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A comparison of four regimens for treatment of iron storage disease using the European starling (Sturnus vulgaris) as a model.

Abstract: European starlings (Sturnus vulgaris) were fed an iron loading diet (3235 ppm) for 31 days to induce nonheme liver iron concentrations approaching those in birds that died with iron storage disease. All birds then were fed a low-iron diet (32-48 ppm) and assigned to 4 treatment groups: 1) low-iron diet only, 2) low-iron diet with phytate (inositol) and tannic acid, 3) low-iron diet and deferoxamine (100 mg/kg SC q24h), and 4) low-iron diet and phlebotomy (1% of body weight q7d). Starlings were treated for 16 weeks. In the groups treated with phlebotomy or with deferoxamine and a low-iron diet, nonheme liver iron concentrations decreased to safe levels after 16 weeks of treatment at similar rates (190 ppm/wk and 163 ppm/wk, respectively). The low-iron diet alone reduced stored liver iron levels at a slower rate (45 ppm/wk). The addition of inositol and tannic acid to the low-iron diet had no impact on stored liver iron concentrations. These results suggest that both phlebotomy and treatment with deferoxamine are effective treatment options for birds with iron storage disease.

Key words: iron storage disease, deferoxamine, phlebotomy, inositol, tannic acid, diet, avian, birds, European starling, Sturnus vulgaris

Introduction

Iron storage disease (ISD) is a life-threatening disease of captivity that occurs in several species of toucans (Rhamphastidae), mynahs (Sturnidae), birds-of-paradise (Paradisaeidae), curassow (Cracidae), and quetzals (Pharomachrus species), (l-5) Many of the species susceptible to ISD are kept as pets and some are highly endangered in the wild. (1-3) Iron storage disease is characterized by a massive accumulation of iron in the liver, primarily as hemosiderin in hepatocytes. (6) Clinical signs associated with ISD are ascites, hepatomegaly, cardiomegaly, dyspnea, abdominal distension, depression, and, if untreated, death. (7-9) Currently the only way to diagnose and monitor treatment for ISD is by liver biopsy. The pathogenesis of ISD is poorly understood but ultimately appears to result in heart failure and, in some cases, liver disease.

In people, ISD occurs in 2 forms that are collectively referred to as hemochromatosis. The primary and most common form of hemochromatosis is a recessive genetic disorder that results in excessive liver iron regardless of iron intake. (10) The second form of hemochromatosis develops in situations of excess iron intake such as repeated blood transfusions or high dietary iron. Studies in mynahs suggest that ISD in susceptible species of birds resembles the primary form of hemochromatosis in people in that iron absorption is not down regulated when the bird is iron replete. (11)

The most common treatment for hemochromatosis in people is whole blood phlebotomy. (12) Phlebotomy is performed weekly or twice weekly, and a blood volume equal to approximately 0.7% of the body weight is removed with each bleeding. (10) If anemia develops, then parental chelation therapy with the drug deferoxamine is substituted for phlebotomy. (10,13)

Deferoxamine is a chelator that forms stable complexes with iron, preventing the iron from being involved with further reactions. It binds preferentially with ferritin and hemosiderin forms of iron but not transferrin, cytochromes, or hemoglobin. The chelated iron is metabolized by plasma membranes and is excreted in urine and feces.

Traditionally, phlebotomy, deferoxamine therapy, and low-iron diets have been used individually or in combination as treatments for birds with ISD. (14-18) These case reports, describing ISD treatment in a small number of birds, suggest that phlebotomy, deferoxamine treatment, or a combination of the two should effectively lower stored liver iron concentrations in birds with ISD. However, because these reports span multiple species and describe a range of treatments that varied in duration, it remains unclear as to which treatment is most effective and how long a treatment is necessary to decrease liver iron levels to a normal concentration. Recently, results of one study showed that the oral chelator deferiprone may also be effective at reducing liver iron stores in birds. (19)

