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N-Methyladrenaline: age-dependent urinary excretion, perinatal organ content and relation with ‘classical’ catecholamines
Clinica Chimica Acta 238 (1995) 137-150
ELSEVIER
N-Methyladrenaline: age-dependent urinary excretion, perinatal organ content and relation with ‘classical’ catecholamines Ido P. Kerna*“, Coen J.F. Schootsb, Corrie E.M. Gidding’, Albert Okkenc, Jan G. Aarnoudsed, Frits A.J. Muskiet” “Central
Laboratory
for
Clinical
Chemistry,
P.O. Box 30001, bDepartment
of Pathology,
University
Universiry
9700 RB Groningen. and University
and University
Hospital
of Groningen,
The Netherlands
Hospital
of Groningen,
9700 RB Groningen.
of Groningen,
9700 RB Groningen.
The Netherlancis ‘Departmeni
of Paediatrics.
University
and University
Hospital
The Netherlands dDepartment
of Obstetrics
and Gynaecology,
University
9700 RB Groningen,
and University
Hospital
of Groningen,
The Netherlands
Received 9 January 1995; accepted 30 March 1995
Abstract Using high performance liquid chromatography with electrochemical detection we determined free dopamine, noradrenaline, adrenaline and N-methyladrenaline in: (1) urines from newborns (n = 32), children (n = 45) and adults (n = 19) and (2) adrenals, organ of Zuckerkandl, dorsal roots and perirenal brown adipose tissue from deceased fetuses (n = 2), very premature (n = 6) and term (n = 2) newborns and infants (n = 2). Data from children and adults showed that contributions of adrenaline and N-methyladrenaline to the sum of urinary free catecholamines increase with age. Relative amounts of adrenaline and N-methyladrenaline increased in both adrenal and extra adrenal chromafin tissues from late gestation up to several months of postnatal life. Increase of adrenal N-methyladrenaline content follows endocrine maturation of the medulla, phenylethanolamine-N-methyltransferase induction and subsequent adrenaline synthesis. Relative amounts of N-methyladrenaline in extra adrenal chromaffin tissue increase in a period that is associated with its regression. Further investigations are necessary to elucidate the function and possible clinical chemical usefulness of Nmethyladrenaline.
-. Corresponding author.
??
0009-8981/95/%09.50 0 1995 Elsevier Science B.V. All rights reserved SD1 0009-898 I (95)06082-O
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Keywords: Adrenaline; Phenylethanolamine-Nmethyltransferase; Extra adrenal chromaffin tissue; High performance liquid chromatography
1. Introduction
N-methyladrenaline (N-MA) is formed by N-methylation of adrenaline (A). The reaction is catalysed by phenylethanolamine-Nmethyltransferase (PNMT, EC 2.1.1.6). In 1960, Axelrod [l] was the first to show that N-MA is a naturally occurring catecholamine, demonstrable in the adrenal medulla of a variety of animal species. Following intravenous N-MA administration to rats, large quantities of N-methylmetanephrine (N-MM; the 3-0-methylated metabolite of N-MA) were recovered from urine in both free and conjugated forms. Axelrod suggested the synthesis and metabolism of N-MA to occur according to: A - N-MA - N-MM. Early pharmacological experiments revealed that N-MA exhibits properties that are at best described as a mixture of those of dopamine (DA), noradrenaline (NA) and A. These effects, e.g. increases of blood glucose and diastolic and systolic blood pressures and decrease of pulse rate, were observed at lo-40 times higher doses than A and NA and at comparable DA dose [3]. N-methyladrenaline effects on systolic blood pressure lasted longer than those of A [2]. Mueller and Horwitz [3] showed that at an intravenous dose of 180 &kg body weight of the L-enantiomer of NMA, blood glucose levels and both systolic and diastolic blood pressures increased, whereas pulse rate decreased. No significant influences on plasma free fatty acid levels were found. Evans [4] showed that, in contrast to its metabolite N-MM, N-MA is incapable of passing the blood brain barrier in rats. Since its first demonstration, the number of clinical chemical studies on N-MA has been scanty. Apart from measurements of N-MA in urine samples from normotensive and hypertensive adults and patients with phaeochromocytoma [5-71, and determinations of urinary N-MM in postoperative patients [8] and certain psychiatric patients [9], virtually no data on its (patho)physiological significance or clinical chemical importance have been reported. Urinary N-MA levels of normotensive and hypertensive adults were usually found to correlate with those of A [7]. However, high urinary excretion levels of A did not necessarily coincide with increased N-MA levels. Some investigators pointed to the difficulty, or inability, of routinely employed catecholamine analyses (e.g. trihydroxyindole and radioenzymatic (COMT) methods) to detect or quantify N-MA [7,10]. Since urinary N-MA and A levels are similar [7], incomplete separation of N-MA from A in commonly applied catecholamine assays by high performance liquid chromatography (HPLC) with electrochemical detection leads to overestimation of A. Using an HPLC-electrochemical detection method that enables complete separation of N-MA and A, we determined age-dependent urinary excretion levels of NMA and related the results to those of the ‘classical’ catecholamines, DA, NA and A. Assays were performed in samples collected from newborns, children and adults. In an attempt to disclose the origin of N-MA and its synthesis in relation to the other
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catecholamines, determinations were additionally performed in adrenals, organs of Zuckerkandl, dorsal roots and perirenal brown adipose tissue from deceased fetuses, very premature neonates, neonates and infants.
2. Materials and methods 2.1. Reagents N-MA was kindly donated by Dr. M. Schussler van Hees (University of Leiden, The Netherlands). DA, NA, A and 3,4-dihydroxybenzylamine were obtained from Sigma #(St.Louis, MO, USA), HPLC grade octanesulphonic acid was from Fisons (Loughborough, UK), citric acid monohydrate and Na2HP04.2H20 from Merck (Darmstadt, Germany) and acetonitrile from Rathburn (Walkbum, UK). Ethylenediamine tetraacetic acid (EDTA) and Na2Sz05 were purchased from BDH (Poole, UK). Pentafluoropropionic anhydride (PFPA) was from Pierce (Rockford, IL, USA). All reagents were of analytical grade. Glass distilled water was used throughout. 2.2. Urine samples For assessment of age-dependent reference values of urinary free DA, NA, A and N-MA, samples from neonates, children and adults were collected without dietary restrictions and in an undefined metabolic state. Using sterile paediatric urine collection bags (Urinocol, Biotrol Pharma, Paris, France), untimed urine samples were collected from 32 premature and term hospital&d neonates (14 females, 18 males; median gestational age 32 weeks; range 27-40). Twenty-three neonates were spontaneously delivered, whereas eight were born by caesarean section. Hospitalisation took place for a variety of reasons, including caesarean section, premature birth, respiratory distress syndrome, hyperbilirubinaemia, generalised infections, cardiovascular disease, gastrointestinal disease and combinations of these. Urines were collected for approximately 12 h on the first day (n = 2) up to the 77th day after birth (median 27 days). Immediately after c:ompletion of collection Na2S205 and EDTA were added to final concentrations of about 0.5 g/l. Twenty-four hour urine collections of 45 apparently healthy children (22 females, 23 males; median age 9 years; range 4- 12) and 19 healthy adults ( 10 females, 9 males; median age 41 years; range 27-63 years) were completed in 2-l brown polypropylene bottles (Sarstedt, Nuembrecht, Germany), containing about 250 mg each of Na,S205 and EDTA as preservatives. All urine specimens were acidified to pH 4 with acetic acid before freezing. Samples were stored at -2O”C, or -80°C and analysed within 1 month, or 1 year, respectively, after collection. Addition of Na,S205 and EDTA and subsequent storage at -80°C stabilises all catecholamines and metabolites for a year or more [ 111. Urinary creatinine levels, used to quantify excretion in terms of creatinine, were measured by a picric acid method on an SMA-2 analyser (Technicon Instruments, Tarrytown, NY, I-ISA).
