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Aminoaciduria in the nephrotic syndrome in children
805
CLINICA CHIMICA ACTA
AMINOACIDURIA
IN THE
NEPHROTIC
J. OPIEikKA-BLAUTH
AND H. KOWALSKA
Department of Biochemistry, (Received
IN CHILDREN
SYNDROME
Medical School, Lublin (Poland)
February 3rd. 1961)
INTRODUCTION WOOLF
AND GILES~,
hyperaminoaciduria BERGER~, 6 cases have
however,
examined.
a distinct Conflicting
also been reported In the present
pathological in urine with
HOOFT
in the
aminoaciduria the clinical
of lipoid
aminoaciduria results
by other
study
picture
occurred
concerning
in only
one case from
aminoaciduria
in lipoid
among
nephrosis
authors.
we have and
and others almost always observed nephrosis in children. According to
AND HERPOL~
course
attempted
to compare
to establish the
of the nephrotic
criteria
intensity
for what
of amino
acid
is called excretion
syndrome.
propavrol water-
propanol water +
I
C A Fig. I. (,4) Amino acids in physiological urine (IOO ~1) R.J. 9 years old. (B) Amino acids in urine (IOO ~1) R.J. after hormonal treatment. (C) Amino acids in urine R.J. after hormonal treatment and high protein diet. MATERIAL
AND METHODS
15 children, between the ages of 3 and 13 years, with lipoid nephrosis were investigated. Aminoaciduria, as well as aminoacidaemia, was followed by twodimensional chromatography. Urine (from 24-h samples) and blood, after the removal of salts on cation exchangers, were applied to Whatman No. I filter paper and developed in the solvents: (A) Propanol-water (7 : 3), phenol-water (7 : 3). (B) sec.butanol-3% ammonia (150 : 60), sec.-butanol-formic acid-water (150 : 30 : 20). Clin. Chinz.Acta, 6 (1961) 805-813
806
J. OPIEtiSXA-BLAUTH,
H. XOWALSXA
-
propand water -4
Fig. 2. of spots of 24-h of q-h
propnnoi water C
propan w&r D
(d) NormaJ urine “C” not desalted from a .+-year old child. 3.3 ~‘1of q-h urine. Number 5. (B) Normal urine “C” desalted on cation exchanger SDW, from a q-year child. 33 ~1 urine. Number of spots 17, (C) Normal urine “C” desalted from a +-year old child. 66 ,ul urine. Number of spots 16. (D) Normal urine “C” desalted on cation exchanger SDW, from a. qyear old child. 332 ~1 of q-h urine. Number of spots 17.
The spots were revealed with 0.2% ninhydrin in acetone at room temperature. cc-Amino-nitrogen was determined according to SPIER-PASCHER’s method4. The fourteen cases of lipoid nephrosis were examined several times in various stages of the illness. In order to establish the effect of high protein diet and hormone therapy (ACTH, prednisone) a control group of healthy children aged j to IO years was followed in parallel. The clinical state was evaluated mainly on the basis of oedema, the intensity of
AMINOACIDURIS
IN NEPHROTIC
SYNDROME
807
which was expressed arbitrarily with : &, I_, + +, + + +. Pathological aminoaciduria and aminoacidaemia were always evaluated with respect to physiological urine and serums, the following criteria being taken into consideration: (u) cc-Amino-nitrogen in urine/kg/24 h/mg. (b) Increased or decreased concentration of common amino acids as compared to physiological urines. (c) Increased or decreased number of ninhydrin-positive spots on two-dimensional chromatograms. (d) Appearance of “rare” amino acids. (e) Appearance of “strange” amino acids which never occur in physiological (under established experimental conditions). (f) Lack of some common
urines.
amino acids.
-------+
Fig. 3. Amino acids in lipoid nephrotic urine M.B. Type “R” from IOO ,ul of 24-h urine specimen.
Fig. 4. “Rare” amino acids in lipoid nephrotic urine.
RESULTS
The results are summarised in Tables I-V and Figs. 2-4. Table I shows the relationship between a-amino-nitrogen of urine and the gravity of the illness in several selected cases. Fig. 2, A, B, C, D, shows control chromatograms from urine of a 4-year old healthy child, carried out under standard conditions. Fig. 3 presents a chromatogram from urine of a case of lipoid nephrosis. Fig. 4 shows a chromatogram with clear-cut ethanolamine from urine of a patient, developed in butanol solvents. Table II gives the frequency of occurrence of rare and strange amino acids on chromatograms in 14 cases (25examinations). It has been observed that in the majority of cases two common amino acids e.g. serine and threonine are lacking on the chromatograms. This finding is correlated with the clinical status in Table III. Some cases were chosen for a systematic study of aminoaciduria and aminoacidaemia in various stages of the illness and therapy, the results are recorded in Table IV. The results of 15 cases of lipoid nephrosis are summarized in Table V.
J. OPIEkKA-BLAUTH,
808
H. KOWALSKA
TABLE
I
a-AMINO-NITROGENOF URINE IN THE CASES OF LIPOID NEPHROSIS Case
Oedema
NNH~~&?/+
R.K. R.K.
