ClinicalScience(1986) 71, 367-374
367
Ribonucleic acid turnover in man: RNA catabolites in urine as
measure for the metabolism of each of the three major species
of RNA
GERNOT SANDER, HEINRICH TOPP, GESA HELLER-SCHOCH,
JAN WIELAND AND GERHARD SCHOCH
Forschungsinstitutf i r Kinderernahrung, Dortmund, Federal Republic of Germany
(Received 18 November 1985/1OMarch1986; accepted 11 April 1986)
Summary
1. Urinary excretion of the non-reusable modified RNA catabolites pseudouridine ( V), 7-methylguanine (m7Gua) and N2,N2-dimethylguanosine
(m;Guo) was measured in preterm infants and in
adults. The values (in pmoVmmo1 of creatinine)
were: for preterm and ‘small for gestational age’
infants (n= 26; number of samples = 38) 11 = 164
(SD 32), m7Gua = 39.1 (SD 9.0), m;Guo = 10.6 (SD
2.1); for adults ( n= 32) 11 = 25.3 (SD 3.1))m7Gua
= 4.8 (SD 0.89), m;Guo = 1.53 (SD 0.42).
2. Our measurements were compared with an
expectation derived from the average cellular distribution of 11, m7Gua and m;Guo between rRNA,
tRNA and mRNA. m;Guo occurs exclusively in
tRNA, 11in both rRNA and tRNA, and m7Gua in
all three RNA classes, in proportions which can be
estimated for the steady state. Urinary excretion of
qb and m$Guo should reflect their steady-state distribution, since rRNA and tRNA have been shown
to have similar turnover rates in mammalian tissues.
3. We conclude that we can use the excretion of
m;Guo to assess whole-body tRNA turnover. Since
tRNA contains 11 in a constant proportion to
m;Guo, the proportion of urinary ly stemming from
tRNA can be estimated, and the remainder ( 60-65%) is an indicator of rRNA turnover. Finally,
the excretion of m7Gua far exceeds the proportion
predicted to come from rRNA and tRNA. We
ascribe this excess (-60-70% of the total) to the
turnover of the mRNA ‘cap’-structure, which is
typical for all higher organisms. mRNA turnover is
Correspondence: Professor Dr G. Schoch, Forschungsinstitut fiir Kinderemiihrung, Heinstiick 11, D-4600
Dortmimd 50, FRG.
known to be much higher than that of rRNA or
tRNA.
4. Calculated average RNA turnover rates (in
pmol day-’ kg-’ body weight) were: for preterm
infants 0.1 rRNA, 1.93 tRNA, 2.44 mRNA-‘cap’;
for adults 0.037 rRNA, 0.63 tRNA, 0.62 mRNA‘cap’.
5. Estimation of RNA turnover rates may supplement the hitherto more widely used ISN tracer
studies in assessing whole-body metabolic state.
Results obtained up to now closely agree with the
findings from 15N tracer studies (approximately
threefold higher turnover rates in preterm infants
than in adults). This is probably correlated with the
fact that RNA is intimately involved in all steps of
protein synthesis.
Key words: adults, N2,N2-dimethylguanosine,messenger RNA, 7-methylguanine, premature infants,
pseudouridine, ribonucleic acid, ribosomal RNA,
transfer RNA, urine.
Abbreviations: 11, pseudouridine;m7Gua,7-methylguanine; mBGuo, N2,
P-dimethylguanosine.
Introduction
Biochemical markers for the non-invasive measurement of whole-body metabolism are thought to constitute a potentially powerful means for assessing
metabolic state [l].In analogy to 3-methylhistidine
(a marker for actin and myosin turnover [2]) we
have been looking for non-reusable RNA catabolites which are not metabolized but are quantitatively excreted in urine and can thus serve as
markers for the turnover of their parent macro-
368
G. Sander et al.
molecules [3]. The central role of RNA in protein
synthesis suggests that measurement of RNA turnover may be used to complement the better-known
lSNtracer studies used to estimate whole-body protein turnover.
Protein synthesis implies the co-ordinate functioning of the three major RNA species found in all
cells, rRNA, tRNA and &A.
