Articles in PresS. Am J Physiol Renal Physiol (April 11, 2012). doi:10.1152/ajprenal.00084.2012
1
Low plasma carnosinase activity promotes carnosinemia following
2
carnosine ingestion in humans
3 4
Running title: Supplementation-induced carnosinemia in humans
5 6
Inge Everaert1, Youri Taes2, Emile De Heer3, Hans Baelde3, Ana Zutinic3, Benito Yard4, Sibylle
7
Sauerhöfer4 , Lander Vanhee1, Joris Delanghe5, Giancarlo Aldini6, Wim Derave1*
8 1
9
Department of Movement and Sport Sciences, Ghent University, Ghent, Belgium 2
10 3
11 12
4
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands
Department of Medicine V, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany
13 5
14 15
Department of Endocrinology, Ghent University Hospital, Ghent, Belgium
6
Department of Clinical Chemistry, Ghent University Hospital, Ghent, Belgium
Department of Pharmaceutical Sciences “Pietro Pratesi”, Univerity of Milan, Milan, Italy
16 17
* Contact information: Wim Derave, Department of Movement and Sports Sciences, Ghent
18
University, Watersportlaan 2, B-9000 Ghent, Belgium.
[email protected], 0032 (09) 264 63 26
19 20
Copyright © 2012 by the American Physiological Society.
21
Abstract
22
Objectives A polymorphism in the CNDP1 gene, resulting in decreased plasma carnosinase activity,
23
is associated with a reduced risk for diabetic nephropathy. Because carnosine, a natural
24
scavenger/suppressor of reactive oxygen species, AGE’s and reactive aldehydes, is readily degraded
25
in blood by the highly active carnosinase enzyme, it has been postulated that low serum carnosinase
26
activity might be advantageous to reduce diabetic complications. The aim of this study was to
27
examine whether a low carnosinase activity promotes circulating carnosine levels following
28
carnosine supplementation in humans.
29
Methods Blood and urine was sampled, in 25 healthy subjects following acute supplementation
30
with 60mg/kg BW carnosine. Pre-cooled EDTA tubes were used for blood withdrawal and plasma
31
samples were immediately deproteinized and analyzed for carnosine and beta-alanine by HPLC.
32
CNDP1 genotype, baseline plasma carnosinase activity and protein content were assessed.
33
Results Upon carnosine ingestion, 8 of the 25 subjects (= responders) displayed a measurable
34
increase in plasma carnosine up to 1h following supplementation. Subjects with no measurable
35
increment in plasma carnosine (= non-responders) had an approximately 2-fold higher plasma
36
carnosinase protein content and an approximately 1.5-fold higher activity compared to the
37
responders. The urinary carnosine recovery was 2.6-fold higher in the responders versus non-
38
responders and was negatively dependent on both the activity and protein content of the plasma
39
carnosinase enzyme.
40
Conclusion A low plasma carnosinase activity promotes the presence of circulating carnosine upon
41
an oral challenge. These data may further clarify the link between CNDP1 genotype, carnosinase
42
and diabetic nephropathy.
43
Keywords CNDP1, beta-alanine, diabetic nephropathy
44
Introduction
45
Adequate glycemic and blood pressure control are the most effective therapeutic modalities in
46
diabetic patients to delay the onset of microvascular complications (1). Nonetheless, an increasing
47
number of patients will continue to develop microvascular complications despite these therapeutic
48
measures. Ample evidence indicates that susceptibility to develop diabetic nephropathy, the most
49
common cause of renal failure in the western world, is genetically determined (23). One of the
50
genes that have recently been linked to diabetic nephropathy is carnosinase dipeptidase-1 (CNDP1),
51
encoding the serum carnosinase enzyme (16). Diabetic nephropathy is strongly associated with a
52
(CTG)n polymorphism in the CNDP1 gene, affecting serum carnosinase secretion (30). Diabetic
53
patients homozygous for (CTG)5 have a lower risk to develop diabetic nephropathy and have a
54
lower plasma carnosinase activity (16). Although the association between the CNDP1
55
polymorphism and diabetic nephropathy have been confirmed in an independent study in European
56
Americans (11), other studies did not show an association in type 1 diabetic patients (37) or showed
57
that the association in type 2 diabetic patients is sex specific (25). Inconsistent findings may be
58
explained by differences in ethnicity (22) or alternatively, by assuming that protection from diabetic
59
nephropathy afforded by (CTG)5 homozygosity in CNDP1 may be masked by additional risk
60
haplotypes (9; 22).
61
Glycation and oxidative stress (glycoxidative stress) and accelerated formation of advanced
62
glycation end-products (AGE’s) during hyperglycemia are implicated in the development of
63
diabetic complications (8). Carnosine (β-alanyl-L-histidine) is a versatile dipeptide, mainly present
64
in neuronal tissue and skeletal muscle, that has the propensity to suppress several aspects of
65
glycoxidative stress, such as inhibition of AGE formation (14), quenching of reactive aldehydes (3)
66
and suppression of oxidative stress (18). Moreover, it has been shown that carnosine inhibits
67
angiotensin converting enzyme (ACE) (15), albeit only at high concentrations. The influence of
68
carnosine on blood pressure control has thusfar not been demonstrated.
69
In humans, circulating carnosine is readily degraded by the highly active serum carnosinase
70
enzyme, which is secreted from the liver into the plasma (30). Therefore, plasma carnosine
71
concentrations in fasted subjects are in general below the detection limits of current quantitative
72
carnosine assays. Also the presence of carnosine in plasma (carnosinemia) following dietary intake
73
of pure carnosine or meat, which is a rich source of carnosine, is controversial. Both Asatoor et al.
