Dicarboxylic aciduria: the response to fasting.

The urine of a child who presented with an episode of a disease resembling Reye's syndrome was found to contain large quantities of the dicarboxylic acids adipic and suberic acids, as well as the glycine conjugate of suberic acid, suberyl glycine. A variety of other dicarboxylic acids, both saturated and unsaturated, were also found in the urine at the time of the attack. It was found that the excretion of these unusual metabolites could be markedly increased by fasting for periods of greater than 10 h. These results indicate that the patient may have a defect in fatty acid oxidation which becomes clinically significant during periods of prolonged fasting.     

Suberylglycine excretion in the urine from a patient with dicarboxylic aciduria

Suberylglycine (HOOC(CH₂)₆CONHCH₂COOH) was found in the urine from a patient with C₆-C₁₀-ω-dicarboxylic aciduria and unexplained episodes of lethargy and unconsciousness. The total excretion of adipic, suberic and sebacic acid ranged from 0.77 to 1.3 mg/mg creatinine after episodes of acute attack of the disease. Suberylglycine, identified by gas chromatography/mass spectrometry, was repeatedly found in the urine samples. The amount of this conjugate ranged from 0.2 to 0.5 mg/mg creatinine. The precursors of the dicarboxylic acids are suggested to be long chain monocarboxylic acids, oxidized through ω- and β-oxidation to adipic, suberic and sebacic acid. Suberylglycine is subsequently formed by glycine-N-acylase-catalyzed conjugation.     

Non-ketotic C6-C10-dicarboxylic aciduria: biochemical investigations of two cases

Two boys, who are not related, with hypoglycemia and C6-C10-dicarboxylic aciduria were investigated. Besides substantial amounts of adipic, suberic and sebacic acids, the urinary metabolic profile of organic acids contained 5-OH-caproic acid and caproylglycine. During acute attacks the concentrations of adipic, suberic and sebacic acids were 300--530, 160--200 and 35--200 micrograms/mg creatinine, respectively, and the excretions of 5-OH-caproic acid and caproylglycine were 75--330 and 41--260 micrograms/mg creatinine, respectively. It is argued that the biosynthesis of adipic acid passes through an omega-oxidation, that the production of 5-OH-caproic acid is caused by an omega-1-oxidation, and that caproylglycine formation passes through a glycine-N-acylase catalysed conjugation of accumulated caproic acid in the patients. Suberic acid and sebacic acid are in the same way omega-oxidation products of accumulated caprylic acid and capric acid, respectively. From the excretion pattern presented it is hypothesized that the patients suffer from a defect in the dehydrogenation of fatty acids in the beta-oxidation pathway. The biological significance of the findings is discussed.     

General (medium-chain) acyl-CoA dehydrogenase deficiency (non-ketotic dicarboxylic aciduria): quantitative urinary excretion pattern of 23 biologically significant organic acids in three cases

Urinary analysis of the pattern of 23 organic acid metabolites derived from fatty acids in three patients with general (medium-chain) acyl-CoA dehydrogenase deficiency was performed. Although there exist quantitative differences in the excreted amounts of the different metabolites in the three patients the qualitative picture was the same. The excretion of adipic, suberic and sebacic acids was substantial, whereas that of dodecanedioic acid was within or just above control limit. The monounsaturated C6-C10-dicarboxylic acid excretion was only marginally or not increased. 5-OH-hexanoic acid and hexanoylglycine were excreted in excessive amounts, whereas 7-OH-octanoic acid, 9-OH-decanoic acid, octanoylglycine and decanoylglycine were excreted in limited amounts. The excreted amounts of 6-OH-hexanoic, 8-OH-octanoic and 10-OH-decanoic acids were not or only marginally elevated compared to controls. In one of the patients the excretion of ethylmalonic and methylsuccinic acids was enhanced, whereas the excretion of these two acids in the two other patients was comparable to that in controls. The urinary excretion of hexanoic, octanoic, decanoic and dodecanoic acids was just a little above the control limit, whereas the esterified hexanoic and octanoic acids were excreted in appreciable amounts. It is argued that the microsomal omega- and omega-1-oxidation systems are involved in the dicarboxylic and omega-1-OH-monocarboxylic acids formation at C10 and C12 level and that the C8-C6-dicarboxylic and omega-1-OH-monocarboxylic acids are formed from higher chained acids by beta-oxidation in both mitochondria and peroxisomes.     

