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Serum carnitine levels in patients with tumoral cachexia
European Journal of Internal Medicine 16 (2005) 419 – 423 www.elsevier.com/locate/ejim
Original article
Serum carnitine levels in patients with tumoral cachexia Ernesto Vinci, Elvira Rampello, Luca Zanoli, Giovanni Oreste, Giovanni Pistone, Mariano Malaguarnera * Department of Senescence, Urological and Neurological Sciences, University of Catania, Cannizzaro Hospital, Via Messina, 829-95126 Catania, Italy Received 14 October 2004; received in revised form 22 February 2005; accepted 25 February 2005
Abstract Background: Cachexia is a serious complication of many cancers that is common in cancer and AIDS patients. However, the key factors and mechanisms involved in the development of cachexia are not yet understood. There is little data currently available regarding carnitine metabolism in patients with neoplasm and cachexia. Methods: Forty-six neoplastic patients with different localizations of their primary disease gave signed, informed consent before enrolling in the present study. They underwent routine laboratory investigation, including examination of the levels of the various forms of carnitine present in serum (i.e., long-chain acylcarnitine, short-chain acylcarnitine, soluble acid acylcarnitine, free carnitine, and total carnitine). These values were compared with those found in 30 cancer patients in good nutritional status as well as with those of 30 healthy control subjects. Results: In the comparison of serum plasma carnitine of cachectic patients versus controls, the difference in free carnitine was 8.20 Amol/L ( p = 0.000); the difference in short-chain acylcarnitine 2.60 Amol/L ( p = 0.029); the difference in soluble acid carnitine 10.80 Amol/L ( p = 0.000); the difference in long-chain acylcarnitine 0.40 Amol/L ( p = 0.036); and the difference in total carnitine 11.20 Amol/L ( p = 0.000). In the comparison of serum plasma carnitine of cachectic versus neoplastic patients in good nutritional status, the difference in free carnitine was 5.80 Amol/L ( p = 0.006); the difference in soluble acid carnitine 7.20 Amol/L ( p = 0.000); and the difference in total carnitine 7.50 Amol/L ( p = 0.000). Conclusion: Our study showed that, in the multifactorial pathogenesis of cachexia, the low serum levels of carnitine in terminal neoplastic patients, which are due to a decreased dietary intake as well as to an impaired endogenous synthesis of this substance, could play an important role. These low serum carnitine levels may also contribute to the development of cachexia in cancer patients. D 2005 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved. Keywords: Carnitine; Cachexia; Cancer; Acetyl-carnitine; Stress; Metabolism; Cytokine; Sarcopenia; Nutrition
1. Introduction Cachexia, derived from the Greek ‘‘kakos’’ (bad) and ‘‘hexis’’ (condition), is characterized by an inevitable progression of nutritional deterioration, which occurs in approximately 50% of all patients with malignant disorders [1]. A decrease in food intake, combined with a decrease in physical exercise, leads to a decline in muscle mass and power. Clinically, the cancer cachexia syndrome is characterized by anorexia, early satiety, wasting, weight loss, weakness, * Corresponding author. Tel.: +39 95 7262008; fax: +39 95 7262011. E-mail address: [email protected] (M. Malaguarnera).
fatigue, poor mental and physical performance, decreased capacity for wound healing, impaired immunological function, and a compromised quality of life, none of which are resolved by forced nutrient intake [2]. The pattern of weight loss seen in cachexia differs from that seen in pure nutrient deficiency, suggesting that several metabolic alterations are involved in triggering off the protein catabolism and muscle wasting. Cachexia may be suspected if there has been an unexpected weight loss of greater than 5% within a 6month period of time, combined with muscle wasting. Body compartment analysis has shown that patients with cachexia lose approximately equal amounts of fat-free mass. These losses occur primarily in skeletal muscle and reflect
0953-6205/$ - see front matter D 2005 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ejim.2005.02.014
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E. Vinci et al. / European Journal of Internal Medicine 16 (2005) 419 – 423
decreases in both cellular mass and intracellular potassium concentration [3]. l-carnitine is a hydrosoluble-polarized substance (3hydroxy-4-trimethylaminobutirric acid) synthesized from lysine. This latter amino acid is methylated using adenosylmethionine and produces epsilon-trimethyl-lysine, which is transformed into deoxycarnitine in all tissues. However, only the kidney and the liver have an enzymatic pattern that enables them to produce endogenous carnitine. From these sites, carnitine is absorbed and carried out to various tissues. The greatest amounts are found in the most metabolically active tissues, such as myocardium, skeletal muscle (as l-carnitine), and neurons (as lacetylcarnitine) [4,5]. Carnitine plays a key role in the production and distribution of cell energy, ensuring fatty acid transfer throughout the mitochondrial membrane to their metabolic oxidation sites. This last biochemical step is fundamental for the production of energy, stored as ATP. The carnitine system in cancer has been studied in different experimental and clinical models. Although much information is still lacking, it is clear that the carnitine system is modified. It presents abnormalities in the modulation and expression of its components in different ways in the various forms of cancer [6]. Although food generally contains a significant amount of carnitine, the endogenous synthesis of this substance is important in order to keep serum carnitine levels within the normal range. When there is a malignancy, several metabolic changes take place throughout the body. One of these may involve a reduction in endogenous synthesis of carnitine. Little is known about intracellular carnitine enzymatic abnormalities in human cancers. The data that are available confirm that the carnitine system is affected in a specific manner in different forms of human cancer. The aim of the present study was to evaluate levels of serum of patients with neoplasm and cachexia and to provide new insights into the mechanisms of cachexia.