Dietary modification is an important element in treating ISD. If the bird has been fed a high-iron diet, changing it to a diet with the minimum requirement of dietary iron (approximately 50-100 ppm) (20) may be beneficial by preventing the additional iron from accumulating. Experimentally, in starlings fed a low-iron diet (32 ppm), nonheme liver iron concentrations decreased at a rate of 60 ppm/ wk. (20) This rate of iron loss is assumed to be low, and although a low-iron diet may be of benefit when used in combination with other treatments, dietary modification alone is not expected to be effective therapy. Previous experimental work has shown that adding a phytate (inositol) and tannic acid to a high-iron diet prevents excess iron uptake in European starlings (Sturnus vulgaris). (20) These results suggest that these additives may also reduce iron absorption further if added to low-iron diets and, therefore, their incorporation into a diet may be a useful part of treatment for ISD.

This study had 2 objectives: 1) to induce liver iron concentrations in European starlings approaching those seen in other species with naturally occurring ISD by feeding a high-iron diet, and 2) to compare the efficacy of dietary modification alone or in combination with either phlebotomy or chelation therapy on the rate of decrease of liver iron concentration in these starlings.

Materials and Methods

Starlings (N = 64) were trapped on the Texas A&M University campus (College Station, TX, USA) in an open-air building housing swine in December 2003. Before experimental manipulation, 7 starlings were euthanatized and evaluated to determine stored liver iron concentrations. The Texas A&M University Laboratory Animal Care Committee approved all animal care and handling.

All diets in this study were formulated to contain 15% protein, 3.8% fat, and 53.5% carbohydrates (Harlan Teklab, Madison, WI, USA). (20) Water was provided ad libitum. Starlings were initially fed a loading iron diet formulated to contain 3000 ppm of iron for 31 days. At the end of this period, starlings were then randomized into 4 treatment groups of 12 birds each, and the remaining 13 birds were euthanatized and evaluated to determine pretreatment stored liver iron concentrations. For the therapeutic trial, all 4 treatment groups were fed a low-iron diet formulated to contain 23 ppm of iron. Treatment group 1 was treated with the low-iron diet only. In treatment group 2, inositol (20 gm/kg wet weight) and tannic acid (20 gm/kg wet weight) were added to the low-iron diet. In treatment group 3, birds were treated with deferoxamine mesylate (100 mg/kg SC q24h; Desferal, Novartis Pharmaceuticals Corp, East Hanover, N J, USA) in addition to the low-iron diet. In these birds, the skin region covering the pectoral muscles was divided into 4 quadrants, and the injection sites were rotated so that an injection was given in each quadrant once every 4 days. In treatment group 4, birds were treated with weekly phlebotomy and low-iron diet. A blood volume of approximately 1% of the body weight (0.7 ml) was collected from the right jugular vein of each starling on the first day and weekly thereafter for the remainder of the trial. At 6, 11, and 16 weeks after treatment began, 4 starlings from each of the treatment groups were euthanatized for evaluation. Blood samples were collected from these birds immediately before euthanasia. Packed cell volumes (PCV) were determined from blood samples collected from the birds treated by phlebotomy (group 4) and from all samples collected before euthanasia.

Dietary iron concentration was verified by the Trace Element Research Laboratory (College of Veterinary Medicine, Texas A&M University) by digesting samples in ultrapure nitric acid, 30% hydrogen peroxide, and hydrochloric acid. Each digestion was then analyzed by a SpectroCiros inductively coupled plasma-optical emissions spectroscopy (SpectroCiro, ICE Spect Kleve, Germany) for total iron content.

All starlings were euthanatized by first administering a 50/50 vol/vol dose of ketamine (100 mg/ ml) and xylazine (20 mg/ml) (0.15 ml IM). Next, when unresponsive to a toe pinch and single-feather extraction, starlings were exsanguinated. Euthanasia of the starlings was necessary in this study because the quantity of liver needed for analysis could not be collected by serial biopsy. The right liver lobe of each bird was frozen (-20[degrees]C) for nonheme iron determination.

