Iron, Zinc, and Copper Concentration in Serum ... - Springer Link

Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001), pp. 1444 –1457

Iron, Zinc, and Copper Concentration in Serum, Various Organs, and Hair of Dogs with Experimentally Induced Exocrine Pancreatic Insufficiency KATERINA ADAMAMA-MORAITOU, DVM, PhD,* TIMOLEON RALLIS, DVM, PhD,* ACHILLES PAPASTERIADIS, DVM, PhD,* NIKOLAOS ROUBIES, D Chem, PhD,* and HELEN KALDRIMIDOU, DVM, PhD†

The concentration of iron, zinc, and copper in serum, pancreas, liver, duodenum, kidneys, myocardium, brain, and hair was studied in dogs with experimentally induced exocrine pancreatic insufficiency. Exocrine pancreatic insufficiency was performed surgically in eight healthy, 8-month-old, mongrel dogs (group I). An equal number of dogs, of the same breed and age, were used as controls (group II). One month postoperatively, the dogs in group I showed symptoms of exocrine pancreatic insufficiency, confirmed by the serum Trypsin-like immunoreactivity test, and on autopsy, by histological examination of the pancreas. At the end of the experiment (20-week duration) the dogs in both groups were sacrificed. The values of serum iron, percentage transferrin saturation, and iron concentration in pancreas, duodenum, and kidneys in group I dogs were significantly higher than those in control animals. The concentrations of zinc in serum, pancreas, and myocardium and of copper in serum, pancreas, duodenum, myocardium, and hair in group I dogs were significant lower than those in control animals. Histological examination of various organs of group I dogs revealed severe atrophy and fibrosis of the pancreas, fatty infiltration of the liver, destruction and reduction in height of the villi of the duodenal epithelium, and diffuse infiltration of the duodenal lamina propria with lymphocytes and plasmocytes. KEY WORDS: exocrine pancreatic insufficiency; dog; iron; zinc; copper.

There is a dearth of information on the absorption of trace elements from the gastrointestinal tract, as well as on their concentration in serum, various organs, and hair during exocrine pancreatic insufficiency (EPI) in dogs. Manuscript received June 29, 1995; accepted January 16, 1996. From the *Department of Internal Medicine and †Department of Pathology, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece. Address for reprint requests: Katerina Adamama-Moraitou, DVM, Ph.D., Department of Internal Medicine, School of Veterinary Medicine, Aristotle University of Thessaloniki, 11 St. Voutyra Street, 546 27 Thessaloniki, Greece.

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Increased iron absorption, due to the possible inhibitory effect of secretin, has been reported in dogs with experimental pancreatic duct ligation (1). Ligation of the pancreatic ducts in dogs is followed by an increase in the iron concentration in the liver (2). Adults suffering from chronic pancreatitis and children with cystic fibrosis absorb more iron from the gastrointestinal tract than healthy persons do (3– 8). On the other hand, in healthy men, iron absorption is diminished by stimulating the exocrine pancreatic function with secretin and pancreozymin (9). The serum iron concentration was decreased in healthy Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)

0163-2116/01/0700-1444$19.50/0 © 2001 Plenum Publishing Corporation

Fe, Zn, AND Cu IN DOGS WITH PANCREATIC INSUFFICIENCY

men after oral administration of pancreas ferment preparations (10). In a depancreatized cat, extensive deposition of hemosiderin in hepatocytes, spleen, and lymph nodes was observed 32 weeks postoperatively (11). Kinney et al (12) found a significant increase in iron deposition of rat and mouse hepatocytes following damage of the exocrine pancreas due to ingestion of DLethionine with their diet. An inhibitory effect of pancreatic extract (13) or lyophilized sheep pancreatic juice (14) on iron absorption was observed in the isolated rat jejunal loop. On the contrary, no significant change in iron absorption was noted in rats with pancreatic duct ligation (15) or rats fed pancreatic extract (16, 17). There are some controversial aspects regarding the possible effects of exocrine pancreatic function on zinc (Zn) absorption. No effect on Zn absorption from the small intestine was noted in dogs with common bile duct ligation. The net absorption of Zn from segments of duodenum, jejunum, and ileum was determined and led to the conclusion that pancreatic secretions do not appear to be necessary for adequate Zn absorption (18). In a group of German shepherd dogs with pancreatic acinar atrophy, serum concentrations of Zn were determined both before and after treatment, and there were no differences from the concentrations in healthy dogs (19). On the contrary, Evans et al (20) reported that a low molecular weight Zn-binding factor is present in dog pancreatic secretions as well as in rat pancreas which facilitates Zn transport into the small intestinal epithelial cells. Humans with EPI demonstrated significantly depressed absorption of zinc sulfate from the small intestine in comparison with healthy controls (21–23). However, the mean absorption of Zn was still significantly lower than in healthy controls when pancreatic extract was given to patients with EPI (23). In humans with exclusion of biliopancreatic secretions from the functioning gastrointestinal tract to treat morbid obesity, the hair and plasma Zn concentration remained unchanged (24). On the contrary, 8 – 44 months postoperatively the serum Zn concentration was decreased as a result of a jejunoileal bypass operation (25). In children suffering from cystic fibrosis without growth retardation, the mean plasma Zn concentration was within normal limits. With moderate to severe growth retardation (and moderate to severe chronic pulmonary infection), the serum Zn concentration was significantly low (8, 26, 27). In another Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)

