From the Department of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
Received for publication, October 25, 2000, and in revised form, January 18, 2001
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ABSTRACT |
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Isolation and structure analysis of two amino
acids from bovine ligamentum nuchae elastin hydrolysates revealed the
presence of pyridine cross-links in elastin. The structures of these
amino acids were determined to have 3,4,5- and 2,3,5-trisubstituted pyridine skeletons both with three carboxylic acids and a mass of
396 (C18H28N406)
identified as
4-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)-pyridine and
2-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)-pyridine. We have named these pyridine cross-links desmopyridine (DESP) and
isodesmopyridine (IDP), respectively. Structure analysis of these
pyridine cross-links implied that the formation of these cross-links
involved the condensation reaction between ammonia and allysine. The
elastin incubated with ammonium chloride showed that DESP and IDP
levels increased as the allysine content decreased. DESP and IDP were
measured by high pressure liquid chromatography (HPLC) with UV
detection and were found in a variety of bovine tissues. The
DESP/desmosine (DES) and IDP/isodesmosine (IDE) ratios in aorta elastin
were higher than in other tissues. DESP and IDP contents in human aorta
elastin were found to be gradually increased with age. The
concentration of IDP was significantly elevated in aorta elastin of rat
with chronic liver cirrhosis induced by carbon tetrachloride (mean ± S.D.; 11.1 ± 0.9 nmol/mg elastin) when compared with normal
rats (5.9 ± 1.5 nmol/mg elastin). Although DESP and IDP are
present at only trace concentrations in the tissue elastin, these
pyridine cross-links may be useful biomarkers for the aortic elastin
damaged by ammonia.
The elastic properties of several vertebrate tissues such as lung,
skin, and large blood vessels are mainly due to the presence of elastic
fibers. Elastin, the major component of these fibers, is synthesized by
mesenchymal cells as a soluble precursor, tropoelastin, which undergoes
posttranslational modifications leading to the formation of specific
cross-links that join together several tropoelastin chains (1).
All of the cross-links in elastin are formed spontaneously after
oxidative deamination of specific lysine residues of tropoelastin by
lysyl oxidase in the extracellular space. Formed reactive aldehyde, allysine, reacts with lysine and/or another allysine to form
polyfunctional cross-links such as allysine-aldol, lysinonorleucine,
merodesmosine, desmosine
(DES),1 isodesmosine (IDE),
and cyclopentenosine (2-10).
Elastin and collagen are the key structural components of the mammalian
artery wall, particularly in the aorta (11). In concert with other
extracellular matrix macromolecules, these two proteins provide the
structural integrity and resiliency required of this specialized blood
vessel (12). Extracellular matrix proteins, such as collagen and
elastin, are known to be greatly affected by age because of impaired
functional properties and increased susceptibility to diseases (13).
Collagen undergoes progressive changes that are characterized by
decreased solubility, decreased proteolytic digestibility, increased
heat denaturation time, and accumulation of yellow and fluorescent
material (14-16). These changes are thought to result from the
formation of age-related intermolecular cross-links (17, 18). Also,
age-related changes of elastin are its fragmentation and progressive
proteolysis (19). These changes are accompanied by a reduction of
elastin in itself and by content of elastin cross-links. Previously, we
isolated two new dihydrooxopyridine cross-links, oxodesmosine (OXD) and isooxodesmosine (IOXD), from the acid hydrolysates of the bovine aortic
elastin (20). Recently, we found that OXD and IOXD are oxidative
metabolic intermediates generated from DES and IDE in vitro,
respectively, by reactive oxygen species (21). Structures of DES, IDE,
OXD, and IOXD are shown in Fig. 7. Little is known about the formation
of cross-links associated with the damaged elastin in
vivo.
This study was undertaken to elucidate the chemical nature of
cross-linking structures of elastin initiated by lysyl oxidase. We
isolated two pyridine cross-links, desmopyridine (DESP) and isodesmopyridine (IDP) from ligamentum nuchae elastin hydrolysates. Using a reverse-phase HPLC assay, we measured the levels of these pyridine cross-links in bovine tissue elastin and showed that the
concentrations of both cross-links increased in concert with age in
human aorta elastin. In the present study, we found that DESP and IDP
were formed during incubation of ammonium chloride with ligamentum
nuchae elastin consistent with the decrease of allysine residues.
Compared with normal rats, the IDP concentration was found to increase
in rat aorta elastin with liver cirrhosis induced by carbon
tetrachloride, consistent with the decrease of allysine. These results
suggested that allysine residues undergo a Chichibabin reaction
(22, 23) with ammonia to form DESP and IDP in elastin. IDP may be
an abnormal cross-link in elastin.
Materials--
Acetonitrile (HPLC-grade), p-cresol,
sodium dihydrogen phosphate, hydrochloric acid, EDTA, carbon
tetrachloride, mineral oil (all analytical grade), and diphosphorous
pentaoxide (P2O5) were purchased from
Nacalai Tesque (Kyoto, Japan). Activated charcoal (60-150 mesh)
and Silica Gel 60 (70-230 mesh) for column chromatography were
obtained from Nacalai Tesque, and silica gel (Art. 7734) for
column chromatography was obtained from Merck (Darmstadt, Germany).
Deuterium oxide (D2O) and dioxane D8 for
NMR spectroscopy were obtained from EURISO-TOP (Saint Aubin Cedex, France).
