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Plasma amino acid profiles in dogs with closed extrahepatic portosystemic shunts are only partially improved 3 months after successful gradual attenuation

Abstract Background Dogs with portosystemic shunts have an altered blood amino acid profile, with an abnormal branched‐chained amino acid (BCAA)‐to‐aromatic amino acid (AAA) ratio being the most common abnormality. Different liver diseases have distinctive amino acid profiles. Objectives Determine the changes in plasma amino acid profiles in dogs with extrahepatic portosystemic shunts (EHPSS) from diagnosis to complete closure. Animals Ten client‐owned dogs with EHPSS closed after surgical attenuation. Methods Prospective cohort study. Medical treatment was instituted in dogs diagnosed with EHPSS. At least 4 weeks later, gradual surgical attenuation was performed. Three months postoperatively, EHPSS closure was confirmed by transsplenic portal scintigraphy. Clinical signs were scored and blood was taken before institution of medical treatment, at time of surgery, and 3 months postoperatively. At the end of the study, the plasma amino acid profiles were analyzed in batch. Results The median BCAA‐to‐AAA ratio was extremely low (0.6) at time of diagnosis and remained low (0.5) at time of surgery, despite the fact that median neurological score significantly improved from 22 to 2 after starting medical treatment (P = .04). Three months after surgical attenuation, a significantly higher BCAA‐to‐AAA ratio (1.5) was observed (P < .001). Conclusions and Clinical Importance Medical treatment does not improve the BCAA‐to‐AAA ratio in dogs with EHPSS, despite substantial clinical improvement. Although the ratio significantly increased after EHPSS closure, it was still indicative of moderate to severe hepatic dysfunction in all dogs.


| INTRODUCTION
The liver is an important organ involved in many essential metabolic functions, including metabolism and synthesis of proteins, carbohydrates, and fats. 1 Portosystemic shunts (PSS) are vascular anomalies that connect the portal system to the systemic circulation, bypassing the liver parenchyma. 2 Clinical signs associated with PSS are growth retardation, hepatic encephalopathy (HE), gastrointestinal signs, and urinary signs secondary to the development of ammonium urate urolithiasis. 3 Gastrointestinal signs can occur because of decreased bile production secondary to hepatic dysfunction, leading to maldigestion and malabsorption. 3 Longterm malabsorption can cause deficiencies in proteins, fatty acids, and vitamins, resulting in growth retardation and skin and hair coat changes. 3,4 In dogs with hepatobiliary disease, an adequate amount of high quality protein should be provided in the diet to avoid negative nitrogen balance. 5,6 It is, however, important to assure that dogs with HE do not receive an excessive amount of protein, which can exacerbate clinical signs. 6 Blood ammonia concentrations often are linked to the presence of HE but ammonia concentrations can be normal despite the presence of HE. 7 Other substances such as increased amounts of glutamine, glutamate, manganese, and systemic inflammation can contribute to HE. [8][9][10] Also a decreased ratio of branched-chain amino acids (BCAA)-toaromatic amino acids (AAA) can induce derangements in central neurotransmitter function that trigger HE. 11 Two experimental studies, in which a portocaval shunt was created in adult mongrel dogs clearly showed that amino acid profiles changed over time. The most important alterations were a significant increase of AAAs and a significant decrease of BCAAs, changes that were linked to the development of HE. 11,12 Similarly, a recent study that assessed blood amino acid concentrations in dogs with congenital PSS found lower concentrations of BCAA and higher concentrations of AAA in dogs with PSS compared with dogs with chronic hepatitis and healthy dogs. 13 Normal BCAA-to-AAA ratios in dogs are reported to be 3.0 to 4.0, whereas dogs with severe hepatic insufficiency have ratios <1.5. 14 In an experimental study in dogs in which a portocaval shunt was created after partial hepatectomy, a BCAA-enriched diet did not have a beneficial effect on the severity of HE. 15 No research has been performed to identify the effect of medical or surgical treatment on the amino acid profiles in dogs with congenital PSS receiving medical treatment and undergoing surgical attenuation. Yet, this information is important because surgical treatment is not always performed or successful, and consequently a number of dogs with congenital PSS need life-long medical treatment.
Our aim was to determine whether the plasma amino acid profile changes after starting medical treatment and if the plasma amino acid profile normalizes in dogs with extrahepatic PSS (EHPSS) after complete EHPSS closure.

