Testosterone synthesis was inhibited in the testis metabolomics of a depression mouse model.

INTRODUCTION: Depression is a common emotional disorder. Previous studies have suggested that depression is associated with the central nervous system. Recent studies have suggested that reduced testosterone level is the core inducement of depression. Testis is the vital organ for the synthesis of testosterone. How does testis mediate depression is still unknown. OBJECTIVES: We adopted a classical depression model of mouse caused through chronic mild stress (CMS). The metabolomics liquid chromatography-mass spectrometry was adopted to analyse the influence of CMS on testis metabolism. Then we confirmed the possible abnormal metabolism of the testis in depression mice by pathway analysis and molecular biological technique. RESULTS: Compared with control mice, 16 differential metabolites were found in CMS mice by multivariate statistical analysis. In comparison with control mice, CMS mice showed higher levels for campesterol, ribitol, citric acid, platelet activating factor, guanosine, cytosine and xanthine and lower levels for docosahexaenoic acid, hippuric acid, creatine, testosterone, dehydroepiandrosterone, progesterone, l-carnitine, acetyl carnitine and propionyl carnitine. The pathway analysis indicated that these differential metabolites are associated with steroid hormone synthesis, purine metabolism and phenylalanine metabolism. In addition, we also first discovered that testicular morphology in depression mice was damaged and steroid hormone synthetases (including steroidogenic acute regulatory protein and P450 cholesterol side chain cleavage) were inhibited. CONCLUSION: These findings may be helpful to parse molecular mechanisms of pathophysiology of depression. It also pointed out the direction to search for potential therapy schedules for male depression and provide novel insights into exploring the pathogenesis of male depression.

Analysis of Choline, Carnitine, Acetylcarnitine and Acetylcholine in Animal Feeds, Blood and Urine Using Ion Chromatography Coupled with Tandem Mass Spectrometry.

A simple and sensitive method was proposed for choline, carnitine, acetylcarnitine (ACa) and acetylcholine (ACh) separation in feed, blood and urine of animals by using ion chromatography (IC) and detection by tandem mass spectrometry (MS/MS). Analytes were extracted using a mixture of acetonitrile and water, purified by C18 solid-phase extraction columns, separated via IC with an IonPac SCS-1 column and detected by an MS/MS detector by using isotopic internal standards for quantification. The effects of different chromatographic parameters on the separation were also investigated. Under optimal conditions, the recovery was >90%, with the relative standard deviations of <15%. The proposed method was highly reliable for the simultaneous determination of choline, carnitine, ACa and ACh in feed, blood and urine.

The association between newborn screening analytes as measured on a second screen and childhood autism in a Texas Medicaid population.

Autism (or autism spectrum disorder [ASD]) is an often disabling childhood neurologic condition of mostly unknown cause. We previously explored whether there was an association of ASD with any analyte measured in the first newborn screening blood test. Here we explore the second screen. Our matched case-control study examined data on 3-5 year-old patients with any ASD diagnosis in the Texas Medicaid system in 2010-2012. Subjects were linked to their 2007-2009 newborn screening blood test data, which included values for 36 analytes or analyte ratios. Data were available for 3,005 cases and 6,212 controls. The most compelling associations were evident for fatty acid oxidation analytes octanoylcarnitine (C8) and octanoylcarnitine/acetylcarnitine (C8/C2). Their adjusted odds ratios comparing 10th versus first analyte deciles were between 1.42 and 1.54 in total births, term births, and males. C8 was consistent with first screen results. Adipylcarnitine (C6DC), an organic acid analyte, showed opposite results in the two screens. Several other analytes exhibiting significant associations in the first screen did not in the second. Our results provide evidence that abnormal newborn blood levels of some carnitines may be associated with risk of later ASD, possibly related to their involvement with mitochondrial function in the developing brain.

Spatial Metabolomics of the Human Kidney using MALDI Trapped Ion Mobility Imaging Mass Spectrometry.

