The essential amino acid L-methionine is a potential compound in the prophylaxis of recurrent or relapsing urinary tract infection due to acidification of urine. As an intermediate of L-methionine metabolism, homocysteine is formed. The objective was to study the metabolism of L-methionine and homocysteine, and to assess whether there are differences between patients with chronic urinary tract infection and healthy control subjects.
A randomized placebo-controlled double-blind intervention study with cross-over design.
Department of Nutritional Physiology, Institute of Nutrition in cooperation with the Department of Internal Medicine III, Friedrich Schiller University of Jena, Germany.
Eight female patients with chronic urinary tract infection and 12 healthy women (controls).
After a methionine-loading test, the volunteers received 500 mg L-methionine or a placebo three times daily for 4 weeks.
Main outcome measures:
Serum and urinary concentrations of methionine, homocysteine, cystathionine, cystine, serine, glycine and serum concentrations of vitamin B12, B6 and the state of folate.
Homocysteine plasma concentrations increased from 9.4±2.7 μmol/l (patients) and 8.9±1.8 μmol/l (controls) in the placebo period to 11.2±4.1 μmol/l (P=0.031) and 11.0±2.3 μmol/l (P=0.000), respectively, during L-methionine supplementation. There were significant increases in serum methionine (53.6±22.0 μmol/l; P=0.003; n=20) and cystathionine (0.62±0.30 μmol/l; P=0.000; n=20) concentrations compared with the placebo period (33.0±12.0 and 0.30±0.10 μmol/l; n=20). Simultaneously, renal excretion of methionine and homocysteine was significantly higher during L-methionine intake.
Despite an adequate vitamin status, the supplementation of 1500 mg of L-methionine daily significantly increases homocysteine plasma concentrations by an average of 2.0 μmol/l in patients and in control subjects. An optimal vitamin supplementation, especially with folate, might prevent such an increase.
Urinary tract infection is the most common disease caused by microorganisms. However, women suffer more often from this disease than men. Especially recurrent or relapsing infections are important because they might lead to renal insufficiency. Treatment with antibiotics may be associated with the risk of developing resistance. One alternative to antibiotic drug administration is provided by the essential amino acid L-methionine (in short, methionine). The beneficial effect of methionine is due to an excretion of acidic valences with urine (Wilms, 1984) and a decrease in bacterial cytoadherence (Fünfstück et al, 1997).
The metabolism of methionine includes activation to S-adenosylmethionine (SAM), the most important donor of methyl groups. After demethylation to S-adenosylhomocysteine (SAH) and hydrolytic splitting homocysteine is formed. Elevated plasma concentrations of this cytotoxic amino acid are known to be an independent risk factor for vascular diseases (Hankey & Eikelboom, 1999). Several mechanisms have been discussed, including the production of reactive oxygen species, the binding of the endothelium-derived relaxing factor nitric oxide, the production of homocysteinylated proteins, and the inhibition of transmethylation reactions through the accumulation of its precursor SAH (Perna et al, 2003).
To avoid an overload of homocysteine and its pathophysiological consequences, homocysteine has to be metabolised quickly. Two metabolic pathways are known: remethylation and transsulphuration (Figure 1). The remethylation of homocysteine into methionine requires 5-methyltetrahydrofolate for methyl group donation and cobalamine as coenzyme. 5-Methyltetrahydrofolate is formed from serine. Tetrahydrofolate, FAD and pyridoxalphosphate act as coenzymes. Serine, itself, is generated by glycine. Alternatively, betaine may act as methyl group donor. The second pathway results in the formation of cysteine via cystathionine. Cysteine plays an important role as a precursor of glutathione and taurine. This transsulphuration pathway requires serine as cofactor and pyridoxalphosphate as coenzyme.
Owing to the fact that elevated plasma total homocysteine is a causal risk factor for cardiovascular disease (Ueland et al, 2000), this study focused on the effect of a 4-week intake of methionine on the metabolism of this amino acid and the intermediate homocysteine. Since there are different factors involved in the metabolism of methionine and homocysteine, this study additionally concentrated on the levels of certain amino acids (cystathionine, cystine, serine, glycine) and the individual vitamin supply.
Subjects and methods
Two groups of volunteers were enrolled in this study: 11 female patients with chronic urinary tract infection, aged 26–69 y, and 12 healthy women, aged 25–61 y. All volunteers were carefully informed of the purpose, course, and possible risks of the study before they gave their written consent to participate.
