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Aliphatic C6−C14 dicarboxylic acids in urine from an infant with fatal congenital lactic acidosis
363
CLINICA CHIMICA ACTA
CCA 5255
ALIPHATIC WITH
LENA
C,-C,
FATAL
BORG,
DICARBOXYLIC
CONGENITAL
SVEN
LINDSTEDT
ACIDS
IN URINE
LACTIC
ACIDOSIS
AND GijRAN
STEEN
FROM
AN INFANT
University of Gothenburg, Department of Clinical Chemistry, Sahlgven’s Hospital, S-413 45 Gothenburg AND OLA H JALMARSON Department of Pediatrics, Gothenburg (Sweden) (Received
June 14, 1972)
SUMMARY
A newborn male infant with a severe metabolic acidosis who died one day after birth was found to have high levels of lactic acid in his urine, together with a series of dicarboxylic acids with 6 to 14 carbon atoms chain length. Adipic and suberic acids which occurred in a concentration of N 0.05 mmoles/l were also present in the urine of normal newborns. The concentration in the urine of dicarboxylic acids with IO, 12 and 14 carbon atoms was 0.1, 0.5 and 0.2 mmoles/l. These acids were saturated, monoenic and dienic. The monounsaturated acids were mainly of the &-A5 configuration. It is suggested that the findings could be consistent with a defect in the normal /?-oxidation of fatty acids located at the acyl-CoA dehydrogenase step. The parents to the patient were second cousins; a previous child died 26 h after birth in acidosis. Three cousins to the father also died soon after birth. These facts support the contention that the present case represents an inborn error of metabolism.
INTRODUCTION
We wish to report a case of fatal congenital lactic acidosis, which excreted C,-C,, dicarboxylic acids in the urine. The most probable etiology is a defect in the ,&oxidation of fatty acids. Only one inborn error of fatty acid fi-oxidation has been known previously, a condition in which butyric and caproic acids were excretedl. Case history The infant studied was child number two of healthy parents, who are second cousins. The father had three cousins, who died during the first days of life, and two other cousins are mentally retarded. The first child of these parents, a boy, was born at term and was small for date. Following a transient cyanosis after birth, the infant appeared well during the first 12 h, but then developed cyanosis and respiratory distress. Hypoglycemia and a severe metabolic acidosis was found. The acidosis progressed rapidly and the infant Clin. Chim. Acta, 41 (1972) 363-366
mm
364
et al.
died 26 h old. Autopsy revealed areas of bronchopneumonia in the lungs. All other internal organs including the liver were normal. The second child was the object of this study. He was born at term and delivery was normal. The birth weight was 3990 g and he appeared healthy during the first 15 h of life. After this, the respiratory frequency increased, and retractions and grunting were noted. A severe metabolic acidosis developed, with blood pH 7.01, @CO, 19 mm Hg and standard bicarbonate 7.5 mmoles/l. Blood glucose was 53 mg/roo ml. During the following 12 h, the infant was given large amounts of sodium bicarbonate and THAM (trishydroxymethylaminomethane) through the umbilical vein, with only slight and temporary effect on the acidosis. The infant was then transferred to the Children’s Hospital in Gothenburg. At admission the infant was pale and irritable, and the primitive reflexes could not be elicited. The respiratory frequency was 7o/min and rales were heard on both lungs. The liver was not palpable. Shortly after admission the infant developed bradycardia and cardiac arrest resisting all trials of resuscitation. Autopsy revealed a haemorrhage in the falx cerebri, and a subdural hematoma in the parietal region. The liver was not enlarged and no lipid storage was seen. METHODS The urine was analyzed for its content of low-molecular organic compounds, using a gas chromatograph coupled to a mass spectrometer (LKB 9000). Organic acids were extracted into diethyl ether from acidified urine, and methyl esters were prepared with diazomethanez. Amino acids in the aqueous phase were converted into N-TFA butyl esters3. Gas-liquid chromatography was performed on a 6-ft 1% OV-17 column, with linear temperature programming (5’/min) from 70~ to 290°, or on a 6-ft 10% EGSS-X column at zoo’ (isothermal). Double bond positions in the fatty acids were determined by hydroxylation with osmium tetroxide4, trimethylsilylation of the hydroxyl groups5 and gas chromatography-mass spectrometry of the products. Double bond configuration was determined by infrared spectroscopy of a total dicarboxylic acid fraction, isolated by preparative thin-layer chromatography. The urine concentrations of the acids were calculated from peak area measurements using n-eicosane as internal standard. No detector response corrections were made. RESULTS The diethyl-ether extract, when analyzed on the OV-17 column, showed several compounds, not found in urine from normal newborns. The identity of the compounds, as given in Table I, is based on information from different derivatizations of the total diethyl-ether extract. The major peakin the chromatogram, eluted at low temperature, was identical to lactic acid in retention times and mass spectra, when compared first as methyl esters, and then as trimethylsilylated methyl esters. A series of five peaks, eluted in the temperature interval 150”-250”, showed spectra, characteristic for aliphatic compounds, with intense ions at m/e 74 and 87. The two first GLC peaks gave spectra, essentially identical to the spectra of authentic dimethyl adipate and dimethyl suberate. The three peaks eluted later in the chromatogram seemed to be C,,, C,, and C,, homologues, and the spectra indicated the presence of unsaturation (mass deficits of C&n. China.
