Serum Neutrophil Gelatinase-associated Lipocalin(NGAL)Is Elevated in Patients with Asthma and Airway Obstruction

Neutrophilic airway inflammation is one of the features of severe asthma.Neutrophil gelatinase-associated lipocalin(NGAL),or lipocalin-2,is a glycoprotein associated with neutrophilic inflammation and can be detected in blood.Recently,blood NGAL levels have been reported to be elevated in chronic obstructive pulmonary disease.However,the clinical significance of serum NGAL levels in patients with asthma has not been elucidated.The aim of this study was to explore the association between serum NGAL level and clinical parameters in patients with asthma.Sixty.one non-smoking people with stable asthma were enrolled in this study.All patients underwent blood ollction and pulmonary function tests.The associations between serum NGAL levels and clinical parameters were analyzed retrospectively.Serum NGAL levels in patients with asthma and obstructive ventilatory defect were higher than those in patients with asthma without obstructive ventilatory defect(76.4±51.4 ng/mL vs.39.3±27.4 ng/mL,P=0.0019).Serum NGAL levels were correlated with forced expired flow at 50%of vital capacity%predicted and forced expired flow at 75%of vital capacity%predicted(r=-0.3373,P=0.0078 and r=0.2900,P=0.0234,respectively).Results of a multiple regression analysis demonstrated that serum NGAL level was independently associated with obstructive ventilatory defect.Serum NGAL levels were elevated in patients with asthma and obstructive ventilatory defect.NGAL may be involved in airway remodeling possibly mediated by neutrophilic inflammation in asthma.

Neutrophil gelatinase-associated lipocalin does not predict acute kidney injury in heart failure

BACKGROUND Acute cardiorenal syndrome type 1(CRS-1)is defined by a rapid cardiac dysfunction leading to acute kidney injury(AKI).Neutrophil gelatinaseassociated lipocalin(NGAL)is expressed on the surface of human neutrophils and epithelial cells,such as renal tubule cells,and its serum(sNGAL)and urinary have been used to predict AKI in different clinical settings.AIM To characterize CRS-1 in a cohort of patients with acute heart diseases,evaluating the potentiality of sNGAL as an early marker of CRS-1.METHODS We performed a retrospective cohort,multi-centre study.From January 2010 to December 2011,we recruited 202 adult patients admitted to the coronary intensive care unit(CICU)with a diagnosis of acute heart failure or acute coronary syndrome.We monitored the renal function to evaluate CRS-1 development and measured sNGAL levels within 24 h and after 72 h of CICU admission.RESULTS Overall,enrolled patients were hemodynamically stable with a mean arterial pressure of 92(82-107)mmHg,55/202(27.2%)of the patients developed CRS-1,but none of them required dialysis.Neither the NGAL delta value(AUC 0.40,95%CI:0.25-0.55)nor the NGAL peak(AUC 0.45,95%CI:0.36-0.54)or NGAL cutoff(≥140 ng/mL)values were statistically significant between the two groups(CRS-1 vs no-CRS1 patients).The area under the ROC curve for the prediction of CRS-1 was 0.40(95%CI:0.25-0.55)for the delta NGAL value and 0.45(95%CI:0.36-0.54)for the NGAL peak value.Finally,in multivariate analysis,the risk of developing CRS-1 was correlated with age>60 years,urea nitrogen at admission and 24 h-urine output(AUC 0.83,SE=60.5%SP=93%),while sNGAL was not significantly correlated.CONCLUSION In our population,sNGAL does not predict CRS-1,probably as a consequence of the mild renal injury and the low severity of heart disease.So,these data might suggest that patient selection should be taken into account when considering the utility of NGAL measurement as a biomarker of kidney damage.

Combination of neutrophil gelatinase-associated lipocalin and matrix metalloproteinase-9 are biomarkers for the detection of colon tubular adenocarcinoma

