Blood Β-hydroxybutyrate Levels Research Articles

Identification of genetic markers associated with hyperketonemia patterns in early lactation Holstein cows.

Ketosis, evidenced by hyperketonemia with elevated blood β-hydroxybutyrate (BHB) levels, is a significant metabolic disorder of dairy cattle, typically diagnosed within the first 6 weeks post-calving when high energy levels are essential to milk production. Our study aimed to identify genetic markers linked to hyperketonemia (HYK) patterns in Holstein cows during early lactation and compare these to HYK-negative cows. We screened 964 cows for HYK using a threshold of BHB ≥1.2 mmol/L during the first 2 weeks postpartum (screening period, SP). Cows that tested negative initially were retested the following week. Cows were deemed HYK-negative (CON group) if BHB levels were below 1.2 mmol/L in both tests, while those with BHB levels exceeding this threshold at any test were treated and classified as HYK-positive (HYK+). Post-treatment, HYK+ cows were monitored for two-week follow-up period (FP) and classified based on their recovery: cured (CUR; consistently low BHB), recurrent (REC; fluctuating BHB levels), severe (SEV; high initial BHB that decreased), or chronic (CHR; persistently high BHB). Using 489 cows that were genotyped, a GWAS was conducted using GCTA software, revealing significant associations of several SNPs across different HYK patterns when compared to the CON group. These SNPs were primarily linked to genes affecting milk traits and were enriched in biological pathways relevant to protein glycosylation, inflammatory response, glucose homeostasis, and fatty acid synthesis. Our findings highlight genomic regions, potential candidate genes, and biological pathways related to ketosis, underscoring potential targets for improving health management in dairy cattle. These insights could lead to better strategies for managing ketosis through genetic selection, ultimately enhancing dairy cattle welfare and productivity. Further research with a larger number of cows is recommended to validate these findings and help confirm the implicated SNPs and genes.

Changes in lipids and inflammation in adults with super-refractory status epilepticus on a ketogenic diet.

Introduction: This study aims to test the hypothesis that increased ketone body production resulting from a ketogenic diet (KD) will correlate with reductions in pro-inflammatory cytokines and lipid subspecies and improved clinical outcomes in adults treated with an adjunctive ketogenic diet for super-refractory status epilepticus (SRSE). Methods: Adults (18years or older) were treated with a 4:1 (fat: carbohydrate and protein) ratio of enteral KD as adjunctive therapy to pharmacologic seizure suppression in SRSE. Blood and urine samples and clinical measurements were collected at baseline (n = 10), after 1week (n = 8), and after 2weeks of KD (n = 5). In addition, urine acetoacetate, serum β-hydroxybutyrate, lipidomics, pro-inflammatory cytokines (IL-1β and IL-6), chemokines (CCL3, CCL4, and CXCL13), and clinical measurements were obtained at these three time points. Univariate and multivariate data analyses were performed to determine the correlation between ketone body production and circulating lipids, inflammatory biomarkers, and clinical outcomes. Results: Changes in lipids included an increase in ceramides, mono-hexosylceramide, sphingomyelin, phosphocholine, and phosphoserines, and there was a significant reduction in pro-inflammatory mediators, IL-6 and CXCL13, seen at 1 and 2weeks of KD. Higher blood β-hydroxybutyrate levels at baseline correlated with better clinical outcomes; however, ketone body production did not correlate with other variables during treatment. Higher chemokine CCL3 levels following treatment correlated with a longer stay in the intensive care unit and a higher modified Rankin Scale score (worse neurologic disability) at discharge and 6-month follow up. Discussion: Adults receiving an adjunctive enteral ketogenic diet for super-refractory status epilepticus exhibit alterations in select pro-inflammatory cytokines and lipid species that may predict their response to treatment.

Donkey Oil-Based Ketogenic Diet Prevents Tumor Progression by Regulating Intratumor Inflammation, Metastasis and Angiogenesis in CT26 Tumor-Bearing Mice.

