Reduced plasma arginine (ARG) concentrations are found in various types of cancer. ARG and its product nitric oxide (NO) are important mediators in the immune function and the defense against tumour cells. It remains unclear whether the diminished systemic ARG availability in cancer is related to insufficient endogenous ARG synthesis, negatively affecting NO synthesis, and whether a dietary amino acid mixture is able to restore this. In 13 patients with advanced non-small cell lung cancer (NSCLC) and 11 healthy controls, whole body ARG and CIT (citrulline) rates of appearance were measured by stable isotope methodology before and after intake of a mixture of amino acids as present in whey protein. The conversions of CIT to ARG (indicator of de novo ARG synthesis) and ARG to CIT (marker of NO synthesis), and ARG clearance (reflecting ARG disposal capacity) were calculated. Plasma isotopic enrichments and amino acid concentrations were measured by LC–MS/MS. Conversions of CIT to ARG and ARG to CIT (P<0.05), and CIT rate of appearance (P=0.07) were lower in NSCLC. ARG rate of appearance and clearance were comparable suggesting no enhanced systemic ARG production and disposal capacity in NSCLC. After intake of the mixture, ARG rate of appearance and concentration increased (P<0.001), and ARG to CIT conversion was restored in NSCLC. In conclusion, an impaired endogenous ARG synthesis plays a role in the reduced systemic ARG availability and NO synthesis in advanced NSCLC. Nutritional approaches may restore systemic ARG availability and NO synthesis in cancer, but the clinical implication remains unclear.

CLINICAL PERSPECTIVES

  • Arginine (ARG) and its product nitric oxide (NO) play a role in the immune function and the defense against tumour cells in cancer, although a role in disease progression has recently been suggested in some cancer types particularly at low concentrations. As cancer patients are characterized by reduced systemic arginine availability, insight is required into the underlying disturbances in ARG metabolism and whether it affects NO synthesis rate.

  • Whole body arginine to citrulline (CIT) conversion (indirect indicator of NO synthesis) was reduced in advanced NSCLC due to impaired endogenous ARG rate of appearance related to a diminished endogenous production of ARG precursors. A dietary amino acid mixture resulted in normalization of ARG to CIT conversion in NSCLC.

  • Nutritional approaches containing ARG and/or its precursors are able to restore ARG availability and NO synthesis in patients with cancer, although the exact clinical implications remain unclear and need further investigation.

INTRODUCTION

Arginine (ARG) and its product nitric oxide (NO) play an important role in cancer as both are important mediators in the immune function and the defense against tumour cells [1,2]. Adequate levels of ARG in the extracellular milieu are crucial for immune function in relation to T-cell activation and proliferation [3]. We and others found reduced plasma ARG concentrations in various cancer types including lung cancer [46], irrespective of weight loss, tumour stage, or body mass index (BMI), suggesting a reduced systemic ARG availability in cancer.

Higher circulating myeloid derived suppressor cells (MDSCs) have been found in both non-malignant and malignant chronic inflammatory conditions including lung cancer [7]. MDSC are immune suppressors, which down-regulate the T-cell receptor chain through ARG deprivation, leading to T-cell dysfunction and impaired anti-tumour immunity. The mechanism by which MDSC deplete ARG levels and alter NO production is by producing the ARG catabolic enzymes arginase and inducible nitric oxide synthase (iNOS). Elevated serum arginase levels have been recently observed in lung cancer patients [7] while arginase also is expressed by human lung tumour tissue [8,9]. However, local arginase expression does not relate to escape of lung tumour cells from immune cells in contrast with other cancer types, and does not negatively affect disease progression or patient survival [9]. Despite lack of NOS expression in lung tumour cells [9,10], up-regulation of iNOS activity has been observed in alveolar macrophages of patients with lung cancer [11].

Besides a potential role of increased arginase and iNOS activity likely caused by the MDSC on plasma ARG levels, the reduced systemic ARG availability in cancer might also be related to impaired endogenous ARG production. ARG is considered a conditionally essential amino acid, especially during stressful metabolic inflammatory conditions, when endogenous ARG production is impaired and therefore may not be sufficient to meet requirements [12]. Although we recently observed an up-regulation of protein breakdown in advanced non-small cell lung cancer (NSCLC) [13], ARG appearance from protein breakdown as well as from the conversion of citrulline (CIT), an indirect marker of ARG de novo synthesis, might not be sufficient to balance the increased ARG catabolism, leading to a reduced systemic ARG availability in these patients.

