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Plasma amino acid abnormalities in chronic renal failure
?15
CLINICAc.HIMICA ACTA
CC,4 Report
Plasma amino acid abnormalities
in chronic
renal failure
A.J. ROBINS,B.K.MILE\VCZYK. E.M BOOTH and N.P.MALLICK Departments
of Biochemistry.
Dietetics
and Medicine.
The R~~,valInfirmary.
hfanchester
Ml.7 9WL (L1.K.I
(Received July 17, 1972)
SUMMARY
Amino acid analysis of the plasma from 3 patients with chronic renal failure revealed several abnormalities. In particular, the presence of homocysteine-cysteine disulphide appeared td accompany the elevation of plasma cystine levels in these patients. Levels of each of these sulphur-containing amino acids fell after a 10-h period of dialysis.
Abnormal plasma levels of several amino acids have been described in uraemic patients with chronic renal insufficiency’ -‘. Significant elevations of aspartic acid, cystine and 3-methylhistidine were noted in a series of 6 non-dialysed uraemic patients; the elevation supplementation. of cystine concentration appeared to be associated with dietary methionine Abnormalities of the ratio of plasma concentrations of phenylalanine to tyrosine and histidine to 3methylhistidine have also been noted4. Three uraemic patients have been studied inatnis hospital as part of a preliminary study of amino acid metabolism in subjects with chronic renal failure who are undergoing dialysis. Details of these patients are: Mrs. E.B. receives 20 h dialysis weekly with a daily sodium intake of 80 mequiv. Mrs. S.H. and Mr. D.B. each receive 30 h dialysis weekly, the former having a daily intake of 20 mequiv. and the latter 50 mequiv. sodium. All three patients receive 75 g protein daily, of which 61% is high biological value. Fasting heparinised blood samples were obtained immediately prior to dialysis, a further sample being obtained at the end of the 10-h dialysis period at least 5 h after any food received during this period. The plasma was deproteinised immediately with sulphosalicylic acid. Plasma amino acid analysis was carried out by the Stein-Moore technique using an automated gradient elution device’ modified for physiological fluids and with ninhydrin iodoplatinate6 as detection reagents. The results are shown in Table I.
Clin Chim Acla,
42
and
(1972) 215-217
216
CCA REPORT
TABLE I -~ Potienr
I_-_
--...-I__
Plasma amino acid levels ~rng~~O0ml)
Age Methionine
Cystine HCD* ..-..-__._
Mrs. E.B. 46 y. Pre-dialysis Post-dialysis
0.36 0.49
2.90 1.40
0.43 0.23
1.00 1.50
0.74 1.25
Mr.D.B. 30 y, Pre-dialysis Post-dialysis
0.38 0.75
3.40 2.25
0.35 0.27
1.17 2.00
0.41 0.90
Mrs.S.H.45 y. Pre-dialysis 0.51 Postdiaiysis 0.64
4.30 3.50
0.30 0.20
1.80 2.00
1.40 1.66
*Homocysteine-cysteine
disulphide.
-~_.__--_.
Phenylalanine __. _
Tyrosine
These studies confirm the elevation of pfasma cystine levels in patients with chronic renal failure, particuIarly in predialysis specimens. These patients were receiving a normal intake of sulphur amino acids so that the in%reasein cystine could not be attributed to dietary methionine supplementation with which it appeared to be associated in the patients described by Condon and Asatoor’, who received 250 mg supplemental methionine per day. The abnormal ratio of phenyl~~ine to tyrosine in plasma was also apparent, confirming the suggestion that hydroxylation of phenyl~a~ne to tyrosine is reduced in uraemic patients4. This may be due to inhibition of the enzyme phenylalanine hydroxylase in uraemic patients’. In addition, and in agreement with a previous report4, the ratio of histidine to 3.methylhistidine in plasma was abnormally low in the one patient (Mrs. E.B.) for whom optimal separation for the imidazole amino acids was achieved. Several unusu~ peaks were observed in the plasma amino acid chromatogram. In particular, a peak occurred in the isoleucine-leucine region which was both ninhydrinpositive and iodoplatinate-reactive, suggesting a sulphur-containing amino acid, probably homocysteine-cysteine disulphide (HCD). It was in this region that Condon and Asatoor’ noted an unidentified peak, absent from the plasma of normal subjects. HCD has been detected in cystinuric urine’ and occurs in the physiolo~c~ fluids of individuals with homo~ystinu~a where homocyst(e)ine accumulates due to cystathionine synthase deficiency9 but is below the limit of detection in normal subjects. HCD apparently arises in these uraemic patients by interaction of the excess cyst(e)ine with homocysteine formed after demethylation of methionine, although in this situation the increased cyst(e)ine concentration has not been attributed to an enzyme deficiency. The use of the iodoplatinate analytical system, together with the elution time of the peak, provides good evidence that this compound is HCD. The iodoplatinate system has CIin Chim Acta- 42 (1972)
215-217
217
CCA REPORT
been utilised for the detection HCD concentration
of heterozygotes
in physiological
for homocystinuria
by measurement
of the
fluids following a load of L-methionine”.