Colon cancer is one of the typical malignant tumors, and its prevalence has increased yearly. The ketogenic diet (KD) is a low-carbohydrate and high-fat dietary regimen that inhibits tumor growth. Donkey oil (DO) is a product with a high nutrient content and a high bioavailability of unsaturated fatty acids. Current research investigated the impact of the DO-based KD (DOKD) on CT26 colon cancer in vivo. Our findings revealed that DOKD administration significantly lowered CT26+ tumor cell growth in mice, and the blood β-hydroxybutyrate levels in the DOKD group was significantly higher than those in the natural diet group. Western blot results showed that DOKD significantly down-regulated Src, hypoxia inducible factor-1α (HIF-1α), extracellular signal-related kinases 1 and 2 (Erk1/2), snail, neural cadherin (N-cadherin), vimentin, matrix metallopeptidase 9 (MMP9), signal transducer and activator of transcription 3 (STAT3), and vascular endothelial growth factor A (VEGFA), and it significantly up-regulated the expressions of Sirt3, S100a9, interleukin (IL)-17, nuclear factor-kappaB (NF-κB) p65, Toll-like receptor 4 (TLR4), MyD88, and tumor necrosis factor-α. Meanwhile, in vitro validation results showed that LW6 (a HIF-1α inhibitor) significantly down-regulated the expressions of HIF-1α, N-cadherin, vimentin, MMP9, and VEGFA, which supported those of the in vivo findings. Furthermore, we found that DOKD inhibited CT26+ tumor cell growth by regulating inflammation, metastasis, and angiogenesis by activating the IL-17/TLR4/NF-κB p65 pathway and inhibiting the activation of the Src/HIF-1α/Erk1/2/Snail/N-cadherin/Vimentin/MMP9 and Erk1/2/HIF-1α/STAT3/VEGFA pathways. Our findings suggest that DOKD may suppress colon cancer progression and help prevent colon cancer cachexia.

A ketogenic diet containing medium-chain triglycerides reduces REM sleep duration without significant influence on mouse circadian phenotypes

Ketogenic diets (KDs) affect the circadian rhythms of behavior and clock gene expression in experimental animals. However, these diets were designed to simulate a fasting state; thus, whether these effects are caused by diet-induced ketogenesis or persistent starvation is difficult to distinguish. The present study aimed to define the effects of a KD containing medium-chain triglycerides (MCT-KD) that increase blood ketone levels without inducing carbohydrate starvation, on circadian rhythms and sleep regulation. Mice were fed with a normal diet (CTRL) or MCT-KD for 2 weeks. Blood β-hydroxybutyrate levels were significantly increased up to 2 mM by the MCT-KD, whereas body weight gain and blood glucose levels were identical between the groups, suggesting that ketosis accumulated without carbohydrate starvation in the MCT-KD mice. Circadian rhythms of wheel-running activity and core body temperature were almost identical, although wheel-running was slightly reduced in the MCT-KD mice. The circadian expression of the core clock genes, Per1, Per2, Bmal1, and Dbp in the hypothalamus, heart, liver, epididymal adipose tissues, and skeletal muscle were almost identical between the CTRL and MCT-KD mice, whereas the amplitude of hepatic Per2 and adipose Per1 expression was increased in MCT-KD mice. The MCT-KD reduced the duration of rapid-eye-movement (REM) sleep without affecting the duration of non-REM sleep and the duration of wakefulness. These findings suggested that the impact of ketone bodies on circadian systems are limited, although they might reduce locomotor activity and REM sleep duration.

Short- and long-term cognitive and metabolic effects of medium-chain triglyceride supplementation in rats

Medium-chain triglycerides (MCT) possess neuroprotective properties. However, the long-term metabolic consequences of supplementing a regular diet with cognition-enhancing doses of MCT are largely unknown. We studied the effects of chronic (28 days) supplementation of regular diet with different doses of MCT oil (1, 3, or 6 g/kg/day) or water (control) on working memory (Y-maze), behavior in the Open Field, spatial learning (Morris water maze), and weight of internal organs in male Wistar 2.5-m.o. Rats. In a separate experiment, we evaluated acute (single gavage) and chronic (28 days) effects of MCT or lard supplementation (3 g/kg) on blood biochemical parameters. MCT-1 and MCT-3 doses improved working memory in YM. In MWM, MCT-6 treatment improved spatial memory. Chronic MCT-1 or MCT-3 treatment did not affect internal organ weight, while MCT-6 dose increased liver weight and the brown/white adipose tissue ratio. Acutely, MCT administration elevated blood β-hydroxybutyrate and malondialdehyde levels. Chronic MCT administration (3 g/kg) did not affect the blood levels of glucose, lactate, pyruvate, acetoacetate, β-hydroxybutyrate, total and HDL cholesterol, triglycerides, malondialdehyde, and aspartate transaminase and alanine transaminase activities. Therefore, daily supplementation of standard feed with MCT resulted in mild intermittent ketosis. It improved working memory at lower concentrations without significant adverse side effects. At higher concentrations, it improved long-term spatial memory but also resulted in organ weight changes and is likely unsafe. These results highlight the importance of monitoring the metabolic effects of MCT supplementation alongside cognitive assessment in future studies of MCT's neuroprotective properties.

