Glycation of antibodies: Modification,methods and potential effects on biological functions

Glycation is an important protein modification that could potentially affect bioactivity and molecular stability, and glycation of therapeutic proteins such as monoclonal antibodies should be well characterized. Glycated protein could undergo further degradation into advance glycation end (AGE) products. Here, we review the root cause of glycation during the manufacturing, storage and in vivo circulation of therapeutic antibodies, and the current analytical methods used to detect and characterize glycation and AGEs, including boronate affinity chromatography, charge-based methods, liquid chromatography-mass spectrometry and colorimetric assay. The biological effects of therapeutic protein glycation and AGEs, which ranged from no affect to loss of activity, are also discussed.     

血清AGEs、sRAGE水平与急性脑梗死后出血性转化的相关性

摘要 目的：探讨血清晚期糖基化终末产物（AGEs）及可溶性期糖基化终末产物受体（sRAGE）与急性脑梗死后出血性转化（hemorrhagic transformation,HT）的相关性。方法：将2017年1月至2019年12月于我院诊治的131例急性脑梗死患者纳入研究对象,根据患者是否出现出血性转化将其分为HT组及NHT组,比较两组患者AGEs、sRAGE等实验室指标,采用多因素Logistic回归模型对影响急性脑梗死出血性转化的因素进行分析,并采用Pearson相关模型对血清中AGEs水平与sRAGE水平的相关性进行分析。结果：HT组患者合并糖尿病史、心房颤动病史比例、脑栓塞比例、梗死面积、采用抗凝治疗比例及血清中IL-1β、TNF-α、AGEs水平明显高于NHT组（均P＜0.05）;多因素Logistic回归分析示大梗死面积、高IL-1β、TNF-α及AGEs水平是患者出现急性脑梗死后出血性转换的保护因素（OR=0.625, 0.832, 0.874, 0.708;均P＜0.05）,而无抗凝治疗、高sRAGE则是患者出现急性脑梗死后出血性转换的危险因素（OR=10.901, 1.004;均P＜0.05）;相关性分析示血清中sRAGE水平与AGEs、IL-1β及TNF-α水平呈明显负相关（ρ=-0.852,-0.828,-0.826;均P＜0.05）。结论：血清AGEs是急性脑梗死患者出现出血性转化的危险因素,而sRAGE则是保护因素,sRAGE可能通过抑制RAGE与AGEs结合,从而减少释放炎症介质,减轻血管损伤,降低HT发生风险。     

The impact of vitamin D3 supplementation on muscle function among HIV-infected children and young adults: a randomized controlled trial

Objectives:

We tested the hypothesis that daily vitD₃ supplementation increases neuromuscular motor skills, jump power, jump energy, muscular force, and muscular strength.

Methods:

This was a secondary analysis of a randomized controlled trial of 12-months of oral 7,000 IU/day vitD₃ supplementation or placebo among 56 persons living with HIV aged 9-25 years. Neuromuscular motor skills were quantified using the Bruininks-Oseretsky Test of Motor Proficiency. Power was quantified using peak jump power, and energy was quantified using peak jump height. Muscular force was quantified using isometric ankle plantar- and dorsiflexion, isokinetic knee flexion and extension. Muscular strength was quantified using isometric handgrip strength.

Results:

After 12-months, serum 25-hydroxyvitamin D [25(OH)D] was higher with supplementation versus placebo (β=12.1 ng/mL; P<0.001). In intention-to-treat analyses, supplementation improved neuromuscular motor skills versus placebo (β=1.14; P=0.041). We observed no effect of supplementation on jump power, jump energy, muscular force, or muscular strength outcomes versus placebo.

Conclusions:

Among HIV-infected children and young adults supplementation with daily high-dose vitD₃ increased concentration of serum 25(OH)D and improved neuromuscular motor skills versus placebo.     

