American Journal of Clinical Nutrition, Vol. 81, No. 5, 1142-1146,
May 2005
© 2005 American Society for Clinical Nutrition
ORIGINAL RESEARCH COMMUNICATION |
Plasma arginine concentrations are reduced in cancer patients: evidence for arginine deficiency?1,2,3
Yvonne LJ Vissers,
Cornelis HC Dejong,
Yvette C Luiking,
Kenneth CH Fearon,
Maarten F von Meyenfeldt and
Nicolaas EP Deutz
1 From the Departments of Surgery, Nutrition, and Toxicology, Research Institute Maastricht, Maastricht University, and University Hospital Maastricht, Maastricht, Netherlands (YLJV, YCL, CHCD, MFvM, and NEPD), and the Royal Infirmary, Edinburgh, United Kingdom (CHCD and KCHF)
2 Supported by grants from the Netherlands Organisation for Health Research and Development (ZonMw, grant numbers 907-00-033 and 920-03-240) and the Niels Stensen Foundation.
3 Address reprint requests to NEP Deutz, Department of Surgery, Maastricht University, PO Box 616, 6200 MD Maastricht, Netherlands. E-mail: nep.deutz{at}ah.unimaas.nl.
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ABSTRACT
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Background: The disturbances leading to cancer cachexia remain to be unraveled. Preliminary evidence suggests that arginine availability in cancer is reduced. However, no valid data are available on plasma arginine concentrations in cancer patients.
Objective: We aimed to determine whether there is evidence for disturbed arginine metabolism in cancer.
Design: We measured plasma arginine concentrations postabsorptively in patients with various types of tumors, hypothesizing that arginine concentrations would be lower than those in age- and sex-matched control subjects. Patients with localized tumors with a range of metabolic implications were studied: breast cancer (no weight loss), colonic cancer (sometimes weight loss), and pancreatic cancer (usually weight loss). Plasma amino acid concentrations were measured by HPLC.
Results: Plasma arginine concentrations were lower in patients with cancer (breast cancer: 80 ± 3 compared with 103 ± 9 µmol/L; colonic cancer: 80 ± 3 compared with 96 ± 7 µmol/L; pancreatic cancer: 76 ± 5 compared with 99 ± 7 µmol/L; P < 0.05 versus respective age- and sex-matched control subjects), irrespective of tumor type, weight loss, tumor stage, or body mass index (correlations with P > 0.05).
Conclusions: Malignant tumors associated with various degrees of metabolic derangements are all associated with decreased plasma arginine concentrations, even without weight loss. This suggests that decreased arginine availability is a specific feature of the presence of cancer. These disturbances in arginine metabolism could contribute to the cascade of metabolic events leading to cancer cachexia.
Key Words: Cachexia • arginine • tumor • amino acids • malignancy • breast cancer • colonic cancer • pancreatic cancer
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INTRODUCTION
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Up to one-third of cancer patients die from cachexia rather than from cancer itself, even after surgical removal of the tumor (1). This suggests that cancer-related alterations in metabolism of the host are an important factor in determining mortality. Unfortunately, attempts to reverse cachexia by supplying large amounts of protein or calories have been unsuccessful. Therefore, more detailed insight into the disturbances in host metabolism of cancer patients is necessary.
Evidence is accumulating that the amino acid arginine is of importance in cancer. Both arginine and its product nitric oxide (NO) are important mediators in the defense against tumor cells, because both influence T cell–mediated immunity (2, 3), cytokine induction (4), and macrophage-mediated tumor toxicity (5). Besides, various malignant tumor tissues contain considerable amounts of the enzyme arginase (6-8), which converts arginine to ornithine and urea. It was recently shown that the high arginase activity of tumors is a mechanism of tumor-induced immunosuppression through depletion of arginine concentrations in the microenvironment of the tumor (9). However, it is not known whether this disturbs the arginine metabolism of the host as well. Changes in systemic arginine concentrations of cancer patients could indicate whether the high arginase activity in the proximity of the tumor affects arginine metabolism on the whole-body level as well.
