Human fibroblast growth factor 21 (hFGF21) has become a candidate drug for regulating blood glucose and lipid metabolism. The poor stability and short half-life of hFGF21 resulted in low target tissue availability, which hampers its clinical application. In this study, the hFGF21 was fused with a recombinant human serum albumin (HSA), and the resulted fusion protein rHSA-hFGF21 was expressed in Pichia pastoris. After codon optimization, the recombinant gene fragment rHSA-hFGF21 was inserted into two different vectors (pPIC9k and pPICZαA) and transformed into three different strains (X33, GS115 and SMD1168), respectively. We investigated the rHSA-hFGF21 expression levels in three different strains and screened an engineered strain X33-pPIC9K-rHSA-hFGF21 with the highest expression level. To improve the production efficiency of rHSA-hFGF21, we optimized the shake flask fermentation conditions, such as the OD value, methanol concentration and induction time. After purification by hollow fiber membrane separation, Blue affinity chromatography and Q ion exchange chromatography, the purity of the rHSA-hFGF21 protein obtained was 98.18%. Compared to hFGF21, the biostabilities of rHSA-hFGF21, including their resistance to temperature and trypsinization were significantly enhanced, and its plasma half-life was extended by about 27.6 times. Moreover, the fusion protein rHSA-hFGF21 at medium and high concentration showed a better ability to promote glucose uptake after 24 h of stimulation in vitro. In vivo animal studies showed that rHSA-hFGF21 exhibited a better long-term hypoglycemic effect than hFGF21 in type 2 diabetic mice. Our results demonstrated a small-scale production of rHSA-hFGF21, which is important for large-scale production and clinical application in the future.
Animals ; Blood Glucose/metabolism* ; Diabetes Mellitus, Experimental ; Fibroblast Growth Factors ; Humans ; Hypoglycemic Agents/metabolism* ; Methanol/metabolism* ; Mice ; Pichia/metabolism* ; Recombinant Fusion Proteins ; Recombinant Proteins/metabolism* ; Saccharomycetales ; Serum Albumin/metabolism* ; Serum Albumin, Human/metabolism*

Albumin is the most abundant protein in human serum. A dye-binding method is commonly used in clinical laboratories for its estimation using different types of dyes. However, all these dye methods were interfered by a variety of compounds. Here we present a method for the detection of albumin in human serum and other biological fluids. The principle is based on the fact that lactate dehydrogenase isoenzyme-5 (LDH-5) binds specifically to Dextran-Blue (DB). Albumin inhibits the binding of LDH-5 with DB. Absence of LDH activity in DB fraction after gel filtration indicates the presence of albumin in sample and vice versa.
Chemistry, Clinical/*methods ; Chromatography, Gel ; Human ; Isoenzymes/metabolism ; Lactate Dehydrogenase/metabolism ; Protein Binding ; Sepharose/chemistry ; Serum Albumin/*analysis

BACKGROUND: The effect of the redox state of human serum albumin (HSA) on the antioxidant properties of the entire body has been a focus of recent research. The usefulness of HSA redox state as a biomarker for reducing oxidative stress has been investigated in clinical settings; however, evidence for its significance as a health index in non-clinical settings is yet to be established. This study aimed to examine the associations between HSA redox state and the atherosclerotic indices of carotid intima-media thickness (IMT) and plaque formation in a rural Japanese population. METHODS: We conducted a cross-sectional study as part of a health check-up program in the rural area of Hokkaido, Japan, at the end of August 2013. A total of 281 residents (124 men and 157 women) were included in the final analysis. Lifestyle-related data were obtained through a self-reported questionnaire, and ultrasound examinations were performed to measure IMT and determine plaque formation. The high-performance liquid chromatography postcolumn bromocresol green method was used to separate HSA into human nonmercaptalbumin and human mercaptalbumin (HMA). RESULTS: We found a significant negative relationship between the fraction of HMA [f(HMA)] and IMT (standardized β = - 0.132, p = 0.03). Moreover, f(HMA) was significantly associated with plaque formation (p < 0.01) with an odds ratio of 0.89 (95% confidence interval, 0.81-0.97) for every 10% increment in f(HMA). CONCLUSIONS We found that the HSA redox state, as determined by f(HMA), was associated with atherosclerotic indices in Japanese subjects. These results suggest that the HSA redox state indicates the risk of developing atherosclerosis.
Adult ; Aged ; Aged, 80 and over ; Atherosclerosis ; epidemiology ; etiology ; Biomarkers ; Carotid Intima-Media Thickness ; statistics & numerical data ; Cross-Sectional Studies ; Female ; Humans ; Japan ; epidemiology ; Male ; Middle Aged ; Oxidation-Reduction ; Risk Factors ; Serum Albumin ; metabolism ; Serum Albumin, Human ; metabolism

