Thyroid Function in Dolphins: Radioimmunoassay Measurement of Thyroid Hormones

Br. vel.j. ( 1979), 135,96

THYROID FUNCTI O N IN DO LPHI NS: RADIOIMMUN OASSAY MEASUREMENT O F THYROID H ORMO NES By

A.

G . GREENWOOD AND

C. E.

BARLOW

Hainsworth House, Damens Lane, Keighley and Radio-Isotope Unit , Airedale General Hospital, Keighley, West Yorkshire

SUMMARY

Serum samples from Atlantic Bottlenosed dolphins (Tursiops truncatus ) were examined by radioimmunoassay for circulating thyroid hormones and the results compared with human values and standard cu rves. Values for dolphin T4 and free T4 index were twice those for human controls, but T3 and THBC values were similar. The results are in agreement with earlier work using non-immune methods. Human radioimmune tests provide an indicator of thyroid function in the dolphin. INTROD U CTION

Dolphins, and other members of the suborder Odontoceti (toothed whales ), are recognized to have larger thyroid glands than terrestrial mammals (Crile & Quiring, 1940; Ridgway, 1968; Harrison, 1969). Basal metabolic rate and plasma thyroxine (T 4) levels are also increased, probably as a response to life in cold waters, although there are some discrepancies between species in the correlation of increased thyro id size, metabolic rate and circulating hormone levels (Kanwisher & Sundness, 1966; Ridgway & Patton, 1971). Plasma thyroxine (T 4 ), bound and free iodine, thyroid-binding globulin (TBG) and thyroid hormone binding capacity (THBC ), as indicated by the triiodothyronine (T 3) uptake test, have been reported by Ridgway & Patton ( 1971) and Ridgway (1972) for 50 odontocetes, including 31 Atlantic Bottlenosed dolphins (Tursiops truncatus ). These represent the only published records of thyroid function tests in dolphins, and employed measurements and methods whi ch are rapidly being replaced by direct radioimmunoassay of thyroid hormones (T 4 and T 3) Ongbar & Woeber, 1974 ; Evered , Vice & Clark, 1976). Thyroid pathology, including atrophy, adenoma, colloid depletion, colloid goitre and thyroiditis, has been reported in both stranded and captive dolphins (Cowan, 1966; Harrison, 1969; Sweeney & Ridgway, 1975; Greenwood & Taylor, 1977), but thyroid disease has not been clinically recognized or treated . Whilst many thyroid lesions may be secondary to intercurrent disease and starvation (Harrison , 1969 ), apparent primary pathology has been observed (Sweeney & Ridgway, 197 5; Greenwood & Taylor, 1977). The concern of this study, therefore, has been to

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estab lish a working range of measurements of th yro id function m dolphins under captive conditions for routine clinical diagnosis . Accurate methods which are in current use in human medicine were employed, allowing a degree of comparison with the normal human range. Further studies are in progress on thyroid pathology and thyroid function in diseased dolphins. MATERIALS AND METHODS

