Engineered acetoacetate-inducible whole-cell biosensors based on the AtoSC two-component system.

Whole-cell biosensors hold potential in a variety of industrial, medical, and environmental applications. These biosensors can be constructed through the repurposing of bacterial sensing mechanisms, including the common two-component system (TCS). Here we report on the construction of a range of novel biosensors that are sensitive to acetoacetate, a molecule that plays a number of roles in human health and biology. These biosensors are based on the AtoSC TCS. An ordinary differential equation model to describe the action of the AtoSC TCS was developed and sensitivity analysis of this model used to help inform biosensor design. The final collection of biosensors constructed displayed a range of switching behaviours at physiologically relevant acetoacetate concentrations and can operate in several Escherichia coli host strains. It is envisaged that these biosensor strains will offer an alternative to currently available commercial strip tests and, in future, may be adopted for more complex in vivo or industrial monitoring applications.

Validation of a Gas Chromatography-Mass Spectrometry Method for the Measurement of the Redox State Metabolic Ratios Lactate/Pyruvate and ß-Hydroxybutyrate/Acetoacetate in Biological Samples.

The metabolic ratios lactate/pyruvate and ß-hydroxybutyrate/acetoacetate are considered valuable tools to evaluate the in vivo redox cellular state by estimating the free NAD+/NADH in cytoplasm and mitochondria, respectively. The aim of the current study was to validate a gas-chromatography mass spectrometry method for simultaneous determination of the four metabolites in plasma and liver tissue. The procedure included an o-phenylenediamine microwave-assisted derivatization, followed by liquid-liquid extraction with ethyl acetate and silylation with bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane 99:1. The calibration curves presented acceptable linearity, with a limit of quantification of 0.001 mM for pyruvate, ß-hydroxybutyrate and acetoacetate and of 0.01 mM for lactate. The intra-day and inter-day accuracy and precision were within the European Medicines Agency's Guideline specifications. No significant differences were observed in the slope coefficient of three-point standard metabolite-spiked curves in plasma or liver and water, and acceptable recoveries were obtained in the metabolite-spiked samples. Applicability of the method was tested in precision-cut liver rat slices and also in HepG2 cells incubated under different experimental conditions challenging the redox state. In conclusion, the validated method presented good sensitivity, specificity and reproducibility in the quantification of lactate/pyruvate and ß-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.

[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.

A new hyperpolarized 13C ketone body probe reveals an increase in acetoacetate utilization in the diabetic rat heart.

Emerging studies have recently shown the potential importance of ketone bodies in cardio-metabolic health. However, techniques to determine myocardial ketone body utilization in vivo are lacking. In this work, we developed a novel method to assess myocardial ketone body utilization in vivo using hyperpolarized [3-13C]acetoacetate and investigated the alterations in myocardial ketone body metabolism in diabetic rats. Within a minute upon injection of [3-13C]acetoacetate, the production of [5-13C]glutamate and [1-13C] acetylcarnitine can be observed real time in vivo. In diabetic rats, the production of [5-13C]glutamate was elevated compared to controls, while [1-13C]acetylcarnitine was not different. This suggests an increase in ketone body utilization in the diabetic heart, with the produced acetyl-CoA channelled into the tricarboxylic acid cycle. This observation was corroborated by an increase activity of succinyl-CoA:3-ketoacid-CoA transferase (SCOT) activity, the rate-limiting enzyme of ketone body utilization, in the diabetic heart. The increased ketone body oxidation in the diabetic hearts correlated with cardiac hypertrophy and dysfunction, suggesting a potential coupling between ketone body metabolism and cardiac function. Hyperpolarized [3-13C]acetoacetate is a new probe with potential for non-invasive and real time monitoring of myocardial ketone body oxidation in vivo, which offers a powerful tool to follow disease progression or therapeutic interventions.

