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J Physiol 593.17 (2015) pp 3959–3971
Can creatine supplementation form carcinogenic heterocyclic amines in humans? Renato Tavares dos Santos Pereira1 , Felipe Augusto D¨orr2 , Ernani Pinto2 , Marina Yazigi Solis1 , Guilherme Giannini Artioli1 , Alan Lins Fernandes1 , Igor Hisashi Murai1 , Wagner Silva Dantas1 , Antˆonio Carlos Seguro3 , Mirela Aparecida Rodrigues Santinho3 , Hamilton Roschel1,3 , Alain Carpentier4 , Jacques Remi Poortmans4 and Bruno Gualano1,3 1
School of Physical Education and Sport, University of Sao Paulo, Sao Paulo, Brazil Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil 3 School of Medicine, University of Sao Paulo, Sao Paulo, Brazil 4 Faculty of Motor Sciences, Universit´e of libre de Bruxelles, Belgium
The Journal of Physiology
2
Key points
r There is a long-standing concern that creatine supplementation could be associated with cancer, possibly by facilitating the formation of carcinogenic heterocyclic amines (HCAs).
r This study provides compelling evidence that both low and high doses of creatine r r
supplementation, given either acutely or chronically, does not cause a significant increase in HCA formation. HCAs detection was unrelated to creatine supplementation. Diet was likely to be the main factor responsible for HCAs formation after either placebo (n = 6) or creatine supplementation (n = 3). These results directly challenge the recently suggested biological plausibility for the association between creatine use and risk of testicular germ cell cancer.
Abstract Creatine supplementation has been associated with increased cancer risk. In fact, there is evidence indicating that creatine and/or creatinine are important precursors of carcinogenic heterocyclic amines (HCAs). The present study aimed to investigate the acute and chronic effects of low- and high-dose creatine supplementation on the production of HCAs in healthy humans (i.e. 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-(1,6-dimethylfuro[3,2-e] imidazo[4,5-b])pyridine (IFP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8DiMeIQx)). This was a non-counterbalanced single-blind crossover study divided into two phases, in which low- and high-dose creatine protocols were tested. After acute (1 day) and chronic supplementation (30 days), the HCAs PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx were assessed through a newly developed HPLC–MS/MS method. Dietary HCA intake and blood and urinary creatinine were also evaluated. Out of 576 assessments performed (from 149 urine samples), only nine (3 from creatine and 6 from placebo) showed quantifiable levels of HCAs (8-MeIQx: n = 3; 4,8-DiMeIQx: n = 2; PhIP: n = 4). Individual analyses revealed that diet rather than creatine supplementation was the main responsible factor for HCA formation in these cases. This study provides compelling evidence that both low and high doses of creatine supplementation, given either acutely or chronically, did not cause increases in the carcinogenic HCAs PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx in healthy subjects. These findings challenge the long-existing notion
J.-R. Poortmans and B. Gualano contributed equally to this paper. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
DOI: 10.1113/JP270861
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that creatine supplementation could potentially increase the risk of cancer by stimulating the formation of these mutagens. (Received 5 May 2015; accepted after revision 24 June 2015; first published online 6 July 2015) Corresponding author B. Gualano: Escola de Educac¸a˜o F´ısica e Esporte, Departamento de Biodinˆamica do Movimento Humano, Av. Mello de Moraes, 65, S˜ao Paulo, SP, Brazil, 05508-030. Email: [email protected] Abbreviations CR, creatine; 4,8-DiMeIQx, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline; 4,8-DiMeIQx-D3, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline-d3; HCA, carcinogenic heterocyclic amine; IFP, 2-amino-(1,6dimethylfuro[3,2-e]imidazo[4,5-b])pyridine; LOD, limit of detection; 8-MeIQx, 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline; 8-MeIQx-D3, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline-d; ND, inferior to levels of detection; NQ, inferior to levels of quantification; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine3; PhIP-D3, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-d3; PL, placebo.
Introduction Ingestion of high amounts of heterocyclic amines (HCAs), which may be found in food processing and culinary preparations such as grilling and smoking, has been often associated with increased risk for lung, stomach, bladder, colon and breast cancer (Breslow et al. 2000). Indeed, their carcinogenicity has been experimentally demonstrated in animal studies (Ohgaki et al. 1991; Sugimura et al. 2004). HCAs are among the most potent mutagens assessed by the Ames Salmonella test (Ames et al. 1975), providing biological plausibility for association studies. HCAs, including 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (8-MeIQx), 2-amino-(1,6-dimethylfuro[3,2-e]imidazo[4,5-b])pyridine (IFP) and 2-amino-3, 4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), are formed by the condensation of creatine/creatinine with D-glucose and amino acids (Fig. 1). These compounds share a common imidazole-ring structure with an exocyclic amino group and, therefore, are known chemically as amino-imidazoazaarenes. Although the major exposure to PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx is through the consumption of cooked meats, these mutagens have also been detected in other products such as processed food flavourings, beverages (e.g. beer and wine) and cigarette smoke (Wyss & Kaddurah-Daouk, 2000). In addition, it has been speculated that dietary creatine may have a role in the production of HCAs, as creatine itself and its by-product creatinine have been closely implicated in HCA synthesis. There is compelling evidence indicating that creatine and/or creatinine are important precursors of HCAs, as elegantly pointed out by Wyss & Kaddurah-Daouk (2000). Firstly, in different fried bovine tissues and meat extracts, beef flavours, bouillons, and gravies, mutagenicity was correlated with the creatine and/or creatinine content of the samples (Vikse & Joner, 1993; Robbana-Barnat et al. 1996). Secondly, the addition of creatine or phosphorylcreatine to meat samples or beef extracts before the cooking process increased mutagenicity up to 40-fold
and HCA contents up to 9-fold (Overvik et al. 1989; Skog, 1993), whereas the treatment of beef meat with creatinase before frying reduced the creatine content and the mutagenicity by 65% and 73%, respectively (Vikse & Joner, 1993). Thirdly, the addition of creatine to meat before cooking had no influence on the number or relative proportions of HCA mutagens formed, but increased the level of all mutagens to approximately the same extent, suggesting that creatine is involved in the formation of all HCAs (Overvik et al. 1989). Finally, as previously mentioned, most HCAs could be generated artificially in simple model systems containing creatine or creatinine, amino acids and glucose (Wyss & Kaddurah-Daouk, 2000). Even though the heating process appears to enhance HCA formation, there is evidence that these mutagens could also be formed at physiological temperatures. For instance, patients with chronic renal failure, who are subject to an increased cancer risk, experience high creatinine concentrations, thereby creating ‘favourable’ conditions for the formation of HCAs (Yanagisawa et al. 1986). In this study, 8-MeIQx was detected in the dialysis fluid of all uraemic patients examined. The notion that 8-MeIQx does not originate from meat consumption, but from de novo synthesis, is corroborated by the formation of HCAs in a model system at temperatures as low as 37°C (Manabe et al. 1992). Taken together, these observations confer some degree of biological plausibility for the speculation that dietary creatine could increase HCA formation in vivo. However, this needs to be confirmed in humans. Creatine has become one of the most popular dietary supplements used by athletes and exercise practitioners in order to improve physical capacity and lean mass (Kreider et al. 1998; Terjung et al. 2000; Gualano et al. 2012). Furthermore, creatine supplementation has been also applied as an adjuvant therapeutic tool in a variety of conditions characterized by muscle dysfunction, atrophy, frailty, mental disturbances and bioenergetics disorders (for a comprehensive review, see Gualano et al. (2012)). Despite the beneficial effects of creatine supplementation in sports and C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Creatine and heterocyclic amines
clinical settings, there has been a long-standing concern, raised by a number of review articles and guidelines (Terjung et al. 2000; Wyss & Kaddurah-Daouk, 2000; Agence Franc¸aise de S´ecurit´e Sanitaire et Alimentaire, 2001; Brudnak, 2004), that this dietary supplement could cause adverse outcomes, including HCA-induced carcinogenic/mutagenic effects. Recently, a case-control study showed that ‘muscle-building’ supplements, including those containing creatine, were associated with increased risks of testicular germ cell cancer (odds ratio: 1.65, 95% confidence interval: 1.11–2.46) (Li et al. 2015). However, this study did not control for important confounding factors, such as use of anabolic steroid and supplement contaminants. Thus, the potential carcinogenic effect of creatine supplementation remains highly unexplored, despite speculation. To shed light on this topic, the present study aimed to investigate the acute and chronic effects of low- and high-dose creatine supplementation on the formation of carcinogenic HCAs (i.e. PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx) in healthy humans.
Methods Experimental design and participants
This was a non-counterbalanced single-blind crossover study divided into two phases, in which low- and high-dose creatine protocols were tested (phases I and II, respectively). Healthy males and females (age: 29 ± 4 years) were selected according to the following exclusion criteria: tobacco use, use of creatine supplements for at least 4 months before the commencement of the study, current use of any medication, vegetarian diet, and pre-existing chronic diseases. All procedures were approved by the local ethical committee and were in accordance with the Declaration of Helsinki revised in 2008. The participants were fully informed about risks and benefits associated with participation before signing the informed consent. Participants received placebo and, following a 14-day washout period, creatine monohydrate; each experimental condition (i.e. placebo and creatine) lasted 30 days, which
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consisted of a 7-day ‘loading’ period followed by a 23-day ‘maintenance’ period (details on the supplementation regimens can be seen in ‘Supplementation protocols’). Fasting blood and 12 h urine samples were collected on the first day of the ‘loading’ period (urine collection commenced immediately after the ingestion of the last daily dose) and on the 30th day of supplementation. Samples were immediately frozen at −80ºC until further analysis of PhIP, 8-MeIQx, IFP, 4,8-DiMeIQx (through a HPLC–MS/MS method), and creatinine (through a colorimetric method). Phases I (n = 11 males and 10 females) and II (n = 13 males and 3 females) were interspaced by a 4-month period in order to avoid any carry-over effect of creatine. To mitigate the impact of dropouts that occurred between phases I and II (n = 11), seven additional subjects were selected to participate exclusively in phase II. Participants were strongly instructed to refrain from fried or grilled meat consumption 4 days before urine collections to avoid any interference of the diet on HCA formation. Dietary intake of energy, macronutrients, creatine, and PhIP, 8-MeIQx and 4,8-DiMeIQx was assessed by food records taken 4 days before the commencement of ‘loading’ and during the last 4 days of ‘maintenance’. Figure 2 illustrates the experimental design.
Supplementation protocols
Subject were given 7 g day−1 (phase I) or 20 g day−1 (phase II) of creatine monohydrate (Creapure , Alzchem AG, Germany) or placebo (same doses of dextrose) for 7 days divided into four equal doses, followed by single daily doses of 2 g day−1 (phase I) or 5 g day−1 (phase II) for the next 23 days. During ‘loading’, supplements were given in four packages and the participants were instructed to ingest the supplement packages at breakfast, lunch, dinner and before bedtime. During ‘maintenance’, subjects consumed the supplement as a single dose during their lunch. Creatine and placebo supplements were formulated in undistinguishable tablets with identical appearance, R
NH2 CH3
N N
CH3
N
glucose
N
CH3
NH2 N
N
N
PhlP
Creatine or Creatinine
8-MelQx NH2 N
N CH3
N
N
amino acids Figure 1. Illustrative model of HCA formation from creatine/creatinine, D-glucose and amino acids C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
N
CH3
NH2
CH3 O
N CH3
N
4,8-DiMelQx
CH3
IFP
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taste, and smell, and the packages were coded so that the participants were not aware of the contents until completion of the analyses. Supplements were provided by a staff member from our research team who did not have any participation in the data acquisition, analyses or interpretation. Compliance to the supplementation was monitored weekly by asking the patients personally.