ACKNOWLEDGEMENTS
The analytical
facilities of the Willink Biochemical Genetic Laboratory,
Royal
Manchester Children’s Hospital, are gratefully acknowledged. REFERENCES 1 J.R. Condon and A.M. Asatoor, Clin. chim. Acta, 32 (1971) 333. 2 Z. Czerniak and S. Burzynski, Clin. Chim. Acta, 24 (1969) 367. 3 Z. Czerniak, Clin. Chim. Acta, 28 (1970) 403. 4 C. Giordano, C. de Pascale, D. de Cristofaro, G. Capodicasa, C. Balestrieri and K. Baczyk, in G.M. BeI (Ed.), Nutrition in Renal Disease, Livingstone, Edinburgh, 1969, p. 23. 5 A.J. Thomas in A. Baillie and R.J. Gilbert (Eds.), Automation, Mechanisation and Data Handling in Microbiology, Academic Press, London, 1970, p. 107. 6 B. Fowler and A.J. Rob&J. Chromatog., 72 (1972) 105. I J.D. Kopple, Proceedings of the International Conference on Uremia, Freiburg, 1971, in press. 8 G.W. Frimpter,J. Bioj. Chem., 236 (1961) PC 52. 9 T.L. Perry, S. Hansen, L. MacDougaJl and P.D. Warrington, C/in. Chim. Acta, 15 (1967) 409. 10 B. Fowler, LB. Sardharwalla and A.J. Robins, Biochem. J., 122 (1971) 23P.
Clin Chim Acta, 42 (1972) 215-217
CLINICAc.HIMICA ACTA
CC,4 Report
Plasma amino acid abnormalities
in chronic
renal failure
A.J. ROBINS,B.K.MILE\VCZYK. E.M BOOTH and N.P.MALLICK Departments
of Biochemistry.
Dietetics
and Medicine.
The R~~,valInfirmary.
hfanchester
Ml.7 9WL (L1.K.I
(Received July 17, 1972)
SUMMARY
Amino acid analysis of the plasma from 3 patients with chronic renal failure revealed several abnormalities. In particular, the presence of homocysteine-cysteine disulphide appeared td accompany the elevation of plasma cystine levels in these patients. Levels of each of these sulphur-containing amino acids fell after a 10-h period of dialysis.
Abnormal plasma levels of several amino acids have been described in uraemic patients with chronic renal insufficiency’ -‘. Significant elevations of aspartic acid, cystine and 3-methylhistidine were noted in a series of 6 non-dialysed uraemic patients; the elevation supplementation. of cystine concentration appeared to be associated with dietary methionine Abnormalities of the ratio of plasma concentrations of phenylalanine to tyrosine and histidine to 3methylhistidine have also been noted4. Three uraemic patients have been studied inatnis hospital as part of a preliminary study of amino acid metabolism in subjects with chronic renal failure who are undergoing dialysis. Details of these patients are: Mrs. E.B. receives 20 h dialysis weekly with a daily sodium intake of 80 mequiv. Mrs. S.H. and Mr. D.B. each receive 30 h dialysis weekly, the former having a daily intake of 20 mequiv. and the latter 50 mequiv. sodium. All three patients receive 75 g protein daily, of which 61% is high biological value. Fasting heparinised blood samples were obtained immediately prior to dialysis, a further sample being obtained at the end of the 10-h dialysis period at least 5 h after any food received during this period. The plasma was deproteinised immediately with sulphosalicylic acid. Plasma amino acid analysis was carried out by the Stein-Moore technique using an automated gradient elution device’ modified for physiological fluids and with ninhydrin iodoplatinate6 as detection reagents. The results are shown in Table I.
