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Increased urinary excretion of LTB4 and ω-carboxy-LTB4 in patients with Zellweger syndrome
Clinica Chimica Acta 282 (1999) 151–155
Increased urinary excretion of LTB 4 and v-carboxyLTB 4 in patients with Zellweger syndrome Ertan Mayatepek*, Bianca Flock Division of Metabolic Diseases, University Children’ s Hospital, Im Neuenheimer Feld 150, D-69120 Heidelberg, Germany Received 12 October 1998; received in revised form 8 January 1999; accepted 14 January 1999
Abstract The metabolic inactivation of leukotrienes proceeds by b-oxidation from the v-end. We investigated the importance of peroxisomes and mitochondria in LTB 4 oxidation in vivo. LTB 4 and its oxidation products were analysed after high-performance liquid chromatography separation by immunoassays and gas chromatography–mass spectrometry in the urine of patients with Zellweger syndrome, patients with long-chain acyl CoA dehydrogenase deficiency, and healthy controls. LTB 4 (median 97; range 35–238 nmol / mol creatinine) and its v-oxidation product v-carboxy-LTB 4 (median 898; range 267–4583 nmol / mol creatinine) were present and significantly increased in the urine of all patients with Zellweger syndrome compared to the controls (P , 0.01). In contrast, LTB 4 and v-carboxy-LTB 4 were below the detection limit ( , 5 nmol / mol creatinine) in patients with long-chain acyl CoA dehydrogenase deficiency and healthy controls. The b-oxidation product v-carboxy-tetranor-LTB 3 was neither detectable in the urine of patients with Zellweger syndrome, patients with long-chain acyl CoA dehydrogenase deficiency nor in the controls ( , 5 nmol / mol creatinine). Analysis of urinary leukotrienes represents an additional diagnostic tool in peroxisome deficiency disorders. Furthermore, these results clearly underline the essential role of peroxisomes in the oxidation of LTB 4 in humans. 1999 Elsevier Science B.V. All rights reserved. Keywords: Leukotrienes; Long-chain acyl CoA dehydrogenase deficiency; Mitochondria; Peroxisomes; Zellweger syndrome
*Corresponding author. Tel.: 1 49-6221-562-311; fax: 1 49-6221-563-651. E-mail address: ertan [email protected] (E. Mayatepek) ] 0009-8981 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 99 )00015-7
152
E. Mayatepek, B. Flock / Clinica Chimica Acta 282 (1999) 151 – 155
1. Introduction Leukotrienes (LTs) are potent lipid mediators derived from arachidonate in the 5-lipoxygenase pathway. The metabolic inactivation and degradation of LTs is of major importance because of the biological potency of these mediators which are generated under various pathophysiological conditions [1]. LTB 4 is chemotactically active, whereas the cysteinyl LTs (LTC 4 , LTD 4 , LTE 4 ) increase microvascular permeability and induce smooth muscle contraction. The inactivation of LTs is initiated by oxidation of the fatty acid chain at the v-end and followed by b-oxidation from the v-end [2,3] (Fig. 1). The increased
Fig. 1. Catabolism of LTB 4 . v-Oxidation of LTB 4 yields v-hydroxy-LTB 4 and v-carboxy-LTB 4 . Subsequent stepwise shortening of the fatty acid chain by b-oxidation from the v-end leads to v-carboxy-dinor-LTB 4 , v-carboxy-tetranor-LTB 3 , and more polar b-oxidation products.
E. Mayatepek, B. Flock / Clinica Chimica Acta 282 (1999) 151 – 155
153
degradation of LTs in the b-oxidation pathway after treatment of rats with clofibrate, an inducer of peroxisome proliferation, led to the suggestion that b-oxidation of LTs is localized in peroxisomes [4]. Recently, we have shown that the degradation of LTs is impaired in patients with peroxisome deficiency disorders [5]. However, in vitro studies have also shown that the cysteinyl LTs are exclusively degraded in peroxisomes, whereas v-carboxy-LTB 4 is oxidized both in isolated peroxisomes and mitochondria [4]. The aim of the present study was to investigate the importance of peroxisomes and mitochondria in LTB 4 oxidation in vivo. We therefore performed quantitative analyses of LTB 4 and its oxidation products in patients with Zellweger syndrome (ZS), a peroxisomal b-oxidation deficiency disorder, in patients with LCAD (long-chain acyl CoA dehydrogenase) deficiency, a mitochondrial b-oxidation disorder, and healthy controls.
