Relation of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 levels to growth retardation in extrahepatic portal vein obstruction

Background  Growth retardation has been described in patients with extrahepatic portal vein obstruction (EHPVO). An abnormal growth hormone (GH)–insulin-like growth factor (IGF) axis has been postulated as a possible etiology. We compared anthropometric parameters and IGF-1 and insulin-like growth factor binding protein-3 (IGFBP-3) levels in patients with EHPVO with their siblings as controls. Methods and patients  Consecutive patients diagnosed with EHPVO who presented to out-patient clinic in Department of Gastroenterology between February 2005 and February 2006 were enrolled along with their siblings whenever possible. After detailed history and clinical examination, anthropometric parameters such as age, height, weight, and mid-parental height were measured in patients and controls. IGF-1 and IGFBP-3 levels were also estimated. Results  Fifty-two patients (40 males, 32 adults) were enrolled. Sibling controls were available for 28 patients. Variceal bleeding was the presenting symptom in 41 of 52 (78.8%) patients. Target height was not achieved in 7 of 32 (22.6%) adults and 6 of 20 (30%) children, showing evidence of growth retardation. The mean IGF-1 levels in patients and controls were 124.71 ± 65.49 ng/ml and 233 ± 76.98 ng/ml (P < 0.01), respectively. The mean IGFBP-3 levels in patients and controls were 2.90 ± 1.07 μg/ml and 4.22 ± 0.77 μg/ml (P < 0.01), respectively. Hormonal levels between those with and without evidence of growth retardation did not differ significantly. Duration of symptoms, spleen size, platelet count, and age of presentation did not correlate with anthropometry and hormonal levels. Conclusions  Growth retardation by anthropometry was documented in a quarter of patients with EHPVO. All patients had significantly low IGF-1 and IGFBP-3 levels in comparison with controls despite normal anthropometry in majority of patients (75%).     

Increased 24h mean insulin-like growth factor binding protein-3 proteolytic activity in pubertal type 1 diabetic boys

Hyperglycaemia and increased variability of blood glucose in pubertal children with type 1 diabetes may be related to increased growth hormone (GH) secretion and insulin resistance. The role of changes in insulin-like growth factor-I (IGF-I) bioavailability for the glycaemic control in these patients has not been completely elucidated. In particular, the possible role of increased IGF binding protein-3 (IGFBP-3) proteolysis reported in other insulin resistant states awaits further characterization. The aims of this study were to assess if hyperglycaemia in children with type 1 diabetes was associated with changes in free dissociable IGF-I (fdIGF-I) and IGF binding protein-3 protease activity (IGFBP-3-PA) and if increased insulin resistance during puberty was associated with changes in IGFBP-3-PA in healthy and diabetic children. In diabetic boys in the period of maximal linear growth (Tanner stage 3, n = 5), the mean level and the variability of IGFBP-3-PA, determined every second hour throughout 24 h, were significantly higher both compared to postpubertal diabetic boys (n = 6; P = 0.003 and P = 0.001, respectively), and to age matched healthy boys (n = 4; P = 0.006 and P < 0.001 respectively). This activation of IGFBP-3-PA was most prominent during the day time.The mean 24 h blood glucose level (determined hourly) was the only parameter studied that significantly predicted the changes in mean 24 h IGFBP-3-PA in the diabetes group. The mean 24 h concentrations of fdIGF-I were decreased in the diabetic boys compared to the healthy controls but statistical significance was only achieved in Tanner Stage 5 (p = 0.03). We speculate that the elevated levels of IGFBP-3-PA in Tanner 3 diabetic boys are related to deteriorated glucose homeostasis and that it may be a compensatory mechanism to attenuate the decrease in fdIGF-I in order to partly restore insulin sensitivity and glycemic control.     