Animal Experiments--
Male Wistar rats (200-230 g, 8 weeks of
age) were used in the experiments. The animal studies were in strict
accordance with the guidelines established by the Canadian Council on
Animal Care. Chronic liver injury was induced in rats by repeated
intraperitoneal injections of carbon tetrachloride/mineral oil (1:1) at
a dose of 1 ml/kg of body weight three times a week (on Monday,
Wednesday, and Friday) for a total of 10 injections (24). Rats were
killed under diethyl ether anesthesia 4 days after the last injection. Rats were used for this study only when evidence of cirrhosis was
confirmed histologically.
Preparation of the Elastin--
The elastin from various bovine
tissues was purified by the modified technique with 1 M
NaCl treatment (described previously (25)) as follows. After removing
peripheral connective tissue and lipids, bovine tissues (wet weight
10 g) cut into small segments with a mixer were suspended in 1 M NaCl (50 ml) for 24 h. The supernatant was discarded
after centrifugation, and then the pellet was re-suspended in 1 M NaCl. This extraction process was repeated three times.
The insoluble residue was washed with distilled water. After
centrifugation, the insoluble residue was washed with an excess of
distilled water and defatted with chloroform/methanol (2:1, v/v) for
48 h, and washed with ethanol and ether. The prepared elastin was
dried in vacuo under P2O5. The human
aorta was kindly supplied by the Department of Pathology, Tohoku
University Hospital. Human aortic samples were obtained from 11 autopsy
cases, which had no particular aortic diseases but had sclerotic
changes corresponding with their ages. The range of age was from 19 to
75 years old. The materials were taken from nonatherosclerotic areas of
the thoracic aorta and were preserved in methanol at Isolation of DESP and IDP from Acid Hydrolysates of
Elastin--
The elastin for the isolation of DESP and IDP was
prepared from fresh bovine ligamentum nuchae (about 1 kg). After acid
hydrolysis of elastin with 6 N HCl (110 °C, 48 h),
an aliquot of 30 g of its acid hydrolysates was dissolved in
distilled water (40 ml) and then charged on a large scale charcoal
column (180 × 65 mm). Major lysine-diethyl cross-linking amino
acids were fractionated with water (3 liters) followed by elution with
20% aqueous methanol solution (3 liters). The 20% aqueous methanol
fraction was evaporated at 50 °C. The residue was dissolved with 40 ml of ethyl acetate/acetic acid/water (1.5:1:1, v/v) and then charged
on a large scale silica gel column (130 × 65 mm). After the
elution of neutral amino acids with a solvent (1.5 liters) of ethyl
acetate/acetic acid/water (1.5:1:1, v/v), most of the lysine-derived
cross-links were eluted with water (0.5 liter). The water elution was
evaporated at 50 °C. The residue was dissolved with 40 ml of ethyl
acetate/acetic acid/water (1.5:1:1, v/v), and then an aliquot of 2 ml
was charged on a LiChroprep Si60 (40-63 µm) preparative silica gel
column (310-25 mm, Merck) and was fractioned using ethyl
acetate/acetic acid/water (1.5:1:1, v/v) as a solvent at a flow rate of
3.0 ml/min. DESP and IDP were eluted at 160-180 and 190-210 min,
respectively. After removing the solvent by evaporation at 50 °C,
the residue containing DESP or IDP was dissolved with water (1.0 ml)
containing 0.02 N HCl and then charged on a preparative ODS
column (40-63 µm) (LiChroprep RP-18, 310-25 mm, Merck). Both DESP
and IDP were eluted at 20-24 min using a solvent of water (1.0 ml)
containing 0.02 N HCl, at a flow rate of 3.0 ml/min. After
this method was repeated five times, DESP and IDP were purified by
rechromatography with a preparative silica gel column, respectively.
The purity of DESP and IDP was confirmed by silica gel TLC and
analytical HPLC. TLC was conducted by precoated Kieselgel 60 on an
aluminum sheet (Merck) using ethyl acetate/acetic acid/water (2:1:1,
v/v) as a solvent. DESP and IDP were eluted just before DES and IDE revealed characteristic strong ninhydrin-positive spots both
with Rf of 0.08 (DES and IDE; origin) on TLC and
elution before IDE with Rt of 20.2 and 19.0 min (DES
and IDE; Rt of 34.7 and 24.7 min, respectively) on
analytical HPLC.
DESP and IDP are not artifacts formed from DES and IDE, respectively,
by acid hydrolysis. To elucidate this problem, prolonged hydrolysis of
elastin or pure DES and IDE was carried out under the condition of
110 °C for 240 h in 6 N HCl. DES and IDE were isolated from acid hydrolysates of bovine ligamentum nuchae elastin by
the method of Nakamura and Suyama (26).
In Vitro Incubation of Elastin with Ammonium Chloride--
Dried
elastin powder (50 mg) was incubated with 1.0 M ammonium
chloride in 5 ml of 0.2 M phosphate buffer (pH 7.4)
containing 10 µl each of toluene and chloroform to prevent microbial
growth. As a control experiment, dried elastin powder (50 mg) was
incubated without ammonium chloride in 5 ml of 0.2 M
phosphate buffer (pH 7.4) containing 10 µl each of toluene and
chloroform to prevent microbial growth. The incubation was carried out
at 37 °C or 60 °C for the time indicated in Fig. 8. After
incubation, insoluble elastin was washed free of excess ammonium
chloride three times with distilled water, and then twice with
ether/ethanol. The resulting precipitate was dried in vacuo
under P2O5 overnight, and an aliquot of 25 mg
was subjected to acid hydrolysis with 6 N HCl for 48 h
at 110 °C.