The role of each of
these RNA classes is distinct, from largely structural
(as in ribosomes) via mostly informational (mRNA)
to what might be called ‘translational’ (indeed it is
the proper charging of tRNA with its cognate amino
acid that constitutes the main informational link
between proteins and nucleic acids). These differences in function are reflected in different turnover
rates: mRNA is synthesized and degraded rapidly
[4, 51 whereas both rRNA and tRNA are rather
stable, with half-lives of e.g. 3-5 days in rat liver
[6-81.
Apart from the major nucleosides uridine, cytidine, adenosine and guanosine, all of the three
major RNA species contain modified components,
formed post-transcriptionally by the action of modifying (mostly methylating) enzymes [9-161. Since
most of these modified compounds cannot be reutilized, they are excreted in urine [17, 181. For
pseudouridine [19, 201 and m7Gua [21] it has been
shown that excretion is quantitative; the same is
likely for mZGuo (see the Results section).
It has been shown in this laboratory [22-251 and
has recently been confirmed by others for children
aged 4-13 years [26] that the excretion of RNA
catabolites in urine is a function of age. Indeed it
closely parallels the growth rate: infants excrete
about six to ten times the amounts (in relation to
creatinine) than do adults of a number of RNA
catabolites [22-251. This finding correlates with the
higher protein turnover rates found in newborn or
preterm infants compared with values in adults, and
indeed RNA and protein turnover are closely
linked [27].
In the present communication we compare the
RNA turnover rates of preterm infants and adults.
Methods
Subjects
Fourteen preterm infants appropriate for gestational age (32.8f 1.3 weeks, birth weight
1643+ 248 g) and five preterm infants small for
gestational age (34.3 2.0 weeks, birth weight
1 4 9 0 f 349 g) were studied. Sample collection in
these two groups was done 7-61 days postpartum.
Seven full-term infants small for gestational age
(birth weight 2061 f 203 g) were also studied and
sample collection was 2-29 days post partum.
+
Throughout this paper, these 26 infants are collectively called ‘preterm infants’. Average weight for all
infants at the time of sample collection was
1 8 9 7 f 3 2 1 g. The infants were in the unit for
premature infants of the Children’s Hospital B of
the University of Dusseldorf.
At the time of sample collection all 26 infants (38
samples) were in a stable clinical state and needed
only routine care without any medication (most of
them recovered from initial respiratory distress syndrome in part after caesarean section).
The adults for the RNA catabolite measurements were healthy volunteers from our staff (14
men and 18 women aged 17-55 years).
Urine samples
All urine samples from infants were 24 h urine
specimens collected in Urinocol bags (lo biotrol, 1
rue du foin, Paris cedex 03, France). The urine
samples from adults were single voidings.
Analytical methods
Materials, methods and equipment were the
same as described previously [28, 291 and only an
outline of the current procedure is given here.
Urine (600 pl) was deproteinized with methanol
and the sample was adjusted to pH 10.5 with 25 pl
of aqueous ammonia solution (sp. gr. 0.91) 25%; at
this pH nucleosides as well as nucleobases were
adsorbed on to an anion exchange column. The two
groups of compounds were then eluted together
with 0.15 mol/l ammonium formate in 0.1 mol/l
formic acid, at pH 3.6. The eluate was mixed via a
three-way valve with 1mol/l ammonia in 0.15 mol/l
formic acid, pH 10.5 (yielding a final pH of 9.9,
and passed through a boronate affinity gel column
(Affi-gel, BioRad) which selectively binds compounds bearing cis-diol groups (like the nucleosides) but not the free nucleobases. After passage of
the latter, the nucleosides were eluted with 0.1
mol/l formic acid, pH 2.4.
Nucleobases and nucleosides were quantified
independently by HPLC analysis with a cation
exchange resin and a reversed-phase resin respectively [28]; pseudouridine was measured by cation
exchange HPLC chromatography of diluted native
urine [29].
Measurement of the free bases is essential for
our considerations because 7-methylguanine, the
second most abundant modified RNA catabolite in
urine, is excreted as free base [30] and would go
undetected if we followed the most widely applied
method of Gehrke et al. [31,32].