Dark fermentation: isolation and characterization of hydrogen-producing strains from sludges

To improve bacterial hydrogen production, ten hydrogen-producing strains belonging to Clostridium spp. were isolated from various sludges under low vacuum. Hydrogenogenesis by dark fermentation in batch cultures of these strains was optimal at about 35 ºC and an initial pH of 6.5, which for all strains gradually dropped to ca. pH 4 during the fermentation. Clostridium roseum H5 and C. diolis RT2 had the highest hydrogen yields per total substrate (120 ml H2/g initial COD). Substrate consumption alone by C. beijerinckii UAM and C. diolis RT2 reached 573 and 475 ml H2/g consumed COD, respectively. Butyric acid fermentation was predominant, with butyrate and acetate as the major by-products and propionate, ethanol, and lactate as secondary metabolites. The acetate: butyrate ratios and fermentation pathways varied depending on the strains and environmental conditions. Hydrogenogenesis was studied in greater detail in C. saccharobutylicum H1. In butyric acid fermentation by this representative strain, acetoacetate was detected as an intermediate metabolite. Hydrogenogenesis was also analyzed in an enrichment culture, which behaved similarly to the axenic cultures (AU)

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Elevated acetoacetate and monocyte chemotactic protein-1 levels in cord blood of infants of diabetic mothers.

BACKGROUND: Infants of diabetic mothers (IDMs) are at increased risk for metabolic complications. Type 1 and some type 2 diabetic patients have elevated levels of the ketone bodies acetoacetate (AA) and ß-hydroxybutyrate (BHB). OBJECTIVE: The aim of this study was to examine how hyperketonemia in diabetic mothers affects markers of inflammation and oxidative stress in their offspring. METHODS: Blood was obtained from 23 diabetic mothers and 13 healthy mothers and their infants' umbilical cords at delivery. Interleukin-8, monocyte chemotactic protein-1 (MCP-1) and protein carbonyl (protein oxidation) levels were determined by ELISA. U937 human monocyte cell culture was used to examine the effect of AA and BHB on secretion of MCP-1. RESULTS: There was a significant increase in the levels of AA in cord blood of IDMs compared with cord blood of infants of healthy mothers. A significant increase in the levels of protein oxidation (p < 0.05) and MCP-1 levels (p < 0.05) was observed in the cord blood of IDMs. The level of MCP-1 correlated significantly (r = 0.51, p = 0.01) with the concentration of AA in the IDMs. In further experiments with cultured monocytes treated with exogenous AA (0-4 mM), a significant increase in MCP-1 secretion was observed in AA- but not BHB-treated monocytes. CONCLUSION: Blood levels of AA and MCP-1 are elevated in IDMs, which may contribute to the development of the metabolic complications seen in IDMs.

Blood, urine and vitreous isopropyl alcohol as biochemical markers in forensic investigations.

Isopropyl alcohol (IPA) is widely used as an industrial solvent and cleaning fluid. After ingestion or absorption, IPA is converted into acetone by alcohol dehydrogenase. However, in ketosis, acetone can be reduced to IPA. The aim of this study was to investigate blood IPA and acetone concentrations in a series of 400 medico-legal autopsies, including cases of diabetic ketoacidosis, hypothermia and alcohol misuse-related deaths, to illustrate the extent of ketosis at the time of death. Vitreous glucose, blood 3-ß-hydroxybutyrate (3HB) and acetoacetate (AcAc) concentrations were also determined systematically. Additionally, vitreous and urine IPA, acetone, 3HB and AcAc concentrations as well as other biochemical markers, including glycated hemoglobin and carbohydrate-deficient transferrin (CDT) were also determined in selected cases. The results of this study indicate that ketosis is characterized by the presence of IPA resulting from the acetone metabolism and that IPA can be detected in several substrates. These findings confirm the importance of the systematic determination of IPA and acetone levels that is used to quantify biochemical disturbances and the importance of ketosis at the time of death.

1H NMR-based metabonomic analysis of metabolic changes in streptozotocin-induced diabetic rats.

Diabetes mellitus is a complex metabolic disorder characterized by chronic hyperglycemia, hypoinsulinemia, and ketosis. To access the biochemical process of diabetes, we applied quantitative (1)H NMR-based metabonomics to analyze urine, serum, and liver extracts from streptozotocin-induced diabetic rats. Principle component analysis (PCA) of (1)H NMR spectra disclosed metabolic pattern differences between diabetic and control rats, and identified the related metabolic changes. The PCA scores plot demonstrated that the diabetic group could be distinguished from the control group, indicating that the metabolic characteristics of the two groups were markedly different. Our work reveals the accumulation of triglycerides, fatty acids and acetoacetate in diabetic rats, and may provide an efficient, convenient way for evaluating the pathological state and biochemical process of diabetes mellitus.

Automated synthesis of 11C-acetoacetic acid, a key alternate brain fuel to glucose.