study the Zn serum concentration was not changed (28). Serum and liver concentrations of Zn were studied in the rat after ligation of the pancreatic duct resulted in a moderate EPI. The serum Zn level was unaffected by duct ligation except for a slight decrease after 2 weeks. Zn concentrations in liver tissue were significantly decreased after 19 weeks (29). Evans et al (20) reported that ligature of the rat common bile duct resulted in a marked decrease in 65Zn absorption. On the contrary, the exclusion of bile and pancreatic secretions from the lumen of the rat small intestine resulted in increased Zn absorption in the duodenum (30). In a group of German shepherd dogs with pancreatic acinar atrophy, the serum concentrations of copper (Cu) examined both before and after treatment were not different from the concentrations in healthy dogs (19). In young patients with jejunoileal bypass operation performed for the purpose of treating obesity, serum Cu concentrations were decreased (25). The mean plasma Cu concentration was found to be high in patients with cystic fibrosis, but not significantly increased, while erythrocyte Cu concentrations were significantly increased compared with age-matched and adult controls (27). Ligation of the bile and pancreatic duct in the rat resulted in a significantly enhanced serum Cu concentration, suggesting that pancreatic secretions limit Cu absorption (29, 31, 32). In addition, the liver Cu concentration and serum Cu/Zn ratio were markedly increased in duct-ligated rats (29). The above data and the absence of references on the consequences in dogs with EPI as regards their Fe, Zn, and Cu status motivated this experimental work. MATERIALS AND METHODS Animals and Management Sixteen young mongrel dogs, 8 months old, were divided into the following groups. Group I. EPI was experimentally induced in these eight dogs (Figure 1). Four of them were male and the rest female. Group II. Eight dogs were used as controls (Figure 2). The dogs in both groups came from four litters. Before the beginning of the experiment they remained in cages at the Department of Internal Medicine for 7 months. The animals were vaccinated, and every 3 months they were given parasiticides. At the beginning of the experiment the body weight (BW) of the dogs was 14.5 ⫾ 3.97 kg (mean ⫾ SD) for group I and 15.6 ⫾ 5.46 kg for group II.

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ADAMAMA-MORAITOU ET AL

Experimentally Induced EPI The dogs in group I were given general anesthesia, and pancreatic duct ligation and transection were performed via ventral midline laparotomy. Additionally, the pancreatic branch of the pancreaticoduodenal artery was ligated and transected and the pancreas was bluntly dissected from the duodenum. The integrity of the common bile duct was maintained. The omentum was wrapped around the duodenum. Biochemical Determinations Fig 1. A dog in group I with EPI.

Diet All animals were maintained on a diet consisting of industrial canned food (Waltham Co.) and dry food (Friskies Co.). The quantity of food administered daily was determined from their BW and activity (33). The canned diet consisted of 20% proteins, 7% lipids, 1.5% plant fibers, 6% ash, 9% hydration, 1000 IU/g vitamin A, 40 IU/g vitamin D, and 10 IU/g vitamin E. The composition of the dry diet was 20.5% proteins, 9.9% lipids, 5.8% ash, 2.8% fiber substances, 9.5% hydration, 500 IU/g vitamin A, 50 IU/g vitamin D, and 5 IU/g vitamin E. The iron, zinc, and copper concentration in canned ៮ ⫾ SD), 78.66 ⫾ 10.20, food was 140.08 ⫾ 38.01 (X and 13.22 ⫾ 3.72 ␮g/g dry matter (DM), respectively, and that in dry food was 92.41 ⫾ 9.52, 74.38 ⫾ 4.12, and 7.54 ⫾ 0.68 ␮g/g DM, respectively (determination from 240 samples).

