Serum, urinary, and salivary nitric oxide in rheumatoid arthritis: complexities of interpreting nitric oxide measures

Nitric oxide (NO) may play important roles in rheumatoid arthritis (RA). RA is an inflammatory disease involving joints and other systems including salivary glands. To assess NO production in RA patients, we compared levels of serum, urine, and salivary nitrite and nitrate (NOx) in patients with RA and normal subjects, and we examined the relationships of these measures to disease activity. Serum, urine, and NOx levels as well as renal creatinine, NOx clearance and fractional excretion rates were compared in 25 RA patients and 20 age- and gender-matched healthy controls. Subjects were hospitalized for 3 days and placed on a NOxrestricted diet. NOx was assayed using nitrate reductase and the Griess reagent. RA activity was assessed using standard clinical and laboratory measures. While consuming a restricted diet for 3 days to eliminate the effects of oral intake of NOx, 24 hour urinary NOx excretion decreased in both RA patients and healthy controls. Urine NOx levels at all time points were not significantly different between RA patients and normal subjects. Serum NOx levels also decreased during the 3 days of NOx restriction, but RA patients had higher serum NOx levels at all time points compared with the control group. Likewise, serum NOx/creatinine ratios were higher in RA patients than in controls. Although basal salivary flow rate and tear flow were lower in RA patients, salivary NOx levels did not differ between normal and RA subjects. While renal creatinine clearance was not different between the two groups, we found that RA patients had lower renal NOx clearance and lower renal NOx fractional excretion. After correction of p values for multiple comparisons, there were no significant relationships for the RA group between measures of disease activity and the urinary NOx, serum NOx, or urinary NOx clearance. Despite interest in the use of NO as a marker of disease activity, alterations in renal NOx clearance and fractional excretion in RA make it difficult to assess in vivo NO production even with strict dietary restriction of NOx intake.     

Pancreatitis-Associated Protein Does Not Predict Disease Relapse in Inflammatory Bowel Disease Patients

Background

The pancreatitis-associated protein (PAP) is increased in the serum of active inflammatory bowel disease (IBD) patients and its levels seem to be correlated with disease activity. Our aim was to evaluate the usefulness of serum and fecal PAP measurements to predict relapse in patients with inactive IBD.

Materials and Methods

We undertook a 12-month prospective study that included 66 Crohn''s disease (CD) and 74 ulcerative colitis (UC) patients. At inclusion, patients were in clinical remission, defined by a Harvey-Bradshaw (HB) Index≤4 (CD) or a partial Mayo Score (MS)<3 (UC), along with a normal serum C reactive protein (CRP) and fecal calprotectin. Patients were followed every 3 months. Blood and stool samples were collected and a clinical evaluation was performed at each visit. Serum PAP and CRP levels as well as fecal concentrations of PAP and calprotectin were assessed.

Results

Active CD patients had an increased mean serum PAP at the diagnosis of the flare (104.1 ng/ml) and 3 months prior to activity (22.68 ng/ml) compared with patients in remission (13.26 ng/ml), p<0.05. No significant change in serum PAP levels in UC and fecal PAP levels in CD and UC were detected during disease activity. In CD, serum PAP was a poor diagnostic predictor of disease activity, with an AUC of 0.69. In patients in remission, fecal PAP was barely detectable in UC compared with CD patients.

Conclusion

Serum PAP is increased only in active CD patients, but this marker does not predict disease activity. Inactive UC patients have marked low levels of PAP in fecal samples compared with CD patients.     

The relationship between serum prolactin levels and disease activity in patients with Behcet's disease

The aim of this study was to determine the serum levels of prolactin in patients with Behcet's disease and to evaluate its correlation with disease activity. Serum prolactin levels were measured by a chemiluminescence method in 32 patients with Behcet's disease and compared with 20 age-and sex-matched healthy controls. The patients with Behcet's disease were subdivided into two groups according to disease activity: active (18 patients; 13 men and five women, average age 34.0 +/- 6.5 (28-48) years), and inactive (14 patients; 10 men and four women, average age 32.7 +/- 3.1 (22-49 years). Patients with active Behcet's disease had higher serum prolactin levels than the inactive and control groups. Prolactin levels in patients with active Behcet's disease differed significantly from the healthy control subjects (p < 0.05) only, but not the inactive group. Four patients out of 32 (12.5%) Behcet's disease patients showed mild hyperprolactinemia. All four of these cases were from the active Behcet's disease group. Prolactin levels were correlated with ESR (p < 0.05) and CRP (p < 0.05) levels in the active BD group, but not in the inactive BD and control groups. Our results suggest a possible role for this immunoregulatory hormone in the disease expression and pathogenesis of Behcet's disease.     

