Life Sciences Vol . 20, pp . 955-960, 1977 . Printed in the U .S .A .
THE IFEET OF
Pergamon Prese
CAIIMIUK OB I1
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H . T . Davier and J . â. Campbell Eowett Eesearoh Institute, Baclrsburn, Aberdeen AB2 9sB (Received in final fora February 1, 1977) Summary A study has been made on the effects of dietary Cd on On absorption in rate . When Cd was included in diets containing 2 .6 mg Cu/kg to give Cd:Ca moles ratios of 0:1, 1 :1, 2 :1 and 4 :1, the absorption of a tracer dose of 64Ca added to the diets was reduced -at the highest level of Cd egpple~gtation . At all levels tested dietary Cd increased the amount of c9 retained by the intestinal tissue . In a subsequent aariment it was found that recovered in a low molecular dietary Cd increased the amount of form (mol wt 10,000) although these was a4~inverse relationship between dietary Cd content and amount of °4t .ß bond . The possible roles of this Cu binding fraction in intestinal Ca absorption are discussed . Van Campen(1) first demonstrated that Cd2+ inhibited the absorption of 64Ca by rat duodenm . Staroher(2) confirmed these findings in the ohioi and vivo or added showed that Cd either administered by gastric intubation in vitro to muooeal homogenates inhibited the binding of to a low mole Since Zu similarly reduced oular weight protein in the mnoosal oytosol . both 646u absorption and binding to the duodenal protein, he suggested that binding of On to this fraction is an "important step in the proosse of Ca absorption" . A similar conclusion was reached by Bvans and his oo-wortere from studies of 640a binding to duodenal fraotione in both the bovid(3) and rat(4), and he suggested that this low molecular weight Ctt-binding protein . may be metallothionsin . In the studies of both Van Campen(1) and Starcher(2) the lowest molar ratios of Cd :Cu used (150 :1 and 20 :1 respectively) were far higher tban those used in dietary studies in rate (Campbell and Mills(5)) in which ratios as low as 1 :1-4 :1 were found to induce a conditioned Cu deficiency . The current experiments were therefore oonduoted to determine , firstly whether low levels of dietary Cd could adversely affect Cu absorption and secondly to investigate further a possible role for the low-molecular weight metal-b id protein in Cu absorption . Materials and Methods Dietary treatments Eats were Male Water rats (100-160 g) were used in all experiments . trained to eat two meala per day -(Davies and Hightingale(6)) of a semi-synthetic diet(7) containing 2 .6 mg CnAg . Once the meal feeding pattern was established 955
956
Effect of Cadmium on Copper Absorption
Vol . 20, No . 6, 1977
the rate were allocated at random into four groups and offered meals of the same diets supplemented with Cd (as 30804 .8420) to give Cd contents of 0, 4 " 4 . 8 .8 and 17 .6 mg 04/ire, hence the respeati*e Cd :Cu molar ratios were 0 :1, 1 :1, 2 :1 and 4 :1 . In all experiments the rate were maintained on the Cdsupplemented diet (offered as-meals) for 7 days prior to receiving a meal containing a tracer dose of 64Ca (1 Wi op . activity 25 PCi/üg Cu: Radioohemioal Centre, Amersham, Books, U.K .) . Mete containing radioactivity were prepared as described by Davies and Nightingale(6) . Measarement of
ebsortion and duodenal binding
r hours after receiving the radioactive On. the rate were assayed for C64Cu u in a whole-body monitor (Nuclear Enterprises, 83abthill, Mdinb gh, U.K.), killed undsr ether anaesthesia mad the entire gastrointestinal tract removed and. the carcasses assayed mania for 64Ca. The difference in radioactivity was taken to represent whole body retention (absorption) of the 64Ca. Results were expressed as % absorption of the ingested 64Cn . Binding of 64Ca to intestinal tissue was measured by determining the radioactivity remaining is 15 om lengths of duodena which had been washed with Win n (9 These ). were oven dried, weighed and. the results expressed as opm g dry wt of tissue . Binding cf
640a
to mnooaal gtosol fractions
