Validation of plasma amino acid profile using UHPLC-mass spectrometer (QDa) as a screening method in a metabolic disorders reference centre: Performance and accreditation concerns.

INTRODUCTION: Amino acid (AA) analysis in plasma is essential for diagnosis and monitoring of inborn errors of metabolism (IEM). The efficacy of patient management is governed by the rapidity of AA profile availability, along with the robustness of the method. French quality guidelines and progress made in analytical techniques have led biologists to develop AA profile exploration via mass spectrometry (MS). OBJECTIVES: The aim of this study was to validate an analytical method with a single quadrupole mass spectrometer (MS) and to suggest reference values in regard to French quality and IEM society recommendations. DESIGN AND METHODS: Plasma samples from patients were deproteinised and derivatised with AccqTag™ reagent. Analysis was performed by reverse-phase chromatography coupled to QDA detector. We evaluated accuracy, intra-days and inter-days precision and limit of quantification by the ß-expectation tolerance interval method for 27 AA. Method comparison was performed with the standard method (ion exchange chromatography, IEC) on Jeol Aminotac® and to tandem MS. Reference values were established on AA concentrations of the cohort of patients who had no IEM. RESULTS: Our method allowed the separations of almost all amino acids with a total run time of 12 min. Separation of isoleucine and alloisoleucine was incomplete (R = 0.55) but without impact on biological interpretation. Precision, accuracy and quantification were satisfactory (intra-days coefficient of variation (CV) was <5%, inter-days precision CV <10% and accuracy <15%). The limits of quantification were validated between 1 and 900 µmol/L. Results were comparable between the new method and IEC. CONCLUSION: Ultimately, we validated a rapid method on plasma for quantifying 27 amino acids that can be used in routine practice, according to French quality laboratories and SFEIM (French Society of Inborn Error of Metabolism) recommendations. Furthermore, estimated reference values were similar to those found in published studies focusing on other methods. Despite a lower specificity compared to tandem MS, the simplicity and rapidity of our method are the main advantage of this technique and place it as a major tool in IEM diagnosis and management.

Behavioral, Hormonal, Inflammatory, and Metabolic Effects Associated with FGF21-Pathway Activation in an ALS Mouse Model.

In amyotrophic lateral sclerosis (ALS), motor neuron degeneration occurs simultaneously with systemic metabolic dysfunction and neuro-inflammation. The fibroblast growth factor 21 (FGF21) plays an important role in the regulation of both phenomena and is a major hormone of energetic homeostasis. In this study, we aimed to determine the relevance of FGF21 pathway stimulation in a male mouse model of ALS (mutated SOD1-G93A mice) by using a pharmacological agonist of FGF21, R1Mab1. Mice (SOD1-WT and mutant SOD1-G93A) were treated with R1Mab1 or vehicle. Longitudinal data about clinical status (motor function, body weight) and biological parameters (including hormonal, immunological, and metabolomics profiles) were collected from the first symptoms to euthanasia at week 20. Multivariate models were performed to identify the main parameters associated with R1Mab1 treatment and to link them with clinical status, and metabolic pathways involving the discriminant metabolites were also determined. A beneficial clinical effect of R1Mab1 was revealed on slow rotarod (p = 0.032), despite a significant decrease in body weight of ALS mice (p < 0.001). We observed a decrease in serum TNF-α, MCP-1, and insulin levels (p = 0.0059, p = 0.003, and p = 0.01, respectively). At 16 weeks, metabolomics analyses revealed a clear discrimination (CV-ANOVA = 0.0086) according to the treatment and the most discriminant pathways, including sphingolipid metabolism, butanoate metabolism, pantothenate and CoA biosynthesis, and the metabolism of amino acids like tyrosine, arginine, proline, glycine, serine, alanine, aspartate, and glutamate. Mice treated with R1Mab1 had mildly higher performance on slow rotarod despite a decrease on body weight and could be linked with the anti-inflammatory effect of R1Mab1. These results indicate that FGF21 pathway is an interesting target in ALS, with a slight improvement in motor function combined with metabolic and anti-inflammatory effects.

Author Correction: A ferroptosis-based panel of prognostic biomarkers for Amyotrophic Lateral Sclerosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A ferroptosis-based panel of prognostic biomarkers for Amyotrophic Lateral Sclerosis.

