Early pregnancy urinary biomarkers of fatty acid and carbohydrate metabolism in pregnancies complicated by gestational diabetes.

AIMS: Alterations in organic acid biomarkers from fatty acid and carbohydrate metabolism have been documented in type 2 diabetes patients. However, their association with gestational diabetes mellitus (GDM) is largely unknown. METHODS: Participants were 25 GDM cases and 25 non-GDM controls. Biomarkers of fatty acid (adipate, suberate and ethylmalonate) and carbohydrate (pyruvate, l-lactate and ß-hydroxybutyrate) metabolism were measured in maternal urine samples collected in early pregnancy (17 weeks) using liquid chromatography-mass spectrometry methods. Logistic regression were used to calculate odds ratios (OR) and 95% confidence intervals (CI). RESULTS: GDM cases and controls differed in median urinary concentrations of ethylmalonate (3.0 vs. 2.3µg/mg creatinine), pyruvate (7.4 vs. 2.1µg/mg creatinine), and adipate (4.6 vs. 7.3µg/mg creatinine) (all p-values <0.05). Women in the highest tertile for ethylmalonate or pyruvate concentrations had 11.4-fold (95%CI 1.10-117.48) and 3.27-fold (95%CI 0.72-14.79) increased risk of GDM compared with women in the lowest tertile for ethylmalonate and pyruvate concentrations, respectively. Women in the highest tertile for adipate concentrations, compared with women in the lowest tertile, had an 86% reduction in GDM risk (95%CI 0.02-0.97). CONCLUSIONS: These preliminary findings underscore the importance of altered fatty acid and carbohydrate metabolism in the pathogenesis of GDM.

Do omega-6 and trans fatty acids play a role in complex regional pain syndrome? A pilot study.

OBJECTIVES: The study aims to compare the omega-6 (n-6) and omega-3 (n-3) highly unsaturated fatty acids (HUFA), and trans fatty acid (trans FA) status of Complex Regional Pain Syndrome (CRPS) patients to pain-free controls. DESIGN: Case control study. Setting. The setting was at a multidisciplinary rehabilitation center. PATIENTS: Twenty patients that met the Budapest research diagnostic criteria for CRPS and 15 pain-free control subjects were included in this study. Outcome Measures. Fasting plasma fatty acids were collected from all participants. In CRPS patients, pain was assessed using the McGill Pain Questionnaire-Short Form. In addition, results from the perceived disability (Pain Disability Index), pain-related anxiety (Pain Anxiety Symptom Scale Short Form), depression (Center for Epidemiologic Studies Depression Scale Short Form), and quality of life (Short Form-36 [SF-36]) were evaluated. RESULTS: Compared with controls, CRPS patients demonstrated elevated concentrations of n-6 HUFA and trans FA. No differences in n-3 HUFA concentrations were observed. Plasma concentrations of the n-6 HUFA docosatetraenoic acid were inversely correlated with the "vitality" section of the SF-36. Trans FA concentrations positively correlated with pain-related disability and anxiety. CONCLUSION: These pilot data suggest that elevated n-6 HUFA and trans FA may play a role in CRPS pathogenesis. These findings should be replicated, and more research is needed to explore the clinical significance of low n-6 and trans FA diets with or without concurrent n-3 HUFA supplementation, for the management of CRPS.

Microencapsulated foods as a functional delivery vehicle for omega-3 fatty acids: a pilot study.

It is well established that the ingestion of the omega-3 (N3) fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) positively benefit a variety of health indices. Despite these benefits the actual intake of fish derived N3 is relatively small in the United States. The primary aim of our study was to examine a technology capable of delivering omega-3 fatty acids in common foods via microencapsulation (MicroN3) in young, healthy, active participants who are at low risk for cardiovascular disease. Accordingly, we randomized 20 participants (25.4 +/- 6.2 y; 73.4 +/- 5.1 kg) to receive the double blind delivery of a placebo-matched breakfast meal (~2093 kJ) containing MicroN3 (450-550 mg EPA/DHA) during a 2-week pilot trial. Overall, we observed no differences in overall dietary macronutrient intake other than the N3 delivery during our treatment regimen. Post-test ANOVA analysis showed a significant elevation in mean (SE) plasma DHA (91.18 +/- 9.3 vs. 125.58 +/- 11.3 umol/L; P < 0.05) and a reduction in triacylglycerols (89.89 +/- 12.8 vs. 80.78 +/- 10.4 mg/dL; P < 0.05) accompanying the MicroN3 treatment that was significantly different from placebo (P < 0.05). In post study interviews, participants reported that the ingested food was well-tolerated, contained no fish taste, odor or gastrointestinal distress accompanying treatment. The use of MicroN3 foods provides a novel delivery system for the delivery of essential fatty acids. Our study demonstrates that MicroN3 foods promote the absorption of essential N3, demonstrate bioactivity within 2 weeks of ingestion and are well tolerated in young, active participants who are at low risk for cardiovascular disease.

