Nutritional interventions for patients with alkaptonuria: A minireview.

Alkaptonuria (AKU, OMIM, No. 203500) is a rare, slow-progressing, irreversible, multisystemic disease resulting from a deficiency of the homogentisate 1,2-dioxygenase enzyme, which leads to the accumulation of homogentisic acid (HGA) and subsequent deposition as pigment in connective tissues called ochronosis. As a result, severe arthropathy of large joints and spondyloarthropathy with frequent fractures, ligament ruptures, and osteoporosis develops in AKU patients. Since 2020, the first-time treatment with nitisinone has become available in the European Union. Nitisinone significantly reduces HGA production and arrests ochronosis in AKU patients. However, blocking of the tyrosine metabolic pathway by the drug leads to tyrosine plasma and tissue concentrations increase. The nitisinone-induced hypertyrosinemia can lead to the development of corneal keratopathy, and once it develops, the treatment needs to be interrupted. A decrease in overall protein intake reduces the risk of the keratopathy during nitisinone-induced hypertyrosinemia in AKU patients. The low-protein diet is not only poorly tolerated by patients, but over longer periods, leads to a severe muscle loss and weight gain due to increased energy intake from carbohydrates and fats. Therefore, the development of novel nutritional approaches is required to prevent the adverse events due to nitisinone-induced hypertyrosinemia and the negative impact on skeletal muscle metabolism in AKU patients.

Effects of Nitisinone on Oxidative and Inflammatory Markers in Alkaptonuria: Results from SONIA1 and SONIA2 Studies.

Nitisinone (NTBC) was recently approved to treat alkaptonuria (AKU), but there is no information on its impact on oxidative stress and inflammation, which are observed in AKU. Therefore, serum samples collected during the clinical studies SONIA1 (40 AKU patients) and SONIA2 (138 AKU patients) were tested for Serum Amyloid A (SAA), CRP and IL-8 by ELISA; Advanced Oxidation Protein Products (AOPP) by spectrophotometry; and protein carbonyls by Western blot. Our results show that NTBC had no significant effects on the tested markers except for a slight but statistically significant effect for NTBC, but not for the combination of time and NTBC, on SAA levels in SONIA2 patients. Notably, the majority of SONIA2 patients presented with SAA > 10 mg/L, and 30 patients in the control group (43.5%) and 40 patients (58.0%) in the NTBC-treated group showed persistently elevated SAA > 10 mg/L at each visit during SONIA2. Higher serum SAA correlated with lower quality of life and higher morbidity. Despite no quantitative differences in AOPP, the preliminary analysis of protein carbonyls highlighted patterns that deserve further investigation. Overall, our results suggest that NTBC cannot control the sub-clinical inflammation due to increased SAA observed in AKU, which is also a risk factor for developing secondary amyloidosis.

Lipoprotein profiling in early multiple sclerosis patients: effect of chronic inflammation?

BACKGROUND: Inflammatory cytokines contribute to proatherogenic changes in lipid metabolism by reduction of HDL-cholesterol (HDL-C) levels, impairment of its antiinflammatory and antioxidant functions. Therefore, the protective actions of HDL-C can be limited in chronic inflammatory diseases such as multiple sclerosis (MS). The aim of this study was to assess the association between lipoprotein subfractions and inflammatory status in early stages of multiple sclerosis. METHODS: Polyacrylamide gel electrophoresis Lipoprint© System was used for lipoprotein profile analysis in 19 newly diagnosed MS patients, and in matched 19 healthy controls. Serum levels of interleukin (IL) 1ß, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, interferon-γ and TNF-α were measured by multiplex bead assay. RESULTS: Concentrations of the measured cytokines and lipoprotein subclasses were comparable between MS patients and controls. Male, but not female MS patients had significantly higher total HDL-C and small HDL-C subfraction than healthy controls. Large HDL-C negatively correlated with all measured cytokines except IL-17 in MS but not in controls. Intermediate HDL-C subfractions correlated positively with all measured cytokines except G-CSF in MS females but not in MS males or controls. CONCLUSION: Our results of higher HDL-C and mainly its small HDL-C subfraction suggest that male MS patients are at higher risk of atherosclerosis and the subtle dyslipidemia is present in early stages of the disease. The correlations between specific HDL-C subfractions and the inflammatory cytokines demonstrate mutual links between systemic inflammation and lipid metabolism in MS. TRIAL REGISTRATION: ClinicalTrials.gov, Identifier: NCT03052595 Registered on Feb 14, 2017.

