Evaluation of plasma atrial natriuretic peptide concentration in healthy bottlenose dolphins (Tursiops truncates)

Abstract

There are currently no standard methods for diagnosing cardiac diseases in dolphins. These diseases may consequently be overlooked and go undiagnosed. The presence and severity of cardiac diseases in humans can be determined using blood tests. Atrial natriuretic peptide (ANP) used in human cardiac examinations has low species specificity. There have already been reports of homology between dolphin and human ANP; however, its potential for clinical application in dolphins has not been tested. This study was conducted to establish a reference for ANP levels in healthy bottlenose dolphins. Healthy bottlenose dolphins (seven females; estimated to be 7–30 years of age) at an aquarium in Japan were sampled. Each animal was tested for ANP at least three times, and the mean value and standard deviation were calculated to be 43.4 ± 19.2 pg/mL. In humans, patients with high plasma ANP levels have a poor prognosis. In veterinary medicine, cutoff values for the diagnosis of mitral regurgitation and heart failure in dogs have been established and used to predict prognosis. The results of the present study may contribute to the health management of bottlenose dolphins, particularly in the early detection and treatment of cardiac diseases.

There are few reports of cardiac diseases in dolphins. Previous reports of cardiac diseases in cetaceans have mainly described acute myocardial infarction and myocardial necrosis in stranded dolphins [2, 37]. In these studies, myocarditis, myocardial degeneration, and necrosis were assessed histologically.

Nine striped dolphins (Stenella coeruleoalba) and one bottlenose dolphin (Tursiops truncatus) were stranded. One dolphin had a dilated right ventricle, thickened tricuspid leaflets, and consequent left ventricular dilation. Mitral valve changes were observed in three Stenella coeruleoalba, with mitral fibrosis, mitral valve leaflet thickening, and left ventricular hypertrophy [32]. Although a number of reports have been published on wild dolphins, few clinical cardiac examinations have been conducted on dolphins in captivity in aquaria, indicating that the presence of cardiac diseases in these animals may have been overlooked. There are currently no standard clinical methods for cardiac testing or for diagnosing cardiac diseases in dolphins.

In humans, the presence and severity of cardiac diseases can be determined using blood tests. One of the most important blood tests is that for atrial natriuretic peptide (ANP). ANP is released from a granule in atrial myocytes [13, 17]. In 1979, de Bold found that the number of special atrial granules fluctuated significantly with changes in body fluid volume and salt intake [3]. In 1981, de Bold et al. confirmed that intravenous administration of atrial muscle extract to rats markedly increased urine volume and sodium (Na) excretion and had an antihypertensive effect [4]. In 1984, Kangawa et al. isolated a peptide consisting of 28 amino acids from human atrial cell extracts and named it atrial natriuretic peptide [14]. ANP is a peptide hormone released into the blood by atrial myocytes stimulated by myocardial stretching; this occurs when the blood volume in the atrium increases excessively [19]. ANP acts as a vasorelaxant and reduces blood pressure by decreasing the peripheral vascular resistance and vascular volume. The main functions of ANP are vasodilation, inhibition of renal Na⁺ reabsorption, and suppression of aldosterone secretion from the adrenal glands. During heart failure, the renin-aldosterone, sympathetic nervous, and other systems are activated, increasing vasoconstriction and Na⁺ storage, and maintaining blood pressure. However, this response leads to increased cardiac preload and afterload, and eventually, the deterioration of cardiac function. ANP is secreted to alleviate this pathological reaction [22, 24, 28]. The progression of heart failure increases ANP secretion.

