The Effect of Heavy Metal-Induced Metallothionein on Zn,Cu and Cd Accumulation in Rat Kidney

Abstract: To examine the role of metallothionein on heavy metal accumulation in kidneys of rats after Zn, Cd or Cu injection, the relative Zn, Cd or Cu-binding capacity of heavy metal-induced metallothionein in renal cytosol of rats after Zn, Cd or Cu injection was determined. The Zn or Cu increment in renal cytosol after Zn or Cu injection was attributable to low and high molecular weight proteins, while most of the Cd increment was attributable to a low molecular weight protein. The low molecular weight, metal-binding protein was identified as metallothionein using a competitive ELISA. There was a close relationship between heavy metal contents in the renal cytosol and metallothionein of all heavy metal-injected rats. In dose-response and time-course studies, approximately 45, 40 and 85% of the Zn, Cu and Cd increments in renal cytosol were bound to metallothionein after Zn, Cu and Cd injection, respectively. Therefore the order of relative binding capacities of Zn, Cu and Cd-induced metallothionein in kidney was determined to be Cd>Zn Cu. These results suggest that the role of metallothionein in Zn or Cu accumulation in the kidney after Zn or Cu injection is different from that of metallothionein in Cd accumulation in the kidney after Cd injection.     

Role of metallothionein on Ag accumulation in hepatic and renal cytosol after Ag injection to rats.

To examine the role of metallothionein on Ag accumulation in liver or kidney of rat after Ag injection, the relative Ag-binding capacity of Ag-induced metallothionein in hepatic or renal cytosol of rat after Ag injection was determined. The greater part of Ag increment in hepatic cytosol was attributable to a low molecular weight protein, while the main part of Ag increment in renal cytosol was ascribed to high molecular weight proteins. The low molecular weight, metal-binding protein was identified as metallothionein using ELISA. The maximal levels of hepatic and renal metallothionein mRNA induced by Ag occurred at 7 hr after Ag injection. There was a close relationship between Ag contents in the hepatic or renal cytosol and metallothionein after Ag injection. In dose-response and time-course studies, approximately 60-70% of the Ag increments in hepatic cytosol and approximately 30% of the Ag increments in renal cytosol were bound to metallothionein. These results suggest that the role of metallothionein on Ag accumulation in the liver after Ag injection is different from that of metallothionein on Ag accumulation in the kidney.     

Role of Metallothionein on Ag Accumulation in Hepatic and Renal Cytosol after Ag Injection to Rats

Abstract: To examine the role of metallothionein on Ag accumulation in liver or kidney of rat after Ag injection, the relative Ag-binding capacity of Ag-induced metallothionein in hepatic or renal cytosol of rat after Ag injection was determined. The greater part of Ag increment in hepatic cytosol was attributable to a low molecular weight protein, while the main part of Ag increment in renal cytosol was ascribed to high molecular weight proteins. The low molecular weight, metal-binding protein was identified as metallothionein using ELISA. The maximal levels of hepatic and renal metallothionein mRNA induced by Ag occurred at 7 hr after Ag injection. There was a close relationship between Ag contents in the hepatic or renal cytosol and metallothionein after Ag injection. In dose-response and time-course studies, approximately 60–70% of the Ag increments in hepatic cytosol and approximately 30% of the Ag increments in renal cytosol were bound to metallothionein. These results suggest that the role of metallothionein on Ag accumulation in the liver after Ag injection is different from that of metallothionein on Ag accumulation in the kidney.     

Effect of dimercaptosuccinate on the accumulation and distribution of cadmium in the liver and kidney of the rat

