Am J Physiol Endocrinol Metab 290: E1131–E1139, 2006.
First published December 13, 2005; doi:10.1152/ajpendo.00339.2005.
Ascorbic acid-independent synthesis of collagen in mice
Kelly K. Parsons,1 Nobuyo Maeda,2 Mitsuo Yamauchi,3 Albert J. Banes,4 and Beverly H. Koller1
Departments of 1Genetics, 2Pathology, 3Periodontology, and 4Orthopaedics,
The University of North Carolina Chapel Hill, Chapel Hill, North Carolina
Submitted 26 July 2005; accepted in final form 12 December 2005
ASCORBIC ACID, ALSO KNOWN AS VITAMIN C, was first discovered
in the mid-1700s when a Scottish naval surgeon determined
that he could cure scurvy with a diet containing citrus fruits.
Scurvy is a condition characterized in humans by bleeding
gums, tooth loss, weakening of bones, swelling of joints, and
anemia. Ascorbic acid is a cofactor for the enzyme prolyl
hydroxylase, which catalyzes the hydroxylation of peptidyl
proline in a posttranslational process (39). In this reaction, it is
thought that ascorbic acid reacts with oxidized iron bound to
prolyl hydroxylase, reducing the iron and facilitating the release of the enzyme (26). Hydroxyproline is an important
amino acid in collagen, a prominent structural protein found in
connective tissues such as bones, tendons, ligaments, teeth,
skin, cartilage, and blood vessels. Inadequate maintenance of
these connective tissues leads to the symptoms associated with
scurvy and eventually death in humans deficient in vitamin C.
Collagen is formed by the association of three collagen monomers to form a triple helix (32). At least 35% of the prolines in
a collagen monomer must be hydroxylated to assure thermal
stability (4), and underhydroxylated molecules are usually
degraded within the cell without being excreted in the extracellular matrix (3).
The theory that ascorbic acid is required for proline hydroxylation was established based on results of experiments in
cell-free systems (35) and in cells and tissues in culture (3, 37).
However, it has not been supported by experiments in animal
models, in part, because of the lack of a mouse model with a
demonstrated dependence on dietary vitamin C. Virtually all
animals other than primates and guinea pigs are able to synthesize ascorbic acid in the liver and transport it to other parts
of the body. Studies in guinea pigs, animals that are naturally
unable to synthesize ascorbic acid, have produced conflicting
results concerning the requirement for ascorbic acid in hydroxyproline synthesis in collagen. To determine whether
ascorbic acid is essential for collagen formation in vivo, we
used a mouse lacking an enzyme in the ascorbic acid synthesis
pathway, making it dependent on exogenous vitamin C for
survival.
The process of creating new blood vessels, known as angiogenesis, involves the deposition of an extracellular matrix that
is rich in collagen. Given its role in the production of collagen,
ascorbic acid is likely to be important in the formation of new
blood vessels. Results of previous in vitro experiments have
supported this theory. Angiogenesis assays using a rat aortic
ring model showed that microvessels grown in the absence of
ascorbic acid were large and dilated, whereas vessels grown in
the presence of ascorbic acid resembled normal capillaries.
Presumably, collagen serves as a scaffold to maintain the shape
of the newly formed vessels. In the same study, cells incubated
with ascorbic acid increased their synthesis and extracellular
deposition of collagen, measured by the incorporation of
[3H]proline in collagen monomers (29).
Angiogenesis is rare in normal tissue, occurring mostly
during embryogenesis and in adults in the female reproductive system. In pathological tissues, angiogenesis occurs
during wound healing and during tumor growth (13). Angiogenesis takes place in the normal mammary gland during
puberty, pregnancy, and lactation and also during the
growth of mammary tumors (9). Because proliferating mammary glands and tumors both depend on the generation of
blood vessels, and therefore the generation of collagen, we
chose to investigate the effect of ascorbic acid deficiency on
the growth of mammary glands and mammary tumors in
mice. Mammary glands were induced to proliferate by the
pituitary implant model, and tumors were induced by the
injection of tumor cells in the mammary fat pad. Both
mammary glands and tumors proliferated in the presence
and absence of ascorbic acid. Tumors from vitamin Cdeficient animals contained an abundance of hydroxyproline
and collagen despite a lack of ascorbic acid within the
tumors. Collagen extracted from the skin of young mice
raised in the absence of ascorbic acid contained normal
Address for reprint requests and other correspondence: B. H. Koller, Dept.
of Genetics, Univ. of North Carolina at Chapel Hill, 4341 MBRB, Chapel Hill,
NC 27599 (e-mail: [email protected]).
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.
ascorbic acid; collagen; angiogenesis; prolyl hydroxylase
http://www.ajpendo.org
0193-1849/06 $8.00 Copyright © 2006 the American Physiological Society
E1131
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 18, 2017
Parsons, Kelly K., Nobuyo Maeda, Mitsuo Yamauchi, Albert J. Banes, and Beverly H. Koller. Ascorbic acid-independent synthesis of collagen in mice. Am J Physiol Endocrinol Metab
290: E1131–E1139, 2006. First published December 13, 2005;
doi:10.1152/ajpendo.00339.2005.—The mouse has become the most
important model organism for the study of human physiology and
disease. However, until the recent generation of mice lacking the
enzyme gulanolactone oxidase (Gulo), the final enzyme in the ascorbic acid biosynthesis pathway, examination of the role of ascorbic
acid in various biochemical processes using this model organism has
not been possible. In the mouse, similar to most mammals but unlike
humans who carry a mutant copy of this gene, Gulo produces ascorbic
acid from glucose. We report here that, although ascorbic acid is
essential for survival, its absence does not lead to measurable changes
in proline hydroxylation. Vitamin C deficiency had no significant
effect on the hydroxylation of proline and collagen production during
tumor growth or in angiogenesis associated with tumor or mammary
gland growth. This suggests that factors other than ascorbic acid can
support proline hydroxylation and collagen synthesis in vivo. Furthermore, the failure of Gulo⫺/⫺ mice to thrive on a vitamin C-deficient
diet therefore suggests that ascorbic acid plays a critical role in
survival other than the maintenance of the vasculature.
E1132
ASCORBIC ACID-INDEPENDENT COLLAGEN SYNTHESIS
amounts of hydroxyproline, indicating that proline hydroxylation also occurred in other tissues. These results led to the
conclusion that ascorbic acid is not required for the development of blood vessels in mice.
