Metabolomic analysis of human plasma reveals that arginine is depleted in knee osteoarthritis patients.

OBJECTIVE: To identify novel biomarker(s) for knee osteoarthritis (OA) using a metabolomics approach. METHOD: We utilized a two-stage case-control study design. Plasma samples were collected from knee OA patients and healthy controls after 8-h fasting and metabolically profiled using a targeted metabolomics assay kit. Linear regression was used to identify novel metabolic markers for OA. Receiver operating characteristic (ROC) analysis was used to examine diagnostic values. Gene expression analysis was performed on human cartilage to explore the potential mechanism for the novel OA marker(s). RESULTS: Sixty-four knee OA patients and 45 controls were included in the discovery stage and 72 knee OA patients and 76 age and sex matched controls were included in the validation stage. We identified and confirmed six metabolites that were significantly associated with knee OA, of which arginine was the most significant metabolite (P < 3.5 × 10(-13)) with knee OA patients having on average 69 µM lower than that in controls. ROC analysis showed that arginine had the greatest diagnostic value with area under the curve (AUC) of 0.984. The optimal cutoff of arginine concentration was 57 µM with 98.3% sensitivity and 89% specificity. The depletion of arginine in OA patients was most likely due to the over activity of arginine to ornithine pathway, leading to imbalance between cartilage repair and degradation. CONCLUSION: Arginine is significantly depleted in refractory knee OA patients. Further studies within a longitudinal setting are required to examine whether arginine can predict early OA changes.

NMR and isotope ratio mass spectrometry studies of in vivo uptake and metabolism of polyunsaturates by the developing rat brain.

This article describes the application of in vivo 13C-nuclear magnetic resonance (NMR) spectroscopy and gas chromatography (GC)-combustion-isotope ratio mass spectrometry to the study of brain uptake and metabolism of polyunsaturated fatty acids in the suckling rat model. NMR spectroscopy is uniquely suited to the non-invasive detection of nonradioactive metabolites in living animals. We applied this approach to the noninvasive detection of 13C-arachidonate in brain and liver of living suckling rats but found that technical limitations in our model, mainly poor signal-to-noise, largely prevent useful results at this time. However, in a tracer study using simultaneous doses of 13C-gamma-linolenate and 13C-arachidonate, 13C-NMR of tissue lipid extracts quantitatively demonstrated a 10-fold greater (liver) or 17-fold greater (brain) accumulation of pre-formed vs newly synthesized arachidonate. GC-combustion-isotope ratio mass spectrometry was used to trace the utilization of [U-13C]-alpha-linolenate into three products in the brain: docosahexaenoate, cholesterol, and palmitate. The rationale was that although alpha-linolenate is used in de novo lipogenesis, the quantitative importance of this pathway is unknown. Our results in the suckling rat show that 2-13% of carbon from [U-13C]-alpha-linolenate appearing in brain lipids is in docosahexaenoate while the rest is in brain lipids synthesized de novo. Overall, these results indicate that the suckling rat brain prefers pre-formed to newly synthesized arachidonate and that alpha-linolenate is readily utilized in brain lipid synthesis. These methods are suited to comparative studies of the metabolism of polyunsaturates and they support previous observations that the metabolism of some polyunsaturates such as alpha-linolenate extends well beyond the traditional desaturation-chain elongation pathway.

Lipid and fatty acid profiles in rats consuming different high-fat ketogenic diets.

High-fat ketogenic diets are used to treat intractable seizures in children, but little is known of the mechanism by which these diets work or whether fats rich in n-3 polyunsaturates might be beneficial. Tissue lipid and fatty acid profiles were determined in rats consuming very high fat (80 weight%), low-carbohydrate ketogenic diets containing either medium-chain triglyceride, flaxseed oil, butter, or an equal combination of these three fat sources. Ketogenic diets containing butter markedly raised liver triglyceride but had no effect on plasma cholesterol. Unlike the other fats, flaxseed oil in the ketogenic diet did not raise brain cholesterol. Brain total and free fatty acid profiles remained similar in all groups, but there was an increase in the proportion of arachidonate in brain total lipids in the medium-chain triglyceride group, while the two groups consuming flaxseed oil had significantly lower arachidonate in brain, liver, and plasma. The very high dietary intake of alpha-linolenate in the flaxseed group did not change docosahexaenoate levels in the brain. Our previous report based on these diets showed that although ketosis is higher in rats consuming a ketogenic diet based on medium-chain triglyceride oil, seizure resistance in the pentylenetetrazol model is not clearly related to the degree of ketosis achieved. In combination with our present data from the same seizure study, it appears that ketogenic diets with widely differing effects on tissue lipids and fatty acid profiles can confer a similar amount of seizure protection.

