Upsala J Med Sci 85: 5 9 4 6 ,  1980 

Early Changes in Zinc and Copper Metabolism in Rats with 
Alloxan Diabetes of Short Duration after Local 

Traumatization with Heat 
Goran Hallmans and Folke Lithner 

Departments qf Pailwlogy und Medicine, Univer.uty qf UmeTr, Umeri, S<<Beden 

ABSTRACT 

Local heat trauma was induced in rats with alloxan diabetes of 3 days dura- 
tion. Zinc and copper concentrations in serum, liver, heart and pancreas were 

estimated. 

After traumatization there was a decrease of the serum zinc concentration 

in all animals. The lowest concentration was found in the diabetic animals. 

Serum copper concentrations were lower both in traumatized and nontraumatized 

diabetic animals compared with controls. The serum copper concentration was 

th nontraumatized animals in both slightly lower in traumatized compared w 

diabetic animals and controls. 

In the liver there was an increase of zinc and copper concentration in both 

groups of traumatized animals, but especially in the diabetic animals. 

I n  pancreas there was a decrease in the zinc concentration in traumatized 

and nontraumatized diabetic animals compared with controls. 

INTRODUCTION 

The effects of experimental local thermal cutaneous traumatization in non- 

diabetic humans and animals have earlier been studied by others (see Lithner, 

1975). The skin of the lower legs in diabetics has an altered reaction compared 

with controls (10, 12, 13). This altered reaction is related to the occurrence 

of late diabetic lesions, such as microangiopathy and neuropathy, but not to 

the diabetic metabolic derangement per se. 

To determine the importance of the metabolic derangement, we have studied by 

different means the early inflammatory reaction to local heat trauma in alloxan 

diabetic rats and rabbits (5, 11, 13, 14) without finding any obvious differ- 
ences between diabetic and nondiabetic animals. 

It is plausible, however, to suppose not only a late altered inflammatory 

reaction in diabetes (10, 11, 12, 13) but also an early one, and we have found 
a decrease of serum zinc concentrations after local thermal trauma to be sig- 

nificantly lower in diabetic compared to nondiabetic rats (6). 

59 



In the present study, we have reported in more detail about the metabolism 

of zinc and copper in serum, liver, pancreas and heart of alloxan diabetic rats 

after local traumatization with heat. 

MATERIAL AND METHODS 

Thirty male albino rats of the highly inbred R-strain were used ( 4 ) .  Diabe- 

tes was induced in 16 animals at the age of 3 months with an intravenous injec- 

tion of alloxan, 0.34 mmol/kg, as described earlier (9). No insulin was given 
after the injection of alloxan. All animals had blood glucose concentrations 

higher than 1 4  mmol/l, polyuria and glucosuria. No animal had ketonuria. 

Methods of depilation and anesthesia were described previously (5, 1 4 ) .  

Traumatization was performed 3 days after the alloxan injection, the tempera- 

ture used was 6OoC for 5, 10 and 15 secs. Traumatization was induced in the 

anesthetized animals by placing the end surface of an electrically heated 

cylindrical brass rod, 18 mm in diameter, against the skin ( 1 0 ) .  Inspection of 
the skin injury was performed as described previously (5, 6). 

Immediately before the alloxan injection, 4 hrs after alloxan injection, 

immediately before the traumatization and 8 hrs after traumatization 0.5 ml 

blood was obtained from the tail of each rat. After clotting, samples of serum 

were taken. The animals were killed 24 hrs after the traumatization and samples 

were then taken from serum, liver, heart (myocardium, left ventricle) and pan- 

creas. The samples were removed with stainless steel instruments, placed in 

weighed, acid-washed, borosilicate tubes and the wet weights were determined. 

All samples, except serum, were dried at llO°C for 3 days to constant weight. 

The dry weights were determined and the samples were ashed for 24 hrs at 55OoC. 

The ash was dissolved in 0.5 ml 3 mol/l HC1 overnight and then diluted with 

2 ml of distilled water. If necessary, the samples were further diluted with 

0.6 mol/l HC1 for zinc and copper determinations by atomic absorption spectro- 

photometry. The analyses were performed on a Varian AA-6DB spectrophotometer 

at 213.9 nm and 324.7 nm for zinc and copper, respectively. Reference samples 

of the minerals in 0.6 mol/l HC1 were used. Samples of serum were diluted 10 
times in 0.1 - M HC1 before atomic absorption spectrophotometry with reference 
samples in 0.1 M HC1. 