The diagnosis of chronic urinary tract infection was established on the basis of clinical history as well as clinical, laboratory, radiological and sonographic findings. The decision for the prophylaxis was made on the basis of the number of infection episodes per year, with three or more episodes annually as criterion. In all patients, vesicoureteral reflux and an obstruction due to lithiasis or other causes were ruled out. Patients with a glomerulonephritis, gynaecological diseases, or immunocompromised condition were excluded. Further exclusion criteria were liver insufficiency, metabolic acidose, diabetes mellitus and lactose intolerance. None of the participants received vitamin supplements prior to the study or during the two periods.
To exclude possible hyperhomocysteinemic or hyperuricaemic persons as well as patients suffering from a decline in renal function, a screening for plasma homocysteine, serum uric acid and serum creatinine was performed.
The study protocol was approved by the Ethics Committee of the Friedrich Schiller University of Jena. The study protocol adhered to the principles of GCP (Good Clinical Practice).
At the beginning, a methionine-loading test was performed: after a fasting period of 8–10 h, blood was collected from each volunteer. In all, 100 mg methionine per kg body weight were administered orally in 200 ml tea, and blood samples were drawn again 4 h afterwards. A wash-out period of a minimum of 3 days followed the loading test.
The study population was randomly divided into two groups. In a double-blind, cross-over fashion, participants received a supplement of 500 mg methionine or placebo (lactose) three times daily and were advised to take it in an 8-h rhythm for 4 weeks. Afterwards, the groups were reversed, that is, the group that was first administered placebo now received methionine, and vice versa. Before starting supplementation (baseline) as well as at the end of each period, blood samples were taken from fasting subjects and 24-h urine was collected.
For the analysis of homocysteine, blood was collected in tubes containing EDTA. These samples were immediately centrifuged (3500 r.p.m.; 10 min) and plasma was stored at −80°C until analysis. Blood for analysis of pyridoxal-5-phosphate was incubated on ice for coagulation, centrifuged (3500 r.p.m.; 10 min), and serum was stored at −80°C until further use. For the analysis of red blood cell folate (RBC-folate), 50 μl of EDTA-blood was added to 1 ml of reconstituted ascorbic acid, carefully mixed and incubated for 30 min at room temperature in the absence of light. Afterwards, samples were stored at −80°C until analysis. For other parameters, blood was collected in serum tubes, centrifuged (3500 r.p.m.; 10 min), and serum was stored at −80°C.
After determination and documentation of 24-h volume, an aliquot of each urine sample was drawn in monovettes and stored at −80°C until analysis.
Homocysteine was determined using an RP-HPLC (Shimadzu, LC-10, Kyoto, Japan) with fluorescence-detection according to the method described by Vester and Rasmussen (1991). In brief, a specific amount of plasma was mixed with internal standard solution. Tri-n-buthylphosphine in dimethylformamide (v:v, 1:10) was added and the reduction of protein-bound and disulphide-bound homocysteine was allowed to proceed for 30 min at 4°C, resulting in the determination of the total amount of homocysteine (tHcy). Afterwards, perchloric acid (0.6 mol/l) was added to the samples, mixed and incubated at room temperature for 10 min, then centrifuged at 13000 r.p.m. for 12 min to separate plasma proteins. A specific amount of the supernatant was then mixed with borate buffer (2 mol/l) and the derivatisation agent SBD-F (7-fluorobenzo-2-oxa-1,3-diazol-4-sulphonic acid) was added. The mixture was incubated for 60 min at 60°C. After cooling, the samples were transferred into vials. In all, 10 μl was used for each injection.
Probe processing for the analysis of amino acids in serum and urine included precipitation of protein with 10% sulphosalicylic acid and centrifugation. The supernatant was mixed with dilution buffer (pH 2.2.) and with 10% trifluoracetic acid. Amino acids were separated and detected using the amino-acid analyser LC-3000 (Eppendorf-Biotronik, Germany) with postcolumn derivatisation with ninhydrine. Cystathionine concentrations in serum were measured using the stable isotope dilution method by Stabler et al (1993) in a modified form and a GC-MS (Hewlett Packard, Waldbronn, Germany) assay.
