Changes in pulmonary Cu−Zn contents in superior mesenteric artery occlusion shock of rabbit

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Changes in Pulmonary Cu-Zn Contents in Superior Mesenteric Artery Occlusion

Shock of Rabbit

YAN MING, *'l LING YIHLING, ~ HUANG SHANSHENG, 1

GONG SHUQING, 2 AND XU ZHENX1NG 2

IDepartment of Pathophysiology, Hebei Medical College, Shijiazhuang, PRC; and 2Department of Chemistry,

Hebei Normal University, Shijiazhuang, PRC

Received July 3, 1990; Accepted August 21, 1990

ABSTRACT

Contents of Cu and Zn of into-pulmonary blood (IPB), out- pulmonary blood (OPB), Lung tissue, and supernatant and macrophages of Lung Lavage were determined in superior mesenteric artery occlusion (SMAO) shock of rabbits. Zn of pulmonary tissue was 11.42 ___ 0.60 and 14.52 ___ 1.78 (IJ-g/g wet wt) in SMAO shock and control groups, respectively. Content of Zn was found to be lower, Cu was not changed, and Cu/Zn ratio increased in lung tissue in SMAO shock. Contents of Cu and Zn in other samples were not changed. The results suggest that lower Zn in lung tissue related to acute lung injury.

Index Entries: Trace elements; Cu; Zn; shock; rabbit.

INTRODUCTION

The re la t ionship be tween the trace elements and the diseases is being more a t tended to, but it has not been reported that lung injury is related to the trace elements in superior mesenteric artery occlusion (SMAO) shock.

*Author to whom all correspondence and reprint requests should be addressed.

Biological Trace Element Research 281 Voi. 29, 1991

282 Ming et at

We have recently obtained evidence that the ultrastructure of lung is damaged in rabbit SMAO shock (1). One of the most important factors in the lung injury is free radicals damage, which is derived from leukocytes and injured cells of the lung (2). It is now believed that trace elements can prevent the lung from injury by stabilizing cell membranes and scaveng- ing free radicals (3). Therefore, it is important to determine content changes of the trace elements and to study relationships between them in SMAO shock, so that mechanism of acute lung injury may be better understood.

The aim of the study was to evaluate effect of pulmonary Cu and Zn metabolism on acute lung injury in SMAO shock.

MATERIALS AND METHODS

Animals DMded Groups

New Zealand healthy purebred rabbits weighing 2.0-3.0 kg were randomly divided into two groups. Each group had seven animals. They were raised under the same conditions with adequate dietary levels of Cu and Zn

Control Group The jugular vein and artery were separated under local anesthesia

(1% procaine), and catheters were inserted into both vessels to the site of aorta and right artium, allowing blood flowing into or out of the lung to be collected. The animals were sacrificed by exsanguination. The trachea was exposed, and the bronchoalveolar lavage was performed immediately by the catheter inserted into the trachea, with 50 mL deionized saline each time, three times in all. The volume of Iavage fluid recovered from the lung lavage was recorded. The supernatant and macrophages can be obtained by centrifugation of the lavage fluid. The macrophages were counted by microscope. After taking off the thorax, the catheter was immediately inserted into the main trunk of the pulmonary artery. The lung was washed with deionized saline to clear the pulmonary circulation, and a piece of lung tissue was taken at the same site.

S / ~ O Shock Group After the abdomen was opened, the superior mesenteric artery of

rabbits was clipped with the hemostatic forceps under local anesthesia. The neck operation and the way to collect the samples were similar to those of the control group. After one hour, the hemostatic forceps were loosened and blood flow recovered. Another hour later, when the animals appeared to be breathing faster with blood pressure below 8.00 kPa (60 mmHg), samples of the into- and out-pulmonary blood, lung tissue, and lung lavage fluid were taken.