Acta,
41 (1972)
363-366
URINE
DICARBOXYLIC
ACIDS
IN LACTIC
ACIDOSIS
3%
TABLE I ORGANIC ACIDSIN DIETHYL-ETHER EXTRACT FROM
HEALTHY
OF URINE
Lactic acid /3-Hydroxybutyric acid Dicarboxylic acid (carbon atoms : double bonds) 6:o
10:0,
10:1
12:0,
12:1,
12:2
14:0,
14:‘.
14:2
THE
PRESENT
Normal newborn
Patient in this report
Cornpound
8:0
FROM
CASE
AND
IN
URINE
INFANTS
sam$de I
sample
vnmole 11
wwole
9.6
2
11
infants
(n
=
mmole/l
4)
Normal infants 112-1
2.8
(0.01
0.9
-
0.08
0.04
0.05
0.03
0.13
0.13
0.51 0.21
0.01
year
old (n = 2)
9nmole~l
NO.002
(0.01-0.02)
0.03 (0.02-0.04)
N 0.005 ~0.002
0.49
0.02 (0.01-0.03)
<0.002
0.17
two, sometimes four, mass units on most fragments in the upper part of the spectra). When ethyl esters were prepared with diazoethane, the molecular ions increased by 28 mass units, thus confirming the dicarboxylic acid nature of these compounds. Trimethylsilylation did not change retention times or spectra, as would have been the case for compounds with hydroxyl groups. The mass spectra gave no evidence for chain branching or keto groups. GLC on an EGSS-X column showed, that the C,,carboxylic acid fraction was 73 o/Osaturated and 27 o/o monoenic and the C,,-carboxylic acid fraction 31% saturated, 52% monoenic and 16% dienic. The double bond position was almost exclusively Ag among the monoenes. The lack of any infrared absorbance at 10.3 ,u showed that the double bonds were mainly of cis configuration. The organic acids of the urine from the parents were normal. The urine from normal newborn infants contained adipic and suberic acids at the same concentrations as in this case. The urine amino acid chromatogram of this case showed no large
unusual peaks. DISCUSSION
Compared to our patient, most reported cases of lactic acidosis in infancy started later in life, and the course was less rapid and malignant7~10 (for complete references see ref. 6). However, the two cases, recently reported by Lie et a1.6, both died within one week after birth. Congenital lactic acidosis has in a few cases been attributed to specific enzymic deficiencies (glucose 6-phosphatase7, fructose 1,6-diphosphatases and pyruvate carboxylases) . The disease in patients with these enzymic defects does not run the same rapid course as in our case. In most cases reported, the enzymic defect has not been identified6ylo. The parents of the child described here were consanguineous, and they previously had one child, who also developed a lethal metabolic acidosis. These facts strongly suggest an inborn error of metabolism. As shown in Table I, the urine of this patient contained not only lactic acid, but also a series of aliphatic dicarboxylic acids with 6 to 14 carbon atoms, saturated as well as unsaturated. Dicarboxylic acids are Clin. Chim Ada,
41 (1972) 363-366
BORG et al.
