Analytical performance evaluation of two automated urine chemistry analysers using two levels of commercially available quality control material.

OBJECTIVE: Evaluate two point-of-care urine chemistry analysers, VetScan SA and VetLab UA using assayed, bilevel (two concentrations) urine quality control material to determine if performance is acceptable for semiquantitative clinical urine chemistry analysis. MATERIALS AND METHODS: Normal and abnormal urine quality control material sent to 23 veterinary practices was evaluated three times by each clinic on in-clinic automated urinalysis instruments. Accuracy, precision and clinical utility were evaluated. RESULTS: Normal urine quality control material: Results for blood, glucose, ketones and bilirubin were 100% accurate and precise for both analysers, and pH values were accurately acidic to neutral. However, pH from VetScan SA had clinically significant negative bias. Abnormal urine quality control material: VetScan SA: blood, microalbumin and bilirubin were 100% accurate; glucose, ketones, and protein demonstrated ≤10% inaccuracy; pH demonstrated 34% inaccuracy. VetLab UA: blood, ketones and bilirubin were 100% accurate; glucose and protein demonstrated ≤10% inaccuracy; pH was 100% accurately neutral to alkaline. CLINICAL SIGNIFICANCE: VetScan SA had marked negative pH bias versus VetLab UA resulting in clinically significant, overly acidic results. Specific gravity, nitrite, and leukocyte test pads should not be used. Both instruments had excellent performance in normal quality control material. While blood, glucose, protein and bilirubin are correctly identified as present in abnormal quality control material, exact concentrations cannot be interpreted due to imprecision. Only semiquantitative results, not numerical values implying quantification, should be reported from urine test strips.

Comparing Finger-stick Βeta-hydroxybutyrate with Dipstick Urine Tests in the Detection of Ketone Bodies in the Diagnosis of Children with Diabetic Ketoacidosis.

OBJECTIVE: To compare the finger-stick ß-hydroxybutyrate (ß-OHB) method accuracy with dipstick urine test for the detection of ketone bodies to diagnose diabetic ketoacidosis in children. STUDY DESIGN: Cross-sectional study. PLACE AND DURATION OF STUDY: Department of Pediatrics, National Institute of Child Health, Karachi, from March to August 2021. METHODOLOGY: Ninety-six known or newly diagnosed diabetic patients aged 2-15 years with suboptimal glycemic control and diabetic ketoacidosis were included in the study. A urine dipstick test was utilised to evaluate the absence or presence of ketones in the urine. In point-of-care, blood ß-OHB levels were recorded. RESULTS: Among 96 children, with median age of 10 years (IQR=6-11), 11 (11.5%) children had traces of urine ketones, 7 (7.3%) had + urine ketones, 19 (19.8%) had ++ urine ketones, 26 (27.1%) had +++ ketones and 19 (19.8%) had ++++ ketones. In 66 patients (68.75%), capillary blood ketone was observed to be positive by a finger-stick ß-OHB method. The finger-stick ß-OHB method had a higher sensitivity (90.4% vs. 84.9%), specificity (100% vs. 91.3%), and accuracy (92.7% vs. 86.5%) than the dipstick urine test. CONCLUSION: Finger-stick ß-OHB method can serve as a more accurate alternative to the urinary dipstick method for the measurement of ketones and to exclude ketosis and diagnosis of diabetic ketoacidosis (DKA) in hyperglycemic children. KEY WORDS: Diabetes mellitus, Hyperglycemia, Diabetic ketoacidosis, Point-of-care testing, Ketosis, Urine ketones, Acetoacetates.

Effects of a Non-Energy-Restricted Ketogenic Diet on Clinical Oral Parameters. An Exploratory Pilot Trial.

