Evaluation of dipsticks for the assessment of microhaematuria.

Three types of dipstick were evaluated for the detection of microhaematuria. One type showed no false positives with erythrocyte-free urine, and no false negatives with a urine erythrocyte concentration of 20 X 10(6)/l using 5-10 X 10(6)/l as limit. This pattern of readings was produced by all the observers, thus indicating that method variation was satisfactory. The two other types of dipstick were clearly less sensitive, and had less desirable characteristics.

Lack of effect of exogenous or endogenous gastric inhibitory polypeptide on the elimination rate of Intralipid in man.

Six healthy subjects were studied by means of an intravenous fat tolerance test on three occasions, in the fasting state, in the postprandial state, and during an intravenous infusion of gastric inhibitory polypeptide (GIP). No effects on the elimination rate of Intralipid were seen either by endogenous or exogenous GIP.

Normal 2-aminobutyrate oxidation and increased valine oxidation in fibroblasts deficient in pyruvate dehydrogenase.

Human skin fibroblasts deficient in pyruvate dehydrogenase and five normal control strains were incubated with one of the following labelled substrates: DL-[1-14C]-2-amino-n-butyric acid, DL-[3-14C]-2-amino-n-butyric acid, L-[1-14C]leucine, L-[1-14C]valine, L-[1-14C]alanine, and [1-14C]pyruvate. The rate of 14CO2-production in the deficient cells was normal from 2-aminobutyrate and leucine, increased from valine, and decreased from alanine and pyruvate. These results indicated that in human skin fibroblasts the decarboxylation of 2-oxobutyrate is catalysed by an enzyme system different from the pyruvate dehydrogenase complex.

Identification of 2-chlorothioxanthen-9-one in gastric aspirate in a case of chlorprothixene poisoning.

In gastric aspirate from a case of severe chlorprothixene poisoning, large amounts (approximately 30% of the chlorprothixene) of a previously unrecognized compound were found and identified tentatively as 2-chlorothioxanthen-9-one by combined GLC-low-resolution mass spectrometry and high-resolution mass spectrometry. The identity of the unknown compound was verified after synthesis of 2-chlorothioxan-then-9-one by two procedures. Only negligible amounts of 2-chlorothioxanthen-9-one were formed when chlorprothixene, dissolved in acids, bases, chloroform-isopropanol, methanol, or gastric fluid, was stored in the dark. However, large amounts of the drug were converted to 2-chlorothioxanthen-9-one upon exposure to UV light. Moreover, considerable quantities of unidentified degradation products were formed when chlorprothixene was exposed to lamp light as well as to UV light. Therefore, samples from cases of acute drug poisoning should be protected from light until analysis.

Dicarboxylic aciduria during ketotic phases in various types of glycogen storage disease.

Urine samples were collected before and after a starvation period of 14-16 h from patients with glycogen storage disease, one with type III (amylo-1,6-glucosidase deficiency), four with type VIII (phosphorylase-b-kinase deficiency), and one with an unclassified type. The excretion of adipic, suberic, and 3-hydroxybutyric acid was measured by combined gas chromatography-mass spectrometry. The tendency towards ketosis seemed to decline with age in the patients with type VIII. In the non-ketotic patients no excess amounts of dicarboxylic acids were excreted. Therefore, glycogen storage disease per se seems to have no direct relationship to the excretion of adipic or suberic acid. A positive correlation was, however, found between the urinary excretion of on one side 3-hydroxybutyric and on the other adipic (correlation coefficient (Kendall's tau) +0.64, P less than 0.002 (one-sided test)) or suberic (+0.61, P less than 0.003) acid. The two dicarboxylic acids are most probably formed from long-chain monocarboxylic acids by omega- and beta-oxidation. It is speculated that succinyl-CoA formed by this pathway may counteract the tendency to ketosis in patients with glycogen storage disease.

Urinary metabolites of 2-ethyl-2-methylsuccinimide (ethosuximide) studied by combined gas chromatography mass spectrometry.

Metabolites of 2-ethyl-2-methylsuccinimide (ethosuximide) have been studied by combined gas chromatography mass spectrometry in a urine sample from a patient treated for petit mal epilepsy with ethosuximide. A new metabolite, 2-carboxymethyl-2-methylsuccinimide, was identified in the urine. It is presumably formed by omega 1-hydroxylation of the ethyl sidechain of the drug followed by a further oxidation of the primary alcohol to a carboxyl group. A previously identified metabolite, 2-ethyl-3-hydroxy-2-methylsuccinimide, was shown in the present study to yield two gas chromatographic peaks (as the N-methylated compound), indicating the existence of a diastereoisomeric pair. Thus, the enzyme responsible for the ring hydroxylation is probably not stereospecific, or alternatively two enzymes with different stereospecificity may exist.

