMEL, WV. (1975). Gastrointestinal absorption of heavy metals. In International Encyclopaedia of Pharmacology and Therapeutics, section 39B, 2,599-746. Oxford: Pergamon. GARBER, B. T. & WEI, E. (1974). Influence of dietary factors on the gastrointestinal absorption of lead. Toxic. apple. Pharmac. 27, 685-691. GOODMAN, L. S. & GILMAN, A. (1965). The Pharmacological Basui of Therapeutic8, 3rd edn, pp. 934-939. New York: Macmillan. GRUDEN, N. & STANTIC, M. (1975). Transfer of lead through the rat's intestinal wall. Sci. total Environ. 3, 288-292. HILBURN, M. E. (1977). Environmental lead in perspective. British Association Meeting, University of Aston, Section B. (Chem.). KOENIG, C. S. & VIAL, J. C. D. (1970). A bistochemical study of adenosine triphosphate in the toad gastric mucosa. J. Histochem. Cytochem. 18, 340-353. KOsTIAL, K., SImONOvIC, I. & Pisomc, M. (1971). Lead absorption from the intestine in new born rats. Nature, Lond. 233, 564. KREBS, H. A. & HENSELEIT, K. (1932). Untersuchungen fiber dieHarnstoffbildung in Tierkorper. Hoppe-Seyler's Z. physiol. Chem. 210, 33-66. RABINOWITZ, M., WETHERILL, G. & KoPPLr, J. (1975). Absorption, storage and excretion of lead by normal humans. Trace Subst. environ. Hlth 9, 361-368. SAHAGIAN, B. M., HARDING-BARLOW, I. & PERRY, H. M. (1967). Transmural movements of zinc, manganese, cadmium and mercury by rat small intestine. J. Nutr. 93, 291-300. SALWAY, J. G. (1969). The simultaneous determination of acetoacetate and glucose in capillary blood. Clinical chim. Acta, 25, 109-116. WILSON, T. H. & WISEMAN, G. (1954). The use of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. J. Physiol. 123, 116-125.
343
THE TRANSPORT OF THE LEAD CATION ACROSS THE INTESTINAL MEMBRANE
BY J. A. BLAIR, I. P. L. COLEMAN AND M. E. HILBURN From the Department of Chemistry, University of Aston in Birmingham, Gosta Green, Birmingham B4 7ET (Received 18 April 1978) SUMMARY
1. The transport of the lead cation has been investigated using the everted sac preparation of Wilson & Wiseman (1954). 2. Only a small percentage of lead was transported into the serosal compartment but there was a rapid and massive uptake onto the tissue. There was no significant difference in the amount of lead cations transported across different regions of the small intestine. 3. Both the rate of transport into the serosal compartment and the tissue uptake increased linearly with increasing concentration of the lead cation, from 10-7 M to 5 x 10-5 M. Little evidence for saturation of serosal transport or tissue uptake was found. 4. Lead transport into the serosal compartment appeared to be related to water movement, but was little affected by changes in glucose concentration and temperature. 5. It is concluded that lead is transported into the serosal space by a process of passive diffusion linked to water transport. 6. The interaction between lead ions and the intestinal tissue was extremely tenacious and displayed characteristics of covalent bonding. 7. It is suggested that the lead cation interacts with tissue phosphate ions thus removing lead ions from the transport pool. Chelation of lead to form a neutral species reduces this interaction and also promotes transport. INTRODUCTION
Adults normally absorb only 5-10% of ingested lead compounds (Rabinowitz, Wetherill & Kopple, 1975). However the absorption of dietary lead is the most important factor in determining the body burden of lead. Clinically evident lead poisoning usually results from the absorption of lead across the gastrointestinal tract rather than the respiratory tract (Hilburn, 1977). There is also evidence that age (Kostial, Simonovic & Pisonic, 1971) and dietary factors (Barltrop & Khoo, 1975; Garber & Wei, 1974) affect lead absorption. Nevertheless nothing is known about the mechanism involved in the transport of the lead cation, or how these factors could affect a transport mechanism. Nor is it known whether lead affects the transport of other substances across the intestine. The object of this study is
J. A. BLAIR, I. P. L. COLEMAN AND M. E. HILBURN 344 to establish the site and mechanism of absorption of the lead cation in the rat intestine using a concentration of lead comparable to dietary levels in normal man. METHODS
Intestinal preparation Male Wistar rats (190-220 g) were starved for 18 hr before anaesthesia by injection with (100 mg/kg). The duodenum was excised at the pyloric sphincter and the duodenaljejunal flexure. The remainder of the small intestine was removed by severing at the ileo-caecal junction. Both sections were transferred to Krebs-Henseleit bicarbonate buffer (Krebs & at 0 0C, gassed continuously with 5 % C02/95 % 02. Henseleit, 1932) containing 20 The duodenum was divided into two parts at the point of entry of the bile and the pancreatic ducts (DI proximal, D2 distal). The jejunum and ileum were flushed through with buffer and divided into twelve sections of approximately 8 cm in length (I proximal to XII distal); only three sections were taken from any one animal. The sections were everted and sacs prepared as described by Wilson & Wiseman (1954).