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2.3. Tissue samples
The studied group of deceased fetuses, neonates and infants was composed of three subgroups: (1) two fetuses and six very premature neonates (5 females, 3 males, median gestational age 27 weeks; range 18-30 weeks); the two fetuses were aborted because of chromosomal anomalies, diagnosed by amniocentesis; the six very Qremature neonates were spontaneously live born but died within 48 h (two following intrauterine infections and four from hyaline membrane disease and/or lung hypoplasia); (2) two spontaneously born term neonates (one female and one male, both delivered at a gestational age of 40 weeks); they died within 24 h from congenital heart disease and multiple congenital abnormalities, respectively; (3) two male infants, who died at 1 and 9 postnatal months from congenital heart disease and sudden infant death syndrome, respectively. Consent was obtained from the parents. The study protocol conformed to local ethical standards and the Helsinki declaration of 1975, as revised in 1983. As soon as possible after death the bodies were stored in a refrigerator at 4°C. Gestational age was calculated from the time of last menstruation and confirmed by anthropometric characteristics (crown-heel length, body weight and skeletal- and brain maturation). Specimens from adrenals, organ of Zuckerkandl, dorsal roots and Qerirenal brown adipose tissue were taken within 24 h after death. Histological characterisation was performed by macroscopic and microscopic examination. Dissected samples were weighed and transferred to 0.01 moY1acetic acid, containing 10 g/l each of Na,S205 and EDTA as preservatives. They were stored at -2O’C until analysis. 2.4. Prepurification Isolation of free catecholamines from loo-p1 aliquots of urine from infants was performed by Sephadex G-10 minicolumn extraction, as described by Westerink et al. [12]. The Sephadex eluates were further purified by paired ion extraction, according to Smedes et al. [13]. Prepurification of 5-ml urine samples from children and adults was accomplished by atmospheric pressure cation exchange chromatography (‘catecholamine columns’, Bio-Rad Laboratories, Munich, Germany). In all cases 3,4-dihydroxybenzylamine served as an internal standard. After the addition of 1 ng 3,4-dihydroxybenzylamine (internal standard), 50-100mg amounts of frozen tissue were sonicated in l-2 ml 0.01 mol/l acetic acid (containing 10 g/I each of Na2S20S and EDTA) for 30 s at 0°C. Free catecholamines were isolated from the homogenates by paired ion extraction [ 131. 2.5. Analysis and quantification Free catecholamine (DA, NA, A and N-MA) contents in Qrepurified urines and tissues were determined by HPLC with electrochemical detection. The HPLC system consisted of an LKB type 2150 pump (Bromma, Sweden) and a 220 x 4.6 (i.d.) mm RP-8 reversed phase column filled with 5-pm spherical particles (Brownlee Labs, Santa Clara, CA, USA). The mobile phase was composed of 34 mmol/l citric acid, 42 mmol/I Na2HP04, 5.5 mrnol/l octanesulphonic acid and 160 ml methanol per litre (QH 4.2). The electrochemical detector (type Coulochem II, ESA, Bedford, MA, USA) was equipped with a 5011 high sensitivity cell, set at 0.35 V.
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2.6. Mass spectrometric identification of N-methyladrenaline
Extracts of adrenal tissue (see above) were lyophilised overnight. Aliquots of 100 ~1 each of acetonitrile (distilled and stored over anhydrous sodium sulphate) and PFPA were added. The mixtures were derivatised for 15 min at 60°C in sealed vials. Samples, were cooled to room temperature and evaporated to dryness in a stream of nitrogen at 40°C. Residues were dissolved in 50 ~1 ethyl acetate/PFPA (250:1, v/v). Mass fragmentographic identification of PFP-derivatised N-MA was performed with an HP-5890 gas chromatograph, (Hewlett Packard, Avondale, PA, USA) directly coupled to a VG-70 250-S mass spectrometer (VG Instruments, Manchester, UK). The gas chromatograph was equipped with a 25 m x 0.25 mm (i.d.) CP-Sil 19 coated capillary column (Chrompack, Middelburg, The Netherlands) and operated under the following conditions: injection temperature 250°C oven temperature programme lOO”C,lO”C/min to 230°C. The ion source temperature of the mass spectrometer was 250°C. Samples were monitored in the electron-impact mode at 70 eV, using two fragment ions at m/z 458 [M minus 0-PFP minus CH2]+ and 472 [M minus 0-PFP]+ of the N-MA-(PFP)s derivative. The 4581472 peak area ratio at the retention time corresponding with PFP-derivatised authentic N-MA was calculated by a VG 1l-250 data system. 2.7. Data processing and statistics Urinary free catecholamine levels were expressed in PmoYmol creatinine and nmol/24 h (children and adults only). Tissue levels were calculated in nmol/g wet tissue. Both urinary and tissue levels were additionally expressed in amounts relative to the sum of free catecholamines (in mol/lOO mol). Age-dependency of urinary free catecholamines was investigated with the Spearman rank correlation test [14]. Postconceptional age dependency of tissue catecholamines was investigated with the Kruskal-Wallis one-way analysis of variance test (141. Urinary data were grouped according to age (neonates, children and adults). Tissue data were summed according to fetuses and very premature neonates, term neonates and infants. Median and non-parametric 95% confidence intervals for urinary catecholamine data of the three age groups were calculated according to Solberg (151; for tissue levels medians and ranges for the three postconceptional age groups are reported. Relations between urinary excretion levels of N-MA and A were investiga.ted with orthogonal regression analysis, according to Deming [16]. Sex related differences were studied within each age group (urine) and postconceptional age group (tissues), using the Kruskal-Wallis one-way analysis of variance [14].
3. Results Fig. 1 shows HPLC-electrochemical detection chromatograms of samples from a 24 h urine of an apparently healthy adult (aged 63 years; panel A), fetal adrenal tissue (gestational age 22 weeks; panel B) and the dorsal roots of an infant (aged 9 monthls; panel C).
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3oc
A
B
100
C
75
2
L- L 2
50
’ 3
25
4
a
,
5
lb
15
2b
0
Retention time (min)
4
1
5
10
15
0:
20
0
5
10
15
Fig. 1. High performance liquid chromatography-electrochemical detection of free catecholamines in urine (A), adrenals (B) and dorsal roots (C). Samples were obtained from an apparently healthy adult (panel A, age 63 years), fetus (panel B, gestational age 22 weeks) and infant (panel C, 9 months). I, noradrenaline; 2, adrenaline; 3, N-methyladrenaline; 4, dihydroxybenzylamine (internal standard); 5, dopamine.
3.1.
Urinary free catecholamines
Table 1 shows median and 95% non-parametric confidence intervals for urinary free catecholamine levels of 32 neonates, 45 children and 19 adults. Since data for neonates were obtained from untimed collections they are only given in pmol/mol creatinine. Data for 24 h collections of children and adults are given in both pmollmol creatinine and nmo1/24 h.
Table 1 Urinary free catecholamines levels of neonates, children and adults nmoV24 h
pmollmol creatinine
DA NA A N-MA
Neonatesa (it = 32)
Childrenb (n = 45)
Adultsc (n = 19)
Children (n = 45)
Adults (n = 19)
291 (33-754) 53 (9-215) 4 (O-18) 0 (O-4)
56 (6-160) 12 (3-28) I (O-4) 1 (O-7)
35 (14-94) II (6-18) I (O-4) I (O-4)
322 (34-808) 56 (13-158) 4 (O-24) 6 (O-40)
519 (200-1500) I65 (94-314) 20 (O-58) I9 (O-58)
Data represent medians (95% non-parametric confidence intervals) in pmollmol creatinine and nmoV24 h. aMedian gestational age 32 weeks, range 27-40 weeks. bMedian age 9 years, range 4-12 years. CMedian age 41 years, range 27-63 years.
I
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143
Urine _ ..
‘JNA
?? DA
~ Neonates
Children
I
Adults
Fig. 2. Contributions of dopamine (DA), noradrenaline (NA), adrenaline (A) and N-methyladrenaline (N-MA) to the sum of free catecholamines in urines from neonates, children and adults. Data are in mol/lOO mol free catecholamines. Neonates (n = 32, median gestational age 32 weeks, range 27-40 weeks); children (n = 45, median age 9 years, range 4-12 years); adults (n = 19, median age 41 years, range 27-63 years).