All the samples used in this stud y were obtained from captive Atlanti c Bottlenosed dolphins, the commonest species in European dolphinaria . The a nimals all originated from the Florida/Gulf of Mexico coas tal zone, were estimated as between seven and 20 years old and had been in captivity from four to 11 years. The 33 individuals studied were housed under varying conditions in 12 different facilities, mostly using closed water systems with artificial sea water (manufactured from purified sodium ch loride). Five a nimals were in closed natural sea water systems with regular replacement of water. All water systems were treated by sand or diatomaceous earth filters and chlorinatio n. All the animals received a similar diet of thawed frozen Atlanti c herring (Clupea harengus harengus ) and Atlantic mackerel (Scomber scombrus ), with added vitamins and minerals. Food intake was between 5·S and 9 kg daily for all animals. Maximum dai ly iodine supplementation was 0·1 mg as KI (Gevral tablets; Lederle), although in most facilities poo l areas and food utensils were cleaned with iodine-based disinfectants (e.g. Pevidine Antiseptic; Berk). This possibly provided an additional source of iodine. Pool temperatures varied from 12°C to 22°C and air temperatures from SOC to 33°C, and samples were co llected throughout the year. None of the animals were pregnant. Forty-five samples from 33 animals were used for T 4, THBC and free T4 index determinations, a nd 32 samples from 30 animals for Tg. Blood was drawn from the central vein in the tail fluke for routine health monitoring and clotted samples retained for this study. Time of collection varied through the day, so some animals were fasted overnight whereas others had been recently fed . The samples were allowed to clot at room temperature and then stored at 4 ° C. Serum was separated if storage lime was more than 24 h. T4 and Tg were determ ined in unextracted serum by radioimmunoassay methods (Chopra, 1972; Hesch & Evered, 197 3), which depend on the competition between T4 (or Tg ) in serum a nd T4 (orTg) labelled with radioactive 1m for a controlled number of bind ing sites on a specific antibody (T 4 RIA Kit and Tg RIA Kit, Radiochemical Centre, Amersham ) to the ho rmone. The proportion of labelled hormone bound to the antibody is inversely related to the concen tration of unlabell ed hormone in the serum, rad ioactivity being m eas ured after separation of the free and bound isotope by a buffered res in solution. Results were compared with a curve based on standard human refere nce sera. Interference by hormone-binding proteins in the unextracted serum was minimized by the add ition of barbitone buffer and a TBG blocking agent (th io mersa late). The anti- T4 antibody shows less than S% cross reactivity to Tg and less than O·S% to o ther tyros ine co mpounds. The anti-Tg antibody shows less than 2% cross reactivity to T4 and other compo unds. An assessment of TB G levels was made by measurement of thyroid hormone

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binding capacity (THBC) by the T3 uptake test. After inhibition of thyroid binding prea lbumen (TBPA) and thyroid binding albumen (TBA) by a phosphate/acetate buffer, serum was added to a mixture of buffered Sephadex gel (Pharmacia) and 1125 T 3. Radioactivity in the supernatant at equilibrium is proportional to the amount of 1125 T3 taken up by the serum and is inversely proportional to thyro id activity, reHecting the amo unt of T3 already bound (there is no displacement of T.). Thus THBC is emp irically proportional t~ TBG. The availability offree T. in the serum was assessed by calculation of the free T. index (Clark & Brown, 1970) from the T. and THBC measurements thus : · d _ total measured T4 (nmol/i) free T 41n exTHBC The free T. index gives an indication ofT 4 levels corrected for variations in TBG levels. A range of values from clinically normal men a nd women had already been obta ined for reference use in this laboratory, using the same analytical methods . The range for T3 was determined from 104 individuals and for the other parameters from 200 individuals (age range 16 to 65 ). Females taking oral contraceptives or who were pregnant were excl ud ed. Dolphin samples were run in mixed batches with clinical materia l from patients. For co mparison of standard curves, serum from one dolphin and pooled human serum were extracted by mixing I g charcoal with 10 ml serum and storing at 4°C for five hours. This process was repeated and the centrifuged serum was then sterilized by membrane filtration. After this double extraction, the dolphin serum contained 12 nmol/l T4 a nd the human 15 nmol/l T 4. A stock T4 solution was prepared by dissolving 57 mg sod ium thyroxine in 10 ml phosphate buffer at pH 7·4, containing I g bovine a lbumen per litre. The th yroxine was dissolved by adding 0· 1 M NaOH and made up to a I mg/ml solution with buffer. This solution was then diluted and added to the ex tracted sera to give 270 nmol/I T4 in the human serum and 268 nmol/l T4 in the dolphin serum. Five standard values were obtained by double dilution and these were assayed by radioimmunoassay, in comparison with a series of known human standard sera. The res ults were plotted as graphs (Fig. I). RESULTS