Effect of 8-weeks intensive lifestyle intervention on LDL and HDL subfractions.

OBJECTIVE: Atherogenic dyslipidemia is a cardinal feature of obesity and the metabolic syndrome, which increases the risk of cardiovascular diseases. Many interventional studies, describing the influence of weight loss on cardiometabolic risks, are bariatric surgery studies. The aim of our study was to analyze the effect of intensive lifestyle changes on LDL- and HDL-cholesterol subfractions and cardiometabolic risk factors in obese subjects. METHODS: A group of 41 patients with obesity (11M/30F; 44.1±12.4 years; BMI 30.2±6.3kg/m2) participated in an 8-week weight loss interventional program (NCT02325804), consisting of caloric intake reduced by 30% and physical activity (150min/week). Insulin sensitivity was evaluated according to the homeostasis model assessment of insulin resistance (HOMA-IR) and physical fitness was measured using bicycle ergometry. Lipid subfractions were measured using the Lipoprint system (Quantimetrix Corp., CA, USA). RESULTS: After the intervention, body weight was reduced by 5.4±4.5kg, as well as body fat mass and waist circumference. Physical fitness improved, systolic and diastolic blood pressure as well as heart rate decreased after the intervention. Insulin sensitivity improved after the intervention. Total, LDL, HDL cholesterol, as well as triglycerides decreased after the intervention. Regarding the lipoprotein subfractions, LDL2 and small HDL subfractions decreased, while others have not changed. CONCLUSION: Eight weeks of diet and physical activity intervention led to weight and fat mass loss and induced improvement of insulin sensitivity, as well as atheroprotective changes of lipid profile. However, the weight loss associated changes in cholesterol subfractions as cardiovascular risk biomarkers deserve further studies.

Hyperinsulinemia in newly diagnosed patients with multiple sclerosis.

There are limited data regarding glucose metabolism dysregulation in multiple sclerosis (MS). Present study investigates glucose and insulin response during oral glucose tolerance test (oGTT) in MS patients. We examined 19 MS patients and 19 age, sex and body mass index (BMI) matched healthy controls. MS patients were newly diagnosed, untreated and with low Expanded Disability Status Scale (EDSS) score (1.1 ± 0.7). Plasma glucose, lactate, insulin and GLP-1 during oGTT, and fasting adipokines, lipid and inflammatory parameters were analyzed. Insulin sensitivity indices (ISI) were calculated. MS patients had comparable fasting (5.2 ± 0.3 vs. 5.0 ± 0.4 mmol/l, p = 0.05) and post-load glucose concentrations as controls. Insulin response to oral glucose load in MS was increased (p = 0.022). Insulin sensitivity was lower in MS compared to controls [ISI(Matsuda) 6.95 ± 3.44 vs. 10.60 ± 4.81, p = 0.011 and ISI(Cederholm) 49.9 ± 15.3 vs. 61.3 ± 16.3, p = 0.032]. We did not find any difference in lactate, GLP-1, total, HDL and LDL cholesterol, triglycerides, interleukin 6, tumor necrosis factor, C-reactive protein, resistin, leptin, adiponectin levels between groups. We found decreased insulin sensitivity with postprandial hyperinsulinemia in MS patients, which seems not to be related to chronic inflammation or physical inactivity. The role of hyperinsulinemia in CNS function impairment should be further investigated.

