BHB Levels Research Articles

Ketone and pyruvate Ringer's solutions decrease pulmonary apoptosis in a rat model of severe hemorrhagic shock and resuscitation

Background. Resuscitation fluids containing β-hydroxybutyrate (BHB) have been shown to decrease cellular injury after hemorrhagic shock and resuscitation through an unknown mechanism. We tested whether this effect was related to BHB-induced metabolic modulations. Methods. Male Sprague Dawley rats (n=30) were subjected to volume-controlled hemorrhage (27 mL/kg during 10 minutes followed by 75 minutes of shock during which another 8 mL/kg of blood was withdrawn). Experimental groups included the following: (1) sham, (2) no resuscitation (NR), (3) racemic lactated Ringer's (DL-LR) solution, (4) LR containing L-isomer only (L-LR), (5) ketone Ringer's solution with lactate substituted by BHB (KR), and (6) pyruvate Ringer's solution with lactate substituted by pyruvate (PR). The resuscitation fluids were infused during 45 minutes simultaneously with additional hemorrhage of 8 mL/kg. Hemodynamic and physiologic parameters and the plasma levels of BHB were serially measured. The animals were killed 2 hours after resuscitation, and tissues were frozen instantaneously for cellular adenylate extraction and adenosine triphosphate (ATP) and adenosine diphosphate analysis. Pulmonary apoptosis was studied using Western blotting, immunohistochemistry, and reverse transcriptase-polymerase chain reaction. Expression of enzymes involved in ketogenesis and ketolysis was analyzed by reverse transcriptase-polymerase chain reaction. Results. NR and resuscitation with DL-LR increased the expression of apoptotic markers, whereas resuscitation with KR and PR significantly decreased the expression of apoptotic markers in rat lungs. Resuscitation with KR was followed by a profound increase in plasma BHB levels; however, the expression levels of ketolytic enzymes were essentially unaffected. KR infusion did not induce significant improvements in tissue ATP levels. Conclusion. Resuscitation with KR and PR protects against pulmonary apoptosis without improving tissue ATP content. Therefore, metabolic modulation is unlikely to be the major mechanism by which BHB exerts its protective effects during reperfusion. (Surgery 2003;134:267-74.)

13C‐[2,4]‐b‐hydroxybutyrate metabolism in human brain

Although ketone metabolism is generally believed to be qualitatively similar to glucose consumption in the human brain, this has never been explicitly determined. We used 13C‐MR spectroscopy with infusions of 13C‐[2,4]‐b‐hydroxybutyrate (BHB, 200 mm, 100% enrichment) in overnight fasted adult subjects (n = 4) to examine the fate of this ketone. This was performed on a 2.1T Bruker spectrometer, volume size 6 × 4 × 6 cc3, previously determined to contain ∼ 56% gray, 39% white matter in normal adults. Results: The 2 h infusion increased plasma ketone levels from near negligible (baseline) to 2.25 ± 0.24 mm at steady state. Steady state 13C BHB levels were measured at 0.18 ± 0.04 mm. Similar to glucose, the 13C label appeared rapidly in the amino acid pools. The steady state fractional enrichment reached were: 13C‐4‐glutamate (4‐Glu), 6.78 ± 1.71%, 13C‐4‐glutamine (4‐Gln), 5.68 ± 1.84 and 13C‐3‐aspartate at 3.99 ± 0.57%. Discussion: The relatively low [BHB] of 0.18 ± 0.04 mm is an upper bound for tissue BHB, and is in agreement with the view that ketone consumption is largely regulated at the blood brain barrier in these nonfasted acutely hyperketonemic subjects. The general pattern of labeling of ketones is similar to that which has been previously seen with glucose. Steady state analysis of the ratio of 4‐Gln/4‐Glu suggests that ketones are predominantly used by neurons, and are consumed at a rate of 0.032 ± 0.009 mm/kg/min. This rate includes gray and white matter consumption. Dynamic modelling of the time courses of the brain 4‐Glu and 13C‐BHB, where gray matter ketone consumption is taken as three times greater than white matter (similar to that seen with glucose) gives an estimated upper bound for the flux of ketone oxidation in pure gray matter to be 0.041 ± 0.007 mm/kg/min.