In this communication we have (except for
m7Gua) always taken the sum of each nucleoside
Ribonucleic acid turnover in man
and the corresponding nucleobase whenever possible. Indeed this is a necessity for our interpretation, at least in the cases of 1-methylguanosine
(m'Guo)/l-methylguanine (m'Gua), where - 80%
is excreted as m'Gua, and that of 2-methylguanosine (m2Guo)/2-methylguanine (m2Gua),where 35% appears in urine as mZGua.Measuring both
the nucleosides and the corresponding free bases is
feasible when studying large collectives. For individual samples it has often proved impossible to
detect e.g. m2Gua or N2,N2-dimethylguanine
(m:Gua), in the former case due to interfering peaks
and in the latter case because the free base m$Gua
is very often below the detection limit. Indeed
2 9 5 % of the m:Guo appears to be excreted as
such (i.e. the nucleobide), whose analysis is thus
normally sufficient.
Results
Table 1 shows urinary excretion values for some of
the major modified RNA catabolites found in
human urine, all of them expressed as pmol/mmol
of creatinine and also as per cent of pseudouridine,
the most abundant RNA derivative. Despite the
large differences between preterm infants and
adults when the figures relative to creatinine excretion are compared, there are some striking regularities when the data are reduced to pseudouridine
= 100%. This phenomenon prompted us to probe
into the molecular origin of these substances, a task
which has become feasible by the large amount of
structural data which have become available in
recent years.
In Table 2 ('residues per cell') a summary of
current knowledge on the specific molecular origin
of some of the modified RNA catabolites appearing
in human urine is given (small nuclear RNAs, which
constitute about 1% of the total cellular FWA and
369
are metabolically stable, are omitted here). The distribution is non-random: thus m2,Guo and m'Guo
are found exclusively in tRNA, pseudouridine in
rRNA and tRNA but not in mRNA; only m7Guo is
present in all three major RNA classes (m2Guo
which from Table 2 would appear to be restricted to
tRNA actually is present also in small nuclear
RNA).
Half-lives for rRNA and tRNA are similar to
each other in all mammalian tissues studied so far
[6, 7, 331. We should therefore find in urine the
modified compounds present only in these two
RNA classes in approximately the proportions in
which they are present in the average cell.
What is an average mammalian cell? The
assumption that a few well-defined experimental
systems, such as rat liver, rabbit reticulocytes, HeLa
cells and the like, constitute a sufficiently good
model for the whole organism is rather doubtful,
even though data presented in textbooks are frequently of such origin. When measuring urinary
excretion, we are dealing with products from the
whole organism.
We have therefore tried to obtain a more complete picture by recalculating the data of Gunning et
al. [33], who have measured among other things the
tRNA and rRNA contents of seven different rat
tissues. We think we can be reasonably confident
that the boxed numbers for tRNA in Table 2 give a
good approximation for an average mammalian
cell; even if we are wrong we should not be very far
from reality, as the measured extremes to the left
and to the right of the boxed columns show.
We can expect the situation in human tissues to
be similar, since even widely divergent systems such
as Escherichia coli and mouse L cells have similar
tRNNribosome ratios in conditions of rapid
growth, of the order of 10 and 16, respectively [40,
411. Our adopted value for the tRNNribosome
TABLE1. Urinary excretion of some modified RNA catabolites by preterm infants and
adults
Measurements (mean values) are given in pmoVmmol of creatinine, and are corrected
for known systematic losses (maximally 20Y0, for m2Gua). Each of the n samples (preterm infants, n = 38; adults, n = 32) was analysed at least in duplicate (usually four analyses per sample).
Substance
Preterm infants
Mean -1SD (%)
Pseudouridine
164f 32
m7Gua
39.1 -19
m:Gua+m:Guo
10.6f2.1
m'Gua+m'Guo 5.5-11.95
m2Gua+mZGuo 6.0-12.2
(20)
(23)
(20)
(35)
(37)
Adults
Normalized to
Mean f SD
pseudouridine = 100
(mean f SEM)
100
23.8 f0.63
6.5-10.15
3.4f0.16
3.7k0.17
25.3 k 3.1
4.80 -10.89
1.53f0.42
1.34k0.54
0.67f0.24
("/o)
Normalized to
pseudouridine = 100
(mean f SEM)
(12)
100
(19)
(27)
(40)
(36)
19.0 i 0.53
6.0-10.22
5.3k0.31
2.6-10.14
~
95
1
0
0
0
Residues
Per
molecule
143
1.5
0
0
0
10-6x No.
of residues
per cell
Ribosomes
( 1.5 x 1O6 molecules/ceU)
2.9
0.58
0.51
0.58
0.87
Residues
per molecule
(average)*
11
17
57
109
33
50
165
10-6X No.
