Liquid Chromatography-Mass Spectrometry-Based Metabolomics of Nonhuman Primates after 4 Gy Total Body Radiation Exposure: Global Effects and Targeted Panels.

Rapid assessment of radiation signatures in noninvasive biofluids may aid in assigning proper medical treatments for acute radiation syndrome (ARS) and delegating limited resources after a nuclear disaster. Metabolomic platforms allow for rapid screening of biofluid signatures and show promise in differentiating radiation quality and time postexposure. Here, we use global metabolomics to differentiate temporal effects (1-60 d) found in nonhuman primate (NHP) urine and serum small molecule signatures after a 4 Gy total body irradiation. Random Forests analysis differentially classifies biofluid signatures according to days post 4 Gy exposure. Eight compounds involved in protein metabolism, fatty acid ß oxidation, DNA base deamination, and general energy metabolism were identified in each urine and serum sample and validated through tandem MS. The greatest perturbations were seen at 1 d in urine and 1-21 d in serum. Furthermore, we developed a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) method to quantify a six compound panel (hypoxanthine, carnitine, acetylcarnitine, proline, taurine, and citrulline) identified in a previous training cohort at 7 d after a 4 Gy exposure. The highest sensitivity and specificity for classifying exposure at 7 d after a 4 Gy exposure included carnitine and acetylcarnitine in urine and taurine, carnitine, and hypoxanthine in serum. Receiver operator characteristic (ROC) curve analysis using combined compounds show excellent sensitivity and specificity in urine (area under the curve [AUC] = 0.99) and serum (AUC = 0.95). These results highlight the utility of MS platforms to differentiate time postexposure and acquire reliable quantitative biomarker panels for classifying exposed individuals.

Exploration of the Fecal Microbiota and Biomarker Discovery in Equine Grass Sickness.

Equine grass sickness (EGS) is a frequently fatal disease of horses, responsible for the death of 1 to 2% of the U.K. horse population annually. The etiology of this disease is currently uncharacterized, although there is evidence it is associated with Clostridium botulinum neurotoxin in the gut. Prevention is currently not possible, and ileal biopsy diagnosis is invasive. The aim of this study was to characterize the fecal microbiota and biofluid metabolic profiles of EGS horses, to further understand the mechanisms underlying this disease, and to identify metabolic biomarkers to aid in diagnosis. Urine, plasma, and feces were collected from horses with EGS, matched controls, and hospital controls. Sequencing the16S rRNA gene of the fecal bacterial population of the study horses found a severe dysbiosis in EGS horses, with an increase in Bacteroidetes and a decrease in Firmicutes bacteria. Metabolic profiling by 1H nuclear magnetic resonance spectroscopy found EGS to be associated with the lower urinary excretion of hippurate and 4-cresyl sulfate and higher excretion of O-acetyl carnitine and trimethylamine-N-oxide. The predictive ability of the complete urinary metabolic signature and using the four discriminatory urinary metabolites to classify horses by disease status was assessed using a second (test) set of horses. The urinary metabolome and a combination of the four candidate biomarkers showed promise in aiding the identification of horses with EGS. Characterization of the metabolic shifts associated with EGS offers the potential of a noninvasive test to aid premortem diagnosis.

Discovery and validation of urinary metabotypes for the diagnosis of hepatocellular carcinoma in West Africans.

