Menoufia Medical Journal

ORIGINAL ARTICLE
Year
: 2016  |  Volume : 29  |  Issue : 4  |  Page : 895--903

Plasma free amino acid profile changes in hepatocellular carcinoma patients


Ahmed A Raouf1, Hatem M El-Sebaey2, Ayman K Abd El-Hamead2, Ashraf Y El-Fert1, Yasmin E El-Gendy1,  
1 Department of Clinical Biochemistry, National Liver Institute, Shebin El-kom, Egypt
2 Department of Medical Biochemistry, Faculty of Medicine, Menoufia University, Shebin El-kom, Egypt

Correspondence Address:
Yasmin E El-Gendy
Department of Clinical Biochemistry, National Liver Institute, Menoufia University, Yassin Abdel-Ghaffar Street, Shebin El-kom, Menoufia, 32511
Egypt

Abstract

Objective The aim of this study was to investigate plasma amino acid profile as a possible diagnostic/prognostic method in patients with hepatocellular carcinoma. Background Many previous reports have shown that metabolism is notably altered in cancer cells. Thus, various methods for cancer diagnosis and prognosis have been developed based on metabolite analysis. Among whole metabolites, amino acids were proposed as suitable candidates for focused metabolomics as they are either ingested or synthesized endogenously and they play an essential physiological role both as basic metabolites and metabolic regulators. Patients and methods This study included 71 hepatocellular carcinoma patients (60 male and 11 female) and 30 chronic hepatitis C virus patients (20 male and 10 female) in addition to 30 healthy controls (25 male and 5 female). Laboratory investigations including complete blood picture, liver function tests, serum α-fetoprotein, hepatitis viral markers (HBsAg and anti-HCVAb) were carried out for all participants. Amino acid assay was carried out using ultra performance liquid chromatography–electrospray ionization-mass spectrometry. Results The study showed a statistically significant increase in methionine, tyrosine, ornithine, citrulline, glycine, phenylalanine, alanine, glutamate, proline, and arginine and a decrease in valine, aspartate, leucine/isoleucine, branched tyrosine ratio (BTR), and Fischer's ratio in hepatocellular carcinoma compared with the control group. Moreover, the study indicated that there was a statistically significant correlation between BTR and platelets, alanine transaminase, albumin, bilirubin, and α-fetoprotein and significant differences in BTR among Child-Pugh A, B, and C. Conclusion Our results suggested that the plasma free amino acid profile is considered valuable for diagnosis and nutritional care in cancer patients. Furthermore, BTR reflects the pathological liver background with a high degree of correlation with other liver functional markers.



How to cite this article:
Raouf AA, El-Sebaey HM, Abd El-Hamead AK, El-Fert AY, El-Gendy YE. Plasma free amino acid profile changes in hepatocellular carcinoma patients.Menoufia Med J 2016;29:895-903


How to cite this URL:
Raouf AA, El-Sebaey HM, Abd El-Hamead AK, El-Fert AY, El-Gendy YE. Plasma free amino acid profile changes in hepatocellular carcinoma patients. Menoufia Med J [serial online] 2016 [cited 2020 Mar 28 ];29:895-903
Available from: http://www.mmj.eg.net/text.asp?2016/29/4/895/202493


Full Text

 Introduction



Hepatocellular carcinoma (HCC) is considered the fifth in frequency among all types of cancers worldwide and the third cause of cancer-related deaths [1]. It is usually asymptomatic in the early stages and tends to be invasive. Therefore, most patients present with an incurable disease at the time of detection, which makes early diagnosis of HCC critical for a good prognosis. Surgical resection remains the treatment of choice for HCC, but unfortunately only 10–20% of primary HCCs are resectable at the time of diagnosis. Continuous research studies are ongoing worldwide to find out and evaluate sensitive and specific new markers for hepatocellular diagnosis [2].

The most common cause of HCC is chronic hepatitis or liver cirrhosis caused by hepatitis C virus (HCV) and hepatitis B virus (HBV) infection. Therefore, early detection of patients at risk, such as chronic carriers of HBV and HCV, is justified to improve the outcome of treatment of hepatocellular malignancy [3],[4].

In Egypt, the incidence rate of HCC has increased sharply in the last decade [5]. This could be attributed to HBV and HCV epidemic infection. Furthermore, improvements in diagnostic tools and healthcare led to an increased survival rate among cirrhotic patients, allowing time for some of them to develop HCC [6]. However, not all individuals infected with HBV/HCV develop HCC, thus indicating the implication of other environmental and genetic risk factors in the multistage process of this complex disease [7].