Concentrations of nonheme iron in the liver were determined by a modification of the Torrance-Bothwell nonheme method. (20) Liver samples were acid digested and incubated at 65[degrees]C for 20 hours. After the digestions were allowed to cool, 0.1 ml and 0.05 ml of the extract were evaluated by colorimetric technique (Biomate 3 Spectrophotometer, ThermoSpectronic, Rochester, NY, USA) for optical density. In some cases when liver iron concentrations were high, an additional dilution of 0.010 ml was tested. The iron concentration of each solution was determined by an equation based on a standard curve and the optical density of each acid digestion. The original liver iron concentration (ppm wet weight) of each liver sample was determined by the following equation:

Liver iron concentration = (TC) x (DF)

x [5ml + sample weight [g]/sample weight [g]],

where TC = nonheme iron concentration of sample digestion determined from standard curve and DF = dilution factor (volume of acid digest/1 ml [volume of 5 ppm iron control]).

Stored liver iron concentrations of controls and each treatment group were compared by the Wilcoxon rank sum test, with significance set at P < .05. (21) Packed cell volumes among groups were similarly compared. Nonheme liver iron concentrations were unexpectedly high or low in 3 starlings. Dixon's test was used to determine if these values were outliers. Values determined to be outliers were excluded from the data analysis (Q > 0.33). (22)

Results

The starlings adapted readily to captivity. Decreased food consumption was observed when the starlings were changed to experimental diets, but birds were eating normally within 1 week. Each bird consumed an estimated 30 g of pelleted diet per day during the study period. The actual dietary iron concentration was determined to be 3235 ppm for the loading iron diet, 32 ppm for the low-iron diet, and 49 ppm for the low-iron diet containing inositol and tannic acid.

The mean concentration of nonheme liver iron in the starlings (n = 7) collected before iron loading was 1389 ppm. Starlings euthanatized after 31 days of iron loading had mean liver iron concentrations of 3132 ppm. This corresponded to an average increase of 393 ppm/wk (Table 1).

Nonheme liver iron concentrations for all treatment groups at all collection times are shown (Table 1). After 16 weeks, starlings that were fed the low-iron diet and treated with phlebotomy (group 4) had the lowest mean nonheme liver iron concentration (mean 163 ppm). The liver iron concentrations in this group were significantly different (P < .05) from those in the control group. The population of starlings fed the low-iron diet and treated with deferoxamine (group 3) also had significantly lower nonheme liver iron concentrations than did the control group. After the 16-week treatment, the decrease in the mean concentration of nonheme liver iron was small (827 ppm) for birds fed only the low-iron diet (group 1), but the difference in the populations was significant when compared with control starlings. No significant differences were found in liver nonheme iron concentrations between the starlings fed the low-iron diet containing inositol and tannic acid (group 2) and the control starlings.

Nonheme liver iron concentrations declined linearly over time for the phlebotomy ([R.sup.2] = 0.97), deferoxamine ([R.sup.2] = 0.98), and low-iron diet ([R.sup.2] = 0.82) treatment groups. The rate of decrease was greatest for the starlings treated with the low-iron diet and phlebotomy (190 ppm/wk), followed by the starlings treated with deferoxamine (163 ppm/wk), and finally the starlings treated with the low-iron diet only (45 ppm/wk). The trend line for the phytate and tannin group had a near-zero slope (Fig 1).

[FIGURE 1 OMITTED]

During the first week of treatment, the starlings treated with phlebotomy (group 4) had a mean PCV of 50% (range, 43%-52.5%). After 16 weeks of treatment, the mean PCV in this group was 46% (range, 45%-47%), a drop of 4% (range, 0%7.5%). One starling in the phlebotomy treatment group had an initial PCV of 43%, which was the lowest before treatment and which decreased to 31% after 6 weeks. On physical examination, this starling was found to be heavily infested with mites (Ornithonyssus species). It was euthanatized, and the data were removed from the trial because of confounding associated with blood loss caused by the mites. This bird had the lowest liver iron of all starlings (85 ppm). The mean PCV for the other 3 treatment groups (groups 1, 2, and 3) after 16 weeks of treatment was 49.9% (range, 43%-58%). After 16 weeks of treatment, there was no detectable difference in PCV values between treatment groups and the control group.

