VALPROIC ACID HAS TEMPORAL VARIABILITY IN URINARY CLEARANCE OF METABOLITES

The reasons for the intra- and interindividual variability in the clearance of valproic acid (VPA) have not been completely characterized. The aim of this study was to examine day–night changes in the clearance of 3-oxo-valproate (3-oxo-VPA), 4-hydroxy-valproate (4-OH-VPA), and valproic acid glucuronides under steady state. Six diurnally active healthy male volunteers ingested 200 mg sodium valproate 12 hourly, at 0800 and 2000, for 28 days. On the last study day, two sequential 12-h urine samples were collected commencing at 2000 the evening before. Plasma samples were obtained at the end of each collection. Following alkaline hydrolysis, urine was analyzed for concentrations of VPA, 3-oxo-VPA, and 4-OH-VPA. A separate aliquot was assayed for creatinine (CR). The plasma concentrations of VPA, 3-oxo-VPA, 2-en-VPA, and CR were determined. The analysis of VPA and its metabolites was performed by GC–MS. There was an increase in plasma 3-oxo-VPA concentration at 0800, sampling as compared to 2000 sampling (p <. 05). The urinary excretion of 3-oxo-VPA and VPA glucuronides were decreased between 2000 and 0800, compared to between 0800, and 2000, by 40% and 50% respectively (p <. 05). These results indicate a nocturnal decrease in renal clearance of 3-oxo-VPA rather than a decrease in the β-oxidation of VPA at night. These differences were not explained by differences between the sampling periods in CR excretion. These results indicate the importance of collecting samples of 24-h duration when studying metabolic profiles of VPA. (Chronobiology International, 18(1), 123–129, 2001)     

Effect of Ginkgo biloba extract on oxidative metabolism of valproic acid in hepatic microsomes from donors with the CYP2C9*1/*1 genotype

We investigated the effect of Ginkgo biloba extracts and some of its individual constituents on the oxidative metabolism of valproic acid (VPA) in hepatic microsomes from donors with the CYP2C9*1/*1 genotype. G. biloba extract decreased 4-ene-VPA, 3-OH-VPA, 4-OH-VPA, and 5-OH-VPA formation with mean (+/- SE) IC50 values of 340 +/- 40 microg/mL, 370 +/- 100 microg/mL, 180 +/- 30 microg/mL, and 210 +/- 20 microg/mL, respectively. This was associated with inhibition of not only CYP2C9*1, but also CYP2A6 and CYP2B6. Bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, and isorhamnetin-3-O-rutinoside were not responsible for the inhibition of VPA metabolism by the extract. When analyzed as the sum of the aglycone and total glycosides present in the extract, quercetin decreased 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by 76%, 51%, and 70%, respectively, kaempferol decreased 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by 65%, 46%, and 49%, respectively, and isorhamnetin decreased 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by 29%, 26%, and 31%, respectively. The 3 aglycones did not affect 3-OH-VPA formation. In summary, G. biloba extract decreased hepatic microsomal formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA, but the effect was not due to the terpene trilactones or flavonol glycosides investigated in our study.     

Hypotension and bradycardia during caloric restriction in mice are independent of salt balance and do not require ANP receptor

We hypothesized that caloric restriction (CR)-induced hypotension would correlate with increased sodium excretion through an atrial natriuretic peptide (ANP)-dependent mechanism. To test this hypothesis, the cardiovascular parameters of c57/Bl mice were measured with radiotelemetry while urine was collected. The 23-h mean blood pressure (BP) dropped from 108.6 +/- 1.8 to 92.7 +/- 2.4 mmHg, and 23-h heart rate dropped from 624 +/- 5 to 426 +/- 13 beats/min over 7 days of CR at 29 degrees C. Contrary to our hypothesis, urine sodium excretion decreased by 55% by day 7 of CR. Consistent with decreased sodium excretion was the drop in plasma ANP (from 82.4 +/- 4.3 to 68.0 +/- 5.8 pg/ml). To explore the possibility that CR lowers BP through an ANP receptor-dependent mechanism that is independent of its effect on sodium retention, we measured the cardiovascular parameters of mice deficient in the ANP receptor (NPR1(-/-)) or the ANP clearance receptor (NPR3(-/-)). Mean BP fell from 117.1 +/- 3.9 to 108.0 +/- 4.7 mmHg in the NPR1(-/-) mice and from 87.0 +/- 2.4 to 78.4 +/- 1.7 mmHg in the NPR3(-/-) mice during CR. These data indicate that the hypotension induced by CR does not depend on increased sodium excretion. Rather, it appears that the mouse responds to the low BP induced by CR with an increase in sodium reabsorption. Furthermore, circulating ANP levels and data from NPR1(-/-) and NPR3(-/-) mice suggest that the ANP pathway may not be involved in the cardiovascular response to CR.     