Characterisation of recombinant glycosylation variants of insulin-like growth factor binding protein-3

There are three potential N-glycosylation sites in the non-conserved central region of the insulin-like growth factor binding protein-3 (IGFBP-3) sequence (N89AS, N109AS, N172FS). IGFBP-3 exists as two glycoforms which reduce to a single form on enzymatic deglycosylation. To determine the functional significance of the carbohydrate chains, the N-glycosylation sites were mutated singly and in combinations by substituting Asn residues with Ala. Each recombinant glycoform was detected by radioimmunoassay, indicating that glycosylation is not essential for secretion in Chinese hamster ovary cells. Ligand blotting of the conditioned media using [125I]IGF-I indicated that all seven mutants are active. On the basis of the number and molecular masses of the bands detected for each glycoform, there is approximately 4, 4.5 and 5 kDa of carbohydrate on Asn89, Asn109 and Asn172 respectively, with variable occupancy of Asn172. Ternary complex formation by the glycovariants in the presence of ALS and excess IGF-I was not significantly different from that of fully glycosylated recombinant human (rh)IGFBP-3 [Ka (fully glycosylated)=12.5+/-4.1 l/nmol; mean Ka (all mutants)=22.1+/-3.0 l/nmol]. In contrast, Asn to Asp substitutions decreased acid-labile subunit (ALS) binding activity. Cell-surface association experiments indicate that glycosylation may influence the partitioning of IGFBP-3 between the extracellular milieu and the cell surface. Therefore, while the carbohydrate units appear to be non-essential to ALS or IGF binding, they may modulate other biological activities of IGFBP-3.     

Regulation of insulin-like growth factor binding protein-3 expression by dexamethasone.

Dexamethasone (DXM), a potent long acting glucocorticoid results in growth retardation when administered to children and experimental animals. We have used ligand blotting and RNA blotting techniques to examine the effects of DXM on serum insulin-like growth factor binding protein 3 (IGFBP-3) levels and hepatic IGFBP-3 mRNA abundance. A time- and dose-dependent increase in the 39-42 kDa serum IGF binding proteins and in IGFBP-3 mRNA abundance was observed in DXM-treated rats. A significant increase in serum IGF binding capacity was seen with as little as 0.1 microgram/100 g body weight. A significant increase in IGFBP-3 mRNA abundance was apparent as early as 1 h following DXM administration. IGFBP-3 mRNA levels reached a peak at 3 h (1.9 +/- 0.2 fold, p less than 0.005) and declined to normal levels in 6-12 h after DXM administration. Since the IGF binding proteins may be able to inhibit the action of the IGFs, enhanced expression of IGFBP-3 may be one of the mechanisms involved in DXM-induced growth retardation.     

Serum concentrations of insulin-like growth factor-I and insulin-like growth factor binding protein-3 in patients with chronic hepatitis

BACKGROUND: Insulin-like growth factor-I is a liver-derived humoral factor, which has important anabolic and metabolic actions and is predominantly bound by insulin-like growth factor binding protein-3. Low serum concentrations of both insulin-like growth factor-I and insulin-like growth factor binding protein-3 have been reported in patients with chronic liver disease, especially cirrhosis, but their conditions in chronic hepatitis are uncertain. The aim of this study was to evaluate the effect of chronic hepatitis on serum concentrations of insulin-like growth factor-I and insulin-like growth factor binding protein-3 and their association with hepatic inflammation activity and fibrosis. METHODS: Serum insulin-like growth factor-I and insulin-like growth factor binding protein-3 were measured by RIA (ng/ml) in 17 patients with mild to severe chronic viral hepatitis (12 chronic hepatitis C, 5 chronic hepatitis B) and 16 healthy subjects. The hepatic inflammation activity and the severity of fibrosis were evaluated using Desmet classification. RESULTS: Both insulin-like growth factor-I and insulin-like growth factor binding protein-3 levels did not correlate with inflammation activity, fibrosis or transaminase levels. In the chronic hepatitis group, insulin-like growth factor-I levels were significantly higher than the control group (mean, 263.8 +/- 27.33 versus 127.14 +/- 10.83 ng/ml, P < 0.001, respectively), whereas insulin-like growth factor binding protein-3 levels were significantly lower when compared with the controls (1643.47 +/- 60.68 versus 2728.87 +/- 284.61 ng/ml, P < 0.05, respectively). CONCLUSIONS: These results suggest that the concomitant states of serum insulin-like growth factor-I and insulin-like growth factor binding protein-3 levels in patients with chronic hepatitis may be different from cirrhotic patients and high serum IGF-I levels may be a specific finding of the stage of chronic hepatitis before developing cirrhosis.     