Twenty rate on the same dietary treatment as described above were similarly dosed with 64Cu . Three hours after receiving the radioactive diets the rate were killed and samples of mamma from the first 30 om of the small intestine were scraped from the underlying muscle layers, pooled within treatment gro ~and separately homogenised in 5 volumes of Trio buffer (0 .01 M pH 7 " 41 " Gel filtration on Sepbadex G.75 and estimation of oytosol metal binding proteins were carried out as described for liver homogenates(7), except that prior to metal analysis each fraction was assayed for 64Cu is a well-type grime counter (Tracer Ieb, Instruments Division, Horsham, Surrey, All counts were corrected for background and radio-decay. U .K .) . Trowe_ ele e~at analvoi e All analyses vere performed on as atomic absorption spectrophotometer (AA5 Teohtron Pty, Melbourne, Australia) . Results The combined results of two experiments in which whole body retention and duodenal b inding of 64Cu were measured are shown in Table 1 . The results indicated that at the two higher dose, Cd inhibited b4CU absorption although a statistically signifi cant effect vas only observed in the group receiving In contrast to 64Cu absorption, the the diet with the highest Cd content . binding of 64Ca to dnod@Dal. tissue was significantly increased in all groups receiving Cd supplemented diets . In the final experiment, rate maintained on the same dietary treatments as in the first exparinente were similarly dosed with 64Ca and the cytosol fraction In all groups 64Cu of the duodenal naoosa ohrromatographed on Sephadsx G.75 . eluted as one major traction with a molecular weight corresponding to 10,000 The remaining daltons, which accounted for 65-75% of the oytosolio 64Cu . radioactivity recovered from the column did not resolve into discrete peaks but me fairly unifomly distributed amongst the fractions of higher molecular weight as illustrated in the elution profile of 64Cu for rate receiving the diet containing 4.4 mg Cdrkg (Pigure 1) .
Effect of Cadmium on Copper Absorption
Vol . 20, No . 6, 1977
957
The effects of dietary Cd as absorption and bines by rat intestine Treatment mg Cd/kg diet CdsCu ratio
1
.2
3
4
0.1
4.4
8.8
17 .6
0
1
2
4
96 ingested 64Cu absorbed
38 .7 ± 2.0
40 .3 ± 3 .5
33 .1 ± 2 .7
21 .6 ± 2 .9***
96 ingested 64Cu bound/9 dry duodena
11 .0 ± 0.79
19 .8 ± 2 .77**
18 .9 ± 1 .64
20 .2 ± 2.18
10
5
10
&o of animals
5
Significantly different from control (treatment 1) **, P0 .01 ;
, P0 .001
The amount of 64Cu bound in the major Cti-binding fraction and the Zn and Cd contents of this fraction are shoes in Table 2. TABLR 2 The amount of 640a bound and the Zn and Cd contents of the lox-molecular weight metal-binding fraction Treatment
Cd :Cu in diet
1
0 :1
2
64Cu bound,/ g mucosa
Zn (Moles)/ g muooea
Cd (Wwle@)/ g mucosa
4852
0 .022
0 .01
181
22055
0.019
0 .023
3
20
13148
0 .013
0.030
4
491
8798
0.010
0 .046
The amounts of stable On bound in this fraction were in most instances exceedingly small being near or below the limit of detection by atomic motion spectrophotometry. _ Increasing the dietary_ Cd contents caused increasse in the t of Od and of Sn present in this form At all levels tested, increased the asio nttof 640a associated with this low molecular fraction although the amount bound was inversely rs].e`ted to the dietary Cd level. However, even at the highest Cd level of 17 .6 rig which we previously showed (Table 1) ceased a significant reduction in absorption, the 64Ca in this fraction van 8196 greater than in the controls not receiving Cd . The amount of insoluble 64Cu (associated with 100,000 g sediment) acoaunted for 45-5296 of the total On recovered in the ssooosal homogenates and the Cd treatments similarly increased the total amount of In this fraction.
Vol . 20, No . 6, 1977
Effect of Cadmium on Copper Absorption
958
14
6
10
14
18
.
i
.
i
.
i
,
i
22 26 30 34 FRACTION No .
,
I
38
,
42
Distribution of 640a among soluble proteins of duodenal muoosa separated by gel filtration on Sephades G.75
Discussion The studies on the effects of Cd on On absorption reported here demonstrate that at the higüeet dietary CdzCu ratio tested (481) there was a eigflifi0ant Those findings are in agreement with the reduction in Ca absorption. observations of Van Campen et al (1) and Staraher(2) who both used much higher In a previous etmW5) in which similar dietary Cd and Ca doses of Cd . contents were fed to rate as those employed in this current investigation, prolonged exposure to Cd caused reduced p3a®a On concentration, oaernloplaamin activity and liver Cu contents and clearly as inhibition of Cu absorption may EM-41 ar effects have been observed at least in part have been responsible . in pregnant ewes (Mills and Dalgarmo(S)) exposed to low levels of dietary Cd such as could be encountered in contaminated herbage and again it seems likely that a direct effect of Cd on Ca absorption my have been partly responsible .