Urinary metabolites of 4-isobutylphenylacetic acid studied by combined gas chromatography mass spectrometry.

After ingestion of 4-isobutylphenylacetic acid three metabolites were identified in the urine, viz. 4-(2-carboxypropyl)phenylacetic acid, 4-(2-hydroxy-2-methylpropyl)phenylacetic acid and 4-(1-hydroxy-2-methylpropyl)phenylacetic acid. These metabolites were identified by gas chromatography and mass spectrometry. In the in vivo formation of the metabolites the isobutyl sidechain of the drug is attacked by omega1-hydroxylation, followed by further oxidation of the primary alcohol to a carboxyl group, by omega2-hydroxylation and by omega3-hydroxylation, respectively. It has been shown previously that two other drugs with an isobutyl sidechain, viz. 2-p-methoxybenzenesulphonamido-5-isobutyl-1,3,4-thiadiazole and 2,4'-isobutylphenylpropionic acid, are metabolized in an analogous way.

Studies on the metabolism of 2,4'-isobutylphenylpropionic acid (ibuprofen) by gas chromatography and mass spectrometry. Dialysis fluid, a convenient medium for studies on drug metabolism.

2,4'-Isobutylphenylpropionic acid (ibuprofen) has previously been demonstrated to yield four urinary metabolites, formed by omega 1-, omega 2- and omega 3-hydroxylation and by a further oxidation of the primary alcohol of the omega 1-hydroxylated metabolite to a carboxyl group. By synthesis and gas chromatography--mass spectrometry the suggested structure of the omega 3-hydroxylated metabolite was verified in the present study. Moreover, a new metabolite, 2,4'-carboxyphenylpropionic acid, was demonstrated to be present in substantial amounts in dialysis fluid from a nephrectomized patient. In such patients ingested drugs cannot be excreted in the urine, but are metabolized to end products. Thus, dialysis fluid may be a convenient medium for studies on drug metabolism.

A biochemical analysis of subchondral bone of the medial tibial condyle in the normal state and in osteoarthritis and rheumatoid arthritis.

A chemical analysis has been carried out on specimens from the subchondral weightbearing area of the medial tibial condyle from 22 normal individuals, 14 individuals with osteoarthritis and 12 individuals with rheumatoid arthritis. In the normal group there was a decrease in density with advancing age. Over the age of 50 there was no significant difference between the groups. The content of collagen, calcium, phosphorus and magnesium in each bone specimen was calculated. When expressed in per cent of dry fat free bone there was no significant difference between the three groups. When calculations were made on the basis of content per volume tissue wet bone some differences were found. There was a tendency for a higher content of collagen in rheumatoid bone than in normal and osteoarthritic bone. The content of calcium was significantly higher in rheumatoid arthritis than in osteoarthritis; the same result was found in the analysis of phosphorus. In the normal group there was a decrease in phosphorus content with advancing age, this was also seen in the magnesium analysis.

Clinical conditions associated with urinary excretion of 2-hydroxybutyric acid.

Urine and blood samples from patients with different clinical disorders were examined for 2-hydroxybutyric acid (2-HB) by gas chromatography and mass spectrometry. All patients with combined lactic acidosis and ketoacidosis excreted 2-HB in the urine, in amounts up to 2.3 mmol/mmol creatinine. In blood from these patients, 2-HB was present only in trace amounts. Hard physical exercise resulted in lactic acidosis and accumulation of 2-HB. The lactate to pyruvate ratio in blood and urine was markedly increased in the patients excreting 2-HB but was normal in two patients with lactic acidosis who did not excrete 2-HB. It is concluded that an increased NADH2/NAD ratio is the most important factor for the production of 2-HB.

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.

Subcellular localization of hexadecanedioic acid activation in human liver.

The activation of hexadecanedioic acid has been studied in subcellular fractions of human liver. The activation capacity in a total homogenate of human liver was found to be 0.5 micro mole/min/g wet wt of tissue, about 10% of that for palmitic acid. Hexadecanedioic acid was activated by the mitochondrial and microsomal fractions. The mitochondrial enzyme is probably localized outside the inner mitochondrial compartment. The subcellular distribution of the hexadecanedioic acid activation was almost identical with the distribution of palmitic acid activation. Hexadecanedioic and palmitic acids seemed to compete for the same enzyme.