Physiologic, Metabolic, and Toxicologic Profile of 1,3-Butanediol.

Ketone bodies are essential energy substrates in the absence of exogenous nutrients, and more recently, they have been suggested to prevent disease and improve longevity. ß-hydroxybutyrate (ßHB) is the most abundant ketone body. The secondary alcohol, 1,3-butanediol (1,3-BD), is commonly administered to raise ßHB bioavailability in vivo and in the absence of nutrient deprivation. However, the concentration of 1,3-BD that yields a systemic concentration of ßHB similar to that observed after a 24-hour fast has yet to be determined. To evaluate this knowledge gap, we administered 5%, 10%, or 20% 1,3-BD via the drinking water to adult, male Wistar-Kyoto rats for four weeks. In addition to systemic and excreted ßHB concentration, physiologic, metabolic, and toxicologic parameters were measured. We report that only 20% 1,3-BD significantly elevates the systemic and urinary concentrations of ßHB. Rats treated with 20% 1,3-BD had a rapid and sustained reduction in body mass. All concentrations of 1,3-BD decreased food consumption, but only the 20% concentration decreased fluid consumption. Urine volume, red blood cell count, and hematocrit suggested dehydration in the 10% and 20% 1,3-BD-treated rats. Finally, 20% 1,3-BD-treated rats presented with indicators of metabolic acidosis and sinusoidal dilation, but no evidence of fatty liver or hepatotoxicity. In summary, we report that 20% 1,3-BD, but not 5% or 10%, produces a systemic concentration of ßHB similar to that observed after a 24-hour fast. However, this concentration is associated with deleterious side effects such as body mass loss, dehydration, metabolic acidosis, and sinusoidal dilation. SIGNIFICANCE STATEMENT: 1,3-Butanediol (1,3-BD) is often administered to stimulate the biosynthesis of the most abundant ketone body, ß-hydroxybutyrate (ßHB), and its purported salubrious effects. This article reports that suprapharmacological concentrations of 1,3-BD are necessary to yield a systemic concentration of ßHB similar to that observed after a 24-hour fast, and this is associated with undesirable side effects. On the other hand, low concentrations of 1,3-BD were better tolerated and may improve health independent of its conversion into ßHB.

Development of an Integrated Optical Sensor for Determination of ß-Hydroxybutyrate Within the Microplatform.

Ketone bodies (acetoacetate, beta-hydroxybutyrate (ßHB), acetone) are generated as a result of fatty acid oxidation in the liver and exist at low concentrations in urine and blood. Elevated concentrations can indicate health problems such as diabetes, childhood hypoglycemia, alcohol, or salicylate poisoning. Development of portable and cost-effective bedside point-of-care (POC) tests to detect such compounds can help to reduce the risk of disease progression. In this study, ßHB was chosen as a model molecule for developing an optical sensor-integrated microplatform. Prior to sensor optimization, ßHB levels were measured at a concentration range of 0.02 and 0.1 mM spectrophotometrically, which is far below the reported elevated ranges of 1-2 mM and resulting absorbance changes were converted into an Arduino microcontroller code for the correlation. Measurements performed with the designed integrated microplatform were found significant. Integrated microplatform was verified with the benchtop spectrophotometer. Measurements between 0.02 and 0.1 mM substrate concentration were found highly sensitive with "y = 0.7347x + 0.00184" with R2 value of 0.9796, and the limit of detection was determined as 0.02 mM. Based on these results, the proposed system will allow on-site and early intervention.

The Effect of Blood Ketone Concentration and Exercise Intensity on Exogenous Ketone Oxidation Rates in Athletes.

