Activation of stress-activated protein kinase in osteoarthritic cartilage: evidence for nitric oxide dependence

OBJECTIVE: We have demonstrated in bovine chondrocytes that nitric oxide (NO) mediates IL1 dependent apoptosis under conditions of oxidant stress. This process is accompanied by activation of c-Jun NH2-terminal kinase (JNK; also called stress-activated protein kinase). In these studies we examined activation of JNK in explant cultures of human osteoarthritic cartilage obtained at joint replacement surgery and we characterized the role of peroxynitrite to act as an upstream trigger. DESIGN: A novel technique to isolate chondrocyte proteins (<10% of total cartilage protein) from cartilage specimens was developed. It was used to analyse JNK activation by a western blot technique. To examine the hypothesis that chondrocyte JNK activation is a result of increased peroxynitrite, in vitro experiments were performed in which cultured chondrocytes were incubated with this oxidant. RESULTS: Activated JNK was detected in the cytoplasm of osteoarthritis (OA) affected chondrocytes but not in that of controls. In vitro, chondrocytes produce NO and superoxide anion. IL-1 (48 h), which induces nitric oxide synthase, resulted in an activation of JNK; this effect was reversed by N-monomethylarginine (NMA). TNFalpha treated chondrocytes at 48 h produce superoxide anion (EPR method). Exposure of cells to peroxynitrite led to an accumulation of intracellular oxidants, in association with JNK activation and cell death by apoptosis. CONCLUSION: We suggest that JNK activation is among the IL-1 elicited responses that injure articular chondrocytes and this activation of JNK is dependent on intracellular oxidant formation (including NO peroxynitrite). In addition, the extraction technique here described is a novel method that permits the quantitation and study of proteins such as JNK involved in the signaling pathways of chondrocytes within osteoarthritic cartilage.     

Neuronal nitric oxide synthase: its role and regulation in macula densa cells

Macula densa (MD) cells detect changes in distal tubular sodium chloride concentration ([NaCl](L)), at least in part, through an apical Na:2Cl:K co-transporter. This co-transporter may be a site for regulation of tubuloglomerular feedback (TGF), and recently angiotensin II (Ang II) was shown to regulate the MD Na:2Cl:K co-transporter. In addition, nitric oxide (NO) produced via neuronal NO synthase (nNOS) in MD cells attenuates MD-TGF signaling. This study investigated [NaCl](L)-dependent MD-NO production, the regulation of co-transporter activity by NO, and the possible interaction of NO with Ang II. MD cell Na(+) concentration ([Na(+)](i)) and NO production were measured using sodium-binding benzofuran isophthalate and 4-amino-5-methylamino-2',7'-difluorescein diacetate, respectively, using fluorescence microscopy. Na:2Cl:K co-transport activity was assessed as the initial rate of increase in [Na(+)](i) when [NaCl](L) was elevated from 25 to 150 mM. 10(-4) M 7-nitroindazole, a specific nNOS blocker, significantly increased by twofold the initial rate of rise in [Na(+)](i) when [NaCl](L) was increased from 25 to 150 mM, indicating co-transporter stimulation. There was no evidence for an interaction between the stimulatory effect of Ang II and the inhibitory effect of NO on co-transport activity, and, furthermore, Ang II failed to alter MD-NO production. NO production was sensitive to [NaCl](L) but increased only when [NaCl](L) was elevated from 60 to 150 mM. These studies indicate that MD-NO directly inhibits Na:2Cl:K co-transport and that NO and Ang II independently alter co-transporter activity. In addition, generation of MD-NO seems to occur only at markedly elevated [NaCl](L), suggesting that NO may serve as a buffer against high rates of MD cell transport and excessive TGF-mediated vasoconstriction.     