Low molecular weight metabolites are essential for defining the molecular phenotypes of cells. However, spatial metabolomics tools often lack the sensitivity, specify, and spatial resolution to provide comprehensive descriptions of these species in tissue. MALDI imaging mass spectrometry (IMS) of low molecular weight ions is particularly challenging as MALDI matrix clusters are often nominally isobaric with multiple metabolite ions, requiring high resolving power instrumentation or derivatization to circumvent this issue. An alternative to this is to perform ion mobility separation before ion detection, enabling the visualization of metabolites without the interference of matrix ions. Additional difficulties surrounding low weight metabolite visualization include high resolution imaging, while maintaining sufficient ion numbers for broad and representative analysis of the tissue chemical complement. Here, we use MALDI timsTOF IMS to image low molecular weight metabolites at higher spatial resolution than most metabolite MALDI IMS experiments (20 µm) while maintaining broad coverage within the human kidney. We demonstrate that trapped ion mobility spectrometry (TIMS) can resolve matrix peaks from metabolite signal and separate both isobaric and isomeric metabolites with different distributions within the kidney. The added ion mobility data dimension dramatically increased the peak capacity for spatial metabolomics experiments. Through this improved sensitivity, we have found >40 low molecular weight metabolites in human kidney tissue, such as argininic acid, acetylcarnitine, and choline that localize to the cortex, medulla, and renal pelvis, respectively. Future work will involve further exploring metabolomic profiles of human kidneys as a function of age, sex, and race.

Prospective randomized multicentre comparison on sibling oocytes comparing G-Series media system with antioxidants versus standard G-Series media system.

RESEARCH QUESTION: Does the inclusion of three antioxidants (A3), acetyl-l-carnitine (ALC), N-acetyl-l-cysteine (NAC) and alpha-lipoic acid (ALA) improve human embryo development and pregnancy potential? DESIGN: Prospective randomized multicentre comparison of sibling oocytes. A total of 1563 metaphase II oocytes from 133 patients in two IVF centres. Day 3 embryo and day 5/6 blastocyst quality were assessed. Good embryo quality on day 3 was defined as 8 to 10 cells with even cells and low fragmentation; good quality blastocysts as 3BB or greater. Clinical outcome was assessed on transfers of fresh or vitrified-warmed blastocyst on day 5. RESULTS: Of the two-pronuclei, 40.7% (G-Series) and 50.2% (G-Series with A3 group) resulted in good quality embryos on day 3 (P < 0.05). The implantation rate by fetal sac was 39.2% and 50.6%, and by fetal heartbeat was 37.8% and 47.1% for the G-Series and G-Series with A3 group, respectively. When stratified by female patient age, patients 35-40 years had an implantation rate by fetal sac and heart of 23.5% in the G-Series compared with 57.5% (P < 0.05) and 50.0% (P < 0.05) in the A3 group. The ongoing pregnancies in patients 35-40 years were significantly higher in the A3 group (50%) compared with the control (25.8%) (P < 0.05). CONCLUSIONS: The presence of antioxidants during IVF and embryo culture for patients 35-40 years resulted in a significant increase in implantation and pregnancy rate. Supplementation of antioxidants to IVF and culture media may therefore improve the viability of human embryos in assisted reproductive technologies, plausibly through the reduction of oxidative stress.

Association between 4-year all-cause mortality and carnitine profile in maintenance hemodialysis patients.

BACKGROUND: Patients on dialysis are in a chronic carnitine-deficient state. This condition may be associated with abnormalities of the fatty acid and organic acid metabolisms. Carnitine is required for ß-oxidation of the long-chain fatty acids; therefore, carnitine deficiency decreases the efficiency of ATP synthesis and may incur death. However, the details of this association remain unknown. We examined the relationship between ß-oxidation efficiency represented by the carnitine profile and 4-year all-cause mortality in hemodialysis patients. METHODS: The carnitine profiles of 122 hemodialysis patients were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The associations between the 4-year all-cause mortality and carnitine profile as well as the clinical backgrounds of the patients were investigated. A survival analysis was conducted by the Kaplan-Meier survival method and multivariable Cox proportional hazard analysis. The bootstrap method was performed to confirm the stability and robustness of our model. RESULTS: Of the 122 subjects analyzed, 111 were selected and 24 died during the observation period. Stepwise multivariable Cox regression demonstrated that diabetes state [Hazard ratio (95% confidence interval), 4.981 (2.107-11.77)], age [HR (95% CI), 1.052 (1.014-1.091)], and the acetylcarnitine/(palmitoylcarnitine+octadecenoylcarnitine) [C2/(C16+C18:1)] ratio [HR (95% CI), 0.937 (0.904-0.971)] were independent significant factors of 4-year all-cause mortality. The bootstrap method confirmed the significance of these three factors. CONCLUSION: The 4-year all-cause mortality negatively correlated with the C2/(C16+C18:1) ratio. Improvement of the impaired ß-oxidation state after L-carnitine administration may ameliorate prognosis.