Therefore, ANP levels reflect the severity of the cardiac load and are used to predict the severity of cardiac diseases and determine prognosis. ANP levels are also used to measure risk factors for cardiac diseases, including cardiac capacitance load and myocardial damage [6, 22, 40]. In veterinary medicine, elevated ANP levels have been reported in dogs with mitral regurgitation [1, 6, 8, 9, 35]. Hori reported that the severity of cardiac diseases in dogs may be predicted using ANP. Comparing with higher and lower ANP concentrations in dogs, specifically, heart rates, cardiothoracic ratios, vertebral heart scores, fractional shortening, left atrial diameter/aortic diameter ratios (LA/AO ratios) and mitral early diastolic flow velocity were significantly higher, whereas their relative left ventricular free wall thickness was significantly reduced in higher ANP dogs [12]. There is a strong positive correlation between the ANP concentration and mean pulmonary artery wedge pressure during experimental acute cardiac volume loading in dogs [1]. Therefore, ANP is considered a useful noninvasive parameter for the monitoring of acute hemodynamic changes in the circulatory system.

For these reasons, ANP concentration measurements are generally used as a marker of cardiac diseases severity in canines and felines [11, 26, 27].

The amino acid sequence of ANP is conserved across species. In mature ANP, which consists of 28 amino acids, human and rat ANP differ only by a single amino acid at position 12. Human peptide contains methionine, and rat peptide contains isoleucine [28]. Mature ANP-28 has been detected only in the plasma, which means that pro-ANP stored in the atria is cleaved to mature ANP when secreted into the blood. Mature circulating ANP is the same in humans, chimpanzees, dogs, pigs, horses, and sheep. In addition, it is similar in rats, mice, and rabbits. Thus, ANP has a relatively low specificity across animal species [28].

In cetaceans, high mature ANP amino acid homology exists among Dall’s porpoises, bottlenose dolphins, Pacific white-sided dolphins, and fin whales, as well as among camels, pigs, dogs, rats and humans [25]. ANP in dogs, cats, and cattle is reportedly not species-dependent. This confirms that human ANP assay kits are highly cross-reactive with canine ANP and can be used in various clinical analyses.

All 28 peptide sequences of bottlenose dolphin and human mature ANP are the same. Pacific white-sided dolphin and human mature ANP peptide sequences differ by only one of the 28 amino acids.

Although 19 of the 26 bottlenose dolphins and human mature brain natriuretic peptide (BNP) sequences are identical, they are less homologous than ANP. Therefore, dolphin BNP levels cannot be measured using human BNP kits [25].

We focused on the high homology of dolphin mature ANP with that of other mammals to determine the mean plasma ANP values in healthy bottlenose dolphins.

One previous report has been published on ANP measurements in dolphins. Five healthy bottlenose dolphins were tested while floating, and 10 min after removing the water from the pool as the landing condition. The results showed that ANP was higher when the animals were landed than when they were floating. However, the number of measurements was insufficient to detect any significant differences. To our knowledge, this is the only report of the measurement of ANP levels in dolphins [25]. However, this measurement method is difficult to perform in general clinical practice. The measurements were performed at an external laboratory. The current study is the first clinical study of ANP in bottlenose dolphins.

MATERIALS AND METHODS

Animal participants

The seven healthy bottlenose dolphins used in this study (Nos. 1–7, all female: with estimated ages 7–30 years) were bred in the same environment at the Enoshima Aquarium (Fujisawa, Kanagawa, Japan).

All dolphins were held in open-air pools of sufficient space with each 5 m-deep open-air pool containing 1,350 m³ of natural seawater and a semi-closed life support system [36]. The participants underwent daily health checkups during husbandry training. Body weight, estimated age, complete blood count (CBC), blood biochemistry, and abdominal echocardiography were assessed. During blood sample collection, the dolphins would voluntarily put their caudal fins out of the water. Blood samples were collected by inserting a winged needle into a flake-like vein of the caudal fin.

The present situation regarding the dolphin breeding environment

In Japan, dolphins in aquaria are protected as breeding animals under the law on The Protection and Management of Animals and Standards for the Care and Maintenance of Prohibited Animals. However, because each animal facility is regarded as being maintained and managed under its own rules, there is no requirement to report its management status, and no standards have been determined for specific breeding environments [39].

Methods

Blood was collected in a relaxed state during morning health checks, every 3 or 4 weeks. ANP deactivates quickly and had to be frozen promptly. Three blood samples were collected from each dolphin; however, for numbers 2 and 3, the opportunity arose to take five samples; these were collected and added to the data.