Exhaled nitric oxide and asthma: complex interactions between atopy, airway responsiveness, and symptoms in a community population of children

BACKGROUND: Exhaled nitric oxide (FE(NO)) is raised in asthmatic children, but there are inconsistencies in the relationship between FE(NO) and characteristics of asthma, including atopy, increased airway responsiveness (AR), and airway inflammation. The aim of this study was to investigate the relationship between FE(NO) and asthma, atopy, and increased AR in children. METHODS: One hundred and fifty five children (79 boys) of mean age 11.5 years underwent an assessment that included FE(NO) measurements, spirometric tests, inhaled histamine challenge, and a skin prick test. Blood was collected for eosinophil count. Current and past asthma like symptoms were determined by questionnaire. RESULTS: In multiple linear regression analyses FE(NO) was associated with atopy (p<0.001), level of AR (p = 0.005), blood eosinophil count (p = 0.007), and height (p = 0.002) but not with physician diagnosed asthma (p = 0.1) or reported wheeze in the last 12 months (p = 0.5). Separate regression models were conducted for atopic and non-atopic children and associations between FE(NO) and AR, blood eosinophils and height were only evident in atopic children. Exhaled NO was raised in children with a combination of atopy and increased AR independent of symptoms. CONCLUSION: Raised FE(NO) seems to be associated with an underlying mechanism linking atopy and AR but not necessarily respiratory symptoms.     

Carbon monoxide lung diffusion capacity improves risk stratification in patients without airflow limitation: evidence for systematic measurement before lung resection.

OBJECTIVE: In many centers, carbon monoxide lung diffusion capacity (DLCO) is still not routinely measured in all patients but only in patients with airflow limitation. The objective of the study was to assess the degree of correlation between forced expiratory volume in 1s (FEV1) and DLCO, and verify whether a low predicted postoperative DLCO (ppoDLCO) could have a role in predicting complications in patients without airflow limitation. METHODS: We analyzed 872 patients submitted to lung resection between January 2000 and December 2004 in two units measuring systematically DLCO before operation. Correlation between FEV1 and DLCO was assessed in the entire dataset and in different subsets of patients. A number of variables were then tested for a possible association with postoperative cardiopulmonary complications in patients with FEV1>80% by univariate analysis. Variables with p<0.10 at univariate analysis were used as independent variables in a stepwise logistic regression analysis (dependent variable: presence of cardiopulmonary morbidity), which was in turn validated by bootstrap analysis. RESULTS: The correlation coefficients between FEV1 and DLCO in the entire dataset and in different subsets of lung resection candidates (stratified by age, gender, cause of operation, airflow limitation) were all below 0.5, showing a modest degree of correlation. Two hundred and nineteen of the 508 patients (43%) with FEV1>80% had DLCO<80%. Moreover, in patients with FEV1>80%, logistic regression analysis showed that ppoDLCO<40% was a significant and reliable predictor of postoperative complications (p=0.004). CONCLUSION: The modest correlation between FEV1 and DLCO and the capacity of ppoDLCO to discriminate between patients with and without complications in subjects with a normal FEV1, warrants the routine measurement of DLCO in all candidates for lung resection, irrespective of their FEV1 value, in order to improve surgical risk stratification.     

Regulation of inducible nitric oxide synthase expression in advanced glycation end product-stimulated raw 264.7 cells: the role of heme oxygenase-1 and endogenous nitric oxide

Advanced glycation end products (AGEs) are closely linked to the development of diabetic atherosclerosis. The current study examines the induction of inducible nitric oxide (NO) synthase (iNOS) and heme oxygenase (HO)-1 expression by AGEs, as well as the signaling pathways involved and the interplay between these two enzymes. The stimulation of RAW 264.7 cells with 6.64 or 33.2 microg/ml AGEs leads to HO-1 protein expression, iNOS protein expression, and nitrite accumulation. AGEs lead to the phosphorylation of p42/44 and p38 mitogen-activated protein kinase (MAPK). The inhibition of p42/44 MAPK and protein kinase C prevented, whereas inhibition of p38 MAPK augmented, AGE-induced nitrite release and iNOS expression. In contrast, HO-1 expression was downregulated by inhibition of p38 MAPK. Furthermore, the expression of both proteins was prevented by coincubation with acetovanillone (NADPH oxidase inhibitor). AGE-induced iNOS expression was negatively regulated by stimulation of HO-1 expression with cadmium chloride or endogenous NO. Tin-protoporphyrin IX (HO-1 inhibitor) partially reversed the cadmium chloride-mediated downregulation of iNOS expression. The current study demonstrates that multiple signaling molecules are involved in AGE-stimulated iNOS and HO-1 expression. There also exists a downregulation of iNOS by its own product as well as the products of HO-1.     