i.P.
Inactin
mM-glucose
Measurement of transmural potential difference and glucose transport Transmural p.d. was measured in Krebs-Henseleit bicarbonate buffer containing 20 mMglucose by the method of Barry, Dikstein, Matthews, Smyth & Wright (1964). Using a Pye Unicam of1
hr. The effect of digital voltmeter, readings were taken every 10 min over a period p.d. was investigated by adding lead acetate (10- to 5 x 10-5 M) to both sides of the intestinal preparation. The concentration of glucose in the mucosal and serosal solutions after 1 hr was measured by an automated Technicon Autoanalyser according to the method of Salway
lead on the
(1969).
Measurement of lead transport and water transport The everted sacs D2, V and XI were selected for studies of the transport of lead ions, as being with representative of the distal duodenum, mid-jejunum and distal ileum respectively, and 500 #1. Krebs-Henseleit bicarbonate buffer containing 20 Each sac was incubated, for various periods of time up to hr at either 37 or 27 'C in shaking at 60 oscillations per 10 ml. Krebs-Henseleit bicarbonate buffer and gassed continuously with 5 % C02/95 % 02. 1 of the radioisotope 203Pb was used as a tracer and added to the mucosal buffer solution Lead was estimated in the total ing between 10-7 and 5 x 10-5 lead acetate and 20 serosal fluid and the entire sac by counting the 279 keV gamma emission of in a Nuclear
min,
M
filled
mM-glucose.1
#ac
contain-
mM-glucose.
203Pb
Enterprises NE 8312 counter. Total counts were corrected at 30 min intervals to account for isotope decay. Water transport in the presence and absence of Pb2+ was assessed from appropriate weighings of the sacs on a Whites Instruments torsion balance. The effect of glucose on the transport of in the presence of zero, 10, 20, 30 and 40 mM-glucose. The effect of the Pb2+ was investigated chelating agent trisodium Mcalcium diethylenetriaminepentaacetate (DTPA) was measured in the presence of 10-6 and 10-5
DTPA.
The effect of washing the incubated sacs
The everted sacs D2, V and XI were incubated as described previously at 37 'C for 30 min in Krebs-Henseleit bicarbonate buffer containing 20 acetate and 1 10-6 as tracer. After incubation the sacs were transferred for a further 30 min incubation period at 37 'C in 10 ml. of either the same buffer or Krebs-Henseleit bicarbonate buffer containing but no radioactive 10-6 M-lead or 10-6 M-DTPA. The washing solutions contained 20 tracer. Aliquots were taken from the washing medium at 5 min intervals and the transport of
mM-glucose,
lead
M-lead mM-glucose
eac 203Pb
away from the tissue compartment was assessed as described previously.