74
(5) and Gardner et al. (12) could not detect carnosine in plasma after administration of a high dose
75
of carnosine (respectively ~ 60mg/kg BW and 4g). Cooling of the samples following blood
76
withdrawal however, resulted in the detection of a small amount of plasma carnosine in one subject
77
in the latter study of Gardner et al. (12). Despite the use of EDTA blood tubes, which have been
78
shown to inhibit the hydrolysis of carnosine in plasma, no plasma carnosine was detected after oral
79
supplementation with 450mg carnosine (38). In line with this, both Harris et al. (13) and Yeum et
80
al. (38) could not detect any carnosine in plasma after the ingestion of beef, chicken breast nor
81
chicken broth. These results are in sharp contrast to the findings of Park et al. (27) who reported a
82
peak plasma carnosine concentration of 32.7mg/L 3.5h after ingestion of ground beef. The
83
discrepancy between these studies could be due to differences in blood handling or due to different
84
carnosinase expression of the volunteers as Yeum et al. (38) used female subjects (40-60 years)
85
which are characterized by higher carnosinase activity compared to males (10).
86
The current working hypothesis to explain the physiological mechanism for the protective effect of
87
(CTG)5 homozygosity puts forward that 1) a CNDP1 genetic predisposition leads to low serum
88
carnosinase activity (16) 2) low carnosinase activity promotes higher concentrations of circulating
89
carnosine, and 3) high circulating carnosine levels protect against hyperglycemia-induced cytotoxic
90
metabolites, resulting from oxidative stress and glycation. Evidence for the latter has been provided
91
in a number of animal studies were carnosine supplementation could delay the development and
92
progression of diabetes in db/db mice (32) and resulted in reduced urinary markers of oxidative
93
stress and AGE’s in obese Zucker rats (4). There is currently however insufficient experimental
94
evidence that supports the assumption that low carnosinase activity promotes higher concentrations
95
of circulating carnosine. Therefore, the current study aims to explore the impact of variation
96
between humans in carnosinase activity on circulating carnosine concentrations. We hypothesize
97
that higher circulating carnosine concentrations can be detected in subjects with low carnosinase
98
activity, following a single large oral dose of carnosine (60 mg/kg BW).
99
Methods
100
Subjects
101
Twenty-five subjects (age: 20-31 years, body weight: 70.9 ± 9.8 kg), both male (n = 15) and female
102
(n = 10), participated in this study. All subjects were in good health and none of the participants was
103
vegetarian. The study protocol was approved by the local ethical committee (Ghent University
104
Hospital, Belgium) and written informed consent was obtained from all participants prior to the
105
study.
106 107
Study design
108
Heparin plasma was obtained prior to the experiments and on the morning before the
109
supplementation to quantify plasma carnosinase protein content and activity. After an overnight fast
110
(at least 8 h), an indwelling catheter was inserted in an antecubital vein and blood was withdrawn
111
before and 20, 30, 40, 60 and 120 minutes following oral supplementation of 60 mg/kg BW
112
carnosine (mean ± SD: 4209 ± 577 mg), dissolved in 330 ml water. Blood samples for
113
determination of carnosine were collected in pre-cooled (4°C) EDTA tubes and immediately
114
centrifuged (4°C) to separate the plasma. The anticoagulant EDTA was chosen for its ability to
115
chelate Zn2+ ions which are essential for the catalytic activity of carnosinase (38). Plasma samples
116
were deproteinized with SSA (35%) and stored immediately at -20 °C until further analysis. Urine
117
was collected in EDTA-coated tubes prior and 45, 90, 135, 180 and 240 minutes after the carnosine
118
supplementation. The subjects were allowed to drink water and received a carnosine-free meal after
119
blood collection.
120 121
Determination of carnosine and beta-alanine by HPLC
122
100 µL of deproteinized EDTA plasma and urine was dried under vacuum (40 °C). Dried residues
123
were resolved with 40 μL of coupling reagent: methanol-triethylamine-H2O-phenylisothiocyanate
124
(PITC) (7:1:1:1) and allowed to react for 20 minutes at room temperature. The samples were dried
125
again and resolved in 100 μL of sodium acetate buffer (10 mM, pH 6.4). The same method was
126
applied to the standard solutions of beta-alanine (Sigma) and carnosine (Flamma, dissolved in
127
deionized-distilled water). The derivatized samples (20 μL) were applied to a Waters HPLC system
128
with an Hypersilica column (4.6 x 150 mm, 5 μm) and UV detector (wavelength 210 nm). The
129
column was equilibrated with buffer A [10 mM sodium acetate adjusted to pH 6.4 with 6 % acetic
130
acid] and buffer B [60 % acetonitrile – 40 % buffer A] at a flow rate of 0.8 ml/min at room
131
temperature. Limit of detection and quantification were respectively 3 and 10 µM.
