Proline Effects on Wound Healing in Rats: Local vs Systemic

Journal of Investigative Surgery
ISSN: 0894-1939 (Print) 1521-0553 (Online) Journal homepage: www.tandfonline.com/journals/iivs20
The Effects of Local and Systemic Administration
of Proline on Wound Healing in Rats
Husnu Aydin, Cihad Tatar, Osman Anil Savas, Tamer Karsidag (Associate
Professor), Bahri Ozer, Nevra Dursun, Aylin Bekem (Research Assistant),
Ahmet Unal (Professor) & Ishak Sefa Tuzun (Professor)
To cite this article: Husnu Aydin, Cihad Tatar, Osman Anil Savas, Tamer Karsidag (Associate
Professor), Bahri Ozer, Nevra Dursun, Aylin Bekem (Research Assistant), Ahmet Unal
(Professor) & Ishak Sefa Tuzun (Professor) (2019) The Effects of Local and Systemic
Administration of Proline on Wound Healing in Rats, Journal of Investigative Surgery, 32:6,
523-529, DOI: 10.1080/08941939.2018.1441342
To link to this article: https://doi.org/10.1080/08941939.2018.1441342
Published online: 01 Mar 2018.
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Journal of Investigative Surgery, 32, 523–529, 2019
C 2018 Taylor & Francis Group, LLC
Copyright ISSN: 0894-1939 print / 1521-0553 online
DOI: 10.1080/08941939.2018.1441342
ORIGINAL RESEARCH
The Effects of Local and Systemic Administration of Proline
on Wound Healing in Rats
Husnu Aydin, MD,1 Cihad Tatar, MD,2 Osman Anil Savas, MD,3 Tamer Karsidag, Associate
Professor,4 Bahri Ozer, MD,5 Nevra Dursun, MD,6 Aylin Bekem, Research Assistant,7
Ahmet Unal, Professor,7 Ishak Sefa Tuzun, Professor2
1
Department of General Surgery, Erciyes University, Istanbul, Turkey, 2 Department of General Surgery, Istanbul Training
and Research Hospital, Istanbul, Turkey, 3 Department of General Surgery, Altinbas University, Istanbul, Turkey,
4
Department of General Surgery, Acibadem University, Istanbul, Turkey, 5 Department of General Surgery, Abant Izzet
Baysal University, Bolu, Turkey, 6 Department of Pathology, Istanbul Training and Research Hospital, Istanbul, Turkey,
7
Department of Metallurgical and Materials Engineering, Yildiz Technical University, Istanbul, Turkey
ABSTRACT
Purpose: Wound healing consists of a sequence of complex molecular and cellular events. Collagen is composed
mainly of proline and hydroxyproline. Proline and hydroxyproline constitute 1/3 of the amino acids in collagen,
which makes up approximately 30% of the proteins within the body. The hydroxylation of proline found in
collagen determines the stability of the triple helical structure of collagen. In this study, we examined the effects of
local and systemic administration of proline on wound healing. Materials and Methods: 24 female Sprague-Dawley
rats were used in the study and divided into three groups. Group 1: The defect created in the backs of the subjects
was left to secondary healing. Group 2: 200 µl proline per day was administered topically for 30 days on the defect
in the backs of the subjects. Group 3: 200 µl per day was administered intraperitoneally for 30 days on the defect
in the backs of the subjects. Results: On day 21, there was a statistically significant difference between the groups
in terms of the mean re-epithelialization score. On days 7 and 14, there was a statistically significant difference
between the groups in terms of the mean granulation score. On days 7, 14, and 21, there was a statistically
significant difference between the groups in terms of the mean collagen accumulation score. On day 30, there was
a statistically significant difference between Groups 1 and 3 in terms of the mean E-mode score on mechanical
tensile test. Conclusion: Our study confirmed that proline has positive effects on wound healing. However, it
revealed that systemic administration of proline is more effective than local administration of proline.