INTRODUCTION: Exogenous ketones potentially provide an alternative, energetically advantageous fuel to power exercising skeletal muscle. However, there is limited evidence regarding their relative contribution to energy expenditure during exercise. Furthermore, the effect of blood ketone concentration and exercise intensity on exogenous ketone oxidation rates is unknown. METHODS: Six athletes completed cycling ergometer exercise on three occasions within a single-blind, random-order controlled, crossover design study. Exercise duration was 60 min, consisting of 20-min intervals at 25%, 50%, and 75% maximal power output (WMax). Participants consumed (i) bitter flavored water (control), (ii) a low-dose ß-hydroxybutyrate (ßHB) ketone monoester (KME; 252 mg·kg BW-1, "low ketosis"), or (iii) a high-dose ßHB KME (752 mg·kg BW-1, "high ketosis"). The KME contained a 13C isotope label, allowing for the determination of whole-body exogenous ßHB oxidation rates through sampled respiratory gases. RESULTS: Despite an approximate doubling of blood ßHB concentrations between low- and high-ketosis conditions (~2 mM vs ~4.4 mM), exogenous ßHB oxidation rates were similar at rest and throughout exercise. The contribution of exogenous ßHB oxidation to energy expenditure peaked during the 25% WMax exercise intensity but was relatively low (4.46% ± 2.71%). Delta efficiency during cycling exercise was significantly greater in the low-ketosis (25.9% ± 2.1%) versus control condition (24.1% ± 1.9%; P = 0.027). CONCLUSIONS: Regardless of exercise intensity, exogenous ßHB oxidation contributes minimally to energy expenditure and is not increased by elevating circulating concentrations greater than ~2 mM. Despite low exogenous ßHB oxidation rates, exercise efficiency was significantly improved when blood ßHB concentration was raised to ~2 mM.

Urinary ketone body loss leads to degeneration of brain white matter in elderly SLC5A8-deficient mice.

SLC5A8 is a sodium-coupled monocarboxylate and ketone transporter expressed in various epithelial cells. A putative role of SLC5A8 in neuroenergetics has been also hypothesized. To clarify this issue, we studied the cerebral phenotype of SLC5A8-deficient mice during aging. Elderly SLC5A8-deficient mice presented diffuse leukoencephalopathy characterized by intramyelinic oedema without demyelination suggesting chronic energetic crisis. Hypo-metabolism in the white matter of elderly SLC5A8-deficient mice was found using 99mTc-hexamethylpropyleneamine oxime (HMPAO) single-photon emission CT (SPECT). Since the SLC5A8 protein could not be detected in the mouse brain, it was hypothesized that the leukoencephalopathy of aging SLC5A8-deficient mice was caused by the absence of slc5a8 expression in a peripheral organ, i.e. the kidney, where SLC5A8 is strongly expressed. A hyper-excretion of the ketone ß-hydroxybutyrate (BHB) in the urine of SLC5A8-deficient mice was observed and showed that SLC5A8-deficient mice suffered a cerebral BHB insufficiency. Elderly SLC5A8-deficient mice also presented altered glucose metabolism. We propose that the continuous renal loss of BHB leads to a chronic energetic deficiency in the brain of elderly SLC5A8-deficient mice who are unable to counterbalance their glucose deficit. This study highlights the importance of alternative energetic substrates in neuroenergetics especially under conditions of restricted glucose availability.

[Overview on the relationship between changes in activity and ketosis in dairy cows]. / Übersicht zu Zusammenhängen zwischen Änderungen der Bewegungsintensität und Ketose bei Milchkühen.

With a prevalence of up to 43 % subclinical ketosis is one of the most common diseases in dairy cows in their transition period. In itself, this may cause subsequent diseases such as clinical ketosis or lameness. Therefore, monitoring of animals in this stage is of importance. In addition to the measurement of ß-hydroxybutyrate or acetoacetate in blood, milk, and urine as well as the observation of the animals, computer-assisted systems are suitable means of monitoring. Information such as animal identification and activity data are recorded on a data logger and transmitted to a computer. A change in activity may be an indication of an underlying disease days before the onset of additional clinical signs. In cases of ketosis, a decrease in activity may be observed 5 days before the clinical diagnosis is made. Thus, these data are a valuable contribution in monitoring the cattle herd's health status for both the farmer and the veterinarian. Activity measurement may also be employed for the detection of a beginning lameness. In the presence of lameness, the individual's activity decreases and periods of lying are longer. Activity measurement via transponder as a part of the herd monitoring provides important information on lameness prevalence in the herd. In the presence of a lameness a visual assessment should additionally be made. Lameness scores (Locomotion score, Gait score) have been developed for this purpose and add to determining the lameness status of the herd. This way the animals are divided into different lameness classes. Based on this classification those individuals in need of claw trimming or further treatment may be identified leading to amelioration or prevention of secondary diseases. Due to lameness and subsequent reduction of activity and feed intake, the animals may develop subclinical or clinical ketosis. Therefore, under consideration of both animal welfare and economic factors early disease detection and prophylaxis is desirable and should be a main objective of herd monitoring.