BACKGROUND Tubular adenocarcinoma of the colon,which originates from the epithelium of the glands,is a major health concern worldwide.However,it is difficult to detect at an early stage.The lack of biomarkers is a main barrier to the diagnosis and treatment of tubular adenocarcinoma.Neutrophil gelatinase-associated lipocalin(NGAL)is a secreted protein that induces the expression of matrix metalloproteinase-9(MMP-9)and is involved in various tumors.NGAL and MMP-9 have been reported to be associated with tumorigenesis and development.They may have potential as biomarkers for diagnosis of tubular adenocarcinoma of the colon.AIM To determine whether NGAL and MMP-9 can be used as potential biomarkers to indicate the progression of tubular adenocarcinoma of the colon.METHODS Samples were collected from surgically excised tissue from various patients.The content of pro-gastrin-releasing peptide(pro-GRP)in the serum was measured by an electrochemiluminescence immunoassay.The expression patterns of NGAL and MMP-9 and the relationship between NGAL and MMP-9 were examined by quantitative real-time PCR,Western blotting and immunohistochemical analysis.RESULTS In this study,we found that NGAL and MMP-9 can be used as biomarkers for the detection of tubular adenocarcinoma of the colon and that their combination improved diagnostic accuracy.By analyzing the expression of NGAL in tubular adenocarcinoma at different levels,we found that NGAL expression was significantly upregulated in primary tubular adenocarcinoma tissues compared with normal tissues.The upregulation of NGAL expression was strongly correlated with both the degree of differentiation and the disease stage(I–III),indicating that NGAL could serve as a diagnostic biomarker for tubular adenocarcinoma.When using NGAL as a biomarker for diagnosis,the accuracy was similar to that achieved with the widely used biomarker pro-GRP,suggesting that NGAL is reliable.Moreover,the expression of MMP-9 was also strongly correlated with the differentiation stage,demonstrating that MMP-9 could be used as a biomarker to indicate the progression of tubular adenocarcinoma of the colon.More importantly,the combination of NGAL and MMP-9 produced a more accurate diagnosis of tubular adenocarcinoma,and these results were further confirmed by immunohistochemical analysis of tissue sections.CONCLUSION Our study demonstrated that both NGAL and MMP-9 can be used as biomarkers for the diagnosis of colon tubular adenocarcinoma and that the results could be further improved by combining them.

The Differences of Urinary Neutrophil Gelatinase-Associated Lipocalin (NGAL) Levels between Asphyxiated and Non-Asphyxiated Neonates

Objective: To evaluate the differences of urinary NGAL levels between asphyxiated and non-as- phyxiated neonates. Methods: This was a cross-sectional observational analytic study, including 34 newborns in Dr. Hasan Sadikin Hospital, Bandung, Indonesia. Sample collection was conducted from December 2014 to March 2015. Urine NGAL levels were evaluated using enzyme-linked immunosorbent assays (ELISA) technique. To determine the differences of urinary NGAL levels between asphyxiated and non-asphyxiated group we used Mann-Whitney U test, and to determine the differences of gestational age and birth weight between these two groups we used Fisher’s exact test. Results: Twenty males (60%) and 14 females (40%) neonates participated in the study. From 34 subjects, 17 neonates were diagnosed with asphyxia and 17 neonates without asphyxia. The results showed that urine NGAL levels had significantly increased in asphyxiated neonates. The median urine NGAL level in asphyxiated group is 95% CI: 506.7 (60.0 - 651.7) ng/mL, while the median urine NGAL level in non-asphyxiated group is 95% CI: 6.7 (0.1 - 53.0) ng/mL. Statistically, there were significant urine NGAL levels differences between asphyxiated and non-asphyxi- ated neonates (p < 0.001). There were no differences in gestational age and birth weight between asphyxiated and non-asphyxiated neonates (p > 0.05). Conclusions: Urinary NGAL levels in asphyxiated neonates were significantly higher than those in non-asphyxiated neonates. There were significant differences of urine NGAL levels between the groups.

Evaluation of Reticulocyte Hemoglobin Content and Serum Neutrophil Gelatinase-Associated Lipocalin as Predictive Biomarkers of Anemia in Children on Hemodialysis

Background: Diagnosis of iron deficiency anemia with currently available tests is rendered difficult in hemodialysis patients. The aim: To investigate the role of reticulocyte Hemoglobin Content (CHr) in the diagnosis of iron deficiency anemia in hemodialysis children in comparison to the used traditional markers and assess the impressiveness and the utility of Neutrophil Gelatinase Associated Lipocalin (NGAL) as a novel biomarker of iron status in those patients. Methods: This study investigated CHr in addition to NGAL serum level in the same line with traditional markers for anemia, including: CBC, serum iron, ferritin, total iron-binding capacity (TIBC), and transferrin saturation (TSAT%). Results: It is more significant that CHr content in hemodialysis children is lower than their controls as they are (27.06 ± 2.90) pg and (32.86 ± 3.59) pg, respectively, p = 0.01. There is no significant difference regarding NGAL between the study groups. Significant negative correlation between CHr with ferritin, urea, creatinine, and positively correlated with iron and RBCS. CHr showed a sensitivity of 90% and specificity of 86.67% to detect iron-deficiency anemia with a cut-off value of 27 pg. Conclusion: CHr is superior to ferritin and TSAT % for the early diagnosis of iron deficiency anemia in hemodialysis children;our results do not support NGAL as a marker of anemia in hemodialysis patients.