Subjects with increased alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), or gammaglutamyl transpeptidase and creatinine serum levels were excluded from the study, as were those with increased serum bilirubin or creatinine concentrations or liver disease. We also enrolled two groups of control subjects. There were 30 cancer patients in good nutritional status (15 males and 15 females, mean age 52.8 T 8.1 years). Their diagnoses were as follows: 10 colorectal carcinoma, 5 lung cancer, 5 breast cancer, 3 gastric cancer, 3 bladder cancer, 2 renal cell carcinoma, and 2 prostate cancer. A second control group consisted of 30 healthy individuals aged 51.3 T 9.2 years. The two patient groups were examined in the morning between 8:00 a.m. and 10:00 a.m. after an overnight fast. Afterwards, venous blood samples were taken and stored in tubes containing ethylenediamine tetra acetic acid (EDTA) or heparin. Serum or plasma was obtained by centrifugation. Serum was analyzed immediately, while plasma and urine were stored at 20 -C before analysis. Serum carnitine levels were determined using the Cederblad and Lindstedt method, modified by Brass and Hoppel [7]. Plasma was treated with perchloric acid (final concentration 3% vol/vol) and centrifuged for 2 min at 10,000 g. Long-chain acylcarnitine was extracted from the pellet after alkaline hydrolysis, while free and short-chain acylcarnitine were extracted from the supernatant. The sum of short-chain acylcarnitine, free carnitine, and long-chain acylcarnitine was considered the serum total carnitine level. Creatinine concentrations were determined using a kinetic colorimetric reaction in the same samples used to measure the carnitine concentrations. All laboratory tests were performed using standard laboratory procedures. All subjects followed a daily diet consisting of 1800 kcal/day with a content of total cholesterol < 300 mg/day; 50% carbohydrates, 20% proteins, and 30% fats (of which 10% was saturated fatty acids, 10% unsaturated fatty acids, and 10% polyunsaturated fatty acids).
2. Patients and methods The patients eligible for this study had advanced malignancies localized in various parts of the body. The majority of patients were in the terminal phase of their disease and only palliation was requested. All of them had experienced a weight loss of more than 5% in the 6 months prior to enrollment in the study. There were 46 cachectics (28 males and 18 females, mean age 54.2 T 7 years) with the following diagnoses: 15 colorectal carcinoma, 6 gastric cancer, 5 lung cancer, 5 renal cell carcinoma, 5 malignant melanoma, 5 bladder cancer, 3 breast cancer, and 2 prostate cancer. Twenty-eight of them had undergone surgical intervention, while 27 had had one or more chemotherapeutic treatments and 9 radiotherapy.
Table 1 Main characteristics of the subjects included in our study Parameter
Cachectic patients
Neoplastic patients
Controls
Sex (M/F) Mean age (years T SD) Height (cm) Weight (kg) Systolic arterial pressure (mm Hg) Diastolic arterial pressure (mm Hg) Heart rate (bpm)
28/18 54.2 T 7.0 162.1 T 5.4 48 T 7.1 154.2 T 14.7
15/15 52.8 T 8.1 160.1 T 5.7 59.2 T 8.7 150.3 T 15.4
15/15 51.3 T 5 160 T 6.4 63.8 T 11.2 150 T 16.8
79.1 T 9.4
78.8 T 9.9
80.2 T 10.6
88 T 11
83.7 T 10.9
81.2 T 10
Cachectic patients vs. neoplastic patients in good nutritional status: weight C.I. 19.38 to 11.02, p = 0.000.
E. Vinci et al. / European Journal of Internal Medicine 16 (2005) 419 – 423 Table 2 Laboratory parameters of the subjects included in our study Parameter
Cachectic patients
Neoplastic patients
Controls
BUN (mg/dl) Glucose (mg/dl) Total cholesterol (mg/dl) Triglycerides (mg/dl) ALT (IU/L) AST (IU/L) ALP (IU/L) Creatinine (mg/dL)
39.6 T 5.4 69.7 T 10.7 168.4 T 12.8 151.8 T 10.7 48.4 T 7.2 49.1 T 8.4 214.4 T 21.4 1.04 T 0.18
40.1 T 5.5 78.2 T 8.7 191 T 9.8 165 T 9.9 42.4 T 6.9 44 T 7.2 187 T 15.9 1.06 T 0.24
32.8 T 7.9 71.2 T 9.4 187.7 T 10.8 170.1 T 9.8 36.7 T 4.4 35.8 T 6.7 165 T 18.2 0.97 T 0.21
Patients with malignancy and cachexia vs. cancer patients in good nutritional status: glucose C.I. 13.16 to 3.84, p = 0.000; total cholesterol C.I. 28.08 to 17.12, p = 0.000; triglycerides C.I. 18.06 to 8.34, p = 0.000; ALT C.I. 2.69 to 9.31, p = 0.000; AST C.I. 1.38 to 8.82, p = 0.008; ALP C.I. 118.31 to 36.49, p = 0.000. Cachectic patients vs. healthy control subjects: BUN C.I. 3.7 to 9.8, p = 0.000; total cholesterol C.I. 24.94 to 13.66, p = 0.000; triglycerides C.I. 23.14 to 13.46, p = 0.000; ALT C.I. 8.7 to 14.6, p = 0.000; AST C.I. 9.66 to 16.94, p = 0.000; ALP C.I. 39.9 to 58.8, p = 0.000. Neoplastic patients in good nutritional status vs. healthy control subjects: BUN C.I. 10.8 to 3.7, p = 0.000; glucose C.I. 11.6 to 2.3, p = 0.004; triglycerides C.I. 0.01 to 10.1, p = 0.05; ALT C.I. 8.7 to 2.7, p = 0.000; AST C.I. 17.8 to 4.6, p = 0.000; ALP C.I. 30.8 to 13.1, p = 0.000.