132 133
Determination of carnosine by LC-ESI-MS/MS
134
Analyses of plasma carnosine of one subject were performed by using a validated LC-ESI-MS/MS
135
method (26). Briefly, aliquots of 100 µl of EDTA-plasma samples were spiked with H-Tyr-His-OH
136
as internal standard (20 µM final concentration), deproteinized by perchloric acid (PCA, 700 mM
137
final concentration) and centrifuged at 18,000 rpm for 10 min. The supernatants were then diluted
138
1:1 with mobile phase A (CH3CN/H2O/HFBA 90/10/0.1 v:v:v), filtered through 0.2 µm filters and
139
then injected into a ThermoFinnigan Surveyor LC system equipped with a quaternary pump and
140
connected through an electrospray interface (ESI) to a TSQ Quantum Triple Quadrupole Mass
141
Spectrometer (ThermoFinnigan Italia, Milan, Italy). Chromatographic separations were done by
142
reverse phase elution with a Phenomenex Sinergy polar-RP column (150 mm x 2 mm i.d.; particle
143
size 4 µm) (Chemtek Analytica, Anzola Emilia, Italy) protected by a polar-RP guard column (4 mm
144
x 2 mm i.d.; 4 µm) kept at 25 °C. Separations were done by gradient elution from 100% phase A to
145
80% phase B (CH3CN) in 12 min at a flow rate of 0.2 ml min-1 (injection volume 10 µl); the
146
composition of the eluent was then restored to 100% A within 1 min, and the system was re-
147
equilibrated for 6 min. Quantitations were performed in multiple reaction monitoring (MRM) mode
148
at 2.00 kV multiplier voltage, and the following MRM transitions of [M + H]+ precursor ion
149
product ions were selected as follows:
150
– H-Tyr-His-OH (IS) m/z 319.2 156.5 + 301.6 (collision energy, 25 eV);
151
– CAR: m/z 227.0 110.6 + 156.5 (collision energy, 25 eV);
152 153
Determination of plasma carnosinase activity and protein content
154
At baseline, the mean plasma carnosinase activity and protein content was quantified based on the
155
activity and protein content of two different blood collections. Plasma carnosinase activity was
156
determined according to the method described by Teufel et al (35). Briefly, the reaction was
157
initiated by addition of substrate (L-carnosine) to a heparin plasma sample and stopped after 10 min
158
of incubation at 37 °C by adding 1 % SSA. Liberated histidine was derivatized with o-
159
phtaldialdehyde (OPA) and the maximum increase was used for determining the maximum activity.
160
Fluorescence was measured by excitation at 360 nm and emission at 460 nm. The intra- and inter-
161
assay variations were respectively 7 % and 25 %. The lowest carnosinase activity detectable was
162
0.117 µmol/ml/h.
163
Plasma carnosinase protein content was measured by ELISA. In brief, a human CN1 ELISA was
164
developed by coating high absorbant microtitre plates (Greiner BioChemia, Flacht, Germany)
165
overnight with 100 µl of goat polyclonal anti-human CN1 (1 µg/ml) (R&D, Wiesbaden Germany).
166
The plates were extensively washed and incubated with 5 % w/v of dry milk powder to avoid
167
unspecific binding. For each sample and standard serial dilution were carried out. The plates were
168
placed on a shaker for 1 hr and subsequently extensively washed with PBS/Tween. Hereafter anti-
169
human carnosinase monoclonal antibody (clone ATLAS, Abcam) was added for 1 hr followed by
170
extensively washing. HRP conjugated goat anti-mouse IgG was added for 1 hr and the plates were
171
washed. After addition of peroxidase substrate (deep-Blue POD) (Roche diagnostics, Mannheim,
172
Germany) the reaction was stopped after 15 minutes by addition of 50 µl of 1 M H2SO4 and read in
173
an ELISA reader at 450 nm. CN1 protein concentrations were assessed in the linear part of the
174
dilution curve. Sensitivity of the ELISA was approximately 20 ng/ml.
175 176
CNDP1 genotyping
177
A more detailed description of the CNDP1 genotype determination is explained in the study of
178
Mooyaart et al. (24). In brief, a standard PCR protocol was used with primers 5-FAM-
179
GCGGGGAGGGTGAGGAGAAC (forward) and GGTAACAGACCTTCTTGAGGAATT-TGG
180
(reverse). The denaturing, annealing and extension temperatures were 94 °C, 60 °C and 72 °C,
181
respectively. After PCR amplification, fragment analysis was performed on the ABI3130 analyzer
182
(Perkin Elmer) to determine the fragment length corresponding with the different genotypes. Each
183
peak corresponded with the number of leucine repeats on each allele. A 157, 160 and 163 base pair
184
product corresponded with 5, 6 and 7 CTG codons encoding for 5, 6 and 7 leucine repeats,
185
respectively.
186 187
Statistics
188
Data are expressed as mean ± SD. Bivariate correlations and independent sample T-tests were used
189
for statistical analysis (SPSS 17).
190 191 192 193 194 195
196
Results
197
Carnosine is detectable in human plasma by HPLC.
198
A HPLC chromatogram of EDTA plasma derivatized with PITC (UV detection) of a subject 30
199
minutes following 60 mg/kg BW carnosine ingestion is depicted in Figure 1A. Spiking the sample
200
with a carnosine standard results in an identical chromatogram except for the higher peak at 15.6
201
min, representing carnosine. If the necessary precautions to block the endogenous carnosinase
202
activity are not taken (heparin instead of EDTA tubes; not pre-cooled; not immediately
203
deproteinized), then the same subject at the same time point does not display a carnosine peak at
204
15.6 min (Figure 1B) and the peak of beta-alanine (the product of the carnosinase reaction)
205
increases, which illustrates that the peak at 15.6 min genuinely represents carnosine. Plasma
206
samples of one subject were analysed by an independent laboratory with LC-ESI-MS/MS method
207
(26) and carnosine was detected at 30 (11.8µM) and 45 (3.4µM) minutes following carnosine
208
supplementation (4g) while at the other time-points no carnosine could be detected.
209 210
Supplementation-induced carnosinemia depends on CN1 protein content and activity.