Fig 2. A group II (control) dog.

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The concentration of serum iron (SI), total iron binding capacity (TIBC), and percentage transferrin saturation (SAT) were determined in both groups every week from the beginning of the fourth week postoperation until the end of the experiment and six times before the beginning of the experiment, by means of a Perkin Elmer Type 430 atomic absorption spectrophotometer. The determination methods were those indicated by the manufacturing company and Olsen and Hamlin (34). The concentrations of Zn and Cu in serum were determined at the same time and by means of the same spectrophotometer by the methods indicated by the manufacturing company. Additionally, serum lipase, amylase, alanine aminotransferase, aspargine aminotransferase, and alkaline phosphatase activities and the concentration of bilirubin, glucose, and total proteins were determined by means of kits from Boehringer Mannheim GmbH Co. in a Guilford Type 240 spectrophotometer for serum lipase and amylase and in a Stassar II for the rest. Urine samples were collected from both groups by cystocentesis; the specific gravity was measured by means of an American Optical Refractometer, and the pH, proteins, ketones, bilirubin, blood, and hemoglobin by means of Boehringer Mannheim’s Combur test. At the end of the experiment (end of the 20th week) the dogs in both groups were euthanized and samples of pancreas, duodenum, liver, kidneys (medulla and cortex), myocardium, brain, and hair were collected for Fe, Zn, and Cu determination, using the method of wet digestion. For this purpose, 700 –900 mg (except for the samples of pancreas) of DM was transferred into a 100-ml Kjeldahl flask. Five milliliters of a digestion mixture of concentrated nitric, sulfuric, and perchloric acid was placed in an ion shelf. After boiling for 3 hr, approximately 1 ml, consisting only of inorganic material, remained, and this was diluted with H2O up to 20 – 40 ml. The Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)


Fig 3. SI concentrations in dogs in both groups. 0, before the experiment; 1, beginning of the fourth week.

solution was analyzed with a Perkin Elmer Type 430 atomic absorption spectrophotometer. The standard reference curve was continuously monitored by means of an external standard solution. Histological Examination The euthanized animals were immediately subjected to necroscopy, and samples of pancreas, liver, and duodenum were fixed in 10% formalin in buffered saline. Paraffin sections were stained with hematoxylin– eosin, Gomori, and periodic acid–Schiff (PAS). Methods of Statistical Analysis For estimation of the time trend of the values for each parameter and examination of its significance, the function Y ⫽ a ⫹ bt (or, in logs, logY ⫽ loga ⫹ logbt) was used, where Y and t are the variables of the specific parameter and the time trend, respectively, and a and b are the Y-intercept and the time trend coefficients to be estimated, respectively. The method of estimation used was ordinary least squares (OLS). For comparison of the mean values of the parameters, we used t tests (a) between the groups at the beginning of the experiment, (b) between the groups TABLE 1. SIGNIFICANCE SI(I) – SI(II)* SI(I) – SI(II)‡ SI(I) – SI(I)¶ SI(I) – SI(II)§ SI(II) – SI(II)**

NS† NS P ⱕ 0.05 P ⱕ 0.05 NS

OF

SI, TIBC,

at the beginning of the 4th week (beginning of our observations), (c) between the groups at the end of the experiment (beginning of the 20th week), and (d) between the first (4th week) and the last (20th week) values within the same group. For comparison of the mean concentrations of Fe, Zn, and Cu in the pancreas, duodenum, liver, kidneys, myocardium, brain, and hair between the two groups, the t test was used. RESULTS Biochemical and Clinical Findings The results of all presurgical laboratory evaluations for the dogs in both groups were within normal limits. The results of laboratory evaluations for the dogs in group II (controls) were within normal limits during the entire experiment. Postoperatively, all dogs in group I developed clinical signs of acute pancreatitis and were treated with a balanced electrolyte solution administered iv for 4 days. Approximately 4 weeks after surgery the dogs in group I developed a ravenous appetite, pica, coprophagy, diarrhea, gross and microscopic evidence of steatorrhea, gradual weight loss, growth retardation, and seborrhea sicca.