2.1. Statistical analysis The results are presented as mean T standard deviation. The following two-tailed tests at the p = 0.05 level of significance were used in the study: the Mann-Whitney Utest in the case of two independent samples and the Spearman’s rank correlation coefficient test to test for univariate relationships between variables. In order to evaluate the independent effects of covariates on carnitine concentration, a stepwise multiple linear regression analysis was performed. Data were analyzed using the statistical package SPSS for Windows 7.5 (SPSS Inc., Chicago, IL, USA).
3. Results
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( p = 0.000). The difference in free carnitine was 5.80 Amol/L ( p =0.006); in soluble acid carnitine 7.20 Amol/ L ( p = 0.000); and in total carnitine 7.50 Amol/L ( p = 0.000) (Tables 2 and 3). When comparing the cachectic patients with the healthy control subjects, the difference in BUN was 7.1 mg/dl ( p = 0.000); in total cholesterol 19.30 mg/dl ( p = 0.000); in triglycerides 18.30 ( p = 0.000); in ALT 11.7 IU/L ( p = 0.000); in AST 13.30 IU/L ( p = 0.000); and in ALP 49.4 (IU/L) ( p = 0.000). The difference in free carnitine was 8.20 Amol/L ( p = 0.000); in short-chain acylcarnitine 2.60 Amol/L ( p = 0.029); in soluble acid carnitine 10.80 Amol/L ( p = 0.000); in long-chain acylcarnitine 0.40 Amol/L ( p = 0.036); and in total carnitine 11.20 Amol/L ( p = 0.000) (Tables 2 and 3). When comparing neoplastic patients in good nutritional status with healthy controls, the difference in BUN was 7.30 mg/dl ( p = 0.000); in glucose 7.00 mg/dl ( p = 0.004); in triglycerides 5.1 ( p = 0.05); in ALT 5.7 IU/L ( p = 0.000); in AST 8.2 IU/L ( p = 0.000); and in ALP 22.0 IU/L ( p = 0.000) (Table 2). In the comparison of serum plasma carnitine in controls versus neoplastic patients, there were no significant differences. The correlation studies showed a strong correlation of total carnitine (r = 0.74), short-chain acyl-carnitine (r = 0.72), long-chain acyl-carnitine (r = 0.73), free carnitine (r = 0.72), and weight loss. No correlation was found between total carnitine or the fractions of carnitine and other biohumoral and demographic characteristics.
4. Discussion In patients with cancer and cachexia, serum carnitine levels are decreased in comparison to levels found in cancer patients in good nutritional status and in healthy control subjects. The cachexia is characterized by various abnormalities in carbohydrate, protein, and fat metabolism [8]. In cancer cachexia, the carbohydrate metabolism presents
3.1. Comparison of baseline characteristics
Table 3 Comparison of serum values in the three subject groups
The three groups of subjects were comparable in age, height, systolic and diastolic pressure, and heart rate. The difference in weight between cachectic patients and neoplastic patients in good nutritional status was 11.2 kg ( p = 0.000) (Table 1).
Parameter
Cachectic patients Neoplastic patients Controls
Free carnitine Amol/L Short-chain acylcarnitine Amol/L Long-chain acylcarnitine Amol/L Total carnitine Amol/L
33.7 T 9.4 7.8 T 4.8
39.5 T 7.8 9.2 T 6.4
41.9 T 8.4 10.4 T 5.2
2.4 T 0.9
2.7 T 0.8
2.8 T 0.6
43.9 T 6.8
51.4 T 7.2
55.1 T 9.7
3.2. Comparison of laboratory parameters When comparing the patients with malignancy and cachexia with the cancer patients in good nutritional status, the difference in glucose was 8.5 mg/dl ( p = 0.000); in total cholesterol 22.7 ( p = 0.000); in triglycerides 13.20 mg/dl ( p = 0.000); in ALT 6.00 IU/L ( p = 0.000); in AST 5.10 IU/L ( p = 0.008); and in ALP 27.40 IU/L
Patients with malignancy and cachexia vs. cancer patients in good nutritional status: free carnitine C.I. 9.92 to 1.68, p = 0.006; soluble acid carnitine C.I. 10.43 to 3.97, p = 0.000; total carnitine C.I. 10.75 to 4.25, p = 0.000. Cachectic patients vs. healthy control subjects: free carnitine C.I. 12.42 to 3.98, p = 0.000; short-chain acylcarnitine C.I. 4.92 to 0.28, p = 0.029; soluble acid carnitine C.I. 14.41 to 7.19, p = 0.000; longchain acylcarnitine C.I. 0.77 to 0.03, p = 0.036; total carnitine C.I. 14.97 to 7.43, p = 0.000.