Ketogenic diets (KDs) may be a helpful complement in the prevention of and therapy for several diseases. Apart from their non-cariogenic properties, it is still unclear how KDs affect oral parameters. The aim of this study was to investigate the influence of a KD on clinical periodontal parameters. Twenty generally healthy volunteers with an average age of 36.6 years underwent a KD for 6 weeks. Their compliance was monitored by measuring their urinary ketones daily and by keeping 7-day food records. Clinical oral parameters included plaque (PI), gingival inflammation (GI), a complete periodontal status (probing depths, bleeding on probing), and general physical and serologic parameters at baseline and after 6 weeks. The results showed a trend towards lower plaque values, but with no significant changes from baseline to the end of the study with regard to the clinical periodontal parameters. However, their body weight and BMI measurements showed a significant decrease. The regression analyses showed that the fat mass and the BMI were significantly positively correlated to periodontal inflammation, while HDL, fiber, and protein intake were negatively correlated to periodontal inflammation. The KD change did not lead to clinical changes in periodontal parameters in healthy participants under continued oral hygiene, but it did lead to a significant weight loss.

Application of liquid chromatography coupled to data-independent acquisition mass spectrometry for the metabolic profiling of N-ethyl heptedrone.

We have investigated the metabolic profile of N-ethyl heptedrone, a new designer synthetic stimulant drug, by using data independent acquisition mass spectrometry. Phase I and phase II metabolism was studied by in vitro models, followed by liquid-chromatography coupled to mass spectrometry, to characterize and pre-select the most diagnostic markers of intake. N-ethyl heptedrone was incubated in the presence of pooled human liver microsomes. The contribution of individual enzymatic isoforms in the formation of the phase I and phase II metabolites was further investigated by using human recombinant cDNA-expressed cytochrome P450 enzymesand uridine 5'-diphospho glucuronosyltransferases. The analytical workflow consisted of liquid-liquid extraction with tert-butyl-methyl-ether at alkaline pH, performed before (to investigate the phase I metabolic profile) and after (to investigate the glucuronidation profile) enzymatic hydrolysis. The separation, identification, and determination of the compounds formed in the in vitro experiments were carried out by using liquid chromatography coupled to either high- or low-resolution mass spectrometry. Data independent acquisition method, namely sequential window acquisition of all theoretical fragment-ion spectra (SWATH®) and product ion scan were selected for high-resolution mass spectrometry, whereas multiple reaction monitoring was used for low-resolution mass spectrometry. Thirteen phase-I metabolites were isolated, formed from reactions being catalyzed mainly by CYP1A2, CYP2C9, CYP2C19 and CYP2D6 and, to a lesser degree, by CYP3A4 and CYP3A5. The phase I biotransformation pathways included hydroxylation in different positions, reduction of the ketone group, carbonylation, N-dealkylation, and combinations of the above. Most of the hydroxylated metabolites underwent conjugation reactions to form the corresponding glucurono-conjugated metabolites. Based on our in vitro observation, the metabolic products resulting from reduction of the keto group, N-dealkylation and hydroxylation of the aliphatic chain appear to be the most diagnostic target analytes to be selected as markers of exposure to N-ethyl heptedrone.

Dietary-Induced Ketogenesis: Adults Are Not Children.

There is increasing interest in the use of a ketogenic diet for various adult disorders; however, the ability of adults to generate ketones is unknown. Our goal was to challenge the hypothesis that there would be no difference between adults and children regarding their ability to enter ketosis. METHODS: Two populations were studied, both treated with identical very low-carbohydrate high-fat diets: a retrospective series of children with epilepsy or/and metabolic disorders (2009-2016) and a prospective clinical trial of adults with glioblastoma. Dietary intake was assessed based upon written food diaries and 24-h dietary recall. Ketogenic ratio was calculated according to [grams of fat consumed]/[grams of carbohydrate and protein consumed]. Ketone levels (ß-hydroxybutyrate) were measured in blood and/or urine. RESULTS: A total of 168 encounters amongst 28 individuals were analyzed. Amongst both children and adults, ketone levels correlated with nutritional ketogenic ratio; however, the absolute ketone levels in adults were approximately one quarter of those seen in children. This difference was highly significant in a multivariate linear regression model, p < 0.0001. CONCLUSIONS: For diets with comparable ketogenic ratios, adults have lower blood ketone levels than children; consequently, high levels of nutritional ketosis are unobtainable in adults.

Neutropenia in Children Treated With Ketogenic Diet Therapy.