Elevated urinary leukotriene E4 excretion in patients with ARDS and severe burns.

Increased synthesis of peptidoleukotrienes may occur in a variety of inflammatory diseases. To test this theory, hospitalized patients with a variety of diseases were studied and urine LTE4 quantitated as an index of total body peptidoleukotriene synthesis. 10 patients with ARDS, 7 of which had additional organ involvement, and 5 patients suffering from severe burn injuries were studied. Patients with uncomplicated ARDS excreted approximately 6-fold higher amounts of LTE4 in urine compared to healthy subjects. When ARDS was complicated by multiple organ failure (MOF), urine LTE4 levels were 2- to 150-fold higher than in healthy volunteers. Patients with severe burn injuries had peak urine LTE4 levels which were approximately 20-fold higher than in healthy volunteers. As additional controls, patients with cardiac arrhythmias (absence of inflammatory disease) and patients with uncomplicated pneumonia (localized inflammation) showed normal or mildly elevated urinary LTE4 levels. The urinary LTE4 levels in ARDS patients did not correlate with serum creatinine, bilirubin, or LDH levels, or with the WBC, nor did renal or liver failure by itself predict extremely elevated urinary LTE4 levels. In conclusion, patients with ARDS or ARDS/MOF and patients with severe injuries and sepsis syndrome excrete higher levels of urinary LTE4 than patients healthy volunteers or patients with limited inflammatory disease. In certain situations, urinary LTE4 levels may be useful as a marker of the degree of inflammation.     

Effects of pH, Nitrite, and Ascorbic Acid on Nonenzymatic Nitric Oxide Generation and Bacterial Growth in Urine

Nitrite may be generated by bacteria in urine during urinary tract infections. Acidification of nitrite results in the formation of nitric oxide (NO) and other reactive nitrogen oxides, which are toxic to a variety of microorganisms. We have studied NO formation and bacterial growth in mildly acidified human urine containing nitrite and the reducing agent vitamin C. Urine collected from healthy subjects was incubated in closed syringes at different pH values with varying amounts of nitrite and/or ascorbic acid added. NO generation was measured in headspace gas using a chemiluminescence technique. A similar setup was also used to study the growth of three strains of bacteria in urine. Mildly acidified nitrite-containing urine generated large amounts of NO and this production was greatly potentiated by ascorbic acid. The growth of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus saprophyticus was markedly reduced by the addition of nitrite to acidified urine. This inhibition was enhanced by ascorbic acid. In conclusion, we show that the growth of three common urinary pathogens is markedly inhibited in mildly acidified urine when nitrite is present. The bacteriostatic effect of acidified nitrite is likely related to the release of NO and other toxic reactive nitrogen intermediates. These results may help to explain the well-known beneficial effects of urinary acidification with, e.g., vitamin C in treatment and prevention of urinary tract infection.     

Increased Nitric Oxide Production in Patients with Systemic Sclerosis

Nitric oxide (NO, nitrogen monoxide) is a messenger molecule whose synthesis can be induced by proinflammatory cytokines. Increased production of NO has been reported in various inflammatory and autoimmune diseases. We studied serum nitrite and citrulline as surrogate markers for NO production in patients with systemic sclerosis (SSc) and looked for correlation with extent of disease, disease duration, age, and systemic involvement. Thirty-four patients were studied against 20 controls. The nitrite levels were significantly higher in the disease group (1588.4 +/- 998.2 nmol/ml compared to 327.8 +/- 137.7 nmol/ml; P < 0.001). The citrulline levels of the disease group were also significantly higher (5490.1 +/- 2518.3 nmol/ml compared to 3264.5 +/- 2509.7 nmol/ml in the controls; P = 0.005). There was no significant difference among limited and diffuse subgroups. There was no significant difference in patients with or without arthritis or interstitial lung disease or with other systemic involvement. On multivariate analysis there was a trend toward a rising level of nitrite with worsening lung functions (P = 0.07). Hence, there is evidence of increased NO production in patients with SSc. There is no difference between NO levels in disease subgroups or those with systemic involvement.     