METHODS
AJP-Endocrinol Metab • VOL
RESULTS
Vitamin C deficiency resulted in significant weight loss. A
phenotype associated with scurvy in guinea pigs is the onset of
weight loss ⬃2 wk after the removal of vitamin C from the diet
(2, 36). To determine whether this phenotype is apparent in the
Gulo⫺/⫺ mice and, if so, how long the these mice can remain
healthy without vitamin C, female Gulo⫺/⫺ mice were weighed
at intervals after removal of vitamin C supplementation.
Gulo⫺/⫺ littermates remaining on vitamin C supplementation
served as controls. No significant weight loss was observed
until the 4th wk without vitamin C supplementation. By this
time, unsupplemented mice had lost nearly 20% of their body
weight (Fig. 1A), and the experiment was discontinued.
Ascorbic acid levels varied in different organs after vitamin
C withdrawal. Ascorbic acid levels were measured at weekly
time points after withdrawal of vitamin C supplementation in
tissues known to store high levels of ascorbic acid. Figure 1B
shows the ascorbic acid levels in Gulo⫺/⫺ mice liver, brain,
and adrenal glands, expressed as the percentage of wild-type
ascorbic acid levels. Ascorbic acid was undetectable in the
liver after 2 wk without supplementation, and levels were
reduced to 20 and 10% of wild type in the brain and adrenal
glands. Because ascorbic acid levels were extremely low after
2 wk and continued to decrease in the brain and adrenal glands,
the 2-wk time point was selected as an appropriate starting
point for future experiments.
Skin collagen levels were normal in vitamin C-deficient
mice. Skin is a tissue that is abundant in collagen. To determine
the effect of vitamin C deficiency on the collagen content of
mouse skin, we measured the amount of hydroxyproline in the
skin from wild-type and Gulo⫺/⫺ mice that had been depleted
of vitamin C. Surprisingly, hydroxyproline measurements with
a colorimetric biochemical assay revealed an ⬃80% increase in
hydroxyproline in a 1-cm2 area of skin from vitamin Cdeficient mice compared with wild-type mice (Fig. 2). However, this elevation may be artifactual, reflecting an increase in
the density of collagen in the skin resulting from shrinkage
caused by decreased fluid intake observed after removal of
vitamin C from the diet.
Collagen levels in stimulated mammary glands of vitamin
C-deficient mice were significantly higher than levels in than
nonstimulated glands. Mammary gland proliferation was stimulated in wild-type and Gulo⫺/⫺ mice by implantation of a
290 • JUNE 2006 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 18, 2017
Animal care. A mouse deficient in gulanolactone oxidase
(Gulo⫺/⫺) and on a mixed genetic background was obtained from
Maeda and backcrossed to BALB/c mice from Jackson Laboratories
for 10 generations. All Gulo⫺/⫺ mice were supplied with water
containing 330 mg L-ascorbic acid/l and 0.01 mM EDTA. Western-type
high-fat diet was modified to contain no ascorbic acid (TD.04073,
modified version of TD.88137; Harlan Tekla, Indianapolis, IN).
Pituitary implants. Female mice (7 wk old) received one pituitary
isograft under the right renal capsule from Balb/c donors, as previously described (25). Gulo⫺/⫺ mice were withdrawn from vitamin C
supplementation 1 wk before pituitary implantation. Animals were
killed 4 wk after surgery; mammary glands were saved for whole
mount and hydroxyproline analysis. Serum was collected for prolactin
measurement. Tissue from nonisografted animals served as controls.
Tumor injection. All Gulo⫺/⫺ mice were removed from vitamin C
supplementation 2 wk before initiation of the experiment unless
otherwise noted. For tumor cell injections, the mouse mammary tumor
cell line 4T1 was obtained from ATCC and grown in RPMI 1640
medium with 2 mM L-glutamine, 10% FBS, 10 mM HEPES, pH 7.2,
1 mM sodium pyruvate, 4.5 g/l glucose, and 1.5 g/l sodium bicarbonate. At 90% confluence, cells were harvested, and 106 cells were
injected in the left, fourth mammary fat pad of each experimental and
control mouse. Mice were monitored daily until tumor onset, and
tumors were measured one time a week. Animals were killed at 3 wk,
and tumors were collected and frozen in Eppendorf tubes for RNA
analysis and hydroxyproline measurements and in OCT compound for
immunohistochemistry and Sirius red staining.
Hydroxyproline assay. For skin analysis, hair was removed from
the back of the neck with Nair, and a 1-cm2 piece of skin was removed
for analysis. Hydroxyproline was measured in dried tissue samples as
previously described (18). Hydroxyproline concentrations were determined based on a generated standard curve.
Sirius red staining. Frozen tissue sections were fixed for 10 min in
Omni-fix (FR Chemicals, Mount Vernon, NY) and stained for 30 min
in saturated picric acid containing 0.1% Sirius red and 0.1% Fast
green. Slides were dipped in 70% ethanol, rinsed in water, and
mounted with Permount. Staining was quantitated using Meta-Morph
(Universal Imaging, Downingtown, PA).
Immunohistochemistry. Frozen tissue sections were fixed for 10
min in Omni-fix (FR Chemicals) and air-dried for 30 min. Sections
were quenched with 0.1% H2O2 for 10 min, rinsed three times in PBS
for 5 min, blocked in 2% goat serum for 10 min, and rinsed three
times in PBS for 5 min. Samples were incubated with CD34 primary
antibody (Pharmingen, San Jose, CA) diluted 1:10 in block for 1 h at
room temperature in a humidified chamber and rinsed three times in
PBS. Samples were incubated with Multiple Adsorbed Biotin-Conjugated Goat anti-Rat secondary antibody (Pharmingen) diluted 1:200 in
block for 1 h at room temperature in a humidified chamber, rinsed
three times in PBS, and incubated with avidin-biotin complex (Vector,
Burlingame, CA) for 1 h at room temperature in a humidity chamber.
Slides were rinsed three times in PBS and stained with diaminobenzidine reagent for 8 min, followed by hematoxylin counterstaining.
Slides were rinsed with tap water, air-dried, and mounted with
Permount. Staining was quantitated using Meta-Morph.