Keywords: mechanical tensile test; proline; rat; wound healing
INTRODUCTION
Collagen, which is the most abundant protein
in the body, is composed mainly of proline and
hydroxyproline.1 Proline and hydroxyproline constitute 1/3 of the amino acids in collagen, which makes up
approximately 30% of the proteins within the body. The
hydroxylation of proline found in collagen determines
the stability of the triple helical structure of collagen.2
Various studies have been conducted on the role
of amino acids in facilitating wound healing.3 Studies
have shown that proline accelerated wound healing,
and that topical administration of proline at the wound
site gave better results than oral administration.4 It
has been observed in patients with protein-energy malnutrition that proline and hydroxyproline levels were
decreased, and that wound healing was delayed.5–7
In collagen synthesis, proline is attached to the peptide chain and then is converted to hydroxyproline via
a reaction catalyzed by prolyl 4-hydroxylase, which
requires oxygen and alpha-ketoglutarate as cosubstrates and ferrous iron and ascorbate as cofactors.
Received 23 November 2017; accepted 12 February 2018.
Address correspondence to Husnu Aydin, Erciyes University, Department of General Surgey, Kayseri, Turkey. 38089. E-mail:
[email protected].
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/iivs.
523
524 H. Aydin et al.
FIGURE 1 Creating full-thickness skin defects on the backs of the animals.
Proline is not essential for the body except in
situations like trauma and burns.8 Proline can be
synthesized from arginine, glutamine, glutamate, and
ornithine in specific cell, tissue, and species types.9
Proline has important roles in collagen synthesis and
wound healing. Moreover, proline performs a variety
of important tasks such as regulation of gene expression and cell differentiation, cell nutrition, growth factor signaling, polyamine glutamate and protein synthesis, arginine synthesis (which is especially important in
newborns fed with breast milk), andexcretion of oxidants from the body.10
Although it is known that proline has positive effects
on wound healing, there is no study on the superiority of local and systemic administration each other.
Although topical application of a drug to be applied for
wound healing is more beneficial due to direct action
and superficial spreading, there is no evidence of its
superiority to systemic administration.11 In this study,
we examined the effects of local and systemic administration of proline on wound healing.
MATERIALS AND METHODS
24 female Sprague-Dawley rats weighing 250–300 g
were used in the study. Female rats were preferred
because of adverse effects of testosterone.12–14 All animals were monitored in standard laboratory conditions. They were maintained in a lighting regime of
12 hours light and 12 hours darkness. 17–25°C temperature and 30% humidity were provided. They were
fed with sufficient food and water. The animals were
placed under anesthesia by 50 mg/kg IM ketamine
hydrochloride (Ketalar, Pfizer, 50 mg/ml solution) and
10 mg/kg IM xylazine hydrochloride (Rompun, Bayer,
2% solution). The depth of anesthesia was assessed
by extremity pulling response. Then, their backs were
shaved with an electric razor tool. After antisepsis with
povidone iodine, a 15 × 15 mm (2.5 cm2 ) full thickness
skin defect of about 5 mm thickness was created to contain skin and subcutaneous tissue on the backs of subjects (Figure 1). Hemostasis was achieved by making
pressure with sterile gauze bandages. Buprenorphine
was used as an anesthetic agent.The wounds were left
to secondary healing for 30 days. The animals were randomily divided into three groups, each consisting of
eight animals.
Group 1: The defect created in the backs of the subjects was left to secondary healing.
Group 2: 200 µl proline which is dissolved in 200 mg
saline solution (0.9% solution of sodium chloride)
(L-proline, C5 H9 NO2 , Merck Millipore) per day was
administered topically for 30 days on the defect in the
backs of the subjects in this group.
Group 3: 200 µl proline (L-proline, C5 H9 NO2 , Merck
Millipore) per day was administered intraperitoneally
for 30 days on the defect in the backs of the subjects in this group. (Such as intravenous administration in humans, drug was given into the intraperitoneal
because it would be the fastest and most effective way
of reaching the systemic circulation in rats). After proline was applied locally on the wound, each rat was
allowed to remain immobilized for 3 minutes until the
absorption occurred. After ensuring that the absorption had been performed, the rats were left in their
cages without covering the wound surfaces, as in group
1 and group 3. To prevent rats from harming their own
wounds, skin defects were formed in the back region
and each was kept in separate cages.
Histopathological Examination
Derived from circulating monocytes, macrophages
achieve significant numbers in the wound by 48 to
96 hours post injury and remain present until wound
Journal of Investigative Surgery
The Effects of Proline on Wound Healing 525
TABLE 1
Histopathological parameters.