J Physiol 593.17
Table 1. Optimized parameters for HCA determination in MRM mode Precursor (m/z)
Ion products (m/z)
Fragmentor (V)
CE (V)
8-MeIQx
214
8-MeIQx-D3
217
4,8-DiMeIQx
228
4,8-DiMeIQx-D3
231
PhIP
225
PhiP-d3
228
IFP
203
131 199 131 199 160 187 212 145 140 210 140 210 188 187
140 140 140 140 140 140 140 140 140 140 140 140 98 98
46 26 44 28 30 25 40 44 58 30 50 32 22 34
Compound
Food intake assessment and control for HCA dietary intake
Food intake was assessed by 4-day food records, using the software Avanutri (Rio de Janeiro, Brazil). Specific diet composition tables were used to calculate dietary intake of PhIP, 8-MeIQx, and 4,8-DiMeIQx (Skog et al. 1998), as well as creatine (Harris et al. 1997). In addition, the ingestion of total energy, carbohydrate, lipids and protein was estimated. To avoid any possible confounding effect of diet upon HCA formation, participants were highly encouraged to abstain from food that could potentially increase HCAs. To that end, they were provided with a list of food preparations (e.g. grilled meat, fried chicken, wine) that they should refrain from 4 days before until the end of urine collection. R
Ions shown in bold were used for quantification; 8-MeIQx-D3 was used as internal standard for IFP determination.
homogenised and centrifuged at 5000 g for 5 min. Metabolic conjugates were hydrolysed by the addition of 12 mol l−1 hydrochloric acid (90 µl) to 1000 µl of urine followed by incubation at 70°C for 3 h. After cooling, 200 µl 10 mol l−1 NaOH and 100 µl of a solution containing internal standards of 8-MeIQx-D3, 4,8-DiMeIQx-D3 and PhIP-D3 at 4 ng ml−1 were added. Samples were thoroughly mixed and submitted to liquid–liquid extraction twice using 5 ml ethyl acetate. The organic layers were applied to ion exchange cartridges (Bond Elut Certify, 130 mg; Agilent, Santa Clara, CA, USA), which were preconditioned with methanol and 2% acetic acid/methanol solution. After washing the cartridge with 0.1 mol l−1 acetic acid and methanol, HCAs were eluted with 2 ml of methanol/ammonium hydroxide 95:5 (v/v). Samples were dried under nitrogen at 30ºC, reconstituted in 100 µl methanol/water (50:50, v/v) and analysed by HPLC–MS/MS. HCA analyses were performed using a 1260 Infinity HPLC system coupled with a 6460 Triple-Quad mass spectrometer (Agilent). Chromatographic separation was
Creatinine measurements
Urinary and serum creatinine were determined using the enzymatic method as per the instructions provided by a commercially available assay kit (Labtest, Sao Paulo, Brazil). HCA determination
The following HCAs were assessed in urine samples: PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx. All compounds and internal standards (i.e. 8-MeIQx-D3, 4,8-DiMeIQx-D3 and PhIP-D3) were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). HCA extraction from urine samples was carried out according to a previously validated method described elsewhere (Fu et al. 2014), with slight modifications. Briefly, urine samples were thawed at room temperature, -4
1
2
placebo (20 g or 7 g/d)
7
26
placebo (5 g or 2 g/d)
40
30
washout
44
45
51
creatine (20 g or 7 g/d)
70
74
creatine (5 g or 2 g/d)
time (days)
4-day food record
12-hour urine fasting blood
4-day food 12-hour urine record fasting blood
4-day food 12-hour urine record fasting blood
4-day food 12-hour urine record fasting blood
Figure 2. The experimental design C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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obtained with a Kinetex EVO C18 column (100 × 2.1 mm; 5 µm, Phenomenex, Torrance, CA, USA) at 0.3 ml min−1 , using (a) 2 mmol l−1 ammonium bicarbonate containing ammonium hydroxide (0.02%) at pH 8.0 and (b) acetonitrile as mobile phases. Gradient elution was performed under the following conditions: 0–15 min, 5 to 25% acetonitrile; 15.5–16.5 min, 95% acetonitrile; 17–20 min, 5% acetonitrile. The mass spectrometer electrospray source was operated in the positive mode under the following conditions: capillary voltage, 3500 V; nebuliser (N2 ), 30 psi; drying gas (N2 ), 6 l min−1 at 300°C; sheath gas (N2 ), 10 l min−1 at 300°C. Multiple reaction monitoring (MRM) optimized parameters are displayed in Table 1. Analyte identification was based on the retention time (±2% when compared to its respective calibrator) and peak area ratios of qualifier/quantifier ion transitions (±20%). The analytical protocol was further evaluated for linear range, limits of detection and quantification, precision and accuracy following standard recommendations. The assessors of urinary HCAs were blinded to the treatment. Statistical analysis
Dependent variables were tested by mixed model with repeated measures using the software SAS 9.3. Group (creatine and placebo) and time (pre and post) were defined as fixed factors and subjects were defined as a random factor. Significance level was previously set at P < 0.05. Results are presented as individual data, mean and standard deviation.
subsequently employed to analyse urinary HCAs after creatine supplementation. Tables 3 (phase I) and 4 (phase II) summarize individual data for urinary HCAs. Out of 576 assessments performed (from 149 urine samples), only 9 (3 from creatine and
x102 3 2.5 2 1.5 1 0.5 x103 1 0.8 0.6 0.4 0.2 0 x102 7 6 5 4 3 2 1 0 x102 2
Whilst the extraction protocol validated by Fu et al. (2014) was successfully employed in this study with recovery rates superior to 90% for all HCAs, chromatographic separation was not promptly adequate, as interfering chromatographic peaks compromised the accurate detection of HCAs. In order to improve resolution, separation at alkaline pH was applied (Cooper et al. 2014). The newly developed chromatographic method was evaluated regarding linear range, limits of detection and quantification, precision and accuracy, showing satisfactory results (Table 2). Typical chromatograms of urine spikes are shown in Fig. 3. Linearity data analysis showed that the method was heteroscedastic, and hence weighted least squares linear regressions were used (Almeida et al. 2002). The weighting factor ‘1/x’ was selected according to the sums of the relative error percentages (%RE). Matrix effects were effectively compensated by the use of matched blank matrix (i.e. pooled urine obtained from placebo condition) and isotopic labelled internal standards. The same method was C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
8-MelQx
4
5
6
7
8
9
10
11
12
13
14
15
8 9 IFP
10
11
12
13
14
15
8-MelQx-D3
4
5
6
7
4
5
6 7 8 4,8-DiMelQx
9
10
11
12
13
14
15
4
5 6 7 8 4,8-DiMelQx-D3
9
10
11
12
13
14
15
4
5
6
7
8
9
10
11
12
13 14 PhIP
15
4
5
6
7
8
9
10
11
12
4
5
6
7
8
9
10
11
12
1.5 1 0.5 x102
Results
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4 3 2 1 0 x102 2.5 2 1.5 1 0.5 x102 2.5 2 1.5 1 0.5
13 14 15 PhIP-D3
Counts vs. Acquisition Time (min) Figure 3. Typical chromatograms of urine spikes
13
14
15
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Table 2. Calibration curves, linear range, limit of detection (LOD), precision and accuracy results for HCAs in urine
Compound 8-MeIQx
4,8-DiMeIQx
IFP
PhIP
Calibration curve Y = 0.869492x + 0.029523 R2 = 0.998 Weight 1/x Y = 0.379978x + 0.00012018 R2 = 0.996 Weight 1/x Y = 2.166107 + 0.065861 R2 = 0.999 Weight 1/x Y = 0.824436 + 0.015107 R2 = 0.997 Weight 1/x
Precision (CV%)
Linear range (pg ml−1 )
LOD (pg ml−1 )
Quality control
Intra-day
Inter-day
Accuracy (%)
31.25–1000
10
31.25–1000
20
31.25–1000
10
31.25–1000
10
Low Medium High Low Medium High Low Medium High Low Medium High
6.2 3.1 3.3 3.9 2.7 4.4 6.6 1.8 8.9 5.6 2.7 2.4
6.4 3.3 3.8 3.8 3.8 4.5 6.4 3.4 9.5 6.5 2.7 2.5
101.8 98.8 99.3 95.5 97.2 100.8 97.6 100.2 95.6 97.6 99.7 102.2
6 from placebo) showed quantifiable levels of HCAs (8-MeIQx: n = 3; 4,8-DiMeIQx: n = 2; PhIP: n = 4). Six participants had quantifiable levels of one or more urinary HCAs, three of them after creatine supplementation (subject V in phase I and subjects VI and XIV in phase II) and three after placebo (subject V in phase I and subjects IV and VI in phase II). The remaining assessments showed non-detectable or non-quantifiable HCA levels. Participant V showed increased 4,8-DiMeIQx levels after low-dose chronic creatine supplementation (48.7 pg ml−1 ). However, similar levels were found after placebo (69.6 pg ml−1 ). In fact, 4,8-DiMeIQx dietary intake was comparable in both creatine and placebo conditions (2.48 ± 2.39 vs. 2.74 ± 2.00 ng g−1 , respectively), which explains the similar urinary excretion between the two interventions. Participant IV showed urinary PhIP levels of 32.9 pg ml−1 after acute high-dose creatine supplementation, but not after placebo. However, her PhIP dietary intake was about 10 times lower during placebo (0.24 ± 0.39 ng g−1 ) than in creatine condition (25.36 ± 43.51 ng g−1 ). Participant XVI showed increased levels of 8-MelQx (108.8 pg ml−1 ) after acute high-dose creatine supplementation; however, dietary analysis revealed he consumed twice as much of this HCA during creatine versus placebo supplementation (4.89 ± 1.96 vs. 2.38 ± 3.18, respectively). Individual analysis of the subjects who showed quantifiable levels of urinary HCAs following creatine supplementation led to the proposition that diet, rather than creatine supplementation, was the main contributing factor to HCA formation in these three cases. Dietary intake of HCAs is expressed in Table 3 and Table 4. Ingestion of 8-MeIQx (range: 0–19.55 ng g−1 ), 4,8-DiMeIQx (range: 0.09–5.58 ng g−1 ), and PhIP (range: 0–136.95 ng g−1 ) were comparable between
creatine and placebo in phases I and II, after acute and chronic supplementation (P > 0.05). In addition, total energy, carbohydrate, lipids and protein intake were not significantly different between creatine and placebo in any condition (P > 0.05; data not shown). Serum (Fig. 4A and B) and urinary (Fig. 4C and D) creatinine data are shown in Fig. 4. No changes in serum and urinary creatinine values were observed after either acute or chronic supplementation of creatine or placebo in phases I and II (P > 0.05). Additionally, none of the participants showed changes in creatinine values beyond the normal range following creatine or placebo supplementation in any condition (i.e. low/high dose, acute/chronic, protocols). Discussion This study is the first to show that creatine supplementation does not significantly contribute to HCA formation (i.e. 8-MeIQx, 4,8-DiMeIQx, PhIP, and IFP) in healthy humans. These results suggest that creatine does not provoke any carcinogenic effect secondary to HCA formation in humans. HCAs can be formed through Maillard reactions involving D-glucose, amino acids and creatinine (Wyss & Kaddurah-Daouk, 2000), especially under high temperatures. However, there is also evidence showing that HCA formation is possible under physiological conditions (i.e. at pH 7.4, 37ºC). In fact, it has been demonstrated that imidazo-quinoxalines (e.g. 8-MeIQx) are preferably formed through de novo synthesis rather than meat intake, which confirms the possibility of HCA formation in a physiological ‘environment’. The role of circulating creatinine in the formation of HCAs appears to be crucial. The presence of 8-MeIQx was reported in the dialytic fluid of uraemic patients, suggesting that C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Table 3. Dietary intake and urinary HCAs following low-dose creatine or placebo supplementation (phase I) Subject (sex) I (M)