Clin Chim Acla,
42
and
(1972) 215-217
216
CCA REPORT
TABLE I -~ Potienr
I_-_
--...-I__
Plasma amino acid levels ~rng~~O0ml)
Age Methionine
Cystine HCD* ..-..-__._
Mrs. E.B. 46 y. Pre-dialysis Post-dialysis
0.36 0.49
2.90 1.40
0.43 0.23
1.00 1.50
0.74 1.25
Mr.D.B. 30 y, Pre-dialysis Post-dialysis
0.38 0.75
3.40 2.25
0.35 0.27
1.17 2.00
0.41 0.90
Mrs.S.H.45 y. Pre-dialysis 0.51 Postdiaiysis 0.64
4.30 3.50
0.30 0.20
1.80 2.00
1.40 1.66
*Homocysteine-cysteine
disulphide.
-~_.__--_.
Phenylalanine __. _
Tyrosine
These studies confirm the elevation of pfasma cystine levels in patients with chronic renal failure, particuIarly in predialysis specimens. These patients were receiving a normal intake of sulphur amino acids so that the in%reasein cystine could not be attributed to dietary methionine supplementation with which it appeared to be associated in the patients described by Condon and Asatoor’, who received 250 mg supplemental methionine per day. The abnormal ratio of phenyl~~ine to tyrosine in plasma was also apparent, confirming the suggestion that hydroxylation of phenyl~a~ne to tyrosine is reduced in uraemic patients4. This may be due to inhibition of the enzyme phenylalanine hydroxylase in uraemic patients’. In addition, and in agreement with a previous report4, the ratio of histidine to 3.methylhistidine in plasma was abnormally low in the one patient (Mrs. E.B.) for whom optimal separation for the imidazole amino acids was achieved. Several unusu~ peaks were observed in the plasma amino acid chromatogram. In particular, a peak occurred in the isoleucine-leucine region which was both ninhydrinpositive and iodoplatinate-reactive, suggesting a sulphur-containing amino acid, probably homocysteine-cysteine disulphide (HCD). It was in this region that Condon and Asatoor’ noted an unidentified peak, absent from the plasma of normal subjects. HCD has been detected in cystinuric urine’ and occurs in the physiolo~c~ fluids of individuals with homo~ystinu~a where homocyst(e)ine accumulates due to cystathionine synthase deficiency9 but is below the limit of detection in normal subjects. HCD apparently arises in these uraemic patients by interaction of the excess cyst(e)ine with homocysteine formed after demethylation of methionine, although in this situation the increased cyst(e)ine concentration has not been attributed to an enzyme deficiency. The use of the iodoplatinate analytical system, together with the elution time of the peak, provides good evidence that this compound is HCD. The iodoplatinate system has CIin Chim Acta- 42 (1972)
215-217
217
CCA REPORT
been utilised for the detection HCD concentration
of heterozygotes
in physiological
for homocystinuria
by measurement
of the
fluids following a load of L-methionine”.
ACKNOWLEDGEMENTS
The analytical
facilities of the Willink Biochemical Genetic Laboratory,
Royal
Manchester Children’s Hospital, are gratefully acknowledged. REFERENCES 1 J.R. Condon and A.M. Asatoor, Clin. chim. Acta, 32 (1971) 333. 2 Z. Czerniak and S. Burzynski, Clin. Chim. Acta, 24 (1969) 367. 3 Z. Czerniak, Clin. Chim. Acta, 28 (1970) 403. 4 C. Giordano, C. de Pascale, D. de Cristofaro, G. Capodicasa, C. Balestrieri and K. Baczyk, in G.M. BeI (Ed.), Nutrition in Renal Disease, Livingstone, Edinburgh, 1969, p. 23. 5 A.J. Thomas in A. Baillie and R.J. Gilbert (Eds.), Automation, Mechanisation and Data Handling in Microbiology, Academic Press, London, 1970, p. 107. 6 B. Fowler and A.J. Rob&J. Chromatog., 72 (1972) 105. I J.D. Kopple, Proceedings of the International Conference on Uremia, Freiburg, 1971, in press. 8 G.W. Frimpter,J. Bioj. Chem., 236 (1961) PC 52. 9 T.L. Perry, S. Hansen, L. MacDougaJl and P.D. Warrington, C/in. Chim. Acta, 15 (1967) 409. 10 B. Fowler, LB. Sardharwalla and A.J. Robins, Biochem. J., 122 (1971) 23P.
Clin Chim Acta, 42 (1972) 215-217