CLINICA
CHIMICA ACTA
N-SUBSTITUTED RENAL
403
AMINO
ACIDS
IN SERUM
OF PATIENTS
Medical
School, Lublin
(Poland)
WITH
CHRONIC
INSUFFICIENCY
2. CZEKNIAK Department {Received
of General Chemistry,
May 5th, 1969; revised February 6th, rgp)
SUMMARY Three
N-substituted
amino
acids
were
determined
in sera from
healthy
sub-
In the serum of uremic patients the levels of these amino acids were significantly increased; moreover, aspartic acid, cystine, phenylalanine and proline (all in trace quantities) were detected.
jects:
glycine,
glutamic
acid
and cc-alanine
(traces
only
of the last).
Next, amino acids found in the N-substituted form were determined quantitatively in blood peptides. The levels of all examined peptide-bound amino acids, except aspartic acid, were significantly changed in uremia: cc-alanine, cystine, phenylalanine and glutamic acid levels were significantly decreased, while the ratio of the N-substituted to peptide-bound bound glycine were significantly
amino acids was elevated, increased.
and the levels of peptide-
INTRODCCTIOiX
There are many metabolic disorders in uremia 192, but their mechanism is not yet sufficiently known. Among the different compounds in body fluids of uremic patients, free5-6 and bound’,X amino acids have been reported on extensively. In particular, great differences have been found in the peptide-bound and nonpeptidic ninhydrin-negative amino acid conjugates8 in healthy subjects and patients. Investigations of nonpeptidic conjugates have been rather difficult because no simple analytical method has been available. In particular, the separation of N-substituted amino acids from peptides and free amino acids presented difficulties. The most common method used so far has been the chromatographic column separation of the material g- Il. The method described in my previous reportI was thirl-layer electrophoresis based on the nonampholytic character of N-substituted amino acids. This method permitted the further study of the compounds investigated. The purpose of the present work was to determine N-substituted amino acids in the serum of patients with chronic renal insufficiency and to clear up the quantitative relations between different forms of bound amino acids.
CZERNIAK
404 MATERIALS
AND
METHODS
Sera from 20 patients with chronic renal insufficiency were used. The control group consisted of IO healthy individuals aged 21-65 years. More exact data were previously
reportede.
Blood samples were taken after overnight
fasting and prepared
as described previouslyr3. The separation of N-substituted amino acids from free and peptide-bound amino acids was performed using thin-layer electrophoresis on silica ge112. In the next step, amino acids bound by ninhydrin-negative Peptide-bound amino acids were examined
conjugates were determinedr2. after separation from the free
amino acids. ITor this purpose, the ninhydrin reaction according to Markovitz and Steinberg’4 was applied. In the present investigations, however, a completely different procedure was employed. The ninhydrin-positive zone was outlined on the developed electrophoretograms, so as to include entirely all spots. ,411 gel was scraped ethanol solution of ninhydrin was added. The off the individual zones, and a 0.3:; samples were brought to a pH of 5.0 and then evaporated to dryness at boiling temperature. Next, 30/hhydrogen peroxide was added to the dry mass for decomposition of the formed dye and of the ninhydrin excessXs. The mixture was heated for 2 min at 100’ and then evaporated at 25O. Gels containing the blocked free amino acids were hydrolyzed with 6 N HCl at a temperature of 105”. The optimal hydrolysis time was 16 11. Qualitative and quantitative determinations of amino acids were performed b? one-directional ascending paper chromatography, using butanol as eluent and ninhydrin as reagent. The identification of some amino acids was facilitated by rechromatography and by specific reactions. Quantitative analysis was done by photometry of the negative prints of the chromatograms16 using the modification described recentlvl”. The results were examined fication
and Tukey’s
confidence
statistically intervals
by analysis of variance
for single classi-
t.
RESULTS
N-substituted amino acids The serum of healthy subjects was found to contain three amino acids bound by ninhydrin-negative conjugates: glycine, glutamic acid and cc-alanine (the last in trace quantities only). The mean level of glycine was 5.2 pg/ml (S.D. 1.1) and of glutamic acid 10.3 ,q/ml (S.D. 1.73), range 3.8-7.2 pg:/ml for glycine and 7.1-12.7 /,lg/ml for glutamic acid. The patients were divided into two groups, as in our previous workG. In the first group there were patients with a serum urea level below 150 mg/roo ml and in the second group urea levels were higher than 150 mg/roo ml. In all cases, an increase in the amino acid concentrations over those of healthy subjects was observed, and in addition, aspartic acid, cystine, phenylalanine and proline were found to be present in trace quantities (Fig. I). Quantitative results are given in Table I. Analysis of variance for glycine and glutamic acid at the significance level of 0.01 showed significant differences between the average concentrations of these amino acids.
N-SUBSTITUTED
AMIRTO ACIDS IN REKAL
405
DISEASE
Pe$tide-bozbnd am’?zo acids In the blood of healthy subjects, the following peptide-bound amino acids were found : cc-alanine, glycine, glutamic acid, cystine, phenylalanine, aspartic acid, arginine, leucine, lysine, threonine and proline (Fig. 2). Results for the two patient groups show only quantitative differences. Quantitative analysis was performed for glycine, cr-alanine, cystine, phenyl-
Fig. I. N-substituted amino acids in serum of a normal subject (A5) and 2 patients (X4 and N14). Chromatography on Whatman No. 3 paper. Solvent system: n-butanol-glacial acetic acid-water (4 : I : I). I, cystine; 2, aspartic acid: 3, glycine : 4, glutamic acid; 5. a-alanine; 6, phenylalanine. Proline is invisible because of yellow colour.
