Breath profiles of children on ketogenic therapy.

Ketogenic diets (KDs) were initially introduced to clinical practices as alimentary approaches with the aim to control drug-resistant epilepsies. Over the decades, a large and growing body of research has addressed the antiseizure effect of various KDs, and worked out KD-based dietary regimens, including their acting factors and modes of action. KDs have also appeared in weight loss therapies. Therapy control, particularly at initiation, happens through regular blood analysis and control of urine ketone levels. However, there is a lack of fast, reliable, and preferably non-invasive methods to accomplish this. The detection of exhaled breath constituents may offer a solution. The exhaled breath contains hundreds of volatile organic compounds (VOCs), which can be modified by diet. VOC detection technology has resulted in low-cost sensors that can facilitate the self-monitoring of patients in the future if reliable breath markers are available. Therefore, it is of interest to investigate the composition of exhaled breath in children on KDs. Twenty-two pediatric patients between 4 and 18 years of age were recruited in this study. Eleven of them received a KD and suffered from epilepsy, with the exception of one child, who was admitted to a weight-reduction therapy. The control group involved 11 patients with neurological disorders but not on KD. Breath volatiles were analyzed using gas chromatography mass spectrometry (GC-MS) after preconcentration of the analytes on needle traps (NTs). We found that the breath concentrations of a number of VOCs, namely acetaldehyde, acetone, 2-methylfuran, methyl-vinyl-ketone, and 2-pentanone were significantly elevated in the breath of children on a KD in comparison to their control counterparts. Interestingly, breath ethanol was lower in patients on a KD than in non-KD patients. Association studies revealed an interrelationship among (i) lipid parameters and ketone bodies, (ii) methacrolein, methyl-vinyl-ketone, and high-density lipoprotein, as well as (iii) methyl-vinyl-ketone, acetone, and 2-pentanone, thus raising the possibility of a common metabolic source. The duration of diet was positively and negatively associated with breath acetone and breath ethanol, respectively. Some of the changes were linked to ß-oxidation, but there are uncertainties in regard to metabolic sources of other metabolites. Lipid peroxidation and alteration of intestinal microbial composition may also be involved in the changes of VOC profiles during KD. Since lipids used for metabolism during KD originate from external sources, the processes occurring cannot simply be compared to and deduced from changes appearing in starvation; however, lipid mobilization is also evident in starvation. To find reliable and sensitive VOC markers that are linked to the respective ketogenic regimen, further investigations are needed to reveal the metabolic background.

Barley sprout extracts reduce hepatic lipid accumulation in ethanol-fed mice by activating hepatic AMP-activated protein kinase.

Chronic alcohol consumption leads to hepatic lipid accumulation and alcoholic fatty liver disease. Previously, we demonstrated that barley sprout extract, which contains saponarin as an active compound, reduces hepatic steatosis. In this study, we investigated the effect of barley sprout extracts (BSE) on hepatic lipid accumulation in a mouse model of alcoholic fatty liver disease. Seven-week-old C57BL/6 mice were fed an alcohol-containing diet (5% ethanol) and a low or high dose of BSE (100 or 200mg/kg body weight, respectively) for 10days. The high dose of BSE significantly decreased hepatic lipid accumulation compared with the ethanol-only control group. In the second animal study, mice were fed an alcohol-containing diet for 10days, followed by a 45% high-fat diet with oral administration of BSE (100 or 200mg/day/kg body weight) for 4weeks. Mice in both BSE-fed groups showed reduced hepatic steatosis. In the livers of mice fed BSE, phosphorylation of AMP-activated protein kinase (AMPK) was increased, and expression of hepatic autophagy markers was elevated. In cultured hepatocytes, BSE (200µg/mL) increased the rate of fatty acid oxidation and reduced that of fatty acid synthesis. Taken together, these findings suggest that BSE promotes degradation of lipid droplets and subsequent activation of fat oxidation by activating AMPK in the liver, thus protecting against development of hepatic steatosis in alcohol-fed mice. Saponarin, a major flavonoid in BSE and an activator of AMPK, increased the activity of microsomal triglyceride transfer protein, which suggests that the reduction in hepatic triglyceride levels was mediated by this component of BSE. In conclusion, BSE ameliorated hepatic steatosis in a mouse model of ethanol-induced fatty liver by activating AMPK, an effect possibly mediated by the saponarin component.

