NDT Advance Access originally published online on October 4, 2005
Nephrology Dialysis Transplantation 2006 21(2):383-388; doi:10.1093/ndt/gfi151
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© The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Original Articles: Clinical Nephrology
An oral load of the early glycation compound lactuloselysine fails to accumulate in the serum of uraemic patients
1 Department of Nephrology, University of Heidelberg, Heidelberg, Germany, 2 Gambro Corporate Research, Hechingen, Germany and 3 Institute of Food Chemistry, Technische Universität Dresden, Dresden, Germany
Correspondence and offprint requests to: Vedat Schwenger, MD, Department of Nephrology, University of Heidelberg, Bergheimerstr. 56a, 69115 Heidelberg, Germany. Email: vedat_schwenger{at}med.uni-heidelberg.de
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Abstract |
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Background. It has been hypothesized that in renal failure, exogenous glycation compounds from food accumulate and play a major pathogenetic role when renal excretion is impaired.
Methods. To address this, a diet containing a defined amount of the lysine Amadori product (AP) lactuloselysine was used. Plasma concentrations and cumulative urinary excretion of AP were assessed in 16 healthy subjects, 12 renal failure patients and 6 continuous ambulatory peitoneal dialysis (CAPD) patients. Amadori product was measured as furosine using reverse phase high performance liquid chromatography (RP-HPLC) after acid hydrolysis.
Results. A diet low in glycation compounds significantly decreased excretion of APs in healthy subjects. In healthy individuals, ingestion of lactuloselysine bound to food proteins caused only a minor acute increase (8.24±1.11 mg/day, 2% of the administered dose) of AP excretion in the urine; in patients with renal failure not yet on dialysis, the increase in AP excretion in the urine was significantly less (4.0±0.51 mg/day) and the same was true in CAPD patients (0.21±0.09 mg/day). The plasma concentration of total APs, i.e. the sum of APs as free amino acids and residues bound to plasma proteins, did not change in any of the three groups, however.
Conclusion. Dietary APs do not accumulate in the blood even in advanced renal failure. The amount of APs measured as furosine excreted in the urine is significantly less, however, in renal failure and CAPD patients compared with healthy subjects. Although the findings exclude accumulation of lactuloselysine in renal failure, they do not generally exclude accumulation of other food-derived advanced glycation end products (AGEs).
Keywords: advanced glycation end products (AGEs); furosine; lactuloselysine; nutrition; renal failure
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Introduction |
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Non-enzymatic browning reactions, ever since they were first described by Maillard (1912), have been of great interest to food scientists, but have largely been ignored in medicine. These reaction products are the result of an interaction between the aldehyde functions of reduced sugars and the amino compounds of amino acids, peptides or proteins during heating or storage of food items [1]. It has been recognized that, as a result of analogous reactions in vivo during ageing, diabetes mellitus and renal failure, advanced glycation end products (AGEs), formed predominantly from peptide-bound lysine and arginine residues, accumulate in plasma and tissues and contribute to end organ damage [2,3]. Such AGEs are derived from endogenously formed dicarbonyls or glucose and are presumably the result of oxidative stress [4,5].
In renal failure, apart from endogenous overproduction by the intermediary metabolism, an alternative source of AGEs has to be considered, i.e. intestinal absorption and reduced renal excretion of AGE precursors or AGE products in food items. It has been claimed that the amount of ingested AGEs contained in certain food items is much higher than the total amount of endogenously formed AGEs [6]. If one assumes that a high proportion of such exogenous food-derived AGEs are absorbed and excreted via the kidneys, the contribution of AGE and Amadori products (APs) in food items to plasma AGE could even be greater in subjects with impaired renal function.
Scant information is available on digestion, absorption and elimination of glycated food proteins in humans. A diet consisting of a heated mixture of fructose and egg-white has been used to address this issue in patients with renal failure. After ingestion of the browned egg protein, increased concentrations of epitopes of unknown chemical identity were measured in the circulation by ELISA. This finding led to the hypothesis that food-derived AGEs accumulate in patients with renal failure [7]. Similar results were recently reported by Uribarri et al. [8]. The authors also reported an increase in inflammatory markers such as TNF-
and C-reactive protein during high AGE diets [9]. For uraemic patients, however, no data are available on resorption, accumulation or elimination of food-derived and chemically defined Maillard compounds.
