|Year : 2019 | Volume
| Issue : 4 | Page : 176-181
Evaluation of serum chitinase-3-like protein 1, pentraxin-3, and neutrophil gelatinase-associated lipocalin levels in diabetic foot cases
Soysal Bas1, Mustafa Durgun2, Huriye Erbak Yilmaz3, Bilge Birlik4, Mubin Hosnuter5
1 Department of Plastic Reconstructive and Aesthetic Surgery, Sisli Hamidiye Etfal Training and Research Hospital, University of Healthy Science, Istanbul, Turkey
2 Department of Plastic Reconstructive and Aesthetic Surgery, Ataturk Training and Research Hospital, University of Izmir Katip Celebi, Izmir, Turkey
3 Department of Biochemistry, Ataturk Training and Research Hospital, University of Izmir Katip Celebi, Izmir, Turkey
4 Department of Radiology, Ataturk Training and Research Hospital, University of Izmir Katip Celebi, Izmir, Turkey
5 Binterplast Plastic Surgery Clinic, Izmir, Turkey
|Date of Submission||12-Dec-2018|
|Date of Acceptance||23-Feb-2019|
|Date of Web Publication||26-Sep-2019|
Dr. Soysal Bas
Department of Plastic Reconstructive and Aesthetic Surgery, Sisli Hamidiye Etfal Training and Research Hospital, Halaskargazi Street, 34371, Sisli/Istanbul
Source of Support: None, Conflict of Interest: None
Introduction: Diabetic foot is a chronic metabolic disease requiring a long duration of treatment and hospitalization in advanced stages. It requires a multidisciplinary approach to the different clinical presentations of the disease that complicate the process. In this study, the effects of serum chitinase-3-like protein 1 (YKL-40), pentraxin-3 (PTX-3), and neutrophil gelatinase-associated lipocalin (NGAL) levels on the prognosis of diabetic foot in different stages were evaluated. Subjects and Methods: A total of 65 patients with diabetes, and 13 healthy controls were included in the study. Wagner stage 0–1 patients were classified as mild disease (n = 26), Wagner stage 2–3 patients were classified as moderate disease (n = 26), and Wagner Stage 4 patients were classified as severe disease (n = 13). Venous blood was taken from the patients, and serum C-reactive protein, erythrocyte sedimentation rate, hemoglobin A1c, YKL-40, PTX-3, and NGAL levels were evaluated. In addition, the relationship between lower extremity vascular status and the markers was investigated. Results: YKL-40 was significantly higher in the severe disease group, PTX-3 was significantly higher in the moderate disease group, and NGAL was significantly higher in the mild disease group. In addition, YKL-40 and PTX-3 levels were found to be high in patients with arterial and venous insufficiency, but this was not observed for NGAL. Conclusions: High levels of serum YKL-40 are associated with advanced stage disease with atherosclerotic process at the forefront, high PTX-3 levels are associated with moderate stage disease with a high level of infection findings, and high NGAL levels are associated with the mild stage of diabetic foot.