A reduced systemic ARG availability could directly impair whole body NO synthesis [14] in patients with lung cancer. Adequate NO synthesis is of importance in cancer as besides its role in immune function, cell regeneration, tissue perfusion and wound healing [15], NO has been suggested to have a pro- and anti-tumour effect dependent on its concentration. Although the effect of NO levels on tumour progression is still debated, there is evidence showing that NO could be characterized as pro-malignant at low concentrations, whereas at high concentrations, NO acts as a potent anti-cancer agent, promoting apoptosis and inhibiting angiogenesis [16,17].

In the past years, several in vitro and in vivo studies have focused on the mechanisms and consequences of reduced systemic ARG availability in lung cancer tissue or immune cells, however insight into underlying changes in whole body ARG, CIT and NO kinetics in lung cancer patients is still lacking. Therefore, we investigated, using innovative stable isotope techniques, whether the diminished systemic ARG availability in patients with advanced NSCLC is related to a reduced endogenous (de novo) ARG synthesis as this may lead to an insufficient up-regulation of ARG rate of appearance to meet requirements, and negatively affect NO synthesis.

Furthermore, we examined whether a mixture of amino acids (as present in whey protein) was able to restore systemic ARG availability in NSCLC and increase whole body NO synthesis. In the postabsorptive and post-prandial state, we studied in vivo whole body rate of appearance of ARG and CIT, and the conversions of ARG into CIT and CIT into ARG as indirect markers of systemic endogenous ARG production and NO synthesis rates in cancer patients and healthy age-matched control subjects. The results may initiate new nutritional approaches for lung cancer patients to restore systemic ARG availability and enhance whole body NO synthesis, although the exact clinical implications need further investigation.

MATERIALS AND METHODS

Study population

The study population consisted of 13 subjects with NSCLC and 11 healthy subjects. The subjects with NSCLC were all diagnosed with Stage III (unresectable) or Stage IV lung cancer and recruited during clinic visits to the University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System as described before [13]. Exclusion criteria were previous anti-cancer therapy (e.g. radiotherapy, chemotherapy) or surgery less than 4 weeks prior to the study, pre-existent cardiovascular, metabolic or renal disease, untreated metabolic diseases including liver or renal disease. The healthy control subjects were recruited via flyers in the community. Written informed consent was obtained from all subjects and the study was approved by the Institutional Review Board, University of Arkansas for Medical Sciences.

Anthropometric data and body composition

Body weight was measured by a digital beam scale and height by a stadiometer. BMI was calculated by dividing body weight by squared height. Whole body fat-free mass (FFM) was obtained in the patients and healthy controls by dual-energy X-ray absorptiometry (Hologic QDR 4500/Version 12.7.3.1) and standardized for height [18].

Study protocol

The NSCLC and healthy subjects were studied at the Clinical Center for Translational Research, UAMS, Little Rock, AR. The study day started in the early morning after an overnight fast and lasted for approximately 6 h [13]. Primed, constant and continuous infusion of the stable isotopes of L-[guanidine-15N2]ARG [prime: 3.75 μmol/kg bw (body weight), infusion rate: 3.75 μmol/kg bw·h] and L-[ureido-13C-2H2] or L-[5-13C,2H4]CIT (prime: 0.88 μmol/kg bw, infusion rate: 0.30 μmol/kg bw·h) (Cambridge Isotopic Laboratories) was done for 6 h to be able to measure whole production rate of ARG and CIT. A second catheter for arterialized venous blood sampling was placed in a superficial dorsal vein of the hand or lower arm of the contralateral arm. The hand was placed in a thermostatically controlled hot box (internal temperature: 60°C), a technique to mimic direct arterial sampling [19]. After three hours of stable isotope infusion in the postabsorptive state, each subject ingested within 5 min a 250 ml non-caloric soft-drink to which 30 g of maltodextrin and 14 g of balanced (essential and non-essential) amino acid mixture were added. Arterialized-venous blood was sampled for analysis of the tracer enrichments, concentrations of amino acids throughout the 6 h study (3 h of postabsorptive followed by 3 h of postprandial state) and for the inflammatory mediator C-reactive protein (CRP).

Amino acid composition of the oral mixture

The amino acid mixture was provided by Ajinomoto Co. as an unrestricted gift, and the composition reflected that of an anabolic mixture as used previously [20]. The balanced amino acid mixture used in the present study contained 30.0 g of maltodextrin and 13.39 g of the following essential and non-essential amino acids : alanine (0.70 g), ARG (0.38 g), aspartic acid (1.44 g), cystine (0.00 g), glutamic acid (2.40 g), glycine (0.26 g), histidine (0.26 g), isoleucine (0.71 g), leucine (1.56 g), lysine (1.58 g), methionine (0.36 g), phenylalanine (0.46 g), proline (0.68 g), serine (0.70 g), threonine (0.83 g), tyrosine (0.44 g) and valine (0.65 g).