Glycoprofiling as a novel tool in serological assays of systemic sclerosis: a comparative study with three bioanalytical methods.

Systemic sclerosis (SSc) is an autoimmune disease seriously affecting patient's quality of life. The heterogeneity of the disease also means that identification and subsequent validation of biomarkers of the disease is quite challenging. A fully validated single biomarker for diagnosis, prognosis, disease activity and assessment of response to therapy is not yet available. The main aim of this study was to apply an alternative assay protocol to the immunoassay-based analysis of this disease by employment of sialic acid recognizing lectin Sambucus nigra agglutinin (SNA) to glycoprofile serum samples. To our best knowledge this is the first study describing direct lectin-based glycoprofiling of serum SSc samples. Three different analytical methods for glycoprofiling of serum samples relying on application of lectins are compared here from a bioanalytical point of view including traditional ELISA-like lectin-based method (ELLA), novel fluorescent lectin microarrays and ultrasensitive impedimetric lectin biosensors. Results obtained by all three bioanalytical methods consistently showed differences in the level of sialic acid present on glycoproteins, when serum from healthy people was compared to the one from patients having SSc. Thus, analysis of sialic acid content in human serum could be of a diagnostic value for future detection of SSc, but further work is needed to enhance selectivity of assays for example by glycoprofiling of a fraction of human serum enriched in antibodies for individual diagnostics.

Increased production of IL-6 and IL-17 in lipopolysaccharide-stimulated peripheral mononuclears from patients with rheumatoid arthritis.

TLR4-mediated inflammatory responses are important for innate immune functions, thus their alterations may participate in the pathogenesis of rheumatoid arthritis (RA). Cortisol is one of the most potent immunomodulatory hormones involved in control of inflammation. In this study, we analyzed TLR4-mediated responses and cortisol effects on the process in peripheral blood mononuclear cells (PBMC) from RA patients. Lipopolysaccharide-stimulated PBMC from 23 female patients and 15 healthy controls were cultured in the presence or absence of cortisol (1 µM) for 24 h. A panel of 17 inflammatory cytokines was analyzed in the cell culture supernatants. Higher (p < 0.05) concentrations of IL-6, IL-17 and MCP-1 were found in lipopolysaccharide-stimulated PBMC from RA patients compared to controls. After normalization of stimulated cytokine secretion to unstimulated cells, a significantly higher (p < 0.05) IL-6 and G-CSF production was found in RA PBMC. Cortisol induced stronger (p < 0.05) suppression of lipopolysaccharide-stimulated secretion of IL-1ß, IL-6, IL-17 and G-CSF in RA group compared to controls. The observed higher production of the key inflammatory cytokines by RA PBMC to lipopolysaccharide stimulation supports involvement of TLR4-mediated processes in RA pathogenesis. The higher sensitivity of LPS-stimulated RA PBMC to immunosuppressive effects of cortisol may reflect adaptive processes to chronic inflammation.

Microparticles: a component of various diseases.

Microparticles (MPs) are phenotypically and functionally heterogeneous population of microvesicles. Although MP formation represents a physiological phenomenon. A multitude of pathologies, including inflammatory and autoimmune diseases, atherosclerosis, and malignancies, are associated with a considerable increase in circulating MPs. Elevated levels of platelet­, endothelial cell­, and monocyte­derived MPs have been documented in a number of clinical conditions in which vascular dysfunction and inflammation are important pathophysiological mechanisms (e.g., coronary artery disease or thrombotic microangiopathies). Knowledge of the functional properties of MPs will contribute to a better understanding of the pathological mechanisms of communication between cells and of the causes of various diseases.

Protein array reveals differentially expressed proteins in subcutaneous adipose tissue in obesity.