The res ults a re presented as a range of values two standard deviations either side of the mean value. These were obtained by excluding values deviating from the overall mean of a ll sa mpl es by more than two standard deviations, establishing a mean value for the remainder, and repeating the process. This was found to exclude four dolphins for T., three lo r free T. index, and one for THBC and T 3. All the values excluded were below the final range. No human controls were excluded by a similar process. The means are presented with the standard error calculated to 95% confidence limits . Dolphins For T4 the range was 158 to 261 nmol/l (mean 210 ± 9·47) in 29 animals. 14 females had a ra nge 143 to 2 75 nmol/l (mean 209 ± 17 ·29) and 15 males 17 5 to 247 nmol/I (mean 211 ± 9· 12).

99

THYROID FUNCTION IN DOLPHINS I x

23

'"Q c:

E ;V c.

20

'" C ::> 0

U

17

100

200

300

T4 nmol/l

Fig. J. Standard c urves. x - - x primary human standa rd sera 0 - - 0 derived dolphin standards +--+ derived human sta ndards.

For Tg the range was 0 ·93 to 4 · 14 nmalll (mean 2·53± 0·29) in 29 animals. 15 females had a range O· 76 to 4·10 nmal/l (mean 2·43 ± 0 ·42) and 14 males 1·5 1 to 4·04 nmal/l (mean 2· 77 ± 0·33 ). For THBC the range was 93 to 103 units (mean 98±0·87 ) for 32 animals , and for free T4 index 1·56 to 2·6 1 (mean 2·08 ± 0 ·09) for 29 animals. The sex differences were not significant for any parameter. Human

For T4 the range was 58 to 158 nmalll (mean 108 ± 3·47) in 200 individuals and for Tg 1· 26 to 3· 08 nmalll (mean 2 · 17 ± 0·09 ) in 104 individuals. For THBe the range was 93 to 104 units (mean 99±0·42) and forfreeT 4 indexO·62 to 1·56 (mean 1·09±0·03), bo th for 200 individu;':s. No significant sex difference occurred.

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The graphs of the standard curves for human standard serum, prepared human serum and prepared dolphin serum gave a good fit. The dolphin curve fitted the standard curve marginally better than the human curve.

DISC USS IO

The use of a specific radioimmune hormone assay developed for human patients in another species has two main dangers. The first is that the antibody may be specific to the hormone as it occurs in man, and not in the dolphin. In this case, however, as the antibody was developed against relatively simple non-protein hormones (T 4 and T 3) wh ich are probably the same molecules in all mammals, the test certainly may be expected to give consistent true values for dolphin T4 and T 3. Whether these hormones have the same importance in dolphin thyroid function and metabolism as in man is a separate question which is discussed later. More weight is lent to the validity of the T4 and T3 results by the failure to find any cross reaction when thyroid stimu lating hormone (TSH ) radioimmune assay was attempted in dolphin serum . TSH, as a more co mplex trophic hormone, is likely to have a species-specific structure and so this test might be expected to be ineffective in the absence of a specific antibody to dolphin TSH . This does not necessarily mean that human TSH could not exert a trophic fun ction on the dolphin thyroid, or vice versa . More important is the danger that dolphin serum might in some way cause a methodological error or distortion, so that the higher T4 levels in dolphins than man might be due to comparison with a standard curve derived in human serum , and some (actor in dolphin serum might cause the test to give persistently higher results . For this reason, a standard curve was constructed simultaneously from both extracted dolphin a nd extracted human serum, using prepared T 4, and compared to a curve constructed with human reference sera. The very accurate fit of these curves and th e fact that the do lphin curve was closer to the reference curve than the human curve, with a maximum error of about 8 nmol/l at the mid-point of the range, support the belief thal this radioimmunoassay method measures T4 accurately in the dolphin . In the absence of any distorting factors, it may be expected that the measurement ofT3 will also be accurate. The values presented here for the Atlantic Bottlenosed dolphin are in agreement with those of Ridgway & Patton ( 197 J) in demonstrating a higher level of circulating T4 co mpared with man. This would appear, at first sight, to be consistent with a higher thyroid weight and a higher metabolic rate, as an adaptation to a marine environment, where a high degree of activity and heat conservation are mandatory. However, the dolphin T3 levels found in this study are very similar to those in man, and it is now believed that T3 is the most metabolically active of the thyroid hormones, and that T 3 is a better indicator of thyroid function at the tissue level (I ngbar & Wo eber, 1974). In other words, it is T3 which maintains th e animal in a clinically euthyro id state, sometimes in the face of a hypo-active g land (Kochupillai et at. , 1973; Tunbridge, Harsoulis & Goolden, 1974). T3 in man is mainly derived by tissue convers ion of circu lating T4 (Surks et at., 1973 ), and it may be that the dolphin maintains a higher circulating pool ofT4 for conversion to T3 when requirements are high, or that T3 has a faster turnover rate and shorter half-life in these animals . Failure

