Monitoring of Plasma 25-Hydroxyvitamin D Concentrations in Two Komodo Dragons, Varanus komodoensis: A Case Study

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1 N u t r i t i o n N o t e s Monitoring of Plasma 25-Hydroxyvitamin D Concentrations in Two Komodo Dragons, Varanus komodoensis: A Case Study Zoltan S. Gyimesi, DVM, Roy B. Bums III, DVM Louisville Zoological Garden, 1100 Trevilian Way, PO Box 37250, Louisville, KY 40233, USA A b s t r a c t : Like many basking lizard species, the Komodo dragon, Varanus komodoensis, is believed to require exposure to ultraviolet light of the appropriate wavelength to allow for vitamin D3 synthesis and calcium absorption and homeostasis. Serum or plasma 25-hydroxyvitamin D (25-OH-D) concentration is considered the most useful assessment of overall vitamin D status in humans and animals. This case study describes the trends in plasma 25-OH-D in two captive Komodo dragons housed both indoors and outdoors in Louisville, KY over a 2.5-yr period. Data suggest that ultraviolet light exposure is critical to allow Komodo dragons to reach plasma 25-OH-D levels comparable to those levels reported in both wild dragons, and captive dragons offered daily sun exposure. Additionally, it appears that in temperate climates, approximately 150 d of sun exposure combined with moderate amounts of dietary vitamin D3 may be adequate for Komodo dragons to develop and maintain appropriate circulating levels of 25-OH-D throughout the year. K e y W o r d s : K om odo dragon, Varanus komodoensis, 25-hydroxyvitam in D, vitam in D, ultraviolet light, photobiogenesis. INTRODUCTION The Komodo dragon, Varanus komodoensis, is the world s largest extant lizard and has become increasingly common in zoological parks in North America. Like many basking lizard species, Komodo dragons are assumed to require exposure to ultraviolet (UV) light to allow for vitamin D3 photobiogenesis and calcium absorption and homeostasis (Allen, et al, 1994). Specifically, ultraviolet light B (UVB) from nm is responsible for the cutaneous conversion of 7-dehydrocholesterol to previtamin D3 in most species studied (Bernard, 1997). In addition to vitamin D3 photobiogenesis, a more poorly understood function of UV light for some reptiles is to initiate and maintain normal agonistic, reproductive, and signaling behaviors (Gehrmann, 1994). In captive specimens, UV light may also act as an appetite stimulant in reluctant feeders (Marcus, 1981). Exposure to direct, unfiltered sunlight is believed to be the ideal, and certainly most natural source of UV light. For many captive basking lizards, direct exposure to sunlight may not be possible since many reptiles are maintained in temperate climates and tend to be housed indoors year-round. In these cases, exposure to natural sunlight through UV-transmissible skylights or the use of artificial UV light sources may be utilized. Some zoological parks that rely on sunlight through UV-transmissible skylights may not be providing their animals with year-round UVB. A human study found that in Boston, Massachusetts (42.2 N), and Edmonton, Alberta (52 N), exposure to sunlight from November through February, and October through March, respectively, will not promote vitamin D3 photobiogenesis (Webb, et al, 1988). As the zenith angle of the sun increases with rising latitude in the winter, it dramatically decreases the amount of UVB radiation that reaches the surface of the earth. Vitamin D may be offered orally, however there is wide species variation in vitamin D metabolism and dietary vitamin D utilization capabilities. For example, nocturnal lizard species such as eyed skinks, Chalcides ocellatus, and leopard geckos, Eublepharus macularius, develop normally when the diet is the only source of vitamin D3, however most diurnal species, including day geckos, Phelsuma madagascariensis, green iguanas, Iguana iguana, and Komodo dragons appear to be more dependent on UVB exposure (Allen, et al, 1994, Allen, et al, 1995, Gehrmann, 