Determination of pyridoxal-5-phosphate was performed using an analytical test kit (IMMUN DIAGNOSTIC GmbH, Bensheim, Germany) and RP-HPLC (Shimadzu, LC-10, Kyoto, Japan) with fluorescence detection. Folate, RBC-folate and cobalamine were analysed using a competitive immunoassay with direct chemiluminescent technology (ADVIA® Centaur™, ACS:180® Systems, Bayer Vital GmbH, Fernwald, Germany).
Data analysis was performed using the statistical software package SPSS for Windows Vs. 10.0. For statistical analysis of effects of the methionine-loading test, post-test values were compared with baseline values by a paired t-test. The paired t-test was also used to compare final values of both supplementation periods with each other. For comparison between groups, t-test was used. Data are reported as mean±s.d. (standard deviation) and P<0.05 indicates significant differences.
After the screening measurement of plasma homocysteine, uric acid and serum creatinine, three patients had to be excluded from the study: two patients had uric acid concentrations above the reference range of 140–340 μmol/l (360 and 422 μmol/l), and in one patient, two exclusion criteria exceeded the reference ranges (uric acid 465 μmol/l, serum creatinine 120 μmol/l (reference: 44–96 μmol/l)).
The two study groups (patients and controls) did not show any significant differences in age, body weight and BMI (Table 1).
History of urinary tract infections
At the time of the study, all patients had suffered from recurrent urinary tract infections for 9 y (median) with 3.4±0.9 infection episodes per year. Of controls, 41.7% reported a maximum of one urinary tract infection per year, while 58.3% had never suffered infection.
Plasma total homocysteine
At the beginning of the study, patients and controls had similar plasma tHcy concentrations of 8.9±3.1 and 9.1±1.8 μmol/l, respectively (NS). At 4 h after oral methionine loading, plasma tHcy increased significantly to 29.1±9.0 μmol/l in patients, and 30.9±9.0 μmol/l in controls (P<0.001).
In both groups, plasma tHcy concentrations increased during methionine supplementation to 11.2±4.1 μmol/l (patients; P<0.05) and 11.0±2.3 μmol/l (controls; P<0.001). In the placebo period, no significant changes of tHcy were determined (Figure 2).
Because the main parameter tHcy showed no significant differences between the two study groups, patients and controls were pooled for analysis of the following parameters.
Free serum amino acids
The serum concentrations of cystathionine, an intermediate in the transsulphuration pathway, significantly increased due to the methionine load from 0.24±0.06 to 1.77±0.76 μmol/l (P<0.001). Serine and glycine levels significantly decreased. Branched-chain and cyclic amino acids also showed a significant reduction (Figure 3).
Supplementation of 1.5 g methionine per day for 4 weeks significantly increased the serum methionine concentration from 33.0±12.0 μmol/l in the placebo period to 53.6±22.0 μmol/l (P<0.01). Furthermore, serum cystathionine concentrations showed a significant increase in the methionine period (P<0.01). Concerning the other amino acids, no differences were observed between methionine and placebo supplementation (Table 2).
A tendency towards higher serum folate concentrations during placebo intake compared with methionine intake (10.5±3.9 vs 8.9±3.1 μg/l; P=0.093) was measured. The status of RBC-folate did not significantly differ between placebo and methionine supplementation (251.8±45.2 and 237.4±59.0 μg/l).
Vitamin B6 status did not change in the course of the study, with 13.2±6.9 ng/ml after methionine and 14.2±8.9 ng/ml after placebo intake (NS). There was no difference in serum vitamin B12 concentrations between the methionine (274.1±70.0 pmol/l) and the placebo (273.9±68.5 pmol/l) period.
Serum folate levels showed a significant inverse correlation with plasma tHcy (r=−0.655, methionine period and r=−0.572, placebo period; P<0.01). However, there were no correlations between RBC-folate, vitamin B6 or B12 and plasma tHcy.
Parameters measured in urine
Methionine supplementation significantly increased renal homocysteine excretion from 6.2±2.7 μmol/l in the placebo period to 8.5±4.6 μmol/l (P<0.05). Renal homocysteine excretion at placebo administration did not differ from baseline values (6.1±2.9 μmol/l).
Excretion of methionine significantly increased during the supplementation period to 53.2±27.7 μmol/l compared to the placebo level of 32.9±23.4 μmol/l (P<0.05). Baseline cystathionine excretion of 38.5±21.4 μmol/l significantly increased during methionine supplementation to 57.0±36.8 μmol/l (P<0.05). However, there was no difference compared to placebo administration (45.3±28.7 μmol/l; P=0.191). The excretion of serine, glycine and cystine was unaffected by the intervention (Table 3).