4 B:
+ + i__.-
4.8 I.8
S.K. S.K.
A. B.
++i __ +++
6.0 4.5 6.1
h(w)
-4.x.
A.
M.B.
A.
J.a.
B.
__
2,s
T.B. T.B.
A. B.
++ ++
0.99 0.99
2.9
~______~_
TABLE THE FREQUENCY
OF
NUMBER
OF
II
“STRANGE”
THE
AND
OCCURRENCES
IN
“RARE”
25
AMINO
ACIDS
SPECIMENS
Rare
strange /%Alanine Hydroxyproline Proline
,%Aminoisobutyric acid “Leucine” * “Valine”* wAminobutyric acid Ethanolamine Arginine
-4 -3 -I
--I I -6 -5 --2 -8 -2
* Leucine = Leu + Ileu + Phe. * Valine = Val + Try + Met. TABLE SERINE
AND
THREONINE
IN
LIPOID
Case
III URINE
IN
THE
OF
Oedema
Serine
Threonine absent present absent absent
S.K. S.K.
A. B.
+++
absent present
R.K. R.K.
A. B.
+++
absent absent
S.B.
A.
+++
K.K.
A.
TABLE CASE
CASES
NEPHROSIS
T.B.
AGE
absent
absent
present
present
IV 6
YEARS ~~~~ ~~~~
_.______~
~.
Date of exami-
nation 14-XI-59yr r9-XII-59yr z5-I-6oyr
Number of * -4 mine .I mine .4 ZbuminL Serine globulin N~~~l24 h ninhydrin acids acids Oedema quotient mg positive spots (‘rare’, “strange” _~~~_____ ~~_~ ~~~~ 18 6 0.6 absent absent absent + + absent 12 BAIB absent + + 0.5 79 6 absent absent absent +++ 0.34 0.28 r.2
e6-II-6oyr 19-III-6oyr I 3-V-6oyr
++
1.3
_-.
Threonine absent absent absent
-
15 r7
absent BAIB
absent absent
present present
94
13
Eth Leu Phe
absent
present present _ __. __..
10s
absent present
* From IOO ~1 of 24-h urine specimen. Clin. Chirn. .4cta. 6 (1961) 805-813
A~~INO~~IDURIA IN NEPHROTIC SYNDROME
go9
DISCUSSION Different views are expressed in the literature concerning pathological aminoaciduria in the nephrotic syndrome. BAROW AND HARTMAN 6, BICKEL AND SOUCHON?, SLATER et al. * and PLUCKTHUN et aZ.@were unable to detect hyperaminoaciduria in mild cases of nephrosis. BERGER~ observed hyperaminoaciduria in only one case of severe nephrosis among six cases examined. MILHAUD AND CO~RVOISIER~~ and MARTIN AND MILHAUD I1 regarded hyperaminoaciduria as a result of high protein diet and SCHWARZ-TIENE et a1.12 as a consequence of hormone therapy. SQUIREI” and BLAINEY I4 reported nephrosis with concomitant excretion of amino acids and glucose. SCHREEVE et aLI used microbiological tests in his study but obtained inconsistent results, WOOLF AND GILES~ described hyperaminoaciduria in 25 cases of nephrosis among 28 cases studied. They have classified aminoaciduria into three types. Type “H” is characterized by a mild course and good prognosis. In the urine occur P-aminoisobutyric acid (BAIB), increased taurine and, less frequently, tyrosine, leucine, valine, ethanolamine. The authors connect this type with an altered activity of the renal tubules and with changes in amino acid metabolism. Type “R” is rather severe in its clinical course; such amino acids as leucine, valine, proline and alanine in particularly high concentration are excreted in urine. WOOLF AND GILES~ reported normal aminoaciduria in three cases of remission, type “H” in II cases, type “R” in 3 cases and one as a mixed type “HR”. Similarly, HOOFT AND HERPOL 2 considered hyperaminoaciduria as a constant sign of nephrosis, and they classified their patients according to the scheme proposed by WOOLF AND GILES.’ Moreover, they reported hyperaminoaciduria to be accompanied by glucosuria and by increased excretion of phosphates, We have investigated 15 cases of lipoid nephrosis in children. Taking into consideration the necessity of performing chromatography of the urinary amino acids under strictly defined conditions, we have set up criteria for pathological aminoaciduria. In some cases we were unable to observe all the indices of pathological aminoaciduria-e.q. hyperaminoacidurias occurred with the excretion of common amino acids only. From among the “rare” amino acids we could identify the “leucine” as well as “valine” group, cr-aminobutyric acid and especially p-aminoisobutyric acid (BAIB) (Table II) and ethanolamine. The “strange” amino acids were: proline, hydrox~roline, and sometimes ,&alanine. Except the last all the above amino acids were also found by WOOLF AND GILES~. Under our experimental conditions we did not find serine and threonine in nephrotic urines. Among 15 cases under study in 18 examinations of II cases there was no detectable serine and in 17 examinations of IO cases, no threonine (Table III). It is of interest, that both serine and threonine are frequently lacking from urines of new-born babiesIs. The levels of these two amino acids, however, remain normal in serum, and the question arises what is the cause of their absence from urine. Our attempts to answer this question by analyzing hydrolyzed urines were unsuccessful. According to MUETING~?, serum proteins of nephrotic patients contain less serine and threonine than the normal ones. Other authors dealing with aminoaciduria in nephrosis did not make similar findings, possibly because of differences of chromatographic technique used. In our work18 we let ninhydrin react with amino acids at room temCfin.C&in..4&z,6 (196x)805-813
I
G.L.