OBJECTIVES: The objectives were to investigate the relationship between ketogenic diet therapy and neutropenia in children with epilepsy. METHODS: A retrospective chart review of children who initiated ketogenic diet at the Hospital for Sick Children between January 1, 2000, and May 1, 2018 was performed. Factors associated with the development of neutropenia during ketogenic diet therapy were evaluated and the relationship between development of a significant or suspected infection and neutrophil count was analyzed. RESULTS: One hundred two children met inclusion criteria and were followed on the diet for up to 24 months. Thirteen of 102 (13%) children were neutropenic at diet initiation. In the remaining 89 children, 27 developed neutropenia. Developing neutropenia was significantly associated with the ketogenic diet at 6 (13%), 12 (23%), and 24 (25%) months follow-up. Developing neutropenia was associated with higher urinary ketones (OR = 4.26, 95% CI: 1.27, 14.15) and longer duration of ketogenic diet therapy (OR = 3.29, 95% CI: 1.42, 7.96). There was no significant association between development of a clinically significant infection and neutropenia. CONCLUSION: Ketogenic diet therapy is associated with neutropenia in children with epilepsy, however, it does not have a significant clinical impact. Concern regarding neutropenia should not discourage the use of the ketogenic diet in children.

A 2 Week Cross-over Intervention with a Low Carbohydrate, High Fat Diet Compared to a High Carbohydrate Diet Attenuates Exercise-Induced Cortisol Response, but Not the Reduction of Exercise Capacity, in Recreational Athletes.

Low carbohydrate, high fat (LCHF) diets are followed by athletes, but questions remain regarding effects of LCHF on metabolic adaptation, exercise-induced stress, immune function and their time-course. In this cross-over study, 14 recreational male athletes (32.9 ± 8.2 years, VO2max 57.3 ± 5.8 mL/kg/min) followed a two week LCHF diet (<10 En% carbohydrates (CHO), ~75En% Fat) and a two week HC diet (>50 En% CHO), in random order, with a wash-out period of >2 weeks in between. After 2 days and 2 weeks on either diet, participants performed cycle ergometry for 90 min at 60%Wmax. Blood samples for analysis of cortisol, free fatty acids (FFA), glucose and ketones, and saliva samples for immunoglobin A (s-IgA) were collected at different time points before and after exercise. The LCHF diet resulted in higher FFA, higher ketones and lower glucose levels compared to the HC diet (p < 0.05). Exercise-induced cortisol response was higher after 2 days on the LCHF diet (822 ± 215 nmol/L) compared to 2 weeks on the LCHF diet (669 ± 243 nmol/L, p = 0.004) and compared to both test days following the HC diet (609 ± 208 and 555 ± 173 nmol/L, both p < 0.001). Workload was lower, and perceived exertion higher, on the LCHF diet compared to the HC diet on both occasions. A drop in s-IgA following exercise was not seen after 2 days on the LCHF diet, in contrast to the HC diet. In conclusion, the LCHF diet resulted in reduced workload with metabolic effects and a pronounced exercise-induced cortisol response after 2 days. Although indications of adaptation were seen after 2 weeks on the LCHF diet, work output was still lower.

Exogenous Ketosis Impairs 30-min Time-Trial Performance Independent of Bicarbonate Supplementation.

PURPOSE: We recently demonstrated that coingestion of NaHCO3 to counteract ketoacidosis resulting from oral ketone ester (KE) intake improves mean power output during a 15-min time trial (TT) at the end of a 3-h cycling race by ~5%. This ergogenic effect occurred at a time when blood ketone levels were low, as ketosis was only induced during the initial ~2 h of the race. Therefore, in the current study, we investigated whether performance also increases if blood ketone levels are increased in the absence of ketoacidosis during high-intensity exercise. METHODS: In a double-blind crossover design, 14 well-trained male cyclists completed a 30-min TT (TT30') followed by an all-out sprint at 175% of lactate threshold (SPRINT). Subjects were randomized to receive (i) 50 g KE, (ii) 180 mg·kg-1 body weight NaHCO3 (BIC), (iii) KE + BIC, or (iv) a control drink (CON). RESULTS: KE ingestion increased blood d-ß-hydroxybutyrate to ~3-4 mM during the TT30' and SPRINT (P < 0.001 vs CON). In KE, blood pH and bicarbonate concomitantly dropped, causing 0.05 units lower pH and 2.6 mM lower bicarbonate in KE compared with CON during the TT30' and SPRINT (P < 0.001 vs CON). BIC coingestion resulted in 0.9 mM higher blood d-ß-hydroxybutyrate (P < 0.001 vs KE) and completely counteracted ketoacidosis during exercise (P > 0.05 vs CON). Mean power output during TT30' was similar between CON and BIC at 281 W, but was 1.5% lower in the KE conditions (main effect of KE: P = 0.03). Time to exhaustion in the SPRINT was ~64 s in CON and KE and increased by ~8% in the BIC conditions (main effect of BIC: P < 0.01). DISCUSSION: Neutralization of acid-base disturbance by BIC coingestion is insufficient to counteract the slightly negative effect of KE intake during high-intensity exercise.