In the main parameter plasma tHcy, no differences were identified between patients with history of chronic urinary tract infection and healthy women. Hence, according to the study protocol, data from both groups were pooled for interpretation.
Plasma tHcy concentrations are affected by genetic, nutritional and lifestyle factors. In the general population, folate status is the most important determinant of plasma tHcy (Nygård et al, 1998; Houcher et al, 2003). Despite adequate serum concentrations of folate, vitamin B12 and pyridoxal-5-phosphate, in the present study a moderate increase in daily methionine intake over 4 weeks resulted in a significant increase in plasma tHcy concentrations compared to the intake of a placebo (by about 20%). Simultaneously, the excretion amount of homocysteine in urine increased by about 36%. To the best of our knowledge, this is the first study that has shown a substantial excretion of homocysteine due to an elevated methionine intake. This might represent a compensatory mechanism to prevent homocysteine accumulation in the body. In healthy subjects, the amount of renal homocysteine excretion seems to be dependent on methionine intake and might correlate with blood tHcy concentrations.
However, this observation stands in contrast to other studies. In a cross-over intervention trial by Ward et al (2000), increased dietary methionine intake (+83%) over 7 days showed no changes in plasma tHcy. Additionally, Haulrik et al (2002) did not find an increase in plasma tHcy after 3 and 6 months of intervention with a protein-rich (22% of total energy) diet containing 2.7 g methionine per day in 23 obese subjects, compared with a low-protein (12% of total energy), low-methionine diet (1.4 g methionine/day). These contrasting findings may be the result of the differing type of methionine intake (matrix effect): most of the studies investigating the relation between methionine intake and plasma tHcy concentration realised the increased methionine intake via an increased intake of animal protein-rich food. Supplementation of isolated methionine in single doses of 25, 50 and 75 mg/kg/day (+2.04, +4.08 and +6.14 g/day, respectively) for 1 week at each doses was administered in a study by Ward et al (2001). Their results showed no significant increase in plasma tHcy at levels of 25 and 50 mg of methionine/kg/day, although plasma tHcy concentrations appeared to rise by 5.91 μmol/l (baseline: 7.46 μmol/l) when 50 mg/kg/day were supplemented. The highest level of 75 mg of methionine/kg/day resulted in a significant increase in plasma tHcy concentration of 10.59 μmol/l. Although not significant, even the lowest level of methionine intake, which is comparable with our supplementation, showed a 15% increase (1.1 μmol/l) in plasma tHcy concentrations. The absence of significance may be due to the small number of subjects (n=6), the short intervention time (1 week), and the different supplementation regime (the entire methionine doses administered once daily).
Concurrent with the increase in plasma tHcy in the methionine period, serum and urine concentrations of methionine rose significantly. However, methionine generated in the course of remethylation cannot be differentiated from the supplemented amounts of methionine that had not yet been metabolised. Balance studies in healthy men showed that circa 53% of homocysteine was transformed into cystathionine in each cycle (Mudd & Poole, 1975). Thus, an increase of methionine intake would favour the transsulphuration rate into cystathionine, and precisely this was found in the present study. However, concentrations of serum cystine, which is the oxidised form of cysteine, did not differ from placebo values. This could possibly be associated with an inhibition of cystathionine-γ-lyase by cysteine (Yamaguchi, 1990). Furthermore, due to the fact that cysteine is a highly toxic amino acid, very low cysteine tissue levels had to be maintained (Yamaguchi, 1990) and effects of the methionine supplementation could not be seen.
With regard to the role of vitamins in the metabolism of methionine, significant inverse relations between at least one of the vitamins involved (folate, B12 and B6) and plasma tHcy have often been found (Andersson et al, 1992; Ubbink et al, 1998; Pavia et al, 2000; Lim & Heo, 2002; Houcher et al, 2003; Huang et al, 2003; Samman et al, 2003). Laboratory indices of folate (serum folate and RBC-folate), vitamin B12 and vitamin B6 showed an adequate supply with these vitamins. In persons not using multivitamin supplements, higher intakes of vegetables and fruits correlated positively with serum folate levels (Kato et al, 1999). The importance of a sufficient folate supply becomes apparent through the significant inverse relation between plasma tHcy and serum folate concentrations. Thus, it can be assumed that an additional vitamin intake and especially the supplementation of folate might prevent the observed increase in plasma tHcy. The beneficial effect of vitamin supplementation in the prevention of hyperhomocysteinaemia has already been shown several times (Ubbink et al, 1994; Miller et al, 1999; Van Guldener et al, 1999; Riddell et al, 2000; Cafolla et al, 2002; Tapola et al, 2004).
