**5. Redox alterations in experimental portacaval anastomosis**

Portacaval anastomosis (PCA) is a pathological condition that usually accompanies the portal hypertension associated to cirrhosis; however, the shunt can occur among a variety of portal and systemic veins [70]. Experimentally, PCA is a surgical procedure that com‐ municates a sectioned porta vein to an oval incision in the inferior cava vein. It results in a straight flow of the full‐of‐nutrients portal vein from the small intestine directly to the systemic circulation. It has been used for a long time to implement experimental models of hyperammonemia and the consequent hepatic encephalopathy (HE) [71]. However, only few reports exist regarding the metabolic and physiological consequences of the PCA in the hepatic tissue.

Ammonium (NH<sup>4</sup> + )‐metabolic handling by the liver involves equilibrium between anabolic (synthesis of proteins, nucleic acids and amination reactions) and catabolic (urea cycle and glutamine synthesis) pathways. Intracellular glutamate plays a key role since high glutamate serves as substrate for the synthesis of N‐acetylglutamate, an essential allosteric activator of carbamyl phosphate synthetase I, a key regulatory enzyme in the urea cycle in the periportal hepatocytes. Nitrogen disposal is complemented by glutamine synthesis (glutamate + NH<sup>4</sup> + ) in the pericentral hepatocytes [72].

In human adults, approximately 1 mol (about 17 g) of NH<sup>4</sup> + is produced daily in the liver. Part is reutilized in biosynthesis, while the rest is a metabolic disposal with the potential of being neurotoxic. Its normal concentration in the portal blood varies from 300 to 600 μM, but in the blood leaving the liver the concentration is clearly reduced to 20–60 μM [73]. Other organs such as the brain, muscle and kidney play a role in regulating the NH<sup>4</sup> + levels. Insult to the liver, whether acute or chronic in nature, reduces its capacity to metabolize NH<sup>4</sup> + with the consequence to promote in hyperammonemic state, with up to five times elevation of circulat‐ ing NH<sup>4</sup> + [72]. Although the brain is partially protected by the blood‐brain barrier from toxic agents such as ammonia, excessive amounts of NH<sup>4</sup> + can pass into the brain, constituting the principal factor in the onset of HE.

PCA in the rat results in liver atrophy, sustained hyperammonemia, and subtle neurological symptoms of HE including abnormal locomotor activity, altered sleep patterns, and modifica‐ tions of neuromuscular coordination. Feeding NH<sup>4</sup> + salts or resins to the shunted rats leads to more severe signs, eventually progressing to coma. Neuropathological examination of these rats reveals Alzheimer type II astrocytosis, the histological characteristic of chronic hyperam‐ monemic syndromes [74].

Oxidative stress is believed to play a role in the pathogenesis of HE because acute doses of NH<sup>4</sup> + are prooxidant [75]. ROS include molecules, such as hydrogen peroxide (H2 O2 ), superox‐ ide (O2 **−** ) and the hydroxyl radical (OH. ). Indeed, there is a physiological role for ROS including cellular proliferation, differentiation and signaling. However, a nonphysiological increase in ROS or a decrease in the antioxidant capacity of the organism can lead to an oxidative stress condition [76]. NH<sup>4</sup> + promotes oxidative stress by increasing ROS [77]. In this context, the brain is susceptible to oxidative stress due to high content of unsaturated fatty acids prone to peroxi‐ dation, high O2 consumption, elevated Fe2+/Fe3+, and low antioxidant systems [78]. However, a polemic issue has arisen since recent report in a model of hyperammonemia using a four‐week PCA rat model did not express any signs of oxidative stress in the frontal cortex and in arterial plasma by 4‐hydroxy‐nonenal (4‐HNE)‐linked proteins and detection of carbonyl moieties [70].

Liver is by excellence the main metabolic organ, and shows an extensive handling of prooxi‐ dant reactions; especially during the biochemical transformation of nutrients and the process‐ ing of xenobiotics [79]. However, no information has been reported characterizing putative prooxidant reactions during the experimental PCA.

