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Elevated urinary excretion of nitric oxide metabolites in young infants with Zellweger syndrome
Clinica Chimica Acta 334 (2003) 111 – 115 www.elsevier.com/locate/clinchim
Elevated urinary excretion of nitric oxide metabolites in young infants with Zellweger syndrome Andrzej Surdacki a,1, Dimitrios Tsikas a,*, Ertan Mayatepek b, Ju¨rgen C. Fro¨lich a a
Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany Department of General Pediatrics, University Children’s Hospital, Mooren-Strasse 5, 40225 Du¨sseldorf, Germany
b
Received 6 January 2003; received in revised form 9 April 2003; accepted 17 April 2003
Abstract Background: Children with Zellweger syndrome (ZS), a rare peroxisome deficiency disorder, excrete into the urine highly elevated amounts of urinary metabolites of the arachidonic acid cascade. This pathway may interact in vivo with the L-arginine/ nitric oxide (NO) pathway. The aim of this study was to investigate NO production in ZS. Methods: We studied 11 infants aged 2 – 12 months with ZS and 30 healthy controls (HC) aged 1 – 12 months. Urinary excretion of nitrite plus nitrate (UNOx), which is a reliable measure of whole body NO formation, was determined by gas chromatography – mass spectrometry (GC – MS) and corrected for creatinine excretion. Results: In the subjects aged 1 – 6 months, UNOx was more than twofolds higher in ZS (median, 666 Amol/mmol creatinine) as compared to HC (median, 257 Amol/mmol creatinine) ( P = 0.014 by Mann – Whitney U-test). In children aged 7 – 12 months, UNOx was similar for ZS subjects and HC ( P = 0.96). UNOx correlated negatively with age in ZS (Kendall’s rank correlation coefficient, s = 0.75, P = 0.001). By contrast, no such correlation was found in HC (s = 0.06, P = 0.6). Conclusions: NO production is highly elevated during the first 6 months of life in infants with ZS and falls to normal levels within the following 6 months, suggesting a dramatic decrease in NO synthesis in ZS. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Zellweger syndrome; Nitric oxide; Urinary nitrate; GC – MS
1. Introduction In Zellweger syndrome (ZS), a rare peroxisome deficiency disorder, urinary excretion of 8-iso-prostaAbbreviations: ZS, Zellweger syndrome; HC, healthy control; NO, nitric oxide; LOX, 5-lipoxygenase; COX, cyclooxygenase; EDRF, endothelium-derived relaxing factor; UNOx, urinary excretion of nitrite plus nitrate; GC – MS, gas chromatography – mass spectrometry; QC, quality control. * Corresponding author. E-mail address: [email protected] (D. Tsikas). 1 Present address: Department of Cardiology, Jagiellonian University, Cracow, Poland.
glandin F2a, a product of nonenzymatic free radicalmediated peroxidation of arachidonyl residues of phospholipids, was found elevated over 120-fold, in comparison with that of healthy children [1]. In addition, the pattern of urinary 5-lipoxygenase (LOX) [2] and cyclooxygenase (COX) [3] metabolites of arachidonic acid in ZS is highly abnormal, presumably resulting from altered degradation pathways due to deficient peroxisomal h-oxidation. As a consequence, excretion of cysteinyl leukotrienes, 6-oxo-prostaglandin F1a (i.e., the stable metabolite of prostacyclin) and thromboxane B2 (i.e., the stable metabolite of thromboxane A2) is highly elevated in ZS [2,3]. Moreover,
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leukotriene B4, which is undetectable in the urine of healthy children and adults, was found only in the urine of ZS children [2]. The biologically highly active 8-iso-prostaglandin F2a [4,5], leukotriene B4 [6], cysteinyl leukotrienes [6,7] and prostacyclin [8] have been shown in vitro to stimulate release of endothelium-derived relaxing factor (EDRF)/nitric oxide (NO). The interplay of the L-arginine/NO pathway with the LOX and COX pathways in vivo is poorly investigated. The ZS offers the unique possibility to investigate this subject in vivo. The aim of the present study was, therefore, to investigate whether endogenous NO formation is altered in patients with ZS. Whole-body NO generation was assessed by measuring urinary excretion of nitrite plus nitrate (UNOx), the stable oxidative metabolites of NO. This measure has been previously reported to be altered in normal neonates vs. adults [9] and to be age-dependent in infants and older children [10,11]. In addition, abnormal values of this parameter were found in idiopathic persistent pulmonary hypertension of the newborn [12], in preterm neonates [10], as well as in children with inflammatory diseases [13 – 15] and, at a borderline significance, in childhood hypertension due to renal parenchymal disease [16]. Passaged cell lines of fibroblasts isolated from subjects with ZS have been recently reported to exhibit reduced activity of the endothelial isoform of NO synthase by about one-half [17].