Measurement of beta-hydroxybutyrate in acute hyperketonemia in human brain.

We report the measurement of D-beta-hydroxybutyrate (BHB) in the brains of six normal adult subjects during acute infusions of BHB. We used high field in vivo (1)H magnetic resonance (MR) spectroscopy in the occipital lobe in conjunction with an acute infusion protocol to elevate plasma BHB levels from overnight fasted levels (0.20 +/- 0.10 mM) to a steady state value of 2.12 +/- 0.30 mM. At this level of hyperketonemia, we determined a tissue BHB level of 0.24 +/- 0.04 mM. No increases in brain lactate levels were seen in these data. The concentrations of BHB and lactate were both considerably lower in comparison with previous data acquired in fasted adult subjects. This suggests that up-regulation of the monocarboxylic acid transporter occurs with fasting.

Hormone and metabolite profiles as well as the onset of ovarian cyclicity in dairy cows suffering from various forms of ketosis

AbstractThe involvement of adrenocortical and thyroid hormones in the pathogenesis of ketosis as well as the ovarian consequences of this metabolic disorder were studied in ≥2 parity cows (n=199) in 3 large scale dairy herds. To compare the plasma / serum concentrations of certain hormones [Cortisol, thyroxin (T4), triiodo-thyronine (T3), insulin, isulin-like growth factor-1 (IGF-l)J and metabolites [glucose (G), acetoacetic acid (ACAC), βOH-butyrate (BHB), non-esterified fatty acid (NEFA), trigliceride (TG), total cholesterol (TCh)J, and the activity of aspartate aminotransferase (AST), blood samples were taken 1 to 3 days after calving and again at 7-day intervals on four other occasions. The ACTH-challenged Cortisol responsiveness and the TRH-induced T4/T3 increase were determined between days 1 to 3 and again between days 28 to 35. The resumption of ovarian cyclicity was followed up by individual progesterone (P4) profiles based on milk samples taken 3 times a week for about 80 to 85 days postpartum. A concentration of 1 mmol/l of BHB level was estimated as a border between hyper- (>1 mmol/l) and normal ketonaemic (<1 mmol/l) conditions. 5 different ketone patterns were distinguished: (1) non-ketotic (n=98; normal ketonaemia in all samples), (2) early type ketosis (n=45; hyperketonemia was detected only in the first week after calving), (3) late type (lacta-tional) ketosis (n=11; after a normal ketonaemic period increasing hyperketonaemia was detected in the 5th, or in the 4th and 5th weeks), (4) temporary ketosis (n=ll; hyperketonaemia was detected for 1-2 weeks in the 2nd and 3rd or in the 3rd and 4th weeks); (5) long-lasting ketosis (n=34; hyperketonaemia had been detected since calving for 4 to 5 weeks or until death or emergency slaughtering). Simultaneously with the hyperketonaemic stage increased NEFA, ACAC, depressed TCh, glucose and decreased insulin, IGF-1, T4 and T3 concentrations were detected in almost all the cases. Obvious metabolic and endocrine alterations were found, however, only in long-lasting ketosis. The TRH-stimulated T4 and T3 responses remained almost unaffected proving intact thyroid function in early and late type as well as in temporary ketosis. Depressed thyroid response and delayed onset of cyclic ovarian function were detected only in cases of long-lasting ketosis. The cows characterized by lower than normal (< mean-SD of non-ketotic cows) ACTH-stimulated cortisol response on days 1-3 after calving showed poorer chancefor spontaneous recovery. There was a significant negative correlation between the IGF-1 level in the 1st week after calving and the duration of the postpartum acyclic period. In late type (lactational) ketosis the cessation of ovarian cyclicity was the most characteristic genital malfunction.