UNLABELLED: There is no clinically applicable biomarker for surveillance of hepatocellular carcinoma (HCC), because the sensitivity of serum alpha-fetoprotein (AFP) is too low for this purpose. Here, we determined the diagnostic performance of a panel of urinary metabolites of HCC patients from West Africa. Urine samples were collected from Nigerian and Gambian patients recruited on the case-control platform of the Prevention of Liver Fibrosis and Cancer in Africa (PROLIFICA) program. Urinary proton nuclear magnetic resonance ((1) H-NMR) spectroscopy was used to metabolically phenotype 290 subjects: 63 with HCC; 32 with cirrhosis (Cir); 107 with noncirrhotic liver disease (DC); and 88 normal control (NC) healthy volunteers. Urine samples from a further cohort of 463 subjects (141 HCC, 56 Cir, 178 DC, and 88 NC) were analyzed, the results of which validated the initial cohort. The urinary metabotype of patients with HCC was distinct from those with Cir, DC, and NC with areas under the receiver operating characteristic (AUROC) curves of 0.86 (0.78-0.94), 0.93 (0.89-0.97), and 0.89 (0.80-0.98) in the training set and 0.81 (0.73-0.89), 0.96 (0.94-0.99), and 0.90 (0.85-0.96), respectively, in the validation cohort. A urinary metabolite panel, comprising inosine, indole-3-acetate, galactose, and an N-acetylated amino acid (NAA), showed a high sensitivity (86.9% [75.8-94.2]) and specificity (90.3% [74.2-98.0]) in the discrimination of HCC from cirrhosis, a finding that was corroborated in a validation cohort (AUROC: urinary panel = 0.72; AFP = 0.58). Metabolites that were significantly increased in urine of HCC patients, and which correlated with clinical stage of HCC, were NAA, dimethylglycine, 1-methylnicotinamide, methionine, acetylcarnitine, 2-oxoglutarate, choline, and creatine. CONCLUSION: The urinary metabotyping of this West African cohort identified and validated a metabolite panel that diagnostically outperforms serum AFP.

Effect of carnitine, acetyl-, and propionylcarnitine supplementation on the body carnitine pool, skeletal muscle composition, and physical performance in mice.

PURPOSE: Pharmacokinetics and effects on skeletal muscle and physical performance of oral acetylcarnitine and propionylcarnitine are not well characterized. We therefore investigated the influence of oral acetylcarnitine, propionylcarnitine, and carnitine on body carnitine homeostasis, energy metabolism, and physical performance in mice and compared the findings to non-supplemented control animals. METHODS: Mice were supplemented orally with 2 mmol/kg/day carnitine, acetylcarnitine, or propionylcarnitine for 4 weeks and studied either at rest or after exhaustive exercise. RESULTS: In the supplemented groups, total plasma and urine carnitine concentrations were significantly higher than in the control group receiving no carnitine, whereas the skeletal muscle carnitine content remained unchanged. The supplemented acylcarnitines were hydrolyzed in intestine and liver and reached the systemic circulation as carnitine. Bioavailability of carnitine and acylcarnitines, determined as the urinary excretion of total carnitine, was in the range of 19 %. Skeletal muscle morphology, including fiber-type composition, was not affected, and oxygen consumption by soleus or gastrocnemius fibers was not different between the groups. Supplementation with carnitine or acylcarnitines had no significant impact on the running capacity, but was associated with lower plasma lactate levels and a higher glycogen content in white skeletal muscle after exhaustive exercise. CONCLUSIONS: Oral supplementation of carnitine, acetylcarnitine, or propionylcarnitine in mice is associated with increased plasma and urine total carnitine concentrations, but does not affect the skeletal muscle carnitine content. Despite better preservation of skeletal muscle glycogen and lower plasma lactate levels, physical performance was not improved by carnitine or acylcarnitine supplementation.

Rapid and sensitive HILIC-MS/MS analysis of carnitine and acetylcarnitine in biological fluids.

Monitoring carnitine and acetylcarnitine levels in biological fluids is a powerful tool for diagnostic studies. Research has recently shown that the analysis of carnitine and related compounds in clinical samples can be accomplished by different analytical approaches. Because of the polar and ionic nature of the analytes and matrix complexity, accurate quantitation is a highly challenging task. Thus, sample processing factors, preparation/cleanup procedures, and chromatographic/ionization/detection parameters were evaluated. On the basis of the results obtained, a rapid, selective, sensitive method based on hydrophilic interaction liquid chromatography-tandem mass spectrometry for the analysis of carnitine and acetylcarnitine in serum and urine samples is proposed. The matrix effect was assessed. The proposed approach was validated, the limits of detection were in the nanomolar range, and carnitine and acetylcarnitine levels were found within the micromolar range in both types of sample.

Effect of short- and long-term treatment with valproate on carnitine homeostasis in humans.