Rapid quantitative determination of fat-soluble vitamins and coenzyme Q-10 in human serum by reversed phase ultra-high pressure liquid chromatography with UV detection.

We are presenting the first ultra-high pressure LC (UHPLC) method for rapid quantitative measurement of vitamin A, E (alpha- and gamma-tocopherol), beta-carotene and CoQ(10) from human serum. The chromatography was performed on Shield RP(18) UHPLC column with UV detection. The method was validated based on linearity, accuracy, matrix effects study, precision and stability. The calibration was linear over the following range: 0.09-10.0 for retinol and gamma-tocopherol, 0.05-5 for beta-carotene, 0.9-100 for alpha-tocopherol and 0.14-15 mg/L for CoQ(10). The limit of detection and quantitation for retinol, gamma-tocopherol, beta-carotene, alpha-tocopherol and CoQ(10) were as follows 0.07/0.024, 0.018/0.06, 0.004/0.12, 0.078/0.261, 0.008/0.028 mg/L. The recoveries were above 85%. The inter- and intra-assay precision was below 10%. Reference intervals were established for children and adults. Because of its low cost, extremely short analysis time (2 min) and excellent chromatographic reproducibility this UHPLC method can easily be adopted for high-throughput clinical and pharmacokinetics studies.

Clinical applications of urinary organic acids. Part I: Detoxification markers.

Modern instrumentation allows the measurement of organic acids in urine in their physiological concentration ranges. Eight of the compounds that are reported can serve as markers for specific toxicant exposure or detoxification challenges. Xylene exposure causes elevation of 2-methylhippurate, and orotic acid elevation reveals ammonia challenge that exceeds the capacity of the urea cycle. General hepatic detoxification stimulation by natural compounds, drugs, or xenobiotic compounds causes elevated levels of glucaric acid. Abnormalities of alpha-hydroxybutyrate, pyroglutamate, and sulfate can indicate up-regulated glutathione biosynthesis, impaired reformation of glutathione in the gamma-glutamyl cycle, and depleted total body glutathione status, respectively. Patterns of these compounds measured in a simple overnight urine specimen help to identify focal areas of clinical concern and monitor patient responses to detoxification interventions.

Endogenous nitric oxide synthase inhibitors, arterial hemodynamics, and subclinical vascular disease: the PREVENCION Study.

Endogenous NO synthase inhibitors (end-NOSIs) have been associated with cardiovascular risk factors and atherosclerosis. In addition, end-NOSIs may directly cause hypertension through hemodynamic effects. We aimed to examine the association between end-NOSI asymmetrical dimethylarginine (ADMA) and N-guanidino-monomethyl-arginine (NMMA), subclinical atherosclerosis, and arterial hemodynamics. We studied 922 adults participating in a population-based study (PREVENCION Study) and examined the correlation between end-NOSI/L-arginine and arterial hemodynamics, carotid-femoral pulse wave velocity, and carotid intima-media thickness using linear regression. ADMA, NMMA, and L-arginine were found to be differentially associated with various classic cardiovascular risk factors. ADMA and NMMA (but not L-arginine) were significant predictors of carotid intima-media thickness, even after adjustment for cardiovascular risk factors, C-reactive protein, and renal function. In contrast, ADMA and NMMA did not predict carotid-femoral pulse wave velocity, blood pressure, or hemodynamic abnormalities. Higher L-arginine independently predicted systolic hypertension, higher central pulse pressure, incident wave amplitude, central augmented pressure, and lower total arterial compliance but not systemic vascular resistance or cardiac output. We conclude that ADMA and NMMA are differentially associated with cardiovascular risk factors, but both end-NOSIs are independent predictors of carotid atherosclerosis. In contrast, they are not associated with large artery stiffness, hypertension, or hemodynamic abnormalities. Our findings are consistent with a role for asymmetrical arginine methylation in atherosclerosis but not in large artery stiffening, hypertension, or long-term hemodynamic regulation. L-arginine is independently associated with abnormal pulsatile (but not resistive) arterial hemodynamic indices, which may reflect abnormal L-arginine transport, leading to decreased intracellular bioavailability for NO synthesis.

