Plasma protein oxidation and antioxidant defense during aging.

BACKGROUND: Oxidative stress is an important process that occurs in vivo during aging and is considered one of the main causes of molecular damage to cellular and tissue structures. These changes can accumulate in biological structures during aging. OBJECTIVE: The aim of this work is to evaluate plasma protein oxidative damage, measured as carbonyl groups content, and the concentration of some antioxidant molecules (vitamins and carotenoids) in 122 healthy volunteers (50 males and 72 females), 25 to 89 years old. RESULTS: Total plasma proteins slightly decreased with age, but the level of carbonyl groups was similar in the adult (< 65 years) and in the old, and was similar in both sexes. Plasma concentration of antioxidant molecules such as alpha-tocopherol, beta-carotene and other carotenoids, increased with age and correlated with the level of lipoproteins; plasma total cholesterol and triglycerides were significantly correlated with age as well. CONCLUSIONS: The surprisingly normal level of plasma protein carbonyl groups in our older subjects suggests two possibilities: a) the older people in our study are healthy and free from pathologies because of better protection against oxidative injury during their lifetimes, i.e., they maintained low-level oxidative damage on plasma proteins; or b) the level of carbonyl groups is normal because of the high turnover in plasma: the oxidized circulating proteins are preferentially and quickly removed; in this case oxidative damage is not discernible in plasma proteins but may proceed silently in other tissues.

Scanning force microscopy reveals structural alterations in diabetic rat collagen fibrils: role of protein glycation.

BACKGROUND: The main functional property of collagen is to provide a supporting framework to almost all tissues: the effects of non-enzymatic glycation on this protein are deleterious and in diabetes mellitus contribute to the mechanism of late complications. The aim of this work is to provide evidence by scanning force microscopy of modifications in collagen structure caused by high glucose concentration, in vivo and in vitro, and to correlate the data with markers of non-enzymatic glycation. METHODS: Tendon fibrils were obtained from the tails of 8-month-old rats (BB/WOR/MOL¿BB) which developed diabetes spontaneously at least 12 weeks before they were killed, and from diabetes-resistant rats of the same strain (BB/WOR/MOL¿WB). A scanning force microscope (SFM; Nanoscope III) equipped with a Contact Mode Head was used for imaging. Band interval, diameter and depth of D-band gap were measured in non-diabetic and diabetic tail tendon fibrils and in fibrils incubated with glucose (0.5 M for 2 weeks). Fructosamine was determined in the tendon fibrils by a colorimetric method and pentosidine was evaluated in acid-hydrolyzed samples by coupled reverse phase-ionic exchange column HPLC. RESULTS: Incubated fibrils revealed modifications in radius (228+/-5 nm) and gap depth (3.65+/-0.10 nm) that closely reproduce diabetes-induced damage (236+/-3 and 3.20+/-0.04 nm respectively) and were significantly different from the pattern seen in non-diabetic fibrils (151+/-1 and 2.06+/-0.03 nm; p<0.001). Both fructosamine and pentosidine were higher in diabetic (3.82+/-1.43 nmol/mg and 2.23+/-0.24 pmol/mg collagen respectively) and in glucose-incubated fibrils (9.27+/-0.55 nmol/mg and 5.15+/-0.12 pmol/mg collagen respectively) vs non-diabetic tendons (1.29+/-0.08 nmol/mg and 0.88+/-0.11 pmol/mg collagen respectively; p<0.01); during the time course of incubation, an early increase in fructosamine was seen, whereas pentosidine increased later. The D-band parameter was similar in all three groups, indicating that axial organization is not modified by non-enzymatic glycation. CONCLUSION: This is the first description obtained with SFM of diabetes-induced ultrastructural changes in collagen fibrils. Moreover, the data presented are consistent with the concept that chronic exposure of collagen to glucose in vivo or in vitro leads to similar structural modifications in collagen fibrils, probably through crosslinks. The correlation between morphologic parameters and both markers of glycation provides strong evidence for a crucial role of this non-enzymatic modification.