1997). While green iguanas appear to have limited ability to use dietary vitamin D3, this species represents a poor model for the Komodo dragon given that the former is a folivore, the latter a strict carnivore. It is not clear at this time to what capacity Komodo dragons can utilize oral sources of vitamin D. It is known that the concentrations of vitamin D3 present in typical captive diets, without dietary supplements, do not appear to be sufficient when UVB is absent (Gillespie, et al, 2000). Whether obtained from diet or synthesized endogenously via UVB, vitamin D may either be stored in fat cells and be utilized as needed or it may be hydroxylated to 25-hydroxyvitamin D (25-OH-D) in the liver. The latter is the major circulating form of vitamin D and has a plasma half-life in humans of approximately d (Holick, 1990, Genuth, 1998). 25-hydroxyvitamin D refers to both 25-hydroxyergocalciferol (25-O H-D 2) and 25-hydroxycholecalciferol 4 Journal of Herpetological Medicine and Surgery Volume 12, No. 2,2002

2 (25-0H-D3). Once hydroxylated, 25-OH-D is transported to the kidney where it is converted to 1,25-dihydroxy vitamin D in the renal tubular cells. 1,25-dihydroxy vitamin D is the active form of vitamin D that works directly on target organs to regulate calcium and phosphorus homeostasis. 25-hydroxyvitamin D serum or plasma concentration is considered the most useful assessment of overall vitamin D status in humans and animals. These concentrations reflect the sum of vitamin D from diet and photobiogenesis over a period of several weeks or months (Holick, 1990, Ullrey and Bernard, 1999). Very low or undetectable concentrations rep resen t im pending or fran k v itam in D deficiency, or hypovitaminosis D, while markedly elevated levels represent vitamin D intoxication or hypervitaminosis D. This case study describes the trends in plasma 25-OH-D in two captive Komodo dragons housed both indoors and outdoors in Louisville, KY over a 2.5-year period (latitude = 38 13' N, longitude = 85 44' W, elevation = 149 m). Observations char acterizing basking behaviors and quantitative measurements of UV irradiance were not made. HISTORY Two Komodo dragons, hatched in August 1993, were trans ferred on loan from the Cincinnati Zoo and Botanical Garden in Ohio to the Louisville Zoological Garden in Kentucky. A male dragon (KD1) was relocated in March 1994, and a female (KD2) in June These dragons were always housed separately in several different indoor enclosures. Between 1994 and 1995, these dragons were maintained in smaller enclosures where artificial UV lighting (Vita-Lite, Duro-Test Corp., Fairfield, NJ) was provided within 1.2 m through screen material. A blacklight lamp was an additional light feature in one of these enclosures, however more specif ic details are unavailable. By 1996, both dragons outgrew their enclosures and were transferred between two larger indoor exhibits measuring 6.0 m x 6.0 m x 4.0 m and 5.8 m x 7.3 m x 3.7 m. Ambient tem perature ranged from C (75-85 F). Both exhibits offered supplemental heat either built into the flooring or in the form of heat pads buried under substrate providing surface temperatures of C ( F). These temperature ranges maintained both Komodo dragons within their pre ferred optimum temperature zone (Walsh and Visser, 1999). Artificial UV lighting in the form of clear finish metal halide lamps (Metalarc, Osram Sylvania Inc, Manchester, NH) was used for general illumination in both of these enclosures but was likely too far away from the exhibited dragon ( m) to be considered effective for vitamin D3 photobiogen esis. Although the newer of the two larger enclosures was originally designed to allow ample unfiltered sunlight in dur ing the warmer m onths, by the end of the construction process, the quality of sunlight that could be offered was suboptimal. In this newer exhibit, high, vertical windows would be kept open from May/June through September. This would allow a narrow band of direct sunlight into