211
The mean CN1 protein levels varied widely between subjects from 24.38 to 148.02 µg/ml (mean ±
212
SD: 77.82 ± 30.98 µg/ml) and the CN1 activity values were situated between 2.79 and 10.90
213
µmol/ml/h (mean ± SD: 5.95 ± 1.91 µmol/ml/h). Furthermore, the CN1 protein content is positively
214
correlated to the activity of the enzyme (p = 0.004, r = 0.58, R² = 0.34, Figure 2), confirming that
215
the activity level is largely determined by the amount of enzyme available in the plasma. Upon
216
carnosine ingestion, 8 of the 25 subjects displayed an increase in plasma carnosine concentration
217
(carnosinemia), which we termed as responders (increase of > 10 µM carnosine after
218
supplementation). The increase in plasma carnosine reached its Cmax (mean ± SD: 73.3 ± 59.7 µM;
219
range: 30.7 - 195 µM) at on average 30 - 40 minutes after supplementation and rapidly declined
220
within 1 - 2 hours (Figure 3). However, the remaining 17 subjects had no measurable increment (=
221
non-responders) in plasma carnosine after oral supplementation with a high dose of carnosine,
222
despite the precautions that were taken to block the carnosinase activity during blood collection.
223
Post-hoc analysis revealed that there was a marked difference in plasma carnosinase protein content
224
(p < 0.001) and activity (p = 0.007) between the responders and non-responders (Figure 4A and B).
225
The mean plasma carnosinase protein levels were approximately 2-fold higher in the non-
226
responders (mean ± SD: 91.57 ± 25.37 µg/ml) compared to the responders group (mean ± SD:
227
44.42 ± 11.22 µg/ml, p < 0.001). Moreover, it seems that there is a clear cut-off value in plasma
228
carnosinase protein content (Figure 4A), as the highest value of the responders was approximately
229
the same as the lowest of the non-responders (55 µg/ml). Likewise, the CN1 activity is
230
approximately 1.5-fold higher in non-responders (mean ± SD: 6.65 ± 1.80 µmol/ml/h) compared to
231
the responders (mean ± SD: 4.53 ± 1.26 µmol/ml/h; p = 0.007, Figure 4B). Also within the
232
responders’ group, the amount of plasma carnosine (AUC) is negatively correlated with the plasma
233
carnosinase protein levels (p = 0.059, r = -0.68, R² = 0.47) and carnosinase activity (p = 0.059, r = -
234
0.73, R² = 0.52). Both women (n = 2) and men (n = 6) and subjects with 5-5 (n = 3), 5-7 (n = 1) and
235
6-6 (n = 4) CNDP1 genotype were represented in the responders group (Table 1). The mean age of
236
responders (mean ± SD: 21.6 ± 0.7 yrs) was significantly lower (p = 0.045) compared to the mean
237
age of the non-responders (mean ± SD: 23.1 ± 2.6 yrs).
238 239
Urinary carnosine is related to supplementation-induced carnosinemia.
240
The urinary carnosine recovery (% of ingested dose) varied largely between 0.23 and 13.27 %. The
241
responders had a significantly (p = 0.006) higher recovery (mean ± SD: 7.7 ± 3.6 %) compared to
242
the non-responders (mean ± SD: 2.9 ± 1.3%, Figure 5A). Furthermore, a strong negative association
243
was observed between the urinary carnosine (% of ingested dose) and the plasma carnosinase
244
activity (p = 0.001, r = -0.64, R² = 0.41, Figure 5B) and protein content (p < 0.001, r = -0.66, R² =
245
0.44, Figure 5C). There was a trend for a positive correlation between the areas under the curve of
246
plasma and urinary carnosine within the responders group (n = 8, p = 0.07, r = 0.66, R² = 0.44). The
247
kinetics of urinary carnosine 4h after carnosine supplementation were comparable between the
248
responders and non-responders. The majority was excreted during the first hour (responders: 45.6
249
%; non-responders: 58.6 %) and thereafter, the concentration slowly began to decline and 90 % - 95
250
% of the increase in urinary carnosine was disappeared 4h after carnosine ingestion.
251 252
Profiling beta-alanine in plasma after carnosine ingestion
253
Figure 6 shows the profile of beta-alanine in plasma after 60 mg/kg carnosine supplementation
254
separated for subjects with or without supplementation-induced carnosinemia. The total amount of
255
beta-alanine detected in plasma (AUC) was significantly higher in the responders group (p = 0.005,
256
+ 30%) versus the non-responders. The Cmax was reached after 30 min for non-responders (mean ±
257
SD: 382.6 ± 122.9 µM), after 40 min for responders (mean ± SD: 468.2 ± 66.0µM) and beta-alanine
258
was almost completely removed from the plasma 2h after supplementation. Furthermore, the plasma
259
beta-alanine content was negatively correlated to the plasma carnosinase protein levels (p = 0.001, r
260
= -0.61, R² = 0.37) and activity (p = 0.019, r = -0.47, R² = 0.22).
261 262
Side effects
263
During the first hour after supplementation with 60 mg/kg BW carnosine, a total of 6 participants
264
transiently suffered from both headache and paraesthesia symptoms, 2 subjects only from
265
paraesthesia and 2 subjects from headache. The presence of these symptoms was not related to
266
supplementation-induced carnosinemia, CNDP1 genotype or gender. However, subjects
267
complaining about paraesthesia and/or headache had a higher total amount of beta-alanine in urine
268
(symptoms: 93.7 ± 66.8 mg, no symptoms: 48.1 ± 45.6 mg, p = 0.05), although the total amount of
269
plasma beta-alanine was not different compared to subjects without paraesthesia and/or headache
270
(symptoms: 1.4 ± 0.2 mM, no symptoms: 1.3 ± 0.4 mM, p > 0.5).
271
Discussion
272
The main finding of this study is that a high carnosinase activity counteracts the presence of
273
circulating carnosine upon an oral challenge. This likely provides a missing link in the
274
pathophysiological mechanism between carnosine, CNDP1 genotype and diabetic
275
complications, as shown in Figure 7. The mechanism of the protective effect of the Mannheim
276
allele in diabetic nephropathy has been attributed to a lower carnosinase activity (16).