Accurate patient stratification into prognostic categories and targeting Amyotrophic Lateral Sclerosis (ALS)-associated pathways may pave the way for promising trials. We evaluated blood-based prognostic indicators using an array of pathological markers. Plasma samples were collected as part of a large, phase III clinical trial (Mitotarget/TRO19622) at months 1, 6, 12 and 18. The ALSFRS-r score was used as a proxy of disease progression to assess the predictive value of candidate biological indicators. First, established clinical predictors were evaluated in all 512 patients. Subsequently, pathologic markers, such as proxies of neuronal integrity (Neurofilament light chain and phosphorylated heavy chain), DNA oxidation (8-oxo-2'-desoxyguanosine), lipid peroxidation (4-hydroxy-2-nonenal, isoprostane), inflammation (interleukin-6) and iron status (ferritin, hepcidin, transferrin) were assessed in a subset of 109 patients that represented the whole cohort. Markers of neuronal integrity, DNA and lipid oxidation, as well as iron status at baseline are accurate predictors of disability at 18-month follow-up. The composite scores of these markers in association with established clinical predictors enable the accurate forecasting of functional decline. The identified four biomarkers are all closely associated with 'ferroptosis', a recently discovered form of programmed cell death with promising therapeutic targets. The predictive potential of these pathophysiology-based indicators may offer superior patient stratification for future trials, individualised patient care and resource allocation.

Lipidomics Reveals Cerebrospinal-Fluid Signatures of ALS.

Amyotrophic lateral sclerosis (ALS), the commonest adult-onset motor neuron disorder, is characterized by a survival span of only 2-5 years after onset. Relevant biomarkers or specific metabolic signatures would provide powerful tools for the management of ALS. The main objective of this study was to investigate the cerebrospinal fluid (CSF) lipidomic signature of ALS patients by mass spectrometry to evaluate the diagnostic and predictive values of the profile. We showed that ALS patients (n = 40) displayed a highly significant specific CSF lipidomic signature compared to controls (n = 45). Phosphatidylcholine PC(36:4), higher in ALS patients (p = 0.0003) was the most discriminant molecule, and ceramides and glucosylceramides were also highly relevant. Analysis of targeted lipids in the brain cortex of ALS model mice confirmed the role of some discriminant lipids such as PC. We also obtained good models for predicting the variation of the ALSFRS-r score from the lipidome baseline, with an accuracy of 71% in an independent set of patients. Significant predictions of clinical evolution were found to be correlated to sphingomyelins and triglycerides with long-chain fatty acids. Our study, which shows extensive lipid remodelling in the CSF of ALS patients, provides a new metabolic signature of the disease and its evolution with good predictive performance.

A Multiplatform Metabolomics Approach to Characterize Plasma Levels of Phenylalanine and Tyrosine in Phenylketonuria.

BACKGROUND: Different pathophysiological mechanisms have been described in phenylketonuria (PKU) but the indirect metabolic consequences of metabolic disorders caused by elevated Phe or low Tyr concentrations remain partially unknown. We used a multiplatform metabolomics approach to evaluate the metabolic signature associated with Phe and Tyr. MATERIAL AND METHODS: We prospectively included 10 PKU adult patients and matched controls. We analysed the metabolome profile using GC-MS (urine), amino-acid analyzer (urine and plasma) and nuclear magnetic resonance spectroscopy (urine). We performed a multivariate analysis from the metabolome (after exclusion of Phe, Tyr and directly derived metabolites) to explain plasma Phe and Tyr concentrations, and the clinical status. Finally, we performed a univariate analysis of the most discriminant metabolites and we identified the associated metabolic pathways. RESULTS: We obtained a metabolic pattern from 118 metabolites and we built excellent multivariate models to explain Phe, Tyr concentrations and PKU diagnosis. Common metabolites of these models were identified: Gln, Arg, succinate and alpha aminobutyric acid. Univariate analysis showed an inverse correlation between Arg, alpha aminobutyric acid and Phe and a positive correlation between Arg, succinate, Gln and Tyr (p < 0.0003). Thus, we highlighted the following pathways: Arg and Pro, Ala, Asp and Glu metabolism. DISCUSSION: We obtain a specific metabolic signature related to Tyr and Phe concentrations. We confirmed the involvement of different pathophysiological mechanisms previously described in PKU such as protein synthesis, energetic metabolism and oxidative stress. The metabolomics approach is relevant to explore PKU pathogenesis.