Biochemical analysis

Arterialized-venous blood was put in Li-heparinized or EDTA tubes (Becton Dickinson Vacutainer system), immediately put on ice to minimize enzymatic reactions, and centrifuged (4°C, 3120 g for 5 min) to obtain plasma. A part of the plasma was put in 50% sulfosalicylic acid for deproteinization. Samples were instantly frozen and stored at −80°C until further analysis. Tracer enrichments and plasma amino acid concentrations were analysed by LC–MS/MS by isotope dilution. All samples were analysed in a batch. CRP concentrations were measured by high-sensitive ELISA.

Calculations for whole body ARG and CIT metabolism

Whole body ARG and CIT rate of appearance, CIT to ARG conversion (indirect measure of de novo ARG synthesis) and ARG to CIT conversion (indirect indicator of NO synthesis) in plasma were calculated from isotope enrichment values as described previously [14]. Plasma ARG clearance was calculated as whole body ARG rate of appearance divided by plasma ARG concentration, and is the volume of plasma that is cleared of ARG per unit of time (reflecting the ARG disposal capacity of the body). Tracer–tracee ratio of ARG and CIT reached an isotopic steady state within the first 3 h of infusion in both groups (postabsorptive state). Sum of analysed amino acids (Sum AA) represents the sum of the concentration of amino acids as is present in dietary protein (asparagine, glutamate, aspartate, serine, proline, glutamine, glycine, threonine, histidine, alanine, ARG, tyrosine, valine, methionine, isoleucine, phenylalanine, tryptophan, leucine and lysine).

Statistical analysis

Results are expressed as mean ± S.E.M. Unpaired Student's t test was used to determine differences in characteristics and metabolic response to amino acid intake between the NSCLC group and the control group. Postabsorptive whole body ARG and CIT rate of appearance, CIT to ARG conversion and ARG to CIT conversion were measured as the median value of the measures at time points 150, 165 and 180 min, and in the postprandial state as the 3 h integral (180–360 min) (μmol/kg FFM/3 h). Two-way repeated-measures analysis of variance (two-way RM ANOVA; general linear model) was performed with group (NSCLC compared with healthy controls) and time (postabsorptive compared with postprandial state) as factors. Bonferroni post hoc test was applied when significant interactions were observed. The level of significance was set at P<0.05. The statistical package within Prism (Version 6.04) and SPSS (Version 22) was used for data analysis.

RESULTS

The study group consisted of 13 male patients with NSCLC and 11 healthy matched control subjects. Eleven patients had Stage IIIA or B and two Stage IV lung cancer (Table 1) [13]. Nine of the patients were diagnosed with adenocarcinoma and 4 with squamous cell carcinoma. All patients had not received anti-cancer therapy or surgery in the past month. Average time period between last treatment and study participation was 5 months. Last treatment was concurrent chemo-radiotherapy (n=11), chemotherapy only (n=1) and radiotherapy only (n=1). Chemotherapy drugs were all platinum based (cisplatin or carboplatin, n=12) with placitaxel (n=4), etoposide (7) or taxol (n=1). Plasma CRP was elevated in the NSCLC group.

Table 1
Patient characteristics
Histology Adenocarcinoma (n=9), squamous cell carcinoma (n=4) 
Stage Type IIIA/B: n=11, Type IV: n=4 
Treatment > 3 months before study days Concurrent chemo-radiotherapy: n=11, chemotherapy: n=1, radiotherapy: n=1 
Plasma CRP (mg/l) 9.8±3.7 
Histology Adenocarcinoma (n=9), squamous cell carcinoma (n=4) 
Stage Type IIIA/B: n=11, Type IV: n=4 
Treatment > 3 months before study days Concurrent chemo-radiotherapy: n=11, chemotherapy: n=1, radiotherapy: n=1 
Plasma CRP (mg/l) 9.8±3.7 

No significant differences were found between the NSCLC and healthy control groups in age (66±2 years compared with 69±2 years respectively), BMI (28±1 kg/m2 compared with 27±1 kg/m2 respectively) and whole body fat-free mass (59±2 kg compared with 54±2 kg respectively). Fat-free mass in the legs, however, was significantly lower in the NSCLC patients as compared with the control subjects (19±1 kg compared with 17±1 kg, respectively, P<0.05).