AIMS: The aim of this study was to identify the mechanisms of hypocarnitinemia in patients treated with valproate. METHODS: Plasma concentrations and urinary excretion of carnitine, acetylcarnitine, propionylcarnitine, valproylcarnitine, and butyrobetaine were determined in a patient starting valproate treatment and in 10 patients on long-term valproate treatment. Transport of carnitine and valproylcarnitine by the proximal tubular carnitine transporter OCTN2 was assessed in vitro. RESULTS: In the patient starting valproate, the plasma carnitine and acetylcarnitine levels dropped for 1-3 weeks and had recovered after 3-5 weeks, whereas the plasma levels of propionyl and valproylcarnitine increased steadily over 5 weeks. The renal excretion and excretion fractions (EFs) of carnitine, acetylcarnitine, propionylcarnitine, and butyrobetaine decreased substantially after starting valproate. Compared with controls, patients on long-term valproate treatment had similar plasma levels of carnitine, acetylcarnitine, and propionylcarnitine, whereas valproylcarnitine was found only in patients. Urinary excretion and renal clearance of carnitine, acetylcarnitine, propionylcarnitine, and butyrobetaine were decreased in valproate-treated compared with that in control patients, reaching statistical significance for carnitine. The EFs of carnitine, acetylcarnitine, and propionylcarnitine were <5% of the filtered load in controls and were lower in valproate-treated patients. In contrast, the EF for valproylcarnitine approached 100%, resulting from a low affinity of valproylcarnitine for the carnitine transporter OCTN2 and competition with concomitantly filtered carnitine. CONCLUSIONS: The initial drop in plasma carnitine levels of valproate-treated patients is most likely due to impaired carnitine biosynthesis, whereas the recovery of the plasma carnitine levels is explainable by an increased renal expression of OCTN2. Renally excreted valproylcarnitine does not affect renal handling of carnitine in vivo.

Carnitine palmitoyltransferase 2 deficiency: the time-course of blood and urinary acylcarnitine levels during initial L-carnitine supplementation.

Carnitine palmitoyltransferase 2 (CPT2) deficiency is one of the most common mitochondrial beta-oxidation defects. A female patient with an infantile form of CPT2 deficiency first presented as having a Reye-like syndrome with hypoglycemic convulsions. Oral L-carnitine supplementation was administered since serum free carnitine level was very low (less than 10 micromol/L), indicating secondary carnitine deficiency. Her serum and urinary acylcarnitine profiles were analyzed successively to evaluate time-course effects of L-carnitine supplementation. After the first two days of L-carnitine supplementation, the serum level of free carnitine was elevated; however, the serum levels of acylcarnitines and the urinary excretion of both free carnitine and acylcarnitines remained low. A peak of the serum free carnitine level was detected on day 5, followed by a peak of acetylcarnitine on day 7, and peaks of long-chain acylcarnitines, such as C16, C18, C18:1 and C18:2 carnitines, on day 9. Thereafter free carnitine became predominant again. These peaks of the serum levels corresponded to urinary excretion peaks of free carnitine, acetylcarnitine, and medium-chain dicarboxylic carnitines, respectively. It took several days for oral L-carnitine administration to increase the serum carnitine levels, probably because the intracellular stores were depleted. Thereafter, the administration increased the excretion of abnormal acylcarnitines, some of which had accumulated within the tissues. The excretion of medium-chain dicarboxylic carnitines dramatically decreased on day 13, suggesting improvement of tissue acylcarnitine accumulation. These time-course changes in blood and urinary acylcarnitine levels after L-carnitine supplementation support the effectiveness of L-carnitine supplementation to CPT2-deficient patients.

Urinary Metabolomics Profiles Associated to Bovine Meat Ingestion in Humans.

SCOPE: The impact of meat consumption on human health is widely examined in nutritional epidemiological studies, especially due to the connection between the consumption of red and processed meat and the risk of colon cancer. Food questionnaires do not assess the exposure to different methods of meat cooking. This study aimed to identify biomarkers of the acute ingestion of bovine meat cooked with two different processes. METHODS AND RESULTS: Non-targeted UPLC-MS metabolite profiling was done on urine samples obtained from 24 healthy volunteers before and 8 h after the ingestion of a single meal composed of intrinsically 15 N labelled bovine meat, either cooked at 55 °C for 5 min or at 90 °C for 30 min. A discriminant analysis extension of independent components analysis was applied to the mass spectral data. After meat ingestion, the urinary excretion of 1-methylhistidine, phenylacetylglutamine, and short- and medium-chained acylcarnitines was observed. 15 N labelling was detected in these metabolites, thus confirming their origin from ingested meat. However, no difference was observed in urinary metabolomic profiles according to the meat cooking process used. CONCLUSION: Meat ingestion led to the excretion of several nitrogen-containing compounds, but although a metabolic signature was detected for meat ingestion, the impact of the cooking process was not detectable at the level of urinary metabolic signature in our experimental conditions.