The effects of vitamin D deficiency on proteoglycan and hyaluronate constituents of chick bone

The effect of vitamin D deficiency on proteoglycan and hyaluronate constituents of cortical diaphyseal chick bone was studied. Proteoglycans in rachitic bone showed no significant change with respect to their size, composition, or amount relative to other extractable macromolecular components. In contrast, bone hyaluronate levels were raised in chicks fed on diets that were either vitamin D-deficient or depleted in calcium or phosphate, a 7-fold increase being seen in hypocalcaemic vitamin D-deficient chicks. This increase in hyaluronate was not directly related either to the absence of vitamin D or to abnormal levels of blood calcium or phosphate per se; hyaluronate levels are probably regulated by another factor, not yet identified, that is responsive to changes in vitamin D and mineral metabolism.     

Vitamin D level and its relation to muscle and fat mass in adult male Arabs

Low levels of vitamin D have been linked with increased adiposity and diminished muscle strength. Whether it is also related to fat deposition in muscle tissues is not studied well. This study explored the associations between circulating 25-hydroxyvitamin D (25(OH)D) and fat deposition in muscle tissues of adult Arab males. A total 465 adult Saudi males were included in this cross-sectional study. Anthropometrics, body composition and muscle strength were assessed. Serum 25(OH)D was determined and quantified enzymatically. They were grouped according to vitamin D status: deficient (25(OH)D < 50 nmol/l) N = 325 (69.9%) and sufficient (25(OH)D > 50 nmol/l)140 (30.1%). Mean level of lean/height², lean-arm-legs and lean-arms-legs/height² were significantly higher in 25(OH)D deficient participants (p-values 0.03; 0.05 and 0.01 respectively). Thigh strength was significantly higher in 25(OH)D sufficient participants than their deficient counterparts (p = 0.02). In all participants, a significant correlation between 25(OH)D was observed with age and thigh-strength (p-values < 0.05), while a significant inverse correlation between 25(OH)D and lean/height², lean-arms-legs, lean-arms-legs/height², fat (%) region, fat arms, fat legs, fat trunk, lean legs were noted. In conclusion, low circulating 25(OH)D is associated with enhanced fat infiltration in muscle tissues of adult Arab males.     

Structure-activity relationship studies on vitamin D lactam derivatives as vitamin D receptor antagonist

Structure–activity relationship studies on 1α,25-dihydroxyvitamin D₃-26,23-lactams (DLAMs), antagonists of vitamin D, were conducted, focusing on the substituents of the phenyl group. One of the derivatives (23S,25S)-DLAM-1P-3,5(OEt)₂, showed potent antagonistic activity with an IC₅₀ of 90 nM.     

Advanced glycation end products impair function of late endothelial progenitor cells through effects on protein kinase Akt and cyclooxygenase-2

Endothelial progenitor cells (EPCs) exhibit impaired function in the context of diabetes, and advanced glycation end products (AGEs), which accumulate in diabetes, may contribute to this. In the present study, we investigated the mechanism by which AGEs impair late EPC function. EPCs from human umbilical cord blood were isolated, and incubated with AGE-modified albumin (AGE-albumin) at different concentrations found physiologically in plasma. Apoptosis, migration, and tube formation assays were used to evaluate EPC function including capacity for vasculogenesis, and expression of the receptor for AGEs (RAGE), Akt, endothelial nitric oxide synthase (eNOS), and cycloxygenase-2 (COX-2) were determined. Anti-RAGE antibody was used to block RAGE function. AGE-albumin concentration-dependently enhanced apoptosis and depressed migration and tube formation, but did not affect proliferation, of late EPCs. High AGE-albumin increased RAGE mRNA and protein expression, and decreased Akt and COX-2 protein expression, whilst having no effect on eNOS mRNA or protein in these cells. These effects were inhibited by co-incubation with anti-RAGE antibody. These results suggest that RAGE mediates the AGE-induced impairment of late EPC function, through down-regulation of Akt and COX-2 in these cells.     