Vol . 20, No . 6, 1977
Effect of Cadmium on Copper Absorption
959
Our finding that at all levels -boated, dietary Cd increased the amount of 4W bound to duodenal mnoosa may be interpreted by the suggestion that Cd blocks the exit of Cu out of the mucosal cells Whilst GXWrting either less or The finding in the final study no inhibitory effect ou its muoosgl -uptake . that Cd treatments increased the 040u. recovered in both the low molecular weight forma and the insoluble phase again supports this conclusion . gvans(9) has speculated on possible roles for intestinal metallothionsin in On absorption although it should be pointed out that in neither this or studies by other work(2,3 9 4) has the identity of these low molecular weight metal-binding fractions been established . In a recent report, Evans and LaBlaao(10) have prodnoed evidence based on amino acid composition that the low molecular weight Ca-binding protein differs mar&edly from metallothionsin. While the results of this current study oonfia that . On Made to a fraction of molecular weight oa 10,000(2,3,4) our fines that Cd increases the amount of Cu associated in this fraction is at variance with the results of previous investigations(2 and 3) . However, there are important differences in the techniques employed in this study ccagared with those of others which may aooount for these disparate results . Possibly the most important of these is that the rats used in this investigation were exposed to dietary Cd for one week before receiving the diet containing 64Ca . Another difference is that the Cdi0u ration used by us were considerably lower than those used by Starcher(2) . Little has been reported about the meohaai®s involved in the bind of to these intestinal metal-binding fraotione, although Evans and LeBlanc (10) have recently presented evidence indicating that binding of 64Cn in this form is associated with the synthesis of a low molecular weight protein . However it is not known whether isotopic exchange may occur with the traoe amounts of non-radioactive On present in this form, what the relative affinities of On, Cd and Zn are for these metal-bid proteins and finally whether or not pre-existing metal-free binding sites occur in these fractions . The results of the present study showed that in all groups exposed to Cd., the amount of stable metal (Cd + Zn) found in this fraotion was greater than in the unexposed controls . Depending upon the extent to which these different processes of 04Cu binding operate may explain both the differences in the results reported here and those of other workers, as well as the apparently paradoxical observation of an inverse relationship between amount of 64Cu bound and the dietary Cd content (Table 2) . Thus the amount of 64Cn bound in this form may have been influenced both by the existing amounts of the metalbi ndiag components in this fraction as well as competition for binding at the higher Cd :Cu ratios . lihil e these oarrent findings do not preclude a role for this low molecular weight On binding component in Cn absorption, it must again be emphasised that at the highest dose of Cd used an inhibition of b4~ and. this was associated with an increase in the amount of bound in thisd form. °tUbarly until more studies have been carried oat on the identity of these intestinal metal-binding components and on the meoh-isme involved in their synthesis, stability and affinities for different metals it is premature to assume a role for them in trace metal absorption . Jd
wledaemento
The helpful. advice and criticism of this work by Dr C . B . Mille is gratefully acknowledged. This study was funded, in part, by Rio Tinto Zino Services Limited, Bristol, U .K.
960
Effect of Cadmium on Copper Absorption
Vol . 20, No . 6, 1977
Beferenoee 1 . D. H. Pan Campen, J . Batr . e,, 125-130 (1966) . 2 . B . C . Staraber, J . ETutr . QL 321-326 (1969) . . Bio~ . Bee . 3 . G. W. Bvane, P. P . Aajore and W. B. Coroataer, Bioohm Commun . e,~0, 1142-1148 (1970) . $, 285-297 (1973~ . 4 . G. W. 8dane and C . J . Baha, Adv. Bzv . Med. Biol . 5 . J. I. .Campbeu and 0 . P . Mille, P=o . Satr . Boo . .31, 15A-17A (1974) . 6 . N. T. Daviee and $ . Bightiagal e, Brit . J. Hatr. J&, 243-258 (1975)7 . I . Brema-r, B. T . Daviee and C . P . Müle, Bioohem. Boo . TX~ . 1, 982985 (19735-' 8 . C . P. M _ illa and AA. C . Mal~amo, Nat=e 2 171-173 (1972) .
0.
r57
1~ G. W B~ and PP . oE . LeBlaao , Hatt . Ben. l Inteezm .
jI 281-288 (1976) .
