Establishing vitreous glucose and beta-hydroxybutyrate thresholds to assist in the diagnosis of hypothermia.

The post-mortem diagnosis of hypothermia is challenging to establish due to the lack of pathognomonic findings and the confounding problem that any comorbidity may account for death. A 4-year retrospective case-control study was performed to compare the vitreous glucose and beta-hydroxybutyrate (BHB) concentrations between hypothermia deaths and controls. Over the study period 34 cases of hypothermia and 39 controls were analyzed. Hypothermia deaths versus controls had higher mean vitreous glucose (2.93 mmol/L vs. 1.14 mmol/L; p < 0.0001), BHB (1.89 mmol/L vs. 1.35 mmol/L; p = 0.01), and combined glucose+BHB (4.83 mmol/L vs. 2.46 mmol/L; p < 0.0001). Receiver operating characteristic (ROC) curves showed that the best model for predicting hypothermia in all cases was a combined vitreous glucose+BHB threshold of 2.03 mmol/L (sensitivity 88.2 %; specificity 56.4 %). A sub-group analysis broken down by detectable levels of blood ethanol showed that cases of hypothermia with and without ethanol maintained higher median vitreous glucose relative to the controls (2.05 vs. 0.35 mmol/L and 2.70 vs. 0.65 mmol/L; p = 0.02), however median BHB was only significantly elevated when ethanol was absent (1.88 vs. 1.42 mmol/L; p < 0.0001). Subsequent ROC curve analysis demonstrated that a better model for predicting hypothermia was in cases when blood ethanol was absent. In those deaths vitreous BHB alone had the best area under the curve, with an optimum threshold of 1.83 mmol/L (sensitivity 83.3 %; specificity 96.3 %). This study shows that post-mortem vitreous glucose and BHB are useful ancillary studies to assist in the diagnosis of hypothermia. Ethanol however is a confounder and can alter the utility of vitreous BHB when diagnosing hypothermia in those who have consumed alcohol prior to death.

[Succinyl CoA:3 oxoacid CoA transferase deficiency: A case report]. / Deficiencia de succinil-CoA acetoacetato transferasa: reporte de un caso.

Background: Succinyl-CoA:3 oxoacid CoA transferase deficiency (SCOTD) is a rare autosomal recessive disease, characterized by altered utilization of ketone bodies, with acute episodes of ketoacidosis. Clinical case: It is presented the case of a patient with SCOTD, with a first atypical episode accompanied by hyperglycemia, with 4 subsequent episodes with classic manifestations of the disease, presenting with a biochemical pattern of permanent ketonuria with marked elevation of ketone bodies (acetoacetate, 3 beta-hydroxybutyrate) in the study of urinary organic acids by gas chromatography and mass spectrometry, together with the clinical picture granting the diagnosis. It was started a maintenance therapy with a characteristic feeding plan; it was shown an adequate response to treatment, and the absence of permanent ketosis was surmised. Conclusion: Being a rare disease, the categorization of these patients as diabetic ketoacidosis is frequent. The clinical and biochemical characteristics with ketosis or persistent ketonuria should be analyzed very carefully, especially in patients presenting with hyperglycemia, which is an atypical manifestation of the disease, in order to make an early diagnosis and treatment, positively impacting the prognosis of patients.

Introducción: la deficiencia de succinil-CoA acetoacetato transferasa (SCOT) es una enfermedad rara, autosómica recesiva, caracterizada por alteración en la utilización de cuerpos cetónicos, con episodios agudos de cetoacidosis. Caso clínico: se presenta el caso de un paciente con deficiencia de SCOT, con un primer episodio atípico acompañado con hiperglucemia, con 4 episodios posteriores con manifestaciones clásicas de la enfermedad, que presentó patrón bioquímico de cetonuria permanente con marcada elevación de cuerpos cetónicos (acetoacetato, 3 beta-hidroxibutirato) en estudio de ácidos orgánicos urinarios por cromatografía de gases y espectrometría de masas, aunado a cuadro clínico que otorgó el diagnóstico. Se inició terapia de mantenimiento con plan de alimentación característico; se demostró una adecuada respuesta al tratamiento, y se infirió una ausencia de cetosis permanente. Conclusiones: al ser una enfermedad rara, la categorización de estos pacientes como cetoacidosis diabética es frecuente. Se deben analizar de forma muy minuciosa las características clínicas y bioquímicas con cetosis o cetonuria persistente, sobre todo en pacientes que se presenten con hiperglucemia, que es una manifestación atípica de la enfermedad, para realizar un diagnóstico y tratamiento temprano que impacte de forma positiva el pronóstico de los pacientes.