Keywords: Chitinase-3-like protein 1, diabetic foot, neutrophil gelatinase-associated lipocalin, pentraxin-3, Wagner classification
|How to cite this article:|
Bas S, Durgun M, Yilmaz HE, Birlik B, Hosnuter M. Evaluation of serum chitinase-3-like protein 1, pentraxin-3, and neutrophil gelatinase-associated lipocalin levels in diabetic foot cases. Turk J Plast Surg 2019;27:176-81
|How to cite this URL:|
Bas S, Durgun M, Yilmaz HE, Birlik B, Hosnuter M. Evaluation of serum chitinase-3-like protein 1, pentraxin-3, and neutrophil gelatinase-associated lipocalin levels in diabetic foot cases. Turk J Plast Surg [serial online] 2019 [cited 2020 Jul 15];27:176-81. Available from: http://www.turkjplastsurg.org/text.asp?2019/27/4/176/267936
| Introduction|| |
Diabetic foot requires a multidisciplinary approach because the clinical presentation is variable, and its pathogenesis is complex. The process beginning with peripheral neuropathy is compounded by the addition of ischemia and infection. Glycemic control, infection management, prevention of trauma, and regulation of abnormal pressure distribution in the feet form the basis of treatment. Quality of life is reduced in patients who are not treated properly, the duration of hospitalization and treatment costs increase, and the risk of amputation is encountered. The costliest complication of diabetes, as well as lower extremity amputation due to diabetic foot that occurs every 30 s in the worldwide, reveals the seriousness of this condition.,
Chitinase-3-like protein 1 (YKL-40), also called cartilage glycoprotein-39, is a 40 kDa glycoprotein released from macrophages, neutrophils, chondrocytes, vascular smooth muscle cells, and cancer cells., It's a potent angiogenic factor that plays a key role in the innate immune system, extracellular matrix remodeling, and monocyte macrophage differentiation., Pentraxins (PTX) are divided into two groups as short and long. C-reactive protein (CRP) and serum amyloid protein A are among the short PTX. Pentraxin-3 (PTX-3), first described in long PTX, is released from various cells, such as macrophages, myeloid dendritic cells, endothelial cells, epithelial cells, and neutrophils in response to lipopolysaccharide, tumor necrosis factor alpha, and interleukin 1 beta in inflammatory conditions. It has similar properties to antibodies and plays a role in humoral immunity. It is also involved in thrombogenesis by increasing tissue factor release. Lipocalin 2, or neutrophil gelatinase-associated lipocalin (NGAL), is a 25 kDA acute phase protein in neutrophil granules., NGAL has been shown to bind to matrix metalloproteinase-9 (MMP-9) by forming a stable dimeric complex and to decrease inactivation of MMP-9 by tissue inhibitory metalloproteinase 1. It is an important mediator involved in vascular remodeling and atherosclerotic plaque instability.
In this study, the effect of serum YKL-40, PTX-3, and NGAL levels on the prognosis of diabetic foot was investigated. To the best knowledge of the authors, these markers have not been studied before in this condition.
| Subjects and Methods|| |
This prospective study started with the approval of the ethics committee. Between April 2015 and June 2015, 65 patients with diabetic feet and healthy controls (n = 13) were included in the study. Age, gender, serum CRP, erythrocyte sedimentation rate (ESR), hemoglobin A1c (HgA1c), YKL-40, PTX-3, and NGAL levels were evaluated. In addition, the patients' lower extremity vascular status was examined with the relationship of markers. In the patient group, lower extremity Doppler ultrasonography (USG) was applied to the ulcer side. In the control group, the leg selected randomly was examined. To evaluate disease prognosis, Wagner stage 0–1 patients were classified as mild disease (n = 26), Wagner stage 2–3 patients were classified as moderate disease (n = 26), and Wagner stage 4 patients were classified as severe disease (n = 13). Wagner Stage 5 patients who required amputation were not included in the study because of the small number of clinical presentations. In addition, patients under the age of 18 and above 90 years, pregnant and breastfeeding women, patients with cancer, acute renal failure, myocardial infarction, cerebrovascular disease, autoimmune or rheumatological disease, hepatic impairment, ovulation induction and infertility treatment, and substance addiction were not included in the study.
For the analysis of CPR, YKL-40, PTX-3, and NGAL, venous blood was collected in three plain tubes. One anticoagulant tube was used for HgA1c analysis, and one Na3-citrate tube was used for ESR analysis. CRP, ESR, and HgA1c levels were studied on the same day at the biochemistry laboratory. The remaining two flat tubes were centrifuged at 3000 rpm for 10 min. Serum portions were transferred to Eppendorf tube and stored at −20°C. When the number of targeted cases was reached, the serum was brought to room temperature. Hemolyzed and lipemic samples were not included in the study. YKL-40, PTX-3, and NGAL (Bosterbio, CA, USA) were measured by sandwich enzyme-linked immunosorbent assay after 90 min incubation at 37°C. Analysis was performed according to the manufacturer's instructions. The intra-assay and interassay precision for YKL-40 were 5.3%–5.5% and 6.7%–7.1%, for PTX-3, they were 5%–6.9% and 5.4%–6.9%, and for NGAL, they were 5.2%–7.5% and 6.3%–7.5%.