Dietary nitrite inhibits early glomerular injury in streptozotocin-induced diabetic nephropathy in rats.

Increased production of reactive oxygen species (ROS) is a key event leading to microvascular complications, including nephropathy, in diabetes mellitus (DM). Excessive ROS and oxidative stress in DM have been reported to be associated with subsequent impaired nitric oxide (NO) bioavailability. The aim of this study is to examine the beneficial function of dietary nitrite supplementation as an interventional NO donor to attenuate early progression of diabetic nephropathy. To test this hypothesis, male Sprague-Dawley rats were randomly divided into four groups: non-diabetic rats given water with or without nitrite (nitrite-treated or untreated, respectively), and streptozotocin-induced diabetic rats given water with or without nitrite (nitrite-treated or untreated, respectively). After a 4 week experimental period, untreated diabetic rats exhibited significantly higher malondialdehyde (MDA) levels in the kidney compared with untreated non-diabetic rats, accompanied by a reduction in levels of endogenous NO synthase-derived nitrite. However, dietary nitrite supplementation to diabetic rats not only decreased MDA levels but also increased nitrite levels in the kidney to the same levels as in the non-diabetic kidney. These improvements accompanied an improvement in the parameters of glomerular injury, including urinary protein and albumin excretion, histopathological glomerular hypertrophy, and mesangial matrix accumulation. These results indicate that dietary nitrite is effective in the prevention of early diabetic glomerular injury in which NO bioavailability is impaired.     

Inflammation associated anemia and ferritin as disease markers in SLE

Introduction

In a recent screening to detect biomarkers in systemic lupus erythematosus (SLE), expression of the iron storage protein, ferritin, was increased. Given that proteins that regulate the storage, transfer and release of iron play an important role in inflammation, this study aims to determine the serum and urine levels of ferritin and of the iron transfer protein, transferrin, in lupus patients and to correlate these levels with disease activity, inflammatory cytokine levels and markers of anemia.

Methods

A protein array was utilized to measure ferritin expression in the urine and serum of SLE patients and healthy controls. To confirm these results as well as the role of the iron transfer pathway in SLE, ELISAs were performed to measure ferritin and transferrin levels in inactive or active SLE patients and healthy controls. The relationship between ferritin/transferrin levels and inflammatory markers and anemia was next analyzed.

Results

Protein array results showed elevated ferritin levels in the serum and urine of lupus patients as compared to controls, which were further validated by ELISA. Increased ferritin levels correlated with measures of disease activity and anemia as well as inflammatory cytokine titers. Though active SLE patients had elevated urine transferrin, serum transferrin was reduced.

Conclusion

Urine ferritin and transferrin levels are elevated significantly in SLE patients and correlate with disease activity, bolstering previous reports. Most importantly, these changes correlated with the inflammatory state of the patients and anemia of chronic disease. Taken together, altered iron handling, inflammation and anemia of chronic disease constitute an ominous triad in SLE.     

Urinary proteomics revealed prostaglandin H(2)D-isomerase, not Zn-α2-glycoprotein, as a biomarker for active lupus nephritis

Although renal histopathology is the gold standard for the diagnosis and prognosis of lupus nephritis (LN), the invasiveness of renal biopsy warrants the discovery of novel non-invasive diagnostic and prognostic biomarkers. In the present study, urine samples from 10 LN patients (5 active and 5 inactive) were analyzed by two-dimensional gel electrophoresis (2-DE) to screen for potential biomarkers of active LN. Quantitative analysis and statistics revealed 16 protein spots whose levels significantly differed between groups. These proteins were successfully identified by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS). Among these potential candidates, differential levels of urinary Zn-α2-glycoprotein (ZA2G) and prostaglandin H(2)D-isomerase (PGDS) were further validated by enzyme-linked immunosorbent assay (ELISA) in another independent group of 78 subjects, including 30 active LN, 26 inactive LN, 14 non-LN glomerular diseases, and 8 healthy normal individuals. Whereas ZA2G levels were elevated in urine of patients with active LN and non-LN glomerular diseases, PGDS was elevated only in the urine of the active LN group. Urinary PGDS, not ZA2G, may serve as a biomarker for active LN and upon validation in larger studies, may become the non-invasive test to evaluate the disease activity in future management of LN.     

