Tissue levels of ascorbic acid in marine mammals

0300.9629’80,‘0801-06OSSO2.OWO

TISSUE

LEVELS OF ASCORBIC MARINE MAMMALS

ACID IN

D. J. ST. AWN and J. R. GERACI Wildlife Diseases Section, Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph. Ontario NlG 2W1, Canada (Received 13 Noremher 1979) Abstract-1.

Ascorbic acid concentrations were determined in the tissues of six cetacean and four phocid seal species. 2. Adrenal glands typically contained the highest levels: concentrations in muscle tissues and blubber were low. 3. Cetacean epidermis was a particularly rich depot of the vitamin, which may be linked to the comparatively high metabolic activity of this organ. 4. Estimates of total body pool of ascorbate were made from mean tissue levels and organ weights. 5. A 150 kg cetacean and a 70 kg phocid seal have at least 12.42 g and 2.89 g of ascorbate, respectively.

INTRODUCTION

Ascorbic acid (Vitamin C) is an essential nutrient for all organisms, and has been a focus of investigation since the early realization that dietary deficiency of the vitamin produces disease in a wide variety of animal species (Chick, 1953). Most animals are capable of synthesizing ascorbic acid, but the list of those which require it in the diet is expanding (Chatterjee et al.. 1975; Birney rr al., 1976). Studies on ascorbate lebels in tissues provide both essential nutritional data for assessing potential dietary sources (Rodahl, 1949; Geraci & Smith, 1979), and insights into the physiological role of the vitamin (Yavorsky, 1934; Kirk, 1962; Hornig, 1975). Information on vitamin requirements for marine mammals is limited to experimental work on thiamin, sodium, and tocopherol in phocid seals (Geraci, 1972a,b; Engelhardt & Geraci, 1978), as well as empirlcal data gained from experience with captive exhibit animals. Ascorbate requirements, if any, have not yet been determined. As a prelude to studies on ascorbic acid metabolism in marine mammals, we determined tissue concentrations of the vitamin in six cetacean and four phocid seal species in order to quantify total body reserves and identify specific tissue stores. The data also served as a baseline for subsequent studies on uptake, excretion and requirements in these animals. MATERIALS

AND

METHODS

Tissue collection

Twelve (7M, 5F) 2-3 week old harp seals, Phoca groenin the Gulf of St. Lawrence, and transported by helicopter to outdoor holding facilities on the Iles de la Madeleine, Quebec. The seals were held without feeding for up to 12 days. Nine of the pups were killed during this period, as part of an ancillary study. Adrenals and liver samples were collected immediately after death, held on ice at ambient outdoor temperatures (-5 to - 1O’C) for 44 hr, and then frozen at -25’C. Three pups were transported by commercial carrier to the University landica, were captured

of Guelph. They were kept in indoor pens with access to a spray hose, and fed small amounts of herring, CIupea harengus. After 10 days the seals were killed, and samples of all major tissues were frozen immediately. The tissues were held at -25°C for 4 months prior to analysis. Four adult (3F, 1M) and three pup (sex undetermined) harp seals were killed on the ice near the Iles de la Madeleine, Quebec. Samples of liver, adrenal, gonad and thyroid were collected within 15-30 min after death and held on ice for 4-8 hr before freezing at -25°C. The tissues were assayed after 1 month storage at -25°C. Tissues were collected from three strandling harbor seals, P. &dim, which died at the New England Aquarium (NEA), Boston, Massachusetts. Two of the seals were in captivity for 1 and 4 days and did not eat; the third seal was held for 11 days and was fed herring, C. harengus, and capelin, Mallorus oillosus. Samples were frozen at -25°C within l-3 hr after death, and assayed within 4-14 days. Seven adult (4F. 3M) grey seals, Halichoerus grypus, were killed by gunshot in the Gulf of St. Lawrence near Nova Scotia. Tissue samples were collected within 6@90 min after death, held on ice for 4-8 hr, then frozen at -25°C for 3 months. A subadult male bearded seal, Erignathus barbafus, was shot near Cape Parry, N.W.T. Tissue samples were frozen within 2650 min after death, and stored for 3 months at -25°C. Seven (IF, 6M) beluga whales, Delphinapterus leucas, in Mackenzie Bay, N.W.T., and four (lF, 3M) harbor porpoises, Phocoena phocoenn, in the Bay of Fundy were shot, and tissues were frozen in liquid N, within 9&120min, and 1@20min, respectively. Samples were stored at -25°C for l-4 months prior to analysis. A juvenile female pilot whale, Globicephda meluena, and a juvenile male sperm whale, Physeter catodon, stranded along the northeast coast of the United States in January, 1977 and March, 1976, respectively. The pilot whale was held in captivity at NEA for 1.5 days, and died soon after eating; the sperm whale was a pre-weaned calf which died after 2 days in captivity at NEA. Tissues were collected within 15-60 min after death, frozen at -25”C, and stored for 1-2 months. A pigmy sperm whale, Kogia breuiceps, died within 1 hr after stranding near Jacksonville, FL, and samples were collected 2 hr after death, held on ice for 12 hr, then frozen at -25°C for 1 month prior to analysis. Tissues were obtained from three bottlenosed dolphins, Tursiops truncatus, held at research and exhibit institutions m Ontario, Canada and California, USA. Two dolphins

