Impact of intestinal mannitol on hyperammonemia, oxidative stress and severity of hepatic encephalopathy in the ED

a b s t r a c t

Hyperammonemia results from hepatic inability to remove nitrogenous products generated by protein metabolism of intestinal microbiota, which leads to Hepatic encephalopathy (HE) in chronic liver disease (CLD). In ammonium neurotoxicity, oxidative stress (OxS) plays a pathogenic role. Our objective was to evaluate if intestinal mannitol is as effective and safe as conventional treatment for diminishing hyperammonemia, OxS, and HE in patients with CLD. Material and methods: We included 30 patients with HE classified by “Haven Criteria for Hepatic Encephalopathy”. They were randomized into two groups: 1) Mannitol Group (MG) with mannitol 20% administered into the intes- tine by an enema, 2) conventional group (CG) with lactulose 40 g enema both substances were diluted in 800 mL of double distilled solution every 6 h; all patients received neomycin. We evaluated ammonia concentration, plasma oxidative stress, HE severity, intestinal discomfort and adverse effects.

Results: Hyperammonemia (171 +- 104 vs 79 +- 49 umol ammonia/L, p b 0.01), and oxidative stress (MDA 29 vs 27%, formazan 15 vs 11%, carbonyls 16 vs 9% and dityrosines 10 vs 5%) were reduced in MG and CG respectively. The HE severity decreased by two degrees compared to baseline values in both groups. Intestinal discomfort and electrolyte plasma alterations were less frequent (p b 0.05) in MG than CG.

Conclusions: Intestinal mannitol is as effective and safe as conventional treatment for reducing hyperammonemia, oxidative stress, and hepatic encephalopathy of CLD patients in the emergency room. Likewise, mannitol is better tolerated than conventional treatment.

Introduction

Hepatic Encephalopathy (HE) as acute complication of Chronic Liver Disease (CLD), is characterized by hyperammonemia [1] due to inability of the liver to remove nitrogenous products. These products derive from proteins which are produced by intestinal macrobiota, then are absorbed by the intestine, and remain for a long time in systemic circu- lation. The concentration of ammonia in healthy subjects is 16.6-47.3 for men and 11.5-38.0 for women umol/L and along with OxS inside the brain cause neuronal damage and perpetuate the HE. They are also associated with neurotransmitter dysregulation and cerebral edema in astrocytes, clinically manifested by alterations in behavior, character, in- telligence, and motricity [2].

* Corresponding author at: Instituto Mexicano del Seguro Social-Hospital de Especialidades Centro Medico Nacional “La Raza”, Seris y Zaachila S/N. Col. La Raza, Ciudad de Mexico 02990, Mexico.

E-mail address: maria.cruzdo@imss.gob.mx (M.P. Cruz-Dominguez).

In biological systems, the OxS is characterized by imbalance towards excessive production of Reactive oxygen species or Nitrogen (RNS) and accelerated consumption of antioxidant capacity [3,4]. Be- cause special chemical properties of ROS and RNS (unstable and highly reactive) can oxidize any biological lipid molecules (lipoperoxidation) [5], proteins (hormones) [6], and DNA chain breakdown, they result in acute or chronic damage to hepatic tissue [7].

The treatment of HE comprises protein restriction, antibiotics, non- absorbable disaccharides of action in intestinal lumen [1]. The non- absorbable disaccharides have been a mainstay of therapy for HE for de- cades and have been extensively studied in several small clinical trials since the late 1970s. Of these non-absorbable disaccharides, lactulose remains as standard treatment for HE in patients with CLD. Mannitol belongs to sugar alcohols or polyols (prebiotics) similar to lactitol [8], sorbitol [9], or maltitol with the same efficacy in this kind of patients [10].