Correlation between Serum Neutrophil Gelatinase Associated Lipocalin and Burn Severity: A Pilot Study

The severity of an initial burn injury is critical for determining the treatment plan and prognosis of burn patients. Here, we measured serum neutrophil gelatinase-associated lipocalin (NGAL) levels to determine whether NGAL can be used as a biomarker for severity of burn injuries. A study of the demographic, clinical, and laboratory markers for various organ damage was performed at Bestian Burn Center (n = 10 healthy people, n = 31 patients). NGAL and organ damage marker levels were measured in 31 patients with severe burns within 2 - 3 days following their admission to the intensive care unit. Serum NGAL level of the expired patients was 788.5 (685.0 - 998.0) pg/mL, whereas that of the discharged patients was 421.2 (356.2 - 480.6) pg/mL, showing that the initial serum NGAL level can be used to estimate mortality. We also determined the correlation between serum NGAL level and the currently used severity markers (total body surface area burned and abbreviated burn severity index) and confirmed that serum NGAL level could be used as a severity marker. We also found that serum NGAL level was correlated with damage of organs such as the liver, kidney, heart, and respiratory organs in patients with severe burns.

Plasma neutrophil-gelatinase-associated lipocalin and cystatin C could early diagnose contrast-induced acute kidney injury in patients with renal insufficiency undergoing an elective percutaneous coronary intervention

Background Contrast induced acute kidney injury （CIAKI） is an important complication in the use of iodinated contrast media （CM）. Our study was to evaluate the neutrophil gelatinase-associated lipocalin （NGAL） and cystatin C for early diagnosis of CIAKI. Methods The patients with established or suspected coronary artery disease （CAD） with the estimated glomerular filtration rate （eGFR） was more than 30 ml·min^-1·1.73 m^-2 and nor more than 90 ml·min^-1·1.73 m^-2 were continuously enrolled. The blood samples of the first 50 patients were obtained before and at 2, 4, 8, 24 and 48 hours after procedure to identify the time points at which the biomarkers reached peaks and at which the blood samples of the rest of patients were obtained. The plasma NGAL and cystatin C measure used enzyme-linked immunosorbent assay （ELISA） kit. The diagnostic characteristics of absolute and relative increasing NGAL and cystatin C for CIAKI were evaluated. Results Total 311 patients were enrolled, among whom 39 （12.5%） developed CIAKI. Plasma NGAL increased at 2 hours and reached peak at 4 hours after procedure, while plasma cystatin C increased at 2 hours and reached peak at 24 hours after procedure. Thus, we determine rational point of time at 4 hours for NGAL and at 24 hours after procedure for cystatin C, respectively. The plasma NGAL at 4 hours after CM exposure showed largest area under curve （AUC） of 0.662 （95% confidence interval （CI）： 0.565-0.758, P=0.002） with 51.5% sensitivity and 80.6% of specificity. The relative increasing 25% of NGAL showed the best sensitivity and specificity of 0.872 and 0.808, respectively, with maximum Youden index of 0.680, while cystatin C with relative increasing more than 25% had 76.9% of sensitivity and 81.2% of specificity. Combined two biomarkers might get more than 90% of specificity. Conclusions Single measurement of NGAL or cystatin C had poor sensitivity and specificity; however, the relative increasing 25% of NGAL at 4 hours after CM exposure demonstrated higher diagnostic values for CIAKI. Combining relative increasing plasma NGAL with relative increasing plasma cystatin C might perform better for early diagnosis of CIAKI.