Determination of DESP and IDP by HPLC--
Elastin hydrolysates
were evaporated to dryness in vacuo at 50 °C. The
residues were dried over P2O5 and diluted with
water (1.0 ml), and then a 20-µl portion of solutions was injected
onto the reverse-phase HPLC column. The HPLC system consisted of a Shimadzu (Kyoto, Japan) LC-6A pump, an SPD-6AV UV-VIS
spectrophotometric detector, a C-R5A data station, and a Hitachi
(Tokyo, Japan) L-7300 column oven. Separation was performed with a
Mightysil RP-18 150-4.6 (5 µm) reverse-phase (Kanto Chemical, Tokyo,
Japan). The flow rate was 1.0 ml/min. All HPLC chromatographic
operations were carried out at 40 °C. Determination of pyridine
cross-links was performed with analytical HPLC using a solvent of 0.1 M phosphate buffer/acetonitrile (5:1, v/v) containing 20 mM SDS (pH 3.6). Concentrations of DESP and IDP were
determined by comparison with DES and IDE, respectively, using
analytical HPLC and monitoring at 275 nm.
Determination of Allysine as p-Cresol Derivative by
HPLC--
The allysine concentrations in elastin were determined as
allysine-bis-p-cresol derivative (APC) as described
previously (27). Derivatization of allysine in elastin was carried out
under the condition of acid hydrolysis (6 N HCl, 110 °C,
48 h). Twenty five ml of elastin was precisely weighed and then
dissolved in 5 ml of 6 N HCl containing 5% (w/v)
p-cresol in a flame-sealed Pyrex glass. Derivatization of
allysine in elastin was carried out under the condition of acid
hydrolysis at 110 °C for 48 h. Five ml of n-hexane/ether (8:2, v/v) was added to the solution after
the reaction, and then the organic layer was discarded. Excess
p-cresol was extracted with n-hexane/ether (8:2,
v/v). APC was not detected by HPLC in the organic layer. The resulting
solution was evaporated to dryness in vacuo at 50 °C. The
residue was dried over P2O5 and dissolved with
HPLC solvent (10 ml); then a 20-µl portion of solution was injected
into the reverse-phase HPLC column. The HPLC system consisted of a
Shimadzu (Kyoto, Japan) LC-6A pump, an SPD-6AV UV-VIS
spectrophotometric detector, a C-R5A data station, and a Hitachi
(Tokyo, Japan) L-7300 column oven. Separation was performed with a
LiChrosper 100 RP-18 125-4 reverse-phase column (Merck). A guard
column (LiChrosper 100 RP-18, 4 × 4 mm inner diameter, 5 µm
particle size; Merck) was placed just before the inlet of the
analytical column to reduce contamination of the analytical column. The
mobile phase was composed of 0.05 M sodium dihydrogen
phosphate(pH 2.2)/acetonitrile (3:1). The flow rate was 1.0 ml/min, and
the column temperature was 30 °C. The UV signal was monitored at 282 nm, which corresponds to an absorption maximum for APC.
Determination of Allysine-aldol by HPLC--
Allysine-aldol was
determined as the pyridine derivative,
6-(3-pyridyl)piperidine-2-carboxylic acid by the method of Nakamura and
Suyama (25). The HPLC system consisted of a Shimadzu (Kyoto, Japan)
LC-6A pump, an SPD-6AV UV-VIS spectrophotometric detector, and a C-R5A
data station. Separation was performed with a LiChrosper 100 RP-18
125-4 reverse-phase column (Merck). A guard column (LiChrosper 100 RP-18, 4 × 4 mm inner diameter, 5 µm particle size; Merck) was
placed just before the inlet of the analytical column to reduce contamination of the analytical column. The mobile phase was composed of 0.1 M phosphate buffer/acetonitrile (5:1, v/v)
containing 20 mM SDS (final pH 3.95). The flow rate was 1.0 ml/min. The UV signal was monitored at 260 nm, which corresponds to an
absorption maximum for 6-(3-pyridyl)piperidine-2-carboxylic acid.
Mass Spectrometry--
Positive ion fast atom bombardment (FAB)
mass spectra were obtained using a JEOL JMS700 mass spectrometer
operated at an accelerating voltage of +10 kV. The FAB gun was operated
at an accelerating voltage of 6 keV with an emission current of 10 mA
using xenon atoms as the bombarding gas. The sample was mixed with
glycerol/water.
UV Spectrometry--
The UV spectra for DESP and IDP were
obtained with a UV-2100S spectrophotometer (Shimadzu, Kyoto, Japan).
NMR Spectroscopy--
Structural assignments were made from
sample solutions in D2O. NMR measurements were performed at
20 °C using a Varian Inova 600 MHz spectrometer operating at 600 and
150 MHz for 1H and 13C NMR experiments,
respectively. 1H and 13C chemical shifts were
internally referenced to dioxane at 3.5 and 66.3 ppm, respectively. The
water resonance was attenuated by presaturation during the 0.5-s
relaxation delay. 1H resonance assignments were obtained
using 1H-1H correlated spectroscopy
(1H-1H COSY). Data matrix was 512 real by 2048 complex for the 1H-1H COSY experiment.
Simultaneous 1H and 2H decoupling of
13C was achieved with a WALTZ-16 pulse sequence during
relaxation (1H) and acquisition (1H,
2H). The 13C resonance from carbon with a
directly attached proton was assigned using heteronuclear single
quantum coherence (HSQC) spectroscopy. HSQC spectroscopy was the pulse
field gradient standard Varian program GHSQC. The 13C
resonances from carbons without a directly attached proton were assigned using heteronuclear multiple-bond coherence (HMBC)
spectroscopy. HMBC spectroscopy was the pulse field gradient standard
Varian program GHMQC. Carbon multiplicity was established by
distortionless enhancement by polarization transfer.