366
formed by w-oxidation of fatty acids or their CoA ester+. This reaction has been considered to be of very small physiological significance; it is of interest however that Wada et ~1.1~have recently suggested that under conditions of inadequate glycolysis o-oxidation is increased to produce succinyl-CoA from fatty acids and thus to maintain the tricarboxylic acid cycle. Petterson and co-worker+ have recently reported that patients with ketosis excrete large amounts (i.e. up to 0.5 g/day) of adipic and suberic acid in the urine. When examining normal newborns we found these acids to occur in about the same concentration as in the patient. The major dicarboxylic acids in this case had longer chain lengths (C,,-C,,) and unsaturated as well as saturated acids were present. It appears probable, therefore, that this is not another example of deranged carbohydrate metabolism with ketosis and increased fatty acid metabolism but represents a situation with a primary defect in fatty acid catabolism. The catabolism of fatty acids occurs by successive P-oxidation in the mitochondria. Fatty acids in the FFA fraction are mainly C,, and C,,, i.e. a few t%oxidation steps must have occurred to give the dicarboxylic acids. The location of the double bond in the monoene fraction (cis-As) is thus consistent with two /Soxidations of oleic acid (ci.49). A defect in the transport of fatty acids into the mitochondria is unlikely since one would then have expected a predominance of acids with longer chain length. The same argument would seem to exclude a defect in the acyl-CoA synthetase reaction. A defect in the following dehydrogenase reaction which is catalyzed by fatty acyl-CoA dehydrogenases (EC 1.X.99.3) might lead to an accumulation of acids of intermediate chain length which would be substrates for the o-oxidation system. It is of interest in this context that in pig liver Crane St aLx4 have found at least two different acyl-CoA dehydrogenases with different chain length specificity. A block in the normal p-oxidation of fatty acids at the suggested step would presumably lead to a trapping of CoA with resulting effects on carbohydrate metabolism. It is tempting to speculate on the possibility that a low availability of acetylCoA would lead to a decreased formation of oxalacetate from pyruvate15 and consequently to an accumulation of lactate. This interpretation is supported by a recent report by Nordmann and NordmannlG who found increased liver pyruvate and lactate concentrations on inhibition of fatty acid B-oxidation by ~-mercaptoethanol. REFERENCES
8 9 10 II I2 13 I4 =s 16
J. B. SIDBURY, E. K. SNIITH AND W. HARLAN,J. Pediat., 70 (1967) 8. E.JELLUM,O.STOKKEA~DL.ELDJARN,SC~~~.J.~~~~.~~~.I~V~~~., 27(197X} 273. D. ROACH ANU C. W. GEHRXB, J.Chrowatog., 44fr96g) 269. W.G.NIEHAUS, J~.~NDR.RYHAGE, AnaZ.Chem., 40(1968) 1840. K. A. KARLSSON; Chem. Phys. Lipids, 5 (rg7o) 6. S.O. LIE, A.C,LGKEN,J.H.STRBMME AND 0. AAGENAES. ActaPediat..%and.,60 (1971)x29. R.&~AHLER,~~R.H.S. THOMPSON_~DI.D. P.WOTTON(E~~.), Bioche~icaEI)isov~e~s~iaHzanzan Disease, 3rd. ed.,J. & A. Churchill,London, 1970, p. 95. W. CH~LSMANN AND J.FERNANDES, Pediat.Res.,5 (~971) 633. I?. A. HOMMES, II.A. POLMAN AND J. D. REERINK, Arch. Diseases Childhood, 43 (1968) 423. A. F. HARTIMANN, H. J, WOBLTMANN, M. L. PURK~KSON AND M. E.WESLEV, J.Pediat., 61 (1962) 165. B. PREISS ANU K. BLOCH, J. Biol. Chem., 239 (1964) 85. F. WADA,M.USAMI,M.GOTOAND T.HAYASHI,J. Biochem., 70(1971) 1065. J. E. PETTERSEN,E.JELLUMANDL.ELDJARN,C~~~. Chim.Acta,38(Ig72) 17. F.L. CRANE, J. G.HANGEAND H.BEINERT, Biochirn.Biophys. Acta, 17(1955)293. R. BRESSLER, in S. WAKIL (Ed.),Lipid Metabolism, Academic Press,New York, 1970, p. 60. R. NORDMANR AXD J. ~oRD~A~N,~~oc~~~~~, 53 (1971)705.