of residues
per cell
tRNA
(37.5 x lo6molecules/cell
for the boxed column)
0
1
0
0
0
Residues
Per
molecule
0
0.5
0
0
0
10-6X No.
of residues
per cell
mRNA
(0.5 x lo6molecules/ceU)
11
17
10'6XZ
Residues/cell
As%ofWt
*Numbers of modified residues per molecule differ only in tRNA; therefore, average values from 57 selected eukaryotic and (wherever possible) mammalian tRNA sequences
have been compiled from published data [16]. Figures for rRNA and mRNA are from refs. [ll-131.
t Normalized figures independent of the absolute number of ribosomes per cell, which may indeed be considerably different for the whole-body average of the rat, and which may
also be different in rat and in man. By normalizing to I$ = 100, interspecies differences should be minimized, since the ratio tRNNribosomes can be expected to vary relatively
little from one mammalian species to another.
Pseudouridme
m7Guo
&Guo
m'Guo
m2Guo
~~
Nucleoside
TABLE2. Modified nucleosides in eukaryotic RNA
Modified RNA building blocks within their respective macromolecules are discussed in terms of the nucleosides denoted by the ending -0,as in Guo. Upon
degradation of the parent macromolecule, modified building blocks may be degraded to the nucleoside or (with the exception of q )they may be further
deribosylated to the free base denoted by the ending -a, as in Gua. In urine, often both nucleoside and the corresponding free base are found. The number of
1.5 x lo6 ribosomes per cell is an average from seven rat tissues; it has been recalculated after Gunning et al. [33] assuming 6 pg of DNNcell [34] and a
molecular weight for total rRNA (28 S, 18 S, 5.8 S and 5 S rRNA) of 2.23 X lo6 (calculated from refs. [35-381). The tRNNribosome ratio depends on the
tissue analysed. The boxed numbers represent the averages of seven rat tissues, corresponding to 37.5 X lo6molecules of tRNA per cell (25 tRNA molecules/
ribosome). To the left and to the right of the boxed numbers are shown corresponding values for the lowest (thymus = 13) and highest (muscle = 38) measured tRNNrRNA ratios in the rat [33]. The number of mRNA molecules is an average of eight measurements in five different mammalian tissues (range
190000-880000) [39].
!-
E,
.%
m
b
2
3
3
9
4
0
W
Ribonucleic acid turnover in man
molar ratio of 25, averaged from all tissues, is
higher than that of Alberts et al. [4] ( - 17). Muscle
has the highest measured tRNNribosome ratio ( 38) but relatively low RNA contents. Other tissues
contain between 13 (thymus) and 291 (heart,
cerebral cortex) tFWA molecules per ribosome,
with rat liver being near the lower end, at - 19.
Table 2 ( 2 residues/cell’) also shows the total
number of residues per cell for the modified RNA
building blocks considered here (the most likely
figures, with 25 tRNA molecules per ribosome, are
again boxed), and the same data normalized to
pseudouridine = 100. This latter column is the one
to be compared with the normalized experimental
data in Table 1. Pseudouridine is excreted quantitatively in urine [19, 201 and is therefore an excellent reference substance. The values for miGuo +
m$Gua agree fairly closely with the expectation: 6.0
(adults)and 6.5 (preterm infants) from Table 1compared with a theoretical value of 7.5, corresponding
to 80 and 87% of the expected excretion, respectively. Even the least favourable assumption, i.e. the
highest amount of m$Guo relative to pseudouridine
in Table 2 (9.4%), yields, when compared with the
corresponding figures in Table 1, excretion rates for
miGuo + m$Gua which are > 60% of the theoretical expectation (63% for adults, 69% for preterm
infants).
We conclude that at least 60% and possibly all of
the m$Guo present in the body is excreted in urine
in a measurable form. As this substance occurs
exclusively in tRNA, it is therefore a good marker
for tRNA turnover. The other two modified RNA
catabolites occurring only or mostly in tRNA,
mlGuo and particularly m2Guo, appear to be partially lost to analysis, possibly by degradation or
further modification.
7-Methylguanine is found in all three major
-
-
371
classes of RNA, including mRNA (Table 2). If the
turnover rate of mRNA were similar to that of
rRNA and tRNA, then one would expect urinary
excretion levels only about 1.3-fold higher than the
ones for miGuo + m$Gua, since the contribution
from mRNA would be negligible. What we and
others [42] find, however, are values for m7Gua
three- to four-fold those for m$Guo + miGua.