II
J.P.
III
DZ.
IV
B.M.
-
4
5 me
6 8
4
114
15.Y
3 =’
I’
z0.u
4 Y3
RS
I 111
R
7..X1-58
i-XI-58
I, >.
I.9
L3.j
IO-Ill-jr)
2,
0.9
13.5
lo-III-j9
Ij.6
I fl-XII-
j9
M.B.
13
I m
D6
53.5
9-I\‘- j9
VI
-4.5.
9
2 v.
R
3’ 0
15-X- j9
31.7
21.XII-5Y
12.8
28-X-59
16.1
r%>iII-59
VIII
IX
x
H.N.
J.S.
T.B.
R.K.
3
3 6
8
I m
4m 2m
4Y
U
K
R
u
0.5
,o
,,
0.38
+++ ceased after treatment
1.4 T.2 0.86
25.0
14-X1-5’,
++
20.0
19-X11-59
22.8
25-1-60
18.5
4.TM0
0.6
.- +
o-5
+++
0.34
CfSW?d after treatment
o.28
L
Ic).b
IL-III-Go
21.3
13-V-60
IT
‘-3
23.1
23-X-59
+ + +-
0.36
+XII-59
IL 2
30.x1-59
XII
S.K.
3
1m
R
14.0
II-XII-59
rj.0
8-I-60
27.5
7-I-60
U
ceased after treatment
O.Lj
rt
Ii
rm
.I-
+
I m
S.B.
+i
i-XI-59
3
XIV
0.7 o..i
28-X- j9
K.K.
-.K-6 XV
,, *
16.6
XI
K.Z.
1.9
17.0
23.j
XIII
1.7
13-11-59
V
VII
ceased after treatment
,,
ceased after treatment ,. ++
0.7 t
ceased
after treatment
++
I I
o.rj I. 2
cl.24
811 THE
CASES
EXAMfNED
Urine PVOl&
nwia %o
NNH&~
in mg
h
Chromatograms (no .__II_
-I_-
Number
of spots
0.29
20
AB common
IllCP%XX
_~_ >I
-.-_
--
“Leu” BAIB
.._ ,I
&Ala
Serine
Threw&e
absent
present -._
present
_,,
,,Val”
absent
I,
S0rnKil
absent
‘I
present
present
Great increase
“Leu” “Val”
Pro
absent
absent
14
Normal
BAIB
,,
>I
,I
2.8
20
Increase
Arg BAIB
B-Ala
,,
>*
6.2
‘4
Great increase
Eth BAIB
absent
I,
I9
,.
lifb;ee,?‘;g
B-Ala
,,
,,
Normal
absent
>,
>I
-
4
700
2.64
4o*
I.33
2.9
700
2.64
-
0.03 3.50
1.65
500
0.20
400
r.g6
5oo
4.95
350
3,30
._ 0.8
r7 6
900
3‘40
3.9
460
2.60
-
450
0.33
850
I .48
-
700
2.90
.-
600
T.65
800
a.01
_
pl urine)
AA strangs
Decrease-
2.g3
800
A‘4 Yale
13 -_____. 16
,>
>2
“Val”
-
9
absent
~__. Decrease
BAIB
present
,,
absent
absent
,,
12
Increase
BAIB
>,
,t
6
Normal
absent
>I
,*
16
Increase
,,
I.