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to measure T3 may account for the apparent lack of correlation between increased metabolic rate or gland weight and increased thyroid activity across the range of odontocete species (Ridgway, 1972). Any comparative study of circulating hormone levels must take into account the levels and functions of binding proteins, particularly thyroid-binding globulin (TBG), albumen (TBA) and pre-albumen (TBPA). In man, the major part of circulating T4 is bound to TBG, and it is likely, although by no means certain, that this applies to the dolphin. In pregnant women high T4 levels are found, which are referable to raised TBG. Whilst this does not indicate thyroid disease, it must indicate some physiological need for an increased level of bound T4 in the blood. Dolphin TBG could not be directly measured in this study, but Ridgway & Patton (197 I) recorded a higher level than man. An indirect measure ofTBG is given here by THBC estimation (equivalent to percent T3 uptake in previous studies) with exactly comparable results between the dolphins and man. Thus the dolphin free T4 index is found to be considerably higher, indicating that the additional T4 in the dolphin is in the unbound state and metabolically active. This is in agreement with the higher levels of directly measured free T4 found by Ridgway & Patton (I9 7 I). Measurement of the T 4 binding capacity of dolphin serum as TBG or THBC may be of limited value if the dolphin utilizes albumen or pre-albumen extensively to carry T 4, but this is unknown. It is interesting to note that in a previously reported case of combined thyroid/adrenal atrophy in a do lphin (Greenwood & Taylor, 1977) the only significant abnormality in life was a persistent hyperalbuminaemia, which was inexplicable in terms of known serum chemistry changes in disease. In retrospect, this could possibly have been a response to low T4 output by the failing thyroid gland. All of the dolphin values excluded by the method of calculation of the final normal ranges were from four individuals, and all were below normal. Of these animals, one was an adu lt male with a proven breeding history, one was an old lethargic female wh ich has since died in an accident without thyroid tissue becoming available for examination, one was an active trained female which died from bacterial septicaemia (again without examination of thyroid tissue) and the last was an adult female which had chronic lung abscesses and has since died from this condition . Examination of thyro id gland from this latter dolphin revealed a normal size, with cuboidal follicular epithelium. The follicles contained colloid of variable staining and scalloped edges, probably within normal limits. Three other animals used in this study have since died, one of which showed a fall in T4 to below 20 nmol/l before death, which was from candidiasis. The two other animals , which died from pregnancy toxaemia and apparent heavy metal poisoning, had normal thyroid tissue. It is hoped that further investigations into thyroid pathology and the role of the thyroid gland in intercurrent disease will serve to increase our knowledge of proper dolphin husbandry and to improve the survival rate of captive animals. ACK

OWLEDGEME TS

The authors are gratefu l to Prof. R. J. Harrison, FRS for reading the manuscript, and to Dr E. Tinsley for examining the thyroid gland sections.

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(Accepted for publication 27 june 1978)