Time dependent impact of perinatal hypoxia on growth hormone,insulin-like growth factor 1 and insulin-like growth factor binding protein-3

Hypoxic-ischemia (HI) is a widely used animal model to mimic the preterm or perinatal sublethal hypoxia, including hypoxic-ischemic encephalopathy. It causes diffuse neurodegeneration in the brain and results in mental retardation, hyperactivity, cerebral palsy, epilepsy and neuroendocrine disturbances. Herein, we examined acute and subacute correlations between neuronal degeneration and serum growth factor changes, including growth hormone (GH), insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) after hypoxic-ischemia (HI) in neonatal rats. In the acute phase of hypoxia, brain volume was increased significantly as compared with control animals, which was associated with reduced GH and IGF-1 secretions. Reduced neuronal survival and increased DNA fragmentation were also noticed in these animals. However, in the subacute phase of hypoxia, neuronal survival and brain volume were significantly decreased, accompanied by increased apoptotic cell death in the hippocampus and cortex. Serum GH, IGF-1, and IGFBP-3 levels were significantly reduced in the subacute phase of HI. Significant retardation in the brain and body development were noted in the subacute phase of hypoxia. Here, we provide evidence that serum levels of growth-hormone and factors were decreased in the acute and subacute phase of hypoxia, which was associated with increased DNA fragmentation and decreased neuronal survival.     

Structural and biological characterization of bovine insulin-like growth factor binding protein-3.

Insulin-like growth factor binding protein-3 (IGFBP-3) purified from bovine serum shares 19 of 25 amino-terminal amino acid residues with IGFBP-3 purified from human, rat, and porcine sources. A newly characterized bovine fibroblast model was used to investigate the biological effects of purified bovine IGFBP-3 (bIGFBP-3). Coincubation of insulin-like growth factor I (IGF-I) with increasing concentrations of bIGFBP-3 produced a dose-dependent inhibition of IGF-I-stimulated [3H]aminoisobutyric acid (AIB) uptake in cultured bovine fibroblasts. Inhibition was complete at equimolar concentrations of IGF-I and bIGFBP-3. Inhibition of IGF-I-stimulated [3H]AIB uptake paralleled the ability of bIGFBP-3 to prevent [125I]IGF-I cell surface binding. In contrast, preincubation with bIGFBP-3 resulted in a dose-dependent enhancement of IGF-I-stimulated [3H]AIB uptake; a 32-86% increase in IGF-I bioactivity was seen after a 24 h preexposure to 10 nM bIGFBP-3, and a 2- to 6-fold potentiation was seen after a 72 h preincubation. Preincubation with bIGFBP-3 increased both the sensitivity and maximal responsiveness of the cells to IGF-I. The potentiating effects of bIGFBP-3 were associated with increased [125I]IGF-I binding to cultured bovine fibroblasts. Affinity cross-linking experiments indicated that the increase in IGF-I binding was due to increased membrane-associated bIGFBP-3 rather than to a bIGFBP-3-induced increase in type I IGF receptors. bIGFBP-3 had no effect on insulin stimulation of [3H]AIB uptake under either experimental condition. These data suggest that soluble bIGFBP-3 inhibits IGF-I action by sequestering and preventing IGF-I receptor binding, whereas surface-associated bIGFBP-3 enhances the growth-promoting effects of IGF-I in bovine fibroblasts. We propose that IGFBP-3 serves a dual function in modulating IGF action in vivo.     