Postabsorptive whole body ARG metabolism, NO synthesis and plasma amino acid concentrations

Whole body ARG rate of appearance and clearance (Figure 1) were comparable in the NSCLC and healthy groups suggesting no stimulated ARG use. Lower values were found in NSCLC for CIT to ARG conversion (as a marker of de novo ARG synthesis) (P<0.01) and ARG to CIT conversion rates (reflecting NO synthesis) (P<0.05), and whole body CIT rate of appearance tended to be lower (P=0.07).

Postabsorptive whole body arginine rate of appearance (a), citrulline rate of appearance (b), citrulline to arginine conversion (indirect marker of arginine de novo synthesis) (c), arginine clearance (d) and arginine to citrulline conversion (indirect marker of nitric oxide synthesis (e) in the healthy control (open bars) and advanced NSCLC group (black bars)

Figure 1
Postabsorptive whole body arginine rate of appearance (a), citrulline rate of appearance (b), citrulline to arginine conversion (indirect marker of arginine de novo synthesis) (c), arginine clearance (d) and arginine to citrulline conversion (indirect marker of nitric oxide synthesis (e) in the healthy control (open bars) and advanced NSCLC group (black bars)

Values are mean (± S.E.M.). Significance of difference between the groups: **P<0.01; *P<0.05. Whole body citrulline rate of appearance tended to be lower in the NSCLC group (P= 0.07).

Figure 1
Postabsorptive whole body arginine rate of appearance (a), citrulline rate of appearance (b), citrulline to arginine conversion (indirect marker of arginine de novo synthesis) (c), arginine clearance (d) and arginine to citrulline conversion (indirect marker of nitric oxide synthesis (e) in the healthy control (open bars) and advanced NSCLC group (black bars)

Values are mean (± S.E.M.). Significance of difference between the groups: **P<0.01; *P<0.05. Whole body citrulline rate of appearance tended to be lower in the NSCLC group (P= 0.07).

Furthermore, plasma concentrations of CIT (Figure 2) and sum of all analysed amino acids (P<0.05) were lower in the NSCLC group and there was a tendency towards lower values for plasma ARG and glutamine (GLN) concentrations (P=0.08) in NSCLC. No differences were found in plasma ornithine concentration, ratio plasma ARG to ornithine concentration (Figure 2), ratio plasma ARG to proline + lysine concentration or ratio plasma ARG to ornithine + CIT concentration (results not shown), as indirect markers of systemic arginase activity, between the groups.

Postabsorptive plasma concentrations of arginine (a), citrulline (b), glutamine (c), ornithine (d), sum of all measured amino acids (e) and ratio plasma arginine to ornithine (f) in the healthy control (open bars) and advanced NSCLC group (black bars)

Figure 2
Postabsorptive plasma concentrations of arginine (a), citrulline (b), glutamine (c), ornithine (d), sum of all measured amino acids (e) and ratio plasma arginine to ornithine (f) in the healthy control (open bars) and advanced NSCLC group (black bars)

Values are mean (± S.E.M.). Significance of difference between the groups: *P<0.05. Plasma arginine and glutamine tended to be lower in the NSCLC group (P= 0.08).

Figure 2
Postabsorptive plasma concentrations of arginine (a), citrulline (b), glutamine (c), ornithine (d), sum of all measured amino acids (e) and ratio plasma arginine to ornithine (f) in the healthy control (open bars) and advanced NSCLC group (black bars)

Values are mean (± S.E.M.). Significance of difference between the groups: *P<0.05. Plasma arginine and glutamine tended to be lower in the NSCLC group (P= 0.08).

Response of whole body ARG metabolism, NO synthesis and plasma amino acid concentrations to intake of the balanced amino acid mixture

Intake of the balanced amino acid mixture resulted in an increase in whole body ARG rate of appearance, plasma ARG and ornithine concentrations (P<0.001) with the peak approximately 45 min after intake, and in a reduction in CIT rate of appearance and plasma CIT concentration (P<0.001) (Figures 3 and 4). Furthermore, whole body CIT to ARG conversion rate and ARG clearance reduced after intake (P<0.001) with the lowest value obtained after 45–60 min followed by a gradual increase towards baseline levels. No significant meal effect was observed for ARG to CIT conversion. Plasma GLN concentration decreased below baseline 90 min after intake (P<0.001).