A ketogenic diet in combination with gemcitabine increases survival in pancreatic cancer KPC mice.

Pancreatic ductal adenocarcinoma (PDAC) continues to be a major health problem. A ketogenic diet (KD), characterized by a very low carbohydrate and high fat composition, has gained attention for its anti-tumor potential. We evaluated the effect and mechanisms of feeding a strict KD alone or in combination with gemcitabine in the autochthonous LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx1-Cre (KPC) mouse model. For this purpose, both male and female pancreatic tumor-bearing KPC mice were allocated to a control diet (CD; %kcal: 70% carb, 14% protein, 16% fat), a KD (%kcal: 14% protein, 1% carb, 85% fat), a CD + gemcitabine (CG), or a KD + gemcitabine (KG) group. Mice fed a KD alone or in combination with gemcitabine showed significantly increased blood β-hydroxybutyrate levels compared to mice fed a CD or CG. KPC mice fed a KG had a significant increase in overall median survival compared to KPC mice fed a CD (increased overall median survival by 42%). Interestingly, when the data was disaggregated by sex, the effect of a KG was significant in female KPC mice (60% increase in median overall survival), but not in male KPC mice (28% increase in median overall survival). Mechanistically, the enhanced survival response to a KD combined with gemcitabine was multifactorial, including inhibition of ERK and AKT pathways, regulation of fatty acid metabolism and the modulation of the gut microbiota. In summary, a KD in combination with gemcitabine appears beneficial as a treatment strategy in PDAC in KPC mice, deserving further clinical evaluation.

Oral administration of a novel, synthetic ketogenic compound elevates blood β‐hydroxybutyrate levels in mice in both food‐restricted and ab‐libitum conditions

Elevated ketone levels have been shown to lead to improvement in diseased contexts including epilepsy, diabetes, obesity, cancer, Alzheimer’s, and Parkinson’s Disease. It can be difficult for many patients, especially children, to adhere to the strict and intense ketogenic diet necessary to achieve such elevation via dietary manipulation, hence why the field of metabolic therapy continues to explore the use of exogenous ketone supplementation. This preliminary pharmacodynamic study characterizes the effects of a glycerol acetoacetate tri‐ester on the blood β‐hydroxybutyrate levels in VmDk mice, a model often used in cancer studies in both fasted and non‐restricted states. Promising β‐hydroxybutyrate levels were observed in both conditions along with a hypoglycemic effect when fasted. No detrimental side effects have been observed. Future studies will include characterizing the compound's effects on metabolomics and cytokines.

Effects of delaying increase in milk allowance and type of gradual weaning program on performance and health of calves fed lower levels of milk

The objective of this study was to investigate the effects of delayed increase in milk allowance and type of gradual weaning program on the feed intake, health, and growth of male Holstein calves. Using a 2 × 2 factorial design, each male Holstein calf was randomly assigned to 1 of 2 milk level treatments and 1 of 2 weaning treatments upon arrival to a rearing facility. Calves were either assigned to a moderate (MM) or lower (LM) milk level treatment. All calves received the same milk program from arrival until d 12, increasing from 4 to 5 L/d (115 to 125 g/L) over the 12 d. On d 13, MM calves began receiving additional milk compared with LM calves until they were weaned, with MM offered milk increasing to a peak of 9 L/d (140 g/L). Calves on the MM treatment received a total of 35 kg of milk replacer over 45 d, compared with LM calves, who received a total of 25 kg of milk replacer over 45 d. Calves were also assigned to be weaned by either a 2-step gradual program or a 4-step gradual program. Calves assigned to the 2-step program had their milk reduced in 2 steps from d 30 to 45, and 4-step calves had their milk reduced in 4 steps from d 30 to 45. After d 46, calves received no milk but continued to be monitored until d 59. Calves had ad libitum access to water from arrival, and solid feed was offered from d 5 onward. No interactions of effects of milk level and weaning treatment were detected. By design, calves on the MM treatment consumed greater amounts of milk than LM calves in the preweaning phase from d 13 to 29 (6.1 vs. 4.8 L/d) and weaning phases from d 30 to 45 (4.7 vs. 3.2 L/d). The LM calves consumed greater amounts of solid feed than MM calves from d 22 to 29 of the preweaning phase, and from d 30 to 40 and d 43 to 45 of the weaning phase, whereas postweaning feed consumption was similar for both milk treatments. Calves in both milk treatments had similar blood β-hydroxybutyrate levels, indicating similar rumen development. Body weight and size measurements were similar between milk treatments across the study, although LM calves had a greater average daily gain than MM calves between d 38 and 46 (0.96 vs. 0.71 kg/d). Both gradual weaning treatments resulted in similar feed consumption, growth, and health parameters. Overall, the results indicate that delaying a moderate increase in milk until 12 d after arrival at a rearing facility provided no overall beneficial effect on growth or rumen development.