Plasma arginine concentrations have been studied in cancer patients before, but interpretation of these data may not be valid because of incorrect collection of samples and the use of improper control groups. Thus, to determine whether there is evidence for disturbed arginine metabolism in cancer, we measured arginine concentrations in venous blood samples collected postabsorptively from patients with various types of tumors and compared these with values obtained for age- and sex-matched controls collected previously in our laboratory. We aimed to collect blood samples in a way that prevented decomposition of samples and minimized variations in concentrations due to confounding variables. Three tumor types with a range of metabolic implications were studied: breast tumors (no weight loss), colonic tumors (sometimes weight loss), and pancreatic tumors (often weight loss).
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SUBJECTS AND METHODS
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Patients
Patients with breast cancer (n = 22) or colonic cancer (n = 9) were recruited from the Department of Surgery of the University Hospital Maastricht from 2003 to 2004. Patients with pancreatic cancer (n = 21) and contemporary age- and sex-matched control subjects (n = 17) were recruited from the Department of Surgery of the Royal Infirmary of Edinburgh from 1999 to 2000. Exclusion criteria were previous therapy (surgery, chemotherapy, or radiotherapy), metastatic disease, and known metabolic or endocrine disease. The Medical Ethical Committee of the University Hospital Maastricht and the Royal Infirmary of Edinburgh approved the study. Written informed consent was obtained from all patients. Diagnosis of malignancy was confirmed by histologic examination of tumor tissue after surgical removal. Tumor stages were classified according to the guidelines of the American Joint Committee on Cancer. Patient characteristics are summarized in Table 1
. Amino acid concentrations of all patient groups were compared with a data set of age- and sex-matched control subjects collected previously by our laboratory (10) and referred to as historical controls.
Handling of samples
All blood samples were collected in the morning after the subjects had fasted overnight. Blood was collected from a peripheral vein into precooled heparin-containing tubes and was immediately put on ice. Body weight and length were measured, and patients were asked about any weight loss in the previous 6 mo. Within 0.5 h of sampling, the samples were centrifuged for 10 min at 4000 x g, 4 °C, and the plasma was deproteinized by using sulfosalicylic acid (8 mg per 100 µL plasma). Samples were subsequently put into liquid nitrogen and stored at –80 °C until analyzed. Amino acid concentrations were measured in plasma by using HPLC as described by van Eijk (11) with variation coefficients of <3%.
Statistical analysis
Mann-Whitney U tests were used to test for differences between groups with malignant tumors and their age- and sex-matched controls, hypothesizing that cancer would decrease amino acid concentrations. Correlations between amino acid concentrations and tumor stage were tested by using Kendall's 
; correlations between amino acid concentrations and weight loss or body mass index (BMI) were tested by using Spearman's 
. Significance was defined as P < 0.05.
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RESULTS
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Patient characteristics
Of the patients with breast cancer, 4 of 22 had experienced weight loss in the past 6 mo. Four of the 9 patients with colonic cancer had lost weight in the past 6 mo. None of the patients in these 2 groups had lost >10% of their body weight, and all subjects had BMIs (in kg/m2) greater than 21. Of the patients with pancreatic cancer, all but one had lost weight in the past 6 mo. Twelve of 21 had lost >10% of their initial body weight, and 5 patients had BMIs < 19. All age- and sex-matched control groups were weight stable, and BMI averaged 25 ± 1 in each group.
Amino acid concentrations
Most plasma amino acid concentrations of pancreatic cancer patients were significantly lower than those of the contemporary and historical controls (Table 2). Values did not differ significantly between the contemporary and historical pancreatic cancer controls for most amino acids. In all patients with malignant disease, plasma arginine concentrations were lower than in age- and sex-matched controls (Figure 1). Branched-chain amino acid concentrations were also lower in all cancer patients. Total amino acid concentrations were lower only in patients with pancreatic cancer (Table 2).