Inhibition of nitric oxide synthase activity and nitric oxide-dependent calcium influx in renal epithelial cells by cyclic adenosine monophosphate: implications for cell injury.

Cell injury frequently occurs in the setting of tissue destruction and inflammation and is associated with a rise in intracellular calcium (Cai) and increased NO production. The mechanisms that trigger rises in Cai and NO during cell injury are not fully defined, but they may involve activation of G protein-coupled receptors for substances such as bradykinin, Ang II, thromboxane, and thrombin. These receptors act through G proteins from different families that have distinct functions. Receptors for bradykinin and Ang II act through members of the G alpha i and G alpha q families, whereas receptors for thrombin and thromboxane act through members of the G alpha i, G alpha q, and G alpha 12/13 families. These G proteins cooperate to regulate Cai and NO in epithelial cells through distinct mechanisms. In a number of experimental settings, activators of the adenylyl cyclase system reduce the severity of cell injury. To understand the mechanisms by which G protein-dependent signaling systems may contribute to cell injury and to define the role of adenylyl cyclase in ameliorating cell injury, the effects of adenylyl cyclase on bradykinin-stimulated Ca influx and NO in cultured renal epithelial cells that stably overexpress G alpha q and G alpha 13 were studied. This system allowed for the separation of different components of the signals initiated by receptors for thromboxane and thrombin. G alpha 13 increased bradykinin-stimulated Ca influx by a mechanism that depends on NO and cGMP. The increased Ca influx was blocked by inhibitors of NO synthase and guanylyl cyclase and by activation of adenylyl cyclase. NO production was inhibited by activators of cAMP-dependent protein kinase, which indicated that cAMP blocks Ca influx by inhibiting NO production. Expression of G alpha q, the G protein that regulates phospholipase C, also increased bradykinin-stimulated Ca influx, but by an NO, cGMP-independent mechanism that was insensitive to inhibition by adenylyl cyclase. The authors conclude that Ca influx is modulated by NO-dependent and independent mechanisms, and that to the extent that increased NO production contributes to increased Ca influx and cell injury, cell injury may be reduced by agents that activate adenylyl cyclase.     

Increased circulating nitric oxide in young patients with type 1 diabetes and persistent microalbuminuria: relation to glomerular hyperfiltration

Hyperglycemia has been causally linked to vascular and glomerular dysfunction by a variety of biochemical mechanisms, including a glucose-dependent abnormality in nitric oxide (NO) production and action. NO is a candidate for mediating hyperfiltration and the increased vascular permeability induced by diabetes. Serum nitrite and nitrate (NO2-+ NO3-) concentrations were assessed as an index of NO production in 30 adolescents and young adults with type 1 diabetes, 15 with and 15 without microalbuminuria (albumin excretion rate [AER] between 20 and 200 microg/min), compared with a well-balanced group of healthy control subjects. In all subjects, glomerular filtration rate (GFR) was determined by radionuclide imaging. Our study showed that NO2- + NO3- serum content and GFR values were significantly higher in microalbuminuric diabetic patients than in the other 2 groups. GFR was significantly and positively related to AER levels (r2 = 0.75, P < 0.0001), whereas NO2- + NO3- serum content was independently associated with both AER and GFR values (beta = 2.086, P = 0.05, beta = 1.273, P = 0.0085, respectively), suggesting a strong link between circulating NO, glomerular hyperfiltration, and microalbuminuria in young type 1 diabetic patients with early nephropathy. Interestingly, mean HbA1c, serum concentration was significantly higher in microalbuminuric than in normoalbuminuric diabetic subjects (P < 0.05) and was independently associated with AER values, suggesting a role for chronic hyperglycemia in the genesis of diabetic nephropathy. Moreover, HbA1c serum concentration was significantly and positively related to NO2 + NO3 serum content (r2 = 0.45, P = 0.0063) and GFR values (r2 = 0.57, P = 0.0011), suggesting that chronic hyperglycemia may act through a mechanism that involves increased NO generation and/or action. In conclusion, we suggest that in young type 1 diabetic patients with early nephropathy, chronic hyperglycemia is associated with an increased NO biosynthesis and action that contributes to generating glomerular hyperfiltration and persistent microalbuminuria.     