Pattern of aliphatic dicarboxylic acids in uremic serum including a new organic acid, 2,4-dimethyladipic acid.

(1) 2,4-Dimethyladipic acid was first identified in normal human urine using gas chromatography-mass spectrometry. Urinary excretion of 2,4-dimethyladipic acid in 7 healthy adults ranged from 4.9 mumol to 14 mumol per 24 h. (2) Succinic acid, adipic acid, 3-methyladipic acid, 2,4-dimethyladipic acid, pimelic acid and azelaic acid were identified in the ultrafiltrate of the blood obtained from a chronic uremic patient using a hemodialyzer. (3) Levels of succinic acid, adipic acid, 3-methyladipic acid, 2,4-dimethyladipic acid, pimelic acid and azelaic acid in uremic serum were determined using a mass fragmentographic technique. Concentration of succinic acid in uremic serum was comparable to that in normal serum, whereas concentrations of adipic acid, 3-methyladipic acid, 2,4-dimethyladipic acid, pimelic acid and azelaic acid were highly elevated in uremic serum.     

In vitro fibroblast studies in a patient with C6-C10-dicarboxylic aciduria: evidence for a defect in general acyl-CoA dehydrogenase

Fatty acid oxidation studies were performed on cultured fibroblasts from a patients who had suffered from a Reye's syndrome-like episode, and who excreted adipic acid, suberic acid, sebacic acid, 5-OH-hexanoic acid, and hexanoylglycine. Assays using fatty acids of different chain length on both intact and disrupted cells indicated that the patient had a defect of the general (medium-chain) acyl-CoA dehydrogenase apoenzyme.     

Aliphatic C6−C14 dicarboxylic acids in urine from an infant with fatal congenital lactic acidosis

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 ~ 0.05 mmoles/1 were also present in the urine of normal newborns. The concentration in the urine of dicarboxylic acids with 10, 12 and 14 carbon atoms was 0.1, 0.5 and 0.2 mmoles/1. These acids were saturated, monoenic and dienic. The monounsaturated acids were mainly of the cis-Δ₅ configuration. It is suggested that the findings could be consistent with a defect in the normal β-oxidation of fatty acids located at the acyl-CoA dehydrogenase step.     

Urinary C6-C12 dicarboxylic acylcarnitines in Reye's syndrome

C6-C12 dicarboxylic acylcarnitines have been identified for the first time in urine from a 2-year-old girl presenting with Reye's syndrome. The acylcarnitines were extracted by ion-exchange chromatography and analysed, both underivatised and as methyl esters using high-resolution fast-atom-bombardment mass spectrometry and B/E-linked scanning. The acylcarnitines were quantified by capillary gas chromatography of the acids extracted after hydrolysis of the acylcarnitine esters. Dodecandioylcarnitine was present in the highest concentration (35.9 mmol/mol creatinine) which exceeded the urinary free dodecandioic acid concentration. The adipic, suberic and sebacic acylcarnitine concentrations were less than 10% of the respective free acid concentrations. It is possible that beta-oxidation of dicarboxylic acids is partially inhibited in Reye's syndrome leading to accumulation of precursor dodecandioyl CoA which is metabolised to dodecandioylcarnitine. The accumulation of these metabolic intermediates may be significant in the pathogenesis of Reye's syndrome.     

Minimal effects of free fatty acids on the competitive protein-binding assay of serum thyroxine on reusable sephadex columns

In rats, following oral administration of cream and intravenous heparin, plasma free fatty acids and triiodothyronine Sephadex uptake were elevated. Simultaneously apparent serum thyroxine, measured by the Tetrasorb-E kit, was increased, but not when measured using reusable Sephadex columns. Addition of oleate to a human serum albumin solution or to human serum produced similar effects; appreciable rises in apparent thyroxine were noted in the kit assay but only minor changes occurred when Sephadex columns were used. In the latter procedure almost all [¹⁴C]oleic acid added to serum was removed in the first barbital buffer wash, before application of the thyroxine-binding globulin solution.In the serum thyroxine assay on reusable Sephadex columns, there is minimal interference by free fatty acids because they are removed by the first barbital buffer wash.     