2. Patients and methods Excretion of endogenous LTB 4 and its metabolites was studied in the urine of 10 patients with ZS (age range 3–20 months), four patients with LCAD deficiency (age range 1–8 months), and 25 healthy control subjects (age range 1–24 months). All patients with ZS exhibited the characteristic clinical and biochemical abnormalities described for ZS [6]. 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 biosynthesis in cultured skin fibroblasts. Mitochondrial b-oxidation activity was assayed in cultured fibroblasts using [1- 14 C]palmitic acid and found to be in the range of normal subjects. All patients with a defect of mitochondrial long-chain fatty acid oxidation had LCAD deficiency as measured in cultured skin fibroblasts. Urine was obtained from spontaneous micturition and stored at 2 808C until analysis. Urinary LTB 4 and its v- and b-oxidation metabolites were separated by reversed-phase HPLC and subsequently quantified by immunoassays and gas chromatography–mass spectrometry using [ 18 O]-labeled LTs as internal standards [5].
3. Results and discussion The urinary concentrations of LTB 4 and its oxidation products for the patients with ZS, patients with LCAD deficiency and healthy controls are summarized in Table 1. In general, LTB 4 and its v-oxidation product v-carboxy-LTB 4 were present
154
E. Mayatepek, B. Flock / Clinica Chimica Acta 282 (1999) 151 – 155
Table 1 Concentrations of LTB 4 , v-carboxy-LTB 4 and v-carboxy-tetranor-LTB 3 in urine of patients with Zellweger syndrome, LCAD deficiency and normal subjects. Leukotriene (nmol / mol creatinine)
Zellweger syndrome (n 5 10)a
LCAD deficiency (n 5 4)
Normal subjects (n 5 25)
LTB 4
97 (35–238)b
, 5c
,5
v-Carboxy-LTB 4
898 (267–4583)b
,5
,5
v-Carboxy-tetranor-LTB 3
,5
,5
,5
a
Values given are median; range is given in parentheses. P , 0.01 compared to patients with LCAD deficiency and normal subjects (Wilcoxon–Mann– Whitney test). c 5 nmol / mol creatinine is the limit of detection calculated from urines with up to 10 mmol / l creatinine. b
and significantly increased in the urine of all patients with ZS compared to the controls. In contrast, analyses of urine samples from LCAD-deficient patients and normal subjects showed that both LTB 4 and v-carboxy-LTB 4 were below the detection limit in these groups. The b-oxidation product v-carboxy-tetranorLTB 3 was neither detectable in the urine of patients with ZS, patients with LCAD deficiency nor in the controls. After intravenous administration of [ 3 H]LTB 4 , this LT and its metabolites are not detectable in the urine due to rapid and extensive degradation [7]. Additionally, under physiological conditions endogenous LTB 4 and its metabolites are not detectable in human urine (Table 1). The increased levels of the biologically active, proinflammatory mediator LTB 4 in patients with ZS might be of pathophysiological significance in the course of the disease [5]. We here present for the first time quantitative levels of v-carboxy-LTB 4 in human urine. Peroxisomal deficiency is the first and so far only condition with a pronounced urinary excretion of LTB 4 and v-carboxyLTB 4 . Analyses of these metabolites may serve as an additional diagnostic tool. In contrast to previous in vitro data [4], our results provide further evidence that the b-oxidation process of LTB 4 occurs in the peroxisomes rather than in the mitochondria. As opposed to the LTB 4 and v-carboxy-LTB 4 excretion by ZS patients, the mitochondrial long-chain fatty acid b-oxidation-deficient patients showed, in analogy to normal subjects, no detectable amounts of these metabolites. Thus, patients with LCAD deficiency are able to oxidize LTB 4 and v-carboxy-LTB 4 , indicating that in vivo this oxidation process does not take place in the mitochondria. All together, these results clearly underline the essential role of peroxisomes in the catabolism of LTB 4 in humans.