In vivo collagen labeling. Mice were injected intravenously in the
tail vein with 100 ␮Ci [3H]proline in sterile PBS. After 18 h, animals
were killed by CO2 asphyxiation, and tissues were harvested and
frozen at ⫺80°C. Tissues were analyzed for hydroxyproline content as
previously described, using a toluene extraction to remove proline and
other factors that may result in false positives (22).
Collagen extraction. Tumor tissue (400 mg) was homogenized in
0.5 M acetic acid, and collagen was extracted by incubating homogenized tissue in 20 ml 0.5 acetic acid containing 0.005% pepsin
(Sigma-Aldrich, St. Louis, MO) at 4°C with shaking. After 24 h,
samples were sedimented, and lipids were removed from the supernatant fluid by chloroform/methanol extraction. The acid phase was
filtered using Centriplus 50-kDa cutoff filters (Millipore, Billerica,
MA) and lyophilized, and protein was resuspended in HNTG buffer
(50 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and 10%
glycerol) for SDS-PAGE. Tail collagen was isolated from tail tendon
and treated in a similar manner. For HPLC analysis, dried collagen
samples were hydrolyzed with 6 N HCl at 105°C overnight, dried,
dissolved in distilled water, and filtered. An aliquot of the hydrolysate
was applied to the Varian HPLC system 9050/9012 (Varian, Walnut
Creek, CA) on a cation-exchange column (AA-911; Transgenomic) to
determine hydroxyproline content as described previously (43).
Ascorbic acid measurement. Tissue ascorbic acid levels were
determined as previously described (31).
ASCORBIC ACID-INDEPENDENT COLLAGEN SYNTHESIS
donor pituitary gland under the renal capsule. Collagen content
of the mammary glands was measured by hydroxyproline
analysis. Total hydroxyproline per dried gland increased significantly, 46% in wild-type mice and 24% vitamin C-deficient
mice, compared with controls (P ⬍ 0.05; Fig. 3A). However, a
significant difference was also detected between wild-type and
vitamin C-deficient controls (P ⬍ 0.005) and between wildtype and vitamin C-deficient isografts (P ⬍ 0.001). The possibility existed that the difference in collagen content between
the mammary glands from isografted wild-type and vitamin
C-deficient mice was not directly because of an impaired
ability to synthesize collagen but indirectly because of insufficient prolactin production. Therefore, serum prolactin levels
were measured by ELISA. Average serum prolactin levels in
Gulo⫺/⫺ mice that were vitamin C deprived but did not receive
an isograft were 5.4 ng/ml. Pituitary isografting increased
average serum prolactin levels in Gulo⫺/⫺ mice to 18.7 ng/ml
(Fig. 3B). Serum prolactin levels in wild-type mice were 22.1
ng/ml for control mice and 40.6 ng/ml for isografted mice.
Although pituitary isografting significantly increased serum
prolactin levels in both wild-type and Gulo⫺/⫺ animals, levels
in vitamin C-deficient mice were significantly lower than those
in wild-type controls. In addition, baseline measurements were
significantly decreased in knockout animals compared with the
wild type. These data parallels the differences in hydroxyproline seen in the mammary glands of these animals. These
results indicate that prolactin production or secretion was
AJP-Endocrinol Metab • VOL
impaired during vitamin C deficiency and that prolactin deficiency, rather than impaired collagen synthesis, most likely
caused the decreased hydroxyproline levels in Gulo⫺/⫺
isografted animals. The lower serum prolactin levels of the
Gulo⫺/⫺ mice could also explain the difference in hydroxyproline between the two control nonisografted groups. The importance of prolactin in the normal maturation of the mouse
mammary gland during puberty has been well documented.
Studies in prolactin receptor knockout mice have shown that,
in the absence of prolactin signaling, mammary glands fail to
develop normally during puberty (5). The isograft surgeries
were performed at 7 wk of age, during puberty and mammary
gland development; therefore, some angiogenesis would be
expected to occur in these animals, even those not obtaining
grafts.
Vitamin C-deficient mice developed tumors at the same rate
as wild-type controls. To determine whether vitamin C deficiency affects the production of collagen in the blood vessels of
mammary tumors, 4T1 mouse mammary tumor cells were
injected in the mammary fat pads of 6-wk-old wild-type and
Gulo⫺/⫺ mice after 2 wk of vitamin C withdrawal. The 4T1
tumor cell line was chosen because it was shown to be
responsive to anti-angiogenic therapy and therefore should be
highly dependent on the formation of blood vessels for growth
(16). All 14 experimental and 15 wild-type mice developed
tumors, with an average onset of 3.9 and 4.4 days, respectively.
Tumors were measured weekly, and at the time of death 3 wk
after injection, the average tumor size was not statistically
different between the two groups of mice (data not shown).
Therefore, vitamin C deficiency did not affect the incidence,
onset, or growth of mammary tumors in mice.
Tumors in vitamin C-deficient mice had lower levels of
hydroxyproline but similar amounts of collagen and blood
vessels as those in wild-type controls. After animals were
killed, tumor tissue was collected for histological and biochemical analysis. An initial experiment analyzing tumors from 14
wild-type and 14 Gulo⫺/⫺ mice suggested that ascorbic acid
could enhance collagen synthesis in these tumors. In this
setting, a 20% decrease in hydroxyproline was observed in
tumors obtained from vitamin C-deficient mice (P ⫽ 0.04).
However, expansion of the analysis to include data from all
harvested tumors (n ⫽ 20 wild type, n ⫽ 22 Gulo⫺/⫺) failed to
support this conclusion. Although the combined data continued
to show a small decrease in collagen levels in the tumors from
Fig. 2. Hydroxyproline content of mouse skin was not decreased by vitamin C
deficiency. Collagen content after 4 wk of vitamin C withdrawal was estimated
by biochemical measurement of hydroxyproline. Results are expressed as mg
of hydroxyproline/cm2 of tissue ⫾ SE. *Significantly different from wild type
(P ⬍ 0.05) by Student’s t-test.
