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Changes of the Cu and Zn Contents in Lung and Liver in Intestinal Ischemic

Reperfusion and General Ischemic Reperfusion in Rabbits

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

WANG DIANHUANG, 1 GONG SHUQING, 2 AND XU ZHENXING 2

IDepartment of Pathophysiology, Hebei Medical College, Shijiazhuang, P.R.C; and 2Department of Chemistry, Hebei Normal University,

Shijiazhuang, P.R. C

Received January 5, 1993; Accepted February 12, 1993

ABSTRACT

The changes of pulmonary and liver Cu-Zn contents were deter- mined and evaluated in intestinal ischemic reperfusion (IIR) and general ischemic reperfusion (GIR) of rabbits. The contents of pul- monary Zn and liver Cu were found to be lower, and Cu/Zn ratio increased in lung tissue and decreased in liver tissue in IIR. The contents of pulmonary Zn were increased, and the contents of liver Cu were decreased; Cu/Zn ratio also decreased in both tissues in GIR. Pulmonary Cu and liver Zn contents were not changed in IIR and GIR. These results showed that lower or higher Zn in lung tissue and lower Cu in liver tissue were related to the acute tissue injury during IIR and GIR, suggesting that regulating the state of pulmonary Zn and liver Cu should be attempted during the prevention and treat- ment of both ischemic reperfusions.

Index Entries: Trace elements; Cu; Zn; intestinal ischemic reper- fusion; general ischemic reperfusion; shock; rabbit.

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

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42 Ming et aL

INTRODGCTION

Cu and Zn have been proven to be the essential nutrients for a variety of animal species (1). They are known to be the essential cofactors for a variety of Cu and Zn metalloenzymes and known to be an antioxi- dant (2,3). It is clear that Cu and Zn are changed in chronic diseases, but Cu and Zn are not completely understood in acute diseases, especially in the shock during intestinal ischemic reperfusion (IIR) and general isch- emic reperfusion (GIR) (4).

The liver and lung tissue may become ischemic during general circulatory disturbances; however, the reperfusion of the blood did not reduce the aggravation of the tissue injury, such as hemorrhagic shock (GIR) and occlusion mesenteric vascular diseases (IIR) (5). Zn contents and Cu-Zn superoxide dismutase activity, which provides the first line of defense against free radicals in lung and liver, were decreased in the lung injury caused by the intestinal ischemia during the reperfusion (6). Therefore, it is important to study further the changes of Cu and Zn in different organs and in different ischemic types during ischemic reperfu- sions. The purpose of this comparative study was to evaluate the effect of pulmonary and liver Cu and Zn metabolism on acute tissue injury during IIR and GIR.

/~TERIAL AND METHODS

Animal Groups __ Healthy pure-bred New Zealand rabbits weighing 2.48 _+ 0.27 kg

(X - SD) were randomly divided into three groups (five in each) and were raised in the same conditions with adequate dietary levels of Cu and Zn.

Control Group Under local anesthesia (5 mL of 1% procaine), catheters were insert-

ed into the jugular artery to record the blood pressure. After the animals were sacrificed by exsanguination, the trachea was exposed, and the catheter was immediately inserted into the pulmonary artery to wash the blood of the pulmonary circulation with deionized saline. A piece of lung tissue was taken at the same site. After the abdomen was opened, a piece of liver was taken at the same site. The homogenate of the lung and liver was made and centrifuged; then the supernatant of lung and liver tissue was taken to be measured.

HI? Group After opening the abdomen under local anesthesia (1% procaine),

the superior mesenteric artery was clipped with hemostatic forceps to

Biological Trace Element Research Vol. 39, 1993

Cu and Zn in Rabbits 43

make it ischemic. One hour later, the hemostatic forceps were loosened to cause the reperfusion of blood. Another hour later, when the animals started to breathe faster with the lower blood pressure below 8.00 kPa (60 mmHg), samples of the lung and liver tissue were taken as the control group.