[Relationship between metabolic profiles in herd health and 4 scoring systems used in dairy herd health programs: Statistical analysis with an additive Bayesian network]. / Zusammenhänge zwischen metabolischen Profilen und 4 Bestandsbetreuung-Scores bei Milchkühen: Statistische Analyse anhand eines additiven Bayesian Netzwerkes.

OBJECTIVE: The aim of the present study was to describe the relationship between the biochemical variables used in metabolic profiles and various scores commonly used in herd health management of dairy cows. The Bayesian network was used to determine the relationship between metabolic blood profiles and the scores for body condition (BC), rumen fill (RF), faecal consistency (FC) and undigested fraction (UF) on a herd basis. MATERIALS AND METHODS: In each of 10 dairy herds, blood was collected from a minimum of ten lactating cows for biochemical analysis and generation of metabolic profiles. This yielded a total of 106 blood samples. The biochemical results in the metabolic profiles were stratified by days in milk and compared with the scores of BC, RF, FC and UF using an additive Bayesian network. RESULTS: The blood glucose concentration directly affected the FC score. The was an effect of beta-hydroxybutyrate (BHB) on free fatty acids (FFS). An additional effect of BHB on the concentration of urea was detected. The concentration of urea affected the concentration of phosphorus and GOT activity. Urea concentration also affected the blood concentrations of calcium and subsequently magnesium. Rumen fill had an effect on BC score and liver enzyme activity. Glutathione peroxidase, which is used to assess the levels of selenium in cattle, had no significant relationship with the other variables and was therefore isolated in the model. CONCLUSION: The use of a multidimensional model, which in this study was an additive Bayesian network, showed the relationships between the biochemical variables in the metabolic profiles and the scoring systems commonly used for the management of dairy cow herds. The relationships between the biochemical variables and the four scoring systems can be used to manage dairy herds more effectively. CLINICAL RELEVANCE: The biochemical variables used in metabolic profiles were correlated with health scoring systems commonly used in dairy herds. The latter can be carried out more rapidly and at a lower cost than metabolic profiles. In dairy cows with metabolic diseases or fertility disorders, scoring systems do not replace detailed evaluations that include metabolic profiles.

Hyperketonemia: A Marker of Disease, a Sign of a High-Producing Dairy Cow, or Both?

This review covers the history and nomenclature of ketosis, the source and use of ketones in transition cows, and the controversial role of hyperketonemia's association with health and production outcomes in dairy cows. With the goal of assisting veterinarians with on-farm diagnostic and treatment methods, the authors present current and evolving means of direct and indirect hyperketonemia detection as well as a summary of treatment modalities and their efficacy. They encourage veterinarians to include hyperketonemia testing as part of their routine physical examinations and contemplate day in milk at hyperketonemia diagnosis when designing treatment and management strategies.

Association of residual feed intake with peripartal ruminal microbiome and milk fatty acid composition during early lactation in Holstein dairy cows.

