Original Paper Biol Neonate 1992;62:96-99
Department of Pediatrics, University of Munich, FRG, and Charles University of Plzen. CSFR
Keywords Urodilatin Atrial natriuretic peptide Human neonates
Urodilatin and Atrial Natriuretic Peptide Are Present in the Urine of Healthy Neonates and Young Infants Abstract Urine sampling has been proposed as noninvasive monitoring of plasma atrial natriuretic peptide (ANP) in neonates, assum ing urine contains only filtered plasma ANP. Recently, urodi latin, another natriuretic peptide, which is cross-reactive to ANP in the ANP radioimmunoassay (ANP-RIA), has been isolated from the urine of adults. We studied the urine of healthy term neonates and young infants for the presence of urodilatin. In all urine samples, we found three peaks of ANPRIA-reactive material: the first one in the position of urodila tin, the second one coeluting with synthetic ANP and a third late eluting peak, possibly containing degradation products. The physiological significance of these findings remains to be investigated.
Introduction Plasma ANP [a-human atrial natriuretic peptide, ANP-(99-l 26)], a diuretic, natri uretic and vasorelaxing hormone released by the heart, has been studied during the postna tal diuresis of neonates [ 1-4] and in newborns with cardiac disease [5, 6]. In these studies, material reactive to ANP radioimmunoassay (ANP-RIA) was also demonstrated in the urine of these patients [3, 4], and urine sam pling has been proposed as noninvasive moni toring of plasma ANP [7, 8], assuming that
urine contained only filtered plasma ANP. But a correlation between plasma and urine levels could not be demonstrated. The isola tion of another diuretic and natriuretic pep tide from the urine of adults, called urodila tin, ANP-(95-126) [9], which is cross-reactive to ANP in the ANP-RIA, suggests that urine contains not only filtered plasma ANP. Up to now, there are no pediatric data on urodilatin. This report presents the first results on the separation of urodilatin and ANP in the urine of healthy neonates and young infants.
Karl Bauer, MD Freie Universität Berlin Kinderklinik. Universitätsklinikum Steglitz Hindenburgdamm 30 D-W-1000 Berlin 45 (FRG)
© 1992 S. Karger AG. Basel 0006-3126/92/ 0623-0096S2.75/0
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/14/2019 8:15:50 AM
K. Bauera ./. Solch A. Timnik a A. Solcovab J. Weila
Urodilatin
a-hANP
■ Neonate □ Standards
Fig. 1. Separation of urinary urodilatin and ANP with HPLC followed by ANP-RIA. The figure shows chromatographies of stan dards (50 pg synthetic urodilatin and 50 pg ANP) added to preex tracted urine and of a representa tive urine sample of a neonate.
Subjects We studied the urine of 6 neonates and 4 young infants. The 6 healthy term neonates (gestational age 37-40 weeks) had a median body weight of 2,910 g (range 2,670-4,300 g) and a median postnatal age of 9 days (range 2-19 days). The 4 infants had a median body weight of 5,972 g (range 3,300-8,8 lOg) and a median age of 4 months (range 1-10 months). At the time of the investigation, all subjects were clinically stable with no signs of renal or cardiac disease. Nutri tional intake was ad libitum and ranged from exclusive breast feeding to formula feeding. The neonates showed an initial postnatal weight loss with normal subsequent weight gain. Serum sodium concentrations and blood pressure measurements were within the nor mal range. Urine was collected over a 12-hour period from 8 p.m. to 8 a.m. with adhesive plastic collection bags. Methods The method to separate urodilatin and ANP has previously been described by us [10]. Briefly, one ali quot of urine was used to measure total ANP-RIA reactivity with a rabbit antibody (Hedor, Herrsching, FRG) and another aliquot was used for the separation of the urodilatin and ANP with high performance liq uid chromatography (HPLC Merck Hitachi 665A-11 liquid chromatograph) using a reverse phase column
(HP Gel RP 7S 300, 150 X 4 mm internal diameter; Opergen, Heidelberg, FRG) and 8% isopropanol and 4% methanol in 0.01 M HC1 as a solvent (flow rate 0.7 ml/min). The eluate after HPLC was collected in fractions of 10 s, which were then separately analyzed by ANP-RIA. Synthetic ANP (Sigma, Deisenhofen, FRG) and urodilatin (Bissendorf Biochcmicals, Han nover, FRG) added to preextracted urine were used as standards. The recovery after HPLC was 68 ± 6% (range 60-74%) for 50 pg urodilatin or ANP (n = 5). We also measured sodium and creatinine in the urine samples.