290 • JUNE 2006 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 18, 2017
Fig. 1. Weight loss followed vitamin C withdrawal. A: wild-type (n ⫽ 6) and
gulanolactone oxidase-deficient (Gulo⫺/⫺; n ⫽ 13) females, ages 6 – 8 wk,
were weighed at weekly time points after supplementation withdrawal (day 0),
and average weight is shown ⫾ SE. *Significantly different from Gulo⫺/⫺
average at day 21 (P ⬍ 0.001) by Student’s t-test. B: time course of tissue
ascorbic acid content. Ascorbic acid concentration was measured biochemically at weekly time points after withdrawal of supplementation. Ascorbic acid
content was determined per mg of tissue for wild-type and Gulo⫺/⫺ mice, with
n ⫽ 3/time point. Values were normalized for background, averaged, and
expressed as %wild-type ascorbic acid ⫾ SE. *All 3 tissues were significantly
different from day 0 (P ⬍ 0.05) **Brain and adrenals were significantly
different from day 7 (P ⬍ 0.005) by Student’s t-test.
E1133
E1134
ASCORBIC ACID-INDEPENDENT COLLAGEN SYNTHESIS
vitamin C-deficient animals (19% decrease), the difference
failed to achieve statistical significance (P ⫽ 0.1)
The failure to observe a biochemical difference in hydroxyproline levels was consistent with histological analysis of
the tumors. Sections of tumors from both wild-type and
Gulo⫺/⫺ mice were stained for collagen using Sirius red and
for blood vessels using an antibody to the endothelial cell
marker CD34. Tumors from both vitamin C-deficient and
wild-type mice contained an abundance of collagen (Fig. 4, B
and C). Quantitation of collagen staining showed no difference
between the two groups (Fig. 4D). Sections of tumors that were
immunostained for vasculature showed similar staining in both
groups of mice (Fig. 5, A and B), and quantitation showed no
difference in the amount of staining between the vitamin
C-deficient and wild-type controls (Fig. 5C). Thus collagen and
blood vessels were produced in tumors despite vitamin C
deficiency.
Proline incorporation and hydroxylation occurred at
the same rate in wild-type and vitamin C-deficient mice.
3
L-[5- H]proline was injected intravenously in wild-type and
vitamin C-deficient animals with mammary tumors to determine proline incorporation and hydroxylation. Radioactive
counts in total tissue hydrolysates were comparable in wildtype and vitamin C-deficient animals (Fig. 6A), indicating that
protein synthesis was equivalent in the two groups. Radioactive counts in hydroxyproline fractions separated by toluene
extraction were also equivalent (Fig. 6B); therefore, the conversion of proline to hydroxyproline occurred at the same rate
in wild-type and vitamin C-deficient mice.
The proline-to-hydroxyproline ratio was equivalent in tail
and tumors from wild-type and vitamin C-deficient mice. Collagen was extracted by acid hydrolysis and examined by
SDS-PAGE and HPLC. Collagen extracted from vitamin Cdeficient tumors (Fig. 7A, lanes 6 – 8) did not appear different
from those of wild-type mice (lanes 3–5) when visualized on
an SDS-PAGE gel. Total amino acid content of acid-hydrolyzed collagen samples revealed that the ratio of proline to
hydroxyproline was not significantly different in samples from
Gulo⫺/⫺ mice (Fig. 7B). However, the observed ratios (15:1)
were higher than the expected proline-to-hydroxyproline ratio
of 1.5:1, indicating that the samples contained large amounts of
AJP-Endocrinol Metab • VOL
noncollagen protein. Because the dilution of collagen by noncollagen proteins could impair our ability to observe small but
significant changes in hydroxyproline, we chose to verify these
results using collagen extracted from mouse tail. Because
collagen is a major constituent of tail protein, small differences
in hydroxylation are more likely to be observed. Collagen was
extracted from the tails of wild-type and 2-wk unsupplemented
mice at age 4 wk. Between the ages of 3 and 4 wk, body weight
increases by 40%, and a proportional increase in tail weight is
observed. Tail collagen was subjected to HPLC analysis, and
the resulting proline-to-hydroxyproline ratios were close to the
expected 1.5:1 ratio. No significant difference in proline-tohydroxyproline ratios between vitamin C-deficient and wildtype animals was detected (Fig. 7C) These results again fail to
support a dependence of hydroxylation on vitamin C in the
mouse.
Ascorbic acid uptake does not differ between tumors in
Gulo⫺/⫺ and wild-type mice. Ascorbic acid can be transported
in cells through glucose transporters in addition to the two
sodium-dependent ascorbic acid transporters (40), and glucose
transporters tend to be upregulated in tumor cells (1). Therefore, we investigated whether the tumors increased their ascorbic acid concentrations by absorbing any ascorbic acid that
may have been circulating or stored in neighboring tissues.
Ascorbic acid measurements in tumor samples revealed very
low levels of ascorbic acid in tumors of vitamin C-deficient
mice compared with the wild type (Fig. 8A). The difference
correlates with the disparity of ascorbic acid levels in the livers
of wild-type and Gulo⫺/⫺ unsupplemented mice (Fig. 8B),
indicating that the tumors did not concentrate ascorbic acid
against the gradient. Pretreatment of samples with ascorbate
oxidase before ascorbic acid measurement showed that the
small amount of activity seen in the Gulo⫺/⫺ samples could be
attributed to background likely from other reducing agents. To
show that ascorbate oxidase was actually destroying ascorbate
in this complex system, purified ascorbic acid was added to
liver homogenates before ascorbate oxidase treatment and
ascorbate measurement. Ascorbate oxidase degraded the ascorbate in the samples to the same irreducible amount regardless
of the quantity of purified ascorbate added (Fig. 8C).
290 • JUNE 2006 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 18, 2017
Fig. 3. Mammary gland development and hydroxyproline content were decreased in vitamin C-deficient mice. After pituitary isograft surgery (5 wk), animals
were killed and examined for mammary gland development and collagen content. A: collagen content was estimated by hydroxyproline measurement in wild-type
controls (n ⫽ 8), Gulo⫺/⫺ controls (n ⫽ 6), wild-type isografted mice (n ⫽ 12), and Gulo⫺/⫺ isografted mice (n ⫽ 12) ⫾ SE. *Significant difference between
wild-type isografts and nonisograft controls (P ⬍ 0.05). **Significant difference between Gulo⫺/⫺ isografts and Gulo⫺/⫺ nonisograft controls (P ⬍ 0.05).