AND SAT BETWEEN THE TWO THE END OF THE EXPERIMENT

TIBC(I) – TIBC(II)* TIBC(I) – TIBC(II)‡ TIBC(I) – TIBC(I)¶ TIBC(I) – TIBC(II)§ TIBC(II) – TIBC(II)**

GROUPS,

NS P ⱕ 0.05 P ⱕ 0.05 NS NS

AT THE

BEGINNING

SAT(I) – SAT(II)* SAT(I) – SAT(II)‡ SAT(I) – SAT(I)¶ SAT(I) – SAT(II)§ SAT(II) – SAT(II)**

AND

NS P ⱕ 0.05 NS P ⱕ 0.05 NS

*Before the beginning of the experiment. †Nonsignificant. ‡Beginning of the fourth week. ¶Beginning of the fourth week– end of the experiment. §End of the experiment. **Beginning of the fourth week– end of the experiment. Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)

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Fig 4. TIBC concentrations in dogs in both groups. 0, before the experiment; 1, beginning of the fourth week.

Serum Iron (SI) The SI values of animals in both groups and the differences between the two groups are shown in Figure 3 and Table 1. The dogs in group I showed a significant increase in SI, while the dogs in group II did not show any significant differences (Figure 3). The time trend of the increment was determined to be about 2.004% per week. Total Iron Binding Capacity (TIBC) The TIBC values of animals in both groups and the differences between the two groups are shown in Figure 4 and Table 1. The dogs in both groups showed a significant development of TIBC related with time. However the “behavior” of variable TIBC was different between the two groups. Percentage Transferrin Saturation (SAT) The results of laboratory evaluation of SAT are shown in Figure 5 and Table 1. The dogs in group I showed a significant increase in SAT, while the dogs

in group II did not show any significant differences (Figure 5). The time trend of the increment was determined to be about 1.075% per week. Serum Zinc The results of laboratory evaluation of Zn are shown in Figure 6 and Table 2. The dogs in group I showed a significant decrease in Zn concentration, while the dogs in group II did not show any significant differences (Figure 6). Serum Copper The results of laboratory evaluation of Cu are shown in Figure 7 and Table 2. The dogs in group I showed a significant decrease in Cu concentration, while the dogs in group II did not show any significant differences (Figure 7). The time trend of the diminition was determined to be about 1.501% per week.

Fig 5. SAT values in dogs in both groups. 0, before the experiment; 1, beginning of the fourth week.

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Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)


Fig 6. Zn concentrations in dogs in both groups. 0, before the experiment; 1, beginning of the fourth week.

Concentrations of Fe, Zn, and Cu in Various Organs and Hair The concentration of Fe, Zn, and Cu and its significant differences in pancreas, duodenum, liver, kidneys, myocardium, brain, and hair between the two groups are shown in Tables 3, 4, and 5, respectively. The dogs in group I showed an increased concentration of Fe in pancreas, duodenum, and kidneys and a decreased concentration of Zn in pancreas and myocardium and of Cu in pancreas, duodenum, myocardium, and hair. Trypsin-Like Immunoreactivity (TLI) Test

Postmortem Findings The pancreas of the dogs in group I was firmer than normal and extremely small in size (Figure 8), the liver was hyperemic, and the small intestine was dilated, with pulpy contents, mucosal erosions, and ulcers (Figure 9). Pathological examination of the dogs in group II revealed no pathological lesions. TABLE 2. SIGNIFICANCE OF Zn AND Cu BETWEEN THE TWO GROUPS, AT THE BEGINNING AND THE END OF THE EXPERIMENT NS† NS P ⱕ 0.05 P ⱕ 0.05 NS

Cu(I) – Cu(II)* Cu(I) – Cu(II)‡ Cu(I) – Cu(I)¶ Cu(I) – Cu(II)§ Cu(II) – Cu(II)**

NS P ⱕ 0.05 P ⱕ 0.05 P ⱕ 0.05 NS

*Before the beginning of the experiment. †Nonsignificant. ‡Beginning of the fourth week. ¶Beginning of the fourth week– end of the experiment. §End of the experiment. **Beginning of the fourth week– end of the experiment. Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)

The histopathological lesions of the dogs in group I were atrophy and fibrosis of the pancreas (Figures 10 and 11), foci of fatty infiltration in the liver (Figure 12), destruction of the epithelium of the villi, with a reduction in their height, and infiltration of the duodenum lamina propria with lymphocytes and plasmocytes (Figure 13). The histopathological examination of the pancreas, liver and duodenum of the dogs of group II revealed no pathological lesions. DISCUSSION

Results of the TLI test for the dogs in group I showed values lower than 3 ␮g/L of serum. Normal values range between 5 and 35 ␮g/L of serum.