Urinary neutrophil gelatinase-associated lipocalin(NGAL)and serum cystatin C measurements for early diagnosis of acute kidney injury in children admitted to PICU

Background Acute kidney injury(AKI)is common in critically ill children with significant mortality and morbidity.Serum creatinine is an insensitive and late biomarker compared to newly proposed AKI biomarkers.Methods Prospective study in pediatric intensive care unit(PICU)over three months to compare between serum cystatin-C(s-Cys-C)and urinary neutrophil gelatinase-associated lipocalin(uNGAL)as AKI biomarkers at multiple time points with pediatric risk,injury,failure,loss,end-stage renal disease(pRIFLE)classification in diagnosing AKI.Results Forty children were recruited.Of these 40 children,22 developed AKI according to pRIFLE criteria.There was no significant difference between AKI and non-AKI in age(P=0.29).Post cardiac surgery,renal insult was the main cause of AKI(27.3%).There was a twofold increased risk of incident AKI in those patients with high baseline uNGAL at PICU admission and almost a fourfold increased risk in patients with high baseline s-Cys-C at PICU admission.uNGAL levels were highly predictive of AKI during the follow-up period[area under the curve(AUC)=0.76,95%confidence interval(CI)0.61-0.92].The cutoff point with the highest correctly classified proportion was 223 ng/mL(≥12 centiles)which correctly predict 80.0%patients with AKI,with a corresponding sensitivity of 72.7%and a specificity of 89.9%.AUC for s-Cys-C was 0.86(95%CI 0.75-0.97),and the highest correctly classified proportion was 1009μg/L(≥13 centiles);75%of patients with AKI,with a corresponding sensitivity of 63.6%and a specificity of 88.9%.Conclusion uNGAL and s-Cys-C predicts AKI early in critically ill children.

Effect of Rhizoma Polygoni Cuspidati and Ramulus Cinnamomi Compatibility on Uric Acid Metabolism and Urinary Neutrophil Gelatinase-Associated Lipocalin and Kidney Injury Molecule-1 in Rats with Hyperuricemia

Objective：To explore the effects of Rhizoma Polygoni Cuspidati and Ramulus Cinnamomicompatibility（PR） on uric acid metabolism and the expression of urinary neutrophil gelatinase-associated lipocalin（NGAL） and kidney injury molecule-1（KIM-1） in rats with hyperuricemia. Methods：Seventy male Sprague Dawley（SD） rats were randomly divided into 7 groups with 10 rats per group, including the normal group, model group, allopurinol group, benzbromarone group and PR groups at 3 doses（3.5, 7, 14 g/kg）. Except the normal group, rats of the other groups were intragastrically administered 100 mg/kg hypoxanthine and 250 mg/kg ethambutol, and subcutaneously injected with 200 mg/kg potassium oxonate. All rats were continuously modeled for 17 days, and gavaged with corresponding drugs. The rats of the normal and model groups were gavaged with saline, once a day, for 2 weeks. The levels of serum uric acid（SUA）, blood urea nitrogen（BUN） and creatinine（Cr） were determined. In addition, the contents of NGAL and KIM-1 in urine and the m RNA and protein expressions of xanthine oxidase（XOD） in liver of hyperuricemia rats were measured by reverse transcription polymerase chain reaction（RT-PCR） and Western blot, respectively. Moreover, the pathological changes of kidney were analyzed by hematoxylin and eosin（HE） stain method. Results：Compared with the normal group, the levels of SUA, BUN, NGAL and KIM-1 and the expressions of hepatic XOD m RNA and protein in the hyperuricemia rats were increased significantly（P〈0.01）. PR significantly decreased the levels of SUA, BUN, NGAL and KIM-1 and down-regulated the m RNA and protein expressions of hepatic XOD（P〈0.05 or P〈0.01）. In addition, the pathological changes of kidney were significantly suppressed by oral administration of PR. Conclusions：PR ameliorated uric acid metabolism and protected renal function, the underlying mechanism was mediated by decreasing the levels of SUA, BUN, NGAL and KIM-1, inhibiting the expression of hepatic XOD and ameliorating the pathological change of kidney.

Urine neutrophil gelatinase-associated lipocalin at SICU admission predicts for cardiac surgery-associated acute kidney injury