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glucose intolerance, insulin resistance, abnormal insulin secretion, increased glucose production and turnover, and increased Cori cycle activity. The protein metabolism presents increased whole body protein turnover, increased protein fractional synthesis rates in the liver, decreased functional synthesis rates in muscle, and increased hepatic protein synthesis [8]. In addition to recalcitrant muscle protein breakdown and decreased plasma levels of branched-chain amino-acids, the fat metabolism presentsexcess body fat depletion relative to body protein loss, increased lipolysis, increased free fatty acids, increased glycerol turnover, decreased lipogenesis, hyperlipidemia, and failure of glucose to suppress oxidation of free fatty acids. Most studies investigating the role of carnitine in immunity have been done on patients with HIV infection and they have concluded that carnitine improves immune cell functions and downregulates levels of TNF-alpha [9]. Cachexia has a multifactorial pathogenesis and involves several neuronal systems that modulate production and transport of cell energy, such as hormones (leptine), neuropeptides, cytokines (IL-1, IL-6, TNF), and neurotransmitters (serotonin and dopamine). Consequently, an association between acute phase response and increased hepatic protein synthesis, as well as the high-energy expenditure and wasting, may be observed in cancer patients [10 – 12]. Cachexia is characterized by fast weight loss with wasting of fat tissue and a severe decrease in skeletal-muscular mass. Carnitine is involved in the metabolism of branchedchain amino acids. The formation of branched-chain acylcarnitine from branched-chain amino acids has been detected in peroxisomes. Branched-chain amino acids have been detected in mitochondria and peroxisomes, and branched-chain acyl-carnitine oxidation has been demonstrated in both organ cells [13]. Branched-chain amino acids may modulate the peripheral muscle proteolysis that occurs in cancer cachexia by providing protein-sparing substrate for both muscle metabolism and gluconeogenesis [14]. In these two processes, carnitine plays a key role because of its involvement in the metabolism of fat acids and skeletal muscle. The direct relationship between total serum carnitine and short-chain and long-chain acylcarnitine levels in cachectic patients suggests that the observed decrease in the acylcarnitine concentration is secondary to the decrease in the total amount of this substance. This decrease may be due to a decreased availability of carnitine in the diet or to altered endogenous biosynthesis. Although the relationship is still unclear, several studies suggest that specific alimentary factors may influence serum carnitine levels [15 – 18]. Both gender-and age-related factors, as well as physical activity, may also influence serum carnitine concentrations. The dietary alterations in neoplastic patients may have been a limitation of our study, even though we tried to reduce the effects of diet on serum carnitine concentration by administering a similar diet to all subjects enrolled in the study.
Decreased dietary absorption of carnitine could be due to elevated cytokine production associated with cachexia. In fact, interleukin-6 induces anorexia and an increase in muscular catabolism, resulting in weight loss and decreased muscle strength. Malnutrition triggers a vicious circle in which neoplastic patients produce additional amounts of cytokines. With regard to low serum carnitine levels, one must remember that the first step in the biosynthesis of this substance is methylation of protein-bound lysine to trimethyl-lysine, which is subsequently cleaved from the proteins by proteolysis. Most of the protein-bound trimethyl-lysine is found in skeletal muscle [19], and the rate of carnitine biosynthesis depends on the availability of trimethyl-lysine. Skeletal muscle turnover is reported to be the rate-limiting step of carnitine biosynthesis [20,21]. Carnitine deficiency is associated with impaired fat oxidation and a number of functional abnormalities. Carnitine can also bind acetyl residues in the mitochondrial matrix, where specific carnitine acetyltransferase activity is present, and export them to the cytosolic compartment. As such, carnitine is important in both lipid oxidation and lipid synthesis; from an energy standpoint, it is involved in both the liberation and storage of metabolic energy. A decrease in serum carnitine explains not only the sarcopenia but also the physical and mental fatigue detectable in patients [22]. A decrease in acylcarnitine, a fundamental substance in brain metabolism, induces behavioral and cognitive changes (anxiety, depression, and malignancy or treatment-related anorexia). One of many theories regarding the cachexia syndrome includes heightened cytokine activity (tumor necrosis factor, interleukin 1, and interferon). An increase in resting energy expenditure may contribute to weight loss in cancer patients. This change in energy expenditure may also explain the increased oxidation of fat. The effect that these opposite actions of carnitine have on glucose metabolism has not been determined. One could predict that an enhancement of fat oxidation, whether induced by increased lipolysis or by larger carnitine availability or both, would depress carbohydrate oxidation by substrate competition, according to Randle’s cycle [23]. In vitro studies confirm the ability of carnitine to reduce Fas-mediated apoptosis [24]. Signal transduction via the surface glycoprotein FAS, also known as CD95, is considered the most important pathway for the regulation of programmed cell death (i.e., apoptosis). Carnitine inhibits apoptosis by interacting with the FAS ligand and the FAS receptor systems [25]. Signal transduction via the FAS receptor activates acid sphingomyelinase; as a result, there is a breakdown of sphingomyelin and a release of ceramide [26]. An anti-apoptotic effect has been demonstrated by treating cultured neurons and myocardial cells with carnitine. Involvement of carnitine in balancing cell volume at the cytoskeletal level and phospholipid amount at the cell surface has also been demonstrated.
E. Vinci et al. / European Journal of Internal Medicine 16 (2005) 419 – 423
One may question the value of serum carnitine determination with respect to consequences and interpretation. This is due to the fact that serum carnitine represents approximately 3% of total body carnitine. In fact, skeletal muscles are the main reservoir in the body, with a carnitine concentration that is at least 50 –200 times higher than in blood plasma, where average concentrations range from 40 AM/L to 60 AM/L [27]. The patients in our study had many different types of malignancies. Unfortunately, the numbers of patients with each type of malignancy were too small to allow conclusions to be drawn with respect to the potential differences in serum carnitine levels between the different malignancies. Cachexia is a serious and common problem in cancer management. A better understanding of its causes may lead to increased treatment possibilities.
