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. 1998 Jun;42(6):861–867. doi: 10.1136/gut.42.6.861

Endogenous benzodiazepine-like compounds and diazepam binding inhibitor in serum of patients with liver cirrhosis with and without overt encephalopathy

R Avallone 1, M Zeneroli 1, I Venturini 1, L Corsi 1, P Schreier 1, M Kleinschnitz 1, C Ferrarese 1, F Farina 1, C Baraldi 1, N Pecora 1, M Frigo 1, M Baraldi 1
PMCID: PMC1727144  PMID: 9691927

Abstract

Background/Aim—Despite some controversy, it has been suggested that endogenous benzodiazepine plays a role in the pathogenesis of hepatic encephalopathy. The aim of the present study was to evaluate the concentrations of endogenous benzodiazepines and the peptide, diazepam binding inhibitor, in the blood of patients with liver cirrhosis with and without overt encephalopathy, and to compare these levels with those of consumers of commercial benzodiazepines. 
Subjects—Normal subjects (90), benzodiazepine consumers (14), and cirrhotic patients (113) were studied. 
Methods—Endogenous benzodiazepines were measured by the radioligand binding technique after high performance liquid chromatography (HPLC) purification. The presence of diazepam and N-desmethyldiazepam was assayed by HPLC-electrospray tandem mass spectrometry. Diazepam binding inhibitor was studied in serum by radioimmunoassay. 
Results—Endogenous benzodiazepines were below the limit of detection in 7% of patients with encephalopathy. When detectable, their levels were at least comparable with those of benzodiazepine consumers and correlated with the liver dysfunction but not the stage of encephalopathy. Serum levels of diazepam binding inhibitor tended to decrease when endogenous benzodiazepines levels increased. 
Conclusions—Endogenous benzodiazepines may accumulate in patients with liver cirrhosis during the course of the disease, and the phenomenon appears to be independent of the presence or absence of encephalopathy. 



Keywords: benzodiazepine consumers; diazepam binding inhibitor; endogenous benzodiazepines; liver cirrhosis; overt hepatic encephalopathy

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Figure 1 .

Figure 1

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Serum levels of benzodiazepine (BZD)-like compounds measured by the radioligand binding technique after HPLC purification in liver cirrhotic patients without encephalopathy (stage 0), with encephalopathy (OHE; stages I, II, III and IV) and in BZD consumers. The horizontal bar represents the mean value. Kruskal-Wallis one way analysis of variance shows a significant difference between groups (p<0.0001). The Mann-Whitney U test adjusted using the Bonferroni correction shows that patients with liver cirrhosis without OHE (stage 0) differed from all the patients with OHE (p<0.001). The levels found in patients with OHE stages I, II, III, and IV were not different from each other and not different from the levels found in BZD consumers. 


Figure 2 .

Figure 2

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Serum levels of benzodiazepine (BZD)-like compounds measured by the radioligand binding technique after HPLC purification in control subjects and in liver cirrhotic patients divided according to the severity of the disease by the Child-Pugh classification. The horizontal bar represents the mean values. Kruskal-Wallis one way analysis of variance shows a significant difference between groups (p<0.0001). The Mann-Whitney U test adjusted using the Bonferroni correction shows that the BZD concentrations found in Pugh-Child class A patients did not differ from controls, those found in Pugh-Child class B differed from those in Pugh-Child class A (p<0.001), and those found in Pugh-Child class C differed from those in Pugh-Child class A (p<0.001) and those in Pugh-Child class B (p<0.05). 


Figure 3 .

Figure 3

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Mass chromatograms of serum samples from two patients (A and B) with stage IV hepatic encephalopathy with increased benzodiazepine-like compounds measured by the radioligand binding technique after HPLC purification. The upper panel for patient A shows the presence of molecules undergoing fragmentation at 271/140 m/z, which is characteristic of N-desmethyldiazepam, and the central panel shows the presence of molecules undergoing fragmentation at 285/257 m/z, which is characteristic of diazepam, obtained by selected reaction monitoring (SRM) mass scan. The bottom panel for patient A represents the reconstructed ion chromatograms (RIC) which confirms the presence of both benzodiazepines. Scanning the sample of patient B for molecules with the same fragmentation patterns as above showed the absence of both benzodiazepines, and the RIC confirms this result. 


Figure 4 .

Figure 4

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Plasma levels (mean (SD)) of diazepam binding inhibitor-like immunoreactivity (DBI-LI) in control subjects, in liver cirrhotic patients without overt hepatic encephalopathy (OHE), in patients with different stages of OHE, and in benzodiazepine (BZD) consumers. Kruskal-Wallis one way analysis of variance shows a significant difference between groups (p<0.0001). The Mann-Whitney U test adjusted using the Bonferroni correction shows that the levels found in liver cirrhotic patients with or without OHE were statistically different from controls (p<0.05 and p<0.001 respectively). The DBI-LI in BZD consumers was different from controls (p<0.005) and practically equal to those found in cirrhotic patients with or without OHE. 


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