Zn(I) – Zn(II)* Zn(I) – Zn(II)‡ Zn(I) – Zn(I)¶ Zn(I) – Zn(II)§ Zn(II) – Zn(II)**

Histopathological Findings

Various experimental methods to induce exocrine pancreatic acinar atrophy and symptoms of EPI in dogs have been described. Some of them are pancreatic duct ligation (2, 35, 36), pancreatic duct ligation with placement of the omentum between the pancreas and the duodenum (37, 38), ligation of the pancreatic duct and dissection of all attachments between the duodenum and the pancreas (39), partial pancreatectomy (36), and experimentally induced acute pancreatitis by injection of various chemicals (40 – 44) or biological products (45, 46) into the pancreatic mass or duct. According to the results of our preliminary experiments for production of EPI in experimental animals in group I, we chose ligation and transection of the pancreatic ducts and the pancreatic branch of the pancreaticoduodenal artery and dissection of the pancreas from the duodenum. After surgery, the dogs in group I developed signs of acute pancreatitis. This condition was confirmed by dramatically increased serum lipase and amylase activities (up to 10,150 and 21,160 U/L, respectively). Although clinical signs of acute pancreatitis disappeared 3.5 days postoperatively, serum lipase and

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Fig 7. Cu concentrations in dogs in both groups. 0, before the experiment; 1, beginning of the fourth week.

gested that secretin has a possible inhibitory effect on iron absorption. The absorption of iron salts by children suffering from cystic fibrosis, a disease accompanied by EPI (27), is far above the normal average (4, 5). The investigators proposed that lack of pancreatic secretion results in a significant increase in iron salt intake. The same behavior was shown by adult patients with chronic pancreatitis (3, 6). Especially, Bernstein and Herbert (6) failed to prove that pancreatic enzymatic action or bicarbonate can decrease the transport of iron from the upper intestinal lumen. Davis and Badenoch (3) suggested that an unknown pancreatic factor might be responsible for decreasing the absorption of iron from the intestine. Friedel et al (9, 10) proposed that pancreatic secretions can diminish iron absorption in humans. In the isolated jejunal loop of rats the uptake of iron can be inhibited by the addition of pancreatic extract (13) or pure sheep pancreatic juice (14). These investigators supported the hypothesis that a pancreatic factor may play an important role in the control of iron (Fe) absorption. There was a significant increase in the deposition of Fe in the liver of rats and mice following damage to the exocrine cells of the pancreas due to DL-ethionine added to the diet (12). They concluded that animals with pancreatic damage absorbed more Fe from the gastrointestinal

amylase activities, which remained high for 15–20 days, suggested relapsing episodes of acute pancreatitis. Approximately 1 month following surgery the dogs in group I developed signs of EPI, such as a ravenous appetite, pica, coprophagy, diarrhea, gross and microscopic evidence of steatorrhea, gradual weight loss, growth retardation, and seborrhea sicca. For confirmation of EPI in group I dogs the TLI test was performed. The serum TLI assay is a highly sensitive and specific test for the identification of EPI in dogs (47– 49). For this purpose blood serum from fasted animals was collected at the end of the 17th week. Concentrations of serum TLI measured by radioimmunoassay were lower than 3 ␮g/L in all animals. Normal TLI range values are 5–35 ␮g/L (50 –55). The dogs in group I showed a significant increase in SI and SAT values, while those in group II did not show any significant differences (Figures 3 and 5 and Table 1). Iron absorption in dogs without pancreatic secretion was studied by measurement of the percentage of radioactivity retained in red cells in two groups of dogs given 59Fe in the stomach (1). One group was used as controls and the other was subjected to pancreatic duct ligation. Increased absorption was observed in the latter group and the investigators sugTABLE 3. MEAN ⫾ SD Group I

II

AND

SIGNIFICANCE

OF

Fe

IN

VARIOUS ORGANS

Kidney

AND

Pancreas

Duodenum

Liver

331.9 ⫾ 27.3

81.3 ⫾ 20

262.9 ⫾ 21.4

P ⱕ 0.05

P ⱕ 0.05

NS

P ⱕ 0.05

NS

69.8 ⫾ 9.2

58.1 ⫾ 4.6

237.9 ⫾ 25.8

129.4 ⫾ 16.7

162.1 ⫾ 22.7

181.3 ⫾ 26

Myocardium 153.8 ⫾ 26

HAIR* Brain

Hair

55.1 ⫾ 6.4

16.3 ⫾ 8.9

NS

NS

52.8 ⫾ 5

11.6 ⫾ 3.5

*Values are expressed as ␮g/g of dry matter. NS, nonsignificant.