Indeed our figures are probably still about 20% too
low since one-fifth of the m7Gua appears to be
excreted as 7-methyl-8-hydroxyguanine [43,44].
The m7Gua excreted in excess of what can maximally come from rRNA+ tRNA must be attributed
to the turnover of the ‘cap’-structure of mRNA, a
substantial portion of which appears to be rapidly
degraded shortly after synthesis [4]. According to
Alberts et al. [4] 58% of the RNA synthesis of fibroblast cells in culture is used for producing the 3%
mature mRNA found, whereas 42% of the total
RNA synthesis is devoted to rRNA and tRNA
together, which constitute 86% of the total cellular
RNA in this system, 11% being precursors.
Based on the data and arguments outlined above,
we can now try to obtain a quantitative idea about
the turnover of rRNA (ribosomes), tRNA and
mRNA in preterm infants and adults. We have
based our calculations on the fact that miGuo is
only found in tRNA, and further on the assumption
that it is quantitatively excreted in urine. These two
premises allow us to calculate the fraction of
pseudouridine in urine coming from rRNA, as well
as the fraction of m7Gua coming from mRNA
(Table 3).
Taking the figures from Table 3 and the known
molecular weights of total rRNA (2230000)
[35-381, total ribosomal protein (1790 000) [45]
and tRNA (25000) [16] we arrive at the turnover
rates given in Table 4.
TABLE
3. Calculation of the amounts (pmoUmmol of creatinine) of pseudouridine and
m7Gua comingfrom the turnover of rRNA and mRNA respectively
The fraction of pseudouridine stemming from tRNA turnover is calculated by multiplying the urinary value of m;Guo + m;Gua from Table 1by 5.7, pseudouridine being more
abundant in tRNA than miGuo by this factor (Table 2). Similarly, the fraction of m7Gua
from tFWA is obtained by multiplying the value for miGuo+m$Gua by 1.14
(0.58:0.51). The m7Gua fraction from rRNA is calculated accordingly, taking the most
likely figure of 0.08 for the ratio m7Guo,,,:m~Gu0,,,.
This ratio is obtained from the
respective figures in Table 2: 1.5 x lo6m7Guo:19 x lo6m$Guo residues per cell.
Pseudouridine
Total
Fraction from tRNA
Fraction from rRNA
Fraction from mRNA
m7Gua
Preterm infants
Adults
Preterm infants
Adults
164
60
104
-
25.3
8.7
16.6
39.1
12.1
0.8
26.2
4.80
1.74
0.12
2.94
-
372
G. Sander et al.
TABLE
4. Calculated turnover rates for rRNA, tRNA
and mRNA in preterm infants and adults
The turnover rates have been calculated by first
converting the values from Table 1 (m2,Guo +
m2,Gua, for tRNA) or Table 3 (11, for rRNA and
m7Gua for mRNA) into pmol day-' kg-' by using
the following average creatinine excretion values:
preterm infants, 0.093 mmol day-' kg-' (our data);
adults, 0.21 mmol day-' kg-' [46]. The nucleoside
+ nucleobase excretion values have then been
divided by 95 for 11, from rRNA (one ribosome contains - 95 11, residues) and by 0.51 for tRNA (one
molecule of tRNA contains on the average 0.51
residue of mfGuo). For mRNA no further correction is necessary since there usually is one 'cap'structure for each molecule. Molar turnover rates
have been converted to mg day-' kg-', taking a
molecular weight for rRNA of 2.23 x lo6 (Table 2),
for ribosomes a molecular weight of 4.02 x lo6
[35-38,45]andfortRNAof 25000 [16].
Turnover rates
Preterm infants Adults
~~~~~~~
pmol of rRNA day-l kg-'
mg of rRNA day-' kg-'
mg of ribosomes day-' kg'l*
pmol of tRNA day-' kg-'
mg of tRNA day-' kg-'
pmol of mRNA-cap day-' kg-'
0.1
223
402
1.93
48
2.44
0.037
82.5
149
0.63
15.8
0.62
*Assuming similar half-lives for ribosomal proteins as for
rRNA [6] and a total protein mass per ribosome of
1.79 X lohdaltons [45].
Discussion
The most reliable figures in Table 4 are those for
tRNA turnover since they are based on the
measurement of only one parent compound
(mfGuo),which from our data is almost exclusively
excreted as such, and which occurs only in tRNA.