Valproic acid disposition in rabbits during chronic treatment with Escherichia coli endotoxin

The disposition of valproic acid (VPA) in rabbits was studied after chronic treatment with Escherichia coli endotoxin. Endotoxin (1-2 micrograms/kg) was administered daily to 10 male New Zealand white rabbits for 5 days. On day 5, 50 mg/kg of VPA was given iv during the time of the peak febrile response. Blood samples were drawn at appropriate time intervals and analyzed for free and total VPA levels as well as plasma proteins and free fatty acids. The data were compared with similar control experiments performed 2 weeks before and after endotoxin treatment. Pharmacokinetic analysis indicated that the changes in free VPA clearance after endotoxin were related to the change in the febrile response during chronic treatment (r = 0.77; p less than 0.05); that is, animals which developed tolerance to the febrile response showed elevated drug clearance, whereas nontolerant animals showed decreased clearance of VPA.     

An integrated study of acute effects of valproic acid in the liver using metabonomics, proteomics, and transcriptomics platforms

An integrated omics approach was undertaken in order to elucidate a systems biology level understanding of the acute hepatotoxcity of valproic acid (VPA). Metabonomics, proteomics and gene expression microarray platforms were employed in this systems biology study. CD-1 female pregnant mice were injected subcutaneously with 600 mg/kg VPA or vehicle control. Urine, serum, and liver tissue were collected at 6, 12, and 24 h after dosing. Principal component analysis (PCA) of the metabonomics data showed clustering of the dosed groups away from the controls for the urine samples. Looser clustering was seen in the other sample sets investigated. However, VPA administration resulted in altered glucose concentrations in urine samples at 12 and 24 h and in aqueous liver tissue extracts at 12 h after VPA administration. Proteomics studies identified two proteins, glycogen phosphorylase and amylo-1,6-glucosidase, which were increased in dosed animals relative to control. Both of these proteins are involved in converting glycogen to glucose. Examination of the expression of 20,000 liver genes did not reveal significantly altered expression at 6, 12, or 24 h after VPA exposure. The combined studies indicated a perturbation in the glycogenolysis pathway following administration of VPA.     

Red wine anthocyanins are rapidly absorbed in humans and affect monocyte chemoattractant protein 1 levels and antioxidant capacity of plasma

Epidemiological studies suggest that a moderate consumption of anthocyanins may be associated with protection against coronary heart disease. The main dietary sources of anthocyanins include red-coloured fruits and red wine. Although dietary anthocyanins comprise a diverse mixture of molecules, little is known how structural diversity relates to their bioavailability and biological function. The aim of the present study was to evaluate the absorption and metabolism of the 3-monoglucosides of delphinidin, cyanidin, petunidin, peonidin and malvidin in humans and to examine both the effect of consuming a red wine extract on plasma antioxidant status and on monocyte chemoattractant protein 1 production in healthy human subjects. After a 12-h overnight fast, seven healthy volunteers received 12 g of an anthocyanin extract and provided 13 blood samples in the 24 h following the test meal. Furthermore, urine was collected during this 24-h period. Anthocyanins were detected in their intact form in both plasma and urine samples. Other anthocyanin metabolites could also be detected in plasma and urine and were identified as glucuronides of peonidin and malvidin. Anthocyanins and their metabolites appeared in plasma about 30 min after ingestion of the test meal and reached their maximum value around 1.6 h later for glucosides and 2.5 h for glucuronides. Total urinary excretion of red wine anthocyanins was 0.05+/-0.01% of the administered dose within 24 h. About 94% of the excreted anthocyanins was found in urine within 6 h. In spite of the low concentration of anthocyanins found in plasma, an increase in the antioxidant capacity and a decrease in MCP-1 circulating levels in plasma were observed.     