Blood samples were collected in EDTA2Na tubes (containing aprotinin) and immediately centrifuged (3,000 rpm, 5 min). Plasma was retrieved and immediately frozen at −20°C until analysis. Because ANP is unstable at room temperature, the process from blood sampling to freezing was conducted as quickly as possible. A chemical luminescence enzyme immunoassay (CLEIA) was used to analyze the samples (Detaminer CL ANP, MINARIS Medical Co., Ltd., Tokyo, Japan).

Statistical analysis

We checked whether the null hypothesis held for the ANP levels of the seven dolphins (Smirnolov–Grubbs test). Analyses of the correlations between ANP values and age and between ANP values and body weight were conducted.

RESULTS

Based on the blood test results and abdominal echocardiography, the veterinary staff confirmed that all dolphins had a low likelihood of having ongoing cardiac or kidney diseases that could affect ANP concentrations. Table 1 presents the results of the study. The results of the CBC and blood biochemistry tests have been included in Tables 2 and 3. The results of the blood tests (n=7) performed at the same time as the ANP measurements showed that none of the participants had cardiac or other diseases affecting ANP (Tables 2 and 3). The mean ANP level was calculated for each individual. Statistical analysis confirmed the null hypothesis. In other words, the data were confirmed to be normally distributed. Therefore, the mean ANP value was 43.4 ± 19.2 pg/mL (mean ± 2 SD) (Table 1). Correlations between ANP and age and between ANP and body weight were determined using scatter plots. The Pearson’s correlation coefficient between ANP and age was r=0.805, P=0.029, indicating a significantly strong correlation. The Pearson’s correlation coefficient between ANP and weight was r=0.631, P=0.129, which is a moderate correlation compared with the correlation between ANP and age.

Table 1. Average values and standard deviations of atrial natriuretic peptide in seven bottlenose dolphins.

No.	Estimated age	Sex	Body weight (kg)	No. of blood samples	Mean ANP (pg/mL)
1	30	Female	238.5	3	61.0
2	28	Female	259.5	5	50.7
3	21	Female	196.0	5	47.6
4	19	Female	237.0	3	36.0
5	9	Female	165.5	3	30.2
6	6	Female	188.0	3	37.8
7	6	Female	215.0	3	40.6
Mean	17	−	−	−	43.4 ± 19.2

*Mean ± 2SD.

Table 2. Complete blood count (n=7).

	Unit	Mean value	Average	± SD
WBC	/μL	5,000–9,000	7,351.4	1,015
RBC	106/mm3	3.00–3.74	3.80	0.20
Hb	g/dL	13.5–15.5	17.3	1.0
Ht	%	38–44	50	3
PLT	103/mm3	80–150	111	29
MCV	fL	115–135	131	3
MCH	pg	38–48	46	1
MCHC	%	34–36	35	1
Stab	%	0	2	0
Seg	%	62–68	60	11
Eosino	%	11–13	17	8
Baso	%	0	0	0
Mono	%	2–4	3	1
Lympho	%	18–21	18	7
FIB	mg/dL	170–280	205	28

Table 3. Biochemical blood test (n=7).

	Unit	Mean value	Average	± SD
TP	g/dL	6.0–7.8	7.8	0.7
A/G Ratio		2.12–3.3	1.4	0.2
CPK	U/L	100–250	102	20
AST (GOT)	U/L	190–300	191	56
ALT (GPT)	U/L	28–60	47	18
γ-GPT	U/L	30–50	36	22
CRE	mg/dL	1.0–2.0	1.3	0.1
BUN	mg/dL	42–58	53	7
Glu	mg/dL	90–170	89	7
T-Cho	mg/dL	150–260	204	50
Na	mEq/L	153–158	154	1
K	mEq/L	3.2–4.2	3.2	0.6
Cl	mEq/L	113–125	119	1
Ca	mg/dL	8.5–10.0	8.8	0.5
IP	mg/dL	4.0–6.0	5.6	0.7
Fe	μg/dL	120–340	230	40

