Pressure/Temperature Combined Treatments of Precursors Yield Hormone-like Peptides with Pyroglutamate at the N Terminus

Article abstract of DOI:10.1021/jf0348471

Peptides containing the cyclic product of glutamine at the N terminus are usually biologically active. If the cyclization of glutamine was associated with a volume reduction, pressure should displace the equilibrium in the direction of the lower volume. Here, results in model solutions and in whey are discussed, showing that the theorized cyclization of glutamine in Gln-His-ProNH₂ or Gln-Leu-ProNH₂ is significantly accelerated during the application of heat and even more strongly when elevated temperature and pressure combinations are used. The reaction rate depended on the intensity of the pressure treatment, the pH, and the nature of the amino acids adjacent to glutamine. The products of the reaction were identified as thyrotropin-releasing hormone (TRH) and [Leu(2)]TRH. The reported reactions could affect the naturally balanced concentration of short-chain peptides in foods and therefore induce unpredictable biological effects.

Full text of DOI:10.1021/jf0348471

J. Agric. Food Chem. 2003, 51, 8093−8097 8093
Pressure/Temperature Combined Treatments of Precursors
Yield Hormone-like Peptides with Pyroglutamate at the N
Terminus
AVELINA FERNAÄ NDEZ GARCIÄA,*,† PETER BUTZ,† BERNHARD TRIERWEILER,†
HEIKE ZO¨ LLER,† JOACHIM STA¨ RKE,† EBERHARD PFAFF,§ AND
†
BERNHARD TAUSCHER
Institute of Chemistry and Biology, Federal Research Centre for Nutrition, Haid-und-Neustrasse 9,
D-76131 Karlsruhe, Germany, and Institute of Immunology, Federal Research Centre for Virus
Diseases of Animals, Paul-Ehrlichstrasse 28, D-72076 Tu¨bingen, Germany
Peptides containing the cyclic product of glutamine at the N terminus are usually biologically active.
If the cyclization of glutamine was associated with a volume reduction, pressure should displace the
equilibrium in the direction of the lower volume. Here, results in model solutions and in whey are
discussed, showing that the theorized cyclization of glutamine in Gln-His-ProNH2 or Gln-Leu-ProNH2
is significantly accelerated during the application of heat and even more strongly when elevated
temperature and pressure combinations are used. The reaction rate depended on the intensity of
the pressure treatment, the pH, and the nature of the amino acids adjacent to glutamine. The products
of the reaction were identified as thyrotropin-releasing hormone (TRH) and [Leu(2)]TRH. The reported
reactions could affect the naturally balanced concentration of short-chain peptides in foods and
therefore induce unpredictable biological effects.
KEYWORDS: Pressure; pyroglutamate; thyrotropin-releasing hormone; TRH; TRH analogue
INTRODUCTION
Within minimal food-processing technologies, a treatment
with high hydrostatic pressure is an attractive technique because
of its gentle effect on the sensorial and nutritional characteristics
of the processed products (1). Despite this, chemical reactions
governed by the Le Chatelier principle are usually influenced
by a high-pressure treatment. Those reactions might lead to the
formation of undesirable compounds and, therefore, should not
be disregarded. For instance, it has been reported that high
pressure accelerates the formation of a diketopiperazine after
hydrolysis of the ester bond at the C terminus of certain peptides
(2, 3) and induces the cyclization of the amino acid glutamine
(4) under neutral or basic pH.
Structures containing the cyclic product of glutamine at the
N terminus (pyroglutamate) are not uncommon in nature. Those
peptides have usually an endocrine and/or regulative function
in the body and are of highest interest because their concentra-
tion in the blood will influence the synthesis pathways of
important hormones. Here we investigate the stability of
glutamine-containing peptides under combined pressure and
temperature conditions. If pressure induced chemical changes
in Gln-His-ProNH2 or Gln-Leu-ProNH2, the newly formed
Figure 1. Chemical structure of pGlu-His-ProNH () thyrotropin-releasing
hormone, or TRH) with pyroglutamate at the N terminus.