Differential mobility spectrometry (DMS) reveals the elevation of urinary acetylcarnitine in non-human primates (NHPs) exposed to radiation.

Acetylcarnitine has been identified as one of several urinary biomarkers indicative of radiation exposure in adult rhesus macaque monkeys (non-human primates, NHPs). Previous work has demonstrated an up-regulated dose-response profile in a balanced male/female NHP cohort. As a contribution toward the development of metabolomics-based radiation biodosimetry in human populations and other applications of acetylcarnitine screening, we have developed a quantitative, high-throughput method for the analysis of acetylcarnitine. We employed the Sciex SelexIon DMS-MS/MS QTRAP 5500 platform coupled to flow injection analysis (FIA), thereby allowing for fast analysis times of less than 0.5 minutes per injection with no chromatographic separation. Ethyl acetate is used as a DMS modifier to reduce matrix chemical background. We have measured NHP urinary acetylcarnitine from the male cohorts that were exposed to the following radiation levels: control, 2, 4, 6, 7, and 10 Gy. Biological variability, along with calibration accuracy of the FIA-DMS-MS/MS method, indicates LOQ of 20 µM, with observed biological levels on the order of 600 µM and control levels near 10 µM. There is an apparent onset of intensified response in the transition from 6 to 10 Gy. The results demonstrate that FIA-DMS-MS/MS is a rapid, quantitative technique that can be utilized for the analysis of urinary biomarker levels for radiation biodosimetry.

Carnitine and acylcarnitine metabolism during exercise in humans. Dependence on skeletal muscle metabolic state.

Carnitine metabolism has been previously shown to change with exercise in normal subjects, and in patients with ischemic muscle diseases. To characterize carnitine metabolism further during exercise, six normal male subjects performed constant-load exercise on a bicycle ergometer on two separate occasions. Low-intensity exercise was performed for 60 min at a work load equal to 50% of the lactate threshold, and high-intensity exercise was performed for 30 min at a work load between the lactate threshold and maximal work capacity for the individual. Low-intensity exercise was not associated with a change in muscle (vastus lateralis) carnitine metabolism. In contrast, from rest to 10 min of high-intensity exercise, muscle short-chain acylcarnitine content increased 5.5-fold while free carnitine content decreased 66%, and muscle total carnitine content decreased by 19% (all P less than 0.01). These changes in skeletal muscle carnitine metabolism were present at the completion of 30 min of high-intensity exercise, and persisted through a 60-min recovery period. With 30 min of high-intensity exercise, plasma short-chain and long-chain acylcarnitine concentrations increased by 46% and 23%, respectively. Neither exercise state was associated with a change in the urine excretion rates of free carnitine or acylcarnitines. Thus, alterations in skeletal muscle carnitine metabolism, characterized by an increase in acylcarnitines and a decrease in free and total carnitine, are dependent on the work load and, therefore, the metabolic state associated with the exercise, and are poorly reflected in the plasma and urine carnitine pools.

Urinary excretion of L-carnitine and its short-chain acetyl-L-carnitine in patients undergoing carboplatin treatment.