Mannitol was authorized by the Food and Drug Administration since 1972 [11], with over 3200 registered products; 92 for direct pharmacological use in medicine [12] including all known routes of

https://doi.org/10.1016/j.ajem.2018.01.032

administration in humans. It has been cataloged as a prebiotic and wide- ly used as a sweetener for beverages (juices, soft drinks, and coffee), cakes, sweet cookies, and manufacturing of cosmetics (additive creams and personal hygiene products) [13-15]. Due to its hyperosmolar intra- vascular effect, mannitol has been used for treatment of migraine [16], intraocular or Intracranial hypertension [17,18], kidney injury by ische- mia/reperfusion [19,20], Parkinson’s disease [21] and cystic fibrosis [22]. With respect to the gastrointestinal use of mannitol, it has been admin- istered orally in combination with activated charcoal by gastroclysis for promote digestive and urinary excretion of toxins in patients with poi- soning or overdose drug [23]. Also by enemas to prepare the bowel for gastrointestinal procedures such as colon surgery, colonoscopy and bar- ium enema [24].

In patients with liver cirrhosis it is also useful in the prevention of de- velopment of post – hemorrhagic encephalopathy [25], although until now it has not been evaluated in the treatment of established encepha- lopathy. When mannitol is administered into the intestine, it also in- creases the molarity of intestinal lumen, decreasing the concentration of ammonia in CLD. On the other hand, it is also able to reduce the OxS and therefore renders the possibility of reducing neuronal damage by ammonia.

The aim of the study was to demonstrate that intestinal mannitol is equally effective and just as safe as conventional treatment (lactulose) to reduce hyperammonemia, decrease OxS and improve HE, in patients with CLD.

Methods

This research protocol was approved by the Ethics Committee of Gen- eral Hospital of National Medical Center “La Raza”, Mexican Social Securi- ty Institute. All participants signed a written informed consent. The project was developed in accordance with the ethical principles embod- ied in the Helsinky Declaration of 1975 and up until its last revision in 2013.

There are few studies that measured ammonia levels before and after some treatments. Therefore, we took as reference ammonia levels that corresponded to the degree of encephalopathy which we expected at the beginning and after of either of two interventions in our study. For calculating sample size we used the formula of differences of two aver- ages “n= 2[(Z? – Z?)?]/u1 – u2”. If average venous ammonia concen- tration is 120 umol/L and standard deviation 15 umol/L in patients with HE grade III (u1) without treatment (Ong JP et al. 2003) [26], and after in- terventions with disaccharides (u2) (lactulose or mannitol) venous am- monia was proposed on average of 102 umol/L, the sample size was calculated in 15 patients per group. We considered alpha = 0.05, and Beta = 0.10.

Patients

We included 30 patients with CLD in this clinical trial, HE was classified based on clinical criteria of severity of West-Haven [27]. In- clusion criteria were: patients with HE grade II, III and IV, both gen- ders, 30-60 years of age, admitted to the emergency room, who signed an informed consent. The criteria for non-inclusion were: al- lergy to mannitol, Severe anemia, hypoglycaemia, hypovolemic shock, sepsis, recent intracranial lesions (hematomas, abscesses, hemorrhage-ischemia-type cerebral vascular disease and tumors), meningitis-encephalitis, hypoxemia-hypercapnia, alcohol-related syndromes (acute ethanol intoxication, Wernicke’s encephalopa- thy), hypnotic sedative ingestion, antioxidants, antidepressants and antipsychotics. Clinical data included age, gender, height, weight, body mass index, diabetes type 2, systemic Arterial hypertension, chronic kidney disease, HE causes, etiology of cirrhosis and Child- Pugh score.