A Ketogenic & Low-Protein Diet Slows Retinal Degeneration in rd10 Mice.

PurposeTreatments that delay retinal cell death regardless of genetic causation are needed for inherited retinal degeneration (IRD) patients. The ketogenic diet is a high-fat, low-carbohydrate diet, used to treat epilepsy, and has beneficial effects for neurodegenerative diseases. This study aimed to determine whether the ketogenic diet could slow retinal degeneration.MethodsEarly weaned, rd10 and wild-type (WT) mice were placed on either standard chow, a ketogenic diet, or a ketogenic & low-protein diet. From postnatal day (PD) 23 to PD50, weight and blood β-hydroxybutyrate levels were recorded. Retinal thickness, retinal function, and visual performance were measured via optical coherence tomography, electroretinography (ERG), and optokinetic tracking (OKT). At PD40, serum albumin, rhodopsin protein, and phototransduction gene expression were measured.ResultsBoth ketogenic diets elicited a systemic induction of ketosis. However, rd10 mice on the ketogenic & low-protein diet had significant increases in photoreceptor thickness, ERG amplitudes, and OKT thresholds, whereas rd10 mice on the ketogenic diet showed no photoreceptor preservation. In both rd10 and WT mice, the ketogenic & low-protein diet was associated with abnormal weight gain and decreases in serum albumin levels, 27% and 56%, respectively. In WT mice, the ketogenic & low-protein diet was also associated with an ∼20% to 30% reduction in ERG amplitudes.ConclusionsThe ketogenic & low-protein diet slowed retinal degeneration in a clinically relevant IRD model. In WT mice, the ketogenic & low-protein diet was associated with a decrease in phototransduction and serum albumin, which could serve as a protective mechanism in the rd10 model. Although ketosis was associated with protection, its role remains unclear.Translational RelevanceNeuroprotective mechanisms associated with the ketogenic & low-protein diet have potential to slow retinal degeneration.

The distinctive short-term response of late-pregnant prolific ewes to propylene glycol or glycerol drenching

Pregnancy toxemia is the most frequent metabolic disorder of ewes in late pregnancy. Although propylene glycol (PG) and glycerol (GLY) are common glucogenic supplements for treating pregnancy toxemia in ewes, the relative benefit of these 2 supplements is not entirely clear. Therefore, the objectives of the present study were to determine the changes during 24 h in key blood metabolites and insulin in response to PG or GLY drenching in prolific ewes. To this end, 36 multiparous late-pregnant Afec-Assaf ewes (∼132.4 d pregnant) bearing 2 to 4 fetuses, divided into 2 blocks (18 ewes in each block), with a blood β-hydroxybutyrate (BHB) concentration of 0.5 to 1.6 mmol/L were included. Ewes were divided into 3 groups (12 ewes each; 6 ewes in each experimental day), according to their BHB levels, expected litter size, body weight, and body condition score, and were drenched with the following: (1) control group (CTL), 55 mL of water; (2) PG, 106 mL of PG (100% PG, 448 calories); or (3) GLY, 108 mL of Koforin 80 (80% GL; 448 calories). Blood samples were taken before drenching and every hour after drenching for 24 h. Plasma concentration of glucose, BHB, nonesterified fatty acids, lactate, glycerol, and insulin were determined. Because there were no effects of treatments after 12 h in the first block, the data were analyzed for 12 h after drenching rather than 24 h. The plasma glucose concentration during the first 5 h after drenching was the highest in the GLY, BHB concentration was the lowest in the PG, and the nonesterified fatty acid levels were lower in the PG compared with the CTL ewes during the first 5 h after drenching. However, glucose concentration was higher in the PG ewes at 9, 11, and 12 h after drenching than in CTL or GLY ewes. The mean lactate concentration in plasma for 12 h was 2.5- and 1.9-fold higher in the PG compared with the CTL and GLY ewes, respectively, and except at 11 h after drenching, it was significantly higher at each time point. The insulin concentration was higher in the GLY than in both other groups at 2 to 5 h after drenching. These results suggest that during the first few hours after drenching the effect of PG was more effective in reducing the BHB concentration, whereas the GLY effect was more effective in enhancing glucose concentration. The increased concentration in lactate following PG treatment suggests that the PG contribution to gluconeogenesis is mediated through its metabolism to lactate. In contrast, the lack of an effect on lactate, and the faster increase in blood glucose in response to GLY suggest that GLY has a more advanced entry point to gluconeogenesis, which influences the immediate response in enhancing the glucose blood concentration.