Clinical utility of beta-hydroxybutyrate measurement in the management of physiological ketosis at home in children under 5.

AIM: To verify the possible advantages of 3- ß-hydroxybutyrate (3HB) measurement compared to urinary assay of ketones during an intercurrent disease managed at home. METHODS: Twelve Pediatricians were asked to enroll at least 4 patients aged 3 to 5 years, affected by an intercurrent illness and showing at least one of symptoms reliable to ketosis. Recruited patients were submitted to the simultaneous assay of 3HB in capillary blood and ketones in urine at 3 (T3) and 6 hours (T6) from the first measurement (T0). For urinary and blood ketone detection commercial tests were used. RESULTS: Thirty-eight children (4.36±2.60 years old; 25 boys) were enrolled into the study. At T0 all children showed 3HB levels (1.2-3.2 mmol/L), but only 10 of them (26.3%) associated also urinary ketone bodies (2 to 4+). In response to 3 hour treatment (T3) with a glucose solution, 3HB values decreased in 19 (0,8-1,8 mmol/L) and normalized in 13 children (<0.2 mmol/L); while ketonuria disappeared in only 2 patients, it was confirmed in 8 and appeared (4+) the first time in the remaining 28 children. At T6 3HB levels fell definitively within the normal range in all children, while ketonuria was still present (2+) in 9 patients (23%). The pediatricians reported two limitations about blood 3HB dosage compared to the urinary test: the invasiveness of capillary blood collection, and the cost of supplies for finger pricking, reagent strips and reflectance meter. CONCLUSIONS: 3HB monitor in capillary blood is more effective and clinically more useful in diagnosing and managing of an ongoing ketosis in children with a mild infective disease than ketones detection in the urine. These advantages are mitigated by the cost of 3HB measurement.

Sodium-glucose cotransporter 2 inhibitors regulate ketone body metabolism via inter-organ crosstalk.

AIM: To investigate sodium-glucose cotransporter 2 inhibitor (SGLT2i)-induced changes in ketogenic enzymes and transporters in normal and diabetic mice models. MATERIALS AND METHODS: Normal mice were randomly assigned to receive either vehicle or SGLT2i (25 mg/kg/d by oral gavage) for 7 days. Diabetic mice were treated with vehicle, insulin (4.5 units/kg/d by subcutaneous injection) or SGLT2i (25 mg/kg/d by intra-peritoneal injection) for 5 weeks. Serum and tissues of ketogenic organs were analysed. RESULTS: In both normal and diabetic mice, SGLT2i increased beta-hydroxybutyrate (BHB) content in liver, kidney and colon tissue, as well as in serum and urine. In these organs, SGLT2i upregulated mRNA expression of ketogenic enzymes, 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 and 3-hydroxy-3-methylglutaryl-coenzyme A lyase. Similar patterns were observed in the kidney, ileum and colon for mRNA and protein expression of sodium-dependent monocarboxylate transporters (SMCTs), which mediate the cellular uptake of BHB and butyrate, an important substrate for intestinal ketogenesis. In diabetic mice under euglycaemic conditions, SGLT2i increased major ketogenic enzymes and SMCTs, while insulin suppressed ketogenesis. CONCLUSIONS: SGLT2i increased systemic and tissue BHB levels by upregulating ketogenic enzymes and transporters in the liver, kidney and intestine, suggesting the integrated physiological consequences for ketone body metabolism of SGLT2i administration.

The urinary organic acids profile in single large-scale mitochondrial DNA deletion disorders.