Dietary fat, ketosis, and seizure resistance in rats on the ketogenic diet.

PURPOSE: Fat is the major component of the ketogenic diet (KD), yet no studies have examined whether the type of fat used in the diet can be optimized to provide additional benefits. The purpose of the present experiments was to compare the efficiency of different fats in inducing ketosis and affording seizure resistance. METHODS: The effects of KDs that incorporate lard, butter, medium-chain triglycerides (MCT), or flaxseed oil or a mixture of the latter three fats were examined in rats fed KD for up to 98 days. The maximal electroshock (MES) or pentylenetetrazole (PTZ) threshold tests were used to assess seizure susceptibility in two separate experiments. RESULTS: The rank order of induced ketosis was MCT > mixture > or = flaxseed oil > or = lard = butter > or = control. MES failed to reveal anticonvulsant effects, but the PTZ test indicated that up to 50% of rats fed the KD were seizure protected (p < 0.05). The measures of seizure protection, seizure incidence and score, did not correlate, however, with the level of ketosis in the range of 0. 7-5.2 mmol/L for beta-hydroxybutyrate. In the long-term study, flaxseed oil KD maintained stable ketosis throughout 98 days, whereas ketones declined with lard and butter KD to the control level. CONCLUSIONS: Seizure protection with the versions of the KD did not improve with the higher level of ketosis. The focus of the KD improvement, therefore, is not the achievement of higher ketosis per se but rather designing a diet that provides steady ketosis, exploits advantages of certain fats for neurological development or seizure protection via a nonketogenic mechanism, and is nutritionally balanced.

The MCT ketogenic diet: effects on animal seizure models.

Male Wistar rat pups were weaned at 20 days of age and placed on either a control diet or a ketogenic diet containing medium-chain triglyceride (MCT) oil. After 10 days on the diets, they were subjected to one of four seizure tests-maximal electric shock, threshold electroconvulsive shock, threshold pentylenetetrazol, or maximal pentylenetetrazol. After testing, subjects were sacrificed and blood samples were analyzed for beta-hydroxybutyrate concentration. It was found that the MCT diet produced blood levels of beta-hydroxybutyrate that were comparable to or higher than those commonly reported in clinical studies. However, no anticonvulsant effects were seen in any of the seizure tests. In fact, the tests involving maximal seizures actually showed proconvulsant effects. It appears that clinical levels of ketones may be present in the bloodstream without suppressing seizures.

Carbon recycling into de novo lipogenesis is a major pathway in neonatal metabolism of linoleate and alpha-linolenate.

Recent reports indicate that recycling of the beta-oxidized carbon skeleton of linoleate and alpha-linolenate into newly synthesized cholesterol and fatty acids in the brain is quantitatively significant in both suckling rats and pre- and postnatally in rhesus monkeys. The recycling appears to occur via ketones which are not only readily produced from these 18 carbon polyunsaturates but are also the main lipogenic precursors for the developing mammalian brain. Since the neonatal rat brain appears not to acquire cholesterol or long chain saturated or monounsaturated fatty acids from the circulation, ketones and ketogenic precursors seem to be crucial for normal brain synthesis of these lipids. Cholesterol is plentiful in brain membranes and it has also been discovered to be the essential lipid adduct of the 'hedgehog' family of proteins, the appropriate expression of which determines normal embryonic tissue patterning and neurological development. Insufficient cholesterol or inappropriate expression of 'sonic hedgehog' has major adverse neurodevelopmental consequences typified in humans by Smith-Lemli-Optiz syndrome. Hence, we propose that the importance of alpha-linolenate and linoleate for normal neural development arises not only from being precursors to longer chain polyunsaturates incorporated into neuronal membranes but, perhaps equally importantly, by being ketogenic precursors needed for in situ brain lipid synthesis.

Uptake of 13C-tracer arachidonate and gamma-linolenate by the brain and liver of the suckling rat observed using 13C-NMR.

Arachidonic acid (AA; 20:4n-6) is one of the principal components of the phosphoglycerides in neural cell membranes. During the critical period of postnatal development in mammals, AA is supplied preformed, directly from the milk or derived from precursor fatty acids such as gamma-linolenic acid (GLA; 18:3n-6). In this study, 13C-NMR spectroscopy was applied to investigate the incorporation of [1-(13)C]AA and [3-(13)C]GLA into liver and brain lipids of 7-15-day-old rats. The main objective was to establish the importance of dietary GLA for tissue AA accretion relative to the contribution from preformed dietary AA. [1-(13)C]AA and [3-(13)C]GLA were injected into the stomach of 7-day-old rats as a mixture. 13C-NMR spectroscopy of lipid extracts revealed incorporation of [1-(13)C]AA and [5-(13)C]AA (the latter derived from metabolism of the injected [3-(13)C]GLA) into phosphoglycerides and triacylglycerols. Preformed AA was 10 (liver)-17 (brain) times more efficient in contributing to tissue AA than AA derived from precursor GLA. In separate experiments, NMR spectroscopy was used to assess uptake of [1-(13)C]AA directly in living rats and intact organs. Results showed that intact liver and brain contain an appreciable amount of NMR-detectable lipids. The in vivo/in vitro information obtained from organs provided details on the mobility and turnover of tissue lipids.