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Original article
Serum carnitine levels in patients with tumoral cachexia Ernesto Vinci, Elvira Rampello, Luca Zanoli, Giovanni Oreste, Giovanni Pistone, Mariano Malaguarnera * Department of Senescence, Urological and Neurological Sciences, University of Catania, Cannizzaro Hospital, Via Messina, 829-95126 Catania, Italy Received 14 October 2004; received in revised form 22 February 2005; accepted 25 February 2005
Abstract Background: Cachexia is a serious complication of many cancers that is common in cancer and AIDS patients. However, the key factors and mechanisms involved in the development of cachexia are not yet understood. There is little data currently available regarding carnitine metabolism in patients with neoplasm and cachexia. Methods: Forty-six neoplastic patients with different localizations of their primary disease gave signed, informed consent before enrolling in the present study. They underwent routine laboratory investigation, including examination of the levels of the various forms of carnitine present in serum (i.e., long-chain acylcarnitine, short-chain acylcarnitine, soluble acid acylcarnitine, free carnitine, and total carnitine). These values were compared with those found in 30 cancer patients in good nutritional status as well as with those of 30 healthy control subjects. Results: In the comparison of serum plasma carnitine of cachectic patients versus controls, the difference in free carnitine was 8.20 Amol/L ( p = 0.000); the difference in short-chain acylcarnitine 2.60 Amol/L ( p = 0.029); the difference in soluble acid carnitine 10.80 Amol/L ( p = 0.000); the difference in long-chain acylcarnitine 0.40 Amol/L ( p = 0.036); and the difference in total carnitine 11.20 Amol/L ( p = 0.000). In the comparison of serum plasma carnitine of cachectic versus neoplastic patients in good nutritional status, the difference in free carnitine was 5.80 Amol/L ( p = 0.006); the difference in soluble acid carnitine 7.20 Amol/L ( p = 0.000); and the difference in total carnitine 7.50 Amol/L ( p = 0.000). Conclusion: Our study showed that, in the multifactorial pathogenesis of cachexia, the low serum levels of carnitine in terminal neoplastic patients, which are due to a decreased dietary intake as well as to an impaired endogenous synthesis of this substance, could play an important role. These low serum carnitine levels may also contribute to the development of cachexia in cancer patients. D 2005 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved. Keywords: Carnitine; Cachexia; Cancer; Acetyl-carnitine; Stress; Metabolism; Cytokine; Sarcopenia; Nutrition
1. Introduction Cachexia, derived from the Greek ‘‘kakos’’ (bad) and ‘‘hexis’’ (condition), is characterized by an inevitable progression of nutritional deterioration, which occurs in approximately 50% of all patients with malignant disorders [1]. A decrease in food intake, combined with a decrease in physical exercise, leads to a decline in muscle mass and power. Clinically, the cancer cachexia syndrome is characterized by anorexia, early satiety, wasting, weight loss, weakness, * Corresponding author. Tel.: +39 95 7262008; fax: +39 95 7262011. E-mail address: [email protected] (M. Malaguarnera).
fatigue, poor mental and physical performance, decreased capacity for wound healing, impaired immunological function, and a compromised quality of life, none of which are resolved by forced nutrient intake [2]. The pattern of weight loss seen in cachexia differs from that seen in pure nutrient deficiency, suggesting that several metabolic alterations are involved in triggering off the protein catabolism and muscle wasting. Cachexia may be suspected if there has been an unexpected weight loss of greater than 5% within a 6month period of time, combined with muscle wasting. Body compartment analysis has shown that patients with cachexia lose approximately equal amounts of fat-free mass. These losses occur primarily in skeletal muscle and reflect
0953-6205/$ - see front matter D 2005 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ejim.2005.02.014
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E. Vinci et al. / European Journal of Internal Medicine 16 (2005) 419 – 423
decreases in both cellular mass and intracellular potassium concentration [3]. l-carnitine is a hydrosoluble-polarized substance (3hydroxy-4-trimethylaminobutirric acid) synthesized from lysine. This latter amino acid is methylated using adenosylmethionine and produces epsilon-trimethyl-lysine, which is transformed into deoxycarnitine in all tissues. However, only the kidney and the liver have an enzymatic pattern that enables them to produce endogenous carnitine. From these sites, carnitine is absorbed and carried out to various tissues. The greatest amounts are found in the most metabolically active tissues, such as myocardium, skeletal muscle (as l-carnitine), and neurons (as lacetylcarnitine) [4,5]. Carnitine plays a key role in the production and distribution of cell energy, ensuring fatty acid transfer throughout the mitochondrial membrane to their metabolic oxidation sites. This last biochemical step is fundamental for the production of energy, stored as ATP. The carnitine system in cancer has been studied in different experimental and clinical models. Although much information is still lacking, it is clear that the carnitine system is modified. It presents abnormalities in the modulation and expression of its components in different ways in the various forms of cancer [6]. Although food generally contains a significant amount of carnitine, the endogenous synthesis of this substance is important in order to keep serum carnitine levels within the normal range. When there is a malignancy, several metabolic changes take place throughout the body. One of these may involve a reduction in endogenous synthesis of carnitine. Little is known about intracellular carnitine enzymatic abnormalities in human cancers. The data that are available confirm that the carnitine system is affected in a specific manner in different forms of human cancer. The aim of the present study was to evaluate levels of serum of patients with neoplasm and cachexia and to provide new insights into the mechanisms of cachexia.