Treatment of hepatic encephalopathy

All patients who met the selection criteria were included in a simple randomization (1:1) with an automated online system, ensuring that the research team was unable to affect randomization. We used the single-blind for those who measured the blood biomarkers and clinical degree of encephalopathy. The attending physician was aware of the treatment at all times thus, allowing him to change the management when he considered it convenient for the patient. The first group (MG) received an enema of 20% mannitol (PiSA(R), SA de CV Mexico City/Mexico: Osmorol 20(R)) calculated to 1 g/kg, titrated to 800 mL with double distilled water (PiSA(R)). The second group (CG) received enema of 40 g/60 mL of lactulose (Cetus(R)) diluted in 800 mL of double distilled water. In both groups the retention enema lasted 15 min (5 min ventral position, 5 min left lateral position and 5 min right lateral position), every 6 h for 48 h [1]. All patients were also treated with Neomicine (Neomixen(C)) 250 mg orally every 6 h, and according to guidelines of HE [1]. In addition, the precipitating factors of HE were identified and treated in all patients.

Biochemical measurement in venous blood

Peripheral venous blood was collected at the time of diagnosis of HE (baseline) and at 48 h of follow-up (final). Four milliliters of the blood sample were deposited in a test tube with a red empty BD Vacutainer

(R) stopper with coagulation activator on the walls of the tube to acceler- ate the formation and retraction of the clot, each sample was stamped with the patient’s name and centrifuged for 10 min at 3000 rpm. A 100 uL aliquot of plasma was taken to measure the ammonia concentra- tion by enzymatic methods, using the glutamate dehydrogenase reac- tion with reagents obtained from Roche Diagnostics. The electrolytes, glucose and creatinine in venous blood were analyzed according to the manufacturer’s Protocol on Roche Diagnostics Cobas(R) 4000 analyz- er series automatic (Indianapolis, Indiana USA).

Oxidative stress biomarkers

We measured several biomarkers of lipid and protein oxidation in pa- tients with HE in both MG and CG. An aliquot of 100 uL of plasma was used to determine systemic lipid damage by measuring thiobarbitUric acid-reacting products, such as Malondialdehyde [28]. The MDA was measured at 532 nm; and 1,1,3,3-tetramethoxypropane (Sigma-Al- drich, St Louis, MO) was used as standard. The capacity of oxidized pro- teins to react with nitroblue tetrazolium (NBT), producing formazan, was used as an indirect means of analyzing the degree to which proteins had been modified by oxidative stress. The NBT reaction was carried out with 10 uL of plasma, and the absorbance was measured at 530 nm to de- tect the formazan. The molar extinction coefficient for formazan (E = 15 mmol/L^-1 cm^-1) was used to calculate its concentration [29]. Tyrosine dimers (dityrosine) were determined by measuring fluorescence at exci- tation and emission wavelengths of 320 and 410 nm, respectively, from a 100 uL aliquot of plasma [30]. The final concentration of dityrosine was calculated from a standard curve constructed using dityrosine synthe- sized in the laboratory [31]. Free carbonyl groups were measured using 100-uL aliquots of plasma and 1 mL of 10 mmol/L 2,4-diphenylhydrazine (DPNH) [32]. The absorbance was measured at 370 nm to detect the for- mation of dinitrophenylhydrazones. The molar extinction coefficient for DPNH (E = 22,000/mol/L^-1 cm^-1) was used to calculate the concentra- tion of carbonyl [6]. Total protein was measured as a reference parameter according to Lowry et al. [33].

Assesment severity of degree HE, safe and intestinal discomfort

We evaluated the degree of HE before and after treatment with man- nitol/lactulose, using the clinical criteria and severity of HE by “West Haven criteria for hepatic encephalopathy”. We established that the

reduction by each degree of HE was equivalent to 20%. Electrolytes, hy- perglycemia, creatinine, plasma osmolarity, systemic blood pressure and gastrointestinal discomfort were recorded in all patients.

Statistical analysis

Data are expressed as means +- SD or percentages. Student t tests, ?2 tests and Fisher’s test were used to compare clinical characteristics of the study groups. We used paired t test to compare initial and final values intragroup for ammonia, oxidative stress biomarkers and other quantitative data. The correlation between ammonia levels and the se- verity of HE was analyzed with Spearman Correlation coefficients and 95% confidence intervals. The p values b 0.05 were considered to be sta- tistically significant. All tests were performed using Prism 5 software (GraphPad, San Diego, CA).