In contrast to a moderate daily methionine intake, the loading test represents a more drastic intervention. The methionine-loading test is routinely used to identify persons with genetically impaired homocysteine metabolism, despite normal fasting levels (Andersson et al, 1990). Following oral methionine loading, plasma tHcy concentrations showed a three-fold increase in both study groups. This seems to reflect physiologically nonpathogenic conditions, as our data are consistent with the results of others. Investigating acute impairment of flow-mediated endothelium-dependent vasodilatation, Bellamy et al (1998) observed a three-fold increase in plasma tHcy concentrations after an oral methionine-loading test in 24 healthy subjects. Candito et al (1997) measured fasting plasma tHcy concentrations of 9.3±2.3 μmol/l and postload tHcy concentrations of 29.2±5.5 μmol/l in 20 healthy volunteers. Similar preload and postload tHcy concentrations were reported by Andersson et al (1990) in six individuals before, as well as after a period of excessive methionine intake.
Regarding changes in serum-free amino acids, a 25-fold increase of free methionine was measured 4 h after the oral administration of 100 mg/kg body weight; similar increases were measured by Andersson et al (1990). The rapidly occurring serum methionine overload has to be compensated for through intracellular metabolism or renal excretion. Serum concentrations of free serine and glycine significantly decreased after acute methionine loading. This points to an increased intracellular demand for these amino acids for homocysteine catabolism. The observed significant increase in serum cystathionine concentration represents its intracellular formation in the course of homocysteine catabolism via transsulphuration. However, a corresponding increase in the transsulphuration end product cystine in serum has not been measured.
Changes in concentrations of other free serum amino acids as a consequence of the nonphysiological methionine intake were anticipated. Therefore, a screening for free serum amino acids was performed, showing a significant decline in serum concentrations of branched-chain and cyclic amino acids. To our knowledge, none of the studies that included a methionine-loading test investigated its effects on free serum amino acids. However, Abe et al (1999) reported a significant decrease in branched-chain amino acids and phenylalanine after administration of a mixture of 0.333 g of DL-methionine and 0.111 g of L-lysine-HCl/kg of initial body weight in calves. Thus, methionine loading induces an amino acid imbalance. This imbalance might also be the reason for the dizziness that was reported by some subjects in our study. Krupková-Meixnerová et al (2002), in their evaluation of adverse effects and safety of the methionine-loading test, also singled out dizziness as the most frequent adverse reaction. Their data showed that dizziness was not caused by impaired cerebral blood flow. Therefore, further clarification in terms of the methionine-loading test and its acute physiological consequences is required.
In conclusion, the present study shows that an additional intake of 1.5 g of isolated methionine daily for 4 weeks distinctly influences the metabolism of methionine, resulting in a significant increase in serum methionine itself, in homocysteine, and in its transsulphuration metabolite cystathionine. Furthermore, renal excretion of methionine and homocysteine significantly increased. The Hordaland Homocysteine Study revealed plasma total homocysteine as a strong predictor of both cardiovascular and noncardiovascular mortality (Vollset et al, 2001). Thus, elevations of homocysteine plasma concentrations should generally be prevented. In view of the potiential increased risk for the development of atherosclerosis, special attention should be directed at the vitamin supply of patients, and folate supplementation, in particular, is advisable.
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Guarantor: G Jahreis.
Contributors: BD: conception, design, conduct of the study, analysis, statistics, data interpretation, writing the manuscript; RF: conception, design, medical attendance; MB: analysis, data interpretation; RS: analysis, data interpretation, statistics; JG: medical attendance; GJ: conception, design, data interpretation.
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Ditscheid, B., Fünfstück, R., Busch, M. et al. Effect of L-methionine supplementation on plasma homocysteine and other free amino acids: a placebo-controlled double-blind cross-over study. Eur J Clin Nutr 59, 768–775 (2005). https://doi.org/10.1038/sj.ejcn.1602138
- serum amino acids
- methionine-loading test
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