Subjects with increased alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), or gammaglutamyl transpeptidase and creatinine serum levels were excluded from the study, as were those with increased serum bilirubin or creatinine concentrations or liver disease. We also enrolled two groups of control subjects. There were 30 cancer patients in good nutritional status (15 males and 15 females, mean age 52.8 T 8.1 years). Their diagnoses were as follows: 10 colorectal carcinoma, 5 lung cancer, 5 breast cancer, 3 gastric cancer, 3 bladder cancer, 2 renal cell carcinoma, and 2 prostate cancer. A second control group consisted of 30 healthy individuals aged 51.3 T 9.2 years. The two patient groups were examined in the morning between 8:00 a.m. and 10:00 a.m. after an overnight fast. Afterwards, venous blood samples were taken and stored in tubes containing ethylenediamine tetra acetic acid (EDTA) or heparin. Serum or plasma was obtained by centrifugation. Serum was analyzed immediately, while plasma and urine were stored at 20 -C before analysis. Serum carnitine levels were determined using the Cederblad and Lindstedt method, modified by Brass and Hoppel [7]. Plasma was treated with perchloric acid (final concentration 3% vol/vol) and centrifuged for 2 min at 10,000 g. Long-chain acylcarnitine was extracted from the pellet after alkaline hydrolysis, while free and short-chain acylcarnitine were extracted from the supernatant. The sum of short-chain acylcarnitine, free carnitine, and long-chain acylcarnitine was considered the serum total carnitine level. Creatinine concentrations were determined using a kinetic colorimetric reaction in the same samples used to measure the carnitine concentrations. All laboratory tests were performed using standard laboratory procedures. All subjects followed a daily diet consisting of 1800 kcal/day with a content of total cholesterol < 300 mg/day; 50% carbohydrates, 20% proteins, and 30% fats (of which 10% was saturated fatty acids, 10% unsaturated fatty acids, and 10% polyunsaturated fatty acids).
2. Patients and methods The patients eligible for this study had advanced malignancies localized in various parts of the body. The majority of patients were in the terminal phase of their disease and only palliation was requested. All of them had experienced a weight loss of more than 5% in the 6 months prior to enrollment in the study. There were 46 cachectics (28 males and 18 females, mean age 54.2 T 7 years) with the following diagnoses: 15 colorectal carcinoma, 6 gastric cancer, 5 lung cancer, 5 renal cell carcinoma, 5 malignant melanoma, 5 bladder cancer, 3 breast cancer, and 2 prostate cancer. Twenty-eight of them had undergone surgical intervention, while 27 had had one or more chemotherapeutic treatments and 9 radiotherapy.
Table 1 Main characteristics of the subjects included in our study Parameter
Cachectic patients
Neoplastic patients
Controls
Sex (M/F) Mean age (years T SD) Height (cm) Weight (kg) Systolic arterial pressure (mm Hg) Diastolic arterial pressure (mm Hg) Heart rate (bpm)
28/18 54.2 T 7.0 162.1 T 5.4 48 T 7.1 154.2 T 14.7
15/15 52.8 T 8.1 160.1 T 5.7 59.2 T 8.7 150.3 T 15.4
15/15 51.3 T 5 160 T 6.4 63.8 T 11.2 150 T 16.8
79.1 T 9.4
78.8 T 9.9
80.2 T 10.6
88 T 11
83.7 T 10.9
81.2 T 10
Cachectic patients vs. neoplastic patients in good nutritional status: weight C.I. 19.38 to 11.02, p = 0.000.
E. Vinci et al. / European Journal of Internal Medicine 16 (2005) 419 – 423 Table 2 Laboratory parameters of the subjects included in our study Parameter
Cachectic patients
Neoplastic patients
Controls
BUN (mg/dl) Glucose (mg/dl) Total cholesterol (mg/dl) Triglycerides (mg/dl) ALT (IU/L) AST (IU/L) ALP (IU/L) Creatinine (mg/dL)
39.6 T 5.4 69.7 T 10.7 168.4 T 12.8 151.8 T 10.7 48.4 T 7.2 49.1 T 8.4 214.4 T 21.4 1.04 T 0.18
40.1 T 5.5 78.2 T 8.7 191 T 9.8 165 T 9.9 42.4 T 6.9 44 T 7.2 187 T 15.9 1.06 T 0.24
32.8 T 7.9 71.2 T 9.4 187.7 T 10.8 170.1 T 9.8 36.7 T 4.4 35.8 T 6.7 165 T 18.2 0.97 T 0.21
Patients with malignancy and cachexia vs. cancer patients in good nutritional status: glucose C.I. 13.16 to 3.84, p = 0.000; total cholesterol C.I. 28.08 to 17.12, p = 0.000; triglycerides C.I. 18.06 to 8.34, p = 0.000; ALT C.I. 2.69 to 9.31, p = 0.000; AST C.I. 1.38 to 8.82, p = 0.008; ALP C.I. 118.31 to 36.49, p = 0.000. Cachectic patients vs. healthy control subjects: BUN C.I. 3.7 to 9.8, p = 0.000; total cholesterol C.I. 24.94 to 13.66, p = 0.000; triglycerides C.I. 23.14 to 13.46, p = 0.000; ALT C.I. 8.7 to 14.6, p = 0.000; AST C.I. 9.66 to 16.94, p = 0.000; ALP C.I. 39.9 to 58.8, p = 0.000. Neoplastic patients in good nutritional status vs. healthy control subjects: BUN C.I. 10.8 to 3.7, p = 0.000; glucose C.I. 11.6 to 2.3, p = 0.004; triglycerides C.I. 0.01 to 10.1, p = 0.05; ALT C.I. 8.7 to 2.7, p = 0.000; AST C.I. 17.8 to 4.6, p = 0.000; ALP C.I. 30.8 to 13.1, p = 0.000.