2
structures would probably have the sequence pGlu-His-ProNH2
or pGlu-Leu-ProNH2 as a consequence of the cyclization of
glutamine. The first compound, pGlu-His-ProNH2 [) thyrotro-
pin-releasing hormone, or TRH (see Figure 1)], is normally
produced in the hypothalamus and regulates the metabolism of
thyrotropin-stimulating hormone (TSH), prolactin, triiodothy-
ronine (T3), and thyroxine (T4). After the oral intake of TRH,
changes in the concentration of TSH, T3, and T4 have been
reported (5). The second compound, pGlu-Leu-ProNH2
[) thyrotropin-releasing hormone analogue, or [Leu(2)]TRH],
* Corresponding author [telephone + 49 (0) 721 6625 516; fax + 49
(0) 721 6625 503; e-mail veli.fernandez@bfe.uni-karlsruhe.de].
† Federal Research Centre for Nutrition.
§ Federal Research Centre for Virus Diseases of Animals.
10.1021/jf0348471 CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/25/2003

8094 J. Agric. Food Chem., Vol. 51, No. 27, 2003
Ferna´ndez Garc´ıa et al.
an atmospheric pressure electrospray ionization mode (API-ES). Peaks
were preparatively collected after HPLC loads and rechromatographed
in the LC-MS running an isocratic gradient (100% A). Injection volume
was 10 µL.
High-Pressure Treatment. Experiments up to 600 MPa were
conducted in a high-pressure device consisting of a series of thermo-
stated microautoclaves (i.d. ) 16 mm, ∼10 mL, 700 MPa) connected
by valves. Pressure was generated by an air-driven pump in combination
with a pressure intensifier. The pressure-transmitting medium was a
mixture of water and glycol (80:20).
Pressure experiments up to 800 MPa were conducted in a hydraulic
press U101 from the High Pressure Research Centre, Polish Academy
of Science (Warsaw, Poland) for up to 11000 bar. U101 is a manually
operated twin-piston hydraulic press with an automatic piston return
(80 mm piston movement; 100 mm piston length) equipped with
specialized accessories for pressurizing vessels. The vessel is a cylinder
made of steel with an inside diameter of 16 mm and a 150 mm height.
The piston position is monitored with a linear transformer transducer,
and the pressure-measuring unit is an in-vessel manganin pressure
gauge; both are digitally displayed. The transmitting medium was
petroleum ether (80-100 °C boiling point). Pressure rise required ∼120
s.
Samples were pressurized in polyethylene ampules (250 µL) and
sealed with Teflon. To ensure complete isolation, the ampules were
introduced in a polyethylene-coated aluminum bag, which was heat
sealed. The temperature in the vessels was controlled by a thermostat
Polystat from Huber (Offenburg, Germany).
is known to have anticataleptic activity and to enhance the
secretion of acetylcholine (6) but is inactive in the regulation
of TSH release. Oral intake of [Leu(2)]TRH increased the
endogenous levels of TRH, showing an inhibition of the
metabolism of this substance (7).
This study was carried out to explore the effects of pressure
and temperature on reactions that are possibly leading to the
formation of hormone-like substances. These reactions might
have a certain relevance for food processing, because the formed
substances might cause physiological disorders. Experiments
at different pH values were performed in model solutions, in
water, and in whey.
MATERIALS AND METHODS
The standard substance pyroglutamyl-histidyl-proline-NH2 was
purchased from Calbiochem (Schwalbach, Germany) and the standard
compound pyroglutamyl-leucyl-proline-NH2 from Sigma-Aldrich Che-
mie (Taufkirchen, Germany). The glutamine-containing tripeptides
glutaminyl-histidyl-proline-NH2 and glutaminyl-leucyl-proline-NH2
were synthesized at EMC Microcollections (Tu¨bingen, Germany). All
other chemicals used to carry out the analytical studies by high-
performance liquid chromatography (HPLC) were obtained from Merck
(Darmstadt, Germany) and were of LiChrosolv grade.
