ABSTRACT

Ceruloplasmin (Cp) is an important α-2-glicoprotein acute phase protein (APP). It plays an important role in protecting host tissues from toxic oxygen metabolites released from phagocytic cells during inflammatory states. Additionally, it is involved in copper transport and antioxidant defense, the latter by inhibiting copper ion-stimulated formation of reactive oxidants and by scavenging hydrogen peroxide and superoxide.¹ Ceruloplasmin is an important regulator of iron homeostasis by catalyzing the conversion of Fe (II) to Fe (III) for binding of apo-transferrin through ferroxidase.⁷

The acute phase inflammatory response is a prominent systemic reaction of the organism to local or systemic disturbances in its homeostasis caused by infection and tissue injury. Induction of acute phase response proteins is mediated by specific cytokines. Serum levels of Cp increase in animal models of inflammation, including following an injection of TNF-α, IL-1 and IL-6. These cytokines play a key role in humans and animals acute phase response.⁴

Plasma concentrations of cytokines have been studied in animals with inflammatory conditions. Until further studies are available, the high cost, limited availability, and absence of standardization argue against the measurement of plasma cytokines in clinical practice.³ Acute phase proteins are part of a non-specific inflammatory reaction of the host that occurs shortly after any tissue injury. Origin of the response can be attributed to infection, immune abnormalities, neoplasia, trauma, parasites or endocrine abnormalities, and therefore assay of the APP is an equivalent of a "molecular thermometer" of inflammation.²

Determination of serum concentration of APP can also be used as direct measure of inflammation in cetaceans. Although most APPs are measured by use of immunoassay, it is possible to determine the concentration of a few by utilizing their biological activities. The advantage of these biochemical methods is that they are not species specific. The biochemical assay of haptoglobin is most widespread.^5,8 Ceruloplasmin a copper containing moderate to minor APP can be estimated by measuring its endogenous oxidase activity, a method which is cheap, easy, and rapid.¹

The objective of the present report was to implement and validate a manual method for determination of cetacean serum Cp by the use of the p-phenylenediamine (PPD) substrate. The main task of our investigation was determination of the level of Cp in the serum of the bottlenose dolphins, Tursiops truncatus (Tt, animals, n=32, 14 males, 18 females, samples n=109); and beluga whale, Delphinapterus leucas (Dl, animals, n=28, 16 males, 12 females, samples n=66) in normal conditions and some clinical cases.

The precision of assay was tested within a laboratory. We made analyses of the double samples from 24 randomly chosen animals of each sex (Tt, n=14, Dl, n=10). The coefficient of variation was lower than 10%. The stability of the cetaceans' Cp over time during storing with repeated thawing and freezing was determined (-70°C to room temperature). Application to biological samples was also tested. Three types of samples (control, diseased, pregnant) were collected and used for the assay in the two groups (Tt and Dl):

1.  Control - Samples obtained from clinically healthy animals (healthy samples: Tt, n=63, Dl, n=48). These were clinically healthy, presenting no abnormal findings at physical examination, routine hematological and biochemical test results were unremarkable, and none of these animals was pregnant.

2.  Diseased - Samples from bottlenose dolphins and beluga whales determined to have a pathological condition in the first day of illness. All samples were collected before any treatment. The animals were classified according to clinical examinations.

3.  Pregnant - Samples obtained only from bottlenose dolphins during pregnancy (pregnant samples Tt, n=16).

Diseased animals were gathered on the basis of the category of pathologies:

I - pneumonia (I-Tt, n=7 and I-Dl, n=4 for bottlenose dolphins and beluga whale respectively);

II - skin lesions after bites (II-Tt, n=4 only for bottlenose dolphins);

III - iron deficiency anemia (IDA) without inflammation (III-Tt, n=5 and III-Dl, n=4 for bottlenose dolphins and beluga whale, respectively).

The findings were expressed as mean + SD. The minimum and maximum values were presented. Statistical and correlation analyses were undertaken. A P value < 0.05 was accepted statistically significant. No significant differences were found between males and females and for animals of different ages, so samples were pooled for all subsequent analyses. The ANOVA test was used to compare ceruloplasmin values from different types of samples. Correlations between progesterone levels and ceruloplasmin levels during pregnancy were checked with Pearson correlation coefficient. Mean values of all ceruloplasmin variables and ranges are given in Table 1.

Table 1 shows that ceruloplasmin values from two cetaceans species with pneumonia, skin lesions and iron deficiency anemia were significantly higher then in healthy animals (P<0.05). For bottlenose dolphins, the highest Cp concentrations were found in animals with pneumonia and with skin lesions (50-100% increasing). For IDA cases the increasing of Cp concentrations was in range 25-50%. For pregnant animals the difference was not statistically significant (P>0.05); we made correlations test between Cp and progesterone levels. Pearson correlation coefficient was 0.82. Cp positively correlated with the progesterone levels during pregnancy. For beluga whales, the highest Cp concentrations were found in animals with pneumonia (50-100% increasing).

Table 1. Serum concentration of ceruloplasmin in cetaceans.

Significant increases in Cp were observed in bottlenose dolphins and beluga whales with inflammatory diseases when compared to healthy animals, supporting the results of previous studies revealing increases in diseased animals. The inflammatory mechanisms in these diseases are similar to those seen in other conditions that lead to systemic inflammatory response and multiple cytokine-mediated organ failure syndromes.

Research has shown that iron depletion increases hepatic cell transcription of Cp via activation of hypoxia-inducible factor (HIF)-1.⁶ This data correlates with increasing of Cp level in IDA cases. Furthermore the relationship between Cp and progesterone during pregnancy was verified. These data can help with monitoring pregnancy. The quantification of the concentration of Cp has been established as valuable tool providing diagnostic information in cetaceans with different pathologies. Additionally, it allows differentiating cetaceans with inflammation and Iron Deficiency Anemia from healthy subjects. The value of Cp during pregnancy was determined.

Acknowledgements

The authors would like to thank the training staff at Utrish Dlophinarium. We would also like to thank Dr. Elena I. Rozanova, Dr. Elena S. Rodionova for assisting with the samples.

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