Single large-scale mitochondrial DNA deletions disorders are classified into three main phenotypes with frequent clinical overlap: Pearson marrow-pancreas syndrome (PMS), Kearns-Sayre syndrome (KSS) and chronic progressive external ophtalmoplegia (PEO). So far, only few anecdotal studies have reported on the urinary organic acids profile in this disease class. In this single-center retrospective study, we performed quantitative evaluation of urinary organic acids in a series of 15 pediatric patients, 7 with PMS and 8 with KSS. PMS patients showed an organic acids profile almost constantly altered, whereas KSS patients frequently presented with normal profiles. Lactate, 3-hydroxybutyrate, 3-hydroxyisobutyrate, fumarate, pyruvate, 2-hydroxybutyrate, 2-ethyl-3-hydroxypropionate, and 3-methylglutaconate represented the most frequent metabolites observed in PMS urine. We also found novel metabolites, 3-methylglutarate, tiglylglycine and 2-methyl-2,3-dihydroxybutyrate, so far never reported in this disease. Interestingly, patients with a disease onset as PMS evolving overtime into KSS phenotype, presented persistent and more pronounced alterations of organic acid signature than in patients with a pure KSS phenotype. Our study shows that the quantitative analysis of urinary organic acid profile represents a helpful tool for the diagnosis of PMS and for the differential diagnosis with other inherited diseases causing abnormal organic acidurias.

Enantioselective and simultaneous determination of lactate and 3-hydroxybutyrate in human plasma and urine using a narrow-bore online two-dimensional high-performance liquid chromatography system.

For the enantioselective and simultaneous analysis of lactate and 3-hydroxybutyrate, a validated online two-dimensional high-performance liquid chromatography system using 4-nitro-7-piperazino-2,1,3-benzoxadiazole as a fluorescent derivatization reagent has been developed. For the reversed-phase separation in the first dimension, a Capcell Pak C18 ACR column (1.5 × 250 mm, particle size 3 µm) was used, and the target fractions were isolated by their hydrophobicity. In the second dimension, a polysaccharide-coated enantioselective column, Chiralpak AD-H (2.0 × 250 mm, 5 µm), was used. The system was validated by the calibration curve, intraday precision, interday precision, and accuracy using standards and real human samples, and satisfactory results were obtained. The present method was applied to human plasma and urine, and in the plasma, trace amounts of d-lactate (8.4 µM) and l-3-hydroxybutyrate (1.0 µM), besides high levels of l-lactate (860.9 µM) and d-3-hydroxybutyrate (59.4 µM), were successfully determined. In urine, trace levels of d-lactate (3.7 µM), d-3-hydroxybutyrate (2.3 µM), and l-3-hydroxybutyrate (3.3 µM) in addition to a relatively large amount of l-lactate (15.4 µM) were observed. The present online two-dimensional high-performance liquid chromatography system is useful for the simultaneous determination of all the lactate and 3-hydroxybutyrate enantiomers in human physiological fluids, and further clinical applications are ongoing.

Metabolic phenotyping in the mouse model of urinary tract infection shows that 3-hydroxybutyrate in plasma is associated with infection.

Urinary tract infection is one of the most common bacterial infections worldwide. Current diagnosis of urinary tract infection chiefly relies on its clinical presentation, urine dipstick tests and urine culture. Small molecules found in bio-fluids related with both infection and recovery would facilitate diagnosis and management of UTI. Mass spectrometry-based fingerprinting of plasma and urine at 3 time points, pre-infection (t = -24h), infection (t = 24h) and post 3-day treatment (t = 112h), were acquired in the following four groups: mice which were healthy, infected but not treated, infected and treated with ciprofloxacin, and infected and treated with Relinqing® granules (n = 6 per group). A metabolomics workflow including multivariate analysis and ROC regression was employed to select metabolic features that correlated with UTI and its treatment. Circa 4,000 molecular features were acquired for each sample. The small acid 3-hydroxybutyrate in plasma was found to be differentiated for urinary tract infection, with an area under the curve = 0.97 (95% confidence interval: 0.93-1.00, accuracy = 0.91, sensitivity = 0.92 and specificity = 0.91). The level of 3-hydroxybutyrate in plasma was depleted after infection with a fold change of -22 (q < 0.0001). Correlation between plasma 3-hydroxybutyrate and urine bacterial number in all groups and time points was r = -0.753 (p < 0.0001). The findings show that 3-hydroxybutyrate is depleted in blood and strongly associated with UTI at both infection and post-treatment stage in a UTI mouse model. Further work is envisaged to assess the clinical potential of blood tests to assist with UTI management.