Doppler utrasonography examination
The examination was performed continuously with a 10 MHz high-resolution linear probe from the main femoral artery to the dorsum of the foot. Gray scale was performed first, followed by color Doppler and spectral waveform analysis. Occlusion and plaque formation, which cause stenosis in gray scale and monophasic or biphasic flow in color Doppler examination, were evaluated as arterial insufficiency. Venous structures were evaluated in the supine position with the Valsalva maneuver. The presence of reflux flow (2–3 s or more), deep vein thrombosis, subcutaneous torticosis varicose veins, and subcutaneous edema findings were evaluated as venous insufficiency.
Statistical analysis was performed using SPSS 20.0 (IBM, Chicago, Illinois, USA) software. Categorical variables were determined using frequency and percentage and numerical variables using mean and standard deviation. The relationship between categorical variables was examined by Chi-square test and the relationship between numerical variables was investigated by Spearman correlation analysis. Kruskal–Wallis test was used for comparison of two or more groups, and Mann–Whitney U-test was used for comparison of two groups. The ability of the markers to determine the disease and stage was examined by receiver operating characteristic (ROC) analysis. Youden Index method was used in the threshold value research. The study was performed at a confidence level of 95% (P < 0.05 was considered statistically significant).
| Results|| |
There were 65 (83.3%) patients and 13 (16.7%) controls included in the study. The mean age was 58.3 ± 14.3. There were 43 (55.1%) male and 35 (44.9%) female. Diabetic foot stage and markers evaluated during the first recording do not include changes over time. Mean serum values obtained from patients and control group: CRP 5.8 ± 3.6 mg/L, ESR 46.2 ± 33.6 mm/h, HgA1c 7.8 ± 2.2%, YKL-40 122.0 ± 121.3 ng/mL, PTX-3 1262.1 ± 437.7 ng/mL, and NGAL 280.4 ± 119.2 ng/mL. Of the 65 patients included in the study, 39 (60%) had arterial insufficiency and 26 (40%) had no arterial insufficiency. While 19 (29.2%) of the 65 patients had venous insufficiency, 46 (70.8%) had no venous insufficiency [Table 1].
All of the markers were statistically significantly higher in the patient group compared to the control group [Table 2]. There was no statistically significant difference between the genders. Mean values of markers are shown in [Table 3] paired group comparison according to disease severity.
|Table 3: Characteristics of markers examined on the basis of disease degree|
Click here to view
When the predictive values of all patients were examined, the following significant relationships were found: a positive low significant relationship between YKL-40 and PTX-3, a negative low significant relationship between YKL-40 and NGAL, a positive moderate significant relationship between YKL-40 and CRP, a positive low significant relationship between YKL-40 and ESR, a positive low significant relationship between PTX-3 and NGAL, a positive moderate significant relationship between PTX-3 and CRP, and a negative low significant relationship between NGAL and ESR [Table 4].
|Table 4: The relation of chitinase-3-like protein 1, pentraxin-3, and neutrophil gelatinase-associated lipocalin with each other and with other markers|
Click here to view
In ROC analysis, NGAL for mild, moderate YKL-40 and PTX-3 for mild disease and YKL-40 for severe disease were found to be valuable in clinical decision. The following cutoff values were found: for NGAL in mild disease, it was 239.8, with 88.5% sensitivity and 55.8% specificity; for YKL-40 in moderate disease, it was 55.7, with 84.6% sensitivity and 59.6% specificity; for PTX-3 in moderate disease, it was 1640.1, with 34.6% sensitivity and 96.2% specificity; and for YKL-40 in severe disease, it was 1269.3, with 61.5% sensitivity and 73.8% specificity.