Increased asymmetric dimethylarginine (ADMA) dimethylaminohydrolase (DDAH) activity in childhood hypercholesterolemia type II

Asymmetric dimethylarginine (ADMA) systemic concentrations are elevated in hypercholesterolemic adults and contribute to nitric oxide (NO) dependent endothelial dysfunction. Decreased activity of the key ADMA-hydrolyzing enzyme dimethylarginine dimethylaminohydrolase (DDAH) may be involved. Yet, the ADMA/DDAH/NO pathway has not been investigated in childhood hypercholesterolemia. We studied 64 children with hypercholesterolemia type II (HCh-II) and 54 normocholesterolemic (NCh) children (mean ± SD; age, years: 11.1 ± 3.5 vs. 11.9 ± 4.6). Plasma and urine ADMA was measured by GC-MS/MS. Dimethylamine (DMA), the ADMA metabolite, creatinine, nitrite and nitrate in urine were measured by GC-MS. The DMA/ADMA molar ratio in urine was calculated to estimate whole body DDAH activity. ADMA plasma concentration (mean ± SD; nM: 571 ± 85 vs. 542 ± 110, P = 0.17) and ADMA urinary excretion rate (mean ± SD: 7.1 ± 2 versus 7.2 ± 3 μmol/mmol creatinine, P = 0.6) were similar in HCh-II and NCh children. Both DMA excretion rate [median (25th-75th percentile): 56.3 (46.4-109.1) vs. 45.2 (22.2-65.5) μmol/mmol creatinine, P = 0.0004] and DMA/ADMA molar ratio [median (25th-75th percentile): 9.2 (6.0-16.3) vs. 5.4 (3.8-9.4), P = 0.0004] were slightly but statistically significantly increased in HCh-II children compared to NCh children. Plasma and urinary nitrite and nitrate were similar in both groups. In HCh-II whole body DDAH activity is elevated as compared to NCh. HCh-II children treated with drugs for hypercholesterolemia had lower plasma ADMA levels than untreated HCh-II or NCh children, presumably via increased DDAH activity. Differences between treated and untreated HCh-II children were not due to differences in age. In conclusion, HCh-II children do not have elevated ADMA plasma levels, largely due to an apparent increase in DDAH activity. While this would tend to limit development of endothelial dysfunction, it is not clear whether this might be medication-induced or represent a primary change in HCh-II children.     

Dietary nitrite restores NO homeostasis and is cardioprotective in endothelial nitric oxide synthase-deficient mice

Endothelial production of nitric oxide (NO) is critical for vascular homeostasis. Nitrite and nitrate are formed endogenously by the stepwise oxidation of NO and have, for years, been regarded as inactive degradation products. As a result, both anions are routinely used as surrogate markers of NO production, with nitrite as a more sensitive marker. However, both nitrite and nitrate are derived from dietary sources. We sought to determine how exogenous nitrite affects steady-state concentrations of NO metabolites thought to originate from nitric oxide synthase (NOS)-derived NO as well as blood pressure and myocardial ischemia-reperfusion (I/R) injury. Mice deficient in endothelial nitric oxide synthase (eNOS-/-) demonstrated decreased blood and tissue nitrite, nitrate, and nitroso proteins, which were further reduced by low-nitrite (NOx) diet for 1 week. Nitrite supplementation (50 mg/L) in the drinking water for 1 week restored NO homeostasis in eNOS-/- mice and protected against I/R injury. Nitrite failed to alter heart rate or mean arterial blood pressure at the protective dose. These data demonstrate the significant influence of dietary nitrite intake on the maintenance of steady-state NO levels. Dietary nitrite and nitrate may serve as essential nutrients for optimal cardiovascular health and may provide a novel prevention/treatment modality for disease associated with NO insufficiency.     

Can measuring urinary biomarkers improve the management of lupus nephritis?