Background Acute Kidney Injury （AKI） is a common and serious complication of cardiovascular surgery. There is a need to find biomarkers that are involved in the etiology of cardiac surgery-associated acute kidney injury （CSA-AKI） and have an earlier response to acute kidney injury. The association between urine neutrophil gelatinase-associated lipocalin （NGAL） concentrations and AKI progression is not well established. Methods The prospective-cohort study included 1631 consecutive adult patients undergoing cardiac surgery at Fuwai Hospital between September 2012 and November 2013. AKI defined by Acute Kidney Injury Network （AKIN） criteria with a postoperative increase in plasma creatinine 〉/50% baseline or/〉0.3 mg/dL. Urine NGAL was measured us- ing latex particle-enhanced turbidimetric immunoassay. Associations between Urine NGAL levels and AKI were determined by estimating areas under receiver operating characteristic curves （AUC）. Results A total of 438 （26.9%） patients developed CSA-AKI. And the patients were divided into four groups： 1193 non-AKI patients, 368（22.6%） patients with AKIN stage I AKI, 49（3.0%） with AKIN stage 2 AKI and 21（1.3%） with AKIN stage 3 AKI. urine NGAL concentrations at surgical intensive care unit （SICU） admission were significantly related to AKI severity. The AUCs for urine NGAL were for AKIN stage 1 （0.54±0.02）, AKIN stage 2 （0.67±0.04）, and AKIN stage 3 （0.76±0.06）, respectively. Conclusions Urinary NGAL is associated with CSA-AKI and its progression, indicating their potential use as prognostic markers. Urine NGAL level measured at SICU admission predicts the development of severe AKI after cardiac surgery.

Research progress on markers of urine tubular damage in idiopathic membranous nephropathy

Idiopathic membranous nephropathy is a common primary glomerular disease.Recently,it has been found that the progression and prognosis of glomerular diseases are not only related to the glomeruli itself,but also to the severity of renal tubular injury.Although blood creatinine and urinary protein are recognized biomarkers,they appear late,are insensitive,lack specificity,and are difficult to respond to IMN conditions in a timely and accurate manner.Markers such as N-acetyl-β-D-glucosaminidase,kidney injury molecule 1,neutrophil gelatinase-associated lipoprotein in urine can dynamically reflect the progress of kidney injury in the early stage,and are detected as noninvasive,so It has been applied in IMN,but such research is lacking,and the scope needs to be further expanded and discussed in depth.

Value of serum NGAL and HE4 content change for diagnosing lung cancer and evaluating disease progression

Objective: To study the value of serum NGAL and HE4 content change for diagnosing non-small cell lung cancer (NSCLC) and evaluating disease progression. Methods: Patients who were diagnosed with NSCLC and underwent surgical resection in the First Affiliated Hospital of Chengdu Medical College between February 2015 and March 2017 were selected as the NSCLC group, and healthy volunteers who received physical examination over the same period were selected as control group. The NGAL and HE4 contents in serum of NSCLC group and control group as well as the expression of proliferation, apoptosis and invasion genes in NSCLC lesion of NSCLC group were detected. Results: Serum NGAL and HE4 levels of NSCLC group were significantly higher than those of control group, serum NGAL and HE4 levels of patients with TNM II and TNM III NSCLC were significantly higher than those of patients with TNM I NSCLC, and serum NGAL and HE4 levels of patients with TNM III NSCLC were significantly higher than those of patients with TNM II NSCLC;c-myc, Survivin, CatL, Vimentin, Slug and Snail mRNA expression in NSCLC lesion were significantly higher than those in adjacent lesion and positively correlated with serum NGAL and HE4 levels while PTEN, LATS1 and E-cadherin mRNA expression were significantly lower than those in adjacent lesion and negatively correlated with serum NGAL and HE4 levels. Conclusion: Abnormally elevated NGAL and HE4 in the serum of NSCLC patients can evaluate the proliferation and invasion of cancer cells to a certain extent.

Renal Doppler and Novel Biomarkers to Assess Acute Kidney Injury in a Swine Model of Ventricular Fibrillation Cardiac Arrest