Levels of carbonyl groups in plasma proteins of type 2 diabetes mellitus subjects.

Carbonyl groups result from protein oxidation and their level in tissues and plasma is a relatively stable marker of oxidative damage. Carbonyl content of plasma proteins in 43 type 2 diabetic subjects, 30-87 years of age (25 males and 18 females) and in 20 age-matched healthy controls (31-89 years of age, 12 males and 8 females) was evaluated with 2,4-dinitro-phenyl-hydrazine method. In both groups, lipids, tocopherols (HPLC) and glycated hemoglobin (HPLC) were studied. Fasting blood glucose, glycated hemoglobin and lipids were significantly higher in the diabetic group; carbonyl content and alpha-tocopherol were slightly, but not significantly higher in the diabetic group (1.06 +/- 0.03 vs. 0.97 +/- 0.04 nmol/mg protein, 27. 07 +/- 2.82 vs. 31.55 +/- 2.11 micromol/l, respectively). Significant relationships between age and lipids, alpha-tocopherol and proteins were found in controls, but not in diabetics. Alpha-tocopherol correlated with lipids in both groups; glycated hemoglobin, a marker of glycemic control, was related to lipids, alpha-tocopherol and protein carbonyl groups in diabetics, while only the correlation with carbonyls was found in controls. These results suggest that impaired glycemic control is connected to protein oxidation. Glycation cascade also releases free radicals, becoming responsible for further oxidative attacks. In conclusion, increased oxidative stress, if any, in the diabetic group, is doubtlessly induced by hyperglycemia, and the tocopherols are not seriously affected by a worsening of the metabolic control.

Oxidative stress in subjects affected by celiac disease.

In order to study the role of oxidative stress in celiac disease, protein carbonyl groups, thiobarbituric acid-reactive substance and pentosidine were evaluated in the plasma of nine patients with asymptomatic celiac disease and in a control group (n = 25). Plasma alpha-tocopherol, retinol and lipids were determined in the same samples. The levels of markers of oxidative stress derived from both protein (carbonyl groups) and lipids (thiobarbituric acid-reactive substances) were significantly higher in celiac disease patients, whereas lipoproteins and alpha-tocopherol were significantly lower. These data indicate that in celiac disease, even when asymptomatic, a redox imbalance persists; this is probably caused by an absorption deficiency, even if slight. Dietary supplementation with antioxidant molecules may offer some benefit and deserves further investigation.

Role of advanced glycation end products in aging collagen. A scanning force microscope study.

The collagen structure of young and old rats was examined using a scanning force microscope (SFM). Rat tail tendons of 8- and 24-month-old Wistar rats were frayed by two blades and examined using a Nanoscope III SFM. In the same tendons, the pentosidine concentrations, a marker of the Maillard reaction, were determined by HPLC. The SFM inspection of native fibrils produces images of collagen bundles, with parallel fibrils. The diameters of old rat collagen fibrils were large in comparison to the young ones. Moreover, fibrils obtained from old rats exhibited the same band interval, while the depth of the gap between two overlap zones showed a higher mean value with respect to young collagen. The pentosidine concentration was also higher in the old than in the young tendons. In conclusion, in the presence of an increased concentration of advanced glycation end products, significant structural alterations have been observed in old fibrils.

Protein oxidation in hemodialysis and kidney transplantation.