Although renal biopsy is the most accurate way of assessing renal inflammation in patients with lupus nephritis (LN), the technique is invasive and cannot be performed frequently. Currently used blood and urine biomarkers have limited utility in monitoring the activity of nephritis. In a previous issue of Arthritis Research and Therapy, Singh and colleagues showed that measuring urinary levels of vascular cell adhesion molecule 1 could be useful in both diagnosing and monitoring LN. These levels are higher in patients with lupus than controls, are higher in lupus patients who have active renal disease compared with those who do not, and correlate significantly with the histological activity index in renal biopsies of patients with LN.Lupus nephritis (LN) is one of the most severe forms of systemic lupus erythematosus (SLE). If not treated adequately, the disease can result in renal failure or death. In its early stages, LN may be almost asymptomatic and picked up only by carrying out routine blood and urine tests. Measurements of urine protein are especially helpful in this regard. In a previous issue of Arthritis Research and Therapy, Singh and colleagues described tests for three different urinary biomarkers in patients with SLE, investigating which is likely to be most helpful for monitoring renal disease activity [1].Treatment of LN was revolutionized by the introduction of combined corticosteroid/cyclophosphamide regimes and has advanced more recently with the replacement of cyclophosphamide by mycophenolate in many cases [2]. Early diagnosis and treatment has a major impact on clinical outcomes in patients with LN [3], but can also cause treatment-related adverse effects. Being able to identify exactly when treatment is needed during the course of the disease and when maintenance treatment needs to be increased is therefore important. The most accurate way to assess nephritis in patients with SLE is by carrying out a renal biopsy. The histological type of nephritis can be defined and the degree of inflammation can be quantified using an activity index [4]. A high activity index signifies active yet reversible disease, whereas the chronicity index shows irreversible damage.Renal biopsy is an invasive procedure, however, which cannot be repeated whenever a flare of renal lupus is suspected. We would therefore like to have biomarkers, measurable in blood or urine, which rise and fall with renal disease activity and are closely associated with the degree of inflammation in the kidney. Blood markers such as anti-double-stranded DNA, anti-nucleosome and anti-α-actinin antibodies have been studied. There is some evidence that increases in these markers are associated with renal disease activity [5], as measured by indices such as blood albumin and urine protein, but very little evidence for their association with renal biopsy scores.Given that the kidney is the main site of inflammation in LN, however, biomarkers in urine may reflect this inflammation more closely than those in the blood. There has been some interest in using urinary neutrophil gelatinase-associated lipocalin as a marker of renal inflammation in SLE. Studies have shown that urinary (but not serum) neutrophil gelatinase-associated lipocalin levels correlate with measures of renal disease activity and that a rise in urinary neutrophil gelatinase-associated lipocalin at one visit predicts flare of nephritis at the next visit [6,7]. These studies were cross-sectional and did not look at the association between urinary neutrophil gelatinase-associated lipocalin and renal histology.The current paper by Singh and colleagues is also a cross-sectional study but includes data on renal histology and compares three different urinary assays; CXCL16, monocyte chemoattractant protein 1 (MCP-1) and vascular cell adhesion molecule 1 (VCAM-1) [1]. Since all three of these molecules can play a role in the recruitment of inflammatory cells to the nephritic kidney, it would be entirely reasonable to predict that their levels would rise in the urine when active renal inflammation is at its height. The authors review both clinical and murine evidence supporting this prediction for each of these markers [8]. In this study they report creatinine-normalized urinary CXCL16, MCP-1 and VCAM-1 levels in 73 patients with SLE, 13 healthy volunteers and 22 patients with other forms of glomerulonephritis.The study results showed some evidence of utility for all three of these urinary markers. All markers were elevated in patients with SLE compared to healthy controls and correlated with the level of proteinuria. However, the comparison with healthy controls is complicated by the fact that 92% of these controls were Asian whereas none of the patients were. Disease activity in the study was assessed using the Systemic Lupus Erythematosus Disease Activity Index and correlated with MCP-1 and VCAM-1 but not CXCL16. When patients were defined as having active or inactive renal disease, on the basis of the renal components of the Systemic Lupus Erythema tosus Disease Activity Index score alone, MCP-1 and VCAM-1 but not CXCL16 distinguished those two groups. However, this result was based on a relatively small number of patients with inactive renal disease (n = 14), probably because there was a low threshold for considering a patient to have active renal disease - being positive for any single criterion of hematuria, pyuria, proteinuria or casts was sufficient. This difficulty in defining active LN based on disease activity measures is common to many papers of this type and is a major reason why the availability of renal biopsy data from the date of the urine sample in 24 of these patients is so important. These histological data provide a direct objective measure of nephritis and particularly support the utility of urinary VCAM-1 as a biomarker, since only VCAM-1 was significantly correlated with the activity index and also differentiated class IV LN from the other histological types.This study thus provides compelling evidence that measuring urinary VCAM-1 could be an important new biomarker in patients with LN, and some evidence supporting urinary CXCL16 and MCP-1. Investigation into whether an index combining the values of all three markers, along the lines of the serum chemokine index reported by Bauer and colleagues [9], would have more potential to distinguish active LN from inactive LN would be interesting. As suggested by the authors, however, the most important thing is to study levels of these urinary markers longitudinally in patients with LN to see whether they predict changes in activity of nephritis in individual patients over time.     