BAIB
17
Normal
‘3
Increase
4.8
21
Great increase
Phe “Leu”
I.8
14
Increase
BAIB
Normal
Eth, Phe “Leu”
present
present
,1
‘I
”
,,
>, absent
absent
“Vsl”
----
.--..___ ______-.__
560
0.06
4.3
I8
300
4.62
6.0
I2
600
0.09
4.5
16
800
g.ro
-
77
~_~ 300 800
_--.__ trace 0.6
--
12 12
2,
,,
,,
I> present
>> present
absent
I>
absent
absent
Eth
0
present
present
BAIB “Lell” “Val”
,,
I,
I,
,I
absent
I,
absent
>,
,I
I,
Increase
Normal _--
21
absent present
812
J. OPIEtiSKA-BLAUTH, H. KOWALSKA
perature which allows identification of the violet spot of serine appearing after the orange coloured glycine. In this study we have been interested in the relationship between aminoaciduria and the clinical status of lipoid nephrosis, taking oedema as an index of the gravity of the disease and in several cases we were able to follow the whole course of it (Table IV). In parallel a healthy child and a group of healthy children on high protein diet were examined (Figs. I and 2). When larger volumes of urine were chromatographed, it was possible to detect besides common amino acids, the “leucine” and “valine” group as well as aminobutyric acids, which WOOLF AND GILES regard as index of pathological aminoaciduria. Neither high protein diet nor hormone therapy seem to exert any influence on the type of amino acids excreted, their only effect being to produce an increased excretion of common amino acids such as glycine, alanine, serine, histidine, glutamine or glutamic acic. If we compare the results obtained on urine and serum it becomes evident, that the albumin/globulin ratio and the serum cholesterol concentration are the only biochemical indices consistent with the clinical status. (Cases: M.B. ; H.N. ; T.B. ; R.K. ; S.K.). Other indices involving the pathological aminoaciduria (e.g. cr-amino-nitrogen, occurrence of “rare” or “strange” amino acids or the absence of serine and threonine) proved to be rather irregular. Of course, the more grave was the condition (oedema +++) the more intense appeared the indices for the common amino acids and for the “rare” or “strange” ones. (proline, hydroxyproline in cases: M.B. (T. “RI’); H.N.; T.B.). As the condition improved the signs of pathological aminoaciduria tended to disappear. Neither serine nor threonine were detectable in severe periods of the disease. On the contrary, they reappeared during remission. In three cases there even was a parallelism between the clinical status and the serine-threonine index (Table IV). Among 15 cases examined we could classify one as type “RI’. It was a fatal case. Other cases did not belong to either type “H” or “HR”. We have never observed glucosuria. The behaviour of serine and threonine will be the object of further studies.
ACKNOWLEDGEMENTS Routine determinations of protein and cholesterol in urine and serum were performed in the Clinic of Pediatrics, Medical School, in Lublin. The patients were treated and their clinical status was evaluated by Dr. T. GERKOWICZ and Dr. B. URBA~~SKA,Assistants in the Clinic. SPMMARY Aminoaciduria in 15 evaluation of pathological performed in various stages except one, to be classified GILES. The fact that serine and discussed.
cases of lipoid nephrosis is described. Criteria for the aminoaciduria are proposed. The investigations were of the disease. The results do not allow the cases studied, as type “HI’, “ R” or “HR” as suggested by WOOLF AND and threonine are absent from nephrotic urines is noted
(‘litz.(‘hirn. .-l&z,6 (1961) 805-S1j
AMINOACIDURIA
IN NEPHROTIC
SYNDROME
813
REFERENCES 1 L. J. WOOLF AND H. MC. C. GILES, A4cta Paediat., 45 (1956) 489. 2 C. HOOFT AND J. HERPOL, Acfa Paediat., 48 (1959) 135. 3 H. BERGER, Aminoacidurie und Hyperaminoacidurie, S. Karger, Basel, Ig5g. 4 H. SPIER AND G. PASHER, Z. Physiol. Chem., 296 (1954) 147. 5 J. OPIE&KA-BLAUTH, Clin. Chim. Acta, 4 (1959) 860. 6 R. BAROW AND F. HARTMAN. Deut. Arch. klin. Med., 201_. (1956) 260. -7 H. BICKEL AND F. SOLJCHON, .4rch. fiir h’inderheilk.,.Beiheft 31 (1955). * R. SLATER. N. KRETCHMER. H. MCNAMARA AND H. L. BARNETT, Protein Metabolism in Nephrosis, Studies on Proteinuria, i\bstracts of combined Meeting of American Pediatric Society for Ped. Research, Canadian Paediatric Society, 1955. p. 126. 9 H. PLUECKTHUN, K. SCHRERER AND H. HAUSS, I
Clin. Chirrt.Acta, 6 (1961) 805-813
THE
RENAL
CLEARANCE
OF
PHENYLPYRUVATE
C. L. J. VINK St. Joseph-Hospital,
Eixdhouen
with technical assistence of MISS A. A. KROES
Pediatrics Department of the University (Received
Hospital, Leiden (The Netherlands)
February
I3th, Ig6I)
INTRODUCTION
Phenylpyruvate (PP, a salt of phenylpyruvic acid) is a substance found in the blood and urine of patients with oligophrenia phenylpyruvica (OP). It is the result and occurs through a deficiency of the enzyme of an “inborn error of metabolism”, phenylalanine hydroxylase. In normal subjects this enzyme catalyses the oxidation of phenylalanine to tyrosinel at such a rate that very little or no PP is detectable in the body fluids. An interesting phenomenon in OP is the high concentration of PP in the urine (up to 3000 mg/l) at a very low blood level (up to zo “g/l). This combination may point to a rather high clearance of PP in this disease. The purpose of this article is to help to explain the mechanism of the renal clearance of PP in patients with OP. As PP has often been regarded as a toxic substance in the development of the cerebrum, and as a cause of the mental retardation in these patients, our experiments were performed without PP-loading. Clin. Ckint. Acta, 6 (1961) 813818
CLINICA CHIMICA ACTA
AMINOACIDURIA
IN THE
NEPHROTIC
J. OPIEikKA-BLAUTH
AND H. KOWALSKA
Department of Biochemistry, (Received
IN CHILDREN
SYNDROME
Medical School, Lublin (Poland)
February 3rd. 1961)
INTRODUCTION WOOLF
AND GILES~,
hyperaminoaciduria BERGER~, 6 cases have
however,
examined.