Isolation of DESP and IDP--
DESP and IDP were isolated from
acid hydrolysates of bovine ligamentum nuchae elastin by normal- and
reversed-phase chromatography. DESP and IDP were well separated by
reverse-phase ODS column chromatography using a solvent of water
containing 0.02 N HCl, which is the general solvent system
for peptides, suggesting that those compounds are polyfunctional amino
acids. The purities of DESP and IDP were confirmed by TLC and
analytical HPLC analyses. DESP and IDP were completely separated by TLC
as heavy ninhydrin single spots (Rf of both
compounds was 0.08; Rf of both DES and IDE was 0.0).
With HPLC elution systems that will resolve IDE and DES, DESP and IDP
were (Rt = 20.2 and 19.0 min, respectively) eluted
before DES (Rt = 34.7 min) and IDE
(Rt = 24.7 min) and fractionated as single peaks
(Fig. 1). DESP and IDP were hygroscopic,
white solids with a faint yellow tinge and were soluble in aqueous
solvents but not in dry methanol.
Structural Elucidation of DESP and IDP--
The UV spectra of DESP
and IDP are shown in Fig. 2. DESP
exhibited absorption maximum at 264.8 nm in 0.1 N HCl. The
UV spectrum was reversibly shifted in 0.1 N NaOH. The UV
absorption spectrum of DESP was characteristic of 3,4,5-trisubstituted
pyridine, such as 4-ethyl-3,5-dimethylpyridine (28). IDP exhibited
absorption maximum at 272.0 nm in 0.1 N HCl. The absorption
maximum was reversibly shifted in 0.1 N NaOH to 271.1 nm.
The absorption spectrum of IDP was characteristic of
2,3,5-trisubstituted pyridine, such as 2-ethyl-3,5-dimethylpyridine
(28). Crucial structural information was obtained from the
1H NMR (in D2O). Peaks in the aromatic region
are sharp singlets; comparison with DES and IDE indicated that DESP and
IDP are 3,4,5- and 2,3,5-trisubstituted pyridines, respectively. A
schematic drawing of DESP and IDP is shown in Fig.3, respectively and
indicates the numbering system used. The proton assignments were
completed using 1H-1H COSY experiments, shown
in Fig.3, B and D. The 1H NMR
spectrum of DESP showed the following signals for Formation of DESP and IDP from the Chichibabin Reaction between
Allysine and Ammonia--
The cross-linking in elastin involves
allysine (the aldehyde produced by oxidative deamination of lysine
residues) as well as intact lysine residues. A mechanism featuring the
formation of DES and IDE has been proposed by some investigators
(29-32). In the present study, two novel cross-linking amino acids,
DESP and IDP, have three amino acid groups and pyridine skeletons. DESP
and IDP were determined to be 3,4,5- and 2,3,5-trisubstituted pyridine,
respectively, by structural analyses. These structures were similar to
those of DES and IDE, respectively, as shown in Fig.
7. The studies with model systems have
shown the detail cross-linking mechanism of DES and IDE (29, 30). DES
can be formed from the condensation of the
In vitro experiments at 60 °C were also carried out. As
shown in Fig. 8C, DESP and IDP were formed linearly with the
incubation; the formation of 0.80 nmol/mg DESP and 1.66 nmol/mg IDP was
observed after 14 days of incubation. A time-dependent
decrease of allysine was observed; the allysine content decreased to
about 76% of the control level after 14 days of incubation (Fig.
8D). In this experiment the decrease of allysine-aldol was
not detected after 14 days of incubation by the HPLC analysis.
Formation of DESP and IDP in Vivo--
From in vitro
experiments, two pyridine cross-links (DESP and IDP) may be formed from
the Chichibabin reaction between 3 mol of allysine and 1 mol of
ammonia. The main source of ammonia for the formations of pyridine
cross-links in vivo, however, has remained obscure. In
vivo, deamination of the
As shown in Table I, the IDP content in
the rat aorta elastin after long-term CCl4 treatment (4 weeks) was significantly higher (p < 0.05) than that
observed in the control rat. Also, the allysine content decreased as
compared with that in the control rat (Table I). No DESP could be
detected in aorta elastin of either control or cirrhotic rats by use of
the reverse phase-HPLC analytical procedure described in Fig. 1.
Detection of DESP and IDP in Vivo--
Bovine tissue elastin was
analyzed for DESP and IDP content using the reverse phase-HPLC
analytical procedure described in Fig. 1. The peak identities were
confirmed by coelution with the standard. Contents of DESP, IDP,
allysine, DES, IDE, and allysine-aldol in bovine tissues from different
organs are shown in Table II. DESP and
IDP were found to be distributed in all of the bovine tissues
containing DES and IDE. To assess the biological meaning of pyridine
cross-links, we investigated the ratio of DESP:DES and IDP:IDE in
bovine tissue elastin. Both ratios exhibited a high value in aorta
elastin. We investigated the age-related changes of human aorta elastin
cross-links, such as DESP, IDP, DES, and IDE, and of possible
precursors of pyridine cross-links, allysine and allysine-aldol (Fig.
9). The DES, IDE, allysine, and
allysine-aldol contents of aorta elastin decreased gradually with age.
The DES and IDE contents were decreased gradually with age.