Acute Chronic
II (F)
Acute Chronic
III (F)
Acute Chronic
IV (M)
Acute Chronic
V (F)
Acute Chronic
VI (M)
Acute Chronic
VII (F)
Acute Chronic
VIII (M)
Acute Chronic
IX (M)
Acute Chronic
X (M)
Acute Chronic
XI (M)
Acute Chronic
XII (F)
Acute Chronic
XIII (M)
Acute Chronic
8-MeIQx
4.8-DiMeIQx
PhIP
(ng g−1 ) (pg ml−1 )
Placebo
Creatine
Placebo
Creatine
Placebo
Creatine
Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion
2.39 ± 1.17 ND 2.37 ± 3.67 ND 3.04 ± 3.00 ND 1.08 ± 1.50 ND 7.66 ± 1.22 ND 7.77 ± 4.45 ND 7.27 ± 0.28 ND 7.46 ± 0.60 ND 0.87 ± 1.27 ND 2.51 ± 2.34 NQ 10.03 ± 7.71 ND 10.38 ± 4.44 ND 1.00 ± 1.73 ND 5.07 ± 3.74 ND 1.55 ± 2.47 ND 1.62 ± 2.58 ND 3.88 ± 3.17 ND 7.09 ± 0.79 ND 19.55 ± 9.39 ND 10.14 ± 4.31 ND 5.28 ± 4.25 ND 2.63 ± 1.03 ND 4.33 ± 1.74 ND 4.08 ± 3.46 ND 5.03 ± 5.06 ND 5.13 ± 8.80 ND
2.64 ± 2.57 ND 1.20 ± 2.08 ND 1.08 ± 1.50 ND 0.31 ± 0.54 ND 8.84 ± 3.26 ND 7.05 ± 4.30 ND 7.28 ± 0.27 ND 6.17 ± 2.07 ND 2.46 ± 2.53 ND 3.24 ± 1.35 ND 10.69 ± 7.66 ND 3.04 ± 4.64 ND 5.40 ± 3.17 ND 3.90 ± 3.46 ND 5.73 ± 7.48 ND 5.94 ± 10.29 ND 7.16 ± 0.73 ND 5.24 ± 4.46 ND 17.68 ± 9.02 ND 11.74 ± 6.40 ND 2.74 ± 1.14 ND 6.46 ± 5.47 ND 4.10 ± 3.42 ND 3.43 ± 3.31 ND 3.19 ± 2.96 ND 7.37 ± 9.32 ND
1.35 ± 1.30 ND 1.22 ± 2.00 ND 0.17 ± 0.16 ND 0.32 ± 0.56 ND 0.92 ± 0.82 ND 1.78 ± 0.71 ND 2.65 ± 0.33 ND 2.59 ± 0.22 ND 0.29 ± 0.45 ND 2.48 ± 2.39 69.6 0.79 ± 0.29 ND 1.40 ± 0.68 ND 0.09 ± 0.15 ND 0.51 ± 0.26 ND 0.44 ± 0.76 ND 0.33 ± 0.57 ND 0.52 ± 0.22 ND 2.89 ± 4.42 ND 1.40 ± 0.98 ND 1.02 ± 0.68 ND 0.64 ± 0.73 ND 0.94 ± 0.21 ND 1.57 ± 1.78 ND 1.02 ± 0.88 ND 1.08 ± 1.08 ND 1.10 ± 1.90 ND
0.92 ± 0.89 ND 0.36 ± 0.62 ND 0.32 ± 0.56 ND 0.11 ± 0.19 ND 0.98 ± 0.77 ND 1.16 ± 0.83 ND 2.71 ± 0.29 ND 2.24 ± 0.92 ND 0.42 ± 0.41 ND 2.74 ± 2.00 48.7 0.85 ± 0.18 ND 1.03 ± 1.63 ND 0.54 ± 0.23 ND 0.48 ± 0.56 ND 2.93 ± 2.90 ND 1.88 ± 3.25 ND 2.92 ± 4.40 ND 3.07 ± 4.96 ND 1.28 ± 0.80 ND 1.10 ± 0.63 ND 0.97 ± 0.26 ND 1.91 ± 1.32 ND 1.04 ± 0.85 ND 0.48 ± 0.56 ND 1.65 ± 1.37 ND 0.76 ± 0.55 ND
11.20 ± 19.40 ND 45.41 ± 78.63 ND 27.05 ± 35.63 ND 0.07 ± 0.13 ND 10.90 ± 9.60 ND 14.42 ± 12.50 ND 2.80 ± 4.85 ND 0 ND 30.00 ± 51.96 ND 34.54 ± 33.64 ND 0.46 ± 0.45 ND 0.37 ± 0.37 ND 4.55 ± 7.88 ND 3.97 ± 6.26 ND 32.68 ± 43.82 ND 17.44 ± 30.10 ND 2.35 ± 2.84 ND 37.55 ± 64.47 ND 4.85 ± 6.86 ND 0.42 ± 0.36 ND 3.70 ± 5.81 ND 34.33 ± 48.44 ND 13.16 ± 22.55 ND 6.20 ± 5.38 ND 7.74 ± 8.01 ND 45.50 ± 59.25 ND
0 ND 34.20 ± 59.24 ND 0.07 ± 0.13 ND 7.50 ± 12.99 ND 10.95 ± 9.52 ND 11.36 ± 10.71 ND 2.82 ± 4.84 ND 2.74 ± 4.73 ND 37.70 ± 46.74 ND 34.57 ± 33.59 ND 0.48 ± 0.49 ND 12.51 ± 21.65 ND 0.36 ± 0.04 ND 2.78 ± 4.53 ND 38.27 ± 36.54 ND 14.00 ± 24.25 ND 37.64 ± 64.39 ND 41.18 ± 71.03 ND 1.94 ± 1.86 ND 0.50 ± 0.46 ND 37.00 ± 53.04 ND 9.00 ± 7.94 ND 6.27 ± 5.43 ND 24.31 ± 31.51 ND 12.14 ± 21.02 ND 0.31 ± 0.51 ND
(Continued)
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Table 3. Continued Subject (sex) XIV (F)
Acute Chronic
XV (F)
Acute Chronic
XVI (F)
Acute Chronic
XVII (F)
Acute Chronic
XVIII (M)
Acute Chronic
XIX (F)
Acute Chronic
XX (M)
Acute Chronic
XXI (M)
Acute Chronic
8-MeIQx
4.8-DiMeIQx
PhIP
(ng g−1 ) (pg ml−1 )
Placebo
Creatine
Placebo
Creatine
Placebo
Creatine
Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion
3.35 ± 3.46 ND 5.38 ± 4.39 ND 4.56 ± 4.11 ND 7.87 ± 1.46 ND 3.83 ± 2.32 ND 6.32 ± 4.72 ND 2.77 ± 2.37 ND 4.69 ± 0.91 ND 6.91 ± 6.70 ND 7.25 ± 6.35 ND 0 ND 0.85 ± 1.48 ND 8.19 ± 2.01 NQ 9.75 ± 1.58 ND 10.99 ± 7.22 NQ 10.08 ± 4.82 ND
2.31 ± 1.57 ND 3.93 ± 0.57 ND 4.85 ± 3.87 ND 5.27 ± 4.49 ND 4.52 ± 2.93 ND 5.44 ± 3.57 ND 2.41 ± 3.35 ND 1.23 ± 2.14 ND 2.02 ± 1.81 ND 2.69 ± 0.76 ND 0 ND 1.55 ± 1.35 ND 10.09 ± 2.02 ND 4.64 ± 7.86 ND 5.89 ± 6.49 NQ 9.62 ± 5.61 ND
0.36 ± 0.34 ND 0.88 ± 0.40 ND 1.09 ± 0.80 ND 1.34 ± 0.70 ND 1.22 ± 0.03 ND 0.47 ± 0.09 ND 0.37 ± 0.46 ND 1.59 ± 0.52 ND 1.54 ± 0.43 ND 2.20 ± 1.56 ND 0 ND 0.30 ± 0.51 ND 4.01 ± 1.86 ND 2.14 ± 0.46 ND 2.10 ± 1.99 NQ 3.61 ± 0.95 ND
0.78 ± 0.56 ND 0.45 ± 0.47 ND 1.24 ± 0.74 ND 0.69 ± 0.54 ND 0.92 ± 0.50 ND 1.14 ± 1.23 ND 0.63 ± 0.86 ND 0.25 ± 0.43 ND 0.70 ± 0.63 ND 2.04 ± 1.71 ND 0 ND 0.54 ± 0.47 ND 2.24 ± 0.30 ND 1.52 ± 2.63 ND 1.84 ± 1.78 NQ 3.11 ± 1.81 ND
1.72 ± 2.68 ND 5.01 ± 3.85 ND 33.39 ± 42.90 ND 3.50 ± 4.62 ND 10.00 ± 6.29 ND 20.29 ± 34.39 ND 19.70 ± 28.57 ND 15.20 ± 11.20 ND 10.87 ± 15.02 ND 12.53 ± 17.87 ND 31.20 ± 44.77 ND 50.00 ± 86.60 ND 37.34 ± 42.78 ND 19.17 ± 6.00 87.7 15.90 ± 26.76 NQ 25.53 ± 19.40 ND
0.77 ± 1.27 ND 2.52 ± 4.05 ND 6.26 ± 7.14 ND 23.10 ± 38.54 ND 8.65 ± 8.23 ND 25.25 ± 31.02 ND 8.32 ± 8.00 ND 3.15 ± 5.46 ND 2.71 ± 4.70 ND 3.05 ± 5.28 ND 100.00 ± 14.56 ND 0 ND 19.83 ± 5.57 ND 10.07 ± 8.72 ND 35.94 ± 54.79 NQ 18.54 ± 24.86 ND
ND: inferior to levels of detection; NQ: inferior to levels of quantification; all IFP values were inferior to levels of detection (data not shown); dietary intake of IFP was not measured.
kidney failure-induced elevation in circulating creatinine could predispose to HCA formation (Yanagisawa et al. 1986). Since creatine can be irreversibly and spontaneously converted into creatinine, one could speculate that creatine loading could enhance HCA formation (Wyss & Kaddurah-Daouk, 2000). However, the current results indicate that this is not the case in humans. In this study, two different creatine supplementation protocols were tested, one low (i.e. 7 g day−1 for 7 days followed by 2 g day−1 for the next 23 days) and the other high dose (i.e. 20 g day−1 for 7 days followed by 5 g day−1 for the next 23 days), with urinary HCAs being assessed both acutely (i.e. 1 day after creatine ‘loading’) and chronically (i.e. 30 days after supplementation). This experimental design allowed us to explore a potential dose–response pattern in HCA formation following
creatine intake. In order to avoid any interference of habitual dietary intake on HCAs, subjects were instructed not to consume any foods that could possibly contribute to increased HCA formation. Apparently, the majority of the subjects were able to follow this restriction successfully, as reflected by non-detectable or non-quantifiable levels of HCAs in most of the urine samples. The general absence of HCAs in urine in this study was expected and it is in agreement with previous observations of no HCA detection in subjects who temporarily refrained from meat consumption (Murray et al. 2001; Holland et al. 2005). This experimental condition effectively allowed us to distinguish any potential effect of creatine supplementation (low/high and acute/chronic regimens) from that of the regular diet on HCA formation. Interestingly, HCAs were detected at quantifiable levels in six C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Table 4. Dietary intake and urinary HCAs following high-dose creatine or placebo supplementation (phase II)
Subject (sex) I (M)
Acute Chronic
II (F)
Acute Chronic
III (M)
Acute Chronic
IV (M)
Acute Chronic
V (M)
Acute Chronic
VI (F)
Acute Chronic
VII (M)
Acute Chronic
VIII (M)
Acute Chronic
IX (M)
Acute Chronic
X (M)
Acute Chronic
XI (M)
Acute Chronic
XII (M)
Acute Chronic
XIII (F)
Acute Chronic
XIV (M)
Acute Chronic
XV (M)
Acute Chronic
XVI (M)
8-MeIQx
(ng g−1 ) (pg ml−1 )
Acute Chronic
Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion
4.8-DiMeIQx
PhIP
Placebo
Creatine
Placebo
Creatine
Placebo
Creatine
1.71 ± 1.48 ND 2.85 ± 4.30 ND 1.28 ± 1.98 ND 1.80 ± 3.12 ND 2.26 ± 3.02 ND 5.98 ± 4.54 ND 3.55 ± 2.94 72.1 3.10 ± 3.26 ND 6.07 ± 1.90 ND 9.96 ± 3.60 ND 7.28 ± 6.54 NQ 5.62 ± 7.31 46.2 6.07 ± 2.55 ND 2.60 ± 2.42 ND 10.85 ± 3.69 ND 8.93 ± 6.43 ND 18.00 ± 9.00 ND 12.56 ± 13.03 ND 7.26 ± 4.02 ND 9.69 ± 7.81 ND 1.73 ± 1.33 ND 2.16 ± 0.68 ND 2.00 ± 3.46 ND 10.17 ± 9.97 ND 3.30 ± 2.86 ND 2.49 ± 1.58 ND 2.38 ± 3.18 ND 3.55 ± 1.37 ND 10.76 ± 2.33 ND 13.21 ± 2.30 ND 3.07 ± 3.14 ND 7.47 ± 5.95 ND
0.33 ± 0.58 ND 2.49 ± 3.58 ND 5.05 ± 2.34 ND 3.57 ± 4.53 ND 1.48 ± 1.72 ND 9.59 ± 5.24 ND 3.77 ± 4.35 ND 1.08 ± 1.67 ND 3.95 ± 3.33 ND 4.04 ± 3.32 ND 6.03 ± 4.64 ND 10.73 ± 14.22 ND 7.07 ± 2.76 ND 4.49 ± 4.34 ND 3.99 ± 6.08 ND 4.83 ± 7.53 ND 16.76 ± 10.91 ND 17.01 ± 10.52 ND 4.68 ± 5.66 ND 9.26 ± 1.61 ND 2.25 ± 0.94 ND 3.25 ± 0.60 ND 6.53 ± 10.03 ND 6.43 ± 10.11 ND 4.93 ± 0.59 ND 5.24 ± 5.65 ND 4.89 ± 1.96 108.8 2.41 ± 3.63 ND 3.73 ± 0.23 ND 6.13 ± 0.78 ND 5.03 ± 6.50 ND 9.40 ± 10.48 ND
0.59 ± 0.51 ND 0.71 ± 0.77 ND 0.28 ± 0.42 ND 0.09 ± 0.16 ND 1.18 ± 1.94 ND 0.42 ± 0.27 ND 0.46 ± 0.57 ND 0.30 ± 0.27 ND 1.82 ± 0.58 ND 1.87 ± 0.47 ND 1.17 ± 1.17 ND 0.36 ± 0.42 NQ 3.92 ± 4.40 ND 1.48 ± 1.50 ND 1.25 ± 1.31 ND 0.78 ± 0.44 ND 0.90 ± 0.45 ND 0.88 ± 0.45 ND 3.50 ± 0.81 ND 2.80 ± 1.74 ND 1.63 ± 1.37 ND 0.52 ± 0.05 ND 0.10 ± 0.17 ND 0.88 ± 0.71 ND 0.45 ± 0.58 ND 0.56 ± 0.35 ND 0.13 ± 0.15 ND 0.75 ± 0.89 ND 1.99 ± 2.48 ND 5.58 ± 1.26 ND 0.83 ± 1.02 ND 1.01 ± 0.88 ND
0.33 ± 0.58 ND 0.18 ± 0.17 ND 0.51 ± 0.34 ND 0.90 ± 1.41 ND 0.12 ± 0.12 ND 0.54 ± 0.15 ND 0.33 ± 0.28 ND 0.07 ± 0.08 ND 1.18 ± 0.76 ND 1.23 ± 0.76 ND 2.07 ± 3.06 ND 1.91 ± 2.07 ND 4.40 ± 1.97 ND 2.35 ± 2.92 ND 0.40 ± 0.48 ND 0.33 ± 0.36 ND 1.36 ± 0.46 ND 1.43 ± 0.58 ND 2.02 ± 1.74 ND 2.65 ± 2.03 ND 0.78 ± 0.33 ND 1.91 ± 1.49 ND 0.42 ± 0.49 ND 0.40 ± 0.46 ND 1.06 ± 0.13 ND 0.81 ± 1.40 ND 0.57 ± 0.49 NQ 0.16 ± 0.15 ND 2.80 ± 1.39 ND 3.11 ± 2.55 ND 1.27 ± 0.99 ND 1.46 ± 2.12 ND
22.50 ± 38.97 ND 58.26 ± 50.68 ND 4.34 ± 3.86 ND 27.59 ± 47.55 ND 11.67 ± 16.70 ND 6.87 ± 8.20 ND 40.26 ± 41.55 35.9 1.45 ± 2.22 ND 21.00 ± 7.00 ND 16.89 ± 10.05 ND 0.24 ± 0.39 ND 8.48 ± 6.87 163.4 52.73 ± 63.45 ND 19.60 ± 21.14 ND 7.00 ± 11.26 ND 5.05 ± 7.76 ND 5.83 ± 8.16 ND 6.23 ± 7.82 ND 44.27 ± 13.53 ND 33.90 ± 29.78 ND 79.91 ± 58.83 ND 15.17 ± 22.89 ND 35.74 ± 42.25 ND 3.92 ± 6.48 ND 15.25 ± 19.66 ND 2.70 ± 2.31 ND 70.60 ± 117.50 ND 55.90 ± 49.70 ND 21.00 ± 35.55 ND 68.19 ± 5.46 ND 136.95 ± 77.22 ND 109.67 ± 112.47 ND
57.17 ± 49.69 ND 27.62 ± 47.81 ND 0.21 ± 0.19 ND 50.68 ± 58.74 ND 51.46 ± 85.36 ND 5.33 ± 8.13 ND 1.48 ± 2.19 ND 25.05 ± 21.69 ND 3.00 ± 4.36 ND 3.03 ± 4.32 ND 25.36 ± 43.51 32.9 19.54 ± 21.88 ND 60.37 ± 29.65 ND 30.43 ± 22.16 ND 0.29 ± 0.46 ND 0.24 ± 0.38 ND 0.75 ± 0.68 ND 0.76 ± 0.68 ND 62.17 ± 43.87 ND 30.42 ± 29.21 ND 50.00 ± 68.05 ND 16.80 ± 29.10 ND 7.75 ± 12.53 ND 7.72 ± 12.55 ND 7.73 ± 0.92 ND 14.07 ± 3.29 ND 28.51 ± 38.67 ND 27.62 ± 47.54 ND 90.77 ± 69.92 ND 37.05 ± 42.87 ND 61.99 ± 54.27 ND 116.92 ± 95.95 ND
ND: inferior to levels of detection; NQ: inferior to levels of quantification; all IFP values were inferior to levels of detection (data not shown); dietary intake of IFP was not measured.