@Significant difference between wild-type and Gulo⫺/⫺ isografts (P ⬍ 0.001). #Significant difference between wild-type and Gulo⫺/⫺ nonisograft controls (P ⬍
0.005) by Student’s t-test. B: serum prolactin was measured by HPLC in wild-type controls (n ⫽ 8), Gulo⫺/⫺ controls (n ⫽ 6), wild-type isografted mice (n ⫽
12), and Gulo⫺/⫺ isograft mice (n ⫽ 12) ⫾ SE. *Significant difference between wild-type isografts and nonisograft controls (P ⬍ 0.01). **Significant difference
between Gulo⫺/⫺ isografted mice and Gulo⫺/⫺ controls (P ⬍ 0.001). @Significant difference between wild-type and Gulo⫺/⫺ isografts (P ⬍ 0.0005).
#Significant difference between wild-type and Gulo⫺/⫺ nonisograft controls (P ⬍ 0.005) by Student’s t-test.
ASCORBIC ACID-INDEPENDENT COLLAGEN SYNTHESIS
E1135
DISCUSSION
Fig. 4. Hydroxyproline content but not collagen content of 4T1 tumors was
reduced in tumors from vitamin C-deficient mice. A: collagen content of
tumors was analyzed indirectly through hydroxyproline measurement. Hydroxyproline amounts are represented as average mg of hydroxyproline/mg of
dry tissue ⫾ SE. No significant difference was detected in samples from
wild-type (n ⫽ 20) and Gulo⫺/⫺ (n ⫽ 22) mice by Student’s t-test. Frozen
tumor sections were immunostained with Sirius red and counterstained with
Fast green. Staining was quantitated using Meta-Morph and is presented as
average %staining ⫾ SE (D). No significant difference in staining was seen
between wild-type (n ⫽ 14) and Gulo⫺/⫺ tumors from (n ⫽ 15) by Student’s
t-test. Representative samples are shown from wild-type (B) and Gulo⫺/⫺ (C)
mice.
AJP-Endocrinol Metab • VOL
290 • JUNE 2006 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 18, 2017
Ascorbic acid is involved in numerous biological processes,
including scavenging free radicals to prevent oxidative damage, acting as a cofactor for reactions such as the hydroxylation
of proline necessary for collagen needed for blood vessels and
wound healing, hormone synthesis, cholesterol metabolism,
neurotransmitter synthesis, iron absorption, and immune system function, among others. Its role in the formation of
collagen led us to investigate whether ascorbic acid is required
for collagen synthesis in mouse skin and the development of
blood vessels during mouse mammary gland proliferation and
tumorigenesis.
The use of animal models for the study of the roles of
ascorbic acid has been limited by the fact that all animals other
than primates and guinea pigs are able to synthesize ascorbic
acid, rather than having to rely on a dietary source of this
nutrient. Recently, this problem has been addressed by the
generation of a mouse line in which the gene for L-gulono-␥lactone oxidase, the final enzyme in the ascorbic acid synthesis
pathway, was mutated by homologous recombination (23).
Because this mutation leads to a dependence on dietary vitamin
C, we used these mice to determine the role of ascorbic acid in
the production of collagen in various organs.
The collagen content of skin from adult mice did not
decrease in Gulo⫺/⫺ mice after vitamin C supplementation was
removed. Because this result might reflect low turnover of
collagen in this tissue, we also examined tissues that were
actively proliferating and thus developing new blood vessels in
vitamin C-deficient, growing animals. Angiogenesis, the development of new blood vessels, is rare in normal adult tissue
but does occur in the mammary gland during puberty, pregnancy, and lactation. Examination of pregnant mammary
glands in vitamin C-deficient mice proved to be difficult
because of the fact that these mice do not often maintain
pregnancies. Therefore, we chose to induce mammary gland
proliferation by performing pituitary implant surgeries. When
the pituitary gland from a donor mouse is placed under the
renal capsule, prolactin is released continuously in the bloodstream, stimulating mammary gland growth. Pituitary isografting led to budding of the epithelium in wild-type glands and in
Gulo⫺/⫺ glands in spite of vitamin C deficiency. Collagen
content of proliferating glands, measured by hydroxyproline
analysis, increased in both groups, although to a lesser extent
in the glands from vitamin C-deficient mice. From these data,
we conclude that collagen production is reduced but not abolished in the mammary gland as a result of vitamin C deficiency. However, a significant decrease in serum prolactin
concentration in vitamin C-deficient mice compared with wild-
E1136
ASCORBIC ACID-INDEPENDENT COLLAGEN SYNTHESIS
Fig. 5. Formation of vascular structures was not reduced in tumors from
vitamin C-deficient mice. Frozen tumor sections were immunostained with
anti-CD34 and counterstained with hematoxylin. Staining was quantitated
using Meta-Morph and is presented as average %staining ⫾ SE (C). No
significant difference in staining was seen between wild-type (n ⫽ 14) and
Gulo⫺/⫺ tumors from (n ⫽ 15) by Student’s t-test. Representative samples are
shown from wild-type (A) and Gulo⫺/⫺ (B) mice.
type controls suggests that this reduction in collagen is the
result of decreased prolactin production rather than reduced
proline hydroxylation. These results suggest that the Gulo⫺/⫺
mouse may serve as a model for studying the role of ascorbic
acid in the regulation of prolactin expression.
AJP-Endocrinol Metab • VOL
Fig. 6. Incorporation and hydroxylation of [3H]proline was not decreased in
tumors from vitamin C-deficient mice. Wild-type and Gulo⫺/⫺ mice with 4T1
tumors were injected iv with [3H]proline. Tumors were minced and hydrolyzed
and analyzed for total 3H incorporation (A) and [3H]hydroxyproline (Hypro;
B). Data are expressed as average disintegrations 䡠 min⫺1 (dpm) 䡠 mg tissue⫺1 ⫾
SE. No significant difference was detected in total 3H incorporation between
the wild type (n ⫽ 6) and Gulo⫺/⫺ (n ⫽ 10) or in [3H]hydroxyproline (n ⫽ 6,
8) by Student’s t-test.