Biomarkers in amyotrophic lateral sclerosis: combining metabolomic and clinical parameters to define disease progression.

BACKGROUND AND PURPOSE: The objectives of this study were to define the metabolomic profile of cerebrospinal fluid in amyotrophic lateral sclerosis (ALS) patients, to model outcome through combined clinical and metabolomic parameters and independently to validate predictive models. METHODS: In all, 74 consecutive newly diagnosed patients were enrolled into training (Tr, n = 49) and test (Te, n = 25) cohorts. Investigators recorded clinical data and the metabalomic profile of cerebrospinal fluid at baseline was analyzed with (1)H nuclear magnetic resonance spectroscopy. Markers of disease progression, collected in 1-year prospective follow-up, included change in ALS Functional Rating Scale (var_ALSFRS), change in weight (var_weight) and survival time. Stepwise multiple regression selected from metabolomic and clinical parameters to model rate of progression in the Tr cohort. Best fit models were validated independently in the Te cohort. RESULTS: The best-fit statistical models, using both metabolomic and clinical covariates, predicted outcome with 70.8% (var_weight), 72% (var_ALSFRS) and 76% (survival) accuracy in the Te cohort. Models that used metabolomics or clinical data alone predicted outcome less well. Highlighted metabolites are involved in pathophysiological pathways previously described in ALS. CONCLUSION: Cerebrospinal fluid metabolomics can aid in predicting the clinical course of ALS and tap into pathophysiological processes. The precision of predictive models, independently reproduced in this study, is enhanced through inclusion of both metabolomic and clinical parameters. The findings bring the field closer to a clinically meaningful disease marker.

Biological follow-up in amyotrophic lateral sclerosis: decrease in creatinine levels and increase in ferritin levels predict poor prognosis.

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal disorder of the motor neuron system, with a median survival of 2 to 4 years and a wide variety of prognosis. Thus, there is a critical need for diagnosis and prognosis biomarkers to improve the care of patients in routine practice. In this study, we aimed to determine prognostic value of routine biochemical markers in sporadic ALS (SALS). METHODS: We retrospectively collected clinical and biological data obtained during the systematic routine monitoring of 216 sporadic ALS patients. The main outcomes were disease duration and annual decline of Revised ALS Functional Rating Scale (ALSFRS-R). Changes to these biological variables over time were assessed, in link with disease progression. RESULTS: We found that concentrations of creatinine (P=0.0166) and ferritin (P=0.0306) changed significantly during the progression of ALS. A reduction of creatinine levels and an increase of ferritin levels were associated with disease progression. Multivariate analysis showed that early variation of ferritin was an independent predictive factor of patient survival (P=0.0048). CONCLUSION: Changes to ferritin and creatinine levels with time are associated with ALS progression. This is the first study describing the changes to these biological variables during ALS progression.

Advances in cellular models to explore the pathophysiology of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disorder, is fatal for most patients less than 3 years from when the first symptoms appear. The aetiologies for sporadic and most familial forms of ALS are unknown, but genetic factors are increasingly recognized as causal in a subset of patients. Studies of disease physiology suggest roles for oxidative stress, glutamate-mediated excitotoxicity or protein aggregation; how these pathways interact in the complex pathophysiology of ALS awaits elucidation. Cellular models are being used to examine disease mechanisms. Recent advances include the availability of expanded cell types, from neuronal or glial cell culture to motoneuron-astrocyte co-culture genetically or environmentally modified. Cell culture experiments confirmed the central role of glial cells in ALS. The recent adaptation of induced pluripotent stem cells (iPSC) for ALS modeling could allow a broader perspective and is expected to generate new hypotheses, related particularly to mechanisms underlying genetic factors. Cellular models have provided meaningful advances in the understanding of ALS, but, to date, complete characterization of in vitro models is only partially described. Consensus on methodological approaches, strategies for validation and techniques that allow rapid adaptation to new genetic or environmental influences is needed. In this article, we review the principal cellular models being employed in ALS and highlight their contribution to the understanding of disease mechanisms. We conclude with recommendations on means to enhance the robustness and generalizability of the different concepts for experimental ALS.

Deficiency in the NADPH oxidase 4 predisposes towards diet-induced obesity.