GIR Group

The jugular artery and vein were separated under local anesthesia (1% procaine), and the catheters inserted into both vessels. The artery catheter was connected with the sphygmometrograph and a bottle in order to maintain the level of Bp at 5. 32 kPa (40 mmHg). The blood flowed into the bottle through the catheter of the jugular artery to cause general ischemia. One hour later, the blood was transfused into the animal's body through the jugular vein to cause reperfusion. Another hour later, samples of the lung and liver tissue were taken as they were from the control group.

Determination of Cu and Zn

After placing in 1:1 HNO for 24 h, all instruments and catheters were washed with distilled and deionized water to avoid influencing the Cu and Zn determinations. Cu and Zn in acidolyzed samples were determined with a Hitachi 180-80 flame atomic absorption spec- trophotometer. The data were processed by a microcomputer. The detec~ tion limits of Cu and Zn were 0.002 and 0.014 ~/mL, respectively. The reclamation was 95-105% (7).

RESULTS

Changes of Cu and Zn contents in the supernatant of lung tissue during the IIR and GIR: In the control group, Zn contents in the superna- tant of lung tissue were higher about 18 times than Cu contents (Zn: 23.16 + 1.54 i~g/g wet wt; Cu: 1.32 -+ 0.20 i~g/g wet wt). The Cu/Zn ratio was 0.056.

Compared with the control group, Cu contents in the supernatant of lung tissue were not changed between IIR and GIR (p > 0.05). Zn contents in the supernatant of lung were remarkably lower in IIR (p < 0.01), and pulmonary Zn was significantly higher in GIR (p < 0.01). Cu/Zn was 0.063 and 0.038 in IIR and GIR groups, respectively (Table 1).

The results presented here show that Zn contents in lung tissue were significantly changed between IIR and GIR groups. Cu contents were not remarkably different among control, IIR, and GIR groups.

Changes of Cu and Zn contents in the supernatant of liver tissue during the IIR and GIR reperfusion: In the control group, Zn contents of liver tissue were slightly higher by about 1.6 times than Cu contents (Zn: 486.24 --_ 23. 79; Cu: 292.16 + 47.50). The Cu/Zn ratio was 0.601.

Biological Trace Element Research Vol. 39, 1993

44 /vling et aL

Table 1 The Cu and Zn Contents in the Supernatant of Lung Tissue between

IIR and GIR (X -+ SD, p,g/g wet wt)

Cu Zn Cu/Zn

Control group 1.32 _+ 0.20 23.16 ___ 1.54 0.057 IIR group 1.10 + 0.28 17.50 _ 2.23 a 0.063 GIR group 1.22 ___ 0.27 32.08 + 4.69 ~ 0.038

IIR: intestinal ischernic reperfusion, GIR: general ischemic reperfusion, aCompared with control Zn, p < 0.01.

Compared with the control group, Zn contents in liver tissue were not changed (p > 0.05); Cu contents in liver were remarkably lower in IIR and GIR groups (IIR: p < 0.001; GIR: P < 0.05). The Cu/Zn ratio was lower. It was 0.090 and 0.411 in IIR and GIR, respectively (Table 2). The results show that lower Cu in liver tissue is an important feature in IIR and GIR groups.

DISCUSSION

Copper and zinc play an important role in the maintenance of the structure and function of biomembranes to prevent the damage (8). In the normal condition, Cu and Zn are transported across the brush-border membrane surface of the small intestine bound to one or more absorbable ligands (9). Albumin is believed to be the main ligand that transports Cu and Zn to the portal circulation (10). It is metabolized in the liver in which Cu secreted from hepatocytes is principally in the form of ceruloplasmin, and transported to the lung and other organs through blood circulation (11). Both Cu and Zn are distributed throughout the various subcellular fractions. Clearly, the supernatant fraction contained the highest per- centage of both metals (12).

The results show that Cu and Zn contents in the supernatant of liver tissue were higher by about 213 and 21 times, respectively than those in lung tissue. Liver Zn contents were only higher about two times than liver Cu. It is suggested that Zn and Cu are accumulated by the liver in the normal conditions.