血清免疫炎症相关蛋白复合物、25-羟维生素D、脂肪细胞因子与炎症性肠病患者疾病活动性和肠道菌群的相关性研究

摘要 目的：探讨血清免疫炎症相关蛋白复合物（IIRPCs）、25-羟维生素D[25（OH）D]、脂肪细胞因子（Chemerin）与炎症性肠病（IBD）患者疾病活动性和肠道菌群的相关性。方法：选取2020年12月～2021年12月我院收治的150例IBD患者,其中溃疡性结肠炎（UC）组65例、克罗恩病（CD）组85例,另取同期健康体检者70例作为对照组,检测并比较三组血清IIRPCs、25（OH）D、Chemerin水平。此外,UC组和CD组患者分别根据克罗恩病活动指数（CDAI）和溃疡性结肠炎的改良梅奥（Mayo）评分分为活动期组、缓解期组,分别比较UC组和CD组患者活动期组与缓解期组间的血清IIRPCs、25（OH）D、Chemerin水平、肠道菌群差异,并作相关性分析。结果：IBD患者的血清IIRPCs、Chemerin水平高于对照组,而25（OH）D水平低于对照组（P＜0.05）;UC组血清IIRPCs、Chemerin水平高于CD组,25（OH）D水平低于CD组（P＜0.05）。活动期UC、CD患者的血清IIRPCs、Chemerin水平以及肠球菌、肠杆菌、酵母菌、拟杆菌数量均高于缓解期UC、CD患者,而血清25（OH）D水平以及双歧杆菌、乳酸杆菌数量均低于缓解期UC、CD患者（P＜0.05）。Pearsonn相关性分析结果显示,UC、CD患者的血清IIRPCs、Chemerin水平与肠球菌、肠杆菌、酵母菌、拟杆菌数量呈正相关,与双歧杆菌、乳酸杆菌数量呈负相关（P＜0.05）;UC、CD患者的血清25（OH）D水平与肠球菌、肠杆菌、酵母菌、拟杆菌数量呈负相关,与双歧杆菌、乳酸杆菌数量呈正相关（P＜0.05）。结论：血清IIRPCs、25（OH）D、Chemerin与IBD患者的疾病活动性、肠道菌群有关,检测上述指标对评估IBD患者病情程度有一定价值。     

Investigation of protein oxidation and lipid peroxidation in patients with rheumatoid arthritis

We aimed to determine the importance of neutrophil activation and the source of oxidative stress in the pathogenesis of rheumatoid arthritis (RA) by quantification of advanced oxidation protein products (AOPP) and total thiol levels as markers of oxidative protein damage, malondialdehyde (MDA) levels as a marker of lipid peroxidation and myeloperoxidase (MPO) activity as a marker of neutrophil activation in patients with RA. Fifty-seven rheumatoid arthritis patients were included in the study and sub-grouped according to disease activity (active, n = 31; inactive, n = 26) and compared with healthy controls (n = 25). Serum MPO activity, AOPP, MDA, and thiol levels were measured by an enzymic spectrophotometric method. Serum MPO activity (p < 0.001), AOPP (p < 0.001), MDA (p < 0.001) and levels of thiol (p < 0.002), were higher in the patient group than the controls. Active and inactive RA groups were compared with the control group and there were significant differences between each parameter. MPO activity, AOPP, MDA and thiol levels were significantly higher in both active and inactive RA patients than the controls. On the other hand, when a comparison was made between active and the inactive stage, a statistically significant difference was present only in MDA (p < 0.05) and AOPP levels (p < 0.05). There was also a significant positive correlation between all parameters. These data strongly suggest that neutrophils, which constitute the most important source of chlorinated oxidants due to their high MPO content, may be involved in serum AOPP formation and therefore the production of a novel class of pro-inflammatory mediators of oxidative stress in RA patients and that protein oxidation could play an important role in the pathogenesis of RA as does lipid peroxidation.     