Results

Patient’s clinical characteristics

During the study period, 42 patients with HE were treated in the emergency room, 5 did not meet inclusion criteria, 7 had non- inclusion criteria. The remaining 30 participants were admitted and randomized. The baseline clinical characteristics were similar between the two groups (Table 1). The main cause of HE was gastrointestinal bleeding in both CG and MG (6 vs 4, respectively). Alcoholism was the main etiology of cirrhosis, whereas B-stage of Child-Pugh was the most common in both groups as shown in Table 1.

Severity of hepatic encephalopathy and hyperammonemia

In both groups the severity of HE decreased 48 h after treatment, in MG 13% reduced to grade 2, 40% to grade 1 and 47% were completely re- versed. In CG 20% reduced to grade 2, 40% to grade 1, and in 40% HE was reverted to grade 0 (Table 2).

Plasma ammonia concentrations were positively related to HE severity (Table 2). On arrival at the hospital (before treatment), in both groups the highest concentration of ammonia corresponded to the highest degree of HE. When treated with mannitol or conventional- ly, both groups reduced ammonia concentration and improved encephalopathy.

Correlation of the hepatic encephalopathy severity and hyperammonemia

The blood ammonia reference in healthy subjects is 16.6-47.3 for men and 11.5-38.0 umol/L for women. The highest ammonia concen- tration in venous blood was 401 umol/L for basal HE degree 4. The am- monia was well correlated (rs 0.86, 95% CI: 0.72-0.93) with basal HE, as shown in Fig. 1A. At the end of the treatment (48 h), the correlation de- creased (rs 0.40, 95% CI: 0.32-0.51) to lower degrees of ammonia and HE as shown in Fig. 1B.

Biomarkers of oxidative stress before and after treatment

The Table 3A, B and Fig. 2A, B, C, D show baseline (0 h) and final (48 h) concentrations of biomarkers in plasma of patients with MG and CG treatment. The data show high levels in OxS biomarkers related to lipid and protein damage in both groups. The decline of OxS plasma levels after 48 h of treatment was more evident for MDA (MG 29% and CG 27%, p b 0.001), while formazan was moderate and protein carbonyls, dityosines had a lower but significant reduction (p b 0.05) for MG and CG.

Safety and intestinal discomfort

The safety of mannitol through the intestine, assessed by concentra- tions of electrolytes, blood glucose, creatinine, osmolarity, and systolic/ diastolic blood pressure, was excellent for mannitol and slightly better than for lactulose. Gastrointestinal discomfort occurred less in MG in comparison with CG (p b0.05), especially a lower incidence of meteorism as shown in Table 4.

Discussion

We found that intestinal Mannitol is a convenient option for reduc- ing ammonia, oxidative stress, and HE with less intestinal discomfort and less adverse events than a conventional treatment in the emergen- cy room.

We selected mannitol for our study taking into account their natural ingestion in berries, fruits, vegetables and mushrooms, although the ex- traction of these products is not commercially profitable [13]. It has been considered safe by the FDA [11], by the European Polyols Associa- tion (EPA) [14] and by the European Union [15]. So far, it is non-

Table 1

Clinical characteristics and comorbidity of patients with hepatic encephalopathy.