Bicarbonate Unlocks the Ergogenic Action of Ketone Monoester Intake in Endurance Exercise.

PURPOSE: We recently reported that oral ketone ester (KE) intake before and during the initial 30 min of a 3 h 15 min simulated cycling race (RACE) transiently decreased blood pH and bicarbonate without affecting maximal performance in the final quarter of the event. We hypothesized that acid-base disturbances due to KE overrules the ergogenic potential of exogenous ketosis in endurance exercise. METHODS: Nine well-trained male cyclists participated in a similar RACE consisting of 3 h submaximal intermittent cycling (IMT180') followed by a 15-min time trial (TT15') preceding an all-out sprint at 175% of lactate threshold (SPRINT). In a randomized crossover design, participants received (i) 65 g KE, (ii) 300 mg·kg-1 body weight NaHCO3 (BIC), (iii) KE + BIC, or (iv) a control drink (CON), together with consistent 60 g·h-1 carbohydrate intake. RESULTS: KE ingestion transiently elevated blood D-ß-hydroxybutyrate to ~2-3 mM during the initial 2 h of RACE (P < 0.001 vs CON). In KE, blood pH concomitantly dropped from 7.43 to 7.36 whereas bicarbonate decreased from 25.5 to 20.5 mM (both P < 0.001 vs CON). Additional BIC resulted in 0.5 to 0.8 mM higher blood D-ß-hydroxybutyrate during the first half of IMT180' (P < 0.05 vs KE) and increased blood bicarbonate to 31.1 ± 1.8 mM and blood pH to 7.51 ± 0.03 by the end of IMT180' (P < 0.001 vs KE). Mean power output during TT15' was similar between KE, BIC, and CON at ~255 W but was 5% higher in KE + BIC (P = 0.02 vs CON). Time to exhaustion in the sprint was similar between all conditions at ~60 s (P = 0.88). Gastrointestinal symptoms were similar between groups. DISCUSSION: The coingestion of oral bicarbonate and KE enhances high-intensity performance at the end of an endurance exercise event without causing gastrointestinal distress.

SGLT2 inhibition versus sulfonylurea treatment effects on electrolyte and acid-base balance: secondary analysis of a clinical trial reaching glycemic equipoise: Tubular effects of SGLT2 inhibition in Type 2 diabetes.

Sodium-glucose transporter (SGLT)2 inhibitors increase plasma magnesium and plasma phosphate and may cause ketoacidosis, but the contribution of improved glycemic control to these observations as well as effects on other electrolytes and acid-base parameters remain unknown. Therefore, our objective was to compare the effects of SGLT2 inhibitors dapagliflozin and sulfonylurea gliclazide on plasma electrolytes, urinary electrolyte excretion, and acid-base balance in people with Type 2 diabetes (T2D). We assessed the effects of dapagliflozin and gliclazide treatment on plasma electrolytes and bicarbonate, 24-hour urinary pH and excretions of electrolytes, ammonium, citrate, and sulfate in 44 metformin-treated people with T2D and preserved kidney function. Compared with gliclazide, dapagliflozin increased plasma chloride by 1.4 mmol/l (95% CI 0.4-2.4), plasma magnesium by 0.03 mmol/l (95% CI 0.01-0.06), and plasma sulfate by 0.02 mmol/l (95% CI 0.01-0.04). Compared with baseline, dapagliflozin also significantly increased plasma phosphate, but the same trend was observed with gliclazide. From baseline to week 12, dapagliflozin increased the urinary excretion of citrate by 0.93 ± 1.72 mmol/day, acetoacetate by 48 µmol/day (IQR 17-138), and ß-hydroxybutyrate by 59 µmol/day (IQR 0-336), without disturbing acid-base balance. In conclusion, dapagliflozin increases plasma magnesium, chloride, and sulfate compared with gliclazide, while reaching similar glucose-lowering in people with T2D. Dapagliflozin also increases urinary ketone excretion without changing acid-base balance. Therefore, the increase in urinary citrate excretion by dapagliflozin may reflect an effect on cellular metabolism including the tricarboxylic acid cycle. This potentially contributes to kidney protection.