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Fe, Zn, AND Cu IN DOGS WITH PANCREATIC INSUFFICIENCY TABLE 4. MEAN ⫾ SD

AND

SIGNIFICANCE

OF

Zn

IN

VARIOUS ORGANS

Group

Pancreas

Duodenum

Liver

I

87.7 ⫾ 7.1

101.6 ⫾ 12.3

120.8 ⫾ 27.8

P ⱕ 0.05

NS

NS

NS

114.7 ⫾ 18.2

77.7 ⫾ 10.1

104.9 ⫾ 3.7

II

99 ⫾ 7.5

Kidney 85 ⫾ 11.7

AND

HAIR*

Myocardium

Brain

Hair

72.3 ⫾ 5

52.2 ⫾ 2.3

136.3 ⫾ 20.7

P ⱕ 0.05

NS

NS

78.1 ⫾ 4.6

50.8 ⫾ 3.4

146.6 ⫾ 28.3

*Values are expressed as ␮g/g of dry matter. NS, nonsignificant.

tract. On the contrary, neither pancreatic duct ligation (15) nor administration of pancreatic extract (16, 17) had an effect on iron absorption or deposition in rats. In the case of duodenal contents of low pH, inorganic Fe is able to form soluble complexes that are easily absorbed (56 –58). The fall in pH of duodenal contents in patients or animals with EPI due to the absence of bicarbonates can enhance the absorption of iron (3, 11). On the other hand, in gallbladder dysfunction and in duodenal ulceration, where the pH of the duodenal juice is lower than normal, no increased deposition of Fe in the tissues has been reported (3). There is no information on the concentration of SI and the SAT values in dogs with naturally occurring EPI. In a 14-month-old male Doberman pinscher dog with EPI, admitted to our clinic, the values of SI and SAT were 230 ␮g/100 ml and 48%, respectively, similar to those in our dogs with experimentally induced EPI. The increased iron concentration in the pancreas, duodenum, and kidneys in the group I dogs (Table 3) is perhaps due to the increase in SI and SAT values. Kinney et al (2) found an increased Fe concentration in the liver of dogs with ligation of pancreatic ducts, which were fed raw pancreas daily in addition to Purina dog chow. They concluded that ligation of the pancreatic ducts in dogs is followed by increased absorption of Fe from the gastrointestinal tract, with a marked increase in liver Fe values. Additionally, extensive deposits of Fe were noted in the liver, spleen, and lymph nodes of a depancreatized cat, 32 weeks after the operation (11). TABLE 5. MEAN ⫾ SD Group I

II

AND

The increased SI and SAT values in group I dogs may be due to a lack of pancreatic juice from the duodenum, the fall in the pH of duodenal contents, or the lack of a pancreatic factor that inhibits Fe absorption (11, 56, 57). The increased concentration of Fe in the pancreas, duodenum, and kidneys in dogs with EPI may be due to the increased deposition of Fe, when there is an excess of it in the blood (56, 57, 59). Significant development of the TIBC related with time was observed in the dogs in both groups (Figure 4 and Table 1). The time trend development of the TIBC in blood of dogs could be due to the different needs of animals in Fe or to the rate of transferrin production in the liver. The differences in TIBC values of animals in both groups could be related to the rate of Fe absorption and to the different concentrations of SI. Generally, the behavior of the TIBC cannot be determined exactly in cases of Fe deficiency or overload. Investigations in various species and in humans showed increased, diminished, or normal values of TIBC in cases of Fe deficiency or overload (59, 60 – 63). A significant decrease in the serum Zn concentration was observed among the dogs in group I, while the dogs in group II did not show any significant differences (Figure 6 and Table 2). A low molecular weight zinc-binding factor is present in dog pancreatic secretions which facilitates zinc transport into the epithelial cells (20). On the contrary, ligation of the common bile duct in dogs did not make any significant change in zinc absorption, suggesting that pancreatic secretions do not appear to be necessary for adequate zinc absorption in the canine duodenum