Use of metabolic profiles in dairy cattle in tropical and subtropical countries on smallholder dairy farms

Metabolic profile testing has generally been used as part of a multidisciplinary approach for dairy herds in temperate climates. Our goal was to evaluate the effectiveness of the technique for identifying constraints on productivity in small herds in environments less favorable for milk production. Metabolites tested were chosen for stability in the sample after collection of blood, ease of analysis and practical knowledge of the meaning of the results. Blood levels of five different metabolites in low-producing dairy cows belonging to smallholders in tropical and subtropical environments were measured. The study involved 13 projects with 80 cows in each, carried out in six Latin American, six Asian, and one southern European countries. Data were also collected on feeding, body condition score (BCS) and weight change, parasitism, and reproduction. In Chile, Mexico, Paraguay, Philippines, Uruguay, and Venezuela, globulin levels were high in >17% of cows sampled on each occasion. Globulin levels were also high in Turkey and Vietnam on one or more occasions. In Paraguay, 49% of cows had high globulin levels at two to three months after calving. These results suggest that inflammatory disease was present to a potentially important degree, although this was not always investigated and not always taken into account. In all countries except Mexico and Venezuela, high β-hydroxybutyrate (BHB) levels before calving in many cows highlighted the presence of condition loss in late pregnancy, an important potential constraint on productivity and fertility. Fewer cows showed high BHB levels in lactation, whereas change in BCS and weight was more sensitive for measuring negative energy balance. Urea concentrations were low in only small numbers of cows suggesting that dietary protein shortages were not common. Albumin values were low mainly in cows where globulin values were high and, hence, did not generally provide additional information. The exception was in China where pregnant yaks over winter had high BHB and low albumin values, suggesting that they were seriously underfed. This observation stimulated a successful nutritional intervention in the following winter. Inorganic phosphate values were within the reference range in most countries a majority of the time suggesting, contrary to expectation, that this mineral was not commonly a constraint. The use of metabolic profile testing proved valuable in drawing attention to important potential constraints on productivity in dairy cows in tropical and subtropical environments and in confirming those which were not.

Hyperketonemia (Acetoacetate) Increases the Oxidizability of LDL + VLDL In Type-I Diabetic Patients

The reason for the disproportionately higher level of vascular disease in patients with diabetes is not known. Oxidative modification of low-density lipoproteins has been implicated in impaired cholesterol uptake and its deposition in the arterial wall and atherosclerosis. The present study has examined the effects of hyperketonemia, glycemic control and duration of diabetes on the in vitro oxidative susceptibility to Cu ++ of low-density lipoprotein (LDL) + very low-density lipoprotein (VLDL) from 34 Type-I diabetic patients without any clinical sign of vascular disease and 22 age-matched normal individuals. LDL + VLDL was isolated from plasma using a micro-affinity column. LDL + VLDL isolated from diabetic patients and age-matched normal individuals was treated with 25 mM CuCl2 for 1.5, 3 and 5 h. The ketone bodies acetoacetate (AA) and β-hydroxybutyrate (BHB), as well as glycated hemoglobin (HbA 1), were measured in the blood by standard methods. There was no difference in the in vitro oxidative susceptibility of LDL + VLDL at all time periods between Type-I diabetics ( n = 34) and age-matched normal individuals ( n = 22). However, among diabetics, when patients were separated into normoketonemic (NK) and hyperketonemic (HK) groups, in vitro oxidation of LDL + VLDL at 1.5 h from hyperketonemic diabetics was a 69% greater ( p < .02) compared with that of normoketonemic diabetics and 80% greater ( p < .02) compared with that of normal individuals. There was a significant correlation ( r = 0.38, p < .03) between the in vitro oxidation of LDL + VLDL at 1.5 h and AA levels in diabetic patients. The level of in vitro oxidizability of LDL + VLDL did not have any correlation with levels of BHB ( r = 0.20, p > .26), HbA 1 ( r = 0, p > .99), glucose ( r = 0.06, p > .75) or duration of diabetes ( r = 0.15, p > .40) in diabetic patients. In vitro incubation of normal plasma with AA resulted in an increase in the Cu ++ induced lipid peroxidation of LDL + VLDL. This study suggests that frequent episodes of ketosis and elevated levels of AA constitute a risk factor for the oxidative modification of low-density lipoproteins and development of vascular disease in diabetic patients.