Use of capillary ketones monitoring in treatment of mild ketotic crisis in people with ketosis-prone atypical diabetes.

This study was carried out to assess the potential reduction in duration of intensive diabetic ketoacidosis treatment in adults with ketosis-prone atypical diabetes (KPD) when using capillary versus urinary ketones. In this cross-sectional study, we included 20 people with KPD presented at the National Obesity Center of the Yaoundé Central Hospital with hyperglycemic decompensation (random capillary glucose ≥13 mmol/L) and significant ketosis (ketonuria≥++) requiring intensive insulin treatment. In all subjects, intensive insulin treatment was initiated at 10 UI per hour with simultaneous measurement of capillary beta-hydroxybutyrate and ketonuria every 2 hours until disappearance of ketonuria. Time-to-disappearance of urine ketones was compared with the time-to-normalization of capillary ß-hydroxybutyrate concentrations. Subjects were aged 46±13 years with a median duration of diabetes of 1.5 (IQR: 0-2.5) years. On admission, the mean blood glucose was 22.8±5 mmol/L and capillary ketones level was 2.9±2.7 mmol/L. The median time-to-disappearance of ketonuria was 5 (IQR: 3-8) hours compared with the time-to-normalization of capillary ß-hydroxybutyrate of 4 (IQR: 2-6) hours, p=0.0002. The absolute difference in time-to-normalization of ketonuria versus ketonemia was 2 (IQR: 1-3) hours and the relative time reduction of treatment was 32.5%±18.0%. Our results suggested that the use of capillary ketones versus ketonuria would allow a significant reduction in duration of intensive insulin treatment by one third in people with KPD.

Sensitive mass spectrometric analysis of carbonyl metabolites in human urine and fecal samples using chemoselective modification.

Metabolites with ketone or aldehyde functionalities comprise a large proportion of the human metabolome, most notably in the form of sugars. However, these reactive molecules are also generated through oxidative stress or gut microbiota metabolism and have been linked to disease development. The discovery and structural validation of this class of metabolites over the large concentration range found in human samples is crucial to identify their links to pathogenesis. Herein, we have utilized an advanced chemoselective probe methodology alongside bioinformatic analysis to identify carbonyl-metabolites in urine and fecal samples. In total, 99 metabolites were identified in urine samples and the chemical structure for 40 metabolites were unambiguously validated using a co-injection procedure. We also describe the preparation of a metabolite-conjugate library of 94 compounds utilized to efficiently validate these ketones and aldehydes. This method was used to validate 33 metabolites in a pooled fecal sample extract to demonstrate the potential for rapid and efficient metabolite detection over a wide metabolite concentration range. This analysis revealed the presence of six metabolites that have not previously been detected in either sample type. The constructed library can be utilized for straightforward, large-scale, and expeditious analysis of carbonyls in any sample type.

Untargeted UPLC-MS metabolomics reveals multiple changes of urine composition in healthy adult volunteers after consumption of curcuma longa L. extract.