An automated, one-pot radio-synthesis module for the routine preparation of 1-[(11)C]acetoacetic acid has been developed. The enolate anion of acetone was reacted with [(11)C]CO(2) in tetrahydrofuran (THF), followed by hydrolysis and purification by ion-exchange chromatography. The total synthesis time was 18 min and radiochemical yield was 34% after decay correction. HPLC analysis showed < or =3% impurities while residual THF (< or =200 ppm) and ethanol (< or =500 ppm) were well under the tolerable limits for human studies.

Screening for subclinical ketosis in dairy cattle by Fourier transform infrared spectrometry.

Subclinical ketosis is a metabolic disorder in high-producing dairy cattle that can be detected by ketone bodies in milk: acetone (Ac), acetoacetate (AcAc), and beta-hydroxybutyrate (BHBA). Fourier transform infrared (FTIR) spectrometry is to a growing extent used for determination of milk constituents in milk recording, but as yet there is no calibration for ketone bodies available. The objective of this study was therefore to build a calibration for the MilkoScan FT6000 (FOSS Analytical A/S, Hillerød, Denmark) for Ac, AcAc, and BHBA and to evaluate the FTIR predictions for detection of subclinical ketosis. From 217 herds, 1,080 milk samples were taken from fresh multiparous dairy cows. The Ac, AcAc, and BHBA concentrations were determined by chemical methods using segmented flow analysis. Because of its low concentration, AcAc seemed to be hardly detectable and was therefore not considered further. The correlation between the chemical method results of Ac and BHBA was 0.82, indicating that both ketone bodies were elevated in milk during subclinical ketosis. In wk 1 postpartum, however, most samples with a high Ac concentration did not have a high BHBA concentration, whereas after wk 5 postpartum most samples with a high BHBA concentration did not have a high Ac concentration. For Ac and BHBA, the correlation coefficients between the FTIR predictions and the chemical results were around 0.80 with standard error of cross validation values of 0.184 and 0.064 mM for Ac and BHBA, respectively. Using thresholds of 0.15 mM for Ac and 0.10 mM for BHBA, high values for Ac or BHBA were detected with a sensitivity of 69 to 70%, a specificity of 95%, with 25 to 27% false positives and 6 to 7% false negatives. It is argued that FTIR predictions for Ac and BHBA are valuable for screening cows on subclinical ketosis, especially when used in combination with other indicators, and can serve in the evaluation of the herd health status with respect to subclinical ketosis.

Synergistic potent insulin release by combinations of weak secretagogues in pancreatic islets and INS-1 cells.

Insulin secretion by the beta cell depends on anaplerosis in which insulin secretagogues are metabolized by mitochondria into molecules that are most likely exported to the extramitochondrial space where they have signaling roles. However, very little is known about the products of anaplerosis. We discovered an experimental paradigm that has begun to provide new information about these products. When various intracellular metabolites were applied in combination to overnight-cultured rat or human pancreatic islets or to INS-1 832/13 cells, they interacted synergistically to strongly stimulate insulin release. When these same metabolites were applied individually to these cells, insulin stimulation was poor. Discerning the contributions of the individual compounds to metabolism has begun to allow us to dissect some of the pathways involved in insulin secretion, which was not possible from studying individual secretagogues. Monomethyl succinate (MMS) combined with a barely stimulatory concentration of alpha-ketoisocaproate (KIC) (2 mm) stimulated insulin release in cultured rat islets 18-fold (versus 21-fold for 16.7 mm glucose). MMS plus low glucose (2 mm) or pyruvate (5 mm) gave 11- and 9-fold stimulations. These agents also potentiated MMS-induced insulin release in fresh islets, and KIC plus MMS gave synergistic insulin release in cultured human islets. In INS-1 cells, neither MMS nor KIC (10 mm) was an insulin secretagogue, but when added together KIC (2 mm) and MMS stimulated insulin release 7-fold (versus 12-fold for glucose). In islets and INS-1 cells, conditions that stimulated insulin release caused large relative increases in acetoacetate, which is a precursor of pathways to short chain acyl-CoAs. Liquid chromatography-tandem mass spectrometry measurements of acetyl-CoA, acetoacetyl-CoA, succinyl-CoA, hydroxymethylglutaryl-CoA, and malonyl-CoA confirmed that they were increased by insulin secretagogues. The results suggest a new mechanism of insulin secretion in which anaplerosis increases short chain acyl-CoAs that have roles in insulin exocytosis.