277
However, the further link with higher circulating carnosine has never been established.
278
Carnosine has been shown to be a natural scavenger of the hyperglycemia-induced
279
overproduction of reactive oxygen species, AGE’s and reactive aldehydes and is therefore a
280
promising candidate therapeutic molecule in reducing the risk to develop diabetic
281
complications. More interestingly, Riedl et al. (31) recently showed that carnosinase activity
282
is increased by hyperglycemia through N-glycosylation, and is elevated in type-2 diabetic
283
patients. Therefore, hyperglycemia not only directly induces glycoxidative stress, but also
284
indirectly suppresses the endogenous protective mechanism through carnosine, which
285
probably further speeds the development of complications (Figure 7).
286
The findings of this study implicate that the quantification of the plasma carnosinase protein
287
content could be a reliable tool to determine the risk for developing nephropathy in diabetic
288
patients as there is almost no overlap between subjects with or without supplementation-
289
induced carnosinemia with respect to plasma carnosinase protein content. Interestingly, this
290
indirectly takes the glycemic control into account, as carnosinase protein levels are elevated in
291
hyperglycemic conditions (31). In 2005, Janssen et al. (16) recommended to investigate the
292
potential of the CNDP1 variants in predicting the risk for developing diabetic nephropathy.
293
However, the potential of the CNDP1 gene may be limited as 1) not all patients with
294
homozygosity for the Mannheim allele were protected against diabetic nephropathy (16) and
295
2) the subjects with supplementation-induced carnosinemia in this study were characterized
296
with both 5-5, 5-7 and 6-6 CNDP1 genotype. Therefore, it would be interesting to investigate
297
whether diabetic patients with a low plasma carnosinase protein content will be more
298
protected against diabetic nephropathy compared to patients with a higher carnosinase protein
299
content (irrespective of CNDP1 genotype).
300
The carnosinase activity and protein content was shown to be a discriminating factor for
301
supplementation-induced carnosinemia. Consequently, a potential therapeutic strategy to
302
reduce the risk of diabetic nephropathy could be the inactivation of the carnosinase enzyme.
303
In addition to the (CTG)n polymorphism in the CNDP1 gene (10; 16; 24), female gender (6;
304
10; 29) and increasing age (29) are determinants associated with higher carnosinase activity
305
that can not be manipulated. In vitro experiments, however, revealed that the activity of
306
carnosinase enzyme was markedly decreased in the presence of both homocarnosine (gamma-
307
aminobutyryl-L-histidine) (28; 29) and anserine (beta-alanyl-N1-methylhistidine) (28).
308
Though, the correlation between circulating homocarnosine or anserine levels in fasted
309
subjects and plasma carnosinase activity in vivo is less obvious (28; 29). Importantly, a good
310
glycemic control, either by intervention with insulin, exercise or diet, seems to be crucial for
311
diabetic patients as this not only directly influences oxidative and glycation stress, but also
312
indirectly affects carnosinase activity (31). Besides the inactivation of the carnosinase
313
enzyme, the development of a carnosine-analogue resistant to the carnosinase enzyme would
314
be a promising strategy to prevent patients from diabetic complications. In this light, Aldini et
315
al. (4) recently showed that the enantiomer D-carnosine has the same protective effect as L-
316
carnosine in obese Zucker rats, while it is more resistant to carnosinase activity. However, D-
317
carnosine has a lowered bioavailability as it is less absorbed in respect to L-carnosine and
318
therefore the relevance for human use may be limited (4).
319
In the assumption that dietary intake of carnosine can diminish the risk for diabetic
320
complications, the lack of carnosine in the diet, as is the case in a vegetarian diet, could be
321
deleterious for the protection against glycation and oxidative stress. Indeed, plasma AGE
322
content of healthy vegetarians has been reported to be higher compared to omnivores (19; 33).
323
Although a low-fat carbohydrate-rich vegetarian diet is often recommended for diabetic
324
patients (for its positive impact on insulin sensitivity, blood pressure, serum lipid profile, etc
325
(21)), the dietary intake of carnosine, perhaps by supplementation, should not be neglected.
326
Carnosine is, even after meat or carnosine supplementation, hard to detect in human plasma,
327
as a result of the high carnosinase activity. Yet, when several precautions are taken (cooling,
328
use of EDTA tubes and the immediate deproteinization), when pharmacological doses of
329
carnosine (60mg/kg BW) are ingested and when subjects with low carnosinase activity are
330
studied, plasma carnosine levels can be clearly quantified up to 1h after ingestion. In contrast
331
to plasma, urinary carnosine is more easily detectable, even in subjects ingesting their usual
332
diets (0.2µM - 18.6µM) (2; 36). This urinary carnosine excretion is increased after carnosine
333
or meat supplementation with a urinary recovery up to 14% of the ingested amount (12; 38).
334
As there is no increase in urinary carnosine after beta-alanine (2g) and histidine (2g) intake,
335
Gardner et al. (12) hypothesized that the excreted carnosine is not arisen from resynthesis of
336
beta-alanine and histidine after hydrolysis in intestine and/or plasma and that the amount of
337
urinary carnosine is a reflection of plasma carnosine. This hypothesis is confirmed in the
338
present study by the positive correlation between urinary and plasma carnosine in the
339
responders group and by the strong negative relation between urinary carnosine and plasma
340
carnosinase activity and protein content (Figure 5B and 5C). Following carnosine
341
supplementation, urinary carnosine excretion seems to be a reliable and easier measurable
342
estimation of plasma carnosine content, which is equally dependent on serum carnosinase
343
activity and content.