Clinical evaluation of nervous ketosis induced by isopropanol in sheep / Manifestações clínicas da cetose nervosa induzida por isopropanol em ovinos

Twelve healthy Santa Ines sheep, non-pregnant and non-lactating underwent a protocol of experimental induction of nervous ketosis for studying the symptoms in ketosis and Pregnancy Toxaemia (PT) disease. Eight animals were subjected to infusion of 150 mL of isopropanol (IPA) at 35% in the jugular vein and four sheep were treated identically with isotonic saline solution (NaCl 0.9%) during 40 minutes. The animals treated with IPA showed increased heart rate (HR) after 40 minutes of infusion and decreased ruminal movement from the 10 minutes infusion with IPA and remaining low up to 10 minutes from the end of the infusion. Ruminal atony appeared in three animals, which showed slight meteorism gas. Infusion of IPA caused the appearance of nervous symptoms as depression, staggering, adduction of hind limbs, head pressing, teeth grind and blindness almost always accompanied by a decreased pupillary reflex and nystagmus. All the animals exhaled ketosis breath with strong odor within few minutes of the start of induction that helped the elimination of the IPA from the organism. The present results strongly suggest that part of the nervous symptoms observed in PT may be derived from the action of IPA. These present findings open new perspectives for a better understanding of the pathogenesis of PT in sheep...

Doze ovelhas hígidas, não prenhes e não lactentes foram submetidas a um protocolo de indução experimental de cetose nervosa para a avaliação de sintomatologia nos quadros de cetose e Toxemia da Prenhez. Neste protocolo, oito animais foram submetidos a infusão de 150 mL de solução de isopropanol a 35% na veia jugular, constituindo o grupo Tratado (GT), e quatro ovinos foram tratados identicamente com solução salina isotônica (NaCl 0,9%), constituindo o grupo Controle (GC) no decorrer de 40 minutos. Os animais do GT apresentaram aumento da frequência cardíaca (FC) aos 40 minutos de infusão e redução no movimento ruminal a partir dos 10 minutos de infusão com isopropanol, o qual permaneceu diminuído ate 10 minutos do termino da infusão. A atonia ruminal ocorreu em três animais, que manifestaram em seguida leve meteorismo gasoso. A infusão de isopropanol provocou o surgimento de sintomas nervosos como, depressão e sonolência, cambaleios, adução de membros posteriores, pressão da cabeça em obstáculo, ranger de dentes e cegueira quase sempre acompanhada de diminuição do reflexo pupilar e nistagmo. Todos os animais exalaram forte odor cetótico com poucos minutos do inicio da indução, fato que ajudou a eliminação do isopropanol do organismo. Os presentes resultados sugerem que parte dos sintomas nervosos verificados na Toxemia da Prenhez possa ser oriunda da ação do isopropanol. Estes resultados abrem novas perspectivas para o melhor entendimento da patogenia da Toxemia da Prenhez em ovinos...

Effects of long-term feeding of a diet supplemented with clinoptilolite to dairy cows on the incidence of ketosis, milk yield and liver function.

Fifty-two clinically healthy Holstein cows were randomly assigned to one of three groups according to their age and parity. The first group (A) consisted of 17 cows that were fed a concentrate ration supplemented with 1.25 per cent clinoptilolite, the second group (B) consisted of 17 cows fed a ration supplemented with 2.5 per cent clinoptilolite, and the third group (C) consisted of 18 cows, which were fed the basal ration containing no clinoptilolite. The rations were fed from four weeks before the cows' expected parturition dates until the beginning of the next dry period. Blood samples were collected from each animal at the start of the experiment, on the day of calving and then monthly, and analysed for serum glucose, ketone bodies, liver enzymes, blood urea nitrogen (BUN) and total proteins. The milk yield of each cow was recorded monthly. The cows in group B had significantly fewer cases of clinical ketosis during the first month after calving and a higher total milk yield. Feeding the cows with clinoptilolite for a long period had no apparent adverse effects on their liver function, and did not significantly affect the concentrations of glucose, ketone bodies, BUN and total proteins in their serum.