THE IFEET OF
Pergamon Prese
CAIIMIUK OB I1
w&T" COPPER ABSOEPTrIOH AIM BIS-IN WE Elf
H . T . Davier and J . â. Campbell Eowett Eesearoh Institute, Baclrsburn, Aberdeen AB2 9sB (Received in final fora February 1, 1977) Summary A study has been made on the effects of dietary Cd on On absorption in rate . When Cd was included in diets containing 2 .6 mg Cu/kg to give Cd:Ca moles ratios of 0:1, 1 :1, 2 :1 and 4 :1, the absorption of a tracer dose of 64Ca added to the diets was reduced -at the highest level of Cd egpple~gtation . At all levels tested dietary Cd increased the amount of c9 retained by the intestinal tissue . In a subsequent aariment it was found that recovered in a low molecular dietary Cd increased the amount of form (mol wt 10,000) although these was a4~inverse relationship between dietary Cd content and amount of °4t .ß bond . The possible roles of this Cu binding fraction in intestinal Ca absorption are discussed . Van Campen(1) first demonstrated that Cd2+ inhibited the absorption of 64Ca by rat duodenm . Staroher(2) confirmed these findings in the ohioi and vivo or added showed that Cd either administered by gastric intubation in vitro to muooeal homogenates inhibited the binding of to a low mole Since Zu similarly reduced oular weight protein in the mnoosal oytosol . both 646u absorption and binding to the duodenal protein, he suggested that binding of On to this fraction is an "important step in the proosse of Ca absorption" . A similar conclusion was reached by Bvans and his oo-wortere from studies of 640a binding to duodenal fraotione in both the bovid(3) and rat(4), and he suggested that this low molecular weight Ctt-binding protein . may be metallothionsin . In the studies of both Van Campen(1) and Starcher(2) the lowest molar ratios of Cd :Cu used (150 :1 and 20 :1 respectively) were far higher tban those used in dietary studies in rate (Campbell and Mills(5)) in which ratios as low as 1 :1-4 :1 were found to induce a conditioned Cu deficiency . The current experiments were therefore oonduoted to determine , firstly whether low levels of dietary Cd could adversely affect Cu absorption and secondly to investigate further a possible role for the low-molecular weight metal-b id protein in Cu absorption . Materials and Methods Dietary treatments Eats were Male Water rats (100-160 g) were used in all experiments . trained to eat two meala per day -(Davies and Hightingale(6)) of a semi-synthetic diet(7) containing 2 .6 mg CnAg . Once the meal feeding pattern was established 955
956
Effect of Cadmium on Copper Absorption
Vol . 20, No . 6, 1977
the rate were allocated at random into four groups and offered meals of the same diets supplemented with Cd (as 30804 .8420) to give Cd contents of 0, 4 " 4 . 8 .8 and 17 .6 mg 04/ire, hence the respeati*e Cd :Cu molar ratios were 0 :1, 1 :1, 2 :1 and 4 :1 . In all experiments the rate were maintained on the Cdsupplemented diet (offered as-meals) for 7 days prior to receiving a meal containing a tracer dose of 64Ca (1 Wi op . activity 25 PCi/üg Cu: Radioohemioal Centre, Amersham, Books, U.K .) . Mete containing radioactivity were prepared as described by Davies and Nightingale(6) . Measarement of
ebsortion and duodenal binding
r hours after receiving the radioactive On. the rate were assayed for C64Cu u in a whole-body monitor (Nuclear Enterprises, 83abthill, Mdinb gh, U.K.), killed undsr ether anaesthesia mad the entire gastrointestinal tract removed and. the carcasses assayed mania for 64Ca. The difference in radioactivity was taken to represent whole body retention (absorption) of the 64Ca. Results were expressed as % absorption of the ingested 64Cn . Binding of 64Ca to intestinal tissue was measured by determining the radioactivity remaining is 15 om lengths of duodena which had been washed with Win n (9 These ). were oven dried, weighed and. the results expressed as opm g dry wt of tissue . Binding cf
640a
to mnooaal gtosol fractions