Materials
was supplied by the M.R.C. Cyclotron Unit, Hammersmith Hospital, London, W. 12. 203PbCl2 grade lead acetate and buffer salts were supplied by BDH Chemicals; Inactin by Pro-
Analar
INTESTINAL ABSORPTION OF LEAD
345
monta; DTPA by Ciba-Geigy. Wistar rats were maintained on Heygates Diet 41B and water ad lib. in an animal house at 25 'C. All results presented are the mean of six observations and are expressed as mean + s.E. of mean. Student's t test was used to analyse the results and regression lines were achieved using the method of least squares. Statistical calculations were performed on an Olivetti Programma 101 computer.
RESULTS
The viability of everted sac preparations, taken from the whole of the small intestine was confirmed over a 1 hr experimental period by the presence of a steady transmural p.d., active glucose transport and a linear uptake of water. Over a range of lead concentrations (1I0 to 5 x 1O-5 M) these same parameters were largely unaffected indicating that the preparations were suitable for lead transport studies.
Lead transport The entry of lead into the serosal compartment and uptake by the intestinal tissue were assessed separately at different lead concentrations. Only a small percentage of lead was transported into the serosal compartment but there was a rapid and massive uptake on to the tissue (Table 1). Although generally the amount of lead transported across the duodenum was greater than across other regions, this difference was not statistically significant. The jejunum was observed to take up more lead onto the tissue than the other sacs but again the difference was not significant (Table 1). Table 1. Tissue uptake (n-mole Pb/g initial wet wt.) and serosal transport (p-mole Pb/g initial wet wt.) of the lead cation after 30 min incubation of the everted sac preparation at 37 0C in Krebs-Henseleit buffer containing 10-7-5 x 10- M-lead labelled with 203Pb. Each value is the mean of six experimental observations + s.E. of mean.
Sac D2 V XI
Compartment Serosal (p-mole) Tissue (n-mole) Serosal (p-mole) Tissue (n-mole) Serosal (p-mole)
10-7 m-Pb 1-92 + 0-28
5 x 10-7 M-Pb
0-23+0-03 1-54+0-24 0-33 ± 0-03 1-68 + 0-34
1-99±0-49 7-69 ± 0-67 2-98 ± 0-26 6-73 ± 0-67
6-30±2-36 20-0 + 2-8 5-14 + 0-63 15-4 ± 1-0
Tissue
0-34 ± 0-05
2-75 + 0-55
4-28 + 0-87
5 x 106 M-Pb 96-6± 25 23-8 ± 4-8
10-5 M-Pb 136 ± 27-0 26-7 + 2-8 150±49-0 39-0 + 6-4 110± 18-0 38-8+5-8
5 x 10-5 M-Pb 462 ± 115 102-4 ± 13-5 410± 63 166-3 ± 12-5 423±96
(n-mole)
Sac
Compartment
D2
Serosal (p-mole) Tissue (n-mole) Serosal (p-mole) Tissue (n-mole) Serosal (p-mole) Tissue (n-mole)
V XI
68-8+ 9-0
26-5 + 13-5 78-3+ 14-0
23-4+9-1
9-13±1-35
10-6 M-Pb 23-6 ± 3-0
121-6+26-0
Time based studies indicated that there was a linear transport of Pb2+ into the serosal compartment (Fig. 1). Extrapolation of the regression line to the Y axis produces an intercept some distance from the origin. The probable interpretation of the data is that the maximum rate of lead transport to the serosal compartment occurs during the first 10 min of incubation. Little evidence of saturation was
346 J. A. BLAIR, I. P. L. COLEMAN AND M. E. HILBURN observed after 60 min incubation over a lead concentration range of 10-7-10-5 M. A good correlation between lead transport and water movement into the serosal compartment over the incubation period of 1 hr, for all regions of the small intestine, was also observed (r > 0.92) (as illustrated for the mid-jejunum in Fig. 2). The maximum rate of transport to the serosal compartment coincided with a very rapid uptake of lead onto the intestinal tissue, equilibration of tissue and mucosal lead occurring after approximately 20 min incubation (Fig. 3). The same pattern of lead uptake by the intestinal tissue occurred at all lead concentrations studied. 55r 50
r>096
-
45 Fa)
40
3
[-
n
CA
35 1-
._
30
-
4-
.0
E1 25 F0 4,
20
-
15
-
10 5 BE
I
I
I
I
10
20
30 Time (min)
45
60
Fig. 1. Transport of 10-1 M-lead labelled with 2'Pb into the mid-jejunal serosal compartment of the everted sac after incubation for periods up to 60 min at 37 IC. Each value is the mean of six experimental observations ± S.E. of mean represented by vertical bars.