Results HPLC followed by ANP-RIA resulted in a clear separation of standards (fig. 1). In all urine samples of neonates and infants, we found three peaks of ANP-RIA reactivity: one in the expected position of urodilatin, one coeluting with synthetic ANP and a small third one with a longer retention time (fig. 1). Urodilatin was always slightly less than ANP, i.e. the urodilatin/ANP ratio was < 1 (ta ble 1).
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Subjects and Methods
Table 1. Data of renal func tion, glomerular filtration rate (GFR) estimated by creatinine clearance, urinary excretion of total ANP-RIA-reactive material (ANPir excretion) and urodilatin/ ANP ratio in neonates and infants
Parameters
Neonates (n -6 )
Infants (n = 4)
Urine output, ml/1.73 m2/min GFR, ml/1.73 m2/min Na+excretion, pmol/1.73 m2/min ANPir excretion, pg/1.73 m2/min Urodilatin/ANP ratio
1.46 ±0.35 54 ±13 25± 12 1.1 ±0.5 0.9 ±0.1
1.30±0.70 ND ND 0.56±0.23 0.8±0.2
ND = Not determined.
Discussion We could demonstrate urodilatin and ANP in the urine of healthy term neonates and young infants. ANP and urodilatin belong to the same polypeptide family and have diuretic, natri uretic and smooth muscle relaxant properties. ANP is a peptide of 28 amino acids and urodi latin a peptide of 32 amino acids, containing the 28 amino acids of ANP plus an N-terminal extension by 4 amino acids. Urodilatin was isolated from urine and is most likely of renal origin [9], It produces a stronger diuresis and natriuresis than ANP [11] and takes part in the circadin regulation of sodium excretion [12], The separation of physiological amounts (< 1 0 0 pg) of urodilatin and ANP is difficult, because HPLC and detection by ultraviolet light, which can separate ANP and urodilatin, cannot detect picogram amounts, whereas conventional ANP-RIA, which can detect pi cogram amounts of peptides, cannot distin guish between ANP and urodilatin. By com bining HPLC for the separation and ANPRIA for the detection of the peptides, we
98
could study physiological amounts of ANP and urodilatin in urine with an acceptable recovery of about 70% [ 10]. Other attempts to characterize ANP-RIA-reactive material in urine are rare. In the urine of 2 neonates ahANP and fi-hANP, the antiparallel dimer of ANP, were found using gel permeation chro matography [4], We could not find a correlation between urinary ANP or urodilatin and urine output. Plasma ANP levels have been investigated with regard to postnatal diuresis/natriuresis in neonates. Plasma ANP showed a peak dur ing day 2-4 of life, but a significant correla tion between plasma levels and postnatal di uresis or natriuresis has not been substan tiated so far [ 13], ANP-RIA-reactive material in urine did not show postnatal changes corre sponding to those of plasma ANP [3], We could demonstrate the presence of ANP and urodilatin in the urine of term neo nates and young infants. The significance of this finding under various physiological or pathological conditions remains to be investi gated.
Acknowledgements This study was supported by a grant from the ‘Deutsche Forschungsgemeinschaft’ (We 1130/1-2). J.S. was a research fellow of the Alexander von Humboldt Foundation, Bonn, FRG.
Bauer/Solc/Timnik/Solcova/Weil
Urodilatin and ANP in Urine
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/14/2019 8:15:50 AM
We could not find a correlation between urine output or sodium excretion, and the uri nary excretion of ANP-RIA-reactive material or the urodilatin/ANP quotient (table 1).
References 6 Andersson S, Tikkanen I, Pesonen E. Wallgren El. Fyhrquist F: Atrial natriuretic peptide and atrial pres sures in newborns with transposi tion of the great arteries. Acta Pae diatr Scand 1988:77:72-75. 7 Marumo F, Sakamoto H, Ando K, Ishigami T, Kawakami M: A highly sensitive radioimmunoassay of atrial natriuretic peptide (ANP) in human plasma and urine. Biochem Biophys Res Commun 1986; 137: 231-236. 8 Ando K, Umetani N, Kurosawa T, Takeda S, Katoh Y, Marumo F: Atrial natriuretic peptide in human urine. Klin Wochenschr 1988:66: 768-772. 9 Schulz-Knappe P, Forssmann K, Hcrbst F. Hock D. Pipkorn R. Forssman WG: Isolation and struc tural analysis of ‘urodilatin’, a new peptide of the cardiodilatin-(ANP)family, extracted from human urine. Klin Wochenschr 1988:66:752— 759.