The discovery of pyridine cross-links in bovine ligamentum nuchae
elastin is the first molecular evidence for the involvement of ammonia
and allysine in elastin cross-linking. We have named these pyridine
cross-links, desmopyridine (DESP) and isodesmopyridine (IDP),
respectively, because these pyridine cross-links were 3,4,5- and
2,3,5-trisubstituted pyridine and were similar to quaternary pyridinium
skeletons of DES and IDE. The condensation of aldehydes, ketones,
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C until analysis. Human aorta elastin was purified by the technique as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Representative HPLC profiles of bovine
ligamentum nuchae elastin (A), DESP
(B), and IDP standards (C).
Bovine ligamentum nuchae elastin was hydrolyzed in 6 N HCl.
The analytical HPLC condition was performed with a reverse-phase column
(Mightysil RP-18 150 × 4.6) using a solvent of 0.1 M
sodium phosphate buffer/acetonitrile (5:1) containing 20 mM
SDS at pH 3.6.
H
(D2O): 1.624 (H-12, 2H), 1.818 (H-13, 2H), 2.090 (H-8, 2H),
2.090 (H-17, 2H), 2.818-2.927 (H-7, 2H), 2.818-2.916 (H-16, 2H),
2.807-2.916 (H-11, 2H), 3.810 (H-9, 1H, t), 3.810 (H-14,
1H, t), 3.908 (H-18, 1H), 8.374 (H-2, 1H, s),
8.374 (H-6, 1H, s). 1H NMR (in D2O)
for DESP is shown in Fig. 3A.
The triplet at 3.908 ppm and the proton signals at 3.810 ppm suggested
the presence of three
-protons compatible with the presence of three
amino acids. As shown in Fig. 3B, these
-protons showed a
spin-spin interaction between aliphatic protons at 2.090 and 1.818 ppm, respectively. The proton signals at 2.818-2.927 and 2.818-2.916 ppm
suggested the presence of methyl groups on the pyridine ring. These
methyl proton signals showed a spin-spin interaction between aliphatic
protons at 2.090 and 1.624 ppm, respectively. The proton singlet at
8.374 ppm suggested the presence of two aromatic protons in the
pyridine molecule with substitutions in symmetric positions 2 and 6 by
comparing the ratio of the areas under 1H NMR. The
1H NMR spectrum of IDP showed the following signals for
H (D2O): 1.592-1.605 (H-8, 2H), 1.823 (H-9, 2H), 2.018 (H-17, 2H), 2.030 (H-13, 2H), 2.705 (H-12, 2H), 2.722 (H-16, 2H), 2.892 (H-7, 2H, t), 3.744 (H-10, 1H), 3.758 (H-14, 1H), 3.817 (H-18, 1H, t), 8.144 (H-4, 1H, s), 8.248 (H-6,
1H, s). 1H NMR (in D2O) for IDP is
shown in Fig.3C. The three coupled triplets at 3.744, 3.758, and 3.817 ppm suggested the presence of three
-protons compatible
with the presence of three amino acids. These three
-protons showed
a spin-spin interaction between aliphatic protons at 1.823, 2.030, and
2.018 ppm. The triplet at 2.892 ppm and the proton signals at 2.705 and
2.722 ppm suggested the presence of three methyl groups on the pyridine
ring. These methyl protons showed a spin-spin interaction between
aliphatic protons at 1.592 and 1.605, 2.030, and 2.018 ppm,
respectively, as revealed by a 1H-1H COSY
experiment. The two proton singlets in the aromatic region (
8.144 and
8.248) suggested the presence of two aromatic protons in the
pyridine molecule with substitutions in asymmetric positions 4 and 6. The 13C NMR resonances of DESP and IDP were assigned using
a combination of GHSQC and GHMBC spectra. The 13C NMR
spectra of DESP and IDP are shown in Fig.
4A and Fig.
5A, respectively. All the
resonances of proton-attached carbons were readily assigned based on
the one-bond 1H-13C correlation cross-peaks in
the GHSQC spectrum shown in Figs. 4B and 5B. The
assignments were confirmed by two-bond and three-bond 1H-13C correlation cross-peaks in the GHMBC
spectra, as shown in Figs.4C and 5C. The
13C resonance of the nonproton-attached carbons was also
assigned based on the cross-peaks, in the GHMBC spectra. The
13C NMR spectra of DESP showed the following signals for
C (D2O): 25.853 (C-12), 26.350 (C-7, C-16), 30.332 (C-13), 31.349 (C-8, C-17), 47.462 (C-11), 54.225 (C-9, C-18), 54.393 (C-14), 139.577 (C-2, C-6), 139.789 (C-3, C-5), 160.836 (C-4), 173.538 (C-10), 173.830 (C-15, C-19). In DESP, all of the carbons on the
aromatic ring showed resonance in the chemical shift range of
139.577-160.836 ppm. The 13C resonances at 139.577 ppm had
GHSQC cross-peaks to the H-2 and H-6 protons. The only GHMBC cross-peak
from 13C resonances at 139.577 ppm was to the H-7 proton
resonance. This demonstrated that the resonance at 139.577 ppm was from
the C-2 carbon. The only GHMBC cross-peak from 13C
resonances at 139.577 ppm was to the H-16 proton resonance. This
demonstrated that the resonance at 139.577 ppm was from the C-6 carbon.