HPLC Specifications for TRH Detection. The effects of pressure
on the cyclization of Gln-His-ProNH2 (∼2 mM) were examined in
water, buffered systems, and whey. Buffered experiments were carried
out with Tris-HCl buffers (50 mM) adjusted to pH 7 or 9. Experiments
in whey were performed as follows: previous to the pressure experi-
ments, 50 mL of milk (1.5% fat) was centrifuged at 9000g in a Sigma
2 K15 centrifuge (Sigma Laborzentrifugen, Osterrode, Germany) at 4
°C, and the supernatant (mainly the whey fraction) was filtered for
HPLC through a 0.45 µm pore size filter from Schleicher & Schuell
(Dassel, Germany). The pH of the whey fraction remained unaltered
and slightly above 7. Afterward, a certain amount of the tripeptide was
mixed with the supernatant to achieve a concentration of approximately
∼2 mM. Baselines of chromatograms became noisy due to the presence
of proteins in the whey fraction, which, however, did not interfere with
the identification of additives by retention times and cochomatrography
with standards.
Gln-His-ProNH2 and pGlu-His-ProNH2 were detected in a high-
performance liquid chromatograph Lachrom pump with a detector
L-4200 UV-vis from Merck. A reverse phase Prodigy ODS3 C18
column, 250 mm length, 4.6 mm i.d., 5 µm pore size, from Phenomenex
(Aschaffenburg, Germany) was used. Detection was at 220 nm, and
the elution rate was 1.0 mL/min. The injection volume was 10 µL.
Data on the decay of the glutamine and the increase in the
concentration of the pyroglutamate-containing tripeptides were analyzed.
Calibration was done by comparing peak areas after the collection of
glutamine- and pyroglutamate-containing peaks.
HPLC Specifications for Detection of [Leu(2)]TRH. Gln-Leu-
ProNH2 and pGlu-Leu-ProNH2 were detected in a high-performance
liquid chromatograph Lachrom pump with a detector L-4200 UV-vis
from Merck. A reverse phase Nucleosil 100 C18 column, 250 mm
length, 4 mm i.d., from Knauer (Berlin, Germany) was used.
Effects of pressure on the cyclization of Gln-Leu-ProNH2 (∼1.7 mM)
were followed in Tris-HCl buffer, pH 7. Detection was at 220 nm, and
the elution rate was 1.0 mL/min. The injection volume was 30 µL.
Calibration of [Leu(2)]TRH was done by comparing peak areas with
the standard chemical purchased from Sigma. Only the data on the
increase in the concentration of the pyroglutamate-containing tripeptide
were analyzed.
RESULTS
TRH. The concentration of the glutamine-containing tripep-
tide Gln-His-ProNH2 (2 mM in Tris-HCl, pH 7) decreased
rapidly when the buffered samples were heated under pressure
at 600 or 800 MPa (upper panels of Figures 2 and 3,
respectively). Parallel to the decay in the concentration of Gln-
His-ProNH2, a new product was detected and identified as TRH
(lower panels of Figures 2 and 3) by LC-MS [molecular weights
[(362.1 + H)+ and (362.1 + Na)+ in standard and synthesized]
and retention times were identical]. The identified pyro-
glutamate-containing tripeptide is the product of the cyclization
of the amino acid glutamine situated at the N terminus of Gln-
His-ProNH2. Furthermore, among processing time and treatment
conditions, the molar concentrations of TRH were a symmetrical
reflection of the degradation lines of Gln-His-ProNH2 and
indicated an equimolar reaction. No other new peak could be
identified at this wavelength.
The logarithmic representation of C/C0 (where C is the
concentration of Gln-His-ProNH2 at time x and C0 is the Gln-
His-ProNH2 concentration at time 0) versus the time (in minutes)
decayed following a linear function (r2 > 0.98 in Table 1),
and the reaction constant (k) could be calculated from the slope.
Results presented in Table 1 revealed that the higher pressure
intensities used, combined with the higher temperatures, were
able to accelerate the rate of conversion of glutamine into
pyroglutamate: 800 MPa was more effective than 600 MPa,
and k for the degradation of Gln-His-ProNH2 increased when
the treatment intensity was increased. Remarkable is the yield
of ∼80% TRH after reaction for 30 min at 800 MPa and 80
°C. Moreover, 120 min under the highest pressure/temperature
intensities led to a roughly complete conversion into TRH. High-
pressure sterilization of foods requires the application of high
temperatures to inactivate spores: the interpolation of values
at 10 min, a holding time typically required during commercial
pressurization treatments to achieve a convenient inactivation
of microorganisms, would indicate a decrease of >30% in the
concentration of the initial Gln-His-ProNH2 if samples were
pressurized at 800 MPa and 80 °C.