Efflux experiments Everted sacs incubated for 30 min in buffer containing 106 M-lead labelled with mPb were transferred to non-radioactive buffer and the amount of activity appearing in the bathing medium was measured during a further period of 30 min. Efflux of lead from the tissue was not affected by either the presence or absence of lead in the bathing medium or the presence of DTPA (106 M). Approximately 25 % of the lead was removed from each region of the intestine after 30 min incubation. Half
347 INTESTINAL ABSORPTION OF LEAD of this amount was removed during the first 5 min, after which the appearance of lead in the bathing medium followed a linear trend. A greater percentage of lead was removed from the duodenum than from the jejunum or ileum but the difference was not statistically significant. 60 rm
r>092
5050
0.-
* 40 ¢ 30 C cm
20
0
E 6. 10
0
100
500
1000
Water movement (mg HO/g initial wet wt. of tissue)
Fig. 2. Correlation between the transport of water and lead across the mid-jejunum after incubation periods of either 10, 20, 30, 45 or 60 min at 37 00 in 104 M-lead labelled with 20SPb. Each value is the mean of six experimental observations + s.E. of mean appropriately represented by horizontal and vertical bars. The incubation time associated with each experimental point is indicated in parentheses. an
r-
03
149
'0
8
C 0.
5 4
+.
_6 5 0,6 6.
32
-0 0o
i CL
I
I
I
I
I
10
20
30 Time (min)
45
60
Fig. 3. Uptake of 10-6 M-lead labelled with 203Pb by the mid-jejunal tissue after incubation for time periods up to 60 min at 37 'C. Each value is the mean of six experimental observations ± s.E. of mean represented by vertical bars.
Factors affecting lead transport The transport of the lead cation onto the tissue and into the serosal compartment was measured over a range of glucose concentrations from zero to 40 mm. No significant effect was observed on lead transport into the serosal compartment during
348 J. A. BLAIR, I. P. L. COLEMAN AND M. E. HILBURN a 30 min incubation period. Both lead transport and water movement into the duodenal and jejunal serosal compartments were depressed however after 1 hr when glucose was absent from the bathing medium (Table 2). The passage of lead into the serosal and tissue compartments was not significantly affected by a depression of the incubation temperature. Transport of lead into the serosal compartment over a lead concentration range 106-10-5 M yielded Q10 Table 2. Effect of zero glucose and 20 mM-glucose on transport of lead and water across the duodenum (D2), jejunum (V) and ileum (XI) after 60 min incubation at 37 0C in Krebs-Henseleit buffer containing 10-6 M-Pb labelled with 203Pb. Lead transport expressed as p-mole Pb/g initial wet wt. Water movement expressed as mg H20/g initial wet wt. Each value is the mean of six experimental observations ± s.E. of mean. Transfer of lead to serosal compartment Glucose concentration (mM) Sac D2 V XI
0
25-9 ± 2-79 21-2 ± 3-31
25-9 + 6-11
20 347 +2-40 393 +4-81 278 +4-42
P value 0-05 0.02
0-8
Transfer of water to serosal compartment Glucose concentration (mM)
Sac D2 V XI
0 -104+ 59 120 +87 528 +174
20
59± 79 581+ 152 741 + 151
P value 0-05 0-02 0-3
Table 3. Effect of 10-i M-DTPA on transport of lead across the duodenum (D2), jejunum (V) and ileum (XI) after 30 min incubation at 37 'C in Krebs-Henseleit buffer containing 10-6 M-lead labelled with 203Pb. Lead transport expressed as p-mole Pb/g initial wet wt. Each value is the mean of six experimental observations + S.E. of mean. Sac D2 V XI
Transfer of lead into serosal compartment 10-5 M-DTPA Control 29-8 ± 6-25 49-0 6-73 13-2 + 1-83 44-8 +7-69 14-7 ± 3-61 38-9 5-77
Sac D2
Uptake of lead into tissue 10-5 M-DTPA Control 2-28+0-42 1-83+0-29
V XI
4-10 0-57 3-04 + 0-61
2-47± 035 1-64± 0-25
P