Differences between groups were tested using Student's t-test for unpaired 

observations. The test was modified if the variances were significantly differ- 

ent (p < 0.01; F-test). Differences between groups were also tested with the 
nonparametric rank sum test of Wilcoxon. P < 0.05 was chosen as the level for 
statistical significance. 

60 



RESULTS 

Zinc and copper concentrations 

Alloxan injection did not significantly affect the serum zinc concentrations 

(Table 1). 

Table 1. Zinc concentration in serum before and after injection of alloxan or 
sodium chloride. The results are given as the mean +_ SE. No significant dif- 
ferences were observed between the four groups. 

Before NaCl injection 

4 hrs after NaCl injection 

Before alloxan injection 

4 hrs after alloxan injection 

Concentrations Number 
pg Znlg serum n 

1.33 _t 0.06 14 
1 . 3 1  0.06 14 

1.41 0 . 0 5  16 

1.25 _t 0.09 16 

Eight hrs after traumatization a decrease in serum zinc concentration was 

found both in controls (p < 0.05) and in diabetic animals (p < 0.001). The de- 
crease was most pronounced in the diabetic animals (Fig. 1) and 24 hrs after 

traumatization the decrease persisted in them. 

0 8 2 L  
hours after traumatization 

Fig. 1. The effect of cutaneous traumatization (arrow) by heat on serum zinc 
concentration. The results are presented as the mean SE. T-test results, 
control animals - diabetic animals: 8 hrs p = 0.06. 24 hrs p < 0 . 0 2 .  

The copper concentrations in serum were only registered 24 hrs after trau- 

matization and they were significantly lower both in traumatized and nontrau- 

matized diabetic rats compared with controls (Fig. 2). Lower concentrations of 

serum copper were seen in traumatized diabetic and nondiabetic animals compared 

with nontraumatized animals, but only significantly lower in controls. 

61 



~ l y  Cu/g serum 

0.5 1.5 2.0 

contra( animals 
n on-traumatized 

control animals 
traumatized 

diabetic animals 
non-traumatized 

diabetlc animals 
traumatized 

control animals 
non-traumatized 

control animals 
traumatized 

Fig. 7 
on -erurn copper concentration. The results are presented as the mean 2 SE. 
'I-test results are indicated in the figure. 

The effect of alloxan injection and cutaneous traumatization by heat 

1 1 , ///////f/m& +,05 p.o.001 
I 

An increase of the zinc concentrations in the liver was observed in diabetic 

animals compared with controls and in traumatized animals compared with non- 

traumatized (Fig. 3). The changes of the copper concentrations in the liver 

were similar to those of zinc, however, they were not significant except in 
traumatized diabetic animals compared with controls (Fig. 4 ) .  

diabetic animals 
non-traumatize d' 

$401 phooi 
diabetic animals 
traumatized /////Mu//- 

Fig. 3. The effect of cutaneous traumatization by heat on the zinc concentra- 
tion in the liver. The results are presented as the mean 2 SE. T-test results 
are indicated in the figure. 

62 



~ l y  Cu/g Liver dry weight 
5 10 15 20 25 

control animals 

contrd animals 
non-traumatized 

control animals 
traumatized 

diabetic animals 
non-traumatized 

diabetic animals 

diabetic animals 
traumatized ' 

'ancreas 

3eart 

Fig. 4. The effect of cutaneous traumatization by heat on the copper concentra- 
tion in liver. The results are presented as the mean : SE. 

non- traumatized non- 
traumatized traumatized 

110.8 4.4 101.4 5.6 81.0 3.5*** 
77.8 1.2 72.3 z 0.8 71.0 1.7 

.ug Culg dry weight 

In diabetic animals after traumatization there was a decrease in the 

pancreatic zinc concentrations compared with controls (Table 2). Only a slight 
decrease was seen in the copper concentrations of diabetic animals compared 

with controls. 