Mobile Phases. Mobile phases for both experiments were (A)
trifluoroacetic acid (0.1%) in water and (B) trifluoroacetic acid (1%)
in water, mixed with acetonitrile, ratio 2:3. A gradient was run from
100% A to 50% B in 20 min. Peak areas were measured by the
integration software Kroma System 2000 from Kontron (Milan, Italy).
Peak Identification. Peaks containing pyroglutamate were identified
by cochromatography with authentic standards and by means of a liquid
chromatograph coupled to a mass spectrometer (HP 1100 series), using

Pressure Yields Hormone-like Peptides
J. Agric. Food Chem., Vol. 51, No. 27, 2003 8095
Figure 2. (Top) Decay of 2 mM Gln-His-ProNH in 50 mM Tris-HCl buffer,
Figure 3. (Top) Decay of 2 mM Gln-His-ProNH in 50 mM Tris-HCl buffer,
2
2
pH 7; (bottom) formation of TRH (pGlu-His-ProNH ) from 2 mM Gln-
pH 7; (bottom) formation of TRH (pGlu-His-ProNH ) from 2 mM Gln-
2
2
His-ProNH in 50 mM Tris-HCl buffer, pH 7: ([) 600 MPa, 20 °C; (4)
His-ProNH in 50 mM Tris-HCl buffer, pH 7: ([) 800 MPa, 20 °C; (4)
2
2
0.1 MPa, 60 °C; (2) 600 MPa, 60 °C; (0) 0.1 MPa, 80 °C; (9) 600
MPa, 80 °C. Data points are means of three independent replications;
the average standard deviation was always <10%.
0.1 MPa, 60 °C; (2) 800 MPa, 60 °C; (0) 0.1 MPa, 80 °C; (9) 800
MPa, 80 °C. Data points are means of three independent replications;
the average standard deviation was always <8%.
Table 1. Reaction Constants (min-1) for the Decay of Gln-His-ProNH
under Isobaric−Isothermal Conditions in 50 mM Tris-HCl Buffer, pH 7
The concentration of TRH after pressure treatment at pH 7
(lower panels of Figures 2 and 3) was comparable to the
concentration of TRH after pressurization of 2 mM Gln-His-
ProNH2 in whey (Figure 4). The reaction rate under those
conditions was similar, and no additional matrix effects could
be reported. Milk is a complex matrix with several potent
buffering systems that seem to be stable under pressure. On
the other hand, solutions of 2 mM Gln-His-ProNH2 pressurized
in Tris-HCl buffer at pH 9 (Figure 4) showed that, surprisingly,
the reaction rate for the conversion of the glutamine-containing
tripeptide into TRH was slower at basic pH, as the levels of
TRH at pH 7 or in whey were not reached. Following the Le
Chatelier principle, the achieved equilibrium toward the depro-
tonated amine should be pressure sensitive. However, it has been
probably compensated by the amino acids situated at the second
and third positions of the tripeptide.
The experiments explained above were carried out in 50 mM
Tris-HCl buffer, pH 7 and 9, a pressure-resistant buffer because
of its very small ionization volume, or in whey. However,
aqueous solutions of Gln-His-ProNH2 were stable under pressure
(Figure 4), and the cyclization could not take place, probably
due to the self-ionization of water, which is promoted by
pressure. Self-ionization of water results in a lowering of the
pH and, therefore, the stabilization of the tripeptide by proto-
nation of the N-terminal amine.
2
treatment
k (× 10-3 min-1
)
r 2
600 MPa, 60 °C
800 MPa, 60 °C
600 MPa, 80 °C
800 MPa, 80 °C
7.6
8.6
26.7
39.1
0.99
0.98
0.99
0.99
panels of Figures 2 and 3) because 40% of 2 mM Gln-His-
ProNH2 in Tris-HCl, pH 7, was converted into TRH after 30
min and only 10% of the original substance remained after 120
min. Furthermore, experiments performed in whey under
conditions necessary to fulfill a long pasteurization (60 °C, 30
min) showed that at least 10% of the glutamine-containing
tripeptide was converted into the hormone-like structure (TRH).
Contrary to these results, the amino acid glutamine itself was
rather stable when heated at 50 °C (4), and only negligible losses
have been reported.