344
Since renal tubular epithelium is equipped with oligopeptide transporters with a high affinity
345
for carnosine (17; 34), circulating carnosine can be accumulated by the kidney and may
346
provide an additional exogenous source of protective peptides against diabetic metabolites in
347
patients with low levels of carnosinase.
348
One would expect that beta-alanine, the degradation product of the plasma carnosinase
349
enzyme, would be positively related to the carnosinase activity. However, the opposite is true
350
as the responders group showed a higher total amount of plasma beta-alanine compared to the
351
non-responders. This may suggest that the majority of carnosine is in fact degraded in other
352
compartments and tissues, than the circulation. Thus, low carnosinase activity favors both
353
carnosinemia and beta-alaninemia following carnosine supplementation, although the
354
mechanism for the latter is unclear.
355
It can be concluded that 1) carnosine can be detected in human plasma following oral
356
ingestion and 2) a high carnosinase activity and content potently counteracts the presence of
357
circulating carnosine. In a diabetic environment, this could impede the ability of carnosine to
358
exert its protective effects against cytotoxic agents leading to diabetic complications. The
359
inhibition of the carnosinase enzyme and the development of a carnosine analogue resistant to
360
carnosinase have to be investigated as potential therapeutic strategies for reducing the risk for
361
diabetic complications.
362
Acknowledgements
363
The practical contribution of Anneke Volkaert, Sam Beelprez, Joren Biesbrouck and Fiona
364
Albers is greatly acknowledged. We thank Flamma (Italy) for generously providing carnosine.
365
Grants
366
This study was financially supported by grants from the Research Foundation – Flanders
367
(FWO 1.5.149.08 and G.0046.09).
368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386
Reference List
387 388 389
1.
UK Prospective Diabetes Study (UKPDS) Group: Intensive blood-glucose control
390
with sulphonylureas or insulin compared with conventional treatment and risk of
391
complications in patients with type 2 diabetes (UKPDS 33). Lancet 352: 837-853,
392
1998.
393
2. Abe H, Okuma E, Sekine H, Maeda A and Yoshiue S. Human urinary excretion of
394
L-histidine - related compounds after ingestion of several meats and fish muscle. Int J
395
Biochem 25: 1245-1249, 1993.
396
3. Aldini G, Granata P and Carini M. Detoxification of cytotoxic alpha,beta-
397
unsaturated aldehydes by carnosine: characterization of conjugated adducts by
398
electrospray ionization tandem mass spectrometry and detection by liquid
399
chromatography/mass spectrometry in rat skeletal muscle. J Mass Spectrom 37: 1219-
400
1228, 2002.
401
4. Aldini G, Orioli M, Rossoni G, Savi F, Braidotti P, Vistoli G, Yeum KJ, Negrisoli
402
G and Carini M. The carbonyl scavenger carnosine ameliorates dyslipidemia and
403
renal function in zucker obese rats. J Cell Mol Med 15:1339-1354, 2011.
404 405
5. Asatoor AM, Bandoh JK, Lant AF, Milne MD and Navab F. Intestinal absorption of carnosine and its constituent amino acids in man. Gut 11: 250-254, 1970.
406
6. Bando K, Shimotsuji T, oyoshima H and Miyae K. Fluoremetric assay of human
407
serum carnosinase activity in normal children, adults and patients with myopathy. Ann
408
Clin Biochem 21: 510-514, 1984.
409
7. Bauchart C, Savary-Auzeloux I, Patureau MP, Thomas E, Morzel M and
410
Remond D. Carnosine concentration of ingested meat affects carnosine net release
411
into the portal vein of minipigs. J Nutr 137: 589-593, 2007.
412 413
8. Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. Diabetes 54: 1615-1625, 2005.
414
9. Chakkera HA, Hanson RL, Kobes S, Millis MP, Nelson RG, Knowler WC and
415
Distefano JK. Association of variants in the carnosine peptidase 1 gene (CNDP1)
416
with diabetic nephropathy in American Indians. Mol Genet Metab 103:185-190, 2011.
417
10. Everaert I, Mooyaart A, Baguet A, Zutinic A, Baelde H, Achten E, Taes Y, de HE
418
and Derave W. Vegetarianism, female gender and increasing age, but not CNDP1
419
genotype, are associated with reduced muscle carnosine levels in humans. Amino
420
Acids 40: 1221-1229, 2011.
421
11. Freedman BI, Hicks PJ, Sale MM, Pierson ED, Langefeld CD, Rich SS, Xu J,
422
McDonough C, Janssen B, Yard BA, van der Woude FJ and Bowden DW. A
423
leucine repeat in the carnosinase gene CNDP1 is associated with diabetic end-stage
424
renal disease in European Americans. Nephrol Dial Transplant 22: 1131-1135, 2007.
425
12. Gardner M, Illingworth, Kelleher and Wood. Intestinal absorption of the intact
426
peptide carnosine in man, and comparison with intestinal permeability of lactulose. J
427
Physiol 439: 411-422, 1991.
428
13. Harris RC, Tallon MJ, Dunnett M, Boobis L, Coakley J, Kim HJ, Fallowfield JL,
429
Hill CA, Sale C and Wise JA. The absorption of orally supplied beta-alanine and its
430
effect on muscle carnosine synthesis in human vastus lateralis. Amino Acids 30: 279-
431
289, 2006.
432 433
14. Hipkiss AR, Michaelis J and Syrris P. Non-enzymatic glycosylation of the dipeptide L-carnosine, a potential anti-protein-cross-linking agent. FEBS Lett 371: 81-85, 1995.
