Crohn's disease (CD) is a chronic relapsing inflammatory disease affecting the lower end of the ileum and often involving the colon and other parts of the gastrointestinal tract. The development of this disease is known to be influenced by both environmental factors and genetic predisposition. The most important susceptibility gene for CD was the CARD15 (NOD2) gene on chromosome 16 (Hampe et al. Reference Hampe, Cuthbert and Croucher2001; Hugot et al. Reference Hugot, Chamaillard and Zouali2001; Ogura et al. Reference Ogura, Bonen and Inohara2001). Three disease-associated variants, the R702W, G908R and the 1007finsC, have been found frequently in different populations.
l-Carnitine plays an essential role in mitochondrial β-oxidation of fatty acids, since it is a cofactor for shuttling long-chain fatty acids across the inner mitochondrial membrane (Fritz & Yue, Reference Fritz and Yue1963; Bremer, Reference Bremer1983). Moreover, it is also capable of forming esters with several medium-chain fatty acids and SCFA of both endogenous and exogenous origins (Melegh et al. Reference Melegh, Kerner and Bieber1987; Bieber, Reference Bieber1988). In mammals, carnitine homeostasis is maintained by endogenous synthesis, absorption from dietary sources and efficient tubular reabsorption by the kidney.
Recently Peltekova et al. (Reference Peltekova, Wintle and Rubin2004) found two polymorphisms in the carnitine/organic cation transporter gene cluster (SLC22A4 and SLC22A5, encoding OCTN1 and OCTN2, respectively), that gave rise to susceptibility for CD. As the functional variants in general can have an effect on the carnitine homeostasis, the goal of the present study was to investigate the possible influence of the SLC22A genotypic variants on the circulating carnitine ester profile in CD patients.
Materials and methods
Patients
We examined 100 patients with CD (forty-seven males, fifty-three females, mean age 37·3 years, range 18–87 years), and ninety-four carefully selected clinically healthy age-, sex-, weight- and height-matched control subjects (forty-seven males, forty-seven females, mean age 45·6 years, range 23–80 years).
Diagnosis of the disease relied upon the history of the patients, clinical symptoms, negative stool examination for bacteria and parasites; and histology results of colonoscopy. Exclusion criteria in both groups were as follows: secondary causes of small intestine and/or colonic disease, systemic diseases, any malformations, evidence of intestinal bacterial or other infection, history or evidence of any inherited metabolic disease, hepatic or renal disease, and pregnancy.
The clinical and laboratory data are results of measurements performed from sample aliquots of blood collected after an overnight fast precisely between 08.00 and 08.30 hours, both in the CD patients and in the healthy control subjects. This strict post-alimental time scheduling was introduced to prevent the diet- or fasting time-induced dynamic changes of carnitine esters in the circulation (Costa et al. Reference Costa, de Almeida, Jakobs, Poll-The and Duran1999).
Informed consent was obtained from each participant of the study and the study design was approved by the local Ethics Committee.
ESI–MS–MS analysis
In the majority of the CD patients (n 85) and in a group of the controls (n 45) we determined the acylcarnitines as butyl esters by an isotope dilution MS method using a Micromass Quattro Ultima ESI triple–quadrupole mass spectrometer (Manchester, UK). The procedure was principally the method described previously (Bene et al. Reference Bene, Komlosi, Gasztonyi, Juhasz, Tulassay and Melegh2005). Briefly, 10 μl plasma was spotted and dried on to a filter paper and free carnitine and acylcarnitines were extracted with 200 μl methanol containing internal deuterium-labelled standards (0·76 μmol/l [2H3]free carnitine, 0·04 μmol/l [2H3]propionylcarnitine, 0·04 μmol/l [2H3]octanoylcarnitine and 0·08 μmol/l [2H3]palmitoylcarnitine). After 20 min of agitation the supernatant was evaporated to dryness under nitrogen at 40°C, and subsequently derivatized in 100 μl 3 mol/l butanolic HCl for 15 min at 65°C. Samples were evaporated to dryness again under nitrogen at 40°C and redissolved in 100 μl of the mobile phase (acetonitrile–water, 80 : 20). A 10 μl aliquot of each sample was introduced at a flow rate of 100 μl/min into the ESI cone using the Waters (Milford, MA, USA) 2795 HPLC system. Free carnitine and all acylcarnitines were measured by positive precursor ion scan of m/z 85 (scan range 200–550 m/z) using the following settings: capillary voltage 2·50 kV, cone voltage 55 V and collision energy 26 eV. Total analysis time was 4 min per sample and each sample was measured in triplicate starting with the injection step, and the results are the means of the three determinations.