2. Subjects and methods 2.1. Subjects and study design We studied a total of 11 patients with ZS (7.6 F 3.6 months, mean F S.D., range: 2 – 12 months) and 30 control healthy (HC) children (mean age, 6.2 F 5.0 months, range: 1– 12 months). Neither the ZS infants nor the HC children were on a special diet or parenteral alimentation. The subjects received their normal diet appropriate for age. All patients with ZS exhibited the characteristic clinical and biochemical abnormalities described for ZS [18] and had no evidence of disturbed renal or hepatic function. Specific biochemical analyses in these patients included very long-chain fatty acids
in plasma and fibroblasts as well as plasma bile acid intermediates and de novo plasmalogen synthesis in cultured skin fibroblasts. As the study protocol was based on the analysis of urine samples previously taken for routine diagnostic purposes, the approval of an ethical committee was not required. Written consent was obtained from the parents of all children studied. 2.2. Analytical methods 2.2.1. Determination of urinary nitrite and nitrate Urine samples from spontaneous micturition were collected into a sterile bag and frozen at 80 jC until assayed for nitrite and nitrate by means of gas chromatography – mass spectrometry (GC – MS) as previously described in detail [19]. Briefly, urine aliquots (100 Al) were spiked with the internal standard, i.e., [15N]nitrate (98 at.% at 15N, SigmaAldrich Chemie, Deisenhofen, Germany), at a final concentration of 800 Amol/l. In addition, 100Al aliquots of a 5% (w/v) ammonium chloride buffer (pH 8.8) were also spiked with [15N]nitrate at a final concentration of 800 Amol/l. Aliquots (100 Al) of the urine and the buffer samples were diluted with aliquots (900 Al) of the same buffer. Accurately weighed cadmium powder (10 mg) was then added to the samples; reduction of nitrate to nitrite was achieved by shaking for 90 min at room temperature. The suspensions were centrifuged at 800g for 5 min. Aliquots (100 Al) of the supernatants were treated with acetone (400 Al) and 2,3,4,5,6-pentafluorobenzyl bromide (10 Al; Aldrich, Steinheim, Germany), and the reaction mixtures were incubated at 50 jC for 60 min. Acetone was then removed under a stream of nitrogen and the reaction products were extracted by vortex mixing with toluene (0.8 ml) for 1 min. Following phase separation by centrifugation (800g, 5 min), an aliquot (200 Al) of the organic phase was transferred into a clean glass vial. Aliquots (0.5 Al) from toluene (200 Al) were injected into the GC – MS apparatus (MS Engine 5989, Hewlett-Packard, Waldbronn, Germany) in the splitless mode. Selected ion monitoring at a mass/charge ratio (m/z) of 46 for nitrite (from reduced nitrate) and m/z of 47 for [15N] nitrite (from reduced [15N]nitrate) was performed in the negative-ion chemical ionization mode.
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2.2.2. Quality control samples Urine samples were analysed alongside three quality control (QC1, QC2, QC3) samples within two runs (A and B) using urine from two different subjects. Concentrations of nitrite plus nitrate in the unspiked control urine samples were 420 Amol/l (RSD, 1.4%) (A) and 1560 Amol/l (RSD, 3.7%) (B), respectively. The QC1 sample was analysed without addition of nitrate, while QC2 and QC3 samples were spiked with 400 Amol/l and 800 Amol/l of nitrate, respectively. Urine samples obtained from the children were analyzed once, while all QC samples were analysed in duplicate. Mean recovery was 105.5% (A) and 102.5% (B) for QC2, and 105.5% (A) and 101.9% (B) for QC3. Imprecision (RSD) in all samples was below 3.7%. In order to limit the effect of the variability of renal function, urinary excretion of nitrite plus nitrate was corrected for urinary creatinine [9– 11,13 – 16] and expressed in Amol/mmol creatinine. Creatinine was measured with the picric acid method. 2.3. Statistical methods
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was 666 Amol/mmol creatinine (range: 564– 861 Amol/ mmol creatinine), more than twofolds higher as compared to the HC children of corresponding age (257 Amol/mmol creatinine, range: 44 – 877 Amol/mmol creatinine) (P = 0.014, Fig. 1). The three HC children with the highest UNOx values were 1, 4 and 6 months old. In the children aged 7 – 12 months, UNOx was similar in the ZS subjects (326 Amol/mmol creatinine, range: 174 – 532 Amol/mmol creatinine) and the HC children (360 Amol/mmol creatinine, range: 152 –827 Amol/mmol creatinine) (P = 0.96, Fig. 1). Urinary creatinine did not differ significantly between the ZS patients and HC children within the subjects aged 1 – 6 months (1.1 mmol/l, range: 0.2 – 2.0 mmol/l vs. 1.1 mmol/l, range: 0.6– 2.6 mmol/l for ZS and HC, respectively; P = 0.46) as well as within the children aged 7– 12 months (2.05 mmol/l, range: 0.9 – 5.3 mmol/l vs. 2.5 mmol/l, range: 1.1 – 10.6 mmol/l for ZS and HC, respectively; P = 0.35). In the ZS infants, UNOx correlated negatively with age (s = 0.75; P = 0.001, Fig. 2), which was not found in the HC group (s = 0.06; P = 0.6; not shown).
