Safety study of polyunsaturated fatty acids extracted from New Zealand green lipped mussel and krill on hematology and blood chemistry profiles of healthy

Abstract

New Zealand green-lipped mussel (Perna canaliculus) is a shellfish with a green shell containing many types of fatty acids, especially polyunsaturated fatty acids (PUFAs).

The aim of the present study was to investigate the safety of dietary PUFAs supplementation on blood hematology, chemistry and coagulogram pancreatic enzymes in dogs and cats.

Twelve healthy beagle dogs and healthy mature cats were used in these studies. Complete randomized block and triplicate 4×4 Latin square designs were used for these dog and cat safety studies, respectively. Experimental diets composed of sham control (0X) and 3 treatment groups, including 1X, 3X, and 10X of PUFAs capsules containing extracted mussel (60% or 30 mg/capsule) and krill (40% or 20 mg/capsule), were given consecutively for 56 days. Blood samples were collected every 28 days for hematology, chemistry and coagulogram, and pancreatic enzyme analysis. Hematology and blood chemistry results were in normal ranges and no animals appeared to reveal any abnormal signs.

Dogs fed 3X had significantly (P<0.05) higher in creatine compared to 0X and 1X at D28. Likewise, there were statistically significant differences in the time of cat’s ALT and lipase. Group of cats fed 10X significant reduction in ALT at D56 compared to D0 (P<0.01) and D28 (P<0.05).

In the group of cats fed at 1X, there was a significant within-group difference between the values of ALT levels for baseline (D0) and D56 (P<0.05). At D56, cats in all groups had a significantly higher amount of lipase levels than those at D0 (P<0.05). The prothrombin time of cats in the 0X and 10X groups showed a significant within-group difference between the values for D0 and D56 (P<0.05). PLI of dogs and cats showed good pancreatic function based on precision accuracy.

Overall, EAB-277 contains PUFAs and appears to be safe and improves liver function, especially in cats receiving 10 X administration of EAB-277.

Keywords: blood chemistry, cat, dog, hematology, New Zealand green lipped mussel, PUFA

1Department of Animal Husbandry, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand

*Correspondence: Uttra.J@chula.ac.th (U. Jamikorn)

Received September 19, 2024

Accepted March 10, 2025

Discussion

This safety study demonstrated the various dose effects of EAB-277 supplementation on blood hematology, chemistry and coagulogram in healthy dogs and cats. EAB-277 has been developed from PCSO-524, which is composed of 91 fatty acids, including omega 3 fatty acid, DHA, EPA, DPA, ETA as the key components. These components have been used as an anti-inflammatory treatment in many disorders, especially arthritis in dogs and cats, during the past decade (Kampa et al., 2023). The omega-3 fatty acids from marine sources typically reduce inflammatory processes and result in several other desirable effects (such as gastrointestinal bleeding); they are also considered good fats for dog and cat (Bauer, 2006). Adverse reactions which concern both veterinarians and pet owners mostly involve physiological function, e.g., weight gain, serum cholesterol and triglyceride, tendency to bleed, and pancreatic function e.g., pancreatitis (Xenoulis et al., 2020). Recently, Mektrirat et al. (2022) found that PUFA EAB-277 supplementation improves clinical signs and attenuates Human rhinovirus (HRV) impairment by reducing oxidative stress and inflammation in tracheal collapse dogs. This might increase the satisfaction of pet ownership. Additionally, EAB-277 shows promise as a substitute for current immune-mediated hemolytic anemia (IMHA) medications (Mektrirat et al., 2023). It also contains sterol esters, polar lipids, and carotenoids, which play roles in cellular functions and possess anti-inflammatory properties, cell membrane integrity, and antioxidant properties that contribute to the extract’s health benefits (Tsiantas et al., 2022). As nutraceutical are prescribed by many veterinarians, not only efficacy, but also safety is important to confirm when used with companion animals. Dogs and cats are classified as carnivores even though only cats are obligatory carnivores. This means that cats may respond differently to PUFAs supplement than dogs.

The aim of this study was to evaluate any possible adverse reaction regarding the concern in dogs and cats. One concern for use of PUFAs supplement is the possibility of body weight gain. All animals demonstrated neither weight gain nor loss, with body condition scores being in ideal range (score 2/5 to 3/5). The US National Research Council (2006) had the recommended allowance levels of EPA and DHA for adult dogs and cats at 0.11 and 0.025 g/1000 kcal, respectively. Association of American Feed Control Official (AAFCO) have not determined the requirement of EPA and DHA for adult dog and cat (Lenox, 2016). Suggested supplemental doses of EPA and DHA for dogs’ weights 10 kg and cats’ weights 5 kg were 500-750 (50-75 mg/kg×10 kg) and 150-250 (30-50 mg/kg×5 kg) mg, respectively, with the safe upper limit (dog) at 2080 mg (0.37×100.75) (Lenox, 2016). Each capsule of PUFAs supplement (Antinol®, EAB 277TM) used in this study contains 50 mg of active ingredients. Even though the 20 capsules (5X of a maximum dose or 10X groups) of the supplement provided a total of 1,000 mg PUFAs (about 9 kilocalories, kcal). Approximate daily energy requirement (DER) of dog weights 10 kg and cat weights 3 kg are 742 and 209 kcal/day, respectively. Therefore, the PUFAs supplement provides only 1.21 and 4.31% of DER for dog and cat, respectively. In addition, serum cholesterol and triglyceride concentration of all treatment groups for both dog and cat did not differ significantly when compared between time or treatment.