Monitoring ovarian function in marmosets and tamarins by the measurement of urinary estrogen metabolites

Practical aspects of urinary estrogen analysis were considered with regard to establishing simple and reliable methods for monitoring ovarian function in marmosets and tamarins. Changes in the hormone:creatinine ratio in small volumes of urine from the common marmoset were significantly correlated with changes in 24-h excretion. Comparison of the metabolism and excretion of estrogens during the ovarian cycle in the common marmoset and cottontop tamarin revealed interesting species differences. High concentrations of conjugated estrone were measured in marmoset plasma, but estradiol 17β was the predominant estrogen in urine. In contrast, estrone was the most abundant estrogen measured in tamarin urine. Both species excreted very little estriol. Sulfates and glucuronides were present in urine in similar proportions before ovulation in the marmoset, although after ovulation sulfates were the more abundant. Conversely, most of the estrogens in tamarin urine appeared to be conjugated as glucuronides. Direct assay for estrone sulfate was applied to the measurement of urinary estrogen excretion during the ovarian cycle in a marmoset. The results compared well with those for total estradiol 17β after hydrolysis and ether extraction. The use of direct assays for conjugated estrogens in small volumes of urine is suggested as a practical method for monitoring ovarian function in marmosets and tamarins.     

Effects of fresh and seawater ingestion on osmoregulation in Atlantic bottlenose dolphins (Tursiops truncatus)

Bottlenose dolphins (Tursiops truncatus) are marine mammals with body water needs challenged by little access to fresh water and constant exposure to salt water. Osmoregulation has been studied in marine mammals for a century. Research assessing the effects of ingested fresh water or seawater in dolphins, however, has been limited to few animals and sampling times. Nine 16- to 25-h studies were conducted on eight adult dolphins to assess the hourly impact of fresh water, seawater, and seawater with protein ingestion on plasma and urine osmolality, urine flow rate (ufr), urinary and plasma solute concentrations, and solute clearance rates. Fresh water ingestion increased ufr. Fresh water ingestion also decreased plasma and urine osmolality, sodium and chloride urine concentrations, and solute excretion rates. Seawater ingestion resulted in increased ufr, sodium, chloride, and potassium urine concentrations, sodium excretion rates, and urine osmolality. Seawater with protein ingestion was associated with increased ufr, plasma osmolality, sodium excretion, and sodium, chloride, potassium, and urea urine concentrations. In conclusion, bottlenose dolphins appear to maintain water and plasma solute balance after ingesting fresh water or seawater by altering urine osmolality and solute clearance. Ingestion of protein with seawater appears to further push osmoregulation limits and urine solute concentrations in dolphins.     

F2-isoprostane excretion rate and diurnal variation in human urine

8-Iso-PGF2alpha is formed in vivo by non-enzymatic free radical catalysed oxidation of arachidonic acid. Urinary measurement of this compound has previously been shown to reflect the oxidative stress of the body in human and animal studies. To investigate the normal excretion rate and a possible diurnal variation of 8-iso-PGF2alpha excretion in humans urinary samples were collected from ten healthy volunteers of both sexes at different times during a day and as a 24-h urine sample. The samples were analyzed by a newly developed radioimmunoassay with a specific antibody against free 8-iso-PGF2alpha. There was no diurnal variation in the urinary levels of 8-iso-PGF2alpha during the day in this study. Neither was there any statistically significant difference between the 8-iso-PGF2alpha levels at any time of the day or in the morning urine samples compared to the 24-h urine samples. In conclusion, all urine samples collected at any time of the day, preferably a morning urine sample (representative urine from 6-8 hours), can thus be used to obtain a reliable and adequate value of the amount of the 8-iso-PGF2alpha excretion in urine in healthy individuals.     

Circadian time-dependent hepatic and renal toxicities to valproic acid in mice

This study aims to investigate whether hepatic and renal valproic acid (VPA) toxicities varied according to the dosing time in the 24-h scale in mice. VPA was administered by i.p. route to different groups of animals at four different circadian stages (1, 7, 13, and 19 h after light onset (HALO)). Biochemical study and histopathological examinations on liver and kidney sections were performed. The results showed that the hepatic and renal toxicity induced by VPA was time related. Animals treated at 19 HALO showed vacuolar degenerative changes, congestions, and inflammatory areas on liver parenchyma. Lesions within proximal tubules were observed in the kidney in groups treated at 19 HALO. The largest increases in alanine aminotransferase, alkaline phosphatase and plasma creatinine activities were also observed at 19 HALO. The obtained data indicate that the optimal hepatic and renal tolerance is observed when VPA was injected in the middle of the light-rest span of mice.     