Oral beta-hydroxybutyrate supplementation in two patients with hyperinsulinemic hypoglycemia: monitoring of beta-hydroxybutyrate levels in blood and cerebrospinal fluid, and in the brain by in vivo magnetic resonance spectroscopy.

In persistent hyperinsulinemic hypoglycemia of infancy, ketone body concentrations are abnormally low at times of hypoglycemia, depriving the brain of its most important alternative fuel. The neuroprotective effect of endogenous ketone bodies is evidenced by animal and human studies, but knowledge about exogenous supply is limited. Assuming that exogenous ketone body compounds as a dietetic food might replace this alternative energy source for the brain, we have monitored the fate of orally supplemented DL sodium beta-hydroxybutyrate (beta-OHB) in two 6-mo-old infants with persistent hyperinsulinemic hypoglycemia for 5 and 7 mo, while on frequent tube-feedings and treatment with octreotide. Near total (95%) pancreatectomy had been ineffective in one patient and was refused in the other. In blood, concentrations of beta-OHB increased to levels comparable to a 16- to 24-h fast while on DL sodium beta-OHB 880 to 1000 mg/kg per day. In cerebrospinal fluid, concentrations of beta-OHB increased to levels comparable to a 24- to 40-h fast, after single dosages of 4 and 8 g, respectively. High ratios of beta-OHB to acetoacetate indicated exogenous origin of beta-OHB. An increase of intracerebral concentrations of beta-OHB could be demonstrated by repetitive single-voxel proton magnetic resonance spectroscopy by a clear doublet at 1.25 ppm. Oral DL sodium beta-OHB was tolerated without side effects. This first report on oral supplementation of DL sodium beta-OHB in two patients with persistent hyperinsulinemic hypoglycemia demonstrates effective uptake across the blood-brain barrier and could provide the basis for further evaluation of the neuroprotective effect of beta-OHB in conditions with hypoketotic hypoglycemia.

Food restriction and stimulation of monooxygenation of p-nitroanisole in perfused rat liver.

This study assessed the effect of food restriction on the metabolism of model monooxygenase substrates in the perfused rat liver. Female Sprague-Dawley rats has access ad lib. to a Purina 5001 nonpurified diet (control) or were given 65% of the intake of controls for 3 weeks. Livers were perfused with oxygenated Krebs-Henseleit buffer using a non-recirculating system, and the rates of monooxygenation of p-nitroanisole and 7-ethoxycoumarin were measured. The results indicate that food restriction stimulated p-nitroanisole O-demethylation from 2.9 +/- 0.2 to 4.6 +/- 0.5 mumol/(g.hr) when saturating concentrations of p-nitroanisole were infused. Concomitantly, the ratio of beta-hydroxybutyrate to acetoacetate (B/A) and the rates of ketogenesis (B + A) were increased significantly by food restriction. Further, p-nitroanisole (200 mumol/L) increased hepatic malate concentration nearly 3-fold in liver extracts from food-restricted rats. However, infusion of either a low concentration of p-nitroanisole (50 mumol/L) or 7-ethoxycoumarin (200 mumol/L) did not alter these parameters. On the other hand, food restriction did not alter rates of monooxygenation in isolated microsomes supplemented with excess NADPH. Taken together, these data support the hypothesis that high concentrations of p-nitroanisole increased monooxygenation in food-restricted rats by stimulating fatty acid oxidation, which elevates the mitochondrial NADH/NAD+ ratio. This, in turn, increases the availability of reducing equivalents in the form of NADPH by a malate-pyruvate exchange system, leading to increased drug metabolism.