Residual feed intake (RFI) is a moderately heritable trait of feed efficiency in dairy cows. The main objective of the present study was to assess potential differences in the ruminal microbiome, milk fatty acid (FA) composition, and plasma concentrations of glucose, nonesterified fatty acids (NEFA), and ß-hydroxybutyrate between the most (M-EFF) and the least efficient (L-EFF) dairy cows during early lactation. Forty-seven multiparous Holstein dairy cows with daily ad libitum access to a total mixed ration from 30 d before calving to 30 d in milk were used. Cows were retrospectively classified into M-EFF (i.e., low RFI, n = 29) and L-EFF (high RFI, n = 18) based on a linear regression model. Ruminal digesta and milk samples were collected from each cow at 15 and 30 d in milk for microbiome analysis using 16S rRNA gene sequencing. Microbiome sequencing data were analyzed with the QIIME 2 platform (http://qiime.org/), whereas the microbiome statistical analyses and visual explorations were performed using the web-based MicrobiomeAnalyst platform. Milk FA composition was measured via gas chromatography-mass spectrometry. The statistical model used in SAS 9.4 (SAS Institute Inc.) included RFI, time, and their interactions as fixed effects. The cor() function in R programming was used to determine Pearson correlations between relative abundance of significant bacteria and milk FA. Overall, daily milk yield did not differ due to RFI and averaged 42 ± 1.6 kg for L-EFF and 43 ± 1.3 kg for M-EFF cows. However, M-EFF cows had lower overall dry matter intake (14.9 ± 0.5 kg/d) compared with L-EFF cows (19.2 ± 0.6 kg/d). No incidence of clinical disease was recorded for cows in the study. Compared with L-EFF, overall glucose concentration was lower, whereas NEFA and ß-hydroxybutyrate concentrations were greater in M-EFF cows. Ruminal digesta from both RFI groups had similar bacterial composition, but differed in the relative abundance of some bacteria. Compared with L-EFF, M-EFF cows had greater relative abundance of Lachnospiraceae, Lachnoclostridium, Papillibacter, Desulfovibrio, Sphaerochaeta, Acetobacter, and Histophilus. In contrast, relative abundance of Bifidobacterium, Ruminiclostridium, Prevotellaceae, and Erysipelotrichaceae bacterium was lower in M-EFF cows. Compared with L-EFF, M-EFF cows had greater proportions of long-chain monounsaturated FA, including 16:1 trans-9, 16:1 cis-9, 17:1 trans-10, 17:1 cis-10, 18:1 cis-9, 18:1 cis-11, whereas proportions of medium-chain saturated and 16:0 were lower in M-EFF. Acetate-producing bacteria (Sphaerochaeta and Acetobacter) were positively and significantly correlated (r ≥ 0.24) with concentrations of 16:1 cis-9 and 17:1 cis-10, whereas Prevotellaceae was significantly and negatively correlated (r = -0.25) with these FA. Butyrate-producing bacterium (Papillibacter) had a significant negative correlation (r = -0.27) with concentration of 15:0. Overall, data suggested that feed-efficient cows have unique profiles of ruminal microbiota, some of which are correlated with concentrations of milk FA during early lactation.

Clinicopathologic Appearance of Advanced Ketoacidosis With Basal Vacuolation in Renal Tubules.

CONTEXT.­: Basal vacuolization (BV) in renal tubules is a histopathologic hallmark of advanced ketoacidosis that enables us to retrospectively diagnose these cases. OBJECTIVE.­: To clarify the pathologic background and serologic findings of ketoacidosis with BV, and to reveal the pathologic findings by each pathologic background. DESIGN.­: We examined 664 serial autopsy cases. A systemic histopathologic examination and measurement of serum ß-hydroxybutyrate concentration were performed for the cases with BV. The extent of steatosis and fibrosis in the organs and the degree of coronary artery stenosis were semiquantitatively investigated. Immunohistochemistry for adipophilin was also performed to analyze its usefulness for the pathologic diagnosis. RESULTS.­: Basal vacuolization was found in 16 cases, all of which showed a pathologic serum ß-hydroxybutyrate concentration. The main background of ketoacidosis was considered as alcohol abuse in 6 cases, diabetes in 5, malnutrition in 3, and hypothermia and infection in 1 case each. Severe hepatic fibrosis was observed only in the alcohol-abuser group. Moreover, cardiac steatosis was more severe in patients with possible alcohol abuse than in those with other causes. Immunohistochemistry for adipophilin showed immunoreactivity consistent with BV in 13 of 16 cases. There was no correlation between ß-hydroxybutyrate concentration and either the postmortem or storage interval. CONCLUSIONS.­: Basal vacuolization may be a useful finding for detecting ketoacidosis cases in a postmortem investigation. Serum ß-hydroxybutyrate was a stable and reliable compound for the definitive diagnosis of ketoacidosis in such cases. The present study showed that pathologic changes in some organs may vary by each pathologic background of ketoacidosis with BV.

Markers of hyperglycemia in the vitreous humor. A systematic review and meta-analysis.