Expre,wed in pmol/mol creatinine. For the whole group there were significant (Spearman rank test, P < 0.05) decreases of urinary free DA, NA and A with advancing age. On the other hand, free N-MA excretion increased with age. No agedependent changes were observed when only children and adults were considered. None of the age groups showed sex-related differences between urinary free catecholamine levels. There was a significant correlation between N-MA and A (expressed :inkmol/mol creatinine) in urines of children (A at x-axis and N-MA at y-axis for 45 subjects: y = 1.68x-0.23; r = 0.9376; P < 0.0001). No significant correlations between urinary N-MA and A were found in neonates and adults. Expressed in nmoU24 h. Adults showed higher 24 h urinary excretion rates for all free catecholamines, compared with children (Spearman P < 0.05). None of the age groups showed sex-related differences between urinary free catecholamine levels. There was a significant correlation between N-MA and A (expressed in nmd/24 h) in urines of children (A at x-axis and N-MA at y-axis for 44 subjects: y = 1.61x-0.60; r = 0.9502; P < 0.0001). No significant correlations between N-MA and A were found in urines of adults. Expressed in moMO0 mol. Fig. 2 shows relative amounts of free catecholamines (in mol/lOO mol catecholamines) in urines of neonates, children and adults. With advancing age there was a significant (Spearman rank test, P < 0.05) increase of NMA and a significant decrease of DA. When only children and adults were considered the contributions of both A and N-MA were found to increase significantly with ag,e.
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Table 2 Tissue catecholamine contents of fetuses and very premature neonates, term neonates and infants
Adrenals Very premature neonatess Term neonatesb Infantsc Dorsal roots Very premature neonates Term neonates Infants
DA
NA
0.4 (0.0-1.2)
17.2 (1.5-66.1)
0.7 (0.6-0.8) 1.0 (0.7-1.4)
25.1 (14.5-35.7) 77.1 (61.5-92.7)
0.0 (0.0-1.2)
2.4 (0.1-14.2)
0.0 (0.0-5.4)
0.0 (O.O- 1.9)
0.0 (0.0-0.0) 0.0 (0.0-0.0)
7.3 (1.9-12.6) 0.4 (0.2-0.6)
0.1 (0.1-0.2) 0.5 (0.0-0.9)
0.0 (0.0-0.0) 0.5 (0.4-0.6)
0.3 (0.1-3.9)
0.0 (0.0-0.2)
0.0 (0.0-0.0)
3.5 (1.4-5.7) 0.3 (0.0-0.6)
0.1 (0.0-O. 1) 0.8 (0.6-1.1)
0.0 (0.0-0.0) 0.2 (0.0-0.4)
0.0 (0.0-0.4)
0.0 (0.0-0.0)
0.0 (0.0-0.0) 0.3 (0.1-0.3)
0.0 (0.0-0.0) 0.2 (0.1-0.3)
Brown adipose tissue Verypremature 0.0 (0.0-o. 1) neonates Term neonates 0.0 (0.0-0.0) Infants 0.0 (0.0-0.0) Zuckerkandl Very premature neonates Term neonates Infants
O.l(O.O-0.8) 0.0 (0.0-0.0) 0.0 (0.0-0.0)
32.2 (0.5-l 12.4) 0.1 (0.1-0.1) 0.9 (0.0-l .7)
A
N-MA
10.6 (0.0-166.2)
0.0 (0.0-0.0)
53.2 (28.8-77.7) 0.2 (0.0-0.5) 333.1 (219.6-446.5) 7.5 (3.0-12.0)
Data represent medians (ranges) in mnol/g wet weight. *Including two fetuses, n = 8, median gestational age 27 weeks, range 18-30 weeks. bn = 2, gestational ages 40 weeks. ‘II = 2, 1 and 9 months.
3.2. Tissue catecholamines Table 2 shows medians and ranges for the catecholamine contents (in nmol/g wet tissue) of various post-mortem tissues obtained from deceased very premature newborns, term newborns and infants. Data were grouped according to fetuses and very premature neonates (n = 8; median gestational age 27 weeks; range 18-30 weeks), term neonates (n = 2; both born at a gestational age of 40 weeks) and infants (n = 2; ages 1 and 9 months). Expressed in nmoilg. With advancing postconceptional age there were significant (Kruskal-Wallis, P < 0.05) increases of A and N-MA in adrenal tissues and of NMA in the organ of Zuckerkandl. The highest N-MA levels were observed in adrenal tissue. Expressed in mol/lOO mol. Fig. 3 shows relative amounts of free catecholamines (in mol/lOO mol catecholamines) in adrenals, organ of Zuckerkandl, dorsal roots and perirenal brown adipose tissue from fetuses and very premature neonates, term neo-
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I.P. Kema et al. /Clinica Chimica Acta 238 (1995) 137-150 Organ of Zuckerkandl loo
m 0
.c
z ;i; 0 0 J
oc-i--
’
$nature neonates
FGates
T T--T
/
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infants
$?%ature neonates
Dorsal roots
term neonates
infants
Perirenal brown adipose tissue
1 •n N-MA /HA
, 1DNA 1ODA
01-I
I
i!FF$rs
1 Ehates
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Fig. 3. Contributions of dopamine (DA), noradrenaline (NA), adrenaline (A) and N-methyladrenaline (N-MA) to the sum of catecholamine contents in tissues from fetuses and very premature neonates, term neonates and infants. Data are in mol/lOO mol free catecholamines. Fetuses and very premature neonates (n = 8, median gestational age 27 weeks, range 18-30 weeks); term neonates (n = 2, gestational ages 40 weeks); infants (n = 2, 1 and 9 months).
nates and infants. With advancing postconceptional age, notably after birth, there was a significant (Kruskal-Wallis, P < 0.05) decrease of NA in adrenal tissue, whereas, A and N-MA increased in adrenal tissue and organ of Zuckerkandl. The highest relative amounts of N-MA were observed in extra adrenal tissues. 3.3. Mass fragmentographic identification of N-methyladrenaline Mass fragmentograms obtained from three PFP-derivatised adrenal tissue extracts (one telm neonate and two infants) showed peaks at m/z 458 and 472 at the same retention time as authentic N-MA(PFPh. The m/z 4581472 peak area ratios of adrenal tissue extracts and N-MA(PFP), standards did not differ by more than 4%. 4. Diwmion
Interpretation of the present data in terms of ‘normal’ urinary and tissues catecholamine levels is hampered by several factors. We collected urine samples from
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hospitalised neonates. Tissue samples were obtained from fetuses, newborns and infants who were aborted for medical reasons or died from a variety of diseases, respectively. Some neonatal conditions, notably those known to cause abnormal extrauterine energy metabolism (e.g. prematurity, infection) or otherwise evoke abnormal hormonal responses (e.g. cardiovascular disease, stress), and treatments (e.g. glucose infusion, sympathomimetics) may affect urinary catecholamine levels. Death per se may cause catecholamine release from tissues. Post-mortem changes in tissue catecholamine levels may occur and contributions of catecholamine-containing tissue to the collected specimen, notably in adrenals and brown adipose tissue, is uncertain. Interpretation of reported urinary and tissue levels in terms of normal perinatal sympathoadrenal development should therefore be exercised with appropriate reserve. To allow interpretation of the capacity to synthesise or store certain catecholamines we additionally converted concentrations into relative amounts, i.e. levels were additionally expressed in terms of percentages of the sum of free catecholamine contents. These results are not necessarily unaffected by the above mentioned variables, since neonatal disease and abnormal fetal development may affect the activities of catecholamine biosynthetic enzymes. For instance, prolonged stimulation of the sympathoadrenal system causes tyrosine hydroxylase induction, whereas abnormal corticosteroid levels may affect PNMT expression [ 171. 4.1. Urine Comparison of urinary free DA, NA and A excretion levels with data obtained by similar HPLC techniques [l&23] revealed that for neonates, children and adults our data are at the lower range. Differences in clean-up procedures and detection techniques may account for the discrepancy. Incomplete separation between A and N-MA may explain the somewhat higher A levels reported by others, but not the discrepancy with data from Gerlo et al. [20]. Age-dependent decreases of urinary DA, NA and A excretion, expressed in terms of creatinine, have previously been established [ 18,21,23,24]. Decrease of the catecholamine/creatinine ratio is at least partially caused by increasing urinary creatinine output with age [24-261. Increases of 24-h urinary free catecholamine excretion values with age have also been noted [ 18,211. We did not observe any sex-related differences in catecholamine excretion. When expressed in terms of creatinine, Gerlo et al. [20] found higher urinary A excretion by men, whereas excretions of NA and DA by women were somewhat higher. However, investigating single urine voidings from 482 school children, Reed et al. [23] found no significant sex-related differences in urinary free catecholamine excretion. Urinary N-MA levels have merely been reported for normotensive and hypertensive adults and patients with phaeochromocytoma [5,7]. Using HPLC with electrochemical detection, Gerlo and Malfait [7] established N-MA in urine of 150 hypertensive non-phaeochromocytoma patients. They found N-MA excretion levels to be slightly lower than those of A. Our data (O-58 nmol/24 h, Table 1) proved in excellent agreement with their results (2-65 nmol/24 h). We could not confirm the correlation between urinary free N-MA and A in adults. In the present study urinary free N-MA and A levels of children were, however, significantly related.