The aim of this cross-over study was to investigate whether albumin infusion before furosemide administration could potentiate the diuretic action of furosemide. Seven patients with nephrotic syndrome were given the following infusions in random order on two separate days: 1) a sham solution followed by 160 mg of furosemide, 2) 100 ml of 20% human albumin followed by 160 mg of furosemide. Urine and serum furosemide concentrations were measured by high-performance liquid chromatography. The increment of urine volume was greater in albumin preinfusion than in furosemide alone. However, the increments of sodium and chloride excretions between furosemide alone and albumin preinfusion were not different. No significant differences in the pharmacokinetic parameters between the two treatments were observed: area under the concentration-time curve (AUC: 12.7+/-2.2 vs 15.1+/-4.4 g/ml hr), total plasma clearance (253+/-41 vs 256+/-54 ml/min), volume of distribution (341+/-34 vs 494+/-153 ml/kg), elimination half life (4.0+/-1.1 vs 4.6+/-0.8 hr), and urine furosemide excretion of the administered amount (16.5+/-7.3 vs 7.5+/-1.6%). In conclusion, these data show that albumin preinfusion potentiated diuresis, but not natriuresis, of furosemide without any change in the pharmacokinetics of the agent in patients with nephrotic syndrome.
Adolescence ; Adult ; Aged ; Albumins/*pharmacology ; Cross-Over Studies ; Diuretics/*pharmacology ; Drug Synergism ; Female ; Furosemide/*pharmacology ; Human ; Male ; Middle Age ; Nephrotic Syndrome/*drug therapy/metabolism ; Serum Albumin/analysis

Adult ; Bone Marrow Cells ; cytology ; metabolism ; Cells, Cultured ; Culture Media ; Female ; Hepatitis, Viral, Human ; blood ; Humans ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Serum ; Serum Albumin ; biosynthesis ; genetics ; alpha-Fetoproteins ; biosynthesis ; genetics

OBJECTIVETo explore the effect of minimally invasive Ivor-Lewis esophagectomy on acute phase responses in patients with esophageal carcinoma.

METHODSForty-eight patients with middle or low thoracic esophageal carcinoma underwent Ivor-Lewis esophagectomy. The patients were divided into small incision group (n = 25) and conventional group (n = 23) according to the patients' will. Serum levels of acute phase proteins C reactive protein (CRP), haptoglobin (HPT), α₁-acid glycoprotein (α₁-AG), ceruloplasmin (CER), transferrin (TRF), β₂-microglobulin (β₂-MG), album protein (ALB), interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) were measured and compared on 1st day before operation, at 18 hours as well as 3rd and 7th day after operation.

RESULTSThere was no significant difference in all the acute phase proteins indicators and IL-6 between the small incision and conventional groups at each time points after operation (P > 0.05). In both groups the levels of CRP, α₁-AG and HPT were significantly higher after operation than before operation (P < 0.05). The levels of ALB and TRF were significantly lower after operation than before operation (P < 0.05). The levels of CER and β₂-MG were not significantly different during perioperative period (P > 0.05). The level of TNF-α was significantly higher in the small incision group than that in the conventional group at the 18 hours postoperationally (P < 0.05), and were not significantly different on the other time points between the two groups (P > 0.05).

CONCLUSIONCompared with conventional operation, the small incision Ivor-Lewis esophagectomy do not significantly alleviate the stress of the surgical trauma in patients. Unchanging the essence of operation, if one is trying to minimize the stress caused by surgery on patients, the key factor is not the size of incision. An effective approach should be found in other operation-related factors.

Acute-Phase Proteins ; metabolism ; Aged ; C-Reactive Protein ; metabolism ; Carcinoma, Squamous Cell ; blood ; surgery ; Ceruloplasmin ; metabolism ; Esophageal Neoplasms ; blood ; surgery ; Esophagectomy ; methods ; Female ; Haptoglobins ; metabolism ; Humans ; Interleukin-6 ; blood ; Male ; Middle Aged ; Minimally Invasive Surgical Procedures ; methods ; Orosomucoid ; metabolism ; Perioperative Period ; Serum Albumin ; metabolism ; Serum Albumin, Human ; Transferrin ; metabolism ; Tumor Necrosis Factor-alpha ; blood ; beta 2-Microglobulin ; blood

OBJECTIVETo investigate the accumulation of advanced glycation end products (AGE) and the inflammatory response of skin and wound in diabetic patients, and to analyze their relationship in vitro.