the exhibit for a short period each day. The windows in this enclosure and sky lights in the other large indoor enclosure were not made of UV-transmissible material. Diet for both dragons consisted of whole rodent prey in the form of rats and mice. No vitamin or mineral supplements were offered. Volume 12, No. 2,2002 In April 1998, KD1 developed a rear foot pododermatitis. Evaluation with the aid of a restraint crate included physical examination of the affected foot, cytology, culture and sensi tivity, radiographs, and blood collection for hematology, plasma biochemistries, and vitamin profile. Radiographs were interpreted as normal. Complete blood count and plasma bio chemistries were unremarkable. Vitamin and mineral analyses at the Animal Health Diagnostic Laboratory at Michigan State University revealed a markedly low 25-OH-D level - 2 ng/ml (4 nmol/l). The reference range for 25-OH-D utilized in this case study is ng/ml ( nmol/l) which is the typical range observed in both wild Komodo dragons, and captive dragons offered daily sun exposure (Gillespie, et al, 2000). Although this is the typical range, Gillespie, et al, found that 25% of this group of dragons exhibited plasma 25OH-D levels greater than 100 ng/ml (>250 nmol/l). The level of plasma 25-OH-D was also determined to be very low in KD2, 13 ng/ml (32 nmol/l). Ultraviolet light B irradiance was not measured in either indoor exhibit, however the pre sumption was that levels were inadequate for this species given the low baseline 25-OH-D plasma concentrations. Based on this information, both Komodo dragons were offered unlimited exposure to unfiltered sunlight as weather permitted. This was accomplished by moving the dragons to off-exhibit enclosures with outdoor access. These enclosures offered areas of direct sun as well as shade, allowing the dragons to self regulate their basking. A dietary change was also instituted. Both dragons continued to receive a whole prey rodent diet (KD1 averaged 2.2 kg of whole rodents per week, KD2 averaged 1.0 kg of whole rodents per week), but beginning May 1998, this was supplemented weekly with one tab lesp o o n (approxim ately g) o f a balanced calcium /phosphorus and vitam in A and D 3 supplem ent (Osteo-form, Vet-A-Mix, Shenandoah, IA) per dragon. With this supplementation, each dragon received an additional 424 IU of oral vitamin D 3 per week. With the vitamin and mineral supplementation, the concentration of vitamin D3 in the diets of the dragons during this 2.5-year case study was 1,600 3,000 IU/kg dry matter (DM). Dietary calcium and phospho rus co n ten t was % DM and % DM respectively. Venipuncture was performed on both dragons opportunisti cally over the next 2.5 years utilizing the restraint crate and accessing the ventral tail vein via a lateral approach. 25hydroxyvitamin D concentrations were measured with each blood collection (KD1, n=12, KD2, n=13). Complete blood counts, biochemistry panels, and additional vitamin and min eral panels were repeated periodically, depending in part on the amount of blood obtained. During this time period, both KD1 and KD2 were moved back and forth from off-exhibit outdoor enclosures, to one of the two larger indoor exhibits. RESULTS Plasma 25-OH-D was measured using a commercially available acetonitrile extraction/equilibrium radioimmunoas say kit (25-hydroxyvitamin D l25i RIA Kit, DiaSorin Inc, Stillwater, MN). This technique measures both 25-OH-D2 and 25-OH-D3 equally and is not affected by sample lipemia, hemolysis, or icterus. Limit of detection (sensitivity) was determined as the concentration equivalent to two standard Journal of Herpetological Medicine and Surgery 5