Curcuma longa L. is used as food supplement to prevent diseases, although limited studies have been performed on healthy subjects up to now. In the present work, an untargeted UPLC-MS metabolomics approach was applied to study the changes of 24-hours urinary composition on healthy volunteers due to a 28-days daily consumption of a dried C. longa extract containing a standardized amount of curcuminoids. Changes in the excretion of different metabolites were observed after supplementation. Curcumin and two metabolic derivatives (hexahydrocurcumin and dihydrocurcumin) were detected in urine, indicating the absorption of the main curcuminoid from the extract and its further metabolism by liver and gut microbiota. For the first time ar-turmerone, the main apolar constituent of curcuma, was detected in urine in intact form, and its presence was confirmed by a targeted GC-MS analysis. The increase of tetranor-PGJM and tetranor-PGDM, two prostaglandin-D2 metabolites, was observed, being related to the anti-inflammatory effect exerted by curcuma. The variation of the amounts of HPAG, PAG, proline-betaine and hydroxyphenyllactic acid indicate that the supplementation induced changes to the activity of gut microbiota. Finally, the reduced excretion of niacin metabolites (nicotinuric acid, trigonelline and 2PY) and medium- and short-chain acylcarnitines suggests that curcuma could induce the mitochondrial ß-oxidation of fatty acids for energy production in healthy subjects. Overall, the results indicate that a prolonged daily consumption of a dried curcuma extract exerts multiple effects on healthy subjects, furthermore they show the opportunity offered by untargeted metabolomics for the study of the bioactivity of natural extracts in healthy human volunteers.

Serum vitamin D concentrations at dry-off and close-up predict increased postpartum urine ketone concentrations in dairy cattle.

Vitamin D is commonly supplemented to dairy cows as vitamin D3 to support calcium homeostasis and in times of low sunlight exposure. Vitamin D has beneficial immunomodulatory and anti-inflammatory properties. Serum 25-hydroxyvitamin D [25(OH)D] concentrations fluctuated during lactation, with the lowest concentrations measured in healthy cows within 7 d of calving. However, it is unknown if serum 25(OH)D concentrations measured during the previous lactation are associated with transition diseases or health risk factors in dairy cattle. We collected serum samples from 279 dairy cattle from 5 commercial dairy herds in Michigan at dry-off, close-up, and 2-10 d in milk (DIM). Vitamin D concentrations were determined by measuring serum 25(OH)D by radioimmunoassay. Total serum calcium was measured by colorimetric methods. Body condition scores (BCS) were assigned at the time of blood collection. Clinical disease incidence was monitored until 30 d postparturition. Separate bivariable logistic regression analyses were used to determine if serum 25(OH)D at dry-off, close-up, and 2-10 DIM was associated with various clinical diseases including mastitis, lameness, and uterine disorders (classified as metritis, retained placenta, or both) and increased urine ketone concentrations at P < 0.05. Among all significant bivariable analyses, multivariable logistic regression analyses were built to adjust for potential confounding variables including parity, BCS, season, and calcium. Receiver operator characteristic (ROC) curve analyses were used to determine optimal concentrations of serum 25(OH)D. We found that higher serum 25(OH)D concentrations at dry-off and close-up predicted increased urine ketone concentrations in early lactation, even after adjustment for confounders. Alternatively, we found that lower serum 25(OH)D at 2-10 DIM was associated with uterine diseases. Optimal concentrations for serum 25(OH)D at dry-off and close-up for lower risk of increased urine ketone concentrations were below 103.4 and 91.1 ng/mL, respectively. The optimal concentration for serum 25(OH)D at 2-10 DIM for uterine diseases was above 71.4 ng/mL. These results indicate that serum 25(OH)D at dry-off and close-up may be a novel predictive biomarker for increased urine ketone concentrations during early lactation. Increased urine ketone concentrations are not necessarily harmful or diagnostic for ketosis but do indicate development of negative energy balance, metabolic stress, and increased risk of early lactation diseases. Predicting that dairy cattle are at increased risk of disease facilitates implementation of intervention strategies that may lower disease incidence. Future studies should confirm these findings and determine the utility of serum 25(OH)D concentrations as a predictive biomarker for clinical and subclinical ketosis.

Evaluation of the accuracy of urine analyzers in dogs and cats.