a distinct Conflicting
also been reported In the present
pathological in urine with
HOOFT
in the
aminoaciduria the clinical
of lipoid
aminoaciduria results
by other
study
picture
occurred
concerning
in only
one case from
aminoaciduria
in lipoid
among
nephrosis
authors.
we have and
and others almost always observed nephrosis in children. According to
AND HERPOL~
course
attempted
to compare
to establish the
of the nephrotic
criteria
intensity
for what
of amino
acid
is called excretion
syndrome.
propavrol water-
propanol water +
I
C A Fig. I. (,4) Amino acids in physiological urine (IOO ~1) R.J. 9 years old. (B) Amino acids in urine (IOO ~1) R.J. after hormonal treatment. (C) Amino acids in urine R.J. after hormonal treatment and high protein diet. MATERIAL
AND METHODS
15 children, between the ages of 3 and 13 years, with lipoid nephrosis were investigated. Aminoaciduria, as well as aminoacidaemia, was followed by twodimensional chromatography. Urine (from 24-h samples) and blood, after the removal of salts on cation exchangers, were applied to Whatman No. I filter paper and developed in the solvents: (A) Propanol-water (7 : 3), phenol-water (7 : 3). (B) sec.butanol-3% ammonia (150 : 60), sec.-butanol-formic acid-water (150 : 30 : 20). Clin. Chinz.Acta, 6 (1961) 805-813
806
J. OPIEtiSXA-BLAUTH,
H. XOWALSXA
-
propand water -4
Fig. 2. of spots of 24-h of q-h
propnnoi water C
propan w&r D
(d) NormaJ urine “C” not desalted from a .+-year old child. 3.3 ~‘1of q-h urine. Number 5. (B) Normal urine “C” desalted on cation exchanger SDW, from a q-year child. 33 ~1 urine. Number of spots 17, (C) Normal urine “C” desalted from a +-year old child. 66 ,ul urine. Number of spots 16. (D) Normal urine “C” desalted on cation exchanger SDW, from a. qyear old child. 332 ~1 of q-h urine. Number of spots 17.
The spots were revealed with 0.2% ninhydrin in acetone at room temperature. cc-Amino-nitrogen was determined according to SPIER-PASCHER’s method4. The fourteen cases of lipoid nephrosis were examined several times in various stages of the illness. In order to establish the effect of high protein diet and hormone therapy (ACTH, prednisone) a control group of healthy children aged j to IO years was followed in parallel. The clinical state was evaluated mainly on the basis of oedema, the intensity of
AMINOACIDURIS
IN NEPHROTIC
SYNDROME
807
which was expressed arbitrarily with : &, I_, + +, + + +. Pathological aminoaciduria and aminoacidaemia were always evaluated with respect to physiological urine and serums, the following criteria being taken into consideration: (u) cc-Amino-nitrogen in urine/kg/24 h/mg. (b) Increased or decreased concentration of common amino acids as compared to physiological urines. (c) Increased or decreased number of ninhydrin-positive spots on two-dimensional chromatograms. (d) Appearance of “rare” amino acids. (e) Appearance of “strange” amino acids which never occur in physiological (under established experimental conditions). (f) Lack of some common
urines.
amino acids.
-------+
Fig. 3. Amino acids in lipoid nephrotic urine M.B. Type “R” from IOO ,ul of 24-h urine specimen.
Fig. 4. “Rare” amino acids in lipoid nephrotic urine.
RESULTS
The results are summarised in Tables I-V and Figs. 2-4. Table I shows the relationship between a-amino-nitrogen of urine and the gravity of the illness in several selected cases. Fig. 2, A, B, C, D, shows control chromatograms from urine of a 4-year old healthy child, carried out under standard conditions. Fig. 3 presents a chromatogram from urine of a case of lipoid nephrosis. Fig. 4 shows a chromatogram with clear-cut ethanolamine from urine of a patient, developed in butanol solvents. Table II gives the frequency of occurrence of rare and strange amino acids on chromatograms in 14 cases (25examinations). It has been observed that in the majority of cases two common amino acids e.g. serine and threonine are lacking on the chromatograms. This finding is correlated with the clinical status in Table III. Some cases were chosen for a systematic study of aminoaciduria and aminoacidaemia in various stages of the illness and therapy, the results are recorded in Table IV. The results of 15 cases of lipoid nephrosis are summarized in Table V.
J. OPIEkKA-BLAUTH,
808
H. KOWALSKA
TABLE
I
a-AMINO-NITROGENOF URINE IN THE CASES OF LIPOID NEPHROSIS Case
Oedema
NNH~~&?/+
R.K. R.K.