[Leu(2)]TRH. The glutamine-containing tripeptide Gln-Leu-
ProNH2 (∼1.7 mM in Tris-HCl, pH 7) disappeared slowly when
the buffered samples were heated under pressure at 600 MPa
(Figure 5). Only the higher pressure intensities used in
combination with the higher temperatures accelerated the rate
of conversion of glutamine into pyroglutamate: 800 MPa was
considerably more effective, and the reaction rate for the
degradation of Gln-Leu-ProNH2 increased with increasing
treatment intensity. Remarkable is the loss of ∼30% Gln-Leu-
ProNH2 after reaction for 120 min at 800 MPa and 80 °C.
In addition, heat treatments at normal pressure triggered the
cyclization of glutamine in buffered systems as well. Heating
at 60 °C for 30 min helped the reaction of ∼6% of 2 mM Gln-
His-ProNH2. The values found at 80 °C were remarkable (lower

8096 J. Agric. Food Chem., Vol. 51, No. 27, 2003
Ferna´ndez Garc´ıa et al.
natural matrix. Those molecules were identified as TRH and
[Leu(2)]TRH. Conditions approximating parameters necessary
for a pressure pasteurization or sterilization (600 or 800 MPa
at 60 or 80 °C) would result in the accelerated formation of
two hormone-like structures with probable endocrine and
regulatory functions. The expected results showed that heating
the samples at mild temperatures (60 or 80 °C) at neutral or
basic pH would also lead to a considerable enhancement in the
production of such bioactive peptides. Other authors (11) have
identified pyroglutamate-containing short-chain peptides after
hydrolysis of protein-rich preparations. They concluded that
certain (nonspecified) processing conditions would induce the
formation of the nondigestible pyroglutamate at the N-terminal
glutamine. We have shown here that the application of heat
alone, and of heat accompanied by high pressure, might greatly
accelerate the cyclization of the N-terminal glutamine at neutral
pH.
Figure 4. Formation of pGlu-His-ProNH from 2 mM Gln-His-ProNH in
2
2
water, 50 mM Tris-HCl buffer, pH 9, and whey. Data points are means of
three independent replications; the average standard deviation was always
<5%.
Yields of glutamine cyclization under pressure were deter-
mined by the nature of the amino acids situated at positions
close to the R-amine. The glutamine at the N terminus of Gln-
His-ProNH2 was rapidly cycled in buffers at neutral pH and in
whey but more slowly at basic pH. Contrarily, glutamine itself
had proven to be more stable to pressure-induced cyclization
at neutral than at basic pH (4). The tripeptide illustrates an effect
probably marked by the presence of the imidazole group of
histidine at the second position. The imidazole is weakly acid
at basic pH, thus negatively charged, and might weaken the
nucleophilic character of the free amine at the N terminus by
competing with the available electron par, avoiding cyclization.
In the same way Gln-Leu-ProNH2 was rather pressure stable at
neutral pH, and only pressures of up to 800 MPa applied at 80
°C for at least 120 min were able to induce a remarkable reaction
to pyroglutamate, showing that the lateral chain of leucine, an
unpolar aliphatic amino acid, might protect the adjacent
glutamine from cyclization. The influence of pressure treatments
on other peptides containing glutamine at the N terminus
remains to be investigated and will be the subject of forthcoming
works. However, and in contrast to the results presented here,
Butz et al. (3) showed that peptides containing glutamine at
the C terminus are stable after application of pressure combined
with temperature, in the same way as they are generally stable
in solution, and therefore regularly used as glutamine supple-
mentation.
Parallel to the degradation of Gln-Leu-ProNH2, a new product
was detected and identified as [Leu(2)]TRH [molecular weights
[(338.2 + H)+ and (338.2 + Na)+ in standard and synthesized]
and retention times were identical]. The pyroglutamate-contain-
ing tripeptide would be the product of the cyclization of the
amino acid glutamine situated at the N terminus of Gln-Leu-
ProNH2. No other new peak was identified at this wavelength.
DISCUSSION
Generally, any reaction in food to which the principle of Le
Chatelier applies is of interest under hyperbaric conditions.