Kinetics of whole body arginine rate of appearance (a), citrulline rate of appearance (b), citrulline to arginine conversion (indirect marker of arginine de novo synthesis (c), arginine clearance (d) and arginine to citrulline conversion (indirect marker of nitric oxide synthesis) (e) in the NSCLC (green circles) and healthy control (black circles) groups after intake of a dietary amino acid mixture at t=180 min

Figure 3
Kinetics of whole body arginine rate of appearance (a), citrulline rate of appearance (b), citrulline to arginine conversion (indirect marker of arginine de novo synthesis (c), arginine clearance (d) and arginine to citrulline conversion (indirect marker of nitric oxide synthesis) (e) in the NSCLC (green circles) and healthy control (black circles) groups after intake of a dietary amino acid mixture at t=180 min

Values are mean (± S.E.M.). Two-factor repeated-measures ANOVA was used to test the Group (G) and Time (T) effect. There was a Time effect for whole body arginine and citrulline rate of appearance, citrulline to arginine conversion and arginine clearance (P<0.001). There was a Group effect for whole body citrulline rate of appearance (P<0.05) and citrulline to arginine conversion (P<0.001) and a tendency for arginine clearance (P=0.06). There was no Time × Group interaction.

Figure 3
Kinetics of whole body arginine rate of appearance (a), citrulline rate of appearance (b), citrulline to arginine conversion (indirect marker of arginine de novo synthesis (c), arginine clearance (d) and arginine to citrulline conversion (indirect marker of nitric oxide synthesis) (e) in the NSCLC (green circles) and healthy control (black circles) groups after intake of a dietary amino acid mixture at t=180 min

Values are mean (± S.E.M.). Two-factor repeated-measures ANOVA was used to test the Group (G) and Time (T) effect. There was a Time effect for whole body arginine and citrulline rate of appearance, citrulline to arginine conversion and arginine clearance (P<0.001). There was a Group effect for whole body citrulline rate of appearance (P<0.05) and citrulline to arginine conversion (P<0.001) and a tendency for arginine clearance (P=0.06). There was no Time × Group interaction.

Plasma concentrations of arginine (a), citrulline (b), glutamine (c) and ornithine (d) in the NSCLC (green circles) and healthy control (black circles) group after intake of a dietary amino acid mixture at t=180 min

Figure 4
Plasma concentrations of arginine (a), citrulline (b), glutamine (c) and ornithine (d) in the NSCLC (green circles) and healthy control (black circles) group after intake of a dietary amino acid mixture at t=180 min

Values are mean (± S.E.M.). Two-factor repeated-measures ANOVA was used to test the Group (G) and time (T) effect. There was a Time effect for plasma arginine, citrulline, glutamine and ornithine (P<0.001). There was a Group effect for plasma arginine concentration (P<0.05) and a tendency for citrulline concentration (P=0.08). There was no Time × Group interaction.

Figure 4
Plasma concentrations of arginine (a), citrulline (b), glutamine (c) and ornithine (d) in the NSCLC (green circles) and healthy control (black circles) group after intake of a dietary amino acid mixture at t=180 min

Values are mean (± S.E.M.). Two-factor repeated-measures ANOVA was used to test the Group (G) and time (T) effect. There was a Time effect for plasma arginine, citrulline, glutamine and ornithine (P<0.001). There was a Group effect for plasma arginine concentration (P<0.05) and a tendency for citrulline concentration (P=0.08). There was no Time × Group interaction.

Most of the postabsorptive differences between the groups remained after meal intake. In the postprandial state, lower values were found in the NSCLC group for plasma ARG concentration, whole body CIT rate of appearance (P<0.05) and CIT to ARG conversion rate (P<0.001) (Figure 3 and Table 2). Whole body ARG clearance tended to be higher (P=0.06) and plasma CIT lower (P=0.08) in the NSCLC group. The significantly lower whole body ARG to CIT conversion rate in the NSCLC group in the postabsorptive state disappeared after intake of the amino acid mixture. No differences were observed in postprandial response in whole body ARG rate of appearance, and plasma GLN and ornithine concentrations between the groups.

Table 2
Whole body arginine metabolism after intake of a dietary amino acid mixture in the healthy control and the NSCLC groups

Values are means ± S.E.M. Citrulline to arginine conversion is indirect marker of arginine de novo synthesis, arginine to citrulline conversion is indirect marker of nitric oxide synthesis. Whole body arginine metabolism is expressed as the integral of the 3 h postprandial state in μmol/kg FFM/3 h, arginine clearance in l/kg FFM/3 h. Unpaired Student's t test was used to determine differences between the NSCLC group and the control group ***P<0.001, *P<0.05. There was no Time × Group interaction.