The 13C resonance at 139.789 ppm had no cross-peaks in the
GHSQC spectrum. The resonance at 139.789 ppm was assigned to both the
C-3 and C-5 carbons because it is the only 13C resonance
that has GHMBC cross-peaks to both the H-8 and H-17. The
13C resonance at 160.836 ppm was assigned to the C-4 carbon
because it is the only 13C resonance that has GHMBC
cross-peaks to the following protons: H-2, H-6, H-7, and H-16. The
13C NMR spectra of IDP showed the following signals for
C (D2O): 25.054 (C-8), 27.045 (C-16), 28.009 (C-12), 30.363 (C-9), 30.537 (C-7), 31.152 (C-17), 31.363 (C-13), 53.879 (C-10),
54.104 (C-14, C-18), 139.007 (C-5), 139.584 (C-3), 139.420 (C-6),
148.025 (C-4), 153.058 (C-2), 173.415 (C-19), 173.714 (C-15), 173.512 (C-11). In IDP, all of the carbons on the aromatic ring showed
resonance in the chemical shift range of 139.007-153.058 ppm. The
resonances of the C-4 and the C-6 carbons were easily assigned based on
the GHSQC. That is, the resonances at 148.025 and 139.420 ppm were assigned to the C-4 and C-6 carbon, respectively. The resonance at
153.058 ppm was assigned to the C-2 carbon because it is the only
13C resonance that has GHMBC cross-peaks to both the H-4
and H-6 protons on the aromatic ring. The resonance at 139.007 ppm was assigned to the C-5 carbon because it is the only 13C
resonance that has GHMBC cross-peaks to both the H-6 and H-17 protons.
The resonance at 139.584 ppm was assigned to the C-3 carbon because it
is the only 13C resonance that has GHMBC cross-peaks to
both the H-4 and H-13 protons. The FAB mass spectrum on glycerol matrix
showed a molecular ion at m/z of 397 (M + H+) as shown in Fig. 6. High
resolution FAB-mass spectroscopy in the positive ion mode showed the
molecular mass of 397.2. This corresponded to the molecular
formula C18H28N406 and
was in agreement with the proposed structure. Based on these
characteristics, DESP and IDP were determined as 3,4, 5- and 2, 3, 5-trisubstituted pyridine cross-links, respectively.
View larger version (23K):
[in a new window]
Fig. 2.
UV absorption spectra of DESP (upper
panel) and IDP (lower panel). UV
absorption spectra were monitored in 0.1 N HCl, 0.1 N NaOH, and 0.1 N HCl in this order.
View larger version (41K):
[in a new window]
Fig. 3.
1H (A),
1H-1H COSY (B), NMR spectra of
DESP and 1H (C), and
1H-1H COSY (D) NMR spectra of
IDP. NMR measurements were performed at 20 °C using a Varian
Inova 600 MHz spectrometer. 1H chemical shift was
internally referenced to dioxane at 3.5 ppm. The water resonance was
attenuated by presaturation during the 0.5-s relaxation delay. The
insets on A and C show the structures
of DESP and IDP, respectively.
View larger version (19K):
[in a new window]
Fig. 4.
13C (A),
1H-13 C HSQC (B), and
1H-13C HMBC (C) NMR spectra of
DESP. NMR measurements were performed at 20 °C using a Varian
Inova 600 MHz spectrometer operating at 600 and 150 MHz for
1H and 13C NMR experiments, respectively.
1H and 13C chemical shifts were internally
referenced to dioxane at 3.5 and 66.3 ppm, respectively. The water
resonance was attenuated by presaturation during the 0.5-s relaxation
delay. 1H-13C HSQC spectroscopy was the pulse
field gradient standard Varian program GHSQC.
1H-13C HMBC spectroscopy was the pulse field
gradient standard Varian program GHMQC.
View larger version (20K):
[in a new window]
Fig. 5.
13C (A),
1H-13C HSQC (B), and
1H-13C HMBC (C) NMR spectra of
IDP. NMR measurements were performed at 20 °C using a Varian
Inova 600 MHz spectrometer operating at 600 and 150 MHz for
1H and 13C NMR experiments, respectively.
1H and 13C chemical shifts were internally
referenced to dioxane at 3.5 and 66.3 ppm, respectively. The water
resonance was attenuated by presaturation during the 0.5-s relaxation
delay. 1H-13C HSQC spectroscopy was the pulse
field gradient standard Varian program GHSQC.
1H-13C HMBC spectroscopy was the pulse field
gradient standard Varian program GHMQC.
View larger version (22K):
[in a new window]
Fig. 6.
FAB mass spectra of DESP (upper
panel) and IDP (lower panel). DESP and
IDP were purified and analyzed by positive FAB-mass spectroscopy. The
signal patterns of both compounds were extremely similar, and FAB high
resolution mass spectra gave the molecular formula
C18H28N406 for each
compound.
,
-unsaturated imine,
dehydromerodesmosine, and the aliphatic aldimine,
dehydrolysinonorleucine (29, 30). IDE can be formed from the
condensation of 2 mol of allysine and the aliphatic aldimine,
dehydrolysinonorleucine (29, 30). Judging from the mechanisms
for the formation of DES and IDE, we hypothesized that the formation of
DESP and IDP involved ammonia, allysine, and allysine aldol. To
determine whether DESP and IDP are formed during the reaction of
ammonia with the elastin, the elastin powder (50 mg) was incubated with
1 M NH4Cl in 5 ml of 0.2 M
phosphate buffer (pH 7.4, 37 °C). Following acid hydrolysis (6 N HCl, 48 h at 110 °C), the formation of DESP and
IDP was detected by HPLC analysis based on the coelution with the
standard of DESP and IDP, respectively. As shown in Fig.