290 • JUNE 2006 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 18, 2017
Angiogenesis is common in pathological tissues such as
tumor development. Using the injection of a tumor cell line in
the mammary gland fat pad as a model, we have shown that
tumors grow at the same frequency and rate in vitamin Cdeficient mice as in wild-type controls. Also, tumors in the
experimental and control groups contain equivalent amounts of
hydroxyproline, collagen, and bloods vessels. Because underhydroxylated collagen is more sensitive to intracellular degradation (3), a significant decrease in hydroxylation should be
reflected by a reduction in extracellular collagen deposition.
The observation that amounts of extracellular matrix collagen
in tumors from wild-type mice did not differ significantly from
Gulo⫺/⫺ levels suggests that collagen is not severely underhydroxylated despite the state of vitamin C deficiency. Further
analysis of these tumors revealed that hydroxyproline is actively synthesized in vitamin C-deficient animals, even after 4
wk without vitamin C supplementation. The ratio of hydroxyproline to proline was not measurably affected by the
absence of tissue ascorbic acid. Theses studies suggest that the
production of collagen, presumably required for stable blood
vessel development, is not compromised even during severe
ascorbic acid deficiency.
In humans, plasma ascorbic acid levels measuring ⬍11% of
normal are indicative of scurvy (15). Therefore, connective
tissue damage occurs in humans even when ascorbic acid is
detectable. In contrast, Gulo⫺/⫺ mice display no evidence of
connective tissue injury even when ascorbic acid was undetectable in tissue. This observation, along with our results
showing normal levels of proline hydroxylation in the tail
collagen, mammary gland, and tumors of vitamin C-deprived
Gulo⫺/⫺ mice, suggests that ascorbic acid is not required for
proline hydroxylation in the mouse. However, it is possible that
small amounts of ascorbic acid, below the level of detection,
ASCORBIC ACID-INDEPENDENT COLLAGEN SYNTHESIS
E1137
C-supplemented animals that were acutely starved showed the
same reduction in collagen synthesis as the vitamin C-deficient
animals (7, 33, 38). These data suggest that, although ascorbic
acid does play a role in proline hydroxylation, the substantial
decreases in collagen seen during scurvy in guinea pigs are the
indirect consequence of weight loss accompanying vitamin C
deficiency and may not reflect a defect in proline hydroxylation. In this same study, weight loss was shown to affect
collagen production through inhibition of insulin-like growth
factor I, a molecule known to stimulate production of collagen.
Our data demonstrating a moderate decrease in hydroxyproline
supports the idea that mice, similar to guinea pigs, utilize
ascorbic acid as a cofactor for the production of hydroxyproline but do not rely solely on ascorbic acid for this function.
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 18, 2017
Fig. 7. Ratio of proline to hydroxyproline was not increased in tumors from
vitamin C-deficient mice. Collagen was extracted from tumors, visualized by
SDS-PAGE, and analyzed by HPLC for total amino acid content. A: lane 1,
ladder; lane 2, collagen control extracted from mouse tail; lanes 3–5, collagen
from wild-type tumors; lanes 6 – 8, collagen from Gulo⫺/⫺ tumors. B: average
ratio of hydroxyproline to proline in mouse tumors, determined by HPLC, ⫾
SE. No significant difference was detected between the wild type (n ⫽ 3) and
Gulo⫺/⫺ (n ⫽ 3) by Student’s t-test. C: average ratio of hydroxyproline to
proline in mouse tail collagen, determined by HPLC, ⫾ SE. No significant
difference was detected between the wild type (n ⫽ 3) and Gulo⫺/⫺ (n ⫽ 3)
by Student’s t-test.
are sufficient to catalyze the hydroxylation reaction in mice. In
cell-free systems in vitro, only 1 ␮mol of ascorbic acid is
required to catalyze proline hydroxylation (19). In addition,
ascorbic acid is not consumed during the reaction and can
therefore be recycled for additional hydroxylations (41). If this
hypothesis is correct, our results would suggest that mice are
better able than humans to concentrate ascorbic acid at the sites
of collagen synthesis.
Studies in guinea pigs, which are unable to synthesize
ascorbic acid, have not proven a direct connection between
vitamin C deficiency and defects in proline hydroxylation.
Experiments separating collagen production from proline hydroxylation in guinea pigs have shown that vitamin C-deficient
animals have a 50% reduction in collagen synthesis but only a
30% decrease in proline hydroxylation. Furthermore, vitamin
AJP-Endocrinol Metab • VOL
Fig. 8. Tumors in vitamin C-deficient mice did not concentrate ascorbic acid.
To eliminate the possibility that tumors in Gulo⫺/⫺ are able to accumulate
ascorbic acid, ascorbate content was measured in tumors (A) and liver (B) and
expressed as ␮g of ascorbic acid/g of tissue. Wild-type samples contain high
levels of ascorbate (n ⫽ 3), whereas ascorbic acid levels in tumor and liver of
Gulo⫺/⫺ mice (n ⫽ 3) are not significantly different from samples treated with
ascorbate oxidase. *Significantly different from untreated Gulo⫺/⫺ samples
and ascorbate oxidase-treated samples (P ⬍ 0.005) by Student’s t-test. C: to
show that ascorbate oxidase was actually destroying ascorbate, ascorbic acid
was measured in liver samples that had been supplemented with purified
ascorbic acid before ascorbate oxidase treatment.
290 • JUNE 2006 •
www.ajpendo.org
E1138
ASCORBIC ACID-INDEPENDENT COLLAGEN SYNTHESIS
AJP-Endocrinol Metab • VOL
animals lost less weight than the vitamin C-deficient animals
(36). These results point toward inefficient digestion or metabolism during vitamin C deficiency. The possibility of ascorbic
acid aiding in digestion is supported by experiments with
guinea pigs that have demonstrated reduced absorption of
certain amino acids by the small intestine of vitamin Cdeficient animals (10). Because ascorbic acid is involved in so
many different pathways, it is possible that the weight loss
could be the result of breakdowns in multiple physiological
pathways. The Gulo⫺/⫺ mouse should provide a valuable
model for distinguishing between these various pathways.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-068141 to B. H. Koller.
REFERENCES
1. Agus DB, Vera JC, and Golde DW. Stromal cell oxidation: a mechanism
by which tumors obtain vitamin C. Cancer Res 59: 4555– 4558, 1999.
2. Alfano MC, Miller SA, and Drummond JF. Effect of ascorbic acid
deficiency on the permeability and collagen biosynthesis of oral mucosal
epithelium. Ann NY Acad Sci 258: 253–263, 1975.