Rapid Detection of Small Molecule Metabolites in Serum of Hepatocellular Carcinoma Patients Using Ultrafast Liquid Chromatography-Ion Trap-Time of Flight Tandem Mass Spectrometry.

A method was developed for analyzing broad spectrum small molecule metabolites in the serum of hepatocellular carcinoma (HCC) patients based on ultrafast liquid chromatography-ion trap-time of flight tandem mass spectrometry (UFLC-IT-TOF MS). Serum samples were collected from 80 HCC patients and healthy persons. After pretreatment process for protein precipitation, the supernatant was analyzed with the UFLC-IT-TOF MS to obtain information on the metabonomics of small molecules. The eight compounds of glycocholic acid, choline glycerophosphate, acetyl-L-phenylalanine, oleamide, tetradecanamide, acetylcarnitine, lysolecithin and glycochenodeoxycholic acid in the HCC group were identified with significant differences from those in the health group (P <0.01). By using multidimensional analysis of variation coefficient and principal component analysis for the repeatability and 48 h stability, the method was demonstrated to have good repeatability, excellent precision, and high stability, which can satisfy the metabonomics research requirement. The high throughput and practical usability of the method further shows perspective for metabonomic analysis of large-batch serum samples.

[Application of Metabolomics to Multiple Chemical Sensitivity Research].

Multiple chemical sensitivity (MCS) is an acquired chronic disorder characterized by nonspecific symptoms in multiple organ systems associated with exposure to low-level chemicals. Diagnosis of MCS can be difficult because of the inability to assess the causal relationship between exposure and symptoms. No standardized objective measures for the identification of MCS and no precise definition of this disorder have been established. Recent technological advances in mass spectrometry have significantly improved our capacity to obtain more data from each biological sample. Metabolomics comprises the methods and techniques that are used to determine the small-level molecules in biofluids and tissues. The metabolomic profile-the metabolome-has multiple applications in many biological sciences, including the development of new diagnostic tools for medicine. We performed metabolomics to detect the difference between 9 patients with MCS and 9 controls. We identified 183 substances whose levels were beyond the normal detection limit. The most prominent differences included significant increases in the levels of both hexanoic acid and pelargonic acid, and also a significant decrease in the level of acetylcarnitine in patients with MCS. In conclusion, using metabolomics analysis, we uncovered a hitherto unrecognized alteration in the levels of metabolites in MCS. These changes may have important biological implications and may have a significant potential for use as biomarkers.

Triptolide disrupts fatty acids and peroxisome proliferator-activated receptor (PPAR) levels in male mice testes followed by testicular injury: A GC-MS based metabolomics study.