The accuracy of urine analyzers used for dogs and cats has remained uncertain. This study examines the agreement between results of urine analysis obtained using two devices marketed for animals and for humans and the results of quantitative biochemical analysis. The degrees of concordance for bilirubin and ketones in the same category were ~80%, but for pH these were only ~60% in dogs and cats. Degrees of concordance for protein and the UP/C ratio clearly differed between the devices for animals and humans. We found that values for bilirubin and ketones obtained using urine analyzers may be reliable, but pH is unlikely to be accurate enough to be clinically useful for dogs and cats.

Ketone ester supplementation blunts overreaching symptoms during endurance training overload.

KEY POINTS: Overload training is required for sustained performance gain in athletes (functional overreaching). However, excess overload may result in a catabolic state which causes performance decrements for weeks (non-functional overreaching) up to months (overtraining). Blood ketone bodies can attenuate training- or fasting-induced catabolic events. Therefore, we investigated whether increasing blood ketone levels by oral ketone ester (KE) intake can protect against endurance training-induced overreaching. We show for the first time that KE intake following exercise markedly blunts the development of physiological symptoms indicating overreaching, and at the same time significantly enhances endurance exercise performance. We provide preliminary data to indicate that growth differentiation factor 15 (GDF15) may be a relevant hormonal marker to diagnose the development of overtraining. Collectively, our data indicate that ketone ester intake is a potent nutritional strategy to prevent the development of non-functional overreaching and to stimulate endurance exercise performance. ABSTRACT: It is well known that elevated blood ketones attenuate net muscle protein breakdown, as well as negate catabolic events, during energy deficit. Therefore, we hypothesized that oral ketones can blunt endurance training-induced overreaching. Fit male subjects participated in two daily training sessions (3 weeks, 6 days/week) while receiving either a ketone ester (KE, n = 9) or a control drink (CON, n = 9) following each session. Sustainable training load in week 3 as well as power output in the final 30 min of a 2-h standardized endurance session were 15% higher in KE than in CON (both P < 0.05). KE inhibited the training-induced increase in nocturnal adrenaline (P < 0.01) and noradrenaline (P < 0.01) excretion, as well as blunted the decrease in resting (CON: -6 ± 2 bpm; KE: +2 ± 3 bpm, P < 0.05), submaximal (CON: -15 ± 3 bpm; KE: -7 ± 2 bpm, P < 0.05) and maximal (CON: -17 ± 2 bpm; KE: -10 ± 2 bpm, P < 0.01) heart rate. Energy balance during the training period spontaneously turned negative in CON (-2135 kJ/day), but not in KE (+198 kJ/day). The training consistently increased growth differentiation factor 15 (GDF15), but ∼2-fold more in CON than in KE (P < 0.05). In addition, delta GDF15 correlated with the training-induced drop in maximal heart rate (r = 0.60, P < 0.001) and decrease in osteocalcin (r = 0.61, P < 0.01). Other measurements such as blood ACTH, cortisol, IL-6, leptin, ghrelin and lymphocyte count, and muscle glycogen content did not differentiate KE from CON. In conclusion, KE during strenuous endurance training attenuates the development of overreaching. We also identify GDF15 as a possible marker of overtraining.

A Validated HPLC-MS/MS Assay for 14-O-[(4,6-Diaminopyrimidine-2-yl)thioacetyl] Mutilin in Biological Samples and Its Pharmacokinetic, Distribution and Excretion via Urine and Feces in Rats.