DISCUSSION

The mean ANP level in healthy humans ranges from 34–38 pg/mL, with a normal mean value of 43 pg/mL or less [10, 34, 38]. In veterinary medicine, the mean ANP value in healthy dogs has been reported to be 16.4 ± 7.8 pg/mL [12]. The mean ANP levels in this study were slightly higher than those in terrestrial animals such as humans and dogs. The variation in ANP between individuals was likely to be high, especially considering that dolphin No. 1 was an extremely old animal (30 years old) (Table 1). In humans, there are reports of age-related changes as cardiac fibrosis increases from mature to old age and ANP and BNP levels increases [29]. Dolphins are always exposed to water pressure; therefore, their peripheral blood vessels are constantly under pressure. Blood is consequently more easily pooled in the right heart, causing right ventricular dilation [16]. It is also necessary to increase the right ventricular capacity to maintain sufficient cardiac output while simultaneously withstanding water pressure (Frank–Starling law). In this situation, not only the right ventricle but also the right atrial muscles are stretched. Thus, ANP is released owing to the capacitive load on the right atrium caused by the increased return flow. These characteristics may be associated with the slightly higher ANP levels in bottlenose dolphins than in terrestrial animals. Cardiac hormone levels in marine mammals may be affected by water pressure.

Studies have reported elevated cardiac hormone levels in recreational divers. Right ventricular dilation has been reported in recreational divers. Right ventricular system dilation in these divers causes right axis deviation on electrocardiography [23]. Moreover, as described above, the effects of water pressure due to diving can cause a right ventricular capacitive load [5, 20, 21, 30, 33].

Kurabayashi showed that the correlation between water pressure and ANP is also present in mammals, that is humans, in a water-immersion environment. In one study, 12 healthy male volunteers aged 22–35 years were immersed in water up to the jaw while sitting at the bottom of a bathtub. Blood samples were collected before sitting, and 15 and 30 min after the start of immersion. The ANP levels increased significantly after 10 min of immersion. This suggests that the right atrial load increases due to increased venous return [18]. This reaction has been applied in human medicine, and warm-water immersion treatment is used for patients with cardiac and pulmonary diseases. During this treatment, cardiac output, ejection fraction, and heart rate increase. In contrast, the left ventricular volume and pulmonary artery wedge pressure decrease, and the venous return and right atrial volume load increase. This immersion treatment also increases ANP levels [18]. Based on these findings, the high ANP levels in bottlenose dolphins compared with those in other mammals may be due to physiological reasons for the high right atrial volume load caused by high venous return.

In human cardiac diseases, ANP measurement is valuable in predicting the severity and prognosis of cardiac diseases because patients with high ANP values have a poor prognosis [7, 31, 41]. In dogs with cardiac diseases, ANP concentrations can exceed 100 pg/mL [12]. These values can be used to determine the severity of mitral regurgitation and right-sided congestive heart failure (RCHF) in dogs [12, 15]. It may also be possible to measure ANP levels in bottlenose dolphins with cardiac diseases and compare them with the mean ANP levels of healthy individuals to determine which individuals are susceptible to cardiac diseases.

To date, it has been difficult to perform auscultation and echocardiography on dolphin hearts in aquaria. Echocardiography requires a long acquisition time and the most effective way to visualize dolphin hearts has not yet been determined. Therefore, these examinations are not performed routinely. Conversely, many aquaria routinely perform blood tests. In recent years, most aquaria have undertaken dolphin husbandry, and blood tests have been routinely performed with minimal stress.

By adding ANP to routine blood tests, undetected cardiac diseases could be identified. Although all samples in this study were collected from females, it is important to maintain the health of female animals to continue breeding in aquaria. These seven healthy dolphins were housed in the same environment and were trained in husbandry. Therefore, this is the first report of mean ANP values for normal dolphins in the same environment. The average ANP values of healthy dolphins determined in this study may be useful for early detection and treatment of cardiac diseases in dolphins.

POTENTIAL CONFLICTS OF INTEREST

The authors declare no conflicts of interest. The authors have no financial or proprietary interests in any of the materials discussed in this article.