The purpose of the present study is to assess the kinetics of the early glycation product lactuloselysine in the diet. This lysine derivative is generated in milk products due to a reaction between lactose and the 
-amino group of protein-bound lysine, and represents the quantitatively dominating Maillard compound occurring in milk-protein-containing foods [10]. In order to assess the kinetic behaviour of lactuloselysine, we measured the plasma concentration as well as the urinary excretion of APs as furosine equivalents after ingestion of a milk sample containing a standard amount of lactuloselysine. Furosine is a reaction product which is formed in definite amounts from lactuloselysine during acid hydrolysis, thus representing a widely accepted tool for the assessment of the extent of the early Maillard reaction in food and in vivo [11].
We compared healthy controls, non-dialysis-dependent patients with renal failure and CAPD patients. CAPD patients were selected because of the quasi-continuous mode of elimination. Furthermore, elimination of APs by the dialysis procedure can be reliably quantified in CAPD patients.
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Patients and methods |
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Test milk
Test milk was prepared by heating a commercial roller-dried milk powder at 70°C for 2 h, resulting in a glycated product containing 906 mg furosine per 100 g protein in the hydrolysate. The lysine content was 1153 mg per 100 g protein. Using the well-established conversion factors for the formation of furosine during acid hydrolysis of lactuloselysine [12], a lysine blockage of 76.1% was calculated. The test milk samples were prepared by reconstituting 50 g of this heated milk powder to 500 ml of milk.
Patients
Three groups were included in the study: 12 non-diabetic patients with renal failure not yet on dialysis (median measured creatinine clearance 37.5±8.2 ml/min), 6 non-diabetic CAPD patients (5 high-average transporters, 1 high transporter) and 16 healthy volunteers (the study was approved by the local ethics committee and written informed consent was obtained from each patient).
The 16 healthy volunteers were randomly allocated to four parallel protocols: 4 individuals served as controls ingesting unmodified milk, and 4 individuals received one single portion of a heated milk sample with lactuloselysine corresponding to 76 mg, 152 mg or 453 mg of furosine, respectively.
Similarly, the non-dialysed renal patients were randomly allocated either to a control group ingesting unmodified milk (n = 4) or an experimental group ingesting 453 mg furosine (n = 8).
Finally, to examine elimination kinetics on dialysis, six CAPD patients were examined before and after ingestion of lactuloselysine corresponding to 453 mg furosine. The relevant characteristics of the participants are shown in Table 1.
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Study protocol
The patients were instructed to self-assess their food habits by a dietitian. The individuals were first advised to stick to their habitual foods for 2 days. They were then asked to follow to an isocaloric diet virtually free of APs (‘AP-free diet’), consisting mainly of unheated food, i.e. no boiled, baked or roasted food items. The participants kept a dietary protocol. On the morning of day 5, participants in the test group drank one 500 ml portion of reconstituted milk, prepared as described above, containing a single dose of lactuloselysine corresponding to 453 mg furosine. The AP-free diet was discontinued on day 7, and the participants were subsequently switched back to their customary diet.
Heparinized plasma samples were drawn each morning during the entire study period. In addition, samples were drawn immediately before and every 6 h after ingestion of the test milk. Samples of 24 h urine and dialysate (in CAPD patients) were collected daily. All samples were stored frozen until analysis.
Analytical procedures
Furosine was measured after acid hydrolysis using ion-pair RP-HPLC and UV detection [13]. Conditions for hydrolysis using 6 N hydrochloric acid, resulting in a reproducible formation of furosine from lactuloselysine, have recently been described in detail [12]. In plasma hydrolysates, total furosine is formed both from the minor fraction of APs occurring as free amino acids (1%) and AP residues of plasma peptides and proteins (99%). ‘Low-molecular mass Amadori products’ (LMW-APs; APs as free amino acid or bound to peptides with molar masses below 5000 Da) were measured in the filtrate of samples after filtering 500 µl of plasma or 1000 µl of urine or spent PD dialysate, respectively, through an Ultrafree centrifugal filter (cut-off 5000 Da). ‘High molecular mass Amadori products’ (HMW-APs) were calculated as the difference in furosine values obtained between the whole sample and LMW-APs.