The role of fetal urinary excretion in the transfer of ethanol into amniotic fluid after maternal administration of ethanol to the near-term pregnant ewe

The objective of this study was to determine whether fetal urinary excretion is a major route of ethanol transfer into the amniotic fluid surrounding the fetus following maternal administration of ethanol. Conscious instrumented pregnant ewes between 130 and 137 days' gestation (term, 147 days) with (n = 3) or without (n = 3) a catheter in the fetal bladder were administered 1 g ethanol/kg maternal body weight as a 1-h maternal intravenous infusion. Maternal blood, fetal blood, and amniotic fluid samples were collected at selected times, and fetal urine was collected continuously from the bladder-cannulated fetus during the 14-h study for the determination of ethanol concentrations. Fetal urinary excretion of ethanol occurred, and the total amount of ethanol excreted represented 0.30 +/- 0.07 (SD)% of the maternal ethanol dose. The renal clearance of ethanol by the fetus was 0.43 +/- 0.06 mL/min. The pharmacokinetics of ethanol in the maternal-fetal unit and the amniotic fluid for the bladder-cannulated fetal preparation were similar to the data for the nonbladder-cannulated preparation. The data indicate that fetal urinary excretion of ethanol is a secondary route of ethanol transfer into the amniotic fluid. It would appear that diffusion of ethanol across membranes from the maternal and fetal circulations is a major route of ethanol transfer into this intrauterine compartment.     

Validation and Assessment of Three Methods to Estimate 24-h Urinary Sodium Excretion from Spot Urine Samples in Chinese Adults

24-h urinary sodium excretion is the gold standard for evaluating dietary sodium intake, but it is often not feasible in large epidemiological studies due to high participant burden and cost. Three methods—Kawasaki, INTERSALT, and Tanaka—have been proposed to estimate 24-h urinary sodium excretion from a spot urine sample, but these methods have not been validated in the general Chinese population. This aim of this study was to assess the validity of three methods for estimating 24-h urinary sodium excretion using spot urine samples against measured 24-h urinary sodium excretion in a Chinese sample population. Data are from a substudy of the Prospective Urban Rural Epidemiology (PURE) study that enrolled 120 participants aged 35 to 70 years and collected their morning fasting urine and 24-h urine specimens. Bias calculations (estimated values minus measured values) and Bland-Altman plots were used to assess the validity of the three estimation methods. 116 participants were included in the final analysis. Mean bias for the Kawasaki method was -740 mg/day (95% CI: -1219, 262 mg/day), and was the lowest among the three methods. Mean bias for the Tanaka method was -2305 mg/day (95% CI: -2735, 1875 mg/day). Mean bias for the INTERSALT method was -2797 mg/day (95% CI: -3245, 2349 mg/day), and was the highest of the three methods. Bland-Altman plots indicated that all three methods underestimated 24-h urinary sodium excretion. The Kawasaki, INTERSALT and Tanaka methods for estimation of 24-h urinary sodium excretion using spot urines all underestimated true 24-h urinary sodium excretion in this sample of Chinese adults. Among the three methods, the Kawasaki method was least biased, but was still relatively inaccurate. A more accurate method is needed to estimate the 24-h urinary sodium excretion from spot urine for assessment of dietary sodium intake in China.     

Prediction and detection of ovulation by the measurement of urinary pregnanetriol-3 alpha-glucuronide. A multicentre study

The urine excretion pattern of pregnanetriol 3 alpha-glucuronide (PT-3G) throughout the menstrual cycle in 26 normal ovulating women was evaluated in a multicentre study. The concentration of PT-3G was measured by radioimmunoassay in daily samples of early morning urine (EMU) from 20 women for three consecutive cycles and from 6 women who conceived during the period of study. PT-3G was also measured in 24-h urine samples from 5 additional women. The peak of urine LH was used as a reference point for ovulation (Day 0). The EMU concentration of PT-3G in the follicular phase of 60 normal ovulatory cycles was 5.10 mumol/l +/- 0.11 (arithmetic mean +/- SE). The first PT-3G defined rise (CUSUM analysis) occurred during the late follicular phase (Days -3 to 0) with a PT-3G maximum excretion (9.69 mumol/1 +/- 0.55) on Day 0, whereas a PT-3G excretion peak occurred during mid-luteal phase (Days +5 to +9). The overall PT-3G excretion during the luteal phase (8.06 mumol/1 +/- 0.17) was significantly higher than that of the follicular phase (P less than 0.001). A further sustained increase in PT-3G excretion was noted after day +11 in the conceptional cycles. The 24-h excretion profile of PT-3G was similar to that obtained in EMU samples. No inter-centre significant variation was noticed in terms of PT-3G concentration values. The results were interpreted as demonstrating that the PT-3G excretion profile throughout the cycle exhibits a close resemblance to that of serum 17-OH progesterone. The data also indicates that although the immunoanalytical measurement of this urine steroid metabolite does not give an early sign for the occurrence of ovulation, it can be used for both the immediate prediction and the detection of ovulation.     