BACKGROUND: Vitreous humor has been extensively used in forensic practice to assess hyperglycemia after death. The results from different articles, for various hyperglycemia markers are highly variable, and a systematic analysis of the results from studies currently used in forensic practice as landmarks has not yet been performed. Therefore, we aimed to evaluate to usefulness and limits of using the values of vitreous glucose, lactic acid, beta-hydroxybutyrate, and 1,5 Anhydro-d-glucitol to detect postmortem hyperglycemia. MATERIALS AND METHODS: For this purpose, we performed a systematic review and a meta-analysis using the random-effects model to identify the threshold values and average differences for the markers mentioned above in the vitreous humor of diabetic versus nondiabetic subjects. RESULTS: We included eleven studies in the meta-analysis and found the following mean differences between the diabetic and nondiabetic groups: for glucose - 91.4 mg/dl, for lactate - 34.17 mg/dl, for the Traub formula - 111 mg/dl, for fructosamine - 0.71 mmol/L, for beta-hydroxybutyrate - 36.55 mg/dl and 1,5 Anhydro-d-glucitol - -15.2 mg/dl. We also gave practical recommendations, based on the range of values and 95% confidence intervals in normal subjects and controls to identify antemortem hyperglycemia and evaluated, whenever possible, threshold values for fatal diabetes. CONCLUSIONS: Glucose, Traub formula, fructosamine, and beta-hydroxy-butyrate can be used to detect postmortem hyperglycemia with some limitations; 1,5 Anhydro-d-glucitol can only be used to suggest the absence of a hyperglycemic status before death.

Genome-wide association study and functional analyses for clinical and subclinical ketosis in Holstein cattle.

Ketosis is one of the most frequent metabolic diseases in high-yielding dairy cows and is characterized by high concentrations of ketone bodies in blood, urine, and milk, causing high economic losses. The search for polymorphic genes, whose alleles have different effects on resistance to developing the disease, is of extreme importance to help select less susceptible animals. The aims of this study were to identify genomic regions associated with clinical and subclinical ketosis (ß-hydroxybutyrate concentration) in North American Holstein dairy cattle and to investigate these regions to identify candidate genes and metabolic pathways associated with these traits. To achieve this, a GWAS was performed for 4 traits: clinical ketosis lactation 1, clinical ketosis lactation 2 to 5, subclinical ketosis lactation 1, and subclinical ketosis lactation 2 to 5. The estimated breeding values from 77,277 cows and 7,704 bulls were deregressed and used as pseudophenotypes in the GWAS. The top-20 genomic regions explaining the largest proportion of the genetic variance were investigated for putative genes associated with the traits through functional analyses. Regions of interest were identified on chromosomes 2, 5, and 6 for clinical ketosis lactation 1; 3, 6, and 7 for clinical ketosis lactation 2 to 5; 1, 2, and 12 for subclinical ketosis lactation 1; and 20, 11, and 25 for subclinical ketosis lactation 2 to 5. The highlighted genes potentially related to clinical and subclinical ketosis included ACAT2 and IGF1. Enrichment analysis of the list of candidate genes for clinical and subclinical ketosis showed molecular functions and biological processes involved in fatty acid metabolism, lipid metabolism, and inflammatory response in dairy cattle. Several genomic regions and SNPs related to susceptibility to ketosis in dairy cattle that were previously described in other studies were confirmed. The novel genomic regions identified in this study aid to characterize the most important genes and pathways that explain the susceptibility to clinical and subclinical ketosis in dairy cattle.

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.

Role of Haptoglobin as a Marker of Muscular Improvement in Patients with Multiple Sclerosis after Administration of Epigallocatechin Gallate and Increase of Beta-Hydroxybutyrate in the Blood: A Pilot Study.

Here, we report on the role of haptoglobin (Hp), whose expression depends on the synthesis of interleukin 6 (IL-6), related to the pathogenesis of multiple sclerosis (MS), as a possible marker of muscle improvement achieved after treatment with the polyphenol epigallocatechin gallate (EGCG) and an increase in the ketone body beta-hydroxybutyrate (BHB) in the blood. After 4 months of intervention with 27 MS patients, we observed that Hp does not significantly increase, alongside a significant decrease in IL-6 and a significant increase in muscle percentage. At the same time, Hp synthesis is considerably and positively correlated with IL-6 both before and after treatment; while this correlation occurs significantly reversed with muscle percentage before treatment, no correlation is evident after the intervention. These results seem to indicate that Hp could be a marker of muscle status and could be a diagnosis tool after therapeutic intervention in MS patients.

