J. Inher. Metab. Dis. 15 (1992) 413-415 9 SSIEM and KluwerAcademicPublishers. Printed in the Netherlands
Short Communication
Screening for Defects of Dihydropyrimidine Degradation by Analysis of Amino Acids in Urine Before and After Acid Hydrolysis A. H. VAN GENNIP, P. C. DRIEDIJK, A. ELZINGA and N. G. G. M. ABELING Divisions of Pediatrics and Clinical Chemistry, University Hospital AMC, Meibergdreef 9, 1105 A Z Amsterdam, The Netherlands In man pyrmidines are degraded in four steps catalysed by dihydropyrimidine dehydrogenase (DHPD, EC 1.3.1.2), dihydropyrmidinase (DHP, EC 3.5.2.2), ureidopropionase (UP, EC 3.5.1.6) and a transaminase. Deficiencies of the first and the last enzymes are well known and adequate methods for their detection are available. We present a simple method based on routine amino acid analysis which enables screening of the two remaining defects. The usefulness of the method is illustrated in a case of presumed DHP deficiency. EXPERIMENTAL
Principle of the method Defective activity of DHP is expected to be discovered by increased excretion of the dihydropyrimidines dihydrouracil (DHU) and dihydrothymine (DHT), and deficiency of UP by increased excretion of the N-carbamylamino acids, N-carbamyl-fl-alanine (N-C-fl-ala) and N-carbamyl-fl-aminoisobutyric acid (N-C-fl-AIB). In the last condition the dihydropyrimidines may be increased. It is not clear to what extent thymine-uraciluria will occur in these defects. The dihydropyrimidines and Ncarbamylamino acids in the urine can be measured after acid hydrolysis as fl-alanine (fl-ala), and fl-aminoisobutyric acid (fl-AIB). However, as either amino acid may occur in unhydrolysed urine and the fl-ala-containing dipeptides carnosine, anserine and balenine may also be present, one has to correct for these contributions.
Procedures
Hydrolysis of urine." To 0.5 ml of urine 2.5 ml 6mol/L HCI was added and 0.05 ml internal standard solution-I (IS-l: 2.5 mmol/L norvaline in double-distilled water). The mixture was hydrolysed at 150~ for 18h in a closed tube. After cooling the sample was vacuum dried and redissolved in 0.5 ml of an aqueous solution of 1.5% sulphosalicylic acid. After centrifugation at 11 000g the supernatant was ready for amino acid analysis. 413
414
van Gennip et al.
Urine (1 ml) was deproteinized with 100#1 of a 15% aqueous solution of sulphosalicylic acid and centrifuged (11 000g). To 150#1 supernatant (urine or hydrolysate), 15#1 IS-II (2.5mmol/L S-2-aminoethylcysteine in doubledistilled water) and 165 #1 of the first elution buffer (Li-buffer, pH 2.3) were added. For amino acid analysis we used a Chromakon-500 system (Tegimenta AG, Rotkreuz, Switzerland) based on cation-exchange chromatography with lithium citrate buffers and post-column ninhydrin detection. Amino acid analysis:
Calculation: For calculation of the sum of DHU and N-C-fl-ala, the concentration of fl-ala measured after hydrolysis was corrected by subtraction of the values for flala and fl-ala-containing dipeptides (carnosine, anserine and balenine) determined in the unhydrolysed urine.