434
15. Hou WC, Chen HJ and Lin YH. Antioxidant peptides with Angiotensin converting
435
enzyme inhibitory activities and applications for Angiotensin converting enzyme
436
purification. J Agric Food Chem 51: 1706-1709, 2003.
437
16. Janssen B, Hohenadel D, Brinkkoetter P, Peters V, Rind N, Fischer C, Rychlik I,
438
Cerna M, Romzova M, de HE, Baelde H, Bakker SJ, Zirie M, Rondeau E,
439
Mathieson P, Saleem MA, Meyer J, Koppel H, Sauerhoefer S, Bartram CR,
440
Nawroth P, Hammes HP, Yard BA, Zschocke J and van der Woude FJ. Carnosine
441
as a protective factor in diabetic nephropathy: association with a leucine repeat of the
442
carnosinase gene CNDP1. Diabetes 54: 2320-2327, 2005.
443
17. Kamal MA, Jiang H, Hu Y, Keep RF and Smith DE. Influence of genetic knockout
444
of Pept2 on the in vivo disposition of endogenous and exogenous carnosine in wild-
445
type and Pept2 null mice. Am J Physiol Regul Integr Comp Physiol 296: R986-R991,
446
2009.
447
18. Kohen R, Yamamoto Y, Cundy KC and Ames BN. Antioxidant activity of
448
carnosine, homocarnosine, and anserine present in muscle and brain. Proc Natl Acad
449
Sci U S A 85: 3175-3179, 1988.
450 451
452 453
19. Krajcovicova-Kudlackova M, Sebekova K, Schinzel R and Klvanova J. Advanced glycation end products and nutrition. Physiol Res 51: 313-316, 2002.
20. Lenney JF, Peppers SC, Kucera-Orallo CM and George RP. Characterization of human tissue carnosinase. Biochem J 228: 653-660, 1985.
454
21. McCarty MF. The low-AGE content of low-fat vegan diets could benefit diabetics -
455
though concurrent taurine supplementation may be needed to minimize endogenous
456
AGE production. Med Hypotheses 64: 394-398, 2005.
457
22. McDonough CW, Hicks PJ, Lu L, Langefeld CD, Freedman BI and Bowden DW.
458
The influence of carnosinase gene polymorphisms on diabetic nephropathy risk in
459
African-Americans. Hum Genet 126: 265-275, 2009.
460
23. Mooyaart AL, Valk EJ, van Es LA, Bruijn JA, de HE, Freedman BI, Dekkers
461
OM and Baelde HJ. Genetic associations in diabetic nephropathy: a meta-analysis.
462
diabetologia 54: 544-553, 2011.
463
24. Mooyaart AL, van Valkengoed IG, Shaw PK, Peters V, Baelde HJ, Rabelink TJ,
464
Bruijn JA, Stronks K and de HE. Lower frequency of the 5/5 homozygous CNDP1
465
genotype in South Asian Surinamese. Diabetes Res Clin Pract 85: 272-278, 2009.
466
25. Mooyaart AL, Zutinic A, Bakker SJ, Grootendorst DC, Kleefstra N, van
467
Valkengoed IG, Bohringer S, Bilo HJ, Dekker FW, Bruijn JA, Navis G, Janssen
468
B, Baelde HJ and de HE. Association between CNDP1 genotype and diabetic
469
nephropathy is sex specific. Diabetes 59: 1555-1559, 2010.
470
26. Orioli M, Aldini G, Beretta G, Facino RM and Carini M. LC-ESI-MS/MS
471
determination of 4-hydroxy-trans-2-nonenal Michael adducts with cysteine and
472
histidine-containing peptides as early markers of oxidative stress in excitable tissues. J
473
Chromatogr B Analyt Technol Biomed Life Sci 827: 109-118, 2005.
474 475
27. Park Y, Volpe SL and Decker EA. Quantification of carnosine in human plasma after dietary consumption of beef. J Agric Food Chem 53: 4736-4739, 2005.
476
28. Peters V, Jansen EE, Jakobs C, Riedl E, Janssen B, Yard BA, Wedel J,
477
Hoffmann GF, Zschocke J, Gotthardt D, Fischer C and Koppel H. Anserine
478
inhibits carnosine degradation but in human serum carnosinase (CN1) is not correlated
479
with histidine dipeptide concentration. Clin Chim Acta 412: 263-267, 2011.
480
29. Peters V, Kebbewar M, Jansen EW, Jakobs C, Riedl E, Koeppel H, Frey D,
481
Adelmann K, Klingbeil K, Mack M, Hoffmann GF, Janssen B, Zschocke J and
482
Yard BA. Relevance of allosteric conformations and homocarnosine concentration on
483
carnosinase activity. Amino Acids 38: 1607-1615, 2010.
484
30. Riedl E, Koeppel H, Brinkkoetter P, Sternik P, Steinbeisser H, Sauerhoefer S,
485
Janssen B, van der Woude FJ and Yard BA. A CTG polymorphism in the CNDP1
486
gene determines the secretion of serum carnosinase in Cos-7 transfected cells.
487
Diabetes 56: 2410-2413, 2007.
488
31. Riedl E, Koeppel H, Pfister F, Peters V, Sauerhoefer S, Sternik P, Brinkkoetter
489
P, Zentgraf H, Navis G, Henning RH, Van Den Born J, Bakker SJ, Janssen B,
490
van der Woude FJ and Yard BA. N-glycosylation of carnosinase influences protein
491
secretion and enzyme activity: implications for hyperglycemia. Diabetes 59: 1984-
492
1990, 2010.
493
32. Sauerhofer S, Yuan G, Braun GS, Deinzer M, Neumaier M, Gretz N, Floege J,
494
Kriz W, van der Woude F and Moeller MJ. L-carnosine, a substrate of carnosinase-
495
1, influences glucose metabolism. Diabetes 56: 2425-2432, 2007.