胰岛素样生长因子基因在非酒精性脂肪变性肝细胞模型中的表达及其意义

目的 研究胰岛素样生长因子-1 (IGF-1)及胰岛素样生长因子结合蛋白-3(IGFBP-3)基因在非酒精性脂肪变性肝细胞模型中的表达变化及其意义. 方法 用油酸诱导永生化人肝细胞(IHH)建立非酒精性脂肪性肝病(NAFLD)细胞模型,油红O染色和细胞内甘油三酯含量检测观察细胞脂肪变情况.IHH细胞分为对照组和NAFLD组,对照组细胞以DMEM/F12培养基培养,NAFLD组给予油酸0.5 mmol/L处理72 h.采用逆转录酶-聚合酶链反应及Western blot和免疫荧光染色方法检测IGF-1和IGFBP-3在两组细胞中的mRNA及蛋白质表达变化.组间均数比较采用t检验. 结果 0.5 mmol/L油酸可成功诱导IHH细胞脂肪变性,油红O染色显示细胞内脂肪滴明显增多,细胞内甘油三酯含量从对照组的(150.2±15.6)μg/ng升高到(275.7±27.2) μg/mg (t=21.67,P＜0.01).油酸诱导后,NAFLD组细胞中IGF-1和IGFBP-3的mRNA相对表达量(分.别为0.76±0.04和1.58±0.93)均较对照组(分别为4.82±1.51和5.41±1.37)明显下降,t值分别为17.915和12.893,P值均＜0.01;IGF-1和IGFBP-3的蛋白质相对表达量(分别为1.00±0.29和0.65±0.36)也较对照组(分别为2.56±0.71和1.23±0.91)明显下降,t值分别为29.17和32.12,P值均＜0.01.免疫荧光染色结果也证实NAFLD组细胞中IGF-1和IGFBP-3的蛋白质表达较对照组明显下降. 结论 非酒精性脂肪变性肝细胞模型的IGF-1和IGFBP-3表达下降,为深入研究临床上部分非酒精性脂肪肝病儿童身高受限的机制提供了实验基础.     

非霍奇金淋巴瘤患者血清胰岛素样生长因子-1及其结合蛋白-3表达水平的初步观察

目的:探讨非霍奇金淋巴瘤(NHL)患者血清胰岛素样生长因子-1(IGF-1)及其结合蛋白-3(IGFBP-3)表达水平及其临床意义.方法:选择28例诊断初发NHL患者(淋巴瘤组)及28例健康志愿者(对照组),化学发光法测定血清IGF-1及IGFBP-3水平并计算IGF- 1/IGFBP-3值,分析组间的差异.结果:血清IGF-1及IGFBP-3水平淋巴瘤组显著低于正常对照组(均P＜0.01);IGF-1/IGFBP-3淋巴瘤组与正常对照组差异无统计学意义(P＞0.05);不同亚型的淋巴瘤组的血清IGF-1、IGFBP-3水平及IGF-1/IGFBP-3比值的差异均未达到统计学意义(P＞0.05).结论:N HL患者血清IGF-I及IGFBP-3水平明显降低,可能与NHL相关.IGF-1/IGFBP-3比值与NHL无明显相关性,IGF-1及IGFBP-3水平与NHL的分型无明显相关.     

老年住院病人血清胰岛素样生长因子-1、胰岛素样生长因子结合蛋白-3与衰弱的相关性研究

目的探讨老年住院病人血清胰岛素样生长因子-1(IGF-1)、胰岛素样生长因子结合蛋白-3(IGFBP-3)水平与衰弱的相关性。方法纳入2018年1月至2019年6月老年医学科年龄≥65岁的住院病人195例,进行衰弱表型评估和老年综合评估,采用ELISA法检测血清IGF-1、IGFBP-3水平,分析其与衰弱的相关性。结果与无衰弱、衰弱前期病人相比,衰弱病人血清IGF-1、IGFBP-3水平显著下降,差异有统计学意义(P<0.05)。有序多分类Logistic回归分析显示血清IGF-1(OR=0.943,95%CI0.894~0.994,P<0.05)、IGFBP-3(OR=0.397,95%CI0.259~0.607,P<0.001)水平与衰弱显著相关。结论血清IGF-1、IGFBP-3水平与老年住院病人衰弱显著相关,可能成为老年人衰弱的预测指标。     

Increased levels of insulin-like growth factor binding protein-2 in sera and tumours from patients with colonic neoplasia with and without acromegaly