Twenty rate on the same dietary treatment as described above were similarly dosed with 64Cu . Three hours after receiving the radioactive diets the rate were killed and samples of mamma from the first 30 om of the small intestine were scraped from the underlying muscle layers, pooled within treatment gro ~and separately homogenised in 5 volumes of Trio buffer (0 .01 M pH 7 " 41 " Gel filtration on Sepbadex G.75 and estimation of oytosol metal binding proteins were carried out as described for liver homogenates(7), except that prior to metal analysis each fraction was assayed for 64Cu is a well-type grime counter (Tracer Ieb, Instruments Division, Horsham, Surrey, All counts were corrected for background and radio-decay. U .K .) . Trowe_ ele e~at analvoi e All analyses vere performed on as atomic absorption spectrophotometer (AA5 Teohtron Pty, Melbourne, Australia) . Results The combined results of two experiments in which whole body retention and duodenal b inding of 64Cu were measured are shown in Table 1 . The results indicated that at the two higher dose, Cd inhibited b4CU absorption although a statistically signifi cant effect vas only observed in the group receiving In contrast to 64Cu absorption, the the diet with the highest Cd content . binding of 64Ca to dnod@Dal. tissue was significantly increased in all groups receiving Cd supplemented diets . In the final experiment, rate maintained on the same dietary treatments as in the first exparinente were similarly dosed with 64Ca and the cytosol fraction In all groups 64Cu of the duodenal naoosa ohrromatographed on Sephadsx G.75 . eluted as one major traction with a molecular weight corresponding to 10,000 The remaining daltons, which accounted for 65-75% of the oytosolio 64Cu . radioactivity recovered from the column did not resolve into discrete peaks but me fairly unifomly distributed amongst the fractions of higher molecular weight as illustrated in the elution profile of 64Cu for rate receiving the diet containing 4.4 mg Cdrkg (Pigure 1) .
Effect of Cadmium on Copper Absorption
Vol . 20, No . 6, 1977
957
The effects of dietary Cd as absorption and bines by rat intestine Treatment mg Cd/kg diet CdsCu ratio
1
.2
3
4
0.1
4.4
8.8
17 .6
0
1
2
4
96 ingested 64Cu absorbed
38 .7 ± 2.0
40 .3 ± 3 .5
33 .1 ± 2 .7
21 .6 ± 2 .9***
96 ingested 64Cu bound/9 dry duodena
11 .0 ± 0.79
19 .8 ± 2 .77**
18 .9 ± 1 .64
20 .2 ± 2.18
10
5
10
&o of animals
5
Significantly different from control (treatment 1) **, P0 .01 ;
, P0 .001
The amount of 64Cu bound in the major Cti-binding fraction and the Zn and Cd contents of this fraction are shoes in Table 2. TABLR 2 The amount of 640a bound and the Zn and Cd contents of the lox-molecular weight metal-binding fraction Treatment
Cd :Cu in diet
1
0 :1
2
64Cu bound,/ g mucosa
Zn (Moles)/ g muooea
Cd (Wwle@)/ g mucosa
4852
0 .022
0 .01
181
22055
0.019
0 .023
3
20
13148
0 .013
0.030
4
491
8798
0.010
0 .046
The amounts of stable On bound in this fraction were in most instances exceedingly small being near or below the limit of detection by atomic motion spectrophotometry. _ Increasing the dietary_ Cd contents caused increasse in the t of Od and of Sn present in this form At all levels tested, increased the asio nttof 640a associated with this low molecular fraction although the amount bound was inversely rs].e`ted to the dietary Cd level. However, even at the highest Cd level of 17 .6 rig which we previously showed (Table 1) ceased a significant reduction in absorption, the 64Ca in this fraction van 8196 greater than in the controls not receiving Cd . The amount of insoluble 64Cu (associated with 100,000 g sediment) acoaunted for 45-5296 of the total On recovered in the ssooosal homogenates and the Cd treatments similarly increased the total amount of In this fraction.
Vol . 20, No . 6, 1977
Effect of Cadmium on Copper Absorption
958
14
6
10
14
18
.
i
.
i
.
i
,
i
22 26 30 34 FRACTION No .
,
I
38
,
42
Distribution of 640a among soluble proteins of duodenal muoosa separated by gel filtration on Sephades G.75
Discussion The studies on the effects of Cd on On absorption reported here demonstrate that at the higüeet dietary CdzCu ratio tested (481) there was a eigflifi0ant Those findings are in agreement with the reduction in Ca absorption. observations of Van Campen et al (1) and Staraher(2) who both used much higher In a previous etmW5) in which similar dietary Cd and Ca doses of Cd . contents were fed to rate as those employed in this current investigation, prolonged exposure to Cd caused reduced p3a®a On concentration, oaernloplaamin activity and liver Cu contents and clearly as inhibition of Cu absorption may EM-41 ar effects have been observed at least in part have been responsible . in pregnant ewes (Mills and Dalgarmo(S)) exposed to low levels of dietary Cd such as could be encountered in contaminated herbage and again it seems likely that a direct effect of Cd on Ca absorption my have been partly responsible .