PURPOSE: To evaluate the effect of the anti-cancer drug carboplatin on plasma concentrations and urinary excretion of L-carnitine (LC) and its main ester, acetyl-L-carnitine (ALC), in cancer patients. METHODS: Plasma and urine concentrations of LC and ALC from 11 patients on carboplatin therapy (1 h intravenous infusion; AUC dose 4.8 +/- 1.1 mg/ml min) in combination with docetaxel, paclitaxel or vinorelbine, were determined by high-performance liquid chromatography with fluorimetric detection. RESULTS: Before carboplatin therapy, the mean +/- SD plasma concentrations of LC and ALC were 47.8 +/- 10.9 and 7.04 +/- 1.04 nmoles/ml, respectively, and remained constant throughout the entire study period. In contrast, urinary excretion of LC and ALC, increased significantly during the chemotherapy from 115 +/- 105 to 480 +/- 348 micromoles/day (P < 0.01) and from 41 +/- 41 to 89 +/- 52 micromoles/day (P < 0.05) for LC and ALC, respectively, subsequently reverting to normal 6 days after the end of chemotherapy. Similarly, the renal clearance of LC and ALC increased substantially during chemotherapy from 1.67 +/- 1.43 to 9.05 +/- 9.52 ml/min (P < 0.05) and from 4.02 +/- 4.51 to 7.97 +/- 5.05 ml/min (P = not significant) for LC and ALC, respectively, reverting to normal 6 days after the end of chemotherapy. Plasma concentrations and urinary excretion of glucose, phosphate and urea nitrogen and creatinine clearance, however, were not affected by carboplatin therapy, indicating no impaired kidney function. CONCLUSION: Treatment with carboplatin was associated with a marked urinary loss of LC and ALC, most likely due to inhibition of carnitine reabsorption in the kidney.

Effect of fasting on free and esterified carnitine levels in human serum and urine: correlation with serum levels of free fatty acids and beta-hydroxybutyrate.

Serum levels of free L-carnitine, acylcarnitines, creatinine, beta-hydroxybutyrate, free fatty acids, cholesterol, triglycerides, and glucose were determined in healthy volunteers during a 24-36-hr fast. The effect of oral administration of free L-carnitine (1 g/person) on these parameters was studied. Urinary excretion of carnitine and creatinine was monitored throughout. Serum and urine levels of free carnitine and its renal clearance decreased during the fast. However, the serum concentration and urinary excretion of acylcarnitines increased during the same interval. Following the ingestion of free L-carnitine, both serum and urinary levels of free L-carnitine rose. Within 6 hr of ingestion, 10% of the administered dose could be accounted for by urinary excretion. No significant effect on the other serum constituents under study was seen following the oral L-carnitine dose. A significant negative correlation was found between serum levels of free L-carnitine and beta-hydroxybutyrate and free fatty acids (r equal -0.567, p less than 0.001 and r equal -0.607, p less than 0.001, respectively) during the fast.

A new simple screening method for the diagnosis of medium chain acyl-CoA dehydrogenase deficiency.

We describe a simple inexpensive method for the detection of octanoyl-carnitine in urine by reverse-phase high performance thin-layer chromatography of the p-bromophenacyl derivative. This method provides a rapid and reliable means for the diagnosis of medium-chain acyl-CoA dehydrogenase deficiency which is suitable for routine laboratory use.

Enzyme reactor for urinary acylcarnitines assay by reversed-phase high-performance liquid chromatography.

An immobilized enzyme reactor, made up acylcarnitine hydrolase, carnitine dehydrogenase and diaphorase in sequence, was developed for the sensitive and selective determination of urinary free and individual acylcarnitines by a reversed-phase high-performance liquid chromatography. A 100-microliter urine sample was directly injected onto the TSKgel ODS 80Ts column and eluted by a step-gradient procedure. The eluent was mixed with the substrate solution of beta-NAD+ (1.0 mmol/l), resazurin (25 mumol/l) and Tris acetate (0.2 mol/l, pH 9.0). The mixture was passed through the immobilized enzyme reactor at 40 degrees C. Acylcarnitines were hydrolyzed and the converted to rezorufin which was measured by monitoring the fluorescence intensity at lambda EX = 560 nm and lambda EM = 580 nm. Free, acetyl-, glutaryl-, propionyl-, butyryl-, isobutyryl-, valeryl- and isovalerylcarnitine were determined within 55 min with detection limits (< 1 mumol/l) and within-run and day-to-day imprecision (C.V. < 6%). Free, acetyl- and isobutyrylcarnitine were found in normal urine. On the other hand, propionylcarnitine was detected in the urine of children with propionic aciduria and methylmalonic aciduria and multiple acylcarnitines were found in the urine of children with glutaric aciduria (type II).

