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Beynen AC, 2022. Pentosidine in petfood

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Bonny Canteen 2022; 3: 219-225.

Anton C. Beynen

Pentosidine in petfood

Pentosidine (Pen) may be seen as the product of heat-induced reactions between a simple sugar,
such as glucose, and two amino acids, namely lysine and arginine. Pen may thus be formed during
thermal processing of foods, including commercial dog and cat foods (cf. Note 1). Data on Pen
analyses in petfood were not found. Using analysed Pen contents in foodstuffs, the maximum level
of Pen in dry petfood was put at 8 mg/kg dietary dry matter, or per kg of the food’s residue after
removal of its moisture.

Ingested Pen can cross the intestinal wall, but the magnitude is unknown. Data on metabolism of
Pen by dogs and cats are unavailable. It is expected, as based on extrapolation, that ingested and
absorbed Pen ends up in urine as such and in the form of uncharacterized metabolites. Pen is most
probably synthesized by bodily tissues, but it appears that direct proof and details are unavailable
(cf. Note 1).

When it comes to health of dogs and cats, dietary Pen does not seem to be a matter of immediate
concern (cf. Note 2). That statement relates to a two-year study in which rats consumed drinking
water mixed with coffee brew. The drinking fluid, which did not induce signs of toxicity, brought
about an intake of Pen that was equivalent to 8 mg/kg dietary dry matter, or equal to the
estimated, maximum level in petfood.

The surmise that Pen is likely harmless should be considered against the background of
uncertainties. The proposed, maximum amount of Pen in petfood is doubtful, which means that
Pen analyses in petfood are required. Furthermore, it cannot be excluded that dogs and/or cats
are more sensitive to Pen than rats. Thus, assessment of the upper safe limit of dietary Pen in dogs
and cats is desirable.

Pentosidine

Pentosidine (Pen, C17H26N6O4) was first reported and named by Sell and Monnier (1) in 1989. The
fluorescent molecule was isolated from human extracellular matrix (dura mater) and its structure
was elucidated by mass spectrometry. The compound was described as an imidazo[4,5-b]pyridinium
ring with a lysine and arginine side chain. The investigators suggested that the cross-links with the
two amino acids were the result of Maillard reactions with a pentose (cf. Note 3).

Imidazopyridinium (C6H5N3) reflects a pyridine ring (C5H5N) fused with an imidazole ring (C3H4N2). In
Pen, pyridine’s nitrogen atom is connected with a configuration [-(CH2)4-CHNH2COOH] that has four
methylene groups and a carbon with both a carboxylic and amino group. That side chain becomes
lysine if it has an amino group [H2N-] as end. The carbon in between the two nitrogen atoms of the
imidazole moiety is bound to a configuration [-NH-(CH2)3-CHNH2COOH] that has an amine group,
three methylene groups and a carbon with both a carboxylic and amino group. That side chain
becomes ornithine (Note 4) if it has a hydrogen atom [H-] as end.

Formation and pathway

Different sugars were incubated for 6 hours with NƐ-acetyl-arginine, NƐ-acetyl-lysine and phosphate
at pH 9.0 and 60 oC (2, cf. Note 5). Pen concentrations, as quantitated by reversed-phase HPLC, in
the solutions with xylose and ribose (pentoses) were 22.1 and 48.0 nmol/ml. For threose and
erythrose (tetroses), the amounts were 1.1 and 4.7 nmol/l. For glucose, fructose and galactose
(hexoses), the Pen yields were 0.1, 0.4 and 0.7 nmol/l. In the lysine-arginine model system, Pen was
formed in greatest quantities with ribose.

Rough sketches of the formation pathway of Pen in model systems have been advanced (3-5), but
details of reactions and intermediates were not provided. It is acknowledged that the formation of
Pen’s imidazopyridinium moiety, in terms of reaction mechanisms, may be difficult to understand.
Miyazaki et al. (6) have described the possible synthetic pathway for a Pen-like structure in which
the arginine residue was replaced by creatine.