3 deviations below the mean of the radioactive counts of the zero standard. Intra- and inter-assay precision were assessed by ten replicate analyses in one assay and in ten consecutive daily assays using pooled Komodo dragon plasma containing a mid-range concentration of 25-OH-D (58 ng/ml, 145 nmol/l). The limit of detection for 25-OH-D measurement was calculated to be 1 ng/ml (3nmol/L). Intra- and inter-assay coefficients of variation were 2.3% and 7.6% respectively. Plasma 25-OH-D concentrations over time for KD1 are graphed linearly (Figure 1). This animal s plasma 25-OH-D level was 2 ng/ml (4 nmol/l) shortly before it was first given access to direct sunlight. After 35 d of being outdoors, this level rose to 101 ng/ml (252 nmol/l), and after a total of 154 d of being outdoors, plasma 25-OH-D reached 112 ng/ml (279 nmol/l). This dragon was placed back in its indoor exhibit for the next 299 d. While indoors from October 8, 1998 to August 3, 1999, plasma 25-OH-D levels waned from 112 ng/ml to 44 ng/ml (109 nmol/l). KD1 was again given outdoor access during the late summer/early fall of Over 64 d, plasma 25-OH-D levels rose from 44 ng/ml to 89 ng/ml (222 nmol/l). KD1 was then transferred indoors for 278 d. During this period, plasma 25-OH-D levels waned from 89 ng/ml to 38 ng/ml (95 nmol/l). KD1 was moved outdoors from July 11, 2000 to October 12, 2000 (93 d) and plasma 25-OH-D levels rose from 38 ng/ml to 75 ng/ml (187 nmol/l). Similar trends were observed with KD2. Plasma 25-OH-D concentrations over time for KD2 are graphed linearly* (Figure 2). This dragon s plasma 25-OH-D level was 13 ng/ml (32 nmol/l) at the time it was first given access to direct sun. After 36 d of being outdoors, this level rose to 109 ng/ml (271 nmol/l). This dragon was moved indoors on October 8, 1998 and blood was drawn 35 days after this move. 25-Hydroxyvitamin D levels rose further to 180 ng/ml (450 nmol/l). KD2 was indoor housed for 257 d and plasma 25-OH-D levels waned to 87 ng/ml (217 nmol/l) at the time this dragon was moved outdoors for part of the second summer. Over 42 d of exposure to sun, plasma 25-OH-D levels rose from 87 ng/ml to 94 ng/ml (234 nmol/l). The dragon was moved indoors for 288 d. Plasma 25-OH-D levels were 84 ng/ml (210 nmol/l) at the time KD2 was again given access to direct sunlight during the late spring/early summer of Over 54 d, plasma 25-OH-D levels rose from 84 ng/ml to 133 ng/ml (333 nmol/l). On July 11, 2000, KD2 Figure 1. Male Komodo dragon, Varanus komodoensis, (KD1) plasma 25-hydroxyvitamin D levels over time. Shaded regions represent periods when KD1 was housed indoors with minimal ultraviolet light exposure. Non-shaded regions represent periods when housed outdoors with daily access to direct sunlight. The area between the two horizontal lines represents the range of plasma 25-OH-D typically seen in Komodo dragons with daily sun exposure (captive and wild). Journal of Herpetological Medicine and Surgery Volume 12, No. 2,2002

4 was moved indoors. Sampling on October 12, 2000 revealed that plasma 25-OH-D levels had fallen from 133 ng/ml to 77 ng/ml (192 nmol/l). Complete blood counts, biochemistry profiles, and additional vitamin and mineral analyses evaluated during this time period from KD1 and KD2 were unremarkable, including plasma levels of calcium, phosphorus, and alkaline phosphatase. In December 2000, KD2 was found dead in its enclosure. Aside from two weeks of anorexia which herpetological staff interpreted as normal for this individual during this time of year, no outward clinical signs were observed leading up to death. Whole body radiographs were obtained before necropsy. There was no evidence of angular deformities or historic pathologic fractures, and subjectively, bone mineralization appeared radiographically normal. This seven-year-old female was in good body condition at the time of death. Pathologic evaluation revealed the cause of death was generalized yolk em boli associated with fo llicular degeneration. Yolk embolism was secondary to severe, generalized, vascular degeneration associated with amyloid deposition. The bones examined were grossly and microscopically normal. DISCUSSION This 2.5-year study encompassed three summers. During the first summer, an effort was made to maximize the time period both dragons were offered access to