缺血预处理对糖尿病大鼠在体心肌NO-cGMP表达的影响

目的 通过糖尿病大鼠心肌在缺血预处理(IPC)后环磷酸鸟苷(cGMP)及一氧化氮(NO)、一氧化氮合酶(NOS)表达的变化,探讨糖尿病抑制IPC心肌保护作用的机制.方法 取糖尿病及非糖尿病SD大鼠各30只,各分为3组(每组10只).(1)假手术组(Sham组):开胸后穿线做套环,但不收紧结扎线;持续155 min,全程旷置作为基础对照.(2)缺血再灌注组(I/R组):穿线平衡35 min后,持续收紧结扎造成缺血30 min,放松后再灌注90 min.(3)IPC组:穿线平衡35 min后,缺血5min,再灌注5 min,反复3次,而后重复I/R组操作.比较各组血清肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)及乳酸脱氢酶(LDH)的变化,心肌组织丙二醛(MDA)含量和超氧化物岐化酶(SOD)活性及心肌组织cGMP、NO、NOS含量的变化.电镜标本行线粒体Flameng评分.结果 非糖尿病IPC组与I/R组比较,心肌酶漏出明显减少,MDA含量明显降低,SOD含量明显增加,线粒体损伤明显减轻,cGMP、NO、NOS含量明显增加(P＜0.05);而IPC在糖尿病大鼠未表现出明显心肌保护作用,cGMP、NO、NOS含量无明显增加(P＞0.05).结论 糖尿病抑制IPC的心肌保护作用,其机制可能与糖尿病大鼠心肌NO-cGMP通路表达受抑制有关.     

The spread of cell death from impact damaged cartilage: lack of evidence for the role of nitric oxide and caspases

Over 21 days in culture, cell death spreads, both radially and transversely, from loaded to surrounding cartilage. This spread was prevented by physical separation and separate culture post-impact. OBJECTIVE: One aim was to determine if nitric oxide (NO) is the intercellular signal mediating cell death. Another aim was to clarify the nature of the cell death, whether caspase mediated apoptosis or necrosis. DESIGN: Cyclic impacts were applied to the central 2 mm core of 4 mm canine articular cartilage discs. Post-impact culturing was for 21 days in the presence or absence of the iNOS inhibitor, L-NAME, or the broad-spectrum caspase inhibitor, Z-VAD FMK. Cell death was quantified using the TUNEL assay. Culture media were collected every 2 days for measurements of glycosaminoglycan (GAG) and NO release. RESULTS: Cell death spread from the loaded core into the surrounding ring over 21 days in culture. Although L-NAME significantly reduced nitrite release into the culture media of both loaded and control cartilage, the spread of cell death was not prevented. Neither was the spread of cell death prevented by Z-VAD FMK. CONCLUSIONS: These data indicate that NO is not acting as an intercellular signalling factor in this in vitro system and that the cell death post-impact is not caspase mediated.     