#### **5.1. Lipid peroxidation**

by increasing mitochondrial volume per hepatocyte and possible augmentation of extrahe‐ patic ATP production, as an effort for maintaining mitochondrial function in the cirrhotic liver [68]. These reports agree with the statement that in perfused cirrhotic livers, a reduced cytoplasmic and mitochondrial redox states occur accompanied by a diminished activity in

Portacaval anastomosis (PCA) is a pathological condition that usually accompanies the portal hypertension associated to cirrhosis; however, the shunt can occur among a variety of portal and systemic veins [70]. Experimentally, PCA is a surgical procedure that com‐ municates a sectioned porta vein to an oval incision in the inferior cava vein. It results in a straight flow of the full‐of‐nutrients portal vein from the small intestine directly to the systemic circulation. It has been used for a long time to implement experimental models of hyperammonemia and the consequent hepatic encephalopathy (HE) [71]. However, only few reports exist regarding the metabolic and physiological consequences of the PCA in the

(synthesis of proteins, nucleic acids and amination reactions) and catabolic (urea cycle and glutamine synthesis) pathways. Intracellular glutamate plays a key role since high glutamate serves as substrate for the synthesis of N‐acetylglutamate, an essential allosteric activator of carbamyl phosphate synthetase I, a key regulatory enzyme in the urea cycle in the periportal hepatocytes. Nitrogen disposal is complemented by glutamine synthesis (glutamate + NH<sup>4</sup>

is reutilized in biosynthesis, while the rest is a metabolic disposal with the potential of being neurotoxic. Its normal concentration in the portal blood varies from 300 to 600 μM, but in the blood leaving the liver the concentration is clearly reduced to 20–60 μM [73]. Other organs

consequence to promote in hyperammonemic state, with up to five times elevation of circulat‐

PCA in the rat results in liver atrophy, sustained hyperammonemia, and subtle neurological symptoms of HE including abnormal locomotor activity, altered sleep patterns, and modifica‐

more severe signs, eventually progressing to coma. Neuropathological examination of these rats reveals Alzheimer type II astrocytosis, the histological characteristic of chronic hyperam‐

[72]. Although the brain is partially protected by the blood‐brain barrier from toxic

+

+

)‐metabolic handling by the liver involves equilibrium between anabolic

+

+ )

is produced daily in the liver. Part

levels. Insult to the

+

with the

+

can pass into the brain, constituting the

salts or resins to the shunted rats leads to

**5. Redox alterations in experimental portacaval anastomosis**

the mitochondrial electron‐transport chain [69].

166 Redox - Principles and Advanced Applications

hepatic tissue.

ing NH<sup>4</sup> +

Ammonium (NH<sup>4</sup>

+

in the pericentral hepatocytes [72].

principal factor in the onset of HE.

monemic syndromes [74].

In human adults, approximately 1 mol (about 17 g) of NH<sup>4</sup>

agents such as ammonia, excessive amounts of NH<sup>4</sup>

tions of neuromuscular coordination. Feeding NH<sup>4</sup>

such as the brain, muscle and kidney play a role in regulating the NH<sup>4</sup>

liver, whether acute or chronic in nature, reduces its capacity to metabolize NH<sup>4</sup>

Oxygen is needed for proper energetic metabolism and correct mitochondrial function, but at the same time, it promotes the formation of ROS and, in consequence, oxidation of biomol‐ ecules. Lipid peroxidation is a suitable assay to estimate prooxidant reactions. By measuring the presence of conjugated dienes, it is possible to infer the rate of peroxidative activity under "*in vivo*" conditions, and it is also feasible to deduce the balance between prooxidant reactions and antioxidant defenses using the thiobarbituric acid reactive substances (TBARS) assay. When the TBARS test is done with Fe2+ supplementation, it offers another set of information: Because it enhances the breakdown of hydroperoxides, the Fe2+‐induced lipid peroxidation is maximum and gives an idea about the total antioxidant mechanisms and the presence of global unsaturated fatty acids present in the studied membrane [80].

Initial observations in hepatic subcellular fractions, liver homogenate and serum from sham (*n* = 10) and PCA (*n* = 23) operated rats after 8 to 13 weeks of surgery were used to test lipid peroxidative activity (data not published). Conjugated dienes and TBARS were quantified by standard techniques [81]. Strikingly, rats with PCA showed reduced TBARS levels in the liver homogenate and most of the subcellular fractions (**Figure 4**), whereas conjugated dienes showed no changes with lower levels in the mitochondrial fractions (**Figure 5**). The reduction of TBARS was also observed when the assay was supplemented with Fe2+ (**Figure 6**).

PCA is an experimental protocol to generate a hypofunctional liver condition. The above‐ mentioned information strongly suggests that redox equilibrium within the liver under PCA surgery shows a reduction in the prooxidant reactions and/or increase in antioxidant defense. More focused experiments are needed to elucidate the underlying mechanism(s), but it is interesting to consider the biochemical consequences that the alteration in the portal blood flow can promote within the hepatocytes' redox equilibrium.

**Figure 4.** Effect of portacaval anastomosis on thiobarbituric acid reactive substances (TBARS) assay of liver homogenate, subcellular fractions and serum. Sham, false‐operated rats (white bars); shunt, rats with portacaval anastomosis for 6‐8 weeks (black bars). Data are average ± SEM from at least eight independent observations. \* Significant statistical difference by *t*‐student test, *p* < 0.05.