The effect of dimercaptosuccinic acid (DMSA) on the tissue distribution, renal and hepatic subcellular localization of Cd2+, Zn2+ and Cu2+ in Cd(2+)-pretreated male rats and on the tissue distribution of Zn2+ and Cu2+ in the normal rat was studied. Cd(2+)-pretreated rats which received 3 x 1 mg Cd2+/kg body wt s.c. at 48 hr intervals followed after 7 days by DMSA (50 mg/kg body wt i.p.) daily for 17 days had total hepatic Cd2+ concentrations 25 per cent lower than Cd(2+)-pretreated controls (P < 0.01). DMSA did not influence the concentration or distribution of Cd2+ in the liver cytosol whereas in the mitochondrial-lysosomal and nuclei + cell debris fractions the Cd2+ concentration was reduced by 54 and 48 per cent respectively. Total renal and hepatic Cu2+ concentrations were increased by Cd2+ treatment and reduced by treatment of the Cd(2+)-exposed animal with DMSA. In the liver cytosol Cu2+, Zn2+ and Cd2+ accumulated in the metallothionein fraction and none was mobilized from the cytosol by DMSA. In the kidney cytosol Cu2+ accumulated in fractions in addition to metallothionein and was eliminated from each of these fractions following treatment with DMSA. It is concluded that the high affinity of metallothionein for these cations prevented their elimination from the cytosol and that the interaction of Cd2+ and Cu2+ with DMSA occurred in the particulate fraction and therefore delayed the response to DMSA treatment. It is suggested that long term treatment with DMSA, although ineffective in mobilizing Cd2+ from the kidney may provide a useful therapeutic measure to reduce the liver burden of Cd2+ and the high renal Cu2+ concentrations in the Cd(2+)-exposed animal.     

The effect of sodium aurothiomalate (myochrysin) on the distribution of calcium, magnesium, copper, zinc and iron in the rat

Sodium aurothiomalate was given to male Wistar rats (initial body weights: 150 g) by subcutaneous (s.c.) injection at doses of up to 7.5 mg/kg (corresponding to 4.27 mg gold/kg), twice a week, for 4-5 weeks. The concentrations of Ca, Mg, Fe, Cu and Zn were measured in serum, urine, faeces and in the liver, kidney, spleen, heart, lung, testis, bone and muscle. Kidney cytosol was separated by gel chromatography and the fractions analysed for protein, copper, zinc, iron and gold concentrations. The concentration of copper was increased 5-fold in kidney while smaller increases of zinc in kidney, copper in muscle, iron in muscle and testis and calcium in spleen were found. There was a significant reduction in the concentration of copper in serum. Kidney cytosol from gold-treated but not from control animals contained a low molecular weight protein which was associated with copper, zinc and gold. The rats developed proteinuria and microscopic changes to renal tubular cell structure were also observed. It is suggested that the gold-induced accumulation of copper may follow from an increased rate of synthesis of metallothionein and could be responsible for the renal dysfunction which develops in a proportion of rheumatoid arthritis patients who are treated with gold.     

Induction of metallothionein synthesis by zinc in cadmium pretreated rats

The ability of zinc (Zn) salts to induce the synthesis of metallothionein (MT) in liver, kidney and pancreas of rats pretreated with cadmium (Cd) salts was investigated. Twenty-four hours after either CdCl2 (2.0 mg Cd/kg, s.c.) or saline pretreatment, rats were injected with saline, CdCl2 (2.0 mg Cd/kg, s.c.) or ZnSO4 (20 mg Zn/kg, s.c.) and the concentrations of MT and MT-1 mRNA in tissues subsequently measured. After a single injection of Cd salts, concentrations of MT and MT-1 mRNA were significantly increased in liver as compared to control. With two injections of Cd, the accumulation of MT in liver was approximately twice the levels of MT following a single injection of Cd. In kidney, MT and MT-1 mRNA expression were significantly increased only after two injections of Cd and in the pancreas, Cd injections did not alter either MT content or MT-1 mRNA expression. Treatment with Zn salts increased MT concentrations in both liver and pancreas. However, the pancreas was the most responsive to injections of Zn salts as compared to the liver in terms of increases in both protein concentration and MT-1 mRNA expression. When Zn injection was preceded by a Cd injection, induction as measured by MT-1 mRNA and MT concentrations were approximately additive in liver. In kidney, although Cd or Zn treatment separately had no effect on MT or MT-1 mRNA content, injection of Cd followed by Zn resulted in significantly increased levels of renal MT and MT-1 mRNA. Fractionation of liver cytosols on a Sephadex G-75 column revealed that in animals receiving two injections of Cd, virtually all the Cd was associated with MT whereas Zn was distributed between both high molecular weight (HMW) proteins and MT. In animals receiving both Cd and Zn injections, cytosolic Cd was still bound predominantly to the MT fraction, while the proportion of cytosolic Zn associated with MT increased. The results of this study suggest that, treatment with Cd salts followed by Zn salt injection can induce further synthesis of MT in liver, kidney and pancreas with subsequent binding of both Zn and Cd to the intracellular MT.     