Identification of ethylmalonic acid in urine of two patients with the vomitting sickness of Jamaica.

Large amounts of ethylmalonic acid have been identified in urines from two patients with the vomitting sickness of Jamaica. The amounts were 178 and 882 mug per mg creatinine which are 70 and 350 times, respectively, over control values. Other short and medium chain dicarboxylic acids including glutaric and adipic acids and those with eight and ten carbon chain, saturated and cis-unsaturated, were also detected in large quantities as in the case of hypoglycin treated rats; urine. However, the large increase of urinary ethylmalonic acid in these two human cases is in a sharp contrast to the findings in hypoglycin treated rats in which urinary ethylmalonic acid increased only 3 times over control. It appears that ethylmalonic acid is produced in the cases with the vomiting sickness of Jamaica by carboxylation of n-butyryl-CoA which is not oxidized further due to the inhibition by hypoglycin A. In case of hypoglycin-treated rats, n-butyryl-CoA is mainly conjugated with glycine or deacylated to free butyric acid.     

Ethylmalonic-Adipic Aciduria: IN VIVO AND IN VITRO STUDIES INDICATING DEFICIENCY OF ACTIVITIES OF MULTIPLE ACYL-CoA DEHYDROGENASES

The mechanisms underlying ethylmalonic-adipic aciduria were studied in a 5-yr-old girl. Oxidation of radioactive substrates by cultured skin fibroblasts from the proband and asymptomatic family members was also determined and compared to that by normal fibroblasts and that by cells from a patient with glutaric aciduria type II.Feeding medium-chain triglycerides promptly induced vomiting and lethargy accompanied by a pronounced increase of urinary ethylmalonate. Significant increases of serum isovalerate and urinary isovalerylglycine were observed after leucine feeding, but urinary glutarate increased only slightly after lysine feeding. Thus, the results from clinical investigation remained equivocal as to whether pathways other than fatty acid oxidation were blocked in our patient.Oxidation of [1-(14)C]butyrate by cultured skin fibroblasts from the proband was reduced to 14% of control. In vitro oxidation of [2-(14)C]lysine and [2-(14)C]leucine was also reduced to 28 and 23% of control, respectively. Much more severe reduction in oxidation of these three substrates (3, 9, and 9%, respectively) was observed in glutaric aciduria type II cells.These results indicated that in the proband, degradative pathways of fatty acids, lysine, and leucine are blocked at the steps of butyryl-CoA, glutaryl-CoA, and isovaleryl-CoA dehydrogenases, respectively, as in the case of glutaric aciduria type II. Because activities of multiple acyl-CoA dehydrogenases are reduced, a deficiency of electron-transferring flavoprotein, which serves as a hydrogen-acceptor for these dehydrogenases, is postulated as the underlying mechanisms of these two diseases, but a genetic heterogeneity was indicated by significant differences in the residual activities in these two types of cells. The hypothesis of more than one mutant allele of an autosomal recessive gene was also suggested by the study on cells from asymptomatic members of the family.     

Metabolism of alpha-aminoadipic and alpha-ketoadipic acids: studies using rat and beef liver, and human leukocytes.

The reported studies have shown that alpha-DL-amino [1-14C]adipic acid is metabolized to radioactive alpha-ketoadipic acid and then to 14CO2 in rat and beef liver homogenates. It was also demonstrated that alpha-keto [1-14C]adipic acid is decarboxylated to 14CO2. The effects of several co-factors and other variables, i.e. alpha-ketoglutarate, pyridoxal phosphate, NAD, thiamine pyrophosphate (TPP), FAD, LA, and pH have also been delimited. These reactions and the effects of the co-factors were also established in freshly drawn leukocytes. Using the methods outlined, a technique for studying the metabolism of alpha-amino-adipate and alpha-ketoadipate in the leukocytes from 10 ml of blood was developed.     