Renal Handling of Ketones in Response to Sodium-Glucose Cotransporter 2 Inhibition in Patients With Type 2 Diabetes.

OBJECTIVE: Pharmacologically induced glycosuria elicits adaptive responses in glucose homeostasis and hormone release, including decrements in plasma glucose and insulin levels, increments in glucagon release, enhanced lipolysis, and stimulation of ketogenesis, resulting in an increase in ketonemia. We aimed at assessing the renal response to these changes. RESEARCH DESIGN AND METHODS: We measured fasting and postmeal urinary excretion of glucose, ß-hydroxybutyrate (ß-HB), lactate, and sodium in 66 previously reported patients with type 2 diabetes and preserved renal function (estimated glomerular filtration rate ≥60 mL · min-1 · 1.73 m-2) and in control subjects without diabetes at baseline and following empagliflozin treatment. RESULTS: With chronic (4 weeks) sodium-glucose cotransporter 2 inhibition, baseline fractional glucose excretion (<2%) rose to 38 ± 12% and 46 ± 11% (fasting vs. postmeal, respectively; P < 0.0001) over a range of BMIs (range 23-41 kg/m2) and creatinine clearance (65-168 mL · min-1 · m-2). Excretion of ß-HB (median [interquartile range]: 0.08 [0.10] to 0.31 [0.43] µmol · min-1), lactate (0.06 [0.06] to 0.28 [0.25] µmol · min-1), and sodium (0.27 [0.22] to 0.36 [0.16] mEq · min-1) all increased (P ≤ 0.001 for all) and were each positively related to glycosuria (P ≤ 0.001). These parameters changed in the same direction in subjects without diabetes, but changes were smaller than in the patients with diabetes. Although plasma N-terminal pro-B-type natriuretic peptide levels were unaltered, plasma erythropoietin concentrations increased by 31 (64)% (P = 0.0078). CONCLUSIONS: We conclude that the sodium-glucose cotransporter 2 inhibitor-induced increase in ß-HB is not because of reduced renal clearance but because of overproduction. The increased lactate excretion contributes to lower plasma lactate levels, whereas the increased natriuresis may help in normalizing the exchangeable sodium pool. Taken together, glucose loss through joint inhibition of glucose and sodium reabsorption in the proximal tubule induces multiple changes in renal metabolism.

Metabolomic analysis of urine with Nuclear Magnetic Resonance spectroscopy in patients with idiopathic sudden sensorineural hearing loss: A preliminary study.

OBJECTIVE: Idiopathic sudden sensorineural hearing loss is a frequent emergency, with unknown aetiology and usually treated with empiric therapy. Steroids represent the only validated treatment but prognosis is unpredictable and the possibility to select the patients who will not respond to steroids could avoid unnecessary treatments. Metabolomic profiling of the biofluids target the analysis of the final product of genic expression and enzymatic activity, defining the biochemical phenotype of a whole biologic system. METHODS: We studied the metabolomics of the urine of a cohort of patients with idiopathic sudden sensorineural hearing loss, correlating the metabolic profiles with the clinical outcomes. Metabolomic profiling of urine samples was performed by 1H Nuclear Magnetic Resonance spectroscopy in combination with multivariate statistical approaches. RESULTS: 26 patients were included in the study: 5 healthy controls, 13 patients who did not recover after treatment at 6 months while the remaining 8 patients recovered from the hearing loss. The orthogonal partial least square-discriminant analysis score plot showed a significant separation between the two groups, responders and non-responders after steroid therapy, R2Y of 0.83, Q2 of 0.38 and p value <0.05. The resulting metabolic profiles were characterized by higher levels of urinary B-Alanine, 3-hydroxybutyrate and Trimethylamine N-oxide, and lower levels of Citrate and Creatinine in patients with worst outcome. CONCLUSION: Idiopathic sudden sensorineural hearing loss is a specific disease with unclear systemic changes, but our data suggest that there are different types of this disorder or patients predisposed to effective action of steroids allowing the recover after treatment.

