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The Journal of Nutrition Vol. 128 No. 8 August 1998,
pp. 1355-1360
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Isotope Biochemistry Laboratory, SURRC, East Kilbride, Glasgow G75 0QF, UK; * University Department of Surgery, Royal Infirmary, Glasgow G31 2ER, UK; University Department of Surgery, Royal Infirmary, Edinburgh EH3 9YW, UK; ** Department of Clinical Chemistry, Royal Liverpool University Hospital, Liverpool L69 3GA, UK
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The unusual amino acid composition of acute phase proteins may be relevant to our understanding of the mechanism of tissue wasting in chronic inflammatory disease. During periods in which demand for amino acids outstrips dietary supply, skeletal muscle protein may be mobilized to meet this demand. An imbalance in the amino acid composition of these proteins may thus be detrimental to the body's nitrogen economy. To address this problem, we have measured the synthetic rate of fibrinogen (perhaps the major acute phase protein) and plasma amino acid profiles in a group of patients with adenocarcinoma of the pancreas and an ongoing inflammatory response (serum C-reactive protein >10 mg/L in the absence of any other obvious infective or inflammatory cause). These were also measured in a control group with no evidence of inflammation. Fibrinogen synthesis was measured after an overnight fast, using a flooding dose of 2H5-phenylalanine. The fractional rate of fibrinogen synthesis was significantly elevated in the cancer group compared with healthy controls [39.3 (20.0-49.9) and 21.9 (13.2-37.7) %/d, respectively; median (range), P < 0.05]. The absolute rate of fibrinogen synthesis was also elevated [84 (33-143) and 26 (15-43) mg/(kg·d), respectively; median (range), P < 0.01]. We calculated that, in cancer patients with anorexia-cachexia (i.e., documented ongoing weight loss in the absence of an obvious cause such as obstruction or malabsorption), aromatic amino acid supply (predominantly tryptophan) most limits fibrinogen synthesis from skeletal muscle reserves. Demand for the nonessential amino acids serine and glycine was elevated. Assuming that tryptophan is limiting, up to 2.6 g muscle protein (~12 g skeletal muscle tissue) may be wasted to synthesize 1 g fibrinogen. Interpretation of the observation that circulating free tryptophan concentrations were significantly reduced in the cancer patients will have to await flux measurements. The metabolic changes accompanying the inflammatory response suggest that down-regulation of this process may be beneficial.
KEY WORDS: amino acids · fibrinogen
2H5-phenylalanine ·
pancreatic cancer
protein synthesis rates ·
humans
Restoration of lean body mass in patients with gastrointestinal malignancies has proven extremely difficult despite extensive investigations over the last two decades (Fearon 1992 There have been a number of studies that have compared circulating amino acid concentrations in healthy subjects and cancer patients (Bennegard et al. 1984 A number of procedures for measuring protein synthesis in the clinical situation have been described. The flooding dose protocol (Garlick et al. 1989 The aim of this study was to examine the fibrinogen synthesis rates and amino acid concentrations in normal subjects and in cancer patients with an acute phase response. Experience of the use of the flooding dose technique for measuring the fibrinogen synthetic rate is also reported. The importance of these findings is discussed in relation to the body's nitrogen economy.
Patients.
Six patients with histologically proven adenocarcinoma of the pancreas were entered into the study. None of these patients had undergone surgery in the preceding 2 mo, but all had an ongoing inflammatory response and had a weight loss of >10% of their pre-illness weight. Serum C-reactive protein (CRP) was >10 mg/L; other obvious infective or inflammatory causes were not apparent. Seven patients, who had been admitted to hospital for minor procedures (e.g., inguinal hernia repair), were also studied as normal subjects. These subjects were weight stable, had normal biochemical liver function tests and had no evidence of an acute phase response (CRP <5 mg/L) or any other metabolic or endocrine disorder. None of the patients were jaundiced, pyrexial or had clinical or radiological evidence of infection or were severely anemic. The study was approved by the local ethical committee. All patients were informed of the purpose and procedure of the study and all gave written informed consent.
Experimental design.
After an overnight fast, a venous catheter was inserted into each anticubital fossa. One catheter was used for the administration of a bolus solution of 2H5-labeled phenylalanine, over 10 min (3.5 g of phenylalanine, 10 mol % excess phenyl-2H5-L-phenylalanine, 2 g/L in saline, Tracer Technologies, Sommerville, MA). The catheter in the opposite arm was used to sample 10 mL blood before and 10, 20, 40, 60, 80 and 120 min after receiving the 2H5-phenylalanine dose, for preparation and storage as plasma and serum ( Analytical methods.
Albumin concentrations were measured using the bromocresol green method (Doumas et al. 1971 Sample preparation and isotope analysis.