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Studying children and adults, we found age-dependent increases of the relative amounts of both urinary A and N-MA (Fig. 2). Relative amounts of urinary catecholamines in newborns were highly diverse. The former data suggest that with advancing age both A and N-MA are increasingly formed. Urinary free catechol,amine levels do, however, not necessarily reflect production rates. Differences in metabolic pressure and reuptake efficiency are possible confounders. In an experiment with rats that were intraperitoneally injected with N-MA, we noted a significant increase in urinary output of its 0-methylated metabolite N-MM, thus confirming earlier observations [l]. However, no changes were found in urinary levels of oxidatively deaminated catecholamine metabolites (I.P. Kema et al., unpublished observations). It suggests that, in contrast to ‘classical’catecholamines, N-MA is not an adequate substrate for monoamine oxidase. Differences between urinary levels of N-MA
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believed to compensate for the endocrine immaturity of the adrenal medulla in fetal and early neonatal life, thus enabling the fetus and newborn to maintain internal homeostasis. Using bioassay and fluorescent analysis, West et al. [30] determined ‘pressor amine’ contents of both adrenal tissue and organ of Zuckerkandl from nearly 100 infants and children. It was concluded that the organ of Zuckerkandl reaches maturity at birth, that it mainly contains NA and that at birth its total pressor amine content exceeds that of the adrenal gland. After birth, concomitant with adrenal medulla development, extra adrenal chromaffin bodies regress, showing cellular degeneration, fibrosis and loss of catecholamines [30,31]. Our study shows gestational-age dependent increases of both the levels and relative amounts of N-MA in the organ of Zuckerkandl, with highest values in infants (Table 2, Fig. 3). Several studies [32-341 demonstrated the presence of PNMT in extra adrenal chromaffin tissues, including the organ of Zuckerkandl, albeit at low activities. Whether in these tissues A and N-MA syntheses merely result from PNMT activity remains to be established, since aspecilic N-methylation activity independent from glucocorticoid induction has also been noted [35]. Increasing levels of N-MA in extra adrenal chromaffm tissue during its regression raises the question whether it reflects production of a metabolite of A without any functional role. Fetal brown adipose tissue is located in areas surrounding major vessels [36]. It has an important role in neonatal thermoregulation through non-shivering thermogenesis [37]. Innervation takes place by the sympathetic nervous system. NA is believed to be the principal neurotransmitter in stimulation of non-shivering thermogenesis [31,37] and an increase in urinary catecholamine excretion, notably NA, has been noted after exposure to cold [38]. Moreover, it has been suggested that catecholamines have trophic effects on brown adipose tissue development and maturation [31]. Our data confirm the notion that in brown adipose tissue of very premature and term neonates NA is the main neurotransmitter. Results from the two newborns suggest that after birth A and N-MA become increasingly important (Table 2, Fig. 3). 4.4. Conclusion Analyses in urines from children and adults show that contributions of both A and N-MA to the sum of free catecholamines increase with age. Relative amounts of A and N-MA increase in both adrenal and extra adrenal chromaffin tissues from late gestation up to several months of postnatal life. N-MA content of adrenal tissue follows endocrine maturation of the medulla and, more specifically, PNMT induction and subsequent A synthesis. Relative amounts of N-MA in extra adrenal chromaffin tissue increase in a period in which these structures regress. The function of N-MA, as either a long-acting catecholamine or an A-metabolite, is as yet unclear. Acknowledgements
We thank Prof. Dr. N. Seiler for providing us with the pharmacological profile of N-methyladrenaline and A.M. Bmgman, G. Meiborg, G.T. Nagel and Dr. J. Prins for their skilful technical assistance and co-operation.
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References [l] Axelrod J. N-Methyladrenaline, a new catecholamine in the adrenal gland. Biochim Biophys Acta 1%0$5:6145-6155. [2] Verly W. Proprietes pharmacologiques de la dimethyl-noradrenaline. Arch Int Phannacodyn 1948;77:375-382. [3] Mueller PS, Horwitz D. Plasma free fatty acid and blood glucose responses to analogues of norepinephrine in man. J Lipid Res 1962;3:251-255. [4] Evans BD, Vogel WH. Penetration of some 0- and/or N-methylated norepinephrine derivatives through the rat blood brain barrier. Res Commun Chem Pathol Pharmacol 1977;17:61-76. [5] Itoh C, Yoshinaga K, Sato T, Ishida N, Wada Y. Presence of N-methylmetadrenaline in human urine and tumour tissue of phaeochromocytoma. Nature 1962;193:477-478. [6] Robiuson R, Smith P, Whittaker SRF. Secretion of catecholamines in malignant pheochromocytoma. Br Med J 1964;1:1422-1424. [7] Gerla’ J, Malfait R. Identification of N-methylepinephrine in human urine. J Chromatogr 1987;414:301-311. [8] Coward RF, Smith P. Excretion of metanephrines in post-operative stress. Clin Chim Acta 1966,14:833-835. [9] Perry TL. N-methylmetanephrine: excretion by juvenile psychotics. Science 1963;139:587-589. (10) Kagedal B, Goldstein DS. Catecholamines and their metabolites (review). J Chromatogr 1988;429:177-233. [I l] Moleman P, Borstrok JJM. Analysis of urinary catecholamines and metabolites with high performance liquid chromatography and electrochemical detection. Biogenic Amines 1985;3:31-71. (121 West~erink BHC, Bosker FJ, O’Hanlon JF. Use of alumina, Sephadex GlO and ion exchange columns~to purify samples for determination of epinephrine, norepinephrine, dopamine, homovanillic acid and 5-hydroxyindoleacetic acid in urine. Clin Chem 1982;28:1745-1748. [13] Smedes F, Kraak JC, Poppe H. Simple and fast solvent extraction system for selective and quantitative isolation of adrenaline, noradrenaline and dopamine from plasma and urine. J Chromatogr 1982:,231:25-39. [14) Conover WJ. In: Practical nonparametric statistics, 2nd edition. New York: John Wiley, 1980. [ 151 Solberg HE. Approved recommendation (1987) on the theory of reference values. Part 5: Statistical treat:ment of collected reference values. Clin Chim Acta 1987;17O:Sl3-S32. 1161 Comlbleet PJ, Gochman N. Incorrect least-squares regression coefficients in method comparison analysis. Clin Chem 1979;25:432-438. [17] Landsberg L, Young JB. Catecholamines and the adrenal medulla. In: Wilson JD, Foster DW, eds. Williams Textbook of Endocrinology. Philadelphia: Saunders, 1985;891-965. [18] Abehng NG, Gennip AH van, Overmars H, Voute PA. Simultaneous determination of catecholamines and metanephrines in urine by HPLC with fluorometric detection. Clin Chim Acta 1984;137:211-226. [19] Ross GA, Newbould EC, Thomas J et al. Plasma and 24-h urinary catecholamine concentrations in normal and patient populations. Ann Clin Biochem 1993;30:38-44. [20] Gerlo EAM, Schoors DF, Dupont AG. Age- and sex-related differences for the urinary excretion of tmrepinephrine, epinephrine, and dopamine in adults. Clin Chem 1991;37:875-878. 1211 Pr&nel-Cabic A, Turcant A, Allain P. Normal reference intervals for free catecholamines and their acid metabolites in 24-h urines from children, as determined by liquid chromatography with amperometric detection. Clin Chem 198632: I585- 1587. [22] Moyer TP, Jiang NS, Tyce GM, Sheps SG. Analysis for urinary catecholamines by liquid chromatography with amperometric detection: methodology and clinical interpretation of results. Clin Chelm 1979;25:256-263. [23] Reed P, Bu’Lock D, Jones A, Layton T. Reference ranges for free catechohunine/creatinine ratios in random urines from normal children aged 5 to 16 years. Ann Clin Biochem 1991;28:297-298. [24] Gdink J, Thijssen TNM, Zeegers JH, Schreurs WHP. Diurnal variations in, and age and sex dependency on, the excretion of free catecholamines, homovanillic acid, vanilmandehc acid and 5hydroxyindole-3-acetic acid in urine of children and adolescents. Biogenic Amines 1986;3:257-265.