Triptolide is the major active ingredient of Tripterygium Glycosides (TG), a traditional Chinese medicine with very potent anti-inflammatory effects and has been used in China for the treatment of rheumatoid arthritis and many other inflammatory diseases. However, clinical application of triptolide is restricted due to its multiple side effects, especially male infertility. The mechanism of triptolide on reproduction toxicity remains unclear. In the present study, a GC-MS based metabolomic approach was employed to evaluate the mechanism of triptolide-induced reproductive toxicity as well as identify potential novel biomarkers for the early detection of spermatogenesis dysfunction. In brief, male mice were divided into two groups with or without triptolide intraperitoneal injection at 60 µg/kg/day for 2 weeks and toxic effect of triptolide on testicular tissues were examined by biochemical indicator analysis, testis histopathologic analysis, and sperm quantity analysis. Metabolomics technology was then performed to evaluate systematically the endogenous metabolites profiling. Our results demonstrated that triptolide suppressed the marker-enzymes of spermatogenesis and testosterone levels, decreased sperm counts, reduced the gonad index and destroyed the microstructure of testis. Multivariate data analysis revealed that mice with triptolide induced testicular toxicity could be distinctively differentiated from normal animals and 35 and 39 small molecule metabolites were changed significantly in testis and serum, respectively (Fold-changes >1.5, P<0.05), in triptolide-treated mice. Abnormal level of fatty acids, an important energy source of sertoli cells with critical role in maintaining normal function of the testis tissue, was observed in triptolide-treated mice. Additionally, the protein expressions of PPAR, a transcription factor known to play a pivotal role in lipid and energy metabolism was significantly decreased in the testis tissue of triptolide-treated mice. In summary, our study represents the first comprehensive GC-MS based metabolomics analysis of triptolide-induced testicular toxicity. We reported for the first time that exposure to triptolide led to marked changes of a panel of endogenous metabolites in both testis and serum. The impairment of spermatogenesis may be caused by abnormal lipid and energy metabolism in testis via the down-regulation of PPARs mediated by triptolide. The presence of research suggested that PPARs and its related fatty acids metabolism may serve as potential targets for intervention or treatment of male infertility induced by triptolide.

Metabolic imaging of hyperpolarized [1-(13) C]acetate and [1-(13) C]acetylcarnitine - investigation of the influence of dobutamine induced stress.

PURPOSE: The metabolism of acetate in the heart resembles fatty acid metabolism, which is altered in several diseases like ischemia, diabetes mellitus, and heart failure. A signal-to-noise ratio (SNR) optimized imaging framework for in vivo measurements of hyperpolarized [1-(13) C]acetate and its metabolic product [1-(13) C]acetylcarnitine (ALCAR) in rats at 3 Tesla (T) is presented in this work. METHODS: A spectrospatial pulse was combined with IDEAL encoding to acquire well separated metabolic maps. The influence of dobutamine induced stress onto this metabolic system was investigated in spectra and in an imaging study. RESULTS: An increase of the ALCAR to acetate ratio with dobutamine induced stress was shown in slice selective spectra containing the rat hearts and skeletal muscles. Metabolic maps of acetate and ALCAR were acquired with an acceptable SNR. Quantification of the apparent conversion rate showed stable results in the heart in a time-window of 30 s. The effect of dobutamine on the signal intensities was shown to originate mainly from skeletal than cardiac muscles. CONCLUSION: The acetate activation was mapped with hyperpolarized [1-(13) C]acetate in a clinical 3T system. Quantitative measurement of the activity was possible in the heart, indicating that dobutamine induced stress does not improve the ALCAR SNR in the heart.

An high-performance liquid chromatographic method for the simultaneous analysis of acetylcarnitine taurinate, carnosine, asparagine and potassium aspartate and for the analysis of phosphoserine in alimentary supplements.

A RP-HPLC method with pre-column derivatization was developed and validated for the simultaneous quantification of carnosine (Carn), acetylcarnitine taurinate (AC-Tau), asparagine (Asn), potassium aspartate (Asp) and for the determination of phosphoserine (p-Ser) in new and commercial alimentary supplements. The effect of complex matrices was evaluated by the study of the amino acid derivatization reaction with 2,4-dinitrofluorobenzene (DNFB) both in standard and placebo solutions. The reaction was carried out for 20 min at 70 °C in alkaline medium (pH10) for p-Ser analysis, whereas for 60 min in the case of Carn, AC-Tau, Asn and Asp analysis. The adducts have been separated on a Discovery RP Amide C16 (250 mm×4.6mm, i.d.) column using a mobile phase consisting of acetonitrile (ACN) and triethylammonium (TEA) phosphate buffer (pH 3, 0.05 M) under gradient elution conditions at a flow-rate of 0.8 mL/min. Detection was set at λ=360 nm. The validation parameters such as linearity, sensitivity, accuracy, precision and specificity were found to be highly satisfactory. Linear responses were observed by placebo solutions (determination coefficient ≤0.9996). Intra-day precision (relative standard deviation, RSD) was ≤1.06% for corrected peak area and ≤0.99% for retention times (tR) without significant differences between intra- and inter-day data. Recovery studies showed good results for all examined compounds (from 97.7% to 101.5%) with RSD ranging from 0.5% to 1.3%). The high stability of derivatized compound solutions at room temperature means an undoubted advantage of the method allowing the simultaneous preparation of a large number of samples and consecutive chromatographic analyses by the use of an autosampler. The developed method can be considered suitable for the quality control of new and commercial products.