OBJECTIVE: Patients with acromegaly are at increased risk of developing colorectal carcinoma and premalignant tubulovillous adenoma. The pathogenesis of these neoplasms could involve a stimulatory effect of serum growth factors on colonic epithelial cell proliferation. The aim of this study was to evaluate changes in (1) serum IGF-I, IGF-II, IGFBP-3 and IGFBP-2 and (2) changes in local expression of IGFBPs and p53 in colonic epithelium in patients with colonic neoplasia with and without acromegaly. DESIGN: A cross-sectional retrospective study was performed. Fasting serum samples were obtained at the time of colonoscopy for patients with acromegaly and at the time of surgery for patients with colonic neoplasia without acromegaly. MEASUREMENTS: Serum IGF-I, IGF-II, IGFBP-2 and IGFBP-3 were measured using specific immunoassays. Tissue expression of IGFBP-2, IGFBP-3 and p53 status were determined by immunohistochemistry. PATIENTS: Group 1: 26 age- and sex-matched control subjects (range 40-69 years); group 2: 18 patients with acromegaly without colonic neoplasia (range 39-68 years); group 3: 18 patients with acromegaly and colonic neoplasia (range 41-74 years, 11 = adenoma, seven = carcinoma); group 4: 19 patients with colonic neoplasia without endocrine disease (range 43-91 years, four = adenoma, 15 = carcinoma). Immunohistochemical staining of colonic biopsies was performed for IGFBP-2, IGFBP-3 and p53 in groups 3 and 4. RESULTS: Mean serum IGF-I and IGFBP-3 levels were significantly elevated in group 2 (371 +/- 131 microg/l and 6.5 +/- 1.8 mg/l, respectively) and group 3 (379 +/- 174 microg/l and 5.8 +/- 1.6 mg/l, respectively), and significantly reduced in group 4 (103 +/- 36 microg/l and 2.4 +/- 1 mg/l) compared to controls (165 +/- 40 microg/l and 4.7 +/- 1 mg/l; P < 0.0001, P < 0.001, respectively). However, median serum IGFBP-2 levels were significantly elevated in group 3 (P < 0.01) and group 4 (P < 0.0001). Immunostaining for IGFBP-2 showed strong areas of immunoreactivity in the cytoplasm of malignant colonic epithelium compared to benign epithelium. IGFBP-3 immunostaining showed strong areas of immunoreactivity in the cytoplasm and in the nucleus of malignant and benign colonic epithelium compared to the normal epithelium. Nuclear staining for p53 was observed in three patients from group 3 (two carcinoma, one adenoma) and four patients from group 4 (all carcinoma). CONCLUSION: Our results describe changes in IGFBP-2 expression in colonic neoplasia in patients with and without acromegaly, which suggest that this binding protein may regulate local bioavailability of IGF, which in turn could modulate colonic cell proliferation and/or differentiation.     

Hepatic regeneration in insulin-like growth factor binding protein-1 transgenic mice

BACKGROUND/AIMS: Partial hepatectomy results in activation of genes in the residual liver tissue which serve to restore glucose homeostasis and regenerate liver mass. Expression of insulin-like growth factor binding protein-1 (IGFBP-1) is up-regulated following partial hepatectomy and IGFBP-1 can modulate both the metabolic and mitogenic effects of insulin-like growth factor-1 (IGF-I). The aim of the study was to compare the effects of partial hepatectomy on blood glucose levels and hepatic regeneration in wild-type and transgenic mice which constitutively overexpress IGFBP-1. METHODS: Hepatic IGFBP-1 mRNA, blood glucose concentrations, liver mass and hepatic DNA synthesis were compared in sham-operated and partially hepatectomized transgenic and wild-type mice. RESULTS: Hepatic IGFBP-1 mRNA was higher in sham-operated transgenic than wild-type mice, but in both groups of mice, partial hepatectomy was associated with a significant rise in IGFBP-1 mRNA. The absolute decline in blood glucose levels following partial hepatectomy was greater in transgenic mice. Basal DNA synthesis and the response to IGF-I in isolated hepatocytes from both groups of mice were similar, and DNA synthesis in the regenerating liver in vivo was not significantly different in transgenic as compared to wild-type mice: 449.3 +/- 63.9 vs. 321.6 +/- 52.3 cpm/microgram DNA. Hepatic regeneration as measured by liver weight after hepatectomy was not different between transgenic and wild-type mice. CONCLUSIONS: Constitutive overexpression of IGFBP-1 does not enhance hepatic regeneration and does not prevent the decline in blood glucose following partial hepatectomy.     

Octreotide stimulates insulin-like growth factor binding protein-1 (IGFBP-1) levels in acromegaly.