A systematic review and meta-analysis of the diagnostic accuracy of point-of-care tests for the detection of hyperketonemia in dairy cows.

Several rapid tests for use on farm have been validated for the detection of hyperketonemia (HK) in dairy cattle, however the reported sensitivity and specificity of each method varies and no single study has compared them all. Meta-analysis of diagnostic test accuracy is becoming more common in human medical literature but there are few veterinary examples. The objective of this work was to perform a systematic review and meta-analysis to determine the point-of-care testing method with the highest combined sensitivity and specificity, the optimal threshold for each method, and to identify gaps in the literature. A comprehensive literature search resulted in 5196 references. After removing duplicates and performing relevance screening, 23 studies were included for the qualitative synthesis and 18 for the meta-analysis. The three index tests evaluated in the meta-analysis were: the Precision Xtra(®) handheld device measuring beta-hydroxybutyrate (BHB) concentration in whole blood, and Ketostix(®) and KetoTest(®) semi-quantitative strips measuring the concentration of acetoacetate in urine and BHB in milk, respectively. The diagnostic accuracy of the 3 index tests relative to the reference standard measurement of BHB in serum or whole blood between 1.0-1.4mmol/L was compared using the hierarchical summary receiver operator characteristic (HSROC) method. Subgroup analysis was conducted for each index test to examine the accuracy at different thresholds. The impact of the reference standard threshold, the reference standard method, the prevalence of HK in the population, the primary study source and risk of bias of the primary study was explored using meta-regression. The Precision Xtra(®) device had the highest summary sensitivity in whole blood BHB at 1.2mmol/L, 94.8% (CI95%: 92.6-97.0), and specificity, 97.5% (CI95%: 96.9-98.1). The threshold employed (1.2-1.4mmol/L) did not impact the diagnostic accuracy of the test. The Ketostix(®) and KetoTest(®) strips had the highest summary sensitivity and specificity when the trace and weak positive thresholds were used, respectively. Controlling for the source of publication, HK prevalence and reference standard employed did not impact the estimated sensitivity and specificity of the tests. Including only peer-reviewed studies reduced the number of primary studies evaluating the Precision Xtra(®) by 43% and Ketostix(®) by 33%. Diagnosing HK with blood, urine or milk are valid options, however, the diagnostic inaccuracy of urine and milk should be considered when making economic and treatment decisions.

The effect of a moderate zinc deficiency and dietary fat source on the activity and expression of the Δ(3)Δ (2)-enoyl-CoA isomerase in the liver of growing rats.

Auxiliary enzymes participate in ß-oxidation of unsaturated fatty acids. The objective of the study was to investigate the impact of a moderate zinc deficiency and a high intake of polyunsaturated fat on Δ(3)Δ(2)-enoyl-CoA isomerase (ECI) in the liver and other tissues. Five groups of eight weanling rats each were fed moderately zinc-deficient (ZD) or zinc-adequate (ZA) semisynthetic diets (7 or 50 mg Zn/kg) enriched with 22 % cocoa butter (CB) or 22 % safflower oil (SO) for 4 weeks: (1) ZD-CB, fed free choice; (2) ZA-CBR, ZA-CB diet fed in equivalent amounts consumed by the ZD-CB group; (3) ZD-SO, fed free choice; (4) ZA-SOR, ZA-SO diet fed in equivalent amounts consumed by the ZD-SO group; and (5) ZA-SO, fed free choice. Growth and Zn status markers were markedly reduced in the ZD groups. ECI activity in the liver of the animals fed the ZD- and ZA-SO diets were significantly higher (approximately 2- and 3-fold, respectively) as compared with the CB-fed animals, whereas activities in extrahepatic tissues (kidneys, heart, skeletal muscle, testes, adipose tissue) were not altered by dietary treatments. Transcript levels of the mitochondrial Eci gene in the liver did not significantly differ between ZD and ZA rats, but were 1.6-fold higher in the ZA-SO- than in the ZD-CB-fed animals (P < 0.05). It is concluded that diets enriched with safflower oil as a source high in linoleic acid induce markedly increased hepatic ECI activities and that a moderate Zn deficiency does not affect transcription of the mitochondrial Eci gene in the liver.