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Appelgarth DA, Ross PM. The unsuitability of creatinine excretion as a basis for assessing the excretion of other metabolites by infants and children. Clin Chim Acta 1975;64:83-85. 1-W Muskiet FAJ, Thomasson CG, Gerding AM, Fremouw-Ottevangers DC, Nagel GT. Wolthers BG. Determination of catecholamines and their 3-0-methylated metabolites in urine by mass fragmentography with use of deuterated internal standards. Clin Chem 1979;25:453-456. 1271 Greenberg RE, Lind J. Catecholamines of the human fetus. Pediatrics 1961;27:904-91 I. WI Axelrod J. Methylation reactions in the formation and metabolism of catecholamines and other biogenic amines. Pharmacol Rev 1966;18:95-I 13. (291 Wurtman RJ, Axelrod J. Adrenaline synthesis: control by the pituitary gland and adrenal glucocorticoids. Science 1965;150:1464-1465. 1301 West GB, Shepherd DM, Hunter RB, Macgregor AR. The function of the organs of Zuckerkandl. Clin Sci 1953;12:317-325. [311 Philippe M. Fetal catecholamines. Am J Obstet Gynecol 1983;146:840-855. and [321 Gennser G, Studnitz v. W. Monoamine oxidase, catechol-O-methyltransferase phenylethanolamine-Nmethyltransferase activity in para-aortic tissue of the human fetus. Stand J Clin Invest 1969;24:169-171. 1331 Eranko 0, Lempinen M, Raisanen L. Adrenaline and noradrenaline in the organ of Zuckerkandl and adrenals of newborn rats treated with hydrocortisone. Acta Physiol Stand 1966;66:253-254. 1341 Kennedy B, Ziegler MG. Cardiac epinephrine synthesis. Regulation by a glucocorticoid. Circulation 1991;84:891-895. I351 Elayan H, Kennedy B, Ziegler MG. Epinephrine synthesis by an N-methyltransferase in rat liver. Gastroenterology 1990;98:152-155. 1361 Merklin RJ. Growth and distribution of human fetal brown fat. Anat Ret 1973;178:673-646. I371 Cannon B. Nedergaard J. The function and properties of brown adipose tissue in the newborn. In: Jones CT, ed. Biochemical development of the fetus and neonate. Amsterdam: Elsevier, 1982;697-730. 1381 Stern L. Lees MH, LeDuc J. Environmental temperature, oxygen consumption, and catecholamine excretion in newborn infants. Pediatrics 1965;37:367-373.
ELSEVIER
N-Methyladrenaline: age-dependent urinary excretion, perinatal organ content and relation with ‘classical’ catecholamines Ido P. Kerna*“, Coen J.F. Schootsb, Corrie E.M. Gidding’, Albert Okkenc, Jan G. Aarnoudsed, Frits A.J. Muskiet” “Central
Laboratory
for
Clinical
Chemistry,
P.O. Box 30001, bDepartment
of Pathology,
University
Universiry
9700 RB Groningen. and University
and University
Hospital
of Groningen,
The Netherlands
Hospital
of Groningen,
9700 RB Groningen.
of Groningen,
9700 RB Groningen.
The Netherlancis ‘Departmeni
of Paediatrics.
University
and University
Hospital
The Netherlands dDepartment
of Obstetrics
and Gynaecology,
University
9700 RB Groningen,
and University
Hospital
of Groningen,
The Netherlands
Received 9 January 1995; accepted 30 March 1995
Abstract Using high performance liquid chromatography with electrochemical detection we determined free dopamine, noradrenaline, adrenaline and N-methyladrenaline in: (1) urines from newborns (n = 32), children (n = 45) and adults (n = 19) and (2) adrenals, organ of Zuckerkandl, dorsal roots and perirenal brown adipose tissue from deceased fetuses (n = 2), very premature (n = 6) and term (n = 2) newborns and infants (n = 2). Data from children and adults showed that contributions of adrenaline and N-methyladrenaline to the sum of urinary free catecholamines increase with age. Relative amounts of adrenaline and N-methyladrenaline increased in both adrenal and extra adrenal chromafin tissues from late gestation up to several months of postnatal life. Increase of adrenal N-methyladrenaline content follows endocrine maturation of the medulla, phenylethanolamine-N-methyltransferase induction and subsequent adrenaline synthesis. Relative amounts of N-methyladrenaline in extra adrenal chromaffin tissue increase in a period that is associated with its regression. Further investigations are necessary to elucidate the function and possible clinical chemical usefulness of Nmethyladrenaline.
-. Corresponding author.
??
0009-8981/95/%09.50 0 1995 Elsevier Science B.V. All rights reserved SD1 0009-898 I (95)06082-O
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Keywords: Adrenaline; Phenylethanolamine-Nmethyltransferase; Extra adrenal chromaffin tissue; High performance liquid chromatography
1. Introduction
N-methyladrenaline (N-MA) is formed by N-methylation of adrenaline (A). The reaction is catalysed by phenylethanolamine-Nmethyltransferase (PNMT, EC 2.1.1.6). In 1960, Axelrod [l] was the first to show that N-MA is a naturally occurring catecholamine, demonstrable in the adrenal medulla of a variety of animal species. Following intravenous N-MA administration to rats, large quantities of N-methylmetanephrine (N-MM; the 3-0-methylated metabolite of N-MA) were recovered from urine in both free and conjugated forms. Axelrod suggested the synthesis and metabolism of N-MA to occur according to: A - N-MA - N-MM. Early pharmacological experiments revealed that N-MA exhibits properties that are at best described as a mixture of those of dopamine (DA), noradrenaline (NA) and A. These effects, e.g. increases of blood glucose and diastolic and systolic blood pressures and decrease of pulse rate, were observed at lo-40 times higher doses than A and NA and at comparable DA dose [3]. N-methyladrenaline effects on systolic blood pressure lasted longer than those of A [2]. Mueller and Horwitz [3] showed that at an intravenous dose of 180 &kg body weight of the L-enantiomer of NMA, blood glucose levels and both systolic and diastolic blood pressures increased, whereas pulse rate decreased. No significant influences on plasma free fatty acid levels were found. Evans [4] showed that, in contrast to its metabolite N-MM, N-MA is incapable of passing the blood brain barrier in rats. Since its first demonstration, the number of clinical chemical studies on N-MA has been scanty. Apart from measurements of N-MA in urine samples from normotensive and hypertensive adults and patients with phaeochromocytoma [5-71, and determinations of urinary N-MM in postoperative patients [8] and certain psychiatric patients [9], virtually no data on its (patho)physiological significance or clinical chemical importance have been reported. Urinary N-MA levels of normotensive and hypertensive adults were usually found to correlate with those of A [7]. However, high urinary excretion levels of A did not necessarily coincide with increased N-MA levels. Some investigators pointed to the difficulty, or inability, of routinely employed catecholamine analyses (e.g. trihydroxyindole and radioenzymatic (COMT) methods) to detect or quantify N-MA [7,10]. Since urinary N-MA and A levels are similar [7], incomplete separation of N-MA from A in commonly applied catecholamine assays by high performance liquid chromatography (HPLC) with electrochemical detection leads to overestimation of A. Using an HPLC-electrochemical detection method that enables complete separation of N-MA and A, we determined age-dependent urinary excretion levels of NMA and related the results to those of the ‘classical’ catecholamines, DA, NA and A. Assays were performed in samples collected from newborns, children and adults. In an attempt to disclose the origin of N-MA and its synthesis in relation to the other
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catecholamines, determinations were additionally performed in adrenals, organs of Zuckerkandl, dorsal roots and perirenal brown adipose tissue from deceased fetuses, very premature neonates, neonates and infants.