[3-13C] gamma-linolenic acid: a new probe for 13C nuclear magnetic resonance studies of arachidonic acid synthesis in the suckling rat.

Our objective was to develop a suitable probe to study metabolism of polyunsaturated fatty acids by 13C nuclear magnetic resonance (NMR) in the suckling rat pup. [3-13C] gamma-Linolenic acid was chemically synthesized, and a 20 mg (Experiment 1) or 5 mg (Experiment 2) dose was injected into the stomachs of 6-10-day-old suckling rat pups that were then killed over a 192 h (8 d) time course. 13C NMR showed that 13C in gamma-linolenate peaked in liver total lipids by 12-h post-dosing and that [5-13C]-arachidonic acid peaked in both brain and liver total lipids 48-96 h post-dosing. 13C enrichment in brain gamma-linolenic acid was not detected by NMR, but gas chromatography-combustion-isotope ratio mass spectrometry showed that its mass enrichment in brain phospholipids at 48-96 h post-dosing was 1-2% of that in brain arachidonic acid. 13C was present in liver and brain cholesterol and in perchloric acid-extractable water-soluble metabolites in the brain, liver and carcass. We conclude that low but measurable amounts of exogenous gamma-linolenic acid do access the suckling rat brain in vivo. The slow time course of [5-13C] arachidonic acid appearance in the brain suggests most of it was probably transported there after synthesis elsewhere, probably in the liver. Some carbon from gamma-linolenic acid is also incorporated into lipid products other than n-6 long-chain polyunsaturated fatty acids.

13C NMR spectroscopy and gas chromatograph--combustion--isotope ratio mass spectrometry: complementary applications in monitoring the metabolism of 13C-labelled polyunsaturated fatty acids.

This review aims to describe some recent and novel applications of stable isotope tracer technology to study the metabolism of 13C-polyunsaturated fatty acids. Stable isotope methodology has existed for several decades, and in that sense, it is not novel per se. However, in the past 10 years, developments in the fields of isotope ratio mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and biological approaches to labelling tracer compounds with stable isotopes have provided new opportunities in fatty acid research. Sample preparation for isotope ratio mass spectrometry has been converted from a manual combustion method to an on-line or continuous flow method, making it much more versatile and easier to use. Similarly, 13C-NMR spectroscopy has recently developed as a remarkably useful method for monitoring metabolic steps and pathways both in vivo and at the molecular level. Coincident with these improvements in instrumentation, the commercial availability of numerous uniformly 13C-labelled compounds has made these studies more affordable. The application of some of these developments to questions in the field of polyunsaturated fatty acid metabolism is described.

In vivo 13C nuclear magnetic resonance: applications and current limitations for noninvasive assessment of fatty acid status.

As a noninvasive method, in vivo 13C nuclear magnetic resonance has potentially important applications in understanding the metabolism of long chain fatty acids in organs of living humans. At present, this methodology is most advanced for research on glucose utilization. However, the main 13C signals visible in vivo are from fatty acids in adipose tissue and the olefinic signals can be used to noninvasively estimate adipose tissue content and relative dietary intake of polyunsaturates and monounsaturates. The low natural abundance of 13C improves the utility of this isotope for fatty acid tracer studies. Due to excessive signal broadening, uniform 13C-labelling seems to have limited application in in vivo fatty acid studies. Tracer fatty acids with 13C enrichment at a specific carbon position, i.e., [13-13C] gamma-linolenate, appear to be the most useful for in vivo tracer studies. Development of methods permitting resolution of 13C enrichment in structural lipids of lean tissues will be an important breakthrough which may make human tracer studies feasible and worthwhile.

Utilization of carbon from dietary polyunsaturates for brain cholesterol synthesis during early postnatal development in the rat: a 13C NMR study.

