Jon Elling Pettersen

The occurrence of adipic and suberic acid in urine from ketotic patients

Abstract 1. 1. Urine samples from 53 healthy and diseased persons of both sexes aged 2 days to 52 years have been examined for dicarboxylic acids of chain-length C 3 –C 10 . The identity of the acids was verified by combined gas-liquid chromatography and mass spectrometry technique. 2. 2. In urine samples from ketotic patients adipic and suberic acid were always present in substantial amounts, corresponding to a daily excretion of as much as 750 and 140 mg, respectively. Succinic and glutaric acid were found only in a few of these samples (up to 1150 and 170 mg per day, respectively). 3. 3. The amount of adipic and suberic acid excreted may change rapidly in the course of a few hours and seem to parallel the degree of ketosis. 4. 4. Acidotic patients without ketosis, non-acidotic patients and healthy controls do not excrete dicarboxylic acids in the urine, apart from trace amounts of adipic and succinic acid. 5. 5. Possible explanations for the occurrence of the dicarboxylic acids, adipic and suberic acid, in the urine of ketotic patients are discussed, and it is concluded that they are most probably of endogenous origin.

Formation of n-hexanedioic acid from hexadecanoic acid by an initial ω oxidation in ketotic rats

Abstract 1. 1. Urine samples from normal and ketotic rats (streptozotocin-induced ketosis) have been analysed for dicarboxylic acids using gas-liquid chromatography and mass spectrometry. 2. 2. Normal rats were found to excrete small amounts of hexanedioic acid (0.2–0.5 μmoles/24 h) and only traces of octanedioic acid (less than o.1 μmoles/24 h) in the urine. Ketotic rats, on the other hand, excreted considerable quantities of both hexanedioic and octanedioic acid (up to 8.7 and 1.8 μmoles/24 h, respectively) in addition to large amounts of 3-hydroxybutyrate. 3. 3. There was a positive correlation between the excretion of hexanedioic and octanedioic acid and the excretion of 3-hydroxybutyrate. 4. 4. Urine samples from two ketotic rats given [1- 14 C]- or [16- 14 C]hexadecanoic acid, respectively, have been analysed for radioactive label in the hexanedioic acid by a procedure including extractions, thin-layer chromatography and preparative gas chromatography. Equal amounts of radioactivity (about 0.02% of the injected dose) were found in the hexanedioic acid fraction from both rats. 5. 5. These results indicate that long-chain fatty acids may be precursors of the hexanedioic and octanedioic acid excreted in urine during ketosis, and, furthermore, that most or all of the dicarboxylic acids derived from long-chain fatty acids are formed by an initial co oxidation followed by β oxidations.

The identification and metabolic origin of 2-furoylglycine and 2,5-furandicarboxylic acid in human urine

Abstract 1. 1. Combined gas-liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy have demonstrated the occurrence of 2-furoylglycine and 2,5-furandicarboxylic acid as normal constituents of human urine. 2. 2. The amount of 2-furoylglycine in urine samples from 20 normal adults was 3–28 μg/mg of creatinine, and the excretion of 2,5-furandicarboxylic acid was 0.5–8.2 μg/mg of creatinine. 3. 3. Dietary studies show that the furan derivatives or their precursors are of exogenous origin. Most probably they are derived from furan derivatives found in food prepared by strong heating. This may explain the absence of 2-furoylglycine and 2,5-furandicarboxylic acid in urine of breastfed children, and the absence of these acids in the urine of rat, pig, cow, ox, rabbit and monkey.

The occurrence of 2-hydroxybutyric acid in urine from patients with lactic acidosis

Abstract 1. 1. Urine samples from severely ill children have been examined for organic acids by combined gas-liquid chromatography—mass spectrometry technique. 2. 2. In all urine samples that contained large amounts of lactic acid (5.4–70.8 μmole/ml) considerable quantities of 2-hydroxybutyric acid (0.3–3.4 μmole/ml) were also present. 3. 3. Possible explanations for the occurrence of 2-hydroxybutyric acid in lactic acidosis are discussed. It is concluded that this metabolite is probably formed by lactate dehydrogenase from 2-oxobutyrate, a normal intermediate in the metabolism of some amino acids.

Branched short-chain dicarboxylic acids in human urine

Abstract 1. 1. Urine samples from healthy humans have been analysed for branched shortchain dicarboxylic acids using combined gas-liquid chromatography-mass spectrometry. 2. 2. Methylsuccinic, 3-methylglutaric, 3-methylhexanedioic, 3-methylheptanedioic, and 3-methyloctanedioic acid were present in all urine samples examined. 3-Methylhexanedioic acid was excreted in amounts of 0.10–0.19 mmole in 24 h, the others in much smaller quantities (from traces to 0.03 mmole in 24 h). 3. 3. The amounts of branched short-chain dicarboxylic acids excreted in urine were not significantly influenced by a period of three days on a synthetic diet consisting of tripalmitin, triolein, sucrose, and water. Ketotic patients showed a normal or only a slightly raised urinary excretion of 3-methylhexanedioic acid, whereas the excretion of the straight-chained hexanedioic acid was markedly increased. Thus, neither the diet nor the fat depots seem to contain the precursors of the branched dicarboxylic acids. 4. 4. Since even trace amounts of 2,6-dimethyloctanedioic acid were lacking in the urine samples, 3-methylhexanedioic acid does probably not stem from the degradation of isoprenoid compounds. Feeding of 3-methylhexadecanoic acid to rats resulted in a slight increase of urinary 3-methylhexanedioic acid. 5. 5. It is suggested that the branched short-chain dicarboxylic acids are formed from 3-methyl-substituted or ante-iso medium- or long-chain fatty acids of even or uneven chain-length by a primary Ω-oxidation followed by β-oxidations. Microorganisms in the intestinal tract, which are known to produce monomethyl-substituted fatty acids, may possibly function as a precursor pool for the urinary branched dicarboxylic acids.