10 Sole J. Bauer K, Timnik A. Solcova A, Döhlemann C, Strom TM. Weil J: Combination of high-perfor mance liquid chromatography and radioimmunoassay for the measure ment of urodilatin and alpha-hANP in the urine of healthy males. Life Sei 1991;48:2451-2456. 11 Saxenhofer H, Raselli A, Weidmann P, Forssmann WG, Bub A, Ferrari P, Shaw SG: Urodilatin, a natri uretic factor from kidneys, can mod ify renal and cardiovascular func tion in men. Am J Physiol 1990:259: F832-F838. 12 Drummer C, Fiedler F, König A, Gerzer R: Urodilatin, a kidney-de rived natriuretic factor, is excreted with a circadian rhythm and is stim ulated by saline infusion in man. J Am Soc Nephrol 1990; 1:1109— 1113. 13 Semmekrot B, Guignard JP: Atrial natriuretic peptide during early hu man developement. Biol Neonate 1991:60:341-349.
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1 Kojima T, Hirata Y. Fukuda Y, Iwase S, Kobayashi Y: Plasma atrial natriuretic peptide and spontaneous diuresis in sick neonates. Arch Dis Child 1987;62:667-670. 2 Shaffer SG, Meade VM: Sodium balance and extracellular volume regulation in very low birth weight infants. J Pediatr 1989; 115:285— 290. 3 Timnik A, Weil J. Briickmann M. Strom T, Hauffe M, Heim JM. Gerzer R: Atrial natriuretic peptide in term newborn infants; in Forssmann WG. Scheuermann DW. Ait J (eds): Functional Morphology of the Endocrine Heart. Darmstadt. Steinkopff Verlag, 1989. pp 205-210. 4 Ito Y, Marumo F, Ando K. Hayashi M. Yamashita F: The physiological and biological significances of hu man atrial natriuretic peptide in ne onates. Acta Paediatr Scand 1990; 79:26-31. 5 Andersson S, Tikkanen I. Pesonen E. Meretoja O. Hynynen M. Fyhrquist F: Atrial natriuretic peptide in patent ductus arteriosus. Pediatr Reg 1987;21:396-398.
Department of Pediatrics, University of Munich, FRG, and Charles University of Plzen. CSFR
Keywords Urodilatin Atrial natriuretic peptide Human neonates
Urodilatin and Atrial Natriuretic Peptide Are Present in the Urine of Healthy Neonates and Young Infants Abstract Urine sampling has been proposed as noninvasive monitoring of plasma atrial natriuretic peptide (ANP) in neonates, assum ing urine contains only filtered plasma ANP. Recently, urodi latin, another natriuretic peptide, which is cross-reactive to ANP in the ANP radioimmunoassay (ANP-RIA), has been isolated from the urine of adults. We studied the urine of healthy term neonates and young infants for the presence of urodilatin. In all urine samples, we found three peaks of ANPRIA-reactive material: the first one in the position of urodila tin, the second one coeluting with synthetic ANP and a third late eluting peak, possibly containing degradation products. The physiological significance of these findings remains to be investigated.
Introduction Plasma ANP [a-human atrial natriuretic peptide, ANP-(99-l 26)], a diuretic, natri uretic and vasorelaxing hormone released by the heart, has been studied during the postna tal diuresis of neonates [ 1-4] and in newborns with cardiac disease [5, 6]. In these studies, material reactive to ANP radioimmunoassay (ANP-RIA) was also demonstrated in the urine of these patients [3, 4], and urine sam pling has been proposed as noninvasive moni toring of plasma ANP [7, 8], assuming that
urine contained only filtered plasma ANP. But a correlation between plasma and urine levels could not be demonstrated. The isola tion of another diuretic and natriuretic pep tide from the urine of adults, called urodila tin, ANP-(95-126) [9], which is cross-reactive to ANP in the ANP-RIA, suggests that urine contains not only filtered plasma ANP. Up to now, there are no pediatric data on urodilatin. This report presents the first results on the separation of urodilatin and ANP in the urine of healthy neonates and young infants.