E. Mayatepek, B. Flock / Clinica Chimica Acta 282 (1999) 151 – 155
155
Acknowledgements The authors are grateful to R.B.H. Schutgens and R.J.A. Wanders for performing the specific biochemical diagnosis in the patients with Zellweger syndrome. This study was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn, Germany, to Dr. Mayatepek (Ma 1314 / 2-2).
References [1] Mayatepek E, Hoffmann GF. Leukotrienes: biosynthesis, metabolism and pathophysiologic significance. Pediatr Res 1995;37:1–9. [2] Harper TW, Garrity MJ, Murphy RC. Metabolism of leukotriene B 4 in isolated rat hepatocytes: identification of a novel 18-carboxy-19,20-dinor leukotriene B 4 metabolite. J Biol Chem 1986;261:5414–8. [3] Sala AN, Voelkel N, Maclouf J, Murphy RC. Leukotriene E 4 elimination and metabolism in normal human subjects. J Biol Chem 1990;265:21771–8. ¨ [4] Jedlitschky G, Huber M, Volkl A et al. Peroxisomal degradation of leukotrienes by b-oxidation from the v-end. J Biol Chem 1991;266:24763–72. [5] Mayatepek E, Lehmann WD, Fauler J et al. Impaired degradation of leukotrienes in patients with peroxisome deficiency disorders. J Clin Invest 1993;91:881–8. [6] Lazarow PB, Moser HW. Disorders of peroxisome biogenesis. in: Scriver CR, Beaudet AL, Sly WS, Valle D, editors, The metabolic and molecular bases of inherited disease, 7th ed., McGraw-Hill, New York, 1995, pp. 2287–324. [7] Serafin WE, Oates JA, Hubbard WC. Metabolism of leukotriene B 4 in the monkey. Identification of the principal nonvolatile metabolite in the urine. Prostaglandins 1984;27:899–911.
Increased urinary excretion of LTB 4 and v-carboxyLTB 4 in patients with Zellweger syndrome Ertan Mayatepek*, Bianca Flock Division of Metabolic Diseases, University Children’ s Hospital, Im Neuenheimer Feld 150, D-69120 Heidelberg, Germany Received 12 October 1998; received in revised form 8 January 1999; accepted 14 January 1999
Abstract The metabolic inactivation of leukotrienes proceeds by b-oxidation from the v-end. We investigated the importance of peroxisomes and mitochondria in LTB 4 oxidation in vivo. LTB 4 and its oxidation products were analysed after high-performance liquid chromatography separation by immunoassays and gas chromatography–mass spectrometry in the urine of patients with Zellweger syndrome, patients with long-chain acyl CoA dehydrogenase deficiency, and healthy controls. LTB 4 (median 97; range 35–238 nmol / mol creatinine) and its v-oxidation product v-carboxy-LTB 4 (median 898; range 267–4583 nmol / mol creatinine) were present and significantly increased in the urine of all patients with Zellweger syndrome compared to the controls (P , 0.01). In contrast, LTB 4 and v-carboxy-LTB 4 were below the detection limit ( , 5 nmol / mol creatinine) in patients with long-chain acyl CoA dehydrogenase deficiency and healthy controls. The b-oxidation product v-carboxy-tetranor-LTB 3 was neither detectable in the urine of patients with Zellweger syndrome, patients with long-chain acyl CoA dehydrogenase deficiency nor in the controls ( , 5 nmol / mol creatinine). Analysis of urinary leukotrienes represents an additional diagnostic tool in peroxisome deficiency disorders. Furthermore, these results clearly underline the essential role of peroxisomes in the oxidation of LTB 4 in humans. 1999 Elsevier Science B.V. All rights reserved. Keywords: Leukotrienes; Long-chain acyl CoA dehydrogenase deficiency; Mitochondria; Peroxisomes; Zellweger syndrome