Biological Trace Element Research Vot 29, 1991

Changes in Pulmonary Cu-Zn Contents 283

Table 1 Cu Contents of Into- and Out-Pulmonary Blood, Lung Tissue, Supernatant,

and Macrophages of Lung Lavage in SMAO Shock (X +__ SD)

Control SMAO shock t p

IPB (l~g/mL plasma) 0.91 + 0.11 0.87 _ 0.15 0.5714 > 0.05 OPB (p,g/mL plasma) 0.93 + 0.15 0.87 _ 0.16 0.7500 > 0.05 S (la/mL) 0 0 - - - - M (l~g/10 7 cellstmL) 0 0 - - - - T (Dg/g wet wt) 0.88 __+ 0.13 0.96 +_ 0.11 1.2308 > 0.05

IPB: Into-pulmonary blood, OPB: Out-pulmonary blood, S: Supernatant of lung lavage, M: Macrophages of lung lavage, T: Tissue of lung.

Determination of Cu and Zn

After placing in 1:1 HNO3 for 24 h, all ins t ruments and catheters were w a s h e d wi th distilled and de ionized water to avoid other factors f rom inf luencing the Cu and Zn de terminat ions .

The samples , consist ing of 0.5 mL plasma in to-pulmonary , 0.5 mL plasma ou t -pu lmonary , 0.5 ~g lung tissue, 2 mL supernatant , and all mac rophages (1 x 106-1 • 10J cells/mL), were placed in acid-washed test tubes and d iges ted in 2 mL mixed acid (HNO3:HC104:I--I2SO4 = 3:1:1), respectively. Samples were placed into a heat ing block and boiled until approx 0.5 mL remained . Digested samples were b rought to 10 mL with de ionized water and the conten t of Cu and Zn was de te rmined with a f lame-atomic-absorpt ion spec t ropho tome te r (Hitachi 180-80). Data were processed on microcompute r .

The detec t ion limit of Cu and Zn was 0.002 t~g/mL and 0.014 I~g/mL, respectively. The reclamation was 95-105% (4).

RESULTS

Changes of Cu and Zn Contents in lntro- and Out-Pulmonary Blood, Supernatant, and ~lacrophages of Lung Lavage

In the control group, Cu conten ts of in to -pu lmonary blood (IPB) and o u t - p u l m o n a r y blood (OPB) were no t different (IPB: 0.91 _+ 0.11 t~g/mL plasma; OPB: 0.93 --- 0.15 ~g/mL plasma). No Cu was detected in the supe rna t an t and the macrophages . In SMAO shock group, Cu Conten t s of into- and ou t -pu lmona ry b lood were slightly increased, Cu was not detectable in the supe rna tan t and macrophages (Table 1).

In control group, Zn contents in into- and ou t -pu lmonary blood and mac rophages of lung lavage (M) were not significantly different (IPB:

Biological Trace Element Research VoL 29, 1991

284 /Hing et al.

Table 2 Zn Contents of Into- and Out-Pulmonary Blood, Lung Tissue, Supernatant,

and Macrophages of Lung Lavage in SMAO Shock (X _+ SD)

Control SMAO shock t p IPB (lag/mL plasma) OPB (l~g/mL plasma) S (~g/mL) M (l~g/10" cells/mL) T (t~g/g wet wt)

1.73 ___ 0.33 1.69 ___ 0.51 0.1740 > 0.05 1.70 +_ 0.47 1.84 +__ 0.69 0.44,!9 > 0.05 0.42 + 0.09 0.51 + 0.17 1.2568 > 0.05 1.19 ___ 0.50 1.08 _+ 0.31 0.A.A.'!7 > 0.05

14.52 +--- 1.78 11.42 --+ 0.60 4.0330 > 0.01

Abbreviations of IPB, OPB, S, M, and T are defined in Table 1.

1.73 _ 0.33 ~g/mL plasma; OPB: 1.70 _ 0.47 lag/mL plasma; M: 1.19 _ 0.50 i~g/107 cells/mL). Zn was lower in the superna tan t (0.42 _ 0.09 lag/mL). In SMAO shock group , Zn contents in in to -pu lmonary blood were slightly lower, and that of ou t -pu lmona ry blood higher , but the differences be tween them were not significant (Table 2).