Detection of cerebral {beta}-hydroxy butyrate, acetoacetate, and lactate on proton MR spectroscopy in children with diabetic ketoacidosis.

BACKGROUND AND PURPOSE: Ketone bodies provide important alternate fuel for brain metabolism, and their transport into the brain increases with prolonged fasting. During diabetic ketoacidosis (DKA), serum ketone concentrations markedly increase; however, little is known about whether ketone bodies accumulate in cerebral tissues during DKA. We used proton MR spectroscopy (MRS) to detect cerebral beta-hydroxy butyrate (betaOHB) and acetone/acetocaetate (AcAc) in children with DKA. METHODS: Twenty-five children underwent brain MRS: nine within 4 hours of the start of treatment for DKA; 11, at 4-8 hours; and five, at 8-12 hours. MRS was repeated after their recovery from the DKA episode at > or =72 hours after the start of treatment. MRS was evaluated for peaks corresponding to betaOHB (doublet centered on 1.20 ppm) and lactate (doublet centered on 1.33 ppm). Difference spectroscopy was used to identify the AcAc peak at 2.22-2.26 ppm. RESULTS: betaOHB was detected in 13 children (52%), more frequently within 4 hours (eight children, 89%) than after 4 hours (five children, 31%). AcAc was detected in 15 children (60%), more frequently at >4 hours after the start of treatment (12 patients, 75%) than in the first 4 hours (three patients, 33%). Lactate was detected in five children (18%), all within the first 8 hours of treatment. CONCLUSION: In children, betaOHB and AcAc accumulate in the brain during DKA, and they can be detected on MRS. Care should be taken in interpreting MRS results in patients with DKA to avoid erroneously attributing betaOHB peaks to lactate.

Urinary acetoacetate or capillary beta-hydroxybutyrate for the diagnosis of ketoacidosis in the Emergency Department setting.

OBJECTIVES: We compared the semiquantitative measurement of acetoacetate using urinary dipsticks with the bedside quantitative fingerprick measurement of the principal ketone bodies 3-beta-hydroxybutyrate, for the diagnosis of ketoacidosis. METHODS: This is a one year retrospective study of patients who presented with hyperglycemia levels of 250 mg/l or greater in the Emergency Department setting. We compared the sensitivity, specificity, and predictive value of ketonuria and ketonemia for the diagnosis of ketoacidosis (urine or blood ketone bodies, blood bicarbonates <20 mmol/l, anion gap >16 meq/l) in a sample of patients for whom the levels of ketone bodies in the blood and urine as well as serum electrolytes were available. RESULTS: We studied 355 hyperglycemic patients. The median time between arrival and dipstick testing was 21 min, and was greater than 2 h in more than 10% of cases. Comparison between ketonuria and ketonemia was performed in 173 patients (6% with diabetic ketoacidosis). Ketonuria equal to or less than one cross or a 3-beta-hydroxybutyrate value lower than 3 mmol/l enabled ketoacidosis to be excluded (negative predictive value 100%). At two-cross cutoff points for ketonuria and at the 3 mmol/l cutoff point for ketonemia, the two tests had the same sensitivity (100%), but the specificity of 3-beta-hydroxybutyrate (94%) was significantly higher (P<0.0001) than that of ketonuria (77%). The best positive predictive value for ketonemia was obtained at the 5 mmol/l cutoff point (100%) and for ketonuria at the three-cross cutoff point (26%). At the three-cross cutoff point for ketonuria and at the 5 mmol/l cutoff point for ketonemia, the two tests had the same negative likelihood ratio (0.1), but the positive likelihood ratio of 3-beta-hydroxybutyrate (infinite) was higher than that of ketonuria. CONCLUSION: The measurement of 3-beta-hydroxybutyrate in capillary blood is faster and more effective than the use of dipsticks in the urine to detect ketoacidosis in the Emergency Department setting.

Effect of a monensin controlled release capsule on metabolic parameters in transition dairy cows.