*Corresponding author. Tel.: 1 49-6221-562-311; fax: 1 49-6221-563-651. E-mail address: ertan [email protected] (E. Mayatepek) ] 0009-8981 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 99 )00015-7
152
E. Mayatepek, B. Flock / Clinica Chimica Acta 282 (1999) 151 – 155
1. Introduction Leukotrienes (LTs) are potent lipid mediators derived from arachidonate in the 5-lipoxygenase pathway. The metabolic inactivation and degradation of LTs is of major importance because of the biological potency of these mediators which are generated under various pathophysiological conditions [1]. LTB 4 is chemotactically active, whereas the cysteinyl LTs (LTC 4 , LTD 4 , LTE 4 ) increase microvascular permeability and induce smooth muscle contraction. The inactivation of LTs is initiated by oxidation of the fatty acid chain at the v-end and followed by b-oxidation from the v-end [2,3] (Fig. 1). The increased
Fig. 1. Catabolism of LTB 4 . v-Oxidation of LTB 4 yields v-hydroxy-LTB 4 and v-carboxy-LTB 4 . Subsequent stepwise shortening of the fatty acid chain by b-oxidation from the v-end leads to v-carboxy-dinor-LTB 4 , v-carboxy-tetranor-LTB 3 , and more polar b-oxidation products.
E. Mayatepek, B. Flock / Clinica Chimica Acta 282 (1999) 151 – 155
153
degradation of LTs in the b-oxidation pathway after treatment of rats with clofibrate, an inducer of peroxisome proliferation, led to the suggestion that b-oxidation of LTs is localized in peroxisomes [4]. Recently, we have shown that the degradation of LTs is impaired in patients with peroxisome deficiency disorders [5]. However, in vitro studies have also shown that the cysteinyl LTs are exclusively degraded in peroxisomes, whereas v-carboxy-LTB 4 is oxidized both in isolated peroxisomes and mitochondria [4]. The aim of the present study was to investigate the importance of peroxisomes and mitochondria in LTB 4 oxidation in vivo. We therefore performed quantitative analyses of LTB 4 and its oxidation products in patients with Zellweger syndrome (ZS), a peroxisomal b-oxidation deficiency disorder, in patients with LCAD (long-chain acyl CoA dehydrogenase) deficiency, a mitochondrial b-oxidation disorder, and healthy controls.
2. Patients and methods Excretion of endogenous LTB 4 and its metabolites was studied in the urine of 10 patients with ZS (age range 3–20 months), four patients with LCAD deficiency (age range 1–8 months), and 25 healthy control subjects (age range 1–24 months). All patients with ZS exhibited the characteristic clinical and biochemical abnormalities described for ZS [6]. 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 biosynthesis in cultured skin fibroblasts. Mitochondrial b-oxidation activity was assayed in cultured fibroblasts using [1- 14 C]palmitic acid and found to be in the range of normal subjects. All patients with a defect of mitochondrial long-chain fatty acid oxidation had LCAD deficiency as measured in cultured skin fibroblasts. Urine was obtained from spontaneous micturition and stored at 2 808C until analysis. Urinary LTB 4 and its v- and b-oxidation metabolites were separated by reversed-phase HPLC and subsequently quantified by immunoassays and gas chromatography–mass spectrometry using [ 18 O]-labeled LTs as internal standards [5].