Ketogenic Diet Elicits Antitumor Properties through Inducing Oxidative Stress, Inhibiting MMP-9 Expression, and Rebalancing M1/M2 Tumor-Associated Macrophage Phenotype in a Mouse Model of Colon Cancer.

Many advanced cancers are characterized by metabolic disorders. A dietary therapeutic strategy was proposed to inhibit tumor growth through administration of low-carbohydrate, average-protein, and high-fat diet, which is also known as ketogenic diet (KD). In vivo antitumor efficacy of KD on transplanted CT26+ tumor cells in BALB/c mice was investigated. The results showed that the KD group had significantly higher blood β-hydroxybutyrate and lower blood glucose levels when compared with the normal diet group. Meanwhile, KD increased intratumor oxidative stress, and TUNEL staining showed KD-induced apoptosis against tumor cells. Interestingly, the distribution of CD16/32+ and iNOS+ M1 tumor-associated macrophages (TAMs) increased in the KD-treated group, with concomitantly less arginase-1+ M2 TAMs. Moreover, KD treatment downregulated the protein expression of matrix metalloproteinase-9 in CT26+ tumor-bearing mice. Western blot analysis demonstrated that the expression levels of HDAC3/PKM2/NF-κB 65/p-Stat3 proteins were reduced in the KD-treated group. Taken together, our results indicated that KD can prevent the progression of colon tumor via inducing intratumor oxidative stress, inhibiting the expression of the MMP-9, and enhancing M2 to M1 TAM polarization. A novel potential mechanism was identified that KD can prevent the progression of colon cancer by regulating the expression of HDAC3/PKM2/NF-κB65/p-Stat3 axis.

Clinical Implications of Ketosis in Children with Benign Convulsions with Mild Gastroenteritis.

Background and PurposeThe pathophysiologic mechanisms underlying benign convulsions with mild gastroenteritis (CwG) in children remain unclear. We investigated the incidence of ketosis in CwG and whether this is related to seizures.MethodsThis retrospective study included children aged from 6 months to 6 years who visited our emergency department and were diagnosed as CwG between June 2015 and December 2018. The clinical and laboratory data were analyzed for these cases. Ketosis and severe ketosis were defined as blood β-hydroxybutyrate levels of ≥0.6 and ≥4.5 mmol/L, respectively.ResultsWe enrolled 42 pediatric CwG patients aged 21.0±11.5 months (mean±SD) whose blood β-hydroxybutyrate level was 3.65±1.51 mmol/L. Ketosis was observed in 95.2% of these children, while 35.7% had severe ketosis. Compared to the non-severe-ketosis group (n=27), the severe-ketosis group (n=15) demonstrated significantly lower blood glucose levels (68.8 vs. 82.6 mg/dL, p=0.020) and sodium levels (134.2 vs. 135.6 mEq/L, p=0.018), and included a larger proportion of low-body-weight children (defined as adjusted weight <50th percentile for age and sex) (53.3% vs. 18.5%, p=0.019). However, the incidence of repetitive seizures (two or more during an illness period) did not differ between these groups. Moreover, severe ketosis was not associated with the risk of seizure recurrence in the emergency department.ConclusionsChildren with CwG are in a state of considerable ketosis. Severe ketosis in CwG may be associated with low blood glucose and sodium levels but does not reduce seizure recurrence.