METHODSHistological staining and immunohistochemical staining was respectively performed on skin and wound tissue specimens collected from 10 patients with Type II diabetes mellitus (diabetes group) and 12 non-diabetic patients with skin injury (control group) to observe the arrangement of collagen and the distribution of inflammatory cells, and to determine the expression levels of AGE and its receptor (RAGE). Malondialdehyde (MDA) levels in skin and wound tissue homogenates were assayed by enzyme-linked immunosorbent assay. In vitro, human neutrophils were isolated and treated with RPMI-1640 culture medium or that containing AGE-human serum albumin in the concentration of 0.315, 0.625, 1.250 mg/mL, and they were identified as normal control (NC) group, low concentration (L) group, moderate concentration (M) group, and high concentration (H) group. Cell viability in each group was determined by MTT colorimetric assay, and the reactive oxygen species (ROS) in cell was measured with 2', 7'-dichlorofluorescein-diacetate. Data were processed with t test.

RESULTSCompared with those of skin in control group, collagens of skin tissues in diabetes group atrophied and disorderly arranged. Inflammatory cells in wounds in diabetes group were dispersed, in which collagens arranged loosely and irregularly, as compared with those of wounds in control group. Expression levels of AGE and RAGE of skin in diabetes group were higher than those in control group. In diabetes and control groups, especially in diabetes group, the numbers of RAGE-positive cells in wound tissue were more than those in skin tissue. Large amount of inflammatory cells with positive expression of RAGE were observed in diabetes group. MDA level of skin and wound tissue in diabetes group was respectively (6.3 ± 1.0), (7.1 ± 2.4) nmol per milligram protein, which were obviously higher than those in control group [(2.9 ± 1.0), (3.6 ± 1.4) nmol per milligram protein, with t value respectively 8.017, 4.349, P < 0.05 or P < 0.01]. Cell viability and ROS levels in neutrophils were increased in L, M, and H groups [(59 ± 8)%, (77 ± 5)%, (67 ± 6)% and 1.67 ± 0.14, 2.13 ± 0.17, 3.48 ± 0.48] as compared with those in NC group [(34 ± 5)% and 0.58 ± 0.06, with t value respectively 7.195, 14.890, 11.130 and 20.195, 24.905, 16.864, P < 0.05 or P < 0.01].

CONCLUSIONSAbnormal oxidative stress in diabetic skin leads to an atypical origin of wound repair. AGE-RAGE effect is a critical mediator for oxidative stress in diabetic wound tissue during wound healing.

Aged ; Case-Control Studies ; Diabetes Mellitus, Type 2 ; metabolism ; pathology ; Female ; Glycation End Products, Advanced ; metabolism ; Humans ; Male ; Middle Aged ; Oxidative Stress ; Reactive Oxygen Species ; metabolism ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; metabolism ; Serum Albumin ; metabolism ; Serum Albumin, Human ; Skin ; metabolism ; pathology ; Wound Healing

The present study aimed to determine the role of Rho/Rho kinase (Rho/ROCK) phosphorylation on advanced glycation end products (AGEs)-induced morphological and functional changes in human dermal microvascular endothelial cells (HMVECs). HMVECs were respectively incubated with different concentrations of AGEs-modified human serum albumin (AGEs-HSA) for different time. In some other cases, HMVECs were pretreated with ROCK inhibitors (H-1152 or Y-27632). The morphological changes of F-actin cytoskeleton were visualized by rhodamine-phalloidin staining and the phosphorylation of Rho and ROCK were determined by Western blot. Endothelial monolayer permeability was assessed by measuring the flux of FITC-albumin across the endothelial cells. The results showed that the distribution of F-actin was significantly altered by AGEs-HSA in time and dose-dependent patterns. These effects were inhibited by ROCK inhibitors. The phosphorylation of Rho and RCOK was remarkably increased by AGEs-HSA treatment while total Rho and ROCK protein levels were not affected. The permeability of endothelial monolayer was dramatically increased by AGEs-HSA, and both ROCK inhibitors (H-1152 or Y-27632) attenuated these hyperpermeability responses. The results obtained suggest that the phosphorylation of Rho/ROCK plays an important role in AGEs-induced morphological and functional alterations in HMVECs.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Actin Cytoskeleton ; metabolism ; Actins ; metabolism ; Amides ; pharmacology ; Endothelial Cells ; metabolism ; Endothelium, Vascular ; cytology ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; metabolism ; Glycation End Products, Advanced ; pharmacology ; Humans ; Phalloidine ; analogs & derivatives ; Phosphorylation ; Pyridines ; pharmacology ; Rhodamines ; Serum Albumin ; metabolism ; pharmacology ; Serum Albumin, Human ; Signal Transduction ; rho-Associated Kinases ; metabolism

OBJECTIVETo investigate the effect of synthesized advanced glycation end products (AGE) on apoptosis of human gingival fibroblasts (HGF) and the possible role of caspase-dependent pathway in the process of AGE-induced apoptosis.