Characteristics	Mannitol Group n = 15	Conventional Group n = 15	p Value
Age (years).	63 +- 2	56 +- 3	p = 0.06^a
Gender (Male/Female).	11/4	10/5	p = 0.69^b
Height (m).	1.59	1.60	p = 0.86^a
Weight (Kg).	67 +- 13	69 +- 10	p = 0.74^a
Body mass index (kg/m²)	28 +- 4	27 +- 4	p = 0.48^a
Diabetes Type 2	4/15 (27)	6/15 (40)	p = 0.43^b
Systemic Arterial Hypertension	3/15 (20)	1/15 (7)	p = 0.59^c
Hepatic Encephalopathy Causes: Infection	4/15 (27)	4/15 (27)	p = 1.31^c
Dietary protein overload	4/15 (27)	3/15 (20)	p = 1.00^c
Gastrointestinal bleeding	4/15 (27)	6/15 (40)	p = 0.43^b
Idiopathic	3/15 (20)	2/15 (13)	p = 1.00^c
Etiology of Cirrhosis:
Alcohol	10/15 (67)	12/15 (80)	p = 0.40^b
Infectious Hepatitis C	2/15 (13)	2/15 (13)	p = 1.00^c
Nonalcoholic steatohepatitis	3/15 (20)	1/15 (7)	p = 0.59^c
Child-Pugh class: A	3/15 (20)	4/15 (27)	p = 1.00^c
B	6/15 (40)	8/15 (53)	p = 0.71^b
C	6/15 (40)	3/15 (20)	p = 0.42^c

Data presented number of patients and (%), mean +- SD.

^a Student t test.

^b X² test.

^c Fisher’s test.

Table 2

Comparison between groups of hepatic encephalopathy and plasma ammonia.

	Mannitol Group		Conventional Group
Severity of Hepatic Encephalopathy	Plasma Ammonia (umol/L)	n (%)	Plasma Ammonia (umol/L)	n (%)	p Value
Before treatment (0 h)
Grade 0	–	–	–	–	–
Grade 1	–	–	–	–	–
Grade 2	102 +- 10	3/15 (20)	99 +- 18	5/15 (33)	0.783^a
Grade 3	145 +- 42	7/15 (47)	185 +- 37	6/15 (40)	0.137^a
Grade 4	312 +- 86	5/15 (33)	310 +- 49	4/15 (27)	0.972^a
After treatment (48 h) Grade 0	71 +- 50	7/15 (47)	75 +- 24	6/15 (40)	0.875^a
Grade 1	113 +- 38	6/15 (40)	81 +- 25	6/15 (40)	0.119^a
Grade 2	170 +- 18	2/15 (13)	94 +- 35	3/15 (20)	0.069^a
Grade 3	–	–	–	–	–
Grade 4	–	–	–	–	–

Decreased severity of HE and plasma concentration of ammonia in patients before (0 h) and after (48 h) of treatment. Data presented: mean +- SD, number of patients and (%).

^a Student t test.

carcinogenic, not-genotoxic and non-teratogenic; cataloged as safe in the pharmaceutical field and as a food sweetener. With respect to ad- verse effects, parenteral mannitol at high doses could cause hyperosmolality, decreased electrolytes (sodium and chloride), osmotic nephrosis, and seizures. But when is administered orally, could cause minimal intestinal discomfort, increased bowel sounds, flatulence, ab- dominal distension, and decrease in the consistency of feces and diar- rhea. Mannitol is a non-absorbable disaccharide that has not only a laxative effect, but also acts separating (filtration) harmful or toxic sub- stances from the blood through the enterovascular membrane as a dial- ysis. In this study we found a high correlation between ammonia and the severity of HE and a corresponding reduction post treatment with lactulose and mannitol.

The hepatic encephalopathy is a serious complication of chronic liver disease, a cause of frequent attention and reentry in the emergency room. The precise pathophysiological mechanism responsible for HE is not completely understood. However, increased glutamate, glutamine, ammonia [34] and oxidative stress have been demonstrated in plasma and brain tissue to be synergically associated to the development of HE [26,35].

The systemic ammonia increases in HE because mitochondrial or cy- tosolic Urea cycle is deficient in hepatocytes is surpassed by intestinal, renal or muscular production. Ammonia is generated as a cellular me- tabolite, by myocyte reactions that are catalyzed by the enzymes gluta- mate dehydrogenase, glutaminase and AMP-deaminase [34]. The ammonia generated by intestinal microbiota is produced by bacterial urease predominantly by gram-negative anaerobes, but enterocytes also generate large amounts of ammonia via intestinal glutaminase. A small amount of ammonia derives from the kidney by metabolization of glutamine via glutaminase. It is difficult to know whether the

elevated ammonia plasma levels come from kidney, muscle or intestine in patients with CLD.