OBJECTIVE: Many adipokines, inflammatory cytokines, and other proteins produced by adipose tissue have been shown to be involved in the development of obesity-related insulin resistance. Nevertheless, new factors that play an important role in these processes are still emerging. Therefore, we screened the level of 120 different proteins in biopsies of subcutaneous adipose tissue (ScAT) of lean and obese subjects. RESEARCH METHODS AND PROCEDURES: All studied volunteers (12 obese with BMI >30 and 6 lean with BMI <25 kg/m(2)) were young, clinically healthy, and drug-naive males with normal glucose tolerance. The ScAT was obtained by a needle biopsy from the umbilical region. Protein levels were assessed in adipose tissue lysates using protein arrays; mRNA levels were determined with the aid of real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The obese subjects had higher fasting plasma glucose (although within the normal range) and insulin levels, increased high sensitivity C-reactive protein (hsCRP) in circulation, and decreased in vivo insulin action. Using the protein array technique, it was shown that of 120 proteins measured, 27 showed higher levels (leptin, HGF, EGF-R, FGF-6, IGF-1sR, Fas/Apo-1, ENA-78, PARC, lymphotactin, HCC-4, IL-10, IL-1a, IL-1R1, IL-1R4, IL-12p70, angiopoietin-2, Axl, Dtk, MIF, MIP-1a, -1b, -3b, MSP-a, osteoprotegerin, TECK, TIMP-1, -2) and only one (RANTES) showed a lower level in ScAT of obese subjects when compared with the lean controls (p < 0.05). The real-time RT-PCR confirmed the results of protein arrays for leptin, MIF, MIP-1a, TIMP-2, adiponectin, IL-6, and TNF-alpha but not for RANTES. DISCUSSION: To our knowledge, this is the first protein array data on a very early dysregulation of ScAT protein levels in insulin-resistant obese, but apparently healthy, subjects with normal glucose tolerance.

Thyroid ultrasound volume, structure and function after long-term high exposure of large population to polychlorinated biphenyls, pesticides and dioxin.

We examined 2,046 adults (834 males and 1,212 females aged 20-75 years) from polluted district in East Slovakia (POLL) and two neighboring upstream and upwind located districts of background pollution (BCGR). By ultrasound we estimated the thyroid volume (ThV), hypoechogenicity (HYE), nodules and cysts. Serum levels of thyrotropin (TSH), thyroperoxidase antibodies (TPOab) and thyroglobulin were estimated by electrochemiluminiscent assay and these of 15 PCB congeners, p,p'-DDE, p,p'-DDT, hexachlorobenzene (HCB) and hexachlorocyclohexane by high-resolution gas chromatography. In 320 subjects also selected hydroxylated and methylsulfonated PCB metabolites, polychlorinated dibenzo-dioxins (PCDDs), -furans (PCDFs), five dioxin-like coplanar and eight mono-ortho PCB congeners were estimated. Urinary iodine was measured by automatic microplate method. Reciprocal positive association was found between three major POPs (PCBs, DDE and HCB), the levels of these and also PCDDs plus PCDFs in polluted area being considerably higher than in background pollution area. ThV in groups of males and females from POLL with high PCBs level was significantly higher (p<0.001 by t-test) then in age and sex matched groups from BCGR with low PCBs level. In 1,048 males and females aged <60 years with serum PCBs level >1,000 ng g(-1) lipid (median=1,756 ng g(-1)) a significant effect of age on ThV was found (p<0.01 by ANOVA), while in 921 respective subjects with PCBs level <1,000 ng g(-1) (median=661 ng g(-1)) it was not. These findings supported the view on the additional effect of PCBs on ThV other than that of age. Since the urinary iodine in both districts showed optimal range, any interfering effect of unsatisfactory iodine intake on ThV may be excluded. The frequency of autoimmune thyroiditis signs such as HYE, increased serum level of TPOab and TSH resulting in subclinical or overt thyroid hypofunction was positively associated with sex, age and organochlorine levels. The increase of such frequency in males with POPs levels was much more abrupt than that in females. No considerable differences in the frequency of thyroid nodules as related to PCBs level were found.