4 B:
+ + i__.-
4.8 I.8
S.K. S.K.
A. B.
++i __ +++
6.0 4.5 6.1
h(w)
-4.x.
A.
M.B.
A.
J.a.
B.
__
2,s
T.B. T.B.
A. B.
++ ++
0.99 0.99
2.9
~______~_
TABLE THE FREQUENCY
OF
NUMBER
OF
II
“STRANGE”
THE
AND
OCCURRENCES
IN
“RARE”
25
AMINO
ACIDS
SPECIMENS
Rare
strange /%Alanine Hydroxyproline Proline
,%Aminoisobutyric acid “Leucine” * “Valine”* wAminobutyric acid Ethanolamine Arginine
-4 -3 -I
--I I -6 -5 --2 -8 -2
* Leucine = Leu + Ileu + Phe. * Valine = Val + Try + Met. TABLE SERINE
AND
THREONINE
IN
LIPOID
Case
III URINE
IN
THE
OF
Oedema
Serine
Threonine absent present absent absent
S.K. S.K.
A. B.
+++
absent present
R.K. R.K.
A. B.
+++
absent absent
S.B.
A.
+++
K.K.
A.
TABLE CASE
CASES
NEPHROSIS
T.B.
AGE
absent
absent
present
present
IV 6
YEARS ~~~~ ~~~~
_.______~
~.
Date of exami-
nation 14-XI-59yr r9-XII-59yr z5-I-6oyr
Number of * -4 mine .I mine .4 ZbuminL Serine globulin N~~~l24 h ninhydrin acids acids Oedema quotient mg positive spots (‘rare’, “strange” _~~~_____ ~~_~ ~~~~ 18 6 0.6 absent absent absent + + absent 12 BAIB absent + + 0.5 79 6 absent absent absent +++ 0.34 0.28 r.2
e6-II-6oyr 19-III-6oyr I 3-V-6oyr
++
1.3
_-.
Threonine absent absent absent
-
15 r7
absent BAIB
absent absent
present present
94
13
Eth Leu Phe
absent
present present _ __. __..
10s
absent present
* From IOO ~1 of 24-h urine specimen. Clin. Chirn. .4cta. 6 (1961) 805-813
A~~INO~~IDURIA IN NEPHROTIC SYNDROME
go9
DISCUSSION Different views are expressed in the literature concerning pathological aminoaciduria in the nephrotic syndrome. BAROW AND HARTMAN 6, BICKEL AND SOUCHON?, SLATER et al. * and PLUCKTHUN et aZ.@were unable to detect hyperaminoaciduria in mild cases of nephrosis. BERGER~ observed hyperaminoaciduria in only one case of severe nephrosis among six cases examined. MILHAUD AND CO~RVOISIER~~ and MARTIN AND MILHAUD I1 regarded hyperaminoaciduria as a result of high protein diet and SCHWARZ-TIENE et a1.12 as a consequence of hormone therapy. SQUIREI” and BLAINEY I4 reported nephrosis with concomitant excretion of amino acids and glucose. SCHREEVE et aLI used microbiological tests in his study but obtained inconsistent results, WOOLF AND GILES~ described hyperaminoaciduria in 25 cases of nephrosis among 28 cases studied. They have classified aminoaciduria into three types. Type “H” is characterized by a mild course and good prognosis. In the urine occur P-aminoisobutyric acid (BAIB), increased taurine and, less frequently, tyrosine, leucine, valine, ethanolamine. The authors connect this type with an altered activity of the renal tubules and with changes in amino acid metabolism. Type “R” is rather severe in its clinical course; such amino acids as leucine, valine, proline and alanine in particularly high concentration are excreted in urine. WOOLF AND GILES~ reported normal aminoaciduria in three cases of remission, type “H” in II cases, type “R” in 3 cases and one as a mixed type “HR”. Similarly, HOOFT AND HERPOL 2 considered hyperaminoaciduria as a constant sign of nephrosis, and they classified their patients according to the scheme proposed by WOOLF AND GILES.’ Moreover, they reported hyperaminoaciduria to be accompanied by glucosuria and by increased excretion of phosphates, We have investigated 15 cases of lipoid nephrosis in children. Taking into consideration the necessity of performing chromatography of the urinary amino acids under strictly defined conditions, we have set up criteria for pathological aminoaciduria. In some cases we were unable to observe all the indices of pathological aminoaciduria-e.q. hyperaminoacidurias occurred with the excretion of common amino acids only. From among the “rare” amino acids we could identify the “leucine” as well as “valine” group, cr-aminobutyric acid and especially p-aminoisobutyric acid (BAIB) (Table II) and ethanolamine. The “strange” amino acids were: proline, hydrox~roline, and sometimes ,&alanine. Except the last all the above amino acids were also found by WOOLF AND GILES~. Under our experimental conditions we did not find serine and threonine in nephrotic urines. Among 15 cases under study in 18 examinations of II cases there was no detectable serine and in 17 examinations of IO cases, no threonine (Table III). It is of interest, that both serine and threonine are frequently lacking from urines of new-born babiesIs. The levels of these two amino acids, however, remain normal in serum, and the question arises what is the cause of their absence from urine. Our attempts to answer this question by analyzing hydrolyzed urines were unsuccessful. According to MUETING~?, serum proteins of nephrotic patients contain less serine and threonine than the normal ones. Other authors dealing with aminoaciduria in nephrosis did not make similar findings, possibly because of differences of chromatographic technique used. In our work18 we let ninhydrin react with amino acids at room temCfin.C&in..4&z,6 (196x)805-813
I
G.L.