605

606

D. 1. ST. AUBIN and J. R. GERACI died of stress following capture and transportation. Samples were collected and frozen within 2WOmin of death. The third dolphin drowned accidentally. and tissues were obtained and frozen 3 hr after death. All samples were stored at -25°C for up to 1 month. Tissue homogenization

and analysis

Portions of tissues ranging from 0.5 to 2.Og were weighed on an analytical balance and homogenized in acid (37; HP03 in 0.3 NHISOI and 87; H,CCOOH) using either a hand operated Potter-Elvehjem type homogenizer for 5-lOmin, or a Polytron blender (Brinkmann Instruments, Rexdale, Ontario) at about 15,OOOrpm for 15-20 sec. The type of homogenizer did not affect ascorbic acid recovery, as determined in a test using six replicate liver samples from the same animal. The homogenates were centrifuged for 10 min at 15109 and 22’C, and the supernatants were assayed for ascorbic acid using a modification of the Roe and Keuther (1943) method. A 1 ml aliquot of supernatant was added to 4 ml of a I”,, suspension of acid washed charcoal (Fisher Scientific Co., Fairlawn, New Jersey) in 5”” CI,CCOOH. The tubes were mixed and then centrifuged at 1510 9 for 5 min at 22 C. One ml of 2,4-dinitrophenylhydrazine reagent (2”,, DNPH and 0.25:; thiourea in 9 N H,SO,) and 3 ml of supernatant were incubated for 3 hr at 37-C. The tubes were placed in an ice bath while 5 ml of HzS04 (9 vol concentrated HzSO, and 1 vol H,O) was added. After 3&40 min at room temperature, the solutions were read at 540 nm in 1 cm cuvettes using a Bausch and Lomb Spectronic 70 (Bausch and Lomb Inc., Rochester, New York). Ascorbate concentrations were determined from a standard curve. Estimates of tissue stores and total body ascorbate were made from organ weight data determined in 13 ringed seals, Phocu hispida (Geraci, unpublished), and 41 Atlantic white-sided dolphins, Layenorhynchus ~cutus (Geraci et ul. 1978). Relative organ weights for seals were initially calculated as a percent of lean body weight, which represented 66.2% of total weight. Estimates of relative organ weights in seals using total body weight were highly variable, due to the dynamic nature of the blubber layer. Data from pilot whales. G. melaena, and belugas. D. leucus, were also used to estimate cetacean skin and blubber weights (Vladykov. 1944; Sergeant. 1962).

RESULTS AND DISCUSSION

The distribution pattern of ascorbic acid in marine mammal tissues is similar to that reported for humans, guinea-pigs and rats (Yavorsky et ul., 1934; Damron et a[., 1952) with some important differences (Tables 1 and 2). Adrenal glands typically contained the highest concentrations, up to 158 mg/lOOg; muscle tissues and blubber had the lowest levels. Of particular interest were the high concentrations in cetacean epidermis. The antiscorbutic properties of “muktuk” (epidermis) have long been recognized by the Inuit, and Rodahl (1949) reported similar but slightly lower values in the epidermis of a narwhal, Monodon monoceros. related to analytical

The discrepancy seems to be technique, since concentrations

reported for up to 15 tissues in four seal species are correspondingly lower in Rodahl’s study. Tissue ascorbate levels may be influenced by several factors, including age (Kirk, 1962), sex (Stubbs & McKernan, 1967) and diet (Penney & Zilva, 1946). Its role in physical stress is equivocal (Ryer et al., 1954; Hughes et al., 1971). The difficulty in acquiring marine mammal specimens for analysis has made it impossible to control such variables, and the vari-