Insulin-like growth factors (IGFs) circulate in a complexed state with several binding proteins (BPs). Of these, IGFBP-1 is regulated by hormonal and nutritional factors. The somatostatin analogue, octreotide, has been used to effectively control hypersomaototropism in acromegaly. IGFBP-1 levels were measured by RIA in 17 acromegalic patients receiving octreotide. Serum hormone sampling was conducted hourly for 8 hr periods. Among 13 octreotide responders, mean pre-treatment basal GH, IGF-1, and IGFBP-1 levels were 19 +/- 5 micrograms/L, 1021 +/- 168 micrograms/L, and 36 +/- 8 micrograms/L respectively. One month following octreotide treatment, an acute subcutaneous injection (100 micrograms) maximally attenuated GH to 3 +/- 0.6 microgram/L (18% of control, P less than 0.03) and IGF-1 to 467 +/- 75 micrograms/L (46% of control, P less than 0.008) after 4 hrs. IGFBP-1 levels, however, were stimulated to 95 +/- 16 micrograms/L (297% of control, P less than 0.003) during the same time period. A significant increase in IGFBP-1 levels occurred within 2 hrs (158% of baseline, P less than 0.03), and was sustained until the 7th hr following injection. Insulin, a known suppressor of IGFBP-1, did not change during this time. Among the 4 octreotide non-responders, mean basal IGFBP-1 levels were 42 +/- 4 micrograms/L, and 4 hrs following octreotide administration IGFBP-1 was 40 +/- 7 micrograms/L. Octreotide induced a dynamic inverse relationship between circulating GH and IGFBP-1 levels (r = -0.73, P less than 0.001). The absence of IGFBP-1 changes in octreotide non-responders and the non-suppression of insulin in octreotide responsive patients, suggest a direct GH-mediated mechanism of IGFBP-1 regulation in octreotide treated patients with acromegaly. IGFBP-1 may be another useful marker in evaluating the response of acromegaly to octreotide treatment in patients who experience clinical benefit but equivocal GH and IGF-1 attenuation.     

Association between insulin-like growth factor binding protein-2 levels and cardiovascular risk factors in Korean children

Emerging evidence has indicated that insulin-like growth factor binding protein-2 (IGFBP-2) may be involved in the development of obesity and insulin resistance like IGFBP-1. The aim of this study was to measure serum IGFBP-2 levels in overweight and obese children and to compare these levels with those of controls. We also analyzed the associations between IGFBP-2 and insulin sensitivity indices and cardiovascular risk factors. 134 Korean children including 55 overweight and 59 obese subjects were enrolled. We measured anthropometric values and determined fasting serum levels of IGFBP-2, glucose, insulin, lipid profiles, and insulin sensitivity indices including the homeostatic model assessment of insulin resistance (HOMA-IR) and the Quantitative Insulin Sensitivity Check Index (QUICKI). The subjects were subgrouped based on body mass index (BMI) and pubertal stage, and association analyses between IGFBP-2 levels and measured factors were performed in each group. Serum IGFBP-2 levels in overweight or obese children were significantly lower than those of controls regardless of pubertal development. Serum IGFBP-2 levels were negatively correlated with weight, BMI, waist circumference, fasting insulin levels, and HOMA-IR but were positively correlated with QUICKI. The associations were stronger in pubertal children than those in prepubertal children. However, no association was observed between serum IGFBP-2 levels and auxological or metabolic parameters in children with normal BMIs. These results suggested that IGFBP-2 might be a promising marker for early recognition of insulin resistance, particularly in overweight or obese children, regardless of pubertal stage.     

Lack of insulin-like growth factor binding protein-3 protease activation by venous cannulation.

Disruption of the endothelium activates thrombogenic and fibrinolytic enzymes that cleave insulin-like growth factor binding protein-3 (IGFBP-3) in vitro. The aim of the present human study was to determine whether blood sampling, i.e., venous stasis and cannulation increase IGFBP-3 proteolysis before and/or after surgery by activating these enzymes. Serum samples obtained immediately after cannulation were compared with samples obtained from a previously inserted venous catheter. Cannulation did not increase serum IGFBP-3 proteolytic activity pre- and post-operatively, as determined by in vitro degradation of 125I-IGFBP-3. Furthermore, there was no effect on in vivo IGFBP-3 fragmentation assessed by western immunoblot. In addition, a standardized venous stasis did not affect IGFBP-3 proteolytic activity or fragmentation. Comparison of IGFBP-3 proteolytic activity before and after surgery demonstrated a significant post-operative increase. However, this could not be demonstrated immediately after the initial cannulation, due to a large individual variation at this time-point before surgery.