Inducible nitric oxide synthase mediates delayed cardioprotection induced by morphine in vivo: evidence from pharmacologic inhibition and gene-knockout mice

BACKGROUND: It is not known whether morphine induces delayed cardioprotection against ischemia and reperfusion. The authors measured the delayed preconditioning induced by morphine and determined the role of inducible nitric oxide synthase (iNOS) in mediating this effect using a pharmacological inhibitor and iNOS gene-knockout mice. METHODS: Adult male wild-type and iNOS gene-knockout (B6, 129) mice were treated with morphine (0.3 or 0.1 mg/kg intraperitoneal) or saline. Twenty-four hours later, mice were subjected to 45 min of coronary artery occlusion followed by 120 min of reperfusion. S-methylthiourea sulfate (3 mg/kg, intraperitoneal) was given 30 min before the occlusion to block iNOS. Infarct size as a percentage of the area at risk was determined by triphenyltetrazolium chloride staining. iNOS and endothelial nitric oxide synthase expression were measured by Western blot. RESULTS: Infarct size was significantly reduced in wild-type mice from 43.1 +/- 5.3% in the saline group to 22.4 +/- 4.4% in the higher-dose morphine group (0.3 mg/kg) (P < 0.05). This cardioprotective effect was abolished by S-methylthiourea sulfate (43.3 +/- 3.9%) and was absent in iNOS gene-knockout mice (42.3 +/- 4.7%). Pretreatment with the lower dose of morphine (0.1 mg/kg) did not reduce infarct size (41.1 +/- 5.4%). A significant increase in myocardial iNOS expression was observed 24 h after morphine administration (0.3 mg/kg but not 0.1 mg/kg; P < 0.05), whereas endothelial nitric oxide synthase remained unchanged. CONCLUSIONS:: Pretreatment with morphine induces delayed cardioprotection in mice. The authors demonstrated an obligatory role for iNOS in mediating morphine-induced delayed cardioprotection.     

The interaction of nitric oxide and prostaglandins in the control of corporal smooth muscle tone: evidence for production of a cyclooxygenase-derived endothelium-contracting factor.

OBJECTIVE: To investigate the interaction of endothelium-derived nitric oxide (NO) and prostaglandins (PGs) in regulating corporal smooth muscle tone in vitro. Materials and methods Strips of rabbit corpus cavernosum were mounted in organ chambers for the measurement of isometric tension. Strips were submaximally contracted with noradrenaline and concentration-response curves (CRCs) to acetylcholine (ACh) were constructed before and after treatment with 5 micromol/L atropine, 20 micromol of the cyclooxygenase inhibitor indomethacin and 10 micromol of the PGH2/thromboxane A2 receptor antagonist SQ29548. The NO synthase (NOS) inhibitors L-NG-monomethyl arginine (L-NMMA) and L-NG-nitroarginine (L-NOARG) were added to strips at tonic tension in the presence and absence of indomethacin, and after this CRCs to ACh were constructed. RESULTS: The addition of ACh to strips produced a concentration-dependent relaxation which was inhibited by atropine. Indomethacin, but not SQ29548, significantly increased relaxation to ACh. Relaxation to ACh was impaired by L-NMMA, but adding ACh to strips treated with L-NOARG resulted in contractile responses, whilst both effects were reversed by indomethacin. L-NMMA and L-NOARG led to increases in tonic tone which were unaffected by indomethacin. CONCLUSIONS: In rabbit corpus cavernosum there is a tonic release of NO which does not appear to be inhibited by a vasoconstrictor prostanoid. Endothelium-dependent relaxation to ACh results in the dual production of NO and a cyclooxygenase-derived endothelium contracting factor which acts in opposition to NO; this factor is unlikely to act on PGH2/TXA2 receptors.     

Traumatic axonal injury in the optic nerve: evidence for axonal swelling, disconnection, dieback, and reorganization