Identifying tissue-specific signal variation in MALDI mass spectrometric imaging by use of an internal standard.

Generating analyte-specific distribution maps of compounds in a tissue sample by matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) has become a useful tool in numerous areas across the biological sciences. Direct analysis of the tissue sample provides MS images of an analyte's distribution with minimal sample pretreatment. The technique, however, suffers from the inability to account for tissue-specific variations in ion signal. The variation in the makeup of different tissue types can result in significant differences in analyte extraction, cocrystallization, and ionization across a sample. In this study, a deuterated internal standard was used to account for these signal variations. Initial experiments were performed using pure standards and optimal cutting temperature compound (OCT) to generate known areas of ion suppression. By monitoring the analyte-to-internal-standard ratio, differences in ion signal were taken into account, resulting in images that better represented the analyte concentration. These experiments were then replicated using multiple tissue types in which the analyte's MS signal was monitored. In certain tissues, including liver and kidney, the analyte signal was attenuated by up to 90%; however, when the analyte-to-internal-standard ratio was monitored, these differences were taken into account. These experiments further exemplify the need for an internal standard in the MSI workflow.

Quantitative tandem mass spectrometric imaging of endogenous acetyl-L-carnitine from piglet brain tissue using an internal standard.

Matrix-assisted laser desorption/ionization (MALDI) based mass spectrometric imaging (MSI) is increasingly being used as an analytical tool to evaluate the molecular makeup of tissue samples. From the direct analysis of a tissue section, the physical integrity of sample is preserved; thus, spatial information of a compound's distribution may be determined. One limitation of the technique, however, has been the inability to determine the absolute concentration from a tissue sample. Here we report the development of a quantitative MSI technique in which the distribution of acetyl-L-carnitine (AC) in a piglet brain sample is quantified with MALDI MSI. An isotopically labeled internal standard was applied uniformly beneath the tissue section, and wide-isolation tandem mass spectrometry was performed. Normalizing the analyte ion signal by the internal standard ion signal resulted in significant improvements in MS images, signal reproducibility, and calibration curve linearity. From the improved MS images, the concentration of AC was determined and plotted producing a concentration-scaled image of the distribution of AC in the piglet brain section.

Systematic analysis of adaptations in aerobic capacity and submaximal energy metabolism provides a unique insight into determinants of human aerobic performance.

It has not been established which physiological processes contribute to endurance training-related changes (Delta) in aerobic performance. For example, the relationship between intramuscular metabolic responses at the intensity used during training and improved human functional capacity has not been examined in a longitudinal study. In the present study we hypothesized that improvements in aerobic capacity (Vo(2max)) and metabolic control would combine equally to explain enhanced aerobic performance. Twenty-four sedentary males (24 +/- 2 yr; 1.81 +/- 0.08 m; 76.6 +/- 11.3 kg) undertook supervised cycling training (45 min at 70% of pretraining Vo(2max)) 4 times/wk for 6 wk. Performance was determined using a 15-min cycling time trial, and muscle biopsies were taken before and after a 10-min cycle at 70% of pretraining Vo(2max) to quantify substrate metabolism. Substantial interindividual variability in training-induced adaptations was observed for most parameters, yet "low responders" for DeltaVo(2max) were not consistently low responders for other variables. While Vo(2max) and time trial performance were related at baseline (r(2) = 0.80, P < 0.001), the change in Vo(2max) was completely unrelated to the change in aerobic performance. The maximal parameters DeltaVe(max) and DeltaVeq(max) (DeltaVe/Vo(2max)) accounted for 64% of the variance in DeltaVo(2max) (P < 0.001), whereas Deltaperformance was related to changes in the submaximal parameters Veq(submax) (r(2) = 0.33; P < 0.01), muscle Deltalactate (r(2) = 0.32; P < 0.01), and Deltaacetyl-carnitine (r(2) = 0.29; P < 0.05). This study demonstrates that improvements in high-intensity aerobic performance in humans are not related to altered maximal oxygen transport capacity. Altered muscle metabolism may provide the link between training stimulus and improved performance, but metabolic parameters do not change in a manner that relates to aerobic capacity changes.