Three starlings, 2 treated with the low-iron diet (group 1) and 1 treated with low-iron diet and phlebotomy (group 4), had liver iron values that were unexpectedly high or low compared with other starlings within their treatment group. Dixon's test showed the values from these starlings were outliers, and they were removed from all statistical comparisons (Q = 0.63, 0.51, and 0.86). (22)

The mean body weight ([+ or -] standard deviation) for all starlings was 76 [+ or -] 5.6 g (range, 62-88 g). Weights did not differ significantly among treatment groups when compared by the Wilcoxan rank sum test. At necropsy, no irritation or induced inflammation from the repeated subcutaneous injections of deferoxamine was evident.

Discussion

The first objective of this study was to induce concentrations of nonheme liver iron in the experimental birds that approached those found in birds with ISD but not to cause disease itself. Preliminary work in our laboratory has shown that birds dying with ISD have nonheme liver iron concentrations that range from 4000 to 8000 ppm (G. P. O., unpublished data, July 2004). Therefore, we believe the mean nonheme iron concentrations (3132 ppm) in the iron-loaded starlings approached toxic iron concentrations. Our results show that liver iron concentrations can be safely and quickly increased (393 ppm/wk) in starlings fed a loading iron diet.

The second objective of this study was to determine which of the previously reported treatments would be most effective in reducing stored liver iron in experimental birds. In this study, both phlebotomy and deferoxamine, in combination with a low-iron diet, were effective in decreasing liver iron concentrations, and the rate of decrease was similar for both. Similar to results reported previously, (20) a low-iron diet alone resulted in a significant but small rate of decrease in stored liver iron. Therefore, though a low-iron diet appears to be beneficial when used in combination with other treatments for ISD, a rapid decline in liver iron concentration that is desirable in treating a clinical case would not be expected if dietary modification is used alone.

The treatment of choice for a bird with ISD will depend on the cost of treatment, stress of treatment, and the ability of the bird's owner to treat the bird. All birds with ISD should be switched to a diet that contains less than 100 ppm of iron. Commercial diets with these concentrations are available from some suppliers. A low-iron diet similar to that used in this study must be custom made. Deferoxamine treatment appeared to be more stressful to the birds than phlebotomy. Although deferoxamine treatment resulted in increased excitability in birds before treatment, many bird owners could administer injections at home. In contrast, phlebotomy would usually require that the bird be transported weekly to the veterinarian. This may be more stressful than having the bird bled where it is housed, as was done in this experiment. The current veterinary cost of treating a 100-g bird with deferoxamine to reduce its nonheme liver iron concentration by 3000 ppm during a 16-week period is approximately $70. If the bird were a privately owned pet, weekly trips to the veterinary office would add additional expense.

If either the phlebotomy or deferoxamine treatment, in combination with a low-iron diet, is chosen, then the expected rate of decrease in nonheme liver iron concentration is 163 to 190 ppm/wk. Therefore, to achieve a minimal goal of decreasing the nonheme liver iron concentrations by 3000 ppm, a treatment time of at least 16 weeks is required. We did not study the effectiveness of combining phlebotomy and deferoxamine treatments, but it is likely that this combination would result in a more rapid decrease in liver iron concentrations than either treatment alone. Use of this combination during the initial phase of treatment may be indicated to rapidly decrease concentrations of toxic liver iron. In people, phlebotomy with ISD is sometimes done twice per week. This may also prove to be beneficial in birds with clinical manifestations of ISD.

Adverse effects of treatment were not seen in the birds that we treated. However, a more rapid induction of iron loss could be detrimental. People treated with phlebotomy will sometimes become anemic; therefore, the PCV of birds treated with phlebotomy should be monitored. If birds become anemic during treatment, phlebotomy should be discontinued and the PCV should be allowed to return to normal before treatment is reinstituted. Anemia may also indicate that nonheme liver iron concentrations have dropped to a suboptimal level. The only bird in this study to develop anemia was heavily infested with mites and at necropsy was found to have nonheme liver iron concentration of only 85 ppm.