Based on structural characterization of products from model reactions, Biemel et al. (3) have
proposed that Pen formation starts from native carbohydrates and includes Amadori products and
3-deoxyosones as intermediates, and has a direct precursor, which was named pentosinane. After
dehydrogenation and dehydration that compound would be transformed into Pen. However, the
reaction pathway for the formation of pentosinane was not elucidated.

Dietary content

Henle et al. (7) have analysed Pen in foods using ion-exchange chromatography with direct
fluorescence detection and subsequent ninhydrin derivatization. The results are expressed as mg
Pen/kg protein. Grains and meats were not analysed. The amounts of Pen in bread crumbs and crust
were < 0.05 and 0.4-2.6 mg/kg protein. The highest levels were detected in roasted coffee, the range
being 10.8-39.9 mg Pen/kg protein (n=10). Other studies found that the analysed amounts of Pen in
cooked beef ranged from < 0.2 to 6.7 mg/kg product (Note 6).

Data on analysed amounts of Pen in dog and cat foods were not found. To estimate Pen levels in dry
petfood, the analysed contents of bread crumbs and baked beef were used. The contents
correspond with 0.01 and 13.6 mg/kg dry matter (Note 7). For a mixture containing, on a dry matter
basis, 30% beef, 50% wheat and 20% Pen-free ingredients, the level of Pen would be 4.1 mg/kg
dietary dry matter (ddm).

To take into account possible formation of Pen during petfood production, the level could be put at
8 mg/kg ddm, or almost twice that in the beef-and-wheat mixture. It cannot be excluded that
significant amounts of Pen are formed during the production of dry petfoods. Extruded kibbles have
undergone high temperature (± 130 oC) and high pressure for < 1 minute under moist condition,
followed by drying at 120-150 oC for ± 15 minutes. For the time being, it is tentatively assumed that
the estimated, maximum content of Pen in dry food holds for wet food also.

Metabolism

The Pen level in skin collagen of dogs increases with age (8). At 2, 6 and 10 years of age, the Pen
concentrations may be about 6, 16 and 32 pmol Pen/mg collagen. It is generally accepted that Pen is
synthesized in the body, but tissues involved and quantitative details are unknown. Clearly, it is
unresolved to what relative extent dietary intake and bodily synthesis of Pen contribute to aging-
associated accumulation of Pen in skin collagen.
A study in rats (9, Note 8) has shown indirectly that orally administered Pen is absorbed by the
gastrointestinal tract. In normal rats, the concentration of plasma free Pen peaked at 3-hours post-
dosing. When Pen was given intravenously, the compound was eliminated rapidly from the
circulation and had disappeared within 2 hours. Comparison of the data in normal rats with those
obtained in nephroectomized rats indicated that adequate renal function was crucial for rapid
elimination of Pen from plasma.

For 6 days, 9 human volunteers consumed a diet without cooked or roasted foods and did not drink
coffee (10, Note 9). On Day 5, the diet remained either unchanged (n=5) or was supplemented with
600 ml coffee brew (n=4), containing 20 mg Pen/l. On Day 6, group-mean urinary excretions of Pen
in the control and test subjects were about 5 and 11 μg. In coffee brew and urine, Pen was analysed
by HPLC. The coffee brew contained 15 mg free Pen and 5 mg protein-bound Pen/l (cf. Note 1).

About 60% of free Pen ingested with coffee brew was recovered in urine (10, Note 10). It follows
that urinary excretion of unmetabolized Pen is relevant for balancing the influx and efflux of Pen’s
metabolic pool in the body. However, there is evidence for significant, post-absorptive metabolism
of Pen in rats. After intravenous injection of radiolabeled, free Pen into rats, 16 and 64% of the
radioactivity in urine was associated with intact and uncharacterized, modified Pen (11).

Toxicity

Data on the toxicity of oral Pen in laboratory animals were not found. Roasting of coffee beans not
only generates Pen, but many more, simultaneous chemical changes do occur (cf. Note 11). On that
account, toxicity of coffee brew as observed in experiments is not necessarily caused by Pen.
However, lack of observed toxicity of ingested, Pen-containing coffee brew speaks to Pen’s safety.