Children’s age is presented as mean F S.D. UNOx was compared between four groups of subjects, i.e., ZS children (n = 5) and HC children (n = 18) aged 1– 6 months, as well as ZS children (n = 6) and HC children (n = 12) aged 7 – 12 months. Since neither UNOx nor urinary creatinine exhibited normal distribution (checked by both Shapiro– Wilk’s W-test and Lilliefors’ test) within all the analyzed groups, their values are shown as median and range. Kruskal– Wallis ANOVA for ranks was used to test the presence of significant intergroup differences in UNOx taking into consideration the four above-mentioned groups. Comparisons of UNOx or creatinine between particular groups were performed with Mann –Whitney U-test. In order to assess the relationship between UNOx and age, Kendall’s rank correlation coefficient (s) was computed. A probability value of below 0.05 was considered significant.
3. Results Kruskal – Wallis ANOVA for ranks revealed the presence of a significant intergroup difference in UNOx (P = 0.035). In the ZS children aged 1 –6 months, UNOx
Fig. 1. Scatter plots of urinary excretion of nitrite plus nitrate (UNOx) in children with Zellweger syndrome (ZS) and in healthy children (HC) of two different age groups. Horizontal bars are medians. P values represent statistical significance (by Mann – Whitney U-test) vs. healthy children of corresponding age. Aditionally, UNOx was significantly lower in the older vs. younger ZS infants (P = 0.006), whereas in the control children, UNOx was comparable for both age goups (P = 0.5).
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Fig. 2. Correlation betweeen urinary excretion of nitrite plus nitrate (UNOx) and age in children with Zellweger syndrome (ZS). UNOx and age were negatively correlated in 11 children with Zellweger syndrome: Kendall’s rank correlation coefficient, s = 0.75, P = 0.001.
Accordingly, UNOx was significantly higher in the ZS children aged 7 – 12 months vs. the younger ZS subjects (P = 0.006, Fig. 1), whereas healthy infants had comparable UNOx in both age groups (P = 0.5, Fig. 1).
4. Discussion A highly accurate GC – MS method was used to determine UNOx in children suffering from ZS and healthy subjects. In the present study, UNOx in control subjects was comparable to the values measured by Tsukahara et al. [10] in the urine samples collected by a similar procedure from term healthy infants of comparable age (means: 410 and 420 Amol/mmol creatinine at the age of 4 and 7 months, respectively) kept on their normal diet, as our study subjects. In the first 6 months of life, UNOx was significantly elevated by a factor of 2 in ZS with the reference to the control group of similar age. As a result of the age-dependent fall of UNOx in ZS, the intergroup difference disappeared when UNOx was compared in the ZS infants aged 7 – 12 months and age-matched controls. It has been shown that metabolism of prostaglandins, thromboxane and leukotrienes is disturbed in patients with ZS most likely resulting in accumulation of the biologically highly active eicosanoids in blood and tissue [1– 3]. 8-iso-Prostaglandin F2a [4,5] as well
as products of the COX [8] and LOX [6,7] pathways have been reported to stimulate NO synthesis in vitro. It could, therefore, be speculated that the interplay between the numerous arachidonic acid pathways, notably COX and LOX, with the L-arginine/NO pathway might have resulted in the elevated NO generation found in the young ZS children. It might be further assumed that in older ZS infants the efficacy of these mechanisms and/or the responsiveness of NOS would be diminished, e.g., due to prolonged stimulation or other factors, thus, explaining the negative correlation between UNOx and age in ZS. To date, there are no data available on age-dependent excretion of 8-iso-prostaglandin F2a and products of the COX and LOX pathways in the ZS syndrome. To the best of our knowledge, there are no data on a tendency to hypotension during the first months of life in ZS children. UNOx, being an index of wholebody NO formation, might reflect increased NO production in nonvascular tissues. Alternatively, NO in the vascular tree of ZS children might be unable to produce enhanced vasodilation due to its scavenging by superoxide [20], enhanced ability of erythrocytes to oxidize NO into nitrate as shown for erythrocytes of normal neonates vs. erythrocytes isolated from adults [9] or simultaneous presence of an excess of vasoconstrictors, e.g., 8-iso-prostaglandin F2a [1,5] or prostaglandin F2a, the latter as yet not quantified in ZS in vivo. Peroxisomes contain enzymes which are involved not only in the formation of reactive oxygen species but they are also the main intracellular compartment of catalase and copper – zinc superoxide dismutase [21]. In addition, the following data suggest a pathogenetic role of oxidative stress in ZS. Fibroblasts from patients with ZS exhibit raised susceptibility to oxidative stress despite similar levels of superoxide and hydrogen peroxide, a very likely consequence of defective membrane plasmalogen synthesis [22]. If oxidative stress might be relevant to the pathophysiology of ZS, it seems plausible to assume that NO produced in excessive amounts in young infants with ZS, as suggested by the present study, could interact with superoxide forming toxic peroxynitrite [23], thus, exacerbating damage and possibly contributing to the progression of the disease. In addition, modulatory effect of NO on peroxisomal enzymes has been demonstrated [24]. Further studies are required to
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prove enhanced formation of peroxynitrite in early stages of ZS, e.g., by measuring plasma 3-nitrotyrosine, a product of peroxynitrite-mediated nitration of tyrosine residues in biomolecules [25].
[11]
[12]
Acknowledgements Dr. Mayatepek is supported by the Deutsche Forschungsgemeinschaft (Ma 1314/2-2 and Ma 1314/2-3). Dr. Surdacki was a recipient of a research fellowship from the Alexander von Humboldt Foundation. The laboratory and technical assistance of I. Fuchs and F.-M. Gutzki is gratefully acknowledged.
[13]
[14]
[15]
References [1] Tsikas D, Schwedhelm E, Fauler J, Gutzki F-M, Mayatapek E, Fro¨lich JC. Specific and rapid quantification of 8-iso-prostaglandin F2a in urine of healthy humans and patients with Zellweger syndrome by gas chromatography – tandem mass spectrometry. J Chromatogr B 1998;716:7 – 17. [2] Mayatepek E, Lehmann WD, Fauler J, Tsikas D, Fro¨lich JC, Schutgens RB, et al. Impaired degradation of leukotrienes in patients with peroxisome deficiency disorders. J Clin Invest 1993;91:881 – 8. [3] Fauler J, Tsikas D, Mayatepek E, Keppler D, Fro¨lich JC. Impaired degradation of prostaglandins and thromboxane in Zellweger syndrome. Pediatr Res 1994;36:449 – 55. [4] Jourdan KB, Evans TW, Curzen NP, Mitchell JA. Evidence for a dilator function of 8-iso-prostaglandin F2a in rat pulmonary artery. Br J Pharmacol 1997;120:1280 – 5. [5] Wilson SH, Best PJ, Lerman LO, Holmes Jr DR, Richardson DM, Lerman A. Enhanced coronary vasoconstriction to oxidative stress product, 8-iso-prostaglandin F2a, in experimental hypercholesterolemia. Cardiovasc Res 1999;44:601 – 7. [6] Larfars G, Lantoine F, Devynck MA, Palmblad J, Gyllenhammar H. Activation of nitric oxide release and oxidative metabolism by leukotrienes B4, C4, and D4 in human polymorphonuclear leukocytes. Blood 1999;93:1399 – 405. [7] Walch L, Norel X, Gascard JP, Brink C. Functional studies of leukotriene receptors in vascular tissues. Am J Respir Crit Care Med 2000;16:S107 – 11. [8] Shimokawa H, Flavahan NA, Lorenz RR, Vanhoutte PM. Prostacyclin releases endothelium-derived relaxing factor and potentiates its action in coronary arteries of the pig. Br J Pharmacol 1988;95:1197 – 203. [9] Honold J, Pusser NL, Nathan L, Chaudhuri G, Ignarro LJ, Sherman MP. Production and excretion of nitrate by human newborn infants: neonates are not little adults. Nitric Oxide 2000;4:35 – 46. [10] Tsukahara H, Hiraoka M, Hori C, Tsuchida S, Hata I, Nishida K, et al. Urinary nitrite/nitrate excretion in infancy: compar-
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ison between term and preterm infants. Early Hum Dev 1997; 47:51 – 6. Tsukahara H, Hiraoka M, Hori C, Miyanomae T, Kikuchi K, Sudo M. Age-related changes of urinary nitrite/nitrate excretion in normal children. Nephron 1997;76:307 – 9. Dollberg S, Warner BW, Myatt L. Urinary nitrite and nitrate concentrations in patients with idiopathic persistent pulmonary hypertension of the newborn and effect of extracorporeal membrane oxygenation. Pediatr Res 1995;37:31 – 4. Trachtman H, Gauthier B, Frank R, Futterweit S, Goldstein A, Tomczak J. Increased urinary nitrite excretion in children with minimal change nephrotic syndrome. J Pediatr 1996;128: 173 – 6. Uzuner N, Islekel H, Ozkan H, Sen A, Yenice S, Cevik N. Urinary nitrite excretion in low birth weight neonates with systemic inflammatory response syndrome. Biol Neonate 1997; 71:362 – 6. van Straaten EA, Koster-Kamphuis L, Bovee-Oudenhoven IM, van der Meer R, Forget PP. Increased urinary nitric oxide oxidation products in children with active coeliac disease. Acta Paediatr 1999;88:528 – 31. Goonasekera CDA, Shah V, Rees DD, Dillon MJ. Nitric oxide activity in childhood hypertension. Arch Dis Child 1997;77: 11 – 6. Koeck T, Kremser K. Peroxisomal deficiences are associated with altered activity of endothelial NOS in human fibroblasts. Nitric Oxide 2001;5:213 – 8. Lazarow PB, Moser HW. Disorders of peroxisome biogenesis. In: Scriver CR, Beaudet CL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill; 1995. p. 2287 – 324. Tsikas D, Gutzki FM, Rossa S, Bauer H, Neumann C, Dockendorff K, et al. Measurement of nitrite and nitrate in biological fluids by gas chromatography – mass spectrometry and by the Griess assay: problems with the Griess assay-solutions by gas chromatography – mass spectrometry. Anal Biochem 1997;244:208 – 20. Gryglewski RJ, Palmer RM, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 1986;320:454 – 6. Keller GA, Warner TG, Steimer KS, Hallewell RA. Cu,Zn superoxide dismutase is a peroxisomal enzyme in human fibroblasts and hepatoma cells. Proc Natl Acad Sci U S A 1991;88:7381 – 5. Spisni E, Cavazzoni M, Griffoni C, Calzolari E, Tomasi V. Evidence that photodynamic stress kills Zellweger fibroblasts by a nonapoptotic mechanism. Biochim Biophys Acta 1998; 1402:61 – 9. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am J Physiol 1996;271:C1424 – 37. Kremser K, Stangl H, Pahan K, Singh I. Nitric oxide regulates peroxisomal enzyme activities. Eur J Clin Chem Clin Biochem 1995;33:763 – 4. Ischiropoulos H. Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species. Arch Biochem Biophys 1998;356:1 – 11.
Elevated urinary excretion of nitric oxide metabolites in young infants with Zellweger syndrome Andrzej Surdacki a,1, Dimitrios Tsikas a,*, Ertan Mayatepek b, Ju¨rgen C. Fro¨lich a a
Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany Department of General Pediatrics, University Children’s Hospital, Mooren-Strasse 5, 40225 Du¨sseldorf, Germany
b
Received 6 January 2003; received in revised form 9 April 2003; accepted 17 April 2003
Abstract Background: Children with Zellweger syndrome (ZS), a rare peroxisome deficiency disorder, excrete into the urine highly elevated amounts of urinary metabolites of the arachidonic acid cascade. This pathway may interact in vivo with the L-arginine/ nitric oxide (NO) pathway. The aim of this study was to investigate NO production in ZS. Methods: We studied 11 infants aged 2 – 12 months with ZS and 30 healthy controls (HC) aged 1 – 12 months. Urinary excretion of nitrite plus nitrate (UNOx), which is a reliable measure of whole body NO formation, was determined by gas chromatography – mass spectrometry (GC – MS) and corrected for creatinine excretion. Results: In the subjects aged 1 – 6 months, UNOx was more than twofolds higher in ZS (median, 666 Amol/mmol creatinine) as compared to HC (median, 257 Amol/mmol creatinine) ( P = 0.014 by Mann – Whitney U-test). In children aged 7 – 12 months, UNOx was similar for ZS subjects and HC ( P = 0.96). UNOx correlated negatively with age in ZS (Kendall’s rank correlation coefficient, s = 0.75, P = 0.001). By contrast, no such correlation was found in HC (s = 0.06, P = 0.6). Conclusions: NO production is highly elevated during the first 6 months of life in infants with ZS and falls to normal levels within the following 6 months, suggesting a dramatic decrease in NO synthesis in ZS. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Zellweger syndrome; Nitric oxide; Urinary nitrate; GC – MS
1. Introduction In Zellweger syndrome (ZS), a rare peroxisome deficiency disorder, urinary excretion of 8-iso-prostaAbbreviations: ZS, Zellweger syndrome; HC, healthy control; NO, nitric oxide; LOX, 5-lipoxygenase; COX, cyclooxygenase; EDRF, endothelium-derived relaxing factor; UNOx, urinary excretion of nitrite plus nitrate; GC – MS, gas chromatography – mass spectrometry; QC, quality control. * Corresponding author. E-mail address: [email protected] (D. Tsikas). 1 Present address: Department of Cardiology, Jagiellonian University, Cracow, Poland.