The prognosis of HCC has improved dramatically with the identification of high-risk populations and the advancement of diagnostic imaging and treatment [8]. For further improvement of the prognosis of HCC, it is necessary to assess not only cancer progression but also hepatic functional reserve. Hepatic functional reserve is recognized as a factor affecting survival in the treatment of HCC [9].

To date, the Child-Pugh classification system, which determines the severity of the liver disease according to the degree of ascites, the plasma concentrations of bilirubin and albumin, the prothrombin time, and the degree of encephalopathy, has been the most widely used system for assessing hepatic functional reserve in chronic liver disease (CLD) and in patients undergoing treatment for HCC [10]. In the Child-Pugh classification, the serum albumin level is used to achieve accurate assessment of the status of protein metabolism.

Serum albumin is a protein that is synthesized and secreted by hepatocytes, and is used as an index of hepatic synthetic capacity for protein. This parameter is particularly important for evaluating the severity and prognosis of cirrhosis [8]. However, to date, no attention has been given to the status of amino acid (AA) metabolism in CLD and HCC [11]. Protein malnutrition of HCC patients is a result of amino acid imbalance. Thus, accurate assessment of protein metabolism in HCC patients with a background of CLD needs not only the determination of the serum albumin level but also the status of amino acid metabolism [12].

The Fischer ratio is the molar ratio of branched-chain amino acids (BCAAs) (leucine, valine, and isoleucine) to aromatic amino acids (phenylalanine and tyrosine), and is important for assessing hepatic metabolism and hepatic functional reserve, as well as for judging the severity of liver damage [12].

The branched tyrosine ratio (BTR) is a simpler method and can be a substitute for the Fischer ratio [13]. Furthermore, it is reported that patients with a low BTR, even patients in Child-Pugh class A, have already begun to demonstrate a potential decrease in hepatic functional reserve [14]. In addition, in the event of malnutrition, BTR declines before the serum albumin declines; therefore, determining BTR is useful for the early detection of potential hypoalbuminemia [15].

The significance of amino acid analysis for assessing hepatic functional reserve has not been elucidated in patients with HCC. BTR decreases in advance of decreases in serum albumin level. Thus, early identification of patients at risk for hypoalbuminemia is possible; specifically, determination of BTR enables the prediction of changes in the serum albumin level [15],[16], in turn allowing the prediction of the need for administration of BCAAs. Moreover, because of the existence of that time lag, monitoring of BTR separately from albumin is necessary when considering prognostic factors for HCC. A large-scale clinical study has demonstrated the usefulness of administering oral BCAA preparations for patients showing decreased BTR. In other words, there is a strong possibility that determining BTR provides a prognostic factor for hepatocellular carcinoma [17].

The aim of this study was to investigate plasma free amino acid profile as a possible diagnostic/prognostic factor in HCC patients.

 Patients and Methods



Study population

This study was conducted on 131 participants, including 71 diagnosed untreated HCC patients. They were presented to the Hepatology Department, National Liver Institute, Menoufia University. Diagnosis was based on clinical examination, laboratory tests, ultrasound, and computed tomography. Another group comprised 30 chronic HCV patients in addition to a control group comprising 30 age and sex matched individuals.

All patients and controls were subjected to the following: full history taking, complete clinical examination, abdominal ultrasonography and or computed tomography, and laboratory investigations including complete blood picture, liver function tests (aspartate transaminase, alanine transaminase (ALT) [18], serum bilirubin, serum albumin, serum alkaline phosphatase, serum total proteins, and prothrombin concentration), viral markers (HBsAg and HCVAb) [19], estimation of serum α-fetoprotein (AFP) [20], and amino acid assay.

Written consent was obtained from each individual and the protocol was approved by the ethical committee of Menoufia Faculty of Medicine.

Sample collection

Ten ml of venous blood was collected from all participants included in this study through venipuncture from the cubital vein. Blood was collected as follows: 4 ml was collected into EDTA-containing tubes, for CBC and amino acid profiles (blood is dripped onto a filter paper and dried using 903 Whatman filter paper); and 2 ml was collected into a citrated tube for prothrombin time and concentration. The remaining 4 ml was collected in a plain vacutainer tube, left 15 min for coagulation, and then centrifuged at 3000 rpm for 10 min. The sera were then separated into aliquots for measurement of liver function tests, viral markers (HBsAg and anti-HCVAb), and AFP.