Monitoring urinary excretion of 5-hydroxymethyluracil for assessment of oxidative DNA damage and repair

Urinary excretion of oxidized nucleobases and nucleosides has been used as a biomarker of oxidative DNA damage and repair. Most studies have focused on the measurements of 8-oxo-7,8-dihydro-2'-deoxyguanosine; however, the urinary levels of other DNA modifications may represent useful indicators of oxidative stress. We developed a method for the determination of 5-hydroxymethyluraciI (5-HMUra), consisting of the separation of the modified base in urine by HPLC and quantification by GC/MS in the selective ion monitoring mode. This experimental approach was subsequently validated in human samples, with the effect of storage and the inter- and intra-individual variations in 5-HMUra excretion being evaluated. Results showed that 5-HMUra is stable in samples frozen at-80 °C for at least 4 months. Inter-individual variations in 5-HMUra excretion were observed when the results were expressed either as nmoles excreted per kg per day (1.2-2.4) or corrected by creatinine values (7.2-12.2 nmoles 5-HMUra per mmoles creatinine). Intra-individual variability was low, varying slightly at different time collections for several individuals. Differences in the excretion of 5-HMUra in urine collected at three different 8-h intervals during the day were not significant and, in particular, the levels of 5-HMUra calculated from the overnight or the 24-h samples were highly correlated. These results indicate that monitoring urinary levels of 5-HMUra could be a suitable indicator of oxidative damage in human studies.     

Monitoring urinary excretion of 5-hydroxymethyluracil for assessment of oxidative DNA damage and repair

Abstract

Urinary excretion of oxidized nucleobases and nucleosides has been used as a biomarker of oxidative DNA damage and repair. Most studies have focused on the measurements of 8-oxo-7,8-dihydro-2′-deoxyguanosine; however, the urinary levels of other DNA modifications may represent useful indicators of oxidative stress. We developed a method for the determination of 5-hydroxymethyluraciI (5-HMUra), consisting of the separation of the modified base in urine by HPLC and quantification by GC/MS in the selective ion monitoring mode. This experimental approach was subsequently validated in human samples, with the effect of storage and the inter- and intra-individual variations in 5-HMUra excretion being evaluated. Results showed that 5-HMUra is stable in samples frozen at-80 °C for at least 4 months. Inter-individual variations in 5-HMUra excretion were observed when the results were expressed either as nmoles excreted per kg per day (1.2–2.4) or corrected by creatinine values (7.2–12.2 nmoles 5-HMUra per mmoles creatinine). Intra-individual variability was low, varying slightly at different time collections for several individuals. Differences in the excretion of 5-HMUra in urine collected at three different 8-h intervals during the day were not significant and, in particular, the levels of 5-HMUra calculated from the overnight or the 24-h samples were highly correlated. These results indicate that monitoring urinary levels of 5-HMUra could be a suitable indicator of oxidative damage in human studies.     

Direct high-performance liquid chromatography determination of propofol and its metabolite quinol with their glucuronide conjugates and preliminary pharmacokinetics in plasma and urine of man

Propofol (P) is metabolized in humans by oxidation to 1,4-di-isopropylquinol (Q). P and Q are in turn conjugated with glucuronic acid to the respective glucuronides, propofol glucuronide (Pgluc), quinol-1-glucuronide (Q1G) and quinol-4-glucuronide (Q4G). Propofol and quinol with their glucuronide conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic hydrolysis. The glucuronide conjugates were isolated by preparative HPLC from human urine samples. The glucuronides of P and Q were present in plasma and urine, P and Q were present in plasma, but not in urine. Quinol in plasma was present in the oxidised form, the quinone. Calibration curves of the respective glucuronides were constructed by enzymic deconjugation of isolated samples containing different concentrations of the glucuronides. The limit of quantitation of P and quinone in plasma are respectively 0.119 and 0.138 μg/ml. The limit of quantitation of the glucuronides in plasma are respectively: Pgluc 0.370 μg/ml, Q1G 1.02 μg/ml and Q4G 0.278 μg/ml. The corresponding values in urine are: Pgluc 0.264 μg/ml, Q1G 0.731 μg/ml and Q4G 0.199 μg/ml. A pharmacokinetic profile of P with its metabolites is shown, and some preliminary pharmacokinetic parameters of P and Q glucuronides are given.     

Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays

The ATP-binding cassette (ABC) genes encode the largest family of transmembrane proteins. ABC transporters translocate a wide variety of substrates across membranes, but their physiological function is often incompletely understood. We describe a new method to study the substrate spectrum of ABC transporters: We incubate extracts of mouse urine with membrane vesicles prepared from Spodoptera frugiperda Sf9 insect cells overproducing an ABC transporter and determine the compounds transported into the vesicles by LC/MS-based metabolomics. We illustrate the power of this simple "transportomics" approach using ABCC2, a protein present at sites of uptake and elimination. We identified many new substrates of ABCC2 in urine. These included glucuronides of plant-derived xenobiotics, a class of compounds to which humans are exposed on a daily basis. Moreover, we show that the excretion of these compounds in vivo depends on ABCC2: compared to wild-type mice, the urinary excretion of several glucuronides was increased up to 20-fold in Abcc2(-/-) mice. Transportomics has broad applicability, as it is not restricted to urine and can be applied to other ATP-dependent transport proteins as well.     

Urinary excretion of epsilondA is not predictive of cancer development: a prospective nested case-control study

Human biomonitoring of the lipid peroxidation DNA modification 1,N⁶-ethenodeoxyadenosine (εdA) excreted into urine is thought to be a potential marker for oxidative stress-related DNA damage and human cancer. We have tested this hypothesis in a prospective, nested case-control study. During the years 1984-1989, 24-h urines were collected from 1956 men in the Kuopio Ischaemic Heart Disease (KIHD) Risk Factor Study. εdA concentrations were measured by LC-MS/MS in 24-h urine samples from 47 men with cancer diagnosed at follow up until 2001 and from 31 cancer free smoking-matched control subjects. Odds ratio for having higher than control median εdA excretion rate and cancer, estimated by binary logistic regression, was 0.73 (95% CI 0.29-1.80, p=0.49). In this study, the urinary excretion of εdA provides no additional prediction of cancer development in males after controlling for smoking.     

Single valproic acid treatment inhibits glycogen and RNA ribose turnover while disrupting glucose-derived cholesterol synthesis in liver as revealed by the [U-13C6]-d-glucose tracer in mice

Previous genetic and proteomic studies identified altered activity of various enzymes such as those of fatty acid metabolism and glycogen synthesis after a single toxic dose of valproic acid (VPA) in rats. In this study, we demonstrate the effect of VPA on metabolite synthesis flux rates and the possible use of abnormal ¹³C labeled glucose-derived metabolites in plasma or urine as early markers of toxicity. Female CD-1 mice were injected subcutaneously with saline or 600 mg/kg) VPA. Twelve hours later, the mice were injected with an intraperitoneal load of 1 g/kg [U-¹³C]-d-glucose. ¹³C isotopomers of glycogen glucose and RNA ribose in liver, kidney and brain tissue, as well as glucose disposal via cholesterol and glucose in the plasma and urine were determined. The levels of all of the positional ¹³C isotopomers of glucose were similar in plasma, suggesting that a single VPA dose does not disturb glucose absorption, uptake or hepatic glucose metabolism. Three-hour urine samples showed an increase in the injected tracer indicating a decreased glucose re-absorption via kidney tubules. ¹³C labeled glucose deposited as liver glycogen or as ribose of RNA were decreased by VPA treatment; incorporation of ¹³C via acetyl-CoA into plasma cholesterol was significantly lower at 60 min. The severe decreases in glucose-derived carbon flux into plasma and kidney-bound cholesterol, liver glycogen and RNA ribose synthesis, as well as decreased glucose re-absorption and an increased disposal via urine all serve as early flux markers of VPA-induced adverse metabolic effects in the host.     

The early effects of valproic acid in low doses in liver metabolism

The effect of low doses of valproic acid (VPA), 0.6 mM in arterial blood, in liver metabolism was studied. Twenty four hour fasted rats were infused into the jugular vein with VPA at a dose of 4 mg/kg/min during 50 min. The right carotid artery was also catheterized in order to draw arterial blood samples for determining VPA concentrations and acid-base parameters. After VPA infusion, a tissue sample of liver was obtained and freeze-clamped. VPA did not change the arterial blood acid-base parameters. The liver tissue concentration of pyruvate and alanine increased in VPA group while lactate concentrations did not change. Concentration of glutamine, glutamate, malate, citrate and aspartate in the liver fell significantly. These results suggest that VPA in low doses may modify the hepatic metabolism of the rat in vivo.