direct sunlight by moving them outdoors in the spring and continuing outdoor access for as long as weather permitted. Outdoor access was offered when ambient temperature was a minimum of 16 C (60 F). For the second and third summer of the study, it was decided to give each dragon half the time outdoors as the first summer. This was based on several factors including limited outdoor holding space, the desire by the institution to have a Komodo dragon on exhibit during peak attendance, and the first summers experience of plasma 25-OH-D levels normalizing rapidly and waning slowly. The data suggest that exposure to ultraviolet light is critical in order for Komodo dragons to reach appropriate plasma 25- OH-D levels. The benefits of ultraviolet light are not surprising as Komodo dragons will seek out sunlight and freeranging dragons in Indonesia have been documented to bask daily (Auffenberg, 1981, Walsh and Visser, 1999). Marked and predictable increases in 25-OH-D levels occurred in both May Oct-8-98 Jun Aug-3^9 May Jul Figure 2. Female Komodo dragon, Varanus komodoensis, (KD2) plasma 25-hydroxy vitamin D levels over time. Shaded regions represent periods when KD2 was housed indoors with minimal ultraviolet light exposure. Non-shaded regions represent periods when housed outdoors with daily access to direct sunlight. The area between the two horizontal lines represents the range of plasma 25-OH-D typically seen in Komodo dragons with daily sun exposure (captive and wild). Volume 12, No. 2,2002 Journal of Herpetological Medicine and Surgery 7

5 Komodo dragons in response to sun exposure. This is consis tent with observations made by other researchers (Allen, et al, 1994, Gillespie, et al, 2000, Nijboer, 2001). In the case of KD1 and KD2, their original plasma 25-OH-D deficiencies were corrected within 35 and 36 d of temperate sun exposure, respectively. Average outdoor temperatures during the months of May through October in Louisville, Kentucky range from 15 C (59 F) to 26 C (78 F) (National Weather Service, May 2002, In terms of approximate cloud cover during these months, 30% of the days are typically clear, 33% of the days are considered part ly cloudy, and 36% of the days are typically cloudy (National C lim atic D ata Center, M ay 2002, There were instances with both Komodo dragons when 25OH-D concentrations rose while the lizards were housed indoors in a UVB-insufficient environment. Since excess vit amin D that is absorbed in the gastrointestinal tract or produced in the skin is stored in the body fat, it is possible that some of these rises represent times of increased mobi lization and hydroxylation of vitamin D3 from these depots. The length of time appropriate plasma 25-OH-D levels may be maintained after ultraviolet light access is denied has been poorly documented in basking reptiles. In one unpub lished report, 25-OH-D levels dropped rapidly (within one to two months) in a Komodo dragon after appropriate UV light was no longer provided (Gillespie, 2001). The dragon in that report was offered a whole prey diet with no oral vitamin D3 supplement (Gillespie, 2001). Based on the two animals tracked in this study, it appears that 25-OH-D levels wane rel atively slowly. For example, following the first summer of sun exposure, KD1 and KD2 were still able to maintain nor mal circulating levels of 25-OH-D after being housed indoors for over six months and over eight months, respectively. It is possible that the moderate amount of oral vitamin D 3 supplementation assisted in prolonging the amount of time plasma 25-OH-D could be maintained at appropriate levels. Oral vitamin D3 supplementation may be a useful adjunct to UV light exposure in Komodo dragons kept in similar hous ing situations. In tem perate clim ates, it appears that approximately 150 d of sun exposure combined with moder ate amounts of dietary vitamin D 3 may be adequate for Komodo dragons to develop and maintain appropriate circu lating levels of 25-OH-D throughout the year. We