Changes in Extracellular and Rat Brain Tissue Concentrations of D‐β‐Hydroxybutyrate After 1, 3‐Butanediol Treatment

1,3-Butanediol (BD) treatment was previously shown to produce a dose-related increase of the plasma levels of D-beta-hydroxybutyrate (BHB) and to protect brain tissue against hypoxia and ischemia. The purpose of this study was to test whether BD-induced hyperketonemia was associated with changes in brain extracellular and tissue concentrations of BHB. Changes in extracellular levels of BHB were continuously monitored in anesthetized rats before and after intraperitoneal injection of BD (25 mmol/kg), using intracerebral microdialysis coupled to online analysis of BHB in the dialysate. Cortical tissue concentrations of BHB were determined in control and BD-treated rats (25 and 50 mmol/kg, i.p.) after freezing of the brain in situ. Butanediol produced a rapid increase in dialysate levels of BHB, with a linear relationship between dialysate and plasma BHB concentrations (r = 0.81, p < 0.001). In contrast, and although brain tissue levels of BHB were markedly increased after BD treatment, they were not related to the plasma concentration of BHB. Our results suggest that BHB produced from BD did not accumulate in brain and that BD protects against hypoxia or ischemia by increasing brain BHB availability.

A new quick-test for semiquantitative determination of beta-hydroxybutyric acid in bovine milk.

A new test strip for semiquantitative determination of beta-hydroxybutyric acid (BHB) in bovine milk has been tested in experimental investigations in three lactating cows. After intraruminal administration of butyric acid, the following parameters were determined during 7 hours: milk concentrations of BHB (quantitatively and semiquantitatively) and acetone (quantitatively), BHB concentration in blood and urine (quantitatively), acetone + acetic acid concentration in urine (semiquantitatively). The new quick-test proved to be highly sensitive and even subclinical or physiological levels of BHB in milk could be determined. It seems to be suitable not only for the diagnosis of clinically manifest ketosis in dairy cows, but also for the control of the energy metabolism on a herd basis and as an indicator of ketogenic feedstuffs in the ration.

β-Hydroxybutyrate and response to hypoxia in the ground squirrel, Spermophilus tridecimlineatus

1. 1. Previous studies have suggested that elevated ketone levels are associated with increased survival time in rodents exposed to hypoxia. In this study the association between whole blood BHB (β-hydroxybutyrate) and hypoxic surivaval time was investigated in hibernating and non-hibernating ground squirrels and in rats. 2. 2. Non-hibernating ground squirrels and rats were exposed to hypoxia (4.5% O 2). One hundred per cent of ground squirrels survived 1 hr of hypoxia vs 20% of rats. 3. 3. Ketone levels were significantly higher in ground squirrels than rats during hypoxia, and rats surviving the longest had the highest ketone levels. 4. When hibernation was induced in ground squirrels there was a significant increase in β-hydroxybutyrate from 0.45 to 1.6 mM ( P = 0.0005). 4. 5. Ground squirrel heart mitochondrial respiratory control ratios and ATP synthesis rates indicated no preferential ketone utilization which might suggest a possible extramitochondrial role of BHB during hypoxia. 5. 6. We conclude that elevated blood BHB levels are associated with increased hypoxic survival and they may have evolved in response to life-threatening hypoxia as experienced during hibernation.