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participants only. Since these cases were evenly distributed among treatments (each intervention arm), we can safely conclude that supplementary creatine does not contribute to HCA synthesis in humans. These results directly contrast with the recently suggested biological plausibility for the association between creatine use and risk of testicular germ cell cancer (Li et al. 2015). An apparent explanation for the inability of creatine to result in HCA formation in this study may be due to the fact that creatine supplementation did not provoke any increase in serum or urinary creatinine beyond the normal range. In fact, a number of studies have shown that creatine supplementation normally does not increase creatinine levels (Poortmans et al. 1997, 2005; Poortmans & Francaux, 1999; Kreider et al. 2003; Gualano et al. 2009, 2010; Neves et al. 2011; Lugaresi et al. 2013), although a few conflicting findings do exist (Gualano et al. 2008). For instance, we showed increased creatinine levels (although within normal range) in creatine-supplemented healthy subjects, without any effect in serum cystatin C (a surrogate of glomerular filtration rate that is influenced by creatine ingestion) (Gualano et al. 2008). It is unknown as to whether those subjects experiencing creatinine
J Physiol 593.17
increases after creatine supplementation are more prone to form HCAs. However, it is unlikely that fluctuations in creatinine levels within the physiological range induced by creatine supplementation is a major source of HCAs. In a previous study with meat eaters and vegetarians, 8-MeIQx, Iso-MeIQx and PhIP were remarkably increased in urine after the consumption of 275 g of cooked minced beef patties (containing roughly 1.1 g of creatine) that had been fried for approximately 6 min at 300ºC (Holland et al. 2005). Given the very limited number of participants who had quantifiable levels of HCAs in the present study after creatine (which, in fact, is the same number as in placebo), one may suggest that conventional creatine supplementation regimens (up to 20 g day−1 ) may be less ‘harmful’ than a 275 g grilled meat in terms of HCA formation. Further supporting the safety of creatine, supplementing pigs with up to 50 g day−1 for 5 days did not increase HCA formation upon frying of pork (Pfau et al. 2006). The safety of creatine supplementation has frequently been questioned throughout the years, with several review papers and food agencies raising the possibility that creatine could impair kidney function and/or
Serum
B
2.0
2.0
1.5
1.5 mg/dL
mg/dL
A
1.0
0.5
0.5
0.0
0.0 PL
CR
PL
Acute
CR
PL
Chronic
CR
PL
Acute
Urine
C
CR Chronic
D
300
300
200
200
mg/12h
mg/12h
1.0
100
100
0
0 PL
CR Acute
PL
CR Chronic
PL
CR Acute
PL
CR Chronic
Figure 4. Blood and urinary creatinine after creatine or placebo supplementation in phases I (A and C) and II (B and D) Results expressed as individual data and mean ± SD. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Creatine and heterocyclic amines
result in formation of genotoxic HCAs (Benzi, 2000; Terjung et al. 2000; Wyss & Kaddurah-Daouk, 2000; Agence Franc¸aise de S´ecurit´e Sanitaire et Alimentaire, 2001; Brudnak, 2004; Yoshizumi & Tsourounis, 2004). However, a number of well-controlled studies have refuted the putative nephrotoxic effect of creatine in a variety of populations, including athletes (Poortmans & Francaux, 1999; Kreider et al. 2003), recreationally trained subjects (Poortmans et al. 1997, 2005; Lugaresi et al. 2013), sedentary individuals (Gualano et al. 2008), elderly people (Neves et al. 2011; Gualano et al. 2014), patients with pre-existing chronic kidney disease (Gualano et al. 2010), and children (Hayashi et al. 2014). The safety variables assessed in this study add to this solid literature clearly showing that creatine supplementation, regardless of protocol length and dose, does not affect conventional laboratory variables. More importantly, this study provides novel evidence that creatine supplementation does not elicit HCA formation in humans, contradicting empirical concerns that this dietary supplement could be carcinogenic by raising genotoxic HCAs. This study is not without limitations. Firstly, the conclusions must be confined to the supplementation regimens tested in this study. The abusive use of creatine supplements over the recommended dose (i.e. up to 20 g day−1 ) may not be free of risks. Secondly, in an attempt to distinguish the effect of creatine supplementation from that of diet, creatine supplementation was given to subjects who were advised to refrain from food preparations that could potentially increase HCAs. Therefore, one cannot rule out the possible additive effect of creatine supplementation along with other HCA-rich foods (e.g. grilled meats) on HCA formation in the long term. Thirdly, the HPLC–MS/MS method used in the current study was sufficiently robust to quantify HCAs within the picogram range, with LODs ranging from 10 to 20 pg ml−1 . Thus, slight increases in HCA below this level were not detectable in this study, although the physiological significance of such minor changes would likely be trivial. Finally, four selected HCAs were evaluated in this study due to their well-known relationship with cancer. Nonetheless, there are other less studied HCAs (e.g. 2-amino-3-methyl-imidazo[4,5-f]-quinoline) that could potentially be formed through creatinine and, at least in theory, affected by creatine intake (Holland et al. 2005). Further studies should track a larger number of HCAs in order to advance the knowledge on the effects of creatine supplementation on HCA formation. In conclusion, this study provides compelling evidence that both low and high doses of creatine supplementation, given either acutely or chronically, did not cause increases in the genotoxic HCAs 4,8-DiMeIQx, 8-MeIQx, PhIP and IFP in healthy subjects. These findings challenge the long-existing notion that creatine supplementation could C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Gualano B, Ugrinowitsch C, Novaes RB, Artioli GG, Shimizu MH, Seguro AC, Harris RC & Lancha AH Jr (2008). Effects of creatine supplementation on renal function: a randomized, double-blind, placebo-controlled clinical trial. Eur J Appl Physiol 103, 33–40. Harris RC, Lowe JA, Warnes K & Orme CE (1997). The concentration of creatine in meat, offal and commercial dog food. Res Vet Sci 62, 58–62. Hayashi AP, Solis MY, Sapienza MT, Otaduy MC, deSa Pinto AL, Silva CA, Sallum AM, Pereira RM & Gualano B (2014). Efficacy and safety of creatine supplementation in childhood-onset systemic lupus erythematosus: a randomized, double-blind, placebo-controlled, crossover trial. Lupus 23, 1500–1511. Holland RD, Gehring T, Taylor J, Lake BG, Gooderham NJ & Turesky RJ (2005). Formation of a mutagenic heterocyclic aromatic amine from creatinine in urine of meat eaters and vegetarians. Chem Res Toxicol 18, 579–590. Kreider RB, Ferreira M, Wilson M, Grindstaff P, Plisk S, Reinardy J, Cantler E & Almada AL (1998). Effects of creatine supplementation on body composition, strength, and sprint performance. Med Sci Sports Exerc 30, 73–82. Kreider RB, Melton C, Rasmussen CJ, Greenwood M, Lancaster S, Cantler EC, Milnor P & Almada AL (2003). Long-term creatine supplementation does not significantly affect clinical markers of health in athletes. Mol Cell Biochem 244, 95–104. Li N, Hauser R, Holford T, Zhu Y, Zhang Y, Bassig BA, Honig S, Chen C, Boyle P, Dai M, Schwartz SM, Morey P, Sayward H, Hu Z, Shen H, Gomery P & Zheng T (2015). Muscle-building supplement use and increased risk of testicular germ cell cancer in men from Connecticut and Massachusetts. Br J Cancer 112 Suppl, 1247–1250. Lugaresi R, Leme M, deSalles Painelli V, Murai IH, Roschel H, Sapienza MT, Lancha Junior AH & Gualano B (2013). Does long-term creatine supplementation impair kidney function in resistance-trained individuals consuming a high-protein diet? J Int Soc Sports Nutr 10, 26. Manabe S, Kurihara N, Wada O, Tohyama K & Aramaki T (1992). Formation of PhIP in a mixture of creatinine, phenylalanine and sugar or aldehyde by aqueous heating. Carcinogenesis 13, 827–830. Murray S, Lake BG, Gray S, Edwards AJ, Springall C, Bowey EA, Williamson G, Boobis AR & Gooderham NJ (2001). Effect of cruciferous vegetable consumption on heterocyclic aromatic amine metabolism in man. Carcinogenesis 22, 1413–1420. Neves M Jr, Gualano B, Roschel H, Lima FR, Lucia de Sa-Pinto A, Seguro AC, Shimizu MH, Sapienza MT, Fuller R, Lancha AH Jr & Bonfa E (2011). Effect of creatine supplementation on measured glomerular filtration rate in postmenopausal women. Appl Physiol Nutr Metab 36, 419–422. Ohgaki H, Takayama S & Sugimura T (1991). Carcinogenicities of heterocyclic amines in cooked food. Mutat Res 259, 399–410. Overvik E, Kleman M, Berg I & Gustafsson JA (1989). Influence of creatine, amino acids and water on the formation of the mutagenic heterocyclic amines found in cooked meat. Carcinogenesis 10, 2293–2301.
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Pfau W, Rosenvold K & Young JF (2006). Formation of mutagenic heterocyclic aromatic amines in fried pork from Duroc and Landrace pigs upon feed supplementation with creatine monohydrate. Food Chem Toxicol 44, 2086–2091. Poortmans JR, Auquier H, Renaut V, Durussel A, Saugy M & Brisson GR (1997). Effect of short-term creatine supplementation on renal responses in men. Eur J Appl Physiol Occup Physiol 76, 566–567. Poortmans JR & Francaux M (1999). Long-term oral creatine supplementation does not impair renal function in healthy athletes. Med Sci Sports Exerc 31, 1108–1110. Poortmans JR, Kumps A, Duez P, Fofonka A, Carpentier A & Francaux M (2005). Effect of oral creatine supplementation on urinary methylamine, formaldehyde, and formate. Med Sci Sports Exerc 37, 1717–1720. Robbana-Barnat S, Rabache M, Rialland E & Fradin J (1996). Heterocyclic amines: occurrence and prevention in cooked food. Environ Health Perspect 104, 280–288. Skog K (1993). Cooking procedures and food mutagens: a literature review. Food Chem Toxicol 31, 655–675. Skog KI, Johansson MA & Jagerstad MI (1998). Carcinogenic heterocyclic amines in model systems and cooked foods: a review on formation, occurrence and intake. Food Chem Toxicol 36, 879–896. Sugimura T, Wakabayashi K, Nakagama H & Nagao M (2004). Heterocyclic amines: Mutagens/carcinogens produced during cooking of meat and fish. Cancer Sci 95, 290–299. Terjung RL, Clarkson P, Eichner ER, Greenhaff PL, Hespel PJ, Israel RG, Kraemer WJ, Meyer RA, Spriet LL, Tarnopolsky MA, Wagenmakers AJ & Williams MH (2000). American College of Sports Medicine roundtable. The physiological and health effects of oral creatine supplementation. Med Sci Sports Exerc 32, 706–717. Vikse R & Joner PE (1993). Mutagenicity, creatine and nutrient contents of pan fried meat from various animal species. Acta Vet Scand 34, 363–370. Wyss M & Kaddurah-Daouk R (2000). Creatine and creatinine metabolism. Physiol Rev 80, 1107–1213. Yanagisawa H, Manabe S, Kitagawa Y, Ishikawa S, Nakajima K & Wada O (1986). Presence of 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx) in dialysate from patients with uremia. Biochem Biophys Res Commun 138, 1084–1089. Yoshizumi WM & Tsourounis C (2004). Effects of creatine supplementation on renal function. J Herb Pharmacother 4, 1–7.