glandin F2a, a product of nonenzymatic free radicalmediated peroxidation of arachidonyl residues of phospholipids, was found elevated over 120-fold, in comparison with that of healthy children [1]. In addition, the pattern of urinary 5-lipoxygenase (LOX) [2] and cyclooxygenase (COX) [3] metabolites of arachidonic acid in ZS is highly abnormal, presumably resulting from altered degradation pathways due to deficient peroxisomal h-oxidation. As a consequence, excretion of cysteinyl leukotrienes, 6-oxo-prostaglandin F1a (i.e., the stable metabolite of prostacyclin) and thromboxane B2 (i.e., the stable metabolite of thromboxane A2) is highly elevated in ZS [2,3]. Moreover,
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leukotriene B4, which is undetectable in the urine of healthy children and adults, was found only in the urine of ZS children [2]. The biologically highly active 8-iso-prostaglandin F2a [4,5], leukotriene B4 [6], cysteinyl leukotrienes [6,7] and prostacyclin [8] have been shown in vitro to stimulate release of endothelium-derived relaxing factor (EDRF)/nitric oxide (NO). The interplay of the L-arginine/NO pathway with the LOX and COX pathways in vivo is poorly investigated. The ZS offers the unique possibility to investigate this subject in vivo. The aim of the present study was, therefore, to investigate whether endogenous NO formation is altered in patients with ZS. Whole-body NO generation was assessed by measuring urinary excretion of nitrite plus nitrate (UNOx), the stable oxidative metabolites of NO. This measure has been previously reported to be altered in normal neonates vs. adults [9] and to be age-dependent in infants and older children [10,11]. In addition, abnormal values of this parameter were found in idiopathic persistent pulmonary hypertension of the newborn [12], in preterm neonates [10], as well as in children with inflammatory diseases [13 – 15] and, at a borderline significance, in childhood hypertension due to renal parenchymal disease [16]. Passaged cell lines of fibroblasts isolated from subjects with ZS have been recently reported to exhibit reduced activity of the endothelial isoform of NO synthase by about one-half [17].
2. Subjects and methods 2.1. Subjects and study design We studied a total of 11 patients with ZS (7.6 F 3.6 months, mean F S.D., range: 2 – 12 months) and 30 control healthy (HC) children (mean age, 6.2 F 5.0 months, range: 1– 12 months). Neither the ZS infants nor the HC children were on a special diet or parenteral alimentation. The subjects received their normal diet appropriate for age. All patients with ZS exhibited the characteristic clinical and biochemical abnormalities described for ZS [18] and had no evidence of disturbed renal or hepatic function. Specific biochemical analyses in these patients included very long-chain fatty acids
in plasma and fibroblasts as well as plasma bile acid intermediates and de novo plasmalogen synthesis in cultured skin fibroblasts. As the study protocol was based on the analysis of urine samples previously taken for routine diagnostic purposes, the approval of an ethical committee was not required. Written consent was obtained from the parents of all children studied. 2.2. Analytical methods 2.2.1. Determination of urinary nitrite and nitrate Urine samples from spontaneous micturition were collected into a sterile bag and frozen at 80 jC until assayed for nitrite and nitrate by means of gas chromatography – mass spectrometry (GC – MS) as previously described in detail [19]. Briefly, urine aliquots (100 Al) were spiked with the internal standard, i.e., [15N]nitrate (98 at.% at 15N, SigmaAldrich Chemie, Deisenhofen, Germany), at a final concentration of 800 Amol/l. In addition, 100Al aliquots of a 5% (w/v) ammonium chloride buffer (pH 8.8) were also spiked with [15N]nitrate at a final concentration of 800 Amol/l. Aliquots (100 Al) of the urine and the buffer samples were diluted with aliquots (900 Al) of the same buffer. Accurately weighed cadmium powder (10 mg) was then added to the samples; reduction of nitrate to nitrite was achieved by shaking for 90 min at room temperature. The suspensions were centrifuged at 800g for 5 min. Aliquots (100 Al) of the supernatants were treated with acetone (400 Al) and 2,3,4,5,6-pentafluorobenzyl bromide (10 Al; Aldrich, Steinheim, Germany), and the reaction mixtures were incubated at 50 jC for 60 min. Acetone was then removed under a stream of nitrogen and the reaction products were extracted by vortex mixing with toluene (0.8 ml) for 1 min. Following phase separation by centrifugation (800g, 5 min), an aliquot (200 Al) of the organic phase was transferred into a clean glass vial. Aliquots (0.5 Al) from toluene (200 Al) were injected into the GC – MS apparatus (MS Engine 5989, Hewlett-Packard, Waldbronn, Germany) in the splitless mode. Selected ion monitoring at a mass/charge ratio (m/z) of 46 for nitrite (from reduced nitrate) and m/z of 47 for [15N] nitrite (from reduced [15N]nitrate) was performed in the negative-ion chemical ionization mode.