Healthy controls (n=11)NSCLC (n=13)s
Whole body ARG metabolism 
 Arginine rate of appearance 247±8 253±9  
 Citrulline rate of appearance 34±3 29±1 
 Citrulline to arginine conversion 21±1 16±1 *** 
 Arginine clearance 6.4±0.1 7.3±0.4 P=0.08 
 Arginine to citrulline conversion 4.6±0.7 4.6±0.4  
Healthy controls (n=11)NSCLC (n=13)s
Whole body ARG metabolism 
 Arginine rate of appearance 247±8 253±9  
 Citrulline rate of appearance 34±3 29±1 
 Citrulline to arginine conversion 21±1 16±1 *** 
 Arginine clearance 6.4±0.1 7.3±0.4 P=0.08 
 Arginine to citrulline conversion 4.6±0.7 4.6±0.4  

DISCUSSION

In the present study, we found a reduced systemic ARG availability in normal-weight patients with advanced NSCLC which was related to a reduced endogenous ARG synthesis. Our results extend our previous observation that a reduced systemic ARG availability is present in cancer independent of tumour type, tumour stage, weight loss, and nutritional status [6]. Furthermore, the reduced systemic ARG availability in NSCLC was associated with an impaired ARG to CIT conversion, an indirect indicator of whole body NO synthesis.

What are the possible causes of reduced systemic ARG availability in lung cancer?

Tumours develop a protection mechanism against the specific anti-tumour attack of the immune system by recruiting MDSCs. Circulating MDSCs are up-regulated in lung cancer [7] and are known to lead to T-cell dysfunction and deficient anti-tumour immunity through ARG depletion via producing arginase I, and iNOS. Serum arginase I is higher in lung cancer patients as compared with non-smoking controls [7] but is also up-regulated in other chronic diseases characterized by enhanced systemic inflammation [7,21]. In macrophages, the presence of high levels of the enzyme arginase can limit the availability of ARG for NO production suggesting a competition between arginases and NO synthases for their common substrate [22]. The NSCLC patients that we studied were characterized by an increased systemic inflammatory response (CRP: 9.8±3.7 mg/l) and reduced levels for whole body ARG to CIT conversion rate (as indirect marker of NO synthesis). Although plasma arginase was not measured in the present study, no significant increase was observed in postabsorptive ARG clearance or plasma ornithine concentration, and no differences were found in ratio plasma concentration of ARG to ornithine, ARG to proline + lysine, or ARG to ornithine + CIT (as indirect measures of plasma arginase activity).

A high arginase activity in tumours is a mechanism of tumour-induced immunosuppression through depletion of ARG concentrations in the microenvironment of the tumour [2,23]. Preliminary results show efficacy of ARG deprivation in inducing anti-tumour activity in some tumours [24], decreasing cancer cell survival, and inducing autophagy and cell death [25]. Recombinant human arginase that catabolizes ARG has entered clinical trials as anti-cancer treatment in some cancer types (i.e. melanoma and leukaemia) [24]. Also pegylated ARG deiminase produces depletion of ARG, and certain tumours (i.e. melanoma, hepatocellular carcinoma, sarcoma) are auxotrophic for ARG. Although ARG deiminase has been shown to inhibit growth of small cell lung cancers lacking expression of argininosuccinate synthase [26], no data are available on potential immune suppression by ARG deprivation in NSCLC. Although arginase is known to be expressed in lung tumour (including non-small cell lung carcinoma) [8,9], it is not related to tumour immune escape or disease progression [9]. This suggests that there is no clear evidence in NSCLC that ARG deprivation is of clinical importance, although more research is needed to confirm this.

Other metabolic pathways that might be affected in patients with NSCLC are that of symmetric dimethylarginine (ADMA) and of NO. ADMA is known to inhibit cellular ARG uptake and NO synthase activity. Elevated ADMA plasma levels were found in patients with haematological malignancies, however, these patients were also characterized by elevated plasma ARG concentrations [27] which is in contrast with the reduced plasma ARG levels observed in our NSCLC patients as well as in those with other solid tumours (i.e. breast, colon and pancreas) [6]. It remains to be established whether plasma arginase and ADMA levels are indeed increased in NSCLC and, if so, to what extent these as well as the lung tumour arginase activity contribute to the reduced systemic ARG availability and NO production as observed in our patients with NSCLC. Besides its role in immune function, cell regeneration, tissue perfusion and wound healing [15], NO has been suggested to have a pro- and anti-tumour effect dependent on its concentration. Evidence shows that at low concentrations, NO could be characterized as pro-malignant, whereas at high concentrations, NO acts as a potent anti-cancer agent, promoting apoptosis and inhibiting angiogenesis [16,17]. The exact contribution of NO levels to tumour progression in NSCLC still needs to be established. Despite the fact that serum arginase level and arginase activity in lung tumour tissue, as well as iNOS activity in the alveolar macrophages are increased [79,11], likely caused by the MDSC, whole body production of NO was not elevated but decreased suggesting that the reduced systemic NO production in patients with NSCLC is mainly related to a diminished systemic ARG availability due to lower ARG production relative to its need.