Karl Bauer, MD Freie Universität Berlin Kinderklinik. Universitätsklinikum Steglitz Hindenburgdamm 30 D-W-1000 Berlin 45 (FRG)
© 1992 S. Karger AG. Basel 0006-3126/92/ 0623-0096S2.75/0
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/14/2019 8:15:50 AM
K. Bauera ./. Solch A. Timnik a A. Solcovab J. Weila
Urodilatin
a-hANP
■ Neonate □ Standards
Fig. 1. Separation of urinary urodilatin and ANP with HPLC followed by ANP-RIA. The figure shows chromatographies of stan dards (50 pg synthetic urodilatin and 50 pg ANP) added to preex tracted urine and of a representa tive urine sample of a neonate.
Subjects We studied the urine of 6 neonates and 4 young infants. The 6 healthy term neonates (gestational age 37-40 weeks) had a median body weight of 2,910 g (range 2,670-4,300 g) and a median postnatal age of 9 days (range 2-19 days). The 4 infants had a median body weight of 5,972 g (range 3,300-8,8 lOg) and a median age of 4 months (range 1-10 months). At the time of the investigation, all subjects were clinically stable with no signs of renal or cardiac disease. Nutri tional intake was ad libitum and ranged from exclusive breast feeding to formula feeding. The neonates showed an initial postnatal weight loss with normal subsequent weight gain. Serum sodium concentrations and blood pressure measurements were within the nor mal range. Urine was collected over a 12-hour period from 8 p.m. to 8 a.m. with adhesive plastic collection bags. Methods The method to separate urodilatin and ANP has previously been described by us [10]. Briefly, one ali quot of urine was used to measure total ANP-RIA reactivity with a rabbit antibody (Hedor, Herrsching, FRG) and another aliquot was used for the separation of the urodilatin and ANP with high performance liq uid chromatography (HPLC Merck Hitachi 665A-11 liquid chromatograph) using a reverse phase column
(HP Gel RP 7S 300, 150 X 4 mm internal diameter; Opergen, Heidelberg, FRG) and 8% isopropanol and 4% methanol in 0.01 M HC1 as a solvent (flow rate 0.7 ml/min). The eluate after HPLC was collected in fractions of 10 s, which were then separately analyzed by ANP-RIA. Synthetic ANP (Sigma, Deisenhofen, FRG) and urodilatin (Bissendorf Biochcmicals, Han nover, FRG) added to preextracted urine were used as standards. The recovery after HPLC was 68 ± 6% (range 60-74%) for 50 pg urodilatin or ANP (n = 5). We also measured sodium and creatinine in the urine samples.
Results HPLC followed by ANP-RIA resulted in a clear separation of standards (fig. 1). In all urine samples of neonates and infants, we found three peaks of ANP-RIA reactivity: one in the expected position of urodilatin, one coeluting with synthetic ANP and a small third one with a longer retention time (fig. 1). Urodilatin was always slightly less than ANP, i.e. the urodilatin/ANP ratio was < 1 (ta ble 1).
97
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/14/2019 8:15:50 AM
Subjects and Methods
Table 1. Data of renal func tion, glomerular filtration rate (GFR) estimated by creatinine clearance, urinary excretion of total ANP-RIA-reactive material (ANPir excretion) and urodilatin/ ANP ratio in neonates and infants
Parameters
Neonates (n -6 )
Infants (n = 4)
Urine output, ml/1.73 m2/min GFR, ml/1.73 m2/min Na+excretion, pmol/1.73 m2/min ANPir excretion, pg/1.73 m2/min Urodilatin/ANP ratio
1.46 ±0.35 54 ±13 25± 12 1.1 ±0.5 0.9 ±0.1
1.30±0.70 ND ND 0.56±0.23 0.8±0.2
ND = Not determined.
Discussion We could demonstrate urodilatin and ANP in the urine of healthy term neonates and young infants. ANP and urodilatin belong to the same polypeptide family and have diuretic, natri uretic and smooth muscle relaxant properties. ANP is a peptide of 28 amino acids and urodi latin a peptide of 32 amino acids, containing the 28 amino acids of ANP plus an N-terminal extension by 4 amino acids. Urodilatin was isolated from urine and is most likely of renal origin [9], It produces a stronger diuresis and natriuresis than ANP [11] and takes part in the circadin regulation of sodium excretion [12], The separation of physiological amounts (< 1 0 0 pg) of urodilatin and ANP is difficult, because HPLC and detection by ultraviolet light, which can separate ANP and urodilatin, cannot detect picogram amounts, whereas conventional ANP-RIA, which can detect pi cogram amounts of peptides, cannot distin guish between ANP and urodilatin. By com bining HPLC for the separation and ANPRIA for the detection of the peptides, we
98
could study physiological amounts of ANP and urodilatin in urine with an acceptable recovery of about 70% [ 10]. Other attempts to characterize ANP-RIA-reactive material in urine are rare. In the urine of 2 neonates ahANP and fi-hANP, the antiparallel dimer of ANP, were found using gel permeation chro matography [4], We could not find a correlation between urinary ANP or urodilatin and urine output. Plasma ANP levels have been investigated with regard to postnatal diuresis/natriuresis in neonates. Plasma ANP showed a peak dur ing day 2-4 of life, but a significant correla tion between plasma levels and postnatal di uresis or natriuresis has not been substan tiated so far [ 13], ANP-RIA-reactive material in urine did not show postnatal changes corre sponding to those of plasma ANP [3], We could demonstrate the presence of ANP and urodilatin in the urine of term neo nates and young infants. The significance of this finding under various physiological or pathological conditions remains to be investi gated.