Studies on the copper and zinc content of the rat kidney after treatment with cisplatin

The administration of cis-dichlorodiammine platinum II (cisplatin), at a dose of 5 mg/kg, subcutaneously (s.c.), to adult male Wistar rats, inhibited the normal accumulation of Cu and Zn in the kidneys. Cisplatin also produced a small increase in the concentration of Cu in the brain but had no significant effect on total Cu and Zn concentrations in the heart, thymus, liver, intestine, spleen, testes and muscle. Treatment of rats with 2 mg Cu2+/kg, intraperitoneally (i.p.), increased the renal Cu content whilst cisplatin pretreatment inhibited the uptake of Cu by the kidneys. The administration of cisplatin to the Cd2+-pretreated rat decreased the content of metallothionein-bound Cu in parallel with a reduction in total renal Cu content, but had no significant effect on the total renal content of Cd and Zn. Pretreatment with cisplatin decreased the subsequent uptake of Cd by 42% in the whole kidney and by 23% in the metallothionein fraction, but failed to reduce the Cd2+-induced accumulation of Cu in the kidneys. The significance of these observations in relation to the possible mechanism of the cisplatin-induced change in the Cu and Zn content of the kidney is discussed.     

Urinary metallothionein and tissue metal levels of rats injected with cadmium, mercury, lead, copper or zinc

Since Cd exposure results in a dose dependent increase in metallothionein level in urine, the present investigation was conducted to examine whether exposure to other divalent cations would also cause an elevation in urinary metallothionein. Female Sprague-Dawley rats were injected subcutaneously with either saline, 5 mumol/kg/day of CdCl2, HgCl2, Pb(C2H3O2)2, CuSO4 or ZnCl2 for 5 days. Significant increases in hepatic Cu levels in rats treated with not only Cu, but also Zn, Cd, or Hg, and in hepatic Zn levels in rats treated with Zn or Cd were noted. Similarly, renal Cu and Zn levels were elevated significantly in all groups except the Pb-injected group. These increases in tissue metal levels were presumably due to induction of metallothionein. The urinary metallothionein level in control rats on day 0, determined by radioimmunoassay, was 0.85 +/- 0.17 mg/g creatinine. There was no significant change in urinary metallothionein level in rats given up to 5 injections of saline or Pb. Hg-injected rats showed 25-fold increase in urinary metallothionein after 5 injections, whereas Cd-injected rats had 9-fold increase. There were also 2- and 3-fold increases of urinary metallothionein by Cu and Zn treatments for 5 days, respectively. Thus, urinary metallothionein levels were elevated in response to Cd, Hg, Cu and Zn, but not Pb; Hg had the most profound effect at equimolar doses.     

Stimulation of cadmium uptake by estradiol in the kidney of male rats treated with cadmium

The present study was carried out to analyze the sex differences in the retention of Cd in rats treated with a small amount of Cd, and its mechanisms. Cd and Zn concentrations in the kidney and liver of female rats treated with 28 nmol Cd or 1 nmole Zn were significantly higher than those in male rats. Pretreatment with estradiol (1.8 mumol/kg of b.w., twice a day, 6 consecutive days) increased the Cd and Zn concentrations in the kidney of male rats treated with Cd or Zn. Incubation of MDCK cells with 10(-5) M estradiol, 10(-5) M stilboestrol and 10(-5) M progesterone caused a significant increase in Cd uptake. These results suggest that endogenous female sex hormones may play a role in a higher concentration of Cd and Zn in the kidney of female rats than that in male rats. The basal level of metallothionein (MT) in the liver and kidney of control female rats was within the same range as that in the control male rats. Cd and Zn accumulations caused by pretreatment with estradiol in the kidney of male rats treated with Cd or Zn were so low (Cd: 38 ppb, Zn: 1.0 ppb) as to be probably unable to induce the synthesis of MT. An increase in the concentration of Cd in the cultured renal cells occurred 1 hr after treatment with estradiol and Cd. Pretreatment with estradiol alone also resulted in a modification of the concentration of Na and K, which cannot be bound to MT. Together, all of the above findings suggest that estradiol directly increases the accumulation of Cd into the renal cells without inducing the synthesis of MT.     