Applications
The method was applied on urines from 50 patients suspected for an inborn error of metabolism, a patient with DHPD deficiency who has previously been described (Van Gennip et al 1989) and her relatives, a patient with presumed DHP deficiency (Duran et al 1990) and his brother. The excretion values of the sum of the dihydropyrimidines and their corresponding N-carbamyl-fl-amino acids, expressed in #mol/g creatinine, are given in Table 1. RESULTS AND DISCUSSION As can be seen in Table 1, we found a strongly increased excretion of the sum of DHU + N-C-fl-ala as well as the sum of DHT + N-C-fl-AIB in the patient suspected to be affected with DHP deficiency, but normal values were found in the patient's brother and the family with DHPD deficiency. Differential analysis (Van Gennip et al 1991) revealed that the dihydropyrimidines, but not the corresponding Ncarbamylamino acids, were elevated in the urine of the 'DHP' patient. These results are in agreement with the abnormalities expected in the various conditions. This leads to the conclusion that the method is a reliable and useful contribution to the detection of new inborn errors of pyrimidine degradation. Table 1 Excretion values (pmol/g creatinine) of the sum of the dihydropyrimidines and their corresponding N-carbamyi-fl-amino acids in a family with D H P D deficiency, a patient with presumed D H P deficiency and control subjects (n = 50) DHU + N-C-fl-ala
D H T + N-C-fl-AIB
Control range
0-885
0-334
DHPD-patient Mother Brother
229 143 150
0 0 0
12 200 324
8 630 148
'DHP'-patient Brother
J. Inher. Metab. Dis. 15 (1992)
Defects of Dihydropyrimidine Degradation
415
The results also show the presence of dihydropyrimidines and/or N-carbamyl-flamino acids in control urines. It should be mentioned that for some urines the analysis of fl-ala and/or fl-AIB is complicated by interfering compounds which disappear after hydrolysis and can lead to overcorrection. The urines are not always collected on a standardized diet and some samples are from patients under medication. The effect of the diet and medication has not yet been established and therefore we recommend that the urine is collected while the patient is on a standardized diet and does not use medication. REFERENCES
Duran M, Rovers P, De Bree PK et al (1990) Dihydropyrimidinuria. Lancet 336: 817-818. Van Gennip AH, Abeling NG, Elzinga-Zoetekouw L, Scholten EG, Van Cruchten A, Bakker HD (1989) Comparative study of thymine and uracil metabolism in healthy persons and in a patient with dihydropyrimidine dehydrogenase deficiency. Adv Exp Med Biol 153A: 111-118. Van Gennip AH, Busch S, Scholten EG, Stroomer AEM, Abeling NG (1991) Simple method for the quantitative analysis of dihydropyrimidines and N-carbamyl-fl-amino acids in urine. Adv Exp Med Biol 3098: 15-19.
J. Inher. Metab. Dis. 15 (1992)
Short Communication
Screening for Defects of Dihydropyrimidine Degradation by Analysis of Amino Acids in Urine Before and After Acid Hydrolysis A. H. VAN GENNIP, P. C. DRIEDIJK, A. ELZINGA and N. G. G. M. ABELING Divisions of Pediatrics and Clinical Chemistry, University Hospital AMC, Meibergdreef 9, 1105 A Z Amsterdam, The Netherlands In man pyrmidines are degraded in four steps catalysed by dihydropyrimidine dehydrogenase (DHPD, EC 1.3.1.2), dihydropyrmidinase (DHP, EC 3.5.2.2), ureidopropionase (UP, EC 3.5.1.6) and a transaminase. Deficiencies of the first and the last enzymes are well known and adequate methods for their detection are available. We present a simple method based on routine amino acid analysis which enables screening of the two remaining defects. The usefulness of the method is illustrated in a case of presumed DHP deficiency. EXPERIMENTAL
Principle of the method Defective activity of DHP is expected to be discovered by increased excretion of the dihydropyrimidines dihydrouracil (DHU) and dihydrothymine (DHT), and deficiency of UP by increased excretion of the N-carbamylamino acids, N-carbamyl-fl-alanine (N-C-fl-ala) and N-carbamyl-fl-aminoisobutyric acid (N-C-fl-AIB). In the last condition the dihydropyrimidines may be increased. It is not clear to what extent thymine-uraciluria will occur in these defects. The dihydropyrimidines and Ncarbamylamino acids in the urine can be measured after acid hydrolysis as fl-alanine (fl-ala), and fl-aminoisobutyric acid (fl-AIB). However, as either amino acid may occur in unhydrolysed urine and the fl-ala-containing dipeptides carnosine, anserine and balenine may also be present, one has to correct for these contributions.