OBJECTIVE: NADPH oxidase 4 (NOX4) is a reactive oxygen species (ROS) producing NADPH oxidase that regulates redox homeostasis in diverse insulin-sensitive cell types. In particular, NOX4-derived ROS is a key modulator of adipocyte differentiation and mediates insulin receptor signaling in mature adipocytes in vitro. Our study was aimed at investigating the role of NOX4 in adipose tissue differentiation, whole body metabolic homeostasis and insulin sensitivity in vivo. DESIGN: Mice with genetic ablation of NOX4 (NOX4-deficient mice) were subjected to chow or high-fat-containing diet for 12 weeks. Body weight gain, adiposity, insulin sensitivity, and adipose tissue and liver gene and protein expression were analyzed and compared with similarly treated wild-type mice. RESULTS: Here, we report that NOX4-deficient mice display latent adipose tissue accumulation and are susceptible to diet-induced obesity and early onset insulin resistance. Obesity results from accelerated adipocyte differentiation and hypertrophy, and an increase in whole body energy efficiency. Insulin resistance is associated with increased adipose tissue hypoxia, inflammation and adipocyte apoptosis. In the liver, more severe diet-induced steatosis was observed due to the lack of proper upregulation of mitochondrial fatty acid ß-oxidation. CONCLUSION: These findings identify NOX4 as a regulator of metabolic homeostasis. Moreover, they indicate an anti-adipogenic role for NOX4 in vivo and reveal its function as a protector against the development of diet-induced obesity, insulin resistance and hepatosteatosis.

Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats.

BACKGROUND AND PURPOSE: mTOR inhibitors are currently used as immunosuppressants in transplanted patients and as promising anti-cancer agents. However, new-onset diabetes is a frequent complication occurring in patients treated with mTOR inhibitors such as rapamycin (Sirolimus). Here, we investigated the mechanisms associated with the diabetogenic effects of chronic Sirolimus administration in rats and in in vitro cell cultures. EXPERIMENTAL APPROACH: Sirolimus was administered to rats fed either a standard or high-fat diet for 21 days. Metabolic parameters were measured in vivo and in ex vivo tissues. Insulin sensitivity was assessed by glucose tolerance tests and euglycaemic hyperinsulinaemic clamps. Rapamycin effects on glucose metabolism and insulin signalling were further evaluated in cultured myotubes. KEY RESULTS: Sirolimus induced a decrease in food intake and concomitant weight loss. It also induced specific fat mass loss that was independent of changes in food intake. Despite these beneficial effects, Sirolimus-treated rats were glucose intolerant, hyperinsulinaemic and hyperglycaemic, but not hyperlipidaemic. The euglycaemic hyperinsulinaemic clamp measurements showed skeletal muscle is a major site of Sirolimus-induced insulin resistance. At the molecular level, long-term Sirolimus administration attenuated glucose uptake and metabolism in skeletal muscle by preventing full insulin-induced Akt activation and altering the expression and translocation of glucose transporters to the plasma membrane. In rats fed a high-fat diet, these metabolic defects were exacerbated, although Sirolimus-treated animals were protected from diet-induced obesity. CONCLUSIONS AND IMPLICATIONS: Taken together, our data demonstrate that the diabetogenic effect of chronic rapamycin administration is due to an impaired insulin action on glucose metabolism in skeletal muscles.

Regulation of the pituitary growth hormone-releasing hormone receptor in ageing male and female LOU rats: new insights into healthy ageing.