A two-year toxicity study in rats (12, Note 12) showed that drinking fluid containing 25% fresh-
brewed regular coffee did not affect food intake, body weight and survival in rats. The drinking fluid
may have contained 5 mg Pen/l (cf. 10), which is equivalent to 8 mg Pen/kg ddm (Note 13). That
amount is identical to the estimated, maximum Pen content in petfood (see under Dietary content).

Note 1

Pen is considered an advanced glycation end-product (AGE). Glycation refers to non-enzymatic

bonding of sugar(-like) compounds to proteins or amino acids. Within that context, Pen is formed as
result of a cascade of reactions between a sugar and lysine and arginine, all in free form (1, 2, Note
5, see under Formation and pathway). The side chains of protein-bound lysine and arginine, which
have a terminal amino group may also react with a sugar.

In coffee brew, both free and protein-bound Pen have been detected (10, see under Metabolism).
Details of the reactions that lead to free and protein-bound Pen are unknown (see under Formation
and pathway).

AGEs may be formed in foods, especially during thermal processing, but they also arise in the body
(13). Thus, AGEs in the body may be of endogenous and exogenous origin. AGEs are under scrutiny
on suspicion of enhancing aging-related pathological conditions, such as diabetes, cardiovascular
complications and cancer. However, there is no solid evidence for a causal relation between AGEs
and aging-related disease.
Sell et al (8) have quantitated Pen in skin collagen from eight mammalian species, including dogs.
Within species, Pen concentrations correlated positively with age. Between species, the rate of Pen
formation in skin correlated inversely with maximum life-span. The rate for each species was
calculated from the positive correlation between Pen content in skin collagen and age.

In patients with insulin-dependent diabetes mellitus, Pen concentrations in skin were significantly
elevated when compared with healthy subjects (14). Within patients, Pen levels were significantly
correlated with the severity of complications.

Note 2

Ames (15) has published an article entitled “Evidence against dietary advanced glycation
endproducts being a risk to human health”. The paper discusses carboxymethyllysine (CML) and Pen
as examples of AGEs, implying that the two compounds fall under the title. However, toxicity of CML
and Pen is not addressed by the article.

As to CML in petfood the following conclusion was made: “The long-term safety or danger of CML in
dog and cat food cannot be determined” (16). The judgment of this text reads: “When it comes to
health of dogs and cats, dietary PEN does not seem to be a matter of immediate concern.”

Note 3

In Pen, the epsilon nitrogen atom of lysine* is part of the molecule’s pyridine moiety. In the AGEs
pyrraline and pyridosine, lysine’s epsilon nitrogen is a component of the pyrrole and pyridine
moieties of the molecules (cf. 17, 18). Other AGEs, including CML, lysinealanine, fructose-lysine and
carboxyethyllysine can be considered lysine molecules joined with a distinctive group. The link is
shaped by a -NH-CH2- configuration that involves lysine’s epsilon amino group. The four lysine
adducts have been described in relation to petfood (16, 19-21).

*In lysine (C6H12N2O2), the epsilon amino group is located at carbon 5 when counted from carbon 1
that has both the alpha amino group and a carboxyl group.

Note 4

Ornithine (C5H12N2O2) is a non-proteinogenic amino acid that acts as start and end of the urea cycle.
The compound has a delta amino group (cf. Note 3).

Note 5

To confirm the pentose-derived nature of Pen, Sell and Monnier (1) have heated an equimolar (100
mM) mixture of L-lysine, L-arginine, and D-ribose for 1 hour at 80 oC. Injection of the reaction
mixture on HPLC revealed a major fluorescent peak that co-eluted with Pen isolated from human
dura mater. Synthetic and isolated Pen showed the same UV and fluorescence spectra.

Note 6.