In patients with arterial and venous insufficiency, the mean YKL-40 and PTX-3 levels were found to be significantly higher than those without. There was no statistically significant difference in NGAL [Table 5].
|Table 5: Chitinase-3-like protein 1, pentraxin-3, and neutrophil gelatinase-associated lipocalin properties in patients with and without arterial and venous insufficiency|
Click here to view
| Discussion|| |
There are many classification systems in diabetic foot. The Wagner classification, which was created by Meggitt in 1976 and popularized by Wagner, emphasizes the depth of ulcers. Its use is simple and straightforward. It is a clinical classification. There is no skin lesion in Stage 0, localized superficial ulcer in Stage 1, deep ulcer and soft-tissue infection in Stage 2, deep ulcer and osteomyelitis in Stage 3, ischemic ulcer in the forefoot and heel in Stage 4, and ischemic ulcer of the entire foot in Stage 5.
YKL-40 has been shown to increase in inflammatory diseases such as diabetes mellitus (DM), gestational diabetes, chronic obstructive pulmonary disease, and inflammatory bowel disease.,,, It has also been reported that atherosclerotic plaque progression and the number of vessels involved in coronary artery disease are closely related.
In this study, the highest YKL-40 level was detected in the severe group of patients with arterial insufficiency. Batinic et al. found a high YKL-40 association with the severity of generalized atherosclerosis in peripheral arterial disease (PAD). In addition, high levels of YKL-40 in patients with type 2 DM reflect increased cardiovascular complications and disease mortality. A high level of YKL-40 in the severe disease group with predominant arterial insufficiency is consistent with the literature. In diabetic foot, high levels of YKL-40 show atherosclerotic and indirectly ischemic foot.
It has been shown that PTX-3 levels are increased in diseases, such as systemic inflammation, sepsis, vasculitis, acute myocardial infarction, heart failure, and psoriasis. Huttunen et al. have shown that PTX-3 is a prognostic marker in patients with Staphylococcus aureus, Streptococcus pneumoniae, β-hemolytic Streptococcus, and Escherichia More Details coli bacteremia and is more valuable than CRP.
It has been reported that PTX-3 is elevated in many infections, and there is a correlation between the increase of PTX-3 and the severity of infection. In this study, the highest PTX-3 levels were found in the moderate disease group. In the moderate disease group, diabetic foot is manifested more by infection. The cutoff value calculated for PTX-3 is 1640.1 ng/mL, with a sensitivity of 34.6% and a specificity of 96.2%. PTX-3 and CRP are found in atherosclerotic plaques. In this study, PTX-3 levels were higher in patients with arterial insufficiency than those without arterial insufficiency. Zhou et al. showed that PTX-3 is a more sensitive marker than high sensitivity CRP in PAD. A positive moderate correlation between PTX-3 and CRP is similar to the literature. In this study, the fact that PTX-3 is effective in demonstrating moderate disease indicates the progression to infection.
NGAL is easily filtered from the glomerular membrane due to its small molecular structure, and it accumulates in the cortical tubules and urine of the kidney after nephrotoxic and ischemic injuries. It is an early, sensitive, noninvasive biomarker for acute renal injury. Serum and urinary NGAL levels have been shown to be markers in the evaluation of diabetes in normoalbuminuria individuals and in the early diagnosis of diabetic nephropathy. A positive correlation between diabetic retinopathy and NGAL levels has also been shown. High levels of serum and atherosclerotic plaques are indicative of coronary artery stenosis in diabetic patients. They indicate prognosis in acute myocardial infarction, heart failure, and stroke.
Unlike other markers, NGAL levels decreased as the disease severity increased, and diabetic foot determination (88.5% sensitivity and 55.8% specificity) was detected in mild disease. There was no significant difference in patients with arterial and venous insufficiency. Thus, NGAL may be an effective marker in the early stages of diabetic foot.
The serum YKL-40 level was found to be high in the severe and moderate disease groups, high in the PTX-3 moderate disease group, and high in the NGAL mild and moderate disease groups. Increased serum YKL-40 and PTX-3 levels indicate progression to advanced disease in the Wagner classification (positive low correlation between YKL-40 and PTX-3 [r = 0.260, P= 0.036]). In addition, the high YKL-40 level and low NGAL levels further support the progression to the advanced stage (negative low significant relationship between YKL-40 and NGAL [r = −0.328, P= 0.008]). The high serum NGAL level indicates the initial stage of diabetic foot, and the addition of a high level of PTX-3 indicates that the disease will be exacerbated (positive low correlation between PTX-3 and NGAL [r = 0.309, P= 0.012]).