Recent research in chickens has shown that the oral chelator deferiprone may be an effective treatment for birds that are susceptible to ISD. (19) The rate of decrease in total liver iron in the deferiprone study was 3 to 20 times faster than the highest treatment group in our study. However, some of the chickens died during the course of the study. The cause of death was not determined but was postulated to be a result of the zinc chelating effects of deferiprone. Deferoxamine at higher doses or with more frequent administration could possibly cause the same effect.

The addition of inositol and tannic acid to a diet containing 1730 ppm of iron has been shown to prevent increases in nonheme liver iron concentrations. (20) We anticipated that adding a phytate and tannin to a low-iron diet would inhibit iron absorption from the digestive tract and hasten the decrease in nonheme liver iron concentration. The reason this did not occur may have been because the actual dietary iron concentration (49 ppm) of the diet was higher than the calculated concentration (23 ppm). We have proposed from previous research that the minimum dietary iron concentration for ISD-sensitire species is near 50 ppm. (20) Thus, this diet may meet the minimum dietary iron requirements of the starlings, keeping their liver iron stores elevated. Although these additives may be beneficial in preventing ISD when birds are fed a high-iron diet, the additives may not affect iron absorption when iron concentrations in the diet are low.

In this study we did not account for intraspecies variability to iron deposition and depletion but worked with a single species of bird, the European starling. Individual birds may also deposit or remove iron from liver stores at different rates. Additionally, all birds in our study were wild caught, which may have increased variability of baseline liver stored iron concentrations. Another potential limitation of this study is that liver iron stores were increased acutely, which may not replicate what happens in captive situations.

Acknowledgments: Support for this research was provided by the Morris Animal Foundation and the Schubot Exotic Bird Health Center. We thank Dr Chuck Benton at Harlan Teklab for manufacturing the diets, the Veterinary Medical Park staff for their help in trapping the starlings, and Dr Gerald Bratton and Bryan Britton of the Trace Element Research Laboratory (College of Veterinary Medicine, Texas A&M University) for diet analysis.

References

(1.) Crissey SD, Ward AM, Block SE, Maslanka MT. Hepatic iron accumulation time in European starlings (Sturnus vulgaris) fed two levels of iron. J Zoo Wildl Med. 2000;31:491-496.

(2.) Sheppard C. Hemosiderosis and dietary iron in birds. J Nutr. 1994;124:2685S-2686S.

(3.) Dorrestein GM, Mete A, Lemmens I, Beynen AC. Hemochromatosis/iron storage: new developments. Proc Annu Conf Assoc Avian Vet. 2000;233-238.

(4.) Turner R. Iron storage disease (hemochromatosis) in the curassow. Proc Annu Conf Assoc Avian Vet. 1994;265-267.

(5.) Ensley PK, Osborn K. Hemosiderosis in a wild caught bird of paradise and other species in Papua New Guinea. Proc Am Assoc Zoo Vet. 1993;27.

(6.) Klasing K. Minerals--iron. In: Comparative Avian Nutrition. Wallingford, United Kingdom: CAB International; 1998:259-262.

(7.) Cork SC. Iron storage disease in birds. Avian Pathol. 2000;29:7-12.

(8.) Gosselin S J, Kramer LW. Pathophysiology of excessive iron storage in mynah birds. J Am Vet Med Assoc. 1983;183:1238-1240.

(9.) Sheppard C, Dierenfeld E. Iron storage disease in birds: speculation on etiology and implications for captive husbandry. J Avian Med Surg. 2002; 16:192197.

(10.) Whittington C, Kowdley K. Review article: haemochromatosis. Aliment Pharmacol Ther. 2002;16: 1963-1975.

(11.) Mete A, Hendriks H, Klaren P, et al. Iron metabolism in mynah birds (Gracula religiosa) resembles human hereditary haemochromatosis. Avian Pathol. 2003; 32:625-632.

(12.) Bolan C, Conry-Cantilena C, Mason G, et al. MCV as a guide to phlebotomy therapy for hemochromatosis. Transfusion. 2001;41:819-827.

(13.) Nielsen E Fisher R, Buggisch P, Janka-Schaub G. Effective treatment of hereditary haemochromatosis with desferrioxamine in selected cases. Br J Haematol. 2003;123:952-953.