2.1. Statistical analysis The results are presented as mean T standard deviation. The following two-tailed tests at the p = 0.05 level of significance were used in the study: the Mann-Whitney Utest in the case of two independent samples and the Spearman’s rank correlation coefficient test to test for univariate relationships between variables. In order to evaluate the independent effects of covariates on carnitine concentration, a stepwise multiple linear regression analysis was performed. Data were analyzed using the statistical package SPSS for Windows 7.5 (SPSS Inc., Chicago, IL, USA).
3. Results
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( p = 0.000). The difference in free carnitine was 5.80 Amol/L ( p =0.006); in soluble acid carnitine 7.20 Amol/ L ( p = 0.000); and in total carnitine 7.50 Amol/L ( p = 0.000) (Tables 2 and 3). When comparing the cachectic patients with the healthy control subjects, the difference in BUN was 7.1 mg/dl ( p = 0.000); in total cholesterol 19.30 mg/dl ( p = 0.000); in triglycerides 18.30 ( p = 0.000); in ALT 11.7 IU/L ( p = 0.000); in AST 13.30 IU/L ( p = 0.000); and in ALP 49.4 (IU/L) ( p = 0.000). The difference in free carnitine was 8.20 Amol/L ( p = 0.000); in short-chain acylcarnitine 2.60 Amol/L ( p = 0.029); in soluble acid carnitine 10.80 Amol/L ( p = 0.000); in long-chain acylcarnitine 0.40 Amol/L ( p = 0.036); and in total carnitine 11.20 Amol/L ( p = 0.000) (Tables 2 and 3). When comparing neoplastic patients in good nutritional status with healthy controls, the difference in BUN was 7.30 mg/dl ( p = 0.000); in glucose 7.00 mg/dl ( p = 0.004); in triglycerides 5.1 ( p = 0.05); in ALT 5.7 IU/L ( p = 0.000); in AST 8.2 IU/L ( p = 0.000); and in ALP 22.0 IU/L ( p = 0.000) (Table 2). In the comparison of serum plasma carnitine in controls versus neoplastic patients, there were no significant differences. The correlation studies showed a strong correlation of total carnitine (r = 0.74), short-chain acyl-carnitine (r = 0.72), long-chain acyl-carnitine (r = 0.73), free carnitine (r = 0.72), and weight loss. No correlation was found between total carnitine or the fractions of carnitine and other biohumoral and demographic characteristics.
4. Discussion In patients with cancer and cachexia, serum carnitine levels are decreased in comparison to levels found in cancer patients in good nutritional status and in healthy control subjects. The cachexia is characterized by various abnormalities in carbohydrate, protein, and fat metabolism [8]. In cancer cachexia, the carbohydrate metabolism presents
3.1. Comparison of baseline characteristics
Table 3 Comparison of serum values in the three subject groups
The three groups of subjects were comparable in age, height, systolic and diastolic pressure, and heart rate. The difference in weight between cachectic patients and neoplastic patients in good nutritional status was 11.2 kg ( p = 0.000) (Table 1).
Parameter
Cachectic patients Neoplastic patients Controls
Free carnitine Amol/L Short-chain acylcarnitine Amol/L Long-chain acylcarnitine Amol/L Total carnitine Amol/L
33.7 T 9.4 7.8 T 4.8
39.5 T 7.8 9.2 T 6.4
41.9 T 8.4 10.4 T 5.2
2.4 T 0.9
2.7 T 0.8
2.8 T 0.6
43.9 T 6.8
51.4 T 7.2
55.1 T 9.7
3.2. Comparison of laboratory parameters When comparing the patients with malignancy and cachexia with the cancer patients in good nutritional status, the difference in glucose was 8.5 mg/dl ( p = 0.000); in total cholesterol 22.7 ( p = 0.000); in triglycerides 13.20 mg/dl ( p = 0.000); in ALT 6.00 IU/L ( p = 0.000); in AST 5.10 IU/L ( p = 0.008); and in ALP 27.40 IU/L
Patients with malignancy and cachexia vs. cancer patients in good nutritional status: free carnitine C.I. 9.92 to 1.68, p = 0.006; soluble acid carnitine C.I. 10.43 to 3.97, p = 0.000; total carnitine C.I. 10.75 to 4.25, p = 0.000. Cachectic patients vs. healthy control subjects: free carnitine C.I. 12.42 to 3.98, p = 0.000; short-chain acylcarnitine C.I. 4.92 to 0.28, p = 0.029; soluble acid carnitine C.I. 14.41 to 7.19, p = 0.000; longchain acylcarnitine C.I. 0.77 to 0.03, p = 0.036; total carnitine C.I. 14.97 to 7.43, p = 0.000.