2401-PUB: High Rates of Unrecognized Ketosis in Youth with T1D and Elevated A1c Levels

Youth with type 1 diabetes (T1D) who have A1c levels ≥ 9% have a 13-fold higher rate of diabetic ketoacidosis (DKA) than youth with A1c &amp;lt;7.5% and the rate of DKA is 2-fold higher with multiple daily injections (MDI) than with pump therapy. However, the frequency of mild to moderate ketosis (the gateway to DKA in these high-risk patients) is largely unknown and such knowledge is essential to evaluate strategies aimed at DKA prevention. Methods: Youth ages 8-18 years with uncontrolled T1D (A1c&amp;gt; 8.5%) on MDI treatment were enrolled in the study. Blood glucose (BG) and blood β-hydroxybutyrate (BHB) levels were measured by school personnel in the AM on arrival to school. Results: 14 patients (age 15 ± 2.3 years) enrolled in the study had A1c 10.7 ± 1.5% (range 8.8 - 14.0%). BHB or urine ketones were measured on 125 school days; ketone levels were small (0.6-0.9 mmol/L) on 6.4% of days (mean BHB 0.7+0.1 mmol/L) and moderate/large (&amp;gt;0.9 mmol/L) on 10.4% of days (mean BHB 2.1+1.4 mmol/L) (Figure). BG ranged between 147 to &amp;gt;600 mg/dL on days with elevated BHB levels; all were without other signs or symptoms of DKA. Conclusions: Unrecognized mild to moderate ketosis during regular school attendance is common in youth with uncontrolled T1D on MDI therapy. Novel strategies, such as having school personnel supervise injections of long-acting basal insulin, are needed to support reliable insulin dosing to prevent ketosis in youth with uncontrolled T1D at risk for DKA Disclosure L.M. Nally: None. S. Elahi: None. M.A. Van Name: Research Support; Self; Novo Nordisk Inc. A. Steffen: None. E.M. Tichy: None. A.D. Urban: None. K. Weyman: None. W.V. Tamborlane: Consultant; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Medtronic MiniMed, Inc., Novo Nordisk Inc., Sanofi, Takeda Pharmaceutical Company Limited. Funding Novo Nordisk

Caloric restriction promotes cell survival in a mouse model of normal tension glaucoma.

Glaucoma is characterized by progressive degeneration of retinal ganglion cells (RGCs) and their axons. We previously reported that loss of glutamate transporters (EAAC1 or GLAST) in mice leads to RGC degeneration that is similar to normal tension glaucoma and these animal models are useful in examining potential therapeutic strategies. Caloric restriction has been reported to increase longevity and has potential benefits in injury and disease. Here we investigated the effects of every-other-day fasting (EODF), a form of caloric restriction, on glaucomatous pathology in EAAC1−/− mice. EODF suppressed RGC death and retinal degeneration without altering intraocular pressure. Moreover, visual impairment was ameliorated with EODF, indicating the functional significance of the neuroprotective effect of EODF. Several mechanisms associated with this neuroprotection were explored. We found that EODF upregulated blood β-hydroxybutyrate levels and increased histone acetylation in the retina. Furthermore, it elevated retinal mRNA expression levels of neurotrophic factors and catalase, whereas it decreased oxidative stress levels in the retina. Our findings suggest that EODF, a safe, non-invasive, and low-cost treatment, may be available for glaucoma therapy.

Post-mortem β-hydroxybutyrate determination in synovial fluid

β-hydroxybutyrate concentrations were determined in blood and synovial fluid in a series of medico-legal cases including hypothermia fatalities, individuals found dead in a cold environment and non-hypothermia cases with various, non-traumatic causes of death. Hypothermia was considered to be the cause of death according to circumstantial elements indicating exposure to cold, autopsy findings, biochemical investigation results and exclusion of other causes of death. The intention of this study was to characterize β-hydroxybutyrate distribution in synovial fluid and assess its usefulness for the postmortem diagnosis of antemortem abnormalities in blood β-hydroxybutyrate levels. Unenhanced CT scans, autopsies, histology, neuropathology, toxicology, and biochemistry were systematically performed. Within the limited number of subjects included in the study, the results indicate that abnormalities in antemortem β-hydroxybutyrate blood levels, as may be observed in hypothermia fatalities, are reflected in postmortem synovial fluid values. These preliminary findings notwithstanding, synovial fluid analysis to determine β-hydroxybutyrate is unlikely to be generally applied due to the more invasive collection technique it requires and could be limited to special cases in which biological fluids systematically collected upon autopsy are unavailable.