2. Materials and methods 2.1. Reagents N-MA was kindly donated by Dr. M. Schussler van Hees (University of Leiden, The Netherlands). DA, NA, A and 3,4-dihydroxybenzylamine were obtained from Sigma #(St.Louis, MO, USA), HPLC grade octanesulphonic acid was from Fisons (Loughborough, UK), citric acid monohydrate and Na2HP04.2H20 from Merck (Darmstadt, Germany) and acetonitrile from Rathburn (Walkbum, UK). Ethylenediamine tetraacetic acid (EDTA) and Na2Sz05 were purchased from BDH (Poole, UK). Pentafluoropropionic anhydride (PFPA) was from Pierce (Rockford, IL, USA). All reagents were of analytical grade. Glass distilled water was used throughout. 2.2. Urine samples For assessment of age-dependent reference values of urinary free DA, NA, A and N-MA, samples from neonates, children and adults were collected without dietary restrictions and in an undefined metabolic state. Using sterile paediatric urine collection bags (Urinocol, Biotrol Pharma, Paris, France), untimed urine samples were collected from 32 premature and term hospital&d neonates (14 females, 18 males; median gestational age 32 weeks; range 27-40). Twenty-three neonates were spontaneously delivered, whereas eight were born by caesarean section. Hospitalisation took place for a variety of reasons, including caesarean section, premature birth, respiratory distress syndrome, hyperbilirubinaemia, generalised infections, cardiovascular disease, gastrointestinal disease and combinations of these. Urines were collected for approximately 12 h on the first day (n = 2) up to the 77th day after birth (median 27 days). Immediately after c:ompletion of collection Na2S205 and EDTA were added to final concentrations of about 0.5 g/l. Twenty-four hour urine collections of 45 apparently healthy children (22 females, 23 males; median age 9 years; range 4- 12) and 19 healthy adults ( 10 females, 9 males; median age 41 years; range 27-63 years) were completed in 2-l brown polypropylene bottles (Sarstedt, Nuembrecht, Germany), containing about 250 mg each of Na,S205 and EDTA as preservatives. All urine specimens were acidified to pH 4 with acetic acid before freezing. Samples were stored at -2O”C, or -80°C and analysed within 1 month, or 1 year, respectively, after collection. Addition of Na,S205 and EDTA and subsequent storage at -80°C stabilises all catecholamines and metabolites for a year or more [ 111. Urinary creatinine levels, used to quantify excretion in terms of creatinine, were measured by a picric acid method on an SMA-2 analyser (Technicon Instruments, Tarrytown, NY, I-ISA).
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2.3. Tissue samples
The studied group of deceased fetuses, neonates and infants was composed of three subgroups: (1) two fetuses and six very premature neonates (5 females, 3 males, median gestational age 27 weeks; range 18-30 weeks); the two fetuses were aborted because of chromosomal anomalies, diagnosed by amniocentesis; the six very Qremature neonates were spontaneously live born but died within 48 h (two following intrauterine infections and four from hyaline membrane disease and/or lung hypoplasia); (2) two spontaneously born term neonates (one female and one male, both delivered at a gestational age of 40 weeks); they died within 24 h from congenital heart disease and multiple congenital abnormalities, respectively; (3) two male infants, who died at 1 and 9 postnatal months from congenital heart disease and sudden infant death syndrome, respectively. Consent was obtained from the parents. The study protocol conformed to local ethical standards and the Helsinki declaration of 1975, as revised in 1983. As soon as possible after death the bodies were stored in a refrigerator at 4°C. Gestational age was calculated from the time of last menstruation and confirmed by anthropometric characteristics (crown-heel length, body weight and skeletal- and brain maturation). Specimens from adrenals, organ of Zuckerkandl, dorsal roots and Qerirenal brown adipose tissue were taken within 24 h after death. Histological characterisation was performed by macroscopic and microscopic examination. Dissected samples were weighed and transferred to 0.01 moY1acetic acid, containing 10 g/l each of Na,S205 and EDTA as preservatives. They were stored at -2O’C until analysis. 2.4. Prepurification Isolation of free catecholamines from loo-p1 aliquots of urine from infants was performed by Sephadex G-10 minicolumn extraction, as described by Westerink et al. [12]. The Sephadex eluates were further purified by paired ion extraction, according to Smedes et al. [13]. Prepurification of 5-ml urine samples from children and adults was accomplished by atmospheric pressure cation exchange chromatography (‘catecholamine columns’, Bio-Rad Laboratories, Munich, Germany). In all cases 3,4-dihydroxybenzylamine served as an internal standard. After the addition of 1 ng 3,4-dihydroxybenzylamine (internal standard), 50-100mg amounts of frozen tissue were sonicated in l-2 ml 0.01 mol/l acetic acid (containing 10 g/I each of Na2S20S and EDTA) for 30 s at 0°C. Free catecholamines were isolated from the homogenates by paired ion extraction [ 131. 2.5. Analysis and quantification Free catecholamine (DA, NA, A and N-MA) contents in Qrepurified urines and tissues were determined by HPLC with electrochemical detection. The HPLC system consisted of an LKB type 2150 pump (Bromma, Sweden) and a 220 x 4.6 (i.d.) mm RP-8 reversed phase column filled with 5-pm spherical particles (Brownlee Labs, Santa Clara, CA, USA). The mobile phase was composed of 34 mmol/l citric acid, 42 mmol/I Na2HP04, 5.5 mrnol/l octanesulphonic acid and 160 ml methanol per litre (QH 4.2). The electrochemical detector (type Coulochem II, ESA, Bedford, MA, USA) was equipped with a 5011 high sensitivity cell, set at 0.35 V.
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2.6. Mass spectrometric identification of N-methyladrenaline
Extracts of adrenal tissue (see above) were lyophilised overnight. Aliquots of 100 ~1 each of acetonitrile (distilled and stored over anhydrous sodium sulphate) and PFPA were added. The mixtures were derivatised for 15 min at 60°C in sealed vials. Samples, were cooled to room temperature and evaporated to dryness in a stream of nitrogen at 40°C. Residues were dissolved in 50 ~1 ethyl acetate/PFPA (250:1, v/v). Mass fragmentographic identification of PFP-derivatised N-MA was performed with an HP-5890 gas chromatograph, (Hewlett Packard, Avondale, PA, USA) directly coupled to a VG-70 250-S mass spectrometer (VG Instruments, Manchester, UK). The gas chromatograph was equipped with a 25 m x 0.25 mm (i.d.) CP-Sil 19 coated capillary column (Chrompack, Middelburg, The Netherlands) and operated under the following conditions: injection temperature 250°C oven temperature programme lOO”C,lO”C/min to 230°C. The ion source temperature of the mass spectrometer was 250°C. Samples were monitored in the electron-impact mode at 70 eV, using two fragment ions at m/z 458 [M minus 0-PFP minus CH2]+ and 472 [M minus 0-PFP]+ of the N-MA-(PFP)s derivative. The 4581472 peak area ratio at the retention time corresponding with PFP-derivatised authentic N-MA was calculated by a VG 1l-250 data system. 2.7. Data processing and statistics Urinary free catecholamine levels were expressed in PmoYmol creatinine and nmol/24 h (children and adults only). Tissue levels were calculated in nmol/g wet tissue. Both urinary and tissue levels were additionally expressed in amounts relative to the sum of free catecholamines (in mol/lOO mol). Age-dependency of urinary free catecholamines was investigated with the Spearman rank correlation test [14]. Postconceptional age dependency of tissue catecholamines was investigated with the Kruskal-Wallis one-way analysis of variance test (141. Urinary data were grouped according to age (neonates, children and adults). Tissue data were summed according to fetuses and very premature neonates, term neonates and infants. Median and non-parametric 95% confidence intervals for urinary catecholamine data of the three age groups were calculated according to Solberg (151; for tissue levels medians and ranges for the three postconceptional age groups are reported. Relations between urinary excretion levels of N-MA and A were investiga.ted with orthogonal regression analysis, according to Deming [16]. Sex related differences were studied within each age group (urine) and postconceptional age group (tissues), using the Kruskal-Wallis one-way analysis of variance [14].