Clin.Chim. Acta,
41 (1972)363-366
CLINICA CHIMICA ACTA
CCA 5255
ALIPHATIC WITH
LENA
C,-C,
FATAL
BORG,
DICARBOXYLIC
CONGENITAL
SVEN
LINDSTEDT
ACIDS
IN URINE
LACTIC
ACIDOSIS
AND GijRAN
STEEN
FROM
AN INFANT
University of Gothenburg, Department of Clinical Chemistry, Sahlgven’s Hospital, S-413 45 Gothenburg AND OLA H JALMARSON Department of Pediatrics, Gothenburg (Sweden) (Received
June 14, 1972)
SUMMARY
A newborn male infant with a severe metabolic acidosis who died one day after birth was found to have high levels of lactic acid in his urine, together with a series of dicarboxylic acids with 6 to 14 carbon atoms chain length. Adipic and suberic acids which occurred in a concentration of N 0.05 mmoles/l were also present in the urine of normal newborns. The concentration in the urine of dicarboxylic acids with IO, 12 and 14 carbon atoms was 0.1, 0.5 and 0.2 mmoles/l. These acids were saturated, monoenic and dienic. The monounsaturated acids were mainly of the &-A5 configuration. It is suggested that the findings could be consistent with a defect in the normal /?-oxidation of fatty acids located at the acyl-CoA dehydrogenase step. The parents to the patient were second cousins; a previous child died 26 h after birth in acidosis. Three cousins to the father also died soon after birth. These facts support the contention that the present case represents an inborn error of metabolism.
INTRODUCTION
We wish to report a case of fatal congenital lactic acidosis, which excreted C,-C,, dicarboxylic acids in the urine. The most probable etiology is a defect in the ,&oxidation of fatty acids. Only one inborn error of fatty acid fi-oxidation has been known previously, a condition in which butyric and caproic acids were excretedl. Case history The infant studied was child number two of healthy parents, who are second cousins. The father had three cousins, who died during the first days of life, and two other cousins are mentally retarded. The first child of these parents, a boy, was born at term and was small for date. Following a transient cyanosis after birth, the infant appeared well during the first 12 h, but then developed cyanosis and respiratory distress. Hypoglycemia and a severe metabolic acidosis was found. The acidosis progressed rapidly and the infant Clin. Chim. Acta, 41 (1972) 363-366
mm
364
et al.
died 26 h old. Autopsy revealed areas of bronchopneumonia in the lungs. All other internal organs including the liver were normal. The second child was the object of this study. He was born at term and delivery was normal. The birth weight was 3990 g and he appeared healthy during the first 15 h of life. After this, the respiratory frequency increased, and retractions and grunting were noted. A severe metabolic acidosis developed, with blood pH 7.01, @CO, 19 mm Hg and standard bicarbonate 7.5 mmoles/l. Blood glucose was 53 mg/roo ml. During the following 12 h, the infant was given large amounts of sodium bicarbonate and THAM (trishydroxymethylaminomethane) through the umbilical vein, with only slight and temporary effect on the acidosis. The infant was then transferred to the Children’s Hospital in Gothenburg. At admission the infant was pale and irritable, and the primitive reflexes could not be elicited. The respiratory frequency was 7o/min and rales were heard on both lungs. The liver was not palpable. Shortly after admission the infant developed bradycardia and cardiac arrest resisting all trials of resuscitation. Autopsy revealed a haemorrhage in the falx cerebri, and a subdural hematoma in the parietal region. The liver was not enlarged and no lipid storage was seen. METHODS The urine was analyzed for its content of low-molecular organic compounds, using a gas chromatograph coupled to a mass spectrometer (LKB 9000). Organic acids were extracted into diethyl ether from acidified urine, and methyl esters were prepared with diazomethanez. Amino acids in the aqueous phase were converted into N-TFA butyl esters3. Gas-liquid chromatography was performed on a 6-ft 1% OV-17 column, with linear temperature programming (5’/min) from 70~ to 290°, or on a 6-ft 10% EGSS-X column at zoo’ (isothermal). Double bond positions in the fatty acids were determined by hydroxylation with osmium tetroxide4, trimethylsilylation of the hydroxyl groups5 and gas chromatography-mass spectrometry of the products. Double bond configuration was determined by infrared spectroscopy of a total dicarboxylic acid fraction, isolated by preparative thin-layer chromatography. The urine concentrations of the acids were calculated from peak area measurements using n-eicosane as internal standard. No detector response corrections were made. RESULTS The diethyl-ether extract, when analyzed on the OV-17 column, showed several compounds, not found in urine from normal newborns. The identity of the compounds, as given in Table I, is based on information from different derivatizations of the total diethyl-ether extract. The major peakin the chromatogram, eluted at low temperature, was identical to lactic acid in retention times and mass spectra, when compared first as methyl esters, and then as trimethylsilylated methyl esters. A series of five peaks, eluted in the temperature interval 150”-250”, showed spectra, characteristic for aliphatic compounds, with intense ions at m/e 74 and 87. The two first GLC peaks gave spectra, essentially identical to the spectra of authentic dimethyl adipate and dimethyl suberate. The three peaks eluted later in the chromatogram seemed to be C,,, C,, and C,, homologues, and the spectra indicated the presence of unsaturation (mass deficits of C&n. China.