3. Berg RA, Steinmann B, Rennard SI, and Crystal RG. Ascorbate
deficiency results in decreased collagen production: under-hydroxylation
of proline leads to increased intracellular degradation. Arch Biochem
Biophys 226: 681– 686, 1983.
4. Berisio R, Granata V, Vitagliano L, and Zagari A. Characterization of
collagen-like heterotrimers: implications for triple-helix stability. Biopolymers 73: 682– 688, 2004.
5. Brisken C, Kaur S, Chavarria TE, Binart N, Sutherland RL, Weinberg RA, Kelly PA, and Ormandy CJ. Prolactin controls mammary
gland development via direct and indirect mechanisms. Dev Biol 210:
96 –106, 1999.
6. Chauhan U, Assad R, and Peterkofsky B. Cysteinyl-cysteine and the
microsomal protein from which it is derived act as reducing cofactor for
prolyl hydroxylase. Biochem Biophys Res Commun 131: 277–283, 1985.
7. Chojkier M, Spanheimer R, and Peterkofsky B. Specifically decreased
collagen biosynthesis in scurvy dissociated from an effect on proline
hydroxylation and correlated with body weight loss. In vitro studies in
guinea pig calvarial bones. J Clin Invest 72: 826 – 835, 1983.
8. Christov K, Swanson SM, Guzman RC, Thordarson G, Jin E, Talamantes F, and Nandi S. Kinetics of mammary epithelial cell proliferation in pituitary isografted BALB/c mice. Carcinogenesis 14: 2019 –
2025, 1993.
9. Djonov V, Andres AC, and Ziemiecki A. Vascular remodelling during
the normal and malignant life cycle of the mammary gland. Microsc Res
Tech 52: 182–189, 2001.
10. Dulloo RM, Majumdar S, Chakravarti RN, Mehta SK, and Mahmood
A. Effect of vitamin C deficiency in guinea pigs on intestinal functions and
chemical composition of brush border membrane. Ann Nutr Metab 25:
213–220, 1981.
11. Eipper BA, Stoffers DA, and Mains RE. The biosynthesis of neuropeptides: peptide alpha-amidation. Annu Rev Neurosci 15: 57– 85, 1992.
12. Evans CA and Peterkofsky B. Ascorbate-independent proline hydroxylation resulting from viral transformation of Balb 3T3 cells and unaffected
by dibutyryl cAMP treatment. J Cell Physiol 89: 355–367, 1976.
13. Ferrara N. Role of vascular endothelial growth factor in the regulation of
angiogenesis. Kidney Int 56: 794 – 814, 1999.
14. Hodges RE, Baker EM, Hood J, Sauberlich HE, and March SC.
Experimental scurvy in man. Am J Clin Nutr 22: 535–548, 1969.
15. Hodges RE, Hood J, Canham JE, Sauberlich HE, and Baker EM.
Clinical manifestations of ascorbic acid deficiency in man. Am J Clin Nutr
24: 432– 443, 1971.
16. Huang X, Wong MK, Yi H, Watkins S, Laird AD, Wolf SF, and
Gorelik E. Combined therapy of local and metastatic 4T1 breast tumor in
mice using SU6668, an inhibitor of angiogenic receptor tyrosine kinases,
and the immunostimulator B7.2-IgG fusion protein. Cancer Res 62:
5727–5735, 2002.
17. Huseby RA, Soares MJ, and Talamantes F. Ectopic pituitary grafts in
mice: hormone levels, effects on fertility, and the development of adeno-
290 • JUNE 2006 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 18, 2017
Although most cell lines tested in culture require the addition of ascorbic acid for proline hydroxylation, several cell
lines, such as L-929 and virally transformed 3T3 cells, efficiently synthesize hydroxyproline in the absence of ascorbic
acid (12, 34). The microsomal fractions of these cell lines
contain a protein with a cysteinyl-cysteine active sequence that
is able to function as a reducing agent in place of ascorbic acid
in the proline hydroxylation reaction (6, 24). Additionally, in
hydroxylation assays in cell-free systems, other reductants
such as dithiothreitol, L-cysteine, tetrahydrofolate, and tetrahydropteridine, hydroxylated proline with 16 –56% of the efficiency of ascorbic acid (26, 35). It is possible that this same
microsomal protein or a similar molecule is produced in vivo
in the mouse and guinea pig but is not available in vitro.
Another possibility is that mice express an alternate prolyl
hydroxylase enzyme that does not rely on ascorbic acid as a
cofactor.
Surprisingly, the initial characterization of Gulo⫺/⫺ mice
revealed hemorrhages and disrupted aortic vasculature after
vitamin C withdrawal that were not observed in our studies
(23). In addition, atherosclerotic plaques in Gulo⫺/⫺ Apoe⫺/⫺
mice contained significantly less collagen, measured by Sirius
red staining (28). These apparently conflicting results could be
the result of the difference in genetic background of the
Gulo⫺/⫺ in our study (BALB/c) and those in previous studies
(B6/129 mixed and C57BL/6, respectively). A second possible
explanation is that these phenotypes are secondary effects of
weight loss and are not directly caused by vitamin C deficiency, similar to the results in guinea pigs. Unpublished
preliminary data in the study of atherosclerotic plaques indicating that hydroxyproline measurements were equivalent in
tissues from Gulo⫺/⫺ mice receiving high and low vitamin C
supplementation when these values were normalized to total
proline content supports this hypothesis (28). The decreased
collagen content in the plaques may be because of a downregulation of collagen production during starvation rather than
impaired proline hydroxylation.
The observations that proline hydroxylation can still occur
and connective tissues remain intact in vitamin C-deficient
mice indicate that the eventual death that results in these mice
is not because of scurvy. The only obvious phenotype that
develops in the Gulo⫺/⫺ mice after the removal of vitamin C
supplementation is weight loss, a phenotype that has also been
reported in studies on scorbutic guinea pigs. Several studies
have reported that weight loss in vitamin C-deficient guinea
pigs is the result of a decrease in food intake (2, 36), indicating
that ascorbic acid plays some role in appetite regulation.