The study protocol involved the measurement of 2H5-phenylalanine enrichment in the plasma free phenylalanine pool and in plasma fibrinogen. For free phenylalanine analysis, 1.5 mL plasma was diluted with 5 mL distilled deionized water and cycloleucine (250 nmol) was added as an internal standard. Samples were then deproteinized by ultrafiltration (25,000 MW cut-off Centrifree cone, Amicon, Gloucestershire, UK), acidified and the amino acids purified by cation exchange. 2H5-Phenylalanine enrichment was measured by gas chromatography-mass spectrometry as its tert-butyldimethylsilyl derivative (Slater et al. 1995 Calculations.
Fractional synthesis rates of fibrinogen were calculated by dividing the rate of change of 2H5-phenylalanine enrichment of fibrinogen by the area under the curve of precursor enrichment vs. time as described by Ballmer et al. (1990) Statistics.
Data are presented as medians and ranges. Where appropriate, data were tested for statistical significance using the Mann-Whitney U-test (Mintab, State College, PA). Correlations between appropriate variables were tested using Spearman's Rank Correlation (Minitab).
The clinical characteristics of the control and cancer patients are shown in Table 1. The normal subject and cancer patient groups did not differ in terms of age and body mass index. There were significantly lower circulating albumin concentrations and significantly greater fibrinogen and C-reactive protein concentrations in the cancer patients compared with the control group (P < 0.01).
It has been assumed that elevated fibrinogen concentrations frequently observed in cancer patients (Dvorak 1987
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Heys et al. 1992). Chronic inflammation, evidenced by an ongoing acute phase response, has been associated with the prevention of such restoration (McMillan et al. 1994b and 1994c). The unusual amino acid composition of acute phase proteins may constitute part of the mechanism. Grimble (1990) suggested that acute phase protein synthesis may introduce an additional demand for the metabolically interrelated amino acids glycine, serine, methionine and cysteine. Body composition studies (Preston et al. 1987) have shown that skeletal muscle protein is the most likely reserve of amino acids that are mobilized in the anorectic-cachectic patient. Reeds et al. (1994) argued that, because acute phase proteins are not rich in sulfur amino acids, it is the supply of aromatic amino acids (tryptophan, phenylalanine and tyrosine) from skeletal muscle that most likely limits acute phase protein production.
, Clarke et al. 1978, Levin et al. 1983, Norton et al. 1985). However, such studies have yielded conflicting results and comparison of findings is difficult because there has been no apparent attempt to relate these to the presence of an acute phase response. Furthermore, the potential effect of acute phase protein synthesis on skeletal muscle protein reserves in cancer patients is unclear because data on the magnitude of acute phase protein synthesis are scarce. Fibrinogen is one of the most important acute phase proteins, accounting for ~10% of liver export protein synthesis in normal fasting subjects (McMillan et al. 1996), a figure that appears to be significantly greater in cancer patients (Stein et al. 1978b).
) has a number of practical advantages, including being well accepted by patients and clinical staff. Despite this, its use in humans has been limited. In particular, there are few data to establish protocols for repeat measurements or data on the reproducibility of the measured rates. With respect to the measurement of fibrinogen synthesis, no such data have been reported.
SUBJECTS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
20°C). In one normal volunteer, the protocol was repeated with additional blood samples taken at 6, 12 and 24 h and 2, 3, 4, 7, 10 and 22 d. The blood sample collected before the "flooding" dose was analyzed for baseline phenylalanine enrichment and albumin, fibrinogen, C-reactive protein and amino acid concentrations. Blood samples taken at the time points after the flooding dose were analyzed for 2H5-phenylalanine enrichment in fibrinogen and in the plasma free phenylalanine pool. Plasma volume was predicted from body weight and height (Retzlaff et al. 1969). Because the protocol was to be performed in fasting subjects, no dietary history or nutrient intake was recorded for the period before the investigation.
) on a Technicon RA-1000 automated analyzer (Technicon Instruments, Tarrytown, NY).
). Within-assay variability was <5%. Between assay variability was <10%. The mid-range of the assay was 2.5-4.5 g/L. The limit of detection was 1.0 g/L.
).
). For fibrinogen analysis, the sample in the ultrafiltration cone was then washed three times with 5 mL distilled deionized water to remove traces of free phenylalanine. Fibrinogen was then extracted by diluting the plasma proteins to 20 mL with saline and 0.5 mL calcium chloride (0.5 mol/L), adding 15 units of human (albumin-free) thrombin (Sigma-Aldrich, Poole, UK); after 10 min, the fibrin was collected on a glass rod. The fibrin was then hydrolyzed under vacuum at 145°C for 4 h with 6 mol/L HCl, and its 2H5-phenylalanine enrichment was measured as previously described (Slater et al.1995). The remaining serum sample could then be used for analysis of the synthetic rates of other plasma proteins (McMillan et al. 1996).