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FIGURE 1.. Mean (±SEM) postabsorptive venous plasma arginine concentrations from patients with various types of malignant tumors and from age- and sex-matched historical control subjects. Breast cancer patients and historical controls, n = 22 per group; colonic cancer patients and historical controls, n = 9 per group; pancreatic cancer patients and historical controls, n = 21 per group. For pancreatic cancer patients, a contemporary group of age- and sex-matched controls was also included (n = 17). *Significantly different from age- and sex-matched controls without malignant disease, P < 0.05 (Mann-Whitney U test).
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Correlations
There was no significant correlation between arginine or total amino acid concentrations and tumor stage, weight loss, or BMI in any patient group.
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DISCUSSION
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The results of the present study show that plasma arginine concentrations are reduced in patients with cancer, even without weight loss being present and irrespective of tumor type, tumor stage, or BMI. Our results suggest that high arginase activity around the tumor affects host arginine metabolism on a whole-body level, indicating a prominent role for altered arginine metabolism in cancer.
Plasma arginine concentrations in various types of cancer have been investigated before. These have been reported to be decreased in lung cancer (12), higher in breast cancer (7, 13), unchanged (13) or higher (14) in gastrointestinal cancer, unchanged in head and neck cancer (13), and unchanged in esophageal cancer (12), as shown in Table 3. However, none of these studies reported that plasma samples were deproteinized before storage, which leaves the enzymatic activity of arginase (which converts arginine to ornithine) intact. As we have shown before, ongoing arginase activity in plasma can decrease arginine concentrations up to 94% (10). This could explain why plasma arginine concentrations were already relatively low in control groups reported in the literature (Table 3). Thus, interpretation of these data may be invalid. To prevent decomposition by enzymatic activity, samples must be deproteinized immediately and stored at –80 °C until analyzed. Moreover, the sampling site must be equal in all subjects; we observed that the arterial and venous pools give different results for some amino acids, including arginine, varying from 8% to 38% (YC Luiking, unpublished observations, 2003). Furthermore, all subjects must be studied postabsorptively to overcome acute effects of amino acid intake by food. Finally, to enable interpretation of differences in amino acid concentrations between subjects, control groups should be at least age- and sex-matched, because we showed that amino acid concentrations vary with the sex of the individual and increase with age (10). Only Naini et al's study used age-matched controls and observed that plasma arginine concentrations were lower in patients with lung cancer and unchanged in patients with esophageal cancer (12).
In a clinical situation, however, it can be difficult to obtain samples from healthy control groups with age- and sex-matched subjects. Therefore, the use of historical controls of various age groups is an acceptable alternative. Most amino acid concentrations in contemporary controls of the pancreatic cancer patients were not significantly different from the concentrations in historical controls (Table 2
). This indicates that there are no major differences in amino acid patterns between the 2 control groups and that the historical controls can be used for comparison with current patient data. Thus, our study is the first to report plasma arginine concentrations in cancer patients with the use of strict methods for collection and storage of samples and with the use of proper control groups.
Evidence for abnormal arginine metabolism in cancer has accumulated. As such, various malignant tissues, such as lung (6), skin (15), prostate (16), colon, and breast tumors (7, 8), contain high amounts of arginase. It was shown recently that arginase activity in tumors induces arginine deficiency in the microenvironment of the tumor, which enables tumors to escape the immune response (9). However, it is not known whether this has systemic effects on arginine metabolism. The low arginine concentrations in cancer patients suggest that the presence of a tumor indeed affects arginine metabolism on a whole-body level. Whether this is due to increased arginine uptake by the tumor or the secretion of arginase into the circulation remains to be determined.