In the postabsorptive state, ARG rate of appearance comes both from protein breakdown and endogenous ARG synthesis. Although whole body protein breakdown was enhanced in NSCLC [13], ARG production from protein breakdown was apparently not sufficient to balance the reduced endogenous ARG production in these patients, leading to an impaired whole body ARG to CIT conversion.

Systemic NO production is dependent on the availability of ARG and the activity of the various NO synthase enzymes. Chronic diseases are generally characterized either by a lack or excess of NO synthesis [28]. In the chronic inflammatory wasting diseases chronic obstructive pulmonary disease and cystic fibrosis, we recently observed up-regulated whole body de novo ARG synthesis and CIT rate of appearance rates, whereas whole body ARG to CIT conversion (as measure of NO synthesis) was unaltered or even increased in those with nutritional failure [12]. Systemic inflammation in cancer can limit the availability of ARG for NO production in macrophages, by causing a competition between arginases and NO synthases for their common substrate [22]. An impaired whole body ARG to CIT conversion and inadequate CIT to ARG rate of appearance secondary to reduced CIT rate of appearance, as observed in the our NSCLC group, has previously also been found in patients with sepsis [14,29,30]. A relation was observed between ARG de novo synthesis and whole body NO synthesis in sepsis [14]. In the present study, whole body ARG to CIT conversion was related to CIT rate of appearance (r: 0.46, P=0.02) and a tendency was present with CIT to ARG conversion (r: 0.36, P=0.08), indicating an important role of the diminished endogenous ARG production as explanation for the reduced whole body NO synthesis in NSCLC.

The observed reduced whole body de novo ARG synthesis and CIT rate of appearance in NSCLC is in line with our previous animal data showing that cancer negatively affects de novo ARG synthesis likely through diminished intestinal CIT production [31], which is related to a diminished supply of GLN from muscle and/or a reduced intestinal GLN to CIT conversion (suppressed gut functional capacity). We hypothesize that changes in GLN metabolism could be related to the observed lower CIT production. For instance, we found lower plasma GLN concentration in our studied NSCLC patients. Overall GLN deprivation is common in lung cancer [32] and is associated with systemic inflammation, malnutrition and cancer related fatigue [4,33]. Because GLN is a preferred respiratory fuel for rapidly proliferating cells such as enterocytes and lymphocytes and fibroblasts [34], suppressed gut functional capacity might be related to previous anti-cancer treatment in the NSCLC group as 12 out of 13 had undergone platinum based chemotherapy, and 11 concurrent radiotherapy > 4 weeks before the study day. Although surgery is known to reduce plasma ARG and GLN levels in lung cancer [5], none of the studied NSCLC patients had undergone recent surgery.

Effect of dietary amino acid mixture on whole body ARG and CIT kinetics and NO synthesis in NSCLC

As we anticipated that the NSCLC patients would be characterized by a reduced systemic ARG availability [6], we extended our protocol with measurements of ARG kinetics in response to intake of a meal containing amino acids as present in whey protein. The provided amino acid mixture was anabolic in NSCLC [13] as it contained besides non-essential amino acids also high amounts of essential amino acids. Furthermore, the amino acid mixture increased plasma ARG concentration and whole body ARG rate of appearance independent of the presence of NSCLC. Although the plasma ARG concentration after intake remained lower in the NSCLC as compared with the healthy group, the increased ARG availability after intake of 15 g of amino acid mixture was able to restore whole body ARG to CIT conversion in the NSCLC patients. This observation is in line with previous human data showing that restoring ARG availability by ARG intake is able to drive NO production [30,35]. As mentioned above, there are conflicting findings regarding NO and its role in carcinogenesis and tumour progression [17]. As NO could be pro-malignant at low concentrations and anti-malignant at high concentrations [16,17], this suggests that restoring the impaired NO synthesis in NSCLC by nutrition is beneficial in these patients. More research is needed to confirm this.