In the hepatic acinus, the enzyme glutaminase and the urea cycle are heterogeneously distributed; ammonia detoxification occurs largely via the urea cycle in Zone 1 hepatocytes, and to a lesser degree, via conver- sion to glutamine in Zone 3 hepatocytes [36]. The urea cycle and the en- zyme glutaminase located in periportal region have a high ammonia detoxification capacity. Hepatic glutamine synthetase acts as an ammo- nia scavenger agent, and when there is acute or chronic liver disease there is a decrease in this capacity, causing a prolonged permanence of circulating ammonia thus, favoring cerebral toxicity [36]. We includ- ed patients with chronic liver disease in cirrhosis, therefore all enzyme systems should be reduced.

We managed to reduce significantly and comparably the severity of encephalopathy and hyperammonemia in CLD with two non- absorbable disaccharides by enemas. We chose lactulose (CG) as the standard treatment and compared it with mannitol (MG) as a therapeu- tic alternative based on its polyol mechanism of action and its prebiotic effect. Mannitol is metabolized by intestinal microbiota and by the colonocyte producing short-chain fatty acids and intestinal gases. The Lactobacilli spp. and Bifidobacterium spp. (Firmicutes) of the intestinal microbiota through the cycle of hexoses (glycolysis) metabolize manni- tol into pyruvate [37]. This produced pyruvate is used by bacterial cross- feeding (e.g. Eubacterium hallii/Anaerostipes spp./Coprococcus catus/ Veillonella spp./Phascolarcto bacterium succinatutens/Bacteroidetes), lead to intermediate metabolites (lactate, oxaloacetate, succinate, and Acetyl CoA) to generate short chain fatty acids, (propionate, butyrate and acetate, respectively) [38] and gases (hydrogenions and carbon di- oxide). Second, the reduction of intestinal and fecal pH (less than six) due to short chain fatty acids, gases, and mannitol itself, inhibits the

Fig. 1. Correlation between hepatic encephalopathy and plasma ammonia before and after treatment. spearman correlation of individual values for plasma total ammonia with the degree severity of hepatic encephalopathy, A. before (?p b 0.01) and B. after treatment (?p b 0.05).

Table 3

Plasma concentration and porcentual reduction of oxidative stress biomarkers

3A
Biomarkers in plasma	MDA (umol / L) 0 h	MDA (umol / L) 48 h	p value	Formazan (nmol / mg protein)	Formazan (nmol / mg protein) 48 h	p value
				0h
Mannitol Group	16.91 +- 5.03	12.00 +- 5.38 ^a	pb0.0001	8.65 +- 1.97	7.39 +- 1.71 ^b	pb0.001
Conventional Group	16.47 +- 7.36	12.02 +- 6.39 ^a	* (29%) pb0.0001	10.59 +- 1.87	9.44 +- 2.02 ^c	* (15%) pb0.01
			* (27%)			* (11%)
3B
Biomarkers in	Protein carbonyls	Protein carbonyls	p	Dityrosines (nmol / mg protein) 0	Dityrosines (nmol / mg protein) 48	p
plasma	(osazone nmol / mg	(osazone nmol / mg	value	h	h	value
	protein)	protein)
	0 h	48 h
Mannitol Group	4.25 +- 1.51	3.58 +- 1.12 ^d	pb0.01	1.03 +- 0.16	0.93 +- 0.15 ^d	pb0.05
			*			*
Conventional Group	3.61 +- 1.96	3.28 +- 1.69 ^d	(16%) pb0.05	0.97 +- 0.18	0.92 +- 0.16 ^d	(10%) pb0.05
			* (9%)			* (5%)