Comparative renal toxicity of metallothioneins with different cadmium/zinc ratios in rats

The comparative renal toxicity of rats after injection of cadmium (Cd) and zinc (Zn)-metallothioneins (MTs) with different Cd/Zn ratios at the same dose of 200 micrograms MT-bound Cd/kg was studied. From determination of the urinary excretion of protein, aspartate aminotransferase (AST) and glucose, which are indices of Cd-induced renal damage, the extent of the renal toxicity of the MTs used here was in the order (1 Cd/0Zn)-MT = (2Cd/1Zn)-MT greater than (1Cd/2Zn)-MT greater than (1Cd/6Zn)-MT. The characterization of Cd, Zn and Cu in the urine after injection of MTs was examined using a Sephadex G-75 column. (1Cd/0Zn)-MT injection showed that Cd was present mainly in lower-molecular-weight fractions, with only small amounts of Cd in the MT fraction. Upon injection of other MTs, Cd was present mainly in the MT fraction and increased with decreasing Cd/Zn ratio. Zn was present mainly in lower-molecular-weight fractions and Cu mainly in the MT fraction, indicating the replacement of MT-bound Zn by Cu. The cumulative urinary excretion of Cd during 12 days after injection of MTs decreased with increasing Cd/Zn ratio. The Cd content of the kidney and liver increased with increasing Cd/Zn ratio. The results of this study indicate that in rats injected with MTs with different Cd/Zn ratios, the renal uptake of Cd increases with increasing Cd/Zn ratio, resulting in more severe renal damage.     

Effect of metallothionein core promoter region polymorphism on cadmium, zinc and copper levels in autopsy kidney tissues from a Turkish population

Metallothioneins (MTs) are metal-binding, low molecular weight proteins and are involved in pathophysiological processes like metabolism of essential metals, metal ion homeostasis and detoxification of heavy metals. Metallothionein expression is induced by various heavy metals especially cadmium, mercury and zinc; MTs suppress toxicity of heavy metals by binding themselves to these metals. The aim of this study was to investigate the association between the − 5 A/G metallothionein 2A (MT2A) single nucleotide polymorphism (SNP) and Cd, Zn and Cu levels in the renal cortex from autopsy cases. MT2A core promoter region − 5 A/G SNP was analyzed by PCR-RFLP method using 114 autopsy kidney tissues and the genotype frequencies of this polymorphism were found as 87.7% homozygote typical (AA), 11.4% heterozygote (AG) and 0.9% homozygote atypical (GG). In order to assess the Cd, Zn and Cu levels in the same autopsy kidney tissues, a dual atomic absorption spectrophotometer system was used and the average levels of Cd, Zn and Cu were measured as 95.54 ± 65.58 µg/g, 181.20 ± 87.72 µg/g and 17.14 ± 16.28 µg/g, respectively. As a result, no statistical association was found between the − 5 A/G SNP in the MT2A gene and the Zn and Cu levels in the renal cortex (p > 0.05), but considerably high accumulation of Cd was monitored for individuals having AG (151.24 ± 60.21 µg/g) and GG genotypes (153.09 µg/g) compared with individuals having AA genotype (87.72 ± 62.98 µg/g) (p < 0.05). These results show that the core promoter region polymorphism of metallothionein 2A increases the accumulation of Cd in human renal cortex.     

Metallothionein production: similar responsiveness of avian liver and kidney to chronic cadmium administration

The accumulation of hepatic and renal Cd, Zn, Cu, and metallothionein (MT) was investigated in ringed turtle doves (Streptopelia risoria) chronically exposed to 3 different concentrations of dietary Cd. When only tissue-Cd was considered as an inducer of MT, kidney was found to be 35% as responsive as liver in producing MT. However, when all potentially relevant inducing metals (Cd + Zn + Cu) were taken into account, kidney was found to be 85% as responsive as liver. The greater production of MT/mol Cd in liver was accounted for mainly by a greater co-accumulation of Zn/mol Cd in liver than in kidney. We conclude that the apparent tissue specificity in expression of MT may be overestimated by failure to consider fluctuations in multiple inducers. Variability in tissue-MT concentrations after chronic dietary Cd administration is best accounted for by a consideration of tissue-Cd, -Zn, and -Cu, rather than tissue-Cd alone.     