Peiretti et al. (22, 23) have analysed Pen in raw and cooked beef. Pen was analysed by HPLC/MS.
Commercial beef samples were cooked in five different ways. Meat slices were broiled at 200 °C for
10 min (5 min per side) in a meat grill or broiled at a medium temperature (140 °C) for 10 min (5 min
per side). Meat slices were also cooked by microwave for 10 min (5 min per side) or 3 min and then
grilled (7 min). In addition, meat slices were boiled in water (100 °C) for 10 min. In both raw and
cooked beef samples the concentrations of Pen were < 0.20 mg/kg.

Chao et al. (24) have analysed Pen in beef that was cooked in various ways without or with sauce.
Deproteinized and reconstituted samples were analysed for Pen by HPLC. The Pen contents in beef
that was either boiled (15 min at 100 oC), fried in soybean oil (15 min at 180 oC) or baked (15 min at
230 oC) were 1.82, 2.91 and 3.40 mg/kg. When beef was treated with sour-sweet sauce, the
respective amounts were 1.86, 2.54 and 3.62 mg/kg. After treatment with barbeque sauce, the three
cooking conditions yielded 4.86, 5.62 and 6.72 mg Pen/kg beef.

Note 7

The analysed amount of Pen in bread crumbs was < 0.05 mg/kg protein (7). When assuming that
bread crumbs contain 0.05 mg Pen/kg, 8% protein and 40% moisture, the Pen level is 0.007 mg/kg
dry matter (0.05 x 80/1000 x 100/60). That estimate may be equated with 0.01 mg/kg dry matter.

Because beef cooked in sauce seems incompatible with petfood, the content of Pen in baked beef
was used, or 3.40 mg/kg (Note 6). When assigning a moisture content of 25%, the Pen concentration
is equivalent to 13.6 mg/kg dry matter.

Note 8

Rats (n=10) were fed a casein-based diet containing 0.3 μmol (0.11 mg) Pen/kg as based on Pen
analysis with HPLC (9). After one week and a 24-hour fast, bilateral nephrectomy was performed in 5
rats. Both nephrectomized and intact rats were then given an oral (0.15 mg/rat) or an intravenous
(3.78 μg/rat) load of synthetic Pen. After both oral and intravenous dosing, the disappearance of Pen
from plasma was much slower in the nephrectomized rats.

The casein-based diet fed to the rats was labeled as AIN-76 (9). Thus, the diet comprised 20% casein,
0.3% DL-methionine, 15% corn starch, 50% sucrose, 5% fiber, 5% corn oil, 3.5% AIN mineral mix, 1%
AIN vitamin mix and 0.2% choline bitartrate (25).

Note 9

Förster et al. (10) summarized the design of their experiment as follows. “A nine-day dietary study
involving 18 healthy volunteers was performed in order to investigate the influence of nutrition on
the urinary excretion of the Maillard reaction products (MRPs) fructoselysine, pyrraline, and
pentosidine. From day two through day eight, most types of Maillard product–containing food had
to be avoided. On day five, participants were divided into four groups, three of them receiving a test
meal (pretzel sticks, brewed coffee, or custard) containing defined amounts of MRPs. The fourth
group served as a control. Urine samples taken over a 24-h period were analyzed for MRPs using
chromatographic means.”

Note 10

Most probably, intestinal absorption of free Pen in coffee brew is much higher than that of protein-
bound Pen. Förster et al. (10, Note 9) found that only 2% of protein-bound Pen ingested with pretzel
sticks was recovered in urine.

Note 11
A review article (26) shows that there are at least 45 furan-based compounds in roasted-ground
coffee. Furan in petfood has been described elsewhere (27).

Note 12

Palm et al. (12) summarized the design of their study as follows. “Fresh-brewed regular coffee at
concentrations of 25, 50, and 100% was consumed ad libitum as the sole fluid intake of F1 Sprague-
Dawley rats (55♂ and 55♀/group), derived from P0-treated females which were provided 50% coffee
for about 5 weeks prior to copulation and throughout gestation and lactation. P0 males, P0 control
females, and two groups of F1 control rats received tap water.”

Note 13

For rats, the intake ratio of water and dietary dry matter is about 1.6:1 (28). Thus, 5 mg Pen/l of
drinking fluid corresponds with 8 mg Pen/kg ddm.

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