Apart from arginase, arginine can be converted by NO synthase (NOS) to citrulline and NO. NO is a highly reactive molecule to which many of the functions of arginine are ascribed. For example, NO is the effector molecule of the tumor-cytotoxic mechanism of macrophages (5). Therefore, arginine deficiency could decrease NO production and have adverse effects in cancer patients. On basis of the Michaelis constant (Km) for NOS (2–20 µmol/L), the observed decrease in arginine concentrations in cancer patients should not limit NO production. However, the existence of caveolar complexes between NOS and membrane transporters of arginine (17) suggests that arginine transporters may be more important than the Km of NOS for regulation of NO production. Because these arginine transporters have Km values in the range of physiologic arginine concentrations (18), decreases in arginine concentrations such as observed in the present study may indeed limit NO production.
Disturbances in arginine and NO metabolism in the presence of cancer can be unfavorable, especially when cancer patients undergo treatments that result in tissue damage, such as surgical removal of the tumor or radio- or chemotherapy. Because arginine has been shown to stimulate the immune system (19) and anabolism (3, 20), abnormal arginine metabolism can disturb repair responses such as the acute-phase protein response. We recently observed that low arginine concentrations in tumor-bearing mice prohibit the postoperative increase in arginine and NO production, concomitant with a suppressed acute-phase response (YLJ Vissers, unpublished observation, 2004). This finding indicates that metabolic disturbances become even more pronounced when metabolism is challenged to respond to stress.
There was no relation between arginine or total amino acid concentrations and tumor stage or previous weight loss. Strikingly, plasma arginine concentrations were decreased in all tumor types studied, including breast and colonic cancer, which have always been assumed to cause little metabolic derangements. Moreover, the decreased arginine concentrations were specific, because total amino acid concentrations were maintained. Furthermore, previous studies from our group investigating amino acid metabolism in patients with chronic obstructive pulmonary disease did not show changes in plasma arginine concentrations compared with healthy age- and sex-matched controls (21, 22). Moreover, there was no relation between plasma arginine concentrations and BMI, weight loss, or fat-free mass (21), which indicates that the observed arginine depletion in the presence of a tumor is probably specific for cancer. In the pancreatic cancer patients, in whom weight loss was present, all amino acid concentrations were decreased. This suggests that arginine concentrations change early in the course of tumor development and could indicate that this contributes to the initiation of the cascade of metabolic disturbances leading to cachexia. Other factors playing a role may well be branched-chain amino acid or essential amino acid metabolism. Obviously, further studies are necessary to elucidate whether derangements in arginine metabolism are causally related to the phenotypic characteristics of cachexia.
Over the past decade, it has become clear that the host's metabolism in cancer is different from that in simple starvation or fasting, and that mere supplementation of calories cannot reverse cachexia. This knowledge resulted in studies investigating the metabolic changes accompanying weight loss in cancer. Still, the mechanism of cancer cachexia remains to be unraveled, which might be because of the complexity of the metabolic abnormalities that are present when symptoms such as weight loss or anorexia become obvious. Therefore, studying early changes in cancer may provide insight into the onset of the events that ultimately lead to cachexia. In this respect, it is clear from the present data that metabolic changes were already apparent in patients with breast or colonic cancer without weight loss being present. Our observation supports the idea of cancer inducing disturbances before anorexia and loss of muscle and fat become obvious. In fact, data from noncachectic patients may give better clues to the factors involved in initiating cachexia rather than accompanying it. In this respect, disturbances in arginine metabolism may play a role.
In conclusion, plasma arginine concentrations are decreased in cancer patients both with and without weight loss, which indicates that arginine metabolism is disturbed in the presence of a malignant tumor. Further study of in vivo production rates of arginine and NO in cancer patients is indicated.
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ACKNOWLEDGMENTS
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We thank DJH Haagen, P de Boer, and HMH van Eijk for expert HPLC measurements and JM Maessen and MMH Hendrikx for assistance with collection of blood samples taken in Maastricht.
YLJV and CHCD collected the patient's blood samples; YLJV and NEPD wrote the manuscript; and all authors contributed to study design, data interpretation, and final setup of the manuscript. None of the authors had advisory board affiliations or financial interests in organizations sponsoring the research.
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Received for publication November 10, 2004.
Accepted for publication December 14, 2004.
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ABSTRACT
INTRODUCTION