. Similarly, the secretion times for fibrinogen were calculated as described by Ballmer et al. (1990).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 1.
Patient characteristics1
View this table:
Table 2.
Fibrinogen synthesis in fasting healthy subjects and
cancer patients1
0.60, P < 0.05) and glycine (r = 0.53, P < 0.05).
View this table:
Table 3.
Serum free amino acid concentrations in fasting healthy subjects and cancer patients
View larger version (9K):
[in a new window]
Fig 1.
Plasma free phenylalanine and fibrinogen-bound phenylalanine over a period of 22 d, after a flooding dose of 2H5-phenylalanine in a healthy volunteer.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
) that are associated with an ongoing inflammatory response (Preston et al. 1987 and 1995) are the result of increased synthetic rate, rather than reduced breakdown or reduced pool size. However, confirmation of this hypothesis and determination of the magnitude of any increase have only once been attempted previously (Stein et al. 1978a). In this study, we have determined the increase in fibrinogen fractional and absolute synthetic rate to be ~79 and 220%, respectively, compared with healthy subjects. Furthermore, we have demonstrated that there are significant correlations between the fibrinogen kinetic parameters (i.e., fractional and absolute synthetic rate) and its circulating concentration. This would support the hypothesis that increased fibrinogen synthesis largely determines the observed increase in circulating concentrations. Cytokines, especially interleukin-6 (IL6), are thought to be responsible for the mediation of enhanced acute phase protein synthesis by the hepatocytes (Heinrich et al. 1990). Indeed, we have previously reported the association of elevated circulating IL-6 with the acute phase response in patients with cancer (Fearon et al. 1991, Scott et al. 1996).
. These authors reported the fractional synthetic rate of fibrinogen to be ~15%/d in healthy subjects and 26%/d in cancer patients. Although these values are similar to those from this study (21.9%/d and 39.3%/d, respectively), they are consistently lower. The earlier study had a number of design and methodological limitations. A heterogeneous group of cancer patients was studied and the presence of an acute phase response was not established. Use of plasma total 
-amino acid 15N enrichment as the precursor after administration of 15N-glycine, although measuring fibrinogen-bound -amino acid 15N enrichment, ignores any differences in 15N enrichment between plasma and the intrahepatic free amino acid pool and also ignores differences in amino acid composition between the plasma precursor pool and fibrinogen. Although debate continues concerning the site of the precursor pool for liver export protein synthesis (Connell et al. 1997), the earlier approach may have overestimated precursor amino acid enrichment and have led to an underestimate of fibrinogen synthetic rate. The flooding dose protocol used in this study gives greater confidence in the assumption that the free amino acid pool at the site of protein synthesis is in isotopic equilibrium with that measured in the plasma.
), which may incorporate more tracer in response to a transient increase in insulin concentrations or to elevated intracellular concentrations of essential amino acids. To our knowledge, there is no evidence to suggest that this may be the case with acute phase proteins whose synthesis seems to be regulated largely by cytokines (IL-6, Heinrich et al. 1990). Furthermore, this study was designed as a comparative study. When measuring liver export protein synthesis, plasma free tracer enrichment is assumed to reflect precursor enrichment. In this study, plasma free phenylalanine came into isotopic equilibrium with fibrinogen-bound phenylalanine within 7 h and remained so for 22 d (mean fibrinogen-bound 2H5-phenylalanine enrichment was 101% of that in the plasma free pool between 7 h and 22 d; see Fig. 1), indirectly validating the equivalence of the plasma and the precursor pool within the flooding dose protocol.
suggested that there may be increased demand for the amino acids glycine, serine, methionine and cysteine in fasting subjects with an acute phase protein response. In this study, an increase in fibrinogen synthesis in the cancer group of ~2.4 g/d (the mean change in fibrinogen total synthetic rate) was associated with a significant increase in circulating concentrations of glycine and cysteine with no change in serine or methionine concentrations. As noted by Reeds et al. (1994), fibrinogen is not particularly rich in sulfur amino acids; however, it is relatively rich in glycine and serine. De novo synthesis of these amino acids via glutamate transamination must compensate, but failure to meet this extra demand may lead to conditional essentiality of these amino acids (Slater et al. 1996). With all subjects included, there were significant rank correlations between the concentrations of glycine and cysteine and the absolute fibrinogen synthetic rates. It can be estimated (assuming that albumin and fibrinogen together account for 80-90% of liver export protein synthesis) that there was an overall increase of ~35% in the liver's requirement for these interrelated amino acids. It is therefore of interest that despite a likely increase in demand, the circulating concentrations of these amino acids were maintained or increased and would suggest that there was a considerable increase in the flux of these amino acids and in particular, glycine and cysteine. It is not know whether this could be related to increased degradation of albumin, a protein rich in cysteine whose synthesis is maintained at normal levels while its concentration is significantly lowered (Fearon et al, 1998). A large increase in glycine flux may compromise whole-body protein turnover measurements when using 15N-glycine as tracer in cancer patients with an acute phase protein response, although this tracer may be a sensitive indicator of the phenomenon. Indeed, we have previously questioned the accuracy of such measurements (McMillan et al. 1994a, Preston et al. 1995). The finding that circulating C-reactive protein concentrations rank correlate significantly with fibrinogen absolute synthetic rate parallels our previous observation of the correlation of C-reactive protein concentration and whole-body protein synthesis (Preston et al. 1995). These findings support our assertion that rates of whole-body protein synthesis, measured using 15N-glycine as tracer, tend to be overestimated in subjects with an acute phase protein response.