Microbial oversecretion of (R)-3-hydroxybutyrate oligomer with diethylene glycol terminal as a macromonomer for polyurethane synthesis.

Poly((R)-3-hydroxybutyrate) (P(3HB)) is a polyester that is synthesized and accumulated in many prokaryotic cells. Recently, a new culture method for the secretion of the intracellularly synthesized (R)-3-hydroxybutyrate oligomer (3HBO) from recombinant Escherichia coli cells was developed. In this study, we attempted to produce microbial 3HBO capped with a diethylene glycol terminal (3HBO-DEG) as a macromonomer for polymeric materials. First, we prepared recombinant E. coli strains harboring genes encoding various polyhydroxyalkanoate (PHA) synthases (PhaC, PhaEC or PhaRC) that can incorporate chain transfer (CT) agents such as DEG into the polymer's terminal and generate CT end-capped oligomers. To this end, each strain was cultivated under DEG supplemental conditions, and the synthesis of 3HBO-DEG was confirmed. As a result, the highest secretory production of 3HBO-DEG was observed for the PHA synthase derived from Bacillus cereus YB-4 (PhaRCYB4). To evaluate the usability of the secreted 3HBO-DEG as a macromonomer, 3HBO-DEG was purified from the culture medium and polymerized with 4,4'-diphenylmethane diisocyanate as a spacer compound. Characterization of the polymeric products revealed that 3HBO-based polyurethane was successfully obtained and was a flexible and transparent noncrystalline polymer, unlike P(3HB). These results suggested that microbial 3HBO-DEG is a promising platform building block for synthesizing polyurethane and various other polymers.

Ketogenic Diet: Impact on Cellular Lipids in Hippocampal Murine Neurons.

BACKGROUND: The mechanism of action of the ketogenic diet (KD), an effective treatment for pharmacotherapy refractory epilepsy, is not fully elucidated. The present study examined the effects of two metabolites accumulating under KD-beta-hydroxybutyrate (ßHB) and decanoic acid (C10) in hippocampal murine (HT22) neurons. METHODS: A mouse HT22 hippocampal neuronal cell line was used in the present study. Cellular lipids were analyzed in cell cultures incubated with high (standard) versus low glucose supplemented with ßHB or C10. Cellular cholesterol was analyzed using HPLC, while phospholipids and sphingomyelin (SM) were analyzed using HPTLC. RESULTS: HT22 cells showed higher cholesterol, but lower SM levels in the low glucose group without supplements as compared to the high glucose groups. While cellular cholesterol was reduced in both ßHB- and C10-incubated cells, phospholipids were significantly higher in C10-incubated neurons. Ratios of individual phospholipids to cholesterol were significantly higher in ßHB- and C10-incubated neurons as compared to controls. CONCLUSION: Changes in the ratios of individual phospholipids to cholesterol in HT22 neurons suggest a possible alteration in the composition of the plasma membrane and organelle membranes, which may provide insight into the working mechanism of KD metabolites ßHB and C10.

Genetic and nongenetic profiling of milk ß-hydroxybutyrate and acetone and their associations with ketosis in Holstein cows.