In vitro studies on the metabolism of hexadecanedioic acid and its mono-L-carnitine ester.

Abstract 1. 1. A method for the synthesis of the mono-L-carnitine ester of hexadecanedioic acid (hexadecanedioylcarnitine) is described. A thin-layer chromatography system for the separation of long-chain monocarboxylic and long-chain dicarboxylic acylcarnitines was developed. 2. 2. Hexadecanedioic acid can be activated by rat liver mitochondria in the presence of CoA, ATP, and Mg2+. Since Triton X-Ioo does not increase the activation capacity, the enzyme(s) activating long-chain dicarboxylic acids seems to be localized outside the inner mitochondrial compartment. 3. 3. Hexadecanedioylcarnitine is substrate for a solubilized preparation of hexadecanoyl-CoA:carnitineO-hexadecanoyltransferase (EC 2.3.1.-), but the reaction rate is only about 1/10 of that with palmitylcarnitine. Accordingly, mitochondrial CoA can be acylated by hexadecanoylcarnitine, but much slower than by palmitylcarnitine. 4. 4. Hexadecanedioylcarnitine competes with palmitylcarnitine in the reaction catalyzed by the enzyme. 5. 5. In fasted, ketotic streptozotocin-diabetic, and clofibrate-fed rats the carnitine acyltransferase activity was significantly increased. The relative increases were about the same whether hexadecanedioylcarnitine or palmitylcarnitine were used as substrates. 6. 6. The observations under points 4 and 5 indicate that the same enzyme (hexadecanoyl-CoA:carnitineO-hexadecanoyltransferase, EC 2.3.1.-) may transport activated hexadecanedioic as well as palmitic acid across the inner mitochondrial membrane. 7. 7. The O2 uptake of rat heart and liver mitochondria with hexadecanedioylcarnitine as substrate was very low and transitory. Hexadecanedioylcarnitine did not inhibit the oxidation of palmitylcarnitine. 8. 8. The present in vitro studies demonstrate a possible mechanism for the degradation of long-chain dicarboxylic acids, viz. activation by CoA and transportation into the inner mitochondrial compartment as carnitine esters followed by β-oxidation.

ATP-dependent activation of dicarboxylic acids in rat liver

Abstract 1. 1. The activation of nonanedioic, decanedioic, dodecanedioic, tetradecanedioic, and hexadecanedioic acid was demonstrated in rat liver mitochondria. By our technique the activation of dicarboxylic acids shorter than nonanedioic acid could not be demonstrated. 2. 2. The activation capacity for hexadecanedioic acid in rat liver total homogenate was found to be 1.o μmole/min per g wet wt of tissue, i.e. about 10% of that for palmitic acid. 3. 3. Hexadecanedioic acid is activated by the mitochondrial and microsomal fractions. The subcellular distribution of this enzyme activity is almost identical to the distribution of palmitic acid activation. 4. 4. Hexadecanedioic and palmitic acid seemed to compete for the same enzyme. 5. 5. It is suggested that palmitic and hexadecanedioic acid are activated by the same enzyme(s).

In vivo studies on the metabolism of hexanedioic acid

1. Using the combined gas-liquid chromatography-mass spectrometry technique it was shown that ketotic patients excreted up to 273 mg of hexanedioic acid daily in their urine, whereas serum samples from these patients contained only trace amounts of this acid. Healthy humans excreted 2-5 mg daily. Hexanedioic acid was not detectable in normal serum. 2. An experiment with the infusion of large amounts of 3-hydroxybutyrate into a dog indicated that the increased urinary hexanedioic acid excretion in ketosis is not due to a competition between 3-hydroxybutyrate and hexanedioic acid for the same renal reabsorption mechanism. 3. [ 1,6-14-C]Hexanedioic acid intravenously injected into a dog was at first distributed in the extracellular space, followed by a partial equilibration with the intracellular space. About 11% of the injected dose was expired as 14-CO2 in 220 min. The maximal 14-CO2 production rate was obtained after about 20 min. In 240 min, 47% of the injected radioactivity was recovered in the urine. The large urinary excretion of labeled hexanedioic acid observed in the presence of only trace amounts in serum, showed that the high excretion by ketotic patients of the dicarboxylic acid may be explained without postulating an exclusive renal synthesis for hexanedioic acid.