The valuable finding is that Zn contents in the supernatant of lung and Cu in liver were remarkably decreased, pulmonary Cu and liver Zn were not changed (Figs. 1, 2), Cu/Zn ratio increased in lung tissue, and it decreased in liver tissue at 1 h after IIR. Our experiment has proven that pulmonary Zn contents and Cu-Zn superoxide dismutase activity, which provides the first line of defense against free radicals, were lower in the acute pulmonary injury of superior mesenteric artery occlusion shock (6). This is different from the chronic Zn deficiency. Dietary Zn deficiency has been shown to cause an increasing generation of free radicals in lung microsomes, but not to change lung Zn contents (13). These results

Biological Trace Element Research VoL 39, 1993

Cu and Zn in Rabbits

Table 2 The Cu and Zn Contents in the Supematant of Liver Tissue between

IIR and GIR (X + SD, ~g/g wet wt)

45

Cu Zn Cu/Zn

Control group 292.16 + 47.50 486.24 _ 23.79 0.601 IIR group 45.46 _+ 4.82 a 504.50 +_ 42.09 0.090 GIR group 204.94 _+ 38.18 b 498.42 _ 44.54 0.411

Abbreviations of IIR and GIR are defined in Table 1. aCompared with control Cu, p < 0.001, bCompared with control Cu, p < 0.05.

Cu, Zn ( ppm )

4 0 .

30

20 �84

10

r---" N.S..---, - -T - - ! " ] ' - .,,-]~H,q

lung Cu

r"- p<0.01 "-1

r- p_T~0.01'1~/,

lung Zn

Fig. 1. Comparison of Cu and Zn contents in lung tissue among control, IIR, and GIR groups. Abbreviations of IIR and GIR are defined in Table 1. D, con- trol; E, IIR; [], GIR. N.S.: Not signifi- cant.

suggest that Zn combination with lipoprotein and membrane protein in lung and Cu accumulation in liver may be interfered with, and Zn in the lung and Cu in the liver were reduced in IIR.

During GIR, selective vasoconstriction occurs, which affects the blood vessels of abdominal organs, such as liver, in the first place. Owing to the ischemia and hypoxias, ATP is decomposed in liver, and free radical reaction may have increased, because a late reperfusion of the intravascular volume adds to the severity of GIR and causes reperfusion damage (14). The data show that lung Zn contents were remarkably increased (p < 0.01) and liver Cu contents were significantly decreased (p < 0.05); however , lung Cu and liver Zn were not changed, and the Cu/Zn ratio decreased in both tissues at I h after GIR. The higher Zn in

Biological Trace Element Research Vol. 39, 1993

46 Ming et at.

Cu, Zn ~-- N.S. ( ppm )

500-

400

300.

200-

100,

i---p<0.05

rP<0.0011

Liver Cu Liver Zn

Fig. 2. Comparison of Cu and Zn contents in liver tissue among control, IIR, and GIR groups. Abbreviations of IIR and GIR are defined in Table 1 . 5 , control ; . , IIR; [], GIR. N.S.: Not significant.

lung may be related to the transfusion of the blood through the jugular vein after GIR.

The comparative results between IIR and GIR show that Cu contents in liver were slightly higher in GIR than in IIR, although they were remarkably lower in GIR than in control group (Fig. 2). Zn contents in lung and Cu contents in liver were changed in both ischemic reperfu- sions; there were lower Zn or higher Zn in lung and lower Cu in liver during IIR and GIR. It is possible that the metabolism of lung to Zn and Cu is different from liver. Therefore, in the IIR group, the loss of mucosal integrity could cause a release of toxic substances from the intestine to the general circulation into liver and lung, and injury of liver and lung was more severe in IIR than in GIR (15). The results suggest that the state of pulmonary Zn and liver Cu should be at tended to in order to prevent and treat both ischemic reperfusions.

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Cu and Zn in Rabbits 47

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Biological Trace Element Research Vol. 39, 1993