Additional information Competing interests The authors declare no competing interests related to this study. Author contributions Conception and design of the experiments: J.R.P., F.A.D., G.G.A., B.G.; collection, assembly, analysis and interpretation of data: R.T.S.P., F.A.D., E.P., M.Y.S., A.L.F., I.H.M., W.S.D., A.C.S., M.A.R.S., G.G.A., H.R., B.G.; drafting the article or revising it critically for important intellectual content: R.T.S.P., A.C., H.R., G.G.A., J.R.P., B.G. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Funding
Acknowledgements
This study was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de S˜ao Paulo (FAPESP). B.G. and H.R. are supported by Conselho Nacional de Pesquisa e Tecnologia (CNPq).
The authors are highly grateful to Prof. Arkadiusz Szterk, who kindly donated the reagents for PhIP analysis, and Dr Bryan Saunder, who carefully proofread the manuscript.
Translational perspective Creatine has been anecdotally associated with a number of adverse events, including cancer. In this regard, a recent case-control study suggested increased risk of testicular germ cell cancer (Li et al. 2015) in users of muscle-building supplements, including creatine. Despite serious limitations found in this study, such as lack of control for concomitant use of banned sports-enhancing substances, creatine has made the highlights as ‘guilty as charged’. The biological plausibility underlying creatine-induced cancer lies in the fact that creatine and its byproduct creatinine are possible precursors of heterocyclic amines (HCAs), a class of potent carcinogenic compounds. Surprisingly, the role of dietary creatine in the increased formation of HCAs has been overlooked so far and never tested before. In the present study, we used a newly developed HPLC–MS/MS method for showing that creatine supplemented at different dosages (low and high) is innocuous in terms of HCA formation, either acutely (1 day) or chronically (30 days). This novel finding contradicts unattested growing rumours that creatine could be potentially carcinogenic and strengthens the scientifically driven notion that this dietary supplement is safe, warranting its widespread application, from sports to clinical settings.
C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Can creatine supplementation form carcinogenic heterocyclic amines in humans? Renato Tavares dos Santos Pereira1 , Felipe Augusto D¨orr2 , Ernani Pinto2 , Marina Yazigi Solis1 , Guilherme Giannini Artioli1 , Alan Lins Fernandes1 , Igor Hisashi Murai1 , Wagner Silva Dantas1 , Antˆonio Carlos Seguro3 , Mirela Aparecida Rodrigues Santinho3 , Hamilton Roschel1,3 , Alain Carpentier4 , Jacques Remi Poortmans4 and Bruno Gualano1,3 1
School of Physical Education and Sport, University of Sao Paulo, Sao Paulo, Brazil Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil 3 School of Medicine, University of Sao Paulo, Sao Paulo, Brazil 4 Faculty of Motor Sciences, Universit´e of libre de Bruxelles, Belgium
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Key points
r There is a long-standing concern that creatine supplementation could be associated with cancer, possibly by facilitating the formation of carcinogenic heterocyclic amines (HCAs).
r This study provides compelling evidence that both low and high doses of creatine r r
supplementation, given either acutely or chronically, does not cause a significant increase in HCA formation. HCAs detection was unrelated to creatine supplementation. Diet was likely to be the main factor responsible for HCAs formation after either placebo (n = 6) or creatine supplementation (n = 3). These results directly challenge the recently suggested biological plausibility for the association between creatine use and risk of testicular germ cell cancer.
Abstract Creatine supplementation has been associated with increased cancer risk. In fact, there is evidence indicating that creatine and/or creatinine are important precursors of carcinogenic heterocyclic amines (HCAs). The present study aimed to investigate the acute and chronic effects of low- and high-dose creatine supplementation on the production of HCAs in healthy humans (i.e. 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-(1,6-dimethylfuro[3,2-e] imidazo[4,5-b])pyridine (IFP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8DiMeIQx)). This was a non-counterbalanced single-blind crossover study divided into two phases, in which low- and high-dose creatine protocols were tested. After acute (1 day) and chronic supplementation (30 days), the HCAs PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx were assessed through a newly developed HPLC–MS/MS method. Dietary HCA intake and blood and urinary creatinine were also evaluated. Out of 576 assessments performed (from 149 urine samples), only nine (3 from creatine and 6 from placebo) showed quantifiable levels of HCAs (8-MeIQx: n = 3; 4,8-DiMeIQx: n = 2; PhIP: n = 4). Individual analyses revealed that diet rather than creatine supplementation was the main responsible factor for HCA formation in these cases. This study provides compelling evidence that both low and high doses of creatine supplementation, given either acutely or chronically, did not cause increases in the carcinogenic HCAs PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx in healthy subjects. These findings challenge the long-existing notion
J.-R. Poortmans and B. Gualano contributed equally to this paper. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
DOI: 10.1113/JP270861
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that creatine supplementation could potentially increase the risk of cancer by stimulating the formation of these mutagens. (Received 5 May 2015; accepted after revision 24 June 2015; first published online 6 July 2015) Corresponding author B. Gualano: Escola de Educac¸a˜o F´ısica e Esporte, Departamento de Biodinˆamica do Movimento Humano, Av. Mello de Moraes, 65, S˜ao Paulo, SP, Brazil, 05508-030. Email: [email protected] Abbreviations CR, creatine; 4,8-DiMeIQx, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline; 4,8-DiMeIQx-D3, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline-d3; HCA, carcinogenic heterocyclic amine; IFP, 2-amino-(1,6dimethylfuro[3,2-e]imidazo[4,5-b])pyridine; LOD, limit of detection; 8-MeIQx, 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline; 8-MeIQx-D3, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline-d; ND, inferior to levels of detection; NQ, inferior to levels of quantification; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine3; PhIP-D3, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-d3; PL, placebo.
Introduction Ingestion of high amounts of heterocyclic amines (HCAs), which may be found in food processing and culinary preparations such as grilling and smoking, has been often associated with increased risk for lung, stomach, bladder, colon and breast cancer (Breslow et al. 2000). Indeed, their carcinogenicity has been experimentally demonstrated in animal studies (Ohgaki et al. 1991; Sugimura et al. 2004). HCAs are among the most potent mutagens assessed by the Ames Salmonella test (Ames et al. 1975), providing biological plausibility for association studies. HCAs, including 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (8-MeIQx), 2-amino-(1,6-dimethylfuro[3,2-e]imidazo[4,5-b])pyridine (IFP) and 2-amino-3, 4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), are formed by the condensation of creatine/creatinine with D-glucose and amino acids (Fig. 1). These compounds share a common imidazole-ring structure with an exocyclic amino group and, therefore, are known chemically as amino-imidazoazaarenes. Although the major exposure to PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx is through the consumption of cooked meats, these mutagens have also been detected in other products such as processed food flavourings, beverages (e.g. beer and wine) and cigarette smoke (Wyss & Kaddurah-Daouk, 2000). In addition, it has been speculated that dietary creatine may have a role in the production of HCAs, as creatine itself and its by-product creatinine have been closely implicated in HCA synthesis. There is compelling evidence indicating that creatine and/or creatinine are important precursors of HCAs, as elegantly pointed out by Wyss & Kaddurah-Daouk (2000). Firstly, in different fried bovine tissues and meat extracts, beef flavours, bouillons, and gravies, mutagenicity was correlated with the creatine and/or creatinine content of the samples (Vikse & Joner, 1993; Robbana-Barnat et al. 1996). Secondly, the addition of creatine or phosphorylcreatine to meat samples or beef extracts before the cooking process increased mutagenicity up to 40-fold
and HCA contents up to 9-fold (Overvik et al. 1989; Skog, 1993), whereas the treatment of beef meat with creatinase before frying reduced the creatine content and the mutagenicity by 65% and 73%, respectively (Vikse & Joner, 1993). Thirdly, the addition of creatine to meat before cooking had no influence on the number or relative proportions of HCA mutagens formed, but increased the level of all mutagens to approximately the same extent, suggesting that creatine is involved in the formation of all HCAs (Overvik et al. 1989). Finally, as previously mentioned, most HCAs could be generated artificially in simple model systems containing creatine or creatinine, amino acids and glucose (Wyss & Kaddurah-Daouk, 2000). Even though the heating process appears to enhance HCA formation, there is evidence that these mutagens could also be formed at physiological temperatures. For instance, patients with chronic renal failure, who are subject to an increased cancer risk, experience high creatinine concentrations, thereby creating ‘favourable’ conditions for the formation of HCAs (Yanagisawa et al. 1986). In this study, 8-MeIQx was detected in the dialysis fluid of all uraemic patients examined. The notion that 8-MeIQx does not originate from meat consumption, but from de novo synthesis, is corroborated by the formation of HCAs in a model system at temperatures as low as 37°C (Manabe et al. 1992). Taken together, these observations confer some degree of biological plausibility for the speculation that dietary creatine could increase HCA formation in vivo. However, this needs to be confirmed in humans. Creatine has become one of the most popular dietary supplements used by athletes and exercise practitioners in order to improve physical capacity and lean mass (Kreider et al. 1998; Terjung et al. 2000; Gualano et al. 2012). Furthermore, creatine supplementation has been also applied as an adjuvant therapeutic tool in a variety of conditions characterized by muscle dysfunction, atrophy, frailty, mental disturbances and bioenergetics disorders (for a comprehensive review, see Gualano et al. (2012)). Despite the beneficial effects of creatine supplementation in sports and C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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clinical settings, there has been a long-standing concern, raised by a number of review articles and guidelines (Terjung et al. 2000; Wyss & Kaddurah-Daouk, 2000; Agence Franc¸aise de S´ecurit´e Sanitaire et Alimentaire, 2001; Brudnak, 2004), that this dietary supplement could cause adverse outcomes, including HCA-induced carcinogenic/mutagenic effects. Recently, a case-control study showed that ‘muscle-building’ supplements, including those containing creatine, were associated with increased risks of testicular germ cell cancer (odds ratio: 1.65, 95% confidence interval: 1.11–2.46) (Li et al. 2015). However, this study did not control for important confounding factors, such as use of anabolic steroid and supplement contaminants. Thus, the potential carcinogenic effect of creatine supplementation remains highly unexplored, despite speculation. To shed light on this topic, the present study aimed to investigate the acute and chronic effects of low- and high-dose creatine supplementation on the formation of carcinogenic HCAs (i.e. PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx) in healthy humans.
Methods Experimental design and participants
This was a non-counterbalanced single-blind crossover study divided into two phases, in which low- and high-dose creatine protocols were tested (phases I and II, respectively). Healthy males and females (age: 29 ± 4 years) were selected according to the following exclusion criteria: tobacco use, use of creatine supplements for at least 4 months before the commencement of the study, current use of any medication, vegetarian diet, and pre-existing chronic diseases. All procedures were approved by the local ethical committee and were in accordance with the Declaration of Helsinki revised in 2008. The participants were fully informed about risks and benefits associated with participation before signing the informed consent. Participants received placebo and, following a 14-day washout period, creatine monohydrate; each experimental condition (i.e. placebo and creatine) lasted 30 days, which
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consisted of a 7-day ‘loading’ period followed by a 23-day ‘maintenance’ period (details on the supplementation regimens can be seen in ‘Supplementation protocols’). Fasting blood and 12 h urine samples were collected on the first day of the ‘loading’ period (urine collection commenced immediately after the ingestion of the last daily dose) and on the 30th day of supplementation. Samples were immediately frozen at −80ºC until further analysis of PhIP, 8-MeIQx, IFP, 4,8-DiMeIQx (through a HPLC–MS/MS method), and creatinine (through a colorimetric method). Phases I (n = 11 males and 10 females) and II (n = 13 males and 3 females) were interspaced by a 4-month period in order to avoid any carry-over effect of creatine. To mitigate the impact of dropouts that occurred between phases I and II (n = 11), seven additional subjects were selected to participate exclusively in phase II. Participants were strongly instructed to refrain from fried or grilled meat consumption 4 days before urine collections to avoid any interference of the diet on HCA formation. Dietary intake of energy, macronutrients, creatine, and PhIP, 8-MeIQx and 4,8-DiMeIQx was assessed by food records taken 4 days before the commencement of ‘loading’ and during the last 4 days of ‘maintenance’. Figure 2 illustrates the experimental design.
Supplementation protocols
Subject were given 7 g day−1 (phase I) or 20 g day−1 (phase II) of creatine monohydrate (Creapure , Alzchem AG, Germany) or placebo (same doses of dextrose) for 7 days divided into four equal doses, followed by single daily doses of 2 g day−1 (phase I) or 5 g day−1 (phase II) for the next 23 days. During ‘loading’, supplements were given in four packages and the participants were instructed to ingest the supplement packages at breakfast, lunch, dinner and before bedtime. During ‘maintenance’, subjects consumed the supplement as a single dose during their lunch. Creatine and placebo supplements were formulated in undistinguishable tablets with identical appearance, R
NH2 CH3
N N
CH3
N
glucose
N
CH3
NH2 N
N
N
PhlP
Creatine or Creatinine
8-MelQx NH2 N
N CH3
N
N
amino acids Figure 1. Illustrative model of HCA formation from creatine/creatinine, D-glucose and amino acids C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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CH3 O
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taste, and smell, and the packages were coded so that the participants were not aware of the contents until completion of the analyses. Supplements were provided by a staff member from our research team who did not have any participation in the data acquisition, analyses or interpretation. Compliance to the supplementation was monitored weekly by asking the patients personally.