Our mass spectrometry facility is in the International Newborn Screening Quality Assurance Program organized by the Center for Disease Control and Prevention, USA (http://www2.cdc.gov/nceh/NewbornScreening/). This activity involves controlled measurements of selected acylcarnitines in two quality control regimens and four proficiency tests per year.
Statistics
For statistics the Student's t-test for unpaired samples was used. The values are expressed as means and their standard errors, in three decimals for the carnitine esters with respect to the low levels in the case of the long-chain carnitine esters.
DNA sequencing
To detect the OCTN and NOD2 variants we carried out direct sequencing by means of a BigDye Terminator labelling system with ABI 3100 automatic sequencer (Foster City, CA, USA) with a slightly modified method described previously. Genomic DNA from index patients and controls was isolated from peripheral blood using a standard desalting procedure. For the PCR amplification as well as for the sequencing the following primers were used: for R702W the forward primer was 5′-GAGCCGCACAACCTTCAGATC-3′, and the reverse primer was 5′-ACTTGAGGTGCCCAACATTCAG-3′; for G908R the forward primer was 5′-GTTCATGTCTAGAACACATATCAGG-3′, and the reverse primer was 5′-GTTCAAAGACCTTCAGAACTGG-3′; for 1007finsC the forward primer was 5′-CCTTGAAGCTCACCATTGTATC-3′, and the reverse primer was 5′-GATCCTCAAAATTCTGCCATTC-3′; for SLC22A4 C1672T the forward primer was 5′-AGAGAGTCCTCCTATCTGATTG-3′, and the reverse primer was 5′-TCCTAGCTATTCTTCCATGC-3′; for SLC22A5 G-207C the forward primer was 5′-AGTCCCGCTGCCTTCCTAAG-3′, and the reverse primer was 5′-GTCACCTCGTCGTAGTCCCG-3′. The PCR was carried out in a final volume of 50 μl containing 200 μm of each dNTP, 2 units of Taq polymerase, 5 μl reaction buffer (100 mm-Tris-HCl, pH 9·0; containing 500 mm-KCl, 15 mm-MgCl2), 0·2 μm of each primer and 1 μg DNA to be amplified. The amplification was performed for a total of thirty-five cycles with the use of an MJ Research (Waltham, MA, USA) PTC-200 thermal cycler. The conditions were: predenaturation for 2 min at 95°C, denaturation 30 s at 95°C, annealing 30 s at 50°C for CARD15 mutations, 54°C for SLC22A4 and 58°C for SLC22A5 variants, primer extension 30 s at 72°C, the final extension at 72°C lasted for 5 min.
For statistics the χ2 method (cross-table analyses) was used to analyse the possible associations with mutations.
Results
The allele frequencies are shown in Table 1. A total of 32 % of CD patients carried at least one NOD2 mutation compared to 12·8 % of controls (P < 0·005). With respect to the 1007finsC mutation, 17 % of CD patients were either heterozygous or homozygous compared to 4·3 % of controls (P < 0·005). Generally, the R702W and G908R were also observed in higher frequencies in patients compared to controls. There was no statistically significant difference in the prevalence of TC haplotype between the CD patients and controls (Table 1).
Table 1 Comparison of the alleles of OCTN cation transporters and NOD2/CARD15 genes in patients with Crohn's disease (n 100) and controls (n 94)
The plasma circulating carnitine ester profiles in the mixed CD population are shown in Table 2. The plasma level of free carnitine and acetyl carnitine did not differ between the CD patients and the controls. By contrast, significant decreases were found in fasting propionyl-, butyryl- and isovalerylcarnitine ester levels in CD patients as compared with the controls. There was no significant difference in the level of total short-chain carnitine esters (C2–C5 : 1) between the CD patients (17·301 (sem 0·155) μmol/l) and the healthy controls (16·966 (sem 0·146) μmol/l).
Table 2 Plasma carnitine ester profiles (in μmol/l) in the mixed group of Crohn's disease patients (n 85) and controls (n 48)
Mean values were significantly different from those of the control group: *P < 0·05.