Data presented: Mean +- SD. ^a,b,c,d = paired t test. *(% OxS reduction).

production of ammonia by the microbiota and the colonocyte, traps ammonia as non-diffusable ammonia in the formed stools [39,40]. Third, an acid intestinal environment favors the survival of Lactobacillus and Bifidobacteria that do not produce ammonia on the remaining “positive urease microbiota” generators of ammonia by hydrolysis of in- testinal urea (e.g. Bacteroidetes: Enterobacteriaceae, Bacteroidaceae, Veillonellaceae, Alcaligeneceae, Fusobacteriaceae and Streptococcaceae) [40]. Finally, any non-absorbable disaccharides (as mannitol) that is ad- ministered orally or by enema, have a laxative mechanic effect, favoring fast colonic transit, reducing ammonia absorption from the colon, and the excretion of fecal nitrogen [41]. All these effects resulted in less pro- duction of intestinal ammonia by enterocyte glutaminase, reducing bacte- rial translocation, modulating proinflammatory responses, and gut permeability [42,43]. Residual ammonia inside the intestine that escapes into the porto-systemic circulation and crosses the blood-brain barrier, will be metabolized by glutamine synthetase to glutamine into the astro- cytes. This event will result in detoxification of the brain and

improvement of encephalopathy [44]. We found an important reduction of ammonia plasma concentration after treatment with mannitol or lactulose. Mannitol is a good choice when lactulose is not available or is contraindicated in the therapy of HE.

On the other hand, it is important to reduce OxS in HE because this is one of the most important mechanisms for the initiation and progres- sion of the disease [45,46]. Liver damage caused by OxS induces irreversible alterations in lipid, protein, DNA integrity, and more impor- tantly in pathways that control biological functions [45]. Exposure to high concentrations of ammonia in cell cultures (astrocytes) and animal models (rats), induces production of free radicals [47], generation of cerebral nitric oxide [48] and superoxide, an decrease of antioxidant en- zymes activity (Glutathione peroxidase, superoxide dismatase, catalase) in both brain and liver tissue [49]. This oxidative damage by ROS and ERN in patients with CLD, includes lipid peroxidation (cell membranes), DNA break down, and potentially indiscriminated oxidation of all struc- tural molecules and functional proteins [50]. Stable products such as

Fig. 2. Oxidative stress biomarkers: mannitol vs conventional groups. A) MDA: Malondialdehyde, B) Formazan, C) Protein carbonyls: Ozasone,2D) Dityrosines. Mean +- SD. ? paired t test.

Table 4

Adverse effects and intestinal discomfort before and after treatment in both groups.

Adverse effects and Intestinal discomfort	Mannitol Group			Conventional Group
	0 h	48 h	p Value	0 h	48 h	p Value
Blood electrolytes:
Sodium (mEq/L)	143 +- 2	142 +- 2	0.438^a	142 +- 6	140 +- 5	0.79^a
Potassium (mEq/L)	4.4 +- 1	4.2 +- 1	0.414^a	3.9 +- 1	3.5 +- 1	0.76^a
Chlorine (mEq/L)	108 +- 7	106 +- 5	0.576^a	107 +- 6	106 +- 5	0.40^a
Glycaemia (mg/dL)	114 +- 26	106 +- 24	0.235^a	119 +- 27	123 +- 26	0.93^a
Blood creatinine (mg/dL)	1.3 +- 0.4	1.2 +- 0.3	0.191^a	1.4 +- 0.6	1.3 +- 0.5	0.19^a
Clearance creatinine (mg/min/m²SC)	55 +- 27	58 +- 15	0.540^a	67 +- 24	69 +- 38	0.73^a
Osmolality (mosmol/kg)	302 +- 9	299 +- 5	0.311^a	298 +- 14	295 +- 11	0.48^a
Systolic blood pressure (mmHg)	111 +- 7	109 +- 6	0.674^a	107 +- 11	106 +- 11	0.76^a
Diastolic blood pressure (mmHg)	75 +- 6	72 +- 9	0.104^a	71 +- 8	69 +- 10	0.86^a
Intestinal discomfort (total):
Flatulence (%)		6/15 (40)	13/15 (86)			?0.01^c
Borborygmos (%)		5/15 (33)	7/15 (47)			0.45^b
Meteorism (%)		1/15 (7)	2/15 (15)			0.54^c
Adverse effects		0/15 (0)	4/15 (27)			?0.03^c
Hypokalemia		4/15 (27)	10/15 (66)			?0.02^c
Hyperglycemia		2/15 (14)	3/15 (20)			0.06^c
Hypernatremia		0/15 (0)	0/15 (0)			1.00^c
Hyperchloremia		1/15 (7)	3/15 (20)			0.28^c
Hyperosmolar state		1/15 (7)	3/15 (20)			0.28^c
Increase Cr N 0.3 mg/dL		0/15 (0)	0/15 (0)			1.00^c
		0/15 (0)	1/15 (7)			0.30^c
Data presented are (%) and number of patients. Mean +- SD. Cr = Creatinine.
^a Paired t test.