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2.2.2. Quality control samples Urine samples were analysed alongside three quality control (QC1, QC2, QC3) samples within two runs (A and B) using urine from two different subjects. Concentrations of nitrite plus nitrate in the unspiked control urine samples were 420 Amol/l (RSD, 1.4%) (A) and 1560 Amol/l (RSD, 3.7%) (B), respectively. The QC1 sample was analysed without addition of nitrate, while QC2 and QC3 samples were spiked with 400 Amol/l and 800 Amol/l of nitrate, respectively. Urine samples obtained from the children were analyzed once, while all QC samples were analysed in duplicate. Mean recovery was 105.5% (A) and 102.5% (B) for QC2, and 105.5% (A) and 101.9% (B) for QC3. Imprecision (RSD) in all samples was below 3.7%. In order to limit the effect of the variability of renal function, urinary excretion of nitrite plus nitrate was corrected for urinary creatinine [9– 11,13 – 16] and expressed in Amol/mmol creatinine. Creatinine was measured with the picric acid method. 2.3. Statistical methods
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was 666 Amol/mmol creatinine (range: 564– 861 Amol/ mmol creatinine), more than twofolds higher as compared to the HC children of corresponding age (257 Amol/mmol creatinine, range: 44 – 877 Amol/mmol creatinine) (P = 0.014, Fig. 1). The three HC children with the highest UNOx values were 1, 4 and 6 months old. In the children aged 7 – 12 months, UNOx was similar in the ZS subjects (326 Amol/mmol creatinine, range: 174 – 532 Amol/mmol creatinine) and the HC children (360 Amol/mmol creatinine, range: 152 –827 Amol/mmol creatinine) (P = 0.96, Fig. 1). Urinary creatinine did not differ significantly between the ZS patients and HC children within the subjects aged 1 – 6 months (1.1 mmol/l, range: 0.2 – 2.0 mmol/l vs. 1.1 mmol/l, range: 0.6– 2.6 mmol/l for ZS and HC, respectively; P = 0.46) as well as within the children aged 7– 12 months (2.05 mmol/l, range: 0.9 – 5.3 mmol/l vs. 2.5 mmol/l, range: 1.1 – 10.6 mmol/l for ZS and HC, respectively; P = 0.35). In the ZS infants, UNOx correlated negatively with age (s = 0.75; P = 0.001, Fig. 2), which was not found in the HC group (s = 0.06; P = 0.6; not shown).
Children’s age is presented as mean F S.D. UNOx was compared between four groups of subjects, i.e., ZS children (n = 5) and HC children (n = 18) aged 1– 6 months, as well as ZS children (n = 6) and HC children (n = 12) aged 7 – 12 months. Since neither UNOx nor urinary creatinine exhibited normal distribution (checked by both Shapiro– Wilk’s W-test and Lilliefors’ test) within all the analyzed groups, their values are shown as median and range. Kruskal– Wallis ANOVA for ranks was used to test the presence of significant intergroup differences in UNOx taking into consideration the four above-mentioned groups. Comparisons of UNOx or creatinine between particular groups were performed with Mann –Whitney U-test. In order to assess the relationship between UNOx and age, Kendall’s rank correlation coefficient (s) was computed. A probability value of below 0.05 was considered significant.