Ascorbic acid in marine mammals Table 2. Tissue concentrations

607

of ascorbic acid (mg/lC@g) in four species of pinnipeds

P. groenlandica

P. vitulina

H. grypus E. barbatus

II

Adrenal 18 Liver 19 Spleen 3 Kidney 3 Lung 3 Brain 2 Muscle 3 Skin 3 B,ubber 3 Pancreas 3 Heart 3 Thyroid 10 Lymph node 3 Stomach 3 Tongue 3 Draphragm 3 Intestine 2 Ovary 4 Testis 3

x + SD

121.3 k 22.0 32.0 i 9.7 44.5 14.2 22.1 24.0 5.8 4.8 2.7 32.5 5.8 30.7 f 9.7 45.5 22.5 8.3 5.5 35.0 37.0 78.8

(range) (80.5-157.5) (lS.Cr51.8) (42.0-46.5) (10.5-18.0) (16.4-26.0) (21.0-27.0) (5.0-7.5) ([email protected]) (1.8-3.8) (28.0-35.0) (5.5-6.5) (13.8-45.0) (25.0-79.5) (20.G24.0) (4.8-13.5) (3.5-7.5) (27.0-43.0) (22.G70.3) (26.5-142.0)

n

x

(range)

3 3 2 3 3 3 2 2 3 2 --~ --

54.7 27.0 17.6 9.8 7.3 -2.9 4.0 2.8 11.8 2.8 ~ .~ -

(210-119.0) (15641.5) (8.1-27.0) (6.8-11.5) (3.5-10.5)

ation in reported values may reflect some of these factors. In humans and rats, ascorbate concentrations in selected tissues decrease with age (Yavorsky, 1934; Schaus, 1957; Patnaik & Kanungo, 1966; Adlard et it/., 1973). Our only basis for assessing this trend in marine mammals comes from samples of adrenal, liver and thyroid from four adult and three pup harp seals killed on the floe ice (Table 3). The small number of samples does not permit statistical analysis. but the tissue levels in pups were consistently greater than those in adults. Most of the animals in this study were young or subadult, and therefore the reported tissue concentrations may represent the upper range for these species. The influence of sex on tissue ascorbate could only be assessed for the grey seals. Ascorbic acjd concentration in adrenals from three male seals (X = 96 mg/ lOOg, range 92-100) was somewhat greater than that in four female seals (X = 73 mg/lOOg, range 20-96); there were no apparent differences in the other 14 tissues analyzed. Male rats have significantly higher ascorbate concentrations in plasma and visceral organs, but not in adrenals or bone (Stubbs & McKernan, 1967). A larger sample of seals would be required to elucidate similar subtle differences. Dietary ascorbate intake significantly influences tissue levels in species such as the guinea pig, which is incapable of synthesizing the vitamin (Penney & Zilva, 1946). In the present study, the animals that were shot in the wild had presumably been recently feeding, while the stranded harbor seals, pilot whale and sperm whale were debilitated, suggesting an extended period of fasting or reduced food consumption. In this context, it is noteworthy that tissue levels in the strandlings did not differ appreciably from those in shot animals. These findings suggest one or more of the following: these animals synthesized the vitamin; ascorbic acid has an unusually long half-life in marine mammal tissues; or stress and disease do not accelerate the catabolism of the vitamin.

(1.745) (3.0-5.1) (2.0-3.5) (8.0-13.8) (1.7-3.9) --

(n=l)

n

108.0 30.0 6.0

x&SD

(range)

7 82.6 k 28.4 7 36.4 + 7.6 7 38.1k7.2 7 9.8 + 5.0 7 7.0 f 3.0 6 11.8 k 1.7 7 1.2 + 0.5 7 2.3k1.2 6 12.8 _+ 1.7 7 3.3 + 1.5 7 25.8k6.1 6 8.0 & 2.8 7 2.4 &- 1.2 7 1.7 * 1.1 7 15.5 + 2.7 3 24.3 3 54.7

3.0 4.6 3.8 19.0 24.0

-

(20.&100.0) (27.0-48.0) (29.0-50.0) (4.C18.5) (3.5-l 1.5) (10.0-15.0) (0.5-2.0) (0.4G.O) (10.5-15.0) (1.5-6.0) (19.0-37.0) (5.0-12.6) (1.0-4.4) (1.0-4.0) (13.5-19.0) (20.0-31 .O) (46.5-62.0)