Urinary excretion of metallothionein-I and its degradation product in rats treated with cadmium, copper, zinc or mercury

The metallothionein-I (MT-I) content of urine following administration of cadmium (Cd), copper (Cu), mercury (Hg) or zinc (Zn) to rats was determined by radioimmunoassay. Urinary excretion of MT-I was increased significantly after injection of each of these metals. Fractionation of urine from Cd-treated rats on Sephadex G-50 showed a single immunoreactive component corresponding to native MT-I, whereas in urine from Cu, Zn or Hg-treated rats 2 immunoreactive components corresponding to MT-I and a possible degradation production were observed. Since a comparable low molecular weight component corresponding to this degradation product was not detected to the same extent on fractionation of plasma from Cu-exposed rat, it seemed to be derived from degradation of MT in the kidney.     

Endogenous metallothionein as determinant of intestinal cadmium absorption: a reevaluation

Isolated segments of rat jejunum were perfused in situ with glucosesaline containing varying amounts of CdC12. At low Cd concentrations the endogenous Cd-binding capacity sufficed to trap almost all Cd taken up in the mucosa. At higher Cd levels, induction of metallothionein synthesis by Zn increased fractional trapping of Cd. Pretreatment with Zn did not induce protein synthesis in any fraction other than the low molecular weight heatstable cytosol fraction. Acutely Zn did not affect Cd extrusion from the mucosa. Finally, Cd-metallothionein itself crosses the mucosa only very slowly. An earlier conclusion that metallothionein does not contribute to Cd trapping was based on low Cd concentrations in the perfusate; at higher concentrations metallothionein appears to be a major determinant of mucosal Cd retention.     

Protection against cadmium toxicity by zinc: Decrease in the Cd-high molecular weight protein fraction in rat liver and kidney on Zn pretreatment

The amounts of cadmium, associated with high molecular weight proteins, metallothionein and low molecular weight fractions obtained on Sephadex G-75 gel filtration, were determined in the liver and kidneys of rats treated with Cd. When rats were pretreated with zinc 24 h prior to the Cd injection, Cd associated with the high molecular weight proteins was decreased in both the liver and kidneys. Although the Cd concentration in the liver was increased, the liver showed less morphological damage in Zn-pretreated rats. The above results suggest that Cd-toxicity toward the liver and kidneys may be related to the accumulation of Cd in the high molecular weight proteins.     

Developmental and sex differences in cadmium distribution and metallothionein induction and localization

Age- and sex-related differences in hepatic and renal distribution of cadmium (Cd) and the effect of Cd injection (10 mumol/kg) on tissue zinc (Zn), copper (Cu) and metallothionein (MT) levels were investigated in 2- to 84-day old rats. Renal Cd accumulation increased with age of the animal. Sex differences in renal Cd accumulation were noted in young animals where the 2- and 8-day old males had significantly greater concentration than the females. There were no clear effects of Cd on renal Zn. Renal Cu levels, however, were elevated in the adults. The adult females contained about twice as much MT as the adult males. Cd treatment had no effect on renal MT levels of 8- to 84-day old animals but depressed the levels in 2-day old. Age-related increase in hepatic Cd accumulation was also found; the pattern was more clear cut in females than in males. In addition, in the females the hepatic Cd concentration was significantly higher than in the males. Cd-injection significantly increased hepatic Zn and MT concentrations only in weaned animals. While there were no sex differences in MT levels in the young animals, the weaned females had significantly more MT than the corresponding males. Immunohistochemical staining for MT showed positive staining in both cytoplasm and nuclei of the parenchymal cells. The number of MT-positive nuclei was dependent on the relative MT concentration of the liver. In spite of the intense nuclear staining in 2-day old controls and 84-day old Cd-injected rats, less than 1% of the hepatic MT was present in the nuclear fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

A possible mechanism of resistance to cadmium toxicity in male Long-Evans rats

The susceptibility to cadmium (Cd)-induced toxicity in male Long–Evans (LE) rats was compared with that in male Fischer 344 (Fischer) and Wistar–Imamichi (WI) rats, which are sensitive and resistant, respectively, to Cd toxicity. All rats of the LE and WI strains survived for 7 days after the treatment with a toxic dose of Cd (6.5 mg/kg b.w.). However, all rats of the Fischer strain died by the following day. The strong resistance to Cd toxicity in the LE strain was confirmed to be independent of metallothionein synthesis induced by Cd. The hepatic and renal Cd contents after its administration were significantly lower in the LE strain than in the Fischer strain. Furthermore, the hepatic and renal zinc (Zn) contents after its administration were significantly lower in the LE strain than in the Fischer strain. These limited data suggest that the strong resistance to Cd toxicity in male LE rats results from, at least in part, the lower accumulation of the metal in the liver and kidney, in a similar mechanism as the lower Zn accumulation.     