Quantitation of serum bile acids by isotope dilution with 13C-labelled homologs

Quantitation of individual bile acids in serum can be carried out with radioimmunoassays or with gas chromatography. The most specific measurements are obtained with combined gas chromatography/mass spectrometry employing stable isotope labelled bile acids as internal standards. So far only the use of deuterated internal standards has been described for this purpose. 24-13C-labelled bile acids have not been used since correction for the natural abundance of the 13C contribution has to be made. Furthermore, the maximal degree of labelling of 13C-labelled bile acids is about 90%. This requires additional correction for the percentage of unlabelled molecules. Using 13C-labelled bile acids as internal standards and adequate corrections for isotope interferences we have measured individual serum bile acids in healthy volunteers by inverse isotope dilution with coefficient of variation (CV) values of 5.4-6.2% determined for the total procedure of sample preparation and analytical technique. In fasting serum of 15 healthy volunteers chenodeoxycholic acid averaged 0.98 +/- SD 0.77 mumol/l, cholic acid 0.49 +/- 0.39 mumol/l and deoxycholic acid 1.07 +/- 0.68 mumol/l. Two hours postprandially these values increased to 2.42 +/- 1.46 mumol/l for chenodeoxycholic acid, 0.81 +/- 0.45 mumol/l for cholic acid and 1.66 +/- 1.02 mumol/l for deoxycholic acid. These data agree well with those described in the literature obtained with deuterated internal standards. It may be concluded that 24-13C-labelled bile acids are suitable as internal standards for quantitation of serum bile acids, if corrections for isotope interferences are made.     

血清激活素A对胎儿窘迫的预测价值

目的:探讨分娩时母血、脐血的激活素A(ACTA)水平预测胎儿窘迫的意义。方法:对335例正常产妇分娩前30min内抽取肘静脉血检测乳酸(LA)、ACTA水平,分娩时测定脐动脉血气pH值以及母、脐血ACTA和LA值,结合羊水性状和新生儿1minApgar评分情况,分析它们之间的关系。结果:母血、脐血ACTA水平与pH值呈显著负相关(r=-0.834,r=-0.657,P<0.01),优于LA水平(r=-0.393,r=0.718,P<0.01);母血、脐血中ACTA水平和脐血LA水平,酸血症组和窒息组较正常组差异显著,P<0.01,而母血中LA水平差异不显著,P>0.05;母血ACTA水平仅在羊水清亮组与Ⅲ度浑浊组之间有明显差异P<0.05;其余各羊水不同性状组间的ACTA和LA值均无明显差异,P>0.05。结论:母血、脐血ACTA水平对胎儿窘迫的预测价值明显优于LA,可作为预测胎儿窘迫的一个新敏感指标。     

Noninvasive urinary organic acids test to assess biochemical and nutritional individuality in autistic children

ObjectivesQuantitative organic acid testing can give information about potential problems, especially with energy production, neurotransmitter metabolism, intestinal dysbiosis and nutritional individuality which is very important in autistic children. The aim of this study was to find out potential differences between the levels of organic acids in the urine of autistic and non-autistic children.Design and methodsThe organic acids in the urine were determined by capillary gas chromatography/mass spectrometry (GC/MS). All overnight urine samples were collected from 35 autistic children and 36 neurologically normal children as healthy controls (4–10 years).ResultsSignificant differences were found between the autistic children and the control group in organic acids: 2-oxoglutaric, isocitric, citric, 4-hydroxybenzoic, 4-hydroxyphenylacetic, hippuric, adipic, suberic (all with p < 0.05).ConclusionOrganic acids test can be used to assess an individual need for nutrient and biochemical abnormalities, especially important for autistic children.     

以肾脏损伤为首发症状甲基丙二酸血症患儿17例临床分析

目的分析以肾脏损伤为首发症状的甲基丙二酸血症（methylmalonic acidemia,MMA）患儿临床特点及实验室检查结果。方法以肾脏损伤为首发症状的MMA患儿17例,检测血、尿常规、尿有机酸分析、血气分析及生化相关指标。结果 17例MMA患儿14例合并同型半胱氨酸血症,3例为单纯MMA;尿液甲基丙二酸水平为（0.100-214.143）mmol/（mmol·Cr）;其中11例尿潜血阳性,9例尿蛋白阳性;尿β2-微球蛋白、微量白蛋白、微量IgG、视黄醇结合蛋白、N-乙酰-β-D-氨基葡萄糖苷酶异常例数分别为10、6、8、10、11例;血氧、血乳酸、尿素氮的异常例数分别为9、11、10例;尿中甲基丙二酸水平与患儿血pH呈负相关（r=-0.493,P=0.022）,与血清谷草转氨酶和谷丙转氨酸水平呈正相关（r=0.455,P=0.000;r=0.826,P=0.033）。结论以肾损伤为首发症状的甲基丙二酸患儿多合并同型半胱氨酸升高;以尿潜血和尿蛋白异常为主;肾小管早期损伤指标中N-乙酰-β-D-氨基葡萄糖苷酶最灵敏,β2-微球蛋白、视黄醇结合蛋白次之;血氨、血乳酸可作为诊断该病的辅助指标;当尿中甲基丙二酸水平明显增高时,应警惕酸中毒和肝损伤发生。     