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Table 1. Optimized parameters for HCA determination in MRM mode Precursor (m/z)
Ion products (m/z)
Fragmentor (V)
CE (V)
8-MeIQx
214
8-MeIQx-D3
217
4,8-DiMeIQx
228
4,8-DiMeIQx-D3
231
PhIP
225
PhiP-d3
228
IFP
203
131 199 131 199 160 187 212 145 140 210 140 210 188 187
140 140 140 140 140 140 140 140 140 140 140 140 98 98
46 26 44 28 30 25 40 44 58 30 50 32 22 34
Compound
Food intake assessment and control for HCA dietary intake
Food intake was assessed by 4-day food records, using the software Avanutri (Rio de Janeiro, Brazil). Specific diet composition tables were used to calculate dietary intake of PhIP, 8-MeIQx, and 4,8-DiMeIQx (Skog et al. 1998), as well as creatine (Harris et al. 1997). In addition, the ingestion of total energy, carbohydrate, lipids and protein was estimated. To avoid any possible confounding effect of diet upon HCA formation, participants were highly encouraged to abstain from food that could potentially increase HCAs. To that end, they were provided with a list of food preparations (e.g. grilled meat, fried chicken, wine) that they should refrain from 4 days before until the end of urine collection. R
Ions shown in bold were used for quantification; 8-MeIQx-D3 was used as internal standard for IFP determination.
homogenised and centrifuged at 5000 g for 5 min. Metabolic conjugates were hydrolysed by the addition of 12 mol l−1 hydrochloric acid (90 µl) to 1000 µl of urine followed by incubation at 70°C for 3 h. After cooling, 200 µl 10 mol l−1 NaOH and 100 µl of a solution containing internal standards of 8-MeIQx-D3, 4,8-DiMeIQx-D3 and PhIP-D3 at 4 ng ml−1 were added. Samples were thoroughly mixed and submitted to liquid–liquid extraction twice using 5 ml ethyl acetate. The organic layers were applied to ion exchange cartridges (Bond Elut Certify, 130 mg; Agilent, Santa Clara, CA, USA), which were preconditioned with methanol and 2% acetic acid/methanol solution. After washing the cartridge with 0.1 mol l−1 acetic acid and methanol, HCAs were eluted with 2 ml of methanol/ammonium hydroxide 95:5 (v/v). Samples were dried under nitrogen at 30ºC, reconstituted in 100 µl methanol/water (50:50, v/v) and analysed by HPLC–MS/MS. HCA analyses were performed using a 1260 Infinity HPLC system coupled with a 6460 Triple-Quad mass spectrometer (Agilent). Chromatographic separation was
Creatinine measurements
Urinary and serum creatinine were determined using the enzymatic method as per the instructions provided by a commercially available assay kit (Labtest, Sao Paulo, Brazil). HCA determination
The following HCAs were assessed in urine samples: PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx. All compounds and internal standards (i.e. 8-MeIQx-D3, 4,8-DiMeIQx-D3 and PhIP-D3) were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). HCA extraction from urine samples was carried out according to a previously validated method described elsewhere (Fu et al. 2014), with slight modifications. Briefly, urine samples were thawed at room temperature, -4
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placebo (20 g or 7 g/d)
7
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placebo (5 g or 2 g/d)
40
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washout
44
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creatine (20 g or 7 g/d)
70
74
creatine (5 g or 2 g/d)
time (days)
4-day food record
12-hour urine fasting blood
4-day food 12-hour urine record fasting blood
4-day food 12-hour urine record fasting blood
4-day food 12-hour urine record fasting blood
Figure 2. The experimental design C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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obtained with a Kinetex EVO C18 column (100 × 2.1 mm; 5 µm, Phenomenex, Torrance, CA, USA) at 0.3 ml min−1 , using (a) 2 mmol l−1 ammonium bicarbonate containing ammonium hydroxide (0.02%) at pH 8.0 and (b) acetonitrile as mobile phases. Gradient elution was performed under the following conditions: 0–15 min, 5 to 25% acetonitrile; 15.5–16.5 min, 95% acetonitrile; 17–20 min, 5% acetonitrile. The mass spectrometer electrospray source was operated in the positive mode under the following conditions: capillary voltage, 3500 V; nebuliser (N2 ), 30 psi; drying gas (N2 ), 6 l min−1 at 300°C; sheath gas (N2 ), 10 l min−1 at 300°C. Multiple reaction monitoring (MRM) optimized parameters are displayed in Table 1. Analyte identification was based on the retention time (±2% when compared to its respective calibrator) and peak area ratios of qualifier/quantifier ion transitions (±20%). The analytical protocol was further evaluated for linear range, limits of detection and quantification, precision and accuracy following standard recommendations. The assessors of urinary HCAs were blinded to the treatment. Statistical analysis
Dependent variables were tested by mixed model with repeated measures using the software SAS 9.3. Group (creatine and placebo) and time (pre and post) were defined as fixed factors and subjects were defined as a random factor. Significance level was previously set at P < 0.05. Results are presented as individual data, mean and standard deviation.
subsequently employed to analyse urinary HCAs after creatine supplementation. Tables 3 (phase I) and 4 (phase II) summarize individual data for urinary HCAs. Out of 576 assessments performed (from 149 urine samples), only 9 (3 from creatine and
x102 3 2.5 2 1.5 1 0.5 x103 1 0.8 0.6 0.4 0.2 0 x102 7 6 5 4 3 2 1 0 x102 2
Whilst the extraction protocol validated by Fu et al. (2014) was successfully employed in this study with recovery rates superior to 90% for all HCAs, chromatographic separation was not promptly adequate, as interfering chromatographic peaks compromised the accurate detection of HCAs. In order to improve resolution, separation at alkaline pH was applied (Cooper et al. 2014). The newly developed chromatographic method was evaluated regarding linear range, limits of detection and quantification, precision and accuracy, showing satisfactory results (Table 2). Typical chromatograms of urine spikes are shown in Fig. 3. Linearity data analysis showed that the method was heteroscedastic, and hence weighted least squares linear regressions were used (Almeida et al. 2002). The weighting factor ‘1/x’ was selected according to the sums of the relative error percentages (%RE). Matrix effects were effectively compensated by the use of matched blank matrix (i.e. pooled urine obtained from placebo condition) and isotopic labelled internal standards. The same method was C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
8-MelQx
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8 9 IFP
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6 7 8 4,8-DiMelQx
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4 3 2 1 0 x102 2.5 2 1.5 1 0.5 x102 2.5 2 1.5 1 0.5
13 14 15 PhIP-D3
Counts vs. Acquisition Time (min) Figure 3. Typical chromatograms of urine spikes
13
14
15
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Table 2. Calibration curves, linear range, limit of detection (LOD), precision and accuracy results for HCAs in urine
Compound 8-MeIQx
4,8-DiMeIQx
IFP
PhIP
Calibration curve Y = 0.869492x + 0.029523 R2 = 0.998 Weight 1/x Y = 0.379978x + 0.00012018 R2 = 0.996 Weight 1/x Y = 2.166107 + 0.065861 R2 = 0.999 Weight 1/x Y = 0.824436 + 0.015107 R2 = 0.997 Weight 1/x
Precision (CV%)
Linear range (pg ml−1 )
LOD (pg ml−1 )
Quality control
Intra-day
Inter-day
Accuracy (%)
31.25–1000
10
31.25–1000
20
31.25–1000
10
31.25–1000
10
Low Medium High Low Medium High Low Medium High Low Medium High
6.2 3.1 3.3 3.9 2.7 4.4 6.6 1.8 8.9 5.6 2.7 2.4
6.4 3.3 3.8 3.8 3.8 4.5 6.4 3.4 9.5 6.5 2.7 2.5
101.8 98.8 99.3 95.5 97.2 100.8 97.6 100.2 95.6 97.6 99.7 102.2
6 from placebo) showed quantifiable levels of HCAs (8-MeIQx: n = 3; 4,8-DiMeIQx: n = 2; PhIP: n = 4). Six participants had quantifiable levels of one or more urinary HCAs, three of them after creatine supplementation (subject V in phase I and subjects VI and XIV in phase II) and three after placebo (subject V in phase I and subjects IV and VI in phase II). The remaining assessments showed non-detectable or non-quantifiable HCA levels. Participant V showed increased 4,8-DiMeIQx levels after low-dose chronic creatine supplementation (48.7 pg ml−1 ). However, similar levels were found after placebo (69.6 pg ml−1 ). In fact, 4,8-DiMeIQx dietary intake was comparable in both creatine and placebo conditions (2.48 ± 2.39 vs. 2.74 ± 2.00 ng g−1 , respectively), which explains the similar urinary excretion between the two interventions. Participant IV showed urinary PhIP levels of 32.9 pg ml−1 after acute high-dose creatine supplementation, but not after placebo. However, her PhIP dietary intake was about 10 times lower during placebo (0.24 ± 0.39 ng g−1 ) than in creatine condition (25.36 ± 43.51 ng g−1 ). Participant XVI showed increased levels of 8-MelQx (108.8 pg ml−1 ) after acute high-dose creatine supplementation; however, dietary analysis revealed he consumed twice as much of this HCA during creatine versus placebo supplementation (4.89 ± 1.96 vs. 2.38 ± 3.18, respectively). Individual analysis of the subjects who showed quantifiable levels of urinary HCAs following creatine supplementation led to the proposition that diet, rather than creatine supplementation, was the main contributing factor to HCA formation in these three cases. Dietary intake of HCAs is expressed in Table 3 and Table 4. Ingestion of 8-MeIQx (range: 0–19.55 ng g−1 ), 4,8-DiMeIQx (range: 0.09–5.58 ng g−1 ), and PhIP (range: 0–136.95 ng g−1 ) were comparable between
creatine and placebo in phases I and II, after acute and chronic supplementation (P > 0.05). In addition, total energy, carbohydrate, lipids and protein intake were not significantly different between creatine and placebo in any condition (P > 0.05; data not shown). Serum (Fig. 4A and B) and urinary (Fig. 4C and D) creatinine data are shown in Fig. 4. No changes in serum and urinary creatinine values were observed after either acute or chronic supplementation of creatine or placebo in phases I and II (P > 0.05). Additionally, none of the participants showed changes in creatinine values beyond the normal range following creatine or placebo supplementation in any condition (i.e. low/high dose, acute/chronic, protocols). Discussion This study is the first to show that creatine supplementation does not significantly contribute to HCA formation (i.e. 8-MeIQx, 4,8-DiMeIQx, PhIP, and IFP) in healthy humans. These results suggest that creatine does not provoke any carcinogenic effect secondary to HCA formation in humans. HCAs can be formed through Maillard reactions involving D-glucose, amino acids and creatinine (Wyss & Kaddurah-Daouk, 2000), especially under high temperatures. However, there is also evidence showing that HCA formation is possible under physiological conditions (i.e. at pH 7.4, 37ºC). In fact, it has been demonstrated that imidazo-quinoxalines (e.g. 8-MeIQx) are preferably formed through de novo synthesis rather than meat intake, which confirms the possibility of HCA formation in a physiological ‘environment’. The role of circulating creatinine in the formation of HCAs appears to be crucial. The presence of 8-MeIQx was reported in the dialytic fluid of uraemic patients, suggesting that C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Table 3. Dietary intake and urinary HCAs following low-dose creatine or placebo supplementation (phase I) Subject (sex) I (M)