Comparison of hormone transfer to pleural and synovial exudates.

OBJECTIVES: Local effects of hormones on immune and connective tissues could play some role in the development of local inflammation processes. The aim of this study was to investigate the levels of selected hormones in pleural exudates of patients with pleurisy and lung tumours, and compare these levels with hormone concentration in knee synovial fluid. SUBJECTS AND METHODS: Eleven patients with pleural exudate (mean age 62+/-3) and l9 subjects with rheumatoid arthritis (of the same mean age) participated in the observations. Plasma, pleural exudates and synovial fluid levels of cortisol, prolactin, aldosterone, testosterone, 17-beta-estradiol, dehydroepiandrosterone, progesterone, insulin and C-peptide were determined by specific radioimmunoassay. RESULTS: It was noted that all estimated hormones are transferred into pleural exudates and synovial fluid. Higher levels of dehydroepiandrosterone and C-peptide were observed in pleural exudates as compared to plasma. The concentrations of testosterone, prolactin and estradiol in males were lower in exudates as compared to plasma. Mean levels of cortisol, aldosterone, progesterone and insulin in plasma were similar to these found in pleural exudates. The comparison of hormone levels in pleural exudates and synovial fluid showed that the levels of cortisol, progesterone and dehydroepiandrosterone tended to be higher in the exudates as compared to synovial fluid. However, the levels of insulin, testosterone and estradiol in exudates were lower than these in inflammatory synovial fluid from patients with rheumatoid arthritis. CONCLUSIONS: This study showed the presence of hormones in pleural exudates. The differences in hormone concentrations in pleural exudates and synovial fluid were observed suggesting a specificity of hormone transfer from plasma to these exudates.

Lower adrenocortical and adrenomedullary responses to hypoglycemia in premenopausal women with systemic sclerosis.

OBJECTIVES: To evaluate function of the hypothalamic-pituitary-adrenal (HPA) axis, adrenomedullary hormonal system (AMHS), and sympathetic noradrenergic system (SNS) in premenopausal women with systemic sclerosis (SSc). METHODS: Insulin-induced hypoglycemia (0.1 IU/kg) was performed in 17 longterm, glucocorticoid-naive SSc patients with low disease activity and in 18 healthy women matched for age and body mass index (BMI). Concentrations of glucose, adrenocorticotrophic hormone (ACTH), cortisol, androstenedione (ASD), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 17a-hydroxyprogesterone (17OHP), epinephrine (EPI), norepinephrine (NE), interleukin 1ss (IL-1ss), IL-6, and tumor necrosis factor-a (TNF-a) were analyzed in plasma. RESULTS: Basal plasma levels of cortisol, ASD, 17OHP, DHEAS, IL-1ss, IL-6, and TNF-a were not significantly different in SSc compared to controls. Patients had higher basal ACTH (6.76 +/- 1.0 pmol/l in SSc vs 4.14 +/- 0.45 pmol/l in controls; p < 0.05), lower basal DHEA (9.02 +/- 1.64 nmol/l in SSc vs 17.0 +/- 2.8 nmol/l in controls; p < 0.05), and lower basal NE (1.61 +/- 0.26 nmol/l in SSc vs 2.57 +/- 0.38 nmol/l in controls; p < 0.05). Patients had comparable responses of glucose and ACTH to hypoglycemia. General linear model for repeated measurements, with BMI and age as covariates, revealed that the responses of 17OHP (p < 0.05), ASD (p < 0.05), DHEA (p < 0.01), EPI (p < 0.001), and NE (p < 0.001) to hypoglycemia were lower in SSc compared to controls. Cortisol response to hypoglycemia tended to be lower in SSc patients (p = 0.06) compared to controls. CONCLUSION: Our data indicate decreased adrenocortical and adrenomedullary functions in premenopausal women with SSc. Whether the observed changes in the neuroendocrine system are secondary to chronic disease deserves further investigation.