Analysis of urinary aromatic acids by liquid chromatography tandem mass spectrometry.

The separation and detection of 11 urinary aromatic acids was developed using HPLC-MS/MS. The method features a simple sample preparation involving a single-step dilution with internal standard and a rapid 8 min chromatographic separation. The accuracy was evaluated by the recovery of known spikes between 87 and 110%. Inter- and intra-assay precision (CV) was below 11% in all cases and the analytes were observed to be stable for up to 8 weeks when stored at -20 degrees C. The method was validated based upon linearity, accuracy, precision and stability and was used to establish reference intervals for children and adults.

Clinical applications of urinary organic acids. Part 2. Dysbiosis markers.

Part 1 of this series focused on urinary organic acids as markers of detoxification; part 2 focuses on dysbiosis markers. Intestinal microbial growth is accompanied by the release of products of their metabolism that may be absorbed and excreted in urine. Several organic acids are known to be specific products of bacterial metabolic action on dietary polyphenols or unassimilated amino acids or carbohydrates. Associated gastrointestinal or neurological symptoms may result from irritation of the intestinal mucosa or systemic distribution of absorbed neurotoxic products. Detection of abnormally elevated levels of these products is a useful diagnostic tool for patients with gastrointestinal or toxicological symptoms. Test profiles of urinary organic acids associated with microbial overgrowth can include benzoate, hippurate, phenylacetate, phenylpropionate, cresol, hydroxybenzoate, hydroxyphenylacetate, hydroxyphenylpropionate and 3,4-dihydroxyphenylpropionate, indican, tricarballylate, D-lactate, and D-arabinitol. Effective treatments for the associated microbial overgrowths may be directed at reducing microbial populations, introducing favorable microbes, and restoring intestinal mucosal integrity.

A simple and cost effective method for the quantification of 8-hydroxy-2'-deoxyguanosine from urine using liquid chromatography tandem mass spectrometry.

A rapid and high-throughput method for the determination of urinary levels of the oxidative stress biomarker, 8-hydroxy-2'-deoxyguanosine (8-OH-dG), has been developed and validated using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-MS/MS). The assay features a cheap and readily available non-isotopic internal standard, a single-step filtration sample preparation, and a total analysis time of 6 min including column re-equilibration. The method was validated based on linearity, accuracy (100-106%), precision (CV < 7%), sample preparation stability (< or =5%, 72 h). Intra-laboratory patient ranges were established comparing children and adults (n = 345).

A simple and selective method for the measurement of leucine and isoleucine from plasma using electrospray ionization tandem mass spectrometry.

An analytical method was developed for the rapid and accurate quantification of leucine (LEU) and isoleucine (ILE) from plasma using electrospray ionization tandem mass spectrometry (ESI-MS/MS). The two isomeric amino acids were selectively detected using fragment ions unique to each compound. As a result, the need for chromatographic separation was avoided allowing for faster analysis (3 min). The possibility of any considerable interference between the two transitions was closely monitored, with no significant interference being observed. The presence of interfering compounds in plasma was also evaluated and found to minimal. The method was evaluated based upon linearity, with r2>or=0.995 for both compounds, and accuracy, with no more than 8% deviation from the theoretical value.

High performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) assay for chiral separation of lactic acid enantiomers in urine using a teicoplanin based stationary phase.

A novel method for the separation and simultaneous determination of urinary D- and L-lactic acid enantiomers by high performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) is presented. The chiral separation was optimized on a Chirobiotic teicoplanin aglyocone (TAG) column. Most interestingly, the addition of water in small volume fraction to the polar organic mobile phase was found to significantly improve the chromatography. Calibration curves were linear (r2>0.9950) over the range 3-1000 mg/L for L-lactic acid and 0.5-160.8 mg/L for D-lactic acid. The limit of detection (LOD) (S/N=3) and limit of quantification (LOQ) (S/N=10) were determined experimentally (n=3) to be 0.2 and 0.5mg/L for L-lactic acid and 0.4 and 1.3 mg/L for D-lactic acid, respectively. The normal patient range of L-lactic acid was 1-20 microg/mg creatinine with an elevated value of 85 microg/mg creatinine. For D-lactic acid, the range of normal values were between 0 and 5 microg/mg creatinine with an elevated value of 40 microg/mg creatinine. Finally, the validated method allows for rapid analysis with a total run time of 7.5 min.