The level of octenoylcarnitine was decreased in the healthy subjects; and the level of total medium-chain acylcarnitines (C6–C12) was 0·672 (sem 0·007) in the CD patients v. 0·627 (sem 0·007) μmol/l in the control subjects. In the long-chain acylcarnitine group the plasma levels of myristoleyl-, palmitoyl- and oleylcarnitine were significantly decreased in the healthy group. The level of total long-chain carnitine esters (C14–C18 : 1OH) was 0·612 (sem 0·006) in the patients with CD v. 0·547 (sem 0·005) μmol/l in the controls.
The level of total carnitine esters (the sum of the individual esters) was 47·255 (sem 0·236) μmol/l in the CD patients and 48·779 (sem 0·243) μmol/l in the controls.
The plasma carnitine ester profiles with different SLC22A4 1672 C and T genotypes are shown in Table 3 and with different SLC22A5 -207 G and C genotypes in Table 4. Albeit there were some trends for change for some individual esters (C4 and C5 carnitines), there were no statistically significant differences in the levels of any carnitine esters for any genetic variants when the heterozygous or the homozygous populations were compared. Similarly, TC haplotype did not modify the carnitine ester profile either in the CD group (Table 5) or in the controls (not shown).
Table 3 Comparison of the plasma carnitine ester profiles (in μmol/l) in Crohn's disease patients with different SLC22A4 C1672T genotypes
Table 4 Comparison of the plasma carnitine ester profiles (in μmol/l) in Crohn's disease patients with different SLC22A5 G-207C genotypes
Table 5 Comparison of the plasma carnitine ester profiles (μmol/l) in Crohn's disease patients with different SLC22A4 C1672T and SLC22A5 G-207C genotypes
Discussion
Several studies have reported an association between three CARD15 coding variants (R702W, G908R and 1007finsC) and CD in Caucasian populations, whereas these mutations have not been found at all in Asian patients (Inoue et al. Reference Inoue, Tamura and Kinouchi2002; Leong et al. Reference Leong, Armuzzi, Ahmad, Wong, Tse, Jewell and Sung2003). In the present study the carriage rate for the three common CD-associated CARD15 mutations was 32 % in the patients group and 12·8 % in healthy controls. The results are in concordance with previously reported rates of 30–50 % in CD and 7–20 % in controls from other European CD populations (Ahmad et al. Reference Ahmad, Armuzzi and Bunce2002; Lesage et al. Reference Lesage, Zouali and Cezard2002; Vermeire et al. Reference Vermeire, Wild and Kocher2002; Brant et al. Reference Brant, Picco and Achkar2003; Mendoza et al. Reference Mendoza, Murillo, Fernandez, Pena, Lana, Urcelay, Cruz-Santamaria, de la Concha, Diaz-Rubio and Garcia-Paredes2003; Arnott et al. Reference Arnott, Nimmo and Drummond2004; Vind et al. Reference Vind, Vieira and Hougs2005). The allele frequencies of R702W, G908R and 1007finsC mutations we have found (7·0, 3·0 and 9·5 % in patients and 4·8, 0·5 and 2·1 % in controls, respectively) are in agreement with other studies from Europe (6·7–12·9, 3·3–6·1 and 6·6–16 % in CD and 3·5–6·9, 0·6–3·0 and 1·0–4·4 % in controls) (Lesage et al. Reference Lesage, Zouali and Cezard2002; Vermeire et al. Reference Vermeire, Wild and Kocher2002; Mendoza et al. Reference Mendoza, Murillo, Fernandez, Pena, Lana, Urcelay, Cruz-Santamaria, de la Concha, Diaz-Rubio and Garcia-Paredes2003; Arnott et al. Reference Arnott, Nimmo and Drummond2004).
By resequencing five genes in the IBD5 interval, Peltekova et al. (Reference Peltekova, Wintle and Rubin2004) identified two novel polymorphisms in the SLC22A4 and SLC22A5 genes. These mutations (SLC22A4 C1672T and SLC22A5 G-207C) appeared to form a two-allele risk haplotype (OCTN-TC) for development of CD and showed significant interactions with CD-associated CARD15 mutations (Peltekova et al. Reference Peltekova, Wintle and Rubin2004). This observation has been confirmed in some studies (Newman et al. Reference Newman, Gu, Wintle, Cescon, Yazdanpanah, Liu, Peltekova, Van Oene, Amos and Siminovitch2005; Noble et al. Reference Noble, Nimmo, Drummond, Ho, Tenesa, Smith, Anderson, Arnott and Satsangi2005; Torok et al. Reference Torok, Glas and Tonenchi2005), but no association was found in others (Yamazaki et al. Reference Yamazaki, Takazoe, Tanaka, Ichimori, Saito, Iida, Onouchi, Hata and Nakamura2004; Noble et al. Reference Noble, Nimmo, Drummond, Ho, Tenesa, Smith, Anderson, Arnott and Satsangi2005; Torok et al. Reference Torok, Glas and Tonenchi2005; Vermeire et al. Reference Vermeire, Pierik and Hlavaty2005). In the present study we investigated a larger group of Hungarian patients, first in the literature, and could not detect the accumulation of either SLC22A4 and SLC22A5 susceptibility variants or the TC haplotype in the CD patients; thereby, the present findings do not support the susceptibility role of these haplotypes in the development of CD in this population.