Hypophosphatemia is responsible for skeletal muscle weakness of vitamin D deficiency

A deficiency of vitamin D results in muscle weakness as well as rickets in children and osteomalacia in the adult. To study the basis for this weakness, severe vitamin D deficiency was produced in rats as revealed by a low level or absence of 25-hydroxyvitamin D₃ in the serum. Vitamin D deficiency was achieved by feeding purified diets to weanlings for 16 weeks. Muscle force, peak contraction (P), time-to-half contraction (T_1/2), time-to-peak contraction (T_P), and time-to-half recovery (T_1/2r) were measured. A significant reduction in muscle force was found when vitamin D deficiency was accompanied by hypophosphatemia. Within 2 days of correcting the hypophosphatemia, muscle strength was normalized. When serum calcium and serum phosphorus were maintained in the normal range in vitamin D-deficient rats, muscle weakness did not develop. Further, hypocalcemia together with vitamin D deficiency did not produce muscle weakness. These results strongly suggest that muscle weakness noted in rachitic patients is the result of the hypophosphatemia of vitamin D deficiency.     

Cathepsin D is one of the major enzymes involved in intracellular degradation of AGE-modified proteins

Abstract

Oxidized and cross-linked modified proteins are known to accumulate in ageing. Little is known about whether the accumulation of proteins modified by advanced glycation end products (AGEs) is due to an affected intracellular degradation. Therefore, this study was designed to determine whether the intracellular enzymes cathepsin B, cathepsin D and the 20S proteasome are able to degrade AGE-modified proteins in vitro. It shows that AGE-modified albumin is degraded by cathepsin D, while cathepsin B was less effective in the degradation of aldehyde-modified albumin and the 20S proteasome was completely unable to degrade them. Mouse primary embryonic fibroblasts isolated from a cathepsin D knockout animals were found to have an extensive intracellular AGE-accumulation, mainly in lysosomes, and a reduction of AGE-modified protein degradation compared to cells isolated from wild type animals. In summary, it can be assumed that cathepsin D plays a significant role in the removal of AGE-modified proteins.     

Synthesis of VS-105: A novel and potent vitamin D receptor agonist with reduced hypercalcemic effects

We have synthesized a novel vitamin D receptor agonist VS-105 ((1R,3R)-5-((E)-2-((3αS,7αS)-1-((R)-1-((S)-3-hydroxy-2,3-dimethylbutoxy)ethyl)-7α-methyldihydro-1H-inden-4(2H,5H,6H,7H,7αH)-ylidene)ethylidene)-2-methylenecyclohexane-1,3-diol). Preparation of a-ring phenylphosphine oxide 11, followed by Wittig–Horner coupling of 11 with the protected 25-hydroxy Grundmann’s ketone 22 generated the precursor 12. Deprotection of the TBDMS groups of 12 produced the target compound VS-105. The biological profiles of VS-105 were evaluated using in vitro assays (VDR receptor binding, VDR reporter gene and HL-60 differentiation) in comparison to calcitriol (the endogenous hormone) or paricalcitol. Furthermore, the PTH suppressing potency and hypercalcemic side effects of VS-105 were evaluated in the 5/6 nephrectomized uremic rats in comparison to paricalcitol. Combining various changes at 20-epi, 22-oxa, 24-methyl, and 2-methylene yielded VS-105 that not only is highly potent in inducing functional responses in vitro, but also effectively suppresses PTH in a dose range that does not affect serum calcium in the 5/6 nephrectomized uremic rats.     

Plasma protein advanced glycation end products, carboxymethyl cysteine, and carboxyethyl cysteine, are elevated and related to nephropathy in patients with diabetes

In Diabetes Mellitus (DM), glucose and the aldehydes glyoxal and methylglyoxal modify free amino groups of lysine and arginine of proteins forming advanced glycation end products (AGEs). Elevated levels of these AGEs are implicated in diabetic complications including nephropathy. Our objective was to measure carboxymethyl cysteine (CMC) and carboxyethyl cysteine (CEC), AGEs formed by modification of free cysteine sulfhydryl groups of proteins by these aldehydes, in plasma proteins of patients with diabetes, and investigate their association with the albumin creatinine ratio (ACR, urine albumin (mg)/creatinine (mmol)), an indicator of nephropathy. Blood was collected from forty-two patients with type 1 and 2 diabetes (18–36 years) and eighteen individuals without diabetes (17–35 years). A liquid chromatography-mass spectrophotometric method was developed to measure plasma protein CMC and CEC levels. Values for ACR and hemoglobin A1C (HbA1C) were obtained. Mean plasma CMC (μg/l) and CEC (μg/l) were significantly higher in DM (55.73 ± 29.43, 521.47 ± 239.13, respectively) compared to controls (24.25 ± 10.26, 262.85 ± 132.02, respectively). In patients with diabetes CMC and CEC were positively correlated with ACR, as was HbA1C. Further, CMC or CEC in combination with HbA1C were better predictors of nephropathy than any one of these variables alone. These results suggest that glucose, glyoxal, and methylglyoxal may all be involved in the etiology of diabetic nephropathy.     