Fasted harp seal pups by contrast, did show some reduction in liver ascorbate. Linear regression analysis of liver concentration over the fasting period indicated a half-life of 11-12 days. The stress of captivity, however, may have influenced the decline in levels of ascorbate (Hughes et al., 1971). This trend was apparent in three harp seal pups transported to Guelph and held for a total of 19 days. These pups had lower adrenal ascorbate concentrations (U-106 mg/lOO g) than all other harp seal pups (115158mg/lOOg), there was no apparent relationship with fasting. Lowest adrenal ascorbate levels were observed in two strandling harbor seals (21 and 24 mg/lOO g), one of which was held and fed in captivity for 11 days, and a postpartum grey seal (20 mg/lOO g) shot on the ice. By contrast, a third emaciated harbor seal had an adrenal ascorbate concentration of 119 mg/lOO g, and three other post partum gray seals had levels of 79.5-96 mg/lOO g. While it appears that stress may influence adrenal ascorbate in seals, the exact relationship cannot be described at this time. The tissue concentration data were combined with organ weight ratios determined in ringed seals, P. hispida, and Atlantic white-sided dolphins, L. acutus, in order to estimate total body ascorbate and identify tissue stores of the vitamin (Table 4). A hypothetical seal weighing 70 kg, and a cetacean weighing 150 kg would have total body ascorbate levels of 2.89g (41.3 mg/kg) and 12.42 g (82.8 mg/kg), respectively. These calculations represent minimum values, since Table 3. Tissue concentrations of ascorbic acid (mg/lOO g) in four adult and three pup harp seals Adult

Pup

Tissue

R

Range

R

Range

Adrenal Liver Thyroid

108 25 27

8&120 21-28 1437

142 34 32

133-152 33-35 24-44

608

D. J. ST. AURIN and J. R. GERAC~ Table 4. Total organ

ascorbic

acid contribution

in a hypothetical

70 kg phocid

Cetacean

Phocid

Ascorbate* (mg/lOO g)

Organ Blubber Muscle Skin GI tractt Liver Lung Brain Heart Kidneys Spleen Pancreas Adrenals Thyroids Miscellaneousf

2.4 2.7 4.5 16.3 32.5 10.7 14.8 3.8 10.8 36.3 17.5 105.0 29.7

seal and a 150 kg cetacean

Relative weight (?A) 33.8 29.1 9.4 4.9 2.3 1.7 0.7 0.6 0.5 0.3 0.2 0.01 0.01 15.9

Total ascorbate (mg) 570 560 297 554 520 128 76 16 40 102 24 2 1

Ascorbate* (mg/lOO g) 3.7 3.9 44.4 16.4 22.0 12.5 17.4 5.7 7.8 34.3 19.6 117.1 20.5

Relative weight (“,A) 21.0 40.0 6.0 12.0 2.4 3.0 1.2 0.8 0.8 0.04 0.2 0.01 0.03 12.5

Total ascorbate (mg) 1166 2340 3996 2952 792 562 313 68 94 21 78 23 10

* Mean concentration for all species. t Ascorbate concentration date combined for stomach and intestine, when available. $ Includes bone, tissue fluids, and miscellaneous connective and lymphoid tissue.

only 8487.5”; of total body weight was accounted for in the models. Nevertheless, several important features emerge from these data: in both seals and cetaceans, vitamin stores were greatest in blubber, muscle and the gastrointestinal tract; the liver ascorbate pool represented IS”,: of total body ascorbate in seals, but only 61; in cetaceans. The most striking feature of the study was the affirmation that cetacean skin is a uniquely rich depot of ascorbic acid. This finding is consistent with other lines of evidence which demonstrate considerable metabolic activity in this tissue (Tinyakov et al., 1973; Dargoltz et al., 1978; Geraci & St. Aubin, 1979). The antioxidant properties of ascorbic acid therefore may serve as a vital component in protecting the metabolically sensitive environment

of this tissue.

Acknowledgemenrs--Several individuals and institutions assisted in the collection of tissue samples for this study, We thank Dr D. E. Gaskin. G. Smith, T. Austin and A. Gilman (University of Guelph), J. Pearson and C. Skinder (New England Aquarium), Dr J. Sweeney and T. Otten (Marineland of the Pacific) and S. Dudka, B. Beck, W. Hoek and Dr D. Sergeant (Fisheries and Marine Service). Mrs A. deFreitas performed the laboratory analyses. This study was funded by NSERC grant A6130 to J.R.G.

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