0-05 0-001 0-01 P 0-4 0-02 0-02
values between 1-43 (distal duodenum) and 0-68 (distal ileum). The uptake of lead by the intestinal tissue was partially reduced over the same concentration range, giving Q10 values between 1-8 (distal duodenum) and 1-2 (distal ileum). When the everted sac preparation was incubated for 30 min in the presence of an equimolar quantity of lead and DTPA (106 M) there was an increase in the transport
INTESTINAL ABSORPTION OF LEAD
349 of lead to the serosal compartment across all regions of the intestine. In the presence of a tenfold excess of DTPA the increase in the transport of Pb2+ became significant. At the same time there was a significant reduction in the amount of lead taken up by the jejunal and ileal tissue (Table 3). DISCUSSION
The transport of Pb2+ into the serosal compartment across selected sites of the small intestine is slow and dependent on the initial concentration of lead in the mucosal bathing medium. Lead transport appears to proceed in a linear manner over the 1 hr experimental period, although it is probable that there is a more rapid passage of Pb2+ into the serosal compartment during the first 10 min of incubation. There is no significant difference in the amount of Pb2+ transported across different regions of the small intestine. On the other hand there is a rapid massive uptake of lead by the intestinal tissue which again is dependent on the initial mucosal concentration of lead. More than half the total lead content is taken up by the tissue during the first 10 min of incubation. The data are comparable to those of Sahagian, Harding-Barlow & Perry (1967) who demonstrated by means of an in vitro perfusion of the intestine that there is a rapid uptake in zinc, cadmium and mercury by the intestinal tissue but only a slow rate of transport to the serosal compartment. It would appear that the passage of certain trace metals across the intestinal wall may be dependent upon two separate rate-limiting steps. The first is the uptake and binding of the metal by cellular surfaces, and the second, the transport of the metal across the intestinal membrane and subsequent accumulation in the serosal compartment. Therefore the transport of lead may largely depend upon (a) the affinity of lead for binding sites on the tissue, (b) the number and availability of particular binding sites and (c) the nature of the interaction between the lead cation and the intestinal tissue. The rapid massive binding of lead to the intestinal tissue suggests a surface adsorption process. If the interaction between the lead ions and the tissue is ionic a rapid exchange between the lead in the tissue and that in the mucosal medium would be expected. However the exchange is slow and incomplete. Neither is a large proportion of the tissue lead chelatable. Both facts are suggestive of a predominantly covalent bonding of lead to the tissue. The external surface of the mucosal epithelial cell is rich in phosphate ions due to the hydrolysis of ATP (Koenig & Vial, 1970). The lead ion in the tissue could therefore be sequestered in a covalent form as lead phosphate. This interaction is analogous to that which occurs between erythrocytes and Pb2+ (Clarkson & Kench, 1958). A consequence of lead being sequestered as lead phosphate would be that the Pb2+ would be less available for transport. Although it is often difficult to extrapolate from in vitro experimentation to the normal physiological condition (Forth & Rummel, 1975), the intestinal membrane may act as a protective mechanism against the passage of a large proportion of the lead present in the normal diet. Sequestered lead phosphate along the entire length of the small intestine may also reduce membrane permeability. The passage of the lead ion into the serosal compartment does not display saturation kinetics, nor does incubation at different temperatures appear to affect any