According to this principle, under equilibrium, a process
associated with a decrease in volume will be favored by
pressure. Among others, cyclization reactions involving short-
chain peptides are generally governed by the Le Chatelier
principle and are of highest interest because the cyclization
products might cause off-flavors in foods, as is known in the
case of glutamine (8), or have a certain physiological relevance
(9).
The accelerated cyclization of the amino acid glutamine at
high temperatures has been known for a very long time (10).
Here we communicate that two new molecules have been rapidly
generated when glutamine-containing tripeptides were heated
while pressurized in buffers at neutral or basic pH and in a
Figure 5. Formation of [Leu(2)]TRH (pGlu-Leu-ProNH ) from 1.7 mM Gln-Leu-ProNH in 50 mM Tris-HCl buffer, pH 7: (white) residual concentration of
2
2
Gln-Leu-ProNH ; (black) concentration of pGlu-Leu-ProNH after treatment. Data points are means of three independent replications; the average standard
2
2
deviation was always <5%.

Pressure Yields Hormone-like Peptides
J. Agric. Food Chem., Vol. 51, No. 27, 2003 8097
(2) Butz, P.; Ferna´ndez Garc´ıa, A.; Fister, H.; Tauscher, B. Influence
of high hydrostatic pressure on aspartame: Instability at neutral
pH. J. Agric. Food Chem. 1997, 45, 302-303.
(3) Butz, P.; Ferna´ndez Garc´ıa, A.; Schneider, T.; Sta¨rke, J.;
Tauscher, B.; Trierweiler, B. The influence of high pressure on
the formation of diketopiperazine and pyroglutamate rings. High-
Pressure Res. 2002, 22, 697-700.
(4) Schneider, T.; Butz, P.; Ludwig, H.; Tauscher, B. Pressure
induced formation of pyroglutamic acid from glutamine in neutral
and alkaline solutions. Lebensm.-Wiss. -Technol. 2003, 36, 365-
367.
The typical parameters necessary for a pressure pasteurization
range from 400 to 700 MPa, at temperatures not higher than
40-50 °C. Under those conditions we expect that the related
reactions would have only a negligible importance. However,
pressure/temperature combinations to achieve sterilization pa-
rameters use continuous pressure or pressure pulses up to 1000-
1200 MPa at temperatures of ∼115 °C. In this case a rapid
yield of bioactive peptides containing pyroglutamate at the N
terminus could be expected in foods at neutral pH or in highly
proteolytic environments.
The principal question arising from the discussed results is
whether the target peptides could be naturally available in foods
and, therefore, whether the formed hormone-like substances
could be relevant in the natural matrices. Indeed, the generation
of short-chain peptides with glutamine at the N terminus should
not be disregarded because glutamine and glutamic acid are,
for example, the major amino acids in milk, quantified as ∼20%
of all amino acids in milk proteins (12). For instance, the
concentration of pyroglutamate has been acceptably correlated
to the ripening state of hard-cooked cheeses (13). On the other
hand, TRH and TRH derivatives have been reported to exist
naturally in milk (14). Those compounds are usually the product
of enzymatic activity in the matrix, meaning that the precursor
molecules are present in the food. Consequently, the formation
of precursor peptides during food processing should be a
question of probability.
Roberts et al. (15) concluded that short peptides generated
in the diet can be absorbed intact through the intestine and
probably play a role in the modulation of organ functions and
disease. Moreover, evidence has been reported on the resistance
to degradation during digestion of forms containing pyro-
glutamic acid at the N terminus (16). Because of that, the
products of the investigated reactions could have effects at the
metabolic level. On the one hand, the pyroglutamate ring present
at the N terminus is not digested by regular aminopeptidases,
with the consequent reduction in the concentration of free
glutamine and the possible effects on the bioavailability of this
amino acid, relevant for persons with intestinal disorders. If TRH
was absorbed in the gut, concentrations found in the blood might
have, for example, consequences on the liberation of TRH
derivatives. Indeed, an oral intake of TRH and TRH analogues,
among them pGlu-Leu-ProNH2, raises the levels of TRH in the
spinal cord and the brain, whereas the levels of cyclo(His-Pro)
decrease (7). The physiological effects might also be positive,
as, for example, pyroglutamic acid has been found to improve
age-associated memory impairment (17).