Acute Chronic
II (F)
Acute Chronic
III (F)
Acute Chronic
IV (M)
Acute Chronic
V (F)
Acute Chronic
VI (M)
Acute Chronic
VII (F)
Acute Chronic
VIII (M)
Acute Chronic
IX (M)
Acute Chronic
X (M)
Acute Chronic
XI (M)
Acute Chronic
XII (F)
Acute Chronic
XIII (M)
Acute Chronic
8-MeIQx
4.8-DiMeIQx
PhIP
(ng g−1 ) (pg ml−1 )
Placebo
Creatine
Placebo
Creatine
Placebo
Creatine
Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion
2.39 ± 1.17 ND 2.37 ± 3.67 ND 3.04 ± 3.00 ND 1.08 ± 1.50 ND 7.66 ± 1.22 ND 7.77 ± 4.45 ND 7.27 ± 0.28 ND 7.46 ± 0.60 ND 0.87 ± 1.27 ND 2.51 ± 2.34 NQ 10.03 ± 7.71 ND 10.38 ± 4.44 ND 1.00 ± 1.73 ND 5.07 ± 3.74 ND 1.55 ± 2.47 ND 1.62 ± 2.58 ND 3.88 ± 3.17 ND 7.09 ± 0.79 ND 19.55 ± 9.39 ND 10.14 ± 4.31 ND 5.28 ± 4.25 ND 2.63 ± 1.03 ND 4.33 ± 1.74 ND 4.08 ± 3.46 ND 5.03 ± 5.06 ND 5.13 ± 8.80 ND
2.64 ± 2.57 ND 1.20 ± 2.08 ND 1.08 ± 1.50 ND 0.31 ± 0.54 ND 8.84 ± 3.26 ND 7.05 ± 4.30 ND 7.28 ± 0.27 ND 6.17 ± 2.07 ND 2.46 ± 2.53 ND 3.24 ± 1.35 ND 10.69 ± 7.66 ND 3.04 ± 4.64 ND 5.40 ± 3.17 ND 3.90 ± 3.46 ND 5.73 ± 7.48 ND 5.94 ± 10.29 ND 7.16 ± 0.73 ND 5.24 ± 4.46 ND 17.68 ± 9.02 ND 11.74 ± 6.40 ND 2.74 ± 1.14 ND 6.46 ± 5.47 ND 4.10 ± 3.42 ND 3.43 ± 3.31 ND 3.19 ± 2.96 ND 7.37 ± 9.32 ND
1.35 ± 1.30 ND 1.22 ± 2.00 ND 0.17 ± 0.16 ND 0.32 ± 0.56 ND 0.92 ± 0.82 ND 1.78 ± 0.71 ND 2.65 ± 0.33 ND 2.59 ± 0.22 ND 0.29 ± 0.45 ND 2.48 ± 2.39 69.6 0.79 ± 0.29 ND 1.40 ± 0.68 ND 0.09 ± 0.15 ND 0.51 ± 0.26 ND 0.44 ± 0.76 ND 0.33 ± 0.57 ND 0.52 ± 0.22 ND 2.89 ± 4.42 ND 1.40 ± 0.98 ND 1.02 ± 0.68 ND 0.64 ± 0.73 ND 0.94 ± 0.21 ND 1.57 ± 1.78 ND 1.02 ± 0.88 ND 1.08 ± 1.08 ND 1.10 ± 1.90 ND
0.92 ± 0.89 ND 0.36 ± 0.62 ND 0.32 ± 0.56 ND 0.11 ± 0.19 ND 0.98 ± 0.77 ND 1.16 ± 0.83 ND 2.71 ± 0.29 ND 2.24 ± 0.92 ND 0.42 ± 0.41 ND 2.74 ± 2.00 48.7 0.85 ± 0.18 ND 1.03 ± 1.63 ND 0.54 ± 0.23 ND 0.48 ± 0.56 ND 2.93 ± 2.90 ND 1.88 ± 3.25 ND 2.92 ± 4.40 ND 3.07 ± 4.96 ND 1.28 ± 0.80 ND 1.10 ± 0.63 ND 0.97 ± 0.26 ND 1.91 ± 1.32 ND 1.04 ± 0.85 ND 0.48 ± 0.56 ND 1.65 ± 1.37 ND 0.76 ± 0.55 ND
11.20 ± 19.40 ND 45.41 ± 78.63 ND 27.05 ± 35.63 ND 0.07 ± 0.13 ND 10.90 ± 9.60 ND 14.42 ± 12.50 ND 2.80 ± 4.85 ND 0 ND 30.00 ± 51.96 ND 34.54 ± 33.64 ND 0.46 ± 0.45 ND 0.37 ± 0.37 ND 4.55 ± 7.88 ND 3.97 ± 6.26 ND 32.68 ± 43.82 ND 17.44 ± 30.10 ND 2.35 ± 2.84 ND 37.55 ± 64.47 ND 4.85 ± 6.86 ND 0.42 ± 0.36 ND 3.70 ± 5.81 ND 34.33 ± 48.44 ND 13.16 ± 22.55 ND 6.20 ± 5.38 ND 7.74 ± 8.01 ND 45.50 ± 59.25 ND
0 ND 34.20 ± 59.24 ND 0.07 ± 0.13 ND 7.50 ± 12.99 ND 10.95 ± 9.52 ND 11.36 ± 10.71 ND 2.82 ± 4.84 ND 2.74 ± 4.73 ND 37.70 ± 46.74 ND 34.57 ± 33.59 ND 0.48 ± 0.49 ND 12.51 ± 21.65 ND 0.36 ± 0.04 ND 2.78 ± 4.53 ND 38.27 ± 36.54 ND 14.00 ± 24.25 ND 37.64 ± 64.39 ND 41.18 ± 71.03 ND 1.94 ± 1.86 ND 0.50 ± 0.46 ND 37.00 ± 53.04 ND 9.00 ± 7.94 ND 6.27 ± 5.43 ND 24.31 ± 31.51 ND 12.14 ± 21.02 ND 0.31 ± 0.51 ND
(Continued)
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Table 3. Continued Subject (sex) XIV (F)
Acute Chronic
XV (F)
Acute Chronic
XVI (F)
Acute Chronic
XVII (F)
Acute Chronic
XVIII (M)
Acute Chronic
XIX (F)
Acute Chronic
XX (M)
Acute Chronic
XXI (M)
Acute Chronic
8-MeIQx
4.8-DiMeIQx
PhIP
(ng g−1 ) (pg ml−1 )
Placebo
Creatine
Placebo
Creatine
Placebo
Creatine
Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion
3.35 ± 3.46 ND 5.38 ± 4.39 ND 4.56 ± 4.11 ND 7.87 ± 1.46 ND 3.83 ± 2.32 ND 6.32 ± 4.72 ND 2.77 ± 2.37 ND 4.69 ± 0.91 ND 6.91 ± 6.70 ND 7.25 ± 6.35 ND 0 ND 0.85 ± 1.48 ND 8.19 ± 2.01 NQ 9.75 ± 1.58 ND 10.99 ± 7.22 NQ 10.08 ± 4.82 ND
2.31 ± 1.57 ND 3.93 ± 0.57 ND 4.85 ± 3.87 ND 5.27 ± 4.49 ND 4.52 ± 2.93 ND 5.44 ± 3.57 ND 2.41 ± 3.35 ND 1.23 ± 2.14 ND 2.02 ± 1.81 ND 2.69 ± 0.76 ND 0 ND 1.55 ± 1.35 ND 10.09 ± 2.02 ND 4.64 ± 7.86 ND 5.89 ± 6.49 NQ 9.62 ± 5.61 ND
0.36 ± 0.34 ND 0.88 ± 0.40 ND 1.09 ± 0.80 ND 1.34 ± 0.70 ND 1.22 ± 0.03 ND 0.47 ± 0.09 ND 0.37 ± 0.46 ND 1.59 ± 0.52 ND 1.54 ± 0.43 ND 2.20 ± 1.56 ND 0 ND 0.30 ± 0.51 ND 4.01 ± 1.86 ND 2.14 ± 0.46 ND 2.10 ± 1.99 NQ 3.61 ± 0.95 ND
0.78 ± 0.56 ND 0.45 ± 0.47 ND 1.24 ± 0.74 ND 0.69 ± 0.54 ND 0.92 ± 0.50 ND 1.14 ± 1.23 ND 0.63 ± 0.86 ND 0.25 ± 0.43 ND 0.70 ± 0.63 ND 2.04 ± 1.71 ND 0 ND 0.54 ± 0.47 ND 2.24 ± 0.30 ND 1.52 ± 2.63 ND 1.84 ± 1.78 NQ 3.11 ± 1.81 ND
1.72 ± 2.68 ND 5.01 ± 3.85 ND 33.39 ± 42.90 ND 3.50 ± 4.62 ND 10.00 ± 6.29 ND 20.29 ± 34.39 ND 19.70 ± 28.57 ND 15.20 ± 11.20 ND 10.87 ± 15.02 ND 12.53 ± 17.87 ND 31.20 ± 44.77 ND 50.00 ± 86.60 ND 37.34 ± 42.78 ND 19.17 ± 6.00 87.7 15.90 ± 26.76 NQ 25.53 ± 19.40 ND
0.77 ± 1.27 ND 2.52 ± 4.05 ND 6.26 ± 7.14 ND 23.10 ± 38.54 ND 8.65 ± 8.23 ND 25.25 ± 31.02 ND 8.32 ± 8.00 ND 3.15 ± 5.46 ND 2.71 ± 4.70 ND 3.05 ± 5.28 ND 100.00 ± 14.56 ND 0 ND 19.83 ± 5.57 ND 10.07 ± 8.72 ND 35.94 ± 54.79 NQ 18.54 ± 24.86 ND
ND: inferior to levels of detection; NQ: inferior to levels of quantification; all IFP values were inferior to levels of detection (data not shown); dietary intake of IFP was not measured.
kidney failure-induced elevation in circulating creatinine could predispose to HCA formation (Yanagisawa et al. 1986). Since creatine can be irreversibly and spontaneously converted into creatinine, one could speculate that creatine loading could enhance HCA formation (Wyss & Kaddurah-Daouk, 2000). However, the current results indicate that this is not the case in humans. In this study, two different creatine supplementation protocols were tested, one low (i.e. 7 g day−1 for 7 days followed by 2 g day−1 for the next 23 days) and the other high dose (i.e. 20 g day−1 for 7 days followed by 5 g day−1 for the next 23 days), with urinary HCAs being assessed both acutely (i.e. 1 day after creatine ‘loading’) and chronically (i.e. 30 days after supplementation). This experimental design allowed us to explore a potential dose–response pattern in HCA formation following
creatine intake. In order to avoid any interference of habitual dietary intake on HCAs, subjects were instructed not to consume any foods that could possibly contribute to increased HCA formation. Apparently, the majority of the subjects were able to follow this restriction successfully, as reflected by non-detectable or non-quantifiable levels of HCAs in most of the urine samples. The general absence of HCAs in urine in this study was expected and it is in agreement with previous observations of no HCA detection in subjects who temporarily refrained from meat consumption (Murray et al. 2001; Holland et al. 2005). This experimental condition effectively allowed us to distinguish any potential effect of creatine supplementation (low/high and acute/chronic regimens) from that of the regular diet on HCA formation. Interestingly, HCAs were detected at quantifiable levels in six C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Table 4. Dietary intake and urinary HCAs following high-dose creatine or placebo supplementation (phase II)
Subject (sex) I (M)
Acute Chronic
II (F)
Acute Chronic
III (M)
Acute Chronic
IV (M)
Acute Chronic
V (M)
Acute Chronic
VI (F)
Acute Chronic
VII (M)
Acute Chronic
VIII (M)
Acute Chronic
IX (M)
Acute Chronic
X (M)
Acute Chronic
XI (M)
Acute Chronic
XII (M)
Acute Chronic
XIII (F)
Acute Chronic
XIV (M)
Acute Chronic
XV (M)
Acute Chronic
XVI (M)
8-MeIQx
(ng g−1 ) (pg ml−1 )
Acute Chronic
Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion Ingestion Excretion
4.8-DiMeIQx
PhIP
Placebo
Creatine
Placebo
Creatine
Placebo
Creatine
1.71 ± 1.48 ND 2.85 ± 4.30 ND 1.28 ± 1.98 ND 1.80 ± 3.12 ND 2.26 ± 3.02 ND 5.98 ± 4.54 ND 3.55 ± 2.94 72.1 3.10 ± 3.26 ND 6.07 ± 1.90 ND 9.96 ± 3.60 ND 7.28 ± 6.54 NQ 5.62 ± 7.31 46.2 6.07 ± 2.55 ND 2.60 ± 2.42 ND 10.85 ± 3.69 ND 8.93 ± 6.43 ND 18.00 ± 9.00 ND 12.56 ± 13.03 ND 7.26 ± 4.02 ND 9.69 ± 7.81 ND 1.73 ± 1.33 ND 2.16 ± 0.68 ND 2.00 ± 3.46 ND 10.17 ± 9.97 ND 3.30 ± 2.86 ND 2.49 ± 1.58 ND 2.38 ± 3.18 ND 3.55 ± 1.37 ND 10.76 ± 2.33 ND 13.21 ± 2.30 ND 3.07 ± 3.14 ND 7.47 ± 5.95 ND
0.33 ± 0.58 ND 2.49 ± 3.58 ND 5.05 ± 2.34 ND 3.57 ± 4.53 ND 1.48 ± 1.72 ND 9.59 ± 5.24 ND 3.77 ± 4.35 ND 1.08 ± 1.67 ND 3.95 ± 3.33 ND 4.04 ± 3.32 ND 6.03 ± 4.64 ND 10.73 ± 14.22 ND 7.07 ± 2.76 ND 4.49 ± 4.34 ND 3.99 ± 6.08 ND 4.83 ± 7.53 ND 16.76 ± 10.91 ND 17.01 ± 10.52 ND 4.68 ± 5.66 ND 9.26 ± 1.61 ND 2.25 ± 0.94 ND 3.25 ± 0.60 ND 6.53 ± 10.03 ND 6.43 ± 10.11 ND 4.93 ± 0.59 ND 5.24 ± 5.65 ND 4.89 ± 1.96 108.8 2.41 ± 3.63 ND 3.73 ± 0.23 ND 6.13 ± 0.78 ND 5.03 ± 6.50 ND 9.40 ± 10.48 ND
0.59 ± 0.51 ND 0.71 ± 0.77 ND 0.28 ± 0.42 ND 0.09 ± 0.16 ND 1.18 ± 1.94 ND 0.42 ± 0.27 ND 0.46 ± 0.57 ND 0.30 ± 0.27 ND 1.82 ± 0.58 ND 1.87 ± 0.47 ND 1.17 ± 1.17 ND 0.36 ± 0.42 NQ 3.92 ± 4.40 ND 1.48 ± 1.50 ND 1.25 ± 1.31 ND 0.78 ± 0.44 ND 0.90 ± 0.45 ND 0.88 ± 0.45 ND 3.50 ± 0.81 ND 2.80 ± 1.74 ND 1.63 ± 1.37 ND 0.52 ± 0.05 ND 0.10 ± 0.17 ND 0.88 ± 0.71 ND 0.45 ± 0.58 ND 0.56 ± 0.35 ND 0.13 ± 0.15 ND 0.75 ± 0.89 ND 1.99 ± 2.48 ND 5.58 ± 1.26 ND 0.83 ± 1.02 ND 1.01 ± 0.88 ND
0.33 ± 0.58 ND 0.18 ± 0.17 ND 0.51 ± 0.34 ND 0.90 ± 1.41 ND 0.12 ± 0.12 ND 0.54 ± 0.15 ND 0.33 ± 0.28 ND 0.07 ± 0.08 ND 1.18 ± 0.76 ND 1.23 ± 0.76 ND 2.07 ± 3.06 ND 1.91 ± 2.07 ND 4.40 ± 1.97 ND 2.35 ± 2.92 ND 0.40 ± 0.48 ND 0.33 ± 0.36 ND 1.36 ± 0.46 ND 1.43 ± 0.58 ND 2.02 ± 1.74 ND 2.65 ± 2.03 ND 0.78 ± 0.33 ND 1.91 ± 1.49 ND 0.42 ± 0.49 ND 0.40 ± 0.46 ND 1.06 ± 0.13 ND 0.81 ± 1.40 ND 0.57 ± 0.49 NQ 0.16 ± 0.15 ND 2.80 ± 1.39 ND 3.11 ± 2.55 ND 1.27 ± 0.99 ND 1.46 ± 2.12 ND
22.50 ± 38.97 ND 58.26 ± 50.68 ND 4.34 ± 3.86 ND 27.59 ± 47.55 ND 11.67 ± 16.70 ND 6.87 ± 8.20 ND 40.26 ± 41.55 35.9 1.45 ± 2.22 ND 21.00 ± 7.00 ND 16.89 ± 10.05 ND 0.24 ± 0.39 ND 8.48 ± 6.87 163.4 52.73 ± 63.45 ND 19.60 ± 21.14 ND 7.00 ± 11.26 ND 5.05 ± 7.76 ND 5.83 ± 8.16 ND 6.23 ± 7.82 ND 44.27 ± 13.53 ND 33.90 ± 29.78 ND 79.91 ± 58.83 ND 15.17 ± 22.89 ND 35.74 ± 42.25 ND 3.92 ± 6.48 ND 15.25 ± 19.66 ND 2.70 ± 2.31 ND 70.60 ± 117.50 ND 55.90 ± 49.70 ND 21.00 ± 35.55 ND 68.19 ± 5.46 ND 136.95 ± 77.22 ND 109.67 ± 112.47 ND
57.17 ± 49.69 ND 27.62 ± 47.81 ND 0.21 ± 0.19 ND 50.68 ± 58.74 ND 51.46 ± 85.36 ND 5.33 ± 8.13 ND 1.48 ± 2.19 ND 25.05 ± 21.69 ND 3.00 ± 4.36 ND 3.03 ± 4.32 ND 25.36 ± 43.51 32.9 19.54 ± 21.88 ND 60.37 ± 29.65 ND 30.43 ± 22.16 ND 0.29 ± 0.46 ND 0.24 ± 0.38 ND 0.75 ± 0.68 ND 0.76 ± 0.68 ND 62.17 ± 43.87 ND 30.42 ± 29.21 ND 50.00 ± 68.05 ND 16.80 ± 29.10 ND 7.75 ± 12.53 ND 7.72 ± 12.55 ND 7.73 ± 0.92 ND 14.07 ± 3.29 ND 28.51 ± 38.67 ND 27.62 ± 47.54 ND 90.77 ± 69.92 ND 37.05 ± 42.87 ND 61.99 ± 54.27 ND 116.92 ± 95.95 ND
ND: inferior to levels of detection; NQ: inferior to levels of quantification; all IFP values were inferior to levels of detection (data not shown); dietary intake of IFP was not measured.