SIGNIFICANCE

OF

Cu

IN

VARIOUS ORGANS

AND

HAIR*

Pancreas

Duodenum

Liver

Kidney

Myocardium

Brain

Hair

2 ⫾ 0.3

4.3 ⫾ 0.7

150.1 ⫾ 6.9

12.5 ⫾ 3.7

12 ⫾ 1.3

13.8 ⫾ 1.02

7.3 ⫾ 0.9

P ⱕ 0.05

P ⱕ 0.05

NS

NS

P ⱕ 0.05

NS

P ⱕ 0.05

3.8 ⫾ 0.4

5.8 ⫾ 0.3

143.3 ⫾ 9.3

14 ⫾ 1.3

13.3 ⫾ 1.6

8.4 ⫾ 0.9

16 ⫾ 4.5

*Values are expressed as ␮g/g of dry matter. NS, nonsignificant. Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)

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Fig 8. Pancreata of a group II (M4) dog and a group I (⌸4) dog.

(18). Similarly, examination of the serum Zn concentration in a group of German shepherd dogs with pancreatic acinar atrophy, both before and after treatment, did not show any differences from the concentrations observed in healthy dogs (19). In children with cystic fibrosis the mean plasma (26 –28) and hair Zn level was significantly low (28). Patients with EPI had depressed absorption of zinc, which suggested that normal pancreatic function may play a role in zinc metabolism in humans (21, 22). When pancreatic extract was given to a group of patients with EPI, the mean absorption of zinc was within the normal range, but still significantly lower than normal (23). Complete surgical diversion of biliary and pancreatic secretions from the functioning small intestine has been utilized to treat morbid obesity (24). Hair and serum Zn levels were measured in patients having undergone complete biliopancreatic bypass 12–56 months earlier, and these values were compared with those obtained from other patients subjected to only partial biliopancreatic bypass, as

Fig 9. Duodenum of a group I dog with ulcers and hemorrhages.

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well as to those of morbidly obese controls. No differences were observed in either hair or serum Zn levels between any of these groups of patients, suggesting that pancreatic secretions do not appear to be necessary for adequate zinc absorption. On the contrary, in a similar investigation, low serum Zn levels in the majority of patients suggested that malabsorption of zinc was induced by the operation (25). Ligation of the pancreatic duct in rats leading to “moderate” EPI resulted in a moderate decrease in liver Zn concentrations, but no appreciable changes were observed in serum Zn concentration or whole blood during the experiment (29). The investigators concluded that adequate function is a prerequisite for the maintenance of normal Zn homeostasis. On the other hand, Antonson et al (30) observed increased absorption of zinc from duodenal segments of rats in which bile and pancreatic ducts were excluded, and they suggested that the presence of pancreatic secretions reduces zinc absorption. In our experiment there was a reduction of serum Zn in dogs with EPI. This could be due to the lack of a factor in pancreatic juice that enhances zinc absorption or to the hypoproteinemia that was observed in the dogs in group I from the 15th week onward. Dogs with decreased values of total serum proteins showed decreased serum Zn values (64). Rats fed a lowprotein diet had decreased concentrations of Zn in plasma, liver, and small intestine (65). This might indicate that one important consequence of protein depletion is a secondary zinc deficiency. Evans and Johnson (66), who additionally supplemented the low-protein diet with picolinic acid, a component of pancreatic juice in these species, obtained the same results. Rats fed a supplemental picolinic acid diet absorbed zinc more efficiently than rats fed only the low-protein diet, suggesting that a pancreatic factor facilitates zinc absorption. In a dog with naturally occurring EPI admitted to our clinic, the Zn concentration in serum (56 ␮g/100 ml) was low and similar to that in group I dogs. This dog did not have hypoproteinemia. The decreased Zn concentration in pancreas and myocardium (Table 4) in the group I dogs might be due to mobilization of the trace element to the serum to compensate for the primary deficiency from its reduced absorption. Montgomery et al (67) indicated that canine pancreas can deposit a large quantity of radioactive zinc administered intravenously. A significant decrease in copper (Cu) concentration was observed in the dogs in group I, while the dogs in group II did not show any significant differDigestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)


Fig 10. Pancreas of a group I dog with EPI; light microscopy. Exocrine cells are degranulated, acinar structure is lost, and there is development of connective tissue. H–E; original magnification, ⫻140.