argued that increased demands for amino acids from immune system proliferation and glutathione synthesis, in addition to the acute phase protein response, are also likely to contribute to muscle protein breakdown. Park et al. (1994) studied peripheral blood lymphocyte protein synthesis in patients with colorectal cancer, using a flooding dose of 1-13C-leucine, and concluded that although lymphocyte protein fractional synthetic rate may be 9%/d, posing a similar amino acid demand to that of constitutive liver proteins, lymphocyte protein FSR of cancer patients was significantly reduced. Glutathione is formed by condensation of glutamate, cysteine and glycine. Cysteine can be supplied from the essential amino acid methionine, provided this is available. In this study, circulating concentrations of cysteine and glycine were significantly greater in the cancer patients, whereas those of glutamate and methionine were unaffected, providing little evidence for a failure of their supply. However, interpretation of changes in circulating amino acid concentration is problematic, and the current observations would have to be supported by flux measurements before any conclusions can be drawn.
). Such calculations highlight the limited supply of aromatic amino acids, especially phenylalanine, in such conditions. This calculation was based on the increase of a number of acute phase proteins after acute injury (surgery), whereas fibrinogen appears to be the major acute phase protein in chronic inflammation (cancer). By using acute phase protein changes immediately after surgery, Reeds et al. (1994) calculated that C-reactive protein, 1-antitrypsin and fibrinogen contributed 30, 24 and 24%, respectively, to total acute phase protein synthesis. In contrast, when analyzing longitudinal concentration changes of acute phase proteins after intervention with anti-inflammatory agents in cancer patients (Preston et al. 1995), we have calculated that these three proteins contribute 1, 25 and 59%, respectively, to total acute phase protein synthesis. Fibrinogen thus may be the most important acute phase protein in chronic inflammation, possibly contributing 15% of total liver export protein. With the use of our fibrinogen synthesis data, we have calculated that in fasting subjects, ~7.9 g of skeletal muscle protein (36 g skeletal muscle tissue) would be required daily to support the synthesis of an extra 3 g of fibrinogen (Slater et al. 1996). This would be a major contribution to tissue wasting if it continued unabated. This calculation showed that tryptophan (followed by serine and then tyrosine) was the most limiting amino acid. It is of interest that a negative correlation was observed between circulating concentrations of tryptophan and the absolute synthetic rate of fibrinogen and that tryptophan concentrations were reduced the most compared with the control group. This would appear to support the observation that tryptophan can be a limiting amino acid in cancer patients with an acute phase protein response.
, McMillan et al. 1994a), although there has been no measure of the rate of amino acid return from fibrinolysis. Should recycling be incomplete, or separated temporarily or spatially from synthesis, then acute phase protein synthesis could promote tissue wasting and continued negative nitrogen balance in the anorexia-cachexia patient.
analyzed the data of Fleck et al. (1985) and argued that at the peak of the catabolic response to infection, acute phase protein production could amount to 30% of the total body protein synthesis. Acute phase protein synthesis is unlikely to be of this magnitude in chronic inflammation, but our estimates show that it may contribute 15-25% to liver protein synthesis or 3-5% to whole-body protein synthesis. The realization that an imbalance in the amino acid composition between the supply (skeletal muscle) and demand (acute phase proteins) can exacerbate the negative nitrogen balance may have a profound effect on the nitrogen economy of the cachectic cancer patient with an ongoing acute phase protein response. Clearly, however, for this to be the case the patient would have to be profoundly anorectic.
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FOOTNOTES |
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Manuscript received 21 July 1997. Initial reviews completed 26 September 1997. Revision accepted 15 April 1998.
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ACKNOWLEDGMENTS |
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We thank C. S. McArdle, University Department of Surgery, Royal Infirmary, Glasgow and D. C. Carter, University Department of Surgery, Royal Infirmary, Edinburgh for their support throughout this work. We thank J. A. Ross, University Department of Surgery, Royal Infirmary, Edinburgh for his help and advice on biochemical assays and C. Lane, Department of Clinical Chemistry, University of Liverpool for his help with amino acid analysis.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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an optimal approach.
Am. J. Physiol.
1994;
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