A prospective field study involving 251 Holstein cows and heifers from five dairy farms near Guelph, Ontario, Canada, was conducted to measure the effect of monensin delivered in a controlled release capsule 3 wk precalving on metabolic function in dairy cows immediately pre- and postcalving. At 3 wk before expected calving, cows were randomly assigned to receive either a controlled release capsule containing monensin or to serve as negative controls. Cows were blood sampled once per week precalving and once in the week following calving, at the same time of day and the same day of the week. Serum was evaluated for beta-hydroxybutyrate (BHBA), nonesterified fatty acids (NEFA), cholesterol, urea, glucose, calcium, and phosphorus. Monensin-treated cows had significantly decreased NEFA and BHBA and significantly increased concentrations of serum cholesterol and urea in the week immediately precalving. No effect of treatment was observed for calcium, phosphorus, or glucose in the precalving period. After calving, concentrations of phosphorus were lower and BHBA tended to be lower, and cholesterol and urea were higher in monensin-treated cows. There was no effect of treatment on NEFA, glucose, or calcium in the first week postcalving. Monensin treatment administered precalving significantly improved indicators of energy balance in both the immediate precalving and postcalving periods. The findings indicate better energy metabolism in monensin-treated cows as they approach calving. Improvement of energy balance before calving is important for the prevention of energy associated metabolic diseases, such as retained placenta, clinical ketosis, and displaced abomasum, which might occur immediately postcalving.

High performance liquid chromatographic determination of acetoacetate by post-column derivatization with p-nitrobenzene diazonium fluoroborate.

We have applied a color-developing reagent, p-nitrobenzene diazonium fluoroborate (diazo reagent) as a post-column derivatization tool for the specific determination of acetoacetate (AcAc) in high performance liquid chromatography (HPLC). A mobile phase consisting of 50 mM KH(2)PO(4), 4 mM tetra-n-butylammonium phosphate (TBAP) as an ion-pair reagent and 2 v/v% methanol, pH 3.5, diazo reagent solution with 0.2% triton X-100, and alkaline solution of 1.5 mol/l NaOH were pumped using three independent pumps. Specific color development on-line was monitored at 645 nm. A calibration curve for AcAc standard solution with an injection volume of 20 microl showed a good linearity in the range 0.01-2.5 mM with a correlation coefficient of 0.999. For the determination of 3-hydroxybutyrate (3-HOBA), 3-HOBA was converted to AcAc by an enzymatic-coupling method using 3-HOBA dehydrogenase and lactate dehydrogenase. Analytical recoveries of AcAc and 3-HOBA added to serum and urine were satisfactory.

Time empty and ketone body status in the early postpartum period of dairy cows.

This study aimed to assess the effect of ketone body status early postpartum on time empty in 74 multiparous dairy cows under field conditions. Animals were equally distributed across eight farms and were controlled by the same herd-fertility-monitoring programme. Cows were visited twice antepartum and six times postpartum at weekly intervals between 0530 and 0830 a.m. On these occasions, body condition scores and milk yields were measured, blood and milk samples were taken, cows were gynaecologically examined, and parameters of reproduction were recorded. Cows with a time empty of less or more than 80 days were classified as early and late conceiving cows (EC and LC, respectively). A time empty of 80 days results in calving-to-calving intervals of 1 year and classification based on this threshold value resulted in groups of equal size and equal distribution of EC and LC within farms. Ketone bodies measured were beta-hydroxybutyrate in blood and acetoacetate and acetone in blood and milk. Blood and milk ketone body concentrations, as well as the ratios of acetoacetate and acetone to beta-hydroxybutyrate, over the first 6 weeks postpartum were higher in LC than in EC, whereas plasma glucose and non-esterified fatty acids and milk fat, protein and urea concentrations did not exhibit clear differences between groups. Ketone body concentrations were as good predictors of time empty as ketone body ratios and might have practical impact in herd-fertility-monitoring programmes.

Glucagon effects on brain carbohydrate and ketone body metabolism of rainbow trout.

The levels of glycogen in brain, lactate and acetoacetate in brain and plasma, glucose in plasma and the activities of brain key enzymes of glycogen metabolism (glycogen phosphorylase, GPase, glycogen synthetase, GSase), gluconeogenesis (fructose 1,6-bisphosphatase, FBPase), and glycolysis (6-phosphofructo 1-kinase, PFK) were evaluated in rainbow trout, Oncorhynchus mykiss, from 0.5 to 3 hr after intraperitoneal injection of 1 ml/kg(-1) body weight of saline alone (controls) or containing bovine glucagon at three different doses: 10, 50, and 100 ng/g(-1) body weight. The results obtained demonstrate, for the first time in a teleost fish, the existence of changes in brain carbohydrate and ketone body metabolism following peripheral glucagon treatment. A clear stimulation of brain glycogenolytic potential was observed after glucagon treatment, as judged by the time- and dose-dependent changes observed in brain glycogen levels (up to 88% decrease), and GPase (up to 30% increase) and GSase (up to 42% decrease) activities. In addition, clear time- and dose-dependent increased and decreased levels were observed in brain of glucagon-treated rainbow trout for lactate (up to 60% increase) and acetoacetate (up to 67% decrease), respectively. In contrast, no significant changes were observed after glucagon treatment in those parameters related to glycolytic/gluconeogenic capacity of rainbow trout brain. Altogether, these in vivo results suggest that glucagon may play a role (direct or indirect) in the regulation of carbohydrate and ketone body metabolism in brain of rainbow trout.