Periostin as a Tissue and Urinary Biomarker of Renal Injury in Type 2 Diabetes Mellitus

Background

Improving the early detection of diabetic nephropathy remains a great challenge in disease management. Periostin is a marker of renal tubular injury and related to progressive kidney injury in animal models of chronic kidney disease. The clinical implications of urinary periostin activities in patients with type 2 diabetes have not been evaluated.

Methods

Urine samples were obtained from 30 healthy volunteers and 328 type 2 diabetic patients with normoalbuminuria (n=114), microalbuminuria (n=100) and macroalbuminuria (n=114). The excretion levels of urinary periostin were quantified with enzyme-linked immunosorbent assay. Immunohistochemical periostin expression was determined in kidney tissues from overt diabetic nephropathy.

Results

Increased periostin expression in glomeruli and tubular epithelium in diabetic renal pathology was observed. Urinary periostin levels were significantly elevated in the patients of the normoalbuminuria [3.06 (IQR: 1.12, 6.77) ng/mgCr], microalbuminuria [8.71 (IQR: 5.09, 19.29) ng/mgCr] and macroalbuminuria [13.58 (IQR: 3.99, 16.19) ng/mgCr] compared with healthy controls [1.15 (IQR: 0.60, 1.63) ng/mgCr] (P<0.01).Increased urine periostin level significantly correlated with aging, high albuminuria and decline of GFR. Urine periostin ELISA also demonstrated high performance for the diagnosis of established normoalbuminuric, microalbuminuric and macroalbuminuric type 2 diabetes (AUC 0.78 (95%CI, 0.71 to 0.86), 0.99 (95%CI, 0.98 to 1.00) and 0.95 (95%CI, 0.91 to 0.98), respectively).

Conclusion

The study indicates that increased urine periostin levels can be detected in patients with type 2 diabetes before the onset of significant albuminuria. Urinary periostin is an associated renal derangement in patients with established diabetic nephropathy and it may be used as an early marker of diabetic renal injury.     

The Association Between Effective Dose of Magnesium and Mild Compulsive Exercise on Spatial Learning,Memory, and Motor Activity of Adult Male Rats

The relationship of minerals and trace elements with inflammatory bowel disease (IBD) is complex. Alterations in their metabolism can be induced by the diseases and their complications. To study the role of trace elements in IBD patients’ serum zinc and copper and their related enzymes, including superoxide dismutase (SOD), activity were measured in patients with IBD patients as well as in healthy subjects. In addition, the correlation between serum trace element levels, albumin, total protein, urea level, copper/zinc ratio, and disease activity (DA) was determined in these subjects. Serum samples were obtained from 35 patients (19 ulcerative colitis (UC) and 16 Crohn’s disease (CD)) in the active phase of the disease and 30 healthy control subjects. Serum levels of zinc, copper, SOD activity, albumin, total protein, and urea were measured. The results were compared between the two groups using independent Student’s t test in statistical analysis. Serum levels of zinc, SOD activity, albumin, and total protein were significantly lower (P < 0.05) in patients than controls, while serum urea level was significantly higher in patients compared to controls. Copper concentrations did not differ between patients with IBD (mean ± SD, 58.8 ± 20.7 μg/d) and controls (55.57 ± 12.6 μg/d). Decreased levels of zinc and SOD activity are associated with increased inflammatory processes indicating inappropriate antioxidant system in patients with IBD. Additionally, lower levels of albumin and total protein with higher level of urea reflect metabolic problems in liver system.     

Inorganic nitrate is a possible source for systemic generation of nitric oxide

Nitrate and nitrite have been considered stable inactive end products of nitric oxide (NO). While several recent studies now imply that nitrite can be reduced to bioactive NO again, the more stable anion nitrate is still considered to be biologically inert. Nitrate is concentrated in saliva, where a part of it is reduced to nitrite by bacterial nitrate reductases. We tested if ingestion of inorganic nitrate would affect the salivary and systemic levels of nitrite and S-nitrosothiols, both considered to be circulating storage pools for NO. Levels of nitrate, nitrite, and S-nitrosothiols were measured in plasma, saliva, and urine before and after ingestion of sodium nitrate (10 mg/kg). Nitrate levels increased greatly in saliva, plasma, and urine after the nitrate load. Salivary S-nitrosothiols also increased, but plasma levels remained unchanged. A 4-fold increase in plasma nitrite was observed after nitrate ingestion. If, however, the test persons avoided swallowing after the nitrate load, the increase in plasma nitrite was prevented, thereby illustrating its salivary origin. We show that nitrate is a substrate for systemic generation of nitrite. There are several pathways to further reduce this nitrite to NO. These results challenge the dogma that nitrate is biologically inert and instead suggest that a complete reverse pathway for generation of NO from nitrate exists.     