Traumatic axonal injury (TAI) is a major feature of traumatic brain injury (TBI) and is associated with much of its morbidity. To date, significant insight has been gained into the initiating pathogenesis of TAI. However, the nature of TAI within the injured brain precludes the consistent evaluation of its specific anterograde and retrograde sequelae. To overcome this limitation, we used the relatively organized optic nerve in a central fluid percussion injury (cFPI) model. To improve the visualization of TAI, we utilized mice expressing yellow fluorescent protein (YFP) in their visual pathways. Through this approach, we consistently generated TAI in the optic nerve and qualitatively and quantitatively evaluated its progression over a 48-h period in YFP axons via confocal microscopy and electron microscopy. In this model, delayed axonal swelling with subsequent disconnection were the norm, together with the fact that once disconnected, both the proximal and distal axonal segments revealed significant dieback, with the proximal swellings showing regression and reorganization, while the distal swellings persisted, although showing signs of impending degeneration. When antibodies targeting the C-terminus of amyloid precursor protein (APP), a routine marker of TAI were employed, they mapped exclusively to the proximal axonal segments without distal targeting, regardless of the survival time. Concomitant with this evolving axonal pathology, focal YFP fluorescence quenching occurred and mapped precisely to immunoreactive loci positive for Texas-Red-conjugated-IgG, indicating that blood-brain barrier disruption and its attendant edema contributed to this phenomenon. This was confirmed through the use of antibodies targeting endogenous YFP, which demonstrated the retention of intact immunoreactive axons despite YFP fluorescence quenching. Collectively, the results of this study within the injured optic nerve provide unprecedented insight into the evolving pathobiology associated with TAI.     

Inhaled nitric oxide for acute hypoxic respiratory failure in children and adults: a meta-analysis

We systematically reviewed randomized controlled trials examining inhaled nitric oxide (INO) for the treatment of acute respiratory distress syndrome or acute lung injury in children and adults. Qualitative assessments of identified trials were made, and metaanalyses were performed according to Cochrane methodology. Five randomized controlled trials (n = 535) met entry criteria. One study demonstrated significant improvement in oxygenation in the first 4 days of treatment, with no difference after this. There was no difference in ventilator-free days between treatment and placebo groups, and no specific dose of INO was more advantageous than any other. INO had no effect on mortality in trials without crossover of treatment failures to open-label INO (relative risk, 0.98; 95% confidence interval, 0.66-1.44). Other clinical indicators of effectiveness, such as duration of hospital and intensive care stay, were inconsistently reported. Lack of data prevented assessment of all outcomes. If further trials assessing INO in acute respiratory distress syndrome or acute lung injury are to proceed, they should be stratified for primary etiology, incorporate other modalities that may affect outcome, and evaluate clinically relevant outcomes before any benefit of INO can be excluded.     

Essential role for membrane lipid rafts in interleukin-1beta-induced nitric oxide release from insulin-secreting cells: potential regulation by caveolin-1+

We recently reported that the activation of H-Ras represents one of the signaling steps underlying the interleukin-1beta (IL-1beta)-mediated metabolic dysfunction of the islet beta-cell. In the present study, we examined potential contributory roles of membrane-associated, cholesterol-enriched lipid rafts/caveolae and their constituent proteins (e.g., caveolin-1 [Cav-1]) as potential sites for IL-1beta-induced nitric oxide (NO) release in the isolated beta-cell. Disruption of lipid rafts (e.g., with cyclodextrin) markedly reduced IL-1beta-induced gene expression of inducible NO synthase (iNOS) and NO release from beta-cells. Immunologic and confocal microscopic evidence also suggested a transient but significant stimulation of tyrosine phosphorylation of Cav-1 in beta-cells briefly (for 15 min) exposed to IL-1beta that was markedly attenuated by three structurally distinct inhibitors of protein tyrosine phosphorylation. Overexpression of an inactive mutant of Cav-1 lacking the tyrosine phosphorylation site (Y14F) or an siRNA-mediated Cav-1 knock down also resulted in marked attenuation of IL-1beta-induced iNOS gene expression and NO release from these cells, thus further implicating Cav-1 in this signaling cascade. IL-1beta treatment also increased (within 20 min) the translocation of H-Ras into lipid rafts. Here we provide the first evidence to suggest that tyrosine phosphorylation of Cav-1 and subsequent interaction among members of the Ras signaling pathway within the membrane lipid microdomains represent early signaling mechanisms of IL-1beta in beta-cells.     