Ketone bodies in milk and blood of dairy cows: relationship between concentrations and utilization for detection of subclinical ketosis.

The objective of this study was to investigate the relationship between the concentrations of the different ketone bodies in milk and blood and to evaluate these concentrations for the detection of subclinical ketosis. A total of 60 multiparous cows were used. Concentrations of acetone, acetoacetate, and beta-hydroxybutyrate were analyzed quantitatively in blood and milk, and the Ketolac strip test was used for semiquantitative determination of beta-hydroxybutyrate in milk. Cows were defined subclinically ketotic when their concentration of blood beta-hydroxybutyrate was over 1200 micromol/L. High correlation coefficients were observed between blood acetone and blood acetoacetate, and between blood and milk acetone. On the contrary, concentrations of milk and blood beta-hydroxybutyrate were poorly correlated with the other concentrations of ketone bodies. The Ketolac strip test overestimated the concentrations of beta-hydroxybutyrate in milk. For the detection of subclinical ketosis, the best sensitivity-specificity combination was obtained with the determination of acetoacetate in blood or milk, with threshold concentrations of 125 and 50 micromol/L, respectively. Determination of beta-hydroxybutyrate in the milk via an enzymatic analysis or via the Ketolac strip test provided valuable results, with threshold concentrations of 70 and 100 micromol/L, respectively. The simplicity of use of the Ketolac strip test makes it a valuable way to investigate subclinical ketosis.

First ovulation and ketone body status in the early postpartum period of dairy cows.

The effect of ketone body status on occurrence of first ovulation during early lactation was assessed in 84 multiparous dairy cows under field conditions. Animals were equally distributed across 8 farms and were controlled by the same herd fertility monitoring program. Cows were visited twice antepartum and 6 times postpartum at weekly intervals between 5:30 and 8:30 AM. On these occasions, body condition scores and milk yields were measured, blood and milk samples were taken, cows were gynecologically examined, and parameters of reproduction were determined. The onset of first ovulation was specified by milk progesterone determination and rectal palpation. Cows starting postpartum ovarian cyclicity within or after 30 d were classified as early and late responders (ER and LR, respectively). Resumption of the estrous cycle within 30 d postpartum is considered optimal under practical conditions, and classification based on this threshold value resulted in groups of equal size and equal distribution of ER + LR cows within farms. Ketone bodies measured were beta-hydroxybutyrate in serum and acetoacetate and acetone in serum and milk. Blood serum and milk ketone body concentrations during the first 6 wk of lactation were higher in LR than in ER, whereas plasma glucose and nonesterified fatty acid and milk fat, protein and urea concentrations did not differ between groups. Maximal concentrations of ketone bodies from parturition to first ovulation were better predictors of the onset of the estrous cycle than mean or minimal concentrations over the same period. Milk acetone and serum beta-hydroxybutyrate concentrations provided the most reliable information with regard to resumption of ovarian activity of all ketone bodies.

Evaluation of eight cow-side ketone tests in milk for detection of subclinical ketosis in dairy cows.

The objective of this study was to evaluate eight cowside ketone tests when used with milk for detection of subclinical ketosis. A total of 469 dairy cows in the first week of lactation were studied. Twelve percent of these cows had subclinical ketosis, defined as >1400 micromol of beta-hydroxybutyrate/L of blood serum. The Pink test liquid and the Ketolac test strip were highly sensitive for subclinical ketosis when used with milk. The Uriscan and Rapignost test strips were poorly sensitive; the Ketostix, Ketur-Test, and Medi-Test-Keton test strips and the Acetonreagenz test tablet were insensitive for subclinical ketosis when used with milk. Pink and Ketolac milk ketone tests are potentially useful tools for use in a routine monitoring program to detect subclinical ketosis in early postpartal dairy cows.