Oxidative stress in neonates: evaluation using specific biomarkers

Increased oxidative stress has been implicated in pathogenesis of serious diseases in neonates. We measured urinary levels of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative DNA damage), acrolein-lysine adduct (a marker of lipid peroxidation and oxidative protein damage), and nitrite/nitrate (a marker of endogenous nitric oxide formation) in one-month-old neonates to examine the status of oxidative stress and its relationship to the degree of prematurity and clinical condition in neonates. Study subjects comprised three groups: healthy term neonates, clinically stable preterm neonates requiring no supplemental oxygen, and clinically sick preterm neonates requiring supplemental oxygen and ventilator support. Urinary levels of 8-hydroxy-2'-deoxyguanosine and acrolein-lysine adduct were significantly higher in sick preterm neonates than those of stable preterm and healthy term neonates. In the sick preterm group, neonates developing active retinopathy showed significantly higher levels of acrolein-lysine adduct than the other neonates without retinopathy. There were no significant differences in both urinary markers of oxidative stress between stable preterm and healthy term neonates. The urinary nitrite/nitrate levels were not significantly different among the three groups, suggesting no difference in endogenous nitric oxide formation. Collectively, these results provide evidence of augmentation of oxidative damage to DNA, lipids and proteins, especially in clinically sick preterm neonates.     

Endogenous nitric oxide formation correlates negatively with circulating matrix metalloproteinase (MMP)-2 and MMP-9 levels in black subjects

Deficient formation of endogenous nitric oxide (NO) contributes to cardiovascular diseases, and this may be associated with increased circulating levels of matrix metalloproteinase-9 (MMP-9), as previously shown in white subjects. Because interethnic differences exist with respect to risk factors, prevalence, and severity of cardiovascular diseases, we designed this study to examine whether the circulating levels of nitrites (a marker of endogenous NO formation) are associated with the plasma levels of MMP-9 and MMP-2 in healthy black subjects. We studied 198 healthy subjects self-reported as blacks not taking any medications. Venous blood samples were collected and plasma and whole blood nitrite levels were measured using an ozone-based chemiluminescence assay. Plasma MMP-2 and MMP-9 levels were determined by gelatin zymography. We found a positive correlation between plasma MMP-9 and MMP-2 levels (P < 0.0001, rs = 0.556). Interestingly, we found a negative relationship between the plasma MMP-9 levels and the plasma or whole blood nitrites levels (P = 0.04, rs = -0.149; and P < 0.0001, rs = -0.349, respectively). In parallel, we found similar negative relationships between plasma MMP-2 levels and plasma or whole blood nitrites levels (P = 0.02, rs = -0.172; and P < 0.0001, rs = -0.454, respectively). This is the first study to show that endogenous nitric oxide formation correlates negatively with the circulating levels of both MMP-2 and MMP-9 in black subjects. Our findings suggest a mechanistic link between deficient NO formation and increased MMPs levels, which may promote cardiovascular diseases.     

The Role of Arginine–Nitric Oxide Pathway in Patients with Alzheimer Disease

There is a reciprocal regulation of arginase and nitric oxide synthase in l-arginine-metabolizing pathways. There are various evidences of the role of nitric oxide in several neuropsychiatric disorders including Alzheimer’s disease. However, there is no study that has investigated the role of arginase as an important part of the arginine regulatory system affecting nitric oxide synthase activity in Alzheimer’s disease. This study aims to investigate arginase, manganese (a cofactor of arginase), and total nitrite levels (a metabolite of NO) and their relationship to the arginine–NO pathway in patients with Alzheimer’s disease. Arginase activities, Mn, and total nitrite levels were measured in plasma from 47 patients with Alzheimer’s disease and 43 healthy control subjects. Plasma arginase activities and manganese were found to be significantly lower and total nitrite level higher in patients with Alzheimer’s disease compared with controls. Our results suggest that the arginine–NO pathway is involved in the pathogenesis of Alzheimer’s disease.