3. Results and discussion The urinary concentrations of LTB 4 and its oxidation products for the patients with ZS, patients with LCAD deficiency and healthy controls are summarized in Table 1. In general, LTB 4 and its v-oxidation product v-carboxy-LTB 4 were present
154
E. Mayatepek, B. Flock / Clinica Chimica Acta 282 (1999) 151 – 155
Table 1 Concentrations of LTB 4 , v-carboxy-LTB 4 and v-carboxy-tetranor-LTB 3 in urine of patients with Zellweger syndrome, LCAD deficiency and normal subjects. Leukotriene (nmol / mol creatinine)
Zellweger syndrome (n 5 10)a
LCAD deficiency (n 5 4)
Normal subjects (n 5 25)
LTB 4
97 (35–238)b
, 5c
,5
v-Carboxy-LTB 4
898 (267–4583)b
,5
,5
v-Carboxy-tetranor-LTB 3
,5
,5
,5
a
Values given are median; range is given in parentheses. P , 0.01 compared to patients with LCAD deficiency and normal subjects (Wilcoxon–Mann– Whitney test). c 5 nmol / mol creatinine is the limit of detection calculated from urines with up to 10 mmol / l creatinine. b
and significantly increased in the urine of all patients with ZS compared to the controls. In contrast, analyses of urine samples from LCAD-deficient patients and normal subjects showed that both LTB 4 and v-carboxy-LTB 4 were below the detection limit in these groups. The b-oxidation product v-carboxy-tetranorLTB 3 was neither detectable in the urine of patients with ZS, patients with LCAD deficiency nor in the controls. After intravenous administration of [ 3 H]LTB 4 , this LT and its metabolites are not detectable in the urine due to rapid and extensive degradation [7]. Additionally, under physiological conditions endogenous LTB 4 and its metabolites are not detectable in human urine (Table 1). The increased levels of the biologically active, proinflammatory mediator LTB 4 in patients with ZS might be of pathophysiological significance in the course of the disease [5]. We here present for the first time quantitative levels of v-carboxy-LTB 4 in human urine. Peroxisomal deficiency is the first and so far only condition with a pronounced urinary excretion of LTB 4 and v-carboxyLTB 4 . Analyses of these metabolites may serve as an additional diagnostic tool. In contrast to previous in vitro data [4], our results provide further evidence that the b-oxidation process of LTB 4 occurs in the peroxisomes rather than in the mitochondria. As opposed to the LTB 4 and v-carboxy-LTB 4 excretion by ZS patients, the mitochondrial long-chain fatty acid b-oxidation-deficient patients showed, in analogy to normal subjects, no detectable amounts of these metabolites. Thus, patients with LCAD deficiency are able to oxidize LTB 4 and v-carboxy-LTB 4 , indicating that in vivo this oxidation process does not take place in the mitochondria. All together, these results clearly underline the essential role of peroxisomes in the catabolism of LTB 4 in humans.
E. Mayatepek, B. Flock / Clinica Chimica Acta 282 (1999) 151 – 155
155
Acknowledgements The authors are grateful to R.B.H. Schutgens and R.J.A. Wanders for performing the specific biochemical diagnosis in the patients with Zellweger syndrome. This study was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn, Germany, to Dr. Mayatepek (Ma 1314 / 2-2).
References [1] Mayatepek E, Hoffmann GF. Leukotrienes: biosynthesis, metabolism and pathophysiologic significance. Pediatr Res 1995;37:1–9. [2] Harper TW, Garrity MJ, Murphy RC. Metabolism of leukotriene B 4 in isolated rat hepatocytes: identification of a novel 18-carboxy-19,20-dinor leukotriene B 4 metabolite. J Biol Chem 1986;261:5414–8. [3] Sala AN, Voelkel N, Maclouf J, Murphy RC. Leukotriene E 4 elimination and metabolism in normal human subjects. J Biol Chem 1990;265:21771–8. ¨ [4] Jedlitschky G, Huber M, Volkl A et al. Peroxisomal degradation of leukotrienes by b-oxidation from the v-end. J Biol Chem 1991;266:24763–72. [5] Mayatepek E, Lehmann WD, Fauler J et al. Impaired degradation of leukotrienes in patients with peroxisome deficiency disorders. J Clin Invest 1993;91:881–8. [6] Lazarow PB, Moser HW. Disorders of peroxisome biogenesis. in: Scriver CR, Beaudet AL, Sly WS, Valle D, editors, The metabolic and molecular bases of inherited disease, 7th ed., McGraw-Hill, New York, 1995, pp. 2287–324. [7] Serafin WE, Oates JA, Hubbard WC. Metabolism of leukotriene B 4 in the monkey. Identification of the principal nonvolatile metabolite in the urine. Prostaglandins 1984;27:899–911.