Score
Re-epithelialization
Granulation tissue deposition
Collagen deposition
Inflammatory cell
0
1
2
3
No
Partial
Completed immature
Completed mature
No or immature
Small amount
Moderately mature
Mature
No
Small amount
Moderate
Abundant
No
Small amount
Moderate
Abundant
healing is complete. The proliferative phase is the second phase of wound healing and roughly spans days
4 through 12. The major glycosaminoglycans present
in wounds are dermatan and chondroitin sulfate.
Fibroblasts synthesize these compounds, increasing
their concentration greatly during the first 3 weeks of
healing.15 Because of these overlapping phases biopsies were taken on days 7, 14, and 21 for microscopic
examination after the formation of skin defects in the
subjects. Biopsy materials were fixed in 10% buffered
formalin. After fixation, the tissues were washed in tap
water. They were passed through a rising alcohol series
and were subjected to paraffin inclusion.After a blocking procedure, 5–7 µm thick sections were taken from
the tissues. These specimens were examined under a
light microscope and were stained with hematoxylineosin (HE) by the same pathologist.Histopathological
evaluation was performed according to wound healing
assessment score shown in Table 1.
Angiogenesis
No
Less than 5 vessels
6-10 vessels
More than 10 vessels
FIGURE 2 Mechanical tensile test.
Protocol was approved by the committee for animal
research and the study strictly conformed to the animal
experiment guidelines of the Committee for Human
Care.
Statistical Analysis
Measurement of the Tensile Strength of
Wounds
Tensile strength tests were made in the Composite
Materials Laboratory of the Department of Metallurgical and Materials Engineering at Yildiz Technical University. It was performed using a 500 N (∼50 kg) load
cell by Mares brand universal tensile machine with a
capacity of 20 kN.
In order to measure tensile strength, 30 × 5 mm
strip-shaped skin was excised from incision scars as the
scar tissue remaining in the central area on the 30th
postoperative day after sacrificing the subjects.The
obtained tissue samples were kept in the physiological saline solution. Tensile strength measurements were
made within 6 hours following sample collection. For
this purpose, the width and thickness in the scar region
of strip-shaped tissue samples were measured. These
data were entered into the computer for each sample.
After tissue strips were placed in the tensiometer, the
measurement was started. The newton per square millimeter (N/mm²) at the time of rupture of the strips
was calculated and then was expressed in megapascals (N/mm² = MPa) (Figure 2). Stress-strain graphics
were drawn with the help of software. Moreover, values that are known as modulus of elasticity (E-mode)
were obtained by taking the slope of the stress-strain
curve. E-mode value was used to measure the tissue
durability.
C 2018 Taylor & Francis Group, LLC
The IBM SPSS Statistics 15.0 for Windows package
program was used to analyze the data. Categorical
variables were expressed as number and percentage.
Numerical variables were expressed as mean ± standard deviation (SD). The Kruskal-Wallis test was used
to compare multiple independent groups if parametric
tests could not be applied. Subgroup analyzes were
performed by the Mann-Whitney U test and then were
interpreted by the Bonferroni correction. The Friedman
test was used to compare multiple dependent groups
if parametric tests could not be applied. Subgroup
analyzes were performed by the Wilcoxon test and
then were interpreted by the Bonferroni correction. A
p-value of less than 0.05 was considered statistically
significant.
RESULTS
Macroscopic Findings
On the examination made on day 28, it was seen that
healing and hairing were similarly largely completed
in all groups. After shaving the backs, it was observed
that incision scars were difficultly detected especially
in Group 3 (Figure 3). Although there is no significant
difference between the groups as seen in the photomicrographs there are some histological differences which
explained below.
526 H. Aydin et al.
FIGURE 3 Macroscopic findings (a: Group 1; b: Group 2; c:Group 3).
TABLE 2
Histopathological examinations and mechanical tensile test results. One-Way ANOVA or Kruskal-Wallis test.