Serum unconjugated bile acids in patients with small bowel bacterial overgrowth

Concentrations of total and unconjugated bile acids in serum were measured fasting and 2 h postprandially in 9 patients with a positive [14C]glycocholate breath test consistent with small bowel bacterial overgrowth and in 13 controls. Gas-liquid chromatography-mass spectrometry (GLC-MS) and enzymatic-fluorometric assays were both used. In contrast to previous work, total serum bile acids were only occasionally elevated in patients with bacterial overgrowth. Total 2 h postprandial unconjugated bile acids, however, were elevated in 7/9 patients when measured by GLC-MS and in 6/9 when measured by the enzymatic-fluorometric method. The best separation between patients and controls was achieved by GLC-MS determinations of 2 h postprandial unconjugated cholic acid or primary bile acids, which were abnormal in 8/9 patients. This study indicates that measurement of serum bile acids may be a useful approach to the diagnosis of bacterial overgrowth, but would require accessible methods for separating and measuring cholic acid or unconjugated primary bile acids in post-prandial sera.     

Fructose-1,6-diphosphatase deficiency: another enzyme defect which can present itself with the clinical features of "tyrosinosis"

An infant with a picture of hereditary liver disease corresponding in many respects with so-called “tyrosinosis” is described. The primary defect appeared to be fructose-l,6-diphosphatase deficiency, which was not recognized during the patient's life.Many abnormalities of amino acid metabolism and transport occurred when the patient was on a diet containing saccharose. At that time tyrosyluria was excessive and serum tyrosine strongly elevated. Serum methionine was moderately increased, but a striking cystathioninuria was present and once even homocystine was traced in the urine. In the serum alanine was excessive, but also phenylalanine, lysine, proline, valine, threonine, leucine, serine and other amino acids were increased. There was a generalized amino aciduria and a massive lactic aciduria. Once even phenyllactic acid was found in the urine.When the diagnosis “tyrosinosis” is proposed, fructose-1,6-diphosphatase deficiency as a primary cause should also be kept in mind.     

The degree of conversion of alpha-keto acids to valine and phenylalanine in health and uraemia

13C-labelled alpha-keto acid analogues of valine and phenylalanine were given by mouth and by intravenous route to three healthy and four uraemic individuals who were used as their own controls. The excretion of 13CO2 in expired air and of 13C-labelled keto acid in urine was measured together with plasma and, in some cases, intramuscular concentrations of total and 13C-labelled free valine and phenylalanine. A minimum and maximum limit of degree of conversion of these two alpha-keto acids to essential amino acids were calculated and was of the order of 25-50 per cent for both keto acids given by either route in health and uraemia.     

Diagnostic significance of determining the blood content of lactic and uric acids in chronic liver lesions of alcoholic and viral etiologies

The concentration of lactic and uric acids in blood serum was measured in 115 patients with chronic liver lesions of virus (30 patients) and alcoholic (85 patients) etiology. The highest rise of lactic and uric acid concentrations was recorded in patients with alcoholic hepatitis and active alcoholic liver cirrhosis. The blood concentration of lactic and uric acids in alcoholic hepatopathy increased in parallel with an increment of the gravity of the pathological process, being the highest in patients with alcoholic hepatitis (lactic acid) and liver cirrhosis associated with the edematous ascites syndrome (uric acid). In inactive liver cirrhosis of alcoholic etiology, the concentrations of lactic and uric acids tended toward reduction, however, they were significantly higher than in the group of patients with virus liver lesions. Normal or negligible rises in the content of lactic and uric acids were seen in the groups of patients with chronic liver diseases of virus etiology. It is concluded that the measurement of the content of lactic and uric acids plays a diagnostic part in different patterns of alcoholic hepatopathies.