ences (Figure 7 and Table 2). Dogs with pancreatic acinar atrophy showed no alterations in serum Cu concentrations either before or after treatment and

the values were not different from those demonstrated by healthy dogs (19). In children who suffered from cystic fibrosis, the

Fig 11. Pancreas of a group I dog with EPI; light microscopy. There is acinar atrophy with the presence of connective tissue. PAS; original magnification, ⫻350. Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)

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Fig 12. Liver of a group I dog with EPI; light microscopy. Micronodular lipidosis. H–E; original magnification, ⫻200.

plasma Cu was high, but not significantly increased, while the erythrocyte Cu concentration was also high (27). This finding could have resulted from the increased activity of a copper-containing enzyme, superoxide dismutase, in erythrocytes of patients with cystic fibrosis. In patients with chronic pancreatitis an increased intestinal absorption rate of copper was observed, suggesting that an unknown factor in pancreatic juice limits its absorption (68). Bell et al (69) came to the same conclusion after they had observed, in patients with chronic pancreatitis, increased concentrations of Cu and ceruloplasmin activity in the serum. The same authors demonstrated that this state is reversible when they studied patients before and 6 months after pancreatic supplements were administered. On the contrary, depressed serum Cu concentrations in patients with jejunoileal bypass for morbid obesity were found 8 – 44 months after the operation, suggesting that this finding could be due to impaired intestinal absorption (25). Serum Cu concentrations were markedly enhanced in rats with “moderate” EPI, while Cu concentrations in liver tissue were moderate decreased (29). Increased copper absorption was observed in rats in which the common bile–pancreatic duct was ligated (31, 32). On the contrary, in our experiment we found that dogs with EPI demonstrated decreased serum Cu

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concentrations. This may be due to either lack of pancreatic secretions from the duodenum, to hypoproteinemia, in which EPI results, or to their combination. Dogs with low total serum protein, which was generally associated with malabsorption or protein-losing enteropathies, had low serum Cu values (64). The dog with naturally occurring EPI admitted to our clinic did not show any reduction in serum Cu concentration. The reduction in Cu concentration in pancreas, duodenum, myocardium, and hair (Table 5) of dogs with EPI might be a consequence of the reduced serum Cu concentrations, as a result of mobilization of the Cu deposits to the serum. At the postmortem examination the pancreas of dogs with EPI was firmer than normal and extremely small in size, and its weight was one-tenth that of the controls. On histopathological examination, loss of structure, with fibrosis and acinar atrophy, was found. These findings are compatible with those of other authors (35, 36, 70 –72), confirming that the dogs were suffering from EPI. Some investigators suggest that EPI can be accompanied by diabetes mellitus (19, 50, 55, 73). During the experiment there was no clinical or biochemical evidence of diabetes mellitus in our dogs with EPI. This could be due to the existence of some islets of Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)


that it was dilated with pulpy contents, mucosal erosions, and ulcers. Histopathological examination revealed destruction of the epithelium of its villi, reduction of their height, and infiltration of the duodenum lamina propria with lymphocytes and plasmocytes. These changes are caused by bacterial overgrowth due to the lack of pancreatic enzymes and bicarbonates from the duodenum (47–50, 52, 54, 55, 79, 80) or by loss of the trophic influence of pancreatic secretions on the small intestine (49, 52). REFERENCES

Fig 13. Duodenum of a group I dog with EPI; light microscopy. Destruction of the villous epithelium and diffuse infiltration of the lamina propria with lymphocytes and plasma cells. H–E; original magnification, ⫻70.

normal architectural structure found on histopathological examination. As mentioned earlier, the liver of the dogs in group I was hyperemic, with foci of fatty infiltration. This finding was reported in dogs with EPI only by Montgomery et al (74) and in humans by Woldman et al (75). Alterations in the functional activity of the liver was suspected in this experiment, as the activities of serum alanine aminotransferase and alkaline phosphatase were elevated. This finding is possibly a consequence of EPI, because of the inability of animals suffering from this condition to absorb proteins normally. Especially in humans, protein deficiency can cause fatty infiltration of liver cells (76). Fatty infiltration of the liver is reported to be a consequence of diabetes mellitus (76 –78). As our dogs with EPI did not suffer from this condition, fatty infiltration of the liver might be due to the lack of protein caused by EPI. Gross examination of the small intestine showed Digestive Diseases and Sciences, Vol. 46, No. 7 (July 2001)

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