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.

Diagnostic accuracy of fingerstick ß-hydroxybutyrate for ketonuria in pregnant women with nausea and vomiting.

OBJECTIVES: The aim of this study was to determine whether a rapid, fingerstick ß-hydroxybutyrate (ß-OHB) test predicts ketonuria in pregnant women with nausea and vomiting. METHODS: A convenience sample of 82 pregnant women who presented to the Barnes-Jewish Hospital emergency department (ED) with complaints of nausea or vomiting were enrolled into an institutional review board-approved, prospective, observational study. Exclusion criteria were temperature of >38.3°C, altered mental status, prisoners, and >1 L of any intravenous (IV) fluid before screening. Subjects had fingerstick ß-OHB tests, with results reported in increments of 0.1 mmol/L. Urine ketone results were made available once reported as part of standard care and varied from 0 (trace) to +4 values. Ketonuria was defined as a urine ketone value of 3+ or 4+ and was analyzed as a dichotomous, categorical variable. A contingency table and receiver operator characteristic (ROC) curve were constructed for comparing the ß-OHB values to those urine ketone values for each patient. RESULTS: Mean (±SD) ß-OHB was 0.43 (±0.52) mmol/L, and median urine was 1+ for the cohort as a whole. The area under the ROC curve equaled 0.95. The sensitivity and specificity for a fingerstick test of >0.45 mmol/L were 89 and 94.5%, respectively. The positive likelihood ratio (LR) was 16, and the negative LR was 0.12. CONCLUSIONS: Fingerstick ß-OHB is a rapid and reliable diagnostic tool that correlates well with ketonuria and identifies ketonemia in pregnant women with nausea and vomiting. Fingerstick ß-OHB testing has the potential to increase triage efficiency, shorten length-of-stay times, and positively affect patient outcomes in an ED setting.

Urinary metabolomics in newborns infected by human cytomegalovirus: a preliminary investigation.

Human cytomegalovirus (HCMV) represents one of the most significant viral risks of birth defects and long-term sequelae. The severity of the infection depends on the form of the disease, which can be symptomatic or asymptomatic with or without sequelae. The aim of this study was to investigate in a population of newborns the impact of HCMV infection on the urine metabolome by using (1)H-nuclear magnetic resonance (NMR) spectroscopy combined with multivariate statistical analysis. Twenty-three children born from women with a primary HCMV infection during pregnancy were recruited. Twelve were HCMV infected at birth whereas eleven were not infected (control). The (1)H-NMR spectra were analyzed using a PLS-DA mathematical model in order to identify the discriminant metabolites between the asymptomatic and the control group. The most important metabolites characterizing the clustering of the samples were: myoinositol, glycine, 3-hydroxybutyrate, 3-aminoisobutyrate, creatine, taurine and betaine. These findings suggest the use of metabolomics as a useful new tool in the investigation of HCMV congenital infection.

GC/TOFMS analysis of metabolites in serum and urine reveals metabolic perturbation of TCA cycle in db/db mice involved in diabetic nephropathy.