J. A. BLAIR, I. P. L. COLEMAN AND M. E. HILBURN transport processes. There is also a good correlation between lead transport and water movement across the intestinal wall. Over a range of glucose concentrations the movement of lead is unaffected during a 30 min incubation period; after 60 min incubation in the absence of glucose the transfer of lead across the duodenum and the jejunum is reduced, due to reduced water movement. The experimental data strongly suggest therefore, that the movement of lead across the intestinal membrane occurs by a passive diffusion process linked to some degree to the concomitant movement of water. Further indication of a non-active transfer system has been provided by Gruden & Stantic (1975) who reported serosal-mucosal ratio values of unity after incubating everted sacs for 60 min with equal concentrations of lead on both sides of the membrane. The presence of DTPA in the bathing medium increases the amount of lead transported to the serosal spaces but reduces tissue uptake of lead. Other investigators (Goodman & Gilman, 1965) have shown that DTPA is poorly absorbed by the intestine of man and experimental animals. It may be expected therefore that when the lead cation is complexed to form a neutral species, subsequent interaction with tissue phosphate ions will be reduced, whereas transport of the completed organo-lead moiety to the serosal compartment will be enhanced. 350
The work is supported by the European Economic Community (grant no. 167-77-1-NEV-UK), the Science Research Council and the City of Birmingham Environmental Department.
REFERENCES BARLTROP, D. & KHOO, H. E. (1975). The influence of nutritional factors on lead absorption. Post-grad. med. J. 51, 795-800. BARRY, R. J. C., DIKSTEIN, S., MATTHEWS, J., SMYTH, D. H. & WRIGHT, E. H. (1964). Electrical potentials associated with intestinal sugar transfer. J. Physiol. 171, 316-338. CLARKSON, T. W. & KENCH, J. E. (1958). Uptake of lead by human erythrocytes in vitro. Biochem. J. 69, 432-439. FORTH, W. & RUMMEL, WV. (1975). Gastrointestinal absorption of heavy metals. In International Encyclopaedia of Pharmacology and Therapeutics, section 39B, 2,599-746. Oxford: Pergamon. GARBER, B. T. & WEI, E. (1974). Influence of dietary factors on the gastrointestinal absorption of lead. Toxic. apple. Pharmac. 27, 685-691. GOODMAN, L. S. & GILMAN, A. (1965). The Pharmacological Basui of Therapeutic8, 3rd edn, pp. 934-939. New York: Macmillan. GRUDEN, N. & STANTIC, M. (1975). Transfer of lead through the rat's intestinal wall. Sci. total Environ. 3, 288-292. HILBURN, M. E. (1977). Environmental lead in perspective. British Association Meeting, University of Aston, Section B. (Chem.). KOENIG, C. S. & VIAL, J. C. D. (1970). A bistochemical study of adenosine triphosphate in the toad gastric mucosa. J. Histochem. Cytochem. 18, 340-353. KOsTIAL, K., SImONOvIC, I. & Pisomc, M. (1971). Lead absorption from the intestine in new born rats. Nature, Lond. 233, 564. KREBS, H. A. & HENSELEIT, K. (1932). Untersuchungen fiber dieHarnstoffbildung in Tierkorper. Hoppe-Seyler's Z. physiol. Chem. 210, 33-66. RABINOWITZ, M., WETHERILL, G. & KoPPLr, J. (1975). Absorption, storage and excretion of lead by normal humans. Trace Subst. environ. Hlth 9, 361-368. SAHAGIAN, B. M., HARDING-BARLOW, I. & PERRY, H. M. (1967). Transmural movements of zinc, manganese, cadmium and mercury by rat small intestine. J. Nutr. 93, 291-300. SALWAY, J. G. (1969). The simultaneous determination of acetoacetate and glucose in capillary blood. Clinical chim. Acta, 25, 109-116. WILSON, T. H. & WISEMAN, G. (1954). The use of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. J. Physiol. 123, 116-125.