II
J.P.
III
DZ.
IV
B.M.
-
4
5 me
6 8
4
114
15.Y
3 =’
I’
z0.u
4 Y3
RS
I 111
R
7..X1-58
i-XI-58
I, >.
I.9
L3.j
IO-Ill-jr)
2,
0.9
13.5
lo-III-j9
Ij.6
I fl-XII-
j9
M.B.
13
I m
D6
53.5
9-I\‘- j9
VI
-4.5.
9
2 v.
R
3’ 0
15-X- j9
31.7
21.XII-5Y
12.8
28-X-59
16.1
r%>iII-59
VIII
IX
x
H.N.
J.S.
T.B.
R.K.
3
3 6
8
I m
4m 2m
4Y
U
K
R
u
0.5
,o
,,
0.38
+++ ceased after treatment
1.4 T.2 0.86
25.0
14-X1-5’,
++
20.0
19-X11-59
22.8
25-1-60
18.5
4.TM0
0.6
.- +
o-5
+++
0.34
CfSW?d after treatment
o.28
L
Ic).b
IL-III-Go
21.3
13-V-60
IT
‘-3
23.1
23-X-59
+ + +-
0.36
+XII-59
IL 2
30.x1-59
XII
S.K.
3
1m
R
14.0
II-XII-59
rj.0
8-I-60
27.5
7-I-60
U
ceased after treatment
O.Lj
rt
Ii
rm
.I-
+
I m
S.B.
+i
i-XI-59
3
XIV
0.7 o..i
28-X- j9
K.K.
-.K-6 XV
,, *
16.6
XI
K.Z.
1.9
17.0
23.j
XIII
1.7
13-11-59
V
VII
ceased after treatment
,,
ceased after treatment ,. ++
0.7 t
ceased
after treatment
++
I I
o.rj I. 2
cl.24
811 THE
CASES
EXAMfNED
Urine PVOl&
nwia %o
NNH&~
in mg
h
Chromatograms (no .__II_
-I_-
Number
of spots
0.29
20
AB common
IllCP%XX
_~_ >I
-.-_
--
“Leu” BAIB
.._ ,I
&Ala
Serine
Threw&e
absent
present -._
present
_,,
,,Val”
absent
I,
S0rnKil
absent
‘I
present
present
Great increase
“Leu” “Val”
Pro
absent
absent
14
Normal
BAIB
,,
>I
,I
2.8
20
Increase
Arg BAIB
B-Ala
,,
>*
6.2
‘4
Great increase
Eth BAIB
absent
I,
I9
,.
lifb;ee,?‘;g
B-Ala
,,
,,
Normal
absent
>,
>I
-
4
700
2.64
4o*
I.33
2.9
700
2.64
-
0.03 3.50
1.65
500
0.20
400
r.g6
5oo
4.95
350
3,30
._ 0.8
r7 6
900
3‘40
3.9
460
2.60
-
450
0.33
850
I .48
-
700
2.90
.-
600
T.65
800
a.01
_
pl urine)
AA strangs
Decrease-
2.g3
800
A‘4 Yale
13 -_____. 16
,>
>2
“Val”
-
9
absent
~__. Decrease
BAIB
present
,,
absent
absent
,,
12
Increase
BAIB
>,
,t
6
Normal
absent
>I
,*
16
Increase
,,
I.