METHODSHGF were incubated with AGE-human serum albumin (AGE-HSA). The activity of caspase-8, caspase-9 and caspase-3 were detected by microplate reader after 12 and 24 hours. HGF were incubated with caspase inhibitors for 1 hour, and then incubated with AGE-HSA for 24 hours, HGF was first stained by Hoechst33258 and observed under inverted microscope, and then double stained by annexin V and propidine iodide (PI) and observed by flow cytometry (FCM). The activity of caspase-3 was determined by caspase-3 assay kit and observed by microplate reader.

RESULTSCaspases activity of caspase-8, -9, -3 was 0.1097 ± 0.0051, 0.0965 ± 0.0051 and 0.1280 ± 0.0103 after 12 h of incubation with AGE-HSA and HGF, respectively, and 0.1558 ± 0.0053, 0.1308 ± 0.0035 and 0.1954 ± 0.0051 after 24 h of incubation with AGE-HSA and HGF, respectively (P < 0.05). Positive cells number was 247.7 ± 32.4, 200.1 ± 14.6, 154.1 ± 14.4 and 131.3 ± 14.6 in caspase inhibitor groups by Hoechst33258 staining, respectively. Apoptotic rate was (25.57 ± 2.20)%, (38.87 ± 3.31)%, (17.17 ± 2.24)% and (14.73 ± 2.48)% in caspase inhibitor groups by annexin V-PI staining, respectively. The difference between different groups was significant (P < 0.05). Caspase-3 activity was reduced to 0.1274 ± 0.0076, 0.1465 ± 0.0062, 0.1044 ± 0.0051 in caspase inhibitor groups, respectively. The difference between different groups was significant (P < 0.05).

CONCLUSIONSApoptosis of HGF induced by AGE-HSA may be mainly through activation of caspase-dependant pathway in which cytoplasmic pathway may play a predominant role.

Adolescent ; Adult ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Caspase Inhibitors ; pharmacology ; Caspases ; metabolism ; Cells, Cultured ; Child ; Fibroblasts ; cytology ; metabolism ; Gingiva ; cytology ; Glycation End Products, Advanced ; pharmacology ; Humans ; Oligopeptides ; pharmacology ; Serum Albumin ; pharmacology ; Serum Albumin, Human ; Signal Transduction ; Young Adult

Increased formation of advanced glycation end-products (AGEs) is occurred in hyperglyceamia and diabetes, leading to oxidative stress and progression of diabetic vascular diseases. Glucose-6-phosphate dehydrogenase (G6PD), the principal source of NADPH, serves as an antioxidant enzyme to modulate the redox milieu. Deficiency of G6PD activity is associated with increased endothelial cell oxidative stress. Current study is designed to investigate the effects of AGEs on G6PD activity and expression in human umbilical vein endothelial cells. Treatment of AGE-modified bovine serum albumin (AGE-BSA, 100 µg/mL, 24 h), but not native BSA, to human umbilical vein endothelial cells increased ROS generation by (48.89 ± 5.28)%. G6PD activity was decreased by AGE-BSA treatment by (61.25 ± 11.2)%. The expression of G6PD at mRNA and protein levels was also decreased by AGE-BSA treatment by (27.92 ± 6.73)% and (23.72 ± 2.44)%, respectively. These results suggest that AGEs could result in G6PD deficiency in human umbilical vein endothelial cells by inhibiting the expression of G6PD at mRNA and protein levels and G6PD activity.
Antioxidants ; metabolism ; Cells, Cultured ; Glucosephosphate Dehydrogenase ; antagonists & inhibitors ; metabolism ; Glycation End Products, Advanced ; pharmacology ; Human Umbilical Vein Endothelial Cells ; drug effects ; enzymology ; Humans ; Oxidative Stress ; Reactive Oxygen Species ; metabolism ; Serum Albumin, Bovine ; pharmacology