Statistical analysis
Data are reported as mean±SEM. The data were analyzed using the Mann–Whitney test for random samples or paired differences as appropriate. The zero hypothesis was rejected at a P-value < 0.05.
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Results |
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Cumulative urinary excretion and plasma concentrations of APs in healthy volunteers
In the first two days, before the AP-free diet was started, the baseline urinary AP excretion measured as furosine for healthy volunteers was 3.50±1.10 mg/24 h (range 0.96–9.38). Upon institution of the AP-free diet, the excretion decreased significantly to 0.71±0.11 mg/24 h (P<0.0001). After ingestion of the test milk (APs equivalent to 453 mg furosine, Figure 1) the excretion rate increased significantly to 8.24±1.11 mg/24 h (range 6.20–11.26, P<0.003). No increase was observed in individuals who ingested APs corresponding to 76 mg (1.44±0.18 mg/24 h; range 1.08–1.79) or 152 mg furosine (1.88±0.12 mg/24 h; range 1.55–2.07).
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, 453 mg) urinary excretion was significantly higher in healthy volunteers (P<0.003).At baseline, the plasma concentration of Amadori products was equivalent to 14.81±0.79 mg furosine/100 g of plasma protein and did not change significantly with intake of lactuloselysine-containing milk.
Cumulative urinary excretion and plasma concentration of APs in patients with renal failure
In the first two days, before the AP-free diet was started, the baseline urinary excretion of APs was equivalent to 1.6±0.30 mg furosine/24 h (range 0.7–3.1 mg/24 h). Upon administration of the AP-free diet, only a slight, non-significant decrease was observed in the AP excretion rate (1.4±0.24 mg furosine/24 h; range 1.2–1.9 mg/24 h). Following ingestion of the lactuloselysine-containing milk corresponding to 453 mg furosine, the excretion rate of APs increased significantly to 4.0±0.51 mg furosine/24 h (range 2.0–5.1 mg/24 h, P<0.009) (Figure 2).
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Interestingly, the urinary AP excretion rate as reflected by furosine was three times higher in healthy volunteers than in non-dialysed patients with renal failure (increase 74% vs 250%). The absolute amount of urinary AP excretion was markedly lower in patients with renal failure as compared with healthy volunteers (mean of furosine values 8.24 vs 4 mg/24 h).
Nevertheless, the plasma concentration of AP was lower than in healthy volunteers (11.0±0.28 mg furosine/100 g of plasma protein) and did not change significantly with administration of AP-containing milk (Figure 3).
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CAPD patients
In the first two days, before the AP-free diet was started, the baseline urinary furosine excretion was 0.13±0.07 mg/24 h (range 0.03–0.53). Following administration of the AP-free diet, no significant decrease was observed in the AP excretion rate (0.07±0.04 mg furosine/24 h; range 0.02–0.29 mg/24 h). After ingestion of the test milk (lactuloselysine corresponding to 453 mg furosine), the excretion rate increased to 0.21±0.09 mg furosine/24 h (range 0.02–0.77, Figure 4a) without reaching statistical significance (P = 0.06).
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In the first two days, before the AP-free diet was started, the baseline AP concentration in the CAPD fluid measured as furosine was 0.41±0.06 mg furosine/24 h (range 0.03–0.85 mg/24 h, Figure 4b). Following administration of the AP-free diet, no significant decrease was observed in the furosine values measured after acid hydrolysis in the CAPD fluid (0.33±0.09 mg/24 h; range 0.05–1.06). Conversely, after ingestion of the milk corresponding to 453 mg furosine, no measurable increase in furosine in CAPD fluid was observed (0.31±0.10 mg/24 h; range 0.16–1.03).
Interestingly, although the amount of APs excreted in the urine of CAPD patients was significantly lower compared with that in renal failure patients, and although no increase in CAPD fluids was observed, plasma concentrations did not change significantly (11.0±2.05 mg furosine/100 g of plasma protein).