Muscle metabolites: functional MR spectroscopy during exercise imposed by tetanic electrical nerve stimulation.

Permission from the ethics committee and informed consent were obtained. The purpose of this study was to prospectively evaluate a method developed for the noninvasive assessment of muscle metabolites during exercise. Hydrogen 1 magnetic resonance (MR) spectroscopy peaks were measured during tetanic isometric muscle contraction imposed by supramaximal repetitive nerve stimulation. The kinetics of creatine-phosphocreatine and acetylcarnitine signal changes (P < .001) could be assessed continuously before, during, and after exercise. The control peak (trimethylammonium compounds), which served as an internal reference, did not change. This technique-that is, functional MR spectroscopy-opens the possibility for noninvasive diagnostic muscle metabolite testing in a clinical setting.

Compensatory responses to pyruvate carboxylase suppression in islet beta-cells. Preservation of glucose-stimulated insulin secretion.

We have previously reported that glucose-stimulated insulin secretion (GSIS) is tightly correlated with pyruvate carboxylase (PC)-catalyzed anaplerotic flux into the tricarboxylic acid cycle and stimulation of pyruvate cycling activity. To further evaluate the role of PC in beta-cell function, we constructed a recombinant adenovirus containing a small interfering RNA (siRNA) specific to PC (Ad-siPC). Ad-siPC reduced PC mRNA levels by 83 and 64% and PC protein by 56 and 35% in INS-1-derived 832/13 cells and primary rat islets, respectively. Surprisingly, this manipulation did not impair GSIS in rat islets. In Ad-siPC-treated 832/13 cells, GSIS was slightly increased, whereas glycolytic rate and glucose oxidation were unaffected. Flux through PC at high glucose was decreased by only 20%, suggesting an increase in PC-specific activity. Acetyl carnitine, a surrogate for acetyl-CoA, an allosteric activator of PC, was increased by 36% in Ad-siPC-treated cells, suggesting a mechanism by which PC enzymatic activity is maintained with suppressed PC protein levels. In addition, the NADPH:NADP ratio, a proposed coupling factor for GSIS, was unaffected in Ad-siPC-treated cells. We conclude that beta-cells activate compensatory mechanisms in response to suppression of PC expression that prevent impairment of anaplerosis, pyruvate cycling, NAPDH production, and GSIS.

Monitoring of ketogenic diet for carnitine metabolites by subcutaneous microdialysis.

The ketogenic diet (KD) provides ketones from the degradation of free fatty acids for energy metabolism. It is a therapeutic option for pharmacoresistant epilepsies. Carnitine is the carrier molecule that transports fatty acids across the mitochondrial membrane for degradation into ketones. The integrity of this transport system is a prerequisite for an adequate ketogenic response. For monitoring of tissue metabolism with KD, we used the sampling method of s.c. microdialysis (MD), which permits minimally invasive, frequent, and extensive metabolic monitoring independent of blood tests. By using this new method, we monitored changes in carnitine metabolism induced by KD, particularly in free carnitine (C0), acetylcarnitine (C2), and hydroxybutyrylcarnitine (C4OH). Correlation of microdialysate and tissue concentrations for carnitines in vitro was about 85%. Carnitine metabolism was monitored in seven children started on a KD for pharmacoresistant epilepsy after a conventional initial fasting period. Detected metabolic changes consisted of a slight decrease in s.c. C0 and a marked increase in C2/CO and C4OH/CO levels. The levels of s.c. C4OH strongly correlate with beta-hydroxybutyrate (beta-OHB) levels in plasma providing an additional parameter for the carnitine reserve of the body and reflect an optimal ketogenic energy supply. Subcutaneous MD allows close and extensive monitoring of metabolism with a KD.

Enantiomeric purity determination of acetyl-L-carnitine by NMR with chiral lanthanide shift reagents.