The results p resen ted here show that Cu and Zn contents in into- and o u t - p u l m o n a r y blood, the superna tan t , and the macrophages of lung lavage were not remarkably different be tween control and SMAO shock groups .

Changes of Cu and Zn Contents in Lung 77ssue

Cu con ten t in lung tissue (0.88 _ 0.13 lag/g wet wt) was the same as that in blood, Zn in lung tissue (14.52 + 1.78 I~g/g wet wt) was about ten t imes that in lung lavage and blood in control group. Zn con ten t in lung tissue was remarkably lower than in control g roup (p < 0.01), and Cu did not change significantly in SMAO shock group. Cu/Zn ratio in lung tissue was 0.06 and 0.08 in control and SMAO shock groups , respectively. Cu/Zn ratio increase was the result of lower Zn in lung tissue (Tables 1,2).

The results show that lower Zn in lung tissue is an impor tan t feature in SMAO shock.

DISCUSSION

An adul t male h u m a n body contains 1.4-2.3 g of Zn and 0.08 g of Cu, 16 ~g/g wet wt of Zn, and 1.3 ~g/g wet wt of Cu in lung tissue, and 1.2 + 0.19 ~g/mL of Zn and 0.9 ~g/mL of Cu in plasma (5). In lung tissue, Zn conten ts are about 12 t imes h igher than those of Cu. Lung Cu and Zn contents rank sixth, c o m p a r e d to o ther organs in the body. The result of the s tudy also indicates that Cu and Zn contents rank sixth c o m p a r e d to o ther organs in the body. The result of the s tudy also indicates that Cu and Zn contents in in to -pu lmonary blood and lung tissue of rabbits were as m u c h as those of h u m a n . O t h e r researchers believed that Cu and Zn

Biological Trace Element Research VoL 29, 1991

Changes in Pulmonary Cu-Zn Contents

Table 3 Cu Contents of Blood, Lung Tissue, Supernatant

and Macrophages of Lung Lavage in Human and Rabbit

285

Rabbit

Purebred Human New Zealand Nonpurebred

IPB (Ixg/mL plasma) OPB (Ixg/mL plasma) T (Ixg/g wet wt) S (Ixg/mL) M (Ixg/10" cells/mL)

0.9* 0.91 + 0.11 - - 0.93 +_ 0.15

1.3 0.88 --- 0.13 - - 0

- - 0

5.67 • 2.51"*

2.15 +_ 0.70

Abbreviations of IPB, OPB, S, M, and T are defined in Table 1. *Plasma of vena. **Blood.

Table 4 Zn Contents of Blood, Lung Tissue, Supernatant

and Macrophages of Lung Lavage in Human and Rabbit

Rabbit

Purebred Human New Zealand Nonpurebred

IPB (txg/mL plasma) OPB (txg/mL plasma) T (Ixg/g wet wt) S (Ixg/mL) M (txg/10" cells/mL)

1.2" 1.73 _+ 0.33 6.30 • 1.34"* - - 1.70 + 0.47 - - 16 14.5 + 1.78 20.18 • 2.90 - - 0.42 • 0.09 - - -- 1.19 _+ 0.50 --

Abbreviations of IPB, OPB, S, M, and T are defined in Table 1. *Plasma of vena. **Blood.

con ten t s of the n o n p u r e b r e d rabbit lung were h igher than those of h u m a n (6). In p rev ious ly pub l i shed report , Cu and Zn contents in into- a n d o u t - p u l m o n a r y blood, lung tissue, supe rna tan t , and m a c r o p h a g e s of lung lavage were first p r e s e n t e d he re in N e w Zea land pu reb red rabbits (Tables 3,4).