Safety of Nighttime 2-Hour Suspension of Basal Insulin in Pump-Treated Type 1 Diabetes Even in the Absence of Low Glucose

OBJECTIVEAn integrated sensor-augmented pump system has been introduced that interrupts basal insulin infusion for 2 h if patients fail to respond to low-glucose alarms. It has been suggested that such interruptions of basal insulin due to falsely low glucose levels detected by sensor could lead to diabetic ketoacidosis. We hypothesized that random suspension of basal insulin for 2 h in the overnight period would not lead to clinically important increases in blood β-hydroxybutyrate levels despite widely varying glucose values prior to the suspension.RESEARCH DESIGN AND METHODSSubjects measured blood glucose and blood β-hydroxybutyrate levels using a meter each night at 9:00 p.m., then fasted until the next morning. On control nights, the usual basal rates were continued; on experimental nights, the basal insulin infusion was reprogrammed for a 2-h zero basal rate at random times after 11:30 p.m.RESULTSIn 17 type 1 diabetic subjects (mean age 24 ± 9 years, diabetes duration 14 ± 11 years, A1C level 7.3 ± 0.5% [56 mmol/mol]), blood glucose and blood β-hydroxybutyrate levels were similar at 9:00 p.m. on suspend nights (144 ± 63 mg/dL and 0.09 ± 0.07 mmol/L) and nonsuspend nights (151 ± 65 mg/dL and 0.08 ± 0.06 mmol/L) (P = 0.39 and P = 0.47, respectively). Fasting morning blood glucose levels increased after suspend nights compared with nonsuspend nights (191 ± 68 vs. 141 ± 75 mg/dL, P < 0.0001), and the frequency of fasting hypoglycemia decreased the morning following suspend nights (P < 0.0001). Morning blood β-hydroxybutyrate levels were slightly higher after suspension (0.13 ± 0.14 vs. 0.09 ± 0.11 mmol/L, P = 0.053), but the difference was not clinically important.CONCLUSIONSSystems that suspend basal insulin for 2 h are safe and do not lead to clinically significant ketonemia even if the blood glucose level is elevated at the time of the suspension.

Fgf21 Impairs Adipocyte Insulin Sensitivity in Mice Fed a Low-Carbohydrate, High-Fat Ketogenic Diet

BackgroundA low-carbohydrate, high-fat ketogenic diet (KD) induces hepatic ketogenesis and is believed to affect energy metabolism in mice. As hepatic Fgf21 expression was markedly induced in mice fed KD, we examined the effects of KD feeding on metabolism and the roles of Fgf21 in metabolism in mice fed KD using Fgf21 knockout mice.Methodology/Principal FindingsWe examined C57BL/6 mice fed KD for 6 or 14 days. Blood β-hydroxybutyrate levels were greatly increased at 6 days, indicating that hepatic ketogenesis was induced effectively by KD feeding for 6 days. KD feeding for 6 and 14 days impaired glucose tolerance and insulin sensitivity, although it did not affect body weight, blood NEFA, and triglyceride levels. Hepatic Fgf21 expression and blood Fgf21 levels were markedly increased in mice fed KD for 6 days. Blood β-hydroxybutyrate levels in the knockout mice fed KD for 6 days were comparable to those in wild-type mice fed KD, indicating that Fgf21 is not required for ketogenesis. However, the impaired glucose tolerance and insulin sensitivity caused by KD feeding were improved in the knockout mice. Insulin-stimulated Akt phosphorylation was significantly decreased in the white adipose tissue in wild-type mice fed KD compared with those fed normal chow, but not in the muscle and liver. Its phosphorylation in the white adipose tissue was significantly increased in the knockout mice fed KD compared with wild-type mice fed KD. In contrast, hepatic gluconeogenic gene expression in Fgf21 knockout mice fed KD was comparable to those in the wild-type mice fed KD.Conclusions/SignificanceThe present findings indicate that KD feeding impairs insulin sensitivity in mice due to insulin resistance in white adipose tissue. In addition, our findings indicate that Fgf21 induced to express by KD is a negative regulator of adipocyte insulin sensitivity in adaptation to a low-carbohydrate malnutritional state.

The effect of administering ketoprofen on the physiology and behavior of dairy cows following surgery to correct a left displaced abomasum