14-O-[(4,6-Diaminopyrimidine-2-yl)thioacetyl] mutilin (DPTM), a novel pleuromutilin candidate with a substituted pyrimidine moiety, has been confirmed to possess excellent antibacterial activity against Gram-positive bacteria. To illustrate the pharmacokinetic profile after intravenous (i.v.), intramuscular (i.m.) and oral (p.o.) administrations with DPTM, as well as tissue distribution and excretion via urine and feces in vivo, a specific, sensitive and robust HPLC-MS/MS method was first developed to determine DPTM in rat plasma, various tissues, urine and feces. The plasma, tissues, urine and feces samples were treated by protein precipitation with acetonitrile using tiamulin fumarate as an internal standard (IS). This method which was achieved on an HPLC system detector equipped with an ESI interface, was sensitive with 5 ng/mL as the lower limit of detection and exhibited good linearity (R² > 0.9900) in the range of 5⁻4000 ng/mL for plasma, various tissues, urine and feces, as well as intra-day precision, inter-day precision and accuracy. The matrix effects ranged from 94.2 to 109.7% with RSD ≤ 9.4% and the mean extraction recoveries ranged from 95.4 to 109.5% in plasma, tissue homogenates, urine and feces (RSD ≤ 9.9). After i.v., i.m. and p.o. administrations, DPTM was rapidly absorbed and metabolized in rats with the half-life (t1/2) of 1.70⁻1.86, 3.23⁻3.49 and 4.38⁻4.70 for 10, 25 and 75 mg/kg doses, respectively. The tissue distribution showed that DPTM was diffused into all the tested tissues, especially into the intestine and lung. Excretion via urine and feces studies demonstrated that DPTM was mainly excreted by feces after administration.

Diabetic Ketoacidosis in Adolescents and Children: A Prospective Study of Blood versus Urine Ketones in Monitoring Therapeutic Response.

BACKGROUND: diabetic ketoacidosis (DKA) is a potentially lethal complication of diabetes mellitus (DM). There is no study in Indonesia that compares the much-preferred capillary beta hydroxybutirate (ß-OHB) measurement to urine acetoacetate in monitoring therapeutic response of DKA in adolescents. METHODS: a prospective study of 37 adolescents and children with DKA in Cipto Mangunkusumo Hospital was done between June 2006 and March 2011. The patients were followed until the time of DKA resolution. Hourly measurement of random blood glucose, capillary ß-OHB concentration, and urine ketones were done, while blood gas analysis and electrolyte were measured every four hours. RESULTS: median time to resolution was 21 (9-52) hours. Compared to urine ketones, capillary ß-OHB concentration showed stronger correlation with pH (r= -0,52, p= 0,003 vs r= -0,49, p= 0,005) and bicarbonate level (r=-0,60, p=0.000 vs r= -0.48, p=0.007) during the median time of DKA resolution. All capillary ß-OHB measurement yielded negative results at median time of DKA resolution, while urine ketones were still detected up to 9 hours after resolution. CONCLUSION: blood ketone concentration showed better correlation with pH and bicarbonate level, as a tool to monitor therapeutic response in DKA in adolescent, compared to traditional urine ketones test in adolescents.

Urinary excretion of heptanones, heptanoles and 2,5-heptanedione after controlled acute exposure of volunteers to n-heptane.

A lack of well-established parameters and assessment values currently impairs biomonitoring of n-heptane exposure. Using controlled inhalation experiments, we collected information on urinary n-heptane metabolite concentrations and the time course of metabolite excretion. Relationships between external and internal exposure were analysed to investigate the suitability of selected metabolites to reflect n-heptane uptake. Twenty healthy, non-smoking males (aged 19-38 years, median 25.5) were exposed for 3 h to 167, 333 and 500 ppm n-heptane, each. Spot urine samples of the volunteers, collected before exposure and during the following 24 h, were analysed for heptane-2-one, 3-one, 4-one, 2,5-dione, 1-ol, 2-ol, 3-ol, and 4-ol using headspace solid phase dynamic extraction gas chromatography/mass spectrometry (HS-SPDE-GC/MS). Starting from median pre-exposure concentrations between <0.5 (3-one) and 82.9 µg/L (4-one), exposure increased the concentrations for all parameters except for 4-one. Median post-exposure concentrations ranged up to 840.4 µg/L (2-ol) and decreased with half-lifes <3 h after exposure. Non-parametric correlation analyses (n = 47, p < 0.05) revealed weak to moderate associations of volume related metabolite excretion with external exposure for 2-one, 3-one and 2,5-dione (R = 0.332-0.753). Heptanol excretion was moderately associated with exposure (R ≥ 0.509) only after creatinine adjustment. Lacking association with external exposure impedes the use of 4-one as heptane biomarker, whereas 2-ol and 3-ol turned out to be sensitive indicators of exposure if creatinine correction is applied. By providing fundamental data on a panel of eight potential heptane metabolites, our study can help to promote biological monitoring of n-heptane exposure.