8A, a
time-dependent increase in the amount of DESP and IDP was
observed. After 28 days of incubation, 0.44 and 0.17 nmol/mg protein of
DESP and IDP, were formed. After 28 days of incubation, 1.87 nmol/mg of
allysine was decreased (Fig. 8B). In this experiment, the
decrease of allysine-aldol was not detected after 28 days of incubation
by the HPLC analysis.
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[in a new window]
Fig. 7.
Comparisons of the structures of normal
cross-links (DES and IDE) and abnormal cross-links (DESP, IDP, OXD, and
IOXD) found in elastin. OXD and IOXD were isolated from the acid
hydrolysates of the bovine aortic elastin, and identified to have
N-substituted 1,2-dihydro-2-oxopyridine and
N-substituted 1,4-dihydro-4-oxopyridine skeletons,
respectively, with three -amino acid groups and a mass of 495 (C23H37N5O7) (20, 21).
Recently, we found that OXD and IOXD are oxidative metabolic
intermediates generated in vitro from DES and IDE,
respectively, by reactive oxygen species (21).
View larger version (28K):
[in a new window]
Fig. 8.
Changes of DESP, IDP, and allysine in bovine
ligamentum nuchae elastin (10 mg/ml) incubated with 1 M
NH4Cl under physiological conditions (A
and B) (pH 7.4, 37 °C)
and at 60 °C (C and
D) (pH 7.4). DESP (open circles), IDP
(solid circles), and allysine (solid triangles)
were assayed as described under "EXPERIMENTAL PROCEDURES." The
control experiments without NH4Cl showed no changes of
DESP, IDP, and allysine contents under both conditions (37 and
60 °C). Other experimental details are described under
"EXPERIMENTAL PROCEDURES." Values shown are means ± S.D. of
three independent determinations.
-amino group of amino acids produces
ammonia, a toxic metabolite that is detoxified by conversion into urea
via the urea cycle in the liver. In general, the concentration of
ammonia is highest in the portal vein. Under chronic liver failure,
liver cirrhosis, the blood flow of the portal vein is drained in the
systemic circulation after portal-systemic shunting; it is well known
that the blood ammonia levels are elevated in liver cirrhotic patients.
CCl4-induced cirrhotic rats represent a valuable model for
studying the causes and possible therapeutic prevention of
hyperammonemia (33, 34). In our study, we have used rats with
CCl4-induced liver cirrhosis to further investigate the
potential mechanism for the formations of pyridine cross-links in
vivo.
Content of IDP and allysine in the aorta elastin from control and liver
cirrhotic rats
Cross-links and cross-link precursors in elastin from different bovine
tissues
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Fig. 9.
Cross-links and cross-link precursor contents
in human aorta elastin as a function of age. The inset
shows the enlarged view of age-related changes of DESP and IDP.
Measurements of DES, IDE, DESP, and IDP were conducted as described in
the legends of Fig. 1, and other experimental details are described
under "EXPERIMENTAL PROCEDURES." The assays for allysine and
allysine-aldol were conducted as described under "EXPERIMENTAL
PROCEDURES."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
-unsaturated carbonyl compounds or various compounds with
ammonia or its derivatives to form substituted pyridines is known as
the Chichibabin reaction (22, 23). In vitro experiments under physiological conditions showed that allysine was decreased with
an increase in the amount of DESP and IDP in elastin after 28 days of
incubation with 1 M NH4Cl. If DESP and IDP are
formed from the Chichibabin reaction between 3 mol of allysine and 1 mol of ammonia, the decrease of allysine is estimated as 1.83 nmol/mg
from the formation of DESP (0.17 nmol/mg) and IDP (0.44 nmol/mg):
0.17 × 3 + 0.44 × 3 = 1.83. This estimated value is close to the experimental value (1.87 nmol/mg). After 14 days of
incubation at 60 °, the ratio of formed IDP to DESP was 2.0. In the
Chichibabin reaction with phenylacetaldehyde, the ratio of 2,3,5- to
3,4,5-trisubstituted pyridine formed was 2.0 (23). This value is close
to that of IDP/DESP (0.44/0.17) formed after 28 days of incubation at
37 °, that of IDP/DESP (1.66/0.80) formed after 14 days of
incubation at 60 °, and also that of IDP/DESP (0.50/0.29) in elastin
from bovine ligamentum nuchae. Two pyridine cross-links (DESP and IDP)
may be formed from the Chichibabin reaction between 3 mol of allysine
and 1 mol of ammonia. The overall reaction is shown in Fig.
10.
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Fig. 10.
The overall reaction for the formation of
DESP (upper) and IDP (lower).
R, side chain of amino acid.
1, 4-Dihydrodesmosine and 1, 2-Dihydroisodesmosine are dihydropyridines
that are believed to be the immediate biosynthetic precursors of DES
and IDE, respectively (30). 1, 2-Dihydroisodesmosine can, in theory,
arise via two different pathways: by addition of allysine to
dehydrolysinonorleucine or by Michael addition of lysine to the
allysine-aldol. Similarly, 1,2-dihydrodesmosine can, in theory, arise
by a pathway involving aldol addition of dehydrolysinonorleucine to
allysine or by a pathway involving the ,
-unsaturated imine,
dehydromerodesmosine, formation between lysine and the allysine-aldol.
Akagawa and Suyama (29) have proposed that allysine-aldol is a possible
precursor of DES and IDE by the studies with the model system. Thus
allysine-aldol can be a possible precursor of DESP and IDP. However,
the involvement of allysine-aldol in the formation of DESP and IDP
could not be shown in this study.