Identification of glutarylcarnitine in glutaric aciduria type 1 by carboxylic acid analyzer with an ODS reverse-phase column.

A technique for the identification of glutarylcarnitine in urine from a patient with glutaric aciduria type 1 is described. The patient's urine sample was partially purified using an anion exchange column and analyzed by a carboxylic acid analyzer fitted with an ODS reverse-phase column. The chromatogram of the patient's urine sample revealed 3 different peaks, which corresponded respectively to those of carnitine with amino acids, acetylcarnitine and glutarylcarnitine. Following hydrolysis of the sample, the chromatogram had no peaks of acetylcarnitine and glutarylcarnitine but had remarkably amplified peaks of carnitine, acetic acid and glutaric acid. The eluent fraction of glutarylcarnitine from the non-hydrolyzed sample was hydrolyzed and analyzed again. It no longer had the glutarylcarnitine peak on the chromatogram, but had only two separate peaks of carnitine and glutaric acid. This technique simplifies the identification of glutarylcarnitine, in that it requires only removal of organic acids for preparation of samples, and does not require radioisotope or mass spectrometry.

Determination of acylcarnitines in urine of patients with inborn errors of metabolism using high-performance liquid chromatography after derivatization with 4'-bromophenacylbromide.

A high-performance liquid chromatographic method is presented for the determination of urinary acylcarnitines. After solid phase extraction on silica columns the acylcarnitines are converted to 4'-bromophenacyl esters with 4'-bromophenacylbromide in the presence of N,N-diisopropylethylamine. Complete derivatization was achieved at 37 degrees C within 30 min. The 4'-bromophenacyl esters were separated by high-performance liquid chromatography on a Hypersil BDS C8 reversed-phase column with a binary gradient containing varying proportions of acetonitrile, water and 0.1 M triethylamine phosphate buffer. Essentially baseline separation was obtained with a standard mixture containing 4'-bromophenacyl esters of carnitine and synthetic acylcarnitines of increasing chain length ranging from acetyl- to palmitoylcarnitine. The method was used to obtain urinary acylcarnitine profiles from patients with propionic, methylmalonic and isovaleric acidemia and with medium-chain and multiple acyl-CoA dehydrogenase deficiency. Quantification of the acylcarnitines was achieved using undecanoylcarnitine as internal standard.

Alterations of urinary acetylcarnitine in valproate-treated rats: the effect of L-carnitine supplementation.

Urinary excretion of acetylcarnitine was measured by high-performance liquid chromatography in two experimental groups of valproate-treated rats. In the urine of mature rats weighing 180 to 200 g treated with valproate (500 mg/kg/day), acetylcarnitine levels were higher than those in controls on days 4 and 7, while L-carnitine-supplemented rats showed lower levels than the valproate group. The valproate-treated rats showed an increased acetylcarnitine/acylcarnitine ratio on and after day 4, while the L-carnitine-supplemented rats showed no significant change compared to the controls on any days. In the urine of immature rats weighing 80 to 90 g treated with valproate (50 mg/kg/day), acetylcarnitine levels were increased after the 14th day of treatment. These results suggest that an increase in urinary acetylcarnitine occurs when small doses of valproate are administered for a longer time. We speculate that increased acetylcarnitine is not a product of beta-oxidation in mitochondria, because L-carnitine supplementation decreases the acetylcarnitine levels. Although the mechanism of acetylcarnitine excretion during valproate administration is not clear, L-carnitine supplementation is effective in decreasing the level of urinary acetylcarnitine and keeping the acetylcarnitine/acylcarnitine ratio normal.

The influence of kidney transplantation on carnitine metabolism.