The study was planned prospectively because YKL-40, PTX-3, and NGAL markers were not evaluated as routine laboratory tests. Furthermore, there are some limitations in this study. The sample size was small, so similar research should be conducted with more patients. It is also a study that could not investigate the potential mechanisms of the studied markers. As multiple comparisons are made, errors due to multiple tests cannot be ignored.
Wagner classification is a clinical classification system and requires additional tests for prognosis and treatment planning. The serum levels of these markers in diabetic foot will provide more detailed information about the disease and will reveal the disease stage and prognostic process more clearly.
| Conclusion|| |
Consequently, high levels of serum YKL-40 in diabetic patients will show progression to advanced stage disease as wagner Grade 4 where the atherosclerotic process is at the forefront. High PTX-3 levels indicate moderate disease as Wagner Garde 2 and 3 with a high rate of infection. High NGAL levels indicate a mild stage as Wagner Grade 0 and 1 of diabetic foot.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Singh N, Armstrong DG, Lipsky BA. Preventing foot ulcers in patients with diabetes. JAMA 2005;293:217-28.
Buggy A, Moore Z. The impact of the multidisciplinary team in the management of individuals with diabetic foot ulcers: A systematic review. J Wound Care 2017;26:324-39.
Rehli M, Krause SW, Andreesen R. Molecular characterization of the gene for human cartilage Gp-39 (CHI3L1), a member of the chitinase protein family and marker for late stages of macrophage differentiation. Genomics 1997;43:221-5.
Michelsen AE, Rathcke CN, Skjelland M, Holm S, Ranheim T, Krohg-Sørensen K, et al.
Increased YKL-40 expression in patients with carotid atherosclerosis. Atherosclerosis 2010;211:589-95.
Rathcke CN, Vestergaard H. YKL-40, a new inflammatory marker with relation to insulin resistance and with a role in endothelial dysfunction and atherosclerosis. Inflamm Res 2006;55:221-7.
Shao R. YKL-40 acts as an angiogenic factor to promote tumor angiogenesis. Front Physiol 2013;4:122.
Steel DM, Whitehead AS. The major acute phase reactants: C-reactive protein, serum amyloidPcomponent and serum amyloid A protein. Immunol Today 1994;15:81-8.
Mantovani A, Garlanda C, Bottazzi B, Peri G, Doni A, Martinez de la Torre Y, et al.
The long pentraxin PTX3 in vascular pathology. Vascul Pharmacol 2006;45:326-30.
Doni A, Michela M, Bottazzi B, Peri G, Valentino S, Polentarutti N, et al.
Regulation of PTX3, a key component of humoral innate immunity in human dendritic cells: Stimulation by IL-10 and inhibition by IFN-gamma. J Leukoc Biol 2006;79:797-802.
Introna M, Alles VV, Castellano M, Picardi G, De Gioia L, Bottazzai B, et al.
Cloning of mouse ptx3, a new member of the pentraxin gene family expressed at extrahepatic sites. Blood 1996;87:1862-72.
Liu Q, Nilsen-Hamilton M. Identification of a new acute phase protein. J Biol Chem 1995;270:22565-70.
Kjeldsen L, Johnsen AH, Sengeløv H, Borregaard N. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J Biol Chem 1993;268:10425-32.
Yan L, Borregaard N, Kjeldsen L, Moses MA. The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL. J Biol Chem 2001;276:37258-65.
Hemdahl AL, Gabrielsen A, Zhu C, Eriksson P, Hedin U, Kastrup J, et al.
Expression of neutrophil gelatinase-associated lipocalin in atherosclerosis and myocardial infarction. Arterioscler Thromb Vasc Biol 2006;26:136-42.
Ghotaslou R, Memar MY, Alizadeh N. Classification, microbiology and treatment of diabetic foot infections. J Wound Care 2018;27:434-41.
Wagner FW Jr. The diabetic foot. Orthopedics 1987;10:163-72.
Rathcke CN, Johansen JS, Vestergaard H. YKL-40, a biomarker of inflammation, is elevated in patients with type 2 diabetes and is related to insulin resistance. Inflamm Res 2006;55:53-9.