The effect of nonenzymatic glycation on the stability and conformation of two deoxyoligonucleotide duplexes: a spectroscopic analysis by circular dichroism

Advanced glycation end products (AGEs) play a significant role in the pathophysiology of diabetes leading to such conditions as atherosclerosis, cataract formation, and renal dysfunction. While the formation of nucleoside AGEs was previously demonstrated, no extensive studies have been performed to assess the effect of AGEs on DNA structure and folding. The objective of this study was to investigate the nonenzymatic glycation of two DNA oligonucleotide duplexes with one duplex consisting of deoxy-poly(A)15 and deoxy-poly(T)15 and the other consisting of deoxy-poly(GA)15 and deoxy-poly(CT)15. With D-glucose, D-galactose, D/L-glyceraldehyde, and D-glucosamine serving as the model glycating carbohydrates, D-glucosamine was found to exhibit the greatest effect on the stability and structure of the oligonucleotide duplexes, a finding that was confirmed by circular dichroism. The nonenzymatic glycation of deoxy-poly(AT) by D-glucosamine destabilized the deoxy-poly(AT) structure and changed its conformation from A form to X form. D-glucosamine also altered the conformation of deoxy-poly(GA)15 and deoxy-poly(CT)15 from A form to B form. Capillary electrophoresis and ultraviolet and fluorescence spectroscopy revealed that, of the various purines and pyrimidines, 2'-deoxyguanosine and guanine were most reactive with D-glucosamine. The nonenzymatic modification of nucleic acids warrants further investigation because this phenomenon may occur in vivo, altering DNA structure and/or function.     

Phytoscreening of Vochysiaceae species: Molecular identification by HPLC-ESI-MS/MS and evaluating of their antioxidant activity and inhibitory potential against human α-amylase and protein glycation