Reepithelialization
Granulation tissue Deposition
Collagen Deposition
Inflammatory cell
Angiogenesis
Mechanical Tensile Test
Group 1
Group 2
Group 3
Post-Hoc Test (p)
Day
Mean
Mean
Mean
p
7
14
21
7
14
21
7
14
21
7
14
21
7
14
21
E-mod
0.00
0.33
1.29
1.63
2.83
1.41
0.50
1.83
2.00
2.75
2.17
1.71
3.00
3.00
2.29
0.10
0.00
0.50
2.33
2.13
2.00
1.00
0.88
1.00
1.33
2.25
2.25
1.17
2.88
3.00
2.33
0.16
0.00
0.83
2.86
2.60
2.83
0.43
1.40
2.17
2.86
2.60
1.50
0.71
3.00
2.67
2.00
0.27
1000
0.41
0.0038
0.034
0.002
0.377
0.025
0.002
0.006
0.273
0.076
0.172
0.444
0.085
0.550
0.020
Group 1 vs 2
Group 1 vs 3
0.026
0.001
0.002
Group 2 vs 3
0.002
0.023
0.002
0.002
0.002
0.020
Post Hoc Test ∗ Mann Whitney U Bonferroni Correction p < 0.017 ∗∗ Tukey HSD Test
Histological Findings
Biopsies taken from wounds of the subjects on days 7,
14, and 21 were subjected to histological examination.
They were examined according to Table 2 in terms of reepithelization, granulation tissue formation, collagen
accumulation, amount of inflammatory cells, angiogenesis, and ulcer formation (Figure 4).
re-epithelialization score. On day 21, there was a
statistically significant difference between the
groups in terms ofthe mean re-epithelialization score
(p = 0.003). The mean re-epithelialization score was
statistically significantly lower in Group 1 than in
Groups 2 and 3 (Group 1 vs. Group 2 p = 0.026; Group
1 vs. Group 3 p = 0.001)
Granulation
Re-Epithelialization
On days 7 and 14,there was no statistically significant
difference between the groups in terms ofthe mean
On days 7 and 14, there was a statistically significant
difference between the groups in terms of the mean
granulation score (p = 0.034, p = 0.002). On day 21,
FIGURE 4 a. Complated mature epithelialization in Group 3, day 21. b. Marked granulation tissue and angiogenesis in Group 2, day 14. c. Marked inflamation in Group 1,
day 7.
Journal of Investigative Surgery
The Effects of Proline on Wound Healing 527
there was no statistically significant difference between
the groups in terms of the mean granulation score (p =
0.377).
Collagen Accumulation
On days 7, 14, and 21, there was a statistically significant difference between the groups in terms of
the mean collagen accumulation score (p = 0.025,
p = 0.002, and p = 0.006). On days 7 and 21, the mean
collagen accumulation score was statistically significantly higher in Group 3 than in Group 1(p = 0.023,
p = 0.002). On day 14, the mean collagen accumulation
score was statistically significantly lower in Group 2
than in Groups 1 and 3 (p = 0.002 for both of them).
Inflammatory cell
On days 7, 14, and 21, there was no statistically significant difference between the groups in terms of in
terms of inflammatory cell accumulation (p = 0.273,
p = 0.076, and p = 0.172).
Angiogenesis
On days 7, 14, and 21, there was no statistically significant difference between the groups in terms of in terms
ofangiogenesis (p = 0.444, p = 0.085, and p = 0.550).
Ulcer
On days 7 and 14, there was a statistically significant
difference between the groups in terms of ulcer formation. On day 7, the mean ulcer formation score was statistically significantly lower in Group 3 than in Group 1
(p = 0.011). On day 14, the mean ulcer formation score
was statistically significantly lower in Group 3 than in
Groups 1 and 2 (p = 0.030, p = 0.002).
Mechanical Tensile Test
On day 30,there was a statistically significant difference
between Groups 1 and 3 in terms of the mean E-mode
score on mechanical tensile test (p = 0.020). Histopathological examinations and mechanical tensile test results
are shown in Table 2.
DISCUSSION
Wound healing consists of a sequence of complex
molecular and cellular events including inflammation,
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cell migration, angiogenesis, matrix synthesis, collagen accumulation, and re-epithelization. A close and
coordinated interaction between biochemical mediators, inflammatory cells, extracellular matrix proteins,
and neighboring cells is required for this process to
function properly.16–17
Proline is a non-essential amino acid and is synthesized from glutamic acid. It is a structural component
of collagen and plays important roles in wound healing
as well as in protein synthesis, metabolism, nutrition,
immune response, and antioxidative reactions.2
Prolidase deficiency is a rare autosomal recessive
disorder. Although the symptoms of the disease may
vary between individuals, low proline levels and various wound healing problems are seen.18–19
Although the effects of proline precursors such as
arginine, glutamic acid, ornithine, and citrulline on
wound healing have been extensively studied, there is
a limited number of studies on the efficacy of proline.
