PATHOGENESIS OF METABOLIC BONE DISEASE IN CAPTIVE POLAR BEARS (Ursus maritimus)

¹San Francisco Zoological Society, One Zoo Road, San Francisco, CA 94132
²LeBonheur Children’s Medical Center, 50 N Dunlap Room 306, Memphis, TN 38103

Abstract

Metabolic bone disease is a condition that may result from deficiencies in Vitamin D or calcium or improper ratios of calcium to phosphorus. Clinical signs are generally evident by acute lameness caused by fractures. An alarming number of captive polar bears (Ursus maratimus) are presenting with fractures of long bones. At times, it is necessary for zoo personnel to hand-rear polar bear cubs. These bears are generally fed a canine formula or may eat a dog food diet from an early age. Two cases in U.S. zoos (Denver, San Diego) describe infant polar bear cubs with metabolic bone disease. Rickets is unknown in polar bear cubs in the wild, raising the possibility that changes in the diet or environment of cubs raised in captivity is a potential cause. Captive-born polar bear cubs have a disturbingly high mortality rate. The International Species Inventory System (ISIS) records for the past 25 years show that more than 50% of polar bear cubs born in captivity fail to survive beyond age 3 months.

Taurine, an osmolyte, is important in cell volume regulation since, as an b-amino acid, it is not incorporated into protein synthesis and resides free in the intracellular fluid. Aside from cell volume regulation, its major biologic role is the conjugation of bile acids. Taurine-conjugated bile acids are important in triglyceride and fat-soluble vitamin absorption from the gut lumen into the blood stream. Taurine-conjugated bile acids absorb vitamin D more efficiently than glycine-conjugates.

The taurine content of some dry dog foods is negligible, as these feeds are routinely not supplemented with taurine. The commercial canine milk formula used in hand feeding bears is taurine-free and the cow’s milk cream-fed to these animals also lacks taurine. Taurine may also be required by very young infants of mammals and, thus should be included in milk replacers. Human infants fed a taurine-free milk replacer have developed rickets.

While taurine deficiency is usually found in feline species because they lack the critical biosynthetic enzyme – cysteine sulfinic acid decarboxylase (CSAD) – to synthesize this amino acid, it is interesting to speculate that the lack of dietary taurine may have a nutritional consequence in bear cubs.

Full Text

Metabolic bone disease is a condition that may result from calcium deficiency, improper ratios of calcium to phosphorus, or diseases causing malabsorption. Absorption of calcium is reduced in the absence of bile salts, which may occur in biliary stasis. If fat absorption is impaired, dietary vitamin D absorption is also reduced leading to the development of metabolic bone disease. (Fowler, 1986). Rickets is a disease due to Vitamin D deficiency and characterized by overproduction and deficient calcification of osteoid tissue, with associated skeletal deformities (Hoskins, 1990). Clinical signs are generally evident by acute lameness caused by fractures.

An alarming number of captive polar bears (Ursus maratimus) are presenting with fractures of long bones. At times, it is necessary for zoo personnel to hand-rear polar bear cubs. These bears are generally fed a canine formula or may eat a dog food diet from an early age. In U.S. zoos (Denver, San Diego), several of these cubs have developed metabolic bone disease. (Hedberg, 2002) (Kenny, 1999). Metabolic bone disease is unknown in polar bear cubs in the wild, raising the possibility that changes in the diet or environment of cubs raised in captivity is a potential cause. Captive born polar bear cubs have a disturbingly high mortality rate. The International Species Inventory System (ISIS) records for the past 25 years show that more than 50% of polar bear cubs born in captivity fail to survive beyond age 3 months.

Taurine is an amino acid that is not found in any plant-based food and is an essential nutrient in all mammalian cells. Taurine, an osmolyte, is important in cell volume regulation and as an -amino acid that is not incorporated into protein synthesis that resides free in the intracellular fluid (Chesney, 1998). Aside from cell volume regulation, taurine’s major biologic role is the conjugation of bile acids.

Carnivores tend to be exclusive taurine-conjugators (Huxtable, 1992). Taurine-conjugated bile acids are important in triglyceride and fat-soluble vitamin absorption from the gut lumen into the blood stream (Hoffman, 1999). Taurine-conjugated bile acids absorb vitamin D more efficiently than glycine-conjugates (Hoffman, 1999) (Zamboni, 1993).