In this study, we examined all three plasma carnitine fractions, free carnitine (FC), short-chain acyl- (SCC), and long-chain acylcarnitine (LCC), as well as the urinary excretion of FC and SCC in 62 patients with functioning renal allografts 1-70 months following the kidney transplantation (KT). Patients were classified into three groups according to their transplant function, as characterized by the serum creatinine concentration (CR). A comparison with normal subjects (n = 20) and with patients on chronic hemodialysis (HD, n = 46) revealed a complete normalization of all carnitine plasma fractions for group I patients (Cr less than 120 mumol/l). In contrast, significant elevations of plasma free and esterified carnitine were found in group II (Cr greater than 120, less than 200 mumol/l; with FC + 27%, SCC + 82%, and LCC + 49% and group III patients (Cr greater than 200 mumol/l; with FC + 39%, SCC + 122%, and LCC + 106%), as compared to healthy subjects (p less than 0.001). The elevations in concentrations of SCC were more pronounced than those of FC; consequently, we found a higher ratio of acylcarnitine (AC) to FC in group III than in group I patients (+ 41%, p less than 0.001). Yet even in the former group, this ratio was found to be markedly reduced when compared to HD patients (-41%, p less than 0.001). We found no significant differences in the urinary excretion of FC and SCC between the 3 groups of KT patients. It is thus to conclude that in patients with a well-functioning transplant, the pattern of carnitine fractions in plasma is fully normalized. The decrease in the ratio of AC to FC following a successful KT might suggest a better availability of FC and thereby be associated with an enhanced fatty acid oxidation.

Urinary carnitine excretion in patients with heart failure.

To evaluate the fatty acid metabolism in heart failure, the semiquantitative analysis of urinary free carnitine and acylcarnitine was made by fast atom bombardment mass spectrometry (FABMS) in 22 patients (mean age 67.3 years) with heart failure and 19 age-matched healthy controls (average age 60.4 years). Urinary excretion of free carnitine was 0.20 +/- 0.118 ratio/mg creatinine in the healthy controls and 1.32 +/- 1.170 ratio/mg creatinine in the patients with heart failure. The latter value was significantly higher (p < 0.01). Patients with heart failure were classified into two groups according to the urinary free carnitine concentration. One was the high excretion group (2.19 +/- 0.102 ratio/mg creatinine, 12 cases) and the other was the low excretion group (0.37 +/- 0.212 ratio/mg creatinine, 10 cases). In the high excretion group, urinary acetylcarnitine was also increased, but no significant abnormalities were observed in the urinary organic acid profile. In the high group, 1 patient was classified as NYHA class III and 11 as NYHA class IV. Four patients died in the hospital. In the low excretion group, five patients were classified as NYHA class III and five as NYHA class IV. Only one patient died in the hospital. In the high group, patients with severe and prolonged heart failure tended to maintain higher values of urinary free carnitine. We could not find any abnormalities in fatty acid metabolism in patients with heart failure, but it is suspected that the patients who excrete large amounts of free carnitine into the urine, namely the patients with severe heart failure, have some possibility of carnitine deficiency.

Urinary profile of L-carnitine and its derivatives in starved normal persons and ACTH injected patients with myopathy.

Effect of starvation or ACTH injection on the urinary level and profile of L-carnitine and its derivatives was studied in four healthy adult men or in a normal child and two patients with myopathy, respectively. Mean total L-carnitine level in the control urine sample obtained before starvation was 389 +/- 34 mumol . man . day. The percentage distribution was found to be 46% for free-, 9% for acetyl- and 45% for acyl-L-carnitine. The acyl-L-carnitine fraction contained short-chain (65%) and long-chain acyl-L-carnitine (35%). With 2-day starvation urinary excretion of free-L-carnitine was slightly decreased and, in contrast, that of acetyl-L-carnitine was considerably increased, resulting in a significant increase in urinary total L-carnitine levels. Urinary excretion of acyl-L-carnitine was increased two-folds with starvation, but that of long-chain acyl-L-carnitine was not changed. In a normal child (female, 3.5 yr) and two patients (female, 4.5 yr and male, 23 yr) with myopathy, ACTH injection induced a significant elevation of urinary total L-carnitine levels, being mainly caused by an increased excretion of free-L-carnitine and, in the adult patient, acyl-L-carnitine. Muscle total L-carnitine contents were normal in two children but abnormally low in the adult patient, who had simultaneously very low urinary total L-carnitine level before ACTH injection. Thus, in the adult patient myopathy might be possibly caused in part by carnitine deficiency. Starvation and ACTH-induced changes in urinary level and profile of L-carnitine and its derivatives were discussed in relation to carnitine biosynthesis as well as renal regulation of carnitine clearance.