Amino acid assay using HPLC tandem mass spectrometry (LC–MS/MS)

The blood spots were analyzed for amino acids using a triple-quadrupole tandem mass spectrometer (ACQUITY UPLC H-Class; Waters corporation, Milford, Massachusetts, USA) with a positive electrospray ionization probe, utilizing Mass Chrom Amino acids and Acylcarnitines from Dried Blood/Nonderivatised kit (Chromsystems Instruments & Chemicals GmbH, München, Germany). This method uses stable, isotopically labeled (deuterated) internal standards for calibration and measurement [21].

Sample preparation procedure

Punch a 3 mm dried blood spot disk out of the filter card into a well of a microtiter plate. Thereafter, add 100 μl of the reconstituted internal standard. Seal the microtiter plate with a protective sheet and agitate at 600 rpm for 20 min at ambient temperature. Thereafter, remove the protective sheet from the microtiter plate and transfer the supernatant into a new microtiter plate. Seal the microtiter plate with an aluminum foil protective sheet. Inject 10 μl of the eluate into the LC–MS/MS system. Additional controls in each analytical run were added for monitoring the precision and accuracy of the analyses.

Chromatography conditions and instrument parameters

The instrument used for analysis is a liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and Masslynx V4.1 software (Waters Corporations, Milford, USA). No HPLC column or column oven is required. The MS/MS system contains two mass spectrometers connected in series. In the first mass spectrometer (MS 1), the ions are separated on the basis of their mass-to-charge ratio. Subsequently, the ions reach a collision cell, where they dissociate into fragments induced by colliding with an inert gas (argon or nitrogen). After this, the second mass spectrometer (MS 2) analyzes the characteristic fragmentation again on the basis of their mass-to-charge ratio [21].

Instrument settings

10 μl of elute was injected into the LC–MS/MS system, where the flow of mobile phase (provided by the kit) was adjusted to 200 μl/min to be reduced to 20 μl/min at 0.25 min and up again to 600 μl/min at 1.25 min to be reduced again to 200 μl/min as the scan time of the tandem MS system has to be set at 1.25 min.

Statistical methods

Data were statistically analyzed using SPSS (statistical package for social science) program, version 13 for windows for all analyses. P value less than 0.05 was considered statistically significant.

 Results



The present study showed a statistically significant difference between the HCC and the control group, with a statistically significant increase in methionine (P < 0.001), tyrosine (P < 0.001), ornithine (P < 0.001), citrulline (P < 0.001), glycine (P < 0.001), phenylalanine (P < 0.001), alanine (P < 0.001), glutamate (P < 0.001), proline (P = 0.001), and arginine (P < 0.001), and a decrease in valine (P < 0.001), aspartate (P < 0.001), leucine/ileucine (P < 0.001), BTR (P < 0.001), and Fischer's ratio (P < 0.001) in the HCC group compared with the control group.

There was a statistically significant difference between the CLD and the control group, with a statistically significant increase in methionine (P < 0.001), tyrosine (P < 0.001), citrulline (P = 0.003), proline (P = 0.005), and arginine (P < 0.001), and a decrease in ornithine (P = 0.04), valine (P < 0.001), aspartate (P = 0.003), leucine/ileucine (P < 0.001), BTR (P < 0.001), and Fischer's ratio (P < 0.001) in the CLD compared with the control group. No statistically significant difference was found as regards phenylalanine (P = 0.37), alanine (P = 0.48), glycine (P = 0.11), and glutamate (P = 0.06).

There was a statistically significant difference between the HCC and the CLD group, with a statistically significant increase in methionine (P < 0.001), tyrosine (P = 0.01), ornithine (P < 0.001), citrulline (P = 0.002), glycine (P < 0.001), phenylalanine (P < 0.001), alanine (P = 0.002), and glutamate (P = 0.01), and a decrease in valine (P = 0.004), aspartate (P = 0.05), leucine/isoleucine (P = 0.003), arginine (P = 0.001), BTR (P = 0.001), and Fischer's ratio (P < 0.001) in the HCC group compared with the CLD group.