Therefore, we wanted to compare the effects of local
and systemic administration of proline on wound healing in this study. In this respect, our study is the only
study in this area.
Epithelization is defined that keratinocytes multiply
through cell division in the lower skin layers and cover
over the granulation tissue. Ponrasu et al. showed that
re-epithelization increased in proline-treated rats.4 In
our study, it was found that re-epithelization significantly increased in the groups receiving systemic and
local administration of proline compared to the control
group. In addition, it was found that re-epithelization
significantly increased in the group receiving systemic
administration of proline compared to the group
receiving local administration of proline.
Fibroblasts and vascular endothelial cells proliferate between 1 and 5 days after the wound. They produce granulation tissue, which is an essential feature
of wound healing. It is thought that proline accelerates
the transformation from fibroblast to myofibroblast,
and thus that wound contraction occurs earlier.4,20 In
our study, it was found that granulation tissue formation significantly increased in the group receiving systemic administration of proline compared to the other
groups.
Ponrasu et al.4 have shown that proline accelerated
wound healing, and that topical administration of
proline at the wound site gave better results than
oral administration. However, they did not show the
systemic effect clearly because proline amino acid was
administered orally. Therefore, the local bioavailability of proline amino acid is higher in their study. It
would be more appropriate to consider our work as
continuation rather than as a copy of Ponrasu and
his colleagues’ work. Our study showed that in the
case of systemic administration via intraperitoneal
route, proline amino acid is more effective than local
administration.
528 H. Aydin et al.
The accumulation of newly synthesized collagen at
the wound site increases collagen concentration per
unit area and subsequently tissue tensile strength. The
fact that collagen accumulation significantly increased
in the group receiving systemic administration of
proline compared to the group receiving local administration of proline and the control group shows that
systemic administration of proline is more effective on
collagen synthesis and storage.
Macrophages and neutrophils are predominant during the inflammatory phase (They peak at 2–3 days).
Lymphocytes appear after several days (They peak at
7 days). In our study, there was no statistically significant difference between the groups in terms of inflammatory cells.
Angiogenesis provides nutrients needed by tissues
during wound healing and also serves for structural repair via granulation tissue formation. In our
study, there was no statisticallysignificant difference between the groups in terms of the amount
of angiogenesis.This was attributed to the fact that
growth factors, which trigger and sustain angiogenesis, are secreted by inflammatory cells, mainly
macrophages.
In our study, ulcer formation was found to be significantly lower in the group receiving systemic administration of proline compared to the other groups. There
was no statistically significant difference between the
group receiving local administration of proline and the
control group in terms of ulcer formation.
The most important consequence of all morphological and chemical events that occur in wound healing
in terms of surgical biology is that wound tensile
strength reaches normal tissue value.One of the factors
that accelerate the recovery of wound tensile strength
is the occurrence of collagen fibrils. The most important factor in increasing tensile strength and separation
resistance is that intramolecular and intermolecular
covalent bonds rather than the amount of collagen in
the wound are increased.
The mean elastic modulus was found to be significantly higher in the group receiving systemic administration of proline than in the control group. This
demonstrates that administration of proline increases
tissue resistance and prevents deformity.
Although the amount of collagen was not significantly different in the group receiving local administration of proline and the control group, tensile strength
was found to be higher in the group receiving local
administration of proline. This suggests that proline
increases intramolecular and intermolecular covalent
bonds in collagen.
In conclusion, our study confirmed that proline
has positive effects on wound healing. However,
it revealed that systemic administration of proline is more effective than local administration of
proline.
DECLARATION OF INTEREST
The authors report no conflicts of interest. The authors
alone are responsible for the content and writing of the
paper.
This research did not receive any specific grant from
any funding agency in the public, commercial, or notfor-profit sector.
REFERENCES
1. Hu CA, Khalil S, Zhaorigetu S. Human D1-pyrroline-5carboxylate synthase: function and regulation. Amino Acids.
2008;35:665–672. doi:10.1007/s00726-008-0075-0. PMID:
18401542.
2. Guoyao WU, Bazer Fuller W, Burghardt Robert C.
Proline and hydroxyproline metabolism: implications
for animal and human nutrition. Amino Acids. 2010;
40:1053–1063.
doi:10.1007/s00726-010-0715-z.