(14.) Worrell A. Diagnosis and management of iron storage disease in toucans. Semin Avian Exotic Pet Med. 1994;3:37-39.

(15.) Morris PJ, Avgeris SE, Baumgartner RE. Hemochromatosis in a greater Indian hill mynah (Gracula religiosa): case report and review of the literature. J Assoc Avian Vet. 1989;3:87-92.

(16.) Cornelissen H, Ducatelle R, Roels S. Successful treatment of a channel-billed toucan (Ramphastos vitellinus) with iron storage disease by chelation therapy: sequential monitoring of the iron content of the liver during the treatment period by quantitative chemical and image analyses. J Avian Med Surg. 1995;9:131-137.

(17.) Drews AV, Redrobe SP, Patterson-Kane JC. Successful reduction of hepatocellular hemosiderin content by dietary modification in toco toucans (Rhamphastos toco) with iron storage disease. J Avian Med Surg. 2004;18:101-105.

(18.) Loomis MR, Wright JE Treatment of iron storage disease in a Bali mynah. Proc Am Assoc Zoo Vet. 1993; 28.

(19.) Whiteside DE Barker IK, Mehren KG, et al. Clinical evaluation of the oral iron chelator deferiprone for the potential treatment of iron overload in bird species. J Zoo Wildl Med. 2004;35:40-49.

(20.) Olsen GP, Russell K, Dierenfeld E, et al. Impact of supplements on iron absorption from diets containing high and low iron concentrations in the European starling (Sturnus vulgaris). J Avian Med Surg. 2006; 20:67-73.

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(22.) Dixon W. Processing data for outliers. Biometrics. 1953;9:74-89.

From the Department of Veterinary Pathobiology (Olsen, Phalen, Russell), the Schubot Exotic Bird Health Center (Olsen, Phalen), and the Department of Large Animal Medicine and Surgery (Phalen), College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4475, USA; and the St Louis Zoo, One Government Drive, St Louis, MO 63110, USA (Dierenfeld).
Table 1. Stored liver iron concentrations (wet weight) and mean
body weights of European starlings euthanatized before treatment,
after loading with a high-iron diet, and after treating with 4
treatment regimens.

 Birds Time Mean nonheme Standard
 Treatment (n) iron (ppm) deviation (ppm)

Preloading only 7 0 1389 586
High-iron diet 13 31 d 3132 722
 (all birds)
Group 1 (Low-iron 3 6 wk 2767 543
 diet only) 3 11 wk 2848 657
 4 16 wk 2305 180
Group 2 (Low-iron 4 6 wk 2820 548
 diet w/phytate and 4 11 wk 2833 422
 tannic acid) 4 16 wk 2999 792
Group 3 (Low-iron 4 6 wk 1810 621
 diet/deferoxamine) 4 11 wk 1236 194
 4 16 wk 450 228
Group 4 (Low-iron 3 6 wk 1708 292
 diet/phlebotomy) 4 11 wk 623 534
 3 16 wk 163 75

 Range Mean body Range
 Treatment weight (g) (g)

Preloading only 656-2119 72.7 71-81
High-iron diet 1882-4233 72 62-78.5
 (all birds)
Group 1 (Low-iron 2163-3214 81 68-88
 diet only) 2090-3265 79 74-85
 2115-2477 71 68-71
Group 2 (Low-iron 2149-3358 77 73-81
 diet w/phytate and 2567-3456 76 73-78
 tannic acid) 2243-3963 71 65-78
Group 3 (Low-iron 979-2346 75 64-86
 diet/deferoxamine) 1035-1492 75 71-77
 186-731 71 69-73
Group 4 (Low-iron 1491-2040 76 75-77
 diet/phlebotomy) 108-1117 77 67-82
 109-251 72 68-77
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Title Annotation:Original Study
Author:Olsen, Geoffrey P.; Russell, Karen E.; Dierenfeld, Ellen; Phalen, David N.
Publication:Journal of Avian Medicine and Surgery
Geographic Code:1USA
Date:Jun 1, 2006
Words:4116
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