In the heart no differences were seen, neither in zinc nor in copper con- 

centrations between groups (Table 2). 

control animals 

Table 2. Zinc and copper concentrations in pancreas and heart bg/g dry weight). 
The results are given as the mean : SE. The results of comparisons with non- 
traumatized control animals (t-test) are indicated. 

diabetic animals 

I u g  Znlg dry weight 

'ancreas 

leart 

non- traumatized non- traumatized 
traumatized traumatized 

7.4 : 0.7 5.3 0.3* 6.6 0.4 6.1 : 0.5 
21.8 0.6 20.1 : 0.9 20.3 1.7 19.4 1.0 

traumatized 

85.3 7.7* 

70.2 1.2 

* 
p < 0.05 

*** 
p < 0.001 

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Macroscopic and microscopic findings in traumatized areas 

A slightly higher, but not statistically significant, extent of purpura was 

seen in controls compared with diabetic animals 8 and 2 4  hrs after the trau; 

matization. Similar findings were described previously (6). 

Microscopically, no differences were found between controls and diabetic 

animals. 

There was extensive tissue damage to the skin after 10 and 15 secs of trau- 
matization, often with necrosis through the entire thickness of the skin in- 

cluding the panniculus carnosus. There was an accumulation of granulocytes and 

mononuclear cells surrounding this devitalized area. After 5 secs of traumati- 
zation the epithelium showed signs of disruption and necrosis with accumulation 

of inflammatory cells, mostly granulocytes, in the upper dermis. Signs of in- 

flammation were sometimes seen to extend to the panniculus carnosus. 

DISCUSSION 

The serum zinc concentration decreased after heat trauma both in diabetic 

animals and in controls while the liver concentration of zinc increased. This 

relationship has been postulated to be due to the liberation of a leucocytic 

endogenous mediator (LEM) from activated phagocytic cells (6) which, in the 
present study, were located in the traumatized areas. Several functional dis- 

turbances in diabetic granulocytes have been reported (see Bagdade et al. 

1978). 
The decrease of serum zinc concentrations was more pronounced in the dia- 

betic animals, and this has been reported previously (6). This can explain the 

more pronounced increase of zinc concentrations in the liver of traumatized 

diabetic animals. However, an increase of the zinc concentrations in the liver 

was also seen in nontraumatized diabetic animals when compared with controls. 

This indicates that an increased concentration of zinc binding sites is found 

in the liver of the diabetic animal. This may be due to an increased synthesis 

or an incorporation of a zinc binding ligand in diabetic animals in response 

to the metabolic derangement or to the injection of alloxan. 

The increase of zinc concentrations in the liver in traumatized controls and 

in nontraumatized diabetic animals suggests that these animals need more zinc 

than what is available from the loosely bound serum zinc. Partly, it comes from 

an increased absorption from the intestine ( 7 )  and partly probably from zinc 

mobilized from tissues of other organ systems. I n  this study, however, no sig- 

nificant decreases were observed in the zinc concentrations of pancreas or 

heart after traumatization, but the zinc concentrations of nontraumatized dia- 

betic animals were decreased compared with the corresponding controls. 

Most of the serum copper is strongly bound to ceruloplasmin, a n d  -2-globu- 
lin with oxidase activity ( 3 ,  8 ) .  A minor part of the serum copper is weakly 

64 



bound to albumin and amino acids (3). Ceruloplasmin belongs to the group of 

acute phase proteins. Elevation of ceruloplasmin and serum copper is usually 

induced by acute stress (1) but we should'nt expect it to be present as soon 

as 24 hrs after the thermal trauma, as in our study. The decrease of the serum 

copper concentration in the diabetic animals probably is due to a decrease of 

ceruloplasmin concentration. The liver plays an important role in copper homeo- 

stasis and 3 of it's functions in this respect are: storage of copper, cerulo- 

plasmin synthesis and excretion of copper through the bile (3). At least one of 

these functions has to be altered after traumatization in the diabetic animals 

which had increased copper concentrations in their livers. 

Acknowledgements 

This study was supported by the Swedish Medical Research Council (No. 718). 
We wish to thank Ann-Marie Nilsson for expert technical assistance. 

1. 

2. 

3. 
4. 

5. 

6. 

7. 

8 .  

9. 

1 0 .  

11. 

12. 

13. 

14. 

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Received November 5, 1979. 

Address for reprints: 

Folke Lithner, M.D. 

Department of Medicine 

University of Umea 

S-901 85 UmeH 

Sweden 

66