Acknowledgements This study was supported by a grant from the ‘Deutsche Forschungsgemeinschaft’ (We 1130/1-2). J.S. was a research fellow of the Alexander von Humboldt Foundation, Bonn, FRG.
Bauer/Solc/Timnik/Solcova/Weil
Urodilatin and ANP in Urine
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/14/2019 8:15:50 AM
We could not find a correlation between urine output or sodium excretion, and the uri nary excretion of ANP-RIA-reactive material or the urodilatin/ANP quotient (table 1).
References 6 Andersson S, Tikkanen I, Pesonen E. Wallgren El. Fyhrquist F: Atrial natriuretic peptide and atrial pres sures in newborns with transposi tion of the great arteries. Acta Pae diatr Scand 1988:77:72-75. 7 Marumo F, Sakamoto H, Ando K, Ishigami T, Kawakami M: A highly sensitive radioimmunoassay of atrial natriuretic peptide (ANP) in human plasma and urine. Biochem Biophys Res Commun 1986; 137: 231-236. 8 Ando K, Umetani N, Kurosawa T, Takeda S, Katoh Y, Marumo F: Atrial natriuretic peptide in human urine. Klin Wochenschr 1988:66: 768-772. 9 Schulz-Knappe P, Forssmann K, Hcrbst F. Hock D. Pipkorn R. Forssman WG: Isolation and struc tural analysis of ‘urodilatin’, a new peptide of the cardiodilatin-(ANP)family, extracted from human urine. Klin Wochenschr 1988:66:752— 759.
10 Sole J. Bauer K, Timnik A. Solcova A, Döhlemann C, Strom TM. Weil J: Combination of high-perfor mance liquid chromatography and radioimmunoassay for the measure ment of urodilatin and alpha-hANP in the urine of healthy males. Life Sei 1991;48:2451-2456. 11 Saxenhofer H, Raselli A, Weidmann P, Forssmann WG, Bub A, Ferrari P, Shaw SG: Urodilatin, a natri uretic factor from kidneys, can mod ify renal and cardiovascular func tion in men. Am J Physiol 1990:259: F832-F838. 12 Drummer C, Fiedler F, König A, Gerzer R: Urodilatin, a kidney-de rived natriuretic factor, is excreted with a circadian rhythm and is stim ulated by saline infusion in man. J Am Soc Nephrol 1990; 1:1109— 1113. 13 Semmekrot B, Guignard JP: Atrial natriuretic peptide during early hu man developement. Biol Neonate 1991:60:341-349.
99
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/14/2019 8:15:50 AM
1 Kojima T, Hirata Y. Fukuda Y, Iwase S, Kobayashi Y: Plasma atrial natriuretic peptide and spontaneous diuresis in sick neonates. Arch Dis Child 1987;62:667-670. 2 Shaffer SG, Meade VM: Sodium balance and extracellular volume regulation in very low birth weight infants. J Pediatr 1989; 115:285— 290. 3 Timnik A, Weil J. Briickmann M. Strom T, Hauffe M, Heim JM. Gerzer R: Atrial natriuretic peptide in term newborn infants; in Forssmann WG. Scheuermann DW. Ait J (eds): Functional Morphology of the Endocrine Heart. Darmstadt. Steinkopff Verlag, 1989. pp 205-210. 4 Ito Y, Marumo F, Ando K. Hayashi M. Yamashita F: The physiological and biological significances of hu man atrial natriuretic peptide in ne onates. Acta Paediatr Scand 1990; 79:26-31. 5 Andersson S, Tikkanen I. Pesonen E. Meretoja O. Hynynen M. Fyhrquist F: Atrial natriuretic peptide in patent ductus arteriosus. Pediatr Reg 1987;21:396-398.