Ageing is characterised by a decrease of somatotroph functionality, involving growth hormone-releasing hormone receptor (GHRH-R). The present study was conducted in LOU/C/jall (LOU) rats, a strain described as a model of healthy ageing, which is characterised by a low adiposity and long life expectancy without developing severe pathologies. Effects of age and diet (chow versus self-selection), on levels of anterior pituitary GHRH-R mRNA transcripts, were assessed in male and female LOU rats. The effect of age on pituitary GHRH-R functionality was examined in the anterior pituitary of both males and females fed chow diet. Moreover, serum insulin-like growth factor-I (IGF-I), T4 and leptin were measured because changes in their concentration could affect GHRH-R expression. In the pituitary of 18-month-old male and female LOU/C/jall rats fed standard chow, the level of 2.5-kb GHRH-R mRNA transcript, coding for functional GHRH-R, was significantly decreased. In 24- to 34-month-old males and females, it progressively returned to the level of younger animals, suggesting an enrichment of the group with survivors maintaining functional GHRH-R. In males and females repeatedly submitted to self-selection, this phenomenon was not observed. Studies with the GHRH-R agonist, Fluo-GHRH, revealed that 73% of 16-18-month-old male and female rats studied did not show an increase of fluorescence density characteristic of receptor-mediated internalisation upon incubation at 37 degrees C. In the other 27%, the increase of fluorescence was identical to that observed in pituitaries of young rats, suggesting the presence of an optimal level of functional GHRH-R. Serum levels of leptin, free T4 and total IGF-I decreased more drastically in ageing males and in rats fed a self-selection diet. A positive correlation was demonstrated between leptin and IGF-I levels in ageing males and females fed standard chow and ageing females submitted to a self-selection regimen. In conclusion, healthy ageing in LOU rats fed chow diet appears to be associated with a maintenance of functional pituitary GHRH-R levels found in younger rats but not necessarily with those of serum leptin, T4 and IGF-I.

Effects of peripheral and central administration of GHRH on feeding in aging LOU rats.

In aging LOU rats, a decreased protein intake is restored by GH administration. To study the contribution of GHRH to macronutrient selection, hGHRH NH(2) was administered sc. (1 mg/kg B.W./day/14 days) or icv. (4 and 40 pmol/rat) to 11-, 19-, 24- and 28-month-old rats. Sc. administration induced a decreased food and lipid intakes from 24 months of age and a transient stimulation of protein intake in 19-month-old and older low protein eaters (<10% protein/total intake). Icv. administration induced decreased food and lipid intakes in all age groups. These results suggest that GHRH may regulate feeding through pituitary and/or hypothalamic GHRH receptor mechanisms.

Age-related changes in nociception and effect of morphine in the Lou rat.

Little is known about how the ageing process affects pain sensitivity and a relevant animal model is therefore required. The effect of age on pain reactivity in animals has been investigated by several experimenters but the results are conflicting. Four groups of male and female Lou/C/Jall rats (4-29 months old) were used for our study. Four pain tests based on evaluation of reflex or more integrated behaviours after a thermal (tail immersion test) or mechanical (paw pressure test and von Frey test) stimulation were used. With mechanical stimulus, a significant decrease in the pain threshold across age was observed, females were more sensitive than males. This increase in nociceptive sensitivity to mechanical stimulation was more pronounced on integrated behaviours (struggle reaction) than on withdrawal reflex. An age-related increase in sensitivity was found on von Frey test. No effect on the latency of reflex induced by thermal stimulation was observed. In addition, a decrease in the spontaneous motor activity during exploration was observed across ageing; this effect was more marked for the females. The effect of morphine at doses of 1, 3 and 9 mg/kg (s.c.) decreased in intensity across ageing. These data demonstrate the need to use (1) various noxious stimuli because differences were observed in the modification of pain reactivity according to the nature of the stimulus; (2) various pain parameters and particularly integrated behaviours; (3) several age groups. In addition, Lou/C/Jall rat could be a useful model for studying of effect of age on pain.

Age-related changes in adaptive macronutrient intake in swimming male and female Lou rats.

To evaluate the age-related changes in capacity to adjust the nutrient intake to needs, self-selecting male and female Lou/C/jall rats of 4, 6, 12, 16 and 23 months of age were submitted to a swimming exercise. They were given 6 consecutive days of moderate intensity training (3 x 15 minutes per day). Exercise and postexercise periods were compared with results from the pretraining period. During swimming, a body weight loss and a decrease in both caloric intake and fat selection were observed. This effect was more marked in older groups compared to 4 month-old groups. An increase in protein intake was observed in females, specially in older groups, whereas no effect was seen in males. The ability to increase caloric ingestion and regain weight during the postexercise period decreased with advancing age and was better in females than in males. We also showed an age-related effect on the recovery of initial nutrient intake rate that was more pronounced and more precocious for males. Moreover, males tended to decrease their protein intake, whereas females significantly increased it. The present findings suggest a decrease of capacity of adjusting feeding behavior to metabolic needs in aged rats, may be due to a deterioration of the central control of food intake.

Peripheral injection of growth hormone stimulates protein intake in aged male and female Lou rats.