Létuvé S, Kozhich A, Arouche N, Grandsaigne M, Reed J, Dombret MC, et al.
YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages. J Immunol 2008;181:5167-73.
Koutroubakis IE, Petinaki E, Dimoulios P, Vardas E, Roussomoustakaki M, Maniatis AN, et al.
Increased serum levels of YKL-40 in patients with inflammatory bowel disease. Int J Colorectal Dis 2003;18:254-9.
Li J, Niu G, Wang H, Wang K, Huang B, Li M, et al.
Serum YKL-40 levels in gestational diabetes mellitus. Gynecol Endocrinol 2016;32:412-5.
Kucur M, Isman FK, Karadag B, Vural VA, Tavsanoglu S. Serum YKL-40 levels in patients with coronary artery disease. Coron Artery Dis 2007;18:391-6.
Batinic K, Höbaus C, Grujicic M, Steffan A, Jelic F, Lorant D, et al.
YKL-40 is elevated in patients with peripheral arterial disease and diabetes or pre-diabetes. Atherosclerosis 2012;222:557-63.
Persson F, Rathcke CN, Gall MA, Parving HH, Vestergaard H, Rossing P, et al.
High YKL-40 levels predict mortality in patients with type 2 diabetes. Diabetes Res Clin Pract 2012;96:84-9.
Karakas MF, Buyukkaya E, Kurt M, Motor S, Akcay AB, Buyukkaya S, et al.
Serum pentraxin 3 levels are associated with the complexity and severity of coronary artery disease in patients with stable angina pectoris. J Investig Med 2013;61:278-85.
Huttunen R, Hurme M, Aittoniemi J, Huhtala H, Vuento R, Laine J, et al.
High plasma level of long pentraxin 3 (PTX3) is associated with fatal disease in bacteremic patients: A prospective cohort study. PLoS One 2011;6:e17653.
Liu S, Qu X, Liu F, Wang C. Pentraxin 3 as a prognostic biomarker in patients with systemic inflammation or infection. Mediators Inflamm 2014;2014:421429.
Matsuura Y, Hatakeyama K, Imamura T, Tsuruda T, Shibata Y, Kodama T, et al.
Different distribution of pentraxin 3 and C-reactive protein in coronary atherosclerotic plaques. J Atheroscler Thromb 2012;19:837-45.
Zhou Y, Ni Z, Zhang J, Zhang W, Wu Q, Shen G, et al.
Plasma pentraxin 3 may be a better marker of peripheral artery disease in hemodialysis patients than C-reactive protein. Vasc Med 2013;18:85-91.
Mishra J, Ma Q, Kelly C, Mitsnefes M, Mori K, Barasch J, et al.
Kidney NGAL is a novel early marker of acute injury following transplantation. Pediatr Nephrol 2006;21:856-63.
Mishra J, Dent C, Tarabishi R, Mitsnefes MM, Ma Q, Kelly C, et al.
Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet 2005;365:1231-8.
Lacquaniti A, Donato V, Pintaudi B, Di Vieste G, Chirico V, Buemi A, et al.
“Normoalbuminuric” diabetic nephropathy: Tubular damage and NGAL. Acta Diabetol 2013;50:935-42.
Chung JO, Park SY, Cho DH, Chung DJ, Chung MY. Plasma neutrophil gelatinase-associated lipocalin levels are positively associated with diabetic retinopathy in patients with type 2 diabetes. Diabet Med 2016;33:1649-54.
Eilenberg W, Stojkovic S, Piechota-Polanczyk A, Kaider A, Kozakowski N, Weninger WJ, et al.
Neutrophil gelatinase associated lipocalin (NGAL) is elevated in type 2 diabetics with carotid artery stenosis and reduced under metformin treatment. Cardiovasc Diabetol 2017;16:98.
Helanova K, Spinar J, Parenica J. Diagnostic and prognostic utility of neutrophil gelatinase-associated lipocalin (NGAL) in patients with cardiovascular diseases – Review. Kidney Blood Press Res 2014;39:623-9.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]