^b X² test.

^c Fisher’s test.

* p b 0.05.

malondialdehyde [51], protein carbonyls [52], formazan [53], dityrosines [54] and nitrotyrosines [55] are biomarkers of lipid and pro- tein oxidation [56], and are elevated in encephalopathy and CLD. We found high levels of these oxidative stress biomarkers in our patients, however lactulose or mannitol in the intestine reduced OxS in direct Declaration of interest“>proportion to ammonia in plasma. This OxS reduction was slightly greater in MG than CG, therefore mannitol can be a good alternative for HE. Mannitol that has showed an scavenger effect on the free radi- cals in vitro [57,58] and is probably the same mechanism in vivo.

Regarding safety and adverse effects, such as Electrolyte disorders, hy- perglycemia, hyperosmolar state, acute renal injury and hypotension, these occurred in two thirds of patients in the lactulose group, compared with less than one third in the mannitol group. Another important differ- ence was intestinal complaints as flatulence and borborygmos which fre- quently occurred in almost all patients on lactulose, but was reduced to about one half when we used mannitol. Therefore, mannitol showed bet- ter tolerance than conventional treatment.

The current study had some limitations: a) the small size of the sam- ple was according with the intention to know safety and patients were followed for only 48 h, b) the biomarkers were not measured in brain tissue, cerebrospinal fluid, or liver because of ethic principles although plasma measures a systemic profile, c) third, we did not include patients with degree 0 or 1 HE severity because treatment was ambulatory,

d) levels of free iron in plasma were not quantified, since they could favor an endogenous Fenton reaction thus contributing to the develop- ment of oxidative stress, e) although mannitol and lactulose are not absorbed from the intestine, the total body water deficit and hourly di- uresis were not evaluated to confirm that this did not happen.

Conclusions

We confirm direct relationship between ammonia levels with the severity of HE, and plasma levels of OxS to lipids and proteins. Therefore, we confirm hyperamonnemia and oxidative stress in our patients with functional neuronal damage and the possibility to reduce both in order to protect the central nervous system. Also our results confirm that in- testinal mannitol is an option for HE treatment, because reduce the

oxidative stress, plasma ammonia and HE with less intestinal discom- fort. It is necessary to include more patients with different characteris- tics in order to obtain external validity. Intestinal mannitol is a secure and well tolerated therapeutic alternative for improve the encephalop- athy in CLD in the emergency room.

Declaration of interest

The authors of this study have no actual or potential conflict of inter- est to declare that could inappropriately influence, or be perceived to in- fluence, their work.

Funding

This research did not receive any specific grant from funding agen- cies in the pharmacist, commercial, or not-for-profit sectors.

References

AJEM