Early diagnosis of diabetic nephropathy (DN) is difficult although it is of crucial importance to prevent its development. To probe potential markers and the underlying mechanism of DN, an animal model of DN, the db/db mice, was used and serum and urine metabolites were profiled using gas chromatography/time-of-flight mass spectrometry. Metabolic patterns were evaluated based on serum and urine data. Principal component analysis of the data revealed an obvious metabonomic difference between db/db mice and controls, and db/db mice showed distinctly different metabolic patterns during the progression from diabetes to early, medium, and later DN. The identified metabolites discriminating between db/db mice and controls suggested that db/db mice have perturbations in the tricarboxylic acid cycle (TCA, citrate, malate, succinate, and aconitate), lipid metabolism, glycolysis, and amino acid turnover. The db/db mice were characterized by acidic urine, high TCA intermediates in serum at week 6 and a sharp decline thereafter, and gradual elevation of free fatty acids in the serum. The sharp drop of serum TCA intermediates from week 6 to 8 indicated the downregulated glycolysis and insulin resistance. However, urinary TCA intermediates did not decrease in parallel with those in the serum from week 6 to 10, and an increased portion of TCA intermediates in the serum was excreted into the urine at 8, 10, and 12 wk than at 6 wk, indicating kidney dysfunction occurred. The relative abundances of TCA intermediates in urine relative to those in serum were suggested as an index of renal damage.

Ketoacidosis and trace amounts of isopropanol in a chronic alcoholic patient.

BACKGROUND: Alcohol ketoacidosis is a frequently missed diagnosis, but is well described in the literature. We present a case of ketoacidosis, likely alcohol ketoacidosis, in a 40 y-old chronic alcoholic patient. The detection of trace serum isopropanol prompted a discussion of alcohol ketoacidosis versus toxic isopropanol ingestion or a combination of both, including comparisons with citations in current literature. METHODS: The automated instruments used to analyze the patient's urine, blood, and serum samples are described. RESULTS: The initial impression was severe metabolic acidosis with an increased anion gap and normal serum glucose and whole blood lactate. Testing for potential toxic ingestions detected only increased serum acetone and trace serum isopropanol. A urinalysis positive for ketones and an increased serum ß-hydroxybutyrate concentration clenched the diagnosis of ketoacidosis. CONCLUSION: Ketoacidosis with an increased anion gap in the absence of hyperglycemia or glycosuria in a chronic alcoholic patient should prompt the evaluation for alcohol ketoacidosis. Trace serum isopropanol may be worrisome for a toxic ingestion, but this finding in severe ketoacidosis may be explained by the reversible action of the enzyme alcohol dehydrogenase. Markedly increased serum isopropanol with a low serum acetone:isopropanol ratio would be more indicative of a toxic isopropanol ingestion.

NMR-based metabolomics study of canine bladder cancer.

Bladder cancer is one of the leading lethal cancers worldwide. With the high risk of recurrence for bladder cancer following the initial diagnoses, lifelong monitoring of patients is necessary. The lack of adequate sensitivity and specificity of current noninvasive monitoring approaches including urine cytology, other urine tests, and imaging, underlines the importance of studies that focus on the detection of more reliable biomarkers for this cancer. The emerging area of metabolomics, which deals with the analysis of a large number of small molecules in a single step, promises immense potential for discovering metabolite markers for screening and monitoring treatment response and recurrence in patients with bladder cancer. Since naturally-occurring canine transitional cell carcinoma of the urinary bladder is very similar to human invasive bladder cancer, spontaneous canine transitional cell carcinoma has been applied as a relevant animal model of human invasive transitional cell carcinoma. In this study, we have focused on profiling the metabolites in urine from dogs with transitional cell carcinoma and healthy control dogs combining nuclear magnetic resonance spectroscopy and statistical analysis methods. (1)H NMR-based metabolite profiling analysis was shown to be an effective approach for differentiating samples from dogs with transitional cell carcinoma and healthy controls based on a partial least square-discriminant analysis of the NMR spectra. In addition, there were significant differences in the levels of six individual metabolites between samples from dogs with transitional cell carcinoma and the control group based on the Student's t-test. These metabolites were selected to build a separate partial least square-discriminant analysis model that was then used to test the classification accuracy. The result showed good classification between transitional cell carcinoma and control groups with the area under the receiver operating characteristic curve of 0.85. The sensitivity and specificity of the model were 86% and 78%, respectively. These results suggest that urine metabolic profiling may have potential for early detection of bladder cancer and of bladder cancer recurrence following treatment, and may enhance our understanding of the mechanisms involved.