INCREASED β- AND ω-OXIDATION OF FATTY ACIDS IN THE SILVER RUSSELL SYNDROME

We found evidence for an increased oxidation of fatty acids in two Silver-Russell patients (dwarfism of prenatal onset, typical craniofacial appearance and dystrophy).Fasting studies showed elevated blood and urine levels of β-hydroxybutyrate (BHB) and aceto-acetate (AA) and a massive urinary excretion of C6-C12 dicarboxylic acids. After 20 hrs of fasting BHB in blood was 3.50 and 3.50 mmol/1 while AA was 1.02 and 1.12 mmol/1 in the two Silver-Russell dwarfs, respectively. Seven controls of comparable age (1-2½ yr) had significanly lower blood levels of BHB (mean: 1.68 mmol/l) and AA (mean: 0.63 mmol/1). GC-MS analysis of urine organic acids of both Silver-Russell patients showed a very similar pattern with a massive excretion of BHB (<10 mmol/1) and C6-C12 dicarboxylic acids (adipic acid, suberic acid, sebacic acid, C12-dicarboxylic acid, cis/trans unsaturated suberic acid and sebacic acid, cis unsaturated 3-OH-derivates of sebacic acid and C12dicarboxylic acid). In contrast controls had a low excretion of BHB (<1 mmol/l), while C6-C12 dicarboxylic acids were present in very small amounts or not detectable. The combination of both an activated β-oxidation resulting in high blood and urine levels of BHB and AA, and an activated ω-oxidation resulting in a massive urinary excretion of dicarboxylic acids is very typical.We suggest that this increased oxidation of fatty acids is a distinct feature in the Silver-Russell syndrome and might provide an explanation for the dystrophy which is so characteristic.

Effects of energy levels and sources on plasma metabolites and live weight of Holstein cows

The objective of the study was to evaluate the effects of nutritional treatments, which differed after calving, on energy levels and sources on plasma metabolite profiles and live weight (LW) changes as an indication of the nutritional status in Holstein cows. During the dry period, pregnant heifers (n = 69) and dry cows (n = 153) from Elsenburg Research Farm were maintained under similar feeding and management conditions. After parturition, cows had ad libitum access to cultivated irrigated kikuyu-ryegrass pastures, and were assigned to three concentrate groups, according to calving date, parity, LW and the milk yield of their previous lactation. The groups were supplemented with various levels and types of concentrate, of which the energy was provided by starch and fat. The control group was offered 7 kg/cow/day of a control concentrate supplement for both primiparous and multiparous groups, while concentrates in treatment groups were fed at 11.6 and 12.6 kg/cow/day for primiparous and multiparous groups, respectively. The control supplement was a maize-based concentrate, which contained low levels of starch. The concentrate components of the treatments consisted of high starch-low fat (HSLF) and a high starch-low fat/low starch-high fat (HSLF-LSHF) combination. The HSLF supplement was a glucogenic concentrate, which contained maize as the energy source. The HSLF-LSHF supplements consisted of a glucogenic concentrate, which was offered for the first 60 days in milk (DIM) as per the HSLF treatment, and was followed from 61 DIM with a lipogenic concentrate containing wheat bran and calcium (Ca) salts of long-chain fatty acids as the energy sources. The results showed that all cows mobilized their body fat reserves, as was evident in changes in plasma non-esterified fatty acids (NEFA) before and after calving. Postpartum plasma NEFA and β-hydroxybutyrate (BHB) did not differ significantly between nutritional treatments in multiparous cows. However, the postpartum levels of plasma NEFA and BHB were significantly higher for the control, indicating a status of advanced negative energy balance (NEB) and possible subclinical ketosis compared with HSLF and HSLF-LSHF treatments in primiparous cows. Postpartum plasma urea levels decreased significantly in both primiparous and multiparous animals in the control group, compared with the HSLF and HSLF-LSHF groups. As affected by time, postpartum LW was significantly lowest and LW loss was significantly highest in cows that received the control supplements compared with HSLF and HSLF-LSHF supplements for primiparous and multiparous cows. In addition, LW lossnadir and the number of days to reach it significantly increased in primiparous cows that received the control concentrate, compared with those of the HSLF and HSLF- LSHF treatments. However, this trend was not observed for multiparous cows. The findings of this study showed that HSLF and HSLF-LSHF treatments improved the nutritional status, as was evident in the reduced extent of NEB and limited LW loss of dairy cows, compared with those in the control group.Keywords: Concentrate, dairy cattle, energy nutrients, nutritional status, pasture