496
33. Sebekova K, Krajcoviova-Kudlackova M, Schinzel R, Faist V, Klvanova J and
497
Heidland A. Plasma levels of advanced glycation end products in healthy, long-term
498
vegetarians and subjects on a western mixed diet. Eur J Nutr 40: 275-281, 2001.
499
34. Shen H, Smith DE, Yang T, Huang YG, Schnermann JB and Brosius FC 3rd.
500
Localization of PEPT1 and PEPT2 proton-coupled oligopeptide transporter mRNA
501
and protein in rat kidney. Am J Physiol 276 (5 Pt 2): F658-F665, 1999.
502
35. Teufel M, Saudek V, Ledig JP, Bernhardt A, Boularand S, Carreau A, Cairns
503
NJ, Carter C, Cowley DJ, Duverger D, Ganzhorn AJ, Guenet C, Heintzelmann
504
B, Laucher V, Sauvage C and Smirnova T. Sequence identification and
505
characterization of human carnosinase and a closely related non-specific dipeptidase. J
506
Biol Chem 278: 6521-6531, 2003.
507
36. Tsuruta Y, Maruyama K, Inoue H, Kosha K, Date Y, Okamura N, Eto S and
508
Kojima E. Sensitive determination of carnosine in urine by high-performance liquid
509
chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl
510
chloride as a fluorescent labeling reagent. J Chromatogr B Analyt Technol Biomed
511
Life Sci 878: 327-332, 2010.
512
37. Wanic K, Placha G, Dunn J, Smiles A, Warram JH and Krolewski AS. Exclusion
513
of polymorphisms in carnosinase genes (CNDP1 and CNDP2) as a cause of diabetic
514
nephropathy in type 1 diabetes: results of large case-control and follow-up studies.
515
Diabetes 57: 2547-2551, 2008.
516
38. Yeum KJ, Orioli M, Regazzoni L, Carini M, Rasmussen H, Russell RM and
517
Aldini G. Profiling histidine dipeptides in plasma and urine after ingesting beef,
518
chicken or chicken broth in humans. Amino Acids 38:847-858, 2009.
519 520
Tables & figures
521
522
Table I
523
CN1 activity and protein content (mean ± SD) separated for CNDP1 genotype and gender (* p <
524
0.05 vs. male, † p < 0.1 vs. male). The subjects in the responders group were both males and
525
females and were characterized with both 5-5, 5-7 and 6-6 CNDP1 genotype.
526
Figure 1
527
A. HPLC chromatogram of human plasma (subject from the responders group) withdrawn 30
528
minutes after ingestion of 60mg/kg BW carnosine in a cooled EDTA tube which was immediately
529
deproteinized (solid line) and the same sample spiked with standard carnosine (dotted line). The
530
peak at 15.6 min (carnosine) increased, while all other peaks remained the same (including beta-
531
alanine at 12.1 min).
532
B. HPLC chromatogram from human plasma (subject from the responders group) withdrawn 40
533
minutes after ingestion of 60mg/kg BW carnosine in 1/ a cooled EDTA tube which was
534
immediately deproteinized (solid line) and in 2/ a non-cooled heparin tube (not deproteinized before
535
storage, dotted line). The carnosine peak at 15.6 minutes has disappeared and beta-alanine (12.1
536
min), taurine (13.2 min) and the peak at 14.2 min (partially histidine) were higher in heparin versus
537
EDTA plasma.
538
Figure 2
539
The activity of plasma carnosinase enzyme is dependent on the amount of plasma carnosinase
540
protein content as there is a strong positive correlation between CN1 activity and protein content (p
541
= 0.004, r = 0.58, R² = 0.34).
542
Figure 3
543
Time course of plasma carnosine of the 8 responders (individual values, mean = solid line) up to 2h
544
after oral administration of 60mg/kg BW carnosine. The increase reached a peak (range: 30.7 –
545
195.0 µM) at 20 - 60 minutes and rapidly declined within 1-2 hours.
546
547
Figure 4
548
Boxplot, representing the minimum/maximum values, lower/upper quartile and median of CN1
549
protein content (A) and CN1 activity (B) of the non-responders compared to responders. The
550
plasma carnosinase protein content (A) and activity (B) was significantly lower in the subjects
551
characterized with supplementation-induced carnosinemia after carnosine supplementation
552
(60mg/kg BW) compared to the non-responders group (p < 0.001 and p = 0.007, respectively; ° =
553
outlier).
554
Figure 5
555
Urinary carnosine and beta-alanine: A/ The amount of urinary carnosine (black bars, expressed as
556
% of ingested carnosine) is 2.6-fold higher (p = 0.006) in the responders group while there is no
557
difference in urinary beta-alanine (white bars) between the responders and non-responders
558
(respectively 1.6 ± 0.9 vs. 1.4 ± 1.3, p > 0.05). B,C/ Relationship between CN1 activity (B, p =
559
0.001, r = -0.64, R² = 0.41) / CN1 protein content (C, p < 0.001, r = -0.66, R² = 0.44) and urinary
560
carnosine (% of ingested dose) during the 4h after carnosine supplementation (60mg/kg BW).
561
Figure 6
562
The mean (± SD) area under the curve of plasma beta-alanine (µM) is significantly higher in the
563
responders (dotted line) versus the non-responders group (solid line, p = 0.005, +30%).
564
Figure 7
565
Proposed pathophysiological mechanism linking carnosine and CNDP1 genotype to diabetic
566
complications. * denotes effects that are now supported by the findings of the current study in
567
humans.