It is well established that growth hormone (GH) induces growth rate and food efficiency and stimulates protein accretion in young mammals. Senescence is characterized by metabolic and hormonal disorders, particularly a decrease in protein turnover, which could be correlated to a decrease in GH and insulin-like growth factor I (IGF-I) secretion. We have shown that body weight, protein intake, and IGF-I plasma levels are greatly decreased with aging in Lou/C rats, particularly in males. In order to specify the GH effect on protein intake during aging, males and females (6, 19, and 24 mo) placed on a self-selection regimen were injected daily with a physiological dose of human GH (0.023 mg/rat sc). No GH effect on caloric intake was observed. Nevertheless, GH treatment stimulated body weight in older rats. It also increased protein intake in females and older males (19-24 mo). This stimulating effect was positively correlated with the degree of weight loss in senescent rats, suggesting that the decrease in protein intake observed with aging could be a marker of senescence.

Changes in 20S proteasome activity during ageing of the LOU rat.

Muscular functions decline and muscle mass decreases during ageing. In the rat, there is a 27% decrease in muscle protein between 18 and 34 months of age. We examined age-related changes in the proteasome-dependent proteolytic pathway in rats at 4, 18, 24, 29 and 34 months of age. The three best characterised activities of the proteasome (chymotrypsin-like, trypsin-like and peptidylglutamyl peptide hydrolase) increased to 29 months and then decreased in the senescent animal. These variations in activity were accompanied by an identical change in the quantity of 20S proteasome measured by Western blot, whereas the S4 subunit of the 19S regulator and the quantity of ubiquitin-linked proteins remained constant. mRNA of subunits C3, C5, C9, and S4 increased in the senescent animal, but ubiquitin mRNA levels were unchanged. These findings suggest that the 20S proteasome may be partly responsible for the muscular atrophy observed during ageing in the rat.

Age-related changes in adaptive mechanisms of macronutrient self-selection: evidence for a sexual dimorphism.

The effect of aging on patterns of food intake and nutrient selection was investigated using a longitudinal study. Male (n = 10) and female (n = 10) Wistar-Lou rats from 4 to 28 months of age were repeatedly submitted to a macronutrient self-selection (S-S) regimen while controls were maintained under a standard chow diet (Std). An age-related shift of preferences from CHO to fat diets, and a decrease in protein intake for both males and females were evident. Nevertheless, all these modifications were more pronounced and precocious for males. Physical exercise (45 min/day of swimming, on 6 days) induced a body weight loss and an hypophagia more pronounced for males than for females. S-S regimen results revealed that hypophagia concerned exclusively fat intake and that females significantly increased protein intake during and after the exercise period. Study of longevity curves showed a decrease of the mortality in S-S submitted male rats compared to control rats. This study shows that aging induces a sex-difference in feeding patterns which undoubtedly reveals a sex-difference metabolic requirements. Moreover, these results suggest that allowing rats to select macronutrient intakes could delay the process of senescence.

Dietary self-selection can compensate an age-related decrease of rat liver 20 S proteasome activity observed with standard diet.

Aged Lou female rats (33 months) submitted to a self-selection regimen showed a decrease in protein intake (down to 11% of the total intake), whereas mature rats (18 months) selected a high percentage of protein (20% of the total intake) similar to the protein content of the standard diet. To find out if this decrease in protein intake would prevent an observed age-related decrease in proteasome activity, four peptidase activities and oxidized protein degradation were tested with proteasome purified from the liver of 18- and 33-month-old rats. The peptidylglutamyl-peptide hydrolase activity, which is decreased with age for rats fed the standard diet, was restored in the self-selecting old rats to the level observed for the mature rats. Degradation of oxidized glutamine synthetase, which is also decreased with age for rats fed the standard diet, was partly restored. Proteasome from self-selecting old rats showed a slight increase in trypsin-like and chymotrypsin-like activities as compared to proteasome from old rats fed the standard diet. Two-dimensional gel electrophoresis followed by quantitative analysis of the pattern of proteasome subunits revealed an increase in the intensity of two protein spots for proteasome from old rats fed the standard diet as compared with proteasome from either mature rats or self-selecting old rats. These findings may have important implications in aging for proteasome-mediated proteolysis and subsequent accumulation of oxidatively damaged protein.