3. Results Kruskal – Wallis ANOVA for ranks revealed the presence of a significant intergroup difference in UNOx (P = 0.035). In the ZS children aged 1 –6 months, UNOx
Fig. 1. Scatter plots of urinary excretion of nitrite plus nitrate (UNOx) in children with Zellweger syndrome (ZS) and in healthy children (HC) of two different age groups. Horizontal bars are medians. P values represent statistical significance (by Mann – Whitney U-test) vs. healthy children of corresponding age. Aditionally, UNOx was significantly lower in the older vs. younger ZS infants (P = 0.006), whereas in the control children, UNOx was comparable for both age goups (P = 0.5).
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Fig. 2. Correlation betweeen urinary excretion of nitrite plus nitrate (UNOx) and age in children with Zellweger syndrome (ZS). UNOx and age were negatively correlated in 11 children with Zellweger syndrome: Kendall’s rank correlation coefficient, s = 0.75, P = 0.001.
Accordingly, UNOx was significantly higher in the ZS children aged 7 – 12 months vs. the younger ZS subjects (P = 0.006, Fig. 1), whereas healthy infants had comparable UNOx in both age groups (P = 0.5, Fig. 1).
4. Discussion A highly accurate GC – MS method was used to determine UNOx in children suffering from ZS and healthy subjects. In the present study, UNOx in control subjects was comparable to the values measured by Tsukahara et al. [10] in the urine samples collected by a similar procedure from term healthy infants of comparable age (means: 410 and 420 Amol/mmol creatinine at the age of 4 and 7 months, respectively) kept on their normal diet, as our study subjects. In the first 6 months of life, UNOx was significantly elevated by a factor of 2 in ZS with the reference to the control group of similar age. As a result of the age-dependent fall of UNOx in ZS, the intergroup difference disappeared when UNOx was compared in the ZS infants aged 7 – 12 months and age-matched controls. It has been shown that metabolism of prostaglandins, thromboxane and leukotrienes is disturbed in patients with ZS most likely resulting in accumulation of the biologically highly active eicosanoids in blood and tissue [1– 3]. 8-iso-Prostaglandin F2a [4,5] as well
as products of the COX [8] and LOX [6,7] pathways have been reported to stimulate NO synthesis in vitro. It could, therefore, be speculated that the interplay between the numerous arachidonic acid pathways, notably COX and LOX, with the L-arginine/NO pathway might have resulted in the elevated NO generation found in the young ZS children. It might be further assumed that in older ZS infants the efficacy of these mechanisms and/or the responsiveness of NOS would be diminished, e.g., due to prolonged stimulation or other factors, thus, explaining the negative correlation between UNOx and age in ZS. To date, there are no data available on age-dependent excretion of 8-iso-prostaglandin F2a and products of the COX and LOX pathways in the ZS syndrome. To the best of our knowledge, there are no data on a tendency to hypotension during the first months of life in ZS children. UNOx, being an index of wholebody NO formation, might reflect increased NO production in nonvascular tissues. Alternatively, NO in the vascular tree of ZS children might be unable to produce enhanced vasodilation due to its scavenging by superoxide [20], enhanced ability of erythrocytes to oxidize NO into nitrate as shown for erythrocytes of normal neonates vs. erythrocytes isolated from adults [9] or simultaneous presence of an excess of vasoconstrictors, e.g., 8-iso-prostaglandin F2a [1,5] or prostaglandin F2a, the latter as yet not quantified in ZS in vivo. Peroxisomes contain enzymes which are involved not only in the formation of reactive oxygen species but they are also the main intracellular compartment of catalase and copper – zinc superoxide dismutase [21]. In addition, the following data suggest a pathogenetic role of oxidative stress in ZS. Fibroblasts from patients with ZS exhibit raised susceptibility to oxidative stress despite similar levels of superoxide and hydrogen peroxide, a very likely consequence of defective membrane plasmalogen synthesis [22]. If oxidative stress might be relevant to the pathophysiology of ZS, it seems plausible to assume that NO produced in excessive amounts in young infants with ZS, as suggested by the present study, could interact with superoxide forming toxic peroxynitrite [23], thus, exacerbating damage and possibly contributing to the progression of the disease. In addition, modulatory effect of NO on peroxisomal enzymes has been demonstrated [24]. Further studies are required to
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prove enhanced formation of peroxynitrite in early stages of ZS, e.g., by measuring plasma 3-nitrotyrosine, a product of peroxynitrite-mediated nitration of tyrosine residues in biomolecules [25].
[11]
[12]
Acknowledgements Dr. Mayatepek is supported by the Deutsche Forschungsgemeinschaft (Ma 1314/2-2 and Ma 1314/2-3). Dr. Surdacki was a recipient of a research fellowship from the Alexander von Humboldt Foundation. The laboratory and technical assistance of I. Fuchs and F.-M. Gutzki is gratefully acknowledged.
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