Hematology and blood chemistry values of all dose treatment groups of dogs and cats obtained from the current study were all in the reference range (Merck, 2023). For dog blood creatinine analyses, significant differences were observed in dogs receiving 3X, which had higher creatinine levels than other treatments at D28. Administration of 3X of dietary PUFAs could be appropriate to build up muscle mass and health conditions, while low level blood creatinine might indicate malnutrition or lower muscle mass (Ruaux et al., 2012). Excessive blood creatine level (over than 1.4 mg/dl) can be used as a marker of kidney disease as acute kidney injury (AKI) and chronic kidney disease (CKD) in dogs (Babyak et al., 2017). Even so, the 3X dog’s rising creatine level (less than 1.4 mg/dL) is still within the standard range and indicates no health risks.

For cat blood chemistry analyses, significant differences were observed between times within treatments for concentration of ALT (1X and 10X) and lipase (3X and 10X). During the period of safety study, many extraneous factors such as photoperiod, humidity, ventilation, ambient temperature (Fox, 1986) and even routine procedures such as removing test animals from their cage (Reinhardt, 2004) may possibly affect animal physiology including the dependent variable, blood hematology and serum chemistry values. In addition, a slight change in blood chemistry that remains within reference interval could be due to clinical variation. Aside from these, hemolysis could also interfere with the analysis of some serum chemistry such as creatinine kinase, aspartate dehydrogenase, lipase, and albumin (O’Neill and Feldman, 1989). However, there was no blood hemolysis detected in this study.

Prolonged bleeding time with PUFAs supplements is another adverse reaction which concerns veterinarian and pet owners. This condition can possibly increase risk (for example, trauma, fracture, burn) and affect many practical procedures such as any surgery, scratch of tartar etc. Blood coagulation factors of the studied dogs and cats mostly were not significantly difference for comparison neither between treatment nor time. Thin blood condition was not observed on any of the studied animals. The range of blood coagulation varies between Merck (2023) and Chulalongkorn university laboratory (CU lab) index. Regarding Merck (2023) reference, the range of TT, PT, and aPTT are 10-21, 6.7-16.6, and 15-20 seconds while CU lab values are 11.5-30.6, 5.8-15.6, and 10.0-21.8seconds, respectively. However, Prihirunkij et al. (2003) reported the mean +SD of TT of cats at 22.2+2.9. Therefore, using Merck as the baseline for the comparison seem to be appropriate. Cats fed 1X and 10X of dietary PUFA had higher PT level during day 56 compared to day 1. An increase in PT alone may be interpreted as an extrinsic pathway issue (e.g., Factor VII deficiency, early rodenticide poisoning), but 16 Jamikorn U. and Kongpanna P. / Thai J Vet Med. 2025. 55(1): 15.

prolongation of both aPTT and PT mostly occurs in cases of severe liver disease, vitamin K deficiency or antagonism and is typically caused by an absence of multiple variables (Parry, 1989). In associated with our ALT result, as expected, the ALT level at day 56 of cat fed 1X and 10X was significantly lower than day 1. This result has also confirmed the negative correlation between blood PT and ALT, which is associated with hepatic dysfunction (increased ALT or ALP, and in most cases other possible biomarkers of hepatic dysfunction of damage). A coagulogram’s variation may be triggered by an array of factors, including sample time and technique, anticoagulant transfer delays, and insufficient anticoagulant mixing in the glass tube (Mischeke et al., 2005). Other possible causes of the prolongation include a deterioration of factors because of the delay between blood collection and analysis, inadvertent thawing and incorrect plasma to anticoagulant ratio. Alternatively, factor VII deficiency may be responsible for prolongation of the PT. Factor VII is the coagulation protease responsible for starting a cascade of proteolytic events that lead to thrombin generation, fibrin deposition, and platelet activation (Eigenbrot, 2002).