Scientific research based on medicinal plants has been highlighted as a complementary treatment to T2DM, stand out the Vochysiaceae family, which have been widely used in folk medicine by traditional South American communities to treat some diseases. Our study aimed to investigate the antioxidant and antiglycation activities of ethanol extracts of leaves (LF) and stem barks (SB) of Vochysiaceae species, evaluated their capacities to inhibit glycoside and lipid hydrolases related to T2DM and molecular identification by HPLC-ESI-MS/MS. Our main findings indicate that the ethanolic extract of four of eight analyzed plants such as LF and SB of Q. grandiflora, Q. parviflora, V. elliptica and Calisthene major exhibited, respectively, potential of α-amylase inhibition (IC₅₀ of LF: 5.7 ± 0.6, 4.1 ± 0.5, 5.8 ± 0.5, 3.2 ± 0.6 and IC₅₀ of SB: 3.3 ± 0.7, 6.2 ± 2.0, 121.0 ± 8.6 and 11.2 ± 2.8 μg/mL), capacities of antioxidant (ORAC of LF: 516.2 ± 0.1, 547.6 ± 4.9, 544.3 ± 6.1, 442.6 ± 2.4 and ORAC of SB: 593.6 ± 22.3, 497.7 ± 0.8, 578 ± 12.3, 593.6 ± 19.5 µmol trolox eq/g; FRAP of LF: 796.1 ± 0.9, 427.7 ± 22.0, 81.0 ± 1.9, 685 ± 37.9 and FRAP of SB: 947.4 ± 24.9, 738.6 ± 24.3, 98.8 ± 7.9, 970.8 ± 13.9 µmol trolox eq/g; DPPH IC₅₀ of LF: 14.2 ± 1.8, 36.3 ± 6.9, 11.8 ± 1.9, 13.3 ± 1.2 and DPPH IC₅₀ of SB: 16.0 ± 3.0, 15.5 ± 1.9, 126.1 ± 23. 6, 5.3 ± 0.3 μg/mL, respectively) and antiglycation (BSA/Frutose IC₅₀ of LF: 43.1 ± 3.4, 52.1 ± 6.0, 175.5 ± 32, 8, 111.8 ± 14.7 and BSA/Frutose IC₅₀ of SB:, 40.1 ± 11.9, 51.2 ± 16. 7, 46.6 ± 5.7, 53.5 ± 13.6 μg/mL) and presence of polyphenols, such as flavonoids and condensed tannins. The extracts presented low ability to inhibit α-glycosidase and lipase enzymes in the initial assays, with values below 40% of inhibition. In BSA/methylglyoxal, only Q. grandiflora SB, V. eliptica LF and V. tucanorum LF showed activity (IC₅₀: 655.5 ± 208.5, 401.9 ± 135.2 and 617.1 ± 80.6 μg/mL, respectively) and only C. major LF and SB, in Arg/methylglyoxal (IC₅₀: 485.1 ± 130.8 and 468.0 ± 150.5 μg/ml, respectively). This study presented new findings about the biological and pharmacological potential of some species of Vochysiaceae family, contributing to the understanding of the action and efficacy in use of these plants, in their management of postprandial hyperglycemia and in glycation and oxidative processes that contribute to managing diabetes mellitus.     

Reduced cell replication and induction of apoptosis by advanced glycation end products in rat Schwann cells

We investigated the effects of advanced glycation end products (AGEs) derived from glucose, glyceraldehyde, and glycolaldehyde (designated as AGE-1, -2, and -3, respectively) on the viability, replication rate, and cytokine production of cultured Schwann cells. AGE-2 and -3, but not AGE-1, induced apoptosis, and significantly decreased the viability measured by MTT assay. The decrease was prevented completely by antioxidant alpha-lipoic acid and was prevented partially by p38 mitogen-activated protein kinase inhibitor SB202190. The decrease in mitochondrial membrane potential by AGE-2 and -3 was also observed. In addition, AGE-2 and -3 significantly suppressed the replication rate as shown by reduced bromodeoxyuridine uptake, whereas they enhanced the release of TNF-alpha and IL-1beta into the medium and activated nuclear factor-kappaB. The effects of AGE-1 on these measures were equivocal. The series of events elicited by AGE-2 and -3 may be responsible for some of the aspects of pathogenetic mechanisms in patients with diabetic neuropathy.     

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.

Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.

We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

葛根素注射液对糖尿病大鼠牙周炎的治疗效果及血清AGEs、炎性因子表达的影响

摘要 目的：分析葛根素注射液对糖尿病大鼠牙周炎的治疗效果及血清晚期糖基化终末产物（advanced glycosylation end products,AGEs）、炎性因子表达的影响。方法：随机选取10只大鼠作为正常对照组,另选40只建立糖尿病牙周炎大鼠,将建模成功的大鼠随机分成模型组、实验组和阳性组,每组10只。给药处理6周后,取血清检测AGEs、白细胞介素-6（interleukin-6,IL-6）、IL-8及肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）表达情况;取牙周组织观察压槽骨丧失程度。结果：与健康对照组相比,模型组大鼠胰岛素水平显著降低,血糖、AGEs、IL-6、IL-8和TNF-α水平显著升高,压槽骨降低高度显著增大（P＜0.05）。与模型组相比,实验组和阳性组大鼠水平显著升高,血糖、AGEs、IL-6、IL-8和TNF-α水平显著降低,压槽骨降低高度升高减小（P＜0.05）。结论：葛根素注射液可通过促进胰岛素表达,减少AGEs积聚,降低血糖及炎性因子IL-6、IL-8、TNF-α的表达抑制糖尿病大鼠牙周炎的发展。