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glucose intolerance, insulin resistance, abnormal insulin secretion, increased glucose production and turnover, and increased Cori cycle activity. The protein metabolism presents increased whole body protein turnover, increased protein fractional synthesis rates in the liver, decreased functional synthesis rates in muscle, and increased hepatic protein synthesis [8]. In addition to recalcitrant muscle protein breakdown and decreased plasma levels of branched-chain amino-acids, the fat metabolism presentsexcess body fat depletion relative to body protein loss, increased lipolysis, increased free fatty acids, increased glycerol turnover, decreased lipogenesis, hyperlipidemia, and failure of glucose to suppress oxidation of free fatty acids. Most studies investigating the role of carnitine in immunity have been done on patients with HIV infection and they have concluded that carnitine improves immune cell functions and downregulates levels of TNF-alpha [9]. Cachexia has a multifactorial pathogenesis and involves several neuronal systems that modulate production and transport of cell energy, such as hormones (leptine), neuropeptides, cytokines (IL-1, IL-6, TNF), and neurotransmitters (serotonin and dopamine). Consequently, an association between acute phase response and increased hepatic protein synthesis, as well as the high-energy expenditure and wasting, may be observed in cancer patients [10 – 12]. Cachexia is characterized by fast weight loss with wasting of fat tissue and a severe decrease in skeletal-muscular mass. Carnitine is involved in the metabolism of branchedchain amino acids. The formation of branched-chain acylcarnitine from branched-chain amino acids has been detected in peroxisomes. Branched-chain amino acids have been detected in mitochondria and peroxisomes, and branched-chain acyl-carnitine oxidation has been demonstrated in both organ cells [13]. Branched-chain amino acids may modulate the peripheral muscle proteolysis that occurs in cancer cachexia by providing protein-sparing substrate for both muscle metabolism and gluconeogenesis [14]. In these two processes, carnitine plays a key role because of its involvement in the metabolism of fat acids and skeletal muscle. The direct relationship between total serum carnitine and short-chain and long-chain acylcarnitine levels in cachectic patients suggests that the observed decrease in the acylcarnitine concentration is secondary to the decrease in the total amount of this substance. This decrease may be due to a decreased availability of carnitine in the diet or to altered endogenous biosynthesis. Although the relationship is still unclear, several studies suggest that specific alimentary factors may influence serum carnitine levels [15 – 18]. Both gender-and age-related factors, as well as physical activity, may also influence serum carnitine concentrations. The dietary alterations in neoplastic patients may have been a limitation of our study, even though we tried to reduce the effects of diet on serum carnitine concentration by administering a similar diet to all subjects enrolled in the study.
Decreased dietary absorption of carnitine could be due to elevated cytokine production associated with cachexia. In fact, interleukin-6 induces anorexia and an increase in muscular catabolism, resulting in weight loss and decreased muscle strength. Malnutrition triggers a vicious circle in which neoplastic patients produce additional amounts of cytokines. With regard to low serum carnitine levels, one must remember that the first step in the biosynthesis of this substance is methylation of protein-bound lysine to trimethyl-lysine, which is subsequently cleaved from the proteins by proteolysis. Most of the protein-bound trimethyl-lysine is found in skeletal muscle [19], and the rate of carnitine biosynthesis depends on the availability of trimethyl-lysine. Skeletal muscle turnover is reported to be the rate-limiting step of carnitine biosynthesis [20,21]. Carnitine deficiency is associated with impaired fat oxidation and a number of functional abnormalities. Carnitine can also bind acetyl residues in the mitochondrial matrix, where specific carnitine acetyltransferase activity is present, and export them to the cytosolic compartment. As such, carnitine is important in both lipid oxidation and lipid synthesis; from an energy standpoint, it is involved in both the liberation and storage of metabolic energy. A decrease in serum carnitine explains not only the sarcopenia but also the physical and mental fatigue detectable in patients [22]. A decrease in acylcarnitine, a fundamental substance in brain metabolism, induces behavioral and cognitive changes (anxiety, depression, and malignancy or treatment-related anorexia). One of many theories regarding the cachexia syndrome includes heightened cytokine activity (tumor necrosis factor, interleukin 1, and interferon). An increase in resting energy expenditure may contribute to weight loss in cancer patients. This change in energy expenditure may also explain the increased oxidation of fat. The effect that these opposite actions of carnitine have on glucose metabolism has not been determined. One could predict that an enhancement of fat oxidation, whether induced by increased lipolysis or by larger carnitine availability or both, would depress carbohydrate oxidation by substrate competition, according to Randle’s cycle [23]. In vitro studies confirm the ability of carnitine to reduce Fas-mediated apoptosis [24]. Signal transduction via the surface glycoprotein FAS, also known as CD95, is considered the most important pathway for the regulation of programmed cell death (i.e., apoptosis). Carnitine inhibits apoptosis by interacting with the FAS ligand and the FAS receptor systems [25]. Signal transduction via the FAS receptor activates acid sphingomyelinase; as a result, there is a breakdown of sphingomyelin and a release of ceramide [26]. An anti-apoptotic effect has been demonstrated by treating cultured neurons and myocardial cells with carnitine. Involvement of carnitine in balancing cell volume at the cytoskeletal level and phospholipid amount at the cell surface has also been demonstrated.
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One may question the value of serum carnitine determination with respect to consequences and interpretation. This is due to the fact that serum carnitine represents approximately 3% of total body carnitine. In fact, skeletal muscles are the main reservoir in the body, with a carnitine concentration that is at least 50 –200 times higher than in blood plasma, where average concentrations range from 40 AM/L to 60 AM/L [27]. The patients in our study had many different types of malignancies. Unfortunately, the numbers of patients with each type of malignancy were too small to allow conclusions to be drawn with respect to the potential differences in serum carnitine levels between the different malignancies. Cachexia is a serious and common problem in cancer management. A better understanding of its causes may lead to increased treatment possibilities.
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