All bile acids must be conjugated in order to form bile salts, which are important in the formation of mixed micelles that enhance diffusion through the unstirred layer of the small intestine (Hoffman, 1999) (Zamboni, 1993).

The amino acid taurine is sometimes in short supply in infant formulas (Chesney,1998). Infants fed a taurine-free formula have been reported to have Vitamin D deficiency with nutritional rickets (Zamboni, 1993). The commercial canine milk formula that is often used in hand-rearing carnivores is free of taurine, as is cow’s milk.

Feline taurine deficiency leads to retinal degeneration, tapetal degeneration, cardiomyopathy and, in F1 generation kittens, fetal wastage and renal abnormalities (Sturman, 1995) (Chesney, 2000) (Hayes, 1975). These disorders have been noted in domestic and barnyard cats, (Morris, 1990) (Lippincott, 1988) (Kenny, 1998) captive Bengal tigers and leopards (Pickett, 1990). The major reasons for feline taurine deficiency in cats are due to a vegetable-based canine diet which is taurine free or a canned feline diet labeled to contain adequate taurine (Morris, 1990) (Hayes, 1975)). The heating process of the canned feline diet produces a Maillard reaction with the sugars in the chow, rendering the taurine in the chow incapable of being absorbed from the gut of the feline (Morris, 1990). A recent study indicates a taurine deficiency in the domestic dog fed commercial lamb and rice based dry dog food (Stratton-Phelps ,2002).

Most animals can produce taurine from methionine, cystenine, or inorganic sulfate and serine, but cats cannot. The occurrence of taurine synthesis in most animals and the loss of this ability in cats occurs because taurine does not occur in plants, but is abundant in meat. Consequently, all animals except a strict carnivore must produce taurine (Robbins, 1993).

All of this background information led to a review of taurine values in both blood and diet of polar bears. Little information is available about taurine and its relationship to infant and adult polar bear nutrition.

Plasma taurine values are significantly lower (60%) in captive bears. The mechanism of taurine reduction in plasma is unknown but may well involve a taurine-insufficient diet (see prelim results).

If the mechanism of low taurine plasma values is unclear, its relationship to rickets is even more obscure. The investigators speculate that it may relate to the unique bile acids of ursid species – Urosodeoxycholic acid (UCDA). UCDA (3, 7-dehydroxy-5B-colanic acid) was first identified as a major constituent of dried bile in the Chinese black bear (Beuers, 1998) and has been used for centuries in China for liver disorders. (Shoda, 1927). UCDA has been found particularly effective in the treatment of cholestratic liver disease in man and in the therapy of primary bleary cirrhosis (Hoffmann, 1999).

Bile acids are necessary for cholesterol elimination, stimulation of bile flow, stimulation of biliary phosphalidylcholine secretion and enhancement of lipid absorption as well as other actions. All bile acids must be conjugated in order to form bile salts that are important in the formation of mixed micelles that enhance diffusion through the unstirred layer of the small intestine (Hoffman 1999) (Zamboni, 1993). Conjugated bile acids are impermeable to most membranes and thus can have high intraluminal (i.e., within the gut) concentrations. They are also less likely to precipitate in the presence of intraluminal calcium. The two amino acids that are important in conjugation are glycine and taurine. Taurine conjugates of primary and secondary bile acids are always associated with greater lipid absorption. (Hoffman, 1999) Unless bile acids are present in this micellar form, the fat-soluble vitamins (A, D, E and K) will not undergo absorption and deficiency can occur (Pickett, 1990).

The investigators speculate that the taurine conjugation of UCDA is impaired in polar bear cubs fed dog formula or dog chow because of relative taurine deficiency in the diet. This, in turn, results in poor micellar formation and reduced vitamin D absorption as has been shown in pre-term human infants (Zamboni, 1993). Reduced vitamin D absorption will manifest as vitamin D-deficiency rickets. Cubs raised in the wild have a high taurine diet, because of the high fish and seal content and because taurine-sufficient mothers may well have high bear milk taurine content. It is further speculated that the conjugation of UCDA bile acid with glycine cannot support normal micellar formation and adequate vitamin D absorption. This has been shown in human infants fed a taurine-free formula (Zamboni, 1993); their 25 (OH) vitamin D levels were significantly reduced.