BAIB
17
Normal
‘3
Increase
4.8
21
Great increase
Phe “Leu”
I.8
14
Increase
BAIB
Normal
Eth, Phe “Leu”
present
present
,1
‘I
”
,,
>, absent
absent
“Vsl”
----
.--..___ ______-.__
560
0.06
4.3
I8
300
4.62
6.0
I2
600
0.09
4.5
16
800
g.ro
-
77
~_~ 300 800
_--.__ trace 0.6
--
12 12
2,
,,
,,
I> present
>> present
absent
I>
absent
absent
Eth
0
present
present
BAIB “Lell” “Val”
,,
I,
I,
,I
absent
I,
absent
>,
,I
I,
Increase
Normal _--
21
absent present
812
J. OPIEtiSKA-BLAUTH, H. KOWALSKA
perature which allows identification of the violet spot of serine appearing after the orange coloured glycine. In this study we have been interested in the relationship between aminoaciduria and the clinical status of lipoid nephrosis, taking oedema as an index of the gravity of the disease and in several cases we were able to follow the whole course of it (Table IV). In parallel a healthy child and a group of healthy children on high protein diet were examined (Figs. I and 2). When larger volumes of urine were chromatographed, it was possible to detect besides common amino acids, the “leucine” and “valine” group as well as aminobutyric acids, which WOOLF AND GILES regard as index of pathological aminoaciduria. Neither high protein diet nor hormone therapy seem to exert any influence on the type of amino acids excreted, their only effect being to produce an increased excretion of common amino acids such as glycine, alanine, serine, histidine, glutamine or glutamic acic. If we compare the results obtained on urine and serum it becomes evident, that the albumin/globulin ratio and the serum cholesterol concentration are the only biochemical indices consistent with the clinical status. (Cases: M.B. ; H.N. ; T.B. ; R.K. ; S.K.). Other indices involving the pathological aminoaciduria (e.g. cr-amino-nitrogen, occurrence of “rare” or “strange” amino acids or the absence of serine and threonine) proved to be rather irregular. Of course, the more grave was the condition (oedema +++) the more intense appeared the indices for the common amino acids and for the “rare” or “strange” ones. (proline, hydroxyproline in cases: M.B. (T. “RI’); H.N.; T.B.). As the condition improved the signs of pathological aminoaciduria tended to disappear. Neither serine nor threonine were detectable in severe periods of the disease. On the contrary, they reappeared during remission. In three cases there even was a parallelism between the clinical status and the serine-threonine index (Table IV). Among 15 cases examined we could classify one as type “RI’. It was a fatal case. Other cases did not belong to either type “H” or “HR”. We have never observed glucosuria. The behaviour of serine and threonine will be the object of further studies.
ACKNOWLEDGEMENTS Routine determinations of protein and cholesterol in urine and serum were performed in the Clinic of Pediatrics, Medical School, in Lublin. The patients were treated and their clinical status was evaluated by Dr. T. GERKOWICZ and Dr. B. URBA~~SKA,Assistants in the Clinic. SPMMARY Aminoaciduria in 15 evaluation of pathological performed in various stages except one, to be classified GILES. The fact that serine and discussed.
cases of lipoid nephrosis is described. Criteria for the aminoaciduria are proposed. The investigations were of the disease. The results do not allow the cases studied, as type “HI’, “ R” or “HR” as suggested by WOOLF AND and threonine are absent from nephrotic urines is noted
(‘litz.(‘hirn. .-l&z,6 (1961) 805-S1j
AMINOACIDURIA
IN NEPHROTIC
SYNDROME
813
REFERENCES 1 L. J. WOOLF AND H. MC. C. GILES, A4cta Paediat., 45 (1956) 489. 2 C. HOOFT AND J. HERPOL, Acfa Paediat., 48 (1959) 135. 3 H. BERGER, Aminoacidurie und Hyperaminoacidurie, S. Karger, Basel, Ig5g. 4 H. SPIER AND G. PASHER, Z. Physiol. Chem., 296 (1954) 147. 5 J. OPIE&KA-BLAUTH, Clin. Chim. Acta, 4 (1959) 860. 6 R. BAROW AND F. HARTMAN. Deut. Arch. klin. Med., 201_. (1956) 260. -7 H. BICKEL AND F. SOLJCHON, .4rch. fiir h’inderheilk.,.Beiheft 31 (1955). * R. SLATER. N. KRETCHMER. H. MCNAMARA AND H. L. BARNETT, Protein Metabolism in Nephrosis, Studies on Proteinuria, i\bstracts of combined Meeting of American Pediatric Society for Ped. Research, Canadian Paediatric Society, 1955. p. 126. 9 H. PLUECKTHUN, K. SCHRERER AND H. HAUSS, I
Clin. Chirrt.Acta, 6 (1961) 805-813
THE
RENAL
CLEARANCE
OF
PHENYLPYRUVATE
C. L. J. VINK St. Joseph-Hospital,
Eixdhouen
with technical assistence of MISS A. A. KROES
Pediatrics Department of the University (Received
Hospital, Leiden (The Netherlands)
February
I3th, Ig6I)
INTRODUCTION
Phenylpyruvate (PP, a salt of phenylpyruvic acid) is a substance found in the blood and urine of patients with oligophrenia phenylpyruvica (OP). It is the result and occurs through a deficiency of the enzyme of an “inborn error of metabolism”, phenylalanine hydroxylase. In normal subjects this enzyme catalyses the oxidation of phenylalanine to tyrosinel at such a rate that very little or no PP is detectable in the body fluids. An interesting phenomenon in OP is the high concentration of PP in the urine (up to 3000 mg/l) at a very low blood level (up to zo “g/l). This combination may point to a rather high clearance of PP in this disease. The purpose of this article is to help to explain the mechanism of the renal clearance of PP in patients with OP. As PP has often been regarded as a toxic substance in the development of the cerebrum, and as a cause of the mental retardation in these patients, our experiments were performed without PP-loading. Clin. Ckint. Acta, 6 (1961) 813818