3. Results Fig. 1 shows HPLC-electrochemical detection chromatograms of samples from a 24 h urine of an apparently healthy adult (aged 63 years; panel A), fetal adrenal tissue (gestational age 22 weeks; panel B) and the dorsal roots of an infant (aged 9 monthls; panel C).
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3oc
A
B
100
C
75
2
L- L 2
50
’ 3
25
4
a
,
5
lb
15
2b
0
Retention time (min)
4
1
5
10
15
0:
20
0
5
10
15
Fig. 1. High performance liquid chromatography-electrochemical detection of free catecholamines in urine (A), adrenals (B) and dorsal roots (C). Samples were obtained from an apparently healthy adult (panel A, age 63 years), fetus (panel B, gestational age 22 weeks) and infant (panel C, 9 months). I, noradrenaline; 2, adrenaline; 3, N-methyladrenaline; 4, dihydroxybenzylamine (internal standard); 5, dopamine.
3.1.
Urinary free catecholamines
Table 1 shows median and 95% non-parametric confidence intervals for urinary free catecholamine levels of 32 neonates, 45 children and 19 adults. Since data for neonates were obtained from untimed collections they are only given in pmol/mol creatinine. Data for 24 h collections of children and adults are given in both pmollmol creatinine and nmo1/24 h.
Table 1 Urinary free catecholamines levels of neonates, children and adults nmoV24 h
pmollmol creatinine
DA NA A N-MA
Neonatesa (it = 32)
Childrenb (n = 45)
Adultsc (n = 19)
Children (n = 45)
Adults (n = 19)
291 (33-754) 53 (9-215) 4 (O-18) 0 (O-4)
56 (6-160) 12 (3-28) I (O-4) 1 (O-7)
35 (14-94) II (6-18) I (O-4) I (O-4)
322 (34-808) 56 (13-158) 4 (O-24) 6 (O-40)
519 (200-1500) I65 (94-314) 20 (O-58) I9 (O-58)
Data represent medians (95% non-parametric confidence intervals) in pmollmol creatinine and nmoV24 h. aMedian gestational age 32 weeks, range 27-40 weeks. bMedian age 9 years, range 4-12 years. CMedian age 41 years, range 27-63 years.
I
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143
Urine _ ..
‘JNA
?? DA
~ Neonates
Children
I
Adults
Fig. 2. Contributions of dopamine (DA), noradrenaline (NA), adrenaline (A) and N-methyladrenaline (N-MA) to the sum of free catecholamines in urines from neonates, children and adults. Data are in mol/lOO mol free catecholamines. Neonates (n = 32, median gestational age 32 weeks, range 27-40 weeks); children (n = 45, median age 9 years, range 4-12 years); adults (n = 19, median age 41 years, range 27-63 years).
Expre,wed in pmol/mol creatinine. For the whole group there were significant (Spearman rank test, P < 0.05) decreases of urinary free DA, NA and A with advancing age. On the other hand, free N-MA excretion increased with age. No agedependent changes were observed when only children and adults were considered. None of the age groups showed sex-related differences between urinary free catecholamine levels. There was a significant correlation between N-MA and A (expressed :inkmol/mol creatinine) in urines of children (A at x-axis and N-MA at y-axis for 45 subjects: y = 1.68x-0.23; r = 0.9376; P < 0.0001). No significant correlations between urinary N-MA and A were found in neonates and adults. Expressed in nmoU24 h. Adults showed higher 24 h urinary excretion rates for all free catecholamines, compared with children (Spearman P < 0.05). None of the age groups showed sex-related differences between urinary free catecholamine levels. There was a significant correlation between N-MA and A (expressed in nmd/24 h) in urines of children (A at x-axis and N-MA at y-axis for 44 subjects: y = 1.61x-0.60; r = 0.9502; P < 0.0001). No significant correlations between N-MA and A were found in urines of adults. Expressed in moMO0 mol. Fig. 2 shows relative amounts of free catecholamines (in mol/lOO mol catecholamines) in urines of neonates, children and adults. With advancing age there was a significant (Spearman rank test, P < 0.05) increase of NMA and a significant decrease of DA. When only children and adults were considered the contributions of both A and N-MA were found to increase significantly with ag,e.
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Table 2 Tissue catecholamine contents of fetuses and very premature neonates, term neonates and infants
Adrenals Very premature neonatess Term neonatesb Infantsc Dorsal roots Very premature neonates Term neonates Infants
DA
NA
0.4 (0.0-1.2)
17.2 (1.5-66.1)
0.7 (0.6-0.8) 1.0 (0.7-1.4)
25.1 (14.5-35.7) 77.1 (61.5-92.7)
0.0 (0.0-1.2)
2.4 (0.1-14.2)
0.0 (0.0-5.4)
0.0 (O.O- 1.9)
0.0 (0.0-0.0) 0.0 (0.0-0.0)
7.3 (1.9-12.6) 0.4 (0.2-0.6)
0.1 (0.1-0.2) 0.5 (0.0-0.9)
0.0 (0.0-0.0) 0.5 (0.4-0.6)
0.3 (0.1-3.9)
0.0 (0.0-0.2)
0.0 (0.0-0.0)
3.5 (1.4-5.7) 0.3 (0.0-0.6)
0.1 (0.0-O. 1) 0.8 (0.6-1.1)
0.0 (0.0-0.0) 0.2 (0.0-0.4)
0.0 (0.0-0.4)
0.0 (0.0-0.0)
0.0 (0.0-0.0) 0.3 (0.1-0.3)
0.0 (0.0-0.0) 0.2 (0.1-0.3)
Brown adipose tissue Verypremature 0.0 (0.0-o. 1) neonates Term neonates 0.0 (0.0-0.0) Infants 0.0 (0.0-0.0) Zuckerkandl Very premature neonates Term neonates Infants
O.l(O.O-0.8) 0.0 (0.0-0.0) 0.0 (0.0-0.0)
32.2 (0.5-l 12.4) 0.1 (0.1-0.1) 0.9 (0.0-l .7)
A
N-MA
10.6 (0.0-166.2)
0.0 (0.0-0.0)
53.2 (28.8-77.7) 0.2 (0.0-0.5) 333.1 (219.6-446.5) 7.5 (3.0-12.0)
Data represent medians (ranges) in mnol/g wet weight. *Including two fetuses, n = 8, median gestational age 27 weeks, range 18-30 weeks. bn = 2, gestational ages 40 weeks. ‘II = 2, 1 and 9 months.