Preliminary Work

Serum and blood samples have been collected from polar bears from several zoos, including the San Diego Zoo, the San Francisco Zoo, Sea World San Diego, and the Detroit Zoo. The American Zoo and Aquarium Association Bear Taxonomic Advisory Group have been approached to investigate collaboration among a wider group of zoos. Collaboration has been developed with Dr. Andrew Derocher, Professor at University of Alberta, Edmonton, Canada; Norwegian Polar Institute, Tromso, Norway; and Dr. Robert J. Letcher of the Great Lakes Institute for Environmental Research (University of Windsor, Ontario Canada), who has collected blood and milk samples from free-ranging polar bears. The investigators have also collaborated with Dr. Quinton Rogers, Professor of Nutrition and Veterinary Sciences at University of California Davis, who has measured blood and plasma taurine values in 13 captive polar bears. Rogers’ lab has shown a 60% reduction in plasma taurine in captive bears as compared to values in two recent wild-caught captive bear cubs.

The values for plasma and whole blood have been obtained in 13 animals. Plasma taurine in bears in captivity was 97±34 SE n moles/ml (n=9) and in free-ranging bears was 237±10 SE n moles/ml (n=4) p<.02. In whole blood, which permits taurine to leak from platelets and white cells, the values were 248±71 SE n moles/ml (n=13) in captive vs. 453±8 SE n moles/ml (n=4) p05. These preliminary results indicate that both plasma and whole blood taurine concentrations were higher than those in man (40-100 nM/ml), dog (60-120 nM/ml), rat (50-95 nM/ml), and cat (60-120 nM/ml)(Chesney, Helms et al. 1998) and may reflect the maritime diet of polar bears. Clearly more values from wild and captive animals are needed. Nonetheless, the captive animals have a 60% reduction in plasma and a 45% reduction in whole blood taurine values.

Hypothesis

Rickets found in polar bear cubs raised in captivity is dependent upon:

• Reduced concentrations of taurine as present in canine artificial formula or dry dog food result in reduced plasma and breast milk taurine concentrations.

• Reduced quantities of taurine are available for taurine conjugation of UCDA, the unique Ursid bile acid.

• Predominate glycine conjugates of UCDA results in inferior micellar production and impaired vitamin D absorption and vitamin D deficiency that will result in nutritional rickets.

• Chronic reduction of plasma taurine will also result in up regulation of the gene for the Na-dependent (TauT) in all cell membranes taurine transporter.

Specific Aims

• Measure plasma and whole blood taurine content in 100 wild and captive polar bears of different ages.

• Measure bear milk taurine concentration in 10 each wild and captive polar bear to seek differences

• Measure plasma bile acids and determine the percent of UCDA and other bile acids conjugated with taurine and with glycine in 10 each wild and captive polar bear to seek differences.

• Measure plasma 25 hydroxyvitamin D in 30 captive and wild polar bear plasma samples to seek differences.

• Measure the level of mRNA and TauT protein in the membrane of lymphocytes from wild and captive polar bears by Northern and Western blot technology to seek up regulation of the transporter in captive bears. This could indicate an adaptive response based on a gene-nutrient interaction.

• Measure Vitamin A-Retinol in 30 captive and wild polar bear serum samples to seek differences. Hypovitaminosis A can lead to abnormal bone development in fetal and neonatal life. Spontaneous fractures can occur in Hypervitaminosis A.

Methods

Animals and Biological Fluid Samples

The investigators have been in contact with Drs. Letcher and Derocher to assist in obtaining plasma samples, blood samples, and milk samples from animals in the wild. Dr. Drencher has already obtained more than 200 plasma samples and 60 milk samples that are stored in a frozen state (-20º to -80º C).

Plasma samples are preferable to serum samples because of the high content of taurine in platelets.[16] Both whole blood and plasma samples are obtained in heparinized tubes. Each sample would be 1.0 ml and, hence, would not pose a great risk to the animal.