Incorporation of 13C from a dietary precursor into cholesterol was studied in neonatal rats. Rats were given uniformly 13C-enriched polyunsaturated fatty acids intragastrically and total lipid extracts of liver and brain were analyzed by 13C-NMR 1, 4, 8, and 15 days later. 13C-enrichment was detected in brain but not in liver cholesterol. Maximal 13C-labeling was observed 4 days after injection of the label. Spectra revealed that 70% of newly incorporated 13C had 13C as an adjacent neighbor, the other 30% had 12C as the neighbor. Double quantum NMR revealed the arrangement in the cholesterol skeleton of the 13C-13C pairs transferred from precursors to cholesterol. Desmosterol, an intermediate of cholesterol synthesis, was identified in the spectra of brain lipids. Comparison of 13C-13C unit arrangements in both cholesterol and desmosterol allowed carbons 26 and 27 of desmosterol to be unambiguously assigned.

[The effect of gamma-hydroxybutyric acid on the reaction rate of phosphate-containing metabolites in the rat brain during ischemia estimated from (31)P-NMR spectroscopic data]. / Vliianie GOMK na tempy snizheniia makroérgicheskikh fosfatov mozga krys vo vremia ishemii po dannym prizhiznennoi (31)P-YaMR-spektroskopii.

Protective effects of gamma-hydroxybutyric acid on bioenergetic reactions were studied in brain of rats with ischemia using 31P-NMR spectroscopy in vivo. Intraperitoneal preadministration of gamma-hydroxybutyric acid at a dose of 400 mg/kg within 30-40 min before ischemia led to a decrease in the ATP pool in ischemic brain tissue, to alteration in the PCr/ATP ratio in the 31P-NMR spectrum, to prevention of Pi concentration increase and to increase in the intracellular acidosis development rate. Possible mechanisms of the gamma-hydroxybutyric acid effects on bioenergetic reactions in nervous tissue during ischemia are discussed.

[Prognostically significant indices of the bioenergetic metabolism of the brain in experimental ischemia (a NMR spectroscopic study)]. / Nekotorye prognosticheski znachimye pokazateli bioénergeticheskogo metabolizma mozga pri ego ishemii v éksperimente (IaMR-spektroskopicheskoe issledovanie).

Initial levels of phosphate brain metabolites were measured using 31P NMR spectroscopy in rats which subsequently died or survived under bilateral ligation of common carotid arteries. A multidimensional analysis was applied. In the rats which died after the brain ischemia: (1), NAD and NADH+ concentrations were much higher than those of creatine phosphate or ATP (i.e. baseline dysbalance existed between the systems of hydrogen acceptors and major macroergic substances); (2), the force of relationships between parameters of NMR spectra in each correlation matrix were 10 times higher and the variability of elements in each matrix was significantly lower than those of the surviving group. These regularities can be used in detection of special groups at high professional risk and in designing individual procedures of prevention and treatment of cerebral circulation disorders. The data are valuable in terms of development of drugs which would correct the dysbalance between hydrogen acceptors and macroergic systems thus providing a metabolic defense for the ischemic brain.

[Changes in energy metabolism in the brain in experimental cerebral ischemia of different degree of severity (nuclear magnetic resonance-spectroscopic study)]. / Izmeneniia énergeticheskogo metabolizma mozga pri raznoi tiazhesti ego ishemii v éksperimente (IaMR-spektroskopicheskoe issledovanie).

The peculiarities of brain energy metabolism were studied in male rats before and during cerebral ischemia of various severity elicited by bilateral common carotid arteries ligation. A multidimensional analysis was applied. In the rats which died after the ischemia, the NAD + NADH+/phosphocreatine (PCr) ratio and ATP content before ligation were higher than those in the surviving group. Also the strength of relationships between parameters of NMR spectra in each correlation matrix were 10 times higher and the variability of elements in each matrix was significantly lower in victims than those in the surviving group. The development of severe ischemia and the animals death were accompanied by an increase in the inorganic phosphate content, decrease in pH and stepwise disappearing of PCr and ATP. In animals surviving the same brain ischemia model, the changes in 31P spectra parameters pointed to some increase in the ratio of NAD + NADH+ only to ATP + ADP but not to PCr, and to an increase in summarized strength of correlation between 31P spectra parameters with the variability of elements decreased within each correlation matrix. Detection of these changes can be helpful in the diagnosis of mild ischemia without neurological deficit which already needs preventive therapy against more severe ischemia.

[The effect of verapamil on the dynamics of decrease in the brain levels of phosphorus macroergs during ischemia studied by 31P-NMR in vivo]. / Vliianie verapamila na dinamiku snizheniia urovnei makroérgicheskikhfosfatov mozga pri ishemii po dannym 31P-IaMR spektroskopii in vivo.

Protective effects of verapamil on dynamics of phosphorus-containing metabolites were studied during 30 min complete ischemia by means of 31P NMR. Verapamil appears to decrease the ATP and creatine phosphate pools consumption in ischemic brain tissue. The efficiency of the drug depended on the administration procedure and was not similar in two different models of ischemia. Possible mechanisms of the verapamil effect on bioenergetics of nervous tissue are discussed.