Prolactin and growth hormone responses to hypoglycemia in patients with systemic sclerosis and psoriatic arthritis.

This study compared prolactin (PRL) and growth hormone (GH) responses to hypoglycemia in premenopausal females with systemic sclerosis (SSc) and psoriatic arthritis (PsA) with those in matched healthy controls. No differences were found in glucose and GH responses to hypoglycemia in both groups of patients compared to controls. SSc patients had lower PRL response (P < 0.05) to hypoglycemia compared to controls. PRL response tended to be lower also in PsA patients, however the difference did not reach level of statistical significance (P = 0.11). The present study showed decreased PRL response to hypoglycemia in premenopausal females with SSc.

Prolactin and growth hormone responses to hypoglycemia in patients with rheumatoid arthritis and ankylosing spondylitis.

OBJECTIVE: Prolactin (PRL) and growth hormone (GH) are pituitary hormones with immunomodulating properties. Their upregulated secretion may play a role in the pathogenesis of chronic inflammatory diseases. We evaluated PRL and GH responses to secretion stimulus in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). METHODS: Insulin hypoglycemia (0.1 IU/kg) was induced in 15 women with RA, 18 men with AS, and healthy controls matched for age, sex and body mass index. Plasma concentrations of glucose, PRL, GH, interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-a) were analyzed. RESULTS: RA patients had significantly lower area under the curve (AUC) of PRL (p = 0.049) compared to RA controls. During hypoglycemia double or higher increase of plasma PRL occurred in 5 RA (33%) patients and in 8 RA controls (57%). Using the General Linear Model procedure, no significant differences in PRL or GH responses were observed in patients with RA and AS. TNF-a was higher in patients with RA compared to RA controls (p < 0.05). There was no significant difference in TNF-a concentrations between AS patients and AS controls. IL-6 was higher in RA patients compared to controls (p < 0.05) and in AS patients compared to controls (p < 0.01). Significant positive correlation was found between TNF-a levels and AUC of PRL in AS patients (r = 0.46, p = 0.047), but not in the 2 control groups or in RA patients. CONCLUSION: Our results indicate no upregulated PRL or GH responses to stimulation in premenopausal women with RA or men with AS.

Gonadal and adrenal steroid hormones in plasma and synovial fluid of patients with rheumatoid arthritis.

OBJECTIVES: Gonadal and adrenal steroids were shown to affect multiple immune processes including inflammatory response. These effects were documented, specifically, through an influence on local productions of cytokines and the functions of synovial cells at the site of inflammatory processes. The aim of this study was to investigate the levels of selected hormones in synovial fluid of knee joints of patients with rheumatoid arthritis (RA) and with osteoarthrosis (OS, control group). METHODS: The concentrations of cortisol (CORT), 17-beta-estradiol (ES), dehydroepiandrosterone (DHEA), testosterone (TE), progesterone (PRG), and aldosterone (ALD) were determined in plasma and synovial fluid. RESULTS: Significant positive correlations between the levels in plasma and synovial fluids were observed in hormones ES, PRG, TE, DHEA and ALD. In most hormones, the levels in synovial fluids were similar as in plasma; however, the content of ALD was higher in synovial fluid as compared to plasma. Higher levels of ES (in females), DHEA (in males), and ALD were observed in plasma and synovial fluids of RA patients as compared to OS patients. After adjustment to age, no significant RA vs. OS difference was noted in ES, TE, DHEA, PRG, and CORT in plasma and synovial fluid. Age-adjusted ALD concentration tended to be higher in synovial fluid of RA patients as compared to OS patients. The ratio of ES/TE concentrations in synovial fluid was significantly higher in male RA patients compared to OS group. Also the ES/CS and ES/DHEA ratios in synovial fluid were elevated in RA patients in comparison to controls. CONCLUSIONS: These results demonstrated the prevalence of pro-inflammatory hormones in synovial fluid of RA patients, suggesting the possible role of these steroid hormones in inflammatory processes.