Ascorbic acid is known to be required for the synthesis of
neurotransmitters such as neuropeptide Y (11), norepinephrine
(27), serotonin, and dopamine (20). Depression and changes in
eating behaviors are often associated with deficiencies in these
biogenic amines. In addition, depression is often a symptom of
scurvy in humans (14, 21, 42). The anti-obesity drug fenfluramine, which acts through the serotonin pathway, has been
shown in guinea pigs to reduce the levels of ascorbic acid in the
brain. Also, vitamin C supplementation reduces the weight loss
associated with fenfluramine administration, indicating that
ascorbic acid and serotonin may work together in appetite
regulation (30).
In guinea pigs, control animals that were fed an amount
equivalent to the food consumed by the vitamin C-deficient
ASCORBIC ACID-INDEPENDENT COLLAGEN SYNTHESIS
18.
19.
20.
21.
22.
23.
25.
26.
27.
28.
29.
AJP-Endocrinol Metab • VOL
30. Odumosu A. Anorectic drugs and vitamin C: role in appetite and brain
ascorbic acid in guineapigs. Int J Vitam Nutr Res 51: 247–253, 1981.
31. Omaye ST, Turnbull JD, and Sauberlich HE. Selected methods for the
determination of ascorbic acid in animal cells, tissues, and fluids. Methods
Enzymol 62: 3–11, 1979.
32. Ottani V, Martini D, Franchi M, Ruggeri A, and Raspanti M. Hierarchical structures in fibrillar collagens. Micron 33: 587–596, 2002.
33. Peterkofsky B. Ascorbate requirement for hydroxylation and secretion of
procollagen: relationship to inhibition of collagen synthesis in scurvy.
Am J Clin Nutr 54: 1135S–1140S, 1991.
34. Peterkofsky B, Kalwinsky D, and Assad R. A substance in L-929 cell
extracts which replaces the ascorbate requirement for prolyl hydroxylase
in a tritium release assay for reducing cofactor; correlation of its concentration with the extent of ascorbate-independent proline hydroxylation and
the level of prolyl hydroxylase activity in these cells. Arch Biochem
Biophys 199: 362–373, 1980.
35. Peterkofsky B and Udenfriend S. Conversion of proline to collagen
hydroxyproline in a cell-free system from chick embryo. J Biol Chem 238:
3966 –3977, 1963.
36. Richmond V and Stokstad EL. Effect of ascorbic acid on guinea pig skin
collagen synthesis. I. Total collagen. J Dent Res 48: 863– 871, 1969.
37. Roach HI, Hillier K, and Shearer JR. Ascorbic acid requirements for
collagen synthesis (proline hydroxylation) during long-term culture of
embryonic chick femurs. Biochim Biophys Acta 842: 139 –145, 1985.
38. Spanheimer RG and Peterkofsky B. A specific decrease in collagen
synthesis in acutely fasted, vitamin C-supplemented, guinea pigs. J Biol
Chem 260: 3955–3962, 1985.
39. Stetten MR. Some aspects of the metabolism of hydroxyproline, studied
with the aid of isotopic nitrogen. J Biol Chem 181: 31–37, 1949.
40. Tsukaguchi H, Tokui T, Mackenzie B, Berger UV, Chen XZ, Wang Y,
Brubaker RF, and Hediger MA. A family of mammalian Na⫹-dependent L-ascorbic acid transporters. Nature 399: 70 –75, 1999.
41. Tuderman L, Myllyla R, and Kivirikko KI. Mechanism of the prolyl
hydroxylase reaction. 1. Role of co-substrates. Eur J Biochem 80: 341–
348, 1977.
42. Walker A. Chronic scurvy. Br J Dermatol 80: 625– 630, 1968.
43. Yamauchi M and Shiiba M. Lysine hydroxylation and crosslinking of
collagen. Methods Mol Biol 194: 277–290, 2002.
290 • JUNE 2006 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on June 18, 2017
24.
myosis uteri, prolactinomas, and mammary carcinomas. Endocrinology
116: 1440 –1448, 1985.
Huszar G, Maiocco J, and Naftolin F. Monitoring of collagen and
collagen fragments in chromatography of protein mixtures. Anal Biochem
105: 424 – 429, 1980.
Hutton JJ, Tappel AL, and Udenfriend S. Cofactor and substrate
requirements of collagen proline hydroxylase. Arch Biochem Biophys 118:
231–240, 1967.
Kaufman S and Friedman S. Dopamine-beta-hydroxylase. Pharmacol
Rev 17: 71–100, 1965.
Kinsman RA and Hood J. Some behavioral effects of ascorbic acid
deficiency. Am J Clin Nutr 24: 455– 464, 1971.
LeRoy EC, Harris ED Jr, and Sjoerdsma A. A modified procedure for
radioactive hydroxyproline assay in urine and tissues after labeled proline
administration. Anal Biochem 17: 377–382, 1966.
Maeda N, Hagihara H, Nakata Y, Hiller S, Wilder J, and Reddick R.
Aortic wall damage in mice unable to synthesize ascorbic acid. Proc Natl
Acad Sci USA 97: 841– 846, 2000.
Mata JM, Assad R, and Peterkofsky B. An intramembranous reductant
which participates in the proline hydroxylation reaction with intracisternal
prolyl hydroxylase and unhydroxylated procollagen in isolated microsomes from L-929 cells. Arch Biochem Biophys 206: 93–104, 1981.
Medina D. Mammary tumorigenesis in chemical carcinogen-treated mice.
I. Incidence in BALB-c and C57BL mice. J Natl Cancer Inst 53: 213–221,
1974.
Myllyla R, Kuutti-Savolainen ER, and Kivirikko KI. The role of
ascorbate in the prolyl hydroxylase reaction. Biochem Biophys Res Commun 83: 441– 448, 1978.
Nakashima Y, Suzue R, Sanada H, and Kawada S. Effect of ascorbic
acid on hydroxylase activity. I. Stimulation of tyrosine hydroxylase and
tryptophan-5-hydroxylase activities by ascorbic acid. J Vitaminol (Kyoto)
16: 276 –280, 1970.
Nakata Y and Maeda N. Vulnerable atherosclerotic plaque morphology
in apolipoprotein E-deficient mice unable to make ascorbic acid. Circulation 105: 1485–1490, 2002.
Nicosia RF, Belser P, Bonanno E, and Diven J. Regulation of angiogenesis in vitro by collagen metabolism. In Vitro Cell Dev Biol 27A:
961–966, 1991.
E1139