ALT is important in the metabolism of nitrogen and is most often associated with the liver function. High levels of ALT indicate either liver disorders, damage, or toxin ingestion. In the present study, cat’s ALT level was significantly decreased when receiving 10X of dietary PUFAs at D56 compared to D0 and D28. This possibly concluded that not only as an energy source but also helps to carry out various biological reactions involved in the development and maintenance of normal renal functions (Katakura et al., 2015). Dietary PUFAs can influence the immune response through changes in metabolic status and coagulation (Ferroni et al., 2012). In contrast, PUFAs can decrease the levels of several coagulation factors (V, VII, and X) and reduce thrombin generation and does not improve coagulation, metabolic, and inflammatory status (Macchia et al., 2012; Poreba et al., 2017). The possible mechanism could relate to the anti-inflammatory and antioxidant properties and may have beneficial effects on liver health. The role of dietary PUFAs in inhibiting proliferation, inducing apoptosis, and promoting differentiation in antioxidant enzymes has been recently studied (Thérien et al., 2021). Generally, oxidative damage may be the primary cause of lipid peroxidation and cellular damage. Negative correlation between malondialdehyde (MDA) and glutathione (GSH) indicated the occurrence of an oxidative insult that caused hepatic and renal damage (Abdou and Hassan, 2014).

Our study showed that cats at D56 had higher serum lipase than D0 even in control group. Regarding digestive physiology, cats could adjust to dietary fat better than dogs, as a result, their serum lipase remained stable no matter what doses ware applied. Additionally, supplementation of PUFAs in continuous for two weeks up could possibly enhance pancreatic lipase secretion although after terminating the supplement. Furthermore, serum lipase and fPLI observed in the current study were all in normal range as serum lipase range was 13-108 U/L and fPLI was below 3.5 ng/mL.

Pancreatitis is a potential problem in animals fed with high-fat diets or high doses of fatty acid supplementation (Lenox and Bauer, 2013). Variables indicating pancreatic function of the animals include serum amylase and lipase. These two enzyme concentrations were found in the normal range for dogs, and no significant difference was observed overtreatment or time. Polynomial contrast found neither linear, cubic nor quadratic relationships between dose treatments and any parameters. Evaluation of pancreatic function of dogs using PLI values mainly resulted in normal status at every point of time. PLI values of all studied cats demonstrated the normal status of pancreas, and no difference was observed over the dose, treatment or time. Furthermore, no clinical sign of pancreatitis was observed on any animals in this study. Bauer (2011) reported the dosages of EPA and DHA when use in clinical treatment are approximate from 120 up to 310 mg/kg0.75. The maximum dose of the supplement used in this safety study (10X groups) was total of PUFAs 1,000 mg for 10 kg BW dog or 500 mg for 3-4 kg BW cat. The PUFAs supplement extracted from New Zealand green-lipped mussels, Perna canaliculus, contained not only omega 3 fatty acids, EPA and DHA, but also some other omega 6, monounsaturated and saturated fatty acids (Taylor and Candida, 2006). These inconsistent effects demonstrate the effective dose of the lipid extract, EPA and DHA, have not been clearly identified. However, the data from the current safety study verify that the fatty acids composition and dose of the PUFAs supplement used in the current study showed no influence on pancreatic function.

Current results are supported by the findings of our previous safety study with healthy beagle dogs, where the blood hematology, chemistry and coagulogram were not significant among the various groups considered when administered with 0X, 1X, 3X and 10X dose of PCSO-524 (Antinol®) (Jamikorn and Yibchok-anun, 2014). This was also demonstrated by no change in food intake and behavior, which suggested that this supplement did not have a harmful effect. However, the current data could not exclude that there were other factors such as previous dietary fatty acid composition and source or formulation of the basal diet that might influence the results. To the best of our knowledge, this is the first study evaluating 10 times overdose administration as safety study in healthy dogs and cats with dietary PUFAs (EAB-277). All dependent variables were in normal ranges referenced by Merck (2023). Therefore, interpreting the results of blood hematology, chemistry and coagulogram should be approached with recommendation. This is because they may fluctuate greatly due to factors such as age, nutrition, and environmental conditions, as well as regulations of technology and analytical methodologies utilized, all of which must be addressed before drafting final results.

In conclusion, supplementation of the PUFA containing EAB 277 extract from New Zealand green lipped mussel to the dogs and the cats at 0X, 1X, 3X and 10X resulted in the normal ranges for most of the blood hematology and blood chemistry values. No abnormal signs were observed during the safety study period including amylase, lipase and PLI values for all animals. For blood coagulation factors, only the PT of all treatment groups were in the normal range of Chulalongkorn university lab. However, the TT, PT and aPTT of all treatment groups demonstrated similar patterns with no significant difference except between the 1X and 10X at D56 (P<0.05). These could indicate the normal ranges of the TT, PT and aPTT of animals at the kennels used in the current study. Overall, this safety study found that PUFA supplementation using EAB-277 derived from New Zealand green lipped mussel appeared to be safe for use in dogs and cats.

Chula Digital Collection: https://digital.car.chula.ac.th/tjvm/vol55/iss1/15/