Glutathione species and metabolomic prints in subjects with liver disease as biological markers for the detection of hepatocellular carcinoma

Abstract

Background: The incidence of liver disease is increasing in USA. Animal models had shown glutathione species in plasma reflects liver glutathione state and it could be a surrogate for the detection of hepatocellular carcinoma (HCC).

Methods: The present study aimed to translate methods to the human and to explore the role of glutathione/metabolic prints in the progression of liver dysfunction and in the detection of HCC. Treated plasma from healthy subjects (n = 20), patients with liver disease (ESLD, n = 99) and patients after transplantation (LTx, n = 7) were analyzed by GC- or LC/MS. Glutathione labeling profile was measured by isotopomer analyzes of ²H₂O enriched plasma. Principal Component Analyzes (PCA) were used to determined metabolic prints.

Results: There was a significant difference in glutathione/metabolic profiles from patients with ESLD vs healthy subjects and patients after LTx. Similar significant differences were noted on patients with ESLD when stratified by the MELD score. PCA analyses showed myristic acid, citric acid, succinic acid, l-methionine, d-threitol, fumaric acid, pipecolic acid, isoleucine, hydroxy-butyrate and glycolic, steraric and hexanoic acids were discriminative metabolites for ESLD-HCC⁺ vs ESLD-HCC^- subject status.

Conclusions: Glutathione species and metabolic prints defined liver disease severity and may serve as surrogate for the detection of HCC in patients with established cirrhosis.

Figure 1

a. Glutathione species (GSH = glutathione reduced; GSSG = glutathione oxidized and bound; ratio = GSH/GSSG-bound²; OA = ophthalmic acid)) in healthy controls (n = 10) fasted for 8 h or after subjects were fed an 800 calories meal. Results are expressed as mean on relative concentration units. There was a statistical significant difference between the fast vs fed status in the relative concentrations of GSH and OA (t-test, p < 0.05). b. Tier 1 metabolites (glucose, glycerol and lactate) in healthy controls (n = 10) fasted for 8 h or after subjects were fed standardized 800 calories meal. There was a statistical significant difference between the fast status vs fed in the relative concentrations of glucose, glycerol and lactate (ANOVA followed by t-test, p < 0.05)

Figure 2

Turn over of glutathione species. (GSH = reduced, GSSG = oxidized and bound and OA = ophthalmate) in healthy subjects (n = 10) by isotopomer analyses (M1) on²H₂O enriched plasma. There was not a difference in the synthesis of glutathione sp. on healthy subjects a) Fasted for 8 h vs b) Fed a standardized 800 calorie meal (p > 0.05 by ANOVA)

Figure 3

a. Glutathione species (GSH = glutathione reduced; GSSG = glutathione oxidized and bound; ratio = GSH/GSSG-bound²; OA = ophthalmic acid)) in fasted subjects from healthy controls (n = 20) and patients with end stage liver disease stratified by the MELD score (<14; n = 72) and ≥14; n = 27). Results are expressed as mean on relative concentration units. There was a statistical significant difference between the glutathione species print from patients with ESLD (either lower or higher MELD scores) compared to the print of healthy individuals (p < 0.05, by t-test). In addition there was a significant difference in the glutathione species print of patients with ESLD and lower vs higher MELD score (p < 0.05). b. Tier 1 metabolites (glucose, glycerol and lactate) in fasted subjects from healthy controls (n = 20) and form patients with end stage liver disease stratified by the MELD score (<14; n = 72) and ≥14; n = 27). There was a statistical significant difference between the Tier 1 metabolic print from patients with ESLD (either lower or higher MELD scores) compared to the print of healthy individuals (p < 0.05). In addition there was a significant difference in the Tier 1 metabolic print of patients with ESLD and lower vs higher MELD score (p < 0.05, by t-test)

Figure 4

Principal Component Analyses (PCA) and Volcano Plot of metabolites in patients with ESLD. a. Tier 2 metabolites were measured in patients with end stage liver disease (ESLD) and healthy subjects. In was found pyro-glutamic acid, pyruvate, cholesterol and glucose significantly discriminate patients with ESLD and MELD ≥14 when compared to healthy controls. Results are displayed in b as a Volcano plot. In addition, PLS-DA, found lysine and fructose as additional discriminant metabolites between compared groups (VP scores no shown)

Figure 5

a. Glutathione species (GSH = glutathione reduced; GSSG = glutathione oxidized and bound; ratio = GSH/GSSG-bound²; OA = ophthalmic acid)) in fasted subjects from three groups: healthy controls (n = 20), patients with end stage liver disease and no primary malignancy (HCC⁻, n = 83) and patients with end stage liver disease and primary malignancy (HCC⁺, n = 16). Results are expressed as mean on relative concentration units. There was a statistical significant difference between the glutathione species from patients with ESLD with or without malignancy compared to the print of healthy individuals (p < 0.05, by ANOVA followed by t-test). b. Tier 1 metabolites (glucose, glycerol and lactate) in fasted subjects from healthy controls (n = 20) and patients with ESLD by tumor status. There was a statistical significant difference between the Tier 1 metabolic print from patients with ESLD with or without malignancy compared to the print of healthy individuals (p < 0.05). Furthermore, there was a significant difference in the Tier 1 metabolic print of patients with ESLD and HCC⁻ vs HCC⁺ (p < 0.05)

Figure 6

Volcano Plot of metabolites by PCA & PLS-DA in patients with ESLD. a. Tier 2 metabolites were measured in patients with end stage liver disease (ESLD) and tumor (n = 16)) vs healthy subjects (n = 20). In was found cholesterol, pyroglutamic acid, inositol, ethanolamine, fructose, pyruvate, citric acid and lysine were discriminative metabolites. Additional metabolites displayed in the volcano plot included glycolic acid, galacturonic acid and ribose. Importance in projection (VIP) Scores for each metabolite found by PLS-DA are displayed. b. Tier 2 metabolites were measured in patients with ESLD-HCC⁺ (n = 16)) vs ESLD-HCC⁻(n = 68). It was found myristic acid, citric acid, succinic acid, l-methionine, d-threitol, fumaric acid, pipecolic acid, isoleucine, glycolic acid, hydroy-butirate, steraric acid and hexanoic acid were discriminative metabolites. No additional metabolites were found in the volcano plot. Importance in projection (VIP) Scores for each metabolite found by PLS-DA are displayed

Figure 7

Synthesis of glutathione sp. (GSH = reduced and GSSG = oxidized and bound) in fasted subjects from three groups: a) healthy controls (n = 10),b) patients with end stage liver disease (ESLD, n = 7) andc) patients after liver transplantation (LTx, n = 7). There was a statistical significant difference between the glutathione species labeling from patients with ESLD when compared to the glutathione synthesis from healthy individuals (p < 0.05 by ANOVA). The glutathione labeling from patients after liver transplantation were similar to the one from healthy patients