Acta,
41 (1972)
363-366
URINE
DICARBOXYLIC
ACIDS
IN LACTIC
ACIDOSIS
3%
TABLE I ORGANIC ACIDSIN DIETHYL-ETHER EXTRACT FROM
HEALTHY
OF URINE
Lactic acid /3-Hydroxybutyric acid Dicarboxylic acid (carbon atoms : double bonds) 6:o
10:0,
10:1
12:0,
12:1,
12:2
14:0,
14:‘.
14:2
THE
PRESENT
Normal newborn
Patient in this report
Cornpound
8:0
FROM
CASE
AND
IN
URINE
INFANTS
sam$de I
sample
vnmole 11
wwole
9.6
2
11
infants
(n
=
mmole/l
4)
Normal infants 112-1
2.8
(0.01
0.9
-
0.08
0.04
0.05
0.03
0.13
0.13
0.51 0.21
0.01
year
old (n = 2)
9nmole~l
NO.002
(0.01-0.02)
0.03 (0.02-0.04)
N 0.005 ~0.002
0.49
0.02 (0.01-0.03)
<0.002
0.17
two, sometimes four, mass units on most fragments in the upper part of the spectra). When ethyl esters were prepared with diazoethane, the molecular ions increased by 28 mass units, thus confirming the dicarboxylic acid nature of these compounds. Trimethylsilylation did not change retention times or spectra, as would have been the case for compounds with hydroxyl groups. The mass spectra gave no evidence for chain branching or keto groups. GLC on an EGSS-X column showed, that the C,,carboxylic acid fraction was 73 o/Osaturated and 27 o/o monoenic and the C,,-carboxylic acid fraction 31% saturated, 52% monoenic and 16% dienic. The double bond position was almost exclusively Ag among the monoenes. The lack of any infrared absorbance at 10.3 ,u showed that the double bonds were mainly of cis configuration. The organic acids of the urine from the parents were normal. The urine from normal newborn infants contained adipic and suberic acids at the same concentrations as in this case. The urine amino acid chromatogram of this case showed no large
unusual peaks. DISCUSSION
Compared to our patient, most reported cases of lactic acidosis in infancy started later in life, and the course was less rapid and malignant7~10 (for complete references see ref. 6). However, the two cases, recently reported by Lie et a1.6, both died within one week after birth. Congenital lactic acidosis has in a few cases been attributed to specific enzymic deficiencies (glucose 6-phosphatase7, fructose 1,6-diphosphatases and pyruvate carboxylases) . The disease in patients with these enzymic defects does not run the same rapid course as in our case. In most cases reported, the enzymic defect has not been identified6ylo. The parents of the child described here were consanguineous, and they previously had one child, who also developed a lethal metabolic acidosis. These facts strongly suggest an inborn error of metabolism. As shown in Table I, the urine of this patient contained not only lactic acid, but also a series of aliphatic dicarboxylic acids with 6 to 14 carbon atoms, saturated as well as unsaturated. Dicarboxylic acids are Clin. Chim Ada,
41 (1972) 363-366
BORG et al.