By sequence studies of elastin, Mecham and Foster (35) have shown that some or perhaps all allysine residues are not restricted to the alanine-enriched sites for other elastin cross-links and exist in the sequence, Gly-Ala-Glu-Allysine-(Glu), creating regions of negative charge from glutamic acid residues. Because ammonia exists as an ammonium ion of positive charge in solution, it seems to access easily to the sites containing allysine residues. In elastin, the amount of allysine residues are also much higher than that of allysine-aldol as seen in Table II. Thus the formation of DESP and IDP may be associated with the Chichibabin reaction between 3 mol of allysine and ammonia. In this study, however, the involvement of allysine-aldol in these formation cannot be denied as described above.
It is known that pyridine compounds are formed by the Hoffmann degradation of quaternary pyridinium compounds under high temperature (36, 37). No DESP or IDP were found to be formed from DES or IDE, respectively, by prolonged acid hydrolysis under the condition of 110 °C for 240 h in 6 N HCl (results not shown). DESP and IDP, therefore, are not artifacts formed from DES and IDE, respectively, by acid hydrolysis. This may also suggest that assembled DES and IDE in elastin are very stable and are not subject to the Hoffmann degradation.
To obtain more information on the formation of pyridine cross-links, in vivo experiments were performed using CCl4-induced liver cirrhotic rats. This animal model seems to be well suited for hyperammonemia in patients with liver cirrhosis. Hyperammonemia causes dysfunction of multiple organs in patients with cirrhosis. We have investigated the changes of IDP and allysine content in the aorta elastin. IDP was found to be formed in the rat aorta elastin by long-term CCl4 treatment (4 weeks), consistent with the decrease of allysine residue (Table I). IDP may be formed by the Chichibabin reaction between both allysine residues in elastin and ammonia elevated in blood associated with chronic liver failure; IDP may be an abnormal cross-link in elastin.
Kidney failure frequently accompanies liver failure. Although in cirrhotic patients the kidney continued to release ammonia into the circulation (38, 39), this release decreased at elevated ammonia concentrations (38). Similarly, artificial hyperammonemia in healthy volunteers turned the kidney into an organ of net ammonia uptake from the circulation (40), also suggesting enhanced renal ammonia excretion. Urine production is markedly diminished as cirrhosis worsens. In end-stage cirrhosis, complete kidney shut down (hepatorenal syndrome) is usually fatal. Therefore, under this condition DESP and IDP contents may increase in the aorta elastin, and the aorta elastin may be damaged by ammonia.
Connective tissues in the aorta are known to be greatly affected by age because of impaired functional properties and increased susceptibility to diseases. Elastin and collagen are major important extracellular matrix proteins that provide the aorta with tensile strength and elasticity. The ratio between elastin and collagen plays a key role in conditioning the morphological/functional properties of the aorta. During aging and senescence, the aorta becomes stiffer, and its elasticity is reduced. The age-related changes in stiffness and elasticity have been ascribed to these changes in collagen and elastin concentrations in the aorta. In general, the elastin concentration decreases, and the collagen concentration increases until a certain age and then reaches a plateau (41-45). However, few studies have investigated the biochemical properties and the contents of elastin and collagen in the same vessel, and the results have been conflicting (41, 42, 45). The changes in the biochemical properties of collagen are characterized by the formation of cross-links, especially those derived from nonenzymatic glycation (19, 20). Unlike collagen, elastin is formed only in developing tissue, with little or no synthesis in adults (46). Thus, human aorta elastin shifts from an anabolic state to a catabolic state with aging (47). All elastin cross-links are gradually reduced with age (47). The age-related formation of cross-links in aorta elastin has not been reported. We found that the pyridine cross-links reported in the present paper were increased with age (Fig. 9). Thus, the distribution and gradual increased levels may indicate the functional changes of the aorta elastin damaged by ammonia with aging and senescence.
Studies on the distribution of DESP and IDP and on the DESP/DES and
IDP/IDE ratios in the aorta elastin under various conditions may
provide new insights into the chemical changes of elastin in the aorta.
We have been investigating the biological meaning of the presence of
higher pyridine cross-links in the aorta rather than in other tissue
elastin. In normal cross-linking of elastin, it is well known that 1 mol of ammonia is produced when 1 mol of allysine is formed from
1 mol of lysine by lysyl oxidase. Thus additional studies containing
the participation of lysyl oxidase will be also needed to determine the
source of ammonia, which is involved in the formation of the pyridine
cross-links DESP and IDP.
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ACKNOWLEDGEMENTS |
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We are deeply grateful to Dr. M. Watanabe from the Department of Pathology, Tohoku University Hospital for supplying aorta samples.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Applied
Bioorganic Chemistry, Div. of Life Science, Graduate School of Agricultural Science, Tohoku Univ., 1-1 Tsutsumidori-Amamiya, Aoba-ku,
Sendai 981-8555 Japan. Tel.: 81-22-717-8818; Fax: 81-22-717-8820; E-mail: suyama@bios.tohoku.ac.jp.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M009744200
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ABBREVIATIONS |
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The abbreviations used are: DES, desmosine; IDE, isodesmosine; OXD, oxodesmosine; IOXD, isooxodesmosine; DESP, desmopyridine; IDP, isodesmopyridine; HPLC, high pressure liquid chromatography; APC, allysine-bis-p-cresol derivative; FAB, fast atom bombardment; HSQC, heteronuclear single quantum coherence; GHSQC, pulse field gradient heteronuclear single quantum coherence; HMBC, heteronuclear multiple-bond coherence; GHMBC, pulse field gradient heteronuclear multiple-bond coherence.
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