As regards BTR, the results showed that there was a significant correlation between BTR and albumin level among HCC patients (r = 0.65; P < 0.001), CLD patients (r = 0.51; P = 0.004), and controls (r = 0.62; P < 0.001), with a tendency for the low BTR group to have lower serum albumin levels. The study also revealed a significant correlation between BTR and total bilirubin, ALT, prothrombin time, and AFP in HCC patients (r=−0.65, P < 0.001; r=−0.33, P = 0.005; r = 0.69, P < 0.001; and r = 0.28, P = 0.01, respectively) with a tendency for the low BTR group to have a higher level of total bilirubin, ALT, prothrombin time, and AFP. Moreover, there was a statistically significant difference in BTR among Child-Pugh A, B, and C (Child-Pugh A: 1.509 ± 0.324, Child-Pugh B: 1.303 ± 0.774, and Child-Pugh C: 0.8357 ± 0.194) among patients in the HCC group ([Table 1],[Table 2],[Table 3],[Table 4],[Table 5]).{Table 1}{Table 2}{Table 3}{Table 4}{Table 5}

 Discussion



HCC is a major challenge in contemporary medicine. The incidence of HCC is on increase and it is becoming more and more significant both clinically and epidemiologically [1].

It has been reported that metabolism, including that of amino acids, is not only altered in cancer cells but also in plasma [22].

Although a tumor tissue is restricted to a certain organ, changes in the levels of amino acids are affected by their metabolism in multiple organs of the body. Thus, the plasma of patients with cancer mainly contains all information concerning the pathogenic changes caused by the disease in the content of the metabolites, which reflect aberrant alterations at the level of gene expression and regulation, as well as abnormalities in the function of multiple organs and tissues [23].

In view of the previous observations, the aim of the present work was to investigate plasma amino acid profile as a possible diagnostic/prognostic method in patients with HCC.

In this study, plasma arginine level was significantly increased in patients with HCC compared with the control group. Plasma arginine concentrations have been investigated in various types of cancer, and the results seem inconsistent. Kubota et al. [24] have reported that plasma arginine concentrations were increased in breast cancer. In contrast, Vissers et al. [25], who investigated 21 patients with pancreatic cancer and nine patients with colonic cancer, showed that plasma arginine level was decreased in pancreatic and colonic cancer patients. However, Naini et al. [26] reported that plasma arginine level was unchanged in lung cancer and head and neck cancer.

The present study also revealed a significantly increased concentration of plasma level of proline in HCC patients. This is consistent with the findings of Miyagi et al. [27], who reported that the proline level was increased in lung cancer, gastric cancer, colorectal cancer, breast cancer, prostate cancer, and acute leukemia. However, a study carried out by Hasim et al. [22] revealed that plasma proline levels in cervical cancer were significantly lower than those in controls.

In the present study, the BCAA (valine, leucine, and isoleucine) levels in the plasma of patients with HCC were significantly lower than those in patients with CLD and controls. This is in agreement with the findings of Watanabe et al. [28], who investigated plasma free amino acid concentrations in 14 patients with HCC. They reported a significant decrease in BCAA level in HCC patients. Lee et al. [29] also agreed with the results of the current study, as they reported that BCAA were decreased in HCC patients. Further, a study conducted by Hasim et al. [22], which evaluated the plasma free amino acid profile profiles in 22 cervical squamous cell carcinoma patients and 35 healthy controls revealed significant reductions in BCAA levels in cervical cancer patients.

The present study also revealed a significantly higher level of glutamate amino acids in patients with HCC compared with the control and CLD groups. This was explained by Holecek. [30], who reported an increased level of glutamate amino acid in patients with cirrhosis compared with the control group; they attributed the increased levels to the stimulatory effect of hyperammonemia on glutamate synthesis in extrahepatic tissue as a result of hepatocyte dysfunction and portosystemic shunting Holecek et al. [31]. The findings of Huang et al. [32] are in line with the results of this work as they reported an increase in the level of glucogenic amino acids, including glutamate, in hepatocellular carcinoma tissue compared with distal noncancerous tissue.

The present study revealed a significantly lower level of aspartic acid in HCC patients compared with CLD patients and controls. This is in agreement with the findings of Wang et al. [33], who reported that aspartic acid was too low to be detected in most patients with cancer colon. Leichtle et al. [34] also agreed with the results of the current study, as they showed a decreased level of aspartic acid in patients with colorectal cancer compared with controls. A study conducted by Chen et al. [35] also demonstrated that aspartic acid was consistently depleted at each of the four stages of HCC.