| Animals
The study was approved by the local ethical (EC2017-49) and deontological committee (2017N06), and, before inclusion of their dog, all owners signed an informed consent. Client-owned dogs with congenital EHPSS were prospectively enrolled. Inclusion criteria were dogs that had not yet received medical treatment (including a liversupportive diet) at the time of diagnosis and that would undergo surgical attenuation of the EHPSS.
After confirmation of the diagnosis of EHPSS, medical treatment was given for a minimum of 4 weeks. All dogs received a strict liversupportive diet (Royal Canin hepatic, Royal Canin, Zaventem, Belgium) divided into several small meals per day. Additional medication consisted of lactulose (0.5 mL/kg q8h), metronidazole (7.5-10 mg/kg q12h) or both lactulose and metronidazole. All dogs underwent gradual attenuation of the EHPSS using either an ameroid constrictor or thin film banding, always placed as close as possible to the systemic circulation. Postoperatively, medical treatment was continued until the 1-month follow-up visit. In dogs that had clinical signs compatible with PSS or dogs with fasting ammonia concentration above the upper limit 1 month postoperatively, lactulose was continued; in all other dogs only the liver-supportive diet was continued until the 3-month follow-up visit. Three months postoperatively, transsplenic portal scintigraphy was performed. After ultrasound-guided injection of 99m Tc pertechnetate into the spleen, the percentage of blood bypassing the liver (shunt fraction) was calculated. Dogs with a shunt fraction <4.3% were considered to have a closed EHPSS. 16 Dogs that had persistent shunting were excluded from the study.  (Table 1). 17

| Blood tests
At diagnosis, at the day of surgery and 1 and 3 months after surgery, a blood sample was taken from a jugular vein. Owners were asked to fast their dogs for 12 hours before blood sampling. On all occasions, 1 drop of fresh blood was used to immediately measure fasting ammonia concentration using a hand-held ammonia analyzer Sample analyses were carried out in 96-well plates containing 2 calibration curves and 6 quality samples per plate to monitor assay performance. Accuracy was evaluated using high-, medium-, and lowquality controls based on historical values from TAM146 validation.
Precision was evaluated using the quality control replicates in each sample run.
The ratio of BCAAs-to-AAAs was determined for each dog by dividing the sum of all BCAA plasma concentrations by the sum of all AAA plasma concentrations at all 3 sampling points.  Table 2. Both body weight and BCS significantly increased over time (P = .001 and P = .01, respectively). Pairwise comparisons, however, only identified a significant increase in body weight from diagnosis to 3 months postoperatively (P = .001) and from 1 to 3 months postoperatively (P = .004).

| Statistical analyses
Within dogs, all clinical scores (gastrointestinal, urinary, and neurological scores) significantly decreased over time (P < .001, P = .02, and P < .001, respectively). Pairwise comparisons showed a significant decrease in gastrointestinal scores from diagnosis to 3 months postoperatively (P < .001), and a significant decrease in neurological scores from diagnosis to 1 month postoperatively (P = .001) and from diagnosis to 3 months postoperatively (P < .001).
Fasting ammonia concentrations significantly decreased over time within dogs (P < .001). Pairwise comparisons indicated that fasting ammonia concentration significantly decreased from diagnosis to 1 and to 3 months postoperatively (P = .002 and P < .001, respectively) and from surgery to 1 and to 3 months postoperatively (P = .02 and P = .01, respectively).