The numbers given in Table 4 represent the turnover of mature tFWA since miGuo is probably
formed only in those portions of the tRNA precursor molecules which are conserved in the mature
tRNA [47]. The actual whole-body turnover of
mature tRNA would be higher if excretion of
mfGuo were not quantitative.
Part of the pseudouridine excretion (mfGuo x
5.7) can be ascribed to tRNA turnover with a high
degree of certainty, since pseudouridine is present
in tRNA in 5.7 times the amount as m;Guo. Our
values for rRNA and ribosome turnover could be
too high if the correction for the pseudouridine
coming from tRNA were too low (the case of less
than quantitative excretion of mfGuo). We have
again restricted ourselves to the turnover of completed rRNA. This is justified because pseudouri-
dine is apparently formed only in the conserved
part of the precursor molecule [48].
In the case of mRNA only molar quantities can
be calculated with some certainty since the average
length of mRNA precursor (HnRNA)is not known
for the whole body. Model studies suggest that an
average HnRNA may consist of 6000-8000
nucleotides, each molecule containing one m7Guo
residue as 'cap'-structure; the cap appears to be
conserved during HnRNA processing, which yields
mRNA molecules with an average chain length of
1200 translated nucleotides [4],or a total of - 2000
nucleotides when the untranslated 5' and 3'
sequences are included. Taking the average figure of
7000 nucleotides from above, we obtain -5500
mg of mRNA precursor produced per day per kg in
preterm infants; for adults, the corresponding figure
is 1500 mg'day-I kg'. Such a calculation presupposes that all or most mRNA precursor molecules are in fact processed to mature mRNA, which
is not an established fact. Indeed, the actual size
distribution of HnRNA and mRNA in the various
tissues is not known, nor is it evident that the processing events are similar in all tissues. Therefore
these figures should not be taken as definitive.
Our data suggest quite a close quantitative correlation between the turnover of all three major RNA
species and that of whole-body protein: RNA turnover as estimated by us is 2.7 (rRNA), 3.1 (tFWA)
and 3.9 times (mRNA) higher in preterm infants
than in adults (cf. [49]). Protein turnover in preterm
infants has likewise been estimated to be three to
four times higher in preterm infants than in adults:
adults synthesize about 4 g of protein day-] kg-1
[27], preterm infants about 15 [50]. Assuming an
average protein molecular weight of 40000 [4], we
find (together with the mRNA data in Table 4)
almost identical protein/mRNA-'cap' molar turnover ratios for adults and preterm infants, around
155 mol of proteidmol of mRNA-'cap' [511.
The practical interest of our approach may be
twofold. 1. Measurement of urinary RNA catabolites may in healthy individuals be used to supplement the classical 15Ntracer studies for measuring
whole-body protein turnover. Our method is noninvasive and relatively easy to follow (especially
when restricting oneself to pseudouridine as a combined marker for rRNA and tRNA turnover). The
quantitative interpretation of the data is fairly
straightforward. 2. For diseased individuals, urinary
excretion of modified RNA catabolites can be
expected to be altered, as has already been
observed in certain malignancies [22, 23, 26, 30,
52-60]. Pilot studies seem to indicate changed
urinary excretion of certain RNA catabolites in different states of failure to thrive [61]. If substantiated, these findings might pave the way for using
-
Ribonucleic ac:id turnover in man
urinary RNA catabolites as non-invasive markers
for the metabolic state of individuals.
Acknowledgments
This work has been funded by the Ministerium fiir
Wissenschaft und Forschung des Landes Nordrhein-Westfalen and by the Bundesministerium fiir
Jugend, Familie und Gesundheit. We gratefully
acknowledge the excellent technical assistance of
Mss E. Steiner, V. Iontcheva, J. Rzychon and A.
Junghardt. We thank Ms E. Kohler and Ms E.
Hrdina for typing the manuscript. We thank Professor Dr med. Eberhard Schmidt and cand. med.
A.-B. Gehlhar and U. Kordass from the Children’s
Hospital B, University of Dusseldorf, for providing
the specimens. This paper includes results from the
Ph.D. thesis of H.T.
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