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participants only. Since these cases were evenly distributed among treatments (each intervention arm), we can safely conclude that supplementary creatine does not contribute to HCA synthesis in humans. These results directly contrast with the recently suggested biological plausibility for the association between creatine use and risk of testicular germ cell cancer (Li et al. 2015). An apparent explanation for the inability of creatine to result in HCA formation in this study may be due to the fact that creatine supplementation did not provoke any increase in serum or urinary creatinine beyond the normal range. In fact, a number of studies have shown that creatine supplementation normally does not increase creatinine levels (Poortmans et al. 1997, 2005; Poortmans & Francaux, 1999; Kreider et al. 2003; Gualano et al. 2009, 2010; Neves et al. 2011; Lugaresi et al. 2013), although a few conflicting findings do exist (Gualano et al. 2008). For instance, we showed increased creatinine levels (although within normal range) in creatine-supplemented healthy subjects, without any effect in serum cystatin C (a surrogate of glomerular filtration rate that is influenced by creatine ingestion) (Gualano et al. 2008). It is unknown as to whether those subjects experiencing creatinine
J Physiol 593.17
increases after creatine supplementation are more prone to form HCAs. However, it is unlikely that fluctuations in creatinine levels within the physiological range induced by creatine supplementation is a major source of HCAs. In a previous study with meat eaters and vegetarians, 8-MeIQx, Iso-MeIQx and PhIP were remarkably increased in urine after the consumption of 275 g of cooked minced beef patties (containing roughly 1.1 g of creatine) that had been fried for approximately 6 min at 300ºC (Holland et al. 2005). Given the very limited number of participants who had quantifiable levels of HCAs in the present study after creatine (which, in fact, is the same number as in placebo), one may suggest that conventional creatine supplementation regimens (up to 20 g day−1 ) may be less ‘harmful’ than a 275 g grilled meat in terms of HCA formation. Further supporting the safety of creatine, supplementing pigs with up to 50 g day−1 for 5 days did not increase HCA formation upon frying of pork (Pfau et al. 2006). The safety of creatine supplementation has frequently been questioned throughout the years, with several review papers and food agencies raising the possibility that creatine could impair kidney function and/or
Serum
B
2.0
2.0
1.5
1.5 mg/dL
mg/dL
A
1.0
0.5
0.5
0.0
0.0 PL
CR
PL
Acute
CR
PL
Chronic
CR
PL
Acute
Urine
C
CR Chronic
D
300
300
200
200
mg/12h
mg/12h
1.0
100
100
0
0 PL
CR Acute
PL
CR Chronic
PL
CR Acute
PL
CR Chronic
Figure 4. Blood and urinary creatinine after creatine or placebo supplementation in phases I (A and C) and II (B and D) Results expressed as individual data and mean ± SD. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Creatine and heterocyclic amines
result in formation of genotoxic HCAs (Benzi, 2000; Terjung et al. 2000; Wyss & Kaddurah-Daouk, 2000; Agence Franc¸aise de S´ecurit´e Sanitaire et Alimentaire, 2001; Brudnak, 2004; Yoshizumi & Tsourounis, 2004). However, a number of well-controlled studies have refuted the putative nephrotoxic effect of creatine in a variety of populations, including athletes (Poortmans & Francaux, 1999; Kreider et al. 2003), recreationally trained subjects (Poortmans et al. 1997, 2005; Lugaresi et al. 2013), sedentary individuals (Gualano et al. 2008), elderly people (Neves et al. 2011; Gualano et al. 2014), patients with pre-existing chronic kidney disease (Gualano et al. 2010), and children (Hayashi et al. 2014). The safety variables assessed in this study add to this solid literature clearly showing that creatine supplementation, regardless of protocol length and dose, does not affect conventional laboratory variables. More importantly, this study provides novel evidence that creatine supplementation does not elicit HCA formation in humans, contradicting empirical concerns that this dietary supplement could be carcinogenic by raising genotoxic HCAs. This study is not without limitations. Firstly, the conclusions must be confined to the supplementation regimens tested in this study. The abusive use of creatine supplements over the recommended dose (i.e. up to 20 g day−1 ) may not be free of risks. Secondly, in an attempt to distinguish the effect of creatine supplementation from that of diet, creatine supplementation was given to subjects who were advised to refrain from food preparations that could potentially increase HCAs. Therefore, one cannot rule out the possible additive effect of creatine supplementation along with other HCA-rich foods (e.g. grilled meats) on HCA formation in the long term. Thirdly, the HPLC–MS/MS method used in the current study was sufficiently robust to quantify HCAs within the picogram range, with LODs ranging from 10 to 20 pg ml−1 . Thus, slight increases in HCA below this level were not detectable in this study, although the physiological significance of such minor changes would likely be trivial. Finally, four selected HCAs were evaluated in this study due to their well-known relationship with cancer. Nonetheless, there are other less studied HCAs (e.g. 2-amino-3-methyl-imidazo[4,5-f]-quinoline) that could potentially be formed through creatinine and, at least in theory, affected by creatine intake (Holland et al. 2005). Further studies should track a larger number of HCAs in order to advance the knowledge on the effects of creatine supplementation on HCA formation. In conclusion, this study provides compelling evidence that both low and high doses of creatine supplementation, given either acutely or chronically, did not cause increases in the genotoxic HCAs 4,8-DiMeIQx, 8-MeIQx, PhIP and IFP in healthy subjects. These findings challenge the long-existing notion that creatine supplementation could C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Pfau W, Rosenvold K & Young JF (2006). Formation of mutagenic heterocyclic aromatic amines in fried pork from Duroc and Landrace pigs upon feed supplementation with creatine monohydrate. Food Chem Toxicol 44, 2086–2091. Poortmans JR, Auquier H, Renaut V, Durussel A, Saugy M & Brisson GR (1997). Effect of short-term creatine supplementation on renal responses in men. Eur J Appl Physiol Occup Physiol 76, 566–567. Poortmans JR & Francaux M (1999). Long-term oral creatine supplementation does not impair renal function in healthy athletes. Med Sci Sports Exerc 31, 1108–1110. Poortmans JR, Kumps A, Duez P, Fofonka A, Carpentier A & Francaux M (2005). Effect of oral creatine supplementation on urinary methylamine, formaldehyde, and formate. Med Sci Sports Exerc 37, 1717–1720. Robbana-Barnat S, Rabache M, Rialland E & Fradin J (1996). Heterocyclic amines: occurrence and prevention in cooked food. Environ Health Perspect 104, 280–288. Skog K (1993). Cooking procedures and food mutagens: a literature review. Food Chem Toxicol 31, 655–675. Skog KI, Johansson MA & Jagerstad MI (1998). Carcinogenic heterocyclic amines in model systems and cooked foods: a review on formation, occurrence and intake. Food Chem Toxicol 36, 879–896. Sugimura T, Wakabayashi K, Nakagama H & Nagao M (2004). Heterocyclic amines: Mutagens/carcinogens produced during cooking of meat and fish. Cancer Sci 95, 290–299. Terjung RL, Clarkson P, Eichner ER, Greenhaff PL, Hespel PJ, Israel RG, Kraemer WJ, Meyer RA, Spriet LL, Tarnopolsky MA, Wagenmakers AJ & Williams MH (2000). American College of Sports Medicine roundtable. The physiological and health effects of oral creatine supplementation. Med Sci Sports Exerc 32, 706–717. Vikse R & Joner PE (1993). Mutagenicity, creatine and nutrient contents of pan fried meat from various animal species. Acta Vet Scand 34, 363–370. Wyss M & Kaddurah-Daouk R (2000). Creatine and creatinine metabolism. Physiol Rev 80, 1107–1213. Yanagisawa H, Manabe S, Kitagawa Y, Ishikawa S, Nakajima K & Wada O (1986). Presence of 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx) in dialysate from patients with uremia. Biochem Biophys Res Commun 138, 1084–1089. Yoshizumi WM & Tsourounis C (2004). Effects of creatine supplementation on renal function. J Herb Pharmacother 4, 1–7.
Additional information Competing interests The authors declare no competing interests related to this study. Author contributions Conception and design of the experiments: J.R.P., F.A.D., G.G.A., B.G.; collection, assembly, analysis and interpretation of data: R.T.S.P., F.A.D., E.P., M.Y.S., A.L.F., I.H.M., W.S.D., A.C.S., M.A.R.S., G.G.A., H.R., B.G.; drafting the article or revising it critically for important intellectual content: R.T.S.P., A.C., H.R., G.G.A., J.R.P., B.G. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Funding
Acknowledgements
This study was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de S˜ao Paulo (FAPESP). B.G. and H.R. are supported by Conselho Nacional de Pesquisa e Tecnologia (CNPq).
The authors are highly grateful to Prof. Arkadiusz Szterk, who kindly donated the reagents for PhIP analysis, and Dr Bryan Saunder, who carefully proofread the manuscript.
Translational perspective Creatine has been anecdotally associated with a number of adverse events, including cancer. In this regard, a recent case-control study suggested increased risk of testicular germ cell cancer (Li et al. 2015) in users of muscle-building supplements, including creatine. Despite serious limitations found in this study, such as lack of control for concomitant use of banned sports-enhancing substances, creatine has made the highlights as ‘guilty as charged’. The biological plausibility underlying creatine-induced cancer lies in the fact that creatine and its byproduct creatinine are possible precursors of heterocyclic amines (HCAs), a class of potent carcinogenic compounds. Surprisingly, the role of dietary creatine in the increased formation of HCAs has been overlooked so far and never tested before. In the present study, we used a newly developed HPLC–MS/MS method for showing that creatine supplemented at different dosages (low and high) is innocuous in terms of HCA formation, either acutely (1 day) or chronically (30 days). This novel finding contradicts unattested growing rumours that creatine could be potentially carcinogenic and strengthens the scientifically driven notion that this dietary supplement is safe, warranting its widespread application, from sports to clinical settings.
C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