Ketosis is a metabolic disorder of increasing importance in high-yielding dairy cows, but accurate population-wide binary health trait recording is difficult to implement. Against this background, proper Gaussian indicator traits, which can be routinely measured in milk, are needed. Consequently, we focused on the ketone bodies acetone and ß-hydroxybutyrate (BHB), measured via Fourier-transform infrared spectroscopy (FTIR) in milk. In the present study, 62,568 Holstein cows from large-scale German co-operator herds were phenotyped for clinical ketosis (KET) according to a veterinarian diagnosis key. A sub-sample of 16,861 cows additionally had first test-day observations for FTIR acetone and BHB. Associations between FTIR acetone and BHB with KET and with test-day traits were studied phenotypically and quantitative genetically. Furthermore, we estimated SNP marker effects for acetone and BHB (application of genome-wide association studies) based on 40,828 SNP markers from 4,384 genotyped cows, and studied potential candidate genes influencing body fat mobilization. Generalized linear mixed models were applied to infer the influence of binary KET on Gaussian-distributed acetone and BHB (definition of an identity link function), and vice versa, such as the influence of acetone and BHB on KET (definition of a logit link function). Additionally, linear models were applied to study associations between BHB, acetone and test-day traits (milk yield, fat percentage, protein percentage, fat-to-protein ratio and somatic cell score) from the first test-day after calving. An increasing KET incidence was statistically significant associated with increasing FTIR acetone and BHB milk concentrations. Acetone and BHB concentrations were positively associated with fat percentage, fat-to-protein ratio and somatic cell score. Bivariate linear animal models were applied to estimate genetic (co)variance components for KET, acetone, BHB and test-day traits within parities 1 to 3, and considering all parities simultaneously in repeatability models. Pedigree-based heritabilities were quite small (i.e., in the range from 0.01 in parity 3 to 0.07 in parity 1 for acetone, and from 0.03-0.04 for BHB). Heritabilites from repeatability models were 0.05 for acetone, and 0.03 for BHB. Genetic correlations between acetone and BHB were moderate to large within parities and considering all parities simultaneously (0.69-0.98). Genetic correlations between acetone and BHB with KET from different parities ranged from 0.71 to 0.99. Genetic correlations between acetone across parities, and between BHB across parities, ranged from 0.55 to 0.66. Genetic correlations between KET, acetone, and BHB with fat-to-protein ratio and with fat percentage were large and positive, but negative with milk yield. In genome-wide association studies, we identified SNP on BTA 4, 10, 11, and 29 significantly influencing acetone, and on BTA 1 and 16 significantly influencing BHB. The identified potential candidate genes NRXN3, ACOXL, BCL2L11, HIBADH, KCNJ1, and PRG4 are involved in lipid and glucose metabolism pathways.

Ketoacidosis associated with ketogenic diet in a non-diabetic lactating woman.

A 37-year-old woman who had 8 weeks post partum, breast feeding and on a low carbohydrate and high protein (ketogenic) diet, was admitted to the hospital with acute onset of nausea, vomiting and abdominal pain of 1-day duration. On admission, she was found to have high anion gap metabolic acidosis, elevated beta-hydroxybutyric acid level, normal glucose level and evidence of ketoacidosis. She was treated with lactated Ringer solution, along with dextrose 5% solution with the resolution of symptoms and metabolic derangement.

Opportunities and limitations of milk mid-infrared spectra-based estimation of acetone and ß-hydroxybutyrate for the prediction of metabolic stress and ketosis in dairy cows.

Subclinical (SCK) and clinical (CK) ketosis are metabolic disorders responsible for big losses in dairy production. Although Fourier-transform mid-infrared spectrometry (FTIR) to predict ketosis in cows exposed to great metabolic stress was studied extensively, little is known about its suitability in predicting hyperketonemia using individual samples, e.g. in small dairy herds or when only few animals are at risk of ketosis. The objective of the present research was to determine the applicability of milk metabolites predicted by FTIR spectrometry in the individual screening for ketosis. In experiment 1, blood and milk samples were taken every two weeks after calving from Holstein (n = 80), Brown Swiss (n = 72) and Swiss Fleckvieh (n = 58) cows. In experiment 2, cows diagnosed with CK (n = 474) and 420 samples with blood ß-hydroxybutyrate [BHB] <1.0 mmol/l were used to investigate if CK could be detected by FTIR-predicted BHB and acetone from a preceding milk control. In experiment 3, correlations between data from an in farm automatic milk analyser and FTIR-predicted BHB and acetone from the monthly milk controls were evaluated. Hyperketonemia occurred in majority during the first eight weeks of lactation. Correlations between blood BHB and FTIR-predicted BHB and acetone were low (r = 0.37 and 0.12, respectively, P < 0.0001), as well as the percentage of true positive values (11.9 and 16.6%, respectively). No association of FTIR predicted ketone bodies with the interval of milk sampling relative to CK diagnosis was found. Data obtained from the automatic milk analyser were moderately correlated with the same day FTIR-predicted BHB analysis (r = 0.61). In conclusion, the low correlations with blood BHB and the small number of true positive samples discourage the use of milk mid-infrared spectrometry analyses as the only method to predict hyperketonemia at the individual cow level.