366
formed by w-oxidation of fatty acids or their CoA ester+. This reaction has been considered to be of very small physiological significance; it is of interest however that Wada et ~1.1~have recently suggested that under conditions of inadequate glycolysis o-oxidation is increased to produce succinyl-CoA from fatty acids and thus to maintain the tricarboxylic acid cycle. Petterson and co-worker+ have recently reported that patients with ketosis excrete large amounts (i.e. up to 0.5 g/day) of adipic and suberic acid in the urine. When examining normal newborns we found these acids to occur in about the same concentration as in the patient. The major dicarboxylic acids in this case had longer chain lengths (C,,-C,,) and unsaturated as well as saturated acids were present. It appears probable, therefore, that this is not another example of deranged carbohydrate metabolism with ketosis and increased fatty acid metabolism but represents a situation with a primary defect in fatty acid catabolism. The catabolism of fatty acids occurs by successive P-oxidation in the mitochondria. Fatty acids in the FFA fraction are mainly C,, and C,,, i.e. a few t%oxidation steps must have occurred to give the dicarboxylic acids. The location of the double bond in the monoene fraction (cis-As) is thus consistent with two /Soxidations of oleic acid (ci.49). A defect in the transport of fatty acids into the mitochondria is unlikely since one would then have expected a predominance of acids with longer chain length. The same argument would seem to exclude a defect in the acyl-CoA synthetase reaction. A defect in the following dehydrogenase reaction which is catalyzed by fatty acyl-CoA dehydrogenases (EC 1.X.99.3) might lead to an accumulation of acids of intermediate chain length which would be substrates for the o-oxidation system. It is of interest in this context that in pig liver Crane St aLx4 have found at least two different acyl-CoA dehydrogenases with different chain length specificity. A block in the normal p-oxidation of fatty acids at the suggested step would presumably lead to a trapping of CoA with resulting effects on carbohydrate metabolism. It is tempting to speculate on the possibility that a low availability of acetylCoA would lead to a decreased formation of oxalacetate from pyruvate15 and consequently to an accumulation of lactate. This interpretation is supported by a recent report by Nordmann and NordmannlG who found increased liver pyruvate and lactate concentrations on inhibition of fatty acid B-oxidation by ~-mercaptoethanol. REFERENCES
8 9 10 II I2 13 I4 =s 16
J. B. SIDBURY, E. K. SNIITH AND W. HARLAN,J. Pediat., 70 (1967) 8. E.JELLUM,O.STOKKEA~DL.ELDJARN,SC~~~.J.~~~~.~~~.I~V~~~., 27(197X} 273. D. ROACH ANU C. W. GEHRXB, J.Chrowatog., 44fr96g) 269. W.G.NIEHAUS, J~.~NDR.RYHAGE, AnaZ.Chem., 40(1968) 1840. K. A. KARLSSON; Chem. Phys. Lipids, 5 (rg7o) 6. S.O. LIE, A.C,LGKEN,J.H.STRBMME AND 0. AAGENAES. ActaPediat..%and.,60 (1971)x29. R.&~AHLER,~~R.H.S. THOMPSON_~DI.D. P.WOTTON(E~~.), Bioche~icaEI)isov~e~s~iaHzanzan Disease, 3rd. ed.,J. & A. Churchill,London, 1970, p. 95. W. CH~LSMANN AND J.FERNANDES, Pediat.Res.,5 (~971) 633. I?. A. HOMMES, II.A. POLMAN AND J. D. REERINK, Arch. Diseases Childhood, 43 (1968) 423. A. F. HARTIMANN, H. J, WOBLTMANN, M. L. PURK~KSON AND M. E.WESLEV, J.Pediat., 61 (1962) 165. B. PREISS ANU K. BLOCH, J. Biol. Chem., 239 (1964) 85. F. WADA,M.USAMI,M.GOTOAND T.HAYASHI,J. Biochem., 70(1971) 1065. J. E. PETTERSEN,E.JELLUMANDL.ELDJARN,C~~~. Chim.Acta,38(Ig72) 17. F.L. CRANE, J. G.HANGEAND H.BEINERT, Biochirn.Biophys. Acta, 17(1955)293. R. BRESSLER, in S. WAKIL (Ed.),Lipid Metabolism, Academic Press,New York, 1970, p. 60. R. NORDMANR AXD J. ~oRD~A~N,~~oc~~~~~, 53 (1971)705.
Clin.Chim. Acta,
41 (1972)363-366