Enantiomer signal separation of acetyl-carnitine chloride was obtained on a 500 MHz Nuclear Magnetic Resonance (1H NMR) analysis by fast diastereomeric interaction with chiral shift reagents such as chiral lanthanide-camphorato or chiral samarium-pdta shift reagents. Effects of the kinds of chiral shift reagents and the molar ratio of chiral shift reagent to acetyl-carnitine chloride on enantiomer signal separation were investigated and evaluated. Optimization of the experimental conditions provided two significant split signals for the enantiomers, leading to the successful quantitative analysis. Distinguishment of 0.5% of the minor enantiomer (D-form) in acetyl-L-carnitine chloride was found to be possible by 1H NMR with tris[3-(heptafluoropropylhydroxymethylene)-D-camphorato] and praseodymium derivative, (Pr[hfc]3), as chiral shift reagents.

Strategy for the isolation, derivatization, chromatographic separation, and detection of carnitine and acylcarnitines.

A strategy for detection of carnitine and acylcarnitines is introduced. This versatile system has four components: (1) isolation by protein precipitation/desalting and cation-exchange solid-phase extraction, (2) derivatization of carnitine and acylcarnitines with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase chromatography using a single non-end-capped C8 column, and (4) detection of carnitine and acylcarnitine pentafluorophenacyl esters using an ion trap mass spectrometer. Recovery of carnitine and acylcarnitines from the isolation procedure is 77-85%. Derivatization is rapid and complete with no evidence of acylcarnitine hydrolysis. Sequential ion-exchange/reversed-phase HPLC results in separation of reagent byproducts from derivatized carnitine and acylcarnitines, followed by reversed-phase separation of carnitine and acylcarnitine pentafluorophenacyl esters. Detection by MS/MS is highly selective, with carnitine pentafluorophenacyl ester yielding a strong product ion at m/z 311 and acylcarnitine pentafluorophenacyl ester fragmentation yielding two product ions: (1) loss of m/z 59 and (2) generation of an ion at m/z 293. To demonstrate this analytical strategy, phosphate buffered serum albumin was spiked with carnitine and 15 acylcarnitines and analyzed using the described protein precipitation/desalting and cation-exchange solid-phase extraction isolation, derivatization with pentafluorophenacyl trifluoromethanesulfonate, chromatography using the sequential ion-exchange/reversed-phase chromatography HPLC system, and detection by MS and MS/MS. Successful application of this strategy to the quantification of carnitine and acetylcarnitine in rat liver is shown.

Effect of L-carnitine administration on the seminal characteristics of oligoasthenospermic stallions.

The effect of orally administered l-carnitine on the quality of semen obtained from stallions with different semen qualities was investigated. Four stallions with proven fertility (high motility group, HM) and with normal seminal characteristics (>50% progressive motility and > 80 x 10(6) spermatozoa/ml), and four questionable breeders (low motility group, LM) with <50% of sperm progressive motility and < 80 x 10(6) spermatozoa/ml, received p.o. 20 g of l-carnitine for 60 days. Blood and semen samples were collected before treatment (T0) and after 30 (T1) and 60 days (T2). Semen evaluation were performed on five consecutive daily ejaculates (n = 120 ejaculates) and conventional semen analysis was carried out on each ejaculate, both at collection and after refrigeration for 24, 48, and 72 h. Furthermore l-carnitine, acetylcarnitine, pyruvate, and lactate concentrations, and carnitine acetyltransferase activity (CAT) were determined both in raw semen and seminal plasma. There were an increase in progressive motile spermatozoa only in the LM group (26.8 +/- 12.9, 39.1 +/- 15.5, and 48.8 +/- 8.6 for T0, T1, and T2, respectively). Free seminal plasma carnitine concentration was higher in the LM group compared to the HM one. Both pyruvate and lactate were higher in the LM group. Raw semen and seminal plasma carnitine and acetylcarnitine levels correlate positively with both sperm concentration and progressive motility; moreover, acetylcarnitine content was positively correlated with total motile morphologically normal spermatozoa. In conclusion, oral administration of l-carnitine to stallions with questionable seminal characteristics may improve spermatozoa kinetics and morphological characteristics; whereas, it seem to be ineffective in normospermic animals.