Vol . 20, No . 6, 1977
Effect of Cadmium on Copper Absorption
959
Our finding that at all levels -boated, dietary Cd increased the amount of 4W bound to duodenal mnoosa may be interpreted by the suggestion that Cd blocks the exit of Cu out of the mucosal cells Whilst GXWrting either less or The finding in the final study no inhibitory effect ou its muoosgl -uptake . that Cd treatments increased the 040u. recovered in both the low molecular weight forma and the insoluble phase again supports this conclusion . gvans(9) has speculated on possible roles for intestinal metallothionsin in On absorption although it should be pointed out that in neither this or studies by other work(2,3 9 4) has the identity of these low molecular weight metal-binding fractions been established . In a recent report, Evans and LaBlaao(10) have prodnoed evidence based on amino acid composition that the low molecular weight Ca-binding protein differs mar&edly from metallothionsin. While the results of this current study oonfia that . On Made to a fraction of molecular weight oa 10,000(2,3,4) our fines that Cd increases the amount of Cu associated in this fraction is at variance with the results of previous investigations(2 and 3) . However, there are important differences in the techniques employed in this study ccagared with those of others which may aooount for these disparate results . Possibly the most important of these is that the rats used in this investigation were exposed to dietary Cd for one week before receiving the diet containing 64Ca . Another difference is that the Cdi0u ration used by us were considerably lower than those used by Starcher(2) . Little has been reported about the meohaai®s involved in the bind of to these intestinal metal-binding fraotione, although Evans and LeBlanc (10) have recently presented evidence indicating that binding of 64Cn in this form is associated with the synthesis of a low molecular weight protein . However it is not known whether isotopic exchange may occur with the traoe amounts of non-radioactive On present in this form, what the relative affinities of On, Cd and Zn are for these metal-bid proteins and finally whether or not pre-existing metal-free binding sites occur in these fractions . The results of the present study showed that in all groups exposed to Cd., the amount of stable metal (Cd + Zn) found in this fraotion was greater than in the unexposed controls . Depending upon the extent to which these different processes of 04Cu binding operate may explain both the differences in the results reported here and those of other workers, as well as the apparently paradoxical observation of an inverse relationship between amount of 64Cu bound and the dietary Cd content (Table 2) . Thus the amount of 64Cn bound in this form may have been influenced both by the existing amounts of the metalbi ndiag components in this fraction as well as competition for binding at the higher Cd :Cu ratios . lihil e these oarrent findings do not preclude a role for this low molecular weight On binding component in Cn absorption, it must again be emphasised that at the highest dose of Cd used an inhibition of b4~ and. this was associated with an increase in the amount of bound in thisd form. °tUbarly until more studies have been carried oat on the identity of these intestinal metal-binding components and on the meoh-isme involved in their synthesis, stability and affinities for different metals it is premature to assume a role for them in trace metal absorption . Jd
wledaemento
The helpful. advice and criticism of this work by Dr C . B . Mille is gratefully acknowledged. This study was funded, in part, by Rio Tinto Zino Services Limited, Bristol, U .K.
960
Effect of Cadmium on Copper Absorption
Vol . 20, No . 6, 1977
Beferenoee 1 . D. H. Pan Campen, J . Batr . e,, 125-130 (1966) . 2 . B . C . Staraber, J . ETutr . QL 321-326 (1969) . . Bio~ . Bee . 3 . G. W. Bvane, P. P . Aajore and W. B. Coroataer, Bioohm Commun . e,~0, 1142-1148 (1970) . $, 285-297 (1973~ . 4 . G. W. 8dane and C . J . Baha, Adv. Bzv . Med. Biol . 5 . J. I. .Campbeu and 0 . P . Mille, P=o . Satr . Boo . .31, 15A-17A (1974) . 6 . N. T. Daviee and $ . Bightiagal e, Brit . J. Hatr. J&, 243-258 (1975)7 . I . Brema-r, B. T . Daviee and C . P . Müle, Bioohem. Boo . TX~ . 1, 982985 (19735-' 8 . C . P. M _ illa and AA. C . Mal~amo, Nat=e 2 171-173 (1972) .
0.
r57
1~ G. W B~ and PP . oE . LeBlaao , Hatt . Ben. l Inteezm .
jI 281-288 (1976) .