Induction and binding of Cd, Cu, and Zn to metallothionein in carp (Cyprinus carpio) using HPLC-ICP-TOFMS.

The binding of Cd, Cu, and Zn to metallothionein in carp was studied under control and acute Cd exposure scenarios. Carp were exposed to different Cd concentrations for 96 h. Total (Cu, Cd, Zn)-MT levels were determined by the cadmium thiomolybdate saturation assay. Total tissue and cytosolic Cd, Cu, and Zn concentrations were determined by ICP-MS. The cytosolic metal speciation was determined by high pressure liquid chromatography (size-exclusion [SE] in combination with anion exchange [AE]) directly coupled to an inductively coupled plasma time of flight mass spectrometer (ICP-TOFMS). This coupled technique allows the chromatographic separation and online determination of the metals associated to the protein fractions separated. Very strong differences in the tissue compartmentalization and cytosolic speciation of the metals were observed. For example, over 30% of cytosolic zinc was bound to MT in liver while this was only 2% in the kidneys although total cytosolic levels were considerably higher. Induction of metallothionein during cadmium exposure was also tissue specific, displaying different response patterns in gills, liver, and kidney. Cadmium accumulated much stronger in liver and kidney compared to the gills and the latter also showed much lower MT levels. The renal MT-induction was more sensitive to Cd exposure than the hepatic MT induction since a significant increase of Cd-MT and total MT levels occurred at lower tissue Cd concentrations in the kidney in comparison to the liver, except for the highest Cd exposure level where a drastic 10-fold increase in hepatic Cd-MT was observed. At this Cd exposure level also an apparent spill over of zinc to the high molecular weight fraction was observed in the kidneys.     

Role of intestinal metallothionein in absorption and distribution of orally administered cadmium

The effects of mucosal metallothionein (MT) preinduced by Zn on the intestinal absorption and tissue distribution of Cd were studied. 109CdCl2 was administered to control and Zn-pretreated rats. The total amount of Cd distributed to the liver and the kidney in the group pretreated with 100 mg/kg of Zn was about 70% that of the control group. In the control group, the Cd concentration in the intestinal mucosa reached a maximum 16-24 hr after its administration and then gradually decreased with time, unlike that in the liver and the kidney. The concentration of intestinal Cd in the pretreated group reached a maximum earlier than it did in the control group and most of the Cd was in the MT fraction. Pretreatment with Zn (100 mg/kg or higher, po) caused a reduction in the Cd concentration in the liver and an increase in the kidney. Pretreatment with Zn (5 X 10 mg/kg, sc) or Cd (5 mg/kg, po) also increased renal Cd concentration. This was effective at 24 hr but not at 0.5 hr after pretreatment. These effects of pretreatment with Zn (100 mg/kg, po) on tissue distribution of Cd were also observed after an intraintestinal injection of Cd but not after an iv injection. The results indicate that MT in intestinal mucosa plays a significant role not only in the absorption of Cd but also in its transport to the kidney.     

Effects of zinc deficiency of pre-existing cadmium-metallothionein in the pancreas

Although the injection of cadmium salts increases the metallothionein (MT) concentration in a number of organs, including pancreas, the induced synthesis of pancreatic metallothionein was specifically decreased in zinc difeciency. The fate of C-induced metallothionein and the change in tissue distribution of Zn and Cd were studied with the onset of Zn deficiency. Rats were rapidly injected with CdCl₂ to induce MT synthesis, and were subsequently made Zn deficient. The lability of the pancreatic zn pool, as compared with that of liver and kidney, was demonstrated. During Zn deficiency, there eas little change in Cd concentration or MT level in either liver or kidney. When Zn dificiency led to a decrease in pre-existing pancreatic MT, Cd was not elimited from the pancreas, but was transferred to a high molecular weight fraction. On the basis of these results we suggest that the presence of Cd-Zn-MT has little effect on the eventual pancreatic Zn and MT contents, during subsequent Zn deficiency.