N-SUBSTITUTED
A&no
AMINO
acid
Glycinc Glutamic acid a-Alanine Cystine Aspartic acid Phenylalanine S.1).
=
ACIDS
IN
SERUM
OFPATIENTS
WITH
CHRONIC
RENAL
(in pg/ml)
IKSUFFICIENCY
standard
Patient
group
I
Mean
S.D.
S.8 22.1 2.1 1.1 4.’
1.4 2.2
0.8
0.2
Patient i?IelZVl
‘5.1 3;:;
group
2 S.D.
1.6 2.3 0.2
0.2
1.6
0.4
4.7
0.4
0. I
I.1
0.2
0.2
deviation. Clin. Chim.
Acta,
28 (1970)
403-408
406
CZERNIAK
alanine, aspartic acid and glutamic acid (all found previously in ninhydrin-negative conjugates). Analysis of variance demonstrated significant differences in the average concentrations of all these amino acids, except aspartic acid. Tukey’s confidence intervals allowed the detection of the significant rise of glycine in cases with urea levels higher than 150 mg/roo ml. The mean concentrations of cr-alanine, cystine and phenylala-
Fig. 2. Peptide-bound amino acids in serum of a normal subject (-45) and 2 patients (X4 and NI$. Chromatography on Whatman No. 3 paper. Solvent system : n-butanol-glacial acetic acid-water (4: I : I). I, cystinc; 2, lysine ; 3, arginine; 4, aspartic acid; 5, glycine; 6, glutamic acid; 7. threonine; 8, a-alanine; 9, phenylalaninc; IO, leucine. Proline is invisible because of yellow colonr.
Amino
acid
Glycine Glutamic acid cr.Alanine Cystine Aspartic acid Phenylalanine
Patient
group
I S.D.
M&Wl
6.2 37.4 2.6 1.8 6.9 0.9
0.6 2.4 0.3
9.1 35.3 2.0
group ~._
2
S.D.
0.7 2.5 0.2
0.4
1.6 7.’
0.2 0.5
0.1
0.8
0. I
0.2
S.D. = standard deviation. Clzn. Chiua. Acta,
Patient
Mean
28 (1970) 403-408
4,s 41.2
0.4 2.2
3.8
0.3
2.4
0.1
6.5
0.4 0.3
2.1
N-SUBSTITUTED
AMINO
ACIDS IX RESAL
407
DISEASE
nine were significantly decreased in patients with urea levels higher than 150 mg/roo ml (confidence intervals of 50/b and I"/;).The glutamic acid concentration decreased significantly
with an elevation
when the urea level rose above
of urea level above 45 mg/roo ml and did not change
150 mg/roo ml.
DISCUSSION
Investigations of the problem of N-substituted amino acids have, so far, been concerned with human urine onlyg-ll. Blood, however, also contains amino acids bound with ninhydrin-negative conjugates, e.g. hippuric acid and phenylacetylglutamine. These two compounds may be the source of the N-substituted glycine and glutamic acid found in normal serum. However, liberated amounts of these amino acids seem to be too high to derive from two sources only. No compounds bound with a-alanine are known. The increases of N-substituted amino acids in uremia depend probably on two factors: the retention of ninhydrin-negative compounds in the blood due to renal failure and increased synthesis. The latter factor is especially important. Increased catabolism in uremia1 leads to the production of many organic acids (e.g. benzoic acid) which can conjugate increase
with amino acids. Therefore,
Frimpter
et aL8 observed
an
of substances
such as phenylacetylglutamine. The results obtained are only partly similar to those of other authors’ reports 8r7p17.Frimpter et ~1.~ found increases in bound glutamic acid, aspartic acid and glycine, and so did Bock and Gerok’ and Gerokl7. However, only Frimpter et al.* considered the probability of the existence of these amino acids in nonpeptide conjugates. In the present work, peptide-bound amino acids have also been examined. Our analysis showed a decrease in peptide-bound amino acid levels, except for aspartic acid, the concentration of which was unchanged, and the glycine concentration, which increased with the elevation of the blood urea level. The results obtained are not comparable with data in the literature, because no reports are available on the determination of separated peptide-bound amino acids. Protein catabolism, renal failure, retention and protein-free diet have a great
influence on peptide-bound amino acid levels in uremia. Other important factors are disorders of liver function and the intensification of some enzymatic process (especially oxidative deamination). Protein catabolism and protein-free diet seem to be the most important factors. Studies on the quantitative relations in uremia between N-substituted and peptide-bound amino acids demonstrated an increase in the ratio of the N-substituted amino acids to the total amount of peptide-bound amino acids. The most marked changes existed in the levels of aspartic and glutamic acids. The great changes in N-substituted amino acid levels observed in sera of uremic patients indicate the importance of these compounds in the metabolism of uremia. Further investigations of the chemical character and structure of these conjugates are necessary to clarify their role in human metabolism. Such studies will be helpful in revealing the pathomechanism of uremia.
Clin.Chim. Acta, 28 (1970) 403-408
408
CZERNIAK
ACKNOWLEDGEMENT
I wish to thank Dot. Dr. Jan Kowalewski, Head of The Second Department of Medicine, Medical School, Lublin (Poland) for the samples of blood obtained from patients with uremia.
REFERENCI
Ital. Biol.
Sprr.,
40
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