caution that there are inherent risks when relying on oral vitamin supple ments to meet the physiologic needs of Komodo dragons and the importance and safety of providing ultraviolet light, ideal ly sunlight, cannot be overemphasized. The true value of dietary vitamin D3 in Komodo dragon husbandry requires fur ther investigation. During the 2.5-year study period, plasma 25-OH-D levels in the two Komodo dragons ranged from as low as 2 ng/ml (4 nmol/l) to as high as 180 ng/ml (450 nmol/l). In humans, circulating levels below 20 ng/ml (50 nmol/l) are typically considered to indicate impending or frank hypovitaminosis D (Holick, 2000). Given the suboptimal UVB exposure before outdoor access was offered, it is likely that both dragons had severely low 25-OH-D levels for at least two years before the deficiencies were first recognized. Signs of hypovitaminosis D include defects in bone mineralization, hypotonia, muscle Journal of Herpetological Medicine and Surgery weakness, poor growth, hypocalcemia, hypophosphatemia and elevation in alkaline phosphatase (Ullrey and Bernard, 1999). Sequelae may include pathologic fractures. Interestingly, neither dragon ever exhibited recognizable clin ical signs or blood abnorm alities co n sisten t with hypovitaminosis D or metabolic bone disease, other than low 25-OH-D levels. This is particularly surprising as these were fairly young, growing reptiles where one might expect chron ic vitamin imbalances to lead to growth retardation and clinical disease. The Komodo dragons discussed in this paper demonstrated growth rates comparable to those housed at numerous other zoological parks (Walsh, 2001). In addition, KD1 and KD2 demonstrated weight to total length relation ships com parable to w ild Komodo dragons surveyed (Auffenberg, 1981). In humans, hypervitaminosis D is usually associated with 25-OH-D levels above 125 ng/ml (312 nmol/l) (Holick, 2000). Reptiles likely have a higher 25-OH-D tolerance as there are reports of healthy green iguanas developing levels greater than 400 ng/ml (1000 nmol/l) (Allen, et al, 1995). The female Komodo dragon in this report reached circulating levels as high as 180 ng/ml (450 nmol/l). The highest 25OH-D concentration reported in one study which included 43 samples obtained from both wild Komodo dragons, and cap tive dragons offered daily sun exposure, was 130 ng/ml (324 nmol/l) (Gillespie, et al, 2000). During the 2.5-year study period, KD1 had over 60 more days with exposure to sunlight than KD2. Interestingly, the limited data subjectively suggest that KD2 was able to devel op higher plasma levels of 25-OH-D and appeared more able to maintain adequate levels than KD1. This may be explained by the fact that numerous factors affect vitamin D3 photobio genesis. For example, KD1 and KD2 were not always given access to sunlight during the same time of year. Ultraviolet light quality and intensity, as well as skin temperature, are dynamic variables that all influence the rate at which vitamin D 3 is synthesized (Gehrmann, 1994, Tian, et al, 1994). It is also possible that there were differences in the amount of time spent basking between these two Komodo dragons, but this factor was not quantified. Lastly, dietary factors may have contributed to the perceived differences in the ability to maintain appropriate plasma 25-OH-D levels. The larger, male dragon (average body weight during case study kg) was offered and consumed more food than the female (average body weight during case study kg) while receiving the same amount of vitamin D3 supplementation, diluting the concentrations of calcium, phosphorus, and vita min D3 from the supplement in the diet of KD1 compared to the diet of KD2. Therefore KD2 consistently consumed a diet with a higher concentration of vitamin D3. Differences in basking behavior and diet may also explain why KD2 was able to reach higher plasma 25-OH-D levels than the Komodo dragons sampled by Gillespie, et al, The loss of KD2 was unexpected, however the premature death of female Komodo dragons is a phenomenon that has been experienced by several other zoological parks in North America (Gillespie, 2001). Vascular amyloidosis appears to have been involved in at least one other case. Amyloidosis has been rarely reported in reptiles and current efforts focus on identification and classification of the amyloid protein(s) Volume 12, No. 2,2002