Oxidative damage of plasma proteins determined with the markers carbonyl group (CG) content and thiobarbituric acid-reactive substances (TBARS) was studied in 13 hemodialyzed and eight kidney-transplanted patients. The level of CGs was 38% higher in hemodialysis (HD) patients (1.49 +/- 0.05 nmol/mg protein) than in the healthy subjects (1.08 +/- 0.03 nmol/mg protein); the TBARS level was also higher in HD patients than in the control group (2.64 +/- 0.15 v 1.81 +/- 0.09 nmol/mL, P < .001). These data confirm that in end-stage renal failure, an increased oxidative stress is present and is able to induce protein damage. After transplantation, the CG content in protein was reduced (1.34 +/- 0.08 nmol/mg protein), but it was not significantly different from the level in the HD group. The failure to return to the normal range suggests that an impaired redox status is maintained, resulting in a sustained elevation of CG. Conversely, the level of TBARS in transplanted patients (1.99 +/- 0.22 nmol/mL) was not significantly different from that in the control group (1.81 +/- 0.09), suggesting that lipoperoxidation may be inhibited. These results may be explained by the different turnover rates of the molecules and by the distinct origin of the two markers, resulting from the damage of proteins or lipids. Thus, lipoperoxidation would produce rapidly removable molecules, whereas protein oxidation damage would tend to accumulate. However, the significant correlation found between CGs and TBARS indicates that a common cause (oxidative stress) binds the two markers of damage.

Structure of rat tail tendon collagen examined by atomic force microscope.

The Atomic Force Microscope (AFM) was used to inspect collagen fibrils deposited on mica sheets at different fibrillogenesis times. Collagen was obtained from rat tail tendon fibers. Various fibril forms were observed, together with the characteristic periodic intra-fibril structure (D-bands). The fibril thickness, width, D-band periodicity and depth were measured and the statistical distribution of these parameters at 1, 2, 5, 10 and 15 days of in vitro fibril formation time was calculated. The fibrils showed an increasing size with time, but the band interval measure remained stable. The band depth, after an initial increase, exhibited a relative steadiness. The results indicate that AFM offers, at low resolution, images qualitatively similar to those obtained with electron microscopy, but with less manipulation of the sample. A quantitative evaluation of collagen structural features in the nanometer scale is made possible by AFM.

Study of aging rat tail collagen using atomic force microscope.

Collagen structure of young and old rats was examined by using atomic force microscope (AFM) images. Rat tail tendons of eight and twenty-four month-old Wistar rats were digested enzymatically (pepsin), and allowed to refibrillate for 24 hours at 37 degrees C. The samples were examined using a Nanoscope III (Digital Instruments, Santa Barbara, CA, U.S.A.) with a J scanning head and a 200 microns silicon nitride cantilever. The study was performed in air and without filters. The AFM inspection of refibrillated collagen produced images showing long fibrils with relatively homogeneous heights and widths, characterized by clear banding with a periodic interval (D band) of 67 nm. With respect to collagen extracted from young rats, collagen extracted from old rats revealed fibrils exhibiting the same band interval, but with lower widths and heights. Furthermore, the depth of gap between two overlaps showed a higher mean value in the aged rats. These data are consistent with biochemical reports of collagen modifications during aging; we suggest that post-synthetic reactions might be responsible for this as they interfere with the refibrillation process and also modify the three-dimensional structure of fibrils.

Relationships between protein carbonyls, retinol and tocopherols level in human plasma.

Free radical oxidation has been claimed as one of the most important mechanism of damage in aging and in several diseases. Carbonyl content in tissue and circulating proteins is a stable marker of this attack. In 29 apparently healthy subjects (25-89 years old) carbonyl content of plasma proteins and retinol and tocopherols (alpha- and gamma-) were studied. Carbonyls level did not show an increase with age. A good correlation between carbonyls content and gamma-tocopherol (r = 0.44, P < 0.05) and a trend with retinol (r = 0.34, P = 0.07) was found, but not with alpha-tocopherol. An inverse correlation was observed between carbonyls and plasma proteins (r = -0.63, P < 0.01) and the natural antioxidant studied showed an increase with age and a good relationship with lipids. These data suggest that retinol and tocopherols, well known scavengers of free radicals, are involved, at least partially, in the prevention of oxidative damage of circulating proteins.