Amino Acid Analysis

Plasma taurine will be measured in the lab of Quinton Rogers, Ph.D.(Morris, 1990). Dr Rogers uses a phenylisothiocynate precolumn derivatization following perchlorate precipitation of 0.25 ml of plasma.(Lippincott, 1988). Because taurine is highly acidic in its Zwitterionic form and at pK1, it is the first amino acid that comes off the column. This fact means that samples can be run rapidly, and the column cleaned before the second plasma sample. Hence, 100 samples can be determined in 100 hours run time. Milk taurine content can be measured by a modification of the plasma sample technique. (Lippincott, 1988). Dr. Rogers’ lab has a Beckman Amino Acid analyzer. Plasma taurine concentrations are expressed in terms of m moles/L of plasma or n moles/ml plasma.

If plasma levels are very low, we can derivatize taurine with fluorescamine as our lab has done in small samples for 28 years. (Lippincott, 1988) (Chesney, 1976). We will measure plasma taurine in young and adult bears, both male and female.

Bile Acid Measurements

Plasma samples will be measured in the laboratory of Dr. Kenneth Setchel at the University of Cincinnati. A rate of $35 per sample has been negotiated. This laboratory, a national reference lab, employs High Pressure Liquid Chromatography and GC-Mass Spec technology. They have used Gas Chromatography – Mass Spectroscopy to determine the glycine and taurine conjugates of all major bile acids including UCDA.

25 Hydroxyvitamin D Measurements

The major circulating form of vitamin D is 25 (OH) vitamin D that is found in man at concentrations of 20-80 ng/ml (Chesney, 1980). Measurements of this metabolite are very low in TPN-induced cholestasis[(Chesney, 1975) (Chesney, 1981)and in nutritional rickets in infants (Chesney 1981). Plasma will be measured 25(OH) vitamin D values in the laboratory of Dr. Leon LeVan at Bone Care International, a vitamin D pharmaceutical company in Madison, WI. Dr. LeVan uses HPLC methodology after Cep Pack separation to measure this metabolite in plasma. Dr. Chesney is a consultant for Bone Care International and has used this laboratory for other studies(Frazao, 1998).

Vitamin A – Retinol Measurements

On August 3, 2002, the working team reviewed potential Hypovitaminosis and Hypervitaminosis A conditions. Hypovitaminosis A can lead to abnormal bone development in fetal and neonatal life. Spontaneous fractures can occur in Hypervitaminosis A. Dr. Chesney will review available laboratory resources for testing.

Statistics

This is a cross-sectional study of plasma, blood and milk of bears in the wild and in captivity. A comparison of mean and SD using the T-statistic will be run and a correlate of plasma taurine with each of bile acid values, % taurine conjugated UCDA, 25 (OH) vitamin D concentration, and with taurine content using the least squares method to determine correlation coefficients. Each of those parameters can be compared by the least squares method.

The details of power analysis show that 100 plasma samples are adequate. A measurement of the relative density of Northern and Western Blots will be done to determine if the taurine transporter is unregulated.

Results that would support the investigators hypothesis

• Plasma and whole blood taurine will be significantly lower in bears in captivity than in the wild.

• Female bears in captivity will have low plasma taurine values as compared to their counterparts in the wild.

• Milk concentrations in female adult bears will correlate with plasma taurine concentrations being lower in captive vs. wild bears.

• Captive bears will show lower percentages of taurine-conjugated bile acids than wild bears.

• 25(OH) vitamin D values will demonstrate that wild bears have improved vitamin D sufficiency than captive bears.

• Some bears may have values of 25 (OH) vitamins D in the range associated with vitamin D-deficiency in other mammalian species. We suspect these will be younger bears.

• Taurine transporter mRNA and membrane protein abundance will be higher in those animals with evidence of reduced taurine concentrations in plasma. This indicates transporter adaptation to overcome nutritional inadequacy.

Future Studies

Should the investigators’ hypotheses prove correct, it will be necessary to define the quantity of taurine to supplement bears raised in captivity. If plasma taurine values are extremely low in bears raised in captivity, it may also be important to examine the retina and measure an electroretinogram. These studies, however, are not part of this research.

Case studies of captive polar bears with fractures have been reviewed along with the recommendation to evaluate dietary intake of vitamin D. A complete captive nutritional assessment of both blood and diet elements of this species would further define the specific requirements for Vitamin A, Vitamin D, and Taurine.

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