A prospective study of early-pregnancy plasma malondialdehyde concentration and risk of preeclampsia.

OBJECTIVES: Preeclampsia is associated with elevated plasma malondialdehyde concentration, but prospective data are scarce. We examined the relation between early-pregnancy plasma malondialdehyde and subsequent preeclampsia risk. DESIGN AND METHODS: In a nested case-control analysis, we measured malondialdehyde concentrations in 22 women who developed preeclampsia and 711 who remained normotensive during pregnancy. We calculated adjusted odds ratios (OR) and 95% confidence intervals (CI). We repeated analyses after adjustment for early-pregnancy plasma lipid concentrations, which are related to preeclampsia risk and lipid peroxidation measures. RESULTS: After adjustment for confounders, preeclampsia risk increased across tertiles of malondialdehyde concentration (trend P = 0.04). Further adjustment for triglyceride concentration slightly strengthened the association. Middle- and high-tertile malondialdehyde ORs were 3.2 (CI 0.8-12.2) and 4.2 (1.1-16.0) versus low-tertile exposure. CONCLUSIONS: Early-pregnancy plasma malondialdehyde concentration is positively associated with subsequent preeclampsia risk independent of plasma lipid concentrations. These results support lipid peroxidation as an etiologic component of preeclampsia.

Erythrocyte omega-3 and omega-6 polyunsaturated fatty acids and preeclampsia risk in Peruvian women.

OBJECTIVE: This case-control study was conducted in Lima, Peru, from June 1997 through January 1998 to assess whether alteration in maternal erythrocyte omega-3 (n-3) and omega-6 (n-6) fatty acids was associated with increased risk of preeclampsia. METHODS: A total of 99 preeclampsia and 100 normotensive pregnant women were included. Maternal erythrocyte n-3 and n-6 fatty acids were determined using capillary gas chromatography/mass spectrometry and expressed as micromolar (mM) concentrations. We employed logistic regression procedures to estimate odds ratios (ORs) and 95% confidence intervals (CIs). RESULT: n-3 fatty acids were consistently lower in preeclampsia cases than controls. After adjusting for confounders, the corresponding ORs for preeclampsia across decreasing quartiles of sum of long-chain n-3 fatty acids were 1.0, 3.3, 2.4, and 3.3, respectively (P=0.07 for trend). A similar pattern was observed for eicosapentenoic acid (20:5n-3, EPA) and docosahexenoic acid (22:6n-3, DHA). There was no clear evidence of an association between arachidonic acid (20:4n-6, AA) and preeclampsia risk, the ORs in successively lower quartiles were 1.0, 1.1, 1.0, and 1.5 (P=0.48 for trend). A similar pattern was seen for the sum of long-chain n-6 fatty acids. CONCLUSION: In Peruvian women, low erythrocyte n-3 fatty acids appeared to be associated with an increased risk of preeclampsia.

Estrogen metabolism and the diet-cancer connection: rationale for assessing the ratio of urinary hydroxylated estrogen metabolites.

Estrogens are known for their proliferative effects on estrogen-sensitive tissues resulting in tumorigenesis. Results of experiments in multiple laboratories over the last 20 years have shown that a large part of the cancer-inducing effect of estrogen involves the formation of agonistic metabolites of estrogen, especially 16-alpha-hydroxyestrone. Other metabolites, such as 2-hydroxyestrone and 2-hydroxyestradiol, offer protection against the estrogen-agonist effects of 16-alpha-hydroxyestrone. An ELISA method for measuring 2- and 16-alpha-hydroxylated estrogen (OHE) metabolites in urine is available and the ratio of urinary 2-OHE/16-alpha-OHE (2/16-alpha ratio) is a useful biomarker for estrogen-related cancer risk. The CYP1A1 enzyme that catalyzes 2-hydroxyestrone (2-OHE1) formation is inducible by dietary modification and supplementation with the active components of cruciferous vegetables, indole-3-carbinol (I-3-C), or diindolylmethane (DIM). Other dietary components, especially omega-3 polyunsaturated fatty acids and lignans in foods like flax seed, also exert favorable effects on estrogen metabolism. Thus, there appear to be effective dietary means for reducing cancer risk by improving estrogen metabolism. This review presents the accumulated evidence to help clinicians evaluate the merit of using tests that measure estrogen metabolites and using interventions to modify estrogen metabolism.