(5) Kauppila, A.; Ylikorkala, O. Effects of oral and intravenous TRH
and metoclopramide on PRL and TSH secretion in women. Clin.
Endocrinol. 1982, 17, 617-623.
(6) Prokai, L.; Zharikova, A. D. Neuropharmacodynamic evaluation
of the centrally active thyrotropin-releasing hormone analogue
[Leu2]TRH and ist chemical brain-targeting system. Brain Res.
2002, 952 (2), 268-274.
(7) Prasad, C.; Eloby-Childress, S.; Iriuchijima, T.; Wilber, J. B.;
Szirtes, T.; Kisfaludy, L. TRH analog administration increases
endogenous TRH levels in the central nervous system. Neuroen-
docrinology 1989, 49 (2), 219-222.
(8) Shallenberger, R. S.; Pallesen, H. R.; Moyer, J. C. Formation of
pyrrolidoncarboxylic acid during the production of beet puree.
Food Technol. 1957, Feb, 92-93.
(9) Prasad, C. Bioactive cyclic dipeptides. Peptides 1995, 16 (1),
151-164.
(10) Archibald, R. M. Chemical characteristics and physiological roles
of glutamine. Chem. ReV. 1945, 37, 161-208.
(11) Higaki-Sato, N.; Sato, K.; Esumi, Y.; Okumura, T.; Yoshikawa,
H.; Tanaka-Kuwajima, C.; Kurata, A.; Kotaru, M.; Kawabata,
M.; Nakamura, Y.; Ohtsuki, K. Isolation and identification of
indigestible pyroglutamyl peptides in an enzymatic hydrolysate
of wheat gluten prepared on an industrial scale. J. Agric. Food
Chem. 2003, 51, 8-13.
(12) Davis, T. A.; Nguyen, H. V.; Garcia-Bravo, R.; Fiorotto, M. L.;
Jackson, E. M.; Lewis, D. S.; Lee, R. L.; Reeds, P. J. Amino
acid composition of human milk is not unique. J. Nutr. 1997,
124, 1126-1132.
(13) Mucchetti, G.; Locci, F.; Gatti, M.; Neviani, E.; Addeo, F.;
Dossena, A.; Marchelli, R. Pyroglutamic acid in cheese: pres-
ence, origin, and correlation with ripening time of Grana Padano
cheese. J. Dairy Sci. 2000, 83, 659-665.
(14) Ghilchik, M. W.; Tobaruela, M.; Rio-Garcia, J.; Smyth, D. G.
Characterization of neutral TRH-like peptides in mammary gland,
mammary tumors and milk. Biochim. Biophys. Acta 2000, 1475,
55-60.
(15) Roberts, P. R.; Burney, J. D.; Black, K. W.; Zaloga, G. P. Effect
of chain length on absorption of biologically active peptides from
the gastrointestinal tract. Digestion 1999, 60, 332-337.
(16) Masson, M. A.; Moreau, O.; Debuire, B.; Han, K.-K. Evidence
for the resistance of thyrotropin-releasing hormone (TRH) and
pseudo-hormone, pyroglutamyl-histidyl-amphetamine, to deg-
radation by enzymes of the digestive tract in vitro. Biochimie
1979, 61, 847-854.
The increment in the concentration of pyroglutamate-contain-
ing peptides in buffers and in a natural matrix discussed
throughout this paper would therefore affect the naturally
balanced concentration of short-chain peptides when occurring
in foods and might induce unpredictable biological effects that
should be further studied in natural peptides contained in food
matrices.
(17) Grioli, S.; Lomeo, C.; Quattropani, M. C.; Spignoli, G.; Villardita,
C. Pyroglutamic acid improves the age associated memory
impairment. Fundam. Clin. Pharmacol. 1990, 4, 169-173.
LITERATURE CITED
(1) Ferna´ndez Garc´ıa, A.; Butz, P.; Bognar, A.; Tauscher, B.
Antioxidative capacity, nutrient content and sensory quality of
orange juice and an orange-lemon-carrot juice product after high-
pressure treatment and storage in different packaging. Eur. Food
Res. Technol. 2001, 213, 290-296.
Received for review July 28, 2003. Revised manuscript received October
22, 2003. Accepted October 27, 2003.
JF0348471