| Amino acids
Median plasma concentrations of the different amino acids at different time points are presented in Table 3. Both BCAAs and AAAs are considered to be key factors in the pathogenesis of HE. 20 In contrast to other amino acids, BCAAs are primarily metabolized in muscle tissue, 21 where they serve as precursors of other amino acids and as an energy substrate. 22 In the presence of hyperammonemia, BCAA metabolism in muscle tissue changes and helps to detoxify ammonia by forming glutamine. 20,23 Also in brain tissue, BCAAs are used as an alternative pathway to detoxify  (Continues) ammonia, 24 which becomes the main route of detoxification in case of malnutrition. 25 The glutamine that is formed, however, causes astrocyte swelling, which in turn can aggravate HE. 26 Previous experimental studies, in which portocaval shunts were created in adult mongrel dogs that subsequently developed HE, indeed found a decrease in plasma BCAA concentrations, which might be the consequence of increased BCAA metabolism secondary to hyperammonemia. 11,12 Nevertheless, BCAA concentrations in cerebrospinal fluid remained stable. 11 Meanwhile, it was found that plasma AAA concentrations A previous experimental study in dogs concluded that an overall improved nutritional state is more important in improving clinical signs than the proportions of amino acids that are present within the food. 11 In contrast to BCAAs, AAAs typically have a very high extraction ratio and are rapidly metabolized by the liver, 31 which can explain why AAAs significantly decreased after surgical attenuation of the EHPSS. In our study, gradual attenuation devices were used to obtain EHPSS closure. Although ameroid constrictors are used for gradual attenuation, complete EHPSS closure can occur as early as 10 days postoperatively. 32 Thin film banding typically is reported to cause slower EHPSS closure, which can take up to 8 weeks if the internal diameter of the vessel is decreased to <2.5 mm. 33 Although liver perfusion is normalized once the EHPSS is completely closed, it might take longer for liver function to completely normalize and to restore amino acid balance. Further research is needed to verify if the BCAA-to-AAA ratio will completely normalize in the long-term.
In our cohort of dogs, the installed medical treatment device did not improve the overall amino acid profile. This is an important After at least 4 weeks of medical treatment using a liversupportive diet, only methionine and serine concentrations increased significantly. Surprisingly, both amino acid concentrations decreased again to the concentration observed initially at diagnosis, 3 months after surgical attenuation, despite any change in diet. Both methionine and serine have a high liver extraction ratio. 31 The most likely explanation for this finding is the relatively high intake of both amino acids in the liver-supportive diet that could only be metabolized efficiently after surgical attenuation of the EHPSS. Methionine is an essential amino acid that is converted into S-adenosylmethionine in the liver, and has important physiological roles such as decreasing tissue oxidative stress. 34 In human medicine, controversy exist as to whether methionine supplementation is useful in patients with chronic liver disease. 35 Our findings suggest that the liver-supportive diet used might not be ideally balanced in terms of amino acids for dogs with portosystemic shunting. A double-blind cross-over study found that clinical signs of HE significantly improved in dogs with PSS that received a diet based on soy instead of meat protein. 35 Yet, the BCAA-to-AAA ratio of the soy-based diet was only 2.05 compared to 2.30 for the meat-based diet. Despite a lower BCAA-to-AAA ratio in the food, improved HE scores in that study were attributed to the fact that vegetable proteins are highly digestible leading to a higher absorption of amino acids in the small intestine and subsequently less nitrogen reaching the colon. 36 Blood amino acid concentrations were not examined in that study. The digestibility of amino acids in food is dependent not only on the protein source, but also on the way the food is processed and on the presence of other components in the food. It remains to be investigated how the composition of diets can influence blood amino acid profiles in dogs with impaired hepatic function. [37][38][39] Our study had some limitations. Only a small number of dogs was included; different small dog breeds were represented and half of the dogs were young at the time of inclusion. In dogs, it has been shown that blood amino acid concentrations differ between small and large breeds. 40 Because only small breed dogs were included in the study, no influences are expected based on the breed. In people, it is well known that blood amino acid concentrations are age-specific, with histidine, arginine, cysteine, tyrosine and taurine being semi-essential amino acids in children because these are important in normal development and growth. 41,42 Similar age-specific blood amino acid concentrations would be expected in dogs. Nevertheless, no age-specific differences were identified in our study between dogs with EHPSS <1 year of age and those that were older.
An age-and size-matched healthy control group could have been included to strengthen our findings. Blood amino acid concentrations in healthy dogs however vary among studies, with all studies describing relatively wide ranges. 18,19,30 In addition, differences in reported normal values can be explained by the fact that most studies only included a small number of apparently healthy dogs that received different types of food, and also by the fact that amino acids were analyzed using different laboratory techniques.
The fact that dogs were fed different foods at inclusion makes interpretation of concentrations obtained at diagnosis difficult. Nevertheless, our study clearly showed that even if dogs were subsequently placed on a standardized liver-supportive diet, no differences in the amino acid profile occurred, apart from the increases in methionine and serine concentrations. Therefore, the influence of diets the dogs were receiving before treatment did not seem to have much impact.
In accordance with previous studies, dogs with EHPSS had a marked decreased BCAA-to-AAA ratio. Medical treatment did not influence this ratio, notwithstanding the fact that clinical signs attributable to HE improved substantially. Despite the fact that the BCAA-to-AAA ratio increased significantly after surgery, it still indicated moderate to severe liver dysfunction in all dogs with closed EHPSS, suggesting that functional recovery requires more time than does clinical recovery.

ACKNOWLEDGMENT
Funding partially provided by Royal Canin Benelux.