Procedures
Hydrolysis of urine." To 0.5 ml of urine 2.5 ml 6mol/L HCI was added and 0.05 ml internal standard solution-I (IS-l: 2.5 mmol/L norvaline in double-distilled water). The mixture was hydrolysed at 150~ for 18h in a closed tube. After cooling the sample was vacuum dried and redissolved in 0.5 ml of an aqueous solution of 1.5% sulphosalicylic acid. After centrifugation at 11 000g the supernatant was ready for amino acid analysis. 413
414
van Gennip et al.
Urine (1 ml) was deproteinized with 100#1 of a 15% aqueous solution of sulphosalicylic acid and centrifuged (11 000g). To 150#1 supernatant (urine or hydrolysate), 15#1 IS-II (2.5mmol/L S-2-aminoethylcysteine in doubledistilled water) and 165 #1 of the first elution buffer (Li-buffer, pH 2.3) were added. For amino acid analysis we used a Chromakon-500 system (Tegimenta AG, Rotkreuz, Switzerland) based on cation-exchange chromatography with lithium citrate buffers and post-column ninhydrin detection. Amino acid analysis:
Calculation: For calculation of the sum of DHU and N-C-fl-ala, the concentration of fl-ala measured after hydrolysis was corrected by subtraction of the values for flala and fl-ala-containing dipeptides (carnosine, anserine and balenine) determined in the unhydrolysed urine.
Applications
The method was applied on urines from 50 patients suspected for an inborn error of metabolism, a patient with DHPD deficiency who has previously been described (Van Gennip et al 1989) and her relatives, a patient with presumed DHP deficiency (Duran et al 1990) and his brother. The excretion values of the sum of the dihydropyrimidines and their corresponding N-carbamyl-fl-amino acids, expressed in #mol/g creatinine, are given in Table 1. RESULTS AND DISCUSSION As can be seen in Table 1, we found a strongly increased excretion of the sum of DHU + N-C-fl-ala as well as the sum of DHT + N-C-fl-AIB in the patient suspected to be affected with DHP deficiency, but normal values were found in the patient's brother and the family with DHPD deficiency. Differential analysis (Van Gennip et al 1991) revealed that the dihydropyrimidines, but not the corresponding Ncarbamylamino acids, were elevated in the urine of the 'DHP' patient. These results are in agreement with the abnormalities expected in the various conditions. This leads to the conclusion that the method is a reliable and useful contribution to the detection of new inborn errors of pyrimidine degradation. Table 1 Excretion values (pmol/g creatinine) of the sum of the dihydropyrimidines and their corresponding N-carbamyi-fl-amino acids in a family with D H P D deficiency, a patient with presumed D H P deficiency and control subjects (n = 50) DHU + N-C-fl-ala
D H T + N-C-fl-AIB
Control range
0-885
0-334
DHPD-patient Mother Brother
229 143 150
0 0 0
12 200 324
8 630 148
'DHP'-patient Brother
J. Inher. Metab. Dis. 15 (1992)
Defects of Dihydropyrimidine Degradation
415
The results also show the presence of dihydropyrimidines and/or N-carbamyl-flamino acids in control urines. It should be mentioned that for some urines the analysis of fl-ala and/or fl-AIB is complicated by interfering compounds which disappear after hydrolysis and can lead to overcorrection. The urines are not always collected on a standardized diet and some samples are from patients under medication. The effect of the diet and medication has not yet been established and therefore we recommend that the urine is collected while the patient is on a standardized diet and does not use medication. REFERENCES
Duran M, Rovers P, De Bree PK et al (1990) Dihydropyrimidinuria. Lancet 336: 817-818. Van Gennip AH, Abeling NG, Elzinga-Zoetekouw L, Scholten EG, Van Cruchten A, Bakker HD (1989) Comparative study of thymine and uracil metabolism in healthy persons and in a patient with dihydropyrimidine dehydrogenase deficiency. Adv Exp Med Biol 153A: 111-118. Van Gennip AH, Busch S, Scholten EG, Stroomer AEM, Abeling NG (1991) Simple method for the quantitative analysis of dihydropyrimidines and N-carbamyl-fl-amino acids in urine. Adv Exp Med Biol 3098: 15-19.
J. Inher. Metab. Dis. 15 (1992)