Despite a reduction in whole body CIT rate of appearance and ARG de novo synthesis, ARG rate of appearance and plasma ARG concentration increased after meal intake in NSCLC, indicating that the amount of ARG in the mixture was sufficient. We attribute the reduction in CIT rate of appearance to the lack of GLN in the mixture because of the instability of GLN in water. In line with this, a previous study in patients with lung cancer [33] also showed a significantly lower plasma GLN concentration during hyperaminoacidemia as induced by an intravenous amino acid solution as compared with healthy control subjects. Another explanation for the lower CIT production is that we used free glutamate (instead of GLN) in the mixture. Previously we found that plasma CIT increases after free GLN ingestion but reduces after free glutamate intake [36], suggesting that dietary glutamate decreases the GLN conversion to CIT in the gut. Choice of a meal containing GLN would likely have prevented the reduction in CIT flux in cancer. Furthermore, there is evidence that GLN enriched diets are able to replete host GLN stores and support muscle GLN metabolism without stimulating tumour growth [37]. Oral GLN administration may even decrease tumour growth by enhancing immune function [38]. Despite the fact that the provided amino acid mixture has its limitation regarding the composition, it was still able to induce a high anabolic response in cancer patients [13] due to its high essential amino acid levels and was able to increase the whole body ARG rate of appearance.

Supplemental ARG has been shown to improve immunological status and survival in severely malnourished patients with head and neck cancer and even decreased cancer recurrence [39]. We previously showed that the response of whole body ARG appearance and ARG to CIT conversion can be increased with a protein-energy enriched meal in critically ill children [35], often characterized by diminished ARG availability or reduced NO synthesis [35]. In contrast with our data in NSCLC, the protein energy enriched meal in the critically ill children did not lead to a reduction in whole body CIT rate of appearance, ARG de novo synthesis, plasma CIT and GLN concentrations. This suggests that a well-balanced protein formula might be even a more preferable meal than the amino acid mixture used to increase whole body NO synthesis in patients with lung cancer. The provided dietary amino acid mixture, although used in other studies to successfully induce anabolism [21,40], is not complete as a few very important amino acids (i.e. GLN) are lacking.

Other limitations of the present study is the small study group size although sufficient to answer the aims of our study, and that the fact that only male subjects (mostly veterans) were enrolled. As most patients had Stage IIIa NSCLC, a potential selection bias toward the less sick cannot be excluded. However in these patients already pronounced disturbances were observed in whole body ARG and CIT kinetics. We acknowledge that measurement of whole body NO synthesis lacks specific organ information. Only measurements across organs can give more insight into where in the body the changes in NO metabolism take place, however this is not feasible in human clinical populations like cancer patients.

In conclusion, besides a potential role of increased arginase and iNOS activity likely caused by the MDSC on plasma ARG levels, the present study shows that reduced systemic ARG availability in NSCLC and the reduced whole body NO synthesis in these patients are mainly caused by impaired endogenous ARG production. Further studies are warranted to examine the effects of anti-cancer therapy (i.e. chemotherapy and radiotherapy), known to compromise gut function, on whole body CIT and ARG metabolism. An amino acid mixture is able to increase systemic ARG availability and restore whole body NO production in advanced cancer patients, suggesting that incorporating sufficient ARG and/or CIT as well as GLN in the diet is important. However, the exact clinical implications of these new nutritional approaches for cancer patients remain unclear and need further investigation.

AUTHOR CONTRIBUTION

Nicolaas Deutz and Mariëlle Engelen designed the research and were involved in the conduct of the research, data analysis and writing of the manuscript. Fari Koeman was involved in the conduct of the research. Ahmed M. Safar and Thaddeus Bartter were involved in the recruitment of study participants. All authors reviewed the manuscript.

We thank the NSCLC patients and the healthy control subjects for their willingness to participate in this research study and who have made this work possible.

FUNDING

This work was supported by the American Institute for Cancer Research [grant number #09A051]; the National Institutes of Health Clinical and Translational Science Award [grant number UL1RR029884]; and the National Institutes of Health [grant numbers S10RR027047 and UL1RR029884].

Abbreviations

     
  • ARG

    arginine

  •  
  • BMI

    body mass index

  •  
  • CIT

    citrulline

  •  
  • CRP

    C-reactive protein

  •  
  • FFM

    fat-free mass index

  •  
  • GLN

    glutamine

  •  
  • iNOS

    inducible nitric oxide synthase

  •  
  • MDSC

    myeloid derived suppressor cell

  •  
  • NO

    nitric oxide

  •  
  • NSCLC

    non-small cell lung cancer

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