Surgical correction of left displaced abomasum (LDA) is common in lactating dairy cattle. Despite the growing acceptance that abdominal surgery is painful, few cows are administered analgesia following LDA surgery. The objective of this research was to examine the effect of administering a label dose of ketoprofen on physiological and behavioral indicators of pain in dairy cattle. Holstein cows were enrolled in a field study following LDA surgery. Surgery was performed using the standing right flank (RF) approach or the paramedian (PARA) approach. Using a triple-blind randomized trial, each animal was assigned to receive either 3mg of ketoprofen/kg of body weight or saline (the equivalent volume) by intramuscular injection immediately following surgery and 24h postoperatively. Physiological parameters (heart rate, respiration rate, and rumen motility), blood β-hydroxybutyrate (BHBA) levels, and an assessment of cow attitude were measured on the day of surgery, and at 2 follow-up visits (visit 1=3±0.9 d and visit 2=9±1.2 d postsurgery; n=175). Milk production and culling were recorded for all cows enrolled in the study. Producers assessed their cows’ attitudes and appetites daily for the first 3 d following surgery. A subset of cows (n=37) were fitted on the day of surgery with a 3-axis accelerometer on the hind leg to assess lying behavior. Continuous and binary outcome data were analyzed using multivariable mixed linear and mixed logistic models, respectively, with cow modeled as a random effect. Ketoprofen did not alter the physiological measures, BHBA levels, or behavioral outcomes measured. Cows subjected to RF surgery had longer lying times [model coefficient β=228.9min; 95% confidence interval (CI): 122.2 to 335.6] in the first 3 d following surgery, and lower heart rates (β=−9.4 beats/min; 95% CI: −12 to −6.9 beats/min) at the follow-up visits, compared with animals that underwent PARA surgery. Regardless of surgical procedure, BHBA decreased from surgery day to visit 1 (β=−1.9mmol/L; 95% CI: −2.1 to −1.7) and visit 2 (β=−2.0mmol/L; 95% CI: −0.2.2 to −1.8). Producer reports indicated that animals that received ketoprofen were more likely to begin eating when provided fresh feed during the first 3 d following surgery compared with those that received saline (odds ratio=4.8; 95% CI: 0.97 to 23.8). These results indicate that PARA surgery may be more painful relative to lying down than the RF approach. The apparent differences in appetite or attitude in cows that received ketoprofen reported by producers warrant further investigation.

Functional redundancy of mitochondrial enoyl‐CoA isomerases in the oxidation of unsaturated fatty acids

Mitochondrial enoyl-CoA isomerase (ECI1) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild-type (WT) and Eci1-deficient knockout (KO) mice to explore a potential presentation of human ECI1 deficiency. Upon food withdrawal, Eci1-deficient mice displayed normal blood β-hydroxybutyrate levels (WT 1.09 mM vs. KO 1.10 mM), a trend to lower blood glucose levels (WT 4.58 mM vs. KO 3.87 mM, P=0.09) and elevated blood levels of unsaturated acylcarnitines, in particular C12:1 acylcarnitine (WT 0.03 μM vs. KO 0.09 μM, P<0.01). Feeding an olive oil-rich diet induced an even greater increase in C12:1 acylcarnitine levels (WT 0.01 μM vs. KO 0.04 μM, P<0.01). Overall, the phenotypic presentation of Eci1-deficient mice is mild, possibly caused by the presence of a second enoyl-CoA isomerase (Eci2) in mitochondria. Knockdown of Eci2 in Eci1-deficient fibroblasts caused a more pronounced accumulation of C12:1 acylcarnitine on incubation with unsaturated fatty acids (12-fold, P<0.05). We conclude that Eci2 compensates for Eci1 deficiency explaining the mild phenotype of Eci1-deficient mice. Hypoglycemia and accumulation of C12:1 acylcarnitine might be diagnostic markers to identify ECI1 deficiency in humans.

Use of a hand-held meter for detecting subclinical ketosis in dairy cows

The Optium Xceed is a new hand-held meter for determining blood β-hydroxybutyrate (BHBA) and glucose in human medicine. The objective of this study was to compare BHBA and glucose results obtained using the hand-held meter with those results made with a laboratory method and to evaluate its usefulness as a cowside test in the diagnosis of subclinical ketosis (SCK) in dairy cows. Seventy-eight blood samples from clinically healthy Holstein cows between 5 and 60 days post-calving were analysed. BHBA and glucose values were significantly higher with the hand-held meter versus laboratory methods. Correlation coefficients ( r) for BHBA and glucose with the Optium Xceed versus laboratory methods were 0.97 and 0.63, respectively. Based on Bland–Altman plot and Passing–Bablok regression, agreement between two methods was good for BHBA but the agreement for glucose was only fair. When SCK was defined as plasma BHBA levels ⩾1200 μmol/L, the sensitivity and specificity of the hand-held meter ketone testing in determining SCK were 85% and 94%, respectively. Raising the threshold of the laboratory method to ⩾1400 μmol/L, the sensitivity and specificity incremented to 0.90 and 0.98, respectively. In conclusion, the blood ketone-monitoring device can be used as a rapid and reliable diagnostic test to detect SCK under field conditions.