ß-Hydroxybutyrate dehydrogenase decorated MXene nanosheets for the amperometric determination of ß-hydroxybutyrate.

MXene nanosheets of type Ti3C2Tx were modified with ß-hydroxybutyrate dehydrogenase and then used as a biosensor for amperometric sensing of ß-hydroxybutyrate. The MXene and the nanocomposite were characterized by X-ray photoelectron spectroscopy, field-emission scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The MXene has a layered structure and proved to be an excellent immobilization matrix providing good compatibility with the enzyme ß-hydroxybutyrate dehydrogenase. The MXene-based biosensor, best operated at a potential of - 0.35 V (vs. Ag/AgCl), displays a wide linear range (0.36 to 17.9 mM), a sensitivity of 0.480 µA mM-1 cm-2, and a low detection limit (45 µM). The biosensor was successfully applied to the determination of ß-hydroxybutyrate in (spiked) real serum samples. Graphical abstract Schematic representation of the synthesis and decoration of Mxene 2D sheets with ß-hydroxybutyrate dehydrogenase for the amperometric determination of ß-hydroxybutyric acid.

Predictive ability of host genetics and rumen microbiome for subclinical ketosis.

Subclinical metabolic disorders such as ketosis cause substantial economic losses for dairy farmers in addition to the serious welfare issues they pose for dairy cows. Major hurdles in genetic improvement against metabolic disorders such as ketosis include difficulties in large-scale phenotype recording and low heritability of traits. Milk concentrations of ketone bodies, such as acetone and ß-hydroxybutyric acid (BHB), might be useful indicators to select cows for low susceptibility to ketosis. However, heritability estimates reported for milk BHB and acetone in several dairy cattle breeds were low. The rumen microbial community has been reported to play a significant role in host energy homeostasis and metabolic and physiologic adaptations. The current study aims at investigating the effects of cows' genome and rumen microbial composition on concentrations of acetone and BHB in milk, and identifying specific rumen microbial taxa associated with variation in milk acetone and BHB concentrations. We determined the concentrations of acetone and BHB in milk using nuclear magnetic resonance spectroscopy on morning milk samples collected from 277 Danish Holstein cows. Imputed high-density genotype data were available for these cows. Using genomic and microbial prediction models with a 10-fold resampling strategy, we found that rumen microbial composition explains a larger proportion of the variation in milk concentrations of acetone and BHB than do host genetics. Moreover, we identified associations between milk acetone and BHB with some specific bacterial and archaeal operational taxonomic units previously reported to have low to moderate heritability, presenting an opportunity for genetic improvement. However, higher covariation between specific microbial taxa and milk acetone and BHB concentrations might not necessarily indicate a causal relationship; therefore further validation is needed before considering implementation in selection programs.

Symposium review: Big data, big predictions: Utilizing milk Fourier-transform infrared and genomics to improve hyperketonemia management.

Negative animal health and performance outcomes are associated with disease incidences that can be labor-intensive, costly, and cumbersome for many farms. Amelioration of unfavorable outcomes through early detection and treatment of disease has emphasized the value of improving health monitoring. Although the value is recognized, detecting hyperketonemia (HYK) is still difficult for many farms to do practically and efficiently. Increasing data streams available to farms presents opportunities to use data to better monitor cow and herd health; however, challenges remain with regard to validating, integrating, and interpreting data. During the transition to lactation period, useful data are presented in the form of milk production and composition, milk Fourier-transform infrared (FTIR) wavelength absorbance, cow management records, and genomics, which have been employed to monitor postpartum onset of HYK. Attempts to predict postpartum HYK from test-day milk and performance variables incorporated into multiple linear regression models have demonstrated sufficient accuracy to monitor monthly herd prevalence; however, they lacked the sensitivity and specificity for individual cow diagnostics. Subsequent artificial neural network prediction models employing FTIR data and milk composition variables achieved 83 and 81% sensitivity and specificity for individual cow diagnostics. Although these results fail to reach the diagnostic goals of 90%, they are achieved without individual cow blood samples, which may justify acceptance of lower performance. The caveat is that these models depend on milk analysis, which is traditionally done every 4 weeks. This infrequent sampling allows for a single diagnostic sample for about half of the fresh cows. Benefits to farms are greatly improved if postpartum cows can be milk-tested weekly. Additionally, this allows for close monitoring of somatic cell count and may open the door for use of other herd health monitoring tools. Future improvements in these models may be achievable by maximizing sensitivity at the expense of specificity and may be most economical in disorders for which the cost of treatment is less than that of mistreating (e.g., HYK). Genomic predictions for HYK may be improved by incorporating genome-wide associated SNP and further utilized for precision management of HYK risk groups. Development and validation of HYK prediction models may provide producers with individual cow and herd-level management tools.