PMID:
20697752.
3. Karsıdag T, Assensio J, Kabukcuoglu F. Preliminary study
comparing the effects of locally and systemically applied Lcarnitine on the healing of full-thickness skin defects. Scand
J Surg. 2010;99:147–152. doi:10.1177/145749691009900309.
PMID: 21044932.
4. Ponrasu T, Jamuna S, Mathew A. Efficacy of L-proline
administration on the early responses during cutaneous
wound healing in rats. Amino Acids. 2013 45:179–189.
doi:10.1007/s00726-013-1486-0. PMID: 23508578.
5. Williams JZ, Barbul A. Nutrition and wound healing.
Surg Clin North Am. 2003;83:571–574. doi:10.1016/S00396109(02)00193-7. PMID: 12822727.
6. Baker DH. Advances in protein-amino acid nutrition of
poultry. Amino Acids. 2009; 37:29–41. doi:10.1007/s00726008-0198-3. PMID: 19009229.
7. Barbul A. Proline precursors to sustain Mammalian
collagen synthesis. J Nutr. 2008; 138:2021–2024.
doi:10.1093/jn/138.10.2021S.
8. Elango R, Ball RO, Pencharz PB. Amino acid requirements in humans: with a special emphasis on the
metabolic availability of amino acids. Amino Acids.
2009;37:19–27.
doi:10.1007/s00726-009-0234-y.
PMID:
19156481.
9. Wu G, Bazer FW, Datta S. Proline metabolism in the conceptus: Implications for fetal growth and development.
Amino Acids. 2008;35:691–702. doi:10.1007/s00726-008-00527. PMID: 18330497.
10. Townsend CM, Beauchamp DR, Evers MB. Sabiston textbook
of surgery: the biological basis of modern surgical practice. 17th
ed. Philadelphia: Elsevier Saunders; 2004.
11. Ponrasu T, Suguna L. Efficacy of Annona Squamosa
on wound healing in streptozotocin induced diabetic
rats. Int Wound J. 2012;9:613–623 doi:10.1111/j.1742481X.2011.00924.x. PMID: 22233431.
12. Ashcroft GS, Mills SJ. Androgen receptor-mediated inhibition of cutaneous wound healing. J Clin Invest. 2002;110:
615–624. doi:10.1172/JCI0215704. PMID: 12208862.
13. Ashcroft GS, Mills SJ, Ashworth JJ. Ageing and wound
healing. Biogerontology. 2002;3:337–345. doi:10.1023/
A:1021399228395. PMID: 12510172.
14. Gilliver SC, Ruckshanthi JP, Atkinson SJ. Androgens influence expression of matrix proteins and proteolytic factors during cutaneous wound healing. Lab Invest. 2007;87:
871–881. doi:10.1038/labinvest.3700627. PMID: 17607299.
Journal of Investigative Surgery
The Effects of Proline on Wound Healing 529
15. Barbul A, Efron DT, Kavalukas SL. Schwartz Principles of Surgery. In: Brunicardi FC editör. Wound
Healing. New York, USA: Mc Graw Hill; 2015.
p. 241–273.
16. Wu G, Bazer FW, Burghardt RC. Impacts of amino acid
nutrition on pregnancy outcome in pigs: mechanisms and
implications for swine production. J Anim Sci. 2010;88:195–
204. doi:10.2527/jas.2009-2446.
17. Tatar C, Aydin H, Karsidag T, et al. The effects of plateletrich plasma on wound healing in rats. Int J Clin Exp Med.
2017;10:7698–7706.
C 2018 Taylor & Francis Group, LLC
18. Trent JT, Kirsner RS. Leg ulcers secondary to prolidase deficiency. Adv Skin Wound Care. 2004;17:468–472
doi:10.1097/00129334-200411000-00011. PMID: 15632738.
19. Özcan Ö, Gültepe M, İpçioğlu OM. Prolidazın Mutlak
Aktivitesini Değerlendirmede Fotometrik Enzim Aktivitesi
Ölçüm Metodunun Optimizasyonu. Turk J Biochem.
2007;32;12–16.
20. Dunphy JE, Udupa KN. Chemical and histochemical
sequences in the normal healing of wounds. N Engl J
Med. 1955;253:847–851. doi:10.1056/NEJM195511172532002.
PMID: 13272801.