The primary biochemical function of carnitine is its involvement in the energy production of cells (Bieber, Reference Bieber1988). Moreover, via ester formation it facilitates the removal of SCFA and medium-chain fatty acids, which accumulate as a result of normal and abnormal metabolism in the mitochondria. The circulating carnitine ester spectrum can reflect affected cellular metabolism of the SCFA, medium- and long-chain fatty acids (Rashed, Reference Rashed2001).
Very limited data are available on the carnitine status in CD; these studies are from the enzymatic radiochemical carnitine determination era. Using this approach, Demirkol et al. (Reference Demirkol, Sewell and Bohles1994) found a significant decrease of free and total carnitine concentrations in paediatric patients with CD. In another study a dramatically decreased free carnitine and significantly increased long- and short-chain acylcarnitine concentrations were observed, resulting in a net increase in total carnitine. However, in that study patients with different types of inflammatory disorders after multiple trauma or head injury were examined (Adlouni et al. Reference Adlouni, Katrib and Ferard1988). Therefore, these data are not really comparable to the findings of the present study of a mixed population of CD-affected patients. The data presented here show a complex change of the carnitine esters, and the current knowledge is not enough for the full understanding of the findings revealed.
Proinflammatory cytokines are essential in the pathogenesis of CD. Activation of NF-κB, which plays a central role in immune and inflammatory responses and is involved in the proinflammatory cytokine gene transcription, is increased in the intestinal mucosa of CD patients (Schreiber et al. Reference Schreiber, Nikolaus and Hampe1998). Butyrate inhibits the translocation of NF-κB transcription factor to the nucleus, thereby preventing the activation of proinflammatory genes (Segain et al. Reference Segain, Raingeard de la Bletiere, Bourreille, Leray, Gervois, Rosales, Ferrier, Bonnet, Blottiere and Galmiche2000; Di Sabatino et al. Reference Di Sabatino, Morera, Ciccocioppo, Cazzola, Gotti, Tinozzi, Tinozzi and Corazza2005). Thus, the observed butyrylcarnitine level can be an element of the development of inflammatory processes. However, another explanation cannot be ruled out.
The OCTN1 and OCTN2 play an essential role in the plasmalemmal carnitine uptake, thereby regulating the normal tissue/circulating carnitine balance (Tamai et al. Reference Tamai, Ohashi, Nezu, Yabuuchi, Oku, Shimane, Sai and Tsuji1998; Wu et al. Reference Wu, Prasad, Leibach and Ganapathy1998, Reference Wu, George, Huang, Wang, Conway, Leibach and Ganapathy2000). In fibroblasts with C1672T missense substitution in exon 9 of the SLC22A4 gene resulting in marked changes in OCTN1 transporter activity, and also in fibroblasts with G-207C transversion in the SLC22A5 promoter region causing OCTN2 promoter function impairment, the carnitine uptake was affected showing that these genetic variations can associate with functional consequences (Peltekova et al. Reference Peltekova, Wintle and Rubin2004). The authors suggested that these genes had a role in chronic inflammatory disorders, moreover, they supposed that these gene variants may cause disease by impairing OCTN activity or expression, reducing carnitine transport in a cell-type and disease-specific manner. Here we analysed the plasma carnitine ester profiles of the patients according to the different SLC22A4 C1672T and SLC22A5 G-207C genotypes. To our surprise, the variations of the genotypes were not associated with influenced carnitine ester profiles. In the current study we could exclude therefore any major effects of the genotype on the carnitine esters.
Acknowledgements
This work was supported by grants from the Hungarian Science Foundation (OTKA T 49 589), the Ministry of Health (ETT 497/2006) and the National Office for Research Technology of Hungary (Pazmany Peter Program, RET-II-08/2005, MEDIPOLISZ). The authors are grateful to Ilona Szántó, Judit Oksai and Edit Papp for their help in the technical management of the study.