Comparison of the beta-hydroxybutyrate, glucose, and lactate concentrations derived from postmortem proton magnetic resonance spectroscopy and biochemical analysis for the diagnosis of fatal metabolic disorders.

PURPOSE: The detection and quantification of metabolites relevant for the diagnosis of fatal metabolic disorders by proton magnetic resonance spectroscopy (1H-MRS) was recently demonstrated. This prospective study aimed to compare the concentrations of beta-hydroxybutyrate (BHB), glucose (GLC), and lactate (LAC) derived from both biochemical analyses and 1H-MRS for the diagnosis of fatal metabolic disorders. METHODS: In total, 20 cases with suspected fatal metabolic disorders were included in the study. For the agreement based on thresholds, the concentrations of BHB and GLC in the vitreous humor (VH) from the right vitreous and in cerebrospinal fluid (CSF) from the right lateral ventricle were derived from 1H-MRS and biochemical analyses. The predefined thresholds for pathological elevations were 2.5 mmol/l for BHB and 10 mmol/l for GLC based on the literature. In addition, concentrations of the same metabolites in white matter (WM) tissue from the corona radiata of the right hemisphere were analyzed experimentally using both methods. To enable the biochemical analysis, a dialysate of WM tissue was produced. For all three regions, the LAC concentration was determined by both methods. RESULTS: The conclusive agreement based on thresholds was almost perfect between both methods with only one disagreement in a total of 70 comparisons due to the interference of a ferromagnetic dental brace. The differences in the concentrations between both methods showed high standard deviations. Confidence intervals of the bias not including 0 were found in CSF-GLC (- 3.1 mmol/l), WM-GLC (1.1 mmol/l), and WM-LAC (- 6.5 mmol/l). CONCLUSION: Despite a considerable total error attributable to both methods, MRS derives the same forensic conclusions as conventional biochemical analyses. An adaptation of the protocol to reduce the detected errors and more data are needed for the long-term validation of MRS for the diagnosis of fatal metabolic disorders. The production of WM dialysates cannot be recommended due to high glycolytic loss.

Polyhydroxyalkanoate (PHA) biosynthesis from directly valorized ragi husk and sesame oil cake by Bacillus megaterium strain Ti3: Statistical optimization and characterization.

Polyhydroxyalkanoates (PHAs) signify the most promising biological substitute to petrochemical plastics. Renewable and inexpensive agro-industrial by-products can be used as potent fermentation feedstocks for sustainable PHA biosynthesis. This study aimed at using a wild type B. megaterium strain Ti3 innate hydrolytic enzyme/s for eco-friendly valorization of 16 lignocellulosic agrowastes to PHA without pretreatments. Initial hydrolytic screening PHA concentration of (0.04-0.17 g/L), highlighted the strain's metabolic versatility. Pareto ranking of Taguchi orthogonal array (TOA) established ragi husk (RH), sesame oil cake (SOC) and KH2PO4 as the most influential factors (p < 0.05). The optimized and validated Response surface methodology (RSM) model (R2, 0.979; desirability, 1) resulted in 3.8 and 3.6 fold increased PHA production, 4.3 and 3.25 fold increased PHA productivity. A positive correlation (r2, 0.5-0.97) was observed amid the producer innate hydrolytic enzymes (lipase, amylase and cellulase) and PHA production. The PHA was characterized by 1H and 13C NMR, GPC, TGA. The polymer was identified as a scl-mcl copolyester with 92% 3HB (3-hydroxybutyrate) and 8% 3HHp (3-hydroxyheptanoate) monomers by NMR. This the first report on B. megaterium self-enzyme reliant non-food agrowastes bioconversion to PHA with 3HHp (3-hydroxyheptanoate) monomers excluding precursor addition, commercial enzymes, pure carbon and nitrogen sources.