It has b e e n p r o p o s e d that there are two forms of trace e l emen t s in the body , O n e that is combined to prote ins , nucleic acids or o the r c o m p o n e n t s , and the o the r that compr i ses the me ta l loenzymes . Cu in p lasma is ma in ly ceruloplasmic, a n d acts as an oxidase in Fe metabol i sm. The Cu e n z y m e in the l ung is supe rox ide d i smutase (SOD), con ta in ing Cu a n d Zn. C u - Z n S O D is located in var ious cells of the body . There is little d i f ference a m o n g C u - Z n S O D of the species. C u - Z n S O D is able to scavenge the free radical, w h e r e a s Zn in p lasma is Zn b o n d to protein . Zn e n z y m e s are k n o w n to be essential cofactors of at least 200 e n z y m e s ,

Biological Trace Element Research Vol. 29, 1991

286 Ming et al.

Fig. 1. Comparison of Cu contents in into- and out-pulmonary blood, lung tissue, supernatant, and macrophages of lung lavage between control and SMAO shock. Abbreviations of IPB, OPB, T, S, and M are defined in Table l. [Z], control; m, SMAO shock. N.S.: No significant difference.

regulating the synthesis of proteins and nucleic acids, as well as other metabolism. Zn has a special affinity to lipoproteins and enzyme proteins of the biomembranes , stabilizing the membranes (7,8).

Trace elements (Cu, Zn) are absorbed mainly in the digestive tract. They are metabolized in the liver and transported to the lung and other organs through blood circulation. The main excretory route for Cu and Zn is the feces (7). Also, no report showed the metabolic change in lung Cu or Zn and the lung effect on Cu or Zn metabolism in SMAO shock.

The results show that blood either into- and out-pulmonary or lung lavage (supernatant, macrophages) were not remarkably changed between control and SMAO shock groups. This suggests that lung metabolism did not affect Cu and Zn contents of into- or out -pulmonary after one hour of SMAO shock. However , contents of Zn in lung tissue were remarkably decreased in the acute lung injury of SMAO shock (Figs. 1,2). We have presented that the cell membrane was damaged by the products of metabolism and free radicals resulting from ischemia and hypoxia in pu lmonary microcirculation in SMAO shock of rabbits (1), Therefore, Zn- combination with l ipoprotein and membrane protein was interfered with, and Zn in the lung tissue was reduced.


Changes in Pulmonary Cu-Zn Contents 287

Fig. 2. Comparison of Zn contents in into- and out-pulmonary blood, lung tissue, supernatant, and macrophages of lung lavage between control and SMAO shock. Abbreviations of IPB, OPB, T, S, and M are defined in Table 1. 71, control; I , SMAO shock. N.S.: No significant difference.

Two main changes might be considered for lowering Zn of the lung:

1. Cell membrane damage; shortage of Zn lowers the stability and integrity of the cell membrane , leading to release of en- zymes from lysosome and histamine from mastocyte. The ability of the membrane to resist free radicals is impaired (9). 2. C u - Z n S O D structure changes; lower Zn damages the structure and affects the activity of Cu-ZnSOD, consequently re- duces the ability of Cu-ZnSOD to scavenge free radicals (10).

The mechan i sm of pu lmonary lower Zn and its clinical significance should be further studied in the acute tung injury.


288 /H ing et al.

REFERENCES

1. Z. Y. Cui and Y. L. Ling, Chine. J. Crit. Care Med. 2, 16 (1982). 2. A. Peter, Acta. Physiol. Scand. 79, 548 (1986). 3. J. W. C. Peereboom, Sci. Total. Environ. 1, 42 (1985). 4. S. Q. Gong, J. Heb. Norm. Univ. Nature Sci. Edit. 142, 2 (1987). 5. P. J. Aggett, Arch. Dis. Child. 909, 54 (1979). 6. T. Y. Li, J. Trace Elem. 1, 1 (1985). 7. R. J. Cousins, Phsiol. Rev. 238, 222 (1985). 8. T. F. Slater, Biochim. ]. 3, 15 (1984). 9. W. J. Bettger, Lift, Sci. 1425, 28 (1981).

10. A. E. Cass, Biochem. J. 477, 177 (1979).


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Changes in pulmonary Cu−Zn contents in superior mesenteric artery occlusion shock of rabbit