Urinary ortho-tyrosine excretion in diabetes mellitus and renal failure: evidence for hydroxyl radical production

BACKGROUND: Phenylalanine is converted to para- and ortho-tyrosine by hydroxyl free radical, or to para-tyrosine by the phenylalanine hydroxylase enzyme. The aim of this study was to measure para- and ortho-tyrosine in the urine and plasma of patients with chronic renal disease and/or diabetes, to obtain information on the renal handling of the different tyrosine isomers and, furthermore, to measure urinary levels of 8-epi-prostaglandin-F(2alpha), a marker of lipid peroxidation. METHODS: In our cross-sectional study we measured para-, ortho-tyrosine, and phenylalanine levels, using high performance liquid chromatography and 8-epi-prostaglandin-F(2alpha) with enzyme-linked immunosorbent assay (ELISA). We compared 4 groups: (1) controls (CONTR, N = 14), (2) patients with chronic kidney disease (CKD, N = 12), (3) patients with type 2 diabetes mellitus (DIAB, N = 17), (4) patients with chronic kidney disease and type 2 diabetes (DIAB-CKD, N = 19). RESULTS: We found a decreased plasma para-tyrosine level and decreased urinary para-tyrosine excretion in CKD patients, while the fractional excretion of para-tyrosine was similar in all 4 groups, approximately 1%. There was no difference in the plasma ortho-tyrosine levels between the groups. However, urinary ortho-tyrosine excretion was higher in all 3 groups of patients than in the CONTR group, and higher in DIAB and in DIAB-CKD patients than in CKD patients. The fractional excretion of ortho-tyrosine was significantly higher in DIAB and in DIAB-CKD patients than in the CONTR group. The fractional excretion of ortho-tyrosine exceeded 100% in the 2 diabetic groups. Urinary 8-epi-prostaglandin-F(2alpha)/creatinine ratio did not correlate with urinary ortho-tyrosine excretion. CONCLUSION: The difference between para-tyrosine levels of the groups is probably due to renal impairment, while there is indirect evidence for an increased tubular secretion or production of ortho-tyrosine in the kidney in diabetic patients with or without CKD.     

Contribution of known and unknown susceptibility genes to early-onset diabetes in scandinavia: evidence for heterogeneity

In an attempt to identify novel susceptibility genes predisposing to early-onset diabetes (EOD), we performed a genome-wide scan using 433 markers in 222 individuals (119 with diabetes) from 29 Scandinavian families with > or =2 members with onset of diabetes < or =45 years. The highest nonparametric linkage (NPL) score, 2.7 (P < 0.01), was observed on chromosome 1p (D1S473/D1S438). Six other regions on chromosomes 3p, 7q, 11q, 18q, 20q, and 21q showed a nominal P value <0.05. Of the EOD subjects in these 29 families, 20% were GAD antibody positive and 68% displayed type 1 diabetes HLA risk alleles (DQB*02 or 0302). Mutations in maturity-onset diabetes of the young (MODY) 1-5 genes and the A3243G mitochondrial DNA mutation were detected by single-strand conformation polymorphism and direct sequencing. To increase homogeneity, we analyzed a subsample of five families with autosomal dominant inheritance of EOD (greater than or equal to two members with age at diagnosis < or =35 years). The highest NPL scores were found on chromosome 1p (D1S438-D1S1665; NPL 3.0; P < 0.01) and 16q (D16S419; NPL 2.9; P < 0.01). After exclusion of three families with MODY1, MODY3, and mitochondrial mutations, the highest NPL scores were observed on chromosomes 1p (D1S438; NPL 2.6; P < 0.01), 3p (D3S1620; NPL 2.2; P < 0.03), 5q (D5S1465; NPL 2.1; P < 0.03), 7q (D7S820; NPL 2.0; P < 0.03), 18q (D18S535; NPL 1.9; P < 0.04), 20q (D20S195; NPL 2.5; P < 0.02), and 21q (D21S1446; NPL 2.2; P < 0.03). We conclude that considerable heterogeneity exists in Scandinavian subjects with EOD; 24% had MODY or maternally inherited diabetes and deafness, and approximately 60% were GAD antibody positive or had type 1 diabetes-associated HLA genotypes. Our data also point at putative chromosomal regions, which could harbor novel genes that contribute to EOD.