Although Chen et al. [35] showed that ornithine and citrulline were significantly decreased in HCC patients compared with healthy controls, this study showed a significant increase in the level of ornithine and citrulline amino acids. These amino acids participate in the urea cycle, which allows for the disposal of excess nitrogen that takes place only in mammalian hepatocyte [36]. Similar studies that have been conducted on a few other carcinomas reported an increase in the level of these amino acids. For example, Poschke et al. [37] demonstrated an increased level of these amino acids in patients with breast cancer compared with healthy controls, and Proenza et al. [38] reported a significant increase in patients with lung cancer. A study conducted by Cascino et al. [39] reported a significant increase in breast cancer patients. Similar results have been reported by Kubota et al. [24] in head and neck cancer in female patients.

This study also showed a significant increase in glucogenic amino acids (methionine, alanine, and glycine) in HCC patients compared with other studied groups. The explanation of this is that tumor growth consumes a large amount of energy. Glycolysis was ascertained as the major source of energy [40]. Moreover, the increased consumption of glucose may upregulate gluconeogenesis from lipids and proteins. Because of the increased catabolism, the quantity of glucogenic amino acids and free fatty acids consequently increases [41],[42]. Similarly, Huang et al. [32] reported that most of the amino acids and their related metabolites significantly increased in the hematocrit group, with special interest on the levels of these glucogenic amino acids, which can be transformed into glucose for energy production. Similar studies have been conducted on a few other carcinomas. For example, Liu et al. [43] demonstrated the difference in amino acids between cancer and normal gastric tissue, Wang et al. [33] measured the serum of normal and cancerous colon tissues, and Poschke et al. [37] measured the serum amino acid levels in breast cancer patients. All of the above studies showed an increased level of these amino acids in cancer patients compared with controls.

As regards aromatic amino acids, the present study showed a significant increase in aromatic amino acids (phenylalanine and tyrosine) in HCC patients compared with controls and CLD patients and an increase in these amino acids in CLD patients compared with controls. These findings are in line with the results of a recent study conducted by Holecek. [30], who showed an increase in the levels of plasma aromatic amino acids as a result of the reduced ability of diseased liver, portal systemic shunting, and activated protein breakdown in cirrhotic patients. Another study by Huang et al. [32], which was conducted on 50 HCC patients reported a significant increase in aromatic amino acids in the HCC compared with the control group. Lee et al. [29] showed that HCC patients had high levels of the aromatic amino acids and low levels of BCAA.

As regards BTR, the results showed that there is a significant positive correlation between BTR and albumin level among HCC patients and controls, with a tendency for the low BTR group to have lower serum albumin levels. This can be explained according to Ishikawa et al. [9], who conducted a cohort study of 50 patients with HCC. They reported that BTR has a significant correlation with albumin level and that BTR calculation can be used for the prediction of serum albumin changes, allowing the early identification of individuals at risk for hypoalbuminemia and prediction of when to begin treatment with BCAA. Similarly, Ishikawa [8] reported that BTR decreases in advance of decreases in serum albumin level; therefore, monitoring of BTR separately from albumin is necessary when considering prognostic factors for HCC. These findings are in line with the results of Mizuguchi et al. [44], who conducted a study on 105 HCC patients. They reported a significant correlation between BTR and serum albumin level. Suzuki et al. [15] also reported that measurement of BTR level is a useful method in the prediction of serum albumin level change in CLD patients.

The present study also revealed a significant negative correlation between BTR and total bilirubin, ALT, prothrombin time, and AFP in HCC patients with a tendency for the low BTR group to have a higher level of total bilirubin, ALT, prothrombin time, and AFP. This is in agreement with the findings of Ishikawa et al. [9], who reported that total bilirubin levels and prothrombin time were higher in HCC patients with low BTR value.

This present study also revealed statistically significant differences in BTR among Child-Pugh A, B, and C (Child-Pugh A: 1.509 ± 0.324, Child-Pugh B: 1.303 ± 0.774, and Child-Pugh C: 0.8357 ± 0.194).

These findings are in line with the results of a study by Hasan et al. [45], who conducted a study on 52 patients; there were 26 patients with Child-Pugh A, 19 with Child-Pugh B, and 7 with Child-Pugh C. This study had demonstrated a significant level of BTR according to the Child-Pugh criteria. More severe liver cirrhosis had lower BTR.

 Conclusion



Cancer cells are hypermutable and may result in amino acid changes in certain protein sequences. Thus, the plasma free amino acids profile is considered valuable for diagnosis and for nutritional care in cancer patients.

Recommendations

Screening of amino acid profile would be useful in clinical practice to detect new cases of HCC in high-risk groups, and hence further investigations with a larger sample size is required to confirm these results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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