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Discussion |
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The measurement of furosine as a hallmark for lysine APs is a widely accepted tool for evaluating the generation of early Maillard reaction products in food items [14]. During acid hydrolysis, furosine is formed in stoichiometric amounts from lysine-linked APs [12]. Using chromatographic techniques, it can be reliably quantified in hydrolysates. Such precisely measured concentrations of furosine in hydrolysates of plasma and urine allow one to calculate the elimination kinetics of an orally administered AP (i.e. lactuloselysine). Earlier reports yielded interesting information on handling of AGEs in uraemic patients [7], but used immunological methods which left the exact chemical nature of the AGE undefined. This consideration prompted us to adopt a method which allows specific detection and quantification of the early glycation compound that is quantitatively dominant in common food items [15]. The results concerning the kinetics of lactuloselysine in healthy individuals are in perfect agreement with earlier reports in the literature [16].
The main observation of the present study is that in patients with renal failure, either pre-endstage or endstage, a smaller proportion of the oral load of lactuloselysine is excreted in the urine. Calculating the clearance based on the amount of APs excreted in the urine may yield erroneous estimates. Amadori products predominantly occur as residues of plasma proteins, so that the total amount of furosine in acid hydrolysates of the proteinuric urine does not allow one to calculate the renal clearance of free APs.
In view of the reduced total excretion of APs in the urine, one would anticipate accumulation of this early glycation compound leading to increased plasma concentrations at baseline, particularly after an oral load. Nevertheless, the baseline concentration was not elevated, nor was there any change in the plasma concentration at different time intervals after oral ingestion of the lactuloselysine-containing milk sample. It is unlikely that this results from a failure of lactuloselysine to be absorbed, but we cannot entirely exclude this possibility. Recovery of lactuloselysine in the faeces was very low, around 1–2% of ingested amounts [16]. This does not necessarily prove, however, that all lactuloselysine was absorbed, since it may also be lost by enzymatic or bacterial degradation in the gut. We are aware of the fact that diet-derived lactuloselysine in plasma or urine is present predominantly in the free form or as a residue of di- or tri-peptides (the latter cannot be distinguished using our method). This fraction was not found to accumulate after ingestion of a diet containing this early Maillard product. We considered the possibility that after ingestion, the increase of the free form in plasma – if present in plasma at all – might be too low to be measured, but we tried to circumvent this possibility by administering a very high dose of lactuloselysine per os, which was calculated to cause a significant increase in the total amount of APs as measured via furosine.
The measured increase in urinary excretion of APs suggests that a certain amount has been absorbed in renal failure patients. To explain the failure, to see changes in the plasma concentration, one also has to consider the possibility that these predominantly protein-bound compounds (99%) may have shifted into the extravascular compartment and have been enzymatically degraded there or sequestered in deep compartments, e.g. vessel wall.
In the present study, the AP lactuloselysine, which represents an early glycation compound and is an established parameter used for analyses in food chemistry, was evaluated. While it can be accurately measured and is thought to be biologically relevant as possible precursor for AGEs [6], the present study has its limitations.
First, in healthy individuals, no more than 2% of the administered dose of lactuloselysine is excreted in the urine. Second, the compound is mainly protein-bound, so that – in contrast with the steady state concentrations – acute transient changes may not be readily detectable (although we had deliberately selected a high dose per os to minimize this risk). The increments after the oral load, if anything, were lower in patients with renal failure. Third, to explain the absence of an increase in plasma concentration despite diminished urinary excretion, one must also consider the possibility that the known deglycating enzymes which eliminate lactuloselysine from circulation are upregulated [17,18]. This hypothesis is currently under investigation. Finally, the failure to detect relevant accumulation of this specific Maillard product does not exclude the possibility that alternative Maillard products do indeed accumulate in renal failure patients.
In conclusion, we found that the total amount of APs excreted in the urine (in the peritoneal dialysis fluid in CAPD patients) was significantly lower in renal patients compared with healthy volunteers. This observation suggests that a certain degree of renal retention does in fact occur under normal circumstances. The failure of plasma concentrations to increase is compatible with the notion that the early glycation compound lactuloselysine, measured as furosine, fails to accumulate in the circulation – unless our method missed a transient early increase and rapid transit of furosine into the extraplasmatic compartment of pathogenetic relevance.
Conflict of interest statement. None declared.
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Accepted in revised form: 26. 8.05
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