6 involved so that the pathogenesis can be elucidated. Whether a lack of year-round, quality ultraviolet light or other environmental stressors play a role in these mortalities remains to be seen. Today, plasma 25-OH-D levels are opportunistically monitored in KD1. A perm anent outdoor exhibit has been constructed to maximize the time this Komodo dragon may reap the full benefits of natural ultraviolet light. ACKNOWLEDGMENTS We thank Dr. Peter Graham, Susan Lombardini, and the Animal Health Diagnostic Laboratory at Michigan State University for performing the 25-OH-D analyses, Dr. Don Gillespie for his guidance with this study, Bill McMahan, Gary Johnson, and the rest of the herpetological staff at the Louisville Zoo for their assistance and support, and Virginia Crossett and Kalman Gyimesi for their technical assistance. REFERENCES Allen ME, Bush M, Oftedal OT, Rosscoe R, Walsh T, Holick MF Update on vitamin D and ultraviolet light in basking lizards. Proc AAZV, Allen ME, Oftedal OT, Horst RL Remarkable differences in the response to dietary vitamin D among species of reptiles and primates: Is ultraviolet B light essential? In Holick MF, Jung EG (eds): Biologic Effects of Light Walter de Gruyter, Berlin, Germany: Auffenberg W The Behavioral Ecology of the Komodo Monitor. University Presses of Florida, Gainesville, FL. Bernard JB Vitamin D and ultraviolet radiation: Meeting lighting needs for captive animals. Nutrition Advisory Group Handbook, Fact Sheet 002. Gehrmann WH Light requirements of captive amphibians and reptiles. In Murphy JB, Adler K, Collins JT (eds): Captive Management and Conservation of Amphibians and Reptiles. Society for the Study of Amphibians and Reptiles, Ithaca, NY: Gehrmann WH Reptile lighting: A current perspective. The Vivarium, 8(2):44-45, 62. Genuth SM Endocrine regulation of calcium and phosphate metabolism. In Berne RM, Levy MN (eds): Physiology. Mosby, Inc, St. Louis, MO: Gillespie D, DVM. Personal communication Nashville Zoo at Grassmere, 3777 Nolensville Road, Nashville, TN 37211, USA. Gillespie D, Frye FL, Stockham SL, Fredeking T Blood values in wild and captive Komodo dragons (Varanus komodoensis). Zoo Biol, 19: Holick MF The use and interpretation of assays for vitamin D and its metabolites. J Nutr, 120: Holick MF Calcium and vitamin D. Diagnostics and therapeutics. Clin Lab Med, 20: Marcus LC Veterinary Biology and Medicine of Captive Amphibians and Reptiles. Lea & Febiger, Philadelphia, PA. Nijboer J, BSc. Personal communication Rotterdam Zoo, Veterinary Department, Van Aerssenlaan 49, 3039 KE Rotterdam, The Netherlands. Tian XQ, Chen TC, Allen M, Holick MF Photosynthesis of previtamin D3 and its isomerization to vitamin D3 in the savanna monitor lizard. In Norman AW, Bouillon R, Thomasset M (eds): Vitamin D, a Pluripotent Steroid Hormone: Structural Studies, Molecular Endocrinology and Clinical Applications. Walter de Gruyter, Berlin, Germany: Ullrey DE, Bernard JB Vitamin D: Metabolism, sources, unique problems in zoo animals, meeting needs. In Fowler ME, Miller RE (eds): Zoo & W An Med, Cur Ther 4. WB Saunders Co, Philadelphia, PA: Walsh T. Personal communication National Zoological Park, 3001 Connecticut Avenue, NW, Washington, DC 20008, USA. Walsh T, Visser G Taxon Management Account, Komodo dragon, Varanus komodoensis. Webb AR, Kline L, Holick MF Influence of season and latitude on the cutaneous synthesis of vitamin D3: Exposure to winter sunlight in Boston and Edmonton will not promote vitamin D3 synthesis in human skin. J Clin Endocrinol Metab, 67(2): Volume 12, No. 2,2002 Journal of Herpetological Medicine and Surgery 9