Background： Majority of the research on cardiac arrest （CA） have focused on post-CA brain injury and myocardial dysfunction, the renal dysfunction and acute kidney injury （AKI） in other critical illnesses after CA have not been well described. This study was designed to assess AKI with renal Doppler and novel AK1 biomarkers in a swine model of ventricular fibrillation cardiac arrest （VFCA）. Methods： Thirty healthy piglets were divided into VFCA group （17 = 22） and Sham group （n = 8） in a blinded manner. Mean arterial pressure, heart rate. and cardiac output were recorded continuously. Cardiac arrest （CA） was induced by programmed electric stimulation in the VFCA group, and then cardiopulmonary resuscitation was performed. Twenty piglets returned of spontaneous circulation （ROSC） and received intensive care. Blood and urine samples were collected for AKI biomarkers testing, and Color Doppler flow imaging was performed at baseline, 6 h, 12 h, and 24 11. respectively after ROSC. At ROSC 24 h, the animals were sacrificed and a semi-quantitative evaluation of pathologic kidney injury was performed. Results： In the VFCA group, corrected resistive index （cRl） increased from 0.47 ± 0.03 to 0.64 ± 0.06, and pulsatility index （PI） decreased from 0.82 ± 0.03 to 0.68 ± 0.04 after ROSC. Cystatin C （CysC） in both serum and urine samples increased at ROSC 6 h, but neutrophil gelatinase-associated lipocalin （NGAL） in serum increased to 5.34 ± 1.68 ng/ml at ROSC 6 h, and then decreased to 3.16 ± 0.69 ng/ml at ROSC 24 h while CysC increasing constantly. According to the renal histopathology, 18 of 20 animals suffered from kidney injury. The grade of renal injury was highly correlated with RI, cRI, NGAL, and CysC. Linear regression equation was established： Grade of renal injury = 0.002× serum CysC ＋ 6.489 × PI ＋ 4.544 × cRI - 8.358 （r^2 = 0.698, F = 18.506, P 〈 0.001）. Conclusions： AKI is common in post-CA syndrome. Renal Doppler and novel AKI biomarkers in serum and urine are of significant importance as early predictors of post-CA AKI.

Value of urine IL-8, NGAL and KIM-1 for the early diagnosis of acute kidney injury in patients with ureteroscopic lithotripsy related urosepsis

Purpose::To investigate the clinical value of urine interleukin-18(IL-8),neutrophil gelatinase-associated lipocalin(NGAL)and kidney injury molecule-1(KIM-1)for the early diagnosis of acute kidney injury(AKI)in patients with ureteroscopic lithotripsy(URL)related urosepsis.Methods::A retrospective study was carried out in 157 patients with urosepsis after URL.The patients were divided into AKI group and non-AKI group according to the Kidigo guideline and urine IL-8,NGAL and KIM-1 levels were detected by enzyme-linked immunosorbent assay at 0,4,12,24 and 48 h after the surgery.Receiver operating characteristic curve(ROC)was used to evaluate the diagnostic value of these three biomarkers for postoperative AKI.Results::The level of urine IL-8,NGAL and KIM-1 in AKI group was significantly higher than that in non-AKI group at 4,12,24 and 48 h(p<0.01).The ROC analysis showed the combined detection of urine IL-8,NGAL and KIM-1 at 12 h had a larger area under curve(AUC)than a single marker(0.997,95%CI:0.991-0.998),and the sensitivity and specificity were 98.2%and 96.7%,respectively.Pearson correlation analysis showed that the levels of urine NGAL at 4,12,24 and 48 h in AKI patients were positively correlated with the levels of urine KIM-1 and IL-18(p<0.01).Conclusion::AKI could be quickly recognized by the elevated level of urine IL-8,NGAL and KIM-1 in patients with URL-related urosepsis.Combined detection of the three urine biomarkers at 12 h after surgery had a better diagnostic performance,which may be an important reference for the early diagnosis of AKI.

Detection of weak non-covalent cation-π interactions in NGAL by single-molecule force spectroscopy

Cation-π interaction is an electrostatic interaction between a cation and an electron-rich arene.It plays an essential role in many biological systems as a vital driving force for protein folding,stability,and receptor-ligand interaction/recognition.To date,the discovery of most cation-π interactions in proteins relies on the statistical analyses of available three-dimensional(3D)protein structures and corresponding computational calculations.However,their experimental verification and quantification remain sparse at the molecular level,mainly due to the limited methods to dynamically measure such a weak non-covalent interaction in proteins.Here,we use atomic force microscopy-based single-molecule force spectroscopy(AFM-SMFS)to measure the stability of protein neutrophil gelatinase-associated lipocalin(also known as NGAL,siderocalin,lipocalin 2)that can bind iron through the cation-π interactions between its three cationic residues and the iron-binding tri-catechols.Based on a site-specific cysteine engineering and anchoring method,we first characterized the stability and unfolding pathways of apo-NGAL.Then,the same NGAL but bound with the iron-catechol complexes through the cation-π interactions as a holo-form was characterized.AFM measurements demonstrated stronger stabilities and kinetics of the holo-NGAL from two pulling sites,F122 and F133.Here,NGAL is stretched from the designed cysteine close to the cationic residues for a maximum unfolding effect.Thus,our work demonstrates high-precision detection of the weak cation-π interaction in NGAL.