3.2. Tissue catecholamines Table 2 shows medians and ranges for the catecholamine contents (in nmol/g wet tissue) of various post-mortem tissues obtained from deceased very premature newborns, term newborns and infants. Data were grouped according to fetuses and very premature neonates (n = 8; median gestational age 27 weeks; range 18-30 weeks), term neonates (n = 2; both born at a gestational age of 40 weeks) and infants (n = 2; ages 1 and 9 months). Expressed in nmoilg. With advancing postconceptional age there were significant (Kruskal-Wallis, P < 0.05) increases of A and N-MA in adrenal tissues and of NMA in the organ of Zuckerkandl. The highest N-MA levels were observed in adrenal tissue. Expressed in mol/lOO mol. Fig. 3 shows relative amounts of free catecholamines (in mol/lOO mol catecholamines) in adrenals, organ of Zuckerkandl, dorsal roots and perirenal brown adipose tissue from fetuses and very premature neonates, term neo-
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I.P. Kema et al. /Clinica Chimica Acta 238 (1995) 137-150 Organ of Zuckerkandl loo
m 0
.c
z ;i; 0 0 J
oc-i--
’
$nature neonates
FGates
T T--T
/
I
infants
$?%ature neonates
Dorsal roots
term neonates
infants
Perirenal brown adipose tissue
1 •n N-MA /HA
, 1DNA 1ODA
01-I
I
i!FF$rs
1 Ehates
I .J
I
infank
Fig. 3. Contributions of dopamine (DA), noradrenaline (NA), adrenaline (A) and N-methyladrenaline (N-MA) to the sum of catecholamine contents in tissues from fetuses and very premature neonates, term neonates and infants. Data are in mol/lOO mol free catecholamines. Fetuses and very premature neonates (n = 8, median gestational age 27 weeks, range 18-30 weeks); term neonates (n = 2, gestational ages 40 weeks); infants (n = 2, 1 and 9 months).
nates and infants. With advancing postconceptional age, notably after birth, there was a significant (Kruskal-Wallis, P < 0.05) decrease of NA in adrenal tissue, whereas, A and N-MA increased in adrenal tissue and organ of Zuckerkandl. The highest relative amounts of N-MA were observed in extra adrenal tissues. 3.3. Mass fragmentographic identification of N-methyladrenaline Mass fragmentograms obtained from three PFP-derivatised adrenal tissue extracts (one telm neonate and two infants) showed peaks at m/z 458 and 472 at the same retention time as authentic N-MA(PFPh. The m/z 4581472 peak area ratios of adrenal tissue extracts and N-MA(PFP), standards did not differ by more than 4%. 4. Diwmion
Interpretation of the present data in terms of ‘normal’ urinary and tissues catecholamine levels is hampered by several factors. We collected urine samples from
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hospitalised neonates. Tissue samples were obtained from fetuses, newborns and infants who were aborted for medical reasons or died from a variety of diseases, respectively. Some neonatal conditions, notably those known to cause abnormal extrauterine energy metabolism (e.g. prematurity, infection) or otherwise evoke abnormal hormonal responses (e.g. cardiovascular disease, stress), and treatments (e.g. glucose infusion, sympathomimetics) may affect urinary catecholamine levels. Death per se may cause catecholamine release from tissues. Post-mortem changes in tissue catecholamine levels may occur and contributions of catecholamine-containing tissue to the collected specimen, notably in adrenals and brown adipose tissue, is uncertain. Interpretation of reported urinary and tissue levels in terms of normal perinatal sympathoadrenal development should therefore be exercised with appropriate reserve. To allow interpretation of the capacity to synthesise or store certain catecholamines we additionally converted concentrations into relative amounts, i.e. levels were additionally expressed in terms of percentages of the sum of free catecholamine contents. These results are not necessarily unaffected by the above mentioned variables, since neonatal disease and abnormal fetal development may affect the activities of catecholamine biosynthetic enzymes. For instance, prolonged stimulation of the sympathoadrenal system causes tyrosine hydroxylase induction, whereas abnormal corticosteroid levels may affect PNMT expression [ 171. 4.1. Urine Comparison of urinary free DA, NA and A excretion levels with data obtained by similar HPLC techniques [l&23] revealed that for neonates, children and adults our data are at the lower range. Differences in clean-up procedures and detection techniques may account for the discrepancy. Incomplete separation between A and N-MA may explain the somewhat higher A levels reported by others, but not the discrepancy with data from Gerlo et al. [20]. Age-dependent decreases of urinary DA, NA and A excretion, expressed in terms of creatinine, have previously been established [ 18,21,23,24]. Decrease of the catecholamine/creatinine ratio is at least partially caused by increasing urinary creatinine output with age [24-261. Increases of 24-h urinary free catecholamine excretion values with age have also been noted [ 18,211. We did not observe any sex-related differences in catecholamine excretion. When expressed in terms of creatinine, Gerlo et al. [20] found higher urinary A excretion by men, whereas excretions of NA and DA by women were somewhat higher. However, investigating single urine voidings from 482 school children, Reed et al. [23] found no significant sex-related differences in urinary free catecholamine excretion. Urinary N-MA levels have merely been reported for normotensive and hypertensive adults and patients with phaeochromocytoma [5,7]. Using HPLC with electrochemical detection, Gerlo and Malfait [7] established N-MA in urine of 150 hypertensive non-phaeochromocytoma patients. They found N-MA excretion levels to be slightly lower than those of A. Our data (O-58 nmol/24 h, Table 1) proved in excellent agreement with their results (2-65 nmol/24 h). We could not confirm the correlation between urinary free N-MA and A in adults. In the present study urinary free N-MA and A levels of children were, however, significantly related.
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Studying children and adults, we found age-dependent increases of the relative amounts of both urinary A and N-MA (Fig. 2). Relative amounts of urinary catecholamines in newborns were highly diverse. The former data suggest that with advancing age both A and N-MA are increasingly formed. Urinary free catechol,amine levels do, however, not necessarily reflect production rates. Differences in metabolic pressure and reuptake efficiency are possible confounders. In an experiment with rats that were intraperitoneally injected with N-MA, we noted a significant increase in urinary output of its 0-methylated metabolite N-MM, thus confirming earlier observations [l]. However, no changes were found in urinary levels of oxidatively deaminated catecholamine metabolites (I.P. Kema et al., unpublished observations). It suggests that, in contrast to ‘classical’catecholamines, N-MA is not an adequate substrate for monoamine oxidase. Differences between urinary levels of N-MA
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believed to compensate for the endocrine immaturity of the adrenal medulla in fetal and early neonatal life, thus enabling the fetus and newborn to maintain internal homeostasis. Using bioassay and fluorescent analysis, West et al. [30] determined ‘pressor amine’ contents of both adrenal tissue and organ of Zuckerkandl from nearly 100 infants and children. It was concluded that the organ of Zuckerkandl reaches maturity at birth, that it mainly contains NA and that at birth its total pressor amine content exceeds that of the adrenal gland. After birth, concomitant with adrenal medulla development, extra adrenal chromaffin bodies regress, showing cellular degeneration, fibrosis and loss of catecholamines [30,31]. Our study shows gestational-age dependent increases of both the levels and relative amounts of N-MA in the organ of Zuckerkandl, with highest values in infants (Table 2, Fig. 3). Several studies [32-341 demonstrated the presence of PNMT in extra adrenal chromaffin tissues, including the organ of Zuckerkandl, albeit at low activities. Whether in these tissues A and N-MA syntheses merely result from PNMT activity remains to be established, since aspecilic N-methylation activity independent from glucocorticoid induction has also been noted [35]. Increasing levels of N-MA in extra adrenal chromaffm tissue during its regression raises the question whether it reflects production of a metabolite of A without any functional role. Fetal brown adipose tissue is located in areas surrounding major vessels [36]. It has an important role in neonatal thermoregulation through non-shivering thermogenesis [37]. Innervation takes place by the sympathetic nervous system. NA is believed to be the principal neurotransmitter in stimulation of non-shivering thermogenesis [31,37] and an increase in urinary catecholamine excretion, notably NA, has been noted after exposure to cold [38]. Moreover, it has been suggested that catecholamines have trophic effects on brown adipose tissue development and maturation [31]. Our data confirm the notion that in brown adipose tissue of very premature and term neonates NA is the main neurotransmitter. Results from the two newborns suggest that after birth A and N-MA become increasingly important (Table 2, Fig. 3). 4.4. Conclusion Analyses in urines from children and adults show that contributions of both A and N-MA to the sum of free catecholamines increase with age. Relative amounts of A and N-MA increase in both adrenal and extra adrenal chromaffin tissues from late gestation up to several months of postnatal life. N-MA content of adrenal tissue follows endocrine maturation of the medulla and, more specifically, PNMT induction and subsequent A synthesis. Relative amounts of N-MA in extra adrenal chromaffin tissue increase in a period in which these structures regress. The function of N-MA, as either a long-acting catecholamine or an A-metabolite, is as yet unclear. Acknowledgements
We thank Prof. Dr. N. Seiler for providing us with the pharmacological profile of N-methyladrenaline and A.M. Bmgman, G. Meiborg, G.T. Nagel and Dr. J. Prins for their skilful technical assistance and co-operation.
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